Pharmacological characterization of chick and frog beta adrenergic receptors in primary cultures of myocardial cells. In membrane preparations derived from primary cultures of chick myocardial cells, beta adrenergic receptors modeled for a single low-affinity site for both betaxolol (beta-1-selective) and ICI 118551 (beta-2-selective) displacement of [125I]iodocyanopindolol (ICYP), indicating that the chick beta receptor is pharmacologically distinct from both mammalian beta-1 and beta-2 adrenergic receptors with respect to these antagonists. However, the highly beta-1-selective compound CGP 20712A was able to distinguish two binding sites on ICYP competition curves, a high-affinity "beta-1 site" (75%) and a low-affinity "beta-2 site" (25%). Also, in chick heart cell membranes the relative ability of agonists to displace ICYP produced a profile typical of beta-1 adrenergic receptors with a rank order of potency or efficacy of: isoproterenol greater than epinephrine = norephinephrine. When agonist-mediated adenylyl cyclase stimulation was assessed the order of potency was slightly different, isoproterenol greater than epinephrine greater than or equal to norepinephrine. Additionally, antagonism of isoproterenol stimulation of adenylyl cyclase by CGP 20712A yielded a Kb value (1.16 +/- 0.35 x 10(-7) M) intermediate between the high and low-affinity binding sites of CGP 20712A, suggesting that the low-affinity site is coupled to adenylyl cyclase. In membrane preparations of frog myocardial cells, ICYP/antagonist competition curves modeled for a mixed population of receptors, with subtype percentages varying from 50:50 beta-1:beta-2 to 100% beta-2 depending on the specific antagonist used and the individual cell preparation. For ICYP/agonist competition binding experiments the relative ability to displace ICYP was isoproterenol greater than epinephrine = norepinephrine, a profile typical of beta-1 adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)


Cytochrome P-450 mediates bioactivation of organic nitrates. The cellular mechanism of bioactivation underlying guanylate cyclase activation by organic nitrates was investigated. In cultured rat lung fibroblasts (RFL-6 cells), the inhibitor of cytochrome P-450 proadifen (0.1 mM) decreased cyclic GMP stimulation by glyceryl trinitrate (GTN, 1-100 microM) by up to 81%. Cyclic GMP stimulation by isoidide dinitrate was inhibited to a similar degree under these conditions. However, proadifen did not affect cyclic GMP stimulation by sodium nitroprusside that spontaneously releases nitric oxide. Cyclic GMP stimulation in RFL-6 cells by GTN remained unaltered in the presence of the inhibitor of glutathione S-transferase sulfobromophthalein. In the same cell type, a 24-hr pretreatment with the inducer of cytochrome P-450 3-methylcholanthrene (10 microM) augmented cyclic GMP stimulation by GTN or isoidide dinitrate by up to 102%. Cultured porcine aortic endothelial cells were found to be without a cyclic GMP response to GTN, although sodium nitroprusside produced a marked cyclic GMP elevation in these cells. The endothelial cells remained unresponsive to GTN even in the presence of N-acetylcysteine (5 mM). Moreover, in a cell-free preparation from rat liver, glutathione-dependent biotransformation of GTN was not accompanied by activation of soluble guanylate cyclase. These findings suggest that in intact cells bioactivation of, i.e., nitric oxide formation from organic nitrates is mediated by a cytochrome P-450 enzyme system rather than by glutathione S-transferase or free thiols.


Expression of the multidrug-resistant gene in human musculoskeletal tumors. We measured the levels of messenger RNA of the human multidrug-resistant (MDR) gene in 15 human musculoskeletal tumors. In metastatic tumors and those which did not respond to combination chemotherapy, there was an increased expression of this gene. No evidence of expressions of the MDR gene was found in the benign tumors. The high expression of the MDR gene from musculoskeletal tumors apparently induced a multidrug resistance, and this acquired resistance may be due to outgrowth of the P-glycoprotein-expressing MDR tumor. Elucidation of expression of the MDR gene is an important step in malignant musculoskeletal tumors research.


Central effects of quinpirole on blood pressure of spontaneously hypertensive rats. The i.v. administration of the dopamine D-2 receptor agonist quinpirole induced a rapid increase in blood pressure in spontaneously hypertensive rats (SHR). Heart rate showed little change. The pressor response to quinpirole was similar in SHR and normotensive Wistar-Kyoto rats (WKY) at doses of 0.03 to 0.3 mg/kg but, at 1 mg/kg, quinpirole induced a greater increase in blood pressure in SHR than in WKY. In contrast, although both strains showed a decreased locomotor activity after administration of 0.01 to 0.05 mg/kg of quinpirole, only in WKY was activity enhanced by 0.25 to 1.25 mg/kg of quinpirole. The i.v. administration of the dopamine agonists apomorphine, N-propylnorapomorphine and (R)-(+)-3-(3-hydroxyphenyl)-N-propylpiperidine, but not the putative presynaptic D-2 agonist (S)-(-)-3-(3-hydroxyphenyl)-N- propylpiperidine, induced pressor responses in SHR comparable to those after quinpirole administration. The pressor effect of quinpirole was enhanced by pretreatment with the peripheral D-2 antagonist domperidone, but blocked by the centrally acting dopamine antagonists haloperidol or sulpiride. In SHR, which were pretreated centrally with pertussis toxin, quinpirole induced a significantly smaller increase in blood pressure than in control SHR. Pretreatment centrally with 6-hydroxydopamine had no effect on the pressor action of quinpirole in SHR. Thirty minutes after i.v. administration of quinpirole, an additional injection of quinpirole did not significantly change blood pressure. Increasing the interval between two subsequent injections of quinpirole showed that this desensitization slowly reversed, but only after 24 hr had the pressor response to quinpirole fully recovered.(ABSTRACT TRUNCATED AT 250 WORDS)


Serotonin-stimulated release of [3H]dopamine via reversal of the dopamine transporter in rat striatum and nucleus accumbens: a comparison with release elicited by potassium, N-methyl-D-aspartic acid, glutamic acid and D-amphetamine. Release of preloaded radiolabeled dopamine ([3H]DA) elicited by several agents from terminal fields of mesolimbic and nigrostriatal projections in rats was compared. Several similarities between the two areas were observed. For example, potassium, which stimulates release both directly, through altering the potential across the membrane of the dopaminergic neuron, as well as indirectly, presumably by releasing endogenous excitatory neurotransmitters, exhibited some similarities to release stimulated by L-glutamate and N-methyl-D-aspartic acid. These included sensitivity to tetrodotoxin (TTX), Mg++ and Ca++. In contrast, release of [3H]DA stimulated by serotonin (5-HT), like that stimulated by D-amphetamine, depended upon a functional dopamine transport system and was less sensitive to TTX, Mg++ and Ca++. 5-HT-stimulated [3H]DA release in striatum (STR) and nucleus accumbens (NACC) was not modified by antagonists at 5-HT2 or 5-HT3 receptors. Differences were observed in release of [3H]DA from STR and NACC. Elevated potassium (20 mM) released about twice as much [3H]DA from NACC as it did from STR. 5-HT was also able to release more [3H]DA from NACC than from STR. Conversely, D-amphetamine released more [3H]DA from STR than from NACC. TTX increased release stimulated by potassium in STR, but decreased release stimulated by potassium in NACC. These observations suggest that receptor- and non-receptor-mediated mechanisms may contribute to regulation of [3H]DA release in mesolimbic and nigrostriatal areas of the brain. It is possible that endogenous 5-HT in STR or NACC acts as a local regulator of DA release acting via a transport-dependent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)


Dihydrexidine, a novel full efficacy D1 dopamine receptor agonist. The present work provides a detailed pharmacological characterization of dihydrexidine (DHX) (trans-10,11-dihydroxy- 5,6,6a,7,8,12b-hexahydrobenzo[a]phenanthridine), the first high-potency, full efficacy, bioavailable D1 dopamine receptor agonist. DHX represents a new conformationally rigid structural class of dopamine receptor ligands. It competes stereoselectively and potently for D1 binding sites in rat striatal membranes labeled with [3H]SCH23390 with an IC50 of about 10 nM compared to about 30 nM for the prototypical D1 agonist SKF38393. Like other dopamine agonists, DHX has a shallow competition curve (nH = ca. 0.7) that can be fitted by a two-site model consisting of high-affinity (63%; KD = 3 nM) and low-affinity (37%; KD = 75 nM) sites. DHX was screened for activity against 40 other binding sites, and was inactive (IC50 greater than 10 microM) against all except D2 dopamine receptors (IC50 = 130 nM) and alpha 2 adrenoreceptors (IC50 = ca. 230 nM). Functionally, DHX is a full efficacy dopamine D1 agonist. In homogenates of rat striatum, DHX or dopamine doubles the rate of cyclic AMP synthesis, whereas SKF38393 only causes a maximal increase of about 50%. These effects of DHX are blocked by the selective D1 antagonist SCH23390, but are not affected by D2, 5-hydroxytryptamine2, muscarinic, or alpha or beta adrenergic antagonists. Because DHX is known to cause D2-like behavioral effects at high doses, the nature of its D2 activity was characterized using prolactin release as an end-point. DHX and the prototypical D2 agonist quinpirole both caused a significant inhibition of the prolactin release induced by 5-hydroxytryptophan. These effects of DHX are not due to "indirect" alterations at the presynaptic terminal, because DHX is essentially inactive at inhibiting the dopamine uptake system, and does not cause the release of dopamine. These data demonstrate the utility of DHX for probing the biochemistry and function of D1 dopamine receptors.


Relationship between brain levels of 3-(3-hydroxyphenyl)-N-n-propylpiperidine HCl enantiomers and effects on locomotor activity in rats. After s.c. administration of 3-(3-hydroxyphenyl)-N-n-propylpiperidine HCl (3-PPP) to rats, plasma and brain levels were monitored in relation to the amount of spontaneous locomotor activity. Based on time-course relationships, concentration-effect curves were elaborated after administration of (-)3-PPP and (+)3-PPP in the dose range of 1 to 256 mumol.kg-1. Both enantiomers were readily absorbed and distributed, as evidenced by a close correlation between brain and plasma levels, brain levels being 7 to 9 times higher than those found in plasma. Plasma half-lives were 25 and 32 min for (-)3-PPP and (+)3-PPP, respectively. Plotting brain concentrations of (-)3-PPP against locomotor activity resulted in a good fit to a declining two-phase curve, in all probability reflecting preferential actions at pre- and postsynaptic dopamine receptors, respectively. These two underlying mechanisms could also be identified from the biphasic effects produced by (+)3-PPP on locomotor activity: suppression followed by stimulation, respectively. Together, these observations provide further support for the contention that at low doses, both enantiomers have sedative actions due to stimulation of inhibitory autoreceptors. With increasing doses, however, a postsynaptic receptor blockade will predominate for a partial agonist like (-)3-PPP, producing suppression of locomotion, whereas the full agonist, (+)3-PPP, will produce behavioral activation due to stimulation of postsynaptic receptors.


Neuroanatomical sites mediating the central actions of beta-endorphin as mapped by changes in glucose utilization: involvement of mu opioid receptors. Local cerebral glucose utilization, which is a correlate of neuronal activity, was measured to obtain information on the neuroanatomical sites mediating the different behaviors elicited by i.c.v. administration of the opioid peptide beta-endorphin (beta-END). The selective mu and delta opioid receptor antagonists d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) and ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH), respectively, were used to characterize the opioid receptor type involved in the actions of beta-END. beta-END was found to produce profound increases in glucose utilization in limbic regions such as the lateral septal nucleus, the amygdalo-hippocampal transition area, the nucleus accumbens and the hippocampal formation. The ventral hippocampus proved the most sensitive structure, displaying increases in glucose utilization of up to 200%; changes in the dorsal part amounted up to 100%. Only moderate effects were induced by beta-END in motor areas, such as the substantia nigra, pars reticulata and the nucleus ruber. This regional pattern of changes is assumed to underlie the epileptogenic-, motivational-, mood- and possibly memory-modulating actions of beta-END. The effects of beta-END on local cerebral glucose utilization were blocked by pretreatment with the mu antagonist, CTOP, whereas the selective delta opioid antagonist ICI 174,864 was less effective. An involvement of predominantly mu opioid receptors in the central actions of beta-END is, therefore, suggested.


Profile of action of a novel 5-hydroxytryptamine1A receptor ligand E-4424 to inhibit aversive behavior in the mouse, rat and marmoset. E-4424 (2-(4-[4-(4-chloro-1-pyrazolyl)butyl]-1-piperazinyl)pyrimidine) was shown to be a 5-hydroxytryptamine1A receptor ligand in radioligand binding assays and in an in vitro guinea pig ileum preparation had both 5-hydroxytryptamine1A antagonist and agonist effects. The antagonist/agonist ratio of E-4424 was greater than in the case of buspirone and ipsapirone. E-4424 was compared to diazepam, buspirone and ipsapirone to inhibit the behavioral response to an aversive situation in the mouse black and white test box, the rat social interaction test and a marmoset human threat test. The acute administration of E-4424 (0.0001-0.5 mg/kg, i.p.) to the mouse decreased aversion to the white area of the test box and was as effective as diazepam (0.125-1.0 mg/kg, i.p.) and much more potent than buspirone (0.25-1.0 mg/kg, i.p.) or ipsapirone (0.5-5.0 mg/kg, i.p.). E-4424 was also effective in enhancing rat social interaction and reducing anxiety-related behaviors in the marmoset and was again more potent than diazepam, buspirone or ipsapirone. Withdrawal from a 14-day administration of diazepam, cocaine, nicotine or alcohol exacerbated the response to the aversive situation in the mouse test. This was not observed after withdrawal from a chronic treatment with E-4424, buspirone or ipsapirone. However, E-4424 administered during drug withdrawal prevented the response caused by withdrawal from cocaine, alcohol, nicotine and diazepam: buspirone was ineffective and ipsapirone only attenuated that syndrome after alcohol withdrawal.(ABSTRACT TRUNCATED AT 250 WORDS)


Mosquito and arbovirus ecology in southeastern California, 1986-1990. Mosquito abundance and western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) virus activity were monitored in five valleys in southeastern California from June 1986 through April 1990 to study virus overwintering and possible dissemination from south to north along geographically defined corridors. Culex tarsalis Coquillett predominated in CO2 trap collections and was the only species repeatedly infected with WEE and SLE viruses. Abundance peaked during April-May and August-October. WEE virus infections in Cx. tarsalis generally were detected after the spring peak and were followed approximately 1 mo later by seroconversions in sentinel chickens. SLE virus infections occurred later in the summer but before the fall peak in Cx. tarsalis abundance. Peak Cx. tarsalis abundance occurred when monthly temperatures averaged 25 degrees C, whereas virus infections generally were detected most frequently when temperatures exceeded 29 degrees C. Although the spring increase in Cx. tarsalis abundance occurred earlier in southern valleys, the onset of virus activity was variable among valleys and did not follow a south to north progression.


Coronary ostial endarterectomy in Takayasu's aortitis--confirmation of patency nine years postsurgically. A case of Takayasu's aortitis with severe bilateral coronary ostial stenosis is reported. A transaortic coronary endarterectomy was performed and sufficient patency was confirmed angiographically 9 years after the operation. This is the first report of late coronary angiography after a transaortic coronary ostial endarterectomy in Takayasu's aortitis. The efficacy of this procedure for coronary ostial stenosis in Takayasu's aortitis is emphasized.


Neuroleptic malignant syndrome.

Neuroleptic malignant syndrome. NMS is a life-threatening, hyperpyrexic syndrome that follows the blockage of certain central dopaminergic receptor sites; it is commonly associated with the use of neuroleptic medications. Clinical signs usually include hyperthermia, altered mental status, muscle rigidity, and autonomic instability. Treatment is mainly supportive. Dopamine agonists and muscle relaxants are often used in therapy; however, their effectiveness has never been adequately determined in controlled studies.


Assessment of X-chromosome inactivation patterns using the hypervariable probe M27 beta in normal hemopoietic cells and acute myeloid leukemic blasts. The value of the hypervariable X-linked probe M27 beta for use in the analysis of X-chromosome inactivation patterns in normal blood and bone marrow cells and in the assessment of clonality in acute myeloid leukemia (AML) blast cells has been determined. By electrophoresing samples for 30 h, heterozygosity of the M27 beta locus was demonstrable in 324/415 females (78%) and this value could be increased to 93% by electrophoresing for 50 h. Determination of the X-chromosome inactivation patterns in blood and bone marrow samples from hematologically normal females was possible in approximately 90% of heterozygous individuals. The X-chromosome inactivation ratios obtained with M27 beta were comparable with phosphoglycerate kinase or hypoxanthine phosphoribosyl transferase in 46 individuals heterozygous for one of these genes in addition to M27 beta. The results obtained were closely correlated (r = 0.89). In 21 of 35 (60%) AML blasts, however, there was hypermethylation of the M27 beta locus and clonal analysis was not possible. Hypermethylation was not related to FAB type.


Cell-cycle-associated expressions of proliferating cell nuclear antigen and Ki-67 reactive antigen of bone marrow blast cells in childhood acute leukemia. To investigate the growth characteristics of human leukemia cells, the expression of proliferation-associated nuclear antigens was examined in relation to cell cycle phases in marrow blast cells obtained from 37 untreated children with acute leukemia. Ki-67 monoclonal antibody reactive antigen and proliferating cell nuclear antigen (PCNA) were measured by the simultaneous flow cytometric analysis of DNA and nuclear antigens. The percentage of PCNA-positive cells was always higher than that of Ki-67-positive cells in individual patients. The level of PCNA was greatly increased in G1 or early S phase, but was generally stable in S and G2 phases. Accordingly, most of the cells in the proliferative compartments (greater than 2C DNA) showed a high expression of PCNA. In contrast, expression of Ki-67 antigen varied greatly from patient to patient, and differed significantly in different subtypes of the disease. The level of Ki-67 antigen increased with the cell cycle progression, showing maximum expression in late S and G2 phases. However, in most of the patients, a distinct population of Ki-67-negative cells was found not only in G1 phase, but also in the proliferative compartments. These results appear to reflect differences in the proliferative activity of bone marrow blast cells in childhood acute leukemia.


High level of HTLV-I specific protein expression in a patient with adult T-cell leukemia, chronic progressive myelopathy and Kaposi's sarcoma. Analysis was made of serum anti-HTLV-I antibodies, virus-specific proteins in peripheral blood lymphocytes (PBL) and proviruses in lymphocyte DNA of a patient with adult T-cell leukemia (ATL), Kaposi's sarcoma, and chronic myelopathy. Using Western blot and PCR (with HIV-1 specific primers), it was shown that Kaposi's sarcoma was not linked to HIV infection. Western blot analysis of serum revealed antibodies against p19, p24 and Pr 53 of HTLV-I. Examination of proteins in fresh PBL by Western blot revealed a high level of HTLV-I specific protein expression. Southern blot analysis of the patient's DNA revealed two different sites for HTLV-I provirus integration.


Cellular and humoral effects of naftopidil in man. The effects of naftopidil on the intracellular concentration and transmembrane fluxes of Na+ and K+ in erythrocytes and on the intracellular Na+, K+ and free cytosolic Ca2+ concentration in platelets were studied in twenty-four normal male subjects, using a double-blind study design. After a run-in period on placebo for 1 week, the subjects were treated with either placebo (n = 8) or naftopidil 25 mg (n = 8) or 50 mg (n = 8) once a day for 4 weeks. Intraerythrocyte Na+ concentration and the erythrocyte anion carrier were decreased during naftopidil administration. No significant effect of naftopidil could be demonstrated on ouabain-sensitive Na+ efflux, bumetanide-sensitive Na+ efflux, ouabain, bumetanide-resistant Na+ and K+ efflux and Na+, Li(+)-countertransport activity in red blood cells or on the intraerythrocyte K+ and Mg2+ concentration. The free cytosolic Ca2+ and Na+ concentration in platelets was also decreased during naftopidil administration while no effect of naftopidil was found on the intracellular K+ and Mg2+ concentration in platelets.


Effect of preservation solution on results of cadaveric kidney transplantation. The European Multicentre Study Group. University of Wisconsin (UW) preservation solution has been reported to be beneficial for canine organ transplants and for human liver and pancreas transplants. To examine whether it affects renal graft survival, a randomised multicentre trial was conducted to compare its effect with that of EuroCollins solution on delayed graft function, renal function, and patient and graft survival in 695 recipients of cadaveric renal transplants. 352 kidneys were preserved with UW and 343 with EuroCollins solution. Delayed graft function occurred in 23% of the UW group and in 33% of the EuroCollins group (p = 0.003). Three factors other than type of preservation fluid were associated with a higher incidence of delayed graft function: older donor age, intracerebral haemorrhage in the donor, and oliguria in the donor. Renal function as indicated by serum creatinine concentration was better in the UW than in the EuroCollins group. Patient survival in the UW and EuroCollins groups after 1 year was 95% and 94%, respectively. In both groups, delayed graft function reduced 1-year graft survival by 15% (p = 0.0001). 1-year graft survival of UW-preserved kidneys was 6% higher than that of controls (88.2% vs 82.5%, p = 0.04). Delayed graft function is significantly associated with a reduction in 1-year graft survival. The preservation solution is the most important factor influencing development of delayed graft function, and UW solution is superior to EuroCollins solution in reducing occurrence of delayed graft function, improving graft function, extending graft survival.


Schizophrenia and city life.

Schizophrenia and city life. Prevalence of schizophrenia and rates of first admission to hospital for this disorder are higher in most modern industrialised cities, and in urban compared with rural areas. The "geographical drift" hypothesis (ie, most schizophrenics tend to drift into city areas because of their illness or its prodrome) has remained largely unchallenged. We have investigated the association between place of upbringing and the incidence of schizophrenia with data from a cohort of 49,191 male Swedish conscripts linked to the Swedish National Register of Psychiatric Care. The incidence of schizophrenia was 1.65 times higher (95% confidence interval 1.19-2.28) among men brought up in cities than in those who had had a rural upbringing. The association persisted despite adjustment for other factors associated with city life such as cannabis use, parental divorce, and family history of psychiatric disorder. This finding cannot be explained by the widely held notion that people with schizophrenia drift into cities at the beginning of their illness. We conclude that undetermined environmental factors found in cities increase the risk of schizophrenia.


Loss of heterozygosity on chromosome 17p and mutant p53 in HPV-negative cervical carcinomas. Inactivation of the protein product of the wild-type tumour suppressor gene p53 through complexing of the protein with the E6 oncoprotein of human papillomaviruses (HPV) in HPV-infected cells is thought to be important in the aetiology of cervical carcinoma. Mutations of p53 have also been reported in HPV-negative carcinomas, and we now demonstrate loss of heterozygosity (LOH) of chromosome region 17p13 (in which p53 is located) in such tumours. Immunocytochemical staining with monoclonal antimutant-p53 antibody revealed that the carcinomas with LOH on 17p and completely lacking HPV DNA sequences had mutant p53. Thus the LOH had apparently resulted in the loss of the wild-type allele. Consequently, in both HPV-positive and HPV-negative tumours there is loss of function of wild-type p53, in the former because the protein product of the p53 gene complexes with that of the viral E6 gene, in the latter because the protein is altered, presumably as a result of a direct alteration of the p53 gene but possibly because of other post-translational changes. That this mutant allele of the tumour suppressor gene may sometimes behave like an oncogene is suggested by the presence of more than the expected number of copies of the remaining chromosome 17 homologue in some carcinomas.


Low-dose aspirin and subsequent peripheral arterial surgery in the Physicians' Health Study. In the US Physicians' Health Study the early termination of the aspirin arm has provided the opportunity to test the hypothesis that low-dose aspirin (325 mg on alternate days) might affect the subsequent occurrence of peripheral arterial surgery. In the study, a randomised double-blind placebo-controlled trial among 22,071 healthy US male physicians aged 40-84, there were, during an average of 60.2 months of treatment and follow-up, 56 participants who underwent peripheral arterial surgery (20 aspirin, 36 placebo). The relative risk of peripheral artery surgery in the aspirin group was 0.54 (95% confidence intervals 0.30-0.95; p = 0.03). These data indicate that chronic administration of low-dose aspirin to apparently healthy men reduced the need for peripheral arterial surgery.


Low-molecular-weight heparin versus standard heparin in general and orthopaedic surgery: a meta-analysis. Low-molecular-weight heparins (LMWHs) have theoretical advantages over standard heparin as postoperative thromboprophylactic agents. We conducted a meta-analysis of studies reported between 1984 and April, 1991, in which LMWHs were compared with standard heparin for postoperative prophylaxis. We included only randomised studies (reported in English, French, or German) in which investigators compared currently recommended doses of the agents and used adequate screening techniques for deep vein thrombosis. For all surgical studies the relative risk (LMWH versus standard heparin) for deep vein thrombosis was 0.74 (95% Cl 0.65-0.86), for pulmonary embolism 0.43 (95% Cl 0.26-0.72), and for major bleeding 0.98 (95% Cl 0.69-1.40). Comparable relative risks were observed for the general and orthopaedic surgery studies separately. When the analysis for the general surgery studies was limited to those of strong methodology, assessed by eight criteria defined in advance, the benefit/risk ratio was less favourable--relative risk for deep vein thrombosis 0.91 (95% Cl 0.68-1.23), for major bleeding 1.32 (95% Cl 0.69-2.56). There is at present no convincing evidence that in general surgery patients LMWHs, compared with standard heparin, generate a clinically important improvement in the benefit to risk ratio. However, LMWHs may be preferable for orthopaedic surgery patients, in view of the larger absolute risk reduction for venous thrombosis.


Protein processing in lysosomes: the new therapeutic target in neurodegenerative disease. A little recognised feature of neurons is their large complement of lysosomes. Studies of the accumulation of the abnormal isoform of the prion protein (PrPSC) in the prion encephalopathies and the formation of beta/A4 protein from its precursor in Alzheimer's disease suggest that generation of these key proteins takes place in lysosome-related organelles. The release of hydrolytic enzymes from lysosomes may be a primary cause of neuronal damage. Although molecular genetic approaches have identified protein mutations central to the main neurodegenerative disease, cell biological observations are now beginning to unravel the intracellular pathways involved in the molecular pathogenesis of neurodegeneration: as a result, it is now appropriate to consider therapeutic manipulation of the lysosomal system as an approach to treatment.


Primary aldosteronism: hypertension with a genetic basis. Exciting developments in knowledge of primary aldosteronism include description of new subtypes and elucidation of the genetic basis of one variety. Furthermore, relatively simple biochemical screening (aldosterone/renin ratio) has disclosed that primary aldosteronism is more common than previously thought, by diagnosing patients at an earlier, normokalaemic stage. The mutant gene discovered in the glucocorticoid-suppressible variety (FHI) codes for an aldosterone biosynythetic enzyme normally controlled by angiotensin II, and now controlled by corticotropin. The zona fasciculata is hyperplastic and makes aldosterone and "hybrid steroids" 18-oxocortisol and 18-hydroxycortisol in excess, in response to ACTH but not to angiotensin II. Adrenal tumours have not yet been described in this condition. Aldosterone-producing adenomas (Conn's syndrome) are also commonly composed of zona fasciculata-like cells, make "hybrid steroids" in excess and are very sensitive to ACTH but not to angiotensin II. We have described a new variety of aldosterone-producing adenoma which is responsive to angiotensin II (AII-responsive APA), consists of at least 20% zona glomerulosa-like cells, and does not make "hybrid steroids" in excess. We have also described a new familial variety of primary aldosteronism that includes tumours and is not glucocorticoid-suppressible (FHII). We propose that primary aldosteronism is a spectrum of genetic diseases expressed as either hyperplasia or neoplasia, and that morphological and genetic diversity explains biochemical and clinical behaviour.


Regulation of somatostatin synthesis by GABAA receptor stimulation in mouse brain. Neuroanatomical data have documented the existence of synaptic contacts between gamma-aminobutyric acid (GABA) terminals and periventricular hypothalamic somatostatin (SRIF) neurons. In other brain regions, like the cortex or hippocampus, GABA and SRIF are colocalized in short interneurons. These observations suggest that GABA modulates SRIF neuronal activity. In order to test this hypothesis, we studied the effects of the in vivo stimulation of the GABAA receptor (muscimol, 0.75 mg/kg + diazepam, 2.5 mg/kg) on SRIF content and preproSRIF mRNA levels, in mouse brain. Chronic (7 days), but not acute, treatment induced a 38% decrease in hypothalamic SRIF content (as estimated by RIA), a 20% decrease in cortex and no effect in the striatum. The decrease in hypothalamic and cortical SRIF levels lasted until 24 h after cessation of the treatment. In the hypothalamus, prosomatostatin mRNA levels were estimated by Northern blot analysis using a 32P-labeled 45-mere oligoprobe. ProSR1F mRNA hypothalamic levels were equally (48%) decreased by the acute and chronic treatments and remained lower than controls 48 h after the last injection. Quantitative in situ hybridization was used to examine the regional distribution of GABA-induced acute inhibition of proSR1F mRNA densities, using the same oligomere labeled with 35S. ProSR1F mRNA levels were decreased by 35% in the periventricular hypothalamic nucleus. In contrast, no significant modification was observed in cortex, striatum and hilus of the dentate gyrus of the dorsal hippocampus. The present data demonstrate a regionally selective inhibitory action of GABA, mediated by GABAA receptors stimulation, on the biosynthetic mechanisms of the long projecting neuroendocrine SRIF neurons of the anterior periventricular nucleus of the hypothalamus.


Ethanol blocks the cold-induced increase in thyrotropin-releasing hormone mRNA in paraventricular nuclei but not the cold-induced increase in thyrotropin. The effects of a single intraperitoneal injection of ethanol (3 g/kg b.wt.) on the hypothalamic-pituitary-thyroid system was explored as a possible explanation of the hypothermic effect of ethanol. Serum thyroid hormones were significantly reduced by ethanol injection, but ethanol did not affect the cold-induced increase in serum thyroid hormones or thyroid-stimulating hormone (TSH). Since cold-exposure stimulates serum levels of TSH and thyroid hormones by stimulating thyroid-releasing hormone (TRH) release from neurons of the PVN, these findings demonstrate that ethanol did not block pituitary response to TRH or thyroid response to TSH. Paradoxically, ethanol increased cellular levels of TRH mRNA in the paraventricular nucleus (PVN), and blocked the cold-induced increase in TRH mRNA, suggesting that ethanol uncouples the regulation of TRH gene expression from the regulation of TRH release specifically in neurons of the PVN. Measurements of the effects of ethanol on TRH mRNA in thalamus, and beta-actin, vasopressin, somatostatin and corticotropin-releasing hormone (CRH) mRNAs in the PVN in addition to TRH mRNA revealed very specific effects of ethanol on the TRH neuronal system.


Dopamine transporter mRNA: dense expression in ventral midbrain neurons. Oligonucleotides and a full-length cDNA encoding a functional dopamine transporter (DAT1) hybridize to a 3.7 kb mRNA that is concentrated in mRNA prepared from midbrain and absent in specimens from cerebellum or cerebral cortex. In situ hybridization reveals substantial hybridization densities overlying neurons of the substantia nigra, pars compacta, and the parabrachialis pigmentosus region of the ventral tegmental area (VTA). Neurons in the linear and paranigral VTA regions display lower levels of expression. Preliminary studies in arcuate neurons suggest modest hybridization. Different dopaminergic cell groups display different levels of DAT1 dopamine transporter expression.


Involvement of a possible chaperonin in the efficient expression of a cloned CryIIA delta-endotoxin gene in Bacillus thuringiensis. The Bacillus thuringiensis cryIIA delta-endotoxin gene is found as the third-gene in a three-gene operon, with a sporulation-dependent promoter lying upstream of the first gene, orf1. We show here that the polypeptide product of the middle gene (orf2) is required for efficient expression of the toxin gene. In the absence of a functional ORF2 polypeptide the toxin does not form the crystalline inclusions characteristic of other known Bacillus thuringiensis toxins. We discuss the importance of this finding with respect to the possible role of chaperonins in the crystallization of these proteins.


The vasa vasorum and the paradox of beta-blocker therapy. The use of beta-blockers in the treatment of angina, claudication or hypertension is a therapeutic paradox. All those conditions feature increased constrictor tone, so it appears to make little sense to treat them with drugs which block the action of vasodilators. The paradox would disappear, however, if vasodilators could be shown to have the ability to increase constrictor tone in certain circumstances. This paper argues that they have. It presents evidence that isoprenaline, a potent dilator of the dog's saphenous vein, is a powerful constrictor of the vein when it is released from the vasa vasorum of the vein. Indeed, on a molar basis, it appears to be a more powerful constrictor of the vein than exogenous noradrenaline is. Since there is no reason to suppose that isoprenaline is unique among dilators in demonstrating this type of bimodal behaviour, it is possible to justify the proposal that compounds which are normally classified as endogenous dilators may, when released from the pathological vasa vasorum which neoproliferate in atherosclerosis, be responsible for the constrictor effects associated with claudication, and some forms of hypertension and angina. If true then beta-blockade would not be a paradoxical choice of treatment for those conditions.


Management of a major box jellyfish (Chironex fleckeri) sting. Lessons from the first minutes and hours. To report the management of a serious box jellyfish (Chironex fleckeri) envenomation from the first minutes of bystander first aid and treatment by ambulance personnel to subsequent treatment in hospital. A 14-year-old girl sustained a serious Chironex fleckeri sting. There was no loss of consciousness, but the patient suffered severe pain, myocardial irritability, acute pulmonary oedema and mild systemic hypotension, due to the direct toxic effects of the venom. Thirst was a dominant symptom. Management involved rapid bystander action and call for ambulance assistance; and early intervention with oxygen/nitrous oxide administration, compression bandaging, antivenom administration and electrocardiographic monitoring at the site by ambulance personnel. Echocardiography in hospital three hours after the sting showed a normal myocardium. In hospital management resulted in recovery. Nocturnal itching of the sting persisted for six weeks. (i) Vinegar dousing may irritate freshly stung skin, but as a nematocyst inhibitor vinegar remains an essential part of the first aid treatment for cubozoan jellyfish stings. (ii) Compression/immobilisation bandaging was not associated with long-term harm to the sting area. (iii) The pain of an intramuscular antivenom injection may not be felt by a chirodropid sting victim, so safe injection protocols must be strictly observed. (iv) Ambulance services in other States whereas there is a risk of box jellyfish (Chironex fleckeri or Chiropsalmus quadrigatus) stings should be similarly trained and equipped to deal with serious jellyfish envenomations.


Characterization of 3-carboxy-5-phosphono-1,2,3,4-tetrahydroisoquinoline (SC-48981), a potent competitive N-methy-D-aspartate (NMDA) receptor antagonist, in vitro and in vivo. (+/-)-3-Carboxy-5-phosphono-1,2,3,4-tetrahydroisoquinoline (SC-48981), a conformationally restricted analog of the potent competitive N-methyl-D-aspartate (NMDA) antagonist, 2-amino-5-phosphonopentanoate (AP-5), potently inhibited the binding of [3H]glutamate to the N-methyl-D-aspartate (NMDA) receptors with a Ki of 1.6 mcM, but with minimal affinity for kaininate and quisqualate receptors (Ki greater than 50 mcM), in vitro. Consistent with its ability to antagonize the NMDA receptor, SC-48981 decreased the binding of [3H]glycine and [3H]TCP [1-(2-thienyl)cyclohexylpiperidine] to the NMDA-associated glycine and phencyclidine (PCP) recognition sites, in vitro. SC-48981 attenuated levels of basal cGMP and harmaline-induced increases in levels of cGMP in the mouse cerebellum, in vivo, in a competitive manner, with ED50 values of 5.5 and 8.7 mg/kg, i.p. Direct intracerebellar injection of SC-48981 (0.5 microgram) attenuated increases in levels of cGMP induced by central injection of the NMDA-associated glycine receptor agonist, D-serine and by NMDA itself. Parenteral administration of SC-48981 (25 mg/kg, s.c.) decreased basal levels of cGMP for up to 3 h. These results indicate that SC-48981 represents a novel bioavailable competitive NMDA antagonist with a long duration of action.


Carbamazepine-induced neurotoxicity and its prevention by NMDA in cultured cerebellar granule cells. Long-term neurotoxicity of carbamazepine was studied in cultured cerebellar granule cells. Treatment of cells with carbamazepine for 3 days induced a dose-dependent neurotoxicity detected by a loss of [3H]ouabain binding to Na+,K(+)-ATPase, and [3H]N-methyl scopolamine binding to muscarinic cholinergic receptors as well as by direct morphologic examination. NMDA protected against carbamazepine-induced toxicity and this protection was blocked by 2-amino-5-phosphono-valerate (APV). The neurotoxicity induced by carbamazepine may be involved in the teratogenic and adverse effects of overdose associated with the treatment of manic-depressive illness and seizures.


Induction of giant depolarizing potentials by zinc in area CA1 of the rat hippocampus does not result from block of GABAB receptors. The possibility that zinc (Zn2+) induces giant depolarizing potentials (GDPs) by blocking pre- and postsynaptic gamma-aminobutyric acidB (GABAB) receptors in area CA1 of rat hippocampal slices was investigated. Monosynaptic GABAA receptor-mediated fast and GABAB receptor-mediated late inhibitory postsynaptic potentials (IPSPs) were evoked in the presence of the excitatory amino acid (EAA) receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and D,L-amino-5-phosphonovalerate (APV). Addition of Zn2+ (0.3 mM) resulted in the appearance of long-lasting GDPs which obscured monosynaptic late IPSPs. The GABAA receptor antagonist bicuculline methiodide (BMI; 30 microM) blocked fast monosynaptic IPSPs and GDPs, revealing a monosynaptic late IPSP that was prolonged in the presence of Zn2+ and blocked by the GABAB receptor antagonist CGP 35,348 (100 microM). The selective GABAB receptor agonist baclofen (10 microM) depressed monosynaptic IPSPs and population excitatory postsynaptic potentials (pEPSPs) by acting at presynaptic GABAB receptors. Depression of synaptic potentials by baclofen was unaffected by Zn2+. These results suggest that induction of GDPs in area CA1 does not result from an action of Zn2+ at GABAB receptors. We suggest instead that Zn2+ induces GDPs by inducing synchronized discharge of GABAergic interneurons.


Cat retinal ganglion cells show transient responses to acetylcholine and sustained responses to L-glutamate. The neuropharmacological basis for the different receptive field properties of cat retinal ganglion cells was investigated using whole cell voltage-clamp recordings from acutely dissociated adult tissue. Subclasses of physiologically characterised ganglion cells were determined on the basis of (i) their soma diameters and (ii) their projection to central visual nuclei (identified by microinjection of fluorescent dyes into the lateral geniculate and/or superior colliculus). The sensitivities of all categories of ganglion cells, prepared from peripheral retina were found to be similar for gamma-amino butyric acid, glycine, acetylcholine and glutamate. The kinetics of desensitisation differed among receptor subtypes, revealing possible physiologically significant molecular specialisations that could be involved in shaping synaptic transmission.


Selectivity of Thy-1 monoclonal antibodies in enhancing neurite outgrowth. Thy-1 monoclonal antibodies (MAbs) have previously been shown to promote neurite outgrowth from retinal ganglion cells and a variety of other neurons. We have studied the effect on neurite outgrowth of several Thy-1 MAbs with quantitatively similar binding properties and found that only certain Thy-1 MAbs promote neurite outgrowth. This finding suggests that the antibody effects depend on specific interactions with one or more active sites on the Thy-1 glycoprotein.


Concomitant increases in the extracellular concentrations of excitatory and inhibitory amino acids in the rat hippocampus during forebrain ischemia. The extracellular concentrations of aspartate, glutamate, glutamine, taurine and gamma-aminobutyric acid in the hippocampus were determined during and after forebrain ischemia (4-vessel model) in the unanaesthetized rat. Ischemia led to a large increase in both inhibitory (taurine and gamma-aminobutyric acid) and excitatory amino acids (aspartate, glutamate). These results suggest that in this model, as previously proposed in other models of ischemia, the large increase of inhibitory amino acids could counterbalance the excitotoxicity due to aspartate and glutamate.


[An unusual association: panarteritis nodosa and rheumatoid arthritis as an overlapping collagenous syndrome]. The clinical record of a woman with two connective tissue diseases is presented. It started as a necrotizing arteritis with hepatic involvement and chronic multineuritis consistent with PAN. After a long period of clinical evolution the patient developed a real rheumatoid arthritis with positive RF. This clinical picture overlapped clinically and serologically the former one. A good response to the steroid therapy and a careful follow-up permitted a satisfactory life to this patient for about ten years.


[Bladder neoplasm in a patient with panarteritis nodosa treated with cyclophosphamide]. Cyclophosphamide is used both in the treatment of malignant and non-malignant diseases. Urinary neoplasms secondary to its use have been described. We discuss the case of a patient with panarteritis nodosa treated with cyclophosphamide during 63 months, with a total dose of 210 grams, and that showed a bladder neoplasm 8 years after beginning of the treatment. In patients receiving a total dose of cyclophosphamide over 85 grams, a follow-up of ten years minimum should be performed aimed to the early detection of secondary neoplasms.


Assistants-at-surgery: recognition of the role of the registered nurse. In the Omnibus Budget Reconciliation Act of 1989 (OBRA '89), Congress directed the Physician Payment Review Commission (PPRC or "the Commission") to make recommendations on payment policies for assistants-at-surgery, including physicians, physician assistants (PAs) and registered nurses (RNs). The National Association of Orthopaedic Nurses (NAON), via the Government Relations Committee and Executive Board, participated in the public hearing on this issue and submitted testimony on the role of the RN first assistant during orthopaedic surgery. In its 1991 report to Congress, the Commission recommended that inappropriate utilization of assistants-at-surgery could be reduced by implementing "profiling"--a variety of techniques to examine the use of assistants. PPRC failed to comment on policies related to non-physician providers, determining that this was a coverage issue, not a payment issue and thus outside the scope of their jurisdiction. However, as global surgical payment policy is further defined by the Health Care Financing Administration (HCFA) and Congress, consideration will again be given to incorporating payment for assistants-at-surgery into a comprehensive fee schedule. Recognition of the registered nurse as an assistant-at-surgery will continue to be a primary goal of NAON.


Inhibition of fibroblast proliferation in L-valine reduced selective media. A selective cell culture medium, D-valine minimal essential medium (92 mg/l), has been developed to inhibit the proliferation of fibroblasts in cell culture (Gilbert & Migeon 1975). Substitution of D-valine for L-valine prevents fibroblast growth due to the absence of D-amino acid oxidase in these cells. Most cell cultures require foetal bovine serum as an essential component of the culture media, however foetal bovine serum contains L-valine, negating the value of D-valine selective media. To overcome this difficulty, we have produced a modified selective media for cell culture, by the dialysis of foetal bovine serum and confirmed its ability to inhibit fibroblast growth whilst still allowing the proliferation of epithelial cells in culture.


Effectiveness of alpha 2-adrenergic receptor stimulation in reducing the central toxicity following cholinesterase inhibition. Previous studies in this laboratory have demonstrated that the centrally-acting alpha 2-adrenergic agonist clonidine can offer significant protection against both the acute and chronic toxicity following irreversible cholinesterase inactivation with soman. The purpose of this study was to estimate the contribution of central mechanisms to soman toxicity in a rat model; and to determine the effectiveness of clonidine and a series of related agonists to offer protection against the acute and chronic manifestations of this toxicity. To investigate the central component of soman toxicity, animals were pretreated with the peripherally selective reversible cholinesterase inhibitor pyridostigmine, a standard protective agent. Pyridostigmine pretreatment resulted in significant improvement in survival following soman administration. However, pyridostigmine was not able to inhibit the signs of central soman toxicity, including convulsive behavior. Clonidine and several related drugs produced both a further reduction in lethality and a significant reduction in the central signs of soman toxicity. Signs of delayed toxicity to soman were apparent in rats surviving 48 h after administration as measured in open-field locomotor monitoring. Again, pyridostigmine did not offer protection against such delayed toxicity. When clonidine was included in the regimen, however, significant improvement in performance in this measure was observed. These results are consistent with our earlier findings of significant protection provided by clonidine and related drugs against acute and chronic manifestations of soman toxicity and provide further evidence that 1) central toxicity is an important contributor to soman's actions, and 2) stimulation of central alpha 2-adrenergic receptors limits the expression of this central toxicity.


Role of diethylnitrosamine, 2-acetylaminofluorene and partial hepatectomy in the expression of glutathione-S-transferase-P and gamma-glutamyltranspeptidase in the early steps of rat liver carcinogenesis. Three factors involved in the Solt and Farber model of rat liver carcinogenesis were studied alone and in various combinations: diethylnitrosamine (DEN) initiating dose, 2-acetylaminofluorene (2-AAF) feeding and partial hepatectomy. The administration of DEN alone (200 mg/kg) was able to switch on glutathione-S-transferase, placental type (GST-P) expression 3 weeks later at a low level (85 U/micrograms protein) which was stable for 10 weeks in the absence of histopathological lesions. During the same time, gamma-glutamyl transpeptidase (GGT) activity presented 2 waves of increase. The feeding of 0.03% 2-AAF for 2 weeks appeared as a determinant factor in the expression of GST-P protein as well as GGT induction (15- and 7-fold versus DEN alone, respectively). The addition of partial hepatectomy enhanced again GST-P expression (1.5-fold) and GGT induction (2-fold). However, GST-P foci increased in size, not in number while GGT foci increased both in size and in number. These data indicated that 2-AAF was a crucial component of the selection procedure since partial hepatectomy alone, with or without DEN initiation was inefficient in promoting GST-P expression. Therefore, 2-AAF would be able to promote the growth of GST-P-positive cells initiated by DEN, a mechanism likely responsible for its tumor-promoting effect.


Biological activity of subfractions from scrapie-associated fibrils. Subfractions, a nucleic acid fraction and a PrP fraction consisting of PrP17-25, a core fragment of PrPsc, were prepared from the scrapie-associated fibril-enriched fraction from scapie-affected mouse brains. The nucleic acid fraction consisted mainly of variously fragmented DNA and no scrapie-specific nucleic acid was detected in the fraction by SDS polyacrylamide gel electrophoresis. To examine the biological activity, the nucleic acid fraction was either first introduced into mouse L-929 cells before or after nuclease treatments, then transfected cell lysates prepared 2 weeks later were inoculated into mice, or directly inoculated into mice with or without the PrP fraction. The PrP fraction alone was also inoculated into mice. Mice inoculated with the transfected cell lysates or with the nucleic acid fraction alone showed no scrapie signs during their lifespan or the observation period. While 60% of the mice inoculated with the PrP fraction alone and 67% of those inoculated with the fraction together with the nucleic acid fraction showed clinical signs of scrapie. A nucleic acid molecule bound covalently to PrP17-25 was not detected. The results obtained by the present procedures so far suggest scrapie infectivity to be associated with PrPsc, which does not contain any detectable scrapie-genome molecule as either free or covalently bound nucleic acid.


[Effects of MPTP on the mouse retina].

[Effects of MPTP on the mouse retina]. MPTP is known as a selective neurotoxin which destroys dopamine-containing neurons, and induces a model of Parkinson's disease. In the retina, MPTP acts on the amacrine cells which contain dopamine. In the present study, C57BL/6J mice were treated i.p. with MPTP (cumulative dose, 150 mg/kg), and the role of dopamine in the mouse retina was investigated electrophysiologically and immunohistochemically. ERGs were recorded before and 10, 30 and 50 days after MPTP injection. The amplitude of the oscillatory potentials was greatly reduced, and that of the b-wave to a lesser degree, while the a-wave was slightly reduced 10 days after injection. These ERG changes tended to return to the control level 50 days after injection. Immunohistochemical analysis showed that 10 days after MPTP injection the number of tyrosine hydroxylase positive amacrine cells was reduced by approximately 50%, and that these changes had lasted at least until 50 days after MPTP injection.


Current status of non-digitalis positive inotropic drugs. Considerable effort and resources have been directed at the development and study of positive inotropic drugs over the past 10-15 years. Much has been learned about the physiology and pharmacology of myocardial contraction, the application of agents to augment contractility, and, importantly, the general and specific limitations of positive inotropic therapy. Studies on acute inotropic intervention have now shown that a drug's ability to augment overall cardiac performance is heavily dependent on its effects on vasculature, vascular control, and ventricular-vascular coupling. The clinical research on new agents has served to remind us how difficult it is to formulate the "ideal" positive inotropic or cardiovascular support drug for the critical care setting. The vast effort to develop a chronically and orally administrable drug to replace or even supplement digitalis has generally been disappointing. The dopaminergic agents (e.g., ibopamine, levodopa) act primarily via vasodilation and their effectiveness and role in managing heart failure remain unresolved. The initial excitement about the phosphodiesterase III inhibitors (e.g., amrinone, milrinone, enoximone) has been tempered by the results of large well-designed trials indicating variable effectiveness and a prominent adverse effect profile. During long-term oral administration none of these agents has been shown to improve clinical status or exercise capacity beyond that achieved by digoxin, when administered either separately or in combination with digoxin. The Prospective Randomized Milrinone Survival Evaluation (PROMISE) trial, showing that repeated oral administration of milrinone can increase mortality in heart failure, is having a devastating effect on the further development of this class of drugs.(ABSTRACT TRUNCATED AT 250 WORDS)


Redefining the role of digoxin in the treatment of atrial fibrillation. Atrial fibrillation (AF) encompasses a variety of discrete clinical syndromes, including paroxysmal, chronic, acute, and postoperative. Digoxin, long considered the mainstay of therapy for rate control in all types of AF, appears to have only modest electrophysiologic effects, which are mediated primarily by the autonomic nervous system. Digoxin has less potency than the calcium antagonists or beta-blocking drugs with respect to atrioventricular nodal blockade. Although less potent than calcium antagonists or beta-blocking drugs on the atrioventricular node, digoxin provides positive inotropic support, whereas the other 2 agents can suppress left ventricular function. Thus, digoxin is the agent of choice in patients with AF in the setting of significant left ventricular dysfunction. However, in the absence of left ventricular dysfunction, digoxin should be considered second-line therapy for the treatment of all AF syndromes.


Beta-adrenergic blockade for the treatment of hyperthyroidism. To review the clinical and biochemical effects of beta-adrenergic blocking drugs on hyperthyroidism. Studies published since 1972 were identified through a computerized search of MEDLINE and extensive searching of the bibliographies of the articles identified. Based on an understanding of the differences in beta-blocker metabolism in euthyroid and hyperthyroid patients, we reviewed the differences in pharmacokinetics and metabolic and clinical outcomes during their use in hyperthyroidism, as reported in the articles reviewed. beta Blockers have been used to modify the severity of the hyperadrenergic symptoms of hyperthyroidism for the past 20 years. The clinical efficacy of these agents is affected by hyperthyroid-induced alterations in their gastrointestinal absorption, hepatic metabolism, and renal excretion. The mechanisms whereby these clinical changes are effected is unknown. The agents differ in their beta 1 cardioselectivity, membrane-stabilizing activity, intrinsic sympathomimetic activity, and lipid solubility. They do not appear to alter synthesis or secretion of thyroid hormone by the thyroid gland. Their effects on thyroxine metabolism are contradictory. Decreased thyroxine to triiodothyronine conversion is caused by some, but not all, beta blockers, and this appears to correlate with membrane-stabilizing activity. There does not appear to be any alteration in catecholamine sensitivity during beta-adrenergic blockade. The principal mechanism of action of beta blockers in hyperthyroidism is to antagonize beta-receptor-mediated effects of catecholamines. beta Blockers are effective in treating hypermetabolic symptoms in a variety of hyperthyroid states. Used alone, they offer significant symptomatic relief. They are also useful adjuvants to antithyroid medications, surgery, and radioactive iodide treatment in patients with Graves' disease and toxic nodular goiters.


Classical genetic analyses of responses to sedative-hypnotic drugs in crosses derived from long-sleep and short-sleep mice. A classical (Mendelian) genetic analysis of responses to eight sedative-hypnotic compounds (ethanol, urethane, trifluoroethanol, chloral hydrate, barbital, paraldehyde, methyprylon, pentobarbital) was conducted in crosses derived from mouse lines that were selectively bred for differential duration of anesthesia following ethanol. The sleep-time responses of these mice, the long-sleep (LS) and short-sleep (SS) mouse lines, as well as the F1, F2 and backcross (F1 x LS, F1 x SS) generations were measured. Generally, differences in responses among the generations were greater for water soluble compounds than were differences for more lipid soluble compounds. Also, the inheritance of responses to water soluble compounds could be explained primarily by additive effects of alleles while the inheritance patterns for more lipid soluble compounds were more complex. Genetic correlation with ethanol response decreased with increasing lipophilicity. These results suggest that the selection of the LS-SS mouse lines was specific for water soluble anesthetic agents. Because several of these agents are known to act at GABA receptors, examination of the interactions of compounds which differ in lipid solubility at GABA receptors from LS and SS mice may prove useful in elucidating the mechanism of the anesthetic actions of ethanol and other drugs.


Circulatory responses during electroconvulsive therapy. The comparative effects of placebo, esmolol and nitroglycerin. Electroconvulsive therapy is usually accompanied by activation of the autonomic nervous system, which may be harmful in patients with cerebrovascular or ischaemic heart disease. We have compared this haemodynamic response in a series of 82 electroconvulsive treatments randomly assigned to receive either nitroglycerin 3 micrograms.kg-1, esmolol 2 mg.kg-1 or placebo. These drugs were given shortly after the suxamethonium and 2 min before the electroconvulsive therapy in all cases. Heart rate was significantly lower with esmolol 1 min after therapy as was blood pressure (systolic and diastolic). The pulse rate was higher following nitroglycerin than placebo, which in turn was higher than esmolol. The three groups did not differ with regard to seizure duration. The results demonstrate that esmolol is more effective than nitroglycerin in controlling the haemodynamic response to electroconvulsive therapy. With recent emphasis on stabilisation of heart rate in preference to blood pressure in at-risk cardiac patients, our study suggests that, in the doses selected, esmolol is preferred to nitroglycerin to control the heart rate response to electroconvulsive therapy.


Diagnostic implications of detection of proteinase K-resistant protein in spleen, lymph nodes, and brain of sheep. Brain, spleen, and selected lymph nodes from sheep with clinical signs of scrapie were analyzed for presence of proteinase K-resistant protein (PrP-res). Diagnosis of scrapie on the basis of detection of PrP-res was compared with diagnosis on the basis of histologic evaluation of the brain from clinically affected or exposed sheep. Proteinase K-resistant protein was found in every brain that was histologically positive for scrapie, and in addition, was found in the brain of several clinically positive sheep that were not diagnosed as scrapie-positive by histologic evaluation. Proteinase K-resistant protein was also found in 87% of the spleens and lymph nodes from sheep that had PrP-res detected in brain homogenates. Therefore, analysis of sheep brain, spleen, or lymph nodes for PrP-res provided a diagnostic approach that was superior to histologic examination alone for detection of naturally scrapie agent-infected sheep.


Short-term interaction of airway and tissue oxygen tensions on ciliary beat frequency in dogs. The responses of canine tracheal ciliary beat frequency (CBFt) to total lung, tracheal lumen, and peripheral lung hyperoxia, to tracheal lumen anoxia with or without peripheral lung hypoxia, and to isolated tracheal lumen hyperoxia combined with a beta-antagonist were delineated in anesthetized beagle dogs. CBFt was measured using a heterodyne laser light-scattering technique. When oxygen mixtures were delivered to the whole lung, a dose-dependent increase in maximal CBFt was observed from 6.3 +/- 1.0 Hz on air to 13.8 +/- 1.2 Hz on 100% oxygen. When oxygen mixtures were delivered to the isolated tracheal lumen, a dose-dependent increase in maximal CBFt from 6.9 +/- 1.3 Hz on air to 25.7 +/- 6.3 Hz on 100% oxygen was observed. CBFt was unchanged under conditions of peripheral lung hyperoxia. There were no significant changes from room air baseline CBFt of 7.6 +/- 1.5 Hz due to either isolated tracheal anoxia alone or in combination with alveolar hypoxia. CBFt stimulated with 100% oxygen insuffiated to the isolated tracheal lumen decreased from 14.1 +/- 3.2 to 9.5 +/- 1.9 Hz and from 16.5 +/- 1.2 to 9.7 +/- 1.2 Hz in response to 6 and 18 micrograms/kg of intravenous esmolol, respectively. This study demonstrates that short-term, local hyperoxia stimulates CBFt and that a pulmonary or systemically derived factor can be activated to inhibit this stimulation. It indicates that acute airway anoxia and alveolar and blood hypoxia do not suppress ciliary beat frequency. It also suggests that the adrenergic system is involved in the oxygen-induced stimulation of ciliary beat.


Effects of broxaterol and theophylline on fatigued canine diaphragm in vivo. A randomized, controlled study. Since broxaterol, a new beta 2-agonist, has been shown to improve contractility of fatigued canine diaphragm in vitro, a controlled, randomized study was designed to assess its effects on fatigued canine diaphragm in vivo, and compare these to the expected inotropic effects of aminophylline. Diaphragm fatigue was induced in 21 dogs using electrophrenic stimulation at 20 Hz until transdiaphragmatic pressure (Pdi) at 20 Hz was reduced to about 50% of its original value. After stabilization of fatigue, animals were randomized in three groups. Aminophylline-treated animals received an intravenous bolus of 20 mg/kg, broxaterol-treated animals were given an initial bolus of 100 micrograms/kg, and control animals obtained an equal load of saline. After 3 h, aminophylline-treated animals and broxaterol-treated animals received a second dose of 20 mg/kg and 200 micrograms/kg, respectively, whereas control animals received a second dose of saline. Pdi was measured every 30 min for 6 h. At therapeutic serum levels, theophylline did not affect Pdi at any stimulation frequency compared with control conditions. In contrast, broxaterol administration resulted in a significant (p less than 0.05) and long-lasting increase in Pdi at low stimulation frequencies. Pdi at 20 Hz thus increased by 20 +/- 16% 90 min after the first bolus, and by 36 +/- 18% 90 min after the second dose. We conclude that (1) broxaterol promotes recovery of low-frequency fatigue in a dose-dependent way, and (2) theophylline does not improve the force output of fatigued canine diaphragm in vivo.


A case of xeroderma pigmentosum group A diagnosed with a polymerase chain reaction (PCR) technique. Usefulness of PCR in the detection of point mutation in a patient with a hereditary disease. The gene responsible for the xeroderma pigmentosum (XP) group A gene was recently identified and isolated. Preliminary study with fibroblasts from patients with XP group A revealed that most of the Japanese patients with XP group A have a point mutation at the 3' splice acceptor site of intron 3. This mutation site in the XP group A-complementing gene can be recognized by a restriction enzyme, AlwNI. This article describes the usefulness of the polymerase chain reaction diagnosis on XP group A. We tried a polymerase chain reaction diagnosis on genomic DNA from a sporadic case of XP group A and this child's parents. AlwNI restriction fragment length polymorphism of the polymerase chain reaction product showed three bands for his father's and mother's DNA, one corresponding to normal and two corresponding to a point-mutated gene, respectively. A DNA sample from our patient showed two fragments indicating that only he has the mutated gene. This study demonstrated that we can know that this patient has group A XP without a complementation test that needs a lot of time. Moreover, our study revealed that we can detect the hidden mutated XP group A-complementing gene in the pedigree in which there is no patient with XP. Furthermore, it may be available soon for prenatal diagnosis.


Late diagnosis of cryptorchidism: a failure of medical screening? A retrospective review of hospital and available community records of 47 children undergoing orchidopexy in a district hospital was undertaken to determine adequacy of screening for cryptorchidism and factors associated with late referral. Twenty eight of these boys were previously examined on 108 occasions. Diagnosis was missed on 32 occasions and the record of 38 clinical examinations did not include position of testes. In the case of 16 boys (four under school age and 12 of school age) appropriate action was not taken once the diagnosis was made. Diagnosis was reliably made in school aged children but in children under the age of 1 year cryptorchidism was frequently missed by the examining doctor. It is suggested that criteria for diagnosis and referral should be agreed in any surveillance programme. Junior doctors in hospital responsible for routine clinical examination of children during admission and clinical medical officers or general practitioners during routine clinical examination of boys should be clearly instructed to examine and record the position of the testes.


The impact of human T-lymphotrophic virus type I/II infection on the prognosis of sexually acquired cases of acquired immunodeficiency syndrome. Twenty (18%) of 111 Peruvian men with sexually acquired human immunodeficiency virus infection were found also to be infected with human T-lymphotrophic virus type I or II in a retrospective study. At the time of data evaluation, 75 patients had reached Centers for Disease Control stage IV (clinical acquired immunodeficiency syndrome) and had not received antiviral medication; mortality in this group was 63.3% (38/60) among patients infected with human immunodeficiency virus alone and 80% (12/15) in the dually infected group. Of the 50 patients who had died, survival time from onset of stage IV to death was shorter in the dually infected group (5.02 +/- 3.27 months) than in those with human immunodeficiency virus infection alone (10.07 +/- 4.42 months). In Peru, sexually acquired human immunodeficiency virus infection in men is often accompanied by human T-lymphotrophic virus type I/II infection, and dual retrovirus infection is associated with a shorter survival after onset of clinical acquired immunodeficiency syndrome.


CD4 lymphocyte count as an indicator of delay in seeking human immunodeficiency virus-related treatment. As many as half of patients infected with the human immunodeficiency virus who are medically eligible for Pneumocystis prophylaxis and zidovudine treatment have not received these treatments. We used the CD4 lymphocyte count as an indicator of delay in seeking treatment among patients infected with human immunodeficiency virus and assessed whether insurance status was associated with the stage of illness when care is initiated. Data from 96 patients who initiated medical care at a university acquired immunodeficiency syndrome clinic from August 1989 to January 1991 were retrospectively reviewed. Patients initiated care at a relatively late stage of illness (mean CD4 lymphocyte count, 0.37 x 10(9)/L [369/mm3]), and 29% were below the threshold for Pneumocystis prophylaxis. Patients with private insurance had significantly lower CD4 counts (mean, 0.27 x 10(9)/L) than did individuals with public insurance (mean, 0.46 x 10(9)/L). CD4 counts did not increase during the 18-month study period. The majority of patients infected with human immunodeficiency virus are eligible for medical therapy and could benefit by initiating care sooner. Private insurance was not associated with initiating early care, supporting anecdotal reports that some privately insured individuals may be reluctant to seek care for a human immunodeficiency virus-related condition.


Positron emission tomographic analysis of central D1 and D2 dopamine receptor occupancy in patients treated with classical neuroleptics and clozapine. Relation to extrapyramidal side effects. Positron emission tomography and selective radioligands were used to determine D1 and D2 dopamine receptor occupancy induced by neuroleptics in the basal ganglia of drug-treated schizophrenic patients. In 22 patients treated with conventional dosages of classical neuroleptics, the D2 occupancy was 70% to 89%. Patients with acute extrapyramidal syndromes had a higher D2 occupancy than those without side effects. This finding indicates that neuroleptic-induced extrapyramidal syndromes are related to the degree of central D2 occupancy induced in the basal ganglia. In five patients treated with clozapine, the prototype atypical antipsychotic drug, a lower D2 occupancy of 38% to 63% was found. This finding demonstrates that clozapine is also "atypical" with respect to the central D2 occupancy in patients. During treatment with clozapine, there is a low frequency of extrapyramidal syndromes, which accordingly may reflect the comparatively low D2 occupancy induced by clinical doses of clozapine. Classical neuroleptics, like haloperidol or sulpiride, did not cause any evident D1 occupancy, but the thioxanthene flupentixol induced a 36% to 44% occupancy. In four patients treated with clozapine, the D1 occupancy was 38% to 52%. The D1 occupancy induced by clozapine and flupentixol may contribute to the antipsychotic effect of these drugs.


Functional expression of soluble ICAM-1 by baculovirus-infected Sf9 cells. Inflammation, metastasis and ischemia are processes that require lymphocyte or leukocyte cell recognition and adherence to endothelial counter receptors such as ICAM-1. Mapping the sites of interaction of ICAM-1 with LFA-1, the receptor for ICAM-1 on lymphocytes, may lead to the design of novel inhibitors of inflammation or metastasis. To this end, recombinant soluble ICAM-1 cDNA was engineered into the baculovirus expression system, which is capable of expressing large amounts of proteins. These constructs were designed to contain a protein leader sequence so that the transfected insect cells would secrete the recombinant polypeptide into the culture media for ease of isolation. We engineered four constructs of ICAM-1 into the baculovirus system and obtained relatively high expression of two soluble forms of ICAM-1, a two domain and a five domain form. These truncated proteins were isolated and shown to promote adherence of HL-60 cells and Molt-4 cells. These recombinant soluble proteins also inhibited cell adherence to purified intact ICAM-1 isolated from K562 cells.


Interaction between phosphoinositide turnover system and cyclic AMP pathway for the secretion of pancreastatin and somatostatin from QGP-1N cells. It is found that secretion of pancreastatin and somatostatin from QGP-1N cells is regulated through muscarinic receptor-mediated activation of phosphatidylinositide hydrolysis system. In this report, whether the cAMP pathway interacts with the phosphoinositide turnover system for the secretion of pancreastatin and somatostatin from QGP-1N cells through muscarinic receptors was studied. Stimulation of QGP-1N cells with carbachol increased intracellular cAMP levels. The carbachol-induced increase in cAMP levels was inhibited by atropine. Calcium ionophore (A23187) and phorbol 12-myristate 13-acetate increased cAMP synthesis. Dibutyryl cAMP, forskolin and theophylline stimulated secretion of pancreastatin and somatostatin. When either dibutyryl cAMP, forskolin or theophylline was added in culture medium with A23187, phorbol ester or carbachol, a synergistic effect was found on pancreastatin and somatostatin secretion. These results suggest that interaction between the phosphoinositide turnover system and the cAMP pathway occurs in QGP-1N cells through muscarinic receptor stimulation for the secretion of pancreastatin and somatostatin.


Structures and properties of seven isoforms of the NMDA receptor generated by alternative splicing. We here report the existence of 6 additional isoforms of the NMDA receptor generated via alternative splicing by molecular analysis of cDNA clones isolated from a rat forebrain cDNA library. These isoforms possess the structures with an insertion at the extracellular amino-terminal region or deletions at two different extracellular carboxyl-terminal regions, or those formed by combinations of the above insertion and deletions. One of the deletions results in the generation of a new carboxyl-terminal sequence. All these isoforms possess the ability to induce electrophysiological responses to NMDA and respond to various antagonists selective to the NMDA receptor in the Xenopus oocyte expression system. In addition, a truncated form of the NMDA receptor also exists that contains only the extreme amino-terminal sequence of this protein molecule. These data indicate that the NMDA receptor consists of heterogeneous molecules that differ in the extracellular sequence of the amino- and carboxyl-terminal regions.


Nitroprusside-sensitive and insensitive guanylate cyclases in retinal rod outer segments. Rod outer segments of retina contain guanylate cyclase activity both in the cytosol and membrane fractions. Though the activity in the cytosol is a small fraction of the total activity, it is highly activated by nitroprusside, a nitric oxide generating agent. The membrane guanylate cyclase on the other hand is unaffected by nitroprusside both before and after solubilization. The effects of nitroprusside or nitric oxide on photoreceptor function should therefore be mediated by the cytosolic and not the membrane guanylate cyclase.


Inhibition of cellular glutathione biosynthesis by rifampicin in Mycobacterium smegmatis. This study was carried out to investigate the mechanism of Rifampicin (RIF) induced glutathione (GSH) depletion in M. smegmatis. RIF at various concentrations decreased the activities of gamma glutamyl cysteine synthetase (GGCS) and GSH synthetase. Maximum decrease in the activities of biosynthetic enzymes of GSH was observed when 15 micrograms RIF ml-1 medium was incorporated in the growth medium before performing inoculations. The activity of GGCS was also decreased when three day grown M. smegmatis was exposed to 60 micrograms RIF ml-1 medium for a period of 6 h and 9 h. RIF did not alter the activity of gamma glutamyl transferase. The results of the present study demonstrate that the depletion caused by RIF in cellular GSH is due to its decreased biosynthesis whereas its degradation is not affected in M. smegmatis.


Amyl nitrite: use as a smooth muscle relaxant in difficult preterm cesarean section. Amyl nitrite is a smooth muscle relaxant that has been used clinically to facilitate uterine relaxation in difficult deliveries. In this retrospective study, we evaluate the safety of amyl nitrite use during preterm cesarean deliveries, and we assess possible advantageous effects on surgical incision choice. Women who received amyl nitrite cesarean section were compared to a control group matched for gestational age, fetal presentation, and mode of delivery who did not receive amyl nitrite. There were no statistical differences between the groups in the independent variables (maternal age, parity, medical or obstetric history, type of anesthesia, anesthesia or obstetric attending physician, antepartum hematocrit, or neonatal weight). Outcome (dependent) variables (estimated blood loss, Apgar scores, postpartum hematocrit, cord gases, or postpartum complications) were assessed, and there were no significant differences between the groups. Low transverse cesarean section was performed more frequently in the amyl nitrite group (58 of 64) than in the comparison group (48 of 64) (p less than 0.03). Considering the 128 women with and without amyl nitrite together, the decrease in hematocrit observed postpartum was greater after classic section (7%) than after low transverse section (4%) (p less than 0.002). We conclude that the use of amyl nitrite during preterm cesarean section poses no threat to mother or fetus and may facilitate delivery by allowing the performance of a low transverse rather than a classic cesarean section without maternal or neonatal complications.


C4 polymorphism in multiplex families with insulin dependent diabetes in the Tunisian population: standard C4 typing methods and RFLP analysis. The polymorphism of C4A and C4B genes was investigated in Tunisian patients with insulin dependent diabetes (IDDM) and compared to family members (sibs) and to healthy controls. Multiplex families were analysed. A significant increase in C4AQO (26.86% vs 6.90%) and C4BQO (40.29% vs 8.28%) phenotypes was noted in IDDM patients compared with controls. Using RFLP analysis, we confirmed the high frequency of C4 null alleles. We also observed that most of these alleles were genes deleted in IDDM patients (72.23% vs 20% for CA4QO and 74.07% vs 16.70% for C4BQO). A significant decrease in the C4B long (14.92% vs 67.12%) form of the gene was also demonstrated by RFLP analysis compared with controls. Two haplotypes were frequently associated with IDDM patients in whom the C4A and C4B were deleted genes.


Activation of CD4+ and CD8+ lymphocyte subsets by streptozotocin in murine popliteal lymph node (PLN) test. Time-related and dose-related popliteal lymph node (PLN) enlargement and lymphocyte subset alterations owing to subcutaneous (s.c.) injection of streptozotocin (STZ) into the foot pad were determined in (C57BL/6 x DBA/2)F1 [B6D2F1] mice. Early cell activation and time-related changes in T and B lymphocyte subsets were monitored during the onset of STZ-induced lymphoproliferative reaction by flow cytometry and immunophenotyping of lymphocyte subsets stained with a panel of monoclonal antibodies. Examination of cell size and chromatin decondensing for T and B cell subsets revealed differences in their activation profiles during the early phase of STZ-induced lymph node enlargement. The kinetics of the reaction showed initial activation and proliferation of CD4+ cells associated with accumulation of CD8+ and B cells. Subsequently CD8+ cells were activated and proliferated, but there was no evidence of early or late B cell activation as shown by the lack of increase in cell size or nuclear decondensation and the low and transient synthesis of immunoglobulins. The activation characteristics for CD4+ and CD8+ cell subsets in STZ-induced node enlargement were found to be analogous with T cell activation in acute allogeneic graft-versus-host (GVH) reaction in which the F1 recipient differed from the parent at both class I and II MHC loci. Our data support a central role for T cell activation in the induction of STZ-related PLN enlargement and suggest that recirculatory host B cells can play a major role in early node enlargement.


Expression of c-erbB-2 in in situ and in adjacent invasive ductal adenocarcinomas of the female breast. Several lines of evidence have demonstrated that expression of the c-erbB-2 gene product contributes to the malignant phenotype. We and others have determined that c-erbB-2 is substantially expressed in most ductal in situ carcinomas of the comedo type, but not in other patterns of ductal carcinoma in situ or in atypical ductal hyperplasia of the breast. In the present investigation, by immunohistochemistry we inquired whether invasive ductal adenocarcinomas retained the c-erbB-2 expression status of the in situ carcinomas from which they derived. Of twelve specimens containing both cribriform/micropapillary in situ and derivative invasive adenocarcinomas in the same section, all tumor cells were negative for c-erbB-2 expression. In thirteen in situ carcinomas of the comedo type, with identifiable invasive components, ten had definite c-erbB-2 expression, and in every case there was comparable c-erbB-2 protein staining of in situ and invasive components; in three of these ten cases the staining in the in situ component tended to be more intense. These findings imply that a significant proportion of invasive mammary adenocarcinomas expressing c-erbB-2 protein is derived from ductal in situ carcinomas of the comedo type.


Reactivity-based hydrocarbon controls: scientific issues and potential regulatory applications. The California Air Resources Board and the South Coast Air Quality Management District hosted a conference on April 8-9, 1991 to examine the scientific issues associated with reactivity-based hydrocarbon controls, and to identify the obstacles to potential regulatory applications. Owing to residual uncertainties in the underlying science, and the complex emission measurement capabilities required for enforcement, a general consensus emerged on the need for further research before application of reactivity-based controls. A number of recommendations were made for research on the remaining scientific, enforcement, and policy issues, many of which have led to cooperative efforts initiated since the conference.


Regulation of type I and type II transglutaminase in normal human bronchial epithelial and lung carcinoma cells. In cultured, undifferentiated normal human bronchial epithelial (HBE) cells, transglutaminase activity was localized predominantly in the cytosolic fraction of cell lysates. Upon squamous differentiation, this cytosolic activity declined and was replaced by a 40-fold increase in the activity of particulate (membrane-associated) transglutaminase. Immunoblot analysis demonstrated that the cytosolic transglutaminase was Type II (tissue) transglutaminase and that squamous differentiation shifted gene expression to the Type I (epidermal) transglutaminase. Retinoic acid, an inhibitor of squamous cell differentiation, suppressed the increase in Type I transglutaminase. The decrease in Type II transglutaminase activity was unaffected by retinoic acid. Transforming growth factor-beta 1 (TGF-beta 1) enhanced Type II transglutaminase activity about 10-fold in the undifferentiated cells but did not increase Type I transglutaminase or cholesterol sulfate, two early markers of squamous differentiation. TGF-beta 2 was equivalent to TGF-beta 1 in inducing Type II transglutaminase and in inhibiting the growth of HBE cells. The differentiation-related and TGF-beta-induced changes in transglutaminase activity were reflected in the level of transglutaminase Type I and Type II protein and mRNA. Expression of transglutaminases in lung carcinoma cell lines was variable. No correlation was observed between the expression of Type I transglutaminase and the classification of the cells as squamous cell carcinoma. Several lung carcinoma cell lines exhibited high levels of Type II transglutaminase activity that were increased several-fold by TGF-beta 1 treatment. Retinoic acid was ineffective in altering transglutaminase expression in most cell lines but induced Type II transglutaminase in a time- and dose-dependent manner in NCI-HUT-460 cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Detection of mutations in human DNA.

Detection of mutations in human DNA. Efficient methods for the detection of mutations are of fundamental importance in research and in diagnostics. By detection of a DNA sequence alteration that cosegregates with a clinical phenotype in an affected family, the gene at fault may be identified and assigned a function. Mutation detection methods are also a rate-limiting factor for the clinical application of DNA diagnostics. Currently a large number of techniques are in use to scan for new mutations and to distinguish among previously established sequence variants. Here, some of the problems connected with mutation detection are discussed together with principles on which current and future mutation detection assays can be based.


Optimized centrifugation for rapid elution of DNA from agarose gels. A simple method has been developed for the isolation of DNA from agarose gels. Centrifugation in a low-cost device for 45 s at low speed provides a high yield of DNA suitable for further manipulation by restriction enzymes, T4 DNA ligase, Taq polymerase, Klenow fragment, and T4 polynucleotide kinase, and also for sequencing. In contrast, centrifugation for greater than 1 min leads to coelution of substances that inhibit DNA ligase.


Mechanism of multidrug resistance in human tumour cell lines and complete reversion of cellular resistance. The biochemical basis of the multidrug-resistant (MDR) phenotype has been investigated in drug-resistant sublines derived from LoVo and SW984 human colon carcinoma cell lines by doxorubicin selection. Besides drug extrusion through the plasma membrane, two further observations, both ascribable to the drug transport property of the gp170 glycoprotein, were made. First drug deposition into cytoplasmic membranous structures which allows cells to tolerate a high intracellular drug concentration since it prevents drugs from reaching their cellular target site(s). Secondly drug removal from the complexes formed by interaction of drug with target cellular macromolecules, a phenomenon which extends its an effect that continues after treatment and appears to be the most important resistance mechanism in MDR cells. Treatments based on the gp170 inhibitory property of verapamil were developed that allowed abrogation of resistance in MDR cell lines, a strategy that may be applicable to therapy treatments.


Segmental determination in Drosophila central nervous system: analysis of the abdominal-A region of the bithorax complex. The bithorax complex (BX-C) comprises several genes required for the diversification of posterior segments in Drosophila. The BX-C genes control segment differences not only in the epidermis but in other tissues as well, especially in the central nervous system. We have examined the control of one segment-specific neural structure: the lateral dots, a paired structure present in the first abdominal segment of the larval CNS and absent in all following abdominal segments. Our results show that the suppression of lateral dots in segments A3 and A4 requires the presence of two active copies of one of the BX-C genes, abdominal-A (abd-A). We also show that the adjacent BX-C regions, iab-3 and iab-4, can act in trans on abd-A not only when the two copies of BX-C are paired but also, at least to some extent, when pairing is disturbed.


Ancestral haplotypes carry haplotypic and haplospecific polymorphisms of BAT1: possible relevance to autoimmune disease. The human BAT1 gene, located in the central MHC region (approximately 170 kb centrometric of HLA-B), is polymorphic and the polymorphism correlates with MHC ancestral haplotypes. Allelic RFLP patterns have been assigned to several ancestral haplotypes and have been shown to be 'haplotypic' (i.e. found on all examples of the same ancestral haplotype) and in some cases 'haplospecific' (i.e. unique to one ancestral haplotype). The relevance of the BAT1 polymorphism to susceptibility to Myasthenia Gravis (MG) has been investigated. The frequency of the BAT1 B allelic pattern is increased in patients with MG (n = 16) compared to an equal number of control subjects. The increase is due to the association between MG and the 8.1 ancestral haplotype (HLA A1, Cw7, B8, BfS, C4AQ0, C4B1, DR3, DQw2).


Functional analysis of chimeric proteins constructed by exchanging homologous domains of two P-glycoproteins conferring distinct drug resistance profiles. P-Glycoproteins (P-gps) encoded by the mouse mdr1 and mdr3 (Phe939, mdr3F) genes confer distinct drug resistance profiles. While the mdr1 and mdr3F clones confer comparable levels of vinblastine (VBL) resistance, mdr3F confers actinomycin D (ACT) resistance levels 2-fold greater than mdr1, while mdr1 confers resistance to colchicine at levels 7-fold greater than mdr3F. We wished to identify in chimeric proteins discrete protein domains responsible for the distinct drug resistance profiles of mdr1 and mdr3F. Homologous protein domains were exchanged in hybrid cDNA clones, and the specific drug resistance profiles conferred by chimeric proteins were determined in stably transfected cell clones expressing comparable amounts of wild-type or chimeric P-gps. Immunoblotting experiments showed that all chimeras were found expressed in membrane-enriched fractions of transfected cell clones and all conveyed cellular drug resistance at levels above the background of nontransfected drug-sensitive LR73 cells. For VBL, all chimeric constructs were found to convey similar levels of resistance. For COL and ACT, the levels of resistance conferred by the various chimeras were heterogeneous, being similar to either the parental mdr1 or the parental mdr3F clones, or in many cases being intermediate between the two. The preferential COL and ACT resistance phenotypes of mdr1 and mdr3F, respectively, did not segregate in chimeric proteins with any specific protein segment. Taken together, our results suggest that the preferential drug resistance phenotypes encoded by the mdr1 and mdr3F clones implicate complex interactions between the two homologous halves of the respective P-gp.


The effects of a dopamine antagonist on luteinizing hormone and prolactin release in women with anorexia nervosa and in normal controls. To evaluate the possible role of central dopaminergic suppression of gonadotropin secretion in the genesis of amenorrhea associated with anorexia nervosa (A.N.), a central D-2 dopamine receptor blocker was administered to 10 women with A.N. and 10 regularly menstruating age-matched controls. Serum prolactin and luteinizing hormone (LH) levels were measured at -15, 0, 30, 60, 120, and 180 min after administration of metoclopramide (10 mg orally). Mean basal prolactin (p less than 0.001) and estradiol levels (p less than 0.02) were significantly lower in women with A.N. The prolactin response to metoclopramide was significantly impaired in women with anorexia nervosa. No correlation was found between the prolactin response and percentage ideal body weight. Basal and post-stimulation prolactin levels were correlated with estradiol levels. After adjusting for the effects of estradiol, significant differences between patients with A.N. and controls remained in prolactin levels at baseline (p less than 0.01), 120 min (p less than 0.02) and 180 min (p less than 0.05). Metoclopramide did not induce a significant rise in LH levels in either the A.N. or control groups. These data are consistent with central dopaminergic inhibition of prolactin secretion in anorexia nervosa but do not support the hypothesis that central dopaminergic inhibition is related to diminished LH release in this state.


Molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine proteinase on the cell surface. The cDNA coding for the human dipeptidyl peptidase IV (DPPIV) has been isolated and sequenced. The nucleotide sequence (3465 bp) of the cDNA contains an open reading frame encoding a polypeptide comprising 766 amino acids, one residue less than those of rat DPPIV. The predicted amino acid sequence exhibits 84.9% identity to that of the rat enzyme, and contains nine potential N-linked glycosylation sites, one site more than those in the rat enzyme. A putative catalytic triad for serine proteinases, serine, aspartic acid and histidine, are found in a completely conserved COOH-terminal region (positions 625-752).


P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines. MDR has been studied extensively in mammalian cell lines. According to usual practice, the MDR phenotype is characterized by the following features: cross resistance to multiple chemotherapeutic agents (lipophilic cations), defective intracellular drug accumulation and retention, overexpression of P-gp (often accompanied by gene amplification), and reversal of the phenotype by addition of calcium channel blockers. An hypothesis for the function of P-gp has been proposed in which P-gp acts as a carrier protein that actively extrudes MDR compounds out of the cells. However, basic questions, such as what defines the specificity of the pump and how is energy for active efflux transduced, remain to be answered. Furthermore, assuming that P-gp acts as a drug transporter, one will expect a relationship between P-gp expression and accumulation defects in MDR cell lines. A review of papers reporting 97 cell lines selected for resistance to the classical MDR compounds has revealed that a connection exists in most of the reported cell lines. However, several exceptions can be pointed out. Furthermore, only a limited number of well characterized series of sublines with different degrees of resistance to a single agent have been reported. In many of these, a correlation between P-gp expression and transport properties can not be established. Co-amplification of genes adjacent to the mdr1 gene, mutations [122], splicing of mdr1 RNA [123], modulation of P-gp by phosphorylation [124] or glycosylation [127], or experimental conditions [26,78] could account for some of the complexity of the phenotype and the absence of correlation in some of the cell lines. However, both cell lines with overexpression of P-gp without increased efflux [i.e., 67,75] and cell lines without P-gp expression and accumulation defects/increased efflux [i.e., 25,107] have been reported. Thus, current results from MDR cell lines contradict--but do not exclude--that P-gp acts as multidrug transporter. Other models for the mechanism of resistance have been proposed: (1) An energy-dependent permeability barrier working with greater efficacy in resistant cells. This hypothesis is supported by studies of influx which, although few, all except one demonstrate decreased influx in resistant cells; (2) Resistant cells have a greater endosomal volume, and a greater exocytotic activity accounts for the efflux.(ABSTRACT TRUNCATED AT 400 WORDS)


Protein folding and chaperonins.

Protein folding and chaperonins. The folding of polypeptide chains in cells, following either translation or translocation through membranes, must take place under conditions of extremely high protein concentrations. In addition, folding into a correct structure must occur in the presence of other rapidly folding species, and at temperatures known to destabilize aggregation-prone folding intermediates. To facilitate folding in vivo, molecular chaperones have evolved that stabilize protein folding intermediates, thus partitioning them towards a pathway leading to the native state rather than forming inactive aggregated structures.


Intrathymic activation events and the generation of IL-4 producer CD4+8- thymocytes. The generation of mouse CD4+8- thymocytes appears to involve an activation process. CD69, an early activation marker, is first expressed on 3% of TCRlo/med double-positive thymocytes and peaks (70-90%) at the HSAhi TCRmed CD4+8lo and the HSAhi TCRhi CD4+8- stages, before being downmodulated. CD44, a marker of a later stage of activation is selectively expressed at the HSAlo CD4+8- stage. Functional changes associated with the late activated state, such as the transient acquisition of the potential to secrete IL-4 upon stimulation, are also induced at the HSAlo CD4+8- stage and are still evident after export to the periphery. During thymic selection, interactions of different affinities between TCR/CD4 and self ligand/MHC class II, are likely to induce different degrees of activation. This may impart different functional capabilities--such as the potential to secrete IL-4--to a subset of emerging CD4+ T lymphocytes that express TCRs with intermediate affinities for self. Such a pathway may be involved in the maintenance of tolerance and the prevention of some deleterious autoimmune reactions.