Production and characterization of a monoclonal antibody to dopamine D2 receptor: comparison with a polyclonal antibody to a different epitope. A monoclonal antibody (Mab) that recognizes the rat dopamine D2 receptor (DAR) has been generated using DAR specific peptide. The Mab, IgM isotype recognizes five proteins (Mr 220, 145, 95, 66 and 47 kDa) in striatal membrane on Western blot. Preincubation of Mab with free peptide blocked the labeling of all five bands. A polyclonal antibody against peptide from a different region of the DAR, reacted with three out of five proteins (220, 66, and 47 kDa) in these membranes. The DAR antagonist NAPS-biotinyl binds to a 220 kDa protein in striatal membrane on ligand blotts; the labeling can be blocked by the addition of 2 microM sulpride. The 220 kDa Mab reactive protein was less in cerebellum and was absent in the liver. Neither the Mab nor polyclonal antibody inhibited binding of a DAR antagonist, [3H]YM09151-2, to the striatal membranes. These antibodies will enable us to study the structure/function and regulation of the synthesis of DAR protein.


Glial cells as a model for the role of cobalamin in the nervous system: impaired synthesis of cobalamin coenzymes in cultured human astrocytes following short-term cobalamin-deprivation. The conversion of cyanocobalamin to adenosyl- and methylcobalamin is impaired in cobalamin-deficient cultured human glial cells. In contrast cultured human skin fibroblasts retained their ability to synthesize coenzyme forms when grown in cobalamin-deficient medium. Cells were pre-conditioned by growing in cobalamin-deficient media for six weeks and then subcultured in medium containing either free or transcobalamin II-bound 57Co-cyanocobalamin. Although both coenzyme levels were low in cobalamin-deficient glial cells, the decrease in methylcobalamin was more marked than that of adenosylcobalamin. Methionine synthase and Cb1 reductase activities were markedly decreased in cobalamin-deficient glial cells but were unchanged in fibroblasts cultured in cobalamin-deficient medium. Our data suggest that in glial cells, cobalamin coenzyme synthesis and function is exquisitely sensitive to short-term cobalamin deprivation. Glial cells apparently synthesize and secrete transcobalamin II since antibodies directed against the transport protein inhibit the uptake of free cobalamin.


Resistance of CHO cells expressing P-glycoprotein to cyclopropylpyrroloindole (CPI) alkylating agents. Several new antitumor agents belonging to the class of minor groove binders that are able to form covalent bonds with DNA via a cyclopropylpyrroloindole (CPI) group are susceptible to a multidrug resistance (MDR) phenotype in Chinese hamster ovary (CHO) cells. The multidrug resistant CCHR-C5 cell line was 16-, 23- and 13-fold more resistant to the analogs U-73,975, U-77,779 and U-80,244, respectively, although its cytotoxic response to the parent compound CC-1065 was similar to the response of the drug-sensitive wild-type cells (AuxB1). For a sequence of MDR cell lines showing increasing expression of P-glycoprotein (Pgp) there were corresponding increments in the level of resistance to U-73,975, arguing that Pgp is the key determinant in resistance of the MDR cells to CPI agents. MDR cells treated with U-73,975 showed diminished generation of covalent adducts on DNA as well as increased resistance to cytotoxicity.


HLA antigens in North American patients with Takayasu arteritis. To determine the frequency of HLA class I and II antigens in Caucasian North American patients with Takayasu arteritis (TA). Seventy-three class I antigens and 13 class II antigens were examined in 21 Caucasian North American patients with TA, and their frequencies were compared with findings in 243 healthy, untreated controls. HLA typing was performed by microlymphocytotoxicity assay. We found no statistically significant positive association of TA with DR2, DR4, DQw3, or any of the other class I or class II antigens studied. A negative association between TA and DR1 was noted. There was no significant association between any of the HLA antigens and the severity of the disease or the presence of complications. The negative association between TA and the DR1 antigen suggests that this antigen may be protective against the development of the disease.


Dubbo Study of the elderly: hypertension and lipid levels. Untreated hypertension in age groups below 60 years has been shown to be associated with significant elevations in serum cholesterol and triglyceride levels. Drug therapy of hypertension has also been shown to have adverse effects on lipoproteins. We have investigated lipid and lipoprotein levels in a community-based sample of men and women 60 years and older belonging to one of the following groupings: (a) normal blood pressure (n = 1075); (b) untreated hypertension (n = 329); (c) drug-treated hypertension (n = 880). Serum lipid, lipoprotein, apolipoprotein or plasma glucose levels did not vary significantly between untreated hypertensives and normotensives of either sex. In a multiple regression model controlling for possible influences of age, overweight, alcohol and tobacco usage, and presence of coronary heart disease, anti-hypertensive drug therapy significantly predicted increased serum triglycerides (P less than 0.001) and reduced high density lipoprotein (HDL) cholesterol levels (P less than 0.01) in both sexes, reduced apolipoprotein A-I levels in males (P less than 0.001), and increased apolipoprotein B (P less than 0.01) and plasma glucose levels (P less than 0.001) in females. Adjusted triglycerides were 20% higher and HDL cholesterol was 7% lower in the presence of anti-hypertensive drug therapy. These effects were partially consistent with the known actions of thiazide diuretics and beta-blockers which were used by more than 50% and 40% of subjects, respectively.


The role of cell division in the induction of clonal anergy. The interaction of antigen with lymphocyte antigen receptors can result in either clonal expansion or unresponsiveness (anergy). In 1970, Bretscher and Cohn proposed a two-signal model of lymphocyte activation to explain this paradox: antigen receptor occupancy alone could induce unresponsiveness whereas antigen receptor occupancy plus a costimulatory signal could induce immunity. Here, Marc Jenkins reviews in vitro and in vivo manifestations of clonal anergy and evaluates the ability of the two-signal model to explain these phenomena.


Quality of life in treatment of hypertension. A metaanalysis of clinical trials. A metaanalysis was performed to determine the effects on quality of life (QL) in hypertension as reported in published clinical trials of antihypertensive drug therapy. All studies included compared active treatment to baseline (placebo or no treatment) with the patients as their own control and used blinded, randomized trials. Change was measured by self and/or interviewer-assisted evaluation, standardized psychomotor/cognitive tests, or sleep laboratory observations. After an exhaustive literature search (1970 to 1990), nine published trials of 27 population groups (n = 1620) using 14 drugs from six pharmacological groups met selection criteria and were analyzed for five QL constructs: sexual function, sleep, psychomotor, general well-being, and mood. Small positive effect size (d) improvement with treatment was seen for sleep (d = 0.106), psychomotor (d = 0.283), general well-being (d = 0.139), and mood (d = 0.167) while no effect could be determined for sexual function (d = -0.030) based on 95% confidence intervals. Either a comparably small improvement with treatment or no effect was seen among various pharmacological drug groups; no negative effect with treatment was identified. A larger positive effect could be postulated if the drug choice was individualized to the patient rather than randomized as in clinical trial methodology. Although none of the drug groups had a clearly superior effect, a more frequent positive effect with angiotensin converting enzyme inhibitors and beta-blockers was seen for all constructs. Narrower demographics and smaller sample sizes may have biased similar positive effects in calcium-channel blockers and diuretics.


An evaluation of self-measured blood pressure in a study with a calcium-channel antagonist versus a beta-blocker. In recent years self-measurement of blood pressure at home has gained increasing importance but there have been only a few studies comparing casual, ambulatory, and self-measured blood pressure determinations during a single clinical trial. We therefore compared treatment-induced blood pressure-reductions in a double-blind, placebo-controlled, parallel study design with a single morning dose of either 10 mg bisoprolol (n = 26) or 20 mg nitrendipine (n = 27) with casual blood pressure readings in the morning before the dose, ambulatory 24-h monitoring, and self-recorded measurements in the morning before the dose and in the evening. Mean reductions for systolic and diastolic blood pressure after 4 weeks of therapy were significantly greater for bisoprolol than for nitrendipine. The treatment-induced blood pressure reductions were most pronounced as assessed by casual readings but showed good agreement between casual, ambulatory, and self-measured blood pressure for group comparisons. In some patients, however, marked individual differences between the three methods were observed. Correlation coefficients between ambulatory and self-measured blood pressure were 0.4 for systolic blood pressure (P less than .05) and 0.6 for diastolic blood pressure (P less than .0005). Under the conditions of this parallel study design and the usual statistical risks, a difference of 5 mm Hg in diastolic blood pressure can be detected in 118 patients at the clinic, in 70 patients if ambulatory blood pressure is used, or in 56 patients if self-measured blood pressure is used. In conclusion, bisoprolol was more effective over 24 h than nitrendipine at the doses studied.(ABSTRACT TRUNCATED AT 250 WORDS)


Efficacy of conventional and sustained-release verapamil in stable angina pectoris. The efficacy of sustained-release (s.r.) verapamil and conventional verapamil were compared in a double blind, crossover study in 20 patients (age 53 +/- SD6 years) who had stable effort angina and had used betablockers and long acting nitrates for at least two weeks. All patients received s.r. verapamil 200 mg b.i.d. and conventional verapamil 120 mg t.i.d. in a randomised order for two weeks. A symptom limited bicycle exercise test was performed at the end of the patients' previous medication period with betablocker plus long acting nitrate and at the end of both verapamil treatments in the morning before drug administration and three hours thereafter. All the patients improved subjectively during both verapamil regimens according to NYHA classification and they had fewer anginal attacks. The time to onset of ST-segment depression during exercise remained shorter during beta-blockade and long acting nitrates than during both verapamil regimens (P less than 0.05). During the peak action three hours after drug administration conventional verapamil was most effective at comparable workloads (P less than 0.05), whereas the exercise time was slightly prolonged with s.r. verapamil before drug administration.


H1 antagonists in the management of the itch of urticarias. This article reviews the effect of H1 antihistamines on the pruritus of urticaria, from articles in which their therapeutic effect on chronic idiopathic and physical urticaria is assessed. In limited studies available pruritus improved concomitantly with wealing by an average of two thirds, though the response in individual patients was variable. In some physical urticarias the pruritus and wealing showed disproportionate improvement compared to erythema. The minimally sedating H1 antihistamines were as effective or more effective than classical H1 antihistamines. The dose of antihistamines that totally abolished a histamine weal only partly reduced urticarial weals (therefore by inference also of the associated pruritus). Additional therapy aimed at pruritogenic mediators other than histamine would be expected to improve urticarial pruritus.


Effect of H1-receptor blockade on anthralin inflammation. The effect of terfenadine, an H1-receptor antagonist, on anthralin inflammation was studied in 12 subjects. Subjects were randomised to receive terfenadine 60 mg or placebo b.d. in a double-blind, cross-over study. Anthralin 5, 10 and 20 micrograms in 10 microliters chloroform was pipetted onto flexor forearm skin. Anthralin inflammatory oedema and erythema were measured using Harpenden calipers and a reflectance photometer, 48 h after anthralin application. After a 3-day washout period, placebo and terfenadine were restarted and the procedures repeated on the opposite arm. Anthralin erythema and oedema increased with dose but the dose-response curves showed no significant difference with or without terfenadine (p greater than 0.05). This study shows that H1-receptor blockade does not inhibit anthralin inflammation.


DNA hybridization analysis of mycobacterial DNA using the 18-kDa protein gene of Mycobacterium leprae. DNA hybridization studies using a 611-base pair (bp) probe, encoding the entire 18-kDa protein of Mycobacterium leprae, demonstrated that M. simiae, M. intracellulare, M. kansasii, M. terrae, ADM-2, M. avium, M. scrofulaceum, M. gordonae and M. chelonei appear to possess DNA sequences homologous to the 18-kDa protein gene of M. leprae. RFLP analysis revealed that the restriction sites in the M. leprae 18-kDa gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare. The restriction patterns observed with the 611-bp probe were useful in differentiating M. intracellulare, M. simiae, and M. leprae from each other, as well as in distinguishing strains of M. simiae serovar 1. Finally, the presence of homologous sequences in various mycobacteria did not affect the specificity of a previously described PCR test for detection of M. leprae, based on the M. leprae 18-kDa protein gene.


Beta-adrenergic blocking agents: their current status. Use of beta (beta)-adrenergic blocking agents began in the 1960s and has risen dramatically since then. Newer agents offer more actions and fewer side effects. Uses for the drugs continue to increase as their safety and efficacy become increasingly apparent. beta block, today, has widespread application in cardiovascular disease. In addition, these agents are used in the management of endocrine disorders, neurologic situations, psychiatric disorders, gastrointestinal problems, and sensory disorders. Many new beta blockers are still investigational, and some show even greater promise for therapeutic applications in the future.


Dose-response study with high-dose inhaled salmeterol in healthy subjects. A double-blind, placebo-controlled, cross-over study in 10 healthy male subjects has been carried out to investigate the non-pulmonary effects of single inhaled doses of salmeterol 100 micrograms, 200 micrograms and 400 micrograms and salbutamol 400 micrograms from a metered-dose inhaler. At all doses tested, salmeterol produced statistically significant changes in pulse rate, tremor, blood glucose and plasma potassium concentrations, compared with placebo. All changes were dosed related. A number of dose-related adverse events including tremor, awareness of heart beat and headache were reported after salmeterol administration.


Dose-dependent alpha 1-adrenoceptor antagonist activity of the anti-arrhythmic drug, abanoquil (UK-52,046), without reduction in blood pressure in man. 1. The dose-dependency of the alpha 1-adrenoceptor antagonist activity of the anti-arrhythmic abanoquil (UK-52,046) was investigated in 10 healthy male subjects who received serially increasing infusions of phenylephrine before and 2, 4, 8, 12, 24 and 48 h after single oral doses of abanoquil 0.25, 0.5 and 1 mg and placebo in a double-blind randomised manner. 2. The doses of phenylephrine required to increase systolic BP by 20 mm Hg (PS20) were calculated using a quadratic fit to the individual dose-response curves. 3. Abanoquil 0.25, 0.5 and 1 mg increased the PS20 in a dose-dependent manner with effects which were maximal at 2 to 8 h and lasted for 24 to 48 h (P less than 0.05). Maximal dose ratios were: abanoquil 0.25 mg 2.0 +/- 0.9, 0.5 mg 2.4 +/- 1.3, 1 mg 3.4 +/- 1.1. 4. No change occurred in supine BP but a small increase (P less than 0.01) occurred in supine HR 8 h post-dosing (64 +/- 9, 58 +/- 6 beats min-1 for abanoquil 1 mg and placebo respectively). 5. Therefore abanoquil 0.25, 0.5 and 1 mg showed dose-dependent alpha 1-adrenoceptor antagonist activity with no effect on supine BP.


Evaluation of in vivo partial beta 1/beta 2-agonist activity: a dose-ranging study with carteolol. 1. The aims of this study were to investigate the partial agonist profile of carteolol and evaluate methodology for differentiating relative beta 1 and beta 2 partial agonist activity (PAA) in vivo. 2. Eight normal subjects received single oral doses of carteolol 10 mg, 30 mg and 60 mg; nadolol 40 mg; pindolol 30 mg and placebo, given in a single-blind, randomised crossover design. 3. beta 1-PAA was demonstrated with carteolol by dose-related increases in resting heart rate and systolic blood pressure, and a plateau in the dose-response curve for attenuation of exercise tachycardia. beta 2-PAA with carteolol was evidenced by a dose-related increase in resting finger tremor and progressive attenuation of exercise-induced hyperkalaemia. beta 2-adrenoceptor antagonism was shown by attenuation of terbutaline induced hypokalaemic, chronotropic and finger tremor responses. 4. Carteolol behaved as a non-selective beta-adrenoceptor antagonist with both beta 1 and beta 2-PAA components. In the standard clinical dose range of 10-30 mg, its in vivo PAA effects were relatively beta 1-selective. Thus at low doses, there appeared to be a dissociation between selectivity of antagonist and partial agonist activity. 5. Attenuation of exercise hyperkalaemia appears to be a novel and sensitive method for the evaluation of beta 2-PAA in vivo.


Inhibition of histamine-induced nasal obstruction by cetirizine in allergic rhinitis. This double-blind randomized crossover placebo-controlled study was designed to assess objectively the nasal antihistamine effect of cetirizine in patients with allergic rhinitis and control subjects. Nasal challenge was performed by nebulization of increasing doubling doses of histamine (0; 0.04 to 1.28 mg/nostril) in six patients with allergic rhinitis and six control subjects on cetirizine (2 x 10 mg daily for 3 days) or placebo. Sneezings were counted and nasal obstruction was assessed by subjective scoring and by objective measurement of nasal airway resistance by posterior rhinomanometry. Histamine induced sneezing and a dose-dependent increase in nasal airway resistance and in perceived sensation of obstruction. Responses were greater in patients with allergic rhinitis compared with controls, although of borderline significance for nasal obstruction. Cetirizine totally abolished sneezing and significantly reduced increase in nasal airway resistance and perceived sensation of nasal obstruction both in normal and rhinitic subjects. Our results demonstrate by an objective measurement the nasal antihistamine effect of cetirizine. We propose this simple provocation test to assess the time-course of the effect of antihistamines and to compare the relative potency of related compounds.


Pharmacokinetics of dipipanone after a single oral dose. A single oral dose of Diconal (dipipanone HCl 10 mg, cyclizine HCl 30 mg) was given to six volunteers. The mean peak plasma dipipanone concentration was 29 ng ml-1, the time to peak plasma concentration was 1-2 h, the mean elimination half-life was 3.5 h and the mean AUC was 156 ng ml-1 min. Less than 1% of the dose was excreted in urine unchanged over 24 h.


The effect of celiprolol on glomerular filtration rate and renal blood flow in patients with chronic renal impairment and healthy volunteers. A double-blind, placebo controlled study investigated the effects of celiprolol, 200 mg daily for 7 days, on glomerular filtration rate (GFR) and estimated renal blood flow (ERBF) in eight healthy volunteers and eight patients with chronic renal insufficiency. In healthy volunteers the mean difference in GFR was 4.8 ml min-1 (95% CI -8.2 to 17.7 ml min-1) and the mean difference in ERBF was 49.8 ml min-1 (95% CI -47.5 to 147 ml min-1) after celiprolol. In patients with chronic renal insufficiency the mean difference in GFR was -2.1 ml min-1 (95% CI -64.6 to 65.8 ml min-1). The study had sufficient power to detect a 15% change in GFR for normals and 10% for patients, and for ERBF, changes of 14% and 23% were detectable. Celiprolol at a dose of 200 mg daily for 7 days can be used in patients with chronic renal insufficiency without adversely affecting GFR or ERBF.


Pharmacotherapy of dilated cardiomyopathy: current status and future directions. The current status and future directions of the pharmacotherapy of dilated cardiomyopathy are reviewed. The Japanese multicenter study on the effect of beta-blockers revealed significant improvement of NYHA functional classification, LV end-diastolic dimension, ejection fraction, and exercise tolerance time in patients with dilated cardiomyopathy. From our study using normal rabbits, metoprolol augmented adenylate cyclase activity without upregulation of the beta-adrenergic receptor number. Carteolol, a beta-adrenergic blocker with intrinsic sympathomimetic action, prevented the development of dilatation and hypertrophy of the heart in the chronic stage following murine encephalomyocarditis (EMC) viral myocarditis. Metoprolol exerted no such effect. Animal experiments indicated that immunosuppressive treatment for inflammatory myocarditis may aggravate the clinical course of the disease. However, immunosuppressive treatment in acute myocarditis should be reevaluated with the use of newly developed antiviral agents. A new synthetic immunoactive peptide FK 565, given before or simultaneously with viral inoculation, proved effective in inhibiting myocardial virus replication and myocardial damage in murine EMC viral myocarditis. Beneficial effects of captopril on survival rate and myocardial injury were demonstrated dose dependently in murine EMC viral myocarditis, even when the treatment was started around the peak of virus replication, namely, on day 4-14 after inoculation. Captopril may be promising for the treatment of acute myocarditis, and hopefully for prevention of the progression from myocarditis to dilated cardiomyopathy.


[Polyarteritis nodosa in a woman with temporal arteritis]. A case of a 74-year-old woman, with a histological diagnosis of Transient Arteritis two years before and Nodose Polyarteritis, is described. Although the coexistance of these two vasculitis in a same patient has been rarely described in the literature, we considered that the diagnostic suscpiction of Nodose Polyarteritis should be maintained in a patient with Transient Arteritis developing multisystemic signs.


Micro-effects of language on risk perception in drug prescribing behavior. The decision to prescribe neuroleptics for the treatment of psychosis involves a potentially tragic choice between, on the one hand, a probability of psychosis and a probability of side effects, such as tardive dyskinesia, on the other. In an experimental paradigm, we examined this decision process. We hypothesized that linguistic factors considered irrelevant under classical formulations of individual choice behavior would have a significant effect on this decision. All subjects were presented with a case vignette involving a potentially psychotic patient. Subjects were then asked what probability of tardive dyskinesia they would either "accept" or "risk" in order to prevent psychotic decompensation. In addition this factor was crossed with a contextual factor that varied the patient's age. The effect of "risk" versus "accept" language was evident in significantly different patterns of decision making across age groups. The data have important implications for clinical decision making, the elicitation of informed consent, and the directions that the courts have taken in malpractice and patient's rights cases.


Typing of Listeria monocytogenes by restriction polymorphism of the ribosomal ribonucleic acid gene region. Ninety four strains of Listeria monocytogenes of different serovars and phagovars as well as of varying origins were characterized by ribosomal RNA gene restriction polymorphism. After digestion by EcoRI or HindIII, chromosomal DNAs were hybridized with a cloned rDNA probe from Bacillus subtilis that included the 16S rRNA gene. The 94 strains were divided into 14 ribovars according to the different hybridization patterns generated by cleavage with EcoRI. Less important genomic heterogeneity could be detected when DNAs were digested by HindIII. EcoRI ribovars analysis allowed to describe a new typing scheme which did not corroborate routine typings such as serotyping and phage typing. It also confirmed a new view of this species in exhibiting a clone gathering most human strains, as first inferred from multilocus enzyme analysis (Piffaretti et al., 22).


Agonists of cholinergic and noradrenergic receptors facilitate synergistically the induction of long-term potentiation in slices of rat visual cortex. Acetylcholine (ACh) and noradrenaline (NA) have been shown to facilitate experience-dependent modifications of synaptic connectivity during postnatal development of the kitten visual cortex. To further investigate the mechanisms of this facilitation we studied the effects of these neuromodulators in an in vitro model of use-dependent synaptic plasticity. We have chosen long-term potentiation (LTP) in rat visual cortex slices because it shares several features with the in vivo model. In both cases induction of synaptic modifications requires that postsynaptic activation reaches a critical threshold and in both cases changes are induced more easily in young animals and when N-methyl-D-aspartate (NMDA) receptor-gated conductances are activated. Intracellular recordings were obtained from regular spiking cells in supragranular layers of rat visual cortex and LTP was induced by tetanic stimulation of the underlying white matter. Both cholinergic and noradrenergic agonists raised the probability that tetanic stimuli induced LTP and as in vivo they acted synergistically. These effects were mediated by agonists of muscarinic and beta-receptors, respectively. The agonists of both receptor systems enhanced the depolarizing response to the tetanus and increased NMDA receptor-gated conductances during this response. We suggest that this mode of action also accounts for the facilitatory effects which ACh and NA have on use-dependent synaptic plasticity in the developing visual cortex.


[Changes in excitatory amino acids and N-methyl-D-aspartate receptors in the brain in aged mice]. By means of chromatography and radioligand binding assay, changes of excitatory amino acids (EAA) and N-methyl-D-aspartate(NMDA)receptors in the cerebral cortex of aged mice were investigated. It has been demonstrated that the maximal binding capacity (Bmax) of NMDA-sensitive 3H-L-glutamic acid (3H-L-Glu) in the cerebral cortex of aged mice (14 months) was obviously decreased as compared with that of young mice (2 months), but no significant differences in KD values between young and aged mice were observed. The content of L-Glu in the cerebral cortex of aged mice was reduced 18.7% when compared with that of young mice. However, no change in the content of L-aspartic acid was found. Long-term potentiation (LTP) of synaptic transmissions in the hippocampus is mediated by NMDA receptors. We also confirmed that the slope and amplitude of hippocampal population spikes were significantly lower in aged mice than in young mice after induction of LTP by tetanus (100 Hz, ls), and persistent time of LTP in aged mice was shorter as compared with that in young mice. The results showed that the functions of the central EAA neurotransmitter systems are decreased, and such changes may be related to the decline of learning and memory capacities in the aged.


Assay of stanozolol for tumor initiating and promoting activity in two rat liver foci bioassays. In this study stanozolol, one of the most abused anabolic steroids, was investigated for tumor initiating and promoting activity in two rat liver foci bioassays, using gamma-glutamyltranspeptidase (GGT) as marker for detection of putative preneoplastic foci. Stanozolol, orally administered for 2 weeks, at a dose level approximately 400-times larger than the human therapeutic dose, in rats initiated with N-nitroso-diethylamine according to the Solt-Farber system assay, did not produce any increase in the number and volume of GGT-positive liver foci. A 6-week oral treatment with stanozolol (430 ppm in the diet) followed by 2 weeks of 2-acetylaminofluorene (AAF) diet (200 ppm), carried out according to the Tatematsu assay system to evaluate the initiating activity, did not provoke any significant modification of the number and volume of GGT-positive foci as compared to the controls. In the rats receiving AAF (200 ppm in the diet for 2 weeks) followed by 6 weeks of stanozolol, to evaluate the promoting activity, an increase in number and volume of the GGT-positive foci was observed at the highest oral dose, but the differences from the corresponding control values which resulted were not statistically significant. Taken as a whole the results of this study do not provide any substantial evidence of carcinogenic activity of stanozolol in rat liver, even when orally administered at high doses.


Antiproliferative effect of the garlic compound S-allyl cysteine on human neuroblastoma cells in vitro. A variety of compounds derived from garlic bulbs have been shown in animal systems to possess anticancer properties. However, little information is available regarding the effectiveness of garlic in the prevention or treatment of human cancers. In the current study, we have assessed the ability of S-allyl cysteine (SAC), a derivative of aged garlic extract, to affect the proliferation and differentiation of LA-N-5 human neuroblastoma cells in vitro. Time-and dose-dependent inhibition of cell grow was observed in cultures treated with SAC for at least 2 days, with a half-maximal response at approximately 600 micrograms/ml. SAC treatment was unable to induce differentiation in neuroblastoma cells as assessed by morphological, biochemical and molecular markers. In addition, SAC was unable to potentiate the effects of retinoic acid and 8-bromo-cyclic AMP, agents known to promote differentiation of LA-N-5 cells. Our results indicate that SAC can inhibit human neuroblastoma cell growth in vitro. However, the apparent inability of this compound to induce differentiation may limit its therapeutic potential.


Homozygosity of the short arm of chromosome 17 in cervical carcinoma. We have determined the frequency of heterozygosity of the short arm of chromosome 17 in 20 cervical tumours using the highly polymorphic probe pYNZ22. Only 25% of the tumours were heterozygous at this locus. This is significantly lower than the level of 86% heterozygosity for this locus in the general population indicating that loss of one allele occurs in cervical cancer. Heterozygosity for a locus on the long arm of the same chromosome showed no significant difference between the tumours and the general population indicating that genetic loss was confined to the short arm of the chromosome. The analysis of premalignant lesions showed 70% of patients were heterozygous suggesting that loss of material from the short arm of chromosome 17 took place at a late stage in tumour development. This report confirms predictions made from previous karyotypic analysis and is the first indication of allele loss on the short arm of chromosome 17 in cervical cancer.


Parkinsonism associated with calcium channel blockers: a prospective follow-up study. Parkinsonism is a well-known side effect of some calcium channel blockers (CCB). Its long-term evolution, however, is unknown. To clarify this issue, we performed a prospective follow-up study involving 32 patients diagnosed with CCB-induced parkinsonism. After the baseline examination, the CCB were discontinued and serial evaluations were carried out according to the same protocol. Despite a global improvement, cognitive and mood disturbances subsided slowly, and tremor persisted in most patients. After 18 months of CCB withdrawal, 44% of patients had depression, 88% had tremor, and 33% still had criteria for diagnosis of parkinsonism. During the survey, only three patients were found to be fully recovered. The improvement of some clinical symptoms was related to age: Patients younger than 73 years recovered better than older patients did. Our data indicate that CCB-induced parkinsonism is not the benign condition previously thought, and suggest an age-related prognosis of this entity.


A survey of tardive dyskinesia in psychiatric inpatients in Japan. To investigate the prevalence of tardive dyskinesia (TD) in a group of psychiatric patients receiving low doses of antipsychotic drugs, we examined 647 Japanese inpatients (361 men and 286 women) with a mean age of 49.8 years, receiving a mean dose of antipsychotic drugs of 276.8 mg of chlorpromazine equivalent. TD was diagnosed according to the criteria of Schooler and Kane with the Abnormal Involuntary Movement Scale. The overall prevalence of TD was 22.3%. Mild TD was found in 67.4% of TD patients, moderate TD in 29.2%, and severe TD in 3.5%. The TD patients were older (59.0 years) than those without this condition (47.2 years). The prevalence of TD increased with advancing age until the 7th decade, when it reached a plateau. The dose of daily antipsychotic drugs was lower in the TD patients (207.6 mg) than in the patients without TD (296.6 mg). The duration of primary illness was longer in the TD patients (28.9 years) than in the patients without TD (20.4 years). Patients receiving antiparkinsonism drugs showed TD less frequently (20.7%) than those not receiving such drugs (31.0%). No significant associations were found between the presence of TD and sex or primary illness.


Motor response to repeated dopaminergic stimulation in Parkinson's disease. Recent studies giving subcutaneous apomorphine or intravenous levodopa boluses have not found clear evidence of behavioral hyposensitivity to repeated dopaminergic stimulation in Parkinson's disease (PD). Here we analyze that data, and review experimental findings in animal models and our previous experience with parkinsonian patients. We conclude that acute tolerance to pulsatile stimulation is likely to play a role in the pathophysiology of motor fluctuations in PD.


Accessory function of human mononuclear phagocytes for lymphocyte responses to the superantigen staphylococcal enterotoxin B. The role of the cytokines IL-1 alpha, IL-1 beta, and IL-6 and the cell adhesion molecules ICAM-1, LFA-1 (alpha and beta), and Mac-1 as accessory molecules for stimulation of T cells by the superantigen staphylococcal enterotoxin B (SEB) was examined. Both blood monocytes and alveolar macrophages were used as accessory cells because these cells differ in patterns of cytokine expression and thus potentially in accessory cell function for superantigens. The blastogenic response of highly purified T cells to SEB was reconstituted with either monocytes or alveolar macrophages. IL-1 secretion was increased comparably in monocytes and alveolar macrophages by SEB, but IL-6 was not stimulated by SEB. IL-1 alpha plus IL-1 beta reconstituted the response of T cells to SEB but required the addition of accessory cells. The cell adhesion molecules ICAM-1 and LFA-1 but not Mac-1 also functioned as accessory molecules for SEB-induced cluster formation and lymphocyte blastogenesis. Thus, not only must this superantigen bind to Class II MHC on accessory cells as is well known, but also SEB requires at least certain cytokines (IL-1 alpha and IL-1 beta) produced by accessory cells and cell adhesion molecules (ICAM-1 and LFA-1) for activation of T lymphocytes.


Mycophenolic acid inhibits the degranulation of rat peritoneal mast cells. The effect of mycophenolic acid (MPA), a potent inhibitor of inosine monophosphate dehydrogenase, on the degranulation of rat peritoneal mast cells (RMC) was studied. RMC were pretreated for 48 hr with 0.1-10 microM MPA before the cells were sensitized with IgE and triggered with specific Ag. The net amount of [3H]5-HT released from granules was decreased by 44 and 32% with 1 and 10 microM MPA treatment, respectively. MPA inhibition of degranulation was completely reversed by the addition of 30 microM guanosine to the incubation medium. There was no difference in the apparent number or affinity of IgE binding sites between control and MPA-treated RMC. MPA pretreatment also had no effect on the IgE receptor-mediated production of PGD2 in RMC. These results suggest that depletion of intracellular GTP pools by MPA can disrupt the signaling between the IgE receptor and the secretory granules and that, under these same conditions, the release and metabolism of arachidonic acid are unaffected.


Synthesis and biological activities of optical isomers of 2-(4-chlorophenyl)-5,6-dihydro-(1)benzothiepino[5,4-c]pyridazin-3(2H)-o ne 7-oxide. Two enantiomers of 2-(4-chlorophenyl)-5,6-dihydro-(1)benzothiepino[5,4- c]pyridazin-3(2H)-one 7-oxide ((+/-)-1: Y-23684) were synthesized in high yields by asymmetric oxidation of the synthetic precursor (2) using modified Sharpless reagent. Among the oxidants tested, cumene hydroperoxide (CHP) gave the highest optical and chemical yields, while tert-butyl, tert-amyl, and 1,1,3,3-tetramethylbutyl hydroperoxides did not show such high enantio-selectivities. The absolute configuration of (+)-1 enantiomer synthesized from 2, Ti(O-iso-Pr)4, (-)-diethyl tartarate, and CHP was determined to be S by X-ray crystallographic analysis. Both enantiomers, S-(+)-1 and R-(-)-1, and (+/-)-1 had approximately equivalent in vivo activities to antibicuculline test in mice and anticonflict test in rats, although S-( + )-1 showed about three times higher affinity to benzodiazepine receptor than R-(-)-1 in [3H]diazepam binding assay.


Association between growth stimulation by phenobarbital and expression of cytochromes P450 1A1, 1A2, 2B1/2 and 3A1 in hepatic hyperplastic nodules in male F344 rats. This study was conducted to examine relationships between phenobarbital (PB) treatment, specific cytochrome P450 gene expression patterns and growth rates of hepatic hyperplastic nodules. Nodules were induced in 8 week old male F344 rats by a Solt-Farber resistance protocol. Six weeks after diethylnitrosamine (DEN) initiation, subgroups of rats were either kept on control chow diet or transferred to a chow diet containing 0.05% PB, then killed 2 weeks later. [3H]Thymidine was delivered continuously via osmotic minipump during the final 3 days of the experimental to label dividing cells. PB treatment resulted in a 89% increase in the number of persistent gamma-glutamyl transpeptidase (GTT) nodules per cm2 section, a 278% increase in the area of persistent GGT nodules per cm2 section, and a 116% increase in the average area per persistent nodule. PB increased the number of [3H]thymidine-labeled persistent GGT nodules but did not significantly change the labeling index (LI) distribution pattern or the average LI. A slight but uniform increase in CYP1A2 expression (relative to surrounding, non-nodular tissue) was observed in 50% (23/46) and 59% (60/102) of persistent nodules in control and PB-treated animals respectively. In contrast, for nodules undergoing remodeling, CYP1A2 expression was elevated in only 9% (2/22) and 0% (0/24) in control and PB groups respectively. In the PB group, CYP2B1/2 was underexpressed in 53% (54/102) of persistent GGT nodules and in 0% (0/24) of the remodeling nodules. Comparing LI among the persistent GGT nodules, those that displayed simultaneous increases in CYP1A2 and decreases in CYP2B1/2 had the highest LI, and were followed in level by those expressing either increases in CYP1A2 or decreases in CYP2B1/2. Nodules that expressed both CYP1A2 and 2B1/2 in a manner similar to the surrounding tissue had the lowest LI. Thus, these data suggest that expression of specific forms of cytochrome P450 may be an important factor in determining other phenotypic characteristics, e.g. rate of cell proliferation and GGT expression, within specific nodules.


Relative efficacy and safety of loratadine, hydroxyzine, and placebo in chronic idiopathic urticaria and atopic dermatitis. The subjects of this double-blind study were 59 patients with chronic idiopathic urticaria or atopic dermatitis randomly assigned to receive 10 mg of loratadine once daily and placebo twice daily (n = 20), 25 mg of hydroxyzine thrice daily (n = 20), or placebo thrice daily (n = 19). The patients (15 men, 44 women) were aged 18 to 65 years. Among the 18 patients with urticaria and 41 with atopic dermatitis, daily symptom scores decreased 43% and 57% in those receiving loratadine, 47% and 38% in those receiving hydroxyzine, and 0% and 33% in the placebo patients. The difference between the treated and placebo patients was significant among the urticaria patients. According to a global evaluation of treatment effects, more treated than placebo patients reported marked or complete symptom relief; among the patients with atopic dermatitis, the difference was significant between the loratadine and placebo patients. Somnolence or sedation during treatment was reported by one of the patients receiving loratadine, by eight of the hydroxyzine patients, and by two of the placebo patients; the difference between the loratadine and hydroxyzine patients was significant. It was concluded that loratadine is as effective as hydroxyzine in the treatment of urticaria and demonstrates a significant antipruritic effect in atopic dermatitis, but does not have the central nervous system effects of hydroxyzine.


The effects of monensin on blood-borne arrest and glycosylation of BL/VL3 lymphoma cells. We have previously shown that inhibitors of N-glycan processing alter both the cell surface carbohydrates and the homing properties in lymphoid cells. We have now studied the effects of the ionophore monensin (MON) on these parameters. Arrest in the spleen of [111In]-labelled BL/VL3 murine T lymphoma cells, injected intravenously was clearly reduced if the cells had been cultured for 24 h in the presence of monensin (0.1-1.0 microgram ml-1). We have characterized glycopeptides from BL/VL3 murine T lymphoma cells. Following labelling with tritiated precursors (fucose, mannose, galactose, glucosamine), surface glycopeptides from BL/VL3 murine T lymphoma cells, were released by trypsin and separated by gel filtration on Bio-Gel P6 and by affinity chromatography on immobilized lectins. After treatment with MON, a class of high molecular mass glycopeptides was no longer found. There were less complex and more high mannose glycans, as a consequence of a reduction of terminal glycosylation (sialylation, fucosylation or incorporation of N-acetyl-glucosamine). Similar findings were obtained with immunoprecipitated Thy-1 antigen. However, as estimated by flow cytometry analysis, the cell surface expression of Thy-1 was not reduced in MON-treated cells. Taken together our results show that cell surface oligosaccharides are modified dramatically, but that at least, certain cell surface antigens are present in normal amounts. It is tempting to speculate that changes in glycosylation account for the abnormal homing properties of MON-treated cells.


Electrical field stimulation-mediated relaxation of rabbit middle cerebral artery. Evidence of a cholinergic endothelium-dependent component. The effects of electrical field stimulation (EFS) of rabbit middle cerebral arteries were examined using wire-mounted arterial segments. EFS of segments maintained at rest tension caused a tetrodotoxin-sensitive sympathetic contraction. In agonist-contracted segments maintained at approximately 60% of tissue maximum force, EFS caused a relaxation in two thirds of the preparations. Maximum response (mean +/- SEM) was 33 +/- 3.5% of maximal relaxation. The EFS relaxation was tetrodotoxin-sensitive but was not blocked by either chronic surgical sympathectomy or exposure to guanethidine (5 microM). Electron microscopy of chromaffin-fixed arterial sections showed the presence of chromaffin-positive large and small vesicles. Within the same sheath of Schwann were also a smaller number of nerve profiles containing many small clear vesicles. Removal of the vascular endothelium or treatment with atropine (10 nM) eliminated the EFS relaxation in approximately 50% of the segments and reduced the response in another 35-40%; in the remainder, relaxation was unaffected. Combined data for endothelium removal and atropine treatment showed that each caused a significant (p less than 0.01) reduction in the EFS relaxation. Atropine also significantly reduced EFS relaxation in guanethidine-treated segments. There was no reduction in EFS relaxation after procedures that antagonized ATP- or substance P-mediated relaxations. These results indicate that EFS of precontracted rabbit middle cerebral artery causes a neurogenic nonadrenergic relaxation. The neuroeffector mechanism mediating this response has a predominantly cholinergic endothelium-dependent component as well as a noncholinergic endothelium-independent component.


Cholinesterases exhibiting aryl acylamidase activity in human amniotic fluid. Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) in human amniotic fluid were estimated in the presence of selective inhibitors. Amniotic fluid cholinesterases (mixture of acetylcholinesterase and butyrylcholinesterase) purified by procainamide-Sepharose affinity chromatography exhibited aryl acylamidase activity which was sensitive to serotonin inhibition (a property of aryl acylamidases associated with both acetyl- and butyrylcholinesterases) and tyramine activation (shown exclusively by aryl acylamidase associated with butyrylcholinesterase). Tyramine activation was unaffected in the presence of the selective acetylcholinesterase inhibitor BW284C51 whereas it was abolished in the presence of the selective butyrylcholinesterase inhibitor ethopropazine, suggesting the presence of both types of aryl acylamidases in amniotic fluid, one associated with acetylcholinesterase and the other associated with butyrylcholinesterase. Butyrylcholinesterase and the associated aryl acylamidase activity in the affinity purified enzyme was selectively immunoprecipitated by a polyclonal antibody raised against human serum butyrylcholinesterase. Estimation of the activity ratio of acetylcholinesterase to butyrylcholinesterase in a few samples of amniotic fluid showed that this could vary depending on the butyrylcholinesterase arising from contaminating blood in the samples. Gel electrophoresis under non-denaturing conditions and enzyme staining showed that butyrylcholinesterase band was detectable on the gel in all the samples whereas acetylcholinesterase band was below detectable levels in normal samples but visible in samples from pregnancies of neural tube defect fetuses. It is suggested that the use of selective cholinesterase inhibitors along with gel electrophoresis and immunoprecipitation studies may be useful in the assessment of cholinesterase activities in human amniotic fluid.


Clearance of purified human liver gamma-glutamyltransferase after intravenous injection in the rat. The clearance of gamma-glutamyltransferase was studied by injecting the purified human liver enzyme intravenously in the rat. The results show a biphasic clearance, with a rapid initial rate of removal. The initial uptake is more rapid for neuraminidase-treated GT. Liver accounts for the bulk organ uptake and the enzyme is almost exclusively taken up into the parenchymal cells. We suggest that the uptake of circulating GT is receptor mediated, most likely by the galactose receptor of the parenchymal cells.


Biochemical investigation of a Brazilian patient with a defect in mitochondrial acetoacetylcoenzyme-A thiolase. A case report of 3-ketothiolase deficiency due to a defect of mitochondrial acetoacetyl-CoA thiolase protein in a Brazilian boy and its biochemical investigation is presented. The child had moderate generalized hypotonia, EEG alterations and crises of metabolic acidosis following infections. Hypotonia and EEG abnormalities disappeared with a low protein diet, and physical and mental development are normal. Urinary organic acid excretion was typical of 3-ketothiolase deficiency, showing consistently high levels of 2-methyl-3-hydroxybutyric acid and tiglylglycine. Activation of acetoacetyl-CoA thiolase activity by potassium (K) ion in cultured fibroblasts was not observed, demonstrating the lack of activity of mitochondrial acetoacetyl-CoA thiolase. In addition, the signal for the mitochondrial acetoacetyl-CoA thiolase protein was undetectable in the immunoblot analysis. In the pulse-chase experiments, the signal for mitochondrial acetoacetyl-CoA thiolase was detected after a 1-h pulse but not after a 24-h chase. These results indicate that the deficiency was caused by an unstable mitochondrial acetoacetyl-CoA thiolase protein.


Role of neurohumoral control of the circulation in determining exercise capacity in patients with heart failure. The weak relationship between left ventricular function and exercise capacity in heart failure has stimulated interest in neurohumoral mechanisms as possibly contributing to exercise intolerance. Although chronic activation of neuroendocrine systems is characteristic of heart failure and is accompanied by impaired reflex responsiveness to physiologic stimuli, data from clinical trials do not support the hypothesis that the abnormal neurohumoral state is directly related to exercise intolerance. Thus, these systems may play an important role in the natural history of the disease and its high mortality, but they may not be critical to the impaired exercise capacity.


[The relationship between metastatic phenotype and steady expression of BGC-Ha-ras oncogene from metastasis cell lines in nude mice (abstract)]. NIH/3T3 cells transformed by activated BGC-Ha-ras (6.6 kb) with a point mutation at codon 12 were able to induce tumor in nude mice with lung metastasis. The metastatic phenotype seemed stable in vivo metastasis assay. After two round subculture of the successively induced metastasis foci, two cell lines, GCM-1/3T3 and GCM-2/3T3, were established. In Southern blot analysis it was found that the bands from GCM-1/3T3 and GCM-2/3T3 were the same. Based on Southern analysis and polymerase chain reaction-restriction fragment length polymorphism (PCR-RPLF), it was proved that the activated c-Ha-ras (6.6 kb) existed all along in the genomes of the transformed and metastatic culture cells. Amplification and over-expression of activated c-Ha-ras were shown by DNA and RNA dot blot hybridization in transformed and metastatic culture cells. The metastatic phenotype might be related to the existence and steady expression of the point mutated ras.


[Detection of human papillomavirus (HPV) DNA in carcinoma of uterine cervix and genital tract diseases in Jiangsu province, China]. Pst-1 cleaved DNA restriction fragment length polymorphism (RFLP) of biopsy samples from 41 patients with squamous cell carcinoma of uterine cervix collected in Jiangsu province, China were examined for HPVs by Southern blot hybridization using HPV 16 DNA as a probe. 20 of the 41 samples were positive for HPVs when hybridized under non-stringent condition. HPV was not detectable in samples collected in the same time period from patients with cervical adenocarcinoma (N = 2), vaginal carcinoma (N = 3), vulval carcinoma (N = 1) and benign cervicitis (N = 8). Of the 20 positive samples, 7 (17.1%) had CHPV X1, a new type of NPV previously discovered is China, 6 (14.6%) had HPV 16, 4 (9.9%) had HPV 31, and in 3 (7.2%) the HPV type is as yet undetermined. Our data indicate that HPV 16 and CHPV X1 may be more closely related to cancer of the uterine cervix in Jiangsu province.


Organogenesis in Drosophila melanogaster: embryonic salivary gland determination is controlled by homeotic and dorsoventral patterning genes. We have investigated Drosophila salivary gland determination by examining the effects of mutations in pattern forming genes on the salivary gland primordium. We find that the anterior-posterior extent of the primordium, a placode of columnar epithelial cells derived from parasegment 2, is established by the positive action of the homeotic gene Sex combs reduced (Scr). Embryos mutant for Scr lack a detectable placode, while ectopic Scr expression leads to the formation of ectopic salivary glands. In contrast, the dorsal-ventral extent of the placode is regulated negatively. Functions dependent on the decapentaplegic product place a dorsal limit on the placode, while dorsal-dependent genes act to limit the placode ventrally. We propose a model in which these pattern forming genes act early to determine the salivary gland anlage by regulating the expression of salivary gland determining genes, which in turn control genes that are involved in salivary gland morphogenesis.


Hyperprolactinemia. Evaluation and management. Hyperprolactinemic syndromes are a diverse group of disorders that present in widely different age groups. They are common disorders in both men and women and require management over a lifetime. The past 20 years have greatly increased our knowledge about these disorders and simplified their management. Yet, we have made little progress in understanding the origin and dynamics of pituitary tumors and their natural histories, and as yet we have been unable to develop effective tumoricidal therapy or medication that corrects, at the central axis level, neurotransmitter disorders that may produce hyperprolactinemia. Perhaps these issues will be the subject of such reviews in the future.


An investigation of large inhibitors binding to phosphoglycerate kinase and their effect on anion activation. This study extends, to a series of larger anions, our earlier investigation of the interaction of the trypanocidal drug suramin and other small negatively charged molecules with yeast phosphoglycerate kinase. 1H-NMR structural studies of phosphoglycerate kinase in the presence of varying concentrations of these large molecules (designed to mimic, at one end, the anionic charge distribution in the substrate 3-phosphoglycerate, while possibly being able to interact across the cleft of the enzyme) including inositol 1,4,5-triphosphate, 4-amino-6-trichloroethenyl-1,3- benzenedisulphonamide, gallic acid and sulphasalazine are described. The anion activation and/or inhibition of the enzyme by these molecules are also reported. Evidence that binding to the general anion site in the 'basic patch' region of the protein may be responsible for either the activating or inhibiting effects, while binding at the hydrophobic (catalytic) site leads to inhibition only is presented. A reaction scheme which explains these observations is given.


A novel 24-kDa microtubule-associated protein purified from sea urchin eggs. Chromatographic fractionation of a crude extract of sea urchin eggs on a hydrophobic column enabled us to find a new 24-kDa microtubule-associated protein (SU-MAP24) that bound tightly to the column and was eluted under alkaline conditions. Biochemical studies using the purified protein showed its direct binding to microtubules reconstituted from tubulin purified from starfish sperm outer fibers. SU-MAP24 promoted tubulin polymerization in a dose-dependent manner. Immunoblotting analysis showed that SU-MAP24 is present in a microtubule protein fraction obtained from a crude extract using taxol, and immunostaining of paraffin-sectioned metaphase eggs showed its localization in the mitotic apparatus. These results show that SU-MAP24 is a newly identified microtubule-associated protein.


Polyclonal immunoglobulin therapy protects against cardiac damage in experimental coxsackievirus-induced myocarditis. Balb/c male mice infected i.p. with 2 x 10(5) plaque forming units (PFU) of coxsackievirus B3 (CVB3) develop severe myocarditis 7 days later. Studies were performed to determine whether therapy with normal polyclonal immunoglobulin would prevent cardiac inflammation. Partially purified immunoglobulin was derived from pooled mouse serum by ammonium sulphate precipitation. Infected animals given either 100 or 1000 micrograms of this preparation for 2 days prior to infection showed greater than 50% reduction in myocarditis compared to control animals which were infected and given either phosphate buffered saline, human immunoglobulin or monoclonal mouse IgG to an extraneous antigen. Protection did not depend upon inhibition of virus infection since cardiac viral titres were frequently equivalent in control and immunoglobulin-treated groups.


Disposition kinetics and concentration-effect relationship of metipranolol in patients with cirrhosis and healthy subjects. The disposition kinetics and heart rate reducing effect of deacetylmetipranolol (DMP), the active form of the beta-adrenoreceptor blocking agent metipranolol (MP), administered as a single 40 mg oral dose have been compared in 6 patients with cirrhosis and 6 healthy volunteers. The mean maximal DMP concentration was significantly higher and the time to reach the peak level shorter in the patients compared to the healthy subjects. There was also a significantly higher AUC of DMP, a shorter half-life of the rapid phase of the decline in DMP concentrations, a smaller central compartment and lower apparent DMP clearance in patients. A correlation with the albumin level was observed in cirrhotics for individual values of apparent DMP clearance (r = 0.92) and AUC (r = -0.89). The maximal reduction in heart rate was recorded in patients at plasma DMP levels which were already significantly lower than the peak levels. Median inhibitory concentrations (IC50) and maximum possible heart rate reductions (delta HRmax), obtained by fitting individual plots of the plasma DMP concentration-effect relationship to the inhibitory Emax model in the postdistributional phase of DMP disposition were significantly higher in cirrhotics than in healthy subjects. It is conjectured that down-regulation of adrenoreceptors due to chronic sympathetic activation in hepatic cirrhosis contributes to decreased sensitivity to the reduction in heart rate following a single dose of the beta-blocker.


Membranes of activated CD4+ T cells expressing T cell receptor (TcR) alpha beta or TcR gamma delta induce IgE synthesis by human B cells in the presence of interleukin-4. In the present study it is demonstrated that human B cells can be induced to switch to IgE production following a contact-mediated signal provided by activated T cell receptor (TcR) gamma delta+, CD4+ and TcR alpha beta+, CD4+ T cell clones and interleukin (IL)-4. The signal provided by these T cell clones was antigen nonspecific, indicating that the TcR alpha beta/CD3 or TcR gamma delta/CD3 complexes were not involved in these T-B cell interactions. Activated TcR alpha beta+, CD8+, and TcR gamma delta+, CD4-CD8-, or resting CD4+ T cell clones were ineffective. Intact TcR alpha beta+ or TcR gamma delta+, CD4+ T cell clones could be replaced by plasma membrane-enriched fractions isolated from these activated CD4+ T cell clones. In contrast, membranes isolated from resting TcR alpha beta+, CD4+, TcR gamma delta+, CD4+ T cell clones or an Epstein-Barr virus (EBV)-transformed B cell line (EBV-LCL) failed to provide the costimulatory signal that, in addition to IL-4, is required for induction of IgE synthesis. As described for intact CD4+ T cells, CD4+ T cell membranes induced purified surface IgM+ B cells to switch to IgG4- and IgE- but not to IgA-producing cells, excluding the possibility of a preferential outgrowth of IgG4- and IgE-committed B cells. The membrane activity was inhibited by protease or heat treatment. Induction of IgE synthesis by B cells co-cultured with both TcR alpha beta+, CD4+ and TcR gamma delta+, CD4+ T cell clones and membrane preparations of these cells was blocked by anti-class II major histocompatibility complex (MHC) monoclonal antibodies (mAb), whereas various anti-CD4 mAb had differential blocking effects. Murine L cells, or EBV-LCL transfected with CD4 could not replace CD4+ T cell clones. These results indicate that, although CD4 and class II MHC antigens are required for productive CD4+ T cell clone-B cell interactions, an additional signal, provided by a membrane associated (glyco)protein that is induced by activation of both TcR alpha beta and TcR gamma delta, CD4+ T cells, is needed for induction of IgE production in the presence of IL-4.


Molecular cloning of a novel member of the immunoglobulin gene superfamily homologous to the polymeric immunoglobulin receptor. The CMRF35 monoclonal antibody recognizes a cell membrane antigen present on the surface of monocytes, neutrophils, a proportion of peripheral blood T and B lymphocytes and lymphocytic cell lines. Initial studies with CMRF35 suggested an unusual pattern of serological reactivity which did not correspond to any of the known leukocyte differentiation antigen clusters. We describe here the cloning and sequencing of a cDNA encoding the CMRF35 antigen by means of expression in COS cells and immunoselection with the CMRF35 monoclonal antibody. The cDNA encodes a novel integral membrane glycoprotein of 224 amino acids that represents a new member of the immunoglobulin (Ig) gene superfamily. The molecule comprises (a) a single extracellular Ig variable domain remarkably similar to the Fc receptor for polymeric IgA and IgM, (b) a membrane-proximal domain containing a high proportion of proline, serine and threonine residues that was predicted to be heavily O-glycosylated, (c) an unusual transmembrane anchor that contained a glutamic acid and a proline residue and (d) a short cytoplasmic tail. Transcripts encoding the CMRF35 protein were detected in early monocytic cell lines, in peripheral blood T cells and in some B lymphoblastoid cell lines, confirming the results of immunocytological staining. However, the level of CMRF35 expression on peripheral blood T cells was shown to decrease in response to mitogenic stimulation. The likelihood that the CMRF35 antigen shares a common evolutionary ancestor with the poly Ig Fc receptor is discussed.


Interleukin (IL)-4 production by human T cells: differential regulation of IL-4 vs. IL-2 production. We have examined the regulation of interleukin (IL)-4 production by human peripheral blood T cells. Production of IL-4 was shown to be regulated differently from IL-2 and interferon(IFN)-gamma production. Stimulation of peripheral blood lymphocytes with anti-CD3, anti-CD2, anti-CD28, Phorbol 12-myristate 13-acetate (PMA) or IL-2 as a single stimulant did not induce IL-4 production. However, combinations of anti-CD2 with either anti-CD28 or IL-2 resulted in IL-4 production, peaking at days 3-4. Stimulation with anti-CD3 instead of anti-CD2 gave similar results, but was less potent. After days 3-4, IL-4 levels decreased, most likely due to consumption of IL-4. PMA profoundly affected cytokine production, it enhanced IL-2 production by at least tenfold, whereas, in the same cell population, IL-4 production was almost completely inhibited. This was observed at the protein as well as at the mRNA level. In contrast, agents that increase intracellular cAMP levels inhibited IL-2 production but left IL-4 production unaffected. IFN-gamma production behaved similar to IL-2 production but the effects were less outspoken.


Is there a dopaminergic projection from the A11 catecholamine cell group to the amygdala? The dopaminergic nature of the pathway from the subparafascicular thalamic nucleus and its adjacent region to the amygdala was reexamined by means of retrograde fluorescent tracers coupled with tyrosine hydroxylase (TH) immunofluorescence. After injecting a small amount of tracer into the amygdala, retrogradely labeled cells were found in the subparafascicular thalamic nucleus and its adjacent periventricular region. TH immunofluorescence showed that these labeled cells completely lacked TH immunoreactivity. Similar results were obtained when a larger amount of tracer was applied to the amygdala. The present study, in contrast to the previous report describing the dopaminergic innervation of the amygdala by the cells in and around the subparafascicular area (A11 catecholamine cell group), indicates that the A11 cell group does not contribute to a dopaminergic input to the amygdala.


Quercetin, a bioflavonoid, inhibits the increase of human multidrug resistance gene (MDR1) expression caused by arsenite. Expression of the MDR1 gene, which encodes P-glycoprotein, is increased under some stress conditions. We have reported that quercetin, a bioflavonoid, inhibits the expression of heat-shock proteins. We have identified the effects of quercetin on the MDR1 gene expression in the human hepatocarcinoma cells line, HepG2. The increase of P-glycoprotein synthesis and MDR1 mRNA accumulation caused by exposure to arsenite were inhibited by quercetin. The CAT assay suggested that quercetin suppressed the transcriptional activation of the MDR1 gene after exposure to arsenite. Although many drugs that prevent the P-glycoprotein function have been reported, this is the first report to describe the inhibition of MDR1 expression by a reagent.


Analysis of mouse Evx genes: Evx-1 displays graded expression in the primitive streak. Two mouse genes, Evx-1 and Evx-2, each encoding a homeodomain closely related to that of the Drosophila even-skipped gene were isolated using a PCR-based strategy. The structure and sequence of these genes are described. Mapping studies localized Evx-1 to chromosome 6, near the Hox-1 gene cluster, and Evx-2 to chromosome 2, near the Hox-4 cluster. The evolutionary implications of these linkages are discussed. RNA in situ hybridization analysis of Evx expression in embryos demonstrated a striking pattern of Evx-1 expression during gastrulation, whereas Evx-2 RNA could not be detected at any stage by this technique. Evx-1 RNA is first detected shortly before the onset of gastrulation in a region of ectoderm containing cells that will soon be found in the primitive streak. This localized expression of Evx-1 provides the first molecular evidence for regional differences in the mouse embryonic ectoderm before gastrulation. Throughout gastrulation, Evx-1 expression is limited to cells near and within the streak and that expression is graded, with a posterior-to-anterior decrease in the level of RNA. Based on fate-mapping studies indicating that different types of mesoderm emerge from different regions of the primitive streak and our observation that high levels of expression are localized to the region that will give rise to extraembryonic and ventral mesoderm, we speculate that Evx-1 plays a role in the dorsoventral specification of mesodermal cell fate.


Effect of luminal administration of thyrotropin-releasing hormone or somatostatin on gastric pH and interaction of these peptides in rats. The effect of intraluminal administration of thyrotropin-releasing hormone (TRH) on gastric pH and release of luminal somatostatin, and a possible interrelationship between TRH and somatostatin in the rat stomach were studied. TRH was administered into the stomach via an intragastric tube at various doses (50 pg/kg-10 micrograms/kg) and gastric pH was measured after 15 min. The intraluminal administration of TRH significantly decreased gastric pH at doses over 1.0 ng/kg. Time-course studies at a dose of 100 ng/kg TRH exhibited a significant decrease in gastric pH at 15, 30 and 60 min. Furthermore, TRH administration caused a significant increase in immunoreactive-somatostatin (ir-somatostatin) concentrations in the gastric wall and a significant decrease in ir-somatostatin concentrations in the gastric juice. On the other hand, intraluminal administration of somatostatin caused a significant increase in ir-TRH concentrations in the gastric wall and a significant decrease in ir-TRH concentrations in the gastric juice, and significantly raised gastric pH at 5 min. These findings suggest that luminal TRH may exert a regulatory effect on gastric acid secretion, and that TRH may have a possible interaction with somatostatin in the modulation of gastric acid secretion.


Production of hemagglutinins and pili by Vibrio mimicus and its adherence to human and rabbit small intestines in vitro. Clinical isolate of Vibrio mimicus were examined for production of cell-associated hemagglutinin (HA) and pili and for adherence to formalin-fixed human intestinal mucosa. V. mimicus grown on CFA agar for 3 h at 37 degrees C possessed HA and adhered better to the mucus layer than to the epithelial cell surface. A significant correlation was found between the HA titers and adherence ability to the epithelial cell surface of villi (P less than 0.05); adherence to the ileal lymphoid follicle-associated epithelium occurred at higher levels. In contrast, V. mimicus grown on CFA agar for 20 h at 37 degrees C exhibited lower levels of HA and reduced adherence ability. The production of pili was more pronounced after 20 h of incubation than after 3 h of incubation. In comparison with V. cholerae 01 and V. cholerae non-01 cultured under similar conditions, V. mimicus showed inferior adherence, but with similar HA production or piliation.


Clonal differences within M-types of the group A Streptococcus revealed by pulsed field gel electrophoresis. Digestion of chromosomal DNA with the rare cutting restriction enzyme SfiI in association with pulsed field gel electrophoresis was used to observe restriction fragment length polymorphisms (RFLP) among isolates of group A Streptococcus. Streptococci examined included isolates belonging to the same M-type (epidemiologically related and unrelated), and isolates from other M-types. RFLP patterns were quite distinct between all serotypes tested. More importantly, isolates from within a serotype could be differentiated by this technique.


Physician extenders: who is using them?

Physician extenders: who is using them? Nurse practitioners and physician assistants (physician extenders) are playing an increased role in medical care. The purpose of this research was to determine the proportion of adults who have received health care from physician extenders. This study used the subject population of the 1990 Kentucky Health Survey, a probability survey of all households in Kentucky. Study personnel contacted subjects using random digit telephone dialing. Subjects were then interviewed to ascertain whether subjects had received health care from physician extenders and whether they were satisfied with that care. Of 687 participating subjects, 25% had received care from physician extenders during the previous two years, primarily for minor problems and routine checkups. More than 90% of these subjects reported satisfaction with the care they received. Users of physician extenders did not differ from nonusers with respect to income, education, insurance status, self-assessment of health status, or rural versus urban location. Men used physician extenders more frequently than women. A substantial proportion of the population has used physician extenders. Patient satisfaction with physician extenders is high. Use of physician extenders may be an effective strategy for improving delivery of primary care.


apterous, a gene required for imaginal disc development in Drosophila encodes a member of the LIM family of developmental regulatory proteins. The apterous (ap) gene is required for the normal development of the wing and haltere imaginal discs in Drosophila melanogaster. ap encodes a new member of the LIM family of developmental regulatory genes. The deduced amino acid sequence of ap predicts a homeo domain and a cysteine/histidine-rich domain known as the LIM domain. In these domains ap is highly similar to the mec-3 and lin-11 proteins of Caenorhabditis elegans and to the vertebrate insulin enhancer-binding protein isl-1. ap is presumably required for transcriptional regulation of genes involved in wing and haltere development. The nature of the defects in homozygous null mutant flies is consistent with the pattern of ap expression in the larval imaginal discs. ap is also expressed in a complex pattern in the embryo, including portions of the peripheral nervous system (PNS) and central nervous system (CNS). A requirement for ap expression in the larval and adult CNS may be the underlying cause of the defects in hormone production and vitellogenesis described for ap mutations.


[Psychopharmacology and clinical aspects of benzodiazepine dependence]. Recent aspects of mechanism of benzodiazepines (BDZ) via indirect GABA-mimetic inhibitory effects at brain-specific BDZ-GABA receptors may contribute to an understanding of pathophysiological mechanisms of anxiety disorders and drug dependencies. The development of various BDZ receptor ligands with diverse psychopharmacological properties may lead to safer drugs with regard to "drug seeking" and "maintaining" properties. This paper reviews recent research of BDZ receptor pharmacology and clinical aspects of abstinence syndromes including also prospective studies.


[Drug-induced headache].

[Drug-induced headache]. Migraine or tension-type headache lasting for several years is frequently complicated by chronic daily headaches. Those patients recurrently take analgesics or ergotamines. Headache induced by chronic ergotamine use is well known. Recent studies, however, have shown that a similar form of headache is caused by analgesic abuse. Treatment of drug-induced headaches envisages withdrawal of the headache medication.


Lessons from animal experiments in myocarditis. We have developed murine models of viral myocarditis induced by encephalomyocarditis (EMC) virus in which severe myocarditis, congestive heart failure and dilated cardiomyopathy occur in high incidence. From these models, we have learned the natural history and pathogenesis and assessed new diagnostic methods and therapeutic and preventive interventions. Mural thrombi in the atria and ventricles, ventricular aneurysms, conduction disturbance and various arrhythmias were seen in these models. Anti-heart antibody were found in sera of mice and myosin isoenzyme were changed during the course of EMC virus myocarditis. Atrial natriuretic polypeptide was markedly increased in the ventricles in these mice. Successive infection with coxsackievirus and EMC virus developed lesions similar to chronic myocarditis. The myocardial uptake of antimyosin antibody was proved to be a useful method of diagnosis of myocarditis. Treatment with the nucleoside analogue, ribavirin and recombinant alpha interferon effectively inhibited myocardial virus replication and reduced myocardial damage. Passive immunization and virus-specific vaccine prevented development of myocarditis. The use of immunosuppressive therapy was associated with greater mortality when administered early in illness and beneficial effects were not seen by later administration. Angiotensin-converting enzyme inhibitor improved myocardial injury and congestive heart failure. A nonselective beta-adrenergic blocker with intrinsic sympathomimetic activity, carteolol, prevented the development of myocardial lesions similar to those in dilated cardiomyopathy after myocarditis in the chronic stage.


Medical treatment of gastroesophageal reflux disease: options and priorities. Gastroesophageal reflux disease (GERD) represents a spectra of symptoms and of reflux damage to the esophagus. This reflux damage is due to a prolonged acid exposure of the esophagus arising from an imbalance between protective motility factors and aggressive acid secretory factors. Initially, patients may be managed by modifying their food intake and by supportive anti-gravity measures. However, many individuals will require drug therapy. Symptomatic relief can be achieved with pro-kinetic agents, antacids, sucralfate suspension, H2-receptor antagonists and H(+)-K+ ATPase pump blockers. There are limitations in the study design of experiments which have compared one agent with another. Accepting these design limitations, it would appear that pump blockers lead to higher rates of endoscopic healing than the use of standard doses of H2-receptor antagonists. However, higher doses of H2-receptor antagonists will likely give higher rates of symptomatic relief and endoscopic healing of GERD. Recurrence of symptoms and esophagitis occur in a high proportion of patients with GERD, and some patients may need to be considered for maintenance therapy.


Potent versus mild acid suppression in peptic ulcer disease. In the consideration of potent versus mild suppression of gastric acid secretion in the treatment of peptic ulcer disease, it must be remembered that pharmacological effect in terms of degree of suppression of acidity is closely linked to therapeutic efficacy in terms of numbers of ulcers healed. More potent degrees of suppression of acidity result in faster ulcer healing. Moderate suppression of acidity, such as is achieved by the H2RA, can heal just as many ulcers if treatment is continued longer. Mild acid suppression will take still longer to produce the same healing rates. Therapeutic benefit of any treatment must be assessed also in terms of the side-effect profile of the drug or drugs used. There is an enormous cumulative experience with the H2RA as a group and they are known to have an excellent safety profile.


Short- and long-term management of peptic ulcer disease: current role of H2-antagonists. With numerous active agents available to treat peptic ulcer disease both acutely and long-term, this paper reviews various aspects of these agents and their effect on the disease process, reviewing efficacy, compliance, incidence of relapse, side-effect profile, drug interactions and the effect on the natural history of peptic ulcer disease. Helicobacter pylori is obviously a factor in the occurrence of peptic ulcer disease, but with the high prevalence of asymptomatic infection and evidence suggesting that duodenal ulcer disease may be self-limiting, widespread treatment with bismuth and antibiotics may do more harm than good. Maintenance therapy is recommended for the patient with previous bleeding or other complication, the patient with severe symptoms, and the patient with frequent recurrences. Maintenance therapy appears to reduce the complication rate in the patient with previous bleeding and possibly in the patient with uncomplicated disease, although the incidence of complications in this group is quite low.


Induction by cortisol of aminopeptidases production from the human placenta in tissue culture. Human placental aminopeptidase M and A and post-proline endopeptidase are known to act as degrading enzymes of bioactive peptides such as angiotensin II, oxytocin and endogenous opioids. We tested the effects of cortisol on the activities of human placental aminopeptidase A and M and post-proline endopeptidase using short-cultured placental tissues. From 34.5 nM to 3.45 microM of cortisol significantly increased the activities of 3 enzymes. Our present data suggest a possible important role of cortisol in the growth of human placenta via induction of placental aminopeptidases.


Parathyroid hyperplasia in multiple endocrine neoplasia type 1: a pathological and immunohistochemical reappraisal. Twenty-nine parathyroid glands from nine patients with multiple endocrine neoplasia type 1 (MEN 1) syndrome were examined histopathologically and immunocytochemically to characterize better the nature of the accompanying parathyroid hyperplasia. The parathyroids showed varying degrees of nodular and diffuse (partial and total) hyperplastic involvement as well as apparently normal tissue. The nodules were usually multiple within any one gland and showed a varied cytoarchitectural pattern. All glands studied showed both cellular argyrophilia and parathyroid hormone immunoreactivity. The staining pattern for parathyroid hormone ranged from negative or weak to strong and from patchy to diffuse in hyperplastic tissue from different glands and within the same gland. Apparently normal areas usually showed the strongest positive reaction. Amyloid material was observed within glandular lumens from hyperplastic areas in over half of the studied cases and stained positively for parathyroid hormone. This suggests that the hormone could be the precursor molecule of parathyroid amyloid as occurs with hormone-derived amyloid from other endocrine tumours. The overall findings indicate that the most striking feature of the parathyroid glands in MEN 1 is their variability, both morphological and functional, as indicated by their parathyroid hormone immunoreactivity.


An empirical review of the impact of triplicate prescription of benzodiazepines. In 1989 New York became the first state to add benzodiazepines to the list of controlled substances requiring a triplicate prescription, allowing the state to track prescribing patterns and target providers, pharmacies, and patients for investigation when misuse is suspected. Studies by the state reporting that regulation has significantly reduced inappropriate prescribing and illicit diversion of benzodiazepines without affecting legitimate prescribing practices are being challenged by other studies showing that patients with legitimate needs for benzodiazepines are being denied them, often after abrupt discontinuation. Several reports indicate a significant increase in the prescribing of benzodiazepine substitutes that are less safe and effective, along with increased overdoses of some substitute drugs. Changes in physicians' legitimate prescribing practices may reflect their fears of the damage to career and peace of mind that follows investigations by regulatory agencies.


Prenatal diagnosis of 21-hydroxylase deficiency congenital adrenal hyperplasia using the polymerase chain reaction. We present an improved method for the prenatal diagnosis of congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. The polymerase chain reaction (PCR) was used to analyze DNA from an affected index case, the parents, and a cultured chorionic villus sample, for point mutations in the steroid 21-hydroxylase (CYP21) gene. We can predict that the fetus is an unaffected carrier.


Glucose-6-phosphate dehydrogenase (G6PD) electrophoretic variants and the PvuII polymorphism in southern African populations. Southern African Bantu-speaking negroid and San populations were examined with regard to the glucose-6-phosphate dehydrogenase (G6PD) PvuII restriction fragment length polymorphism (RFLP) showing alleles of 4 kb and 1.6 kb, called Type 1 and Type 2, respectively. The standardized disequilibrium coefficient for the electrophoretic G6PD types and PvuII alleles in the Southern African population was 0.28. The molecular lesion causing the GdA mutation is the same in the San and Southern African negroid populations. GdA chromosomes are found in association with both the Type 1 and Type 2 alleles, whereas none of the 62 GdB chromosomes from the Southern African populations had the Type 2 allele. Five of the 44 GdB chromosomes studied in the American Black population had the Type 2 allele, indicating that the GdB allele in the two populations may have different origins. The presence of all 3 A- deficiency mutations in the G6PD A gene, in a region where the ancestral population was thought to have predominantly G6PD B, may be explained by their origin in Africa after the divergence of the races.


Genotype mosaicism in fragile X fetal tissues. The fragile X syndrome is one of the most common familial causes of mental retardation. It is associated with the expression of a fragile site at Xq27.3, although not all individuals carrying the mutation are fragile-X-positive. Recently, the mutation causing this disease has been identified as the amplification of, or insertion into, a CGG repeat sequence at the fragile site. The mutated chromosome can be recognised by the decrease in mobility of the EcoRI fragment that covers the mutated region. Analysis of lymphocytes of affected males often gives a number of different sized fragments indicating somatic heterogeneity. We have investigated this mosaicism in various tissues of an affected fetus in order to determine the extent of the variation between tissues, and to ascertain how to interpret the results in lymphocytes. Our results suggest that the heterogeneity occurs in all fetal tissues, but that the pattern of fragments observed varies between tissues. Methylation across the region also varies. These differences may be reflected in the cellular phenotypes and may influence the ultimate expression of the clinical phenotype.


Highly polymorphic region of the human prothrombin (F2) gene. We have found a highly polymorphic region in the human prothrombin gene. Our sequence differed from that previously reported at as many as 6 positions in a 225-bp stretch spanning exon 6 and its flanking regions; four of these positions were related to endonuclease restriction sites for AluI, HpaII(MspI), MboII, and NcoI. AluI and HpaII digested all alleles of the Japanese tested. MboII and NcoI restriction fragment length polymorphisms are highly heterozygous and not in linkage disequilibrium; they thus serve as good human DNA markers.


Anonymous markers located on chromosome 6 in the HLA-A class I region: allelic distribution in genetic haemochromatosis. Two yeast artificial chromosomes of the HLA class I region were subcloned. Four of the subclones studied displayed restriction polymorphisms that corresponded to six bi-allelic series. Allelic distribution of the anonymous markers was then studied by comparing a control population with a group of patients with familial haemochromatosis. Only one marker presents an unequivocal association with the haemochromatosis gene and is 100 kb centromeric to HLA-A. This association however is not as strong as with HLA-A3. The results suggest two possible locations for the haemochromatosis gene: less than 100 kb centromeric to the HLA-A locus, or on the telomeric side.


Alpha alpha alpha alpha anti-3.7 type II: a new alpha-globin gene rearrangement suggesting that the alpha-globin gene duplication could be caused by intrachromosomal recombination. We report here a new human alpha-globin gene rearrangement carrying the two normal, alpha 2 and alpha 1, and two hybrid, alpha 1/alpha 2, globin genes in the order 5'-alpha 2-alpha 1/alpha 2-alpha 1/alpha 2-alpha 1-3'. Both the hybrid genes, subtyped with ApaI and RsaI restriction enzymes, were found to be of the uncommon anti 3.7 type II. The hybrid genes were expressed at the biosynthetic level and their interaction with the beta-thalassaemia IVS 1 nt 1 G----A mutation caused thalassaemia intermedia. We also report a case of an alpha alpha alpha-globin gene rearrangement in the twin of one of the alpha alpha alpha alpha-globin gene carriers; the duplicated gene was of the anti 4.2 type and was associated with the absence of RsaI polymorphism. The singular finding of an alpha alpha alpha alpha-anti 3.7 cluster with two identical rare hybrid genes suggests that the reciprocal unequal recombination causing the alpha-globin gene rearrangements could be of the intrachromosomal rather than the interchromosomal type.


Allotype distribution of human T cell receptor beta and gamma chain genes in Caucasians, Asians and Australian aborigines: relevance to chronic hepatitis B. RFLPs of TCR beta and gamma genes have been analyzed in chronic HBV carriers of three different ethnic populations to determine if there is an association of TCR allotypes with the development of chronic hepatitis B. The RFLPs of TCR beta and gamma genes were defined respectively by BglII and PvuII genomic fragments on Southern blots. These methods allow allotype assignment. The distribution of TCR beta alleles showed ethnic variation, with one allele significantly decreased in Australian Aborigines, but there was no association with chronic hepatitis B. The distribution of TCR gamma alleles did not show ethnic variation. However, a significant frequency decrease of one allele occurred in Aboriginal HBV carriers, suggesting the possibility of involvement of TCR gamma allotypes in the development of the chronic HBV carrier state in Australian Aborigines.


Linkage disequilibrium between phenylketonuria and RFLP haplotype 1 at the phenylalanine hydroxylase locus in Portugal. RFLPs of 36 normal and 41 mutant alleles at the phenylalanine hydroxylase locus were determined in 31 Portuguese kindreds. A total of 14 haplotypes including 10 normal and 7 mutant alleles were observed. Almost 75% of all mutant alleles were confined within only two haplotypes, namely haplotype 9 (17.1%) and haplotype 1 (56.1%). This frequency of mutant haplotype 1 in Portugal is, to our knowledge, the highest for this mutant haplotype in all studies reported to date. Other mutant haplotypes were either rare (haplotype 2, 9.7%) or totally absent (haplotype 3, 0%). Only 24.5% of all mutant alleles were found to consistently carry identified mutations, particularly R261Q (9.8%), R252W (3.3%), R408W (1.6%) and delta I94 (3.3%). A new mutation, L249F, located in the seventh exon of the gene, accounted for 6.5% of all mutant alleles in our series. Interestingly, this mutant genotype was consistently associated with mutant haplotype 1 (P less than 0.01), as also observed for the R261Q mutation. It appears, therefore, that mutant haplotype 1 is genotypically heterogeneous in Portugal and that more than two mutations account for its prevalence in this country.


Screening for nonsense mutations in patients with severe hemophilia A can provide rapid, direct carrier detection. Despite marked genetic heterogeneity in families with hemophilic patients, transition mutations in CG dinucleotides occur frequently. Of 71 CG dinucleotides in the factor VIII cDNA, a C-to-T transition in 12 would lead to a new Stop codon (CGA to TGA). Using restriction enzyme digestion of 11 amplified DNA fragments, seven point mutations were localized among 60 patients with severe hemophilia A. Five were detected as loss of a natural or introduced TaqI site at codons -5, 583, 1941, 2116, and 2209 and were confirmed as CGA (Arg) to TGA (Stop) nonsense mutations by DNA sequencing. A novel C-to-T nonsense mutation was detected as loss of the RsaI site at codon 1966 and confirmed by sequence in two unrelated individuals. Two partial gene deletions were detected as selective failure to amplify exon 1 and exons 15-22, respectively. In an additional (61st) patient who was subsequently found to have mild (instead of severe) hemophilia, digests suggested a mutation in codon 1696. Upon sequencing, this codon contained a novel missense mutation, a C-to-G transversion changing CGA (Arg 1696) to GGA (Gly). In four families with women available for testing, carrier status was rapidly determined by direct screening for the point mutation. In two of three with sporadic occurrences, the mother was a carrier as were two of four sisters. In the other family, the mother and a sister were homozygous for the TaqI cleavage site in their amplified exon 24 fragment, indicating a de novo C-to-T transition in codon 2209 in the patient's factor VIII gene. This final patient's sister was a noncarrier even though by linkage analysis she inherited the same factor VIII gene as her brother.


Polymerase chain reaction-based genotyping for allotypic markers of immunoglobulin kappa shows allelic association of Km with kappa variable segment. Allotypic markers of immunoglobulin kappa (Km) may be determined using a novel method of amplification of the constant segment (C kappa) (IGKC) by polymerase chain reaction (PCR) followed by restriction enzyme digestion. Restriction sites in the C kappa PCR product correlate with allotypic differences among Km(1), Km(1,2), and Km(3) alleles. An AccI site in the PCR product correlates with Km(3); and presence or absence of a MaeII site correlates with the Km(1) or Km(1,2) allele, respectively. Km allelic frequencies were determined in a Caucasian population and compared to genotypic frequencies of nearby polymorphic markers. Among unrelated individuals with rheumatoid arthritis (RA) and controls, there is no evidence of allelic association between CD8A and polymorphic markers of the immunoglobulin kappa region [a V kappa (IGKV) BglII polymorphism about 24 kb centromeric to C kappa, Km allotype, and a SacI polymorphism 3.5 kb telomeric to the C kappa segment]. Similarly, there is no allelic association of the SacI C kappa polymorphism with Km or with the BglII V kappa polymorphism. However, there is evidence of allelic association of V kappa B3 and Km, specifically between the V kappa BglII 2.2-kb allele and Km(3) and also between the V kappa 3.5-kb allele and Km(1,2). Therefore, Km typing by PCR-based methods suggests the presence of allelic association between polymorphisms within the coding region of the C kappa segment and the nearest V kappa segment.


A high-resolution cytogenetic map of 168 cosmid DNA markers for human chromosome 11. We have constructed a high-resolution cytogenetic map with 168 DNA markers, including 90 RFLP markers for human chromosome 11. The cosmid clones were mapped by fluorescence in situ suppression hybridization, in which discrete fluorescent signals can be detected directly on prometaphase R-banded chromosomes. Although these cosmid clones were distributed throughout the chromosome, they had some tendency to localize in the regions of R-positive band, such as 11p15, 11p11.2, 11q13, 11q23, and 11q25. Since these regions of chromosome 11 are considered to contain genes responsible for certain genetic diseases, cancer breakpoints involved in chromosome rearrangements, and tumor-suppressor genes, this high-resolution cytogenetic map will contribute to the molecular characterization of such genes. This map will also provide many landmarks essential for construction of the complete physical map with contigs of cosmid and YAC clones.


Fine genetic localization of the gene for autosomal dominant polycystic kidney disease (PKD1) with respect to physically mapped markers. PKD1, the gene for the chromosome 16-linked form of autosomal dominant polycystic kidney disease, has previously been genetically mapped to an interval bounded by the polymorphic loci Fr3-42/EKMDA2 distally and O327hb/O90a proximally. More recently, 26.6PROX was identified as the closest proximal flanking locus. We set out to refine the localization of PKD1 by identifying a series of single recombinant events between the flanking markers Fr3-42/EKMDA2 and O327hb/O90a and analyzing them with a new set of polymorphic loci that have been physically mapped within the PKD1 interval. We identified 11 such crossovers in eight families; 6 of these fell into the interval between GGG1 and 26.6PROX, a distance of less than 750 kb. Three of these crossovers placed PKD1 proximal to GGG1 and two crossovers placed PKD1 distal to 26.6PROX. Both of the latter also placed PKD1 telomeric to a locus 92.6SH1.0, which lies 200-250 kb distal to 26.6PROX. The sixth recombinant, however, placed the disease mutation proximal to the locus 92.6SH1.0. Several possible explanations for these observations are discussed. An intensive study to locate deletions, insertions, and other chromosomal rearrangements associated with PKD1 mutations failed to detect any such abnormalities. Thus we have defined, in genetic and physical terms, the segment of 16p13.3 where PKD1 resides and conclude that a gene-by-gene analysis of the region will be necessary to identify the mutation(s).


A 14-Mb physical map of the region at chromosome 11q13 harboring the MEN1 locus and the tumor amplicon region. We have constructed a physical map of chromosome 11q13, using 54 DNA markers that had been localized to 11q13.1----q13.5 by means of somatic hybrid cell panels. Although the map has some gaps, it spans nearly 14 Mb and includes the region containing the gene responsible for multiple endocrine neoplasia type 1 (MEN1) and also the region that is amplified in several types of malignant tumors. As the estimated average distance between each locus is roughly 300 kb, the markers reported here will be valuable resources for construction of contig maps with yeast artificial chromosomes and/or cosmid clones. Furthermore, these clones will be useful in efforts to identify the MEN1 gene and in analyses of the amplification units present at 11q13 in certain tumors.


Physical mapping of 14 new DNA markers isolated from the human distal Xp region. We have isolated 14 new DNA markers from the human Xpter-Xp21 region distal to the Duchenne muscular dystrophy gene by targeted cloning, employing two somatic cell hybrids containing this region as their sole human material. High-resolution physical localization of these markers within this region was obtained by hybridization to two mapping panels consisting of DNA from patients carrying various translocations and deletions in distal Xp. Five markers were assigned to the pseudoautosomal region where their position on the long-range map of this region was further determined by pulsed-field gel electrophoresis. The other nine markers map to the X-specific region. Informative TaqI restriction fragment length polymorphisms were observed for four loci. One of these represents a region-specific low-copy repeated element. These 14 new markers represent useful tools for the understanding of distal Xp deletion and translocation mechanisms and for the positional cloning of disease genes in the region.