Patterns of substance use among methadone maintenance patients. Indicators of outcome. Critics often cite the use of illicit drugs among methadone maintenance patients as evidence of this treatment modality's failure. The fact that methadone maintenance treatment does not lead quickly to abstinence in most clients can be a disappointment to treatment professionals and the public. By studying a cohort of 41 patients for their first year in a methadone maintenance program, this study revealed significant trends of diminished narcotic and cocaine use. However, increased benzodiazepine use was also identified. More importantly, this study patterns of drug using that predicted less drug use at the end of one year of treatment. These findings reflect the need for additional research to clarify the meaning of illicit drug use by methadone maintenance patients. In addition, the implications for providing, stopping, and determining effective drug treatment are discussed.


Alcoholism in relatives of primary cocaine-dependent patients. Inherited susceptibility is believed by some to be a factor in cocaine dependence. In this study, nonalcoholic cocaine-dependent patients (N = 175) were screened to identify alcoholic first-degree relatives. One or more first-degree relatives are also reported to be alcoholic by 25% to 50% of alcoholic patients. Approximately the same percentage of these cocaine patients (34%) also report having an alcoholic first-degree relative. This lends credence to the hypothesis that the apparent inherited susceptibility to alcoholism may be a more general susceptibility to addictive disease. A much higher proportion of females than males have an alcoholic first-degree relative.


Swiss heroin-addicted females. Career and social adjustment. Studies on gender-related differences among heroin-addicted individuals are uncommon because women usually make up only one quarter of any one sample group. In our sample group of 248 Swiss-German heroin-addicted individuals from different therapeutic programs and prisons, 70 were women (28.2%). Follow-up investigations were conducted after a lapse of 2 years, and after a lapse of 7 years, respectively. Men and women appear to differ with respect to why they begin to use drugs, as well as why they relapse back into drug use. Prior to the commencement of drug abuse, social background concerns and social adjustment dynamics appear to constitute a heavier burden for women than for men. Observations of social functioning and adjustment phenomena made during their drug careers and in the follow-up investigations, however, failed to yield significant statistical differences. The predominant effects of drug use appear to eclipse the gender-related role-pattern. On the basis of our background data, as well as our therapeutic experience, we postulate that for an individual whose sex-role identity is threatened, drug abuse has a stabilizing function, and it carries a message.


Has Malaysia's antidrug effort been effective? It is a common belief that a massive effort in law enforcement, preventive education and rehabilitation will result in the elimination of a country's drug problem. Based on this premise. Malaysia in 1983 implemented such a multifaceted anti-drug strategy, and the results of a 1987 study by the author suggested that Malaysia's effort had begun to contribute to a steady decrease in the number of identified drug abusers. Although the number of drug-addicted individuals declined, the country's recidivism rates were still high. Because of this high relapse rate, Malaysia expanded their rehabilitation effort and developed a community transition program. In order to determine the impact of these changes on the country's battle against drug abuse, a follow-up study was conducted in 1990. The results of this study did not clearly demonstrate that the Malaysian effort had been successful in eliminating the problem of drug abuse, and raised some questions concerning the effectiveness of the country's drug treatment programs.


Enucleation technique for children with retinoblastoma. Based on personal experience with approximately 350 enucleations for retinoblastoma, the authors describe their current surgical technique for removal of the eye, opening the globe to harvest fresh tissue for research including DNA analysis, and placement of the new hydroxyapatite orbital implant. Enucleation should be performed as gently as possible. A hemostat is placed on the stump of the severed medial rectus muscle for traction purposes, and long, minimally curved scissors are used to cut the optic nerve near the orbital apex. Clamps and snares are not advised and cautery of the orbital tissue is avoided. A piece of optic nerve is first submitted separately for histopathologic study and the globe is opened with a specific technique to harvest fresh tumor tissue. A hydroxyapatite implant covered by sterilized sclera is placed in the anophthalmic socket to provide orbital volume and improved motility of the prosthesis.


A multicomponent model for substance abuse treatment. The Addiction Research and Treatment Corporation. This paper reviews the activities of the Addiction Research and Treatment Corporation (ARTC), a substance abuse treatment agency, located in Brooklyn, New York, which services approximately 2,300 patients annually. ARTC's history, treatment, and research activities are first described. We then proceed to detail the ARTC's employee health maintenance and community relations programs. A multicomponent treatment model is then outlined, which emphasizes the interactive role of the above functions within an optimal treatment network.


The intermediate syndrome in organophosphate poisoning: presentation of a case and review of the literature. A dimethoate-poisoned woman gradually developed a moderately severe cholinergic crisis that was readily treated by atropine. After being symptom-free for nearly two days, she suffered from sudden life-threatening respiratory paresis and weakness of the facial, extraocular, neck flexor and proximal limb muscles. Muscarinic symptoms were absent. Cholinesterase inhibition was severe, and EMG revealed marked decrements at low rates of repetitive nerve stimulation, and increments at a high rate. The clinical course was compatible with the Intermediate Syndrome. This syndrome seems due to persistent cholinesterase inhibition presumably leading to combined pre- and postsynaptic impairment of neuromuscular transmission. Inadequate pralidoxime therapy is proposed but not established as contributory. Prolonged monitoring of respiratory function in patients poisoned by particular organophosphate agents is mandatory.


Effect of estrogen treatment on the peroxidase activity and estrogen receptors in the female rabbit urogenital tissues. The effect of one, 4 and 8 weeks of continuous estrogenization of rabbit on peroxidase (PO) activity and on both cytoplasmic and nuclear estrogen receptors was studied in the uterus, vagina, urethra and urinary bladder. Whereas the peroxidase activity in the urogenital tissue of untreated controls was near zero, after one week of estrogen treatment it increased very substantially in the uterus and vagina and much less dramatically in the urethra or urinary bladder. With continuation of estrogen treatment for 4 or 8 weeks the PO decreased by 80-90% in the uterus and vagina and by only 40% in the urethra. After one week of estrogen treatment the density of both cytosolic and nuclear estrogen receptors decreased by several fold in both the uterus and vagina, whereas it decreased by 30-50% in the urethra and bladder. The concentration of both cytosolic and nuclear receptors decreased further although less dramatically with continuing estrogen treatment, up to 8 weeks, in all tissues. These data suggest that although the general pattern of responses to estrogen showing an initial increase in PO followed by a reduction with continuing estrogen treatment is the same in all urogenital tissues, the responses seem to have a prolonged time scale in the case of lower urinary tract tissues. The quantitative aspect of the response generally corresponds with the density of estrogen receptors in the urogenital tissues.


Using ribosomal RNA gene restriction patterns in distinguishing isolates of Pasteurella haemolytica from bighorn sheep (Ovis canadensis). Pasteurella haemolytica isolates (n = 31) from two isolated captive herds of Rocky Mountain bighorn sheep (Ovis canadensis canadensis) were characterized and compared phenotypically (biotype, serotype, hemolytic activity) and by a genomic fingerprinting method known as ribotyping. Seven to nine distinct phenotypes were observed. Depending on the method used for serotyping, one to three phenotypes were common to both herds. Eighteen isolates, recovered from both herds, were non-hemolytic, biotype T, indirect hemagglutination assay serotype 4. Ribotyping, a method for highlighting genetically conserved deoxyribonucleic acid restriction site heterogeneity with a 32P-labelled Escherichia coli ribosomal ribonucleic acid probe, produced six to eight distinct ribotype pattern groups within the 31 P. haemolytica isolates, depending on the restriction enzyme used. In contrast to phenotypes, ribotypes appeared unique to each herd, and ribotyping helped to further differentiate some isolates of the same biotype and serotype. In addition, ribotyping provided an alternative means for evaluating relationships between isolates differing in hemolytic activity but which were otherwise phenotypically identical. We propose that ribotyping may be a useful adjunct to other bacterial characterization methods in studying the epizootiology of pasteurellosis in bighorn sheep.


[DNA analysis by polymerase chain reaction]. Polymerase chain reaction (PCR) is a technique to amplify only a specific segment of DNA without using a plasmid or a phage vector. It is a powerful tool for genetic analysis of various diseases including inherited and viral diseases, and is now being applied to clinical diagnosis. Here, presented are several methods using PCR mainly for diagnosis of hemoglobinopathy which we have been engaged in. Some other diseases are also included.


[A study on reaction mechanism of sodium lauryl sulfate-hemoglobin (SLS-Hb), Part 1]. The cyanmethemoglobin (HiCN) method has been adopted as the international standard procedure for hemoglobin (Hb) determinations due to the accuracy and stability of result. However, the presence of potassium cyanide (KCN) and potassium ferricyanide (K3-Fe(CN)6) in the reagents has raised problems of laboratory and environmental pollution. In 1981, Oshiro and colleagues developed a cyanide free method of Hb determination that is based on a low toxicity compound Sodium Lauryl Sulfate (SLS). The SLS-Hb method provides stable SLS-Hb formation through the following steps. 1) Reaction of SLS to erythrocytic membrane (disruption of the erythrocytic membrane). 2) Conformation change of Hb by SLS. 3) Iron oxidation by oxygen (Fe2+----Fe2+). 4) Formation of stable SLS-Hb (coordination of SLS). The paper presents several findings on the reaction mechanism of the SLS-Hb method.


[Usefulness of segmental Lp-TAE using lipiodol mixed with anticancer agent for inoperable hepatocellular carcinoma]. To determine the effect of appraising subsegmental or segmental transarterial embolization with Lipiodol mixed with anticancer drugs followed by gelatin particles (Segmental Lp-TAE) on inoperable hepatocellular carcinoma, we examined CT patterns and therapeutic results in 57 patients after Segmental Lp-TAE. Fifty-six tumors including 47 tumors less than 5 cm in size were the nodular type and 1 tumor was the massive type. The mean tumor size was 3.6 cm and the mean amount of Lipiodol was 4.4 ml. Portal veins in the embolized segment were highly visualized by injected Lipiodol on plain film immediately after Segmental Lp-TAE. On the follow-up CT, the size of the tumor with dense Lipiodol accumulation were reduced in all cases, and atrophy of the embolized segment was recognized. Forty-four of the 57 patients are alive, with the longest surviving patient still alive at 4 years and 5 months. Seventeen patients have survived for more than 2 years (direct crude survival rate: 65.0%), with the cumulative survival rates 93.2% at 1 year, 71.6% at 2 years. No recurrence was recognized in 33 of the 41 patients (80.0%) that were followed up for more than 1 year after Segmental Lp-TAE is a useful therapeutic method for hepatocellular carcinoma.


Histopathological studies of a case of cytomegalovirus retinochoroiditis. A case of cytomegalovirus (CMV) retinochoroiditis initially misdiagnosed as fungal endophthalmitis is reported. An 83-year-old man who was suspected of having cholangiocarcinoma presented uveitis in both eyes. Candida endophthalmitis was suspected on the basis of ophthalmic findings and past history, which included systemic corticosteroid administration and intravenous hyperalimentation. Intravenous treatment with miconazole was not effective. At autopsy, 3 months after the initial ophthalmological examination, the right eye was enucleated and examined histologically and histochemically. Light microscopic examination showed extensive retinal necrosis and numerous cytomegalic cells, so-called owl's eye cells, with intranuclear and intracytoplasmic inclusion bodies. CMV particles were seen by electron microscopy, and CMV-infected cells were observed by immunohistochemical staining by the direct method with fluorescein-labeled antibodies. These findings indicate that in suspected cases of fungal endophthalmitis various tests should also be carried out for CMV.


Protein increases glomerular eicosanoid production and activity of related enzymes. We examined the in vitro production of PGE2, 6-keto PGF1 alpha and TxB2 by isolated glomeruli from rats fed a low (6% casein) or a high (40% casein) protein diet for approximately eight weeks. Glomeruli from high protein-fed rats produced significantly greater amounts of PGE2, 6-keto PGF1 alpha and TxB2 under basal conditions and in response to the addition of 100 nM angiotensin II (Ang II) than glomeruli from low protein-fed rats. To elucidate the mechanisms by which greater protein intake enhanced the glomerular production of eicosanoids, we explored phospholipase (A2 and C) and cyclooxygenase activity in glomeruli isolated from low- or high-protein fed rats. PE-specific PLA2 activities were significantly increased in glomeruli from rats fed a high protein diet when compared to a low protein diet. On the other hand, PC-specific PLA2 activities were significantly decreased in glomeruli from rats fed a high protein diet. No significant difference in PIP2-PLC activities was detected between glomeruli of the two dietary groups. The cyclooxygenase content and activity was significantly greater in glomeruli from rats fed a high protein diet than in glomeruli from rats fed a low protein diet. Glomeruli of rats fed a 50/50 mixture of the diets (23% casein) had amounts and activity of cyclooxygenase and activities of PE-specific PLA2 intermediate between those of high and low protein-fed animals. In conclusion, increased synthesis of eicosanoids by glomeruli from rats fed a high protein diet may be mediated by increases in the amount and activity of cyclooxygenase coupled with enhanced activity of PE-specific PLA2.


Lipids and cellular Na+/H+ antiport activity in diabetic nephropathy. Increased cellular Na+/H+ antiport activity has been documented in various cell types from hypertensive humans and rats. This membrane abnormality may be associated with the thickening of the vascular media of resistance vessels. Such an abnormality has also been demonstrated in cells from type I diabetic patients with nephropathy, and may indicate the predisposition to essential hypertension in such patients. We now demonstrate the importance of the rate-limiting enzyme for cholesterol and isoprenoid synthesis, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, in determining cellular Na+/H+ antiport activity. This finding may have application in the future treatment of diabetic patients with proteinuria.


Treatment of arterial hypertension in diabetic humans: importance of therapeutic selection. This study was undertaken to test the hypothesis that, given equal arterial pressure reductions, the combination of an angiotensin converting enzyme (ACE) inhibitor and calcium antagonist slows declines in renal function and yields greater reductions in albuminuria over either agent alone. This hypothesis was evaluated in four groups of hypertensive, non-insulin dependent, diabetic subjects with renal insufficiency (N = 30). Renal hemodynamics, albuminuria and metabolic parameters were evaluated for a period of one year. Subjects were all placed on a 90 mEq sodium, 0.8 g/kg protein, 1500 calorie American Diabetes Association diet for the entire length of the study. Subjects were followed for two weeks off antihypertensive medications and were subsequently randomized to either lisinopril, alone (group I), sustained release verapamil, alone (group II), reduced doses of both lisinopril and sustained release verapamil (group III), and hydrochlorothiazide with guanfacine (group IV). At the end of one year group III had the greatest reduction in albuminuria (78 +/- 7%, group III vs. 59% +/- 4, group I: P less than 0.05). In addition, the decline in glomerular filtration rate (GFR) was the lowest in this group (0.28 +/- 0.07, group III vs. 0.69 +/- 0.12, group I; P less than 0.05) although there was no significant difference between groups II and IV. The highest side effect profiles were noted in group IV, the least in group III. The greatest reductions in renal hemodynamics occurred in all groups within the first month; however, striking differences between groups were noted (7.4 +/- 2%, group I vs. 1.4 +/- 2%, group III; P less than 0.05). We conclude that the combination of reduced doses of an ACE inhibitor and calcium antagonist attenuate both albuminuria and the rate of decline in glomerular filtration rate. Furthermore, the combination of these classes of agents appear to yield the lowest side effect profile over either agent alone. Lastly, high doses of ACE inhibition alone may be detrimental to renal function in late stage diabetics with renal insufficiency.


[Adenoid cystic carcinoma of the lacrimal gland]. A 38-year-old female presented with a left-sided peripheral N. VI-paresis and negative neuroradiologic work-up. After one year she developed an incomplete N. III paresis, numbness of her face correlating to N. V1 and V2, reduced lacrimal secretion as well as a palpable mass at the lateral orbital rim. Magnetic resonance imaging revealed an orbital tumor extending into the cavernous sinus. Biopsy disclosed an adenoid cystic carcinoma of the lacrimal gland with basaloid, cribriform and tubular pattern. Therefore, an exenteration with postoperative radiation therapy was performed. Immunohistochemical findings included positive reactions for keratin (KL-1 and AE 1 + 3), Vimentin and Egp 34. By determination of Ki 67 the proliferative activity of the tumor was found to be 15%. Our case report demonstrates that even in absence of an exophthalmus or pain a malignant orbital tumor may be assumed as the underlying cause for a combined monolateral oculomotor and trigeminal paresis. Immunohistochemical results support the notion, that the adenoid cystic carcinoma of the lacrimal gland may arise from precursor cells of the terminal duct system.


Noninvasive estimation of total body water in critically ill children after cardiac operations. Validation of a bioelectric impedance method. The understanding of fluid fluxes in pediatric cardiac critical care is crucial to effective management. Knowledge of variations in total body water in this situation would aid this understanding, but most available methods are unsuitable for routine use. Recently, estimation of total body water by a tetrapolar bioelectric impedance has been validated in older children and adolescents. We undertook a study to validate the method in the taxing conditions of pediatric cardiac critical care. A prospective comparative study was done in 16 children whose ages ranged from 6 days to 10 years (mean 23 months) after a variety of cardiac operations. Total body water was estimated by a standard isotope dilution method (deuterium oxide) and by bioelectric impedance by means of a Holtain body composition analyzer. Individual estimations of total body water were made on two successive days on each patient at varying intervals after a cardiac operation, bioelectric impedance being measured hourly during 4 hourly urine collections for the deuterium oxide method. Thirty-two simultaneous values of total body water (by isotope and by impedance) were collected. Population-specific regression relationship was established by plotting total body water (isotope) against height2/bioelectric impedance. From this data plot r = 0.911, giving this equation: total body water = 0.158 +/- 0.662 x (height2/bioelectric impedance). Levels of agreement of -1.771 to +1.725 were observed, with a standard error of measurement of 16% across the range. The data suggest that bioelectric impedance is a satisfactory and reliable method of estimating total body water in children requiring cardiac critical care. The standard error of 16% suggests that the method may be more useful for measuring trends than absolute values, but the technique should be a valuable noninvasive tool both for continuous monitoring of total body water and in longitudinal research studies of rapid fluid flux and in the assessment of capillary leak.


Heparin-coated circuits reduce activation of granulocytes during cardiopulmonary bypass. A clinical study. Activated granulocytes release highly active enzymes such as myeloperoxidase and lactoferrin, which can be involved in tissue destruction mediated by oxygen free radicals. Cardiopulmonary bypass has been reported to activate granulocytes. Bypass circuits coated with heparin have been shown to reduce release of granulocyte factors in experimental studies. In the present study, heparin-coated circuits were compared with noncoated circuits. In seven patients undergoing coronary bypass, heparin-coated circuits were used (group HC), and seven served as control patients (group C). In group HC the heparin dose was reduced to 75% (225 IU/kg). Group C had the standard dose of 300 IU/kg. No preoperative differences in myeloperoxidase and lactoferrin were observed between the groups. At the end of bypass in both groups, there was a significant increase of these enzymes (p less than 0.001) followed by a later decrease. In group HC, however, the release of myeloperoxidase was significantly lower than in group C (215 +/- 24 versus 573 +/- 133 micrograms/L, mean +/- standard error of the mean). The release of lactoferrin was significantly lower in group HC than in group C both at the end of cardiopulmonary bypass (659 +/- 79 versus 1448 +/- 121 micrograms/L) and 3 hours after bypass (224 +/- 37 versus 536 +/- 82 micrograms/L). Granulocytes as well as total number of leukocytes continued to increase until 1 hour after bypass (p less than 0.001) and then manifested a slow decrease. It was concluded that the use of heparin-coated circuits reduced the release of granulocyte factors because of lower activation of leukocytes.


Prolonged hypothermic cardiac storage for transplantation. The effects on myocardial metabolism and mitochondrial function. Cardiac storage for transplantation is currently limited to 6 hours. To better understand the metabolic changes that occur during hypothermic (4 degrees C) storage, we monitored the morphologic and metabolic changes in the canine myocardium at 0, 12, and 24 hours of storage in University of Wisconsin solution. Attempts to isolate cardiac mitochondria resulted in a progressive decline in the yield (milligrams of mitochondria per gram of heart tissue), which decreased (p less than 0.05) from 9.2 +/- 0.4 at 0 hours (control) to 4.0 +/- 0.3 after 12 hours and further decreased (p less than 0.05) to 1.9 +/- 0.2 after 24 hours of cold storage. Mitochondrial state 3 respiration fell to 64% of control after 12 hours and 28% of control after 24 hours of cold storage (p less than 0.05). Citrate synthetase activity, but not cytochrome C oxidase activity, was significantly depressed after 12 and 24 hours of cold storage. Adenosine triphosphate content decreased to 67% of control after 12 hours and 50% of control after 24 hours. After 12 hours of storage, sufficient adenosine diphosphate and monophosphate were present to permit some restoration of adenosine triphosphate, provided mitochondrial function was normal after transplantation. However, restoration of mitochondrial function and adenosine triphosphate levels sufficient to support myocardial contractility was unlikely after 24 hours of storage. This study suggests that a return of adequate cardiac function after transplantation may be possible after 12 hours of cold storage in University of Wisconsin solution but not after 24 hours of cold storage.


ET-3 sensitive reduction of tissue blood flow in rat liver. Changes in gastric mucosal and hepatic tissue blood flow were simultaneously determined using a laser-Doppler blood flow meter in rats given i.v. injection of endothelin-1 (ET-1) and endothelin-3 (ET-3), both at 2 nmol/kg. Gastric mucosal blood flow decreased significantly after administration of ET-1 compared to after administration of ET-3. Decreases in blood flow due to ET-1 were reversed by pre-treatment with 10 mg/kg of BQ-123 (sodium salt), an ETA receptor antagonist, to levels comparable to those induced by ET-3, but BQ-123 had no effects on decreases in blood flow due to ET-3. On the other hand, decreases in hepatic tissue blood flow by ET-3 were significant compared to those by ET-1. Decreases in hepatic tissue blood flow due to ET-1 were slightly inhibited by pre-treatment with 10 mg/kg of BQ-123, but it had no effect at all on decreases due to ET-3. These findings indicate that decreases in gastric mucosal blood flow are mainly caused by ET-1 via ETA receptors inhibited by BQ-123, while decreases in hepatic tissue blood flow are caused mainly by ET-3 via non-ETA receptors not inhibited by BQ-123. The fact that ET-3 decreases blood flow more significantly than ET-1 suggests the involvement of ET-3 selective receptors (ETc). The findings obtained in the present study indicate that complex mechanisms are involved in the regulation of tissue blood flow by ET, with different receptor subtypes and ET family peptides being involved according to the type of tissue.


Differential effects of chronic agonist administration on mu-opioid receptor- and muscarinic receptor-regulated adenylate cyclase in rat striatal neurons. In cultured rat striatal neurons exposed to 10 microM morphine or oxotremorine for 24 hours, we observed an increased (about 30%) dopamine D1 receptor-stimulated cyclic AMP production, whereas no desensitization of mu-opioid receptor or muscarinic cholinergic receptor was found. However, whereas upregulation of dopamine D1 receptor-stimulated adenylate cyclase activity upon 7 days morphine exposure was at least as pronounced as observed after 24 hours of exposure to the opioid, this adaptive phenomenon was virtually absent following one week of oxotremorine treatment. This reduced adenylate cyclase sensitization upon 7 days oxotremorine exposure appeared to coincide with desensitization of muscarinic cholinergic receptors. A possible role of the resistance of mu receptors to desensitization and the (resulting) upregulation of the neuronal adenylate cyclase system upon chronic receptor activation in the development of opiate tolerance and dependence is suggested.


Peripheral 5-carboxamidotryptamine induces hindlimb scratching by stimulating 5-HT1A receptors in rats. Treatment of rats with 5-carboxamidotryptamine (5-CT) or 5-methoxy-tryptamine (5-MeOT) induces a hindlimb scratch response. These compounds have high affinity for 5-HT1A and 5-HT1D receptors. The selective 5-HT1A receptor agonist N,N-dipropyl-5-CT (DP-5-CT) also induced hindlimb scratching while the selective 5-HT1D receptor agonist, sumatriptan, did not. 5-CT-induced hindlimb scratching was inhibited dose-dependently by several 5-HT1A antagonists (BMY 7378, NAN-190, MDL 73005EF and pindobind-5-HT1A) as well as the non-selective 5-HT antagonist, methiothepin. Pretreatment of rats with the serotonin (5-HT) synthesis inhibitor, p-chlorophenylalanine (PCPA) or the 5-HT depleting agent, reserpine, markedly attenuated 5-CT-induced hindlimb scratching. These data suggest that hindlimb scratching induced by 5-HT agonists may not be centrally mediated but rather may be mediated by a neuronal 5-HT1A receptor localized outside the blood-brain barrier.


L-dopa administration enhances exocytotic dopamine release in vivo in the rat striatum. Peripheral administration of L-3,4-dihydroxyphenylalanine (L-DOPA) methylester increased extracellular levels of DOPA and dopamine (DA) in the rat striatum monitored by in vivo brain microdialysis. The increase in DA levels persisted after inhibition of DA reuptake by nomifensine. Administration of blockers of voltage-dependent Na+ (tetrodotoxin) or Ca2+ (NKY-722) channels through the dialysis membrane completely eliminated the increase in DA levels. These results demonstrate that the L-DOPA-induced DA release is exocytotic in nature and hence, derived from neurons in the striatum.


Studies on the functional role of alpha-adrenoceptors in the cardiac sympathetic ganglia of the dog. Inhibitory alpha-adrenoceptors were studied in cardiac ganglia of pentobarbital-anesthetized dogs. Blockade of alpha 1- or alpha 2-adrenoceptors augmented preganglionic nerve stimulation induced tachycardia without altering the response to postganglionic nerve stimulation. The effect produced by blockade of ganglionic alpha 1-adrenoceptors with terazosin had different frequency-response characteristics from, was of smaller magnitude than, and was additive with the effect produced by blockade of ganglionic alpha 2-adrenoceptors with rauwolscine. The response to activation of ganglionic nicotinic cholinergic receptors in the absence of electrical stimulation of the preganglionic nerve was not affected by blockade of either alpha 1- or alpha 2-adrenoceptors. The response to nicotinic cholinergic receptor activation during periods of continuous preganglionic nerve stimulation was augmented following blockade of alpha 2-adrenoceptors but unaffected by alpha 1-adrenoceptor blockade. These results suggest that there are two different inhibitory pathways involving alpha-adrenoceptors in mammalian sympathetic ganglia and provide evidence that these inhibitory pathways are operative under the experimental conditions of ganglionic transmission.


Evidence for two binding sites on membrane-bound angiotensin-converting enzymes (ACE) for exogenous inhibitors except in testis. It was possible to assess that ACE inhibitors bound to two different sites on the enzymes which consist in two homologous domains. Accordingly, it was also shown that testis ACE which consists in a single domain exhibits only one site for inhibitors. The common binding site of both enzymes contains Zn2+, bears the enzymatic activity, is less sensitive to added Zn2+ and Cl- and has a lower affinity for inhibitors than the first one, found only in duplicated domain enzymes and highly sensitive to the effect of ions.


Fabry's disease--a rare cause of sudden death. A case of Fabry's disease, an X-linked in-born error of metabolism, presenting as a sudden death in an elderly female, is reported, along with a review of the literature. The pathological findings of the cellular vacuolation and the characteristic inclusion bodies in the tissues are described.


The impact of the ISIS experiment order on spatial contamination. When performing volume-localized spectroscopy measurements, the amount of spatial contamination is an important quality criterion. With the ISIS localization technique contamination cannot only arise from the transition regions around the volume of interest, but also from remote regions of the sample. The latter contamination component is a consequence of inhomogeneous excitation pulses, if short repetition times TR are used. Its severity depends both on the order of the eight phase cycling experiments needed for an ISIS measurement, and on the ratio TR/T1. Here it is theoretically discussed from which regions of the sample contamination can arise for a specific phase cycling order. For the worst orders the contaminating regions are almost three times as large as for the optimal orders. The ratio for the effectively measured contamination, however, can be moderated in real experiments, because cancellation effects occur due to the phase distribution of the contaminating signals. 31P phantom experiments clearly demonstrate that contamination is present even if adiabatic excitation pulses are applied and that spatial contamination can be reduced to about a third by an optimal choice of the phase cycling order.


Pulsed saturation transfer contrast.

Pulsed saturation transfer contrast. In vivo 1H conventional NMR image contrast generation usually relies on the macroscopic T1 and T2 relaxation parameters of the tissues of interest. Recently cross-relaxation related image contrast has been reported by Wolff and Balaban in animal models. Due primarily to the broad lineshape of the intended saturation spin pool and the use of off-resonance irradiation, high specific absorption rate and an auxiliary RF amplifier have been necessary to produce these images. The relatively long spin-lattice relaxation property of this spin pool, however, suggests the use of pulse methods to achieve saturation. In this paper, we show that short-T2 spin pools can be selectively saturated with short intense RF pulses. Cross-relaxation time constants can be measured using the technique of saturation recovery. In vivo magnetization-transfer-weighted images can be produced using pulses on commercial whole-body imagers without additional hardware.


The magnetic properties and water dynamics of the red blood cell: a study by proton-NMR lineshape analysis. The magnetic properties and water dynamics of human red blood cells were examined by analysis of the water proton spectra of suspensions of oxygenated, deoxygenated, carbon monoxide-treated, and methemoglobin-containing cells at a magnetic field strength of 7.05 T. Total lineshape analysis of spectra from deoxygenated red blood cell suspensions was performed to determine the transmembrane water exchange rate, the contribution of diffusion in local magnetic field gradients to the transverse relaxation rate, and the difference between the intra- and extracellular chemical shifts of water protons. Mathematical models are proposed to account for the dependence of the chemical shifts and linewidths of these spectra on the magnetic susceptibility or density of the cells.


Induced CD25 expression in a human B-lymphoma cell line transfected with the Epstein-Barr virus nuclear antigen 2 gene. Two EBV-negative human B-lymphoma cell lines, BJAB and DG75, were transfected with an Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) gene, which plays a critical role in the EBV-induced immortalization of primary B lymphocytes. Furthermore, DG75 cells were co-transfected with the EBNA-2 gene and a latent membrane protein (LMP) gene. Expression of eight surface antigens on the resultant EBNA-2-expressing cell clones was analyzed by flowcytometry. None of the EBNA-2-expressing cell clones derived from BJAB and DG75 showed a significant increase in the expression of cell surface marker CD23, of which enhancement by EBNA-2 in a different EBV-negative human B cell line, Louckes, was previously reported. Expression of CD25 (IL-2R/Tac) on cell surface, however, was induced in two of six DG75-derived cell clones. One of the two CD25-induced cell clones was expressing EBNA-2 only, and the other was co-expressing EBNA-2 and LMP. The results suggest that EBNA-2 has a potential to up-regulate CD25 independently of CD23 on human B cells.


IgM neutralizing antibody responses to human herpesvirus-6 in patients with exanthem subitum or organ transplantation. The assay for detecting IgM neutralizing (NT) antibody activity to human herpesvirus-6 (HHV-6) was developed by using pretreatment of blood sample with staphylococcal protein A. The activity was mostly present in IgM fractions of serum but not in IgA fractions separated by ultracentrifugation. The assay was used for seroepidemiological studies for HHV-6 infection. In primary HHV-6 infection, IgM NT antibodies appeared 5 to 7 days after onset of exanthem subitum, reached maximum titers at 2 to 3 weeks, and tended to decline to undetectable levels after 2 months. In contrast, reactivation of HHV-6 observed in organ transplants showed somewhat greater degree of IgM NT antibody responses that persisted for 2 to 3 months and became undetectable 5 to 6 months after transplantation. The level and persistence of NT antibody titers measured by the conventional method was generally greater than those of the IgM titers. The prevalence of the IgM NT antibodies was examined in healthy individuals. The antibody was first detected at 4 to 7 months of age (5%), reached maximum level at 8 to 11 months (40%), and was detectable by 4 to 6 years (17%). A few (4 to 5%) of adolescents and adults were positive for the antibody.


Novel amiloride analog allosterically modulates the alpha 2-adrenergic receptor but does not inhibit Na+/H+ exchange. Two novel amiloride analogs have been synthesized during the course of efforts to develop a photoaffinity label for the amiloride allosteric domain on alpha 2-adrenergic receptors. One of these, 5-[N-2'-aminoethyl-N'-isopropyl]amiloride-N-[4"-azidosalicylamide] (A-EIA-AS), markedly accelerates the rate of dissociation of [3H]yohimbine from affinity-purified alpha 2-adrenergic receptors, an assay for allosteric modulation of receptor-adrenergic ligand interactions. In contrast, this agent does not appreciably inhibit Na+/H+ exchange, measured as 5-(N-ethyl-N-isopropyl)amiloride (EIA)-inhibitable 22Na+ uptake into cultured renal epithelial cells. A second analog, 5-[N-2'-(4"-azidosalicylamidino)ethyl-N'- isopropyl]amiloride (ASA-EIA), does not foster an accelerated rate of dissociation of [3H]yohimbine binding from the alpha 2 receptor but does block the ability of A-EIA-AS to do so, suggesting that ASA-EIA and A-EIA-AS interact at a common binding site. Interestingly, the ability of EIA to accelerate [3H]yohimbine dissociation is not blocked by ASA-EIA, a finding that may indicate that EIA and A-EIA-AS allosterically modulate alpha 2 receptor-ligand interactions via distinct or nonoverlapping binding sites.


Action of thrombin receptor polypeptide in gastric smooth muscle: identification of a core pentapeptide retaining full thrombin-mimetic intrinsic activity. We have used a guinea pig gastric longitudinal (LM) smooth muscle bioassay system to evaluate the contractile activities of a previously described thrombin receptor-derived polypeptide, S42FLLRNPNDKYEPF55 (one-letter amino acid code) (TRP42-55) and of a series of peptides derived from this sequence. The contractile activities of the polypeptides were compared with the actions of thrombin. Shortened peptides of the sequences S42FLLRNPND50, S42FLLRN47, and S42FLLR46 (TRP42-46) all exhibited contractile activities that were equivalent to or greater than those of the parent polypeptide, TRP42-55. Both TRP42-55 and TRP42-46 mimicked the action of thrombin, in terms of two different signal transduction pathways that were activated either in the LM preparation or in the related but distinct gastric circular muscle assay. In the LM preparation, the peptide FSLLR also exhibited appreciable, but much reduced, activity. Minimal activity was exhibited in the LM by the sequence SFLLA, but the lysine-containing analogue S42FLLK46 was about one fifth as potent as TRP42-46. In contrast, the receptor-derived sequences S42FLL45, S42FL44-NH2, F43LLR46, and S42ALLR46, as well as arginine-containing polypeptides beginning with the SF motif, SFRG and SFRGHITR, were inactive in the LM bioassay system, at concentrations of greater than or equal to 200 microM, as either agonists or antagonists against TRP42-55. In addition to its actions in the LM and circular muscle preparations, the active pentapeptide, TRP42-46, also exhibited thrombin-mimetic intrinsic activity in a rat aortic arterial ring relaxation bioassay, whereas the pentapeptide S42FLLA46 and the tetrapeptide S42FLL45 were inactive. We conclude that the intrinsic biological activity of the thrombin receptor-derived peptide resides in the pentapeptide TRP42-46 and that the phenylalanine and arginine residues at positions 43 and 46 play key roles in the activity of this pentapeptide in smooth muscle systems.


Immortalized hypothalamic GT1-7 neurons express functional gamma-aminobutyric acid type A receptors. Neuronal cell lines provide a source of pure populations of neurons and allow the properties of many neurotransmitter receptors to be studied. However, none of these cells have been reported to express functional gamma-aminobutyric acid (GABA)A receptors. Indeed, there have been no reports of cell lines expressing functional amino acid receptors. Using biochemical and electrophysiological techniques, we have identified a neuronal cell line expressing functional GABAA receptors. Membranes from immortalized hypothalamic (GT1-7) neurons bound [3H]muscimol but not [3H]flunitrazepam. GABA-activated chloride currents, recorded from GT1-7 cells, were blocked by bicuculline and Zn2+ but were insensitive to diazepam. These results suggest that GABAA receptors on GT1-7 cells lack gamma subunits. The neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one and pentobarbital both modulated GABAA receptors in these cells. Polymerase chain reaction analysis of the cells revealed the presence of mRNAs encoding alpha 1, beta 1, and beta 3 polypeptides. GT1-7 cells provide a useful model system for studying the regulation of GABAA receptor polypeptide expression.


Two binding sites on angiotensin-converting enzyme: evidence from radioligand binding studies. Purified angiotensin-converting enzyme (ACE) from rat lung and testis, membrane preparations of ACE from lung, kidney, and testis, and ACE from plasma were used in radioligand binding studies, to seek evidence for two binding sites in ACE of somatic origin, as predicted by molecular biology studies. 125I-Ro 31-8472, a radioiodinated hydroxy derivative of cilazaprilat, and 125I-351A, a radioiodinated p-hydroxybenzamidine analogue of lisinopril, were used as radioligands. Autoradiographic study of renal ACE using 125I-Ro 31-8472 or 125I-351A showed the same distribution of radioligand binding across kidney sections and identical radioligand binding for purified lung and testis ACE by Western blot, confirming that the same protein bound both radioligands. Analysis of displacement of 125I-Ro 31-8472 binding from ACE by ACE inhibitors 351A and lisinopril yielded biphasic curves for pulmonary, renal, and plasma ACE and a monophasic curve for ACE from the testis. Analysis by LIGAND suggested two binding sites in ACE from plasma or somatic sources and one binding site in ACE of testicular origin, as predicted by molecular biology studies. Displacement of 125I-351A binding from lung and testis ACE by Ro 31-8472 and 351A was monophasic. LIGAND analysis revealed interaction with a single class of binding sites on lung and testis ACE, in agreement with previous studies using 125I-351A. Equilibrium dissociation constants (Kd) for the carboxyl-terminal (KdI) and amino-terminal (KdII) binding sites of purified ACE, using 125I-Ro 31-8472, were similar for Ro 31-8472 (lung kdI, 65 +/- 7 pM; KdII, 175 +/- 38 pM) but were clearly different for 351A (lung KdI, 19 +/- 2 pM; KdII, 2771 +/- 489 pM; p less than 0.01). Cell membrane-associated somatic ACE and plasma ACE, which is devoid of a carboxyl-terminal hydrophobic anchor region, had similar absolute values and differences in 351A binding affinities at the two ACE binding sites. Kd in testis was 46 +/- 6 pM for Ro 31-8472 and 16 +/- 3 pM for 351A, corresponding to estimates at the carboxyl-terminal binding site of somatic ACE. The 65-200-fold reduction in 351A binding affinity at the amino binding site may limit the detection of two binding sites on somatic ACE by 125I-351A radioligand binding analysis.(ABSTRACT TRUNCATED AT 400 WORDS)


Identification of endothelin receptor subtypes in rat kidney cortex using subtype-selective ligands. 125I-Endothelin (ET)-1 and 125I-ET-3 displayed specific, saturable, and high affinity binding to membranes prepared from rat kidney cortex. Saturation binding experiments using 125I-ET-1 and 125I-ET-3 revealed that 125I-ET-3 binding sites were 40-50% less abundant than 125I-ET-1 binding sites. The dissociation constants (Kd) and maximum binding (Bmax) for 125I-ET-1 and 125I-ET-3 with these membranes were 218 +/- 23 pM and 275 +/- 20 fmol/mg of protein and 207 +/- 19 pM and 113 +/- 17 fmol/mg of protein, respectively. In the presence of 10 nM sarafotoxin 6c, a selective agonist for ETb receptors, 125I-ET-1 binding was decreased by 45-50% and 125I-ET-3 binding was totally abolished, suggesting that approximately 40-50% of kidney cortex ET receptors are of the ETB subtype and that 125I-ET-1 binds to both ETA and ETB receptors with the same high affinity, whereas 125I-ET-3 binds to only ETB receptors with high affinity. In addition, in the presence of BQ123 [cyclo(D-Trp,D-Asp,L-Pro,D-Val,L-Leu)], a selective antagonist for ETA receptors, 125I-ET-1 binding was decreased by 50%, whereas 125I-ET-3 binding was unaffected. Our results strongly suggest that rat kidney cortex contains ETA and ETB receptors in a 50:50 ratio and that sarafotoxin 6c and BQ123 are valuable tools in identifying the subtypes of ET receptors in various tissues.


Receptor occupancy and adenylate cyclase activation in AR 4-2J rat pancreatic acinar cell membranes by analogs of pituitary adenylate cyclase-activating peptides amino-terminally shortened or modified at position 1, 2, 3, 20, or 21. In AR 4-2J rat pancreatic acinar cell membranes, receptors for the two pituitary adenylate cyclase-activating peptides (PACAP) PACAP-27 (the short version of PACAP) and PACAP-38 [the long version, with a carboxyl-terminal (residues 28-38) extension] can be subdivided into (a) type A receptors, with high affinity (Kd, 0.3-0.5 nM) for both PACAP-27 and PACAP-38, and (b) type B receptors, with high affinity for PACAP-38 (Kd, 0.3 nM) but low affinity for PACAP-27 (Kd, 20 nM). Determinants of agonist/antagonist activity in 47 PACAP-27 and PACAP-38 analogs (mono- or disubstituted in positions 1, 2, 3, 20, and 21) or amino-terminally shortened were tested by (a) the occupancy of PACAP-A receptors, preferentially labeled with [125I-N-acetyl-His1]PACAP-27, and that of PACAP-A and -B receptors, both labeled with 125I-PACAP-38, and (b) the resulting activation or inhibition of adenylate cyclase. For PACAP-A receptor recognition, deprotonated His1 was a major determinant for PACAP-27 but not PACAP-38; the Kd of 125I-PACAP-27 decreased 2.4-fold at 37 degrees between pH 6.0 and 7.5 and 3.6-fold at 15 degrees, whereas the IC50 of [N-acetyl-His1]PACAP-27 was less affected and that of PACAP(2-27), PACAP(2-38), and PACAP(1-38) was pH independent. In addition, PACAP-A receptors coupled to adenylate cyclase were much more sensitive to PACAP-38 derivatives than to PACAP-27 derivatives; for instance, [D-Phe2]PACAP-38 was a more potent antagonist (Ki, 5 nM) than [D-Phe2]PACAP-27 (Ki, 350 nM), and PACAP(6-38) was a more potent antagonist (Ki, 7 nM) than PACAP(6-27) (Ki, 300 nM). PACAP-B receptors, apart from showing high affinity for PACAP-38, displayed relatively high affinity for amino-terminally shortened PACAP-38 fragments and poor affinity for PACAP-27 and PACAP-27 fragments.


Rapid, simple and sensitive microassay for skeletal and cardiac muscle myoglobin and hemoglobin: use in various animals indicates functional role of myohemoproteins. A novel, simple, rapid, sensitive and reproducible microassay is described for determination of myoglobin and hemoglobin content of myocardial and skeletal muscle biopsy specimens from various mammals, birds and fish. As little as 50 mg of tissue is needed and myoglobin concentrations lower than 1 mg% can be detected. Myoglobin and hemoglobin are separated at alkaline pH by ammonium sulfate extraction followed by ultrafiltration. Heme content is determined by absorption of the Soret band when the hemoprotein extract is visibly colored or more sensitively by its peroxidase activity when the extract has low color. The heme reacts with tertiary-butyl hydroperoxide and orthotolidine to generate a blue color. Hemoglobin content is correlated with myoglobin content and is related to aerobic capacity and blood flow to the tissue. Myoglobin content varied over 5 orders of magnitude up to 7 per cent of the weight of tissue, whereas hemoglobin content varied over 2 orders of magnitude up to 6 per cent of tissue weight. Myoglobin content is increased in species with high basal metabolic rate, high physical activity, prolonged diving capacity, fatigue resistance, and red muscle, whereas it is decreased in white muscle, iron-deficient animals, animals with sedentary lifestyles, and in animals and tissues with small fiber diameters such as avian or fish hearts.


Prevalence of hepatitis C virus RNA in organ donors positive for hepatitis C antibody and in the recipients of their organs. There is a high prevalence of liver disease among the recipients of organs from donors with antibodies to hepatitis C virus (HCV). We undertook a study to determine the frequency of persistent HCV infection, as indicated by the presence of HCV RNA, among both cadaveric organ donors positive for antibodies to HCV (anti-HCV) and the recipients or organs from these donors. Serum samples from donors and recipients were tested for HCV RNA with the reverse transcriptase polymerase chain reaction, with use of primers from the 5' untranslated region of the HCV genome, and for anti-HCV with the first-generation enzyme-linked immunosorbent assay (ELISA) and two second-generation tests. HCV RNA was detected in 9 of the 11 organ donors (82 percent) with a positive first-generation ELISA for anti-HCV. Among the organ recipients, the prevalence of HCV RNA increased after transplantation: 7 of 26 patients (27 percent) had positive samples before transplantation, as compared with 23 of 24 patients (96 percent) after transplantation (P less than 0.001). Among 13 recipients who were HCV RNA-negative before receiving organs from the nine HCV RNA-positive donors, HCV infection was detected in all 13 after transplantation, and anti-HCV developed in 8 (62 percent). On the basis of a positive test for HCV RNA, the maximal sensitivity of the three anti-HCV tests was 57 percent (positive in 4 of 7 patients with end-stage organ failure) before transplantation and 70 percent (positive in 16 of 23 patients) after transplantation. Nearly all the recipients of organs from anti-HCV-positive donors become infected with HCV. The current tests for anti-HCV antibodies underestimate the incidence of transmission and the prevalence of HCV infection among immunosuppressed organ recipients.


A controlled trial of a single dose of azithromycin for the treatment of chlamydial urethritis and cervicitis. The Azithromycin for Chlamydial Infections Study Group. Currently, there is no single-dose therapy that is effective in the treatment of urethral or endocervical infections with Chlamydia trachomatis. Azithromycin is a new azalide antibiotic that has substantial activity against C. trachomatis, is concentrated intracellularly, and has a long half-life in serum and tissue. We conducted a trial in which 299 female patients and 158 male patients with uncomplicated genital infection and a positive C. trachomatis antigen test were randomly assigned to receive either azithromycin (1 g once orally) or doxycycline (100 mg orally twice daily for seven days). Only patients subsequently determined to have a culture positive for C. trachomatis at base line were included in the evaluation of efficacy. Among the patients who could be evaluated, 5 of the 141 patients (4 percent) treated with azithromycin did not respond to treatment, as compared with 3 of the 125 patients (2 percent) treated with doxycycline (difference between groups, 2 percent; 95 percent confidence interval, 0 to 6 percent). Of the patients evaluated 21 to 35 days after treatment, none of 112 treated with azithromycin and 1 of 102 treated with doxycycline had a positive culture. The rates of bacteriologic cure were similar for the 98 female patients (97 percent) and the 43 male patients (95 percent) treated with azithromycin. Seventeen percent of the patients who received azithromycin and 20 percent of those treated with doxycycline had mild-to-moderate drug-related side effects, mainly gastrointestinal symptoms. A single 1-g dose of azithromycin is as effective for the treatment of uncomplicated genital chlamydial infections as a standard seven-day course of doxycycline.


Relationship between fumonisin contamination of feed and mystery swine disease. A case-control study. Fumonisin is a recently identified mycotoxin that has been shown to be the cause of pulmonary edema disease in swine and leukoencephalomalacia in horses. Mystery Swine Disease (MSD), is an economically devastating disease complex of unknown etiology that has been reported to have occurred in several swine producing states since 1988. To determine the relationship between MSD and fumonisin, a case-control study was carried out in Illinois in mid-1990. Feed samples collected from 12 case and 9 control farms were analyzed for fumonisin. Sera from swine on all farms was screened for titers against encephalomyocarditis (EMC) virus and concentrations of alpha-1 acid glycoprotein (an acute phase reactive protein). Fumonisin concentrations greater than or equal to 20 ppm were found on 1 control farm (1/9) and 8 case farms (8/12). Titers against EMC virus (greater than or equal to 1:16) were found on 5 control farms (5/9) and on 6 case farms (6/12). Farms with greater than or equal to 20 ppm fumonisin in the feed were at significantly increased risk (OR = 11.2, Fisher's exact test p = 0.037) for MSD. Furthermore, the pi2 test for trend was (p = 0.017), meaning that as the level of fumonisin in the feed increased, the risk of MSD also increased. The presence of EMC virus titers in the sow herd was not a significant risk for MSD (OR = 1.25, Fisher's exact test p = 0.75). Alpha-1 acid glycoprotein concentrations obtained from a 2-week old nursing pigs differed significantly (p = 0.0005) between MSD case and control herds.


[Electron-cytochemical study of carbohydrate components in the surface membrane of cultured Helix pomatia neurons]. The structure and topography of carbohydrates on the surface of nerve cells of snail Helix pomatia cultured in vitro have been characterized with a series of colloidal gold-labelled lectins of different sugar specificity. The analysis of the lectin binding has shown substantial differences in the carbohydrate pattern between the soma of monoaminergic and peptidergic neurons. It has been found that the surface of monoaminergic and peptidergic neurons contains N-acetylglucosamine (WGA+) and N-acetyllactosamine (RCA-1-) determinants and does not contain neuraminic acid (LPA-) and complex branched N-glycosyl chains (PVA-). At the same time N-acetylgalactosamine (HPA+) was detected on the peptidergic neuron membrane only. It has been concluded that terminal residues of sialic acid are absent on the most of snail nerve cells. Differences in lectin binding between monoaminergic and peptidergic neurons can serve as a basis for formation of specific connections of cells by different types in the developing brain.


Radiobiology of radiosurgery: Part II. The rat C6 glioma model. We developed an experimental animal model to evaluate the potential role of stereotactic radiosurgery for glial neoplasms. Rats were randomized to control or treatment groups after implantation of C6 glioma cells into the right frontal region; 14 days later, 19 rats underwent stereotactic radiosurgical treatment of the induced tumor, using the 4-mm collimator of the gamma unit. Both groups were observed for up to 65 days after implantation. Treated animals had a mean survival of 39.2 days; the 22 control animals lived a mean of 29.4 days before death from tumor growth (P = 0.07). Six treated animals (32%), but only one control animal, survived the full observation period (P = 0.07). The mean tumor diameter in the control group was 9.64 mm; in the radiosurgery group, it was 6.47 mm (P = 0.001). Compared with tumors in control animals, treated tumors had a hypocellular appearance (P less than 0.001) and demonstrated cellular edema (P less than 0.005) under light microscopy, indicating a direct cytotoxic response to treatment. No difference was identified in the amount of tumor necrosis, intratumor hemorrhage, or degree of brain invasion between the two groups. Variations in the maximum treatment dose (30, 40, 50, 70, or 100 Gy) did not result in observed differences in tumor response. This in vivo rat malignant glioma model is a valuable tool to evaluate the tumoricidal effects of single-fraction, focused irradiation. Additional studies are warranted to evaluate dose-response relationships, radiation sensitizers, and use of radiosurgery with other adjuvant treatments.


Progressive parkinsonism associated with Rosenthal fibers: senile-onset Alexander's disease? Rosenthal fibers are present in several neurologic disorders. Their occurrence in widespread subependymal, subpial, and perivascular locations is thought to be diagnostic of Alexander's disease, but they have also been reported in rare adult cases. Some of these patients were neurologically intact with severe systemic illnesses. In symptomatic cases, the neurologic disorder has simulated multiple sclerosis. We report an elderly woman with progressive parkinsonism, dementia, and hallucinations whose brain showed severe Rosenthal fiber proliferation.


Atypical herpes simplex encephalitis: clinical, virologic, and neuropathologic evaluation. An atypical form of herpes simplex encephalitis produced by HSV-1 documented in the present article demonstrates that (1) prominent EEG abnormality may correlate with subtle increase in signal intensity on MRI; (2) the disease may start with prominent involvement of the cingulate gyri; and (3) viral infection of the brainstem may cause early onset of severe neurologic dysfunction and coma.


Radiation maculopathy after proton beam irradiation for choroidal melanoma. Radiation maculopathy is a microangiopathy of the retina, which is often observed after irradiation of the eye. To quantitatively determine the frequency of anatomic and functional features of this condition, the authors reviewed a large series of patients with choroidal melanomas who were treated by proton beam irradiation. Color photographs and/or fluorescein angiograms of 218 patients with paramacular choroidal melanomas were graded by two independent masked readers to determine the frequency of the various lesions of radiation maculopathy. Overall, 194 (89%) of the 218 patients in this study developed some degree of radiation maculopathy. The earliest and most common finding was macular edema, which was observed in 87% of patients, overall, by the end of year 3. Microvascular changes (microaneurysms and/or telangiectasia), intraretinal hemorrhages, and capillary nonperfusion were noted in 76%, 70%, and 64% of eyes, respectively, by the end of postirradiation year 3. Visual acuity was retained at 20/200 or better in 90% of eyes 1 year after irradiation and in 67% of eyes 3 years after treatment. Radiation maculopathy is common after proton beam irradiation of paramacular choroidal melanomas. However, ambulatory vision is preserved in many eyes.


Selective immunohistochemical staining in the paraneoplastic retinopathy syndrome. The mechanism leading to visual loss in paraneoplastic retinopathy is not known. An autoimmune process has been imputed based on immunologic investigations of several patients and by analogy to certain other paraneoplastic syndromes. Two patients with documented small cell carcinoma of the lung who had clinical evidence of paraneoplastic retinopathy are described. Histopathologic examination of the retina from one patient and immunohistochemical staining of human retina with serum from control subjects and both patients were performed. Electroretinograms demonstrated dysfunction of photoreceptors in both patients, with predominant loss of rod function in one patient. Post mortem examination showed patchy loss of photoreceptors of the extramacular retina and relative sparing of cones, findings consistent with the clinical and electrophysiologic test results. Serum from both patients stained the retina in an identical manner, with restriction of the stain to the outer retina. Stain was present over the outer plexiform layer, the outer nuclear layer, and the inner and outer segments of most photoreceptors. A sharp demarcation was present between those areas that did and did not stain. All rod inner and outer segments appeared to stain, and many cone inner segments were not stained. Immunologic tests obtained elsewhere did not show serum antibody to the 23 kD protein. These findings support the concept of an autoimmune pathogenesis by showing selectivity of the immune response and correlation between the apparent target of the immune response and the clinical and pathologic findings. The mechanism by which cell loss occurs in the retina is not answered by this study. The absence of antibody to the 23 kD protein does not exclude the diagnosis of paraneoplastic retinopathy.


Serological differentiation of five bluetongue virus serotypes in indirect ELISA. The serological reactivity in indirect ELISA of five different bluetongue virus (BTV) serotypes (4, 10, 15, 16 & 20) was compared using polyclonal antisera raised against virus particles and an outer structural protein, VP2. Rabbit and sheep antisera against BTV-10 produced higher ELISA values with their homologous antigens than with heterologous serotypes. A hyperimmune rabbit serum specific for virus particles was able to distinguish heterologous serotypes from each other, but a sheep serum from an infected animal was not. An antiserum directed against VP2, the protein responsible for serotype specificity in neutralization tests, was not serotype-specific in ELISA and cross-reacted with other serotypes. The discriminatory ability of a BTV-4 antiserum was improved by cross-absorption with heterologous antigens. This greatly reduced the ELISA signals with heterologous serotypes and produced an antiserum that was effectively serotype-specific.


Characterization of ovine strains of Mycobacterium paratuberculosis by restriction endonuclease analysis and DNA hybridization. Restriction endonuclease analysis and DNA hybridization revealed five ovine strains of Mycobacterium paratuberculosis from South Africa had identical DNA patterns to an ovine strain from Canada. Genetically this strain type has features in common with the two major groups of M. paratuberculosis.


A time course analysis of the changes in spontaneous and evoked behaviour in a rat model of neuropathic pain. We have previously demonstrated that scratching was significantly increased in a rat model of polyarthritis and that this could be reversed by morphine and electrical stimulation of pain-modulating brain areas. We therefore proposed that scratching might represent a parameter of chronic pain. In this study, we examined the spontaneous behaviour of rats in a model of peripheral neuropathy induced by loosely tying 4 ligatures around the right common sciatic nerve. In half of the animals (N = 7), the ligatures were made with resorbable sutures and, in the other half (N = 7), with non-resorbable sutures of the same size. Postoperatively, scratching was significantly increased at the ligated side. This increase was already observed on the first postoperative day, and maximal effects were reached on the 3rd day. We also observed a qualitative change in the scratching behaviour; postoperatively, scratching was often a vibratory-like shaking of the hind paw in the air. The time course of the increased scratching was time-locked with the development of allodynia to thermal stimulation. No differences were found either in the time course of the increased scratching behaviour or in the time course of the thermal allodynia between the rats ligated with resorbable and with non-resorbable sutures. However, a difference in the walking pattern, as measured by the sciatic functional index (SFI), was observed between the two groups: whereas the SFI normalized after 4 weeks in rats ligated with resorbable sutures, it remained disturbed until the end of the 16-week observation period in the rats ligated with non-resorbable sutures. Morphine 1, 2 and 5 mg/kg dose-dependently reduced the increased scratching behaviour. This was not due to a general depressant effect on the rats' behaviour. This finding is discussed in light of the debate on opioid sensitivity of neuropathic pain. The present results add new evidence that scratching is a possible sign of chronic pain in the animal.


Spatial patterns of spinal cord [14C]-2-deoxyglucose metabolic activity in a rat model of painful peripheral mononeuropathy. Spatial patterns of spinal cord glucose metabolic activity were examined in unanesthetized rats with painful peripheral mononeuropathy produced by sciatic nerve ligation (chronic constrictive injury, CCI). Spinal cord metabolic activity was assessed 10 days after nerve ligation by using the fully quantitative [14C]2-deoxyglucose technique. This technique allows simultaneous examination of both neural activity inferred from local glucose utilization and its spatial distribution in multiple spinal regions previously implicated in nociceptive processing. Rats used in the experiment exhibited thermal hyperalgesia to radiant heat applied to the hind paw ipsilateral to nerve ligation and behaviors indicative of spontaneous pain. Sciatic nerve ligation produced a significant increase in spinal cord metabolic activity in four sampling regions (laminae I-IV, V-VI, VII and VIII-IX) of lumbar segments compared to sham-operated rats. The pattern of altered metabolic activity in CCI rats presented 3 distinct features. (1) The spinal cord grey matter both ipsilateral and contralateral to nerve ligation exhibited substantial increases in metabolic activity compared to sham-operated rats. (2) This increase in metabolic activity was somatotopically specific, i.e., higher metabolic rates were observed on the side ipsilateral to nerve ligation than on the contralateral side, and higher metabolic rates were seen in the medial portion of the ipsilateral spinal cord dorsal horn than in the lateral portion. The peak metabolic activity occurred in laminae V-VI of CCI rats, a region involved in nociceptive processing. (3) The increase in spinal cord metabolic activity of CCI rats extended from lumbar segment L1 to L5 in all 4 sampling regions. The substantial increase in metabolic activity in both the ipsilateral and contralateral spinal cord that occurs over an extensive rostro-caudal area in CCI rats may represent a unique pattern of spinal cord metabolic activity distinct from that observed in rats exposed to acute thermal pain. This pattern of spinal cord neural activity in CCI rats may reflect possible radiation of neuropathic pain. In addition, the procedure of curare-induced paralysis in a separate group of CCI rats did not change the extent and patterns of metabolic activity seen in non-paralyzed CCI rats, reflecting a minimal influence of the afferent feedback from flexor motor reflexes on spinal cord metabolic activity following sciatic nerve ligation. This chronic increase in spinal cord neural activity in the absence of overt peripheral stimulation suggests a spinal cord hyperactive state and may account for behaviors suggestive of spontaneous pain in CCI rats.(ABSTRACT TRUNCATED AT 400 WORDS)


[State of lipid peroxidation processes and cyclase system components in lymphocytes and blood plasma of children with aplastic anemia]. Lipid peroxidation, antioxidant defence enzymes in lymphocytes and the content of cyclonucleotides in blood plasma were examined in children (3 groups) with aplastic anemia. The groups were formed depending on the disease stages in the examined. Disorders in the distribution of the activity of antioxidant defence enzymes, in the content of hydroperoxides and malonic aldehyde were discovered, with these disorders being diverse and dependent on the disease stage. The cAMP/cGMP in blood plasma was changed towards considerable rise of the cAMP level. During a remission, a majority of the characteristics did not return to normal.


Avoiding tropical infections and parasitic disease. The incidence of tropical disease among people returning from long distance journeys is increasing and nurses need to improve their knowledge of these conditions. It is more important to know where to obtain accurate and up-to-date information on tropical diseases. Travellers need advice on prophylactic measures, and this may involve altering lifestyle patterns while abroad. Nurses should be alert to the possibility of tropical or infectious diseases when patients return from abroad, and should consider measures such as barrier nursing where appropriate.


Their future in your hands. Infant heart transplantation. Recent advances in techniques have meant that infant heart transplant programmes are more assured of success. This article describes one such programme in America, and highlights the ethical and nursing dilemmas involved.


Drug-drug heart rate conditioning in rats: effective USs when pentobarbital is the CS. Injections of two drugs in sequence may be considered the Pavlovian pairing of one drug as a conditioned stimulus (CS) with a second drug as an unconditioned stimulus (US). If pentobarbital was the CS and d-amphetamine or nicotine sulfate the US, then after about four drug-drug pairings the pentobarbital CS produced a higher heart rate (HR) than control conditions. With the same pentobarbital CS, HR conditioning was not obtained with the following USs: atropine, caffeine, lithium chloride, continuous foot-shock, and intermittent foot-shock. Although amphetamine and nicotine are pharmacologically different, a common conditioning mechanism seems indicated because of striking similarities in their parametric effects as USs. There also were strong similarities in these two USs when the conditioned response was a reduced capacity of the CS drug to produce conditioned taste aversions.


[Serum testosterone level in the diagnosis of virilizing ovarian neoplasms]. The paper is concerned with the results of investigation of 15 patients with virile ovarian tumors (VOT). The following methods were employed for instrumental diagnosis: USI, computerized tomography and laparoscopy. Hormonal investigation included the determination of basal levels of serum testosterone (T), excretion with urine of 17-ketosteroids and their time course against a background of suppression tests with dexamethasone and stimulation tests with CG. The results of hormonal investigations were compared with those of similar investigations of 14 patients with stromal ovarian thecomatosis and 8 patients with androgen producing adrenal tumors. Ovarian tumors were detected in 40% by instrumental and clinical methods. Hormonal studies revealed a high level of T in patients with virile ovarian and adrenal tumors exceeding 3-3.8-fold a similar indicator in patients with stromal thecomatosis. All patients with a "tumor" level of T underwent laparotomy after excluding an adrenal lesion. Ovarian tumors of 1.0-2.5 cm in diameter were detected.


Effect of adrenal and sex hormones on opioid analgesia and opioid receptor regulation. The role of endocrine factors on opioid analgesia (antinociception) and opioid receptors was studied in male and female Swiss-Webster mice. Morphine was more potent in male than in female mice, although this difference appears to be due to greater availability of morphine to the brain in males. Saturation binding studies indicated that the density and affinity of brain mu- and delta-opioid binding sites were equivalent in males and females. Males and females were implanted SC with naltrexone (NTX) or placebo pellets for 8 days, and then the pellets were removed. This treatment increased the density of mu and delta binding sites in brain and increased the potency of morphine for both sexes, although the increase in antinociceptive effects for males was greater than for females. Adrenalectomy (ADX) in male mice increased the potency of morphine and methadone but did not alter the brain levels of either drug. ADX did not alter brain opioid binding of either mu or delta ligands. When male ADX and control mice were treated with NTX, the potency of morphine and brain opioid binding sites were increased equivalently in both groups. Gonadectomy (GDX) in male mice tended to decrease morphine potency, although this was not found to be a very reliable effect. When male GDX and control mice were implanted with NTX, brain opioid binding was increased similarly in both groups, although morphine potency was increased less in GDX mice. Overall, these studies show that sex differences and hormones of the adrenals and gonads in male mice do not alter brain opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)


Ligand-dependent effects of ethanol and diethylether at brain benzodiazepine receptors. The GABAA receptor chloride channel complex interacts with various categories of sedatives, including the benzodiazepines, and possibly ethanol and volatile general anesthetics. Thus, specific binding of tritiated derivatives of a benzodiazepine antagonist, flumazenil, and an agonist, flunitrazepam, to rat brain membrane fragments was monitored at equilibrium in the presence and absence of anesthetizing concentrations of ethanol and diethylether. Ethanol produced a concentration-dependent inhibition of [3H]flumazenil binding, which was not reversed by the GABAA receptor competitive antagonist bicuculline, but had no effect on [3H]flunitrazepam binding. Both ethanol and diethylether decreased the affinity of the benzodiazepine site for [3H]flumazenil. These data indicate that ethanol and diethylether have GABA-independent effects at the benzodiazepine sites of the GABAA receptor. These findings are inconsistent with a two-state functional model of the benzodiazepine site and, instead, support a model containing a specific, antagonist-favored conformation.


Pharmacological mechanisms in the cardiovascular effects of methamphetamine in conscious squirrel monkeys. The effects of methamphetamine were studied on cardiovascular function in conscious squirrel monkeys. Methamphetamine (0.1-3.0 mg/kg, IV) produced a dose-dependent increase in blood pressure. Its effects on heart rate were more complex, with lower doses (0.1-0.3 mg/kg) producing increases in heart rate and higher doses (1.0-3.0 mg/kg) producing decreases. To determine the pharmacological mechanisms involved in methamphetamine's effects, a number of drugs were tested as pretreatments to an injection of 0.2 mg/kg methamphetamine. This dose produced the maximal heart rate increase. The alpha 1-antagonist prazosin completely antagonized the effects of methamphetamine on blood pressure, while the nonselective beta-antagonist propranolol and beta 1-selective antagonist atenolol completely antagonized the tachycardiac effect of methamphetamine. The dopaminergic antagonists SCH 23390 and haloperidol antagonized some of the cardiovascular effects of methamphetamine. These results indicate that the pressor and tachycardiac effects of methamphetamine are mediated via alpha 1- and beta 1-adrenoceptor mechanisms, respectively. Dopaminergic mechanisms are also involved in methamphetamine's cardiovascular effects.


Isolation and characterization of IS1165, an insertion sequence of Leuconostoc mesenteroides subsp. cremoris and other lactic acid bacteria. We have cloned and characterized an insertion sequence from Leuconostoc mesenteroides subsp. cremoris strain DB1165. This element, designated IS1165, is 1553 bp, has imperfect inverted repeat ends, contains an open reading frame of 1236 bp, and is not related to any previously described insertion sequence. The copy number of IS1165 varies from 4 to 13 in L. mesenteroides subsp. cremoris strains allowing genetic fingerprinting of strains based on location and number of bands on hybridization. IS1165 or closely related elements have been detected by hybridization in L. lactis, L. oenos, Pediococcus sp., Lactobacillus helveticus, and Lb. casei but not in Lactococcus.


Complete nucleotide sequence and gene organization of plasmid NTP16. We have determined the complete nucleotide sequence of the 8.3-kb multicopy plasmid NTP16 and produced a functional map of its gene organization. Sixty percent of the plasmid DNA comprises transposon-derived sequences; in the remaining 3320 bp, we have identified three protein coding regions. NTP16 has a ColE1-type replication system, a cis-acting stability locus and a mobilization system comprising an oriT site and one mobilization protein. The roles of the other two protein products of this plasmid are unknown, but they are possibly involved in the plasmid incompatibility system.


Transposition of Tn4351 in Porphyromonas gingivalis. Genetic analysis of Porphyromonas gingivalis, an obligately anaerobic gram-negative bacterium, has been hindered by the apparent lack of naturally occurring bacteriophages, transposable elements, and plasmids. Plasmid R751::*omega 4 has previously been used as a suicide vector to demonstrate transposition of Tn4351 in B. uniformis. The erythromycin resistance gene on Tn4351 functions in Bacteroides and Porphyromonas. Erythromycin-resistant transconjugants were obtained at a mean frequency of 1.6 x 10(-7) from matings between Escherichia coli HB101 containing R751::*omega 4 and P. gingivalis 33277. Southern blot hybridization analysis indicated that about half of the erythromycin-resistant P. gingivalis transconjugants contained simple insertions of Tn4351 and half contained both Tn4351 and R751 sequences. The presence of R751 sequences in some P. gingivalis transconjugants most likely occurred from Tn4351-mediated cointegration of R751, since we were unable to detect autonomous plasmid in these P. gingivalis transconjugants. The P. gingivalis-Tn4351 DNA junction fragments from different transconjugants varied in size. These results are consistent with transposition of Tn4351 and with insertion at several different locations in the P. gingivalis chromosome. Tn4351 may be useful as a mutagen to isolate well-defined mutants of P. gingivalis.


Conjugative plasmids in Bordetella avium. Three of five antibiotic-resistant plasmids isolated from virulent strains of Bordetella avium were found to be conjugative. A physical and genetic map of one of these plasmids, the 51.5-kb plasmid p4093, revealed that the area of p4093 responsible for streptomycin and tetracycline resistance was located in a region consisting of a cluster of restriction enzyme recognition sites, whereas the remainder of p4093 contained relatively few restriction sites. Additionally, the genes involved in the conjugative ability of p4093 were clustered in at least two widely separated regions of the plasmid.


Osteogenin, a bone morphogenetic protein, adsorbed on porous hydroxyapatite substrata, induces rapid bone differentiation in calvarial defects of adult primates. Osteogenin, a bone morphogenetic protein, in conjunction with insoluble collagenous bone matrix initiates local endochondral bone differentiation by induction in vivo. This study, by exploiting the affinity of native osteogenin for hydroxyapatite, was designed to construct a delivery system for the expression of the biologic activity of osteogenin in nonhealing calvarial defects of adult primates. After exposure of the calvaria, 64 cranial defects, 25 mm in diameter, were prepared in 16 adult male baboons (Papio ursinus). Defects were implanted with disks of porous nonresorbable and resorbable hydroxyapatite substrata obtained after hydrothermal conversion of calcium carbonate exoskeletons of corals. In each animal, one disk of each hydroxyapatite preparation was treated with osteogenin isolated and purified from baboon bone matrix after sequential chromatography on heparin-Sepharose, hydroxyapatite, and Sephacryl S-200 gel filtration columns. The remaining two defects were implanted with one disk of each hydroxyapatite preparation without osteogenin as control. Histomorphometry on decalcified sections prepared on days 30 and 90 showed superior osteogenesis in osteogenin-treated nonresorbable hydroxyapatite specimens as compared with controls. On day 90, substantial bone formation also had occurred in control nonresorbable hydroxyapatite specimens. On day 90, but not on day 30, significantly greater amounts of bone had formed in osteogenin-treated resorbable specimens as compared with resorbable controls. Overall, resorbable substrata performed poorly when compared with nonresorbable substrata, perhaps due to a premature dissolution of the implants. These results provide evidence that the biologic activity of osteogenin can be restored and delivered by a substratum other than the organic collagenous matrix, inducing rapid bone differentiation in calvarial defects of adult nonhuman primates. The adsorption strategy of osteogenin on porous inorganic nonimmunogenic substrata may help to design appropriate osteogenic delivery systems for craniofacial and orthopedic applications in humans.


Breast reconstruction in women treated with radiation therapy for breast cancer: cosmesis, complications, and tumor control. The records of 55 patients who had breast cancer treated by mastectomy, irradiation, and breast reconstruction were reviewed for cosmetic outcome, complications, and tumor control. Median follow-up was 35 months. Local control rates were 95 percent in patients treated for high risk factors or breast conservation and 85 percent in patients treated for recurrent breast cancer. Acceptable cosmetic results were obtained in only 42 percent of patients. The incidence of complications was 55 percent. Transverse rectus abdominis muscle (TRAM) reconstructions gave superior cosmetic results compared with all other types of reconstructions. The timing of reconstruction in relation to mastectomy or radiation therapy did not significantly influence cosmetic outcome, although other factors suggest that delayed reconstruction may give better results. A majority of patients were satisfied with cosmetic outcome.


A review of recently-developed ligands for neuronal benzodiazepine receptors and their pharmacological activities. There has recently been a large increase in the number of new benzodiazepine receptor ligands, some with benzodiazepine structures, but many with other chemical structures. The pharmacological activities of these ligands have been interpreted on the basis of a continuum of efficacy ranging from full agonist through different degrees of partial agonism to antagonist and through partial to full inverse agonists. Inconsistencies with this hypothesis are considered in terms of alternative hypotheses, particularly the existence of functionally separable receptor subtypes. The potential of partial agonists as non-sedative anxiolytic agents with reduced potential of dependence and of weak partial inverse agonists as pro-cognitive agents is discussed. A pharmacophore for benzodiazepine receptors is proposed and supporting evidence presented.


Effect of sedative drugs upon receptor binding in vivo. Acute treatment with pentobarbital (PB), ethanol and flunitrazepam significantly decreased 3H-SCH 23390 binding in mouse striatum in a dose dependent manner. In contrast, no significant alterations in 3H-SCH 23390 binding in the cerebral cortex have been observed in mice treated with these sedative and hypnotic drugs. Flumazenil Ro15-1788) reversed the effect of flunitrazepam suggesting the reduction in dopamine D1 receptor binding in the striatum was mediated via GABA-Bz-Cl channel complex. Using kinetic analysis, it was found that such changes in dopamine D1 receptor binding in vivo were mainly due to changes in rates of ligand-receptor binding in vivo. Other non-site-specific drugs such as propanol and butanol also decreased 3H-SCH 23390 binding in vivo, depending on their lipophilicities. These results indicated that micro-environmental factors surrounding receptors, including cell membranes seem to have important roles in receptor binding in vivo. Both PB and flunitrazepam decreased muscarinic acetylcholine receptor binding in mouse cortex, striatum, hippocampus and other regions. Together with the fact that PB also altered 3H-Ro15-1788 binding in vivo, this suggested global changes in microenvironmental factors may occur due to these sedative drugs. In vivo quantitative analysis of neuroreceptors with positron emission tomography (PET) seems to have some potencies to reveal the neurochemical base of benzodiazepine dependence.


RU486 in depression.

RU486 in depression. RU486 is a synthetic glucocorticoid antagonist. The authors used RU486 to examine the hypothesis that the elevated plasma cortisol and ACTH in patients is due to suprahypophyseal stimulation of the anterior pituitary. Seven patients and matched controls were studied before and after the administration of RU486. RU486 produced an increase in HPA activity in depressed patients. Thus providing support for the hypothesis that there is increased suprahypophyseal stimulation of the anterior pituitary.


Novel Ca2+ currents in mammalian CNS neurons. Voltage-dependent Ca2+ currents (ICa) in neurons can be classified into T-, N- and L-types. In the CA1 pyramidal neurons freshly dissociated from rat hippocampus we found an additional tetrodotoxin (TTX)-sensitive Ca2+ current (termed 'TTX-ICa'). The TTX-ICa showed a heterogeneous distribution, preferentially in the dorsal site of CA1 region. Activation and inactivation processes of the TTX-ICa were highly potential-dependent, and the latter was fitted by a double exponential function. The TTX-ICa was activated at a threshold potential of about -55 mV and reached full activation at -30 mV. The steady-state inactivation of TTX-ICa could be fitted by a Boltzmann equation with a slope factor of 6.0 mV and a half-inactivation voltage of -72.5 mV. When the peak amplitudes of TTX-ICa were plotted as a function of extracellular Ca2+ concentration ([Ca2+]o), the current amplitude increased linearly without showing any saturation. The ratio of peak amplitude in the individual I-V relationships of Ca2+, Sr2+ and Ba2+ currents passing through the TTX-sensitive Ca(2+)-conducting channel was 1:0.33:0.05, although the current kinetics were much the same. TTX inhibited the TTX-ICa in time- and concentration-dependent manner without affecting the current kinetics. Lignocaine inhibited the TTX-ICa in a second in a concentration-dependent manner, with accelerating the inactivation process. The concentrations of half-inhibition (IC50) were 3.5 x 10(-9) M for TTX and 3.6 x 10(-4) M for lignocaine. Scorpion toxin prolonged the inactivation phase of TTX-ICa in a time- and concentration-dependent manner. In the toxin-treated neurons, both the slow time constant of inactivation (tau is) and its functional contribution to the total current increased with increasing the toxin concentration.


Charge-dependent renal uptake of beta 2-microglobulin in conscious rats. The influence of molecular charge on the tubular reabsorption of proteins was studied in conscious rats injected intravenously with beta 2-microglobulins of different isoelectric points (pI). Native human beta 2-microglobulin (pI 5.8), two anionized (pI 4.85 and 5.55) and three cationized derivatives (pI 7.2, 8.35 and 8.7) were used. The six forms of beta 2-microglobulin had a molecular radius between 15.7 and 15.9 A. The renal uptake was estimated by measuring the amount excreted in urine with a sensitive immunoassay. The ability of rat kidney to reabsorb beta 2-microglobulin was clearly related to the net charge of the protein. Increasing the pI of the protein significantly reduced the urinary excretion, whereas lowering it had the opposite effect. Anionization was particularly effective in reducing the beta 2-microglobulin uptake, since a decrease of the pI of one unit enhanced the urinary output by two orders of magnitude. This charge-dependency persisted when the tubular reabsorption of proteins was partly inhibited by lysozyme. By contrast, it was practically abolished by lysine, probably because the inhibitory effect of this amino acid on protein tubular reabsorption is not competitive. The administration of ammonium chloride in rats produced an immediate and transient elevation of rat beta 2-microglobulinuria. This phenomenon, which was partly inhibited by the subsequent administration of sodium bicarbonate, presumably results from a competition between the NH4+ ion and beta 2-microglobulin for tubular binding sites. These data support the hypothesis that proteins bind to the luminal membrane of tubular cells mainly via positively charged amino groups.


The detection of human papillomaviruses in cervical biopsies by immunohistochemistry and in situ hybridization. The presence of human papillomavirus (HPV) types 6, 16 and 18 in cervical biopsies can be detected by an immunoperoxidase technique using type-restricted monoclonal antibodies raised against fusion proteins representing the L1 major capsid proteins of these three HPV types. In a retrospective study (n = 54) we have used these antibodies and biotinylated DNA probes of HPV 6, 16 and 18 to detect and type HPV in formalin-fixed material from the cervix. The biopsies were classified histologically into normals, wart infections without dysplasia, cervical intraepithelial neoplasia (CIN) and squamous cell carcinomas. Antibody staining showed that 22% of all CIN was positive for HPV 16 and 40% of cervical warts were positive for HPV 6, 16 and 18. There was no HPV capsid protein detected in the normals and squamous cell carcinomas using these antibodies, whereas 25% of the tumours were positive for HPV 16 by in situ hybridization. Sections of cervical warts and CIN positive for HPV types by in situ hybridization were also positive by antibody staining which suggests that both techniques are detecting replicating virus. We feel these two techniques complement each other in detection and typing of HPV in cervical biopsies from patients with active disease.


[Prevalence of antibodies against CMV in Grenada]. The prevalence of anti-CMV antibodies was studied on 1,552 serum samples by means of latex passive haemagglutination method. Of all the sera studied, 1,084 were positive (69.8%). Amongst them, 523 samples came from women and 561 from men, which represent 74.7% and 65.8%, respectively. Regarding to age, 62.3% of the positive samples were from people under 30 years and 91.3% from subjects over that age. The screening of anti-CMV antibodies is especially important in blood and organ donors, where the high percentage of positivity makes it difficult to select negative blood donations. Thus, taking into account the cost-effectiveness, and the results of this study, the search for CMV-negative blood must be exerted preferentially on the group of blood donors under 30 years of age, regardless of sex.


Subcutaneous low molecular weight heparin versus subcutaneous unfractionated heparin in the treatment of deep vein thrombosis: a Polish multicenter trial. In a prospective multicenter trial, 149 consecutive patients with phlebographically proven proximal and/or distal deep vein thrombosis of the leg were randomly allocated to receive subcutaneously for 10 days either low molecular weight heparin CY 216 (Fraxiparine) in a fixed dose or unfractionated heparin (UFH) in doses adjusted according to the activated partial thromboplastin time. Pre- and post-treatment phlebograms were assessed blindly using the Arnesen's score system in 134 patients available for analysis of the treatment efficacy. The mean phlebographic score after 10 days of treatment was significantly decreased in both groups (p less than 0.001) in comparison with the baseline score but the difference in score changes between the two groups was not statistically significant. There was an improvement in 45/68 patients (66%) in the Fraxiparine group and in 32/66 patients (48%) in the UFH group, and an increase in the thrombus size in 10/68 (15%) and 12/66 (18%), respectively. One symptomatic non-fatal pulmonary embolism and one major bleeding episode were observed in the UFH group. During a follow-up period of 3 months, two rethromboses had occurred in the UFH group and none in the Fraxiparine group. It is concluded that subcutaneous fixed dose Fraxiparine is safe and at least as effective as subcutaneous adjusted UFH in the treatment of deep vein thrombosis.


The effects of vitamin K1 and warfarin on prothrombin expression in human hepatoblastoma (HepG2) cells. Cultures of human hepatoblastoma (HepG2) cells were treated with vitamin K1 or warfarin and prothrombin antigen and mRNA levels were determined. With 3 and 6 h of 10 micrograms vitamin K1 treatment secreted prothrombin antigen levels, relative to total secreted protein levels, were increased 1.5-fold and 2.1-fold, respectively, over ethanol-treated control levels as determined by an enzyme-linked immunosorbent assay. Dose-response analysis with 3 h of 25 micrograms/ml vitamin K1 treatment demonstrated a maximal increase of 2.0-fold in secreted prothrombin antigen levels, relative to total secreted protein levels, over ethanol-treated control levels. Pulse-chase analysis with 35S-methionine and immunoprecipitation of 35S-labelled prothrombin demonstrated that, with vitamin K1 treatment (25 micrograms/ml, 3 h), the rate of prothrombin secretion increased approximately 2-fold and the total amount (intra- and extracellular) of prothrombin synthesized increased approximately 50% over ethanol-treated control levels. Warfarin treatment (1, 5, or 10 micrograms/ml, 24 h) resulted in decreases in secreted prothrombin antigen levels, relative to total protein levels to approximately 85%, 87% or 81% of ethanol-treated control levels. Analysis of total RNA isolated from these cultures by Northern and solution hybridization techniques demonstrated that prothrombin mRNA was approximately 2.1 kb and that neither vitamin K1 nor warfarin treatment affected the quantity of prothrombin mRNA (ranging from 240-350 prothrombin mRNA molecules per cell). These results demonstrate that vitamin K1 and warfarin, in addition to effects on gamma-carboxylation, affect prothrombin synthesis post-transcriptionally, perhaps influencing translation, post-translational processing and/or secretion mechanisms.


Comparison of recombinant tissue factor pathway inhibitors expressed in human SK hepatoma, mouse C127, baby hamster kidney, and Chinese hamster ovary cells. Recombinant tissue factor pathway inhibitor (rTFPI) has been expressed in four mammalian expression systems using human SK hepatoma, mouse C127, baby hamster kidney (BHK), and Chinese hamster ovary (CHO) cells as hosts. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the immunoaffinity purified rTFPIs all show broad bands and the mean molecular weight of SK hepatoma and C127 rTFPIs (M(r) approximately 38,000) appear larger than those of BHK and CHO rTFPIs (M(r) approximately 35,000). All these proteins inhibit factor Xa and appear to bind factor Xa with 1:1 stoichiometry. The ability of these proteins to inhibit tissue factor-induced coagulation in plasma was examined using a prothrombin time assay. The relative activities of SK rTFPI:C127 rTFPI:BHK rTFPI:CHO rTFPI were found to be 28:15:2.1:1. By Western blot using specific antisera against the amino- and carboxy-termini of TFPI as probes, it is found that all the immunoaffinity purified rTFPIs possess approximately equal amounts of the amino terminus, but the C127 and BHK rTFPIs are deficient in carboxy terminus and the CHO rTFPI is essentially devoid of this region of the protein. Mono S chromatography allowed separation of the full-length and the truncated molecules with high and low anticoagulant activities, respectively. The above results suggest that proteolysis of the carboxy terminus of TFPI occurs to different extent when TFPI is expressed in different cells and that the carboxy terminal region of the TFPI molecule is important for the inhibition of tissue factor-induced coagulation.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of gold in urine and bile following administration of gold sodium thiomalate with chelating agents to rats. Gold was characterized in the urine and bile of rats treated with D-penicillamine (D-PEN), 2,3-dimercaptosuccinic acid (DMSA), 2,3-dimercaptopropane sulphonate (DMPS), or N-(2-mercapto-2-methylpropanoyl)-L-cysteine (bucillamine) immediately after gold sodium thiomalate (AuTM) injection by both gel chromatographic and electrophoretic methods. It is suggested that the gold in the urine and bile after AuTM administration was predominantly bound to high molecular weight compounds. The characterization of gold in the urine after administration of AuTM with D-PEN, DMSA, or DMPS showed that most of the gold was bound to the chelating agents. In the treatment with the chelating agents such as D-PEN and DMPS, the gold was mainly excreted as a gold-chelating agent compound in the bile and a minor portion of the gold was present in the form of a gold-L-cysteine compound and high molecular weight compounds. DMSA treatment showed that a major portion of the gold was bound to high molecular weight compounds in the bile and a minor portion of the gold was present in the forms of gold-DMSA and gold-L-cysteine compounds. The administration of AuTM and bucillamine indicated that the gold was mainly present as a gold-Me-bucillamine compound in the urine and a gold-bucillamine compound in the bile.