The Chemical Use, Abuse, and Dependence Scale (CUAD). Rationale, reliability, and validity. This article describes the rationale for the development of the Chemical Use, Abuse, and Dependence Scale (CUAD). The instrument is in a semistructured interview format; it derives both substance use severity scores and DSM-III-R substance use disorder diagnoses and can be administered in a short period with minimal training. The reliability and validity of the CUAD are reported and appear satisfactory. The CUAD is recommended for use as a detection, diagnostic, and treatment selection index in clinical, research, and program evaluation contexts.


Prevalence of psychopathology in drug-addicted Dutch. In Dutch samples of treated heroin addicts, high prevalences of a heterogeneous psychiatric co-morbidity can be found with regard to Diagnostic and Statistical Manual (third edition) (DSM-III) classifications, Zung Depression Inventory, and sum scores of a 90-item Symptom Checklist (SCL-90). A high-threshold (N = 87) and a low-threshold (N = 116) program are compared with regard to psychopathology and severity of psychopathology. A consecutive admissions design was used. More than 50% of the respondents suffered from a lifetime DSM-III Axis I disorder (70% with antisocial personality disorder included), and 40% were still suffering from one of the disorders in the year preceding the interview. Schizophrenia was diagnosed five times as much as in normal population samples (5%). The most frequently diagnosed disorders were recurrent major depression, phobic disorders, alcohol abuse and dependence, dysthymic disorder, and antisocial personality disorder. The prevalences of DSM-III disorders, the total number of symptoms, and the score on the Zung Depression Inventory and 90-item Symptom Checklist were all significantly higher in treatment-seeking drug addicts entering the high-threshold program. Within each program, three clinically meaningful subgroups can be distinguished: one group with DSM-III Axis I lifetime or current psychopathology and/or antisocial personality disorder, one with antisocial personality disorder only, and one with neither DSM-III psychopathology nor antisocial personality disorder. Possibly, self-selection results in patients with more serious conditions entering more treatment-oriented facilities. Odds ratios show that schizophrenia and mood disorders and especially associated on a lifetime and current basis.


Demand for alcohol treatment by problem drinkers. Problem drinkers applying for treatment at both outpatient and inpatient treatment programs were compared with problem drinkers identified in a general population survey. The characteristics in which these populations differ were interpreted as factors contributing to the demand for treatment and, thus, indicative of the need for alcohol treatment. Older problem drinkers appear to apply for treatment more frequently, and the same is true for divorced and unemployed or disabled problem drinkers. More important, age, marital status, and employment status were found to contribute to the demand for alcohol treatment independent of alcohol consumption. It can be concluded from this study that the demand for alcohol treatment is not determined by alcohol consumption alone, suggesting that alcohol treatment should not only focus on alcohol consumption but also focus on problems related to marital and employment status in order to prevent future need for alcohol treatment.


Adapting the chronic disease model in the treatment of dually diagnosed patients. This article suggests that while the focal point of inpatient treatment in the field of addiction should continue to be abstinence and 12-step programs, there should be considerable modification of the process of treatment. Many of the present inpatient psychiatric hospitals that treat addiction seem to be largely unaware of the biochemistry and physiology of early recovery. A model of treatment is proposed that takes into account the neurocognitive impairment and emotional augmentation that is present in early recovery. The focus of active treatment in this program is the treatment of the addictive process. The psychiatric diagnosis is managed through supportive psychotherapy and/or education if appropriate. Active treatment of the psychiatric diagnosis is deferred to outpatient treatment.


Nitric oxide is a potent relaxant of human and rabbit corpus cavernosum. Nitric oxide (NO) caused a potent, marked, and transient relaxation of precontracted strips of corpus cavernosum isolated from humans and rabbits. The relaxation response elicited by NO was very similar to the relaxation evoked by electrical field stimulation via the nonadrenergic-noncholinergic pathway. Sodium nitroprusside, nitroglycerin, and S-nitroso-N-acetylpenicillamine, which are nitrovasodilators known to generate NO, also caused marked concentration-dependent relaxation of corpus cavernosum. Relaxant responses to NO were enhanced by the cyclic GMP phosphodiesterase inhibitor M&B 22,948 and inhibited by oxyhemoglobin. Similarly, relaxation of corpus cavernosum in response to electrical field stimulation or acetylcholine was enhanced by M&B 22,948 and inhibited by oxyhemoglobin. NO stimulated cyclic GMP formation in corpus cavernosum and a close positive correlation was found between the magnitudes of relaxation and cyclic GMP formation. The data suggest that NO-elicited activation of guanylate cyclase and cyclic GMP formation represents the signal transduction mechanism responsible for relaxation and nonadrenergic-noncholinergic-mediated penile erection. These observations indicate that NO is a potent relaxant of human and rabbit corpus cavernosum and support our hypothesis that endogenous NO is the principal mediator of penile erection caused by nonadrenergic-noncholinergic stimulation.


Leucocytes and free radicals in stable angina pectoris. Bicycle ergometer exercise was used to induce ischemia in 20 patients with stable angina pectoris (SAP). Superoxide dismutase (SOD) blood concentrations, free radical generation (by the SOD-inhibitable reaction of ferricytochrome C), malondialdehyde (MDA) plasma concentrations, the unfractionated leucocyte filterability rate and the filterability rates of the granulocyte and mononuclear sub-fractions (using a positive pressure filtration system and 5 mu diameter Nuclepore filters), were monitored before and after exercise in the patients and in 18 matched controls. At the onset of ischemia a significant increase in the level of MDA plasma concentrations and significant decreases in both SOD blood concentrations and the SOD-inhibitable reduction of ferricytochrome C indicated oxygen free radicals had been released in the SAP patients. These changes were associated with significant impairments of granulocyte and unfractionated leucocyte filterabilities and with morphological evidence of granulocyte activation.


[Molecular bases of chronic granulomatous disease--analysis of the involvement of cytosol factors for NADPH oxidase]. During the past 5 years, the discovery of cell-free superoxide generation system (Bromberg Y, Pick E: Cell Immunol 88:213-221, 1984) has been revolutionized our understanding of phagocyte superoxide generation. Using cell-free system, it was clarified that NADPH oxidase for superoxide generation was comprised of components present in both the plasma membrane as well as in the cytosol. This oxidase could be kept inactive by keeping its components separated from each other within the cell and then quickly bringing them together in the plasma membrane upon activation. We analyzed cytosol components with column method, and clarified that 3 neutrophil cytosol factors (NCF-1/-2/-3) was necessary for reconstitute the cytosol activity which was missing in an autosomal recessive type of Chronic Granulomatous Disease (CGD) patients (Nunoi H, et al:Science 242:1298-1301, 1988). NCF-1/-2 were analyzed with B1 antibody and found their molecular weight as 47 and 67 kilodalton respectively. One of autosomal CGD patients was missing NCF-67k and the others were missing NCF-47k. NCF-47k and -67k were cloned with this antibody and sequenced and expressed as recombinant NCF-47k/-67k using baculovirus/insect cell system. Using these recombinants, we are trying to purify NCF-3 which is reported as small G protein in these days. Using monoclonal antibodies against these recombinants, we analyzed tissues with immunohistochemical methods and are trying to classify the type of CGD patients in Japan. In summary, at least five oxidase components now have been identified (alpha and beta chain of cytochrome b558 (gp91-phox, p22-phox) and NCF-47k/-67k/-3 (p47-phox/p67-phox/delta-1 or SOCI)).


[Active oxygen generating system in immune cells]. Oxygen metabolites, such as O2-, H2O2 and HClO formed during the respiratory burst in phagocytes are known to be essential for killing certain microorganisms. In the initial reaction of the respiratory burst, an electron is transferred from NADPH to molecular oxygen to generate O2-. Other active oxygen species are formed secondarily from O2-. Patients with chronic granulomatous disease, whose phagocytes can not generate O2-, often suffer from recurrent, life-threatening infections. We have demonstrated recently that B lymphocytes but not T lymphocytes (nor NK cells) have an O2- generating system identical or very similar to that in phagocytes. This article deals with the present status of the research on O2- generating system in phagocytes and B lymphocytes.


[Determination of human erythrocyte membrane Na+/K(+)-ATPase activity in small volume of blood sample]. Na+/K(+)-ATPase of the cell membrane is considered to be closely related to the pathology of various diseases including hypertension and heart failure. The activity of this enzyme in the erythrocyte membrane has been determined in earlier reports by the assay of inorganic phosphate generated from the substrate ATP or radioimmunoassay after binding 3H ouabain to the erythrocyte membrane, using a large volume of blood samples. However, as neither method was appropriate for wide routine use, we developed a method to assay this enzyme in a small volume (10 ml) of fresh human blood samples with re-evaluation of conditions for the inorganic phosphate assay. In this method, the coefficient value (CV) of membrane protein amount and the NA+/K(+)-ATPase activity were 2.2% and 2.5% respectively, indicating sufficient precision of the assay. Moreover, in 97 subjects without abnormalities in blood biochemical tests (77 males and 20 females) aged 35-59 years, the enzyme activity showed no differences according to age or sex, ranging from 0.217 to 0.071 mumols Pi/mg/hr with a mean of 0.130.


Significant difference in the effect of acute saline loading on plasma Na-K ATPase inhibitor and blood pressure between children and adults with essential hypertension. Twenty-one male children and 3 male adults with essential hypertension were infused with physiological saline solution (15 ml/kg/hr) for 1 hr after they had been supine for 90 min. The blood pressure and heart rate were monitored, and blood was taken twice before and after the infusion to measure the plasma Na-K ATPase inhibitor. After saline infusion, both the plasma Na-K ATPase inhibitor and blood pressure increased significantly in the hypertensive adults, and the number of Na pump sites decreased. However, such changes were not observed in the hypertensive children. These findings suggest that circulating Na-K ATPase inhibitor may not appear following acute saline infusion in hypertensive children unlike in hypertensive adults, and that the mechanisms regulating cell membrane sodium transport and high blood pressure may differ between hypertensive children and adults.


Role of the 5-lipooxygenase pathway in obstructive nephropathy. Leukotrienes are products of the 5-lipooxygenase pathway of arachidonic acid metabolism that possess potent inflammatory properties. We examined the potential role of this pathway in the decrease in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) observed in rats after unilateral release of bilateral ureteral obstruction (BUO) of 24 hours duration. Isolated glomeruli from rats with BUO produced significantly greater amounts of leukotriene B4 (LTB4) than glomeruli from sham-operated rats (SOR; P less than 0.0001). Glomeruli from rats with BUO given MK886, an inhibitor of the 5-lipooxygenase enzyme, or from rats with BUO subjected to both total body irradiation to prevent the leukocyte infiltration of the kidney and also given MK886 prior to obstruction, produced amounts of LTB4 not significantly different from those in glomeruli of SOR. Glomeruli from rats with BUO that had only total body irradiation prior to obstruction produced significantly less LTB4 than glomeruli from untreated BUO rats, but LTB4 production was still significantly greater than in glomeruli from SOR. There were no significant differences in GFR among SOR, SOR given MK886, and SOR subjected to total body irradiation. However, SOR given MK886 had significantly higher ERPF and lower renal vascular resistance (RVR) than SOR not pretreated with the lipooxygenase inhibitor. Rats with BUO given MK886, or subjected to total body irradiation, or both, prior to obstruction had significantly greater GFR and ERPF values and lower RVR than untreated BUO rats. Glomeruli from rats with BUO which were not pretreated had three times the leukocytes of glomeruli from SOR. This leukocyte infiltrate was composed of macrophages (about 55%) and neutrophils (about 45%).(ABSTRACT TRUNCATED AT 250 WORDS)


The vasorelaxant effects of acetate: role of adenosine, glycolysis, lyotropism, and pHi and Cai2+. The mechanism of acetate vasorelaxation is unknown. In the rat caudal artery, acetate has a vasorelaxant effect and also increases cyclic AMP. Here we evaluate the role of adenosine, of possible glycolysis inhibition by acetate, of the lyotropic properties of acetate and other anions, and of intracellular calcium and pH. Adenosine per se did not relax the caudal artery in the range of 10(-8) to 10(-2) M. Preincubation with adenosine deaminase (ADA, 5.0 U/ml) or with 8-phenyltheophylline (8-PT, 10(-6) to 10(-4) M) increased, rather than blocked the vasorelaxant effect of acetate. Oxypurinol (10(-3) M) or the nucleoside transport inhibitor NBMPR (10(-4) M) had no effect on acetate relaxation. Whereas acetate increased tissue cyclic AMP content, 10(-3) M adenosine or 10(-6) M PIA had no effect. In strips studied under conditions of inhibited glycolysis (no glucose, with 11 mM 2-deoxyglucose, 1.0 mM pyruvate, and 0.5 mM 5-iodoacetate), acetate-induced relaxation, as well as acetate-induced cyclic AMP generation, tended to be reduced but not significantly so. Other anions relaxed vascular strips in relation to their lyotropic number, but only at higher doses, and they did not stimulate cyclic AMP formation. Acetate (10 mM) caused a transient fall in Ca2+i followed by a slight, sustained rise. A concomitant decrease in pHi was seen. DIDS, which blocks the relaxant and cyclic AMP effects of acetate, had no effect on the pHi decrease, but did decrease the rate of pHi recovery.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of angiotensin converting enzyme inhibition in adult polycystic kidney disease. The pathogenesis of hypertension in autosomal-dominant polycystic kidney disease (ADPKD) is unclear, but increased activity of the renin-angiotension system may contribute. The renal and systemic hemodynamic response to lisinopril, an angiotension converting enzyme (ACE) inhibitor, in patients with ADPKD without renal failure was compared with the response in matched unaffected family members. Mean blood pressure and renal vascular resistance decreased in the affected group after lisinopril, with no significant change in the unaffected group. Glomerular filtration rate (GFR) was unchanged and therefore filtration fraction fell significantly. Changes in urinary excretion of 6-keto-PGF1 alpha and kallikrein suggested that increased renal synthesis of PGI2 or activation of the renal kallikrein-kinin system were not likely to be responsible for the hemodynamic effects. The acute decrease in renal vascular resistance without change in GFR suggests that ACE inhibition may have a particular value in the treatment of hypertension associated with ADPKD which should be assessed by further long-term studies.


Regulation of atrial natriuretic peptide-stimulated cGMP production in the inner medulla. Studies were performed to examine the regulation of atrial natriuretic peptide- (ANP) stimulated guanylate cyclase in the the inner medulla. Primary cultures of rat inner medullary collecting tubular cells exposed to 10(-7) M ANP increased cGMP formation to 31.2 +/- 1.8 compared to the basal production of 2.1 +/- 0.6 fm/micrograms protein. This response did not appear to be transduced via a Gi protein, as preincubation with pertussis toxin did not alter the response to 10(-7) M ANP, and saponized cells exposed to 10 microM GTP gamma S did not enhance the response to ANP (77.3 +/- 5.9 vs. 86.7 +/- 6.3 g/micrograms). Likewise, changes in extracellular Ca2+ from 0.5 to 3.0 mM, decrements in intracellular Ca2+ with EGTA or increments in intracellular Ca2+ with ionomycin (5 microM) did not significantly alter the response to ANP. Neither activation of protein kinase A with forskolin (36.5 +/- 5.1) nor of protein kinase C with s,n-1,2-dioctanoylglycerol (33.2 +/- 2.5) altered the response to 10(-7) M ANP (32.2 +/- 3.3, NS). As the inner medullary environment was hypertonic, the effect of altering tonicity was studied. Cells grown for 48 hours in hypertonic media (600 mOsm/kg H2O) displayed enhanced response to 10(-8) and 10(-7) M ANP when osmolality was raised by either Na+ alone or in combination with urea, but not by urea alone. Our studies demonstrate that ANP-stimulated guanylate cyclase is insensitive to alterations in either intra- or extracellular Ca2+, is not subject to inhibition by protein kinase, and does not involve a pertussis-sensitive G protein.(ABSTRACT TRUNCATED AT 250 WORDS)


Relationship between intracellular proton buffering capacity and intracellular pH. In a recent publication the widely held view that the intracellular proton buffering power [defined as the amount of acid or base that has to be added to the cytosol to change the intracellular pH (pHi) by one pH unit] increases as the intracellular pH decreases, has been challenged, with the opposite relationship being proposed. In that publication, buffering was defined not in terms of pH change, but in terms of the change in proton concentration. The reason for this re-definition was the fear that the conventional analysis, using as it does a logarithmic function (pHi), could bias the outcome in favor of an increasing buffering power with decreasing pHi. The new system uses a "buffering co-efficient," defined as the number of protons necessary to be added to the cytosol to change the intracellular proton concentration by 1 mM. We report the use of both of these methods to analyze the relationship of pHi and buffering power, using human peripheral leucocytes loaded with the pH-sensitive fluorophore BCECF examined over a very wide range of pHi values (pHi 6.0 to 7.5). The most common method for pHi perturbation for the measurement of buffering is used, the rapid diffusion of ammonia across the cell membrane. In this study, analysis for both a bicarbonate-containing "open" system and for a Hepes-buffered "closed" system was performed.(ABSTRACT TRUNCATED AT 250 WORDS)


Renal tubular epithelial cells express osteonectin in vivo and in vitro. Osteonectin (SPARC, culture shock protein, BM-40) is a widely distributed glycoprotein which binds calcium and several extracellular matrix proteins, including interstitial collagens and thrombospondin, but whose physiologic role remains undefined. In the present studies, we have demonstrated that immunoreactive osteonectin is present in the distal cortical tubule and medullary tubules of murine kidney. We surveyed the renal epithelial cell lines LLC-PK1, MDCK, and OK for the expression of mRNA encoding osteonectin. We found that osteonectin mRNA is expressed by LLC-PK1 and OK cells but not by MDCK cells, as well as by adult kidney from several species. Calcitonin and vasopressin, agents which increase cAMP in these cells, were found to decrease steady-state osteonectin mRNA concentrations. We found that LLC-PK1 cells produced osteonectin protein, that the protein was localized to intracellular granules, and that the protein bound hydroxyapatite in vitro. Pulse-chase analysis revealed that osteonectin was secreted from the cell layer to the medium after a lag time of four to six hours and was secreted preferentially from the basolateral domain of the cell. The preferential secretion of the calcium-binding protein osteonectin from the renal epithelial cell is consistent with several possible functions, including a structural extracellular matrix protein, a participant in transepithelial ion transport, and an inhibitor of extracellular calcification.


Protection from hypoxemia-induced renal dysfunction by the thiophosphate WR-2721. The acute renal effects of hypoxemia-reoxygenation and the putative protective action of WR-2721 [S-, 2-(3-aminopropyl-amino)-, etylphosphosphorothioic acid], a drug with specific properties such as PTH-secretion inhibiting, calcium lowering and free radical scavenging activities, were investigated in anesthetized-ventilated rabbits. Glomerular filtration rate (GFR) and renal blood flow (RBF) were assessed by the clearance of inulin and para-aminohippuric acid, respectively. Each animal acted as its own control. Normoxemic control rabbits showed no changes in renal hemodynamics and function during 150 minutes. The administration of WR-2721 (75 mg/kg body wt i.v.) to normoxemic animals induced a significant decrease in MBP, RBF and diuresis, without affecting GFR. It significantly reduced plasma levels of PTH, decreased calcemia and increased urinary calcium excretion. In untreated hypoxemic-reoxygenated rabbits, 45 minutes of severe hypoxemia (PO2 around 35 mm Hg) induced a significant fall in MBP, GFR, RBF and diuresis, which persisted during the 60-minute reoxygenation period. The administration of WR-2721 before hypoxemia or reoxygenation prevented the hypoxemia-induced decrease in GFR and diuresis. Filtration fraction increased significantly. The renal functional improvement observed in hypoxemic rabbits administered WR-2721 could be mediated by its effect on calcium metabolism and/or its oxygen free radical scavenging properties.


Modulation of lymphatic smooth muscle contractile responses by the endothelium. The endothelium regulates smooth muscle tone in blood vessels through the release of endothelium-deprived relaxing factor (EDRF). We hypothesized that the lymphatics possess endothelium-dependent relaxant properties analogous to those in blood vessels. Fresh porcine tracheobronchial lymphatic vessel rings were mounted in organ baths and connected to force-displacement transducers. Rings were submaximally precontracted with 10(-5) M histamine and exposed to cumulative addition of acetylcholine (ACH; 10(-7)-3 x 10(-4) M) or bradykinin (BRD; 10(-8)-3 x 10(-6) M), both of which are known to promote release of EDRF. ACH caused concentration-dependent relaxation (maximum effect = -2.3 +/- 2.6% of initial histamine-induced active tension), while a similar but less pronounced effect was seen with BRD (39.6 +/- 5.4%). The relaxant effects of ACH and BRD were inhibited by collagenase pretreatment and mechanical endothelial denudation. The results confirm our hypothesis that lymphatic vessels possess endothelium-dependent relaxant properties and suggest that lymph vessels can regulate lymph flow through processes similar to those found in blood vessels.


Anti-neoplastic effects of interleukin-4. Interleukin-4 (IL-4) is a cytokine, with potential anti-neoplastic effects. This study examined the effects of IL-4 on host anti-tumor responses in a murine model. C57/B16 mice (n = 40) were randomized to receive Lewis lung carcinoma (10(6) cells: right flank; sc) or saline, and sacrificed 10 days postinoculation for assessment of peritoneal macrophage (PMO) anti-tumor mechanisms [superoxide anion generation (O2-), tumor necrosis factor (TNF), and TNF-independent (P815) cytotoxicity], splenocyte mixed lymphocyte response (MLR) (Balb/c stimulator), and cytotoxic lymphocyte generation (CTL against P815). Cells were cultured +/- IL-4 (100 U/ml). In a second study, 20 mice received Lewis lung implants (sc) and were randomized on Day 21 to receive daily IL-4 (1000 U/mouse; ip) or saline. Tumor volumes and median survival were assessed. Tumor necrosis factor-independent cytotoxicity (O2-, MLR and CTL) was impaired in the tumor-bearing (TB) study group. Interleukin-4 administered to cultured cells from TB mice enhanced O2-, as well as MLR and CTL (P less than 0.01), and decreased TNF release but did not alter PM phi TNF-independent anti-tumor responses (P815). In vivo administration of IL-4 significantly decreased tumor growth (P less than 0.05) after 10 days of treatment and significantly prolonged median host survival (P less than 0.05). These findings indicate the therapeutic potential of IL-4 in the TB host which may function through downregulation of TNF production while potentiating certain T cell-dependent and independent anti-tumor immune mechanisms.


Endobronchial brachytherapy: a preliminary experience. Fourteen patients with malignant airway obstruction were treated with a placement of a flexible nylon catheter for low dose rate manual afterloading Iridium 192 endobronchial brachytherapy using a flexible fibreoptic bronchoscope. Eight patients had obstructive pneumonitis at initial presentation, while 6 cases were recurrences after previous external irradiation. Six evaluable patients of the former group had complete or partial reinflation of lung and were followed by external radiotherapy. Of the latter group, 3 were evaluable and had moderate to good palliation of their symptoms. No complication was observed. The technique is simple and safe with good patient compliance. Further evaluation is indicated to assess its role in the locoregional management of lung cancer.


The diagnostic significance of gastrin measurement of bronchoalveolar lavage fluid for lung cancer. In this study, determination of gastrin concentration in bronchoalveolar lavage fluid and serum has been detected by radioimmunoassay in 30 cases of lung cancer and 24 cases of non-cancer pulmonary diseases. The results show that the gastrin concentration and its positive rate of lavage fluids from cancer lung are much higher than those from healthy lung and serum in lung cancer patients, and those from serum and both disease and healthy lung in non-cancer pulmonary disease patients (P less than 0.01). The gastrin ratio of lavage fluids from cancer lung to serum is also significantly higher than the ratio of lavage fluid from healthy lung to serum and all the ratios in the non-cancer pulmonary disease group. These results suggest that there is a high gastrin concentration in local tissue of lung cancer, which is signified by the high concentration of gastrin and its high positive rate in lavage fluids from the lung with cancer. Therefore, the gastrin determination in lavage fluids and gastrin ratio of lavage fluids to serum are more reliable in the differential diagnosis of benign from malignant pulmonary diseases than gastrin determination of serum alone.


HIV-1 ancestry primordial expansions of RRE and RRE related sequences. The human T-lymphotropic retroviruses HTLV-I and HIV-1/-2 share a complementary patchwork homology in which the RRE regions of HIV-1 and -2 cover 78.1% of a 169 nucleotide (nt) region (PX1,2) positioned precisely between open reading frames PX I and PX II of ATLV (HTLV-I). The sequence character of the PX1,2 region is shown to be influenced by a primordial expansion, CTC2T, originating several hundred nucleotides upstream. A second primordial expansion, AGCU(U/C), is identified and shown to represent 52.1% of the HIV-1 RRE region. It is argued that prior to the present AIDS pandemic the efficiency of the rev receptor was enhanced by an ancestral recombination event.


Modeling the dynamic features of the electrogenic Na,K pump of cardiac cells. The purpose of this paper is to examine the dynamic features of the electrogenic Na,K pump of cardiac cells, based on a comparative analysis of a mechanistic model and an ad hoc mathematical description of the Na,K pump. Both representations are incorporated into a modified version of the Beeler-Reuter model for the ventricular membrane, and the resulting action potential models are studied under conditions of repetitive stimulation at steady rates between 0 and 3 Hz. The two Na,K pump representations have nearly identical steady-state characteristics of sensitivity to internal Na+ concentration, external K+ concentration, and membrane potential. Rapid voltage-dependent transient pump currents are present in the mechanistic model, while they are absent in the ad hoc mathematical description we used. The stimulation results show that a sizable peak of pump current caused by the action potential upstroke in the mechanistic model affects phase 1 repolarization, and that this effect is relatively independent of the stimulation rate. The pump current generated by our ad hoc mathematical description is constant during the action potential and does not affect directly the repolarization time course. While the two Na,K pump models show similar pumping efficiency at low stimulation rates, the mechanistic pump is more efficient at high rates of activity. In essence, the distinctive features of the mechanistic model are due to an energy barrier expressing the voltage dependence of the translocation step of the mechanism, and to the redistribution of the intermediates of the biochemical reactions during activity. In comparison, the ad hoc mathematical description exhibits a fixed dependence of the pump current on voltage and ionic concentrations.


Expression of the Wilms' tumor gene (WT1) in human leukemias. Leukemic cells from seventy patients with various types of human leukemias were examined for expression of the WT1 gene, the Wilms' tumor gene located at chromosome 11p13. WT1 was expressed in 7 of 16 cases of acute lymphoblastic leukemia, 15 of 22 with acute myelogenous leukemia and 8 of 10 in blast crisis of chronic myelogenous leukemia. No detectable WT1 RNA was found in chronic leukemias, including chronic lymphocytic leukemia, plasma cell leukemia, hairy cell leukemia and chronic myelogenous leukemia in chronic phase. The expression pattern of WT1 in these human leukemia samples indicates the involvement of this gene in the early stage of hematological cell differentiation.


Transformation of large granular lymphocytic leukemia during the course of a reactivated human herpesvirus-6 infection. A patient with CD3+ large granular lymphocytic (LGL) leukemia developed transformation (TF). The phenotype of the leukemic cells was CD3+, CD4+ and CD8-. The leukemic cell count increased rapidly; the cells became large and the nuclear outline, which had been reniform, became lobulated. Anti-HTLV-1 and anti-HIV antibodies were negative in the serum of the patient and no HTLV-1 specific sequences were detected in the cDNA of the leukemic cells by polymerase chain reaction (PCR). Comparison of the karyotype abnormality of the leukemic cells before and after TF revealed an abnormality of the 21 trisomy in 90% of mitotic cells of the patient. Analysis of the cell cycle revealed that 13.7% of the leukemic cells were in DNA synthesis phase which was not previously found. The titer of anti-human herpesvirus-6 (HHV-6) immunoglobulin G which had been high at chronic phase (1:1640 compared to normal titer of less than 1:160), became 1:20,000 at TF. The titer of anti-HHV-6 immunoglobulin M also increased from less than 1:4 at the chronic phase to 1:120 at TF (normal value less than 1:4). A HHV-6-specific DNA sequence was detected by PCR in the peripheral mononuclear cells collected at TF but not at the chronic phase. These data suggests that TF occurs not only in CD3-negative but also in CD3-positive LGL leukemia. HHV-6 reactivation is therefore a possible cause in immunocompromised hosts whose general conditions are deteriorated.


Amplification and sequencing of genomic breakpoints located within the M-bcr region by Vectorette-mediated polymerase chain reaction. The polymerase chain reaction (PCR) cannot be used to amplify the breakpoint in the chimaeric BCR-ABL gene in CML and acute leukaemias due to the large variation in the sites of breakpoint in the BCR gene (within a 5.8 kb region) and in the ABL gene (within a 150 kb region). The disease state is usually monitored using RNA-PCR to monitor abnormal transcripts. We have used a new modification of the PCR to amplify breakpoints within zone 3 of the M-bcr. A synthetic oligonucleotide linker, the Vectorette, is ligated to restriction digested DNA, and amplification is carried out between primers for a known target sequence and the Vectorette linker. Three Philadelphia chromosome Ph1-positive CML patients with breakpoints within the ALU region of zone 3 have been amplified and the sequence immediately around the breakpoint determined. The breaks occurred within 70 bp and two were only 14 bp apart. The Vectorette-PCR technique has the potential to rapidly identify and sequence breakpoints, and will enable the design of patient-specific primers to monitor disease progression, particularly following bone marrow transplantation.


Characterization and scatchard binding analysis of adrenal digitalis-like material. Culture medium conditioned by the adrenocortical tumor cell line Y-1 has been fractionated by HPLC to determine whether the ouabain-like binding activity (OLB) of this medium co-purifies with digoxin immunoreactivity. Furthermore, we have evaluated whether this medium contains the ability to inhibit the ion translocating action of the sodium pump and how this material purifies on HPLC. These activities are not homogenous, however, maximal activity in each assay was found in the same HPLC fraction. The OLB of this fraction was not reduced by alkaline hydrolysis or trypsinization, heating in air at 150 degrees C did reduce activity. Scatchard binding analysis of this fraction indicated the presence of a single class of specific competitive inhibitor of the binding of radiolabelled ouabain to human erythrocytes. The inhibitor showed a Hill coefficient of unity.


Regulation of adrenomedullary preproenkephalin mRNA: effects of hypoglycemia during development. To further evaluate whether transsynaptic mechanisms account for stress-induced changes in adrenomedullary preproenkephalin mRNA (ppEnk mRNA), neonatal rats were made hypoglycemic at a time when synapses are non-functional (less than 10 days postnatal age). While ppEnk mRNA in medullae from adult rats increased as much as 60-fold in this paradigm (insulin 10 U/kg), ppEnk mRNA levels in the newborn increased only 1.6-fold (insulin 20 U/kg). To evaluate whether postsynaptic cholinergic pathways of the neonatal adrenal medulla were functional, we treated 5-day-old pups with cholinergic agonists (nicotine [1 mg/kg, s.c., q 12 h] + carbachol [1.7 mumol/kg, s.c., q 12 h x 4 days]). Combined cholinergic agonist treatment augmented enkephalin prohormone and peptide levels up to 3-fold (P less than 0.05). To determine whether the blunted response to hypoglycemia in the newborn resulted from a deficiency in functional transsynaptic activity, synapses were matured using thyroid hormone pretreatment (postnatal days 2 and 3) before hypoglycemic stress. Hypoglycemia now caused a 40-fold increase in adrenomedullary ppEnk mRNA levels only in the T3/insulin treated group. To exclude other secondary effects of hypoglycemia (eg. hormonal, or insulin treatment-dependent), intracellular glycopenia was produced in the presence of secondary hyperglycemia by injecting adult rats or pups with 2-deoxyglucose (500 mg/kg). Similar to the insulin-hypoglycemia group, a large increase in adrenomedullary ppEnk mRNA resulted in the adult but not in the 5-day-old neonatal adrenal medullae. We conclude that enkephalin biosynthesis, like co-stored catecholamines, is induced by a transsynaptic process.(ABSTRACT TRUNCATED AT 250 WORDS)


Transient ischemia stimulates glial fibrillary acid protein and vimentin gene expression in the gerbil neocortex, striatum and hippocampus. Astrocytic activation plays a major role in homeostatic maintenance of the central nervous system in response to neuronal damage. To assess the reactivity of astrocytes in transient cerebral ischemia of the gerbil, we studied the levels of glial fibrillary acidic protein (GFAP) and its mRNA. GFAP mRNA increased by 4 h after carotid artery occlusion, reached peak levels by 72 h with a 12-fold increase over control and then started declining as early as 96 h postischemia. An examination of the specific regions of the brain revealed an increase in GFAP mRNA associated with the forebrain, midbrain, hippocampus and striatum. GFAP mRNA in the non-ischemic cerebellum however, remained expressed at constitutively low levels. Immunoblot analysis with anti-GFAP antibodies demonstrated a 2- to 3-fold increase in the protein after 24 and 48 h of reperfusion. Pretreatment with pentobarbital and 1-(5'-oxohexyl)-3-methyl-7-propyl xanthine (HWA 285), the drugs that have been shown to protect against ischemic damage, prevented the increase in GFAP mRNA in the cortex following ischemic injury. Forebrain ischemia also induced vimentin mRNA and protein quantities by 12 h of reperfusion in the cortex. The levels of c-fos and preproenkephalin mRNA increased rapidly within 1 h after ischemic injury, demonstrating a temporal difference in mRNA changes following ischemia. These results indicate that an increase in GFAP and vimentin, the two glial intermediate filament proteins in the area of the ischemic lesion may be associated with a glial response to injury.


Primary afferent stimulation acts through a 193 base pair promoter region to upregulate preproenkephalin expression in dorsal horn of transgenic mice. The expression of the principal opioid peptide gene, preproenkephalin A, is exquisitely regulated by primary afferent inputs to the spinal and medullary dorsal horns. This regulated expression in response to neural synaptic activity has been referred to as trans-synaptic regulation. To define which DNA regions could mediate this trans-synaptic regulation, transgenic 'HEC' mice whose genomes include 193 bp of the human preproenkephalin A promoter fused to a chloramphenicol acetyltransferase (CAT) reporter gene were studied. Mice received unilateral electrical stimulation of the trigeminal ganglion or adjuvant injection into the hindpaw, stimuli known to regulate dorsal horn proenkephalin expression in vivo. CAT activity conferred by this promoter displayed trans-synaptic upregulation with both stimuli. Although the level of the upregulation was 2- to 3-fold higher than the change in the wild type gene, several features of this induction paralleled aspects of the behavior of the wild-type gene: the rapidity of responses to trigeminal ganglion stimulation, the stimulation intensity dependence of responses to trigeminal ganglion stimulation and the time course of upregulation noted following adjuvant injection. Regulatory proteins binding to this restricted promoter region are thus likely to mediate aspects of dorsal horn enkephalin regulation by pain and other somatic stimuli.


Positive and negative feedback regulation of choline acetyltransferase mRNA levels in Drosophila: a study using temperature-sensitive mutants and embryo cell cultures. Steady state levels of the mRNA coding for the neurotransmitter biosynthetic enzyme, acetylCoA-choline-O-acetyltransferase (ChAT, EC 2.3.1.6), were measured in wild type Drosophila and two temperature-sensitive mutants (Chats1 and Chats2) using the RNase protection method. At a permissive temperature the relative amounts of ChAT mRNA were: wild type: Chats1:Chats2 = 1:2.09 (+/- 0.39):3.37 (+/- 0.57) (mean +/- S.E.M.) indicating that mutant flies may compensate, for making a thermolabile form of enzyme, by producing and/or maintaining higher levels of ChAT mRNA. At a restrictive temperature the ChAT mRNA levels decreased in both mutants and increased in wild type flies. The regulatory mechanism(s) responsible for increasing ChAT mRNA in wild type flies appears to have failed in the mutants at high temperature. Steady state mRNA levels were also measured in embryonic cell cultures prepared from wild type embryos. Cultures grown in the presence of two pharmacologic agents (carbamylcholine and d-tubocurarine) which should interfere with cholinergic neurotransmission, showed less mRNA resulting from a decrease in levels of ChAT gene transcription. Our results imply that neurotransmission and the rate of neurotransmitter biosynthetic enzyme gene transcription are coupled for the cholinergic system in Drosophila.


Reduction of vitamin D hormone receptor mRNA levels in Alzheimer as compared to Huntington hippocampus: correlation with calbindin-28k mRNA levels. Receptors for vitamin D hormone (VDR) and the calcium binding protein, calbindin-28k, have been localized in many tissues, including brain. In brain, VDR and calbindin-28k were reported to colocalize in hippocampal CA1 cells. We have shown that mRNA pool size for calbindin-28k was reduced, on average, by 35% in Alzheimer hippocampal CA1 cells, as compared to Huntington control (manuscript in preparation). In the present study, in situ hybridization with tritiated antisense RNA probes was used to examine VDR expression in paired Alzheimer and Huntington brain tissue. Message levels for VDR were reduced, on average, by 34% and 31%, respectively, in Alzheimer hippocampal CA1 and CA2 pyramidal cells, as compared to Huntington control. However, VDR message levels were not significantly different from control in Alzheimer temporal cortex or cerebellum. There was no correlation between VDR message levels and brain weight, autopsy interval, patient age or the extent of neurofibrillary degeneration. Instead, VDR mRNA pool size in hippocampal CA1 cells correlated significantly with calbindin-28k message levels (r = 0.52, P less than 0.001). Decreased message levels for VDR and calbindin-28k in these cells were due to an increased percentage of cells expressing lower message levels for these proteins. These results show that in Alzheimer hippocampal CA1 cells, VDR mRNA pool size is downregulated and that this downregulation may play a role in the reduction of calbindin-28k expression.


Calbindin-D28K-containing neurons in animal models of neurodegeneration: possible protection from excitotoxicity. Brain levels of the calcium binding protein Calbindin-D28K (CaBP28K) and CaBP28K mRNA were measured for various animal models of neurodegenerative diseases (MPTP-treated C57BL/6J mice and Sprague-Dawley rats receiving striatal/intraperitoneal kainic acid or quinolinic acid into the nucleus basalis magnocellularis). Brain areas were tested (radioimmunoassay, Western blot, slot blot, and Northern blot) for levels of CaBP28K and CaBP28K mRNA. The various models did not exhibit any changes in protein or mRNA levels from the controls, suggesting that CaBP28K-containing neurons were not lost after exposure to these neurotoxins. Immunocytochemical characterization of the substantia nigra of the MPTP-treated mice revealed that there was significant dopaminergic cell loss in this brain area after MPTP treatment. The majority of dopaminergic neurons that degenerated did not contain CaBP28K. The small percentage of surviving neurons were CaBP28K-positive. These results suggest that the presence of CaBP28K may protect neurons from calcium-mediated neurotoxicity.


Activation of the zinc finger encoding gene krox-20 in adult rat brain: comparison with zif268. Zif268 and krox-20 are transcription regulatory factors that contain highly homologous zinc finger DNA-binding domains. Recent studies have demonstrated that zif268 expression is rapidly regulated in brain by neuronal stimulation. We now report that, like zif268, krox-20 is rapidly and transiently activated by electroconvulsive shock treatment (ECT), D1 dopamine receptor activation, and opiate withdrawal. These studies indicate that, as found for the leucine zipper family of transcription factors, multiple members of the zinc finger family of transcription factors are induced by neuronal stimulation.


GAP-43 mRNA localization in the rat hippocampus CA3 field. Gene expression of the axonal growth-associated protein, GAP-43, has been studied in the adult rat brain by in situ hybridization histochemistry. This protein is synthesized at high levels in neuronal somata in immature and regenerating neurons, but after establishment of mature synaptic relations its synthesis generally declines sharply, thus providing a marker denoting propensity for exhibiting synaptic plasticity. Detailed examination of the distribution of mRNA for GAP-43 in rat hippocampus is selectively and robustly expressed in the pyramidal neurons of field CA3 and, to a lesser extent, the polymorph neurons of the hilus of the dentate gyrus. Additional hippocampal regions of moderate expression include the tenia tecta and the subicular and entorhinal fields, but CA1 and CA2 are strikingly lower in signal. The significance of this pattern of localization is considered in the context of the phosphorylation of GAP-43 and its role in influencing synaptic events underlying the establishment and maintenance of long-term potentiation and plasticity in the hippocampus.


Expression of histone H3 cell cycle-related gene, vimentin and MYC genes in pediatric brain tumors. A preliminary analysis showing the different malignant cell growth potential. Eleven pediatric brain tumors were studied for the histone H3, Vimentin and MYC gene expression. H3, an S phase cell cycle-related gene (ccr), was found prevalently expressed in tumors with a high mitotic index (MI). Vimentin gene, which contributes to maintaining the cell structure but is also demonstrated to be an early responder gene to growth stimulation was found variously expressed. The different expression of Vimentin gene in the examined samples suggests the active proliferation of the tumor cells. Analysis of MYC gene expression was found increased only in a mesenchymal chondrosarcoma while in other samples MYC mRNA was undetectable. Medulloblastoma, chondrosarcoma, and choroid plexus carcinoma have high S phase H3 gene expression associated with a high MI. Differently an astrocytoma shows a low MI associated with high H3 gene expression. This first preliminary report of H3, Vimentin and MYC gene expression in brain tumors demonstrates that malignant cells are characterized by a different gene expression and different growth potentials.


Phosphorus-31 magnetic resonance imaging of hydroxyapatite: a model for bone imaging. One-dimensional 31P nuclear magnetic resonance images (projections) of synthetic calcium hydroxyapatite, Ca10(OH)2(PO4)6, have been obtained for samples on the order of 0.5 to 1.0 cm in linear extent at 7.4 T magnetic field strength. Because of the solid state nature of these samples, short 31P spin-spin relaxation times under 1 ms occur, necessitating echo times of 1 ms and phase-encoding magnetic field gradient pulses shorter than 500 microseconds. Optimal projection quality and shortest acquisition times result from pulsed gradient phase-encoding of the spatial dimension, using a compensating gradient pulse to cancel the distorting effects of gradient waveform transients. The exceedingly long 31P spin-lattice relaxation times could lead to potentially intolerable image acquisition times; these have been reduced with a flipback pulse technique. In addition to holding great potential as a novel research tool in the study of biomineralization of those organisms containing calcium phosphate solid phases, these methods should be of general utility in the multinuclear imaging of a wide variety of solids of interest in biophysics and materials science.


Silicosis among pottery workers--New Jersey. In March 1985, two cases of silicosis in former employees of a sanitary-ware pottery (i.e., a manufacturer of china plumbing fixtures) were identified from death certificates by the New Jersey State Department of Health (NJSDH). A site visit to the pottery in January 1987 revealed potential overexposure of employees to crystalline silica throughout the plant. This report summarizes the investigation of employee exposure to silica.


Surveillance of congenital cytomegalovirus disease, 1990-1991. Collaborating Registry Group. In January 1990, a registry was initiated for surveillance of infants with the often severe symptoms of congenital cytomegalovirus (CMV) disease. In the first 2 years, 100 cases were reported to the registry. Petechiae, the most commonly noted clinical sign, were reported for approximately 50% of infants, usually accompanied by hepatomegaly and splenomegaly. Of the various severe neurologic conditions that can result from congenital CMV infection, the most frequent was intracranial calcifications, which were noted in 43% of the cases. The most common laboratory abnormality was low platelet count, which was observed in 52% of the cases. Infants with severe neurologic damage were about twice as likely as infants with less severe damage to have most other clinical signs and laboratory abnormalities. Databases will be developed to facilitate comparisons among symptomatically infected infants and asymptomatically infected as well as noninfected infants.


Localization of pancreatic endocrine tumors by endoscopic ultrasonography. After a pancreatic endocrine tumor has been diagnosed on the basis of clinical signs and the results of laboratory tests, localization of the tumor by the usual imaging procedures fails in as many as 40 to 60 percent of patients. Endoscopic ultrasonography, a sensitive test for small carcinomas of the pancreas, might also be useful in patients with endocrine tumors of the pancreas that cannot be localized by conventional methods. We studied 37 patients later shown to have 39 endocrine tumors of the pancreas who had negative results on transabdominal ultrasonography and CT. All the patients underwent endoscopic ultrasonography, and 22 also underwent selective angiography. All the tumors were confirmed by surgical excision and immunohistologic examination; they consisted of 31 insulinomas, 7 gastrinomas, and 1 glucagonoma, 0.5 to 2.5 cm (mean, 1.4 cm) in diameter. All but one of the patients were cured of their disease, as ascertained by at least six months of clinical and laboratory follow-up. Using endoscopic ultrasonography, we were able to localize 32 of the 39 tumors (sensitivity, 82 percent); no tumor was incorrectly localized. The size of the tumors was very similar (within 2 mm) to that predicted by endoscopic ultrasonography. Among the 22 patients who underwent both angiography and endoscopic ultrasonography, ultrasonography was significantly more sensitive than angiography for tumor localization (sensitivity, 82 percent vs. 27 percent). Among 19 control patients without pancreatic endocrine tumors, endoscopic ultrasonography was negative in 18 (specificity, 95 percent). Endoscopic ultrasonography is a highly sensitive and specific procedure for the localization of pancreatic endocrine tumors. It should be considered for the preoperative localization of such tumors once the clinical and laboratory diagnosis has been established.


Proteolysis, proteasomes and antigen presentation. Proteins presented to the immune system must first be cleaved to small peptides by intracellular proteinases. Proteasomes are proteolytic complexes that degrade cytosolic and nuclear proteins. These particles have been implicated in ATP-ubiquitin-dependent proteolysis and in the processing of intracellular antigens for cytolytic immune responses.


Regulation of deactivation of photoreceptor G protein by its target enzyme and cGMP. The photoreceptor G protein, transducin, is one of the class of heterotrimeric G proteins that mediates between membrane receptors and intracellular enzymes or ion channels. Light-activated rhodopsin catalyses the exchange of GDP for GTP on multiple transducin molecules. Activated transducin then stimulates cyclic GMP phosphodiesterase by releasing an inhibitory action of the phosphodiesterase gamma-subunits. This leads to a decrease in cGMP levels in the rod, and closure of plasma membrane cationic channels gated by cGMP. In this and other systems, turn-off of the response requires the GTP bound to G protein to be hydrolysed by an intrinsic GTPase activity. Here we report that the interaction of transducin with cGMP phosphodiesterase, specifically with its gamma-subunits, accelerates GTPase activity by several fold. Thus the gamma-subunits of the phosphodiesterase serve a function analogous to the GTPase-activating proteins that regulate the class of small GTP-binding proteins. The acceleration can be partially suppressed by cGMP, most probably through the non-catalytic cGMP-binding sites of phosphodiesterase alpha and beta-subunits. This cGMP regulation may function in light-adaptation of the photo-response as a negative feedback that decreases the lifetime of activated cGMP phosphodiesterase as light causes decreases in cytoplasmic cGMP.


[Non-neoplastic Lambert-Eaton syndrome. A frequently missed diagnosis?]. Three cases of non-paraneoplastic Lambert-Eaton syndrome are presented. There remains no indication of neoplasm after illnesses of nine years, 36 months and 20 months respectively. In one case, the syndrome became manifest during chloroquine medication. The cause of the proximal lower limb weakness had not been recognized during previous hospital stays, although in retrospect indications of a Lambert-Eaton syndrome were found in the electrophysiological tracings. The conclusive diagnosis was made electrophysiologically. The leading sign was an abnormally low compound muscle action potential evoked by supramaximal nerve stimulation. Furthermore, the characteristic increase of amplitude of the compound muscle potential after maximal voluntary contraction or at high frequency stimulation (20 Hz) could be demonstrated. It is concluded that the non-paraneoplastic Lambert-Eaton myasthenic syndrome may frequently remain unrecognized.


Ontogeny and characterization of [125I]Bolton Hunter-eledoisin binding sites in rat spinal cord by quantitative autoradiography. The distribution and characteristics of [125I]Bolton Hunter-eledoisin binding sites in rat lumbar spinal cord were studied during postnatal development by in vitro receptor autoradiography. At three, six and 10 days of age, specific [125I]eledoisin binding was distributed throughout the dorsal and ventral horns of the spinal cord. In contrast, from day 24 onwards, specific binding of [125I]eledoisin was confined to superficial layers of the dorsal horn, with negligible amounts of specific binding in the ventral horn. [125I]Eledoisin binding to neonatal (three day) and adult (eight to 12 weeks) spinal cord sections was characterized using tachykinin agonists. In both dorsal and ventral horns of neonatal spinal cord, the rank order of potency of agonists indicated that the majority (64%) of specific [125I]eledoisin binding was to neurokinin-3 binding sites. The identity of the non-neurokinin-3 sites labelled by [125I]eledoisin remains to be determined. In adult rat spinal cord, [125I]eledoisin appeared to bind exclusively to neurokinin-3 binding sites. These results suggest that major changes take place in the localization of neurokinin-3 receptors during postnatal ontogeny of the rat spinal cord. These changes may reflect an important role for tachykinins in neuronal plasticity of the developing spinal cord.


Monoclonal antibodies for ultrastructural visualization of L-baclofen-sensitive GABAB receptor sites. Monoclonal antibodies were raised against the L-enantiomer of baclofen conjugated by glutaraldehyde to keyhole limpet hemocyanin. Hybridoma clones were selected for their stability and their production of high titers of antibodies directed against the p-chlorophenyl moiety of the L-baclofen molecule. The chosen antibody showed no cross-reactivity with conjugates of GABA and other neurotransmitters to human or bovine serum albumin. Specificity was further confirmed by the ability of L-baclofen-HCl to inhibit the binding of the antibody to L-baclofen-bovine serum albumin conjugate. Immunocytochemical studies were conducted on brain tissue from rats and monkeys injected with baclofen to localize baclofen-sensitive GABAB receptor sites. In these animals, the molecular layer of cerebellar cortex was clearly immunostained and the granular layer showed only some pale immunoreactivity. Ultrastructural observations were conducted in cerebellar cortex, as well as in the substantia nigra and the vestibular nuclei. Discrete labeling of neuronal profiles was observed in these structures, and both immunoperoxidase and colloidal gold methods were employed successfully. Material from saline-injected control animals showed no immunoreactivity at both light and electron microscopic levels. We conclude that the anti-L-baclofen antibody preferentially recognizes the p-chlorophenyl moiety of the baclofen molecule. Antibodies of such specificity are useful tools for the ultrastructural localization of baclofen-sensitive GABAB receptor sites. In general, antibodies directed against accessible moieties of specific neuroactive substances may serve as valuable markers for their sites of action.


Excitatory amino acid binding sites in the basal ganglia of the rat: a quantitative autoradiographic study. Quantitative receptor autoradiography was used to determine the distribution of excitatory amino acid binding sites in the basal ganglia of rat brain. alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid, N-methyl-D-aspartate, kainate, quisqualate-sensitive metabotropic and non-N-methyl-D-aspartate, non-kainate, non-quisqualate glutamate binding sites had their highest density in striatum, nucleus accumbens, and olfactory tubercle. Kainate binding was higher in the lateral striatum but there was no medial-lateral striatal gradient for other binding sites. N-Methyl-D-aspartate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding sites were most dense in the nucleus accumbens and olfactory tubercle. There was no dorsal-ventral gradient within the striatal complex for the other binding sites. Other regions of the basal ganglia had lower densities of ligand binding. To compare binding site density within non-striatal regions, binding for each ligand was normalized to the striatal binding density. When compared to the striatal complex, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and metabotropic binding sites had higher relative density in the globus pallidus, ventral pallidum, and subthalamic nucleus than other binding sites. Metabotropic binding also had a high relative density in the substantia nigra. Non-N-methyl-D-aspartate, non-kainate, non-quisqualate glutamate binding sites had a high relative density in globus pallidus, ventral pallidum, and substantia nigra. N-Methyl-D-aspartate binding sites had a low relative density in pallidum, subthalamic nucleus, substantia nigra and ventral tegmental area. Our data indicate heterogeneous distribution of excitatory amino acid binding sites within rat basal ganglia and suggest that the character of excitatory amino acid-mediated neurotransmission within the basal ganglia is also heterogeneous.


Successful pregnancies in women on renal replacement therapy: report from the EDTA Registry. This study reports the geographical incidence of successful pregnancies in women on renal replacement therapy (RRT) and related information on gestation and clinical status of newborns. The impact of successful pregnancy on graft function was assessed by means of a retrospective case-control study. Since 1977 special questionnaires have been sent to each dialysis and transplant centre which reported babies born to mothers on RRT on the yearly centre questionnaire. After 10 years of data collection, a total of 490 pregnancies and 500 babies were available for analysis. A percentage of 88.4 of the babies were born to mothers with a functioning graft, 11.2% to mothers on chronic haemodialysis, and the remaining 0.4% to mothers on CAPD. Almost 50% of all successful pregnancies were reported from the UK. The number of successful pregnancies increased steadily and in parallel with the increasing number of females of childbearing age with a functioning renal transplant. The majority of mothers delivered at age 24-32. For transplanted mothers delivery occurred most commonly during the 3rd and 4th year after successful transplantation. In approximately 85% of cases the duration of pregnancy was shorter than the lower 10th percentile of normal. Birthweight was reduced in accordance with gestational age. Newborn mortality was 1.8%. Fifty-three mothers with a successful pregnancy in 1984-1987 were computer matched with controls according to a number of criteria. The serum creatinine concentration recorded in coded form at the end of each year on the individual EDTA patient questionnaire was used to assess changes in graft function.(ABSTRACT TRUNCATED AT 250 WORDS)


Functional endothelin 1 receptors on human glomerular podocytes and mesangial cells. To determine if endothelin 1 (Et1) receptors are present in human glomeruli, and which glomerular cells possess these receptors, 125I Et1 binding to isolated glomeruli and cultured glomerular mesangial and epithelial cells was studied. The latter were identified as podocytes. We demonstrated that Et1 binds specifically and reversibly to isolated human glomeruli and to cultured glomerular mesangial and epithelial cells. Scatchard analysis of competitive inhibition of 125I Et1 binding gave the following results (m +/- SEM, n = 3): isolated glomeruli, Kd = 4.2 +/- 2.1 x 10(-10) M, Bmax = 8.1 +/- 1.2 x 10(10) sites/mg protein; mesangial cells, Kd = 5.2 +/- 1.5 x 10(-10) M, Bmax = 1.87 +/- 0.49 x 10(4) sites/cell; epithelial cells, Kd = 7.2 +/- 1.5 x 10(-10) M, Bmax = 2.46 +/- 0.15 x 10(4) sites/cell. These receptors seem to be functional, since in both mesangial and epithelial cells Et1 induces a rapid and transient increase in intracellular [Ca2+]i. All these results indicate that Et1 may regulate glomerular filtration rate through an autocrine-paracrine pathway on mesangial cells and on podocytes.


Presence of circulating antibodies against brush border antigens (Fx1A) in a patient with membranous nephropathy and bilateral pyeloureteral stenosis. Comparison with idiopathic membranous nephropathy. In a patient with membranous nephropathy and bilateral pyeloureteral stenosis with hydronephrosis, we examined the possibility that an increase in the intratubular pressure could facilitate the passage of the Fx1A antigens to the circulation. Elevated serum anti-Fx1A antibodies were detected in this particular patient by ELISA on three occasions during the disease follow-up, even though he was in clinical remission. These antibodies reacted in vitro with the tubular brush border of a normal human kidney. The anti-Fx1A antibodies isolated from the patient's sera by affinity chromatography competed with the rabbit anti-Fx1A antisera binding to plates coated with human Fx1A antigen. In immunoblotting studies the isolated specific IgG antibodies from that patient reacted with a 180 kDa antigen of the human Fx1A and with less intensity with 75 kDa and 50-55 kDa polypeptides. In none of 12 patients with idiopathic membranous nephropathy could the circulating anti-Fx1A antibodies be demonstrated. On the whole, this particular case suggests that on some occasions increased intratubular pressure could cause the release of Fx1A antigens, facilitating an autologous immunocomplex nephritis. These antigens, by contrast, do not seem to play any role in most cases of membranous nephropathy in man.


Endothelin in chronic renal failure.

Endothelin in chronic renal failure. The aims of the present study were to determine plasma endothelin (ET) in chronically uraemic patients, the renal clearance of endogenous ET in normal dog and man, and the effect of acute volaemic expansion on ET. The mean plasma ET concentration in haemodialysis patients was 57.5 +/- 5 pg/ml before haemodialysis and remained unchanged at 52.5 +/- 5 pg/ml after haemodialysis. They were thus significantly elevated both before and after haemodialysis (P less than 0.01) compared with plasma ET in normal subjects of 20.8 +/- 0.8 pg/ml. There was no evidence of ET clearance across the cuprophane membrane of the dialyser. Resting plasma ET values in the 15 non-dialysed uraemic patients ranged between 20 and 52.5 pg/ml (mean 38.2 +/- 2.3 pg/ml), significantly greater than those observed in controls (P less than 0.01). In CAPD patients, plasma ET was also significantly (P less than 0.01), elevated (63 +/- 10 pg/ml) when compared to controls, and similar to those observed in patients before haemodialysis. In dogs, mean ET did not diminish between the aorta and the renal vein (28.1 +/- 1 versus 28.4 +/- 2 pg/ml). In man mean ET did not significantly decline between the renal artery and the renal vein (17 +/- 3 to 13 +/- 0.8 pg/ml). In the seven healthy subjects who received 2000 ml of isotonic saline intravenously ET remained unchanged (24 +/- 2; 23 +/- 1 and 23 +/- 2 pg/ml before and 1 and 2 h after starting hydration respectively). We have thus shown that plasma ET is elevated in patients with chronic renal failure especially those on dialysis and CAPD.(ABSTRACT TRUNCATED AT 250 WORDS)


Hormonal changes in patients with severe chronic congestive heart failure treated by ultrafiltration. Plasma atrial natriuretic peptide (ANP), antidiuretic hormone (ADH), plasma renin activity (PRA), and circulatory haemodynamics were studied in five patients with chronic congestive heart failure undergoing ultrafiltration on two consecutive days. The patients were in the New York Heart Association class IV, and were considered candidates for heart transplantation. A mean of 3.3 +/- 0.5 litres of fluid was removed during each ultrafiltration. Plasma ANP concentration remained unchanged during ultrafiltration: 369 +/- 151 pg/ml at start and 316 +/- 116 pg/ml at the end, while plasma ADH concentration and PRA increased from 5.1 +/- 2.1 to 7.5 +/- 3.4 pg/ml (P less than 0.02), and 5.9 +/- 3.0 to 7.7 +/- 3.2 ng/ml (P less than 0.03) respectively (n = 10). After treatment, plasma ADH and PRA declined to baseline values within 1 h. Pulmonary artery, pulmonary capillary wedge, and right atrial pressures decreased significantly, while blood pressure and heart rate remained constant during ultrafiltration. A volume of 3.3 +/- 0.5 litres of fluid was removed, and caused an increase in colloid osmotic pressure from 22.0 +/- 3.0 to 33.7 +/- 3.9 mmHg (P less than 0.02). It was unexpected that plasma ANP concentration did not decline. Due to long-standing severe heart failure the atrial wall may have lost some of its elastic properties, resulting in less ability to adapt to reduced filling pressures. Accordingly, atrial wall stretch remained unchanged, explaining the constant ANP levels. Ultrafiltration treatment caused an increased responsiveness to diuretic therapy, and four patients survived long enough to receive heart transplants.


Responsiveness to recombinant erythropoietin therapy in end-stage renal disease. An analysis of the predictive value of several biological measurements, including circulating erythroid progenitors. In end-stage renal disease (ESRD), the human recombinant erythropoietin doses required to keep haemoglobin in the target range may vary considerably between patients. Previous studies have failed to find any predictive factor of the response. We thus performed the present investigation in 30 ESRD patients to discover if the haematological response to human recombinant erythropoietin (rHuEpo) was related to the results of circulating erythroid progenitor cultures. Peripheral erythroid burst forming units (BFU-E) were cultured in a plasma clot system in the absence or in the presence of autologous serum just before starting rHuEpo therapy. The results showed a higher BFU-E number in ESRD patients than in controls and a stimulatory effect of autologous serum in both patients and controls. Comparison between culture results and haematological response yielded positive correlation between the BFU-E number and the haemoglobin increase during the first month of treatment, and negative correlation between the increase of BFU-E numbers during the first week of therapy and the rHuEpo doses required for a long-term response. We thus conclude that in ESRD patients the individual response to rHuEpo is linked to the numbers of circulating BFU-E.


Cardiac arrhythmias and electrolyte changes during haemodialysis. Cardiac arrhythmias are a frequent event in chronic haemodialysis patients, and their pathogenesis is still poorly understood. We evaluated plasma K+ (PK), intraerythrocytic K+ (EK) and acid-base changes during haemodialysis in six patients with frequent arrhythmias (A-pts), and in six (used as controls) nonarrhythmic dialysis patients (C-pts). PK decreased significantly (P less than 0.01) during haemodialysis in both groups: A-pts (pre HD: 4.81 +/- 0.52 mM; 1st hour: 3.66 +/- 0.44; end HD: 3.17 +/- 0.38) and C-pts (4.75 +/- 0.80; 3.71 +/- 0.32 and 3.18 +/- 0.18 respectively) without any significant difference at any time between the two groups. Predialysis arterial pH and HCO3 were similar in A-pts (7.33 +/- 0.07 and 22.1 +/- 4.5 mM) and C-pts (7.29 +/- 0.04 and 19.7 +/- 2.6 mM) but an apparently better correction of acidosis within the treatment was seen in A-pts (arterial pH 1st hour: 7.38 +/- 0.07; end HD: 7.39 +/- 0.07) than C-pts (1st hour: 7.31 +/- 0.02, P less than 0.05 versus A-pts; end HD: 7.33 +/- 0.03, P less than 0.05 versus A-pts). EK was significantly (P less than 0.01) greater at all times in C-pts (pre HD: 90.6 +/- 15.7 mmol/l RBC; 1st hour: 93.3 +/- 11.7; end HD 96.6 +/- 10.7) than A-pts (72.1 +/- 9.0; 77.2 +/- 3.7 and 79.3 +/- 8.4, respectively). We conclude that haemodialysis patients with arrhythmias have a decreased intraerythrocytic K content in comparison with other patients despite similar PK values; this finding might constitute a predisposing factor for arrhythmias.


Response to vaccination against tetanus in chronic haemodialysed patients. We studied the response to vaccination against tetanus and the changes in the antibody titers 6 months after this vaccination in 66 haemodialysed patients with chronic renal failure. We also investigated the factors that may affect the quality of this immune response. After the booster injection 96.5% of patients had antitetanus antibody titres considered to be protective (0.06 HU/ml). However, the titre of these antibodies rapidly declined and after 6 months, only 62% of the haemodialysed patients had a titre greater than 0.06 HU/ml. Among the different factors considered, only age significantly impaired or reduced the immune response. In addition, the acquisition of protection against tetanus was independent of the response to vaccination against hepatitis B in the same subjects. This study showed the efficacy and safety of vaccination against tetanus in hemodialysed patients, though the antibody titres should be assayed several months after vaccination to confirm the persistence of immunization.


Endotoxin transfer through dialysis membranes: small- versus large-pore membranes. In this in-vivo study, dialysate and serum endotoxin was evaluated before and after haemodialysis with small-pore (PS400) and large-pore (PS600) polysulphone dialysers, and before and after haemodiafiltration with the PS600 filter. The source of the endotoxin was the presence in dialysate of Pseudomonads at a concentration of 10(3)-10(4) CFU/ml. Endotoxin was measured by a modified chromogenic limulus amoebocyte lysate (LAL) assay. In spite of dialysate endotoxin concentrations greater than 100 pg/ml, no changes in pre- versus posttreatment LAL reactivity were observed in PS400 dialysis and PS600 haemodiafiltration. In contrast, PS600 haemodialysis was related to an increase in serum LAL reactivity from 1.3 +/- 1.5 to 3.8 +/- 2.0 pg/ml (n = 15, P less than 0.01), and five patients (33.3%) showed a post-dialysis value in excess of 5 pg/ml. Our data are consistent with the absence of in-vivo endotoxin transfer during haemodialysis with small-pore dialyser membranes, and during haemodiafiltration with membranes with larger pores. An increase in LAL reactivity during haemodialysis with membranes with larger pores is, however, present, presumably due to the occurrence of backdiffusion/filtration with that specific strategy.


Cellulose-based haemodialysis membranes: biocompatibility and functional performance compared. Regenerated cellulose membranes are widely used in the treatment of renal failure. The presence of hydroxyl (OH) groups on the membrane surface plays an important role in initiating complement activation and also influences thrombogenicity. The OH groups may be masked or reduced by alteration of the manufacturing process of the membrane. We have undertaken a clinical study of four cellulose-based membranes (Cuprophan, Hemophan, cellulose acetate, and cellulose triacetate) in which the hydroxyl groups of the membrane have been replaced and the magnitude of replacement has varied from less than 1% to greater than 80%, to assess the role that these modifications play in functional performance, biocompatibility (neutropenia, leukocyte activation, anaphylatoxin generation, and hypoxaemia). Our findings indicate that there does not appear to be a straightforward correlation between the numbers of hydroxyl groups replaced and modification of biocompatibility, suggesting that not all hydroxyl groups behave in a similar way.


High incidence of post-transplant diabetes mellitus in a single-centre study. Twelve of 29 Saudi patients (41.4%) developed diabetes mellitus following renal transplantation. Post-transplant diabetes mellitus occurred within the first 2 months in eight patients; two others presented with diabetic ketoacidosis associated with severe infections. The diabetic and non-diabetic patients had received similar doses of prednisolone and cyclosporin (CsA) during the initial 2 months post-transplantation, and their mean CsA blood values at 3 months were not significantly different. Increasing patient age (over 40 years), but not sex, donor source, or body mass index, was associated with an increased risk for developing diabetes mellitus. Post-transplant diabetes mellitus was controlled with oral hypoglycaemic agents in most patients, but one-third required insulin. Patients who developed diabetes had significantly decreased mean creatinine clearance/1.73 m2 at a mean graft age of 3.4 years (P less than 0.001). Diabetes mellitus after transplantation may be more common among Saudi patients than elsewhere, especially those aged over 40 years. It develops rapidly, may present with ketosis, and is associated with graft dysfunction.


Ganciclovir effectively treats cytomegalovirus disease after solid-organ transplantation, even during rejection treatment. Ganciclovir (DHPG) was used to treat eight patients with severe cytomegalovirus (CMV) disease after renal transplantation. Four had generalized disease, one had severe recurrent gastrointestinal bleeding and three had interstitial pneumonia. All patients responded to treatment, symptoms disappearing within 7 days (range: 3-22 days). Leukocytopenia and confusion were the main side-effects, both occurring in three patients. Encouraged by the effectiveness and the mild side-effects of ganciclovir we enlarged the indication for treatment. Five patients with CMV disease and concomitant rejection were treated with ganciclovir together with antirejection therapy. Four patients received methylprednisolone, one patient anti thymocyte globulin. CMV-related symptoms disappeared in all five patients. The only side-effect observed was a mild leukocytopenia in one patient. Renal function was preserved in three of the five patients. The virological response to the drug was also evaluated. Twelve patients had positive cultures in blood and/or urine before ganciclovir was started. In three patients the cultures became negative during follow-up. In eight patients cultures remained positive after ganciclovir treatment although in seven of these CMV had initially been absent from blood and/or urine. We conclude that ganciclovir treatment is clinically effective in severe CMV disease and that treatment with ganciclovir makes antirejection therapy during CMV disease possible.


Transmission of herpes simplex virus infection via lacrimal canaliculi. We analyzed the differentiation of two strains isolated from the conjunctiva and rhinorrhea of a patient with herpetic keratitis by the restriction endonuclease digestion method of herpes simplex virus (HSV) DNA. As a result two strains were identified as the same one. This result suggests that HSV contained in tears flows into the nasal cavity via the lacrimal canaliculi.


Biology and molecular aspects of herpes simplex and varicella-zoster virus infections. The herpes simplex and varicella-zoster viruses are members of the subfamily alpha herpesviruses with specific properties of the virion and with the capacity to establish latent infections in humans. The genome of each of these viruses has been determined with an estimate of the number of genes and proteins encoded. The biology and molecular events of the herpes simplex virus productive and latent infection have been detailed with the use of both in vitro and in vivo model systems. The neuron is the site of latency in the ganglia with a limited transcription of genes expressed during the latent period. The specific molecular regulation of latency and reactivation are not well established. There are co-cultivation, electron microscopy, and biochemical studies that support the concept of corneal latency, although this has not been proven conclusively. Details about the varicella-zoster virus biology and molecular events are not as well advanced since animal models have been lacking. The biology of the productive infection (varicella) is different from herpes simplex virus infection since the portal of entry is the respiratory system. Data support the concept of the maintenance of latency within satellite cells in the ganglia rather than within neurons. There are multiple genes expressed during this latency. These features may explain the different clinical presentations and course of reactivation (zoster) compared with herpes simplex virus reactivation.


Exposed hydroxyapatite orbital implants. Report of six cases. Six patients with complications of primary or secondary hydroxyapatite implants were studied. Complications included socket infection and/or conjunctival dehiscence. Complications were detected during regular follow-up examinations, and various treatment approaches were used. The hydroxyapatite implant exposure occurred 4 to 6 weeks (mean, 4.5 weeks) after implantation. Three of the six implants were wrapped in preserved donor sclera before implantation. One of the implants showed wide exposure and chronic infection and was removed. In two cases, scleral patch grafts with a conjunctival pedicle graft were performed, resulting in successful coverage of the implant without further conjunctival dehiscence. In one of the patients, a Tenon's conjunctival flap was advanced to cover the defect, and was unsuccessful with the spicules of the hydroxyapatite eroding through the vascular flap after 1 month. Three of the patients demonstrate a persistent conjunctival epithelial defect. These three patients with chronically exposed hydroxyapatite have remained stable with follow-up intervals ranging from 8 to 12 months. Early exposure of hydroxyapatite orbital implants is a potential problem despite meticulous technique. Implant coverage is difficult, although chronic exposure seems to be tolerated often in the hydroxyapatite orbital implant without migration or extrusion.


The N-terminal and C-terminal domains of a receptor tyrosine phosphatase are associated by non-covalent linkage. We have cloned the rat homolog of the human leukocyte common antigen-related gene (LAR), which encodes a transmembrane receptor phosphotyrosine phosphatase, and raised antibodies against its protein product. We present evidence here for a processing event resulting in a two-chain structure of the mature receptor on the cell surface. The LAR protein is synthesized as a 190-kDa precursor which is subsequently cleaved into 145-kDa and 85-kDa fragments. The 145-kDa fragment, representing the amino terminus of the protein, is exclusively extracellular and is modified by N-linked glycosylation. The 85-kDa component, derived from the C-terminus of the protein, contains the transmembrane region and intracellular phosphotyrosine phosphatase domains. The two products, associated in a non-covalent manner, comprise the functional LAR cell-surface receptor.


Evidence for non-covalent clusters of the c-met proto-oncogene product. The met proto-oncogene is a member of the tyrosine kinase growth factor receptor family and is the receptor for hepatocyte growth factor (HGF) or scatter factor. The primary met product is a 150 kDa precursor that is glycosylated to generate a 170 kDa (p170met) proreceptor protein. The mature form of the receptor is generated by cleavage of p170met to yield a disulfide-linked 140 kDa beta-subunit (p140met) and a 45 kDa alpha-subunit (p45met). Both products are glycosylated. Under non-reducing conditions both p170met and the alpha, beta-disulfide-linked protein are detected as a 185 kDa product (p185met), but only alpha-beta heterodimeric p185met is cross-linked and rendered resistant to disulfide reduction with membrane-impermeable 6.4 A linker length cross-linking reagents. These data indicate that the p170 precursor is not on the cell surface. Cross-linking experiments using 12-A linker reagents yield multimeric forms of alpha-beta heterodimeric p185met greater than 500 kDa in size. These multimeric forms are produced in all cell lines tested regardless of the levels of protein expressed. These data suggest that alpha-beta heterodimeric p185met occurs in clusters or patches on the cell surface. Immunohistochemical analysis of met protein in the absence of ligand reveals a clustered staining pattern on the cell surface.


T-antigen mutant activities in vivo: roles of p53 and pRB binding in tumorigenesis of the choroid plexus. To study the mechanism by which SV40 large T antigen transforms cells under physiological conditions, we analysed several mutant forms of T antigen for their ability to induce cell proliferation and tumorigenesis in transgenic mice. These mutant proteins, which differ in their ability to form complexes with the tumor suppressors pRB and p53, were analysed under conditions in which wild-type T antigen induces choroid plexus papillomas as a result of uniform proliferation of the entire choroid plexus epithelium. The results presented here show that binding of T antigen to p53 is not required for induction of choroid plexus tumors. However, tumorigenesis does appear to require the binding of T antigen to pRB/p107. An additional activity, resident in the amino-terminal one-fifth of the protein, may also play a role. These experiments indicate the importance of whole-animal assays in determining the molecular basis of transformation, since each of these mutants possessed similar transformation phenotypes in culture but showed distinct phenotypes in the choroid plexus of the animal.


Pretreatment with gangliosides reduces abnormal nociceptive responses associated with a rodent peripheral mononeuropathy. A peripheral mononeuropathy was produced in adult male rats by placing loosely constrictive ligatures around the common sciatic nerve. As reported by others, this procedure reliably results in postoperative behavior indicative of hyperalgesia, allodynia, and potentially, spontaneous pain. In these experiments, thermal hyperalgesia was assessed by measuring foot-withdrawal latencies to radiant heat aimed at the plantar surface of rat hind paws. Behaviors potentially indicative of spontaneous pain were assessed by rating spontaneous hind paw guarding positions. Rats with sciatic nerve ligation were divided into 5 groups (n = 6/group). Three groups received injections (i.p.) of either 10, 20 or 40 mg/kg of cerebral ganglioside mixture, GA. The 4th group was injected with 10 mg/kg of the purified ganglioside GM1, and the 5th group received an equal volume of saline. All injections were given daily for 2 days before surgery, the day of surgery and 9 days after surgery. All animals were behaviorally assessed for 2 days prior to surgery, the day of surgery, as well as 1, 3, 5, 7, and 10 days afterwards. All 5 groups had significantly reduced latencies to hind paw withdrawal on the side ipsilateral to sciatic nerve ligation. However, these hyperalgesic responses were significantly attenuated in rats receiving GA or GM1 pretreatment. These data suggest that this animal model of peripheral neuropathic pain is sensitive to pharmacological manipulations useful for understanding mechanisms of neuropathic pain, including mechanisms related to excitotoxic processes. Such studies could lead to development of clinical approaches to treat this disorder.


Antinociceptive and motor effects of delta/mu and kappa/mu combinations of intrathecal opioid agonists. Interactions between selective opioid agonists acting at spinal mu-, delta-, and kappa-opioid receptors were evaluated by co-administering a low-antinociceptive dose of the selective delta-agonist, DPDPE, or the selective kappa-agonist, U50,488H, with sequentially increasing doses of the selective mu-agonist, DAMGO, intrathecally. Antinociceptive synergy (i.e., a more than additive antinociceptive effect) was observed with both combinations of opioid agonists tested. The demonstration of antinociceptive synergy suggests that the subtypes of spinal opioid receptors can act, at least in part, through a common neural circuit. Since our measure of antinociception, the Randall-Selitto paw-withdrawal test, is dependent on a normally functioning motor system, we also evaluated the effects of these same combinations of opioid peptides on motor coordination using a rotarod treadmill. A low-antinociceptive dose of DPDPE or U50,488H co-administered intrathecally, with sequentially increasing doses of DAMGO, did not worsen the decrement in rotarod performance observed with the same doses of DAMGO administered as a single agent. In fact, the low-antinociceptive dose of DPDPE significantly attenuated the decrease in rotarod performance produced when the same dose of DAMGO was administered as a single agent. The results of this study suggest that intrathecal combinations of selective mu- with both delta- or kappa-selective opioid agonists can produce antinociceptive synergy without producing an increase in motor side effects.(ABSTRACT TRUNCATED AT 250 WORDS)


Prolonged potentiation of transmission through a withdrawal reflex pathway after noxious stimulation of the heel in the rabbit. The sural-gastrocnemius reflex of the spinalized rabbit was potentiated to an average of 3-6 times control levels after the application of noxious mechanical, thermal or chemical stimuli to the skin of the heel. Facilitation of the reflex was maximal within 1 min of the noxious stimulus, and in many cases persisted for more than 1 h. Prolonged increases in the excitability of the sural-gastrocnemius reflex were not seen after innocuous mechanical or thermal stimulation of the heel. Repetitive electrical stimulation of the sural nerve (100 shocks given at 0.5 Hz) caused persistent facilitation of the reflex when small myelinated A delta fibres or non-myelinated C-fibres were recruited by the conditioning stimulus. Such protracted increases in the excitability of the sural-gastrocnemius pathway would enhance the protective functions of this reflex. The mechanisms described here have probably evolved to provide a high level of reflex protection to the heel after tissue damage has occurred at that site.


Sensitizing effects of leukotriene B4 on intradental primary afferents. Previous studies have established that leukotriene B4 (LTB4) sensitizes cutaneous nociceptors. In this study the effects of LTB4 on spontaneous and stimulus-evoked nerve activity from primary afferents innervating the dentin of canines in adult cats were examined. LTB4 treatment (25 micrograms/ml) significantly enhanced stimulus-evoked intradental nerve activity (INA) for at least 20 min after the removal of the compound from the recording preparation. Teeth treated with LTB4 demonstrated enhanced spontaneous nerve activity following the removal of hypertonic saline used to evoked INA. These findings provide additional evidence that LTB4 is able to sensitize nociceptors and may be a long-lasting hyperalgesic factor which may contribute to pain of pulpal origin.


What is the legal 'standard of medical care' when there is no standard medical care? A survey of the use of home apnea monitoring by neonatology fellowship training programs in the United States. In treating a patient, a doctor is obliged to use the skill and care that is ordinarily used by reasonably well-qualified doctors in similar cases. In addition, the only way in which a juror may decide whether the defendant used the skill and care which the law required of him or her is from evidence presented by doctors called as expert witnesses (cf Illinois Pattern Jury Instructions). However, what should be done if expert opinions differ concerning the care that is "ordinarily used"? Home apnea monitoring (HAM) is prescribed at times for graduates of neonatal intensive care units despite the fact that indications for its use are not well established and efficacy is completely unknown. The authors attempted to determine standards for HAM as it is currently practiced in neonatology training programs. The primary teaching hospital for each of the 99 neonatology training programs in the United States was identified. Both the medical director (MD) and a neonatal intensive care unit nurse manager (RN) were asked about the use of HAM in their own nursery for four clinical vignettes. Each vignette depicted a 1000-g birth weight infant, currently 7 weeks old and ready for discharge. In three vignettes, the infant had demonstrated no apnea, mild apnea (resolved by 2 weeks of age), or moderate apnea (requiring theophylline therapy at discharge) during the hospital course. In the fourth vignette, the infant had no apnea but was to be discharged home with supplemental oxygen. For 67 of 99 training programs, paired responses of RN managers and MD directors were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)


Constitutive and enhanced expression from the CMV major IE promoter in a defective adenovirus vector. A defective adenovirus (Ad) type 5 E1- vector has been combined with the powerful constitutive cytomegalovirus (CMV) major immediate early (IE) promoter to produce a novel eukaryotic expression system. The Ad vector can replicate to high titres in 293 cells and then be used to infect a wide variety of non-permissive cell types. The Escherichia coli lacZ and CMV IE1 genes have been cloned to generate the Ad recombinants RAd35 and RAd31 respectively. In human fibroblasts infected with RAd35 beta-galactosidase (beta-gal) expression could be detected in virtually 100% of target cells, there was no detectable transcription from the Ad genome and extremely high levels of expression could be achieved with beta-gal representing the predominant cytoplasmic cellular protein. Additionally, a number of agents, including the CMV IE1 gene product (in RAd31) and forskolin, significantly enhanced expression from RAd35-infected human fibroblasts. Lower levels of constitutive beta-gal expression were obtained in RAd35-infected HeLa cells but again expression could be enhanced (up to 60 fold) by chemical inducing agents. Expression from the IE promoter in the Ad vector could be repressed by coinfection with CMV.


Site-directed and transposon-mediated mutagenesis with pfd-plasmids by electroporation of Erwinia amylovora and Escherichia coli cells. The suicide plasmid pfdA31-Tn5 was constructed to mutagenize Erwinia amylovora and Escherichia coli strains by electorporation. This vector carries the bacteriophage fd replication origin, a beta-lactamase gene and the transposon Tn5. For propagation the plasmid depends on host cells producing fd gene-2 protein. Electroporation of E.amylovora or E.coli cells with plasmid pfdA31-Tn5 yielded more than 10(4) transposition events per micrograms DNA. We have produced and characterized transposon mutants of E.amylovora affecting either galactose metabolism or the synthesis of the phytotoxin (L)-2,5-dihydrophenylalanine. A Tn5-insertion in a gene, involved in exopolysaccharide synthesis of E.amylovora strain Ea7/74, was subcloned into vector pfdA31 and used to mutagenize E.amylovora strain Ea1/79 by site-directed recombination.


The swi4+ gene of Schizosaccharomyces pombe encodes a homologue of mismatch repair enzymes. The swi4+ gene of Schizosaccharomyces pombe is involved in termination of copy-synthesis during mating-type switching. The gene was cloned by functional complementation of a swi4 mutant transformed with a genomic library. Determination of the nucleotide sequence revealed an open reading frame of 2979 nucleotides which is interrupted by a 68 bp long intron. The putative Swi4 protein shows homology to Duc-1 (human), Rep-3 (mouse), HexA (Streptococcus pneumoniae) and MutS (Salmonella typhimurium). The prokaryotic proteins are known as essential components involved in mismatch repair. A strain with a disrupted swi4+ gene was constructed and analysed with respect to the switching process. As in swi4 mutants duplications occur in the mating-type region of the swi4 (null) strain, reducing the efficiency of switching.


The 5'-flanking region of the mouse muscle nicotinic acetylcholine receptor beta subunit gene promotes expression in cultured muscle cells and is activated by MRF4, myogenin and myoD. The expression of the nicotinic acetylcholine receptor (AChR) in vertebrate striated muscle is regulated both during development by nerve-evoked muscle activity and by local factors released or associated with the nerve ending. The expression pattern of AChR is achieved by coordinate regulation of four embryonic subunit mRNAs, alpha, beta, gamma and delta. We have taken the approach of identifying the similarities and differences among cis-acting regulatory elements of AChR genes to gain a better understanding of these mechanisms. Thus, to begin to define DNA sequences necessary for the transcriptional regulation of the mouse beta AChR gene, we have analyzed its 5'-flanking region. Primer extension and RNAase protection analyses showed that transcription initiates at one major and two minor sites, all of which are close to the translational initiation site. Using plasmids in which segments of the 5'-flanking region were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, we have demonstrated that 150 bp of the 5'-flanking region is active in C2 myotubes but not C2 myoblasts or NIH3T3 fibroblasts. This region contains a putative binding site for myoD, and when linked to CAT was transactivated by the muscle regulatory factors myoD, myogenin, and MRF4. Thus, a 150 bp sequence of the beta-subunit gene contains information necessary for developmental specificity and responsiveness to myogenic factors.


Malignant renal tumors of childhood.

Malignant renal tumors of childhood. The prognosis in nephroblastoma (Wilms' tumor) has been improved considerably by treatment protocols combining surgery, chemotherapy, radiation therapy, and, in some clinical trials, pre-operative chemotherapy. Cure is now achieved in most patients. All clinical trials have employed treatment strategies tailored to the individual risk of the patient, including the histological subtype of the tumor. In the National Wilms' Tumor Study (NWTS) of the United States these subtypes have been divided into two groups of tumors according to their "favorable" or "unfavorable" histology. At the Kiel Pediatric Tumor Registry we have devised a system which distinguishes three groups of tumors classified according to prognosis. The first group includes tumors with a favorable prognosis, even if only surgery is performed. These comprise congenital mesoblastic nephroma (CMN) and cystic, partially differentiated nephroblastoma (CPDN). The second group consists of tumors posing an intermediate risk, such as typical nephroblastoma and its histological variants characterized by variations in the relative proportions of the histological components. Fetal rhabdomyomatous nephroblastoma (FRN) is also included in this group. The third group comprises tumors of high risk such as anaplastic nephroblastoma, clear cell sarcoma of the kidney (CCSK), and malignant rhabdoid tumor of the kidney (MRTK). Since histological diagnosis plays a crucial role in the assignment of a patient to a particular type of treatment protocol, knowledge of the histological appearance of the various tumor types both with and without preoperative treatment is of utmost importance.