Lidocaine block of human heart sodium channels expressed in Xenopus oocytes. The tertiary amine lidocaine is used clinically for preventing cardiac arrhythmias, and has been widely studied on mammalian tissue. Xenopus oocytes were used as an expression system to study the effect of lidocaine on a sodium (Na) channel, derived from a full-length human heart (hH1) cDNA clone. The concentration dependence of the lidocaine block of hH1 Na current was consistent with a binding stoichiometry of 1:1. At low frequency stimulation, and at holding potentials < or = 100 mV, the IC50 was 226 microM, comparable to values found in mammalian cardiac cells. Lidocaine also shifted the steady-state inactivation of hH1 Na current to hyperpolarized potentials in a dose-dependent manner. Our experiments suggest that lidocaine block is state dependent, with high affinity for an inactivated state (KI = 11 microM) and low affinity for the resting state (KR = 3.9 mM). The quaternary amine derivative of lidocaine, QX-314, had no effect on Na current at an extracellular concentration of 1 mM.


Deferoxamine, an iron chelator, reduces myocardial injury and free radical generation in isolated neonatal rabbit hearts subjected to global ischaemia-reperfusion. The protective action of deferoxamine, an iron chelator, against functional and metabolic deteriorations of ventricular muscle, induced by ischaemia-reperfusion, was investigated in Langendorff-perfused hearts of neonatal rabbits in comparison with superoxide dismutase (SOD) plus catalase. The perfused hearts were subjected to normothermic (37 degrees C) global ischaemia for 45 min following cardiac arrest with St Thomas cardioplegic solution and then reperfused with oxygenated Krebs-Henseleit solution. In control hearts, the recovery of the left ventricular developed pressure (LVDP) after 30 min reperfusion was 50.7 +/- 3.1% (mean +/- SE, n = 5) of the pre-ischaemic value. The LVDP recovery was significantly improved in the hearts treated with deferoxamine at 10-100 microM (89.4 +/- 1.4% at 30 microM, P < 0.01 vs. control). The improvement in LVDP was less prominent when treated with 30 x 10(4) U/l SOD plus 30 x 10(4) U/l catalase (67.9 +/- 2.0%, P < 0.01 vs. deferoxamine at 30 microM). CPK leakage into the coronary effluent during the initial 5 min of reperfusion was reduced to around half of the control value with 30 microM deferoxamine (P < 0.05 vs. control), while unaffected by the addition of SOD plus catalase. Free radicals in the coronary effluent were measured with electron spin resonance spectroscopy in separate experiments by using a spin-trapping agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). A burst of DMPO-OH signal was detected during the initial minutes of reperfusion. The intensity of DMPO-OH signal was significantly reduced by 30 microM deferoxamine to about one-third of control.(ABSTRACT TRUNCATED AT 250 WORDS)


Mechanism of the increase in intracellular sodium during metabolic inhibition: direct evidence against mediation by voltage-dependent sodium channels. During ischemia or metabolic inhibition, intracellular Na+ concentration ([Na+]i) increases considerably. Elevation of [Na+]i figures critically in the mechanism of cellular injury by promoting Ca2+ influx via the Na+-Ca2+ exchanger, but the exact mechanism of this intracellular Na+ accumulation remains unknown. To test directly the hypothesis that voltage-dependent Na+ channels are involved, we measured Na+ currents (INa) in isolated guinea-pig ventricular myocytes using the patch-clamp technique. The cell-attached configuration was used in order to avoid disturbing the intracellular milieu. Metabolic inhibition was induced by exposing the cells to either iodoacetate (IAA, 1 mM) to inhibit glycolysis or 2,4-dinitrophenol (DNP, 0.2 mM) to uncouple oxidative phosphorylation. The amplitude of INa was measured in multichannel patches before and during exposure to IAA or DNP, by depolarizing the cell to different membrane potentials from a holding potential of -135 mV. Analysis of current-voltage relations before and during metabolic inhibition revealed a modest but significant reduction of peak INa at test potentials positive to -40 mV with DNP; no change was observed with IAA. The voltage dependence of steady-state parameters of inactivation was not altered by either intervention; specifically, no steady-state ("window") current was induced. Although we cannot exclude the possibility that other factors not explored here might lead to different conclusions during genuine ischemia, metabolic inhibition alone does not up-regulate the function of Na+ channels. Thus, we conclude that other mechanisms underlie the accumulation of intracellular Na+ observed during metabolic inhibition.


Myocardial beta adrenoceptor and voltage sensitive calcium channel changes in a canine model of chronic heart failure. Effects of chronic heart failure upon receptor binding and cardiac function were studied in mongrel dogs. Heart failure was induced by three to seven, graded, sequential, intracoronary microembolizations performed 1 to 3 weeks apart. Depressed systolic and diastolic left ventricular function, reduced cardiac output, increased systemic vascular resistance, increased plasma norepinephrine concentration, left ventricular hypertrophy, and dilation were associated with the development of heart failure in this model. Three months after the last embolization, the density and affinity of myocardial beta adrenoceptors and voltage sensitive calcium channels were quantified by analyzing saturation isotherms of specific radioligand binding. [3H]Dihydroalprenolol and [3H]nitrendipine bound specifically and with high affinity to cardiac beta adrenoceptors and calcium channels, respectively. Scatchard transformation of the specific binding of these radioligands in membranes prepared following intracoronary embolization demonstrated a 47% decrease in the density of [3H]dihydroalprenolol binding sites (605 +/- 20 fmol/mg, normal, vs. 323 +/- 18 fmol/mg, failed; P < 0.05) and a 20% decrease in [3H]nitredipine binding sites (371 +/- 11 fmol/mg, normal, vs. 298 +/- 17 fmol/mg, failed; P < 0.05). The binding equilibrium dissociation constants for [3H]dihydroalprenolol and [3H]nitrendipine were not significantly different between normal and failed myocardium. There was no difference in the sialic acid content in the sarcolemmal membranes prepared from normal and failed dog hearts (31.07 +/- 0.76 nmol/mg, normal, vs. 30.58 +/- 5.25 nmol/mg, failed). This is inconsistent with the selective purification of membranes utilized in these radioligand binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)


One-year follow-up after multimodal inpatient treatment for cocaine and methamphetamine dependencies. Of a randomly selected sample of 214 patients treated with aversion therapy for cocaine dependence in four chemical dependency units operated by Schick Shadel Hospitals, 156 were followed up 12 to 20 months posttreatment (average 15.2 months). Significant other validation was obtained in 33%. Total abstinence from cocaine for the group overall was 53% at one year post treatment, and current abstinence of at least 6 months at follow-up was 68.6%. Those treating with aversion for cocaine alone had a one-year abstinence of 39% and a current abstinence of 62.4%. Those treating with aversion for alcohol and cocaine had a one-year total abstinence from cocaine of 69% and a current abstinence of 76%. Those treating with aversion for cocaine and marijuana had a one-year total abstinence from cocaine of 50% and a current abstinence of 65%. Those treating with aversion for alcohol, cocaine, and marijuana had a one-year total abstinence from cocaine of 73% and a current abstinence of 73%. One-year total abstinence from alcohol was 54% for those receiving aversion for both alcohol and cocaine and 77% for those receiving aversion for alcohol, cocaine, and marijuana. Current abstinence from alcohol at follow-up was 68% and 81%, respectively. One-year total abstinence from marijuana was 42% for those treating with aversion for cocaine and marijuana and 64% for those treating with aversion for alcohol, cocaine, and marijuana. Current abstinence at follow-up from marijuana was 61% and 81%, respectively. The use of aversion therapy for both alcohol and cocaine in alcoholics who were also using cocaine was associated with higher total abstinence rates (88% vs. 55%) from cocaine when compared with alcoholics who used cocaine but received no aversion as part of their program. The conclusion is tentative since the follow-up rate in this study was lower than that of the previous study (64% vs. 84%). Being around other users accounted for 49% of relapse situations. Family/Work stress was associated with relapse in 33% of cases and unpleasant feelings in 24% of cases. The use of both reinforcement treatments and the use of support following treatment were associated with improved abstinence rates from cocaine. Those patients who reported losing all urges for cocaine after treatment had a total abstinence from cocaine of 90%, those who reported losing all the uncontrollable urges had a total abstinence of 64%, and those who reported still having the urge reported only 33% total abstinence from cocaine.


Training and employment programs in methadone treatment: client needs and desires. Training and employment programs (TEP) in methadone treatment have declined in recent years. Yet heroin abusers who enter methadone treatment have historically high unemployment and low earnings compared to the general population and other treatment modalities. This paper reports on the first phase of a multiyear project to develop and evaluate TEPs for methadone treatment clients. Preliminary findings from the treatment intake survey of the pilot study are used to identify client needs and desires. Specifically, we report descriptive statistics for clients' (a) demographics, (b) education and training, (c) employment status, (d) income and expenditures, (e) interest in a TEP, and (f) labor market expectations thereafter. We conclude with a presentation of our proposed future analyses and a discussion of the policy implications of the project. Our main finding is that most addicts have a strong interest in training and employment services, but their expectations about the impact of such services is often unrealistic.


Family functioning as a predictor of progress in substance abuse treatment. This study tested the hypothesis that the level of family functioning is related to changes in the patient's progress while in treatment. We studied the relation of family functioning, as measured by the Family Adaptability and Cohesion Scales' (FACES II) dimensions of cohesion and adaptability, to 7 dimensions of the severity of patient drug use, as measured by the Addiction Severity index (ASI). The results indicated that family functioning, the cohesion dimension in particular, predicts severity of patient's dysfunction resulting from drug use and family and psychological problems. In particular, 28% of the variability in the ASI Drug Problems, 52% of the variability in the ASI Family Problems, and 29% of the variability in the ASI Psychological Problems were accounted for by the FACES II cohesion score. Although the study has several limitations, it supports the relevance of family factors in the treatment of drug-addicted patients.


Inpatient and outpatient cocaine abusers: clinical comparisons at intake and one-year follow-up. Given controversy about the comparative utility of inpatient and outpatient treatment for substance abusers, we compared samples of cocaine-abusing inpatients (n = 149) and outpatients (n = 149) regarding a range of clinical characteristics both at entrance into treatment and one year following this initial assessment. We wished to assess (a) whether inpatient treatment appeared justified on the basis of more severe clinical problems in this group of patients and (b) the comparative one-year outcome of patients treated in the two conditions. Regarding the presenting clinical picture, inpatients had more severe ratings in numerous areas, with heavier drug use, social impairment, and psychopathology. At one-year follow-up, the direction of clinical ratings had reversed in the two groups, with inpatients showing lower problem severity in several areas, particularly cocaine use and psychopathology. Thus, results of this nonrandomized study of inpatient and outpatient treatment suggest that decisions to hospitalize were made on a rational basis and that inpatient treatment had better long-term efficacy.


A comparison study of treated and untreated pregnant and postpartum cocaine-abusing women. The purpose of this study was to determine whether untreated pregnant and recently post-partum cocaine-abusing women could be differentiated from women who enrolled in drug treatment programs. The experimental sample was selected from women referred to the Georgia Addiction, Pregnancy, and Parenting Project, an intervention program for pregnant and postpartum addicted women, between January 1987 and January 1988 (n = 45). The comparison group was randomly selected from women who were admitted to two (2) day treatment programs during the same time period (n = 50). Groups were compared using the Addiction Severity Index (ASI) and the Psychiatric Symptom Checklist-90 (SCL-90). Results indicated that untreated women were less impaired socially and exhibited fewer symptoms of psychiatric distress. These findings confirm the commonly held belief that the severity of psychosocial distress may be an important motivating factor in the decision to enter drug treatment. Alternatively, the lack of gender-sensitive program components, such as childcare, and the social stigma attached to drug use in pregnancy may also account for the reluctance of pregnant and post-partum mothers to seek drug treatment. Implications for the development of intervention and treatment programs for women are discussed.


Rehabilitation from substance abuse in individuals with a history of sexual abuse. Twenty subjects were randomly selected from 200 intake files in a substance abuse rehabilitation facility. Each subject provided information regarding whether or not any history of sexual abuse existed. Treatment records were compared on seven categories of treatment taken from Daughters and Sons United criteria for successful recovery from sexual abuse. A higher rate of relative success across categories of treatment was demonstrated for those who dealt with issues of sexual abuse. Conversely, greater rates of recidivism and lower progress in recovery from substance abuse was noted in those who failed to address issues of sexual abuse. This study demonstrates that concentrating on substance abuse and failing to address issues of sexual abuse increases the likelihood of lower rates of rehabilitation. It is suggested that information regarding sexual abuse should be obtained during the assessment and intake procedures conducted by addiction rehabilitation programs and should form an important part of treatment itself.


Avoiding the quality assurance boondoggle in drug treatment programs through total quality management. This paper considers limitations to three often-used approaches for improving treatment quality in a drug abuse treatment program: supervisory review, internal program evaluations, and standard quality assurance. Total Quality Management (TQM) is then presented as a technique that maximizes the effectiveness of quality-improvement strategies and builds on them by establishing a more comprehensive approach to improving treatment quality. This paper recommends issues for treatment providers to consider in designing an efficacious TQM approach. Specific organizational characteristics are discussed that can either enhance or defeat TQM. The program should be goal oriented and have the broad commitment of staff and management to quality improvement. It should also have a well-defined and tailored system for monitoring, feedback, and change. Moreover, the program should delegate authority and responsibility to staff for improvements, possess organizational readiness, and contain an evaluative component that can determine whether TQM is meeting the program's needs.


Abnormal lipid and lipoprotein patterns in liver cirrhosis with and without hepatocellular carcinoma. Plasma lipid and lipoprotein profile was determined in 12 cirrhotics, 15 patients with cirrhosis and hepatocellular carcinoma (HCC) and 20 healthy volunteers. When compared with controls plasma total cholesterol (TC), high density lipoprotein cholesterol (HDLC), high density lipoprotein phospholipids (HDLPL), HDLPL/PL levels were low, phospholipid (PL) was normal and HDLC/TC and PL/TC were high in cirrhosis. In cirrhotics with HCC, TC, PL, PL/TC levels were elevated while HDLC, HDPL, HDLC/TC and HDLPL/PL were normal. A comparison within the patient groups showed that in cirrhosis alone, the levels of TC, HDLC, PL, HDLPL and HDLPL/PL were lower and PL/TC level was higher than in cirrhotics with HCC. Plasma albumin levels showed a negative correlation with PL/TC and a positive correlation with HDL-cholesterol in cirrhosis. In cirrhosis with HCC plasma, phospholipid levels showed a significant negative correlation with total bilirubin and alanine aminotransferase. The variations in the level of plasma lipids and lipoproteins may assist in describing the nature of these two forms of liver disease.


Can high blood pressure alone increase erythrocytic intracellular sodium concentration? In order to clarify the direct effects of high blood pressure on erythrocytic intracellular sodium concentration ([Na+]i) and sodium transport systems, a static pressure of 2.5 atm was applied to whole blood in plastic syringes at room temperature for 5 and 24 h. In the control samples, 5 h incubation under atmospheric pressure produced a significant decrease in ouabain-sensitive Na(+)-K+ pump activity and plasma pH, but no change in other parameters. After 24 h incubation, [Na+]i and mean corpuscular volume were significantly increased and intracellular potassium concentration, ouabain-sensitive Na(+)-K+ pump activity, and plasma pH were decreased. The change in [Na+]i during incubation under atmospheric pressure may be due to the increased permeability of the cell membrane and the decrease in ouabain-sensitive Na(+)-K+ pump activity. The pressure load did not increase erythrocytic [Na+]i but did decrease it relative to the control. The pressure load had no apparent effects on sodium transport systems, mean corpuscular volume and pH of plasma relative to the control. Although the mechanisms of the effect of pressure load on [Na+]i were not determined, we did find that high blood pressure alone was unable to increase erythrocytic [Na+]i.


[The comparison of alkalizing methods in dogs during the immediate postresuscitation period]. The purpose of this study is to determine the effects of sodium bicarbonate (SB) on acid-base balance in dogs during immediate post-resuscitation period when tissue blood flow is decreased. Anesthetized dogs underwent 6 min of cardiac arrest plus 8 min of open chest CPR. Treatments with SB (0.1 mEq.kg-1.min-1), THAM (0.1 mEq.kg-1.min-1) and hyperventilation (TV = 20 ml.kg-1, RR = 20) were performed for 1h after restoration of spontaneous heart beat. Immediately after resuscitation, the following data were obtained; arterial (a) pH 7.17, PaCO2 37.8 mmHg, aHCO3- 13.2 mEq.l-1, venous (v) pH 7.09, PvCO2 57.5 mmHg, vHCO3- 16.6 mEq.l-1, CSF (c) pH 7.27, PcCO2 41.4 mmHg, cHCO3- 18.6 mEq.l-1, serum osmotic pressure (SOP) 310 mOsm.kg-1, and serum lactate (SL) 24.8 mg.dl-1. Metabolic acidosis in control group (no alkalizing agent, normoventilation) was not corrected within 120 min. In SB group, apH, aHCO3-, cpH and cHCO3- improved in 15-45 min after starting SB treatment. SL increased markedly (twice control group) which is attributable to CO2 production by SB in low flow state. SOP also increased to 320 mOsm.kg-1 by 120 min, with associated serum Na increase. In THAM group apH, PaCO2, aHCO3-, cpH and cHCO3- improved as in SB group. SOP rose to 324 mOsm.kg-1. No SL increase was observed. In hyperventilation group apH and aHCO3- were not normalized. PcCO2 and cpH were normalized earliest among the groups. SL changes were similar to that of control group. The author concludes that SB administration during immediate postresuscitation period may not be beneficial for treatment of acid-base balance.


[A study of Epstein-Barr virus-associated hemophagocytic syndrome successfully treated with VP16 and analysis of T cell receptor chain genes of the bone marrow cells]. Virus-associated hemophagocytic syndrome (VAHS) is a nonneoplastic, generalized histiocytic proliferation disorder with marked hemophagocytosis associated with a systemic viral infection. We describe a female child with EBV-related VAHS, in whom Southern blot analysis showed monoclonal proliferation of bone marrow cells having EBV genome as detected with Xho-1 fragment of latent infection membrane protein (LMP) genome. The EBV serology showed anti-EBNA, anti-VCA-IgG, anti-VCA-IgA elevation and positive EBNA of SRBC-rosette forming bone marrow cells in late period of her clinical course, which indicated primary infection or secondary alteration of EBV immunity. The DNA analysis of the bone marrow cells also demonstrated monoclonal rearrangement of T cell receptor-beta and -gamma chain genes. Thus our study suggests that EBV might infect T cells and the T cells might proliferate monoclonally. Repeated administration of VP16 was capable of inducing remission of the disease, although adrenocortical steroid, vincristine and cyclophosphamide were administrated unsuccessfully. This is the first case of VAHS in which the monoclonal proliferation of EBV-infected T cells was demonstrated.


[Role of superoxide anion on onset and maintenance of hypertension in spontaneously hypertensive rats]. Endothelium-derived relaxing factor (EDRF) is a substance that is released by the vascular endothelium and mediates vasodilator responses induced by various substances including acetylcholine (AC). Superoxide anion (O2-) inactivates EDRF. It is well known that the endothelium-dependent vascular relaxations to AC are depressed in the aorta of spontaneously hypertensive rats (SHR). We studied the role of O2- on onset and maintenance of hypertension in SHR. Male 4- and 17-week old SHR (4SHR, 17SHR), and enalapril treated 17-week old SHR (5 mg/kg/day for 4 weeks: ETSHR), and age-matched normotensive Wistar-Kyoto rats (WKY; 4WKY, 17WKY) were used. Relaxation responses to AC or superoxide dismutase (SOD) were measured in isolated aortae from rats. Mean arterial pressure (MAP) was measured after injection of SOD in rats under conscious state. Systolic blood pressure of 4SHR, 17SHR, ETSHR, 4WKY, and 17WKY were 129 +/- 2 mmHg, 203 +/- 3 mmHg, 158 +/- 3 mmHg, 97 +/- 1 mmHg, and 138 +/- 2 mmHg, respectively. Although relaxation responses to AC were decreased in aortae from 4SHR, 17SHR, and ETSHR compared with those from age-matched WKY, relaxation responses to SOD dit not differ between SHR and corresponding WKY. Whereas the injection of SOD(10000 U/kg) elicited a significant reduction of MAP in 4SHR (-11 +/- 3 mmHg) and 17SHR (-24 +/- 5 mmHg), it has no effect in WKY. These data suggest that AC mediated endothelium-dependent relaxation is attenuated in SHR and that excessive O2- in the endothelium resulted from hypertension may contributes the decreased response in SHR.


Maximum binding of ouabain to erythrocytes in relation to a family history of essential hypertension, sodium balance and body weight in normotensive children. The number of sodium pump receptors in erythrocytes (maximum binding of ouabain to erythrocytes; Bmax) was examined in relation to a family history of essential hypertension (FH-HT), the body mass index (BMI), urinary sodium excretion and blood pressure in 71 normotensive children (13 to 15 years of age), and in relation to sodium intake in 6 children who had various kidney diseases but displayed a normal renal function (6 to 13 years of age). Bmax was significantly lower in children with FH-HT than in those without FH-HT. However, the BMI, urinary sodium excretion and blood pressure showed no significant differences between the groups. Bmax was not significantly different between the top 20% tile group of BMI or urinary sodium excretion and the bottom 20% tile group of BMI or urinary sodium excretion. Furthermore, Bmax was unchanged during and after restriction of sodium intake in children with nephropathy. These findings suggest that Bmax may be a genetic marker for essential hypertension, since it was suppressed regardless of the BMI or urinary sodium excretion in normotensive children with FH-HT, and since it did not change according to sodium intake in children with renal diseases.


Participation of GABAergic systems in the production of antinociception by various stresses in mice. Based on the data that diazepam, a benzodiazepine (BZP) receptor agonist, antagonized psychological (PSY)-stress induced analgesia (SIA) without prominent action on footshock (FS)- and forced swimming (SW)-SIA and that BZP receptors are coupled with GABA receptors, we examined how the GABAergic system participates in the production of various SIAs. Muscimol, a GABAA receptor agonist, at doses of 0.25 to 1.0 mg/kg, affected each SIA differently, suppressed PSY-SIA at 0.25 mg/kg but tended to potentiate it at 1.0 mg/kg, potentiated SW-SIA dose-dependently and did not affect FS-SIA at the doses employed. Both bicuculline, a GABAA receptor antagonist, 0.5 to 2.0 mg/kg, and picrotoxin, a Cl- channel blocker, 0.25 to 1.0 mg/kg, dose-dependently suppressed PSY- and FS-SIA. Meanwhile, the effects of both drugs on SW-SIA were less than those on PSY- and FS-SIA, namely, bicuculline slightly inhibited it only at 2.0 mg/kg, and picrotoxin did not produce any appreciable effect even at the highest dose. Baclofen, a GABAB receptor agonist, at 5.0 and 10.0 mg/kg had no influence on each SIA. On the contrary, CGP 35348, a GABAB receptor antagonist at 20 to 100 mg/kg caused the dose-dependent blockade of FS-SIA, but affected neither PSY- nor SW-SIA. The production of PSY- and SW-SIA is attributable to the GABAA receptors/Cl- channel mediated mechanism alone, while that of FS-SIA involves both GABAA and GABAB receptor mediated systems. Thus, GABAergic systems play an important role in the production of each SIA; however, the participation of the receptor subtypes in the mechanism was different from each other.


[Experimental studies of urinary incontinence. Effect of tiropramide on the urinary stage and evacuation]. Tiropramide, a tyrosine derivative, has an antispasmodic effect on the gastrointestinal smooth muscles and it is suggested that the effect is mediated by cAMP. Therefore, we studied the effects of tiropramide on the lower urinary tract smooth muscle functions. In animal experiments, the effects of tiropramide on vesicourethral smooth muscle contraction were investigated and cAMP and cGMP levels following tiropramide administration were measured in the vesicourethral and gastrointestinal smooth muscles. In clinical trials, five healthy volunteers and five patients with neurogenic bladder dysfunction were treated orally with tiropramide 300 mg a day for two weeks and examined for urodynamic parameters before and after tiropramide treatment. Tiropramide significantly inhibited contraction of the vesicourethral smooth muscle and the inhibitory effect upon the bladder was remarkable particularly at the lower concentrations. Tiropramide remarkably increased cAMP level but it had no effect on cGMP level in the bladder at the lower concentrations. Tiropramide at the lower concentrations did not affect cAMP and cGMP levels in the smooth muscles of the urethra, stomach and intestines. In the patients with neurogenic bladder dysfunction treated with tiropramide orally, the urine volume at first desire to void and maximum bladder capacity increased significantly, but the volume of residual urine did not significantly increase. In the healthy volunteers, there were no significant changes in urodynamic parameters. These findings indicate that tiropramide relaxes the urinary bladder smooth muscle via cAMP to increase the urinary bladder capacity.


Recent advances in the study of hepatocellular carcinoma. Among the known causes for the occurrence of hepatocellular carcinoma (HCC), chemical carcinogens, chronic alcoholic intake and hepatitis B virus (HBV), especially in Asia, has been emphasized. HBV has been industriously studied and many queries about the relationship between HBV infection and hepatocarcinogenesis have been clarified. Recent discovery of hepatitis C virus (HCV) revealed that there may be the participation of this virus in hepatocarcinogenesis. However, a precise mechanism in such a viral infection has not been known. Host immunological defence mechanisms including the role of cytokines should be also taken into consideration. Cellular gene abnormalities have been noted in the late period of cancer cell progression. The technical development in the clinically available diagnostic procedures have enabled us to detect early phase of HCC. Some new concepts in the pathological diagnosis of precancerous lesions of HCC and also early HCC have been reported recently. We gave an outline of the recent advances and references in the study of HCC.


Double antibody sandwich ELISA for the detection of rubella virus antigen. We developed enzyme linked immunosorbent assay (ELISA) for the detection of rubella virus antigen, using two monoclonal antibodies, MC-7 and MC-22. The double antibody sandwich ELISA method was carefully standardized and found to be sensitive enough to detect as small as 2.5 ng protein of rubella virus. The infective titers by the double antibody sandwich ELISA closely related to those judged by interference of vesicular stomatitis virus in RK-13 cells. The method is simple, sensitive, and readily applicable to the detection of rubella virus.


Sequence analysis and molecular detection of mouse hepatitis virus using the polymerase chain reaction. Sequence analysis of the nucleocapsid protein genes of five strains of mouse hepatitis virus (MHV) disclosed that the 3' region of the nucleocapsid protein gene contains highly conserved sequences unique to MHV. We designed a pair of primers to amplify cDNA from such sequences of MHV by using the polymerase chain reaction (PCR). Six isolates of wild-type MHV, as well as prototype viruses, were amplified successfully and detected in ethidium bromide-stained agarose gels. The sequence identity of PCR products was readily verified by confirming target size and a MflI site within the target. The sensitivity of our PCR assay was estimated to be sufficient to detect a single cell infected with MHV. This new approach may permit more sensitive and rapid detection of MHV in biologic materials than current methods such as virus isolation, the infant mouse bioassay, and the mouse antibody production test.


[Subacute paraneoplastic cerebellar degeneration in microcytic carcinoma of the lung. Presentation of 2 cases]. Subacute paraneoplastic cerebellous degeneration is a rare syndrome which is found in less than 1% of patients with cancer. Small cell cancer of the lung and of the ovary are the two neoplasms most frequently associated to this entity. Two patients with small cell lung cancer who initially had a cerebellous syndrome in which no sign of macroscopic cerebellous lesion could be demonstrated by either computerized tomography or nuclear magnetic resonance of the head are presented. One of the patients was evaluated at autopsy. Both patients were treated with polychemotherapy with which partial response was obtained. Neurologic symptomatology was not alleviated in the first patient with death due to bronchopneumonia at 5.5 months of initiation of the disease, while improvement of the cerebellous paraneoplastic syndrome was achieved in the second patient. The different evolution of subacute paraneoplastic cerebellous degeneration in two patients in whom antibodies were not demonstrated and in whom initial response of the tumor to chemotherapy was achieved may be explained by the second patient having undergone prolonged treatment of 6 cycles suggesting a strict relation ship between the tumor and subacute cerebellous degeneration which, to date, remains unknown.


[Cushing syndrome in a neuroendocrine pancreatic tumor. Clinicopathologic case report]. A 59-year-old female patient with mild clinical features of a Cushing syndrome underwent surgery for a suspected hormonally active tumor of the left adrenal gland. Surprisingly, the adrenal gland was unremarkable, however, a tumor in the pancreatic tail was found. A left pancreatic resection with splenectomy resulted in curative removal of the tumor. The pathohistological examination of the tumor established the diagnosis of an ACTH-producing pancreatic carcinoid. Morphology and pathogenesis of pancreatic carcinoids which are tumors of the APUD-cell-system are discussed in detail.


The 5' to 3' exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae. Three different mutations were introduced in the polA gene of Streptococcus pneumoniae by chromosomal transformation. One mutant gene encodes a truncated protein that possesses 5' to 3' exonuclease but has lost polymerase activity. This mutation does not affect cell viability. Other mutated forms of polA that encode proteins with only polymerase activity or with no enzymatic activity could not substitute for the wild-type polA gene in the chromosome unless the 5' to 3' exonuclease domain was encoded elsewhere in the chromosome. Thus, it appears that the 5' to 3' exonuclease activity of the DNA polymerase I is essential for cell viability in S. pneumoniae. Absence of the polymerase domain of DNA polymerase I slightly diminished the ability of S. pneumoniae to repair DNA lesions after ultraviolet irradiation. However, the polymerase domain of the pneumococcal DNA polymerase I gave almost complete complementation of the polA5 mutation in Escherichia coli with respect to resistance to ultraviolet irradiation.


Clostridium histolyticum collagenases: a new look at some old enzymes. Seven collagenases denoted by the letters alpha, beta, gamma, delta, epsilon, zeta and eta have been purified to homogeneity from the culture filtrate of Clostridium histolyticum. All seven enzymes are zinc proteinases that require calcium ions for activity and have essential carboxyl, tyrosyl and lysyl residues. These enzymes can be divided into two classes on the basis of the sequence homologies in their polypeptide chains, as revealed from a comparison of their tryptic digests. This division into classes is also supported by a comparison of their specificities toward peptide substrates, their interaction with substrate-analog inhibitors, and their mode of attack of triple helical collagens. The sequence specificities of these enzymes have been studied in detail. The specificities of the two classes are similar, but complementary. Both classes exhibit both endopeptidase and tripeptidylcarboxypeptidase activities, where the latter is thought to facilitate removal of Gly-X-Y triplets from the C-terminus of collagen fragments. The mode of attack of these collagenases on triple helical type I, II and III collagens is very similar for the enzymes within each class, but different for the two classes. The class I enzymes first hydrolyze loci near the ends of the triple helical domains of these collagen molecules, while the class II enzymes make their initial cleavages in the interior. The sites of these initial cleavages are being sequenced and preliminary results indicate that they do not resemble the tissue collagenase cleavage site with respect to either their imino acid content or distribution. The kinetic parameters for the hydrolysis of type I, II and III collagens have been measured and are similar in magnitude to those for the tissue collagenases. Synthetic peptide substrate-analog inhibitors have been prepared for both classes of collagenases and shown to be transition-state-analog inhibitors.


Specific members of the Jun protein family regulate collagenase expression in response to various extracellular stimuli. The transcription factor AP1 which regulates expression of collagenase in response to various extracellular signals is a multimeric complex composed of members of the Jun- and Fos families. To examine the biological role of the various components in signal transduction we analyzed the expression of two of them (cJun, JunB) and collagenase in response to phorbol esters, cAMP and TGF-beta. While all three genes are induced by phorbol ester and TGF-beta only JunB is induced by cAMP. In contrast expression of cJun and collagenase is reduced by cAMP indicating that cJun and JunB are not coordinately regulated. In addition JunB is not an efficient activator of the cJun and collagenase promoters although both cJun and JunB exhibit similar DNA binding properties, indicating that the differences in biological activity is due to differences in their activation domains. Our results imply that enhanced expression of collagenase (and cJun) depends on the activation of cJun. Expression of cJun and collagenase is inhibited under certain conditions of high levels of JunB. This suggests a negative regulatory function of JunB which greatly expands the potential of the Jun protein family in changing the transcription of specific genes involved in triggering complex biological processes.


Mosaic structure of the secreted ECM metalloproteases and interaction of the type IV collagenases with inhibitors. SV-40 transformed human lung fibroblasts and HT 1080 fibrosarcoma cells secrete a 92-kDa type IV collagenase (in addition to 72-kDa type IV collagenase identical to that found in macrophages, phorbol ester differentiated U937 cells, and keratinocytes. The expression of this protease is induced by the tumor promoter TPA, and interleukin-1 and was not detected in the parental human lung fibroblast. The 92-kDa preproenzyme has a predicted Mr of 78,426, including a 19 amino acid long hydrophobic signal peptide. The apparent discrepancy between the predicted molecular weight and the molecular weight of the secreted protein is due to a post-translational modification of the enzyme through glycosylation. The 92-kDa type IV collagenase consists of five distinct domains, including a unique 54 amino acid long collagen--like domain, and is a member of the secreted ECM metalloprotease gene family. Both the 72 and 92-kDa type IV collagenase contain a fibronectin-like collagen binding domain. The mosaic structure of the secreted ECM metalloproteases is a result of a recruitment of the functional units from ECM structural macromolecules into an enzyme protein in the process of evolution. The 92-kDa and 72-kDa type IV collagenase proenzymes form a noncovalent complex with inhibitors, which is activatable by APMA, yielding an enzymes with similar if not identical substrate specificity profile. Our results demonstrate that while the 92-kDa type IV collagenase forms a stoichiometric complex with TIMP, the 72-kDa type IV collagenase, purified from the same starting material, contains a novel 24-kDa inhibitor-TIMP-2.


Photoaffinity labeling of the partially purified mitochondrial phenylalkylamine calcium antagonist receptor. Mitochondria contain specific Ca2+ antagonist binding sites that are associated with an inner mitochondrial membrane anion channel. These mitochondrial Ca2+ antagonist receptors can be solubilized with digitonin and partially purified [as assessed by postreversible (+/-)-[3H]nitrendipine binding] using ion exchange chromatography and sucrose density gradient centrifugation. In the present study, reversible binding of the phenylakylamine Ca2+ antagonist [3H]ludopamil, an optically pure photoaffinity analog of verapamil, to the partially purified mitochondrial Ca2+ antagonist receptor complex (Kd, 9 +/- 4 microM; Bmax, 1.2 +/- 0.5 nmol/mg of protein) depended on NaNO3 and was inhibited by the 1,4-dihydropyridine niludipine and by ATP. Accordingly, the unlabeled racemic analog of [3H]ludopamil, (+/-)-LU 47781, dose-dependently inhibited the binding of the 1,4-dihydropyridine (+/-)-[3H]nitrendipine to the purified mitochondrial receptors (IC50, 2.1 +/- 0.1 microM). After UV irradiation, [3H]ludopamil specifically incorporated into two polypeptides of 12.7 +/- 0.1 kDa and 11.7 +/- 0.1 kDa, with the pharmacological profile of [3H]ludopamil photoincorporation stimulation and protection being identical to that of reversible binding.


Pre-steady state study of beta-adrenergic and purinergic receptor interaction in C6 cell membranes: undelayed balance between positive and negative coupling to adenylyl cyclase. Interactions between beta-adrenergic and ADP purinergic receptors in C6 glioma cell membrane preparations were investigated under steady state and then pre-steady state conditions of adenylyl cyclase (EC 4.6.1.1) activity, in order to determine how fast the second receptor antagonizes the transduction mechanism of the first. Cell membranes were washed to deplete them as thoroughly as possible of low molecular weight compounds, especially ATP and ADP, and to ensure better control of both substrate and agonist nucleotide concentrations. ATP concentrations were kept constant with the use of an ATP-regenerating system; the C6 cell line exhibited very active ectonucleotidases. The purinergic agonist ADP was replaced by its nonhydrolyzable congener adenosine 5'-O-(2-thio)diphosphate (ADP beta S), which was demonstrated, like ADP, to inhibit isoproterenol-stimulated adenylyl cyclase activity in intact cells (IC50 for ADP, 0.5 +/- 0.1 microM; IC50 for ADP beta S, 25 +/- 2 microM) and in membrane preparations (IC50 for ADP beta S, 79 +/- 20 microM). In the case of membrane preparations, ADP beta S did not compete with ATP, the substrate of the cyclase-catalyzed reaction, and behaved apparently as a non-competitive inhibitor of the enzyme. The pre-steady state kinetics of isoproterenol-stimulated adenylyl cyclase activity measured with a pulsed quenched-flow apparatus have previously been shown to include two steps, the first very rapid (taking place within 1-2 sec) and giving rise to a burst of cAMP synthesis and the second much slower and corresponding to the steady state reaction. ADP beta S inhibited the occurrence of both steps with comparable IC50 values (mean value, 55 +/- 20 microM). In the presence of increasing concentrations of the purinergic receptor agonist, the time constant of the exponential burst reaction was not affected, but its amplitude progressively decreased to zero. These results showed that the extinction of the beta receptor cAMP response by the purinergic ADP receptor occurred within the dead-time of the pulsed quenched-flow apparatus, which was 50 msec. Such a rapid inhibition of cAMP production excluded modulation of isoproterenol-stimulated adenylyl cyclase activity by the ADP receptor by a pathway other than its direct negative coupling to the cyclase via a Gi protein. In this respect, the P2 purinergic ADP receptor of the C6 glioma cell line appears comparable to the P2t receptor of platelets.


Neuromedin B receptors retain functional expression when transfected into BALB 3T3 fibroblasts: analysis of binding, kinetics, stoichiometry, modulation by guanine nucleotide-binding proteins, and signal transduction and comparison with natively expressed receptors. The receptor that interacts with the mammalian bombesin-related peptide neuromedin B (NMB) is ubiquitous in the gastrointestinal tract and central nervous system. However, little is known regarding its cellular mechanisms of action. This receptor has been recently cloned, sequenced, and stably transfected into BALB 3T3 fibroblasts, permitting detailed study of the pharmacology and coupled biological activities of this receptor. In the present study, we compare the ability of transfected receptors to alter cell function with that of receptors natively expressed in small numbers by the rat glioblastoma cell line C6. NMB inhibited binding of 125I-[D-Tyro]NMB with high affinity in transfected cells (Ki = 3.08 +/- 0.14 nM) and in C6 cells (Ki = 1.90 +/- 1.10 nM), whereas the bombesin-related agonists gastrin-releasing peptide (GRP) and [D-Phe6, D-Ala11, Leu14]bombesin(6-16) (GRP analogue) had 100- and 300-fold lower affinities, respectively, for NMB receptors in either cell type. For both cell systems, maximal binding was observed between 5 and 15 min at 22 degrees. Both cell types internalized NMB at similar rates, with > 70% of bound ligand being internalized by 60 min at 22 degrees. The nonhydrolyzable guanosine analogue guanosine 5'-(beta,gamma-imido)triphosphate was equipotent in causing a decrease in binding of 125I-[D-Tyro]NMB due to decreased receptor affinity in both cell types, without a change in receptor number, demonstrating that the NMB receptor remained coupled to a guanine nucleotide-binding protein in both native and transfected cells. In both cell systems, NMB increased inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in a time-dependent fashion. Inositol phosphates were increased in a dose-dependent fashion, with similar half-maximal values being obtained for NMB in both cell types (transfected, 1.01 +/- 0.09 nM; C6, 2.09 +/- 0.15 nM) and for the GRP analogue (transfected, 1855 +/- 140 nM; C6, 2129 +/- 250 nM). NMB mobilized intracellular Ca2+ in both cell systems, and the dose-response curves were superimposible (EC50 for transfected, 0.10 +/- 0.08 nM; C6, 0.11 +/- 0.02 nM). These data demonstrate that activation of the receptor for NMB stimulates phospholipase C and increases intracellular Ca2+. These results also demonstrate that transfected and native NMB receptors behave similarly, suggesting that the transfected cell line will be useful in future studies investigating ligand-receptor interactions, as well as in molecular biological studies of the structure-function relationship of the receptor.


Dual excitatory and inhibitory effects of opioids on intracellular calcium in neuroblastoma x glioma hybrid NG108-15 cells. The intracellular free calcium concentration ([Ca2+]i) was measured in single NG108-15 cells using indo-1-based microfluorimetry. In cells differentiated for 6-14 days in serum-free, forskolin (5 microM)-supplemented medium, application of micromolar concentrations of [D-Ala2,D-Leu5]-enkephalin (DADLE) inhibited Ca2+ influx mediated by voltage-gated Ca2+ channels. DADLE, at concentrations ranging from 1 nM to 1 microM, also produced rapid transient increases in [Ca2+]i (EC50 = 10 nM). The [Ca2+]i increases elicited by DADLE did not correlate with the inhibitory effects of the peptide. DADLE-induced [Ca2+]i increases were blocked by naloxone. In single cells, sequential application of selective opioid agonists (30 nM) evoked responses of the rank order DADLE = [D-Pen2,D-Pen5]-enkephalin > (trans)-(+-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) benzeneacetamide > [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, consistent with activation of a delta-opioid receptor. The response was completely blocked by removal of extracellular Ca2+ or application of 1 microM nitrendipine, indicating that the increase in [Ca2+]i results from Ca2+ influx via dihydropyridine-sensitive, voltage-gated Ca2+ channels. Substitution of N-methyl-D-glucamine for extracellular Na+ or application of 1 microM tetrodotoxin greatly reduced, and in some cases blocked, the DADLE-induced [Ca2+]i increase, consistent with amplification of the response by voltage-gated Na+ channels. The [Ca2+]i increase was mimicked by both dibutyryl-cAMP and phorbol 12,13-dibutyrate. These findings are consistent with a delta-opioid-induced depolarization, possibly mediated by a second messenger, that subsequently recruits voltage-sensitive Ca2+ channels. In contrast to differentiated cells, undifferentiated cells responded to DADLE with a modest [Ca2+]i increase that was not sensitive to nitrendipine. In these cells, activation of the same second messenger system may elevate [Ca2+]i by mobilization from intracellular stores rather than influx. In addition to previously described inhibitory coupling to adenylyl cyclase and Ca2+ channels in NG108-15 cells, these results suggest that a novel, excitatory, effector system may also couple to opioid receptors.


Coexistence of contractile and relaxant 5-hydroxytryptamine receptors coupled to distinct signaling pathways in intestinal muscle cells: convergence of the pathways on Ca2+ mobilization. Muscle cells were dispersed separately from circular and longitudinal muscle layers of guinea pig intestine, and 5-hydroxytryptamine (5-HT) receptors were characterized in naive cells and in cells in which one receptor type was preserved by selective receptor protection. In naive cells from both regions, 5-HT caused contraction and stimulated increases in cytosolic free calcium concentration ([Ca2+]i) (3-fold; p < 0.01) and cAMP levels (40-60%; p < 0.01) that were inhibited, respectively, by the 5-HT2 antagonist ketanserin and the 5-HT1p antagonist N-acetyl-5-hydroxytryptophyl 5-hydroxytryptophan amide (5-HTP-DP). In circular muscle cells, where agonist-induced increase in [Ca2+]i is mediated by Ca2+ release from inositol (1,4,5)trisphosphate-sensitive stores, 5-HT caused an increase in inositol (1,4,5)trisphosphate levels that was inhibited by ketanserin. In cells maximally contracted with a non-5-HT agonist (cholecystokinin octapeptide), 5-HT caused relaxation when the contractile effect mediated by 5-HT2 receptors was blocked with ketanserin; relaxation and the concomitant increase in cAMP were inhibited by 5-HTP-DP. The singular contributions of the Ca2+ and cAMP signaling pathways were identified in cells where only one receptor type was preserved. In cells with only 5-HT2 receptors, 5-HT caused contraction and an increase in [Ca2+]i but not in cAMP levels; contraction and the increase in [Ca2+]i were inhibited by ketanserin. Conversely, in cells with only 5-HT1p receptors, 5-HT caused relaxation and an increase in cAMP levels but not in [Ca2+]i; relaxation and the increase in cAMP levels were inhibited by 5-HTP-DP. The two signaling pathways were functionally linked, converging to regulate the level of [Ca2+]i. Thus, the increase in [Ca2+]i was augmented 1) when cAMP production was inhibited by 5-HTP-DP in naive cells or 2) when cAMP production was suppressed in cells where 5-HT1p receptors were inactivated and only 5-HT2 receptors were preserved. The results imply that the increase in cAMP levels mediated by 5-HT1p receptors acted to attenuate the increase in [Ca2+]i mediated by 5-HT2 receptors. We conclude that the response to 5-HT in muscle cells is a compound effect involving activation of two receptor types coupled to distinct signaling pathways that converge on [Ca2+]i as the determinant of mechanical activity.


[3H]Phenytoin identifies a novel anticonvulsant-binding domain on voltage-dependent sodium channels. The voltage-dependent sodium channel has been proposed as a specific target for the actions of the anticonvulsant drug phenytoin. Working at 0-4 degrees, we previously reported the existence of specific [3H]phenytoin binding sites in rat brain membranes. In the present study, the binding of [3H]phenytoin was assessed at 22 degrees, a temperature favorable to the binding of sodium channel ligands. At 22 degrees, the site had a Kd of 1.5 microM, which is in the relevant therapeutic concentration range for anticonvulsant activity (1-10 microM), and a calculated Bmax of 4.5 pmol/mg of protein, which is similar to previous estimates of sodium channel concentration in brain membranes. In competition experiments, specific [3H]phenytoin binding was found to be inhibited by drugs that interact with the sodium channel, including antiarrhythmics, local anesthetics, anticonvulsants, and site-specific neurotoxins (the steroidal alkaloid activators, beta-scorpion venoms, and brevetoxin-3). Diazepam, used clinically in the management of tonic-clonic status epilepticus, and flunarizine, a calcium channel blocker with anticonvulsant activity, potentiated [3H]phenytoin binding at micromolar concentrations. Other drugs and ligands, including neurotransmitters, neuromodulators, and ligands for other ion channels, had no effect. Depolarization with KCl showed [3H]phenytoin binding to be voltage sensitive. Experiments with batrachotoxin (a specific site 2 toxin) and anticonvulsants demonstrated that the interactions between these compounds and the [3H]phenytoin binding site are allosteric in nature. These results provide direct evidence that phenytoin interacts with the voltage-dependent sodium channel and indicate that such interactions take place at therapeutic concentrations. They support previous proposals, based on toxin-binding and electrophysiological studies, that the therapeutic effects of phenytoin result from a selective inhibition of voltage-dependent sodium flux.


Interaction of pumiliotoxin B with an "alkaloid-binding domain" on the voltage-dependent sodium channel. The alkaloid pumiliotoxin B (PTX-B) "activates" voltage-dependent sodium channels in synaptoneurosomes and neuroblastoma cells. It appears that PTX-B activates sodium channels by interacting with a site that is allosterically coupled to other sites on the sodium channel, namely two scorpion toxin sites and the brevetoxin site. In guinea pig cortical synaptoneurosomes, alpha-scorpion toxin, beta-scorpion toxin, and brevetoxin induce a dose-dependent potentiation of PTX-B-induced 22Na+ influx. The synergism with beta-scorpion toxin differentiates PTX-B from the alkaloid veratridine, which induces an activation of sodium channels that is not affected by beta-scorpion toxin. PTX-B does not inhibit [3H]batrachotoxinin-A benzoate ([3H]BTX-B) binding to the alkaloid site on sodium channels. On the other hand, aconitine, which activates sodium channels and inhibits [3H]BTX-B binding, induces a 22Na+ influx that, like PTX-B-induced 22Na+ influx, is potentiated by alpha-scorpion toxin, beta-scorpion toxin, and brevetoxin. Inhibition of [3H]BTX-B binding by aconitine is reduced in the presence of PTX-B. Both a type I pyrethroid (allethrin) and a type II pyrethroid (fenvalerate) inhibit PTX-B- and PTX-B/alpha-scorpion toxin-mediated 22Na+ influx. Allethrin and fenvalerate also inhibit aconitine-mediated 22Na+ flux but not BTX-mediated 22Na+ influx. It is proposed that on the sodium channel there is an "alkaloid-binding domain" at which alkaloids exert stimulatory actions. However, depending on the region on the domain to which the binding occurs, different allosteric interactions with other sites can be observed. PTX-B is proposed to interact with a part of the alkaloid-binding domain that is shared by aconitine but not by batrachotoxin or veratridine, whereas aconitine interacts with a part of the domain shared by PTX-B and by batrachotoxin/veratridine.


Rodent and human beta 3-adrenergic receptor genes contain an intron within the protein-coding block. DNA blot analysis of the cloned rat beta 3-adrenergic receptor gene revealed unexpected restriction enzyme cleavage sites that suggested the presence of one or more introns near the end of the coding block. This region of the rat gene was mapped and sequenced and was found to contain two introns. The first intron occurs 12 amino acids from the end of the coding block, as deduced by comparison with the beta 3 receptor cDNA. Sequence analysis of the first intron indicates that it might contain enhancer elements that could be important in the adipose tissue-specific expression of this gene. The mouse and human beta 3 receptor genes have been assumed to be intronless; however, these genes contain potential splice sites that are homologous to those present in the rat gene. The relevant regions of the mouse and human beta 3 receptor cDNAs were cloned and, by comparing them to the respective genomic sequences, it was concluded that these genes also contain one or more introns. Sequence analysis of the mouse and human beta 3 receptor cDNAs indicates that they code for proteins that are, respectively, 12 and 6 amino acids larger than previously deduced from genomic clones.


Effects of chemical and surgical sympathectomy on expression of beta-adrenergic receptors and guanine nucleotide-binding proteins in rat submandibular glands. Expression of beta 1-adrenergic receptors and guanine nucleotide-binding proteins in rat submandibular glands was determined after reserpine administration and sympathetic denervation. Pretreatment of rats with reserpine resulted in up-regulation of the density of beta 1-adrenergic receptors and the immunoreactivity of the 64-kDa species of beta 1-adrenergic receptor in submandibular membranes, by 2.6 +/- 0.3-fold (eight experiments), within 7 days. Steady state levels of beta 1-adrenergic receptor mRNA quantified by DNA-excess solution hybridization were 0.15 +/- 0.03 amol of beta 1-adrenergic receptor mRNA/micrograms of total cellular RNA (six experiments). beta 1-Adrenergic receptor mRNA increased by 50% within 8 hr after pretreatment with reserpine. Maximal levels of 0.37 +/- 0.04 amol of beta 1-adrenergic receptor mRNA/micrograms of RNA were attained by 4 days and these levels were sustained for the next 3 days (six experiments). Northern blot hybridization also revealed a 3-fold increase in the 2.5-kilobase beta 1-adrenergic receptor mRNA transcript, which was equivalent in magnitude to that determined by solution hybridization. Reserpine pretreatment also affected steady state levels of submandibular guanine nucleotide-binding proteins. Two immunoreactive forms of the alpha subunit of Gs, migrating as 42 kDa (major) and 50 kDa (minor), were detected in salivary membranes. The immunoreactivity of the 42-kDa species of Gs alpha declined by 50% after 7 days of continuous daily injections of reserpine. In contrast, steady state levels of Gi alpha 2 (41 kDa), Go (39 kDa), and G beta 2 (35 kDa) and their mRNAs in submandibular membranes were unaffected by reserpine pretreatment. The rate of beta 1-adrenergic receptor gene transcription assessed by nuclear run-on transcription assay in nuclei of submandibular glands was not altered by reserpine pretreatment. However, reserpine had a dramatic effect on the half-life of beta 1-adrenergic receptor mRNA in submandibular glands. The half-life of beta 1-adrenergic receptor mRNA in control submandibular glands was 3.5 hr, whereas it increased to 8 hr in reserpine-pretreated glands. Reserpine-promoted stabilization of beta 1-adrenergic receptor mRNA provides a mechanism for up-regulation of postjunctional beta 1-adrenergic receptors in sympathetically innervated tissues.


Stable expression of type I gamma-aminobutyric acidA/benzodiazepine receptors in a transfected cell line. Expression plasmids were constructed with cDNAs encoding the rat gamma-aminobutyric acid-A (GABAA) receptor alpha 1, beta 2, and gamma 2 subunits and were cotransfected into cultured human embryonic kidney 293 cells. A single cell line (WS-1) was established after G-418 treatment and clonal selection. This cell line contained saturable, high affinity binding sites for the benzodiazepines [3H] Ro 15-4513 and [3H]flunitrazepam that were modulated by GABA. Competition experiments with benzodiazepine receptor ligands suggest a profile characteristic of native "type I" benzodiazepine receptors, because strong correlations were observed between the Ki values of these ligands in WS-1 cells and in both cerebellar homogenates (r = 0.97, p < 0.0001) and 293 cells transiently transfected with the corresponding cDNAs (r = 0.96, p < 0.001). Fluorescence intensity in WS-1 cells loaded with the Cl(-)-specific probe 6-methoxy-N-(3-sulfopropyl)-quinolinium was reliably increased by GABA. This effect was blocked by bicuculline and augmented by midazolam, consistent with the presence of GABA-gated, benzodiazepine receptor-modulated, Cl- channels. Northern blot analysis revealed the presence of mRNAs encoding alpha 1 and gamma 2 receptor subunits. Southern blot analysis confirmed genomic integration of transfected alpha 1 and gamma 2 cDNAs. The beta 2 subunit was not detected in either Northern or Southern blot analysis, indicating that a functional type I GABAA/benzodiazepine receptor complex can be constituted without a beta subunit.


The substrate specificity of phosphoinositide-phospholipase C in rat heart sarcolemma. In rat cardiac sarcolemmal membranes a phosphoinositide-specific phospholipase C (PLC) was found to be present. The enzyme hydrolysed exogenous [3H-]phosphatidylinositol 4,5-biphosphate ([3H-]PtdIns(4,5)P2) in an optimized assay mixture containing 15 micrograms SL protein, 100 mM NaCl, 1 mM free Ca2+, 14 mM Na-cholate and 20 microM [3H-]PtdIns-(4,5)P2 (400-500 dpm/microliter) in 30 mM HEPES-Tris buffer (pH 7.0). The average specific activity was 9.14 +/- 0.55 nmol.mg-1.2.5 min-1. The addition of Mg2+ to the assay mixture did not change PLC activity but increased the relative amounts of dephosphorylated inositol products. In the absence of Na+ and at a low Ca2+ concentration (0.3 microM), Mg2+ also enhanced the intraSL levels of PtdIns4P and PtdIns, and, moreover, inhibited PLC activity (IC50-0.07 mM). PtdIns4P seemed to be a good substrate for th rat SL PLC (23.07 +/- 1.57 nmol.mg-1.2.5 min-1) whereas PtdIns was hydrolysed at a very low rate (0.36 +/- 0.08 nmol.mg-1.2.5 min-1). Unlike PtdIns(4,5)P2, PLC-dependent PtdIns4P and PtdIns hydrolysis was not inhibited by Ca2+ concentrations over 1 mM. The possibility of distinct isozymes being responsible for the different hydrolytic activities is discussed.


Phosphatidylcholine metabolism in ischemic and hypoxic hearts. The rates of phosphatidylcholine biosynthesis in the isolated hamster hearts under ischemic and hypoxic conditions were examined. Global ischemia was produced by perfusion of the heart with a reduced flow, whereas hypoxia was produced by perfusion with a N2-saturated buffer. A 51% reduction in the biosynthesis of phosphatidylcholine was observed in the ischemic heart. The reduction was caused by a severe decrease in ATP level which resulted in a diminished conversion of choline into phosphocholine. A 22% reduction in the biosynthetic rate of phosphatidylcholine was also detected in the hypoxic heart. The reduction was caused by a diminished level of CTP which resulted in a decreased conversion of phosphocholine to CDP-choline. No compensatory mechanism was triggered during ischemia, but the CTP: phosphocholine cytidylyltransferase activity was enhanced in the hypoxic heart. Our results demonstrate the possible rate-limiting role of choline kinase and reconfirm the regulatory role of the cytidylyltransferase in the biosynthesis of phosphatidylcholine.


Effects of lactation on the regulation of hepatic metabolism in the rat and sheep: adrenergic receptors and cyclic AMP responses. The number and coupling efficiency of beta-adrenoceptors in liver membranes and intact hepatocytes of lactating and non-lactating female rats were compared to assess whether or not alterations in this signalling system could contribute towards the changed pattern of hepatic metabolism during lactation. In view of the different adaptations of hepatic metabolism to lactation in ruminants, the adrenergic receptor profile of sheep liver membranes was also determined. Post-receptor responses at two stages 'down-stream' of cyclic AMP generation were also evaluated in rat hepatocytes in response to the beta-adrenergic agonist isoprenaline. No changes in the number of affinity of hepatic beta-adrenoceptors were found in sheep or rats when lactating and non-lactating individuals were compared. Sheep liver was found to have a much greater concentration of beta-adrenoceptors than rat liver, and a much higher ratio of beta:alpha 1. The sensitivity and responsiveness of cyclic AMP generation in response to isoprenaline were similar in hepatocytes prepared from lactating and non-lactating rats, although the response to saturating concentrations of glucagon was diminished in hepatocytes from lactating rats. The activity ratio of cyclic AMP-dependent protein kinase (PK-A) also reacted similarly (in respect of both responsiveness and sensitivity) to isoprenaline in these two groups of hepatocytes. Contrastingly, the sensitivity of rat hepatocyte phosphorylase activity to beta-adrenergic stimulation was greatly diminished during lactation.


Blockade of insulin sensitive steady-state R-type Ca2+ channel by PN 200-110 in heart and vascular smooth muscle. The effect of high K+ concentration, insulin and the L-type Ca2+ channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca2+]i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca(2+)-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K+]o induced a rapid transient increase of [Ca2+]i that was followed by a sustained component. The early transient increase of [Ca2+]i by high [K+]o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca2+ blocker was found to decrease both the early transient and the sustained increase of [Ca2+]i induced by depolarization of the cell membrane with high [K+]o. Insulin at a concentration of 40 to 80 microU/ml only produced a sustained increase of [Ca2+]i that was blocked by PN 200-110 or by lowering the extracellular Ca2+ concentration with EGTA. The sustained increase of [Ca2+]i induced by high [K+]o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na+ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca2+ current and the delayed outward K+ current. These results suggest that the early increase of [Ca2+]i during depolarization of the cell membrane of heart and VSM cells with high [K+]o is due to the opening and decay of an L-type Ca2+ channel. However, the sustained increase of [Ca2+]i during a sustained depolarization is due to the activation of a resting (R) Ca2+ channel that is insensitive to lowering [ATP]i and sensitive to insulin.


Stable expression of the calbindin-D28K complementary DNA interferes with the apoptotic pathway in lymphocytes. The WEHI7.2 thymoma cell line undergoes apoptotic cell death when exposed to glucocorticoids and agents that increase intracellular cAMP. Several lines of evidence indicate that calcium may play an important role in events culminating in lymphocyte apoptosis. In these studies, calbindin-D28K was stably overexpressed in WEHI7.2 cells to determine if increasing the Ca(2+)-binding capacity of the cell interferes with the apoptotic pathway. Indeed, stable expression of calbindin-D28K decreased the apoptotic effects of dexamethasone and forskolin, and the level of resistance to these agents correlated with the relative amount of calbindin expressed in each line. Overexpression of calbindin also increased cell survival in the presence of the calcium ionophore A23187. The stably expressed calcium-binding protein appeared to exert its protective effect subsequent to transcriptional activation, since glucocorticoid- and cAMP-induced gene expression were not affected. These data support the proposal that calcium fluxes are involved in apoptosis and suggest that high level expression of proteins that buffer calcium fluxes can effectively suppress death in apoptosis-susceptible cells.


Stimulation of early gene expression by angiotensin II in bovine adrenal glomerulosa cells: roles of calcium and protein kinase C. The adrenal glomerulosa cell is a major site of action of angiotensin II (AII), which binds to AT1 receptors to stimulate phosphoinositide hydrolysis and Ca2+ mobilization, and the subsequent production of aldosterone. All also influences adrenal growth and proliferation and promotes thymidine incorporation in adrenocortical cells. In primary cultures of bovine glomerulosa cells, AII was found to induce the expression of several early growth response genes (c-fos, c-jun, JunB, and Krox 24). This effect of AII was dose-dependent and was blocked by [Sar1,IIe8] AII and the nonpeptide antagonist DuP 753, indicating that it is mediated by the AT1 subtype of the AII receptor. ACTH, which elevates cAMP in glomerulosa cells, was a relatively weak inducer of c-fos expression but was as potent as AII in stimulating the expression of JunB. ACTH did not further enhance the maximal effect of AII on c-fos expression. The role of the AII-induced cytoplasmic Ca2+ increase in generating the c-fos response was suggested by the ability of the Ca2+ ionophore ionomycin to induce c-fos expression. However, mobilization of intracellular Ca2+ by the Ca2+ ATPase inhibitor thapsigargin, as well as the stimulation of Ca2+ influx by depolarization with potassium, were less potent stimuli of c-fos expression. Omission of Ca2+ from the extracellular medium, which abolishes the plateau phase of the AII-induced Ca2+ signal without affecting the early increase due to Ca2+ mobilization, enhanced the early phase of the AII-induced c-fos response, indicating that Ca2+ also has an inhibitory effect on the early gene response. Activation of protein kinase C by phorbol 12-myristate, 13-acetate (PMA) also stimulated c-fos expression, but the combination of PMA and ionomycin did not further increase the c-fos response. Inhibition of protein kinase C by staurosporine, or its depletion by prolonged exposure to PMA, prevented the c-fos response to PMA but only partially inhibited the response to AII, suggesting the involvement of other factors in stimulus-transcription coupling from the AT1 receptor.


A comparison study of human papillomavirus prevalence by the polymerase chain reaction in low risk women and in a gynaecology referral group at elevated risk for cervical cancer. The reported prevalence of human papillomavirus (HPV) type 16 in the genital tracts of women with various gynaecological conditions is highly variable. In particular, some results with the polymerase chain reaction (PCR) technique have suggested that HPV-16 is a ubiquitous or very common virus. We undertook this study to help clarify the current confusion. PCR with HPV consensus L1 primers and specific E6 primers was used to study 89 women attending two gynaecology referral clinics, as well as 99 women attending a health maintenance organization (HMO) clinic; 70 of these latter women had no current or prior history of genital HPV disease. HPV-16 was detected in less than 5% of cytologically normal women from either group and in 17% (6/36) and 31% (9/29) of women with cervical intraepithelial neoplasia (CIN) from the referral clinic and the HMO, respectively. The other high-risk or intermediate-risk HPVs (types 18, 31, 33 or 35) were less prevalent than HPV 16 in all groups of women. A majority of the HPV types detected by the L1 primers in normal women were uncharacterized HPVs. Overall these uncharacterized HPVs were detected in 37% (46/123) of the normal women and in 48% (31/65) of the women with CIN. Using the most sensitive PCR product detection method employed in the study, HPV DNA was detected in 36% (4/11) of swab specimens obtained from the external abdomen.


Selective amplification of cDNA sequence from total RNA by cassette-ligation mediated polymerase chain reaction (PCR): application to sequencing 6.5 kb genome segment of hantavirus strain B-1. A method, referred to as cassette-ligation mediated polymerase chain reaction (PCR), has been developed to permit selective and specific amplification of cDNA sequence from total cellular RNA. This technique comprises (i) digestion of cDNA with multiple restriction enzymes, (ii) ligation of cleavage products to double-stranded DNA cassettes possessing a corresponding restriction site and (iii) amplification of cassette-ligated restriction fragments containing a short, known sequence (but not all the other ligation products) by PCR using the specific and cassette primers; the specific primer is designed to prime synthesis from the known sequence of the cDNA whereas the cassette primer anneals to one strand of the cassette. Sequencing from the cassette primer provides information to design a new primer for the next walking step. The amplified cDNA fragments are often larger than the maximum DNA fragments (500-600 bp) that can be sequenced without the need of synthesizing internal sequencing primer. Each of such large cDNA fragments is dissected into smaller DNA fragments by repeating cassette-ligation mediated PCR exploiting different restriction sites and different sets of cassette primers. This dissection process reduces the number of specific primers to a minimum, thereby increasing the speed of sequencing and minimizing the overall cost. We have successfully applied this cDNA walking and sequencing by the cassette-ligation mediated PCR to the sequencing of an entire 6.5 kb genome segment of hantavirus strain B-1.(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatitis B virus vaccine in The Gambia, West Africa: synergy between public health research and practice. Hepatitis B virus infection is endemic in West Africa, and as a result, this region has a high rate of hepatoma. The recent development of an effective, safe vaccine against hepatitis B virus that provides durable immunity may now make feasible the prevention of persistent hepatitis B virus infection and thus the prevention of hepatoma. A comprehensive primary health care program in which over 70% of children in The Gambia, West Africa, receive all their childhood immunizations afforded a unique opportunity to carry out a large public health research program to prevent hepatoma and at the same time strengthen the country's primary health care program. In 1986, phased introduction of hepatitis B virus vaccine was initiated in The Gambia's Expanded Programme on Immunization. By 1990, 124,577 children had been carefully identified and recruited into the 35-year longitudinal cohort study; 59,803 of these children received hepatitis B virus vaccine. The other 62,774 unvaccinated children will serve as controls. Preliminary studies have demonstrated that the rate of hepatitis B virus infection has fallen to less than 5% in children who received all four doses of hepatitis B virus vaccine. This study should provide the data from which the protective efficacy of hepatitis B virus vaccine against hepatoma can be accurately determined. In addition, it has energized The Gambia's Expanded Programme on Immunization so that at present more than 90% of all children are receiving all their childhood immunizations. This model cancer prevention program demonstrates the potential synergy between public health research and practice.


In vitro effect of diacetoxyscirpenol and deoxynivalenol on microbicidal activity of murine peritoneal macrophages. Diacetoxyscirpenol and deoxynivalenol, two trichothecene mycotoxins shown previously to exert immunosuppressive effects on the immune system were examined for their in vitro effects on some functions of murine peritoneal macrophages. The cells were pre-incubated for 4 hr with the mycotoxin concentrations of 0.1 ng/ml-1 micrograms/ml. At concentrations that did not affect the cell viability (Specific Lactate Dehydrogenase test), diacetoxyscirpenol and deoxynivalenol suppress microbicidal activity of phagocytic cells. The diacetoxyscirpenol concentrations, which reduce phagocytosis (2 ng/ml), microbicidal activity (1 ng/ml), superoxide anion production (1 ng/ml) and phagosome-lysosome fusion (0.1 ng/ml), indicate that the inhibition of killing mechanism arise from both oxidative and non-oxidative pathways. Phagocytosis, microbicidal activity and superoxide anion production are inhibited by deoxynivalenol at 1 ng/ml whereas phagosome-lysosome fusion is reduce above 100 ng/ml. These results suggest that microbicidal activity inhibition by deoxynivalenol did not depend on non-oxidative pathway (phagosome-lysosome fusion) impairment.


Prevention of deep vein thrombosis in neurosurgical patients: a prospective double-blind comparison of two prophylactic regimen. In a prospective, randomized, double-blind investigation of anticoagulant agents for prevention of deep vein thrombosis in patients undergoing operations at the lumbar-vertebral disc, 179 patients were randomly allocated to two groups. 87 patients received a fixed combination of low-molecular weight heparin 1,500 U-aPTT plus dihydroergotamine 0.5 mg (LMWH/DHE) once a day and additionally one injection of placebo per day, 92 patients received a fixed combination of sodium heparin 5,000 U plus dihydroergotamine 0.5 mg (HDHE) twice a day. Treatment was initiated two hours preoperatively in both groups and continued for at least seven days. Deep vein thrombosis (DVT), detected by the 125Iodine-labelled fibrinogen uptake-test, occurred in four patients treated with LMWH/DHE and in three patients with HDHE. In all seven patients phlebography was performed, confirming the diagnosis of DVT in one patient of the LMWH/DHE group and in two patients of the HDHE group, only. No increased bleeding was found in either group. Especially no neurological complications caused by epidural bleeding were observed. We therefore recommended to treat routineously all patients undergoing operations at the vertebral disc with antithrombotic agents. The advantages of the once daily regimen with low-molecular weight heparin include better patients' acceptance and less nursing time.


Endothelium myeloperoxidase-antimyeloperoxidase interaction in vasculitis. Antibodies to myeloperoxidase (MPO) are found in the sera of patients with microscopic polyarteritis and idiopathic crescentic glomerulonephritis. Their pathogenicity is unknown. Studies were carried out on the binding of MPO to cultured human umbilical vein endothelial cells and the recognition of endothelium-bound MPO by antibody to MPO. Endothelial cells were cultured from human umbilical veins. The binding of MPO to endothelial cells and its inhibition by poly-D-lysine was detected using a monoclonal antibody to MPO and direct staining with APAAP and also by an enzyme-linked immunoassay (ELISA). The binding of anti-MPO antibody in the sera of patients with microscopic polyarteritis to endothelium-coated MPO was detected by ELISA. MPO bound to endothelial cells both on direct staining and ELISA and this binding was inhibited by the polycation poly-D-lysine, suggesting that it was charge mediated. Binding of anti-MPO antibody in the sera of patients with microscopic polyarteritis to endothelium-coated MPO was significantly higher than the binding of sera from normal subjects (P = 0.04), patients with idiopathic glomerulonephritis (P = 0.0005), and patients with lupus nephritis (P = 0.009). MPO binds to cultured human umbilical vein endothelium probably by a charge mechanism, and can react with anti-MPO antibodies in the sera of patients with microscopic polyarteritis as well as with a mouse monoclonal anti-MPO antibody. This binding of anti-MPO antibody to MPO fixed to the endothelial cell surface provides a mechanism by which endothelial injury and inflammation might occur in microscopic polyarteritis.


No association between Alport's syndrome and antithyroid antibodies. The presence of circulating antithyroid antibodies in a small number of patients with Alport's syndrome has led some to suggest that the two abnormalities are associated. If confirmed, such an association could have important diagnostic as well as pathophysiological implications. Using sensitive methods, we assessed the prevalence of antithyroid antibodies in 40 patients with Alport's syndrome from 28 families. Antithyroglobulin antibodies were absent in all patients. Antimicrosomal and antithyroperoxidase antibodies were absent in all but one patient. Three patients with Alport's syndrome from the latter family were negative for antithyroid antibodies. In this large number of patients we found no association between Alport's syndrome and antithyroid antibodies. Screening for the presence of such antibodies in patients with Alport's syndrome is thus not warranted. The previously reported coexistence of the two disorders might be fortuitous.


Determination of functional kidney volume by single-photon emission computed tomography in patients with insulin-dependent diabetes mellitus. Functional parenchymal kidney volume was determined by single-photon emission computed tomography (SPECT) for 99mTc-dimercaptosuccinic acid (DMSA) using a rotating gamma camera in phantom experiments and in patients with insulin-dependent diabetes mellitus (IDDM). The results from the patient examinations were corrected according to the phantom studies and were thereafter set in relation to renal haemodynamics, blood pressure, and urinary albumin excretion. Functional parenchymal kidney volume was significantly greater in diabetic patients compared to that of 11 healthy controls (P < 0.003). Urinary albumin excretion was increased and glomerular filtration rate (GFR) per renal parenchymal volume significantly less in patients with a duration of diabetic disease of more than 15 years compared to patients with shorter duration of disease (P < 0.03 and P < 0.05 respectively). Diabetic patients with a GFR of more than 120 ml/min had greater renal parenchymal volume than patients with lower GFR (P < 0.02). Patients with increased GFR, renal plasma flow (RPF), renal blood flow, or filtration fraction had significantly greater functional parenchymal volume than the healthy subjects (P < 0.01 for all comparisons). We conclude that by application of SPECT for DMSA we were able to show that IDDM patients have greater renal parenchymal volumes than healthy subjects. GFR/kidney volume was increased in IDDM patients with a duration of disease of < 15 years compared to patients with long-standing diabetes. The SPECT technique seems suitable for prospective long-term follow-up studies of functional kidney volume in IDDM patients.


Diabetes mellitus prevalence among dialysed patients in France (UREMIDIAB study). The prevalence of diabetes mellitus among patients treated for end-stage renal failure was studied using a questionnaire mailed to all dialysis units of mainland France in 1989. With a response rate of 80.8%, the study population amounted to 12,903 dialysed patients of whom 884 were declared diabetic (6.9%). In a second phase, the study focused on the diabetic patients treated in the 63 largest units (those with at least four diabetic patients). Seven specially trained physicians completed questionnaires after having interviewed the patients and checked their medical records. All this material was reviewed by the same diabetologist. The conflict of diabetes type declared by both sources of information (the nephrologists and the diabetologist) showed a misclassification rate of 31.2%. Using these new data, the prevalence of type 1 diabetes mellitus was estimated at 1.4% of patients on dialysis therapy in mainland France, and 5.5% for type 2 diabetes mellitus. A north-south declining trend was suggested for type 2 diabetes mellitus. Diabetic nephropathy was the only primary renal diagnosis among 93.9% of type 1 diabetic patients, but only for 36.8% of type 2 diabetic patients. Of the latter, 51.6% had a non-diabetic cause of renal failure. These data show that the proportion of diabetics among patients receiving dialysis, while steadily increasing in France, remains lower than in other countries in Europe and in North America. However, the validity of international comparisons depends on diabetes ascertainment. Heterogeneity in selection of patients and in diabetes type classification by dialysis units may account to a considerable degree for the differences between diabetes mellitus prevalence across countries.


Identification of risk factors for radiographic hyperparathyroidism in 422 patients with end-stage renal disease: development of a clinical predictive index. The hand radiographs of 422 patients with end-stage renal failure were graded for severity of subperiosteal resorption. Two hundred and seventy-three patients (64.7%) had no evidence of resorption; 114 (27.0%) showed resorption, in 32 of whom it was severe (7.6% of the total). Thirty-five patients (8.3%) were assessed as having doubtful evidence of subperiosteal resorption. Age, gender, race, renal diagnosis, duration of renal failure and vitamin D status were assessed as potential risk factors for the development of subperiosteal resorption. Duration of renal failure, female gender, young age, and certain renal diagnostic groups namely obstructive uropathy, unknown diagnosis, presumed glomerulonephritis and tubulointerstitial disease emerged as independent risk factors. Diabetic patients appeared to be least at risk of developing subperiosteal resorption. Patients whose renal failure was of unknown duration showed a degree of risk similar to those whose duration was < 2 years. In order to identify prospectively patients likely to develop subperiosteal resorption by the time they reach renal replacement therapy, the relative risks were used to create a risk index. Use of such an index might allow prophylactic treatment to be given to those particularly at risk. The concept of a risk index requires testing by a prospective study, which is in progress.


Biochemical characterization of serum and urinary beta 2 microglobulin in end-stage renal disease patients. Since the identification of beta 2 microglobulin (beta 2-M) in haemodialysis-associated amyloidosis, the biochemical characterization of the different forms of beta 2-M has been sought by several groups. New beta 2-M isoforms (pI 5.1 and lower) have been identified in amyloid deposits, and it has been suggested that they are of pathogenetic importance. The finding of N-terminal proteolysed beta 2-M in amyloid deposits prompted the hypothesis that proteolysis would render beta 2-M more amyloidogenic. Finally, a 'novel beta 2-M' (pI 5.2) with a single amino acid replacement (Asn by Asp at position 17) has been reported as possibly specific for patients with dialysis associated amyloidosis, and consequently proposed as 'the amyloidogenic' form. We purified beta 2-M from serum of a newly haemodialysed patient and from urine of a transplanted patient in the early recovery period. Both patients were clinically amyloid free. Three pure isoforms were obtained from serum (pI 5.7, 5.3, and 5.1) and only two from urine (5.7 and 5.3). Further purification of each isoform was obtained by HPLC in a C4 column. Sequence analysis showed that all isoforms had an intact N-terminus. Tryptic digestion of the serum isoforms was performed after alkylation with iodoacetic acid and the peptides were isolated by HPLC in a C18 column. The 5.3 and 5.1 isoforms had identical peptide patterns with the appearance of an early peak missing in the 5.7 form. The sequence of this peptide showed a replacement of the D 42 (Asp 42) by N (Asn) after K41 (Lys 41).(ABSTRACT TRUNCATED AT 250 WORDS)


Vascular access surgery: a 2-year study and comparison with the Permcath. The results of a 2-year prospective study of primary and secondary vascular access surgery for haemodialysis have been compared with a retrospective study of central venous access via a flexible silicone catheter (Permcath). Cumulative patency for 61 primary fistulae in 57 patients was 64.8% at 1 year and 57.7% at 2 years. The patency of 55 secondary procedures in 43 patients was 48.1% at 1 and 2 years. Cumulative survival of 64 Permcaths inserted into 51 patients was 74% at 1 year and 43% at 2 years. Surgical complications included explorations for bleeding (2), haematomas (4), swollen arms (4), and inadequately dilated veins (4). Permcath complications included explorations for bleeding (3) and a temporary recurrent laryngeal nerve palsy (1). Exit site infection and septicaemia rates were 4.95 and 3.36 per 1000 catheter days respectively, but 20.6% of septicaemic episodes occurred in a patient who refused catheter removal. For haemodialysis, the Permcath is comparable with secondary vascular access. The Permcath may have a primary access role in patients with limited life expectancy.


Recurrence of SLE in transplanted kidneys: a follow-up transplant biopsy study. Renal transplant biopsies were obtained from 16 patients with systemic lupus erythematosus 6 months to 11 years post-transplant. Eight biopsies were taken on clinical grounds while eight were elective. Histopathological findings suggesting recurrent lupus nephritis were found in seven biopsies, five of which were taken on clinical indication. By light-microscopy, five graft biopsies showed proliferative glomerulopathy and two glomerulosclerosis. Immunofluorescence was positive for IgM and C3 in a finely granular pattern in all biopsies, for C1q in three, but for IgG in only two. Electron-dense deposits were found in all seven biopsies with predominantly subendothelial location. All but one patient had clinical signs of renal involvement, but only three had extrarenal symptoms and three had serological signs of active SLE. Upon increased immunosuppressive therapy, renal and serological signs improved but one graft was later lost due to recurrent SLE nephritis.


Antagonist capacity of felodipine on cyclosporin A nephrotoxicity in the rat. Functional and morphological cyclosporin A (CsA) nephropathy has been attributed to a CsA-induced constriction of the afferent glomerular arteriole. Calcium-channel blockade with nifedipine prevented the development of short-term functional nephrotoxicity in CsA-treated rats. This study investigated whether the calcium antagonist felodopine, a structural analogue of nifedipine, which reduces renal tubular fractional sodium reabsorption, could prevent both short- and long-term functional and long-term morphological CsA nephropathy. In short-term experiments, four groups of Spraque-Dawley rats (n = 39) were given CsA (either 0 or 12.5 mg/kg per day by daily gastric intubation for 2 weeks), and felodipine (0 or 30 mg/kg per day) in the diet. In long-term experiments, rats (n = 39) were given CsA (12.5 mg/kg per day for 16 weeks), and felodipine (0 or 30 mg/kg per day in the diet). Renal function was investigated with clearance methods (inulin, lithium, and sodium), and kidney morphology was studied by light-microscopy. In short-term experiments, CsA treatment reduced GFR (730 versus 1181 microliters/min per g kidney weight (KW), P < 0.05) and CLi (130 versus 271 microliters/min per gKW, P < 0.02). Felodipine decreased proximal fractional reabsorption (PFR) (67.5% versus 71.4%, P < 0.05) and increased CNa (15.9 versus 8.4 microliters/min per gKW, P < 0.02) as compared to controls. In CsA-treated rats felodipine increased C in (1260 versus 730 microliters/min per gKW, P < 0.05) and CLi (319 versus 130 microliters/min per gKW, P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of captopril treatment on allograft survival and induction of unresponsiveness in heart-transplanted, ATG-treated rats. The angiotensin-converting enzyme inhibitor captopril has been suggested to have an immunomodulatory effect both in clinical and experimental studies. To further investigate this possible effect in organ transplantation, captopril was given to inbred rats before transplantation of an allogeneic heart. Captopril was found to prolong graft survival significantly compared to untreated controls. In rats treated with a tolerogenic dose of ATG, additional captopril administration tended to increase the proportion of rats with long-term functioning grafts, but this did not reach statistical significance. It is concluded that captopril displays an immunosuppressive effect that seems to be weak, since no augmentation of ATG-induced transplantation unresponsiveness was seen.


Postsynaptic action of endogenous GABA released by nipecotic acid in the hippocampus. Intracellular and whole-cell recording from CA1 pyramidal cells and dentate granule cells was used to study the release of endogenous GABA by nipecotic acid. Local application of nipecotic acid produced responses that could be entirely blocked by a combination of the GABAA receptor antagonist picrotoxin and the GABAB receptor antagonist CGP 35348. These responses were due to the heteroexchange release of endogenous GABA because they were blocked by low Na+ which blocks the GABA transporter and by SKF 89976 which is a competitive antagonist of the GABA transporter. Local application of nipecotic acid could, depending on the location, evoke pure GABAA or pure GABAB responses supporting proposals that GABAA and GABAB receptors can be segregated at separate inhibitory synapses.


Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localisation of the kinase. Glycogen synthase kinase-3 (GSK-3) reduced the mobility of human tau on SDS-PAGE, prevented binding of the monoclonal antibody (mAb), Tau.1, and induced binding of the mAb 8D8. Recombinant tau phosphorylated by GSK-3 aligned on SDS-PAGE with the abnormally phosphorylated tau (PHF-tau) associated with the paired helical filaments in Alzheimer's disease brain. Phosphorylated serine396 (numbering of the largest human brain tau isoform) was identified as a binding site on tau for mAb 8D8. The localisation of GSK-3 within granular structures in pyramidal cells indicates that GSK-3 alpha and GSK-3 beta may have a role in the production of PHF-tau in Alzheimer's disease.


Inhibitory effects of psychotomimetic sigma ligands on nicotine-induced K+ flux from differentiated PC12 cells. In NGF-treated PC12 cells, nicotine-induced K+ release was measured with a K(+)-sensitive microelectrode. The K+ outflow via nicotinic ACh receptor cation channels was inhibited by various psychotomimetic sigma ligands in the sequence of PCP, dextromethorphan >> DTG, MK 801, (+)SKF10047 >> (+)3-PPP. The K+ release was not affected by the neuroleptic sigma ligand haloperidol nor by the calcium antagonist nifedipine. The results suggest that psychotomimetic sigma ligands inhibit nicotine-stimulated K+ flux by interacting with nicotinic, rather than via sigma 2 receptors.


A hypothesis regarding possible interactions between the pressure-induced disorders in dopaminergic and amino-acidergic transmission. When human divers or experimental animals are exposed to high pressure they develop the high pressure neurological syndrome (HPNS). The main symptoms include electroencephalographic changes and behavioral disturbances such as tremor, myoclonia, and hyperlocomotor activity. Recently, pressure-induced disorders in dopaminergic and amino-acidergic neurotransmission have been reported. In the present theoretical study, we review in vitro and in vivo neurochemical, electrophysiological, and pharmacobehavioral evidence concerning alterations in dopaminergic, glutamatergic, and GABAergic transmission occurring at high pressure, and their possible relationship to the symptoms of HPNS. Moreover, we also examine data concerning interactions, at normal pressure, between dopaminergic, glutamatergic, and GABAergic transmission that we suggest they could apply equally under high pressure between the pressure-induced disorders in dopaminergic and amino-acidergic transmission.


Acyclovir-resistant herpes simplex virus keratouveitis after penetrating keratoplasty. A case of acyclovir-resistant herpes simplex virus keratouveitis after penetrating keratoplasty is reported. Resistance to acyclovir was evident clinically and was confirmed by in vitro susceptibility testing. The susceptibility of the herpes simplex isolates to acyclovir and foscarnet was determined by a dye uptake assay that measured cytopathic effect, and thymidine kinase activity was measured by a plaque autoradiography technique. The viral isolate from postoperative day 22 was susceptible to acyclovir and foscarnet, and showed normal thymidine kinase activity. Isolates from postoperative days 29 and 32 (coinciding with deterioration in clinical appearance) were resistant to acyclovir, susceptible to foscarnet, and deficient in thymidine kinase activity. Practitioners should be aware of the potential for the emergence of resistance in this setting; prophylaxis and rational alternate therapies are discussed.


[Placental development and viral infections]. The stages of placental development are briefly described as well as the cellular and molecular mechanisms involved in the immunologic defense systems at the maternofetal interface. Methods available for morphologic studies of placentas with viral infection are reviewed. Routes of transmission of viruses from mother to fetus, as well as the placental lesions seen in viral infections are discussed. Emphasis is put on the value of "oriented" pathologic studies of the placenta whenever viral fetal disease is suspected.


[Cytomegalovirus and the fetus].

[Cytomegalovirus and the fetus]. The literature on the risks to the fetus and neonate of maternal cytomegalovirus infection, whether primary or reactivated, is briefly reviewed. The various rates of risks reported and the mechanism of fetal infection are discussed. Screening for maternal infection, antenatal diagnosis of fetal infection, and development of new antiviral treatments and immunizations are advocated.


Neonatal herpes simplex virus infections: pathogenesis and therapy. Neonatal herpes simplex virus (HSV) infections are of increasing incidence in North America, now occurring at a rate of approximately one in 3,500 to one in 5,000 deliveries per year. Disease manifests as one of three forms; namely, infection: localized to the skin, eye and mouth (SEM), encephalitis (CNS), or disseminated disease. With the advent of antiviral therapy, it has become possible to decrease mortality and improve morbidity for babies suffering from infection. Advances in antiviral therapy have allowed for prevention of disease progression beyond states of SEM involvement. Furthermore, life threatening infections of the CNS or of multiple organs, have mortality with either acyclovir or vidarabine therapy. Now approximately 15% (CNS) and 50% (disseminated disease) of babies die from neonatal HSV disease. The results of ongoing studies in the United States will summarize the pathogenesis and treatment of neonatal HSV infection.