Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation.

We have developed an experimental paradigm to study the mechanism by which nerve growth factor (NGF) allows the survival of sympathetic neurons. Dissociated sympathetic neurons from embryonic day-21 rats were grown in vitro for 7 d in the presence of NGF. Neurons were then deprived of trophic support by adding anti-NGF antiserum, causing them to die between 24 and 48 h later. Ultrastructural changes included disruption of neurites, followed by cell body changes characterized by an accumulation of lipid droplets, changes in the nuclear membrane, and dilation of the rough endoplasmic reticulum. No primary alterations of mitochondria or lysosomes were observed. The death of NGF-deprived neurons was characterized biochemically by assessing [35S]methionine incorporation into TCA precipitable protein and by measuring the release of the cytosolic enzyme adenylate kinase into the culture medium. Methionine incorporation began to decrease approximately 18 h post-deprivation and was maximally depressed by 36 h. Adenylate kinase began to appear in the culture medium approximately 30 h after deprivation, reaching a maximum by 54 h. The death of NGF-deprived neurons was entirely prevented by inhibiting protein or RNA synthesis. Cycloheximide, puromycin, anisomycin, actinomycin-D, and dichlorobenzimidazole riboside all prevented neuronal death subsequent to NGF deprivation as assessed by the above morphologic and biochemical criteria. The fact that sympathetic neurons must synthesize protein and RNA to die when deprived of NGF indicates that NGF, and presumably other neurotrophic factors, maintains neuronal survival by suppressing an endogenous, active death program.

Increased carbamazepine plasma concentrations after fluoxetine coadministration.

The interaction between fluoxetine and carbamazepine was investigated in six normal, healthy male volunteers (aged 23 to 40 years). Subjects were given carbamazepine, 400 mg every morning, for 3 weeks. Venous carbamazepine blood samples were obtained at baseline and 1, 2, 4, 6, 8, 10, 12, and 24 hours after the morning dose. Fluoxetine, 20 mg every morning, was then coadministered with carbamazepine for 7 days. Venous carbamazepine blood samples were again obtained as described. Carbamazepine and carbamazepine-10,11-epoxide (CBZE) were assayed by HPLC. Addition of fluoxetine resulted in a significant increase in the area under the concentration-time curve of carbamazepine (105.93 +/- 18.05 micrograms/ml.hr versus 134.97 +/- 12.15 micrograms/ml.hr; t = 3.284; df = 5; p = 0.022) and CBZE (11.6 +/- 1.93 micrograms/ml.hr versus 15.2 +/- 2.4 micrograms/ml.hr; t = 2.805; df = 5; p = 0.038). Both oral and intrinsic clearance of carbamazepine was decreased significantly on fluoxetine addition (3.87 +/- 0.68 L/hr versus 2.98 +/- 0.26 L/hr; t = 3.025; df = 5; p = 0.029 and 17.90 +/- 4.9 L/hr versus 11.92 +/- 1.4 L/hr; t = 3.037; df = 5; p = 0.029, respectively). No significant changes were determined for fraction of absorbed dose, volume of distribution, absorption rate constant, and elimination rate constant. These findings suggest that fluoxetine can inhibit the metabolism of carbamazepine. Careful monitoring of patients is recommended when these two drugs are coadministered.

Role of nicotinamide adenine dinucleotide in ethanol-induced depressions in testicular steroidogenesis.

It is rapidly becoming accepted, without direct evidence, that a change in the NAD+/NADH ratio in the testes produced by the metabolism of ethanol is the principal mechanism involved in its now well-established effects on testicular steroidogenesis. The purposes of the present studies were 2-fold: (1) to examine whether, in fact, in vivo or in vitro ethanol exposure alters the NAD+/NADH ratio in the testes; and (2) to examine the validity of previous reports in which it was found that NAD+ prevented the effects of ethanol on testicular steroidogenesis under in vitro conditions. With regard to the first objective, we found that a large dose of ethanol (2.5 g/kg) markedly reduced gonadotropin-stimulated testicular steroidogenesis in vivo in the male rat, but it did not alter the NAD+ and NADH concentrations in the testes. Similarly, extremely high ethanol concentrations (200 mM) substantially suppressed hMG-stimulated testosterone biosynthesis in in vitro Leydig cell preparations but no change in NAD+ concentration occurred; NADH levels were very low in the Leydig cell preparations (less than 2% of NAD+ levels), but did not appear to change as a function of ethanol exposure. Finally, in contrast to previously published results, we found that NAD+ (1 mM) did not prevent the in vitro effects of ethanol on cAMP-stimulated testicular steroidogenesis. Consequently, our results fail to support the hypothesis that acute in vivo or in vitro ethanol administration inhibits the biosynthesis of testosterone by altering the NAD+/NADH ratio in the testes.

Metabolite changes associated with heat shocked avian fibroblast mitochondria.

A previous report from our laboratory (Collier et al 1993) showed that the elongated tubules of mitochondria in the cytoplasm of cultured chicken embryo fibroblasts collapsed to irregularly shaped structures surrounding the nuclear membrane after a 1 h heat shock treatment. The normal mitochondrial morphology reappeared upon removal of the thermal stress. We have now determined that several changes occurred in mitochondrial-related metabolites under these same heat shock and recovery conditions. Among these were significant decreases in the levels of fumarate and malate and increases in the amounts of aspartate and glutamate. In contrast, other intermediates of the tri-carboxylic acid cycle were unaltered as were levels of ATP and phosphocreatine. The changes observed might result from heat shock-induced changes in enzyme activities of the mitochondria, from alterations in the membrane-embedded specialized carrier proteins that transport metabolites between cytosol and mitochondria or from a disorganization of the electron-transport system normally coupled to oxidative metabolism. The rapid recovery, however, suggested that these changes were transient and readily reversible.

Vasomotor dynamics associated with cardiac operations. II. Effect of cardiopulmonary bypass on venous tone.

The effect of cardiopulmonary bypass on venous tone was evaluated in 19 patients undergoing coronary artery bypass graft operations. The use of cardiopulmonary bypass with reduction of mean arterial pressure, nonpulsatile blood flow, and emptying of the heart maximally stimulates the sympathoadrenal system and would be expected to cause intense venous constriction. Venous capacitance, however, was reduced by the general effects of the operation, including anesthesia and surgical trauma, while cardiopulmonary bypass was a minor factor in the venous constriction response.

The effects of cardiopulmonary bypass and cold cardioplegia on coronary flow velocity and the reactive hyperemic response in patients and dogs.

In normal coronary arteries, reactive hyperemic responses to a 20-second occlusion, an index of coronary reserve, usually demonstrate a peak-to-resting flow velocity ratio of 4:1 or more. Most intraoperative studies that have assessed reactive hyperemic responses in bypassed vessels have reported peak-to-resting flow velocity ratios of 2:1 or less following a 20-second occlusion. These decreased reactive hyperemic responses could be due to coronary vasodilatation after cardiopulmonary bypass or to an inadequate physiological result of the surgical procedure. In 14 patients with angiographically normal coronary arteries, the peak-to-resting flow velocity ratio following a 20-second coronary occlusion decreased significantly (p less than 0.05) from 4.4 +/- 0.2 (mean +/- standard error) before bypass to 3.0 +/- 0.3 after bypass. In a similar dog model, the peak-to-resting flow velocity ratio decreased by 36 to 52% during the first hour following one hour of cardiopulmonary bypass and cardioplegia. During the same period, left ventricular perfusion increased 21 to 30%, mean arterial pressure and coronary vascular resistance decreased, and myocardial oxygen consumption was unchanged. In a second group of dogs studied for the effects of duration (200 to 240 minutes) of anesthesia and thoracotomy alone, peak-to-resting flow velocity ratio was significantly lower. These clinical and experimental studies suggest that major coronary vasodilatation occurs early following cardiopulmonary bypass and cold cardioplegia, and may contribute to the blunted coronary reactive hyperemic responses reported during this time. Consequently, an intraoperative peak-to-resting flow velocity ratio of 3:1 for bypassed coronary arteries may represent an excellent physiological result.

Glutamate and potassium stimulation of hippocampal slices metabolizing glucose or glucose and pyruvate.

Using 2-deoxyglucose phosphorylation as an index of glucose use and concentrations of selected intermediates to monitor metabolic pathways, responses of rat hippocampal slices to glutamate and K+ stimulation were examined. With glutamate, the glucose phosphorylation rate (GPR) increased, and the slices accumulated glutamate at a constant rate, for 10 min. The uptake rate at each glutamate level was matched, approximately, by the increase in GPR at that level, with 4 or 5 glutamate molecules accumulated for every glucose molecule phosphorylated. Phosphocreatine and ATP levels fell abruptly, and lactate rose, probably reflecting neuronal activity, found by others to be very brief in the presence of glutamate. K+ stimulation produced responses of phosphocreatine, ATP and lactate levels and of GPR similar to those due to glutamate. There were also prolonged changes in the levels of other metabolites: with both stimulants glucose 6-phosphate fell, and malate rose. The changes in malate may be the result of the participation of mitochondrial malate dehydrogenase in both citrate cycle and malate shuttle. Citrate and alpha-ketoglutarate rose only with K+. When pyruvate was added to the medium, resting GPR was reduced, but for both stimulants the relative increases in GPR with stimulation were the same as without pyruvate. The changes in metabolic intermediates in response to K+ were like those with glucose alone. But with glutamate, the rise in lactate was greatly diminished, and malate fell instead of rising. Glutamate interference with the transfer of both 3-carbon as well as 4- and 5-carbon intermediates from glia to neurons may explain these results. If so, this interference is greater with pyruvate supplementation than with glucose alone.

Personal toll of trichotillomania: behavioral and interpersonal sequelae.

Patients suffering from trichotillomania are at risk for significant mood and interpersonal problems. Using a clinical sample, this study sought to clarify the nature and types of problems experienced by patients and to examine how these problems might be interrelated. The charts of 67 patients who sought treatment for trichotillomania were reviewed. The majority of patients reported problems with affect and interpersonal relationships. Public and social activities (e.g., haircuts, sexual activities) were avoided by a large number of patients. Self-esteem, shame, feelings of unattractiveness, depressed affect, and secretiveness were all interrelated, suggesting that these issues might best be conceptualized as a cluster that needs to be considered in the etiology, effects, and treatment of trichotillomania.

Malignant hyperthermia.

A short review of the history, incidence, and present-day concept of the etiology of malignant hyperthermia is presented. Protocols for diagnosis, treatment of the unexpected occurrence, and management of known malignant hyperthermic patients is presented. The exact etiology of the syndrome is still unknown.

Splicing factor polymorphisms, the control of VEGF isoforms and association with angiogenic eye disease.

PURPOSE: Alternative splicing of the last exon (exon 8) of vascular endothelial growth factor (VEGF) pre-mRNA is a key element in the balance of pro- and anti-angiogenic VEGF isoforms in exudative age-related macular degeneration (exAMD) and proliferative diabetic retinopathy (PDR). Three splicing factors, SRp40, ASF/SF2, and SRp55 are predicted to control alternative splicing by binding to exonic splice enhancers (ESE) in VEGF exon 8. This pilot study examines whether there is an association between angiogenic eye disease and splicing factor polymorphisms, and whether there are sequence variations in the alternative splice sites of the VEGF gene. MATERIALS AND METHODS: A case:control pilot study comparing 163 individuals with angiogenic eye disease (94 exAMD and 69 PDR patients) with 95 age-matched controls. Splicing factor polymorphisms were genotyped by Restriction Fragment Length Polymorphism (RFLP) and sequencing, and the VEGF alternatively spliced region was assessed by denaturing High Performance Liquid Chromatography (dHPLC) using a transgenomic WAVE heteroduplex analyzer. RESULTS: No variations were observed in the alternatively spliced region of VEGF exon 8. ASF/SF2 polymorphisms showed no association with exAMD or PDR. For PDR, we observed a trend in SRp40 (rs6573908) where the 5136CC genotype was more frequent in controls (p = 0.0517) and a significant association of the SRp55 (rs2235611), where the 2994C allele was more common in the PDR group (p = 0.03). This remained strong, but not significant, after logistic regression for age, sex, disease type, and duration (p = 0.06). CONCLUSIONS: The lack of variation in the VEGF alternatively spliced region suggests the importance of sequence conservation in this area in maintaining the balance of pro- and anti-angiogenic VEGF isoforms. The link between PDR and the SRp55 2994 polymorphism suggests a disease-specific association between factors controlling VEGF splicing and ocular angiogenesis.