Genetic analysis of von Hippel-Lindau disease.

Mutations in the von Hippel-Lindau (VHL) gene are responsible for VHL disease, congenital polycythemia, and are found in many sporadic tumor types as well. Reports of VHL mutations are dispersed throughout original articles and databases that have not been recently updated. We compiled a comprehensive mutation table of 1,548 germline and somatic VHL mutations, derived from this protein of only 213 amino acids. We describe detailed phenotype and gene mutation information for 945 VHL families, including 30 previously unpublished kindreds from The Netherlands (six novel mutations). These data represent the most extensive catalog of germline VHL mutations to date. We also review VHL disease, known and theorized pathogenesis of common VHL manifestations, and genotype-phenotype correlations. Analysis of all VHL families, excluding germline mutations resulting in congenital polycythemias, describes the spectrum of mutation types: 52% missense, 13% frameshift, 11% nonsense, 6% in-frame deletions/insertions, 11% large/complete deletions, and 7% splice mutations. This easy-to-use compilation of VHL mutations is intended to facilitate research and function as a necessary adjunct for physicians when providing patient information.

TCF11/Nrf1 overexpression increases the intracellular glutathione level and can transactivate the gamma-glutamylcysteine synthetase (GCS) heavy subunit promoter.

Gamma-glutamylcysteinylglycine or glutathione (GSH) performs important protective functions in the cell through maintenance of the intracellular redox balance and elimination of xenobiotics and free radicals. The production of GSH involves a number of enzymes and enzyme subunits offering multiple opportunities for regulation. Two members of the CNC subfamily of bZIP transcription factors (TCF11/Nrf1 and Nrf2) have been implicated in the regulation of detoxification enzymes and the oxidative stress response. Here we investigate the potential role of one of these factors, TCF11/Nrf1, in the regulation of GSH levels in the cell and particularly its influence on the expression of one of the enzymatic components necessary for the synthesis of GSH, the heavy subunit of gamma-glutamylcysteine synthetase (GCS(h)). Using overexpression of the transcription factor in COS-1 cells we show that TCF11/Nrf1 stimulates GSH accumulation. Using co-transfection with reporter constructs where reporter expression is driven through the GCS(h) promoter we show that this increase may be mediated in part by induced expression of the GCS(h) gene by TCF11/Nrf1. We further show that a distal portion of the promoter including two antioxidant-response elements (AREs) predominantly mediates the TCF11/Nrf1 transactivation and an electromobility shift assay showed that just one of these AREs specifically binds TCF11/Nrf1 as heterodimers with small Maf proteins. We suggest that TCF11/Nrf1 can operate through a subset of AREs to modulate the expression of GCS(h) together with other components of the pathway and in this way play a role in regulating cellular glutathione levels.

VIP-sensitive adenylate cyclase, guanylate cyclase, muscarinic receptors, choline acetyltransferase and acetylcholinesterase, in brain tissue afflicted by Alzheimer's disease/senile dementia of the Alzheimer type.

Biochemical parameters were determined in autopsy material from several brain regions of thirteen patients with Alzheimer's disease/senile dementia of Alzheimer type (AD/SDAT) (mean age 75 years) and from brains of ten age-matched controls (mean age 76 years). Choline acetyltransferase specific activity was significantly lower in the nucleus caudatus, putamen, left thalamus, hippocampus and the cortex from gyrus hippocampus and the temporal lobe in AD/SDAT, acetylcholinesterase specific activity was significantly lower in the hippocampus, parietal and left frontal lobe in AD/SDAT samples than in corresponding samples from aged-matched controls. A compensatory increase of muscarinic receptors was found in the nucleus caudatus and left frontal lobe samples in AD/SDAT. Guanylate cyclase activity was not significantly altered in AD/SDAT in the brain regions examined. The basal, non-stimulated activity of adenylate cyclase was significantly (p less than 0.05) elevated in hippocampus samples from AD/SDAT patients and the enzyme activity stimulated by the vasoactive intestinal polypeptide VIP (2 microM) or forskolin (10 microM) was also elevated in AD/SDAT although not significantly.

Intact human lymphocyte membranes respond to muscarinic receptor stimulation by oxotremorine with marked changes in microviscosity and an increase in cyclic GMP.

The muscarinic agonist oxotremorine produced a linear dose-dependent increase in membrane fluidity of intact and viable human lymphocytes in vitro. This effect proved to be receptor-mediated because preincubation with 10(-5)M atropine shifted the dose-response curve one order of magnitude rightward. Pirenzepine preincubation did not affect membrane fluidity variation. A cGMP increase was also found after oxotremorine treatment. The results are discussed in terms of possible modulation of guanyl cyclase and adenyl cyclase through membrane fluidity variations.

Muscarinic receptors and receptor-mediated actions on rat thymocytes.

Rat thymocytes possess a single class of saturable, high affinity binding sites for muscarinic antagonists of the benzilate type such as [3H]3-quinuclidinyl benzilate ([3H]3-QNB). The average number of receptors per cell is 3000 and the equilibrium dissociation constant of [3H]3-QNB on intact cells is 7.5 nM. In the work reported here we found that perturbation of the thymocyte membrane by addition of phytohemagglutinin (4 micrograms/ml) caused a transient increase in muscarinic antagonist binding, and hydrocortisone (100 mg/kg s.c.) treatment of rats for 2 days prior to sacrifice increased the average number of muscarinic receptor sites on thymocytes by 100%. Atropine treatment, which in other tissues causes increased muscarinic receptor concentration, did not alter the receptor number on thymocytes. Binding of carbachol to the receptor on intact cells resulted in inhibition of cAMP synthesis and stimulation of cGMP synthesis. These muscarinic agonist effects were each inhibited by the simultaneous addition of the muscarinic antagonist atropine (5 X 10(-5) M). No stimulation of phosphatidylinositol turnover by muscarinic agonists was observed.

Increased affinity of choline acetyltransferase for choline in Alzheimer's disease: a steady-state kinetic study.

The steady-state kinetics of choline acetyltransferase (CAT) from autopsy samples of human caudate nucleus of aged controls and of patients with Alzheimer's disease was studied. In 10 samples from Alzheimer's disease-afflicted brains the affinity for the limiting substrate choline (Ch) was significantly higher: Michaelis constant KmCh was for these samples 1.93 +/- 0.72 mM while in the samples from 9 age-matched controls KmCh was 2.53 +/- 0.78 mM. The difference is statistically significant (P less than 0.05). Endogenous choline concentrations in the samples were 124 +/- 39 (n = 10) nmol/g wet wt. in the Alzheimer's disease-afflicted samples and 180 +/- 57 (n = 9) nmol/g wet weight (n = 9) in the control samples (P less than 0.05). The initial velocity at 70 microM acetyl co-enzyme (AcCoA) in Alzheimer's samples was 171.5 +/- 131.0 pmol [14C]acetyl choline [14C]ACh/mg protein/min as compared to the controls 422.1 +/- 231.0 pmol [14C]ACh/mg protein/min replicating many previous findings about decline of CAT activity in Alzheimer's disease. However, in the same samples the affinity for the other substrate acetyl-CoA (AcCoA) was significantly lower for the Alzheimer patients, KmAcCoA = 61 +/- 40 microM, than for the age-matched control patients, KmAcCoA = 28 +/- 8 microns (P less than 0.01). The data suggest some compensation of the loss of enzyme molecules via changed affinity for the limiting substrate, Ch.

Pre- and postsynaptic muscarinic receptors in surgical samples from human cerebral cortex.

The present study was carried out using fresh surgical material from human cerebral cortex of patients who were not medicated with atropine or other drugs known to affect the cholinergic system. The concentration of [3H]L-quinuclidinyl benzilate binding sites was 0.45 /+- 0.05 pmol/mg protein and the Kd-value of the receptor-[3H]L-QNB-complex was 0.038 /+- 0.005 nM. Agonist binding was studied by varying the concentration of carbamylcholine (10(-8) to 10(-2) M) in the presence of a constant concentration (0.2nM) of [3H]L-quinuclidinyl benzilate. The data revealed the existence of two populations of binding sites for carbamylcholine with different affinities and capacities. Presynaptic muscarinic receptors were studied in slices of the cerebral cortex, which were loaded with [3H]choline. The muscarinic antagonist, atropine (10(-6) and 10(-7) M) acting at the presynaptic muscarinic receptors enhanced the release of [3H] acetylcholine. It was also shown that muscarinic stimulation leads to elevation of cyclic GMP levels in the human cerebral cortical slices.

Disappearance of low affinity adenosine binding sites in aging rat cerebral cortex and hippocampus.

A1 adenosine receptor binding was investigated, using the selective agonist, [3H]cyclohexyladenosine, on membranes prepared from the cerebral cortex and hippocampus of 3- and 24-month-old rats. The Scatchard analysis of the binding results obtained in the cerebral cortex of young animals showed two distinct binding sites with apparent Kd of 2 and 24 nM and Bmax of 259 and 675 fmol/mg protein, respectively. Conversely, in the old rats only one population of high affinity binding sites with a Kd of 2.2 nM and a Bmax of 450 fmol/mg protein was found. Displacement curves of labelled ligand carried out on hippocampal membranes also demonstrate the disappearance of a low affinity subpopulation of A1 receptors in the old rat brain.

Evidence for an inhibitory effect of the peptide galanin on dopamine release from the rat median eminence.

The role of the neuropeptide galanin (GAL) in rat hypothalamus has been studied in different experimental models. Thus, the effect of GAL on potassium-induced dopamine release was analyzed in vitro, and the localization of GAL and GAL binding sites was studied with immunohistochemistry and receptor autoradiography, respectively. In the median eminence GAL and presumably dopamine were found to coexist in nerve endings and this area contained a high density of 125I-GAL binding sites. In vitro experiments revealed that GAL inhibited the release of [3H]dopamine in a dose-dependent manner (IC50 = 7-10 nM), possibly via a presynaptic receptor.

[Qualitative aspects of migration research]. / Kvalitativa aspekter inom migrationsforskningen

PIP: The author suggests that the emphasis in migration studies should be shifted from quantitative to qualitative concerns, particularly in studies of migration in developing countries. He argues that more emphasis should be placed on the environment the migrant left and has now experienced in the new place of residence and on the transfer of knowledge that was an essential accompaniment of the migration process. (summary in ENG)^ieng

Recall during intermittent propofol anaesthesia.

We discontinued temporarily an infusion of propofol for surgical reasons in 20 patients undergoing incontinence surgery. The patients, who had not received neuromuscular blockers, were allowed to regain consciousness to a level enabling them to cough on command, open their eyes, and identify and verbally confirm a randomly assigned digit shown on paper. Thereafter, 5-14 min after discontinuation of the propofol infusion, anaesthesia was reinstituted. Memory of the request to cough, a standard conversation and the digit shown was tested 1 h after anaesthesia and on the following day. Only 35% of patients were able to recall one or more of the stimuli presented during wakefulness or were even able to recall having been "awake", and there were very few differences in memory on the day after surgery compared with 1 h after anaesthesia. In comparison with corresponding stimuli given before anaesthesia, memory of material learned during wakefulness was significantly impaired (P < 0.0001). Thus patients temporarily capable of cognitive action during propofol anaesthesia may have no subsequent explicit recall of intraoperative events.

Muscarinic autoreceptor regulates acetylcholine release in rat hippocampus: in vitro evidence.

Release of 3H-ACh from isolated nerve endings of rat hippocampus was evoked by incubation in Krebs-Ringer's buffer containing 25 or 35 mM potassium. The release was Ca2+-dependent and could be inhibited by Mg2+ (20 mM). The muscarinic antagonist, atropine (10(-10)-10(-6) M), enhanced 3H-ACh-release. The muscarinic agonist, carbachol (10(-5)-10(-3) M) inhibited 3H-ACh release via interaction with muscarinic receptors: this effect could be blocked by atropine (10(-6) M). The presence of the feed-back regulation of 3H-ACh release in a cell-free preparation provides further evidence that the presynaptic regulation is exerted by muscarinic autoreceptors localized on the cholinergic nerve ending itself. The feed back inhibition of the 3H-ACh release does not require the presence of intact neurons or neuronal loops as tetrodotoxin (2.5 x 10(-6) M) does not affect the above results.

Delayed presentation of an extradural abscess in a patient with alcohol abuse.

Three cases of extradural abscess with delayed presentation after extradural analgesia have been reported previously. The present report describes a patient with alcohol abuse who was treated for 5 days with extradural injections of bupivacaine for pain from multiple rib fractures. The first symptoms of an extradural abscess developed approximately 11 days after removal of the extradural catheter, and definite diagnosis was delayed a further 7 days.

Dopamine release is enhanced while acetylcholine release is inhibited by nimodipine (Bay e 9736).

The calcium-channel ligand, nimodipine (Bay e 9736), in submicromolar concentrations (10(-7) to 10(-5) M), enhanced the potassium (25 mM) or electrical stimulation (1 Hz, 1 ms, 180 pulses) evoked release or [3H]dopamine from rat striatal slices, while it inhibited the release of [3H]acetylcholine. Nimodipine had similar effects on slices from the cerebral cortex loaded with [3H]dopamine or [3H]acetylcholine, the electrical stimulation evoked release of the catecholamine was enhanced, while release of [3H]acetylcholine was suppressed. The data indicate that nimodipine may distinguish between Ca2+ channels in dopaminergic and cholinergic nerve-terminals. The simultaneous elevation of dopamine release and suppression of acetylcholine release may prove useful in the treatment of Parkinson's disease.

Incidence of awareness in total i.v. anaesthesia based on propofol, alfentanil and neuromuscular blockade.

BACKGROUND: There is no reliable technique for monitoring drug concentrations in total i.v. anaesthesia (TIVA) with muscle relaxation. An increased risk of awareness with TIVA has been stated as a possible drawback. The present study was conducted in order to assess the incidence of conscious awareness in TIVA based on propofol, alfentanil and neuromuscular blockade. METHODS: One thousand patients anaesthetized with TIVA based on propofol, alfentanil, and neuromuscular blockade were subjected postoperatively to a structured interview for conscious awareness on two separate occasions: on discharge from the post-anaesthesia care unit, and the day after anaesthesia. Five hundred of these patients were also interviewed a third time, approximately 1 week later. RESULTS: Two cases of awareness were detected (0.2%). One of these was identified immediately after extubation. The second patient had no memory of intraoperative events or dreams at the first interview, recalled a bad dream on the day after, and had explicit recall of intraoperative events at the interview 8 days later. In both cases, haemodynamic signs of inadequate anaesthesia were present. The incidence of conscious awareness in this study is similar to the lowest previously reported incidence of awareness following general anaesthesia in patients who had been paralyzed and thereafter subjected to a structured postanesthesia interview. CONCLUSIONS: If the true incidence of conscious awareness is to be determined, interviews must be extended beyond the first postoperative day. Our study indicates that if appropriate dosing of propofol and alfentanil are adhered to, and proper action is taken in case of haemodynamic alterations suggestive of inadequate anaesthesia, the incidence of conscious awareness in non-cardiac TIVA with neuromuscular blockade is low.

In vivo and in vitro studies on the potentiation of muscarinic receptor stimulation by alaproclate, a selective 5-HT uptake blocker.

Alaproclate (10-60 mg/kg) injected i.p. into male mice potentiated and prolonged the oxotremorine and physostigmine-induced tremor in a dose-dependent manner. Atropine completely blocked the tremor caused by oxotremorine or physostigmine both in the presence and absence of alaproclate. Pretreatment with the 5-HT receptor antagonist metitepine completely blocked the enhancement of oxotremorine-induced tremor caused by alaproclate. Biochemical studies indicated that the above effects cannot be explained by assuming that alaproclate a) acts as a cholinergic agonist, b) inhibits the acetylcholine esterase, c) interferes with choline uptake or acetylcholine synthesis, or d) directly potentiates the release of acetylcholine. In ligand binding studies alaproclate was found to be a weak competitive inhibitor of muscarinic antagonist binding to membranes from the rat cerebral cortex, rat striatum, human cerebral cortex and human striatum. (Ki approximately 28-40 microM in all four tissues). The present results suggest that alaproclate may potentiate muscarinic responses by a mechanism involving serotonergic receptor mechanisms rather than by a direct interaction with the muscarinic cholinergic receptors.

Presynaptic antagonist-postsynaptic agonist at muscarinic cholinergic synapses. N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide.

A muscarinic cholinergic ligand, N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)-acetamide (Compound BM-5), was shown to act simultaneously as a presynaptic antagonist and as a postsynaptic agonist at peripheral and central muscarinic synapses. In the presence of eserine it enhanced release of [3H]acetylcholine evoked by electrical stimulation in the guinea pig ileum longitudinal muscle myenteric plexus preparation. It also enhanced the release of [3H]acetylcholine evoked by potassium (25 mM) from rat hippocampal slices. It contracted the ileum and stimulated the synthesis of cyclic guanosine 3',5'-monophosphate in rat hippocampal slices and in human lymphocytes in a manner similar to the muscarinic agonist oxotremorine. This effect of BM-5 could be blocked by atropine.

Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo, and in vitro studies.

A high density of galanin binding sites was found by using 125I-labeled galanin, iodinated by chloramine-T, followed by autoradiography in the ventral, but not in the dorsal, hippocampus of the rat. Lesions of the fimbria and of the septum caused disappearance of a major population of these binding sites, suggesting that a large proportion of them is localized on cholinergic nerve terminals of septal afferents. As a functional correlate to these putative galanin receptor sites, it was shown, both in vivo and in vitro, that galanin, in a concentration-dependent manner, inhibited the evoked release of acetylcholine in the ventral, but not in the dorsal, hippocampus. Intracerebroventricularly applied galanin (10 micrograms/15 microliters) fully inhibited the scopolamine (0.5 mg/kg, s.c.)-stimulated release of acetylcholine in the ventral, but not in the dorsal, hippocampus, as measured by microdialysis technique. In vitro, galanin inhibited the 25 mM K+-evoked release of [3H]acetylcholine from slices of the ventral hippocampus, with an IC50 value of approximately 50 nM. These results are discussed with respect to the colocalization of galanin- and choline acetyltransferase-like immunoreactivity in septal somata projecting to the hippocampus.