An unusual presynaptic action of Bothrops insularis snake venom mediated by phospholipase A2 fraction

A phospholipase A2-containing fraction was isolated from the venom of Bothrops insularis by a combination of gel filtration on Sephadex G-150 and ion exchange chromatography on DEAE-Sephadex. Peak IV of the latter chromatography containing all of the phospholipase A2 (PLA2) activity, was assayed on isolated neuromuscular preparations. In the mouse phrenic nerve-diaphragm incubated in Tyrode at 37 degrees C, the PLA2 fraction produced an initial increase in the twitch tension and in the frequency of the mepps, followed by a dose-dependent, irreversible blockade. The replacement of 1.8 mM Ca2+ by 4 mM Sr2 inhibited the neuromuscular blocking effect of the fraction. In the chick hiventer cervicis preparation incubated with Krebs solution at 37 degrees C, the PLA2 fraction induced blockade but did not affect the response to acetylcholine and K+, excluding the involvement of post-synaptic and direct muscular effects. A low temperature (18-22 degrees C) incubation prevented the neuromuscular effect from developing. These results suggest that the PLA2-containing fraction acts predominantly at presynaptic sites at the neuromuscular junction. This fraction also accounts for most of the pharmacological effects of the crude venom.     

CD5 B cells and B-cell malignancies

Evidence from both animal, and human, studies suggests that repeated administration of 3,4-methylenedioxymethamphetamine (MDMA: "ecstasy") produces lasting decreases in serotonergic activity. Serotonin is believed to play a modulatory role in a variety of psychological processes, including learning and memory. There are recent reports that polydrug users, who have used ecstasy recreationally, exhibit selective impairments in memory. However, these studies did not compare ecstasy users with polydrug users who had not taken ecstasy, leaving open the possibility that the memory deficits may be associated with a history of use of other illicit drugs. The present study used the Rivermead Behavioural Memory test to investigate immediate and delayed recall in: 25 polydrug-users who had taken more than 20 tablets of ecstasy (MDMA group), 22 participants (polydrug controls) who had never taken ecstasy, but, otherwise has personal characteristics (e.g. age, gender, education, height, weight), and illicit drug use histories, that were generally not significantly different from those of the MDMA group, and 19 participants who had not used illicit drugs but who also had similar personal characteristics (non-drug controls). Participants in the MDMA group recalled significantly fewer ideas (approximately 75% of the number of ideas recalled by participants in either of the other two groups), in both immediate and delayed recall conditions. The two illicit drug-using groups did differ in their estimated IQ scores and their duration of use of LSD, but only the latter proved to be a statistically significant covariate, and the difference in recall performance between the MDMA and polydrug controls groups remained statistically significant when this variable was treated as a covariate. The present findings provide the first evidence that deficits in memory performance in recreational ecstasy users are primarily associated with past exposure to ecstasy, rather than with the other legal and illicit drugs consumed by these individuals, and are consistent with reduced serotonergic modulation of mnemonic function as a result of long-term neurotoxic effects of MDMA in humans.     

Repeated intracerebroventricular administration of glucagon-like peptide-1-(7-36) amide or exendin-(9-39) alters body weight in the rat

Central nervous system glucagon-like peptide-1-(7-36) amide (GLP-1) administration has been reported to acutely reduce food intake in the rat. We here report that repeated intracerebroventricular (i.c.v.) injection of GLP-1 or the GLP-1 receptor antagonist, exendin-(9-39), affects food intake and body weight. Daily i.c.v. injection of 3 nmol GLP-1 to schedule-fed rats for 6 days caused a reduction in food intake and a decrease in body weight of 16 +/- 5 g (P < 0.02 compared with saline-injected controls). Daily i.c.v. administration of 30 nmol exendin-(9-39) to schedule-fed rats for 3 days caused an increase in food intake and increased body weight by 7 +/- 2 g (P < 0.02 compared with saline-injected controls). Twice daily i.c.v. injections of 30 nmol exendin-(9-39) with 2.4 nmol neuropeptide Y to ad libitum-fed rats for 8 days increased food intake and increased body weight by 28 +/- 4 g compared with 14 +/- 3 g in neuropeptide Y-injected controls (P < 0.02). There was no evidence of tachyphylaxis in response to i.c.v. GLP-1 or exendin-(9-39). GLP-1 may thus be involved in the regulation of body weight in the rat.     

Manganese toxicity in children receiving long-term parenteral nutrition

BACKGROUND: In patients receiving long-term parenteral nutrition (PN), cholestatic disease and nervous system disorders have been associated with high blood concentrations of manganese. In such patients, the normal homoeostatic mechanisms of the liver and gut are bypassed and the requirement for this trace element is not known; nor has it been certain whether hypermanganesaemia causes the cholestasis or vice versa. We explored the direction of effect by serial tests of liver function after withdrawal of manganese supplements from children receiving long-term PN. We also examined the relation between blood manganese concentrations and brain lesions, as indicated by clinical examination and magnetic resonance imaging (MRI). METHODS: From a combined group of 57 children receiving PN we identified 11 with the combination of hypermanganesaemia and cholestasis; one also had a movement disorder. Manganese supplements were reduced in the first three and withdrawn in the remainder. MRI was done in two of these children. We also looked at manganese concentrations and MRI scans in six children who had received PN for more than 2 years without developing liver disease. FINDINGS: In the hypermanganesaemia/cholestasis group, four of the 11 patients died. In the seven survivors baseline whole-blood manganese was 615-1840 nmol/L, and after 4 months it had declined by a median of 643 nmol/L (p < 0.01). Over the same interval total bilirubin declined by a median of 70 mumol/L (p < 0.05). Two of these children had movement disorders, one of whom survived to have an MRI scan; this showed, with T1 weighted images, bilateral symmetrically increased signal intensity in the globus pallidus and subthalamic nuclei. Such changes were also seen in five other children--one from the hypermanganesaemia/cholestasis group and four of six in the long-term PN group without liver disease (in all of whom blood manganese was above normal). INTERPRETATION: The cholestasis complicating PN is multifactorial, but these results add to the evidence that manganese contributes. In view of the additional hazard of basal ganglia damage from high manganese levels in children receiving long-term PN, we recommend a low dose regimen of not more than 0.018 mumol/kg per 24 h together with regular examination of the nervous system.     

No connection between alcohol use and unsafe sex among gay and bisexual men

The actions of substance P and thyrotropin-releasing hormone (TRH) on neonatal rat spinal motoneurones in vitro were compared using intracellular current and voltage clamp techniques. Like TRH, substance P evoked a slowly-developing, persistent depolarisation plus an increase in input resistance under current clamp conditions. Under voltage clamp conditions, substance P elicited an inward current (mainly due to a conductance block) which peaked near -40 mV and reversed polarity close to the estimated EK. A distinct conductance increase (with a reversal potential near zero) also appeared to contribute to this response. The response to substance P at resting potential was suppressed by 1.5 mM Ba2+, but not by 20 mM tetraethylammonium, 2 mM 4-aminopyridine, 2 mM Cs+ and 0.2 mM Cd2+. In addition, co-application of TRH and substance P mutually occluded each other. Thus, it is suggested that substance P and TRH share a common effector mechanism, which primarily involves the suppression of IK(T), a persistent K+ current recently discovered in these neurones.     

Protein kinase C isotypes in human erythroleukemia (K562) cell proliferation and differentiation. Evidence that beta II protein kinase C is required for proliferation

The human erythroleukemia (K562) cell line undergoes megakaryocytic differentiation and cessation of proliferation when treated with phorbol myristate acetate (PMA). To investigate the role of individual protein kinase C (PKC) isotypes in these events, we have assessed PKC isotype expression during leukemic proliferation and PMA-induced differentiation. Immunoblot analysis using isotype-specific antibodies demonstrates that proliferating K562 cells express the alpha, beta II, and zeta PKC isotypes. PMA-induced differentiation and cytostasis lead to a decrease in beta II PKC and increases in alpha and zeta PKC levels. The role of the alpha and beta II PKC isotypes was further assessed in cells overexpressing these isotypes. K562 cells overexpressing human alpha PKC grew more slowly and were more sensitive to the cytostatic effects of PMA than control cells, whereas cells overexpressing beta II PKC were less sensitive to PMA. PMA-induced cytostasis is reversed upon removal of PMA. Resumption of proliferation is accompanied by reexpression of beta II PKC to near control levels, whereas alpha and zeta PKC levels remain elevated for several days after removal of PMA. Proliferation of PMA-withdrawn cells can be partially inhibited by antisense beta II PKC oligodeoxyribonucleotide. Growth inhibition is dose-dependent, specific for beta II PKC-directed antisense oligonucleotide, and associated with significant inhibition of beta II PKC levels indicating that beta II PKC is essential for K562 cell proliferation. Sodium butyrate, which unlike PMA induces megakaryocytic differentiation without cytostasis, causes increases in both alpha and beta II PKC levels. These data demonstrate that beta II PKC is required for K562 cell proliferation, whereas alpha PKC is involved in megakaryocytic differentiation.     

Relation of exocytotic release of gamma-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.     

Oncocytomas of the upper jaw

Combinations of certain phospholipids and gangliosides increase the specific activity of m calpain and can activate m calpain at 1 to 10 microM Ca2+ concentration. However, this level of calcium is still greater than the normal intracellular calcium level. We have used combinations of lipids to demonstrate the m calpain activity at the physiological Ca2+ level. GD1a (100 microM) and cerebroside (Cerb; 750 microM; 1:7.5) mixture was the most effective. At 0.5 microM to 1.0 microM Ca2+ concentrations, 15-20% of the maximal activity was detected for the purified myelin and cytosolic m calpains. Other combinations were GD1a (100 microM), GM1 (100 microM), Cerb (750 microM), sulfatide (Sulf; 750 microM), and phosphatidylinositol (PI; 300 microM) at a ratio of 1:1: 7.5:7.5:3, respectively. These lipid mixtures stimulated calpain activity at three- to tenfold less calcium concentration than control. The other mixtures, including GD1a:Sulf (1:9) > GD1a:PI (1:4) > PI:Sulf (1:5) > Cerb:Sulf (1:5) and PI:Cerb (1:2.5), also stimulated calpain activity at 1.0 microM Ca2+ concentration. Triton X-100, oxidized glutathione (GSSG), and calpain activator did not affect the Ca2+ requirement. Liposomes containing GD1a, Cerb, and m calpain also showed recognizable calpain activity at a significantly reduced Ca2+ concentration (0.4 microM), confirming the glycolipid-mediated enzyme modulation. These studies indicate that specific lipid mixtures can stimulate m calpain activity at an intracellular level of Ca2+.     

Morphogenesis of the C. elegans hermaphrodite uterus

We have undertaken electron micrographic reconstruction of the Caenorhabditis elegans hermaphrodite uterus and determined the correspondence between cells defined by their lineage history and differentiated cell types. In this organ, many cells do not move during morphogenesis and the cell lineage may function to put cells where they are needed. Differentiated uterine cell types include the toroidal ut cells that make structural epithelium, and specialized utse and uv cells that make the connection between the uterus and the vulva. A cell fate decision in which the anchor cell (AC) induces adjacent ventral uterine intermediate precursor cells to adopt the pi fate, rather than the ground state rho, has profound consequences for terminal differentiation: all pi progeny are directly involved in making the uterine-vulval connection whereas all rho progeny contribute to ut toroids or the uterine-spermathecal valve. In addition to specifying certain uterine cell fates, the AC also induces the vulva. Its multiple inductions thereby function to coordinate the connection of an internal to an external epithelium. The AC induces the pi cells and ultimately fuses with a subset of their progeny. This is an example of reciprocal cell-cell interaction that can be studied at single cell resolution. The AC is thus a transitory cell type that plays a pivotal role in organizing the morphogenesis of the uterine-vulval connection.