Crystal structure determination of basic phospholipase A_2 from venom of Agkistrodon halys pallas by molecular replacement method

Basic phospholipase A2 (BPLA2) from the venom of Agkistrodon halys pallas has a strong ability to hemolyze erythrocytes. The asymmetrical unit of P212121 crystal of BPLA2 contains two molecules. Self-rotation function was used to study the orientation relationship of these two molecules. Cross-rotation and translation functions were then used to determine the orientations and positions of the two molecules in the unit cell. The model building and preliminary structure refinement were carried out. The result shows that the two molecules in the asymmetrical unit of orthorhombic crystal are related by a non-crystallographic 2-fold symmetry axis.     

Catalysis of an isotopic exchange between CO2 and the carboxyl group of acetate by Methanosarcina barkeri grown on acetate

Cell suspensions of Methanosarcina barkeri (strain Fusaro) grown on acetate were found to catalyze the formation of methane and CO₂ from acetate (30–40 nmol/min·mg protein) and an isotopic exchange between the carboxyl group of acetate and ¹⁴CO₂ (30–40 nmol/min·mg protein). An isotopic exchange between [¹⁴C]-formate and acetate was not observed. Cells grown on methanol mediated neither methane formation from acetate nor the exchange reactions. The data indicate that the isotopic exchange between CO₂ and the carboxyl group of acetate is a partial reaction of methanogenesis from acetate. Both reactions were completely inhibited by low concentrations of cyanide (20 M) or of hydrogen (0.5% in the gas phase). Methane formation from acetate was also completely inhibited by low concentrations of carbon monoxide (0.2% in the gas phase) whereas only significantly higher concentrations of CO had an effect on the exchange reaction. In the concentration range tested KCN, H₂ and CO had no effect on methane formation from methanol or from H₂ and CO₂; however, cyanide (20 M) also affected methane formation from CO. The results are discussed with respect to proposed mechanisms of methane and CO₂ formation from acetate.     

In vitro inhibitor studies of vitamin D 25-hydroxylase in rat liver microsomes

The ability of four vitamin D analogs to inhibit the liver microsomal vitamin D-25-hydroxylase was determined. 19-Hydroxy-10(S),19-dihydrovitamin D3,25-fluorovitamin D3, 3 beta-hydroxy-9,10-seco-5,7,10(19)-choletrien-24-oic acid dimethylamide and 25-aza-vitamin D3 were competitive inhibitors with apparent KI values of 44, 137, and 870 nM, and 6.4 microM, respectively. The values for the 19-hydroxy-10(S), 19-dihydrovitamin D3, 25-fluorovitamin D3, and 25-aza-vitamin D3 correspond well to other literature reports with respect to their relative in vivo inhibitory properties. 24-Oxovitamin D3 oxime also proved to be a potent inhibitor but a detailed analysis was prohibited by the lack of material. The 3 beta-hydroxy-9,10-seco-5,7,10(19)-choletrien-24-oic acid dimethylamide was also tested in vivo but had no antagonistic activity when provided at a 2000-fold excess over vitamin D3.     

Depth-dependent reproductive output of the green sea urchin, Strongylocentrotus droebachiensis (O.F. Müller), in relation to the nature and availability of food

Reproductive output of green sea urchins (Strongylocentrotus droebachiensis O.F. Müller) in the field was highest at depths where preferred macro-algae were abundant, and lowest at depths where preferred macro-algae were overgrazed or replaced by non-preferred species (Agarum and Ptilota). Feeding rate and gonad indices of sea urchins in the laboratory were highest on a diet of preferred algae (Fucusdistichus L. subsp. edentatus (Pyl.) Powell, Laminaria longicruris Pyl., Desmarestia spp. and Saccorhizadermatodea (Pyl.) J. Ag.), and lowest on the less preferred Agarum cribrosum (Mert.) Bory, Ptilota serrata Kütz., and crustose corallines. Gamete production/unit area in overgrazed habitats was as great or greater than in kelp beds because of the higher biomass of urchins in overgrazed areas. Gonad weight and reproductive output of urchins from habitats poor in food can be increased by providing preferred foods.     

Translocation of a lysosomal enzyme across the microsomal membrane requires signal recognition particle

Signal recognition particle (SRP), an 11S ribonucleoprotein (Walter and Blobel (1982) Nature 299, 691-698), is required for translocation of secretory proteins across microsomal membranes (Walter and Blobel (1980) Proc. Natl. Acad. Sci. USA 77, 7112-7116) and for asymmetric integration into microsomal membranes of a transmembrane protein (Anderson et al., (1982) J. Cell Biol. 93, 501-506). We demonstrate here that SRP is also required for translocation of the lysosomal protease cathepsin D across microsomal membranes.     

Regulation of adenylate cyclase in cultured cardiocytes from neonatal rats by adenosine and other agonists

The presence of adenosine receptors coupled to adenylate cyclase in cultured cardiocytes from atria and ventricles from neonatal rats is demonstrated in these studies. N-Ethylcarboxamideadenosine (NECA), l-N⁶-phenylisopropyladenosine (PIA), and 2-chloroadenosine (2-cl-Ado) stimulated adenylate cyclase in a concentration-dependent manner in both cultured atrial and ventricular cells. The order of potency of stimulation was NECA > PIA > 2-cl-Ado. The stimulation of adenylate cyclase by NECA was enhanced by guanine nucleotides and was blocked by 3-isobutyl-1-methylxanthine in both these cells. Other agonists such as epinephrine, norepinephrine, dopamine, F^?, and forskolin were also able to stimulate adenylate cyclase, although the extent of stimulation by these agents was higher in ventricular than in atrial cells. The stimulation of adenylate cyclase by epinephrine and norepinephrine was inhibited by propranolol but not by phentolamine. On the other hand, phentolamine, propranolol, and haloperidol inhibited dopamine-stimulated adenylate cyclase activity to the same extent. Forskolin, at its maximal concentration, potentiated the stimulatory effect of epinephrine, norepinephrine, and dopamine on adenylate cyclase in both atrial and ventricular cardiocytes, but the interaction of NECA with epinephrine, norepinephrine, or dopamine was different in atrial and ventricular cells. The stimulation by an optimal concentration of NECA was additive with maximal stimulation by the catecholamines in atrial cells but not in ventricular cells. The data suggest the existence of adenosine “Ra” and catecholamine receptors in cultured atrial and ventricular cardiocytes. It can be postulated that adenosine in addition to its role as a potent vasodilator might regulate cardiac performance through its interaction with “Ra” receptors associated with adenylate cyclase. The difference in the mode of interaction of adenosine with catecholamines in atrial and ventricular cells suggests that the mechanism by which these agents activate adenylate cyclase may be different in these cells.     

Din+ ('invertase') activity in Gram-negative bacteria

Abstract The H₂ region, encoding one of the flagellar antigens of Salmonella typhimurium and the controlling elements of phase variation has been subcloned into the broad-host-range plasmid, RP4. Due to a deletion within the H₂ region, the 'invertase' normally encoded by the hin gene is absent. This plasmid was transferred into a number of Gram-negative bacteria, and a hin complementary ( din ) activity detected in some.     

Individual adaptations in stochastic environments

Summary Many natural populations undergo radical and unpredictable fluctuations, associated with stochastic environmental conditions. Under such circumstances, fitness of a genotype (or strategy) is defined as the geometric mean of the intergenerational genotypic population growth ratel(t). Unfortunately, this population-level criterion has proved difficult to apply at the level of individual organisms.After developing a formula for the variance ofl as the sum of developmental and environmental variance, we discuss several models of individual adaptations, involving clutch size, progeny size and number, and foraging behaviour under risk of predation, based on the geometric-mean fitness concept. We then show how the method of dynamic programming can be extended to deal with facultative behaviour in stochastic environments. Finally we discuss the concept of an evolutionarily stable strategy in a stochastic environment.Our analysis suggests several novel interpretations of field and laboratory observations. Under the geometric mean criterion behaviour may be determined primarily by the worst likely environment; behaviour may appear suboptimal if observed only under normal or average conditions. For example,except under extreme environmental conditions, avian clutches larger than those that are observed might result in increased fecundity, with little if any cost of reproduction in terms of parental survival; however, in unusually bad years such large clutches might be disastrous, in terms of parental survival. This consideration may help explain some recently reported experimental clutch-size manipulation results. Similarly, our analysis indicates that the known phenomenon of seasonal reduction in seed size may constitute a double bet-hedging strategy, determined by parental mortality risk and future seed survival probability. We also discuss circumstances in which phenotypic polymorphism is an adaptation to environmental uncertainty. Thus almost any individual life history or behavioural adaptation may be affected by environmental stochasticity.     

Redox Modulation of N-Methyl-D-Aspartate-Stimulated Neurotransmitter Release from Rat Brain Slices

Rat brain cortical slices released tritiated norepinephrine ([3H]NA) during a 2-min stimulation with N-methyl-D-aspartate (NMDA). Dithiothreitol (DTT; 0.1-5 mM), present for 6 min prior to stimulation, dose-dependently increased the release of [3H]NA from cortical slices stimulated with a maximally effective concentration of NMDA (500 microM). Similar results were observed for [3H]NA release from hippocampal slices and tritiated and endogenous dopamine release from striatal slices. DTT treatment also markedly shifted the dose-response curve of NMDA to the left. Cortical slices released approximately the same amount of [3H]NA with 10 microM NMDA following DTT treatment (about 5%) as non-DTT-treated control slices did with 500 microM NMDA. The effects of DTT were fully reversed by subsequent treatment with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB; 0.5 mM). DTT treatment did not significantly alter the ability of magnesium (1.3 mM) or the polyamine antagonist arcaine to block the NMDA-stimulated release of [3H]NA. In contrast, DTT treatment significantly attenuated the antagonist effects of the competitive glycine antagonist, 7-chlorokynurenic acid, and the competitive NMDA antagonist, 2-aminophosphonopentanoic acid. These results suggest that oxidation and reduction of disulfide bonds located within the NMDA receptor complex might regulate the activation of the NMDA receptor. This could have important consequences in vivo if endogenous oxidizing/reducing systems are found to have similar effects on NMDA-stimulated responses.     

Incorporation of [3H]Arachidonic and [14C]Eicosapentaenoic Acids into Glycerophospholipids and Their Metabolism via Lipoxygenases in Isolated Brain Cells from Rainbow Trout Oncorhaynchus mykaiss

The incorporation of [3H]arachidonate [( 3H]AA) and [14C]eicosapentaenoate [( 14C]EPA) into glycerophospholipids was studied in isolated brain cells from rainbow trout, a teleost fish whose lipids are rich in (n-3) polyunsaturated fatty acids (PUFAs). EPA was incorporated into total lipid to a greater extent than AA, but the incorporation of both PUFAs into total glycerophospholipids was almost identical. The incorporation of both AA and EPA was greatest into phosphatidylethanolamine (PE). However, when expressed per milligram of individual phosphoglycerides, both AA and EPA were preferentially incorporated into phosphatidylinositol (PI), the preference being significantly greater with AA. On the same basis, significantly more EPA than AA was incorporated into phosphatidylcholine (PC). When double-labelled cells were challenged with calcium ionophore A23187, the 3H and 14C released from the cells closely paralleled each other, peaking at 10 min after addition of ionophore. The 12-monohydroxylated derivative was the pre-dominant lipoxygenase product from both AA and EPA with a rank order of 12-hydroxyeicosatetraenoic acid (12-HETE) greater than leukotriene B4 (LTB4) greater than 5-HETE greater than 15-HETE for the AA products and 12-hydroxyeicosapentaenoic acid (12-HEPE) greater than 5-HEPE greater than LTB5 greater than 15 HEPE for EPA products. The 3H/14C (dpm/dpm) ratios in the glycerophospholipids, total released radioactivity, and the lipoxygenase products suggested that PC rather than PI was the likely source of eicosanoid precursors in trout brain cells.