Oxygen-Dependent Inhibition of Neutrophil Respiratory Burst by Nitric Oxide

Effect of nitric oxide (NO) on the respiratory burst of neutrophils was examined under different oxygen tensions. Phorbol myristate acetate (PMA) stimulated oxygen consumption and superoxide (O₂^-) generation in neutrophils by a mechanism which was inhibited reversibly by NO. The inhibitory effect of NO increased significantly with a decrease in oxygen tension in the medium. The inhibitory effect of NO was suppressed in medium containing oxyhemoglobin (HbO₂), a NO scavenging agent. In contrast, 3-morpholinosydnonimine (SIN-1), a compound that rapidly generates peroxynitrite (ONOO^-) from the released NO and O₂^-, slightly stimulated the PMA-induced respiratory burst. These results suggested that NO, but not ONOO, might reversibly inhibit superoxide generation by neutrophils especially at physiologically low oxygen tensions thereby decreasing oxygen toxicity particularly in and around hypoxic tissues.     

Effects of water content on the enantioselectivity of α-chymotrypsin catalysis in organic media

Summary The -chymotrypsin-catalyzed transesterification between N-trifluoroacetyl-DL-phenylalanine trifluoroethyl ester and 1-propanol was carried out in a variety of organic solvents. The addition of small quantities of water enhanced both the rate of reaction and enantioselectivity. A high enantioselectivity was achieved in ethyl acetate (E = 120), diethyl ether (86), or acetonitrile (60). The competing hydrolysis became significant at water content higher than 0.5% (w/w).     

Possible involvement of ethylene-activated {beta}-cyanoalanine synthase in the regulation of cocklebur seed germination

A possible involvement of ß-cyanoalanine synthase(CAS: EC 4.4.1.9 [EC] ) in germination processes of seeds was demonstratedusing pre-soaked upper seeds of cocklebur (Xanthium pennsylvanicumWallr.). Pretreatment in anoxia not only with KCN but also cysteine,as the substrates for CAS, stimulated the subsequent germinationof cocklebur seeds in air. However, the effect of cysteine wasmanifested even in air when applied together with C₂H₄, andits effect was further enhanced in combination with KCN. Thegermination-stimulating effect of KCN was intensified by C₂H₄only when 0₂ was present. In contrast, serine, another substrateof CAS, was effective in air only when combined with C₂H₄ and/orKCN. The addition of cysteine greatly reduced the cyanogenicglycoside content of seeds, but increased HCN evolution. Onthe other hand, glutathione did not have any effect on cockleburseed germination, HCN evolution or bound cyanogen content, suggestingthat cysteine is not acting as a reducing reagent. It is suggestedthat CAS regulates the process of cocklebur seed germinationby the dual action of enlarging the pool of amino acids andsupplying sulphydryl bases, the latter being more determinatelyimportant. Serine is effective only via the former action, whilecysteine would act via both. Key words: Cyanide, cyanogenic glycoside, ß-cyanoalanine synthase, seed germination, Xanthium pennsylvanicum     

Synonymous and nonsynonymous substitutions in mammalian genes and the nearly neutral theory

The nearly neutral theory of molecular evolution predicts larger generation-time effects for synonymous than for nonsynonymous substitutions. This prediction is tested using the sequences of 49 single-copy genes by calculating the average and variance of synonymous and nonsynonymous substitutions in mammalian star phylogenies (rodentia, artiodactyla, and primates). The average pattern of the 49 genes supports the prediction of the nearly neutral theory, with some notable exceptions.The nearly neutral theory also predicts that the variance of the evolutionary rate is larger than the value predicted by the completely neutral theory. This prediction is tested by examining the dispersion index (ratio of the variance to the mean), which is positively correlated with the average substitution number. After weighting by the lineage effects, this correlation almost disappears for nonsynonymous substitutions, but not quite so for synonymous substitutions. After weighting, the dispersion indices of both synonymous and nonsynonymous substitutions still exceed values expected under the simple Poisson process. The results indicate that both the systematic bias in evolutionary rate among the lineages and the episodic type of rate variation are contributing to the large variance. The former is more significant to synonymous substitutions than to nonsynonymous substitutions. Isochore evolution may be similar to synonymous substitutions. The rate and pattern found here are consistent with the nearly neutral theory, such that the relative contributions of drift and selection differ between the two types of substitutions. The results are also consistent with Gillespie's episodic selection theory.     

The Expression of Two Splice Variants of Metabotropic Glutamate Receptor Subtype 5 in the Rat Brain and Neuronal Cells During Development

Abstract: We previously reported that a variant with extra amino acid residues exists in the metabotropic glutamate receptor subtype 5 (mGluR5). Either of the two isoforms, named mGluR5b and mGluR5a for the isoforms with and without the inserted sequence, respectively, generated Ca²⁺-activated Cl⁻ current when expressed in Xenopus oocytes. We herein report that these two isoforms are produced by the alternative splicing of the exon skipping type. When examined during the course of postnatal development, the major mGluR5 isotype mRNA was observed to switch from mGluR5a to mGluR5b in the rat hippocampus and the cerebral cortex. We also investigated two cell lines that could be differentiated into neuron-like cells in vitro. Whereas the mGluR5b mRNA was hardly detectable in either undifferentiated or differentiated NG108-15 cells, the relative amounts of the two variant mRNAs changed after the induction of differentiation in the P19 cells. An extracellular application of trans - d,l -1-amino-1,3-cyclopentanedicarboxylate on the neuron-like P19 cells induced intracellular Ca²⁺ mobilization, thus suggesting that the cells could express functional mGluR(s) coupled to phospholipase C and other components that could mediate the signal transduction pathway. This cell line may thus provide a model system for studying both mGluR5 expression and other mGluR-induced phenomena at the molecular level.     

A mutation at proline-115 in the A-factor receptor protein of Streptomyces griseus abolishes DNA-binding ability but not ligand-binding ability.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) and its specific receptor protein (ArpA) are required for streptomycin production and aerial mycelium formation in Streptomyces griseus. A mutant strain HO1 that produced streptomycin and formed aerial mycelium and spores was derived from an A-factor-deficient mutant, S. griseus HH1. The phenotypes of mutant HO1 were found to result from a single amino acid replacement of ArpA; the proline residue at position 115 in the wild-type ArpA was replaced by serine, yielding mutant ArpA (P115S). The mutant ArpA (P115S) was still able to form a homodimer and possessed A-factor-binding ability but lost the ability to bind DNA. The properties of P115S suggest that ArpA consists of two independently functional domains, one for A-factor binding and one for DNA binding, and that proline-115 plays an important role in DNA binding. This is in agreement with the idea that A-factor binding to the COOH-terminal domain of ArpA causes a subtle conformational change of the distal NH2-terminal DNA-binding domain, resulting in dissociation of ArpA from DNA.     

An Estimate of Divergence Time of Parazoa and Eumetazoa and That of Cephalochordata and Vertebrata by Aldolase and Triose Phosphate Isomerase Clocks

Previously we suggested that four proteins including aldolase and triose phosphate isomerase (TPI) evolved with approximately constant rates over long periods covering the whole animal phyla. The constant rates of aldolase and TPI evolution were reexamined based on three different models for estimating evolutionary distances. It was shown that the evolutionary rates remain essentially unchanged in comparisons not only between different classes of vertebrates but also between vertebrates and arthropods and even between animals and plants, irrespective of the models used. Thus these enzymes might be useful molecular clocks for inferring divergence times of animal phyla. To know the divergence time of Parazoa and Eumetazoa and that of Cephalochordata and Vertebrata, the aldolase cDNAs from Ephydatia fluviatilis, a freshwater sponge, and the TPI cDNAs from Ephydatia fluviatilis and Branchiostoma belcheri, an amphioxus, have been cloned and sequenced. Comparisons of the deduced amino acid sequences of aldolase and TPI from the freshwater sponge with known sequences revealed that the Parazoa–Eumetazoa split occurred about 940 million years ago (Ma) as determined by the average of two proteins and three models. Similarly, the aldolase and TPI clocks suggest that vertebrates and amphioxus last shared a common ancestor around 700 Ma and they possibly diverged shortly after the divergence of deuterostomes and protostomes.     

ET-1-induced bronchoconstriction is mediated via ETB receptor in mice

Nagase, Takahide, Tomoko Aoki, Teruaki Oka, YoshinosukeFukuchi, and Yasuyoshi Ouchi. ET-1-induced bronchoconstriction ismediated via ET_B receptor in mice.J. Appl. Physiol. 83(1): 46-51, 1997.Endothelin (ET)-1 is one of the most potent agonists of airwaysmooth muscle and can act via two different ET receptor subtypes, i.e.,ET_A andET_B. To determine the effects ofET-1 on in vivo pulmonary function and which ET receptors are involved in murine lungs, we investigated 1)the effects of ET and sarafotoxin S6c (S6c), a selectiveET_B agonist, on pulmonary functionand 2) the effects of BQ-123 andBQ-788, specific ET_A- andET_B-receptor antagonists, onET-1-induced bronchoconstriction. ICR mice were anesthetized and mechanically ventilated (frequency = 2.5 Hz, tidalvolume = 8 ml/kg, positive end-expiratory pressure = 3 cmH₂O). Intravenous ET-1, ET-2,and ET-3 increased lung resistance similarly and equipotently, whereasS6c elicited a greater degree of bronchoconstriction. Mice were thenpretreated with saline (Sal), BQ-123 (0.2, 1, and 5 mg/kg), or BQ-788(0.2, 1, and 5 mg/kg) before administration of ET-1(10⁷ mol/kg iv). No dose ofBQ-123 blocked ET-1-induced constriction, whereas pretreatment witheach dose of BQ-788 significantly inhibited ET-1-induced responses.There were significant differences in morphometrically assessed airwayconstriction between Sal and BQ-788 and between BQ-123 and BQ-788,whereas no significant difference was observed between Sal and BQ-123.There were no significant morphometric differences in the airway wallarea among the three groups. These observations suggest that theET_B- but notET_A-receptor subtype may mediatethe changes in murine pulmonary function in response to ET-1. Inaddition, the ET_B-receptorantagonist reduces ET-1-induced airway narrowing by affecting airwaysmooth muscle contraction in mice.