Evolution of structure and substrate specificity ind-alanine:d-Alanine ligases and related enzymes

Thed-alanine:d-alanine-ligase-related enzymes can have three preferential substrate specificities. Usually, these enzymes synthesized-alanyl-d-alanine. In vancomycin-resistant Gram-positive bacteria, structurally related enzymes synthesized-alanyl-d-lactate or Dalanyl-d-serine. The sequence of internal fragments of eight structurald-alanine:d-alanine ligase genes from enterococci has been determined. Alignment of the deduced amino acid sequences with those of other related enzymes from Gram-negative and Gram-positive bacteria revealed the presence of four distinct sequence patterns in the putative substrate-binding sites, each correlating with specificity to a particular substrate (d-alanine:d-lactate ligases exhibited two patterns). Phylogenetic analysis showed different clusters. The enterococcal subtree was largely superimposable on that derived from 16S rRNA sequences. In lactic acid bacteria, structural divergence due to differences in substrate specificity was observed. Glycopeptide resistance proteins VanA and VanB, the VanC-type ligases, and Dd1A and DdlB from enteric bacteria andHaemophilus influenzae constituted separate clusters. Correspondence to: P. Courvalin     

In Vivo and In Vitro Evidence for the Biosynthesis of Testosterone in the Telencephalon of the Female Frog

Abstract: Neurons and glial cells are capable of synthesizing various steroid hormones, but biosynthesis of testosterone in the CNS has never been reported. The aim of the present study was to demonstrate the synthesis of testosterone in the frog brain. The presence of 17β-hydroxysteroid dehydrogenase (17β-HSD)-like immunoreactivity was detected in a population of glial cells located in the telencephalon. Reversed-phase HPLC analysis of brain tissue extracts combined with radioimmunoassay detection revealed the presence of substantial amounts of testosterone and 5α-dihydrotestosterone (5α-DHT) in the telencephalon where 17β-HSD-positive cells were visualized. In male frogs, castration totally suppressed testosterone and 5α-DHT in the blood and in the rhombencephalon but did not affect the concentration of these two steroids in the telencephalon. Chemical characterization of testosterone in female frog telencephalon extracts was performed by coupling HPLC analysis with gas chromatography-mass spectrometry. Using the pulse-chase technique with [³H]pregnenolone as a precursor, the formation of a series of metabolites was observed, including dehydroepiandrosterone, androstenedione, testosterone, 5α-DHT, and estradiol. These data demonstrate the existence of an active form of 17β-HSD in the frog telencephalon, which is likely involved in testosterone biosynthesis within the brain.     

Trends in plant carbon concentration and plant demand for N throughout this century

 Atmospheric CO₂ concentration has increased by 25% over the preindustrial level. A parallel increase in C concentration and decreases in N concentration and δ¹³C of plants grown throughout this century have been observed in plant specimens stored in herbaria. We tested our previous results in a study of 12 more species collected in the western Mediterranean throughout this century (1920–1930, 1945–1955, and 1985–1990) and tree rings of Quercus pubescens from the same area. These changes were accompanied by apparent increases in condensed tannin concentration. A decreasing trend in δ¹⁵N both in herbarium material and tree rings was also found, indicating that ecosystems might cope with higher plant N demand by decreasing N losses and increasing N fixation and mineralization. These results may contribute to a better understanding of the effects of global change on carbon and nitrogen cycling. Received: 12 November 1995 / Accepted: 17 May 1996     

O-glycosylation of dipeptides using β-galactosidase activity of Achatina achatina digestive juice

Summary Enzymatic O-glycosylation of dipeptide derivatives containing a serine residue in the N or C terminal position and alanine or glycine as the second amino acid was achieved using the transgalactosylation activity of -galactosidase from the Achatina achatina digestive juice. Reactions were performed with lactose as glycosyl donor and the dipeptide ethyl (or methyl) esters N-protected by a benzyloxycarbonyl group (Z) as glycosyl acceptors. Yields of galactosyl-dipeptide derivatives were much higher than those obtained with the E.coli -galactosidase as catalyst.     

Changes in distribution of chloroplast adenylate kinase isoforms during floral induction

In the short day plant Chenopodium rubrum and the long day plant Nicotiana tabacum cv. Havana 425, adenylate kinase (EC 2.7.4.3) occurs as a family of isoforms, with at least two members localized in the chloroplast representing the main isoforms. In this work, isoforms were separated by anion exchange chromatography and relative isoform activities were compared between vegetative plants and plants induced to flowering. In both species examined, a light regime leading to floral induction resulted in a significant decrease in the activity of one chloroplast isoform. This decrease modified considerably the relative distribution of isoform activities, especially that between the two chloroplast activities.     

Genetically stable cell lines of cucumber for the large-scale production of diploid somatic embryos

For the initiation of embryogenic cucumber ( Cucumis sativus L.) cell lines, from excised radicles, directly in liquid medium, the culture regime, explant density and type and concentration of hormones were adjusted so that pro-embryogenic masses (PEMs) were formed within about 8 weeks. The established cucumber cell lines were maintained tor several years without loss of embryogenic and genetic stability. The ploidy level of somatic embryos from different cucumber eell lines was either diploid or tetraploid and depended on the ploidy level of Ihe cell line. Cucumber cell lines that produced only diploid embryos were obtained by selecting completely diploid explant material and growing it in the dark during the initiation phase. Mixoploid explains could lead to tetraploid or mixoploid ceil lines. Isolation and additional selection and subculturing of single PEMs resulted in either completely diploid or tetraploid cell lines, indicating that all cells of individual PEMs are either diploid or tetraploid. The ernbryogenic cucumber cell Imes, differing only in ploidy level, were indistinguishable in growth rate and embryogetiic potential and were genetically stable over several years.     

Immunoaffinity Purification of Epitope-Tagged Human β2-Adrenergic Receptor to Homogeneity

To obtain large quantities of pure human β₂-adrenergic receptor (β₂-AR) needed for structural studies, an efficient method for β₂-AR purification was developed using a recombinant receptor with an eight amino acid epitope at its C-terminus. This epitope is recognized by KT3-monoclonal antibody. The epitope tagged β₂-AR was expressed in Sf9 cells with a specific activity of 5–20 pmol/mg of membrane protein. The epitope-tagged and wild-type receptors had identical ligand binding properties. The tagged receptor was solubilized using dodecyl-β-maltoside with a quantitative yield. Solubilized epitope-tagged receptors were partially purified by KT3-mAb immunoaffinity in 60–70% yield. Further purification of the receptors on an alprenolol-affinity column resulted in a homogenous preparation with an overall yield of >30%. The purified receptor was concentrated to >1 mg/ml without loss of ligand binding activity.     

Modulation of ion currents and regulation of transmitter release in short-term synaptic plasticity: The rise and fall of the action potential

Up and down-regulation of calcium and potassium conductances are associated with several forms of short-term synaptic modulation. Detailed investigation of synaptic plasticity in the marine gastropodAplysia, and in other mollusks, indicates that synaptic transmission can be influenced in a number of ways by modulatory neurotransmitters acting through several second-messenger cascades. Modulation at the synapse itself occurs by means of the regulation of calcium current as well as through effects on processes directly involved in transmitter mobilization and exocytosis. Modulation of potassium current plays a major role in controlling neuronal excitability and may contribute to a lesser extent to the regulation of transmitter release through actions on the resting potential and on action potential configuration.     

Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease

The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1 ⁺ gene as a multi-copy suppressor of snm1. The pac1 ⁺ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1 ⁺ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1 ⁺ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.