Biochemical and molecular characterization of poly(aspartic acid) hydrolase-2 from sphingomonas sp. KT-1

Poly(aspartic acid) (PAA) hydrolase-2 was purified from crude soluble cellular extracts of Sphingomonas sp. KT-1 (JCM10459) and characterized to elucidate the mechanism of alpha,beta-poly(d,l-aspartic acid) (tPAA) biodegradation. The molecular mass of PAA hydrolase-2 was 42 kDa, and the isoelectric point was 9.6. The optimum values of pH and temperature for the hydrolysis of alpha-di(l-aspartic acid) by PAA hydrolase-2 were 7.0 and 55 degrees C, respectively. The effect of inhibitors on the hydrolysis of alpha-di(l-aspartic acid) showed that the activity of PAA hydrolase-2 was significantly inhibited by EDTA. Thermally synthesized tPAA was hydrolyzed in the presence of two enzymes, PAA hydrolase-1 and PAA hydrolase-2, to generate aspartic acid. The PAA hydrolase-2 was capable of hydrolyzing alpha-poly(l-aspartic acid) of high molecular weights but had limited activity for tPAA. These results lead us to propose the following mechanism. First, PAA hydrolase-1 hydrolyzes tPAA to yield oligo(aspartic acid) via an endo-mode cleavage, and subsequently, PAA hydrolase-2 hydrolyzes the resultant oligo(aspartic acid) to yield aspartic acid. Analysis of hydrolyzed products from alpha- and beta-penta(l-aspartic acid) revealed that PAA hydrolase-2 catalyzed the exo-mode hydrolysis of alpha- and beta-penta (l-aspartic acid). The gene encoding PAA hydrolase-2 from Sphingomonas sp. KT-1 was cloned, and genetic analysis showed that the deduced amino acid sequence of PAA hydrolase-2 is similar to a putative peptidase, which belongs to the M20/M25/M40 family of proteins, from Caulobacter crescentus CB15.     

Evolution and microsynteny of the apyrase gene family in three legume genomes

Apyrases have been suggested to play important roles in plant nutrition, photomorphogenesis, and nodulation. To help trace the evolution of these genes in the legumes—and possibly, the acquisition of new functions for nodulation—apyrase-containing BACs were sequenced from three legume genomes. Genomic sequences from Medicago truncatula, Glycine max and Lotus japonicus were compared to one another and to corresponding regions in Arabidopsis thaliana. A phylogenetic analysis of apyrase homologs from these regions and sequences from other legume species, as well as other plant families, identified a potentially legume-specific clade that contains a well-characterized soybean ( G. soja) apyrase, Gs52, as well as homologs from Dolichos, Lotus , Medicago and Pisum. Sister clades contain homologs from members of Brassicaceae, Solanaceae, Poaceae and Fabaceae. Comparisons of rates of change at synonymous and nonsynonymous sites in the Gs52 and sister clades show rapid evolution in the potentially legume-specific Gs52 clade. The genomic organization of the apyrase-containing BACs shows evidence of gene duplication, genomic rearrangement, and gene conversion among Gs52 homologs. Taken together, these results suggest a scenario of local apyrase gene duplication in an ancestor of the legumes, followed by functional diversification and increased rates of change in the new genes, and further duplications in the Galegae (which include the genera Medicago and Pisum). The study also provides a detailed comparison of genomic regions between two model genomes which are now being sequenced ( M. truncatula and L. japonicus), and a genome from an economically important legume species ( G. max).Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by A. Kondorosi     

NORs inheritance analysis in crossings including individuals from two stocks of rainbow trout (Oncorhynchus mykiss)

Silver nitrate staining of rainbow trouts (Oncorhynchus mykiss) chromosomes, for the identification of the nucleolar organizing regions (NORs), revealed that in individuals from Núcleo Experimental de Salmonicultura de Campos do Jord?o (Brazil) NORs were located in the long arms of submetacentric pair while in specimens from Mount Shasta (USA) NORs were located in the short arms of a submetacentric pair. Cytogenetic analysis of the offspring, obtained through artificial crosses including individuals from both stocks, allowed the identification of NORs in two submetacentric chromosomes, one in the short arms and the other in the long arms, confirming the effectiveness of the hybridization process. Complementary results obtained using the FISH technique with 18S and 5S rDNA probes showed that NOR-bearing chromosomes exhibited a cluster of 5S genes located in tandem with the 18S gene cluster in both stocks. The results allow us to suggest that the difference in NOR-bearing chromosomes found between the two stocks is likely to be due to pericentric inversion involving the chromosome segment where 18S and 5S rDNA genes are located. The presence of ribosomal genes in the long arms of a submetacentric chromosome is apparently a particular characteristic of the rainbow trout stock of Campos do Jord?o and might be used as a chromosome marker in studies of controlled crosses in this species.     

Two phenolic acids from Lithospermum erythrorhizon cell suspension cultures

Two phenolic acids isolated from colourless cell cultures of Lithospermum erythrorhizon were identified as rosmarinic acid and lithospermic acid. However, these compounds were not detected in pigmented cell cultures producing shikonin derivatives.     

Clustered genes required for the synthesis of heterocyst envelope polysaccharide in Anabaena sp. strain PCC 7120

As demonstrated with alr2835 (hepA) and alr2834 (hepC) mutants, heterocysts of Anabaena sp. strain PCC 7120, a filamentous cyanobacterium, must have an envelope polysaccharide layer (the Hep+ phenotype) to fix dinitrogen in an oxygen-containing milieu (the Fox+ phenotype). Transpositions presumptively responsible for a Fox- phenotype were localized in open reading frames (ORFs) near hepA and hepC. A mutation in each of nine of these ORFs was complemented by a clone bearing only that single, intact ORF. Heterocysts of the nine mutants were found to lack an envelope polysaccharide layer. Complementation of mutations in alr2832 and alr2840 may have resulted from recombination. However, alr2825, alr2827, alr2831, alr2833, alr2837, alr2839, and alr2841, like hepA and hepC, are required for a Hep+ Fox+ phenotype.     

Melatonin binding sites in the brain of European sea bass (Dicentrarchus labrax)

Characteristics, day-night changes, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) modulation, and localization of melatonin binding sites in the brain of a marine teleost, European sea bass Dicentrarchus labrax, were studied by radioreceptor assay using 2-[(125)I]iodomelatonin as a radioligand. The specific binding to the sea bass brain membranes was rapid, stable, saturable and reversible. The radioligand binds to a single class of receptor site with the affinity (Kd) of 9.3 +/-0.6 pM and total binding capacity (Bmax) of 39.08 +/-0.86 fmol/mg protein (mean+/-SEM, n=4) at mid-light under light-dark (LD) cycles of 12:12. Day-night changes were observed neither in the Kd nor in the Bmax under LD 12:12. Treatment with GTPgammaS significantly increased the Kd and decreased the Bmax both at mid-light and mid-dark. The binding sites were highly specific for 2-phenylmelatonin, 2-iodomelatonin, melatonin, and 6-chloromelatonin. Distribution of melatonin binding sites in the sea bass brain was uneven: The Bmax was determined to be highest in mesencephalic optic tectum-tegmentum and hypothalamus, intermediate in telencephalon, cerebellum-vestibulolateral lobe and medulla oblongata-spinal cord, and lowest in olfactory bulbs with the Kd in the low picomolar range. These results indicate that melatonin released from the pineal organ and/or retina plays neuromodulatory roles in the sea bass brain via G protein-coupled melatonin receptors.     

Synthesis and physical characterization of poly(ethylene glycol)-gelatin conjugates

Poly(ethylene glycol) (PEG) with the terminal group of active ester was coupled to the amino group of gelatin to prepare PEG-grafted gelatin (PEG-gelatin). The affinity chromatographic study revealed that the PEG-gelatin with high degrees of PEGylation did not adsorb onto the gelatin affinity column, in remarked contrast to gelatin alone and the PEG-gelatin with low PEGylation degrees. The former PEG-gelatin showed a critical micelle concentration while it had the apparent molecular size of about 100 nm and a surface charge of almost zero. These findings indicate that the PEG-gelatin formed a micelle structure of which the surface is covered with PEG molecules grafted. When the body distribution of 125I-labeled gelatin and PEG-gelatin after intravenous injection was evaluated, the radioactivity of micellar PEG-gelatin was retained in the blood circulation compared with that of gelatin and the PEG-gelatin of no micelle formation. At the same PEGylation degree, the blood concentration was significantly higher for the PEG-gelatin prepared from PEG with a molecular weight of 12 000 than that of molecular weights of 2000 and 5000. It is concluded that the PEG-gelatin is a drug carrier with a micelle structure which retains in the blood circulation.     

Disruption of the novel plant protein NEF1 affects lipid accumulation in the plastids of the tapetum and exine formation of pollen, resulting in male sterility in Arabidopsis thaliana

A novel male-sterile mutant of Arabidopsis thaliana was isolated by means of T-DNA tagging. Pollen abortion of the mutant was evident after microspore release, and pollen grains were completely absent at anthesis. Transmission electron microscope analysis revealed that primexine was coarsely developed, and that although sporopollenin was produced, it was not deposited onto the microspore plasma membrane. The sporopollenin that failed to be deposited aggregated and accumulated within the locule and on the locule wall. Finally, as no exine formation was observed, the mutant was named nef1. The plastoglobuli within the plastids of the tapetum were reduced, and lipid accumulation was considerably decreased. The mutant had a significantly altered leaf chloroplast ultrastructure and showed various growth defects. Lipid analysis revealed that the total lipid content in nef1 was lower than that in the wild type, which indicated that Nef1 was involved in lipid metabolism. Cloning of the full-length Nef1 indicated that the gene encodes a novel plant protein of 1123 amino acids with limited sequence similarities to membrane proteins or transporter-like proteins, and the NEF1 is predicted to be a plastid integral membrane protein. Motif analysis revealed that NEF1 contains prokaryotic membrane lipoprotein lipid attachment sites that are involved in maintaining cell envelope integrity. It is predicted that the Nef1 encodes a membrane protein that maintains the envelope integrity in the plastids.