Characterization of Bacillus thuringiensis isolates from Argentina that are potentially useful in insect pest control

In order to find novel strains of Bacillus thuringiensis that are toxic to some of the major pests that impact economically important crops in Argentina, we initiated a search for B. thuringiensis isolates native to Argentina. We succeeded in assembling a collection of 41 isolates, some of which show a high potential to be used in biological control programs against lepidopteran and coleopteran pests. About 90% of the strains showed toxicity against Spodoptera frugiperda and Anticarsia gemmatalis, two important lepidopteran pests in Argentina. It is noteworthy that only one of these strains contained a cry1-type gene, while another isolate showed a dual toxicity against the lepidopteran and coleopteran insects assayed. Genetic characterization of the strains suggests that the collection likely harbors novel Cry proteins that may be of potential use in biological insect pest control.     

Characterization and delignification activity of a thermostable α-l-arabinofuranosidase from Bacillus stearothermophilus

Bacillus stearothermophilus L1 was isolated by enrichment culture using an alkaline extract of pulp as the carbon source at 65°C and pH 9.0. The bacterium produced extracellular xylanase and -l-arabinofuranosidase (EC 3.2.1.55). The xylanase activity was high when the cells were grown in the presence of d-xylose, whereas the arabinofuranosidase activity was high when grown in media containing l-arabinose. The arabinofuranosidase was purified 59-fold with an 80% yield by DEAE Sephacel and Sephadex G-100 chromatography. The purified enzyme had an apparent molecular mass of 110 000 kDa and consisted of two subunits of 52 500 kDa and 57 500 kDa. Using p-nitrophenyl--l-arabinofuranosidase as the substrate, the enzyme had a Michaelis constant (K _m) of 2.2 × 10^–4 m, maximum reaction velocity (V_max) of 11o mol min^–1 mg^–1, temperature optimum of 70°C and pH optimum of 7.0 (50% activity at pH 8.0). The enzyme was specific for the furanoside configuration. The purified enzyme partially delignified softwood Kraft pulp. Treatment of the pulp with 38 units ml^–1 of -l-arabinofuranosidase at 65°C for 2 h at pH 8.0 and 9.0 led to lignin releases of 2.3% and 2.1%, respectively. The enzyme acted synergistically with a thermophilic xylanase in the delignification process, yielding a 19.2% release of lignin. Correspondence to: Eugene Rosenberg     

Transesterification activity of a novel lipase from Acinetobacter venetianus RAG-1

Transesterification activity and the industrial potential of a novel lipase prepared from Acinetobacter ventiatus RAG-1 were evaluated. Purified lipase samples were dialyzed against pH 9.0 buffer in a single optimization step prior to lyophilization. The enzyme and organic phase were pre-equilibrated (separately) to the same thermodynamic water activities (a _w) ranging from a _w 0.33 to 0.97. Production of 1-octyl butyrate by lipase-catalyzed transesterification of vinyl butyrate with 1-octanol in hexane was monitored by gas chromatography. Production of 1-octyl butyrate and initial rate of reaction depended on water activity. Product synthesis and rate of transesterification increased sharply with increase from a _w 0.33 to 0.55. Highest product concentration (218 mM) and rate of reaction (18.7 μmol h⁻¹ · 10 μg protein) were measured at a _w 0.86. Transesterification activity in hexane represented 32% of comparable hydrolytic activity in aqueous buffer.     

TRP channel–associated factors are a novel protein family that regulates TRPM8 trafficking and activity

TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor–activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named “TRP channel–associated factors” (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity.     

Extragenic suppressor mutations of a β-tubulin mutation in the basidiomyceteCoprinus cinereus: Isolation and genetic and biochemical analyses

In total, 111 revertants were isolated from the oidia of a heat-sensitive, -tubulin mutant BEN193 (benA193) of the basidiomyceteCoprinus cinereus after mutagenesis by ultraviolet. Of the 111 revertants, 48 were genetically analyzed. In 15 of the 48 revertants, reversion was due to mutations at loci unlinked tobenA, whereas in the remaining 33 revertants, reversion was due to mutations withinbenA or at loci closely linked tobenA. The 15 extragenic suppressor mutations comprised three groups in terms of genetic linkage; two of them were designated asmipA andmipB. Suppressor mutations in the third group were found to bebenC, one of the four loci we have previously identified as genes conferring benomyl resistance. Biochemical analysis revealed thatbenC ⁺ is a structural gene for a major -tubulin in vegetative hyphae.     

Evidence that expression of SpαI/65 hereditary elliptocytosis is compounded by a genetic factor that is linked to the homologous α-spectrin allele

Summary Many cases of hereditary elliptocytosis (HE) result from mutated spectrin -chains. It has repeatedly been observed that the amount of a mutant -chain is different in various affected individuals, resulting in clinical pictures of variable severity. The different levels are thought to result from different percentages of the spectrin allele in trans. Such percentages, in turn, could be under genetic control. We tested this hypothesis in a large Algerian family with Sp^I/65 HE. In an informative sibship, we found three persons with a distinctly high level of expression of the Sp^I/65 variant, suggesting the existence, in trans, of a low percentage -allele. The -spectrin gene haplotype associated with the latter was constantly – +–, based on the XbaI, PvuII and MspI polymorphic sites. In contrast, a basal level of expression of the Sp^I/65 variant in the same sibship indicated, in trans, the existence of a normal percentage -allele. The haplotype corresponding to this other -allele was + – +. Study of another generation of the family showed, however, that the – + – haplotype could also be linked to a normal percentage -allele. These results are consistent with the view that the expression level of ^I/65 spectrin (and of other types of -variants) is compounded by a genetic factor that is linked to the normal -allele in trans. The low percentage allele itself remains silent in the simple heterozygous state.     

Design,synthesis, and structure–activity relationships of a series of novel N-aryl-2-phenylcyclopropanecarboxamide that are potent and orally active orexin receptor antagonists

Herein we describe the design, synthesis, and structure–activity relationships (SARs) of a novel phenylcyclopropane series represented by 7 and 33b as antagonists of orexin 1 and orexin 2 receptors. With 4 serving as the initial lead for the development of orexin antagonists, exploration of SAR resulted in improved binding affinity for orexin 1 and orexin 2 receptors. Among the synthesized compounds, 33b ((−)-N-(5-cyanopyridin-2-yl)-2-[(3,4-dimethoxyphenyl)oxymethyl]-2-phenylcyclopropanecarboxamide) exhibited potent in vitro activity and oral efficacy in animal sleep measurement experiments. The results of our study suggest that compound 33b may serve as a valuable template for the development of new orexin receptor antagonists.     

Characterization of a novel β-thioglucosidase CpTGG1 in Carica papaya and its substrate-dependent and ascorbic acid-independent O-β-glucosidase activity

Plant thioglucosidases are the only known S-glycosidases in the large superfamily of glycosidases.These enzymes evolved more recently and are distributed mainly in Brassicales.Thioglucosidase research has focused mainly on the cruciferous crops due to their economic importance and cancer preventive benefits.In this study,we cloned a novel myrosinase gene,CpTGG1,from Carica papaya Linnaeus.and showed that it was expressed in the aboveground tissues in planta.The recombinant CpTGG1 expressed in Pichia pastoris catalyzed the hydrolysis of both sinigrin and glucotropaeolin(the only thioglucoside present in papaya),showing that CpTGG1 was indeed a functional myrosinase gene.Sequence alignment analysis indicated that CpTGG1 contained all the motifs conserved in functional myrosinases from crucifers,except for two aglycon-binding motifs,suggesting substrate priority variation of the non-cruciferous myrosinases.Using sinigrin as substrate,the apparent Km and Vmax values of recombinant CpTGG1 were 2.82 mM and 59.9 μmol min-1 mg protein-1,respectively.The Kcat IKm value was 23 s-1 mM-1.O-β-glucosidase activity towards a variety of substrates were tested,CpTGG1 displayed substrate-dependent and ascorbic acid-independent O-β-glucosidase activity towards 2-nitrophenyl-βD-glucopyranoside and 4-nitrophenyl-β-D-glucopyranoside,but was inactive towards glucovanillin and n-octyl-β-D-glucopyranoside.Phylogenetic analysis indicated CpTGG1 belongs to the MYR II subfamily of myrosinases.     

Broad-specificity GH131 β-glucanases are a hallmark of fungi and oomycetes that colonize plants

Plant-tissue-colonizing fungi fine-tune the deconstruction of plant-cell walls (PCW) using different sets of enzymes according to their lifestyle. However, some of these enzymes are conserved among fungi with dissimilar lifestyles. We identified genes from Glycoside Hydrolase family GH131 as commonly expressed during plant-tissue colonization by saprobic, pathogenic and symbiotic fungi. By searching all the publicly available genomes, we found that GH131-coding genes were widely distributed in the Dikarya subkingdom, except in Taphrinomycotina and Saccharomycotina, and in phytopathogenic Oomycetes, but neither other eukaryotes nor prokaryotes. The presence of GH131 in a species was correlated with its association with plants as symbiont, pathogen or saprobe. We propose that GH131-family expansions and horizontal-gene transfers contributed to this adaptation. We analysed the biochemical activities of GH131 enzymes whose genes were upregulated during plant-tissue colonization in a saprobe (Pycnoporus sanguineus), a plant symbiont (Laccaria bicolor) and three hemibiotrophic-plant pathogens (Colletotrichum higginsianum, C. graminicola, Zymoseptoria tritici). These enzymes were all active on substrates with β-1,4, β-1,3 and mixed β-1,4/1,3 glucosidic linkages. Combined with a cellobiohydrolase, GH131 enzymes enhanced cellulose degradation. We propose that secreted GH131 enzymes unlock the PCW barrier and allow further deconstruction by other enzymes during plant tissue colonization by symbionts, pathogens and saprobes.     

Random mutagenesis suggests that sequence errors are not a major cause of variation in the activity of individual molecules of β-galactosidase

Wild-type Escherichia coli lacZ was subjected to error-prone PCR to generate two plasmid-encoded gene libraries containing approximately 2.6 (SD 1.9) nucleotide exchanges resulting in 1.8 (SD 1.4) amino-acid substitutions. The libraries were used, along with a plasmid containing wild-type lacZ, to transform E. coli lacking genomic lacZ. Cells expressing functional β-galactosidase were identified by blue/white screening. Cell lysates containing the populations of heterogeneously mutagenized β-galactosidase were subjected to single molecule assays using a capillary electrophoresis laser-induced fluorescence-based protocol. There was no significant difference in the average catalytic rate between the random mutagenized and wild-type enzyme populations. Furthermore, there was no clear pattern between error rates and the variances in the population catalytic rates. This suggests that random sequence errors are not a substantial source of the catalytic heterogeneity of this enzyme.     

Characterization and genetic manipulation of primed stem cells into a functional na?ve state with ESRRB

AIM To identify differences between primed mouse embryonic stem cells(ESCs) and fully functional naive ESCs; to manipulate primed cells into a naive state. METHODS We have cultured 3 lines of cells from different mouse strains that have been shown to be naive or primed as determined by generating germline-transmitting chimeras.Cells were put through a battery of tests to measure the different features. RNA from cells was analyzed using microarrays, to determine a priority list of the differentially expressed genes. These were later validated by quantificational real-time polymerase chain reaction. Viral cassettes were created to induce expression of differentially expressed genes in the primed cells through lentiviral transduction. Primed reprogrammed cells were subjected to in-vivo incorporation studies.RESULTS Most results show that both primed and naive cells have similar features(morphology, proliferation rates, stem cell genes expressed). However, there were some genes that were differentially expressed in the na?ve cells relative to the primed cells. Key upregulated genes in na?ve cells include ESRRB, ERAS, ATRX, RNF17, KLF-5, and MYC. After over-expressing some of these genes the primed cells were able to incorporate into embryos in-vivo, re-acquiring a feature previously absent in these cells. CONCLUSION Although there are no notable phenotypic differences, there are key differences in gene expression between these na?ve and primed stem cells. These differences can be overcome through overexpression.     

Characterization of the Fad12 mutant of Synechocystis that is defective in Δ12 acyl-lipid desaturase activity

The Fad12 mutant of Synechocystis sp. PCC 6803 has a defect in the desA gene for Δ12 acyl-lipid desaturase. We identified a change in the nucleotide sequence of the structural gene for the desaturase, in which a leucine codon has been converted to a stop codon. Western blot analysis revealed that the Δ12 acyl-lipid desaturase was localized in both plasma membranes and thylakoid membranes of wild-type cells but was absent from both types of membrane in Fad12 cells. These findings suggest that the desaturation of fatty acids takes place in both types of membrane in Synechocystis sp. PCC 6803. The mutation in the Δ12 desaturase did not affect the lipid composition of thylakoid and plasma membranes, but it changed the fatty acid composition of lipids in similar ways in both types of membrane.     

Dietary rescue of a lethal “null” activity allele of 6-phosphogluconate dehydrogenase in Drosophila melanogaster

The genetic rescue of Pgd ⁿ lethal alleles, accomplished by combining them with mutations lacking glucose-6-phosphate dehydrogenase activity, has led to the hypothesis that Pgd ⁿ lethality may be due to the accumulation of 6-phosphogluconate. In this article we report the rescue of Pgd ⁿ /Y males by dietary supplements (fructose and linolenate) designed to minimize 6-phosphogluconate production.This investigation was supported by Research Grant GM-15691 and Training Grant T01-GM-0685 of the National Institutes of Health.     

Identification of new γ-hydroxybutenolides that preferentially inhibit the activity of mPGES-1

Microsomal prostaglandin E(2) synthase-1 (mPGES-1) has been recognized as novel, promising drug target for anti-inflammatory and anticancer drugs. mPGES-1 catalyzes the synthesis of the inducible prostaglandin E(2) in response to pro-inflammatory stimuli, rendering this enzyme extremely interesting in drug discovery process owing to the drastic reduction of the severe side effects typical for traditional non-steroidal anti-inflammatory drugs. In the course of our investigations focused on this topic, we identified two interesting molecules bearing the γ-hydroxybutenolide scaffold which potently inhibit the activity of mPGES-1. Notably, the lead compound 2c that inhibited mPGES-1 with IC(50)=0.9μM, did not affect other related enzymes within the arachidonic acid cascade.     

Cloning,expression and biochemical characterization of a GH1 β-glucosidase from Cellulosimicrobium cellulans

β-Glucosidase plays an important role in the degradation of cellulose. In this study, a novel β-glucosidase ccbgl1b gene for a glycosyl hydrolase (GH) family 1 enzyme was cloned from the genome of Cellulosimicrobium cellulans and expressed in Escherichia coli BL21 cells. The sequence contained an open reading frame of 1494?bp, encoded a polypeptide of 497?amino acid residues. The recombinant protein CcBgl1B was purified by Ni sepharose fastflow affinity chromatography and had a molecular weight of 57?kDa, as judged by SDS-PAGE. The optimum β-glucosidase activity was observed at 55?°C and pH 6.0. Recombinant CcBgl1B was found to be most active against aryl-glycosides p-nitrophenyl-β-D-glucopyranoside (pNPβGlc), followed by p-nitrophenyl-β-D-galactopyranoside (pNPβGal). Using disaccharides as substrates, the enzyme efficiently cleaved β-linked glucosyl-disaccharides, including sophorose (β-1,2-), laminaribiose (β-1,3-) and cellobiose (β-1,4-). In addition, a range of cello-oligosaccharides including cellotriose, cellotetraose and cellopentaose were hydrolysed by CcBgl1B to produce glucose. The interaction mode between the enzyme and the substrates driving the reaction was modelled using a molecular docking approach. Understanding how the GH1 enzyme CcBgl1B from C. cellulans works, particularly its activity against cello-oligosaccharides, would be potentially useful for biotechnological applications of cellulose degradation.     

Characterization of Staphylococcus epidermidis Polynucleotide phosphorylase and its interactions with ribonucleases RNase J1 and RNase J2

Polynucleotide phosphorylase catalyzes both 3′-5′ exoribonuclease and polyadenylation reactions. The crystal structure of Staphylococcus epidermidis PNPase revealed a bound phosphate in the PH2 domain of each protomer coordinated by three adjacent serine residues. Mutational analysis suggests that phosphate coordination by these serine residues is essential to maintain the catalytic center in an active conformation. We note that PNPase forms a complex with RNase J1 and RNase J2 without substantially altering either exo-ribonuclease or polyadenylation activity of this enzyme. This decoupling of catalytic activity from protein-protein interactions suggests that association of these endo- or exo-ribonucleases with PNPase could be more relevant for cellular localization or concerted targeting of structured RNA for recycling.     

DRB1*LY10 — a newDRB1 allele and its haplotypic association

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M34372.