Heterologous expression of an Aspergillus niveus xylanase GH11 in Aspergillus nidulans and its characterization and application

A xylanase was cloned from Aspergillus niveus and successfully expressed in Aspergillus nidulans (XAN). The full-length gene consisted of 890 bp and encoded 275 mature amino acids with a calculated mass of 31.3 kDa. The deduced amino acid sequence was highly homologous with the xylanase belonging to family 11 of the glycoside hydrolases. The recombinant protein was purified to electrophoretic homogeneity by anion-exchange chromatography and gel filtration. The optima of pH and temperature for the recombinant enzyme were 5.0 and 65 degrees C, respectively. The thermal stability of the recombinant xylanase was extremely improved by covalent immobilization on glyoxyl agarose with 91.4% of residual activity after 180 min at 60 degrees C, on the other hand, the free xylanase showed a half-life of 9.9 min at the same temperature. Affinity chromatography on Concanavalin A- and Jacalin-agarose columns followed by SDS-PAGE analyses showed that the XAN has O- and N-glycans. XAN promotes hydrolysis of xylan resulting in xylobiose, xylotriose and xylotetraose. Intermediate degradation of xylan resulting in xylo-oligomers is appealing for functional foods as the beneficial effect of oligosaccharides on gastrointestinal micro flora includes preventing proliferation of pathogenic intestinal bacteria and facilitates digestion and absorption of nutrients. (C) 2011 Elsevier Ltd. All rights reserved.

TRADE-OFF BETWEEN IMMUNE STIMULATION AND EXPRESSION OF STORAGE PROTEIN GENES

Proteins stored in insect hemolymph may serve (is a source of amino acids and energy for metabolism, and development. The expression of the main storage proteins was assessed in bacterial-challenged honey bees using real-time (RT)-PCH and Western blot.. After ensuring that. the immune system had, been activated by measuring the ensuing expression (, the innate immune response genes, defensin-1 (def-1) and prophenoloxidase (pro PO), we verified the expression of four genes encoding storage proteins. The levels of vitellogenin (vg) mRNA and of the respective protein. were significantly lowered in bees injected with bacteria or water only (injury). An equivalent response was observed in orally-infected bees. The levels of apolipophorin II/I (apoLP-II/I) and hexamerin (hex 70a) mRNAs did not significantly change, but levels of Hex 70a protein subunit showed a substantial decay after bacterial challenge or injury. Infection also caused a strong reduction in the levels of apoLP-III transcripts. Our findings are consistent with a down-regulation, of the express and accumulation of storage proteins as a consequence of activation of the immune system, suggesting that this phenomenon. represents a strategy to redirect resources to combat injury or infection. (C) 2009 Wiley Periodicals, Inc.

Mitochondrial genome data alone are not enough to unambiguously resolve the relationships of Entognatha, Insecta and Crustacea sensu lato (Arthropoda)

An analysis of the relationships of the major arthropod groups Was undertaken using mitochondrial genome data to examine the hypotheses that Hexapoda is polyphyletic and that Collembola is more closely related to branchiopod crustaceans than insects. We sought to examine the sensitivity of this relationship to outgroup choice, data treatment. gene choice and optimality criteria used in the phylogenetic analysis of mitochondrial genome data. Additionally we sequenced the mitochondrial genome of ail archaeognathan, Nesomachilis australica. to improve taxon selection in the apterygote insects, a group poorly represented in previous mitochondrial phylogenies. The sister group of the Collembola was rarely resolved in our analyses with a significant level of support. The use of different outgroups (myriapods, nematodes, or annelids + mollusks) resulted in many different placements of Collembola. The way in which the dataset was coded for analysis (DNA, DNA with the exclusion of third codon position and as amino acids) also had marked affects on tree topology. We found that nodal Support was spread evenly throughout the 13 mitochondrial genes and the exclusion of genes resulted in significantly less resolution in the inferred trees. Optimality criteria had a much lesser effect on topology than the preceding factors; parsimony and Bayesian trees for a given data set and treatment were quite similar. We therefore conclude that the relationships of the extant arthropod groups as inferred by mitochondrial genomes are highly vulnerable to outgroup choice, data treatment and gene choice, and no consistent alternative hypothesis of Collembola's relationships is supported. Pending the resolution of these identified problems with the application of mitogenomic data to basal arthropod relationships, it is difficult to justify the rejection of hexapod monophyly, which is well supported on morphological grounds. (c) The Willi Hennig Society 2004.

Evolutionary History of Arabidopsis thaliana Aminoacyl-tRNA Synthetase Dual-Targeted Proteins

Aminoacyl-transfer RNA (tRNA) synthetases (aaRS) are key players in translation and act early in protein synthesis by mediating the attachment of amino acids to their cognate tRNA molecules. In plants, protein synthesis may occur in three subcellular compartments (cytosol, mitochondria, and chloroplasts), which requires multiple versions of the protein to be correctly delivered to its proper destination. The organellar aaRS are nuclear encoded and equipped with targeting information at the N-terminal sequence, which enables them to be specifically translocated to their final location. Most of the aaRS families present organellar proteins that are dual targeted to mitochondria and chloroplasts. Here, we examine the dual targeting behavior of aaRS from an evolutionary perspective. Our results show that Arabidopsis thaliana aaRS sequences are a result of a horizontal gene transfer event from bacteria. However, there is no evident bias indicating one single ancestor (Cyanobacteria or Proteobacteria). The dual-targeted aaRS phylogenetic relationship was characterized into two different categories (paralogs and homologs) depending on the state recovered for both dual-targeted and cytosolic proteins. Taken together, our results suggest that the dual-targeted condition is a gain-of-function derived from gene duplication. Selection may have maintained the original function in at least one of the copies as the additional copies diverged.

Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins

An Escherichia coli cell-free transcription/translation system was used to explore the high-level incorporation Of L-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI-MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean N-15-HSQC NMR spectra. A redox-staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell-free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and/or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome-mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High-yield cell-free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains.

Mapping of suramin binding sites on the group IIA human secreted phospholipase A(2)

Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A(2) (hsPLA(2)GIIA) was investigated by molecular dynamics simulations (MD) and isothermal titration calorimetry (ITC). MD indicated two possible bound suramin conformations mediated by hydrophobic and electrostatic interactions with amino-acids in three regions of the protein. namely the active-site and residues located in the N- and C-termini, respectively. All three binding sites are located on the phospholipid membrane recognition surface, suggesting that suramin may inhibit the enzyme, and indeed a 90% reduction in hydrolytic activity was observed in the presence of 100 nM suramin. These results correlated with ITC data, which demonstrated 2.7 suramin binding sites on the hsPLA(2)GIIA, and indicates that suramin represents a novel class of phosphohpase A(2) inhibitor. (C) 2009 Elsevier Inc. All rights reserved.

Sucrose Fermentation by Brazilian Ethanol Production Yeasts in Media Containing Structurally Complex Nitrogen Sources

Four Saccharomyces cerevisiae Brazilian industrial ethanol production strains were grown, under shaken and static conditions, in media containing 22% (w/v) sucrose supplemented with nitrogen sources varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Sucrose fermentations by Brazilian industrial ethanol production yeasts strains were strongly affected by both the structural complexity of the nitrogen source and the availability of oxygen. Data suggest that yeast strains vary in their response to the nitrogen source`s complex structure and to oxygen availability. In addition, the amount of trehalose produced could be correlated with the fermentation performance of the different yeasts, suggesting that efficient fuel ethanol production depends on finding conditions which are appropriate for a particular strain, considering demand and dependence on available nitrogen sources in the fermentation medium.

Adsorption of phenylalanine on layered double hydroxides: effect of temperature and ionic strength

In this work we report the adsorption of phenylalanine (Phe) on Magnesium Aluminum Layered Double Hydroxides (Mg-Al-CO(3)-LDH) at two different temperatures (298 and 310 K) and under two distinct ionic strength conditions (with and without the addition 0.1 M of NaCl). The adsorption isotherms exhibit the same profile in all conditions, and they only differ in the amount of removed Phe. At lower ionic strength, the isotherms are almost identical at both temperatures, except for the last points, where the increase in temperature causes a decrease in the amount of adsorbed Phe. An increase in ionic strength results in a decrease in Phe adsorption. The electrokinetic potential decreases as the amount of adsorbed Phe increases, and only positive values are observed. This indicates that the surface of the adsorbent is not totally neutralized and suggests that more Phe could be removed by adsorption. The presence of Phe on the solid is confirmed by FTIR spectra, which present the specific bands assigned to Phe. The hydrophobicity of the amino acid probably contributes to its extraction, thus enabling the removal of a great amount of Phe. In conclusion, LDH is potentially applicable in the removal of Phe from wastewater.

Glucose and Fructose Fermentation by Wine Yeasts in Media Containing Structurally Complex Nitrogen Sources

Glucose and fructose fermentations by industrial yeasts strains are strongly affected by both the structural complexity of the nitrogen Source and the availability of oxygen. In this Study two Saccharomyces cerevisiae industrial wine strains were grown, under shaken and static conditions, in a media containing either a) 20% (w/v) glucose, or b) 10% (w/v) fructose and 10% (w/v) glucose or c) 20% (w/v) fructose, all supplemented with nitrogen Sources varying from a single ammonium salt (ammonium Sulfate) to free amino acids (casamino acids) and peptides (peptone). Data Suggest that 1 complex Structured nitrogen source is not submitted to the same control mechanisms as those involved in the utilization of simpler structured nitrogen Sources, and mutual interaction between carbon and nitrogen Sources, including the mechanisms involved ill the regulation of aerobic/anaerobic metabolism, may play in important role in defining yeast fermentation performance and the differing response to the structural complexity of the nitrogen Source, with a strong impact oil fermentation performance.

Solution structure of χ-conopeptide MrIA, a modulator of the human norepinephrine transporter

The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a hairpin with the two strands connected by an inverse gamma-turn. The residues primarily involved in activity have previously been shown to be located in the turn region (Sharpe, I. A.; Palant, E.: Schroder, C. L; Kaye, D. M.; Adams, D. I.; Alewood, P. F.; Lewis, R. J. J Biol Client 2003, 278, 40317-40323), which appears to be more flexible than the beta-strands based on disorder in the ensemble of calculated structures. Analogues of MrIA with N-terminal truncations indicate that the N-terminal residues play a role in defining a stable conformation and the native disulfide connectivity. In particular, noncovalent interactions between Val3 and Hypl2 are likely to be involved in maintaining a stable conformation. The N-terminus also affects activity, as a single N-terminal deletion introduced additional pharmacology at rat vas deferens, while deleting the first two amino acids reduced chi-conopeptide potency. This article was originally published online as an accepted preprint. The Published Online date corresponds to the preprint version. You can request a copy of the preprint by entailing the Biopolymers editorial office at biopolymers@wiley.com (c) 2005 Wiley Periodicals, Inc.

Absence of GH-Releasing Hormone (GHRH) Mutations in Selected Patients with Isolated GH Deficiency

Context: Although numerous reports of mutations in GH1 and GHRHR (GHRH receptor) causing isolated GH deficiency (IGHD) have been published, mutations in GHRH itself have not been hitherto reported but are obvious candidates for GH deficiency. Objective: The aim of this study was to identify mutations in GHRH in a large cohort of patients with IGHD. Patients and Methods: DNA was isolated from 151 patients diagnosed with IGHD at national and international centers. Seventy-two patients fulfilled all the following criteria: severe short stature (height SD score <= -2.5), low peakGHafter stimulation (peak <= 5 ng/ml), eutopic posterior pituitary lobe, and absence of mutations in GH1 and GHRHR and therefore were strong candidates for GHRH mutations. The coding sequence and splice sites of GHRH were amplified by PCR with intronic primers and sequenced. Results: In five of 151 patients (four of 42 from Brazil), the GHRH c. 223 C>T, p. L75F change was identified in heterozygosity. This variant has been previously reported as a polymorphism and is more frequent in African than European and Asian populations. Six allelic variants (five novel) that do not predict change of amino acids or splice sites were identified in five patients: c. 147 C>T, p.S49S, IVS1 -70 G>A, IVS1 -74 T>C, IVS3 -47 del1, and IVS3 +7 G>A/IVS3 + 41 G>A. No functional mutations were found in this cohort. Conclusions: GHRH mutations were not identified in a selected cohort of patients with IGHD, suggesting that, if they exist, they may be an extremely rare cause of IGHD. Other, as-yet-unidentified genetic factors may be implicated in the genetic etiology of IGHD in our cohort. (J Clin Endocrinol Metab 96: E1457-E1460, 2011)

Identification and characterization of a G protein-coupled receptor homolog encoded by murine cytomegalovirus

This report describes the identification of a murine cytomegalovirus (MCMV) G protein-coupled receptor (GCR) homolog. This open reading frame (M33) is most closely related to, and collinear with, human cytomegalovirus UL33, and homologs are also present in human herpesvirus 6 and 7 (U12 for both viruses). Conserved counterparts in the sequenced alpha- or gammaherpesviruses have not been identified to date, suggesting that these genes encode proteins which are important for the biological characteristics of betaherpesviruses. We have detected transcripts for both UL33 and M33 as early as 3 or 4 h postinfection, and these reappear at late times. In addition, we have identified N-terminal splicing for both the UL33 and M33 RNA transcripts. For both open reading frames, splicing results in the introduction of amino acids which are highly conserved among known GCRs. To characterise the function of the M33 in the natural host, two independent MCMV recombinant viruses were prepared, each of which possesses an M33 open reading frame which has been disrupted with the beta-galactosidase gene. While the recombinant M33 null viruses showed no phenotypic differences in replication from wild-type MCMV in primary mouse embryo fibroblasts in vitro, they showed severely restricted growth in the salivary glands of infected mice. These data suggest that M33 plays an important role in vivo, in particular in the dissemination to or replication in the salivary gland, and provide the first evidence for the function of a viral GCR homolog in vivo.

Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel

Fast synaptic neurotransmission is mediated by transmitter-activated conformational changes in ligand-gated ion channel receptors, culminating in opening of the integral ion channel pore. Human hereditary hyperekplexia, or startle disease, is caused by mutations in both the intracellular or extracellular loops flanking the pore-lining M2 domain of the glycine receptor alpha 1 subunit. These flanking domains are designated the M1-M2 loop and the M2-M3 loop respectively. We show that four startle disease mutations and six additional alanine substitution mutations distributed throughout both loops result in uncoupling of the ligand binding sites from the channel activation gate. We therefore conclude that the M1-M2 and M2-M3 loops act in parallel to activate the channel. Their locations strongly suggest that they act as hinges governing allosteric control of the M2 domain. As the members of the ligand-gated ion channel superfamily share a common structure, this signal transduction model may apply to all members of this superfamily.

Changes in purines concentration in the cerebrospinal fluid of patients experiencing pain: A case-control study

This study analyzes the relationship between extracellular purines and pain perception in humans. Cerebrospinal fluid (CSF) levels of purines and their metabolites were compared between patients displaying acute and/or chronic pain syndromes and control subjects. The CSF levels of IMP, inosine, guanosine and uric acid were significantly increased in the chronic pain group and correlated with pain severity (P<0.05). Patients displaying both chronic and acute pain presented similar changes in the CSF purines concentration (P<0.05). However, in the acute pain group, only CSF inosine and uric acid levels were significantly increased (P<0.05). These findings suggest that purines, in special inosine, guanosine and uric acid, are associated with the spinal mechanisms underlying nociception. (C) 2010 Elsevier Ireland Ltd. All rights reserved.