Expression of genes encoding cytosolic and endoplasmic reticulum HSP90 proteins in the aquatic fungus Blastocladiella emersonii

HSP90 proteins are important molecular chaperones involved in multiple cellular processes. This work reports the characterization of cDNAs encoding two distinct HSP90 proteins (named HSP90A and HSP90B) from the chytridiomycete Blastocladiella emersonii. Deduced amino acid sequences of HSP90A and HSP90B exhibit signatures of the cytosolic and endoplasmic reticulum (ER) HSP90 proteins, respectively. A genomic clone encoding HSP90A was also characterized indicating the presence of a single intron of 184 bp interrupting the coding region, located near the amino-terminus of the protein. Expression of both HSP90A and HSP90B genes increases significantly during heat shock at 38 degrees C, with highest induction ratios observed in cells stressed during germination of the fungus. Changes in the amount of HSP90A transcript were also evaluated during B. emersonii life cycle at physiological temperature (27 degrees C), and its levels were found to increase both during germination and sporulation of the fungus. HSP90A protein levels were analyzed during B. emersonii life cycle and significant changes were observed only during sporulation. Furthermore, during heat stress a large increase in the amount of HSP90A protein was observed. Induction of HSP90A and HSP90B genes during heat stress indicates the importance of both genes in the response to high temperature in B. emersonii. (C) 2008 Elsevier B.V. All rights reserved.

Characterization of a beta-1,3-glucanase active in the alkaline midgut of Spodoptera frugiperda larvae and its relation to beta-glucan-binding proteins

Spodoptera frugiperda beta-1,3-glucanase (SLam) was purified from larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against beta-1,3-glucan (laminarin), but cannot hydrolyze yeast beta-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive beta-1,3-glucanases from insects, SLam is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLam was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. Multiple sequence alignment of beta-1,3-glucanases and beta-glucan-binding protein supports the assumption that the beta-1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the derived beta-1,3-glucanases by the loss of an extended N-terminal region and the beta-glucan-binding proteins by the loss of the catalytic residues. SLam homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLam antiserum reacts with a single protein in the insect midgut. Immunocytolocalization shows that the enzyme is present in secretory vesicles and glycocalyx from columnar cells. (C) 2010 Elsevier Ltd. All rights reserved.

Optimization of the rheological properties of probiotic yoghurts supplemented with milk proteins

This study aimed to optimize the rheological properties of probiotic yoghurts supplemented with skimmed milk powder (SMP) whey protein concentrate (WPC) and sodium caseinate (Na-Cn) by using an experimental design type simplex-centroid for mixture modeling It Included seven batches/trials three were supplemented with each type of the dairy protein used three corresponding to the binary mixtures and one to the ternary one in order to increase protein concentration in 1 g 100 g(-1) of final product A control experiment was prepared without supplementing the milk base Processed milk bases were fermented at 42 C until pH 4 5 by using a starter culture blend that consisted of Streptococcus thermophilus Lactobacillus delbrueckii subsp bulgaricus and Bifidobacterium (Humans subsp lactis The kinetics of acidification was followed during the fermentation period as well the physico-chemical analyses enumeration of viable bacteria and theological characteristics of the yoghurts Models were adjusted to the results (kinetic responses counts of viable bacteria and theological parameters) through three regression models (linear quadratic and cubic special) applied to mixtures The results showed that the addition of milk proteins affected slightly acidification profile and counts of S thermophilus and B animal`s subsp lactis but it was significant for L delbrueckii subsp bulgaricus Partially-replacing SMP (45 g/100 g) with WPC or Na-Cn simultaneously enhanced the theological properties of probiotic yoghurts taking into account the kinetics of acidification and enumeration of viable bacteria (C) 2010 Elsevier Ltd All rights reserved

Microwave-assisted solid-phase peptide synthesis at 60 degrees C: alternative conditions with low enantiomerization

Several conditions have been used in the coupling reaction of stepwise SPPS at elevated temperature (SPPS-ET), but we have elected the following as our first choice: 2.5-fold molar excess of 0.04-0.08 M Boc or Fmoc-amino acid derivative, equimolar amount of DIC/HOBt (1:1)or TBTU/DIPEA(1:3), 25% DMSO/toluene, 60 degrees C, conventional heating. In this study, aimed to further examine enantiomerization under such condition and study the applicability of our protocols to microwave-SPPS, peptides containing L-Ser, L-His, L-Cys and/or L-Met were manually synthesized traditionally, at 60 degrees C using conventional heating and at 60 degrees C using microwave heating. Detailed assessment of all crude peptides (in their intact and/or fully hydrolyzed forms) revealed that, except for the microwave-assisted coupling of L-Cys, all other reactions occurred with low levels of amino acid enantiomerization (<2%). Therefore, herein we (i) provide new evidences that our protocols for SPPS at 60 degrees C using conventional heating are suitable for routine use, (ii) demonstrate their appropriateness for microwave-assisted SPPS by Boc and Fmoc chemistries, (iii) disclose advantages and limitations of the three synthetic approaches employed. Thus, this study complements our past research on SPPS-ET and suggests alternative conditions for microwave-assisted SPPS. Copyright (C) 2009 European Peptide Society and John Wiley & Sons, Ltd.

Stoichiometry and thermodynamics of the interaction between the C-terminus of human 90 kDa heat shock protein Hsp90 and the mitochondrial translocase of outer membrane Tom70

A large majority of the 1000-1500 proteins in the mitochondria are encoded by the nuclear genome, and therefore, they are translated in the cytosol in the form and contain signals to enable the import of proteins into the organelle. The TOM complex is the major translocase of the outer membrane responsible for preprotein translocation. It consists of a general import pore complex and two membrane import receptors, Tom20 and Tom70. Tom70 contains a characteristic TPR domain, which is a docking site for the Hsp70 and Hsp90 chaperones. These chaperones are involved in protecting cytosolic preproteins from aggregation and then in delivering them to the TOM complex. Although highly significant, many aspects of the interaction between Tom70 and Hsp90 are still uncertain. Thus, we used biophysical tools to study the interaction between the C-terminal domain of Hsp90 (C-Hsp90), which contains the EEVD motif that binds to TPR domains, and the cytosolic fragment of Tom70. The results indicate a stoichiometry of binding of one monomer of Tom70 per dimer of C-Hsp90 with a K(D) of 360 30 nM, and the stoichiometry and thermodynamic parameters obtained suggested that Tom70 presents a different mechanism of interaction with Hsp90 when compared with other TPR proteins investigated. (C) 2011 Elsevier Inc. All rights reserved.

Identification of non-zein proteins in BR473 maize protein bodies by LC-nanoESI-MS/MS

The nutritional value of maize seed is limited due to its high content of storage proteins (zeins), which are deficient in essential amino acids such as lysine and tryptophan. In a previous paper, we showed that protein bodies obtained from BR473 maize variety, developed by Embrapa (Brazilian Agricultural Research Corporation), were mainly constituted by Z27 and a smaller quantity of Z50 gamma-zeins. Besides zein proteins, other not identified protein band in the SDS/PAGE was also observed, which could indicate the presence of non-zein proteins additionally to gamma-zeins. In the present paper, we have demonstrated the presence of non-zein proteins in BR473 maize protein bodies by LC-nanoESI-MS/MS and database searching. This fact could be related to the excellent energetic value and higher protein quality of BR473 maize grains, since high lysine concentration in some maize varieties has been related to the presence of cytoskeleton proteins that are non-zeins. We have identified the following proteins: Brittle-1 protein (chloroplast precursor), Legumin-1, glyceroldehyde-3-phosphate dehydrogenase, and elongation factor 1-alpha.