Chitin and chitosan from Basidiomycetes

Chitinous material was isolated from the mycelium of seven species of Basidiomycetes to evaluate the possibility of using fungal biomass as a source of chitin and chitosan. Such material was characterised for its purity, degree of acetylation and crystallinity. Chitin yields ranged between 8.5 and 19.6% dry weight and the chitosan yield was approximately 1%. The characteristics of the fungal chitins were similar to those of commercial chitin. Chitosans, with a low degree of acetylation, comparable with that of commercial chitosan, were obtained by the chemical deacetylation of fungal chitins.     

Determination of Fecal Glucocorticoid Metabolites to Evaluate Stress Response in <Emphasis Type="Italic">Alouatta pigra</Emphasis>

Measuring fecal glucocorticoid metabolites is now a common practice to assess the stress response in primates. Nevertheless, it is important to validate the utilized immunoassay for each primate species before the technique is applied to populations in the wild. We determined the stress response of black howlers (Alouatta pigra) via 2 different group-specific enzyme immunoassays (EIAs). 11-oxoetiocholanolone EIAs are suited to assess the stress response of black howlers via fecal glucocorticoid metabolites. Levels of fecal glucocorticoid metabolites increased after we applied a stressor, i.e. anesthesia, reaching peak concentrations 24–96 h poststressor. Both basal and stress-induced fecal glucocorticoid metabolite levels showed individual variations. The increase of fecal glucocorticoid metabolites after the stressor (paralleling increases in serum) indicates that one can effectively measure adrenocortical activity in Alouatta pigra via these 2 enzyme immunoassays. However, it is important to consider individual variations in the excretion of fecal glucocorticoid metabolites when planning field endocrinological research on Alouatta pigra. Fecal glucocorticoid metabolite excretion takes 1–3 d poststressor depending on the individual. Further, there is an important individual variability in the concentrations of glucocorticoid metabolites, which might reflect differences in stress reactivity or fecal glucocorticoid metabolite metabolism and excretion.     

Anaplasma marginale major surface protein 1a directs cell surface display of tick BM95 immunogenic peptides on Escherichia coli

The surface display of heterologous proteins on live Escherichia coli using anchoring motifs from outer membranes proteins has impacted on many areas of biochemistry, molecular biology and biotechnology. The Anaplasma marginale major surface protein 1a (MSP1a) contains N-terminal surface-exposed repeated peptides (28-289 amino acids) that are involved in pathogen interaction with host cell receptors and is surface-displayed when the recombinant protein is expressed in E. coli. Therefore, it was predicted that MSP1a would surface display on E. coli peptides inserted in the N-terminal repeats region of the protein. The Rhipicephalus (Boophilus) microplus BM86 and BM95 glycoproteins are homologous proteins that protect cattle against tick infestations. In this study, we demonstrated that a recombinant protein comprising tick BM95 immunogenic peptides fused to the A. marginale MSP1a N-terminal region is displayed on the E. coli surface and is recognized by anti-BM86 and anti-MSP1a antibodies. This system provides a novel approach to the surface display of heterologous antigenic proteins on live E. coli and suggests the possibility to use the recombinant bacteria for immunization studies against cattle tick infestations.     

Epigallocatechin-3-gallate (EGCG) inhibits PC-3 prostate cancer cell proliferation via MEK-independent ERK1/2 activation

Epigallocatechin-3-gallate (EGCG), a tea polyphenol, inhibits the proliferation of many cancer cell lines; however, the antiproliferative mechanism(s) are not well-characterized. The objective of this study is to identify the cellular signaling mechanism(s) responsible for the antiproliferative effects of EGCG in the PC-3 prostate cancer cell line. EGCG inhibited PC-3 cell proliferation in a concentration-dependent manner with an IC(50) value of 39.0 microM, but had no effect on the proliferation of a nontumorigenic prostate epithelial cell line (RWPE-1). Treatment of PC-3 cells with EGCG (0-50 microM) resulted in time and concentration-dependent activation of the extracellular signal-regulated kinase (ERK1/2) pathway. EGCG treatment did not induce ERK1/2 activity in RWPE-1 cells. The activation of ERK1/2 by EGCG was not inhibited using PD98059, a potent inhibitor of mitogen-activated protein kinase kinase (MEK), the immediate upstream kinase responsible for ERK1/2 activation; suggesting a MEK-independent signaling mechanism. Pretreatment of PC-3 cells with a phosphoinositide-3 kinase (PI3K) inhibitor partially reduced both EGCG-induced ERK1/2 activation and the antiproliferative effects of this polyphenol. These results suggest that ERK1/2 activation via a MEK-independent, PI3-K-dependent signaling pathway is partially responsible for the antiproliferative effects of EGCG in PC-3 cells.     

Immunosuppression in cardiac graft rejection: a human in vitro model to study the potential use of new immunomodulatory drugs

CXCL10-CXCR3 axis plays a pivotal role in cardiac allograft rejection, so that targeting CXCL10 without inducing generalized immunosuppression may be of therapeutic significance in allotransplantation. Since the role of resident cells in cardiac rejection is still unclear, we aimed to establish reliable human cardiomyocyte cultures to investigate Th1 cytokine-mediated response in allograft rejection. We used human fetal cardiomyocytes (Hfcm) isolated from fetal hearts, obtained after legal abortions. Hfcm expressed specific cardiac lineage markers, specific cardiac structural proteins, typical cardiac currents and generated ventricular action potentials. Thus, Hfcm represent a reliable in vitro tool for allograft rejection research, since they resemble the features of mature cells. Hfcm secreted CXCL10 in response to IFNgamma and TNFalphaalpha; this effect was magnified by cytokine combination. Cytokine synergy was associated to a significant TNFalpha-induced up-regulation of IFNgammaR. The response of Hfcm to some currently used immunosuppressive drugs compared to rosiglitazone, a peroxisome proliferator-activated receptor gamma agonist and Th1-mediated response inhibitor, was also evaluated. Only micophenolic acid and rosiglitazone halved CXCL10 secretion by Hfcm. Given the pivotal role of IFNgamma-induced chemokines in Th1-mediated allograft rejection, these preliminary results suggest that the combined effects of immunosuppressive agents and rosiglitazone could be potentially beneficial to patients receiving heart transplants.     

Deciphering the structural role of histidine 83 for heme binding in hemophore HasA

Heme carrier HasA has a unique type of histidine/tyrosine heme iron ligation in which the iron ion is in a thermally driven two spin states equilibrium. We recently suggested that the H-bonding between Tyr75 and the invariantly conserved residue His83 modulates the strength of the iron-Tyr75 bond. To unravel the role of His83, we characterize the iron ligation and the electronic properties of both wild type and H83A mutant by a variety of spectroscopic techniques. Although His83 in wild type modulates the strength of the Tyr-iron bond, its removal causes detachment of the tyrosine ligand, thus giving rise to a series of pH-dependent equilibria among species with different axial ligation. The five coordinated species detected at physiological pH may represent a possible intermediate of the heme transfer mechanism to the receptor.     

Human cells and cell membrane molecular models are affected in vitro by chlorpromazine

This study presents evidence that chlorpromazine (CPZ) affects human cells and cell membrane molecular models. Human SH-SY5Y neuroblastoma cells incubated with 0.1 mM CPZ suffered a decrease of cell viability. On the other hand, phase contrast microscopy observations of human erythrocytes indicated that they underwent a morphological alteration as 1 μM CPZ changed their discoid normal shape to stomatocytes, and to hemolysis with 1 mM CPZ. X-ray diffraction experiments performed on dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) bilayers, classes of the major phospholipids present in the outer and inner sides of the erythrocyte membrane, respectively showed that CPZ disordered the polar head and acyl chain regions of both DMPC and DMPE, where these interactions were stronger with DMPC bilayers. Fluorescence spectroscopy on DMPC LUV at 18 °C confirmed these results. In fact, the assays showed that CPZ induced a significant reduction of their generalized polarization (GP) and anisotropy (r) values, indicative of enhanced disorder at the polar head and acyl chain regions of the DMPC lipid bilayer.     

Purification and characterization of recombinant lipid storage protein-2 from Drosophila melanogaster

Lipid storage protein 2 (Lsd 2) is a conserved insect protein that belongs to the small PAT family of proteins. PAT proteins are found associated to the lipid droplets of adipocytes and play significant roles in the regulation of triacylglycerides metabolism. Here we describe the expression and purification of Lsd2, its reconstitution in lipoprotein particles, the location of putative lipid binding sites and its secondary structure. This study provides the starting point for future studies on the mechanism of function of Lsd2. The similarities and differences between Lsd1 and Lsd2, the only PAT proteins found in insects, are discussed.     

Monitoring the positioning of short polycationic peptides in model lipid bilayers by combining hydrogen/deuterium exchange and electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to analyze the hydrogen/deuterium exchange properties of the mastoparan peptide Apoica-MP during interactions with lipid vesicle membranes. Synthetic peptide was incorporated into large unilamellar vesicles (LUVs) of L-alpha-phosphatidylcholine (PC), resulting in proteoliposomes which were then diluted with D2O. After quenching deuteration by the addition of formic acid the H/D exchange was directly analyzed by ESI-MS. This strategy was used to investigate the architecture of the peptide in the membranes of PC LUVs. The deuterated peptide ions were analyzed under collision-induced dissociation (CID) mass spectrometry, which permitted the location of deuterons at the amide sites along the peptide backbone. Intramolecular hydrogen scrambling was investigated both in the free peptide and in its proteoliposome form. Some scrambling was observed for the free peptide; however, almost no scrambling occurred in the amide hydrogens of the peptide backbone embedded in the membrane. The CID spectra suggest that the N-terminal moiety of the peptide lies on the polar side of the lipid membrane, while the C-terminal region is embedded in the membrane. The protocol described here may be reliably applied to investigate the interaction of mastoparans with bilayer lipid systems.