Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris.

Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures. This antitoxic protein can serve as an excellent starting template for the design of novel therapeutics against snakebite envenomation, particularly venom-induced local tissue damage. Therefore, the aim of this work was to produce a recombinant DM64 (rDM64) in the methylotrophic yeast Pichia pastoris and to compare its biological properties with those of native DM64. Yeast fermentation in the presence of Pefabloc, a serine protease inhibitor, stimulated cell growth (~1.5-fold), increased the rDM64 production yield approximately 10-fold and significantly reduced the susceptibility of rDM64 to proteolytic degradation. P. pastoris fermentation products were identified by mass spectrometry and Western blotting. The heterologous protein was efficiently purified from the culture medium by affinity chromatography (with immobilized PLA2 myotoxin) and/or an ion exchange column. Although both native and recombinant DM64 exhibit different glycosylation patterns, they show very similar electrophoretic mobilities after PNGase F treatment. rDM64 formed a noncovalent complex with myotoxin II (Lys49-PLA2) from Bothrops asper and displayed biological activity that was similar to that of native DM64, inhibiting the cytotoxicity of myotoxin II by 92% at a 1:1 molar ratio.

Identification of BPI protein produced in different expression system and its association with Escherichia coli F18 susceptibility.

The super antibiotic bactericidal/permeability-increasing (BPI) protein is a member of a new generation of proteins that have been implicated as endotoxin-neutralizing agents. In this study, recombinant porcine BPI protein was obtained by generating porcine BPI encoding prokaryotic, eukaryotic, and yeast expression vectors. Recombinant protein expression was detected in yeast GS115, Escherichia coli, and 293-6E cells by gel electrophoresis and Western blotting. Escherichia coli F18 is the primary Gram-negative bacteria in the gut and the main pathogen leading to diarrhea and edema dis-ease in weaning piglets. Therefore, E. coli F18-resistant and -sensitive Sutai piglets were used to test differential expression of BPI protein by Western blotting and to investigate the potential correlation between BPI protein expression and E. coli F18-susceptibility. Recombinant porcine BPI protein expression was not detected in the prokaryotic and yeast expression systems; however, soluble protein was detected in the eukaryotic expression system. These data indicate the strong bacterio-static action of the BPI protein and confirm the feasibility of obtaining large amounts of recombinant porcine BPI recombinant protein using this eukaryotic expression system. In addition, the BPI protein expres-sion levels in the E. coli F18-resistant group were significantly higher than those in the sensitive group, indicating that high BPI protein ex-pression is associated with resistance to E. coli F18. Our findings pro-vide a basis for further investigations into the development of a drug designed to confer resistance to E. coli F18 in weaning piglets.

Inflammatory, immune and lipid transportation proteins are differentially expressed in spontaneous and proximal deep vein thrombosis patients.

INTRODUCTION: Deep vein thrombosis (DVT) is a multi-causal disease associated with high morbidity and mortality due to complications, and 25% of patients present recurrence within 5 years. The identification of factors involved with DVT can help in the management of patients, prevention of recurrence and in the development of new therapies. The evaluation of plasma components using proteomics potentially provides a window into the individual's state of health. We analyzed the protein profile of plasma samples from 3 DVT patients and compared results to those obtained from 1 sibling and 1 neighbor of each patient. These patients were selected as they presented a personal and family history of spontaneous and recurrent episodes of proximal DVT. MATERIAL AND METHODS: Albumin was removed using Affi-Gel Blue Gel, and the proteins were alkylated, reduced, precipitated and hydrolyzed. The peptides were fractionated by SCX chromatography, the 7 fractions obtained were directed to the ESI Q-TOF Premier mass spectrometer. Protein search was performed using the Mascot engine against the IPI human database. RESULTS: Proteins that were statistically overexpressed in DVT patients included C4-A plasma protease, C1 inter-alpha-trypsin inhibitor, heavy chain H inhibitor and serum amyloid A. Proteins that were statistically reduced in DVT patients included alpha-2-HS-glycoprotein, isoform 2 of inter-alpha-trypsin inhibitor heavy chain H4 and apolipoprotein A-IV. CONCLUSIONS: The evaluation of plasma from patients with spontaneous DVT allows the identification of differently expressed proteins when compared to controls; this expression may be of pathological importance for immune and inflammatory processes in DVT.

Reduction of AHSP synthesis in hemin-induced K562 cells and EPO-induced CD34(+) cells leads to alpha-globin precipitation, impairment of normal hemoglobin production, and increased cell death.

OBJECTIVE: alpha-Hemoglobin stabilizing protein (AHSP) binds alpha-hemoglobin (Hb), avoiding its precipitation and its pro-oxidant activity. In the presence of betaHb, the alphaHb-AHSP complex is dismembered and betaHb displaces AHSP to generate the quaternary structure of Hb. The relationship between Hb formation and alterations in AHSP expression, which may affect human erythropoiesis, has not yet been described in human cells. Hence, in this study, we examined the effects of AHSP knockdown in hemin-induced K562 and erythropoietin-induced CD34(+) cells with particular reference to cellular aspects and gene expression. MATERIALS AND METHODS: Short-hairpin RNA expression vectors aimed at the AHSP mRNA target sequence were cloned and transfected into K562 and CD34(+) cells. K562 and CD34(+) cells were stimulated to erythroid differentiation. Cells were examined in terms of gene expression using quantitative real-time polymerase chain reaction; reactive oxygen species (ROS) production, apoptosis, and Hb production through flow cytometry assays; and immunofluorescence assays for globin chains. RESULTS: RNA interference-mediated knockdown of AHSP expression resulted in considerable alphaHb precipitation, as well as in a significant decrease in HbF formation. AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis. CONCLUSION: These findings strengthen the hypothesis that AHSP stabilizes the alphaHb chain, avoiding its precipitation and its ability to generate ROS, which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.

Molecular cloning, expression analysis and cellular localization of gomesin, an anti-microbial peptide from hemocytes of the spider Acanthoscurria gomesiana.

Gomesin is a cationic anti-microbial peptide of 18 amino acid residues isolated from the hemocytes of unchallenged tarantula spider Acanthoscurria gomesiana. This paper reports the first study of the processing and cellular location of an anti-microbial peptide (AMP) in spiders. Gomesin cDNA sequence analysis indicated that it is processed from a precursor containing a signal peptide (23 amino acid residues) and a negative C-terminal region (43 amino acid residues). The gomesin gene was constitutively transcribed in hemocytes and the gene product localized in hemocyte granules. The constitutive production of gomesin by a spider is discussed in the context of an ancient mechanism of AMP regulation and storage.

Changes in Vitellogenin expression during captivity-induced stress in a tropical anole.

Tropical anoline lizards have been shown to undergo massive reproductive retrogression when held in captivity. The present study focused on the effects generated by captivity-induced stress on the hepatic expression of vitellogenin (Vtg), the precursor of the major egg yolk proteins, in Anolis pulchellus. Several hepatic dysfunctions accompanying the regression of the reproductive organs were detected when mature highly vitellogenic females were kept in captivity for long periods. These included decreased synthesis of Vtg to undetectable levels after 4 days of captivity concomitant with a large reduction in the levels of its cognate mRNA. In addition, a drastic reduction in Vtg plasma levels preceding the conspicuous cessation of follicular growth was observed. Results suggest the activation of a specific mechanism for rapid clearance of vitellogenic and other female-specific proteins from plasma. The effects of captivity, both in the liver and in the reproductive tract, were alleviated or even reversed by treatment with 17 Beta-estradiol. Hepatic protein synthesis increased threefold when animals were maintained under estrogen therapy during the captivity period. Also, the levels of Vtg mRNA and Vtg protein synthesis and plasma levels were similar or even higher to the observed in control vitellogenic females. Animals treated with 17 beta-estradiol after long-term captivity recovered the normal vitellogenic levels after 72 to 96 hr. Therefore, our results im this tropical anole strongly suggest that the stress effects upon reproductive behavior previously reported in anoline lizards results from suppression of the estrogen stimulus for the hepatic vitellogenic response.

Eosinophil cationic protein in nasopharyngeal secretions and serum of infants infected with respiratory syncytial virus.

Eosinophil cationic protein (ECP) was assayed in nasopharyngeal secretion (NPS) and serum from 42 infants, hospitalized with acute lower respiratory infection, in El Salvador and the results analyzed in relation to etiology of the infection. ECP concentrations were high in NPS, at an average 50 times higher than those found in serum. Exceedingly high levels of ECP (> 1000 micrograms/L) were found more frequently in wheezing than in non-wheezing children (30% vs 7%) and, accordingly, were more commonly found in children hospitalized with bronchiolitis than in those with pneumonia. Excessive levels were significantly more common in girls than in boys. Of the 42 cases, 28 were found to be caused by respiratory syncytial virus (RSV) subgroup A, and 3 by RSV-B, by means of detection of RSV antigen in nasopharyngeal cells. ECP serum levels were moderately elevated during the acute phase of the respiratory infection and increased slightly but significantly, in cases with RSV antigen-positive bronchiolitis, but not in those with pneumonia. The ECP levels in NPS from patients in Sweden who, by antigen detection in NPS cells, were diagnosed as either RSV or para-influenza 3 infection or none of these, were similar. These results indicate that elevation of ECP in NPS is associated with acute lower respiratory infection in general, but particularly pronounced in cases of bronchiolitis. Elevation of ECP is not an exclusive consequence of RSV infection, but may occur to an equal extent in infections caused by other agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Elephant seals: genetic variation and near extinction.

Blood samples from northern elephant seals (Mirounga angustirostris), representing five breeding colonies in California and Mexico, were surveyed electrophoretically for protein variation reflecting underlying genetic differences. No polymorphisms were found among 21 proteins encoded by 24 loci. This uniform homozygosity may be a consequence of fixation of alleles brought about by the decimation of this species by sealers in the last century.