Characterization of the RstB2 protein,the DNA-binding protein of CTXϕ phage from Vibrio cholerae

The low abundant protein RstB2, encoded in the RS2 region of CTX?, is essential for prophage formation. However, the only biochemical activity so far described is the single/double-stranded DNA-binding capacity of that protein. In this paper, a recombinant RstB2 (rRstB2) protein was overexpressed in E. coli with a yield of 58.4 mg l^?1 in shaken cultures, LB broth. The protein, purified to homogeneity, showed an identity with rRstB2 by peptide mass fingerprinting. The apparent molecular weight of the RstB2 native protein suggests that occurs mostly as a monomer in solution. The monomers were able of reacting immediately upon exposure to DNA molecules. After a year of storage at ?20 °C, the protein remains biologically active. Bioinformatics analysis of the amino acid sequence of RstB2 predicts the C-end of this protein to be disordered and highly flexible, like in many other single-stranded DNA-binding proteins. When compared with the gVp of M13, conserved amino acids are found at structurally or functionally important relative positions. These results pave the way for additional studies of structure and molecular function of RstB2 for the biology of CTX?.     

Expression and functional identification of the hypothetical adhesin P32 from Mycoplasma genitalium

Mycoplasma genitalium is the main causative agent for non-gonococcal and non-chlamydial urethritis. P32 is the putative surface-exposed membrane protein of M. genitalium and it has substaintial identity in amino acid sequence with adhesin protein P30 from M. pnewnoniae. Since M. pneumoniae mutants lacking P30 protein is defective in cytadherence, P32 protein has been proposed to be an essential adhesin implicated in the adherence of M. genitalium to host cells. The prokaryotic expression vector pET-30 ( )/p32 was constructed in the present study, and the recombinant protein was expressed in E. coli and purified under denaturing condition. As demonstrated by the immunoblotting analysis, the recombinant protein could react with rabbit antisera against M. genitalium, and adherence inhibition assays were petformed with antisera against this recombinant protein. It was demonstrated that P32 protein apperared to be an adhesion protein of M. genitalium, thus providing the experimental basis for better understanding of the pathogenesis of M. genitalium infection and for the development of the related vaccines against the infection.     

Expression of cytokines and chemokines and microvasculature alterations of the tongue from patients with chronic Chagas’ disease

Chagas’ disease is caused by the protozoan Trypanosoma cruzi and continues to be a significant public health problem, since 10 million people are still infected in Latin America. The purpose of this study was to analyze the microvasculature alterations as well the expression of cytokines and chemokines in the tongues from patients with chronic Chagas’ disease (CC; n = 18), comparatively with a non-chagasic group (NC; n = 22). We observed several vascular alterations in the tongue of CC such as a greater vascular diameter, increased vascular wall area, high density of the blood vessels, and increased thickening of the capillary basement membrane. The expression of cytokines interferon gamma and tumor necrosis factor alpha and chemokine macrophage inflammatory protein 1α were significantly down-regulated in the tongue of CC group. These results demonstrated that, in the tongue of chagasic patients, a microvascular abnormality and immunological impairment occurs, probably due to chronic inflammation evoked by T. cruzi antigens.     

Expression and localization of dystrophins and β-dystroglycan in the hypothalamic supraoptic nuclei of rat from birth to adulthood

Dystrophins (Dps) are the sub-membranous proteins that work via the dystrophin-associated proteins complex, which comprises β-dystroglycan (β-DG), a cell surface receptor for extracellular matrix. Recently, we have revealed β-DG decrease and central function impairment of supraoptic nucleus (SON) in Dp71 deficient adult mice, opening the question on the profiles of Dps and β-DG during SON development. At birth and the age of 10, 20 and 60 days, we examined the expression by RT-PCR and Western-blotting, and the distribution by immunohistochemistry of Dps and β-DG. Also, we analyzed, by immunohistochemistry and Western-blotting, the neuropeptide, arginine vasopressin (AVP), in the SON at the different ages.At birth, Dp71 and to a lesser extends, Dp140 and Dp427, and also β-DG are revealed in the SON. They are localized in the magnocellular neurons (MCNs), astrocytes and vessels.From birth to adulthood, the AVP raise in the SON coincides with the progressive increase of Dp71 level while the level of Dp140 and Dp427 increased only at D20, D10 post-natal development, respectively, and β-DG expression did not change. Moreover, the location of Dps or/and β-DG in the cell compartments was modified during development: at D10, Dps appeared in the astrocytes end-feet surrounding MCNs, and at D20, Dps and β-DG codistributed in the astrocytes end-feet, surrounding MCNs and vessels. Such a distribution marks the first steps of post-natal SON development and may be considered essential in the establishment of structural plasticity mechanisms in SON, where astrocyte end-feet, vessels, magnocellular neurons, are physiologically associated. The disappearance of β-DG in the MCNs nucleus marks the adulthood SON and suggests that the complex of Dps associating β-DG is required for the nucleoskeleton function in the post-natal development.     

Effect of Thalidomide on the Expression of TNF-α m-RNA and Synthesis of TNF-α in Cells from Leprosy Patients with Reversal Reaction

Hypersensitivity reactions called reversal reaction (RR) and erythema nodosum leprosum (ENL) occur in leprosy. They are characterized by an increase in tumor necrosis factor-alpha (TNF-α). Thalidomide is an effective treatment for ENL but not RR. Its effectiveness in ENL is attributed to inhibition of TNF-α, and this does not explain its failure to treat RR. We assessed thalidomide's effect on TNF-α in RR. Mononuclear cells from RR and non-RR patients and healthy individuals were treated with thalidomide and M.leprae (AFB), a cytosol fraction of M. leprae or Dharmendra lepromin. Thalidomide suppressed TNF-α, but when some RR patients' cells were stimulated with AFB, it enhanced TNF-α.     

Expression of recombinant β-tubulin alleles from Cylicocyclus nassatus (Cyathostominae)

Small strongyles (Cyathostominae) are common nematode parasites of horses that have developed resistance to the benzimidazole anthelmintics used to control their populations. Evidence suggests that the principal mechanism of resistance involves a phenylalanine-to-tyrosine mutation at codon 200 in the β-tubulin proteins that are components of microtubules. Other works, however, suggest that a phenylalanine-to-tyrosine mutation at codon 167, or alternative mechanisms, may be involved. As part of an ongoing project examining the role that these two β-tubulin mutations may play in benzimidazole resistance, we have cloned the wild-type allele and the two alleles with the phenylalanine-to-tyrosine mutations at codons 167 and 200 of the β-tubulin isotype 1 gene from the small strongyle Cylicocyclus nassatus. In this work, we describe the construction of expression vectors containing these alleles and their expression in Escherichia coli.     

Validity and reliability of the Turkish version of the DSM-5 Dissociative Symptoms Severity Scale–Child Form

The goal of this study was to assess the validity and reliability of the Turkish version of the DSM–5 (Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition) Dissociative Symptoms Severity Scale–Child Form. The scale was prepared by translating and then back-translating the DSM–5 Dissociative Symptoms Severity Scale. The study groups included one group of 30 patients diagnosed with posttraumatic stress disorder who were treated in a child and adolescent psychiatry unit and another group of 83 healthy volunteers from middle and high schools in the community. For assessment, the Adolescent Dissociative Experiences Scale (ADES) was used in addition to the DSM–5 Dissociative Symptoms Severity Scale. Regarding the reliability of the DSM–5 Dissociative Symptoms Severity Scale, Cronbach’s alpha was .824 and item–total score correlation coefficients were between .464 and .648. The test–retest correlation coefficient was calculated to be r = .784. In terms of construct validity, one factor accounted for 45.2% of the variance. Furthermore, in terms of concurrent validity, the scale showed a high correlation with the ADES. In conclusion, the Turkish version of the DSM–5 Dissociative Symptoms Severity Scale–Child Form is a valid and reliable tool for both clinical practice and research.     

Effect of Interleukin-1β on the Expression of Tight Junction Proteins in the Culture of HaCaT Keratinocytes

We studied the effect of IL-1β on the expression of tight junction proteins (occludin and claudins) in cultured HaCaT keratinocytes and changes of transepithelial resistance. Addition of IL-1β had little effect on transepithelial resistance, increased the expression of claudin-1, and did not modify the expression of occludin. In other tissues, IL-1β also increases claudin-1 expression, but significantly decreases occludin expression. These changes are accompanied by the reduction of transepithelial resistance. The IL-1β-induced increase in the expression of claudin-1 in cultured HaCaT keratinocytes simulates the appearance of claudin-1 at the early stage of skin wound healing. It is accompanied by an increase in IL-1β concentration in the wound fluid.     

Expression of TGF-β-like molecules in the life cycle of Schistosoma japonicum

The transforming growth factor (TGF-) family controls an extremely wide range of biological activities, such as the growth and differentiation of cells, and immunological events against infectious agents. Although TGF- homologs appear to be widely present in metazoan animals, studies of parasite-derived molecules are relatively few. Using antibodies against anti-mouse TGF-₁, -₂, and -₃, we show the expression of TGF--like molecules in Schistosoma japonicum cercariae, schistosomula, eggs and adult worms. Intense immunoreactivity was found on the surface of free-living cercarial bodies. In transverse sections of cercariae, the molecules were localized in the tegument and subtegumental cells, and the number and distribution of producing cells significantly differed with each antibody. In the skin-migrating stage, the expression in the tegumental surface gradually decreased and became almost negative within 48 h of exposure. In adult worms and eggs, the reactivity was found in subtegumental cells and in cells of a tubular structure, respectively. In western blot analysis, the detection of conventional TGF- molecules failed. The expression of TGF--like molecules was distinctly regulated at each developmental stage.     

Cloning, Recombinant Expression and Activity Studies of a Major Allergen “Enolase” from the Fungus Curvularia Lunata

Recombinant allergens are required to study allergy at the molecular level and are helpful tools for the improvement of diagnosis and therapy. In the present study, enolase was expressed from Curvularia lunata and analyzed for its immunological reactivity as an allergen. cDNA library was synthesized in λ zap vector and screened with sera obtained from C. lunata allergic patients. A cDNA clone with an ORF of 1.3 kb showed homology to enolases from different fungal sources. It was expressed in E. coli, purified from inclusion bodies yielding 0.5 mg/L and showed enzyme activity of 48 units/mg. It resolved as 48-kDa band on SDS-PAGE and was recognized by all the individual Curvularia positive patient sera in immunoblot and ELISA. r Cur l 2 stimulated patients’ PBMCs and supernatant of these cells showed elevated levels of Th 2 cytokines. Ten B cell epitopes were predicted using computational software and one showed 90% homology to an important IgE epitope of Cla h 6. The various parameters predicted by computational approach can be validated later as a future study to draw conclusive evidence about putative antigenic epitopes. This can further help in generating knowledge about residues important for IgE binding and developing therapeutic modalities.     

Intracellular Trafficking of AIP56, an NF-κB-Cleaving Toxin from Photobacterium damselae subsp. piscicida

AIP56 (apoptosis-inducing protein of 56 kDa) is a metalloprotease AB toxin secreted by Photobacterium damselae subsp. piscicida that acts by cleaving NF-κB. During infection, AIP56 spreads systemically and depletes phagocytes by postapoptotic secondary necrosis, impairing the host phagocytic defense and contributing to the genesis of infection-associated necrotic lesions. Here we show that mouse bone marrow-derived macrophages (mBMDM) intoxicated by AIP56 undergo NF-κB p65 depletion and apoptosis. Similarly to what was reported for sea bass phagocytes, intoxication of mBMDM involves interaction of AIP56 C-terminal region with cell surface components, suggesting the existence of a conserved receptor. Biochemical approaches and confocal microscopy revealed that AIP56 undergoes clathrin-dependent endocytosis, reaches early endosomes, and follows the recycling pathway. Translocation of AIP56 into the cytosol requires endosome acidification, and an acidic pulse triggers translocation of cell surface-bound AIP56 into the cytosol. Accordingly, at acidic pH, AIP56 becomes more hydrophobic, interacting with artificial lipid bilayer membranes. Altogether, these data indicate that AIP56 is a short-trip toxin that reaches the cytosol using an acidic-pH-dependent mechanism, probably from early endosomes. Usually, for short-trip AB toxins, a minor pool reaches the cytosol by translocating from endosomes, whereas the rest is routed to lysosomes for degradation. Here we demonstrate that part of endocytosed AIP56 is recycled back and released extracellularly through a mechanism requiring phosphoinositide 3-kinase (PI3K) activity but independent of endosome acidification. So far, we have been unable to detect biological activity of recycled AIP56, thereby bringing into question its biological relevance as well as the importance of the recycling pathway.     

Expression and Purification of the Bacillus anthracis Protective Antigen Receptor-binding Domain

Anthrax is primarily a disease of herbivores, but all mam mals including humans are susceptible. The infection mayprogress to fatal outcome. Bacillus anthracis is the aetio logical agent of clinical anthrax, and is a Gram positivenon motile, aerobic fa…