Isolation and characterization of outer membrane proteins (OMPs) from Salmonella Gallinarum in chicken and antibiogram of the isolates

Salmonella isolates should be distinguished as it may assist in tracing the source of an outbreak and monitoring trends in antimicrobial resistance associated with a particular type. The specific detection of these Salmonella serotypes is therefore extremely important in order to attribute an isolate to a previously known epidemic outbreak. The present investigation was to isolate and identify S. Gallinarum, to study variation in the profile of outer membrane proteins (OMPs) and to determine in vitro antibiogram of S. Gallinarum in poultry. A total of 228 faecal samples and 22 visceral samples suspected for Salmonellosis were collected, of these 15 samples (6.0%) were found positive for S. Gallinarum. In the present study, rfbS gene sequence was helpful in the serotype-specific detection of S. Gallinarum giving a 187 bp product. Salmonella Gallinarum crude protein extracts determined by SDSPAGE showed migration of OMPs as several bands at approximate moleculer weights of appx. 45 kDa, 55 kDa, 64 kDa, 65 kDa, 74 kDa, 110 kDa, 120 kDa, 135 kDa, 150 kDa,155 kDa, 200 kDa and above 200 kDa. The study indicated a definite variation in the profile of OMPs of various Salmonella Gallinarum strains with major OMPs in the range of appx 80-100 kDa which could be the target for vaccine production. All the isolates tested against 14 antimicrobial agents showed variable susceptibility pattern with highest resistance to nalidixic acid, ampicillin and sulphadiazine and sensitivity to chloramphenicol, gentamicin and enrofloxacin

Stage-dependent changes in localization of a germ cell-specific lamin during mammalian spermatogenesis

We had earlier identified a 110/120-kDa protein specific to nuclear matrix of rat pachytene spermatocytes (Behal, A., Prakash, K., and Rao, M.R.S. (1987) J. Biol. Chem. 262, 10898-10902). This protein is now shown to be a disulfide-linked homodimer of a 60-kDa polypeptide. Indirect immunofluorescence and Western blot analyses using anti-120-kDa polyclonal antibodies have shown that this protein is a component of the pore-complex lamina structure of spermatogonia. As germ cells enter meiotic prophase and the lamina structure disassembles, this polypeptide is redistributed in the nucleus and can be isolated as a component of synaptonemal complexes. Following meiotic division, this 60-kDa protein is relocalized in the lamina, then representing the sole major component of the lamina structure of round spermatids. The identity of the 60-kDa protein in the pore-complex lamina fraction and synaptonemal complexes was further confirmed by two-dimensional analysis of iodinated tryptic peptides. Such an analysis has also shown that the germ cell-specific 60-kDa protein is related but not identical to somatic lamin B

A 39 kDa fragment of endogenous ASK1 suggests specific cleavage not degradation by the proteasome

Transfected human apoptosis signal-regulating kinase 1 (ASK1) produces a 150 kDa protein. However, we have detected endogenous ASK1 predominantly as 39 and 50 kDa C-terminal and 75 and 110 kDa N-terminal fragments in a panel of nontransfected cancer cell lines and HUVEC endothelial cells. This suggests that in nonapoptotic cells, endogenous ASK1 protein is normally cleaved at a number of specific sites, some of which are in the kinase domain. Transfected ASK1 protein is known to be degraded by the proteasome. In contrast, the cleavage of endogenous ASK1 is independent of the proteasome as treatment with the proteasome inhibitor, lactacystin did not inhibit cleavage. Cisplatin treatment decreased the amount of 39 kDa C-terminal ASK1 fragments in mutant p53 cell lines suggesting a decrease in cleavage associated with apoptosis. Transfected ASK1 may, therefore, not accurately reflect the role of endogenous ASK1

A 39 kDa fragment of endogenous ASK1 suggests specific cleavage not degradation by the proteasome

Transfected human apoptosis signal-regulating kinase 1 (ASK1) produces a 150 kDa protein. However, we have detected endogenous ASK1 predominantly as 39 and 50 kDa C-terminal and 75 and 110 kDa N-terminal fragments in a panel of nontransfected cancer cell lines and HUVEC endothelial cells. This suggests that in nonapoptotic cells, endogenous ASK1 protein is normally cleaved at a number of specific sites, some of which are in the kinase domain. Transfected ASK1 protein is known to be degraded by the proteasome. In contrast, the cleavage of endogenous ASK1 is independent of the proteasome as treatment with the proteasome inhibitor, lactacystin did not inhibit cleavage. Cisplatin treatment decreased the amount of 39 kDa C-terminal ASK1 fragments in mutant p53 cell lines suggesting a decrease in cleavage associated with apoptosis. Transfected ASK1 may, therefore, not accurately reflect the role of endogenous ASK1

Identification of a cold shock transcriptional enhancer of the Escherichia coli gene encoding nucleoid protein H-NS

The hns (27 min) gene encoding the 15.4-kDa nucleoid protein H-NS was shown to belong to the cold shock regulon of Escherichia coli, its expression being enhanced 3- to 4-fold during the growth lag that follows a shift from 37 degrees C to 10 degrees C. A 110-base-pair (bp) DNA fragment containing the promoter of hns fused to a promoterless cat gene (hns-cat fusion) conferred a similar cold shock response to the expression of chloramphenicol acetyltransferase (CAT) activity in vivo and in coupled transcription-translation systems prepared with extracts of cold-shocked cells. Extracts of the same cells produce a specific gel shift of the 110-bp DNA fragment and this fragment, immobilized on a solid support, specifically retains a single 7-kDa protein present only in cold-shocked cells that was found to be identical to F10.6 (CS7.4), the product of cspA. This purified protein, which is homologous to human DNA-binding protein YB-1, recognizes some feature of the 110-bp promoter region of hns and acts as a cold shock transcriptional activator of this gene since it stimulates the expression of CAT activity and of cat transcription in in vitro systems programmed with plasmid DNA carrying the hns-cat fusion

Peripheral Lymph Node Addressins are Expressed on Skin Endothelial Cells

The term “peripheral node addressins” describes a set of several endothelial adhesion molecules, which collectively bind to L-selectin and react with monoclonal antibody MECA-79. They regulate lymphocyte recirculation through peripheral nodes. Their expression is thought to be restricted to a specialized vascular segment within the node, called the high endothelial venule. In certain chronic skin diseases, however, postcapillary venules of the skin may also acquire a high endothelial venule-like morphology. Employing immunohistochemistry on cryostat sections, we found these skin endothelial cells – like peripheral node high endothelial venules – to be reactive with monoclonal antibody MECA-79. Tissue lysates from the same specimens were then analyzed by immunoprecipitation using recombinant human L-selectin Fc-chimeras followed by immunoblotting using monoclonal antibody MECA-79. In contrast to peripheral node endothelium, which mainly expressed peripheral node addressin moieties of molecular sizes 90–110 kDa and 160 kDa, endothelial cells in cutaneous T cell lymphoma skin lesions expressed an additional and not yet defined 220 kDa peripheral node addressin-like molecule. Most surprisingly, even in normal skin specimens, we found a distinct subset of endothelial cells located around hair follicles constitutively expressing 90–110 kDa peripheral node addressin-like moieties. It is intriguing to speculate that – in analogy to the role of peripheral node addressins in peripheral nodes – the induced expression of peripheral node addressins in chronic T cell mediated skin diseases is responsible for a sustained lymphocyte recruitment. The constitutive expression of peripheral node addressins on perifollicular endothelium may serve for a continuous lymphocyte recirculation through normal skin

Caracterização antigênica preliminar de preparação de vômito de verme adulto de Fasciola hepatica por soros humanos infectados

Fascioliasis is an emerging/re-emerging vector-borne disease with the widest known distribution. Approximately 17 million people are infected around the world, being the Andean region the most affected area. There is an important necessity to develop sensitive and specific diagnostic tools to treat patients early and to avoid complications. In this paper we evaluated the immune response of infected humans against two antigenic preparations: the total soluble extract (FhTSE) and the adult worm vomit (FhAWV) in order to identify antigenic fractions specific for Fasciola hepatica. Both preparations were processed by SDS-PAGE and Western blot with human sera with fascioliasis (F), other parasitosis and healthy individuals. In the immunoblot of FhTSE, sera F recognised 16 bands with MW between eight and 110 kDa, from which those of 8, 9, 10, 38, 45 and 57 kDa were specific. In the preparation FhAWV, sera F recognised nine bands with MW from eight to 85 kDa, from which those of 8, 12, 15 and 24 kDa were specific. Some bands of cross-reaction were evident with sera from patients with other parasitoses, more frequent with the FhTSE. Bands within the MW mentioned, particularly that of eight kDa, have been shown to be specific by others, and deserve additional characterisation for their potential use in immunodiagnosis.Fasciolíase é uma doença emergente/re-emergente transmitida por vetores com a distribuição sabidamente mais ampla. Existem aproximadamente 17 milhões de pessoas infectadas em todo mundo, sendo a região andina a área mais afetada. Há uma necessidade importante para desenvolver ferramentas diagnósticas sensíveis e específicas para tratar cedo os pacientes e para evitar complicações. Neste trabalho avaliamos a resposta imune de seres humanos infectados comparando a duas preparações antigênicas: o extrato solúvel total (FhTSE) e o vômito (FhAWV) do verme adulto a fim de identificar as frações antigênicas específicas para Fasciola hepatica. Ambas as preparações foram processadas por SDS-PAGE e Western blot com os soros humanos de portadores de fasciolíase (F), outras parasitoses e indivíduos saudáveis. No immunoblot de FhTSE, os soros F reconheceram 16 faixas com PM entre 8 e 110 kDa, das quais as de 8, 9, 10, 38, 45 e 57 kDa foram específicas. Na preparação de FhAWV, os soros F reconheceram 9 faixas com PM entre 8 e 85 kDa, das quais as de 8, 12, 15 e 24 kDa foram específicas. Algumas faixas com reação cruzada foram evidentes com os soros dos pacientes com outras parasitoses, mais freqüentes com o FhTSE. As faixas dentro do PM mencionado, particularmente aquela de 8 kDa, mostraram ser específicas por outros autores, e merecem a caracterização adicional para seu uso potencial no diagnóstico imunológico

The 41-kDa Protein of Human Herpesvirus 6 Specifically Binds to Viral DNA Polymerase and Greatly Increases DNA Synthesis

AbstractWe previously isolated a 41-kDa early antigen of human herpesvirus 6 (HHV-6), which exhibited nuclear localization and DNA-binding activity (Agulnicket al.,1993). In this study, we observed that a 110-kDa protein was coimmunoprecipitated with p41 from HHV-6-infected cells by an anti-p41 antibody. This 110-kDa protein was identified as the HHV-6 DNA polymerase (Pol-6) by an antibody raised against the N terminus of Pol-6. Reciprocal immunoprecipitation and Western blot analyses confirmed that p41 complexes with Pol-6 in HHV-6-infected cells. In addition, both p41 and Pol-6 were expressedin vitroand shown to form a specific complex. Anin vitroDNA synthesis assay using primed M13 single-stranded DNA template demonstrated that p41 not only increased the DNA synthesis activity of Pol-6 but also allowed Pol-6 to synthesize DNA products corresponding to full-length M13 template (7249 nucleotides). By contrast, Pol-6 alone could only synthesize DNA of <100 nucleotides. The functional interaction between Pol-6 and p41 appears to be specific because they could not be physically or functionally substitutedin vitroby their herpes simplex virus 1 homologues. Moreover, as revealed by mutational analysis, both the N and C termini of Pol-6 contribute to its binding to p41. In the case of p41, the N terminus is required for increasing DNA synthesis but not binding to Pol-6, whereas the C terminus is totally dispensable

Karakterisasi Protein Lernaea Cyprinacea Dengan Metode Elektroforesis SDS-PAGE [Characterization of Protein Lernaea Cyprinacea by Using SDS-PAGE Electrophoresis Method]

One of the diseases that often attack are fish is parasitic disease caused by Lernaea cyprinacea, called Lernaeosis. This ectoparasites could be detected on skin, gill, eyes, fin or inside of mouth and nostril of fishes. The aim of the study was to know the character is tic of L.cyprinacea protein based on molecular weight using SDS-PAGE. The results of this study were expected as Lernaeosis vaccine candidate. The experiment used SDS-PAGE with 12% separating gel, 3% stacking gel, voltage 110 volts 28 A and gel staining using coomassie blue. The result showed that, there were eight protein bands with molecular weight 82.3, 73.3, 66.6, 60.5, 54.9, 27.5, 23.1 and 19.8 kDa. Among them, the bands with molecular weight 27.5, 23.1 and 19.8 kDa were thicker and clearer than the others