Serum amyloid A (SAA) mRNA expression in chicken and quails in response to bacterial stress.

Monitoring of acute phase proteins such as serum amyloid A at gene expression level may provide quick information about immune status of the host and its susceptibility towards common infections. Present study was carried out to evaluate and compare the mRNA expression of SAA gene in Rhode Island Red chicken (RIR) and Japanese quails using real time PCR analysis in response to inactivated Salmonella gallinarum culture. The results showed that expression of SAA gene was approximately 17-33 folds higher in case of birds administered with bacterial culture when compared to un-inoculated controls and expression was higher and quicker in case of quails than RIR chicken. The SAA genes from chicken and quail were cloned and upon sequence analysis it was observed that deduced amino acid sequence of SAA from chicken and quails were having approximately seven percent variation which might have significance in function of this protein in these species.

Evaluation of immuno-modulating effect of recombinant heat shock protein 40 of Brucella abortus in mice.

The present study was aimed to evaluate the immuno-modulatory effect of Brucella-specific recombinant HSP40 (rDnaJ) when co-immunized with Brucella rOmp22 in mice. For this, dnaJ of Brucella abortus was cloned, expressed in E. coli, and purified to homogeneity using Ni-NTA agarose columns. Three groups of mice (n = 6 in each group) were used in the study. The control group was immunized with rOmp22 alone, while group 1 mice were injected subcutaneously with rOmp22 along with conventional adjuvants (FCA, FIA), and group 2 mice with rOmp22 mixed with rDnaJ. IgG isotype (IgG1 and IgG2a) response to rOmp22 immunization was evaluated by enzyme-linked immunosorbent assay which was found to be directed towards the cell-mediated arm of immune system (CMI) in group 2 mice in which rOmp22 was co-immunized with rDnaJ. Expression profiling of IL-4 and IL-12 was checked in all the groups by qRT PCR. IL12 mRNA was up-regulated to a greater extent in group2 mice, suggesting that the CMI arm of immune system was stimulated. Hence, it was concluded that CMI response against rOmp22 is stimulated to a greater extent in mice when co-immunized with Brucella rDnaJ.

Analysis of lysyl oxidase as a marker for diagnosis of canine mammary tumors.

Lysyl oxidase (LOX) is an extracellular metalloenzyme which mediates crosslinking of collagen and elastin. It has been reported to play a pivotal role in cancer metastasis especially in women suffering from breast cancer. The present study is the first to evaluate the gene expression levels of LOX by Real time-polymerase chain reaction (Real time-PCR) in dogs with mammary tumor besides molecular cloning and expression of canine lysyl oxidase gene (lox). Real time-PCR studies showed a significant upregulation (threefold higher) of lox in mammary tumor cases as compared to healthy dogs indicating its possible diagnostic and prognostic role in canine mammary tumors (CMTs). Cloning and sequencing of lox gene revealed 1230 bp CDS which is mostly conserved in C-terminal region. Sequence analysis of canine lox showed that it shares 99% homology with the predicted sequence available on NCBI and had greatest identity with the lox gene from cat. Protein structure predicted with homology modelling was validated by Ramachandran plot analysis which revealed most (approximately 95%) of the amino acids in favoured region. Additionally, recombinant lysyl oxidase expressed as His-tagged fusion protein in prokaryotic expression vector (pPROExHTa) was used in an ELISA for detection of circulating protein LOX in serum of CMT subjects. Receiver operating characteristics analysis of the ELISA revealed high sensitivity (90%) and specificity (85%) with histopathology as reference standard. Taken together, we propose LOX as a diagnostic biomarker and a putative prognostic candidate in CMT cases.

Over-expression of mammaglobin-B in canine mammary tumors.

BACKGROUND: Mammaglobin, a member of secretoglobin family has been recognized as a breast cancer associated protein. Though the exact function of the protein is not fully known, its expression has been reported to be upregulated in human breast cancer.We focused on studying the expression of mammaglobin-B gene and protein in canine mammary tumor (CMT) tissue. Expression of mammaglobin-B mRNA and protein were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC), respectively. RESULTS: High levels of mammaglobin-B mRNA expression (6.663 ± 0.841times) was observed in CMT as compared to age and breed matched healthy controls. Further, expression of mammaglobin-B protein was detected in paraffin-embedded mammary tumor tissues from the same subjects by IHC. Mammaglobin-B protein was overexpressed only in 6.67% of healthy mammary glands while, a high level of its expression was scored in 76.7% of the CMT subjects. Moreover, no significant differences in terms of IHC score and qRT-PCR score with respect to CMT histotypes or tumor grades were observed, indicating that mammaglobin-B over-expression occurred irrespective of CMT types or grades. CONCLUSION: Overall, significantly increased expression of mammaglobin-B protein was found in CMTs with respect to healthy mammary glands, which positively correlates to its transcript. These findings suggest that overexpression of mammaglobin-B is associated with tumors of canine mammary glands.

The overexpression of Hsp90B1 is associated with tumorigenesis of canine mammary glands.

Heat shock proteins (Hsp) are molecular chaperones that are responsible for protein folding and maintenance of cellular homeostasis. Hsp90, an important member of HSP family, has an important role in breast cancer. Glucose-regulated protein 94 (Grp94) is the endoplasmic reticulum paralog of Hsp90 encoded by Hsp90B1 gene. To test if this protein is overexpressed in dogs with mammary tumor, we estimated and compared its serum levels in healthy dogs and that of dogs with mammary tumors. Hsp90B1 mRNA expression was measured in tumorous and healthy mammary tissues (from age- and breed-matched dogs) by real-time PCR. The gene was found to be overexpressed in mammary tumors (3.586 ± 0.067 times). Further, it was heterologously expressed in a prokaryotic system as 90 kDa protein. A recombinant Grp94-based sandwich ELISA was developed to quantify serum Grp94 in dogs with mammary tumors. Based on receiver-operating characteristics' analysis, the assay was found to be 90.62% sensitive and 93.75% specific for a cutoff value of 0.35 with respect to histopathological staining in diagnosing the disease. The t test showed that serum Grp94 levels were significantly elevated (92.97 ± 3.62 ng/ml) in dogs with mammary tumors compared with healthy controls (10.30 ± 0.79 ng/ml) (p < 0.0001). These findings suggest that Grp94 might act as a potential biomarker for prognosis of canine mammary tumors and monitoring its therapy.

Cellular thermotolerance is independent of HSF 1 expression in zebu and crossbred non-lactating cattle.

Heat stress is an important domain of research in livestock due to its negative impact on production and disease resistance. The augmentation of stress in the body stimulates the antioxidative activity comprising various enzymes (viz., catalase, superoxide dismutase), metabolites (reduced glutathione, etc.), vitamins, minerals, etc. to combat the situation. The major key players involved in regulation of heat shock response in eukaryotes are the transcription factors, called as heat shock factors (HSF). They activate the heat shock protein (HSP) genes by binding to their promoters. Lymphocytes are considered to be the best model to evaluate the immunity in any living body as it contains plethora of white blood cells (WBCs).In this study, the peripheral blood mononuclear cells (PBMC) obtained from non-lactating Sahiwal vis-à-vis crossbred (Holstein Friesian × Sahiwal) cattle with 75% or more exotic inheritance were subjected to heat shock at 39, 41, and 43 °C in three different incubators, in vitro. The cell count and viability test of pre and post heat stress of concerned PBMCs indicated that the crossbreeds are more prone to heat stress as compared to Sahiwal. The reverse transcription PCR (qRT-PCR) expression data revealed an increment in HSF1 expression at 41 °C which subsequently declined (non-significantly) at 43 °C in both breeds post 1 h heat shock. However, the association between the HSF 1 expression and antioxidative activity through correlation analysis was found to be non-significant (P < 0.05), though enzymatic activity appeared to behave in a similar fashion in both breeds at 5% level of significance (P < 0.05). This rule out the role of HSF1 expression level on the activity of enzymes involved in oxidative stress in vitro in zebu and crossbred cattle.

Anticancer potential of dietary vitamin D and ascorbic acid: A review.

Cancers have been the leading cause of death worldwide and poor diet and physical inactivity are major risk factors in cancer-related deaths. Micronutrients such as vitamins and minerals appear to have preventive properties against cancer. One important mechanism by which dietary changes can exert preventive effects on cancer is via the modulation of micronutrient concentrations in target tissues. Many of these micronutrients are available in the form of dietary supplements, and the intake of these supplements is prevalent in various parts of the world. However, in most cases, it is not known which micronutrient (or combination of micronutrients) is best when it comes to lowering the risk of cancer. The present review illustrates the effect of vitamin D and ascorbic acid intake on preventing cancer.

Expression and characterization of tissue inhibitor of metalloproteinase 4 from complex canine mammary carcinomas.

All four members of the tissue inhibitors of metalloproteinase (TIMP) family have been reported to be over-expressed in breast cancer cells in vitro. Dysregulation of TIMP-4 expression predicts poor prognosis in cancers. The present study evaluated the association of the expression levels of TIMP-4 with mammary tumor development in dogs, measured by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mammary tissue samples were collected from healthy canine mammary gland and from tumor subjects. TIMP-4 expression was found to be upregulated (5.856 times) in complex canine mammary carcinomas. Also, TIMP-4 mature peptide was expressed heterologously in E. coli. The recombinant protein was purified by Ni- NTA affinity chromatography and further confirmed by western blotting. The rTIMP-4 was found to be functionally active and could inhibit matrix metalloproteinase 11(MMP-11) activity. Immunization of mice with rTIMP-4 resulted in increased antigen specific serum antibody titer, and this serum could be suitably used to detect and quantify the protein in sera of dogs with mammary tumors. TIMP-4 could act as a marker of canine mammary tumors. To the best of our knowledge, this is the first report of heterologous expression of TIMP-4 from complex canine mammary carcinomas.

Characterization and Immunogenicity of Outer Membrane Vesicles from Brucella abortus.

Bovine brucellosis is a worldwide spread zoonotic disease. The objectives of this study were characterization of outer membrane vesicles from B. abortus and to evaluate their immunogenicity in mice. For this purpose, OMVs were derived from B. abortus strain 99 using ultracentrifugation method. Isolated OMVs were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transmission electron microscopy which revealed spherical 20-300 nm structures rich in proteins. OMVs also showed immuno-reactivity with mice antisera in Western blot. Further, indirect ELISA showed specific and high-titer immune responses against the antigens present in OMVs suggesting their potential for a safe acellular vaccine candidate.

Heterologous expression and functional characterization of matrix metalloproteinase-11 from canine mammary tumor.

Matrix metalloproteinases (MMPs) are reported to be involved in tumor growth, apoptosis, angiogenesis, invasion, and development of metastases. These are zinc containing metalloproteases, known for their role in extracellular matrix degradation. MMP-11 (stromelysin3) is reported to be highly expressed in breast cancer, therefore it may act as marker enzyme for breast cancer progression. The present work was carried out to produce recombinant canine (Canis lupus familiaris) MMP-11 lacking the signal and propeptide in E. coli by optimizing its expression and purification in biologically active form and to functionally characterize it. A bacterial protein expression vector pPROEX HTc was used. The MMP-11 mature peptide encoding gene was successfully cloned and expressed in E. coli and the purified recombinant enzyme was found to be functionally active. The recombinant enzyme exhibited caseinolytic activity and could be activated by Trypsin and 4-Amino phenyl mercuric acetate (APMA). However Ethylene diamine tertra acetate (EDTA) inhibited the enzyme's caseinolytic activity. The recombinant enzyme degraded extracellular matrix constituents and facilitated migration of MDCK (Madin-Darby canine kidney) cells through BD Biocoat Matrigel invasion chambers. These results suggest that in vivo MMP-11 could play a significant role in the turnover of extracellular matrix constituents.