Isolation, identification and characterization of secretory proteins of IVMFC embryos and blood circulation of estrus and early pregnant goat

693-701The aim of the present study was to isolate, identify and characterize the secretory proteins of IVM oocytes and IVMFC embryos to evaluate its immunogenecity and identify of such proteins if any, in blood circulation of estrus and early pregnant goats. Oocytes were matured in TCM-199 with 1μg/ml, estradiol -17β; 0.5 μg/ml, FSH: 100 IU/ml , LH and 10% FCS on granulosa cell monolayer.  After 18 hr of maturation, oocytes were further cultured in maturation medium containing 3 mg/ml polyvinyl alcohol (PV A) without serum and BSA for 12 hr and medium was collected. The IVF embryos of 4-8 cell stage were cultured in medium containing PVA without serum and BSA. Embryo culture medium was collected after 24 hr of culture and was pooled. The proteins were analyzed on SDS-PAGE (12.5%). Four secretory proteins of oocytes with approximately molecular weight of 45, 55, 65 and 95 kDa and three secretory proteins of embryos 45, 55 and 65 kDa were obtained on SDS-PAGE in silver staining. The protein profile of midluteal, estrus and early pregnant goat serum was similar and no variation was observed among the proteins on SDS-PAGE. Two secretory proteins of 55 and 65 kDa of both IVM oocytes and IVMFC embryos were observed on Western analysis. None of such proteins was observed in midluteal, estrus and early pregnant goat serum on western blotting. It can be concluded that IVM oocytes and IVMFC embryos secrete proteins in medium and two of them can develop antibody.The proteins secreted from embryos till morula stage was similar to that of oocytes. None of these oocyte/embryo released proteins were observed in blood circulation of estrus and early pregnant goats

In vitro culture of functionally active buffalo hepatocytes isolated by using a simplified manual perfusion method.

In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes.Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3 ± 0.66)×107 cells per gram of liver tissue with a viability of 82.3 ± 3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies.We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes for studying gene expression, regulation, hepatic genomics and proteomics in farm animals

Structural and functional insights into the catalytic inactivity of the major fraction of buffalo milk xanthine oxidoreductase.

BACKGROUND: Xanthine oxidoreductase (XOR) existing in two interconvertible forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), catabolises xanthine to uric acid that is further broken down to antioxidative agent allantoin. XOR also produces free radicals serving as second messenger and microbicidal agent. Large variation in the XO activity has been observed among various species. Both hypo and hyper activity of XOR leads to pathophysiological conditions. Given the important nutritional role of buffalo milk in human health especially in south Asia, it is crucial to understand the functional properties of buffalo XOR and the underlying structural basis of variations in comparison to other species. METHODS AND FINDINGS: Buffalo XO activity of 0.75 U/mg was almost half of cattle XO activity. Enzymatic efficiency (k cat/K m) of 0.11 sec(-1) µM(-1) of buffalo XO was 8-10 times smaller than that of cattle XO. Buffalo XOR also showed lower antibacterial activity than cattle XOR. A CD value (Δε430 nm) of 46,000 M(-1) cm(-1) suggested occupancy of 77.4% at Fe/S I centre. Buffalo XOR contained 0.31 molybdenum atom/subunit of which 48% existed in active sulfo form. The active form of XO in buffalo was only 16% in comparison to ∼30% in cattle. Sequencing revealed 97.4% similarity between buffalo and cattle XOR. FAD domain was least conserved, while metal binding domains (Fe/S and Molybdenum) were highly conserved. Homology modelling of buffalo XOR showed several variations occurring in clusters, especially close to FAD binding pocket which could affect NAD(+) entry in the FAD centre. The difference in XO activity seems to be originating from cofactor deficiency, especially molybdenum. CONCLUSION: A major fraction of buffalo milk XOR exists in a catalytically inactive form due to high content of demolybdo and desulfo forms. Lower Fe/S content and structural factors might be contributing to lower enzymatic efficiency of buffalo XOR in a minor way

Not Available

Not AvailableTrinket cattle were introduced to Trinket Island of Nicobar archipelago by Danish people during their early colonization period. These cattle became feral in nature after Great Sumatra earthquake and Indian Ocean Tsunami since 26 December 2004. Negligence has brought the cattle on the brink of extinction. In the present study, we document the complete mitochondrial genome sequence of Trinket Cattle. The mitogenome contains 37 genes including 13 protein-coding genes, 22 tRNAs, and two ribosomal RNA genes. In addition, a control region (D-loop) was also present. Phylogenetic analysis showed that Trinket Cattle belongs to Bos indicus group. The results of the study will be helpful for formulating of conservation strategy of the highly endangered breed.Not Availabl

Recombinant purified buffalo leukemia inhibitory factor plays an inhibitory role in cell growth

<div><p>Leukemia Inhibitory Factor (LIF) is a polyfunctional cytokine, involved in numerous regulatory effects <i>in vivo</i> and <i>in vitro</i>, varying from cell proliferation to differentiation, and has therapeutic potential for treating various diseases. In the current study, a COS-1 cell line overexpressing recombinant Buffalo LIF (rBuLIF) was established. The rBuLIF was purified to homogeneity from the total cell lysate of COS-1 cells using a two-step affinity chromatography. The purified LIF was confirmed by western blot and mass spectrometer (MS/MS). Particularly, high-resolution MS has identified the rBuLIF with 73% of sequence coverage with highest confidence parameters and with the search engine score of 4580. We determined the molecular weight of rBuLIF protein to be 58.99 kDa and 48.9 kDa with and without glycosylation, respectively. Moreover, the purified rBuLIF was verified to be functionally active by measuring the growth inhibition of M1 myeloid leukemia cells, revealing a maximum inhibition at 72 hours and half-maximal effective concentration (EC50) of 0.0555 ng/ml, corresponding to a specific activity of >1.6×10⁷ units/mg. Next, we evaluated the effect of rBuLIF on buffalo mammary epithelial cell lines for its role in involution and also identified the IC50 value for BuMEC migrating cells to be 77.8 ng/ml. Additionally, the treatment of MECs (BuMEC and EpH4) displayed significant (<i>P</i> < 0.05) reduction in growth progression, as confirmed by qRT-PCR analysis, suggesting its strong involvement in the involution of the mammary gland <i>in vivo</i>. Thus, we conclude that the glycosylated rBuLIF, purified from COS-1 cells was found to be functionally active as its natural counterpart.</p></div

Immunostaining of 5 days old cultured buffalo hepatocytes with anti-cytokeratin-18 and anti-albumin.

<p>Immunostaining with (A) CY3 labelled anti-cytokeratin-18 antibodies (fluorescence signal in red); (B) FITC labelled anti-albumin antibodies (green); (C) staining of hepatocytes nuclei with DAPI (blue). Panel D shows the merged images from panels A, B and C. Panel E shows light microscopic image of hepatocytes; panel F shows negative control (Isotype control), and panels G shows staining with DAPI, while panel H shows images merged from panels F and G.</p

Agarose gel electrophoresis of RT-PCR products of hepatocyte-specific marker genes expressed in 5 days old cultured hepatocytes.

<p>Panel A shows 293 bp amplicon of albumin; B—130 bp amplicon of hepatocyte nuclear factor 4α; C—240 bp amplicon of glucose-6-phosphatase; D—136 bp amplicon of CYP1A1; E—164 bp amplicon of CYP3A4; F—142 bp amplicon of tyrosine aminotransferase. Lane 1: 100 bp ladder; Lane 2: RT-PCR of liver tissue (positive control) by using the gene-specific primers; Lane 3: RT-PCR of respective genes from cultured buffalo hepatocytes; Lane 4: RT-PCR from skin fibroblasts (negative control). Amplification of Glyceraldehyde 3–phosphate dehydrogenase (GAPDH) was used as housekeeping gene.</p

Immunostaining of 5 days old cultured buffalo hepatocytes with α1-antitrypsin antibody.

<p>Panel A shows light microscopic image of hepatocytes, and panel B shows immunostained hepatocytes with α1-antitrypsin antibody labelled with FITC (green) and nuclear stain DAPI (blue) dyes.</p

Hepatocyte growth on fibroblast feeder layer.

<p>Hepatocyte proliferation curve represented by the absorbance at 540 nm of BrdU-labelled hepatocytes at different days of culture.</p

Western blot analysis of 5 days cultured buffalo hepatocytes.

<p>Blots of buffalo hepatocytes lysate by using antibodies against albumin (Panel A); lane 1: HepG2 (Positive control), Lane 2: buffalo hepatocytes (test); Lane 3: skin fibroblast (negative control); Lane 4: pre-stained protein marker; cytokeratin-18 (Panel B); Lane 1: pre-stained protein marker; Lane 2: HepG2 cells; Lane 3: buffalo hepatocytes; Lane 4: skin fibroblast; and α1-antitrypsin (Panel C), order of lanes is similar to that shown in panel B.</p