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Urinary peptides are the products of systemic protein turn over which are excreted out of the body and hence can serve as an important biomarker for various pathophysiology. These peptides in other species of bovine have been reported to possess several bioactive properties. To investigate the potential of urinary peptides we generated a peptidome profile from the urine of Murrah Buffaloes (n=10). Urine samples were processed using

BackgroundLeukemia inhibitory factor (LIF) is a multifunctional cytokine which plays multiple roles in different biological processes such as implantation, bone remodeling, and hematopoiesis. The buESCs are difficult to culture due to lack of proper understanding of the culture conditions. LIF is one of the important factors which maintain the pluripotency in embryonic stem cells and commercial LIF from murine and human origin is used in the establishment of buffalo embryonic stem cells (buESCs). The LIF from a foreign origin is not able to maintain pluripotency and proliferation in buESCs for a long term which is contributed by difference in the binding sites on LIF; therefore, culture medium supplemented with buffalo-specific LIF may enhance the efficiency of buESCs by improving the environment of culture conditions. The high cost of LIF is another major drawback which restricts buESCs research, thus limits the scope of buffalo stem cell use. Various methods have been developed to produce human and murine LIF in prokaryotic system. However, Buffalo leukemia inhibitory factor (BuLIF) has not been yet produced in prokaryotic system. Here, we describe a simple strategy for the expression and purification of biologically active BuLIF inEscherichia coli(E. coli).ResultsThe BuLIF cDNA from buffalo (Bubalus bubalis) was cloned into pET22b(+) and expressed inE. coliLemo-21(DE3). The expression of BuLIF was directed into periplasmic space ofE. coliwhich resulted in the formation of soluble recombinant protein. One step immobilized metal affinity chromatography (IMAC chromatography) was performed for purification of BuLIF with ≥ 95% of homogeneity. The recombinant protein was confirmed by western blot and identified by mass spectroscopy. The biological activity of recombinant BuLIF was determined on murine myeloid leukemic cells (M1 cells) by MTT proliferation assay. The addition of BuLIF increased the reduction of MTT by stimulated M1 cells in a dose-dependent manner. The BuLIF induced the formation of macrophage like structures from M1 cells where they engulfed fluorescent latex beads. The recombinant BuLIF successfully maintained pluripotency in buffalo embryonic stem cells (buESCs) and were positive for stem cells markers such as Oct-4, Sox-2, Nanog, and alkaline phosphatase activity.ConclusionsThe present study demonstrated a simple method for the production of bioactive BuLIF inE. colithrough single step purification. BuLIF effectively maintained buffalo embryonic stem cells pluripotency. Thus, this purified BuLIF can be used in stem cell study, biomedical, and agricultural research.

Almost all living organism require to transport molybdenum (Mo) from outside cellular milieu for maintenance of essential life processes. Mo is serving as cofactor for catalytic activity of xanthine dehydrogenase, aldehyde oxidase and sulphite oxidase from bacteria to mammals. Although Mo transport is well established in prokaryotes and some eukaryotes including plants, little information is available on Mo transport system in mammals. Recently, HsMoT2/MFSD5 was reported to transport Mo by its over expression in HEK-293T cells. But the structural and functional annotation of MoT2/MFSD5 gene product is missing in mammals and still no sequence information is available on farm animals, particularly in buffalo. Here we cloned and sequenced buffalo MoT/MFSD5 and analysed the sequence in silico. We predicted that buffalo MoT is unstable, hydrophobic and trans membrane protein with 50KD molecular weight. It is having 11 trans membrane helices to traverse the cell membrane. Functionally the protein shared sequence homology with a sugar porter family, bovine MFSD5. Three-dimensional model of buffalo MoT was also analysed using Raptor X server that predicted the protein is having two-fold pseudo symmetry between N and C terminal domain. Finally, we performed comparative analysis of buffalo MoT with human and cattle which revealed the protein is highly conserved both structurally and functionally. Thus, the structural and functional annotation of buffalo MoT would pave a way to design suitable mutants or inhibitors which could be exploited to manage Mo deficiency and Mo toxicity in ruminants.