Postbiotic Lipoteichoic acid of probiotic Lactobacillus origin ameliorates inflammation in HT-29 cells and colitis mice.

Lipoteichoic acid (LTA) is a key surface component of probiotic lactobacilli that is involved in important cellular functions including cross talk with the host immune cells. In this study, the anti-inflammatory and ameliorative properties of LTA from probiotic lactobacilli strains were assessed in in vitro HT-29 cells and in vivo colitis mice. The LTA was extracted with n-butanol and its safety was confirmed based on its endotoxin content and cytotoxicity in HT-29 cells. In the Lipopolysaccharide stimulated HT-29 cells, the LTA from the test probiotics evoked a visible but non-significant increase in IL-10 and decrease in TNF-α levels. During the colitis mice study, probiotic LTA treated mice showed substantial improvement in external colitis symptoms, disease activity score and weight gain. The treated mice also showed improvements in key inflammatory markers such as the gut permeability, myeloperoxidase activity and histopathological damages in colon, although non-significant improvements were recorded for the inflammatory cytokines. Furthermore, structural studies by NMR and FTIR revealed increased level of D-alanine substitution in the LTA of LGG strain over MTCC5690. The present study demonstrates the ameliorative effect of LTA as a postbiotic component from probiotics which can be helpful in building effective strategies for combating gut inflammatory disorders.

Ameliorative Effect of Surface Proteins of Probiotic Lactobacilli in Colitis Mouse Models.

The increase in concern from viable cells of probiotics specifically in acute inflammatory conditions has led to the emergence of the concept of postbiotics as a safer alternative therapy in the field of health and wellness. The aim of the present study was to evaluate the efficacy of surface proteins from three probiotic strains in dextran sodium sulfate and trinitrobenzenesulphonic acid = induced colitis mouse models. The molecular weight of total surface proteins extracted from the three probiotic strains ranged from ∼25 to ∼250 kDa with the presence of negligible levels of endotoxins. Surface layer proteins (SLPs) (∼45 kDa) were found to be present only in the Lactobacillus acidophilus NCFM strain. In the in vivo study, significant differences were not observed in the weight loss and general appetite, however, the decrease in colon length was apparent in TNBS colitis control mice. Further, the administration of these surface proteins significantly reversed the histopathological damages induced by the colitogens and improved the overall histological score. The oral ingestion of these surface proteins also led to a decrease in myeloperoxidase activity and TNF-α expression while the IL-10 levels significantly increased for the strain NCFM followed by MTCC 5690 and MTCC 5689. Overall, the present study signifies the ameliorative role of probiotic surface proteins in colitis mice, thereby, offering a potential and safer alternative for the management of inflammatory bowel disorders.

TMT based deep proteome analysis of buffalo mammary epithelial cells and identification of novel protein signatures during lactogenic differentiation.

The lactating mammary gland harbours numerous matured alveoli with their lumen surrounded by differentiated mammary epithelial cells (MECs), which are exclusively involved in milk synthesis and secretion. Buffalo (Bubalus bubalis) is the second major milk-producing animal, and its physiology is different from cattle. The complete protein machinery involved in MECs differentiation is still not defined in ruminants, in particular, buffalo. Therefore, we have studied the differential expression of regulated proteins in the in vitro grown buffalo MECs (BuMECs) at different time points (on 3, 6, 12, and 15 days) of their differentiation in the presence of lactogenic hormones. TMT-based MS analysis identified 4,934 proteins; of them, 681 were differentially expressed proteins (DEPs). The principal component analysis suggested a highly heterogeneous expression of DEPs at the four-time points of hormone treatment, with most of them (307) attained the highest expression on 12 days. Bioinformatics analysis revealed the association of DEPs with 24 KEGG pathways. We observed few new proteins, namely ABCA13, IVL, VPS37, CZIB, RFX7, Rab5, TTLL12, SMEK1, GDI2, and TMEM131 in BuMECs. The function of one of the highly upregulated proteins, namely involucrin in the differentiation of BuMECs was confirmed based on biochemical inhibition assay. The results further conclude that the proteins with higher abundance can be considered as the potential biomarkers for differentiation, and they may have a significant association with the lactation process in buffalo too. The proteome dataset obtained can be used to understand the species-specific variations among other lactating animals.

Crystal structure of breast regression protein 39 (BRP39), a signaling glycoprotein expressed during mammary gland apoptosis, at 2.6 Å resolution.

Breast regression protein 39 (BRP39) is a 39 kDa protein that is a member of chitolectin class of glycosyl hydrolase family 18 (GH18). High expression levels of BRP39 have been detected in breast carcinoma. It helps in proliferation of cells during the progression of this disease and may act as a signaling factor. BRP39 may act as a potential candidate for rational structure-based drug design against breast carcinoma. In this study, we report the crystal structure of mouse recombinant BRP39 expressed in E. coli. The structure was solved by molecular replacement and refined to 2.6 Å resolution. The overall structure of BRP39 consisted of two globular domains: a large (ß/α)8 triosephosphate isomerase (TIM) barrel domain and a small (α + ß) domain. Three non-proline cis-peptides were detected in the sugar-binding cleft of BRP39, including Ser57-Phe58, Leu141-Tyr142, and Trp353-Ala354. The latter residues were conserved in other GH18 family members. It was notable that the conformation of critical Trp100 residue within the sugar-binding cleft was oriented away from the barrel. The side-chain conformation was found to be similar to that observed in chitinases, however, it was oriented into the barrel in other chitinase-like proteins (CLPs). The conformation of this critical residue may have significant implications in sugar binding. Further, two amino acid substitutions were observed in the sugar-binding groove of BRP39. The conserved Asn100 and Arg263 in Hcgp39 and other CLPs proteins (SPX-40 structures) were substituted by Lys101 and Lys264 in BRP39 which may have a significant impact on the sugar-binding properties.

In-depth proteome analysis of more than 12,500 proteins in buffalo mammary epithelial cell line identifies protein signatures for active proliferation and lactation.

The mature mammary gland is made up of a network of ducts that terminates in alveoli. The innermost layer of alveoli is surrounded by the differentiated mammary epithelial cells (MECs), which are responsible for milk synthesis and secretion during lactation. However, the MECs are in a state of active proliferation during pregnancy, when they give rise to network like structures in the mammary gland. Buffalo (Bubalus bubalis) constitute a major source of milk for human consumption, and the MECs are the major precursor cells which are mainly responsible for their lactation potential. The proteome of MECs defines their functional state and suggests their role in various cellular activities such as proliferation and lactation. To date, the proteome profile of MECs from buffalo origin is not available. In the present study, we have profiled in-depth proteome of in vitro cultured buffalo MECs (BuMECs) during active proliferation using high throughput tandem mass spectrometry (MS). MS analysis identified a total of 8330, 5970, 5289, 4818 proteins in four sub-cellular fractions (SCFs) that included cytosolic (SCF-I), membranous and membranous organelle's (SCF-II), nuclear (SCF-III), and cytoskeletal (SCF-IV). However, 792 proteins were identified in the conditioned media, which represented the secretome. Altogether, combined analysis of all the five fractions (SCFs- I to IV, and secretome) revealed a total of 12,609 non-redundant proteins. The KEGG analysis suggested that these proteins were associated with 325 molecular pathways. Some of the highly enriched molecular pathways observed were metabolic, MAPK, PI3-AKT, insulin, estrogen, and cGMP-PKG signalling pathway. The newly identified proteins in this study are reported to be involved in NOTCH signalling, transport and secretion processes.

Structural and functional characterization of buffalo oviduct-specific glycoprotein (OVGP1) expressed during estrous cycle.

Oviduct-specific glycoprotein (OVGP1) is a high molecular weight chitinase-like protein belonging to GH18 family. It is secreted by non-ciliated epithelial cells of oviduct during estrous cycle providing an essential milieu for fertilization and embryo development. The present study reports the characterization of buffalo OVGP1 through structural modeling, carbohydrate-binding properties and evolutionary analysis. Structural model displayed the typical fold of GH18 family members till the boundary of chitinase-like domain further consisting of a large (ß/α)8 TIM barrel sub-domain and a small (α+ß) sub-domain. Two critical catalytic residues were found substituted in the catalytic centre (Asp to Phe118, Glu to Leu120) compared with the active chitinase. The carbohydrate-binding groove in TIM barrel was lined with various conserved aromatic residues. Molecular docking with different sugars revealed the involvement of various residues in hydrogen-bonding and non-bonded contacts. Most of the substrate-binding residues were conserved except for a few replacements (Ser13, Lys48, Asp49, Pro50, Asp167, Glu199, Gln272 and Phe275) in comparison with other GH18 members. The residues Trp10, Trp79, Asn80, Gln272, Phe275 and Trp334 were involved in recognition of all six ligands. The α+ß sub-domain participated in sugar-binding through Thr270, Gln272, Tyr242 and Phe275. The binding assays revealed significant sugar-binding with purified native and recombinant OVGP1. Phylogenetic analysis revealed that OVGP1 was closely related to AMCases followed by other CLPs and evolution of OVGP1 occurred through several gene duplications. This is the first study describing the structural characteristics of OVGP1 that will further help to understand its interaction with gametes to perform crucial reproductive functions.

Functional analysis of recombinant buffalo lactoferrin and monoferric lobes and their cytotoxic effect on buffalo mammary epithelial cells.

Lactoferrin (Lf) has been involved in diverse type of cellular activities and its biochemical properties are species specific. Lf is a bilobal molecule in which each lobe binds with one Fe2+/Fe3+ ion. A lot of physiological effects of Lf are regulated by its iron binding and release properties; however these properties are species-specific. To understand the iron-binding, thermal stability and cytotoxic effect of buffalo Lf (buLf) and contribution of individual N- and C-terminal lobes therein, buLf and the truncated monoferric lobes were expressed in Kluyveromyces lactis or Pichia pastoris yeast expression systems. The iron-uptake/release behavior and thermal stability of recombinant buLf was observed similar to the Lf purified from buffalo milk. Supplementation of recombinant buLf to the buffalo mammary epithelial cells (BuMEC) culture decreased their proliferation and the cell viability in a dose dependent manner. The cell growth decreased by 37% at 1.0 mg/ml Lf. C-lobe decreased the viability of BuMEC by 15% at 1 mg/ml. The C-lobe showed greater cytotoxic effect against BuMEC in comparison to N-lobe. buLf caused a reduced expression of the casein in BuMEC. At 1.0 mg/ml of buLf, CSN2 transcript level was reduced by 74% and 78% in the normal and hormone free media, respectively. The expression of IL-1ß gene in BuMEC increased by 4-5 fold in the presence of 1.0 mg/ml of Lf. The effect was similar to that observed in the involutory mammary gland, suggesting the role of elevated level of Lf in remodeling of buffalo mammary tissue during involution.

Comparative transcriptome analysis reveals the genetic basis of coat color variation in Pashmina goat.

The genetics of coat color variation remains a classic area. Earlier studies have focused on a limited number of genes involved in color determination; however, the complete set of trait determinants are still not well known. In this study, we used high-throughput sequencing technology to identify and characterize intricate interactions between genes that cause complex coat color variation in Changthangi Pashmina goats, producer of finest and costly commercial animal fiber. We systematically identified differentially expressed mRNAs and lncRNAs from black, brown and white Pashmina goat skin samples by using RNA-sequencing technique. A pairwise comparison of black, white and brown skin samples yielded 2479 significantly dysregulated genes (2422 mRNA and 57 lncRNAs). Differentially expressed genes were enriched in melanin biosynthesis, melanocyte differentiation, developmental pigmentation, melanosome transport activities GO terms. Our analysis suggested the potential role of lncRNAs on color coding mRNAs in cis and trans configuration. We have also developed online data repository as a component of the study to provide a central location for data access, visualization and interpretation accessible through http://pcd.skuastk.org/ .

New insights into the catalytic inactivity of mammary gland protein-40, a chitinase-like protein expressed during mammary gland involution.

MGP-40 is a mammary gland-specific glycoprotein which is expressed during involution and is an important marker for mammary gland apoptosis. It is an inactive chitinase-like protein belonging to Glycosyl Hydrolase family 18. The present study reports sequence characterization, tissue-specific expression analysis, production of recombinant MGP-40 and its mutant (A117D and L119E) in both E. coli and COS1 cells for their chitin-binding and chitinase activity analysis. The cDNA of buffalo MGP-40 was cloned and sequenced which corresponded to 1803 bp with an open reading frame of 1152 bp (361 aa), signal sequence of 63 bp (21 aa), 5' and 3' UTR of 144 bp and 507 bp, respectively. The 3' UTR analysis revealed potential sites for high level expression and stability during involution. The half-life of buffalo MGP-40 was found to be 11.7 h. MGP-40 was highly expressed in mammary gland followed by small intestine, spleen and mammary epithelial cells. The purified recombinant MGP-40 and its mutant expressed in E.coli were observed to bind chitin efficiently, however, no chitinase activity was observed. Further, chitinase activity was also not observed by expressing mutant recombinant MGP-40 in COS1 cells ruling out the possible role of post-translational modifications. Structure-based in-silico mutagenesis by FoldX algorithm showed a drastic decrease in overall fold stability which might be a possible reason for inability to recover its activity. Therefore, chitinase activity could not be restored in MGP-40 even after reverting back two critical residues in active site which may be due to detrimental effect of mutations on structural stability.

Primary structures of different isoforms of buffalo pregnancy-associated glycoproteins (BuPAGs) during early pregnancy and elucidation of the 3-dimensional structure of the most abundant isoform BuPAG 7.

Pregnancy-associated glycoproteins (PAGs) are expressed during pregnancy by the trophoectodermal cells of fetus. Presence of PAGs in dam's circulation has been widely used in pregnancy diagnosis. The present study reports the identification and characterization of different PAG isoforms in buffalo during early stages of pregnancy. The PAG mRNAs isolated from fetal cotyledons (Pregnancy stages: 45, 75 and 90 days) were successfully cloned in pJET1.2 vector and transformed in E. coli. A total of 360 random clones were sequenced and correlated with their stages of expression. A total of 12 isoforms namely, BuPAG 1, 2, 4, 6, 7, 8, 9, 13, 15, 16, 18 and one new isoform were identified. BuPAG 7 was found as the most abundant isoform in all three stages followed by BuPAG 18. Further, a large number of variants were found for most of these isoforms. Phylogenetic relationship of identified BuPAGs showed that BuPAG 2 belonged to an ancient group while other members clustered with modern group. Three-dimensional (3D) structure of BuPAG 7 was determined by homology modeling and molecular dynamic (MD) simulations which displayed a typical fold represented by other aspartic proteinase (AP) family members. Molecular docking of Pepstatin inhibitor with BuPAG 7 revealed to interact through various hydrogen bonding and hydrophobic interactions. Various amino acid substitutions were observed in peptide-binding cleft of BuPAG 7. Superimposition of BuPAG 7 with homologous structures revealed the presence of a 35-41 amino acid long insertion (alpha helix connected by two loops) near the N- terminus which seems to be a unique feature of BuPAG 7 in AP family. This is the first report on identification and sequence characterization of PAG isoforms in buffalo with unique finding that these isoforms represent many transcript variants. We also report 3D structure of the most abundant isoform BuPAG 7 for the first time.

Effect of recombinant and native buffalo OVGP1 on sperm functions and in vitro embryo development: a comparative study.

BACKGROUND: An oviduct- specific glycoprotein, OVGP1, is synthesized and secreted by non-ciliated epithelial cells of the mammalian oviduct which provides an essential milieu for reproductive functions. The present study reports the effects of recombinant buffalo OVGP1 that lacks post-translational modifications, and native Buffalo OVGP1 isolated from oviductal tissue, on frozen- thawed sperm functions and in vitro embryo development. RESULTS: The proportion of viable sperms was greater (P < 0.05) in the recombinant OVGP1-treated group compared to the native OVGP1-treated group at 2 h, 3 h, and 4 h of incubation. The proportion of motile sperms at 3 h and 4 h of incubation; and membrane- intact sperms at 4 h was greater (P < 0.05) in the native OVGP1-treated group compared to the control and recombinant OVGP1-treated groups. The proportion of capacitated and acrosome- reacted sperms was greater (P < 0.05) in the native OVGP1-treated group compared to the recombinant OVGP1 group at 4 h. The rates of cleavage of embryos and their development to the blastocyst stage were greater (P < 0.05) in the presence of either native or recombinant OVGP1 in comparison to control at 10 µg/mL concentration as compared to 5 or 20 µg/mL. CONCLUSIONS: The study suggests that both native and recombinant OVGP1 impart a positive effect on various sperm features and in vitro embryo development. However, native OVGP1 was found to have a more pronounced effect in comparison to recombinant non-glycosylated OVGP1 on various sperm functions except viability. Hence, our current findings infer that glycosylation of OVGP1 might be essential in sustaining the sperm functions but not the in vitro embryo development.

Buffalo Leukemia Inhibitory Factor Induces Differentiation and Dome-Like Secondary Structures in COS-1 Cells.

This study aimed to understand the molecular characteristics of buffalo leukemia inhibitory factor (BuLIF) and the generation of a stably transfected COS-1_BuLIF cell line for its functional characterization. Cumulus cells, isolated from oocytes, were separated, and total cDNA was prepared. The BuLIF gene was ligated into the cloning vector pJET1.2/blunt and expression vector pAcGFP-N1 which was transfected into COS-1 cells and confirmed by qRT-PCR and Western blot. BuLIF was immunoprecipitated and evaluated through a MTT assay. qRT-PCR of STAT3 was performed. The multiple sequence alignment of BuLIF showed high similarity with sheep (98.77%) and cattle (96.62%) compared with other species. The BuLIF gene has an open reading frame of 609 nucleotides coding for 202 amino acids. BuLIF was integrated into the genome of COS-1 cells and resulted in the formation of dome-like secondary structures which are indicative of its functional role mediated through STAT3 proteins. In conclusion, this cell line is suitable for understanding LIF-mediated biological functions.

Structural, Functional and Phylogenetic Analysis of Sperm Lysozyme-Like Proteins.

Sperm lysozyme-like proteins belonging to c-type lysozyme family evolved in multiple forms. Lysozyme-like proteins, viz., LYZL2, LYZL3 or SLLP1, LYZL4, LYZL5 and LYZL6 are expressed in the testis of mammals. Not all members of LYZL family have been uniformly and unambiguously identified in the genome and proteome of mammals. Some studies suggested a role of SLLP1 and LYZL4 in fertilization; however, the function of other LYZL proteins is unknown. We identified all known forms of LYZL proteins in buffalo sperm by LC-MS/MS. Cloning and sequence analysis of the Lyzl cDNA showed 38-50% identity at amino acid level among the buffalo LYZL paralogs, complete conservation of eight cysteines and other signature sequences of c-type lysozyme family. Catalytic residues in SLLP1, LYZL4 and LYZL5 have undergone replacement. The substrate binding residues showed significant variation in LYZL proteins. Residues at sites 62, 101, 114 in LYZL4; 101 in SLLP1; 37, 62, and 101 in LYZL6 were more variable among diverse species. Sites 63 and 108 occupied by tryptophan were least tolerant to variation. Site 37 also showed lower tolerance to substitution in SLLP1, LYZL4 and LYZL5, but more variable in non-testicular lysozymes. Models of LYZL proteins were created by homology modeling and the substrate binding pockets were analyzed in term of binding energies and contacting residues of LYZL proteins with tri-N-acetylglucosamine (NAG)3 in the A-B-C and B-C-D binding mode. Except LYZL6, LYZL proteins did not show significant difference in binding energies in comparison to hen egg white lysozyme in the A-B-C mode. (NAG)3 binding energy in the B-C-D mode was higher by 1.3-2.2 kcal/mol than in A-B-C mode. Structural analysis indicated that (NAG)3 was involved in making more extensive interactions including hydrogen bonding with LYZL proteins in B-C-D mode than in A-B-C mode. Despite large sequence divergence among themselves and with respect to c-type lysozymes, substrate binding residues as well as hydrogen bonding network between (NAG)3 and proteins were mostly conserved. LYZL5 in buffalo and other mammalian species contained additional 10-12 amino acid sequence at c-terminal that matched with ankyrin repeat domain-containing protein 27. Phylogenetic analysis indicated LYZL2 to be most ancient among all the LYZL proteins and that the evolution of LYZL proteins occurred through several gene duplications preceding the speciation of mammals from other vertebrates as distant as reptiles and amphibians.

Identification of potential protein biomarkers for early detection of pregnancy in cow urine using 2D DIGE and label free quantitation.

BACKGROUND: An early, reliable and noninvasive method of early pregnancy diagnosis is prerequisite for efficient reproductive management in dairy industry. The early detection of pregnancy also help in to reduce the calving interval and rebreeding time which is beneficial for industries as well as farmers. The aim of this work is to identify potential biomarker for pregnancy detection at earlier stages (16-25 days). To achieve this goal we performed DIGE and LFQ for identification of protein which has significant differential expression during pregnancy. RESULTS: DIGE experiment revealed a total of eleven differentially expressed proteins out of which nine were up regulated having fold change ≥1.5 in all time points. The LFQ data analysis revealed 195 differentially expressed proteins (DEPs) out of 28 proteins were up-regulated and 40 down regulated having significant fold change ≥1.5 and ≤0.6 respectively. Bioinformatics analysis of DEPs showed that a majority of proteins were involved in regulation of leukocyte immunity, endopeptidase inhibitor activity, regulation of peptidase activity and polysaccharide binding. CONCLUSION: This is first report on differentially expressed protein during various time points of pregnancy in cow to our best knowledge. In our work, we identified few proteins such MBP, SERPIN, IGF which were differentially expressed and actively involved in various activities related to pregnancy such as embryo implantation, establishment and maintenance of pregnancy. Due to their involvement in these events, these can be considered as biomarker for pregnancy but further validation of is required.

Molecular cloning and production of caprine recombinant Oct4 protein for generation induced pluripotent stem cells.

Oct4, pluripotency marker and transcription factor, expresses in embryonic stem cells. It plays a pivotal role in determination of stem cells fate. Up and down regulation of Oct4 causes differentiation of embryonic stem cells. It is one of the main transcription factors which remained concerned in every study related to induced pluripotent stem cell. Here, we report the production of goat Oct4 protein using plasmid and lentiviral based vectors. Firstly, Oct4 ORF was cloned in pAcGFP1-N1 plasmid vector and positive clones were screened with colony PCR. Oct4 was over-expressed in CHO-K1 cell line and expression was confirmed by observing green florescent protein expression in CHO-K1 cells. Secondly, Oct4 lentiviral expression construct has been prepared using pLenti-gw vector. Oct4 ORF was cloned into pLenti4/V5-DEST vector and viral particles were produced in 293FT cells. Oct4 viral particles were used to infect goat fibroblast cells. Oct4 expression was observed and confirmed in transfected goat fibroblast cells using RT-PCR. Detection of Oct4 protein in western blotting assay affirmed the capacity of over-expression of our Oct4 lentiviral vector. The lentiviral expression construct and recombinant Oct4 protein may be used for reprogramming of somatic cell into induced pluripotent stem cell.

Profiling of urinary proteins in Karan Fries cows reveals more than 1550 proteins.

Urine is a non-invasive source of biological fluid, which reflects the physiological status of the mammals. We have profiled the cow urinary proteome and analyzed its functional significance. The urine collected from three healthy cows was concentrated by diafiltration (DF) followed by protein extraction using three methods, namely methanol, acetone, and ammonium sulphate (AS) precipitation and Proteo Spin urine concentration kit (PS). The quality of the protein was assessed by two-dimensional gel electrophoresis (2DE). In-gel digestion method revealed more proteins (1191) in comparison to in-solution digestion method (541). Collectively, 938, 606 and 444 proteins were identified in LC-MS/MS after in-gel and in-solution tryptic digestion of proteins prepared by AS, PS and DF methods, respectively resulting in identification of a total of 1564 proteins. Gene ontology (GO) using Panther7.0 grouped the majority of the proteins into cytoplasmic (location), catalytic activity (function), and metabolism (biological processes), while Cytoscape grouped proteins into complement and coagulation cascades; protease inhibitor activity and wound healing. Functional significance of few selected proteins seems to play important role in their physiology. Comparative analysis with human urine revealed 315 overlapping proteins. This study reports for the first time evidence of more than 1550 proteins in urine of healthy cow donors. This article is part of a Special Issue entitled: Proteomics in India.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

DIGE based proteome analysis of mammary gland tissue in water buffalo (Bubalus bubalis): lactating vis-a-vis heifer.

Mammary gland is an exocrine and sebaceous gland made up of branching network of ducts that end in alveoli. Milk is synthesized in the alveoli and secreted into alveolar lumen. Mammary gland represents an ideal system for the study of organogenesis that undergoes successive cycles of pregnancy, lactation and involution. To gain insights on the molecular events that take place in pubertal and lactating mammary gland, we have identified 43 differentially expressed proteins in mammary tissue of heifer (non-lactating representing a virgin mammary gland), and lactating buffaloes (Bubalus bubalis) by 2D-difference gel electrophoresis (2D-DIGE) and mass spectrometry. Twenty one proteins were upregulated during lactation whereas 8 proteins were upregulated in heifer mammary gland significantly (p<0.05). Bioinformatics analyses of the identified proteins showed that a majority of the proteins are involved in metabolic processes. The differentially expressed proteins were validated by real-time PCR and Western blotting. We observed differential expressions of certain new proteins including EEF1D, HSPA5, HSPD1 and PRDX6 during lactation which have not been reported before. The differentially expressed proteins were mapped to available biological pathways and networks involved in lactation. This study signifies the importance of some proteins which are preferentially expressed during lactation and in heifer mammary gland. BIOLOGICAL SIGNIFICANCE: This work is important because we have generated information in water buffalo (B. bubalis) for the first time which is the major milk producing animal in Indian Subcontinent. Out of a present production of 133milliontons of milk produced in India, contribution of buffalo milk is around 54%. Its physiology is somewhat different from the lactating cows. Buffalo milk composition varies from cow milk in terms of higher fat and total solid content, which confers an advantage in preparation of specialized cheese, curd and other dairy products. Being a major milk producing animal in India it is highly essential to understand the lactation associated proteins in the mammary gland of buffalo. In the present investigation our attempt has been to identify new protein evidences which are expressed in lactating buffalo mammary gland and have not been reported before. The findings reported in the present study will help in understanding the lactation biology of buffalo mammary gland in particular and the mammary gland biology in general.

Comparative 2D-DIGE proteomic analysis of bovine mammary epithelial cells during lactation reveals protein signatures for lactation persistency and milk yield.

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.