Biochemistry Division

Major Achievements

Salient Achievements

Division of Biochemistry was established in 1972 during the Fifth Five year plan under the guidance of Dr C.M. Singh, the then Director of the institute. It was realized that the basic understanding of chemical processes in the cell is essential for every student of biology. The mandate of the division since its inception is to carry out basic and applied research in the areas of animal health and production and to impart post-graduate education. Presently the division is also imparting education pertaining to biochemistry discipline to BVSc students.

Research Areas: Clinical Biochemistry, Reproductive Biochemistry, Host-pathogen Biology, Biochemical Toxicology, Cancer Biochemistry, Membrane Biochemistry, Disease diagnosis

Salient decadal achievements are as follows:

  • Biochemical studies on buffalo milk – Predominant component of buffalo milk lipid was triglyceride, having both short and long chain fatty acids. Notable feature of buffalo milk phospholipids was the higher concentration of sphingomyelin.
  • Lipids of buffalo spermatozoa – The buffalo spermatozoa do not utilize endogenous lipids for energy generation.
  • Physicochemical properties of buffalo muscles – Buffalo meat had higher moisture content as compared to beef. Moisture and glycogen contents decrease with advancing age. A ~61 kDa meat tenderizing enzyme was isolated from unripe fruits of Cucumispubescens that appears to be related with papain.
  • Angiotensin converting enzyme – The presence of enzyme was studied in different animal species and it released during in vitro capacitation of the spermatozoa and it may have a role in sperm activation.
  • Enzyme inhibitors in buffalo seminal plasma – Trypsin inhibitor was isolated and characterized and suggested to be involved in semen protein metabolism.
  • Buffalo muscle proteins – The skeletal muscle proteins of buffalo were categorized into highly metabolic sarcoplasmic proteins, moderately metabolized myofibr-illar proteins and slow metabolic stroma proteins.
  • Clinical biochemistry of buffalo – The cationic peptides isolated from buffalo PBM cells were active against gram negative (E. coli) and gram positive (S. aureus) bacteria.
  • Parasite biochemistry – An ELISA for Dicyocaulusfilaria was developed.
  • Biochemical studies on buffalo pituitary – The buffalo anterior pituitary showed the presence of a possible pro-hormone of growth hormone and prolactin.
  • Spermatozoa motility inhibitor – The Spermatozoa motility inhibitor was isolated from Fowl semen that showed ant-bacterial property.
  • Biochemical toxicology- The biochemical basis of bracken fern toxicity and the hepato-protective effect of Liv 52 and Kumaryasava were studied. Characterization of goat liver glutathione-S-transferase and its role in de-toxification was elucidated.
  • Host pathogen biology – Defensins from different epithelial linings of buffalo tissues showed anti-bacterial properties. The Ocimum sanctum extracts showed antiviral activity against Bovine herpes virus 1 infected MDBK cells through inhibition of virus adsorption to the cells. The virus was found to induce apoptosis in infected bovine peripheral blood mononuclear cells.
  • Matrix metalloproteases and cancer – Different forms of Matrix metalloproteases were identified in canine cancer and their role in metastasis was investigated. An ELISA was developed for MMP- 7, MMP – 11 as diagnostic marker for dog mammary cancer.
  • Clinical Biochemistry - Further studies on buffalo defensins were carried out to analyse their role in host defense. The genes related to the innate immunity (TLR2, TLR3 and TLR4) in buffalo, Nilgai, blackbuck and goats were studied. Complete cDNA encoding IL2, IL4, IL18, TNFα and IFNγ in Nilgai were sequenced.
  • Host-pathogen biology – Micro array DNA chip for livestock virus detection developed. The feeding of Ocimumtenuiflorum and Curcuma caseia extracts was found to augment immune responses as oral immunomodulators in mice. Calreticulin of Haemocuscontortus was identified as the anti-coagulant factor and shown to act as an immunomodulator by binding to complement C1q and the C-reactive protein.
  • Matrix metalloproteases (MMPs) in cancer-
    1. MMP-9 as xenogenic canine vaccine was tested for antitumor potential in murine breast cancer model induced by 4TI cancer cell line. Immunization with recombinant plasmids encoding canine matrix metalloprotease 7 gene alone and combined with interleukin-18 produced both humoral and cell mediated immune responses, inhibited tumor growth and prolonged survival of the treated BALB/c mice in both the prophylactic and therapeutic settings.
    2. Antimetastatic effects of phytocompounds as inhibitor of MMP’s were studied on breast cancer cell lines. Among urosilic acid, eugenol, apeginin, capcisine and berberine, apeginin was found to be more effective in inhibiting MMP’s gene expression and enzyme activity, cell scratch assay and inducing apoptosis in MDA-MBA 231 cancer cell line.
  • Host-pathogen biology-
    1. One fnr knock out (STM:Δfnr) and one fnr codon deoptimised mutant (STM:dfnr) strain of Salmonella typhimurium were generated and evaluated for in vitro growth kinetics under anaerobic conditions and for their in vivo colonization in mice wherein both mutant strains showed reduced growth and colonization in mice.
    2. Standardized and employed a battery of genomics and proteomics approaches along with live animal studies to determine the roles of Msr and PIMT in the Salmonella virulence. MSR and PIMT deletion mutants of S. Typhimurium had reduced virulence.
    3. Mutants for genes encoding for polyamine synthesis (Spermidine synthase, speE) and transport (potD) as well as involved in repair of Met-SO in periplasmic space (periplasmic methionine sulfoxide reductase, msrP) in S. Typhimurium were generated. ∆speE gene deletion strain showed hypersensitivity to H2O2 and defective survival in mice. ∆potD mutant strain showed hypersensitivity to macrophages and neutrophils; and defective survival in mice.
    4. GAPDH of H. contortus was identified as the complement C3 binding protein; this interaction shuts up the complement cascades and defines a novel survival strategy of the parasite. A 95 amino acids stretch of GAPDH was generated and this smaller fragment had both – the C3 binding site as well as the antigenic region of the enzyme. Further, the fragment stimulated PBMCs in vitro and this protein may find therapeutic application.
    5. Immunization of adult goats free of any parasitic infestations with purified H. contortus GAPDH showed the presence of anti-GAPDH antibody compared to control animals. On challenge with H.contortus larvae, on 7th week post challenge, none of the immunized goats showed fecal egg output whereas control animals showed remarkable egg numbers. The H. contortus GAPDH may serve as a potential vaccine candidate.
    6. Vitronectin (Vn) modulates the activity of complement C9, an important component of innate immunity. In addition, Vn also facilitates bacterial binding to host cells leading to infection. In C9 binding site and the bactericidal property of caprine Vn were mapped by generating defined length fragments of the protein.The residues 101-150 of N-terminal part of caprine vitronectin was found to contribute to antimicrobial activity against E. coli and S. aureus by modulating the activity of complement C9, an important component of innate immunity, may be of therapeutic application.
  • Reproductive biochemistry-
    1. Regucalcin was identified as an anti-capacitatory and cryoprotective protein in frozen-thawed buffalo spermatozoa. In addition, reference genes for qRT-PCR data normalization for frozen-thawed buffalo spermatozoa were identified.
    2. Relaxin based sandwich ELISA was developed and pregnancy diagnosis in canine serum was attempted. The methodology was found useful for detecting the well-being of the fetus but not reliable for pregnancy diagnosis as such.
    3. The presence of Antimicrobial peptide namely, SPAG11B isoforms were detected by RT-PCR from testes and epididymal region of Buffalo.
    4. Study of oxidatively damaged proteins and the measures to reduce the protein damage due to cryopreservation in buffalo semen revealed that the supplementation of 1µg of recombinant methionine sulphoxide reductase A(rMsrA)/ 50 million spermatozoa in 1 ml of semen improved the frozen-thawed semen quality.
    5. Mass spectrometric identification of carbonylated proteins in fresh-extended and frozen-thawed seminal plasma revealed more number of proteins carbonylated in frozen-thawed seminal plasma than the fresh-extended seminal plasma.
  • Membrane biology -
    1. Multidrug resistance protein 4 (MRP4) coding region in Gyps vulture and domestic chicken were characterized and two transcript variants were identified.
    2. Stably transfected MDCK cell lines expressing these MRP4 transcript variants were developed and characterized. Functional evaluation of these cell lines revealed that MRP4 is used as diclofenac efflux transporter in both chicken and vulture, full MRP4 more functional than 19 aa deleted one. Diclofenac was not found to inhibit MRP4-mediated urate efflux in MRP4 expressing cells as compared to mock transfected controls.
    3. Identification of other transporters and predicting their ORF in vulture renal transcriptome analysis suggested that the urate secretion in birds may not be unidirectional.
    4. Membrane cholesterol regulates the cross talk between caspases and machineries of autophagy. Cholesterol modulation by Methyl-β-cyclodextrins was found to induce cell death via down regulating the caspases-8, which facilitate the induction of autophagic cell death. Further, the depleting membrane cholesterol reduced the metastasis potential of cancer cells.
    5. MβCD induced programmed cell death (autophagy) in 4T1 cells was found mediated through P2RX7 hyperactivation after the membrane cholesterol perturbation or lipid raft disruption.

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