Hemen Das

Cloning and characterization of novel cathelicidin cDNA sequence of Bubalus bubalis homologous to Bos taurus cathelicidin-4.

Cathelicidin synthesized by bone marrow cells plays an important role in neutralizing invading pathogens. In the present study, the myeloid cathelicidin cDNA from Bubalus bubalis has been cloned and characterized. RNA from bone marrow of buffalo ribs was extracted, reverse transcribed and amplified using specific pair of primer designed from published cathelicidin-4 cDNA sequences of Bos taurus popularly known as indolicidin. An expected amplified product of 517 bp was obtained, which was cloned and sequenced. Comparison of buffalo cathelicidin and indolicidin sequences reveal that the open reading frames (ORF) of both these two congeners consist of 435 nucleotides with 28 divergent nucleotides and the translated proteins of 144 amino acid residues. Fourteen amino acid residues were found to be dissimilar between these two congeners. The molecular mass of buffalo cathelicidin calculated from the deduced amino acid sequence was 16.23 kDa, which is in close proximity of indolicidin. The sequence comparison with known B. taurus cathelicidin congeners again show 70.8–92.9% identity at nucleotides level and 65–88.3% identity at amino acids level. The maximum similarity of buffalo cathelicidin both at nucleotides level (92.9%) and protein level (88.3%) was found to be with indolicidin. Phylogenetic tree analysis at nucleotides and amino acids level indicate that buffalo, cattle, sheep, pig and equine cathelicidin sequences comprise one clade which are distantly related with human, rabbit and murine cathelicidins. It may be reasonably concluded that buffalo possess the ancestral gene of cathelicidin like that of bovine species.

Molecular characterization of cDNA encoding B. taurus cathelicidin-7 like antibiotic peptide from bone marrow cells of Bubalus bubalis

Cathelicidins represent a diverse family of endogenous cationic antibiotic peptide present in all mammalian species. In the present study, a novel cathelicidin cDNA was identified and characterized from bone marrow cells of buffalo (Bubalus bubalis) using RT-PCR based approach. The cDNA encodes a propeptide of 1.18 kDa with net positive charge at neutral pH. The precursor peptide possesses a signal peptide of 29 amino acids and a biologically active peptide of 34 residues. Comparison of sequences indicates only 66.1 and 64.1% identity at nucleotides and amino acids level respectively, with the already reported cathelicidin congener from the same species. However, high degree of similarity (92.8% nucleotides and 81.9% amino acids) was noticed with cathelicidin 7 sequence of Bos taurus suggesting interspecies conservation of cathelicidin peptides rather than intra-species within bovidae family. Phylogenetic trees analyses also support these data. Our findings, further justify the cloned cDNA as a unique cathelicidin member of B. bubalis, and may reasonably considered to be another example of structural diversity exhibited by cathelicidin-derived peptides as reported from other mammals.

Effect of Zinc Supplementation on Egg Quality of Japanese Quails (Coturnix coturnix japonica)

Zinc is required for many biological processes in avian and mammalian species. Higher Zn supplementation was found to be beneficial as animals had a higher response for total, IgM, and IgG antibody. The deficiency of zinc decreases egg production and causes reproductive performance problems, in bone and skin development and mortality. The present study was conducted to evaluate the effect of dietary supplementation with different levels of zinc (0, 25, 50 and 75 mg/kg diet) on egg quality traits of Japanese quails. The four experimental diets were: C= the basal diet (control) without any addition (0 Zn), T1= 25 mg Zn/ kg diet, T2= 50 mg/kg diet and T3= 75 mg/kg diet. Zinc supplementation had significant effect in T1, T2 and T3 on Albumen index however, no significant differences were observed between treatment groups with respect to egg weight, shape index, yolk index, yolk %, albumen %, shell weight % and Haugh unit.

Molecular Characterization of Mitochondrial D Loop of Indian Red Jungle Fowl

Mitochondrial DNA has the characteristic of quick evolution, matrilineal inheritance, and simple molecular structure, and it serves as the most used marker for molecular study. As an important role of genomics, studying it can help understand the origins, history, and adaptation of domestication. A 1325 bp fragment of the mtDNA D-loop region was amplified, sequenced and phylogenetic study was done. Out of 400 sites about 12 % were polymorphic between the Gallus gallus (G.g.) subspecies and most of them (60%) were transitions. Indian RJF showed very low genetic distances (0.008-0.013) with G.g. gallus birds from Thailand as compared to those from Japan and Indonesia. The G.g. murghi showed comparatively high genetic distance i.e. 031-0.034 with G.g. spadiceus and from 0.061-0.066 with G. gallus bankiva. The phylogenetic tree showed that G.g. banikva is well separated from the other three subspecies as it made a separate cluster. G.g. gallus makes two separate clusters i.e. one those from Thailand and other those from Japan and Indonesia. Within each chicken breed, there is an excess of homozygosity, but there is no significant reduction in the nucleotide diversity. Phenotypic modifications of chicken breeds as a result of artificial selection appear to stem from ancestral polymorphisms at a limited number of genetic loci.

Lingual Antimicrobial Peptide Expresses in Buffalo Mammary Gland

Mammalian β-defensins are innate host defense peptides with potential as a therapeutic agent. Present study reports expression of β-defensin mRNA transcript in teat mucosal layer of Bubalus bubalis, which is homologous to lingual antimicrobial peptide (LAP) of cattle. The PCR-amplified and cloned full-length cDNA encodes for a putative peptide of 64 residues (≈7 kDa) with pI of 11.324 and possess characteristic features of β-defensin family including presence of six evolutionary conserved cysteine residues. The first 21 amino acids constitute a putative signal peptide, suggesting that it is a secretory protein. The peptide shares 85%–100% sequence identity with β-defensins of cattle (Bos taurus). Nonetheless, the maximum identity at nucleotide (100%) and amino acid (98.5%) level was with cattle LAP, which is in consistent with phylogeny analysis report. Hence, the cloned β-defensin has been designated as buffalo LAP. This study envisages role of β-defensin peptides in innate immunity of mammary gland.

Identification and Molecular Characterization of Cathelin-like Prepro Region from Bubalus bubalis

Abstract Das, H., Sharma, B. and Kumar, A. 2007. Identification and molecular characterization of cathelin-like prepro region from Bubalus bubalis. J. Appl. Anim. Res., 31: 213–216. In the present study primers from the conserved region of reported Bos taurus cathelicidins were designed and a cathelin-like prepro region of 273 bp from myeloid cells of water buffalo was amplified which codes for 10.5 kDa peptide of 91 residues. This prepropeptide shares 86.4% and 80.2% sequence identity at nucleotides and amino acids level, respectively, with the prepro region of the earlier reported buffalo cathelicidin amplified using primer designed from cattle indolicidin. The results are suggestive of the presence of a different cathelicidin congener in Bubalus bubalis.