Characterization and Structural Analysis of Hepcidin Like Antimicrobial Peptide From Schizothorax richardsonii (Gray)

Innate immune system is a primary line of defense in fish that protects it from the invading pathogens. Antimicrobial peptides (AMPs) are widely distributed in nature and are essential components of innate immunity. These molecules enable the host’s innate immune system to fight against a variety of infectious agents. One such AMP, hepcidin, is a cysteine rich amphipathic peptide. We have amplified, cloned and characterized hepcidin like AMP from Schizothorax richardsonii that inhabits one of the most difficult aquatic ecosystems in the Indian Himalayas. The cDNA encoding hepcidin like peptide was amplified as a 371 bp fragment with an open reading frame (ORF) of 279 nucleotides flanked by 5′ and 3′ UTRs of 70 and 22 bases respectively. This ORF encodes a peptide of 93 amino acids with a signal peptide of 24 amino acids and a mature peptide of 25 amino acids. The mature hepcidin like peptide of S. richardsonii has eight cystine residues that participate in the formation of four disulfide bonds, a unique feature of hepcidin like AMPs. A 3D model of hepcidin like mature peptide was generated using Modeller 9.10 which was validated using PROCHECK and ERRAT. Phylogenetic analysis of hepcidin like AMP from S. richardsonii revealed that it was closely related to hepcidin from olive barb (Puntius sarana).     

Genetic Polymorphisms in the Bovine Toll-Like Receptor 4 (TLR4) and Monocyte Chemo Attractant Protein-1(CCL2) Genes: SNPs Distribution Analysis in Bos indicus Sahiwal Cattle Breed

Toll-like receptor 4 gene (TLR4) that recognizes the Gram negative bacterial ligand LPS was sequenced in the Bos indicus Sahiwal cattle breed. Ninety four single nucleotide polymorphisms (SNPs) were detected within 10.8 kb gene region. Seventeen of the SNPs were in the coding regions and the one at position 9589(A > G) in exon3 resulted in an amino acid change from Valine to Isoleucine. These SNPs led to generation of 27 TLR4 gene haplotypes. All the Sahiwal animals studied presently showed the occurrence of the genotype CC at gene position 9662, which codes for the amino acid threonine at position 674 of the TLR4 protein, and which had been reported to be associated with lower somatic cell score and, therefore, a lower susceptibility to mastitis, in Taurus cattle. This nucleotide configuration of the Toll-like receptor 4 gene of the Bos indicus Sahiwal cattle breed could possibly indicate toward a lower susceptibility to mastitis in the Sahiwal animals. Monocyte chemo-attractant protein-1 (CCL2) gene encoding for small inducible cytokine A2 that belongs to the CC chemokine family was also sequence characterized in these Sahiwal animals. The CCL2 gene was observed to have 12 polymorphic sites in 3.3 kb region of which one SNP at position 2500 (A > G) in exon 3 resulted in amino acid change from Valine to Isoleucine at position 46 of the mature CCL2 peptide. Seventeen haplotypes of the CCL2 gene were predicted corresponding to 12 genotypes detected.     

Detection of Polymorphism and Sequence Characterization of Toll-Like Receptor 7 Gene of Indian Goat Revealing Close Relationship Between Ruminant Species

In this study, approximately 3.4 kb nucleotide sequence of caprine TLR7 (Toll-like receptor 7) gene was generated from twelve different Indian goat breeds belonging to different geographical regions. Goat TLR7 gene ORF (Open Reading Frame) was found to be 3141 nucleotides long coding for 1046 amino acids similar to sheep. The sequence analysis at nucleotide level revealed goat TLR7 having 99.5% homology with sheep, followed by other livestock species. Simple Modular Architecture Research Tool (SMART) was used for the structural analysis of goat TLR7 that showed the presence of 22 leucine rich repeats (LRRs) along with single Toll/interleukin-1 receptor (TIR) domains. TIR domain, when compared, was found to be similar in ruminant species, goat, sheep, cattle, and buffalo. The phylogenetic analysis also revealed grouping of all ruminant species together, goat being closer to sheep followed by cattle and buffalo. A total of 22 polymorphic sites were observed in TLR7 gene of 24 goats representing 12 different breeds, out of which 19 were present within the coding region and three in 3'UTR. Out of the seven nonsynonymous SNPs, two were in ectodomains and one in TIR domain. Overall our results indicate substantial variation within goat TLR7 gene, which could be exploited for association with disease susceptibility.     

Molecular characterization and expression of type-I interferon gene in Labeo rohita

Genes coding for type-I interferon (I-IFN) has been cloned from Labeo rohita, a commercially important and widely cultured fish in India and South East Asia. In the present study, full-length gene of I-IFN was amplified and sequenced. The sequence analysis revealed that I-IFN consists of 1,786 bp genomic sequence with four introns and five exons and an ORF of 546 bp encoding for a putative protein of 181 amino acids. The mature protein has a molecular weight of 18.97 kDa and consists of 158 amino acids and a signal peptide of 23 amino acids at the N terminus. The sequence carries I-IFN signature motif, one glycosylation site, two conserved cystine amino acids and other conserved amino acids. The sequence showed highest similarity to that of Cyprinus carpio (84 %). In silico analysis of the rohu I-IFN protein was done using various bioinformatic tools. The constitutive expression of I-IFN gene was found to be more in spleen compared to gill and kidney in real time PCR assay. Expression of I-IFN increased about 20-fold in cultured kidney cell 2 h after induction with poly I:C and showed maximum expression at 8 h post-induction.     

Microsatellite Analysis of Genetic Population Structure of Zebu Cattle (Bos indicus) Breeds from North-Western Region of India

The present study aims to understand the existing genetic diversity and structure of six native cattle breeds (Rathi, Tharparkar, Nagori, Mewati, Gir, and Kankrej) adapted to the north-western arid and semi-arid region of India based on microsatellite loci. Various diversity estimates, mean number of alleles (12.84); effective number of alleles (5.02); gene diversity (0.769), and observed heterozygosity (0.667) reflected the existence of substantial within-breed diversity in all the investigated cattle breeds. Mean estimates of F-statistics: F_IT = 0.144 ± 0.023, F_IS = 0.071 ± 0.021, and F_ST = 0.078 ± 0.014 were significantly different from zero (P < 0.05). The interbreed relationships indicated moderate level of breed differentiation between the six cattle breeds with least differentiation between Kankrej-Mewati pair. The phylogeny structuring further supported close grouping of Kankrej and Mewati breeds. Correspondence analysis plotted Rathi, Tharparkar, and Gir individuals into three separate areas of multivariate space; whereas, Kankrej, Mewati, and Nagori cattle showed low breed specific clustering. This reflected the existence of discrete genetic structure for Tharparkar, Rathi, and Gir, the prominent dairy breeds of the region; whereas, admixture was observed for Kankrej, Mewati, and Nagori individuals.     

Toll-Like Receptor 2 Gene Polymorphism and its Relationship with SCS in Dairy Cattle

Toll-like receptor 2 (TLR2) plays an important role in the innate immune response to a variety of pathogens. In this study, bovine TLR2 gene was taken as a candidate gene for mastitis resistance. Through PCR-SSCP analysis and sequencing, three missense mutations at T385 G, G398A, and G1884A were detected in the coding region that encoded extracellular domain. Altogether 240 dairy cattle of three breeds (Holstein, Simmental, and Sanhe cattle) were genotyped and allele frequencies were determined. The effects of TLR2 polymorphisms on somatic cell score (SCS) were analyzed and significant association was found between T385 G and SCS. The mean of genotype GG was significantly lower than those of genotype TT and TG. No significant associations were found with SCS for G398A and G1884A. Information provided in this research will be useful in further studies to determine the role of TLR2 gene in the mastitis resistance.     

Molecular Characterization of a Newly Identified Subfamily Member of Penaeidin from two Penaeid Shrimps, <Emphasis Type="Italic">Fenneropenaeus indicus</Emphasis> and <Emphasis Type="Italic">Metapenaeus monoceros</Emphasis>

Penaeidins are a major group of antimicrobial peptides found in penaeid shrimps. This study reports a new isoform of penaeidin from the hemocytes of Indian white shrimp, Fenneropenaeus indicus (Fi-PEN, JX657680), and the pink shrimp, Metapenaeus monoceros (Mm-PEN, KF275674). Mm-PEN is also the first antimicrobial peptide to be identified from M. monoceros. The complete coding sequences of the newly identified Fi-PEN and Mm-PEN consisted of an ORF of 338 bp encoding 71 amino acids with a predicted molecular weight of 5.66 kDa and a pI of 9.38. The penaeidins had its characteristic signal peptide region (19 amino acids), which was followed by a mature peptide with a proline-rich domain (24 amino acids) at the N-terminal region and a cysteine-rich domain (28 amino acids) at the C-terminal region, designating it to penaeidin-3 subgroup. Structural analysis revealed an alpha-helix in its secondary structure and an extended structure at the proline-rich domain. The newly identified penaeidin isoform showed maximum similarity of 63 % to a penaeidin-3 isoform of P. monodon, which further proves it to be a new isoform. Phylogenetic analysis showed that it possessed similar evolutionary status like other penaeidins, which has subsequently diverged at different phases of evolution. The wide distribution of penaeidins in penaeid shrimps indicates the importance of these AMPs in the innate immunity.     

Seasonal variation in HSP70 expression and oxidative stress in skin of zebu (Tharparkar) and crossbred (Karan Fries) cattle under tropical climate

The study aimed to evaluate inducible HSP70 (HSP70.1 and HSP70.2) gene expression and oxidative stress status in skin of cattle during different seasons. Ten each of Tharparkar (zebu) and Karan Fries (crossbred) heifers were selected from NDRI herd, Karnal. Animals were maintained under standard managemental practices followed at the farm. Skin biopsies were aseptically collected from each animal during winter, spring, and summer. Real time PCR was performed to examine HSP70 expression. Reactive oxygen species (ROS) and antioxidant enzymes (SOD and CAT) were determined by ELISA. In both the breeds, significantly higher (p < 0.05) levels of HSP70 expression, ROS, caspases, and antioxidant enzymes were observed during summer followed by winter and spring. Breeds showed no significant difference during winter and spring. During summer, HSP70 expression, ROS, and antioxidant enzymes were higher (p < 0.05) in Karan Fries than Tharparkar, whereas caspases levels were higher in Tharparker than Karan Fries. The study concludes that levels of HSP70 expression, ROS, caspases, and antioxidant enzymes in skin of cattle were strongly affected by seasonal change in temperature. Differences exist in skin tissue thermotolerance of Tharparkar and Karan Fries cattle. This might be an efficient and centrally important mechanism for better adaptability of zebu cattle to heat stress.     

北京鸭卵磷脂胆固醇酰基转移酶的cDNA和蛋白质的结构分析

为获得不易感动脉粥样硬化动物北京鸭卵磷脂胆固醇酰基转移酶 (LCAT)的cDNA和蛋白质序列 ,分析其结构特点 .以从北京鸭肝脏mRNA反转录获得的cDNA一链为模板 ,应用SMART RACE技术 ,获得了北京鸭LCAT的cDNA序列 ,推导出其蛋白质氨基酸序列 ,应用分子生物学软件对该蛋白的一级、二级结构进行分析和比较 .北京鸭LCATcDNA (在GenBank中的注册号为AF32 4 887)全长 195 3bp ,其中开放阅读框架 135 6bp ,编码 4 5 1个氨基酸 ,包括一个由 2 3个氨基酸构成的疏水性信号肽和一个由 4 2 8个氨基酸组成的成熟蛋白 .该成熟蛋白比人LCAT在C端多 12个氨基酸 ,其与鸡、人、家兔的同源性依次为 98%、83%和 82 % .与其它种属LCAT蛋白序列的比较结果表明 ,北京鸭LCAT蛋白质序列虽然在长度上和结构上与其它种属有一定的差异 ,但序列中与酶催化活性相关的序列均非常保守     

Yak (Bos Grunniens) Stomach Lysozyme: Molecular Cloning,Expression and its Antibacterial Activities

The cDNA coding for stomach lysozyme in yak was cloned. The cloned cDNA contains a 432 bp open reading frame and encodes 143 amino acids (16.24 KDa) with a signal peptide of 18 amino acids. Further analysis revealed that its amino acid sequence shares many common properties with cow milk lysozyme. Expression of this gene was also detected in mammary gland tissue by RT-PCR. Phylogenetic relationships among yak stomach lysozyme and 8 cow lysozymes indicated that the yak enzyme is more closely related to both cow milk lysozyme and the pseudogene ΨNS4 than cow stomach lysozyme. Recombinant yak lysozyme purified by Ni²⁺-column showed a molecular weight of 33.78 kDa and exhibited lytic activity against Staphylococcus aureus, providing evidence of its antibacterial activities.     

Molecular cloning and determination of the nucleotide sequence of raw starch digesting alpha-amylase from Aspergillus awamori KT-11

Complementary DNAs encoding alpha-amylases (Amyl I, Amyl III) and glucoamylase (GA I) were cloned from Aspergillus awamori KT-11 and their nucleotide sequences were determined. The sequence of Amyl III that was a raw starch digesting alpha-amylase was found to consist of a 1,902 bp open reading frame encoding 634 amino acids. The signal peptide of the enzyme was composed of 21 amino acids. On the other hand, the sequence of Amyl I, which cannot act on raw starch, consisted of a 1,500 bp ORF encoding 499 amino acids. The signal peptide of the enzyme was composed of 21 amino acids. The sequence of GA I consisted of a 1,920 bp ORF that encoded 639 amino acids. The signal peptide was composed of 24 amino acids. The amino acid sequence of Amyl III from the N-terminus to the amino acid number 499 showed 63.3% homology with Amyl I. However, the amino acid sequence from the amino acid number 501 to C-terminus, including the raw-starch-affinity site and the TS region rich in threonine and serine, showed 66.9% homology with GA I.     

Effect of toll-like receptor activation on thymosin beta-4 production by chicken macrophages

Thymosin beta-4 (Tβ4) is an actin-binding intracellular peptide that promotes wound healing, tissue remodeling, and angiogenesis. The mechanism of Tβ4 secretion to the extracellular environment is not understood. The macrophage is a rich source of Tβ4 which also participates in wound healing process. The objective of this study was to find how Tβ4 may be externalized. Using activation of macrophage through their toll-like receptors (TLR), the changes in cellular Tβ4 was studied. A naturally transformed chicken macrophage cell line HTC was treated with different TLR agonists and the cellular Tβ4 changes was determined at 6 and 24 h after stimulations using stable isotope labelling of amino acids in cell culture (SILAC) and mass spectrometry. Real time PCR was used to determine changes in gene expression. The results showed that TLR agonists such as peptidoglycan (PGN) or lipopolysacharide (LPS) caused depletions in cellular Tβ4 peptide along with its detection in the cell culture supernatant at 24 h. These TLR agonists also induced the expression of interleukins-1β, -6, and nitric oxide synthase genes at 6 h but failed to modulate Tβ4 gene at that time point indicating that the Tβ4 externalization was not associated with its production. To find whether Tβ4 externalization was associated with cell death, we measured the lactate dehydrogenase (LDH) activity of the conditioned media as an indicator of cell damage. The results showed that the TLR agonists which induced depletion of intracellular Tβ4 at 24 h also increased the LDH content of the conditioned media, suggesting that the Tβ4 in the extracellular media most likely originated from dying macrophages.     

Polymorphism of cytokine and innate immunity genes associated with bovine brucellosis in cattle

Genetic susceptibility to brucellosis is multifactorial, and it is known that impairment of the immune system could contribute to risk for getting brucellosis. The aim of the study was to find association of bovine brucellosis with 20 SNPs pertaining to bovine cytokine (IFNG, IFNGR1, IFNGR2, TNFA) and innate immunity (SLC11A1, TLR1, TLR4, and TLR9) genes using PCR-RFLP genotyping technique and it was observed that SLC11A1 (+1066 C/G), TLR1 (+1446 C/A), TLR1 (+1380 G/A), TLR4 (+10 C/T) and TLR4 (+399 C/T) loci were significantly (P ≤ 0.05) associated with bovine brucellosis. The odds ratios (OR) of CG and CC genotypes versus GG genotype were 0.31 (0.12–0.82; 95 % CI) and 0.18 (0.03–1.06; 95 % CI) at SLC11A1 (+1066 C/G) locus in cases of brucellosis affected cattle. For TLR1 (+1380 G/A) locus, the OR for AG and AA genotypes versus GG genotypes were 0.15 (0.05–0.44; 95 % CI) and 0.26 (0.04–1.47; 95 % CI) which indicated that proportion of GG homozygote was significantly higher in brucellosis affected animals as compared to control. At TLR1 (+1446 C/A) locus the OR of AC genotype versus CC genotype was 0.24 (0.08–0.68; 95 % CI) which revealed that relative proportion CC genotypes was significantly higher in case population. The TLR4 (+10 C/T) locus had three genotypes (TT, CT and CC) where OR of CT and CC genotypes versus TT genotype were near to zero. The OR of CT genotypes versus CC genotypes was 8.25 (0.94–71.92; 95 % CI) at TLR4 (+399 C/T) locus and indicated that CT genotype had higher odds of bovine brucellosis than control animals.     

Polymorphism and Haplotype Structure in River Buffalo (Bubalus bubalis) Toll-Like Receptor 5 (TLR5) Coding Sequence

Most of the 160 million river buffalo in the world are in Asia where they are used extensively, both as a food source and for draught power. Only recently have investigations begun exploring the buffalo genome for variation that might influence health and productivity of these economically important animals. This paper describes the sequence variability of the toll-like receptor 5 (TLR5) gene, which recognizes bacterial flagellin and is a key player in the immune system. TLR5 is comprised of a single exon that is 2577 bp and codes 858 amino acids. We examined single-nucleotide polymorphisms (SNPs) located within the coding region. Overall, 17 SNPs were discovered, seven of which are non-synonymous. Our study population yielded four different haplotypes. We examined predicted protein domain structure and found that river buffalo, swamp buffalo, and African Forest buffalo shared the same protein domain structure and are more similar to each other than they are to cattle and American bison, which are similar to each other. PolyPhen 2 analysis revealed one amino acid substitution in the river buffalo population with potential functional significance.     

Effect of thermal stress on HSP70 expression in dermal fibroblast of zebu (Tharparkar) and crossbred (Karan-Fries) cattle

The present studies were conducted to investigate the difference response of dermal fibroblasts to heat stress in Tharparkar and Karan-Fries cattle. Skin is the most important environmental interface providing a protective envelope to animals. In skin, dermal fibroblasts are the most regular cell constituent of dermis that is crucial for temperature homeostasis. The study aimed to examine the reactive oxygen species (ROS) formation, cytotoxicity (%) and heat shock protein 70 (HSP70) genes expression in dermal fibroblast of Tharparkar and Karan-Fries cattle and to assess whether resistance of dermal fibroblast to heat stress is breed specific. Dermal fibroblasts from ear pinna of Tharparkar and Karan-Fries cattle were exposed at 25 °C, 37 °C, 40 °C and 44 °C for 3 h to measure the ROS, cytotoxicity (%) and HSP 70 (HSPA1A, HSPA2 and HSPA8) genes’ expression. The results showed that ROS formation at low temperature (25 °C) decreased in both breeds as compared to control (37 °C) and the differences were significant (P<0.0001). Heat stress at 40 °C did not increase ROS formation significantly in Tharparkar but increased significantly (P<0.001) in Karan-Fries cattle. The overall cytotoxicity (%) was also found to be significantly different (P<0.001) between Tharparkar and Karan-Fries cattle, and on exposure to different temperatures (P<0.001). The cytotoxicity (%) in dermal fibroblast cells of Karan-fries cows was more than Tharparkar. The expression studies indicated that all HSP70 genes (HSPA8, HSPA1A and HSPA2) were up-regulated at different temperatures in both breeds. In Tharparkar, the relative mRNA expression of HSPA8 gene was higher but HSPA1A and HSPA2 genes were low as compared to Karan-Fries cattle. At 40 and 44 °C, the relative expressions of inducible HSP 70 genes (HSPA1A and HSPA2) were higher in Karan-Fries than Tharparkar. In summary, dermal fibroblast resistance to heat shock differed between breeds. Dermal fibroblasts of Tharparkar were observed to be more heat tolerant than crossbred Karan-Fries cattle. The study concludes that zebu cattle (Tharparkar) dermal fibroblasts are more adapted to tropical climatic condition than crossbreed cattle (Karan-Fries). Differences exist in dermal fibroblasts of heat adapted and non-adapted cattle.     

Evolution of the bovine TLR gene family and member associations with Mycobacterium avium subspecies paratuberculosis infection

Members of the Toll-like receptor (TLR) gene family occupy key roles in the mammalian innate immune system by functioning as sentries for the detection of invading pathogens, thereafter provoking host innate immune responses. We utilized a custom next-generation sequencing approach and allele-specific genotyping assays to detect and validate 280 biallelic variants across all 10 bovine TLR genes, including 71 nonsynonymous single nucleotide polymorphisms (SNPs) and one putative nonsense SNP. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and specialized beef and dairy breeds could not be differentiated despite an average polymorphism density of 1 marker/158 bp. Collectively, 160 tagSNPs and two tag insertion-deletion mutations (indels) were sufficient to predict 100% of the variation at 280 variable sites for both Bos subspecies and their hybrids, whereas 118 tagSNPs and 1 tagIndel predictively captured 100% of the variation at 235 variable sites for B. t. taurus. Polyphen and SIFT analyses of amino acid (AA) replacements encoded by bovine TLR SNPs indicated that up to 32% of the AA substitutions were expected to impact protein function. Classical and newly developed tests of diversity provide strong support for balancing selection operating on TLR3 and TLR8, and purifying selection acting on TLR10. An investigation of the persistence and continuity of linkage disequilibrium (r2≥0.50) between adjacent variable sites also supported the presence of selection acting on TLR3 and TLR8. A case-control study employing validated variants from bovine TLR genes recognizing bacterial ligands revealed six SNPs potentially eliciting small effects on susceptibility to Mycobacterium avium spp paratuberculosis infection in dairy cattle. The results of this study will broadly impact domestic cattle research by providing the necessary foundation to explore several avenues of bovine translational genomics, and the potential for marker-assisted vaccination.     

Molecular cloning of the E1 beta subunit of the rat branched chain alpha-ketoacid dehydrogenase.

We determined the cDNA sequence of the mRNA for antithrombin III (AT III) from sheep liver. It encodes a protein of 465 amino acids, including a signal peptide of 32 amino acids. The amino acid sequence of the mature protein shows a sequence identity of 89.1%, 95.6% and 85.0% to the human, bovine and rabbit equivalents, respectively. Cysteine residues involved in disulfide bonds as well as potential glycosylation sites are conserved between the four species. In contrast, the amino acid sequence of the signal peptide shows a smaller identity, i.e., 68.7% and 56.3% compared to the human and rabbit preprotein, respectively.