Genetic diversity and population genetic analysis of bovine MHC class II DRB3.2 locus in three Bos indicus cattle breeds of Southern India.

The present study was performed to evaluate the genetic polymorphism of BoLA-DRB3.2 locus in Malnad Gidda, Hallikar and Ongole South Indian Bos indicus cattle breeds, employing the PCR-RFLP technique. In Malnad Gidda population, 37 BoLA-DRB3.2 alleles were detected, including one novel allele DRB3*2503 (GenBank: HM031389) that was observed in the frequency of 1.87%. In Hallikar and Ongole populations, 29 and 21 BoLA-DRB3.2 alleles were identified, respectively. The frequencies of the most common BoLA-DRB3.2 alleles (with allele frequency > 5%), in Malnad Gidda population, were DRB3.2*15 (10.30%), DRB3*5702 (9.35%), DRB3.2*16 (8.41%), DRB3.2*23 (7.01%) and DRB3.2*09 (5.61%). In Hallikar population, the most common alleles were DRB3.2*11 (13.00%), DRB3.2*44 (11.60%), DRB3.2*31 (10.30%), DRB3.2*28 (5.48%) and DRB3.2*51 (5.48%). The most common alleles in Ongole population were DRB3.2*15 (22.50%), DRB3.2*06 (20.00%), DRB3.2*13 (13.30%), DRB3.2*12 (9.17%) and DRB3.2*23 (7.50%). A high degree of heterozygosity observed in Malnad Gidda (H(O) = 0.934, H(E) = 0.955), Hallikar (H(O) = 0.931, H(E) = 0.943) and Ongole (H(O) = 0.800, H(E) = 0.878) populations, along with F(IS) values close to F(IS) zero (Malnad Gidda: F(IS) = 0.0221, Hallikar: F(IS) = 0.0127 and Ongole: F(IS) = 0.0903), yielded nonsignificant P-values with respect to Hardy-Weinberg equilibrium probabilities revealing, no perceptible inbreeding, greater genetic diversity and characteristic population structure being preserved in the three studied cattle populations. The phylogenetic tree constructed based on the frequencies of BoLA-DRB3.2 alleles observed in 10 Bos indicus and Bos taurus cattle breeds revealed distinct clustering of specific Bos indicus cattle breeds, along with unique genetic differentiation observed among them. The results of this study demonstrated that the BoLA-DRB3.2 is a highly polymorphic locus, with significant breed-specific genetic diversities being present amongst the three studied cattle breeds. The population genetics and phylogenetic analysis have revealed pivotal information about the population structure and importance of the presently studied three Bos indicus cattle breeds as unique animal genetic resources, which have to be conserved for maintaining native cattle genetic diversity.

Objective sperm motion analysis to assess dairy bull fertility using computer-aided system--a review.

Motility is one of the most important characteristics associated with the fertilizing ability of spermatozoa and is an expression of their viability and structural integrity. Computer-assisted semen analyser (CASA) provides precise and accurate information on different sperm motion characteristics. This article reviews various aspects of computer-aided motility analysis of bull sperm like sample preparation, standardization of instrument settings, importance of various motility parameters evaluated by the system and its impact on basic functional studies of spermatozoa. It gives special emphasis to various aspects of bull sperm motion analysis especially sub-populations of spermatozoa, hyper-activation, motion characteristic in different genetic and age groups, etc. and their utility in predicting the fertility of dairy bulls. The need to fill the gap in research and the necessity of universal standardization of the equipment has been discussed.

Two mouse retinal degenerations caused by missense mutations in the beta-subunit of rod cGMP phosphodiesterase gene.

We report the chromosomal localization, mutant gene identification, ophthalmic appearance, histology, and functional analysis of two new hereditary mouse models of retinal degeneration not having the Pde6brd1("r", "rd", or "rodless") mutation. One strain harbors an autosomal recessive mutation that maps to mouse chromosome 5. Sequence analysis showed that the retinal degeneration is caused by a missense point mutation in exon 13 of the beta-subunit of the rod cGMP phosphodiesterase (beta-PDE) gene (Pde6b). The gene symbol for this strain was set as Pde6brd10, abbreviated rd10 hereafter. Mice homozygous for the rd10 mutation showed histological changes at postnatal day 16 (P16) of age and sclerotic retinal vessels at four weeks of age, consistent with retinal degeneration. Retinal sections were highly positive for TUNEL and activated caspase-3 immunoreactivity, specifically in the outer nuclear layer (ONL). ERGs were never normal, but rod and cone ERG a- and b-waves were easily measured at P18 and steadily declined over 90% by two months of age. Protein extracts from rd10 retinas were positive for beta-PDE immunoreactivity starting at about the same time as wild-type (P10), though signal averaged less than 40% of wild-type. Interestingly, rearing rd10 mice in total darkness delayed degeneration for at least a week, after which morphological and functional loss progressed irregularly. With the second strain, a complementation test with rd1 mice revealed that the retinal degeneration phenotype observed represents a possible new allele of Pde6b. Sequencing demonstrated a missense point mutation in exon 16 of the beta-subunit of rod phosphodiesterase gene, different from the point mutations in rd1 and rd10. The gene symbol for this strain was set as Pde6bnmf137, abbreviated nmf137 hereafter. Mice homozygous for this mutation showed retinal degeneration with a mottled retina and white retinal vessels at three weeks of age. The exon 13 missense mutation (rd10) is the first known occurrence of a second mutant allele spontaneously arising in the Pde6b gene in mice and may provide a model for studying the pathogenesis of autosomal recessive retinitis pigmentosa (arRP) in humans. It may also provide a better model for experimental pharmaceutical-based therapy for RP because of its later onset and milder retinal degeneration than rd1 and nmf137.

Extra-hepatic expression of serum albumin mRNA in mouse retina.

PURPOSE: In some mammals, serum albumin protein exists in the interphotoreceptor space (IPS), the space between photoreceptor cells and the retinal pigment epithelium. Serum albumin is synthesized largely in the liver, though low levels of extra-hepatic expression have been documented in several other tissues, including fetal rat kidney, pancreas, lung, and heart. The purpose of this study was to investigate whether serum albumin protein and mRNA are found in mouse retina. METHODS: Using albumin rabbit antibodies and HRP goat anti-(rabbit IgG), we performed immunoassays on mouse IPS washes to detect the presence of serum albumin protein. Protein extracts from IPS washes were subjected to Affigel Blue chromatography. This resin has an affinity for serum albumin. Reverse transcription-polymerase chain reaction (RT-PCR) of retina total RNA was performed to search for albumin mRNA. Also, real-time reverse transcription polymerase chain reaction (RT-RT-PCR) was employed to look at the levels of expression in different age groups. RESULTS: A constituent of the IPS washes specifically bound and eluted from Affigel Blue column, suggesting that the washes contained serum albumin. SDS PAGE revealed that the size of the constituent was 67 kDa, the size of serum albumin. This 67 kDa band reacted with mouse serum antibody. An RT-PCR amplified fragment of serum albumin mRNA from retina displayed the expected size. The sequence of this fragment is identical to authentic serum albumin cDNA sequence. RPE and choroid were negative for serum albumin mRNA. However, rd1(-)/rd1(-) retina was positive, suggesting that at least some serum albumin is synthesized in the inner layers of the retina. RT-RT-PCR showed that serum albumin mRNA levels in whole retina reached a maximum at about postnatal day 15 and gradually decreased to about one-sixth of maximum at 12 months age. CONCLUSIONS: Serum albumin protein and mRNA were found in mouse IPS and retina, suggesting that the protein is synthesized in the retina. The previously demonstrated ability of serum albumin to bind fatty acids and retinoids and its presence in the mouse IPS suggest a role for serum albumin in transporting retinoids in the retina or IPS, especially at young ages when concentrations appear greatest.

Photoaffinity labeling of human IRBP with all-trans-retinoic acid.

Interphotoreceptor retinoid-binding protein (IRBP), found only in photosensitive tissues, is a large approximately 135-kDa glycoprotein that contains a fourfold repeat structure. IRBP may function as a buffer and prevent retinoid toxicity and retinoid degeneration. Here we asked (i) whether each repeat of IRBP possesses the capability of photo-crosslinking all-trans-retinoic acid (RA), (ii) within Repeat 1 whether a single retinoic acid-binding domain exists, and (iii) whether protease and CNBr digestion of Repeat 1 bound RA indicate the exact location of the binding site. 3H-RA cross-linked to all four repeats, consistent with the current model of multiple binding sites in IRBP. Acetone precipitation was effective in removing unbound 3H-RA. LysC and tryptic digestion of the RA-Repeat 1 detected 18- and 5-kDa bands, respectively. CNBr digestion showed two bands about 9 and 11 kDa in size. Our data suggests a single binding site near positions 151-160 in the center of Repeat 1.

Convergence of redox-sensitive and mitogen-activated protein kinase signaling pathways in tumor necrosis factor-alpha-mediated monocyte chemoattractant protein-1 induction in vascular smooth muscle cells.

Monocyte chemoattractant protein-1 (MCP-1) is an important component of the inflammatory response of the vessel wall and has been shown to be regulated by cytokines, such as tumor necrosis factor-alpha (TNF-alpha). However, the precise signaling pathways leading to MCP-1 induction have not been fully elucidated in vascular smooth muscle cells (VSMCs). Cytokine signal transduction involves protein kinases as well as reactive oxygen species (ROS). The relation between these 2 factors is not clear. In this study, we show that TNF-alpha induces a parallel phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38MAPK) and increases MCP-1 mRNA expression in cultured VSMCs. Inhibition of ERK1/2 but not p38MAPK caused a partial attenuation of MCP-1 induction (43+/-10% inhibition). Incubation of VSMCs with multiple antioxidants (diphenylene iodonium, liposomal superoxide dismutase, catalase, N-acetylcysteine, dimethylthiourea, and pyrrolidine dithiocarbamate) had no effect on TNF-alpha-mediated MCP-1 upregulation. However, simultaneous blockade of the ERK1/2 and ROS pathways by using PD098059 combined with diphenylene iodonium or N-acetylcysteine potently enhanced the ability of MAPK kinase inhibitors to abrogate MCP-1 mRNA expression (100+/-2% inhibition). Thus, parallel ROS-dependent and ERK1/2-dependent pathways converge to regulate TNF-alpha-induced MCP-1 gene expression in VSMCs. These data unmask a complex but organized integration of ROS and protein kinases that mediates cytokine-induced vascular inflammatory gene expression.

Characterization of human B cell proteins binding specifically to uveitopathogenic peptide 1169-1191 of bovine IRBP.

Peptide 1169-1191 is a major uveitopathogenic determinant of bovine Interphotoreceptor Retinoid Binding Protein (IRBP) in Lewis rats. Previously, we identified two proteins with approximate molecular masses of 72 and 74 kDa and one with a molecular mass of 40 kDa from B cells of naive Lewis rats and EBV-transformed B cells from a human patient with ocular Behçet's disease that bind to bovine IRBP peptide 1169-1191. In this study, we have partially characterized these proteins. The two proteins with molecular masses 72 and 74 kDa belong to the HSP 70 family of proteins and the 40-kDa protein is actin.

Identification of heat shock proteins binding to an immunodominant uveitopathogenic peptide of IRBP.

Intracellular binding proteins have been identified and isolated from B cells by their ability to bind to the synthetic peptide (1169-1191), the major immunodominant epitope of bovine interphotoreceptor retinoid-binding protein (IRBP) coupled to cyanogen bromide activated Sepharose 4B. After SDS-PAGE, two discrete protein bands of approximately 72 and 74 kDa, were found to be present in B cells of naive Lewis rats as well as in EBV transformed B cells from a human patient with ocular Behçet's disease. Enhanced expression of these peptide-binding proteins was achieved by incubating the cells with Lipopolysaccharide (LPS) from S. typhimurium. The approximately 72 and 74 kDa peptide-binding proteins reacted in western blot with monoclonal antibodies specific for both constitutively expressed and inducible 72/74 kDa hsp 70 proteins. The demonstration that these proteins bind to the immunodominant epitope of IRBP indicates that they may play a role in the processing and presentation of antigens by antigen-presenting cell (APC).

Biochemical analysis of serum proteins from Eales' patients.

In the present study attempt has been made to identify the possible factor(s) which are responsible for Eales' disease. The serum of Eales' patients and that of age and sex matched healthy controls did not differ in their total protein concentration. Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis did not reveal any difference between the two groups. However, analysis of the serum samples with isoelectric focusing showed the presence of two unique proteins with pI of 5.5 and 5.9 in Eales' patients. Further two dimensional SDS-PAGE analysis indicated the presence of a distinct protein spot with a pI of 5.9 and a molecular weight around 23 KD in the serum of Eales' patients. This 23 KD protein has been partially purified and found to be anionic in nature. Antisera to this partially purified protein have been raised and tested. The implication of this finding is discussed in relation to the aetiology of Eales' disease.

Immunological status of patients of Eales' disease.

To test the hypothesis that altered immune reactivity to an extraneous agent might lead to primary retinal perivasculitis, a study was undertaken to determine the serum immunoglobulin levels, T lymphocyte subsets, antibody responses to BCG and 'S' antigen, and lymphoproliferative response to mitogens. No difference was observed in these parameters between patients and controls. Both Mantoux positive and negative conditions existed in patients with Eales' disease. Mantoux positive patients showed a higher level of lymphoproliferative response in vitro to PPD than Mantoux positive controls, indicating the presence of two populations among Eales' patients.