Candidate SNP of CACNA2D1 Gene Associated with Clinical Mastitis and Production Traits in Sahiwal (Bos taurus indicus) and Karan Fries (Bos taurus taurus × Bos taurus indicus)

The present study was conducted to identify polymorphisms in CACNA2D1 gene and their association with clinical mastitis and production traits. Exon 18 and its flanking regions were screened for the presence of SNPs. Statistical analysis was performed to identify association of period of birth, breed, and genotype with mastitis incidence on randomly selected 103 Sahiwal and 102 Karan Fries cattle. PCR-RFLP analysis revealed that g.38819398G?>?A mutation in exon 18 (269?bp amplicon) of CACNA2D1 gene resolved into AA, AG, and GG genotypes in Sahiwal and Karan Fries cattle. Wald chi-square analysis revealed that the period of birth, breed, and genotype were significantly associated with mastitis incidence. GG genotyped cattle were found to be less susceptible to mastitis. Least square analysis revealed that GG and AG genotype animals of G38819398A SNP of CACNA2D1 gene in Sahiwal as well as in Karan Fries cattle were associated with higher average milk yields during 1st, 2nd, and 3rd lactations (P?<?0.01). These observations and their differential association with the incidence of mastitis and production traits can be utilized as an aid to selection for simultaneous improvement of both antagonistic traits; however, validation of results on large number of animals is warranted.     

Stent implantation through a self-expanding stent

Jailing of a side-branch is a known complication of stent implantation, and makes access to the side-branch difficult, especially if the stent is of the self-expanding type. Although plain balloon angioplasty is feasible for the jailed side-branches, the use of newer devices (a stent, Rotablation or atherectomy) has not been described. We describe a novel way of treating a side-branch jailed by a self-expanding stent by using stent implantation through the strut of a self-expanding stent.     

EDTA chelation therapy for cardiovascular disease: a systematic review

Background

Experimental studies support an important role for endothelial nitric oxide synthase (eNOS) in the regulation of angiogenesis. In humans, a common polymorphism exists in the eNOS gene that results in the conversion of glutamate to aspartate for codon 298. In vitro and in vivo studies have suggested a decreased NOS activity in patients with the Asp²⁹⁸ variant. We hypothesized that a genetic-mediated decreased eNOS activity may limit collateral development in patients with chronic coronary occlusions.

Methods

We selected 291 consecutive patients who underwent coronary angiography and who had at least one chronic (>15 days) total coronary occlusion. Collateral development was graded angiographically using two different methods: the collateral flow grade and the recipient filling grade. Genomic DNA was extracted from white blood cells and genotyping was performed using previously published techniques.

Results

Collateral development was lower in patients carrying the Asp²⁹⁸ variant than in Glu-Glu homozygotes (collateral flow grade: 2.64 ± 0.08 and 2.89 ± 0.08, respectively, p = 0.04; recipient filling grade: 3.00 ± 0.08 and 3.24 ± 0.07, respectively, p = 0.04). By multivariable analysis, three variables were independently associated with the collateral flow grade: female gender, smoking, and the Asp²⁹⁸ variant (p = 0.03) while the Asp²⁹⁸ variant was the sole variable independently associated with the recipient filling grade (p = 0.03).

Conclusion

Collateral development is lower in patients with the Asp²⁹⁸ variant. This may be explained by the decreased NOS activity in patients with the Asp²⁹⁸ variant. Further studies will have to determine whether increasing eNOS activity in humans is associated with coronary collateral development.     

Dose-dependent reduction of apoptosis in nutrient-limited cultures of NS/0 myeloma cells transfected with the E1B-19K adenoviral gene.

It is now well documented that apoptosis represents the prevalent mode of death in lymphoid cultures and occurs spontaneously in late-exponential phase of batch cultures following nutrient exhaustion. In an attempt to enhance the cell survival of these cell lines, we have initially engineered nonproducing NS/0 myeloma cells with a vector expressing the adenoviral E1B-19K protein. NS/0 cells transfected with E1B-19K were found to be more resistant to apoptosis occurring in the late phase of batch culture and under stressful conditions such as cultivation in glutamine-free medium or following heat shock. In this study, we have characterised a number of NS/0 subclones constitutively expressing different levels of E1B-19K, as well as several subclones in which the expression of E1B-19K was regulated by a tetracycline-controllable gene switch. We have found that a threshold E1B-19K level was required in order to achieve protection against apoptosis. The extent of resistance against cell death induced by nutrient deprivation in glutamine-free medium and in the late phase of batch cultures correlated with the level of E1B-19K expression up to an optimal level where further increases in E1B-19K levels did not result in significant additional protection. To assess the effects of E1B-19K on antibody productivity, an apoptosis-resistant NS/0 clone was then transfected with a chimeric antibody construct. Despite their improved viability, the antibody productivity of E1B-19K clones in batch culture was not significantly improved. Moreover, while the use of E1B-19K considerably delayed cell death, cells eventually died by apoptosis. Surprisingly, E1B-19K had no beneficial effect on the efficiency of fusion of NS/0 myelomas and splenocytes for the generation of hybridoma cells. Furthermore, the resulting hybridomas, although expressing E1B-19K at levels comparable to the myeloma parent, were no longer resistant to apoptosis. This indicates that the ability of E1B-19K to prevent apoptosis is not only dose-dependent but also seems to be cell-type dependent.     

Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines

Background  

The use of lactic acid bacteria as vehicles to delivery antigens to immunize animals is a promising issue. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization.     

Affinity purification of active subunit 1 of herpes simplex virus type 1 ribonucleotide reductase exhibiting a protein kinase activity

Herpes simplex virus (HSV) ribonucleotide reductase is formed by the association of two distinct dimeric subunits, R1 and R2. Attempts to purify either the HSV holoenzyme or its R1 subunit in their active form have been unsuccessful until now. The C terminus of the R2 protein being involved in the association with R1, the synthetic nonapeptide corresponding to this terminus, impedes the formation of the holoenzyme by competing with R2 for a critical site on R1. Based upon these observations, we developed an affinity chromatographic procedure to purify the R1 protein from HSV-1-infected baby hamster kidney cells. Specific binding of R1 to an affinity column made by linking the peptide HSV R2-(326-337) to Affi-Gel 10, followed by specific elution with an excess of an analogous peptide exhibiting a higher affinity for R1 yielded, in a single step, highly purified R1 protein. The purified R1 preparations contained approximately 95% of intact R1, the remaining 5% consisting of two R1 copurifying proteolytic breakdown products. The purified R1 protein exhibited a high reductase specific activity when mixed with an excess of the R2 subunit. Moreover, in vitro kinase assays revealed that the purified R1 protein of HSV-1 possesses an autophosphorylating activity also able to phosphorylate alpha-casein and histone II-S. The intrinsic protein kinase activity of HSV R1 is associated with its unique N-terminal domain which is absent from all other reductase subunits 1 and contains consensus motifs found in Ser/Thr protein kinases. A preliminary characterization of the kinase activity of the R1 protein of HSV-1 ribonucleotide reductase is presented.     

Presence of a high-molecular-weight form of catalse in enzyme purified from mouse liver.

Ultracentrifugation studies of purified mouse hepatic catalase revealed that 5-7% of the total material consists of a form with a higher molecular weight than the bulk of the catalase. The two components were separated by sucrose-gradient centrifugation. Polyacrylamide-gel electrophoresis (in borate buffer) demonstrated that high-molecular-weight catalase is enriched in a more slowly migrating component, and sodium dodecyl sulphate/polyacrylamide gel-electrophoresis demonstrated that the molecular weight of the subunits of the high-molecular-weight material is identical with that of the subunits of the major form. These results suggest that high-molecular-weight catalase consists of subunits that are not markedly distinct from those present in the normal catalase tetramer.     

MAP2 IHC detection: a marker of antigenicity in CNS tissues

Immunohistochemistry (IHC) is used to detect antibody-specific antigens in tissues; the results depend on the ability of the primary antibodies to bind to their antigens. Therefore, results depend on the quality of preservation of the specimen. Many investigators have overcome the deleterious effects of over-fixation on the binding of primary antibodies to specimen antigens using IHC, but if the specimen is under-fixed or fixation is delayed, false negative results could be obtained despite certified laboratory practices. Microtubule-associated protein 2 (MAP2) is an abundant microtubule-associate protein that participates in the outgrowth of neuronal processes and synaptic plasticity; it is localized primarily in cell bodies and dendrites of neurons. MAP2 immunolabeling has been reported to be absent in areas of the entorhinal cortex and hippocampus of Alzheimer’s disease brains that were co-localized with the dense-core type of amyloid plaques. It was hypothesized that the lack of MAP2 immunolabeling in these structures was due to the degradation of the MAP2 antigen by the neuronal proteases that were released as the neurons lysed leading to the formation of these plaques. Because MAP2 is sensitive to proteolysis, we hypothesized that changes in MAP2 immunolabeling may be correlated with the degree of fixation of central nervous system (CNS) tissues. We detected normal MAP2 immunolabeling in fixed rat brain tissues, but MAP2 immunolabeling was decreased or lost in unfixed and delayed-fixed rat brain tissues. By contrast, two ubiquitous CNS-specific markers, myelin basic protein and glial fibrillary acidic protein, were unaffected by the degree of fixation in the same tissues. Our observations suggest that preservation of various CNS-specific antigens differs with the degree of fixation and that the lack of MAP2 immunolabeling in the rat brain may indicate inadequate tissue fixation. We recommend applying MAP2 IHC for all CNS tissues as a pre-screen to assess the quality of the tissue preservation and to avoid potentially false negative IHC results.