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Identification of SNPs in Beta 2 Microglobulin (β2M) Gene and their Association with IgG Concentration in Neonatal Buffalo Calves

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... The amplicons of each fragment for all samples were analyzed by SSCP (single strand conformational polymorphism) for identification of allelic variants. Standard procedure for SSCP [8,10,30] was employed. On the basis of their band patterns the fragments were identified as polymorphic or monomorphic. ...
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Background The neonatal Fc receptors (FcRn) mediate the transfer of immunoglobulin G (IgG) molecules from a dam’s circulation to the colostrum produced by it immediately after parturition. In ruminants, the calves born are agammaglobulinemic therefore, ingestion of colostrum with high concentration of IgG imparts passive immunity to the newborn. The FcRn molecule is a heterodimer, coded by FCGRT (Fc fragment of IgG Receptor Transporter neonatal) and B2M (Beta 2 microglobulin) genes. Present study attempted to identify single nucleotide polymorphisms (SNPs) in the FCGRT gene in 40 buffaloes of Murrah breed and evaluated the association of these nucleotide variations and haplotypes with IgG concentration in their colostrum. Methods and results Animals producing colostrum with high IgG and low IgG levels were identified by indirect ELISA and selected. SNPs were detected in the FCGRT gene sequence of selected animals by amplifying it in nine fragments covering all exons (with flanking introns) followed by PCR-single strand conformational polymorphism (PCR-SSCP). A total of nine SNPs were observed of which seven were present in flanking introns and two in exon 4 of the gene. The SNP A75G was non-synonymous and produced an amino acid change from isoleucine to valine. The exonic SNPs and corresponding haplotypes were found to be significantly (P < 0.01 and 0.05 respectively) associated with colostral IgG concentration based on Odds ratios at 95% confidence interval. Conclusion Polymorphism in FCGRT gene is found to be associated with IgG concentration in colostrum and identification of females with desirable variations may prevent failure of passive transfer in neonatal ruminants.
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This study aimed to evaluate passive immunity transfer in healthy buffalo calves. Colostrum samples from heifers (without previous calving) and primiparous and pluriparous dams and blood samples from their offspring were obtained at calving, before colostrum intake, and at 24, 48, and 72 h after calving for determination of serum activities of gammaglutamyltransferase and alkaline phosphatase and serum concentrations of total protein (TP), immunoglobulin A (IgA) and IgG, and lactoferrin. The results were analyzed as repeated measures, and differences were considered statistically significant at P ≤ 0.05. Considering that the buffalo calves were born hypogammaglobulinemic (4.23 ± 0.33 mg/ml) and, at 24 h, the mean serum concentration of IgG was 34.5 ± 1.48 mg/ml, passive immunity transfer was successful. Moreover, colostrum IgG concentrations at 0 h were correlated with serum IgG concentrations at 24 h in buffalo calves. Additionally, TP concentrations were highly correlated with IgG in both colostrum at calving and blood in calves at 24 h. TP is recommended as a reliable indirect parameter to evaluate both colostrum quality and passive immunity transfer in buffalo calves.
Article
The present study was taken up to find out Beta 2 Microglobulin (b2M) gene variants and their association with IgG concentrations in colostrum of Murrah buffaloes. 40 newly parturated Murrah buffaloes dams were included in the present study. Polymerase chain reaction- single stranded conformational polymorphism (PCR-SSCP) technique was used to explore the polymorphism in b2M gene of buffaloes. The PCR-SSCP analysis and sequencing of different patterns obtained by silver staining of fragments pertaining to exon 1 through 4 and partial introns 1 through 3, revealed 9 SNPs which were further used to prepare the haplotypes. 7 different haplotypes were observed. Indirect ELISA was done to estimate IgG concentrations in colostrum (collected 1 hour before first milking) obtained from buffaloes. IgG levels in colostrum ranged from 11.22 to 185.1 mg/ml with a mean IgG concentration of 51.71 ± 5.99 mg/ml. The effect of different haplotypes was evaluated on IgG concentrations. The least square analysis of variance could not exhibit a significant effect of buffalo dam haplotype on colostral IgG concentration.
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Channel activities, particularly those of calcium channels, have vital roles in the process of sperm maturation, motility and sperm-egg interaction. A group of the recently discovered ion channels associated with these processes is four novel channel-like proteins known as CatSper (cation channel sperm) gene family. CatSper1 and CatSper2 show sperm specific expression patterns. However, neither CatSper1 nor CatSper2 have been shown to function as cation channels when transfected into cells, singly or together. It has been suggested that the known CatSper proteins require additional subunits and/or interaction partners to function. Using in silico gene identification and prediction techniques, two further members of the CatSper family, CatSper3 and CatSper4, have been identified. It has been hypothesized that the CatSper proteins may form a functional tetrameric channel. Based on these findings, we evaluated the temporal pattern of CatSper3 and CatSper4 genes during mouse testis development to assess any potential co-incidence of their expression with CatSper1 and CatSper2. In the present study, a total of 55 male BALB/c mice were categorized into 11 age groups: 1 day, 1 week, 2 weeks, 16, 18, 20, 22, 24 and 26 days, 1 month as well as adult mice. Testes were removed from each mouse, total RNA was extracted and RT-PCR was performed with specific primers for CatSper3, CatSper4 and beta-2-microglobin (as an internal control). Our results revealed that CatSper3 and CatSper4 genes are expressed predominantly at 3 week of age. The obtained data demonstrate that: 1) The expression of CatSper3 and CatSper4 in mouse is developmentally regulated. 2) There is a direct correlation between the temporary expressions of these genes and the other two members of the family, CatSper1 and CatSper2. Iran. Biomed. J. 10 (2): 111-115, 2006
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The search for Ca2+ channels residing in sperm has led to the recent cloning and characterization of a novel gene, named CatSper, which codes for a unique Ca2+ channel expressed exclusively in the testis. It plays an essential role in sperm motility, penetration into the oocyte, and ultimately in male fertility. In this study, we assessed the temporal profile of CatSper gene expression during mouse testis development and performed a semi-quantitative evaluation of expression levels in a group of subfertile men which lack sperm motility. A small piece of testicular tissue obtained by either multi-site testicular biopsy or orchidectomy was used for semi-quantitative RT-PCR of CatSper and beta2-microglobulin (beta2m, as an internal control) genes. Our results reveal that: (i) the expression of mouse CatSper is developmentally regulated with a direct correlation between CatSper expression and mouse sexual maturation. CatSper gene expression is first detected at 3 weeks of age and coincides with the appearance of round spermatids in the developing mouse testis. (ii) There is a significant reduction in the level of CatSper gene expression (up to 3.5-fold difference) among patients which lack sperm motility as compared with patients whose infertility cannot be ascribed to a deficiency in motility (used as a control). The data obtained in this study support a potential role for CatSper in sperm motility and fertility in mouse and human. CatSper is therefore implicated as a potential target to explore the molecular mechanisms of male infertility.
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The present study was taken up to find out Beta 2 Microglobulin (b2M) gene variants and their association with IgG concentrations in colostrum of Murrah buffaloes. 40 newly parturated Murrah buffaloes dams were included in the present study. Polymerase chain reaction- single stranded conformational polymorphism (PCR-SSCP) technique was used to explore the polymorphism in b2M gene of buffaloes. The PCR-SSCP analysis and sequencing of different patterns obtained by silver staining of fragments pertaining to exon 1 through 4 and partial introns 1 through 3, revealed 9 SNPs which were further used to prepare the haplotypes. 7 different haplotypes were observed. Indirect ELISA was done to estimate IgG concentrations in colostrum (collected 1 hour before first milking) obtained from buffaloes. IgG levels in colostrum ranged from 11.22 to 185.1 mg/ml with a mean IgG concentration of 51.71 ± 5.99 mg/ml. The effect of different haplotypes was evaluated on IgG concentrations. The least square analysis of variance could not exhibit a significant effect of buffalo dam haplotype on colostral IgG concentration.
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The expression of human beta2-microglobulin (beta2m) on human spermatozoa cell surface was investigated by cytotoxicity and immunofluorescence tests. beta2m was also found in human seminal fluid by use of immunochemical techniques. Seminal fluids obtained from couples with infertility problems were tested for beta2m, albumin and total proteins concentration. A higher level of beta-2m was found in azoospermia compared with that of normal sperms.
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A number of studies have shown a relationship between transferrin levels in seminal plasma and sperm count. In the present study, the levels of transferrin, beta 2-microglobulin and albumin were measured in 171 samples of seminal plasma (50 subjects with normal sperm count; 32 with azoospermia; 49 with oligozoospermia; 10 with polyzoospermia; and 31 vasectomized). All three proteins were related to sperm count. It is concluded that there is a general relationship between seminal plasma proteins and sperm numbers and that some products of the sperm themselves must control the entry of plasma proteins into seminal plasma.
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There is considerable evidence to suggest that an FcR similar in structure to class I MHC Ags, neonatal Fc receptor (FcRn), transports IgG across the intestinal epithelium of suckling mice. However, this has not previously been shown definitively, nor has it been shown whether FcRn is the only, or even the major, IgG transporter in the neonatal mouse gut. We report here that neonatal mice homozygous for a targeted disruption of the beta 2microglobulin (beta 2m) gene, which encodes one subunit of FcRn, had reduced FcRn alpha-chain at the lumenal plasma membrane of intestinal cells. These mice had strikingly lower serum IgG levels during the first month after birth than littermates that possessed functional FcRn. Furthermore, we found by fostering mice on mothers with a different IgG allotype that all of the IgG in sera of beta 2m-/- mice was endogenous, and that none was obtained from milk. We conclude that FcRn is the only transporter of IgG from mother to young in the mouse. The onset of IgG synthesis in mice that received no milk IgG lagged behind that in siblings with normal IgG transport, suggesting that maternal IgG stimulates Ab production in the neonate. We noted no difference between the IgG concentrations in the milk of beta 2m-/- and beta 2m+/- mice, indicating that FcRn is not involved in the secretion of IgG into milk.
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To examine the ability of several commonly used tests for evaluation of passive transfer of immunoglobulin to predict mortality in dairy replacement heifers. Prospective observational study. 246 dairy replacement heifers between 1 and 8 days of age. Using serum samples obtained from each calf, total serum protein concentration and results of zinc sulfate turbidity, sodium sulfite turbidity, radial immunodiffusion, and glutaraldehyde coagulation were determined. Calves were monitored for 100 days, and relative risks for death were calculated. Logistic regression models predicting death also were developed. None of the logistic regression models detected a significant association between test results and mortality. The greatest relative risks of mortality were observed in calves with serum protein concentrations < 4.5 g/dl, serum IgG1 concentrations < 500 mg/dl, and sodium sulfite test scores < 1+. Calves with lower passive transfer values had increased risk of death; however, failure of passive transfer is not an infallible predictor of mortality.
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Among the multiple functions, which have been identified for the neonatal Fc receptor (FcRn), we study its role in the IgG transport in the mammary gland during the colostrum formation. For this reason, we have obtained several mammary gland biopsies from a pregnant sheep around parturition. The presence of the FcRn heavy chain mRNA was detected exclusively in the acinar and ductal epithelial cell by in situ hybridization (ISH). We detected strong signal in samples harvested 24 and 10 days prepartum; however, in samples we collected postpartum was barely detectable. Immunohistochemistry confirmed our ISH data. The cytoplasm of the epithelial cells of the acini and ducts in the mammary gland biopsies stained homogeneously before parturition, although a remarkable difference was observed in the pattern after lambing. The signal indicated uneven distribution of the FcRn alpha chain in the epithelial cells 1 and 5 days postpartum, since the apical sides of the epithelial cells were highlighted. The presence of the FcRn in the acinar and ductal epithelial cells and the obvious change of its distribution before and after parturition suggest that FcRn plays an important role in the IgG transport during colostrum formation. FcRn expression was also found in the lamb duodenal crypt epithelial cells, which have been previously demonstrated to secrete IgG1 in newborn ruminants, suggesting secretory role of the FcRn in ruminant epithelial cells.
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Twenty-four Jersey calves were randomly assigned to 1 of 4 treatment groups (6 calves per group). Pooled colostrum from first milkings (colostrum high in IgG1, 84 mg/mL) of multiparous cows was fed to treatment groups 1 and 2. Pooled colostrums from second and third milkings (colostrum low in IgG1, 31.2 mg/mL) of multiparous Jersey cows were fed to calves in treatment groups 3 and 4. The quality and timing of colostrum feeding was as follows: group 1 were fed (high IgG1 colostrum) 4 L at 0 h (birth); group 2 calves were fed (high IgG1 colostrum) 2 L at 0 h (birth) and 2 L at 12 h; group 3 calves were fed (low IgG1 colostrum) 4 L at 0 h (birth); and group 4 calves were fed (low IgG1 colostrum) 2 L at 0 h (birth) and 2 L at 12 h. Mean serum Ig() was 38.66, 45.66, 13.81 and 9.95 mg/mL in groups 1 to 4, respectively. At 48 h of age, calves fed colostrum with higher concentrations of total ingested IgG1 (groups 1 and 2) had significantly higher serum protein and IgG1 concentrations than calves fed low IgG1 colostrum at 48 h of age (groups 3 and 4). Mean apparent efficiency of IgG1 absorption was measured at 48 h; calves (group 2) receiving 2 L at birth and 2 L at 12 h of high IgG1 colostrum had higher mean apparent efficiency of IgG1 absorption than calves (group 4) fed 2 L of colostrum that was low in IgG1 at birth and 12 h (31.2 and 18.2% in groups 2 and 4, respectively). Results suggest that Jersey calves should receive 2 separate feedings of high quality colostrum to maximize the colostral IgG1 absorption.
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Amyloid fibrils were produced from the full-length mouse prion protein (PrP) under solvent conditions similar to those used for the generation of synthetic prions from PrP 89-230. Analysis of the ultrastructure by atomic force microscopy revealed extremely broad polymorphism in fibrils formed under a single growth condition. Fibrils varied with respect to the number of constitutive filaments and the manner in which the filaments were assembled. PrP polymerization was found to show several peculiar features: (i) the higher-order fibrils/ribbons were formed through a highly hierarchical mechanism of assembly of lower-order fibrils/ribbons; (ii) the lateral assembly proceeded stepwise; at each step, a semi-stable fibrillar species were generated, which were then able to enter the next level of assembly; (iii) the assembly of lower into higher-order fibrils occurred predominantly in a vertical dimension via stacking of ribbons on top of each other; (iv) alternative modes of lateral association co-existed under a single growth condition; (iv) the fibrillar morphology changed even within individual fibrils, illustrating that alternative modes of filament assembly are inter-convertible and thermodynamically equivalent. The most predominant fibrillar types were classified into five groups according to their height, each of which was divided in up to three subgroups according to their width. Detailed analysis of ultrastructure revealed that the fibrils of the major subtype (height 3.61(+/-0.28)nm, width 31.1(+/-2.0)nm) were composed of two ribbons, each of which was composed of two filaments. The molecular volume calculations indicated that a single PrP molecule occupied a distance of approximately 1.2 nm within a single filament. High polymorphism in fibrils generated in vitro is reminiscent of high morphological diversity of scrapie-associated fibrils isolated from scrapie brains, suggesting that polymorphism is peculiar for polymerization of PrP regardless of whether fibrils are formed in vitro or under pathological conditions in vivo.
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