Syntrophin-dependent expression and localization of Aquaporin-4 water channel protein.

The Aquaporin-4 (AQP4) water channel contributes to brain water homeostasis in perivascular astrocyte endfeet where it is concentrated. We postulated that AQP4 is tethered at this site by binding of the AQP4 C terminus to the PSD95-Discs large-ZO1 (PDZ) domain of syntrophin, a component of the dystrophin protein complex. Chemical cross-linking and coimmunoprecipitations from brain demonstrated AQP4 in association with the complex, including dystrophin, beta-dystroglycan, and syntrophin. AQP4 expression was studied in brain and skeletal muscle of mice lacking alpha-syntrophin (alpha-Syn(-/-)). The total level of AQP4 expression appears normal in brains of alpha-Syn(-/-) mice, but the polarized subcellular localization is reversed. High-resolution immunogold analyses revealed that AQP4 expression is markedly reduced in astrocyte endfeet membranes adjacent to blood vessels in cerebellum and cerebral cortex of alpha-Syn(-/-) mice, but is present at higher than normal levels in membranes facing neuropil. In contrast, AQP4 is virtually absent from skeletal muscle in alpha-Syn(-/-) mice. Deletion of the PDZ-binding consensus (Ser-Ser-Val) at the AQP4 C terminus similarly reduced expression in transfected cell lines, and pulse-chase labeling demonstrated an increased degradation rate. These results demonstrate that perivascular localization of AQP4 in brain requires alpha-Syn, and stability of AQP4 in the membrane is increased by the C-terminal PDZ-binding motif.

Heterotetrameric composition of aquaporin-4 water channels.

Aquaporin (AQP) water channel proteins are tetrameric assemblies of individually active approximately 30 kDa subunits. AQP4 is the predominant water channel protein in brain, but immunoblotting of native tissues has previously yielded multiple poorly resolved bands. AQP4 is known to encode two distinct mRNAs with different translation initiating methionines, M1 or M23. Using SDS-PAGE urea gels and immunoblotting with anti-peptide antibodies, four polypeptides were identified in brain and multiple other rat tissues with the following levels of expression: 32 kDa > 34 kDa > 36 kDa > 38 kDa. The 34 and 38 kDa polypeptides react with an antibody specific for the N-terminus of the M1 isoform, and 32 and 36 kDa correspond to the shorter M23 isoform. Immunogold electron microscopic studies with rat cerebellum cryosections demonstrated that the 34 kDa polypeptide colocalizes in perivascular astrocyte endfeet where the 32 kDa polypeptide is abundantly expressed. Velocity sedimentation, cross-linking, and immunoprecipitation analyses of detergent-solubilized rat brain revealed that the 32 and 34 kDa polypeptides reside within heterotetramers. Immunoprecipitation of AQP4 expressed in Xenopus laevis oocytes demonstrated that heterotetramer formation reflects the relative expression levels of the 32 and 34 kDa polypeptides; however, tetramers containing different compositions of the two polypeptides exhibit similar water permeabilities. These studies demonstrate that AQP4 heterotetramers are formed from two overlapping polypeptides and indicate that the 22-amino acid sequence at the N-terminus of the 34 kDa polypeptide does not influence water permeability but may contribute to membrane trafficking or assembly of arrays.

Linkage analysis of asthma, allergic rhinitis and atrophy to markers on chromosome 12q in families from Barbados - abstract

Findings from numerous studies have demonstrated that there is a strong heritable component to asthma and atrophy, although the genetic pathophysiology of these traits is poorly understood. To identify loci in chromosome 12q1s-q24.1 contributing to asthma and asthma-associated traits, we conducted linkage analyses among 29 multiples Barbadian families. Sib-pair analysis of 10 polymorphic micro satellite markers in 345 full and 219 half-sib pairs from Barbados revealed evidence for linkage of certain markers with a gene(s) controlling asthma (D12S379,p=0.001; D12S311,p=0.010; D12S95,p=0.010; D12S360,p=0.018), allergic rhinitis (D12S1052,p=0.040; D12S311,p=0.005; D12S95,p0.021), total serum IgE concentration (D12S1052,p=0.016; D12S311,p=0.007; D12S360,p=0.013; D12S78,p=0.002), and specific IgE antibodies (Alec) to the storage mite Blomia tropicalis (Blot M; D12S311,p=0.006; D12S360,p=0.007; D12S78,p=0.003). Significant evidence of transmission disequilibrium was observe for certain alleles at these loci in addition to high multi allergen IgE Ab. These findings suggest that a gene(s) in the 12q 15-q24.1 region, which contains several candidate genes, including interferon-y (IFNG), is important for asthma and the associated traits of allergic rhinitis, "high" total IgE, and "high" specific IgE (AU).

Linkage of asthma and total serum IgE concentration to markers on chromosome 12q: evidence from Afro-Caribbean and Caucasian populations.

To identify genes potentially relevant in atopic asthma, we analyzed markers in chromosome 12q15-q24.1 for linkage to asthma and total serum IgE concentration. Sib-pair analyses of 10 markers in 345 full- and 219 half-sib pairs from 29 multiplex Afro-Caribbean families provided evidence for linkage to this region for both asthma and total serum IgE. Certain alleles at these loci showed significant evidence of transmission disequilibrium with both asthma and high IgE. Using 6 of these markers and 11 additional markers, evidence for linkage of total IgE to 12q was also found in 12 Caucasian Amish kindreds (24 nuclear families) by both sib-pair and transmission disequilibrium analyses. These findings suggest that the 12q15-q24.1 region may contain a gene(s) controlling asthma and the associated "high total IgE" trait.

Linkage of asthma and total serum IgE concentration to markers on chromosome 12q: evidence from Afro-Caribbean and Caucasian population

To identify genes potentially relevant in atopic asthma, we analyzed markers in chromosome 12q15-q24.1 for linkage to asthma and total serum Ige concentration. Sib-pair analyses of 10 markers in 345 full- and 219 half-sib pairs from 29 multiplex Afro-Caribbean families provided evidence for linkage to his region for both asthma and total serum IgE. Certain alleles at these loci showed significant evidence of transmission disequilibrium with both asthma and high IgE. Using 6 of these markers and 11 additional markers, evidence for linkage of total IgE to 12q was also found in 12 Caucasian Amish kindreds (24 nuclear families) by both sib-pair and transmission disequilibrium analyses. These findings suggest that the 12q15-q24.1 region may contain a gene(s) contolling asthma and the associated high total IgE. trait.(AU)

Genetic basis of IgE responsiveness: relevance to the atopic diseases.

Genetic analysis of 170 subjects in 11 extended Amish families revealed evidence for linkage of five markers in chromosome 5q31.1 with a gene controlling total serum IgE levels. No linkage was found between these markers and specific IgE antibody levels. Analysis of total IgE within a subset of 128 IgE-antibody-negative sib pairs confirmed evidence for linkage to 5q31.1, especially IL4 (p = 4 x 10(-6)). These and other data suggest that IL4 or a nearby gene regulates IgE production in a non-antigen-specific (noncognate) fashion and provide evidence for a possible link between asthma and the IL4 gene.

Linkage analysis of IL4 and other chromosome 5q31.1 markers and total serum immunoglobulin E concentrations.

Sib-pair analysis of 170 individuals from 11 Amish families revealed evidence for linkage of five markers in chromosome 5q31.1 with a gene controlling total serum immunoglobulin E (IgE) concentration. No linkage was found between these markers and specific IgE antibody concentrations. Analysis of total IgE within a subset of 128 IgE antibody-negative sib pairs confirmed evidence for linkage to 5q31.1, especially to the interleukin-4 gene (IL4). A combination of segregation and maximum likelihood analyses provided further evidence for this linkage. These analyses suggest that IL4 or a nearby gene in 5q31.1 regulates IgE production in a nonantigen-specific (noncognate) fashion.

Estimates of heterosis for sexual activity in boars.

Data on 118 Duroc, Yorkshire and reciprocal cross boars were utilized to evaluate the effect of crossbreeding on mating behavior. Boars were penned with an estrous gilt on 2 consecutive days. Number of mounts (proper and improper), sexual interest score and whether a successful mating occurred were recorded. Times at which each of these events occurred were recorded also, allowing calculation of times to first mount, to final mount and to completion of mating. Most importantly, more crossbred boars completed a mating than purebred boars (69 vs 27%). Crossbreds had greater sexual interest (P less than .05), more mounts and a higher proportion of properly oriented mounts (P less than .05). Crossbreds also began mounting activity (first mount, first proper mount and final mount; P less than .05) earlier and required less time to complete a mating (P less than .05) than purebred boars. Heterosis values for number of proper and improper mounts, proportion of proper mounts and sexual interest were 2, 53, 32 and 52%, respectively. Time to first mount, to final mount and to completing mating had heterosis values of -34, -29 and -20%, respectively. Scrotal measures taken at 140 and 168 d and at post-trial castration, as well as excised testes measures and sperm numbers, showed heterosis; however, no consistent associations between these traits and behavior traits were found. These data suggest that crossbred boars were more sexually active than purebred boars, perhaps due to an advanced physiological or behavioral stage.

Genetic parameters for testes size and sperm number in Hereford bulls.

Paternal half-sib heritability and genetic correlation estimates were obtained utilizing data from 578 Hereford bulls from 66 sires. Bulls were maintained in three lines (weaning weight, postweaning gain and control) of an ongoing selection project. Growth performance traits studied were adjusted 205-d weaning weight, 365-d weight, individual feed efficiency, sonoray fat thickness and postweaning gain. Heritability estimates for these traits were .15 +/- .17, .33 +/- .19, .46 +/- .21, .28 +/- .18 and .52 +/- .21, respectively. Scrotal measurements taken were circumference of both testes and length and diameter of right testis at 205 and 365 d of age. Heritability estimates were .08 +/- .20, .07 +/- .20 and .28 +/- .24 at 205 d, and .44 +/- .24, .16 +/- .21 and .40 +/- .24 at 365 d, respectively. Excised testes traits, circumference, right testis length, diameter and weight, total sperm in the testes and sperm/gram of testes had heritability estimates of .21 +/- .26, .19 +/- .26, .02 +/- .24, .63 +/- .27, .14 +/- .21 and -.13 +/- .18, respectively. Genetic correlations of scrotal measurements at 205 d with scrotal measurements at 365 d and excised testes traits were negative. Scrotal measurements at 365 d had high positive genetic correlations with excised testes size, weight and total sperm. These relationships suggest that selection for increase of scrotal size at 365 d should increase testes size and weight and sperm numbers. Genetic correlations of weights and gain with scrotal measurements at 365 d and excised testes characteristics were moderate to high, in a favorable direction. Genetic correlations of testes traits with feed efficiency were essentially zero, while those with fat thickness were moderately positive. These results suggest that increasing testes size should not adversely affect growth performance traits except through the reduction in selection intensity.