Treatment with different antiestrogens in the neonatal period and effects in the cervicovaginal epithelium and ovaries of adult mice: a comparison to estrogen-induced changes

Female mice of the NMRI strain were treated for the first 5 days after birth with the following compounds: diethylstilbestrol (DES), MER-25 (ethamoxytriphetol), tamoxifen, ICI 47.699 (the cis-isomer of tamoxifen, an estrogen agonist), clomiphene, nafoxidine or 17 beta-estradiol-3-benzoate (E2). Females were killed at 8 wk or 6 mo and, in the case of tamoxifen also at 12 mo. The cervicovaginal region and the ovaries were prepared for histological studies. MER-25 had no effect on either the cervicovaginal epithelium or ovarian histology. Tamoxifen, clomiphene and nafoxidine resulted in extensive regions with a heterotopic columnar epithelium (HCE) in the cervicovaginal preparations. At 8 wk these regions were more widespread than those observed after treatment with DES and E2. While earlier studies have shown a progressive development of DES-induced HCE, that induced by the antiestrogens regressed with time. All ovaries from adult females treated with DES or E2 lacked corpora lutea. For the antiestrogens there were ovaries with or without corpora lutea, and this treatment was not incompatible with fertile females. It is concluded that in the neonatal period, the cervicovaginal epithelium is more sensitive to antiestrogens than central structures (hypothalamic nuclei), but for DES the opposite is true.     

The Multi-allelic Genetic Architecture of a Variance-Heterogeneity Locus for Molybdenum Concentration in Leaves Acts as a Source of Unexplained Additive Genetic Variance

Genome-wide association (GWA) analyses have generally been used to detect individual loci contributing to the phenotypic diversity in a population by the effects of these loci on the trait mean. More rarely, loci have also been detected based on variance differences between genotypes. Several hypotheses have been proposed to explain the possible genetic mechanisms leading to such variance signals. However, little is known about what causes these signals, or whether this genetic variance-heterogeneity reflects mechanisms of importance in natural populations. Previously, we identified a variance-heterogeneity GWA (vGWA) signal for leaf molybdenum concentrations in Arabidopsis thaliana. Here, fine-mapping of this association reveals that the vGWA emerges from the effects of three independent genetic polymorphisms that all are in strong LD with the markers displaying the genetic variance-heterogeneity. By revealing the genetic architecture underlying this vGWA signal, we uncovered the molecular source of a significant amount of hidden additive genetic variation or “missing heritability”. Two of the three polymorphisms underlying the genetic variance-heterogeneity are promoter variants for Molybdate transporter 1 (MOT1), and the third a variant located ~25 kb downstream of this gene. A fourth independent association was also detected ~600 kb upstream of MOT1. Use of a T-DNA knockout allele highlights Copper Transporter 6; COPT6 (AT2G26975) as a strong candidate gene for this association. Our results show that an extended LD across a complex locus including multiple functional alleles can lead to a variance-heterogeneity between genotypes in natural populations. Further, they provide novel insights into the genetic regulation of ion homeostasis in A. thaliana, and empirically confirm that variance-heterogeneity based GWA methods are a valuable tool to detect novel associations of biological importance in natural populations.     

Divergence time of porcine reproductive and respiratory syndrome virus subtypes

Porcine reproductive and respiratory syndrome virus (PRRSV) recently emerged in domestic pigs of Western Europe and North America. Although time of emergence was identical on the two continents, genetic composition was markedly different with a clear geographical subtype structure, indicating that subtypes diverged in separate reservoirs prior to emergence. Genetic analyses have shown that the most recent common ancestor (MRCA) of Western European isolates existed around 1980 and that these originate from Eastern European pigs. These findings are challenged by a study of Hanada et al. who place the MRCA of all PRRSV isolates around 1980 and find that no significant subtype divergence occurred before emergence. Here, I discuss problems of information content, methodology, and biological plausibility associated with this study. Using alternative methodology, I reanalyze the existing data and conclude that the MRCA of all PRRSV isolates existed around 1880, 100 years before the date estimated by Hanada et al.     

Involvement of mu- and m-calpains and protein kinase C isoforms in L8 myoblast differentiation

The objectives were to investigate the roles of different calpains and protein kinase C (PKC) isoforms in muscle differentiation. Concentrations of mu- and m-calpain increased significantly whereas PKCalpha and delta declined significantly during L8 myoblast differentiation. Both mu-calpain and m-calpain antisense oligonucleotides inhibited myotube formation and creatine kinase activity during L8 myoblast differentiation. These results implied that both mu- and m-calpain were involved in L8 myoblast differentiation. To investigate the involvement of calpain in regulation of PKC concentrations, mu-calpain antisense oligonucleotides were added to L8 myoblasts. PKCalpha remained unchanged and PKCdelta declined. By adding m-calpain antisense oligonucleotides instead, PKCalpha level remained unchanged and PKCdelta concentrations increased significantly during differentiation. These results suggest that PKCalpha, but not PKCdelta, is the substrate for mu-calpain and PKCalpha and delta are the substrates for the m-calpain. In addition, more phosphorylated myogenin was found in day 2 antisense oligonucleotides treated L8 cells. It is concluded that the decline of PKCalpha mediated by m- and mu-calpain is essential for L8 myoblast differentiation. The decline of PKC during myoblast differentiation may cause hypo-phosphorylation of myogenin, which in turn activates muscle-specific genes during myogenesis.     

Genetic analysis of the yopE region of Yersinia spp.: identification of a novel conserved locus, yerA, regulating yopE expression.

The yopE gene of Yersinia pseudotuberculosis was recently sequenced, and YopE was identified as an indispensable virulence determinant when tested in a mouse model (A. Forsberg and H. Wolf-Watz, Mol. Microbiol. 2:121-133, 1988). In the study described here, the DNA sequences of the yopE genes of Yersinia pestis EV76 and Yersinia enterocolitica 8081 were determined and compared with that of the Y. pseudotuberculosis gene. Only two codons were found to differ, both leading to amino acid replacements, when the gene from Y. pestis was compared. These two replacements were also present in the gene from Y. enterocolitica; in addition, 18 other codons were found to differ. Thirteen of these substitutions led to amino acid replacements. Downstream of the yopE gene, the plasmid partition locus par was found to be conserved in all three species. In Y. enterocolitica 8081, the sequence homology was interrupted by a putative insertion sequence element inserted between the yopE gene and the par region at a position only 5 base pairs downstream of the yopE stop codon. Upstream of the yopE gene, 620 base pairs were conserved in the three species. This region contained a 130-amino-acid-long open reading frame reading in the opposite direction to the yopE gene and expressed a 14-kilodalton protein in minicells. An insertion mutation in this region constructed in Y. pseudotuberculosis expressed significantly lower amounts of YopE protein in vitro than did the corresponding wild type. The expression level could be restored by transcomplementation. This new locus was designated yerA, for yopE-regulating gene A. The yerA mutant was avirulent when mice were challenged by oral infection.     

Affinity of integrin alpha 1 beta 1 from liver sinusoidal membranes for type IV collagen.

Hepatic sinusoidal membranes isolated from adult rats were extracted with detergent and fractionated on a wheat germ agglutinin affinity column. Bound glycoproteins were eluted with N-acetyl glucosamine and chromatographed on a type IV collagen affinity column. Recovery of the bound fraction by EDTA and analysis by SDS-PAGE revealed two glycoproteins with apparent molecular weights of 180,000 and 117,000. These were identified immunologically by Western blotting as the alpha and beta subunits of integrin alpha 1 beta 1.     

Heterologous Radioimmunoassay for Llama and Alpaca Luteinizing Hormone with a Monoclonal Antibody,an Equine Standard and a Human Tracer

A radioimmunoassay for llama and alpaca LH was developed using a human I125LH tracer from a commercial kit, equine LH diluted in human LH free serum as standard, and a monoclonal antibody (518B7) specific for LH but with low species specificity. A 60-min delay in the addition of the tracer and overnight incubation gave a sensitivity of 0.8 μg L−1. The intra-assay coefficient of variation was 37% at 1 μg L−1, declined to 15% at 4 pg L−1 and was below 6% for concentrations up to 32 μg L−1. The inter-assay coefficients of variation for 3 control samples were 20% (2.8 μg L−1), 16% (7.1 μg L−1) and 9.8% (19 μg L−1). In an attempt to increase sensitivity, all tubes were preincubated for 4 h at room temperature before adding the tracer, and the sample volume was increased from 50 μL to 100 μL· (in the standard curve the increased volume was compensated for by human LH free serum). With this protocol, the assay sensitivity was 0.5 μg L−1. The assay was validated clinically and demonstrated increased concentrations of LH after mating in llamas and alpacas. Furthermore, the assay was used to monitor LH responses to a single dose of GnRH in llamas (adult males and females at different ages).