Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D

The vitamin D hormone 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], the biologically active form of vitamin D, is essential for an intact mineral metabolism. Using gene targeting, we sought to generate vitamin D receptor (VDR) null mutant mice carrying the reporter gene lacZ driven by the endogenous VDR promoter. Here we show that our gene-targeted mutant mice express a VDR with an intact hormone binding domain, but lacking the first zinc finger necessary for DNA binding. Expression of the lacZ reporter gene was widely distributed during embryogenesis and postnatally. Strong lacZ expression was found in bones, cartilage, intestine, kidney, skin, brain, heart, and parathyroid glands. Homozygous mice are a phenocopy of mice totally lacking the VDR protein and showed growth retardation, rickets, secondary hyperparathyroidism, and alopecia. Feeding of a diet high in calcium, phosphorus, and lactose normalized blood calcium and serum PTH levels, but revealed a profound renal calcium leak in normocalcemic homozygous mutants. When mice were treated with pharmacological doses of vitamin D metabolites, responses in skin, bone, intestine, parathyroid glands, and kidney were absent in homozygous mice, indicating that the mutant receptor is nonfunctioning and that vitamin D signaling pathways other than those mediated through the classical nuclear receptor are of minor physiological importance. Furthermore, rapid, nongenomic responses to 1,25-(OH)(2)D(3) in osteoblasts were abrogated in homozygous mice, supporting the conclusion that the classical VDR mediates the nongenomic actions of 1,25-(OH)(2)D(3).     

The solvent-inaccessible Cys67 residue of HLA-B27 contributes to T cell recognition of HLA-B27/peptide complexes

Crystallographic studies have suggested that the cysteine at position 67 (Cys(67)) in the B pocket of the MHC molecule HLA-B*2705 is of importance for peptide binding, and biophysical studies have documented altered thermodynamic stability of the molecule when Cys(67) was mutated to serine (Ser(67)). In this study, we used HLA-B27.Cys(67) and HLA-B27.Ser(67) tetramers with defined T cell epitopes to determine the contribution of this polymorphic, solvent-inaccessible MHC residue to T cell recognition. We generated these HLA-B27 tetramers using immunodominant viral peptides with high binding affinity to HLA-B27 and cartilage-derived peptides with lower affinity. We demonstrate that the yield of refolding of HLA-B27.Ser(67) molecules was higher than for HLA-B27.Cys(67) molecules and strongly dependent on the affinity of the peptide. T cell recognition did not differ between HLA-B27.Cys(67) and HLA.B27.Ser(67) tetramers for the viral peptides that were investigated. However, an aggrecan peptide-specific T cell line derived from an HLA-B27 transgenic BALB/c mouse bound significantly stronger to the HLA-B27.Cys(67) tetramer than to the HLA-B27.Ser(67) tetramer. Modeling studies of the molecular structure suggest the loss of a SH ... pi hydrogen bond with the Cys-->Ser substitution in the HLA-B27 H chain which reduces the stability of the HLA-B27/peptide complex. These results demonstrate that a solvent-inaccessible residue in the B pocket of HLA-B27 can affect TCR binding in a peptide-dependent fashion.     

A probabilistic model for the arrangement of a subset of human chromosome territories in WI38 Human fibroblasts

There is growing evidence that chromosome territories have a probabilistic non‐random arrangement within the cell nucleus of mammalian cells. Other than their radial positioning, however, our knowledge of the degree and specificity of chromosome territory associations is predominantly limited to studies of pair‐wise associations. In this study we have investigated the association profiles of eight human chromosome pairs (numbers 1, 2, 3, 4, 6, 7, 8, 9) in the cell nuclei of G₀‐arrested WI38 diploid lung fibroblasts. Associations between heterologous chromosome combinations ranged from 52% to 78% while the homologous chromosome pairs had much lower levels of association (3–25%). A geometric computational method termed the Generalized Median Graph enabled identification of the most probable arrangement of these eight chromosome pairs. Approximately 41% of the predicted associations are present in any given nucleus. The association levels of several chromosome pairs were very similar in a series of lung fibroblast cell lines but strikingly different in skin and colon derived fibroblast cells. We conclude that a large subset of human chromosomes has a preferred probabilistic arrangement in WI38 cells and that the resulting chromosomal associations show tissue origin specificity. J. Cell. Physiol. 221: 120–129, 2009. © 2009 Wiley‐Liss, Inc     

The stability and anti-apoptotic function of A1 are controlled by its C terminus

Most Bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their C-terminal portion. We found that the C terminus of the anti-apoptotic family member A1 did not function as a membrane anchor. Instead, this stretch of the protein rendered A1 highly unstable by mediating its polyubiquitination and rapid proteasomal degradation. Moreover, the domain did not only function independently of its position within the A1 protein but when transferred could even destabilize unrelated proteins like enhanced green fluorescent protein and caspase-3. A1 was, however, much more stable in the presence of the Bcl-2 homology-only protein BimEL, suggesting that direct interaction of A1 with pro-apoptotic members of the Bcl-2 family strongly reduces its rate of turnover. We further show that the C-terminal end of A1 also contributes to the anti-apoptotic capacity of the protein. In conclusion, our data demonstrate that the C terminus serves a dual function by controlling the stability of A1 and by amplifying the capacity of the protein to protect cells against apoptosis.     

Synonymous codon usage pattern among the S,M, and L segments in Crimean-congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) virus is one among the major zoonosis viral diseases that use the Hyalomma ticks as their transmission vector to cause viral infection to the human and mammalian community. The fatality of infectious is high across the world especially in Africa, Asia, Middle East, and Europe. This study regarding codon usage bias of S, M, and L segments of the CCHF virus pertaining to the host Homo sapiens, reveals in-depth information about the evolutionary characteristics of CCHFV. Relative Synonymous Codon Usage (RSCU), Effective number of codons (ENC) were calculated, to determine the codon usage pattern in each segment. Correlation analysis between Codon adaptation index (CAI), GRAVY (Hydrophobicity), AROMO (Aromaticity), and nucleotide composition revealed bias in the codon usage pattern. There was no strong codon bias found among any segments of the CCHF virus, indicating both the factors i.e., natural selection and mutational pressure shapes the codon usage bias.     

Whole-genome scan for guttural pouch tympany in Arabian and German warmblood horses

Equine guttural pouch tympany (GPT) is a hereditary disease in foals of several breeds, including thoroughbreds, Arabian, Quarter and warmblood horses. We performed a whole-genome scan for GPT in 143 horses from five Arabian and five German warmblood families and genotyped 257 microsatellites. Chromosome-wide significant linkage was detected on ECA2 and ECA15 using multipoint non-parametric linkage analyses. Analyses stratified by sex revealed chromosome-wide significant linkage on ECA2 for fillies and chromosome-wide significant linkage on ECA15 for colts. For Arabian colts, the quantitative trait locus (QTL) on ECA15 was genome-wide significant. Haplotypes including two to four microsatellites within the QTL on ECA2 and 15 in fillies and colts, respectively, were significantly associated with GPT for both breeds. Thus, our analysis indicated sex-specific QTL, a fact which is in agreement with a two- to fourfold higher incidence of GPT in females. This is the first report of QTL for equine GPT and a first step towards identifying genes responsible for GPT.