Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Ion-imprinted beads for molecular recognition based mercury removal from human serum

The aim of this study is to prepare ion-imprinted polymers which can be used for the selective removal of mercury ions [Hg(2+)] from human serum. N-Methacryloyl-(L)-cysteine (MAC) was chosen as the complexing monomer. In the first step, Hg(2+) was complexed with MAC and the Hg(2+)-imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-cysteine) (MIP) beads were synthesized by suspension polymerization. After that, the template ions (i.e., Hg(2+)) were removed using thiourea (0.5%, v/v) in 0.05 M HCl. The specific surface area of the MIP beads was found to be 59.04 m(2)/g with a size range of 63-140 micro m in diameter and the swelling ratio was 91.5%. According to the elemental analysis results, the MIP beads contained 87.0 micro mol MAC/g polymer. The maximum adsorption capacity was 0.45 mg Hg(2+)/g beads. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. The relative selectivity coefficients of MIP beads for Hg(2+)/Cd(2+), Hg(2+)/Zn(2+) were 14.7 and 21.5 times greater than the non-imprinted (NIP) matrix, respectively. The MIP beads could be used many times without decreasing in their adsorption capacities significantly.     

A Major Histocompatibility Class I Locus Contributes to Multiple Sclerosis Susceptibility Independently from HLA-DRB1*15:01

Background

In Northern European descended populations, genetic susceptibility for multiple sclerosis (MS) is associated with alleles of the human leukocyte antigen (HLA) Class II gene DRB1. Whether other major histocompatibility complex (MHC) genes contribute to MS susceptibility is controversial.

Methodology/Principal Findings

A case control analysis was performed using 958 single nucleotide polymorphisms (SNPs) spanning the MHC assayed in two independent datasets. The discovery dataset consisted of 1,018 cases and 1,795 controls and the replication dataset was composed of 1,343 cases and 1,379 controls. The most significantly MS-associated SNP in the discovery dataset was rs3135391, a Class II SNP known to tag the HLA-DRB1*15:01 allele, the primary MS susceptibility allele in the MHC (O.R. = 3.04, p<1×10⁻⁷⁸). To control for the effects of the HLA-DRB1*15:01 haplotype, case control analysis was performed adjusting for this HLA-DRB1*15:01 tagging SNP. After correction for multiple comparisons (false discovery rate = .05) 52 SNPs in the Class I, II and III regions were significantly associated with MS susceptibility in both datasets using the Cochran Armitage trend test. The discovery and replication datasets were merged and subjects carrying the HLA-DRB1*15:01 tagging SNP were excluded. Association tests showed that 48 of the 52 replicated SNPs retained significant associations with MS susceptibility independently of the HLA-DRB1*15:01 as defined by the tagging SNP. 20 Class I SNPs were associated with MS susceptibility with p-values ≤1×10⁻⁸. The most significantly associated SNP was rs4959039, a SNP in the downstream un-translated region of the non-classical HLA-G gene (Odds ratio 1.59, 95% CI 1.40, 1.81, p = 8.45×10⁻¹³) and is in linkage disequilibrium with several nearby SNPs. Logistic regression modeling showed that this SNP''s contribution to MS susceptibility was independent of the Class II and Class III SNPs identified in this screen.

Conclusions

A MHC Class I locus contributes to MS susceptibility independently of the HLA-DRB1*15:01 haplotype.     

Common genetic variation in multiple metabolic pathways influences susceptibility to low HDL-cholesterol and coronary heart disease

A low level of HDL-C is the most common plasma lipid abnormality observed in men with established coronary heart disease (CHD). To identify allelic variants associated with susceptibility to low HDL-C and CHD, we examined 60 candidate genes with key roles in HDL metabolism, insulin resistance, and inflammation using samples from the Veterans Affairs HDL Intervention Trial (VA-HIT; cases, n = 699) and the Framingham Offspring Study (FOS; controls, n = 705). VA-HIT was designed to examine the benefits of HDL-raising with gemfibrozil in men with low HDL-C (≤40 mg/dl) and established CHD. After adjustment for multiple testing within each gene, single-nucleotide polymorphisms (SNP) significantly associated with case status were identified in the genes encoding LIPC (rs4775065, P < 0.0001); CETP (rs5882, P = 0.0002); RXRA (rs11185660, P = 0.0021); ABCA1 (rs2249891, P = 0.0126); ABCC6 (rs150468, P = 0.0206; rs212077, P = 0.0443); CUBN (rs7893395, P = 0.0246); APOA2 (rs3813627, P = 0.0324); SELP (rs732314, P = 0.0376); and APOC4 (rs10413089, P = 0.0425). Included among the novel findings of this study are the identification of susceptibility alleles for low HDL-C/CHD risk in the genes encoding CUBN and RXRA, and the observation that genetic variation in SELP may influence CHD risk through its effects on HDL.     

Association and interaction of the IL4R,IL4, and IL13 loci with type 1 diabetes among Filipinos

In the search for genes involved in type 1 diabetes (T1D), other than the well-established risk alleles at the human leukocyte antigen loci, we have investigated the association and interaction of polymorphisms in genes involved in the IL4/IL13 pathway in a sample of 90 Filipino patients with T1D and 94 controls. Ten single-nucleotide polymorphisms (SNPs), including two promoter SNPs in the IL4R locus on chromosome 16p11, one promoter SNP in the IL4 locus on chromosome 5q31, and four SNPs--including two promoter SNPs--in the IL13 locus on chromosome 5q31 were examined for association, linkage disequilibrium, and interaction. We found that both individual SNPs (IL4R L389L; odds ratio [OR] 0.34; 95% confidence interval [CI] 0.17-0.67; P=.001) and specific haplotypes both in IL4R (OR 0.10; 95% CI 0-0.5; P=.001) and for the five linked IL4 and IL13 SNPs (OR 3.47; P=.004) were strongly associated with susceptibility to T1D. Since IL4 and IL13 both serve as ligands for a receptor composed, in part, of the IL4R alpha chain, we looked for potential epistasis between polymorphisms in the IL4R locus on chromosome 16p11 and the five SNPs in the IL4 and IL13 loci on chromosome 5q31 and found, through use of a logistic-regression model, significant gene-gene interactions (P=.045, corrected for multiple comparisons by permutation analysis). Our data suggest that the risk for T1D is determined, in part, by polymorphisms within the IL4R locus, including promoter and coding-sequence variants, and by specific combinations of genotypes at the IL4R and the IL4 and IL13 loci.