Population-based meta-analysis in Caucasians confirms association with COL5A1 and ZNF469 but not COL8A2 with central corneal thickness

Central corneal thickness (CCT) has become an endophenotype of major interest for the genetically complex disorder glaucoma. CCT has a high heritability, and thin CCT is an independent risk factor for the diagnosis and progression of open-angle glaucoma. Genome-wide association studies thus provide genetic loci associated with CCT and potentially related to open-angle glaucoma. The distribution of CCT and prevalence of glaucoma in population-based studies have demonstrated ethnic differences suggesting ethnic-dependent variations in the genetic determinants of CCT. We conducted a genome-wide association study in Caucasians (n?=?3,931) from the Gutenberg Health Study (Germany) followed by replication of 30 genome-wide significant SNPs or SNPs of interest (P?<?10^?5) in the Rotterdam Study (The Netherlands, n?=?1,418). In a combined analysis, we confirmed quantitative trait loci on chromosomes 9q34 and 16q24 for association with CCT. On chromosome 16q24, the locus is located in an intergenic region near the ZNF469 gene (top SNP: rs9938149, P?=?1.45?×?10^?12). ZNF469 missense mutation is involved in a syndrome with very thin cornea (brittle cornea syndrome). The second locus on chromosome 9q34 represents the intergenic region between the RXRA and COL5A1 gene (top SNP: rs3132306, P?=?2.71?×?10^?10). Collagen type 5 determines the diameter of the corneal collagen fibrils. In our Caucasian population-based GWA study, we reinforce the involvement of collagen-related genes influencing CCT in Caucasians. We could not confirm the collagen type 8 locus on chromosome 1 as reported in Asian studies.     

Absence of Filamin A Prevents Cells from Responding to Stiffness Gradients on Gels Coated with Collagen but not Fibronectin

Cell types from many tissues respond to changes in substrate stiffness by actively remodeling their cytoskeletons to alter spread area or adhesion strength, and in some cases changing their own stiffness to match that of their substrate. These cell responses to substrate stiffness are linked to substrate-induced changes in the state, localization, and amount of numerous proteins, but detailed evidence for the requirement of specific proteins in these distinct forms of mechanical response are scarce. Here we use microfluidics techniques to produce gels with a gradient of stiffness to show the essential function of filamin A in cell responses to mechanical stimuli and dissociate cell spreading and stiffening by contrasting responses of a pair of human melanoma-derived cell lines that differ in expression of this actin cross-linking protein. M2 melanoma cells null for filamin A do not alter their adherent area in response to increased substrate stiffness when they link to the substrate only through collagen receptors, but change adherent area normally when bound through fibronectin receptors. In contrast, filamin A-replete A7 cells change adherent area on both substrates and respond more strongly to collagen I-coated gels than to fibronectin-coated gels. Strikingly, A7 cells alter their stiffness, as measured by atomic force microscopy, to match the elastic modulus of the substrate immediately adjacent to them on the gradient. M2 cells, in contrast, maintain a constant stiffness on all substrates that is as low as that of A7 cells on the softest gels examined (1000 Pa). Comparison of cell spreading and cell stiffening on the same gradient substrates shows that cell spreading is uncoupled from stiffening. At saturating collagen and fibronectin concentrations, adhesion of M2 cells is reduced compared to that of A7 cells to an extent approximately equal to the difference in adherent area. Filamin A appears to be essential for cell stiffening on collagen, but not for cell spreading on fibronectin. These results have implications for different models of cell protrusion and adhesion and identify a key role for filamin A in altering cellular stiffness that cannot be compensated for by other actin cross-linkers in vivo.     

Antibody is required for clearance of infectious murine hepatitis virus A59 from the central nervous system, but not the liver.

Intracerebral inoculation with mouse hepatitis virus strain A59 results in viral replication in the CNS and liver. To investigate whether B cells are important for controlling mouse hepatitis virus strain A59 infection, we infected muMT mice who lack membrane-bound IgM and therefore mature B lymphocytes. Infectious virus peaked and was cleared from the livers of muMT and wild-type mice. However, while virus was cleared from the CNS of wild-type mice, virus persisted in the CNS of muMT mice. To determine how B cells mediate viral clearance, we first assessed CD4(+) T cell activation in the absence of B cells as APC. CD4(+) T cells express wild-type levels of CD69 after infection in muMT mice. IFN-gamma production in response to viral Ag in muMT mice was also normal during acute infection, but was decreased 31 days postinfection compared with that in wild-type mice. The role of Ab in viral clearance was also assessed. In wild-type mice plasma cells appeared in the CNS around the time that virus is cleared. The muMT mice that received A59-specific Ab had decreased virus, while mice with B cells deficient in Ab secretion did not clear virus from the CNS. Viral persistence was not detected in FcR or complement knockout mice. These data suggest that clearance of infectious mouse hepatitis virus strain A59 from the CNS requires Ab production and perhaps B cell support of T cells; however, virus is cleared from the liver without the involvement of Abs or B cells.     

A 48 kDa collagen-binding phosphoprotein isolated from bovine aortic endothelial cells interacts with the collagenous domain, but not the globular domain, of collagen type IV.

We have identified collagen-binding proteins in detergent extracts of metabolically labelled bovine aortic endothelial cells (BAEC) by collagen type IV-Sepharose affinity chromatography. The major collagen type IV-binding protein identified by SDS/PAGE had a molecular mass of 48 kDa, which we term the 'collagen-binding 48 kDa protein' (CB48). The pI of CB48 was 8.0-8.3 in a two-dimensional gel system, running non-equilibrium pH gel electrophoresis in the first dimension and SDS/PAGE in the second dimension. Under these conditions CB48 separated into two major (a and b) and one minor isoform (c); a was the most basic of the three isoforms. Two-dimensional chymotryptic peptide maps derived from each individual isoform were virtually identical. The charge differences between the isoforms were due in part to differential H3(32)PO4 incorporation by the protein. CB48 bound to intact collagen type IV and the collagenous region of collagen type IV, but not to the globular NC1 domain. Cell-surface labelling and indirect immunofluorescence experiments localized the bulk of CB48 intracellularly in the endoplasmic reticulum Golgi region, with a minor population of molecules on the cell surface. A specific rabbit polyclonal anti-CB48 serum did not inhibit the attachment or spreading of BAEC to collagen type IV in an 'in vitro' adhesion assay, suggesting that the cell-surface population of CB48 is not involved in BAEC adhesion. We conclude that CB48 is a collagen-binding phosphoprotein that interacts with the collagenous domain of collagen type IV and may be involved in intracellular transport of collagen molecules.     

The monoclonal antibody 69A1 recognizes an epitope found on neurones with axons that fasciculate but not on those with non-fasciculating processes

We have previously reported that the cell-type distribution and pattern of expression of the surface antigen recognized by the monoclonal antibody 69A1, suggests that it may be involved during the period of nerve fibre outgrowth and the formation of fibre bundles in the rat (Pigott & Kelly, 1986). In this current study, we have examined the expression of the epitope recognized by antibody 69A1 in regions of the rat central nervous system in which it is possible to distinguish between neurones with axons that fasciculate to form clearly defined fibre tracts and neurones with non-fasciculating processes. We have also examined antibody 69A1 labelling in several regions of the peripheral nervous system. We report that the 69A1 epitope is expressed on neurones with axons that fasciculate but is not found on neurones with short, non-fasciculating axons or on neurones without a morphologically identifiable axon. The antigen 69A1 has been purified and shown to be immunochemically closely related or identical to the L1 antigen.     

Transfection with the E1A and E1B-19kDa oncogenes does not prevent rat embryo fibroblasts from cell cycle arrest after gamma-radiation

Introduction of the E1A early region of the human adenovirus type 5 impairs the ability of mammalian cells to arrest the cell cycle at G1/S after damage. Two-parameter fluorescent-activated cell sorting (FACS) with iododeoxyuridine revealed the radiation-induced G1/S arrest in rat embryo fibroblasts transformed with the complementing E1A + E1B-19 kDa oncogenes. This was due to selective inhibition of CycIE/Cdk2-associated kinase activity, while activities of type 2 kinase and of CyclA/Cdk2 complexes remained unchanged. The inhibitor of G1-phase cyclin kinases, p21/Waf1, was accumulated and interacted with target kinases both in normal and in transformed cells after irradiation. As shown by immunoprecipitation, p21/Waf1 formed complexes with the E1A on coproducts in the transformants, which possibly accounted for its functional inactivation. Kinase modification in cyclin-kinase complexes was assumed to play a key role in regulation of cyclin-dependent kinases in the transformants with inactivated p21/Waf1.     

Release of arachidonic acid from vascular endothelial cells: fatty acyl specificity is observed with receptor-mediated agonists and with the calcium ionophore A23187 but not with melittin

Vascular endothelial cells respond to a variety of physiological and pharmacological stimuli by releasing free arachidonic acid from membrane phospholipids, thus initiating synthesis of prostacyclin. Previous work in our laboratory has demonstrated that the thrombin-stimulated deacylation is specific for arachidonate and structurally similar polyunsaturated fatty acids that contain a delta-5 double bond. We now report that histamine, bradykinin, and the calcium ionophore A23187 exhibit the same fatty acid specificity as does thrombin. Experiments with both human umbilical vein and calf pulmonary artery endothelial cells indicate that these agonists stimulate the release of previously incorporated [14C]arachidonate but not 8,11,14-[14C]eicosatrienoate or [14C]docosatetraenoate. By contrast, melittin stimulates the release of 8,11,14-eicosatrienoate, docosatetraenoate, and oleate as well as arachidonate. These results suggest that histamine, bradykinin, and A23187 activate a common calcium-dependent phospholipase A2. Melittin appears either to alter the substrate specificity of the receptor-linked phospholipase A2 activity or to activate additional enzymes as well.     

A purified green barley extract with modulatory properties upon TNF alpha and ROS released by human specialised cells isolated from RA patients

Based on the finding that reactive oxygen species (ROS) play important roles in mediating proinflammatory cytokine production (e.g. TNF alpha) and that many plant extracts contain substances with antioxidant properties, we examined the antiinflammatory action of a green barley extract, commercially available as "Natural SOD". The results obtained in this study demonstrate that the antiinflammatory properties of "Natural SOD" due to its micromolecular substances, able to scavenge ROS and to down-regulate TNF alpha production, main inflammation mediators produced by specialised cells from peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA). We prepared and tested a purified green barley extract (PE) containing micromolecular substances under 1 kDa, able to inhibit TNF alpha releasing--measured by an bioassay--from LPS stimulated human mononuclear cells (MNC) isolated both from PB and SF of RA patients. Luminol-dependent chemiluminescence has been used to measure the scavenging activity of PE on ROS releasing from activated neutrophils isolated from PB of RA patients. PE, containing high concentrations of substances with antioxidant and antiinflammatory properties, could be a more efficient natural drug for human use than "Natural SOD" in the treatment of RA patients.     

Nigexine, a phospholipase A2 from cobra venom with cytotoxic properties not related to esterase activity. Purification, amino acid sequence, and biological properties

The venoms of the Naja species are known to be cytotoxic. This toxicity has been attributed to the presence of small nonenzymatic polypeptides of 60 amino acid residues, designated as cardiotoxins or cytotoxins. We investigated the cytotoxic potency of Naja nigricollis venom fractions and isolated another type of cytotoxic component which is even more potent than cardiotoxins. This cytotoxic compound, which was designated as nigexine, was purified to homogeneity and its amino acid sequence was determined. Nigexine is a basic phospholipase A2 consisting of a single chain of 118 amino acids. A detailed investigation of the cytotoxic effects on epithelial FL cells, C-13T neuroblastoma cells, and promyelocytic leukemia HL 60 cells revealed that nigexine not only altered cell viability but also prevented cell proliferation. This is a property that was specific to nigexine since other phospholipases A2 from various sources had no detectable cytotoxic activity. The cytotoxic activity of nigexine was not dependent on the presence of divalent cations, unlike its enzymatic activity. In particular, the cytotoxic activity of nigexine was identical in the presence or absence of either 2 mM Ca2+ or Sr2+, or 6 mM EDTA. We also present evidence based on chemical modifications that cytotoxic activity was not correlated with enzymatic activity. Thus, modification with parabromophenacyl bromide totally abolished the enzymatic activity of nigexine, which nevertheless retained 6-20% of the cytotoxicity of native nigexine. Conversely, treatment with cyanogen bromide gave a compound that retained 7% of the enzymatic activity of the parent molecule but was devoid of detectable cytotoxicity.     

Induction of CYP1A by a diterpene quinone tanshinone IIA isolated from a medicinal herb Salvia miltiorrhiza in C57BL/6J but not in DBA/2J mice

Effects of tanshinone IIA, an active diterpene quinone of the herbal medicine Salvia miltiorrhiza (Danshen), on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in the arylhydrocarbon (Ah)-responsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice. Oral treatment of tanshinone IIA caused a dose-dependent increase of liver microsomal 7-methoxyresorufin O-demethylation (MROD) activity in B6 but not in D2 mice. In B6 mice, tanshinone IIA increased hepatic benzo(a)pyrene hydroxylation (AHH), 7-ethoxyresorufin O-deethylation, MROD, and 7-ethoxycoumarin O-deethylation activities. The levels of Cyp1A2 protein and mRNA were elevated. On the contrary, in D2 mice, tanshinone IIA decreased hepatic AHH and nifedipine oxidation activities and the CYP3A protein level without affecting other activities determined. Cyp1A2 protein and mRNA levels were not affected by tanshinone IIA in D2 mice. Tanshinone IIA had no effects on UGT and GST activities in both B6 and D2 mice. These results demonstrated that induction of CYP1A2 by tanshinone IIA depended on the Ah-responsiveness and occurred at pre-translational level.