Type 1 plasminogen activator inhibitor binds to fibrin via vitronectin

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), circulates as a complex with the abundant plasma glycoprotein, vitronectin. This interaction stabilizes the inhibitor in its active conformation In this report, the effects of vitronectin on the interactions of PAI-1 with fibrin clots were studied. Confocal microscopic imaging of platelet-poor plasma clots reveals that essentially all fibrin-associated PAI-1 colocalizes with fibrin-bound vitronectin. Moreover, formation of platelet-poor plasma clots in the presence of polyclonal antibodies specific for vitronectin attenuated the inhibitory effects of PAI-1 on t-PA-mediated fibrinolysis. Addition of vitronectin during clot formation markedly potentiates PAI-1-mediated inhibition of lysis of (125)I-labeled fibrin clots by t-PA. This effect is dependent on direct binding interactions of vitronectin with fibrin. There is no significant effect of fibrin-associated vitronectin on fibrinolysis in the absence of PAI-1. The binding of PAI-1 to fibrin clots formed in the absence of vitronectin was characterized by a low affinity (K(d) approximately 3.5 micrometer) and rapid loss of PAI-1 inhibitory activity over time. In contrast, a high affinity and stabilization of PAI-1 activity characterized the cooperative binding of PAI-1 to fibrin formed in the presence of vitronectin. These findings indicate that plasma PAI-1.vitronectin complexes can be localized to the surface of fibrin clots; by this localization, they may modulate fibrinolysis and clot reorganization.     

Determination of N- and C-terminal borders of the transmembrane domain of integrin subunits

Previous studies on the membrane-cytoplasm interphase of human integrin subunits have shown that a conserved lysine in subunits alpha(2), alpha(5), beta(1), and beta(2) is embedded in the plasma membrane in the absence of interacting proteins (Armulik, A., Nilsson, I., von Heijne, G., and Johansson, S. (1999) in J. Biol. Chem. 274, 37030-37034). Using a glycosylation mapping technique, we here show that alpha(10) and beta(8), two subunits that deviate significantly from the integrin consensus sequences in the membrane-proximal region, were found to have the conserved lysine at a similar position in the lipid bilayer. Thus, this organization at the C-terminal end of the transmembrane (TM) domain seems likely to be general for all 24 integrin subunits. Furthermore, we have determined the N-terminal border of the TM domains of the alpha(2), alpha(5), alpha(10), beta(1), and beta(8) subunits. The TM domain of subunit beta(8) is found to be 22 amino acids long, with a second basic residue (Arg(684)) positioned just inside the membrane at the exoplasmic side, whereas the lipidembedded domains of the other subunits are longer, varying from 25 (alpha(2)) to 29 amino acids (alpha(10)). These numbers implicate that the TM region of the analyzed integrins (except beta(8)) would be tilted or bent in the membrane. Integrin signaling by transmembrane conformational change may involve alteration of the position of the segment adjacent to the conserved lysine. To test the proposed "piston" model for signaling, we forced this region at the C-terminal end of the alpha(5) and beta(1) TM domains out of the membrane into the cytosol by replacing Lys-Leu with Lys-Lys. The mutation was found to not alter the position of the N-terminal end of the TM domain in the membrane, indicating that the TM domain is not moving as a piston. Instead the shift results in a shorter and therefore less tilted or bent TM alpha-helix.     

High-level production and optimization of monodispersity of 11beta-hydroxysteroid dehydrogenase type 1

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is an intraluminally oriented, endoplasmic reticulum (ER)-bound enzyme catalyzing the interconversion between inactive cortisone and hormonally active cortisol. Heterologous production of 11beta-HSD1, devoid of its N-terminal transmembrane segment, is possible but yields only small amounts of soluble protein. Here we show that the soluble portion of recombinant 11beta-HSD1 produced in E. coli is found mainly as multimeric aggregates in the absence of detergent, and to a large extent associated with the endogenous chaperonin GroEL and other E. coli proteins. By co-overexpressing GroEL/ES and adding an 11beta-HSD1 inhibitor during protein synthesis, we have increased the accumulation of soluble 11beta-HSD1 by more than one order of magnitude. Using monodispersity as a screening criterion, we have also optimized the purification process by evaluating various solubilizing systems for the chromatographic steps, finally obtaining stable monodisperse preparations of both human and guinea pig 11beta-HSD1. By analytical ultracentrifugation, we could demonstrate that 11beta-HSD1 mainly exists as a dimer in the solubilized state. Moreover, active site titration of human 11beta-HSD1 revealed that at least 75% of the protein in a typical preparation represents active enzyme. Equilibrium unfolding experiments indicate that addition of inhibitor and the cofactor NADP(H) can stabilize the conformational stability of this enzyme in an additive manner. The outlined procedure may provide a general method for preparing similar proteins to oligomeric homogeneity and with retained biological activity.     

Linkage of osteoporosis to chromosome 20p12 and association to BMP2

Osteoporotic fractures are a major cause of morbidity and mortality in ageing populations. Osteoporosis, defined as low bone mineral density (BMD) and associated fractures, have significant genetic components that are largely unknown. Linkage analysis in a large number of extended osteoporosis families in Iceland, using a phenotype that combines osteoporotic fractures and BMD measurements, showed linkage to Chromosome 20p12.3 (multipoint allele-sharing LOD, 5.10; p value, 6.3 × 10⁻⁷), results that are statistically significant after adjusting for the number of phenotypes tested and the genome-wide search. A follow-up association analysis using closely spaced polymorphic markers was performed. Three variants in the bone morphogenetic protein 2 (BMP2) gene, a missense polymorphism and two anonymous single nucleotide polymorphism haplotypes, were determined to be associated with osteoporosis in the Icelandic patients. The association is seen with many definitions of an osteoporotic phenotype, including osteoporotic fractures as well as low BMD, both before and after menopause. A replication study with a Danish cohort of postmenopausal women was conducted to confirm the contribution of the three identified variants. In conclusion, we find that a region on the short arm of Chromosome 20 contains a gene or genes that appear to be a major risk factor for osteoporosis and osteoporotic fractures, and our evidence supports the view that BMP2 is at least one of these genes.     

A major susceptibility gene for asthma maps to chromosome 14q24

Asthma is a complex genetic disorder with a heterogeneous phenotype, largely attributed to the interactions among many genes and between these genes and the environment. Numerous loci and candidate genes have been reported to show linkage and association to asthma and atopy. Although some studies reporting these observations are compelling, no gene has been mapped that confers a sufficiently high risk of asthma to meet the stringent criteria for genomewide significance. Using 175 extended Icelandic families that included 596 patients with asthma, we performed a genomewide scan with 976 microsatellite markers. The families were identified by cross-matching a list of patients with asthma from the Department of Allergy/Pulmonary Medicine of the National University Hospital of Iceland with a genealogy database of the entire Icelandic nation. We detected linkage of asthma to chromosome 14q24, with an allele-sharing LOD score of 2.66. After we increased the marker density within the locus to an average of one microsatellite every 0.2 cM, the LOD score rose to 4.00. We designate this locus "asthma locus one" (AS1). Taken together, these results provide evidence of a novel susceptibility gene for asthma on chromosome 14q24.     

Localization of a susceptibility gene for common forms of stroke to 5q12

Stroke is one of the most complex diseases, with several subtypes, as well as secondary risk factors, such as hypertension, hyperlipidemia, and diabetes, which, in turn, have genetic and environmental risk factors of their own. Here, we report the results of a genomewide search for susceptibility genes for the common forms of stroke. We cross-matched a population-based list of patients with stroke in Iceland with an extensive computerized genealogy database clustering 476 patients with stroke within 179 extended pedigrees. Linkage to 5q12 was detected, and the LOD score at this locus meets the criteria for genomewide significance (multipoint allele-sharing LOD score of 4.40, P=3.9×10^-6). A 20-cM region on 5q was physically and genetically mapped to obtain accurate marker order and intermarker distances. This locus on 5q12, which we have designated as “STRK1,” does not correspond to known susceptibility loci for stroke or for its risk factors and represents the first mapping of a locus for common stroke.     

Interferon-gamma-induced oligodendrocyte cell death: implications for the pathogenesis of multiple sclerosis.

BACKGROUND: The histopathology of multiple sclerosis (MS) is characterized by a loss of myelin and oligodendrocytes, relative preservation of axons, and a modest inflammatory response. The reasons for this selective oligodendrocyte death and demyelination are unknown. MATERIALS AND METHODS: In light of the T lymphocyte and macrophage infiltrates in MS lesions and the numerous cytokines these cells secrete, the direct influence of cytokines on survival of cultured oligodendrocytes and sensory neurons was investigated. Expression of cytokines in vivo was determined by immunolabeling cryostat sections of snap-frozen tissue containing chronic active lesions from four different patients. The samples were also analyzed for the presence of apoptotic nuclei by in situ labeling of 3'-OH ends of degraded nuclear DNA. RESULTS: The results showed: (i) interferon-gamma (IFN gamma) to be a potent inducer of apoptosis among oligodendrocytes in vitro and that this effect can be reversed by leukemia inhibitory factor (LIF); (ii) IFN gamma has a minimal effect on the survival of cultured neurons; (iii) IFN gamma at the margins of active MS plaques but not in unaffected white matter; (iv) evidence for apoptosis of oligodendrocytes at the advancing margins of chronic active MS plaques. CONCLUSIONS: Injury to a substantial number of oligodendrocytes in MS is the results of programmed cell death rather than necrotic cell death mechanisms. We postulate that IFN gamma plays a role in the pathogenesis of MS by activating apoptosis in oligodendrocytes.     

Proligodendroblast Antigen (POA), a Developmental Antigen Expressed by A007/O4-Positive Oligodendrocyte Progenitors Prior to the Appearance of Sulfatide and Galactocerebroside

Evidence is presented for the immunological identification of a developmental antigen appearing at a critical point in the oligodendroglial lineage. Specifically, monoclonal antibody A007 recognizes cells in the oligodendrocyte lineage at two distinct stages. Analyses of purified lipid standards and lipid extracts from galactocerebroside-positive (GalC+) oligodendrocytes by enzyme-linked immunosorbent assay, lipid dot blot, and immuno-TLC demonstrated that A007 recognizes sulfatide (SUL) and seminolipid. However, neither 35SO4 incorporation into SUL nor SUL accumulation could be detected in A007-positive cells lacking galactocerebroside (i.e., A007+GalC- progenitor cells) present early in development. These data suggest that A007 also recognizes an antigen, named proligodendroblast antigen (POA), that appears during the late stage of oligodendrocyte progenitor development prior to the expression by oligodendrocytes of SUL and GalC. We have previously reported that monoclonal antibody O4 also recognizes not only SUL and seminolipid, but in addition an antigen that appears prior to the expression of SUL and galactocerebroside. In the present study all A007+ cells were also O4+ (and vice versa), and the developmental patterns of the two antibodies appeared to be identical. We conclude that (1) A007 is similar or identical to O4 with respect to its antigenic specificity, and (2) during oligodendrocyte lineage progression both antibodies react first with antigen POA on the surface of the oligodendrocyte progenitor cell prior to the expression of SUL [i.e., A007+O4+(POA+)SUL-GalC- proligodendroblasts], and only later with SUL as terminally differentiating oligodendrocytes emerge (i.e., A007+O4+SUL+GalC+ oligodendrocytes).