Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin-green fluorescent protein. (33/15188)

Fibronectin (FN) forms the primitive fibrillar matrix in both embryos and healing wounds. To study the matrix in living cell cultures, we have constructed a cell line that secretes FN molecules chimeric with green fluorescent protein. These FN-green fluorescent protein molecules were assembled into a typical matrix that was easily visualized by fluorescence over periods of several hours. FN fibrils remained mostly straight, and they were seen to extend and contract to accommodate movements of the cells, indicating that they are elastic. When fibrils were broken or detached from cells, they contracted to less than one-fourth of their extended length, demonstrating that they are highly stretched in the living culture. Previous work from other laboratories has suggested that cryptic sites for FN assembly may be exposed by tension on FN. Our results show directly that FN matrix fibrils are not only under tension but are also highly stretched. This stretched state of FN is an obvious candidate for exposing the cryptic assembly sites.  (+info)

Mixed agonist-antagonist properties of clozapine at different human cloned muscarinic receptor subtypes expressed in Chinese hamster ovary cells. (34/15188)

We recently reported that clozapine behaves as a partial agonist at the cloned human m4 muscarinic receptor subtype. In the present study, we investigated whether the drug could elicit similar effects at the cloned human m1, m2, and m3 muscarinic receptor subtypes expressed in the Chinese hamster ovary (CHO) cells. Clozapine elicited a concentration-dependent stimulation of [3H]inositol phosphates accumulation in CHO cells expressing either the m1 or the m3 receptor subtype. Moreover, clozapine inhibited forskolin-stimulated cyclic AMP accumulation and enhanced [35S] GTP gamma S binding to membrane G proteins in CHO cells expressing the m2 receptor. These agonist effects of clozapine were antagonized by atropine. The intrinsic activity of clozapine was lower than that of the full cholinergic agonist carbachol, and, when the compounds were combined, clozapine potently reduced the receptor responses to carbachol. These data indicate that clozapine behaves as a partial agonist at different muscarinic receptor subtypes and may provide new hints for understanding the receptor mechanisms underlying the antipsychotic efficacy of the drug.  (+info)

Characterization of the interaction between the herpes simplex virus type I Fc receptor and immunoglobulin G. (35/15188)

Herpes simplex virus type I (HSV-1) virions and HSV-1-infected cells bind to human immunoglobulin G (hIgG) via its Fc region. A complex of two surface glycoproteins encoded by HSV-1, gE and gI, is responsible for Fc binding. We have co-expressed soluble truncated forms of gE and gI in Chinese hamster ovary cells. Soluble gE-gI complexes can be purified from transfected cell supernatants using a purification scheme that is based upon the Fc receptor function of gE-gI. Using gel filtration and analytical ultracentrifugation, we determined that soluble gE-gI is a heterodimer composed of one molecule of gE and one molecule of gI and that gE-gI heterodimers bind hIgG with a 1:1 stoichiometry. Biosensor-based studies of the binding of wild type or mutant IgG proteins to soluble gE-gI indicate that histidine 435 at the CH2-CH3 domain interface of IgG is a critical residue for IgG binding to gE-gI. We observe many similarities between the characteristics of IgG binding by gE-gI and by rheumatoid factors and bacterial Fc receptors such as Staphylococcus aureus protein A. These observations support a model for the origin of some rheumatoid factors, in which they represent anti-idiotypic antibodies directed against antibodies to bacterial and viral Fc receptors.  (+info)

Disulfide bond structure and N-glycosylation sites of the extracellular domain of the human interleukin-6 receptor. (36/15188)

The high affinity interleukin-6 (IL-6) receptor is a hexameric complex consisting of two molecules each of IL-6, IL-6 receptor (IL-6R), and the high affinity converter and signaling molecule, gp130. The extracellular "soluble" part of the IL-6R (sIL-6R) consists of three domains: an amino-terminal Ig-like domain and two fibronectin-type III (FN III) domains. The two FN III domains comprise the cytokine-binding domain defined by a set of 4 conserved cysteine residues and a WSXWS sequence motif. Here, we have determined the disulfide structure of the human sIL-6R by peptide mapping in the absence and presence of reducing agent. Mass spectrometric analysis of these peptides revealed four disulfide bonds and two free cysteines. The disulfides Cys102-Cys113 and Cys146-Cys157 are consistent with known cytokine-binding domain motifs, and Cys28-Cys77 with known Ig superfamily domains. An unusual cysteine connectivity between Cys6-Cys174, which links the Ig-like and NH2-terminal FN III domains causing them to fold back onto each other, has not previously been observed among cytokine receptors. The two free cysteines (Cys192 and Cys258) were detected as cysteinyl-cysteines, although a small proportion of Cys258 was reactive with the alkylating agent 4-vinylpyridine. Of the four potential N-glycosylation sites, carbohydrate moieties were identified on Asn36, Asn74, and Asn202, but not on Asn226.  (+info)

Trimming and readdition of glucose to N-linked oligosaccharides determines calnexin association of a substrate glycoprotein in living cells. (37/15188)

To analyze the role of glucose trimming and reglucosylation in the binding of substrate proteins to calnexin in the endoplasmic reticulum (ER) of living cells, we made use of the thermosensitive vesicular stomatitis virus tsO45 glycoprotein (G protein). At nonpermissive temperature the G protein failed to fold completely and remained bound to calnexin. When the cells were shifted to permissive temperature, complete folding occurred accompanied by glucosidase-mediated elimination of calnexin-G protein complexes. If release from calnexin was blocked during the temperature shift by inhibiting the glucosidases, folding occurred, albeit at a reduced rate. In contrast, when unfolded by a shift from permissive to nonpermissive temperature, the G protein was reglucosylated rapidly and became capable of rebinding to calnexin. The rate at which calnexin binding occurred showed a 20-min delay that was explained by accumulation of the G protein in calnexin-free exit sites of the ER. These contained the glucosyltransferase responsible for reglucosylation of misfolded glycoproteins but had little or no calnexin. After unfolding and reglucosylation, the G proteins moved slowly from these structures back to the ER where they reassociated with the chaperone. Taken together, these results in live cells fully supported the lectin-only model of calnexin function. The ER exit sites emerged as a potentially important location for components of the quality control system.  (+info)

Inflammatory cytokines and vascular endothelial growth factor stimulate the release of soluble tie receptor from human endothelial cells via metalloprotease activation. (38/15188)

Activation of endothelial cells, important in processes such as angiogenesis, is regulated by cell surface receptors, including those in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be modulated by phosphorylation, turnover, or proteolytic release of a soluble extracellular domain. Previously, we demonstrated that release of soluble tie-1 receptor from endothelial cells by phorbol myristate acetate (PMA) is mediated through protein kinase C and a Ca2+-dependent protease. In this study, the release of soluble tie-1 was shown to be stimulated by inflammatory cytokines and vascular endothelial growth factor (VEGF), but not by growth factors such as basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFalpha). Release of soluble tie by tumor necrosis factor alpha (TNFalpha) or VEGF occurred within 10 minutes of stimulation and reached maximal levels within 60 minutes. Specificity was shown by fluorescence-activated cell sorting (FACS) analysis; endothelial cells exhibited a significant decrease in cell surface tie-1 expression in response to TNF, whereas expression of epidermal growth factor receptor (EGF-R) and CD31 was stable. In contrast, tie-1 expression on megakaryoblastic UT-7 cells was unaffected by PMA or TNFalpha. Sequence analysis of the cleaved receptor indicated that tie-1 was proteolyzed at the E749/S750 peptide bond in the proximal transmembrane domain. Moreover, the hydroxamic acid derivative BB-24 demonstrated dose-dependent inhibition of cytokine-, PMA-, and VEGF-stimulated shedding, suggesting that the tie-1 protease was a metalloprotease. Protease activity in a tie-1 peptide cleavage assay was (1) associated with endothelial cell membranes, (2) specifically activated in TNFalpha-treated cells, and (3) inhibited by BB-24. Additionally, proliferation of endothelial cells in response to VEGF, but not bFGF, was inhibited by BB-24, suggesting that the release of soluble tie-1 receptor plays a role in VEGF-mediated proliferation. This study demonstrated that the release of soluble tie-1 from endothelial cells is stimulated by inflammatory cytokines and VEGF through the activation of an endothelial membrane-associated metalloprotease.  (+info)

Interactive effects of inhibitors of poly(ADP-ribose) polymerase and DNA-dependent protein kinase on cellular responses to DNA damage. (39/15188)

DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase (PARP) are activated by DNA strand breaks and participate in DNA repair. We investigated the interactive effects of inhibitors of these enzymes [wortmannin (WM), which inhibits DNA-PK, and 8-hydroxy-2-methylquinazolin-4-one (NU1025), a PARP inhibitor] on cell survival and DNA double-strand break (DSB) and single-strand break (SSB) rejoining in Chinese hamster ovary-K1 cells following exposure to ionizing radiation (IR) or temozolomide. WM (20 microM) or NU1025 (300 microM) potentiated the cytotoxicity of IR with dose enhancement factors at 10% survival (DEF10) values of 4.5 +/- 0.6 and 1.7 +/- 0.2, respectively. When used in combination, a DEF10 of 7.8 +/- 1.5 was obtained. WM or NU1025 potentiated the cytotoxicity of temozolomide, and an additive effect on the DEF10 value was obtained with the combined inhibitors. Using the same inhibitor concentrations, their single and combined effects on DSB and SSB levels following IR were assessed by neutral and alkaline elution. Cells exposed to IR were post-incubated for 30 min to allow repair to occur. WM or NU1025 increased net DSB levels relative to IR alone (DSB levels of 1.29 +/- 0.04 and 1.20 +/- 0.05, respectively, compared with 1.01 +/- 0.03 for IR alone) and the combination had an additive effect. WM had no effect on SSB levels, either alone or in combination with NU1025. SSB levels were increased to 1.27 +/- 0.05 with NU1025 compared with IR alone, 1.02 +/- 0.04. The dose-dependent effects of the inhibitors on DSB levels showed that they were near maximal by 20 microM WM and 300 microM NU1025. DSB repair kinetics were studied. Both inhibitors increased net DSB levels over a 3 h time period; when they were combined, net DSB levels at 3 h were identical to DSB levels immediately post-IR. The combined use of DNA repair inhibitors may have therapeutic potential.  (+info)

MSH3 deficiency is not sufficient for a mutator phenotype in Chinese hamster ovary cells. (40/15188)

In the yeast Saccharomyces cerevisiae, the mutS homolog protein products MSH3 and MSH6, each in cooperation with MSH2, play well-defined and specific roles in the repair of DNA mismatches and nucleotide loops. The discrete functions of the human homologs hMSH3 and hMSH6 are less clear and current evidence suggests that the substrate specificity of these proteins may be less strict. To determine the role of MSH3 in mammalian mismatch repair, we employed MSH3-deficient Chinese hamster ovary (CHO) cell lines. No significant changes in mutation rate were detected in the MSH3-deficient strain and there were no differences in sensitivity to DNA-damaging agents. Further analysis of hprt mutants did not show a MSH3-dependent shift in the mutant spectrum. Interestingly, thorough examination of four dinucleotide microsatellite regions revealed instability at only one locus in one of the MSH3-deficient cell lines. These data support the idea of a high degree of redundancy in the function of the MutS homologs MSH3 and MSH6, at least with respect to the control of microsatellite instability.  (+info)