Modifications to rat lens major intrinsic protein in selenite-induced cataract.
(73/33178)
PURPOSE: To identify modifications to rat lens major intrinsic protein (MIP) isolated from selenite-induced cataract and to determine whether m-calpain (EC 3.4.22.17) is responsible for cleavage of MIP during cataractogenesis. METHODS: Cataracts were induced in rats by a single injection of sodium selenite. Control and cataract lenses were harvested on day 16 and dissected into cortical and nuclear regions. Membranes were washed with urea buffer followed by NaOH. The protein was reduced/alkylated, delipidated, and cleaved with cyanogen bromide (CNBr). Cleavage products were fractionated by high-performance liquid chromatography (HPLC), and peptides were characterized by mass spectrometry and tandem mass spectrometry. MIP cleavage by m-calpain was carried out by incubation with purified enzyme, and peptides released from the membrane were analyzed by Edman sequencing. RESULTS: The intact C terminus, observed in the control nuclear and cataractous cortical membranes, was not observed in the cataractous nuclear membranes. Mass spectrometric analysis revealed heterogeneous cleavage of the C terminus of MIP in control and cataract nuclear regions. The major site of cleavage was between residues 238 and 239, corresponding to the major site of in vitro cleavage by m-calpain. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis indicated that in vivo proteolysis during cataract formation also included sites closer to the C terminus not produced by m-calpain in vitro. Evidence for heterogeneous N-terminal cleavage was also observed at low levels with no differences between control and cataractous lenses. The major site of phosphorylation was determined to be at serine 235. CONCLUSIONS: Specific sites of MIP N- and C-terminal cleavage in selenite-induced cataractous lenses were identified. The heterogeneous cleavage pattern observed suggests that m-calpain is not the sole enzyme involved in MIP C-terminal processing in rat lens nuclei. (+info)
Proliferative responses to human immunodeficiency virus type 1 (HIV-1) gp120 peptides in HIV-1-infected individuals immunized with HIV-1 rgp120 or rgp160 compared with nonimmunized and uninfected controls.
(74/33178)
The proliferative responses to a series of peptides constituting the human immunodeficiency virus type 1 (HIV-1) gp120 sequence were evaluated in 19 HIV-1-infected rgp160 vaccine recipients, 17 HIV-1-infected rgp120 vaccine recipients, 15 HIV-1-infected placebo recipients, and 18 HIV-1-uninfected controls. Many regions of the gp120 molecule were found to contribute proliferative epitopes, although there were clearly regions of relative dominance and silence. Vaccine recipients tended to have broader, more robust, and more frequent peptide recognition than the placebo recipients. Despite the considerable variability in the pattern of peptide recognition among individuals, there was a striking similarity between the rgp160 and rgp120 vaccinee groups as a whole. Low-risk HIV-1-uninfected individuals may react to a few peptides within the gp120 sequence as well, despite a lack of significant response to the whole gp120 protein. (+info)
Conformational changes in the A3 domain of von Willebrand factor modulate the interaction of the A1 domain with platelet glycoprotein Ib.
(75/33178)
Bitiscetin has recently been shown to induce von Willebrand factor (vWF)-dependent aggregation of fixed platelets (Hamako J, et al, Biochem Biophys Res Commun 226:273, 1996). We have purified bitiscetin from Bitis arietans venom and investigated the mechanism whereby it promotes a form of vWF that is reactive with platelets. In the presence of bitiscetin, vWF binds to platelets in a dose-dependent and saturable manner. The binding of vWF to platelets involves glycoprotein (GP) Ib because it was totally blocked by monoclonal antibody (MoAb) 6D1 directed towards the vWF-binding site of GPIb. The binding also involves the GPIb-binding site of vWF located on the A1 domain because it was inhibited by MoAb to vWF whose epitopes are within this domain and that block binding of vWF to platelets induced by ristocetin or botrocetin. However, in contrast to ristocetin or botrocetin, the binding site of bitiscetin does not reside within the A1 domain but within the A3 domain of vWF. Thus, among a series of vWF fragments, 125I-bitiscetin only binds to those that overlap the A3 domain, ie, SpIII (amino acid [aa] 1-1365), SpI (aa 911-1365), and rvWF-A3 domain (aa 920-1111). It does not bind to SpII corresponding to the C-terminal part of vWF subunit (aa 1366-2050) nor to the 39/34/kD dispase species (aa 480-718) or T116 (aa 449-728) overlapping the A1 domain. In addition, bitiscetin that does not bind to DeltaA3-rvWF (deleted between aa 910-1113) has no binding site ouside the A3 domain. The localization of the binding site of bitiscetin within the A3 domain was further supported by showing that MoAb to vWF, which are specific for this domain and block the interaction between vWF and collagen, are potent inhibitors of the binding of bitiscetin to vWF and consequently of the bitiscetin-induced binding of vWF to platelets. Thus, our data support the hypothesis that an interaction between the A1 and A3 domains exists that may play a role in the function of vWF by regulating the ability of the A1 domain to bind to platelet GPIb. (+info)
Inflammatory cytokines and vascular endothelial growth factor stimulate the release of soluble tie receptor from human endothelial cells via metalloprotease activation.
(76/33178)
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)
HLA-DM and invariant chain are expressed by thyroid follicular cells, enabling the expression of compact DR molecules.
(77/33178)
Thyroid follicular cells (TFC) in Graves' disease (GD) hyperexpress HLA class I and express ectopic HLA class II molecules, probably as a consequence of cytokines produced by infiltrating T cells. This finding led us to postulate that TFC could act as antigen-presenting cells, and in this way be responsible for the induction and/or maintenance of the in situ autoimmune T cell response. Invariant chain (li) and HLA-DM molecules are implicated in the antigen processing and presentation by HLA class II molecules. We have investigated the expression of these molecules by TFC from GD glands. The results demonstrate that class II+ TFC from GD patients also express li and HLA-DM, and this expression is increased after IFN-gamma stimulation. The level of HLA-DM expression by TFC was low but sufficient to catalyze peptide loading into the HLA class II molecules and form stable HLA class II-peptide complexes expressed at the surface of TFC. These results have implications for the understanding of the possible role of HLA class II+ TFC in thyroid autoimmune disease. (+info)
Effects of hypertension, diabetes mellitus, and hypercholesterolemia on endothelin type B receptor-mediated nitric oxide release from rat kidney.
(78/33178)
BACKGROUND: Although endothelin-1 is a potent vasoconstrictor peptide, stimulation of endothelin type B receptor (ETBR) causes bidirectional changes in vascular tone, ie, vasodilation and vasoconstriction. Roles of ETBR in pathological conditions are largely unknown. METHODS AND RESULTS: We studied the effect of BQ-3020, a highly selective ETBR agonist, on renal vascular resistance and nitric oxide (NO) release in the isolated, perfused kidney of rats with hypertension, diabetes mellitus, and hypercholesterolemia. Immunohistochemistry of endothelial NO synthase and ETBR was also examined. Infusion of BQ-3020 at concentrations of +info)
Phosphorylation of tyrosine 992, 1068, and 1086 is required for conformational change of the human epidermal growth factor receptor c-terminal tail.
(79/33178)
We reported previously that a conformation-specific antibody, Ab P2, to a 16-amino acid peptide (Glu-Gly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-Leu-Arg) of the cytoplasmic domain of the beta-type platelet-derived growth factor receptor also recognizes the epidermal growth factor (EGF) receptor. Although the antibody is not directed to phosphotyrosine, it recognizes in immunoprecipitation the activated and hence phosphorylated form of both receptors. In P2 peptide, there are two tripeptide sequences, Asp-Glu-Glu and Tyr-Gln-Gln, that are also present in the EGF receptor. Our present studies using either EGF receptor C-terminal deletion mutants or point mutations (Tyr-->Phe) and our previous studies on antibody inhibition by P2-derived peptides suggest that Gln-Gln in combination with Asp-Glu-Glu forms a high-affinity complex with Ab P2 and that such complex formation is dependent on tyrosine phosphorylation. Of the five phosphate acceptor sites in the EGF receptor, clustered in the extreme C-terminal tail, phosphorylation of three tyrosine residues (992, 1068, and 1086) located between Asp-Glu-Glu and Gln-Gln is necessary for Ab P2 binding. In contrast, the acceptor sites Tyr 1173 and 1148 play no role in the conformation change. Asp-Glu-Glu and Gln-Gln are located 169 amino acids apart, and it is highly likely that the interactions among three negatively charged phosphotyrosine residues in the receptor C terminus may result in the bending of the peptide chain in such a way that these two peptides come close to each other to form an antibody-binding site. Such a possibility is also supported by our finding that receptor dephosphorylation results in complete loss of Ab P2-binding activity. In conclusion, we have identified a domain within the cytoplasmic part of the EGF receptor whose conformation is altered by receptor phosphorylation; furthermore, we have identified the tyrosine residues that positively regulate this conformation. (+info)
Cluster of differentiation antigen 4 (CD4) endocytosis and adaptor complex binding require activation of the CD4 endocytosis signal by serine phosphorylation.
(80/33178)
Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56(lck), undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56(lck), we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues. (+info)