Treponema denticola outer membrane enhances the phagocytosis of collagen-coated beads by gingival fibroblasts.
(17/31038)
Human gingival fibroblasts (HGFs) degrade collagen fibrils in physiological processes by phagocytosis. Since Treponema denticola outer membrane (OM) extract perturbs actin filaments, important structures in phagocytosis, we determined whether the OM affects collagen phagocytosis in vitro by HGFs. Phagocytosis was measured by flow cytometric assessment of internalized collagen-coated fluorescent latex beads. Confluent HGFs pretreated with T. denticola ATCC 35405 OM exhibited an increase in the percentage of collagen phagocytic cells (phagocytosis index [PI]) and in the number of beads per phagocytosing cell (phagocytic capacity [PC]) compared with untreated controls. The enhancement was swift (within 15 min) and was still evident after 1 day. PI and PC of HGFs for bovine serum albumin (BSA)-coated beads were also increased, indicating a global increase in phagocytic processes. These results contrasted those for control OM from Veillonella atypica ATCC 17744, which decreased phagocytosis. The T. denticola OM-induced increase in bead uptake was eliminated by heating the OM and by depolymerization of actin filaments by cytochalasin D treatment of HGFs. Fluid-phase accumulation of lucifer yellow was enhanced in a saturable, concentration-dependent, transient manner by the T. denticola OM. Our findings were not due to HGF detachment or cytotoxicity in response to the T. denticola OM treatment since the HGFs exhibited minimal detachment from the substratum; they did not take up propidium iodide; and there was no change in their size, granularity, or content of sub-G1 DNA. We conclude that a heat-sensitive component(s) in T. denticola OM extract stimulates collagen phagocytosis and other endocytic processes such as nonspecific phagocytosis and pinocytosis by HGFs. (+info)
Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the beta 3A subunit of the AP-3 adaptor.
(18/31038)
Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defective lysosome-related organelles. Here, we report the identification of two HPS patients with mutations in the beta 3A subunit of the heterotetrameric AP-3 complex. The patients' fibroblasts exhibit drastically reduced levels of AP-3 due to enhanced degradation of mutant beta 3A. The AP-3 deficiency results in increased surface expression of the lysosomal membrane proteins CD63, lamp-1, and lamp-2, but not of nonlysosomal proteins. These differential effects are consistent with the preferential interaction of the AP-3 mu 3A subunit with tyrosine-based signals involved in lysosomal targeting. Our results suggest that AP-3 functions in protein sorting to lysosomes and provide an example of a human disease in which altered trafficking of integral membrane proteins is due to mutations in a component of the sorting machinery. (+info)
Interferon-alpha does not improve outcome at one year in patients with diffuse cutaneous scleroderma: results of a randomized, double-blind, placebo-controlled trial.
(19/31038)
OBJECTIVE: To determine whether interferon-alpha (IFNalpha) reduces the severity of skin involvement in early (<3 years) diffuse scleroderma. METHODS: In a randomized, placebo-controlled, double-blind trial, 35 patients with early scleroderma received subcutaneous injections of either IFNalpha (13.5 x 10(6) units per week in divided doses) or indistinguishable placebo. Outcomes assessed were the modified Rodnan skin score, as determined by a single observer at baseline, 6 months, and 12 months, as well as data on renal, cardiac, and lung function. Pre- and posttreatment skin biopsy samples were analyzed and blood was obtained for assessment of procollagen peptide levels. RESULTS: There were 11 withdrawals from the IFNalpha group and 3 from the placebo group due to either toxicity, lack of efficacy, or death. In the intent-to-treat analysis, there was a greater improvement in the skin score in the placebo group between 0 and 12 months (mean change IFNalpha -4.7 versus placebo -7.5; P = 0.36). There was also a greater deterioration in lung function in patients receiving active therapy, as assessed by either the forced vital capacity (mean change IFNalpha -8.2 versus placebo +1.3; P = 0.01) or the diffusing capacity for carbon monoxide (mean change IFNalpha -9.3 versus placebo +4.7; P = 0.002). Skin biopsy showed no significant decrease in collagen synthesis in the IFNalpha group, and no significant differences in the levels of procollagen peptides were seen between the 2 groups. CONCLUSION: This study suggests that IFNalpha is of no value in the treatment of scleroderma, and that it may in fact be deleterious. (+info)
Modulation of basal intracellular calcium by inverse agonists and phorbol myristate acetate in rat-1 fibroblasts stably expressing alpha1d-adrenoceptors.
(20/31038)
In rat-1 fibroblasts stably expressing alpha1d-adrenoceptors BMY 7378, phentolamine, chloroethylclonidine and 5-methyl urapidil decreased basal [Ca2+]i. WB 4101 induced a very small effect on this parameter but when added before the other antagonists it blocked their effect. All these agents inhibited the action of norepinephrine. Phorbol myristate acetate also blocked the effect of norepinephrine and decreased basal [Ca2+]i. Staurosporine inhibited these effects of the phorbol ester. Our results suggest that: (1) alpha1d-adrenoceptors exhibit spontaneous ligand-independent activity, (2) BMY 7378, phentolamine, chloroethylclonidine and 5-methyl urapidil act as inverse agonists and (3) protein kinase C activation blocks spontaneous and agonist-stimulated alpha1d-adrenoceptor activity. (+info)
Requirement of FADD for tumor necrosis factor-induced activation of acid sphingomyelinase.
(21/31038)
The generation of mice strains deficient for select members of the signaling complex of the 55-kDa tumor necrosis factor receptor (TNF-R55) has allowed the assignment of specific cellular responses to distinct TNF-R55-associated proteins. In particular, the TNF-R55-associated protein FADD seems to be responsible for recruitment and subsequent activation of caspase 8. In this report we demonstrate the requirement of FADD for TNF-induced activation of endosomal acid sphingomyelinase (A-SMase). In primary embryonic fibroblasts from FADD-deficient mice the activation of A-SMase by TNF-R55 ligation was almost completely impaired. This effect is specific in that other TNF responses like activation of NF-kappaB or neutral (N-)SMase remained unaffected. In addition, interleukin-1-induced activation of A-SMase in FADD-deficient cells was unaltered. In FADD-/- embryonic fibroblasts reconstituted by transfection with a FADD cDNA expression construct, the TNF responsiveness of A-SMase was restored. The results of this study suggest that FADD, in addition to its role in triggering a proapoptotic caspase cascade, is required for TNF-induced activation of A-SMase. (+info)
Identification of a subpopulation of lymphocytes in human peripheral blood cytotoxic to autologous fibroblasts.
(22/31038)
A naturally occurring subpopulation of human peripheral blood lymphocytes is cytotoxic to autologous and/or allogeneic fibroblasts. The autocytotoxic lymphocytes have a receptor for the third component of complement and for aggregated gamma globulin, do not form rosettes with sheep red blood cells, and are not removed by passage through nylon. The autocytotoxic subpopulation is not present in the thymus and tonsils of normal children or in the peripheral blood of individuals with X-linked agammaglobulinemia. Fibroblast absorption experiments demonstrate that the autocytotoxic cells are "sensitized" to antigens expressed on allogeneic fibroblasts in addition to the antigens expressed on autologous cells. Some normal individuals have a second subpopulation of lymphocytes that may "regulate" the autocytotoxic cells. The relevance of these observations to the murine autocytotoxic cells is discussed. (+info)
Conformational difference between nuclear and cytoplasmic actin as detected by a monoclonal antibody.
(23/31038)
Using a reconstituted complex of profilin and skeletal muscle actin as an antigen, we generated a monoclonal mouse antibody against actin, termed 2G2. As revealed by immunoblots of proteolytic actin fragments and by pepscan analysis, the antibody recognises a nonsequential epitope on actin which is located within three different regions of the sequence, consisting of aa131-139, aa155-169, and aa176-187. In the actin model derived from X-ray diffraction, these sequences lie spatially close together in the region of the nucleotide-binding cleft, but do not form a coherent patch. In immunoblots, 2G2 reacts with all SDS-denatured actin isoforms and with actins of many vertebrates. In contrast, its immunofluorescence reactivity is highly selective and fixation-dependent. In fibroblasts and myogenic cells, fixed and extracted by formaldehyde/detergent, stress fibres or myofibrils, respectively, remained unstained. Likewise, after microinjection into living cells, 2G2 did not bind to such microfilament bundles. Extraction of myosin and tropomyosin did not alter this pattern indicating that the lack in reactivity is probably not due to epitope-masking by actin-binding proteins. More likely, the reason for the lack of reactivity with filamentous actin is that its epitope is not accessible in F-actin. However, the antibody revealed a distinct pattern of nuclear dots in differentiated myogenic cells but not in myoblasts, and of fibrillar structures in nuclei of Xenopus oocytes. In contrast, after methanol treatment, a 2G2-specific staining of stress fibres and myofibrils was observed, but no nuclear dot staining. We conclude that 2G2, in addition to binding to SDS- and methanol-denatured actin, recognises a specific conformation of native actin which is present in the nucleus and specified by compaction of the antibody-reactive region into a coherent patch. This conformation is apparently present in differentiated myogenic cells and oocytes, but not in cytoplasmic actin filament bundles. (+info)
Regulation of p190 Rho-GAP by v-Src is linked to cytoskeletal disruption during transformation.
(24/31038)
The v-Src oncoprotein perturbs the dynamic regulation of the cellular cytoskeletal and adhesion network by a mechanism that is poorly understood. Here, we have examined in detail the effects of a temperature-dependent v-Src protein on the regulation of p190 RhoGAP, a GTPase activating protein (GAP) that has been implicated in disruption of the organised actin cytoskeleton, and addressed the dependence of v-Src-induced stress fibre loss on inhibition of Rho activity. We found that activation of v-Src induced association of tyrosine phosphorylated p190 with p120(RasGAP) and stimulation of p120(RasGAP)-associated RhoGAP activity, although p120(RasGAP) itself was not a target for phosphorylation by v-Src in chicken embryo cells. These events required the catalytic activity of v-Src and were linked to loss of actin stress fibres during morphological transformation and not mitogenic signalling. Furthermore, these effects were rapidly reversible since switching off v-Src led to dissociation of the p190/p120(RasGAP) complex, inactivation of p120(RasGAP)-associated RhoGAP activity and re-induction of actin stress fibres. In addition, transient transfection of Val14-RhoA, a constitutively active Rho protein that is insensitive to RhoGAPs, suppressed v-Src-induced stress fibre loss and cell transformation. Thus, we show here for the first time that an activated Src kinase requires the inactivation of Rho-mediated actin stress fibre assembly to induce its effects on actin disorganisation. Moreover, our work supports p190 as a strong candidate effector of v-Src-induced cytoskeletal disruption, most likely mediated by antagonism of the cellular function of Rho. (+info)