Regulation of microtubule-organizing center orientation and actomyosin cytoskeleton rearrangement during immune interactions.

The reorganization of membrane, cytoskeletal and signaling molecules during immune interactions is critical for the generation of immune response. At the initiation of the T cell-antigen presenting cell (APC) interaction, antigen-independent weak adhesion forces allow the scanning of the APC surface by the T cell receptor for specific antigens. The stabilization of T cell-APC conjugates involves the segregation of membrane and intracellular signaling proteins, driven by reorganization of membrane microdomains and cytoskeletal changes. In early T cell-APC cognate interactions, the microtubular cytoskeleton undergoes drastic changes that lead to microtubule-organizing center (MTOC) reorientation to the vicinity of the cell-cell contact area. Recent data on the dynamics of MTOC redistribution and its influence in T cell-APC conjugate stabilization, together with the description of an increasing number of signaling molecules associated to this complex, underscore the key role of MTOC translocation in the T cell response. We focus on the mechanisms that control the early MTOC reorientation during T cell-APC interaction and the relevance of this process to T cell activation.

Antibody directed against the 142-148 sequence of the myosin heavy chain interferes with myosin-actin interaction.

It has been reported recently that the isolated and renatured 23-kDa N-terminal fragment of rabbit skeletal muscle myosin binds tightly to F-actin in an ATP-dependent manner [Muhlrad, A. (1989) Biochemistry 28, 4002-4010]. The binding to actin is of electrostatic nature and may involve a positively charged cluster of residues on the 23-kDa fragment stretching from Arg-143 to Arg-147. An octapeptide containing this positive cluster was synthesized and coupled to BSA through a cysteine residue added to the N-terminus of the peptide. Polyclonal antibody was raised against the BSA-coupled peptide in rabbits which recognized the N-terminal 23-kDa fragment of rabbit skeletal myosin subfragment 1, and a peptide comprised of residues 122-204 of the 23K fragment in Western blots. The purified antibody [IgG and F(ab)] inhibited the actin-activated ATPase activity of S1 without affecting its Mg2(+)- and K+(EDTA)-modulated ATPase activity. Both IgG and F(ab) decreased the binding of S1 to F-actin in a sedimentation assay, and actin inhibited the binding of both IgG and F(ab) to S1 in a competitive binding assay. The cysteine thiol of the synthetic octapeptide was labeled by the fluorescent thiol reagent monobromobimane, and the labeled peptide was found to bind to actin in a sedimentation assay. The results support the possibility that the positively charged Arg-143 to Arg-147 stretch of residues on the 23-kDa fragment participates in actin binding of myosin and may represent an essential constituent of the actin-S1 interface.

[Cytotoxic effect of the lymphocytes in ischemic heart disease]. / Tsitotoksicheskii éffekt limfotsitov pri ishemicheskoi bolezni serdtsa.

Coronary patients were shown to have a pool of sensitized killer T cells that produce a cytotoxic effect on target cells loaded with infarcted and intact myocardial antigens and on actomyosin proteins, and evidence of delayed hypersensitivity developing in association with coronary disease. Two kinds of immune shifts were identified with respect to the time of onset of this effect in acute myocardial infarction: in the first case, the appropriate level was exceeded within 2-3 weeks of the disease, and in the second case, within the first days in hospital. The disease tended to take a more severe course in patients with the second type of immune response.

Preservation of mesangium and immunohistochemically defined antigens in glomerular basement membrane isolated by detergent extraction.

To define the characteristics of isolated glomerular basement membrane (GBM), immunohistochemical and morphometric analyses have been carried out on rat and human tissues. Site-specific arrays of antigens were identified in detergent-isolated GBM in a distribution similar to that observed in intact kidney. In the human, fibronectin, procollagen IV, and collagen V were observed along the internal aspect of GBM continuous with antigenic sites in the mesangium. Another array of antigens was identified in the GBM but not within the mesangium--Goodpasture's antigen, bovine lens capsule type IV collagen, and amyloid P component. In addition, sites reactive with rabbit antiserum to laminin were present on both sides of the lamina densa as well as within the mesangial region. Actomyosin, a presumed mesangial cell antigen persisted in the mesangium of isolated GBM. Mesangial matrix was identified in detergent-isolated GBM in an amount equivalent to that present in intact glomeruli. Sonicated GBM contained the same antigens but it was not possible to quantitate the amount of mesangial material by immunofluorescence or morphometric analysis. The thickness of the lamina densa was greater in sonicated and detergent-treated rat GBM preparations than in native rat kidney. These studies demonstrated that isolated GBM is heterogeneous with respect to its antigenic constituents and in addition contains mesangial matrix, which is morphologically and immunohistochemically distinct from peripheral GBM.

Burn toxin and its competitin.

A "toxic factor' has been isolated and purified from scalded normal human skin; it is lethal to mice and toxic to HeLa and HEP2 cell lines in tissue culture. The toxic factor in both crude and purified form is antigenic in rabbits, producing an antisera that neutralizes the in vivo and in vitro effects of "burn toxin', similar to that of the convalescent sera of burned human subjects. The sonicate of the toxic glycoprotein named competitin is relatively non-lethal and protects against the lethal effect of the toxic factor. The action of the purified burn toxic factor and its competitin is at the ATP site of actomyosin preparations. The data presented suggests that the purified burn toxic factor and its competitin compete for the same receptor sites in the myocardium. A thesis is presented that states that it is vital to neutralize the toxic effects of burn breakdown tissue products in severely burned subjects before the vicious cycle of depressed immunological function and malnutrition ensues. Competitin produced in vitro from toxic factor(s) generated scalded normal human skin is offered as a means of neutralizing the toxic factors(s).

Attempts at detection of actomyosin associated with influenza virus.

Influenza virus strans A/Scotland/74, A/Hong Kong/68, A/Port Chalmers/73 and the MRC-12 recombinant were tested with immune antiserum against actomyosin. As shown by electron microscopy, the serum aggregated virus particles, but only after bromelain treatment (without haemagglutinin and neuraminidase spikes). In rocket electrophoresis the serum gave positive precipitation reaction with all the strains tested, and with virus from various hosts (chick embryo, monkey kidney cell culture, mice after adaptation). There fore the host protein presumably is present in the influenza virus structure irrespective of the strain or the host in which the virus is grown.

Inhibition of energy-dependent Ca2+ accumulation in fibroblasts by smooth muscle antibodies.

An antibody prepared against smooth muscle myosin interferes with active Ca2+ accumulation of fibroblasts. This provides further evidence for the existence of myosin at the cell surface.

Leukocyte migration test for the study of immune reactivity in cases of Graves disease associated with infiltrative ophthalmopathy.

The leukocyte migration test was carried out in 26 untreated cases of Graves disease, using crude thyroid extract, human eye muscle antigen and actomyosin as antigens. A significant reduction in the spontaneous (antigen-free) migration values was demonstrable in the cases of Graves disease, compared with the controls. The migration inhibition test gave positive results with the thyroid antigen in the hyperthyroid cases, in opposition to euthyroid ophthalmopathy, where it was found negative. The results were positive with retrobulbar muscle antigen in hyperthyroid and euthyroid infiltrative ophthalmopathy. In advanced cases of infiltrative ophthalmopathy, the results were positive with actomyosin as well.

Autoantibodies in sera of influenza patients.

Sera of the influenza patients and healthy controls were tested for some types of autoantibody (SMA, ANA, ABBA, AMA). They were detected in 83.8% of the patients' sera and in 16.6% of controls. SMA were present in 77.4%, ANA in 54.8%, and ABBA in 16.1% of the patient's sera. AMA were not detected. A majority of the sera contained more than one autoantibody type. The possible mechanisms of induction of the autoantibodies in virus infection and their possible role in disease are briefly discussed.

Anti-actomyosin. Influence on adhesive behaviour of eukaryotic cells and of Cuvierian tubules.

The effect of antisera against chicken gizzard smooth-muscle actomyosin and against pectoralis striated-muscle actomyosin on adhesive behaviour of eukaryotic cells (from sea urchin embryos and from a silicious sponge) and of Cuvierian tubules has been studied. The results with sea urchin cells, which require divalent cations for aggregation, showed that antiserum to chicken gizzard smooth-muscle actomyosin inhibited reaggreagation of trypsin-treated cells better than mechanically dissociated cells, while anti-chicken pectoralis striated-muscle had no effect. Primary reaggreagation of trypsin-dissociated sponge cells, in the presence of calcium and magnesium, is also inhibitable by anti-gizzard smooth-muscle but not by anti-pectoralis straited muscle. Anti-gizzard smooth-muscle had no effect on secondary reaggregation of sponge cells mediated by a soluble aggregation factor. Anti-gizzard smooth-muscle inhibited Cuvierian tubule adhesion.

A simple method for differentiating vascular smooth muscle cells and fibroblasts in tissue culture.

The morphologic differentiation of vascular smooth muscle cells and fibroblasts in tissue culture is difficult if not impossible. By direct immunofluorescence, it is possible to distinguish between vascular smooth muscle cells and fibroblasts after 6 to 10 days in tissue culture. Microfilaments appear from the 6th to the 10th day. After an incubation period of 30 minutes with antibody against smooth muscle actomyosin at room temperature, microfilaments are demonstrable in smooth muscle cells. In contrast, fibroblasts, if incubated for the same period, show strong nuclear fluorescence and a primary fluorescence of the cytoplasm, but filaments are not visible. If fibroblasts are incubated with antiactomyosin for one hour at 37 degrees C, however microfilaments are easily detectable. With this method it is possible to differentiate in a simple manner vascular smooth muscle cells from fibroblasts in a heterologous tissue culture.

Production of specific antibodies to contractile proteins, and their use in immunofluorescence microscopy. I. Antibodies to smooth and striated chicken muscle myosins.

Antibodies prepared against actomyosins can be shown to behave similarly, if not identically to more recently prepared antibodies against highly purified myosins. Details of the purification of the antigens, and of the production of antibodies to chick myosins from smooth gizzard muscle and from striated pectoral muscle are given. The antibody specificity appears to be directed against the heavy chains of the myosin molecules, since these antibodies specifically inhibit the myosin ATPase reaction, and since in situ staining of myosin polypeptide chains on an SDS gel using the antibodies in indirect fluorescence shows staining only in the heavy band region. Use of the antibodies in immunofluorescence microscopy suggest that the antibodies are tissue, but not species, specific. Example of their use in staining tissue sections are shown.

Human glomerular smooth muscle (mesangial) cells in culture.

Glomerular smooth muscle cells were grown and subcultured from human glomeruli in vitro. These cells, stained with antiserum to smooth muscle actomyosin and examined by immunofluorescence, had brightly stained intracellular fibrils similar to those seen in smooth muscle cells. Actomyosin fibrils were altered by conditions affecting actomyosin in vitro. Glomerular smooth muscle cells lacked the antihemophilic factor and blood group antigens present in endothelial cells, and are, therefore, most likely derived from the smooth muscle cells of the glomerular mesangium. By radio-labeled amino acid analysis, they synthesize a collagen differing from that of fibroblasts, and which probably differs from basement membrane collagen. Other cell types could be grown and subcultured using a different glomerular isolation technique, culture medium and method of subculture.

A solid-liquid biphasic model for characterization of properties of muscle and platelet contractile proteins.

Actomyosin, myosin, and actin from different sources are adsorbed, apparently as a monolayer, by polystyrene particles teins for 1 mg of Lytron were about 10-7 liters mol-1, while heterogeneity indices (alpha) varied from 0.70 to 1.0 presumably as a function of spontaneous aggregation in the liquid phase. Adsorption was irreversible. Orientation of absorbed molecules permitted association of bound muscle actin with platelet or muscle myosin. The association constant of the former reaction was 2.78 times 10-6 liters mol-1. Enzymatic properties of adsorbed actomyosin, Mg2+ATPase activity was abolished, but association of myosin with bound actin, or association of actin with bound myosin was accompanied by restoration of Mg2+ATPase activity. Every subunit of F-actin strands, unless F-actin had been fully depolymerized to G-actin, could bind myosin and activate Mg2+ATPase activity. Immunogenic characteristics of muscle myosin were enhanced by Lytron adsorption. Elicited antibodies showed selective specificity for an antigenic determinant located near or at the actin combining site of muscle myosin. Antibodies did not react with actomyosin. Antibodies prevented association of actin with muscle myosin because they inhibited both superprecipitation and development of Mg2+ATPase activity.