Erythrocyte actin and spectrin. Interactions with muscle contractile and regulatory proteins.

Actin and spectrin were isolated from washed red blood cell membranes. Spectrin bound and polymerized erythrocyte actin in the absence of potassium. Spectrin coated into polystyrene latex particles bound 8--9 mol of erythrocyte actin per mol of spectrin when actin was in its depolymerized state. Spectrin enhanced the interaction of erythrocyte actin with muscle myosin as manifested by changes in Mg2+-ATPase activity. A similar enhancement also was observed with muscle alpha-actinin while muscle tropomyosin abolished these effects. The data suggest that spectrin may play the role of polymerizing factor as well as the anchoring site for erythrocyte actin just as alpha-actinin is the anchoring site for actin filaments in muscle and other non-muscle cells.

Immunological identification of complex proteins resolved by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis.

Immunological identification of an antigen resolved from a protein complex by sodium dodecyl sulfate polyacrylamide gel electrophoresis has been attained. The identification is based on the formation of immunoprecipitin lines after the antigen diffuses laterally from acrylamide gel transverse slices into a surrounding agarose gel. This technique was designed for study of contractile and regulatory protein complexes of non-muscle cells where the scarcity of tissue precludes easy purification or high yield of muscle-like proteins. It complements double-gel immunodiffusion or immunoelectrophoresis and its use may be extended to other protein complexes.

Synthetic guinea pig insulin B-chain C-terminal decapeptide: a novel immunogen for generating immunocytochemical-grade antisera.

Antisera to guinea pig insulin are not commonly available, largely because of the short supply and limited immunogenicity of the intact hormone. To overcome these problems we have employed a novel reagent, synthetic guinea pig insulin B-chain C-terminal decapeptide, as a hapten for raising antibodies that react with intact guinea pig insulin. The decapeptide, coupled to bovine serum albumin, was successfully used as an immunogen in rabbits. The resulting anti-serum was employed for immunocytochemical staining of guinea pig insulin in pancreatic sections. The specificity of the staining was verified by both pre-absorption and pre-immune serum controls. The utility of this new antiserum for investigations of guinea pig insulin physiology is discussed.

Statistical geometry of pancreatic islets.

Quantitative histomorphometric studies of the dynamics of growth and development of pancreatic islets in normal and pathological states pose substantial methodological and conceptual problems. We address these problems with the geometry of random fractals, and apply our methods to the analysis of islet regeneration in the alloxan-treated guinea-pig. In both experimental islet-regenerated and control animals, islet centres are found to cluster in similar fractal subsets of dimension strictly less than 3, in agreement with the postulated origin of islets along a system of ductules, and suggesting that regeneration follows the same mathematical dynamics as original islet formation.

Nonuniform distribution of occult blood in feces.

Inhibition of anti-Rh29 by erythrocytic stroma in feces was devised as a specific test for fecal occult blood. The sensitivity of this test was equivalent to that of a standard Hemoccult test, namely, 10(8) erythrocytes/g feces. Comparison of results of this test with results of Hemoccult tests of random stool specimens and of stools following ingestion of autologous blood revealed nonuniform distribution of occult blood in feces. The extent of nonuniformity was determined by testing samples of stool specimens following ingestion of 51Cr-labeled autologous blood. This allowed comparison of Hemoccult, inhibition of anti-Rh, and radioactivity, and showed that the three labels could separate in the feces and that some single small samples of feces could be relatively free of blood while blood was readily demonstrable in other portions. The variability of standard Hemoccult test was somewhat reduced by dispersing the feces in distilled water before performing the test.

Studies of antiproteolytic effects of glyburide on rat L6 myoblasts: comparisons with insulin.

Cultured L6 myoblasts afford considerable advantages for identifying and studying the insulin-like actions of test substances in a muscle-derived line. We have used this system to examine the interaction of the oral hypoglycemic sulfonylurea glyburide with bovine insulin on protein degradation and synthesis as well as on thymidine incorporation (as a measure of DNA synthesis) in these cells. Bovine insulin, at doses of 0.1 microgram/mL to 10 micrograms/mL, produced a dose-dependent inhibition of protein degradation (measured by release of trichloracetic acid (TCA)-soluble 14C-tyrosine from myoblasts into the culture medium) and increase in total protein content in the cultured myoblasts. At concentrations of 10 micrograms/mL, insulin achieved its maximal suppression of protein degradation (by nearly 50%) and increased cellular protein content (by 15%) over levels observed in the absence of added insulin. Glyburide, at concentrations at or above 1 microgram/mL, significantly suppressed protein degradation (up to 14%) and slightly augmented protein content of the cells. The effects of glyburide on protein degradation were additive with those of submaximally but not maximally effective concentrations of insulin, suggesting a common mechanism of action of the compounds. Both insulin and glyburide, at maximally effective doses, significantly depressed protein degradation as early as 2 to 6 hours after exposure. In addition, in a 24-hour labeling experiment, insulin stimulated tyrosine incorporation into TCA-insoluble protein and thymidine incorporation into DNA in the cells, whereas glyburide did not enhance these processes and, under certain conditions, inhibited them. These results demonstrate that glyburide, either alone or in concert with insulin, is capable of significantly inhibiting protein turnover in skeletal muscle-derived cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocyte troponin inhibitor-like protein: isolation and characterization.

A protein was isolated from a human erythrocyte lysate with an apparent molecular weight of 23,000--24,000 daltons. This protein was purified by batch DEAE cellulose followed by column DEAE cellulose chromatography and a gradient of NaCl. On sodium dodecyl sulfate acrylamide electrophoresis, the erythrocyte protein comigrated with muscle troponin inhibitor. An isoelectric precipitation (pH 9.25) was used for the separation of muscle troponin inhibitor from a complex with another troponin component. Both the erythrocyte protein and the muscle troponin inhibitor partially inhibited muscle myosin Ca2+ and K+-EDTA ATPase activity. Furthermore, they inhibited actin-activated Mg2+-ATPase of muscle myosin. The inhibitory effects were absent in the presence of muscle troponin calcium-binding component. Muscle troponin inhibitor and the erythrocyte troponin inhibitor-like protein bound to muscle myosin when myosin was precipitated twice at low ionic strength. The presence of a troponin inhibitor-like protein in erythrocytes suggests that it may be a component in the regulation of contractile activity.

Expression of a cholecystokinin precursor-related peptide in vertebrate and invertebrate tissues.

We have developed a radioimmunoassay for the nonapeptide predicted by cDNA sequence analysis to reside at the extreme C-terminus of the mouse cholecystokinin (CCK) precursor. Sensitivity of the assay is 1 pg synthetic CCK precursor-related peptide (CCK-PRP)/ml. The antibody has no cross-reactivity with cholecystokinin, gastrin, or a variety of other known neuropeptides. We have employed this assay to demonstrate the presence, in rodent brain, gut, and peripheral plasma, of peptides with immunological properties that are identical to, and gel filtration characteristics that are very similar to, those of the synthetic CCK-PRP. We have also detected a similar peptide in the culture media of a human CCK-producing tumor. The molar ratios of immunoreactive CCK-PRP/CCK vary widely among tissues of origin and during ontogeny, suggesting regional and developmental differences in the turnover rates or in posttranslational modification of the two peptides. Our studies suggest that peptides very similar to intact CCK-PRP are posttranslationally liberated from the cholecystokinin precursor in a variety of tissues and may have neurotransmitter and/or hormonal functions distinct from those of CCK. Relatively high quantities of material immunologically indistinguishable from CCK-PRP were also found in several coelenterate species, indicating that this epitope arose as early in evolution as did CCK.

Platelet cytoskeleton: immunofluorescence studies on thrombin-activated platelets.

Cytoskeletal proteins were isolated from chicken gizzard smooth muscle and from platelets and antibodies prepared against them. It was shown by indirect immunofluorescence technique that actin, alpha-actinin, and vinculin are not present on the surface of platelets. Physiologic concentrations of thrombin (0.04 to 0.5 U/ml) that induce platelet aggregation and release in the presence of calcium from freshly isolated platelets do not induce platelet changes resulting in the availability of the cytoskeleton to antibodies. Because the F(ab')2 fragments of anti-cytoskeletal proteins IgG do not inhibit thrombin-induced aggregation of platelets, the direct role of these proteins in thrombin-induced platelet aggregation, as with ADP and collagen, may be rejected. However, when freshly isolated platelets are treated with thrombin (1 U/ml), antibodies to actin, alpha-actinin, and vinculin stained the platelets; therefore, this demonstrates that thrombin at this high and probably nonphysiologic concentration induces a reorganization of the membrane components with the subsequent exposure of the proteins of the cytoskeleton. We demonstrate interaction between isolated actin and alpha-actinin but not vinculin with fibronectin. After stimulation of platelets by thrombin, certain cytoskeletal proteins may interact with subendothelial fibronectin and thereby promote and consolidate platelet adhesion.

Quantification and characterization of ACTH-related peptides produced by human peripheral blood mononuclear cells.

We have used a sensitive radioimmunoassay to quantify and characterize PBMC-associated immunoreactive ACTH (ACTH-IR). Mean ACTH content of freshly isolated human PBMCs was 3.8 +/- 0.72 pg (SEM) per 10(6) cells. During 3 days of incubation ACTH-IR in conditioned media of control PBMCs increased significantly, p less than 0.02. Gel filtration chromatography revealed a minor peak of ACTH-IR coeluting with ACTH (1-39) and a major peak coeluting with ACTH (11-24). Treatment with 15 nM CRH did not alter the amount of ACTH-IR secreted or its gel pattern. Synthetic ACTH (11-24), was radioiodinated and was used for binding experiments that demonstrated specific high- and low-affinity binding sites for ACTH (11-24) on a human T cell line. These results add support for a role of ACTH and related peptides in immune regulatory systems and suggest that cell-specific post-translational processing of POMC may generate an expanding number of biologically active moieties.

Tetracyclines inhibit intracellular muscle proteolysis in vitro.

Tetracycline antibiotics (TETs) have a recently discovered novel action: inhibition of extracellular metalloproteinase activity, especially that of collagenase and gelatinase. This property, now confirmed in 8 different laboratories using > 40 tissue sources, includes natural and semi-synthetic TETs as well as a chemically modified TET (CMT) devoid of antimicrobial activity. We have used 14C-Tyr biosynthetically labelled intracellular proteins in L-6 myoblast culture as a test system to assess intracellular proteolysis. Starvation accelerates proteolysis, which can be suppressed by agents such as insulin or serum. Minocycline, doxycycline, and CMT all retarded the rate of intracellular protein degradation in a dose dependent manner. These agents also demonstrated marked synergism with insulin. A CMT derivative (pyrazole) stripped of one of its metal chelation sites and lacking anti-collagenase activity, also lost its antiproteolytic effect. CMT at physiologic concentrations (< or = 5 micrograms/ml) had no effect on protein synthesis, but at 15 micrograms/ml (pharmacologic), a suppressive effect was noted. These findings demonstrate that TETs can inhibit protein degradation as well as synthesis in a mammalian muscle-derived cell line.

Expression of the cholecystokinin gene in pediatric tumors.

We have examined a wide range of cultured human tumor cell lines and found that a specific subset of tumors expresses the cholecystokinin (CCK) gene. All neuroepitheliomas (eight) and Ewing sarcoma (eight) cell lines that were tested express CCK RNA. In addition, two of six rhabdomyosarcoma cell lines also express the CCK gene, suggesting that rhabdomyosarcomas are probably heterogenous and that a subset may be similar to Ewing sarcoma and neuroepithelioma. Very few of the positive tumors express completely processed immunoreactive CCK. However, we have used a radioimmunoassay that detects the CCK precursor to demonstrate synthesis of CCK precursor-like peptides by all of the Ewing sarcoma and neuroepithelioma lines that were tested and by the rhabdomyosarcoma cell line that expresses CCK mRNA. These data demonstrate a consistent association of CCK gene expression with a specific group of human neoplasms. The data also add credence to the theory that Ewing sarcoma and neuroepithelioma are derived from the same transformed cell type. Finally, our results suggest that CCK gene expression may serve as a marker to distinguish these tumors, which are considered to be small-round-cell tumors of childhood, from other pediatric tumors.

Platelet cytoskeleton: immunofluorescence studies on ADP and collagen-activated platelets.

Immunofluorescence studies reveal that platelet changes induced by adenosine diphosphate and collagen do not include the reorganization of the cytoskeleton in such a way as to expose actin, alpha-actinin, or vinculin. However, when such platelets were made permeable by saponin, these cytoskeletal proteins were present. In studies with collagen, fluorescence was observed along the fibers at areas of platelet adhesion and where no platelets were seen by phase microscopy. No fluorescence was observed with collagen treated with platelet-poor plasma. Scanning electron microscopy of collagen samples treated with platelet-rich plasma revealed a fibrillar meshwork with single platelets, platelet aggregates, and nodular structures that were smaller in size than individual platelets. These nodular structures may represent remnants of platelets still attached to the collagen after platelet detachment has occurred. These tenacious collagen-platelet membrane-binding sites have associated with them cytoplasmic alpha-actinin and vinculin, proteins that have been proposed by others to anchor actin filaments to the plasma membrane.

alpha-Actinin and tropomyosin interactions with a hybrid complex of erythrocyte-actin and muscle-myosin.

alpha-Actinin isolated from dog muscle was used to incite antibodies in rabbits, Antibodies, purified by affinity chromatography on CNBr-Sepharose coupled with alpha-actinin and then ferritin-labeled were found to localize on the Z disc of muscle sarcomeres. Molecules of alpha-actinin as an adsorbed monolayer on the surface of polystyrene Lytron particles could bind muscle-actin and tropomyosin from solution. Both the ATPase activity and superprecipitation of an erythrocyte-actin and muscle-myosin hybrid actomyosin complex were altered by alpha-actinin, while tropomyosin diminished these alpha-actinin effects. The binding properties of alpha-actinin are consistent with those of an anchoring protein for microfilaments in nonmuscle cells.