The protein phosphatases involved in cellular regulation. Antibody to protein phosphatase-2A as a probe of phosphatase structure and function

Antibody prepared against the catalytic subunit of protein phosphatase-2A from rabbit skeletal muscle, could completely inhibit this enzyme, but did not significantly affect the activities of protein phosphatases-1, 2B and 2C. The antibody was used to establish the following points. The three forms of protein phosphatase-2A that can be resolved by ion-exchange chromatography, termed 2A0, 2A1, and 2A2, share the same catalytic subunit. The antigenic sites on the catalytic subunit of protein phosphatase-2A remain accessible to the antibody, when the catalytic subunit is complexed with the other subunits of protein phosphatases-2A0, 2A1 and 2A2. The catalytic subunits of protein phosphatase-2A from rabbit skeletal muscle and rabbit liver are very similar, as judged by immunotitration experiments. Protein phosphatase-1 and protein phosphatase-2A account for virtually all the phosphorylase phosphatase activity in dilute tissue extracts prepared from skeletal muscle, liver, heart, brain and kidney, and for essentially all the glycogen synthase phosphatase activity in dilute skeletal muscle and liver extracts. Protein phosphatase-2A is almost absent from the protein-glycogen complex prepared from skeletal muscle or liver extracts. Protein phosphatase-2A accounts for a major proportion of the phosphatase activity in dilute liver extracts towards 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, 6-phosphofructo-1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase and phenylalanine hydroxylase, the major phosphorylated enzymes involved in the hormonal control of hepatic glycolysis and gluconeogenesis.     

Heart-reactive antibodies in rabbit anti-Streptococcus mutans sera fail to cross-react with Streptococcus mutans

Immunization of rabbits with Streptococcus mutans antigens results in the production of serum antibodies that bind in vitro to human, rabbit, and monkey cardiac muscle. Antibodies to heart, however, have also been reported to occur at lower titers in the sera of unimmunized rabbits. In this study, the specificities of heart-reactive antibodies (HRA) in sera of unimmunized and S. mutans-immunized rabbits were compared using indirect immunofluorescence, Western blot, and Bio-Dot immunoassays. Both groups of sera gave striational indirect immunofluorescence-staining patterns on thin sections of native human and monkey cardiac muscle. Western blot analyses revealed that antibodies in normal sera bound 9 to 20 components of human, rabbit, and monkey heart. The major bands had Mr of 205,000, 160,000, 135,000, and 70,000. Several of the normal sera did not have antibody activity to S. mutans antigens, indicating that these HRA do not cross-react with these bacteria. Although immunization of rabbits with S. mutans caused increased titers of HRA (two to three doubling dilutions), Western blot assays using anti-S. mutans sera showed banding patterns qualitatively similar to those of normal sera on heart extracts. Antibodies to skeletal muscle myosin were detected in both serum groups. Of eighteen normal rabbit sera sixteen had antimyosin titers of 10 to 40, whereas all eighteen anti-S. mutans sera had titers of 10 to 160. Affinity-purified antimyosin antibodies isolated from anti-S. mutans serum did not bind to S. mutans components. Conversely, affinity-purified antibodies to S. mutans antigens did not bind to myosin or to other cardiac muscle components. Among these were antibodies to the 185-kDa cell wall protein (also known as B, I/II, IF, Spa A, and P1) previously believed to possess antigenic mimicry. HRA were removed from anti-S. mutans sera by absorption with S. mutans but this effect was not specific, because a non-cross-reactive internal standard antibody was also absorbed to the same extent. Because previous evidence for antigenic mimicry between S. mutans and cardiac muscle was based on serum cross-absorption experiments, this immunologic relationship is not substantiated. These results indicated that naturally occurring antibodies to cardiac muscle components are present in the sera of unimmunized rabbits and that immunization with S. mutans does not stimulate production of new heart-reactive antibody, but rather serves to boost antibody production by preexisting clones of self-reactive B-lymphocytes.     

Trypsin-activated complex of human factor B with cobra venom factor (CVF), cleaving C3 and C5 and generating a lytic factor for unsensitized guinea pig erythrocytes. I. Generation of the activated complex.

A complex formed between cobra venom factor (CVF) and isolated human factor B (B) was found to be converted by trypsin to a stable enzyme, CVF-B which cleaved the third component (C3) and the fifth component (C5) of human complement. The formation of CVF-B by trypsin required divalent cations, whereas the formation of the lytic factor from human serum occurred even in the presence of EDTA. CVF-B purified by gel filtration could initiate the hemolysis of unsensitized guinea pig erythrocytes when incubated with human complement components C5 to C9 in 0.01 M EDTA buffer. C3 was not required for the lysis of guinea pig erythrocytes initiated by CVF-B because of the beta1C precipitation line formed between human serum and anti-beta1C antibody did not inhibit the hemolysis by CVF-B in agarose gel. Treatment of beta1C and beta1F globulins in whole human serum with CVF-B in the presence of 0.01 M EDTA converted them to components with higher mobilities on immunoelectrophoresis.     

Specific antibody-mediated detection of Brochothrix thermosphacta in situ in British fresh sausage

S.C. STRINGER, B.J. CHAFFEY, C.E.R. DODD, M.R.A. MORGAN AND W.M. WAITES. 1995. A rabbit polyclonal antibody-linked probe was developed which detected 76% of 800 food isolates of the spoilage bacterium Brochothrix thermosphacta when cells were bound to nitrocellulose. In slide cross-reaction tests all six environmental isolates tested were stained but the type strain was not. The antibody did not cross-react with Listeria grayi, L. monocytogenes, Lactobacillus plantarum, Lactococcus lactis, Streptococcus mutans, Bacillus cereus or B. subtilis.
The antibody-linked probe detected Br. thermosphacta in thin sections of British fresh sausage when the viable count was greater than 10⁶ g⁻¹ Cells were detected mainly within 1 or 2 mm of the surface on the loose starchy material. They were not detected within muscle blocks or in the centre of the sausage. Such results suggest that growth of this organism occurs close to the surface of the sausage.     

Bactericidal activity of C9-deficient human serum.

Escherichia coli B/SM, strain 1-1, was killed dose dependently by human hereditary C9-deficient serum (C9DHS), which was shown to contain no C9 Ag by an ELISA method. On the other hand, human hereditary C7-deficient serum did not kill the bacteria under similar conditions. The bactericidal activity of C9DHS was inhibited by rabbit anti-C5 antibody but not by murine anti-C9 mAb. The anti-C9 antibody decreased the bactericidal activity of normal human serum (NHS) to the level of that with C9DHS. Sheep anti-human lysozyme antibody did not affect the bactericidal activity of C9DHS or NHS even when added at more than twice the concentration required to block the serum lysozyme activity on Micrococcus luteus. After treatment with C9DHS and washing, surviving Escherichia coli were killed by C9, but not by lysozyme, transferrin, or both. Other strains of E. coli (K12 W3110, C600, and NIHJ) and Salmonella typhimurium (strain NCTC 74), all maintained in the laboratory, were also killed by C9DHS. However, pathogenic strains recently isolated from patients with traveler's diarrhea and some strains of S. typhimurium were resistant to both C9DHS and NHS, at least at the serum concentration tested. A concentration of 0.1 M Tris did not increase the susceptibility of serum-resistant strains of bacteria to C9DHS, but made one strain of S. typhimurium tested susceptible to NHS, but not to C9DHS. These results clearly showed that C9DHS kills bacteria that are sensitive to NHS through activation of C up to the step of C8 in the same way that C9-deficient C serum lyzed sensitized erythrocytes.     

Immunological characterization of phosphoprotein phosphatases

Phosphoprotein phosphatases regulate the biological activities of proteins through their involvement in cyclic phosphorylation/dephosphorylation cascades. A variety of multimeric phosphatases have been isolated and grouped into several classes, termed type 1 and types 2A, 2B, and 2C. To elucidate the relationship between the different phosphoprotein phosphatases, highly purified enzymes from soil amoebae, turkey gizzards, bovine heart and brain, and rabbit skeletal muscle and reticulocytes were tested for immunological antigenic relatedness. Two heterologous antibody preparations were employed for this purpose. One was made against an Acanthamoeba type 2A phosphatase and the other was made to bovine brain phosphatase type 2B (calcineurin, holoenzyme). Specific subunit cross-reactivity was examined by protein blot ("Western") analysis. The antibody to the type 2A phosphatase reacted with the catalytic subunits of every type 2 enzyme tested, including both the catalytic and Ca2+-binding subunits of the Ca2+/calmodulin-dependent type 2B phosphatase (calcineurin), bovine cardiac type 2A phosphatase, and turkey gizzard smooth muscle phosphatase-1 (type 2A1). It did not react with any type 1 phosphatase (catalytic subunit or ATP-Mg-dependent). The antigenic relatedness of calcineurin and the bovine cardiac type 2A phosphatase (Mr 38,000) was demonstrated further by protein blot analysis showing that the anti-calcineurin antibody cross-reacted with both enzymes. The mutual cross-reactivity poses an intriguing problem because these enzymes are so different in their molecular structures and modes of regulation. The degree of evolutionary conservation exhibited by the antigenic cross-reactivity of the type 2 enzymes from a broad range of species and tissues suggests a strong selective pressure on maintaining one or more features of these important regulatory enzymes.     

Identification of a glycogen synthase phosphatase from yeast Saccharomyces cerevisiae as protein phosphatase 2A.

A glycogen synthase phosphatase was purified from the yeast Saccharomyces cerevisiae. The purified yeast phosphatase displayed one major protein band which coincided with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis. This phosphatase had a molecular mass of about 160,000 Da determined by gel filtration and was comprised of three subunits, termed A, B, and C. The subunit molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 60,000 (A), 53,000 (B), and 37,000 (C), indicating that this yeast glycogen synthase phosphatase is a heterotrimer. On ethanol treatment, the enzyme was dissociated to an active species with a molecular weight of 37,000 estimated by gel filtration. The yeast phosphatase dephosphorylated yeast glycogen synthase, rabbit muscle glycogen phosphorylase, casein, and the alpha subunit of rabbit muscle phosphorylase kinase, was not sensitive to heat-stable protein phosphatase inhibitor 2, and was inhibited 90% by 1 nM okadaic acid. Dephosphorylation of glycogen synthase, phosphorylase, and phosphorylase kinase by this yeast enzyme could be stimulated by histone H1 and polylysines. Divalent cations (Mg2+ and Ca2+) and chelators (EDTA and EGTA) had no effect on dephosphorylation of glycogen synthase or phosphorylase while Mn2+ stimulated enzyme activity by approximately 50%. The specific activity and kinetics for phosphorylase resembled those of mammalian phosphatase 2A. An antibody against a synthetic peptide corresponding to the carboxyl terminus of the catalytic subunit of rabbit skeletal muscle protein phosphatase 2A reacted with subunit C of purified yeast phosphatase on immunoblots, whereas the analogous peptide antibody against phosphatase 1 did not. These data show that this yeast glycogen synthase phosphatase has structural and catalytic similarity to protein phosphatase 2A found in mammalian tissues.     

小鼠泛素结合酶UBE2W的抗体制备及组织表达谱分析

泛素结合酶(E2)是蛋白泛素化修饰所需的第二个连接酶, 在泛素转移和底物特异性识别过程中发挥着重要作用。UBE2W是一种新发现的E2酶, 其果蝇属同源蛋白可能在光转导或视网膜变性过程中发挥作用, 鼠和人同源蛋白功能未见报道。生物信息学分析UBE2W鼠源氨基酸序列, 发现UBE2W具有典型的UBC结构域并在多种物种高度保守。通过构建UBE2W原核表达质粒, 在大肠杆菌中表达并纯化了GST-UBE2W融合蛋白。以此纯化蛋白作为抗原免疫新西兰白兔制备抗UBE2W多抗血清, 并利用制备的UBE2W抗原柱亲和纯化UBE2W多抗。为了检测纯化抗体特异性, 在真核细胞中瞬时表达了myc-UBE2W融合蛋白, 分别用myc单抗和UBE2W多抗进行Western blotting分析, 结果表明获得了特异性的UBE2W抗体。利用此特异性抗体在小鼠脑、心脏、肾脏、肝脏、肺、肌肉、脾脏和睾丸等组织中均检测到了UBE2W的表达, 且在小鼠睾丸中成年期表达最高。     

小鼠泛素结合酶UBE2W的抗体制备及组织表达谱分析

泛素结合酶(E2)是蛋白泛素化修饰所需的第二个连接酶, 在泛素转移和底物特异性识别过程中发挥着重要作用。UBE2W是一种新发现的E2酶, 其果蝇属同源蛋白可能在光转导或视网膜变性过程中发挥作用, 鼠和人同源蛋白功能未见报道。生物信息学分析UBE2W鼠源氨基酸序列, 发现UBE2W具有典型的UBC结构域并在多种物种高度保守。通过构建UBE2W原核表达质粒, 在大肠杆菌中表达并纯化了GST-UBE2W融合蛋白。以此纯化蛋白作为抗原免疫新西兰白兔制备抗UBE2W多抗血清, 并利用制备的UBE2W抗原柱亲和纯化UBE2W多抗。为了检测纯化抗体特异性, 在真核细胞中瞬时表达了myc-UBE2W融合蛋白, 分别用myc单抗和UBE2W多抗进行Western blotting分析, 结果表明获得了特异性的UBE2W抗体。利用此特异性抗体在小鼠脑、心脏、肾脏、肝脏、肺、肌肉、脾脏和睾丸等组织中均检测到了UBE2W的表达, 且在小鼠睾丸中成年期表达最高。     

S100A1 modulates skeletal muscle contraction by desensitizing calcium activation of isometric tension, stiffness and ATPase

S100, a subfamily of the EF-hand type calcium sensing proteins, is implicated in many cellular functions including muscle contractility. Two isoforms, S100A1 and S100B, at 2-10 microM significantly inhibit active tension, stiffness and ATPase of skinned single rabbit psoas muscle fibers at sub-maximal (pCa approximately 6.1-5.6), but not at maximal levels of activation (pCa 4.0). S100A1 is a more potent inhibitor than S100B. Hill analysis of the ATPase-pCa and tension-pCa curves indicates that these proteins reduce calcium sensitivity and enhance the cooperativity toward calcium. We propose S100A1, and perhaps S100B, are viable candidates as physiological modulators of muscle contraction.     

HIV-1 B’/C亚型Rev蛋白的表达、纯化与抗体制备

目的:为方便实验室工作中对HIV-1 B’/C亚型Rev蛋白的检测,制备相应的Rev蛋白及其抗体。方法:将我国HIV-1 B’/C亚型流行株的rev基因按大肠杆菌优势密码子进行改造后人工合成,在原核系统中与pET30a(+)载体中的His.Tag、Trx.Tag及S.Tag进行融合表达,目的蛋白经Ni2+金属螯合层析柱纯化后用于免疫家兔,制备多克隆抗体。结果与结论:合成基因在原核系统中融合表达得到相对分子质量约18×103的融合蛋白,目的蛋白的表达量约占菌体总蛋白量的36%;用纯化后的融合蛋白免疫家兔,制备了多克隆抗体,Western印迹及间接免疫荧光检测结果显示,获得的多克隆抗体与HIV-1 B’/C亚型的Rev蛋白能产生特异性反应,可用于检测HIV-1 B’/C亚型Rev蛋白的表达。     

Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy

Lowicryl K4M (K4M) was recently introduced as an embedding medium for immunocytochemistry at the electron microscope level (BL Armbruster, E Carlemalm, R Chiovetti, RM Garavito, JA Hobot, E Kellenberger, W Villiger (1982):J Microsc 126:77 and E Carlemalm, M Garavito, W Villiger (1982):J Microsc 126:123). While earlier protocols of fixation and embedding required 4-6 days, the present method has reduced the processing time by accelerating both dehydration of tissues and polymerization of K4M so that tissues can be prepared for sectioning within 4 hr. The immunocytochemical labeling density was quantitated in order to determine relative antigen preservation in tissues embedded by the accelerated protocol as compared to slower K4M embedding techniques and to tissues embedded in glutaraldehyde-cross-linked bovine serum albumin (BSA). Thin sections of Bufo marinus kidney were labeled with rabbit antibody to Na+,K+ATPase alpha chain catalytic subunit isolated from B. marinus kidney microsomes (M Girardet, K Geering, JM Frantes, D Geser, BC Rossier, JP Kraehenbuhl, C Bron (1981):Biochemistry 20:6684). B. marinus retinas were labeled with rabbit anti-opsin. After fixation in paraformaldehyde(3%)-glutaraldehyde(3%), tissues were washed in buffer, dehydrated in 50, 75, and 90% dimethyl-formamide (DMF, 10 min each); K4M:DMF, 1:2 (15 min); K4M:DMF, 1:1, (20 min); K4M (25 min); K4M (30 min) at room temperature and transferred in fresh K4M to BEEM capsules for exposure to ultraviolet light (GE 15 watt, Black-lite, 10 cm, 45 min or less) at 4 degrees C. Thin sections were labeled successively with antibody, biotinylated sheep anti-rabbit F(ab')2 and avidin-ferritin. Ferritin labeling densities were determined by point counting. High labeling densities were observed with both antibodies, equaling or exceeding levels of labeling by slower protocols or embedment in BSA.     

Rho-mediated Contractility Exposes a Cryptic Site in Fibronectin and Induces Fibronectin Matrix Assembly

Many factors influence the assembly of fibronectin into an insoluble fibrillar extracellular matrix. Previous work demonstrated that one component in serum that promotes the assembly of fibronectin is lysophosphatidic acid (Zhang, Q., W.J. Checovich, D.M. Peters, R.M. Albrecht, and D.F. Mosher. 1994. J. Cell Biol. 127:1447–1459). Here we show that C3 transferase, an inhibitor of the low molecular weight GTP-binding protein Rho, blocks the binding of fibronectin and the 70-kD NH₂-terminal fibronectin fragment to cells and blocks the assembly of fibronectin into matrix induced by serum or lysophosphatidic acid. Microinjection of recombinant, constitutively active Rho into quiescent Swiss 3T3 cells promotes fibronectin matrix assembly by the injected cells. Investigating the mechanism by which Rho promotes fibronectin polymerization, we have used C3 to determine whether integrin activation is involved. Under conditions where C3 decreases fibronectin assembly we have only detected small changes in the state of integrin activation. However, several inhibitors of cellular contractility, that differ in their mode of action, inhibit cell binding of fibronectin and the 70-kD NH₂-terminal fibronectin fragment, decrease fibronectin incorporation into the deoxycholate insoluble matrix, and prevent fibronectin's assembly into fibrils on the cell surface. Because Rho stimulates contractility, these results suggest that Rho-mediated contractility promotes assembly of fibronectin into a fibrillar matrix. One mechanism by which contractility could enhance fibronectin assembly is by tension exposing cryptic self-assembly sites within fibronectin that is being stretched. Exploring this possibility, we have found a monoclonal antibody, L8, that stains fibronectin matrices differentially depending on the state of cell contractility. L8 was previously shown to inhibit fibronectin matrix assembly (Chernousov, M.A., A.I. Faerman, M.G. Frid, O.Y. Printseva, and V.E. Koteliansky. 1987. FEBS (Fed. Eur. Biochem. Soc.) Lett. 217:124–128). When it is used to stain normal cultures that are developing tension, it reveals a matrix indistinguishable from that revealed by polyclonal anti-fibronectin antibodies. However, the staining of fibronectin matrices by L8 is reduced relative to the polyclonal antibody when the contractility of cells is inhibited by C3. We have investigated the consequences of mechanically stretching fibronectin in the absence of cells. Applying a 30–35% stretch to immobilized fibronectin induced binding of soluble fibronectin, 70-kD fibronectin fragment, and L8 monoclonal antibody. Together, these results provide evidence that self-assembly sites within fibronectin are exposed by tension.     

Comparison of calcium-binding proteins. Bovine heart and brain protein activators of cyclic nucleotide phosphodiesterase and rabbit skeletal muscle troponin C.

In previous studies we have shown that the activation of bovine heart cyclic nucleotide phosphodiesterase by purified protein activator is completely dependent on the presence of Ca2+ and that the protein activator Ca2+ complex is probably the true activator for the enzyme (Teo, T.S. and Wang, J.H. (1973) J. Biol. Chem. 248, 5930-5955). More recent studies have led us to believe that the mechanism of the Ca2+ activation of phosphodiesterase resembles that of the Ca2+ activation of muscle contraction and that the protein activator may play a role similar to troponin. In the present study we show that the protein activator resembles rabbit muscle troponin C in amino acid composition, molecular weight, isoelectric point, and ultraviolet absorption spectrum. Preliminary structural studies also indicate that these two proteins may have evolved from a common ancestral protein through gene duplication. This argument is strengthened by the finding that the tryptic peptide map of the bovine heart protein activator is indistinguishable from that of the bovine brain phosphodiesterase activator protein for which preliminary sequence information also suggests homology to troponin C (Watterson, D.M., Harrelson, W.G., Jr., Keller, P.M., Sharief, F., and Vanaman, T.C. (1976) J. Biol. Chem. 251, 4501-4513).     

Phosphorylation of phospholemman (FXYD1) by protein kinases A and C modulates distinct Na,K-ATPase isozymes

Phospholemman (FXYD1), mainly expressed in heart and skeletal muscle, is a member of the FXYD protein family, which has been shown to decrease the apparent K(+) and Na(+) affinity of Na,K-ATPase ( Crambert, G., Fuzesi, M., Garty, H., Karlish, S., and Geering, K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11476-11481 ). In this study, we use the Xenopus oocyte expression system to study the role of phospholemman phosphorylation by protein kinases A and C in the modulation of different Na,K-ATPase isozymes present in the heart. Phosphorylation of phospholemman by protein kinase A has no effect on the maximal transport activity or on the apparent K(+) affinity of Na,K-ATPase alpha1/beta1 and alpha2/beta1 isozymes but increases their apparent Na(+) affinity, dependent on phospholemman phosphorylation at Ser(68). Phosphorylation of phospholemman by protein kinase C affects neither the maximal transport activity of alpha1/beta1 isozymes nor the K(+) affinity of alpha1/beta1 and alpha2/beta1 isozymes. However, protein kinase C phosphorylation of phospholemman increases the maximal Na,K-pump current of alpha2/beta1 isozymes by an increase in their turnover number. Thus, our results indicate that protein kinase A phosphorylation of phospholemman has similar functional effects on Na,K-ATPase alpha1/beta and alpha2/beta isozymes and increases their apparent Na(+) affinity, whereas protein kinase C phosphorylation of phospholemman modulates the transport activity of Na,K-ATPase alpha2/beta but not of alpha1/beta isozymes. The complex and distinct regulation of Na,K-ATPase isozymes by phosphorylation of phospholemman may be important for the efficient control of heart contractility and excitability.     

A 42,000-Da protein in rabbit tissues and in a glycogen synthase preparation cross-reacts with antibodies to glycogenin

Rabbit skeletal muscle glycogen previously has been shown to be covalently bound to a 40,000-Da protein ("glycogenin") via a novel glucosyl-tyrosine linkage [I.R. Rodriguez and W.J. Whelan (1985) Biochem. Biophys. Res. Commun. 132, 829-836]. Antibodies raised against rabbit skeletal muscle glycogenin cross-react with a similar protein present in rabbit heart and liver glycogens, as well as with a 42,000-Da "acceptor protein" present in high-speed supernatants of rabbit muscle, heart, retina, and liver. This 42,000-Da protein incorporates [U-14C]Glc when an ammonium sulfate fraction prepared from the tissue supernatants is incubated with UDP-[U-14C]Glc. The [U-14C]Glc incorporated can be removed quantitatively by treatment with amylolytic enzymes, indicating that the [U-14C]Glc incorporation represents elongation of a preexisting glucan attached to the acceptor protein. Furthermore, a commercial preparation of rabbit skeletal muscle glycogen synthase contains this 42,000-Da protein. We propose that the 42,000-Da protein represents the free form of glycogenin in tissues, with its covalently attached glucan chain(s) providing a "primed" elongation site for glycogen synthesis.     

Relationships of the spectrin complex of human erythrocyte membranes to the actomyosins of muscle cells.

Important similarities are reported between human smooth muscle actomyosin and the human erythrocyte spectrin complex, primarily components 1, 2, and 5 (Fairbanks G., Steck, T.L., and Wallach, D.F.H. (1971), Biochemistry 10, 2606). The actin-like protein, component 5, is identical with human uterine actin in its ability to form 50-70-A filaments to stimulate myosin ATPase activity, and to bind rabbit heavy meromyoson specit heavy meromyosin specifically. Antibodies to human smooth muscle myosin(uterine) were prepared which were monospecific. A weak but specific cross-reaction of these antisera with components 1 and/or 2 (spectrin) was characterized and at least 25% of the antimyosin antibodies showed a low affinity reaction iwth spectrin. Antibodies generated against a soluble complex of spectrin components 1 and 2 reacted only with component 1 and did not cross-react with myosin. In addition to these structural similarities between smooth muscle actomyosin and the spectrin complex, we have found that spectrin is involved in ATP-dependent erythrocyte shape changes (Sheetz, M.P., Painter, R.G., AND Singer, S.J. (1976B), Cold Spring Harbor Symp. Cell Motility (in press) and, therefore, the spectrin complex is also a mechanochemical protein system.     

Circulating immunoglobulin G can play a critical role in clearance of intestinal reovirus infection.

Reoviruses are encapsidated double-stranded RNA viruses that cause systemic disease in mice after peroral (p.o.) inoculation and primary replication in the intestine. In this study, we define components of the immune system involved in the clearing of reovirus from the proximal small intestine. The intestines of immunocompetent adult CB17, 129, and C57BL/6 mice were cleared of reovirus serotype 3 clone 9 (T3C9) within 7 days of p.o. inoculation. Antigen-specific lymphocytes were important for the clearance of intestinal infection, since severe combined immunodeficient (SCID) mice failed to clear T3C9 infection. To define specific immune components required for intestinal clearance, reovirus infection of mice with null mutations in the immunoglobulin M (IgM) transmembrane exon (MuMT; B cell and antibody deficient) or beta 2 microglobulin gene (beta 2-/-; CD8 deficient) was evaluated. beta 2-/- mice cleared reovirus infection with normal kinetics, while MuMT mice showed delayed clearance of T3C9 7 to 11 days after p.o. inoculation. Adoptive transfer of splenic lymphocytes from reovirus-immune CB17 mice inhibited growth of T3C9 in CB17 SCID mouse intestine 11 days after p.o. inoculation. The efficiency of viral clearance by adoptively transferred cells was significantly diminished by depletion of B cells prior to adoptive transfer. Results in SCID and MuMT mice demonstrate an important role for B cells or IgG in clearance of reovirus from the intestines. Polyclonal reovirus-immune rabbit serum, protein A-purified immune IgG, and murine monoclonal IgG2a antibody specific for reovirus outer capsid protein sigma 3 administered intraperitoneally all normalized clearance of reovirus from intestinal tissue in MuMT mice. This result demonstrates an IgA-independent role for IgG in the clearance of intestinal virus infection. Polyclonal reovirus-immune serum also significantly decreased reovirus titers in the intestines of SCID mice, demonstrating a T-cell-independent role for antibody in the clearance of intestinal reovirus infection. B cells and circulating IgG play an important role in the clearance of reovirus from intestines, suggesting that IgG may play a more prominent functional role at mucosal sites of primary viral replication than was previously supposed.     

Detection of dystrophin on two-dimensional gel electrophoresis

A protein with MW approximately 350 k daltons and pI approximately 5.5, which was deleted in the dystrophic mouse (C57BL/10ScSn-mdx), was detected on two-dimensional gel electrophoresis with silver staining. Deletion of this protein was uniformly observed in the dystrophic mouse extensor digitus longus, soleus and cardiac muscle. This protein specifically reacted with the monoclonal antibody against the chemically synthesized N-terminal fragment of human dystrophin. The protein reacting with this monoclonal antibody was also detected in rabbit back-muscle, rat extensor digitus longus and human skeletal muscle at the same position as the mouse muscle protein, on the two-dimensional gel electrophoresis. Our results show that dystrophin is solubilized in 8M guanidine HCl and that the modified two-dimensional gel electrophoresis can be applied to separate dystrophin.