Monoclonal antibody specific for the transverse tubular membrane of skeletal muscle activates the dihydropyridine-sensitive Ca2+ channel.

In skeletal muscle, dihydropyridine receptors and dihydropyridine-sensitive Ca2+ channels are preferentially localized in the transverse tubular membranes. Starting with an antigenic membrane fraction enriched in rabbit muscle transverse tubules (T-tubules), several monoclonal antibodies were produced by a fusion of spleen cells from an immunized BALB/c mouse with P3 X 63Ag.8.6.5.3 mouse myeloma cells. Antibodies were screened according to a scheme designed to select IgG immunoglobins that recognized a determinant specifically associated with the T-tubule membrane. Antibodies that fulfilled the screening criteria were used in in vitro planar bilayer recording of the activity of the dihydropyridine-sensitive Ca2+ channel present in T-tubules. Cells producing one antibody (Ab 21) survived cloning dilution and stably produced a monoclonal antibody (mAb21-4) that increased the rate of single channel opening when interacting with the internal side of the channel protein. mAb21-4 immobilized by covalent crosslinking on beads (Affi-Gel 10) consistently immunoprecipitated polypeptide bands with the following electrophoretic mobility: Mr values of greater than or equal to 175,000; 90,000; 55,000; and 34,000.     

Cell-specific activation of the human skeletal alpha-actin by androgens

Although it is evident that androgens increase muscle mass and strength, little is known about the critical molecular targets of androgens in skeletal muscle. In rodents, the skeletal alpha-actin gene is a tissue-specific gene expressed only in the levator ani and other skeletal muscles but not in the prostate or preputial gland, the well-known androgen target tissue. We identified tissue-specific androgen-regulated genes in the skeletal muscle in rats after oral administration of androgens and focused on androgen-dependent up-regulation of the skeletal alpha-actin gene. To investigate the mechanism of action, an in vitro system with various cell lines and a series of deletion mutants of the alpha-actin promoter were used. The human skeletal alpha-actin promoter was activated by androgens in the muscle cell line C2C12 but not in the liver, prostate, or breast cancer cell lines in which exogenous human androgen receptor is expressed. The sequence of the promoter is sufficient for cell-specific androgen response, providing a model for the tissue specificity demonstrated in vivo. Using a series of deletion mutants, the androgen response can be maintained using just the proximal promoter region. The importance of androgen regulation of this small portion of the human skeletal alpha-actin promoter was demonstrated by the correlation between muscle and the alpha-actin promoter activity for an array of selective androgen receptor modulators (SARMs), including an orally active SARM LGD2226. Taken together, the results suggest that the regulation of skeletal alpha-actin by androgens/SARMs may represent an important model system for understanding androgen anabolic action in the muscle.     

Global and specific transcriptional repression by the histone H3 amino terminus in yeast

The yeast CHA1 promoter is activated in the presence of serine or threonine. Activation requires the Cha4p activator, and it results in perturbation of a nucleosome that incorporates the TATA element under noninducing conditions. We show that in yeast lacking the amino terminus of histone H3, the promoter is constitutively active and the chromatin is concomitantly perturbed. This derepression occurs in the absence of elevated intracellular levels of serine or threonine and is not observed in cells lacking Rpd3p, Tup1p, or the amino terminus of histone H4. Furthermore, derepression in the absence of the H3 amino terminus requires the primary activator of this promoter, Cha4p, which we show by chromatin immunoprecipitation to be constitutively bound to the CHA1 promoter in WT yeast. Thus, the H3 amino terminus is required to prevent Cha4p from activating CHA1 in the absence of inducer. We also present results of a microarray experiment showing that the H3 amino terminus has a substantial repressive effect on a genome-wide scale.     

Molecular structure of the human desmoplakin I and II amino terminus.

Desmoplakins (DPs) I and II are closely related proteins found in the innermost region of the desmosomal plaque, which serves as a cell surface attachment site for cytoplasmic intermediate filaments. Overlapping cDNA clones comprising 9.2 kilobases of DP-I, predicted to encode a full-length 310-kDa polypeptide (2677 amino acid residues), have now been identified. Here we report the predicted protein sequence and structural analysis of the N terminus of DP, extending our previous study of the rod and carboxyl domains. The N terminus contains groups of heptad repeats that are predicted to form at least two major alpha-helical-rich bundles. Unlike the rod and carboxyl domains, the N terminus did not display a periodic distribution of charged residues. Northern blot mapping and genomic sequence analysis were also undertaken to examine the organization of the DP mRNAs. A 1-kilobase intron was located at the 3' boundary of a DP-I-specific region; however, instead of an intron at the 5' junction, a possible splice donor site was observed within a potential coding sequence, suggesting alternative RNA splicing from an internal donor site.     

Ontogenetic changes in the content of methylated amino acids in rodent skeletal muscle

Changes in the content of four methylated basic amino acids (3-methylhistidine, N ,N -dimethylarginine, monomethyllysine and trimethyllysine) in myosin of lower extremities in laboratory rodents (Wistar rats, golden hamsters and white mice) were followed. Material from animals aged 4, 6, 14 and 90 days and 1 and 2 yr as well as from foetuses was available by the time of the present study.

3-Methylhistidine in myosin hydrolysates from foetuses was absent. First trace amounts of this amino acid were observed in 6 days old baby animals regardless of the tested rodent species. The age-dependent increase in 3-methylhistidine content is about logarithmic.

Similar trends were observed also with -N-monomethyllysine. First measurable amounts of this amino acid appeared shortly before delivery.

The content of trimethyllysine increased up to the 14th day of the postnatal development. Thenafter its content in myosin hydrolysates declines.

The highest content of N ,N -dimethylarginine (unsym. DMA) was observed in the prenatal stage. Shortly after delivery, its content drops steeply down and last traces of this amino acid were observed at the age of 6 days.     

Similarities and differences in smooth muscle alpha-actin induction by TGF-beta in smooth muscle versus non-smooth muscle cells.

Transforming growth factor-beta (TGF-beta) has been shown to stimulate smooth muscle (SM) alpha-actin expression in smooth muscle cells (SMCs) and non-SMCs. We previously demonstrated that the 2 CArG boxes A and B and a novel TGF-beta control element (TCE) located within the first 125 bp of the SM alpha-actin promoter were required for TGF-beta inducibility of SM alpha-actin in SMCs. The aims of the present study were (1) to determine whether the TCE exhibits SMC specificity or contributes to TGF-beta induction of SM alpha-actin expression in non-SMCs (ie, endothelial cells and fibroblasts) and (2) to determine whether TGF-beta can induce expression of multiple TCE-containing SMC differentiation marker genes, such as SM22alpha, h(1) calponin, and SM myosin heavy chain (SM MHC) in non-SMCs. Results of transient transfection assays demonstrated that mutation of CArG A, CArG B, or the TCE within a 125-bp promoter context completely abolished TGF-beta inducibility of SM alpha-actin in endothelial cells and fibroblasts. However, in contrast to observations in SMCs, inclusion of regions upstream from (-155) completely repressed TGF-beta responsiveness in non-SMCs. Electrophoretic mobility shift assays showed that TGF-beta enhanced binding of a serum response factor to the CArG elements and the binding of an as-yet-unidentified factor to the TCE in endothelial cells and fibroblasts, but to a much lesser extent compared with SMCs. TGF-beta also stimulated expression of the SMC differentiation marker SM22alpha in non-SMCs. However, in contrast to SMCs, TGF-beta did not induce expression of h(1) calponin and SM MHC in non-SMCs. In summary, these results suggest a conserved role for CArG A, CArG B, and the TCE in TGF-beta-induced expression of SM alpha-actin in SMCs and non-SMCs that is modified by a complex interplay of positive- and negative-acting cis elements in a cell-specific manner. Furthermore, observations that TGF-beta stimulated expression of several early but not late differentiation markers in non-SMCs indicate that TGF-beta alone is not sufficient to induce transdifferentiation of non-SMCs into SMCs.     

Endothelin converting enzyme is located on alpha-actin filaments in smooth muscle cells.

OBJECTIVE: Endothelin converting enzyme is the key enzyme in the generation of endothelin-1 from big-endothelin-1. The mature endothelin-1 is a potent vasoconstrictor which also promotes mitogenesis and proliferation of smooth muscle cells. The objectives were to demonstrate in smooth muscle cells the presence of a phosphoramidon-sensitive endothelin converting enzyme activity, reveal the subcellular localization of the enzyme protein and determine the effects of the metalloproteinase inhibitor, phosphoramidon, the lysosomotrophic drug, chloroquine, and colchicine on the cycling pathway of the enzyme. METHODS: Subcellular localization of endothelin converting enzyme on human smooth muscle cells and the rat cell line, A7r5, was by immunofluorescence and confocal microscopy or by biotinylation of cell cultures and immunoblotting, after treatment of cell cultures with cytochalasin D, colchicine, chloroquine and phosphoramidon. Converting enzyme activity was determined by high performance liquid chromatographic assay. RESULTS: We detected phosphoramidon-sensitive endothelin converting enzyme activity in smooth muscle cells. In addition to its plasma membrane location, for the first time we revealed a striking co-localization of endothelin converting enzyme and alpha-actin filaments in smooth muscle cells. Colchicine treatment results in a perinuclear accumulation of endothelin converting enzyme. An increased level of endothelin converting enzyme protein was shown to be present in smooth muscle cells which had been grown in the presence of phosphoramidon or chloroquine. CONCLUSION: The 120 kDa endothelin converting enzyme co-localizes with alpha-actin in smooth muscle cells and resembles that found in endothelial cells in that it is present on both the plasma membrane and intracellularly.     

Pseudomonas exotoxin contains a specific sequence at the carboxyl terminus that is required for cytotoxicity.

Pseudomonas exotoxin (PE), a single-chain polypeptide toxin of 613 amino acids, consists of three functional domains: an amino-terminal receptor-binding domain, a middle translocation domain, and a carboxyl-terminal ADP-ribosylation domain. Deletion of as few as 2 or as many as 11 amino acids from the carboxyl terminus of PE does not affect ADP-ribosylation activity but produces noncytotoxic molecules. Deletions and substitutions between positions 602 and 611 of PE show that the last 5 amino acids of PE are very important for its cytotoxic action. The carboxyl-terminal sequence of PE is Arg-Glu-Asp-Leu-Lys. Mutational analysis indicates that a basic amino acid at 609, acidic amino acids at 610 and 611, and a leucine at 612 are required for full cytotoxic activity. Lysine at 613 can be deleted or replaced with arginine but not with several other amino acids. Mutant toxins are able to bind normally to target Swiss mouse 3T3 cells and are internalized by endocytosis, but apparently they do not penetrate into the cytosol. A PE molecule that ends with Lys-Asp-Glu-Leu, which is a well defined endoplasmic reticulum retention sequence [Munro, S. and Pelham, R. B. (1987) Cell 48, 899-907], is fully cytotoxic, suggesting that a common factor may be involved in intoxication of cells by PE and retention of proteins in the lumen of the endoplasmic reticulum. Sequences similar to those at the carboxyl end of PE are also found at the end of Cholera toxin A chain and Escherichia coli heat-labile toxin A chain.     

Molecular mechanisms and therapeutics of the deficit in specific force in ageing skeletal muscle

The age-related impairment in muscle force is only partially explained by the loss of muscle mass. The loss both in specific and absolute forces contributes to the muscle weakness measured in the elderly and in animal models of ageing. Successful interventions aimed at preventing age-associated functional deficits will require a better insight into the mechanisms underlying the decline in muscle-specific force. The present review article is focused on recent evidence supporting excitation-contraction uncoupling as a key factor underlying fast and slow muscle fiber impairment with ageing. The molecular, functional and structural factors supporting this theory and counteracting measures such as insulin-like growth factor 1 transgenic overexpression are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

A monoclonal antibody specific for mouse dendritic cells.

Dendritic cells (DC) are a small subpopulation of lymphoid cells with distinctive cytologic features, surface properties, and functions. This report describes the DC-specific antibody (Ab) secreted by clone 33DI. Rat spleen cells immune to mouse DC were fused to the P3U myeloma. Hybrid culture supernatants were screened simultaneously against DC, a macrophage (M phi) cell line, and mitogen-stimulated lymphoblasts. 33DI Ab specifically killed 80-90% of DC from spleen and lymph node, but no other leukocytes, including Ia+ and Ia- M phi (Ia, I-region-associated antigen,). Quantitative binding studies with 5H-labeled 33D1 Ab showed that DC had an average of 14,000 binding sites per cell. Binding to DC was inhibited with Fab fragment of 33D1 Ab but not with a panel of other monoclonal antibodies, including anti-Ia Ab. Adherence and flotation procedures that enriched for DC enriched for 3H-labeled 33D1 Ab binding in parallel. 33D1 antigen was not detectable on: M phi from spleen, peritoneal cavity, and blood; three M phi cell lines; lymphocytes; granulocytes; platelets; and erythroid cells. DC continued to express the 33D1 antigen after 4 days in culture, whereas M phi and lymphocytes did not acquire it. Quantitative and autoradiographic studies confirmed that spleen and lymph node suspensions contain less than 1% DC. We conclude that 33D1 Ab detects a stable and specific DC antigen and can be used to monitor DC content in complex lymphoid mixtures.     

Monoclonal antibody specific for an activated RAS protein.

Activated RAS transforming genes that encode proteins (p21s) with amino acid substitutions at positions 12, 13, or 61 have been detected in 10-20% of human neoplasms. This report describes a monoclonal antibody (DWP) raised against a synthetic peptide corresponding to amino acids 5-16 of a mutated RAS gene encoding Val instead of Gly at position 12. DWP reacted in competition assays with peptides containing Val or Cys at position 12, but did not react with peptides containing Gly, Arg, Ser, Ala, Asp, or Glu at position 12. Immunoblot analysis of transformed NIH cells and human carcinoma cell lines showed that DWP reacts specifically with activated RAS proteins containing Val at position 12 and not with normal p21s or p21s activated by other amino acid substitutions at positions 12 and 61. Immunohistochemical studies showed that DWP-labeled transformed NIH cells and human carcinoma cells contained p21s with either Val or Cys at position 12 but not normal or other activated p21s. In contrast to the specificity seen with human carcinoma cell lines, analysis of formalin-fixed, primary carcinoma specimens indicated that positive immunoperoxidase staining with DWP did not necessarily correlate with immunoblot and transfection assays for the presence of activated RAS proteins. Immunohistochemical studies did show, however, that DWP preferentially binds human carcinoma cells.     

An overview of the endocrinology of skeletal muscle.

Endocrine regulation of the balance between skeletal muscle anabolism and catabolism has been investigated extensively. Factors determining whether hormones exert anabolic or catabolic influences are multifaceted and often unclear. Testosterone, growth hormone, insulin and insulin-like growth factor-I have complex anabolic effects, some of which have only recently been elucidated, and are important regulators of muscle remodeling, whereas glucocorticoids have direct catabolic effects and induce muscle protein loss. The effects of estrogen are poorly understood and warrant further study. We review recent literature and evaluate the hormones driving skeletal muscle anabolism and catabolism, which ultimately dictate the endocrinology and metabolism of skeletal muscle in humans. Understanding hormonal regulation of skeletal muscle remodeling might facilitate development of improved hormone-mediated therapies for muscle wasting conditions.     

Contraction characteristics and ATPase activity of skeletal muscle fibers in the presence of antibody to myosin subfragment 2.

To investigate the role of the myosin hinge region in muscle contraction, we examined the contraction characteristics and Mg-ATPase activity of glycerinated muscle fibers prepared from rabbit psoas in the presence and absence of polyclonal antibody directed against the subfragment 2 (S-2) region of myosin. The antibody-induced reduction of Ca(2+)-activated isometric force was always accompanied by a parallel decrease of muscle fiber stiffness, so that the stiffness versus force relation remained unchanged by the antibody treatment. Force-velocity relations of the fibers, obtained by applying ramp decreases in force at steady isometric forces, indicated that the antibody had no effect on maximum shortening velocity or on the shape of force-velocity curves. Simultaneous measurements of Mg-ATPase activity and Ca(2+)-activated force showed that Mg-ATPase activity of the fibers remained unchanged despite the antibody-induced reduction of isometric force even to zero. These results indicate that when anti-S-2 antibody attaches to the S-2 region of myosin molecules, their heads still hydrolyze ATP but no longer contribute to both force generation and muscle fiber stiffness.