Characterization of a developmentally regulated perinatal myosin heavy-chain gene expressed in skeletal muscle

A cDNA clone, labeled pFOD5, isolated from a fetal-rat skeletal-muscle cDNA library, has been characterized and found to contain sequences corresponding to a perinatal-specific skeletal myosin heavy-chain (MHC) mRNA. This MHC cDNA demonstrates a high degree of nucleotide- and amino acid-sequence conservation with other MHC genes, but its carboxyl-terminal peptide and 3'-untranslated region are highly divergent and specific for this gene. S1 nuclease mapping experiments have shown that the perinatal MHC gene represented by this cDNA clone is only transiently expressed during skeletal-muscle development. Perinatal MHC mRNA is first detected late in fetal life, reaches maximal levels of expression at the end of the first postnatal week, and is de-induced thereafter. Its levels are almost undetectable at 28 days of postnatal life. During fetal and early postnatal life, the expression of this perinatal gene in skeletal muscle overlaps with the expression of the embryonic MHC gene. After the first week of extrauterine life, this gene is coexpressed with two adult MHC genes. The transient expression of this perinatal MHC gene raises interesting questions about the physiological significance of the MHC transitions and offers an interesting model for the study of MHC gene regulation.     

Identification of two types of smooth muscle myosin heavy chain isoforms by cDNA cloning and immunoblot analysis

We previously reported the characterization of a rabbit uterus cDNA clone (SMHC29) which encoded part of the light meromyosin of smooth muscle myosin heavy chain (Nagai, R., Larson, D.M., and Periasamy, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1047-1051). We have now characterized a second cDNA clone (SMHC40) which also encodes part of the light meromyosin but differs from SMHC29 in the following respects. Nucleotide sequence analysis demonstrates that the two myosin heavy chain mRNAs are identical over 1424 nucleotides but differ in part of the 3'-carboxyl coding region and a portion of the 3'-nontranslated sequence. Specifically, SMHC40 cDNA encodes a unique stretch of 43 amino acids at the carboxyl terminus, whereas SMHC29 cDNA contains a shorter carboxyl terminus of 9 unique amino acids which is the result of a 39-nucleotide insertion. Recent peptide mapping of smooth muscle myosin heavy chain identified two isotypes with differences in the light meromyosin fragment that were designated as SM1 (204 kDa) and SM2 (200 kDa) type myosin (Eddinger, T. J., and Murphy, R.A. (1988) Biochemistry 27, 3807-3811). In this study we present direct evidence that SMHC40 and SMHC29 mRNA encode the two smooth muscle myosin heavy chain isoforms, SM1 and SM2, respectively, by immunoblot analysis using antibodies against specific carboxyl terminus sequences deduced from SMHC40 and SMHC29 cDNA clones.     

cis-4-Hydroxy-L-proline and ethyl-3,4-dihydroxybenzoate prevent myogenesis of C2C12 muscle cells and block MyoD1 and myogenin expression.

cis-4-Hydroxy-L-proline (cis-OH-Pro) and ethyl-3,4-dihydroxybenzoate (EDHB), two distinct inhibitors of collagen synthesis, prevented myogenesis in C2C12 mouse skeletal muscle cells. Both inhibitors blocked myotube formation and the expression of sarcomeric myosin heavy chain. Northern blot analysis showed that cis-OH-Pro- and EDHB-treated C2C12 muscle cells did not express the myogenic regulatory genes, MyoD1 and myogenin, but continued to express non-muscle isoforms of actin (beta and gamma) and alpha-tropomyosin. 10TFL2-3B cells, a C3H10T1/2 cell line permanently transfected with myogenin cDNA, constitutively expressed exogenous myogenin in the presence of cis-OH-Pro but failed to activate endogenous myogenin and to undergo myogenesis. These results demonstrate that commitment to terminal differentiation and activation of myogenic regulatory genes requires active synthesis of the extracellular matrix component collagen.     

Picosecond rotation of small polar fluorophores in the cytosol of sea urchin eggs.

A new fluorescence method to measure viscosity in cell cytosol [Fushimi, K., & Verkman, A. S. (1991) J. Cell Biol. 112, 719-725] has been applied to determine fluid-phase viscosity in sea urchin eggs. Freshly harvested eggs from Lytechinus pictus were loaded with the dyes 2,7-bis(2-carboxyethyl)-5-(and-6-)carboxyfluorescein (BCECF), 6-carboxyfluorescein (6CF), fluorescein, or calcein. Fluorescence lifetimes and anisotropy decay were measured in single eggs by multiharmonic, frequency-domain microfluorometry using a 1-2-micron focused laser spot and 25x air objective. In calibration solutions consisting of glycerol in pH 8 buffered sea water, probe lifetime was single exponential and probe rotation was isotropic with a single correlation time which increased linearly with viscosity in the range 1-3.6 cP. In eggs at 22 degrees C, there were single lifetimes (in nanoseconds) of 3.6 (BCECF), 3.4 (6CF), 3.2 (fluorescein), and 3.3 (calcein). Probe rotation in eggs had two components, a fast component (in picoseconds, mean +/- SE, 10-18 eggs) of 568 +/- 39 (BCECF), 311 +/- 21 (6CF), 313 +/- 15 (fluorescein), and 516 +/- 44 (calcein) and a slow component of 10-40 ns. The fractional amplitude of the fast component, corresponding to unbound dye, was 0.72-0.81. Apparent viscosities of fluid-phase cytoplasm (centipoises) given by the four different probes were in good agreement: 2.3 +/- 0.2 (BCECF), 2.1 +/- 0.1 (6CF), 2.5 +/- 0.1 (fluorescein), and 2.3 +/- 0.2 (calcein). The viscosity in cytosol of sea urchin eggs (2.1-2.5 cP) is thus relatively low, yet significantly greater than that of water (1 cP) or cytosol in cultured fibroblasts (1.2-1.4 cP).(ABSTRACT TRUNCATED AT 250 WORDS)     

An Extracellullar Nuclease from Bacillus Firmus VKPACU-1: Specificity and Mode of Action

An extracellular nuclease from Bacillus firmus VKPACU-1 was multifunctional enzyme, this nuclease hydrolyzed poly U rapidly and more preferentially than the other homopolyribonucleotides. Hydrolysis of RNA this enzyme released mononucleotides in the order 5′UMP > 5′AMP > 5′GMP where as in hydrolysis of DNA the mononucleotides in the order of 5′dAMP > 5′dGMP > 5′dTMP and oligonucleotides. Uridylic linkages in RNA and adenylic linkages in DNA were preferentially cleaved by the nuclease. Nuclease produced oligonucleotides having only 3’ hydroxyl and 5’ phosphate termini. Present nuclease hydrolyzed RNA and DNA released oligonucleotides as major end products and mononucleotides, suggesting an endo mode of action.     

Ruthenium(II) carbonyl complexes containing S-methylisothiosemicarbazone based tetradentate ligand: synthesis, characterization and biological applications

A series of hexa-coordinated ruthenium(II) complexes of the type [Ru(CO)(B)L ⁿ ] (n = 1–4; B = PPh₃, AsPh₃ or Py) have been synthesized by reacting dibasic quadridentate Schiff base ligands H₂L ⁿ (n = 1–4) with starting complexes [RuHCl(CO)(EPh₃)₂(B)] (E = P or As; B = PPh₃, AsPh₃ or Py). The synthesized complexes were characterized using elemental and various spectral studies including UV–Vis, FT-IR, NMR (¹H, ¹³C and ³¹P) and mass spectroscopy. An octahedral geometry was tentatively proposed for all the complexes based on the spectral data obtained. The experiments on antioxidant activity showed that the ruthenium(II) S-methylisothiosemicarbazone Schiff base complexes exhibited good scavenging activity against various free radicals (DPPH, OH and NO). The in vitro cytotoxicity of these complexes has been evaluated by MTT assay. The results demonstrate that the complexes have good anticancer activities against selected cancer cell line, human breast cancer cell line (MCF-7) and human skin carcinoma cell line (A431). The DNA cleavage studies showed that the complexes have better cleavage of pBR 322 DNA.     

GLUT12 functions as a basal and insulin-independent glucose transporter in the heart

Glucose uptake from the bloodstream is the rate-limiting step in whole body glucose utilization, and is regulated by a family of membrane proteins called glucose transporters (GLUTs). Although GLUT4 is the predominant isoform in insulin-sensitive tissues, there is recent evidence that GLUT12 could be a novel second insulin-sensitive GLUT. However, its physiological role in the heart is not elucidated and the regulation of insulin-stimulated myocardial GLUT12 translocation is unknown. In addition, the role of GLUT12 has not been investigated in the diabetic myocardium. Thus, we hypothesized that, as for GLUT4, insulin regulates GLUT12 translocation to the myocardial cell surface, which is impaired during diabetes. Active cell surface GLUT (-4 and -12) content was quantified (before and after insulin stimulation) by a biotinylated photolabeled assay in both intact perfused myocardium and isolated cardiac myocytes of healthy and type 1 diabetic rodents. GLUT localization was confirmed by immunofluorescent confocal microscopy, and total GLUT protein expression was measured by Western blotting. Insulin stimulation increased translocation of GLUT-4, but not -12, in the healthy myocardium. Total GLUT4 content of the heart was decreased during diabetes, while there was no difference in total GLUT12. Active cell surface GLUT12 content was increased in the diabetic myocardium, potentially as a compensatory mechanism for the observed downregulation of GLUT4. Collectively, our data suggest that, in contrast to GLUT4, insulin does not mediate GLUT12 translocation, which may function as a basal GLUT located primarily at the cell surface in the myocardium.