Hormonal regulation of myosin heavy chain and alpha-actin gene expression in cultured fetal rat heart myocytes

Thyroid hormone regulates the expression of ventricular myosin isoenzymes by causing an accumulation of alpha-myosin heavy chain (MHC) mRNA and inhibiting expression of beta-MHC mRNA. However, the mechanism of thyroid hormone action has been difficult to examine in vivo because of its diverse actions. Accordingly, hormonal control of expression of six MHC isoform mRNAs and cardiac and skeletal alpha-actin mRNAs was studied in primary cultures of fetal rat heart myocytes grown in defined medium. The results indicate that in the absence of thyroid hormone, cultured heart cells express predominantly beta-MHC and cardiac alpha-actin mRNAs. Addition of 3,5,3'-triiodo-L-thyronine (T3) caused a rapid induction of alpha-MHC mRNA and decreased beta-MHC mRNA levels without affecting the skeletal muscle MHC mRNAs. There was an almost parallel change in the myosin isoenzymes. Cardiac alpha-actin mRNA levels were transiently increased by T3 treatment, but skeletal alpha-actin was unaffected. Elimination of insulin and epithelial growth factor from the medium did not alter the effects of T3 on cardiac MHC mRNA expression. Addition of various adrenergic agents to the medium had no appreciable effect on cardiac MHC mRNA expression despite the presence of functionally coupled alpha- and beta-adrenergic receptors. Addition of steroid hormones, muscarinic agents, and glucagon to the medium also had no effect. Thus, under defined conditions, T3 is able to regulate MHC gene expression at a pretranslational level without the need for other exogenous factors.     

alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases

The nucleotide sequence of the alpha-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the alpha-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature alpha-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 alpha-amylase with those from other bacterial and eucaryotic alpha-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca(2+)-binding site (consensus region I) of this Ca(2+)-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the alpha-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the beta-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.     

Prevention of the metabolic effects of 2-tetradecylglycidate by octanoic acid in the genetically diabetic mouse (db/db)

2-Tetradecylglycidate is a specific inhibitor of the enzyme carnitine palmitoyl transferase, the rate-limiting step in long chain fatty acid oxidation. We previously showed that chronic administration of TDGA to genetically diabetic mice caused a dose-dependent decrease in blood glucose, retarded the development of renal immunopathologic lesions, and resulted in significant cardiomegaly. The present study was designed to evaluate whether all the observed consequences of chronic TDGA administration resulted from inhibition of long chain fatty acid oxidation or whether the drug exerted other nonspecific effects. To circumvent the effects of LCFAO inhibition, diabetic mice were dosed with TDGA and given a diet containing 9% octanoic acid. Octanoic acid is a medium chain fatty acid, whose oxidation is not dependent on the carnitine transferase system and is not inhibited by TDGA. Administration of the octanoate diet to diabetics receiving TDGA abrogated all the drug effects, including lowering of blood glucose and prevention of renal immunopathology. Cardiomegaly, a consequence of increased protein accretion associated with TDGA dosing, did not occur in the octanoate-fed animals. These results indicate that all the actions of TDGA are mediated via its inhibitory effects on long chain fatty acid oxidation. The cardiac changes resulting from chronic TDGA administration suggest that long chain fatty acid oxidation and its relationship with myocardial energetics may exert a regulatory role on protein synthesis in the myocardium.     

Biosynthesis of lutropin in ovine pituitary slices: Incorporation of [35S]sulfate in carbohydrate units

Sulfate incorporation into carbohydrate of lutropin (LH) has been studied in sheep pituitary slices using H₂³⁵SO₄. Labeled ovine LH was purified to homogeneity by Sephadex G-100 and carboxymethyl-Sephadex chromatography from both the incubation medium and tissue extract. Autoradiography of the gel showed only two protein bands which comigrated with the α and β subunits of ovine LH in both the purified ovine LH and the immunoprecipitate obtained with LH-specific rabbit antiserum. Furthermore, [³⁵S]sulfate was also incorporated into several other proteins in addition to LH. The location of ³⁵SO₄^2? in the oligosaccharides of ovine LH was evidenced by its presence in the glycopeptides obtained by exhaustive Pronase digestion. The location and the point of attachment of sulfate in the carbohydrate unit were established by the isolation of 4-O-[³⁵S]sulfo-N-acetylhexosaminyl-glycerols and 4-O-[³⁵S]sulfo-N-acetylglucosaminitol from the Smith degradation products and by the release of ³⁵SO₄^2? by chondro-4-sulfatase. Thus, the present line of experimentation indicates the presence of sulfate on both the terminal N-acetylglucosamine and N-acetylgalactosamine in the oligosaccharide chains of the labeled ovine LH.     

Quaternary structure,Mg2+ interactions,and some kinetic properties of the (β-galactosidase fromThermoanaerobacterium thermosulfurigenes EM1

Theβ-galactosidase fromThermoanaerobacterium thermosulfurigenes EM1 was found to be a dimer with a monomer molecular weight of about 85,000. It lacks theα-peptide and an importantα-helix that are both needed for dimer-dimer interaction and there is no homology in other important dimer-dimer interaction areas. These differences in structure probably account for the dimeric (rather than tetrameric) structure. Only 0.19 Mg²⁺ bound per monomer and Mg²⁺ had only small effects on the activity and heat stability. The absence of residues equivalent to Glu-416 and His-418 (two of the three ligands to Mg²⁺ in theβ-galactosidase fromEscherichia coli) probably accounts for the low level of Mg²⁺ binding and the consequent lack of response to Mg²⁺. Both Na⁺ and K⁺ also had no effect on the activity. The enzyme activity witho-nitrophenyl-β-D-galactopyanoside (ONPG) was very similar to that withp-nitrophenyl-β-D-β-D-galactopyranoside (PNPG) and the ONPG pH profile was very similar to the PNPG pH profile. These differences are in contrast to theE. coli β-galactosidase, which dramatically discriminates between these two substrates. The lack of discrimination by theT. thermosulfurigenes β-galactosidase could be due to the absence of the sequence equivalent to residues 910-1023 of theE. coli β-galactosidase. Trp-999 is probably of the most importance. Trp-999 of theE. coli β-galactosidase is important for aglycone binding and ONPG and PNPG differ only in their aglycones. The suggestion that the aglycone site of theT. thermosulfurigenes β-galactosidase is different was strengthened by competitive inhibition studies. Compared toE. coli β-galactosidase, D-galactonolactone was a very good inhibitor of theT. thermosulfurigenes enzyme, while L-ribose inhibited poorly. These are transition-state analogs and the results indicate thatT. thermosulfurigenes β-galactosidase binds the transition state differently than doesE. coli β-galactosidase. Methanol and glucose were good acceptors of galactose, and allolactose was formed when glucose was the acceptor. Allolactose could not, however, be detected by TLC when lactose was the substrate. The differences noted may be due to the thermophilic nature ofT. thermosulfurigenes.     

Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.     

Effect of pH and butyrate concentration on the production of acetone and butanol by Clostridium acetobutylicum grown in continuous culture

Summary When Clostridium acetobutylicum was grown in continuous culture under glucose limitation at neutral pH and varying dilution rates the only fermentation products formed were acetate, butyrate, carbon dioxide and molecular hydrogen. The Y _glucose ^max and (Y _ATP ^max ) _gluc ^exp values were 48.3 and 23.8 dry weight/mol, respectively. Acetone and butanol were produced when the pH was decreased below 5.0 (optimum at pH 4.3). The addition of butyric acid (20 to 80 mM) to the medium with a pH of 4.3 resulted in a shift of the fermentation from acid, to solvent formation.A preliminary report of part of this work was presented at a symposium Trends in the Biology of Fermentations for Fuels and Chemicals held December 7–11, 1980, at Brookhaven National Laboratory, Upton, New York; Gottschalk and Bahl 1981     

Acetone-butanol production by Clostridium acetobutylicum in an ammonium-limited chemostat at low pH values

Clostridium acetobutylicum was grown in continuous culture under ammonium limitation (15.15 mM NH₄ ⁺). At a pH of 6.0 and at various dilution rates only acetate, butyrate and ethanol were formed as non-gaseous products. A decrease of the pH to values between 5.2 and 4.3 resulted in a shift of the fermentation towards acetone-butanol formation.