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M Gubler

Publications and source records attributed to M Gubler.

15 recordsLinked to original sources
PubMed2005

Corticotropin-releasing hormone directly stimulates thermogenesis in skeletal muscle possibly through substrate cycling between de novo lipogenesis and lipid oxidation.

The mechanisms by which CRH and related peptides (i.e. the CRH/urocortin system) exert their control over thermogenesis and weight regulation have until now focused only upon their effects on brain centers controlling sympathetic outflow. Using a method that involves repeated oxygen uptake determinations in intact mouse skeletal muscle, we report here that CRH can act directly on skeletal muscle to stimulate thermogenesis, an effect that is more pronounced in oxidative than in glycolytic muscles and that can be inhibited by a selective CRH-R2 antagonist or blunted by a nonselective CRH receptor antagonist. This thermogenic effect of CRH can also be blocked by interference along pathways of de novo lipogenesis and lipid oxidation, as well as by inhibitors of phosphatidylinositol 3-kinase or AMP-activated protein kinase. Taken together, these studies demonstrate that CRH can directly stimulate thermogenesis in skeletal muscle, and in addition raise the possibility that this thermogenic effect, which requires both phosphatidylinositol 3-kinase and AMP-activated protein kinase signaling, might occur via substrate cycling between de novo lipogenesis and lipid oxidation. The effect of CRH in directly stimulating thermogenesis in skeletal muscle underscores a potentially important peripheral role for the CRH/urocortin system in the control of thermogenesis in this tissue, in its protection against excessive intramyocellular lipid storage, and hence against skeletal muscle lipotoxicity and insulin resistance.

Adenylate Kinase↗
PubMed2004

Substrate cycling between de novo lipogenesis and lipid oxidation: a thermogenic mechanism against skeletal muscle lipotoxicity and glucolipotoxicity.

Life is a combustion, but how the major fuel substrates that sustain human life compete and interact with each other for combustion has been at the epicenter of research into the pathogenesis of insulin resistance ever since Randle proposed a 'glucose-fatty acid cycle' in 1963. Since then, several features of a mutual interaction that is characterized by both reciprocality and dependency between glucose and lipid metabolism have been unravelled, namely: the inhibitory effects of elevated concentrations of fatty acids on glucose oxidation (via inactivation of mitochondrial pyruvate dehydrogenase or via desensitization of insulin-mediated glucose transport),the inhibitory effects of elevated concentrations of glucose on fatty acid oxidation (via malonyl-CoA regulation of fatty acid entry into the mitochondria), and more recentlythe stimulatory effects of elevated concentrations of glucose on de novo lipogenesis, that is, synthesis of lipids from glucose (via SREBP1c regulation of glycolytic and lipogenic enzymes). This paper first revisits the physiological significance of these mutual interactions between glucose and lipids in skeletal muscle pertaining to both blood glucose and intramyocellular lipid homeostasis. It then concentrates upon emerging evidence, from calorimetric studies investigating the direct effect of leptin on thermogenesis in intact skeletal muscle, of yet another feature of the mutual interaction between glucose and lipid oxidation: that of substrate cycling between de novo lipogenesis and lipid oxidation. It is proposed that this energy-dissipating substrate cycling that links glucose and lipid metabolism to thermogenesis could function as a 'fine-tuning' mechanism that regulates intramyocellular lipid homeostasis, and hence contributes to the protection of skeletal muscle against lipotoxicity.

CCAAT-Enhancer-Binding Proteins↗
PubMed1996

The monofunctional glycosyltransferase of Escherichia coli is a member of a new class of peptidoglycan-synthesising enzymes.

Using conserved fingerprints in the glycosyltransferase (GTase) domain of high-molecular-weight penicillin-binding proteins (PBP), a gene (mgt) encoding a putative monofunctional glycosyltransferase has been identified in Haemophilus influenzae and in other bacteria] species. Here we report the cloning of the homologous Escherichia coli gene and show that the solubilised membrane fraction of E. coli cells overexpressing the mgt gene contain a significantly increased peptidoglycan synthesis activity. In contrast to the high-molecular-weight PBPs, this activity is not inhibited by Flavomycin.

Amino Acid Sequence↗
PubMed1996

Overexpression, purification, and characterization of UDP-N-acetylmuramyl:L-alanine ligase from Escherichia coli.

UDP-N-acetylmuramyl:L-alanine ligase from Escherichia coli was overexpressed more than 600-fold and purified to near homogeneity. The purified enzyme was found to ligate L-alanine, L-serine, and glycine, as well as the nonnatural amino acid beta-chloro-L-alanine, to UDP-N-acetylmuramic acid. On the basis of (i) the specificity constants of the enzyme determined for L-alanine, L-serine, and glycine and (ii) the levels of these amino acids in the intracellular pool, it was calculated that the rates of incorporation of L-serine and glycine into peptidoglycan precursor metabolites could maximally amount to 0.1 and 0.5%, respectively, of the rate of L-alanine incorporation.

Amino Acid Sequence↗
PubMed1994

Cloning of the pyruvate kinase gene (pyk) of Corynebacterium glutamicum and site-specific inactivation of pyk in a lysine-producing Corynebacterium lactofermentum strain.

The pyruvate kinase gene pyk from Corynebacterium glutamicum was cloned by applying a combination of PCR, site-specific mutagenesis, and complementation. A 126-bp DNA fragment central to the C. glutamicum pyk gene was amplified from genomic DNA by PCR with degenerate oligonucleotides as primers. The cloned DNA fragment was used to inactivate the pyk gene in C. glutamicum by marker rescue mutagenesis via homologous recombination. The C. glutamicum pyk mutant obtained was unable to grow on minimal medium containing ribose as the sole carbon source. Complementation of this phenotype by a gene library resulted in the isolation of a 2.8-kb PstI-BamHI genomic DNA fragment harboring the C. glutamicum pyk gene. Multiple copies of plasmid-borne pyk caused a 20-fold increase of pyruvate kinase activity in C. glutamicum cell extracts. By using large internal fragments of the cloned C. glutamicum gene, pyk mutant derivatives of the lysine production strain Corynebacterium lactofermentum 21799 were generated by marker rescue mutagenesis. As determined in shake flask fermentations, lysine production in pyk mutants was 40% lower than that in the pyk+ parent strain, indicating that pyruvate kinase is essential for high-level lysine production. This finding questions an earlier hypothesis postulating that redirection of carbon flow at the phosphoenol pyruvate branch point of glycolysis through elimination of pyruvate kinase activity results in an increase of lysine production in C. glutamicum and its close relatives.

Amino Acid Sequence↗
PubMed1992

Recombination of constant and variable modules alters DNA sequence recognition by type IC restriction-modification enzymes.

EcoR124 and EcoDXXI are allelic type I restriction-modification (R-M) systems whose specificity genes consist of common structural elements: two variable regions are separated by a constant, homologous region containing a number of repetitive sequence elements. In vitro recombination of variable and constant elements has led to fully active, hybrid R-M systems exhibiting new and predictable target site specificities. Methylation of synthetic DNA sequences with purified, hybrid modification methylases was used to confirm the proposed recognition sequences. The results clearly demonstrate the correlation between protein domains and target site specificity. Our data suggest that a bacterial population may switch the recognition sequences of its type I R-M system by single recombination events and thus is able to maintain a prokaryotic analogue of the immune system of variable specificity.

Base Sequence↗
PubMed1992

[Shoulder arthroscopy in degenerative and inflammatory diseases].

A new concept of shoulder arthroscopy is presented based on experience with 800 shoulder arthroscopies. All open surgical procedure on the shoulder are preceded by arthroscopy. The procedure is performed with the patient lying on his or her side. The medium is CO2, and the advantages are less bleeding, a natural picture, no fluid edema. After diagnostic arthroscopy (with CO2), operative arthroscopy (with fluid) or open joint surgery follows. The surgery can easily be performed in the same position for good access with any approach. Preliminary diagnostic arthroscopy helps to plan the procedure (approach, additional surgery). Minimal exposure is beneficial for faster rehabilitation.

Acromioclavicular Joint↗
PubMed1991

Increased protein flexibility leads to promiscuous protein--DNA interactions in type IC restriction-modification systems.

We have investigated the role of a four amino acid element that is repeated twice and three times, respectively, in the specificity polypeptides of the two allelic restriction-modification systems EcoR124 and EcoR124/3. We had earlier shown that this difference in amino acid sequence between the two systems is solely responsible for the different DNA sequence specificities of the two systems. The effect of single amino acid substitutions and small insertion and deletion mutations on restriction activity and modification specificity was determined in vivo by phage infection assays and in vitro by methylation of DNA with purified modification methylases. Mutant restriction-modification systems with changes in the number and the length of the central amino acid repeats exhibited decreased restriction activity and in some cases relaxed substrate specificity. Our data strongly support the idea that the repetitive amino acid motif in the specificity polypeptides forms part of a flexible interdomain linker. It may be responsible for positioning on the DNA the two major specificity polypeptide domains which are thought to contact independently the half sites of the split recognition sequences typical for all type I restriction-modification systems.

Alleles↗
PubMed1989

The Bradyrhizobium japonicum fixBCX operon: identification of fixX and of a 5' mRNA region affecting the level of the fixBCX transcript.

The Bradyrhizobium japonicum fixX gene was identified and shown to be essential for symbiotic and free-living, microaerobic nitrogen fixation. The fixX gene encodes a ferredoxin-like protein which may be involved in a redox process (electron transport?) essential for nitrogenase activity. This gene was localized downstream of fixC and its expression was dependent on the fixB promoter, providing evidence for the existence of a fixBCX operon. Mutagenesis and sequence analysis of the unusually long, 709bp leader region between the fixB promoter and the fixB structural gene did not reveal the presence of a nif or fix gene that was absolutely essential for nitrogen fixation. However, a short open reading frame (ORF) within this region encoding a polypeptide of 35 amino acids (ORF35) was shown to be efficiently translated. Chromosomal deletion of a 400bp DNA fragment covering ORF35 resulted in a three-fold reduction of the fixBCX mRNA level, which in turn also reduced the nitrogen fixation activity of this mutant. This suggests a possible post-transcriptional control mechanism for the expression of the fixBCX operon involving the stabilization of fixBCX mRNA by ribosomes actively translating ORF35.

Amino Acid Sequence↗
PubMed1989

Fine-tuning of nif and fix gene expression by upstream activator sequences in Bradyrhizobium japonicum.

The significance of Bradyrhizobium japonicum upstream activator sequences (UASs) for differential NifA-mediated fix and nif gene expression was investigated by two means: (i) hybrid fixA- and fixB-lacZ fusions were constructed by transposing a nifH-UAS cartridge in front of their promoters; and (ii) B. japonicum mutants were generated carrying specific chromosomal deletions or UAS cartridge insertions within the fixA, fixB or nifH promoter-upstream regions. Expression of fixA was not affected, and expression of fixB decreased only to 42%, when the respective fixA and fixB promoter-upstream DNAs were deleted. This shows that in B. japonicum the NifA-dependent activation of at least the fixA promoter does not require the presence of a closely adjacent UAS. Deletion of the UASs in front of the nifH gene not only reduced the expression of nifH down to 2.5% but, surprisingly, also resulted in a reduction of the fixB mRNA level to less than 20%. This suggests that the nifH-UASs may exert a long-range effect on the expression of the 3-kb-distant fixBCX operon in nif cluster I or B. japonicum. Artificial transposition of the nifH-UASs in front of the fixA and fixB promoters strongly enhanced fixA and fixB expression.

Base Sequence↗
PubMed1988

Regulation of the fixA gene and fixBC operon in Bradyrhizobium japonicum.

The transcriptional start site of the Bradyrhizobium japonicum fixBC operon was identified by nuclease S1 mapping. It was located approximately 700 base pairs upstream of fixB and was preceded by a promoter sequence that showed strong homology to the B. japonicum fixA promoter and thus to the general nif consensus promoter sequence. Further transcript mapping experiments revealed that fixA and fixBC transcription in B. japonicum strictly depended on the presence of the regulatory gene nifA and on low oxygen partial pressure. Consistent with these data, chromosomally integrated fixA- and fixB-lacZ fusions expressed beta-galactosidase activity only in the wild type but not in a nifA mutant and only under microaerobic but not aerobic growth conditions. The presence of nifA accounted for a 19-fold and 44-fold activation of the fixA and fixB promoters, respectively. These results show that the fixA and fixBC genes are regulated in a way similar to that of the nitrogenase genes nifH and nifDK. A very peculiar finding was that the fixA and fixB promoters, when they were located on plasmids, could hardly be activated by the NifA protein, irrespective of whether this was tested in Escherichia coli or B. japonicum backgrounds. This is in clear contrast to the situation with nifH and nifD promoters.

Chromosome Mapping↗
PubMed1987

Single amino acid changes that render human IFN-alpha 2 biologically active on mouse cells.

Human IFN-alpha 1 and IFN-alpha 2 differ in 28 of 166 amino acids and show very different specific antiviral activities on human and murine cells. We have identified, by hybrid scanning and site-directed mutagenesis, three residues in IFN-alpha 2, in positions 121, 125 and 132 which, when replaced individually or jointly by their IFN-alpha 1 counterparts, modify its activity on mouse cells by up to 400-fold. We argue that these residues are involved in direct contacts with the mouse interferon receptor.

Amino Acid Sequence↗