Ethanol, glycogen and glucosylglycerol represent competing carbon pools in ethanol-producing cells of Synechocystis sp. PCC 6803 under high-salt conditions.

Cyanobacteria are photoautotrophic micro-organisms, which are increasingly being used as microbial cell factories to produce, for example, ethanol directly from solar energy and CO2. Here, we analysed the effects of different salt concentrations on an ethanol-producing strain of Synechocystis sp. PCC 6803 that overexpresses the pyruvate decarboxylase (pdc) from Zymomonas mobilis and the native alcohol dehydrogenase (adhA). Moderate salinities of 2 % NaCl had no negative impact on ethanol production, whereas the addition of 4 % NaCl resulted in significantly decreased ethanol yields compared to low-salt conditions. Proteomic analysis identified a defined set of proteins with increased abundances in ethanol-producing cells. Among them, we found strong up-regulation of α-1,4 glucan phosphorylase (GlgP, Slr1367) in the producer strain, which consistently resulted in a massive depletion of glycogen pools in these cells regardless of the salinity. The salt-induced accumulation of the compatible solute glucosylglycerol was not affected by the ethanol production. Glycogen and probably compatible solutes could present competing pools with respect to organic carbon, explaining the decreased ethanol production at the highest salinity.

"Short-chain" alpha-1,4-glucan phosphorylase having a truncated N-terminal domain: functional expression and characterization of the enzyme from Sulfolobus solfataricus.

All known alpha-1,4-glucan phosphorylases (GPs) are active as homodimers and use their N-terminal domains for oligomerisation. Structure-based sequence comparison of a putative phosphorylase from the thermophilic crenarchaeon Sulfolobus solfataricus (SsGP) with the well characterized GP from Escherichia coli reveals that SsGP totally lacks the otherwise conserved regions for building the dimer interface. Because all efforts of producing functional SsGP in E. coli failed, we used heterologous gene expression in the hyperthermophilic archaeon Thermococcus kodakaraensis and isolated, in low amounts, SsGP harboring Strep-Tag II fused to the C-terminal Tyr-465 of the enzyme. The recombinant protein eluted in size exclusion chromatography with an apparent molecular mass of approximately 69 kDa, consistent with neither the mass expected for a monomer (55 kDa) nor that of a homodimer (110 kDa). The biochemical properties of SsGP were similar to those seen for other GPs containing the N-terminal elements for dimerisation, suggesting that the "short-chain" format of SsGP is fully appropriate for phosphorylase catalytic function and stability. However, the substrate specificity of SsGP differed from that reported for GPs from other thermophilic microorganisms.

Expression of muscle-gene-specific isozymes of phosphorylase and creatine kinase in innervated cultured human muscle.

Isozymes of creatine kinase and glycogen phosphorylase are excellent markers of skeletal muscle maturation. In adult innervated muscle only the muscle-gene-specific isozymes are present, whereas aneurally cultured human muscle has predominantly the fetal pattern of isozymes. We have studied the isozyme pattern of human muscle cultured in monolayer and innervated by rat embryo spinal cord explants for 20-42 d. In this culture system, large groups of innervated muscle fibers close to the ventral part of the spinal cord explant continuously contracted. The contractions were reversibly blocked by 1 mM d-tubocurarine. In those innervated fibers, the total activity and the muscle-gene-specific isozymes of both enzymes increased significantly. The amount of muscle-gene-specific isozymes directly correlated with the duration of innervation. Control noninnervated muscle fibers from the same dishes as the innervated fibers remained biochemically immature. This study demonstrated that de novo innervation of human muscle cultured in monolayer exerts a time-related maturational influence that is not mediated by a diffusable neural factor.

Synthesis of three deoxy-sophorose derivatives for evaluating the requirement of hydroxy groups at position 3 and/or 3' of sophorose by 1,2-ß-oligoglucan phosphorylases.

Sophorose (Sop2) is known as a powerful inducer of cellulases in Trichoderma reesei, and in recent years 1,2-ß-D-oligoglucan phosphorylase (SOGP) has been found to use Sop2 in synthetic reactions. From the structure of the complex of SOGP with Sop2, it was predicted that both the 3-hydroxy group at the reducing end glucose moiety of Sop2 and the 3'-hydroxy group at the non-reducing end glucose moiety of Sop2 were important for substrate recognition. In this study, three kinds of 3- and/or 3'-deoxy-Sop2 derivatives were synthesized to evaluate this mechanism. The deoxygenation of the 3-hydroxy group of D-glucopyranose derivative was performed by radical reduction using a toluoyl group as a leaving group. The utilization of a toluoyl group that plays two roles (a leaving group for the deoxygenation and a protecting group for a hydroxy group) resulted in efficient syntheses of the three target compounds. The NMR spectra of the two final compounds (3-deoxy- and 3,3'-dideoxy-Sop2) suggested that the glucose moiety of the reducing end of Sop2 can easily take on a furanose structure (five-membered ring structure) by deoxygenation of the 3-hydroxy group of Sop2. In addition, the ratio of the five- and six-membered ring structures changed depending on the temperature. The SOGPs exhibited remarkably lower specific activity for 3'-deoxy- and 3,3'-dideoxy-Sop2, indicating that the 3'-hydroxy group of Sop2 is important for substrate recognition by SOGPs.

Expression of muscle-type phosphorylase in innervated and aneural cultured muscle of patients with myophosphorylase deficiency.

Patients with McArdle's myopathy lack muscle glycogen phosphorylase (M-GP) activity. Regenerating and cultured muscle of patients with McArdle's myopathy presents a glycogen phosphorylase (GP) activity, but it is not firmly established whether M-GP or non-M-GP isoforms are expressed. We have cultured myoblasts from biopsy specimen of five patients with McArdle's myopathy. Skeletal muscle was cultured aneurally or was innervated by coculture with fetal rat spinal cord explants. In the patients' muscle biopsies and in their cultured innervated and aneural muscle we studied total GP activity, isoenzymatic pattern, reactivity with anti-M-GP antiserum, and presence of M-GP mRNA. There was no detectable enzymatic activity, no immunoreactivity with anti-M-GP antiserum, and no M-GP mRNA in the muscle biopsy of all patients. GP activity, M-GP isozyme, and anti-M-GP antiserum reactivity were present in patients' aneural cultures, increased after innervation, and were undistinguishable from control. M-GP mRNA was demonstrated in both aneural and innervated cultures of patients and control by primer extension and PCR amplification of total RNA. Our studies indicate that the M-GP gene is normally transcribed and translated in cultured muscle of patients with myophosphorylase deficiency.

[Mechanism of inhibiting proliferation of human ovarian cancer cells of the line HO-8910 by dexamethasone: the role of RhoB signaling pathway].

OBJECTIVE: To observe the effect of dexamethasone (Dex) on the proliferation of human ovarian cancer cells of the line HO-8910, and explore the role of RhoB signaling pathway in this process. METHODS: Human ovarian cancer cells of the line HO-8910he were cultured in culture fluids with or without different concentrations of Dex. The cell growth levels in anchor-dependent and anchor-independent manner were detected by MTT and soft agar assay. Another HO-8910 cells were inoculated in gel with different concentrations of Dex. HO-8910 was transfected with the eukaryotic expression plasmid RhoB-wt, blank plasmids pcDNA3 and RhoB-RNAi, and then the mRNA expression of RhoB, a small GTPase gene, was examined by semi-quantitative RT-PCR. and the protein expressions of RhoB, p-Akt, and p21(cip1/waf1) and p27, both cyclin kinase inhibitors (CDIs), were detected by Western blotting. HO-8910 cells were co-transfected with the reporter gene p21-luc containing p21 promoter and marker reporter gene pRL-tk-luc, then treated with Dex for 24 h. Western blotting was used to detect the transcription of p21(cip1/waf1) gene. RESULTS: The RhoB mRNA expression was significantly increased 2 hours after the treatment of 100 nM Dex, and peaked 4 hours later as high as 2.5 times that of the control group. Western blotting showed that the RhoB protein expression increased along the increase of the Des concentration. The protein expression of RhoB in the HO-8910 cells transfected with RhoB-wt was 2.02 times that in the HO-8910 cells transfected with blank plasmid, and the protein expression of RhoB in the HO-8910 cells transfected with RhoB-RNAi was 36% of that of the blank plasmid group (P < 0.01). The HO-8910 cell proliferation of the RhoB-RNA1 group was not significantly different from that of the control group, however, the proliferation of the HO-8910 cell treated by 100 nM Dex for 6 days was significantly inhibited with an inhibition rate of 13% (P < 0.01). Western blotting showed that Dex down-regulated the p-Akt protein expression. Dex time and dose-dependently up-regulated the protein expression of p21(cip1/waf1) and p27. The HO-8910 cells co-transfected with p21-luc and pRL-tk-luc and then treated with Dex for 24 h showed an higher p21-luc activity, 1.72 times that of the control group (P < 0.05). CONCLUSION: The mechanism of inhibiting the proliferation by Dex in ovarian cancer cells may involve the depression of PI3K/p-Akt, and then up-regulation of RhoB and its downstream signal molecules p21(cip1/waf1) and p27 proteins.

Histochemical profile of mouse hepatocellular adenomas and carcinomas induced by a single dose of diethylnitrosamine.

In continuation of earlier studies on murine neoplastic liver lesions, we characterized by histochemical methods the phenotype of hepatocellular adenomas and carcinomas induced by single injections of diethylnitrosamine (1.25, 2.5, or 5.0 micrograms/g of body weight) in 15-day-old C57BL/6 x male C3H F1 mice. The hepatocellular adenomas were composed predominantly of basophilic cells but stored excessive amounts of fat and glycogen in large portions of the tumors. Irrespective of the carcinogenic dose, the adenomas showed a consistent histochemical pattern. Glycogen synthase and phosphorylase were highly active in the hepatocytes that stored glycogen. In cells poor in, or free of, this polysaccharide, these enzymes were only moderately active or even inactive. In glycogen-storing parts of the adenomas, the activity of adenylate cyclase was reduced compared with normal liver parenchyma, but in fat-storing portions it was elevated. In a few adenomas, uniform increase in adenylate cyclase activity could be encountered. The levels of ATPase, acid phosphatase, and glucose-6-phosphatase were either increased or decreased. Glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase showed an increased activity in all adenomas compared with preneoplastic foci, which in turn exhibited a higher glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase activity than the surrounding parenchyma or the liver of untreated controls. The hepatocellular carcinomas showed remarkable histochemical changes compared with adenomas. The levels of fat and glycogen and the activities of glycogen synthase, phosphorylase, and in most cases also that of glucose-6-phosphate dehydrogenase, were reduced significantly. In contrast, adenylate cyclase, glucose-6-phosphatase, glyceraldehyde-3-phosphate dehydrogenase, and also alkaline phosphatase showed a striking elevation in developing carcinomas. Similar, although more pronounced, histochemical changes were seen in the advanced hepatocellular carcinomas. These observations indicated that progression from adenomas to hepatocellular carcinomas was associated with a change in the activity of several enzymes involved in cell membrane function, glycogen metabolism, the oxidative pentose phosphate pathway, and glycolysis.

Reduction of the plastidial phosphorylase in potato (Solanum tuberosum L.) reveals impact on storage starch structure during growth at low temperature.

Tubers of potato (Solanum tuberosum L.), one of the most important crops, are a prominent example for an efficient production of storage starch. Nevertheless, the synthesis of this storage starch is not completely understood. The plastidial phosphorylase (Pho1; EC 2.4.1.1) catalyzes the reversible transfer of glucosyl residues from glucose-1-phosphate to the non-reducing end of α-glucans with the release of orthophosphate. Thus, the enzyme is in principle able to act during starch synthesis. However, so far under normal growth conditions no alterations in tuber starch metabolism were observed. Based on analyses of other species and also from in vitro experiments with potato tuber slices it was supposed, that Pho1 has a stronger impact on starch metabolism, when plants grow under low temperature conditions. Therefore, we analyzed the starch content, granule size, as well as the internal structure of starch granules isolated from potato plants grown under low temperatures. Besides wild type, transgenic potato plants with a strong reduction in the Pho1 activity were analyzed. No significant alterations in starch content and granule size were detected. In contrast, when plants were cultivated at low temperatures the chain length distributions of the starch granules were altered. Thus, the granules contained more short glucan chains. That was not observed in the transgenic plants, revealing that Pho1 in wild type is involved in the formation of the short glucan chains, at least at low temperatures.

Temporal regulation of the Dictyostelium glycogen phosphorylase 2 gene.

The product of the glycogen phosphorylase-2 gene in Dictyostelium functions to provide the glucose units that are used to construct the structural components of the terminal stage of development. In this report, we link a 1233 bp upstream gp2 fragment to a luciferase reporter gene in order to study the sequences that are involved in the temporal expression of the gene. Various deletions of the promoter-luciferase fusion were then transformed into Dictyostelium cells. All deletion constructs, from -1216 to -486 nucleotides from the translational start codon, showed the same temporal pattern of expression as the authentic gp2 gene, as well as similar luciferase activities. Removal of an additional 37 nucleotides resulted in nearly 100-fold decrease in activity, yet retained the normal temporal expression of luciferase. Analysis of DNA binding proteins with the gel shift assay revealed a stage-dependent pattern of proteins that bound to the gp2 promoter. A similar pattern of temporal expression of the binding proteins was observed with either the full-length probe or with oligonucleotide probes that contained sequences that were identified as putative regulatory sites. Likewise, the full-length and oligonucleotide probes demonstrated identical binding patterns during several steps of purification of the DNA binding proteins. SDS-PAGE and Southwestern blot analysis of a DNA-affinity purified fraction, identified a 23 kDa peptide as the binding protein.

Expression of glucokinase in cultured human muscle cells confers insulin-independent and glucose concentration-dependent increases in glucose disposal and storage.

Insulin resistance, as is found in skeletal muscle of individuals with obesity and NIDDM, appears to involve a reduced capacity of the hormone to stimulate glucose uptake and/or phosphorylation. The glucose phosphorylation step, as catalyzed by hexokinase II, has been described as rate limiting for glucose disposal in muscle, but overexpression of this enzyme under control of a muscle-specific promoter in transgenic mice has had limited metabolic impact. In the current study, we investigated in a cultured muscle model whether expression of glucokinase, which in contrast to hexokinase II is not inhibited by glucose-6-phosphate (G-6-P), would have a pronounced metabolic impact. We used a recombinant adenovirus containing the cDNA-encoding rat liver glucokinase (AdCMV-GKL) to increase the glucose phosphorylating activity in cultured human muscle cells by fourfold. G-6-P levels increased in AdCMV-GKL-treated cells in a glucose concentration-dependent manner over the range of 1-30 mmol/l, whereas the much smaller increases in G-6-P in control cells were maximal at glucose concentrations <5 mmol/l. Further, cells expressing glucokinase accumulated 17 times more 2-deoxyglucose-6-phosphate than control cells. In AdCMV-GKL-treated cells, the time-dependent rise in G-6-P correlated with an increase in the activity ratio of glycogen synthase. AdCMV-GKL-treated cells also exhibited a 2.5- to 3-fold increase in glycogen content and a four- to fivefold increase in glycolytic flux, proportional to the increase in glucose phosphorylating capacity. All of these observations were made in the absence of insulin. Thus we concluded that expression of glucokinase in cultured human muscle cells results in proportional increases in insulin-independent glucose disposal, and that muscle glucose storage and utilization becomes controlled in a glucose concentration-dependent manner in AdCMV-GKL-treated cells. These results encourage testing whether delivery of glucokinase to muscle in vivo has an impact on glycemic control, which could be a method for circumventing the failure of insulin to stimulate glucose uptake and/or phosphorylation in muscle normally in insulin-resistant subjects.

High expression of a synthetic gene encoding potato alpha-glucan phosphorylase in Aspergillus niger.

We describe the successful heterologous expression of the Solanum tuberosum alpha-glucan phosphorylase (GP) gene in Aspergillus niger. Special attention was paid to the influence of different codon usage and A+T content in the coding region on GP protein expression. Use of A. niger-preferred codon usage and lower A+T content in a synthetic gene (GP-syn) resulted in a significant improvement in the level of the GP mRNA and a dramatic increase in the quantity of GP protein produced such that it accounted for approximately 10% of the total soluble protein. We suggest that redesigning the primary DNA sequence encoding a desired protein product can be an extremely effective method for improving heterologous protein production in filamentous fungi.

Regulation of the rat muscle glycogen phosphorylase-encoding gene during muscle cell development.

The muscle isozyme of glycogen phosphorylase (MGP) catalyzes the hydrolysis hydrolysis of intracellular glycogen in mammalian tissues and is produced in skeletal muscle, brain and heart. The MGP gene is developmentally and neutrally regulated in skeletal muscle, but little is known about the gene's transcriptional regulation. We have isolated and characterized the 5' flanking region of rat MGP. Truncated portions of the MGP 5' flanking region were coupled to the bacterial cat reporter gene and used in transient transfection assays in the mouse muscle C2C12 cell line. The region between -211 and +62 contained the smallest regulatory domain capable of demonstrating developmentally regulated myogenic expression in C2C12 cells. This was in contrast with findings from another investigation that transfected this cell line with human MGP [Lockyer and McCracken, J. Biol. Chem. 266 (1991) 20262-20269]. A 172-nucleotide (nt) region between -839 and -666 functioned as a potent enhancer in C2C12 cells when coupled to its cognate promoter, but not when coupled to a simian virus 40 promoter. This rat MGP enhancer region is 78% identical to a comparable region of the human MGP 5' flanking region, but contains only one putative regulatory element that has been previously identified in other muscle genes. These data suggest that rat MGP transcription in C2C12 muscle cells is modulated by a potent enhancer that utilizes novel regulatory elements.

Molecular cloning and sequencing of the glycogen phosphorylase gene from Escherichia coli.

The glgP gene, which codes for glycogen phosphorylase, was cloned from a genomic library of Escherichia coli. The nucleotide sequence of the glgP gene contained a single open reading frame encoding a protein consisting of 790 amino acid residues. The glgP gene product, a polypeptide of Mr 87,000, was confirmed by SDS-polyacrylamide gel electrophoresis. The deduced amino acid sequence showed that homology between glgP of E. coli and rabbit glgP, human glgP, potato glgP, and E. coli malP was 48.6, 48.6, 42.3, and 46.1%, respectively. Within this homologous region, the active site, glycogen storage site, and pyridoxal-5'-phosphate binding site are well conserved. The enzyme activity of glycogen phosphorylase increased after introduction on a multicopy of the glgP gene.

Gene expression during the differentiation of myogenic cells of the L6 line.

The problem of the mechanistic relationship among the different phenotypic expressions in an established myogenic line was approached by blocking cell fusion at different developmental stages, by addition of cytochalasin B. The addition of the drug to cultures at the time when the first two myotubes appeared on the dish, blocked fusion, but did not affect DNA synthesis, expression of myosin, phosphorylase, phosphocreatine kinase, phosphorylase kinase or glycogen synthetase, nor the organization of the elements of the hexagonal lattice. It is concluded that cell fusion is not a prerequisite for the expression of the differentiated phenotype.

Cloning of bovine muscle glycogen phosphorylase cDNA and identification of a mutation in cattle with myophosphorylase deficiency, an animal model for McArdle's disease.

Genetic defects of myophosphorylase in humans cause a metabolic myopathy (McArdle's disease) characterized by exercise intolerance, cramps, and recurrent myoglobinuria. Recently, a breed of cattle with myophosphorylase deficiency has been identified: this is the first animal model of McArdle's disease. To define the molecular genetic error in the cattle, we cloned and sequenced the wild-type bovine myophosphorylase cDNA. Homology to human cDNA is 95.8% for the amino acid sequence, and 92.0% for the nucleotide sequence. Sequence homology to rabbit cDNA is 97.3% in amino acid, 90.8% in nucleotide. In the cDNA fragments amplified by RT-PCR from muscle RNA of the cattle with myophosphorylase deficiency, we identified a C-to-T substitution, changing an encoded arginine (CGG) to tryptophan (TGG) at codon 489. The mutant residue is adjacent to pyridoxal phosphate binding sites and to an active site residue, and the sequence around this mutation is highly conserved in different species.

Coupled diminished energy turnover and phosphorylase a formation in contracting hypothyroid rat muscle.

Hypothyroidism leads to a diminished phosphorylase a formation and a reduction in both glycogen breakdown and lactate production during tetanic stimulation of fast-twitch skeletal muscle (mixed type). Phosphorylase kinase activity is almost 50% lower in the hypothyroid (Tx) group, and the possibility that this might explain the reduction in phosphorylase a formation is discussed. In both the Tx group and the euthyroid (C) group the reconversion of phosphorylase a to b correlates well with a decrease in the energy cost for contraction.

Reciprocal effects of the protein kinase C inhibitors staurosporine and H-7 on the regulation of glycogen synthase and phosphorylase in the primary culture of hepatocytes.

The effects of the protein kinase C inhibitors staurosporine and H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine] on glucose-induced regulation of glycogen synthase and phosphorylase activities were investigated in the primary culture of hepatocytes. Glycogen synthesis as measured by the incorporation of [14C]glucose into glycogen was enhanced up to 78% (P < .001) by 100 nmol/L staurosporine. In contrast, H-7 inhibited glycogen synthesis in a dose-dependent manner, with an IC50 value of 70 mumol/L. Activation of glycogen synthase by 30 mmol/L glucose was enhanced significantly (P < .02 and less) by staurosporine at 20 nmol/L and higher concentrations whereas the activity of this enzyme was inhibited by H-7 (IC50 = 50 mumol/L). The inactivation of phosphorylase by glucose was significantly greater when staurosporine was included in the medium. However, H-7 increased the phosphorylase activity ratio by 1.5- to 2.5-fold at concentrations of 20 to 100 mumol/L. The time course of synthase activation and phosphorylase inactivation showed that the effect of glucose was enhanced by staurosporine and inhibited by H-7. These novel reciprocal effects of protein kinase C inhibitors were also observed at different concentrations of glucose. The effects of H-8, a compound with structural resemblance to H-7 and an inhibitor of protein kinase A, were similar to those of staurosporine but not to those of H-7. Staurosporine blocked the effects of vasopressin and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), whereas H-7 in combination with these protein kinase C activators acted in the same direction. The effects of staurosporine, a relatively more specific inhibitor of protein kinase C, indicated that this enzyme plays a role in the regulation of glycogen metabolism in liver. However, H-7, which is known to have protein kinase C-independent effects in intact cells, seems to alter the activities of glycogen synthase and phosphorylase by a different mechanism.

Carcinogenesis of intestinal-type gastric cancer and colorectal cancer is commonly accompanied by expression of brain (fetal)-type glycogen phosphorylase.

Our previous studies have demonstrated the significant enzymatic activity of glycogen phosphorylase (GP) in the gastric carcinoma and proliferating cells of particular intestinal metaplasia (IM). This paper reviewed the identification of the GP isoform in the gastrointestinal carcinoma, and the investigation on the role of this molecule in the gastrointestinal carcinogenesis. The only isoform expressed in gastric cancer was brain-type GP (BGP) using polymerase chain reaction (PCR) analysis. The expression of BGP, oncogene products and proliferating cell nuclear antigen in the gastric and colorectal carcinomas, their premalignant lesions, and the normal mucosa were examined using 136 gastric and 96 colorectal surgically resected specimens, and 55 endoscopically resected colorectal adenomas. The BGP visualized by immunohistochemistry was commonly present in intestinal-type gastric (80.6%) and colorectal (83.3%) carcinomas, whereas no BGP expression was seen in the normal human gastric and large intestinal mucosa except in the BGP foci described below. IMs with BGP had close correlation with intestinal-type gastric carcinoma, and some of them coexpressed accumulated p53 protein. The expression of BGP during 'adenoma carcinoma sequence' (ACS) showed excellent correlation with the increased dysplasia and was found prior to p53 expression. Positive staining in overtly normal looking colonic mucosa (BGP foci) was observed mainly around carcinomas without any adenoma component, and frequent p53 mutation (41.2%) was detected in the BGP foci using PCR-single strand conformation polymorphism analysis. It is suggested that BGP is a novel biomarker for carcinogenesis in the intestinal-type gastric carcinoma and in both of the pathways of ACS and the 'de novo' colorectal carcinoma.