scholarly journals CcpC-Dependent Regulation of Citrate Synthase Gene Expression in Listeria monocytogenes

2008 ◽  
Vol 191 (3) ◽  
pp. 862-872 ◽  
Author(s):  
Meghna Mittal ◽  
Silvia Picossi ◽  
Abraham L. Sonenshein
Keyword(s):  
Listeria Monocytogenes ◽  
Citrate Synthase ◽  
De Novo ◽  
Krebs Cycle ◽  
Proximal Promoter ◽  
De Novo Synthesis ◽  
Distal Promoter ◽  
Rate Limiting

ABSTRACT Citrate synthase, the first and rate-limiting enzyme of the tricarboxylic acid branch of the Krebs cycle, was shown to be required for de novo synthesis of glutamate and glutamine in Listeria monocytogenes. The citrate synthase (citZ) gene was found to be part of a complex operon with the upstream genes lmo1569 and lmo1568. The downstream isocitrate dehydrogenase (citC) gene appears to be part of the same operon as well. Two promoters were shown to drive citZ expression, a distal promoter located upstream of lmo1569 and a proximal promoter located upstream of the lmo1568 gene. Transcription of citZ from both promoters was regulated by CcpC by interaction with a single site; assays of transcription in vivo and assays of CcpC binding in vitro revealed that CcpC interacts with and represses the proximal promoter that drives expression of the lmo1568, citZ, and citC genes and, by binding to the same site, prevents read-through transcription from the distal, lmo1569 promoter. Expression of the lmo1568 operon was not affected by the carbon source but was repressed during growth in complex medium by addition of glutamine.

scholarly journals In Salmonella enterica, 2-Methylcitrate Blocks Gluconeogenesis

2009 ◽  
Vol 192 (3) ◽  
pp. 771-778 ◽  
Author(s):  
Christopher J. Rocco ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Salmonella Enterica ◽  
Citrate Synthase ◽  
Krebs Cycle ◽  
Single Amino Acid ◽  
Inborn Errors ◽  
Cycle Enzyme ◽  
Serovar Typhimurium ◽  
Key Enzyme

ABSTRACT Strains of Salmonella enterica serovar Typhimurium LT2 lacking a functional 2-methylcitric acid cycle (2-MCC) display increased sensitivity to propionate. Previous work from our group indicated that this sensitivity to propionate is in part due to the production of 2-methylcitrate (2-MC) by the Krebs cycle enzyme citrate synthase (GltA). Here we report in vivo and in vitro data which show that a target of the 2-MC isomer produced by GltA (2-MCGltA) is fructose-1,6-bisphosphatase (FBPase), a key enzyme in gluconeogenesis. Lack of growth due to inhibition of FBPase by 2-MCGltA was overcome by increasing the level of FBPase or by micromolar amounts of glucose in the medium. We isolated an fbp allele encoding a single amino acid substitution in FBPase (S123F), which allowed a strain lacking a functional 2-MCC to grow in the presence of propionate. We show that the 2-MCGltA and the 2-MC isomer synthesized by the 2-MC synthase (PrpC; 2-MCPrpC) are not equally toxic to the cell, with 2-MCGltA being significantly more toxic than 2-MCPrpC. This difference in 2-MC toxicity is likely due to the fact that as a si-citrate synthase, GltA may produce multiple isomers of 2-MC, which we propose are not substrates for the 2-MC dehydratase (PrpD) enzyme, accumulate inside the cell, and have deleterious effects on FBPase activity. Our findings may help explain human inborn errors in propionate metabolism.

Isotopomer spectral analysis of intermediates of cholesterol synthesis in human subjects and hepatic cells

2002 ◽  
Vol 282 (6) ◽  
pp. E1222-E1230 ◽  
Author(s):  
B. Lindenthal ◽  
T. A. Aldaghlas ◽  
A. L. Holleran ◽  
T. Sudhop ◽  
H. K. Berthold ◽  
...  
Keyword(s):  
Spectral Analysis ◽  
Liver Cell ◽  
Cholesterol Synthesis ◽  
De Novo ◽  
Human Subjects ◽  
Cell Models ◽  
Turnover Rates ◽  
De Novo Synthesis

Steroid intermediates of the cholesterol synthesis pathway are characterized by rapid turnover rates relative to cholesterol due to their small pool size. Because the small pools will label rapidly, these intermediates may provide valuable information about the incorporation of isotopes in de novo synthesis of cholesterol and related compounds. The labeling of cholesterol synthesis intermediates from [1-13C]acetate was investigated in human subjects and in liver cell models by means of isotopomer spectral analysis (ISA). In human subjects, infusing [1-13C]acetate into the duodenum for 12 h demonstrated that ∼50% of the plasma lathosterol pool was derived from de novo synthesis during this interval. The lipogenic acetyl-CoA precursor pool enrichment reached a constant value within 3 h of the start of the infusion. In vitro studies indicated that liver cell models decrease de novo lathosterol synthesis when cholesterol synthesis is inhibited by statins or cholesterol-containing serum. We propose a new calculation to increase the accuracy and precision of cholesterol synthesis estimates in vivo combining the ISA of lathosterol and cholesterol.

De novo synthesis of fructo-oligosaccharides in leaf disks of certain Asteraceae

1972 ◽  
Vol 50 (2) ◽  
pp. 295-303 ◽  
Author(s):  
K. R. Chandorkar ◽  
F. W. Collins
Keyword(s):  
Chain Length ◽  
De Novo ◽  
Close Correlation ◽  
Acid Synthesis ◽  
Degree Of Polymerization ◽  
De Novo Synthesis ◽  
The Relationship ◽  
Leaf Disks

Incubation of leaf disks of certain genera of Asteraceae on phosphate-buffered, 5% sugar solutions resulted in the de novo synthesis of a homologous series of inulin-type fructosans. Fructo-oligosaccharides of degree of polymerization 3 to 21 or 22 were present in dandelion, chicory, lettuce, hawkweed, and sow thistle leaf disks after 72 h, but not in dahlia or sunflower. Synthesis occurred with media containing either fructose, glucose, or sucrose, but not with mannose or galactose. Fructosan formation began after about 36 h and continued with the sequential synthesis of homologs of increasing chain length. After 72 h, the relationship between the amount of polymer synthesized and the chain length appeared to be logarithmically biphasic, consisting of two series of exponentially decreasing values. Incubation for 120 h however, resulted in a distribution more closely resembling that found naturally in fructosan storing tissues. 14C-tracer studies showed that both the endogenous and exogenous carbohydrate sources contribute to fructosan synthesis. Fructo-oligosaccharide formation was blocked by cycloheximide, puromycin, and actinomycin D but not chloramphenicol, indicating that cytoplasmic protein and nucleic acid synthesis was required. Analysis of fructosan formation during incubation suggests a close correlation between transfructosylation mechanisms observed in vitro and the de novo synthesis of fructosans in vivo.

scholarly journals The de novo synthesis of numerous proteins is decreased during vitamin D3 deficiency and is gradually restored by 1, 25-dihydroxyvitamin D3 repletion in the islets of langerhans of rats

1999 ◽  
Vol 162 (1) ◽  
pp. 101-109 ◽  
Author(s):  
PM Bourlon ◽  
A Faure-Dussert ◽  
B Billaudel
Keyword(s):  
Molecular Weight ◽  
Vitamin D3 ◽  
Protein Production ◽  
De Novo ◽  
De Novo Synthesis ◽  
Glucose Stimulation ◽  
Dihydroxyvitamin D3 ◽  
Vitamin D3 Deficiency

Since both the release and de novo biosynthesis of insulin are severely decreased by vitamin D3 deficiency and improved by 1, 25-dihydroxyvitamin D3 (1,25(OH)2D3) repletion following a 6-h delay in the rat, the present experiments investigated the effects of vitamin D3 deficiency on the biosynthesis of heavier molecular weight proteins using electrophoretic separation. Gel protein staining by Coomassie blue showed very different profiles for islets protein production from 4-week vitamin D3-deficient rats compared with normal islets. The pattern was characterised by a decrease in high molecular weight proteins, concomitantly accompanied by an increase in low molecular weight proteins. This tendency was partially reversed in vivo by 1,25(OH)2D3 repletion treatment for 7 days and was evident after only 16 h of treatment. In parallel with these in vivo observations, which represent a static index of islets protein production, a kinetic study was performed in vitro by a double-labelling method allowing us to measure the de novo synthesis of proteins in islets during a strong 16.7 mM glucose stimulation. Comparison of 3H and 14C labelled samples was achieved via coelectrophoresis to avoid experimental artefacts. The study of the ratio of d.p.m. 3H/d.p.m. 14C for each molecular weight protein in islets stimulated by 16.7 mM glucose (versus basal 4.2 mM glucose) showed an increase in the height of certain peaks: 150, 130 and 8.5 kDa. Under the same conditions, islets from 4-week vitamin D3-deficient rats (versus normal islets) presented a large deficit of numerous newly synthesised proteins and particularly those implicated in the response to glucose stimulation. In vitro repletion of 1,25(OH)2D3 tended to reverse, at least in part, the deleterious effect of vitamin D3 deficiency on the de novo protein synthesis of islets but these effects were gradual. Indeed, there was no detectable effect at 2 h incubation, but 1,25(OH)2D3 increased the 60 to 65 kDa, 55 kDa, and 9 to 8 kDa molecular mass proteins at 4 h, and increased the level of most newly synthesised proteins at 6 h. These data support the hypothesis of a beneficial genomic influence of 1,25(OH)2D3 that occurs progressively within the islets of Langerhans and which may prepare the beta cells for an enhanced response to glucose stimulation.

scholarly journals Inosine 5′-monophosphate dehydrogenase binds nucleic acids in vitro and in vivo

2004 ◽  
Vol 379 (2) ◽  
pp. 243-251 ◽  
Author(s):  
Jeremy E. McLEAN ◽  
Nobuko HAMAGUCHI ◽  
Peter BELENKY ◽  
Sarah E. MORTIMER ◽  
Martin STANTON ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Catalytic Domain ◽  
De Novo ◽  
Tritrichomonas Foetus ◽  
Nucleic Acid Binding ◽  
Rate Limiting ◽  
Human Isoforms ◽  
Monophosphate Dehydrogenase

Inosine 5´-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides. In addition to the catalytic domain, IMPDH contains a subdomain of unknown function composed of two cystathione β-synthase domains. Our results, using three different assays, show that IMPDHs from Tritrichomonas foetus, Escherichia coli, and both human isoforms bind single-stranded nucleic acids with nanomolar affinity via the subdomain. Approx. 100 nucleotides are bound per IMPDH tetramer. Deletion of the subdomain decreases affinity 10-fold and decreases site size to 60 nucleotides, whereas substitution of conserved Arg/Lys residues in the subdomain with Glu decreases affinity by 20-fold. IMPDH is found in the nucleus of human cells, as might be expected for a nucleic-acid-binding protein. Lastly, immunoprecipitation experiments show that IMPDH binds both RNA and DNA in vivo. These experiments indicate that IMPDH has a previously unappreciated role in replication, transcription or translation that is mediated by the subdomain.

Regulatory Factors Associated with Synthesis of the Osmolyte Glycine Betaine in the Halophilic MethanoarchaeonMethanohalophilus portucalensis

1999 ◽  
Vol 65 (2) ◽  
pp. 828-833 ◽  
Author(s):  
Mei-Chin Lai ◽  
Daw-Renn Yang ◽  
Ming-Jen Chuang
Keyword(s):  
Glycine Betaine ◽  
De Novo ◽  
Compatible Solutes ◽  
In Vitro Assays ◽  
Intracellular Accumulation ◽  
De Novo Synthesis ◽  
Factors Associated ◽  
High Level

ABSTRACT The halophilic methanoarchaeon Methanohalophilus portucalensis can synthesize de novo and accumulate β-glutamine, N ɛ-acetyl-β-lysine, and glycine betaine (betaine) as compatible solutes (osmolytes) when grown at elevated salt concentrations. Both in vivo and in vitro betaine formation assays in this study confirmed previous nuclear magnetic resonance 13C-labelling studies showing that the de novo synthesis of betaine proceeded from glycine, sarcosine, and dimethylglycine to form betaine through threefold methylation. Exogenous sarcosine (1 mM) effectively suppressed the intracellular accumulation of betaine, and a higher level of sarcosine accumulation was accompanied by a lower level of betaine synthesis. Exogenous dimethylglycine has an effect similar to that of betaine addition, which increased the intracellular pool of betaine and suppressed the levels of N ɛ-acetyl-β-lysine and β-glutamine. Both in vivo and in vitro betaine formation assays with glycine as the substrate showed only sarcosine and betaine, but no dimethylglycine. Dimethylglycine was detected only when it was added as a substrate in in vitro assays. A high level of potassium (400 mM and above) was necessary for betaine formation in vitro. Interestingly, no methylamines were detected without the addition of KCl. Also, high levels of NaCl and LiCl (800 mM) favored sarcosine accumulation, while a lower level (400 mM) favored betaine synthesis. The above observations indicate that a high sarcosine level suppressed multiple methylation while dimethylglycine was rapidly converted to betaine. Also, high levels of potassium led to greater amounts of betaine, while lower levels of potassium led to greater amounts of sarcosine. This finding suggests that the intracellular levels of both sarcosine and potassium are associated with the regulation of betaine synthesis inM. portucalensis.

A micromass culture method for rat embryonic neural cells

1983 ◽  
Vol 61 (1) ◽  
pp. 247-262
Author(s):  
O.P. Flint
Keyword(s):  
De Novo ◽  
Culture Method ◽  
Neural Cells ◽  
Gamma Amino Butyric Acid ◽  
De Novo Synthesis ◽  
Rat Embryo ◽  
Amino Butyric Acid ◽  
Acid Incorporation

A method of culturing early (13-day) rat embryo neural cells is described. Undifferentiated neural epithelium is disaggregated and cultured in small discrete islands. Cells that destined to differentiate as neurons actively segregate from the other cells in the island and aggregate together into small clumps. Other cells flatten and attach to the substrate and resemble typical fibroblasts throughout the culture period. The clumps of preneuron cells spread out forming large irregular foci. Spreading is mediated by active cell movements. Cells in the foci differentiate as a pure population of neurons identifiable by specific inhibition of 3H-labelled gamma-amino butyric acid incorporation or by labelling with a monoclonal antibody to GQ-ganglioside. The ganglioside is not found on the cell surface at the start of culture after trypsinization, but emerges during the 5 days of culture. The antigen is similarly not present in the embryonic mesencephalon in vivo at 13 days post coitum, only emerging later in the differentiated midbrain. There is thus an apparent de novo synthesis, which is paralleled in vivo and in vitro.

scholarly journals Creatine synthesis: hepatic metabolism of guanidinoacetate and creatine in the rat in vitro and in vivo

2009 ◽  
Vol 296 (2) ◽  
pp. E256-E261 ◽  
Author(s):  
Robin P. da Silva ◽  
Itzhak Nissim ◽  
Margaret E. Brosnan ◽  
John T. Brosnan
Keyword(s):  
De Novo ◽  
Rat Hepatocytes ◽  
Hepatic Metabolism ◽  
Creatine Phosphate ◽  
Hepatic Uptake ◽  
De Novo Synthesis ◽  
Creatinine Excretion

Since creatinine excretion reflects a continuous loss of creatine and creatine phosphate, there is a need for creatine replacement, from the diet and/or by de novo synthesis. Creatine synthesis requires three amino acids, methionine, glycine, and arginine, and two enzymes, l-arginine:glycine amidinotransferase (AGAT), which produces guanidinoacetate acid (GAA), and guanidinoacetate methyltransferase (GAMT), which methylates GAA to produce creatine. In the rat, high activities of AGAT are found in the kidney, whereas high activities of GAMT occur in the liver. Rat hepatocytes readily convert GAA to creatine; this synthesis is stimulated by the addition of methionine, which increases cellular S-adenosylmethionine concentrations. These same hepatocytes are unable to produce creatine from methionine, arginine, and glycine. 15N from 15NH4Cl is readily incorporated into urea but not into creatine. Hepatic uptake of GAA is evident in vivo by livers of rats fed a creatine-free diet but not when rats were fed a creatine-supplemented diet. Rats fed the creatine-supplemented diet had greatly decreased renal AGAT activity and greatly decreased plasma [GAA] but no decrease in hepatic GAMT or in the capacity of hepatocytes to produce creatine from GAA. These studies indicate that hepatocytes are incapable of the entire synthesis of creatine but are capable of producing it from GAA. They also illustrate the interplay between the dietary provision of creatine and its de novo synthesis and point to the crucial role of renal AGAT expression in regulating creatine synthesis in the rat.

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