Metabolic adaptation of endothelial cells to substrate deprivation.
(25/4423)
Endothelial cells are known to be metabolically rather robust. To study the mechanisms involved, porcine aortic endothelial cells (PAEC), cultured on microcarrier beads, were perfused with glucose (10 mM) or with substrate-free medium. Substrate-free perfusion for 2 h induced an almost complete loss of nucleoside triphosphates (31P-NMR) and decreased heat flux, a measure of total energy turnover, by >90% in parallel microcalorimetric measurements. Heat flux and nucleoside triphosphates recovered after addition of glucose. Because protein synthesis is a major energy consumer in PAEC, the rate of protein synthesis was measured ([14C]leucine incorporation). Reduction or blockade of energy supply resulted in a pronounced reduction in the rate of protein synthesis (up to 80% reduction). Intracellular triglyceride stores were decreased by approximately 60% after 2 h of substrate-free perfusion. Under basal perfusion conditions, PAEC released approximately 30 pmol purine. mg protein-1. min-1, i.e., 16% of the cellular ATP per hour, while ATP remained constant. Substrate deprivation increased the release of various purines and pyrimidines about threefold and also induced a twofold rise in purine de novo synthesis ([14C]formate). These results demonstrate that PAEC are capable of recovering from extended periods of substrate deprivation. They can do so by a massive downregulation of their energy expenditure, particularly protein synthesis, while at the same time using endogenous triglycerides as substrates and upregulating purine de novo synthesis to compensate for the loss of purines. (+info)
G+C content variation along and among Saccharomyces cerevisiae chromosomes.
(26/4423)
Past analyses of the genome of the yeast Saccharomyces cerevisiae have revealed substantial regional variation in G+C content. Important questions remain, though, as to the origin, nature, significance, and generality of this variation. We conducted an extensive analysis of the yeast genome to try to answer these questions. Our results indicate that open reading frames (ORFs) with similar G+C contents at silent codon positions are significantly clustered on chromosomes. This clustering can be explained by very short range correlations of silent-site G+C contents at neighboring ORFs. ORFs of high silent-site G+C content are disproportionately concentrated on shorter chromosomes, which causes a negative relationship between chromosome length and G+C content. Contrary to previous reports, there is no correlation between gene density and silent-site G+C content in yeast. Chromosome III is atypical in many regards, and possible reasons for this are discussed. (+info)
Incorporation of molybdenum into the iron-molybdenum cofactor of nitrogenase.
(27/4423)
The biosynthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase was investigated using 99Mo to follow the incorporation of Mo into precursors. 99Mo label accumulates on dinitrogenase only when all known components of the FeMo-co synthesis system, NifH, NifNE, NifB-cofactor, homocitrate, MgATP, and reductant, are present. Furthermore, 99Mo label accumulates only on the gamma protein, which has been shown to serve as a chaperone/insertase for the maturation of apodinitrogenase when all known components are present. It appears that only completed FeMo-co can accumulate on the gamma protein. Very little FeMo-co synthesis was observed when all known components are used in purified forms, indicating that additional factors are required for optimal FeMo-co synthesis. 99Mo did not accumulate on NifNE under any conditions tested, suggesting that Mo enters the pathway at some other step, although it remains possible that a Mo-containing precursor of FeMo-co that is not sufficiently stable to persist during gel electrophoresis occurs but is not observed. 99Mo accumulates on several unidentified species, which may be the additional components required for FeMo-co synthesis. The molybdenum storage protein was observed and the accumulation of 99Mo on this protein required nucleotide. (+info)
"Action-at-a-distance" mutagenesis. 8-oxo-7, 8-dihydro-2'-deoxyguanosine causes base substitution errors at neighboring template sites when copied by DNA polymerase beta.
(28/4423)
8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a common oxidative DNA lesion, favors a syn-conformation in DNA, enabling formation of stable 8-oxo-dG.A base mispairs resulting in G.C --> T.A transversion mutations. When human DNA polymerase (pol) beta was used to copy a short single-stranded gap containing a site-directed 8-oxo-dG lesion, incorporation of dAMP opposite 8-oxo-dG was slightly favored over dCMP depending on "downstream" sequence context. Unexpectedly, however, a significant increase in dCMP.A and dGMP.A mispairs was also observed at the "upstream" 3'-template site adjacent to the lesion. Errors at these undamaged template sites occurred in four sequence contexts with both gapped and primed single-stranded DNA templates, but not when pol alpha replaced pol beta. Error rates at sites adjacent to 8-oxo-dG were roughly 1% of the values opposite 8-oxo-dG, potentially generating tandem mutations during in vivo short-gap repair synthesis by pol beta. When 8-oxo-dG was replaced with 8-bromo-2'-deoxyguanosine, incorporation of dCMP was strongly favored by both enzymes, with no detectable misincorporation occurring at neighboring template sites. (+info)
Regulation of UCP3 by nucleotides is different from regulation of UCP1.
(29/4423)
UCP3 is an isoform of UCP1, expressed primarily in skeletal muscle. Functional properties of UCP3 are still largely unknown. Here, we report about the expression of UCP3 and of UCP1 in inclusion bodies of Escherichia coli. On solubilization and reconstitution into proteoliposomes, both UCP3 and UCP1 transport Cl- at rates equal to the reconstituted native UCP1. Cl- transport is inhibited by low concentrations of ATP, ADP, GTP and GDP. However, no H+ transport activity is found possibly due to the lack of a cofactor presents in UCP from mitochondria. The specificity of inhibition by nucleoside tri- and diphosphate is different between UCP1 and UCP3. UCP1 is more sensitive to tri- than diphosphate whereas in UCP3, the gradient is reverse. These results show a new paradigm for the regulation of thermogenesis at various tissues by the ATP/ADP ratio. In brown adipose tissue, the thermogenesis is correlated with a low ATP/ADP whereas in skeletal muscle, non-shivering thermogenesis is active at a high ATP/ADP ratio, i.e. in the resting state. (+info)
A low rate of nucleotide changes in Escherichia coli K-12 estimated from a comparison of the genome sequences between two different substrains.
(30/4423)
Two genome sequences of Escherichia coli K-12 substrains, one partial W3110 and one complete MG1655, have been determined by Japanese and American genome projects, respectively. In order to estimate the rate of nucleotide changes, we directly compared 2 Mb of the nucleotide sequences from these closely-related E. coli substrains. Given that the two substrains separated about 40 years ago, the rate of nucleotide changes was estimated to be less than 10(-7) per site per year. This rate was supported by a further comparison between partial genome sequences of E. coli and Shigella flexneri. (+info)
Thermodynamics of nucleotide binding to the chaperonin GroEL studied by isothermal titration calorimetry: evidence for noncooperative nucleotide binding.
(31/4423)
We characterized the thermodynamics of binding reactions of nucleotides ADP and ATPgammaS (a nonhydrolyzable analog of ATP) to GroEL in a temperature range of 5 degrees C to 35 degrees C by isothermal titration calorimetry. Analysis with a noncooperative binding model has shown that the bindings of nucleotides are driven enthalpically with binding constants of 7x103 M-1 and 4x104 M-1 for ADP and ATPgammaS, respectively, at 26 degrees C and that the heat capacity change DeltaCp is about 100 cal/mol.K for both the nucleotides. The stoichiometries of binding were about 8 and 9 molecules for ADP and ATPgammaS, respectively, per GroEL tetradecamer at 5 degrees C, and both increased with temperature to reach about 14 (ADP) and 12 (ATPgammaS) for both nucleotides at 35 degrees C. The absence of initial increase of binding heat as well as Hill coefficient less than 1.2, which were obtained from the fitting to the model curve by assuming positive cooperativity, showed that there was virtually no positive cooperativity in the nucleotide bindings. Incorporating a difference in affinity for the nucleotide (ADP and ATPgammaS) between the two rings of GroEL into the noncooperative binding model improved the goodness of fitting and the difference in the affinity increased with decreasing temperature. (+info)
Determinants of nucleotide sugar recognition in an archaeon DNA polymerase.
(32/4423)
Vent DNA polymerase normally discriminates strongly against incorporation of ribonucleotides, 3'-deoxyribonucleotides (such as cordycepin) and 2',3'-dideoxyribonucleotides. To explore the basis for this discrimination we have generated a family of variants with point mutations of residues in conserved Regions II and III and assayed incorporation of nucleo-tides with modified sugars by these variants, all of which were created in an exonuclease-deficient form of the enzyme. A Y412V variant incorporates ribonucleotides at least 200-fold more efficiently than the wild-type enzyme, consistent with Y412 acting as a 'steric gate' to specifically exclude ribonucleotides. The most striking variants tested involved changes to A488, a residue predicted to be facing away from the nucleotide binding site. The pattern of relaxed specificity at this position roughly correlates with the size of the substituted amino acid sidechain and affects a variety of modified nucleotide sugars. (+info)