DNA methylation is a reversible biological signal.
(17/1719)
The pattern of DNA methylation plays an important role in regulating different genome functions. To test the hypothesis that DNA methylation is a reversible biochemical process, we purified a DNA demethylase from human cells that catalyzes the cleavage of a methyl residue from 5-methyl cytosine and its release as methanol. We show that similar to DNA methyltransferase, DNA demethylase shows CpG dinucleotide specificity, can demethylate mdCpdG sites in different sequence contexts, and demethylates both fully methylated and hemimethylated DNA. Thus, contrary to the commonly accepted model, DNA methylation is a reversible signal, similar to other physiological biochemical modifications. (+info)
Purification and characterization of phosphoglycerate mutase from methanol-grown Hyphomicrobium X and Pseudomonas AM1.
(18/1719)
Phosphoglycerate mutase has been purified from methanol-grown Hyphomicrobium X and Pseudomonas AMI by acid precipitation, heat treatment, ammonium sulphate fractionation, Sephadex G-50 gel filtration and DEAE-cellulose column chromatography. The purification attained using the Hyphomicrobium X extract was 72-fold, and using the Pseudomonas AMI extract, 140-fold. The enzyme purity, as shown by analytical polyacrylamide gel electrophoresis, was 50% from Hyphomicrobium X and 40% from Pseudomonas AMI. The enzyme activity was associated with one band. The purified preparations did not contain detectable amounts of phosphoglycerate kinase, phosphopyruvate hydratase, phosphoglycerate dehydrogenase or glycerate kinase activity. The molecular weight of the enzymic preparation was 32000 +/- 3000. The enzyme from both organisms was stable at low temperatures and, in the presence of 2,3-diphosphoglyceric acid, could withstand exposure to high temperatures. The enzyme from Pseudomonas AMI has a broad pH optimum at 7-0 to 7-6 whilst the enzyme from Hyphomicrobium X has an optimal activity at pH 7-3. The cofactor 2,3-diphosphoglyceric acid was required for maximum enzyme activity and high concentrations of 2-phosphoglyceric acid were inhibitory. The Km values for the Hyphomicrobium X enzyme were: 3-phosphoglyceric acid, 6-0 X 10(-3) M: 2-phosphoglyceric acid, 6-9 X 10(-4) M; 2,3-diphosphoglyceric acid, 8-0 X 10(-6) M; and for the Pseudomonas AMI ENzyme: 3-4 X 10(-3) M, 3-7 X 10(-4) M and 10 X 10(-6) M respectively. The equilibrium constant for the reaction was 11-3 +/- 2-5 in the direction of 2-phosphoglyceric acid to 3-phosphoglyceric acid and 0-09 +/- 0-02 in the reverse direction. The standard free energy for the reaction proceeding from 2-phosphoglyceric acid to 3-phosphoglyceric acid was -5-84 kJ mol(-1) and in the reverse direction +5-81 kJ mol(-1). (+info)
Characterization of methanol-oxidizing bacteria by their growth response to various chemicals.
(19/1719)
"Fingerprints" of strains of methanol-oxidizing bacteria were obtained by exposing them to a set of chemicals which could stimulate or inhibit the growth. The chemicals gave quantitative results, which were used to calculate the similarities between the strains. The method has been used for establishing identity or nonidentity between isolates, and its use in a search for random mutants is also outlined. (+info)
Alcoholysis reactions from starch with alpha-amylases.
(20/1719)
The ability of alpha-amylases from different sources to carry out reactions of alcoholysis was studied using methanol as substrate. It was found that while the enzymes from Aspergillus niger and Aspergillus oryzae, two well-studied saccharifying amylases, are capable of alcoholysis reactions, the classical bacterial liquefying alpha-amylases from Bacillus licheniformis and Bacillus stearothermophilus are not. The effect of starch and methanol concentration, temperature and pH on the synthesis of glucosides with alpha-amylase from A. niger was studied. Although methanol may inactivate alpha-amylase, a 90% substrate relative conversion can be obtained in 20% methanol at a high starch concentration (15% w/v) due to a stabilizing effect of starch on the enzyme. As the products of alcoholysis are a series of methyl-oligosaccharides, from methyl-glucoside to methyl-hexomaltoside, alcoholysis was indirectly quantified by high performance liquid chromatography analysis of the total methyl-glucoside produced after the addition of glucoamylase to the alpha-amylase reaction products. More alcoholysis was obtained from intact soluble starch than with maltodextrins or pre-hydrolyzed starch. The biotechnological implications of using starch as substrate for the production of alkyl-glucosides is analyzed in the context of these results. (+info)
Structure elucidation of Sch 20562, a glucosidic cyclic dehydropeptide lactone--the major component of W-10 antifungal antibiotic.
(21/1719)
A novel bacterium designated as Aeromonas sp. W-10 produces the antibiotic W-10 complex which comprises of two major and several minor components. The two major components from this complex, Sch 20562 (1) and Sch 20561 (1a), are of biological interest in view of their potent antifungal activity. The chemical degradation studies utilized for the assignment of structure 1 for Sch 20562 are described here. Some of the noteworthy diversity of structural features in this glucosidic cyclic dehydrononapeptide lactone 1 are: an N-terminal (D)-beta-hydroxymyristyl unit, three D-amino acid units, two (E)-alpha-aminocrotonyl units, and an O-alpha-D-glucosyl-N-methyl-L-allo-threonine unit. The structure determination of 1 utilized the selective cleavage of the dehydropeptide units by ozonolysis to form fragments that were sequenced by mass spectrometry. The stereochemistry of the amino acid units were assigned by isolation of the free amino acids from the hydrolysates of the fragments. The stereochemistry of the alpha-aminocrotonyl units and the glucosidic linkage were assigned by nmr spectroscopy and molecular rotation data. (+info)
Modified VP22 localizes to the cell nucleus during synchronized herpes simplex virus type 1 infection.
(22/1719)
The UL49 gene product (VP22) of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) is a virion phosphoprotein which accumulates inside infected cells at late stages of infection. We previously (J. A. Blaho, C. Mitchell, and B. Roizman, J. Biol. Chem. 269:17401-17410, 1994) discovered that the form of VP22 packaged into infectious virions differed from VP22 extracted from infected-cell nuclei in that the virion-associated form had a higher electrophoretic mobility in denaturing gels. Based on these results, we proposed that VP22 in virions was "undermodified" in some way. The goal of this study is to document the biological and biochemical properties of VP22 throughout the entire course of a productive HSV-1 infection. We now report the following. (i) VP22 found in infected cells is distributed in at least three distinct subcellular localizations, which we define as cytoplasmic, diffuse, and nuclear, as measured by indirect immunofluorescence. (ii) Using a synchronized infection system, we determined that VP22 exists predominantly in the cytoplasm early in infection and accumulates in the nucleus late in infection. (iii) While cytoplasmic VP22 colocalizes with the HSV-1 glycoprotein D early in infection, the nuclear form of VP22 is not restricted to replication compartments which accumulate ICP4. (iv) VP22 migrates as at least three unique electrophoretic species in denaturing sodium dodecyl sulfate-DATD-polyacrylamide gels. VP22a, VP22b, and VP22c have high, intermediate, and low mobility, respectively. (v) The relative distribution of the various forms of VP22 derived from infected whole-cell extracts varies during the course of infection such that low-mobility species predominate at early times and high-mobility forms accumulate later. (vi) The highest-mobility forms of VP22 partition with the cytoplasmic fraction of infected cells, while the lowest-mobility forms are associated with the nuclear fraction. (vii) Finally, full-length VP22 which partitions in the nucleus incorporates radiolabel from [32P]orthophosphate whereas cytoplasmic VP22 does not. Based on these results, we conclude that modification of VP22 coincides with its appearance in the nucleus during the course of productive HSV-1 infection. (+info)
Pex22p of Pichia pastoris, essential for peroxisomal matrix protein import, anchors the ubiquitin-conjugating enzyme, Pex4p, on the peroxisomal membrane.
(23/1719)
We isolated a Pichia pastoris mutant that was unable to grow on the peroxisome-requiring media, methanol and oleate. Cloning the gene by complementation revealed that the encoded protein, Pex22p, is a new peroxin. A Deltapex22 strain does not grow on methanol or oleate and is unable to import peroxisomal matrix proteins. However, this strain targets peroxisomal membrane proteins to membranes, most likely peroxisomal remnants, detectable by fluorescence and electron microscopy. Pex22p, composed of 187 amino acids, is an integral peroxisomal membrane protein with its NH2 terminus in the matrix and its COOH terminus in the cytosol. It contains a 25-amino acid peroxisome membrane-targeting signal at its NH2 terminus. Pex22p interacts with the ubiquitin-conjugating enzyme Pex4p, a peripheral peroxisomal membrane protein, in vivo, and in a yeast two-hybrid experiment. Pex22p is required for the peroxisomal localization of Pex4p and in strains lacking Pex22p, the Pex4p is cytosolic and unstable. Therefore, Pex22p anchors Pex4p at the peroxisomal membrane. Strains that do not express Pex4p or Pex22p have similar phenotypes and lack Pex5p, suggesting that Pex4p and Pex22p act at the same step in peroxisome biogenesis. The Saccharomyces cerevisiae hypothetical protein, Yaf5p, is the functional homologue of P. pastoris Pex22p. (+info)
Modulation of host cell membrane fluidity: a novel mechanism for preventing bacterial adhesion.
(24/1719)
Adhesion of bacterial enteropathogens to host mucosal surfaces is a critical primary step in the pathogenesis of diarrheal disease. We investigated the effects of altering the physical properties of eukaryotic cells on bacterial adhesion with the use of a series of three structurally dissimilar membrane fluidizers and several Escherichia coli as test strains. Lipid fluidity of the cell plasma membrane was measured by steady-state fluorescence anisotropy employing the probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3, 5-hexatriene. There was a dose-dependent and reversible inhibition of bacterial adhesion with increasing membrane fluidity. Time course experiments indicated that increasing membrane fluidity during the early stages of bacterial adhesion was essential for inhibition of attachment. None of the fluidizers affected the viability of either eukaryotic or prokaryotic cells. These findings demonstrate, for the first time, that changes in plasma membrane physical properties of epithelial cells can prevent microbial adhesion. This also suggests that altering the membrane properties of host cells could form a basis for novel strategies to prevent bacterial adhesion during infection in vivo. (+info)