Characterisation of Trypanosoma cruzi populations by dna polymorphism of the cruzipain gene detected by single-stranded dna conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

Mdm20 Modulates Actin Remodeling through the mTORC2 Pathway via Its Effect on Rictor Expression

NatB is an N-terminal acetyltransferase consisting of a catalytic Nat5 subunit and an auxiliary Mdm20 subunit. In yeast, NatB acetylates N-terminal methionines of proteins during de novo protein synthesis and also regulates actin remodeling through N-terminal acetylation of tropomyosin (Trpm), which stabilizes the actin cytoskeleton by interacting with actin. However, in mammalian cells, the biological functions of the Mdm20 and Nat5 subunits are not well understood. In the present study, we show for the first time that Mdm20-knockdown (KD), but not Nat5-KD, in HEK293 and HeLa cells suppresses not only cell growth, but also cellular motility. Although stress fibers were formed in Mdm20-KD cells, and not in control or Nat5-KD cells, the localization of Trpm did not coincide with the formation of stress fibers in Mdm20-KD cells. Notably, knockdown of Mdm20 reduced the expression of Rictor, an mTORC2 complex component, through post-translational regulation. Additionally, PKCα^S657 phosphorylation, which regulates the organization of the actin cytoskeleton, was also reduced in Mdm20-KD cells. Our data also suggest that FoxO1 phosphorylation is regulated by the Mdm20-mTORC2-Akt pathway in response to serum starvation and insulin stimulation. Taken together, the present findings suggest that Mdm20 acts as a novel regulator of Rictor, thereby controlling mTORC2 activity, and leading to the activation of PKCα^S657 and FoxO1.     

Genetic analysis of a mutation affecting ribosomal protein S1 in Escherichia coli

Molecular Genetics and Genomics - Ribosomal protein S1 from a newly isolated Escherichia coli mutant has a molecular weight of about 54,000 which is smaller than the wild type S1 (M.W. 65,000). The...     

Purification and Properties of Prostaglandin H-E Isomerase from the Cytosol of Human Brain: Identification as Anionic Forms of Glutathione S-Transferase

Abstract: Prostaglandin H-E isomerase (EC 5.3.99.3) was purified from human brain cytosol. Purification was by ammonium sulfate fractionation, diethylaminoethyl-Sephar-ose chromatography, gel filtration on a BioGel P-100 column, GSH-agarose chromatography, and MonoQ chromatography. The activity was eluted in two peaks from the MonoQ column, which were designated peaks 1 and 2. The molecular weights of peaks 1 and 2, determined by gel filtration, were 42,000 and 44,000, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peak 1 showed two bands at the molecular weights of 24,500 and 25,000, and peak 2 showed a single band at the molecular weight of 25,000, results suggesting that both were dimeric proteins. The pI values of both enzymes were ∼5.4. The enzymes catalyzed selective conversion of prostaglandin H₂ to prostaglandin E₂. The K _m values for prostaglandin H₂ of peaks 1 and 2 were 147 and 308 μ M , respectively, and the V _max values were 380 and 720 nmol/min/mg of protein, respectively. GSH was required for the catalysis of both enzymes, and no other sulfhydryl compounds could support the reaction. A part of glutathione S -transferase (EC 2.5.1.18) was copurified with peaks 1 and 2 of prostaglandin H-E isomerase. Prostaglandin H-E isomerase activity of peak 2 enzyme was competitively inhibited by 1-chloro-2,4-dinitrobenzene, a substrate of glutathione S -transferase. These results suggested that prostaglandin H-E isomerases in human brain cytosol were identical with anionic forms of glutathione S -transferase.     

Site-directed mutagenesis of hepatitis B surface antigen sequence at codon 160 from arginine to lysine for conversion of subtypic determinant from r to w

Site-directed mutagenesis from G to A was induced at nucleotide 479 in the S gene of hepatitis B virus DNA, cloned from an individual carrying the surface antigen of subtype ayr. HepG2 cells were transfected with the plasmid DNA containing the mutant. They produced surface antigen of subtype ayw, unlike HepG2 cells harboring the parent viral DNA that produced surface antigen of subtype ayr. These results indicate that a point mutation from G to A at nucleotide 479 in the S gene, changing codon 160 for arginine to that for lysine, can convert the subtypic determinant of hepatitis B surface antigen from r to its allelic determinant w.     

Mutagenicity of nitro- and amino-substituted phenazines in Salmonella typhimurium

The nitro- and amino-substituted phenazines were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98 and TA98NR. Of 7 tested nitrophenazines, 4 were mutagenic in the absence of a microsomal metabolic activation system (S9 mix) and were more mutagenic in TA98 than in TA98NR. The order of mutagenicity of nitrophenazines in TA98 is 1.7- less than 2- less than 2.8- less than 2.7-substituted phenazine. Of 7 tested amino derivatives, 4 exhibited mutagenic activity with S9 mix in TA98. 1-Nitro-, 1-amino, 1.6-dinitro-, 1.9-dinitro-, 1.6-diamino- and 1.9-diamino-phenazine were not mutagenic. As regards the relationship between mutagenic potency and chemical structure of the phenazines, the results suggested that structural requirements favoring mutagenic activity were the presence of substituents at the 2 and/or 7 position. Furthermore, 2.7-disubstituted phenazines were extremely mutagenic, 2.7-dinitrophenazine and 2.7-diaminophenazine induced 36,450 and 12,110 rev./nmole, respectively. In the preliminary study, 2.7-diaminophenazine was identified by gas chromatography/mass spectrometry from the reaction mixture of m-phenylenediamine and hydrogen peroxide.     

Determination of the primary structure of Paim II, an alpha-amylase inhibitor from Streptomyces coruchorushii, by high-performance tandem mass spectrometry

This study indicates one of the advantages of tandem mass spectrometry; the primary structures of proteins with little structural difference can be determined by using tandem mass spectrometry without prior purification of each component. The primary structure of Paim II, a protein alpha-amylase inhibitor from Streptomyces coruchorushii, was determined by using tandem mass spectrometry. Paim II consists of two component proteins with ragged N-terminus, and was sequenced on the basis of the structure of Paim I, an analogous alpha-amylase inhibitor from the same natural origin.     

The primary structure of human EGF produced by genetic engineering, studied by high-performance tandem mass spectrometry

The primary structure of human epidermal growth factor (hEGF), which was produced by Escherichia coli using recombinant DNA technique, has been studied by tandem mass spectrometry. The molecular weight of hEGF (about 6200 amu) was determined by fast atom bombardment mass spectrometry. Then reduced and carboxymethylated hEGF was digested by chymotrypsin into seven peptides which could cover the whole sequence of hEGF. The amino acid sequences of five of these seven peptides could be confirmed by tandem mass spectrometry with or without isolation by high-performance liquid chromatography (HPLC). After isolation by HPLC, the other two peptides were digested with trypsin or thermolysin into small peptides, and sequenced by tandem mass spectrometry.     

15N]glycine metabolism in normal and cirrhotic subjects

Following a single oral dose of 10 mg/kg of [15N]glycine, plasma [15N]glycine kinetics and urinary 15N excretion were measured in 12 cirrhosis patients and in 6 control subjects. Cirrhosis patients were divided into two groups of 6 patients with and without a history of hepatic encephalopathy designated as group II and group I, respectively. Thirty minutes after oral administration of labeled glycine, the plasma concentration of [15N]glycine was significantly higher in both cirrhosis groups than that in the control group (P less than 0.05 and P less than 0.01). The elimination constant of plasma [15N]glycine slightly decreased in group II, but not significantly. Urinary 15N excretion did not differ among the three groups, but the rate of urinary ammonia 15N in urinary 15N was significantly increased in group II (P less than 0.05). The whole-body protein flux did not differ among the three groups, but whole-body protein breakdown was significantly increased in group II cirrhosis patients (P less than 0.05). These findings indicated that the kinetics of glycine were substantially altered in severe cirrhosis patients. Because hepatic uptake and oxidation of glycine was well maintained even in group II, increased endogenous protein breakdown seemed to be responsible for hyperglycinemia and also for the negative nitrogen balance seen in this group.     

Structural heterogeneity regarding local Shwartzman activity of lipid A

The relation of chemical structure to local Shwartzman activity of lipid A preparations purified by thin-layer chromatography from five bacterial strains was examined. Two lipid A fractions from E. coli F515--Ec-A2 and Ec-A3--exhibited strong activity, similar to that of previous synthetic E. coli-type lipid A (compound 506 or LA-15-PP). The Ec-A3 fraction contained a component that appeared to be structurally identical to compound 506, and the main component of Ec-A2 fraction was structurally similar to compound 506 except that it carried a 3-hydroxytetradecanoyl group at the C-3' position of the backbone in place of a 3-tetradecanoyloxytetradecanoyl group. Free lipid A (12 C) and purified lipid A fractions, Ec-A2 (12 C) and Ec-A3 (12 C), respectively, obtained from bacteria grown at 12 C, exhibited activity comparable to Ec-A2 or Ec-A3. In these preparations, a large part of the 3-dodecanoyloxytetradecanoyl group might be replaced by 3-hexadecenoyloxytetradecanoyl group. Salmonella minnesota R595 free lipid A also contained at least two active lipid A components as seen in E. coli lipid A, but the third component corresponding to the synthetic Salmonella-type lipid A (compound 516 or LA-16-PP) exhibited low activity. A lipid A fraction, Cv-A4 from Chromobacterium violaceum IFO 12614, which was proposed to have two acyloxyacyl groups at the C-2 and C-2' positions with other acyl groups, exhibited weaker activity than the free lipid A or LPS. The purified lipid A fractions from Pseudomonas diminuta JCM 2788 and Pseudomonas vesicularis JCM 1477 contained an unusual backbone with 2,3-diamino-2,3-dideoxy-D-glucose disaccharide phosphomonoester, and these lipid A (Pd-A3 and Pv-A3) exhibited strong activity comparable to the E. coli lipid A. Thus, the present results show that the local Shwartzman reaction can be expressed by partly different lipid A structures in both hydrophilic backbone and fatty acyl residues; when they have the same backbone the potency varies markedly depending on the structure of the acyl residues.