Site-directed mutagenesis of glutathione S-transferase YaYa. Important roles of tyrosine 9 and aspartic acid 101 in catalysis.

The roles of tyrosine 9 and aspartic acid 101 in the catalytic mechanism of rat glutathione S-transferase YaYa were studied by site-directed mutagenesis. Replacement of tyrosine 9 with phenylalanine (Y9F), threonine (Y9T), histidine (Y9H), or valine (Y9V) resulted in mutant enzymes with less than 5% catalytic activity of the wild type enzymes. Kinetic studies with purified Y9F and Y9T mutants demonstrated poor catalytic efficiencies which were largely due to a drastic decrease in kcat. The estimated pK alpha values of the sulfhydryl group of glutathione bound to Y9F and Y9T mutant enzymes were 8.5 to 8.7, similar to the chemical reaction, in contrast to the estimated pK alpha value of 6.7 to 6.8 for the glutathione enzyme complex of wild type glutathione S-transferase. These results indicate that tyrosine 9 is directly responsible for the lowering of the pKa of the sulfhydryl group of glutathione, presumably due to the stabilization of the thiolate anion through hydrogen bonding with the hydroxyl group of tyrosine. To examine the role of aspartic acid in the binding of glutathione to YaYa, 4 conserved aspartic acid residues at positions 61, 93, 101, and 157 were changed to glutamic acid and asparagine. All mutant enzymes retained either full or partial activity except D157N, which was virtually inactive. Kinetic studies with four mutant enzymes (D93E, D93N, D101E, and D101N) indicate that only D101N exhibited a 5-fold increase in Km toward glutathione. Also, the binding of this mutant to the affinity column was greatly reduced. These results demonstrate that aspartic acid 101 plays an important role in glutathione interaction to YaYa. The role of aspartic acid 157 in catalysis remains to be determined.     

Regulation of Hsp70 function by a eukaryotic DnaJ homolog.

We report that a purified cytoplasmic Hsp70 homolog from Saccharomyces cerevisiae, Hsp70SSA1, exhibits a weak ATPase activity, which is stimulated by a purified eukaryotic dnaJp homolog (YDJ1p). Stable complex formation between Hsp70SSA1 and the permanently unfolded protein carboxymethylated alpha-lactalbumin (CMLA) was assayed by native gel electrophoresis. The affinity of Hsp70SSA1 for CMLA appeared to be regulated by YDJ1p. Significant reduction in both CMLA-Hsp70SSA1 complex formation and the release of CMLA pre-bound to Hsp70SSA1 was observed only in the presence of both YDJ1p and ATP. Thus, Hsp70SSA1 and YDJ1p interact functionally in the execution of Hsp70SSA1 chaperone activities in the eukaryotic cell.     

Similar replication capacities of primary human immunodeficiency virus type 1 isolates derived from a wide range of clinical sources.

Numerous studies have suggested that there are significant differences in replication capacities and cytopathicities among human immunodeficiency virus type 1 (HIV-1) isolates and that these differences correlate with the clinical status and geographical origin of infected individuals. However, it has been difficult to assess whether reported distinctions could be attributed to the methods used or whether they imply a true disparity between viral isolates. We thus attempted to characterize the replication properties of HIV-1 isolates directly recovered from infected patients (primary isolates) by using a standardized infection assay. Viruses were isolated from patients' peripheral blood mononuclear cells (PBMC) by a single coculture with normal donor PBMC stimulated with phytohemagglutinin. Replication curves and cytopathic effect of a standard inoculum (1 ng of p24) of 66 primary HIV-1 isolates were similar regardless of clinical stage of the patient (asymptomatic, AIDS-related complex, or AIDS) and evolutive feature (rate of progression to AIDS). There was no difference between viruses derived from patients sensitive to zidovudine and those derived from patients resistant to zidovudine. Moreover, no difference was found among viral isolates of different geographical origins (Central Africa, Zaire, Brazil, or France). Similarly, the replication patterns and cytopathicities of isolates from bronchoalveolar lymphocytes did not differ from those of isolates derived from PBMC. In contrast, the same amount of viral inoculum of five laboratory HIV-1 strains (HIV-1, EL1, SF, MN, and RF) produced different replication curves and were much less cytopathic. In contrast to laboratory viral strains, it appears that the primary HIV-1 isolates tested, whatever their clinical status and source, exhibited similar replication capacities and cytopathicities in allogeneic donor PBMC.     

Site-specific oligodeoxynucleotide binding to maize Adh1 gene promoter represses Adh1-GUS gene expression in vivo

There is a 36 bp tract of extreme homopurine/homopyrimidine (PuPy) asymmetry in the maize Adh1 gene promoter (from –44 to –79) that is S1-hypersensitive in plasmids under supercoil tension. Oligodeoxynucleotides corresponding to the PuPy tract were designed to examine the secondary structure of the region and address the possible role of the tract in gene regulation. On the basis of oligodeoxynucleotide band-shift and DNase I footprinting analyses, it was concluded that the homopyrimidine oligodeoxynucleotide can form a triple helix with the duplex PuPy tract in vitro. Transient assays in protoplasts, suspension cells, and seedling roots show that the homopyrimidine oligodeoxynucleotide is also capable of repressing Adh1-GUS gene expression during co-transformation, presumably by the formation of a triple helix with the PuPy tract in vivo. The complementary homopurine oligodeoxynucleotide would not form a triple helix in vitro, nor would it repress Adh1-GUS in vivo. We propose that triple helix formation is a potential regulatory phenomenon in vivo, and that an intraregion triple helix could occur within the Adh1 promoter via the formation of H-DNA.     

Alcohol and aldehyde dehydrogenase genotypes and alcoholism in Chinese men.

The liver enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), which are responsible for the oxidative metabolism of ethanol, are polymorphic in humans. An allele encoding an inactive form of the mitochondrial ALDH2 is known to reduce the likelihood of alcoholism in Japanese. We hypothesized that the polymorphisms of both ALDH and ADH modify the predisposition to development of alcoholism. Therefore, we determined the genotypes of the ADH2, ADH3, and ALDH2 loci of alcoholic and nonalcoholic Chinese men living in Taiwan, using leukocyte DNA amplified by the PCR and allele-specific oligonucleotides. The alcoholics had significantly lower frequencies of the ADH2*2, ADH3*1, and ALDH2*2 alleles than did the nonalcoholics, suggesting that genetic variation in both ADH and ALDH, by modulating the rate of metabolism of ethanol and acetaldehyde, influences drinking behavior and the risk of developing alcoholism.     

Conformational preference and ligand binding properties of DNA junctions are determined by sequence at the branch

Four-arm DNA branched junctions are stable analogues of Holliday recombinational intermediates. A number of four-arm DNA junctions synthesized from oligonucleotides have now been studied. Gel mobility or chemical footprinting experiments on several immobile four-arm junctions indicate that in the presence of Mg2+, they assume a preferred conformation consisting of two helical domains, each formed by stacking a particular pair of arms on each other. We show here that a junction we designate as J1c that has the same chemical composition as one we have previously studied in detail, J1, but is formed from the four strands complementary to those of the latter, exhibits the reverse stacking preference. The pattern of self-protection of the strands of J1c exposed to Fe(II).EDTA-induced scission reveals that twofold symmetry is preserved, but the opposite pair of strands preferentially cross over. Moreover, the Fe(II).EDTA scission profiles of J1c indicate that this junction exhibits a weaker bias as to which strands cross over than is observed in J1. The preference for the dominant species in J1 is 1.3 times greater than in J1c at 4 degrees C and in the presence of 10 mM Mg2+, based on chemical reactivity data. This is confirmed by a cleavage experiment using the resolvase enzyme, endonuclease I, from bacteriophage T7. This difference could reflect either sequence-dependent differences in the equilibrium among isomers, or in the structure of these junctions. Chemical footprinting experiments using the probes MPE.Fe(II) and (OP)2Cu(I) show that the high-affinity ligand binding site in immobile junctions is determined by junction geometry.     

Hybrid cytokines as hematopoietic growth factors.

A large body of in vitro and in vivo data suggests that combinations of cytokines provide the most effective mechanism for stimulating multilineage acceleration of hematopoiesis. Creation of a granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin 3 (IL-3) fusion protein has yielded a single therapeutic which has enhanced biological activity in comparison to the individual cytokines from which it is composed. In vivo studies with this fusion protein (PIXY321) suggest that it may provide a means to accelerate both neutrophil and platelet recovery in clinical settings in which hematopoiesis is suppressed. The biology of PIXY321 and the potential for other fusion proteins is discussed.     

Immunodetection of thiol proteinase levels in various populations of Artemia cysts and during development

An immunodetection assay on Western blots has been used to determine the thiol proteinase content and composition in cysts from 12 populations of the brine shrimp Artemia. Our results showed no differences in the subunit composition of the thiol proteinase among cysts from eight bisexual strains and four parthenogenic strains, and confirmed an earlier finding that the proteinase is composed of two subunits of 25.9 and 31.5 kilodaltons. In contrast, we found that Artemia cysts from parthenogenic strains contain 17.1 ng/cyst of the thiol proteinase, while cysts from bisexual strains contain 8.2 ng/cyst of the thiol proteinase. Also, there was a good linear correlation (r = 0.863; p less than 0.001) between the thiol proteinase content and cyst mass. Embryo fractionation experiments showed that 82% of the thiol proteinase was in the cytosol, while 14 and 4%, respectively, were in the nuclei/yolk platelets and mitochondria/lysosome fractions. Measurements of the thiol proteinase content of developing Artemia embryos showed that the proteinase content was relatively constant during early development, suggesting that the activity of the thiol proteinase gene(s) may be constitutive and not developmentally regulated in Artemia embryos.