The roles of histidine residues at the starch-binding site in streptococcal-binding activities of human salivary amylase.

Human salivary alpha-amylase participates in the initial digestion of starch and may be involved in the colonization of viridans streptococci in the mouth. To elucidate the role of histidine residues located near the starch-binding site on the streptococcal-binding activity, the wild type and three histidine mutants, H52A, H299A and H305A were constructed and expressed in a baculovirus system. While His52 is located near the non-reducing end of the starch-binding pocket (subsite S3/S4), the residues His299 and His305 are located near the subsites S1/S1'. For the wild type, the cDNA encoding the leader and secreted sequences of human salivary amylase was amplified by polymerase chain reaction from a human submandibular salivary-gland cDNA library, and subcloned into the baculovirus shuttle vector pVL1392 downstream of the polyhedrin promoter. Oligonucleotide-based, site-directed mutagenesis was used to generate the mutants expressed in the baculovirus system. Replacing His52 or His299 or His305 to Ala residue did not alter the bacterial-binding activity significantly, but these mutants did show differences in their catalytic activities. The mutant H52A showed negligible reduction in enzymatic activity compared to that of wild type for the hydrolysis of starch and oligosaccharides. In contrast, the H299A and H305A mutants showed a 12 to 13-fold reduction (90-92%) in starch-hydrolysing activity. In addition, the k(cat) for the hydrolysis of oligosaccharides by H299A decreased by as much as 11-fold for maltoheptaoside. This reduction was even higher (40-fold) for the hydrolysis of p-nitrophenyl maltoside, with a significant change in K(M). The mutant H305A, however, exhibited a reduction in k(cat) only, with no changes in the K(M) for the hydrolysis of oligosaccharides. The reduction in the k(cat) for the H305A mutant was almost 93% for maltoheptaoside hydrolysis. The pH activity profile for the H305A mutant was also significantly different from that of the wild type and the other two mutants. These results suggest that, although histidines at the starch-binding site of salivary amylase are involved in starch binding and catalysis, they may not participate in Streptococcus gordonii G9B binding.

DNA methylation in mycobacteria: absence of methylation at GATC (Dam) and CCA/TGG (Dcm) sequences.

The presence of 6-methyladenine and 5-methylcytosine at Dam (GATC) and Dcm (CCA/TGG) sites in DNA of mycobacterial species was investigated using isoschizomer restriction enzymes. In all species examined, Dam and Dcm recognition sequences were not methylated indicating the absence of these methyltransferases. On the other hand, high performance liquid chromatographic analysis of genomic DNA from Mycobacterium smegmatis and Mycobacterium tuberculosis showed significant levels of 6-methyladenine and 5-methylcytosine suggesting the presence of DNA methyltransferases other than Dam and Dcm. Occurrence of methylation was also established by a sensitive genetic assay.

Genotoxicity studies with Cuman L in Drosophila melanogaster.

Cuman L, a dithiocarbamate fungicide, was tested for its ability to induce sex-linked recessive lethals (SLRLs) and II-III chromosome translocations in Drosophila melanogaster by the larval feeding method. The three concentrations of Cuman L, of 20, 40, and 60 microliters/100 ml, induced significant (P less than 0.01) increases in SLRLs but failed to induce any translocations.

Cytogenetic effects of Cuman L, a dithiocarbamate fungicide.

Cuman L, a dithiocarbamate fungicide, was assessed for its effects in the germ cells and the bone marrow erythrocytes of Swiss Albino male mice. The 3 sublethal doses of 350, 700 and 1050 mg/kg b.w. of Cuman L induced a significant (P less than 0.01) increase in the number of chromosomal aberrations in the germ cells. A significant increase (P less than 0.01) in the percentage of micronuclei in the erythrocytes was also induced by the three doses.

Mutagenicity studies in Drosophila melanogaster with Lannate 20.

Lannate 20 a carbamate pesticide was evaluated for its mutagenicity in Drosophila melanogaster by the sex-linked recessive lethals and chromosome II-III translocation tests by continuous larval feeding. The 3 sublethal doses of 0.2, 0.4 and 0.6 microliter of Lannate per 100 ml of the food medium induced a significant (P less than 0.01) increase in the number of sex-linked recessive lethals over the controls. However, no translocations were observed either in the treated or the control series.

Evaluation of lannate 20, a carbamate pesticide in the germ cells of male mice.

Lannate 20, a carbamate pesticide, was evaluated for its effects on the germ cells of Swiss albino male mice, by the sperm morphology assay and meiotic chromosome preparations. The three sublethal concentrations of 20, 40, and 60 mg/kg body wt administered by the oral intubation method produced significant results (P less than 0.01) with both the above protocols in the said test system under study, thus indicating that lannate 20 is mutagenic in mice.