Substantivity of zinc salts used as rinsing solutions and their effect on the inhibition of Streptococcus mutans

The antimicrobial efficacy of zinc (Zn) salts (sulfate and acetate) against Streptococcus mutans (S. mutans) present in the oral cavity was tested in this study. The substantivity of Zn salts was assessed by determining the concentration of Zn in whole, unstimulated saliva and by measuring the magnitude of suppression of salivary S. mutans, 2h after rinsing. The concentration of Zn was measured by atomic absorption spectrometry (AAS) with electrothermal atomization (ET AAS) in saliva sampled before (basal) and 24h after mouth rinsing with different concentrations of Zn (0.1%, 0.5% and 1%) administrated as sulfate and acetate. The estimation of Zn levels in samples collected 30, 60, 90 and 120 min after rinsing was carried out by AAS with flame atomization (FAAS). Immediately after rinsing, the concentration of Zn in saliva sharply increased with respect to the baseline values (0.055+/-0.017 mg/L), followed by a sustained decrease, probably due to clearance of salivary flow or swallowing during sampling. A significant reduction (>87%) in the total mean S. mutans counts was found 2h after rinsing either with sulfate or acetate solutions, as evidence of the high substantivity and effectiveness of the Zn salts tested. A statistically significant inverse relationship (p<0.001 and the Pearson correlation coefficients between -34% and -50%) was found between Zn levels and the respective pH values measured in the samples collected 60 and 120 min after rinsing, sustaining the theory of bacterial glycolysis inhibition.     

Structural and catalytic characteristics of Escherichia coli adenylate kinase

The adk gene encoding adenylate kinase in Escherichia coli was cloned in pBR322. Adenylate kinase represented about 4% of total proteins in extracts of cells containing the pBR322:adk plasmid. This allowed preparation of more than 90% pure enzyme in a single-step purification procedure. Amino acid analysis, high performance liquid chromatography separation of trypsin digests, sequence analysis of most peptides, and determination of the N-terminal sequence of the whole protein confirmed the primary structure of E. coli adenylate kinase predicted from the nucleotide sequence of the adk gene (Brune, M., Schumann, R., and Wittinghofer, F. (1985) Nucleic Acids Res. 13, 7139-7151). 2-Nitro-5-thiocyanatobenzoic acid reacted with the single cysteine residue of E. coli adenylate kinase. The cyanylated protein was cleaved upon exposure to alkaline pH, yielding two peptides corresponding to residues 1-76 and 77-214, respectively. A mixture of purified peptides tended to reassociate, recovering both catalytic activity and binding properties for adenine nucleotides. E. coli adenylate kinase has a broader specificity for nucleoside monophosphates than does the mammalian enzyme. In addition to 2'-dAMP, other nucleoside monophosphates such as 3'-dAMP, adenine-9-beta-D-arabinofuranoside 5'-monophosphate, and 7-deazaadenosine (tubercidine) 5'-monophosphate were able to replace AMP as substrate.     

Nucleotide sequence of the metH gene of Escherichia coli K-12 and comparison with that of Salmonella typhimurium LT2

The Escherichia coli K-12 metH gene, encoding the vitamin B12-dependent homocysteine transmethylase, is located between iclR and lysC in the 91-min region of the chromosome. The metH gene has been sequenced and reveals an open reading frame of 3600 bp encoding a polypeptide of 1200 amino acids (aa) with a calculated Mr of 132 628. The first 414 aa of the deduced polypeptide sequence are 92% identical to the 414 aa deduced from the partially sequenced Salmonella typhimurium LT2 metH gene. In-frame fusions of metH to lacZ were used to confirm the reading frame of the metH gene and to study its regulation. metH was repressed tenfold, presumably indirectly, by L-methionine and the metJ gene product, while vitamin B12 did not induce de novo synthesis of MetH.     

Identification of minor tightly bound H1 histone subfractions which fail to cleave their initiator methionine

Groups of CBA mice were administered [³⁵S] methionine (1 mCi/mouse). Non-histone proteins, H1 and H1⁰ histones and nucleosomal core histones were isolated from different issues by selective extractions. The measurements of radioactivity of individual bands and autoradiography of dry gels were used to identify methionine-containing and methionine-free histone variants. H1A and H1B histone variants extracted with 5% perchloric acid were methionine-free. However, minor sub-fractions of these histones which are more tightly bound to DNA (and which can be extracted only with 0.25 N HC1) contained [³⁵S] methionine and did show a higher specific activity than methionine-containing nucleosomal hitones. Cyanogen Bromide reaction which destroys non-histone proteins and methionine-containing nucleosomal histones removes radioactivity but does not alter the position of methionine-containing H1 minor bands. This indicates that the radioactive methionine occupies only the N-terminus of the H1 molecules. It is suggested that this methionine is an uncleaved initiator methionine. The presence of these methionine-containing minor H1 subfractions varies in different tissues.     

Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

1H NMR investigation of reduced copper-cobalt superoxide dismutase

Human copper-cobalt superoxide dismutase in the reduced form has been investigated through ¹H NMR techniques. The aim is to monitor the structural properties of this derivative and to compare them with those of reduced and oxidized native superoxide dismutases. The observed signals of the cobalt ligands have been assigned as well as the signals of the histidines bound to copper(I). The latter signals experience little pseudocontact shifts which allow a rough orientation of the magnetic susceptibility tensor in the molecular frame. The connectivities indicate that, although the histidine bridge is broken in the reduced form, the interproton distances between ligands of both ions are essentially the same.Abbreviations WEFT water eliminated Fourier transform - NOE nuclear Overhauser effect - NOESY NOE spectroscopy - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - SOD superoxide dismutase - E₂Co(II)SOD SOD with empty copper site (E=empty) and with cobalt(II) in the Zinc(II) site Offprint requests to: I. Bertini     

Purification and subunit structure of RNA polymerases I and II from Dictyostelium discoideum vegetative cells

Summary A purification procedure to obtain RNA polymerases I (or A) and II (or B) from Dictyostelium discoideum amoeba has been developed. The enzymes were solubilized from purified nuclei and separated by DEAF-Sephadex chromatography. RNA polymerases I and II were further purified by a second chromatography on DEAE-Sephadex followed by chromatographies on phosphocellulose and heparin-sepharose. The specific activities of purified RNA polymerases I and II are 92 units/ mg protein and 70 units/ mg protein, respectively. The subunit structure of both RNA polymerases were analyzed by polyacrylamide gel electrophoresis under denaturing conditions after glycerol gradient centrifugation of the enzymes. The putative subunits of RNA polymerase I have molecular weights of 180 000,125 000,43 000,40 000,34 000, 31 000, 25 000,19 000, 17 000 and 14 000. The putative subunits of RNA polymerase II have molecular weights of 200 000 (170 000), 130 000, 33 000, 25 000, 19 000, 17 000, 15 000, 13 000. There are three polypeptides with common molecular weight in Dictyostelium RNA polymerases I and 11. The subunit of 25 000 daltons of both enzymes has common immunological determinants with RNA polymerase II from crustacean Artemia.Abbreviations TLCK tosyl-lysine-chloromethyl-ketone - DPT diazophenylthioether