Regulation of glucose metabolism in oral streptococci through independent pathways of glucose 6-phosphate and glucose 1-phosphate formation

In vivo rates of glucose uptake and acid production by oral streptococci grown in glucose- or nitrogen-limited continuous culture and batch culture were compared with the glucose phosphorylation activities of harvested, decryptified cells. The strains examined contained significant phosphoenolpyruvate-phosphotransferase system (PTS) activity, measured by a glucose 6-phosphate (G6P) dehydrogenase-linked assay procedure, but this activity was insufficient to account for the in vivo glucose uptake rates. However, ATP was a superior phosphoryl donor to phosphoenolpyruvate, and unlike the PTS, phosphoryl transfer with ATP was insensitive to bacteriostatic concentrations of chlorhexidine, suggesting glucokinase-mediated G6P formation. Again, G6P formation from the PTS and glucokinase reactions was not commensurate with some of the glucose uptake rates observed, implying that other phosphorylation reactions must be occurring. Two novel reactions involving carbamyl phosphate and acetyl phosphate were identified in some of the strains. No G6P formation was detected with these potential phosphoryl donors, but in the presence of phosphoglucomutase, glucose 1-phosphate (G1P) formation was evident, which was insensitive to chlorhexidine. G1P is a precursor of glycogen, and good correlation was obtained between G1P formation activity and endogenous metabolism of washed cells measured either as a rate of acid production at a constant pH 7 or as a decrease in pH with time in the absence of titrant. A "league table" of abilities to synthesize G1P and produce acid from endogenous metabolism was compiled for oral streptococci grown in batch culture. This indicated that Streptococcus mutans Ingbritt and Streptococcus sanguis Challis were unable to form G1P or produce much acid endogenously, whereas increasing activities were obtained with Streptococcus salivarius, Streptococcus sanguis, and Streptococcus mitis. In particular, S. mitis had the highest G1P formation activities and was able to decrease the pH to less than 5 in 15 min by endogenous metabolism alone. The data are consistent with the intracellular accumulation of free glucose driven by proton motive force when PTS activities are low and the subsequent phosphorylation to either G6P for metabolism via glycolysis or G1P for glycogen biosynthesis. The accumulation of acetyl phosphate during glucose-limited growth and the availability of arginine for catabolism to carbamyl phosphate provide an explanation as to why some glucose-limited oral streptococci continue to synthesize glycogen under these conditions, which might prevail in plaque.     

Vasopressin analog (DDAVP) improves memory in human males

One specific analog of arginine vasopression, 1-desamine-8-D-arginine vasopressin (DDAVP), has been shown to improve learning and memory in humans. Healthy young male adult subjects treated with DDAVP demonstrated better memory for implicational sentences than did control subjects. The same treatment had no influence on women given the same memory task. These results suggest that DDAVP may have a sexually dimorphic effect on learning and memory.     

GELRITE as an Agar Substitute in Bacteriological Media

GELRITE gellan gum (formerly known as PS-60 and S-60) is a new naturally derived, highly purified polysaccharide which displays several interesting properties, including selfgelling. The suitability of GELRITE as an agar substitute was tested by evaluating the performance of several media selected from among those most commonly used in the isolation, identification, and enumeration of microorganisms in clinical laboratories. Fifty different bacterial species previously implicated in human infections served as test strains. On the basis of the various parameters considered, namely, colony characteristics, biochemical reactions, hemolytic patterns, and plating efficiency, media gelled by agar and by GELRITE compared quite favorably.     

Bio-techniques for the detection of genetic toxicity in the aquatic environment.

This article reviews the application of sensitive biotechniques in the detection of DNA damage within the aquatic environment as "early warning" indicators of pollution-related genetic toxicity. Bacterial mutagenicity assays have played an important role both in the discovery of mutagens and in the analysis of the ability of aquatic organisms to convert inert substances to reactive genotoxic products. Genetic damage in aquatic species treated with carcinogens has been detected by analysis of DNA adducts, induction of DNA repair, DNA strand breakage and formation of chromosomal aberrations and sister chromatid exchanges. Little information is available on pollution-related DNA damage resulting from field exposure. A 32P-postlabelling technique for the sensitive detection of DNA adducts is a promising development towards this aim and the analysis of changes in oncogene nucleotide sequence in tumour tissue is considered an important future direction.     

[3H]SCH 39166, A New D1-Selective Radioligand: In Vitro and In Vivo Binding Analyses

SCH 39166 [(-)-trans-6,7,7a,8,9, 13b-hexahydro-3-chloro-2-hydroxy-N-methyl-5H-benzo-[d]naphtho[2, 1b]azepine] has recently been described as a selective D1 antagonist and has entered clinical trials for the treatment of schizophrenia. The tritiated analogue of this compound, [3H]SCH 39166, has now been synthesized and characterized for its in vitro and in vivo binding profiles. [3H]SCH 39166 binds to D1 receptors in a saturable, high-affinity fashion, with a KD of 0.79 nM. In competition studies, D1-selective antagonists like SCH 23390 displaced the binding of [3H]SCH 39166 with nanomolar affinities, whereas antagonists of other receptors exhibited poor affinity. In vivo, [3H]SCH 39166 bound to receptors in rat striatum in a fashion suggestive of D1 selectivity. Further, when the time course for the binding of [3H]SCH 39166 was compared with the behavioral time course of the unlabeled compound, the two durations of action were virtually indistinguishable. Similar studies were performed for SCH 23390 and its tritiated analogue, but the in vivo binding of this radioligand exhibited a duration of action far greater than the behavioral activity of the unlabeled drug. In concert, these data demonstrate that [3H]SCH 39166 selectively labels D1 receptors in vitro and in vivo, and that this drug is superior for in vivo imaging of the D1 receptor.     

Differential regulation of antioxidant enzymes in response to oxidants.

We have demonstrated the selective induction of manganese superoxide dismutase (MnSOD) or catalase mRNA after exposure of tracheobronchial epithelial cells in vitro to different oxidant stresses. Addition of H2O2 caused a dose-dependent increase in catalase mRNA in both exponentially growing and confluent cells. A 3-fold induction of catalase mRNA was seen at a nontoxic dose of 250 microM H2O2. Increase in the steady-state mRNA levels of glutathione peroxidase (GPX) and MnSOD were less striking. Expression of catalase, MnSOD, and GPX mRNA was highest in confluent cells. In contrast, constitutive expression of copper and zinc SOD (CuZnSOD) mRNA was greatest in dividing cells and was unaffected by H2O2 in both exponentially growing and confluent cells. MnSOD mRNA was selectively induced in confluent epithelial cells exposed to the reactive oxygen species-generating system, xanthine/xanthine oxidase, while steady-state levels of GPX, catalase, and CuZnSOD mRNA remained unchanged. The 3-fold induction of MnSOD mRNA was dose-dependent, reaching a peak at 0.2 unit/ml xanthine oxidase. MnSOD mRNA increases were seen as early as 2 h and reached maximal induction at 24 h. Immunoreactive MnSOD protein was produced in a corresponding dose- and time-dependent manner. Induction of MnSOD gene expression was prevented by addition of actinomycin D and cycloheximide. These data indicate that epithelial cells of the respiratory tract respond to different oxidant insults by selective induction of certain antioxidant enzymes. Hence, gene expression of antioxidant enzymes does not appear to be coordinately regulated in these cell types.     

Epitope mapping of an HLA-DR-specific monoclonal antibody produced by using human-mouse transfectant cells

A polymorphic HLA-DR mAb, TAL15.1, was produced against L cells transfected with DR alpha- and beta-chain cDNA from a cell line homozygous for HLA-DRw8. This antibody reacted with DRw8 plus all other DR types except DR3 and DRw52. DR3 and DRw52 differ uniquely from other other DR antigens at position 77 in the beta 1-domain of their beta-chains where there is asparagine instead of threonine. In Western blots the antibody reacted with DR alpha/beta-dimer but not with free alpha- or beta-chains. Two-dimensional gel analysis of a DRw11, DRw52 cell line showed that TAL15.1 immunoprecipitated the DR products. Although it also coprecipitated the DQ beta chain products, flow microfluorimetric analysis with various transfectant cell lines showed that TAL15.1 failed to bind the DQ or DP products tested. We conclude that TAL15.1 is a DR-specific polymorphic antibody whose activity correlates with a specific residue. It has already proved to be a valuable reagent for distinguishing DR3 homozygotes from DR3, DRw6 heterozygotes, which have in the past been difficult to separate.     

Cellular processing of a ricin-antibody conjugate. A kinetic analysis of the rate-limiting step

Upon exposure to holoricin-antibody conjugates, target cells display a period of no measurable intoxication followed by a concentration-dependent exponential decline in protein synthesis. The rate of this decline is increased by conjugate structural variables such as addition of a second ricin to the antibody or introduction of a peptide spacer between the toxin and antibody. Additionally, the rate is enhanced by the addition of ammonia or monensin. The relationship between immunotoxin concentration and the rate of protein synthesis inhibition is shown to obey typical Michaelis-Menten kinetics. By displacing bound immunotoxin with native antibody, it was demonstrated that the rate-limiting saturable process is not due to antibody-surface antigen interaction. Although addition of a second ricin as well as addition of ammonia elevated the measured rate of protein synthesis inhibition, curve-fitting techniques indicated that they did not elevate the maximal rate; however, introduction of a peptide spacer into the conjugate or addition of monensin increased the maximal rate of this saturable process. Additionally, the kinetic examination of the potentiation effects of ammonia and monensin indicates that they operate on different cellular processes involved with the intracellular routing toward intoxication. An equational model is developed which helps interpret the known features of the conjugate intoxication process.     

Complete primary structure of a Lolium perenne (perennial rye grass) pollen allergen, Lol p III: comparison with known Lol p I and II sequences

The complete amino acid sequence of a Lolium perenne (rye grass) pollen allergen, Lol p III, determined by the automated Edman degradation of the protein and its selected fragments, is reported in this paper. Cleavage by enzymatic and chemical techniques established unambiguously the sequence for this 97-residue protein (Mr = 10,909), which lacks cysteine and shows no evidence of glycosylation. The sequence of Lol p III is very similar to that of another L. perenne allergen, Lol p II, which was sequenced recently; of the 97 positions in the two proteins, 57 are occupied by identical amino acids (59% identity). In addition, both allergens share a similar structure with an antibody-binding fragment of a third L. perenne allergen, Lol p I. Since human antibody responsiveness to all these three allergens is associated with HLA-DR3, and since the structure common to the three molecules shows high degrees of amphipathicity in Lol p II and III, we speculate that this common segment in the three molecules might contain or contribute to the respectively Ia/T-cell sites.