Characterization of AP lyase activities of Saccharomyces cerevisiae Ntg1p and Ntg2p: implications for biological function

Saccharomyces cerevisiae possesses two Escherichia coli endonuclease III homologs, NTG1 and NTG2, whose gene products function in the base excision repair pathway and initiate removal of a variety of oxidized pyrimidines from DNA. Although the glycosylase activity of these proteins has been well studied, the in vivo importance of the AP lyase activity has not been determined. Previous genetic studies have suggested that the AP lyase activities of Ntg1p and Ntg2p may be major contributors in the initial processing of abasic sites. We conducted a biochemical characterization of the AP lyase activities of Ntg1p and Ntg2p via a series of kinetic experiments. Such studies were designed to determine if Ntg1p and Ntg2p prefer specific bases located opposite abasic sites and whether these lesions are processed with a catalytic efficiency similar to Apn1p, the major hydrolytic AP endonuclease of yeast. Our results indicate that Ntg1p and Ntg2p are equally effective in processing four types of abasic site-containing substrates. Certain abasic site substrates were processed with greater catalytic efficiency than others, a situation similar to Apn1p processing of such substrates. These biochemical studies strongly support an important biological role for Ntg1p and Ntg2p in the initial processing of abasic sites and maintenance of genomic stability.     

beta-Hydroxybutyrate inhibits myocardial fatty acid oxidation in vivo independent of changes in malonyl-CoA content

This study tested the hypothesis that an acute infusion of beta-hydroxybutyrate inhibits myocardial fatty acid uptake and oxidation in vivo. Anesthetized pigs were untreated (n = 6) or treated with an intravenous infusion of fat emulsion (n = 7) to elevate plasma free fatty acid levels. A third group received fat emulsion plus an intravenous infusion of beta-hydroxybutyrate (25 micromol.kg-1.min-1; n = 7) for 60 min. All animals received a continuous infusion of [3H]palmitate, and myocardial fatty acid oxidation was measured from the cardiac production of 3H2O. Plasma free fatty acid concentrations were elevated in the fat emulsion group (0.77 +/- 0.11 mM) compared with the untreated group (0.15 +/- 0.03 mM), which resulted in greater myocardial free fatty acid oxidation. In contrast, the group receiving beta-hydroxybutyrate in addition to fat emulsion had elevated beta-hydroxybutyrate concentration (0.87 +/- 0.11 vs. 0.04 +/- 0.01 mM), but suppressed fatty acid oxidation (0.053 +/- 0.013 micromol.g-1.min-1) (P < 0.05) compared with the fat emulsion group (0.116 +/- 0.029 micromol.g-1.min-1). There were no differences among the three groups in the tissue content for malonyl-CoA, acetyl-CoA, or free CoA or the activity of acetyl-CoA carboxylase; thus the inhibition of fatty acid oxidation by elevated beta-hydroxybutyrate did not appear to be due to malonyl-CoA inhibition of carnitine palmitoyl transferase-I or to an increase in the acetyl-CoA-to-free CoA ratio. In conclusion, fatty acid uptake and oxidation is blocked by an infusion of beta-hydroxybutyrate; this effect was not due to elevated myocardial malonyl-CoA content.     

Effects of pH and temperature on myocardial calcium-activation in Pterygoplichthys (catfish)

Fish are chronically exposed to a wide range of temperatures and acidic environments. Fish hearts have to therefore adapt to these changes in order to maintain contractility. Myofibrillar responsiveness to Ca²⁺ is exquisitely sensitive to both temperature and pH in mammalian myocardium. To evaluate myofilament calcium-activation, we chemically skinned ventricular myocardium from catfish (Pterygoplichthys). A decrease in pH from 7.5 to 6.8, irrespective of temperature change, shifted the calcium-force curve towards higher calcium concentrations without affecting maximal Ca²⁺-activated force. The contractile elements are therefore sensitive to changes in pH. In intact muscle preparations the active twitch force was decreased with increasing temperature (10–22 °C). However, the sensitivity of the myofilaments to Ca²⁺ was independent of temperature. These data suggest a possible role of the sarcoplasmic reticulum (SR) in mediating the effects of temperature. The response of intact muscle preparations to changes in temperature is therefore not likely due to temperature-dependent changes in myofilament calcium responsiveness. Accepted: 11 May 1998     

Vector competence of Coquillettidia linealis (Skuse) (Diptera: Culicidae) for Ross River and Barmah Forest viruses

Abstract Coquillettidia linealis is a severe pest on some of the Moreton Bay islands in Queensland, Australia, but little is known of its breeding habitats and biology. Because of its high abundance and its association with Ross River (RR) and Barmah Forest (BF) viruses by field isolation, its vector competence was evaluated in the laboratory by feeding dilutions of both viruses in blood. For RR, Cq. linealis was of comparable efficiency to Ochlerotatus vigilax (Skuse), recognised as being a major vector. Results were as follows for Cq. linealis and Oc. vigilax , respectively: dose to infect 50%, 10^2.2 and <10^1.7 CCID₅₀/mosquito; 88% and 90% disseminated infection at 4 days postinfection; transmission at 4 days with rates of 68−92% and 25−60%. For BF dose to infect 50%, 10^2.7 and 10^2.0; disseminated infection rates on first transmission day (day 6), 40% and 70%; transmission rates of 8−16% and 0−10%. As a capillary-tube method was used rather than suckling mice to demonstrate transmission, transmission rates may be underestimates. This, the first study of the vector competence of Cq. linealis in Australia, demonstrates that this species deserves control on the southern Moreton Bay islands.     

Herpes simplex virus infection and propagation in a mouse L cell mutant lacking heparan sulfate proteoglycans.

We have isolated a variant line of mouse L cells, termed gro2C, which is partially resistant to infection by herpes simplex virus type 1 (HSV-1). Characterization of the genetic defect in gro2C cells revealed that this cell line harbors a specific defect in the heparan sulfate synthesis pathway. Specifically, anion-exchange high-performance liquid chromatography of metabolically radiolabeled glycosaminoglycans indicated that chondroitin sulfate moieties were synthesized normally in the mutant cells, whereas heparin-like chains were absent. Because of these properties, we have used these cells to investigate the role of heparan sulfate proteoglycans in the HSV-1 life cycle. In this report, we demonstrate that the partial block to HSV-1 infection in gro2C cells occurs in the virus entry pathway. Virus adsorption assays using radiolabeled HSV-1 (KOS) revealed that the gro2C cell surface is a relatively poor target for HSV-1 in that virus attachment was 85% lower in the mutant cells than in the parental L cell controls. A portion of the 15% residual virus adsorption was functional, however, insofar as gro2C cells were susceptible to HSV-1 infection in plaque assays and in single-step growth experiments. Moreover, although the number of HSV-1 plaques that formed in gro2C monolayers was reduced by 85%, the plaque morphology was normal, and the virus released from the mutant cells was infectious. Taken together, these results provide strong genetic evidence that heparan sulfate proteoglycans enhance the efficiency of HSV attachment to the cell surface but are otherwise not essential at any stage of the lytic cycle in culture. Moreover, in the absence of heparan sulfate, other cell surface molecules appear to confer susceptibility to HSV, leading to a productive viral infection.     

A computer-based protocol for semiautomated assignments and 3D structure determination of proteins

Summary A strategy is presented for the semiautomated assignment and 3D structure determination of proteins from heteronuclear multidimensional Nuclear Magnetic Resonance (NMR) data. This approach involves the computer-based assignment of the NMR signals, identification of distance restraints from nuclear Overhauser effects, and generation of 3D structures by using the NMR-derived restraints. The protocol is described in detail and illustrated on a resonance assignment and structure determination of the FK506 binding protein (FKBP, 107 amino acids) complexed to the immunosuppressant, ascomycin. The 3D structures produced from this automated protocol attained backbone and heavy atom rmsd of 1.17 and 1.69 Å, respectively. Although more highly resolved structures of the complex have been obtained by standard interpretation of NMR data (Meadows et al. (1993) Biochemistry, 32, 754–765), the structures generated with this automated protocol required minimal manual intervention during the spectral assignment and 3D structure calculations stages. Thus, the protocol may yield an approximate order of magnitude reduction in the time required for the generation of 3D structures of proteins from NMR data.     

Characterization of human chemotactic lymphokine production induced by mitogens and mixed leukocyte reactions using a new microassay.

Quantification of lymphokine production in vitro can be a useful tool in the evaluation of delayed hypersensitivity in various disease states. A micro-method for the measurement of chemotactic lymphokine production by human mononuclear leukocytes (MNL's) has been developed. MNL's are isolated on Ficoll-Hypaque gradients and cultured without plasma in microtiter plates. Culture supernatants are harvested through glass fibre filter paper under vacuum in a semi-automatic harvester. Chemotactic lymphokine activity in the supernatants is quantified in miniaturized Boyden chambers using human monocytes as responder cells. The production of chemotactic activity can be initiated by mixed leukocyte reactions as well as by soluble antigens or mitogens, and therefore may be a useful adjunct in tissue typing. Studies of lymphokine production in normal individuals indicate that these methods are quantitative, reproducible, and readily applicable to the study of this parameter of immune function in human disease.     

Hoechst 33258 as a vital stain for plant cell protoplasts

A method for staining nuclei in unfixed protoplasts using Hoechst 33258 is described. It was found that the dye was not taken up by all protoplasts except in the presence of a detergent and that the staining of the nuclei was pH- and concentration-dependent. The use of vital fluorescent probes with plant protoplasts may be helpful for the identification, selection and subsequent culture of hybrid fusion products in automatic cell sorting systems.     

Effect of Elicitation and Changes in Extracellular pH on the Cytoplasmic and Vacuolar pH of Suspension-Cultured Soybean Cells

We have employed both ³¹P nuclear magnetic resonance spectroscopy and two intracellular fluorescent pH indicator dyes to monitor the pH of the vacuole and cytoplasm of suspension-cultured soybean cells (Glycine max Merr cv Kent). For the ³¹P nuclear magnetic resonance studies, a flow cell was constructed that allowed perfusion of the cells in oxygenated growth medium throughout the experiment. When the perfusion medium was transiently adjusted to a pH higher than that of the ambient growth medium, a rapid elevation of vacuolar pH was observed followed by a slow (approximately 30 minute) return to near resting pH. In contrast, the concurrent pH changes in the cytoplasm were usually fourfold smaller. These data indicate that extracellular pH changes are rapidly communicated to the vacuole in soybean cells without significantly perturbing cytoplasmic pH. When elicitors were dissolved in a medium of altered pH and introduced into the cell suspension, the pH of the vacuole, as above, quickly reflected the pH of the added elicitor solution. In contrast, when the pH of either a polygalacturonic acid or Verticillium dahliae elicitor preparation was adjusted to the same pH as the ambient medium, no significant change in either vacuolar or cytoplasmic pH was observed during the 35 minute experiment. These results were confirmed in experiments with pH-sensitive fluorescent dyes. We conclude that suspension-cultured soybean cells do not respond to elicitation by significantly changing the pH of their vacuolar or cytoplasmic compartments.