The binding of 1,N6-ethenoNAD to bovine liver glutamate dehydrogenase: studies using the time-correlated single photon counting fluorescence technique

The time-correlated single photon counting (TCPC) fluorescence technique has been used as a novel approach to investigate ligand-protein interaction, for the case of the binding of the fluorescent coenzyme analogue 1,N6-ethenoNAD (epsilon NAD) to bovine liver glutamate dehydrogenase in the presence of glutarate, a substrate analogue which stabilizes the complex. System calibration was performed using solutions of epsilon ADP and carefully purified epsilon NAD mixed at variable molar ratios (pH 7.0, 0.05 M sodium phosphate buffer, 20 degrees C). The fluorescence lifetimes obtained after deconvolution were 2.4 ns (for epsilon NAD) and 23 ns (for epsilon ADP), in good agreement with literature values obtained under similar conditions. epsilon NAD binds to glutamate dehydrogenase in the presence of 50 mM glutarate, with a fluorescence quantum yield enhancement factor, Q, of about 17-fold, as previously reported (Favilla, R. and Mazzini, A. (1984) Biochim. Biophys. Acta 48-57). For this system, fluorescence lifetime values were obtained after deconvolution as 2.4 ns for free epsilon NAD and 21 ns for bound epsilon NAD. These values did not vary appreciably with enzyme concentration nor with degree of saturation, thus reflecting the existence of only one spectroscopically relevant type of complex. Addition of either GTP or ADP did not affect the lifetime of epsilon NAD bound to the enzyme, but only its affinity, thus allowing calculations of binding strengths. In the case of a simple binding (i.e., in the absence of GTP) the dissociation constant of the complex could be derived from a simple relationship, in which only the ratio between the pre-exponential factors and the parameter gamma, which represents the molar fraction of epsilon NAD molecules free in solution in the open conformation, are to be taken into account. The results are in good agreement with those reported by some of us (reference above) using a steady-state fluorescence technique, which by itself is, however, unable to resolve the number of relevant species present in the system.     

Dissection of the role of macrophages in triggering T lymphocytes for interleukin 2 production by monoclonal antibody OKT3

Unfractionated human peripheral blood mononuclear cells produce a small amount of interleukin 2 (IL 2) by stimulation with a monoclonal anti-T3 antibody (OKT3) in vitro. The IL 2 production could be greatly augmented by the addition of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In the presence of TPA, the T cell enriched fraction deprived of macrophages did not produce IL 2, but the T cells pulse-incubated with OKT3 and reconstituted with macrophages efficiently produced IL 2 in subsequent culture in the presence of TPA as did T cells reconstituted with OKT3-pulse-incubated macrophages. The stimulating effect of OKT3 in the presence of macrophages was inhibited dose-dependently by the addition of immunoglobulins, particularly by mouse IgG2a which is the same isotype as that of the OKT3 antibody, showing that it inhibits by blocking the binding of OKT3 to Fc receptors on macrophages. The same extent of IL 2 production was induced in T cells when paraformaldehyde-fixed macrophages were substituted for intact macrophages. Remarkable IL 2 production was also induced by OKT3 when latex beads coated with rabbit anti-mouse IgG2a antibody and TPA were added to the culture. It was confirmed that the production induced by these stimulations was due to an increase of IL 2 mRNA. These results show that effective signals for IL 2 production are generated by efficient crosslinking of T3 molecules which results from multi-interaction of T3 molecules on the T cell membrane and anti-T3 antibody molecules on macrophage membrane or on the surface of the latex particle.     

Rapid Antiretroviral Therapy Initiation for Women in an HIV-1 Prevention Clinical Trial Experiencing Primary HIV-1 Infection during Pregnancy or Breastfeeding

During an HIV-1 prevention clinical trial in East Africa, we observed 16 cases of primary HIV-1 infection in women coincident with pregnancy or breastfeeding. Nine of eleven pregnant women initiated rapid combination antiretroviral therapy (ART), despite having CD4 counts exceeding national criteria for ART initiation; breastfeeding women initiated ART or replacement feeding. Rapid ART initiation during primary HIV-1 infection during pregnancy and breastfeeding is feasible in this setting.     

Degradation of aromatic amino acids by Rhodococcus erythropolis

A Gram-positive Rhodococcus erythropolis strain S1 was shown to assimilate aromatic amino acids such as L-phenylalanine, L-tyrosine, L-tryptophan, D-phenylalanine, D-tyrosine and D-tryptophan, which were utilized not only as the sole carbon source but also as a suitable nitrogen source. The highest growth on these aromatic amino acids occurred at a temperature of 30°C. L-Phenylalanine, L-tyrosine and L-tryptophan degradative pathways would appear to be independent, and to be induced alternatively. The strain S1 also showed the ability to assimilate peptides which consisted of only L-phenylalanine and L-tyrosine.     

Environmental needs and social justice

Despite the growth in awareness and practical action to maintain biodiversity, environmental degradation and ecosystem destruction has continued at a high rate over the last 20 years. The roots of this lie in the predominant international economic order, underpinned by lifestyle demands for increased material consumption. Net flow of wealth from South (less developed) to North (more developed) nations has exacerbated a spiral of increased poverty and environmental degradation in the former. Global environmental conservation depends upon a radical change of direction with the principle of equity as the starting point. Notwithstanding the importance of continuing to add to local and small-scale conservation achievements, the prospect of radical change happening seems small, despite it being in the long-term self-interest of the North. The concept of equity is, apparently, unacceptable to Northern electorates.     

Means for efficient electron beam generation in wide-aperture open-discharge light sources

The interaction of a plasma in the accelerating gap of an open discharge with a strong external electric field and with the cathode surface has been investigated theoretically and experimentally. In a pulsed nanosecond discharge, the ion inertia and plasma screening of the electric field cause a fast growth of the electric field E in the cathode region and a decrease in the length of the latter. Along with a reduction of the electron multiplication factor at high electric fields, this leads to a substantial decrease in the ion flux toward the cathode, which allows one to develop highly efficient open-discharge light sources with a long lifetime and low cathode sputtering. In this respect, continuous and quasi-continuous discharges are less advantageous because of the smaller increase in the electric field in the cathode region. The Townsend coefficients of charge multiplication and electron emission at high electric fields typical of open discharges have been measured for the first time. Fast ions and atoms extracted from the plasma of the accelerating gap significantly affect the cathode emission properties. In particular, photoemission is enhanced by more than one order of magnitude and becomes the main mechanism for electron generation. This also increases the efficiency and lifetime of open-discharge light sources.     

Evaluation of the antimicrobial activity of methylglyoxal bis(guanylhydrazone) analogues, the inhibitors for polyamine biosynthetic pathway

Metabolic and antiproliferative effects of methylglyoxal bis(butylamidinohydrazone) (MGBB) and methylglyoxal bis(cyclopentylamidinohydrazone) (MGBCP), inhibitors for polyamine biosynthetic pathway, on Escherichia coli, Shigella sonnei, Aeromonas sobria, Aeromonas hydrophila and Vibrio cholerae were investigated. MGBB at the concentration of 100 mumol/l depleted intracellular putrescine and spermidine concentrations of E. coli to 25 and 20% of the controls, respectively, while MGBCP depressed their concentrations to 38 and 24%, respectively. In these polyamine-depleted E. coli cells the syntheses of RNA, DNA and protein decreased to 13, 54 and 29% of the control, respectively, with MGBB and to 23, 71 and 55%, respectively, with MGBCP. The minimum inhibitory concentrations (MIC) of MGBB for the growth of A. sobria, E. coli, A. hydrophila, V. cholerae and Sh. sonnei were estimated to be 50, 160, 240, 285 and 320 mumol/l, respectively, whereas those of MGBCP were slightly higher for respective bacteria.