Self-association of glutamic acid-rich fusion peptide analogs of influenza hemagglutinin in the membrane-mimic environments: Effects of positional difference of glutamic acids on side chain ionization constant and intra- and inter-peptide interactions deduced from NMR and gel electrophoresis measurements

Two glutamic acid-rich fusion peptide analogs of influenza hemagglutinin were synthesized to study the organization of the charged peptides in the membranous media. Fluorescence and gel electrophoresis experiments suggested a loose association between the monomers in the vesicles. A model was built which showed that a positional difference of 3, 7 and 4, 8 results in the exposure of Glu3 and Glu7 side chains to the apolar lipidic core. Supportive results include: first, pK_a values of two pH units higher than reference value in aqueous medium for Glu3 and Glu7 CγH, whereas the deviation of pK_a from the reference value for Glu4 and Glu8 CγH is substantially smaller; second, Hill coefficients of titration shift of these protons indicate anti-cooperativity for Glu3 and Glu7 side chain protons but less so for Glu4 and Glu8, implying a strong electrostatic interaction between Glu3 and Glu7 possibly resulting from their localization in an apolar environment; third, positive and larger titration shift for NH of Glu3 is observed compared to that of Glu4, suggesting stronger hydrogen bond between the NH and the carboxylic group of Glu3 than that of Glu4, consistent with higher degree of exposure to hydrophobic medium for the side chain of Glu3.     

Mutational analysis of slyD, an Escherichia coli gene encoding a protein of the FKBP immunophilin family

slyD encodes a 196 amino acid polypeptide that is a member of the FKBP family of cis–trans peptidyl–prolyl isomerases (PPIases). slyD mutations affect plaque formation by the phage φX174 by blocking the action of the phage lysis protein E. Here we describe the selection of a set of spontaneous slyD mutations conferring resistance to the expression of gene E from a plasmid. These mutations occur disproportionately in residues of SlyD that, based on the structure of the prototype mammalian FKBP12, make ligand contacts with immunosuppressing drug molecules or are conserved in other FKBP proteins. A wide variation in the plating efficiency of φX174 on these E  ^R strains is observed, relative to the parental, indicating that these alleles differ widely in residual SlyD activity. Moreover, it is found that slyD mutations cause significant growth rate defects in Escherichia coli B and C backgrounds. Finally, overexpression of slyD causes filamentation of the host. Thus, among the FKBP genes found in organisms across the evolutionary spectrum, slyD is unique in having three distinct drug-independent phenotypes.     

Physiological characteristics of NG2-expressing glial cells

Antibodies against the chondroitin sulfate proteoglycan NG2 label a subpopulation of glial cells within the CNS, which have a small cell body and thin radiating processes. Physiological recordings from these small cells in acute brain slices have revealed that they possess unique properties, suggesting that they may comprise a class of glial cells distinct from astrocytes, oligodendrocytes, or microglia. NG2-expressing glial cells (abbreviated as “NG2 cells” here) have a moderate input resistance and are not dye- or tracer-coupled to adjacent cells. They express voltage-gated Na⁺, K⁺and Ca²⁺conductances, though they do not exhibit regenerative Na⁺or Ca²⁺action potentials due to the much larger K⁺conductances present. In addition to voltage-gated conductances, they express receptors for various neurotransmitters. In the hippocampus, AMPA and GABA_Areceptors on these cells are activated by release of transmitter from neurons at defined synaptic junctions that are formed with CA3 pyramidal neurons and GABAergic interneurons. These rapid forms of neuron-glial communication may regulate the proliferation rate of NG2 cells or their development into mature oligodendrocytes. These depolarizing inputs may also trigger the release of neuroactive substances from NG2 cells, providing feedback regulation of signaling at neuronal synapses. Although the presence of Ca²⁺permeable AMPA receptors provides a pathway to link neuronal activity to Ca²⁺dependent processes within the NG2 cells, these receptors also put these cells at risk for glutamate-associated excitotoxicity. This vulnerability to the sustained elevation of glutamate may underlie ischemic induced damage to white matter tracts and contribute to cerebral palsy in premature infants.     

Evidence for two forms of RNA-dependent DNA polymerase in Visna virus.

The visna viral RNA-dependent DNA polymerase has been resolved into two forms by affinity chromatography. Glycerine gradient centrifugation of the two forms showed that one form sedimented at 6.9 S corresponding to an apparent molecular weight of 135 000 and the other at 6.3 S corresponding to 118 000. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the two forms indicated that the 6.9 S enzyme is composed of 2 molecules of 68 000 mol. wt. chain and the 6.3 S is a single chain enzyme. The latter form has been identified as a glycoprotein. The 6.9 S form can be completely inactivated in 20 min at 45 degrees C, prefers poly(rC) over poly(rA) as template and has high efficiency in utilizing visna 70 S RNA as template. The 6.3 S form is stable at 45 degrees C, active with 70 S viral RNA as template, prefers poly(rA) over poly(rC), and requires higher concentration of Mn2+ (0.4 mM) for maximum activity than the 6.9 S form does (0.1 mM) with synthetic homopolymers as templates. However, both 6.9 S and 6.3 S forms prefer Mg2+ over Mn2+ regardless of the nature of the templates.     

Purification and characterization of RNA polymerase I from a higher plant.

RNA polymerase I was purified from chromatin isolated from auxin-treated soybean hypocotyl. Purification was achieved by using Agarose A-1.5m gel filtration, DEAE-cellulose, CM-sephadex, and phosphocellulose chromatography, and sucrose density gradient centrifugation. With denatured calf thymus DNA as template, the enzyme has a high specific activity (200-300 nmol/mg/30 min at 28 degrees C) which is comparable to other RNA polymerase I enzymes purified from animals and yeast. While the gel profiles indicate that purification to homogeneity (greater than 90%) may not have been achieved, the enzyme appears to be composed of possibly 7 subunits, several of which are similar to the subunits of yeast RNA polymerase I. The putative subunits and molar ratios are 183 000 (1), 136 000 (1), 50 000 (0.5), 46 000 (0.5), 40 000 (0.5), 33 000 (0.2), and 28 000 (2). The purified enzyme strongly prefers a completely denatured template such as poly(dC).     

Reaction mechanism of surfactant‐sensitized chemiluminescence of bis(2,4,6‐trichlorophyenyl) oxalate and hydrogen peroxide induced by gold nanoparticles

The chemiluminescence (CL) of bis(2,4,6‐trichlorophyenyl) oxalate with hydrogen peroxide in the present of cationic surfactant and gold nanoparticles was studied. The CL emission was obviously enhanced in the presence of surfactant at a suitable concentration, with a synergetic catalysis effect exhibited. Different sizes of gold nanoparticles (15 and 50 nm) showed different effects on CL intensity. Mechanisms of the CL reaction and sensitization effect are discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

Construction and expression of nonsense suppressor tRNAs which function in plant cells

An Arabidopsis thaliana L. DNA containing the tRNA(TrpUGG) gene was isolated and altered to encode the amber suppressor tRNA(TrpUAG) or the ochre suppressor tRNA(TrpUAA). These DNAs were electroporated into carrot protoplasts and tRNA expression was demonstrated by the translational suppression of amber and ochre nonsense mutations in the chloramphenicol acetyltransferase (CAT) reporter gene. DNAs encoding tRNA(TrpUAG) and tRNA(TrpUAA) nonsense suppressor tRNAs caused suppression of their cognate nonsense codons in CAT mRNAs, with the tRNA(TrpUAG) gene exhibiting the greater suppression under optimal conditions for expression of CAT. The development of these translational suppressors which function in plant cells facilitates the study of plant tRNA gene expression and will make possible the manipulation of plant protein structure and function.