Site-directed mutagenesis of the ionizable groups in the active site of Zymomonas mobilis pyruvate decarboxylase: effect on activity and pH dependence.

Pyruvate decarboxylase (PDC, EC 4.1.1.1) is a thiamin diphosphate-dependent enzyme about which there is a large body of structural and functional information. The active site contains several absolutely conserved ionizable groups and all of these appear to be important, as judged by the fact that mutation diminishes or abolishes catalytic activity. Previously we have shown [Schenk, G., Leeper, F.J., England, R., Nixon, P.F. & Duggleby, R.G. (1997) Eur. J. Biochem. 248, 63-71] that the activity is pH-dependent due to changes in kcat/Km while kcat itself is unaffected by pH. The effect on kcat/Km is determined by a group with a pKa of 6.45; the identity of this group has not been determined, although H113 is a possible candidate. Here we mutate five crucial residues in the active site with ionizable side-chains (D27, E50, H113, H114 and E473) in turn, to residues that are nonionizable or should have a substantially altered pKa. Each protein was purified and characterized kinetically. Unexpectedly, the pH-dependence of kcat/Km is largely unaffected in all mutants, ruling out the possibility that any of these five residues is responsible for the observed pKa of 6.45. We conjecture that the kcat/Km profile reflects the protonation of an alcoholate anion intermediate of the catalytic cycle.     

Neurofascin: a novel chick cell-surface glycoprotein involved in neurite-neurite interactions

We have identified neurofascin, a novel chick cell-surface glycoprotein involved in neurite-neurite interactions. Neurofascin is defined by its reactivity with monoclonal antibody (MAb) F6, which detects two polypeptides (160 and 185 kd) in immunotransfers of brain plasma membrane proteins. Immunoaffinity chromatography using immobilized MAb F6 yields major molecular mass bands at 185, 160, 135-110, and 92 kd. Fingerprint analyses show that these polypeptides are related. Neurofascin is expressed primarily in fiber-rich areas of embryonic cerebellum, spinal cord, and retina. Fab fragments of polyclonal antibodies to neurofascin interfere with the outgrowth of retinal and sympathetic axons in two different in vitro bioassays. Neurofascin is immunologically distinct from other known neurite-associated surface glycoproteins.     

Expression of the precore region of an avian hepatitis B virus is not required for viral replication.

The core-antigen-coding region of all hepadnaviruses is preceded by a short, in-phase open reading frame termed precore whose expression can give rise to core-antigen-related polypeptides. To explore the functional significance of precore expression in vivo, we introduced a frameshift mutation into this region of the duck hepatitis B virus (DHBV) genome and examined the phenotype of this mutant DNA by intrahepatic inoculation into newborn ducklings. Animals receiving mutant DNA developed DHBV infection, as judged by the presence in hepatocytes of characteristic viral replicative intermediates; molecular cloning and DNA sequencing confirmed that the original mutation was present in the progeny genomes. Infection could be efficiently transmitted to susceptible ducklings by percutaneous inoculation with serum from mutant-infected animals, indicating that infectious progeny virus was generated. These findings indicate that expression of the precore region of DHBV is not essential for genomic replication, core particle morphogenesis, or intrahepatic viral spread.     

Sequence and translation of the murine coronavirus 5''-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase.

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed.     

Optimizing the expression of chimeric genes in plant cells

Summary The Serratia marcescens chiA gene encodes a secreted chitinase activity which contributes to the fungal growth inhibition exhibited by this bacterium. The coding region from the chiA gene was fused to the promoter and 3 polyadenylation region of the Agrobacterium nopaline synthase gene. Site-directed mutagenesis of specific nucleotides surrounding the initiating AUG of the coding sequence of this chimeric gene resulted in up to an eight-fold increase in the amount of chitinase protein detected in transformed plant tissue. Analysis of the chiA mRNA indicated that these nucleotides also affected mRNA levels. At least 50% of the chitinase protein produced in transformed tobacco cells was the same molecular weight as the S. marcescen secreted protein.     

Nitrogen fixation and nitrate reduction in the root nodules of legumes

Published data on, and hypotheses regarding the effect of NO⁻₃ on functioning of legume root nodules are reviewed. It is concluded that a short-term reversible effect of NO⁻₃ may act via an increased resistance to O₂ diffusion in nodules; this is coupled to decreased bacteroid respiration. For longer exposures to NO⁻₃ nodule activity is irreversibly lost, but how this relates to carbohydrate deprivation or NO-₂ accumulation is unclear. Complicating factors include denitrification reactions and the interaction of NO⁻₂ with leghaemoglobin.     

Direct induction of lymphocyte killing by calcium ionophore and phorbol ester treatment

To analyze transduction mechanisms in human lymphocyte killing, intracellular Ca2+ levels were increased by ionophore A23187 treatment and protein kinase C activated by phorbol ester 12-O-tetradecanoylphorbol-acetate (TPA). Drugs were tested either alone or in combinations on effector cells active in natural, antibody-dependent, and lectin-dependent killing. TPA suppressed killing in all systems at 100 ng/ml whereas A23187 was only suppressive for NK killing at concentrations higher than 0.1 microM. TPA combined with A23187, above 10 ng/ml and 0.5 microM, respectively, induced killing of all tested target cell lines with a slower kinetic than NK killing of K562 cells. Drug-induced killing did not increase optimal lectin and antibody-dependent killing and was demonstrated most easily on NK-resistant target cell lines. Fractionation of effector lymphocytes into NK cell-depleted, T3-positive and NK cell-enriched, T3-negative cells demonstrated that similar levels of TPA/A23187-dependent killing could be induced in both fractions. It is concluded that TPA/A23187 induce normal lymphocytes to nonselective killing of different target cells in similarity to the triggering effect these drugs have in many other cell systems. Whether the induced killing is representative of NK killing is discussed in relation to the presence of other potential effector cells and effector molecules in peripheral blood lymphocytes.     

The cyclopoid copepods of a wet campo marsh in central Brazil

Eight species of cyclopoid copepods were recorded from 1979–1983 in the complex microhabitats of a wet campo (campo úmido) marsh in central Brazil. Ectocyclops herbsti and Paracyclops fimbriatus occurred most often in areas with water covering the soil; Muscocyclops therasiae n. sp. occurred mainly in soils with no surface water; while Metacyclops campestris n. sp. showed no distinct microhabitat preference. Occurrence of the remaining four species was too sporadic to determine microhabitat preference. Paracyclops carectum n. sp., Metacyclops campestris n. sp., Muscocyclops therasiae n. sp., Muscocyclops bidentatus n. sp. and Ponticyclops boscoi n. g. n. sp. are described. A key to the New World species of Metacyclops s. str. is provided.