Identification and characterization of the bacterial <Emphasis Type="SmallCaps">d</Emphasis>-gluconate dehydratase in <Emphasis Type="Italic">Achromobacter xylosoxidans</Emphasis>

Achromobacter xylosoxidans is known to utilize d-glucose via the modified Entner-Doudoroff pathway. Although d-gluconate dehydratase produced from this bacterium was purified and partially characterized previously, a gene that encodes this enzyme has not yet been identified. To obtain protein information on bacterial d-gluconate dehydratase, we partially purified d-gluconate dehydratase in A. xylosoxidans and investigated its biochemical properties. Two degenerate primers were designed based on the N-terminal amino acid sequence of the partially purified d-gluconate dehydratase. Through PCR performed using degenerate primers, a 1,782-bp DNA sequence encoding the A. xylosoxidans d-gluconate dehydratase (gnaD) was obtained. The deduced amino acid sequence of A. xylosoxidans gnaD showed strong similarity with that of proteins belonging to the dihydroxy-acid dehydratase/phosphogluconate dehydratase family (COG0129). This is in contrast to the archaeal d-gluconate dehydratase, which belongs to the enolase superfamily (COG4948). The phylogenetic tree showed that A. xylosoxidans d-gluconate dehydratase is closer to the 6-phosphogluconate dehydratase than the dihydroxy-acid dehydratase. Interestingly, a clade containing A. xylosoxidans enzyme was clustered with proteins annotated as a second and a third dihydroxy-acid dehydratase in the genomes of Clostridium acetobutylicum (Cac_ilvD2) and Streptomyces ceolicolor (Sco_ilvD2, Sco_ilvD3), indicating that the function of these enzymes is the dehydration of d-gluconate.     

Pathogen-inducible CaUGT1 is involved in resistance response against TMV infection by controlling salicylic acid accumulation

Capsicum annuum L. Bugang exhibits a hypersensitive response against Tobacco mosaic virus (TMV) P₀ infection. The C. annuumUDP-glucosyltransferase 1 (CaUGT1) gene was upregulated during resistance response to TMV and by salicylic acid, ethephon, methyl viologen, and sodium nitroprusside treatment. When the gene was downregulated by virus-induced gene silencing, a delayed HR was observed. In addition, free and total SA concentrations in the CaUGT1-downregulated hot pepper were decreased by 52% and 48% compared to that of the control plants, respectively. This suggested that the CaUGT1 gene was involved in resistance response against TMV infection by controlling the accumulation of SA.     

Structural and biochemical implications of single amino acid substitutions in the nucleotide-dependent switch regions of the nitrogenase Fe protein from<Emphasis Type="Italic"> Azotobacter vinelandii</Emphasis>

The structures of nitrogenase Fe proteins with defined amino acid substitutions in the previously implicated nucleotide-dependent signal transduction pathways termed switch I and switch II have been determined by X-ray diffraction methods. In the Fe protein of nitrogenase the nucleotide-dependent switch regions are responsible for communication between the sites responsible for nucleotide binding and hydrolysis and the [4Fe-4S] cluster of the Fe protein and the docking interface that interacts with the MoFe protein upon macromolecular complex formation. In this study the structural characterization of the Azotobacter vinelandii nitrogenase Fe protein with Asp at position 39 substituted by Asn in MgADP-bound and nucleotide-free states provides an explanation for the experimental observation that the altered Fe proteins form a trapped complex subsequent to a single electron transfer event. The structures reveal that the substitution allows the formation of a hydrogen bond between the switch I Asn39 and the switch II Asp125. In the structure of the native enzyme the analogous interaction between the side chains of Asp39 and Asp125 is precluded due to electrostatic repulsion. These results suggest that the electrostatic repulsion between Asp39 and Asp125 is important for dissociation of the Fe protein:MoFe protein complex during catalysis. In a separate study, the structural characterization of the Fe protein with Asp129 substituted by Glu provides the structural basis for the observation that the Glu129-substituted variant in the absence of bound nucleotides has biochemical properties in common with the native Fe protein with bound MgADP. Interactions of the longer Glu side chain with the phosphate binding loop (P-loop) results in a similar conformation of the switch II region as the conformation that results from the binding of the phosphate of ADP to the P-loop.     

Distribution of neuronal nitric oxide synthase-immunoreactive neurons in the cerebral cortex and hippocampus during postnatal development

Although many reports have argued a role for nitric oxide (NO) during postnatal development, there has been no combined demonstration in the cerebral cortex and hippocampus. We have investigated the distribution and morphology of neurons and fibers expressing neuronal NO synthase (nNOS) in the cerebral cortex and hippocampal formation of rats during the postnatal development, and correlated these findings with developmental events taking place in these regions. In the cerebral cortex, the nNOS-immunoreactive cells could be divided into two classes : heavily stained neurons and lightly stained neurons. For the lightly stained nNOS-positive neurons, only the cell bodies were observed, whereas for the heavily stained neurons, the cell bodies and their dendrites were visible. During the postnatal days, heavily stained neurons reached their typical morphology in the second week and appeared in all layers except for layer I. In the hippocampus, there was a transient expression of nNOS in the pyramidal cell layer at P3â€P7, and this expression disappeared during following days. The adult pattern of staining developed gradually during the postnatal period. This study suggested that these alterations might reflect a region-specific role of NO and a potential developmental role in the postnatal cerebral cortex and hippocampus     

Retrovirus-mediated gene transfer and expression of EGFP in chicken

Here, we successfully demonstrate expression of the EGFP (enhanced green fluorescence protein) gene in chickens using replication-defective MLV (murine leukemia virus)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G glycoprotein). The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). After 12 days incubation, all of the eight living embryos assayed were found to express this vector-encoded EGFP gene, which was under the control of the RSV (Rous Sarcoma Virus) promoter, in diverse organ tissues, including head, beak, neck, wing, hock, tail, toes, heart, amnion, and yolk sac. Surprisingly, despite the presumed cytotoxicity of EGFP, some embryos hatched and survived and these had prominent green fluorescent spots, both in internal organs and externally.     

Requirement of spermidine for developmental transitions in Aspergillus nidulans

Deletion of the spermidine synthase gene in the fungus Aspergillus nidulans results in a strain, deltaspdA, which requires spermidine for growth and accumulates putrescine as the sole polyamine. Vegetative growth but not sporulation or sterigmatocystin production is observed when deltaspdA is grown on media supplemented with 0.05-0.10 mM exogenous spermidine. Supplementation of deltaspdA with >/= 0.10 mM spermidine restores sterigmatocystin production and >/= 0.50 mM spermidine produces a phenotype with denser asexual spore production and decreased radial hyphal growth compared with the wild type. DeltaspdA spores germinate in unsupplemented media but germ tube growth ceases after 8 h upon which time the spores swell to approximately three times their normal diameter. Hyphal growth is resumed upon addition of 1.0 mM spermidine. Suppression of a G protein signalling pathway could not force asexual sporulation and sterigmatocystin production in deltaspdA strains grown in media lacking spermidine but could force both processes in deltaspdA strains supplemented with 0.05 mM spermidine. These results show that increasing levels of spermidine are required for the transitions from (i) germ tube to hyphal growth and (ii) hyphal growth to tissue differentiation and secondary metabolism. Suppression of G protein signalling can over-ride the spermidine requirement for the latter but not the former transition.     

An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo

pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.     

Regulation of septation and cytokinesis during resumption of cell division requires uvi31+, a UV-inducible gene of fission yeast

uvi31+ is a sequence homolog of Escherichia coli bolA gene in Schizosaccharomyces pombe, identified as a UV-inducible gene. Here, the cellular function of uvi31+ was investigated by null mutant analysis. Deletion of uvi31+ led to a delayed germination of spore and defects in subsequent cell division. However, the uvi31 mutant cell proliferated faster with smaller cell size than the wild-type cell during vegetative growth. In addition, the uvi31 mutant was sensitive to UV-light. It showed a normal cell cycle delay after UV-irradiation but displayed aberrant septum formation and defective cytokinesis when released from the UV damage checkpoint. These results suggest that uvi31+ may be involved in control of cell division, especially during the resumption from cell cycle arrest.     

Phylogenetic Relations among Percid Fishes as Inferred from Mitochondrial CytochromebDNA Sequence Data

Hypotheses of relationship among genera of Percidae have been conflicting. Based on different phylogenetic premises, the evolution of small benthic forms in Percidae has been interpreted as resulting from either convergence or common ancestry. In order to assess various phylogenetic hypotheses of Percidae we collected complete sequences (1140 bp) of mitochondrially encoded cytochromebfor 21 species of percids. Seven species representing four additional families of Perciformes were used as outgroups. Maximum parsimony and minimum evolution analyses both recovered single shortest trees, and the results of these analyses were generally congruent with one another. All analyses consistently recovered three monophyletic groups in Percidae: Etheostomatinae (Ammocrypta, Crystallaria, Etheostoma,andPercina), Percinae (PercaandGymnocephalus), and Luciopercinae (Stizostedion, Zingel,andRomanichthys). As a result of this analysis we present a revised classification of Percidae and discuss the phylogenetic evidence for the independent evolution of small benthic species within Etheostomatinae and Luciopercinae.