Influence of carbon source on wood decay-associated gene expression in sequential hyphal zones of the brown rot fungus Gloeophyllum trabeum.

Brown rot fungi show a two-step wood degradation mechanism comprising oxidative radical-based and enzymatic saccharification systems. Recent studies have demonstrated that the brown rot fungus Rhodonia placenta expresses oxidoreductase genes ahead of glycoside hydrolase genes and spatially protects the saccharification enzymes from oxidative damage of the oxidoreductase reactions. This study aimed to assess the generality of the spatial gene regulation of these genes in other brown rot fungi and examine the effects of carbon source on the gene regulation. Gene expression analysis was performed on 14 oxidoreductase and glycoside hydrolase genes in the brown rot fungus Gloeophyllum trabeum, directionally grown on wood, sawdust-agar, and glucose-agar wafers. In G. trabeum, both oxidoreductase and glycoside hydrolase genes were expressed at higher levels in sections behind the wafers. The upregulation of glycoside hydrolase genes was significantly higher in woody substrates than in glucose, whereas the oxidoreductase gene expression was not affected by substrates.

Colony Suppression and Possible Colony Elimination of the Subterranean Termites Coptotermes formosanus and Reticulitermes speratus by Discontinuous Soil Treatment Using a Diluent of Fipronil Suspension Concentrate.

We assessed the efficacy of a discontinuous soil treatment using a diluent of fipronil suspension concentrate in controlling colonies of Coptotermes formosanus and Reticulitermes speratus. In-ground monitoring stations were installed at Isogi Park and Kindai University, and individual termites inhabiting the stations were collected for four or six years to determine the numbers and locations of colonies present in test areas before and after the discontinuous soil treatment. Microsatellite genotyping indicated that two C. formosanus and two R. speratus colonies in the test area at Isogi Park and five R. speratus colonies in the test area at Kindai University were active and that their territories fluctuated every year. One of the two C. formosanus colonies at Isogi Park and one of the five R. speratus colonies at Kindai University were subjected to discontinuous soil treatments with fipronil and were strongly affected by the treatment at the colony level, resulting in the suppression and possible elimination of colonies. Termite activity of the fipronil-treated colony of C. formosanus was detected within one week after the discontinuous soil treatment and was not found for more than two years (28 months), while termite activity of the fipronil-treated colony of R. speratus was detected within four days and three weeks after the discontinuous soil treatment and was not detected thereafter for three years. Fipronil residue analysis showed that workers of C. formosanus moved at least 28 m and that workers of R. speratus moved 6 m from the treated soil locations for up to three weeks.

Presence of extracellular NAD(+) and NADH in cultures of wood-degrading fungi.

Our previous studies indicated that extracellular glycoproteins produced by some white-rot and brown-rot basidiomycetous fungi reduce Fe(III) to Fe(II) and O2 to H2O2 and produce hydroxyl radicals. The continuous generation of hydroxyl radicals requires a constant supply of O2 and an electron donor for the reduction of oxidized forms of the glycoproteins to the reduced forms. However, electron donors for this reaction, such as NADH, have not been identified. In this study, the amounts of the extracellular pyridine coenzymes, NAD(＋) and NADH, were measured in agar cultures of four white-rot fungi, one brown-rot fungus, and three soft-rot fungi. The sums of NAD(＋) and NADH detected in wood-containing cultures of all five basidiomycetes were greater than those in glucose cultures. The amounts of NAD(＋) were higher than those of NADH in all wood-containing cultures except that of Irpex lacteus, and NAD(＋) was greater than NADH in all glucose cultures except that of Fomitopsis palustris. Significant amounts of pyridine coenzymes were present in glucose and wood-containing cultures of the three soft-rot fungi. The non-wood-degrading fungus, Penicillium funiculosum, did not produce NAD(＋) or NADH in either glucose or wood-containing cultures. The extracellular pyridine coenzyme levels were relatively high compared to the rates of extracellular hydroxyl radical generation in wood-degrading fungal cultures. Thus, white-, brown-, and soft-rot fungi produce pyridine coenzymes that could serve as electron donors for the production of hydroxyl radicals during wood degradation.

Effect of norharmane in vitro on juvenile hormone epoxide hydrolase activity in the lower termite, Reticulitermes speratus.

The aromatic beta-carboline norharmane was determined in workers, nymphs, and ergatoids of Reticulitermes speratus (Kolbe) (Isoptera: Rhinotermitidae) by gas chromatography/mass spectrometry. Norharmane levels in workers, nymphs, and ergatoids collected in May (approximately 4.3 ng/termite) were higher than those in November (approximately 0.2 ng/termite). Fluorescence of norharmane was observed in histological sections in whole animals and in the fat body. Norharmane, at a final concentration of 1 mM, stimulated juvenile hormone epoxide hydrolase (JHEH) activity of enzyme extract from ergatoids, but inhibited JHEH activity at higher concentrations. The elevated JHEH activity stimulated by norharmane should accelerate juvenile hormone metabolism in R. speratus.

Characterization of a hydroxyl-radical-producing glycoprotein and its presumptive genes from the white-rot basidiomycete Phanerochaete chrysosporium.

During wood decay, the white-rot basidiomycete Phanerochaete chrysosporium secretes low-molecular-mass glycoproteins that catalyze a redox reaction between O(2) and electron donors to produce hydroxyl radical. This reaction accounts for most of the hydroxyl radical produced in wood-degrading cultures of P. chrysosporium. In combination with phenol oxidases, hydroxyl radical is believed to play a role in lignin degradation. The secreted glycoproteins also reduce Fe(III) to Fe(II) and strongly bind Fe(II). The partially purified glycoproteins contain 1-amino-1-deoxy-2-ketose (ketoamine) produced by the condensation of side-chain amino groups and carbohydrate. cDNAs and two putative genes encoding these glycoproteins, glp1 and glp2, have been isolated and sequenced. The 875bp glp1 and 864bp glp2 are found on scaffold 2 of the P. chrysosporium genome. These presumptive genes each consist of seven introns and eight exons. The latter encode a predicted protein of 138 amino acids and a 22-amino-acid signal sequence for secretion. The predicted protein sequences are nearly identical to N-terminal and internal sequences obtained from the partially purified glycoprotein. The molecular masses of the deduced mature proteins, 13,981 (glp1) and 13,970 (glp2), coincide with the molecular mass of the glycoprotein as determined by tricine-SDS-PAGE.

Identification of two subterranean termite species (Isoptera: Rhinotermitidae) using cellulase genes.

To identify subterranean termite species, we designed a pair of common primers that amplified 381-bp fragments from cDNAs encoding the endo-beta-1,4-glucanases (EGases) of Coptotermes formosanus Shiraki and Reticulitermes speratus (Kolbe). cDNAs from C. formosanus and R. speratus, and genomic DNA from R. speratus, were amplified by polymerase chain reaction (PCR) by using this primer pair and then cloned and sequenced. Sequences amplified from C.formosanus cDNA displayed 97-99% identity to cDNA encoding the EGase of C. formosanus (CfEG), and 92-94% identity to cDNA encoding the EGase of R. speratus (RsEG). By contrast, cDNA from R. speratus displayed 99-100% identity to RsEG cDNA and 93-94% identity to CfEG cDNA. CfEG and RsEG cDNAs can therefore be used as markers for the identification of these termite species. This is the first report of the successful identification of termite species by using cDNA and genomic DNA sequences of termite origin.

Phylogeny of vertebrate Src tyrosine kinases revealed by the epitope region of mAb327.

Mass fingerprinting and MS/MS analysis demonstrated that Xyk, a 57-kDa Src family tyrosine kinase that is activated within minutes of Xenopus egg fertilization, comprises a mixture of two Src proteins, Src1 and Src2. However, the Xenopus Src protein, denoted as xSrc, is hardly detectable with mAb327, a universal Src-specific antibody, whose target sequence has not yet been determined. We show that a point amino acid substitution in the Src homology 3 domain of xSrc is critical for improvement of the low efficiency of its recognition by mAb327. Namely, a point-mutated xSrc, in which Arg-121 was replaced by His that is conserved among mAb327-reactive Src in mammals and chicken, showed increased recognition by mAb327. On the other hand, a mutant chicken Src, in which the His-122 residue is replaced by Arg, showed decreased recognition by mAb327. Genomic sequencing analysis also demonstrated that reptile Src proteins are of either the R-type (snake) or H-type (caiman, turtle, and tortoise). These studies revealed, for the first time, a critical amino acid in the Src SH3 domain for mAb327 recognition, and suggest a novel scheme for the molecular evolution of Src, in which the H-type Src(s) are monophyletic and derived from the R-type Src.

Regulation of Src kinase activity during Xenopus oocyte maturation.

Expression of constitutively active Src protein tyrosine kinase in Xenopus oocytes has been shown to accelerate oocyte maturation suggesting that Src may be involved in meiotic progression. However, meiotic regulation of endogenous Src kinase in oocytes has not been investigated in detail. To address this problem, we measured the activity, expression level, and phosphorylation state of the endogenous Xenopus Src (xSrc) and overexpressed xSrc mutants in the process of progesterone-induced oocyte maturation. We found that the enzyme is first transiently activated in the plasma membrane-containing fraction of oocytes within 3 min of progesterone administration. This event represents one of the earliest responses of oocytes to the hormone and should be related to triggering some early signaling pathways of maturation. Thereafter, xSrc activity increases again at the time of germinal vesicle breakdown (GVBD) and remains elevated till the completion of maturation. This elevation of xSrc activity is associated with a 2-fold increase of xSrc protein content in the absence of change in its specific activity and xSrc mRNA content. No significant changes in the phosphorylation state of C-terminal regulatory phosphotyrosine can be registered either in endogenous xSrc or in overexpressed kinase-negative and wild-type xSrc proteins during maturation. Altogether, these results indicate that upregulation of xSrc in the meiotic metaphase occurs at the translation level. We also demonstrate here that the expression of constitutively active xSrc in Xenopus oocytes is accompanied by the activation of mitogen-activated protein kinase (MAPK). Our data suggest that the Src kinase acts through the MAPK pathway to accelerate oocyte maturation.

Pyruvate and acetate metabolism in termite mitochondria.

Intact mitochondria have been successfully prepared from body tissues from the termites Nasutitermes walkeri and Coptotermes formosanus. This is the first report of the successful isolation of mitochondria from termites (Isoptera: Termitidae). Using an oxygen electrode, oxygen consumption by the mitochondria during the oxidation of various respiratory substrates was determined and their properties measured in terms of respiratory control index and ADP/O. ADP/O was as expected for substrates such as pyruvate, acetylcarnitine and acetyl-CoA and carnitine. Pyruvate and acetate were the major respiratory substrates in both species. The total activity of the pyruvate dehydrogenase complex (PDHc) in the mitochondria from N. walkeri and C. formosanus was determined to be 72.87+/-8.98 and 8.29+/-0.42 nmol/termite/h, respectively. Mitochondria isolated in the presence of inhibitors of PDHc interconversion were used to determine that about 60% of the PDHc was maintained in the active form in both N. walkeri and C. formosanus. The sufficient PDHc activity and high rate of pyruvate oxidation in mitochondria from N. walkeri suggest that pyruvate is rapidly metabolised, whereas the low mitochondrial PDHc activity of C. formosanus suggests that in this species more pyruvate is produced than can be oxidised in the termite tissues.

Interarm interaction of DNA cruciform forming at a short inverted repeat sequence.

A novel interarm interaction of DNA cruciform forming at inverted repeat sequence was characterized using an S1 nuclease digestion, permanganate oxidation, and microscopic imaging. An inverted repeat consisting of 17 bp complementary sequences was isolated from the bluegill sunfish Lepomis macrochirus (Perciformes) and subcloned into the pUC19 plasmid, after which the supercoiled recombinant plasmid was subjected to enzymatic and chemical modification. In high salt conditions (200 mM NaCl, or 100-200 mM KCl), S1 nuclease cut supercoiled DNA at the center of palindromic symmetry, suggesting the formation of DNA cruciform. On the other hand, S1 nuclease in the presence of 150 mM NaCl or less cleaved mainly the 3'-half of the repeat, thereby forming an unusual structure in which the 3'-half of the inverted repeat, but not the 5'-half, was retained as an unpaired strand. Permanganate oxidation profiles also supported the presence of single-stranded part in the 3'-half of the inverted repeat in addition to the center of the symmetry. Both electron microscopy and atomic force microscopy have detected a thick protrusion on the supercoiled DNA harboring the inverted repeat. We hypothesize that the cruciform hairpins at conditions favoring triplex formation adopt a parallel side-by-side orientation of the arms allowing the interaction between them supposedly stabilized by hydrogen bonding of base triads.

Towards the molecular dissection of fertilization signaling: Our functional genomic/proteomic strategies.

Recent advances in DNA sequencing techniques and automated informatics has led to clarification of all genome sequence of some model organisms in a very short period. The demonstration of the first draft sequence of the human genome has prompted us to elaborate new approaches in biology, pharmacology and medicine. Such new research will focus on high throughput methods to function on collections of genes, and hopefully, on a genome-wide, quantitative modeling of the cell system as a whole. In this review article, we discuss the present status of "post genome sequencing" approaches in line with our strategies for understanding the molecular mechanism of fertilization and activation of development using the African clawed frog, Xenopus laevis, as a model system.