Cellulase and pectinase activities were

Dioxin and related chemicals alter the expression of a number of genes by activating the aryl hydrocarbon receptors (AHR) to produce a variety of disorders including hepatotoxicity. However, it remains largely unknown how these changes in gene expression are linked to toxicity. To address this issue, we initially examined the effect of 2,3,7,8-tetrachrolodibenzo--dioxin (TCDD), a most toxic dioxin, on the hepatic and serum metabolome in male pubertal rats and found that TCDD causes many changes in the level of fatty acids, bile acids, amino acids, and their metabolites. Among these findings was the discovery that TCDD increases the content of leukotriene B4 (LTB4), an inducer of inflammation due to the activation of leukocytes, in the liver of rats and mice. Further analyses suggested that an increase in LTB4 comes from a dual mechanism consisting of an induction of arachidonate lipoxygenase-5, a rate-limiting enzyme in LTB4 synthesis, and the down-regulation of LTC4 synthase, an enzyme that converts LTA4 to LTC4. The above changes required AHR activation, because the same was not observed in AHR knock-out rats. In agreement with LTB4 accumulation, TCDD caused the marked infiltration of neutrophils into the liver. However, deleting LTB4 receptors (BLT1) blocked this effect. A TCDD-produced increase in the mRNA expression of inflammatory markers, including tumor-necrosis factor and hepatic damage, was also suppressed in BLT1-null mice. The above observations focusing on metabolomic changes provide novel evidence that TCDD accumulates LTB4 in the liver by an AHR-dependent induction of LTB4 biosynthesis to cause hepatotoxicity through neutrophil activation.

This study investigated dioxin-induced changes in metabolomes in pubertal rat excrement. The administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or restricting dietary intake (pair-fed group) markedly altered the metabolomic profile including lipids, hormones, and vitamins in the urine and feces. TCDD caused an increase in the fecal chenodeoxycholic acid and taurocholic acid content and in urinary adrenaline and 17β-estradiol, while the urinary melatonin level was reduced by TCDD. These changes were not observed in the pair-fed group. In accordance with the elevated level of fecal bile acids, TCDD reduced the intestinal expression of the apical sodium-dependent bile salt transporter, which plays a role in resorbing bile acids from the bile duct. In addition, CYP7A1, a rate-limiting enzyme for bile acid biosynthesis, was attenuated by TCDD treatment, although TCDD induced hepatic CYP8B1, an enzyme essential for cholic acid synthesis. Supplying cholic acid or chenodeoxycholic acid to TCDD-exposed rats tended to restore the TCDD-produced reduction in serum triglycerides, whereas no similar trend was observed in wasting syndrome and lipid accumulation in the liver. These results suggest that: 1) TCDD alters the circulating levels of bile acids and hormones via a mechanism distinct from an attenuation in dietary intake, although the majority of TCDDinduced changes in nutrient contents in the excrement is due to a reduction in food intake; and 2) TCDD facilitates the excretion of bile acids and disrupts their biosynthesis, resulting in the disturbance of lipid homeostasis.

The production of pectinases and the expression of genes encoding pectinases by conidia and germlings of Blumeria graminis f. sp. tritici were investigated. By pectate plate assay, the activity of polygalacturonase was detected in homogenates from ungerminated conidia and germlings grown on an artificial substratum. We could amplify the fragments of two endo-polygalacturonase genes, two pectin lyase genes and a pectate lyase gene from genomic DNA of the fungus by the polymerase chain reaction (PCR). The genes were designated as pg1 and pg2, pul1 and pnl2, and pel1, respectively. Their nucleotide sequences and the deduced amino acid sequences of their fragments showed high homology to those of the reported sequences of corresponding pectinases of other fungi. The RT-PCR indicated the expression of pg1, pg2 and pnl2 in ungerminated conidia and germlings. The expression of pnll and pell was detected only at the late stage of the infection in wheat leaves but not in ungerminated conidia and germlings on an artificial substratum and at an early stage of infection in wheat leaves. The present results suggested possible involvement of pg1, pg2, pal1, pnl2 and pel1 in morphogenesis and/or pathogenesis of this fungus.

Expression of a xylanase gene and release of xylanases by Blumeria graminis were investigated. Exudates released from ungerminated conidia and germlings on plastic membranes were collected and fractionated through anion-and cation-exchange columns. Xylanase activity was detected in the exudate of both ungerminated conidia and germlings by a p-nitrophenyl-d-xylopyranoside (PNPX) assay, and the fractions were eluted stepwise by 0.2-0.5 M NaCl from an anion-exchange column by a plate assay. The activity detected in the fractions eluted by 0.2 and 0.3 M NaCl from an anion-exchange column by a PNPX assay increased during morphogenesis on the membranes. Genomic Southern blot analysis with a heterologous probe prepared from Aspergillus oryzae xynG1 demonstrated the existence of multiple xylanase genes in the genome of this fungus. Using this probe, one xylanase gene, xyn1, was cloned from its genomic library. The nucleotide and predicted amino acid sequences showed high homology to those of endo-1,4--xylanases produced by other fungi. The reverse transcription-polymerase chain reaction showed that xyn1 expression increased in ungerminated conidia and germlings with the time after inoculation onto plastic membranes.