Cancer chemotherapy and somatic cell mutation

The occurrence of a second neoplasm is one of the major obstacles in cancer chemotherapy. The elucidation of the genotoxic effects induced by anti-cancer drugs is considered to be helpful in identifying the degree of cancer risk. Numerous investigations on cancer patients after chemotherapy have demonstrated: (i) an increase in the in vivo somatic cell mutant frequency (Mf) at three genetic loci, including hypoxanthine–guanine phosphoribosyl-transferase (hprt), glycophorin A (GPA), and the T-cell receptor (TCR), and (ii) alterations in the mutational spectra of hprt mutants. However, the time required for and the degree of such changes are quite variable among patients even if they have received the same chemotherapy, suggesting the existence of underlying genetic factor(s). Accordingly, some cancer patients prior to chemotherapy as well as patients with cancer-prone syndrome have been found to show an elevated Mf. Based on the information obtained from somatic cell mutation assays, an individualized chemotherapy should be considered in order to minimize the risk of a second neoplasm.     

The impact of a single-nucleotide mutation of <Emphasis Type="Italic">bgl2</Emphasis> on cellulase induction in a <Emphasis Type="Italic">Trichoderma reesei</Emphasis> mutant

Background

The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.

Results

To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.

Conclusion

We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

     

Livers with Constitutive mTORC1 Activity Resist Steatosis Independent of Feedback Suppression of Akt

Insulin resistance is an important contributing factor in non-alcoholic fatty liver disease. AKT and mTORC1 are key components of the insulin pathway, and play a role in promoting de novo lipogenesis. However, mTORC1 hyperactivity per se does not induce steatosis in mouse livers, but instead, protects against high-fat diet induced steatosis. Here, we investigate the in vivo mechanism of steatosis-resistance secondary to mTORC1 activation, with emphasis on the role of S6K1-mediated feedback inhibition of AKT. Mice with single or double deletion of Tsc1 and/or S6k1 in a liver-specific or whole-body manner were generated to study glucose and hepatic lipid metabolism between the ages of 6–14 weeks. Following 8 weeks of high-fat diet, the Tsc1-/-;S6k1-/- mice had lower body weights but higher liver TG levels compared to that of the Tsc1-/- mice. However, the loss of S6k1 did not relieve feedback inhibition of Akt activity in the Tsc1-/- livers. To overcome Akt suppression, Pten was deleted in Tsc1-/- livers, and the resultant mice showed improved glucose tolerance compared with the Tsc1-/- mice. However, liver TG levels were significantly reduced in the Tsc1-/-;Pten-/- mice compared to the Pten-/- mice, which was restored with rapamycin. We found no correlation between liver TG and serum NEFA levels. Expression of lipogenic genes (Srebp1c, Fasn) were elevated in the Tsc1-/-;Pten-/- livers, but this was counter-balanced by an up-regulation of Cpt1a involved in fatty acid oxidation and the anti-oxidant protein, Nrf2. In summary, our in vivo models showed that mTORC1-induced resistance to steatosis was dependent on S6K1 activity, but not secondary to AKT suppression. These findings confirm that AKT and mTORC1 have opposing effects on hepatic lipid metabolism in vivo.     

Novel strategy using an adsorbent-column chromatography for effective ethanol production from sugarcane or sugar beet molasses

Molasses-based distilleries generate large volumes of a highly polluted and dark brown-colored wastewater. The present work describes the way in which an adsorbent-column chromatography can effectively remove the colorant and produce biomass ethanol from sugarcane or sugar beet molasses. It was found that the color and chemical oxygen demand of the resulting wastewater was respectively reduced by approximately 87% and 28% as compared with conventional molasses fermentation. Gas chromatography showed that the decolorized molasses maintained good ethanol productivity almost equal to that of the original molasses. Furthermore, it was revealed that the colorant concentrations of about 5 mg ml⁻¹ in the medium were the most favorable for ethanolic fermentation. In summary, we have concluded that this method is the most effective when the adsorbent chromatography is performed just before molasses fermentation and that the decolorized molasses is an ideal substrate for fuel ethanol production.     

Disappearance of deep profundal zoobenthos in Lake Ikeda, southern Kyushu, Japan, with relation to recent environmental changes in the lake

A benthological survey in a deep caldera, Lake Ikeda, southern Kyushu, Japan, in 1998 revealed that no zoobenthos were found in the deep profundal, although two tubificid oligochaetes, Tubifex tubifex and Limnodrilus hoffmeisteri, and a chironomid, Procladius sp., were distributed in the upper profundal zone. This is the first record of oligochaete composition in the lake. Lake Ikeda had been typically oligotrophic until the 1940s, and zoobenthic assemblages were recorded throughout the profundal bottom in the 1920s and 1970s. Recent disappearance of the deep profundal zoobenthos could be caused by the stagnation of anoxic waters in the hypolimnion, in connection with eutrophication triggered by nutrient loading, as well as change in the thermal circulation system presumably caused by global warming.     

Human Parainfluenza Virus Type 2 V Protein Inhibits TRAF6-Mediated Ubiquitination of IRF7 To Prevent TLR7- and TLR9-Dependent Interferon Induction

Paramyxovirus V proteins block Toll-like receptor 7 (TLR7)- and TLR9-dependent signaling leading to alpha interferon production. Our recent study has provided evidence that interaction of the V proteins with IRF7 is important for the blockade. However, the detailed mechanisms still remain unclear. Here we reexamined the interaction of the human parainfluenza virus type 2 (HPIV2) V protein with signaling molecules involved in TLR7/9-dependent signaling. Immunoprecipitation experiments in HEK293T cells transfected with V protein and one of the signaling molecules revealed that the V protein interacted with not only IRF7 but also TRAF6, IKKα, and MyD88. Whereas overexpression of TRAF6 markedly enhanced the level of V protein associating with IRF7, IKKα, and MyD88 in HEK293T cells, the level of V protein associating with TRAF6 was little affected by overexpression of IRF7, IKKα, and MyD88. Moreover, knockdown or knockout of endogenous TRAF6 in HEK293T or mouse embryonic fibroblast cells resulted in dissociation of the V protein from IRF7, IKKα, and MyD88. These results demonstrate that binding of the V protein to IRF7, IKKα, and MyD88 is largely indirect and mediated by endogenous TRAF6. It was found that the V protein inhibited TRAF6-mediated lysine 63 (K63)-linked polyubiquitination of IRF7, which is prerequisite for IRF7 activation. Disruption of the tryptophan-rich motif of the V protein significantly affected its TRAF6-binding efficiency, which correlated well with the magnitude of inhibition of K63-linked polyubiquitination and the resultant activation of IRF7. Taken together, these results suggest that the HPIV2 V protein prevents TLR7/9-dependent interferon induction by inhibiting TRAF6-mediated K63-linked polyubiquitination of IRF7.     

离子交换树脂用于角蒿总生物碱的纯化研究

本文以角蒿总生物碱溶液为对象,以其主要有效成分角蒿酯碱（incarvillateine）为指标,研究了阳离子交换树脂纯化角蒿总生物碱的方法,包括树脂类型的选择、氨水浓度和乙醇浓度对洗脱效果的影响.试验结果表明强酸性阳离子交换树脂DOWEX 50WX2对角蒿生物碱成分的交换能力最强,且被树脂吸附的生物碱成分可用氨性乙醇溶液快速洗脱.验证试验结果表明,用含2.0 N氨水的不同浓度乙醇溶液（60%、70%和80%）进行洗脱,得到的总生物碱含量均在60%以上,且随着乙醇浓度的提高,总生物碱的含量也不断提高.     

Host range expansion by Rhopalomyia yomogicola (Diptera: Cecidomyiidae) from a native to an alien species of Artemisia (Asteraceae) in Japan

In 2001, subconical galls were found on the leaves of an alien Artemisia species (Asteraceae) in Ibusuki City, Kagoshima Prefecture, Japan. These galls were quite similar to those induced by Rhopalomyia yomogicola (Diptera: Cecidomyiidae) on Artemisia princeps, Artemisia montana, and Artemisia japonica in Japan. The morphological features of the pupal head and molecular sequencing data indicated that the gall midge from the alien Artemisia was identical to R. yomogicola. Usually, galling insects do not expand readily their host range to alien plants, but R. yomogicola is considered to have expanded its host range to the alien Artemisia by its multivoltine life history trait and oligophagous habit across two different botanical sections of the genus Artemisia. Adult abdominal tergites and sternites and immature stages of R. yomogicola are described for the first time and detailed biological information is presented.     

Deficiency of SP-B reveals protective role of SP-C during oxygen lung injury.

Although the surface properties of surfactant protein (SP)-B and SP-C are similar, the contributions that either protein may make to lung function have not been identified in vivo. Mutations in SP-B cause lethal respiratory failure at birth; however, SP-B null mice are deficient in both SP-B and SP-C. To identify potential contributions of SP-C to lung function in vivo, the following transgenic mice were generated and exposed to 95% O(2) for 3 days: (SP-B(+/+),SP-C(+/+)), (SP-B(+/+), SP-C(-/-)), (SP-B(+/-),SP-C(+/+)), (SP-B(+/-),SP-C(+/-)), and (SP-B(+/-),SP-C(-/-)). Hyperoxia altered pressure-volume curves in mice that were heterozygous for SP-B, and these values were further decreased in (SP-B(+/-),SP-C(-/-)) mice. Likewise, alveolar interleukin (IL)-6 and IL-1 beta were maximally increased by O(2) exposure of (SP-B(+/-),SP-C(-/-)) mice compared with the other genotypes. Lung hysteresivity was lower in the (SP-B(+/-),SP-C(-/-)) mice. Surfactant isolated from (SP-B(+/+),SP-C(-/-)) and (SP-B(+/-),SP-C(-/-)) mice failed to stabilize the surface tension of microbubbles, showing that SP-C plays a role in stabilization or recruitment of phospholipid films at low bubble radius. Genetically decreased levels of SP-B combined with superimposed O(2)-induced injury reveals the distinct contribution of SP-C to pulmonary function in vivo.