Tsutomu Nishihara

Biodegradation of didecyldimethylammonium chloride by Pseudomonas fluorescens TN4 isolated from activated sludge

Bacteria that degrade didecyldimethylammonium chloride (DDAC) were isolated from activated sludge from a municipal sewage treatment plant by enrichment culture with DDAC as a sole carbon source. One of the isolates, Pseudomonas fluorescens TN4, degraded DDAC to produce decyldimethylamine and subsequently, dimethylamine, as the intermediates. The TN4 strain also assimilated the other quaternary ammonium compounds (QACs), alkyltrimethyl‐ and alkylbenzyldimethyl‐ammonium salts, but not alkylpyridinium salts. TN4 was highly resistant to these QACs and degraded them by an n‐dealkylation process. These data mean that there are some QAC‐resistant and QAC‐degrading bacteria such as TN4 in the environment.

Biodegradation of bisphenol a and disappearance of its estrogenic activity by the green alga Chlorella fusca var. vacuolata

Bisphenol A (BPA) is known as an endocrine disruptor and often is found in landfill leachates. Removal of BPA by green alga, Chlorella fusca, was characterized, because we previously found that various phenols were well removed by this strain, including BPA. Chlorella fusca was able to remove almost all BPA in the concentration range from 10 to 80 microM for 168 h under continuous illumination at 18 W/m2. At the low light intensity of 2 W/m2, 82% of 40 microM BPA was removed, and only 27% was removed in the dark. Moreover, C. fusca could remove 90% of 40 microM BPA under the 8:16-h light:dark condition, which was almost as high as that under the continuous-light condition. The amount of BPA contained in the cells was less than the amount of BPA removed from the medium. Monohydroxybisphenol A was detected as an intermediate of BPA degradation. Moreover, estrogenic activity that originated from BPA in the culture medium also completely disappeared. Based on these results, BPA was finally degraded to compounds having nonestrogenic activity. Therefore, C. fusca can be considered a useful organism to remove BPA from landfill leachates.

Transcriptional regulatory elements in the 5′ upstream and first intron regions of the human smooth muscle (aortic type) α-actin-encoding gene

Abstract We have determined the nucleotide (nt) sequence of 5.5 kb including the 5′ flanking, first untranslated exon and first intron regions of the human smooth muscle (SM) (aortic type) α-actin-(SMαA)-encoding gene. The promoter region and a part of the first intron show remarkably high sequence conservation with equivalent regions of the chicken gene, and contain multiple transcriptional regulatory elements. From transient chloramphenicol acetyltransferase gene (cat) expression assays m SM cells, a DNA fragment from nt − 123 to + 49 containing two CArG boxes showed strong positive promoter activity, whereas a far upstream region from nt − 253 to − 124 showed a negative effect. The conserved region in the first intron also contains the CArG box and showed an enhancer activity. Therefore, the human SMαA gene is controlled under positive and negative mechanisms.

Inactivation of human placenta glutathione S-transferase by SHSS exchange reaction with biological disulfides

The oxidized glutathione inhibited the activity of glutathione S-transferase purified from human placenta just through competitive inhibition. On the other hand, cystine and cystamine inactivated the activity by pseudo first-order in low concentrations, accompanying the stoichiometric incorporation of the radioactivity of [14C]-cystine to the enzyme protein until a half mole per one subunit. This and the protective effect of glutathione analogues suggested that the SH/SS exchange reaction occurred between the disulfide and the SH group near the glutathione binding site of the enzyme to form a mixed disulfide.

Histone acetyltransferase MOZ acts as a co-activator of Nrf2-MafK and induces tumour marker gene expression during hepatocarcinogenesis.

HATs (histone acetyltransferases) contribute to the regulation of gene expression, and loss or dysregulation of these activities may link to tumorigenesis. Here, we demonstrate that expression levels of HATs, p300 and CBP [CREB (cAMP-response-element-binding protein)-binding protein] were decreased during chemical hepatocarcinogenesis, whereas expression of MOZ (monocytic leukaemia zinc-finger protein; MYST3)--a member of the MYST [MOZ, Ybf2/Sas3, Sas2 and TIP60 (Tat-interacting protein, 60 kDa)] acetyltransferase family--was induced. Although the MOZ gene frequently is rearranged in leukaemia, we were unable to detect MOZ rearrangement in livers with hyperplastic nodules. We examined the effect of MOZ on hepatocarcinogenic-specific gene expression. GSTP (glutathione S-transferase placental form) is a Phase II detoxification enzyme and a well-known tumour marker that is specifically elevated during hepatocarcinogenesis. GSTP gene activation is regulated mainly by the GPE1 (GSTP enhancer 1) enhancer element, which is recognized by the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2)-MafK heterodimer. We found that MOZ enhances GSTP promoter activity through GPE1 and acts as a co-activator of the Nrf2-MafK heterodimer. Further, exogenous MOZ induced GSTP expression in rat hepatoma H4IIE cells. These results suggest that during early hepatocarcinogenesis, aberrantly expressed MOZ may induce GSTP expression through the Nrf2-mediated pathway.

CTG Triplet Repeat in Mouse Growth Inhibitory Factor/Metallothionein III Gene Promoter Represses the Transcriptional Activity of the Heterologous Promoters

Growth inhibitory factor/metallothionein III (GIF/MT-III) is expressed specifically in brain, and neither mRNA nor protein is detected in other organs. This tissuespecific expression might be regulated by negative elements as well as positive elements, such as tissue-specific enhancers. To investigate the repression mechanisms of this gene in organs other than the brain, transfection experiments were performed by using various deletion mutants. Interestingly, a 25 CTG repeat in the promoter region seemed to contribute to the repression activity. Moreover, the repression activity of this 25 CTG repeat was also observed in various promoters and in a direction and position independent manner, indicating that this element could act as a silencer. However, no binding protein was detected by gel-shift and footprint analyses. These results strongly suggest that the CTG repeat functions as a negative element, and that this effect is caused by unknown mechanisms, rather than by interactions between specific cis-elements and specific trans-acting factors as reported previously. It is also possible that the CTG repeat functions as a general silencer in many genes.

Isolation of up- or down-regulated genes in PPARγ-expressing NIH-3T3 cells during differentiation into adipocytes

Adipocyte differentiation is a complex process in which the expression of many transcription factors and adipocyte‐specific genes is regulated under a strict program. The peroxisome proliferator‐activated receptor γ (PPARγ), a member of the steroid/thyroid nuclear hormone receptor superfamily of ligand‐activated transcription factors, is an important regulator of adipocyte gene expression and differentiation. In this study, we tried to identify downstream target genes of PPARγ, by using PPARγ‐expressing cells and a subtractive cloning strategy, and isolated cDNA clones which were up‐regulated or down‐regulated by PPARγ. Northern blot analyses revealed that the expression levels of the aldehyde dehydrogenase‐2‐like, type VI collagen alpha 3 subunit, cellular retinoic acid binding protein 1 and thrombospondin 1 are changed during the differentiation of mouse 3T3‐L1 preadipocyte cells, indicating that these genes might be downstream targets of PPARγ in adipocyte differentiation.

Induction of Bach1 and ARA70 gene expression at an early stage of adipocyte differentiation of mouse 3T3-L1 cells

Using a subtraction method, we have isolated genes that are induced early in the differentiation of mouse 3T3-L1 preadipocyte cells into adipocytes. These include the genes encoding transcription factors and signalling proteins, as well as unknown genes. Bach1, a transcription factor, and ARA70, a cofactor, were rapidly induced during differentiation. The induction of these two genes was observed only in growth-arrested 3T3-L1 cells, and not in proliferating cells. In NIH-3T3 cells, no induction was observed under either set of conditions. These results strongly indicate that Bach1 and ARA70 have valuable roles at the onset of adipocyte differentiation.

Identification of DNA binding-site preferences for nuclear factor I-A.

Nuclear factor I (NFI) proteins constitute a large family of DNA binding proteins. These proteins promote the initiation of adenovirus replication and regulate the transcription of viral and cellular genes. The binding sites for NFI have been reported in a wide variety of promoters, and they exhibit flexibility in their sequences. To clarify the DNA binding site of NFI‐A, one of the NFI proteins, we performed a polymerase chain reaction‐mediated random site selection, and determined the optimal sequence as 5′‐TTGGCANNNN(G/T)CCA(G/A)‐3′.

Two nuclear localization signals are required for nuclear translocation of nuclear factor 1-A.

Nuclear factor 1 (NF1) proteins are encoded by at least four genes (NF1‐A, B, C, X). Although DNA‐binding and the transcription regulation domains of these proteins are well characterized, the nuclear localization signals (NLSs) are still unknown in all NF1s. We have identified two NLSs in NF1‐A, and both are required for full translocation to the nucleus, although one of them itself has a partial translocation ability. These two NLSs are conserved in all four NF1s. Interestingly, three isoforms of NF1‐A (NF1‐A1, A2, A4) have two NLSs and translocate completely to the nucleus. In contrast, NF1‐A3 lacks the second NLS and partially stays in the cytoplasm. Since NF1s construct homodimer and heterodimer, these findings indicate the differential regulations of the NF1 translocation.