Role of multifunctional transcription factor TFII-I and putative tumour suppressor DBC1 in cell cycle and DNA double strand damage repair.

BACKGROUND: In multicellular organisms, precise control of cell cycle and the maintenance of genomic stability are crucial to prevent chromosomal alterations. The accurate function of the DNA damage pathway is maintained by DNA repair mechanisms including homologous recombination (HR). Herein, we show that both TFII-I and DBC1 mediate cellular mechanisms of cell-cycle regulation and DNA double strand damage repair. METHODS: Regulation of cell cycle by TFII-I and DBC1 was investigated using Trypan blue dye exclusion test, luciferase assay, and flow cytometry analysis. We also analysed the role of TFII-I and DBC1 in DNA double strand damage repair after irradiation by immunofluorescence study, clonogenicity assay, and HR assay. RESULTS: Flow cytometry analysis revealed a novel function that siRNA-mediated knockdown of endogenous DBC1 resulted in G2/M phase arrest. We also have shown that both endogenous TFII-I and DBC1 activate DNA repair mechanisms after irradiation because irradiation-induced foci formation of TFII-I-γH2AX was observed, and the depletion of endogenous TFII-I or DBC1 resulted in the inhibition of normal HR efficiency. CONCLUSION: These results reveal novel mechanisms by which TFII-I and DBC1 can modulate cellular fate by affecting cell-cycle control as well as HR pathway.

Characterization of an omega-class glutathione S-transferase in the stress response of the silkmoth.

The glutathione S-transferase (GST) superfamily is involved in detoxification of various xenobiotics. Using real-time PCR, mRNA encoding an omega-class GST of Bombyx mori (bmGSTO) was shown to be induced after exposure to various environmental stresses. A soluble form of recombinant protein (rbmGSTO) was functionally overexpressed in Escherichia coli cells and purified to homogeneity. Cys 38 and Pro 39 were found to be highly conserved in omega-class GSTs, and their roles were investigated by site-directed mutagenesis/kinetic analysis. Mutations of Cys 38 and Pro 39 residues affected the catalytic efficiency of enzymes, indicating that the presence of Cys 38 and Pro 39 residues is important for bmGSTO activity. Thus, bmGSTO could contribute to increasing the environmental stress resistance of lepidopteran insects.

TAK1 represses transcription of the human telomerase reverse transcriptase gene.

In human cells, telomerase activity is tightly regulated by the expression of its catalytic subunit, namely, the human telomerase reverse transcriptase (hTERT). However, the molecular mechanisms involved in the regulation of hTERT expression have not been completely clarified. We have previously reported that transforming growth factor beta (TGF-beta) represses the expression of the hTERT gene. In the present study, we demonstrated that TGF-beta-activated kinase 1 (TAK1), originally identified as a mitogen-activated kinase kinase kinase, represses the hTERT core promoter activity in an E-box-independent manner, and it also represses the transcription of the hTERT gene in human lung adenocarcinoma cell line, A549 cells. This TAK1-induced repression was found to be caused by the recruitment of histone deacetylase to Sp1 at the hTERT promoter and a consequent reduction in the amount of acetylated histone H4 at the hTERT promoter. Finally, we demonstrated that TAK1 induces cellular senescence programs in normal human diploid cells. Thus, we assume that TAK1 triggers the repression mechanisms of the hTERT gene as a result of evoking cellular senescence programs. Considered together, TAK1 is thought to play a causative role in the determination of a finite replicative lifespan of normal and cancer cells.

Comparative analysis of transcriptional responses to saline stress in the laboratory and brewing strains of Saccharomyces cerevisiae with DNA microarray.

To construct yeast strains showing tolerance to high salt concentration stress, we analyzed the transcriptional response to high NaCl concentration stress in the yeast Saccharomyces cerevisiae using DNA microarray and compared between two yeast strains, a laboratory strain and a brewing one, which is known as a stress-tolerant strain. Gene expression dynamically changed following the addition of NaCl in both yeast strains, but the degree of change in the gene expression level in the laboratory strain was larger than that in the brewing strain. The response of gene expression to the low NaCl concentration stress was faster than that to the high NaCl concentration stress in both strains. Expressions of the genes encoding enzymes involved in carbohydrate metabolism and energy production in both strains or amino acid metabolism in the brewing strain were increased under high NaCl concentration conditions. Moreover, the genes encoding sodium ion efflux pump and copper metallothionein proteins were more highly expressed in the brewing strain than in the laboratory strain. According to the results of transcriptome analysis, candidate genes for the creation of stress-tolerant strain were selected, and the effect of overexpression of candidate genes on the tolerance to high NaCl concentration stress was evaluated. Overexpression of the GPD1 gene encoding glycerol-3-phosphate dehydrogenase, ENA1 encoding sodium ion efflux protein, and CUP1 encoding copper metallothionein conferred high salt stress tolerance to yeast cells, and our selection of candidate genes for the creation of stress-tolerant yeast strains based on the transcriptome data was validated.

Symptoms in relation to chemicals and dampness in newly built dwellings.

OBJECTIVES: As the airtightness of dwellings has recently increased, problems associated with indoor air pollution and dampness have become important environmental health issues. The aim of this study was to clarify whether symptoms in residents living in newly built dwellings were related to chemicals and dampness. METHODS: Symptoms of 317 residents were surveyed by standardized questionnaires, and the concentrations of formaldehyde, acetaldehyde, and 17 volatile organic compounds (VOCs) in their homes were measured. Dampness (condensation on window panes and/or walls, and mold growth) was identified by questionnaires given to the householders or their partners. RESULTS: Some VOCs (toluene, butyl acetate, ethylbenzene, alpha-pinene, p-dichlorobenzene, nonanal, and xylene) were significantly related to the symptoms, and the sum of all VOCs (all identified VOCs) was significantly related to throat and respiratory symptoms [odds ratio (OR) for eye symptoms =2.4; 95% confidence interval (CI) 1.0-5.5], although the concentrations of VOCs were relatively low. As for the dampness index, condensation on window panes and/or walls was related to all symptoms, and mold growth was related to all symptoms except skin, throat and respiratory and general symptoms. As the number of dampness signs increased, the ORs increased for the symptoms except general symptoms (OR for nose symptoms = 4.4, 95% CI 1.6-11.9). CONCLUSION: Both VOCs and dampness were significantly related to symptoms. We should take measures to reduce the concentrations of VOCs, dampness and microbial growth in dwellings.

Relation between colour vision loss and occupational styrene exposure level.

AIMS: To investigate the relation between colour vision loss and the exposure level of styrene. Exposure level included the current exposure concentration, past cumulative exposure, and the maximum exposure level in the past. METHODS: Colour vision was examined by the Lanthony desaturated panel D-15 test for 76 subjects exposed to styrene in a fibreglass reinforced plastics boat plant (as an exposed group) and 102 non-exposed subjects (as a control group). The current exposure level was expressed by the concentration of atmospheric styrene and end shift urinary mandelic acid (MA) and phenylglyoxylic acid (PGA) levels. The individual cumulative exposure index (CEI) was calculated, based on the exposure frequency and urinary MA concentrations measured for the past eight years. RESULTS: The Colour Confusion Index (CCI) of the exposed group showed a significant difference from the age matched controls. However, only a slight significant relation was found between CCI and the concentration of urinary MA plus PGA. In this study, the exposed group was further divided into two subgroups (as sub-MA+PGA groups) by the median of urinary MA plus PGA of each subject. The dividing line between the subgroups was 0.24 g/g creatinine, which was equivalent to an atmospheric concentration of styrene of about 10 ppm. The CCI values of both the sub-MA+PGA groups were significantly higher than that of the control group. The relation between CCI value and the maximum exposure concentration in the past eight years was examined. It was found that the CCI values of the group with the maximum exposure concentration of styrene over 50 ppm were significantly higher than that of the other groups. CONCLUSIONS: Exposure to styrene would impair colour vision even if the exposure concentration was lower than 10 ppm. Furthermore, if the maximum concentration of styrene exposure transiently exceeded 50 ppm in the past, the styrene related damage might remain. Thus, the safe limit of exposure to styrene and the relation between exposure to styrene and the degree of damage to ocular structure, retina, optic nerve, and brain need to be re-examined.

The progression of aging in klotho mutant mice can be modified by dietary phosphorus and zinc.

Reduction in klotho gene expression causes accelerated senescence in klotho mutant mice. We have now found two key substances, phosphorus and zinc, which affect the appearance of klotho phenotypes. Klotho mutant homozygotes fed nonpurified diet with a phosphorus concentration of 1.03 g/100 g showed typical klotho phenotypes. However, most of the klotho phenotypes no longer appeared in male homozygotes fed a 0.4 g/100 g phosphorus diet. These homozygotes were capable of spermatogenesis. In the kidneys of the rescued male homozygotes, klotho protein expression was clearly detected. On the other hand, female klotho mice required supplementation of 0.25 g/100 g zinc orotate to the 0.4 g/100 g phosphorus diet to be rescued. Unlike in the rescued male mice, klotho protein levels in the kidneys of the rescued females were quite low. Wild-type (C3H/He) mice fed 1.5 or 1.0 g/100 g phosphorus diets had lower klotho protein expression in the kidneys than those fed a 0.4 g/100 g phosphorus diet (Kruskal-Wallis test, P < 0.05). These results indicate that dietary phosphorus and zinc modulate the phenotypes of klotho mice, and that klotho expression in the kidneys is regulated not only in klotho mutant mice, but also in wild-type mice.

Flow cytometry: an improved method for the selection of highly productive gene-amplified CHO cells using flow cytometry.

In previous work, we clarified the relationship between the productivity and stability of gene-amplified cells and the location of the amplified gene. The location of the amplified gene enabled us to classify resistant cells into two types. One type of resistant cell group, in which the amplified genes were observed near the telomeric region, was named the "telomere type." The other type of cell group, in which the amplified genes were observed in other chromosomal regions, was named the "other type." The phenotypes of these two types of cells are very different. In this experiment, using a fluorescein isothiocyanate-labeled methotrexate (F-MTX) reagent with flow cytometry, we were easily able to distinguish between highly productive cells and the other types of cells. The level of fluorescence differed according to the difference in resistance to MTX. Based on this new finding, highly productive gene-amplified cells could be isolated from heterogeneous gene-amplified cell pools more easily than by the method of limiting-dilution assay. The limiting-dilution method requires several months to obtain highly productive gene-amplified cells, while our flow-cytometry-based method of selection requires only a few weeks.

Meningeal carcinomatosis in patients with breast cancer: report of 8 patients.

BACKGROUND: Meningeal carcinomatosis (MC) is an uncommon but aggressive complication of advanced breast cancer with a recently increasing incidence. Although the prognosis is extremely poor for MC patients, early diagnosis and appropriate treatment are important. SUBJECTS AND METHODS: We reviewed 8 cases of MC from breast cancer at Kyoto University Hospital from 1990 to 1999. The median age was 51.5 years. All patients had widespread systemic metastases when diagnosed with MC. clinical symptoms were categorized into 3 groups: cranial nerve symptoms, spinal nerve symptoms, and other symptoms. Imaging studies were positive for MC in only 4 patients. Initial CSF cytology studies were positive in 4 patients, and repeated CSF cytology yielded positive results in the remaining 4 patients. Thus the median interval between the onset of any clinical symptom of MC and the initiation of treatment was 22.5 days (range 7 to 120 days ). All patients received whole brain radiotherapy (WBRT). Four patients were given intrathecal chemotherapy and/or intrathecal immunotherapy in addition to WBRT. RESULTS: Improvement of cranial nerve symptoms, spinal nerve symptoms, and other symptoms were observed in 3/5, 1/3, and 5/7 patients, respectively. Patients with cranial nerve symptoms who started WBRT within 29 days of the onset of the symptoms showed at least partial recovery whereas patients who started WBRT later showed no recovery. The median survival was 123 days (53 to 310 days). MC was the direct cause of death in 1 of 8 patients. CONCLUSION: When MC is clinically suspected, neither a negative imaging study nor a single negative CSF cytology can rule out MC. Prompt initiation of WBRT with or without intrathecal chemotherapy may be important for recovery from cranial nerve symptoms.

Effects of low-level occupational exposure to styrene on color vision: dose relation with a urinary metabolite.

To investigate the threshold effects of chronic low-level occupational exposure to styrene on color vision, we examined color discrimination in 105 male workers exposed to styrene (mean age 37.7 years; mean length of exposure 6.2 years; mean urinary concentration of mandelic acid 0.21 g/L) and in 117 referents (mean age 37.7 years). We also assessed the effects of styrene by examination of the nature of the relation between disorders of nervous function and age, alcohol consumption, and other variables. A standardized questionnaire was adopted to collect information about work history, occupational or nonoccupational solvent exposure, alcohol consumption, and drug use. Color vision was evaluated by the Lanthony desaturated panel D-15 test. The results of the test were expressed as the color confusion index (CCI). There was a dose-dependent relationship between the urinary concentration of mandelic acid and color vision loss. The CCIs of the subgroups whose urinary mandelic acid levels were 0.1-0.2 and >0.2 g/L were significantly higher than those of each referent group (P<0.05 and P<0.01, respectively), but not in the subgroup whose urinary mandelic acid level was lower than 0.1 g/L. Our study suggests that a low level of styrene, presumably 0.1-0.2 g/L, involves the risk of inducing adverse effects on color vision. After confounding factors were adjusted for, the urinary mandelic acid level had a significant positive relationship with color vision.

Effects of prenatal styrene exposure on postnatal development and brain serotonin and catecholamine levels in rats.

Maternal reproductive effects in Wistar rats exposed to 0, 50, or 300 ppm styrene for 6 h/day during gestational days 6 to 20 were evaluated. Their offspring were observed postnatally for neurochemical changes, growth, and physical landmarks of development. Mothers exposed to styrene were compared with pair-fed and ad-lib-fed controls in order to adjust nutrient conditions. Prolongation of the gestational period, food intake, and the number of neonatal deaths or stillbirths in 300-ppm-exposed dams showed evidence of styrene-related effects. Other reproductive parameters, such as litter size, birth weight, and sex ratio, were found to exhibit no effects within the variation range studied. A neurochemical effect was observed in that the 5-HT and HVA concentrations in cerebrum were significantly decreased. Incisor eruption (mandible), eye opening, and the air-righting reflex were delayed in rat pups born to dams receiving 300 ppm styrene exposure compared with the pair-fed and ad lib control groups. Pups born to dams exposed to 50 ppm styrene also had a significantly delayed air-righting reflex compared with ad lib controls. These results suggest that the offspring were susceptible to the effects of styrene on a few developmental landmarks even when nutritional effects were controlled.

Constrained optimization of L-lysine production based on metabolic flux using a mathematical programming method.

Constrained optimization for microbial fermentation was studied. For optimization, we used not the maximum principle but a nonlinear programming method because of the need to consider many metabolic reactions. In the case of L-lysine fermentation, the optimization problem in L-lysine production was formulated as a nonlinear programming problem. In general, the state equations based on material balances are represented as differential equations, but such equations which are dependent on time can not be applied to a nonlinear programming problem. Therefore, the state equations were made discrete in a time base, and a new single vector which is not dependent on time was substituted. From these formulae, the objective function and the constraints using nonlinear programming problem were defined as the amount of L-lysine produced, and as a metabolic reaction model and empirical equations, respectively. Computer program was developed to solve this constrained nonlinear programming problem. The applied algorithm of the computer programming was a sequential quadratic programming method (SQP method). When the constrained nonlinear programming problem is solved using the SQP method, the maximum amount of L-lysine produced and the optimal feeding rate of L-threonine could be calculated. From the calculated results, it was clear that introduction of the equality and inequality constraints was easy. L-Lysine at a concentration up to 75.3 g/l could be produced when the fermentation was carried out under optimal conditions.

A kinetic model for a biopanning process considering antigen desorption and effective antigen concentration on a solid phase.

A phage display library is a powerful tool for screening ligands such as antibodies and peptides that specifically recognize a target. In this study, we established a kinetic model describing the affinity selection process of phage display libraries and verified the model experimentally. Desorption of target molecules from a solid phase and orientation of the epitopes of adsorbed target molecules are taken into account in this model. The ratio of the effective antigen density to the total antigen density was estimated to be 0.0127(+/-)0.0018 when bovine pancreatic ribonuclease A (RNase A) adsorbed on polystyrene beads was recognized by an anti-RNase A single-chain Fv phage antibody. The model can faithfully describe the recovery of the phage antibody in a round of biopanning based on the effective concentration of RNase A on the beads, the desorption rate constant of RNase A from the beads, the dissociation constant and dissociation rate constant of the phage antibody from RNase A, and the time for blocking, equilibrium and washing in the biopanning process. A recommended biopanning protocol based on the model is also described.

Measurement of association rate constant of antibody-antigen interaction in solution based on enzyme-linked immunosorbent assay.

A simple and rapid method based on enzyme-linked immunosorbent assay (ELISA) was developed for measuring the association rate constant of antibody-antigen interactions. An antibody and its antigen are mixed in a solution to initiate the equilibrium reaction. At different time intervals, the amount of the free antibody in the reaction mixture is estimated by an indirect ELISA. The association rate constant was estimated by nonlinear regression against an equation introduced from the derivation of the mass balance of antigen-antibody interaction. This method can determine the association rate constant of antibodies with a dissociation rate constant up to 5 x 10(-3) s(-1). The association rate constant of a single-chain Fv (scFv) to its antigen, bovine pancreatic ribonuclease A (RNase A), determined by the present method agreed well with those determined by the fluorescence polarization method and surface plasmon resonance method. No significant difference in the association rate constant was found between the soluble anti-RNase A scFv and the same scFv displayed on a phage (5.65 +/- 0.54 x 10(4) M(-1) s(-1) and 5.96 +/- 0.56 x 10(4) M(-1) s(-1), respectively).

Availability of oncogene activated production system for mass production of light chain of human antibody in CHO cells.

We previously established a ras-oncogene amplified Chinesehamster ovary (CHO) cell line, named ras clone I, as anuniversal host cell line for oncogene activated production(OAP) system to mass-produce recombinant protein by activationof the cytomegalovirus immediate early (CMV) promoter with ras protein. The lambda light chain(C5lambda) of human monoclonal antibody HB4C5 is expected tobe potentially useful for lung cancer targeting. We generated aC5lambda hyper-producing cell line by transfecting ras cloneI with the C5lambda gene expression plasmid regulated by theCMV promoter, of which productivity was 5.3 times greater thanthe hyper productive CHO cell line generated by using conventional CHO cells. Introduction of the adenovirus E1A geneinto the hyper-producing cell line derived from ras clone I resulted in further 9.5 times enhancement of the productivity,suggesting the synergistic effect of E1A and ras oncogenes on the recombinant protein production driven by the CMV promoter. In addition, intracellular accumulation of C5lambda andupregulation of BiP was found in hyper-producing cell lineswhich were introduced E1A and ras oncogene. This resultsuggests that excessive intracellular accumulation ofC5lambda protein, which might be caused by that the amount of produced C5lambda in ER is beyond the ability of CHO cells to secrete, might signal the BiP promoter. Our data imply that ras clone I is available as a general host cell for establishing the recombinant protein hyper-producing CHOcells by the OAP system, and suggest that further mass production of recombinant proteins in the OAP system can be possible by clarifying the accurate role of upregulated BiP protein.

Subtractive screening of genes involved in cellular senescence.

We attempted to identify the genes involved in cellularsenescence, telomere maintenance and telomerase regulationthrough subtractive screening of cDNA libraries prepared froma human lung adenocarcinoma cell line A549 and its sublinesnamed A5DC7, CK and AST-9. Cell phenotypes of A5DC7, CK andAST-9 are normal cell-like, cancer cell-like and intermediate,respectively. These cell lines have different phenotypes interms of telomerase activity and telomere maintenance, andthus are thought to be useful for identifying the genesinvolved in cellular senescence and telomerase regulation. In this study, we identified 86 independent cDNA clones bysubtractive screening. Among these cDNA clones, subtractingA5DC7 cDNAs from A549 cDNAs and CK cDNAs gave 7 and 3 cDNAclones which highly and specifically expressed in tester celllines. Genes corresponding to these 10 cDNA clones mightparticipate in maintaining cancer-cell phenotypes. As aresult of database searching, each four of A549 specific cDNAclones are found to correspond to known cDNAs. Each two ofA549 specific and two of CK specific cDNA clones have highhomology to independent ESTs. Sequences having homology toeach one of A549 specific and one of CK specific cDNA cloneshave not been deposited in the Genbank database, indicatingthat these two cDNA clones are part of novel genes. Weanticipate that their involvement in telomerase regulationand/or senescence program can be clarified by functionalanalysis using each full-length cDNA.

Molecular analysis of cross-reactive human monoclonal antibody AE6F4 generated by in vitro immunization: Epitope mapping of AE6F4 antibody on 14-3-3 family proteins and cytokeratin 8.

We reported previously that adenocarcinoma-reactive human monoclonal antibody AE6F4, which had been generated by in vitro immunization method, recognizes both 14-3-3protein and cytokeratin 8 (CK8). In this study, to analyze the cross-reactivity of AE6F4 antibody, epitopes of AE6F4 antibody on 14-3-3 proteins and CK8 were studied by using synthetic linear peptide scanning technology. To determine the locations of B cell epitope, 48 and 95 of decapeptides covering the entire 14-3-3 proteins and CK8, respectively,were synthesized and binding to AE6F4 antibody was examined by ELISA. The AE6F4 antibody was strongly reactive to peptides containing amino acid sequences TLWTSDTQGD in 14-3-3 proteins and INFLRQLYEE in CK8. These results indicate that AE6F4 antibody can recognize the different peptide sequences in 14-3-3 proteins and CK8.

Epitope analysis of human monoclonal antibody specific for rice allergenic protein generated by in vitro immunization.

We previously established an in vitro immunization protocol for generating antigen specific human monoclonal antibodies (mAbs). In vitro immunization was performed against the soluble protein of rice allergenic protein (RA), resulting in the generation of three B cell clones, AC7-1/F9, CB7-1/E2 and CB7-8/F5, all of which produce a RA-specific human monoclonal IgM antibody. We attempted to map the epitope regions recognized by thesem Abs to characterize their specificities. We performed two rounds of epitope mapping, rough mapping using 10-mer peptides covering the full-length RA with 5 amino acids overlapping, and fine mapping using 8-mer peptides covering the putative epitope regions from the rough mapping with 1amino acid overlapping. As a result of the fine mapping,we identified the epitope regions of these three mAbs as(45)QVWQDCCRQ(54)L, (56)AVDDGWCRCGA(67)L and(91)FPGCRRG(98)D on the RA molecule and found to be identical. Furthermore, we determined the putative core epitope regions, which are critical for mAb binding to each region, (47)WQDCC(52)R and (60)GWC(63)R. The information about the epitope region on the RA molecule,which might trigger the allergenic response, would be useful to establish a specific immunotherapy against rice allergy.

Neural stem cells lose telomerase activity upon differentiating into astrocytes.

Serum-free mouse embryo (SFME) cells were established by D. Barnes et al., and are known to be a neural stem cell line, which differentiate into astrocytes upon treatment with TGF-beta. Therefore, SFME cells is thought to be a model well suited to analyze the differentiation mechanism of neural stem cells. Until now, we have investigated the regulation mechanisms of telomerase activity and telomere length in human cancer and normal cells. Telomerase is the enzyme responsible for the synthesis and maintenance of telomere repeats located at chromosomal ends and is normally expressed in embryonic and germline cells, but not in most normal cells. Here, using SFME cells, we attempted to analyze the regulation mechanism of telomerase activity in neural stem cells and to detect a change upon differentiation into astrocytes. When SFME cells were cultured in the presence of TGF-beta, cells showed anelongated morphology and decreased its growth to 50% of control culture. Cells also expressed the glial fibrillary acidic protein (GFAP), a marker for astrocytes,indicating that TGF-beta induced differentiation in SFME cells from neural stem cells into astrocytes. At the same time,TGF-beta also inhibited telomerase activity and repressed the expression of the mouse telomerase reverse transcriptase(mTERT), demonstrating that SFME cells was vested with a finite replicative life span upon treatment with TGF-beta. To understand the mechanisms regulating mTERT levels during differentiation into astrocytes, we have estimated the expression level of c-myc, which is known to be a key molecule in activating the TERT promoter. As a result, TGF-beta-treated SFME cells were shown to repress the expression of c-myc. Furthermore, promoter analysis, using the 5'-region of the mTERT gene, which possess two E-box elements bound to c-Myc/Max, demonstrated that mTERT promoter activity greatly decreased in TGF-beta-treated SFME cells as compared to non-treated SFME cells. These suggest that c-myc might play a critical role in the expression of mTERT, and that down-regulation of c-myc dependent upon the astrocytic differentiation in SFME cells might cause the repression of mTERT in TGF-beta-treated SFME cells.