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.

Tryptophan-19 of beta-lactoglobulin, the only residue completely conserved in the lipocalin superfamily, is not essential for binding retinol, but relevant to stabilizing bound retinol and maintaining its structure.

Residue 19 of tryptophan in bovine beta-lactoglobulin (beta-LG) is the only invariant residue throughout the lipocalin superfamily having two characteristic features: binding ability for small hydrophobic molecules and the unique beta-barrel three-dimensional structure. In this study, we investigated whether this strictly conserved Trp-19 of beta-LG would be indispensable for its structure and function such as maintaining the molecular structure and biological activity of beta-LG. Spectroscopic and enzymatic oxidation experiments on retinol bound to W19Y, in which Tyr was substituted for Trp-19, showed that Trp-19 was not critical for this binding, but was important for stably maintaining the environment surrounding retinol and the bound retinol. An using four anti-beta-LG monoclonal antibodies as probes, revealed a structural change in region 20-29, but not in the reverse region of Trp-19. A guanidine hydrochloride-induced unfolding study showed that the conformational stability of W19Y was greatly reduced by 6.9 kcal/mol compared to that of wild-type beta-LG. These facts indicated that Trp-19 is one of the important residues in correctly maintaining the local structure of beta-LG and stably retaining its overall structure, thereby conserving the bound retinol molecule.

Accelerated secretion of mutant beta-lactoglobulin in Saccharomyces cerevisiae resulting from a single amino acid substitution.

Transformed yeasts producing a mutant form of bovine beta-lactoglobulin (beta-LG), W19Y, in which Trp(19) was replaced with Tyr, were shown to secrete 6 times more than those producing wild type beta-LG. Northern blot analysis suggested that the enhanced level of secretion was not the result of upregulated transcription of W19Y. The ratio of the amount of W19Y secreted into the supernatant to the amount of W19Y remaining inside the cells was much larger than that in the case of wild type beta-LG as shown by immunoblot analysis. A pulse/chase experiment revealed that the speed of secretion of W19Y was significantly accelerated, compared to wild type beta-LG. These results indicated that W19Y was more efficiently and rapidly transported in the course of secretion than wild type beta-LG. Our previous study showed that the DeltaG of unfolding of W19Y in water is 6.9 kcal/mol smaller than that of wild type beta-LG. Furthermore, immunoblot analysis of intracellular beta-LG under non-reducing conditions indicated that W19Y as well as wild type beta-LG maintained a specific folded structure inside the yeast cells, whereas other non-secretable mutant beta-LGs with Phe or Ala at position 19 (W19F and W19A, respectively) did not. These data suggest that low molecular stability and the maintenance of a specific folded structure inside the yeast cells are prerequisites for efficient and rapid secretion. W19Y was more efficiently secreted than wild type beta-LG also in transformed ern1 mutant yeast cells expressing only a basal level of BiP which is considered to function in quality control in the endoplasmic reticulum (ER) by playing an important role in determining the secretion efficiency of secretory proteins. Thus, the reason for the enhanced secretion of W19Y is considered to be that the improved folding ability of W19Y can allow the half-life of the W19Y-BiP complex to become shorter than that of the wild type beta-LG-BiP complex, leading to faster translocation of W19Y into transport vesicles, or that W19Y can fold in a BiP-independent manner in the ER of the yeast cells. Our findings demonstrate that the amount of protein secreted can be improved by alteration of a single amino acid residue crucial for its structure.

Fluorescence polarization study of a salt bridge between a single chain Fv and its antigen ribonuclease A.

The interaction between a single chain Fv (sFv) of the monoclonal antibody 3A21 and its antigen, bovine pancreatic ribonuclease A (RNase A), was studied by site-directed mutagenesis of the hypervariable regions and fluorescence polarization analysis. The affinity constants of wild-type sFv and a mutant sFv D31A (Asp31 of heavy chain was replaced by Ala) for RNase A were found to be 2.7 x 10(7) and 4.7 x 10(6) M(-1) in PBS at pH 7.2 and 37 degrees C, respectively. Whereas the affinity constant of D31A is not affected by NaCl concentration, that of wild-type sFv is almost the same as that of D31A in the presence of more than 1 M NaCl. These results demonstrate that Asp31 of the heavy chain interacts electrostatically with a positively charged amino acid residue of RNase A.

Fluorescence polarization study of a salt bridge between a single-chain Fv and its antigen ribonuclease A.

The interaction between a single-chain Fv (sFv) of the monoclonal antibody 3A21 and its antigen, bovine pancreatic ribonuclease A (RNase A), was studied by site-directed mutagenesis of the hypervariable regions and fluorescence polarization analysis. The affinity constants of wild-type sFv and a mutant sFv D31A (Asp31 of heavy chain was replaced by Ala) for RNase A were found to be 2.7 x 10(7) and 4.7 x 10(6) M-1 in PBS at pH 7.2 and 37 degrees C, respectively. While the affinity constant of D31A is not affected by NaCl concentration, that of wild-type sFv is almost the same as that of D31A in the presence of more than 1 M NaCl. These results demonstrate that Asp31 of the heavy chain interacts electrostatically with a positively charged amino acid residue of RNase A.

Purification and characterization of yeast protein disulfide isomerase.

Protein disulfide-isomerase (PDI), which reactivates inactive scrambled RNase, was purified from Saccharomyces cerevisiae. The enzyme was purified 1,850-fold to apparent homogeneity by five purification steps: 30-70% ammonium sulfate fractionation, DEAE Toyopearl-650S and Butyl Toyopearl-650S chromatographies, and differential Phenyl-5PW HPLC with or without cysteine. The native enzyme had an apparent Mr of 140,000 on gel filtration chromatography, and its NH2-terminal was blocked. The Mr of its subunits were estimated to be 70,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the enzyme is probably composed of two identical subunits. The Mr of the subunits changed to 60,000 on endoglucosaminidase H treatment, indicating that the enzyme is transported into the endoplasmic reticulum. The enzyme has a pH optimum of 8.5, and pI of 4.02. Its enzymic properties were compared with those of purified bovine liver PDI. The Km values of yeast and bovine PDIs for scrambled RNase were 1 x 10(-5) and 2 x 10(-5) M, and their Vmax values were 6 and 7 units/mg protein, respectively. The two enzymes showed no significant differences in Km or Vmax values with respect to thiol compounds. Bacitracin inhibited both PDIs in the same fashion. These results indicate that this yeast PDI corresponds to mammalian PDI.

Molecular structure of a yeast gene, PDI1, encoding protein disulfide isomerase that is essential for cell growth.

A genomic DNA clone for protein disulfide isomerase (PDI) of Saccharomyces cerevisiae was isolated by hybridization with synthesized oligonucleotide probes based on a partial amino acid sequence of yeast PDI. The introduction of a multiple copy plasmid carrying this fragment into yeast caused a tenfold increase in PDI specific activity and in the amount of PDI antigen in the extract. The gene on this fragment was named PDI1. The nucleotide sequence of the gene predicts a polypeptide of 522 amino acids with about 30% identity to mammalian PDIs. The predicted amino acid sequence contains an N-terminal signal peptide-like sequence, the C-terminal putative endoplasmic reticulum retention signal of yeast (HDEL), and two putative active site sequences of PDI (WCGHCK). The predicted polypeptide is acidic and contains five putative glycosylation sites, consistent with the molecular properties of the purified yeast PDI [T. Mizunaga et al. (1990) J. Biochem. 108, 846-851]. The PDI1 gene was mapped on chromosome III. A gene disruption experiment revealed that the PDI1 gene is essential for cell growth.

Structure and function of the recombinant fifth domain of human beta 2-glycoprotein I: effects of specific cleavage between Lys77 and Thr78.

In order to elucidate the mechanism of binding of beta 2-glycoprotein I (beta 2-GPI) to cardiolipin (CL), we constructed a high-level expression system for the C-terminal domain (Domain V) of beta 2-GPI using Pichia pastoris and studied its conformation and liposome-binding activity. Purified Domain V was found to have the native disulfide bonds. It had a compactly folded conformation, judging from the circular dichroism spectrum, and exhibited a cooperative unfolding transition induced by pH or urea. Also, it bound liposomes containing CL. Commercially available human beta 2-GPI is known to be selectively cleaved between Lys 317 and Thr 318. We found that bovine factor Xa weakly but specifically cleaves the corresponding site of recombinant Domain V, i.e., the peptide bond between Lys 77 and Thr 78. The conformation of the "nicked" Domain V, which was cleaved at this site, was examined by circular dichroism and fluorescence measurements, and concluded to be similar to that of the intact protein. The stability of the nicked Domain V to urea was slightly lower than that of the intact protein. Although both Domains V bound to liposomes containing CL, the affinity of the nicked Domain V was greatly reduced in comparison with the intact protein, indicating that the cleavage of the peptide bond between Lys 77 and Thr 78 controls the binding to CL. In addition, analysis of the fluorescence spectra in the presence and absence of CL liposomes indicated that Trp 76 is involved in the binding site. These results suggest that the region including Trp 76, Lys 77, and Thr 78 has a critical role in binding to CL.

Amplified gene location in chromosomal DNA affected recombinant protein production and stability of amplified genes.

Previously, we established an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese hamster ovary (CHO) cell line. With a gradual increase in methotrexate (MTX) concentration, gene-amplified cell pools had high and stable specific growth and production rates. Moreover, the phenotype of gene-amplified cells seemed to be affected by the location of the amplified gene in chromosomal DNA. We suspected that various kinds of gene-amplified cells might appear during the long-term selection to construct gene-amplified cell pools. To clarify the behavior of gene-amplified cell pools during a stepwise increase of MTX concentration, we isolated gene-amplified clones derived from gene-amplified cell pools. We compared the characteristics of isolated clones, such as the productivity of recombinant protein, stability of amplified genes, and the location of amplified genes. As a result, telomere-type clones, in which the amplified gene was located near the telomeric region, were found to be more stable and productive than other types of clones. Telomere-type clones had over 100 copies of amplified genes in the chromosomal DNA. In contrast, a large number of other types of clones had less than 10 copies of amplified genes. During long-term cultivation in the absence of MTX, in other types of clones, amplified genes rapidly decreased in the chromosomal DNA.

Effects of prenatal exposure to styrene on neurochemical levels in rat brain.

Styrene was evaluated to determine its neurochemical effects in the offspring of rats exposed during the gestation period. Maternal Wistar rats were exposed to 0, 50 or 300 ppm styrene during gestation days 6 to 20 and the neurochemical effects on their offspring were compared with their pair-feeding and ad lib. feeding controls. The cerebrum weights at birth on day 0 were significantly lower than those for an ad lib. feeding control group. Neurotransmitter analyses showed decreases of neuroamines, especially 5-hydroxytryptamine and homovanillic acid in the cerebrum of newborn offspring of dams receiving a 300 ppm styrene exposure compared with the ad lib. fed control group and homovanillic acid was also decreased compared to the pair-feeding control. On postnatal day 21, the styrene-exposure pups showed a significant decrease of 5-hydroxyindoleacetic acid in the frontal neocortex compared with the ad lib. control group. In the hippocampus a significant decrease of 5-hydroxyindoleacetic acid was observed compared with both control groups. Moreover, the ratio of 5-HIAA/5-HT in the hippocampus was significantly decreased among the styrene-exposure groups. The 50 ppm styrene exposed group induced increase of concentrations of 5-hydroxytryptamine in the striatum. These results suggest that prenatal styrene exposure affects the developing fetal brain in terms of a few signs of neurochemical alteration.

Neurochemical effects in rats following gestational exposure to styrene.

Styrene was evaluated for the reproductive effects of pregnant rats and the neurochemical effects in the offspring of rats exposed during gestation. Pregnant Wistar rats were exposed to 0, 50, or 300 ppm styrene for 6 h/day during days 7 to 21 of gestation. No significant differences in the number of offspring delivered were observed between the exposed and control groups. Body weights at 1 day of age of the offspring whose mothers were exposed to styrene were significantly lower than those of the control group. Although, there were neither statistically significant differences of protein contents nor brain weights among styrene-exposed and their control offsprings of rats, analyses of neurotransmitter studies showed dose-dependent decreases of neuroamines, especially 5-HT (serotonin) and its metabolite 5HIAA (5-hydroxyindoleacetic acid) in the newborn offspring of styrene-exposed rats. The results suggest that gestational exposure to styrene at these concentrations does not produce apparent reproductive toxicity but affects the body weight of pups and causes lowering of the neurotransmitter levels in the brain.

Effect of prenatal exposure to styrene on the neurobehavioral development, activity, motor coordination, and learning behavior of rats.

Maternal Wistar rats were exposed via inhalation to 0, 50, or 300 ppm styrene for 6 h/day during gestation days 7 to 21, and offspring were subsequently evaluated in several neurobehavioral tests. Preliminary results with a small number of litters revealed significant dose-dependent effects in tests performed prior to weaning (surface righting, pivoting locomotion, and bar holding), as well as in tests performed after weaning (motor coordination, open-field behavior, and motor activity). Exposure to low concentrations of styrene (50 ppm) caused disturbances in motor coordination in addition to delaying some motor and reflex developments. Large doses (300 ppm) led to changes in open-field behavior and increases in spontaneous activity in addition to the delay in neurobehavioral developments. Exposure of dams to styrene did not clearly affect the learning behavior of the offspring. It was also observed that age played a role in the differences in styrene's effects on neurobehavioral function. Only subtle effects were found in both open-field behavior and motor-coordination function when compared with control rats at 120 days of age. These results suggest that the functional neurobehavioral development of progeny of dams exposed to styrene (or other solvents) should be further investigated.

Molecular cloning of yeast cytochrome c-like polypeptide expressed in human lung carcinoma: an antigen recognizable by lung cancer-specific human monoclonal antibody.

We previously determined the amino acid sequence to the epitope (ATLFKTR) of cytochrome c from Candida krusei, which is cross-reactive to the lung cancer-specific human monoclonal antibody HB4C5. Here we report that an antigen messenger RNA, which codes for a structure similar to the cytochrome c epitope, is expressed in the human lung adenocarcinoma A549. Sequencing analysis has revealed that this messenger RNA encodes a novel 190 amino acid polypeptide of 21-kDa containing an amino acid sequence (ALLFFT) similar to the cytochrome c epitope, although the total messenger RNA sequence is apparently different from the cytochrome c messenger RNA. Western analysis indicated that an antibody-recognizable 21-kDa antigen which has the same molecular weight as the predicted polypeptide is expressed in the A549 adenocarcinoma. The in vitro translated product of the antigen messenger RNA and synthesized ALLFFT peptide were both shown to be reactive with the monoclonal antibody, indicating that this protein contains the epitope which enables A549 cells to specifically react with the antibody. The antigen mRNA was not expressed in non-transformed fibroblasts, suggesting that the antigen mRNA expression was associated with cellular transformation. Also in part of the antigen nucleotide sequence, there was a segment that had about 90% homology to the long terminal repeat sequence (no. 297-475) of the human endogenous retrovirus HERV-K10, which was related to the mouse mammary tumor virus.

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.

L-Lysine production by exponential feeding of L-threonine.

The effect of L-threonine feeding in the production phase on L-lysine production by Brevibacterium flavum, which requires L-homoserine or L-threonine for cell growth, was investigated considering the concerted inhibition by L-threonine plus L-lysine, and the metabolism related to lysine production. Exponential feeding of L-threonine increased L-lysine production to 70 g/l about three times that without feeding. From the analysis of the metabolic flux, carbon flux of L-lysine synthesis pathway in the production phase after L-threonine feeding was higher than that in the growth phase. The results show that feeding of an inhibitory substance may increase the production, especially when the substance is necessary for the continuation of cell growth and/or production.

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.

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).