Biomedical subjects

K Wakabayashi

Publications and source records attributed to K Wakabayashi.

At least 55 records · Page 3Linked to original sources

PubMed2001

Identification of 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-4) as a potent mutagen in river water in Kyoto and Aichi prefectures, Japan.

We have previously isolated five mutagens in blue rayon-adsorbed substances from water at a site below sewage plants in the Nishitakase River, in Kyoto, Japan, and identified two of them as 2-phenylbenzotriazole derivatives, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-[2-(acetylamino)-4-[(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2). In the present study, we collected adsorbed materials on blue cotton (3 kg x 9 times) at the same location, and isolated a sufficient amount (97 microg) of one of the remaining three mutagens other than PBTA-1 and PBTA-2, for structural analysis, by multiple column chromatography. The structure of mutagen, accounting for 12% of the total mutagenicity of the blue rayon-adsorbed substances, was determined to be a PBTA-1 analogue, 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-4). PBTA-4 is a potent mutagen, inducing 190,000 and 7,800,000 revertants of Salmonella typhimurium TA98 and YG1024 per microgram, respectively, in the presence of S9 mix. In addition to the water of the Nishitakase River, PBTA-4 was detected in water samples from two rivers that flow through other regions where textile-dyeing industries have been developed. Like other PBTA analogues, PBTA-4 might also be produced from azo dyes during industrial processes in dyeing factories and treatment at sewage plants.

Animals↗

PubMed2001

Phenanthroindolizidine alkaloids as cytotoxic substances in a Danaid butterfly, Ideopsis similis, against human cancer cells.

We previously reported the presence of cytotoxic substances in extracts of the Danaid butterfly, Ideopsis similis. In the present study, we isolated cytotoxic substances against a human gastric cancer cell line, TMK-1, in I. similis pupae, with an activity similar to that of the adult butterfly. The basic fraction, prepared from a methanol extract, accounted for 83% of the cytotoxic activity. Two major cytotoxic substances were purified by HPLC, and one was determined to be a new phenanthroindolizidine alkaloid, trans-(+)-3,14alpha-dihydroxy-6,7-dimethoxyphenanthroindolizidine (1), and the other a known compound, trans-(+)-3,14alpha-dihydroxy-4,6,7-trimethoxyphenanthroindolizidine (2). The IC(50) values for TMK-1 cells were 0.5 ng/mL and 0.7 ng/mL, respectively. These two compounds showed similar cytotoxic potential with four other cancer cell lines including cervical, lung, and colon carcinomas and leukemia. Quantitative analyses indicated the presence of each of the two phenanthroindolizidine alkaloids at levels of 11-74 microg in each larva, pupa, or adult of I. similis. However, 1 was not detected in the leaves of Tylophora tanakae, a host plant for larvae of I. similis, and the level of 2 (2 microg per gram of leaves) was far less than that in the larvae. Since the leaves of T. tanakae are known to contain various phenanthroindolizidines, compounds 1 and 2 are presumably metabolically converted from such alkaloids in larvae of I. similis.

Alkaloids↗

PubMed2001

Inflammatory stimuli increase prostaglandin D synthase levels in cerebrospinal fluid of rats.

To examine the possibility that prostaglandin D synthase in CSF is involved in the brain responses to inflammation, we monitored the enzyme levels before and after administration of a bacterial endotoxin lipopolysaccharide (LPS) or proinflammatory cytokines. After an i.p. injection of LPS, the synthase levels began to rise within 1 day, peaked at 2 days and returned to the basal by 5 days after injection. The enzyme levels increased in a similar manner in response to tumor necrosis factor-alpha or interleukin (IL)-1 beta injected i.v. Centrally administered IL-1 beta had a similar effect. These findings support the hypothesis that PGD synthase in CSF participates in the brain responses to inflammation.

Animals↗

PubMed2001

Similarity and variation in gene expression among human cerebral cortical subregions revealed by DNA macroarrays: technical consideration of RNA expression profiling from postmortem samples.

The functional regionality of the human cerebral cortex suggests that a set of genes might be activated in each subregion of the neocortex to support its specific functions. To test this hypothesis, we employed the DNA array technique to compare the mRNA expression profiles of three neocortical subregions of the human brain: prefrontal cortex (Area 46), motor cortex (Area 4) and visual cortex (Area 17). The macroarray analysis on high quality mRNA from postmortem brains revealed that the expression profiles of the different cortical areas are almost similar: only six out of 1088 known genes exhibited significant differences (>2-fold) in their expression. RT-PCR studies with an increased number of samples confirmed that expression of only two genes, annexin II and early growth response protein 1, varied by 2-fold among the regions, whereas expression of the others showed large inter-individual difference. These results suggest that the whole neocortex of humans is more homogeneous than we expected at the level of gross gene expression profiles. In parallel, sensitivity and accuracy of radioisotope-based DNA macroarrays and fluorescence-based DNA microarrays were tested.

Aged↗

PubMed2001

Distinct roles for the N- and C-terminal regions in the cytotoxicity of pierisin-1, a putative ADP-ribosylating toxin from cabbage butterfly, against mammalian cells.

Pierisin-1 is an 850-aa cytotoxic protein found in the cabbage butterfly, Pieris rapae, and has been suggested to consist of an N-terminal region with ADP-ribosyltransferase domain and of a C-terminal region that might have a receptor-binding domain. To elucidate the role of each region, we investigated the functions of various fragments of pierisin-1. In vitro expressed polypeptide consisting of amino acid residues 1-233 or 234-850 of pierisin-1 alone did not show cytotoxicity against human cervical carcinoma HeLa cells. However, the presence of both polypeptides in the culture medium showed some of the original cytotoxic activity. Introduction of the N-terminal polypeptide alone by electroporation also induced cell death in HeLa cells, and even in the mouse melanoma MEB4 cells insensitive to pierisin-1. Thus, the N-terminal region has a principal role in the cytotoxicity of pierisin-1 inside mammalian cells. Analyses of incorporated pierisin-1 indicated that the entire protein, regardless of whether it consisted of a single polypeptide or two separate N- and C-terminal polypeptides, was incorporated into HeLa cells. However, neither of the terminal polypeptides was incorporated when each polypeptide was present separately. These findings indicate that the C-terminal region is important for the incorporation of pierisin-1. Moreover, presence of receptor for pierisin-1 in the lipid fraction of cell membrane was suggested. The cytotoxic effects of pierisin-1 were enhanced by previous treatment with trypsin, producing "nicked" pierisin-1. Generation of the N-terminal fragment in HeLa cells was detected after application of intact entire molecule of pierisin-1. From the above observations, it is suggested that after incorporation of pierisin-1 into the cell by interaction of its C-terminal region with the receptor in the cell membrane, the entire protein is cleaved into the N- and C-terminal fragments with intracellular protease, and the N-terminal fragment then exhibits cytotoxicity.

ADP Ribose Transferases↗

PubMed2001

Induction of liver preneoplastic lesions by aminophenylnorharman, formed from norharman and aniline, in male F344 rats.

9-(4'-Aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), produced by the reaction of norharman with aniline in the presence of S9 mix, is a novel heterocyclic amine (HCA), with mutagenicity to Salmonella typhimurium TA 98 and YG 1024 comparable to that of other HCAs such as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). This experiment was designed to investigate its potential to induce glutathione S-transferase placental form (GST-P) positive foci in the liver. Male F344 rats, 7 weeks old, were fed diet containing 0, 10, 20, or 50 ppm APNH for 4 weeks, killed by ether euthanasia and performed complete necropsy. Numbers of GST-P positive foci larger than 0.1 mm in diameter induced by APNH at the dose of 10, 20, and 50 ppm were increased in a dose dependent manner to 0.52, 1.3, and 21 foci/cm2, respectively, with areas of 0.006, 0.01, and 2.3 mm2/cm2. No such GST-P positive foci were observed in rats fed control diet. These findings suggest that APNH has hepatocarcinogenic potential in male F344 rats.

Animals↗

PubMed2001

Desferrioxamine, an iron chelator, upregulates cyclooxygenase-2 expression and prostaglandin production in a human macrophage cell line.

Prostaglandins (PGs) play regulatory roles in a variety of physiological and pathological processes, including the immune response, cytoprotection and inflammation. Desferrioxamine (DFX), an iron chelator, is known to reduce free radical-mediated cell injury and to upregulate certain inflammatory mediators. We investigated the effects of DFX on the production of PGs and the expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the synthesis of PGs, using a human macrophage cell line, U937. Our results showed that COX-2 expression and PGE(2) production are upregulated by DFX treatment and that this upregulation is dependent on both COX-2 promoter activity and alteration of mRNA stability. COX-2 promoter activity may be, at least in part, mediated by activation of the extracellular signal-regulated kinase pathway. These findings suggest that iron metabolism may regulate inflammatory processes by modulating PGs as well as other inflammatory mediators.

Binding Sites↗

PubMed2001

Effects of heterocyclic amines with mammary gland carcinogenic potential on estrogenic response of uterus in ovariectomized rats.

Heterocyclic amines (HCAs) present in cooked foods are suggested to be involved in human breast cancer development. Estrogen plays a pivotal role in mammary gland carcinogenesis. Therefore, we designed an in vivo experiment to investigate potential estrogenic effects of two HCAs, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which induce mammary gland cancers in rodents, on the uterus of ovariectomized (OVX) Sprague-Dawley (SD) rats. Female SD rats ovariectomized at 35 days of age were given intraperitoneal injections of 17beta-estradiol (E2) at doses of 0, 30 or 50 microg/kg or one of the HCAs at a dose of 50 mg/kg b.w. once a day at 47, 48, and 49 days of age. E2 dramatically increased uterine weights, stromal thickness, epithelial cell height, and 5-bromo-2'-deoxyuridine (BrdU) positive cell counts in a dose dependent manner. Intraperitoneal administration of PhIP or IQ, in contrast, did not produce any estrogenic responses in this assay system. These results indicate that the carcinogenicities of these two HCAs in mammary glands are not associated with estrogenic potential.

Animals↗

PubMed2001

Analysis of the accumulation of mutants in Sabin attenuated polio vaccine viruses passaged in Vero cells.

To confirm the safety of oral poliomyelitis vaccine (OPV) cultured in Vero cells, the genetic stability of cultured polio vaccine viruses was analysed by MAPREC (mutant analysis by PCR and restriction enzyme cleavage). The rates of mutant accumulation of the viruses passaged in Vero cells under a low multiplicity of infection (MOI) condition (approximately 10(-3.5)CCID50/cell; the same as under usual OPV production conditions) were higher than those passaged in secondary cultured monkey kidney cells. However, the rates of mutant accumulation were restrained when the viruses were cultured under a high MOI condition (approximately 10(-1.5)CCID50/cell) in Vero cells. Furthermore, neurovirulence of the passaged viruses in pollovirus susceptible transgenic mice PVR-Tg21 was shown to correlate highly with the results of MAPREC. It is expected that our results will contribute to the large scale preparation of safe and effective OPV using Vero cells.

Animals↗

PubMed2001

Mode of bleaching phytotoxicity of herbicidal diphenylpyrrolidinones.

A mode of action study of herbicidal diphenylpyrrolidinones was carried out through carotenoid analyses in intact Scenedesmus cells and by a cell-free plant-type phytoene desaturase assay using Escherichia coli transformants. A series of forty-eight diphenylpyrrolidinones decreased the carotenoid content of Scenedesmus cells in the light and inhibited phytoene desaturase. The relationship between substituents at various positions and inhibition of phytoene desaturase is discussed. Using very active bleaching diphenylpyrrolidinones, a 10(-5) M concentration affected neither the zeta-carotene desaturase nor the protoporphyrinogen-IX oxidase. Although some differences in their inhibitory activity were found between the in vivo and cell-free assays, it is concluded that the compounds are essentially bleachers affecting carotenoid biosynthesis in plants. Enzyme kinetics studies with recombinant phytoene desaturase revealed a non-competitive inhibition with respect to the substrate phytoene. A competition against the inhibitor was shown by the cofactor NADP+, suggesting an interaction of pyrrolidinones at the cofactor-binding site of phytoene desaturase.

Biological Assay↗

PubMed2001

Transport and arrangement of the outer-dynein-arm docking complex in the flagella of Chlamydomonas mutants that lack outer dynein arms.

The outer dynein arms of Chlamydomonas flagella are attached to a precise site on the outer doublet microtubules and repeat at a regular interval of 24 nm. This binding is mediated by the outer dynein arm docking complex (ODA-DC), which is composed of three protein subunits. In this study, antibodies against the 83- and 62-kD subunits (DC83 and DC62) of the ODA-DC were used to analyze its state of association with outer arm components within the cytoplasm, and its localization in the axonemes of oda mutants. Immunoprecipitation indicates that DC83 and DC62 are preassembled within the cytoplasm, but that they are not associated with outer arm dynein. Both proteins are lost or greatly diminished in oda1 and oda3, mutants in the structural genes of DC62 and DC83, respectively, demonstrating that their association is necessary for their stable presence in the cytoplasm. Immunoelectron microscopy indicates that DC83 repeats at 24-nm intervals along the length of the doublet microtubules of oda6, which lacks outer arms; thus, outer arm periodicity may be determined by the ODA-DC. Flagellar regeneration and temporary dikaryon experiments indicate that the ODA-DC can be rapidly transported into the flagellum and assembled on the doublet microtubules independently of the outer arms and independently of flagellar growth. Unexpectedly, the intensity of ODA-DC labeling decreased toward the distal ends of axonemes of oda6 but not wild-type cells, suggesting that the outer arms reciprocally contribute to the assembly/stability of the ODA-DC.

Animals↗

PubMed2001

Interferon regulatory factor 1 promoter polymorphism and response to type 1 interferon.

The clinical success of interferon-treatment has been found to vary in different individuals. To explain this, we hypothesized that responses to type 1 interferons could be partly determined by interferon regulatory factor-1 gene transcription, because the latter is an important transcription factor in the interferon system. We demonstrated that the antiproliferative effect of type 1 interferons on human liver cancer cells correlates with levels of transcription of the interferon regulatory factor-1 gene in parallel with those of p21(WAF-1) expression. Here, we investigated whether mutations in the interferon regulatory factor-1 gene cause different responses to type 1 interferons. DNA from several human liver cancer cell lines and peripheral blood mononuclear cells was investigated. Nucleotide sequences of the interferon regulatory factor-1 gene and polymerase chain reaction products of its upstream region were determined directly and after cloning. The promoter activity of the upstream region of this gene was measured by the luciferase reporter assay. We found 4 point mutations in the upstream (- 1 approximately - 495) region, and the luciferase promoter assay demonstrated that these mutations did modify promoter activity. Analysis of DNA from healthy volunteers showed that these mutations are single nucleotide polymorphisms. These results suggest that single nucleotide polymorphisms of the interferon regulatory factor-1 promoter contribute, at least in part, to determining responses to type 1 interferons. J. Cell. Biochem. Suppl. 36: 191-200, 2001.

Antineoplastic Agents↗

PubMed2001

Reduction of telomerase activity in human liver cancer cells by a histone deacetylase inhibitor.

The presence of telomerase has been demonstrated recently in many different malignancies. Several reports documented that in human hepatocellular carcinoma, the level of telomerase activity parallels its differentiation stage. In the present study, the effect of the differentiation-inducing agent sodium butyrate on telomerase activity in four human liver cancer cell lines was investigated using the telomeric repeat amplification protocol. We assayed telomerase activity before and after butyrate treatment and in cell cycle synchronized non-dividing quiescent cells. In addition, telomerase reverse transcriptase levels were measured at the mRNA level. All four cell lines possessed high but not identical levels of telomerase activity. Telomerase activity was significantly reduced by treatment with sodium butyrate as well as trichostatin A in a dose- and time-dependent fashion, paralleling the reduction of cell proliferation. Although methotrexate, hydroxyurea, and colchicine synchronized the cell cycle at G1, S, and G2/M, respectively, and thereby also caused proliferating cells to cease dividing and become quiescent, in this case telomerase activity remained essentially unaltered compared to the control cultures. Moreover, levels of mRNA encoding telomerase reverse transcriptase were not always significantly altered by either sodium butyrate treatment or cell cycle synchronization. These results suggest that sodium butyrate, as a histone deacetylase inhibitor, effectively reduces telomerase activity without affecting transcription levels of the reverse transcriptase component.

Antineoplastic Agents↗

PubMed2001

Reduction of c-myc expression by an antisense approach under Cre/loxP switching induces apoptosis in human liver cancer cells.

c-Myc has been documented to be both a positive and a negative signal for the induction of apoptosis. It is well known that overexpression of the c-myc gene induces apoptosis of normal cells, but the result of a reduction in its expression is not fully understood. We examined whether a reduction in c-myc expression would induce apoptosis in human liver cancer cells. Specifically, antisense and sense oligodeoxynucleotides (oligos) against the human c-myc mRNA were synthesized, mixed with a liposome reagent at various ratios, and were applied to the liver cancer-derived cell lines, HCC-T, HepG2, and PLC/PRF/5. To exclude effects resulting from using oligos, plasmid vectors expressing the full-length c-myc cDNA in both sense and antisense orientations under the control of the Cre/loxP system were generated. Monoclonal cell lines including these plasmid vectors were produced and Cre was supplied by adenovirus infection. Apoptosis was determined morphologically and c-Myc and Bcl-2 expression was examined by Western blotting. The antisense myc significantly inhibited the proliferation of the cells within two days, while neither the liposome reagent alone nor sense myc did so. Most of the cells were rounded up by the antisense-treatment and nuclear fragmentation and DNA ladder formation were detected after two days in antisense c-myc-treated cells. Antisense c-myc largely reduced c-Myc and partially Bcl-2 expression; overexpression of Bcl-2 partially rescued from apoptosis in HCC-T and HepG2 cells. These results suggest that the massive reduction in c-myc mRNA induces apoptosis in liver cancer cell lines and consequent decrease in Bcl-2 enhances the cell death. c-Myc reduction under the Cre/loxP switching system may be a useful tool for the clarification of c-myc-related cellular mechanisms in differentiation and proliferation.

Apoptosis↗

PubMed2001

Autosomal recessive spastic paraplegia with hypoplastic corpus callosum, multisystem degeneration and ubiquitinated eosinophilic granules.

We report a 48-year-old woman with familial spastic paraplegia (FSP) showing mental retardation, amyotrophy and sensory disturbance. Her parents were second cousins and there were two other affected siblings in the family. Autopsy revealed degenerative lesions characterized by neuronal loss and gliosis in the upper and lower motor neuron systems, thalamus, lateral geniculate body, dentate nucleus and posterior column of the spinal cord. The remaining neurons often contained ubiquitinated lipofuscin granules. Although the corpus callosum was severely attenuated, it exhibited well-preserved myelination and only minimal gliosis. In the substantia nigra, the number of pigmented neurons was apparently low, but there was slight gliosis and no extraneuronal free melanin pigment in the background. The neurons in this brain region contained much smaller amounts of melanin pigment than might be expected for the patient's age. These findings suggest that this is an example of a family with autosomal recessive FSP with thin corpus callosum, and that maldevelopment of the corpus callosum and substantia nigra is a characteristic feature of the disease.

Agenesis of Corpus Callosum↗

PubMed2001

Co-localization of alpha-synuclein and phosphorylated tau in neuronal and glial cytoplasmic inclusions in a patient with multiple system atrophy of long duration.

Neuronal and glial cytoplasmic inclusions (NCIs and GCIs), which contain alpha-synuclein as a major component, are characteristic cytopathological features of multiple system atrophy (MSA). We report MSA of 19 years' duration in a 73-year-old woman. Her initial symptom was parkinsonism, with dementia appearing about 8 years later. Postmortem examination showed marked atrophy of the frontal and temporal white matter and limbic system, in addition to the pathology typical of MSA. In the limbic system, severe neuronal loss and astrocytosis were observed, and the remaining neurons often had lightly eosinophilic, spherical cytoplasmic inclusions. Interestingly, a double-labeling immunofluorescence study revealed that the NCIs in the dentate gyrus and amygdaloid nucleus, and the GCIs in the frontal and temporal white matter often expressed both alpha-synuclein NACP-5 and phosphorylated tau AT8 epitopes. Double-immunolabeling electron microscopy of the NCIs in the dentate gyrus and the GCIs in the temporal white matter clearly revealed labeling of their constituent granule-associated filaments with NACP-5, and some of them were also labeled with AT8. These findings strongly suggested that some alpha-synuclein filaments were decorated with phosphorylated tau without formation of fibrils such as paired helical filaments. Immunoblotting of sarkosyl-insoluble tau indicated that the accumulated tau consisted mainly of four-repeat tau isoforms of 383 amino acids and 412 amino acids. We consider that the limbic system can be a major site of neurodegeneration in MSA of long duration. The mechanisms of such abnormal tau accumulation in the NCIs and GCIs are unknown.

Aged↗

PubMed2001

Analysis of the expression level of alpha-synuclein mRNA using postmortem brain samples from pathologically confirmed cases of multiple system atrophy.

To determine whether multiple system atrophy (MSA) is associated with altered expression levels of the alpha-synuclein messenger RNA (mRNA), we performed quantitative reverse transcription polymerase chain reaction for alpha-synuclein mRNA using postmortem brain samples from 11 cases of MSA and 14 age-matched control subjects. The brain specimens used in this study contained both the gray matter and white matter, which were dissected from the frontal, temporal or occipital lobe. The expression levels of alpha-synuclein mRNA in the brain specimens of MSA cases were not different from those of the control subjects. These results suggest that the transcriptional regulation of the alpha-synuclein gene is unlikely to be affected in MSA brains.

Aged↗

PubMed2001

Selective insolubility of alpha-synuclein in human Lewy body diseases is recapitulated in a transgenic mouse model.

alpha-Synuclein (alpha-SYN) is deposited in intraneuronal cytoplasmic inclusions (Lewy bodies, LBs) characteristic for Parkinson's disease (PD) and LB dementias. alpha-SYN forms LB-like fibrils in vitro, in contrast to its homologue beta-SYN. Here we have investigated the solubility of SYNs in human LB diseases and in transgenic mice expressing human wild-type and PD-associated mutant [A30P]alpha-SYN driven by the brain neuron-specific promoter, Thy1. Distinct alpha-SYN species were detected in the detergent-insoluble fractions from brains of patients with PD, dementia with LBs, and neurodegeneration with brain iron accumulation type 1 (formerly known as Hallervorden-Spatz disease). Using the same extraction method, detergent-insolubility of human alpha-SYN was observed in brains of transgenic mice. In contrast, neither endogenous mouse alpha-SYN nor beta-SYN were detected in detergent-insoluble fractions from transgenic mouse brains. The nonamyloidogenic beta-SYN was incapable of forming insoluble fibrils because amino acids 73 to 83 in the central region of alpha-SYN are absent in beta-SYN. In conclusion, the specific accumulation of detergent-insoluble alpha-SYN in transgenic mice recapitulates a pivotal feature of human LB diseases.

Amino Acids↗