Biomedical subjects

K Fukuchi

Publications and source records attributed to K Fukuchi.

At least 109 records · Page 6Linked to original sources

PubMed1995

Effects of N-3554S, a polyprenyl phosphate, on B16-F10 mouse melanoma cells.

N-3554S, an optically active S-isomer of alpha-dihydrodecaprenyl phosphate, reduced the tumorigenicity of cultured B16-F10 mouse melanoma cells probably by affecting protein N-glycosylation. Accordingly, membrane glycoprotein samples were prepared from the melanoma cells cultured with or without N-3554S, and amounts and structures of N-linked sugar chains were determined. Analyses of the N-linked oligosaccharides released by hydrazinolysis from these samples and reduced with NaB3H4 revealed that the N-3554S-treated cells contain 1.5-1.8 times as much oligosaccharides as the control cells, and the relative amounts of high-mannose-type and bi-, tri- and tetra-antennary complex-type sugar chains are almost the same between two samples. Western blot analysis, however, showed that binding of L-PHA, which binds to oligosaccharides with the GlcNAc beta 1-->6(GlcNAc beta 1-->2)Man structure, is significantly reduced in 90 K, 96 K, 140 K, 155 K and 180 K glycoproteins in N-3554S-treated cells. Immunoblot analysis showed that the 140 K glycoprotein could be a fibronectin receptor. It was also shown that N-3554S treatment enhances the adhesiveness of the cells to fibronectin. These results indicate that N-3554S affects N-glycosylation of membrane glycoproteins and alters the cell surface properties of B16-F10 cells.

Animals↗

PubMed1995

Molecular analysis of the Cip1/Waf1 (p21) gene in diverse types of human tumors.

We have screened for mutations in the Cip1/Waf1 gene using Southern blot analysis and the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method in diverse human tumors. Seven of 102 (7%) human tumor samples were identified to have point mutations within the coding region of the Cip1/Waf1 gene. Two of the seven mutated cases showed gene rearrangements. These results suggest that the frequency of genetic alterations in the Cip1/Waf1 gene is relatively low in comparison with several known tumor suppressor genes.

Base Sequence↗

PubMed1995

Molecular mimicry: histone H3 and mycobacterial protein epitopes.

A 15-kDa protein detected initially in amyloidotic ileum from a transgenic mouse and subsequently in control (nontransgenic) ileum by various polyclonal rabbit antiserums applied to electroblots of extracts derived from these tissues was identified by partial sequence analysis as histone H3. Antiserums were made against immunogens unrelated to the histone, but they recognized calf thymus histone H3 (14.7 kDa) on Western blots. The bacterial component of the Freund's medium used as an adjuvant for the immunogens was either Mycobacterium butyricum or Mycobacterium smegmatis. Absorption tests with histone H3 and sonicated M. butyricum substantiated the presence of anti-histone H3 activity in the antiserums. These findings indicate that the two mycobacterium species make a protein with epitopes perceived as nonself by recipient rabbits but sufficiently similar to epitopes of mammalian histone H3 that the rabbits produced antibodies cross-reactive with the histone.

Amino Acid Sequence↗

PubMed1995

Structural integrity of the cyclin-dependent kinase inhibitor genes, p15, p16 and p18 in myeloid leukaemias.

The cyclin-dependent kinase inhibitors known as p15, p16 and p18 have been suggested as candidates for tumour suppressor genes. We examined these genes for their alterations in 46 myeloid leukaemias and 15 myeloid leukaemia cell lines. p16 mRNA expression was studied in 41 myeloid leukaemias. The p15 and p16 genes were either deleted or mutated in myeloid leukaemia lines at a high frequency [6/15 (40%) for p15; 8/15 (53%) for p16] but alterations in primary myeloid leukaemias are much less frequent [2/46 (4%) for p15; 3/46 (6%) for p16]. Alterations of p18 were not found in any of the samples. 13 primary myeloid leukaemia samples had negligible levels of p16 mRNA. In summary, the deletions of p15 and p16 genes identified in the myeloid leukaemia cell lines probably occurred during their in vitro immortalization. Alterations of the p16 or p15 gene only occurred in primary acute myeloid leukaemia samples that were of mixed myeloid/lymphoid lineage (CD19/CD20-positive acute myeloid leukaemia [AML], CD2/CD19-positive AML, and lymphoid blastic crisis of chronic myeloid leukaemia). Further studies are required to determine if the absence of mRNA expression results from inactivation of the p16 gene.

Blotting, Northern↗

PubMed1995

Iron deprivation results in an increase in p53 expression.

Deferoxamine (DFO)-induced iron deprivation caused an increase in p53 expression in ML-1 and Raji cells. In ML-1 cells, with express wild type p53, p53 protein levels were transiently increased 6 h after addition of 10(-4)M DFO. In Raji cells, which carry a mutant p53 allele, p53 increased 6 h after addition of 10(-4)M DFO and remained elevated for 24 h. Growth inhibition was observed in both cell types 6 h after addition of 10(-4)M DFO. In both cells, p53 mRNA levels did not increase following incubation with DFO, suggesting that increased p53 expression is the result of a post-transcriptional mechanism. Although increases in wild type p53 protein in ML-1 cells resulted in increases in a p53 target gene, p21cipl/wafl/sdil, this effect was not observed in Raji cells which express a mutant p53 protein.

Apoptosis↗

PubMed1995

[Detection of K-ras mutation from malignant cells in sputum with mutant-allele specific amplification (MASA) method].

Sputum cells from 44 lung cancer patients (22 adenocarcinoma, 19 squamous cell carcinoma and 3 small cell carcinoma cases) were examined to contain mutant K-ras genes. The mutant-allele-specific amplification (MASA) method was used for detection of K-ras point mutations. The reaction was designed to amplify only mutant codons with polymerase chain reaction (PCR). Codons 12, 13 and 61 were studied with synthesized primers. Two cases (primary adenocarcinoma of the lung and pancreas adenocarcinoma with lung metastasis) were positive for point mutations at codon 12. The mutation of GGT to GTT was found in the first case and to GAT in the second one. Further experiments with colony hybridization revealed the mutations were found in 1% of alleles in both cases. There were no point mutations found at codon 13 nor 61. Our experiments showed it is possible to detect K-ras mutation in sputum cells from lung cancer patients, with MASA method.

Adenocarcinoma↗

PubMed1995

[Rearrangement of the c-erbB2 oncogene--analysis of the gastrocolic carcinoma].

We analyzed amplification, rearrangement and point mutation of the c-erbB2 oncogene about 10 cases of the stomach cancer and 30 cases of the colon cancer. Rearrangement and amplification of the c-erbB2 oncogene were found in one case of moderately differentiated tubular adenocarcinoma of the stomach. According to the restriction map, the rearrangement apparently occurred in the region from the 5' promotor to the extracellular domain. In this case, it appeared that the over expression of ErbB2 oncoprotein detected by immunohistochemical staining was associated with rearrangement of the gene. Thus it was suggested that the point mutation of the transmembrane domain, which has been demonstrated in the rat protooncogene neu, a homolog of human c-erbB2, was not detected in this case.

Base Sequence↗

PubMed1995

[Experience with mild exercise 123I-BMIPP myocardial imaging in two cases of ischemic heart disease].

During mild to moderate ischemia, glycolytic flux is enhanced and free fatty acid uptake is reduced in proportion to the reduction in mitochondrial metabolism. We considered that mild exercise may induce the reduction of 123I-BMIPP, reflecting myocardial fatty acid metabolism, in ischemic myocardium compared to normal myocardium. Therefore, mild exercise 123I-BMIPP myocardial imaging was carried out to detect myocardial ischemia in 2 cases of ischemic heart disease. Mild exercise was performed using a bicycle ergometer with 25-50 W loading. At seven minutes before cessation of exercise, 111 MBq of 123I-BMIPP was injected. Case 1 was a 12 year-old boy with Kawasaki's disease. The study showed a reduction of mild exercise 123I-BMIPP uptake in the anteroseptal wall. In contrast, stress 201T1 myocardial imaging did not show perfusion defect in the anteroseptal wall. Case 2 was a 64 year-old female with triple vessels disease. Mild exercise 123I-BMIPP myocardial imaging showed similar with those of stress 99mTc-sestamibi. We conclude that mild exercise 123I-BMIPP myocardial SPECT may be a sensitive method to detect myocardial ischemia.

Child↗

PubMed1994

Characterization of proteoglycans synthesized by murine embryonal carcinoma cells (P19) reveals increased expression of perlecan (heparan sulfate proteoglycan) during neuronal differentiation.

Proteoglycans (PGs) incorporated into cell layer and secreted into media were characterized during retinoic acid-induced neuronal differentiation of cultured P19 murine embryonal carcinoma cells. Heparan sulfate significantly increased (P < 0.01) in cell layer following neuronal differentiation of P19 cells by 3.9-fold. CL-4B gel chromatography revealed the major PGs present in cell layer of stem cells eluted as a broad peak with a Kav = 0.65, and was susceptible to chondroitin ABC lyase. The chondroitin ABC lyase resistant material eluted as a broad peak between Kav = 0.40 and Kav = 0.60, and was only partially digested with heparitinase/heparinase (with resistant material eluting at Kav = 0.70). Therefore, the cell layer of stem cells contained primarily chondroitin sulfate/dermatan sulfate (CS/DS) PGs, with lesser amounts of heparan sulfate proteoglycans (HSPGs). This was confirmed by SDS-PAGE. The CS/DS PGs in the cell layer of stem cells had an apparent M(r) of approximately > 200 kDa, and the HSPGs had an apparent M(r) of approximately 140-230 kDa. In contrast, the major PGs in the cell layer of neurons consisted primarily of HSPGs, with only a minor proportion of CS/DS PGs. Furthermore, both gel filtration chromatography and SDS-PAGE analysis revealed a larger HSPG in the cell layer of neurons (Kav = 0.3-0.6 on CL-4B following chondroitin ABC lyase digestion; M(r) 170 kDa- > 400 kDa on SDS-PAGE) in comparison to stem cells (Kav = 0.4-0.6 on CL-4B following chondroitin ABC lyase digestion; M(r) 140-230 kDa on SDS-PAGE). Likewise, the major PGs secreted into media of stem cells consisted almost exclusively of CS/DS PGs, with lesser amounts of HSPGs, whereas an increase in HSPGs in the media of neurons was apparent. Western, Northern, and immunocytochemical analysis demonstrated that mRNA transcript and protein levels for a specific HSPG (i.e., perlecan) markedly increased in cell layer following P19 neuronal differentiation. Perlecan core protein was identified by Western blot analysis using specific monoclonal and polyclonal antibodies, as a large HSPG with a core protein of apparent M(r) approximately 370-400 kDa, and was observed primarily in extracts from neurons. Northern blot analysis with a cDNA to perlecan revealed a significant (P < 0.01) 12.7-fold increase in expression of perlecan in neurons (day 9) in comparison to stem cells. The increase in perlecan message during P19 neuronal differentiation was concomitant with a significant (P < 0.01) 26.3-fold increase in message for beta-amyloid precursor protein (beta PP).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Iron deprivation-induced apoptosis in HL-60 cells.

Iron deprivation of HL-60 cells with deferoxamine B mesylate (DFO) induced apoptosis. DNA fragmentation became apparent with 10(-6) M DFO after 48 h treatment. The apoptosis peak according to the DNA histogram on flow cytometry and typical nuclear collapse and were observed microscopically after 48 h treatment with 10(-4) M DFO. Cells treated with 10(-4) M DFO for as little as 24 h were shown to be committed to apoptosis, as chromatin condensation progressed gradually thereafter.

Apoptosis↗

PubMed1994

Homozygosity mapping of the Werner syndrome locus (WRN).

Werner syndrome (WS) is an autosomal recessive disorder characterized by the early onset of several age-related diseases. The locus for this disease was recently mapped to 8p12. We studied 27 WS kindreds of mixed ethnic origins, 26 of which were consanguineous. In 24 of these families, the affected subject was given the diagnosis of "definite" WS and affected subjects in the remaining 3 pedigrees were given the diagnosis of "probable" WS. Affected subjects from each kindred were genotyped for 13 short tandem repeat polymorphic sites. Two-point linkage analysis yielded significant evidence for linkage to D8S137, D8S339, D8S87, PLAT, D8S165, and D8S166. The locus yielding a maximum lod score at the smallest recombination fraction was D8S339, suggesting that this marker is the closest to the WS gene (WRN locus) of those tested. D8S339 gave significant lod scores (Zmax > or = 3.0) for both Japanese and non-Japanese (mostly Caucasian) families, demonstrating that a single locus is responsible for WS in both groups. Multipoint analysis of these markers yielded a maximum lod score of 17.05 at a distance of approximately 0.6 cM from D8S339. The combined evidence from 2-point analysis, multipoint analysis, and analysis of regions of homozygosity in subjects from inbred pedigrees indicates that the WRN locus is between D8S131 and D8S87, in an 8.3-cM interval containing D8S339.

Alleles↗

PubMed1994

Oxygen-resistant multipotent embryonic carcinoma cell lines exhibit antimutator phenotypes.

Stable, oxygen-resistant cell lines (O2R) were isolated from P19 and P19H22 (APRT hemizygote) mouse embryonic carcinoma cells by serial exposures of increasing durations to 95% O2. Neurally differentiated progeny were also oxygen-resistant. P19O2R exhibited reduced oxygen-mediated micronucleation and a 10- to 20-fold reduction of the forward mutation rate at the HPRT locus in 20% O2. P19H22O2R cells showed reduced frequencies of colonies resistant to 2,6-diaminopurine. The modal karyotype of P19O2R was identical to that of a nonmodal karyotype present in the parental line [39,X,-Y, add(14)]. There was no evidence of enhanced resistance to ionizing radiation. We conclude that this general approach, when applied to pluripotent embryonic stem cells, has the potential to lead to the synthesis of antimutator strains of mice.

Adenine Phosphoribosyltransferase↗

PubMed1994

Activity assays of nine heterogeneous promoters in neural and other cultured cells.

To express high levels of proteins encoded by transfected DNA constructs in a variety of cultured cells, including neuronal cells, the activities of nine different promoters were evaluated using Escherichia coli beta-galactosidase (beta-gal) (LacZ) as a reporter gene. These nine promoters were categorized into three distinct groups (high, intermediate, and low expresser), in terms of the levels of beta-gal expression. An expression vector containing the cytomegalovirus enhancer and the chick beta-actin promoter (high expresser) showed the highest levels of expression, followed by vectors containing the cytomegalovirus promoter/enhancer and the SV40 promoter/enhancer (intermediate expresser). The rest of the promoters (thymidine kinase, adenovirus, murine proliferative sarcoma virus, nerve growth factor receptor, Rous sarcoma and mouse mammary tumor virus, and beta-amyloid precursor protein) expressed low levels of beta-gal. These results were consistent for eight different cell types. A particularly attractive model is the stem cell, P19; cultures differentiating into progeny consisting predominantly of cholinergic neurons could be readily transfected with expression vectors using liposomes and expressed beta-gal without significant morphologic changes of the differentiated neurons. The systems should be useful for the study of promoters and various expressed proteins, including those involved in axonal transport.

Animals↗

PubMed1994

Cytotoxicity mediated by conditional expression of a carboxyl-terminal derivative of the beta-amyloid precursor protein.

The beta amyloid peptide which accumulates within the brains of patients with Alzheimer's disease (AD) is proteolytically derived from a precursor protein (beta PP). We established and characterized four stably transformed human neuroblastoma cell lines which conditionally expressed a partial beta PP fusion protein (amino-17 residues+carboxyl-99 residues; S beta C). Conditional expression of S beta C was achieved using a tetracycline-responsive promoter system. Expression of this fusion protein in one of the cell lines resulted in pronounced cytotoxicity. Addition of n6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate and/or fetal bovine serum to the culture medium of this cell line further elevated the level of S beta C expression and enhanced the associated cytotoxicity. Conditioned medium, acquired from cells expressing S beta C, was not cytotoxic. These findings suggest that modulation of beta PP expression and/or metabolism can have cytotoxic consequences. This is the first report of cytotoxic effects mediated by conditional expression of a beta PP derivative. This immortal cell line provides a unique opportunity to screen for complementary DNAs which suppress this toxicity. Such cDNAs could help elucidate the processes underlying S beta C mediated cytotoxicity which in turn could further our understanding of the pathogenesis of AD and could also provide additional candidate genes for various forms of familial AD.

Alzheimer Disease↗

PubMed1994

Distribution of cocaine recognition sites in rat brain: in vitro and ex vivo autoradiography with [125I]RTI-55.

The distribution of binding sites of [125I]RTI-55 (3 beta-(4-iodophenyl)tropan-2 beta-carboxylic acid methyl ester), a phenyl tropane analog of cocaine, and the selective labeling of the dopamine transporter (DAT) were studied by in vitro and ex vivo autoradiography in the rat whole brain. Recent evidence has shown that RTI-55 binds to not only DAT but also serotonin transporter (5HTT). In the present study, in vitro autoradiography revealed that [125I]RTI-55 bound to the olfactory tubercle, the caudate putamen, the accumbens nucleus, the midline and lateral geniculate nuclei of the thalamus, the hypothalamic nuclei, the substantia nigra compact part, the subthalamic nucleus, the ventral tegmental area, the superior colliculus, the dorsal raphe nucleus, and the facial nucleus. Further, in the presence of clomipramine, a selective ligand for 5HTT, [125I]RTI-55 binding was remarkably inhibited in the midline and lateral geniculate nuclei of the thalamus, the hypothalamic nuclei, the superior colliculus, the dorsal raphe nucleus, and the facial nucleus, while [125I]RTI-55 binding remained in the olfactory tubercle, the caudate putamen, the accumbens nucleus, the substantia nigra compact part, the subthalamic nucleus, and the ventral tegmental area. These findings suggest that [125I]RTI-55 binds to 5HTT in the former areas and to DAT in the latter areas. It is therefore concluded that RTI-55 is a suitable ligand for studying the action of cocaine in whole brain regions, including the thalamus, the hypothalamus and the dorsal raphe nucleus, regions in which cocaine is thought to act evoking several neurological effects, e.g., analgesia and elevation of adrenocorticotropic hormone. DAT was also labelled selectively both in vitro and in vivo using [125I]RTI-55 combined with clomipramine. Therefore, radiolabelled RTI-55, combined with unlabelled clomipramine, which displaces its binding to 5HTT, also appears to be suitable for the selective imaging of DAT in vivo.

Animals↗

PubMed1994

[Surveillance of multidrug-resistant environmental bacteria].

The multidrug-resistance of Staphylococcus epidermidis and Pseudomonas aeruginosa was surveyed. The multidrug-resistant S.epidermidis isolated accounted for 18% of the total S.epidermidis (1991.4 to 1992.3) and were frequently isolated from specimens of the urine and respiratory system. The polymerase chain reaction revealed the existence of the mec A gene in S.epidermidis showing various degree of antibiotic resistance, suggesting that S.epidermidis is in the process of achieving multidrug resistance. The multidrug-resistant P.aeruginosa were isolated from 14.6% of the total P.aeruginosa (1993.5 to 1993.8) and were most frequently isolated from the urine. Most of the multidrug resistant P.aeruginosa showed serotype E, suggesting the relationship between serotype and acquirement of drug resistance. Pulsefield electrophoresis of SpeI digested P.aeruginosa genomic DNA showed a characteristic pattern and the genome pattern should be applicable for the epidemiology.

Anti-Bacterial Agents↗

PubMed1994

Kinetics of a putative hypoxic tissue marker, technetium-99m-nitroimidazole (BMS181321), in normoxic, hypoxic, ischemic and stunned myocardium.

UNLABELLED: This study focused on the kinetics of the newly developed 99mTc-nitroimidazole, propyleneamine oxime-1,2-nitroimidazole (BMS181321) in the different setting of myocardial perfusion states and oxygenation levels, and compared the kinetics of BMS181321 with those of other technetium analogues. METHODS: The kinetics of BMS181321 were evaluated in isolated perfused rat hearts. Technetium-99m-hexamethylpropyleneamine oxime (HMPAO) and a non-nitroimidazole-containing analogue of BMS181321 (6-methyl propyleneamine oxime; PAO-6-Me) were used to compare their kinetics with those of BMS181321. RESULTS: BMS181321 cleared quickly from normoxic hearts and the retention in the myocardium 10 min after injection was 0.84% +/- 0.04% ID/g wet wt (mean +/- s.e.m.). In contrast, BMS181321 was retained after reperfusion when it was injected before ischemia; the uptake in the myocardium 10 min after reperfusion was significantly greater than in controls (23.9% +/- 3.9% ID/g wt, p < 0.05). CONCLUSIONS: These results indicate that 99mTc-BMS181321 is well trapped in ischemic myocardium and moderately trapped in hypoxic myocardium, but washed out quickly in stunned myocardium. The residence time influences the amount retained.

Animals↗

PubMed1993

Transgenic animal models for Alzheimer's disease.

The neuropathology of Alzheimer's disease is characterized by the deposition of abnormal protein aggregates. The main constituent of the deposition is beta-amyloid protein. A seminal role of this protein is supported by the discovery of point mutations in the gene of its precursor protein in certain forms of familial Alzheimer's disease. In vitro (cultured neuronal cells), overexpression of the precursor protein or a part of the precursor leads to degeneration of neurons, suggesting neurotoxicity of its derivatives. At this time, all of the reported transgenic mice bearing DNA construct for the precursor or a part of the precursor, however, have not developed convincing pathological changes similar to what is observed in patients with Alzheimer's disease. This interesting discrepancy between in vitro and in vivo suggests suppressors in vivo which ameliorate beta-amyloid precursor protein derivative-mediated neurotoxicity.

Alzheimer Disease↗