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Biomedical subjects

A Kikuchi

Publications and source records attributed to A Kikuchi.

At least 253 records · Page 14Linked to original sources
PubMed1996

[A new quantitative assay of heat shock protein 70 by based on an enzyme-linked immunosorbent assay method].

Heat Shock Protein 70 (HSP70) was very important stress protein. HSP70 had been detected by immunoblotting or mRNA of HSP70. Our purpose is to develop an easy quantitative assay of HSP70 and utilize clinical samples, cells, tissues etc. We examined a new quantitative assay of HSP70 that was based on an enzyme-linked immunosorbent assay (ELISA). This quantitative assay showed reproducible standard curve, high reproducibility (CV : 7.1, 6.8%) and recovery (93 approximately 116%). The determination of HSP70 in human umbilical vein endothelial cells (HUVEC) by heat stress was assayed using this method. HSP70 in HUVEC increased according to the heat temperature. It is concluded that the quantity of HSP70 might be possible even in cells and tissues by this method.

Endothelium, Vascular↗
PubMed1996

Decreased DNA topoisomerase II alpha expression and cold-sensitive growth in a mouse mammary cancer cell line resistant to etoposide and doxorubicin.

A mouse mammary carcinoma FM3A cell line resistant to the DNA topoisomerase (topo) II-targeting agent, etoposide (VP-16), FM3A/VP-2B, had a markedly reduced growth rate at a low temperature (33 degrees C). The cells had the following properties: (a) FM3A/VP-2B, which had 24-fold higher resistance to VP-16 than its parental line, FM3A, was cross-resistant to doxorubicin, but not to a camptothecin derivative, CPT-11. (b) Cold-resistant revertants from FM3A/VP-2B, R-6 and R-11, remained 8- to 9-fold more resistant to VP-16 and 2- to 3-fold more resistant to doxorubicin. (c) FM3A/VP-2B had one-fourth the level of topo II activity and one-third of the topo II alpha content and mRNA of FM3A. R-6 and R-11, however, had levels similar to FM3A. (d) FM3A/VP-2B and FM3A had a 3-base deletion at position 4170 on one allele on the topo II alpha cDNA, but expression of the wild-type and the deletion allele was not appreciably changed in both cell lines. Decreased topo II alpha expression might have led to the acquisition of drug resistance to etoposide in FM3A/VP-2B, and appeared to be linked with the cold-sensitive growth. We also present a corrected mouse topo II alpha cDNA sequence.

Amino Acid Sequence↗
PubMed1996

Expression and ultrastructural localization of HMB-45 antigen.

HMB-45 is a monoclonal antibody specific for melanoma cells and premature developing melanocytes. We examined the expression and specific subcellular binding sites of HMB-45 in various types of melanocytes including epidermal melanocytes from fetuses and infants with or without tyrosinase-negative oculocutaneous albinism (type IA), melanin-producing and non-producing melanoma cell lines (G361 and MeWo), and in vivo melanoma cells (melanotic and amelanotic malignant melanoma). Subcellular HMB-45 binding was examined by using post-embedding immunogold electron microscopy with rapid freezing and freeze substitution fixation methods without the use of chemical fixatives to preserve the intracytoplasmic delicate antigen property of HMB-45. HMB-45 antigen was detected not only in in vivo melanoma cells and normal fetal melanocytes, but also in melanocytes in the other conditions. Post-embedding immunogold electron microscopy revealed that HMB-45 antigen was exclusively localized to stages I and II melanosomes in the cytoplasm of neoplastic melanocytes, but was detected mainly on stages II and III melanosomes in the melanocytes from fetuses and infants. In tyrosinase-negative oculocutaneous albinism, only stages I and II melanosomes were detected in the cytoplasm, but both stages of melanosomes were HMB-45 positive. We conclude that HMB-45 appears mainly on the immature melanosomes during melanogenesis in both neoplastic and non-neoplastic melanocytes regardless of their tyrosinase activity, but the intracytoplasmic localization of HMB-45 antigen is different by each condition of melanocytes.

Adolescent↗
PubMed1996

A morphological study of root resorption of the maxillary first deciduous molars.

The aim of this study was to elucidate the relationship between root resorption of the maxillary first deciduous molars and the developmental state of the successive permanent teeth. Twenty-four specimens of the maxilla from 12 dry skulls were classified into four dental eruption stages based upon their deciduous and permanent dentition. Serial sections prepared from the maxilla and embedded in polyester resin were observed with soft X-ray films. The area from the lowest point of the protruded part of the zygomatic process to the alveolar crest of the maxillary first deciduous molar was divided into 6 areas parallel to the Frankfort plane. Specimens from the upper 1/6, 3/6 and 5/6 areas were used. In addition, the shortest distance from the root resorption surface to the bony crypt was measured. The bony crypt containing the successive permanent tooth germ was located closer to the buccal root among the 3 roots of first deciduous molars but grew lingually with the progress of eruption stage. The distance from the root resorption surface to the bony crypt shortened and resorption actively progressed from the deciduous dentition stage to the first molar, and the central incisor reached the occlusal line in the deciduous dentition stage. Three-dimensional reconstruction to reproduce the relationship between the root and bony crypt revealed an increase in the root resorption surface with the eruption phase and growth of the bony crypt in the direction of root furcation.

Alveolar Process↗
PubMed1995

rap1 p21 regulates the interaction of ras p21 with RGL, a new effector protein of ras p21.

We have recently found that ralGDS family members (RGL and ralGDS) are putative effector proteins of ras p21. rap1 p21 is a small GTP-binding protein which has the same amino acid sequence as the effector loop of ras p21. We examined the effect of rap1 p21 on the interaction of ras p21 with RGL. The GTP-bound form of rap1 p21 interacted with RGL as well as did ras p21. rap1 p21 inhibited the interaction of ras p21 with RGL. RGL was phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A). Phosphorylation of RGL did not affect its binding to ras p21 and rap1 p21 under the conditions that phosphorylation of Raf-1 reduced its affinity for ras p21. These results demonstrate that rap1 p21 but not protein kinase A regulates the interaction of ras p21 with RGL and suggest that rap1 p21 and protein kinase A may cooperate to distinguish the signal or ras p21 to RGL from that to Raf-1.

Cloning, Molecular↗
PubMed1995

Characterization of the interaction of Raf-1 with ras p21 or 14-3-3 protein in intact cells.

Several deletion mutants of Raf-1 were expressed with v-ras p21 or 14-3-3 protein in COS-7 cells and Sf9 cells and the interaction of Raf-1 with ras p21 or with 14-3-3 protein in intact cells was examined. Raf(1-135) (residues 1-135) and Raf(1-322) interacted with v-ras p21, but other deletion mutants such as Raf(136-322) or Raf(321-648) did not. Raf(1-322) interacted with 14-3-3 protein much more efficiently than Raf(321-648) did. While Raf(1-135) did not interact with 14-3-3 protein, Raf(136-322) did. These results clearly indicate that Raf-1 simultaneously interacts with both ras p21 and 14-3-3 protein through the distinct binding domains in intact cells.

14-3-3 Proteins↗
PubMed1995

A novel type of protein modification by isoprenoid-derived materials. Diphytanylglycerylated proteins in Halobacteria.

Previous work from this laboratory has shown that a derivative of [3H]mevalonic acid is incorporated into a number of specific proteins in Halobacterium halobium and Halobacterium cutirubrum and that the major radioactive material released by treatment with methyl iodide was neither farnesyl nor geranylgeranyl compound, which have been generally accepted to be prenyl groups of a number of prenylated proteins found in eukaryotic cells, but an unknown compound (Sagami, H., Kikuchi, A., and Ogura, K. (1994) Biochem. Biophys. Res. Commun. 203, 972-978). In the current study, the unknown compound was prepared in a large amount from H. halobium cells and analyzed by reverse and normal phase high performance liquid chromatographies followed by mass spectrometry. The mass spectrum of this compound exhibited a parent ion peak (M+) at m/z 682, suggesting that it is a 1-methylthio-2,3-di-O-(3',7',11',15'-tetramethylhexadecyl)glycerol (diphytanylglyceryl methylthioether). Diphytanylglyceryl methyl thioether was chemically synthesized, and its mass fragmentation pattern was completely coincident with that of the mevalonic acid-derived material from H. halobium. These results indicate that Halobacteria contains specific proteins with a novel type of modification of a cysteine residue of the proteins with a diphytanylglyceryl group in thioether linkage.

Bacterial Proteins↗
PubMed1995

Replication factor encoded by a putative oncogene, set, associated with myeloid leukemogenesis.

DNA replication of the adenovirus genome complexed with viral core proteins is dependent on the host factor designated template activating factor I (TAF-I) in addition to factors required for replication of the naked genome. Recently, we have purified TAF-I as 39- and 41-kDa polypeptides from HeLa cells. Here we describe the cloning of two human cDNAs encoding TAF-I. Nucleotide sequence analysis revealed that the 39-kDa polypeptide corresponds to the protein encoded by the set gene, which is the part of the putative oncogene associated with acute undifferentiated leukemia when translocated to the can gene. The 41-kDa protein contains the same amino acid sequence as the 39-kDa protein except that short N-terminal regions differ in both proteins. Recombinant proteins, which were purified from extracts of Escherichia coli, expressing the proteins from cloned cDNAs, possessed TAF-I activities in the in vitro replication assay. A particular feature of TAF-I proteins is the presence of a long acidic tail in the C-terminal region, which is thought to be an essential part of the SET-CAN fusion protein. Studies with mutant TAF-I proteins devoid of this acidic region indicated that the acidic region is essential for TAF-I activity.

Amino Acid Sequence↗
PubMed1995

Mutations of the p53 and ras genes in childhood t(1;19)-acute lymphoblastic leukemia.

We have investigated the alterations of p53 and ras genes including H-, K-, and N-ras genes in 22 acute lymphoblastic leukemia (ALL) cases and five cell lines carrying t(1;19) by use of polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis and direct sequencing. The mutations of the p53 gene were found in 2 of 20 t(1;19)-ALL cases at diagnosis (10%), all of 4 cases at relapse (100%), and 4 of the 5 cell lines (80%). Four of the five patients who died had missense mutations at codons 49, 177, 179, and 248. In cases examined sequentially, one had the same point mutation at codon 179 at both diagnosis and relapse, and another had the same p53 gene mutation at codon 240 both in leukemic cells at relapse and in a cell line derived at that time. The other case had no mutation at diagnosis but had the mutation at codon 177 at relapse and cell lines derived from blast cells at diagnosis, suggesting that a small number of leukemic cells with the p53 gene mutation at diagnosis might have escaped PCR-SSCP analysis. In cell lines, SCMC-L9 had three point mutations in the p53 gene at codons 175, 248, and 358, whereas SCMC-L10 had frame shift at codons 209-211. One case had a rare polymorphism at codon 11. We found only one mutation of the N-ras gene that was a 2-bp substitution of GGT(Gly) to GTC(Val) at codon 13 among 22 t(1;19)-ALL cases and five cell lines. This case showed no mutation of the p53 gene and has had a good course. These results suggest that in t(1;19)-ALL, mutations of the p53 and ras genes are infrequent at diagnosis and that p53 gene alterations may be associated with relapse phase or progression of t(1;19)-ALL.

Adolescent↗
PubMed1995

Adenovirus E1A-induced apoptosis elicits a steep decrease in the topoisomerase II alpha level during the latent phase.

The human KB derivative cell line MA1, established by introduction of the adenovirus E1A 12S cDNA linked to the hormone-inducible promoter, elicits apoptosis upon treatment with dexamethasone. The cell lines partially refractory to apoptosis were established by introducing the expression plasmid for the adenovirus E1B 19k protein to MA1 cells. After induction of E1A in MA1 cells by dexamethasone, the level of p53 increased to about 10-fold within 24 h, and morphological changes characteristics of apoptosis began to be observed within 48 h. Most of cells were killed at 72 h releasing apoptotic bodies. The level of topoisomerase II alpha began to decrease steeply within 36 h, preceding the onset of DNA degradation while its mRNA level unchanged throughout the apoptotic process. E1B 19k protected the decrease in topoisomerase II alpha as well as DNA fragmentation depending on its expression levels. Topoisomerase II alpha is induced specifically at G2/M, and computer search revealed the presence of cyclin B type destruction box in topoisomerase II alpha. These results strongly suggest that E1A or E1A stabilized p53 induces apoptosis by targeting topoisomerase II alpha to the ubiquitination pathway and E1B 19k alleviates its action.

Adenovirus E1A Proteins↗
PubMed1995

Preparation and evaluation of Eudragit gels. VIII. Rectal absorption of 5-fluorouracil from Eudispert hv gels in rats.

Rectal absorption of the hydrophilic 5-fluorouracil (5-FU) in rats was studied with Eudispert hv gels with or without fatty acids as the rectal bases. In the absence of fatty acids, absolute bioavailabilities of 5-FU for Eudispert hv hydrogel and xerogel preparations increased approximately 2.5 times compared with those of Witepsol H-15 and PEG 2000 suppositories. When n-capric acid or linolenic acid was used as an absorption enhancer, absolute bioavailabilities of 5-FU were, respectively, 25.5 and 30.9% for Witepsol H-15 and 64.4 and 66.1% for PEG 2000. Furthermore, the absolute bioavailabilities of 5-FU for Eudispert hv hydrogel with n-capric acid or linolenic acid were 95.6% and 81.7%. The addition of capric acid or linolenic acid to the hydrogel was a useful method for increasing 5-FU permeability through the rectal membranes. These results are consistent with the observation that the total amounts of 5-FU remaining in the lumenal contents of the rectum and that accumulated in the rectal tissue decreased in relation to the increase in the bioavailabilities. Thus, the Eudispert hv hydrogel containing 5-FU with capric acid may be a useful rectal preparation for increasing the maximum plasma level and improving the absolute bioavailability of 5-FU.

Acrylic Resins↗
PubMed1995

Epidermal melanocytes in normal and tyrosinase-negative oculocutaneous albinism fetuses.

In tyrosinase-negative (type IA) oculocutaneous albinism (tyr(-) OCA) fetuses the development of melanocytes has not been fully elucidated. We analysed the distribution of melanocytes in skin samples from a fetus with tyr(-) OCA and from four normal fetuses. Skin samples obtained from 12 different body sites of each fetus were examined by transmission electron microscopy, an electron microscopic DOPA reaction test and immunohistochemistry. No S100 protein-positive cells were detected in any sample. There were fewer HMB-45-positive melanocytes in the skin of the tyr(-) OCA fetus than in the skin of normal fetuses from all body sites sampled. The greatest number of HMB-45-positive melanocytes was present in samples from the scalp of the normal fetuses and in those from the lower limbs of the tyr(-) OCA fetus. Very few melanocytes were detected immunohistochemically in samples from the soles and palms, though their presence was confirmed by transmission electron microscopy. The electron microscopic DOPA reaction test enhanced the melanization of melanocytes in samples from the normal fetuses but not in those from the tyr(-) OCA fetus. Postembedding immunogold electron microscopy using the HMB-45 antibody revealed that the HMB-45 antigen was localized mainly on stages I and II melanosomes. The presence of epidermal melanocytes in samples from all fetal body areas obtained at 17-21 weeks of gestation justifies the use of the electron microscopic DOPA reaction test in the prenatal diagnosis of tyr(-) OCA.

Albinism, Oculocutaneous↗
PubMed1995

Intrauterine ultrasonography with a high-frequency probe: preliminary report.

Intrauterine ultrasonography was performed using miniature probes and high-frequency ultrasound to examine the possible clinical applications of this technique to gynecology. There were 44 women in the study population (age 19-76; ten with a normal uterus, nine fibromyoma, 15 endometrial cancer, ten cervical cancer). The probe was easily introduced into the endometrial cavity in 36 subjects (81.8%). No notable complications were encountered. In subjects with a normal uterus, the endometrium in the secretory phase showed a clearly demarcated hyperechoic area, and higher resolution was obtained than with transvaginal scanning. In patients with fibromyomas, myoma nodules were not clearly visualized because of the attenuation of ultrasound. Myometrial invasion of endometrial cancer was estimated correctly in ten of the 11 patients in which the lesion was visualized. In the subjects with cervical cancer, intracervical scanning was completed in only five cases, and an endocervical lesion was identified in three cases. Intrauterine ultrasonography with a high-frequency miniature probe may be a useful tool, especially in the preoperative evaluation of endometrial cancer and possibly in infertility practice. However, this modality does not appear to be satisfactory for cervical lesions.

Adult↗
PubMed1995

Spontaneous resolution of transfusion-associated graft-versus-host disease.

BACKGROUND: Transfusion-associated graft-versus-host disease (TA-GVHD) is a serious complication of blood transfusion that is characterized by high fever, a scaly maculopapular erythematous rash, diarrhea, hepatocellular damage with marked elevation of liver function test values, and pancytopenia. It can occur in immunocompetent as well as immunocompromised recipients. The existence of atypical TA-GVHD that resolves spontaneously and does not exhibit all of the manifestations has been suggested, but there has been to date no documented diagnosis of GVHD supported by evidence of engraftment. CASE REPORT: A female patient presented and was diagnosed with acute myelogenous leukemia (AML:M4), and, after unsuccessful combination chemotherapy, she received a transfusion and developed manifestation of TA-GVHD as well as evidence of chimerism. TA-GVHD was proved by demonstrating Y chromosome-specific genes in the skin by polymerase chain reaction. The manifestations of clinical GVHD abated within 4 months. CONCLUSION: Polymerase chain reaction analysis of Y chromosomes in specimens from female patients is useful in the diagnosis of suspected cases of spontaneously resolving TA-GVHD.

Base Sequence↗
PubMed1995

Members of the NAP/SET family of proteins interact specifically with B-type cyclins.

Cyclin-dependent kinase complexes that contain the same catalytic subunit are able to induce different events at different times during the cell cycle, but the mechanisms by which they do so remain largely unknown. To address this problem, we have used affinity chromatography to identify proteins that bind specifically to mitotic cyclins, with the goal of finding proteins that interact with mitotic cyclins to carry out the events of mitosis. This approach has led to the identification of a 60-kD protein called NAP1 that interacts specifically with members of the cyclin B family. This interaction has been highly conserved during evolution: NAP1 in the Xenopus embryo interacts with cyclins B1 and B2, but not with cyclin A, and the S. cerevisiae homolog of NAP1 interacts with Clb2 but not with Clb3. Genetic experiments in budding yeast indicate that NAP1 plays an important role in the function of Clb2, while biochemical experiments demonstrate that purified NAP1 can be phosphorylated by cyclin B/p34cdc2 kinase complexes, but not by cyclin A/p34cdc2 kinase complexes. These results suggest that NAP1 is a protein involved in the specific functions of cyclin B/p34cdc2 kinase complexes. In addition to NAP1, we found a 43-kD protein in Xenopus that is homologous to NAP1 and also interacts specifically with B-type cyclins. This protein is the Xenopus homolog of the human SET protein, which was previously identified as part of a putative oncogenic fusion protein (Von Lindern et al., 1992).

Amino Acid Sequence↗