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Biomedical subjects

K Miki

Publications and source records attributed to K Miki.

At least 253 records · Page 14Linked to original sources
PubMed1993

A paracrine migration-stimulating factor for metastatic tumor cells secreted by mouse hepatic sinusoidal endothelial cells: identification as complement component C3b.

Selective malignant cell invasion at secondary sites mediated by organ-specific (paracrine) motility factors may be of importance in preferential organ colonization of metastatic cells. In this study we isolated and examined a migration-stimulating activity present in mouse hepatic sinusoidal endothelial cell-conditioned medium (HSE-CM). HSE-CM contains migration-stimulating activity for highly liver-metastatic (RAW117-H10) and highly lung- and liver-metastatic (RAW117-L17) mouse large cell lymphoma sublines but not for the poorly metastatic parental line (RAW117-P). A migration-stimulating factor for H10 cells was purified from HSE-CM by hydroxylapatite affinity and DEAE anion exchange chromatography, Sephacryl S-200 gel filtration, and preparative native gel electrophoresis. The activity in each of the purification fractions was measured in a Transwell chamber assay using 3-microns diameter pore filters. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, the component migrated as a single component of M(r) > 200,000 (nonreducing conditions) or as two components or M(r) approximately 110,000 and approximately 67,000 (reducing conditions). The factor was bound to Concanavalin A-Sepharose but not to heparin- or gelatin-Sepharose affinity columns, induced mainly H10 chemotactic cell activity and some chemokinetic activity, and preferentially stimulated the chemotaxis of liver-colonizing RAW117 sublines (H10 > L17 > P). NH2-terminal amino acid sequence analysis of each subunit indicated that the HSE-CM-derived migration-stimulating factor was a proteolytic fragment of complement component C3. HSE-derived migration-stimulating factors may be important in determining the ability of RAW117 tumor cells to invade and colonize the liver.

Amino Acid Sequence↗
PubMed1993

Crystallization and preliminary X-ray diffraction studies of photolyase (photoreactivating enzyme) from the cyanobacterium Anacystis nidulans.

Photolyase (photoreactivating enzyme) from the cyanobacterium Anacystis nidulans was crystallized by the hanging drop vapor diffusion procedure using ammonium sulfate as a precipitant. The pale-yellow crystals were grown to a size of 0.4 mm in length and 0.1 mm in diameter. They belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 90.7 A and c = 135 A. Assuming that the asymmetric unit contains one molecule, the Vm value is calculated as 2.6 A3/dalton. The crystals are stable towards X-ray exposure and diffract beyond 2.5 A resolution.

Crystallization↗
PubMed1993

PDI and glutathione-mediated reduction of the glutathionylated variant of human lysozyme.

A mutant human lysozyme, designated as C77A-a, in which glutathione is bound to Cys95, has been shown to mimic an intermediate in the formation of a disulfide bond during folding of human (h)-lysozyme. Protein disulfide isomerase (PDI), which is believed to catalyze disulfide bond formation and associated protein folding in the endoplasmic reticulum, attacked the glutathionylated h-lysozyme C77A-a to dissociate the glutathione molecule. Structural analyses showed that the protein is folded and that the structure around the disulfide bond, buried in a hydrophobic core, between the protein and the bound glutathione is fairly rigid. Thioredoxin, which has higher reducing activity of protein disulfides than PDI, catalyzed the reduction with lower efficiency. These results strongly suggest that PDI can catalyze the disulfide formation in intermediates with compact structure like the native states in the later step of in vivo protein folding.

Animals↗
PubMed1993

Crystallization and preliminary X-ray diffraction studies of C-1027-AG, the apoprotein of the macromolecular antitumor antibiotic C-1027 from Streptomyces globisporus.

C-1027-AG, the apoprotein of the macromolecular antitumor antibiotic C- 1027, isolated from Streptomyces globisporus, was crystallized by the vapor-diffusion procedure using 2-methyl-2,4-pentanediol as a precipitant. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit-cell dimensions a = 55.1, b = 61.3 and c = 79.1 A. Assuming that the asymmetric unit contains two or three molecules, the V(m) value is calculated as 3.2 or 2.1 A(3) Da(-1), respectively. A total of 7630 independent reflections was obtained up to 2.5 A resolution with synchrotron radiation, the merging R factor being 0.077 for 24 713 measurements.

Journal Article↗
PubMed1993

An antisense oligodeoxynucleotide that depletes RI alpha subunit of cyclic AMP-dependent protein kinase induces growth inhibition in human cancer cells.

Enhanced expression of the RI alpha subunit of cyclic AMP-dependent protein kinase type I has been correlated with cancer cell growth. We provide evidence that RI alpha is a growth-inducing protein that may be essential for neoplastic cell growth. Human colon, breast, and gastric carcinoma and neuroblastoma cell lines exposed to a 21-mer human RI alpha antisense phosphorothioate oligodeoxynucleotide (S-oligodeoxynucleotide) exhibited growth inhibition with no sign of cytotoxicity. Mismatched sequence (random) S-oligodeoxynucleotides of the same length exhibited no effect. The growth inhibitory effect of RI alpha antisense oligomer correlated with a decrease in the RI alpha mRNA and protein levels and with an increase in RII beta (the regulatory subunit of protein kinase type II) expression. The growth inhibition was abolished, however, when cells were exposed simultaneously to both RI alpha and RII beta antisense S-oligodeoxynucleotides. The RII beta antisense S-oligodeoxynucleotide alone, exhibiting suppression of RII beta along with enhancement of RI alpha expression, led to slight stimulation of cell growth. These results demonstrate that two isoforms of cyclic AMP receptor proteins, RI alpha and RII beta, are reciprocally related in the growth control of cancer cells and that the RI alpha antisense oligodeoxynucleotide, which efficiently depletes the growth stimulatory RI alpha, is a powerful biological tool toward suppression of malignancy.

Amino Acid Sequence↗
PubMed1993

Transient gene expression by SV40 promoter characterizes sequential differentiation of embryonal carcinoma F9 cells into primitive and visceral endoderm.

Transient expression of the chloramphenicol acetyltransferase (CAT) gene under the control of simian virus 40 (SV40)y Moloney murine leukemia virus, human T cell leukemia virus, and cytomegalovirus promoters was stimulated by the differentiation of F9 stem cells into primitive endoderm, but repressed again by further differentiation into visceral endoderm. Deletion mutants of the SV40 enhancer showed that a similar set of motifs is critical for CAT expression at all stages of F9 differentiation, but differentiation dependency was observed even in their absence. The stability of transient gene expression under the control of the SV40 promoter was markedly dependent on F9 differentiation. Appreciable expression was detected even in undifferentiated F9 cells immediately after gene transfection, was maximal at 12 h and declined rapidly thereafter. On the other hand, expression in primitive endoderm increased until 72 h. The decline was accelerated again in visceral endoderm. This shift was somewhat specific to the virus promoter since CAT expression in undifferentiated F9 cells under the control of the elongation factor 1 alpha promoter was more stable than for virus promoters tested. Thus, the change in stability of expression is important for differentiation-dependent virus promoter activity.

Animals↗
PubMed1993

Antigenicities of Group I and II hepatitis C virus polypeptides--molecular basis of diagnosis.

Comparative nucleotide sequence studies on the putative NS3 and NS4 regions of the genomes of hepatitis C viruses (HCV) have revealed that there are at least two groups of HCV, group I and group II. The cDNA clone E, corresponding to a boundary between the NS3 and NS4 (NS3-4) region of the group II HCV genome, encodes antigens that react to antibodies specific to group II HCV (Tsukiyama-Kohara et al. (1991) Virus Genes 5, 243-254). To understand the molecular basis of the group-specific antigenicity of HCV peptides, the predicted amino acid sequences around the NS3-4 region of our group II HCV cDNAs were compared with those of other HCV isolates. The analysis revealed the presence of group-specific amino acids in this peptide region. Evolutionary analysis of nucleotide sequences within this region of these HCV isolates also led to the same classification. A similar result was obtained by sequence analysis of cloned cDNAs corresponding to the core region. A cDNA of the group II HCV core region was prepared by polymerase chain reaction from the cDNA synthesized with group II-specific primer complementary to the NS3-4 region. The products directed by the cDNA of the core region did not have group-specific antigenicity. The NS3 peptide region also appeared not to carry group-specific antigens. Our results indicate that most HCV isolates can be classified into either group I or II, and that the existence of two groups of HCV does not disturb HCV diagnosis as long as core and/or NS3 peptides are used to detect HCV antibodies.

Amino Acid Sequence↗
PubMed1993

Severe pulmonary pneumocystosis in simian acquired immunodeficiency syndrome induced by simian immunodeficiency virus: its characterization by the polymerase-chain-reaction method and failure of experimental transmission to immunodeficient animals.

Pneumocystis carinii (Pc) infection was observed in three of five rhesus monkeys infected with simian immunodeficiency virus (SIVmac251). They showed severe symptoms similar to those associated with human acquired immunodeficiency syndrome (AIDS). Histopathology revealed severe pulmonary pneumocystosis in one of three Pc-positive monkeys, and anti-Pc antibodies were detected in sera from two of the three monkeys. Localization of Pc organisms in various organs of the monkeys was examined by the polymerase-chain-reaction (PCR) method, and Pc-specific bands of DNA amplification were detected in the liver, kidney, spleen, adrenal gland, testis, brain, and other organs examined, but no Pc organism was found in these organs by histopathologic examination. These results suggest that the activation of a latent infection of Pc occurs in SIV-infected rhesus monkeys as well as in human AIDS. Experimental transmission of Pc derived from a simian was attempted in severe combined immunodeficiency (SCID) mice and athymic nude (rnu/rnu, F344) rats. These animals were inoculated intranasally with 10(4) Pc cysts, but neither histopathologic changes nor Pc organisms were detected in SCID mice at 4 months after inoculation or in nude rats at 2 months postinoculation, suggesting that simian Pc is species-specific.

Animals↗
PubMed1993

In vitro stability of biphasic calcium phosphate ceramics.

Biphasic calcium phosphate ceramics consist of both beta-tricalcium phosphate and hydroxyapatite. This study was performed to examine the in vitro stability of biphasic calcium phosphate ceramics in deionized distilled water and in physiological solutions. Biphasic calcium phosphates of two different molar ratios and pure hydroxyapatite and pure beta-tricalcium phosphate granules were tested. X-ray diffraction data were obtained before and after immersion. Surface morphology was compared using scanning electron microscopy. The pure beta-tricalcium phosphate and the beta-tricalcium phosphate in the biphasic calcium phosphate ceramics gradually converted to apatite. Needle-like apatite crystals were observed on the surface of biphasic calcium phosphate and beta-tricalcium phosphate, but hydroxyapatite showed no change.

Biocompatible Materials↗
PubMed1993

DNA methylation of the pepsinogen 1 gene during rat glandular stomach carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine or catechol.

The methylation patterns of the rat pepsinogen 1 (Pg1) gene in preneoplastic and neoplastic stomach lesions induced by genotoxic N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or the non-genotoxic carcinogen catechol were investigated. Male WKY/Ncrj rats were given MNNG in their drinking water (50 mg/l) for 30 weeks or 0.8% catechol throughout the experiment (60 weeks). MNNG induced Pg1 altered pyloric glands (PAPG), adenomatous hyperplasias and well-differentiated adenocarcinomas. Catechol also induced PAPG and adenomatous hyperplasias although cancers did not develop. Adenomatous hyperplasias and adenocarcinomas all consisted of gastric type cells resembling surface mucous cells or pyloric gland cells with little or no Pg1 expression. In MNNG-induced stomach cancers generally lacking Pg1, altered Pg1 gene methylation was observed with both CCGG and GCGC sites being methylated more than normal pyloric mucosa. MNNG or catechol-induced adenomatous hyperplasias also demonstrated essentially the same methylation changes in the CCGG, but not in the GCGC sites. In the mucosa containing PAPG in groups treated with MNNG or catechol the methylation patterns of the Pg1 gene were quite similar to those of normal pyloric mucosa, although the CCGG sites tended to demonstrate slightly increased methylation. The results suggest that the altered methylation of the Pg1 gene observed in stomach cancers is acquired early in the carcinogenic process and progressive methylation changes occur with tumor development.

Adenocarcinoma↗
PubMed1993

Further analyses of epitopes for human monoclonal anti-basement membrane zone antibodies produced by stable human hybridoma cell lines constructed with Epstein-Barr virus transformants.

We previously established Epstein-Barr virus (EBV)-transformed bullous pemphigoid (BP) patient lymphoblastoid cell lines, which produced human monoclonal anti-basement membrane zone antibodies. In the present study, we established two independent human-human hybridomas by fusion of these EBV transformants with a human B-cell line. These hybridomas, designated 5E-HY-4B and 10D-HY-8B, were very stable and showed a high yield of monoclonal antibody (MoAb) secretion. Each cell line was tetraploid and showed combined rearranged segments of immunoglobulin heavy-chain gene derived from both an EBV transformant and a parent cell. Immunoblot analysis showed that the 5E-HY-4B MoAb recognized the 230-kDa BP antigen but that the 10D-HY-8B MoAb did not show any reactivity. In contrast, both MoAbs precipitated the 230-kDa BP antigen with immunoprecipitation. These results indicate that the two MoAbs reacted with different epitopes on the 230-kDa BP antigen: a continuous epitope for the 5E-HY-4B MoAb and a conformation-dependent epitope for the 10D-HY-8B MoAb. This speculation was confirmed at the molecular level by the result that the fusion protein produced by a partial cDNA for the 230-kDa mouse BP antigen reacted with the 5E-HY-4B MoAb but not with the 10D-HY-8B MoAb. Furthermore, the study of the reactivity with fusion proteins of a series of deleted clones restricted the epitope for the 5E-HY-4B MoAb within the region with 114 amino acid residues in the C-terminal domain of the 230-kDa BP antigen.

Antibodies, Monoclonal↗
PubMed1993

Clinical application of serum pepsinogen I and II levels for mass screening to detect gastric cancer.

A considerable number of gastric cancers derive from stomach mucosa where chronic atrophic gastritis is severe and extensive. Based on the fact that the serum pepsinogen levels provide a precise measure of the extent of chronic atrophic gastritis, we have devised a mass screening method involving serum pepsinogen measurement to identify subjects at high risk of gastric cancer. In 1991, we screened 4,647 workers (male: 4,113, female: 534, mean age: 49.0 years) at a Japanese company using this method. Out of 875 subjects (18.8%) with a serum pepsinogen I level of less than 50 micrograms/liter and a pepsinogen I/II ratio of less than 3.0, 676 subjects (14.5%) were selected for further investigation by endoscopy. This led to the detection of four subjects (0.086%) with gastric cancer (three in an early stage) and four subjects with adenoma. The cancer detection rate of this new screening method was comparable, and in some respects superior, to that of the traditional barium X-ray screening. Since the incidence of test-positive subjects was as low as 10% amongst subjects aged less than 40, this screening method appears to be especially useful for screening of younger generations. The new method is less expensive than the traditional barium X-ray and subjects experience little discomfort. Further, many serum samples can be quickly measured simultaneously. The results of this study have indicated that serum pepsinogen screening provides a valuable method for detecting gastric cancers.

Adenoma↗
PubMed1993

Markers of surface mucous cell type human gastric cancer cells: galactose oxidase-Schiff reactive mucins, monoclonal antibody SH-9 reactive mucins and cathepsin E.

Cellular differentiation of gastric cancer cells allows the classification of cell type into surface mucous cell, pyloric gland cell, intestinal absorptive cell and goblet cell types by mucin histochemistry and pepsinogen (Pg) immunohistochemistry. Surface mucous cell differentiation of gastric cancers of each histologic type has previously been detected by the galactose oxidase-Schiff (GOS) reaction although this is not always positive in all cases. Mucus granules of surface mucous cells of normal gastric mucosa show an intense reactivity for SH-9 (monoclonal antibody against CA125-bearing antigenic molecule fragments). Cathepsin E is also expressed in the cytoplasm of surface mucous cells, weakly in absorptive cells of duodenal villi and occasionally in pyloric gland cells. Expression of SH-9 reactive mucin and of cathepsin E were therefore investigated as possible additional markers to distinguish between the gastric cancer cell type in 203 primary stomach cancers. SH-9 reactive mucin was found selectively in GOS positive cancer cells of surface mucous cell type and/or cancer cells unclassified by mucin histochemistry. These latter cells were therefore classified into the surface mucous cell category. Cathepsin E was found mainly in cancer cells of the GOS positive surface mucous cell type and occasionally, in intestinal absorptive and pyloric gland cell types. Galactose oxidase-Schiff, SH-9 and cathepsin E reactive or positive cancer cells were found in 145 (71.4%), 151 (74.4%) and 144 (70.9%), respectively, of the 203 primary stomach cancers investigated.

Antibodies, Monoclonal↗
PubMed1993

Expression of sialosyl-Tn in intestinal type cancer cells of human gastric cancers.

A sample of 219 primary stomach cancers, 143 advanced cancers and 76 early cancers were examined for mucin histochemical staining (the paradoxical concanavalin A method, the galactose oxidase-Schiff [GOS] reaction, and the sialidase-GOS reaction) and immunohistochemical reactivity (pepsinogen [Pg] I, Pg II, SH-9 and TKH-2). Gastric cancer cells were clearly classified according to mucin histochemistry into a gastric type, including mucus neck cell, pyloric gland cell and surface mucus cell types, and an intestinal type, including goblet-cell, and intestinal absorptive cell types. TKH-2 monoclonal antibody, which recognizes the mucin-associated sialosyl-Tn antigen, reacted with the mucin of goblet cells in both the normal small intestine and in the intestinal metaplasia of the stomach. Sixty-five of 106 (61%) differentiated adenocarcinomas and 76 of 113 (67%) undifferentiated adenocarcinomas had over 10% of their cancer cells positive for TKH-2. The TKH-2-positive cancers were primarily classified as a goblet-cell type by mucin-histochemical staining and the other immunohistochemical staining methods. Therefore, it is concluded that sialosyl-Tn is an excellent marker of small intestinal mucins and is indicative of a small intestinal type of differentiation in two-thirds of gastric cancers.

Adult↗
PubMed1993

Cardiac-renal-neural reflex plays a major role in natriuresis induced by left atrial distension.

The role of cardiac-renal-neural reflex in the natriuresis induced by left atrial balloon inflation was investigated in conscious dogs. Female mongrel dogs were assigned randomly to 1) sham-operated (n = 8), 2) cardiac-denervated (n = 6), and 3) renal-denervated (n = 8) groups. The dogs were chronically instrumented with a bipolar stainless steel wire electrode for measurement of renal sympathetic nerve activity (RSNA). Balloon inflation induced a step increase in left atrial pressure (Pla) by 7.7 +/- 1.7 mmHg, a step decrease in RSNA (-66.6 +/- 5.5%), and concomitant increases in urine flow (441 +/- 142%), osmolal excretion (60 +/- 12%), and sodium excretion (300 +/- 69%) in sham-operated dogs. Renal denervation abolished the diuresis and natriuresis during balloon inflation. Chronic cardiac denervation abolished also the diuresis and natriuresis in the face of a similar increase in Pla. RSNA did not change significantly throughout the experimental period in cardiac-denervated dogs. It is concluded that a sustained reduction of RSNA originating from left atrial mechanoreceptors plays a major role in the natriuresis during left atrial distension in conscious dogs.

Animals↗
PubMed1993

Desmoyokin, a 680 kDa keratinocyte plasma membrane-associated protein, is homologous to the protein encoded by human gene AHNAK.

We have obtained a monoclonal antibody (33A-3D) that specifically recognize desmoyokin, a 680 kDa desmosomal plaque protein that is well characterized in bovine muzzle epidermis. A cDNA clone (DY6, 3693 bp) was isolated by immunoscreening a mouse keratinocyte expression library with 33A-3D, and it was confirmed that DY6 has a partial coding sequence for desmoyokin. DY6 consists of highly homologous repeats about 128 residues long. Furthermore, the 128-residue repeats exhibit a quasi seven-residue substructure, which we believe will adopt an antiparallel beta-sheet structure. Surprisingly, the amino acid sequence showed a significant homology with AHNAK, a newly identified human gene encoding a 700 kDa protein, which was suggested to be down-regulated in neuroblastoma. From its extensive homology, the similarity in both size and structure, and the identical patterns on Southern blot analysis of genomic DNAs, desmoyokin and AHNAK protein are thought to be identical. Although the desmoyokin/AHNAK protein is detected in a variety of cell types at both protein and mRNA levels, its distribution in keratinocytes (associated closely with cell membrane) is quite different from that in cells other than keratinocytes (distributed diffusely in the cytoplasm). These findings suggest that the desmoyokin/AHNAK protein is a ubiquitous molecule with a unique structure and appears to have different distributions (and probably different functions) among different cells.

Amino Acid Sequence↗