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Biomedical subjects

A Okamoto

Publications and source records attributed to A Okamoto.

At least 127 records · Page 7Linked to original sources
PubMed1995

Characteristics of the antitumor activity of M-16 and M-18, major metabolites of a new mitomycin C derivative KW-2149, in mice.

The cell growth inhibitory activity, antitumor activity and toxicity of M-16 and M-18, the major metabolites of a new mitomycin C (MMC) derivative KW-2149, in mouse and human were compared with those of KW-2149 or MMC in vitro and in vivo. The growth inhibitory activity of M-18, a symmetrical disulfide dimer, against human uterine cervix carcinoma HeLa S3 cells was almost equivalent to that of KW-2149 and their IC50 values were about 10-fold smaller than that of MMC. The activity of M-16, a methyl sulfide form, was almost equivalent to that of MMC. The cell-killing activity of MMC and M-16 was augmented in the hypoxic condition, whereas that of KW-2149 and M-18 was reduced. M-16 also exhibited almost equivalent activities to MMC in vivo in terms of many biological profiles, i.e., antitumor activity against murine P388 leukemia, ascitic or solid B16 melanoma or human lung carcinoma xenograft L-27, and bone marrow toxicity in mice. These in vitro and in vivo results indicate that the antitumor activity and toxicity of KW-2149 might not be mediated by M-16 in mice. On the other hand, M-18 exhibited almost equivalent activities to KW-2149 in these regards, suggesting the involvement of M-18 in the biological activities of KW-2149. However, the small values of the area under the curve of M-18 in mice make this unlikely. Thus the biological activities of KW-2149 in mice are not explained by the M-16 or M-18 concentration in plasma and are postulated to be manifested by KW-2149 itself.

Animals↗
PubMed1995

Hematogenous metastases of pancreatic ductal carcinoma.

To investigate the heterogeneity of hematogenous metastases of pancreatic ductal carcinoma, we investigated carcinomatous spread in 130 autopsy cases. Hepatic metastases occurred most frequently, in 81 cases (62%), which may be explained by the fact that all veins draining the pancreas flow into the portal system. We closely examined the 49 cases without hepatic metastases. Sixteen patients had pulmonary metastases without hepatic metastases, whereas seven had peculiar hematogenous metastases without hepatic or pulmonary metastases. Fifteen of these 23 patients had pancreatic body carcinomas. The unusual patterns of spread might be due to (a) hepatofugal portosystemic shunting induced by splenic vein obstruction, (b) retrograde lymphatic infiltration from metastatic tracheobronchial lymph nodes, or (c) aggressive characteristics of the tumors indicated by peculiar histologic features such as pleomorphic or mucoepidermoid carcinoma, etc. Sixteen patients showed only lymph node metastases and 10 had no distant metastases. Seventeen of these 26 cases had pancreatic head carcinoma. Histologically, two patients had mucinous cystadenocarcinomas, and six had adenocarcinomas producing rich mucin. The average age of the group with no distant metastases was higher.

Aged↗
PubMed1995

Efficient production of casoxin D, a bradykinin agonist peptide derived from human casein, by Bacillus brevis.

We efficiently produced a small peptide by the host-vector system using Bacillus brevis as a host. DNA encoding the physiologically functional casoxin D, composed of seven amino acids, was ligated in tandem. An expression-secretion vector containing DNA, which codes for a fusion protein of epidermal growth factor-casoxin D pentamer, was constructed. B. brevis transformed with this plasmid produced about 0.5 g/liter of the fusion protein in the culture supernatant. The fusion protein was purified with ammonium sulfate fractionation from the supernatant and digested with two kinds of proteinases. A peptide well separated by high pressure liquid chromatography was identified as biologically active casoxin D.

Amino Acid Sequence↗
PubMed1995

Effects of high pressure treatment on Ca2+ release and Ca2+ uptake of sarcoplasmic reticulum.

To clarify the mechanism of pressure-induced meat tenderization or acceleration of meat conditioning, the pressure-induced morphological and biochemical changes in sarcoplasmic reticulum (SR), and Ca2+ release from SR in the rabbit skeletal muscle treated with high pressure (100-300 MPa, 5 min) were investigated in comparison with those of the SR from conditioned muscle. The destruction of the membrane structure of the SR expanded with increasing pressure applied to the muscle. Significant changes in the SDS-PAGE profile were not observed in the SR from the pressurized muscle up to 200 MPa, but a marked decrease of the ATPase protein and high-affinity Ca(2+)-binding protein were observed in the SR from the pressurized muscle at 300 MPa. The ATPase activities increased in the SR isolated from the muscle exposed to high pressure up to 200 MPa. When the muscle was pressurized at 300 MPa, the ATPase activity dropped to the same level with that of the SR from the untreated muscle. Ca2+ uptake ability of the SR vesicles measured using a fluorescent chelating reagent decreased with increasing pressure applied to the muscle. Ultrastructural studies showed that Ca2+, which was mainly localized in the SR region of the untreated fiber bundles, was translocated into myofibrillar space in the pressurized muscle. It is clear that a brief exposure of the muscle to high pressure causes considerable changes in membrane structure and biochemical function of SR as compared with those of SR in the muscle induced by conditioning.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1994

Overexpression of human cyclin D1 reduces the transforming growth factor beta (TGF-beta) type II receptor and growth inhibition by TGF-beta 1 in an immortalized human esophageal epithelial cell line.

Cyclin D1 has been implicated in G1 cell cycle progression and is frequently amplified, overtranscribed, and oversynthesized in human tumors, including esophageal carcinomas. To further address the role of cyclin D1 in cell cycle control and tumorigenesis, we have stably transfected the human cyclin D1 in the nontumorigenic esophageal epithelial cell line HET-1A. These transfected cells, which express increased amounts of cyclin D1, have enhanced colony-forming efficiency and saturation density and are resistant to growth inhibition by TGF-beta 1 compared with the parental cell line or a control vector cell clone. The clones which express increased amounts of cyclin D1 exhibited a decrease in the amount of TGF-beta type II receptor, indicating a plausible mechanism for their diminished response to TGF-beta 1. Therefore, deregulated expression of the cyclin D1 gene can modulate the negative growth factor pathway of TGF-beta 1 and may disturb the control of epithelial cell proliferation in esophageal carcinogenesis.

Cell Cycle↗
PubMed1994

Mutations and altered expression of p16INK4 in human cancer.

Cell cycle arrest at the G1 checkpoint allows completion of critical macromolecular events prior to S phase. Regulators of the G1 checkpoint include an inhibitor of cyclin-dependent kinase, p16INK4; two tumor-suppressor proteins, p53 and RB (the product of the retinoblastoma-susceptibility gene); and cyclin D1. Neither p16INK4 nor the RB protein was detected in 28 of 29 tumor cell lines from human lung, esophagus, liver, colon, and pancreas. The presence of p16INK4 protein is inversely correlated with detectable RB or cyclin D1 proteins and is not correlated with p53 mutations. Homozygous deletions of p16INK4 were detected in several cell lines, but intragenic mutations of this gene were unusual in either cell lines or primary tumors. Transfection of the p16INK4 cDNA expression vector into carcinoma cells inhibits their colony-forming efficiency and the p16INK4 expressing cells are selected against with continued passage in vitro. These results are consistent with the hypothesis that p16INK4 is a tumor-suppressor protein and that genetic and epigenetic abnormalities in genes controlling the G1 checkpoint can lead to both escape from senescence and cancer formation.

Base Sequence↗
PubMed1994

Interactions between neutral endopeptidase (EC 3.4.24.11) and the substance P (NK1) receptor expressed in mammalian cells.

Interactions between neutral endopeptidase-24.11 (NEP) and the substance P receptor (SPR; NK1) were investigated by examining substance P (SP) degradation, SP binding and SP-induced Ca2+ mobilization in epithelial cells transfected with cDNA encoding the rat SPR and rat NEP. Expression of NEP accelerated the degradation of SP by intact epithelial cells and by membrane preparations, and degradation was reduced by the NEP inhibitor thiorphan. In cells expressing SPR alone, specific 125I-SP binding after 20 min incubation at 37 degrees C was 92.2 +/- 3.1% of maximal binding and was unaffected by thiorphan. Coexpression of NEP in the same cells as the SPR markedly reduced SP binding to 13.9 +/- 0.5% of maximal, and binding was increased to 82.7 +/- 2.4% of maximal with thiorphan. Coexpression of NEP in the same cells as the SPR also reduced to undetectable the increase in intracellular Ca2+ in response to low concentrations of SP (0.3 and 0.5 nM), and significantly reduced the response to higher concentrations (1 and 3 nM). The Ca2+ response was restored to control values by inhibition of NEP with thiorphan. In contrast, SP binding and SP-induced Ca2+ mobilization were only slightly reduced when cells expressing SPR alone were mixed with a 3- to 24-fold excess of cells expressing NEP alone. Therefore, in this system, NEP markedly down-regulates SP binding and SP-induced Ca2+ mobilization only when coexpressed in the same cells as the SPR.

Amino Acid Sequence↗
PubMed1994

Characteristics of antitumor activity of KW-2189, a novel water-soluble derivative of duocarmycin, against murine and human tumors.

Methyl(1S)-1-bromomethyl-7-methyl-5-[(4-methylpiperazinyl)-carb onyloxy]- 3-[(5,6,7-trimethoxy-2-indolyl)-carbonyl]-1,2-dihydro-3H-pyrolo[3, 2-e] indole-8-carboxylate hydrobromide (KW-2189), a novel derivative of duocarmycin B2, was selected for extensive evaluation based on its improved antitumor activity, water solubility, and stability in the culture medium, as compared with duocarmycin B2. Although the in vitro cell growth-inhibitory activity of KW-2189 was less potent than that of duocarmycin B2, it significantly inhibited the growth of five murine solid tumors including Colon 26 adenocarcinoma, Colon 38 adenocarcinoma, and B16 melanoma in vivo. KW-2189 was also effective against murine P388 leukemia and L1210 leukemia not only by local administration (i.p.-i.p. system), but also by systemic administration (i.p.-i.v. or i.v.-i.v. system). The most remarkable feature of KW-2189 was its efficacy against various human xenografts, which was observed in 14 tumors among 16 tested tumors including drug-insensitive tumors by single i.v. administration. Tumor regression was observed in mice bearing LC-6 lung, St-4 and St-40 stomach, Li-7 liver, PAN-2 pancreas, and MX-1 breast carcinomas. In many cases, the activities of KW-2189 were more than those of clinically active agents, mitomycin C, Adriamycin, cisplatin, and cyclophosphamide. Delayed lethal toxicity, which was reported in mice treated with CC-1065 whose structure was similar to KW-2189, was not observed in mice treated with KW-2189. KW-2189 inhibited DNA synthesis more significantly than RNA or protein synthesis, although DNA strand breaks were not observed. KW-2189 was activated by porcine liver esterase, mouse liver homogenate or Hep G2 homogenate, and DU-86-DNA adducts were detected in KW-2189-treated HeLa S3 cells, suggesting that KW-2189 was converted to DU-86 in the cells. These results indicate that KW-2189 is an interesting candidate for further development as a novel antitumor agent.

Animals↗
PubMed1994

Construction of aminotransferase chimeras and analysis of their substrate specificity.

Escherichia coli aspartate aminotransferase (AspAT) and E. coli aromatic amino acid aminotransferase (AroAT) have almost identical and high activities toward acidic amino acid substrates. AroAT also has high activity toward aromatic amino acid substrates. The two proteins have 44% amino acid sequence homology. In order to study the mechanism responsible for the different substrate specificities of these aminotransferases, chimeric enzymes of AspAT and AroAT were constructed using homologous recombination in E. coli cells. Five chimeric enzymes were obtained, even though the nucleotide sequence homology between the two parent enzymes was as low as about 50%. The yields of the legitimate chimeric genes were related to the lengths of the homologous region between the two parent genes. Homologous recombination occurred in the region where more than eight nucleotides out of ten were identical. The substrate specificity of the chimeric enzymes suggest that not only the amino acid residues in the active site but also those distant from the active site contribute to the substrate specificity of the parental aminotransferases.

Amino Acid Sequence↗
PubMed1994

X-ray crystallographic study of pyridoxal 5'-phosphate-type aspartate aminotransferases from Escherichia coli in open and closed form.

We determined the three-dimensional structures of aspartate aminotransferase (AspAT) from Escherichia coli and its complex with inhibitor (2-methyl-L-aspartate) at 1.8A resolution. This enzyme reversibly catalyzes the transamination reaction and is a dimer of two identical subunits. Each subunit has 396 amino acid residues and one pyridoxal 5'-phosphate as a cofactor, and is divided into two domains, one large and the other small. Upon binding of the inhibitor, the small domain rotates by 5 degrees toward the large domain to close the active site. This domain movement is caused mainly by small but important main-chain conformational changes in the residues located over the domain interface of the small domain. In chicken mitochondrial AspAT, the domain movement was larger, with a rotational angle of 13 degrees. By comparison of these two structures, the difference in the rotational angles was found to be caused by the larger opening of the domain in the open form of chicken mitochondrial AspAT. Although the overall structures of these two enzymes were almost identical, the surface area of the domain interface in the E. coli enzyme was larger than that in mitochondrial AspAT, suggesting that the structure of the domain interface is responsible for the degree of movement of the small domain.

Aspartate Aminotransferases↗
PubMed1994

Differential effect of duocarmycin A and its novel derivative DU-86 on DNA strand breaks in HeLa S3 cells.

Duocarmycin A (DUMA) and DU-86, a semisynthetic derivative of duocarmycins (DUMs) and a possible active form of KW-2189, both showed potent cell growth-inhibitory and cell-killing activities against human uterine cervix carcinoma HeLa S3 cells. Both drugs showed similar profiles of inhibition of macromolecular synthesis and influence on cell-cycle distribution. Namely, they inhibited [3H]thymidine uptake at lower concentrations than [3H]uridine or [3H]leucine uptake, suggesting that the inhibition of DNA synthesis is the primary site of their actions. Furthermore, they induced the accumulation of cells in early S phase. However, a significant difference was observed between these drugs in terms of DNA-fragmentation activity against HeLa S3 cells by using two independent methods, pulse-field gel electrophoresis and alkaline elution. DNA fragmentation was insignificant in the cells treated with DU-86, in contrast to the cells treated with DUMA. The analysis of DNA adducts in the cells revealed that DU-86 alkylated adenine quite selectively, while DUMA alkylated both adenine and guanine. These results suggest that the pyrrolidone ring of DUMA is responsible for its adduct formation with guanine and the subsequent DNA-fragmentation and inhibition of DNA synthesis, while DU-86 alkylated adenine and inhibited DNA synthesis through mechanisms other than DNA-fragmentation.

Adenine↗
PubMed1994

Temporary change of compound action potential amplitude after intense sound exposure.

Intense sound induces a temporary or permanent threshold shift. The aim of this study is to make an experimental model for temporary threshold shift (TTS) using guinea pigs. In this study, two kinds of sound exposure were used for inducing a threshold shift in the guinea pig; one of 110 dB SPL at 10 kHz for 10 min and the other of 120 dB SPL at 10 kHz for 5 min. The former condition produced different levels of reduction in compound action potential (CAP) immediately after sound exposure and different steady levels of CAP amplitude after recovery (within 120 min) in 10 guinea pigs. The latter condition produced a total loss of CAP amplitude immediately after sound exposure in 8 of 9 guinea pigs, and this condition continued for 120 min in 7 of the 9 guinea pigs. Additionally, in 2 of 3 guinea pigs which were exposed the sound of 120 dB SPL at 10 kHz for 5 min, the CAP amplitudes were almost the same as those of control animals 1 day after the exposure. One the basis of these results, we consider the sound of 120 dB SPL at 10 kHz for 5 min to be the optimal intensity and frequency of sound and duration of exposure for producing a TTS in the guinea pig.

Acoustic Stimulation↗
PubMed1994

In vivo wear pattern of experimental composite resins containing different filler components.

Using a scanning electron microscope (SEM) and an electron probe surface roughness analyzer (ERA), we conducted the in vivo study of the effect of various filler components on the wear of composite resins. Experimental light-cured composite resins were prepared employing three different filler components; -(1) Silica type, (2) Strontium type, and (3) Barium type. The filler content for all three types was 80 wt%, with the mean particlesize being 2.6 microns in diameter. The resin monomers consisted of 40 wt% Bis-GMA, 40 wt% TEGDMA and 20 wt% UTMA. These materials were placed in 2 mm diameter cylindrical cavities located in the OCA (occlusal contact area) or the CFA (contact free area) of cast crowns temporarily set in a volunteer patient's mouth. The crowns were removed at monthly intervals for longitudinal SEM observation. After two months, worn surfaces were also analyzed by ERA. The result showed that the wear patterns of the composites were characterized by the filler components, especially in the OCA.

Adult↗
PubMed1994

Characterization of antibodies to the rat substance P (NK-1) receptor and to a chimeric substance P receptor expressed in mammalian cells.

Antibodies to neuropeptide receptors can be used to localize and characterize the receptors in tissues and cell lines. Two strategies were used to study the rat substance P receptor (SPR, NK-1) by immunological methods. First, a polyclonal antiserum was raised by immunizing rabbits with a peptide corresponding to the 15 amino acid residues (KTMTESSSFYSNMLA, SPR393-407) at the intracellular C-terminus of the rat SPR coupled to bovine thyroglobulin. An antiserum was obtained with a titer for half-maximal binding of 125I-SPR393-407 of 1:70,000. Nonradioactive SPR393-407 inhibited 50% of binding at a concentration of 10 pM. Binding of 125I-SPR393-407 to the antiserum was also displaced in a parallel manner by membrane proteins from tissues expressing high levels of the SPR (brain and submaxillary gland). Second, a chimeric SPR construct of a hydrophilic Flag peptide (DYKDDDDK) genetically engineered in sequence with the extracellular N-terminus of rat SPR was generated by polymerase chain reaction. The Flag-SPR chimera was expressed in rat kidney epithelial cells (KNRK) and judged to be fully functional, assessed by binding of 125I-substance P (apparent Kd of 5.63 nM) and calcium mobilization in response to substance P (EC50 of 0.66 nM). Antibodies to SPR393-407 and the Flag peptide stained the plasma membrane of KNRK cells expressing the native SPR or the Flag-SPR chimera. Staining was abolished by preincubation with SPR393-407 or the Flag peptide. Cells transfected with vector alone were unstained. The SPR antiserum recognized a broad protein band on Western blots of membranes prepared from cells expressing SPR but not from cells transfected with vector alone. The signal was quenched by preincubation of the antiserum with SPR393-407. By immunohistochemistry, the SPR antiserum was found to bind to neurons in the dorsal horn of the rat spinal cord and to ganglion cells in the myenteric plexus of the rat ileum near substance P-immunoreactive nerve fibers. Staining was abolished by preabsorption of the antiserum with SPR393-407. These antibodies can be used to localize the SPR in tissues and cells and to examine the function of the receptor in cell lines.

Amino Acid Sequence↗