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K Inoue

Publications and source records attributed to K Inoue.

At least 1,027 records · Page 57Linked to original sources
PubMed1996

Extended fitness of variable region primers by a novel PCR protocol.

In an attempt to isolate the variable region genes of five monoclonal natural autoantibodies, we failed to amplify all ten variable region cDNAs using a V region primer set and following conventional PCR protocols. A novel PCR procedure was established in which the reaction began with three cycles of low temperature annealing and chain extension using 5'-primer as the only primer present, and continuing with conventional PCR cycles following addition of 3'-primer. Combined with solid-phase cDNA synthesis on oligo(dT)cellulose, this new protocol rescued all failed PCR amplifications based on a conventional protocol, thereby extending the universal utility of the V region primers. A notable increase in amplification specificity was also achieved. Such a protocol could be useful in primer design and the amplification of non-antibody genes whose sequences are not well-understood.

Animals↗
PubMed1996

Release of secretory phospholipase A2 from rat neuronal cells and its possible function in the regulation of catecholamine secretion.

Here we show that secretory phospholipase A2 (sPLA2) that is immunochemically indistinguishable from type II sPLA2 is (i) stored in neuroendocrine cells, (ii) released in response to neurotransmitters or depolarization, and (iii) involved in the regulation of catecholamine secretion by these cells. Rat brain synaptic vesicle fractions contained PLA2 activity, which was neutralized completely by an antibody raised against rat type II sPLA2. sPLA2 immunoreactive with anti-(type II sPLA2) antibody was released from synaptosomes in response to depolarization evoked by a high concentration of potassium in the presence of Ca2+. Rat pheochromocytoma PC12 cells, which differentiated into adherent cells similar to sympathetic neurons in response to nerve growth factor, were used for the detailed analysis of the dynamics and function of sPLA2 in neuronal cells. Antibody against rat type II sPLA2 precipitated approximately 80% of the PLA2 activity in PC12 cell lysates. Transcript for type II sPLA2 was detected in PC12 cells by reverse transcriptase-PCR. When neuronally differentiated PC12 cells were stimulated with carbamylcholine or potassium, sPLA2 was released into the medium and reached a maximal approximately 40% release by 15 min. Inhibitors specific to type II sPLA2 suppressed catecholamine secretion by PC12 cells which had been activated by carbamylcholine. Furthermore, treatment of PC12 cells with exogenous type II sPLA2 alone elicited catecholamine secretion. These observations indicate that sPLA2 released from neuronal cells may regulate the degranulation process leading to release of neurotransmitters and are compatible with our earlier finding that this enzyme is involved in the degranulation of rat mast cells.

Adrenal Gland Neoplasms↗
PubMed1996

Molecular cloning and bacterial expression of a cDNA encoding furostanol glycoside 26-O-beta-glucosidase of Costus speciosus.

Furostanol glycoside 26-O-beta-glucosidase (F26G) purified from Costus speciosus rhizomes was digested with endoproteinase, and several internal peptide fragments were obtained. Degenerate oligonucleotide primers based on amino acid sequences of the peptides were used for amplification of F26G cDNA fragments by applying nested polymerase chain reactions to cDNAs from in vitro cultured plantlets of C. speciosus. Using primers based on sequences of the cDNA fragments, the 5'- and 3'-end clones were isolated by rapid amplification of cDNA ends (RACE) methods. Finally, the entire coding portion of F26G cDNA was cloned by using primers designed from sequences of the RACE products. The deduced amino acid sequence of CSF26G1, the protein encoded by the cloned cDNA, consists of 562 amino acids and shows high homology to a widely distributed family of beta-glucosidases (BGA family). Cell-free homogenate of Escherichia coli expressing CSF26G1 cDNA showed beta-glucosidase activity specific for cleavage of the C-26 glucosidic bond of furostanol glycosides.

Amino Acid Sequence↗
PubMed1996

Cloning, sequencing and sites of expression of genes for the hydroxyarginine-containing adhesive-plaque protein of the mussel Mytilus galloprovincialis.

A segment of Mytilus galloprovincialis foot protein 3 (Mgfp-3) cDNA was amplified by means of reverse-transcription (RT)/PCR with degenerate primers. The 5' and 3' regions of the cloned segment were amplified by means of rapid amplification of cDNA ends. The 5'-region clones had almost identical nucleotide sequences, but two sequences were found among 3'-region clones. The Mgfp-3 coding region was amplified between a 5' untranslated sequence and one of two 3' untranslated sequences. Two cDNA clones which encoded variants Mgfp-3A and Mgfp-3B, were isolated. These two clones encoded proteins with 70 and 77 amino acid residues, of which the first 24 residues are predicted to be signal peptides. The existence of additional variants was suggested by the sequences of other clones. Thus, it was suggested that Mgfp-3 genes constitute a gene family. RT/PCR of RNA from developing larvae indicates that Mgfp-3 genes are transcribed after settlement. RT/PCR of RNA from major organs and in situ hybridization of the foot indicate that Mgfp-3 genes are transcribed in a limited part of the foot, i.e., the phenol gland and a distal part of the accessory gland.

Amino Acid Sequence↗
PubMed1996

Angiosarcoma of the Breast: Immunohistochemical Demonstration of Steroid Receptors and Literature Review.

A 44-year-old female with bilateral angiosarcoma of the breast with multiple skin metastases is reported. She presented with a 6x8 cm and 1x1 cm mass in her left and right breasts, respectively, and multiple skin lesions around the chest. Histologically, the excised tumor was diagnosed as angiosarcoma of the breast with skin metastasis. Tumor cells were immunohistochemically positive with factor VIII-related antigen, CD34, alpha-smooth muscle actin and vimentin, and positive with UEA-I lectin. The majority of tumor cell nuclei expressed progesterone receptor (PgR) protein but estrogen receptro (ER) protein and androgen receptor (AR) protein were nagative. According to the literature, angiosarcoma of the breast mostly affects females in their fourth (33%) and fifth (40%) decades (mean age, 39.5 +/- 10.7 years). The survival rate depends on tumor size and tumor differentiation. The 3-year survival rate is 86% in patients with a tumor size less than 5 cm, and 90% in well-differentiated group I lesions.

Journal Article↗
PubMed1996

Local Recurrence after Conservative Surgery for Breast Cancer.

The advent of limited operative therapy for breast cancer has produced both good cosmetic results and local recurrence. In our department we treated 38 cases with conservation surgery for early stage breast cancers between February 1988 and October 1994, and local recurrence occurred in 2. PATIENT 1. A 56-year-old-woman had a 1 x 0.8 cm tumor in the C region of the left breast. A quadrantectomy with axillary dissection (level 2) was performed as the margin of the nipple side was negative, followed by entire breast irradiation (50 Gy). Approximately four years after the first operation she developed a local recurrence in the CD region of the breast. A salvage mastectomy with subclavicular lymph node dissection was performed. After the second operation she appears to be healthy. PATINENT 2. A 54-year-old-woman noticed a 1 cm tumor located in the EAC region of the left breast. Excisional biopsy showed solid-tubular carcinoma. The distance between the nipple and the tumor was only 0.7 cm. A wide excision with axillary dissection (level 2) was performed as the margin of the nipple side was negative on the frozen section. Neither irradiation of the entire breast nor the adjuvant chemotherapy could be performed. After a three and a half year follow up local recurrence beneath the operative scar was recognized, and an additional wide excision was performed. A half year has passed since the second operation, there have been no signs of recurrence and she appears to be healthy. In conclusion, we experienced two local recurrences in 38 cases of breast conservation surgery for early breast cancers. One patient had multicentric cancers with histologically different types: mucinous and noninvasive ductal carcinoma, and another had a very short distance between the nipple and the tumor and did not undergo chemotherapy or radiotherapy.

Journal Article↗
PubMed1996

Mutant mtDNA at 1555 A to G in 12S rRNA gene and hypersusceptibility of mitochondrial translation to streptomycin can be co-transferred to rho 0 HeLa cells.

Human skin fibroblast line 95-119, which had been isolated from the mother of a Japanese patient with aminoglycoside-induced deafness and a 1555 A to G mutation at 12S rRNA gene in mitochondrial DNA (mtDNA), was used to investigate the relationship between the 1555 mtDNA mutation and its pathogenicity. By the intercellular transfer of mtDNA with or without the 1555 mutation to mtDNA-less (rho 0) HeLa cells, we isolated cybrid clones and found that the mitochondrial translation in a cybrid clone repopulated with the homoplasmic 1555 mutation showed the highest susceptibility to streptomycin. These observations suggest that the genotype of the mutant mtDNA and the phenotype of hypersusceptibility to streptomycin observed in 95-119 fibroblasts were co-transferred simultaneously to rho 0 HeLa cells, supporting the idea that the homoplasmic 1555 mtDNA mutation is involved in the pathogenesis leading to aminoglycoside-induced hearing loss.

Aminoglycosides↗
PubMed1996

Mosaic structures of neurotoxins produced from Clostridium botulinum types C and D organisms.

We isolated the gene encoding a botulinum neurotoxin (BoNT) of 1285 amino acids with a molecular weight of 147,364 from the toxigenic bacteriophage d-sA of Clostridium botulinum type D strain South African (Dsa). The BoNT of Dsa (BoNT/Dsa) is composed of three regions on the basis of the homology to BoNT types C1 (BoNT/C1) and D (BoNT/D). The N-terminal (Met-1 to Val-522) and the C-terminal regions (Trp-945 to Glu-1285) have high identity to corresponding regions of BoNT/D (96% identity) and BoNT/C1 (74% identity), respectively. The core region (Pro-523 to Lys-944) is common to three toxins (83% to 92% identity). These results suggest that neurotoxins produced from Clostridium botulinum types C and D are composed in a mosaic-like fashion.

Amino Acid Sequence↗
PubMed1996

An aggressive therapeutic approach to carcinoma of the body and tail of the pancreas.

BACKGROUND: Prognosis of patients with adenocarcinoma of the pancreatic body and tail is extremely poor. Anatomically, this part of the pancreas is thin, and cancerous invasion to the retropancreatic structures occurs easily. The majority of patients have residual tumor in the retroperitoneal tissues after conventional distal pancreatectomy. METHODS: Between 1962 and 1979, 10 patients with carcinoma of the pancreatic body underwent simple distal pancreatectomy. Between 1980 and 1990, 22 patients including 7 with distant metastasis underwent a more aggressive approach intended to achieve longer survival: distal pancreatectomy with extended dissection of the lymph nodes and adjacent structures, especially into the retropancreatic space. After 1984, intraoperative ŕadiation (IORT) by electron beam and chemotherapy by hepatic infusion plus systemic injection of mitomycin C (MMC) were added for 7 patients without distant metastasis. RESULTS: The 10 patients who underwent pancreatectomy between 1962 and 1979 all died within 20 months after their operations. Conversely, the patients treated with an aggressive approach between 1980 and 1990 survived longer; the 5-year survival rate for 15 patients without distant metastasis was 29%, though the 7 patients with distant metastasis died within 10 months of their operations. There were 4 long term survivors (> or = 5 years); 3 of whom received IORT and chemotherapy with MMC. Invasion to the retropancreatic soft tissues was present in 95% of the resected specimens from the 22 patients. However, invasion to the surgical margin at the posterior surface of the resected specimen was present in only 36% after extended resection of the retropancreatic structures. CONCLUSIONS: Survival improved for this disease after distal pancreatectomy with extended dissection, especially of the retropancreatic structures, adjuvant IORT, and chemotherapy had been performed.

Adenocarcinoma↗
PubMed1996

Analysis of the substrate-recognition mode of aromatic amino acid aminotransferase by combined use of quasisubstrates and site-directed mutagenesis: systematic hydroxy-group addition/deletion studies to probe the enzyme-substrate interactions.

Escherichia coli aromatic amino acid aminotransferase (ArAT) catalyzes transamination reactions of both dicarboxylic amino acids and aromatic amino acids. Because both reactions are supposed to occur in a single reaction center, whether ArAT provides alternative binding sites for the two different types of substrate side chains has been an intriguing question. This was probed by spectroscopic analysis of the complexes of beta-hydroxylated substrates and the wild-type and [Tyr70-->Phe] mutant enzymes. Both L-erythro-3-hydroxyaspartate and L-erythro-3-phenylserine reacted with the wild-type ArAT to give an absorption maximum at around 500 nm, reflecting the formation of the quinonoid intermediate. When the hydroxy group of Tyr70 of ArAT was deleted by replacement of the residue with phenylalanine, the 500-nm absorption greatly decreased in either of the ArAT-beta-hydroxy amino acid complexes, showing the presence of specific interactions, which stabilize the 500-nm absorbing quinonoid intermediates, between the phenolic hydroxy group of Tyr70 and the beta-hydroxy groups of the two quasisubstrates. From these results, it was concluded that the conformations of the two quasisubstrates are essentially identical in their enzyme-bound forms. This implies that the phenyl group of the substrate phenylalanine occupies the same region as that occupied by the beta-carboxyl group of the substrate aspartate, and the region should be near Arg292, the residue that binds the beta-carboxylate group of substrates. The [Arg292-->Ala] or [Arg292-->Leu] mutation increased the Km values for aromatic amino acids 5-10 fold, and the [Arg292-->Lys] mutation increased these values 10-100-fold, without affecting the kcat values. This shows that the side chain of Arg292 is partially involved in the binding of the aromatic ring of substrates to ArAT.

Amino Acid Sequence↗
PubMed1996

Topographic representation in human intraparietal sulcus of reaching and saccade.

Regional cerebral blood flow was measured by positron emission tomography in seven subjects during reaching with saccade, reaching without saccade, saccade and control tasks. The reaching with saccade task activated two spatially distinct areas in the contralateral intraparietal sulcus (IPS) compared with the control condition. The area located in the anterior part of the IPS was also activated during the reaching without saccade but not during the saccade task. The other area, located in the posterior part of the IPS was, in contrast, active during the saccade but not the reaching without saccade task. The results indicate that the human IPS is functionally heterogeneous, and that functional roles of its anterior and posterior parts include control of reaching movements and eye movements, respectively.

Adolescent↗
PubMed1996

Cellular localization of Na+/MYO-inositol co-transporter mRNA in the rat brain.

The distribution of Na+/MYO-inositol co-transporter (SMIT) mRNA in the rat brain was studied by in situ hybridization histochemistry. The highest levels of SMIT mRNA were observed in the choroid plexus. Intense hybridization signals were found in the pineal gland, the area postrema, the hippocampus, the locus coeruleus, the suprachiasmatic nucleus, the olfactory bulb and the Purkinje cell and granule cell layers of the cerebellum. Low to moderate levels of labelling were detected in almost all neurones and small glia-like cells throughout the brain. These results suggest that almost all cells in the brain possess an SMIT-mediated osmotic and ionic regulatory system, and uneven densities of positive SMIT mRNA signals may reflect the differences in sensitivity of the cells to osmotic and ionic changes and also reflect differences in permeability of capillaries.

Animals↗
PubMed1996

Growth inhibition of human leukemic cells by WT1 (Wilms tumor gene) antisense oligodeoxynucleotides: implications for the involvement of WT1 in leukemogenesis.

We have previously reported expression of WT1 in acute leukemia. To elucidate its biological significance, we examined the effect of the suppression of the WT1 expression by WT1 antisense oligomers on the growth of the leukemic cells expressing WT1. When 20 different WT1 antisense (AS) oligomers covering from the 5' cap sites of the WT1 gene to the 3' end were examined for the inhibitory effect on the growth of K562 cells expressing WT1, four WT1 AS oligomers inhibited the cell growth, whereas WT1 sense and random sequence oligomers had no effect on the cell growth of K562. Moreover, WT1 AS oligomers significantly inhibited the growth of the clonogenic cells of fresh leukemic cells in six of 14 patients with acute myeloid leukemia, in one of two patients with chronic myelogenous leukemia (CML) chronic phase, and in one of one patient with CML blastic crisis. However, these oligomers did not inhibit normal colony-forming unit-granulocyte-macrophage. Western blot analysis clearly demonstrated the significant reduction in the WT1 protein levels in the K562 and fresh leukemic cells that were treated with the WT1 AS oligomers, confirming that the inhibitory effect of the WT1 AS oligomers on the cell growth operates via the reduction in the WT1 protein levels. These results show that WT1 plays an important role in leukemogenesis.

Base Sequence↗
PubMed1996

Regulation of energy metabolism by interleukin-1beta, but not by interleukin-6, is mediated by nitric oxide in primary cultured rat hepatocytes.

The effects of inflammatory cytokines (interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha) on energy metabolism were studied in primary cultured rat hepatocytes. Adenine nucleotide (ATP, ADP, and AMP) content, lactate production, the ketone body ratio (acetoacetate/beta-hydroxybutyrate) reflecting the liver mitochondrial redox state (NAD+/NADH), and nitric oxide formation were measured. Insulin increased ATP content in hepatocytes and had a maximal effect after 8-12 h of culture. Both interleukin-1beta and interleukin-6, but not tumor necrosis factor-alpha, significantly inhibited the ATP increase time- and dose-dependently. Interleukin-1beta and interleukin-6 also stimulated lactate production. During the same period, interleukin-1beta but not interleukin-6 decreased the ketone body ratio. Furthermore, interleukin-1beta markedly stimulated nitric oxide formation in hepatocytes, and this increase was blocked by NG-monomethyl-L-arginine (a nitric oxide synthase inhibitor) and by interleukin-1 receptor antagonist. NG-monomethyl-L-arginine reversed inhibition of the ATP increase, decrease in the ketone body ratio, and increase in lactate production, which were induced by interleukin-1beta. Interleukin-1 receptor antagonist completely abolished all of the effects induced by interleukin-1beta. These results demonstrated that interleukin-1beta and interleukin-6 affect the insulin-induced energy metabolism in rat hepatocytes by different mechanisms. Specifically, interleukin-1beta inhibits ATP synthesis by causing the mitochondrial dysfunction, a process which may be mediated by nitric oxide.

Adenine Nucleotides↗
PubMed1996

7-Ethoxycoumarin O-deethylation catalyzed by cytochromes P450 1A2 and 2E1 in human liver microsomes.

7-Ethoxycoumarin O-deethylation has been used widely as a marker activity for assessing substrate specificities of cytochromes P450 (P450) in liver microsomes of mammals, and extensive studies have shown that in rats and mice the major catalysts are P450 1A1, 1A2, and 2B enzymes. In contrast to findings in experimental animal models, P450 2E1 has been reported to be a principal enzyme involved in 7-ethoxy-coumarin O-deethylation in human livers. In this study, we further examined the roles of individual forms of human P450 involved in 7-ethoxycoumarin O-deethylation using microsomes from different human liver samples and from human lymphoblastoid cells expressing human P450 enzymes and purified P450 enzymes isolated from the membrane of Escherichia coli expressing modified P450 proteins. Kinetic analysis showed that there were at least two different enzymes involved in 7-ethoxycoumarin O-deethylation in different human samples. Samples that contained high amounts of P450 2E1 in liver microsomes showed biphasic curves for O-deethylation with relatively high turnover numbers, whereas P450 1A2-rich samples tended to have low Km values with low Vmax values. Anti-human P450 2E1 antibodies inhibited markedly (P < 0.05) the 7-ethoxycoumarin O-deethylation activities catalyzed by human liver microsomes particularly when examined at a high substrate concentration (200 microM). However, we also found that anti-P450 1A2 antibodies suppressed O-deethylation activities only at a low substrate concentration (10 microM). Recombinant human P450 1A2 was found to have a low Km value for 7-ethoxycoumarin O-deethylation, whereas P450 2E1 showed a high Km value. Of the P450 enzymes examined, P450 1A1 gave the highest O-deethylation activities with a low Km value, although this enzyme is reported to be expressed extrahepatically in humans. Other human P450 enzymes, including P450 2A6, 2C10, 2D6, 3A4, and 3A5, did not show significant O-deethylation activities except that P450 2B6, a minor P450 component in human livers, was found to have a Vmax value similar to that of P450 1A2 and a Km value similar to that of P450 2E1. These results suggest that P450 1A2 is a low Km enzyme for 7-ethoxycoumarin O-deethylation in human liver microsome, although it has a low Vmax value than P450 2E1.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1996

Use of 3-(1,8-naphthalimido)propyl-modified silyl silica gel as a stationary phase for the high-performance liquid chromatographic separation of purine derivatives.

The use of a packing material, 3-(1,8-naphthalimido)propyl-modified silyl silica gel (NAIP), as a stationary phase for high-performance liquid chromatography, has been studied. NAIP behaved like a reversed-phase stationary phase with some pi-pi interaction. Purine derivatives, i.e., xanthine, hypoxanthine, uric acid, theobromine, theophylline and caffeine, were separated by a column packed with NAIP using an eluent of borate solution (pH 6.4)-MeOH (50:50, v/v). Of these, caffeine was selected as the target of the subsequent investigation and its determination was examined in commercially available medicinal drinks and pharmaceutical preparations. The average recoveries of caffeine were 98.0-107.4% for five drinks and 99.6-107.8% for five tablets and one powder. Subsequently, determination of caffeine and its metabolites in human plasma was examined. In twelve normal human plasma, caffeine levels ranged from 0.24 to 4.26 micrograms/ml. Time curves of plasma caffeine concentrations and those of its demethylated metabolite, 1,7-dimethylxanthine (1,7-DMX), after an oral ingestion of caffeine (200 mg) were measured by the proposed method and it was found that the maximum concentrations of caffeine and 1,7-DMX were obtained at 1-1.5 h and 3-6 h after ingestion, respectively.

1-Naphthylamine↗