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Biomedical subjects

H Oikawa

Publications and source records attributed to H Oikawa.

At least 19 recordsLinked to original sources
PubMed2000

Detailed reaction mechanism of macrophomate synthase. Extraordinary enzyme catalyzing five-step transformation from 2-pyrones to benzoates.

Macrophomate synthase from the fungus Macrophoma commelinae IFO 9570 is a Mg(II)-dependent dimeric enzyme that catalyzes an extraordinary, complex five-step chemical transformation from 2-pyrone and oxalacetate to benzoate involving decarboxylation, C-C bond formation, and dehydration. The catalytic mechanism of the whole pathway was investigated in three separate chemical steps. In the first decarboxylation step, the enzyme loses oxalacetate decarboxylation activity upon incubation with EDTA. Activity is fully restored by addition of Mg(II) and is not restored with other divalent metal cations. The dissociation constant of 0.93 x 10(-)(7) for Mg(II) and atomic absorption analysis established a 1:1 stoichiometric complex. Inhibition of pyruvate formation with 2-pyrone revealed that the actual product in the first step is a pyruvate enolate, which undergoes C-C bond formation in the presence of 2-pyrone. Incubation of substrate analogs provided aberrant adducts that were produced via C-C bond formation and rearrangement. This strongly indicates that the second step is two C-C bond formations, affording a bicyclic intermediate. Based on the stereospecificity, involvement of a Diels-Alder reaction at the second step is proposed. Incubation of the stereospecifically deuterium-labeled malate with 2-pyrones in the presence of malate dehydrogenase provided information for the stereochemical course of the reaction catalyzed by macrophomate synthase, indicating that the first decarboxylation provides pyruvate (Z)-[3-(2)H]enolate and that dehydration at the final step occurs with anti-elimination accompanied by concomitant decarboxylation. Examination of kinetic parameters in the individual steps suggests that the third step is the rate-determining step of the overall transformation.

Benzoates↗
PubMed2000

Quantitative analysis of pulmonary neuroendocrine cell distribution of the fetal small airways using double-labeled immunohistochemistry.

Pulmonary neuroendocrine cells (PNECs) are supposed to play an essential role in development of fetal lung and neonatal respiratory adaptation. Some previous studies have suggested the close relation between PNECs and sudden infant death syndrome (SIDS). To investigate how PNECs distribute to the thermal bronchioli of fetal lung may be a clue to clarify this relation. Since it is difficult to distinguish bronchiole from alveolus in fetal lung, we performed double immunostaining with antibody against chromogranin A (CGA) and alpha-smooth muscle actin (SMA) which can make clear distinction between them. In this study, formalin-fixed, paraffin-embedded lung tissues from 18 autopsy cases from 16 to 28 weeks of gestation were assessed. CGA immunopositive cells were counted and the length of basement membranes of terminal bronchioli was measured with computed image analyzer. Density of PNECs was expressed as the number of immunopositive cells per millimeter of basement membrane. Terminal bronchiole stained with SMA was clearly distinguished from alveolus at 16 weeks. With gestational age, CGA immunopositive PNECs were gradually increased in 2 folds by the 25th week. After that, their density wasn't changed significantly until termination. It is suggested that PNECs in terminal bronchiole was playing an important role in morphogenesis of alveolar ducts and alveolar sacks.

Actins↗
PubMed2000

Macrophomate synthase: characterization, sequence, and expression in Escherichia coli of the novel enzyme catalyzing unusual multistep transformation of 2-pyrones to benzoates.

Macrophoma commelinae isolated from spots on leaves of Commelina communis has the ability to transform 5-acetyl-4-methoxy-6-methyl-2-pyrone (1) to 4-acetyl-3-methoxy-5-methylbenzoic acid (macrophomic acid, 2). This biotransformation includes the condensation of the 2-pyrone ring with a C3-unit precursor to form a substituted benzoic acid. We optimized conditions for induction of enzyme activity in M. commelinae, identified oxalacetate as a C3-unit precursor with cell extract, and purified the novel enzyme, macrophomate synthase. Oxalacetate inhibited the enzyme activity at a concentration higher than 5 mM, and magnesium chloride stimulated the enzyme activity. Kinetic analyses gave K(m) of 1.7 mM for 1 at 5 mM oxalacetate, K(m) of 1.2 mM for oxalacetate at 5 mM 1, and k(cat) of 0.46 s(-1) per subunit. Pyruvate was a weak substrate, with K(m) of 35.2 mM and k(cat) of 0.027 s(-1) at 5 mM 1. We cloned and sequenced a cDNA encoding the macrophomate synthase. The cDNA of 1,225 bp contained an open reading frame that encoded a polypeptide of 339 amino acid residues and 36,244 Da, the sequence of which showed no significant similarity with known proteins in a homology search with BLAST programs. Transformed E. coli cells carrying the cDNA encoding the mature protein of macrophomate synthase overproduced macrophomate synthase under the control of the T7 phage promoter induced by IPTG. The purified enzyme showed the same values of K(m) and optimum pH as the native macrophomate synthase.

Amino Acid Sequence↗
PubMed2000

Substrate diversity of macrophomate synthase catalyzing an unusual multistep transformation from 2-pyrones to benzoates.

Macrophomate synthase, which we have recently purified, catalyzes an unusual multistep transformation from 5-acetyl-4-methoxy-6-methyl-2-pyrone to 4-acetyl-3-methoxy-5-methyl-benzoic acid (macrophomic acid). To investigate the substrate diversity of the enzyme, 40 analogs of 2-pyrone were prepared and their relative efficiency was examined in the enzymatic conversions. The experimental results reveal the structural requirements of the substrates and the rough size of the enzyme active site, and eliminate the ambiguity caused by contamination by other enzymes in the whole-cell experiments.

Benzoates↗
PubMed2000

[Intranet-based integrated information system of radiotherapy-related images and diagnostic reports].

PURPOSE: To use an intranet technique to develop an information system that simultaneously supports both diagnostic reports and radiotherapy planning images. METHODS: Using a file server as the gateway a radiation oncology LAN was connected to an already operative RIS LAN. Dose-distribution images were saved in tagged-image-file format by way of a screen dump to the file server. X-ray simulator images and portal images were saved in encapsulated postscript format in the file server and automatically converted to portable document format. The files on the file server were automatically registered to the Web server by the search engine and were available for searching and browsing using the Web browser. RESULTS: It took less than a minute to register planning images. For clients, searching and browsing the file took less than 3 seconds. Over 150,000 reports and 4,000 images from a six-month period were accessible. Because the intranet technique was used, construction and maintenance was completed without specialty. CONCLUSION: Prompt access to essential information about radiotherapy has been made possible by this system. It promotes public access to radiotherapy planning that may improve the quality of treatment.

Computer Communication Networks↗
PubMed1999

The spiroketals containing a benzyloxymethyl moiety at C8 position showed the most potent apoptosis-inducing activity.

The spiroketals containing a benzyloxymethyl moiety at the C8 position showed the most potent apoptosis-inducing activity, whereas its analogous compounds lacking any substituent at C8 or possessing ones other than the benzyloxymethyl moiety at C8 were all much less active. These results strongly suggest an important role of the benzyloxymethyl moiety linked to the C8 oxygen atom.

Apoptosis↗
PubMed1999

Primary hepatic lymphoma associated with primary biliary cirrhosis.

We report a case of primary hepatic lymphoma in a 55-yr-old female patient with primary biliary cirrhosis and Sjögren's syndrome. On July 1994, a tumor measuring 11 mm in diameter was detected in the right lobe of the liver by abdominal ultrasonography. A needle biopsy specimen showed the lesion to contain small- and medium-sized lymphoid cells without obvious atypia, and a provisional diagnosis of pseudolymphoma was made. About 2 yr later, the tumor increased to 15 mm in diameter, necessitating a second needle biopsy. Histological and genetic examinations confirmed non-Hodgkin's lymphoma of diffuse, mixed small and large cell, B-cell type. However, the size of the tumor remained almost stable (16 mm in diameter) over a period of 7 months after diagnosis, without any treatment for lymphoma, indicating a low grade malignancy. We document hepatic lymphoma as an additional complication of primary biliary cirrhosis.

Adult↗
PubMed1999

The apoptosis-inducing activity of the two protein phosphatase inhibitors, tautomycin and thyrsiferyl 23-acetate, is not due to the inhibition of protein phosphatases PP1 and PP2A (review).

Thyrsiferyl 23-acetate (TF-23A) has been shown to potently and specifically inhibit PP2A. TF-23A also induced a rapid cell death in various leukemic T- and B-cell lines. The TF-23A induced cell death with a typical apoptotic process. TF-23A and its several analogous compounds showed apoptosis-inducing activity. However, only TF-23A out of these compounds showed an inhibitory activity for PP2A. These results suggest that a portion of TF-23A involved in induction of apoptosis is different from that involved in the PP2A inhibition. Then, the effects of tautomycin and its derivatives on PP1 and PP2A and their apoptosis-inducing activity were examined. The C22-C26 moiety was essential for inhibition of protein phosphatase activity, whereas the C1-C18 moiety was essential for induction of apoptosis. Therefore, different moieties of tautomycin are involved in protein phosphatase inhibition and induction of apoptosis. From these results, it was concluded that the biological effects of phosphatase inhibitors are not necessarily induced by the inhibition of PP1 and PP2A but through other different molecular mechanisms which remain to be elucidated.

Animals↗
PubMed1999

Blood-flow route from the hepatic artery and portal vein to the sinusoid in normal human liver observed by confocal laser scanning microscopy.

OBJECTIVE: To observe the microvasculature in normal human liver. STUDY DESIGN: Four autopsy livers cut into 50-micron-thick sections were observed by confocal laser scanning microscopy. Immunofluorescence was performed using anti-alpha smooth muscle actin (alpha-SMA) antibody. In addition, double immunofluorescence was performed on the other sections using antilysozyme antibody. The routes from the portal vein branches and hepatic artery branches to the sinusoids were defined as follows: portal venule, septal branch, inlet venule, hepatic arteriole and terminal hepatic arteriole. RESULTS: The reactivity of the walls of septal branches and inlet venule was positive for alpha-SMA. Lysozyme-positive cells (Kupffer cells) were dense in the sinusoids but were sparse in the septal branches and absent from the inlet venules. Terminal hepatic arterioles were observed along the septal branch, and the anastomoses between them were observed at the peripheral portion. No routes opening directly from the terminal hepatic arteriole into the sinusoids or arterioportal anastomoses in the portal tract were observed on alpha-SMA-stained sections. CONCLUSION: Regulation of the microcirculation in human liver may be performed by the smooth muscle layer of both peripheral portal and hepatic arterial routes.

Actins↗
PubMed1998

Enzymatic activity and partial purification of solanapyrone synthase: first enzyme catalyzing Diels-Alder reaction.

In cell-free extracts of Alternaria solani, an enzymatic activity converting prosolanapyrone II to solanapyrones A and D via oxidation and subsequent Diels-Alder reaction has been found. Chromatography with DEAE-Sepharose provided two active fractions, pools 1 and 2. The former fraction converted prosolanapyrone II to solanapyrones A and D in a ratio of 2.2:1 with optical purities of 99% and 45% ee, respectively. The latter fraction did so in a ratio of 7.6:1 with 99% and nearly 0% ee, respectively. The enzyme partially purified from pool 2 native molecular weight of 40-62 kD and a pl of 4.25. The high reactivity of prosolanapyrone III in aqueous solution and the chromatographic behavior of the enzyme in pool 2 suggest that a single enzyme catalyzes both the oxidation and Diels-Alder reaction.

Alcohol Oxidoreductases↗
PubMed1998

Different moieties of tautomycin involved in protein phosphatase inhibition and induction of apoptosis.

The effects of tautomycin and its derivatives on protein phosphatases PP1 and PP2A and their apoptosis-inducing activity toward human leukemia Jurkat cells were examined, and the relationship between chemical structure and function was discussed. Among the compounds we examined, tautomycin was the most potent inhibitor and the most effective inducer of apoptosis. It inhibited PP1 and PP2A enzymatic activity concentration-dependently with IC50 values of 20 and 75 pM, respectively, in the presence of 0.01% Brij-35, and an LC50 value of 1 microM. Esterification of the anhydride moiety of tautomycin markedly increased the IC50 for the protein phosphatases. The C1'-C7' fragment of tautomycin had no inhibitory effect, but the fragment containing the C22-C26 moiety was inhibitory. These results suggest that the C22-C26 moiety is essential for inhibition of protein phosphatase activity and that the anhydride moiety enhances the inhibition. However, the esterification of the anhydride did not decrease, nor did the inclusion of the C22-C26 moiety increase the apoptosis-inducing activity. On the other hand, the C1-C18 moiety of tautomycin was essential for induction of apoptosis, and the conformation and the arrangement of functionalities of the C18-C26 carbon chain affected the apoptosis activity. However, modification of C1-C18, C1-C21, or C1-C26 compounds had little effect on phosphatase inhibitory activity. Our results strongly suggest that different moieties of tautomycin are involved in protein phosphatase inhibition and induction of apoptosis.

Antifungal Agents↗
PubMed1998

Correlation of magnetic resonance and oxygen microelectrode measurements of carbogen-induced changes in tumor oxygenation.

PURPOSE: The aim of this work was to test the hypothesis that decreases in the linewidth of magnetic resonance (MR) water signals in tumors caused by oxygenating treatments are due to increases in capillary and venous oxygen saturation of hemoglobin, which are tightly coupled to increases in extravascular oxygen tension (pO2). To establish this link, changes measured by MR were compared to changes in tissue pO2 measured directly by oxygen microelectrodes during carbogen (95% O2/5% CO2) inhalation. METHODS AND MATERIALS: Mammary adenocarcinomas (R3230AC) in nine rats were imaged at 4.7 Tesla. T1-weighted (TR = 200 ms, flip angle = 45 degrees) spectroscopic images of the water resonance in a single slice through each tumor were acquired with spectral resolution of 3.9 Hz and bandwidth of +/-1000 Hz. In the same slices in these tumors, microelectrode measurements were made using a non-Clark style oxygen electrode with a 350-micron tip. MR and microelectrode measurements were made during alternating periods of air and carbogen inhalation. RESULTS: Water resonance linewidth decreased significantly during carbogen-induced hyperoxia. Paired Student's t-test analysis of microelectrode data indicated that pO2 was significantly (p < 0.05) increased as a result of carbogen inhalation. MR and microelectrode data averaged over each tumor demonstrated that decreased MR water signal linewidth is strongly correlated (r = 0.92, p < 0.05) with increased tumor pO2 levels. CONCLUSION: Although tumor oxygenating agents increase response to radiation in rodent tumors, clinical studies have shown only marginal effects on the radiosensitivity of human tumors. This may be, in part, because the effects of tumor oxygenating treatments are highly heterogeneous both within each tumor and among a population of tumors. The noninvasive, high-resolution MR methods that are validated by the present work could guide the design of new and more effective tumor oxygenating agents and optimize treatments for individual patients.

Animals↗
PubMed1998

Changes in lymph vessels and portal veins in the portal tract of patients with idiopathic portal hypertension: a morphometric study.

Little is known about the effects of the pathological process associated with idiopathic portal hypertension (IPH) on hepatic lymph vessels or lymph flow. We used morphometric analysis to examine IPH-associated changes in lymph vessels and branches of the portal vein, with use of immunohistochemical staining for alpha smooth muscle actin. We also quantitated these changes using an image analysis system. The study was conducted with use of liver wedge biopsy material from 10 patients with advanced IPH and 10 control samples from patients with gastric carcinoma without liver disease. The number of lymph vessels, identified by a lack of smooth muscle layer in the wall, and the ratio of the total area of these vessels to that of the portal tract were higher in IPH samples than in the control samples, but the ratio of the area of a single lymph vessel to that of the portal tract in IPH samples was not different from control samples. The number of portal vein branches, characterized by hypertrophy of the smooth muscle layer in IPH samples was not different from control samples. The ratio of the total area of these branches to that of the portal tract, and the ratio of a single portal vein branch to that of the portal tract, were lower in IPH samples than in the control samples. Our results suggest that these morphometric changes in IPH may be associated with a reduction in portal blood flow and increased lymph flow, and that the latter may in turn reduce the high portal vein pressure in idiopathic portal hypertension.

Adult↗
PubMed1998

Electron microscopic examination of oligocilia in naevus of Ota.

The structure of the cilia present in dermal melanocytes of 14 patients with naevus of Ota was examined by electron microscopy. Cilia and basal bodies were found in 10 and 9 lesions, and in 39 and 18 dermal melanocytes, respectively. In each case, 1-12 cells with a single cilium or multiple cilia were observed. In a total of 3 dermal melanocytes from 2 cases, two cilia per cell were observed. The cilia contained 7, 6, 5 and 4 pairs of doublet microtubules in the periphery and no central microtubule. Another pattern with several pairs of doublet microtubules in the periphery and one or two centrally located doublet microtubules were also observed. The latter were not bona fide central microtubules but one and two doublets, which seemed to be displaced to the centre from the periphery of the cilium.

Adolescent↗
PubMed1998

How thick are the paraffin-embedded tissue sections routinely prepared in laboratory? A morphometric study using a confocal laser scanning microscope.

In order to evaluate the differences between cut and measured thicknesses of formalin-fixed, paraffin-embedded tissue sections, formalin-fixed, paraffin-embedded blocks of liver tissue were prepared from four male Wistar rats. The sections were cut at preselected thicknesses of 3, 6, 9, 12, 15 and 18 microns, and observed under confocal laser scanning microscopy. The maximum and minimum section thicknesses were measured using a vertically cut section image constructed by the software incorporated in the microscope. The surface of the section was not smooth but showed fine undulations in addition to a slight inclination. The mean and range of the section thicknesses measured were 4.3 +/- 1.3, 7.0 +/- 1.7, 9.8 +/- 2.1, 12.0 +/- 2.6, 14.1 +/- 3.0, and 17.7 +/- 3.8 microns for the preselected cut thicknesses of 3, 6, 9, 12, 15 and 18 microns. It is stressed that the evaluated parameters of the section (volume or surface densities) include standard errors of up to 10% caused by the differences between the cut and real thicknesses.

Animals↗
PubMed1998

Renal small cell carcinoma (neuroendocrine carcinoma) without features of transitional cell carcinoma.

Seventeen cases of renal small cell carcinoma have been reported in the literature. Approximately half of the reported cases show combined features of transitional cell carcinoma. Presented herein is a case of renal small cell carcinoma in a 37-year-old Japanese male who had been treated for 10 years with famotidine for duodenal ulcer. He suffered from sudden-onset chest pain at presentation and myxoma of the right atrium was suspected. He was treated by atriotomy and a tumor was removed from the right atrium and pulmonary artery. Histological examination, however, revealed it to be small cell carcinoma. Accordingly, a radical operation was performed for the removal of a tumor found in the right kidney. Histological examination of the tumor confirmed the presence of renal small cell carcinoma without any features of transitional cell carcinoma. It is reported that long-term administration of an histamine 2 (H2) receptor antagonist may produce carcinoid tumors in rodents and enterochromaffin-like cell hyperplasia in humans. The possible relationship between neuroendocrine carcinoma and H2 receptor antagonist therapy is discussed.

Adult↗
PubMed1998

[Significance of p53 and VEGF expression in liver metastasis from colorectal cancer].

We examined the significance of expression of p53 and vascular endothelial growth factor (VEGF) on the liver metastasis in 155 patients with colorectal cancer. The expression of p53, VEGF, and microvessel density (MVD) were immunohistochemically studied. There were significant differences between patients positive for p53 and patients negative for p53, between patients positive for VEGF and patients negative for VEGF, with respect to the occurrence of liver metastases. The frequency of patients positive for both p53 and VEGF in the liver metastases group was significantly higher than that in the control group. The frequency of patients positive for both p53 and v, both VEGF and v in the liver metastases group, was significantly higher than in the control group. MVD in patients positive for VEGF was significantly higher than that negative for VEGF. Thus, p53 and VEGF expression affects the formation of liver metastases from colorectal cancer by the activation of neovascularization followed by feasibility of the extravasation of tumor cells in the tumor tissue.

Colorectal Neoplasms↗