Biomedical subjects

M Furuya

Publications and source records attributed to M Furuya.

At least 37 records · Page 2Linked to original sources

PubMed2000

Light-induced nuclear translocation of endogenous pea phytochrome A visualized by immunocytochemical procedures.

Although the physiological functions of phytochrome A (PhyA) are now known, the distribution of endogenous PhyA has not been examined. We have visualized endogenous PhyA apoprotein (PHYA) by immunolabeling cryosections of pea tissue, using PHYA-deficient mutants as negative controls. By this method, we examined the distribution of PHYA in different tissues and changes in its intracellular distribution in response to light. In apical hook cells of etiolated seedlings, PHYA immunolabeling was distributed diffusely in the cytosol. Exposure to continuous far-red (cFR) light caused a redistribution of the immunolabeling to the nucleus, first detectable after 1.5 hr and greatest at 4.5 hr. During this time, the amounts of spectrally active phytochrome and PHYA did not decline substantially. Exposure to continuous red (cR) light or to a brief pulse of red light also resulted in redistribution of immunolabeling to the nucleus, but this occurred much more rapidly and with a different pattern of intranuclear distribution than it did in response to cFR light. Exposures to cR light resulted in loss of immunolabeling, which was associated with PHYA degradation. These results indicate that the light-induced intracellular location of PHYA is wavelength dependent and imply that this is important for PhyA activity.

Biological Transport↗

PubMed2000

Clinical and pharmacological studies on liver diseases treated with Kampo herbal medicine.

Hepatitis C virus (HCV) infection frequently causes chronic hepatitis, which is linked to the development of liver cirrhosis and hepatocellular carcinoma. Most physicians who practice Kampo medicine in Japan have observed that Kampo medicine can be as effective as interferon therapy in the treatment of chronic hepatitis C. In the present study, to evaluate the effect of Kampo medicine on chronic hepatitis C, clinical treatment was assessed in short-term and long-term study, and it was shown that ninjin-yoei-to (Formula ginseng compositae: TJ-108) was very effective. Therefore, to find the most active herbal component of TJ-108 in the treatment of HCV, Citrus Unshiu Peel, Schisandra Fruit, and Polygala Root, which are specific to TJ-108, were screened using an in vitro HCV infection model. Among the three herbs, Schisandra Fruit was found to be most active. In the next step, Gomisin A, an active component of Schisandra Fruit, was studied using an in vitro model with MOLT-4 cells and an animal model of immunologically induced acute hepatic failures. It is concluded that the therapeutic effect of TJ-108 on chronic hepatitis C is from the inhibitory effect on HCV infection, and also from the protective effect on immunological hepatopathy of Schisandra Fruit and its lignan component, Gomisin A.

Adult↗

PubMed2000

Analyses of matrix metalloproteinases and their inhibitors in cyst fluid of serous ovarian tumors.

OBJECTIVES: Expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) in serous tumors of the ovary was investigated to determine whether and how these proteolytic enzymes are associated with the progression of these tumors. METHODS: Cyst fluid of 24 serous ovarian tumors (8 adenocarcinomas, 2 borderline tumors and 14 adenomas) was analyzed using gelatin/casein zymography and enzyme-linked immunosorbent assay. RESULTS: Concentrations of MMP-9 and MMP-2 were statistically higher in serous adenocarcinomas than in serous adenomas (p < 0.01, p < 0.05, respectively), while the concentrations of TIMP-1 and TIMP-2 showed no significant difference between adenocarcinomas and adenomas. The molar ratio of TIMP-2/MMP-2 was lower in adenocarcinomas than in adenomas (p < 0.05). With gelatin zymography, the MMP-9 band was detected in all serous adenocarcinomas, but only in 8 of 14 serous adenomas (p = 0.05). Using casein zymography, MMP-7 was more frequently detected in serous adenocarcinomas (7/8) than in serous adenomas (4/14; p < 0.05). CONCLUSIONS: These observations indicate that matriolytic enzymes such as MMP-2, MMP-7 and MMP-9 are secreted into cyst fluid from serous adenocarcinoma tissues. In part, the aggressive invasion of serous carcinoma cells may be explained by the expression of matriolytic enzymes.

Adenoma↗

PubMed2000

Fluorescent differential display identifies circadian clock-regulated genes in Arabidopsis thaliana.

Circadian rhythms in gene expression were first observed in plants more than 13 years ago, but the underlying mechanism controlling rhythmic gene expression is still not understood. The isolation of novel circadian clock-controlled genes (ccgs) is likely to provide new tools for studying circadian rhythms. Fluorescent differential display (FDD) was used to screen Arabidopsis thaliana mRNAs for cycling transcripts. Seventy PCR primer pairs were screened, and 17 different cycling bands were observed out of an estimated 10,500 bands screened. The identities of 10 bands were determined, and the rhythmic gene expression was confirmed using northern blot analysis. The 10 cycling bands represent 7 different genes, 6 of which are present in the databases and 1 that does not match anything in current databases. The rhythmic expression of the 7 genes is composed of four distinct phases of clock regulation. The results demonstrate that FDD can be used to isolate ccgs. The genes identified in this screen range from known A. thaliana ccgs, as well as genes shown to be clock controlled in other plant species, to a novel gene that may encode a pioneer protein. Further study of these ccgs is likely to increase our understanding of circadian-regulated gene expression.

Arabidopsis↗

PubMed1999

Rare germinal unequal crossing-over leading to recombinant gene formation and gene duplication in Arabidopsis thaliana.

Small, multigene families organized in a tandem array can facilitate the rapid evolution of the gene cluster by a process of meiotic unequal crossing-over. To study this process in a multicellular organism, we created a synthetic RBCSB gene cluster in Arabidopsis thaliana and used this to measure directly the frequency of meiotic, intergenic unequal crossing-over between sister chromatids. The synthetic RBCSB gene cluster was composed of a silent DeltaRBCS1B::LUC chimeric gene fusion, lacking all 5' transcription and translation signals, followed by RBCS2B and RBC3B genomic DNA. Expression of luciferase activity (luc(+)) required a homologous recombination event between the DeltaRBCS1B::LUC and the RBCS3B genes, yielding a novel recombinant RBCS3B/ 1B::LUC chimeric gene whose expression was driven by RBCS3B 5' transcription and translation signals. Using sensitive, single-photon-imaging equipment, three luc(+) seedlings were identified in more than 1 million F2 seedlings derived from self-fertilized F1 plants hemizygous for the synthetic RBCSB gene cluster. The F2 luc(+) seedlings were isolated, and molecular and genetic analysis indicated that the luc(+) trait was caused by the formation of a recombinant chimeric RBCS3B/1B::LUC gene. A predicted duplication of the RBCS2B gene also was present. The recombination resolution break points mapped adjacent to a region of intron I at which a disjunction in sequence similarity between RBCS1B and RBCS3B occurs; this provided evidence supporting models of gene cluster evolution by exon-shuffling processes. In contrast to most measures of meiotic unequal crossing-over that require the deletion of a gene in a gene cluster, these results directly measured the frequency of meiotic unequal crossing-over (approximately 3 x 10(-6)), leading to the expansion of the gene cluster and the formation of a novel recombinant gene.

Arabidopsis↗

PubMed1999

Use of urine samples from healthy humans, nephritis patients or other animals as an alternative to foetal calf serum in the culture of Leishmania (L.) donovani in vitro.

The effect of supplementing in-vitro cultures of Leishmania donovani with urine was investigated. The parasites were isolated from Bangladeshi patients with visceral leishmaniasis. The urine samples used were collected from healthy human donors, patients with nephrotic syndrome, diabetic nephritis (DN) or diabetes mellitus, a dog and a cow. Promastigotes from blood-agar cultures were inoculated into RPMI-1640 basal medium with 10% heat-inactivated foetal calf serum (FCS) and/or 1%-20% urine. The parasites were then counted in a haemocytometer, on days 2, 4, 5, 6, 7, 8, 10, 12 and 14 post-inoculation. From day 4, the numbers of parasites/ml in cultures containing 5% healthy-human urine but no FCS were at least as high as those in cultures containing 10% FCS but no urine (P = 0.191). The wet weights of parasites harvested from mass cultures of the parasites in RPMI-1640 plus 5% healthy-human urine and in RPMI-1640 plus 10% FCS were practically the same. Multiplication of the parasites in the presence of 5% urine from a DN patient was significantly greater (P < 0.01) than that seen with other urine samples at the same concentration or with 10% FCS. The multiplication seen with 8% canine urine was almost the same as with 5% healthy-human urine. Parasites could be maintained in RPMI-1640 plus 5% healthy-human urine for at least 40 days, sub-culturing every 4 days. Urine may be a better and much cheaper stimulant of Leishmania multiplication in vitro than FCS.

Animals↗

PubMed1999

Analysis of matrix metalloproteinases and related tissue inhibitors in cystic fluids of ovarian tumors.

Proteolytic activity of cystic neoplasms of the ovary appears to play a role in destruction of the extracellular matrix and tumor invasion. The purpose of this study was to determine whether the enzymatic activities reflect the degrees of tumor malignancy. The author examined the activity and quantity of matrix metalloproteinases (MMPs) and tissue inhibitors of MMP (TIMPs) in cystic fluids of both benign and malignant epithelial ovarian tumors. The concentration of MMP-9 was statistically higher in mucinous carcinomas (p < 0.05) than in benign ones. TIMP-1, which combines with MMP-9, was also higher (p < 0.05) in malignancies than in benign ones. The ratios of MMP-9/MMP-2 and the concentrations of activated forms of MMPs well associated with the degrees of malignancy, while the mol ratios of TIMP-1/MMP-9 and TIMP-2/MMP-2 inversely correlated. Expressions of MMP-3 and/or trypsin in the fluids were frequently associated with activation of MMP-7 and MMP-9. These observations support the concept that the imbalance of TIMPs/MMPs and the activation of MMPs correlate with the biological malignancy of ovarian cystic tumors.

Adenoma↗

PubMed1999

[Comparison of effects of verapamil and those of nicardipine on myocardial ischemia and reperfusion injury: a study in an in situ rabbit model].

This study was designed to investigate whether two L-type calcium antagonists, verapamil and nicardipine reduce the myocardial necrosis (infarct size) following ischemia and reperfusion. Rabbits (n = 52) were subjected to regional myocardial ischemia by 30 min of the left anterior descending artery occlusion followed by 3 hrs of reperfusion under ketamine/xylazine anesthesia. The animals were randomly assigned to a control group, and verapamil or nicardipine treatment groups. A continuous infusion of verapamil (0.1 mg.kg-1.h-1) or nicardipine (0.06 mg.kg-1.h-1) was initiated 5 min prior to ischemia, immediately after ischemia, 5 min after ischemia, 5 min prior to reperfusion, immediately after reperfusion, and 5 min after reperfusion. A lead II ECG was recorded throughout the experiment for determining ventricular arrhythmias. The area at risk was delineated by Evans blue, and infarct size was determined by triphenyl tetrazolium chloride staining at the end of the experiment in the heart (the left anterior descending artery was religated). The area at risk showed no significant differences among all the groups tested; infarct size in the rabbits to which verapamil was given 5 min before subjecting to ischemia was significantly reduced to 41.7 +/- 4.3% from 65.9 +/- 1.7% in control rabbits. The treatment with nicardipine at any period of time did not reduce infarct size. On the other hand, the incidence of reperfusion-induced arrhythmias was decreased by nicardipine infusion, when started 5 min prior to ischemia, immediately after ischemia, 5 min after ischemia, 5 min prior to reperfusion, and immediately after reperfusion. These results suggest that verapamil given before coronary artery occlusion has an infarct limiting effect in the myocardium, and that nicardipine has an antiarrhythmic effect during reperfusion in the rabbit heart.

Animals↗

PubMed1999

[Nutritional therapy for ulcerative colitis].

Despite its supplementary role for a remedy of the Ulcerative Colitis, nutritional therapy performs the important role as well as medicine. In the active state, the aim of nutrition therapy is to reduce the activity of the disease. In the remission state, the aim is to keep the condition and to supply elements of nutrition which the disease causes these deficiency such as vitamins or minerals. Needless to say, it is required to consider a few points; patient's condition, lesion's digestion disorder and leakage, or side effects of caused by medicines. High calorie, low fat and rich protein is ideal for the nutrition therapy for this disease, however it is suggested to satisfy patient's mental health at the same time. Nutrition therapy for Ulcerative Colitis should be regarded as the long-term treatment that requires the co-operation and comprehension between patients and medical supporting staffs.

Colitis, Ulcerative↗

PubMed1998

Cox multivariate regression models for estimating prognosis of patients with endometrioid adenocarcinoma of the uterine corpus who underwent thorough surgical staging.

The International Federation of Gynecology and Obstetrics (FIGO) adopted surgical staging criteria in 1988. Many studies have shown that histologic grade, nuclear grade, lymph-vascular space invasion and cell type are also important predictors of survival. It has not been clarified, however, how to integrate these histopathologic variables into the process of estimating individual prognosis. We performed Cox multivariate regression analysis to create models that incorporate various histopathologic factors for estimating the prognoses of patients with endometrioid adenocarcinoma of the uterine corpus. Our study was based on data from 206 patients who underwent complete surgical staging, including systematic pelvic and para-aortic lymph node dissection. Two models resulted: one included depth of myometrial invasion, para-aortic node metastasis and the number of sites involved by the tumor among the cervix, ovary and pelvic lymph nodes (which we designated as extracorporeal spread score, ECS) and the other incorporated nuclear grade and lymph-vascular space invasion as variables. These 2 models enabled the prognosis for patients with endometrioid adenocarcinoma to be stratified into several levels according to hazard ratio. Comprehensive integration of the histopathologic prognostic factors, categorized into those relating to tumor extent and those relating to tumor virulence, should facilitate the estimation of individual prognosis more accurately than FIGO staging alone.

Adenocarcinoma↗

PubMed1998

Rapid identification of Leishmania species from formalin-fixed biopsy samples by polymorphism-specific polymerase chain reaction.

The precise identification and classification of Leishmania species is important for public health surveillance since different species cause different clinical features of the disease. A highly specific polymerase chain reaction (PCR) panel was developed to enable the identification of the five major Leishmania species that cause New World cutaneous leishmaniases. The primers used for this panel were designed to distinguish the polymorphism in sequences of commonly amplified DNA bands of the parasites produced by arbitrarily primed PCR. These polymorphism-specific PCR diagnoses were performed with formalin-fixed biopsy specimens of the leishmanial lesions from four patients in Ecuador and one hamster skin lesion, and these lesions were determined to be caused by Leishmania (Viannia) panamensis, L. (Leishmania) mexicana, and L. (L.) amazonensis. The PCR panel may offer an important and practical approach to the standardized identification of Leishmania species in field examinations.

Animals↗

PubMed1998

A 4-year study of plasma ochratoxin A in a selected population in Tokyo by immunoassay and immunoaffinity column-linked HPLC.

Employing a competitive ELISA (cELISA) based on monoclonal antibody and a novel immunoaffinity-column (IAC)-linked HPLC-fluorometry, ochratoxin A (OTA) levels in the plasma of 184 healthy volunteers (130 males, 54 females) were surveyed in Tokyo during the 4 years from 1992 to 1996. It was found that 85% of the cases were positive for OTA except 38% in 1994, and an average value in the positives was estimated as 68 pg/ml. This suggests that the population in Tokyo is exposed to OTA at high frequency, although the level in plasma is far less than that reported in Europe and Canada. A partial contribution of fermented dietary foodstuffs, coffee and wine is suspected as the source of the OTA observed in the human plasma.

Chromatography, Affinity↗

PubMed1998

Mode of phytochrome B action in the photoregulation of seed germination in Arabidopsis thaliana.

Arabidopsis thaliana seeds imbibed for a short duration show phytochrome B (PhyB)-specific photo-induction of germination. Using this system, the relationship was determined between the amount of PhyB in seeds and photon energy required for PhyB-specific germination in two transgenic Arabidopsis lines transformed with either the Arabidopsis PhyB cDNA (ABO) or the rice PhyB cDNA (RBO). Immunochemical detection of PhyB apoprotein (PHYB) showed that the expression level of PHYB in ABO seeds was at least two times higher than that in the wild-type seeds, but in RBO seeds the PHYB level was indistinguishable from that in wild-type seeds. The photon fluence required for induction and photoreversible inhibition of germination was examined using the Okazaki large spectrograph. At the wavelengths of 400-710 nm, the ABO seeds required significantly less photon fluence than wild-type seeds for induction of germination, whereas the RBO seeds required similar fluence to wild-type seeds. A critical threshold wavelength for either induction or inhibition of germination of ABO seeds shifted towards the longer wavelengths relative to wild-type seeds. By assuming that PhyA and PhyB are similar in their photochemical parameters, amounts of Pfr at each wavelength were calculated. The photon fluence required for 50% germination was equivalent to the fluence generating a Pfr/Ptot ratio of 0.21-0.43 in wild-type seeds, and of 0.035-0.056 in ABO seeds. These results indicate that PhyB-specific seed germination is not strictly a function of the Pfr/Ptot ratio, but is probably a function of the absolute Pfr concentration.

Arabidopsis↗

PubMed1998

Photomorphogenic development of the Arabidopsis shy2-1D mutation and its interaction with phytochromes in darkness.

We previously reported a photomorphogenic mutation of Arabidopsis thaliana, shy2-1D, as a dominant suppressor of a hy2 mutation. Here, we report that shy2-1D confers various photo-responsive phenotypes in darkness and the dark phenotypes of the mutant are affected by phytochrome deficiency. Dark-grown seedlings of the mutant developed several photomorphogenic characteristics such as short hypocotyls, cotyledon expansion and opening, and partial differentiation of plastids. When grown further in darkness, the mutant plant underwent most of the developmental stages of a light-grown wild-type plant, including development of foliar leaves, an inflorescence stem with cauline leaves, and floral organs. In addition, two light-inducible genes, the nuclear-encoded CAB and the plastid-encoded PSBA genes, were highly expressed in the dark-grown mutant seedlings. Furthermore, reduced gravitropism, a phytochrome-modulated response, was observed in the mutant hypocotyl in darkness. Thus, shy2-1D is one of the most pleiotropic photomorphogenic mutations identified so far. The results indicate that SHY2 may be a key component regulating photomorphogenesis in Arabidopsis. Surprisingly, double mutants of the shy2-1D mutant with the phytochrome-deficient mutants hy2, hy3(phyB-1) and fre1-1(phyA-201) showed reduced photomorphogenic response in darkness with a longer hypocotyl, a longer inflorescence stem, and a lower level expression of the CAB gene than the shy2-1D single mutant. These results showed that phytochromes function in darkness in the shy2-1D mutant background. The implications of these results are discussed.

Arabidopsis↗

PubMed1998

Control of expression of a gene encoding an extensin by phytochrome and a blue light receptor in spores of Adiantum capillus-veneris L.

In the present study, using a newly developed fluorescent differential display technique, we have carried out large-scale screening for genes whose expression was regulated by phytochrome and antagonistically by a blue light receptor in the spores of the fern Adiantum capillus-veneris L. Spores after imbibition were briefly irradiated with red, red/blue or blue light and collected 8 h after the irradiation. Total RNA was isolated from each sample and used to make cDNA with an oligo-dT primer. The cDNA was then used as a template for PCR with the oligo-dT primer and 80 arbitrary primers. The resulting PCR products were analyzed by an automated fluorescent DNA sequencer. Among 8000 displayed bands, we identified 15 up-regulated and four down-regulated bands by red light, and this red light effect was irreversibly reversed by blue light. We cloned one of the up-regulated cDNA fragments and used it to screen a cDNA library prepared from the spores. The isolated insert is predicted to encode Ser-(Pro)n repeats and showed homology with cell wall-associated extensins. The expression of this cDNA was induced 8 h after a red light treatment and the red light induction was photoreversibly prevented by far-red light and photoirreversibly by blue light. The mRNA of this gene was detectable 4 h after red light irradiation and gradually increased in germinating spores.

Amino Acid Sequence↗

PubMed1998

Genetic identification of FIN2, a far red light-specific signaling component of Arabidopsis thaliana.

Phytochrome A (PhyA) mediates most, if not all, various plant responses to far-red (FR) light. Here, we report a novel genetic mutation that impairs a variety of responses in the PhyA-signaling pathway of Arabidopsis thaliana. The mutation was isolated by screening seedlings that show reduced sensitivity to continuous far-red (FRc) light irradiation, but not to continuous red (Rc) light irradiation. The mutation named fin2-1 is not allelic to a PHYA mutation. Furthermore, immunoblot analysis indicated that the amount of the phytochrome A apoprotein in the fin2-1 mutant was comparable to that in wild type. Seedling of the fin2-1 mutant showed defects in hypocotyl growth inhibition and apical hook and cotyledon opening in FRc light but not in Rc light. The results showed that the mutation occurred in a downstream signaling component potentially specific to PhyA. Other PhyA-mediated responses such as FR-preconditioned blocking of greening, anthocyanin accumulation, reduction of gravitropic response, and expression of the CAB and CHS genes were impaired by the fin2-1 mutation: the degree of the mutant effect on the responses was variable. However, FR light-mediated seed germination and photoperiodic flowering responses were not affected significantly in the mutant. These results showed that FIN2 defines an upstream branch point in the PhyA signaling pathway.

Alleles↗

PubMed1998

A preliminary study aimed at the detection of Leishmania parasites in subjects with cutaneous leishmaniasis using polymerase chain reaction.

As a basic study for future diagnosis of cutaneous leishmaniasis, we tried to detect Leishmania parasites representing different species in the subgenera Leishmania and Viannia from subject patients with cutaneous leishmaniasis by using the polymerase chain reaction (PCR) with the subgenus Viannia specific primer. Four out of the 14 specimens revealed an amplified DNA of 70 bp specific for the subgenus Viannia (L. braziliensis complex). No bands were detected in the rest of the specimens belonging to the subgenus Leishmania and unclassified groups. The base sequences of the amplified DNA corresponded with those of the L. (V). braziliensis kinetoplast minicircle. We concluded that PCR using the present primer specific for the subgenus Viannia would be useful in detecting Leishmania parasites in lesions of cutaneous leishmaniasis caused by the L. braziliensis complex.

Animals↗

PubMed1998

Comparative studies of the detection rates of Leishmania parasites from formalin, ethanol-fixed, frozen human skin specimens by polymerase chain reaction and Southern blotting.

In this study, detection rates of Leishmania parasites from human skin were compared among three different types of specimens, formalin-fixed, ethanol-fixed, and frozen, by polymerase chain reaction (PCR) and Southern blotting. For this purpose, we used biopsy specimens collected from 19 leishmaniasis patients and performed PCR and Southern hybridization with the probe specific for Leishmania (Viannia) braziliensis complex. Among these 19, 16 specimens were from cutaneous leishmaniasis (CL), one, diffuse cutaneous leishmaniasis (DCL) and 2, mucocutaneous leishmaniasis (MCL) and were formalin-fixed and paraffin-embedded. The causative agents for one case of CL and one case of DCL were already identified as L. (Leishmania) complex. Six specimens of CL were preserved in 100% ethanol. Two specimens of MCL were frozen tissues. PCR using the formalin-fixed and paraffin-embedded specimens revealed positive bands at 70 bp in 9 (47.4%) out of 19 specimens of CL, MCL and DCL. Southern blotting detected the signals in 12 (63.2%) out of the 19. PCR using the 100% ethanol-fixed specimens revealed positive bands in 4 (66.7%) out of 6, and Southern blotting also detected the signals in 4 (66.7%) out of the 6. PCR and Southern blotting using 2 frozen specimens of MCL were always positive (100%). Although we failed to detect significant differences by Chi-square test between the results from the formalin-fixed, paraffin-embedded specimens and those from 100% ethanol-fixed ones, we concluded that ethanol-fixed specimens, convenient for transportation and storage, would be more useful for diagnosis of leishmaniasis by PCR in a developing country.

Adolescent↗