Biomedical subjects
Publications and source records attributed to K Inoue.
In the present study serotonin (5-hydroxytryptamine, 5-HT) and monoamine oxidase (MAO) were both found localized in the blood vessel walls of human dental pulp. Our discovery of MAO activity in human dental pulp suggests a functional relationship between serotonin and MAO in this region.
CD9 is an integral membrane protein associated with integrins and other membrane proteins. Mice lacking CD9 were produced by homologous recombination. Both male and female CD9-/- mice were born healthy and grew normally. However, the litter size from CD9-/- females was less than 2% of that of the wild type. In vitro fertilization experiments indicated that the cause of this infertility was due to the failure of sperm-egg fusion. When sperm were injected into oocytes with assisted microfertilization techniques, however, the fertilized eggs developed to term. These results indicate that CD9 has a crucial role in sperm-egg fusion.
We analyzed the serum levels of type IV collagen, a major component of the basement membrane, during various stages of sporadic amyotrophic lateral sclerosis (ALS). Serum concentrations of type IV collagen increased significantly with the duration of illness. Findings indicate that the metabolism of type IV collagen and the basement membrane may be affected in the disease process of ALS.
A highly sensitive fluorometric assay technique was adopted in order to examine the adenylate cyclase activity in the minute right ventricular endomyocardial biopsy samples from patients with chronic congestive heart failure (n = 10). Norepinephrine (10(-4) M) and adenosine (10(-3) M) were incubated for 30 min with 10 microl of membrane preparation (1-2 mg protein/mg) to analyze the extent of the receptor-coupled adenylate cyclase activity. Forskolin (10(-4) M) stimulation was used to estimate the maximum adenylate cyclase activity (pmol/mg protein/min, mean +/- SE). The new microanalytical cyclic AMP assay involves four steps: enzymatic destruction of noncyclic adenine nucleotides and phosphorylated metabolites, conversion of cyclic AMP to ATP, amplification of ATP by enzymatic cycling, and fluorometric measurement of NADPH, which is generated in proportion to initial cyclic AMP levels. Basal and forskolin-stimulated maximum adenylate cyclase activities were 75 +/- 8 and 123 +/- 15, respectively. Norepinephrine increased the adenylate cyclase activity to 107 +/- 14, while adenosine tended to decrease it to 65 +/- 7. In addition, elimination of adenosine by adenosine deaminase (10 U/ml) slightly increased the adenylate cyclase activity to 82 +/- 9. These results indicate that the adenylate cyclase activity can be measured in minute endomyocardial biopsy samples. Use of this new approach shows promise of becoming a new and potentially important way to predict the efficacy of pharmacological treatment.
Mice have been successfully cloned from cumulus cells, fibroblast cells, embryonic stem cells, and immature Sertoli cells only after direct injection of their nuclei into enucleated oocytes. This technical feature of mouse nuclear transfer differentiates it from that used in domestic species, where electrofusion is routinely used for nuclear transfer. To examine whether nuclear transfer by electrofusion can be applied to somatic cell cloning in the mouse, we electrofused tail tip fibroblast cells with enucleated oocytes, and then assessed the subsequent in vitro and in vivo development of the reconstructed embryos. The rate of successful nuclear transfer (fusion and nuclear formation) was 68.8% (753/1094) and the rate of development into morulae/blastocysts was 40.8% (260/637). After embryo transfer, seven (six males and one female; 2.5% per transfer) normal fetuses were obtained at 17.5-21.5 dpc. These rates of development in vitro and in vivo are not significantly different from those after cloning by injection (44.7% to morulae/blastocysts and 4.8% to term). These results indicate that nuclear transfer by electrofusion is practical for mouse somatic cell cloning and provide an alternative method when injection of donor nuclei into recipient oocytes is technically difficult.
The development of astroglial cells and the effect of the retinohypothalamic tract on it were studied by vimentin and glial fibrillary acidic protein (GFAP) immunocytochemistry in the suprachiasmatic nucleus (SCN) of the rat. At the embryonic stage, vimentin-immunoreactive (VIM-IR) radial glia, precursors of astrocytes, were dominant. However, their filaments vanished in the first few postnatal days. Instead of VIM-IR glial filaments, GFAP-immunoreactive (GFAP-IR) astrocytes appeared at E20 and grew rapidly from the P3 stage. GFAP immunoreactivity in the ventrolateral portion of the SCN (VLSCN) was measured using a computer-assisted image analyzing system. In normal rats, GFAP immunoreactivity showed a stepwise pattern with two slopes at P3-P4 and P20-P25. Bilaterally eye-enucleated rats operated on the day of birth showed lower GFAP immunoreactivity than normal rats and the GFAP immunoreactivity did not increase between P20 and P25 when GFAP-IR glial processes rapidly expand. Electron microscopic investigation at P50 (adult stage) revealed that neurons in the VLSCN had often direct apposition without astroglial processes and the frequency of this finding was significantly higher in eye-enucleated rats than in the control rats. These findings strongly suggest that the postnatal development of astroglial elements, particularly the expansion of GFAP-IR processes in the SCN, is regulated by retinohypothalamic projection.
We measured the regional cerebral blood flow (rCBF) in six healthy volunteers with PET (positron emission tomography) and H(15)(2)O to identify the areas of the human brain involved in sensorimotor learning. The learning task was visually guided reaching with sensorimotor discrepancy caused by optical rotation. PET measurements were performed in the early and late stages of the adaptation to the sensorimotor perturbation. Control measurements were obtained during an eye movement task and a reaching task without optical rotation. The rCBF data of each learning stage were compared to those of both control conditions. During the early stage, rCBF increases were detected in the rostral premotor cortex bilaterally, the posterior part of the left superior parietal lobule (SPL), and the right SPL including the intraparietal sulcus (IPS). During the late stage, rCBF increases were detected in the left caudal premotor area, the left supplementary motor area proper, the left SPL, the right SPL including the IPS, and the right postcentral sulcus extending to the inferior parietal lobule. These results reveal that sensorimotor learning accompanies changes in the recruited cortical areas during different stages of the adaptation, reflecting the different functional roles of each area for different components of adaptation, from learning of new sensorimotor coordination to retention or retrieval of acquired coordination.
Four human cytochrome P450 1B1 (CYP1B1) allelic variants were purified from membranes of Escherichia coli in which respective CYP1B1 cDNAs and human NADPH-P450 reductase cDNA have been introduced. Purified CYP1B1 variants were used to reconstitute 7-ethoxycoumarin O-deethylation activities with purified rabbit liver or recombinant (rat) NADPH-P450 reductase in the phospholipid vesicles and compared with those catalyzed by CYP1B1 enzymes in the membranes of E. coli in monocistronic (by adding the reductase) and bicistronic (without addition of extra reductase) systems. In the bicistronic system, the ratio of expression of NADPH-P450 reductase to CYP1B1 proteins was found to range from 0.2 to 0.5. Purified CYP1B1 enzymes (under optimal reconstitution conditions) catalyzed 7-ethoxycoumarin O-deethylation at rates one-third to one-fourth of those catalyzed by membranes of E. coli coexpressing CYP1B1 and the reductase proteins. Full catalytic activities in reconstituted systems were achieved with a twofold molar excess of NADPH-P450 reductase to CYP1B1; in membranes of E. coli with the monocistronic CYP1B1 construct, an eightfold molar excess of reductase to CYP1B1 was required. However, in membranes of bicistronic constructs, there was no additional stimulation of 7-ethoxycoumarin O-deethylation by extra NADPH-P450 reductase, despite the fact that the molar ratio of expression levels of reductase to CYP1B1 was <0.5. These results suggest that NADPH-P450 reductase produced in the bacterial membranes is more active in interacting with CYP1B1 proteins in the bicistronic system than the reductase added to artificial phospholipid vesicles or bacterial membranes.
Malignant lymphoma originating in the bone is rare and is now recognized as being an independent clinicopathologic entity known as primary lymphoma of bone. A 60-year-old man complaining of right chest and back pain consulted our hospital for further examination. Chest X-ray and computed tomogram revealed osteolysis and a surrounding soft tissue mass in the sixth right rib. An ultrasonically-guided needle biopsy of the tumor was performed, and histologic examination indicated the dense proliferation of similar-sized atypical cells with nucleoli and an irregular nuclear border. A diagnosis of diffuse, medium-sized non-Hodgkin's lymphoma, B-cell type was made. En block resection of the tumor and chest wall was performed. Macroscopically, the tumor measured 7.5 x 4.8 x 3.0 cm in diameter, and the histologic findings were similar to those of the preoperative needle biopsy. Unfortunately, postoperative treatment with radiation therapy and chemotherapy was ultimately unsuccessful, and a local recurrence and metastatic lesions appeared in the stomach and para-aortic abdominal lymph nodes 7 months after the first symptom appeared. The patient died 3 months later. Surgery was chosen as the initial therapy as it was considered that a rib resection would not result in serious respiratory compromise and the complete resection of the tumor would be superior to radiation therapy for local control. Some authors have reported that the surgical resection of a primary lymphoma of the bone originating in a rib can yield a good prognosis. However, it is a systemic disease and a more effective therapeutic strategy should be developed.
OBJECTIVE: Lidocaine exhibits a cardioplegic action via acute inhibition of sodium influx into the myocardial cells. In terms of the cardiac function and calcium dynamics in the myocardial cells, we investigated the myocardial protective effect of addition of FC43 of Perfluorochemicals, which has an excellent oxygen transport function to meet the myocardial oxygen demand, on lidocaine-induced cardioplegia. METHODS: Isolated rat hearts were perfused with Langendorff mode and were divided to three experimental groups. During of preservation, these hearts were perfused continuously with the next three solution, potassium chloride was added to Krebs-Henseleit bicarbonate buffer to make potassium concentration of 20 mM in the first group (Group A), 2 mM lidocaine was added to Krebs-Henseleit bicarbonate buffer in the second group (Group B), and 2 mM lidocaine and 20% FC43 were added to Krebs-Henseleit bicarbonate buffer in the third group (Group C). After 60 minutes of continuous perfusion, the cardiac function and the intracellular calcium concentration in Groups A and B during cardioplegia were measured. Furthermore, after 360 minutes of continuous coronary perfusion, the cardiac function were measured in Group B and Group C. RESULTS AND CONCLUSIONS: Lidocaine cardioplegia showed a good recovery of cardiac function, because lidocaine induced prompt cardiac arrest by blocking sodium influx and inhibited the intracellular calcium overload by the following inhibition of sodium-calcium channels. Moreover, our results suggested that combining Perfluorochemicals with lidocaine produced a more effective myocardial-preservation that meets the myocardial oxygen demand during long-term cardiac arrest.
Geranylhydroquinone 3"-hydroxylase, which is likely to be involved in shikonin and dihydroechinofuran biosynthesis, was identified in cell suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae). The enzyme hydroxylates the isoprenoid side chain of geranylhydroquinone (GHQ), a known precursor of shikonin. Proton/proton correlation spectroscopic and proton/proton long-range correlation spectroscopic studies confirmed that hydroxylation takes place specifically at position 3", i.e. at the methyl group involved in the cyclization reaction. The enzyme is membrane-bound and was found in the microsomal fraction. It requires NADPH and molecular oxygen as cofactors, and is inhibited by cytochrome P-450 inhibitors such as cytochrome c and CO. The inhibitory effect of CO is reversed by illumination. These data suggest that the enzyme is a cytochrome P-450-dependent monooxygenase. The optimum pH of GHQ 3"-hydroxylase is 7.4, and the apparent K(m) value for GHQ is 1.5 microM. The reaction velocity obtained with 3-geranyl-4-hydroxybenzoic acid was more than 100 times lower than that obtained with geranylhydroquinone.
To determine whether the CYP2E1 genetic polymorphisms cause alterations in protein expression and enzyme catalytic activities, three CYP2E1 genetic polymorphisms, namely RsaI/PstI, DraI, and MspI types, were determined in liver genomic DNA isolated from 39 Japanese and 45 Caucasians. These genotypes were compared with levels of CYP2E1 and activities of 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation in liver microsomes from these human samples. In combination of three types of CYP2E1 polymorphisms, it was classified into seven genotypes in the Japanese population and four in the Caucasian population. The incidence in the occurrence of RsaI/PstI polymorphism or DraI polymorphism was 0.24 and 0.29 for Japanese, and 0.01 and 0.02 for Caucasians. Ethnic difference was also noted in the MspI polymorphism in which frequencies in Japanese and Caucasian populations were 0.15 and 0.02, respectively. Studies with liver microsomes showed that there were no significant differences in the levels of expression of CYP2E1 protein between wild-type (group A) and other 6 genotypes (B, C, D, E, F, and G) in Japanese and other three genotypes (B, D, and F) in Caucasians. Catalytic activities for 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation by liver microsomes were also found to be less significantly affected by mutations in the CYP2E1 gene in human samples examined in this study. These results support the view that RsaI/PstI, DraI, and MspI types of CYP2E1 genetic polymorphisms may not cause significant alterations in protein expression and enzyme catalytic activities of CYP2E1 enzyme in human livers.
Genotypes of CYP2A6, namely CYP2A6(*)1 (wild-type), CYP2A6(*)2, and CYP2A6(*)3, were examined in liver DNA of 39 Japanese and 43 Caucasians using two-step polymerase chain reaction (PCR) methods. We first amplified a DNA fragment (1725 bp) located between near middle of exon 1 and end of exon 4 of the CYP2A6 gene and further amplified using a forward primer 't' or 'mut' (middle of exon 3) and a reverse primer 'E3R' (middle of intron 3) for the detection of CYP2A6(*)2-genetic polymorphism. The 1725 bp fragment was also used for the amplification between exon 3 and near middle of intron 3 of the CYP2A6 gene and the fragment thus obtained digested with XcmI or DdeI to detect and confirm the CYP2A6(*)2- and CYP2A6(*)3-types, respectively. Only one DNA sample from a Japanese origin (J18) was not amplified by CYP2A6-specific primers; liver microsomes from this individual had very low activity of coumarin 7-hydroxylation and were devoid of protein(s) immunoreactive to anti-CYP2A6 antibody. Thus, this individual was suggested to be due to the gene deletion in CYP2A6. By analyzing the remaining 38 Japanese and 43 Caucasians, we found that there were no cases of CYP2A6(*)3-type polymorphism in the samples examined in this study, and no cases of CYP2A6(*)2-type polymorphism in the Japanese samples. Of Caucasians studied two individuals were classified into heterozygous CYP2A6(*)1/(*)2-type. Liver microsomal coumarin 7-hydroxylation activities in these two Caucasians were found to be lower than those of the other 41 Caucasians. Kinetic analysis showed that two CYP2A6(*)1/(*)2 individuals had a very low ratio of V(max) to K(m) for nicotine C-oxidation as well as coumarin 7-hydroxylation in liver microsomes, compared with those of homozygous CYP2A6(*)1-type. These results suggest that among 39 Japanese and 43 Caucasians examined one Japanese is classified to be CYP2A6 gene deletion and two Caucasians are heterozygous CYP2A6(*)1/(*)2-genotype. Thus the race-related differences in the occurrence of CYP2A6 genetic polymorphisms were supported.
The aim of this study was to examine whether tauroursodeoxycholate (TUDC) and cholestyramine resin (CR) enhance biliary carcinogenesis in the hamster model. A cholecystoduodenostomy with dissection of the extrahepatic bile duct on the distal end of the common duct was performed on Syrian hamsters. The hamsters were then divided randomly into 3 groups: control group, TUDC-treated group, and CR-treated group. All animals received N-nitrosobis(2-oxopropyl)amine (BOP) to initiate pancreaticobiliary cancer. The experiment was terminated at week 16 and the number of neoplastic lesions was counted microscopically. In the TUDC group, the intrahepatic biliary carcinogenesis was more accelerated than that observed in the control group, but no promoting effect was seen in the pancreas, gallbladder, or extrahepatic bile duct. In the CR group, both the intrahepatic biliary and the gallbladder carcinogenesis were inhibited compared with that observed in the control group and the TUDC group. TUDC enhanced the intrahepatic bile duct carcinogenesis, whereas CR inhibited both the intrahepatic bile duct and the gallbladder carcinoma. Bile acids were suggested to promote biliary carcinoma in the hamster model.
The name Enterobacter cowanii sp. nov. is proposed for a group of organisms referred to as NIH Group 42. Members of this species are Gram-negative, motile rods conforming to the definition of the family Enterobacteriaceae. The DNA relatedness of nine strains of NIH Group 42 to the proposed type strain of this species averaged 85% at 70 degrees C, whereas the relatedness to other species within the family Enterobacteriaceae was less than 38%. Because the DNA relatedness (5-38%) is closer to species of the genus Enterobacter than to other species of the family, the members of NIH Group 42 were placed in the genus Enterobacter. The majority of strains of E. cowanii were isolated from clinical specimens. A culture of the type strain (888-76) has been deposited in the Japan Collection of Microorganisms as JCM 10956.
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We have reported that the unique thyroid-stimulating hormone-immunoreactive cells (TSH cells) in the intact adult and fetal rat pars tuberalis (PT) show an intense spot-like TSH immunoreaction in the perinuclear region. The present study was designed to investigate the relationship between melatonin and these unique TSH cells. We classified TSH cells in the PT (PT-TSH cells), on the basis of immunoreactivity, into spot-like stained cells (SC) and whole cytoplasm stained cells (WC), and estimated the proportion of each TSH cell type to total cells in the experimental rats by morphometry. Chronic administration of melatonin to control rats leads to an increase of WC in number but a decrease of SC. On the other hand, the intensity of TSH immunoreactivity and the number of rat PT-TSH cells significantly decreased after pinealectomy and recovered by melatonin administration. Radioimmunoassay showed that melatonin treatment increased the TSH content in the PT. Moreover, electron microscopy showed that the number of TSH secretory granules in the PT-TSH cells increased in the melatonin-replaced rats. These results demonstrated that melatonin stimulates the accumulation of TSH in the rat PT-TSH cells via secretory granule formation and suggest that melatonin regulates TSH release from PT-TSH cells.
UNLABELLED: We investigated serum vascular endothelial growth factor (SVEGF) levels in Kawasaki disease and determined whether these levels had any association with the development of coronary artery lesions. We measured SVEGF levels in 66 patients with Kawasaki disease, 18 patients with active infections and 18 afebrile controls. SVEGF levels of patients in the acute phase of Kawasaki disease (0.0-2003.6 pg/ml, median 59.87 pg/ml) were significantly higher than those of patients with active infections (0.0-45.2 pg/ml, median 8.10 pg/ml; P < 0.05) or afebrile controls (0.0-49.8 pg/ml, median 7.75 pg/ml; P < 0.05) and decreased to undetectable or low levels in the recovery phase (n = 31, acute phase: 0.0-2003.6 pg/ml, median 62.50 pg/ml versus recovery phase: 0.0-146.5 pg/ml, median 26.90 pg/ml; P = 0.0007) of the disease. There existed a positive correlation between SVEGF levels and serum C-reactive protein concentrations in the acute phase of Kawasaki disease (rs = 0.347, P = 0.0051). In addition, SVEGF level and duration of fever were found to be major risk factors for the occurrence of coronary artery lesions by univariate (P = 0.012 and P = 0.003, respectively) and multivariate (P = 0.037, OR 6.16 and P = 0.0059, OR 7.59, respectively) analyses. CONCLUSION: Serum vascular endothelial growth factor level, in combination with persistence of fever, could be a powerful predictor for the development of coronary aneurysms.