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Biomedical subjects

H Fujino

Publications and source records attributed to H Fujino.

At least 37 records · Page 2Linked to original sources
PubMed2001

Suppression of mediastinal metastasis by uracil-tegafur or cis-diamminedichloroplatinum(II) using a lymphogenous metastatic model in a human lung cancer cell line.

PURPOSE AND EXPERIMENTAL DESIGN: The extent of lymphatic metastasis is the most important factor in the prognosis for non-small cell lung cancer (NSCLC). Therefore, suppression of lymphatic metastasis provides an improvement in survival time in lung cancer patients. We established a new patient-like model for lung cancer metastasis by orthotopic implantation in severe combined immunodeficiency (SCID) mice and demonstrated the lymphogenous spread histologically using human NSCLC cell lines. The cardinal features of this model are a simple procedure and a similarity to the metastatic form of human lung cancer. The purpose of this study is to assess the inhibitory action of uracil-tegafur (UFT) and cis-diamminedichloroplatinum(II) (CDDP) on lymphatic metastasis and life span prolongation in our lymphogenous metastatic model system using SCID mice. RESULTS: The inhibition ratios of mediastinal lymph node metastasis were 86.2, 94, and 92.1% for 12 mg/kg body UFT, 17 mg/kg body UFT, and 10 mg/kg body CDDP, respectively. The administration of anticancer drugs prolonged the life span by 4.6 days (17 mg/kg body UFT) and 8 days (10 mg/kg body CDDP) in MST. CONCLUSION: We demonstrated that UFT alone and CDDP alone suppressed mediastinal metastasis and prolonged the life span in our lymphogenous metastatic model. Regardless of the administration route and characteristics of anticancer drugs, cytostatic or cytotoxic, our model is capable of evaluating the inhibitory effect of drugs on lymphatic metastasis. This model should make an important contribution to our understanding of the mechanism and selection of drugs for antilymphatic metastasis in lung cancer.

Animals↗
PubMed2000

Delayed reversal of shape change in cells expressing FP(B) prostanoid receptors. Possible role of receptor resensitization.

Prostaglandin F(2 alpha) (PGF(2 alpha)) receptors are G-protein-coupled receptors consisting of two alternative mRNA splice variants, named FP(A) and FP(B). As compared with the FP(A) isoform, the FP(B) isoform lacks the last 46 amino acids of the carboxyl terminus and, therefore, represents a truncated version of the FP(A). We recently found (Pierce, K. L., Fujino, H., Srinivasan, D., and Regan, J. W. (1999) J. Biol. Chem. 274, 35944-35949) that stimulation of both isoforms with PGF(2 alpha) leads to activation of a Rho signaling pathway, resulting in tyrosine phosphorylation of p125 focal adhesion kinase, formation of actin stress fibers, and cell rounding. Although the activation of Rho and subsequent cell rounding occur at a similar rate for both isoforms, we now report that following the removal of PGF(2 alpha) the reversal of cell rounding is much slower for cells expressing the FP(B) isoform as compared with the FP(A) isoform. Thus, in HEK-293 cells that stably express the FP(A) isoform, the reversal of cell rounding appears to be complete after 1 h, whereas for FP(B)-expressing cells there is essentially no reversal even after 2 h. Similarly, the disappearance of stress fibers and dephosphorylation of p125 focal adhesion kinase following removal of agonist are much slower in FP(B)-expressing cells than in FP(A)-expressing cells. The mechanism of this differential reversal appears to involve a difference in receptor resensitization following the removal of agonist. Based upon whole cell radioligand binding, agonist-induced stimulation of inositol phosphate formation, and mobilization of intracellular Ca(2+), the FP(B) isoform resensitizes more slowly than the FP(A) isoform. These findings suggest that the carboxyl terminus of the FP(A) is critical for resensitization and that the slower resensitization of the FP(B) isoform leads to prolonged signaling. This differential signaling distinguishes the FP(A) and FP(B) receptor isoforms and could be important toward understanding the physiological actions of PGF(2 alpha).

Calcium↗
PubMed2000

Teratogenic effects of bis-diamine on early embryonic rat heart: an in vitro study.

BACKGROUND: Bis-diamine induces cardiac defects, including conotruncal anomalies in rat embryos when the agent is administered to the mother. To evaluate the teratogenic effects and mechanism of bis-diamine, we performed morphological and immunohistochemical analyses of early rat embryos cultured in medium containing bis-diamine. METHODS: The embryos were removed from mother rats on gestational day 10.5 and cultured in medium containing 1 mg of bis-diamine for 6 hr. The embryos were then cultured in medium only for another 6, 12, 18, and 42 hr, corresponding to embryonic day (ED) 11.0, 11.25, 11.5, and 12.5, respectively. Some embryos from the same mothers were used as controls and were cultured in medium only for the corresponding periods to the embryos exposed to bis-diamine. Some mother rats were given a single oral dose of 200 mg of bis-diamine on gestational day 10.5. Embryos from these pregnant rats were removed 6 hr after the oral administration of bis-diamine, and were also cultured in medium only for 6, 12, 18, and 42 hr. RESULTS: No cardiac abnormalities were detected in the controls at any stage of development. Thirty-three of 51 (65%) embryos exposed to bis-diamine and 15 of 20 (75%) embryos removed from bis-diamine-administered mothers showed abnormal cardiac development, including dilated ventricle, elongation of outflow tract, and pericardial defect on ED 11.5. Four of six (67%) embryos exposed to bis-diamine, and five of seven (71%) removed from bis-diamine-administered mothers also presented almost the same cardiac abnormalities on ED 12.5. No cardiac abnormalities were detected in bis-diamine-treated embryos before ED 11.5. In addition, the expression of neural cell adhesion molecule (N-CAM) was examined using immunohistochemical methods. Fewer N-CAM immunoreactive cells were detected in the third and fourth aortic arches in the bis-diamine-treated embryos than in controls on ED 11.5. However, more N-CAM immunoreactive cells were detected in the bis-diamine-treated embryos than in controls on ED 12.5. CONCLUSIONS: These results suggest that bis-diamine induces cardiac anomalies by delaying the migration of neural crest cells into the heart and by disturbing the proliferation of pericardial precursor during early cardiac development.

Abnormalities, Drug-Induced↗
PubMed2000

A new approach to gene mutation analysis using "GFP-Display".

The unique behavior of green fluorescent protein (GFP) on SDS-PAGE was applied to the detection of a single amino acid substitution in GFP-tagged polypeptides. This simple detection method using SDS/urea gels was designated GFP-display. The N-terminal 18 or 37 amino acids of K-Ras was used as a model GFP-tagged polypeptide. K-ras exon 1 was fused to a gfp cDNA at each end and expressed in Escherichia coli. Amino acid number 12 of K-Ras (wild type; Gly) was changed to Ser, Arg, Cys, Asp, Ala, or Val, and the mobility shift of the greenish fluorescent bands in the SDS/urea gel was analyzed. These mutants were easily detected by GFP-display; however, detection depended strongly on the urea concentration and electrophoresis temperature. Subsequently, GFP-display was applied to the 36 amino acids encoding human p53 exon 7. Amino acid number 248 (wild type; Arg) was changed to Gly, Trp, Gln, Pro, or Leu, and similar mobility shifts were observed. GFP-display could be coupled with an in vitro translation system. Fluorescent active GFP and GFP-Ras fusion proteins were synthesized within a few hours. GFP-display shows potential as a modern approach to gene mutation analysis at the protein level, and is a useful method for protein engineering studies.

DNA Mutational Analysis↗
PubMed2000

Effects of intracoronary tissue-type plasminogen activator treatment in kawasaki disease and acute myocardial infarction.

We retrospectively studied 3 patients with Kawasaki disease (KD) and acute myocardial infarction (AMI) who were treated with intracoronary administration of tissue-type plasminogen activator (t-PA). Two-dimensional echocardiogram on the next day of the treatment revealed reduction of thrombus and improvement of the cardiac function in all 3 patients. However, a 12-month-old patient treated with 200,000 U/kg of t-PA at 48 h after the onset of AMI died of recurrent myocardial infarction. The other 2 patients treated with 400,000 and 800,000 U/kg, respectively, showed clear, though not prompt, improvement in clinical symptoms and laboratory data. The intracoronary thrombolytic therapy using high-dose t-PA appears effective in treating AMI associated with KD.

Coronary Angiography↗
PubMed2000

Extracellular signal regulated protein kinase and c-jun N-terminal kinase are involved in ml muscarinic receptor-enhanced interleukin-2 production pathway in Jurkat cells.

We have previously shown that m1 and m2 muscarinic receptors were expressed on human peripheral blood lymphocytes (hPBL) and that pre-stimulation of these receptors enhanced phytohemagglutinin (PHA)-induced interleukin-2 (IL-2) production. Possible intracellular signal pathways of muscarinic receptors to regulate IL-2 production were examined in human T cell line Jurkat cells. Pretreatment of the cells with muscarinic receptor agonist, oxotremorine M (Oxo-M), enhanced IL-2 production induced by phorbol 12-myristate 13-acetate (PMA)/A23187, while Oxo-M by itself did not affect IL-2 production. The enhancement of IL-2 production by Oxo-M was inhibited by 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) an ml/m3 receptor antagonist. When the cells were pretreated with AF-DX116, an m2 antagonist, the IL-2 production enhanced by Oxo-M was further stimulated. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that ml and m2 muscarinic receptors exist on Jurkat cells. The stimulation of ml receptors enhanced the PMA/A23187-induced binding activity to AP-1 consensus sequences in IL-2 promoter and production of c-Fos and c-Jun protein. The stimulation of ml receptors did not modify the DNA binding of NF-kappaB, NF-AT or Oct-1. When ml receptors were stimulated, activities of mitogen-activated protein kinase (MAPK)/extracellular signal regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) were increased, while p38 MAPK was not affected. Incubation with Oxo-M induced a transient increase in [Ca2+]i, which was abolished by pretreatment with 4-DAMP. Treatment with cyclosporin A markedly decreased the PMA/A23187-induced IL-2 promoter activity. This treatment, however, did not affect the enhancement of the promoter activity induced by ml receptor stimulation. The results suggest that transcription factor AP-1 is involved in the ml receptor-mediated enhancement of IL-2 transcript in Jurkat cells, and that pathways via MAPK/ERK and JNK, but not via p38 MAPK, are involved in the ml receptor-mediated enhancement of IL-2 promoter activity.

Blotting, Western↗
PubMed2000

A flexible single-step detection of blotted antigen using a fusion protein between protein A and green fluorescent protein.

A green fluorescent protein mutant (S147P GFP) was fused with protein A and expressed in Escherichia coli. This fusion protein (PA-GFP147) was used in immunoblotting studies as a new detection system, designated as "flexible single-step detection (FSSD)". In FSSD, the detection of blotted antigen was done in one step, and the antigen-antibody reaction can be monitored by UV-irradiation in real time. The reaction time, therefore, can be flexibly controlled by monitoring the green fluorescence.

Animals↗
PubMed2000

Differential regulation of prostaglandin F(2alpha) receptor isoforms by protein kinase C.

Prostaglandin F(2alpha) receptors (FP) are G protein-coupled receptors that bind prostaglandin F(2alpha) (PGF(2alpha)), resulting in the activation of an inositol phosphate (IP) second messenger pathway. Alternative mRNA splicing generates two FP receptor isoforms. These isoforms, designated FP(A) and FP(B), are otherwise identical except for their carboxyl termini. FP(B) is essentially a truncated version of FP(A) that lacks the 46 carboxyl-terminal amino acids, including four putative protein kinase C (PKC) phosphorylation sites. Until now, functional differences between these FP receptor isoforms have not been identified. We now report that pretreatment with the PKC inhibitor bisindolylmaleimide I enhanced PGF(2alpha)-stimulated IP accumulation in transfected cells stably expressing the FP(A) isoform but not in cells stably expressing the FP(B) isoform. Whole-cell phosphorylation experiments showed a strong agonist-dependent phosphorylation of the FP(A) isoform but little or no phosphorylation of the FP(B). Pretreatment of cells with bisindolylmaleimide I decreased PGF(2alpha)-stimulated phosphorylation of the FP(A) isoform consistent with a PKC-dependent phosphorylation. In vitro phosphorylation of an FP(A) carboxyl-terminal fusion protein by recombinant PKCalpha showed that the carboxyl terminus of the FP(A) is a substrate for PKC. These results suggest that PKC-dependent phosphorylation is responsible for differential regulation of second messenger signaling by FP prostanoid receptor isoforms.

Cell Line↗
PubMed1999

Activation of FP prostanoid receptor isoforms leads to Rho-mediated changes in cell morphology and in the cell cytoskeleton.

Prostaglandin F(2alpha) (PGF(2alpha)) exerts its biological effects by binding to and activating FP prostanoid receptors. These receptors, which include two isoforms, the FP(A) and FP(B), have been cloned from a number of species and are members of the superfamily of G-protein-coupled receptors. Previous studies have shown that the activation of FP receptors leads to phosphatidylinositol hydrolysis, intracellular calcium release, and activation of protein kinase C. Here, we demonstrate that PGF(2alpha) treatment of 293-EBNA (Epstein-Barr nuclear antigen) cells that have been stably transfected with either the FP(A) or FP(B) receptor isoforms leads to changes in cell morphology and in the cell cytoskeleton. Specifically, cells treated with PGF(2alpha) show retraction of filopodia and become rounded, and actin stress fibers are formed. Pretreatment of the cells with bisindolylmaleimide I, a protein kinase C inhibitor, has no effect on the PGF(2alpha)-induced changes in cell morphology, although it does block the effects of phorbol myristate acetate on cell morphology. On the other hand, the PGF(2alpha)-induced changes in cell morphology and formation of actin stress fibers can be blocked by pretreatment of the cells with C3 exoenzyme, a specific inhibitor of the small G-protein, Rho. Consistent with FP receptor induced formation of actin stress fibers and focal adhesions, FP(A) receptor activation also leads to rapid (within two minutes) tyrosine phosphorylation of p125 focal adhesion kinase (FAK) which can be blocked by pretreating the cells with C3 exoenzyme. Taken together, these results suggest that the FP receptor isoforms are coupled to at least two second messenger pathways, one pathway associated with protein kinase C activation, and the other with activation of Rho.

ADP Ribose Transferases↗
PubMed1999

Simultaneous determination of NK-104 and its lactone in biological samples by column-switching high-performance liquid chromatography with ultraviolet detection.

A simple and sensitive column-switching HPLC method has been developed for the simultaneous determination of NK-104 (HMG-CoA reductase inhibitor) and its lactone in human and dog plasma. Plasma sample was extracted with methyl tert-butyl ether and then the extract was subjected to methylation with diazomethane to prevent the mutual conversion between NK-104 and its lactone. The extract was injected into the column-switching HPLC system. The calibration curves of NK-104 and NK-104 lactone were linear over the ranges 0.5 to 100 ng/ml for human plasma samples and 0.5 to 500 ng/ml for dog plasma, respectively. The intra-day and inter-day C.V. values of these analytes were less than 13.3%. The intra-day and inter-day accuracies of these analytes were between -14.0 and 6.5%. The proposed method has been applied to plasma samples obtained after oral administration of a single 2 mg dose of NK-104 to volunteers.

Animals↗
PubMed1999

Progression of congenital heart disease in the prenatal period.

BACKGROUND: Prenatal echocardiography has shown evidence of prenatal development of congenital heart disease. Prenatal cardiac anatomy, chamber size and function change during gestation, so that the appearance of cardiac structure in abnormal hearts may be different from that which is usually seen postnatally. METHODS: Published prenatal echocardiographic studies were reviewed and in utero development of congenital heart disease from midtrimester to the early postnatal period is discussed. RESULTS: The growth of the great vessels and ventricles is reduced in fetuses with ventricular outflow obstruction. Valve regurgitation may progress. The foramen ovale and ductus arteriosus have been reported to become restrictive in utero in several settings. Pulmonary vascular obstructive changes may progress prenatally. Fetal arrhythmia (both bradycardia and tachycardia) may develop in utero. Development of congestive heart failure is a very important issue during follow up of fetuses with significant cardiac or extracardiac problems. Some may progress to fetal hydrops and prognosis of the affected fetuses is usually very poor. CONCLUSIONS: Correct knowledge of possible development is important for accurate prenatal diagnosis. Information on prenatal progression of the cardiac anomaly is also important to make plans for follow up and perinatal management, to predict outcomes and to counsel family. Furthermore, the benefits of prenatal treatment instead of postnatal treatment should be assessed by the accurate prediction of the progression of the cardiac problem in utero. Further extensive studies using a large number of cases is required to predict progression accurately. In addition, further studies for elucidating the mechanisms of progression is important to provide better outcomes for fetuses with various congenital heart diseases.

Arrhythmias, Cardiac↗
PubMed1999

Identification of metabolites of NK-104, an HMG-CoA reductase inhibitor, in rat, rabbit and dog bile.

NK-104 is an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase with a very potent lipid-lowering effect. Biotransformation profiles of NK-104 in bile from rat, rabbit and dog given an intravenous infusion of NK-104 were investigated. Structural assignment was made by liquid chromatography (LC)-mass spectrometry (MS)-MS and proton NMR analyses. The predominant component was intact NK-104 in all the animals. At least eight other metabolites were present in rat, four in rabbit, and 10 in dog. These bile metabolites were purified and isolated by preparative HPLC. Biotransformation pathways elucidated for NK-104 were as follows: (a) lactonization ; (b) beta-oxidation of the side-chain; (c) hydroxylation of the quinoline ring; (d) conjugation with Beta-glucuronic acid and taurine. Beta-oxidative degradation of the side-chain in the case of other HMG-CoA reductase inhibitors is necessary for epimerization of the hydroxy group which has an R-configuration. However, M-16, glucuronide of the ketolactone derivative, was obtained as a key metabolite suggesting another beta-oxidation pathway for the side-chain.

Animals↗
PubMed1999

Genetic vulnerability of cortical neurons isolated from stroke-prone spontaneously hypertensive rats in hypoxia and oxygen reperfusion.

Severe hypertension and cerebrovascular diseases develop in stroke-prone spontaneously hypertensive rats (SHRSP). Cortical neurons from SHRSP are more vulnerable than those from Wistar Kyoto rats (WKY) to the effects of nitric oxide (NO)- and N-methyl-D-aspartate (NMDA)-mediated neurotoxic agents. Growth factors, idebenone, and nilvadipine (a Ca2+ channel blocker) can reduce neuronal damage caused by hypoxia or neurotoxic agents. This study was designed to determine 1) whether cortical neurons from SHRSP are more vulnerable than those from WKY and 2) whether neuronal damage is minimized by the so-called neuroprotective agents in cells exposed to hypoxia and oxygen reperfusion. We demonstrated that 6 to 24 h of hypoxia did not increase cell death in either WKY or SHRSP, whereas 36 h of hypoxia significantly increased cell death in SHRSP (p < 0.01). Furthermore, 6 to 36 h of hypoxia and 1.5 to 5 h of reperfusion heavily damaged cells from both strains of rats, and most cells became apoptotic or necrotic. We also verified that the ability to protect neurons in hypoxia and oxygen reperfusion was as follows: idebenone > insulin-like growth factor-1 (IGF-1) > nilvadipine. These data indicate that oxygen radical generation occurs and the free radicals heavily damage neurons in hypoxia and oxygen reperfusion. SHRSP neurons are weaker than WKY neurons in these conditions. Furthermore, we surmise that idebenone, an antioxidant, decreases free radicals, and IGF-I attenuates p53-mediated apoptosis and thereby prevents cell death. We conclude that antioxidants are more potent than IGF-1 in protecting cortical neurons from damage caused by hypoxia and oxygen reperfusion, although both are very useful in minimizing damage to cortical neurons.

Animals↗
PubMed1999

Vitamin E prevents apoptosis in hippocampal neurons caused by cerebral ischemia and reperfusion in stroke-prone spontaneously hypertensive rats.

Cerebral ischemia followed by oxygen reperfusion induced apoptosis in hippocampal neurons in stroke-prone spontaneously hypertensive rats (SHRSP) but not in Wistar Kyoto rats. Oxygen radicals were involved in reoxygenation injury after hypoxia in hippocampal slices. Vitamin E inhibited the reoxygenation injury in cultured cortical neurons. In addition, the temporal cortices in Alzheimer's disease have increased sensitivity to oxygen radicals, and Vitamin E slowed the progression of the disease. Thus we fed Wistar Kyoto and SHRSP rats either a normal diet or a high Vitamin E diet for 3 weeks. We measured Vitamin E concentrations of plasma and brain by applying the HPLC method. Vitamin E increased its concentration in plasma, cerebral cortex, and hippocampus (p < 0.01) during a 3-week pretreatment. In addition, we clipped both common carotid arteries in these rats for 30 minutes. After the blocking, the rats were reperfused for 6 and 9 days, respectively, and then killed. We cut the brains coronally, removed the hippocampal CA1 regions, and examined the neurons using an electron microscope. SHRSP rats with normal cerebral circulation had 30.4+/-8.0 apoptotic neurons per 1000 neurons. Cerebral ischemia followed by 6 and 9 days of reperfusion, respectively, increased apoptotic neurons in SHRSP rats fed a normal diet (6 days: 542.5+/-154.1 per 1000 neurons; 9 days: 657.5+/-110.2 per 1000 neurons). In contrast, apoptotic neurons in SHRSP rats fed a high Vitamin E diet were significantly (p < 0.01) small in number (6 days: 41.3+/-27.5 per 1000 neurons; 9 days: 35.5+/-19.7 per 1000 neurons) even though the rats were treated in the same way. These data demonstrate that oxygen radical generation occurs after reperfusion and that free radicals heavily damage the neurons in SHRSP rats. Vitamin E reacts with the radicals and prevents neuronal apoptosis caused by cerebral ischemia and reperfusion. Therefore, Vitamin E seems to be an important agent in lowering radical damage to hippocampal neurons.

Animals↗
PubMed1999

Doppler echocardiographic evaluation of the hemodynamics in absent aortic valve.

Hemodynamics were estimated by Doppler echocardiography in a neonate with an absent aortic valve and absent or extremely hypoplastic mitral valve. The coronary blood flow depended on the increased end-diastolic pressure of the left ventricle. Pulmonary venous congestion, which was also due to the increased end-diastolic pressure of the left ventricle, may induce decreased oxygen saturation and, subsequently, further myocardial hypoxia, poor contraction and increased end-diastolic pressure of the left ventricle. Finally, hypoxic blood was supplied to each organ from the pulmonary artery through the ductus arteriosus, which induced severe acidosis and differential cyanosis after birth.

Aortic Valve↗
PubMed1998

Suppressive effect of globin digest on postprandial hyperlipidemia in male volunteers.

We have reported previously that various edible protein digests inhibit dietary hyperlipidemia in mice, rats, pigs and dogs. Of the various digests tested, globin digest had the most potent inhibitory activity, and a tetrapeptide extracted from globin digest, Val-Val-Tyr-Pro, had activity 7000-fold greater than that of the parent digest. In this clinical study, we investigated the influence of globin digest on serum chylomicron triglyceride concentrations as an indicator of the effect of globin digest on fat absorption and catabolism in humans. Parallel and crossover trials were conducted in which men consumed a control high fat diet (25 g fat, 7.6 g carbohydrate, 1.9 g protein and 0.7 g sodium chloride) or the same diet supplemented with globin digest. The supplemented dosages were 1 and 4 g globin digest. In the parallel trial, 22 men were divided into three groups: control, globin digest 1 g and globin digest 4 g. The increases in chylomicron triglyceride concentrations at 1 h after ingestion of 1 or 4 g globin digest were significantly lower (P < 0.05) compared with the control group. The crossover trial involved six subjects who consumed the control high fat diet and the same diet supplemented with 4 g globin digest. Serum chylomicron triglyceride levels increased in both groups at 1 and 2 h after ingestion, but when subjects consumed 4 g globin digest the increases were suppressed to 75 (P < 0.05) and 42% (P < 0.05) of the increases in controls at the corresponding times, respectively. The areas under the curves of chylomicron and serum total triglyceride concentrations during the 4 h after ingestion of 4 g globin digest were 46 (P < 0.05) and 34% (P < 0.05) lower, respectively, than when the men consumed the high fat control diet. In these trials, globin digest reduced the increase in serum chylomicron triglyceride concentrations as a result of the ingestion of a high fat diet. This hypotriglyceridemic effect of globin digest may be valuable for preventing obesity and in lowering the incidence of cardiovascular diseases.

Adult↗