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K Furukawa

Publications and source records attributed to K Furukawa.

At least 55 records · Page 3Linked to original sources
PubMed2001

Ganglioside G(D2) in small cell lung cancer cell lines: enhancement of cell proliferation and mediation of apoptosis.

Expression levels of gangliosides and glycosyltransferase genes responsible for their syntheses in human lung cancer cell lines and a normal bronchial epithelial cell line were analyzed. Both non-small cell lung cancers and small cell lung cancers (SCLCs) mainly expressed G(M2) and G(M1), whereas only SCLCs expressed b-series gangliosides, such as G(D2), G(D1b), and G(T1b). Accordingly, many SCLC cell lines showed up-regulation of the G(D3) synthase gene. Consequently, we introduced G(D3) synthase cDNA into a SCLC line with low expression of b-series gangliosides and analyzed the effects of newly expressed gangliosides on tumor phenotypes. The transfectant cells expressing high levels of G(D2) and G(D3) exhibited markedly increased growth rates and strongly enhanced invasion activities. Addition of anti-G(D2) monoclonal antibodies into the culture medium of these cells resulted in the marked growth suppression of G(D2)-expressing cell lines with reduced activation levels of mitogen-activated protein kinases but not of nonexpressants, suggesting that G(D2) plays important roles in cell proliferation. Moreover, G(D2)-expressing cells treated with anti-G(D2) antibodies showed features of apoptotic cell death at 30 min after addition of antibodies, i.e., shrinkage of cytoplasm, binding of Annexin V, and staining with propidium iodide, followed by DNA fragmentation. This G(D2)-mediated apoptosis was associated with caspase-3 activation and partly inhibited by a caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone. The finding that anti-G(D2) antibodies suppressed the cell growth and induced apoptosis of SCLC cells strongly suggested the usefulness of G(D2) as a target for the therapy of disastrous cancer, although the precise mechanisms for apoptosis remain to be clarified.

Antibodies, Monoclonal↗
PubMed2001

Emergence of multifunctional oxygenase activities by random priming recombination.

Biphenyl dioxygenase (Bph Dox) is responsible for the initial dioxygenation of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in determination of substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs). Functional evolution of Bph Dox of Pseudomonas pseudoalcaligenes KF707 was accomplished by random priming recombination of the bphA1 gene, involving two rounds of in vitro recombination and mutation followed by selection for increased activity in vivo. Evolved Bph Dox acquired novel and multifunctional degradation capabilities not only for PCBs but also for dibenzofuran, dibenzo-p-dioxin, dibenzothiophene, and fluorene, the compounds scarcely attacked by the original KF707 Bph Dox. The modes of oxygenation were angular and lateral dioxygenation for dibenzofuran and dibenzo-p-dioxin, sulfoxidation for dibenzothiophene, and mono-oxygenation for fluorene. These enzymes also exhibited enhanced degradation abilities for PCB congeners, retaining 2,3-dioxygenase activity and gaining 3,4-dioxygenase activity, depending on the chlorine substitution of PCB congeners. Further mutation analysis revealed that the amino acid at position 376 in BphA1 is significantly involved in the acquisition of multifunctional oxygenase activities and mode of oxygenation.

Base Sequence↗
PubMed2001

Novel functions of complex carbohydrates elucidated by the mutant mice of glycosyltransferase genes.

Complex carbohydrates consist of carbohydrate moieties and protein or lipid portions, resulting in the formation of glycoproteins, proteoglycans or glycosphingolipids. The polymorphic carbohydrate structures are believed to contain profound biological implications which are important in cell-cell or cell-extracellular matrix interactions. A number of studies to delineate the roles of carbohydrates have been performed, and demonstrated definite changes in their profiles, cellular phenotypic changes or, sometimes, morphological and functional changes in tissues after modification of their structures. Recent successes in the isolation of glycosyltransferase genes and their modification enzyme genes has enabled clearer demonstrations of the roles of complex carbohydrates. In particular, genetic modification of glycosyltransferase genes in mice can elucidate the biological significances of their products in vivo. Here, we summarize recent advances in the understanding of the roles of complex carbohydrates provided from studies of gene knock-out mice of glycosyltransferase and modification enzyme genes focusing on novel functions which had not been expected.

Animals↗
PubMed2001

Halenaquinone, a novel phosphatidylinositol 3-kinase inhibitor from a marine sponge, induces apoptosis in PC12 cells.

In nerve growth factor-treated PC12 cells, 12b-methyl-(S)-1H-benzo[6,7]phenanthro[10,1-bc]furan-3,6,8,11(2H,12bH)-tetrone (halenaquinone) caused cytotoxicity in a concentration-dependent manner (EC(50) value; 10 microM). Gel electrophoretic DNA analysis of PC12 cells treated with halenaquinone (10 microM) and 11-(acetyloxy)-1,6b,7,8,9a,10,11,11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-[1S-(1 alpha,6b alpha,9a beta,11 alpha,11b beta)]-3H-furo[4,3,2-de]indeno[4,5-h]-2-benzopyran-3,6,9-trione (wortmannin) (3 microM) showed a typical apoptotic DNA ladder. In the flow cytometric analysis, halenaquinone caused apoptosis in a concentration- and time-dependent manner (EC(50) value; 10 microM), whereas 2,3-dihydro-12b-methyl-(S)-1H-benzo[6,7]phenanthro[10,1-bc]furan-6,8,11(12bH)-trione (xestoquinone) with the methylene group at the C-3 position failed to cause apoptosis, suggesting that the carbonyl group at the C-3 position in halenaquinone is important for exerting apoptotic effects in PC12 cells. Phosphatidylinositol 3-kinase was inhibited by halenaquinone (IC(50) value; 3 microM) as well as wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. Halenaquinone inhibited phosphatidylinositol 3-kinase activity at lower concentrations than those at which it induced apoptosis in PC12 cells. These results suggest that halenaquinone causes the death of PC12 cells through an apoptotic process and that the mechanism of halenaquinone-induced apoptosis may be partially explained by the inhibition of phosphatidylinositol 3-kinase activity.

Acetylcysteine↗
PubMed2001

Inhibition of insulin-induced activation of Akt by a kinase-deficient mutant of the epsilon isozyme of protein kinase C.

Akt, also known as protein kinase B, is a protein-serine/threonine kinase that is activated by growth factors in a phosphoinositide (PI) 3-kinase-dependent manner. Although Akt mediates a variety of biological activities, the mechanisms by which its activity is regulated remain unclear. The potential role of the epsilon isozyme of protein kinase C (PKC) in the activation of Akt induced by insulin has now been examined. Expression of a kinase-deficient mutant of PKCepsilon (epsilonKD), but not that of wild-type PKCepsilon or of kinase-deficient mutants of PKCalpha or PKClambda, with the use of adenovirus-mediated gene transfer inhibited the phosphorylation and activation of Akt induced by insulin in Chinese hamster ovary cells or L6 myotubes. Whereas the epsilonKD mutant did not affect insulin stimulation of PI 3-kinase activity, the phosphorylation and activation of Akt induced by a constitutively active mutant of PI 3-kinase were inhibited by epsilonKD, suggesting that epsilonKD affects insulin signaling downstream of PI 3-kinase. PDK1 (3'-phosphoinositide-dependent kinase 1) is thought to participate in Akt activation. Overexpression of PDK1 with the use of an adenovirus vector induced the phosphorylation and activation of Akt; epsilonKD inhibited, whereas wild-type PKCepsilon had no effect on, these actions of PDK1. These results suggest that epsilonKD inhibits the insulin-induced phosphorylation and activation of Akt by interfering with the ability of PDK1 to phosphorylate Akt.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2001

Virol A, a toxic trans-polyacetylenic alcohol of Cicuta virosa, selectively inhibits the GABA-induced Cl(-) current in acutely dissociated rat hippocampal CA1 neurons.

The effects of virol A (VA), a toxic component of Cicuta virosa (water hemlock), on the GABA-induced Cl(-) current (I(GABA)) in acutely dissociated rat hippocampal CA1 neurons were investigated using whole-cell patch-clamp techniques. VA reversibly reduced I(GABA) and the muscimol (Mus)-induced current (I(Mus)) in a concentration-dependent manner. The IC(50) values for VA against I(GABA) and I(Mus) were 9.6x10(-7) and 9.8x10(-7) M, respectively. VA shifted the EC(50) value of I(GABA) from 6.5x10(-6) to 2.1x10(-5) M, whereas it had no effect on the maximum response, thereby suggesting that VA inhibited I(GABA) in a competitive manner. VA had no apparent effect on current-voltage relationships for I(GABA), thus indicating the lack of voltage-dependency. On the other hand, application of VA (10(-6) M) did not additionally reduce the I(GABA) suppressed by >10(-5) M picrotoxin. VA but not bicuculline accelerated the decay phase of I(GABA), as was seen with picrotoxin. Moreover, pre-application of 10(-5) M VA reduced I(GABA). VA did not inhibit that induced by glycine (10(-4) M). These results indicate that VA inhibits I(GABA) by acting both on the GABA agonist site and on the Cl(-) channel of the GABA(A) receptor-channel complex. VA is a structurally novel type of compound that selectively inhibits the GABA(A) receptor-Cl(-) channel complexes in mammalian central nervous system neurons.

Animals↗
PubMed2001

Differentiation-associated expression of beta-N-acetylgalactosaminylated N-linked oligosaccharides in mammary epithelial cells.

Not only mammalian pituitary glycoprotein hormones but also many glycoproteins from a variety of animal species have been shown to contain N-linked oligosacchardies with the GalNAcbeta1 --> 4GlcNAc structure. Two types of beta-1,4-GalNAcT were found; one transfers N-acetylgalactosamine to acceptor oligosaccharides, which is stimulated by the hormone peptide and the other simply transfers sugar without such activation. In the case of bovine mammary membrane glycoproteins, the expression of beta-N-acetylgalactosaminylated N-linked oligosaccharides was associated with the functional differentiation of the epithelial cells. In contrast, the expression level of such oligosaccharides was much reduced in glycoprotein samples from human breast tumors compared with those from the unaffected regions. These results strongly suggest that the beta-N-acetylgalactosaminylation is one which is regulated under cellular differentiation and dedifferentiation of the mammary gland. Whether or not beta-N-acetylgalactosaminylated N-linked oligosaccharides have unique functions in addition to clearance of the hormone from the circulation remains to be elucidated.

Acetylgalactosamine↗
PubMed2001

The meroperon of a mercury-resistant Pseudoalteromonas haloplanktis strain isolated from Minamata Bay, Japan.

A mer operon of mercury-resistant Pseudoalteromonas haloplanktis strain M1, isolated from sea water of Minamata Bay, was cloned and analyzed. The mer genes were located in the chromosome and organized as merR-merT-merP-merC-merA-merD, the same order as that in Tn21. However, the orientation of the merR gene is the same as that of other mer genes (opposite direction to Tn21), and merR was cotranscribed with other mer genes, a pattern that has not been previously seen with mer determinants from other Gram-negative bacteria. Furthermore, the amino acid similarities of the corresponding mer gene products between those from strain M1 and Tn21 were unusually low.

Amino Acid Sequence↗
PubMed2001

Venous hemangioma of the mediastinum.

Mediastinal hemangiomas are rare tumors. We present a case of venous hemangioma of the mediastinum. Computed tomography shows anterior mediastinal solid mass without enhancement effects. We believe that it is necessary to include a mediastinal hemangioma for differential diagnosis, when little or no enhancement effects are noted at early and delayed CT examination.

Adult↗
PubMed2001

Occurrence of poly-N-acetyllactosamine synthesis in Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase I, II, III, IV, V and VI cDNAs.

Lectin blot analysis of membrane glycoprotein samples from Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase (beta-1,4-GalT) I, II, III, IV, V et VI cDNAs showed that the endogenous N-linked oligosaccharides are galactosylated (Guo et al., Glycobiology (2001), in press). Further analysis revealed that membrane glycoprotein samples from all the gene-transfected cells are also reactive to Lycopersicon esculentum agglutinin (LEA) et Datura stramonium agglutinin (DSA), both of which bind to oligosaccharides with poly-N-acetyllactosamine chains while no lectin reactive protein bands are detected when blots are pretreated with a mixture of diplococcal beta-1,4-galactosidase et jack bean beta-N-acetylhexosaminidase or N-glycanase. Analysis of endo-beta-galactosidase-digestion products revealed the presence of the Gal1-->GlcNAc1-->Gal and/or GlcNAc1-->Gal structures in the gene-transfected cells. When the homogenates of the gene-transfected cells were used as enzyme sources towards oligosaccharides with the GlcNAc beta 1-->(3Gal beta 1-->4GlcNAc)(1-3) structures, human recombinant beta-1,4-GalTs I et II galactosylated these oligosaccharides more effectively than other beta-1,4-GalTs. These results indicate that beta-1,4-GalTs I-VI can synthesize poly-N-acetyllactosamine chains with beta-1,3-N-acetylglucosaminyltransferase.

Animals↗
PubMed2001

Coronary endothelial damage during off-pump CABG related to coronary-clamping and gas insufflation.

OBJECTIVE: Although off-pump coronary artery bypass grafting (CABG) has been recognized less invasive than conventional CABG on cardiopulmonary bypass, off-pump CABG may be partly invasive especially to the coronary endothelium. The present study was designed to evaluate the adverse effects of coronary snaring with looped sutures and gas insufflation on the coronary endothelium. The protective efficacies on the coronary endothelium of coronary snaring with elastic sutures or humidified gas insufflation with/without heparin and dipyridamole-added were also tested. METHODS: Thirty-six mongrel dogs were used. After systemic heparinization (150 U/kg), a 5 mm longitudinal coronary incision was made with looped non-elastic monofilament sutures or elastic sutures applied proximally and distally. The incised coronary artery was exposed to non-humidified carbon dioxide, humidified carbon dioxide with lactated Ringer solution, or humidified carbon dioxide with heparin and dipyridamole-added lactated Ringer solution for 10 or 20 min in each group. After gas insufflation, the incised coronary artery was repaired, then, the coronary was reperfused. Perfusion-fixation was done for observation of the coronary endothelium by scanning electron microscopy. The adverse effect on the endothelium was graded as follows: grade 1, appeared normal; grade 2, few blood cells deposited; grade 3, many blood cells deposited; grade 4, few endothelial cells delaminated with blood cells deposited; grade 5, many endothelial cells delaminated with blood cells deposited. RESULTS: Non-elastic looping caused much more endothelial tears than elastic looping (P<0.00001). Non-humidified gas blowing for 20 min caused more endothelial cell damage than humidified gas blowing (P=0.00005). Non-humidified gas blowing for 10 min caused less damage than for 20 min (P=0.00326), but still caused more damage than humidified gas blowing (P=0.00253). Heparin and dipyridamole-added humidification reduced coronary endothelial area mottled by the deposited cells when compared with simple humidification (P=0.00120). CONCLUSIONS: Coronary snaring resulted in coronary endothelial injury, which was ameliorated by using elastic sutures instead of non-elastic sutures. Non-humidified gas insufflation made blood cells deposited and endothelial cells delaminated with time. Humidified gas insufflation attenuated these adverse effects. Heparin and dipyridamole-added humidification had potential advantage in terms of reducing deposited blood cells on the endothelium over simple humidification.

Animals↗
PubMed2001

Genetic analysis of the cytochrome P-450c17alpha (CYP17) and aldosterone synthase (CYP11B2) in Japanese patients with 17alpha-hydroxylase deficiency.

OBJECTIVE: To determine the clinical and molecular genetic characterization of two Japanese patients with 17alpha-hydroxylase deficiency, we analysed the 17alpha-hydroxylase/17,20-lyase gene (CYP17). Next, to clarify the mechanism of hypoaldosteronism in 17alpha-hydroxylase deficiency, we analysed the expression of aldosterone synthase (CYP11B2) messenger RNA and sequenced CYP11B2 in these patients. PATIENTS: Patient 1 (46 XY), phenotypically female, sought medical attention for hypertension, amenorrhea and infantile genitalia. Patient 2 (46 XX), phenotypically female, presented for hypertension and amenorrhea. Hormonal data in both patients showed decreased levels of sex steroids, cortisol, aldosterone and plasma renin activity and extreme elevation of deoxycortisol. DESIGN: Direct sequencing of CYP17 and CYP11B2 was performed using genomic DNA from the patients. An expression studies of mutated forms of CYP17 was performed using COS-1 cells. The expression of CYP11B2 messenger RNA in mononuclear leucocytes (MNLs) of these patients and normal subjects was measured using the competitive polymerase chain reaction METHOD: The effect of renin secretion stimulation on the levels of CYP11B2 messenger RNA in MNLs of normal subjects was also studied. RESULTS: We detected two novel genetic defects in 17alpha-hydroxylase. Sequence analysis revealed one base pair deletion (T) at codon 243 in exon 4 in patient 1. CYP17 in patient 2 contained a point mutation (C to T) at position 415 in exon 8. Transfected cells of mutant from patient 1 had no 17alpha-hydroxylase or 17,20-lyase activity. The R415C mutant protein showed very weak activity of 17alpha-hydroxylase or 17,20-lyase activity. In the renin secretion stimulating test, the increase in CYP11B2 messenger RNA levels in MNLs was parallel with that of plasma aldosterone concentration. The expression of CYP11B2 mRNA in NMLs of these patients was lower compared to controls. No mutations in CYP11B2, including the 5' flanking region, were found. CONCLUSIONS: These results indicate that the novel mutations of the CYP17 gene found in these patients inactivate cytochrome P450c17 function, and that hypoaldosteronism in these patients may be partly explained by a decreased activity of aldosterone synthase, which is regulated at the transcriptional level.

Adrenal Hyperplasia, Congenital↗
PubMed2001

Structural study of N-linked oligosaccharides of human intercellular adhesion molecule-3 (CD50).

The N-linked oligosaccharides were released from purified human intercellular adhesion molecule (ICAM)-3 by hydrazinolysis. Approximately 6 mol of oligosaccharides were released from 1 mol of ICAM-3. The oligosaccharides reduced with NaB[3H]4 were separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial lectin column chromatography followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endo-glycosidase digestion and by methylation analysis revealed that N-linked oligosaccharides of ICAM-3 are mainly of tri- and tetra-antennary complex-type, about 60% of which contain two to three poly N-acetyllactosamine chains terminated with the type 1 structure and those without the type 1 structure per oligosaccharide. In addition, a small amount of the high mannose-type oligosaccharide with six alpha-mannose residues, which could act as a ligand for the dendritic cell-specific ICAM-3 grabbing nonintegrin, was detected.

Antigens, CD↗
PubMed2001

Two patients with severe alcoholic hepatitis accompanied by hypercytokinemia and granulocytic hyperelastasemia, successfully treated by intravenous infusion of urinastarine (Miraclid).

Severe alcoholic hepatitis (SAH) is not simply a disease of the liver, but it also causes infection and multiple organ failure, and therefore carries an extremely poor prognosis. We report the successful treatment of two patients with SAH. Case 1: The patient was a 55-year-old man. He was a heavy drinker whose alcohol intake had increased for some 3 years to 1.8 L sake a day. Slight clouding of consciousness, fever, and jaundice were evident on his admission to our hospital. Laboratory data showed leukocytosis with a predominance of polymorphonuclear leukocytes, and a decline in coagulability. He tested negative for various hepatitis virus markers. With the diagnosis of SAH made, steroid pulse therapy and bilirubin adsorption therapy were administered. The jaundice improved and the interleukin-8 (IL-8) level decreased. Continuous intravenous infusion of urinastarine (Miraclid) normalized the granulocyte elastase level. Improvement was also seen in coagulability, ascites, icterus and consciousness. Case 2: The patient was a 49-year-old man. He was a heavy drinker whose alcohol intake had increased for 1 month. Fever, jaundice, ascites, and mild disturbance of consciousness were evident at the time of admission. Examination on admission diagnosed SAH. Bilirubin adsorption and continuous intravenous infusion of urinastarine were initiated. As a result, circulating IL-8 level was decreased and coagulability was improved. Therapy for granulocytic hyperelastasemia and hypercytokinemia supervening on SAH is a new effective approach on improvement in the disease.

Blood Coagulation↗