Biomedical subjects

Koichi Furukawa

Publications and source records attributed to Koichi Furukawa.

At least 19 recordsLinked to original sources

PubMed2007

Identification and expression of a sialyltransferase responsible for the synthesis of disialylgalactosylgloboside in normal and malignant kidney cells: downregulation of ST6GalNAc VI in renal cancers.

Although disialyl glycosphingolipids such as GD3 and GD2 have been considered to be associated with malignant tumours, whether branched-type disialyl glycosphingolipids show such an association is not well understood. We investigated the sialyltransferases responsible for the biosynthesis of DSGG (disialylgalactosylgloboside) from MSGG (monosialylgalactosylgloboside). Among six GalNAc:alpha2,6-sialyltransferases cloned to date, we focused on ST6GalNAc III, V and VI, which utilize sialylglycolipids as substrates. In vitro enzyme analyses revealed that ST6GalNAc III and VI generated DSGG from MSGG with V(max)/K(m) values of 1.91 and 4.16 respectively. Transfection of the cDNA expression vectors for these enzymes resulted in DSGG expression in a renal cancer cell line. Although both ST6GalNAc III and VI genes were expressed in normal kidney cells, the expression profiles of ST6GalNAc VI among 20 renal cancer cell lines correlated clearly with those of DSGG, suggesting that the sialyltransferase involved in the synthesis of DSGG in the kidney is ST6GalNAc-VI. ST6GalNAc-VI and DSGG were found in proximal tubule epithelial cells in normal kidney tissues, while they were downregulated in renal cancer cell lines and cancer tissues. All these findings indicated that DSGG was suppressed during the malignant transformation of the proximal tubules as a maturation arrest of glycosylation.

Cell Line↗

PubMed2006

Heart slice NMR.

Nuclear magnetic resonance (NMR) spectroscopy of the heart is normally carried out using whole heart preparations under coronary perfusion. In such preparations, either radical changes in ionic composition of the perfusate or applications of numerous drugs would affect coronary microcirculation. This report communicates the first (31)P NMR spectroscopy study using a heart slice preparation (left ventricular slices) superfused with extracellular medium. The ratio of phosphocreatine concentration to ATP concentration was approximately 2.1. Also, intracellular pH and Mg(2+) concentration ([Mg(2+)](i)), estimated from the chemical shifts of inorganic phosphate and ATP, were comparable with those under retrograde perfusion. [Mg(2+)](i) was significantly increased by the removal of extracellular Na(+), supporting the essential role of Na(+)-coupled Mg(2+) transport in Mg(2+) homeostasis of the heart. Heart slice preparation could also be used to evaluate the potency of cardiac drugs, regardless of their possible effects on coronary microcirculation.

Adenosine Triphosphate↗

PubMed2006

Genetic mechanisms for the synthesis of fucosyl GM1 in small cell lung cancer cell lines.

Fucosyl GM1 has been reported to be specifically expressed in small cell lung cancer (SCLC) cells. However, the genetic basis for the synthesis of fucosyl GM1 has not been investigated. We analyzed the glycosyltransferases responsible for the synthesis of fucosyl GM1 in SCLC cell lines. In four SCLC cell lines expressing fucosyl GM1, both FUT1 and FUT2 mRNAs were detected, indicating that either one or both of alpha1,2-fucosyltransferases may be involved in the expression of fucosyl GM1. However, three of these four lines contained function-loss mutations in the FUT2 coding region, suggesting that FUT1 is mainly involved in the alpha1,2-fucosylation of GM1. The expression levels of the GM1 synthase gene showed no correlation with those of fucosyl GM1, whereas the co-transfection of GM1 synthase cDNA with FUT1 or FUT2 into SK-LC-17 clearly enhanced the neo-expression of fucosyl GM1, indicating its essential role. In contrast, the co-transfection of GD3 synthase cDNA reduced the expression levels of fucosyl GM1 with FUT1 or FUT2. Consequently, FUT1 seems to mainly contribute to the expression of fucosyl GM1, although both FUT1 and FUT2 are capable of generating the antigen. These results should promote the functional analysis of fucosyl GM1 leading to the development of novel therapies for SCLC.

Blood Group Antigens↗

PubMed2006

Metastatic potential of mouse Lewis lung cancer cells is regulated via ganglioside GM1 by modulating the matrix metalloprotease-9 localization in lipid rafts.

To analyze mechanisms for cancer metastasis, we established high metastatic sublines from mouse Lewis lung cancer (P29) by repeated injection. Sublines established from the two subclones H7 and C4 commonly exhibited increased proliferation and invasion activity and reduced expression of ganglioside GM1, although they showed different preferences in their target organs of metastasis. The high metastatic sublines secreted higher levels of activated matrix metalloprotease (MMP)-9 as well as pro-MMP-9 in the culture medium than the parent lines. Furthermore, they contained MMP-9 at the glycolipid-enriched microdomain (GEM)/rafts fractionated by the sucrose density gradient ultracentrifugation of Triton X-100 extracts, whereas the parent cells showed faint bands at the fraction. When high metastatic sublines were treated with methyl-beta-cyclodextrin, their invasion activities were dramatically suppressed, and the MMP-9 secretion was also suppressed. All these results indicated that GEM/rafts play crucial roles in the increased invasion and high metastatic potential. To clarify the implication of reduced GM1 expression, low GM1-expressing cell lines were established using an RNA interference-expression vector of the GM1 synthase. Low GM1-expressing cell lines showed increased proliferation and invasion, enrichment in the GEM/rafts, and increased secretion of MMP-9. Among adhesion molecules, only integrin beta1 was detected in GEM/rafts with stronger intensity in high metastatic lines and low GM1-expressing cells. Taken together, integrins seemed to be enriched in the GEM/rafts by reduced GM1 levels, and subsequently MMP-9 was recruited to the GEM/rafts, resulting in its efficient secretion and activation, and eventually in the increased invasion and metastatic potentials.

Animals↗

PubMed2006

Targeted disruption of Gb3/CD77 synthase gene resulted in the complete deletion of globo-series glycosphingolipids and loss of sensitivity to verotoxins.

To examine whether globotriaosylceramide (Gb3/CD77) is a receptor for verotoxins (VTs) in vivo, sensitivity of Gb3/CD77 synthase null mutant mice to VT-2 and VT-1 was analyzed. Although wild-type mice died after administration of 0.02 microg of VT-2 or 1.0 microg of VT-1, the mutant mice showed no reaction to doses as much as 100 times that administered to wild types. Expression analysis of Gb3/CD77 in mouse tissues with antibody revealed that low, but definite, levels of Gb3/CD77 were expressed in the microvascular endothelial cells of the brain cortex and pia mater and in renal tubular capillaries. Corresponding to the Gb3/CD77 expression, tissue damage with edema, congestion, and cytopathic changes was observed, indicating that Gb3/CD77 (and its derivatives) exclusively function as a receptor for VTs in vivo. The lethal kinetics were similar regardless of lipopolysaccharide elimination in VT preparation, suggesting that basal Gb3/CD77 levels are sufficient for lethal effects of VTs.

Animals↗

PubMed2006

Roles of glycolipids in the development and maintenance of nervous tissues.

Glycoshingolipids are involved in a wide variety of biological events, including cell proliferation, differentiation, development, regeneration, and apoptosis in vertebrates. Expression profiles of glycolipids during the development and cell differentiation or transformation suggest that glycolipids are largely implicated in the determination of cell fates by directly transducing biosignals as receptors and/or modulating receptors' function. Despite of a number of efforts to clarify the molecular functions of glycolipids, no unambiguous results have been obtained until genetic modification of glycolipids became possible. Recent progress in the isolation of cDNAs of glycosphingolipid synthase genes has enabled us to examine roles of glycosphingolipids and strongly promoted further understanding of significances of glycosphingolipids. In particular, knock-out mice of glycosyltransferases showed quite novel aspects of glycolipid function and also redundancy among similar enzymes and glycolipid structures. Here, we summarize analytical methods with which roles of glycolipids in the development and maintenance of nervous tissues, including techniques to establish transgenic mice and gene knock-out mice, to survey fundamental behavior abnormalities, and to examine fine morphological changes lying under abnormal phenotypes of the glycolipids-modified cells and glycolipid-lacking mutant mice.

Animals↗

PubMed2006

Biosignals modulated by tumor-associated carbohydrate antigens: novel targets for cancer therapy.

Based on the remodeling of glycosphingolipids on the human tumor cell lines with manipulation of glycosyltransferase genes, roles of sugar moieties in tumor-associated carbohydrate antigens have been analyzed. Two main topics, that is, the roles of ganglioside GD3 in human malignant melanomas and those of GD2 in small cell lung cancer (SCLC) were reported. GD3 enhances tyrosine phosphorylation of two adaptor molecules, p130Cas and paxillin, resulting in the increased cell growth and invasion in melanoma cells. GD2 also enhances the proliferation and invasion of SCLC cells. GD2 also mediates apoptosis with anti-GD2 monoclonal antibodies (mAbs) via dephosphorylation of the focal adhesion kinase. These approaches have promoted further understanding of mechanisms by which gangliosides modulate malignant properties of human cancer, and the results obtained here propose novel targets for cancer therapy.

Anoikis↗

PubMed2006

Down-regulation of caveolin-1 in mouse Lewis lung cancer P29 is a causal factor for the malignant properties in a high-metastatic subline.

In order to analyze molecular mechanisms for cancer metastasis, we established a high-metastatic subline H7-Lu from a subline H7 of mouse Lewis lung cancer (P29) by repeated injection into tail veins. H7-Lu exhibited increased proliferation and invasion activity. Analysis of gene expression profiles between the parent H7 and H7-Lu revealed that several genes were down-regulated in H7-Lu. One of them, caveolin-1, was a component of lipid/rafts. After confirming the down-regulation of caveolin-1 mRNA by real-time RT-PCR and reduction of the protein by immunoblotting, respectively, H7 was transfected with siRNA for caveolin-1 to examine the role of caveolin-1 in H7-Lu. mRNA of the caveolin-1 gene was suppressed to approximately one third of the original level in H7 cells transfected with siRNA. The transfectant cells showed significantly increased cell proliferation and motility when analyzed by MTT assay and scratching wound healing assay, respectively. In the siRNA-transfectant cells, both ERK1/2 and Akt showed stronger phosphorylation than the mock-transfectant cells indicating that both of these signaling pathways were activated in caveolin-1-suppressed cells. These situations seem to reflect some aspects of the cellular changes in the high metastatic subline H7-Lu. Thus, down-regulation of caveolin-1 in a high-metastatic subline of Lewis lung cancer as defined by DNA array is really a causal factor for the increased malignant properties.

Animals↗

PubMed2006

[A case of gastric cancer with abdominal wall invasion treated by weekly low-dose paclitaxel therapy].

Here we report a case of gastric cancer with diffuse abdominal wall invasion treated with weekly low-dose paclitaxel therapy. A 62-year-old male visited our hospital because of abdominal distention, prepubic tumor,and testicular hydrocele. Computed tomography revealed diffuse swelling of the abdominal wall and hydronephrosis of the right kidney. Upper gastrointestinal endoscopy demonstrated type 3' advanced gastric cancer. Pathological diagnosis of both gastric tumor and abdominal wall biopsy specimens was poorly-differentiated adenocarcinoma containing signet ring cell carcinoma. Low-dose paclitaxel (90 mg/body) was given once a week for 3 weeks. Abdominal wall swelling like cuirass disappeared after 2 courses of low-dose paclitaxel therapy. Nine repeated courses of this regimen have been given until now; the relapse of the abdominal wall invasion has not become apparent, and primary gastric lesion has been a stable disease. Diffuse abdominal wall invasion of gastric cancer like cuirass without ascites is a rare condition, and low-dose paclitaxel was very effective for this condition.

Abdominal Wall↗

PubMed2005

Complex formation of Plk1 and INCENP required for metaphase-anaphase transition.

Mitotic chromosomal dynamics is regulated by the coordinated activities of many mitotic kinases, such as cyclin-dependent kinase 1 (Cdk1), Aurora-B or Polo-like kinase 1 (Plk1), but the mechanisms of their coordination remain unknown. Here, we report that Cdk1 phosphorylates Thr 59 and Thr 388 on inner centromere protein (INCENP), which regulates the localization and kinase activity of Aurora-B from prophase to metaphase. INCENP depletion disrupts Plk1 localization specifically at the kinetochore. This phenotype is rescued by the exogenous expression of INCENP wild type and INCENP mutated at Thr 59 to Ala (T59A), but not at Thr 388 to Ala (T388A). The replacement of endogenous INCENP with T388A resulted in the delay of progression from metaphase to anaphase. We propose that INCENP phosphorylation by Cdk1 is necessary for the recruitment of Plk1 to the kinetochore, and that the complex formation of Plk1 and Aurora-B on INCENP may play crucial roles in the regulation of chromosomal dynamics.

Amino Acid Sequence↗

PubMed2005

Murine glycosyltransferases responsible for the expression of globo-series glycolipids: cDNA structures, mRNA expression, and distribution of their products.

Biological functions of globo-series glycosphingolipids are not well understood. In this study, murine cDNAs of two glycosyltransferases responsible for the synthesis of globo-series glycolipids and mRNA expression of those genes were analyzed. Distribution of their products was also analyzed. Murine cDNAs for Gb3/CD77 synthase and Gb4 synthase predicted that both of them are type II membrane proteins with 348 and 331 amino acids, respectively. In northern blotting, Gb3/CD77 synthase gene was mainly expressed in kidney and lung but also detected in many other tissues. Gb4 synthase was expressed in brain, heart, kidney, liver, skin, and testis. In the immunohistological analysis, Gb3/CD77 was mainly expressed in the proximal tubules as revealed with coincidental expression with angiotensin-converting enzyme (ACE). In spleen, it was detected in pre-B cells in the peripheral region of the white pulp, as suggested with coincidental expression with CD10. It was also expressed on the endothelia of the alveolar capillaries in lung and on the sebaceous ducts aside of the hair follicles. Gb4 was also detected mainly on the proximal tubules in kidney and on the endothelia of the alveolar capillaries in lung as Gb3/CD77. But it was also detected on the epithelium of the bronchus, seminiferous tubules and tails of spermatozoa in testis, blood vessels of choroids plexus and endothelial cells in brain, and central and hepatoportal veins in liver. The expression patterns of two genes and their products almost corresponded with some exception. The results would provide essential information for the functional studies of globo-series glycolipids.

Amino Acid Sequence↗

PubMed2005

Ganglioside GD3 promotes cell growth and invasion through p130Cas and paxillin in malignant melanoma cells.

Although ganglioside GD3 levels are highly elevated in malignant melanomas, the role of GD3 in melanomas' malignant properties has not been clearly shown. To investigate this problem, we genetically generated GD3-positive (GD3+) transfectant cells from a GD3-negative (GD3-) mutant line SK-MEL-28-N1 and analyzed the phenotypic changes in the transfected cells. GD3+ cells showed markedly increased cell growth and invasive characteristics. Two bands that underwent stronger tyrosine phosphorylation in GD3+ cell lines than in controls after treatment with FCS were found with molecular masses of 130 and 68 kDa. They were identified as p130Cas and paxillin by sequential immunoprecipitation. Their roles in cell growth and invasion were analyzed with a small interfering RNA (siRNA) approach. Cell growth, as analyzed by BrdUrd uptake, was strongly suppressed in GD3+ cells to near the levels of GD3- cells when treated with siRNA for p130Cas but not when treated with siRNA for paxillin. However, treatment with siRNAs of either p130Cas or paxillin resulted in the marked suppression of the invasive activity of GD3+ cells almost to the levels of control cells. These results suggested that these two molecules function as effectors of GD3-mediated signaling, leading to such malignant properties as rapid cell growth and invasion.

Cell Division↗

PubMed2005

Different response of the knockout mice lacking b-series gangliosides against botulinum and tetanus toxins.

We assessed the response in knockout mice lacking the b-series (G(D2), G(D1b), G(T1b) and G(Q1b)) gangliosides against Clostridium botulinum (types A, B and E) and tetani toxins. We found that botulinum toxins were fully toxic, while tetanus toxin was much less toxic in the knockout mice. Combining the present results with our previous finding that tetanus toxin and botulinum types A and B toxins showed essentially no toxic activity in the knockout mice lacking both the a-series and b-series gangliosides (complex gangliosides), we concluded that the b-series gangliosides is the major essential substance for tetanus toxin, while b-series gangliosides may be not the essential substance for botulinum toxins, at the initial step during the intoxication process in mouse.

Animals↗

PubMed2005

RNA polymerase II is required for RNAi-dependent heterochromatin assembly.

In Schizosaccharomyces pombe, the RNA interference (RNAi) machinery converts pericentromeric transcripts into small interfering RNAs (siRNAs) and is required for the assembly of pericentromeric heterochromatin. Here we describe a mutation in the second largest subunit of RNA polymerase II (RNAPII). Both wild-type and mutant RNAPII localized to the pericentromere. However, the mutation resulted in the loss of heterochromatic histone modifications and in the accumulation of pericentromeric transcripts, accompanied by the loss of siRNAs. This phenotype resembles mutants in RNAi and suggests that RNAPII couples pericentromeric transcription with siRNA processing and heterochromatin assembly.

Amino Acid Sequence↗

PubMed2005

Mechanisms for the apoptosis of small cell lung cancer cells induced by anti-GD2 monoclonal antibodies: roles of anoikis.

Anti-GD2 ganglioside antibodies could be a promising, novel therapeutic approach to the eradication of human small cell lung cancers, as anti-GD2 monoclonal antibodies (mAbs) induced apoptosis of small cell lung cancer cells in culture. In this study, we analyzed the mechanisms for the apoptosis of these cells by anti-GD2 mAbs and elucidated the mechanisms by which apoptosis signals were transduced via reduction in the phosphorylation levels of focal adhesion kinase (FAK) and the activation of a MAPK family member, p38, upon the antibody binding. Knock down of FAK resulted in apoptosis and p38 activation. The inhibition of p38 activity blocked antibody-induced apoptosis, indicating that p38 is involved in this process. Immunoprecipitation-immunoblotting analysis of immune precipitates with anti-FAK or anti-integrin antibodies using an anti-GD2 mAb revealed that GD2 could be precipitated with integrin and/or FAK. These results suggested that GD2, integrin, and FAK form a huge molecular complex across the plasma membrane. Taken together with the fact that GD2+ cells showed marked detachment from the plate during apoptosis, GD2+ small cell lung cancer cells seemed to undergo anoikis through the conformational changes of integrin molecules and subsequent FAK dephosphorylation.

Anoikis↗

PubMed2005

Overexpression of GD1a ganglioside sensitizes motor nerve terminals to anti-GD1a antibody-mediated injury in a model of acute motor axonal neuropathy.

Anti-GD1a ganglioside antibodies (Abs) are the serological hallmark of the acute motor axonal form of the post-infectious paralysis, Guillain-Barre syndrome. Development of a disease model in mice has been impeded by the weak immunogenicity of gangliosides and the apparent resistance of GD1a-containing neural membranes to anti-GD1a antibody-mediated injury. Here we used mice with altered ganglioside biosynthesis to generate such a model at motor nerve terminals. First, we bypassed immunological tolerance by immunizing GD1a-deficient, beta-1,4-N-acetylgalactosaminyl transferase knock-out mice with GD1a ganglioside-mimicking antigens from Campylobacter jejuni and generated high-titer anti-GD1a antisera and complement fixing monoclonal Abs (mAbs). Next, we exposed ex vivo nerve-muscle preparations from GD1a-overexpressing, GD3 synthase knock-out mice to the anti-GD1a mAbs in the presence of a source of complement and investigated morphological and electrophysiological damage. Dense antibody and complement deposits were observed only over presynaptic motor axons, accompanied by severe ultrastructural damage and electrophysiological blockade of motor nerve terminal function. Perisynaptic Schwann cells and postsynaptic membranes were unaffected. In contrast, normal mice were not only unresponsive to immunization with GD1a but also resistant to neural injury during anti-GD1a Ab exposure, demonstrating the central role of membrane antigen density in modulating both immune tolerance to GD1a and axonal susceptibility to anti-GD1a Abmediated injury. Identical paralyzing effects were observed when testing mouse and human anti-GD1a-positive sera. These data indicate that anti-GD1a Abs arise via molecular mimicry and are likely to be clinically relevant in injuring peripheral nerve axonal membranes containing sufficiently high levels of GD1a.

Animals↗

PubMed2005

Characterisation of the immunoglobulin variable region gene usage encoding the murine anti-ganglioside antibody repertoire.

Neuropathogenic murine antibodies reactive with terminal disialylgalactose epitopes are innate and preferentially encoded by the VH7183.3b gene. Here we have studied antibodies reactive with internal galactose-linked disialosyl epitopes and the terminal trisaccharide of GT1b. Antibodies were of moderate affinity and unmutated. Anti-GD1b antibodies were often encoded by the VH10.2b heavy and gj38c light chain genes. Anti-GT1b antibodies with broader glycan binding patterns were encoded by VHQ52 and VHJ558 family genes. These data indicate that the discrete specificities of ganglioside-binding antibodies are dictated by particular patterns of V gene usage residing within the innate B cell repertoire.

Amino Acid Sequence↗

PubMed2005

GD3 synthase gene knockout mice exhibit thermal hyperalgesia and mechanical allodynia but decreased response to formalin-induced prolonged noxious stimulation.

Gangliosides are a family of sialic acid-containing glycosphingolipids that are highly enriched in the mammalian nervous system. In particular, b- and c-series gangliosides, all of which contain alpha-2,8 sialic acids, have been considered to play important roles in adhesion, toxin-binding, neurite extension, cell growth and apoptosis. However, the neurobiological functions of these series of gangliosides remain largely unknown. To clarify the function of b- and c-series gangliosides in pain sensation in vivo, we generated mice in whom the gene for the alpha-2,8-sialyltransferase (GD3 synthase), which is responsible for the generation of all b-series gangliosides as well as c-series gangliosides, was disrupted. Compared to the wild-type mice, the mutant mice exhibited increased sensory responses to thermal and mechanical stimuli as measured by a hot plate test and von Frey test. In contrast, the mutant mice showed decreased responses during the late phase of the formalin test. Paw edema and Fos expression in the spinal cord after formalin injection were significantly decreased in the mutant mice compared to the wild-type mice. No significant differences in the conduction velocity of the sciatic nerve, and no apparent morphologic differences in the spinal cord and the sciatic nerve were detected between the wild-type and the mutant mice. These results suggested that b- and c-series gangliosides are critical in the development and/or maintenance of the sensory nervous system responsible for the transmission of acute pain sensation and pain modulation. Moreover, they play an important role in the development of hyperalgesia induced by inflammation.

Analysis of Variance↗