Assignment of human desert hedgehog gene (DHH) to chromosome band 12q13.1 by in situ hybridization.
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Chromosome Banding↗
Biomedical subjects
M Furutani
Publications and source records attributed to M Furutani.
Modified sagittal split ramus osteotomy with a reciprocating saw and new retractors.
Modified sagittal split ramus osteotomy with new instruments and a reciprocating saw is reported. With this modification, the sagittal separation of the ramus is performed by the reciprocating saw with an original wide-blade buccal retractor and a new lingual retractor, instead of the traditional channeled retractor. The wide-blade retractor is inserted to the buccal aspect of the mandibular ramus, which ensures protection from instrumental injury to the adjacent soft tissues and vessels, and the new lingual retractor, instead of the channeled retractor, is inserted to the lingual aspect of the ramus. The osteotomy line follows that of Dal Pont's modification. Close attention must be paid to the direction of the saw blade. The separated bone plane should be located in the external cortical bone layer of the ramus, so as to avoid injury of the inferior alveolar neurovascular bundle. The osteotomy is completed with the smooth osteotomized interface, which facilitates positioning of the bone segments by the surgeon. The new instruments and the reciprocating saw may provide safe and rapid sagittal split ramus osteotomy.
Episode of Care↗
FKBP-type peptidyl-prolyl cis-trans isomerase from a sulfur-dependent hyperthermophilic archaeon, Thermococcus sp. KS-1.
A gene encoding an FK506 binding protein (FKBP)-type peptidyl-prolyl cis-trans isomerase (PPIase) was cloned from a hyperthermophilic archaeon, Thermococcus sp. KS-1, and sequenced. This gene encoded an FKBP with 159 amino-acid residues with a molecular mass of 17.6kDa. Two insertion sequences with 13 and 44 amino acids were found in the regions corresponding to the bulge and flap regions of human FKBP-12, respectively. Comparison with other archaeal FKBP sequences obtained from reported genome sequences revealed that the insertion sequences in the bulge and flap regions were common to archaeal FKBPs. It was also revealed that archaeal FKBPs are classified into two groups: one is approx. 17kDa and the other 27kDa. This Thermococcus FKBP (TcFK) belonged to the smaller archaeal FKBP. In this TcFK, 9 out of 15 amino acid residues forming the FK506 binding pocket of human FKBP12 were found. This gene was expressed in Escherichia coli and the recombinant protein was purified. The purified protein showed PPIase activity and its activity was inhibited by FK506 with an IC50 of 7 microM. This enzyme showed high kinetic stability with a half-life of 40 min at 100 degrees C. Catalytic efficiency of this recombinant PPIase was 1.2-times higher with the substrate N-succinyl-A-L-P-F-p-nitroanilide than with N-succinyl-A-A-P-F-p-nitroanilide.
Amino Acid Sequence↗
Group II chaperonin in a thermophilic methanogen, Methanococcus thermolithotrophicus. Chaperone activity and filament-forming ability.
A gene encoding 544 amino acids for a subunit of group II chaperonin (thermosome) was cloned from a thermophilic methanogen, Methanococcus thermolithotrophicus. The deduced amino acid sequence showed 66.5, 56.1, and 20.1% similarities to those of Methanopyrus kandleri and Thermoplasma acidophilum and group I chaperonin of Escherichia coli, respectively. We call this chaperonin MTTS (M. thermolithotrophicus thermosome). The MTTS gene was expressed in E. coli. The purified recombinant MTTS seemed to be monomeric on gel filtration in the absence of Mg2+ and ATP. The monomer assembled to an oligomer (complex) in the presence of 50 mM MgCl2, 0.25 mM ATP, and 0.3 M (NH4)2SO4. It was eluted immediately before the elution volume of E. coli GroEL tetradecamer on gel filtration with a TSKgel G3000SWXL column. This reconstructed MTTS complex showed the cylindrical structure with two stacked rings in electron microscopy. The MTTS complex formed filamentous structures in the presence of Mg2+ and ATP at the protein concentration above 3.0 mg/ml. This filament formation was reversible. The MTTS filament was dissociated to the complex by dilution to the protein concentration of 0.2 mg/ml, even in the presence of Mg2+ and ATP. The MTTS complex exhibited weak ATPase activity with the hydrolysis rate of 74 mol of ATP hydrolysis/mol of MTTS complex/min at 70 degreesC. The MTTS complex promoted the refolding of chemically denatured thermophilic archaeal citrate synthase and glucose dehydrogenase at 50 degreesC in an ATP-dependent fashion. The analysis of nucleotide specificity of chaperone activity of MTTS suggested that it was coupled with hydrolysis of ATP, CTP, or UTP.
Adenosine Triphosphatases↗
Expression of the Na+/Ca2+ exchanger emerges in hepatic stellate cells after activation in association with liver fibrosis.
Activation of hepatic stellate (Ito) cells is a final common pathway of liver fibrosis. The findings presented in this paper indicate that expression of Na+/Ca2+ exchanger (NCX) emerges in rat hepatic stellate cells after activation in vitro during primary culture or in vivo in response to intoxication with CCl4. NCX mRNA became detectable by Northern blot analysis in cultured stellate cells on day 3, as was alpha-smooth muscle actin, an indicator not only of smooth muscle differentiation but also of stellate cell activation. Western blot analysis showed expression of the exchanger protein in the activated stellate cells. Functional expression of the exchanger, monitored by Ni2+-sensitive, verapamil-insensitive intracellular free Ca2+ increases in response to reduction of extracellular Na+ concentration, became sizable by using Fura-2 in stellate cells by 7 days in culture. Furthermore, increased expression of the exchanger mRNA was found predominantly in stellate cells freshly isolated from the CCl4 model rat of hepatic fibrosis. Thus, it is concluded that NCX expression is closely associated with activation of hepatic stellate cells in vitro and in vivo. Because, even at the whole liver level, increased expression of NCX mRNA became observable after induction of liver fibrosis, it is suggested that NCX expression serves a useful diagnostic marker of liver fibrosis or cirrhosis.
Actins↗
Identification of a neutrophil gelatinase-associated lipocalin mRNA in human pancreatic cancers using a modified signal sequence trap method.
To identify the intercellular signal-transducing proteins and receptors produced by cancer cells, we attempted to clone cDNAs encoding secreted and type I membrane proteins using a signal sequence trap (SST) method with some modifications. By screening an SST library derived from pancreatic cancer cells, we identified two secretory proteins (neutrophil gelatinase-associated lipocalin (NGAL) and lung surfactant protein D) and three membrane proteins (carcinoembryonic antigen, BiP/GRP78 and Hsa4 mitochondrion cytochrome oxidase subunit II). NGAL mRNA was expressed in eight of the pancreatic cancer cell lines and eight pancreatic cancer tissues. The expression of NGAL mRNA was also observed in colorectal and hepatic tumors.
Acute-Phase Proteins↗
A de novo missense mutation (R1623Q) of the SCN5A gene in a Japanese girl with sporadic long QT sydrome. Mutations in brief no. 140. Online.
Two missense mutations and a nine-nucleotide deletion of the cardiac sodium channel (SCN5A) gene have been shown to cause long QT syndrome (LQTS) in several familial cases. We identified a novel missense mutation (R1623Q) of the SCN5A gene in a Japanese girl with sporadic LQTS. We used polymerase chain reaction, single-strand conformation polymorphism analysis and DNA sequence analysis to identify a mutation of the SCN5A gene in the patient. A single nucleotide substitution of guanine to adenine, in codon 1612, changed the coding sense of the SCN5A from arginine to glutamine (R1623Q) in the S4 segment of domain IV which is a highly conserved region of the SCN5A. This mutation was not identified in the unaffected biological parents and brother of the patient, and 100 normal, unrelated individuals. This finding is the first evidence of a de nova mutation in SCN5A associated with LQTS.
Amino Acid Substitution↗
Expression of human macrophage metalloelastase gene in hepatocellular carcinoma: correlation with angiostatin generation and its clinical significance.
Macrophage metalloelastase, a member of the human matrix metalloproteinase family, is believed to play an important role in angiostatin generation, which, in experimental studies, has an antiangiogenic function and is a key molecule in tumor dormancy. However, no clinical studies have been reported regarding the correlation between human macrophage metalloelastase (HME) gene expression and angiostatin production. Therefore, the present study was designed to evaluate the HME messenger RNA (mRNA) expression and angiostatin generation in hepatocellular carcinoma (HCC). Tumorous and contiguous nontumorous tissues were obtained from 40 HCC patients who underwent curative partial hepatectomy. By using Northern blot hybridization, HME mRNA was detected in 25 of the 40 HCC samples and, in all of these cases, the expression in tumorous tissues was stronger than in the nontumorous tissues. In situ hybridization identified the HCC cells as mainly responsible for the signals shown in Northern blot. Angiostatin was detected by Western blot mainly in tumors and showed significant association with HME mRNA expression in tumorous tissues (P = .0008). The patients whose tumors did not express HME mRNA and, thus, did not produce angiostatin, demonstrated poorer survival than those whose tumors showed high expression of HME mRNA and angiostatin generation (P = . 002). The univariate and multivariate analyses revealed that HME mRNA expression is a new and independent variable affecting overall survival (P = .001 and P = .03, respectively). These findings show that the HME gene is expressed in HCC being significantly associated with angiostatin generation by such tumors and that HME mRNA expression may serve as a new molecular prognostic marker in HCC patients after partial hepatectomy.
Adult↗
Novel missense mutation (G601S) of HERG in a Japanese long QT syndrome family.
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Amino Acid Substitution↗
Results of surgical treatment for recurrent hepatocellular carcinoma; comparison of outcome among patients with multicentric carcinogenesis, intrahepatic metastasis, and extrahepatic recurrence.
No consensus has been reached on the indications for and effectiveness of surgery for secondary intrahepatic hepatocellular carcinoma (HCC) and extrahepatic metastasis after macroscopically complete removal of primary HCC. Secondary intrahepatic HCCs, usually regarded as recurrence are classified into those arising as a result of multicentric carcinogenesis or intrahepatic metastases derived from the primary HCC. The present study was designed to evaluate the utility of surgical treatment in relation to the pathogenesis of the secondary HCC: classified as multicentric carcinogenesis (MC), intrahepatic metastasis (IM), and extrahepatic metastasis. Thirty patients underwent extirpation of secondary HCC: 22 patients had secondary HCCs in the remnant liver (MC group; n = 8; IM group, n = 14), 6 patients had extrahepatic metastases, and 2 patients had both intrahepatic and extrahepatic metastases. Survival rates after the re-resection in the 22 patients with the secondary intrahepatic HCCs were 94.7% at 1 year, and 50.2% at 3 years postoperatively, and the 8 patients with extrahepatic metastasis had survival rates of 62.5% at 1 year, 37.5% at 3 years, and at 5 years. The survival rates after re-resection in the MC group were 100% at 1 year and 80.0% at 3 years, whereas those in the IM group were 91.7% at 1 year, and 38.1% at 3 years. Surgery can be indicated not only in patients with localized intrahepatic secondary HCCs but also in those with extrahepatic metastasis. In particular, patients with secondary HCCs arising as a result of multicentric carcinogenesis are expected to have a good prognosis.
Adult↗
Inhibition of tumor growth and microvascular angiogenesis by the potent angiogenesis inhibitor, TNP-470, in rats.
The antiangiogenic effects of TNP-470 on the neovascularization of tumors were studied by examining ultrastructural alterations in the vasculature and interstitial fluid pressure (IFP) of tumors. Wistar rats were first inoculated subcutaneously (s.c.) with the Walker 256 carcinosarcoma cell line, then either vehicle medium or TNP-470, 30 mg/kg, was injected s.c. on day 1. A tumor growth assay, the necrotic area, and the IFP in the tumor were all measured on day 12. The antiangiogenic effects of TNP-470 were studied by scanning electron microscopic images of tumor vascular casts. TNP-470 was observed to inhibit tumor growth and increase the necrotic area significantly. In the TNP-470-treated group, the IFP in the superficial layer, defined as 2-3 mm from the tumor capsule, and in the deep layer, defined as 8-10 mm from the tumor capsule, were significantly higher than the corresponding values in the control. Moreover, vascular casts showed a significant reduction in the budding of sprouts in the superficial layer, and a decrease in the maximum diameter of the tumor vessels in the deep layer. It is possible that the higher IFP in the TNP-470-treated tumors might have prevented tumor vessel dilation. The findings of this study demonstrated that TNP-470 inhibited the budding of tumor vessel sprouts, and increased the IFP. These processes seem to act synergistically to suppress tumor angiogenesis.
Animals↗
Membrane-type matrix metalloproteinase-1(MT1-MTP) gene is overexpressed in highly invasive hepatocellular carcinomas.
BACKGROUND/AIMS: The matrix metalloproteinase (MMP) family play important roles in the invasion of cancer cells by degrading the extracellular matrices. The current study was designed to determine the expression pattern of membrane-type matrix metalloproteinase-1 (MT1-MMP) in hepatocellular carcinomas and its participation in invasion potential. METHODS: MT1-MMP mRNA expression was examined in 25 human hepatocellular carcinoma specimens using Northern blot, and the correlation to clinicopathological features was evaluated. In situ hybridization and immunohistochemistry were performed to study the localization and the cells responsible for the production. RESULTS: Northern blot analysis revealed high levels of MT1-MMP mRNA expression in tumorous portions in all cases, whereas in non-tumorous portions moderate or faint expression was evident in 22/25 cases. In 21/25 cases, the expression levels in tumorous portion were higher than those in non-tumorous portion. In particular, hepatocellular carcinoma with capsule infiltration demonstrated significantly higher expression than those without (p<0.05). In situ hybridization and immunohistochemical study revealed MT1-MMP transcripts and proteins in cancer cells and stromal cells, respectively. MT1-MMP positive cells were preferentially observed in the invading border of tumor nests. The MMP-2 transcript showed a similar pattern to that of MT1-MMP by in situ hybridization. CONCLUSION: The present study showed that the MT1-MMP gene is strongly expressed in hepatocellular carcinoma cells and is involved in the invasion potential of hepatocellular carcinoma, and also that MT1-MMP may be one of the key molecules responsible for the invasion potential of hepatocellular carcinoma. Furthermore, the evidence suggests that MT1-MMP and MMP-2 cooperate in the process of cancer invasion.
Adult↗
Predictive value of vascular endothelial growth factor (VEGF) in metastasis and prognosis of human colorectal cancer.
Vascular endothelial growth factor (VEGF) may affect the phenotype of cancer cells, such as growth velocity and metastatic potential, due to its probable multifunctional property including a mitogenic activity for vascular endothelial cells. The present study was designed to investigate the association of VEGF mRNA expression with progression and metastasis of human colorectal cancer. The level of VEGF mRNA expression was quantified by Northern blot hybridization in tumorous and non-tumorous tissues obtained from 60 primary colorectal cancer patients. The ratio of the former to the latter was defined as the VEGF T/N ratio, and the prognostic significance of this ratio, following surgery, in addition to the relationship to progression and metastatic potential, was evaluated. The value of the VEGF T/N ratio was significantly correlated with the depth of tumour infiltration (P=0.046), the incidence of liver metastasis (P < 0.0001) and lymph node metastasis (P=0.036). Patient prognosis was estimated by the Kaplan-Meier method and the log-rank test. When the VEGF T/N ratio was higher than 4.8 for which the chi2 value of the log-rank test was maximal, the tumour was defined as showing overexpression of VEGF mRNA. Patients with overexpression of VEGF mRNA demonstrated poorer survival than patients without overexpression of VEGF mRNA (P < 0.001). The overall estimated hazard ratio for death in patients with overexpression of VEGF mRNA was 1.94 according to a multivariate analysis (P=0.005). Thus, VEGF is associated with the progression, invasion and metastasis of colorectal cancer, and overexpression of VEGF mRNA in the primary tumour is assumed to be closely correlated with poor prognosis in colorectal cancer patients. Moreover, the VEGF T/N ratio may be used as an independent prognostic marker in colorectal cancer patients.
Adult↗
Neuroleptic-induced Meige's syndrome following akathisia: pharmacologic characteristics.
A 52-year-old schizophrenic patient acutely showed blepharospasm and oromandibular dystonia following neuroleptic-induced akathisia. She had suffered from schizophrenia and been treated with neuroleptics for 15 years and had manifested tardive dyskinesia 5 years ago. Following a change in her neuroleptic medication, severe akathisia developed. Two days after the appearance of akathisia, blepharospasm and oromandibular dystonia appeared. After the disappearance of akathisia, the disorder continued. The frequency of blepharospasm ranged from 30 to 40 (times/min). The oral administration of trihexyphenidyl (6 mg/day), perphenazine (12 mg/day), and fluphenazine (12 mg/day) significantly decreased the frequency of blepharospasm, whereas carbamazepine (600 mg/day) and sulpiride (1200 mg/day) did not. These results suggest that overactivity of both cholinergic and dopaminergic functions in the striatum may be involved in this patient. Our patient, who showed acute onset of Meige's syndrome following neuroleptic-induced akathisia, is of interest to those studying the pathogenesis of Meige's syndrome.
Akathisia, Drug-Induced↗
Biochemical and genetic characterization of an FK506-sensitive peptidyl prolyl cis-trans isomerase from a thermophilic archaeon, Methanococcus thermolithotrophicus.
A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100 degrees C being 90 and 30 min, respectively. The catalytic efficiencies (k(cat)/Km) measured at 15 degrees C for the peptidyl substrates, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 microM(-1) s(-1), respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicus FK506-binding protein). The MTFK gene (462 bp) was cloned from an M. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.
Amino Acid Sequence↗
Characterization of soluble thrombomodulin fragments in human urine.
The soluble thrombomodulin (TM) subspecies in human urine detected by polyclonal anti-human TM IgG were isolated and characterized. 105, 85, 80, 56, 33, 31 and 28 kDa subspecies under reducing conditions was comparable to 78, 66, 56, 200, 52, 30 and 25 kDa under non-reducing conditions, respectively, in the two-dimensional electrophoresis. Each subspecies under non-reducing conditions, except the 200 and 52 kDa molecules, was constituted of single subspecies, whereas the 200 and 52 kDa molecules were constituted of the tetramer of the 56 kDa subspecies of reducing conditions and a dimer of the 33 kDa subspecies, respectively. NH2-terminal amino acid sequences of the 105, 85 and 80 kDa subspecies maintained Ala1-Pro2-Ala3- of intact human TM, however, 56, 33, 31 and 28 kDa subspecies started from Glu137-Gln138-, Gln214-Gly215-, Ser228-Val229- and Ala240-Ile241-, respectively. All subspecies obtained under non-reducing conditions exhibited cofactor activity for thrombin-dependent protein C activation ranging from 58 to 162 pmol APC/min/nmol TM at 0.4 mM Ca2+ indicating that all of the subspecies maintained the fourth to sixth repeat of epidermal growth factor-like structure of intact TM. 85, 80, 56, 33, 31 and 28 kDa subspecies were suggested to lack both chondroitin sulfate glycosaminoglycan (CSGAG), transmembrane and cytoplasmic domains of intact TM, while 105 kDa subspecies lack only CSGAG from the results of kinetic properties and the interaction with phospholipid vesicles composed from phosphatidylcholine and phosphatidylethanolamine.
Humans↗
Gene for a cyclophilin-type peptidyl-prolyl cis-trans isomerase from a halophilic archaeum, Halobacterium cutirubrum.
A gene encoding a cyclophilin (CyP)-type peptidyl prolyl cis-trans isomerase (PPIase) was cloned from a halophilic archaeum, Halobacterium cutirubrum DSM 669, and sequenced. Although amino-acid residues common to CyPs were conserved, an insertion that showed no homology to other CyPs was found in its N-terminal region. Sequence analysis revealed that the amino-acid sequence of this CyP was 40-45% identical to those of eukaryotes and Bacillus subtilis with high cyclosporin A sensitivity, but 27% identical to those of cyclosporin A-insensitive PPIases of Escherichia coli. The gene was also expressed in E. coli. The activity of purified recombinant CyP-type PPIase was stimulated by the addition of KCl, and was suppressed by cyclosporin A.
Amino Acid Sequence↗
NO as an indicator of portal hemodynamics and the role of iNOS in increased NO production in CCl4-induced liver cirrhosis.
It is well known that nitric oxide, a vasodilator, is overproduced in liver cirrhosis. This study was designed to elucidate the role of nitric oxide (NO) in portal hemodynamics and to determine the mechanism underlying the increased serum NO levels in rats with liver cirrhosis induced by the oral intake of CCl4. Using rats, liver cirrhosis was induced by oral administration of CCl4. The serum levels of NO2-/NO3-(NOx) were measured, and portal hemodynamic parameters were evaluated with and without the administration of the NO synthase inhibitor N omega-nitro-L-arginine (NNA). Furthermore, Northern blot analysis was used to detect iNOS and cNOS mRNA, and immunohistochemical methods were used to detect iNOS-like immunoreactivity. In cirrhotic rats, the portal flow had increased significantly and the portal resistance had decreased significantly when compared with normal control rats. Hepatic capillary flow in the cirrhotic rats was similar to the control rats. NNA decreased portal flow and increased portal resistance in both groups, but the change was greater in the cirrhotic rats than in controls. The serum levels of NOx were significantly higher in cirrhotic rats than in normal control rats and were positively correlated with portal flow and negatively correlated with portal resistance. The expression of iNOS mRNA, which was barely detectable in control rats, had increased in all organs of the cirrhotic rats, whereas no significant increase in cNOS mRNA was found in any of the organs from cirrhotic rats. The immunohistochemical analysis was generally consistent with the results of the Northern blot analysis. In the control rats, only the bronchial epithelial cells were stained with the anti-iNOS antibody, but in cirrhotic rats, the bronchial cells in the lungs as well as the histiocytic mesenchymal cells in all organs, and the alveolar epithelial cells of the lungs, were stained. This study demonstrated that NO plays a significant role in portal hypertensive hemodynamics in CCl4-induced liver cirrhosis, and that NO is a useful indicator for the evaluation of portal hypertension. Furthermore, the increased serum levels of NO were found to be derived at least in part from the increased expression of iNOS mRNA in the liver, spleen, and lung.
Animals↗