Search PubMed⌕ Search

Biomedical subjects

R Makino

Publications and source records attributed to R Makino.

At least 19 recordsLinked to original sources
PubMed2003

No major tumorigenic role for beta-catenin in serrated as opposed to conventional colorectal adenomas.

Intracellular redistribution of beta-catenin through mutation of the adenomatous polyposis coli (APC) gene has been proposed as an early tumorigenic event in most colorectal tumours. In serrated adenoma (SA), a newly recognised subtype of colorectal adenoma, APC mutations are uncommon, and the contribution of beta-catenin to tumorigenesis remains unclear. We compared intracellular localisation of beta-catenin and presence of mutations in exon 3 of beta-catenin between 45 SAs, with 71 conventional adenomas (CADs), and eight carcinomas invading the submucosa (SCAs). Widespread or focal nuclear beta-catenin expression was demonstrated in 7% of SAs (three out of 45), 61% of CADs (43 out of 71), and 88% of SCAs (seven out of eight). Cytoplasmic immunostaining for beta-catenin was demonstrated in 16% of SAs (seven out of 45), 77% of CADs (55 out of 71), and 88% of SCAs (seven out of eight). No mutation in exon 3 of beta-catenin was found in SAs or SCAs, while 7% of CADs (five out of 71) had beta-catenin mutations. No nuclear or cytoplasmic expression of beta-catenin was observed in the hyperplastic or conventionally adenomatous epithelium of mixed-type SAs. These findings suggest that beta-catenin mutation is unlikely to contribute to the tumorigenesis in SA, and that intracellular localisation of beta-catenin may not be associated with an early event of the tumour progression in most SAs.

Adenoma↗
PubMed2001

Organic phosphates as a new class of soluble guanylate cyclase inhibitors.

This study aimed to examine effects of varied organic phosphates on activities of soluble guanylate cyclase (sGC). The enzyme was purified from bovine lung. Physiologically relevant concentrations of ATP, 2,3-bisphosphoglyceric acid and inositol hexakisphosphate inhibited its enzyme activities under steady-state conditions as well as those determined under stimulation with S-nitroso-N-acetylpenicillamine, a nitric oxide donor, carbon monoxide or YC-1. Lineweaver-Burk plot analyses revealed that these three organic phosphates act as competitive inhibitors. Other organic phosphates such as cardiolipin and sphingomyelin but not inorganic phosphates exhibited such inhibitory actions. These results suggest that organic phosphates serve as inhibitors for sGC-dependent signaling events.

2,3-Diphosphoglycerate↗
PubMed2001

Prognostic value of p53 mutations in patients with locally advanced esophageal carcinoma treated with definitive chemoradiotherapy.

PURPOSE: A significant correlation has been found between p53 mutation and response to chemotherapy or radiotherapy. To determine the prognostic value of p53 mutation in patients with locally advanced esophageal carcinoma treated with definitive chemoradiotherapy, p53 mutation was analyzed using the biopsied specimens taken for diagnosis. METHODS: Concurrent chemoradiotherapy was performed for 40 patients with severe dysphagia caused by esophageal squamous cell carcinoma associated with T3 or T4 disease. Chemotherapy consisted of protracted infusion of 5-fluorouracil, combined with an infusion of cisplatinum. Radiation treatment of the mediastinum was administered concomitantly with chemotherapy. The p53 gene mutation was detected by fluorescence-based polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) methods. DNA sequences were determined for DNA fragments with shifted peaks by SSCP methods. RESULTS: Of the 40 patients, 15 had T3 disease and 25 had T4 disease; 11 patients had M1 lymph node (LYM) disease. Of the 40 patients, 13 (33%) achieved a complete response. The median survival time was 14 months, and the 2-year survival rate was 20%. Among the 40 tumor samples, p53 mutation was detected in 24 tumors (60%). The survival rate in the 24 patients with p53 mutation did not differ significantly from that in the 16 patients without p53 mutation. In contrast, the 15 patients with T3 disease survived longer than the 25 patients with T4 disease (P = 0.016); however, the survival rate in the 11 patients with M1 LYM disease did not differ significantly from that in the 29 patients without M1 LYM disease. CONCLUSION: Concurrent chemoradiotherapy is potentially curative for locally advanced esophageal carcinoma, but p53 genetic abnormality has no impact on prognosis.

Aged↗
PubMed2001

Effects of a thiolate axial ligand on the pi-->pi* electronic states of oxoferryl porphyrins: a study of the optical and resonance Raman spectra of compounds I and II of chloroperoxidase.

Optical absorption and resonance Raman spectra have been investigated for enzymatic intermediates, compounds I and II, of chloroperoxidase (CPO) which contains a thiolate-ligated iron porphyrin. Compound I of CPO (CPO-I), an oxoferryl porphyrin pi cation radical, gave an apparently asymmetric single-peaked Soret band at 367 nm, for which band fitting analyses revealed the presence of two transition bands around 365 and 415 nm. Compound II of CPO (CPO-II), an oxoferryl neutral porphyrin, gave a split Soret spectrum with two bands (blue and red Soret bands) at 373 and 436 nm. Thus both CPO-I and CPO-II can be categorized as hyperporphyrins. The maximum extinction coefficients (epsilon(b) and epsilon(r)) and energies (Eb and Er) of the blue and red Soret bands of CPO-II were found to fall on an epsilon(b)/epsilon(r) versus Eb-Er correlation line derived from data reported for six-coordinate ferrous derivatives of cytochrome P450 and CPO. Corresponding data for CPO-I did not fall on the correlation line. Resonance enhancement of the FeIV=O stretching (vFeO) Raman band was found for CPO-I when Raman scattering was excited at wavelengths within both transition bands around 365 and 415 nm, while the vFeO Raman band was not identified for CPO-II at any of the excitation wavelengths examined here. These findings suggest that the thiolate axial ligand causes Soret band splitting of CPO-II through configuration interaction between the sulfur-->porphyrin e(g)* charge transfer and porphyrin a1u,a2u-->e(g)* transitions, while the FeO portion is important in determining the shape of the Soret band of CPO-I.

Chloride Peroxidase↗
PubMed2001

Frequent down-regulation of E-cadherin by genetic and epigenetic changes in the malignant progression of hepatocellular carcinomas.

E-cadherin mediates cell-cell adhesion by associating with catenins. Loss of E-cadherin function by genetic or epigenetic alteration of the E-cadherin gene (CDH1) leads to tumorigenesis. To study the involvement of E-cadherin dysfunction in liver tumorigenesis, we examined the allelic loss and methylation of 5'-CpG sites of CDH1 in hepatocellular carcinomas (HCCs). Loss of heterozygosity (LOH) of CDH1 and adjacent 16q22-23 loci was observed in 13 of 30 (43%) HCCs. Methylation of the 5'-CpG of CDH1 was analyzed by Southern blot hybridization, and hypermethylation was observed in 8 of the 24 (33%) HCCs examined. The amount of E-cadherin mRNA was analyzed by RNase protection assay, and a decrease in E-cadherin mRNA was observed in 10 of the 23 cases examined. A reduction in E-cadherin was found in 10 of 21 HCCs using immunoblot analysis. The amount of E-cadherin was comparable to that of E-cadherin mRNA. Down-regulation of E-cadherin was common in cases with LOH but rare in cases with methylated promoter. These results suggest that hypermethylation of the CDH1 promoter is present in a small cell population in the tumor, thus the methylation status is liable to vary according to individual cell condition. Hypermethylation was observed in early stage HCCs, whereas LOH was found frequently in more malignant tumors. Down-regulation of E-cadherin is closely related to the progression of HCCs and is stably induced by LOH of CDH1.

Blotting, Southern↗
PubMed2000

Detection of mutation of the p53 gene with high sensitivity by fluorescence-based PCR-SSCP analysis using low-pH buffer and an automated DNA sequencer in a large number of DNA samples.

Detection of mutations in genes responsible for hereditary diseases or tumors is important clinically. It is necessary to establish a simple technique for screening mutations in large numbers of samples. The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method has proved to be a useful technique for analyzing mutations or DNA polymorphisms. Non-radioisotopic versions using fluorescent dye and an automated DNA sequencer have also been exploited to extend this technique into the clinical field. We have examined mutations of exons 5-9 of the p53 gene in 112 colorectal, 28 esophageal and 33 hepatocellular carcinomas by fluorescence-based PCR-SSCP (F-SSCP) under various conditions. We found 64 types of mutations in 63, 17 and 12 cases of colon, esophageal and hepatocellular carcinomas by F-SSCP. We determined the sequence of all samples, and confirmed that all mutations were successfully detected by F-SSCP. With the low-pH buffer system, 61 types of mutants were detected, while 51 types were detected by TBE and 57 types were detected by TBE with glycerol gel. The polyacrylamide gel in TME or TBE without glycerol was tough and could be used repeatedly, but the glycerol containing gel was fragile and could not stand repeated use. Thus, use of a low-pH buffer in the electrophoresis of F-SSCP is simpler and better at detecting mutations than the conventional TBE buffer system. We believe that low-pH F-SSCP analysis is an efficient and powerful technique for examination of a large number of samples, in particular clinical specimens obtained by biopsy or surgery.

DNA Mutational Analysis↗
PubMed2000

Growth patterns of superficially elevated neoplasia in the large intestine.

BACKGROUND: The growth pattern and malignant potential of superficially elevated neoplastic lesions remain controversial. A flat adenoma is classified as a superficially elevated neoplasm that characteristically demonstrates high-grade dysplasia despite its small size. In contrast, a nodule-aggregating (NA) tumor, which consists of multiple small aggregated nodules, can also be classified as superficially elevated neoplasia. METHODS: In this prospective study, 2720 consecutive patients undergoing total colonoscopy were examined for superficially for elevated lesions. Clinicopathologic characteristics, Ki-ras mutational status, and overexpression of p53 protein were compared in 25 NA tumors and 55 flat adenomas without a central depression (flat tumor). RESULTS: All flat tumors had a tubular pattern, whereas 21 of 25 NA tumors showed a villotubular or tubulovillous pattern. Ki-ras mutation was observed in 44% of NA tumors but in none of the flat tumors. Overexpression of p53 protein was found in 12% of NA tumors and 7% of flat tumors. CONCLUSIONS: NA tumors and flat tumors have different clinicopathologic and genetic characteristics, although both types of tumor are classified as superficially elevated lesions.

Adenocarcinoma↗
PubMed2000

X-ray crystal structure and catalytic properties of Thr252Ile mutant of cytochrome P450cam: roles of Thr252 and water in the active center.

The structure-function relationship in cytochrome P450cam monooxygenase was studied by employing its active site mutant Thr252Ile. X-ray crystallographic analyses of the ferric d-camphor-bound form of the mutant revealed that the mutation caused a structural change in the active site giving an enlarged oxygen-binding pocket that did not contain any hydrophilic group such as the OH group of Thr and H(2)O. The enzyme showed a low monooxygenase activity of ca. 1/10 of the activity of the wild-type enzyme. Kinetic analyses of each catalytic step revealed that the rate of proton-coupled reduction of the oxygenated intermediate of the enzyme, a ternary complex of dioxygen and d-camphor with the ferrous enzyme, decreased to about 1/30 of that of the wild-type enzyme, while the rates of other catalytic steps including the reduction of the ferric d-camphor-bound form by reduced putidaredoxin did not change significantly. These results indicated that a hydrophilic group(s) such as water and/or hydroxyl group in the active site is prerequisite to a proton supply for the reduction of the oxygenated intermediate, thereby giving support for the operation of a proton transfer network composed of Thr252, Asp251, and two other amino acids and water proposed by previous investigators.

Binding Sites↗
PubMed1999

A single nucleotide insertion in the 5'-untranslated region of hepatitis C virus leads to enhanced cap-independent translation.

The 5'-untranslated region (5'-UTR) of hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) that directs translation of the viral open reading frame (ORF). The 5'-UTR consists of 341 nucleotides (nt) in most strains, and multiple segments within this region are important for its IRES activity. Sequencing analysis of a full-length HCV cDNA clone derived from a Japanese HCV1b-positive patient showed the 5'-UTR was 342 nt long due to a nucleotide T insertion at position 207. The influence of this T insertion on the IRES activity in directing cap-independent translation was investigated. The IRES of the 5'-UTR342 was approximately five- and two- to sevenfold more active in directing luciferase expression in monocistronic and bicistronic expression systems, respectively, when compared with the IRES of the 5'-UTR341 of a previously reported HCV1b strain. In addition to the T insertion, another point mutation involving an A to C transition at position 119 was also present in the 5'-UTR342. Simultaneous comparison of the IRES activities in engineered constructs that contained each of the two mutations indicated that the insertion at position 207 is responsible for the enhanced IRES activity of the 5'-UTR342. Further determination of the abilities of the engineered 5'-UTRs harbouring A, G, or C insertions at the same position to initiate translation indicated that both T and non-T nucleotide insertions lead to enhanced cap-independent translation.

5' Untranslated Regions↗
PubMed1999

EPR characterization of axial bond in metal center of native and cobalt-substituted guanylate cyclase.

The nature of the metal-proximal base bond of soluble guanylate cyclase from bovine lung was examined by EPR spectroscopy. When the ferrous enzyme was mixed with NO, a new species was transiently produced and rapidly converted to a five-coordinate ferrous NO complex. The new species exhibited the EPR signal of six-coordinate ferrous NO complex with a feature of histidine-ligated heme. The histidine ligation was further examined by using the cobalt protoporphyrin IX-substituted enzyme. The Co2+-substituted enzyme exhibited EPR signals of a broad g perpendicular;1 component and a g;1 component with a poorly resolved triplet of 14N superhyperfine splittings, which was indicative of the histidine ligation. These EPR features were analogous to those of alpha-subunits of Co2+-hemoglobin in tense state, showing a tension on the iron-histidine bond of the enzyme. The binding of NO to the Co2+-enzyme markedly stimulated the cGMP production by forming the five-coordinate NO complex. We found that N3- elicited the activation of the ferric enzyme by yielding five-coordinate high spin N3- heme. These results indicated that the activation of the enzymes was initiated by NO binding to the metals and proceeded via breaking of the metal-histidine bonds, and suggested that the iron-histidine bond in the ferric enzyme heme was broken by N3- binding.

Animals↗
PubMed1999

Hepatocyte growth factor stimulates cell growth and enhances the expression of transforming growth factor alpha mRNA in AsPC-1 human pancreatic cancer cells.

We investigated the effects of hepatocyte growth factor (HGF) and transforming growth factor alpha (TGF alpha) on cell growth in four human pancreatic cancer cell lines. Changes in the expression of mRNAs of HGF, c-met, TGF alpha, and epidermal growth factor receptor (EGFR) by treatment with HGF and TGF alpha were observed. Cell growth with growth factors was assessed with the MTT assay and compared with basal growth without growth factors. Although HGF stimulated cell growth in AsPC-1, COLO-357, and T3M4 cells, Panc-1 cells showed no response to HGF. TGF alpha stimulated the growth of all the above cells. The expression of c-met mRNA under nonstimulated conditions was detected with Northern blotting in all cells. Treatment with HGF slightly enhanced the expression of c-met mRNA only in COLO-357 cells. The intensity of EGFR expression was consistent, and HGF mRNA was not detected during induction experiments in any cell type. Concomitant treatment with HGF and TGF alpha exerted an effect that was additive or less on the growth of all cells. Expression of TGF alpha was enhanced by HGF treatment only in AsPC-1 cells. These results suggested that HGF and TGF alpha stimulated cell growth through a final common pathway of signal transduction.

Blotting, Northern↗
PubMed1998

Hereditary nonpolyposis colorectal cancer associated with duodenal carcinoma: a case report.

Hereditary nonpolyposis colorectal cancer is an autosomal, dominantly inherited disease, characterized by an early age of onset, right colon predominance and an association with various extracolorectal malignancies. We present a case of a 47-year-old woman who met the clinical criteria for the diagnosis of hereditary nonpolyposis colorectal cancer from her past and family histories. She had undergone operations for uterine cancer (histology not confirmed) at age 35 and for advanced cancer of the ascending colon at age 45. Gastroendoscopy revealed a flat elevated lesion, 20 mm in size, with a protrusion (type IIa + Is) in the second portion of the duodenum in March 1996. Additionally, colonoscopy showed a flat elevated lesion, 30 mm in size, with an irregular and nodular surface (type IIa, laterally spreading tumor) in the descending colon. After the operation, the resected specimen of the duodenum histologically showed a well-differentiated adenocarcinoma associated with a tubulo-villous adenoma which had invaded the submucosal layer. The tumor of the colon was histologically confirmed to be a moderately-differentiated adenocarcinoma with submucosal invasion. A high frequency of replication error positivity (4/5 loci) was detected in both of the tumors. Reports of early cancer of the duodenum, associated with extracolorectal malignancies in hereditary nonpolyposis colorectal cancer, are very rare in the literature. Although it is difficult to determine which extracolorectal tumor sites should be taken into consideration by screening programs, we believe that careful observation by upper gastrointestinal endoscopy, which includes the duodenum, is necessary for patients with hereditary nonpolyposis colorectal cancer.

Colonoscopy↗
PubMed1997

[Interferon treatment for hepatitis G virus infection in patients with chronic hepatitis C].

Clinical features of HGV infection as a novel virus infection have not yet been clarified enough. We studied the implication of HGV in the activity of hepatitis and the sensitivity of HGV to IFN. We treated 10 HGV RNA positive patients with chronic hepatitis C with IFN. HGV RNA was identified in serum by RT-PCR method using the primer derived from the base sequences of the NS5 region of HGV genome. HGV RNA became negative in the all of 10 patients during IFN treatment, but returned to be positive again in the all patients after the completion of IFN treatment. In 5 of 10 patients, HCV RNA became persistently negative. In 3 of these 5 patients, ALT was continuously normal and hepatitis subsided biochemically and clinically after IFN treatment. We assume that HGV is sensitive to IFN, but could not be implicated in hepatitis or liver damage.

Adult↗
PubMed1996

The 5' end sequences and exon organization in rat regucalcin gene.

The 5'-flanking region of the gene for a Ca(2+)-binding protein regucalcin was cloned from a rat genomic library which was constructed in lambda EMBL3 SP6/T7 vector. The genomic library was screened by using the radiolabeled probe with the 5' region (0.5 kb) of rat regucalcin complementary deoxyribonucleic acid (cDNA). Positive clone had the 5.5 kb fragment which was hybridized with the 5'-probe. This fragment contained three exons (I-III) of the gene coding for a rat regucalcin. The nucleotide sequence of exons completely agreed with that of a rat regucalcin cDNA clone. A supposed translational initiation site existed in the exon II. Homology analysis showed that a putative transcription start site in the rat regucalcin gene was located at position 26 downstream from a TATA-box. Another upstream element, a CCAAT box-like sequence, was located at -170. Moreover, there were many regulatory elements (Hox, AP-1, AP-2 and AP-4) in the 5'-flanking region of the rat regucalcin gene. The organization of rat regucalcin gene seemed to be about 18 kb in size and consisted of seven exons and six introns.

Amino Acid Sequence↗
PubMed1996

Role of Arg112 of cytochrome p450cam in the electron transfer from reduced putidaredoxin. Analyses with site-directed mutants.

The mechanism for the reduction of ferric cytochrome P450cam by reduced putidaredoxin, the physiological electron donor for the cytochrome, has been studied by using site-directed mutants of cytochrome P450cam, in which Arg112, an amino acid residue at the presumed binding site for putidaredoxin, was changed to several other amino acid residues. The affinity of reduced putidaredoxin for ferric cytochrome P450cam to form a diprotein complex was decreased greatly by changing Arg112 to a neutral amino acid such as Cys, Met, or Tyr. The rate of intracomplex electron transfer from putidaredoxin to cytochrome P450cam also diminished upon replacing the basic residue with neutral ones, being 42, 18, 4.0, 1.3, and 0. 16 s-1 for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. Furthermore, the oxidation-reduction potential of cytochrome P450cam (Fe3+/Fe2+ couple) decreased in a similar way to the decrease in the rate of electron transfer upon amino acid substitution; the values were -138, -162, -182, -200, and -195 mV for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. These results indicate that the amino acid substitution at position 112 affects the oxidation-reduction potential of the heme iron in cytochrome P450cam, thereby diminishing the rate of electron transfer between the two metal centers. The rate of electron transfer from putidaredoxin to oxyferrous cytochrome P450cam also diminished upon substitution of Arg112 with a neutral amino acid.

Arginine↗
PubMed1996

The structure-function relationship and reduction potentials of high oxidation states of myoglobin and peroxidase.

In these studies, we substitute electron-withdrawing (diacetyl) or -donating (diethyl) groups at the 2- and 4-positions of the heme in sperm whale Mb and HRP, and examine the structural and biochemical consequences. X-ray absorption spectroscopy shows that increased electron density at the heme results in an increased iron-pyrrole nitrogen average distance in both HRP and Mb, while decreased electron density results in shorter average distances. In HRP, the proximal ligand is constrained by a H-bonding network, and axial effects are manifested entirely at the distal site. Conversely, in Mb, where the proximal ligand is less constrained, axial effects are seen at the proximal side. In HRP, electron density at the heme iron depends linearly on pK3, a measure of the basicity of the porphyrin pyrrole nitrogens [Yamada, H., Makino, R., & Yamazaki, I. (1975) Arch. Biochem. Biophys. 169, 344-353]. Using diethyl substitution (pK3 = 5.8) and diacetyl substitution (pK3 = 3.3) in HRP and Mb, we measured the one-electron reduction potentials (E(O)') of HRP compounds I and II and ferryl Mb. Compound I showed a decreased E(O)' with increasing electron density at the heme (pK3), similar to E(O)' of ferric HRP. E(O)' of HRP compound II and ferryl Mb showed an opposite dependence. This behavior of E(O)', while initially surprising, can be explained by the apparent net positive charge on the iron porphyrin in each oxidation state of the hemoproteins.

Animals↗