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Biomedical subjects

Akira Murakami

Publications and source records attributed to Akira Murakami.

At least 127 records · Page 7Linked to original sources
PubMed2002

Effect of chemical features and stability of polyplexes on their gene transfer efficiency.

Chemically modified poly-L-lysine (PL) derivatives with two essential features, which we have recently reported on, have been used to study key factors affecting transgene expression efficiency. PL derivatives having both of N epsilon-trimethyl lysine residue and 25 mole % serine residue showed enhanced transfection efficiency. When PL was modified in either way, no marked enhancement in gene expression was observed. These PL derivatives were found to be able to deliver plasmid DNA into nucleus of the transfected cells with a similar amount. Judging from the loss of EtBr fluorescence intercalating to DNA, the condensing tendency of the DNA were greatly affected by the sequence of the polypeptides used. This loose compaction seems to enhance the transgene expression because of an easy disassembly of the formed complexes or recognition of the DNA molecules in the complexes.

Fluorescence↗
PubMed2002

Selection of RNA-binding peptides containing Arg-rich motif.

In order to search the RNA-binding peptides, selection from randomized peptides containing Arg-rich motif was carried out using rRNA-immobilized column. As a result of the selection for 16S- and 23S-rRNA from E. coli, six peptides were identified to be the rRNA-binding peptide by MALDI-TOF MS. These peptides contained R, N, Q, or G at three randomized positions. This result indicates that amino acids with a positive residue or an amide residue might be essential for peptides to recognize the RNA.

Amino Acid Motifs↗
PubMed2002

[Gelatinous drop-like corneal dystrophy: mutation analysis of membrane component, chromosome 1, surface marker 1].

PURPOSE: To investigate mutation in membrane component, chromosome 1, surface marker 1(M1 S1) gene for patients and unaffected relatives of gelatinous drop-like corneal dystrophy(GDLD). MATERIALS AND METHODS: We performed mutation analysis for 11 patients and 18 unaffected relatives from 7 unrelated families with GDLD. They were followed at the Department of Ophthalmology, Juntendo University, Tokyo, and St. Luke's International Hospital, Tokyo. RESULTS: Ten patients including the affected members of 6 families were detected to be homozygotes by transition of a C to T at nucleotide position 352, replacing a glutamine at codon 118 with a stop codon (Q118X), and unaffected relatives were heterozygous for the mutation. One patient and his parents and brother from one family have had no mutation in open reading frame of the M1S1 gene. CONCLUSIONS: In these 6 families the genotypes of Q118X mutation on M1S1 gene were co-segregated with the phenotype. For the patients who have had no mutation, we need further investigation of the 5' upper site, and it is possible that there is another candidate gene for GDLD.

Antigens, Neoplasm↗
PubMed2002

Suppressive Effects of Edible Thai Plants on Superoxide and Nitric Oxide Generation.

We screened ethanol extracts from a total of 134 species of edible Thai plants for their suppressive effects on superoxide (O2(-)) generation using a xanthine (XA)-xanthine oxidase (XOD) assay system. When the extracts were tested at a concentration of 500 &mgr;/g/ml, 28.4% significantly suppressed O2(-) generation. Of these active extracts, it was found that in 17.9% of cases the action was due to XOD inhibition, in 1.5% due to O2(-) scavenging activity, and in 9% due to both XOD inhibition and O2(-) scavenging. In addition, some plant extracts (25 species) which had been known to possibly possess anti-tumor promoting activity were tested for O2(-) and NO generation in cellular systems. In this test, 13 species exhibited strong inhibitory activity toward both O2(-) and NO generation. From the fruit pods of Oroxylum indicum (Bignoniaceae), a traditional vegetable in Thailand, two flavones, oroxylin A and chrysin, and a triterpene carboxylic acid, ursolic acid (UA), were identified as inhibitors of O2(-) generation in XA/XOD system. These compounds also showed marked inhibitory effects on the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced O2(-) generation in dimethylsulfoxide (DMSO)-differentiated HL-60 cells. Our results suggest that, as we have reported earlier, edible Thai plants are promising sources of antioxidants with chemopreventive potential.

Journal Article↗
PubMed2000

Search for Naturally-occurring Antioxidative Chemopreventors on the Basis of the Involvement of Leukocyte-derived Reactive Oxygen Species in Carcinogenesis.

Chemoprevention with food phytochemicals is currently regarded as one of the most attractive strategies for cancer control. We have been continuously working on the identification and characterization of chemopreventive phytochemicals extracted from a diverse range of edible plants. Recently, we have utilized a convenient assay, the tumor promoter-induced superoxide generation test, with differentiated HL-60 cells for primary screening, and performed chemical studies of antioxidative food phytochemicals. Here, we report our criteria for evaluation of new types of chemopreventors on the basis of the involvement of leukocyte-derived reactive oxygen in carcinogenesis.

Journal Article↗
PubMedDate not supplied

Mutation analysis of the TGFBI gene in Vietnamese with granular and Avellino corneal dystrophy.

PURPOSE: Mutations of the human transforming growth factor beta-induced gene (TGFBI) were reported to cause granular (GCD) and Avellino (ACD) corneal dystrophy in various nationalities. In this study we examined the TGFBI gene in a Vietnamese population with GCD and ACD. METHODS: Eight unrelated Vietnamese families, including 20 affected and 24 unaffected individuals, were examined; 50 normal Vietnamese individuals were used as controls. Genomic DNA was extracted from peripheral blood leukocytes. The TGFBI gene was analyzed using the polymerase chain reaction and direct sequencing. The corneal button was studied. RESULTS: Slit-lamp examination revealed typical features of GCD in most cases. A few features of ACD and a patient with an atypical form of GCD were also seen. Histopathological analysis of a GCD cornea showed deposits that stained bright red with Masson trichrome. Sequencing revealed three distinct mutations: R555W in six families, R124H in one family, and D123H in another. CONCLUSIONS: R555W and R124H mutations were co-segregated with the disease phenotype and thus caused GCD and ACD, respectively, in the families studied. The R555W detected in six of the eight families indicates that the GCD phenotype may be the most common in Vietnamese individuals, unlike in other Asians (Japanese and Korean), where ACD is most common (>90%). The D123H mutation may cause an atypical variant of GCD.

Adolescent↗
PubMedDate not supplied

Analysis of COL8A2 gene mutation in Japanese patients with Fuchs' endothelial dystrophy and posterior polymorphous dystrophy.

PURPOSE: To determine whether Japanese patients with Fuchs' endothelial corneal dystrophy (FECD) and posterior polymorphous dystrophy (PPMD) carry mutations in the COL8A2 gene, and to investigate the possible pathogenicity of the COL8A2 gene in these corneal dystrophies. METHODS: DNA analysis of the COL8A2 gene was performed in 15 unrelated Japanese patients with FECD, and 5 patients with PPMD using polymerase chain reaction and direct sequencing. Mutation screenings were also performed in 36 unrelated normal volunteers as controls, as well as slit-lamp and specular microscopy. RESULTS: Two types of heterozygous missense mutations of the COL8A2 gene (R155Q and T502M) in 5 of 15 FECD probands (R155Q, 3/30 chromosomes, 10.0%; T502M, 3/30 chromosomes, 10.0%) were found. No mutation was detected in the coding region of the COL8A2 gene in the remaining 10 patients with FECD nor in any of the 5 patients with PPMD. These two mutations were also found in normal Japanese volunteers (R155Q, 5/72 chromosomes, 6.9%; T502M, 11/70 chromosomes, 15.7%). The chromosomal frequency of the two mutations was not significant between the patients and normal controls. CONCLUSIONS: The R155Q and T502M mutations of COL8A2 may not be the causative defect in the Japanese FECD and PPMD patients examined in this study.

Aged↗
PubMedDate not supplied

Mutations in the membrane component, chromosome 1, surface marker 1 (M1S1) gene in gelatinous drop-like corneal dystrophy.

PURPOSE: To report mutations in the membrane component, chromosome 1, surface marker 1 ( M1S1) gene in two members of the same family who showed symptoms of gelatinous drop-like corneal dystrophy (GDLD). METHODS: DNA was extracted from leukocytes of peripheral blood of the two affected members of the family and from controls, and the coding region of M1S1 was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by direct sequencing. Normal and mutant M1S1 expression vectors were constructed and transfected into CHO cells to identify the cellular location of the gene products. RESULTS: The affected members had compound heterozygous mutations consisting of a nonsense change at codon 84 (K84X) and a missense mutation resulting in a substitution of arginine for cysteine at codon 108 (C108R). Neither of these mutations was found in the 50 controls. Protein expression analysis showed that the C108R product was distributed diffusely in the cytoplasm, whereas the normal gene product accumulated at cell-to-cell adhesion borders. CONCLUSION: These data indicate that the K84X and C108R mutations in M1S1 cause GDLD.

Adult↗
PubMedDate not supplied

Immunohistochemical characterization of epithelial cells implanted in the flap-stroma interface of the cornea.

PURPOSE: To investigate the expression of extracellular matrix collagens and their relationship to corneal opacities after implantation of epithelial cells in the flap-stroma corneal interface. METHODS: A corneal flap was made on rabbit eyes, and epithelial cells, mechanically scraped from tissue surrounding the flap, were implanted beneath the flap. The corneas were harvested 1, 3, 7, and 30 days following surgery. Histological and immunohistochemical examinations were performed. The expression and localization of types I, III, and IV collagens and gelatinase A were determined. RESULTS: Slit-lamp examination showed corneal opacity in the area where the epithelial cells were implanted. Histological study revealed clusters of epithelial cells between the flap and stromal interface. One week and 1 month after the implantation, intense immunoreactivity for collagen type IV was detected at the perimeters of the intrastromal epithelial islands, but not in the interface outside the implanted epithelial cells. Weak positive staining for gelatinase A was detected in the implanted epithelial cells and surrounding keratocytes. CONCLUSIONS: The heavy deposition of collagen type IV surrounding the implanted epithelial cells indicated that it might be an essential component of the interface haze observed in patients following laser in situ Keratomileusis. Gelatinase A may also play a role in the regulation of stromal remodeling after epithelial ingrowth.

Animals↗
PubMedDate not supplied

Novel mutation (V505D) of the TGFBI gene found in a Chinese family with lattice corneal dystrophy, type I.

PURPOSE: To report a novel V505D mutation of the human transforming growth factor beta-induced (TGFBI) gene found in a Chinese family with lattice corneal dystrophy, type I (LCDI). METHODS: Genomic DNA was extracted from peripheral leukocytes from eight affected and four unaffected members of a Chinese family with LCDI. Exons of the TGFBI gene were amplified by polymerase chain reaction and directly sequenced. Fifty normal Chinese individuals were also analysed as controls. Histopathological examination of a corneal button was performed after keratoplasty of the proband. RESULTS: A heterozygous single-base-pair transversion (GTC to GAC, valine to aspartic acid) at codon 505 in exon 11 of the TGFBI gene (V505D) was detected in all of the affected members. No mutation was found in the unaffected members or in the 50 normal controls. The mutation cosegregated with the disease phenotype throughout three generations. Although a slit-lamp examination showed features of LCDI in most cases, the age at onset of the symptoms was several years later than that in cases of LCDI with an R124C mutation. By histopathological examination, numerous amyloid deposits were observed in the stroma, including beneath Bowman's membrane. CONCLUSION: A novel V505D mutation in the TGFBI gene causes LCDI in this Chinese family. It is the fourth reported mutation of the TGFBI gene associated with LCDI.

Adult↗
PubMedDate not supplied

Disodium cromoglycate inhibits capsaicin-induced eosinophil infiltration of conjunctiva independent of mast cells.

PURPOSE: To investigate whether disodium cromoglycate (DSCG) inhibits capsaicin-induced eosinophil infiltration of the conjunctiva independent of mast cells. METHODS: We administered 5 microl of capsaicin solution (10(-5) M) into the conjunctival sacs of mast cell-deficient W/W(v) mice (12 animals) and wild-type mice (12 animals). As controls, the eyes of 12 wild-type and 12 W/W(v) mice were treated with phosphate-buffered saline. Following treatment, the eyelids and eyeballs were removed en bloc at 3, 9, or 24 h, and were histologically examined. The number of infiltrated eosinophils and the expression of vascular cell adhesion molecule-l (VCAM-1) in the conjunctiva were quantified by the staining method of Hansel and immunohistochemical analysis. We also investigated whether treatment by depletion of neuropeptides or by DSCG administration could suppress the capsaicin-induced eosinophil infiltration of the conjunctiva. RESULTS: In both W/W(v) and wild-type mice, eosinophil infiltration of conjunctival tissues was observed 3 h after capsaicin administration. In both strains of mice, the number of infiltrated eosinophils increased over time, with VCAM-1 expression on vascular endothelial cells peaking at 9 h after treatment, and decreasing gradually within 24 h after treatment. In both the neuropeptide-depleted and the DSCG-treated groups, eosinophil infiltration and VCAM-1 expression were suppressed in comparison with the nontreated group. CONCLUSION: DSCG can directly inhibit neuropeptide-induced eosinophil infiltration of the conjunctiva independent of mast cells.

Animals↗
PubMedDate not supplied

Transferrin-polyethylenimine conjugate, FuGENE6 and TransIT-LT as nonviral vectors for gene transfer to the corneal endothelium.

PURPOSE: To investigate the efficacy and pathogenicity of three commercially available nonviral DNA vectors for gene transfer to the corneal endothelium. METHODS: Corneas obtained from New Zealand White rabbits were cultured ex vivo. For cell culture, the corneal endothelial cells were removed and cultured in vitro under standard conditions. Using the vectors, culture cells or ex vivo corneas were transfected with plasmid DNA coding for green fluorescent protein (GFP). Expression of the transduced gene was monitored by fluorescence microscopy. Transfection efficiency was estimated as the percentage of GFP-positive cells identified. The viability and morphology of the endothelium were also examined. RESULTS: Transferrin-polyethylenimine conjugate was effective in vitro but not ex vivo. FuGENE6 and TransIT-LT mediated the transfer of GFP gene both in in vitro and in ex vivo culture. Their efficiency estimated at day 3 was 28.8% and 38.8% in vitro, and 8.1% and 8.8% ex vivo, respectively. Viability staining revealed no dead cells. Morphological study showed no apparent alteration. CONCLUSIONS: FuGENE6 and TransIT-LT are safe, simple to use, and may be useful alternative methods for gene transfer to the corneal endothelium, avoiding certain side effects of viral vectors. As the efficiency could be enhanced, these nonviral vectors may be promising for practical application.

Animals↗
PubMedDate not supplied

Heterozygous Ala137Pro mutation in keratin 12 gene found in Japanese with Meesmann's corneal dystrophy.

PURPOSE: To report the molecular genetic analysis of a Japanese pedigree with Meesmann's corneal dystrophy (MCD). METHODS: Sequencing of the keratin 3 and keratin 12 genes was performed in 2 patients who were siblings and in an unaffected individual in the same family. The patients had the typical corneal microcysts and recurrent erosions with mild photophobia. RESULTS: A novel mutation resulting in the substitution of alanine to proline in codon 137 of the keratin 12 gene (Ala137Pro) was found in the 2 patients, but not in the unaffected member of the family and the 50 controls. CONCLUSIONS: This novel mutation (Ala137Pro) of the keratin 12 gene found in a Japanese family had caused MCD.

Aged↗
PubMedDate not supplied

Multiplex polymerase chain reaction for detection of herpes simplex virus type 1, type 2, cytomegalovirus, and varicella-zoster virus in ocular viral infections.

PURPOSE: To detect simultaneously herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), cytomegalovirus (CMV), and varicella-zoster virus (VZV) in ocular specimens suspected of indicating viral infection, and to compare the results of multiplex polymerase chain reaction (PCR) with those of uniplex PCR. METHODS: Forty specimens, collected from 33 patients with clinically suspected herpes virus ocular infection, were tested. DNA was extracted from the specimens and amplified by multiplex and uniplex PCR. RESULTS: Both multiplex PCR and uniplex PCR gave the same results. Nineteen (19/33, 57.6%) patients were PCR-positive, among whom HSV-1 was detected in 13 (13/19, 68.4%) patients, and VZV in 6 (6/19, 31.6%) patients. CONCLUSION: These results demonstrated that multiplex PCR is as reliable as uniplex PCR, and is an accurate and a cost-saving method to identify several agents from a single specimen.

Adolescent↗
PubMedDate not supplied

A novel mutation of the TGFBI gene found in a Vietnamese family with atypical granular corneal dystrophy.

BACKGROUND: Mutation of the human transforming growth factor beta-induced (TGFBI) gene causes granular corneal dystrophy (GCD) in various ethnic groups. In this report, we identify the genetic defect on the TGFBI gene in a Vietnamese family with atypical GCD . CASES: The patient and her relatives were examined clinically. Genomic DNA was extracted from blood leukocytes. Fifty normal Vietnamese were used as controls. Analysis of the TGFBI gene was performed using polymerase chain reaction and direct sequencing. OBSERVATIONS: The 42-year-old proband clinically showed multiple white dot-like opacities scattered in the anterior and mid-stroma of the central cornea. Unlike GCD, these deposits were smaller, localized deeper and less severe. DNA analysis revealed a nucleotide transversion at codon 123 (GAC --> CAC), causing Asp --> His substitution (D123H). This mutation was also detected in 3 out of 5 unaffected family members, but was absent in the 50 normal controls. CONCLUSIONS: The novel D123H mutation of the TGFBI gene was not co-segregated with GCD in the family studied, and did not exist in the control population. It probably was a disease-causing mutation, thus expected to cause a novel variant of GCD in the proband. The detection of the D123H mutation in three unaffected family members indicates that it has low penetrance for GCD.

Adult↗
PubMedDate not supplied

OPA1 gene mutations in Japanese patients with bilateral optic atrophy unassociated with mitochondrial DNA mutations at nt 11778, 3460, and 14484.

PURPOSE: To report mutations in the OPA1 gene in Japanese patients with bilateral optic atrophy unassociated with mitochondrial DNA mutations at nt 11778, 3460, and 14484. METHODS: Twelve unrelated patients with bilateral optic atrophy and 100 healthy controls were examined. Each exon of the OPA1 gene was amplified by polymerase chain reaction (PCR). All PCR products were sequenced. RESULTS: Of the 12 patients, 2 had nonsense mutations of the OPA1 gene (nt 1039G --> T and nt 1096C --> T, leading to Glu347Stop and Arg366Stop, respectively). These nonsense mutations were not found in the 100 healthy controls. Two of the patients had silent mutations of OPA1 gene (nt 1177T --> G and nt 1923G --> A causing no amino acid change). CONCLUSIONS: The mutations (Glu347Stop and Arg366Stop) of the OPA1 gene are involved in the pathogenesis of bilateral optic atrophy in Japanese patients.

Adolescent↗