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Biomedical subjects

K Sakagami

Publications and source records attributed to K Sakagami.

At least 37 records · Page 2Linked to original sources
PubMed1997

Genetic recombination through double-strand break repair: shift from two-progeny mode to one-progeny mode by heterologous inserts.

Double-strand break repair models of genetic recombination propose that a double-strand break is introduced into an otherwise intact DNA and that the break is then repaired by copying a homologous DNA segment. Evidence for these models has been found among lambdoid phages and during yeast meiosis. In an earlier report, we demonstrated such repair of a preformed double-strand break by the Escherichia coli RecE pathway. Here, our experiments with plasmids demonstrate that such reciprocal or conservative recombination (two parental DNAs resulting in two progeny DNAs) is frequent at a double-strand break even when there exists the alternative route of nonreciprocal or nonconservative recombination (two parental DNAs resulting in only one progeny DNA). The presence of a long heterologous DNA at the double-strand break, however, resulted in a shift from the conservative (two-progeny) mode to the nonconservative (one-progeny) mode. The product is a DNA free from the heterologous insert containing recombinant flanking sequences. The potential ability of the homology-dependent double-strand break repair reaction to detect and eliminate heterologous inserts may have contributed to the evolution of homologous recombination, meiosis and sexual reproduction.

DNA Damage↗
PubMed1997

Reopening of macular holes in highly myopic eyes with retinal detachments.

BACKGROUND: Gas tamponade with or without vitrectomy is commonly used to treat retinal detachment resulting from a macular hole in a highly myopic eye. Redetachment of the retina occurs frequently, however, and the pathogenesis of redetachment is unclear. One possible mechanism leading to redetachment is tangential traction caused by an epiretinal membrane of the posterior retina. METHODS: Eleven consecutive highly myopic eyes with retinal detachment resulting from a macular hole were treated by vitrectomy, gas or silicone oil tamponade, and removal of an epiretinal membrane. Surgical outcomes and clinical characteristics of these patients were reviewed. RESULTS: An epiretinal membrane was found to be present in all eyes. Of 11 eyes, removal of an epiretinal membrane was complete in six eyes, and macular holes remained closed in all 6 eyes. In five of the 11 eyes, removal of the epiretinal membrane was incomplete. In four of these five eyes, the macular hole reopened. CONCLUSION: Reopening of the macular holes most likely were caused by tangential traction of the remaining epiretinal membranes. It seems that it is important to remove epiretinal membranes as completely as possible during vitrectomy for retinal detachment resulting from a macular hole in a highly myopic eye.

Aged↗
PubMed1997

A new type of illegitimate recombination is dependent on restriction and homologous interaction.

Illegitimate (nonhomologous) recombination requires little or no sequence homology between recombining DNAs and has been regarded as being a process distinct from homologous recombination, which requires a long stretch of homology between recombining DNAs. Under special conditions in Escherichia coli, we have found a new type of illegitimate recombination that requires an interaction between homologous DNA sequences. It was detected when a plasmid that carried 2-kb-long inverted repeats was subjected to type II restriction in vitro and type I (EcoKI) restriction in vivo within a delta rac recBC recG ruvC strain. Removal of one of the repeats or its replacement with heterologous DNA resulted in a reduction in the level of recombination. The recombining sites themselves shared, at most, a few base pairs of homology. Many of the recombination events joined a site in one of the repeats with a site in another repeat. In two of the products, one of the recombining sites was at the end of one of the repeats. Removal of one of the EcoKI sites resulted in decreased recombination. We discuss the possibility that some structure made by homologous interaction between the long repeats is used by the EcoKI restriction enzyme to promote illegitimate recombination. The possible roles and consequences of this type of homologous interaction are discussed.

Base Sequence↗
PubMed1997

[Emulsification experiments with dimethylsiloxane/phenylmethylsiloxane copolymer].

We compared the emulsification tendency of dimethylsiloxane/phenylmethylsiloxane copolymer (DPC; 5%-phenylated, specific gravity 0.984) with that of silicone oil (dimethylsiloxane; SO, specific gravity 0.966) and fluorosilicone oil (methyl-3,3,3-trifluoropropylsiloxane; FSO, specific gravity 1.256), which are clinically used as an intraocular tamponade. After SO, DPC and FSO were placed in a glass container with an equal amount of albumin solution (1 mg/ml) or gamma-globulin solution (1 mg/ml), and shaken SO and DPC, with their specific gravities being closer to water, showed less emulsification than FSO. Following vitrectomy, DPC was injected into the rabbit vitreous cavity, but did not seem to show any severe histological damage. Highly-phenylated DPC, which is slightly heavier than water, may be useful to treat inferior retinal detachment in place of FSO.

Animals↗
PubMed1997

[Treatment of full-thickness macular holes with autologous serum].

A total of 29 eyes in 28 patients with stage 2 to 4 idiopathic full-thickness macular hole were treated with autologous serum. Autologous serum (20-30 microliters) was placed over the macular hole followed by injection of 16% perfluoropropane gas. Postoperatively, twenty-eight eyes (97%) had flattening of the macular hole, and the hole was invisible in 27 eyes (93%). Twenty-two eyes (76%) showed visual improvement of at least two lines or more. Preoperative factors such as good visual acuity, earlier stage, and younger age were correlated with postoperative good visual acuity. These results suggest the benefit of autologous serum in the treatment of full-thickness macular holes.

Adult↗
PubMed1997

Synthesis of cyclodeca-1,5-diyne derivatives having o-nitrophenylseleno group.

7-Benzoyloxy-3-(2-nitrophenylseleno)-1,5-cyclodecadiyne (13) was prepared from 5-hexyn-1-ol and propargyl bromide via 10-iodo-7-(t-butyldiphenylsiloxy)-5,9-decadiynal (10). The facile cyclization of the acyclic precursor 10 would be rationalized in terms of "bulky group (TBDPSO) assisted conformational control". Oxidation of 13 by NaIO4, followed by thermal treatment gave tetrahydronaphthalene derivative 15, indicating the generation of biradical.

Antibiotics, Antineoplastic↗
PubMed1996

Estimation of the optimal cut off point in a new immunological faecal occult blood test in a corporate colorectal cancer screening programme.

OBJECTIVE: To estimate the optimal cut off point in a new immunological method (OC-Hemodia) for faecal occult blood testing (FOBT). SETTING: A corporate colorectal cancer screening programme in Japan. METHOD - The screening programme targeted colorectal cancer and adenomatous polyps > or = 10 mm, and was conducted on 27 860 participants (age > or = 40 ) during 1991-92. The follow up consisted of diagnostic management by total colonoscopy on positive screened subjects exceeding the manufacturer recommended cut off level of 50 ng/ml faecal haemoglobin, and the identification of false negative cases by health insurance claims. The optimal cut off point was estimated by the positive predictive value, receiver operating characteristic (ROC) curve, and a cost effectiveness analysis. In this study evaluation was carried out only for cancer as the target disease. RESULTS: - At the current cut off level of 50 ng/ml the sensitivity and specificity were 86.5% and 94.9%. When the optimal cut off point was estimated the highest positive predictive value was obtained at 250-350 ng/ml. The ROC curve showed that the sum of sensitivity and specificity is maximised at 50 ng/ml, but evaluation of the ratio, change in sensitivity/change in false positive rate, pointed to higher optimal cut off points, showing marked changes occurring at about 200 ng/ml. The average cost per case was lowest at 250-300 ng/ml. Overall, the optimal cut off point was estimated to be about 200 ng/ml, at which the sensitivity and specificity of the test would be 77.5% and 98.9%, respectively. CONCLUSION: The optimal cut off point of the new immunological method of FOBT was estimated to be about 200 ng/ml, a value which, more than the current cut off value, favours specificity over sensitivity.

Adenoma↗
PubMed1996

A new wound healing model of retinal pigment epithelial cells in sheet culture.

PURPOSE: To evaluate some RPE cell functions, such as wound healing, in a preparation more similar to in situ conditions, we developed a method to obtain and culture retinal pigment epithelial (RPE) cells as a sheet. And we assessed the effects of fetal bovine serum (FBS) on the rate of RPE wound healing. METHODS: We prepared RPE sheet cultures by incubating rat eyes in 0.1% proteinase K for 13 min, peeling away the neural retina-RPE complex, and then incubating the tissue for 1 h to promote spontaneous separation of the RPE sheet from the retina. After several days of incubation, the cultured sheets of RPE cells were examined by phase-contrast microscopy, scanning and transmission electron microscopy and immunocytochemistry. We made round defects 1 mm in diameter in cultured RPE sheets and estimated the rate of wound closure in media with different concentrations of FBS (0 to 10%). RESULTS: The RPE cells cultured in sheets retained their in situ features, including microvilli, tight junctions and gap junctions, and the distribution of actin and cytokeratin filaments. A wound was noted to close with restoration of a polygonal configuration. The rate of wound closure depended on serum concentration in the culture medium; when supplemented with 10% fetal bovine serum, wound closure was complete in approximately 40 h. CONCLUSIONS: The RPE sheet-culture technique we developed thus provides a suitable model for studying such RPE cell functions as wound healing or phagocytosis.

Animals↗
PubMed1995

Gene targeting with a replication-defective adenovirus vector.

Wide application of the gene-targeting technique has been hampered by its low level of efficiency. A replication-defective adenovirus vector was used for efficient delivery of donor DNA in order to bypass this problem. Homologous recombination was selected between a donor neo gene inserted in the adenovirus vector and a target mutant neo gene on a nuclear papillomavirus plasmid. These recombinant adenoviruses allowed gene transfer to 100% of the treated cells without impairing their viability. Homologous recombinants were obtained at a level of frequency much higher than that obtained by electroporation or a calcium phosphate procedure. The structure of the recombinants was analyzed in detail after recovery in an Escherichia coli strain. All of the recombinants examined had experienced a precise correction of the mutant neo gene. Some of them had a nonhomologous rearrangement of their sequences as well. One type of nonhomologous recombination took place at the end of the donor-target homology. The vector adenovirus DNA was inserted into some of the products obtained at a high multiplicity of infection. The insertion was at the end of the donor-target homology with a concomitant insertion of a 10-bp-long filler sequence in one of the recombinants. The possible relationship between these rearrangements and the homologous recombination is discussed. These results demonstrate the applicability of adenovirus-mediated gene delivery in gene targeting and gene therapy.

Adenoviridae↗
PubMed1995

A rapid method for isolation of retinal pigment epithelial cells from rat eyeballs.

A new, simple method for isolating retinal pigment epithelial (RPE) cells from rat eyeballs was developed: incubation of the eyeballs in 0.1% proteinase K solution. This method yielded an average of 4 x 10(4) viable RPE cells from each Sprague-Dawley rat eyeball in 1 h. These RPE cells were hexagonal and had cytokeratin in their cytoplasm and numerous microvilli on their surface, similar to RPE cells evaluated in vivo. The results of this study thus show that our method for incubating eyeballs in proteinase K can provide good-quality RPE cells in sufficient quantities for study in a short time.

Animals↗
PubMed1995

[Efficacy of treatment with frequent and low-dose epirubicin in two cases of pulmonary metastases after surgery of liver cancer].

Case 1 was a 55-year-old male with multiple pulmonary metastases after two operations to excise hepatocellular carcinoma. Treatment consisted of initial administration of 5'-DFUR (po), followed by frequent administration of low-dose epirubicin (20 mg/body once every 2 weeks iv). This resulted in disappearance of the pulmonary metastases and a striking decrease in the AFP level. Case 2 was a 68-year-old male with multiple pulmonary metastases after surgery for hepatocellular carcinoma. Treatment with 5-FU (iv; once a week) and epirubicin (20 mg/body once every 2 weeks iv) resulted in disappearance of the pulmonary metastases and a marked decrease in the AFP level. It was concluded that administration of epirubicin in frequent, low doses is a useful method for the treatment of pulmonary metastasis after surgery for liver cancer, and it can be safely performed even on an outpatient basis.

Aged↗
PubMed1994

Homology-associated nonhomologous recombination in mammalian gene targeting.

Nonhomologous (illegitimate) recombination of DNA underlies many changes in the genome. It involves no or little homology between recombining DNAs and has been considered unrelated with homologous recombination, which requires long homology. In mouse cells, however, we found recombination products whose sequences suggest that homologous interaction between DNAs caused nonhomologous recombination with another DNA. The intermediates of homologous recombination were apparently trapped at various stages and shunted to nonhomologous recombination. In one product, the nonhomologous recombination disrupted gene conversion. In another, it took place exactly at the end of long homology shared between two DNAs. This finding explains why gene targeting needs long uninterrupted homology and why mammalian homologous recombination is often nonconservative. We discuss possible consequences and roles of this type of homology-driven gene destruction mechanism.

Animals↗