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Biomedical subjects

A Abe

Publications and source records attributed to A Abe.

At least 181 records · Page 10Linked to original sources
PubMed1994

Retrovirus-mediated transfer of a hygromycin phosphotransferase-thymidine kinase fusion gene into human CD34+ bone marrow cells.

Retrovirus-mediated gene transfer into human hematopoietic stem cells has been proposed as a means of therapy for various inherited diseases and as a method of gene marking. The transduction efficiency of an amphotropic retroviral vector (PA317/HyTK) containing a hygromycin phosphotransferase-thymidine kinase fusion gene was examined with human CD34+ bone marrow cells in the presence of interleukin-3 (IL-3), interleukin-6 (IL-6), and stem cell factor. Transduction efficiencies determined from the ability of transduced granulocyte-macrophage colony forming units (CFU-GM) to grow in hygromycin B and from polymerase chain reaction analysis of individual transduced CFU-GM growing in the presence of hygromycin B were 0.3-3.0% (mean +/- S.D., 1.1 +/- 0.9%) and 0.1-1.2% (mean +/- S.D., 0.5 +/- 0.4%), respectively. Ganciclovir at a dose of approximately 1 microM reduced the number of CFU-GM derived from vector-infected CD34+ cells by 50%. These findings demonstrate that human hematopoietic stem cells infected with this retroviral vector are susceptible to ganciclovir, offering the potential to control transduced gene expression in vivo.

Antigens, CD↗
PubMed1994

Utilization of bloodfed females of Aedes aegypti as a vehicle for the transfer of the insect growth regulator pyriproxyfen to larval habitats.

Bloodfed female Aedes aegypti were exposed to a surface treated with pyriproxyfen at 1.0 g m2 for 30 min and then allowed to lay eggs in cups of water containing 4th-instar larvae. Adult emergence from the immatures was highly inhibited, and transmission of pyriproxyfen from the females to the water was revealed. The transfer of the chemical to the water decreased with time before the blood meal. Chemical analysis for pyriproxyfen on the exoskeleton of treated females demonstrated the rapid disappearance of the compound. Pyriproxyfen obviously affected egg maturation of females treated before blood meals, as the number of eggs deposited decreased concurrently with the number of days before the blood meals.

Aedes↗
PubMed1994

[Relapse of acute leukemia after allogeneic bone marrow transplantation].

The relapses of 45 acute leukemia patients who underwent allogeneic bone marrow transplantation between April 1976 and February 1992 were analyzed clinically. The relapse rates of acute nonlymphocytic leukemia (ANLL) and acute lymphocytic leukemia (ALL) were 4/26 (15%) and 9/19 (47%), respectively. Although multivariate analysis could not be done, a high frequency of leukemia relapse was significantly associated with ALL, a younger age, history of extramedullary leukemia and blood-type identity. Chromosomal abnormalities at diagnosis, interval from diagnosis to bone marrow transplantation (BMT) and graft-versus-host disease (GVHD) prophylaxis were likely to be predictive of relapse. On the other hand, neither stage of leukemia at BMT, acute GVHD, sex, nor WBC count at diagnosis was a predictive factor. Eight of 10 relapsed patients achieved complete remission posttransplant, but the duration of remission was only 138 days on average. Four second BMT procedures for relapsed leukemia were carried out but were associated with many complications. However, one is alive well over 6 years after the second transplantation. There is a high treatment-related mortality following second BMT, but there is some possibility of long survival in some patients.

Adolescent↗
PubMed1993

Rapid and sensitive method for detection of Salmonella strains using a combination of polymerase chain reaction and reverse dot-blot hybridization.

We have developed a reverse dot-blot hybridization assay for detection of Salmonella using Salmonella-specific oligonucleotide probes designed from the base sequence of the 16S rRNA gene (rDNA). The target fragment of 16S rDNA was amplified, and labelled with biotin by the polymerase chain reaction. The amplified fragment was hybridized with the membrane-immobilized probe and the hybridization was detected by chemiluminescence. Amplified fragments from 24 different serovars of Salmonella hybridized with the probes, whereas those of species of Enterobacteriaceae, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus failed to hybridize. By this assay, it was possible to detect in the order of 10(4) bacteria in fish meat homogenate in 10 h.

Base Sequence↗
PubMed1993

Molecular mechanism of the regulation of expression of plasmid-encoded mouse bacteremia (mba) genes in Salmonella serovar Choleraesuis.

The regulation of mouse bacteremia genes (mba genes) encoded by a 6.4 kb region on the 50 kb virulence plasmid (pKDSC50) of Salmonella serovar Choleraesuis was analyzed. The genes mba1, mba2, mba3, and mba4, are arranged in this order, and form a cluster located in the 6.4 kb mba region. We prepared four antibodies, each specific for an individual Mba protein, using synthetic peptides as antigens. Their amino acid sequences were deduced from the DNA sequence of the corresponding mba genes. Each Mba peptide antiserum was able to recognize the corresponding Mba protein produced by Escherichia coli carrying a recombinant plasmid containing individual mba genes. When the recombinant plasmid contained all four mba genes (pMKD601), three Mba proteins (Mba2, Mba3, and Mba4) were identified by Western blotting analysis using Mba antisera. These proteins could not be detected when the recombinant plasmid lacked mba1 (pMKD201). Three species of mRNA for mba2, mba3, and mba4 with different chain length were detected from pMKD601 by Northern blot hybridization, and two start sites were identified by primer extension assay. Gel mobility shift assays demonstrated that Mba1 specifically bound to a fragment containing the start sites of mRNAs. The amino acid sequence of Mba1 had significant homology to the LysR family of DNA binding proteins, possessing a characteristic helix-turn-helix DNA binding motif. The present study provides clear evidence to show that the Mba1 protein binds to the promoter region of mba2, and positively regulates the expression of mba2, mba3, and mba4 genes.

Amino Acid Sequence↗
PubMed1993

The pectic polysaccharide from Bupleurum falcatum L. enhances immune-complexes binding to peritoneal macrophages through Fc receptor expression.

Binding of glucose oxidase-anti-glucose oxidase complexes (GAG), a model of immune complexes, to macrophages was enhanced by treatment with an acidic pectic polysaccharide, bupleuran 2IIb, from Bupleurum falcatum L. GAG binding to macrophages by bupleuran 2IIb increased in a dose-dependent fashion, and was abolished when the Pronase-treated macrophages were incubated with bupleuran 2IIb. The GAG binding enhancing activity of bupleuran 2IIb was reduced by periodate oxidation but not Pronase digestion of bupleuran 2IIb. When bupleuran 2IIb was digested with endo-polygalacturonase, the resulting enzyme resistant carbohydrate portion showed potent activity. Scatchard analysis indicated enhanced expression of the Fc receptor (FcR) on the surface by the action of bupleuran 2IIb. The enhancement of GAG binding by bupleuran 2IIb was inhibited by the presence of actinomycin D or cycloheximide. Bupleuran-2IIb-stimulated cells showed enhanced expression of both FcRI and FcRII mRNA, which were measured as PCR products. These results suggested that the endo-polygalacturonase resistant carbohydrate portion of bupleuran 2IIb is important for the expression of the activity, and that the activity of bupleuran 2IIb on GAG binding was mediated by receptors for polysaccharide on the cells. The up-regulation of the Fc receptor by bupleuran 2IIb was also suggested to mediate by de novo synthesis of the receptor protein.

Animals↗
PubMed1993

Purification of Bacillus subtilis spore coat protein by electrophoretic elution procedure and determination of NH2-terminal amino acid sequences.

Spore coat protein of Bacillus subtilis was purified by electrophoretic elution procedure. Solubilized coat protein components were separated on SDS-PAGE and the desired protein was recovered from the gel pieces under the optimal condition examined. Two purified polypeptides with molecular weights of about 40 kDa were obtained; each of them was in very closed size on SDS-PAGE, both retaining antigenic activity against anti-spore coat protein serum on immunoblot analysis. The N-terminal 23 and 30 amino acid sequences of them were determined, and they were not identical to each other and also not homologous in the sequences of coat proteins previously reported.

Amino Acid Sequence↗
PubMed1993

Gene transfer of herpes simplex virus type I thymidine kinase gene as a drug sensitivity gene into human lung cancer cell lines using retroviral vectors.

One of the recent strategies for gene therapy as a cancer control is the targeted introduction of a drug-sensitivity gene into tumor cells. We investigated the gene transfer of herpes simplex virus type I thymidine kinase (HSV-TK) gene as a drug-sensitivity gene into human lung cancer cell lines. We used a recombinant retroviral vector derived from Moloney murine leukemia virus (MuLV) as one of potential vectors for gene therapy. The amphotropic retroviral vector consisted of the HSV-TK gene and the neomycin-resistant gene under Rous sarcoma virus (RSV) promoter control. The antiherpes drugs, acyclovir (ACV) and ganciclovir (GCV), were chosen for testing the activity of HSV-TK that was transferred into human lung cancer cell lines. ACV and GCV are nucleoside analogs specifically converted by HSV-TK to a toxic form capable of inhibiting DNA synthesis. The cytotoxicity was determined by using a tetrazolium-based colorimetric assay (MTT assay). The results obtained from our experiments demonstrated that the retroviral vector-mediated HSV-TK gene transfer leads to ACV- and GCV-dependent cytotoxicity in human lung cancer cell lines, which were both small cell carcinoma and non-small cell carcinoma established from human specimens. These findings suggest that the gene transfer of HSV-TK gene into tumor cells would be one of the models for the use of gene therapy to control lung cancer.

Drug Resistance, Microbial↗
PubMed1993

Leakage of the fluorescent Ca2+ indicator fura-2 in smooth muscle.

The movement of a fluorescent intracellular Ca2+ indicator, fura-2, in smooth muscle was examined. Strips of rat and rabbit aortas and bovine trachea were loaded with the acetoxymethyl ester of fura-2 (fura-2/AM), followed by washing with normal physiological solution. Not only fura-2/AM but also fura-2 was detected in the washout solution. The amount of fura-2 in the cells, measured fluorometrically, decreased gradually during the washout. The decrease was fastest in rat aorta followed by rabbit aorta > bovine trachea. In rat aorta, fura-2 leakage was inhibited by an inhibitor of anion transport, probenecid, or by a decrease in bath temperature. The Ca2+ ionophore ionomycin (10 microM) increased the leakage of fura-2, which was not inhibited by probenecid, possibly because a high concentration of ionomycin nonselectively increased membrane permeability. These results suggest that fura-2/AM is cleaved to fura-2 in the cell which gradually leaked out of the cell mainly by an anion transport system. The amount of fura-2 in the cell seemed to be determined mainly by the rate of leakage of fura-2, which is the largest in rat aorta followed by rabbit aorta and bovine trachea.

Animals↗
PubMed1993

Synergistic effects of cyclic AMP-related vasodilators and the phosphatase inhibitor okadaic acid.

The phosphatase inhibitor okadaic acid at 100 nM slowly but completely inhibited high K(+)-induced contraction in the rat aorta (t1/2 = 118.9 min). High K(+)-induced contraction was partially inhibited (to 37-65%) by 1 microM forskolin, 100 microM dibutyryl cyclic AMP, 100 nM atrial natriuretic peptide, 1 microM nitroglycerin, 10 nM sodium nitroprusside, 300 pM nicardipine or 100 nM verapamil. The rate of relaxation due to okadaic acid became faster when the contraction was partially inhibited by these compounds. Augmentation of the relaxation was greater with forskolin and dibutyryl cyclic AMP than with the other inhibitors. These results support the suggestion that okadaic acid inhibits phosphatase to augment the phosphorylation due to cyclic AMP-dependent kinase, resulting in smooth muscle relaxation.

Animals↗
PubMed1993

Comparison of purified lens glutathione S-transferase isozymes from rabbit with other species.

Two glutathione S-transferase (GST) isozymes, GST-rl1 and GST-rl2, were purified from rabbit lenses and their properties were compared with those of other animals. GST-rl1 and GST-rl2 are dimeric enzymes whose subunit sizes are 24,000 and 21,500, respectively. The substrate specificities and inhibitor sensitivities of GST-rl1 and GST-rl2 are different from each other and from those of the isozymes from other animals. GST-rl1 immunologically crossreacted with the antibody against class mu GST (rat GST Yb1-Yb1), and GST-rl2 crossreacted with the antibody against class pi GST (rat GST Yp-Yp). N-Terminal amino acid sequences of GST-rl1 and GST-rl2 have great homology with other class mu and class pi enzymes, and thus indicate that they belong to class mu and class pi, respectively. Class pi GST-rl2 is inactivated by 1,2-naphthoquinone, an oxidized metabolite of naphthalene, but class mu GST-rl1 is insensitive to it. These results are similar to those of class pi pig lens GST and class mu bovine lens GST. Thus, the expression pattern of GST isozymes in lens varies with animal species, and may relate to their variation in sensitivity to oxidative stress.

Amino Acid Sequence↗
PubMed1993

Transduction of a drug-sensitive toxic gene into human leukemia cell lines with a novel retroviral vector.

To investigate the possibility of killing tumor cells by the expression of an exogenously introduced toxic gene, we have constructed a novel retroviral vector (LTRNL) which has the polyA signal deleted herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene. The vector becomes toxic by treating cells expressing HSV1-tk with the antiherpetic drugs acyclovir or ganciclovir (GCV). Cells of the human leukemia lines (K562, MEG-01) were infected with this vector and two transduced cell lines (K562/LTRNL, MEG-01/LTRNL) were established. Southern blot analysis confirmed the integration of the HSV1-tk transgene in these cells and Northern blot analysis exhibited the expression of 4.8-kb viral mRNA containing the HSV1-tk gene. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for the in vitro cytotoxic effects of GCV to these cells demonstrated that concentrations of about 2.5 microM for K562/LTRNL and 1.25 microM for MEG-01/LTRNL cells resulted in 50% inhibition of cell growth after 72 hr. Subcutaneous tumors of MEG-01/LTRNL in KSN nude mice, but not those of uninfected MEG-01 cells, showed durable regressions after exposure of the mice to 40 mg/kg of GCV given subcutaneously once a day for 15 days. This study indicates that the LTRNL-infected human leukemia cells exhibit inducible susceptibility to GCV.

Acyclovir↗
PubMed1993

[Lp(a) lipoprotein in patients on maintenance hemodialysis--a study from apo(a) isoform].

It has been suggested that lipid abnormalities may be involved in the development of cardiovascular disorders in patients on maintenance hemodialysis (HD). Hypertriglyceridemia commonly accompanies this condition and is associated with decreased concentrations of HDL-cholesterol. Recent clinical interest has been paid to the disturbances of Lp(a) and apo (a) isoform in relation to cardiovascular disorders. Although high concentrations of Lp(a) are associated with ischemic heart disease, we are unaware of the availability of such data concerning patients on maintenance hemodialysis. We therefore compared levels of Lp(a) and the frequency of occurrence of their isoforms in 310 patients (chronic glomerulonephritis, N = 221; diabetic nephropathy, N = 77; polycystic kidney disease, N = 12) on maintenance hemodialysis and in 212 normal subjects. The following results were obtained. (1) HD patients showed significantly elevated levels of Lp(a) compared to normal subjects. (2) Studies of apo (a) isoform showed that HD patients showed high frequency of S2 and low frequency of S4. (3) HD patients, especially long-term patients, showed high frequency of double band (S2/S3). (4) There were no significant differences in the levels of Lp(a) and in the frequency of apo(a) isoform among 3 different etiological studies.

Adolescent↗
PubMed1992

Metabolic effects of short-chain ceramide and glucosylceramide on sphingolipids and protein kinase C.

Recent studies have identified a potential role for glucosylceramide (GlcCer) in growth promotion and hormonal signalling. In an effort to demonstrate a growth-promoting activity of GlcCer, we prepared a GlcCer having a short-chain acid (octanoyl), in the belief that this glycolipid could be absorbed more readily and more uniformly by cultured cells. By using a mixture of two specific lecithins, dioleoylglycerophosphocholine and 1-stearoyl-2-palmitoylglycerophosphocholine, we were able to prepare dispersions containing a high molar proportion of the GlcCer and the related ceramide, octanoyl sphingosine. Unexpectedly, both sphingolipids inhibited protein and DNA synthesis in Madin-Darby canine kidney cells and produced large increases in the levels of the natural lipids, GlcCer, ceramide, free sphingosine, and an amine that may be glucosylsphingosine (GlcSph). Decreases were seen in the level of sphingomyelin and the proportion of protein kinase C in the cell membranes. The level of lactosylceramide was diminished by octanoyl GlcCer but elevated considerably by octanoyl sphingosine. Diacylglycerols were increased by the lecithins in the liposomes, but the exogenous sphingolipids had no effect. Octanoyl sphingosine labeled in the sphingoid base yielded labeled GlcCer and sphingomyelin labeled in both long-chain and very-long-chain fatty acid families, as well as the octanoyl version. The two families of ceramides, however, had relatively little radioactivity. Some of these changes are attributed to rapid hydrolysis of the added lipids with the formation, particularly from the ceramide, of sphingosine and its anabolic metabolite, GlcSph. Several observations support the idea that the octanoyl sphingosine inhibited the phosphocholinetransferase that synthesizes sphingomyelin while the octanoyl GlcCer inhibited GlcCer beta-glucosidase and GlcCer galactosyltransferase. The use of unnatural short-chain lipids in the study of cell growth and other phenomena may result in unexpected changes in related metabolites and the findings from such experiments should therefore be interpreted cautiously.

Animals↗
PubMed1992

Rapid DNA typing utilizing immobilized oligonucleotide probe and a nonradioactive detection system. Application to HLA-DR typing of the Japanese population.

We established a rapid and simple method of HLA-DR genotyping, and applied it for analysis of the Japanese population. Our method includes rapid preparation of DNA samples from buccal mucosa, incorporation of biotin-dATP into DRB genes during amplification by the polymerase chain reaction, hybridization with sequence-specific oligonucleotide (SSO) probes immobilized on nylon membranes via poly (dT) tails, and detection of the hybridization signal as chemiluminescence. We carried out DR typing of 30 Japanese donors using 20 different immobilized SSO probes, and obtained unambiguous typing signals showing perfect correlation with their serologic DR types. The genotyping also enabled us to identify several DR types unique to the Japanese population, such as DRw12b (DRB1*1202), DRw14c (DRB1*1405), and serology blank type, DR'JX6' (DRB1*1403). The method presented here would be suitable for routine DR typing in tissue-typing laboratories.

Base Sequence↗
PubMed1992

High-performance liquid chromatography-thermospray mass spectrometry of hydroperoxy polyunsaturated fatty acid acetyl derivatives.

A method for the analysis of hydroperoxy polyunsaturated fatty acids was developed. The hydroperoxy groups were acetylated by acetic anhydride, and the mixture was partially purified on a Sep-Pak C18 cartridge and analysed by high-performance liquid chromatography with thermospray mass spectrometry. Generally, the base ion, [M+H - n(60)]+ or [M+H - n(60) - n(H2O)]+, is produced through elimination of acetic acid or water (n = number of hydroperoxy groups). The detection limit for these derivatives was ca. 1 pmol at concentrations of hydroperoxy polyenoic acids prior to derivatization. Using this method, many hydroxy and hydroperoxy polyunsaturated fatty acid derivatives could be detected simultaneously within 30 min on a selected-ion monitoring detection chromatogram without a gradient system. The assay was successfully applied to hydroxy and hydroperoxy polyunsaturated fatty acids from an incubation mixture of rat brain homogenate to which polyunsaturated fatty acids had been added.

Acetylation↗
PubMed1992

High-performance liquid chromatography-thermospray mass spectrometry of hydroxy-polyunsaturated fatty acid acetyl derivatives.

A method for the determination of hydroxy-polyenoic acids has been developed. Hydroxy groups of hydroxy-polyunsaturated fatty acid were acetylated by acetic anhydride, and the mixture was partially purified on a Sep-Pak C18 cartridge and analysed by high-performance liquid chromatography combined with thermospray mass spectrometry. Using this method, fifteen kinds of hydroxy-polyunsaturated fatty acid derivative could be detected simultaneously within 30 min on a selected-ion monitoring detection chromatograph without a gradient system. Generally, the base ion, [M + H - n(60)]+, is produced through elimination of acetic acid (n = number of the hydroxy group of hydroxy-polyunsaturated fatty acid). The detection limit for these derivatives was ca. 0.2 pmol at the levels of hydroxy-polyenoic acids prior to derivatization. They could be analysed in the range 0.5-25 pmol. The assay was successfully applied to hydroxy-polyunsaturated fatty acids from an incubation mixture of rat brain homogenate to which polyunsaturated fatty acid was added.

Animals↗