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Biomedical subjects

Y Sugahara

Publications and source records attributed to Y Sugahara.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cytogenetic analysis of de novo acute myeloid leukemia with trilineage myelodysplasia in comparison with myelodysplastic syndrome evolving to acute myeloid leukemia.

Characteristics of karyotypes were analyzed in de novo acute myeloid leukemia (AML) with trilineage myelodysplasia (AML/TMDS) at initial diagnosis and compared with myelodysplastic syndrome (MDS) cases that had evolved to AML (MDS/AML). Abnormal karyotypes were seen in 11 of 19 patients with AML/TMDS and 13 of 16 MDS/AML cases. Trisomy 8 was observed in 3 AML/TMDS cases as a sole anomaly and was also present in 3 MDS/AML cases but not as a sole finding. Although MDS/AML frequently displayed monosomies or long-arm deletions of chromosome 5, 7 and 9, only one case exhibited long-arm deletion (of chromosome 7) in AML/TMDS. Two or more chromosome aberrations were found in some cases in both groups. These findings suggest that AML/TMDS had passed through several preleukemic stages at diagnosis, as has been well documented in MDS and MDS/AML. Additionally, clonal evolution may have already occurred in AML/TMDS, as MDS transformed to AML is associated with clonal evolution.

Acute Disease↗
PubMed1998

[Drug susceptibility of clinically isolated Helicobacter pylori].

Between January 1995 and March 1997, 78 Helicobacter pylori strains were isolated from patients with gastritis and gastric ulcer and their drug-susceptibilities to 8 antimicrobial agents and 3 anti-ulcer drugs were determined. Imipenem was the most active agent and its MICs to all the strains tested were lower than 0.013 microgram/ml. Amoxicillin, cefaclor and minocycline were active against H. pylori with MIC90s of 0.05 microgram/ml, 0.78 microgram/ml and 0.39 microgram/ml, respectively, and no resistant strains against these drugs were isolated. However, resistant strains to clarithromycin (isolation frequency: 9%), erythromycin (13%), ofloxacin (8%) and metronidazole (13%) were isolated. Triple, double and single resistant strains to above 4 antimicrobial agents were noted. No quadruple resistant strain was isolated. Frequencies of those resistance patterns were 14.3% (triple), 28.6% (double), and 57.1% (single), respectively. Seven erythromycin-resistant strains were shown to be cross-resistant to clarithromycin but 3 erythromycin-resistant strains were susceptible to clarithromycin. It seems likely that this phenomenon is caused by the fact that clarithromycin is more active to H. pylori than erythromycin. The MIC90 value of lansoprazole was lower than those of omeprazole and famotidine.

Amoxicillin↗
PubMed1997

Adaptation of equine herpesvirus 1 to unnatural host led to mutation of the gC resulting in increased susceptibility of the virus to heparin.

Heparin extensively inhibited infection of MDBK cells by equine herpesvirus 1 (EHV-1) strains adapted to bovine cells or hamsters, while the reagent merely reduced infectivity of strains passaged only in equine cells. The gC of two strains adapted to non-equine cells seemed to have higher affinity for heparin, although the reagent bound to both the gC and gB of all strains tested. Amino acid substitutions of the gC of the EHV-1 strains adapted to non-equine cells converged on the hydrophilic regions, amino acid residues 92 to 175, resulting in the glycoprotein becoming more cationic. These results indicate that these hydrophilic regions of the gC may be responsible for binding to heparin.

Adaptation, Physiological↗
PubMed1997

[Serotypes and drug susceptibility of Pseudomonas aeruginosa isolated from clinical specimens].

Between January, 1982 and December, 1994, 236 Pseudomonas aeruginosa strains were isolated from clinical specimens at our division, and were tested for serotypes and drug-susceptibilities to 15 antibiotics. Serotype G strains were isolated at the highest frequency (32.6%), and followed by strains of serotype B (15.7%), A (11.9%), E (9.3%), I (7.2%), F and M (5.5%), non-typable (5.1%), D (3.4%), H (2.1%), C and K (0.8%). We examined the changes of isolation frequencies of different serotypes annually. Isolation frequencies of serotypes E and F showed tendency to decrease, whereas serotype I has been isolated increasingly year by year. MIC90's of the 15 antibiotics were as follows, tosufloxacin: 0.78 microgram/ml, biapenem (BIPM) and ofloxacin (OFLX): 3.13 micrograms/ml, imipenem (IPM), ceftazidime, cefozopran, cefsulodin and gentamicin: 6.25 micrograms/ml, aztreonam and amikacin: 12.5 micrograms/ml, piperacillin, cefoperazone and minocycline (MINO): 25 micrograms/ml, fosfomycin: > 100 micrograms/ml and chloramphenicol: > 200 micrograms/ml. MIC90'S of IPM, BIPM, MINO and OFLX increased 4-fold from stage I (1982-1987) through stage III (1992-1994) and the isolation frequency of drug-resistant strains increased year by year. In other words, antibiotic resistant strains appeared increasing with time. No relationship between serotypes and drug-resistance were observed.

Anti-Bacterial Agents↗
PubMed1996

Construction of human factor IX expression vectors in retroviral vector frames optimized for muscle cells.

Development of a highly refined human factor IX (hFIX) expression vector system is critical for establishing a durable hemophilia B gene therapy. Here we report construction of a series of retroviral vectors and identification of an optimal basic structure and components for expressing hFIX in skeletal muscle cells. These vectors, which are derived from Moloney murine leukemia virus (MoMLV) with its enhancer sequence in the 3' long terminal repeat (LTR) deleted, contained internal hFIX expression units inserted in forward configuration without or with a viral vector intron sequence (pdL or pdLIn vector frame, respectively) or in inverted configuration without a viral vector intron sequence (pdLi frame). Internal expression units contained a hFIX cDNA or hFIX minigene (hIXm1 or hIXm2) derived from the hFIX cDNA by insertion of a shortened first intron sequence of the hFIX gene. Regardless of the promoter and vector frame used, both hIXm1 and hIXm2 gave 10- to 14-fold higher hFIX expression compared to those with hFIX cDNA. Internal hFIX transcriptional control units of these vectors were composed of various promoters linked with or without the muscle creatine kinase enhancer (Me) sequence. Promoters tested included those of alpha-actin (alpha A775), beta-actin (beta A280), cytochrome oxidase (CO1250 and CO650), myogenin (Mg1031 and Mg353), and Rous sarcoma virus (RSV). beta A200, which was derived from beta A280 by eliminating potential polyadenylation sites, was also tested. As extensively examined with the myogenin promoter, presence of one or multiple copies of Me in the vectors elevated the expression activity in myotubes by 4.5- to 19-fold over those without Me, but not significantly in myoblasts. Similar enhancements in expression activity with Me were also observed with other promoters, except those of RSV and CO. The latter two showed only modest enhancements in the presence of Me. As assayed with myotubes in culture, the general order of hFIX expression activity of various promoters with four copies of Me in the three different vector frames was beta A280 approximately beta A200 > Mg353 > Mg1031 approximately RSV approximately CO650 approximately alpha A775 > CO1250. One exception was that CO650 showed significantly less activity in pdLi-type vectors than in the pdLIn vectors. Based on the systematic analyses of various structural components, a group of pdLi vectors consisting of beta A200, two to four copies of Me, and hIXm2 was identified to have the optimal basic vector structure to be used in retrovirus for hFIX expression in differentiated skeletal muscle cells. The present studies provide the critical first step for establishing a highly refined hemophilia B gene therapy based on skeletal muscle-targeted hFIX gene transfer.

Animals↗
PubMed1996

Isolation and characterization of canine factor IX.

Canine plasma factor IX was purified to homogeneity by a combination of barium citrate precipitation and three-step column chromatographies of DEAE sepharose, heparin agarose and a monoclonal antifactor IX antibody-linked agarose. Canine factor IX has an apparent molecular size of 61 kDa, which is slightly smaller than that of human factor IX, as determined by denatured polyacrylamide gel electrophoresis. Its amino acid composition, amino-terminal and carboxyterminal amino acid sequences agreed well with those predicted from the reported cDNA. Unlike purified human factor IX, canine factor IX preparation often showed a discrete smaller molecular species (approximately 50 kDa) which was generated by a specific proteolytic cleavage between Arg310 and Val311. When purified canine factor IX was utilized as a standard for enzyme linked immunosorbent assay, the concentration of canine factor IX in the pooled normal dog plasma was determined to be 5.3 micrograms/ml with 11.2% carbohydrate content (or 4.7 micrograms/ml for its polypeptide chain moiety). Concentration of plasma factor IX antigen was measured in six severely affected, unrelated hemophilia B dogs. Four had factor IX antigen of less than 1% of the normal, and two had undetectable levels. The latter two had gross molecular abnormalities in their factor IX genes. Three obligate carrier females had variable but proportionately reduced factor IX antigen and factor IX coagulant activity levels. These results provide a quantitative method for measuring canine factor IX antigen which is a prerequisite for studying hemostasis and development of gene transfer approaches in the canine model of hemophilia B.

Animals↗
PubMed1995

Transovarian transmission of a foreign gene in the silkworm, Bombyx mori, by Autographa californica nuclear polyhedrosis virus.

We introduced a firefly luciferase gene, expressed under control of Drosophila heat shock protein gene promoter, into Autographa californica nuclear polyhedrosis virus (AcNPV). When the 5th instar larvae of the silkworm, Bombyx mori, were inoculated with the recombinant virus, luciferase activities were detected in the virus-infected larvae and pupae, and in the newly hatched larvae of the next generation. PCR amplification and Southern blot hybridization analysis demonstrated that the luciferase gene was transmitted through at least the F2 generation. In addition, the V-cathepsin gene, encoding a cysteine protease of AcNPV, was also detected in the DNA of all individuals of the F2 generation. These results show that AcNPV can be utilized as vector for the transovarian transmission of foreign genes in the silkworm.

Animals↗
PubMed1995

Evolution of severe aplastic anemia to myelodysplasia with monosomy 7 following granulocyte colony-stimulating factor, erythropoietin and high-dose methylprednisolone combination therapy.

A 19-year-old man was diagnosed as having severe aplastic anemia and received high-dose methylprednisolone treatment without hematological response. A second course of high-dose mPSL treatment together with granulocyte colony-stimulating factor (G-CSF) plus erythropoietin (EPO) was then started and resulted in trilineage blood cell response. Ten months after the combination therapy thrombocytopenia developed and cytogenetic analysis showed 45,XX,-7, indicating an evolution to myelodysplastic syndrome (MDS) associated with monosomy 7.G-CSF and EPO treatment together with immunosuppression may be an effective therapy in SAA patients, but such a therapy may increase the risk of evolution to MDS.

Adult↗
PubMed1994

Complement receptor type 1 (CR1) deficiency on neutrophils in myelodysplastic syndrome.

We present a patient with myelodysplastic syndromes (MDS) whose neutrophils exhibited defective expression of complement receptor type 1 (CR1). A 73-year-old man was admitted with an evolution of MDS from RA into RAEBT according to the FAB classification of MDS. The neutrophil alkaline phosphatase (NAP) score was zero. The surface expression of membrane effector molecules on neutrophils was determined by indirect immunofluorescence using flow cytometry and monoclonal antibodies. The expression of CR1 on neutrophils as identified by staining with CD35 was defective in the patient, and the expression of other complement receptors (CR3 and CR4), Fc receptors and adhesion molecules was normal. CR1 deficiency and defective NAP score on neutrophils in the patient might account for impairment of common storage pool, presumably novel intracellular secretory vesicles.

Aged↗
PubMed1994

Compound heterozygous protein C deficiency caused by two mutations, Arg-178 to Gln and Cys-331 to Arg, leading to impaired secretion of mutant protein C.

The protein C gene in a patient apparently homozygous for protein C deficiency was analyzed. Two different point mutations, each located in a different allele, were detected to reveal that the patient is a compound heterozygote. Mutation of Arg-178 (CGG) to Gln (CAG) [mutation I] was detected in exon VII, in the vicinity of activation peptide cleavage site by thrombin. Mutation of Cys-331 (TGC) to Arg (CGC) [mutation II] was found in exon IX, at one of the sites involved in disulfide bond formation in the catalytic domain of the heavy chain. The alteration of Cys-331 to Arg disables the formation of the disulfide bond and would alter the protein conformation. Transient expression assays using COS-7 cells transfected with protein C expression vectors containing each one of these two mutations suggested that each of the two mutations would lead to the protein C deficiency by an impairment of secretion of the respective mutant proteins.

Alleles↗
PubMed1992

Protein C deficiency Hong Kong 1 and 2: hereditary protein C deficiency caused by two mutant alleles, a 5-nucleotide deletion and a missense mutation.

We characterized a mutant protein C gene from an individual with no detectable protein C antigen in blood plasma. Southern blot hybridization analysis with human protein C cDNA demonstrated neither gross deletion nor rearrangement of the gene. Sequencing all the exons and exon-intron boundaries of the gene except the 3' noncoding region showed two mutant alleles. The one, derived from the mother, represents a deletion of 5 nucleotides (nt) (CCCGC) in the end of exon VI (mutation I), predicted to result in the generation of a new stop codon due to a reading frameshift and the premature termination of translation. The other, derived from the father, represents a point mutation (G to A) in exon IX (mutation II), resulting in an amino acid substitution, Gly-376(GGC) to Asp(GAC), in the catalytic domain of the protein. Allele-specific oligonucleotide probe hybridization confirmed the presence of the two mutations. Mutation I would result in a truncated polypeptide of 169 amino acid residues that lacks the heavy chain. Mutation II gives rise to an alteration of a highly conserved amino acid, Gly-376. These data indicate that this patient is a compound heterozygote of the two mutant alleles, each one inherited from each parent. Transient expression assays using COS-7 cells transfected with mutated protein C expression vectors suggested that each of the two mutations leads to the protein C deficiency by causing an impairment of secretion of the respective mutant proteins.

Amino Acid Sequence↗
PubMed1991

BHV-1 adsorption is mediated by the interaction of glycoprotein gIII with heparinlike moiety on the cell surface.

The gIII glycoprotein of bovid herpesvirus 1 (BHV-1) has been shown to mediate the adsorption of the virions to cells (K. Okazaki, E. Honda, T. Minetoma, and T. Kumagai, 1987, Arch. Virol. 97, 297-307). In this study, the cellular receptor for BHV-1 was investigated. Addition of heparin to the virus inoculum and treatment of the cells with heparinase prevented the virus from adsorbing to and infecting the cells. Of the major glycoproteins of BHV-1 only gIII was found to bind specifically to heparin. The binding of gIII was inhibited by a monoclonal antibody against antigenic site Ia, which interferes with the adsorption of the virus. These findings indicate that the virus adsorption to cells is mediated by interaction of the gIII antigenic site Ia with a heparinlike moiety on the cell surface, which serves as a receptor for BHV-1.

Adsorption↗
PubMed1989

Hereditary alpha 2-plasmin inhibitor deficiency caused by a transport-deficient mutation (alpha 2-PI-Okinawa). Deletion of Glu137 by a trinucleotide deletion blocks intracellular transport.

alpha 2-Plasmin inhibitor is the most important physiological inhibitor of fibrinolysis; hence, its deficiency results in a severe hemorrhagic diathesis. We have cloned and characterized a mutant alpha 2-plasmin inhibitor gene from an individual homozygous for the deficiency. By sequencing all the coding exons and exon-intron boundaries of the gene, a trinucleotide deletion in exon VII that results in deletion of Glu137 was identified. We have designated this variant as alpha 2-plasmin inhibitor Okinawa. Using DNA samples amplified with the polymerase chain reaction, hybridization analysis by oligonucleotide probes confirmed the presence of this mutation in all the affected family members, including both parents. To elucidate the mechanism by which this mutation leads to the deficiency, a eukaryotic expression plasmid for alpha 2-plasmin inhibitor containing this mutation was constructed and transfected into COS-7 cells for transient expression analysis. Immunoprecipitation analysis and enzyme-linked immunosorbent assay revealed that the mutant alpha 2-plasmin inhibitor synthesized is mostly retained within the cells as an endoglycosidase H-sensitive form, and only a small portion of it is secreted into the medium as a neuraminidase-sensitive form. These results suggest that the Glu137 deletion leads to the alpha 2-plasmin inhibitor deficiency by causing a block in its intracellular transport from the endoplasmic reticulum to the Golgi complex.

Animals↗
PubMed1989

Restriction fragment length polymorphism caused by a deletion involving Alu sequences within the human alpha 2-plasmin inhibitor gene.

A restriction fragment length polymorphism within the human alpha 2-plasmin inhibitor gene has been detected by Southern blot hybridization using an alpha 2-plasmin inhibitor cDNA probe. This restriction fragment length polymorphism can be attributed to the presence of two alleles, A and B, that are distributed in Hardy-Weinberg equilibrium with frequencies of 73.5% and 2.65%, respectively, in 66 unrelated Caucasian individuals or with frequencies of 51.0% and 49.0%, respectively, in 50 unrelated Japanese individuals. The minor allele, B, is due to a deletion of about 720 base pairs in intron 8 of the alpha 2-plasmin inhibitor gene. Sequence analysis of the deletion junction in allele B and the corresponding regions of allele A demonstrated the presence of oppositely oriented Alu sequences at the 5' and 3' deletion boundaries. These data suggest that this restriction fragment length polymorphism was caused by intrastrand recombination between Alu sequences.

Alleles↗
PubMed1989

DNA rearrangement and restriction fragment length polymorphism within the first BCR intron in Philadelphia-positive acute leukemia.

In some patients with Philadelphia (Ph)-chromosome positive acute lymphoblastic leukemias, breakpoints on chromosome 22 are reported to occur within the first BCR intron. To analyze the breakpoints in chromosome 22 of Ph-positive acute leukemia patients without rearrangement of the 5.8 kb bcr, we cloned the 3' part of the first BCR intron using a synthetic DNA probe. During the course of study, we mapped the region of the deletion/insertion of 1 kb that causes a restriction fragment length polymorphism (RFLP) and found a racial difference in the frequencies of the alleles giving rise to this RFLP. Analyses of the patients' samples indicated that breakpoints were located within the 8.5 kb EcoRl fragment of the first BCR intron in two of five Ph-positive acute leukemia patients. The data, together with the previous reports, indicate that breakpoints within this approximately 50 kb intron are widely scattered, in contrast to those confined within the 5.8 kb bcr in chronic myelogenous leukemias.

Acute Disease↗