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Biomedical subjects

J Mizoguchi

Publications and source records attributed to J Mizoguchi.

16 recordsLinked to original sources
PubMed1992

A non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridinetriphosphate on primer-immobilized microtiter plates.

We developed a non-radioisotopic (non-RI) reverse transcriptase assay (RTA). The reverse transcriptase (RT) incorporates biotin-11-deoxyuridine-triphosphate (bio-dUTP) using a poly(rA) template hybridized with oligo(dT) primer that is immobilized on the surface of a 96-well microtiter plate. This assay is thus semi-automated by adapting it to an ELISA testing format. The incorporation of bio-dUTP was enhanced by adding cold dTTP to the reaction mixture, optimally in a molar ratio 4:1 (dTTP:bio-dUTP). This non-RI RTA is more sensitive than the conventional RI assay for the detection of purified Rous-associated virus 2 (RAV-2) and of human immunodeficiency virus type 1 (HIV-1) lysate. Because of its simple procedure, higher sensitivity and non-use of RI materials, the assay can be utilized not only for virological studies but also for routine safety screening of biological products for retroviral contamination.

Avian Leukosis Virus↗
PubMed1991

Tissue specific expression of the plasma glutathione peroxidase gene in rat kidney.

Rat plasma glutathione peroxidase (GSH-Px) was purified 1,400-fold from rat serum by a combination of phenyl Sepharose, DEAE Sephacel, blue Sepharose and Sephacryl S-200 column chromatographies. The purified GSH-Px migrated as a single band corresponding to a molecular weight of 22,500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was used for the immunization of chickens to obtain a specific antibody and for determination of its amino acid sequence. Two overlapping cDNA clones for rat plasma GSH-Px were isolated from a placental cDNA library. The composite nucleotide sequence is 1,529 base-pairs long and encodes 226 amino acids. The deduced amino acid sequence completely coincided with the sequences of five individual peptide fragments derived from the purified plasma GSH-Px on digestion with lysyl endopeptidase. In order to identify the tissue(s) generating this plasma GSH-Px, immunoblot analysis was performed on homogenates prepared from 13 tissues. A single immunoreactive band of 22.5 kDa, corresponding to plasma GSH-Px, was detected for the kidney homogenate. A much fainter band was observed for the lung preparation, but liver, spleen, bone marrow, and other tissues examined were negative. Northern blot analysis further revealed that the expression level of the plasma GSH-Px gene was high in kidney and low in lung. No transcript was detected in liver or spleen. These results indicate that plasma GSH-Px is predominantly synthesized and secreted by renal cells.

Amino Acid Sequence↗
PubMed1991

Serum-free cultivation of anchorage-dependent cells on microcarrier: effective production of human macrophage colony-stimulating factor.

For the purpose of establishing a large scale production process of biologically active substances by cultivation of anchorage-dependent mammalian cells, basic studies were carried out on the following items; establishment of a new cell line and derivation of high productivity; construction of optimal serum-free medium; optimization of cultivation method using microcarrier in serum-free medium; and establishment of purification process. The cell line, TRC-29SF, used in this study was newly established from human renal carcinoma with a function of producing macrophage colony-stimulating factor constitutively. Improvement of M-CSF productivity upon TRC-29SF cell line was performed by M-CSF gene amplification with dhfr-MTX system and by truncation of membrane-binding amino acid sequence by recombinant DNA technique. Two kinds of serum-free media, IPEG-85 and IREG-89, were formulated for the growth of TRC-29SF cell and its transformant, respectively. A new cell-adhesion method which permits homogeneous attachment to microcarrier in short term was developed by equalizing the sedimentation velocity between cells and microcarrier by addition of 7% Ficoll into the medium. High cell density perfusion culture of TRC-29SF cells was achieved by microcarrier method using IPEG-85 medium, and final cell density reached over 10(7) cells/ml. Based on the results obtained, long-term perfusion cultures were performed using Mn10-5 and Mn10-5/R600 cell lines, which were created by M-CSF gene transfection and amplification. We found that the productivity of M-CSF per cell began to decrease from the end of logarithmic growth phase. Long-term cultivation with high productivity was accomplished by perfusing medium containing 2 mM sodium butyrate. Purification process for M1-CSF from the culture supernatant of transformed cell line was also established.

Amino Acid Sequence↗
PubMed1990

Primary structure of human plasma glutathione peroxidase deduced from cDNA sequences.

Human plasma glutathione peroxidase (GSHPx) has been shown to be a selenium-containing enzyme immunologically distinct from cellular GSHPx. Oligonucleotide probes, based on the partial amino acid sequence of plasma GSHPx, were synthesized and used to screen a human placenta cDNA library. Nucleotide sequence analysis of the obtained clones revealed that GSHPx consisted of a 678-base pair open reading frame coding for a 226-amino acid polypeptide with a Mr of 25,389. About 50% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of the peptides in a lysine endopeptidase-digest of the purified enzyme. The amino acid sequence exhibited only 44% homology with that of human cellular GSHPx. Northern blot analysis revealed a single transcript of 2.2 kilobases in the poly(A)+ RNA fractions of human placenta and HepG2 (a human hepatic cell line), but not that of human liver and endothelial cells.

Amino Acid Sequence↗
PubMed1990

Molecular cloning of cDNA coding for rat plasma glutathione peroxidase.

The plasma glutathione peroxidase (PGSH-PO), which is different from erythrocyte glutathione peroxidase (EGSH-PO) in immunochemical property and substrate specificity, was purified from male Wistar rat serum. The amino acid sequence of 5 independent peptides were determined and a cDNA clone for this enzyme was isolated from placental cDNA library. The nucleotide sequence of the cDNA revealed that, similar to EGSH-PO cDNA, the seleno-cysteine was genetically encoded by "TGA" codon. On comparing the nucleotide sequences of EGSH-PO and PGSH-PO, no significant homology was found in the vicinities of "TGA" codons of both enzymes.

Amino Acid Sequence↗
PubMed1989

Development and distribution of the major pollen allergen (Cry j I) in male flower buds of Japanese cedar (Cryptomeria japonica).

We investigated the production of the major pollen allergen [Cry j I] of Japanese cedar (Cj) in the course of male flower bud development. We found that most of the pollen was at the tetrad stage in early September, and they developed to the mature stage in mid-October (1987) or early November (1988). Large amounts of Cry j I seemed to be extractable to ABS solution from the mature stage pollen but not from the pollen at other stages, that is, the tetrad and immature stages. Mature pollen could be ruptured with ammonium bicarbonate buffer, but tetrad and immature pollen could not. By immunofluorescent technique, antigen Cry j I was detected in the pollen only at the mature stage of Cj pollen development. Therefore, we think that Cry j I is produced at the time of pollen maturation.

Allergens↗
PubMed1987

Decrease in amylase (EC 3.4.21.4) synthesis in lactating rats.

The amylase (EC 3.4.21.4) and trypsin (EC 3.2.1.1) activities in the pancreas in rats during pregnancy, lactation and after the weaning period, and the secretory responses to a secretagogue (caerulein) in the exocrine pancreas of lactating rats were measured. Trypsin activity increased as lactation progressed and reached twice that of unmated rats in the second half of the lactation period. The amylase activity fell before parturition and failed to recover even after the start of lactation and was significantly decreased throughout the lactation period. The total amount of pancreatic juice produced in the lactating rats was significantly greater than that of unmated rats; the amylase output was significantly less than that of unmated rats. When the pups were removed, amylase activity in the pancreas returned to the value in unmated rats. Furthermore, the amylase activity in lactating rats receiving a daily injection of insulin significantly exceeded that of normal lactating rats. These results indicate that the decrease in amylase activity in lactating rats is due to the reduction of amylase synthesis and there is a possibility that insulin is required for normal or elevated rates of amylase synthesis in lactating rats.

Amylases↗
PubMed1979

Mechanism of synergistic action of a combination of ampicillin and dicloxacillin against a beta-lactamase-producing strain of Citrobacter freundii.

The mechanism of synergistic activity of a combination of ampicillin and dicloxacillin was studied on beta-lactamase-producing Citrobacter freundii GN346 and its derived beta-lactamaseless mutant GN346/16. The synergistic activity was exhibited against the parent strain but not against the mutant strain. Precultivation of the parent strain with the combination reduced the amount of the subsequent binding of [14C]penicillin G to the membrane fraction from the treated cells, but no reduction was observed in the case of cells treated with ampicillin or dicloxacillin alone. On the other hand, the amount of binding of [14C]penicillin G to the membrane fraction from the mutant strain was reduced by ampicillin treatment alone. These results clearly indicated that dicloxacillin inhibited the beta-lactamase activity produced by the parent strain, and, consequently, ampicillin can penetrate through the outer membrane and periplasmic beta-lactamase barrier into its target sites on the cytoplasmic membrane.

Ampicillin↗
PubMed1978

[Synergistic effect of ampicillin and dicloxacillin on penicillin and cephalosporin-susceptible Escherichia coli (author's transl)].

An effect of a combination of ampicillin (ABPC) and dicloxacillin (MDIPC) on penicillins and cephalosporin-susceptible E. coli was determined. Minimum inhibitory concentrations of the combined drug were as same as that of ABPC, but bacterial growth was inhibited by the combined drug stronger than ABPC, low activity of beta-lactamase was detected in sonicated E. coli 0.225 cells, and this activity was inhibited by relatively low concentration of MDIPC. We considered that a weak synergism of the combined drug might attribute to the inhibition of the beta-lactamase activity by MDIPC.

Ampicillin↗
PubMed1977

On the mode of action of a new antifungal antibiotic, aculeacin A: inhibition of cell wall synthesis in Saccharomyces cerevisiae.

The mode of action of a new antifungal antibiotic, aculeacin A, was studied with the cells of Saccharomyces cerevisiae. In the presence of aculeacin A, the distinct decrease of viable cells was observed. The most of cells treated with aculeacin A lysed with releasing intracellular substances at the tips of their buds. This lysis was considered to be due to the inhibition of cell wall synthesis, because the incorporation of glucose into the cell wall glucan was significantly reduced. Aculeacin A also had a weak activity to burst the protoplasts of S. cerevisiae at a relatively high concentration.

Antifungal Agents↗