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Biomedical subjects

T Mizukami

Publications and source records attributed to T Mizukami.

At least 73 records · Page 4Linked to original sources
PubMed1990

Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium.

A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a good host cell line for recombinant DNA technology. We previously reported the expression of human beta-interferon (beta-IFN) in Namalwa KJM-1 (Miyaji, 1989a). The utility of Namalwa KJM-1 for expression of foreign genes was further examined. As a target gene to be expressed, human lymphotoxin (hLT) cDNA was used. It was engineered for expression in Namalwa KJM-1 using a simian virus 40 (SV40)-based expression vector pAGE107 (Miyaji, 1989a). It contains all components necessary for the expression of cDNA in mammalian cells. The expression vector was introduced into Namalwa KJM-1 by electroporation. Among the transformants, clone 7 was further examined for the expression of hLT in serum-free medium. The production level of hLT was augmented with the increase of the cell density. Thus it was further indicated that Namalwa KJM-1 is useful for production of foreign gene products.

Cell Line↗
PubMed1990

Efficient expression of human beta-interferon in Namalwa KJM-1 cells adapted to serum-free medium by a dhfr gene coamplification method.

We previously reported the expression of human beta-interferon (beta-IFN) (Miyaji et al., 1989) and human lymphotoxin (Miyaji et al., 1990) in Namalwa KJM-1 cells adapted to serum-free medium. To establish an efficient gene expression system, a dihydrofolate reductase (dhfr) gene coamplification method was applied to this cell line. A beta-IFN expression plasmid was introduced with a dhfr expression plasmid into KJM-1 and methotrexate (MTX)-resistant derivatives were selected by a stepwise increase of MTX concentration. Among them, derivatives which showed higher expression levels of beta-IFN than that achieved by the parental transformants were obtained, suggesting that a dhfr gene coamplification method can be used for efficient expression of foreign genes in KJM-1 which contains endogenous dhfr genes. Then, an improved beta-IFN expression vector was constructed, which contains a dhfr transcription unit. This plasmid was introduced into KJM-1 and then, MTX-resistant derivatives were selected. Among them, the highest producer, clone 40-10-24, secreted beta-IFN at a level as high as 5 micrograms/ml, which is about 100-fold higher than that obtained by the G418-resistant parental transformants. In addition, beta-IFN produced by recombinant KJM-1 cells had the same molecular weight of that produced by fibroblasts.

Blotting, Western↗
PubMed1990

Synthesis, oligomerization, and biological activity of the human immunodeficiency virus type 2 envelope glycoprotein expressed by a recombinant vaccinia virus.

The full-length envelope gene from an infectious human immunodeficiency virus type 2 (HIV-2) molecular clone was expressed in CD4+ and CD4- cells by a recombinant vaccinia virus vector. Pulse-chase experiments indicated that gp160 was processed into gp120 and gp41 subunits. Although large amounts of gp120 were shed into the medium, the recombinant vaccinia virus-infected cells fused with uninfected CD4+ cells. The receptor binding of HIV-2 gp120 was further analyzed using a panel composed of nine soluble CD4 mutants containing insertions of 2 amino acids within the first and second immunoglobulin-like domains. Of three mutations previously shown to interfere with HIV-1 gp120 binding, two also interfered with binding of the HIV-2 glycoprotein indicating use of the same binding site. Chemical crosslinking, sucrose gradient sedimentation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were employed to study the oligomerization of the envelope protein. The data indicated that gp160 assembles posttranslationally into dimers and higher oligomers that are probably tetramers.

Animals↗
PubMed1990

Bathoiodopsin, a primary intermediate of iodopsin at physiological temperature.

Measurement of the primary photochemical reaction of iodopsin, a chicken red-sensitive cone visual pigment, was carried out at room temperature by using picosecond (ps) laser photolysis. Excitation of iodopsin with a ps green pulse (pulse width, 21 ps) caused the instantaneous formation of a bathochromic product, which was stable on a ps time scale. This product may correspond to "bathoiodopsin," which was detected by low-temperature spectrophotometry. Although bathoiodopsin produced at the temperature of liquid nitrogen or helium reverted to the original pigment (iodopsin) on warming (above -170 degrees C), the bathoiodopsin produced at physiological temperature decayed to all-trans-retinal and R-photopsin (the protein moiety of iodopsin) presumably through several intermediates. The absorption maximum of bathoiodopsin at room temperature was at 625 nm, a wave-length slightly shorter than that measured at low temperature (lambda max, 640 nm). The extinction coefficient of bathoiodopsin at room temperature was lower than that at low temperature and close to that of the original iodopsin at room temperature.

Animals↗
PubMed1989

Isolation and characterization of a novel growth hormone cDNA from chum salmon (Oncorhynchus keta).

A cDNA clone which codes for a novel growth hormone has been isolated from the library of chum salmon pituitaries. The clone encodes a polypeptide of 210 amino-acid residues including 22 amino-acid residues of signal peptide, which is identical in length with known chum salmon growth hormone. In the coding region, there are 30 base substitutions, some of which result in 12 amino-acid substitutions. There are 8 base changes in the 5' untranslated region, and large insertions/deletions are in the 3' non-coding region. These results clearly indicate that there are at least two species of mRNAs for growth hormone in chum salmon pituitary.

Amino Acid Sequence↗
PubMed1988

Selective killing of HIV-infected cells by recombinant human CD4-Pseudomonas exotoxin hybrid protein.

It is projected that in the absence of effective therapy, most individuals infected with human immunodeficiency virus (HIV) will develop acquired immune deficiency syndrome (AIDS) and ultimately succumb to a combination of opportunistic microbial infections, malignancies and direct pathogenic effects of the virus. Anti-viral agents, immunomodulators, and inhibitors of specific HIV functions are being tested as potential treatments to alleviate the high morbidity and mortality. An alternative therapeutic concept involves the development of cytotoxic agents that are targeted to kill HIV-infected cells. Here we describe the purification and characterization of a recombinant protein produced in Escherichia coli that contains the HIV-binding portion of the human CD4 molecule linked to active regions of Pseudomonas exotoxin A. This hybrid protein displays selective toxicity toward cells expressing the HIV envelope glycoprotein and thus represents a promising novel therapeutic agent for the treatment of AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Binding region for human immunodeficiency virus (HIV) and epitopes for HIV-blocking monoclonal antibodies of the CD4 molecule defined by site-directed mutagenesis.

The binding region for human immunodeficiency virus (HIV) and epitopes for a panel of HIV-blocking anti-CD4 monoclonal antibodies of the CD4 molecule were defined by using in vitro site-directed mutagenesis. Codons for two amino acid residues (Ser-Arg) were inserted at selected positions within the region encoding the first and second immunoglobulin-like domains of CD4. A vaccinia virus-based expression system was used to produce soluble full-length extracellular CD4 fragments containing the insertions. The mutant proteins were tested for direct binding to soluble gp120 (the CD4-binding subunit of the viral envelope glycoprotein) and to a series of HIV-blocking anti-CD4 monoclonal antibodies. Impaired gp120 binding activity resulted from insertions after amino acid residues 31, 44, 48, 52, 55, and 57 in the first immunoglobulin-like domain. The epitopes for two HIV-blocking monoclonal antibodies, OKT4A and OKT4D, were also mapped in the gp120-binding region in the first domain. Insertions after amino acid residues 21 and 91 in the first domain had no effect on gp120 binding but impaired the binding of OKT4E, suggesting that this antibody recognizes a discontinuous epitope not directly involved in gp120 binding. Moderate impairment of gp120 binding resulted from the insertion after amino acid residues 164 in the second immunoglobulin-like domain, where the epitopes for monoclonal antibodies MT151 and OKT4B were also mapped.

Amino Acid Sequence↗
PubMed1987

Unusual renal sarcoma in a young adult: its similarities to clear cell sarcoma of the kidney.

We report an unusual case of renal sarcoma in a young adult. Histological examination demonstrated many similarities to the histopathological features of clear cell sarcoma of the kidney. Immunohistochemically, none of the intrinsic tumor cells showed positive staining with the antibodies against the intermediate filament proteins, epithelial membrane antigen, S100 protein, neuron-specific enolase, Leu-7 or myoglobin. The clinical course of this tumor was that of high grade malignancy, resulting in death with generalized metastases 13 months after tumor resection.

Adult↗
PubMed1987

A new SV40-based vector developed for cDNA expression in animal cells.

A useful vector, pAGE103, has been developed for the expression of cDNA in animal cells using the simian virus 40 (SV40) expression signals. cDNA could be expressed easily by inserting it into the multiple cloning sites (HindIII, SalI/AccI, XbaI, BamHI, SmaI/XmaI, KpnI/Asp718, SacI and EcoRI) of the vector, which are located between the SV40 early promoter and the SV40 early RNA processing signals for splicing and polyadenylation. In addition to the above transcription unit, pAGE103 contains the replication origin of ColE1, and a dual KmR/G418R selective gene. Several unique restriction sites are located on the boundaries between the above-mentioned three components of the vector, allowing the easy substitution or insertion of other genetic elements. The human interferon-beta gene was inserted into pAGE103 and shown to be expressed transiently in COS-1 cells and stably in several animal cell lines.

Animals↗
PubMed1987

[Usefulness of the bladder assist device (BLAD)].

We investigated the effectiveness of the Bladder Assist Device on urinary tract infection of patients with indwelling catheters. The patients, except for those with cord bladder and vesicoureteral reflux, could urinate intermittently under the BLAD system. Irrigation of the bladder using BLAD made urine clear and decreased urinary bacteria. Because of intermittent urination in patients who had been using the BLAD system for two weeks, the urine became somewhat clear and the number of bacteria decreased. Combination of bladder irrigation and intermittent urination using this system may inhibit urinary tract infection of an indwelling catheter. BLAD is considered to be a useful device for patients with continuous indwelling catheters.

Adult↗
PubMed1985

Expression of intact Ki-ras p21 protein in Escherichia coli.

We have constructed recombinant plasmids capable of expressing in Escherichia coli the intact ras p21 protein encoded by Kirsten murine sarcoma virus. The Ki-ras gene was inserted into an expression vector carrying the E. coli tryptophan promoter and E. coli lipoprotein transcriptional terminator. The resulting plasmids direct the synthesis of large quantities of p21 protein, which represented 20% of the total cellular protein. The Ki-ras p21 protein is immunoprecipitated with monoclonal antibody to p21, and exhibits guanine nucleotide binding activity and autophosphorylation activity. The purified Ki-ras p21 expressed in E. coli has shown to have intact N-terminal and C-terminal amino acid sequences predicted by the nucleotide sequences and migrate as -23K in SDS/polyacrylamide gels.

Amino Acid Sequence↗
PubMed1985

Cloning and expression of cDNA for salmon growth hormone in Escherichia coli.

cDNA clones encoding chum salmon (Oncorhynchus keta) growth hormone (sGH) have been isolated from a cDNA library prepared from chum salmon pituitary gland poly(A)(+) RNA. Synthetic oligodeoxynucleotide mixtures based on amino acid residues 23-28 of sGH were used as hybridization probes to select recombinant plasmids carrying the sGH coding sequence. The complete nucleotide sequence of sGH cDNA has been determined. The cDNA sequence codes for a polypeptide of 210 amino acids, including a putative signal sequence of 22 amino acids. The 5' and 3' untranslated regions of the message were 64 and 426 bases long, respectively. Mature sGH was efficiently expressed in Escherichia coli carrying a plasmid in which the sGH cDNA was under control of the E. coli trp promoter; sGH comprised about 15% of the total cellular protein in such bacteria. The partially purified sGH from E. coli stimulated the growth of rainbow trout and the activity was indistinguishable from that of natural sGH.

Journal Article↗
PubMed1984

Efficient expression in Escherichia coli of a mature and a modified human interferon-beta 1.

Ten recombinant plasmids were constructed which direct the synthesis of a mature human interferon-beta 1 (IFN-beta 1) under the control of the Escherichia coli tryptophan (trp) promoter. The spacing between the Shine-Dalgarno sequence of the trpL and the ATG initiation codon of the interferon gene was varied from 6 to 23 nucleotides by utilizing a Cla I site located within the spacer region of the plasmids. The optimal spacing for expression of IFN-beta 1 was determined to be 8-13 nucleotides from the results of interferon assay. The E. coli lipoprotein (lpp) promoter was also used for expression of IFN-beta 1 in E. coli. The results with an expression vector carrying a lpp-lac promoter showed that a modified IFN-beta 1 containing an additional 7 amino acids at the amino-terminus might be less active than the mature molecule.

Amino Acid Sequence↗
PubMed1981

Experimental studies on cadaver pancreas preserved by perfusion.

pancreatic transplantation is the most effective therapy of advanced diabetes mellitus. However, the problem is whether the cadaver pancreas can be used as a donor, because the living pancreas cannot be used as a donor from an anatomical standpoint. In case of the cadaver pancreas another problem is coming up that the pancreas proceeds to rapid autolysis. So, brain death is recommended as the most available condition for this transplantation. In this study we investigated the viability of the cadaver pancreas from decapitated rats preserved by the cold perfusion method. The results obtained were as follows: 1) The perfusing time affected the ratio of degenerative islets/normal islets. A large number of degenerative islets appeared with long perfusion time. 2) The cold (4 degrees C) perfusion method could preserve cadaver pancreas for 5 hours. 3) Insulin-secretion of cadaver pancreatic islets preserved for 5 hours under cold perfusion was 60% or more of that of normal pancreatic islets. 4) Three hundred islets from cadaver pancreas preserved for 5 hours under cold perfusion were transplanted into the portal vein of streptozotocin-induced diabetic rats. Four of these 11 rats recovered from diabetic state and maintained the non-diabetic state for 20 weeks after transplantation.

Animals↗