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Biomedical subjects

S Matsuki

Publications and source records attributed to S Matsuki.

At least 19 recordsLinked to original sources
PubMed1992

Chemical modification of recombinant human granulocyte colony-stimulating factor by polyethylene glycol increases its biological activity in vivo.

Recombinant human granulocyte colony-stimulating factor (rHuG-CSF) produced in Escherichia coli was chemically modified by polyethylene glycol (PEG) of molecular weights 4,500 or 10,000. The neutrophils observed at 32 hours after intravenous injection of the rHuG-CSF modified with PEG (4,500) or PEG (10,000) to mice were, respectively, 2.5 times and 5 times more than that observed after the injection of the unmodified rHuG-CSF. These results show that the covalent attachment of PEG to rHuG-CSF enhanced its pharmacological activity in vivo and that the modification with the larger PEG molecule is more effective to enhance the in vivo activity of rHuG-CSF.

Animals↗
PubMed1992

The substitution of cysteine 17 of recombinant human G-CSF with alanine greatly enhanced its stability.

Human recombinant granulocyte-colony stimulating factor (rhG-CSF) has one free cysteine at position 17 and has two disulfide bridges (Cys36-Cys42 and Cys64-Cys74). The Cys17 of rhG-CSF was substituted with Gly, Ala, Ser, Ile, Tyr, Arg, and Pro, or deleted using site-directed mutagenesis in order to improve its thermostability. With the exception of Pro17-rhG-CSF, all mutant proteins retained biological activity which promotes the growth of mouse bone marrow cells in vitro. Among these mutant proteins, Ala17-rhG-CSF had more than 5 times higher stability than rhG-CSF. But Ser17-rhG-CSF had almost same stability as rhG-CSF and other mutant proteins had only lower stability.

Alanine↗
PubMed1992

Is necrosis induction therapy relevant to hepatocellular carcinoma? Prognostic evaluation viewed from necrosing feature in HCC occluding the main portal vein after treatment with DBcAMP and MMC.

Intraarterial infusion therapy combining dibutyryl adenosine 3',5'-monophosphate and mitomycin C was administered to 27 patients with hepatocellular carcinoma (HCC) occluding the main portal vein. Twelve patients (44%) survived more than 6 months, among them nine (75%) whose tumors were non-necrosing before treatment. When patients were compared for the presence or absence of necrosis in the tumor, those with necrosis responded poorly to treatment; only 3 of 11 (27%) survived more than 6 months. Although the combination therapy exerted a salutary effect on HCC occluding the main portal vein, the presence of necrosis in the tumor was an unfavorable sign for the prognosis, which casts doubt upon the use of the necrosis-inducing method for the treatment of HCC.

Adult↗
PubMed1991

Pharmacokinetics of recombinant human granulocyte colony-stimulating factor conjugated to polyethylene glycol in rats.

The pharmacokinetics of recombinant human granulocyte colony-stimulating factor conjugated to polyethylene glycol (PEG-rhG-CSF) and rhG-CSF were studied in male Sprague-Dawley rats. The serum concentration after i.v. administration at a dose of 100 micrograms protein/kg was investigated by a bioassay. The serum rhG-CSF concentration decreased steadily after injection with a terminal half-life of 1.79 h. The PEG-rhG-CSF concentration after injection decreased much more slowly with a half-life of 7.05 h. The slower disappearance of PEG-rhG-CSF resulted in a greater area under the concentration-time curve. The neutrophil count after 100 micrograms of protein/kg of rhG-CSF administration reached a peak 12 h after injection and returned to the control level 48 h after injection. The neutrophil count after 100 micrograms of protein/kg of PEG-rhG-CSF administration was identical to that of rhG-CSF after 12 h but the highest level was maintained for 24 to 72 h after injection and returned to the control level after 168 h. These data indicated that PEG-rhG-CSF administration exerted a sustained biological effect on peripheral blood neutrophils. It is expected that PEG-rhG-CSF may contribute greatly to human G-CSF treatment because it has a prolonged neutrophil-proliferating activity enabling fewer administrations.

Animals↗
PubMed1991

Selective DNA cleavage by elsamicin A and switch function of its amino sugar group.

We report guanine-specific recognition and selective cleavage of DNA by the antitumor antibiotic elsamicin A equipped with an amino sugar and compare these results with cleavage by chartarin and chartreusin antibiotics. The preferential cutting sites of DNA strand scission with elsamicin A are on the bases adjacent to the 3'-side of guanine residues such as 5'-GN sites, in particular 5'-GG sites. The present results also indicate that (1) the aglycon portion binds intercalatively to the 3'-side of guanine in host DNA, (2) the guanine 2-amino group has an important effect on selective DNA binding of elsamicin A, and (3) the amino sugar residue of elsamicin A facilitates the drug binding into the minor groove of B-DNA. In addition, we found that an acetylation of the amino group on the elsamicin A sugar portion plays an interesting switch function for the activity of elsamicin A. The biological implication of this switch has also been discussed.

Aflatoxin B1↗
PubMed1991

Mutant protein of recombinant human granulocyte colony-stimulating factor for receptor binding assay.

A new mutant protein of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was produced for the studies on receptors for human G-CSF. The mutant protein [(Tyr1, Tyr3]rhG-CSF), the biological activity of which was almost equal to that of rhG-CSF, was prepared by the replacement of threonine-1 and leucine-3 of rhG-CSF with tyrosine. The radioiodinated preparation of the mutant protein showed high specific radioactivity and retained full biological activity for at least 3 weeks. The binding capacity of the radioiodinated ligand was compared with that of [35S]rhG-CSF. Both radiolabeled ligands showed specific binding to murine bone marrow cells. Unlabeled rhG-CSF and human G-CSF purified from the culture supernatant of the human bladder carcinoma cell line 5637 equally competed for the binding of labeled rhG-CSFs in a dose-dependent manner, demonstrating that the sugar moiety of human G-CSF made no contribution to the binding of human G-CSF to target cells. In contrast, all other colony-stimulating factors and lymphokines examined did not affect the binding. Scatchard analysis of the specific binding of both labeled ligands revealed a single class of binding site with an apparent dissociation constant (Kd) of 20-30 pM and 100-200 maximal binding sites per cell. These data indicate that the radioiodinated preparation of the mutant protein binds the same specific receptor with the same affinity as [35S]rhG-CSF. The labeled mutant protein also showed specific binding to human circulating neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Site-directed mutagenesis of glutamine residue of calmodulin. Activation of guanylate cyclase of Tetrahymena plasma membrane.

Tetrahymena calmodulin (CaM) differs from mammalian CaM in its ability to activate Tetrahymena guanylate cyclase. Of 12 differences in amino acid sequence, two occur near the carboxyl terminus (Gln-143----Arg and Thr-146----deletion). To investigate the functional significance of the carboxyl-terminal region in activation of the guanylate cyclase, three mutated CaMs were engineered by using cassette mutagenesis of rat CaM cDNA: Gln-143----Arg (CaM.A), Thr-146----deletion (CaM.D), and Gln-143----Arg/Thr-146 deletion (CaM.AD). Recombinant wild type CaM (wCaM), CaM.A, CaM.D, and CaM.AD were indistinguishable in their ability to activate cyclic AMP phosphodiesterase. The two mutated CaMs (CaM.A and CaM.AD) with the Gln-143 replacement activated guanylate cyclase of Tetrahymena plasma membrane in the presence of Ca2+, with the maximal activation being half of that produced by Tetrahymena CaM. In contrast, neither CaM.D nor wCaM could stimulate the cyclase activity. A CaM antagonist, W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), prevented the cyclase activation by either Tetrahymena CaM, CaM.A, or CaM.AD. Thus, we conclude that Arg-143 is in a region of the molecule involved in activation of Tetrahymena guanylate cyclase. The data also suggest that the cyclase activation by Tetrahymena CaM requires complex macromolecular interactions between the entire CaM molecule and the enzyme.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1990

Frequent expression of receptors for granulocyte-macrophage colony-stimulating factor on human nonhematopoietic tumor cell lines.

Receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) were identified on 9 of 35 (26%) human nonhematopoietic tumor cell lines including non-small cell lung cancer, stomach cancer, colon cancer, and osteosarcoma cells. GM-CSF receptors distributed on these human tumor cells were low affinity types with an equilibrium dissociation constant of 1.5-10.0 nM. Cross-linking studies revealed that the molecular weights of the low affinity GM-CSF receptors were 65-85 kilodaltons. The high affinity receptors identified on hematopoietic cells were not detected on human nonhematopoietic tumor cells which we studied, and we could detect no effects of GM-CSF on cell growth of these tumor cells.

Carcinoma, Non-Small-Cell Lung↗
PubMed1990

Identification and cellular distribution of distinct proteins forming human GM-CSF receptor.

Two proteins forming the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF)1 were identified and characterized. One with apparent Mr of about 80,000 was defined as alpha-chain and has Kd of 0.7-2.8 nM. The other binding molecule with apparent Mr of about 135,000 was defined as beta-chain and is related to the high-affinity binding with Kd of 10-40 pM. The binding kinetic studies confirmed that the 125I-GM-CSF associated slower to and dissociated more rapidly from the alpha-chain than the beta-chain. The alpha-chain is expressed not only on hemopoietic cells but also on full-term placental tissues, choriocarcinoma cells, and other solid tumor cells. In contrast, the distribution of the beta-chain is restricted on hemopoietic cells. The alpha-chain probably corresponds to the low-affinity GM-CSF receptor whose cDNA has been cloned and sequenced.

Granulocyte-Macrophage Colony-Stimulating Factor↗
PubMed1990

[Combination chemotherapy of M-VAC (methotrexate, vinblastine, adriamycin and cisplatin) for advanced uroepithelial cancer].

Seventeen patients with advanced uroepithelial cancer were treated with M-VAC regimen. Out of 17 patients, 10 were evaluable but 7 were not. Evaluable 10 patients comprised 7 males and 3 females ranging in age from 50 to 83 (median 69.5). The number of performed cycle ranged from 1 to 3 and the performance status was from 0 to 2. Complete response (CR) was observed in 2, partial response (PR) in 4 and no change (NC) in 4 out of 10 patients. The efficacy rate was 60%. Every site of the tumor responded well and CR was observed, in particular, in nodal and pulmonary metastasis. One of 2 CR patients relapsed and died. One of 4 PR patients died of cancer, 2 are alive with regrowing tumors and the other is alive without disease after surgical removal of all residual tumors. The same regimen was carried out again to 2 patients with regrowing tumors, but no response was observed. As to drug toxicity in total 31 cycles (17 patients), gastrointestinal disturbances, anorexia and alopecia were reversible but hematological toxicity was serious and 3 of 17 (17.6%) died of severe bone marrow suppression. M-VAC is an effective regimen for advanced uroepithelial cancer, but bone marrow suppression was serious. Therefore, special attention should be paid to myelosuppression.

Aged↗
PubMed1990

Structural analysis of the receptors for granulocyte colony-stimulating factor on neutrophils.

We investigated granulocyte colony-stimulating factor (G-CSF) receptors on neutrophils from three patients with chronic myelogenous leukemia (CML) in the chronic phase, in comparison with four normal volunteers. Because we experienced some difficulties in radioiodinating intact recombinant human G-CSF, we developed a new derivative of human G-CSF termed YPY-G-CSF. It was easy to iodinate this protein using the lactoperoxidase method because of two additional tyrosine residues, and its radioactivity was higher than that previously reported. The biological activity of YPY-G-CSF as G-CSF was fully retained. Scatchard analysis demonstrated that CML neutrophils had a single class of binding sites (1400 +/- 685/cell) with a dissociation constant (Kd) of 245 +/- 66 pM. The number of sites and Kd value of CML neutrophils were not significantly different from those of normal neutrophils (p greater than 0.9). Cross-linking studies revealed two specifically labeled bands of [125I]YPY-G-CSF-receptor complexes with apparent molecular masses of 160 and 110 kd on both normal and CML neutrophils. This is the first report describing two receptor proteins on neutrophils. According to the analyses of the proteolytic process of these cross-linked complexes and proteolytic mapping, we assume that alternative splicing or processing from a single gene may generate two distinct receptor proteins that bind specifically to G-CSF but have different fates in intracellular metabolism.

Adult↗
PubMed1990

High-level expression of complementary DNA encoding rat calmodulin in Escherichia coli.

We report the production and characterization of a rat calmodulin made in Escherichia coli. To express the rat calmodulin cDNA in E. coli, we have employed an expression vector containing the E. coli trp promoter and trpA terminator. The cDNA was modified so as to delete the 5' nontranslated sequence and to incorporate a consensus sequence for the E. coli ribosome-binding site. Several codons for the N-terminal amino acids were selected to fit the E. coli consensus nucleotide sequence around the translational initiation codon. After induction of expression in E. coli, rat calmodulin accounted for over 30% of total cellular proteins. About 100 mg of recombinant rat calmodulin, purified to over 90% homogeneity by extraction from bacterial lysate followed by phenyl-Sepharose column chromatography, was obtained from 1 liter of E. coli culture. This recombinant calmodulin activated rat brain cyclic AMP phosphodiesterase to the same extent as the native calmodulin purified from rat brain. These results indicate that the overproduction system of the recombinant calmodulin in E. coli facilitates the study of the structure-function relationship by site-specific mutagenesis.

Amino Acid Sequence↗
PubMed1990

[Two cases of megaureter in children].

We reported 2 cases of megaureter in children. The courses of renal function and ureteral dilatation in these patients were observed by urography and renoscintigraphy to determine the timing of surgery. These two patients with unilateral megaureter were treated by reconstructive operation involving excision of the stenotic portion of the ureter, tapering of the dilated ureter and re-implantation. The courses after reconstruction were excellent after 5 years. Recently, active surgery has been recommended for megaureter together with identification of the cause. Urograms and diuretic renoscintigrams were useful for determining the timing of surgery in our cases.

Child↗
PubMed1989

Characterization of human interleukin-3 receptors on a multi-factor-dependent cell line.

Recombinant human interleukin-3 (hIL-3) was radioiodinated by Bolton-Hunter method with maintenance of biological activity. Using 125I-hIL-3, hIL-3 receptors were characterized on a multi-factor-dependent cell line TF-1. Equilibrium binding studies revealed the existence of a single class of binding sites (667 +/- 306 sites/cell) with a Kd of 173 +/- 25 pM. Affinity labeling of TF-1 cells with 125I-IL-3 yielded two bands of 150 kDa and 85 kDa, implying molecular weights of 135 kDa and 70 kDa for the hIL-3 receptors.

Cell Line↗
PubMed1988

[Clinical study of the effect of cisplatin therapy in unresectable hepatocellular carcinoma].

Twenty-one cases of unresectable hepatocellular carcinoma (HCC), including 15 cases receiving intravenous infusion of CDDP in addition to transcatheter arterial embolization (TAE), and 6 cases receiving intraarterial infusion of CDDP in combination with sodium thiosulfate rescue (STS rescue) were studied. In the 15 cases given intravenous infusion therapy with TAE, favorable effects were observed in 33.3% of patients, and the 50% survival period was 22.5 months. In the 6 cases given intraarterial infusion, favorable effects were obtained in 66.6% of patients, but the 50% survival period was 2 months. The side effects observed most frequently were nausea and vomiting. All the other side effects observed were not so severe. These results suggest that intravenous CDDP infusion in addition to TAE is favorable, producing a life-prolongation effect.

Aged↗
PubMed1987

Molecular cloning and expression in Escherichia coli of a cDNA encoding human pancreatic elastase 2.

We have cloned a DNA that is complementary to the messenger RNA that encodes human pancreatic elastase 2 from a human pancreatic cDNA library using a cloned cDNA for rat pancreatic elastase 2 messenger RNA. This complementary DNA contains the entire protein coding region of 807 nucleotides which encodes preproelastase of 269 amino acids, and 4 and 82 nucleotides of the 5'- and 3'-untranslated sequences, respectively. When this deduced amino acid sequence was compared with known amino acid sequences it showed 82% homology with rat pancreatic elastase 2. This deduced sequence also contains a 16-amino-acid peptide identical with the N-terminal sequence determined for native human pancreatic proelastase 2. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 241 amino acids including 16 and 12 amino acids for a signal peptide and an activation peptide, respectively. Moreover, the predicted key amino acid residues involved in determining the substrate specificity of mammalian pancreatic elastase 2 are retained in the human enzyme. Cloned human pancreatic elastase 2 cDNA was expressed in E. coli as a mature and pro-form protein. Both resulting proteins showed immunoreactivity toward anti-elastase serum and enzymatic activity. We have also cloned and sequenced a porcine pancreatic elastase 2 cDNA.

Amino Acid Sequence↗