Masahiro Nobuhara

Molecular cloning and nucleotide sequence of the HU-1 gene of Escherichia coli

SummaryThe Escherichia coli HU-1 was cloned by use of mixed synthetic oligonucleotides (17-mer) predicted from a portion of its amino acid sequence. The amino acid sequence of the HU-1 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream from the translational initiation codon (GUG) of the HU-1 gene.

Interferon potentiates cytotoxic effects of 5-fluorouracil on cell proliferation of established human cell lines originating from neoplastic tissues.

A potentiation of the cytotoxic effects of 5-fluorouracil (5-FU) on human tumor cells by interferon was examined. The human neoplastic cell lines used were HeLa (uterine cervical cancer), MCF-7 (mammary cancer), WI-38 CT-1 (embryonic lung fibroblasts transformed in culture by Co-60 gamma-ray irradiation), KMM-1 (myeloma) and Raji (Burkitts lymphoma). The normal human cell strain used was WI-38 (normal human lung fibroblasts). The cytotoxic effects were determined by colony formation for HeLa, MCF-7, WI-38 CT-1 and WI-38 cells, and by cell growth for KMM-1 and Raji cells. Each cell line was different in sensitivity to interferon or 5-FU. Interferon potentiated synergistically the cytotoxic effects of 5-FU on HeLa, WI-38 CT-1 and KMM-1 cells. In the case of Raji cells, the cytotoxic effects of the combination of interferon and 5-FU were additive. Neither synergistic nor additive lethal effects of the combination of the 2 agents were observed in MCF-7 and WI-38 cells. The present results indicate a possibility that interferon and 5-FU can mutally reduce the amount of the other needed to treat cancer patients.

In vitro and in vivo studies on potentiation of cytotoxic effects of anticancer drugs or cobalt 60 gamma ray by interferon on human neoplastic cells

A possibility that interferon may potentiate the cytotoxic effects of anticancer drugs or 60Co gamma ray on human neoplastic cells was studied by in vitro and in vivo experimental procedures. The human neoplastic cells used were HeLa (uterine cervical cancer) and WI‐38 CT‐1 (embryonic lung fibroblasts transformed in culture by 60Co gamma ray) cells. As normal human cells, WI‐38 cells were used. Interferon was a preparation of β‐type produced by human fibroblasts. The cytotoxicity was determined by colony formation for in vitro experiments and by tumor growth for animal experiments. Of 17 anticancer drugs, the cytotoxic effects of six drugs, namely, peplomycin, bleomycin, aclacinomycin, cisplatin, 5‐fluorouracil (5‐FU), and Adriamycin (doxorubicin) were potentiated by concomitant application of interferon. The cytolethal effects of 60Co gamma ray were also enhanced by interferon. The growth of tumor induced by transplantation of HeLa cells into a nude mouse was remarkably reduced by combination therapy of interferon and 5‐FU. The current results indicate a possibility that combined therapy of certain types of anticancer drugs or 60Co gamma ray with interferon may be effective in treatment of cancer patients.

In vitro studies on potentiation of cytotoxic effects of anticancer drugs by interferon on a human neoplastic cell line (HeLa)

Experiments were performed to ascertain whether the antitumor effect of various anticancer drugs might be enhanced by interferon, using cultures of HeLa cells originating from a human carcinoma of the uterine cervix. The effects of drugs were assessed by counting cell colonies formed in culture. The drugs studied included 4 metabolic antagonists: cytosine arabinoside (Ara-C), 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP) and methotrexate (MTX), 7 antibiotics: aclacinomycin (ACM), adriamycin (ADM), actinomycin D (ACD), cycloheximide, mitomycin C (MMC), peplomycin (PEP) and puromycin; 2 alkylating agents: nimustine hydrochloride (ACNU) and melphalan, and 3 other drugs, vincristine (VCR), cisplatin (CDDP) and hydroxyurea (HU). Interferon was a preparation of the beta-type produced by human fibroblasts. A specific additive or synergistic potentiation of the cytotoxic effect by concomitant application of interferon was observed with PEP, ACNU, ACM, CDDP, 5-FU and ADM; the drug concentration given a 50% inhibition of cell growth was reduced by one-half or more in cultures with the combination of interferon and these drugs. The treatment of cells with interferon alone caused only 10-30% inhibition of cell proliferation.

Novel factor Xa and plasma kallikrein inhibitory-activities of the second Kunitz-type inhibitory domain of urinary trypsin inhibitor

Urinary trypsin inhibitor is a glycoprotein with a structure in which two Kunitz-type inhibitory domains are linked in a row. We isolated two genes encoding the 70 amino acid sequence from the 78th amino acid (Thr) to the C-terminal and the 68 amino acid sequence from the 80th (Ala) to the C-terminal of human urinary trypsin inhibitor, both which correspond to the second Kunitz-type inhibitory domain, and then constructed expression plasmids by ligating it to the E. coli alkaline phosphatase signal peptide gene. These plasmids under the control of the tryptophan promoter expressed the second domain in E. coli strain JE5505 which lacks the membrane lipoprotein. The recombinant second domain purified from the culture supernatant of the transformant inhibited trypsin, plasmin, leukocyte elastase and chymotrypsin which are known to be inhibited by urinary trypsin inhibitor. In addition it inhibited blood coagulation factor Xa and plasma kallikrein in a concentration dependent and competitive manner, and significantly prolonged the plasma-based activated partial thromboplastin time (APTT). The truncated natural counterpart obtained by a limited degradation of human urinary trypsin inhibitor also revealed the identical inhibitory activities.

Structure of slow-reacting substance of anaphylaxis (SRS-A)

To elucidate the chemical structure of slow-reacting substance of anaphylaxis from rat (SRS-A rat), SRS-A rat were purified by the method of Orange with modification using DEAE-Sephadex A-25 chromatography. Ultraviolet absorption spectrum of purified SRS-A rat indicated the presence of conjugated triene. Arylsulfatase B degradation products and HCl degradation products were subjected to analysis by a gas chromatography and mass spectrometry and a thin layer chromatography. Products obtained by arylsulfatase b catalysis contained 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid. HCl degradation products showed the presence of glycine, glutamic acid and cysteic acid. Furthermore, the analysis of anhydrous hydrazine degradation products of SRS-A rat and of HCl hydrolyzed products of dinitrophenylated SRS-A rat revealed the presence of glycine at C-terminal and glutamine acid at N-terminal. The study of the substrate specificity of arylsulfatase B against various materials including SRS-A rat suggested the presence of sulfone in SRS-A rat. The molecular ion peak of SRS-A rat sodium salt was observed at m/e 680 in field desorption mass spectrum of SRS-A rat. On the basis of these data, we identified the structure of SRS-A rat as [gamma]glutamyl-4(5-hydroxy-7,9,11,14-eicosatetraenoic acid-6-yl)-4,4-dioxyocysteinyl] glycine.

Stable expression of human tissue-type plasminogen activator regulated by β-actin promoter in three human cell lines: HeLa, WI-38 VA13 and KMS-5

A high-level and stable expression system of human tissue-type plasminogen activator (t-PA) was accomplished in human cells by selecting a promoter and a host cell line. First, we have constructed two types of t-PA expression plasmids containing 3 kb of the human beta-actin promoter region or 0.3 kb of SV40 early promoter region and these plasmids were transfected into HeLa cells, respectively, and the resulting transfectants were found to secrete various amounts of t-PA derived from the plasmids to the culture media. Southern blot analysis revealed that the beta-actin promoter was more efficient than the SV40 early promoter with regard to the expression level per single copy of the t-PA gene in the transfected HeLa cells. Next, the t-PA expression plasmid containing the beta-actin promoter was also transfected into WI-38 VA13 cells, a human fibroblastic cell line, and KMS-5 cells, a human lymphoid cell line, in order to compare the expression ability of the promoter among these three cell lines. Some of the transfectants from both cell lines were also found to produce t-PA. It was also found that the expression levels in HeLa and WI-38 VA13 seemed to be more efficient than that in KMS-5.

Potentiation of cytotoxic effects of 5-fluorouracil by inosiplex on cancer cells.

The antitumor effect of 5-fluorouracil (5-FU) was significantly enhanced by inosiplex which has been developed as a drug possessing antiviral activity. The enhancement of antitumor effect of 5-FU was demonstrated by experiments both in vitro and in vivo, viz. depression of the colony formation rate in cultures of HeLa cells (an established cell line of human cervical carcinoma), and prolongation of the survival of mice bearing transplanted Ehrlich ascites tumor of murine mammary carcinoma origin. The HeLa cell colony formation was synergistically decreased in the presence of 0.5-2.0 micrograms/ml of 5-FU combined with 100 micrograms/ml of inosiplex. Inosiplex did not cause any appreciable inhibition of cell growth at this concentration when added alone to the culture. The mean duration of survival of tumor-bearing mice was 18.2, 20.3, 31.9 and 47.1 days in the control group and groups receiving inosiplex, 5-FU, or a combination of 5-FU and inosiplex, respectively; hence significantly prolonged in the combined therapy regimen group as compared with the control and the 5-FU treated group (P less than 0.01).

A synthetic translation-terminator gene: A tool for dissecting the translation direction of a gene

A 41-nucleotide-long duplex DNA, which contains the translation termination codon TAA in six reading frames and lactose operator sequence of Escherichia coli, has been synthesized. This fragment may be useful not only for producing a truncated protein encoded in a plasmid, but also for the identification of the precise coding region and translation direction of a bacterial gene in the cloned chromosomal segment. The synthetic fragment was inserted into beta-lactamase structural gene in pBR322 in order to test the in vivo activity. The plasmid produced mutant beta-lactamase reduced in size, as expected from the insertion site, and rendered the host bacterium constitutive for beta-galactosidase. Thus, termination codons and lactose operator in synthetic nucleotide appear to be functional in vivo.

CONTAMINATING SUBSTANCES IN UROKINASE PREPARATIONS

Urokinase (UK) preparations, obtained at random on the commercial market from ten manufacturers, were investigated for their degree of purification by measurement of their coagulant activity, lysozyme activity, kinin‐ and brady‐kinin‐like activity, endotoxin content and hepatitis B surface antigen (HBs antigen). The results indicated that the UK preparations could be divided into two groups: ‘clean’ UK which contained no detectable biologically active substances, and ‘dirty’ UK which contained abundant biological active substances. When ‘dirty’ UK containing high procoagulant activity was injected into rabbits, the coagulation system was activated. Separation of the pro‐coagulant activity from the UK components may be difficult on an industrial scale by gel filtration. Since ‘clean’ UK contains no detectable biologically active substances, its specific activity was the highest among the UK preparations.