Purification and properties of hydroxymethylpyrimidine kinase from Escherichia coli

Hydroxymethylpyrimidine kinase, which catalyzes the conversion of 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) to its monophosphate, is purified about 3300-fold to apparent homogeneity from the cell-free extracts of E. coli K-12 through four successive steps of column chromatographies. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis and its molecular weight is estimated to be 43 000-44 000. The enzyme phosphorylated each of the pyridoxine substrates, pyridoxine, pyridoxal and pyridoxamine as well as hydroxymethylpyrimidine, and the reaction gave rise to a corresponding 5'-phosphate compound. The Km values of the purified enzyme for hydroxymethylpyrimidine and for pyridoxine are 1.1.10(-4) and 6.6.10(-5) M, respectively. Pyridoxine inhibits competitively the phosphorylation of hydroxymethylpyrimidine with a Ki value of 2.7.10(-6) M and hydroxymethylpyrimidine shows the same for that of pyridoxine with a Ki value of 9.0.10(-5) M. A similarity in enzymic properties between the hydroxymethylpyrimidine kinase and an enzyme which has been characterized as pyridoxal kinase leads to the assumption that both hydroxymethylpyrimidine and pyridoxine might be phosphorylated by the same enzyme species.     

Radioimmunoassay for non-enzymatically glycated serum proteins

We have developed a radioimmunoassay (RIA) for nonenzymatically glycated serum proteins. The polyclonal antibodies prepared against reduced glycated human albumin were specific for the glucitollysine residues of serum proteins. Serum proteins from diabetic patients (n = 25) contained 5.3 +/- 2.8 nmoles of glucitollysine/mg protein, compared to 2.0 +/- 0.2 in controls (n = 20). The intra- and inter-assay variables were 3.2-6.2% and 4.4-8.6%, respectively. Results from this assay procedure correlated well with those from the boronate affinity chromatography procedure (r = 0.94; P less than 0.001). The data suggested that diabetic serum proteins contained at least 2.5 times as much immunochemically detectable glucitollysine residures as normal serum proteins after reduction of the proteins with sodium borohydride.     

The thiM locus and its relation to phosphorylation of hydroxyethylthiazole in Escherichia coli.

A mutant of Escherichia coli lacking hydroxyethylthiazole kinase (EC 2.7.1.50) was produced by a further mutation of a temperature-sensitive, auxotrophic mutant for hydroxyethylthiazole. The parent cells possessed two distinct enzymes capable of phosphorylating hydroxyethylthiazole: one was hydroxyethylthiazole kinase, and the other was a phosphotransferase species that required p-nitrophenylphosphate as a phosphoryl donor. Osmotic shock fluid prepared from the mutant cells phosphorylated hydroxyethylthiazole to an extent comparable to that observed with shock fluid from the parent cells, whereas extracts from shocked cells were unable to catalyze the kinase reaction. Shock fluid from a mutant of the other type obtained as a reduced phosphatase activity against p-nitrophenylphosphate did not show any appreciable activity for the phosphotransferase reaction, while extracts from shocked cells showed full kinase activity. The former mutant had lost its ability to grow on hydroxyethylthiazole at high temperature, but the latter mutant still responded to it. It thus appears that the kinase is an enzyme which might play a role in the biosynthesis of thiamine PPi in situ. By conjugation and P1 transduction, a gene governing hydroxyethylthiazole kinase activity, for which we propose the designation thiM, was mapped on the chromosome close to thiD, a gene specifying phosphomethylpyrimidine kinase activity.     

DNA ploidy pattern and amplification of ERBB and ERBB2 genes in human gastric carcinomas

The DNA ploidy pattern and amplification of ERBB and ERBB2 genes were examined in paraffin-embedded tissue from gastric carcinomas using flow cytometry and a slot-blot hybridization technique. The incidence of aneuploidy in well differentiated adenocarcinomas (56%) was significantly higher (p less than 0.05) than that in poorly differentiated adenocarcinomas (21%). The DNA ploidy pattern was not remarkably different between the primary tumors and metastatic deposits in lymph nodes. Of the nine specimens having an aneuploid stem cell line in the primary tumor and/or in metastases, three showed ERBB2 gene amplification and one showed ERBB gene amplification. The incidence of epidermal growth factor (EGF) immunoreactivity in tumor cells showed no difference between diploid and aneuploid tumors. These findings indicate that aneuploidy is frequently associated with amplification of ERBB and ERBB2 genes.     

Expression of several growth factors and their receptor genes in human colon carcinomas

We have examined the expression of mRNAs for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-α), EGF receptor (EGFR), PDGF-A chain (PDGFA), PDGF-B chain (PDGFB) and PDGF receptor (PDGFR) genes in seven human colorectal carcinoma cell lines and 18 human colorectal carcinomas. In surgically resected specimens of the 18 colorectal tumors, TGF-α, EGFR, PDGFA, PDGFB and PDGFR mRNAs were detected at various levels in 15 (83%), 9 (50%), 18 (100%), 8 (44%) and 12 (67%), respectively. They were also detected in normal tissues. Interestingly, EGF mRNA was detected in only five (28%) of the tumors, but not in normal mucosa. Expression of EGF was also confirmed immunohistochemically in tumor cells. Of the five tumors expressing EGF, four expressed EGFR mRNA and showed a tendency to invade veins and lymphatics. All the colorectal carcinoma cell lines expressed TGF-α mRNA, and five cell lines expressed EGFR mRNA simultaneously. Production of TGF-α protein by DLD-1 and CoLo320DM cells was confirmed by TGF-α specific monoclonal antibody binding assay. The spontaneous³H-thymidine uptake by DLD-1 was suppressed by an anti-TGF-α monoclonal antibody. PDGFA and PDGFB mRNA were also expressed in four cell lines, but PDGFR and EGF mRNA was not detected. These results suggest that human colorectal carcinomas express multi-loops of growth factors and that TGF-α produced by tumor cells functions as an autocrine growth factor in human colonic carcinoma.     

Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.     

Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.     

Kinetic mechanism of pulmonary carbonyl reductase.

An IgG1 monoclonal antibody, Sulph I, reacting with sulphatide (3'-sulphogalactosylceramide), was produced by immunizing Balb/c mice with that glycolipid coated on Salmonella minnesota bacterial membrane. Radioimmunodetection of the binding of the monoclonal antibody to structurally related glycolipids adsorbed to microtitre plates or chromatographed on thin-layer plates was used to determine its binding epitope. The antibody showed similar binding avidity to three sulphated glycolipids: sulphatide, sulpholactosylceramide and seminolipid. Lysosulphatide did bind the antibody, but, compared with sulphatide, 30 times more antigen was needed for half-maximal binding. Bis(sulphogangliotriosyl)ceramide and bis-sulphogangliotetraosylceramide did not bind the antibody. These results suggest that terminal galactose-3-O-sulphate and part of the hydrophobic region of the glycolipid are recognized by the Sulph I antibody.     

Monoclonal Antibodies Specific for NADH-Ferricyanide Reductase Domain of Spinach Leaf Nitrate Reductase

Two monoclonal antibodies, 17(3)9 and 36(79)4, were preparedagainst nitrate reductase from Spinacia oleracea L. leaves.An enzyme-linked immunosorbent assay showed that 17(3)9, butnot 36(79)4, reacted more strongly to heat-denatured than nativeantigen. These antibodies inhibited NADH-nitrate reductase aswell as its various partial activities including reduced methylvilogen-nitrate reductase, reduced flavin mononucleotide-nitratereductase and NADH-cytochrome c reductase activities, but notNADH-ferricyanide reductase activity. Immunoblotting after electrophoreticseparation of nitrate reductase fragments obtained by Staphyrococcusaureus V8 protease digestion of native enzyme revealed thatthe two monoclonal antibodies bind to different epitopes locatedon the 28 kDa of the NADH-ferricyanide reductase domain. (Received October 2, 1987; Accepted June 9, 1988)