New Plasmid-Mediated Phosphorylation of Gentamicin C in Staphylococcus aureus

A clinical isolate of Staphylococcus aureus resistant to gentamicin, kanamycin and 3′,4′-dideoxykanamycin B contained two enzymes capable of inactivating gentamicin, i.e., an aminoglycoside 2″-phosphotransferase and aminoglycoside acetyltransferase.     

Clinical studies on cell-mediated immunity in patients with renal cell carcinoma: interleukin-2 and interferon-γ production of lymphocytes

Summary The authors examined interleukin-2 (IL-2) production and interferon (IFN) production of peripheral blood mononuclear cells in 28 patients with renal cell carcinoma and 17 control subjects. The peripheral blood was obtained prior to the initiation of therapeutic procedures. The patients were divided into two groups according to tumor size, 5 cm and >5 cm. The production of IL-2 and IFN was measured by immunoradiometric assay. As a result, in the patients with tumors >5 cm, IL-2 and IFN production was impaired. However, in the patients with tumors 5 cm, IFN production was enhanced, though IL-2 production was not significantly different from that of the control subjects. There was no significant correlation between IL-2 production and IFN production.     

Immunological detection of the dystrophin molecule with antibody directed against the synthetic peptide.

We synthesized a peptide designated R8 (amino acid residues 1157-1201) based on the primary structure presumed from the nucleotide sequence of the cDNA clone from the gene for Duchenne muscular dystrophy. Antibody to the synthetic R8 generated by immunization of rabbits was tested on human and mouse skeletal muscle by Western blotting analysis. The antibody reacted with a component of the 400K dystrophin of normal human and mouse skeletal muscles, but not with components of the muscles of Duchenne muscular dystrophy patients and mdx mice. Thus we established that this peptide sequence is in fact missing in the protein product 'dystrophin' encoded by the DMD gene. The antibody may prove useful for the diagnosis of the Duchenne types of muscular dystrophy.     

Enhancement of tissue plasminogen activator production from human normal fibroblast IMR-90 cells by limitation of cell growth

Summary Tissue plasminogen activator (t-PA) production induced by proteose peptone from IMR-90 cells was investigated. Cells monolayered on plastic surfaces had a higher ability to produce t-PA per unit cell compared to those grown tri-dimensionally on ceramic pieces. Furthermore, confluent monolayers of the cells, which suffered contact inhibition and resulted in limited growth, were available for t-PA production. Repeated batch production with microcarriers, on which the cells were almost confluent monolayers similar to those in T-flasks, was performed. Utilization of the cells, which had limited serum in the growth phase, resulted in an increase in production. Moreover, dilution of the basal components of the medium at initiation of the production phase markedly promoted t-PA production. The volumetric productivity was stable for 30 days at 100 IU/cm³ per day. The cells were then mostly retained on microcarriers. Thus, an effective and scalable method of t-PA production by normal fibroblast cells was developed. Offprint requests to: S. Mitsuda     

A fibroblast-derived tumor cytotoxic factor/F-TCF (hepatocyte growth factor/HGF) has multiple functions in vitro.

We previously demonstrated that a tumor cytotoxic factor(F-TCF) purified from the culture broth of human embryonic lung diploid fibroblast, IMR-90 cells was one of the human hepatocyte growth factors (hHGFs). In the present report, we demonstrate its biological functions. F-TCF showed moderate cytotoxicity on human tumor cell lines KB, BG-1, MCF-7 and Hs913 T, and strong cytotoxicity on mouse tumor cell lines Sarcoma 180, Meth A sarcoma and P388. On the contrary, F-TCF was a potent mitogen not only for adult rat hepatocytes, but also for human endothelial cells, HUVEC and human melanocytes. Moreover, F-TCF induced the differentiation of premyelocyte leukemia, HL-60 cells into morphologically granulocyte-like cells. These biological functions suggest that F-TCF is an effector molecule responsible for inflammation and repair in injured tissues including tumor and liver.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Effect of changes in thyroid state on metabolism of thyroxine by rat placenta

We studied the effect of the state of the thyroid on T4 monodeiodination in the rat placenta, and it was compared with those in the liver and kidney. The tissues, maternal serum, and amniotic fluid were obtained from pregnant rats. The tissues were homogenized in cold 50 mM Tris-HCl buffer, pH 7.5. The homogenate (1 mg protein) was incubated at 37 degrees C for 60 min with 1 microgram T4 in the presence of 5 mM DTT. The T3 and reverse T3 generated in the reaction mixture were extracted into cold ethanol and measured by RIAs. The conversion of T4 to reverse T3 in rat placenta was not significantly changed in MMI-induced hypothyroidism or T4 induced hyperthyroidism. On the other hand, conversion of T4 to T3 in the liver and kidney were changed in parallel with the thyroid state. The concentration of reverse T3 in the amniotic fluid was increased in accordance with the increase in the maternal serum T4 concentration. These results indicate that the placental T4 inner ring deiodination is not affected by the thyroid state, and that the change in the amniotic fluid reverse T3 concentration in this study is mainly dependent upon the change in maternal thyroid function.     

Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli

Summary DNA fragments of 3.4 kb containing the gyrB gene were cloned from Escherichia coli KL-16 and from spontaneous nalidixic acid-resistant mutants. The mutations (nal-24 and nal-31) had been determined to be in the gyrB gene by transduction analysis. Nucleotide sequence analysis of the cloned DNA fragments revealed that nal-24 was a G to A transition at the first base of the 426th codon of the gyrB gene, resulting in an amino acid change from aspartic acid to asparagine, and nal-31 was an A to G transition at the first base of the 447th codon, resulting in an amino acid change from lysine to glutamic acid. This indicates that mutations in the gyrB gene are responsible for nalidixic acid resistance.     

Postischemic Cerebral Lipid Peroxidation In Vitro: Modification by Dietary Vitamin E

Using an in vitro system, we studied the effect of postischemic reoxygenation on cerebral lipid peroxidation in relation to the dietary intake of vitamin E (VE) in rats. Homogenates prepared from VE-deficient, -normal, and -supplemented brains, which were previously rendered ischemic for 30 min by decapitation, were incubated under air or nitrogen gas for 60 min. The extent of peroxidation in brain tissue was estimated by a thiobarbituric acid (TBA) test and by diene conjugation in total lipid extracts. The brain levels of alpha-tocopherol and of total and free fatty acids (FAs) were also determined. Aerobic incubation increased TBA reactants in all dietary groups; the effect was largest in the VE-deficient group, intermediate in the VE-normal group, and smallest in the VE-supplemented group. In contrast, nitrogen incubation did not alter the basal levels of TBA reactants except for a small rise associated with VE deficiency. Conjugated dienes changed in parallel with TBA reactants. alpha-Tocopherol decreased after aerobic incubation and also, to a lesser degree, after nitrogen incubation in each dietary group. Only in the reoxygenated samples of the VE-deficient group was there a significant fall in total polyunsaturated FAs. The levels of free FAs continuously increased throughout ischemia and subsequent incubation. However, the level of free polyunsaturated FAs was similar after aerobic and nitrogen incubation in each dietary group, and was not affected by VE. Thus, cerebral reoxygenation after ischemia propagates peroxidative reactions within esterified polyunsaturated FAs. The modification by VE of reoxygenation-induced lipid peroxidation suggests free radical mediation.     

Quantitative fluorescence microscopy on dynamic changes of plastid nucleoids during wheat development

Summary Dynamic change of plastid nucleoids (pt nucleoids) was followed by fluorescence microscopy after staining with 46-diamidino-2-phenyl indole (DAPI). The fluorescence image was quantified with a supersensitive photonic microscope system based on photon counting and image analysis. The results showed that small pt nucleoids located in the center of proplastids in the dry seed increased in size after imbibition and formed highly organized ring structures in the dark, which divided into ca. 10 pieces within 3 days. Corresponding to this morphological change, DNA content of a plastid multiplied 7.5 fold. Total increase in DNA content of pt nucleoids per cell was 34 times as that of dry seed, as plastid multiplied 4.6 times in the average during this period. Upon light illumination small pt nucleoids having basic genome size were separated from divided pt nucleoids, suggesting a relationship with the formation of thylakoid system. The significance of the procedure established in this study is discussed in analysing the dynamic changes of intracellular small genomes.On leave from Department of Biology, Faculty of Science, Nagoya University, Furocho, Chikusaku, Nagoya 464, Japan.