New procedure for DNA transfection with polycation and dimethyl sulfoxide.

A new procedure for DNA transfection has been developed in a system of chicken embryo fibroblast cells and cloned Rous sarcoma virus DNA by using a polycation reagent as a mediator to adsorb DNA to the cell surface and dimethyl sulfoxide as an agent to facilitate the uptake of adsorbed DNA by the cells. In this new, simple, and convenient polycation-dimethyl sulfoxide transfection, which requires no carrier DNA even with small amounts of DNA, the number of transformed cell foci induced by Rous sarcoma virus DNA was proportional to the dose of the transfecting DNA, and chicken embryo fibroblast cells were successfully transformed by v-src-containing subgenomic DNA as well.     

Purification and some properties of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Neurospora crassa.

Uridine diphosphate N-acetylglucosamine pyrophosphorylase (EC. 2.7.7.23) of Neurospora crassa has been purified approximately 210-fold with dithiothreitol as the stabilizing agent by use of chromatographic techniques. The enzyme preparation appeared to be homogeneous when subjected to electrophoresis. The molecular weight was estimated as approximately 37 000 by gel filtration. The enzyme had an isoelectric point around pH 4.4. Maximum activity of the enzyme was observed at pH 7.5. The enzyme required Mg2+, which may be replaced by other divalent cations such as Mn2+ and Co2+ for lesser degrees of effectiveness. The enzyme was strictly specific for UDP-N-acetylglucosamine as the substrate. The estimated values of Km were 2.2 mM for UDP-N-acetylglucosamine and 5.4 mM for inorganic pyrophosphate. The enzyme activity was highly stimulated by the addition of dithiothreitol or dithioerythritol but was lost by sulfhydryl inhibitory reagents.     

Different responses to some stimulators of prolyl hydroxylase activities in various rat organs.

Prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) of soluble fraction (105 000 X g supernatant) of rat granulation tissues was markedly enhanced by addition of nucleoside triphosphates to the assay medium. But the stimulatory activities of nucleoside triphosphates were very different in fractions derived from tissues of rat. In skin, lung or whole fetal tissues other than granuloma, GTP enhanced the enzymatic activity by 3-4 fold. On the other hand, in kidney, liver and spleen tissues it brought about no enhancement. The same results were obtained even if ATP regenerating system was added in the assay medium. The stimulatory effect of nucleoside triphosphates was not seen with the soluble fraction of liver, but it appeared with the enzyme fraction purified by affinity column chromatography. The same phenomenon was observed by addition of bovine serum albumin instead of nucleoside triphosphates as stimulator. We discuss the possible reasons as to why the responses of the enzyme to stimulators were quite different among various tissues.     

A case of Turner's syndrome with hyperthyroidism

A female patient with classical gonadal dysgenesis associated with Graves' disease is reported. The karyotype was mosaicism of 45,X/46,X,i(Xq). The relationship among Graves' disease, Hashimoto's thyroiditis and Turner's syndrome is discussed along with a review of the reported cases.     

Co-denitrification by the denitrifying system of the fungus Fusarium oxysporum

Abstract Nitrogen compounds such as azide, salicylhydroxamic acid, and possibly ammonium ions were converted to nitrous oxide (N₂O) or dinitrogen (N₂) by Fusarium oxysporum under denitrifying conditions. Nitrogen atoms in these compounds were combined with another nitrogen atom from nitrite to form a hybrid N₂O species. The fungus exhibited much higher converting activities as compared with similar reactions catalyzed by bacterial denitrifiers. We thus propose the phenomenon be called co-denitrification, which means that such nitrogen compounds are denitrified by the system induced by nitrite (or nitrate) but are incapable by themselves of inducing the denitrifying system.     

A cellular protein is immunologically crossreactive with and functionally homologous to the Fujinami sarcoma virus transforming protein

We obtained a regressing-tumor antiserum specific for the unique sequence of the transforming protein P140 of Fujinami sarcoma virus by injecting Fischer rats with syngeneic embryo cells transformed with Fujinami sarcoma virus. This serum is capable of immunoprecipitating a protein of 98,000 daltons from cell extracts of normal, uninfected chicken bone marrow cells. This normal cellular protein (NCP98) was shown to be structurally related to P140, sharing the majority of ³⁵S-methionine-labeled tryptic peptides with the viral gene product P140. NCP98 is a phosphoprotein in vivo, with an associated in vitro protein kinase activity, capable of phosphorylating specifically at tyrosine residues of NCP98 itself and a-casein, an externally added substrate. This kinase activity is biochemically indistinguishable from the kinase activity associated with P140 by all criteria tested. Moreover, in vitro-phosphorylated NCP98 and P140 shared the same phosphopeptides. The expression of NCP98 is tissue-specific. It is readily detectable in bone marrow cells and detectable to a lesser extent in liver and lung cells from 6–18 day old chickens.     

Formation of the aldehydic choline glycerophospholipids in human red blood cell membrane peroxidized with an azo initiator.

The production of phospholipid hydroperoxide and aldehydic phospholipid was examined in human red blood cell (RBC) membranes after peroxidation with 2,2-azobis(2-amidinopropane)dihydrochloride (AAPH) or xanthine/xanthine oxidase (XO/XOD/Fe3+). Both radical-generation systems caused a profound decrease in the amount of polyunsaturated fatty acid (PUFA) in choline glycerophospholipid (CGP) and induced formation of peroxidized CGP in RBC membranes to different extents. No consistent generation of peroxidized lipids from CGP was evident after peroxidation with XO/XOD/Fe3+, which caused the apparent decomposition of phospholipids and the formation of large amounts of thiobarbituric acid-reactive substance (TBARS). On the other hand, CGP hydroperoxide was formed as a primary product of peroxidation with AAPH. Aldehydic CGP was also detected as a secondary product of hydroperoxide decomposition in AAPH-peroxidized RBC membranes. Aldehydic CGP was preferentially generated from arachidonoyl CGP rather than from linoleoyl CGP in AAPH-peroxidized membranes. AAPH mainly oxidized CGP to hydroperoxide and aldehydic phospholipids. The sum of hydroperoxide and aldehyde of CGP corresponded to the loss of CGP due to peroxidation by AAPH. This result indicates that CGP was mainly converted into these two oxidized phospholipids in AAPH-peroxidized RBC membranes.