Toxoplasma gondii: Microassay to differentiate toxoplasma inhibiting factor and interleukin 2

Using a sensitive, economical, and reproducible microassay, the relationship of toxoplasma inhibiting factor to interleukin 2 has been examined. The assay developed took advantage of the observation that (1) Toxoplasma gondii tachyzoites replicated efficiently in the murine monocytic cell line, RAW 264; (2) treatment of RAW 264 cells with toxoplasma inhibiting factor prevented intracellular replication of the parasite to an extent similar to that observed with identical treatment of freshly isolated murine peritoneal exudate cells; and (3) [³H]uracil incorporation was an efficacious means to quantify replication (or inhibition of replication) of tachyzoites within the cell line. Although toxoplasma inhibiting factor and interleukin 2 were both present in the same lectin -and antigen-stimulated splenocyte supernatant fluids, results from microassays strongly suggested that the molecules were two distinct entities.     

Titration of the binding sites for the oligomycin-sensitivity conferring protein in beef heart submitochondrial particles

The binding parameters of the oligomycin-sensitivity conferring protein (OSCP) in inside-out particles from beef heart mitochondria have been tested by means of two assays, the oligomycin-sensitive ATP-Pi exchange, and the oligomycin-sensitive ATP hydrolysis. The total number of OSCP binding sites in A particles was equal to 220 pmol/mg particle protein. Each mole of ATPase active site was able to bind 1.1 +/- 0.5 mol OSCP with Kd 1.7 nM.     

Possible physiological role of epidermal growth factor in the development of the mouse mammary gland during pregnancy

Epidermal growth factor stimulated cell proliferation in a primary mammary epithelial cell culture derived from mice at different stages of pregnancy. Moreover, the peptide hormone inhibited casein production induced by the synergistic actions of insulin, cortisol and prolactin. The inhibitory effect of epidermal growth factor was influenced by the gestational stages of the mammary gland. These effects of epidermal growth factor were exerted at physiological concentrations. The dual actions of epidermal growth factor on mammary cells implicate its participation in regulation of the growth and differentiation of the mammary gland during pregnancy.     

Structure determination of the major asparagine-linked sugar chain of human factor VIII--von Willebrand factor

N-glycosidically-linked glycans released by hydrazinolysis of human factor VIII/von Willebrand factor (FVIII/vWf) were separated by high-voltage electrophoresis. Five fractions were obtained, one of them representing 60% of the total amount of the N-glycosidically-linked glycans of FVIII/vWf. On the basis of the carbohydrate composition, methylation analysis and 500 MHz 1H-NMR spectroscopy, we describe the primary structure of this major glycan which is of the monosialylated and monofucosylated biantennary N-acetyllactosaminic type.     

Factor XIII-mediated cross-linking of NH2-terminal peptide of alpha 2-plasmin inhibitor to fibrin

The NH2-terminal 12-residue peptide of alpha 2-plasmin inhibitor, Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Gly-Leu-Lys-NH2 . AcOH, was found to be a good substrate for plasma transglutaminase (activated blood coagulation factor XIII) and rapidly incorporated into fibrin by the enzyme. A high concentration of the peptide inhibited the enzyme-mediated cross-linking of alpha 2-plasmin inhibitor to fibrin probably by competing with the inhibitor for the same site of fibrin alpha-chain.     

Isolation of a brain peptide identical to the intestinal PHI (peptide HI)

The isolation of a brain peptide identical to the intestinal peptide PHI (peptide HI) is described. The peptide was isolated from porcine brain extract using a chemical assay method based on its C-terminal isoleucine amide structure. The complete amino acid sequence of the peptide was found to be: His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Phe-Ser-Arg-Leu-Leu-Gly-Gln-Leu-Ser-Ala- Lys-Lys-Tyr-Leu-Glu-Ser-Leu-Ile-NH2. This sequence is identical to the intestinal peptide thus demonstrating PHI to be a brain-gut peptide. The role of PHI in the central nervous system as a neurotransmitter or neuromodulator is discussed.     

A rapid one-step purification of NADPH-cytochrome c (P-450) reductase from rat liver microsomes

NADPH-cytochrome c (P-450) reductase from liver microsomes of phenobarbital-treated rats has been purified in a single step by affinity chromatography on agarose-hexane-adenosine 2',5'-diphosphate. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme assay, and radioimmunoassay the protein obtained by this single step procedure is as pure as that isolated by multicolumn procedures.     

Inactivation of Rhodospirillum rubrum coupling factor by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Modification of a tyrosine protected by phosphate

Chemical modification of Rhodospirillum rubrum chromatophores by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) results in inactivation of photophosphorylation, Mg²⁺-ATPase, oxidative phosphorylation and ATP-driven transhydrogenase, with apparent first-order kinetics. Other energy-linked reactions such as light-driven transhydrogenase and light-dependent proton uptake were insensitive to NBD-Cl. The Ca²⁺-ATPase activity of the soluble coupling factor from chromatophores (R. rubrum F₁) was inactivated by NBD-Cl with kinetics resembling those described for Mg²⁺-ATPase and photophosphorylation activities of chromatophores. Both NBD-chromatophores and NBD-R. rubrum F₁ fully recovered their activities when subjected to thiolysis by dithioerythritol. Phosphoryl transfer reactions of chromatophores and Ca²⁺-ATPase activity of R. rubrum F₁ were fully protected by 5 mM P_i against modification by NBD-Cl. ADP or ATP afforded partial protection. Analysis of the protection of Ca²⁺-ATPase activity by P_i indicated that NBD-Cl and P_i are mutually exclusive ligands. Spectroscopic studies revealed that tyrosine and sulfhydryl residues in R. rubrum F₁ underwent modification by NBD-Cl. However, the inactivation was only related to the modification of tyrosine groups.     

A Highly Sensitive Enzyme Immunoassay for Mouse β Nerve Growth Factor

Abstract: A sensitive two-site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti-mouse β NGF antibody IgG coated to a polystyrene tube and anti-mouse β NGF antibody Fab'-linked β- d -galactosidase (β- d -galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two-site radioimmunoassay; (b) antibody Fab'-β- d -galactosidase complex is more stable than ¹²⁵I-labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice.     

Effect of TPA on the mutagenicity of caffeine in the soybean mutation test

12-O-Tetradecanoylphorbol-13-acetate (TPA) is a well-known tumor promoter in mouse-skin carcinogenesis. Its effects on mutagenesis in a soybean test system were examined, and the effects were judged from the appearance of spots of various colors on the leaves. When soybean seeds were treated with TPA plus 0.03% caffeine, the frequency of spots per leaf decreased significantly and in proportion to the concentration of TPA. TPA alone at concentrations of 1–20 μg/ml did not induce any mutations. Mutations induced by γ-rays were not affected by administration of TPA either before or after exposure to γ-rays. The mechanism of suppression by TPA of mutations induced by caffeine is discussed.