Mitogenic Activity of Cytoplasmic Membranes Isolated from L-Forms of Staphylococcus aureus

Cytoplasmic membranes of L-forms of Staphylococcus aureus exerted a strong mitogenic effect on splenocytes of athymic nude mice as well as normal mice, while a cytoplasmic fraction of the same bacteria did not show definite mitogenicity. The mitogenic principle(s) of the membrane fraction was resistant to treatment with trypsin and was heat stable (at 100 C for 10 min). The active principle(s) in the insoluble residue of the membrane fraction digested with trypsin was not extracted with cold acetone, but could be solubilized by extraction with a cold chloroform-methanol mixture (2:1, v/v). The mitogenic principle(s) in the extract was fractionated by silicic acid column chromatography. Among five fractions separated by chromatography, fractions eluted with chloroform-methanol mixtures (1:1 and 1:20, v/v) were found to be strongly mitogenic. The cytoplasmic membranes of the L-forms also exerted a definite mitogenic effect on guinea pig splenocytes, but not on the thymocytes.     

Nudeotide Pools and Purine Metabolism in Tissue Cultures of Wild-Type Datura innoxia and Adenine-Requiring Auxotroph

Cells of the auxotrophic mutant, Ad1, of Datura innoxia requiredadenine, adenosine, or inosine for their growth on solid agarmedium which contained Murashige-Skoog salts, 2,4-dichloro-phenoxyaceticacid, and sucrose. Thirteen purine and pyrimidine nucleotidesin extracts of wild-type and Ad1 cells were separated and quantifiedby HPLC. Levels of ADP-glucose and UMP were significantly higherin Ad1 than in wild-type cells, but those of other nucleotideswas found when Ad1 cells were transferred to fresh medium withoutadenine. The rate of the biosynthesis de novo of purines, asestimated from the rate of incorporation of ¹⁴C from [2-¹⁴C]-glycine and [¹⁴C]formate into adenine nucleotides, was reducedin Ad1 cells to 21 and 13% of the wild-type rate, respectively.The activities involved in the salvage of adenine and adenosinein Ad1 cells were similar to those in wild-type cells. Ad1 cellshad the capability to convert adenine to guanine nucleotidesand guanine to adenine nucleotides. ¹ Part 27 of the series, "Metabolic Regulation in Plant CellCulture". (Received March 7, 1988; Accepted August 3, 1988)     

The dynamical attainability of ESS in evolutionary games

In this paper, the attainability of ESS of the evolutionary game among n players under the frequency-independent selection is studied by means of a mathematical model describing the dynamical development and a concept of stability (strongly determined stability). It is assumed that natural selection and small mutations cause the phenotype to change gradually in the direction of fitness increasing. It is shown that (1) the ESS solution is not always evolutionarily attainable in the evolutionary dynamics, (2) in the game where the interaction between two species is completely competitive, the Nash solution is always attainable, and (3) one of two species may attain the state of minimum fitness as a result of evolution. The attainability of ESS is also examined in two game models on the sex ratio of wasps and aphids in light of our criterion of the attainability of ESS.     

Northern hybridization analysis of VH gene expression in murine monoclonal antibodies directed to cancer-associated ganglioside antigens having various sialic acid linkages

The expression of the VH genes in 46 murine hybridoma cells that secrete mAb directed to the cancer-associated carbohydrate Ag, especially acidic glycolipids such as gangliosides and sulfated glycoplipids, was analyzed by Northern hybridization of poly(A)+ RNA of hybridoma with cDNA probes for nine VH gene families. Different hybridomas tended to express VH genes of the same family when the cognate Ag had the same or similar carbohydrate structures; i.e., the VH genes of the J558 family (group 1) were preferentially expressed in the mAb directed to various gangliosides that have NeuAc alpha (or NeuGc alpha) 2-3 and/or 2-8 linkage (71%), the most common linkage of sialic acid residues in the gangliosides of higher animals, and the hybridomas directed to sulfated glycolipids also expressed mainly the VH genes of the J558 family (80%). In contrast, the five mAb directed to various gangliosides with NeuAc alpha 2-6 linkage were exclusively encoded by the VH genes of Q52 family (group 2, 100%), and three antibodies directed to gangliosides with a NeuAc alpha 2-9 linkage all expressed genes of J606 family (group 6, 100%). The VH family usage was largely correlated with the linkage of sialic acid residues in the cognate carbohydrate Ag, but was not correlated at all with the difference in the fine specificities toward the core neutral carbohydrate chain, to which the sialic acid residues were attached. These findings suggest that the VH gene family in these anticarbohydrate antibodies is selected, depending primarily on the linkage of the sialic acid residues in carbohydrate Ag; these residues form the immunodominant sugar residue in the respective antigenic determinant.     

Behavioral effects of benzodiazepine antagonists in chlordiazepoxide tolerant and non-tolerant rats

Rats were trained to respond under 3-min fixed-interval schedules of food presentation, and effects of the benzodiazepine-receptor ligands, flumazenil, 2-(4-methoxy-phenyl)-pyrazolo[4,3-c]quinolin-3(5H)-one (CGS 9895), 3-carbo-t-butoxy-beta-carboline (beta-CCtB), and beta-carboline-3-carboxylic acid ethyl ester (beta-CCE) were assessed before and after the induction of tolerance to chlordiazepoxide. Before daily administration of chlordiazepoxide, none of the antagonists produced appreciable effects on rates of responding up to doses of 32.0 mg/kg i.p. beta-CCE was the only antagonist studied at a higher dose (100.0 mg/kg i.p.), which decreased response rates. After 23 days of daily chlordiazepoxide administration (oral doses started at 10 and increased to 100 mg/kg/day by the 17th day), dose-effect curves for chlordiazepoxide were shifted to the right by about one-half log unit. Subjects were also more sensitive to the flumazenil, CGS 9895, and beta-CCtB, however, since these drugs produced only small effects in non-tolerant subjects, precise estimates of the degree of the shift in dose-effect curves could not be estimated. However, there were differences in the changes in the dose-effect curves induced by chlordiazepoxide tolerance. These results suggest differences in mechanism of action of antagonists in tolerant and non-tolerant subjects, and further that the sensitivity that is induced to antagonists in tolerant subjects is not conferred equally to all drugs having benzodiazepine antagonist activity.     

DNA cleavage by hydroxyl radicals generated in a vanadyl ion-hydrogen peroxide system.

Vanadyl ion (+4 oxidation state) has been shown to be an effective agent for chemoprotection of cancers in animals. For understanding the mechanism, distribution of vanadium was studied. More vanadium was found to accumulate in the nuclei of the liver of rats when it was given as vanadyl sulfate than when it was given as sodium vanadate (+5 oxidation state). The reactivity of vanadyl ion with DNA was investigated by the DNA cleavage technique and the reaction mechanism by ESR spectroscopy. Incubation of double-strand DNA with vanadyl ion and hydrogen peroxide resulted in marked concentration- and pH-dependent DNA cleavage. Studies by the ESR spin-trap method demonstrated that hydroxyl radicals are generated during the reactions of vanadyl ion with hydrogen peroxide. Thus the antineoplastic action of vanadyl ion is proposed to be due to DNA cleavage by hydroxyl radicals generated in the cells.     

Synthesis of Serotonin in Traumatized Rat Brain

Abstract: Previous studies have demonstrated that focal freezing lesions in rats cause a widespread decrease of cortical glucose use in the lesioned hemisphere and this was interpreted as a reflection of depression of cortical activity. The serotonergic neurotransmitter system was implicated in these alterations when it was shown that (1) cortical serotonin metabolism was increased widely in focally injured brain and (2) inhibition of serotonin synthesis prevented the development of cortical hypometabolism. In the present studies we applied an autoradiographic method that uses the accumulation of the ¹⁴C-labeled analogue of serotonin α-methylserotonin to assess changes in the rate of serotonin synthesis in injured brain. The results confirmed that 3 days after the lesion was made, at the time of greatest depression of glucose use, serotonin synthesis was significantly increased in cortical areas throughout the injured hemisphere. The increase was also seen in the dorsal hippocampus and area CA3, as well as in the medial geniculate and dorsal raphe, but not in any other subcortical structures including median raphe. Present results suggest that the functional changes in the cortex of the lesioned hemisphere are associated with an increased rate of serotonin synthesis mediated by activation of the dorsal raphe. We also documented by α-[¹⁴C]aminoisobutyric acid autoradiography that there was increased permeability of the blood-brain barrier, but this was restricted to the rim of the lesion.     

The inheritance of natural chromosomal polymorphisms in the aphid Myzus persicae (Sulzer)

Two types of chromosomal abnormality have been found in natural populations of Myzus persicae in Japan. One type is apparently due to an autosome 3 dissociation, giving a 2n=13 karyotype. The other is interpreted as a translocation between autosomes 1 and 3, resulting in a 2n=12 complement with marked structural heterozygosity. In laboratory crosses, both types of abnormality were inherited through the sexual phase. The proportions of each type in the F₁ agreed well with expectations, except that no forms homozygous for the translocation were obtained from crosses between translocation heterozygotes, and no karyotypes with both the translocation and the dissociation were obtained when translocated and dissociated forms were crossed. In the F₁ of one cross a triploid clone with the autosomal 1,3 translocation was obtained.     

New fluorogenic substrates for alpha-thrombin, factor Xa, kallikreins, and urokinase

Twenty peptide-4-methylcoumarin amides (MCA) were newly synthesized and tested as possible substrates for alpha-thrombin, factor Xa, kallikreins, urokinase, and plasmin. These fluorogenic peptides contained arginine-MCA as the carboxyl-terminus. Release of 7-amino-4-methylcoumarin was determined fluorometrically. Of these peptides, the following were found to be specific substrates for individual enzymes: Boc-Val-Pro-Arg-MCA for alpha-thrombin, Boc-Ile-Glu-Gly-Arg-MCA, and Boc-Ser-Gly-Arg-MCA for factor Xa, Z-Phe-Arg-MCA for plasma kallikrein, Pro-Phe-Arg-MCA for pancreatic and urinary kallikreins, and glutaryl-Gly-Arg-MCA for urokinase. Moreover, these peptide-MCA substrates were resistant to plasmin.