Rapid Identification by Polymerase Chain Reaction of Staphylococcal Exfoliative Toxin Serotype A and B Genes

A new system was designed to detect staphylococcal exfoliative toxin A (ETA) and B (ETB) genes by the polymerase chain reaction (PCR). The primer pairs for the ETA gene (eta) were 20 and 20-mer, and its PCR product was a 741-bp eta fragment, while the primer pairs for the ETB gene (etb) were also 20 and 20-mer, and its PCR product was a 629-bp etb fragment. When these primers were simultaneously used in the PCR, the two types of ET were clearly detected as two bands in an ETA and ETB double-producer using only one colony within 3 hr. We examined 66 strains of Staphylococcus aureus isolated from patients with staphylococcal scalded skin syndrome (SSSS) and compared the results obtained by ELISA and PCR. The same results were obtained for 56 of the strains, i.e., 30 strains were ETA producers, 20 strains were ETB producers, and 6 strains were double-producers. However, positive results were obtained for 5 of the 10 non-ET-producing strains. Two of these strains were judged by PCR as ETA producers and three as ETB producers. Thus, PCR is very sensitive and rapid in detecting ETA and ETB gene fragments in colonies isolated from patients with SSSS.     

In vitro microsome-mediated aflatoxin B1-DNA binding and its inhibition by cytosol of various organs of the hamster and quail

We studied thein vitro activation of aflatoxin B1 (B1) by microsomes and its inactivation by the cytosol of various quail and hamster organs, using B1-DNA binding as an index. The microsomal activity of the liver to bind B1 to DNA was not largely different between the two species and was higher than that of the other organs examined in either species. The microsomal activity of the kidney and lung was very low in the quail compared with the hamster, indicating the very small contribution of the lung and kidney microsomes to the activation of B1 in birds. Only the hamster liver cytosol showed strong inhibition of microsome-mediated B1-DNA binding.     

Switching dynamics and the transient memory storage in a model enzyme network involving Ca2+/calmodulin-dependent protein kinase II in synapses

 The signal processing through a chain of phosphorylation-dephosphorylations mediated by a pair of enzymes, Ca²⁺/calmodulin-dependent protein kinase II and the associated phosphatase, is formulated as a non-autonomous dynamical system in the framework of non-autocatalytic, intraholoenzyme reaction dynamics. A classification of switching characteristics of the system is made in the parameter space comprising the three controllable system parameters: an input-pulse intensity and initial concentrations of the two associated enzymes. It is found that a region of parameter space exists termed the transition zone, that exhibits a quasi-switching behaviour characterized by a signal storage time being prolonged by more than several orders of magnitude (10⁴ times in certain cases) for the increase of two orders of magnitude in the input signal intensity. The effect of alterations of certain rate constants on the quasi-switching property is explored. It is numerically demonstrated that the Ca²⁺/calmodulin-dependent kinase II-related phosphatase is the most important key enzyme for regulating the signal storage time. Received: 25 April 1994/Accepted in revised form: 16 December 1994     

Alcohol and aldehyde dehydrogenase genotypes and drinking behavior of Chinese living in Shanghai

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), the principal enzymes responsible for oxidative metabolism of ethanol, exist in multiple, genetically determined molecular forms. Widely different kinetic properties in some of these isozymes account for the individual differences in alcohol sensitivity. In this study we used the polymerase chain reaction/restriction fragment length polymorphism method to determine the genotypes of the ADH2 and ALDH2 loci of alcoholic and nonalcoholic Chinese living in Shanghai. We also investigated the subjects' drinking patterns by means of semistructured interviews. The alcoholics had significantly lower frequencies of the ADH2² and ALDH2² alleles than did the nonalcoholics, suggesting the inhibitory effects of these alleles for the development of alcoholism. In the nonalcoholic subjects, ADH2² had little, if any, effect, despite the significant effect of the ALDH2² allele in decreasing the alcohol consumption of the individual. Taken together, these results fit the proposed hypothesis for the development of alcoholism, i.e., drinking behavior is greatly influenced by the individual's gentoypes of alcohol-metabolizing enzymes, and the risk of becoming alcoholic is proportionate with the ethanol consumption of the individual.     

Characterization of Methanosarcina mazeii TMA isolated from a paddy field soil

We isolated a methanogenic strain, designated as strain TMA (=DSM 9195), from an enrichment culture inoculated with a Japanese paddy field soil. Strain TMA was Gram positive and strictly anaerobic. Cell shape was pseudosarcina-like, and cells were nonmotile. The strain was able to use methylamines, methanol, H₂–CO₂, and acetate as substrates for methanogenesis, but did not utilize formate. The optimum temperature and optimum pH were 30–37°C and 6.5–7.5 respectively. The G+C content of the DNA was 42.1 mol %. Strain TMA had DNA-DNA hybridization values of more than 80% with Methanosarcina mazeii S-6^T (T = type strain). On the basis of phenotypic and genotypic characteristics, we identified strain TMA as M. mazeii. This is the first methylotrophic methanogen isolated from a paddy field soil and identified to the species level.     

Possible participation of Ca2+, Mg2+-dependent endonuclease in liver DNA fragmentation after N-methyl-N-nitrosourea treatment

We examined the fragmentation of DNA treated with N-methyl-N-nitrosourea under conditions in which Ca2+, Mg2+-dependent endonuclease is active. The molecular mass of DNA found in mouse liver slices treated with methylnitrosurea in the presence of Ca2+ plus Mg2+ was 4 X 10(5) Da. Similar results were obtained with a reconstituted system containing partially purified Ca2+, Mg2+-dependent endonuclease and methylnitrosurea-treated DNA. The enzyme extensively cleaved methylnitrosurea-treated DNA, compared with non-treated DNA. The methylnitrosurea-treated nuclear proteins obtained from mouse liver nuclei had no effect on the DNA fragmentation by the enzyme. Using closed-circular DNA treated with methylnitrosurea, the enzyme produced single-strand cuts in the DNA, as was seen in non-treated, closed-circular DNA, however, the rate of hydrolysis was increased. Ca2+, Mg2+-dependent endonuclease thus warrants further investigation, with regard to the precise mechanism of extensive degradation of DNA in cells treated with carcinogenic alkylating agents.     

Sugar Compositions, Intrinsic Viscosities and Molecular Weights of Hemicellulosic Polysaccharides of the Coleoptile Cell Walls in a Semi-Brachytic and a Normal Type Barley

The sugar compositions and intrinsic viscosities of the hemicellulosicpolysaccharides of the coleoptile cell wall were determinedin a normal type barley and a semi-brachytic type which producedless IAA than the normal type. The major sugar components ofhemicelluloses for both strains were arabinose (Ara), xylose(Xyl) and glucose (Glc). The Ara and Xyl content per unit lengthin the normal type did not change during growth, while thosein the semi-brachytic type decreased during growth. The Glccontents per unit length and per coleoptile decreased duringgrowth in both types of barley. The intrinsic viscosity of hemicellulosesfrom the coleoptile of the normal type was lower than that ofthe semi-brachytic type. These results suggested that the synthesis of arabinoxylan keptpace with the growth of the coleoptile in the normal type butnot in the semi-brachytic type, and that the average mol wtof the hemicelluloses in the normal type was lower than thatin the semibrachytic type. These chemical and physical changesin the hemicellulosic polysaccharides may account for the stuntedcoleoptile of the semi-brachytic barley with its less amountof endogenous IAA. (Received March 14, 1984; Accepted June 8, 1984)     

The chromosomes ofCunninghamia konishii,C. lanceolata,andTaiwania cryptomerioides (Taxodiaceae)

Karyotype analyses were conducted onCunninghamia konishii, Cunninghamia lanceolata, andTaiwania cryptomerioides, all members ofTaxodiaceae. The somatic chromosome number was found to be 2n = 2x = 22 in all species which concurrs with previous reports. The karyotypes are generally asymmetrical with the smaller chromosomes being more submedian than the larger ones. Chromosomes with unusual or specific structures, thought to be associated with the nucleolar organizing region, were found in each species.Cunninghamia species have a marker chromosome pair with an unusually long secondary constriction.Taiwania has an unusually long kinetochore region present in a submedian chromosome pair.     

Ribonucleic acid in the immune response

Summary In the studies of experimental salmonellosis, immunization of mice with a live vaccine SER of S. enteritidis was found to be effective against further infection with virulent S. enteritidis 116-54. Macrophages obtained from the peritoneal cavity, subcutaneous tissue or liver of immunized mice inhibited intracellular growth of bacteria and resisted cell degeneration caused by engulfment of virulent 116-54 bacteria. This immunity was called cellular immunity.We discovered by chance in 1961 a transfer agent of immunity (TA) from the culture fluid of immunized macrophages. This agent is RNA in nature and can be extracted from the spleen, peritoneal exudate cells or the lymph node of immunized animals and is called immune (i) RNA. We could demonstrate antibody activity in macrophages treated in vitro or in vivo with iRNA by the immune adherence hemagglutination technique.Cellular immunity against tumor cells could be transferred in vitro or in vivo to lymphocytes through iRNA prepared from the spleen cells of syngeneic, allogeneic and xenogeneic animals immunized with the tumor cells.We prepared iRNA against antigens capable of inducing humoral antibody production in animals, i.e., RBCs, bacterial toxin, bacterial flagella and hapten-protein conjugates. Serum antibody was not demonstrated in recipient animals of iRNAs by single or repeated injections of these agents. However, in these animals an increase in the number of specific antibody-carrying cells was found as rosette-formers. It was found further that prior injection of iRNA could induce immunologic memory and produced a high titer of humoral antibody after a boosting stimulation with a small dose of the corresponding antigen. The required interval between the first iRNA and the second antigenic stimulation, and the minimal effective doses of iRNA and antigen are described.We studied the interaction of iRNA with either T- or B-cells and with both cells using adoptive transfer system, athymic nude mice and neonatally thymectomized (NT) mice. Immune RNAs against T-dependent and T-independent antigens could not induce the proliferation of antibody-carrying cells in cyclophosphamide-treated (B-cell depleted) mice. But these agents could induce the proliferation of rosette-formers, implying that iRNAs can replace some role of T-cells even against T-dependent antigens. B-cells can be directly activated by treatment with iRNA against both T-dependent and T-independent antigens, and they differentiated into rosette-formers.Passive transfers of iRNA were successful in establishing immunity against infection with S. enteritidis, or immunity to Salmonella flagella, RBCs and hapten-protein conjugates. The ability of iRNA to confer a secondary response of antibody formation is serially and passively transmissible in recipient animals. These facts suggest the presence of some mechanism that is responsible for the amplification of antigenic stimulation in the immune response. The RNA-dependent RNA polymerase and RNA-dependent DNA polymerase are presented and their role in the immune response is discussed.     

High-performance liquid chromatographic determination of hippuric acid in human urine

A method is described for the determination of urinary hippuric acid by high-performance liquid chromatography. The method used ethyl acetate extraction for partial clean-up of the urine. The separation was carried out on a reversed-phase column using 20% methanol in 0.01 M aqueous potassium phosphate containing 0.5% acetic acid as a mobile phase. The column effluent was monitored with a UV detector at 254 nm. Hippuric acid was separated from other normal urine constituents in less than 10 min. Metabolites of xylene and styrene did not interfere with the assay. Analytical recoveries from urine were excellent and peak height and concentration were linearly related.