Microbial metabolism of chlorosalicylates: effect of prolonged subcultivation on constructed strains

The hybrid strain Pseudomonas sp. WR4016 was subcultivated with increasing concentrations of 5-chlorosalicylate (510 mM) as sole carbon source over a period of 9 months. At intervals of approximately 3 months derivative strains WR4017, WR4018 and WR4019 were isolated which exhibited higher growth rates and increased substrate tolerance. Comparative analysis of the turnover rates of the key enzymes in chlorosalicylate degradation showed that the adaptation process did not result from structural modifications of these proteins. Instead, balanced over-production of the salicylate hydroxylase and catechol 1,2-dioxygenase prevented the accumulation of toxic chlorocatechols and accounted for the reduction of the doubling times with 4- or 5-chlorosalicylate. A comparative analysis of a genetically engineered chlorosalicylate degrader PL300-1 showed similar regulatory patterns as the most advanced isolate WR4019 from the adaptation series.     

Spontaneous cytotoxicity of macrophages against pancreatic islet cells

Activated peritoneal macrophages were found to lyse syngeneic [3H]leucine-labeled pancreatic islet cells or rat insulinoma cells after 15 h of coculture at 37 degrees C. Lysis was verified by electron microscopic analysis. Islet cell lysis was dependent on the T:E ratio and was comparable with P815 and L929 tumor cells used as targets. The cytotoxic activity was localized in the adherent fraction of Corynebacterium parvum activated peritoneal cells and was destroyed by incubation of cells with macrophage-toxic silica particles. Syngeneic thyrocytes and hepatocytes were found to be resistant to the cytolytic action of activated macrophages. It has been shown previously that macrophages contribute to pancreatic islet inflammation. The present in vitro analysis demonstrates that macrophages can function as effector cells in islet destruction.     

Adaptive response to 2 low doses of X-rays in human blood lymphocytes

Human peripheral blood lymphocytes exposed to a single adaptive dose of 1 cGy X-rays or 2 adaptive doses, each of 1 cGy, were found to be equally resistant to the induction of chromosome damage by subsequent challenge with a high dose of 1 Gy X-rays, as compared to cells that were not pre-exposed. They responded with a significantly reduced incidence of chromatid and isochromatid breaks. These results indicate the presence of an inducible chromosomal repair mechanism in human blood lymphocytes and confirm the observations made by earlier investigators. The incidence of chromosome damage was found to be similar in the lymphocytes pre-exposed to a single or 2 adaptive doses, suggesting that, under the conditions tested, the second adaptive dose did not offer any additional protection against the chromosome damage induced by the challenge dose.     

Metabolism of 2-chloro-4-methylphenoxyacetate by Alcaligenes eutrophus JMP 134

2-Chloro-4-methylphenoxyacetate is not a growth substrate for Alcaligenes eutrophus JMP 134 and JMP 1341. It is, however, being transformed by enzymes of 2,4-dichlorophenoxyacetic acid metabolism to 2-chloro-4-methyl-cis, cis-muconate, which is converted by enzymatic 1,4-cycloisomerization to 4-carboxymethyl-2-chloro-4-methylmuconolactone as a dead end metabolite. Chemically, only 3,6-cycloisomerization occurs, giving rise to both diastereomers of 4-carboxychloromethyl-3-methylbut-2-en-4-olide. Those lactones harbonring a chlorosubstituent on the 4-carboxymethyl side chain were surprisingly stable under physiological as well as acidic conditions.     

The role of adenosine 3',5'-cyclic monophosphate in the density-dependent regulation of growth and tyrosinase activity of B-16 melanoma cells.

Incubation of cultured B-16 melanoma cells with 1-methyl-3-isobutyl xanthine (MIX) produced a sustained rise in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) which preceded an increase in the specific activity of tyrosinase (EC 1.10.3.1). Cultures of two clones of melanoma cells, one having a mean population doubling time twice that of the other, showed density-dependent inhibition of growth. The tyrosinase activity of each line increased progressively during logarithmic growth, reaching maximal values shortly after the cultures achieved confluence. Intracellular cAMP levels fell during logarithmic growth, being minimal in confluent cultures. The stimulatory effects of MIX and confluence on tyrosinase activity were additive. Cells plated at high density had a lower tyrosinase activity than cells allowed to achieve a similar density by successive division from sparsely planted cultures although the intracellular cAMP levels of such cultures were not different. We support the observations of other investigators that agents which increase intracellular cAMP concentrations can both inhibit cell division and stimulate tyrosinase activity. There are, however, mechanisms for increasing tyrosinase activity and inhibiting cell division which are expressed as B-16 melanoma cells approach confluence and which are not mediated by an increase in intracellular cAMP concentrations.     

beta-Phosphorothioate analogs of nucleotide sugars are resistant to hydrolytic degradation and utilized efficiently by glycosyltransferases.

The alpha- and beta-phosphorothioate analogs of UDP-Gal and UDP-Glc, in which a sulfur is exchanged for a non-bridging oxygen at one of the phosphate groups, have been synthesized and tested for their resistance to enzymatic degradation and for their usefulness in glycosyltransferase reactions. The alpha analogs were found to be no more resistant to hydrolysis than the native nucleotide sugars, but as previously reported (R. B. Marchase et al. (1987) Biochim. Biophys. Acta 916: 157) the beta S analogs were approximately 10 times more resistant. The beta S analog and native UDP-Glc were found to have comparable Km's when used in assays for glucosylphosphoryl dolichol synthase with rat liver and hen oviduct microsomes, although the apparent Vmax of the reaction was about twofold higher for the analog, presumably due to its resistance to degradation. Partially purified 4 beta-galactosyltransferase exhibited a Vmax with (beta S)UDP-Gal that was only slightly lower than that with UDP-Gal and a Km that was slightly increased. The effectiveness of the analog was especially apparent in assays for 4 beta-galactosyltransferase on intact sperm and in rat liver homogenates, in which hydrolysis of the normal substrate was very rapid and net incorporation was at least 4 times greater with the beta S analog in each system.     

Hereditary motor endplate disease (med) of the mouse: Observations on dissociated myogenic cells and their development in culture

Summary Myogenic cells from mice homozygous for the lethal mutation motor endplate disease (med/med) were grown in culture. Like muscle cells taken from wild type (+/?) litter mates they fused to form myotubes which contracted, developed cross striations, and exposed acetylcholine receptors (AChR) on their surface. However, a decrease of 30% in the number of mononucleated cells per unit fresh weight of muscle was observed as early as 2–3 days postnatal, i.e., at least one week prior to the onset of physiological symptoms. Hence, in addition to influencing the functional maintenance of motor endplates, the med gene seems to control early events in muscle development.     

Replication and fine mapping of asthma-associated loci in individuals of African ancestry

Asthma originates from genetic and environmental factors with about half the risk of disease attributable to heritable causes. Genome-wide association studies, mostly in populations of European ancestry, have identified numerous asthma-associated single nucleotide polymorphisms (SNPs). Studies in populations with diverse ancestries allow both for identification of robust associations that replicate across ethnic groups and for improved resolution of associated loci due to different patterns of linkage disequilibrium between ethnic groups. Here we report on an analysis of 745 African-American subjects with asthma and 3,238 African-American control subjects from the Candidate Gene Association Resource (CARe) Consortium, including analysis of SNPs imputed using 1,000 Genomes reference panels and adjustment for local ancestry. We show strong evidence that variation near RAD50/IL13, implicated in studies of European ancestry individuals, replicates in individuals largely of African ancestry. Fine mapping in African ancestry populations also refined the variants of interest for this association. We also provide strong or nominal evidence of replication at loci near ORMDL3/GSDMB, IL1RL1/IL18R1, and 10p14, all previously associated with asthma in European or Japanese populations, but not at the PYHIN1 locus previously reported in studies of African-American samples. These results improve the understanding of asthma genetics and further demonstrate the utility of genetic studies in populations other than those of largely European ancestry.     

Preschool Children Fail Primate Prosocial Game Because of Attentional Task Demands

Various nonhuman primate species have been tested with prosocial games (i.e. derivates from dictator games) in order to better understand the evolutionary origin of proactive prosociality in humans. Results of these efforts are mixed, and it is difficult to disentangle true species differences from methodological artifacts. We tested 2- to 5-year-old children with a costly and a cost-free version of a prosocial game that differ with regard to the payoff distribution and are widely used with nonhuman primates. Simultaneously, we assessed the subjects’ level of Theory of Mind understanding. Prosocial behavior was demonstrated with the prosocial game, and did not increase with more advanced Theory of Mind understanding. However, prosocial behavior could only be detected with the costly version of the game, whereas the children failed the cost-free version that is most commonly used with nonhuman primates. A detailed comparison of the children’s behavior in the two versions of the game indicates that the failure was due to higher attentional demands of the cost-free version, rather than to a lack of prosociality per se. Our results thus show (i) that subtle differences in prosociality tasks can substantially bias the outcome and thus prevent meaningful species comparisons, and (ii) that like in nonhuman primates, prosocial behavior in human children does not require advanced Theory of Mind understanding in the present context. However, both developmental and comparative psychology accumulate increasing evidence for the multidimensionality of prosocial behaviors, suggesting that different forms of prosociality are also regulated differentially. For future efforts to understand the evolutionary origin of prosociality it is thus crucial to take this heterogeneity into account.