An Internal Reaction Chamber in Dimethylglycine Oxidase Provides Efficient Protection from Exposure to Toxic Formaldehyde

We report a synthetic biology approach to demonstrate substrate channeling in an unusual bifunctional flavoprotein dimethylglycine oxidase. The catabolism of dimethylglycine through methyl group oxidation can potentially liberate toxic formaldehyde, a problem common to many amine oxidases and dehydrogenases. Using a novel synthetic in vivo reporter system for cellular formaldehyde, we found that the oxidation of dimethylglycine is coupled to the synthesis of 5,10-methylenetetrahydrofolate through an unusual substrate channeling mechanism. We also showed that uncoupling of the active sites could be achieved by mutagenesis or deletion of the 5,10-methylenetetrahydrofolate synthase site and that this leads to accumulation of intracellular formaldehyde. Channeling occurs by nonbiased diffusion of the labile intermediate through a large solvent cavity connecting both active sites. This central “reaction chamber” is created by a modular protein architecture that appears primitive when compared with the sophisticated design of other paradigm substrate-channeling enzymes. The evolutionary origins of the latter were likely similar to dimethylglycine oxidase. This work demonstrates the utility of synthetic biology approaches to the study of enzyme mechanisms in vivo and points to novel channeling mechanisms that protect the cell milieu from potentially toxic reaction products.     

Chemical immobilization of free-ranging dhole (Cuon alpinus), binturong (Arctictis binturong), and yellow-throated marten (Martes flavigula) in Thailand

No published information exists on the chemical immobilization of free-ranging dholes (Cuon alpinus), binturongs (Arctictis binturong), or yellow-throated martens (Martes flavigula). We chemically immobilized these species in Thailand using a mixture of ketamine hydrochloride with xylazine hydrochloride (KH–XH) and tiletamine hydrochloride with zolazepam hydrochloride (TH–ZH). Mean (±SD) dose of KH–XH was 18.1±5.3 and 0.9±0.1 mg/kg for dholes (n=2), 19.7±4.1 and 1.3±0.4 mg/kg for binturongs (n=8), and 28.7±3.3 and 1.1±0.1 mg/kg for yellow-throated martens (n=5), respectively. Mean dose of TH–ZH was 4.1±0.1 mg/kg for dhole (n=2). Induction time, duration of anesthesia, and recovery time were satisfactory for standard field research procedures including radio-collaring, although the effects on yellow-throated martens and binturongs were more variable. Respiration and muscle rigidity were monitored during sedation with no observed adverse physiological effects. Individuals were released after full recovery and monitored via radio telemetry for 4–23 months with no observed detrimental effects due to chemical immobilization. We conclude that KH–XH and TH–ZH are safe and effective immobilization agents for these carnivores; however, we suggest testing different KH–XH ratios and dosages, and other immobilizing agents for these species.     

Map of the IncP1β Plasmid pTSA Encoding the Widespread Genes (tsa) for p-Toluenesulfonate Degradation in Comamonas testosteroni T-2

The catabolic IncP1β plasmid pTSA from Comamonas testosteroni T-2 was mapped by subtractive analysis of restriction digests, by sequencing outwards from the tsa operon (toluenesulfonate degradation), and by generating overlapping, long-distance-PCR amplification products. The plasmid was estimated to comprise 72 ± 4 kb. The tsa region was found to be a composite transposon flanked by two IS1071 elements. A cryptic tsa operon was also present in the tsa transposon. Those backbone genes and regions which we sequenced were in the same order as the corresponding genes in resistance plasmid R751, and identities of about 99% were observed. Enrichment cultures with samples from four continents were done to obtain organisms able to utilize p-toluenesulfonate as the sole source of carbon and energy for aerobic growth. Most (15) of the 16 cultures (13 of them isolates) were obtained from contaminated sites and were attributed to three metabolic groups, depending on their metabolism of p-toluenesulfonate. The largest group contained the tsa transposon, usually (six of seven isolates) with negligible differences in sequence from strain T-2.     

A new method to quantify phagocytosis and intracellular degradation using green fluorescent protein-labeled Escherichia coli: comparison of cord blood macrophages and peripheral blood macrophages of healthy adults.

Interactions between innate and adaptive immune functions in neonatal macrophages (MPhi) are still unclear. We therefore established a method to quantify bacterial phagocytosis and intracellular degradation, using green fluorescent protein (GFP)-labeled Escherichia coli in combination with phenotypic analysis. The kinetics of the proportion of phagocyting MPhi, phagocytosed bacteria per MPhi, and bacterial degradation were comparable for cord blood MPhi of term neonates and MPhi of healthy adults. Phenotyping revealed CD14 and CD16 to be down-modulated within minutes. GFP-labeled E. coli may be useful tools to further study MPhi subpopulations and determinants of phagocytosis in cord blood MPhi.