Toxicology and safety of DHA

Docosahexaenoic acid (DHA) is a long-chain polyunsaturated fatty acid with activities in both infants and adults. The objective of the current work was to evaluate the published literature concerning the toxicological assessment of DHA-rich oils in animals and the safety profile of DHA consumption in humans. Structured literature searches concerning DHA toxicology and DHA effects on platelet function, lipid levels, oxidative potential, glycemic control, and immune function were conducted. The toxicological profile of DHA derived from single-cell organisms demonstrates that these oils are safe in rats (up to a consumption level of 3290 mg/kg body weight/d) in 90-d toxicology evaluations, as well as in reproductive and developmental toxicology studies. The maximum DHA level in human breast milk exceeds 1% of total fatty acids in high-fish-consuming populations. Consumption of DHA-rich human milk as sole source of nutrition provides approximately 315 mg/d in infants 1–6 months of age, and appears to be a safe level of intake. DHA supplementation studies in adults have employed doses ranging from less than 1 to 7.5 g/d, and have not resulted in any consistent adverse responses in platelet function, lipid levels, in vivo oxidation parameters, glycemic control, or immune function. In conclusion, DHA consumption does not result in consistent adverse events in infants or adults. Safe intake levels may be modeled on DHA intake from human milk in infants, and may be at least as high as the upper doses studied in adults.     

RIP1 kinase activity promotes steatohepatitis through mediating cell death and inflammation in macrophages

Hepatocyte cell death and liver inflammation have been well recognized as central characteristics of nonalcoholic steatohepatitis (NASH), however, the underlying molecular basis remains elusive. The kinase receptor-interacting protein 1 (RIP1) is a multitasking molecule with distinct functions in regulating apoptosis, necroptosis, and inflammation. Dissecting the role of RIP1 distinct functions in different pathophysiology has absorbed huge research enthusiasm. Wild-type and RIP1 kinase-dead (Rip1^K45A/K45A) mice were fed with high-fat diet (HFD) to investigate the role of RIP1 kinase activity in the pathogenesis of NASH. Rip1^K45A/K45A mice exhibited significantly alleviated NASH phenotype of hepatic steatosis, liver damage, fibrosis as well as reduced hepatic cell death and inflammation compared to WT mice. Our results also indicated that both in vivo lipotoxicity and in vitro saturated fatty acids (palmitic acid) treatment were able to induce the kinase activation of RIP1 in liver macrophages. RIP1 kinase was required for mediating inflammasome activation, apoptotic and necrotic cell death induced by palmitic acid in both bone marrow-derived macrophage and mouse primary Kupffer cells. Results from chimeric mice established through lethal irradiation and bone marrow transplantation further confirmed that the RIP1 kinase in hematopoietic-derived macrophages contributed mostly to the disease progression in NASH. Consistent with murine models, we also found that RIP1 kinase was markedly activated in human NASH, and the kinase activation mainly occurred in liver macrophages as indicated by immunofluorescence double staining. In summary, our study indicated that RIP1 kinase was phosphorylated and activated mainly in liver macrophages in both experimental and clinical NASH. We provided direct genetic evidence that the kinase activity of RIP1 especially in hematopoietic-derived macrophages contributes to the pathogenesis of NASH, through mediating inflammasome activation and cell death induction. Macrophage RIP1 kinase represents a specific and potential therapeutic target for NASH.Subject terms: Cell death and immune response, Chronic inflammation     

Fluorometric determination of DNA in Chlamydomonas

The diaminobenzoic acid dihydrochloride fluorescence method has been examined to determine optimal conditions for the assay of DNA in perchloric acid extracts and in pretreated Chlamydomonas cells on glass-fiber filters. Maximal fluorescence yield was obtained by allowing: (a) the perchloric acid extract to react with 4–6% diaminobenzoic acid dihydrochloride for 20–40 min at 60–80°C at about pH 3.0; (b) the pretreated cells on glass-fiber filters to react with a 20% solution of diaminobenzoic acid dihydrochloride for 40 min at 60°C.     

Laboratory diagnosis of melioidosis: Past,present and future

Melioidosis is an emerging, potentially fatal disease caused by Burkholderia pseudomallei, which requires prolonged antibiotic treatment to prevent disease relapse. However, difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes. Isolation of B. pseudomallei from clinical specimens has been improved with the use of selective media. However, even with positive cultures, identification of B. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. Commercial identification systems may fail to distinguish between B. pseudomallei and closely related species such as Burkholderia thailandensis. Genotypic identification of suspected isolates can be achieved by sequencing of gene targets such as groEL which offer higher discriminative power than 16S rRNA. Specific PCR-based identification of B. pseudomallei has also been developed using B. pseudomallei-specific gene targets such as Type III secretion system and Tat-domain protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolutionary technique for pathogen identification, has been shown to be potentially useful for rapid identification of B. pseudomallei, although existing databases require optimization by adding reference spectra for B. pseudomallei. Despite these advances in bacterial identification, diagnostic problems encountered in culture-negative cases remain largely unresolved. Although various serological tests have been developed, they are generally unstandardized “in house” assays and have low sensitivities and specificities. Although specific PCR assays have been applied to direct clinical and environmental specimens, the sensitivities for diagnosis remain to be evaluated. Metabolomics is an uprising tool for studying infectious diseases and may offer a novel approach for exploring potential diagnostic biomarkers. The metabolomics profiles of B. pseudomallei culture supernatants can be potentially distinguished from those of related bacterial species including B. thailandensis. Further studies using bacterial cultures and direct patient samples are required to evaluate the potential of metabolomics for improving diagnosis of melioidosis.     

CHARACTERISTICS OF PHOSPHORUS LIMITATION IN CHLAMYDOMONAS REINHARDTH (CHLOROPHYCEAE) AND ITS PALMELLOIDS1

Chlamydomonas reinhardtii Dang, was grown in a chemostat culture under phosphate limitation. The steady state concentration of phosphate was below the detection limit (< 1 μg P/L) in all runs. The cellular content of phosphorus (Q_p), polyphosphate (Q_pp) and chlorophyll a increased with increasing dilution rate, and the growth rate of the alga was described by Q_p as well as Q_pp in the Droop model. The ratio Q_pp/Q_p and the activity of alkaline phosphatase were maximal at high and low growth rates, respectively. Palmelloids of Chlamydomonas were found at high dilution rates (D > 0.12 h^?1) and became attached to the wall of the culture vessel. They differed from the vegetative stage in both chemical composition and growth rate. Their contents of phosphorus and chlorophyll a were low, as in the vegetative cells, which grew at a low growth rate, whereas the ration Q_pp/Q_p and the activity of alkaline phosphatase were comparable with those of fast growing vegetative cells. The growth rate of the palmelloids was 0.03 h^?1 whereas maximum growth rate (μ_m) for the vegetative cells was 0.21 h^?1.     

Enkephalin-stimulated prolactin release

The effect of methionine- and leucine-enkephalina^a on prolactin secretion was studied in vivo and in vitro. Administration of methionine-enkephalin to rats (5 mg/kg) resulted in a consistent increase in plasma prolactin. In monolayer cultures of rat pituitaries both enkephalins released prolactin at concentrations as low as 5 ng/ml.     

In vivo 31P NMR study of early cellular responses to hyperosmotic shock in cultured glioma cells.

Cell volume regulation in the face of osmotic stress is a fundamental homeostatic activity, and is most critical in brain, which is spatially constrained. Despite the importance of this phenomenon, little is known about volume regulation in the brain, primarily because of the cellular heterogeneity in the tissue. We describe here simultaneous in vivo 31P nuclear magnetic resonance (NMR) measurements of cell volume, intracellular pH and phosphate metabolites during early responses to hyperosmotic stress in C6 glioma cells perfused in NMR-compatible bioreactors. Cell volume was measured using dimethyl methylphosphonate (DMMP) as a probe which has an intracellular NMR resonance shifted upfield from the extracellular resonance. The sensitivity of these measurements allowed 31P NMR spectra to be collected every 30 s. Following an increase in osmolarity from 320 to 480 mOsm by addition of NaCl to the perfusate, C6 glioma cells shrank to 67% of their original volume. We also observed a simultaneous increase of intracellular pH coincident with the decrease in cell volume. The signals from ATP decreased by 10%, but those from phosphocreatine (PCr) increased by 31% after hyperosmotic shock. However, correcting the ATP signals for the decrease in cell volume indicated that its intracellular concentrations increased after treatment. Signals from glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) were not changed significantly. This is the first in vivo report of early cellular responses monitored by NMR spectroscopy following hyperosmotic shock in cultured cells.