Intestinal DGAT1 deficiency reduces postprandial triglyceride and retinyl ester excursions by inhibiting chylomicron secretion and delaying gastric emptying

Acyl CoA:diacylglycerol acyltransferase (DGAT) 1 catalyzes the final step of triglyceride (TG) synthesis. We show that acute administration of a DGAT1 inhibitor (DGAT1i) by oral gavage or genetic deletion of intestinal Dgat1 (intestine-Dgat1^−/−) markedly reduced postprandial plasma TG and retinyl ester excursions by inhibiting chylomicron secretion in mice. Loss of DGAT1 activity did not affect the efficiency of retinol esterification, but it did reduce TG and retinoid accumulation in the small intestine. In contrast, inhibition of microsomal triglyceride transfer protein (MTP) reduced chylomicron secretion after oral fat/retinol loads, but with accumulation of dietary TG and retinoids in the small intestine. Lack of intestinal accumulation of TG and retinoids in DGAT1i-treated or intestine-Dgat1^−/− mice resulted, in part, from delayed gastric emptying associated with increased plasma levels of glucagon-like peptide (GLP)-1. However, neither bypassing the stomach through duodenal oil injection nor inhibiting the receptor for GLP-1 normalized postprandial TG or retinyl esters excursions in the absence of DGAT1 activity. In summary, intestinal DGAT1 inhibition or deficiency acutely delayed gastric emptying and inhibited chylomicron secretion; however, the latter occurred when gastric emptying was normal or when lipid was administered directly into the small intestine. Long-term hepatic retinoid metabolism was not impacted by DGAT1 inhibition.     

Tetracyano-2,2-bipyridineiron(iii), an improved electron acceptor for the spectrophotometric assay of beta-oxidation and of succinate dehydrogenase in intact mitochondria.

A recently described direct reading assay for beta-oxidation and for succinate oxidation in intact mitochondria using [Fe(CN)6]3- as final electron acceptor [Osmundsen & Bremer (1977) Biochem. J. 164. 621--633] has been improved by using instead tetracyano-2,2-bipyridineiron(III) [Fe(CN)4(bpy)]-, which gives a 2.6 times greater absorbance change on reduction. Some physical and kinetic properties of [Fe(CN)4(bpy)]- are described. The use of exogenous cytochrome c(III) as electron acceptor was also tested; this gives the largest absorbance change, although the absolute rate of reaction is only approx. one-third of that using [Fe(CN)6]3- or [Fe(CN)4(bpy)]-.     

Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts

The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts by ultraviolet light (UV) were compared under conditions of maximum expression of the 6-thioguanine resistance (TGr) phenotype. Selection was with 20 micrograms TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions of the UV dose but the slope of the line for normal cells extrapolated to zero induced mutants at 3 J/m2. The postreplication repair-defective XP4BE cells showed a higher frequency of TGr colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TGr colonies was not a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than about 50%. The UV doses and induced mutation frequencies corresponding to 37% survival of cloning abilities were 6.7 J/m2 and 6.2 X 10(-5), respectively, for normal cells and 3.75 J/m2 and 17.3 X 10(-5) for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.     

Hydrogen peroxide is generated during the very early stages of aggregation of the amyloid peptides implicated in Alzheimer disease and familial British dementia

Alzheimer disease and familial British dementia are neurodegenerative diseases that are characterized by the presence of numerous amyloid plaques in the brain. These lesions contain fibrillar deposits of the beta-amyloid peptide (Abeta) and the British dementia peptide (ABri), respectively. Both peptides are toxic to cells in culture, and there is increasing evidence that early "soluble oligomers" are the toxic entity rather than mature amyloid fibrils. The molecular mechanisms responsible for this toxicity are not clear, but in the case of Abeta, one prominent hypothesis is that the peptide can induce oxidative damage via the formation of hydrogen peroxide. We have developed a reliable method, employing electron spin resonance spectroscopy in conjunction with the spin-trapping technique, to detect any hydrogen peroxide generated during the incubation of Abeta and other amyloidogenic peptides. Here, we monitored levels of hydrogen peroxide accumulation during different stages of aggregation of Abeta-(1-40) and ABri and found that in both cases it was generated as a short "burst" early on in the aggregation process. Ultrastructural studies with both peptides revealed that structures resembling "soluble oligomers" or "protofibrils" were present during this early phase of hydrogen peroxide formation. Mature amyloid fibrils derived from Abeta-(1-40) did not generate hydrogen peroxide. We conclude that hydrogen peroxide formation during the early stages of protein aggregation may be a common mechanism of cell death in these (and possibly other) neurodegenerative diseases.     

The contribution of threonine 55 to catalysis in aspartate transcarbamoylase.

Heavy-atom isotope effects and steady-state kinetic parameters were measured for the catalytic trimer of an active site mutant of aspartate transcarbamoylase, T55A, to assess the role of Thr 55 in catalysis. The binding of carbamoyl phosphate to the T55A mutant was decreased by 2 orders of magnitude relative to the wild-type enzyme whereas the affinities for aspartate and succinate were not markedly altered. This indicates that Thr 55 plays a significant role in the binding of CbmP. If, as had been suggested previously, Thr 55 assists in the polarization of the carbonyl group of CbmP, the carbon isotope effect for the T55A mutant should increase relative to that observed for the wild-type enzyme. However, the opposite is seen, indicating that Thr 55 is not involved in stabilizing the oxyanion in the transition state. Quantitative analysis of a series of 13C and 15N isotope effects suggested that the rate-determining step in the reaction catalyzed by T55A trimer may be a conformational change in the protein subsequent to formation of the Michaelis complex. Thus, Thr 55 may facilitate a conformational change in the enzyme that is a prerequisite for catalysis. An altered active site environment in the binary and Michaelis complexes with T55A trimer is reflected in the pH profiles for log V, log (V/K)asp, and pK(i) succinate, show a displacement in the pK values of ionizing residues involved in aspartate binding and catalysis relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's beta-secretase

Cleavage of amyloid precursor protein (APP) by the Alzheimer's beta-secretase (BACE1) is a key step in generating amyloid beta-peptide, the main component of amyloid plaques. Here we report evidence that heparan sulfate (HS) interacts with beta-site APP-cleaving enzyme (BACE) 1 and regulates its cleavage of APP. We show that HS and heparin interact directly with BACE1 and inhibit in vitro processing of peptide and APP substrates. Inhibitory activity is dependent on saccharide size and specific structural characteristics, and the mechanism of action involves blocking access of substrate to the active site. In cellular assays, HS specifically inhibits BACE1 cleavage of APP but not alternative cleavage by alpha-secretase. Endogenous HS immunoprecipitates with BACE1 and colocalizes with BACE1 in the Golgi complex and at the cell surface, two of its putative sites of action. Furthermore, inhibition of cellular HS synthesis results in enhanced BACE1 activity. Our findings identify HS as a natural regulator of BACE1 and suggest a novel mechanism for control of APP processing.     

Differential physiological responses to environmental change promote woody shrub expansion

Direct and indirect effects of warming are increasingly modifying the carbon-rich vegetation and soils of the Arctic tundra, with important implications for the terrestrial carbon cycle. Understanding the biological and environmental influences on the processes that regulate foliar carbon cycling in tundra species is essential for predicting the future terrestrial carbon balance in this region. To determine the effect of climate change impacts on gas exchange in tundra, we quantified foliar photosynthesis (A_net), respiration in the dark and light (R_D and R_L, determined using the Kok method), photorespiration (PR), carbon gain efficiency (CGE, the ratio of photosynthetic CO₂ uptake to total CO₂ exchange of photosynthesis, PR, and respiration), and leaf traits of three dominant species – Betula nana, a woody shrub; Eriophorum vaginatum, a graminoid; and Rubus chamaemorus, a forb – grown under long-term warming and fertilization treatments since 1989 at Toolik Lake, Alaska. Under warming, B. nana exhibited the highest rates of A_net and strongest light inhibition of respiration, increasing CGE nearly 50% compared with leaves grown in ambient conditions, which corresponded to a 52% increase in relative abundance. Gas exchange did not shift under fertilization in B. nana despite increases in leaf N and P and near-complete dominance at the community scale, suggesting a morphological rather than physiological response. Rubus chamaemorus, exhibited minimal shifts in foliar gas exchange, and responded similarly to B. nana under treatment conditions. By contrast, E. vaginatum, did not significantly alter its gas exchange physiology under treatments and exhibited dramatic decreases in relative cover (warming: −19.7%; fertilization: −79.7%; warming with fertilization: −91.1%). Our findings suggest a foliar physiological advantage in the woody shrub B. nana that is further mediated by warming and increased soil nutrient availability, which may facilitate shrub expansion and in turn alter the terrestrial carbon cycle in future tundra environments.     

Failure of Sterne- and Pasteur-Like Strains of Bacillus anthracis to Replicate and Survive in the Urban Bluebottle Blow Fly Calliphora vicina under Laboratory Conditions

This study aimed to elucidate the bacteriological events occurring within the gut of Calliphora vicina, selected as the European representative of blow flies held responsible for the spread of anthrax during epidemics in certain parts of the world. Green-fluorescent-protein-carrying derivatives of Bacillus anthracis were used. These lacked either one of the virulence plasmids pXO1 and pXO2 and were infected, or not infected, with a worm intestine phage (Wip4) known to influence the phenotype and survival of the pathogen. Blood meals were prepared for the flies by inoculation of sheep blood with germinated and, in case of pXO2+ strains, encapsulated cells of the four B. anthracis strains. After being fed for 4 h an initial 10 flies were externally disinfected with peracetic acid to ensure subsequent quantitation representing ingested B. anthracis only. Following neutralization, they were crushed in sterile saline. Over each of the ensuing 7 to 10 days, 10 flies were removed and processed the same way. In the absence of Wip4, strains showed steady declines to undetectable in the total B. anthracis counts, within 7–9 days. With the phage infected strains, the falls in viable counts were significantly more rapid than in their uninfected counterparts. Spores were detectable in flies for longer periods than vegetative bacteria. In line with the findings in both biting and non-biting flies of early workers our results indicate that B. anthracis does not multiply in the guts of blow flies and survival is limited to a matter of days.     

An Inflammation Based Score Can Optimize the Selection of Patients with Advanced Cancer Considered for Early Phase Clinical Trials

Background

Adequate organ function and good performance status (PS) are common eligibility criteria for phase I trials. As inflammation is pathogenic and prognostic in cancer we investigated the prognostic performance of inflammation-based indices including the neutrophil (NLR) and platelet to lymphocyte ratio (PLR).

Methods

We studied inflammatory scores in 118 unselected referrals. NLR normalization was recalculated at disease reassessment. Each variable was assessed for progression-free (PFS) and overall survival (OS) on uni- and multivariate analyses and tested for 90 days survival (90DS) prediction using receiving operator curves (ROC).

Results

We included 118 patients with median OS 4.4 months, 23% PS>1. LDH≥450 and NLR≥5 were multivariate predictors of OS (p<0.001). NLR normalization predicted for longer OS (p<0.001) and PFS (p<0.05). PS and NLR ranked as most accurate predictors of both 90DS with area under ROC values of 0.66 and 0.64, and OS with c-score of 0.69 and 0.60. The combination of NLR+PS increased prognostic accuracy to 0.72. The NLR was externally validated in a cohort of 126 subjects.

Conclusions

We identified the NLR as a validated and objective index to improve patient selection for experimental therapies, with its normalization following treatment predicting for a survival benefit of 7 months. Prospective validation of the NLR is warranted.