Prediction of oxidoreductase-catalyzed reactions based on atomic properties of metabolites

MOTIVATION: Our knowledge of metabolism is far from complete, and the gaps in our knowledge are being revealed by metabolomic detection of small-molecules not previously known to exist in cells. An important challenge is to determine the reactions in which these compounds participate, which can lead to the identification of gene products responsible for novel metabolic pathways. To address this challenge, we investigate how machine learning can be used to predict potential substrates and products of oxidoreductase-catalyzed reactions. RESULTS: We examined 1956 oxidation/reduction reactions in the KEGG database. The vast majority of these reactions (1626) can be divided into 12 subclasses, each of which is marked by a particular type of functional group transformation. For a given transformation, the local structures of reaction centers in substrates and products can be characterized by patterns. These patterns are not unique to reactants but are widely distributed among KEGG metabolites. To distinguish reactants from non-reactants, we trained classifiers (linear-kernel Support Vector Machines) using negative and positive examples. The input to a classifier is a set of atomic features that can be determined from the 2D chemical structure of a compound. Depending on the subclass of reaction, the accuracy of prediction for positives (negatives) is 64 to 93% (44 to 92%) when asking if a compound is a substrate and 71 to 98% (50 to 92%) when asking if a compound is a product. Sensitivity analysis reveals that this performance is robust to variations of the training data. Our results suggest that metabolic connectivity can be predicted with reasonable accuracy from the presence or absence of local structural motifs in compounds and their readily calculated atomic features. AVAILABILITY: Classifiers reported here can be used freely for noncommercial purposes via a Java program available upon request.     

Treatment of neutral glycosphingolipid lysosomal storage diseases via inhibition of the ABC drug transporter, MDR1. Cyclosporin A can lower serum and liver globotriaosyl ceramide levels in the Fabry mouse model

We have shown that the ABC transporter, multiple drug resistance protein 1 (MDR1, P-glycoprotein) translocates glucosyl ceramide from the cytosolic to the luminal Golgi surface for neutral, but not acidic, glycosphingolipid (GSL) synthesis. Here we show that the MDR1 inhibitor, cyclosporin A (CsA) can deplete Gaucher lymphoid cell lines of accumulated glucosyl ceramide and Fabry cell lines of globotriaosyl ceramide (Gb3), by preventing de novo synthesis. In the Fabry mouse model, Gb3 is increased in the heart, liver, spleen, brain and kidney. The lack of renal glomerular Gb3 is retained, but the number of verotoxin 1 (VT1)-staining renal tubules, and VT1 tubular targeting in vivo, is markedly increased in Fabry mice. Adult Fabry mice were treated with alpha-galactosidase (enzyme-replacement therapy, ERT) to eliminate serum Gb3 and lower Gb3 levels in some tissues. Serum Gb3 was monitored using a VT1 ELISA during a post-ERT recovery phase +/- biweekly intra peritoneal CsA. After 9 weeks, tissue Gb3 content and localization were determined using VT1/TLC overlay and histochemistry. Serum Gb3 recovered to lower levels after CsA treatment. Gb3 was undetected in wild-type liver, and the levels of Gb3 (but not gangliosides) in Fabry mouse liver were significantly depleted by CsA treatment. VT1 liver histochemistry showed Gb3 accumulated in Kupffer cells, endothelial cell subsets within the central and portal vein and within the portal triad. Hepatic venule endothelial and Kupffer cell VT1 staining was considerably reduced by in vivo CsA treatment. We conclude that MDR1 inhibition warrants consideration as a novel adjunct treatment for neutral GSL storage diseases.     

Alpha-adrenergic receptor-mediated restraint of skeletal muscle blood flow during prolonged exercise.

Sympathetic nervous system restraint of skeletal muscle blood flow during dynamic exercise has been well documented. However, whether sympathetic restraint of muscle blood flow persists and is constant throughout prolonged exercise has not been established. We hypothesized that both alpha1- and alpha2-adrenergic receptors would restrain skeletal muscle blood flow throughout prolonged constant-load exercise and that the restraint would increase as a function of exercise duration. Mongrel dogs were instrumented chronically with transit-time flow probes on the external iliac arteries and an indwelling catheter in a branch of the femoral artery. Flow-adjusted doses of selective alpha1- (prazosin) and alpha2-adrenergic receptor (rauwolscine) antagonists were infused after 5, 30, and 50 min of treadmill exercise at 3 and 6 miles/h. During mild-intensity exercise (3 miles/h), prazosin infusion resulted in a greater (P < 0.05) increase in vascular conductance (VC) after 5 [42% (SD 6)], compared with 30 [28% (SD 6)] and 50 [28% (SD 8)] min of running. In contrast, prazosin resulted in a similar increase in VC after 5 [29% (SD 10)], 30 [24% (SD 9)], and 50 [22% (SD 9)] min of moderate-intensity (6 miles/h) exercise. Rauwolscine infusion resulted in a greater (P < 0.05) increase in VC after 5 [39% (SD 14)] compared with 30 [26% (SD 9)] and 50 [22% (SD 4)] min of exercise at 3 miles/h. Rauwolscine infusion produced a similar increase in VC after 5 [19% (SD 3)], 30 [15% (SD 6)], and 50 [16% (SD 2)] min of exercise at 6 miles/h. These results suggest that the ability of alpha1- and alpha2-adrenergic receptors to produce vasoconstriction and restrain blood flow to active muscles may be influenced by both the intensity and duration of exercise.     

Formation of cristae and crista junctions in mitochondria depends on antagonism between Fcj1 and Su e/g

Crista junctions (CJs) are important for mitochondrial organization and function, but the molecular basis of their formation and architecture is obscure. We have identified and characterized a mitochondrial membrane protein in yeast, Fcj1 (formation of CJ protein 1), which is specifically enriched in CJs. Cells lacking Fcj1 lack CJs, exhibit concentric stacks of inner membrane in the mitochondrial matrix, and show increased levels of F₁F_O–ATP synthase (F₁F_O) supercomplexes. Overexpression of Fcj1 leads to increased CJ formation, branching of cristae, enlargement of CJ diameter, and reduced levels of F₁F_O supercomplexes. Impairment of F₁F_O oligomer formation by deletion of its subunits e/g (Su e/g) causes CJ diameter enlargement and reduction of cristae tip numbers and promotes cristae branching. Fcj1 and Su e/g genetically interact. We propose a model in which the antagonism between Fcj1 and Su e/g locally modulates the F₁F_O oligomeric state, thereby controlling membrane curvature of cristae to generate CJs and cristae tips.     

Fatty acid-dependent globotriaosyl ceramide receptor function in detergent resistant model membranes

Glycosphingolipid (GSL) fatty acid strictly regulates verotoxin 1 (VT1) and the HIV adhesin, gp120 binding to globotriaosyl ceramide within Gb(3)/cholesterol detergent resistant membrane (DRM) vesicle constructs and in Gb(3) water-air interface monolayers in a similar manner. VT2 bound Gb(3)/cholesterol vesicles irrespective of fatty acid composition, but VT1 bound neither C18 nor C20Gb(3)vesicles. C18/C20Gb(3) were dominant negative in mixed Gb(3) fatty acid isoform vesicles, but including C24:1Gb(3) gave maximal binding. VT1 bound C18Gb(3) vesicles after cholesterol removal, but C20Gb(3)vesicles required sphingomyelin in addition for binding. HIV-1gp120 also bound C16, C22, and C24, but neither C18 nor C20Gb(3) vesicles. C18 and C20Gb(3) were, in mixtures without C24:1Gb(3), dominant negative for gp120 vesicle binding. Gp120/VT1bound C18 and C24:1Gb(3) mixtures, although neither isoform bound alone. Monolayer surface pressure measurement showed VT1, but not VT2, bound Gb(3) at cellular DRM surface pressures, and confirmed loss of VT1 and gp120 (but not VT2) specific C18Gb(3) binding. We conclude fatty-acid mediated fluidity within simple model GSL/cholesterol DRM can selectively regulate GSL carbohydrate-ligand binding.     

Vitamin D safety and requirements

Vitamin D an ancient secosteroid is essential for mineral homeostasis, bone remodeling, immune modulation, and energy metabolism. Recently, debates have emerged about the daily vitamin D requirements for healthy and elderly adults, the safety and efficacy of long term supplementation and the role of vitamin D deficiency in several chronic disease states. Since this molecule acts as both a vitamin and a hormone, it should not be surprising that the effects of supplementation are multi-faceted and complex. Yet despite significant progress in the last decade, our understanding of vitamin D physiology and the clinical relevance of low circulating levels of this vitamin remains incomplete. The present review provides the reader with a comprehensive and up-to-date understanding of vitamin D requirements and safety. It also raises some provocative research questions.     

Effects of CNTO 530, an erythropoietin mimetic‐IgG4 fusion protein,on embryofetal development in rats and rabbits

BACKGROUND: CNTO 530is a biopharmaceutical consisting of a novel peptide that mimics the actions of erythropoietin, fused to the Fc fragment of human IgG4. Pharmacokinetic and pharmacodynamic studies showed that CNTO 530 produced sustained increases in red blood cell parameters in rats and rabbits and that the serum half life of CNTO 530 was 2 days in rabbits and 3 days in rats. METHODS: For the evaluation of embryofetal development, CNTO 530 was injected at loading doses of 0, 0.9/1, 6, or 60 mg/kg subcutaneously (SC) on gestation day (GD)7 followed by maintenance doses of 0, 0.3, 2, or 20 mg/kg SC every 3 days through GD16 in rats and every 2 days through GD19 in rabbits (GD0 was the day of mating). Rats were Caesarean sectioned on GD21, rabbits on GD29. RESULTS: Administration of CNTO 530 was associated with an increase in hematocrit at all dose levels and a decrease in maternal body weight gains. Fetuses exhibited reduced body weight and delayed ossification. Soft tissue changes were limited to cardiovascular alterations in the high‐dose rabbits only. Rat and rabbit fetuses were exposed to CNTO 530 in all dose groups. CONCLUSIONS: These studies show that the embryo/fetal development effects observed following CNTO 530 treatment during organogenesis are qualitatively similar to those seen with other erythropoietin agonists and are likely a secondary consequence of increased hematocrit in the dams. Unlike other erythropoietin receptor agonists, CNTO 530 was able to cross the placental barrier, which was considered likely the result of FcRn‐mediated transcytosis. Birth Defects Res (Part B) 89:87–96, 2010. © 2010 Wiley‐Liss, Inc.     

Influence of Mouse Strain on the Assayed Potency (Unitage) of Tetanus Toxoid

The assayed potency of an adsorbed tetanus toxoid C(2), relative to the international standard for tetanus toxoid (adsorbed), varied significantly with the use of different strains of mice. The unitage was highest with NIH mice, and it was not significantly different from that with CFW mice. With CDF(1) and BALB/c mice, however, the assayed potency was significantly lower than with NIH mice. C3H mice failed to respond to the dose range of the toxoids employed with the other four strains. The significance of the influence of the mouse strain on the designation of a prescribed unit requirement for tetanus toxoid, adsorbed, relative to human efficacy is discussed.     

Model-based Analysis of ChIP-Seq (MACS)

We present Model-based Analysis of ChIP-Seq data, MACS, which analyzes data generated by short read sequencers such as Solexa's Genome Analyzer. MACS empirically models the shift size of ChIP-Seq tags, and uses it to improve the spatial resolution of predicted binding sites. MACS also uses a dynamic Poisson distribution to effectively capture local biases in the genome, allowing for more robust predictions. MACS compares favorably to existing ChIP-Seq peak-finding algorithms, and is freely available.