Synthesis and X-Ray Crystal Structure of 2-β-D-Ribofuranosylthiazole-4-Carboxamide-N3-Oxide

Abstract

2-β-D-Ribofuranosylthiazole-4-carboxamide-N₃-oxide (3) has been prepared by oxidation of tiazofurin (2) with hydrogen peroxide in the presence of trifluoroacetic acid. The absolute structure of 3 has been determined by X-ray diffraction techniques employing Mo Kα radiation. The thiazole ring in 3 is planar and the glycon moiety is in the ³E configuration. The N3-C4 bond in 3 is significantly larger (1.412 Å) than that found in tiazofurin (1.376 Å). Similar to tiazofurin, the S…04’ intramolecular contact distance is considerably shorter than the sum of the van der Waals radii, which favors the anti conformation of 3.     

Leptin does not directly affect CNS serotonin neurons to influence appetite

Serotonin (5-HT) and leptin play important roles in the modulation of energy balance. Here we investigated mechanisms by which leptin might interact with CNS 5-HT pathways to influence appetite. Although some leptin receptor (LepRb) neurons lie close to 5-HT neurons in the dorsal raphe (DR), 5-HT neurons do not express LepRb. Indeed, while leptin hyperpolarizes some non-5-HT DR neurons, leptin does not alter the activity of DR 5-HT neurons. Furthermore, 5-HT depletion does not impair the anorectic effects of leptin. The serotonin transporter-cre allele (Sert(cre)) is expressed in 5-HT (and developmentally in some non-5-HT) neurons. While Sert(cre) promotes LepRb excision in a few LepRb neurons in the hypothalamus, it is not active in DR LepRb neurons, and neuron-specific Sert(cre)-mediated LepRb inactivation in mice does not alter body weight or adiposity. Thus, leptin does not directly influence 5-HT neurons and does not meaningfully modulate important appetite-related determinants via 5-HT neuron function.     

Trypanosoma brucei mitochondrial respiratome: composition and organization in procyclic form

The mitochondrial respiratory chain is comprised of four different protein complexes (I–IV), which are responsible for electron transport and generation of proton gradient in the mitochondrial intermembrane space. This proton gradient is then used by F_oF₁-ATP synthase (complex V) to produce ATP by oxidative phosphorylation. In this study, the respiratory complexes I, II, and III were affinity purified from Trypanosoma brucei procyclic form cells and their composition was determined by mass spectrometry. The results along with those that we previously reported for complexes IV and V showed that the respiratome of Trypanosoma is divergent because many of its proteins are unique to this group of organisms. The studies also identified two mitochondrial subunit proteins of respiratory complex IV that are encoded by edited RNAs. Proteomics data from analyses of complexes purified using numerous tagged component proteins in each of the five complexes were used to generate the first predicted protein-protein interaction network of the Trypanosoma brucei respiratory chain. These results provide the first comprehensive insight into the unique composition of the respiratory complexes in Trypanosoma brucei, an early diverged eukaryotic pathogen.Mitochondria are dynamic organelles essential for cellular life, death, and differentiation of virtually every eukaryotic cell. They house systems for energy production through oxidative phosphorylation, synthesis of key metabolites, and iron-sulfur cluster assembly. The oxidative phoshorylation system of eukaryotic mitochondria comprises five major complexes located in the mitochondrial (mt)¹ inner membrane, and often abbreviated as mt complexes I–V. The redox energy of the substrates NADH and succinate is first converted into an electrochemical proton potential across the inner mt membrane by respiratory complexes I (NADH:ubiquinone reductase), II (SDH, succinate:ubiquinone reductase), III (bc₁, ubiquinone:cytochrome c reductase), and IV (cytochrome c oxidase). The electrochemical proton potential is then used by complex V (F_oF₁-ATP synthase) to synthesize ATP from ADP and inorganic phosphate, a mechanism that has essentially remained unchanged from bacteria to human (1). However, parasitic organisms have exploited unique energy metabolic pathways by adapting to their natural host habitats (2). Indeed, the respiratory systems of parasites typically show greater diversity in electron transfer pathways than those of their host, and Trypanosoma brucei is no exception to this rule (3).T. brucei, the causative agent of human African trypanosomiasis (HAT), or sleeping sickness, is a blood-borne pathogenic parasite transmitted by tsetse flies. It has a complex life cycle that alternates between the bloodstream forms (BF) in the mammalian host and several stages in the insect vector starting with the procyclic form (PF) in the midgut. During T. brucei differentiation between the distinct life-cycle stages, the mitochondrion undergoes morphological and functional changes, and the parasite switches its energy metabolism from amino acid to glucose oxidation (4). BF cells, which live in sugar-rich environment, use energy metabolism predominantly through the glycolytic pathway (5). They contain no cytochrome-mediated respiratory chain and they possess a unique electron transport chain in the mitochondria, the glycerol-3-phosphate dehydrogenase and the salicyl hydroxamic acid (SHAM)-sensitive alternative oxidase, which is known as the trypanosome alternative oxidase (TAO) (6). Despite the absence of complete cytochrome-containing complexes III and IV in BF trypanosomes, a mt membrane potential is maintained and involves the hydrolytic activity of the F_oF₁-ATP synthase complex (7). Conversely, PF cells are dependent on the cytochrome-containing respiratory chain and ATP generated by conventional function of the F_oF₁-ATP synthase complex for their energy production (8, 9). The branched electron-transport chain contains four complexes that donate electrons to the ubiquinone pool, two NADH:ubiquinone oxidoreductases (complex I and a rotenone-insensitive enzyme), complex II, and glycerol-3-phosphate dehydrogenase. Reduced ubiquinol can be reoxidized by the transfer of electron to either the TAO, which does not translocate protons, or to the cytochrome-containing complexes III and IV that produce a proton motive force by translocation of protons and thus create essential membrane potential (10).Although the T. brucei genome has been sequenced (11), little information is available on the subunit composition of mt complexes I–V based on similarity searches. However, some respiratory complexes have been partially characterized in other trypanosomatids such as Crithidia fasciculata, T. cruzi, and Leishmania tarentolae (12–15). In recent studies, we have determined the protein composition of complexes IV and V, and part of complex I purified from mitochondria of T. brucei PF cells (8, 16, 17, 25). These analyses revealed the uniqueness of respiratory complexes in trypanosomes, where large numbers of component proteins have no homologs outside of the Kinetoplastida.In this study, we focus on the comprehensive characterization of all respiratory complexes in T. brucei, collectively termed the respiratome. We report the composition of complexes II and III from PF cells, and extend the characterization of complex I by identifying additional protein constituents. This included the identification of two subunits of the respiratory complex IV, both encoded by mt edited RNAs. We also present a predicted protein-protein interaction network of the respiratome, which was generated using proteomics data collected from numerous tagged proteins in each of the complexes I–V. Our results provide a comprehensive insight into the unique composition of the respiratory complexes in one of the life-cycle stages of T. brucei.     

Notes on Scleroderma,Puff balls and Geastrum of Azad Jammu and Kashmir,Pakistan

Six species of mushrooms allied to the Family Sclerodermataceae, Lycoperdaceae and Geastraceae have been described for the first time from Azad Jammu and Kashmir. These are Scleroderma aurantium, Calvatia verrucosia sp. nov., Lycoperdon pedicellaton sp. nov. L. sphaericon sp. nov., L. echinulaton sp. nov., and Geastrum heptaplex sp. nov.     

Resequencing the susceptibility gene,ITGAM, identifies two functionally deleterious rare variants in systemic lupus erythematosus cases

Introduction

The majority of the genetic variance of systemic lupus erythematosus (SLE) remains unexplained by the common disease-common variant hypothesis. Rare variants, which are not detectable by genome-wide association studies because of their low frequencies, are predicted to explain part of this ”missing heritability.” However, recent studies identifying rare variants within known disease-susceptibility loci have failed to show genetic associations because of their extremely low frequencies, leading to the questioning of the contribution of rare variants to disease susceptibility. A common (minor allele frequency = 17.4% in cases) nonsynonymous coding variant rs1143679 (R77H) in ITGAM (CD11b), which forms half of the heterodimeric integrin receptor, complement receptor 3 (CR3), is robustly associated with SLE and has been shown to impair CR3-mediated phagocytosis.

Methods

We resequenced ITGAM in 73 SLE cases and identified two previously unidentified, case-specific nonsynonymous variants, F941V and G1145S. Both variants were genotyped in 2,107 and 949 additional SLE cases, respectively, to estimate their frequencies in a disease population. An in vitro model was used to assess the impact of F941V and G1145S, together with two nonsynonymous ITGAM polymorphisms, A858V (rs1143683) and M441T (rs11861251), on CR3-mediated phagocytosis. A paired two-tailed t test was used to compare the phagocytic capabilities of each variant with that of wild-type CR3.

Results

Both rare variants, F941V and G1145S, significantly impair CR3-mediated phagocytosis in an in vitro model (61% reduction, P = 0.006; 26% reduction, P = 0.0232). However, neither of the common variants, M441T and A858V, had an effect on phagocytosis. Neither rare variant was observed again in the genotyping of additional SLE cases, suggesting that there frequencies are extremely low.

Conclusions

Our results add further evidence to the functional importance of ITGAM in SLE pathogenesis through impaired phagocytosis. Additionally, this study provides a new example of the identification of rare variants in common-allele-associated loci, which, because of their extremely low frequencies, are not statistically associated. However, the demonstration of their functional effects adds support to their contribution to disease risk, and questions the current notion of dismissing the contribution of very rare variants on purely statistical analyses.     

Formyl Peptide Receptor as a Novel Therapeutic Target for Anxiety-Related Disorders

Formyl peptide receptors (FPR) belong to a family of sensors of the immune system that detect microbe-associated molecules and inform various cellular and sensorial mechanisms to the presence of pathogens in the host. Here we demonstrate that Fpr2/3-deficient mice show a distinct profile of behaviour characterised by reduced anxiety in the marble burying and light-dark box paradigms, increased exploratory behaviour in an open-field, together with superior performance on a novel object recognition test. Pharmacological blockade with a formyl peptide receptor antagonist, Boc2, in wild type mice reproduced most of the behavioural changes observed in the Fpr2/3 ^-/- mice, including a significant improvement in novel object discrimination and reduced anxiety in a light/dark shuttle test. These effects were associated with reduced FPR signalling in the gut as shown by the significant reduction in the levels of p-p38. Collectively, these findings suggest that homeostatic FPR signalling exerts a modulatory effect on anxiety-like behaviours. These findings thus suggest that therapies targeting FPRs may be a novel approach to ameliorate behavioural abnormalities present in neuropsychiatric disorders at the cognitive-emotional interface.     

Analysis of a genome-wide association study-linked locus (CCR6) in Asian rheumatoid arthritis

A genome-wide association study in Japan identified the C-C chemokine receptor type 6 gene (CCR6) as associated with rheumatoid arthritis (RA). This finding has not been validated in other Asian populations. A case-control study involving 996 subjects, comprising 440 controls and 556 RA patients, was done to determine their anticyclic citrullinated peptide (anti-CCP) antibody status and CCR6 polymorphism (rs3093024) genotype. Three hundred eighty-seven patients were anti-CCP positive and 153 anti-CCP negative. Logistic regression showed that allele A was likely to increase the risk of developing RA among females via a recessive model (odds ratio [OR]=1.55, 95% confidence interval [CI]=1.01, 2.39), whereas the risk effect appeared to be reduced among males via an additive model (OR=0.60, 95% CI=0.42, 0.85). Considering only subjects who are anti-CCP positive, allele A increased RA risk among females via a recessive model (OR=1.68, 95% CI=1.07, 2.64) but decreased the risk among males via an additive model (OR=0.59, 95% CI=0.39, 0.89). We showed that CCR6 polymorphism was a risk factor among females but a protective factor among males. Functional studies are warranted to unravel the pathophysiological relevance of the gene variant and other linked variants with RA.     

Transgene-mediated and elicitor-induced perturbation of metabolic channeling at the entry point into the phenylpropanoid pathway

3H-l-Phenylalanine is incorporated into a range of phenylpropanoid compounds when fed to tobacco cell cultures. A significant proportion of (3)H-trans-cinnamic acid formed from (3)H-l-phenylalanine did not equilibrate with exogenous trans-cinnamic acid and therefore may be rapidly channeled through the cinnamate 4-hydroxylase (C4H) reaction to 4-coumaric acid. Such compartmentalization of trans-cinnamic acid was not observed after elicitation or in cell cultures constitutively expressing a bean phenylalanine ammonia-lyase (PAL) transgene. Channeling between PAL and C4H was confirmed in vitro in isolated microsomes from tobacco stems or cell suspension cultures. This channeling was strongly reduced in microsomes from stems or cell cultures of transgenic PAL-overexpressing plants or after elicitation of wild-type cell cultures. Protein gel blot analysis showed that tobacco PAL1 and bean PAL were localized in both soluble and microsomal fractions, whereas tobacco PAL2 was found only in the soluble fraction. We propose that metabolic channeling of trans-cinnamic acid requires the close association of specific forms of PAL with C4H on microsomal membranes.     

Oscillatory Activity in the Medial Prefrontal Cortex and Nucleus Accumbens Correlates with Impulsivity and Reward Outcome

Actions expressed prematurely without regard for their consequences are considered impulsive. Such behaviour is governed by a network of brain regions including the prefrontal cortex (PFC) and nucleus accumbens (NAcb) and is prevalent in disorders including attention deficit hyperactivity disorder (ADHD) and drug addiction. However, little is known of the relationship between neural activity in these regions and specific forms of impulsive behaviour. In the present study we investigated local field potential (LFP) oscillations in distinct sub-regions of the PFC and NAcb on a 5-choice serial reaction time task (5-CSRTT), which measures sustained, spatially-divided visual attention and action restraint. The main findings show that power in gamma frequency (50–60 Hz) LFP oscillations transiently increases in the PFC and NAcb during both the anticipation of a cue signalling the spatial location of a nose-poke response and again following correct responses. Gamma oscillations were coupled to low-frequency delta oscillations in both regions; this coupling strengthened specifically when an error response was made. Theta (7–9 Hz) LFP power in the PFC and NAcb increased during the waiting period and was also related to response outcome. Additionally, both gamma and theta power were significantly affected by upcoming premature responses as rats waited for the visual cue to respond. In a subgroup of rats showing persistently high levels of impulsivity we found that impulsivity was associated with increased error signals following a nose-poke response, as well as reduced signals of previous trial outcome during the waiting period. Collectively, these in-vivo neurophysiological findings further implicate the PFC and NAcb in anticipatory impulsive responses and provide evidence that abnormalities in the encoding of rewarding outcomes may underlie trait-like impulsive behaviour.