Assessment of Physicochemical Properties of Biodiesel from African Grapes (Lannea microcarpa Engl.& K.Krause)

The African Grape (Lannea microcarpa) seed oil was extracted and subjected to fuel properties tests according to standard method for oil and fuel analysis to evaluate its suitability as oil crop for biodiesel production in Nigeria. The oil was transesterified using alkali hydrolysis to biodiesel. The yields of the oil and its methyl ester were 41.20 ±1.32% and 85 ± 1.30% respectively. The biodiesel produced was analysed for its  physicochemical properties and yielded the following properties; kinematic viscosity at 40oC (5.80 cSt), acid value (1.66 mgKOHg-1), flash point (96 oC), cloud point (+9oC), sulphur content (0.03 wt %), and free glycerol (0.54%). The results obtained showed that most of the important properties were within the recommended standards for a biofuel grade biodiesel and suggest the potential of L. microcarpa seeds as a source of biodiesel.Keywords: African Grape, Lannea microcarpa, Seeds, Oil, Biodiese

Regional Analgesia for Post-Operative Pain Management – Initial Experience in a Low Resource Setting

Objective: The aim of this study is to demonstrate the use of some regional anaesthetic techniques in effective postoperative pain control in a low resource setting. We also wanted to find out the potential benefits and prospects of regional techniques to achieve effective postoperative analgesia.Design: This study was a prospective observational study in which 25 patients presenting for various orthopaedic and general surgical procedures were recruited randomly.Setting: Ahmadu Bello University Teaching Hospital (ABUTH), Zaria, Nigeria from December, 2008 to May, 2009.Subjects: Eligible patients were males and females aged 21 – 55 years. These included emergency and elective cases.Results: The age range was 21-55 years with a mean age of 34 years. Of the 25 patient studied, 14 of them were men and 11 women constituting 56% and 44% respectively. Our study shows that Hausa/Fulani ethnic group made up 75% of the study population. Intraoperatively, the anaesthetic techniques used were general anaesthesia (only) in 13 patients (52%), Regional techniques consisting of spinals, epidurals, combined spinals and epidurals and brachial plexus blocks in nine patients (36%) and three (12%) of the patients had a combination of general anaesthesia (GA) and regional anaesthesia (RA). For post-operative pain management, nine patients (36%) had continuous brachial plexus block using intermittent injections, 13 (52%) patients had epidural catheters with intermittent top-up injections and three (12%) patients received combined spinal and epidural with an epidural catheter left in-situ for intermittenttop-ups. The drugs used for top-ups included 0.125% plain bupivacaine (15 patients), 0.125% plain bupivacaine + 2.5mcgs/ml Fentanyl (10 patients) in 10ml aliquots. The outcome was good in most patients with 19 patients (82.4%) experiencing only mild pain (numeric pain score 0-3). Onset of post-operative pain was 13-18 hours in most (52%) of patients with majority of patients (80%) requiring only a single dose of opioidin 24 hours. There was no incidence of infection at site of catheter insertion one week after the procedure.Conclusion: Regional techniques if used properly can provide superior pain control in the post-operative period. There is reduction in the requirements of opioids in the immediate post-operative when regional techniques are used for pain management. We need to encourage the use of these techniques especially in our setting where resources are sparse and potent analgesics are not always available

Prolific plant regeneration through organogenesis from scalps of Musa sp cv. Tanduk

A prolific plant regeneration system using scalps derived from shoot tips of Musa spp. cv. Tanduk was developed. Highly proliferating scalps, produced after four monthly subcultures of shoot tip explant on Murashige and Skoog (MS) medium supplemented with 100 mM BAP and 1.0 mM IAA, were placed on MS basal medium supplemented with 1.0, 2.5 and 5.0 mM BAP. Rooting of shoots was assessed on hormone-free half strength and full strength MS media and on MS medium supplemented with 1.0, 5.0and 10 mM IBA. Four types of potting media comprising of sand, peat, sand + top soil + goat dung (3:2:1 v/v) and top soil + sand (1:1 v/v) were evaluated during acclimatization of the plantlets. Prolific shootregeneration from scalps was obtained on MS medium containing 2.5 mM BAP, at 9.61 and 40.6 shoots per explant after 4 and 8 weeks of culture, respectively. Meanwhile, the highest mean shoot height of 2.19 cm was attained on MS medium with 1.0 mM BAP after 8 weeks of culture. Full-strength MS medium supplemented with 5.0 mM IBA produced the highest mean number of roots per explant at 15.08, while the highest mean root length of 11.07 cm was obtained on hormone-free half strength MS medium at week 4 of culture. The highest plant survivability of 77.5% was achieved in potting medium consisting of top soil + sand + goat dung after 6 weeks of acclimatization. The plants were morphologically normalwith vigorous stems and broad green leaves

Structural basis for the RING catalyzed synthesis of K63 linked ubiquitin chains

This work was supported by grants from Cancer Research UK (C434/A13067), the Wellcome Trust (098391/Z/12/Z) and Biotechnology and Biological Sciences Research Council (BB/J016004/1).The RING E3 ligase catalysed formation of lysine 63 linked ubiquitin chains by the Ube2V2–Ubc13 E2 complex is required for many important biological processes. Here we report the structure of the RING domain dimer of rat RNF4 in complex with a human Ubc13~Ub conjugate and Ube2V2. The structure has captured Ube2V2 bound to the acceptor (priming) ubiquitin with Lys63 in a position that could lead to attack on the linkage between the donor (second) ubiquitin and Ubc13 that is held in the active “folded back” conformation by the RING domain of RNF4. The interfaces identified in the structure were verified by in vitro ubiquitination assays of site directed mutants. This represents the first view of the synthesis of Lys63 linked ubiquitin chains in which both substrate ubiquitin and ubiquitin-loaded E2 are juxtaposed to allow E3 ligase mediated catalysis.PostprintPeer reviewe

Crystal Structure of UBA2ufd-Ubc9: Insights into E1-E2 Interactions in Sumo Pathways

Canonical ubiquitin-like proteins (UBLs) such as ubiquitin, Sumo, NEDD8, and ISG15 are ligated to targets by E1-E2-E3 multienzyme cascades. The Sumo cascade, conserved among all eukaryotes, regulates numerous biological processes including protein localization, transcription, DNA replication, and mitosis. Sumo conjugation is initiated by the heterodimeric Aos1-Uba2 E1 enzyme (in humans called Sae1-Uba2), which activates Sumo's C-terminus, binds the dedicated E2 enzyme Ubc9, and promotes Sumo C-terminal transfer between the Uba2 and Ubc9 catalytic cysteines. To gain insights into details of E1-E2 interactions in the Sumo pathway, we determined crystal structures of the C-terminal ubiquitin fold domain (ufd) from yeast Uba2 (Uba2ufd), alone and in complex with Ubc9. The overall structures of both yeast Uba2ufd and Ubc9 superimpose well on their individual human counterparts, suggesting conservation of fundamental features of Sumo conjugation. Docking the Uba2ufd-Ubc9 and prior full-length human Uba2 structures allows generation of models for steps in Sumo transfer from Uba2 to Ubc9, and supports the notion that Uba2 undergoes remarkable conformational changes during the reaction. Comparisons to previous structures from the NEDD8 cascade demonstrate that UBL cascades generally utilize some parallel E1-E2 interaction surfaces. In addition, the structure of the Uba2ufd-Ubc9 complex reveals interactions unique to Sumo E1 and E2. Comparison with a previous Ubc9-E3 complex structure demonstrates overlap between Uba2 and E3 binding sites on Ubc9, indicating that loading with Sumo and E3-catalyzed transfer to substrates are strictly separate steps. The results suggest mechanisms establishing specificity and order in Sumo conjugation cascades

Proinflammatory cytokine levels in fibromyalgia patients are independent of body mass index

<p>Abstract</p> <p>Background</p> <p>Fibromyalgia (FM) is characterized by chronic, widespread muscular pain and tenderness and is generally associated with other somatic and psychological symptoms. Further, circulatory levels of proinflammatory cytokines (IL-1β, TNF-α, and IL-6) may be altered in FM patients, possibly in association with their symptoms. Recently, rises in BMI have been suggested to contribute to increased circulating levels of proinflammatory cytokines in FM patients. Our aim was to measure the circulatory levels of proinflammatory cytokines to determine the influence of BMI on these levels in FM patients and healthy volunteers (HVs). In Spanish FM patients (n = 64) and HVs (n = 25), we measured BMI and serum concentrations of proinflammatory cytokines by capture ELISA.</p> <p>Findings</p> <p>There were significant differences in BMI levels between FM patients (26.40 ± 4.46) and HVs (23.64 ± 3.45) and significant increase in IL-6 in FM patients (16.28 ± 8.13 vs 0.92 ± 0.32 pg/ml) (P < 0.001). IL-1β and TNF-α decreased in FM patients compared with HVs. By ANCOVA, there was no significant association between BMI and TNF-α (F = 0.098, p = 0.75) or IL-6 (F = 0.221, p = 0.63) levels in FM patients.</p> <p>Conclusions</p> <p>Our analysis in FM patients of BMI as a covariate of proinflammatory cytokines levels showed that serum TNF-α and IL-6 levels are independent of BMI. Further studies are necessary to dissect these findings and their implication in future therapeutic approaches for FM patients.</p

A Mechanistic View of the Role of E3 in Sumoylation

Sumoylation, the covalent attachment of SUMO (Small Ubiquitin-Like Modifier) to proteins, differs from other Ubl (Ubiquitin-like) pathways. In sumoylation, E2 ligase Ubc9 can function without E3 enzymes, albeit with lower reaction efficiency. Here, we study the mechanism through which E3 ligase RanBP2 triggers target recognition and catalysis by E2 Ubc9. Two mechanisms were proposed for sumoylation. While in both the first step involves Ubc9 conjugation to SUMO, the subsequent sequence of events differs: in the first E2-SUMO forms a complex with the target and E3, followed by SUMO transfer to the target. In the second, Ubc9-SUMO binds to the target and facilitates SUMO transfer without E3. Using dynamic correlations obtained from explicit solvent molecular dynamic simulations we illustrate the key roles played by allostery in both mechanisms. Pre-existence of conformational states explains the experimental observations that sumoylation can occur without E3, even though at a reduced rate. Furthermore, we propose a mechanism for enhancement of sumoylation by E3. Analysis of the conformational ensembles of the complex of E2 conjugated to SUMO illustrates that the E2 enzyme is already largely pre-organized for target binding and catalysis; E3 binding shifts the equilibrium and enhances these pre-existing populations. We further observe that E3 binding regulates allosterically the key residues in E2, Ubc9 Asp100/Lys101 E2, for the target recognition

Functional Reconstitution of a Tunable E3-Dependent Sumoylation Pathway in Escherichia coli

SUMO (small ubiquitin-related modifier) is a reversible post-translational protein modifier that alters the localization, activity, or stability of proteins to which it is attached. Many enzymes participate in regulated SUMO-conjugation and SUMO-deconjugation pathways. Hundreds of SUMO targets are currently known, with the majority being nuclear proteins. However, the dynamic and reversible nature of this modification and the large number of natively sumoylated proteins in eukaryotic proteomes makes molecular dissection of sumoylation in eukaryotic cells challenging. Here, we have reconstituted a complete mammalian SUMO-conjugation cascade in Escherichia coli cells that involves a functional SUMO E3 ligase, which effectively biases the sumoylation of both native and engineered substrate proteins. Our sumo-engineered E. coli cells have several advantages including efficient protein conjugation and physiologically relevant sumoylation patterns. Overall, this system provides a rapid and controllable platform for studying the enzymology of the entire sumoylation cascade directly in living cells

Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity

Ubiquitination is initiated by transfer of ubiquitin (Ub) from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2), producing a covalently linked intermediate (E2-Ub)(1). Ubiquitin ligases (E3s) of the 'really interesting new gene' (RING) class recruit E2-Ub via their RING domain and then mediate direct transfer of ubiquitin to substrates(2). By contrast, 'homologous to E6-AP carboxy terminus' (HECT) E3 ligases undergo a catalytic cysteine-dependent transthiolation reaction with E2-Ub, forming a covalent E3-Ub intermediate(3,4). Additionally, RING-between-RING (RBR) E3 ligases have a canonical RING domain that is linked to an ancillary domain. This ancillary domain contains a catalytic cysteine that enables a hybrid RING-HECT mechanism(5). Ubiquitination is typically considered a post-translational modification of lysine residues, as there are no known human E3 ligases with non-lysine activity. Here we perform activity-based protein profiling of HECT or RBR-like E3 ligases and identify the neuron-associated E3 ligase MYCBP2 (also known as PHR1) as the apparent single member of a class of RING-linked E3 ligase with esterification activity and intrinsic selectivity for threonine over serine. MYCBP2 contains two essential catalytic cysteine residues that relay ubiquitin to its substrate via thioester intermediates. Crystallographic characterization of this class of E3 ligase, which we designate RING-Cys-relay (RCR), provides insights into its mechanism and threonine selectivity. These findings implicate non-lysine ubiquitination in cellular regulation of higher eukaryotes and suggest that E3 enzymes have an unappreciated mechanistic diversity