Substrate specificities of recombinant murine Golgi alpha1, 2- mannosidases IA and IB and comparison with endoplasmic reticulum and Golgi processing alpha1,2-mannosidases

The catalytic domains of murine Golgi alpha1,2-mannosidases IA and IB that are involved in N-glycan processing were expressed as secreted proteins in P.pastoris . Recombinant mannosidases IA and IB both required divalent cations for activity, were inhibited by deoxymannojirimycin and kifunensine, and exhibited similar catalytic constants using Manalpha1,2Manalpha-O-CH3as substrate. Mannosidase IA was purified as a 50 kDa catalytically active soluble fragment and shown to be an inverting glycosidase. Recombinant mannosidases IA and IB were used to cleave Man9GlcNAc and the isomers produced were identified by high performance liquid chromatography and proton-nuclear magnetic resonance spectroscopy. Man9GlcNAc was rapidly cleaved by both enzymes to Man6GlcNAc, followed by a much slower conversion to Man5GlcNAc. The same isomers of Man7GlcNAc and Man6GlcNAc were produced by both enzymes but different isomers of Man8GlcNAc were formed. When Man8GlcNAc (Man8B isomer) was used as substrate, rapid conversion to Man5GlcNAc was observed, and the same oligosaccharide isomer intermediates were formed by both enzymes. These results combined with proton-nuclear magnetic resonance spectroscopy data demonstrate that it is the terminal alpha1, 2-mannose residue missing in the Man8B isomer that is cleaved from Man9GlcNAc at a much slower rate. When rat liver endoplasmic reticulum membrane extracts were incubated with Man9GlcNAc2, Man8GlcNAc2was the major product and Man8B was the major isomer. In contrast, rat liver Golgi membranes rapidly cleaved Man9GlcNAc2to Man6GlcNAc2and more slowly to Man5GlcNAc2. In this case all three isomers of Man8GlcNAc2were formed as intermediates, but a distinctive isomer, Man8A, was predominant. Antiserum to recombinant mannosidase IA immunoprecipitated an enzyme from Golgi extracts with the same specificity as recombinant mannosidase IA. These immunodepleted membranes were enriched in a Man9GlcNAc2to Man8GlcNAc2- cleaving activity forming predominantly the Man8B isomer. These results suggest that mannosidases IA and IB in Golgi membranes prefer the Man8B isomer generated by a complementary mannosidase that removes a single mannose from Man9GlcNAc2.      

Population parameters and exploitation rate of Sarotherodon galilaeus galilaeus (Cichlidae) in Lakes Doukon and Togbadji,Benin

Growth, mortality, recruitment and relative yield per recruit of Sarotherodon galilaeus galilaeus from Lakes Doukon and Togbadji were studied. Data on total length, total weight and sex were recorded on a monthly basis between January and December 2013 for S. g. galilaeus captured by local fishers. The estimated asymptotic lengths L_∞ were 26.2 and 23.6?cm for Lakes Doukon and Togbadji, respectively, while the growth rate K was 0.73 in Lake Doukon and 0.87 in Lake Togbadji. Estimates of fishing mortality, 0.27 and 0.47 y^?1 for Doukon and Togbadji, respectively, were low relative to natural mortality, 1.51 and 1.74 y^?1, respectively. Sizes at first sexual maturity were 12.8 and 13.2?cm for females and males, respectively, in Lake Doukon, and 11.5 and 12.4?cm for females and males, respectively, in Lake Togbadji. The size at first capture was estimated at 13.3 and 12.7?cm for Lakes Doukon and Togbadji, respectively, which, in the light of the size at maturity estimates, indicates that fish spawn at least once before capture. The current exploitation rates of 0.15 for Lake Doukon and 0.21 for Lake Togbadji suggest that their stocks of S. g. galilaeus are not overexploited in either lake.     

Sweat gland density and response during high-intensity exercise in athletes with spinal cord injuries

Sweat production is crucial for thermoregulation. However, sweating can be problematic for individuals with spinal cord injuries (SCI), as they display a blunting of sudomotor and vasomotor responses below the level of the injury. Sweat gland density and eccrine gland metabolism in SCI are not well understood. Consequently, this study examined sweat lactate (S-LA) (reflective of sweat gland metabolism), active sweat gland density (SGD), and sweat output per gland (S/G) in 7 SCI athletes and 8 able-bodied (AB) controls matched for arm ergometry VO₂peak. A sweat collection device was positioned on the upper scapular and medial calf of each subject just prior to the beginning of the trial, with iodine sweat gland density patches positioned on the upper scapular and medial calf. Participants were tested on a ramp protocol (7 min per stage, 20 W increase per stage) in a common exercise environment (21±1°C, 45-65% relative humidity). An independent t-test revealed lower (p<0.05) SGD (upper scapular) for SCI (22.3 ±14.8 glands · cm⁻²) vs. AB. (41.0 ± 8.1 glands · cm⁻²). However, there was no significant difference for S/G between groups. S-LA was significantly greater (p<0.05) during the second exercise stage for SCI (11.5±10.9 mmol · l⁻¹) vs. AB (26.8±11.07 mmol · l⁻¹). These findings suggest that SCI athletes had less active sweat glands compared to the AB group, but the sweat response was similar (SLA, S/G) between AB and SCI athletes. The results suggest similar interglandular metabolic activity irrespective of overall sweat rate.     

Production of 3-hydroxy-γ-decalactone, the precursor of two decenolides with flavouring properties, by the yeast Yarrowia lipolytica

3-Hydroxy-γ-decalactone is the precursor of dec-2 and dec-3-en-4-olides which are valuable aroma compounds not yet produced. To promote the accumulation of this lactone, the yeast Yarrowia lipolytica was placed in different environmental conditions aiming at altering β-oxidation fluxes. The concentration of substrate, pH, aeration and dissolved oxygen level were modified. We observed an important accumulation at low aeration (0.40 molar yields) and, to a lesser extent, at lower pH (0.15). As oxygen played a key-role, we evaluated its effect at fixed dissolved oxygen and at the pH which was the most favourable to the biotransformation (pH 4.5). At 5% and 30% dissolved oxygen, yields reached 0.50. β-Oxidation fluxes are very dependent on the presence of oxygen and conditions of accumulation of 3-hydroxy-γ-decalactone with very high yields were identified. These results are an important step in the production of the two decenolides. Moreover, they show the high dependence of β-oxidation fluxes on environmental conditions and relate these conditions to the accumulation of intermediates, results that are of interest to all the processes using yeast on lipids or alkanes.     

Functional claudication distance: a reliable and valid measurement to assess functional limitation in patients with intermittent claudication

Background

Pharmacological inhibition of endothelial arginase-II has been shown to improve endothelial nitric oxide synthase (eNOS) function and reduce atherogenesis in animal models. We investigated whether the endothelial arginase II is involved in inflammatory responses in endothelial cells.

Methods

Human endothelial cells were isolated from umbilical veins and stimulated with TNFα (10 ng/ml) for 4 hours. Endothelial expression of the inflammatory molecules i.e. vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin were assessed by immunoblotting.

Results

The induction of the expression of endothelial VCAM-1, ICAM-1 and E-selectin by TNFα was concentration-dependently reduced by incubation of the endothelial cells with the arginase inhibitor L-norvaline. However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC) had no effects. To confirm the role of arginase-II (the prominent isoform expressed in HUVECs) in the inflammatory responses, adenoviral mediated siRNA silencing of arginase-II knocked down the arginase II protein level, but did not inhibit the up-regulation of the adhesion molecules. Moreover, the inhibitory effect of L-norvaline was not reversed by the NOS inhibitor L-NAME and L-norvaline did not interfere with TNFα-induced activation of NF-κB, JNK, p38mapk, while it inhibited p70s6k (S6K1) activity. Silencing S6K1 prevented up-regulation of E-selectin, but not that of VCAM-1 or ICAM-1 induced by TNFα.

Conclusion

The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells. The anti-inflammatory properties of L-norvaline are partially attributable to its ability to inhibit S6K1.     

Use of dietary diversity score as a proxy indicator of nutrient adequacy of rural elderly people in Sri Lanka

ABSTRACT: BACKGROUND: Macro and micro nutrient deficiencies are public health concerns in most developing countries including Sri Lanka, partly due to monotonous, cereal-based diet that lacks diversity. The objective of the study was to assess validity of food variety score (FVS), dietary diversity score (DDS) and dietary serving score (DSS) as indicators of nutrient adequacy of rural elderly people in Sri Lanka. FINDINGS: A sample of 200 apparently healthy elderly people >60y of age were studied. A single 24 h recall was performed to compute dietary diversity indicators. Pearson's correlation was used to assess the utility of FVS, DDS and DSS as indicators of nutrient adequacy. Sensitivity (Se) and specificity (Spe) analysis were done to determine the most appropriate cut-off points for using FVS and DDS to categorize elderly people with adequate nutrient intake. The average (standard deviation) of the food variety score, dietary diversity score and dietary serving score was 8.4 (2), 4.4 (0.9) and 11.4 (2.5), respectively. Mean adequacy ratio (MAR) of 12 nutrients was 0.39 (39 %). Pearson's correlation coefficients between MAR and FVS was 0.45 (P < 0.01), for DDS it was 0.48 (P < 0.01) and for DSS it was 0.58 (P < 0.01). When maximizing sensitivity and specificity, the best cut-off point for achieving 50 % of MAR was about 9 and 4.5 for FVS and DDS, respectively. CONCLUSION: In conclusion, FVS, DDS and DSS were useful proxy indicators of nutrient adequacy of rural elderly people in Sri Lanka. Indeed, the performance of the indicators is improved when considering the quantities of food consumed.     

AtMND1 is required for homologous pairing during meiosis in Arabidopsis

Background  

Pairing of homologous chromosomes at meiosis is an important requirement for recombination and balanced chromosome segregation among the products of meiotic division. Recombination is initiated by double strand breaks (DSBs) made by Spo11 followed by interaction of DSB sites with a homologous chromosome. This interaction requires the strand exchange proteins Rad51 and Dmc1 that bind to single stranded regions created by resection of ends at the site of DSBs and promote interactions with uncut DNA on the homologous partner. Recombination is also considered to be dependent on factors that stabilize interactions between homologous chromosomes. In budding yeast Hop2 and Mnd1 act as a complex to promote homologous pairing and recombination in conjunction with Rad51 and Dmc1.