Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: characterization, kinetic studies of transglucosylation, and application of immobilised enzyme for production of alpha-D-glucose 1-phosphate

Sucrose phosphorylase catalyzes the reversible conversion of sucrose (alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside) and phosphate into D-fructose and alpha-D-glucose 1-phosphate. We report on the molecular cloning and expression of the structural gene encoding sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase) in Escherichia coli DH10B. The recombinant enzyme, containing an 11 amino acid-long N-terminal metal affinity fusion peptide, was overproduced 60-fold in comparison with the natural enzyme. It was purified to apparent homogeneity using copper-loaded Chelating Sepharose and obtained in 20% yield with a specific activity of 190 Umg(-1). LmSPase was covalently attached onto Eupergit C with a binding efficiency of 50% and used for the continuous production of alpha-D-glucose 1-phosphate from sucrose and phosphate (600 mM each) in a packed-bed immobilised enzyme reactor (30 degrees C, pH 7.0). The reactor was operated at a stable conversion of 91% (550 mM product) and productivity of approximately 11 gl(-1)h(-1) for up to 600 h. A kinetic study of transglucosylation by soluble LmSPase was performed using alpha-d-glucose 1-phosphate as the donor substrate and various alcohols as acceptors. D- and L-arabitol were found to be good glucosyl acceptors.     

Radioresistance of human carcinoma cells is correlated to a defect in raft membrane clustering

In addition to DNA damage, exposure to irradiation involves the plasma membrane in the early phases of gamma-ray-induced cell death. The involvement of raft microdomains following gamma-radiation derives essentially from the role of ceramide as a critical component leading to apoptosis. It is demonstrated here that gamma-irradiation of a radiosensitive human head and neck squamous carcinoma cell line (SCC61) results in the triggering of raft coalescence to larger membrane platforms associated with the externalization of an acid sphingomyelinase (A-SMase), leading to ceramide release in raft, 30 min postirradiation. For the first time, we show that this structural rearrangement is defective in the radioresistant SQ20B cells and associated with the lack of A-SMase activation and translocation, a result which could explain in part their resistance to apoptosis following ionizing radiation. Moreover, we show that SQ20B are protected against radiation injury through a fivefold upper level of endogenous glutathione compared to SCC61. Overcoming the endogenous antioxidant defenses of SQ20B through either H(2)O(2) treatment or GSH depletion triggers A-SMase activation and translocation, raft coalescence, and apoptosis. On the contrary, ROS scavengers abolished these events in radiosensitive SCC61 cells. Translation of this concept to tumor biology suggests that manipulation of rafts through redox equilibrium may provide opportunities for radiosensitization of tumor cells.     

Impact of Hypertension on the Association of BMI with Risk and Age at Onset of Type 2 Diabetes Mellitus: Age- and Gender-Mediated Modifications

Aims

Given that BMI correlates with risk of Type 2 diabetes mellitus (T2DM), and that hypertension is a common comorbid condition, we hypothesize that hypertension augments significantly the impact of obesity on T2DM onset.

Methods

We obtained data on T2DM in Kuwaiti natives from Kuwait Health Network Registry. We considered 1339 comorbid individuals with onset of hypertension preceding that of T2DM, and 3496 non-hypertensive individuals but with T2DM. Multiple linear regressions, ANOVA tests, and Cox proportional hazards models were used to quantify the impact of hypertension on correlation of BMI with age at onset and risk of T2DM.

Results

Impact of increasing levels of BMI on age at onset ot T2DM is seen augmented in patients diagnosed with hypertension. We find that the slope of the inverse linear relationship between BMI and onset age of T2DM is much steep in hypertensive patients (−0.69, males and −0.39, females) than in non-hypertensive patients (−0.36, males and −0.17, females). The decline in onset age for an unit increase of BMI is two-fold in males than in females. Upon considering BMI as a categorical variable, we find that while the mean onset age of T2DM in hypertensive patients decreases by as much as 5–12 years in every higher BMI categories, significant decrease in non-hypertensive patients exists only when severely obese. Hazard due to hypertension (against the baseline of non-hypertension and normal weight) increases at least two-fold in every obese category. While males have higher hazard due to hypertension in early adulthood, females have higher hazard in late adulthood.

Conclusion

Pre-existing condition of hypertension augments the association of BMI with Type 2 diabetes onset in both males and females. The presented results provide health professionals directives on the extent of weight-loss required to delay onset of Type 2 diabetes in hypertensive versus non-hypertensive patients.     

Malaria Incidence in Children in South-West Burkina Faso: Comparison of Active and Passive Case Detection Methods

Background

The aim of this study was to determine the incidence and seasonal pattern of malaria in children in South-West Burkina Faso, and to compare, in a randomized trial, characteristics of cases detected by active and passive surveillance. This study also enabled the planning of a malaria vaccine trial.

Methods

Households with young children, located within 5 kilometers of a health facility, were randomized to one of two malaria surveillance methods. In the first group, children were monitored actively. Each child was visited twice weekly; tympanic temperature was measured, and if the child had a fever or history of fever, a malaria rapid diagnostic test was performed and a blood smear collected. In the second group, children were monitored passively. The child’s parent or caregiver was asked to bring the child to the nearest clinic if he was unwell. Follow up lasted 13 months from September 2009.

Results

Incidence of malaria (Fever with parasitaemia ≥5,000/µL) was 1.18 episodes/child/year in the active cohort and 0.89 in the passive cohort (rate ratio 1.32, 95% CI 1.13–1.54). Malaria cases in the passive cohort were more likely to have high grade fever; but parasite densities were similar in the two groups. Incidence was highly seasonal; when a specific case definition was used, about 60% of cases occurred within the 4 months June-September.

Conclusion

Passive case detection required at least a 30%–40% increase in the sample size for vaccine trials, compared to active detection, to achieve the same power. However we did not find any evidence that parasite densities were higher with passive than with active detection. The incidence of malaria is highly seasonal and meets the WHO criteria for Seasonal Malaria Chemoprevention (SMC). At least half of the malaria cases in these children could potentially be prevented if SMC was effectively deployed.     

Oligomerization of acidic fibroblast growth factor is not a prerequisite for its cell proliferation activity

Oligomerization of fibroblast growth factors (FGFs) induced on binding to heparin or heparan sulfate proteoglycan is considered to be crucial for receptor activation and initiation of biological responses. To gain insight into the mechanism of activation of the receptor by FGFs, in the present study we investigate the effect(s) of interaction of a heparin analog, sucrose octasulfate (SOS), on the structure, stability, and biological activities of a recombinant acidic FGF from Notophthalmus viridescens (nFGF-1). SOS is found to bind to nFGF-1 and significantly increase the thermodynamic stability of the protein. Using a variety of techniques such as size-exclusion chromatography, sedimentation velocity, and multidimensional nuclear magnetic resonance (NMR) spectroscopy, it is shown that binding of SOS to nFGF-1 retains the protein in its monomeric state. In its monomeric state (complexed to SOS), n-FGF-1 shows significant cell proliferation activity. (15)N and (1)H chemical shift perturbation and the intermolecular nuclear Overhauser effects (NOEs) between SOS and nFGF-1 reveal that the ligand binds to the dense, positively charged cluster located in the groove enclosed by beta-strands 10 and 11. In addition, molecular modeling based on the NOEs observed for the SOS-nFGF-1 complex, indicates that SOS and heparin share a common binding site on the protein. In conclusion, the results of the present study clearly show that heparin-induced oligomerization of nFGF-1 is not mandatory for its cell proliferation activity.     

Bacteria induce skin regeneration via IL-1β signaling

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Is there a Cl−−OH− exchange (Cl−−H+ cotransport) mechanism in the brush-border membrane of the intestine of the fresh water trout (Salmo gairdneri,R.)?

Summary Experiments were performed to determine the presence of a Cl^––OH^– exchange (Cl^––H⁺ cotransport) in the brush-border membranes isolated from the intestinal epithelium of freshwater trout. Determinations of alkaline phosphatase activities have shown that vesicle suspensions had an enrichment factor of about 17 in this enzyme indicating a high degree of purification of the brush-border membrane preparation. Cl^– uptake by vesicles in the presence of a proton gradient occurs against a concentration gradient with an overshoot ratio of about 2 and is inhibited by SITS. Several lines of evidence suggest that the mechanism involved is electrical in nature: (i) Cl^– uptake is increased when the proton gradient is increased, but there is a linear relationship between the Cl^– uptake and the Nernst potential of protons. (ii) Cl^– uptake is increased when a proton ionophore is added at low concentration and inhibited at high concentration, suggesting that a proton conductance is involved in the Cl^– uptake. (iii) there is a linear relationship between the initial speed of the uptake of increasing Cl^– concentrations and the Cl^– concentration. (iv) Cl^– uptake can be modulated by different potassium gradients with or without valinomycin. It is concluded that the enterocyte of the freshwater trout is not equipped with a Cl^––OH^– exchange and the Cl^– uptake by vesicles is realized by a Cl^– conductance.     

An industrial photosynthetic system for Skeletonema costatum in arid regions

A biocultivator has been developed for large-scale production in arid regions using the diatom Skeletonema costatum. The product is used for the larviculture of aquaculturally important marine food organisms. This system was operated in India with a total solar collection area of 560 m². Operating under a batch regime, this slow-mixing open reactor has a long light path. Starter cultures were supplied from a polythene bag hung in the air with feeble aeration to prevent distortion and rupture of the long-forming chains. Growth in this system with organic nutrients showed the following relationship between cell number (Y) and culture time in hours (χ: Y = 0.03699χ – 0.1540 Under vertical mixing, a high and low cycle of solar energy irradiance attenuated exponentially just beyond the 1.5-m light path, while areal productivity levelled off at 60 g C m^-3 d^-1. This large-scale approach is appropriate for factory-scale production of cost-effective unialgal Skeletonema ( US $ 0.067 kg^-1 dry matter). This revised version was published online in June 2006 with corrections to the Cover Date.     

Insulin enhances protein phosphorylation in isolated hepatocytes by inhibiting an amiloride sensitive phosphatase

The effects of amiloride on basal and hormone-stimulated protein phosphorylation were studied in isolated rat hepatocytes labeled with ³²P-phosphate. Two types of effect on basal phosphorylation were detected: 1. an increase in labeling of two proteins with molecular weights 93,000 and 18,000; 2. a decrease in labeling of proteins with molecular weights 46,000, 34,000, 22,000 and 13,000. All these effects were dose-dependent (maximum with 0,8-to 1 mM) and reached a maximum after 30 to 40 min treatment of the cells with the drug. Amiloride inhibited specifically all insulin effects whereas glucagon specific effects were largely unaffected. In pulse-chase experiments, amiloride increased and insulin decreased the rate of dephosphorylation of the same proteins (Mr 46,000, 34,000 and 22,000). The data support the conclusion that in hepatocytes insulin increases the degree of phosphorylation of proteins by inhibiting an amiloride-sensitive phosphatase.     

Volumetric determination of metatarsal robusticity in a few living primates and in the foot ofOreopithecus

A new technique is described for the volumetric determination of metatarsal robusticity. Since volume was substituted for weight of a previously used technique, that technique is applicable to fossilized material. The comparability between the metric and volumetric techniques was tested and found to be sufficiently close to make the two techniques interchangeable. When the volumetric technique was applied to fourOreopithecus metatarsals, their robusticity was found to follow the 1→5 pattern (in which M5 is the least robust metatarsal), a pattern typical for all higher primates but present also inHomo. Their total robusticity was found to be closest toHomo and the gorilla, suggesting the likelihood of a terrestrial habitat forOreopithecus.