Neurocognitive and Hormonal Correlates of Voluntary Weight Loss in Humans

1. Download high-res image (127KB)
2. Download full-size image

     

Population dynamics of the Wolbachia infection causing cytoplasmic incompatibility in Drosophila melanogaster.

Field populations of Drosophila melanogaster are often infected with Wolbachia, a vertically transmitted microorganism. Under laboratory conditions the infection causes partial incompatibility in crosses between infected males and uninfected females. Here we examine factors influencing the distribution of the infection in natural populations. We show that the level of incompatibility under field conditions was much weaker than in the laboratory. The infection was not transmitted with complete fidelity under field conditions, while field males did not transmit the infection to uninfected females and Wolbachia did not influence sperm competition. There was no association between field fitness as measured by fluctuating asymmetry and the infection status of adults. Infected field females were smaller than uninfecteds in some collections from a subtropical location, but not in other collections from the same location. Laboratory cage studies showed that the infection did not change in frequency when populations were maintained at a low larval density, but it decreased in frequency at a high larval density. Monitoring of infection frequencies in natural populations indicated stable frequencies in some populations but marked fluctuations in others. Simple models suggest that the infection probably provides a fitness benefit for the host in order to persist in populations. The exact nature of this benefit remains elusive.     

Heterologous Expression of a Bioactive β-Hexosyltransferase,an Enzyme Producer of Prebiotics,from Sporobolomyces singularis

Galacto-oligosaccharides (GOS) are indigestible dietary fibers that are able to reach the lower gastrointestinal tract to be selectively fermented by health-promoting bacteria. In this report, we describe the heterologous expression of an optimized synthetically produced version of the β-hexosyltransferase gene (Bht) from Sporobolomyces singularis. The Bht gene encodes a glycosyl hydrolase (EC 3.2.1.21) that acts as galactosyltransferase, able to catalyze a one-step conversion of lactose to GOS. Expression of the enzyme in Escherichia coli yielded an inactive insoluble protein, while the methylotrophic yeast Pichia pastoris GS115 produced a bioactive β-hexosyltransferase (rBHT). The enzyme exhibited faster kinetics at pHs between 3.5 and 6 and at temperatures between 40 and 50°C. Enzyme stability improved at temperatures lower than 40°C, and glucose was found to be a competitive inhibitor of enzymatic activity. P. pastoris secreted a fraction of the bioactive rBHT into the fermentation broth, while the majority of the enzyme remained associated with the outer membrane. Both the secreted and the membrane-associated forms were able to efficiently convert lactose to GOS. Additionally, resting cells with membrane-bound enzyme converted 90% of the initial lactose into GOS at 68% yield (g/g) (the maximum theoretical is 75%) with no secondary residual (glucose or galactose) products. This is the first report of a bioactive BHT from S. singularis that has been heterologously expressed.     

Phenotypic conversion of TK-deficient cells following electroporation of functional TK enzyme.

The ability to phenotypically rescue a mutant (Rat-3, thymidine kinase-deficient) cell line by electroporation of functional TK enzyme has been investigated. Extracts of electroporated cells showed a 35-fold increase in TK enzyme levels under conditions where greater than 90% of the cells remained viable. The electroporated enzyme was intracellular, as demonstrated by the fact that cells were able to utilize exogenous [3H]thymidine for DNA synthesis. By in situ autoradiography, 82% of electroporated cells contained functional enzyme and incorporated [3H]thymidine into DNA. Thus, this technique can efficiently provide a missing metabolic function to cultured mammalian cells.     

Effects of p47phox C terminus phosphorylations on binding interactions with p40phox and p67phox. Structural and functional comparison of p40phox and p67phox SH3 domains

The neutrophil NADPH oxidase produces superoxide anions in response to infection. This reaction is activated by association of cytosolic factors, p47phox and p67phox, and a small G protein Rac with the membranous flavocytochrome b558. Another cytosolic factor, p40phox, is associated to the complex and is reported to play regulatory roles. Initiation of the NADPH oxidase activation cascade has been reported as consecutive to phosphorylation on serines 359/370 and 379 of the p47phox C terminus. These serines surround a polyproline motif that can interact with the Src homology 3 (SH3) module of p40phox (SH3p40) or the C-terminal SH3 of p67phox (C-SH3p67). The latter one presents a higher affinity in the resting state for p47phox. A change in SH3 binding preference following phosphorylation has been postulated earlier. Here we report the crystal structures of SH3p40 alone or in complex with a 12-residue proline-rich region of p47phox at 1.46 angstrom resolution. Using intrinsic tryptophan fluorescence measurements, we compared the affinity of the strict polyproline motif and the whole C terminus peptide with both SH3p40 and C-SH3p67. These data reveal that SH3p40 can interact with a consensus polyproline motif but also with a noncanonical motif of the p47phox C terminus. The electrostatic surfaces of both SH3 are very different, and therefore the binding preference for C-SH3p67 can be attributed to the polyproline motif recognition and particularly to the Arg-368p47 binding mode. The noncanonical motif contributes equally to interaction with both SH3. The influence of serine phosphorylation on residues 359/370 and 379 on the affinity for both SH3 domains has been checked. We conclude that contrarily to previous suggestions, phosphorylation of Ser-359/370 does not modify the SH3 binding affinity for both SH3, whereas phosphorylation of Ser-379 has a destabilizing effect on both interactions. Other mechanisms than a phosphorylation induced switch between the two SH3 must therefore take place for NADPH oxidase activation cascade to start.     

Evaluation of the Anti-Inflammatory,Antioxidant and Immunomodulatory Effects of the Organic Extract of the Red Sea Marine Sponge Xestospongia testudinaria against Carrageenan Induced Rat Paw Inflammation

Marine sponges are found to be a rich source of bioactive compounds which show a wide range of biological activities including antiviral, antibacterial, and anti-inflammatory activities. This study aimed to investigate the possible anti-inflammatory, antioxidant and immunomodulator effects of the methanolic extract of the Red Sea marine sponge Xestospongia testudinaria. The chemical composition of the Xestospongia testudinaria methanolic extract was determined using Gas chromatography-mass spectroscopy (GC-MS) analysis. DPPH (2, 2-diphenyl-1-picryl-hydrazyl) was measured to assess the antioxidant activity of the sponge extract. Carrageenan-induced rat hind paw edema was adopted in this study. Six groups of rats were used: group1: Control, group 2: Carrageenan, group 3: indomethacin (10 mg/kg), group 4–6: Xestospongia testudinaria methanolic extract (25, 50, and 100 mg/kg). Evaluation of the anti-inflammatory activity was performed by both calculating the percentage increase in paw weight and hisopathologically. Assessment of the antioxidant and immunomodulatory activity was performed. GC-MS analysis revealed that there were 41 different compounds present in the methanolic extract. Sponge extract exhibited antioxidant activity against DPPH free radicals. Xestospongia testudinaria methanolic extract (100 mg/kg) significantly decreased % increase in paw weight measured at 1, 2, 3 and 4 h after carrageenan injection. Histopathologically, the extract caused a marked decrease in the capillary congestion and inflammatory cells infiltrate. The extract decreased paw malondialdehyde (MDA) and nitric oxide (NO) and increased the reduced glutathione (GSH), glutathione peroxidase (GPx), and catalase (CAT) activity. It also decreased the inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-1 β(IL-1β) and IL-6. The results of this study demonstrated the anti-inflammatory, antioxidant, and immunomodulatory effects of the methanolic extract of the Red Sea sponge Xestospongia testudinaria (100 mg/kg).     

Assembly of phagocyte NADPH oxidase: A concerted binding process?

Background

The phagocyte NADPH-oxidase is a multicomponent enzyme that generates superoxide anions. It comprises a membrane redox component flavocytochrome b₅₅₈ and four cytosolic proteins (p67^phox, p47^phox, p40^phox and Rac) that must assemble to produce an active system. In this work we focused on the spatio-temporal control of the activation process of phagocyte NADPH oxidase.

Methods

A wide range of techniques including fast kinetics with a stopped-flow apparatus and various combinations of the activating factors was used to test the order of assembly and the role of the p47^phox–p67^phox complex.

Results

The data presented here are consistent with the absence of a catalytic role of the p47^phox–p67^phox interacting state and support the idea of independent binding sites for the cytosolic proteins on the flavocytochrome b₅₅₈ allowing random binding order. However, the formation of the active complex appears to involve a synergistic process of binding of the activated cytosolic subunits to cytochrome b₅₅₈. All partners should be in the vicinity for optimal assembly, a delay or the absence of one of the partners in this process seems to lead to a decrease in the efficiency of the catalytic core.

Conclusion and general significance

The activation and assembly of the NADPH oxidase components have to be achieved simultaneously for the formation of an efficient and optimal enzyme complex. This mechanism appears to be incompatible with continuous fast exchanges of the cytosolic proteins during the production of superoxide ion in the phagosome.     

Pig liver glyceraldehyde-3-P dehydrogenase: purification, crystallization, and characterization.

Glyceraldehyde 3-P dehydrogenase was purified approximately 250-fold from pig liver and crystallized. The purification procedure consisted of treating liver homogenates with zinc chloride, followed by ammonium sulfate fractionation and ion exchange chromatography. The enzyme was monodisperse in the ultracentrifuge with a sedimentation coefficient of s_20,w = 7.85 S. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single subunit band with an approximate molecular weight of 38,000. High-speed sedimentation equilibrium gave a molecular weight of 1.5 × 10⁵. Incubation of the enzyme with ATP at 0 °C caused a loss of its dehydrogenase activity; some of the lost activity was regained upon warming to room temperature. Sucrose density gradient studies of the ATP-treated enzyme revealed a decrease in its sedimentation coefficient from 7.8 to 3.85 S. In the forward reaction direction, the K_m for glyceraldehyde 3-P was 240 μm and the K_m for NAD was 12 μm. In the backward reaction direction, the K_m for NADH was 23 μm and the K_i for NAD was 850 μm. Pig liver glyceraldehyde-3-P dehydrogenase resembles the rabbit muscle enzyme in that it apparently contains 2 to 3 mol of tightly bound NAD. However, it differs strongly from that enzyme in its rate and extent of inactivation by ATP at 0 °C and by urea; the pig liver enzyme, like the yeast enzyme, dissociates much more slowly and much less completely than the rabbit muscle enzyme under comparable conditions.