Heparin stimulates a plasma membrane Ca2+-ATPase of Arabidopsis thaliana

We have studied the effect of heparin, a glycosaminoglycan widely used in releasing tags from fusion proteins, on isoform 8 of Arabidopsis thaliana PM Ca(2+)-ATPase (ACA8) expressed in Saccharomyces cerevisiae strain K616. Heparin stimulates hydrolytic activity of ACA8 with an estimated K(0.5) value for the complex of 15 +/- 1 microg ml(-1), which is unaffected by free [Ca(2+)]. Heparin increases V(max) up to 3-fold while it does not significantly affect the apparent K(m) for free Ca(2+) and for the nucleoside triphosphate substrate. The heparin effect is not additive with that of exogenous calmodulin and heparin is ineffective on a mutant devoid of the N-terminal auto-inhibitory domain (Delta74-ACA8). Altogether, these results indicate that heparin activation is due to partial suppression of the auto-inhibitory function of ACA8 N-terminus. Pull-down assays using heparin-agarose gel show that heparin directly interacts with ACA8. Binding to the heparin-agarose gel occurs also with a peptide reproducing ACA8 sequence (1)M-I(116). Several single-point mutations within ACA8 sequence A56-T63 significantly alter the enzyme response to heparin, suggesting that heparin interaction with this site may be involved in ACA8 activation. These results highlight a new difference between the plant PM Ca(2+)-ATPase and its animal counterpart, which is inhibited by heparin.     

Mapping the UDP-glucuronic acid binding site in UDP-glucuronosyltransferase-1A10 by homology-based modeling: confirmation with biochemical evidence

The UDP-glucuronosyltransferase (UGT) isozyme system is critical for protecting the body against endogenous and exogenous chemicals by linking glucuronic acid donated by UDP-glucuronic acid to a lipophilic acceptor substrate. UGTs convert metabolites, dietary constituents, and environmental toxicants to highly excretable glucuronides. Because of difficulties associated with purifying endoplasmic reticulum-bound UGTs for structural studies, we carried out homology-based computer modeling to aid analysis. The search found structural homology in Escherichia coli UDP-galactose 4-epimerase. Consistent with predicted similarities involving the common UDP moiety in substrate/inhibitor, UDP-glucose and UDP-hexanol amine caused competitive inhibition by Lineweaver-Burk plots. Among predicted binding sites N292, K314, K315, and K404 in UGT1A10, two informative sets of mutants K314R/Q/A/E/G and K404R/E had null activities or 2.7-fold higher/50% less activity, respectively. Scatchard analysis of binding data of the affinity ligand, 5-azidouridine-[beta- (32)P]diphosphoglucuronic acid, to purified UGT1A10-His or UGT1A7-His revealed high- and low-affinity binding sites. 2-Nitro-5-thiocyanobenzoic acid-digested UGT1A10-His bound with the radiolabeled affinity ligand revealed an 11.3 and 14.3 kDa peptide associated with K314 and K404, respectively, in a discontinuous SDS-PAGE system. Similar treatment of 1A10His-K314A bound with the ligand lacked both peptides; 1A10-HisK404R- and 1A10-HisK404E showed 1.3-fold greater and 50% less label in the 14.3 kDa peptide, respectively, compared to 1A10-His without affecting the 11.3 kDa peptide. Scatchard analysis of binding data of the affinity ligand to 1A10His-K404R and -K404E showed a 6-fold reduction and a large increase in K d, respectively. Our results indicate that K314 and K404 are required UDP-glcA binding sites in 1A10, that K404 controls activity and high-affinity sites, and that K314 and K404 are strictly conserved in 70 aligned UGTs, except for S321, equivalent to K314, in UGT2B15 and 2B17 and I321 in the inactive UGT8, which suggests UGT2B15 and 2B17 contain suboptimal activity. Hence our data strongly support UDP-glcA binding to K314 and K404 in UGT1A10.     

Tissue transglutaminase activation modulates inflammation in cystic fibrosis via PPARgamma down-regulation

Cystic fibrosis (CF), the most common life-threatening inherited disease in Caucasians, is due to mutations in the CF transmembrane conductance regulator (CFTR) gene and is characterized by airways chronic inflammation and pulmonary infections. The inflammatory response is not secondary to the pulmonary infections. Indeed, several studies have shown an increased proinflammatory activity in the CF tissues, regardless of bacterial infections, because inflammation is similarly observed in CFTR-defective cell lines kept in sterile conditions. Despite recent studies that have indicated that CF airway epithelial cells can spontaneously initiate the inflammatory cascade, we still do not have a clear insight of the molecular mechanisms involved in this increased inflammatory response. In this study, to understand these mechanisms, we investigated ex vivo cultures of nasal polyp mucosal explants of CF patients and controls, CFTR-defective IB3-1 bronchial epithelial cells, C38 isogenic CFTR corrected, and 16HBE normal bronchial epithelial cell lines. We have shown that a defective CFTR induces a remarkable up-regulation of tissue transglutaminase (TG2) in both tissues and cell lines. The increased TG2 activity leads to functional sequestration of the anti-inflammatory peroxisome proliferator-activated receptor gamma and increase of the classic parameters of inflammation, such as TNF-alpha, tyrosine phosphorylation, and MAPKs. Specific inhibition of TG2 was able to reinstate normal levels of peroxisome proliferator-activated receptor-gamma and dampen down inflammation both in CF tissues and CFTR-defective cells. Our results highlight an unpredicted central role of TG2 in the mechanistic pathway of CF inflammation, also opening a possible new wave of therapies for sufferers of chronic inflammatory diseases.     

Production of an extracellular polysaccharide with emulsifier properties by Penicillium citrinum

Penicillium citrinum produced a glycolipid with emulsifier properties during cultivation on mineral medium with 1% (v/v) olive oil as carbon source. The emulsifier production was growth-associated and reached maximal activity at 60 h of cultivation. The production yield (Y _p/s) was 0.54 and the best emulsifying activity was observed for xylene and diesel oil when compared to other carbohydrates tested. The emulsifier was shown to be stable to a wide range of pH and temperature values and was shown to contain D-galactose, D-glucose and D-xylose (8.2:1.0:5.3) with a total carbohydrate content of 43%. The presence of salts stimulated the emulsification activity, suggesting potential for its application in industrial waste or marine remediation.     

Distributional survey of large branchiopods of rice paddies in Shiga Prefecture,Japan: a Lake Biwa Museum project based on lay amateur participation

A distributional survey of the large branchiopod crustaceans of the rice paddies of Shiga Prefecture, Japan, was conducted by the Lake Biwa Museum in May and June of 1999 and 2000. Collecting kits were issued to over 100 lay Field Reporters, who returned all samples, usually dried, to the museum for processing. Their 242 species-lots were augmented by 146 from Grygier, 46 from Ida, and 41 from other museum staff. Apart from this survey, 50 additional records in 1997–2000 were also taken into account. Seven species were recorded in the prefecture's southern half: Branchinella kugenumaensis (Ishikawa), Caenestheriella gifuensis (Ishikawa), and Lynceus biformis (Ishikawa) with wide distributions; Triops longicaudatus (LeConte) and Leptestheria kawachiensis Uéno limited to paddy areas around the south end of Lake Biwa; and Eulimnadia sp. mostly in remnant paddy areas threatened with urban development in Otsu city, with Triops granarius (Lucas) at a single such site in Otsu. In the prefecture's northern half, only B. kugenumaensis and C. gifuensis occurred to a limited degree. Possible reasons for these differences are discussed. Data pertaining to species co-occurrence and occurrence relative to the ongoing program of paddy field consolidation in the prefecture are presented and discussed. Success of the survey was due to the use of Field Reporters, who were able to provide a high degree of coverage of the study area, as well as the clearly defined habitat, the low number and easy recognizability of species, and the collection of voucher material from all sites. Conservation-related results included the listing of Eulimnadia sp. as a `rare' species by Shiga Prefecture.     

Modeling of the Bacterial Mechanism of Methicillin-Resistance by a Systems Biology Approach

Background

A microorganism is a complex biological system able to preserve its functional features against external perturbations and the ability of the living systems to oppose to these external perturbations is defined “robustness”. The antibiotic resistance, developed by different bacteria strains, is a clear example of robustness and of ability of the bacterial system to acquire a particular functional behaviour in response to environmental changes. In this work we have modeled the whole mechanism essential to the methicillin-resistance through a systems biology approach. The methicillin is a β-lactamic antibiotic that act by inhibiting the penicillin-binding proteins (PBPs). These PBPs are involved in the synthesis of peptidoglycans, essential mesh-like polymers that surround cellular enzymes and are crucial for the bacterium survival.

Methodology

The network of genes, mRNA, proteins and metabolites was created using CellDesigner program and the data of molecular interactions are stored in Systems Biology Markup Language (SBML). To simulate the dynamic behaviour of this biochemical network, the kinetic equations were associated with each reaction.

Conclusions

Our model simulates the mechanism of the inactivation of the PBP by methicillin, as well as the expression of PBP2a isoform, the regulation of the SCCmec elements (SCC: staphylococcal cassette chromosome) and the synthesis of peptidoglycan by PBP2a. The obtained results by our integrated approach show that the model describes correctly the whole phenomenon of the methicillin resistance and is able to respond to the external perturbations in the same way of the real cell. Therefore, this model can be useful to develop new therapeutic approaches for the methicillin control and to understand the general mechanism regarding the cellular resistance to some antibiotics.     

Intestinal Colonization with Enterococcus faecium Does Not Influence Pulmonary Defense against Pseudomonas aeruginosa in Mice

Background

Enterococci, and especially multiresistant Enterococcus faecium, are increasingly found colonizing hospitalized patients. This increased prevalence of colonization is not only associated with an increased prevalence of infections caused by enterococci, but also by infections with other nosocomial pathogens. In this study we investigated the causality of this observed relationship, by determining the influence of intestinal colonization with E. faecium on pulmonary defense against Pseudomonas aeruginosa.

Methodology/Principal Findings

Three groups of mice were tested; 2 groups of mice were pre-treated with vancomycin, of which one group was subsequently treated by oral gavage of vancomycin-resistant E. faecium (VRE). The third group did not receive any pre-treatment. P. aeruginosa pneumonia was induced in all mice. Vancomycin treatment resulted in intestinal gram-negative bacterial overgrowth and VRE treatment resulted in colonization throughout the intestines. All 3 groups of mice were able to clear P. aeruginosa from the lungs and circulation, with comparable lung cytokine responses and lung damage. Mice treated with vancomycin without VRE colonization displayed modestly increased plasma levels of TNF-α and IL-10.

Conclusion

Overgrowth of E. faecium and/or gram-negative bacteria does not impact importantly on pulmonary defense against P. aeruginosa pneumonia.     

Capacity for physical activity predicts weight loss after Roux-en-Y gastric bypass

Despite its overall excellent outcomes, weight loss after Roux-en-Y gastric bypass (RYGB) is highly variable. We conducted this study to identify clinical predictors of weight loss after RYGB. We reviewed charts from 300 consecutive patients who underwent RYGB from August 1999 to November 2002. Data collected included patient demographics, medical comorbidities, and diet history. Of the 20 variables selected for univariate analysis, 9 with univariate P values 相似文献   

Effect of Low‐ and High‐Calcium Dairy‐Based Diets on Macronutrient Oxidation in Humans

Objective: Higher calcium and dairy intakes may be associated with lower body weights, but a mechanism in humans has yet to be elucidated. We compared the effects of a dairy‐based high‐calcium diet and a low‐calcium diet on macronutrient oxidation. Research Methods and Procedures: Subjects (10 men and nine women) consumed a low‐dairy (LD, ～one serving per day, ～500 mg Ca²⁺/d) or high‐dairy (HD, ～three to four servings per day, ～1400 mg Ca²⁺/d) energy balance diet for 1 week. Each diet condition was performed twice. On the 7th day, subjects were studied in a room calorimeter under one of four study conditions, applied in a randomized crossover design. Within each diet condition, subjects were studied under conditions of energy balance and acute energy deficit. The deficit (?600 kcal/d) was induced only for the 24 hours that subjects resided in the room and was achieved by a combination of caloric restriction and exercise. Results: Under energy balance conditions, there was no effect of diet treatment on respiratory quotient or 24‐hour macronutrient oxidation. Under energy deficit conditions, 24‐hour fat oxidation was significantly increased on the HD diet (HD with deficit = 136 ± 13 g/d, LD with deficit = 106 ± 7 g/d, p = 0.02). Discussion: Consumption of a dairy‐based high‐calcium diet increased 24‐hour fat oxidation under conditions of acute energy deficit. We hypothesize that these effects are due to an increased fat oxidation during exercise.     

Conspicuousness,color resemblance,and toxicity in geographically diverging mimicry: The pan‐Amazonian frog Allobates femoralis

Predation risk is allegedly reduced in Batesian and Müllerian mimics, because their coloration resembles the conspicuous coloration of unpalatable prey. The efficacy of mimicry is thought to be affected by variation in the unpalatability of prey, the conspicuousness of the signals, and the visual system of predators that see them. Many frog species exhibit small colorful patches contrasting against an otherwise dark body. By measuring toxicity and color reflectance in a geographically variable frog species and the syntopic toxic species, we tested whether unpalatability was correlated with between‐species color resemblance and whether resemblance was highest for the most conspicuous components of coloration pattern. Heterospecific resemblance in colorful patches was highest between species at the same locality, but unrelated to concomitant variation in toxicity. Surprisingly, resemblance was lower for the conspicuous femoral patches compared to the inconspicuous dorsum. By building visual models, we further tested whether resemblance was affected by the visual system of model predators. As predicted, mimic‐model resemblance was higher under the visual system of simulated predators compared to no visual system at all. Our results indicate that femoral patches are aposematic signals and support a role of mimicry in driving phenotypic divergence or mimetic radiation between localities.