Characterization of the recombinant copper chaperone (CCS) from the plant Glycine (G.) max

The goal of the present work was to characterize the recombinant copper chaperone (CCS) from soybean. Very little is known about plant copper chaperones, which makes this study of current interest, and allows for a comparison with the better known homologues from yeast and humans. To obtain sizeable amounts of pure protein suitable for spectroscopic characterization, we cloned and overexpressed the G. max CCS chaperone in E. coli in the presence of 0.5 mM CuSO(4) and 0.5 mM ZnSO(4) in the broth. A pure protein preparation was obtained by using two IMAC steps and pH gradient chromatography. Most of the proteins were obtained as apo-form, devoid of copper atoms. The chaperone showed a high content (i.e., over 40%) of loops, turns and random coil as determined both by circular dichroism and homology modelling. The homology 3-D structural model suggests the protein might fold in three structural protein domains. The 3-D model along with the primary structure and spectroscopic data may suggest that copper atoms occupy the two metal binding sites, MKCEGC and CTC, within the N-terminal domain I and C-terminal domain III, respectively. But only one Zn-binding site was obtained spectroscopically.     

Insights into the design of a hybrid system between Anabaena ferredoxin-NADP+ reductase and bovine adrenodoxin.

The opportunity to design enzymatic systems is becoming more feasible due to detailed knowledge of the structure of many proteins. As a first step, investigations have aimed to redesign already existing systems, so that they can perform a function different from the one for which they were synthesized. We have investigated the interaction of electron transfer proteins from different systems in order to check the possibility of heterologous reconstitution among members of different chains. Here, it is shown that ferredoxin-NADP+ reductase from Anabaena and adrenodoxin from bovine adrenal glands are able to form optimal complexes for thermodynamically favoured electron transfer reactions. Thus, electron transfer from ferredoxin-NADP+ reductase to adrenodoxin seems to proceed through the formation of at least two different complexes, whereas electron transfer from adrenodoxin to ferredoxin-NADP+ reductase does not take place due because it is a thermodynamically nonfavoured process. Moreover, by using a truncated adrenodoxin form (with decreased reduction potential as compared with the wild-type) ferredoxin-NADP+ reductase is reduced. Finally, these reactions have also been studied using several ferredoxin-NADP+ reductase mutants at positions crucial for interaction with its physiological partner, ferredoxin. The effects observed in their reactions with adrenodoxin do not correlate with those reported for their reactions with ferredoxin. In summary, our data indicate that although electron transfer can be achieved in this hybrid system, the electron transfer processes observed are much slower than within the physiological partners, pointing to a low specificity in the interaction surfaces of the proteins in the hybrid complexes.     

An SNP in the Adiponectin Gene Is Associated with Decreased Serum Adiponectin Levels and Risk for Impaired Glucose Tolerance

Adiponectin is a plasma protein produced by the adipose tissue. Hypoadiponectinemia has been associated with insulin resistance and several components of the metabolic syndrome (MS): type 2 diabetes, obesity, and dyslipidemia. We investigated whether single nucleotide polymorphisms (SNPs) at positions 45 and 276 in the adiponectin gene were associated with features of the MS in 747 unrelated Spanish subjects. The G allele of SNP45 and the G/G genotype of SNP276 were associated with impaired glucose tolerance (p = 0.020 and 0.042, respectively). The G/G genotype for SNP276 was associated with lower serum adiponectin levels as compared with the G/T and T/T genotypes (G/G, 10.10 ± 0.24 μg/mL; G/T, 10.98 ± 0.32 μg/mL; T/T, 12.00 ± 0.92 μg/mL; p = 0.015) even after adjustment for sex, age, BMI, waist‐to‐hip ratio, homeostasis model assessment index, and the degree of glucose tolerance (p = 0.040). We found a significant negative association of circulating adiponectin levels with waist‐to‐hip ratio (r = ?0.42, p < 0.001), sagittal abdominal diameter (r = ?0.24, p < 0.001), triglycerides (r = ?0.32, p < 0.001), homeostasis model assessment index (r = ?0.14, p = 0.001), and uric acid (r = ?0.36, p < 0.001) and positive association with high‐density lipoprotein‐cholesterol (r = 0.41, p < 0.001). Our findings indicate that serum adiponectin levels are associated with several components of the MS. The SNP276 of the adiponectin gene may affect impaired glucose tolerance and hypoadiponectinemia.     

Quercetin ameliorates metabolic syndrome and improves the inflammatory status in obese Zucker rats

The aim of this study was to analyze the effects of chronic administration of high doses of quercetin on metabolic syndrome abnormalities, including obesity, dyslipidemia, hypertension, and insulin resistance. For this purpose, obese Zucker rats and their lean littermates were used. The rats received a daily dose of quercetin (2 or 10 mg/kg of body weight) or vehicle for 10 weeks. Body weight and systolic blood pressure (SBP) were recorded weekly. At the end of the treatment, plasma concentrations of triglycerides, total cholesterol, free-fatty acids (FFAs), glucose, insulin, adiponectin, and nitrate plus nitrite (NOx) were determined. Tumor necrosis factor-alpha (TNF-alpha) production, inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) protein expression were analyzed in visceral adipose tissue (VAT). The raised SBP and high plasma concentrations of triglycerides, total cholesterol, FFA, and insulin found in obese Zucker rats were reduced in obese rats that received either of the doses of quercetin assayed. The higher dose also improved the inflammatory status peculiar to this model, as it increased the plasma concentration of adiponectin, reduced NOx levels in plasma, and lowered VAT TNF-alpha production in obese Zucker rats. Furthermore, chronic intake of the higher dose of quercetin enhanced VAT eNOS expression among obese Zucker rats, whereas it downregulated VAT iNOS expression. In conclusion, both doses of quercetin improved dyslipidemia, hypertension, and hyperinsulinemia in obese Zucker rats, but only the high dose produced antiinflammatory effects in VAT together with a reduction in body weight gain.     

Pregnancy-induced hyperphagia is associated with increased gene expression of hypothalamic agouti-related peptide in rats

Pregnancy is characterized by an increase in food intake that, in turn, produce a positive energy balance in order to face the considerable metabolic demands associated with the challenge of reproduction. Since hypothalamus is a key brain region involved in many peripheral signals and neuronal pathways that control energy homeostasis and food intake, we investigated if during pregnancy the increase in food intake is mediated by stimulating orexigenic and/or inhibiting anorexigenic neural pathways. We examined hypothalamic gene expressions of Ob-Rb, NPY, AgRP, POMC, MC4-R, and preproorexins in pregnant Wistar rats at day 19 of gestation. Food intake and body weight were increased progressively during the pregnancy. Visceral fat mass depots and serum leptin levels were also increased when compared with virgin animals. No differences were found in mRNA expression of Ob-Rb, POMC, MC4-R, NPY or preproorexin between virgin and pregnant animals. However, pregnancy produced a selective increase in AgRP mRNA levels. These results indicate that the positive energy balance that occurred during pregnancy can hardly be explained by changes in Ob-Rb despite hyperleptinemia associated with pregnancy. The enhanced expression of AgRP suggests the involvement of this neuropeptide in mediating pregnancy-associated hyperphagia.     

Curcumin in plasma and urine: quantitation by high-performance liquid chromatography

Curcumin, a derivative of the plant Curcuma longa, is used extensively in the food industry. It is a major component of curry powder, and research has shown that curcumin may prevent cancer and other chronic diseases. We have developed a robust automated analytical method for the determination of curcumin in plasma and urine. The method involves extracting the curcumin from 0.2 ml sample volume with ethyl acetate/methanol organic solvents, and use of an internal standard, beta-17-estradiol acetate. Analysis utilizes a reversed-phase C(18) column and UV detection at 262 nm. Performance characteristics have been assessed. The assay is linear from 0.2 to 7.0 microgram/ml. The coefficient of variation for intra- and inter-day assays is <7.5%. The average recovery of curcumin from plasma and urine is greater than 96%. The data presented in this report demonstrate that the method provides rapid, sensitive, precise and accurate measurements of curcumin concentrations in plasma and urine.     

Structure-function relationships in Anabaena ferredoxin/ferredoxin:NADP(+) reductase electron transfer: insights from site-directed mutagenesis,transient absorption spectroscopy and X-ray crystallography

The interaction between reduced Anabaena ferredoxin and oxidized ferredoxin:NADP(+) reductase (FNR), which occurs during photosynthetic electron transfer (ET), has been investigated extensively in the authors' laboratories using transient and steady-state kinetic measurements and X-ray crystallography. The effect of a large number of site-specific mutations in both proteins has been assessed. Many of the mutations had little or no effect on ET kinetics. However, non-conservative mutations at three highly conserved surface sites in ferredoxin (F65, E94 and S47) caused ET rate constants to decrease by four orders of magnitude, and non-conservative mutations at three highly conserved surface sites in FNR (L76, K75 and E301) caused ET rate constants to decrease by factors of 25-150. These residues were deemed to be critical for ET. Similar mutations at several other conserved sites in the two proteins (D67 in Fd; E139, L78, K72, and R16 in FNR) caused smaller but still appreciable effects on ET rate constants. A strong correlation exists between these results and the X-ray crystal structure of an Anabaena ferredoxin/FNR complex. Thus, mutations at sites that are within the protein-protein interface or are directly involved in interprotein contacts generally show the largest kinetic effects. The implications of these results for the ET mechanism are discussed.     

Chlororespiration is involved in the adaptation of Brassica plants to heat and high light intensity

Two species of Brassica were used to study their acclimation to heat and high illumination during the first stages of development. One, Brassica fruticulosa, is a wild species from south-east Spain and is adapted to both heat and high light intensity in its natural habitat, while the other, Brassica oleracea, is an agricultural species that is widely cultivated throughout the world. Growing Brassica plants under high irradiance and moderate heat was seen to affect the growth parameters and the functioning of the photosynthetic apparatus. The photosystem II (PSII) quantum yields and the capacity of photosynthetic electron transport, which were lower in B. fruticulosa than in B. oleracea, decreased in B. oleracea plants when grown under stress conditions, indicating inhibition of PSII. However, in B. fruticulosa, the values of these parameters were similar to the values of control plants. Photosystem I (PSI) activity was higher in B. fruticulosa than in B. oleracea, and in both species this activity increased in plants exposed to heat and high illumination. Immunoblot analysis of thylakoid membranes using specific antibodies raised against the NDH-K subunit of the thylakoidal NADH dehydrogenase complex (NADH DH) and against plastid terminal oxidase (PTOX) revealed a higher amount of both proteins in B. fruticulosa than in B. oleracea. In addition, PTOX activity in plastoquinone oxidation, and NADH DH activity in thylakoid membranes were higher in the wild species (B. fruticulosa) than in the agricultural species (B. oleracea). The results indicate that tolerance to high illumination and heat of the photosynthetic activity was higher in the wild species than in the agricultural species, suggesting that plant adaptation to these stresses in natural conditions favours subsequent acclimation, and that the chlororespiration process is involved in adaptation to heat and high illumination in Brassica.