Integrin alphaIIbbeta3:ligand interactions are linked to binding-site remodeling

This study tested the hypothesis that high-affinity binding of macromolecular ligands to the alphaIIbbeta3 integrin is tightly coupled to binding-site remodeling, an induced-fit process that shifts a conformational equilibrium from a resting toward an open receptor. Interactions between alphaIIbbeta3 and two model ligands-echistatin, a 6-kDa recombinant protein with an RGD integrin-targeting sequence, and fibrinogen's gamma-module, a 30-kDa recombinant protein with a KQAGDV integrin binding site-were measured by sedimentation velocity, fluorescence anisotropy, and a solid-phase binding assay, and modeled by molecular graphics. Studying echistatin variants (R24A, R24K, D26A, D26E, D27W, D27F), we found that electrostatic contacts with charged residues at the alphaIIb/beta3 interface, rather than nonpolar contacts, perturb the conformation of the resting integrin. Aspartate 26, which interacts with the nearby MIDAS cation, was essential for binding, as D26A and D26E were inactive. In contrast, R24K was fully and R24A partly active, indicating that the positively charged arginine 24 contributes to, but is not required for, integrin recognition. Moreover, we demonstrated that priming--i.e., ectodomain conformational changes and oligomerization induced by incubation at 35 degrees C with the ligand-mimetic peptide cHarGD--promotes complex formation with fibrinogen's gamma-module. We also observed that the gamma-module's flexible carboxy terminus was not required for alphaIIbbeta3 integrin binding. Our studies differentiate priming ligands, which bind to the resting receptor and perturb its conformation, from regulated ligands, where binding-site remodeling must first occur. Echistatin's binding energy is sufficient to rearrange the subunit interface, but regulated ligands like fibrinogen must rely on priming to overcome conformational barriers.     

Models of fibronectin.

The radius of gyration of human plasma fibronectin was determined by light scattering both under conditions in which the molecule is in an extended conformation (ionic strength 1.01 M, pH 8) and close to its native, more compact conformation (ionic strength 0.16 M, pH 8). These values were found to be 17.5 +/- 0.8 nm and 10.7 +/- 0.9 nm respectively, for a constant mol. wt of 533,000 +/- 8000, in excellent agreement with the value of 520,000 deduced from its known composition. A set of models, each made of two identical, end-to-end joined chains of 28 beads, was then constructed, and their calculated physico-chemical parameters were compared with those available for the whole fibronectin molecule and for some of its proteolytic fragments in both conformations. Two possible models for the circulating form are presented here: in both, the fibronectin molecule is in a compact, tangled conformation, with the amino-terminal end of one chain folded over to the carboxy end of itself or of the other chain either in a hairpin or in a circular fashion. With the exception of the carboxy-terminal fibrin(ogen)-binding domains, all the domains appear to be well exposed to the solvent, and thus free to interact with potential ligands.     

High Sodium and Low Potassium Intake among Italian Children: Relationship with Age,Body Mass and Blood Pressure

Background

Hypertension is the leading cause of death in developed countries and reduction of salt intake is recommended as a key preventive measure.

Objective

To assess the dietary sodium and potassium intakes in a national sample of Italian children and adolescents and to examine their relationships with BMI and blood pressure (BP) in the framework of the MINISAL survey, a program supported by the Italian Ministry of Health.

Population and Methods

The study population included 1424 healthy subjects (766 boys, 658 girls) aged 6-18 years (mean age: 10.1±2.9) who were consecutively recruited in participating National Health Service centers in 10 Italian regions. Electrolyte intake was estimated from 24 hour urine collections tested for completeness by the concomitant measurement of creatinine content. Anthropometric indices and BP were measured with standardized procedures.

Results

The average estimated sodium intake was 129 mmol (7.4 g of salt) per day among boys and 117 mmol (6.7 g of salt) among girls. Ninety-three percent of the boys and 89% of the girls had a consumption higher than the recommended age-specific standard dietary target. The estimated average daily potassium intakes were 39 mmol (1.53 g) and 36 mmol (1.40 g), respectively, over 96% of the boys and 98% of the girls having a potassium intake lower than the recommended adequate intake. The mean sodium/potassium ratio was similar among boys and girls (3.5 and 3.4, respectively) and over 3-fold greater than the desirable level. Sodium intake was directly related to age, body mass and BP in the whole population.

Conclusions

The Italian pediatric population is characterized by excessive sodium and deficient potassium intake. These data suggest that future campaigns should focus on children and adolescents as a major target in the framework of a population strategy of cardiovascular prevention.     

Enthalpy/entropy compensation phenomena in the reduction thermodynamics of electron transport metalloproteins

Compensation phenomena between the enthalpy and entropy changes of the reduction reaction for all classes of electron transport metalloproteins, namely cytochromes, iron-sulfur, and blue copper proteins, are brought to light. This is the first comprehensive report on such effects for biological redox reactions. Following Grunwalds approach for the interpretation of H/S compensation for solution reactions, it is concluded that reduction-induced solvent reorganization effects involving the hydration shell of the molecule dominate the reduction thermodynamics in these species, although they have no net effect on the E° values, owing to exact compensation. Thus the reduction potentials of these species are primarily determined by the selective enthalpic stabilization of one of the two oxidation states due to ligand binding interactions and electrostatics at the metal site and by the entropic effects of reduction-induced changes in protein flexibility.     

Food Marketing towards Children: Brand Logo Recognition,Food-Related Behavior and BMI among 3–13-Year-Olds in a South Indian Town

Objectives

To assess exposure to marketing of unhealthy food products and its relation to food related behavior and BMI in children aged 3–13, from different socioeconomic backgrounds in a south Indian town.

Methods

Child-parent pairs (n = 306) were recruited at pediatric clinics. Exposure to food marketing was assessed by a digital logo recognition test. Children matched 18 logos of unhealthy food (high in fat/sugar/salt) featured in promotion material from the food industry to pictures of corresponding products. Children''s nutritional knowledge, food preferences, purchase requests, eating behavior and socioeconomic characteristics were assessed by a digital game and parental questionnaires. Anthropometric measurements were recorded.

Results

Recognition rates for the brand logos ranged from 30% to 80%. Logo recognition ability increased with age (p<0.001) and socioeconomic level (p<0.001 comparing children in the highest and lowest of three socioeconomic groups). Adjusted for gender, age and socioeconomic group, logo recognition was associated with higher BMI (p = 0.022) and nutritional knowledge (p<0.001) but not to unhealthy food preferences or purchase requests.

Conclusions

Children from higher socioeconomic groups in the region had higher brand logo recognition ability and are possibly exposed to more food marketing. The study did not lend support to a link between exposure to marketing and poor eating behavior, distorted nutritional knowledge or increased purchase requests. The correlation between logo recognition and BMI warrants further investigation on food marketing towards children and its potential role in the increasing burden of non-communicable diseases in this part of India.     

p16(MTS-1/CDKN2/INK4a) in cancer progression

Since its discovery as an inhibitor of cyclin-dependent kinases 4 and 6, the tumor suppressor p16 has continued to gain widespread importance in cancer. The high frequency of deletions of p16 in tumor cell lines first suggested an important role for p16 in carcinogenesis. This initial genetic evidence was subsequently strengthened by numerous studies documenting p16 inactivation in kindreds with familial melanoma. Moreover, a high frequency of p16 gene alterations was found in primary tumors, while recent studies have identified p16 promoter methylation as a major mechanism of tumor-suppressor-gene silencing. Additional insight into p16's role in cancer has come from the genetic analysis of precancerous lesions and various tissue culture models. It is now believed that loss of p16 is an early and often critical event in tumor progression. Consequently, p16 is a major tumor-suppressor gene whose frequent loss occurs early in many human cancers.     

Heterologous expression of bovine and porcine beta-lactoglobulins in Pichia pastoris: towards a comparative functional characterisation

Bovine and porcine beta-lactoglobulins were cloned and expressed in host cells with the aim of developing the tools necessary for their structural, functional and conformational characterisation by NMR techniques. Both lipocalins were expressed in Pichia pastoris, where the use of a constitutive promoter turned out to allow the highest productivity. The yield of recombinant proteins was further improved through multiple integration of the encoding genes and by increasing aeration of the transformed cultures. Both proteins were obtained in the culture medium at the concentration of 200 microg/ml. Recombinant lipocalins were purified by ion-exchange chromatography from the culture medium. A preliminary NMR characterisation showed that both proteins were correctly folded.     

Seasonal alterations in adrenocortical cell function associated with stress-responsiveness and sex in the eastern fence lizard (Sceloporus undulatus)

We characterized steroidogenic properties of dispersed adrenocortical cells from field-active male and female eastern fence lizards (Sceloporus undulatus) to investigate whether alterations in cell function could, in part, explain seasonal variation in baseline and stress-induced plasma corticosterone (B). Lizards were collected during the breeding and postbreeding seasons and shortly prior to hibernation. Dispersed cells in vitro produced B, aldosterone (ALDO), and progesterone in response to 8-Br-cAMP, 25-(OH)cholesterol, adrenocorticotropin (ACTH; as little as 100 fM), and angiotensin II. Maximal progesterone, B, and ALDO responses to ACTH were roughly 1000%, 500%, and 100% greater than corresponding basal values. Angiotensin II was an effective steroidogenic stimulant but much less so than ACTH. Corticosteroid production exhibited considerable steroid-specific variation among seasons. Maximal ACTH-induced B production was lower in the postbreeding season than at either of the other two measurement points, essentially opposite to the pattern for ALDO. Males and females generally produced B at similar rates, but ALDO and progesterone showed numerous sex differences that usually covaried between the two steroids. Cellular sensitivity to 25-(OH)cholesterol and angiotensin II showed few sex differences or seasonal changes. In contrast, sensitivity to ACTH decreased markedly from the breeding to the postbreeding season in males, corresponding to the decrease in stress-responsiveness, and in both sexes was considerably lower prior to hibernation than during the breeding season. Under some conditions, plasma B may be limited by the production capacity of adrenocortical cells. In summary, seasonal variations in body condition, reproductive activity, and baseline and stress-induced plasma B may be attributed at least in part to alterations in adrenocortical cell steroidogenic function.