Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity

The monomeric model of rhodopsin-like G protein-coupled receptors (GPCRs) has progressively yielded the floor to the concept of GPCRs being oligo(di)mers, but the functional correlates of dimerization remain unclear. In this report, dimers of glycoprotein hormone receptors were demonstrated in living cells, with a combination of biophysical (bioluminescence resonance energy transfer and homogenous time resolved fluorescence/fluorescence resonance energy transfer), functional and biochemical approaches. Thyrotropin (TSHr) and lutropin (LH/CGr) receptors form homo- and heterodimers, via interactions involving primarily their heptahelical domains. The large hormone-binding ectodomains were dispensable for dimerization but modulated protomer interaction. Dimerization was not affected by agonist binding. Observed functional complementation indicates that TSHr dimers may function as a single functional unit. Finally, heterologous binding-competition studies, performed with heterodimers between TSHr and LH/CG-TSHr chimeras, demonstrated the unsuspected existence of strong negative cooperativity of hormone binding. Tracer desorption experiments indicated an allosteric behavior in TSHr and, to a lesser extent, in LH/CGr and FSHr homodimers. This study is the first report of homodimerization associated with negative cooperativity in rhodopsin-like GPCRs. As such, it may warrant revisitation of allosterism in the whole GPCR family.     

Ultrasound Measurements of Visceral and Subcutaneous Abdominal Thickness to Predict Abdominal Adiposity Among Older Men and Women

Accurate measures of visceral and abdominal subcutaneous fat are essential for investigating the pathophysiology of obesity. Classical anthropometric measures such as waist and hip circumference cannot distinguish between these two fat depots. Direct imaging methods such as computed tomography and magnetic resonance imaging (MRI) are restricted in large‐scale studies due to practical and ethical issues. We aimed to establish whether ultrasound is a valid alternative method to MRI for the quantitative assessment of abdominal fat depots in older individuals. The study population comprised 74 white individuals (41 men and 33 women, aged 67–76 years) participating in the Hertfordshire Birth Cohort Physical Activity trial. Anthropometry included height, weight, waist and hip circumferences. Abdominal fat was measured by ultrasound in two compartments: visceral fat defined as the depth from the peritoneum to the lumbar spine; and subcutaneous fat defined as the depth from the skin to the abdominal muscles and compared to reference measures by MRI (10‐mm single‐slice image). Ultrasound measures were positively correlated with MRI measures of visceral and subcutaneous fat (visceral: r = 0.82 and r = 0.80 in men and women, respectively; subcutaneous: r = 0.63 and 0.68 in men and women, respectively). In multiple regression models, the addition of ultrasound measures significantly improved the prediction of visceral fat and subcutaneous fat in both men and women over and above the contribution of standard anthropometric variables. In conclusion, ultrasound is a valid method to estimate visceral fat in epidemiological studies of older men and women when MRI and computed tomography are not feasible.     

Protein N-Homocysteinylation Induces the Formation of Toxic Amyloid-Like Protofibrils

Previous works reported that a mild increase in homocysteine level is a risk factor for cardiovascular and neurodegenerative diseases in humans. Homocysteine thiolactone is a cyclic thioester, most of which is produced by an error-editing function of methionyl-tRNA synthetase, causing in vivo post-translational protein modifications by reacting with the ?-amino group of lysine residues. In cells, the rate of homocysteine thiolactone synthesis is strictly dependent on the levels of the precursor metabolite, homocysteine. In this work, using bovine serum albumin as a model, we investigated the impact of N-homocysteinylation on protein conformation as well as its cellular actions. Previous works demonstrated that protein N-homocysteinylation causes enzyme inactivation, protein aggregation, and precipitation. In addition, in the last few years, several pieces of evidence have indicated that protein unfolding and aggregation are crucial events leading to the formation of amyloid fibrils associated with a wide range of human pathologies. For the first time, our results reveal how the low level of protein N-homocysteinylation can induce mild conformational changes leading to the formation of native-like aggregates evolving over time, producing amyloid-like structures. Taking into account the fact that in humans about 70% of circulating homocysteine is N-linked to blood proteins such as serum albumin and hemoglobin, the results reported in this article could have pathophysiological relevance and could contribute to clarify the mechanisms underlying some pathological consequences described in patients affected by hyperhomocysteinemia.     

IGFBP3 Methylation Is a Novel Diagnostic and Predictive Biomarker in Colorectal Cancer

Background and Aim

Aberrant hypermethylation of cancer-related genes has emerged as a promising strategy for the development of diagnostic, prognostic and predictive biomarkers in human cancer, including colorectal cancer (CRC). The aim of this study was to perform a systematic and comprehensive analysis of a panel of CRC-specific genes as potential diagnostic, prognostic and predictive biomarkers in a large, population-based CRC cohort.

Patients and Methods

Methylation status of the SEPT9, TWIST1, IGFBP3, GAS7, ALX4 and miR137 genes was studied by quantitative bisulfite pyrosequencing in a population-based cohort of 425 CRC patients.

Results

Methylation levels of all genes analyzed were significantly higher in tumor tissues compared to normal mucosa (p<0.0001); however, cancer-associated hypermethylation was most frequently observed for miR137 (86.7%) and IGFBP3 (83%) in CRC patients. Methylation analysis using the combination of these two genes demonstrated greatest accuracy for the identification of colonic tumors (sensitivity 95.5%; specificity 90.5%). Low levels of IGFBP3 promoter methylation emerged as an independent risk factor for predicting poor disease free survival in stage II and III CRC patients (HR = 0.49, 95% CI: 0.28–0.85, p = 0.01). Our results also suggest that stage II & III CRC patients with high levels of IGFBP3 methylation do not benefit from adjuvant 5FU-based chemotherapy.

Conclusion

By analyzing a large, population-based CRC cohort, we demonstrate the potential clinical significance of miR137 and IGFBP3 hypermethylation as promising diagnostic biomarkers in CRC. Our data also revealed that IGFBP3 hypermethylation may serve as an independent prognostic and predictive biomarker in stage II and III CRC patients.     

Sexual conflict and environmental change: trade-offs within and between the sexes during the evolution of desiccation resistance

Intralocus sexual conflict occurs when males and females experience sex-specific selection on a shared genome. With several notable exceptions, intralocus sexual conflict has been investigated in constant environments to which the study organisms have had an opportunity to adapt. However, a change in the environment can result in differential or even opposing selection pressures on males and females, creating sexual conflict. We used experimental evolution to explore the interaction between intralocus sexual conflict, sexual dimorphism and environmental variation in Drosophila melanogaster. Six populations were selected for adult desiccation resistance (D), with six matched control populations maintained in parallel (C). After 46 generations, the D populations had increased in survival time under arid conditions by 68% and in body weight by 20% compared to the C populations. The increase in size was the result of both extended development and faster growth rate of D juveniles. Adaptation to the stress came at a cost in terms of preadult viability and female fecundity. Because males are innately less tolerant of desiccation stress, very few D males survived desiccation-selection; while potentially a windfall for survivors, these conditions mean that most males’ fitness was determined posthumously. We conjectured that selection for early maturation and mating in males was in conflict with selection for survival and later reproduction in females. Consistent with this prediction, the sexes showed different patterns of age-specific desiccation resistance and resource acquisition, and there was a trend towards increasingly female-biased sexual size dimorphism. However, levels of desiccation resistance were unaffected, with D males and females increasing in parallel. Either there is a strong positive genetic correlation between the sexes that limits independent evolution of desiccation resistance, or fitness pay-offs from the strategy of riding out the stress bout are great enough to sustain concordant selection on the two sexes. We discuss the forces that mould fitness in males under a regimen where trade-offs between survival and reproduction may be considerable.     

Evaluation of the phenotypic characteristics of coliform bacteria recovered with two methods: the European Drinking Water Directive reference method and the Colilert 18/Quanti-Tray™ system

Summary The taxonomy of the species belonging to Enterobacteriaceae has undergone a series of revisions. As a consequence, the new classification has caused the analytical detection methods to be updated. According to the European Drinking Water Directive 98/83/CE coliforms/Escherichia coli have to be determined with the ISO 9308-1 reference method. Many technical drawbacks of the procedure as well as limitations regarding the recent taxonomy of coliforms have been pointed out by laboratories working in water quality control. In our investigation, water was analyzed in parallel with the reference method and the rapid Colilert 18/Quanti-Tray™ system. Phenotypic characteristics of isolates were recorded for the verification of the response of the two methods to the new taxonomic approach. Results obtained with the ISO standard confirmed the limitations of the test. In fact, in addition to difficulties linked to the readability of results, it failed to detect a significant proportion of coliforms and E. coli in water. Furthermore, it allowed the growth of microorganisms belonging to other groups. The Colilert 18/Quanti-Tray™ system detected a qualitatively and quantitatively higher number of target microorganisms. It also provided results in a shorter time, allowing the simultaneous detection of E. coli and coliforms with no further confirmation steps.     

Survey of Neodohrniphora spp. (Diptera: Phoridae) at colonies of Atta sexdens rubropilosa (FOREL) and specificity of attack behaviour in relation to their hosts

Atta sexdens rubropilosa is a leaf-cutting ant that is a significant agricultural and forestry pest in the Neotropical region. This ant is parasitized by flies from the genera Neodohrniphora spp., Apocephalus spp. and Myrmosicarius spp. This study was carried out to determine which species of Neodohrniphora spp. are found near foraging trails of Atta sexdens rubropilosa and to evaluate the specificity of attack behaviour of these parasitoids. From May 2002 to April 2004, we sampled Neodohrniphora spp. hovering over foraging trails of Atta sexdens rubropilosa between 8:00 and 11:00 h and between 15:00 and 18:00 h. To investigate the attacking behaviour against the ants, flies were released individually inside an observation chamber containing a single leaf-cutting ant worker. Each parasitoid was confronted successively with a worker ant of A. sexdens rubropilosa, Atta laevigata Smith, Acromyrmex crassispinus Forel and Acromyrmex subterraneus molestans Santschi. Phorids of three species were identified: Neodohrniphora elongata Brown, Neodohrniphora declinata Borgmeier and Neodohrniphora tonhascai Brown. The three phorid species were active throughout the year and often along the same foraging trails, but N. elongata was the most frequent species. In the laboratory assay, N. elongata, N. declinata and N. tonhascai attacked workers of A. sexdens rubropilosa, A. laevigata and A. crassispinus, but not of A. subterraneus molestans.     

UVA light stimulates the production of cathepsin G and elastase-like enzymes by dermal fibroblasts: a possible contribution to the remodeling of elastotic areas in sun-damaged skin

Solar elastosis is characterized by accumulation of large amounts of material staining similarly to elastin in the dermis. The nature of this material and the process responsible for its accumulation are still unknown. Elastolytic proteases have important functions in the catabolism of the interstitial matrix and can also generate, by the digestion of the interstitial proteins, soluble peptides which can induce collagen and elastin synthesis and deposition. We investigated whether (i) elastolytic enzymes can be detected in samples from sun-exposed and non-exposed skin, and (ii) ultraviolet (UV) rays influence the production of elastolytic activities in cultured dermal fibroblasts. Immunoelectron microscopy showed a positive reaction for neutrophil elastase and cathepsin G in fibroblast-like cells from specimens of sun-exposed areas. Little or no reaction was found in biopsies of sun-protected skin. Fibroblast cultures from sun-exposed skin expressed higher levels of hydrolytic activity against synthetic substrates of elastases and cathepsin G than those obtained from sun-protected areas. Irradiation with UVA strongly stimulated the production of these activities in fibroblasts from sun-protected sites. No significant change was detected in parallel sets of cultures after UVB irradiation. Inhibition experiments indicated that the elastase-like activity expressed by fibroblasts can be attributed to at least two enzymes.     

Effect of novel non-peptidic delta opioid receptor antagonists on human T and B cell activation

The effects of the antagonist naltrindole (NTI) on cells of the immune system have been largely studied although the mechanisms of action are still unclear. The aim of this study is to evaluate, in vitro, the immunomodulatory activity of four new delta-selective opioid compounds structurally related to naltrindole. The effects at different concentrations of these opioid antagonists on proliferative response were studied on normal human peripheral blood mononuclear cells (PBMC) stimulated with different stimuli: mitogens, the antigen PPD, the anti-CD3 monoclonal antibodies (mAb), the superantigen Staphylococcus aureus Cowan strain 1 (SAC) and alloantigens in the mixed lymphocyte cultures (MLR). The immunomodulatory capacity of these compounds was evaluated by determining the interleukin-2 (IL-2) release in mitogen activated PBMC. The present study shows that all the new delta opioid antagonists at 10(-5) M concentration are immunosuppressive. The inhibitory action is also evident at lower concentrations when anti-CD3 mAb and SAC were used as stimulators. In addition, the production of IL-2 was inhibited by the opioid treatment, but this might not be the only mechanism of action.