Prevalence and tracking of weight disorders in Italian primary school students: a three-year follow-up

Obesity has been widely described as the latest epidemic, and in some areas obesity co-exists with undernutrition. The purpose of this study was to assess the variability of weight status of a cohort of 279 Italian primary school students followed longitudinally for three years. Overweight was the most common weight status disorder in both sexes and generally prevailed in females. Underweight was also more frequent in females than males, while males generally showed a higher incidence of obesity. Overweight showed the highest stability in females. In males, tracking of overweight was 62.5%, while that of normal weight was 89.36%. Overweight tracks through the three consecutive years in a high percentage of both sexes. In the whole sample (regardless of sex and weight category), overweight shows the highest increase over the three-year period. This study provides public health professionals with useful data for policy planning in regard to childhood obesity.     

Dephosphorylation of eIF2α is essential for protein synthesis increase and cell cycle progression after sea urchin fertilization

The eukaryotic Initiation Factor 2 (eIF2) is a key regulator of protein synthesis in eukaryotic cells, implicated in the initiation step of translation. Fertilization of the sea urchin eggs triggers a rapid increase in protein synthesis activity, which is necessary for the progress into embryonic cell cycles. Here we demonstrate that fertilization triggers eIF2α dephosphorylation, concomitant with an increase in protein synthesis and that induction of the eIF2α phosphorylation is intimately linked with an inhibition of protein synthesis and cell cycle arrest. Using a phospho-mimetic protein microinjected into sea urchin eggs, we showed that dephosphorylation of eIF2α is necessary for protein synthesis activity and cell division progression following fertilization. Our results demonstrate that regulation of eIF2α plays an important role in the protein synthesis rise that occurs during early development following fertilization.     

A modified method for the purification of active large enzymes using the glutathione S-transferase expression system

The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. However many GST-tagged proteins are insoluble, and the existing procedures, which employ a mixture of detergents to solubilize the molecules, frequently compromise their functional activity. A further limitation is that large proteins (>80 kDa) are poorly isolated by the current methods and are contaminated by truncated forms. To overcome these problems, we provide here an improved method for efficient purification of active large GST-tagged enzymes such as the 180-kDa GST-fused mitochondrial RNA polymerase.     

Structure and orientation of the gH625-644 membrane interacting region of herpes simplex virus type 1 in a membrane mimetic system

Glycoprotein H (gH) of the herpes simplex virus type 1 is involved in the complex mechanism of membrane fusion of the viral envelope with host cells. The virus requires four glycoproteins (gB, gD, gH, gL) to execute fusion and the role played by gH remains mysterious. Mutational studies have revealed several regions of gH ectodomain required for fusion and identified the segment from amino acid 625 to 644 as the most fusogenic region. Here, we studied the behavior in a membrane-mimicking DPC micellar environment of a peptide encompassing this region (gH625-644) and determined its NMR solution structure and its orientation within the micelles.     

Inhibition of protein kinase CK2 by flavonoids and tyrphostins. A structural insight

Sixteen flavonoids and related compounds have been tested for their ability to inhibit three acidophilic Ser/Thr protein kinases: the Golgi apparatus casein kinase (G-CK) recently identified with protein FAM20C, protein kinase CK1, and protein kinase CK2. While G-CK is entirely insensitive to all compounds up to 40 μM concentration, consistent with the view that it is not a member of the kinome, and CK1 is variably inhibited in an isoform-dependent manner by fisetin and luteolin, and to a lesser extent by myricetin and quercetin, CK2 is susceptible to drastic inhibition by many flavonoids, displaying with six of them IC(50) values < 1 μM. A common denominator of these compounds (myricetin, quercetin, fisetin, kaempferol, luteolin, and apigenin) is a flavone scaffold with at least two hydroxyl groups at positions 7 and 4'. Inhibition is competitive with respect to the phospho-donor substrate ATP. The crystal structure of apigenin and luteolin in complex with the catalytic subunit of Zea mays CK2 has been solved, revealing their ability to interact with both the hinge region (Val116) and the positive area near Lys68 and the conserved water W1, the two main polar ligand anchoring points in the CK2 active site. Modeling experiments account for the observation that luteolin but not apigenin inhibits also CK1. The observation that luteolin shares its pyrocatechol moiety with tyrphostin AG99 prompted us to solve also the structure of this compound in complex with CK2. AG99 was found inside the ATP pocket, consistent with its mode of inhibition competitive with respect to ATP. As in the case of luteolin, the pyrocatechol group of AG99 is critical for binding, interacting with the positive area in the deepest part of the CK2 active site.     

Phospholipase A2IVα regulates phagocytosis independent of its enzymatic activity

Group IVα phospholipase A(2) (PLA(2)IVα) is a lipolytic enzyme that catalyzes the hydrolysis of membrane phospholipids to generate precursors of potent inflammatory lipid mediators. Here, the role of PLA(2)IVα in Fc receptor (FcR)-mediated phagocytosis was investigated, demonstrating that PLA(2)IVα is selectively activated upon FcR-mediated phagocytosis in macrophages and that it rapidly translocates to the site of the nascent phagosome. Moreover, pharmacological inhibition of PLA(2)IVα by pyrrophenone reduces particle internalization by up to 50%. In parallel, fibroblasts from PLA(2)IVα knock-out mice overexpressing FcγRIIA and able to internalize IgG-opsonized beads show 50% lower phagocytosis, compared with wild-type cells, and transfection of PLA(2)IVα fully recovers this impaired function. Interestingly, transfection of the catalytically inactive deleted PLA(2)IVα mutant (PLA(2)IVα(1-525)) and point mutant (PLA(2)IVα-S228C) also promotes recovery of this impaired function. Finally, transfection of the PLA(2)IVα C2 domain (which is directly involved in PLA(2)IVα membrane binding), but not of PLA(2)IVα-D43N (which cannot bind to membranes), rescues FcR-mediated phagocytosis. These data unveil a new mechanism of action for PLA(2)IVα, which demonstrates that the membrane binding, and not the enzymatic activity, is required for PLA(2)IVα modulation of FcR-mediated phagocytosis.     

Acclimation of Chlamydomonas reinhardtii to different growth irradiances

We report on the changes the photosynthetic apparatus of Chlamydomonas reinhardtii undergoes upon acclimation to different light intensity. When grown in high light, cells had a faster growth rate and higher biomass production compared with low and control light conditions. However, cells acclimated to low light intensity are indeed able to produce more biomass per photon available as compared with high light-acclimated cells, which dissipate as heat a large part of light absorbed, thus reducing their photosynthetic efficiency. This dissipative state is strictly dependent on the accumulation of LhcSR3, a protein related to light-harvesting complexes, responsible for nonphotochemical quenching in microalgae. Other changes induced in the composition of the photosynthetic apparatus upon high light acclimation consist of an increase of carotenoid content on a chlorophyll basis, particularly zeaxanthin, and a major down-regulation of light absorption capacity by decreasing the chlorophyll content per cell. Surprisingly, the antenna size of both photosystem I and II is not modulated by acclimation; rather, the regulation affects the PSI/PSII ratio. Major effects of the acclimation to low light consist of increased activity of state 1 and 2 transitions and increased contributions of cyclic electron flow.     

The phylogenetic relationships of the Andean swamp rat genus <Emphasis Type="Italic">Neotomys</Emphasis> (Rodentia,Cricetidae, Sigmodontinae) based on mitochondrial and nuclear markers

The aim of this study was to assess the phylogenetic position of the South American cricetid genus Neotomys using two molecular markers: one nuclear (Irbp) and one mitochondrial (mt-cyb). This genus is currently considered as incertae sedis in the Sigmodontinae radiation. The phylogenetic relationships were estimated using three approaches: Bayesian inference, maximum likelihood and parsimony. We found the genus Neotomys closely related to the genera Euneomys and Irenomys, which are also considered incertae sedis. Our results suggest a common origin for this group of genera; this fact should be reflected in the taxonomy as a supra generic group with a tribal level. However, further and deeper analysis of both molecular and morphological data are needed to diagnose and formalize the proposed tribe. The relationships of this clade to the other members of Sigmodontinae were not clear as assessed by these data sets. The three genera are distributed around the Central and Southern Andes in South America evidencing that the Andes have played an important role in the diversification of several tribes of sigmodontine rodents.     

Neurochemical consequence of steroid abuse: stanozolol-induced monoaminergic changes

An extensive literature has documented adverse effects on mental health in anabolic androgenic steroids (AAS) abusers. Depression seems a common adverse reaction in AAS abusers. Recently it has been reported that in a rat model of AAS abuse stanozolol induces behavioural and biochemical changes related to the pathophysiology of major depressive disorder. In the present study, we used the model of AAS abuse to examine possible changes in the monoaminergic system, a neurobiological substrate of depression, in different brain areas of stanozolol-treated animals. Wistar rats received repeated injections of stanozolol (5mg/kg, s.c.), or vehicle (propylene glycol, 1ml/kg) once daily for 4weeks. Twenty-four hours after last injection, changes of dopamine (DA) and relative metabolite levels, homovanilic acid (HVA) and 3,4-dihydroxy phenylacetic acid (DOPAC), serotonin (5-HT) and its metabolite levels, 5-hydroxy indolacetic acid (5-HIAA), and noradrenaline (NA) amount were investigated in prefrontal cortex (PFC), nucleus accumbens (NAC), striatum (STR) and hippocampus (HIPP). The analysis of data showed that after chronic stanozolol, DA levels were increased in the HIPP and decreased in the PFC. No significant changes were observed in the STR or in the NAC. 5-HT and 5-HIAA levels were decreased in all brain areas investigated after stanozolol exposure; however, the 5-HIAA/5-HT ratio was not altered. Taken together, our data indicate that chronic use of stanozolol significantly affects brain monoamines leading to neurochemical modifications possibly involved in depression and stress-related states.     

2D-DIGE analysis of ovarian cancer cell responses to cytotoxic gold compounds

Cytotoxic gold compounds hold today great promise as new pharmacological agents for treatment of human ovarian carcinoma; yet, their mode of action is still largely unknown. To shed light on the underlying molecular mechanisms, we performed 2D-DIGE analysis to identify differential protein expression in a cisplatin-sensitive human ovarian cancer cell line (A2780/S) following treatment with two representative gold(iii) complexes that are known to be potent antiproliferative agents, namely AuL12 and Au(2)Phen. Software analysis using DeCyder was performed and few differentially expressed protein spots were visualized between the three examined settings after 24 h exposure to the cytotoxic compounds, implying that cellular damage at least during the early phases of exposure is quite limited and selective, reflecting the attempts of the cell to repair damage and to survive the insult. The potential of novel proteomic methods to disclose mechanistic details of cytotoxic metallodrugs is herein further highlighted. Different patterns of proteomic changes were highlighted for the two metallodrugs with only a few perturbed protein spots in common. Using MALDI-TOF MS and ESI-Ion trap MS/MS, several differentially expressed proteins were identified. Two of these were validated by western blotting: Ubiquilin-1, responsible for inhibiting degradation of proteins such as p53 and NAP1L1, a candidate marker identified in primary tumors. Ubiquilin-1 resulted over-expressed following both treatments and NAP1L1 was down-expressed in AuL12-treated cells in comparison with control and with Au(2)Phen-treated cells. In conclusion, we performed a comprehensive analysis of proteins regulated by AuL12 and Au(2)Phen, providing a useful insight into their mechanisms of action.