Ligand- and proton-linked conformational changes of the ferrous 2/2 hemoglobin of Pseudoalteromonas haloplanktis TAC125

The spectroscopic and ligand-binding properties of a 2/2 globin from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 have been studied in the ferrous state. It displays two major conformations characterized by CO-association rates that differ by a factor of 20, with relative fractions that depend on pH. A dynamic equilibrium is found between the two conformations, as indicated by an enhanced slower phase when lower CO levels were used to allow a longer time to facilitate the transition. The deoxy form, in the absence of external ligands, is a mixture of a predominant six-coordinate low spin form and a five-coordinate high-spin state; the proportion of low spin increasing at alkaline pH. In addition, at temperatures above the physiological temperature of 1 °C, an enhanced tendency of the protein to oxidize is observed.     

Cardioprotection through autophagy: Ready for clinical trial?

Interventions that reduce infarct size in animal models have largely failed to improve outcome in patients suffering acute myocardial infarction (MI), or 'heart attack'. Our group recently reported a reduction of infarct size by chloramphenicol treatment in a porcine in vivo model of acute MI, through a mechanism involving the induction of autophagy. Since 2005 several studies have implicated autophagy as a target for cardioprotection.     

Autophagy in hypothalamic AgRP neurons regulates food intake and energy balance

Macroautophagy is a lysosomal degradative pathway that maintains cellular homeostasis by turning over cellular components. Here we demonstrate a role for autophagy in hypothalamic agouti-related peptide (AgRP) neurons in the regulation of food intake and energy balance. We show that starvation-induced hypothalamic autophagy mobilizes neuron-intrinsic lipids to generate endogenous free fatty acids, which in turn regulate AgRP levels. The functional consequences of inhibiting autophagy are the failure to upregulate AgRP in response to starvation, and constitutive increases in hypothalamic levels of pro-opiomelanocortin and its cleavage product α-melanocyte-stimulating hormone that typically contribute to a lean phenotype. We propose a conceptual framework for considering how autophagy-regulated lipid metabolism within hypothalamic neurons may modulate neuropeptide levels to have immediate effects on food intake, as well as long-term effects on energy homeostasis. Regulation of hypothalamic autophagy could become an effective intervention in conditions such as obesity and the metabolic syndrome.     

Ensemble-based computational approach discriminates functional activity of p53 cancer and rescue mutants

The tumor suppressor protein p53 can lose its function upon single-point missense mutations in the core DNA-binding domain (“cancer mutants”). Activity can be restored by second-site suppressor mutations (“rescue mutants”). This paper relates the functional activity of p53 cancer and rescue mutants to their overall molecular dynamics (MD), without focusing on local structural details. A novel global measure of protein flexibility for the p53 core DNA-binding domain, the number of clusters at a certain RMSD cutoff, was computed by clustering over 0.7 µs of explicitly solvated all-atom MD simulations. For wild-type p53 and a sample of p53 cancer or rescue mutants, the number of clusters was a good predictor of in vivo p53 functional activity in cell-based assays. This number-of-clusters (NOC) metric was strongly correlated (r² = 0.77) with reported values of experimentally measured ΔΔG protein thermodynamic stability. Interpreting the number of clusters as a measure of protein flexibility: (i) p53 cancer mutants were more flexible than wild-type protein, (ii) second-site rescue mutations decreased the flexibility of cancer mutants, and (iii) negative controls of non-rescue second-site mutants did not. This new method reflects the overall stability of the p53 core domain and can discriminate which second-site mutations restore activity to p53 cancer mutants.     

Cyclodextrin-Based Nanosponges for Delivery of Resveratrol: <Emphasis Type="Italic">In Vitro</Emphasis> Characterisation,Stability, Cytotoxicity and Permeation Study

The aim of this work was to increase the solubility, stability and permeation of resveratrol by complexation with cyclodextrin-based nanosponges (NS). Nanosponges are recently developed hyper-cross-linked cyclodextrin polymers nanostructured to form three-dimensional networks; they are obtained by reacting cyclodextrin with a cross-linker such as carbonyldiimidazole. They have been used to increase the solubility and stability of poorly soluble actives. This study aimed at formulating complexes of resveratrol with β-cyclodextrin nanosponges in different weight ratios. DSC, FTIR and X-ray powder diffraction (XRPD) studies confirmed the interaction of resveratrol with NS. XRPD showed that the crystallinity of resveratrol decrease after encapsulation. The particle sizes of resveratrol-loaded NS are in between 400 to 500 nm with low polydispersity indices. Zeta potential is sufficiently high to obtain a stable colloidal nanosuspension. TEM measurement also revealed a particle size around 400 nm for NS complexes. The in vitro release and stability of resveratrol complex were increased compared with plain drug. Cytotoxic studies on HCPC-I cell showed that resveratrol formulations were more cytotoxic than plain resveratrol. The permeation study indicates that the resveratrol NS formulation showed good permeation in pigskin. The accumulation study in rabbit mucosa showed better accumulation of resveratrol NS formulation than plain drug. These results signify that resveratrol NS formulation can be used for buccal delivery and topical application.     

H ferritin gene silencing in a human metastatic melanoma cell line: a proteomic analysis

Ferritin, the major intracellular iron-storage protein, is made of 24 subunits of two types, H and L. Besides regulating intracellular iron homeostasis, it has been found that ferritin, in particular the H subunit (FHC), is involved in different biological events such as cell differentiation and pathologic states (i.e., neurodegeneration and cancer). This study is aimed at investigating the whole-cell proteome of FHC-expressing and sh-RNA-silenced human metastatic melanoma cells (MM07(m)) in the attempt to identify and classify the highest number of proteins directly or indirectly controlled by the FHC. We identified about 200 differentially expressed proteins and classified them in clusters on the basis of their functions, as proteins involved in metabolic processes, cell adhesion, migration, and proliferation processes. Some of them have captured our attention because of their involvement in metabolic pathways related to tumor progression and metastasis. In vitro assays confirmed that the FHC-silenced MM07(m) cells are characterized by a decreased growth activity, a reduced invasiveness, and a reduced cell adhesion capability. Moreover, nude mice (CD1 nu/nu), subcutaneously injected with FHC-silenced MM07(m) cells, showed a remarkable 4-fold reduction of their tumor growth capacity compared to those who received the FHC-unsilenced MM07(m) counterpart. In conclusion, these data indicate that gene silencing technology, coupled to proteomic analysis, is a powerful tool for a better understanding of H ferritin signaling pathways and lend support to the hypothesis that specific targeting of this gene might be an attractive and potentially effective strategy for the management of metastatic melanoma.     

Exposure to tebuconazol in rice field and laboratory conditions induces oxidative stress in carp (Cyprinus carpio)

Pesticides can have an effect on the biochemical and physiological functions of living organisms. The changes seen in fish and their response to pesticides can be used as an example for vertebrate toxicity. In this study, carp fish (Cyprinus carpio) were exposed to different concentrations of tebuconazol fungicide, by rice field (31.95 μg/L) and laboratory (33.47 and 36.23 μg/L) conditional testing, during a 7 day period. Parameters such thiobarbituric acid-reactive substance levels (TBARS), protein carbonyl, catalase, glutathione S-transferase and acetylcholinesterase activities were studied, using the liver, brain and white muscle of the fish. The field experiment showed that the TBARS levels were increased in all the analyzed tissues. Similarly, the protein carbonyl of the liver and the brain AChE activity increased after 7 days. The laboratory experiment demonstrated that the TBARS levels in the liver were increased in both of the concentration tests. TBARS levels in the muscle increased only by the lowest test concentration. On the other hand, the protein carbonyl was increased only by the highest concentration. The results indicate that the tebuconazol exposure from the field and laboratory conditions directly affected the health of the fish, showing the occurrence of oxidative stress.     

Identification of proteins associated with ligand-activated estrogen receptor α in human breast cancer cell nuclei by tandem affinity purification and nano LC-MS/MS

Estrogen receptor α (ER-α) is a key mediator of estrogen actions in breast cancer (BC) cells. Understanding the effects of ligand-activated ER-α in target cells requires identification of the molecular partners acting in concert with this nuclear receptor to transduce the hormonal signal. We applied tandem affinity purification (TAP), glycerol gradient centrifugation and MS analysis to isolate and identify proteins interacting with ligand-activated ER-α in MCF-7 cell nuclei. This led to the identification of 264 ER-associated proteins, whose functions highlight the hinge role of ER-α in the coordination of multiple hormone-regulated nuclear processes in BC cells.     

Characterization of Salmonella occurring at high prevalence in a population of the land iguana Conolophus subcristatus in Galápagos Islands, Ecuador

The aim of the study was to elucidate the association between the zoonotic pathogen Salmonella and a population of land iguana, Colonophus subcristatus, endemic to Galápagos Islands in Ecuador. We assessed the presence of Salmonella subspecies and serovars and estimated the prevalence of the pathogen in that population. Additionally, we investigated the genetic relatedness among isolates and serovars utilising pulsed field gel electrophoresis (PFGE) on XbaI-digested DNA and determined the antimicrobial susceptibility to a panel of antimicrobials. The study was carried out by sampling cloacal swabs from animals (n = 63) in their natural environment on in the island of Santa Cruz. A high prevalence (62/63, 98.4%) was observed with heterogeneity of Salmonella subspecies and serovars, all known to be associated with reptiles and with reptile-associated salomonellosis in humans. Serotyping revealed 14 different serovars among four Salmonella enterica subspecies: S. enterica subsp. enterica (n = 48), S. enterica subsp. salamae (n = 2), S. enterica subsp. diarizonae (n = 1), and S. enterica subsp. houtenae (n = 7). Four serovars were predominant: S. Poona (n = 18), S. Pomona (n = 10), S. Abaetetuba (n = 8), and S.Newport (n = 5). The S. Poona isolates revealed nine unique XbaI PFGE patterns, with 15 isolates showing a similarity of 70%. Nine S. Pomona isolates had a similarity of 84%. One main cluster with seven (88%) indistinguishable isolates of S. Abaetetuba was observed. All the Salmonella isolates were pan-susceptible to antimicrobials representative of the most relevant therapeutic classes. The high prevalence and absence of clinical signs suggest a natural interaction of the different Salmonella serovars with the host species. The interaction may have been established before any possible exposure of the iguanas and the biocenosis to direct or indirect environmental factors influenced by the use of antimicrobials in agriculture, in human medicine or in veterinary medicine.