A proteomic approach to obesity and type 2 diabetes

The incidence of obesity and type diabetes 2 has increased dramatically resulting in an increased interest in its biomedical relevance. However, the mechanisms that trigger the development of diabetes type 2 in obese patients remain largely unknown. Scientific, clinical and pharmaceutical communities are dedicating vast resources to unravel this issue by applying different omics tools. During the last decade, the advances in proteomic approaches and the Human Proteome Organization have opened and are opening a new door that may be helpful in the identification of patients at risk and to improve current therapies. Here, we briefly review some of the advances in our understanding of type 2 diabetes that have occurred through the application of proteomics. We also review, in detail, the current improvements in proteomic methodologies and new strategies that could be employed to further advance our understanding of this pathology. By applying these new proteomic advances, novel therapeutic and/or diagnostic protein targets will be discovered in the obesity/Type 2 diabetes area.     

Phylogeographic structure of jack pine (Pinus banksiana; Pinaceae) supports the existence of a coastal glacial refugium in northeastern North America

? Premise of the study: The genetic structure of jack pine (Pinus banksiana Lamb.), a North American boreal conifer with large longitudinal distribution, was investigated to test for the possible existence of a genetically distinct lineage in the Maritimes region in northeastern North America, which could be indicative of a mid-latitude coastal refuge during the last glaciation. ? Methods: One maternally inherited mitochondrial DNA (mtDNA) minisatellite marker and four paternally inherited chloroplast DNA (cpDNA) microsatellite markers were used to assess the range-wide geographical structure of jack pine populations with particular focus on northeastern North America. ? Key results: The populations from the Maritimes region presented a unique mtDNA background characterized by very low diversity and the preponderance of a distinctive mitotype. The distribution of cpDNA diversity was not spatially structured, though three chlorotypes were restricted to the east. ? Conclusions: MtDNA data suggest that populations from the Maritimes region derive from a genetically depauperated north-coastal refugium. Contrastingly, the much higher geographical uniformity observed for cpDNA variation indicates that gene flow by pollen had been much more effective than seed gene flow at homogenizing population structure.     

Distance-dependence in two Amazonian palms: effects of spatial and temporal variation in seed predator communities

Animals aid population growth and fitness in tropical forest communities through dispersal and negatively impact populations through seed predation. The interaction between dispersal and seed predation can produce distance- or density-dependence; powerful mechanisms for maintaining species diversity incorporated in the Janzen–Connell model. Large mammals, the highest biomass seed predators of intact Amazonian communities and at risk due to human disturbance, are potentially central to these interactions. This study tests the Janzen–Connell model and investigates the impact of mammalian seed predators on seedling recruitment and maintenance of tree diversity. Patterns of both vertebrate and invertebrate seed predation and seedling recruitment were studied in the two most abundant canopy tree species in western Amazonia (Arecaceae: Astrocaryum murumuru and Iriartea deltoidea). We specifically examined effects of both spatial and temporal variation of the highest biomass seed predator in southwest Amazonian forests, the white-lipped peccary (Tayassu pecari), on recruitment through disturbed and undisturbed sites and through a fortuitous 12 year natural extinction and recolonization event of T. pecari. Distance-dependent seedling recruitment was found in Astrocaryum and Iriartea at both sites. However, the median distance of seedlings was ~1.5× farther from reproductive adults in both palms at the undisturbed site. The number of Iriartea seeds escaping predation increased 6,000% in both space and time due to the decline of T. pecari abundance. The results demonstrate that Janzen–Connell effects are stronger in intact ecosystems and tie these mechanistically to changes in seed predator abundance. This study shows that anthropogenic changes in mammal communities decrease the magnitude of Janzen–Connell effects in Amazonian forests and may result in decreases in tree diversity.     

Characterization of lytic Pseudomonas aeruginosa bacteriophages via biological properties and genomic sequences

Pseudomonas aeruginosa is an important cause of infections, especially in patients with immunodeficiency or diabetes. Antibiotics are effective in preventing morbidity and mortality from Pseudomonas infection, but because of spreading multidrug-resistant bacterial strains, bacteriophages are being explored as an alternative therapy. Two newly purified broad host range Pseudomonas phages, named vB_Pae-Kakheti25 and vB_Pae-TbilisiM32, were characterized as candidates for use in phage therapy. Morphology, host range, growth properties, thermal stability, serology, genomic sequence, and virion composition are reported. When phages are used as bactericides, they are used in mixtures to overcome the development of resistance in the targeted bacterial population. These two phages are representative of diverse siphoviral and podoviral phage families, respectively, and hence have unrelated mechanisms of infection and no cross-antigenicity. Composing bactericidal phage mixtures with members of different phage families may decrease the incidence of developing resistance through a common mechanism.     

Heterodimerization of CCR2 chemokines and regulation by glycosaminoglycan binding

Despite the wide range of sequence diversity among chemokines, their tertiary structures are remarkably similar. Furthermore, many chemokines form dimers or higher order oligomers, but all characterized oligomeric structures are based primarily on two dimerization motifs represented by CC-chemokine or CXC-chemokine dimer interfaces. These observations raise the possibility that some chemokines could form unique hetero-oligomers using the same oligomerization motifs. Such interactions could modulate the overall signaling response of the receptors, thereby providing a general mechanism for regulating chemokine function. For some chemokines, homo-oligomerization has also been shown to be coupled to glycosaminoglycan (GAG)-binding. However, the effect of GAG binding on chemokine hetero-oligomerization has not yet been demonstrated. In this report, we characterized the heterodimerization of the CCR2 ligands MCP-1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), MCP-4 (CCL13), and eotaxin (CCL11), as well as the effects of GAG binding, using electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. Strong heterodimerization was observed between CCL2 and CCL8 at the expense of homodimer formation. Using NMR, we showed that the heterodimer is predominant in solution and forms a specific CC chemokine-like dimer. By contrast, only moderate heterodimer formation was observed between CCL2.CCL13, CCL2.CCL11 and CCL8.CCL13, and no heterodimerization was observed when any other CCR2 ligand was added to CCL7. To investigate the effect of a highly sulfated GAG on the formation of heterodimers, each chemokine pair was mixed with the heparin pentasaccharide, Arixtra, and assayed by ESI-FTICR mass spectrometry. Although no CCL8.CCL11 heterodimer was observed in the absence of GAG, abundant ions corresponding to the ternary complex, CCL8.CCL11.Arixtra, were observed upon addition of Arixtra. Heterodimerization between CCL2 and CCL11 was also enhanced in the presence of Arixtra. In summary, these results indicate that some CCR2 ligands can form stable heterodimers in preference to homodimers and that these interactions, like those of homo-oligomers, can be influenced by some GAGs.     

Glucocorticoid Receptors are Localized to Dendritic Spines and Influence Local Actin Signaling

Glucocorticoids affect learning and memory but the cellular mechanisms involved are poorly understood. The present studies tested if the stress-responsive glucocorticoid receptor (GR) is present and regulated within dendritic spines, and influences local signaling to the actin cytoskeleton. In hippocampal field CA1, 13?% of synapses contained GR-immunoreactivity. Three-dimensional reconstructions of CA1 dendrites showed that GR aggregates are present in both spine heads and necks. Consonant with evidence that GR?? mRNA associates with the translation regulator Fragile X Mental Retardation Protein (FMRP), spine GR levels were rapidly increased by group 1 mGluR activation and reduced in mice lacking FMRP. Treatment of cultured hippocampal slices with the GR agonist dexamethasone rapidly (15?C30?min) increased total levels of phosphorylated (p) Cofilin and extracellular signal-regulated kinase (ERK) 1/2, proteins that regulate actin polymerization and stability. Dexamethasone treatment of adult hippocampal slices also increased numbers of PSD95+ spines containing pERK1/2, but reduced numbers of pCofilin-immunoreactive spines. Dexamethasone-induced increases in synaptic pERK1/2 were blocked by the GR antagonist RU-486. These results demonstrate that GRs are present in hippocampal spines where they mediate acute glucocorticoid effects on local spine signaling. Through effects on these actin regulatory pathways, GRs are positioned to exert acute effects on synaptic plasticity.     

Xanthine oxidase-generated hydrogen peroxide is a consequence, not a mediator of cell death

Oxidative stress has been associated with a wide range of diseases including atherosclerosis, cancer and Alzheimer's disease. When present in excessive concentrations, reactive oxygen species (ROS) can cause deleterious effects. This has led to the notion that the anticancer effects of various chemotherapeutics may be mediated, at least in part, by an increase in ROS. To investigate the role of xanthine oxidase (XO), a source of hydrogen peroxide, in cell death, MCF7, HeLa and 293T cells were treated with various cell-death-inducing drugs in the presence and absence of allopurinol, a specific inhibitor of XO. In the absence of allopurinol, each drug led to a time- and concentration-dependent increase in percent DNA fragmentation and ROS levels, regardless of the mechanism of cell death incurred, i.e. caspase dependent and caspase independent. By contrast, pretreatment with allopurinol led to dramatically lower ROS levels in all cases, suggesting that XO is a major contributor to oxidative stress. However, allopurinol did not exhibit a protective effect against cell death. Similarly, the administration of siRNA against XO also did not exhibit a protective effect against cell death. The level of oxidative stress was recorded using the ROS sensor CM-H(2) DCFDA and a ratiometric bioluminescent assay that takes advantage of the increased sensitivity of Firefly luciferase to hydrogen peroxide, compared with a stable variant of Renilla luciferase (RLuc), RLuc8. Overall, these findings suggest that XO-generated hydrogen peroxide, and perhaps hydrogen peroxide in general, is a consequence, but not a mediator of cell death.