Quantitative proteomic analysis of mouse embryonic fibroblasts and induced pluripotent stem cells using 16O/18O labeling

Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here, we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. With this platform, a total of 2481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex, were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1), and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.     

Opisthorchis felineus and Metorchis bilis are the main agents of liver fluke infection of humans in Russia

Liver fluke infections are gradually transforming from a local problem of individual geographic regions to a widespread problem. The observed expansion is likely to be connected with the ever-increasing intensity of traffic flow and migration of the infected carriers between cities, regions, and countries. Opisthorchis felineus, the trematode belonging to the family Opisthorchiidae, is a well known causative agent of the infection called opisthorchiasis. Metorchis bilis, also a member of the family Opisthorchiidae, causes metorchiasis, a disease very close to opisthorchiasis in symptomatology. Genetic markers can be used to develop methods for differential diagnostics of these diseases. However, the questions connected with epidemiology of these trematode infections, their clinical characteristics, prognosis and therapy remain open. This review briefs the general biological characteristics of O. felineus and M. bilis persisting in various countries of Eurasia, their geographical range, epidemiology and molecular diagnostics of these liver flukes.     

NODULE ROOT and COCHLEATA Maintain Nodule Development and Are Legume Orthologs of Arabidopsis BLADE-ON-PETIOLE Genes

During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.     

Measurement of pulsatile insulin secretion in the rat: direct sampling from the hepatic portal vein

It has previously been shown that insulin is secreted in discrete secretory bursts by sampling directly from the portal vein in the dog and humans. Deficient pulsatile insulin secretion is the basis for impaired insulin secretion in type 2 diabetes. However, while novel genetically modified disease models of diabetes are being developed in rodents, no validated method for quantifying pulsatile insulin secretion has been established for rodents. To address this we 1) developed a novel rat model with chronically implanted portal vein catheters, 2) established the parameters to permit deconvolution of portal vein insulin concentrations profiles to measure insulin secretion and resolve its pulsatile components, and 3) measured total and pulsatile insulin secretion compared with that in the dog, the species in which this sampling and deconvolution approach was validated for quantifying pulsatile insulin secretion. In rats, portal vein catheter patency and function were maintained for periods up to 2-3 wk with no postoperative complications such as catheter tract infection. Rat portal vein insulin concentration profiles in the fasting state revealed distinct insulin oscillations with a periodicity of approximately 5 min and an amplitude of up to 600 pmol/l, which was remarkably similar to that in the dogs and in humans. Deconvolution analysis of portal vein insulin concentrations revealed that the majority of insulin ( approximately 70%) in the rat is secreted in distinct insulin pulses occurring at approximately 5-min intervals. This model therefore permits direct accurate measurements of pulsatile insulin secretion in a relatively inexpensive animal. With increased introduction of genetically modified rat models will be an important tool in elucidating the underlying mechanisms of impaired pulsatile insulin secretion in diabetes.     

Therapeutic role of sirtuins in neurodegenerative disease

The sirtuins are a family of enzymes which control diverse and vital cellular functions, including metabolism and aging. Manipulations of sirtuin activities cause activation of anti-apoptotic, anti-inflammatory, anti-stress responses, and the modulation of an aggregation of proteins involved in neurodegenerative disorders. Recently, sirtuins were found to be disease-modifiers in various models of neurodegeneration. However, almost in all instances, the exact mechanisms of neuroprotection remain elusive. Nevertheless, the manipulation of sirtuin activities is appealing as a novel therapeutic strategy for the treatment of currently fatal human disorders such as Alzheimer's and Parkinson's diseases. Here, we review current data which support the putative therapeutic roles of sirtuin in aging and in neurodegenerative diseases and the feasibility of the development of sirtuin-based therapies.     

Neural control of heartbeat during two antagonistic behaviors: whole body withdrawal and escape swimming in the Mollusk <Emphasis Type="Italic">Clione limacina</Emphasis>

Two cardioexcitatory and one cardioinhibitory neural groups have been previously identified as the central cardioregulatory system in the pteropod mollusk Clione limacina. We describe in this study one additional element of the central cardioregulatory system, which consists of a large intestinal neuron named Z-cell with a novel effect on the heart activity. Intracellular stimulation of the Z-cell induced only auricle contractions with no effect on the ventricle activity. The Z-cell processes were traced down to the heart, and vigorous branching was found in the auricle tissue. Specific patterns of activity of the Z-cell as well as intestinal heart excitatory and inhibitory neurons were studied during initiation of two behaviors-whole body withdrawal and escape swimming. It was found that initiation of both behaviors was accompanied by activation of Z-cell and intestinal heart excitor neurons. The firing rate of neurons induced by sensory stimuli was sufficient to trigger auricle contractions in the semi-intact preparations. Video analysis of heart activity revealed that auricle indeed was activated during both active and passive avoidance reactions, though the intensity and delay of the activation were different. The possible physiological role of the auricle contractions during antagonistic forms of behavior is discussed.     

Crystal structures of the G139A, G139A–NO and G143H mutants of human heme oxygenase-1. A finely tuned hydrogen-bonding network controls oxygenase versus peroxidase activity

Conserved glycines, Gly139 and Gly143, in the distal helix of human heme oxygenase-1 (HO-1) provide the flexibility required for the opening and closing of the heme active site for substrate binding and product dissociation during HO-1 catalysis. Earlier mutagenesis work on human HO-1 showed that replacement of either Gly139 or Gly143 suppresses heme oxygenase activity and, in the case of the Gly139 mutants, increases peroxidase activity (Liu et al. in J. Biol. Chem. 275:34501, 2000). To further investigate the role of the conserved distal helix glycines, we have determined the crystal structures of the human HO-1 G139A mutant, the G139A mutant in a complex with NO, and the G143H mutant at 1.88, 2.18 and 2.08 Å, respectively. The results confirm that fine tuning of the previously noted active-site hydrogen-bonding network is critical in determining whether heme oxygenase or peroxidase activity is observed.     

Analysis of candidate imprinted genes linked to Dlk1-Gtl2 using a congenic mouse line

The study of genomic imprinting requires the use of DNA sequence polymorphisms between interfertile mouse species or strains. Most commonly, crosses between Mus musculus domesticus and Mus musculus castaneus or Mus spretus animals are used. Difficulties arise in the maintenance of these wild-derived mice in conventional animal facilities, however, and can be overcome by the use of a congenic strain for the region under study. We describe here the generation of a new mouse line, congenic for a region on distal Chromosome (Chr) 12 that encompasses the Dlk1–Gtl2 imprinted domain. We have taken a first step towards demonstrating the utility of these animals by assaying known genes located within the congenic interval for imprinted expression. We show that the two genes located immediately proximal to Dlk1, the Yy1 and Wars genes, are expressed in a biallelic manner. In addition, we have analyzed the Dio3 gene, located distal to Gtl2. This gene displays preferential expression of the paternal allele, with approximately 75% of the total message level originating from the paternal allele and 25% originating from the maternal allele. These data delineate the position of the Wars gene as the proximal boundary of the Dlk1–Gtl2 imprinted domain, and identify Dio3 as another potentially imprinted gene within this domain.     

Classification of the Avian Summer Population on the Western Siberian Plain

The proposed classification system reflects the difference between the three population systems: unbuilt land, built-up land, and aquatic–semiaquatic communities. Two superorder groups—north and median—further divided into types were recognized in each of the systems. Most types are divided into subtypes, classes, and subclasses (and sometimes genera of the population). The estimation of the power and generality of the influence of environmental factors (their variability correlates with heterogeneity of the avian population) has demonstrated that forestation of the territory is most significant in the first half of summer on the western Siberian Plain. The composition of the forest-forming species and zoning are less affected. The influence of moisture and hydration is 2–3 times less significant; mesorelief is 4–5 times less significant; and productivity (feeding capacity) and anthropogenic influence are 7–9 times less significant.