Islet amyloid polypeptide (IAPP) transgenic rodents as models for type 2 diabetes

Blood glucose concentrations are maintained by insulin secreted from beta-cells located in the islets of Langerhans. There are approximately 2000 beta-cells per islet, and approximately one million islets of Langerhans scattered throughout the pancreas. The islet in type 2 diabetes mellitus (T2D) has deficient beta-cell mass due to increased beta-cell apoptosis and islet amyloid derived from islet amyloid polypeptide (IAPP). Accumulating evidence implicates toxic IAPP oligomers in the mediation of beta-cell apoptosis in T2D. Humans, monkeys, and cats express an amyloidogenic toxic form of IAPP and spontaneously develop diabetes characterized by islet amyloid deposits. However, longitudinal studies of islet pathology in humans are impossible, and studies in nonhuman primates and cats are costly and impractical. Rodent IAPP is not amyloidogenic, thus commonly used rodent models of diabetes do not recapitulate islet pathology in humans. To investigate the diabetogenic role of human IAPP (h-IAPP), several mouse models and, more recently, a rat model transgenic for h-IAPP have been developed. Studies in these models have revealed that the toxic effect of h-IAPP on beta-cell apoptosis demonstrates a threshold-dependent effect. Specifically, increasing h-IAPP transgene expression by breeding or induction of insulin resistance leads to increased beta-cell apoptosis and diabetes. These transgenic rodent models for h-IAPP provide an opportunity to elucidate the mechanisms responsible for h-IAPP-induced beta-cell apoptosis further and to test novel approaches to the prevention and treatment of T2D.     

Loss of imprinting at the Dlk1-Gtl2 locus caused by insertional mutagenesis in the Gtl2 5' region

Background

The Dlk1 and Gtl2 genes define a region of mouse chromosome 12 that is subject to genomic imprinting, the parental allele-specific expression of a gene. Although imprinted genes play important roles in growth and development, the mechanisms by which imprinting is established and maintained are poorly understood. Differentially methylated regions (DMRs), which carry methylation on only one parental allele, are involved in imprinting control at many loci. The Dlk1-Gtl2 region contains three known DMRs, the Dlk1 DMR in the 3' region of Dlk1, the intergenic DMR 15 kb upstream of Gtl2, and the Gtl2 DMR at the Gtl2 promoter. Three mouse models are analyzed here that provide new information about the regulation of Dlk1-Gtl2 imprinting.

Results

A previously existing insertional mutation (Gtl2lacZ), and a targeted deletion in which the Gtl2 upstream region was replaced by a Neo cassette (Gtl2Δ5'Neo), display partial lethality and dwarfism upon paternal inheritance. Molecular characterization shows that both mutations cause loss of imprinting and changes in expression of the Dlk1, Gtl2 and Meg8/Rian genes. Dlk1 levels are decreased upon paternal inheritance of either mutation, suggesting Dlk1 may be causative for the lethality and dwarfism. Loss of imprinting on the paternal chromosome in both Gtl2lacZ and Gtl2Δ5'Neo mice is accompanied by the loss of paternal-specific Gtl2 DMR methylation, while maternal loss of imprinting suggests a previously unknown regulatory role for the maternal Gtl2 DMR. Unexpectedly, when the Neo gene is excised, Gtl2Δ5' animals are of normal size, imprinting is unchanged and the Gtl2 DMR is properly methylated. The exogenous DNA sequences integrated upstream of Gtl2 are therefore responsible for the growth and imprinting effects.

Conclusion

These data provide further evidence for the coregulation of the imprinted Dlk1 and Gtl2 genes, and support a role for Dlk1 as an important neonatal growth factor. The ability of the Gtl2lacZ and Gtl2Δ5'Neo mutations to cause long-range changes in imprinting and gene expression suggest that regional imprinting regulatory elements may lie in proximity to the integration site.     

Yeast communities in Sphagnum phyllosphere along the temperature-moisture ecocline in the boreal forest-swamp ecosystem and description of Candida sphagnicola sp. nov.

The effects of the temperature-moisture factors on the phylloplane yeast communities inhabiting Sphagnum mosses were studied along the transition from a boreal forest to a swamp biotope at the Central Forest State Biosphere Reserve (Tver region, Russia). We tested the hypothesis that microclimatic parameters affect yeast community composition and structure even on a rather small spatial scale. Using a conventional plating technique we isolated and identified by molecular methods a total of 15 species of yeasts. Total yeast counts and species richness values did not depend on environmental factors, although yeast community composition and structure did. On average, Sphagnum in the swamp biotope supported a more evenly structured yeast community. Relative abundance of ascomycetous yeasts was significantly higher on swamp moss. Rhodotorula mucilaginosa dominated in the spruce forest and Cryptococcus magnus was more abundant in the swamp. Our study confirmed the low occurrence of tremellaceous yeasts in the Sphagnum phyllosphere. Of the few isolated ascomycetous yeast and yeast-like species, some were differentiated from hitherto known species in physiological tests and phylogenetic analyses. We describe one of them as Candida sphagnicola and designate KBP Y-3887^T (=CBS 11774^T = VKPM Y-3566^T = MUCL 53590^T) as the type strain. The new species was registered in MycoBank under MB 563443.     

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.     

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.