Derivation of Human Induced Pluripotent Stem Cells from Patients with Maturity Onset Diabetes of the Young

Maturity onset diabetes of the young (MODY) is an autosomal dominant disease. Despite extensive research, the mechanism by which a mutant MODY gene results in monogenic diabetes is not yet clear due to the inaccessibility of patient samples. Induced pluripotency and directed differentiation toward the pancreatic lineage are now viable and attractive methods to uncover the molecular mechanisms underlying MODY. Here we report, for the first time, the derivation of human induced pluripotent stem cells (hiPSCs) from patients with five types of MODY: MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY5 (HNF1B), and MODY8 (CEL) with a polycistronic lentiviral vector expressing a Cre-excisable human “stem cell cassette” containing the four reprogramming factors OCT4, KLF4, SOX2, and CMYC. These MODY-hiPSCs morphologically resemble human pluripotent stem cells (hPSCs), express pluripotency markers OCT4, SOX2, NANOG, SSEA-4, and TRA-1–60, give rise to derivatives of the three germ layers in a teratoma assay, and are karyotypically normal. Overall, our MODY-hiPSCs serve as invaluable tools to dissect the role of MODY genes in the development of pancreas and islet cells and to evaluate their significance in regulating beta cell function. This knowledge will aid future attempts aimed at deriving functional mature beta cells from hPSCs.     

Functional Characterization of Key Enzymes involved in l-Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter⁻¹ of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low K_m value for ammonium (10 mM) and a high K_m value for l-glutamate (250 mM), and the V_max value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar K_m values (1 to 1.4 mM) and V_max values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism.     

Remodelling of the zero-stress state and residual strains in apoE-deficient mouse aorta

Atherosclerosis is the most frequent cause of death and severe chronic disability in North America and Europe. The atherosclerosis-prone apolipoprotein E (apoE)-deficient mice contain the entire spectrum of lesions observed during atherogenesis. Significant remodelling of the artery occurs in atherosclerosis. The aim was to study the remodelling of the zero-stress state of the aorta in apoE-deficient mice up to 56 weeks of age. Normal wild-type mice served as control groups. The mice were euthanised at ages 10, 28 and 56 weeks and tissue rings where excised from several locations along the aorta. The rings where photographed in the no-load state (without any external forces applied), then cut radially to obtain the zero-stress state and photographed again. The cross-sectional wall area and wall thickness increased over time in apoE-deficient mice compared to controls (P<0.001). The residual strains at the inner and outer surface varied as function of aortic location both in controls and apoE-deficient mice (P<0.001). From age 28 to age 56 weeks a gradual increase in positive strain at the outer surface and negative strain at the inner surface was found in the apoE-deficient mice when compared to age-matched control mice (P<0.001). Furthermore, the inner residual strain in the plaque location was significantly smaller than in the non-plaque location in the rings with atherosclerotic plaques (P<0.001). The change over time of the opening angle was especially pronounced in the aortic arch. The opening angle increased to app. 200 degrees in the aortic arch in apoE-deficient mice at 56 weeks of age whereas it in age-matched controls was app. 125 degrees. Correspondingly, atherosclerotic plaques were prominent in the apoE-deficient mice, especially at week 56 in the ascending aorta and the aortic arch. In conclusion, a pronounced remodelling of the biomechanical properties in aorta was found in apoE-deficient mice. The stress gradient across the vessel wall in the plaque region is likely larger in vivo due to the smaller residual strain in the plaque area.     

Safety and immunogenicity of a new glycoengineered vaccine against Acinetobacter baumannii in mice

Acinetobacter baumannii poses a serious threat to human health, mainly because of its widespread distribution and severe drug resistance. However, no licensed vaccines exist for this pathogen. In this study, we created a conjugate vaccine against A. baumannii by introducing an O-linked glycosylation system into the host strain. After demonstrating the ability of the vaccine to elicit Th1 and Th2 immune responses and observing its good safety in mouse a model, the strong in vitro bactericidal activity and prophylactic effects of the conjugate vaccine against infection were further demonstrated by evaluating post-infection tissue bacterial loads, observing suppressed serum pro-inflammatory cytokine levels. Additionally, the broad protection from the vaccine was further proved via lethal challenge with A. baumannii. Overall, these results indicated that the conjugate vaccine could elicit an efficient immune response and provide good protection against A. baumannii infection in murine sepsis models. Thus, the conjugate vaccine can be considered as a promising candidate vaccine for preventing A. baumannii infection.     

Effects of climatic warming on cold hardiness of some northern woody plants assessed from simulation experiments

Effects of climatic warming on cold hardiness were investigated for some northern woody plants. In the first experiment, seedlings of Norway spruce ( Picea abies [L.] Karst.), Scots pine ( Pinus sylvestris L.) and lodgepole pine ( Pinus contorta Dougl. var. latifolia Engelm.) were exposed to naturally fluctuating temperatures averaging −6°C (ambient) and 0°C (elevated) for 16 weeks in midwinter before they were thawed and re-saturated with water. In lodgepole pine, needle sugar concentrations had decreased by 15%, and the temperature needed to induce 10% injury to needles in terms of electrolyte leakage had increased by 6°C following treatment to elevated as compared with control temperatures. In contrast, Norway spruce and Scots pine showed no effects. The lack of an effect for Scots pine was ascribed to seedlings containing unusually large energy reserves that buffered respiratory expenditure of sugars. A strong, linear relationship between levels of cold hardiness, assessed by the electrolyte leakage method, and sugars was found when combining data from this and previous, similar experiments. In the second experiment, the evergreen dwarf shrub Empetrum hermaphroditum Hagerup was analysed for leaf cold hardiness, using the electrolyte leakage method, and sugar concentrations in late spring and late autumn during the third year of a warming experiment in a subarctic dwarf shrub community. The objective was to test the hypothesis that warming in the growing season alters hardening/dehardening cycles by increasing soil nitrogen mineralization and plant growth. Data found, however, suggested that cold hardening/dehardening cycles were unaffected by warming.     

Thrombin inhibitor reduces myocardial infarction in apoE-/- x LDLR-/- mice

We have previously shown that atherosclerotic apolipoprotein E-deficient (apoE(-/-)) x LDL receptor-deficient (LDLR(-/-)) mice develop myocardial infarction when exposed to hypoxic stress. This study was performed to assess the role of thrombin and thrombosis in this process. ApoE(-/-) x LDLR(-/-) mice were fed a cholesterol-rich diet for 8 mo and were then subjected to hypoxic stress while receiving isoflurane anesthesia. One group received a bolus dose (5.6 micromol/kg) of the thrombin inhibitor melagatran, and control animals received PBS 10 min before the hypoxic stress. The mice were exposed to 10 min of hypoxia followed by normoxia. Ten minutes after the stress, Alzet pumps delivering melagatran (20 nmol x kg x (-1)min(-1)) or PBS were implanted, and the mice were allowed to recover for 48 h. The cardiac response was analyzed by histology, immunohistochemistry, and serum troponin T assay. All animals showed reversible ECG changes as a sign of ischemia during hypoxic stress, and 50% developed infarctions afterward as judged by troponin T levels. The group that received thrombin inhibitor had significantly lower troponin T and smaller myocardial infarctions than the PBS-treated group. These data show that thrombin generation is an important pathogenetic factor and suggest that coronary thrombosis is involved in myocardial infarction in atherosclerotic mice. Exposure of atherosclerotic mice to hypoxia leads to myocardial infarction through a two-phase pathway in which acute transient ischemia is followed by thrombin-dependent, irreversible, myocardial ischemia and myocardial cell death.     

Limited repair of 8-hydroxy-7,8-dihydroguanine residues in human testicular cells

Oxidative damage in testicular DNA is associated with poor semen quality, reduced fertility and increased risk of stillbirths and birth defects. These DNA lesions are predominantly removed by base excision repair. Cellular extracts from human and rat testicular cells and three enriched populations of rat male germ cells (primary spermatocytes, round spermatids and elongating/elongated spermatids) all showed proficient excision/incision of 5-hydroxycytosine, thymine glycol and 2,6-diamino-4-hydroxy-5-formamidopyrimidine. DNA containing 8-oxo-7,8-dihydroguanine was excised poorly by human testicular cell extracts, although 8-oxoguanine-DNA glycosylase-1 (hOGG1) was present in human testicular cells, at levels that varied markedly between 13 individuals. This excision was as low as with human mononuclear blood cell extracts. The level of endonuclease III homologue-1 (NTH1), which excises oxidised pyrimidines, was higher in testicular than in somatic cells of both species. Cellular repair studies of lesions recognised by formamidopyrimidine-DNA glycosylase (Fpg) or endonuclease III (Nth) were assayed with alkaline elution and the Comet assay. Consistent with the enzymatic activities, human testicular cells showed poor removal of Fpg-sensitive lesions but efficient repair of Nth-sensitive lesions. Rat testicular cells efficiently repaired both Fpg- and Nth-sensitive lesions. In conclusion, human testicular cells have limited capacity to repair important oxidative DNA lesions, which could lead to impaired reproduction and de novo mutations.     

Reciprocal "flipping" underlies substrate recognition and catalytic activation by the human 8-oxo-guanine DNA glycosylase

Both 8oxo-guanine and formamidopyrimidines are major products of oxidative DNA damage that can result in the fixation of transversion mutations following replication if left unrepaired. These lesions are targeted by the N-DNA glycosylase hOgg1, which catalyses excision of the aberrant base followed by cleavage of the phosphate backbone directly 5' to the resultant abasic site in a context, dependent manner. We present the crystal structure of native hOgg1 refined to 2.15 A resolution that reveals a number of highly significant conformational changes on association with DNA that are clearly required for substrate recognition and specificity. Changes of this magnitude appear to be unique to hOgg1 and have not been observed in any of the DNA-glycosylase structures analysed to date where both native and DNA-bound forms are available. It has been possible to identify a mechanism whereby the catalytic residue Lys 249 is "primed" for nucleophilic attack of the N-glycosidic bond.     

Human DNA glycosylases of the bacterial Fpg/MutM superfamily: an alternative pathway for the repair of 8-oxoguanine and other oxidation products in DNA

The mild phenotype associated with targeted disruption of the mouse OGG1 and NTH1 genes has been attributed to the existence of back-up activities and/or alternative pathways for the removal of oxidised DNA bases. We have characterised two new genes in human cells that encode DNA glycosylases, homologous to the bacterial Fpg (MutM)/Nei class of enzymes, capable of removing lesions that are substrates for both hOGG1 and hNTH1. One gene, designated HFPG1, showed ubiquitous expression in all tissues examined whereas the second gene, HFPG2, was only expressed at detectable levels in the thymus and testis. Transient transfections of HeLa cells with fusions of the cDNAs to EGFP revealed intracellular sorting to the nucleus with accumulation in the nucleoli for hFPG1, while hFPG2 co-localised with the 30 kDa subunit of RPA. hFPG1 was purified and shown to act on DNA substrates containing 8-oxoguanine, 5-hydroxycytosine and abasic sites. Removal of 8-oxoguanine, but not cleavage at abasic sites, was opposite base-dependent, with 8-oxoG:C being the preferred substrate and negligible activity towards 8-oxoG:A. It thus appears that hFPG1 has properties similar to mammalian OGG1 in preventing mutations arising from misincorporation of A across 8-oxoG and could function as a back-up repair activity for OGG1 in ogg1(-/-) mice.