Loss of autophagy diminishes pancreatic beta cell mass and function with resultant hyperglycemia

Autophagy is a cellular degradation-recycling system for aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in various diseases including neurodegeneration, its role in pancreatic beta cells and glucose homeostasis has not been described. We produced mice with beta cell-specific deletion of Atg7 (autophagy-related 7). Atg7 mutant mice showed impaired glucose tolerance and decreased serum insulin level. beta cell mass and pancreatic insulin content were reduced because of increased apoptosis and decreased proliferation of beta cells. Physiological studies showed reduced basal and glucose-stimulated insulin secretion and impaired glucose-induced cytosolic Ca2+ transients in autophagy-deficient beta cells. Morphologic analysis revealed accumulation of ubiquitinated protein aggregates colocalized with p62, which was accompanied by mitochondrial swelling, endoplasmic reticulum distension, and vacuolar changes in beta cells. These results suggest that autophagy is necessary to maintain structure, mass and function of pancreatic beta cells, and its impairment causes insulin deficiency and hyperglycemia because of abnormal turnover and function of cellular organelles.     

P2Y12 antagonist attenuates eosinophilic inflammation and airway hyperresponsiveness in a mouse model of asthma

Leukotriene E4 (LTE4) that plays a key role in airway inflammation is expressed on platelets and eosinophils. We investigated whether blocking of the P2Y12 receptor can suppress eosinophilic inflammation in a mouse model of asthma because platelets and eosinophils share this receptor to be activated. BALB/c mice were sensitized by intraperitoneal injection of ovalbumin (OVA), followed by OVA nebulization. On each challenge day, clopidogrel, a P2Y12 antagonist was administered 30 min. before each challenge. Forty‐eight hours after the last OVA challenge, mice were assessed for airway hyperresponsiveness (AHR), cell composition and cytokine levels, including chemokine ligand 5 (CCL5), in bronchoalveolar lavage (BAL) fluid. EOL cells were treated with LTE4, with or without clopidogrel treatment, and intracellular and extracellular eosinophil cationic protein (ECP) expressions were measured to find the inhibiting function of P2Y12 antagonist on eosinophilic activation. The levels of P2Y12 expression were increased markedly in the lung homogenates of OVA‐sensitized and ‐challenged mice after platelet depletion. Administration of clopidogrel decreased AHR and the number of airway inflammatory cells, including eosinophils, in BAL fluid following OVA challenge. These results were associated with decreased levels of Th2 cytokines and CCL5. Histological examination showed that inflammatory cells as well as mucus‐containing goblet cells were reduced in clopidogrel‐administered mice compared to vehicle‐treated mice. Clopidogrel inhibited extracellular ECP secretion after LTE4 stimulation in EOL‐1 cells. Clopidogrel could prevent development of AHR and airway inflammation in a mouse model of asthma. P2Y12 can be a novel therapeutic target to the suppression of eosinophils in asthma.     

Reduced proliferative activity of primary POMGnT1-null myoblasts in vitro

Protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) is an enzyme that transfers N-acetylglucosamine to O-mannose of glycoproteins. Mutations of the POMGnT1 gene cause muscle–eye–brain (MEB) disease. To obtain a better understanding of the pathogenesis of MEB disease, we mutated the POMGnT1 gene in mice using a targeting technique. The mutant muscle showed aberrant glycosylation of α-DG, and α-DG from mutant muscle failed to bind laminin in a binding assay. POMGnT1^?/? muscle showed minimal pathological changes with very low-serum creatine kinase levels, and had normally formed muscle basal lamina, but showed reduced muscle mass, reduced numbers of muscle fibers, and impaired muscle regeneration. Importantly, POMGnT1^?/? satellite cells proliferated slowly, but efficiently differentiated into multinuclear myotubes in vitro. Transfer of a retrovirus vector-mediated POMGnT1 gene into POMGnT1^?/? myoblasts completely restored the glycosylation of α-DG, but proliferation of the cells was not improved. Our results suggest that proper glycosylation of α-DG is important for maintenance of the proliferative activity of satellite cells in vivo.     

Design and application of highly responsive fluorescence resonance energy transfer biosensors for detection of sugar in living Saccharomyces cerevisiae cells

A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker(1)-maltose-binding protein-linker(2)-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 microM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).     

A lectin-coupled, targeted proteomic mass spectrometry (MRM MS) platform for identification of multiple liver cancer biomarkers in human plasma

Aberrantly glycosylated proteins related to liver cancer progression were captured with specific lectin and identified from human plasma by multiple reaction monitoring (MRM) mass spectrometry as multiple biomarkers for hepatocellular carcinoma (HCC). The lectin fractionation for fucosylated protein glycoforms in human plasma was conducted with a fucose-specific aleuria aurantia lectin (AAL). Following tryptic digestion of the lectin-captured fraction, plasma samples from 30 control cases (including 10 healthy, 10 hepatitis B virus [HBV], and 10 cirrhosis cases) and 10 HCC cases were quantitatively analyzed by MRM to identify which glycoproteins are viable HCC biomarkers. A1AG1, AACT, A1AT, and CERU were found to be potent biomarkers to differentiate HCC plasma from control plasmas. The AUROC generated independently from these four biomarker candidates ranged from 0.73 to 0.92. However, the lectin-coupled MRM assay with multiple combinations of biomarker candidates is superior statistically to those generated from the individual candidates with AUROC more than 0.95, which can be an alternative to the immunoassay inevitably requiring tedious development of multiple antibodies against biomarker candidates to be verified. Eventually the lectin-coupled, targeted proteomic mass spectrometry (MRM MS) platform was found to be efficient to identify multiple biomarkers from human plasma according to cancer progression.     

Molecular basis of the differences between normal and tumor tissues of gastric cancer

To be able to describe the differences between the normal and tumor tissues of gastric cancer at a molecular level would be essential in the study of the disease. We investigated the gene expression pattern in the two types of tissues from gastric cancer by performing expression profiling of 86 tissues on 17K complementary DNA microarrays. To select for the differentially expressed genes, class prediction algorithm was employed. For predictor selection, samples were first divided into a training (n=58), and a test set (n=28). A group of 894 genes was selected by a t-test in a training set, which was used for cross-validation in the training set and class (normal or tumor) prediction in the test set. Smaller groups of 894 genes were individually tested for their ability to correctly predict the normal or tumor samples based on gene expression pattern. The expression ratios of the 5 genes chosen from microarray data can be validated by real time RT-PCR over 6 tissue samples, resulting in a high level of correlation, individually or combined. When a representative predictor set of 92 genes was examined, pathways of 'focal adhesion' (with gene components of THBS2, PDGFD, MAPK1, COL1A2, COL6A3), 'ECM-receptor interaction' pathway (THBS2, COL1A2, COL6A3, FN1) and 'TGF-beta signaling' (THBS2, MAPK1, INHBA) represent some of the main differences between normal and tumor of gastric cancer at a molecular level.     

Klebsiella pneumoniae KE-1 degrades endosulfan without formation of the toxic metabolite,endosulfan sulfate

For bioremediation of toxic endosulfan, endosulfan degradation bacteria, which do not form toxic endosulfan sulfate, were isolated from various soil samples using endosulfan as sole carbon and energy source. Among the 40 isolated bacteria, strain KE-1, which was identified as Klebsiella pneumoniae by physiological and 16S rDNA sequence analysis, showed superior endosulfan degradation activity. Analysis of culture pH, growth, free sulfate and endosulfan and its metabolites demonstrated that KE-1 biologically degrades 8.72 microg endosulfan ml(-1) day(-1) when incubated with 93.9 microg ml(-1) endosulfan for 10 days without formation of toxic endosulfan sulfate. Our results suggest that K. pneumoniae KE-1 degraded endosulfan by a non-oxidative pathway and that strain KE-1 has potential as a biocatalyst for endosulfan bioremediation.