DJ-1 Protects Pancreatic Beta Cells from Cytokine- and Streptozotocin-Mediated Cell Death

A hallmark feature of type 1 and type 2 diabetes mellitus is the progressive dysfunction and loss of insulin-producing pancreatic beta cells, and inflammatory cytokines are known to trigger beta cell death. Here we asked whether the anti-oxidant protein DJ-1 encoded by the Parkinson’s disease gene PARK7 protects islet cells from cytokine- and streptozotocin-mediated cell death. Wild type and DJ-1 knockout mice (KO) were treated with multiple low doses of streptozotocin (MLDS) to induce inflammatory beta cell stress and cell death. Subsequently, glucose tolerance tests were performed, and plasma insulin as well as fasting and random blood glucose concentrations were monitored. Mitochondrial morphology and number of insulin granules were quantified in beta cells. Moreover, islet cell damage was determined in vitro after streptozotocin and cytokine treatment of isolated wild type and DJ-1 KO islets using calcein AM/ethidium homodimer-1 staining and TUNEL staining. Compared to wild type mice, DJ-1 KO mice became diabetic following MLDS treatment. Insulin concentrations were substantially reduced, and fasting blood glucose concentrations were significantly higher in MLDS-treated DJ-1 KO mice compared to equally treated wild type mice. Rates of beta cell apoptosis upon MLDS treatment were twofold higher in DJ-1 KO mice compared to wild type mice, and in vitro inflammatory cytokines led to twice as much beta cell death in pancreatic islets from DJ-1 KO mice versus those of wild type mice. In conclusion, this study identified the anti-oxidant protein DJ-1 as being capable of protecting pancreatic islet cells from cell death induced by an inflammatory and cytotoxic setting.     

Evaluation of genotoxic effects induced by exposure to antineoplastic drugs in lymphocytes and exfoliated buccal cells of oncology nurses and pharmacy employees

The continuous introduction of new antineoplastic drugs and their use as complex mixture emphasize the need to carry out correct health risk assessment. The aim of this study was to evaluate genotoxic effects of antineoplastic drugs in nurses (n=25) and pharmacy technicians (n=5) employed in an oncology hospital. The nurses administered antineoplastic drugs in the day-care hospital (n=12) and in the wards (n=13), and pharmacy technicians prepared the drugs in the central pharmacy. We performed the micronucleus (MN) test with lymphocytes and exfoliated buccal cells and conducted traditional analysis of chromosomal aberrations (CA). Thirty healthy subjects were selected as controls. Monitoring of surface contamination with cyclophosphamide, 5-fluorouracil, ifosfamide, cytarabine, and gemcitabine showed the presence of detectable levels only for cyclophosphamide, 5-fluorouracil and ifosfamide. In addition, we measured the 5-fluorouracil metabolite alpha-F-betaalanine in the urine of all subjects and found significant concentrations only in 3 out of 25 nurses. The micronucleus assay with lymphocytes did not show significant differences between exposed and control groups, while the same test with exfoliated buccal cells found higher values in nurses administering antineoplastic drugs than in pharmacy employees. In the CA analysis, we detected in exposed groups a significant increase (about 2.5-fold) of structural CA, particularly breaks (up to 5.0-fold). Our results confirm the genotoxic effect of antineoplastic drugs in circulating blood lymphocytes. Moreover, in exfoliated buccal cells the data show more consistent genetic damage induced during administration of the antineoplastic drugs than during their preparation. The data also stress the use of this non-invasive sampling, to assess occupational exposure to mixture of chemicals at low doses.     

Distinct functions of the unique C terminus of LAP2alpha in cell proliferation and nuclear assembly

The non-membrane-bound lamina-associated polypeptide 2 isoform, LAP2alpha, forms nucleoskeletal structures with A-type lamins and interacts with chromosomes in a cell cycle-dependent manner. LAP2alpha contains a LEM (LAP2, emerin, and MAN1) domain in the constant N terminus that binds to chromosomal barrier-to-autointegration factor, and a C-terminal unique region that is essential for chromosome binding. Here we show that C-terminal LAP2alpha fragment efficiently bound to mitotic chromosomes and inhibited assembly of endogenous LAP2alpha, nuclear membranes, and lamins A/C in in vitro nuclear assembly assays. Full-length recombinant LAP2alpha, which bound to chromosomes, and N-terminal fragment, which did not bind, had no effect on assembly. This suggested an essential role for the LAP2alpha C terminus in chromosome association and for the N-terminal LEM domain in subsequent assembly stages. In vivo analysis upon transient expression of GFP-tagged LAP2alpha fragments confirmed that, unlike the N-terminal fragment, the C-terminal fragment was able to bind to chromosomes during mitosis, if expressed weakly. At higher expression levels, C-terminal LAP2alpha fragment and full-length protein led to cell cycle arrest in interphase and apoptosis, as shown by fluorescence-activated cell sorter analysis, time lapse microscopy, and BrdUrd incorporation assays. These data indicated distinct functions of LAP2alpha in cell cycle progression during interphase and in nuclear reassembly during mitosis.     

IFN-beta pretreatment sensitizes human melanoma cells to TRAIL/Apo2 ligand-induced apoptosis

All human melanoma cell lines (assessed by annexin V and TUNEL assays) were resistant to apoptosis induction by TRAIL/Apo2L protein. TRAIL/Apo2L activated caspase-8 and caspase-3, but subsequent apoptotic events such as poly(ADP-ribose) polymerase cleavage and DNA fragmentation were not observed. To probe the molecular mechanisms of cellular resistance to apoptosis, melanoma cell lines were analyzed for expression of apoptosis regulators (apoptotic protease-associated factor-1, FLIP, caspase-8, caspase-9, caspase-3, cellular inhibitor of apoptosis, Bcl-2, or Bax); no correlation was observed. TRAIL/Apo2L was induced in melanoma cell lines by IFN-beta and had been correlated with apoptosis induction. Because IFN-beta induced other gene products that have been associated with apoptosis, it was postulated that one or more IFN-stimulated genes might sensitize cells to TRAIL/Apo2L. Melanoma cell lines were treated with IFN-beta for 16-24 h before treatment with TRAIL/Apo2L. Regardless of their sensitivity to either cytokine alone, >30% of cells underwent apoptosis in response to the combined treatment. Induction of apoptosis by IFN-beta and TRAIL/Apo2L in combination correlated with synergistic activation of caspase-9, a decrease in mitochondrial potential, and cleavage of poly(ADP-ribose) polymerase. Cleavage of X-linked inhibitor of apoptosis following IFN-beta and TRAIL/Apo2L treatment was observed in sensitive WM9, A375, or WM3211 cells but not in resistant WM35 or WM164 cells. Thus, in vitro IFN-beta and TRAIL/Apo2L combination treatment had more potent apoptotic and anti-growth effects when compared with either cytokine alone in melanoma cells lines.     

Dephosphorylation and activation of exogenous glycogen synthase by adipose-tissue phosphatase

Exogenous purified rabbit skeletal-muscle glycogen synthase was used as a substrate for adipose-tissue phosphoprotein phosphatase from fed and starved rats in order to (1) compare the relationship between phosphate released from, and the kinetic changes imparted to, the substrate and (2) ascertain if decreases in adipose-tissue phosphatase activity account for the apparent decreased activation of endogenous glycogen synthase from starved as compared with fed rats. Muscle glycogen synthase was phosphorylated with [gamma-(32)P]ATP and cyclic AMP-dependent protein kinase alone, or in combination with a cyclic AMP-independent protein kinase, to 1.7 or 3mol of phosphate per subunit. Adipose-tissue phosphatase activity determined with phosphorylated skeletal-muscle glycogen synthase as substrate was decreased by 35-60% as a consequence of starvation. This decrease in phosphatase activity had little effect on the capacity of adipose-tissue extracts to activate exogenous glycogen synthase (i.e. to increase the glucose 6-phosphate-independent enzyme activity), although there were marked differences in the activation profiles for the two exogenous substrates. Glycogen synthase phosphorylated to 1.7mol of phosphate per subunit was activated rapidly by adipose-tissue extracts from either fed or starved rats, and activation paralleled enzyme dephosphorylation. Glycogen synthase phosphorylated to 3mol of phosphate per subunit was activated more slowly and after a lag period, since release of the first mol of phosphate did not increase the glucose 6-phosphate-independent activity of the enzyme. These patterns of enzyme activation were similar to those observed for the endogenous adipose-tissue glycogen synthase(s): the glucose 6-phosphate-independent activity of the endogenous enzyme from fed rats increased rapidly during incubation, whereas that of starved rats, like that of the more highly phosphorylated muscle enzyme, increased only very slowly after a lag period. The observations made here suggest that (1) changes in glucose 6-phosphate-independent glycogen synthase activity are at best only a qualitative measure of phosphoprotein phosphatase activity and (2) the decrease in glycogen synthase phosphatase activity during starvation is not sufficient to explain the differential glycogen synthase activation in adipose tissue from fed and starved rats. However, alterations in the phosphorylation state of glycogen synthase combined with decreased activity of phosphoprotein phosphatase, both as a consequence of starvation, could explain the apparent markedly decreased enzyme activation.     

Freeze-etch and thin section studies ofNeurospora crassa ascospores

Summary Mature, dormant ascospores derived from crosses of wild type stocks of the fungusNeurospora crassa were studied using both thin section and freeze-etch techniques for electron microscopy. The spore wall is composed of three major layers with, perhaps, a fourth distinct layer covering these layers. The spore contains several nuclei, many discrete mitochondria, lipid bodies, vacuoles and small pieces of endoplasmic reticulum. Freeze-etch fracture faces of several membranes are described in this paper. Included is a diagram illustrating various fracture faces and surfaces seen following freeze-etching. The thin section results are less than satisfactory but serve to generally confirm the freeze-etch observations and suggest that further work could lead to the development of techniques which will result in high quality thin sections of ascospores.     

Characterization of fibrous compound of Golgi vesicles in tapetal cells ofDelphinium

Summary It was shown that Golgi structures abundantly appearing in tapetal cells ofDelphinium Ajacis L. developing anthers, prior to meiocytes meiosis, show a fine fibrous material within their vesicles. At the time of the formation of tapetal cell wall this fibrous component, released by an exocytotic process, is incorporated into the cell wall. The membrane of dictyosomes derived vesicles participates in the development of plasma membrane. Fibrous material appears to be morphologically similar to the fibrils of tapetal cell wall; this cell wall gives a positive reaction for cellulose and pectins, as visible in the light microscope. Moreover, the fibrous and pectinase resistant compound of dictyosomes derived vesicles and the fibrils of cell wall disappear partly after cellulase digestion which proves their cellulosic character. On the other hand pectinase treatment as well as ruthenium red staining suggest associated with cellulose pectins within Golgi vesicles.     

The Gla domain of factor IXa binds to factor VIIIa in the tenase complex

During blood coagulation factor IXa binds to factor VIIIa on phospholipid membranes to form an enzymatic complex, the tenase complex. To test whether there is a protein-protein contact site between the gamma-carboxyglutamic acid (Gla) domain of factor IXa and factor VIIIa, we demonstrated that an antibody to the Gla domain of factor IXa inhibited factor VIIIa-dependent factor IXa activity, suggesting an interaction of the factor IXa Gla domain with factor VIIIa. To study this interaction, we synthesized three analogs of the factor IXa Gla domain (FIX1-47) with Phe-9, Phe-25, or Val-46 replaced, respectively, with benzoylphenylalanine (BPA), a photoactivatable cross-linking reagent. These factor IX Gla domain analogs maintain native tertiary structure, as demonstrated by calcium-induced fluorescence quenching and phospholipid binding studies. In the absence of phospholipid membranes, FIX1-47 was able to inhibit factor IXa activity. This inhibition is dependent on the presence of factor VIIIa, suggesting a contact site between the factor IXa Gla domain and factor VIIIa. To demonstrate a direct interaction we did cross-linking experiments with FIX1-479BPA, FIX1-4725BPA, and FIX1-4746BPA. Covalent cross-linking to factor VIIIa was observed primarily with FIX1-4725BPA and to a much lesser degree with FIX1-4746BPA. Immunoprecipitation experiments with an antibody to the C2 domain of factor VIIIa indicate that the factor IX Gla domain cross-links to the A3-C1-C2 domain of factor VIIIa. These results suggest that the factor IXa Gla domain contacts factor VIIIa in the tenase complex through a contact site that includes phenylalanine 25 and perhaps valine 46.     

Mek2 is dispensable for mouse growth and development

MEK is a dual-specificity kinase that activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase upon agonist binding to receptors. The ERK/MAP kinase cascade is involved in cell fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single Mek gene is present in Caenorhabditis elegans, Drosophila melanogaster, and Xenopus laevis, two Mek homologs, Mek1 and Mek2, are present in the mammalian cascade. The inactivation of the Mek1 gene leads to embryonic lethality and has revealed the unique role played by Mek1 during embryogenesis. To investigate the biological function of the second homolog, we have generated mice deficient in Mek2 function. Mek2 mutant mice are viable and fertile, and they do not present flagrant morphological alteration. Although several components of the ERK/MAP kinase cascade have been implicated in thymocyte development, no such involvement was observed for MEK2, which appears to be nonessential for thymocyte differentiation and T-cell-receptor-induced proliferation and apoptosis. Altogether, our findings demonstrate that MEK2 is not necessary for the normal development of the embryo and T-cell lineages, suggesting that the loss of MEK2 can be compensated for by MEK1.