Susceptibility to streptozotocin-induced diabetes is mapped to mouse chromosome 11

To study the contribution of beta-cell vulnerability to susceptibility to diabetes, we studied beta-cell vulnerability to a single high dose of streptozotocin (STZ) in an animal model of type 2 diabetes, the NSY mouse, a sister strain of the STZ-sensitive NOD mouse, in comparison with the STZ-resistant C3H mouse. NSY mice were found to be extremely sensitive to STZ. Introgression of a single Chr 11, where STZ-sensitivity was mapped in the NOD mouse, from NSY mice converted STZ-resistant C3H mice to STZ-sensitive. Two nucleotide substitutions were identified in the nucleoredoxin gene, a positional and functional candidate gene for STZ-induced diabetes on Chr 11. These data, together with the co-localization of type 1 (Idd4) and type 2 (Nidd1n) susceptibility genes on Chr 11, suggest that the intrinsic vulnerability of pancreatic beta cells is determined by a gene or genes on Chr 11, which may also contribute to susceptibility to spontaneous diabetes.     

Heat shock induces phosphorylation of histone H2AX in mammalian cells

Heat shock induces a variety of biological events including gene activation, cell cycle arrest, and apoptosis. Heat shock has recently been shown to be potentially useful when combined with radiation in cancer therapy, probably because, in mammalian cells, heat inhibits the repair of double-strand breaks (DSBs) induced by ionizing radiation. It remains unclear, however, whether heat shock by itself induces DSBs. In this communication, we present the first evidence that heat shock induces the phosphorylated form of histone H2AX, which is thought to be generated at the chromatin proximal to DSB sites. These results suggest that heat shock induces DSBs in mammalian cells and may provide direct evidence to explain previous reports on DSB-related events occurring after heat shock treatment.     

MAP1B phosphorylation is differentially regulated by Cdk5/p35, Cdk5/p25, and JNK

Mode I phosphorylated MAP1B is observed in developing and pathogenic brains. Although Cdk5 has been believed to phosphorylate MAP1B in the developing cerebral cortex, we show that a Cdk5 inhibitor does not suppress mode I phosphorylation of MAP1B in primary and slice cultures, while a JNK inhibitor does. Coincidently, an increase in phosphorylated MAP1B was not observed in COS7 cells when Cdk5 was cotransfected with p35, but this did occur with p25 which is specifically produced in pathogenic brains. Our primary culture studies showed an involvement of Cdk5 in regulating microtubule dynamics without affecting MAP1B phosphorylation status. The importance of regulating microtubule dynamics in neuronal migration was also demonstrated by in utero electroporation experiments. These findings suggest that mode I phosphorylation of MAP1B is facilitated by JNK but not Cdk5/p35 in the developing cerebral cortex and by Cdk5/p25 in pathogenic brains, contributing to various biological events.     

Synthesis of phosphatidylcholine: an improved method without using the cadmium chloride complex of sn-glycero-3-phosphocholine

An improved safe method that does not contaminate the environment with cadmium chloride, a toxic heavy metal salt, was developed for the synthesis of phosphatidylcholine (PC). PC was synthesized from sn-glycero-3-phosphocholine (GPC) and fatty acid in one step under mild conditions without the use of cadmium chloride. GPC was prepared from egg yolk PC and adsorbed by kieselguhr in a Teflon vessel. The GPC on kieselguhr was acylated with fatty acid in the presence of two reagents, dicyclohexylcarbodiimide for synthesis of fatty acid anhydride and 4-dimethylaminopyridine as an acylating catalyst, at 30 degrees C overnight. The PC thus produced was purified by silica gel column chromatography. The yield of dioleoyl PC was 90% based on the starting material, GPC.     

Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide

Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe3+-chelates, which initiated reactions that were dependent on membrane charge: Fe3+-EDTA and Fe3+-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe3+-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe3+-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.     

Zinc transporters, ZnT5 and ZnT7, are required for the activation of alkaline phosphatases, zinc-requiring enzymes that are glycosylphosphatidylinositol-anchored to the cytoplasmic membrane

Numerous proteins are properly folded by binding with zinc during their itinerary in the biosynthetic-secretory pathway. Several transporters have been implicated in the zinc entry into secretory compartments from cytosol, but their precise roles are poorly understood. We report here that two zinc transporters (ZnT5 and ZnT7) localized in the secretory apparatus are responsible for loading zinc to alkaline phosphatases (ALPs) that are glycosylphosphatidylinositol-anchored membrane proteins exposed to the extracellular site. Disruption of the ZnT5 gene in DT40 cells decreased the ALP activity to 45% of that in the wild-type cells. Disruption of the ZnT7 gene lowered the ALP activity only by 20%. Disruption of both genes markedly decreased the ALP activity to <5%. Overexpression of human ZnT5 or ZnT7 in DT40 cells deficient in both ZnT5 and ZnT7 genes recovered the ALP activity to the level comparable to that in the wild-type cells. The inactive ALP protein in DT40 cells deficient in both ZnT5 and ZnT7 genes was transported to cytoplasmic membrane like the active ALP protein in the wild-type cells. Thus both ZnT5 and ZnT7 contribute to the conversion of apo-ALP to holo-ALP.     

Fluoride induces endoplasmic reticulum stress in ameloblasts responsible for dental enamel formation

The mechanism of how fluoride causes fluorosis remains unknown. Exposure to fluoride can inhibit protein synthesis, and this may also occur by agents that cause endoplasmic reticulum (ER) stress. When translated proteins fail to fold properly or become misfolded, ER stress response genes are induced that together comprise the unfolded protein response. Because ameloblasts are responsible for dental enamel formation, we used an ameloblast-derived cell line (LS8) to characterize specific responses to fluoride treatment. LS8 cells were growth-inhibited by as little as 1.9-3.8 ppm fluoride, whereas higher doses induced ER stress and caspase-mediated DNA fragmentation. Growth arrest and DNA damage-inducible proteins (GADD153/CHOP, GADD45alpha), binding protein (BiP/glucose-responsive protein 78 (GRP78), the non-secreted form of carbonic anhydrase VI (CA-VI), and active X-box-binding protein-1 (Xbp-1) were all induced significantly after exposure to 38 ppm fluoride. Unexpectedly, DNA fragmentation increased when GADD153 expression was inhibited by short interfering RNA treatment but remained unaffected by transient GADD153 overexpression. Analysis of control and GADD153(-/-) embryonic fibroblasts demonstrated that caspase-3 mediated the increased DNA fragmentation observed in the GADD153 null cells. We also demonstrate that mouse incisor ameloblasts are sensitive to the toxic effects of high dose fluoride in drinking water. Activated Ire1 initiates an ER stress response pathway, and mouse ameloblasts were shown to express activated Ire1. Ire1 levels appeared induced by fluoride treatment, indicating that ER stress may play a role in dental fluorosis. Low dose fluoride, such as that present in fluoridated drinking water, did not induce ER stress.     

A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) κ locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igκ region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras.     

Robustness of hen lysozyme monitored by random mutations

We investigated the robustness of hen lysozyme by using random mutant libraries. Six random mutant libraries containing 1, 1.5, 2, 3, 5 and 14 amino acid mutations per hen lysozyme were systematically constructed by varying the concentrations of Mg(2+) and Mn(2+) on polymerase chain reaction. The mutated genes from the six libraries were cloned to a yeast expression vector and a total of 4000 clones were screened on the basis of lysis activity and ELISA employing monoclonal antibody that recognized only lysozyme with native conformation. About 80% of the clones with an average of two amino acid mutations retained active structure. Almost all clones with an average of five mutations lost active structure. On the other hand, 80% of the clones with an average of two amino acid mutations retained both gross conformation and active structure and 24% of the clones with an average of 14 amino acid mutations retained gross conformation. These results show that gross conformation is robust against mutations and so is active structure to a lesser extent.     

Different molecular mechanics displayed by titin's constitutively and differentially expressed tandem Ig segments

Titin is a giant elastic protein responsible for passive force generated by the stretched striated-muscle sarcomere. Passive force develops in titin's extensible region which consists of the PEVK segment in series with tandemly arranged immunoglobulin (Ig)-like domains. Here we studied the mechanics of tandem Ig segments from the differentially spliced (I65-70) and constitutive (I91-98) regions by using an atomic force microscope specialized for stretching single molecules. The mechanical stability of I65-70 domains was found to be different from that of I91-98 domains. In the range of stretch rates studied (0.05-1.00 microm/s) lower average domain unfolding forces for I65-70 were associated with a weaker stretch-rate dependence of the unfolding force, suggesting that the differences in the mechanical stabilities of the segments derive from differences in the zero force unfolding rate (K(0)(u)) and the characteristic distance (location of the barrier) along the unfolding reaction coordinate (DeltaX(u)). No effect of calcium was found on unfolding forces and persistence length of unfolded domains. To explore the structural basis of the differences in mechanical stabilities of the two fragment types, we compared the amino acid sequence of I65-70 domains with that of I91-98 domains and by using homology modeling analyzed how sequence variations may affect folding free energies. Simulations suggest that differences in domain stability are unlikely to be caused by variation in the number of hydrogen bonds between the force-bearing beta-strands at the domain's N- and C-termini. Rather, they may be due to differences in hydrophobic contacts and strand orientations.