Directed differentiation of rhesus monkey ES cells into pancreatic cell phenotypes

Embryonic stem cells (ES) can self-replicate and differentiate into all cell types including insulin-producing, beta-like cells and could, therefore, be used to treat diabetes mellitus. To date, results of stem cell differentiation into beta cells have been debated, largely due to difficulties in defining the identity of a beta cell. We have recently differentiated non-human primate (rhesus) embryonic stem (rES) cell lines into insulin producing, beta-like cells with the beta cell growth factor, Exendin-4 and using C-peptide as a phenotype marker. Cell development was characterized at each stage by gene and protein expression. Insulin, NKX6.1 and glucagon mRNA were expressed in stage 4 cells but not in early undifferentiated cells. We concluded that rES cells could be differentiated ex vivo to insulin producing cells. These differentiated rES cells could be used to develop a non-human primate model for evaluating cell therapy to treat diabetes. To facilitate the identification of beta-like cells and to track the cells post-transplantation, we have developed a marker gene construct: fusing the human insulin promoter (HIP) to the green fluorescent protein (GFP) gene. This construct was transfected into stage 3 rES derived cells and subsequent GFP expression was identified in C-peptide positive cells, thereby substantiating endogenous insulin production by rES derived cells. Using this GFP detection system, we will enrich our population of insulin producing rES derived cells and track these cells post-transplantation in the non-human primate model.     

Platelet interactions in thrombosis

Patho/physiological platelet aggregate (thrombus) formation is initiated by engagement of platelet surface receptors, glycoprotein (GP)Ib-IX-V and GPVI that bind von Willebrand factor or collagen. Although beneficial in response to vascular injury by preventing blood loss (haemostasis), platelet aggregation in a sclerotic coronary artery or other diseased blood vessel (thrombosis) can cause thrombotic diseases like heart attack and stroke. At the molecular level, ligand interactions with GPIb-IX-V or GPVI trigger signalling responses, including elevation of cytosolic Ca2+, dissociation of calmodulin from their cytoplasmic domains, cytoskeletal actin-filament rearrangements, activation of src-family kinases or PI 3-kinase, and 'inside-out' activation of the integrin, alphaIIbbeta3 (GPIIb-llla), that binds von Willebrand factor or fibrinogen and mediates platelet aggregation. Furthermore, emerging evidence supports a topographical co-association of these receptors of the leucine-rich repeat family (GPIb-IX-V) and immunoglobulin superfamily (GPVI) in an adhesive cluster or 'adhesosome'. This arrangement may underlie common mechanisms of initiating thrombus formation in haemostasis or thrombotic disease.     

Mouse zinc transporter 1 gene provides an essential function during early embryonic development

The SLC30 family of cation diffusion transporters includes at least nine members in mammals, most of which have been documented to play a role in zinc transport. The founding member of this family, Znt1, was discovered by virtue of its ability to efflux zinc from cells and to protect them from zinc toxicity. However, its physiological functions remain unknown. To address this issue, mice with targeted knockout of the Znt1 gene were generated by homologous recombination in embryonic stem cells. Heterozygous Znt1 mice were viable. In contrast, homozygous Znt1 mice died in utero soon after implantation due to a catastrophic failure of embryonic development. Although extraembryonic membranes formed around these embryos, the embryo proper failed to undergo morphogenesis past the egg cylinder stage and was amorphous by d9 of pregnancy. Expression of the Znt1 gene was detected predominantly in trophoblasts and in the maternal deciduum during the postimplantation period (d5 to d8). The failure of homozygous Znt1 embryos to develop could not be rescued by manipulating maternal dietary zinc (either excess or deficiency) during pregnancy. However, embryos in Znt1 heterozygous females were approximately 3 times more likely to develop abnormally when exposed to maternal dietary zinc deficiency during later pregnancy than were those in wildtype females. These studies suggest that Znt1 serves an essential function of transporting maternal zinc into the embryonic environment during the egg cylinder stage of development, and further suggest that Znt1 plays a role in zinc homeostasis in adult mice.     

Aurora B regulates MCAK at the mitotic centromere

Chromosome orientation and alignment within the mitotic spindle requires the Aurora B protein kinase and the mitotic centromere-associated kinesin (MCAK). Here, we report the regulation of MCAK by Aurora B. Aurora B inhibited MCAK's microtubule depolymerizing activity in vitro, and phospho-mimic (S/E) mutants of MCAK inhibited depolymerization in vivo. Expression of either MCAK (S/E) or MCAK (S/A) mutants increased the frequency of syntelic microtubule-kinetochore attachments and mono-oriented chromosomes. MCAK phosphorylation also regulates MCAK localization: the MCAK (S/E) mutant frequently localized to the inner centromere while the (S/A) mutant concentrated at kinetochores. We also detected two different binding sites for MCAK using FRAP analysis of the different MCAK mutants. Moreover, disruption of Aurora B function by expression of a kinase-dead mutant or RNAi prevented centromeric targeting of MCAK. These results link Aurora B activity to MCAK function, with Aurora B regulating MCAK's activity and its localization at the centromere and kinetochore.     

Novel targeting signals mediate the sorting of different isoforms of the tail-anchored membrane protein cytochrome b5 to either endoplasmic reticulum or mitochondria

Tail-anchored membrane proteins are a class of proteins that are targeted posttranslationally to various organelles and integrated by a single segment of hydrophobic amino acids located near the C terminus. Although the localization of tail-anchored proteins in specific subcellular compartments in plant cells is essential for their biological function, the molecular targeting signals responsible for sorting these proteins are not well defined. Here, we describe the biogenesis of four closely related tung (Aleurites fordii) cytochrome b5 isoforms (Cb5-A, -B, -C, and -D), which are small tail-anchored proteins that play an essential role in many cellular processes, including lipid biosynthesis. Using a combination of in vivo and in vitro assays, we show that Cb5-A, -B, and -C are targeted exclusively to the endoplasmic reticulum (ER), whereas Cb5-D is targeted specifically to mitochondrial outer membranes. Comprehensive mutational analyses of ER and mitochondrial Cb5s revealed that their C termini, including transmembrane domains (TMD) and tail regions, contained several unique physicochemical and sequence-specific characteristics that defined organelle-specific targeting motifs. Mitochondrial targeting of Cb5 was mediated by a combination of hydrophilic amino acids along one face of the TMD, an enrichment of branched beta-carbon-containing residues in the medial portion of the TMD, and a dibasic -R-R/K/H-x motif in the C-terminal tail. By contrast, ER targeting of Cb5 depended primarily upon the overall length and hydrophobicity of the TMD, although an -R/H-x-Y/F- motif in the tail was also a targeting determinant. Collectively, the results presented provide significant insight into the early biogenetic events required for entry of tail-anchored proteins into either the ER or mitochondrial targeting pathways.     

Insights into the effects on metal binding of the systematic substitution of five key glutamate ligands in the ferritin of Escherichia coli

Ferritins are nearly ubiquitous iron storage proteins playing a fundamental role in iron metabolism. They are composed of 24 subunits forming a spherical protein shell encompassing a central iron storage cavity. The iron storage mechanism involves the initial binding and subsequent O2-dependent oxidation of two Fe2+ ions located at sites A and B within the highly conserved dinuclear "ferroxidase center" in individual subunits. Unlike animal ferritins and the heme-containing bacterioferritins, the Escherichia coli ferritin possesses an additional iron-binding site (site C) located on the inner surface of the protein shell close to the ferroxidase center. We report the structures of five E. coli ferritin variants and their Fe3+ and Zn2+ (a redox-stable alternative for Fe2+) derivatives. Single carboxyl ligand replacements in sites A, B, and C gave unique effects on metal binding, which explain the observed changes in Fe2+ oxidation rates. Binding of Fe2+ at both A and B sites is clearly essential for rapid Fe2+ oxidation, and the linking of FeB2+ to FeC2+ enables the oxidation of three Fe2+ ions. The transient binding of Fe2+ at one of three newly observed Zn2+ sites may allow the oxidation of four Fe2+ by one dioxygen molecule.     

The beta-subunit of the signal recognition particle receptor is a novel GTP-binding protein without intrinsic GTPase activity

The beta-subunit of the signal recognition particle receptor (SRbeta), a member of the Ras family of small molecular weight GTPases, is involved in the targeting of nascent polypeptide chains to the protein translocation machinery in the endoplasmic reticulum membrane. We purified SRbeta from an expressing strain of Escherichia coli and investigated the properties of the isolated GTPase. We find that, unlike other Ras family GTPases, most SRbeta purifies bound to GTP, and SRbeta-bound GTP is not easily exchanged with solution GTP. SRbeta possesses no detectable GTPase activity. Although a stable interaction between SRbeta and ribosomes is observed, SRbeta is not stimulated to hydrolyze GTP when incubated with ribosomes or ribosome-nascent chains. A GTPase mutant harboring a mutation in a region predicted to be functionally important, based on observations made in related GTPases, binds GTP with faster kinetics and appears to be a less stable protein but otherwise displays similar properties to the wild-type SRbeta GTPase. Our results demonstrate that as an isolated GTPase, SRbeta functions differently from the Arf- and Ras-type GTPases that it is most closely related to by sequence.     

The relationship between side reactions and slow inhibition of ribulose-bisphosphate carboxylase revealed by a loop 6 mutant of the tobacco enzyme

The first directed mutant of a higher plant ribulose-bisphosphate carboxylase/oxygenase (Rubisco), constructed by chloroplast transformation, is catalytically impaired but still able to support the plant's photosynthesis and growth (Whitney, S. M., von Caemmerer, S., Hudson, G. S., and Andrews, T. J. (1999) Plant Physiol. 121, 579-588). This mutant enzyme has a Leu to Val substitution at residue 335 in the flexible loop 6 of the large subunit, which closes over the substrate during catalysis. Its active site was intact, as judged by its barely impaired competency in the initial enolization step of the reaction sequence, and its ability to bind tightly the intermediate analog, 2'-carboxy-D-arabinitol-1,5-bisphosphate. Prompted by observations that the mutant enzyme displayed much less slow inhibition during catalysis in vitro than the wild type, its tendency to catalyze side reactions and its response to the slow inhibitor D-xylulose-1,5-bisphosphate were studied. The lessening in slow inhibition was not caused by reduced production of inhibitory side products. Except for pyruvate production, these reactions were strongly enhanced by the mutation, as was the ability to catalyze the carboxylation of D-xylulose-1,5-bisphosphate. Rather, reduced inhibition was the result of lessened sensitivity to these inhibitors. The slow isomerization phase that characterizes inhibition of the wild-type enzyme by D-xylulose-1,5-bisphosphate was completely eliminated by the mutation, and the mutant was more adept than the wild type in catalyzing the benzylic acid-type rearrangement of D-glycero-2,3-pentodiulose-1,5-bisphosphate (produced by oxidation of the substrate, D-ribulose-1,5-bisphosphate). These observations are consistent with increased flexibility of loop 6 induced by the mutation, and they reveal the underlying mechanisms by which the side reactions cause slow inhibition.     

Impaired membrane resealing and autoimmune myositis in synaptotagmin VII-deficient mice

Members of the synaptotagmin family have been proposed to function as Ca2+ sensors in membrane fusion. Syt VII is a ubiquitously expressed synaptotagmin previously implicated in plasma membrane repair and Trypanosoma cruzi invasion, events which are mediated by the Ca2+-regulated exocytosis of lysosomes. Here, we show that embryonic fibroblasts from Syt VII-deficient mice are less susceptible to trypanosome invasion, and defective in lysosomal exocytosis and resealing after wounding. Examination of mutant mouse tissues revealed extensive fibrosis in the skin and skeletal muscle. Inflammatory myopathy, with muscle fiber invasion by leukocytes and endomysial collagen deposition, was associated with elevated creatine kinase release and progressive muscle weakness. Interestingly, similar to what is observed in human polymyositis/dermatomyositis, the mice developed a strong antinuclear antibody response, characteristic of autoimmune disorders. Thus, defective plasma membrane repair in tissues under mechanical stress may favor the development of inflammatory autoimmune disease.