GLUT2 surface expression and intracellular transport via the constitutive pathway in pancreatic beta cells and insulinoma: evidence for a block in trans-Golgi network exit by brefeldin A.

The biosynthesis, intracellular transport, and surface expression of the beta cell glucose transporter GLUT2 was investigated in isolated islets and insulinoma cells. Using a trypsin sensitivity assay to measure cell surface expression, we determined that: (a) greater than 95% of GLUT2 was expressed on the plasma membrane; (b) GLUT2 did not recycle in intracellular vesicles; and (c) after trypsin treatment, reexpression of the intact transporter occurred with a t1/2 of approximately 7 h. Kinetics of intracellular transport of GLUT2 was investigated in pulse-labeling experiments combined with glycosidase treatment and the trypsin sensitivity assay. We determined that transport from the endoplasmic reticulum to the trans-Golgi network (TGN) occurred with a t1/2 of 15 min and that transport from the TGN to the plasma membrane required a similar half-time. When added at the start of a pulse-labeling experiment, brefeldin A prevented exit of GLUT2 from the endoplasmic reticulum. When the transporter was first accumulated in the TGN during a 15-min period of chase, but not following a low temperature (22 degrees C) incubation, addition of brefeldin A (BFA) prevented subsequent surface expression of the transporter. This indicated that brefeldin A prevented GLUT2 exit from the TGN by acting at a site proximal to the 22 degrees C block. Together, these data demonstrate that GLUT2 surface expression in beta cells is via the constitutive pathway, that transport can be blocked by BFA at two distinct steps and that once on the surface, GLUT2 does not recycle in intracellular vesicles.

PEG-b-PPS diblock copolymer aggregates for hydrophobic drug solubilization and release: cyclosporin A as an example

Micelles formed from amphiphilic block copolymers have been explored in recent years as carriers for hydrophobic drugs. In an aqueous environment, the hydrophobic blocks form the core of the micelle, which can host lipophilic drugs, while the hydrophilic blocks form the corona or outer shell and stabilize the interface between the hydrophobic core and the external medium. In the present work, mesophase behavior and drug encapsulation were explored in the AB block copolymeric amphiphile composed of poly(ethylene glycol) (PEG) as a hydrophile and poly(propylene sulfide) PPS as a hydrophobe, using the immunosuppressive drug cyclosporin A (CsA) as an example of a highly hydrophobic drug. Block copolymers with a degree of polymerization of 44 on the PEG and of 10, 20 and 40 on the PPS respectively (abbreviated as PEG44-b-PPS10, PEG44-b-PPS20, PEG44-b-PPS40) were synthesized and characterized. Drug-loaded polymeric micelles were obtained by the cosolvent displacement method as well as the remarkably simple method of dispersing the warm polymer melt, with drug dissolved therein, in warm water. Effective drug solubility up to 2 mg/mL in aqueous media was facilitated by the PEG- b-PPS micelles, with loading levels up to 19% w/w being achieved. Release was burst-free and sustained over periods of 9-12 days. These micelles demonstrate interesting solubilization characteristics, due to the low glass transition temperature, highly hydrophobic nature, and good solvent properties of the PPS block

Highly efficient DNA incorporation of intratumourally injected [125I]iododeoxyuridine under thymidine synthesis blocking in human glioblastoma xenografts.

Intratumoural (i.t.) injection of radio-iododeoxyuridine (IdUrd), a thymidine (dThd) analogue, is envisaged for targeted Auger electron- or beta-radiation therapy of glioblastoma. Here, biodistribution of [(125)I]IdUrd was evaluated 5 hr after i.t. injection in subcutaneous human glioblastoma xenografts LN229 after different intravenous (i.v.) pretreatments with fluorodeoxyuridine (FdUrd). FdUrd is known to block de novo dThd synthesis, thus favouring DNA incorporation of radio-IdUrd. Results showed that pretreatment with 2 mg/kg FdUrd i.v. in 2 fractions 0.5 hr and 1 hr before injection of radio-IdUrd resulted in a mean tumour uptake of 19.8% of injected dose (% ID), representing 65.3% ID/g for tumours of approx. 0.35 g. Tumour uptake of radio-IdUrd in non-pretreated mice was only 4.1% ID. Very low uptake was observed in normal nondividing and dividing tissues with a maximum concentration of 2.9% ID/g measured in spleen. Pretreatment with a higher dose of FdUrd of 10 mg/kg prolonged the increased tumour uptake of radio-IdUrd up to 5 hr. A competition experiment was performed in FdUrd pretreated mice using i.t. co-injection of excess dThd that resulted in very low tumour retention of [(125)I]IdUrd. DNA isolation experiments showed that in the mean >95% of tumour (125)I activity was incorporated in DNA. In conclusion, these results show that close to 20% ID of radio-IdUrd injected i.t. was incorporated in tumour DNA after i.v. pretreatment with clinically relevant doses of FdUrd and that this approach may be further exploited for diffusion and therapy studies with Auger electron- and/or beta-radiation-emitting radio-IdUrd.

Coupled synthesis and translocation restrains polyphosphate to acidocalcisome-like vacuoles and prevents its toxicity.

Eukaryotes contain inorganic polyphosphate (polyP) and acidocalcisomes, which sequester polyP and store amino acids and divalent cations. Why polyP is sequestered in dedicated organelles is not known. We show that polyP produced in the cytosol of yeast becomes toxic. Reconstitution of polyP translocation with purified vacuoles, the acidocalcisomes of yeast, shows that cytosolic polyP cannot be imported, whereas polyP produced by the vacuolar transporter chaperone (VTC) complex, an endogenous vacuolar polyP polymerase, is efficiently imported and does not interfere with growth. PolyP synthesis and import require an electrochemical gradient, probably as a driving force for polyP translocation. VTC exposes its catalytic domain to the cytosol and carries nine vacuolar transmembrane domains. Mutations in the VTC transmembrane regions, which are likely to constitute the translocation channel, block not only polyP translocation but also synthesis. Given that they are far from the cytosolic catalytic domain of VTC, this suggests that the VTC complex obligatorily couples synthesis of polyP to its import in order to avoid toxic intermediates in the cytosol. Sequestration of otherwise toxic polyP might be one reason for the existence of acidocalcisomes in eukaryotes.

Synthesis of medium-chain-length polyhydroxyalkanoates in arabidopsis thaliana using intermediates of peroxisomal fatty acid beta-oxidation.

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the beta-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the beta-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.

PIDD orchestrates translesion DNA synthesis in response to UV irradiation.

PIDD has been implicated in survival and apoptotic pathways in response to DNA damage, and a role for PIDD was recently identified in non-homologous end-joining (NHEJ) repair induced by γ-irradiation. Here, we present an interaction of PIDD with PCNA, first identified in a proteomics screen. PCNA has essential functions in DNA replication and repair following UV irradiation. Translesion synthesis (TLS) is a process that prevents UV irradiation-induced replication blockage and is characterized by PCNA monoubiquitination and interaction with the TLS polymerase eta (polη). Both of these processes are inhibited by p21. We report that PIDD modulates p21-PCNA dissociation, and promotes PCNA monoubiquitination and interaction with polη in response to UV irradiation. Furthermore, PIDD deficiency leads to a defect in TLS that is associated, both in vitro and in vivo, with cellular sensitization to UV-induced apoptosis. Thus, PIDD performs key functions upon UV irradiation, including TLS, NHEJ, NF-κB activation and cell death.

Highly inducible synthesis of heterologous proteins in epithelial cells carrying a glucocorticoid-responsive vector.

A glucocorticoid-responsive vector is described which allows for the highly inducible expression of complementary DNAs (cDNAs) in stably transfected mammalian cell lines. This vector, pLK-neo, composed of a variant mouse mammary tumor virus long terminal repeat promoter, containing a hormone regulatory element, a Geneticin resistance-encoding gene in a simian virus 40 transcription unit, and a polylinker insertion site for heterologous cDNAs, was used to express the polymeric immunoglobulin (poly-Ig) receptor and the thymocyte marker, Thy-1, in Madin-Darby canine kidney (MDCK) cells and in murine fibroblast L cells. A high level of poly-Ig receptor or Thy-1 mRNA accumulation was observed in MDCK cells in response to dexamethasone with a parallel ten- to 200-fold increase in protein synthesis depending on the recombinant protein and the transfected cell clone.

Synthesis and characterization of a new class of anti-angiogenic agents based on ruthenium clusters.

New triruthenium-carbonyl clusters derivatized with glucose-modified bicyclophosphite ligands have been synthesized. These compounds were found to have cytostatic and cytotoxic activity and depending on the number of bicyclophosphite ligands, and could be tuned for either anti-cancer or specific anti-angiogenic activity. While some compounds had a broad cellular toxicity profile in several cell types others showed endothelial cell specific dose-dependent anti-proliferative and anti-migratory efficacy. A profound inhibition of angiogenesis was also observed in the in vivo chicken chorioallantoic membrane (CAM) model, and consequently, these new compounds have considerable potential in drug design, e.g. for the treatment of cancer.

Spatial and temporal dynamics of jasmonate synthesis and accumulation in Arabidopsis in response to wounding.

A new metabolite profiling approach combined with an ultrarapid sample preparation procedure was used to study the temporal and spatial dynamics of the wound-induced accumulation of jasmonic acid (JA) and its oxygenated derivatives in Arabidopsis thaliana. In addition to well known jasmonates, including hydroxyjasmonates (HOJAs), jasmonoyl-isoleucine (JA-Ile), and its 12-hydroxy derivative (12-HOJA-Ile), a new wound-induced dicarboxyjasmonate, 12-carboxyjasmonoyl-l-isoleucine (12-HOOCJA-Ile) was discovered. HOJAs and 12-HOOCJA-Ile were enriched in the midveins of wounded leaves, strongly differentiating them from the other jasmonate metabolites studied. The polarity of these oxylipins at physiological pH correlated with their appearance in midveins. When the time points of accumulation of different jasmonates were determined, JA levels were found to increase within 2-5 min of wounding. Remarkably, these changes occurred throughout the plant and were not restricted to wounded leaves. The speed of the stimulus leading to JA accumulation in leaves distal to a wound is at least 3 cm/min. The data give new insights into the spatial and temporal accumulation of jasmonates and have implications in the understanding of long-distance wound signaling in plants.

Synthesis of novel biomaterials in plants.

Metabolic engineering of plants allows the possibility of using crops for the synthesis of novel polymers having useful material properties. Strong and flexible protein-based polymers, which are based on the structure of silk and elastin have been synthesized in transgenic plants. A wide range of polyhydroxyalkanoates having properties ranging from stiff plastics to soft elastomers and glues have been synthesized in various compartments of plants, such as the cytoplasm, plastid and peroxisome. These plant biomaterials could replace, in part, the synthetic plastics, fibers and elastomers produced from petroleum, thus offering the advantage of renewability, sustainability and biodegradability.