Glucagon-like peptide-1 receptor signaling modulates beta cell apoptosis

Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and augments beta cell mass via activation of beta cell proliferation and islet neogenesis. We examined whether GLP-1 receptor signaling modifies the cellular susceptibility to apoptosis. Mice administered streptozotocin (STZ), an agent known to induce beta cell apoptosis, exhibit sustained improvement in glycemic control and increased levels of plasma insulin with concomitant administration of the GLP-1 agonist exendin-4 (Ex-4). Blood glucose remained significantly lower for weeks after cessation of exendin-4. STZ induced beta cell apoptosis, which was significantly reduced by co-administration of Ex-4. Conversely, mice with a targeted disruption of the GLP-1 receptor gene exhibited increased beta cell apoptosis after STZ administration. Exendin-4 directly reduced cytokine-induced apoptosis in purified rat beta cells exposed to interleukin 1beta, tumor necrosis fator alpha, and interferon gamma in vitro. Furthermore, Ex-4-treated BHK-GLP-1R cells exhibited significantly increased cell viability, reduced caspase activity, and decreased cleavage of beta-catenin after treatment with cycloheximide in vitro. These findings demonstrate that GLP-1 receptor signaling directly modifies the susceptibility to apoptotic injury, and provides a new potential mechanism linking GLP-1 receptor activation to preservation or enhancement of beta cell mass in vivo.     

Reproductive fitness and quinone content of Caenorhabditis elegans clk-1 mutants fed coenzyme Q isoforms of varying length

Caenorhabditis elegans clk-1 mutants lack coenzyme Q9 and accumulate the biosynthetic intermediate demethoxy-Q9. A dietary source of ubiquinone (Q) is required for larval growth and development of the gonad and germ cells. We considered that uptake of the shorter Q8 isoform present in the Escherichia coli food may contribute to the Clk phenotypes of slowed development and reduced brood size observed when the animals are fed Q-replete E. coli. To test the effect of isoprene tail length, N2 and clk-1 animals were fed E. coli engineered to produce Q7, Q8, Q9, or Q10. Wild-type nematodes showed no change in reproductive fitness regardless of the Qn isoform fed. clk-1(e2519) fed the Q9 diet showed increased egg production; however, this diet did not improve reproductive fitness of the clk-1(qm30) animals. Furthermore, animals with the more severe clk-1(qm30) allele become sterile and their progeny inviable when fed Q7-containing bacteria. The content of Q7 in the mitochondria of clk-1 animals was decreased relative to Q8, suggesting less effective transport of Q7 to the mitochondria, impaired retention, or decreased stability. Additionally, regardless of E. coli diet, clk-1(qm30) animals contain a dysfunctional dense form of mitochondria. The gonads of clk-1(qm30) worms fed Q7-containing food were severely shrunken and disordered. The differential fertility of clk-1 mutant nematodes fed Q isoforms may result from changes in Q localization, altered recognition by Q-binding proteins, and/or potential defects in mitochondrial function resulting from the mutant CLK-1 polypeptide itself.     

Isolation and characterization of unsaturated fatty acids as natural ligands for the retinoid-X receptor

The retinoid-X receptor (RXR) is a ligand activated nuclear receptor that is the heterodimer partner for many class II nuclear receptors. Previously identified natural ligands for this receptor include 9-cis retinoic acid (9cRA), docosahexaenoic acid, and phytanic acid. Our studies were performed to determine if there are any unidentified, physiologically important RXR ligands. Agonists for RXR were purified from rat heart and testes lipid extracts with the use of a cell-based reporter assay to monitor RXR activation. Purified active fractions contained a variety of unsaturated fatty acids and components were quantified by gas-liquid chromatography of derivatized samples. The corresponding fatty acid standards elicited a similar response in the reporter cell assay. Competition binding analysis revealed that the active fatty acids compete with [3H]9cRA for binding to RXR. Non-esterified fatty acids were analyzed from lipid extracts of isolated heart and testes nuclei and endogenous concentrations were found to be within the range of their determined binding affinities. Our studies reveal tissue dependent profiles of RXR agonists and support the idea of unsaturated fatty acids as physiological ligands of RXR.     

Oxidase domains in epothilone and bleomycin biosynthesis: thiazoline to thiazole oxidation during chain elongation

The natural products epothilone and bleomycin are assembled by hybrid polyketide/nonribosomal peptide synthetases. Of note in these assembly lines is the conversion of internal cysteine residues into thiazolines and their subsequent oxidation to heteroaromatic thiazole rings. We have excised the EpoB oxidase domain, EpoB-Ox, proposed to be responsible for thiazoline to thiazole oxidation in epothilone biosynthesis, and expressed it in soluble form in Escherichia coli. The purified domain is an FMN-containing flavoprotein that demonstrates thiazoline to thiazole oxidase activity when incubated with thioester substrate mimics. Kinetic parameters were determined for both thiazoline and oxazoline substrates, with k(cat) values ranging between 48.8 and 0.55 min(-1). While the physiological electron acceptor is not yet known, molecular oxygen is needed in these in vitro assays to mediate reoxidation of reduced FMN. Additionally, the oxidase domain-containing BlmIII from the bleomycin assembly line was heterologously expressed and purified. BlmIII is also an FMN-containing protein with activity similar to EpoB-Ox. This work marks the first direct characterization of nonribosomal peptide synthetase oxidase domain activity and will lead to further exploration of these flavoproteins.     

Introduction of a mutation in the shutter region of antithrombin (Phe77 --> Leu) increases affinity for heparin and decreases thermal stability

The shutter region of serpins consists of a number of highly conserved residues that are critical for both stability and function. Several variants of antithrombin with substitutions in this region are unstable and predispose the carrier to thrombosis. Although most mutations in the shutter region investigated to date are deleterious with respect to serpin stability and function, the substitution of Phe51 by Leu in alpha(1)-antitrypsin results in enhanced stability. Here, we have investigated the effects of introducing an analogous mutation into antithrombin (Phe 77 to Leu). The mutation did not affect the kinetics of interaction with proteases. Strikingly, however, the thermostability of the protein was markedly decreased, with the serpin displaying a 13 degrees C decrease in melting temperature as compared to wild-type recombinant antithrombin. Further studies revealed that in contrast to wild-type antithrombin, the mutant adopted the latent (inactive) conformation upon mild heating. Previous studies on shutter region mutations that destabilize antithrombin revealed that such variants possess enhanced affinity for both heparin pentasaccharide and full-length heparin. The N135A/F77L mutant had unchanged affinity for heparin pentasaccharide, but the affinity for full-length heparin was increased. We suggest that the Phe77Leu mutation causes conformational changes around the top of the D-helix in antithrombin, in particular, to the arginine 132 and 133 residues that may mediate additional antithrombin/heparin interactions. This paper also demonstrates that there are major differences between the shutter regions of antithrombin and alpha(1)-antitrypsin since a stabilizing mutation in antitrypsin has the converse effect in antithrombin.     

Large-scale propagation of a replication-defective adenovirus vector in stirred-tank bioreactor PER.C6 cell culture under sparging conditions

Large-scale propagation of replication-defective adenovirus vectors has not been well studied to date. One of the challenges for efficient propagation at large scale is to overcome the sensitivity of virus infected cells to gas sparging required for oxygenation and CO(2) removal. In our initial experiments, it was observed that productivity of an adenovirus vector was significantly reduced under sparging conditions as compared to nonsparged, i.e., surface-aerated controls in serum-free cultures. Investigations led to the identification of a buffer containing surfactant (Polysorbate-80, PS-80) that was included in the virus seed stock formulation and introduced through virus infection into the culture at a very low concentration as the cause of the reduced virus productivity. This finding was not obvious and trivial, as neither uninfected sparged nor infected nonsparged PER.C6 trade mark cells in serum-free cultures were affected by the buffer at such a low PS-80 concentration of 0.00025% (v/v), which is a common component of serum-free cell culture media. These results strongly suggest that virus-infected cells behave very differently from uninfected cells under sparging conditions. To mitigate the deleterious effects of sparging, the virus seed stock was prepared in the absence of the buffer containing PS-80. At the same time, the concentration of Pluronic-F68 (PF-68) in the serum-free medium was increased to 1 g/L, at which cell growth and metabolism were unaffected, even though this measure alone did not result in virus productivity improvement. Only by implementing the two measures together was virus productivity loss completely eliminated under sparging conditions. After demonstration of the process robustness in 2-L bioreactors, this adenovirus propagation process was successfully scaled up to 250 L in a 300-L bioreactor under the worst-case sparging conditions projected for 10,000-L scale.     

Biological activity of harpin produced by Pantoea stewartii subsp. stewartii

Pantoea stewartii subsp. stewartii causes Stewart's wilt of sweet corn. A hypersensitive response and pathogenicity (Hrp) secretion system is needed to produce water-soaking and wilting symptoms in corn and to cause a hypersensitive response (HR) in tobacco. Sequencing of the hrp cluster revealed a putative harpin gene, hrpN. The product of this gene was overexpressed in Escherichia coli and shown to elicit the HR in tobacco and systemic resistance in radishes. The protein was designated HrpN(Pnss). Like other harpins, it was heat stable and protease sensitive, although it was three- to fourfold less active biologically than Erwinia amylovora harpin. We used antibodies to purified HrpN(Pnss) to verify that hrpN mutants could not produce harpin. This protein was secreted into the culture supernatant and was produced by strains of P. stewartii subsp. indologenes. In order to determine the importance of HrpN(Pnss) in pathogenesis on sweet corn, three hrpN::Tn5 mutants were compared with the wild-type strain with 50% effective dose, disease severity, response time, and growth rate in planta as parameters. In all tests, HrpN(Pnss) was not required for infection, growth, or virulence in corn or endophytic growth in related grasses.     

Serum-free suspension cultivation of PER.C6(R) cells and recombinant adenovirus production under different pH conditions

PER.C6(R) cell growth, metabolism, and adenovirus production were studied in head-to-head comparisons in stirred bioreactors under different pH conditions. Cell growth rate was found to be similar in the pH range of 7.1-7.6, while a long lag phase and a slower growth rate were observed at pH 6.8. The specific consumption rates of glucose and glutamine decreased rapidly over time during batch cell growth, as did the specific lactate and ammonium production rates. Cell metabolism in both infected and uninfected cultures was very sensitive to culture pH, resulting in dramatic differences in glucose/glutamine consumption and lactate/ammonium production under different pH conditions. It appeared that glucose metabolism was suppressed at low pH but the efficiency of energy production from glucose was enhanced. Adenovirus infection resulted in profound changes in cell growth and metabolism. Cell growth was largely arrested under all pH conditions, while glucose consumption and lactate production were elevated post virus infection. Virus infection induced a reduction in glutamine consumption at low pH but an increase at high pH. The optimal pH for adenovirus production was found to be 7.3 under the experimental conditions used in the study. Deviations from this optimum resulted in significant reductions of virus productivity. The results indicate that culture pH is a very critical process parameter in PER.C6(R) cell culture and adenovirus production.     

How aphids lose their marbles

Insects provide examples of many cunning stratagems to cope with the challenges of living in a world dominated by surface forces. Despite being the current masters of the land environment, they are at constant risk of being entrapped in liquids, which they prevent by having waxy and hairy surfaces. The problem is particularly acute in an enclosed space, such as a plant gall. Using secreted wax to efficiently parcel and transport their own excrement, aphids were able to solve this problem 200 Myr ago. Here, we report on the physical and physiological significance of this ingenious solution. The secreted powdery wax has three distinct roles: (i) it is hydrophobic, (ii) it creates a microscopically rough inner gall surface made of weakly compacted wax needles making the gall ultra-hydrophobic, and (iii) it coats the honeydew droplets converting them into liquid marbles, that can be rapidly and efficiently moved.