Distribution of Glut1 in detergent-resistant membranes (DRMs) and non-DRM domains: effect of treatment with azide

We have previously shown that the acute stimulation of glucose transport in Clone 9 cells in response to azide is mediated by activation of Glut1 and that stomatin, a Glut1-binding protein, appears to inhibit Glut1 function. In Clone 9 cells under basal conditions, 38% of Glut1, 70% of stomatin, and the bulk of caveolin-1 was localized in the detergent-resistant membrane (DRM) fraction; a significant fraction of Glut1 is also present in DRMs of 3T3-L1 fibroblasts and human red blood cells (RBCs). Acute exposure to azide resulted in 40 and 50% decreases in the content of Glut1 in DRMs of Clone 9 cells and 3T3-L1 fibroblasts, respectively, whereas the distribution of stomatin and caveolin-1 in Clone 9 cells remained unchanged. In addition, treatment of Clone 9 cells with azide resulted in a 50% decrease in the content of Glut1 in the DRM fraction of plasma membranes. We conclude that 1) a significant fraction of Glut1 is localized in DRMs, and 2) treatment of cells with azide results in a partial redistribution of Glut1 out of the DRM fraction. stomatin; caveolin-1; transferrin receptor; sucrose density fractionation; lipid raft     

Cannabinoids attenuate norepinephrine-induced melatonin biosynthesis in the rat pineal gland by reducing arylalkylamine N-acetyltransferase activity without involvement of cannabinoid receptors

Cannabinoids modulate neuronal and neuroendocrine circuits by binding to cannabinoid receptors acting upon cAMP/Ca(2+)-mediated intracellular signaling cascades. The rat pineal represents an established model to investigate intracellular signaling processes because a well defined input, the neurotransmitter norepinephrine, is transformed via cAMP/Ca(2+)-dependent mechanisms into an easily detectable output signal, the biosynthesis of melatonin. Here we investigated the impact of cannabinoids on norepinephrine-regulated melatonin biosynthesis in the rat pineal. We demonstrated that treatment of cultured rat pineals with 9-carboxy-11-nor-delta-9-tetrahydrocannabinol (THC), cannabidiol or cannabinol significantly reduced norepinephrine-induced arylalkylamine N-acetyltransferase (AANAT) activity and melatonin biosynthesis. These effects were not mimicked by the cannabinoid receptor agonist WIN55,212-2 and were not blocked by cannabinoid 1 and 2 receptor antagonists. The cannabinoids used did not affect norepinephrine-induced increases in cAMP/Ca(2+) levels. Notably, cannabinoids were found to directly inhibit AANAT activity in lysates of the pineal gland. This effect was specific in so far as cannabinoids did not influence the activity of hydroxyindole-O-methyltransferase (HIOMT), the last enzyme in melatonin biosynthesis. Taken together, our data strongly suggest that cannabinoids inhibit AANAT activity and attenuate melatonin biosynthesis through intracellular actions without involvement of classical cannabinoid receptor-dependent signaling cascades.     

Glucose uptake and lactate production in cells exposed to CoCl(2) and in cells overexpressing the Glut-1 glucose transporter

Glut-1-mediated glucose transport is augmented in response to a variety of conditions and stimuli. In this study we examined the metabolic fate of glucose in cells in which glucose transport is stimulated by exposure to CoCl(2), an agent that stimulates the expression of a set of hypoxia-responsive genes including several glycolytic enzymes and the Glut-1 glucose transporter. Similarly, we determined the metabolic fate of glucose in stably transfected cells overexpressing Glut-1. Exposure of Clone 9 liver cell line, 3T3-L1 fibroblasts, and C(2)C(12) myoblasts to CoCl(2) resulted in an increase glucose uptake and in the activity of glucose phosphorylation ("hexokinase") and lactate dehydrogenase. In cells treated with CoCl(2), the net increase in glucose taken up was accounted for by its near-complete conversion to lactate. Cells stably transfected to overexpress Glut-1 also exhibited enhanced net uptake of glucose with the near-complete conversion of the increased glucose taken up to lactate; however, the effect in these cells was observed in the absence of any change in the activity of two glycolytic enzymes examined. These findings suggest that in cells in which glucose transport is rate-limiting for glucose metabolism, enhancement of the glucose entry step per se results in a near-complete conversion of the extra glucose to lactate.     

Structural studies of SNARE complex and its interaction with complexin by molecular dynamics simulation

Since neurotransmitter releasing into the synaptic space delivers electrical signals from presynaptic neural cell to the postsynaptic cell, neurotransmitter secretion must be much orchestrated. Crowded intracellular vesicles involving neurotransmitters present a question of the how secretory vesicles fuse onto the plasma membrane in a fast synchronized fashion. Complexin is one of the most experimentally studied proteins that regulate assembly of fusogenic four‐helix SNARE complex to synchronized neurotransmitter secretion. We used MD simulation to investigate the interaction of complexin with the neural SNARE complex in detail. Our results show that the SNARE complex interacts with the complexin central helix by forming salt bridges and hydrogen bonds. Complexin also can interact with the Q‐SNARE complex instead of synaptobrevin to decrease the Q‐SNARE flexibility. The complexin alpha‐accessory helix and the C‐terminal region of synaptobrevin can interact with the same region of syntaxin. Although the alpha‐accessory helix aids the tight binding of the central helix to the SNARE complex, its proximity with synaptobrevin causes the destabilization of syntaxin and Sn1 helices. This study suggests that the alpha‐accessory helix of complexin can be an inhibiting factor for membrane fusion by competing with synaptobrevin for binding to the Q‐SNARE complex. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 560–570, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Biophysical Optimization of a Therapeutic Protein by Nonstandard Mutagenesis: STUDIES OF AN IODO-INSULIN DERIVATIVE*

Insulin provides a model for the therapeutic application of protein engineering. A paradigm in molecular pharmacology was defined by design of rapid-acting insulin analogs for the prandial control of glycemia. Such analogs, a cornerstone of current diabetes regimens, exhibit accelerated subcutaneous absorption due to more rapid disassembly of oligomeric species relative to wild-type insulin. This strategy is limited by a molecular trade-off between accelerated disassembly and enhanced susceptibility to degradation. Here, we demonstrate that this trade-off may be circumvented by nonstandard mutagenesis. Our studies employed Lys^B28, Pro^B29-insulin (“lispro”) as a model prandial analog that is less thermodynamically stable and more susceptible to fibrillation than is wild-type insulin. We have discovered that substitution of an invariant tyrosine adjoining the engineered sites in lispro (Tyr^B26) by 3-iodo-Tyr (i) augments its thermodynamic stability (ΔΔG_u 0.5 ±0.2 kcal/mol), (ii) delays onset of fibrillation (lag time on gentle agitation at 37 °C was prolonged by 4-fold), (iii) enhances affinity for the insulin receptor (1.5 ± 0.1-fold), and (iv) preserves biological activity in a rat model of diabetes mellitus. ¹H NMR studies suggest that the bulky iodo-substituent packs within a nonpolar interchain crevice. Remarkably, the 3-iodo-Tyr^B26 modification stabilizes an oligomeric form of insulin pertinent to pharmaceutical formulation (the R₆ zinc hexamer) but preserves rapid disassembly of the oligomeric form pertinent to subcutaneous absorption (T₆ hexamer). By exploiting this allosteric switch, 3-iodo-Tyr^B26-lispro thus illustrates how a nonstandard amino acid substitution can mitigate the unfavorable biophysical properties of an engineered protein while retaining its advantages.     

Optimization and characterization of walnut beverage emulsions in relation to their composition and structure

A central composite rotatable design (CCRD) was used to evaluate the effects of walnut oil (WO, 3-6%, w/w) and gum arabic (GA, 5-10%, w/w) on the average droplet size (D(32)), specific surface area (SSA), polydispersity index (span), apparent viscosity, interfacial tension and opacity of walnut-beverage emulsions. The response surface methodology (RSM) showed that the significant second-order polynomial regression equations with high R(2) (>0.95) were successfully fitted for all responses as function of independent variables. The linear effect of WO had a significant term in all reduced models. The overall optimum region was found to be at the combined level of 10% (w/w) GA content and 5.84% (w/w) WO concentration. At this optimum point, D(32), SSA, span, apparent viscosity, interfacial tension and opacity of emulsions were 0.609 μm, 8.236 m(2)/ml, 0.886, 1.336 Pa s, 51.37 mN/m and 0.810, respectively. No significant (p>0.05) difference was found between the actual values and predicted values. Moreover, principal component analysis (PCA), conducted via PCA variable loadings and cluster dendrogram was able to discriminate the emulsions with different formulations into separate classes.     

Characterisation of transverse slice culture preparations of postnatal rat spinal cord: preservation of defined neuronal populations

Spinal cord injury induces degenerative and regenerative processes and complex interactions of neurons with non-neuronal cells. In order to develop an in vitro tool for the investigation of such processes, we prepared and characterised spinal cord slice cultures (SCSC) from Wistar rats (p0–12). SCSC were sustained in vitro up to 12 days and characterised by immunohistochemistry. Calbindin⁺ neurons, distributed across the entire gray matter, were visible also after longer culture periods. NeuN⁺ neurons were best preserved in the dorsal horn whereas large NeuN⁺ and choline acetyltransferase⁺ motoneurons in the ventral horn vanished after 3 days in vitro. Nestin immunoreactivity was found in animals of all age groups, either in cells interspersed in the ependymal lining around the central canal or in cells resembling protoplasmic astrocytes. Glial fibrillary acidic protein⁺ astrocytes, initially restricted to the white matter, invaded the gray matter of SCSC early during the culture period. Microglial cells, stained by Griffonia simplicifolia isolectin B₄, were rapidly activated in the dorsal tract and in the gray matter but declined in number with time. SCSC derived from p0 or p3 animals showed a better preservation of the cytoarchitecture than cultures derived from older animals. In summary, SCSC undergo degenerative changes, but they contain defined neuronal populations, the cytoarchitecture is partially preserved and the glial reaction is limited.     

Insect small nuclear RNA gene promoters evolve rapidly yet retain conserved features involved in determining promoter activity and RNA polymerase specificity

In animals, most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II (Pol II), but U6 snRNA is synthesized by RNA polymerase III (Pol III). In Drosophila melanogaster, the promoters for the Pol II-transcribed snRNA genes consist of approximately 21 bp PSEA and approximately 8 bp PSEB. U6 genes utilize a PSEA but have a TATA box instead of the PSEB. The PSEAs of the two classes of genes bind the same protein complex, DmSNAPc. However, the PSEAs that recruit Pol II and Pol III differ in sequence at a few nucleotide positions that play an important role in determining RNA polymerase specificity. We have now performed a bioinformatic analysis to examine the conservation and divergence of the snRNA gene promoter elements in other species of insects. The 5' half of the PSEA is well-conserved, but the 3' half is divergent. Moreover, within each species positions exist where the PSEAs of the Pol III-transcribed genes differ from those of the Pol II-transcribed genes. Interestingly, the specific positions vary among species. Nevertheless, we speculate that these nucleotide differences within the 3' half of the PSEA act similarly to induce conformational alterations in DNA-bound SNAPc that result in RNA polymerase specificity.     

Effect of culture conditions on canthaxanthin production by Dietzia natronolimnaea HS-1

This study investigated the effects of various culture parameters (carbon sources, temperature, initial pH of culture, NaCl concentration, and light) on the growth and canthaxanthin production by Dietzia natronolimnaea HS-1. The results showed that the most effective carbon source for growth and canthaxantin production was glucose, and the best pH and temperature were 7 and 31 degrees C, respectively. In addition, the biomass and canthaxanthin production increased in a medium without NaCl and in the presence of light. Under the optimized conditions, the maximum biomass, total carotenoid, and canthaxanthin production were 6.12 +/- 0.21 g/l, 4.51 +/- 0.20 mg/l, and 4.28 +/- 0.15 mg/l, respectively, in an Erlenmeyer flask system, yet increased to 7.25 g/l, 5.48 mg/l, and 5.29 mg/l, respectively, in a batch fermenter system.