Quantification of isotope encoded proteins in 2-D gels using surface enhanced resonance Raman

A strategy for quantification of multiple protein isoforms from a complex sample background is demonstrated, combining isotopomeric rhodamine 6G (R6G) labels and surface-enhanced Raman in polyacrylamide matrix. The procedure involves isotope-encoding by lysine-labeling with (R6G) active ester reagents, isoform separation by 2-DGE, fluorescence quantification using internal standardization to water, and silver nanoparticle deposition followed by surface-enhanced Raman detection. R6G sample encoding and standardization enabled the determination of total protein concentration and the distribution of specific isoforms using the combined detection approach of water-referenced fluorescence spectral imaging and ratiometric quantification. A detection limit of approximately 13.5 picomolar R6G-labeled protein was determined for the surface-enhanced Raman in a gel matrix (15-fold lower than fluorescence). High quantification accuracies for small differences in protein populations at low nanogram abundance were demonstrated for human GMP synthetase (hGMPS) either as purified protein samples in a single-point determination mode (3% relative standard deviation, RSD%) or as HCT116 human cancer cellular lysate in an imaging application (with 16% RSD%). These results represent a prototype for future applications of isotopic surface-enhanced resonance Raman scatter to quantification of protein distributions.     

Regulation of lipolytic activity by long-chain acyl-coenzyme A in islets and adipocytes

Intracellular lipolysis is a major pathway of lipid metabolism that has roles, not only in the provision of free fatty acids as energy substrate, but also in intracellular signal transduction. The latter is likely to be particularly important in the regulation of insulin secretion from islet beta-cells. The mechanisms by which lipolysis is regulated in different tissues is, therefore, of considerable interest. Here, the effects of long-chain acyl-CoA esters (LC-CoA) on lipase activity in islets and adipocytes were compared. Palmitoyl-CoA (Pal-CoA, 1-10 microM) stimulated lipase activity in islets from both normal and hormone-sensitive lipase (HSL)-null mice and in phosphatase-treated islets, indicating that the stimulatory effect was neither on HSL nor phosphorylation dependent. In contrast, we reproduced the previously published observations showing inhibition of HSL activity by LC-CoA in adipocytes. The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue relationship between islets and adipocytes with respect to fatty acid metabolism, LC-CoA signaling, and lipolysis. Elevated LC-CoA in islets stimulates lipolysis to generate a signal to increase insulin secretion, whereas elevated LC-CoA in adipocytes inhibits lipolysis. Together, these opposite actions of LC-CoA lower circulating fat by inhibiting its release from adipocytes and promoting fat storage via insulin action.     

BAF57 governs androgen receptor action and androgen-dependent proliferation through SWI/SNF

Androgen receptor (AR) activity is required for prostate cancer development and progression. Thus, there is a major impetus to understand the regulation of AR action. We and others have previously shown that AR transactivation potential is dependent on the presence of an active SWI/SNF chromatin remodeling complex. However, the mechanisms underlying SWI/SNF regulation of the AR remained unsolved. We show here that the BAF57 subunit, an accessory component of the remodeling complex, is a critical regulator of AR function. We show that BAF57 is expressed in the luminal epithelia of the prostate and is required for AR-dependent transactivation in prostatic adenocarcinoma cells. Our data reveal that BAF57 can directly bind to the AR and is recruited to endogenous AR targets upon ligand activation. Loss of BAF57 or inhibition of BAF57 function severely compromised AR activity, as observed with both exogenous and endogenous AR targets. Rescue of BAF57 function restored AR activity, thus demonstrating a specific requirement of BAF57 for AR activity. This action of BAF57 proved to be dependent on SWI/SNF ATPase function. BAF57 has previously been implicated in nuclear receptor coactivator function, and we show that, although BAF57 facilitated coactivator activity, only a selected subset required BAF57 for coactivator function. Lastly, we demonstrate that both BAF57 and BRM are required for the proliferation of AR-dependent prostatic adenocarcinoma cells. In summary, these findings identify BAF57 as a critical modulator of the AR that is capable of altering AR activity, coactivator function, and AR-dependent proliferation.     

Effect of N-cadherin misexpression by the mammary epithelium in mice

N-cadherin is not typically expressed by epithelial cells. However, it is detected in breast cancers and increases tumor cell migration and invasion in vitro. To explore its misexpression, we generated transgenic mice with N-cadherin in the mammary epithelium. Mammary glands appeared normal and no tumors arose spontaneously. To investigate N-cadherin misexpression in mammary tumors, neu was overexpressed through breeding. Tumors developed in +/neu and N-cadherin/neu mice, although few tumors in bitransgenic mice expressed N-cadherin, and they did not differ from N-cadherin-negative tumors.     

Development and application of a brain-specific cDNA microarray for effect evaluation of neuro-active pharmaceuticals in zebrafish (Danio rerio)

The environmental fate and ecotoxicological effect of pharmaceuticals are poorly understood, and standardized tests to detect and evaluate their potential effects in the environment are not available. We developed a zebrafish brain-specific microarray containing 682 neurologically relevant cDNA-fragments. To investigate the applicability of this microarray for studying neurotoxic modes-of-action and impact assessment of neuro-active pharmaceuticals in zebrafish, chlorpromazine was used as a model compound. After exposure to chlorpromazine (75 μg/L) for 2, 4, 14 and 28 days or control treatment RNA was extracted from brains of males and females. Fluorescently labeled cDNA was prepared and hybridized to the custom microarray. In total, 56 genes were differentially expressed in brains of male and/or female zebrafish, of which most genes were down-regulated. A clear difference in response to chlorpromazine exposure between males and females was observed with exposure time as well as in functional classes of affected genes. The presented study is one of the first reports on molecular effects of human neuro-active pharmaceuticals in aquatic non-target organisms. This new genomic tool successfully detected gene expression effects of exposure to chlorpromazine in the brain of zebrafish. Reported gene expression effects are found to be consistent with literature data for other laboratory animals.     

Chlamydomonas IFT172 is encoded by FLA11, interacts with CrEB1, and regulates IFT at the flagellar tip

The transport of flagellar precursors and removal of turnover products from the flagellar tip is mediated by intraflagellar transport (IFT) , which is essential for both flagellar assembly and maintenance . Large groups of IFT particles are moved from the flagellar base to the tip by kinesin-2, and smaller groups are returned to the base by cytoplasmic dynein 1b. The IFT particles are composed of two protein complexes, A and B, comprising approximately 16-18 polypeptides. How cargo is unloaded from IFT particles, turnover products loaded, and active IFT motors exchanged at the tip is unknown. We previously showed that the Chlamydomonas microtubule end binding protein 1 (CrEB1) localizes to the flagellar tip and is depleted from the tips of the temperature-sensitive (ts) mutant fla11ts . We demonstrate here that FLA11 encodes IFT protein 172, a component of IFT complex B, and show that IFT172 interacts with CrEB1. Because fla11ts cells are defective in IFT particle turnaround at the tip, our results indicate that IFT172 is involved in regulating the transition between anterograde and retrograde IFT at the tip, perhaps by a mechanism involving CrEB1. Therefore, IFT172 is involved in the control of flagellar assembly/disassembly at the tip.     

Conversion of proteins from a non-polarized to an apical secretory pattern in MDCK cells

Previously it was shown that fusion proteins containing the amino terminus of an apical targeted member of the serpin family fused to the corresponding carboxyl terminus of the non-polarized secreted serpin, antithrombin, are secreted mainly to the apical side of MDCK cells. The present study shows that this is neither due to the transfer of an apical sorting signal from the apically expressed proteins, since a sequence of random amino acids acts the same, nor is it due to the deletion of a conserved signal for correct targeting from the non-polarized secreted protein. Our results suggest that the polarity of secretion is determined by conformational sensitive sorting signals.     

Exploring the GluR2 ligand-binding core in complex with the bicyclical AMPA analogue (S)-4-AHCP

The X-ray structure of the ionotropic GluR2 ligand-binding core (GluR2-S1S2J) in complex with the bicyclical AMPA analogue (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]-4-isoxazolyl)propionic acid [(S)-4-AHCP] has been determined, as well as the binding pharmacology of this construct and of the full-length GluR2 receptor. (S)-4-AHCP binds with a glutamate-like binding mode and the ligand adopts two different conformations. The K(i) of (S)-4-AHCP at GluR2-S1S2J was determined to be 185 +/- 29 nM and at full-length GluR2(R)o it was 175 +/- 8 nM. (S)-4-AHCP appears to elicit partial agonism at GluR2 by inducing an intermediate degree of domain closure (17 degrees). Also, functionally (S)-4-AHCP has an efficacy of 0.38 at GluR2(Q)i, relative to (S)-glutamate. The proximity of bound (S)-4-AHCP to domain D2 prevents full D1-D2 domain closure, which is limited by steric repulsion, especially between Leu704 and the ligand.     

Aging reduces glycerol-3-phosphate acyltransferase activity in activated rat splenic T-lymphocytes

T-lymphocyte proliferation declines with age. Phosphatidic acid (PA) is the precursor to all glycerophospholipids, which serve as important membrane structural components and signaling molecules. Therefore, we tested the hypothesis that aged T-lymphocyte proliferation may be reduced, in part, suppressing phosphatidic acid (PA) biosynthesis. We showed, for the first time, that anti-CD3 stimulation in rat splenic T-lymphocytes selectively increased mitochondrial glycerol-3-phosphate acyltransferase (GPAT) activity. GPAT activity could be further increased by the addition of recombinant acyl-CoA binding protein (rACBP), but the amplification of GPAT activity was blunted by aging. This is important because PA is the precursor lipid for phospholipid synthesis and GPAT is the rate-limiting enzyme in PA biosynthesis. The mechanism by which stimulation and rACBP increased GPAT activity may involve phosphorylation since incubating Jurkat T-lymphocyte mitochondria with casein kinase 2 in vitro significantly increased GPAT activity. The data presented here suggest a novel mechanism by which aging may reduce activation-dependent mitochondrial GPAT activity. This age-induced alteration would result in reduced PA biosynthesis and could explain, in part, the diminished phospholipid content of the membrane and subsequent loss of proliferative capacity in the aged T-lymphocyte.     

The long-acting glucagon-like peptide-1 analogue, liraglutide, inhibits beta-cell apoptosis in vitro

We here show that GLP-1 and the long-acting GLP-1 analogue, liraglutide, interfere with diabetes-associated apoptotic processes in the β-cell. Studies using primary neonatal rat islets showed that native GLP-1 and liraglutide inhibited both cytokine- and free fatty acid-induced apoptosis in a dose-dependent manner. The anti-apoptotic effect of liraglutide was mediated by the GLP-1 receptor as the specific GLP-1 receptor antagonist, exendin(9-39), blocked the effects. The adenylate cyclase activator, forskolin, had an anti-apoptotic effect similar to those of GLP-1 and liraglutide indicating that the effect was cAMP-mediated. Blocking the PI3 kinase pathway using wortmannin but not the MAP kinase pathways by PD98059 inhibited the effects of liraglutide. In conclusion, GLP-1 receptor activation has anti-apoptotic effect on both cytokine, and free fatty acid-induced apoptosis in primary islet-cells, thus suggesting that the long-acting GLP-1 analogue, liraglutide, may be useful for retaining β-cell mass in both type 1 and type 2 diabetic patients.