Striatal pre- and postsynaptic profile of adenosine A(2A) receptor antagonists

Striatal adenosine A(2A) receptors (A(2A)Rs) are highly expressed in medium spiny neurons (MSNs) of the indirect efferent pathway, where they heteromerize with dopamine D(2) receptors (D(2)Rs). A(2A)Rs are also localized presynaptically in cortico-striatal glutamatergic terminals contacting MSNs of the direct efferent pathway, where they heteromerize with adenosine A(1) receptors (A(1)Rs). It has been hypothesized that postsynaptic A(2A)R antagonists should be useful in Parkinson's disease, while presynaptic A(2A)R antagonists could be beneficial in dyskinetic disorders, such as Huntington's disease, obsessive-compulsive disorders and drug addiction. The aim or this work was to determine whether selective A(2A)R antagonists may be subdivided according to a preferential pre- versus postsynaptic mechanism of action. The potency at blocking the motor output and striatal glutamate release induced by cortical electrical stimulation and the potency at inducing locomotor activation were used as in vivo measures of pre- and postsynaptic activities, respectively. SCH-442416 and KW-6002 showed a significant preferential pre- and postsynaptic profile, respectively, while the other tested compounds (MSX-2, SCH-420814, ZM-241385 and SCH-58261) showed no clear preference. Radioligand-binding experiments were performed in cells expressing A(2A)R-D(2)R and A(1)R-A(2A)R heteromers to determine possible differences in the affinity of these compounds for different A(2A)R heteromers. Heteromerization played a key role in the presynaptic profile of SCH-442416, since it bound with much less affinity to A(2A)R when co-expressed with D(2)R than with A(1)R. KW-6002 showed the best relative affinity for A(2A)R co-expressed with D(2)R than co-expressed with A(1)R, which can at least partially explain the postsynaptic profile of this compound. Also, the in vitro pharmacological profile of MSX-2, SCH-420814, ZM-241385 and SCH-58261 was is in accordance with their mixed pre- and postsynaptic profile. On the basis of their preferential pre- versus postsynaptic actions, SCH-442416 and KW-6002 may be used as lead compounds to obtain more effective antidyskinetic and antiparkinsonian compounds, respectively.     

Identification of enhanced serine kinase activity in insulin resistance

Insulin receptor substrate (IRS) proteins play a crucial role as signaling molecules in insulin action. Serine phosphorylation of IRS proteins has been hypothesized as a cause of attenuating insulin signaling. The current study investigated serine kinase activity toward IRS-1 in several models of insulin resistance. An in vitro kinase assay was developed that used partially purified cell lysates as a kinase and glutathione S-transferase fusion proteins that contained various of IRS-1 fragments as substrates. Elevated serine kinase activity was detected in Chinese hamster ovary/insulin receptor (IR)/IRS-1 cells and 3T3-L1 adipocytes chronically treated with insulin, and in liver and muscle of obese JCR:LA-cp rats. It phosphorylated the 526-859 amino acid region of IRS-1, whereas phosphorylation of the 2-516 and 900-1235 amino acid regions was not altered. Phosphopeptide mapping of the 526-859 region of IRS-1 showed three major phosphopeptides (P1, P2, and P3) with different patterns of phosphorylation depending on the source of serine kinase activity. P1 and P2 were strongly phosphorylated when the kinase activity was prepared from insulin-resistant Chinese hamster ovary/IR/IRS-1 cells, weakly phosphorylated by the kinase activity from insulin-resistant 3T3-L1 adipocytes, and barely phosphorylated when the extract was derived from insulin-resistant liver. In contrast, P3 was phosphorylated by the serine kinase activity prepared from all insulin-resistant cells and tissues of animals. P1 and P2 phosphorylation can be explained by mitogen-activated protein kinase activity based on the phosphopeptide map generated by recombinant ERK2. In contrast, mitogen-activated protein kinase failed to phosphorylate the P3 peptide, suggesting that another serine kinase regulates this modification of IRS-1 in insulin-resistant state.     

Inducible overexpression of the FUM1 gene in Saccharomyces cerevisiae: localization of fumarase and efficient fumaric acid bioconversion to L-malic acid

Cloning of the Saccharomyces cerevisiae FUM1 gene downstream of the strong GAL10 promoter resulted in inducible overexpression of fumarase in the yeast. The overproducing strain exhibited efficient bioconversion of fumaric acid to L-malic acid with an apparent conversion value of 88% and a conversion rate of 80.4 mmol of fumaric acid/h per g of cell wet weight, both of which are much higher than parameters known for industrial bacterial strains. The only product of the conversion reaction was L-malic acid, which was essentially free of the unwanted by-product succinic acid. The GAL10 promoter situated upstream of a promoterless FUM1 gene led to production and correct distribution of the two fumarase isoenzyme activities between cytosolic and mitochondrial subcellular fractions. The amino-terminal sequence of fumarase contains the mitochondrial signal sequence since (i) 92 of 463 amino acid residues from the amino terminus of fumarase are sufficient to localize fumarase-lacZ fusions to mitochondria and (ii) fumarase and fumarase-lacZ fusions lacking the amino-terminal sequence are localized exclusively in the cytosol. The possibility that both mitochondrial and cytosolic fumarases are derived from the same initial translation product is discussed.     

Assistance of maltose binding protein to the in vivo folding of the disulfide-rich C-terminal fragment from Plasmodium falciparum merozoite surface protein 1 expressed in Escherichia coli

The C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 (F19) is a leading candidate for the development of a malaria vaccine. Successful vaccination trials on primates, immunochemistry, and structural studies have shown the importance of its native conformation for its protective role against infection. F19 is a disulfide-rich protein, and the correct pairing of its 12 half-cystines is required for the native state of the protein. F19 has been produced in the Escherichia coli periplasm, which has an oxidative environment favorable for the formation of disulfide bonds. F19 was either expressed as a fusion with the maltose binding protein (MBP) or directly addressed to the periplasm by fusing it with the MBP signal peptide. Direct expression of F19 in the periplasm led to a misfolded protein with a heterogeneous distribution of disulfide bridges. On the contrary, when produced as a fusion protein with E. coli MBP, the F19 moiety was natively folded. Indeed, after proteolysis of the fusion protein, the resulting F19 possesses the structural characteristics and the immunochemical reactivity of the analogous fragment produced either in baculovirus-infected insect cells or in yeast. These results demonstrate that the positive effect of MBP in assisting the folding of passenger proteins extends to the correct formation of disulfide bridges in vivo. Although proteins or protein fragments fused to MBP have been frequently expressed with success, our comparative study evidences for the first time the helping property of MBP in the oxidative folding of a disulfide-rich protein.     

Impacts and Recovery from Severe Tropical Cyclone Yasi on the Great Barrier Reef

Full recovery of coral reefs from tropical cyclone (TC) damage can take decades, making cyclones a major driver of habitat condition where they occur regularly. Since 1985, 44 TCs generated gale force winds (≥17 metres/second) within the Great Barrier Reef Marine Park (GBRMP). Of the hurricane strength TCs (≥H1—Saffir Simpson scale; ≥ category 3 Australian scale), TC Yasi (February, 2011) was the largest. In the weeks after TC Yasi crossed the GBRMP, participating researchers, managers and rangers assessed the extent and severity of reef damage via 841 Reef Health and Impact Surveys at 70 reefs. Records were scaled into five damage levels representing increasingly widespread colony-level damage (1, 2, 3) and reef structural damage (4, 5). Average damage severity was significantly affected by direction (north vs south of the cyclone track), reef shelf position (mid-shelf vs outer-shelf) and habitat type. More outer-shelf reefs suffered structural damage than mid-shelf reefs within 150 km of the track. Structural damage spanned a greater latitudinal range for mid-shelf reefs than outer-shelf reefs (400 vs 300 km). Structural damage was patchily distributed at all distances, but more so as distance from the track increased. Damage extended much further from the track than during other recent intense cyclones that had smaller circulation sizes. Just over 15% (3,834 km²) of the total reef area of the GBRMP is estimated to have sustained some level of coral damage, with ~4% (949 km²) sustaining a degree of structural damage. TC Yasi likely caused the greatest loss of coral cover on the GBR in a 24-hour period since 1985. Severely impacted reefs have started to recover; coral cover increased an average of 4% between 2011 and 2013 at re-surveyed reefs. The in situ assessment of impacts described here is the largest in scale ever conducted on the Great Barrier Reef following a reef health disturbance.     

Enamel matrix derivative is a potent inhibitor of breast cancer cell attachment to bone

This study examined whether enamel matrix derivative (EMD) inhibits the adhesion of cancer cells to bone. A typical breast cancer cell line, MCF-7, was used. Conditioned human osteosarcoma cell (Saos-2) medium was used as extracellular bone matrix (ECBM) to measure cell attachment. MCF-7 cells were incubated on ECBM-coated culture plates with or without soluble EMD, Arg-Gly-Asp (RGD) sequence blocking peptides, recombinant bone sialoprotein (rBSP), or specific integrin antibodies, and the attached cells were quantified using toluidine blue staining. EMD markedly reduced the attachment of MCF-7 cells to ECBM in a dose-dependent manner. An RGD peptide (GRGDSP) and recombinant BSP inhibited cell attachment to the same degree as EMD. Similarly, anti-alphavbeta3 integrin antibody strongly reduced cell attachment, whereas anti-alphavbeta5 and anti-beta1 integrin antibodies had less marked effects on cell attachment. These results show that EMD inhibits MCF-7 cell attachment to a bone matrix and that it might be useful as an anti-adhesive agent for breast cancer cells to bone in vivo.     

Improving evolutionary models of protein interaction networks

MOTIVATION: Theoretical models of biological networks are valuable tools in evolutionary inference. Theoretical models based on gene duplication and divergence provide biologically plausible evolutionary mechanics. Similarities found between empirical networks and their theoretically generated counterpart are considered evidence of the role modeled mechanics play in biological evolution. However, the method by which these models are parameterized can lead to questions about the validity of the inferences. Selecting parameter values in order to produce a particular topological value obfuscates the possibility that the model may produce a similar topology for a large range of parameter values. Alternately, a model may produce a large range of topologies, allowing (incorrect) parameter values to produce a valid topology from an otherwise flawed model. In order to lend biological credence to the modeled evolutionary mechanics, parameter values should be derived from the empirical data. Furthermore, recent work indicates that the timing and fate of gene duplications are critical to proper derivation of these parameters. RESULTS: We present a methodology for deriving evolutionary rates from empirical data that is used to parameterize duplication and divergence models of protein interaction network evolution. Our method avoids shortcomings of previous methods, which failed to consider the effect of subsequent duplications. From our parameter values, we find that concurrent and existing existing duplication and divergence models are insufficient for modeling protein interaction network evolution. We introduce a model enhancement based on heritable interaction sites on the surface of a protein and find that it more closely reflects the high clustering found in the empirical network.     

Opposite modulatory roles for adenosine A1 and A2A receptors on glutamate and dopamine release in the shell of the nucleus accumbens. Effects of chronic caffeine exposure

Previous studies have demonstrated opposing roles for adenosine A1 and A2A receptors in the modulation of extracellular levels of glutamate and dopamine in the striatum. In the present study, acute systemic administration of motor-activating doses of the A2A receptor antagonist MSX-3 significantly decreased extracellular levels of dopamine and glutamate in the shell of the rat nucleus accumbens (NAc) and counteracted both dopamine and glutamate release induced by systemic administration of motor-activating doses of either the A1 receptor antagonist CPT or caffeine. Furthermore, exposure to caffeine in the drinking water (1 mg/mL, 14 days) resulted in tolerance to the effects of systemic injection of CPT or caffeine, but not MSX-3, on extracellular levels of dopamine and glutamate in the NAc shell. The present results show: first, the existence of opposite tonic effects of adenosine on extracellular levels of dopamine and glutamate in the shell of the NAc mediated by A1 and A2A receptors; second, that complete tolerance to caffeine's dopamine- and glutamate-releasing effects which develops after chronic caffeine exposure is attributable to an A1 receptor-mediated mechanism. Development of tolerance to the dopamine-releasing effects of caffeine in the shell of the NAc may explain its weak addictive properties and atypical psychostimulant profile.     

Mitochondrial dysfunction and oxidative damage in parkin-deficient mice

Loss-of-function mutations in parkin are the predominant cause of familial Parkinson's disease. We previously reported that parkin-/- mice exhibit nigrostriatal deficits in the absence of nigral degeneration. Parkin has been shown to function as an E3 ubiquitin ligase. Loss of parkin function, therefore, has been hypothesized to cause nigral degeneration via an aberrant accumulation of its substrates. Here we employed a proteomic approach to determine whether loss of parkin function results in alterations in abundance and/or modification of proteins in the ventral midbrain of parkin-/- mice. Two-dimensional gel electrophoresis followed by mass spectrometry revealed decreased abundance of a number of proteins involved in mitochondrial function or oxidative stress. Consistent with reductions in several subunits of complexes I and IV, functional assays showed reductions in respiratory capacity of striatal mitochondria isolated from parkin-/- mice. Electron microscopic analysis revealed no gross morphological abnormalities in striatal mitochondria of parkin-/- mice. In addition, parkin-/- mice showed a delayed rate of weight gain, suggesting broader metabolic abnormalities. Accompanying these deficits in mitochondrial function, parkin-/- mice also exhibited decreased levels of proteins involved in protection from oxidative stress. Consistent with these findings, parkin-/- mice showed decreased serum antioxidant capacity and increased protein and lipid peroxidation. The combination of proteomic, genetic, and physiological analyses reveal an essential role for parkin in the regulation of mitochondrial function and provide the first direct evidence of mitochondrial dysfunction and oxidative damage in the absence of nigral degeneration in a genetic mouse model of Parkinson's disease.