Immunological and conformation characterization of a phosphorylated immunodominant epitope on the paired helical filaments found in Alzheimer's disease.

The immunological recognition pattern of one of the most commonly used monoclonal antibodies, PHF-1, which detects the paired helical filaments of Alzheimer's disease, exhibits a high degree of similarity with the recognition of a polyclonal antibody, anti-T3P, raised against a synthetic phosphopeptide, GAEIVYKS(Phospho)PVVSGD, corresponding to amino acids 389-402 of the microtubule-associated protein tau. A panel of 16 synthetic non-phosphorylated and phosphorylated peptides, excised from different regions of tau and peptide analogs thereof, were used to show that PHF-1 is indeed directed against the T3 fragment. Circular dichroism spectroscopy shows that the phosphorylated peptide exhibits a limited propensity to form intramolecular beta-pleated sheets, and alteration is found in the reverse-turn structure that dominates the middle section of the molecule. The shift in the turn-forming amino acids may also allow a stacking procedure, may interfere with microtubule assembly, and, consequently, may be accountable for deposit formation.     

Membrane potential-related effect of calcium on reactive oxygen species generation in isolated brain mitochondria

The effect of Ca²⁺ applied in high concentrations (50 and 300 µM) was addressed on the generation of reactive oxygen species in isolated mitochondria from guinea-pig brain. The experiments were performed in the presence of ADP, a very effective inhibitor of mitochondrial permeability transition. Moderate increase in H₂O₂ release from mitochondria was induced by Ca²⁺ applied in 50 µM, but not in 300 µM concentration as measured with Amplex red fluorescent assay starting with a delay of 100-150 sec after exposure to Ca²⁺. Parallel measurements of membrane potential (ΔΨm) by safranine fluorescence showed a transient depolarization by Ca²⁺ followed by the recovery of ΔΨm to a value, which was more negative than that observed before addition of Ca²⁺ indicating a relative hyperpolarization. NAD(P)H fluorescence was also increased by Ca²⁺ given in 50 µM concentration. In mitochondria having high ΔΨm in the presence of oligomycin or ATP, the basal rate of release of H₂O₂ was significantly higher than that observed in a medium containing ADP and Ca²⁺ no longer increased but rather decreased the rate of H₂O₂ release. With 300 µM Ca²⁺ only a loss but no tendency of a recovery of ΔΨm was detected and H₂O₂ release was unchanged. It is suggested that in the presence of nucleotides the effect of Ca²⁺ on mitochondrial ROS release is related to changes in ΔΨm; in depolarized mitochondria, in the presence of ADP, moderate increase in H₂O₂ release is induced by calcium, but only in ≤ 100 µM concentration, when after a transient Ca²⁺-induced depolarization mitochondria became more polarized. In highly polarized mitochondria, in the presence of ATP or oligomycin, where no hyperpolarization follows the Ca²⁺-induced depolarization, Ca²⁺ fails to stimulate mitochondrial ROS generation. These effects of calcium (≤ 300 µM) are unrelated to mitochondrial permeability transition.     

Cardioprotective mechanisms of Prunus cerasus (sour cherry) seed extract against ischemia-reperfusion-induced damage in isolated rat hearts

The effects of kernel extract obtained from sour cherry (Prunus cerasus) seed on the postischemic cardiac recovery were studied in isolated working rat hearts. Rats were treated with various daily doses of the extract for 14 days, and hearts were then isolated and subjected to 30 min of global ischemia followed by 120 min of reperfusion. The incidence of ventricular fibrillation (VF) and tachycardia (VT) fell from their control values of 92% and 100% to 50% (not significant) and 58% (not significant), 17% (P<0.05), and 25% (P<0.05) with the doses of 10 mg/kg and 30 mg/kg of the extract, respectively. Lower concentrations of the extract (1 and 5 mg/kg) failed to significantly reduce the incidence of VF and VT during reperfusion. Sour cherry seed kernel extract (10 and 30 mg/kg) significantly improved the postischemic recovery of cardiac function (coronary flow, aortic flow, and left ventricular developed pressure) during reperfusion. We have also demonstrated that the extract-induced protection in cardiac function significantly reflected in a reduction of infarct size. Immunohistochemistry indicates that a reduction in caspase-3 activity and apoptotic cells by the extract, beside other potential action mechanisms of proanthocyanidin, trans-resveratrol, and flavonoid components of the extract, could be responsible for the cardioprotection in ischemic-reperfused myocardium.     

A PH Domain in the Arf GTPase-activating Protein (GAP) ARAP1 Binds Phosphatidylinositol 3,4,5-Trisphosphate and Regulates Arf GAP Activity Independently of Recruitment to the Plasma Membranes

ARAP1 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃)-dependent Arf GTPase-activating protein (GAP) with five PH domains that regulates endocytic trafficking of the epidermal growth factor receptor (EGFR). Two tandem PH domains are immediately N-terminal of the Arf GAP domain, and one of these fits the consensus sequence for PtdIns(3,4,5)P₃ binding. Here, we tested the hypothesis that PtdIns(3,4,5)P₃-dependent recruitment mediated by the first PH domain of ARAP1 regulates the in vivo and in vitro function of ARAP1. We found that PH1 of ARAP1 specifically bound to PtdIns(3,4,5)P₃, but with relatively low affinity (≈1.6 μm), and the PH domains did not mediate PtdIns(3,4,5)P₃-dependent recruitment to membranes in cells. However, PtdIns(3,4,5)P₃ binding to the PH domain stimulated GAP activity and was required for in vivo function of ARAP1 as a regulator of endocytic trafficking of the EGFR. Based on these results, we propose a variation on the model for the function of phosphoinositide-binding PH domains. In our model, ARAP1 is recruited to membranes independently of PtdIns(3,4,5)P₃, the subsequent production of which triggers enzymatic activity.Pleckstrin homology (PH)² domains are a common structural motif encoded by the human genome (1, 2). Approximately 10% of PH domains bind to phosphoinositides. These PH domains are thought to mediate phosphoinositide-dependent recruitment to membranes (1–3). Most PH domains likely have functions other than or in addition to phosphoinositide binding. For example, PH domains have been found to bind to protein and DNA (4–12). In addition, some PH domains have been found to be structurally and functionally integrated with adjacent domains (13, 14). A small fraction of PH domain-containing proteins (about 9% of the human proteins) have multiple PH domains arranged in tandem, which have been proposed to function as adaptors but have only been examined in one protein (15, 16). Arf GTPase-activating proteins (GAPs) of the ARAP family are phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃)-dependent Arf GAPs with tandem PH domains (17, 18). The function of specific PH domains in regulating Arf GAP activity and for biologic activity has not been described.Arf GAPs are proteins that induce the hydrolysis of GTP bound to Arfs (19–23). The Arf proteins are members of the Ras superfamily of GTP-binding proteins (24–27). The six Arf proteins in mammals (five in humans) are divided into three classes based on primary sequence: Arf1, -2, and -3 are class 1, Arf4 and -5 are class 2, and Arf6 is class 3 (23, 24, 27–29). Class 1 and class 3 Arf proteins have been studied more extensively than class 2. They have been found to regulate membrane traffic and the actin cytoskeleton.The Arf GAPs are a family of proteins with diverse domain structures (20, 21, 23, 30). ARAPs, the most structurally complex of the Arf GAPs, contain, in addition to an Arf GAP domain, the sterile α motif (SAM), five PH, Rho GAP, and Ras association domains (17, 18, 31, 32). The first and second and the third and fourth PH domains are tandem (Fig. 1). The first and third PH domains of the ARAPs fit the consensus for PtdIns(3,4,5)P₃ binding (33–35). ARAPs have been found to affect actin and membrane traffic (21, 23). ARAP3 regulates growth factor-induced ruffling of porcine aortic endothelial cells (31, 36, 37). The function is dependent on the Arf GAP and Rho GAP domains. ARAP2 regulates focal adhesions, an actin cytoskeletal structure (17). ARAP2 function requires Arf GAP activity and a Rho GAP domain capable of binding RhoA·GTP. ARAP1 has been found to have a role in membrane traffic (18). The protein associates with pre-early endosomes involved in the attenuation of EGFR signals. The function of the tandem PH domains in the ARAPs has not been examined.Open in a separate windowFIGURE 1.ARAP1 binding to phospholipids. A, schematic of the recombinant proteins used in this study. Domain abbreviations: Ank, ankyrin repeat; PLCδ-PH, PH domain of phospholipase C δ; RA, Ras association motif; RhoGAP, Rho GTPase-activating domain. B, ARAP1 phosphoinositide binding specificity. 500 nm PH1-Ank recombinant protein was incubated with sucrose-loaded LUVs formed by extrusion through a 1-μm pore filter. LUVs contained PtdIns alone or PtdIns with 2.5 μm PtdIns(3,4,5)P₃, 2.5 μm PtdIns(3)P, 2.5 μm PtdIns(4)P, 2.5 μm PtdIns(5)P, 2.5 μm PtdIns(3,4)P₂, 2.5 μm PtdIns(3,5)P₂, or 2.5 μm PtdIns(4,5)P₂ with a total phosphoinositide concentration of 50 μm and a total phospholipid concentration of 500 μm. Vesicles were precipitated by ultracentrifugation, and associated proteins were separated by SDS-PAGE. The amount of precipitated protein was determined by densitometry of the Coomassie Blue-stained gels with standards on each gel. C, PtdIns(3,4,5)P₃-dependent binding of ARAP1 to LUVs. 1 μm PH1-Ank or ArfGAP-Ank recombinant protein was incubated with 1 mm sucrose-loaded LUVs formed by extrusion through a 1-μm pore size filter containing varying concentration of PtdIns(3,4,5)P₃. Precipitation of LUVs and analysis of associated proteins were performed as described in B. The average ± S.E. of three independent experiments is presented.Here we investigated the role of the first two PH domains of ARAP1 for catalysis and in vivo function. The first PH domain fits the consensus sequence for PtdIns(3,4,5)P₃ binding (33–35). The second does not fit a phosphoinositide binding consensus but is immediately N-terminal to the GAP domain. We have previously reported that the PH domain that occurs immediately N-terminal of the Arf GAP domain of ASAP1 is critical for the catalytic function of the protein (38, 39). We tested the hypothesis that the two PH domains of ARAP1 function independently; one recruits ARAP1 to PtdIns(3,4,5)P₃-rich membranes, and the other functions with the catalytic domain. As predicted, PH1 interacted specifically with PtdIns(3,4,5)P₃, and PH2 did not. However, both PH domains contributed to catalysis independently of recruitment to membranes. None of the PH domains in ARAP1 mediated PtdIns(3,4,5)P₃-dependent targeting to plasma membranes (PM). PtdIns(3,4,5)P₃ stimulated GAP activity, and the ability to bind PtdIns(3,4,5)P₃ was required for ARAP1 to regulate membrane traffic. We propose that ARAP1 is recruited independently of PtdIns(3,4,5)P₃ to the PM where PtdIns(3,4,5)P₃ subsequently regulates its GAP activity to control endocytic events.     

PapA3 Is an Acyltransferase Required for Polyacyltrehalose Biosynthesis in Mycobacterium tuberculosis

Mycobacterium tuberculosis possesses an unusual cell wall that is replete with virulence-enhancing lipids. One cell wall molecule unique to pathogenic M. tuberculosis is polyacyltrehalose (PAT), a pentaacylated, trehalose-based glycolipid. Little is known about the biosynthesis of PAT, although its biosynthetic gene cluster has been identified and found to resemble that of the better studied M. tuberculosis cell wall component sulfolipid-1. In this study, we sought to elucidate the function of papA3, a gene from the PAT locus encoding a putative acyltransferase. To determine whether PapA3 participates in PAT assembly, we expressed the protein heterologously and evaluated its acyltransferase activity in vitro. The purified enzyme catalyzed the sequential esterification of trehalose with two palmitoyl groups, generating a diacylated product similar to the 2,3-diacyltrehalose glycolipids of M. tuberculosis. Notably, PapA3 was selective for trehalose; no activity was observed with other structurally related disaccharides. Disruption of the papA3 gene from M. tuberculosis resulted in the loss of PAT from bacterial lipid extracts. Complementation of the mutant strain restored PAT production, demonstrating that PapA3 is essential for the biosynthesis of this glycolipid in vivo. Furthermore, we determined that the PAT biosynthetic machinery has no cross-talk with that for sulfolipid-1 despite their related structures.Mycobacterium tuberculosis, the bacterium that causes tuberculosis in humans, has a complex cell wall that contains a number of unique glycolipids intimately linked to mycobacterial pathogenesis (1, 2). The biosynthesis of many of these virulence factors, including the trehalose mycolates, phenolic glycolipids, and sulfolipid-1 (SL-1),³ is largely understood (3–5). In contrast, relatively little is known about the biosynthesis of other prominent M. tuberculosis glycolipids, such as di-, tri-, and polyacyltrehaloses. These acyltrehaloses are located in the outer surface of the cell wall and contain di- and tri-methyl branched fatty acids that are only found in pathogenic species of mycobacteria (6, 7). Previous studies suggest a role for these glycolipids in anchoring the bacterial capsule, which impedes phagocytosis by host cells (6).The major polyacyltrehalose (PAT) of M. tuberculosis, also referred to as pentaacyl or polyphthienoyl trehalose, consists of five acyl chains, four mycolipenic (phthienoic) acids and one fully saturated fatty acid, linked to trehalose (Fig. 1A) (8). The mycolipenic acid side chains of PAT are products of the polyketide synthase gene pks3/4 (7). Disruption of pks3/4 (also referred to as msl3 (7)) abolishes PAT biosynthesis and causes cell aggregation. At present, the remaining proteins required for PAT assembly have not been characterized.Open in a separate windowFIGURE 1.PAT and SL-1 share related structures and biosynthetic gene clusters. A, structure of PAT. B, structure of SL-1. C, genomic arrangement of the PAT and SL-1 biosynthetic gene clusters.Interestingly, the PAT biosynthetic gene cluster strongly resembles that of SL-1, which is a structurally similar trehalose-based glycolipid unique to pathogenic mycobacteria (Fig. 1B) (9). Both gene clusters contain polyketide synthase (pks), acyltransferase (pap), and lipid transport (mmpL) genes in a similar genomic arrangement (Fig. 1C). The SL-1 locus encodes two acyltransferase genes, papA1 and papA2, which are required for SL-1 biosynthesis (5, 10). These proteins belong to the mycobacterium-specific polyketide-associated protein (Pap) family of acyltransferases, which share a conserved HX₃DX₁₄Y motif that is required for activity (11). The PapA2 enzyme catalyzes the esterification of the 2′-position of trehalose 2-sulfate with a saturated fatty acid. PapA1 mediates the subsequent esterification of this intermediate with a hydroxyphthioceranoyl group produced by Pks2 (5). Interestingly, the PAT locus contains a gene, Rv1182, that is homologous to both papA1 and papA2 (55 and 53% amino acid identity, respectively). This gene is annotated as papA3 in the genome and was previously shown to encode a protein bearing the signature Pap motif (11).Here we demonstrate that papA3 encodes an acyltransferase essential for the biosynthesis of PAT. Deletion of the papA3 gene resulted in loss of the glycolipid from M. tuberculosis lipid extracts, as determined by high resolution mass spectrometry. Moreover, the purified enzyme was shown to selectively and sequentially acylate trehalose in vitro, generating a diacylated product similar to the 2,3-diacyltrehaloses of M. tuberculosis. Together, these data confirm that PapA3 plays a crucial role in PAT biosynthesis and highlight its potential involvement in the biosynthesis of related M. tuberculosis acyltrehaloses.     

The impact of plant associations on Macrosteles quadrilineatus management in carrots

Habitat diversification can influence the interactions of insects with plants and this can be used in agroecosystems for the management of pest populations. Plant diversification can be achieved through planting crops, such as trap crops, or by adjusting weed management. Aster leafhopper, Macrosteles quadrilineatus Forbes (Hemiptera: Cicadellidae), is a polyphagous species that uses cereals, vegetables, and weeds as host plants. The influence of weeds on M. quadrilineatus abundance was investigated experimentally in carrot [Daucus carota L. cv. Canada (Apiaceae)] field plots by adjusting the level of management of two groups of weeds (broadleaf and grass) and by comparing it to weed‐free plots. The preference of M. quadrilineatus for different cereal and weed species relative to carrots was tested in choice test assays. Habitat context influenced the abundance of M. quadrilineatus in the field experiments. The presence of border crops such as oat, rye, barley, wheat, and triticale did not significantly attract or repel this insect to carrot plots compared to the no‐border treatment. However, spelt‐bordered plots had 42% fewer M. quadrilineatus than three treatments, triticale, wheat, and barley, that had the highest insect abundance. The type of weed management affected M. quadrilineatus abundance in carrot plots, but not the frequency of herbicide application. Plots that had carrot growing with broadleaf‐weeds had about 59% fewer M. quadrilineatus compared with those growing with crabgrass or carrot alone. In the greenhouse choice tests, grasses (e.g., cereals) attracted and broadleaf‐weeds repelled M. quadrilineatus relative to carrots. In summary, carrot growers may be able to manage this pest by reducing the interaction of cereal cover crops with carrots and eliminating grassy weeds in commercial production fields.