Gibberellin A<Subscript>3</Subscript> detection in plants with high-performance liquid chromatography

A new method of gibberellin A₃ detection based on high-performance liquid chromatography has been elaborated. It combines high accuracy and low cost, and includes the following stages: fixation with liquid nitrogen; extraction of alcohol-soluble substances; purification with freezing, reextraction in ethyl acetate, and thin-layer chromatography; and detection with HPLC. Chromatographic conditions and methods of substance identification have been determined. The method allows the use of modest amounts of plant material (5–20 g), at which variability does not exceed 7%, and the total loss of substance is approximately 3%. The results of gibberellin A₃ detection in buckwheat are presented.     

Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

Neuroprotection by adenosine in the brain: From A<Subscript>1</Subscript> receptor activation to A<Subscript>2A</Subscript> receptor blockade

Adenosine is a neuromodulator that operates via the most abundant inhibitory adenosine A₁ receptors (A₁Rs) and the less abundant, but widespread, facilitatory A_2ARs. It is commonly assumed that A₁Rs play a key role in neuroprotection since they decrease glutamate release and hyperpolarize neurons. In fact, A₁R activation at the onset of neuronal injury attenuates brain damage, whereas its blockade exacerbates damage in adult animals. However, there is a down-regulation of central A₁Rs in chronic noxious situations. In contrast, A_2ARs are up-regulated in noxious brain conditions and their blockade confers robust brain neuroprotection in adult animals. The brain neuroprotective effect of A_2AR antagonists is maintained in chronic noxious brain conditions without observable peripheral effects, thus justifying the interest of A_2AR antagonists as novel protective agents in neurodegenerative diseases such as Parkinsons and Alzheimers disease, ischemic brain damage and epilepsy. The greater interest of A_2AR blockade compared to A₁R activation does not mean that A₁R activation is irrelevant for a neuroprotective strategy. In fact, it is proposed that coupling A_2AR antagonists with strategies aimed at bursting the levels of extracellular adenosine (by inhibiting adenosine kinase) to activate A₁Rs might constitute the more robust brain neuroprotective strategy based on the adenosine neuromodulatory system. This strategy should be useful in adult animals and especially in the elderly (where brain pathologies are prevalent) but is not valid for fetus or newborns where the impact of adenosine receptors on brain damage is different.     

Distribution of calcium-independent phospholipase A<Subscript>2</Subscript> (iPLA<Subscript>2</Subscript>) in monkey brain

The present study was carried out to elucidate the distribution of calcium-independent phospholipase A₂ (iPLA₂) in the normal monkey brain. iPLA₂ immunoreactivity was observed in structures derived from the telencephalon, including the cerebral neocortex, amygdala, hippocampus, caudate nucleus, putamen, and nucleus accumbens, whereas structures derived from the diencephalon, including the thalamus, hypothalamus and globus pallidus were lightly labeled. The midbrain, vestibular, trigeminal and inferior olivary nuclei, and the cerebellar cortex were densely labeled. Immunoreactivity was observed on the nuclear envelope of neurons, and dendrites and axon terminals at electron microscopy. Western blot analysis showed higher levels of iPLA₂ protein in the cytosolic, than the nuclear fraction, but little or no protein in the membrane fraction. Similarly, subcellular fractionation studies of iPLA₂ activity in rat brain cortical cell cultures showed greater enzymatic activity in the cytosolic, than the nuclear fraction, and the least activity in non-nuclear membranes. The association of iPLA₂ with the nuclear envelope suggests a role of the enzyme in nuclear signaling, such as during neuronal proliferation and differentiation or death. In addition, the localization of iPLA₂ in dendrites and axon terminals suggests a role of the enzyme in neuronal signaling.     

A<Subscript>2A</Subscript> and A<Subscript>3</Subscript> adenosine receptor expression in rheumatoid arthritis: upregulation,inverse correlation with disease activity score and suppression of inflammatory cytokine and metalloproteinase release

Introduction  

The reduction of the inflammatory status represents one of the most important targets in rheumatoid arthritis (RA). A central role of A_2A and A₃ adenosine receptors (ARs) in mechanisms of inflammation has been reported in different pathologies. The primary aim of this study was to investigate the A_2A and A₃ARs and their involvement in RA progression measured by Disease Activity Score in 28 or 44 joints (DAS28 or DAS).     

Adenosine A<Subscript>2A</Subscript> agonist and A<Subscript>2B</Subscript> antagonist mediate an inhibition of inflammation-induced contractile disturbance of a rat gastrointestinal preparation

Adenosine can show anti-inflammatory as well as pro-inflammatory activities. The contribution of the specific adenosine receptor subtypes in various cells, tissues and organs is complex. In this study, we examined the effect of the adenosine A_2A receptor agonist CGS 21680 and the A_2BR antagonist PSB-1115 on acute inflammation induced experimentally by 2,4,6-trinitrobenzenesulfonic acid (TNBS) on rat ileum/jejunum preparations. Pre-incubation of the ileum/jejunum segments with TNBS for 30 min resulted in a concentration-dependent inhibition of acetylcholine (ACh)-induced contractions. Pharmacological activation of the A_2AR with CGS 21680 (0.1–10 μM) pre-incubated simultaneously with TNBS (10 mM) prevented concentration-dependently the TNBS-induced inhibition of the ACh contractions. Stimulation of A_2BR with the selective agonist BAY 60-6583 (10 μM) did neither result in an increase nor in a further decrease of ACh-induced contractions compared to the TNBS-induced inhibition. The simultaneous pre-incubation of the ileum/jejunum segments with TNBS (10 mM) and the selective A_2BR antagonist PSB-1115 (100 μM) inhibited the contraction-decreasing effect of TNBS. The effects of the A_2AR agonist and the A_2BR antagonist were in the same range as the effect induced by 1 μM methotrexate. The combination of the A_2AR agonist CGS 21680 and the A_2BR antagonist PSB-1115 at subthreshold concentrations of both agents found a significant amelioration of the TNBS-diminished contractility. Our results demonstrate that the activation of A_2A receptors or the blockade of the A_2B receptors can prevent the inflammation-induced disturbance of the ACh-induced contraction in TNBS pre-treated small intestinal preparations. The combination of both may be useful for the treatment of inflammatory bowel diseases.     

Adenosine A<Subscript>2A</Subscript> receptor as a drug target for treatment of sepsis

Sepsis is a generalized infection accompanied by response of the body that manifests in a clinical and laboratory syndrome, namely, in the systemic inflammatory response syndrome (SIRS) from the organism to the infection. Although sepsis is a widespread and life-threatening disease, the assortment of drugs for its treatment is mostly limited by antibiotics. Therefore, the search for new cellular targets for drug therapy of sepsis is an urgent task of modern medicine and pharmacology. One of the most promising targets is the adenosine A_2A receptor (A_2AAR). The activation of this receptor, which is mediated by extracellular adenosine, manifests in almost all types of immune cells (lymphocytes, monocytes, macrophages, and dendritic cells) and results in reducing the severity of inflammation and reperfusion injury in various tissues. The activation of adenosine A_2A receptor inhibits the proliferation of T cells and production of proinflammatory cytokines, which contributes to the activation of the synthesis of anti-inflammatory cytokines, thereby suppressing the systemic response. For this reason, various selective A_2AAR agonists and antagonists may be considered to be drug candidates for sepsis pharmacotherapy. Nevertheless, they remain only efficient ligands and objects of pre-clinical and clinical trials. This review examines the molecular mechanisms of inflammatory response in sepsis and the structure and functions of A_2AAR and its role in the pathogenesis of sepsis, as well as examples of using agonists and antagonists of this receptor for the treatment of SIRS and sepsis.     

Signaling pathways involving adenosine A<Subscript>2A</Subscript> and A<Subscript>2B</Subscript> receptors in wound healing and fibrosis

Collagen and matrix deposition by fibroblasts is an essential part of wound healing but also contributes to pathologic remodeling of organs leading to substantial morbidity and mortality. Adenosine, a small molecule generated extracellularly from adenine nucleotides as a result of direct stimulation, hypoxia, or injury, acts via a family of classical seven-pass G protein-coupled protein receptors, A_2A and A_2B, leading to generation of cAMP and activation of downstream targets such as PKA and Epac. These effectors, in turn, lead to fibroblast activation and collagen synthesis. The regulatory actions of these receptors likely involve multiple interconnected pathways, and one of the more interesting aspects of this regulation is opposing effects at different levels of cAMP generated. Additionally, adenosine signaling contributes to fibrosis in organ-specific ways and may have opposite effects in different organs. The development of drugs that selectively target these receptors and their signaling pathways will disrupt the pathogenesis of fibrosis and slow or arrest the progression of the important diseases they underlie.     

Species differences and mechanism of action of A<Subscript>3</Subscript> adenosine receptor allosteric modulators

Activity of the A₃ adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A₃AR species homologs used in preclinical drug development. In guanosine 5′-[γ-[³⁵S]thio]triphosphate ([³⁵S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A₃ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A₃ARs but provided only weak activity at mouse A₃ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A₃AR agonist 2-chloro-N ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide as well as adenosine > 2-fold, while slightly reducing potency in human and dog. Based on results from N ⁶-(4-amino-3-[¹²⁵I]iodobenzyl)adenosine-5′-N-methylcarboxamide ([¹²⁵I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A₃AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [¹²⁵I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A₃AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.     

Plant phospholipases A<Subscript>2</Subscript>: perspectives on biotechnological applications

The recent progress in knowledge on biochemical properties and functions of phospholipases A₂ in plants paved the way for approving the suitability of these enzymes for commercial use now. The secreted phospholipases A₂, representing one type of phospholipases A₂ occurring in plants, show distinct differences in substrate specificities with respect to headgroup and acyl chains of the glycerophospholipids in comparison to their counterparts from animal sources. The other type of phospholipases A₂ in plants, the patatin-related phospholipases A₂, is characterized by broad substrate specificity. Accordingly, the unique properties of the plant enzymes open new horizons to engineered biocatalysts with improved performance, e.g., for vegetable oil refinement by degumming and for targeted modification of phospholipids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Interisland Mutation of a Novel Phospholipase A<Subscript>2</Subscript> from <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom and Evolution of Crotalinae Group II Phospholipases A<Subscript>2</Subscript>

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of Amami-Oshima T. flavoviridis venom led to isolation of a novel phospholipase A₂ (PLA₂). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA₂, which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD₅₀ for mice was 1.34 µg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the Amami-Oshima and the Tokunoshima T. flavoviridis venom-gland cDNA libraries. Screening of the Okinawa T. flavoviridis venom-gland cDNA library with PLA-N cDNA led to isolation of the cDNA encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), suggesting that interisland mutation occurred and that Okinawa island was separated from a former island prior to dissociation of Amami-Oshima and Tokunoshima islands. Construction of a phylogenetic tree of Crotalinae venom group II PLA₂s based on the amino acid sequences revealed that neurotoxic PLA₂s including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA₂ groups such as PLA₂ type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA₂s showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA₂s, their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution. It is likely that ancestral genes of neurotoxic PLA₂s evolved in an accelerated manner until they had acquired neurotoxic function and since then they have evolved with less frequent mutation, possibly for functional conservation. The nucleotide sequences reported in this paper are available from the GenBank/EMBL/DDBJ databases under accession numbers AB102728 and AB102729.     

New Phospholipase A<Subscript>1</Subscript>-producing Bacteria from a Marine Fish

Phospholipase A1 is a hydrolytic enzyme that catalyzes the removal of the acyl group from position 1 of glycerophospholipids to form 2-acyl lysophospholipids. Lysophospholipids are used in foods, cosmetics, and pharmaceuticals as surfactants. Novel forms of phospholipase A1 that function at low temperatures are desirable for use in lipophilic systems in food processing. However, there is currently little variety in the available sources of phospholipase A1. Given this situation, we screened the intestinal contents of marine animals for phospholipase A1-producing bacteria. Colonies that formed a halo on K28CP screening medium and that grew in K28 medium were cultured in liquid K28 medium, and the supernatant was retrieved for analysis. Phosphatidylcholine was added to the culture supernatant, and the product of the reaction was analyzed by using TLC. For culture supernatants that were able to generate lysophosphatidylcholine, synthetic phosphatidylcholines were added, and the site of the reaction was determined by analyzing the fatty acid compositions of the lysophosphatidylcholines generated by GLC. A bacterial isolate from a flatfish, which we named HFKI0020, was found to have phospholipase A1 activity at low temperatures. We determined that the isolate HFKI0020 is closely related to Pseudomonas by using 16S rDNA sequence analysis and by characterizing the isolate with respect to its physiologic and biochemical properties. From the intestinal contents of a marine fish, we successfully isolated a bacterium that secretes phospholipase A1 that is active at low temperatures.     

F<Subscript>1</Subscript>-ATPase as an auto-oscillatory system

In a relatively simple mathematical model we analyze the possibility of auto-oscillatory modes of F₁-ATPase during ATP hydrolysis.     

Theoretical study of the novel sandwich compound [Au<Subscript>3</Subscript>Cl<Subscript>3</Subscript>Tr<Subscript>2</Subscript>]<Superscript>2+</Superscript>

A theoretical study of a sandwich compound with a metal monolayer sheet between two aromatic ligands is presented. A full geometry optimization of the [Au₃Cl₃Tr₂]²⁺ (1) compound, which is a triangular gold(I) monolayer sheet capped by chlorines and bounded to two cycloheptatrienyl (Tr) ligands was carried out using perturbation theory at the MP2 computational level and DFT. Compound (1) is in agreement with the 18–electron rule, the bonding nature in the complex may be interpreted from the donation interaction coming from the Tr rings to the Au array, and from the back-donation from the latter to the former. NICS calculations show a strong aromatic character in the gold monolayer sheet and Tr ligands; calculations done with HOMA, also report the same aromatic behavior on the cycloheptatrienyl fragments giving us an insight on the stability of (1). The Au –Au bond lengths indicate that an intramolecular aurophilic interaction among the Au(I) cations plays an important role in the bonding of the central metal sheet. Figure (a) Ground state geometry of complex 1; (b) Top view of compound 1 and Wiberg bond orders computed with the MP2/B1 computational method; (c) Lateral view of compound 1 and NICS values calculated with the MP2/B1 method; the values in parenthesis were obtained at the VWN/TZP level     

An endophytic bacterium isolated from roots of the halophyte <Emphasis Type="Italic">Prosopis strombulifera</Emphasis> produces ABA,IAA, gibberellins A<Subscript>1</Subscript> and A<Subscript>3</Subscript> and jasmonic acid in chemically-defined culture medium

This paper informs the characterization by 16SrDNA partial sequence analysis of an endophytic diazotrophic bacterium isolated from roots of the halophyte shrub Prosopis strombulifera. The bacterium produced ABA, IAA, GA₁, GA₃ and jasmonic acid in chemically-defined culture medium as assessed by GC-EIMS. The results emphasize the role of phytohormones produced by endophytic bacteria in the association host-beneficial microorganisms, especially under conditions of adverse environments.     

Inventing an arsenal: adaptive evolution and neofunctionalization of snake venom phospholipase A<Subscript>2</Subscript> genes

Background  

Gene duplication followed by functional divergence has long been hypothesized to be the main source of molecular novelty. Convincing examples of neofunctionalization, however, remain rare. Snake venom phospholipase A₂ genes are members of large multigene families with many diverse functions, thus they are excellent models to study the emergence of novel functions after gene duplications.     

Immobilization of glucose oxidase on Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>/SiO<Subscript>2</Subscript> magnetic nanoparticles

Glucose oxidase (GOD) was covalently immobilized onto Fe₃O₄/SiO₂ magnetic nanoparticles (FSMNs) using glutaraldehyde (GA). Optimal immobilization was at pH 6 with 3-aminopropyltriethoxysilane at 2% (v/v), GA at 3% (v/v) and 0.143 g GOD per g carrier. The activity of immobilized GOD was 4,570 U/g at pH 7 and 50°C. The immobilized GOD retained 80% of its initial activity after 6 h at 45°C while free enzyme retained only 20% activity. The immobilized GOD maintained 60% of its initial activity after 6 cycles of repeated use and retained 75% of its initial activity after 1 month at 4°C whereas free enzymes retained 62% of its activity.     

Role of extracellular cysteine residues in the adenosine A<Subscript>2A</Subscript> receptor

The G protein-coupled A_2A adenosine receptor represents an important drug target. Crystal structures and modeling studies indicated that three disulfide bonds are formed between ECL1 and ECL2 (I, Cys71^2.69-Cys159^45.43; II, Cys74^3.22-Cys146^45.30, and III, Cys77^3.25-Cys166^45.50). However, the A_2BAR subtype appears to require only disulfide bond III for proper function. In this study, each of the three disulfide bonds in the A_2AAR was disrupted by mutation of one of the cysteine residues to serine. The mutant receptors were stably expressed in Chinese hamster ovary cells and analyzed in cyclic adenosine monophosphate (cAMP) accumulation and radioligand binding studies using structurally diverse agonists: adenosine, NECA, CGS21680, and PSB-15826. Results were rationalized by molecular modeling. The observed effects were dependent on the investigated agonist. Loss of disulfide bond I led to a widening of the orthosteric binding pocket resulting in a strong reduction in the potency of adenosine, but not of NECA or 2-substituted nucleosides. Disruption of disulfide bond II led to a significant reduction in the agonists’ efficacy indicating its importance for receptor activation. Disulfide bond III disruption reduced potency and affinity of the small adenosine agonists and NECA, but not of the larger 2-substituted agonists. While all the three disulfide bonds were essential for high potency or efficacy of adenosine, structural modification of the nucleoside could rescue affinity or efficacy at the mutant receptors. At present, it cannot be excluded that formation of the extracellular disulfide bonds in the A_2AAR is dynamic. This might add another level of G protein-coupled receptor (GPCR) modulation, in particular for the cysteine-rich A_2A and A_2BARs.