Cultivation modes affect the morphology, biochemical composition, and antioxidant and anti-inflammatory properties of the green microalga Neochloris oleoabundans.

Microalgae are considered promising sustainable sources of natural bioactive compounds to be used in biotechnological sectors. In recent years, attention is increasingly given to the search of microalgae-derived compounds with antioxidant and anti-inflammatory properties for nutraceutical or pharmacological issues. In this context, attention is usually focused on the composition and bioactivity of algae or their extracts, while less interest is driven to their biological features, for example, those related to morphology and cultivation conditions. In addition, specific studies on the antioxidant and anti-inflammatory properties of microalgae mainly concern Chlorella or Spirulina. The present work was focused on the characterization of the Chlorophyta Neochloris oleoabundans under two combinations of cultivation modes: autotrophy and glucose-induced mixotrophy, each followed by starvation. Biomass for morphological and biochemical characterization, as well as for extract preparation, was harvested at the end of each cultivation phase. Analyses indicated a different content of the most important classes of bioactive compounds with antioxidant/anti-inflammatory properties (lipids, exo-polysaccharides, pigments, total phenolics, and proteins). In particular, the most promising condition able to prompt the production of antioxidant algal biomass with anti-inflammatory properties was the mixotrophic one. Under mixotrophy, beside an elevated algal biomass production, a strong photosynthetic metabolism with high appression of thylakoid membranes and characteristics of high photo-protection from oxidative damage was observed and linked to the overproduction of exo-polysaccharides and lipids rather than pigments. Overall, mixotrophy appears a good choice to produce natural bioactive extracts, potentially well tolerated by human metabolism and environmentally sustainable.

PKCε as a novel promoter of skeletal muscle differentiation and regeneration.

INTRODUCTION: Satellite cells are muscle resident stem cells and are responsible for muscle regeneration. In this study we investigate the involvement of PKCε during muscle stem cell differentiation in vitro and in vivo. Here, we describe the identification of a previously unrecognized role for the PKCε-HMGA1 signaling axis in myoblast differentiation and regeneration processes. METHODS: PKCε expression was modulated in the C2C12 cell line and primary murine satellite cells in vitro, as well as in an in vivo model of muscle regeneration. Immunohistochemistry and immunofluorescence, RT-PCR and shRNA silencing techniques were used to determine the role of PKCε and HMGA1 in myogenic differentiation. RESULTS: PKCε expression increases and subsequently re-localizes to the nucleus during skeletal muscle cell differentiation. In the nucleus, PKCε blocks Hmga1 expression to promote Myogenin and Mrf4 accumulation and myoblast formation. Following in vivo muscle injury, PKCε accumulates in regenerating, centrally-nucleated myofibers. Pharmacological inhibition of PKCε impairs the expression of two crucial markers of muscle differentiation, namely MyoD and Myogenin, during injury induced muscle regeneration. CONCLUSION: This work identifies the PKCε-HMGA1 signaling axis as a positive regulator of skeletal muscle differentiation.

Allosteric enhancers of A1 adenosine receptors: state of the art and new horizons for drug development.

Adenosine is an important autocoid, exerting its physiological effects on the human body by activation of four different G-protein-coupled-receptors (GPCRs) classified as A(1), A(2A), A(2B), and A(3). These receptors are coupled to secondary messenger systems including adenylate cyclase, inositol phosphate metabolism, and K(+), K(ATP) and Ca(2+) channels. Pharmacological agents that increase the activation of A(1) adenosine receptors in response to adenosine would be useful for treatment of cardiovascular, central nervous system, and inflammatory pathologies. Compounds that are able to enhance the activity of the A(1)-adenosine receptors by the endogenous ligand within specific tissues may have potential therapeutic advantages over non-endogenous agonists. Such an opportunity for intervention is provided by the concept of allosteric modulation of GPCRs. Therefore the use of allosteric enhancers to increase the responsiveness of the A(1) receptors to endogenous adenosine at sites of its production is an appealing alternative to activation by exogenous agonists. This approach minimizes side effects because allosteric enhancers amplify the action of the agonist by stabilizing the agonist-A(1)-receptor-G protein ternary complex. The allosteric enhancement of the GABA(A) receptor by benzodiazepines is the most famous and successful example of this strategy. The aim of this article is to give an overview of the results obtained in this field and discuss the opportunities and challenges that this class of ligands might offer for medicinal chemistry and pharmacology.

Pharmacological characterization of P2X1 and P2X3 purinergic receptors in bovine chondrocytes.

OBJECTIVE: The aim of the present study is that of characterizing, for the first time in a quantitative way, from a biochemical, physico chemical and functional point of view P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes. The affinity and the potency of typical purinergic ligands were studied through competition binding experiments and their role in modulating chondrocyte actvities was investigated by analyzing nitric oxide (NO) and prostaglandin E2 (PGE(2)) release. METHODS: Saturation, competition binding experiments, western blotting and immunohistochemistry assays on the P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes were performed. Thermodynamic analysis of the P2X(1) and P2X(3) purinergic binding was studied to investigate the forces driving drug-receptor coupling. In the functional assays (NO and PGE(2) release) the potency of purinergic agonists and antagonists was evaluated. RESULTS: Bovine chondrocytes expressed P2X(1) and P2X(3) purinergic receptors and thermodynamic parameters indicated that purinergic binding is enthalpy- and entropy-driven for agonists and totally entropy-driven for antagonists. Typical purinergic agonists such as adenosine 5'-triphosphate (ATP) and alpha,beta-methyleneATP were able to increase NO and PGE(2) release. A purinergic antagonist, A317491, was able to block the stimulatory effect on functional experiments mediated by the agonists. CONCLUSIONS: These data demonstrate for the first time the presence of functional P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes. Agonists and antagonists are thermodynamically discriminated and are able to modulate functional responses such as NO and PGE(2) release. These results suggest the potential role of novel purinergic antagonists in the treatment of pathophysiological diseases linked to the inflammation and involved in articular cartilage resorption.

Characterization of adenosine receptors in bovine chondrocytes and fibroblast-like synoviocytes exposed to low frequency low energy pulsed electromagnetic fields.

OBJECTIVE: The present study describes the presence and binding parameters of the A1, A2A, A2B and A3 adenosine receptors in bovine chondrocytes and fibroblast-like synoviocytes. The effect of low frequency low energy pulsed electromagnetic fields (PEMFs) on the adenosine receptor affinity and density was studied. METHODS: Saturation, competition binding experiments and Western blotting assays in the absence and in the presence of PEMFs on the adenosine receptors in bovine chondrocytes or fibroblast-like synoviocytes were performed. Thermodynamic analysis of the A2A or A3 binding was studied to investigate the forces driving drug-receptor coupling. In the adenylyl cyclase and proliferation assays the potency of typical high-affinity A2A or A3 agonists in the absence and in the presence of PEMFs was evaluated. RESULTS: Bovine chondrocytes and fibroblast-like synoviocytes expressed all adenosine receptors. PEMFs evoked an up-regulation of A2A and A3 receptors and thermodynamic parameters indicate that adenosine binding is enthalpy and entropy driven. In PEMF-treated cells the potency of typical A2A or A3 agonists on cyclic AMP assays was significantly increased when compared with the untreated cells. PEMFs potentiated the effect of A2A or A3 agonists on cell proliferation in both cell types. CONCLUSIONS: PEMFs mediate an up-regulation of A2A and A3 receptors related to an increase of their functional activities in bovine chondrocytes and fibroblast-like synoviocytes. No differences are present in adenosine affinity and in the drug-receptor interactions. Our data could be used as a trigger to future studies addressed to PEMFs and adenosine therapeutic intervention in inflammatory joint diseases.

Caffeine intake induces an alteration in human neutrophil A2A adenosine receptors.

Caffeine is the most widely used drug in the world and acts mainly through antagonism of the effects mediated by the adenosine receptor subtypes A1, A2A, A2B and A3. We determined whether repeated caffeine administration at different doses and for different periods of time (400 or 600 mg/day for 1 week and 400 mg/day for 2 weeks) alters human neutrophil A2A adenosine receptor density and function. Saturation binding assays showed an increase in affinity (K(D)) and density (B(max)) of A2A adenosine receptors after caffeine intake. These changes were accompanied by increases in cAMP accumulation and decreases in superoxide anion production after stimulation of the A2A receptor subtype using the agonist 5'-N-ethylcarboxamidoadenosine (NECA). Binding and functional changes of A2A receptors returned to baseline after 48 h of caffeine withdrawal. The findings are consistent with a potential anti-inflammatory effect of caffeine mediated by neutrophil A2A receptors.

Pharmacological and biochemical characterization of adenosine receptors in the human malignant melanoma A375 cell line.

1. The present work characterizes, from a pharmacological and biochemical point of view, adenosine receptors in the human malignant melanoma A375 cell line. 2. Adenosine receptors were detected by RT - PCR experiments. A1 receptors were characterized using [3H]-DPCPX binding with a KD of 1.9+/-0.2 nM and Bmax of 23+/-7 fmol x mg(-1) of protein. A2A receptors were studied with [3H]-SCH 58261 binding and revealed a KD of 5.1+/-0.2 nM and a Bmax of 220+/-7 fmol x mg(-1) of protein. A3 receptors were studied with the new A3 adenosine receptor antagonist [3H]-MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with KD of 3.3+/-0.7 nM and Bmax of 291+/-50 fmol x mg(-1) of protein. 3. The pharmacological profile of radioligand binding on A375 cells was established using typical adenosine ligands which displayed a rank order of potency typical of the different adenosine receptor subtype. 4. Thermodynamic data indicated that radioligand binding to adenosine receptor subtypes in A375 cells was entropy- and enthalpy-driven. 5. In functional assays the high affinity A2A agonists HE-NECA, CGS 21680 and A2A - A2B agonist NECA were able to increase cyclic AMP accumulation in A375 cells whereas A3 agonists Cl-IB-MECA, IB-MECA and NECA were able to stimulate Ca2+ mobilization. In conclusion, all these data indicate, for the first time, that adenosine receptors with a pharmacological and biochemical profile typical of the A1, A2A, A2B and A3 receptor subtype are present on A375 melanoma cell line.

Pharmacological and biochemical characterization of A3 adenosine receptors in Jurkat T cells.

1. The present work was devoted to the study of A3 adenosine receptors in Jurkat cells, a human leukemia line. 2. The A3 subtype was found by means of RT-PCR experiments and characterized by using the new A3 adenosine receptor antagonist [3H]-MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with K(D) of 1.9+/-0.2 nM and B(max) of 1.3+/-0.1 pmol mg(-1) of protein. 3. The pharmacological profile of [3H]-MRE 3008F20 binding on Jurkat cells was established using typical adenosine ligands which displayed a rank order of potency typical of the A3 subtype. 4. Thermodynamic data indicated that [3H]-MRE 3008F20 binding to A3 subtype in Jurkat cells was entropy- and enthalpy-driven, according with that found in cells expressing the recombinant human A3 subtype. 5. In functional assays the high affinity A3 agonists Cl-IB-MECA and IB-MECA were able to inhibit cyclic AMP accumulation and stimulate Ca(2+) release from intracellular Ca(2+) pools followed by Ca(2+) influx. 6. The presence of the other adenosine subtypes was investigated in Jurkat cells. A1 receptors were characterized using [3H]-DPCPX binding with a K(D) of 0.9+/-0.1 nM and B(max) of 42+/-3 fmol mg(-1) of protein. A2A receptors were studied with [3H]-SCH 58261 binding and revealed a K(D) of 2.5+/-0.3 nM and a B(max) of 1.4+/-0.2 pmol mg(-1) of protein. 7. In conclusion, by means of the first antagonist radioligand [3H]-MRE 3008F20 we could demonstrate the existence of functional A3 receptors on Jurkat cells.

Effects of two-carbon bridge region methoxylation of benztropine: discovery of novel chiral ligands for the dopamine transporter.

6-Methoxylated and 8-oxygenated benztropines were prepared and evaluated for their DAT and SERT activity (binding and uptake inhibition). Methoxylation at the two-carbon bridge of benztropine produced a novel class of potent and selective DAT ligands. An interesting enantioselectivity was also observed for this new class of chiral benztropines. The inactivity of the 8-oxygenated analogues seems to point out that, unlike cocaine and its analogues, interactions of benztropine ligands with DAT may be strongly governed by the nitrogen atom.

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as highly potent and selective human A(3) adenosine receptor antagonists: influence of the chain at the N(8) pyrazole nitrogen.

An enlarged series of pyrazolotriazolopyrimidines previously reported, in preliminary form (Baraldi et al. J. Med. Chem. 1999, 42, 4473-4478), as highly potent and selective human A(3) adenosine receptor antagonists is described. The synthesized compounds showed A(3) adenosine receptor affinity in the sub-nanomolar range and high levels of selectivity evaluated in radioligand binding assays at human A(1), A(2A), A(2B), and A(3) adenosine receptors. In particular, the effect of the chain at the N(8) pyrazole nitrogen was analyzed. This study allowed us to identify the derivative with the methyl group at the N(8) pyrazole combined with the 4-methoxyphenylcarbamoyl moiety at the N(5) position as the compound with the best binding profile in terms of both affinity and selectivity (hA(3) = 0.2 nM, hA(1)/hA(3) = 5485, hA(2A)/hA(3) = 6950, hA(2B)/hA(3) = 1305). All the compounds proved to be full antagonists in a specific functional model where the inhibition of cAMP generation by IB-MECA was measured in membranes of CHO cells stably transfected with the human A(3) receptor. The new compounds are among the most potent and selective A(3) antagonists so far described. The derivatives with higher affinity at human A(3) adenosine receptors proved to be antagonists, in the cAMP assay, capable of inhibiting the effect of IB-MECA with IC(50) values in the nanomolar range, with a trend strictly similar to that observed in the binding assay. Also a molecular modeling study was carried out, with the aim to identify possible pharmacophore maps. In fact, a sterically controlled structure-activity relationship was found for the N(8) pyrazole substituted derivatives, showing a correlation between the calculated molecular volume of pyrazolo[4,3-e]1,2, 4-triazolo[1,5-c]pyrimidine derivatives and their experimental K(i) values.

Synthesis and binding of 3-aminopyridine derivatives at central nicotinic receptors.

A series of secondary and tertiary pyridyl amides as potential central nicotinic acetylcholine receptors (nAChRs) ligands were prepared. Amides displayed negligible or very low affinity, whereas two amines achieved by reduction of corresponding secondary amides, showed affinity in the nanomolar range for nAChRs.

Dose and time effects of caffeine intake on human platelet adenosine A(2A) receptors : functional and biochemical aspects.

BACKGROUND-We determined whether repeated caffeine administration at different dosages and for different periods of time (400 or 600 mg/d for 1 week or 400 mg/d for 2 weeks) upregulates human platelet adenosine A(2A) receptors and is accompanied by increases in cAMP accumulation and decreases in aggregation and calcium levels after stimulation of A(2A) receptors by the selective agonist 2-hexynyl-5'-N-ethylcarboxamidoadenosine (HE-NECA). METHODS AND RESULTS-Platelets were obtained from peripheral venous blood of 45 healthy human volunteers at the end of 2 weeks of caffeine abstinence and at 12, 60, and 108 hours after the last dose of caffeine. The lowest dose of caffeine, when given for only 7 days, had no effect. Increasing the total dose, either by giving 400 mg/d for 14 days or giving 600 mg/d, resulted in binding assays performed with the adenosine A(2A) receptor radioligand [(3)H]SCH 58261 [5-amino-7(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1, 5-c]pyrimidine], in the upregulation of A(2A) receptors. Moreover, the potency of HE-NECA to produce antiaggregatory effects, a rise in cAMP accumulation, and a decrease in calcium levels was significantly increased. CONCLUSIONS-Chronic caffeine intake can lead to upregulation of adenosine A(2A) receptors, which is accompanied by sensitization, in a time- and dose-dependent manner, to the actions of the agonist HE-NECA.

[(3)H]MRE 3008F20: a novel antagonist radioligand for the pharmacological and biochemical characterization of human A(3) adenosine receptors.

The lack of a radiolabeled selective A(3) adenosine receptor antagonist is a major drawback for an adequate characterization of this receptor subtype. This paper describes the pharmacological and biochemical characterization of the tritiated form of a new potent A(3) adenosine receptor antagonist, the pyrazolo triazolo pyrimidine derivative [(3)H]5N-(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-furyl )pyrazolo [4,3-e] -1,2,4- triazolo[1,5-c]pyrimidine ([(3)H]MRE 3008F20). [(3)H]MRE 3008F20 bound specifically to the human adenosine A(3) receptor expressed in CHO cells (hA(3)CHO), and saturation analysis revealed a single high affinity binding site, K(D) = 0.80 +/- 0.06 nM, with a B(max) = 300 +/- 33 fmol/mg protein. This new ligand displayed high selectivity (1294-, 165-, and 2471-fold) in binding assay to human A(3) versus A(1), A(2A), and A(2B) receptors, respectively, and binds to the rat A(3) receptors with a K(i) > 10 microM. The pharmacological profile of [(3)H]MRE 3008F20 binding to hA(3)CHO cells was evaluated using known adenosine receptor agonists and antagonists with a rank order of potency consistent with that typically found for interactions with the A(3) adenosine receptors. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency identical with that observed in binding experiments. Thermodynamic data indicated that [(3)H]MRE 3008F20 binding to hA(3)CHO is entropy- and enthalpy-driven in agreement with the typical behavior of other adenosine antagonists to A(1) and A(2A) receptors. These results show that [(3)H]MRE 3008F20 is the first antagonist radioligand with high affinity and selectivity for the human A(3) adenosine receptor and may be used to investigate the physiopathological role of A(3) adenosine receptors.

Synthesis and preliminary biological evaluation of [3H]-MRE 3008-F20: the first high affinity radioligand antagonist for the human A3 adenosine receptors.

The synthesis and the preliminary biological evaluation of the first high affinity radioligand antagonist for the human A3 adenosine receptor, named [3H]-MRE 3008-F20 are reported. [3H]-MRE 3008-20 bound human A3 receptors expressed in CHO cells with K(D) and Bmax value of 0.82 +/- 0.08 nM and 297 +/- 28 fmol/mg of protein, respectively. [3H]-MRE 3008-F20 represents a useful tool for a further characterization of A3 adenosine receptor subtype.

A(3) adenosine receptor ligands: history and perspectives.

Adenosine regulates many physiological functions through specific cell membrane receptors. On the basis of pharmacological studies and molecular cloning, four different adenosine receptors have been identified and classified as A(1), A(2A), A(2B), and A(3). These adenosine receptors are members of the G-protein-coupled receptor family. While adenosine A(1) and A(2A) receptor subtypes have been pharmacologically characterized through the use of selective ligands, the A(3) adenosine receptor subtype is presently under study in order to better understand its physio-pathological functions. Activation of adenosine A(3) receptors has been shown to stimulate phospholipase C and D and to inhibit adenylate cyclase. Activation of A(3) adenosine receptors also causes the release of inflammatory mediators such as histamine from mast cells. These mediators are responsible for processes such as inflammation and hypotension. It has also been suggested that the A(3) receptor plays an important role in brain ischemia, immunosuppression, and bronchospasm in several animal models. Based on these results, highly selective A(3) adenosine receptor agonists and/or antagonists have been indicated as potential drugs for the treatment of asthma and inflammation, while highly selective agonists have been shown to possess cardioprotective effects. The updated material related to this field of research has been rationalized and arranged in order to offer an overview of the topic.

Synthesis and activity of 3-pyridylamine ligands at central nicotinic receptors.

A series of thirty 2-(3-pyridylaminomethyl)azetidine, pyrrolidine and piperidine analogues as nicotinic acetylcholine receptor (nAChR) ligands was explored. In general, pyrrolidinyl and many azetidinyl compounds were found to bind with enhanced affinity relative to the piperidines. In the three series, the parallel structural changes (stereochemistry, N-methylation and/or chloro substitution) do not consistently lead to parallel shifts in affinity. The more active compounds (K(i) affinity values ranging from 8.9 to 90 nM) were about as analgesic as nicotine in a tail-flick assay in mice after subcutaneous injections.

Caffeine alters A2A adenosine receptors and their function in human platelets.

BACKGROUND: Caffeine acts mainly via blockade of adenosine receptors, which have been classified into A1, A2A, A2B, and A3 subtypes. We determined whether repeated caffeine administration (750 mg/d for 1 week) upregulates the human platelet A2A adenosine receptor and is accompanied by sensitization of platelet responses (increase in cAMP accumulation and decrease in platelet aggregation) to selective stimulation of the A2A receptors. METHODS AND RESULTS: Platelets were obtained from peripheral venous blood of 9 human volunteers at the end of 1 week of caffeine abstinence (control) and at 12 and 60 hours after the last dose of caffeine (withdrawal). The A2A receptor radioligand [3H]SCH 58261 (5-amino-7(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1, 5-c]-pyrimidine) bound to a single affinity class of sites in platelet membranes from controls with a Bmax of 98+/-2 fmol/mg protein and a KD of 1.29+/-0.05 nmol/L. At 12 and 60 hours after caffeine withdrawal, the radioligand bound with similar affinity (KD=1.36+/-0.06 and 1.21+/-0.05 nmol/L, respectively), but the Bmax was increased (P<0.01) to 128+/-3 and 132+/-2 fmol/mg protein. The A2A receptor agonist 2-hexynyl-5'-N-ethylcarboxamidoadenosine (HE-NECA) increased cAMP accumulation (EC50=59+/-3 nmol/L) and inhibited (IC50=90+/-6 nmol/L) aggregation of control platelets. The EC50 values for HE-NECA to increase cAMP accumulation of platelets were reduced (P<0.01) at 12 and 60 hours after caffeine withdrawal (31+/-3 and 21+/-2 nmol/L, respectively). The IC50 values for HE-NECA to inhibit ADP-induced platelet aggregation were 50+/-5 and 30+/-2 nmol/L at 12 and 60 hours after caffeine withdrawal, respectively. CONCLUSIONS: Chronic caffeine intake leads to upregulation of A2A receptors and is accompanied by sensitization to the actions of the agonist HE-NECA.

Temporal mapping of transcripts in herpesvirus 6 variants.

To define the molecular features characteristic of the early stages of infection of lymphocytes with human herpesvirus 6 (HHV-6) variant A or B, we studied the temporal regulation of expression of selected sets of viral genes. Thus, U42, U94, U89-U90, U73, and U39 are alpha genes since their transcripts (i) were made in the presence of inhibitors of protein synthesis and (ii) were detected 3 h after infection of untreated cells. U41, U53, U31, and U19 are beta genes since their expression is inhibited by cycloheximide but not by phosphonoacetate, an inhibitor of DNA synthesis. U100 is a gamma gene since its spliced transcript encoding the structural glycoprotein gp82/105 was first detected 16 h after infection of untreated cells but could not be detected in cells treated with phosphonoacetate. HHV-6 variants differ in the transcription patterns of their genes. U16-U17 originates a splice transcript and is regulated as alpha in HHV-6B and as beta in HHV-6A. U91 generates two transcripts, amplified as 476- and 374-bp PCR fragments. The 476-bp fragment is alpha in HHV-6A-infected cells but beta in HHV-6B-infected cells. Conversely, the 374-bp fragment is beta in HHV-6A-infected cells and alpha in HHV-6B-infected cells. Furthermore, the spliced product of U18-U19-U20 (526 bp) is beta in HHV-6A-infected cells, but only a partially spliced form (1.9 kb) was detected at late stages of infection in HHV-6B. HHV-6 transcription was also studied in nonproductive lymphoid cells, and the same transcription pattern detected during lytic infection was observed. Also, HHV-6 variants maintain the differences in U91, U16-17, and U18-U19-U20. We conclude that, as expected from the sequencing data, gene expression is generally similar in HHV-6 variants. However, transcription of selected genes in HHV-6A and HHV-6B differs with respect to temporal regulation and splicing pattern. Furthermore, the identification of viral functions expressed during the different stages of lytic replication suggests that reverse transcription-PCR for HHV-6 genes is a useful diagnostic approach to differentiate between latent and productive HHV-6 infection.