Characterization of extracellular trap production and release by equine neutrophils in response to different stimuli.

This study explores Neutrophil Extracellular Trap (NET) formation in equine neutrophils, which is crucial for eliminating infections and is implicated in various equine inflammatory diseases. We investigated the molecular pathways involved in NET release by equine neutrophils in response to stimuli. We use PMA, A23187, LPS, PAF, OZ, and cytokines, observing NET release in response to PMA, PAF, and A23187. In contrast, LPS, OZ, and the cytokines tested did not induce DNA release or did not consistently induce citrullination of histone 4. Peptidyl-arginine deiminase inhibition completely halted NET release, while NADPH oxidase and mitochondrial reactive oxygen species only played a role in PMA-induced NETs. Neutrophil elastase inhibition modestly affected PAF-induced NET liberation but not in PMA or A23187-induced NET, while myeloperoxidase did not contribute to NET release. We expect to provide a foundation for future investigations into the role of NETs in equine health and disease and the search for potential therapeutic targets.

The phenotype of cryopreserved platelets influences the formation of platelet-leukocyte aggregates in an in vitro model.

Cryopreservation significantly alters the phenotype of platelets; generating distinct subpopulations, which may influence the formation of platelet leukocyte aggregates (PLA). PLAs are immunomodulatory and have been associated with transfusion-associated adverse events. As such, the aim of this study was to examine the effect of cryopreservation on the ability of platelets to form PLAs, using a monocyte-like cell line (THP-1). Platelets were tested pre-freeze, post-thaw and following stimulation with TRAP-6 or A23187, both alone and following co-culture with THP-1 cells for 1 and 24 hours (n = 6). Platelet subpopulations and platelet-THP-1 cell aggregates were analyzed using multi-color imaging flow cytometry using Apotracker Green (ApoT), CD42b, CD62P, CD61, and CD45. Cryopreservation resulted in the generation of activated (ApoT-/CD42b+/CD62P+), procoagulant (ApoT+/CD42b+/CD62P+) and a novel (ApoT+/CD42b+/CD62P-) platelet subpopulation. Co-incubation of cryopreserved platelets with THP-1 cells increased PLA formation compared to pre-freeze but not TRAP-6 or A23187 stimulated platelets. P-selectin on the surface membrane was correlated with increased PLA formation. Our findings demonstrate that cryopreservation increases the interaction between platelets and THP-1 cells, largely due to an increase in procoagulant platelets. Further investigation is required to determine the immunological consequences of this interaction.

What do we know? Cryopreserved platelets are an alternative to overcome issues with the short shelf-life of room-temperature stored plateletsAfter thawing, cryopreserved platelets exhibit changes in cell structure and receptor abundanceActivated platelets can attach to leukocytes, forming platelet-leukocyte aggregates and altering their immune functionPlatelet-leukocyte aggregates can increase inflammation, which is associated with adverse events after transfusion, which can negatively affect patient outcomesWhat did we discover? Cryopreservation results in a heterogenous mix of platelet subpopulationsCryopreserved platelets display increased adherence to a monocyte-like cell line (THP-1 cells). Platelet-THP-1 aggregate formation was linked to expression of CD62P on the surface of the plateletsThe increase in cryopreserved platelet-THP-1 cell aggregates was largely due to an increase in procoagulant plateletsWhat is the impact? Our data demonstrate that cryopreservation increases platelet interaction with a monocyte-like cell lineThis may mediate immune responses and/or circulation time of transfused platelets.

Calcium ionophore-activated platelets induce eosinophil extracellular trap formation.

BACKGROUND: Platelets play a modulatory role in inflammatory response by secreting a vast array of granules and disintegrating into membrane-bound microparticles upon activation. The interplay between eosinophils and platelets is postulated to be implicated in the pathology of allergic airway inflammation. In this study, we investigated whether activated platelets can induce eosinophil extracellular trap (EET) formation, a cellular process by which activated eosinophils release net-like DNA fibers. METHODS: Platelets were stimulated with the calcium ionophore, A23187, and the platelet agonists, thrombin and adenosine diphosphate (ADP). Platelet cultures were fractionated into conditioned medium (CM) and pellet, which were then overlaid on eosinophils to examine EET formation. RESULTS: The CM and pellet from A23187-activated platelets stimulated eosinophils to generate EET, whereas those from thrombin- or ADP-activated platelets failed to induce such generation. The EET-inducing activity of the A23187-activated platelet culture was linearly proportional to the number of activated platelets. Interestingly, while EET formation induced by the direct stimulation of eosinophils with A23187 was NADPH oxidase (NOX)-dependent, EET formation induced by A23187-activated platelets was NOX-independent and significantly inhibited by necroptosis pathway inhibitors. CONCLUSIONS: Activated platelets and their products may induce EET formation, thereby potentiating their role in eosinophilic airway inflammation.

Effects of platelets activated by different agonists on fibrin formation and thrombin generation.

Activated platelets possess procoagulant activity expressing on their surface phosphatidylserine (PS), a substrate for assembling coagulation complexes. We examined the effects of platelets activated by different agonists on fibrin formation and thrombin generation and compared these effects with each other and with PS expression. Modified plasma recalcification assay was developed to assess platelet effects on fibrin formation. Washed human platelets were left intact or activated by A23187 ionophore, collagen, arachidonic acid, ADP or TRAP (Thrombin Receptor Activating Peptide) and spun down in 96-well plates. Plasma was then added, recalcified, and fibrin formation was monitored by light absorbance. Platelets prepared in the same way were tested for their effect on thrombin generation. PS expression was evaluated by flow cytometry using annexin V staining. Platelets significantly accelerated fibrin formation and thrombin generation. They shortened lag phase and increased maximum rate of plasma clotting, and increased peak and maximum rate of thrombin generation. In both tests platelets were presumably activated by endogenous thrombin formed in plasma after triggering coagulation reactions. However, pretreatment with exogenous agonists additionally increased platelet procoagulant activity. It reached the maximum after incubation with A23187, being lower with collagen and arachidonic acid and minimum with ADP and TRAP (the latter might be ineffective due to competition with endogenous thrombin). The effects of platelets activated by different agonists on fibrin formation and thrombin generation correlate with each other and correspond to PS expression on their surface.

Why was the study done? Platelets and blood coagulation system interact with each other in hemostasis and intravascular thrombosis.Direct platelet effects on fibrin formation (plasma clotting), the final stage of blood coagulation cascade, have been insufficiently studied.The work is aimed at developing a method for studying platelet participation in fibrin formation in blood plasma and investigating the influence of platelet agonists on this reaction.What is new? Platelets significantly accelerate fibrin formation and their activation with various agonists (thrombin, collagen, arachidonic acid) enhances these effects.Effects of platelets on fibrin formation correlated with their ability to stimulate thrombin generation in blood plasmaEffects of platelets on fibrin formation and thrombin generation correlated with the level of phosphatidylserine exposure on their surfaceWhat is the impact? This study provides further evidence that platelet procоagulant effects on fibrin formation should be considered in investigations of platelet involvement in hemostatic and thrombotic reactions and in the evaluation of the efficacy of antiplatelet drugs.

Induction and characterization of extracellular traps by gilthead seabream (Sparus aurata L.) head-kidney leucocytes.

The aim of this study was the induction and characterization of extracellular traps (ETs) produced by gilthead seabream (Sparus aurata L.) head-kidney leucocytes. The cells were incubated several times (10, 30, 60, 120, and 180 min) with different concentrations of the stimulants diluted in RPMI-1640 culture medium: RPMI-1640 (control), ß-glucan from Saccharomyces cerevisiae (BG, 0-400 µg mL-1), lipopolysaccharide from Escherichia coli (LPS, 0-10 µg mL-1), calcium ionophore A23187 (CaI, 0-5 µg mL-1), Phorbol 12-myristate 13-acetate (PMA, 0-1000 ng mL-1) and polyinosinic-polycytidylic acid sodium salt (Poly I:C, 0-200 µg mL-1). BG, LPS and CaI exerted only weak stimulatory activity, while PMA and poly I:C exerted a potent one. After stimulation of the leucocytes, ETs structures were quantified and visualised through staining of the chromatin with nucleic acid-specific dyes and immunocytochemical probing of characteristic proteins expected to decorate the structure. ETs structures had DNA and myeloperoxidase. The ETs morphology was studied by light and scanning electron microscopy. These data confirm that seabream leucocytes form ETs with different morphological properties, depending on the used stimulant. These results will be the basis for new studies to analyse the implication of this mechanism in fish immunity. All this new knowledge will have its application in fish farms when we learn to manipulate the innate immune response in order to mitigate microbial infections.

Extracellular vesicles from A23187-treated neutrophils cause cGAS-STING-dependent IL-6 production by macrophages.

In response to several types of bacteria, as well as pharmacological agents, neutrophils produce extracellular vesicles (EVs) and release DNA in the form of neutrophil extracellular traps (NETs). However, it is unknown whether these two neutrophil products cooperate to modulate inflammation. Consistent with vital NETosis, neutrophils challenged with S. aureus, as well as those treated with A23187, released significantly more DNA relative to untreated or fMLF-treated neutrophils, with no lysis occurring for any condition. To test the hypothesis that EVs generated during NETosis caused macrophage inflammation, we isolated and characterized EVs from A23187-treated neutrophils (A23187-EVs). A23187-EVs associated with neutrophil granule proteins, histone H3, transcription factor A, mitochondrial (TFAM), and nuclear and mitochondrial DNA (mtDNA). We showed that DNA from A23187-EVs, when transfected into macrophages, led to production of IL-6 and IFN-α2, and this response was blunted by pre-treatment with the STING inhibitor H151. Next, we confirmed that A23187-EVs were engulfed by macrophages, and showed that they induced cGAS-STING-dependent IL-6 production. In contrast, neither EVs from untreated or fMLF-treated cells exhibited pro-inflammatory activity. Although detergent-mediated lysis of A23187-EVs diminished IL-6 production, removal of surface-associated DNA with DNase I treatment had no effect, and A23187-EVs did not induce IFN-α2 production. Given these unexpected results, we investigated whether macrophage mtDNA activated the cGAS-STING signaling axis. Consistent with mitochondrial outer membrane permeabilization (MOMP), a defined mechanism of mtDNA release, we observed macrophage mitochondrial membrane depolarization, a decrease in cytosolic Bax, and a decrease in mitochondrial cytochrome c, suggesting that macrophage mtDNA may initiate this EV-dependent signaling cascade. All together, these data demonstrate that A23187-EVs behave differently than transfected NET- or EV-DNA, and that neutrophil-derived EVs could be used as a model to study NF-κB-dependent STING activation.

Calcimycin Inhibits Cryptococcus neoformans In Vitro and In Vivo by Targeting the Prp8 Intein Splicing.

Drug resistance is a significant concern in the treatment of diseases, including cryptococcosis caused by Cryptococcus neoformans (Cne) and Cryptococcus gattii (Cga). Alternative drug targets are necessary to overcome drug resistance before it attains a critical stage. Splicing of inteins from pro-protein precursors is crucial for activities of essential proteins hosting intein elements in many organisms, including human pathogens such as Cne and Cga. Through a high-throughput screening, we identified calcimycin (CMN) as a potent Prp8 intein splicing inhibitor with a minimum inhibitory concentration (MIC) of 1.5 µg/mL against the wild-type Cne-H99 (Cne-WT or Cne). In contrast, CMN inhibited the intein-less mutant strain (Cne-Mut) with a 16-fold higher MIC. Interestingly, Aspergillus fumigatus and a few Candida species were resistant to CMN. Further studies indicated that CMN reduced virulence factors such as urease activity, melanin production, and biofilm formation in Cne. CMN also inhibited Cne intracellular infection in macrophages. In a target-specific split nanoluciferase assay, the IC50 of CMN was 4.6 µg/mL. Binding of CMN to recombinant Prp8 intein was demonstrated by thermal shift assay and microscale thermophoresis. Treating Cne cells with CMN reduced intein splicing. CMN was fungistatic and showed a synergistic effect with the known antifungal drug amphotericin B. Finally, CMN treatment at 20 mg/kg body weight led to 60% reduction in lung fungal load in a cryptococcal pulmonary infection mouse model. Overall, CMN represents a potent antifungal with a novel mechanism of action to treat Cne and possibly Cga infections.

Ca2+ participates in programmed cell death by modulating ROS during pollen cryopreservation.

KEY MESSAGE: After cryopreservation, the Ca2+ content increased, which affected the intracellular ROS content, then participated in the occurrence of programmed cell death in pollen. Programmed cell death (PCD) is one of the reasons for the decline in pollen viability after cryopreservation. However, the role of calcium ions (Ca2+) in PCD during pollen cryopreservation has not been revealed in the existing studies. In this study, Paeonia lactiflora 'Fen Yu Nu' pollen was used as the research material for investigating the effects of Ca2+ changes on PCD indices and reactive oxygen species (ROS) during pollen cryopreservation. The results showed that after cryopreservation, with the decrease of pollen viability, the Ca2+ content significantly increased. The regulation of Ca2+ content had a significant effect on PCD indices, which showed that the Ca2+ carrier A23187 accelerated the decrease of mitochondrial membrane potential level and increased the activity of caspase-3-like and caspase-9-like proteases and the apoptosis rate. The expression levels of partial pro-PCD genes were upregulated, the anti-PCD gene BI-1 was downregulated, and the addition of Ca2+-chelating agent EGTA had the opposite effect. The addition of the Ca2+ carrier A23187 after cryopreservation significantly increased the ROS content of pollen, the addition of the Ca2+-chelating agent EGTA had the opposite effect, and Ca2+ regulators also had significant effects on the contents of ROS production and clearance-related substances. Ca2+ affected intracellular ROS content by acting on the ROS production and clearance system during the cryopreservation of pollen and is thus involved in the occurrence of PCD.

17-ß-estradiol enhances neutrophil extracellular trap formation by interaction with estrogen membrane receptor.

Cell death-associated neutrophil extracellular trap formation (NETosis) occurs during various autoimmune diseases including systemic lupus erythematosus and rheumatoid arthritis, as well as during gestation. Although increasing estrogen concentrations associated with pregnancy might induce NETosis via nuclear estrogen receptor (ERα/ERß), little is known about the mechanisms associated with estrogen-induced NETosis. Here, we investigated the effects of estrogen (17-ß-estradiol; E2) on NETosis, focusing on mechanisms associated with estrogen membrane receptor (GPR30) in neutrophil-like HL-60 cells. Our results show that E2 and the GPR30 agonist G-1 increases level of NETosis and NET formation. Moreover, NETosis-associated intracellular and extracellular histone citrullination and peptidyl arginine deiminase 4 (PAD4) expression were also increased by E2 or G-1 treatment. Furthermore, GPR30 antagonist pre-treatment inhibited increases in NETosis and PAD4 expression mediated by G-1 and partially inhibited these effects mediated by E2. These results demonstrate that E2 treatment induces NETosis via not only ERα/ERß but also GPR30 in neutrophil-like HL-60 cells.

Cezomycin Is Activated by CalC to Its Ester Form for Further Biosynthesis Steps in the Production of Calcimycin in Streptomyces chartreusis NRRL 3882.

Calcimycin, N-demethyl calcimycin, and cezomycin are polyether divalent cation ionophore secondary metabolites produced by Streptomyces chartreusis A thorough understanding of the organization of their encoding genes, biosynthetic pathway(s), and cation specificities is vitally important for their efficient future production and therapeutic use. So far, this has been lacking, as has information concerning any biosynthetic relationships that may exist between calcimycin and cezomycin. In this study, we observed that when a Cal- (calB1 mutant) derivative of a calcimycin-producing strain of S. chartreusis (NRRL 3882) was grown on cezomycin, calcimycin production was restored. This suggested that calcimycin synthesis may have resulted from postsynthetic modification of cezomycin rather than from a de novo process through a novel and independent biosynthetic mechanism. Systematic screening of a number of Cal-S. chartreusis mutants lacking the ability to convert cezomycin to calcimycin allowed the identification of a gene, provisionally named calC, which was involved in the conversion step. Molecular cloning and heterologous expression of the CalC protein along with its purification to homogeneity and negative-staining electron microscopy allowed the determination of its apparent molecular weight, oligomeric forms in solution, and activity. These experiments allowed us to confirm that the protein possessed ATP pyrophosphatase activity and was capable of ligating coenzyme A (CoA) with cezomycin but not 3-hydroxyanthranilic acid. The CalC protein's apparent Km and kcat for cezomycin were observed to be 190 µM and 3.98 min-1, respectively, and it possessed the oligomeric form in solution. Our results unequivocally show that cezomycin is postsynthetically modified to calcimycin by the CalC protein through its activation of cezomycin to a CoA ester form.IMPORTANCE Calcimycin is a secondary metabolite divalent cation-ionophore that has been studied in the context of human health. However, detail is lacking with respect to both calcimycin's biosynthesis and its biochemical/biophysical properties as well as information regarding its, and its analogues', divalent cation binding specificities and other activities. Such knowledge would be useful in understanding how calcimycin and related compounds may be effective in modifying the calcium channel ion flux and might be useful in influencing the homeostasis of magnesium and manganese ions for the cure or control of human and bacterial infectious diseases. The results presented here unequivocally show that CalC protein is essential for the production of calcimycin, which is essentially a derivative of cezomycin, and allow us to propose a biosynthetic mechanism for calcimycin's production.

Diverse stimuli engage different neutrophil extracellular trap pathways.

Neutrophils release neutrophil extracellular traps (NETs) which ensnare pathogens and have pathogenic functions in diverse diseases. We examined the NETosis pathways induced by five stimuli; PMA, the calcium ionophore A23187, nigericin, Candida albicans and Group B Streptococcus. We studied NET production in neutrophils from healthy donors with inhibitors of molecules crucial to PMA-induced NETs including protein kinase C, calcium, reactive oxygen species, the enzymes myeloperoxidase (MPO) and neutrophil elastase. Additionally, neutrophils from chronic granulomatous disease patients, carrying mutations in the NADPH oxidase complex or a MPO-deficient patient were examined. We show that PMA, C. albicans and GBS use a related pathway for NET induction, whereas ionophores require an alternative pathway but that NETs produced by all stimuli are proteolytically active, kill bacteria and composed mainly of chromosomal DNA. Thus, we demonstrate that NETosis occurs through several signalling mechanisms, suggesting that extrusion of NETs is important in host defence.

Atomic modelling and systematic mutagenesis identify residues in multiple drug binding sites that are essential for drug resistance in the major Candida transporter Cdr1.

The ABC (ATP-Binding Cassette) transporter Cdr1 (Candida drug resistance 1) protein (Cdr1p) of Candida albicans, shows promiscuity towards the substrate it exports and plays a major role in antifungal resistance. It has two transmembrane domains (TMDs) comprising of six transmembrane helices (TMH) that envisage and confer the substrate specificity and two nucleotide binding domains (NBDs), interconnected by extracellular loops (ECLs) and intracellular loops (ICLs) Cdr1p. This study explores the diverse substrate specificity spectrum to get a deeper insight into the structural and functional features of Cdr1p. By screening with the variety of compounds towards an in-house TMH 252 mutant library of Cdr1p, we establish new substrates of Cdr1p. The localization of substrate-susceptible mutants in an ABCG5/G8 homology model highlights the common and specific binding pockets inside the membrane domain, where rhodamines and tetrazoliums mainly engage the N-moiety of Cdr1p, binding between TMH 2, 11 and surrounded by TMH 1, 5. Whereas, tin chlorides involve both N and C moieties located at the interface of TMH 2, 11, 1 and 5. Further, screening of the in house TMH mutant library of Cdr1p displays the TMH12 interaction with tetrazolium chloride, trimethyltin chloride and a Ca2+ ionophore, A23187. In silico localization reveals a binding site at the TMH 12, 9 and 10 interface, which is widely exposed to the lipid interface. Together, for the first time, our study shows the molecular localization of Cdr1p substrates-binding sites and demonstrates the participation of TMH12 in a peripheral drug binding site.

Potential interactions of calcium-sensitive reagents with zinc ion in different cultured cells.

BACKGROUND: Several chemicals have been widely used to evaluate the involvement of free Ca(2+) in mechanisms underlying a variety of biological responses for decades. Here, we report high reactivity to zinc of well-known Ca(2+)-sensitive reagents in diverse cultured cells. METHODOLOGY/PRINCIPAL FINDINGS: In rat astrocytic C6 glioma cells loaded with the fluorescent Ca(2+) dye Fluo-3, the addition of ZnCl2 gradually increased the fluorescence intensity in a manner sensitive to the Ca(2+) chelator EGTA irrespective of added CaCl2. The addition of the Ca(2+) ionophore A23187 drastically increased Fluo-3 fluorescence in the absence of ZnCl2, while the addition of the Zn(2+) ionophore pyrithione rapidly and additionally increased the fluorescence in the presence of ZnCl2, but not in its absence. In cells loaded with the zinc dye FluoZin-3 along with Fluo-3, a similarly gradual increase was seen in the fluorescence of Fluo-3, but not of FluoZin-3, in the presence of both CaCl2 and ZnCl2. Further addition of pyrithione drastically increased the fluorescence intensity of both dyes, while the addition of the Zn(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) rapidly and drastically decreased FluoZin-3 fluorescence. In cells loaded with FluoZin-3 alone, the addition of ZnCl2 induced a gradual increase in the fluorescence in a fashion independent of added CaCl2 but sensitive to EGTA. Significant inhibition was found in the vitality to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide in a manner sensitive to TPEN, EDTA and BAPTA in C6 glioma cells exposed to ZnCl2, with pyrithione accelerating the inhibition. Similar inhibition occurred in an EGTA-sensitive fashion after brief exposure to ZnCl2 in pluripotent P19 cells, neuronal Neuro2A cells and microglial BV2 cells, which all expressed mRNA for particular zinc transporters. CONCLUSIONS/SIGNIFICANCE: Taken together, comprehensive analysis is absolutely required for the demonstration of a variety of physiological and pathological responses mediated by Ca(2+) in diverse cells enriched of Zn(2+).

Lysosomal NEU1 deficiency affects amyloid precursor protein levels and amyloid-ß secretion via deregulated lysosomal exocytosis.

Alzheimer's disease (AD) belongs to a category of adult neurodegenerative conditions, which are associated with intracellular and extracellular accumulation of neurotoxic protein aggregates. Understanding how these aggregates are formed, secreted and propagated by neurons has been the subject of intensive research, but so far no preventive or curative therapy for AD is available, and clinical trials have been largely unsuccessful. Here we show that deficiency of the lysosomal sialidase NEU1 leads to the spontaneous occurrence of an AD-like amyloidogenic process in mice. This involves two consecutive events linked to NEU1 loss-of-function--accumulation and amyloidogenic processing of an oversialylated amyloid precursor protein in lysosomes, and extracellular release of Aß peptides by excessive lysosomal exocytosis. Furthermore, cerebral injection of NEU1 in an established AD mouse model substantially reduces ß-amyloid plaques. Our findings identify an additional pathway for the secretion of Aß and define NEU1 as a potential therapeutic molecule for AD.

Calcium ameliorates renal cyst growth in metanephric organ culture: a morphological study.

Polycystic kidney disease (PKD) is associated with alterations in developmental processes that severely affect kidney integrity, often leading to fatal consequences. It has been suggested that dysfunctional calcium (Ca2+) regulation associated with the PKD phenotype is consequent to mutations affecting the pkd1 gene. Previously, it has been observed that blocking calcium along with cAMP allowed tubular epithelial cells to enter the proliferative phase that culminated in a cyst-like phenotype. In this regard, mouse metanephroi, (embryonic day 13.5, E13.5) were used to study morphological and ultrastructural effects of calcium replenishment on 8-bromocyclic 3'5'cyclic adenosine monophosphate (8-Br-cAMP)-induced cyst-like tubular dilations. Phase contrast microscopy of 8-Br-cAMP-treated metanephroi exhibited numerous dilated tubules that continued to increase in size for 4 days in culture. The effects of 8-Br-cAMP on renal tubular epithelia were assessed by histopathological and electron microscopic analyses. Transmission electron microscopy revealed changes such as increased vacuolation, swollen mitochondria, chromatin condensation, and disrupted cell membrane in tubular epithelia of 8-Br-cAMP-treated metanephroi. Concurrent treatments with calcium-channel agonists (calcium ionophore A23187 and phorbol-12-myristate-13-acetate) and 8-Br-cAMP abolished cAMP-induced morphometric and ultrastructural alterations. Calcium replenishment rescued tubular epithelial cells from mitogenic effects of cAMP and restored normal morphology at cellular and sub-cellular levels as verified by histopathological and ultrastructural examinations.

Involvement of PI3K/Akt signaling in PTTH-stimulated ecdysteroidogenesis by prothoracic glands of the silkworm, Bombyx mori.

The prothoracicotropic hormone (PTTH) stimulates ecdysteroidogenesis by prothoracic gland in larval insects. Previous studies showed that Ca(2+), cAMP, extracellular signal-regulated kinase (ERK), and tyrosine kinase are involved in PTTH-stimulated ecdysteroidogenesis by the prothoracic glands of both Bombyx mori and Manduca sexta. In the present study, the involvement of phosphoinositide 3-kinase (PI3K)/Akt signaling in PTTH-stimulated ecdysteroidogenesis by B. mori prothoracic glands was further investigated. The results showed that PTTH-stimulated ecdysteroidogenesis was partially blocked by LY294002 and wortmannin, indicating that PI3K is involved in PTTH-stimulated ecdysteroidogenesis. Akt phosphorylation in the prothoracic glands appeared to be moderately stimulated by PTTH in vitro. PTTH-stimulated Akt phosphorylation was inhibited by LY294002. An in vivo PTTH injection into day 6 last instar larvae also increased Akt phosphorylation of the prothoracic glands. In addition, PTTH-stimulated ERK phosphorylation of the prothoracic glands was not inhibited by either LY294002 or wortmannin, indicating that PI3K is not involved in PTTH-stimulated ERK signaling. A23187 and thapsigargin, which stimulated B. mori prothoracic gland ERK phosphorylation and ecdysteroidogenesis, could not activate Akt phosphorylation. PTTH-stimulated ecdysteroidogenesis was not further activated by insulin, indicating the absence of an additive action of insulin and PTTH on the prothoracic glands. The present study, together with the previous demonstration that insulin stimulates B. mori ecdysteroidogenesis through PI3K/Akt signaling, suggests that crosstalk exists in B. mori prothoracic glands between insulin and PTTH signaling, which may play a critical role in precisely regulated ecdysteroidogenesis during development.

Chronic administration of BMS309403 improves endothelial function in apolipoprotein E-deficient mice and in cultured human endothelial cells.

BACKGROUND AND PURPOSE: Adipocyte fatty acid-binding protein (A-FABP) is up-regulated in regenerated endothelial cells and modulates inflammatory responses in macrophages. Endothelial dysfunction accompanying regeneration is accelerated by hyperlipidaemia. Here, we investigate the contribution of A-FABP to the pathogenesis of endothelial dysfunction in the aorta of apolipoprotein E-deficient (ApoE(-/-) ) mice and in cultured human endothelial cells. EXPERIMENTAL APPROACH: A-FABP was measured in aortae of ApoE(-/-) mice and human endothelial cells by RT-PCR, immunostaining and immunoblotting. Total and phosphorylated forms of endothelial nitric oxide synthase (eNOS) were measured by immunoblotting. Changes in isometric tension were measured in rings of mice aortae KEY RESULTS: A-FABP was expressed in aortic endothelium of ApoE(-/-) mice aged 12 weeks and older, but not at 8 weeks or in C57 wild-type mice. Reduced endothelium-dependent relaxations to acetylcholine, UK14304 (selective α(2) -adrenoceptor agonist) and A23187 (calcium ionophore) and decreased protein presence of phosphorylated and total eNOS were observed in aortae of 18 week-old ApoE(-/-) mice compared with age-matched controls. A 6 week treatment with the A-FABP inhibitor, BMS309403, started in 12 week-old mice, improved endothelial function, phosphorylated and total eNOS and reduced plasma triglyceride levels but did not affect endothelium-independent relaxations. The beneficial effect of BMS309403 on UK14304-induced relaxations was attenuated by Pertussis toxin. In cultured human microvascular endothelial cells, lipid-induced A-FABP expression was associated with reduced phosphorylated eNOS and NO production and was reversed by BMS309403. CONCLUSIONS AND IMPLICATIONS: Elevated expression of A-FABP in endothelial cells contributes to their dysfunction both in vivo and in vitro.

In vivo reorganization of the actin cytoskeleton in leaves of Nicotiana tabacum L. transformed with plastin-GFP. Correlation with light-activated chloroplast responses.

BACKGROUND: The actin cytoskeleton is involved in the responses of plants to environmental signals. Actin bundles play the role of tracks in chloroplast movements activated by light. Chloroplasts redistribute in response to blue light in the mesophyll cells of Nicotiana tabacum. The aim of this work was to study the relationship between chloroplast responses and the organization of actin cytoskeleton in living tobacco cells. Chloroplast movements were measured photometrically as changes in light transmission through the leaves. The actin cytoskeleton, labeled with plastin-GFP, was visualised by confocal microscopy. RESULTS: The actin cytoskeleton was affected by strong blue and red light. No blue light specific actin reorganization was detected. EGTA and trifluoperazine strongly inhibited chloroplast responses and disrupted the integrity of the cytoskeleton. This disruption was reversible by Ca(2+) or Mg(2+). Additionally, the effect of trifluoperazine was reversible by light. Wortmannin, an inhibitor of phosphoinositide kinases, potently inhibited chloroplast responses but did not influence the actin cytoskeleton at the same concentration. Also this inhibition was reversed by Ca(2+) and Mg(2+). Magnesium ions were equally or more effective than Ca(2+) in restoring chloroplast motility after treatment with EGTA, trifluoperazine or wortmannin. CONCLUSION: The architecture of the actin cytoskeleton in the mesophyll of tobacco is significantly modulated by strong light. This modulation does not affect the direction of chloroplast redistribution in the cell. Calcium ions have multiple functions in the mechanism of the movements. Our results suggest also that Mg(2+) is a regulatory molecule cooperating with Ca(2+) in the signaling pathway of blue light-induced tobacco chloroplast movements.

The development of methodology for clinical measurement of 5-lipoxygenase pathway intermediates from human peripheral blood mononuclear cells.

Recent studies have shown a correlation between 5-lipoxygenase (5-LO) pathway up-regulation and cardiovascular risk. Despite the existence of several assays for products of the 5-LO pathway, a reliable method for clinical determination of 5-LO activity remains to be established. In the present communication, we report conditions that allow measurement of 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B(4) (LTB(4)) in peripheral blood mononuclear cells (PBMCs) isolated from the blood of atherosclerosis patients before and after stimulation by the calcium ionophore, A23187. LTB(4), a potent mediator of inflammation-linked cardiovascular disease, was measured using an existing competitive enzyme immunoassay (EIA) kit after making specific methodological improvements that allowed PBMCs to be used in this format for the first time. LTB(4) was also measured by LC/MS/MS along with 5-HETE, a direct by-product of the action of 5-LO on arachidonic acid and a molecule for which no commercial EIA kit exists. The LC/MS/MS assay was validated over a range of 0.025-25ng/mL for LTB(4) and 0.1-25ng/mL for 5-HETE. The EIA method has a validated range covering 0.025-4ng/mL. When both assays were applied to analyze LTB(4) from stimulated PBMCs isolated from 25 subjects with various degrees of atherosclerosis, a high correlation was obtained (r=0.9426, Pearson's correlation coefficient). A high correlation was also observed between the levels of LTB(4) and 5-HETE measured by LC/MS/MS after ionophore stimulation (r=0.9159). Details are presented for optimized sample collection, processing, storage, and analysis in accordance with the logistical demands of clinical analysis.

Caffeine treatment prevents age-related changes in ovine oocytes and increases cell numbers in blastocysts produced by somatic cell nuclear transfer.

Maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are key regulators of both meiotic and mitotic cycles. Oocytes arrested at metaphase of the second meiotic division (MII) contain high levels of both kinases; however, these activities decline with age. Caffeine (an inhibitor of Myt1/Wee1 activity) can increase MPF and MAPK activities in ovine oocytes; however, the effects of caffeine treatment on the activation, nuclear configuration and developmental potential of ovine SC nuclear transfer (SCNT) embryos were unknown. We examined the effects of aging and caffeine treatment on MPF and MAPK activities, activation, development, and nuclear remodeling of SCNT embryos. Both kinases reached maximum activities at 24-h postonset of maturation (hpm) and then decreased with time. The decline in MPF activity occurred rapidly, whereas MAPK activity declined more slowly. Caffeine treatment (10.0 mM) of aging oocytes prevented the decline in activities associated with both kinases and prevented the acquisition of activation competence by a single activation stimulus. However, treatment of aged oocytes with caffeine could not increase kinase activities or reverse the acquisition of activation competence. Enucleation did not affect kinase activities, but caffeine treatment significantly increased both. Caffeine treatment did not affect the decline in MPF or MAPK activities following activation or significantly affect development of parthenogenetically activated oocytes. When SCNT reconstructed embryos were treated with caffeine following fusion, no increase in the frequency of development to blastocyst was observed; however, a significant increase in the occurrence of nuclear envelope break-down (NEBD) and an increase in total cell numbers occurred.