Phorbol Ester Research Articles

Hydroxy-octadecenoic acids instead of phorbol esters are responsible for the Jatropha curcas kernel cake\u2019s toxicity

The toxic kernel cake of Jatropha curcas (KCakeJ) is an emerging health and environmental concern. Although phorbol esters are widely recognized as the major toxin of KCakeJ, convincing evidence is absent. Here, we show that rather than phorbol esters an isomeric mixture of 11-hydroxy-9E-octadecenoic acid, 12-hydroxy-10E-octadecenoic acid and 12-hydroxy-10Z-octadecenoic acid (hydroxy-octadecenoic acids, molecular formula C18H34O3) is the major toxic component. The toxicities of hydroxy-octadecenoic acids on experimental animals, e.g. acute lethality, causing inflammation, pulmonary hemorrhage and thrombi, allergies, diarrhea and abortion, are consistent with those on human/animals caused by Jatropha seed and/or KCakeJ. The hydroxyl group and the double bond are essential for hydroxy-octadecenoic acids’ toxicity. The main pathway of the toxicity mechanism includes down-regulating UCP3 gene expression, promoting ROS production, thus activating CD62P expression (platelet activation) and mast cell degranulation. The identification of the major toxin of KCakeJ lays a foundation for establishing an environmentally friendly Jatropha biofuel industry.

Human cytomegalovirus major immediate early transcripts arise predominantly from the canonical major immediate early promoter in reactivating progenitor-derived dendritic cells.

Human cytomegalovirus latency and reactivation is a major source of morbidity in immune-suppressed patient populations. Lifelong latent infections are established in CD34+progenitor cells in the bone marrow, which are hallmarked by a lack of major lytic gene expression, genome replication and virus production. A number of studies have shown that inhibition of the major immediate early promoter (MIEP) – the promoter that regulates immediate early (IE) gene expression – is important for the establishment of latency and that, by extension, reactivation requires reversal of this repression of the MIEP. The identification of novel promoters (termed ip1 and ip2) downstream of the MIEP that can drive IE gene expression has led to speculation over the precise role of the MIEP in reactivation. In this study we show that IE transcripts arise from both the MIEP and ip2 promoter in the THP1 cell macrophage cell line and also CD14+monocytes stimulated with phorbol ester. In contrast, we show that in in vitro generated dendritic cells or macrophages that support HCMV reactivation IE transcripts arise predominantly from the MIEP and not the intronic promoters. Furthermore, inhibition of histone modifying enzyme activity confirms the view that the MIEP is predominantly regulated by the activity of cellular chromatin. Finally, we observe that ip2-derived IE transcription is cycloheximide-sensitive in reactivating DCs, behaviour consistent with an early gene designation. Taken together, these data argue that MIEP activity is still important for HCMV reactivation but ip2 activity could play cell-type-specific roles in reactivation.

Phorbol ester degradation using chemical treatment in jatropha kernel meal

Detoxification of jatropha (Jatropha curcas) kernel meal is of major interest for the biodiesel industry to add economic value to this residue and also to reduce the environmental damage caused by its inappropriate disposal. Anti-nutritional factors present in jatropha kernel meal are phorbol esters, lectins, trypsin inhibitor, phytate and saponins. Toxicity of meal is mainly due to the presence of phorbol esters. Several methods have been tried for detoxifying kernel meal that includes physical, chemical, biological and radiation methods. In the study, four different samples, i.e., raw, defatted, one-time mechanically oil expressed and two-times mechanically oil expressed samples were prepared from jatropha kernels. These samples were subjected to chemical treatment for phorbol ester degradation. Chemical treatment involved heating the samples with 90% methanol and 4% NaOH twice. Phorbol ester content was estimated using standard analytical procedure before and after the treatment. In chemically treated kernel meal, phorbol esters were found to be in range of 0.034-0.052 mg/g.

Abstract A31: Germline RASopathy mutations provide functional insights into the Raf cysteine-rich domain (CRD)

Abstract The three-member RAF kinase family (A-, B- and C-Raf) are direct Ras effectors and key mediators of the proproliferative MAPK pathway. Somatic driver mutations in the B-Raf kinase domain occur frequently in melanoma and cancers of the thyroid and large intestine. These are predominantly the V600E mutation, which allows B-Raf to signal constitutively as a monomer. Germline mutations in B-Raf also occur and are present in ~67% of the childhood developmental RASopathy-cardiofaciocutaneous (CFC) syndrome. Interestingly, many of these CFC mutations cluster around the cysteine-rich domain (CRD) in the B-Raf regulatory region. CRDs are zinc stabilized structures with two flexible loops that are shown to bind phosphatidylserine (PS), DAG and pro-oncogenic phorbol esters in several PKC family members. Although the B-Raf CRD has not been well characterized, prior studies examining the C-Raf CRD show that it binds to PS through its first flexible loop but does not bind DAG or phorbol esters, likely due to its truncated second loop, which is found in all Raf members. In addition to PS binding, C-Raf CRD may also interact directly with Ras, suggesting a second Ras binding site outside of its Ras binding domain (RBD). Interestingly, B-Raf RASopathy mutations occur in both CRD loops and commonly introduce a positively charged residue suited to interacting with negatively charged lipids in the plasma membrane (PM). Of note, Q257R, which is the most common mutation in CFC, maps to the truncated second loop. Our in-cell studies with full-length B-Raf have shown that RASopathy mutations in both loops enhance biologic activity, PM localization and Ras interaction. However, the biologic activity of loop 1 mutations requires an intact RBD, which is not the case for the loop 2 mutant, Q257R. These data suggest that the Q257R mutant either has increased binding to Ras or allows B-Raf to signal independently of Ras. To further investigate these findings, we are examining differences in localization, Ras interaction and lipid binding using the isolated CRDs of C-Raf, B-Raf and the B-Raf RASopathy mutants. As may be predicted, the RASopathy mutations increased membrane localization over WT B-Raf, with Q257R being the most PM localized. Somewhat surprisingly, expression of the wild-type B-Raf CRD in cells showed greatly enhanced PM localization compared to that of wild-type C-Raf, suggesting functional differences between their CRDs, which we are currently investigating. Citation Format: Russell Spencer-Smith, Elizabeth M. Terrell, Constance Agamasu, Daniel A. Ritt, Alyson K. Freeman, Andrew G. Stephen, Deborah K. Morrison. Germline RASopathy mutations provide functional insights into the Raf cysteine-rich domain (CRD) [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A31.

Single-Cell RNA Sequencing Identifies a Novel Population of CD4+CD8+ T Cells that are Regulated by COX-2 During Allergic Lung Inflammation

Abstract To better understand the relative contributions of different cell types during allergic lung inflammation and the role of cyclooxygenase (COX)-derived eicosanoids in this process, we compared vehicle (control) and ovalbumin (OVA)-exposed lungs from wild type (WT) and COX-2−/− mice by single-cell RNA sequencing (scRNA-Seq). Twenty-two unique cell clusters were identified in allergic lungs based on cell signature markers. In addition to known cell types, we identified 2 clusters of CD4+CD8+. Both populations were mitotically active (high % in S/G2/M phases) and exhibited a unique molecular identifier (UMI) index. FACS analysis confirmed the presence of these two distinct cell populations (CD4+HCD8+H and CD4+LCD8+H) that were increased after OVA-induced allergic lung inflammation. Interestingly, isolated CD4+HCD8+H T cells stimulated with phorbol ester expressed IL-4, IL-5 and IL-13, but not IFNγ. In contrast, isolated and stimulated CD4+LCD8+H T cells primarily expressed IL-5. Mass cytometry (CyTOF) using a 37-antibody panel confirmed the increase in CD4+CD8+ T cells after OVA sensitization/exposure, as well as the induction of IL-4, IL-5 and IL-13. Importantly, CD4+CD8+-1 and CD4+CD8+-2 cell populations identified by scRNA-Seq were increased in lungs from OVA-treated COX-2−/− mice compared to lungs from OVA-treated WT mice. Furthermore, COX-2−/− CD4+CD8+ T cells preferentially produced more IL-4 and IL-5 than WT CD4+CD8+ T cells. Thus, using scRNA-Seq, CyTOF and FACS analyses, we identified two populations of CD4+CD8+ T cells that produce immunoregulatory cytokines and are regulated by COX-2. These cells may play an important, previously unappreciated role in the pathogenesis of allergic lung disease.

Identification and characterization of human PEIG-1/GPRC5A as a 12-O-tetradecanoyl phorbol-13-acetate (TPA) and PKC-induced gene

Homo sapiens orphan G protein-coupling receptor PEIG-1 was first cloned and characterized by applying differential display to T84 colonic carcinoma cells incubated in the presence of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (GenBank AF506289.1). Later, Lotan's laboratory found the same gene product in response to retinoic acid analogues, naming it with the symbol RAIG1. Now the official HGNC symbol is GPRC5A. Here, we report the extension of its original cDNA fragment towards the 5' and 3' end. In addition, we show that TPA (100 ng/ml, 162 nM) strongly stimulated GPRC5A mRNA in T84 colonic carcinoma cells, with maximal expression at 4 h and 100 ng/ml (162 nM). Western blots showed several bands between 35 and 50 kDa, responding to TPA stimulation. Confocal microscopy confirmed its TPA upregulation and the location in the plasma membrane. The PKC inhibitor Gö 6983 (10 μM), and the Ca2+ chelator BAPTA-AM (150 μM), strongly inhibited its TPA induced upregulation. The PKA inhibitor H-89 (10 μM), and the MEK1/2 inhibitor U0126 (10 μM), also produced a significant reduction in the TPA response (~50%). The SGK1 inhibitor GSK650394 stimulated GPRC5A basal levels at low doses and inhibit its TPA-induced expression at concentrations ≥10 μM. The IL-1β autocrine loop and downstream signalling did not affect its expression. In conclusion, RAIG1/RAI3/GPRC5A corresponds to the originally reported PEIG-1/TIG1; the inhibition observed in the presence of Gö 6983, BAPTA and U0126, suggests that its TPA-induced upregulation is mediated through a PKC/Ca2+ →MEK1/2 signalling axis. PKA and SGK1 kinases are also involved in its TPA-induced upregulation.

Biocide Potential of Jatropha curcas L. Extracts

Jatropha curcas L., a multipurpose oilseed plant, is very important for biodiesel production; it also has a wide range of bioactive compounds with medicinal properties and biocidal activity for control of crop pests and diseases. This review presents the state-of-the-art of the biocidal activity of J. curcas extracts. Chemical constituents such as phorbol esters are responsible for high bioactivity of J. curcas, due to their toxicity to humans and animals and to their high fungicidal and insecticidal activity. The fungicidal activity of these constituents may be due to destroy endoplasmatic reticulum and hyphae cell walls. The activity of these compounds on insect pest metabolism is well known, leading to an antifeedant effect, repellency, mating inhibition, oviposition inhibition or suppression and/or induction of infertile egg production, and inhibition of larva, nymph, and pupa development. Several studies have shown that although all organs of J. curcas plant are toxic, the degree of toxicity varies in accordance with the extract formulation, the nature of the active substance, the administration rate and procedure, and the individual sensitivity of pests and diseases. Thus, J. curcas stands out as a promising species for bioenergy purposes, as well as for control of pests and diseases that affect agriculture production.

Expression of long pentraxin 3 in human nasal mucosa fibroblasts, tissues, and secretions of chronic rhinosinusitis without nasal polyps.

Numerous studies have shown that microbiomes play an important role in the pathogenesis of chronic rhinosinusitis (CRS). In addition to a known short pentraxin, C-reactive protein, long pentraxin 3 (PTX3) belongs to pentraxin family which detects conserved microbial pentraxin motifs and mobilizes early defense against foreign invaders, but its participation in CRS remains unclear. In the present study, through an intensive screening, peptidoglycan (PGN) was selected as a main material to investigate the action mechanism of a cell wall component on CRS without nasal polyps (CRSsNP) nasal mucosa-derived fibroblasts and the PTX3 expression in human nasal mucosa tissue and discharge. The PGN not only enhanced PTX3 mRNA and protein production in cells but also caused marked PTX3 secretion into extracellular space. The pharmacological interventions indicated that the PTX3 induction was mediated mainly through toll-like receptor 2 (TLR2), phosphoinositide-phospholipase C (PI-PLC), protein kinase C (PKC), NF-κB, and cAMP response element binding protein (CREB), which was further confirmed by the observations that a direct PKC activator (phorbol ester) had a similar inductory effect on PTX3 expression/production and the siRNA interference knockdown of PKCμ/δ, NF-κB, and CREB compromised PTX3 production. Meanwhile, PTX3 was found to be overexpressed/produced in nasal mucosa and discharge/secretion of the CRSsNP patients. Collectively, we first demonstrated here that PGN enhances PTX3 expression and release in nasal fibroblasts through TLR2, PI-PLC, PKCμ/δ, NF-κB, and CREB signaling pathways. The PTX3 is overexpressed in nasal mucosa and discharge/secretion of CRSsNP patients, revealing its possible importance in CRSsNP development and progression. KEY MESSAGES: Long pentraxin 3 (PTX3) is highly expressed in nasal mucosa and discharge/secretion of patients of chronic rhinosinusitis without nasal polyps (CRSsNP). The bacteria cell wall component-peptidoglycan (PGN) causes PTX3 expression in CRSsNP nasal mucosa-derived fibroblasts, contributing to the PTX3 increase in tissues. PGN induces PTX3 expression through a previously known IκB/NF-κB and a novel PKCμ/δ and CREB signaling pathway. The PTX3 may be used as a biomarker for CRS.

Impaired Autoinhibition of Protein Kinase Cγ in Spinocerebellar Ataxia Type 14

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline mutations in the diacylglycerol (DG)/Ca2+‐regulated protein kinase C gamma (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. Curiously, the majority of the approximately 50 missense mutations identified in PKCγ cluster to the DG‐sensing C1A and C1B domains. Here, we use a genetically‐encoded FRET‐based C Kinase Activity Reporter (CKAR) to show that ataxia‐associated PKCγ mutants have higher basal activity in cells, and thus are less autoinhibited, than wild‐type enzyme. However, whereas reduced autoinhibition generally renders PKC sensitive to degradation, we show that mutations in the C1B domain allow translocation to membranes but protect PKCγ from phorbol ester‐induced down‐regulation. Indeed, deletion of the C1B domain prevents PKCγ down‐regulation with phorbol esters, potent ligands for the C1 domains. Strikingly, the degree of impaired autoinhibition correlates inversely with age of disease onset. Patients with the most severe mutation we examined (V138E) present with symptoms as young children, whereas symptoms in patients with the least severe mutation examined (D115Y) manifested in their 40s. To understand the structural basis of mutations outside the C1 domains, we generated a model of PKCγ using homology modeling and molecular docking. Mutations outside the C1 domains occur in regions also predicted to disrupt autoinhibition, including the pseudosubstrate, a predicted interface between the kinase and C1B domains, and the C‐terminal tail of PKCγ. Taken together, our data support a model in which SCA14 mutations enhance PKCγ activity without compromising stability. Furthermore, because many of the genetic causes of the 40+ types of SCA alter Ca2+ homeostasis, deregulated PKCγ activity may be a common cause for the disease. This raises the possibility that inhibition of PKCγ will be a potentially viable therapeutic target for SCA.Support or Funding InformationNIH T32 GM007752

Structural Basis of Protein Kinase C Tumor‐Promoting Response And Membrane Recruitment

Protein Kinase C (PKC) is a family of Ser/Thr kinases that regulate a multitude of cellular processes through participation in the phosphoinositide signaling pathway. The hallmark of their activation is translocation to lipid membranes. Dysregulation of PKC signaling has been implicated in cancer progression, cardiac disease, and Alzheimer’s disease. One of the major challenges in the field is to understand the structural basis of PKC activation and regulation ‐‐ at the level of atomic resolution.Here, we applied NMR and FRET spectroscopy to gain insight into the structure and protein‐membrane interactions of the regulatory domain of PKC that consist of three peripheral membrane modules: the tandem conserved homology‐1 (C1) and conserved homology‐2 (C2) domains. The C1 domains undergo membrane insertion upon binding their natural agonist, diacylglycerol, as well as tumor‐promoting phorbol esters and other small‐molecule agonists. C2 associates with anionic membranes upon binding Ca2+. Simultaneous association of all domains with membranes ensures the coincidence of four signals: Ca2+ ions, diacylglycerol, phosphatidylserine, and phosphatidylinositol‐4,5‐bisphosphate.Here, we report the first structure of the C1 domain complexed to a potent tumor‐promoting agent, phorbol 12,13‐dibutyrate (PDBu), in the membrane‐mimicking environment. The structure of the complex shows that C1 has to undergo a conformational change in order to capture the membrane‐embedded ligand. The structure also revealed the role of individual amino acids in the interactions with PDBu and membrane‐mimicking environment. To probe how different agonists modulate the interaction of C1 with membranes, we conducted NMR paramagnetic relaxation enhancement (PRE) experiments with bicelles doped with paramagnetic lipid. The PRE patterns revealed that the C1 domains adopt tilted rather than vertical orientation in the membrane. Our data on four different agonists indicate that C1 domains dictate the general orientation of the agonist complexes in the membrane, but the agonists determine the depth of insertion. This provides a plausible explanation for the differences in the potency of these ligands to achieve the acute and chronic activation response of PKC.To gain insight into the mechanism of coincidence detection, we developed protein‐to‐membrane FRET assay that we used to characterize the interplay of the C1 and C2 domains in the membrane association step. The FRET experiments revealed a clear picture of how tumor‐promoting agents can elicit constitutive activation of PKC through simultaneous recruitment of C1 domains to membranes and sensitization of the C2 domain to Ca2+.Support or Funding InformationNIH R01 GM108998; NSF CHE‐1905116; Welch Foundation A‐1784Agonist‐guided membrane interaction of C1B domain of PKCδ studied by paramagnetic NMR. (A) Schematic representation of the approach. (B) Chemical representations of the different agonists used in the study.Figure 1

Mitochondria Amplify TRPV4‐dependent Calcium Dynamics within the Pulmonary Microvascular Endothelium

Acute respiratory distress syndrome (ARDS) is an inflammatory lung disease common in critically ill patients marked by diffuse alveolar damage and increased pulmonary endothelial barrier permeability. A large body of evidence supports the role of mitochondrial dysfunction in ARDS, which has been shown to mediate endothelial permeability. For example, in cultured rat pulmonary microvascular endothelial cells (PMVECs), the mitochondrial complex I inhibitor rotenone increases endothelial permeability and in isolated perfused lung studies, rotenone increases the pulmonary vascular filtration coefficient. In both cell culture and isolated perfused lungs, the amphipathic quinone coenzyme Q1 (CoQ1) rescues rotenone‐dependent increases in endothelial permeability by bypassing complex I to restore mitochondrial bioenergetics.The transient receptor potential vanilloid protein 4 (TRPV4), a mechanically gated calcium channel, has also been implicated in mediating endothelial permeability by increasing intracellular calcium in response to high pressure ventilation. The phorbol ester 4α‐phorbol‐12,13‐didecanoate (4αPDD) provides a pharmacological means of TRPV4 activation and stimulates endothelial permeability responses in both cell culture and animal models of lung injury. Given that mitochondria actively participate in calcium buffering, we hypothesized that TRPV4‐dependent calcium signals may be compartmentalized by mitochondria. To test this hypothesis, we used confocal microscopy to measure calcium signals in PMVECs in response to TRPV4 activation via 4αPDD (5 μM) after treatment with vehicle, rotenone (0.5 μM), or rotenone in combination with CoQ1 (10 μM), and developed a sensitive, automated technique for intracellular calcium signal transient analysis.Using our novel analysis approach, we found that rotenone significantly decreased the number of calcium signal transients stimulated by 4αPDD over a 15 minute interval (mean ± SEM: 22 ± 4 rotenone vs 96 ± 14 vehicle, p <0.01), and that CoQ1 in combination with rotenone reversed this effect (60 ± 15, p = n.s. vs vehicle). Rotenone also dampened the amplitude of calcium responses to TRPV4, an effect that was partially restored by CoQ1. These findings support the hypothesis that mitochondria regulate TRPV4‐mediated calcium dynamics by amplifying calcium signals, and that mitochondrial dysfunction impairs this phenomenon. This suggests that mitochondrial regulation of endothelial calcium signaling is essential to the maintenance of barrier integrity and provides a possible therapeutic target in ARDS.Support or Funding InformationNIH K25HL136869 and NIH P01 HL066299‐17

Monocyte/Macrophage - like cell differentiation induced by TPA in HL60 cells leads to loss of Histone H4 Lysine 16 Acetylation

12-O-Tetradecanoylphorbol-13-acetate (TPA) is a phorbol ester and induces monocyte/macrophage like cell differentiation in HL60 cells. The levels and patterns of gene expression differ greatly between differentiated and undifferentiated HL60 cells. Epigenetic histone modifications play a crucial role in transcriptional activation and alterations in the modification levels greatly affect the gene expression pattern. Acetylation of Histone H4 Lysine 16 (H4K16ac) is one such modification and has a significant role in transcriptional activation. Changes in its acetylation level either due to a physiological or pathological effect will have a dramatic effect on cellular gene expression. Here, a study was done to see the effect of TPA induced differentiation on H4K16ac levels in HL60 cells. Results obtained from flow cytometric analysis showed expression of macrophage cell surface marker CD11b on TPA differentiated HL60 cells and the western blots revealed a drastic downregulation of H4K16ac in differentiated cells. Immunoblotting and co-immunoprecipitation assay revealed DNA damage dependent enhancement of H4K16 acetylation and its co-localization with γH2AX in undifferentiated cells. Whereas, TPA differentiated cells (CD11B+ve) didn’t show any such enhancement in H4K16acetylation levels in the presence of DNA damage. The present study shows that TPA induced differentiation of HL60 cells into macrophages leads to the downregulation of H4K16 acetylation.

Total saponin of Dioscoreacollettii attenuates MSU crystal‑induced inflammation via inhibiting the activation of the NALP3 inflammasome and caspase‑1 in THP‑1 macrophages.

Total saponins extracted from Dioscorea collettii (TSD), extracts of the Chinese herb Dioscorea, are thought to exhibit therapeutic benefit in gouty arthritis. However, its exact mechanism remains unclear. The current study aimed to elucidate the underlying mechanisms by investigating the effects of TSD on the inflammation induced by monosodium urate (MSU) crystals in THP-1 macrophages. The viability of THP-1 macrophages was examined using the MTT assay and the levels of inflammatory cytokines, including interleukin (IL)-1β, IL-18 and tumor necrosis factor (TNF)-α, released by the cells were quantitatively measured using ELISA kits. The results revealed that the protein level of cluster of differentiation 11b increased in THP-1 cells treated with 100 ng/ml phorbol ester, suggesting that monocytic THP-1 cells were successfully differentiated into macrophages. TSD decreased the levels of inflammatory cytokines, including TNF-α, IL-18 and IL-1β, secreted by THP-1 macrophages. As the release of IL-1β and IL-18 is dependent on the NLR family pyrin domain containing 3 (NALP3) inflammasome and caspase-1, the current study investigated the effect of TSD on the aforementioned proteins. The results revealed that TSD decreased the protein levels of NALP3 and apoptosis-associated speck-like, which serve important roles in the assembly of the NALP3 inflammasome. Furthermore, NALP3 inflammasome-related proteins were also decreased by TSD in rotenone induced THP-1 macrophages, TSD inhibited the activation of caspase-1 and rotenone-induced NALP3 inflammasome activation in THP-1 macrophages. The results obtained in the current study revealed that TSD attenuated MSU crystal-induced inflammation by inhibiting rotenone-induced activation of the NALP3 inflammasome and caspase-1, suggesting that these two proteins may be novel targets for the treatment of gouty arthritis.

Protein Kinase Cα Promotes Proliferation and Migration of Schwann Cells by Activating ERK Signaling Pathway

Wallerian degeneration (WD) and axon regeneration generally take place following peripheral nerve injury (PNI). Schwann cells (SCs) and macrophages play major role in WD. SCs, acting as repair cells and primary signal mediators, dedifferentiate and proliferate to remove the debris, form Büngner’s bands and secrete trophic factors during these processes. However, the underlying mechanisms remain poorly understood. Here, we found that protein kinase Cα (PKCα), a serine/threonine kinase, expressed in SCs was significantly up-regulated after PNI. Activating PKCα with phorbol 12-myristate 13-acetate (PMA), a phorbol ester binds and activates PKCα) promoted SCs proliferation and migration. While, silence of PKCα by siRNAs inhibited these processes. PD184352, an inhibitor of MEK1, reversed the effect induced by PMA on SCs. Mechanism studies revealed that PKCα functioned through activating the ERK signaling pathway. Furthermore, PKCα also exhibited a neuroprotective role by upregulating the expression of neurotrophic factors in SCs. To sum up, this study offers novel insights for clarifying our understanding of the involvement of PKCα in the mechanism of peripheral nerve degeneration as well as regeneration.

Neopterin formation through radical scavenging of superoxide by the macrophage synthesised antioxidant 7,8-dihydroneopterin

Clinical measurement of neopterin has been extensively used as a marker of inflammation but the in vivo mechanism generating neopterin is poorly understood. Neopterin is described as the oxidation product of 7,8-dihydroneopterin, a potent antioxidant generated by monocyte/macrophages in response to interferon-γ. While peroxyl and hydroxyl scavenging generates dihydroxanthopterin, hypochlorite efficiently oxidises 7,8-dihydroneopterin into neopterin, but this reaction alone does not explain the high levels of neopterin seen in clinical data. Here, we examine whether superoxide scavenging by 7,8-dihydroneopterin generates neopterin.U937 cells incubated with oxLDL showed a time dependent increase superoxide and 7,8-dihydroneopterin oxidation to neopterin. Neopterin generation in oxLDL or phorbol ester treated U937 cells or human monocytes was inhibited by apocynin and PEG-SOD. Addition of the myeloperoxidase inhibitor 4-aminobenzoic acid hydrazide (ABAH) had no effect of the superoxide generation or neopterin formation. 7,8-Dihydroneopterin reacted with superoxide/hydroxy radical mixtures generated by X-ray radiolysis to give neopterin. Formation of neopterin by superoxide derived from the xanthine/xanthine oxidase system was inhibited by superoxide dismutase. Neopterin formation was inhibited by apocynin in phorbol ester treated human carotid plaque rings in tissue culture.These results indicate that 7,8-dihydroneopterin scavenges superoxide and is subsequently oxidised into neopterin in cellular and cell-free experimental systems.

Identification, characterization, and comparison of n-alkanols and anesthetics binding to the C1b subdomain of protein kinase cα: similar function with different binding sites

Protein kinase C (PKC) is a family of lipid-activated enzymes involved in anesthetic preconditioning signaling pathways. Previously, n-alkanols and general anesthetics have been found to activate PKC by binding to the kinase C1B subdomain. In the present study, we attempt to ascertain the molecular mechanism and interaction mode of human PKCα C1B subdomain with a variety of exogenous n-alkanols and volatile general anesthetics as well as endogenous activator phorbol ester (PE) and co-activator diacylglycerol (DG). Systematic bioinformatics analysis identifies three spatially vicinal sites on the subdomain surface to potentially accommodate small-molecule ligands, where the site 1 is a narrow, amphipathic pocket, the site 2 is a wide, flat and hydrophobic pocket, and the site 3 is a rugged, polar pocket. Further interaction modeling reveals that site 1 is the cognate binding region of natural PE activator, which can moderately simulate the kinase activity in an independent manner. The short-chain n-alkanols are speculated to also bind at the site to competitively inhibit PE-induced kinase activation. The long-chain n-alkanols and co-activator DG are found to target site 2 in a nonspecific manner, while the volatile anesthetics prefer to interact with site 3 in a specific manner. Since the site 1 is composed of two protein loops that are also shared by sites 2 and 3, binding of n-alkanols, DG and anesthetics to sites 2 and 3 can trigger a conformational displacement on the two loops, which enlarges the pocket size and changes the pocket configuration of site 1 through an allosteric mechanism, consequently enhancing kinase activation by improving PE affinity to the site.

Subunit-Specific Augmentation of AMPA Receptor Ubiquitination by Phorbol Ester.

Excitatory neurotransmission relies on the precise targeting of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors to the neuronal plasma membrane. Activity-dependent ubiquitination of AMPA receptor (AMPAR) subunits sorts internalised receptors to late endosomes for degradation, which ultimately determines the number of AMPARs on neuronal membrane. Our recent study has demonstrated a functional cross-talk between the phosphorylation and ubiquitination of the GluA1 subunit in mammalian central neurons. However, the existence of such a cross modulation for the GluA2 subunit remains unknown. Here, we have shown that bicuculline induced GluA2 ubiquitination on the same lysine residues (Lys-870 and Lys-882) in the C-terminal as those elicited by the AMPA treatment. Interestingly, bicuculline-induced ubiquitination was markedly enhanced by the phospho-mimetic GluA2 S880E mutant. Pharmacological activation of protein kinase C (PKC) by phorbol ester, which mediates the phosphorylation of GluA2 at Ser-880, augmented bicuculline-induced ubiquitination of GluA2 in cultured neurons. This effect was specific for the GluA2 subunit because phorbol ester did not alter the level of GluA1 ubiquitination. However, phorbol ester-induced enhancement of GluA2 ubiquitination did not require Ser-880 phosphorylation. This suggests that pseudo-phosphorylation of Ser-880 is sufficient but is not necessary for the augmentation of bicuculline-induced GluA2 ubiquitination. Collectively, these data provide the first demonstration of subunit-specific modulation of AMPAR ubiquitination by the PKC-dependent signalling pathway in mammalian central neurons.

NKp46+ lamina propria natural killer cells undergo metabolic reprogramming in a mouse experimental colitis model.

Both innate and adaptive immune system play important roles in the onset and progression of inflammatory bowel diseases (IBDs). However, the significance of natural killer (NK) cells for IBDs remains unclear. To understand the biology of colonic lamina propria natural killer (LPNK) cells in IBDs, we characterized LPNK cell metabolism in a murine acute colitis model. C57BL/6J mice were fed with 3% dextran sulfate sodium to establish the acute colitis model. Colonic LPNK cells were isolated from mice through flow cytometry. The expression of metabolic genes in LPNK cells was analyzed by transcriptome sequencing and quantitative RT-PCR. Glucose uptake, Seahorse assay, and ATP assay were conducted to assess the metabolic status of LPNK cells. Phos-flow assay was performed to evaluate cell signaling pathways in LPNK cells. In vitro stimulation and cytotoxicity assay were conducted to measure the function of LPNK cells. In acute colitis, LPNK cells upregulated the expression of genes related to glycolysis and oxidative phosphorylation (oxphos), and enhanced glucose uptake capability. Intracellular ATP production, glycolysis and oxphos in LPNK cells were also promoted in acute colitis. mTORC1 signaling was essential for the metabolic reprogramming in LPNK cells in acute colitis. Although LPNK cells of diseased mice exhibited equivalent cytokine profile to normal LPNK cells upon stimulation with phorbol ester or IL-2, LPNK cells of diseased mice were more cytotoxic to target cells than normal LPNK cells. LPNK cells undergo metabolic reprogramming which might be a response to upcoming microbial infection in acute colitis.

Nuclear Translocation of Glutaminase GLS2 in Human Cancer Cells Associates with Proliferation Arrest and Differentiation

Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase.

Genetic Potential of Agronomic Traits and Seed Physicochemical Properties of Novel Jatropha Lines Developed from Complex Interspecific Hybridization

Fifteen jatropha lines were obtained from complex crossing between progenies derived from interspecific hybridization of Jatropha curcas and J . integerrima . They were evaluated for variability and association in yield, yield components, oil content, fatty acid composition, phorbol esters content, and growth characters . The major fatty acids in seed were oleic and linoleic acids, with a negative correlation among them. Seed yield showed a positive correlation with number of fruits per inflorescence and seeds per fruit, 100 seed weight and canopy size . A path coefficient analysis showed that number of fruits per inflorescence and canopy width had high positive direct relationship with seed yield per plant . Most characters showed high broad - sense heritability . Clustering by traits classified the breeding lines into five groups with a large distance between groups . The members in each group comprised lines of the same or similar pedigrees . Cluster I was the high seed yield group comprising two accessions (KUJL23 and KUJL18) . Clustering based on principle components classified them into four groups . The lines in cluster II and III from cluster analysis were included in one group, while members of the other groups were the same in both clustering methods .