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The seeds and cake of a new variety of jatropha (J. curcas L. var. Sevangel) were characterized through the quantification of their nutrients and minority components (phorbol esters and secondary compounds) to evaluate their possible use in diets. The dietary fiber was the main nutritional component in both samples (29.4–33.4%). The Jatropha curcas L. var. Sevangel is a source of protein (18% in the seeds, and 32.2% in the cake) with high values of glutamine, histidine, and tyrosine (13.09, 20.74, and 10.17 g/100 g of protein). The most abundant minerals in both samples were Ca, K and Mg. The main fatty acids detected were palmitic and linoleic, and the total content of unsaturated fatty acids was 304.94 mg/100 g and 61.46 mg/100 g in the seed and cake, respectively. Phorbol esters were not detected, so the analyzed plant can be considered as harmless, compared to other toxic varieties. The content of phytic acid was higher in the seeds than in the cake (8.1 vs. 5.0 mg mL−1); which may favor the potential as a functional ingredient. The tannins content, trypsin inhibitors and saponins with hemolytic activity do not represent a risk for the consumption of this plant. Jatropha curcas L. var. Sevangel is a rich source in dietary fiber, high-quality protein, some essential amino acids, and calcium, which makes it suitable for use in diets. [ABSTRACT FROM AUTHOR]

Introduction: Psoriasis is an immune-mediated systemic disease. Neutrophils are enriched in psoriasis lesions and can form neutrophil extracellular traps (NETs) to activate keratinocytes. Receptor-interacting protein kinase RIPK1 and RIPK3 are involved in necroptosis and NET formation. Aim: To elucidate whether RIPK1 regulates circulating neutrophils to form NETs and inflammation in psoriasis. Material and methods: Blood samples of psoriasis patients (n = 20) and healthy controls (n = 20) were detected by flow cytometry. The expression level of RIPK1/3 in isolated circulating neutrophils from psoriasis patients (n = 17) and healthy controls (n = 17) was examined by quantitative real-time PCR. SYTOX Green dye and PicoGreen reagent were used to detect NET formation and DNA release in neutrophils under the stimulation of phorbol 12-myristate 13-acetate (PMA) and necrostain-1 (Nec-1). Correlation analysis was performed between RIPK1/3 expression and Psoriasis Area Severity Index (PASI), neutrophil-to-lymphocyte ratio (NLR). Results: RIPK1 and RIPK3 expression in protein levels were decreased in monocytes and neutrophils from peripheral blood of psoriasis patients. In isolated psoriasis neutrophils, RIPK1 and Caspase8 mRNA were downregulated while RIPK3 and MLKL mRNA were elevated, leading to the necroptosis pathway. In addition, RIPK1-inhitor-necrostatin-1 (Nec-1) enhanced NETosis in psoriasis neutrophils in vitro. More importantly, there is a negative correlation between RIPK1 and psoriasis disease severity. Conclusions: Our data demonstrated that downregulated RIPK1 expression in psoriasis neutrophils may enhance NET generation. RIPK1 may be identified as a novel therapeutic target in psoriasis. [ABSTRACT FROM AUTHOR]

Chemical investigation of Euphorbia wallichii Hook. f. led to the isolation of four undescribed rearranged diterpenoids, euphwanoids I-IV (1-4), and six unreported tigliane diterpenoids walliglianes A-F (5-10). Euphwanoids I-III (1-3) possess a rare 6/6/7/3 ring scaffold, euphwanoid IV (4) is the first spiro[tricyclo[5.4.0.0 2,4 ]undecane-8,1'-cyclopentane] skeleton to be found in the tigliane family. The structures of compounds 1-10 were established by utilizing spectroscopic data analysis, experimental electronic circular dichroism measurements, 13 C NMR calculations, and single-crystal X-ray diffraction. In the preliminary bioassay, compounds 3, 4, and 7 were found to protect BV-2 cells against H 2 O 2 -induced cell injury in a dose-dependent manner by the CCK8 assay., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Understanding the platelet activation molecular pathways by characterizing specific protein clusters within platelets is essential to identify the platelet activation state and improve the existing therapies for hemostatic disorders. Here, we employed various state-of-the-art super-resolution imaging and quantification methods to characterize the platelet spatiotemporal ultrastructural change during the activation process due to phorbol 12-myristate 13-acetate (PMA) stimuli by observing the cytoskeletal elements and various organelles at nanoscale, which cannot be done using conventional microscopy. Platelets could be spread out with the guidance of actin and microtubules, and most organelles were centralized probably due to the limited space of the peripheral thin regions or the close association with the open canalicular system (OCS). Among the centralized organelles, we provided evidence that granules are fused with the OCS to release their cargo through enlarged OCS. These findings highlight the concerted ultrastructural reorganization and relative arrangements of various organelles upon activation and call for a reassessment of previously unresolved complex and multi-factorial activation processes. [ABSTRACT FROM AUTHOR]

Objectives: Blau syndrome is a distinct class of autoinflammatory syndrome presenting with early-onset systemic granulomatosis. Blau syndrome-causing NOD2 mutations located in the central nucleotide-oligomerization domain induce ligand-independent basal NF-κB activation in an in vitro reporter assay. However, the precise role of this signaling on granuloma formation has not yet been clarified. Methods: Blau syndrome-causing NOD2 mutations were introduced into human monocytic THP-1 cells, and their morphological and molecular changes from parental cells were analyzed. Identified molecules with altered expression were examined in the patient's lesional skin by immunostaining. Results: Although the production of proinflammatory cytokines was not altered without stimulation, mutant NOD2-expressing THP-1 cells attached persistently to the culture plate after stimulation with phorbol myristate acetate. Sustained surface ICAM-1 expression was observed in association with this phenomenon, but neither persistent ICAM-1 mRNA expression nor impaired ADAM17 mRNA expression was revealed. However, the transient induction of PDGF-B mRNA expression was specifically observed in stimulated THP-1 derivatives. In the granulomatous skin lesion of a Blau syndrome patient, ICAM-1 and PDGF-B were positively immunostained in NOD2-expressing giant cells. Conclusions: Sustained surface ICAM-1 expression and transient PDGF-B production by newly differentiating macrophages harboring mutant NOD2 might play a role in granuloma formation in Blau syndrome. [ABSTRACT FROM AUTHOR]

Tigliane diterpenoids possess exceptionally complex structures comprising common 5/7/6/3-membered ABCD-rings and disparate oxygen functionalities. While tiglianes display a wide range of biological activities, compounds with HIV latency-reversing activity can eliminate viral reservoirs, thereby serving as promising leads for new anti-HIV agents. Herein, we report collective total syntheses of phorbol ( 13 ) and 11 tiglianes 14 - 24 with various acylation patterns and oxidation states, and their evaluation as HIV latency-reversing agents. The syntheses were strategically divided into five stages to increase the structural complexity. First, our previously established sequence enabled the expeditious preparation of ABC-tricycle 9 in 15 steps. Second, hydroxylation of 9 and ring-contractive D-ring formation furnished phorbol ( 13 ). Third, site-selective attachment of two acyl groups to 13 produced four phorbol diesters 14 - 17 . Fourth, the oxygen functionalities were regio- and stereoselectively installed to yield five tiglianes 18 - 22 . Fifth, further oxidation to the most densely oxygenated acerifolin A ( 23 ) and tigilanol tiglate ( 24 ) was realized through organizing a 3D shape of the B-ring. Assessment of the HIV latency-reversing activities of the 12 tiglianes revealed seven tiglianes ( 14 - 17 and 22 - 24 ) with 20- to 300-fold improved efficacy compared with prostratin ( 12 ), a representative latency-reversing agent. Therefore, the robust synthetic routes to a variety of tiglianes with promising activities devised in this study provide opportunities for advancing HIV eradication strategies.

Background/aim: Tetrazolium-based cell proliferation assays using MDA-MB-231 and HeLa cells revealed that 3,4-dihydro-lactucin (3,4-DHL), a compound isolated from Microbispora rosea AL22, possesses anticancer properties. Apoptotic cell death was observed in 3,4-DHL-treated cells. Lactucopicrin, a related compound, reportedly exerts anticancer activity against different cancer types. However, data on the anticancer mechanism of lactucins are limited. This study aimed to investigate apoptosis induction in MDA-MB-231 cells treated with 3,4-DHL., Materials and Methods: Morphological changes, changes in mitochondrial membrane potential, and apoptosis induction in MDA-MB-231 cells treated with 3,4-DHL were investigated. Furthermore, molecular docking and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis of anti-apoptotic proteins were performed to determine the effector mechanism of 3,4-DHL., Results: 3,4-DHL induced cytotoxicity at a half-maximal inhibitory concentration of 37.62 μg/ml, along with various morphological alterations in apoptotic and viable cells. Furthermore, 3,4-DHL-treated cells showed mitochondrial membrane potential depolarization, intense annexin V-fluorescein isothiocyanate staining, and increased caspase 3 and 8 activities. Molecular-docking studies demonstrated that 3,4-DHL should bind to the active site of various anti-apoptotic proteins, forming stable complexes., Conclusion: Our findings revealed that 3,4-DHL has great potential to be used as an apoptosis-inducing agent in cancer therapy. However, further in-vivo confirmation is required in evaluation of 3,4-DHL as an anticancer agent in cancer chemotherapy., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Thirteen new Euphorbia diterpenoids, euphylonanes A-M ( 1 - 13 ), and eight known ones were isolated from the whole plants of Euphorbia hylonoma . Compounds 1 and 2 are two rearranged ingenanes bearing a rare 6/6/7/3-fused ring system. Compound 3 represents the first example of a 9,10-epoxy tigliane, while 4 - 21 are typical ingenanes varying with substituents. Structures were elucidated using a combination of spectroscopic, computational, and chemical methods. Most ingenanes exerted a significant antiadipogenic effect in 3T3-L1 adipocytes, among which 4 was the most active with an EC 50 value of 0.60 ± 0.27 μM. Mechanistic study revealed that 4 inhibited the adipogenesis and lipogenesis in adipocytes via activation of the AMPK signaling pathway.

To investigate the role of the secondary 5-hydroxy group in the activity of the anticancer drug tigilanol tiglate ( 2b ) (Stelfonta), oxidation of this epoxytigliane diterpenoid from the Australian rainforest plant Fontainea picrosperma was attempted. Eventually, 5-dehydrotigilanol tiglate ( 3a ) proved too unstable to be characterized in terms of biological activity and, therefore, was not a suitable tool compound for bioactivity studies. On the other hand, a series of remarkable skeletal rearrangements associated with the presence of a 5-keto group were discovered during its synthesis, including a dismutative ring expansion of ring A and a mechanistically unprecedented dyotropic substituent swap around the C-4/C-10 bond. Taken together, these observations highlight the propensity of the α-hydroxy-β-diketone system to trigger complex skeletal rearrangements and pave the way to new areas of the natural products chemical space.

Pepluanol B is a new Euphorbia diterpene with an unprecedented tetracyclic backbone. However, its biogenetic relationship with known Euphorbia diterpenes is unclear. We report herein that its β-hydroxyl ketone motif could undergo a base-promoted retro-aldol/aldol process in two pathways and afford the skeletons of tigliane- and myrsinane-type Euphorbia diterpenes through the formation of the C8-C14 and C7-C13 bonds, respectively. The retro-aldol/aldol cascade indicates that pepluanol B is possibly a biosynthetic precursor of lathyranes and other relevant dipterpenes.

The Jatropha curcas plant (Jatropha) has been proposed as a source of biodiesel fuel, as it yields crude glycerol as an abundant by-product. Its by-products could serve as a starting material in making glycerol for FDA-regulated products. Jatropha is not regarded as a source of edible vegetable oil since it contains phorbol esters (PEs). PEs, even at very low exposure concentrations, demonstrate various toxicities in humans and animals, but may not be detected by routine impurity analyses. Here, we demonstrate the development of a rapid and simplified method for the detection and quantification of Jatropha-derived PE toxins using ambient ionization mass spectrometry. To do this, we successfully coupled a paper spray ambient ionization source with an ion trap portable mass spectrometer. The paper spray source was assembled using chromatography papers, and analyte ions were generated by applying a high voltage to a wetted paper triangle loaded with PE standards. For method development, we used commercially available PE standards on an ion trap portable mass spectrometer. Standard solutions were prepared using ethanol with PE concentrations ranging from 1.0 to 0.0001 mg mL−1. Spike and recovery experiments were performed using USP grade and commercially available glycerol. To discern chemical differences between samples, we applied multivariate data analysis. Based on the results obtained, paper spray coupled with a portable mass spectrometric method can be successfully adopted for the analysis of toxic contaminants present in glycerol-based consumer products with LOD and LOQ of 0.175 μg mL−1 and 0.3 μg mL−1 respectively. This direct, simple design, and low-cost sampling and ionization method enables fast screening with high sensitivity in non-laboratory settings. [ABSTRACT FROM AUTHOR]

Abstract Diabetes-induced neutrophil NETosis impairs wound healing through neutrophil extracellular traps (NETs). Reactive oxygen species (ROS)-triggered activation of mitogen-activated protein kinase (MAPK) ERK1/2 and p38 is involved in NETosis. Hydrogen sulfide (H 2 S), an endogenous signaling molecule, accelerates diabetic wound healing (DWH), and inhibits ROS production, ERK1/2 and p38 activation, while its level is decreased in diabetes. However, it remains unknown whether H 2 S could accelerate DWH through inhibition of NETosis, and whether this inhibitory effect was associated with blockage of ROS-induced ERK1/2 and p38 activation. In order to solve these problems, serum NETs content was measured in diabetic foot patients and healthy individuals. Wound was created in dorsal skin of LepR db/db and control mice and NETs content in wound tissues was tested. An in vitro NETosis model was induced by phorbol 12-myristate 13-acetate (PMA) in isolated neutrophils. Effects of H 2 S in form of Na 2 S on skin wound healing and NETosis were investigated both in vivo and in vitro. It was found that NETs level was highly increased in diabetic foot patients. Comparing with LepR m+/db mice, DWH was delayed in LepR db/db mice, accompanied with high NETs level. In PMA-induced NETosis model, peptidylarginine deiminase (PAD)-4 and citrullinated histone H 3 , as well as NETs components dsDNA framework, myeloperoxidase and neutrophil elastase, were significantly increased. PMA-induced neutrophil NETosis and NETs formation were abolished by treatment with H 2 S. The delayed DWH of diabetic mice was partially restored by intraperitoneal injection of H 2 S, meanwhile, the highly expressed NETosis and NETs release were also down-regulated. The treatment with H 2 S not only attenuated ROS production but also abolished MAPK ERK1/2 and p38 activation. Like the effects of H 2 S, inhibition of MAPK ERK1/2 or p38 could decrease NETs release. These findings suggests that H 2 S attenuates NETosis and primes diabetic wound to heal through blockage of ROS-mediated MAPK ERK1/2 and p38 activation. Highlights • H 2 S inhibits neutrophil NETosis and NETs release. • Through inhibition of NETosis H 2 S can improve diabetic wound healing. • The inhibitory effects of H 2 S on NETosis are associated with blockage of ROS-induced MAPK ERK1/2 and p38 activation. [ABSTRACT FROM AUTHOR]

Abstract Macrophages play an important role in the generation of host immune responses against H. pylori. In this study, we investigated the effect of a functionally uncharacterized H. pylori secreted protein HP1173 on macrophage responses. In a screen of eight H. pylori strains, similar expression levels of the HP1173 protein were observed in the whole-cell extracts, however; the amount of protein released into the culture medium varied significantly among strains. Recombinant purified HP1173 (rHP1173) was found to bind to THP-1 cells that were differentiated into macrophages via phorbol-12-myristate-13-acetate (PMA) treatment. The exposure of macrophages to rHP1173 led to the production of the proinflammatory cytokines TNF and IL-1β and the chemokine CXCL8 in a dose- and time-dependent manner. Under similar conditions, rHP1173 failed to induce apoptosis in macrophages. Furthermore, rHP1173-induced expression of TNF, IL-1β and CXCL8 was observed at the level of gene transcription. Incubation of macrophages in the conditioned medium from a mutant H. pylori strain 26695 lacking HP1173 protein expression resulted in the reduced induction of TNF, CXCL8 and IL-1β, confirming the role of the endogenous protein. Intracellular signaling involving MAPKs, NF-κB and the AP-1 family of transcription factors was required for rHP1173-induced TNF, CXCL8 and IL-1β release from macrophages. The blocking of MyD88, which is an adaptor for multiple toll-like receptors (TLRs), had no effect on rHP1173-induced TNF, CXCL8 and IL-1β release from macrophages, suggesting that Myd88-dependent TLR signaling was not involved in the recognition of and responses to rHP1173. These findings provide novel insights into the potential role of HP1173 in H. pylori infection-associated disease development. [ABSTRACT FROM AUTHOR]

Activation of P388D1 macrophages by phorbol myristate acetate (PMA) resulted in the translocation of the protein kinase C (PKC) isoforms alpha, delta, and epsilon from the cytosol to membranes. Furthermore, PMA activated phospholipase D (PLD) in these cells, and potentiated the effect of the inflammatory lipid mediator platelet-activating factor (PAF) on PLD activation. PAF also activated phospholipase A2 (PLA2) and enhanced arachidonic acid (AA) release in P388D1 macrophages, and bacterial lipopolysaccharide (LPS) increased the responsiveness of these cells to PAF. In contrast with PLD, PLA2 activation in P388D1 macrophages was found to take place independently of PKC. This was supported by the following evidence: (i) PMA neither induced AA release nor enhanced the PAF response; (ii) inclusion of PMA along with LPS during priming did not have any effect on PAF-stimulated AA release; (iii) down-regulation of PMA-activatable PKC isoforms by chronic treatment with the phorbol ester had no effect on the PAF response; and (iv) the PKC inhibitor staurosporine did not alter the PAF-induced AA release. The present study provides an example of cells in which the direct activation of PKC by phorbol esters does not lead to a primed and/or enhanced AA release. As a unique example in which PKC activation is neither necessary nor sufficient for AA release to occur, this now allows study of the separate and distinct roles for PLD and PLA2 in signal-transduction processes. This has hitherto been difficult to achieve because of the lack of specific inhibitors of these two phospholipases.

Tigliane type macrocyclic diterpenoids with special structures and diverse bioactivities are mainly extracted from plants of Euphorbiaceae and Thymelaeaceae. According to the different functional groups, they can be classified into types of phorbol esters, C-4 deoxyphorbol esters, C-12 deoxyphorbol esters, C-16 or C-17 substituted phorbol esters and others. Most of them present promising antiviral activities and cytotoxic activities and are expected to be developed as candidates for anti-AIDS, anti-tuberculosis, and anti-tumor clinical trials, demonstrating great potential for the application in healthcare. This paper reviews 115 novel tigliane-type diterpenoids discovered since 2013 and summarize their chemical structures and bioactivities, aiming to lay a foundation for further development and utilization of these compounds and provide new ideas for the development of clinical drugs.

Tigliane and daphnane diterpenoids are characteristically distributed in plants of the Thymelaeaceae family as well as the Euphorbiaceae family and are structurally diverse due to the presence of polyoxygenated functionalities in the polycyclic skeleton. These diterpenoids are known as toxic components, while they have been shown to exhibit a wide variety of biological activities, such as anti-cancer, anti-HIV, and analgesic activity, and are attracting attention in the field of natural product drug discovery. This review focuses on naturally occurring tigliane and daphnane diterpenoids from plants of the Thymelaeaceae family and provides an overview of their chemical structure, distribution, isolation, structure determination, chemical synthesis, and biological activities, with a prime focus on the recent findings., (© 2023. The Author(s).)

In this study, two tigliane diterpenoids, 12-deoxyphorbol-13-hexadecanoate and 12-deoxyphorbol-13-acetate (prostratin), were identified from the methanol extract of the roots of Euphorbia fischeriana and were found to have the ability to significantly reduce the survival of Caenorhabditis elegans . It was determined that exposure to these two compounds had toxic effects on the growth, reproduction, locomotion behavior, and accumulation of lipids and lipofuscin of the nematodes. Moreover, the transcription levels of the genes associated with lipid accumulation, apoptosis, insulin, and nuclear hormone synthesis in C. elegans were significantly influenced. Interestingly, 12-deoxyphorbol-13-hexadecanoate produced exposure toxicity at lower concentrations than that of prostratin. Pearson correlation analysis indicates that the elevated exposure toxicity of 12-deoxyphorbol-13-hexadecanoate may be the result of differing transcription levels, which result from the differential expression of fat-6 , egl-38 , and cep-1 . These results reveal that esterification with a long-chain fatty acid elevates the exposure toxicity of this tigliane diterpenoid, thus providing a basis for the application of tigliane diterpenoids in plant-derived nematicides.

Established analytic methods for the quantification of phorbol esters (PE), which are some toxic components in Jatropha curcas L., include HPLC with UV‐detection with the commercially available phorbol myristate acetate (PMA) as internal standard or HPLC coupled with MS detection with an external calibration, mostly also with PMA. The differences in the fatty acid side chains and connection to the base structure of PMA compared to PE leads to different UV absorption and MS ionization effects and cause problems for exact quantitative measurements. In this paper, a method is presented which combines both detection types and shows differences between both results. For this purpose, an extraction routine is performed on a PE‐containing seed oil to get a PE standard in high purity, which was used for a standard addition method on two real J. curcas oil samples, derived from Ghana and Mexico. Furthermore, a detection window of ±10 ppm for the high accurate ToF‐MS detection is set to eliminate isobaric interferences from co‐eluting material. Method evaluation of inter‐ and intra‐day variance as well as the recovery rate are performed and determined. With this method a limit of detection of 62 ng mL−1 (UV) and 11 ng mL−1 (MS) can be achieved. Practical Applications: Due to the good biological and technical properties of Jatropha curcas L., its seed oil seems perfect for the application as biodiesel feedstock. The toxicity on the other hand could cause problems when converting side products from the oil production to products of higher value. With the here described method an accurate and precise analysis procedure for the quantification of the toxic compounds namely, phorbol esters, could be applied for toxicity studies or routine checks in industry which is converting plant material from J. curcas, so that no toxic material is used for example as animal feed. In this paper, an exact and robust analysis method is described for the quantification of phorbol esters (PE) in Jatropha curcas L. seed oil. This method procedure includes the extraction of PE in methanol, chromatographic separation on a reverse phase C18 HPLC column and the quantification by standard addition method. For the standard addition method a highly pure PE standard is used, which is extracted and purified by semi preparative HPLC right before the measurements. The used detector for identification and quantification is UV set at 280 nm and ESI‐ToF‐MS with a ±10 ppm mass difference of the deprotonated and formate adduct pseudo molecular ion of PE. [ABSTRACT FROM AUTHOR]

Neutrophils operate at the site of injury or inflammation in the periodontal pocket to ensure periodontal health and clearance of bacterial pathogens. Filifactor alocis is recently identified as a potential periodontal pathogen, and in this study, we assessed the formation of neutrophil extracellular traps (NETs), in response to the presence of the organism. NET formation by human neutrophils was not induced when challenged with F. alocis, independent of opsonization, viability, time, or bacterial dose. F. alocis also failed to induce NETs from TNF-α-primed neutrophils and did not induce the release of extracellular neutrophil elastase. However, significant NET induction was observed when neutrophils were challenged with Streptococcus gordonii or Peptoanaerobacter stomatis, In addition, co-infection studies revealed that the presence of F. alocis with S. gordonii or P. stomatis does not enhance or reduce NETs. Additionally, F. alocis failed to impact pre-formed NETs induced by either S. gordonii or P. stomatis. Pretreatment with F. alocis prior to stimulation with phorbol 12-myristate 13-acetate (PMA), S. gordonii, or P stomatis revealed that the bacterium is capable of reducing only PMA but not S. gordonii or P. stomatis NET formation. These results indicate that F. alocis manipulates neutrophils, inhibiting the triggering of NET induction. [ABSTRACT FROM AUTHOR]

(E)-2-Methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) phenol (MMPP), a new (E)-2,4-bis(p-hydroxyphenyl)-2 - butenal derivative, reportedly has therapeutic effects such as anti-arthritic properties. Although previous studies showed that MMPP has anti-arthritic effects on rheumatoid arthritis (RA), the anti-inflammation mechanism of MMPP remains unclear. In this study, phorbol-12-myristate 13-acetate (PMA) was used as an inflammatory stimulus to evaluate the detailed mechanism of the MMPP-mediated anti-inflammatory effect in human monocytic THP-1 cells. We investigated the effects of MMPP on inflammation-related pathways including protein kinase Cδ (PKCδ), mitogen-activated protein kinase, and activator protein-1 (AP-1). PMA induced the translocation of PKCs from the cytosol to the membrane and phosphorylated JNK. MMPP inhibited PMA-induced membrane translocation of PKCδ, phosphorylation of JNK, and nuclear translocation of AP-1, resulting in downregulation of cyclooxygenase-2 and chemokine ligand 5 production. These findings indicate that MMPP inhibits inflammatory responses in THP-1 cells by mitigating PMA-induced activation of PKCδ and JNK and nuclear translocation of AP-1. Therefore, MMPP may be useful as an anti-inflammatory drug. [ABSTRACT FROM AUTHOR]

Astrocytes are glial cells that have an intimate physical and functional association with synapses in the brain. One of their main roles is to recycle the neurotransmitters glutamate and gamma-aminobutyric acid (GABA), as a component of the glutamate/GABA-glutamine cycle. They perform this function by sequestering neurotransmitters and releasing glutamine via the neutral amino acid transporter SNAT3. In this way, astrocytes regulate the availability of neurotransmitters and subsequently influence synaptic function. Since many plasma membrane transporters are regulated by protein kinase C (PKC), the aim of this study was to understand how PKC influences SNAT3 glutamine transport in astrocytes located immediately adjacent to synapses. We studied SNAT3 transport by whole-cell patch-clamping and fluorescence pH imaging of single astrocytes in acutely isolated brainstem slices, adjacent to the calyx of the Held synapse. Activation of SNAT3-mediated glutamine transport in these astrocytes was reduced to 77 ± 6% when PKC was activated with phorbol 12-myristate 13-acetate (PMA). This effect was very rapid (within ~20 min) and eliminated by application of bisindolylmaleimide I (Bis I) or 7-hydroxystaurosporine (UCN-01), suggesting that activation of conventional isoforms of PKC reduces SNAT3 function. In addition, cell surface biotinylation experiments in these brain slices show that the amount of SNAT3 in the plasma membrane is reduced by a comparable amount (to 68 ± 5%) upon activation of PKC. This indicates a role for PKC in dynamically controlling the trafficking of SNAT3 transporters in astrocytes in situ. These data demonstrate that PKC rapidly regulates the astrocytic glutamine release mechanism, which would influence the glutamine availability for adjacent synapses and control levels of neurotransmission. [ABSTRACT FROM AUTHOR]

THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) are widely used as a model for function and biology of human macrophages. However, the conditions used for differentiation, particularly the concentration of PMA and the duration of treatment, vary widely. Here we compare several differentiation conditions and compare the ability of THP-1 macrophages to interact with the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. The results show that THP-1 macrophages differentiated in high concentrations of PMA rapidly died following infection whereas those differentiated in low concentrations of PMA survived and were able to control the intracellular bacteria similar to primary human macrophages. [ABSTRACT FROM AUTHOR]

Gelatin, denatured collagen, temporarily exists in tissues and may well be pathophysiologically involved in tissue remodeling, inflammation or tissue damage. The present study is aimed to investigate possible biological roles of gelatin by examining its effects on monocyte-like histiocytic lymphoma cell line U937. Once stimulated by phorbol 12-myristate 13-acetate (PMA), U937 cells differentiate into macrophage-like cells, changing from non-adherent to adherent cells with extended pseudopodia. Here we pre-treated the cell dishes with gelatin solution for cell culture. Interestingly, we found that PMA-stimulated U937 cells formed multicellular aggregates on gelatin-coated dishes, accompanying NF-κB-mediated production of pro-inflammatory cytokines, whereas cell aggregation was not detected on non-coated dishes. Moreover, differentiated U937 cells on gelatin-coated dishes showed increased autophagy level and endocytosis. Surprisingly, formation of multicellular aggregates and pro-inflammatory cytokine production were both attenuated by either down-regulation of autophagy with inhibitors, such as 3-methyladenine (3MA) or chloroquine (CQ), or repression of endocytosis with siRNA targeting Endo180. Moreover, autophagy was inhibited by si-Endo180, and endocytosis was suppressed by 3MA, suggesting a positive feedback loop between autophagy and endocytosis. The results revealed that gelatin-coating induced differentiated U937 cells to form cell aggregates and promote NF-κB-mediated pro-inflammatory cytokine production at least partially through an endocytosis-autophagy pathway. [ABSTRACT FROM AUTHOR]

Inflammation-associated damage may occur in any tissue following infection, exposure to toxins, following ischemia, and in allergic and auto-immune reactions. Inflammation may also result from mast cell degranulation induced by the intracellular calcium concentration. The inflammatory process may be inhibited by compounds that affect mast cells. Bisdemethoxycurcumin [1,7-bis(4-hydroxyphenyl) hepta-1,6-diene-3,5-dione, BDCM] is the active component of turmeric. It has anticancer, antioxidant and antibacterial properties. To investigate the molecular mechanism associated with the anti-inflammatory activity of BDCM, human mast cell line 1 (HMC-1) cells were treated with phorbol-12-myristate-13-acetate (PMA) and calcium ionophore A23187 (A23187) to induce the inflammatory process. Various HMC-1 cells were pretreated with BDCM prior to stimulation of inflammation. BDCM inhibited the inflammation-triggered production of cytokines including interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α. BDCM inhibition extended to the gene level. In activated HMC-1 cells, phosphorylation levels of extracellular signal-regulated kinase, c-jun N-terminal kinase and p38 mitogen-activated protein kinase were decreased by treatment with BDCM. BDCM also inhibited nuclear factor-(NF)-κB activation and IκB degradation. In conclusion, BDCM suppresses the expression of TNF-α, IL-8, and IL-6 by inhibiting the NF-κB and mitogen activated protein kinase signaling pathways. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) has a central role in the fine-tuning of environmental and internal stimuli. We herein report a ratiometric fluorescent probe, α-Naph, capable of determining basal H2O2 in the ER. The probe specifically responds to H2O2. The limit of detection of the probe is as low as 38 nM, making it a feasible sensor to image intracellular basal H2O2. In addition, utilizing its ratiometric property, we are able to measure the concentration of H2O2 in the ER quantitatively, eliminating the error caused by the probe concentration and environment. The intracellular concentration of H2O2 in the ER is calculated to be 0.692 µM under normal conditions and 1.26 µM under the stimulation of phorbol myristate acetate. [ABSTRACT FROM AUTHOR]

In LNCaP cells that stably express α 1A -adrenergic receptors, oxymetazoline increased intracellular calcium and receptor phosphorylation, however, this agonist was a weak partial agonist, as compared to noradrenaline, for calcium signaling. Interestingly, oxymetazoline-induced receptor internalization and desensitization displayed greater effects than those induced by noradrenaline. Phorbol myristate acetate induced modest receptor internalization and minimal desensitization. α 1A -Adrenergic receptor interaction with β-arrestins (colocalization/coimmunoprecipitation) was induced by noradrenaline and oxymetazoline and, to a lesser extent, by phorbol myristate acetate. Oxymetazoline was more potent and effective than noradrenaline in inducing ERK 1/2 phosphorylation. Mass spectrometric analysis of immunopurified α 1A -adrenergic receptors from cells treated with adrenergic agonists and the phorbol ester clearly showed that phosphorylated residues were present both at the third intracellular loop and at the carboxyl tail. Distinct phosphorylation patterns were observed under the different conditions. The phosphorylated residues were: a) Baseline and all treatments: T233; b) noradrenaline: S220, S227, S229, S246, S250, S389; c) oxymetazoline: S227, S246, S381, T384, S389; and d) phorbol myristate acetate: S246, S250, S258, S351, S352, S401, S402, S407, T411, S413, T451. Our novel data, describing the α 1A -AR phosphorylation sites, suggest that the observed different phosphorylation patterns may participate in defining adrenoceptor localization and action, under the different conditions examined. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are a highly heterogeneous population of innate lymphocytes that constitute our first line of defense against several types of tumors and microbial infections. Understanding the heterogeneity of these lymphocytes requires the ability to integrate their underlying phenotype with dynamic functional behaviors. We have developed and validated a single-cell methodology that integrates cellular phenotyping and dynamic cytokine secretion based on nanowell arrays and bead-based molecular biosensors. We demonstrate the robust passivation of the polydimethylsiloxane (PDMS)-based nanowells arrays with polyethylene glycol (PEG) and validated our assay by comparison to enzyme-linked immunospot (ELISPOT) assays. We used numerical simulations to optimize the molecular density of antibodies on the surface of the beads as a function of the capture efficiency of cytokines within an open-well system. Analysis of hundreds of individual human peripheral blood NK cells profiled ex vivo revealed that CD56dimCD16+ NK cells are immediate secretors of interferon gamma (IFN-γ) upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h), and that there was no evidence of cooperation between NK cells leading to either synergistic activation or faster IFN-γ secretion. Furthermore, we observed that both the amount and rate of IFN-γ secretion from individual NK cells were donor-dependent. Collectively, these results establish our methodology as an investigational tool for combining phenotyping and real-time protein secretion of individual cells in a high-throughput manner. [ABSTRACT FROM AUTHOR]

The recent attention given to diseases associated with memory B-cell (mBC)- produced antibodies (Abs) suggests the need for a similar in vitro assay to evaluate the functions of mBCs. Here, we cultured peripheral blood mononuclear cells (PBMCs) with the intent to collect mBC-derived Abs in vitro and maintain their cell-cell contactdependent interactions with helper T-cells. PBMCs were cultured with interleukin (IL)-21, CpG-oligodeoxynucleotides (ODN), phorbol myristate acetate (PMA), and phytohemagglutinin/leucoagglutinin (PHA-L) in 24-well flat-bottom plates (5 × 105 cells/well). A culture supernatant analysis of PBMCs from healthy donors (n = 10) indicated that antigen-specific IgM Ab levels in a PBMC culture supernatant might be better able to demonstrate the antigen sensitization status in a smaller peripheral blood sample, compared to IgG because Epstein-Barr virus-specific IgM mBCs circulate peripherally at a significantly higher frequency once antiviral humoral immunity has stabilized. Thus, our in vitro assay demonstrated the potential significance of antigen-specific IgM Ab production in the culture supernatants. Furthermore, an analysis of cultured PBMCs from allograft kidney recipients (n = 16) sensitized with de novo donor-specific human leukocyte antigen (HLA)-specific Abs (DSAs) showed that IgM-type HLA-specific Abs were detected mainly from the culture supernatants from PBMCs of patients with stable graft function, whereas IgG isotype HLA Abs were detectable only from patients with biopsy-proven antibody-mediated rejection. In other words, these IgG isotype Abs also represented an activated humoral immune response in vivo. Additionally, IgM- and IgGexpressing mBCs from healthy donors (n = 5) were cultured with IL-21, CpG-ODN, and a supernatant produced by stimulating CD19+ B-cell-depleted PBMCs with PHA-L and PMA in 24-well flat-bottom plates (1 × 105 cells/well), and the resulting in vitro analysis provided some information regarding the biological processes of IgG and IgM mBCs in peripheral blood. Taken together, our findings suggest that antigen-specific Ab subtype analyses of supernatants from cultured PBMCs might more effectively and accurately reflect a patient's Ab-associated pathological condition vs. than serum IgG and IgM levels. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis (TB) is one of the most common infectious diseases worldwide. During active tuberculosis, T helper (Th) 17 cells are decreased, however the association with inhibitory immune regulation is unclear.Methods: We enrolled 27 patients with TB and 20 age- and sex-matched controls and studies their lymphocyte status. Peripheral blood lymphocytes were isolated and programmed death-1 (PD-1) and programmed death ligand 1 (PD-L1) were measured on Th17 cells by using flow cytometry after the cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin for 6 h. In addition, Th2 and regulatory T cells were measured and analyzed.Results: The TB group had lower levels of Th17 cells but higher levels of Th2 and Treg cells than the controls. In Th17 cells, the percentage of PD-L1 was higher in the TB group than that in the controls. In Th2 and Treg cells, the percentage of cytotoxic T-lymphocyte associated protein 4 (CTLA-4) was lower in the TB group and PD-1 was higher in Treg cells in the TB group. In the patients with extra-pulmonary TB, levels of Th1, Th2 and T17 cells were lower than those with pulmonary TB. The percentage of PD-1 on Th1 lymphocytes positively correlated with radiographic score.Conclusions: Lower level of Th17 in TB patients may be associated with increased percentage of PD-L1 and increasing levels of Th2 and Treg cells which influenced by CTLA-4. [ABSTRACT FROM AUTHOR]

Objective To compare the protein profile of culture supernatants in stimulated and unstimulated human fibroblasts to find some proteins indicating the presence of fibroblasts and their activation status. Methods Dermal fibroblasts were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycine for 72 h. MTT assay was done to determine cell viability and A/E fluorescent staining was used to evaluate the cell death pattern. Protein analysis was performed by gradient SDS polyacrylamide gel electrophoresis 8%–16%. Results The supernatant of 24 h cultured both stimulated and unstimulated fibroblasts showed two bands in SDS-PAGE analysis with relative molecular weights of 8.59 and 78.8 kDa. These bands density was decreased during the next 48 h in unstimulated cells while their expression was continued in PMA or PMA/ionomycine stimulated cells and a new 85.3 kDa band was appeared in unstimulated and 72 h PMA stimulated cells. Moreover, we found another seven small size (10–19.5 kDa) proteins in supernatants of 48 h and 72 h unstimulated but not in PMA or PMA/Ionomycine stimulated fibroblasts. Most of these proteins expression were down regulated following fibroblast activation. This down-regulation is consistent with our finding that PMA or PMA/ionomycine stimulated cells exhibited a significant level of apoptosis cell death. Conclusions Human fibroblasts produce some small to intermediate sized proteins with specific SDS-PAGE profile upon cell activation. Most of these proteins can be excreted in urine and can be immunogen theoretically so this data provided a reliable clue for fibrosis biomarker screening based on designation of an appropriated immunoassay. [ABSTRACT FROM AUTHOR]

Rho GDP-dissociation inhibitor β (RhoGDIβ), a regulator of the Rho family of proteins, is expressed abundantly in the hematopoietic cell lineage. During apoptosis of hematopoietic cells, RhoGDIβ is cleaved by caspase-3 at Asp19 and this cleaved form (Δ19-RhoGDIβ) has been implicated in the apoptotic pathway. To clarify the role of RhoGDIβ in hematopoietic cells, the present study performed immunoblotting and immunofluorescence staining to examine the expression of RhoGDIβ and Δ19-RhoGDIβ during phorbol 12-myristate 13-acetate (PMA)-stimulated differentiation of human THP-1 monocytic cells to macrophages. During differentiation of the THP-1 cells to macrophages, the expression of RhoGDIβ remained stable; however, the expression of Δ19-RhoGDIβ increased, particularly in well-spreading, non-apoptotic cells, which differentiated into macrophages. These results suggested that Δ19-RhoGDIβ has an apoptosis-independent role in the PMA-induced differentiation of THP-1 cells to macrophages. [ABSTRACT FROM AUTHOR]

The liver is the most important link between the circadian system and metabolism. As a food-entrainable oscillator, the hepatic clock needs to be entrained by food-related signals. The objective of the present study was to investigate the possible role of ghrelin (an orexigenic peptide mainly synthesized in the gastrointestinal tract) as an endogenous synchronizer of the liver oscillator in teleosts. To achieve this aim, we first examined the presence of ghrelin receptors in the liver of goldfish. Then, the ghrelin regulation of clock gene expression in the goldfish liver was studied. Finally, the possible involvement of the phospholipase C/protein kinase C (PLC/PKC) and adenylate cyclase/protein kinase A (AC/PKA) intracellular signalling pathways was investigated. Ghrelin receptor transcripts, ghs-r1a, are present in the majority of goldfish hepatic cells. Ghrelin induced the mRNA expression of the positive (gbmal1a, gclock1a) and negative (gper genes) elements of the main loop of the molecular clock machinery, as well as grev-erbα (auxiliary loop) in cultured liver. These effects were blocked, at least in part, by a ghrelin antagonist. Incubation of liver with a PLC inhibitor (U73122), a PKC activator (phorbol 12-myristate 13-acetate) and a PKC inhibitor (chelerythrine chloride) demonstrated that the PLC/PKC pathway mediates such ghrelin actions. Experiments with an AC activator (forskolin) and a PKA inhibitor (H89) showed that grev-erbα regulation could be due to activation of PKA. Taken together, the present results show for the first time in vertebrates a direct action of ghrelin on hepatic clock genes and support a role for this hormone as a temporal messenger in the entrainment of liver circadian functions. [ABSTRACT FROM AUTHOR]

It is known that electrochemical energy can be harvested from mammalian cells, more specifically from white blood cells (WBC). This study focuses on an improved biofuel cell operating on phorbol myristate acetate (PMA) activated THP-1 human monocytic cells. Electrochemical investigation showed strong evidence pointing towards hydrogen peroxide being the primary current source, confirming that the current originates from NADPH oxidase activity. Moreover, an adequate substrate for differentiation and activation of THP-1 cells was examined. ITO, gold, platinum and glass were tested and the amount of superoxide anion produced by NADPH oxidase was measured by spectrophotometry through WST-1 reduction at 450 nm and used as an indicator of cellular activity and viability. These substrates were subsequently used in a conventional two-compartment biofuel cell where the power density output was recorded. The material showing the highest cell activity compared to the reference cell culture plate and the highest power output was ITO. Under our experimental conditions, a power density of 4.5 μW/cm 2 was reached. To the best of our knowledge, this is a threefold higher power output than other leukocyte biofuel cells. [ABSTRACT FROM AUTHOR]

Oncostatin-M (OSM), an IL-6 family cytokine, exhibits varied roles in different patho-physiological conditions. Differential expression of OSM in response to varying stimuli indicates importance of its regulation of expression. The present study illustrated transcriptional induction of osm on treatment with chemical inducer, phorbol-12-myristate-13-acetate (PMA). Following initial hours of PMA treatment, a nuclear protein C/EBP-β binds specifically to the CCAAT consensus sequence at the proximal end of the OSM promoter. Genistein (a specific Tyr phosphorylation inhibitor) leads to the interaction of CHOP (C/EBP Homologous Protein) with C/EBP-β, thus negatively regulating it. Knockdown of C/EBP-β also leads to inhibition of PMA-mediated OSM induction. [ABSTRACT FROM AUTHOR]