Glycosylphosphatidylinositols (GPIs) are glycolipids described as toxins of protozoan parasites due to their inflammatory properties in mammalian hosts characterized by the production of interleukin (IL)-1, IL-12 and tumor necrosis factor (TNF)-α. In the present work, we studied the cytokines produced by antigen presenting cells in response to ten different GPI species extracted from Babesia divergens, responsible for babesiosis. Interestingly, B. divergens GPIs induced the production of anti-inflammatory cytokines (IL-2, IL-5) and of the regulatory cytokine IL-10 by macrophages and dendritic cells. In contrast to all protozoan GPIs studied until now, GPIs from B. divergens did not stimulate the production of TNF-α and IL-12, leading to a unique Th1/Th2 profile. Analysis of the carbohydrate composition of the B. divergens GPIs indicated that the di-mannose structure was different from the evolutionary conserved tri-mannose structure, which might explain the particular cytokine profile they induce. Expression of major histocompatibility complex (MHC) molecules on dendritic cells and apoptosis of mouse peritoneal cells were also analysed. B. divergens GPIs did not change expression of MHC class I, but decreased expression of MHC class II at the cell surface, while GPIs slightly increased the percentages of apoptotic cells. During pathogenesis of babesiosis, the inflammation-coagulation auto-amplification loop can lead to thrombosis and the effect of GPIs on coagulation parameters was investigated. Incubation of B. divergens GPIs with rat plasma ex vivo led to increase of fibrinogen levels and to prolonged activated partial thromboplastin time, suggesting a direct modulation of the extrinsic coagulation pathway by GPIs.
The sodium leak channel NALCN is poorly understood, but is reported as a Na+-permeable, nonselective cation leak channel which regulates resting membrane potential and electrical excitability. Previous work has indicated that NALCN currents can be stimulated by activation of several G protein coupled receptors, including the M3 muscarinic receptor. We undertook a study using voltage clamp electrophysiology to investigate NALCN currents. We compared currents elicited from untransfected control HEK239 cells in response to M3R agonists muscarine or Oxotremorine M to currents elicited from cells transfected with M3R only or the M3R plus NALCN and cDNA encoding accessory proteins UNC-80 and Src. Currents with similar properties were observed in all three groups of cells in response to muscarine agonists, in similar proportions of cells tested, from all three groups of cells. Our findings do not support previous electrophysiological studies suggesting that heterologously expressed NALCN functions as a Na+ leak channel in HEK293 cells. More research will be required to determine the molecular requirements for successful expression of the NALCN channel.
Vanadate, a protein tyrosine phosphatase inhibitor which elicits insulin-like effects, has previously been shown to inhibit expression of the insulin receptor gene at the transcriptional level in rat hepatoma cells. In an attempt to identify the DNA sequence and transcription factors potentially involved in this effect, a fragment of the proximal 5'flanking region of the IR gene (-1143/-252 upstream the ATG codon) has been cloned and functionally characterized. RNase protection allowed the identification of several transcription start sites in the conserved region of the gene, among which two major sites at -455 and -396. Upon fusion to the luciferase gene and transient transfection into hepatoma cells, the -1143/-252 fragment showed promoter activity. This was unaffected by deletion of the -1143/-761 sequence, but markedly decreased (90%) by additional deletion of the -760/-465 sequence. Treatment of hepatoma cells with vanadate led to a dose-dependent decrease in promoter activity of the 1143/-252, -760/-252 and -464/-252 constructs (change relative to untreated cells, 40, 55 and 23% at 125 μM, and 70, 85 and 62% at 250 μM, respectively). These data suggest that although the entire DNA sequence upstream the transcription start sites is probably involved in vanadate-induced inhibition, the short sequence downstream of position -464 and is sufficient for inhibition. Potential targets of vanadate are the transcription factors FoxO1 and HMGA1, two downstream targets of the insulin signaling pathway which have been shown to mediate the inhibitory effect of insulin on IR gene expression.
An infectious retroviral particle contains 1000–1500 molecules of the nucleocapsid protein (NC) that cover the diploid RNA genome. NC is a small zinc finger protein that possesses nucleic acid chaperone activity that enables NC to rearrange DNA and RNA molecules into the most thermodynamically stable structures usually those containing the maximum number of base pairs. Thanks to the chaperone activity, NC plays an essential role in reverse transcription of the retroviral genome by facilitating the strand transfer reactions of this process. In addition, these reactions are involved in recombination events that can generate multiple drug resistance mutations in the presence of anti-HIV-1 drugs. The strand transfer reactions rely on base pairing of folded DNA/RNA structures. The molecular mechanisms responsible for NC-mediated strand transfer reactions are presented and discussed in this review. Antiretroviral strategies targeting the NC-mediated strand transfer events are also discussed.
Animals and humans are exposed each day to a multitude of chemicals in the air, water and food. They have developed a battery of enzymes and transporters that facilitate the biotransformation and elimination of these compounds. Moreover, a majority of these enzymes and transporters are inducible due to the activation of xenobiotic receptors which act as transcription factors for the regulation of their target genes (such as xenobiotic metabolizing enzymes, see below §4 for the AhR). These receptors include several members of the nuclear/steroid receptor family (CAR for Constitutive Androstane Receptor, PXR for Pregnane X Receptor) but also the Aryl hydrocarbon Receptor or AhR, a member of the bHLH-PAS family (basic Helix-Loop-Helix - Period/ARNT/Single minded). In addition to the regulation of xenobiotic metabolism, numerous alternative functions have been characterized for the AhR since its discovery. These alternative functions will be described in this review along with its endogenous functions as revealed by experiments performed on knock-out animals.
Herbal drugs, on which 80% of the world's population rely, are relatively safe over conventional drugs. Conventional drugs are costly, have serious side effects and hence over the past few decades researchers have focused on drug discovery from herbal medicines or botanical sources. The majority of new herbal drugs have been generated from secondary metabolites (alkaloids, terpenoids and phenolic compounds) of plant metabolism. Till date, only a small fraction of the vast diversity of plant metabolism has been explored for the production of new medicines and other products. The emergence of new herbal genomics research, medicinal plant genomics consortium, together with advances in other omics information may help for the speedy discovery of previously unknown metabolic pathways and enzymes. This review highlights the importance of genomics research in the discovery of some previously unknown enzymes/pathways which may make significant contributions in plant metabolic biology and may be used for the future discovery of many new pharmaceutical agents.
•Acute exercise promotes an increase in F2-IsoPs plasma level in older athletes.•The F2-IsoPs plasma level significantly decreased after recovery in older athletes.•This kinetic of F2-IsoPs could reflect a decrease of glutathione (GSH).•Oxidative stress status determination should be challenged by exercise.•Assessment of F2-IsoPs plasma level should be paired to GSH assessment.
The recombinant fucolectin-related protein (FRP) of unknown function, encoded by the SP2159 gene of Streptococcus pneumoniae, was expressed in E. coli. In this study, its glycan-recognition epitopes and their binding potencies were examined by enzyme-linked lectinosorbent and inhibition assays. The results indicate that FRP reacted strongly with human blood group ABH and l-Fucα1→2-active glycotopes and in their polyvalent (super) forms. When expressed by mass relative potency, the binding affinities of FRP to poly-l-Fucα1→glycotopes were about 5.0 × 105 folds higher than that of the mono-l-Fucα1→glycotope form. This unique binding property of FRP can be used as a special tool to differentiate complex forms of l-Fucα1→2 and other forms of glycotopes.
Exosomes are 40–100 nm nanovesicles containing RNA and different proteins. Exosomes containing proteins, lipids, mRNAs, and microRNAs are important in intracellular communication and immune function. Exosomes from different sources are usually obtained by combination of centrifugation and ultracentrifugation and according to published data can contain from a few dozens to thousands of different proteins. Crude exosome preparations from milk of eighteen horses were obtained for the first time using several standard centrifugations. Exosome preparations were additionally purified by FPLC gel filtration. Individual preparations demonstrated different profiles of gel filtration showing well or bad separation of exosome peaks and one or two peaks of co-isolating proteins and their complexes. According to the electron microscopy, well purified exosomes displayed a typical exosome-like size (30–100 nm) and morphology. It was shown that exosomes may have several different biological functions, but detection of their biological functions may vary significantly depending on the presence of exosome contaminating proteins and proteins directly into exosomes. Exosome proteins were identified before and after gel filtration by MALDI MS and MS/MS spectrometry of protein tryptic hydrolyzates derived by SDS PAGE and 2D electrophoresis. The results of protein identification were unexpected: one or two peaks co-isolating proteins after gel-filtration mainly contained kappa-, beta-, alpha-S1-caseins and its precursors, but these proteins were not found in well-purified exosomes. Well-purified exosomes contained from five to eight different major proteins: CD81, CD63 receptors, beta-lactoglobulin and lactadherin were common to all preparations, while actin, butyrophilin, lactoferrin, and xanthine dehydrogenase were found only in some of them.
In humans the steroid 5α-reductase (SRD5A) family comprises five integral membrane enzymes that carry out reduction of a double bond in lipidic substrates: Δ4-3-keto steroids, polyprenol and trans-enoyl CoA. The best-characterized reaction is the conversion of testosterone into the more potent dihydrotestosterone carried out by SRD5A1-2. Some controversy exists on their possible nuclear or endoplasmic reticulum localization.
There is a constant need for better stroke treatments. Neurons at the periphery of an ischemic stroke affected brain tissue remains metabolically active for several hours or days after stroke onset. They later undergo mitochondrion-mediated apoptosis. It has been found that inhibiting apoptosis in the peripheral ischemic neurons could be very effective in the prevention of stroke progression. During stroke associated apoptosis, cytosolic c-Jun N-terminal Kinases (JNKs) and Bcl-2 family proteins translocate towards mitochondria and promote cytochrome c release by interacting with the outer mitochondrion membrane associated proteins. This review provides an overview of the plausible interactions of the outer mitochondrial membrane Voltage Dependent Anion Channel, Bcl-2 family proteins and JNKs in cytochrome c release in the peripheral ischemic stroke associated apoptotic neurons. The review ends with a note on designing new anti-stroke treatments.
The use of oregano (Origanum vulgare) essential oil (OEO) has become popular in skin care products. However, scientific research regarding its effects on human skin cells is scarce. In this study, we investigated the biological activity of a commercially available OEO, which is high in carvacrol content, in a human skin cell disease model. OEO induced marked antiproliferative effects and significantly inhibited several inflammatory biomarkers, including monocyte chemoattractant protein 1 (MCP-1), vascular cell adhesion molecule 1 (VCAM-1), intracellular cell adhesion molecule 1 (ICAM-1), interferon gamma-induced protein 10 (IP-10), interferon-inducible T-cell alpha chemoattractant (I-TAC), and monokine induced by gamma interferon (MIG). OEO also significantly inhibited tissue remodeling biomarkers, namely collagen I, collagen III, epidermal growth factor receptor (EGFR), matrix metalloproteinase 1 (MMP-1), plasminogen activator inhibitor 1 (PAI-1), tissue inhibitor of metalloproteinase (TIMP) 1 and 2. An immunomodulatory biomarker, macrophage colony-stimulating factor (M-CSF), was also strongly inhibited by OEO treatment. In addition, OEO significantly modulated global gene expression and altered signaling pathways, many of which are critical in inflammation, tissue remodeling, and cancer signaling processes. These findings along with existing studies largely support the anti-inflammatory, tissue remodeling, immunomodulatory, and anticancer activities of OEO. In conclusion, this study provides the first evidence of the biological activity of OEO in human dermal fibroblasts. We suggest that OEO, with carvacrol as the major active component, is a promising candidate for use in skin care products with anti-inflammatory and anticancer properties.
Research on the biological effects of essential oils on human skin cells is scarce. In the current study, we primarily explored the biological activities of 10 essential oils (nine single and one blend) in a pre-inflamed human dermal fibroblast system that simulated chronic inflammation. We measured levels of proteins critical for inflammation, immune responses, and tissue-remodeling processes. The nine single oils were distilled from Citrus bergamia (bergamot), Coriandrum sativum (cilantro), Pelargonium graveolens (geranium), Helichrysum italicum (helichrysum), Pogostemon cablin (patchouli), Citrus aurantium (petitgrain), Santalum album (sandalwood), Nardostachys jatamansi (spikenard), and Cananga odorata (ylang ylang). The essential oil blend (commercial name Immortelle) is composed of oils from frankincense, Hawaiian sandalwood, lavender, myrrh, helichrysum, and rose. All the studied oils were significantly anti-proliferative against these cells. Furthermore, bergamot, cilantro, and spikenard essential oils primarily inhibited protein molecules related to inflammation, immune responses, and tissue-remodeling processes, suggesting they have anti-inflammatory and wound healing properties. Helichrysum and ylang ylang essential oils, as well as Immortelle primarily inhibited tissue remodeling-related proteins, suggesting a wound healing property. The data are consistent with the results of existing studies examining these oils in other models and suggest that the studied oils may be promising therapeutic candidates. Further research into their biological mechanisms of action is recommended. The differential effects of these essential oils suggest that they exert activities by different mechanisms or pathways, warranting further investigation. The chemical composition of these oils was analyzed using gas chromatography–mass spectrometry.
Tumor-associated macrophages (TAMs) are a significant component of the microenvironment of any solid tumors in the majority of cancers, associated with unfavorable prognosis. TAMs emerge as attractive targets for therapeutic strategies aimed at reprogramming their protumor phenotype into an effective antitumor activity. In this review article, we present an overview of mechanisms responsible for TAMs recruitment and highlight the roles of TAMs in the regulation of tumor angiogenesis, invasion, metastasis, immunosuppression, and chemotherapeutic resistance. We describe the interplay between Th17 cells and other immune cells in the tumor microenvironment, and we assess both the potential antitumorigenic and pro-tumorigenic activities of Th17 cells and their associated cytokines. Understanding the nature of Th17 cell responses in the tumor microenvironment will be important for the design of more efficacious cancer immunotherapies. Finally, we discuss TAM-targeting therapy as a promising novel strategy for an indirect cancer therapy.
Lemongrass (Cymbopogon flexuosus) essential oil (LEO), which has citral as its main component, has exhibited anti-inflammatory effect in both animal and human cells. In this study, we evaluated the anti-inflammatory activity of a commercially available LEO in pre-inflamed human dermal fibroblasts. We first studied the impact of LEO on 17 protein biomarkers that are critically associated with inflammation and tissue remodeling. LEO significantly inhibited production of the inflammatory biomarkers vascular cell adhesion molecule 1 (VCAM-1), interferon gamma-induced protein 10 (IP-10), interferon-inducible T-cell alpha chemoattractant (I-TAC), and monokine induced by gamma interferon (MIG); decreased levels of the tissue remodeling biomarkers collagen-I and III, epidermal growth factor receptor (EGFR), and plasminogen activator inhibitor (PAI-1); and inhibited the immunomodulatory biomarker macrophage colony-stimulating factor (M-CSF). Furthermore, we studied the impact of LEO on genome-wide gene expression profiles. LEO significantly modulated global gene expression and robustly impacted signaling pathways, many of which are critical for inflammation and tissue remodeling processes. This study provides the first evidence of the anti-inflammatory activity of LEO in human skin cells and indicates that it is a good therapeutic candidate for treating inflammatory conditions of the skin.
Studies show that the continuous consumption of fructose can lead to nonalcoholic fatty liver disease (NAFLD) and steatohepatitis. We aimed to investigate the role of Metformin in an animal model of liver injury caused by fructose intake, focusing on the molecular markers of lipogenesis, beta-oxidation, and antioxidant defenses. Male three months old C57BL/6 mice were divided into control group (C) and fructose group (F, 47% fructose), maintained for ten weeks. After, the groups received Metformin or vehicle for a further eight weeks: control (C), control + Metformin (CM), fructose (F), and fructose + Metformin (FM). Fructose resulted in hepatic steatosis, insulin resistance and lower insulin sensitivity in association with higher mRNA levels of proteins linked with de novo lipogenesis and increased lipid peroxidation. Fructose diminished mRNA expression of antioxidant enzymes, and of proteins responsible for mitochondrial biogenesis. Metformin reduced de novo lipogenesis and increased the expression of proteins related to mitochondrial biogenesis, thereby increasing beta-oxidation and decreasing lipid peroxidation. Also, Metformin upregulated the expression and activity of antioxidant enzymes, providing a defense against increased reactive oxygen species generation. Therefore, a significant reduction in triglyceride accumulation in the liver, steatosis and lipid peroxidation was observed in the FM group. In conclusion, fructose increases de novo lipogenesis, reduces the antioxidant defenses, and diminishes mitochondrial biogenesis. After an extended period of fructose intake, Metformin treatment, even in continuing the fructose intake, can reverse, at least partially, the liver injury and prevents NAFLD progression to more severe states.
Bax is a pro-apoptotic cytosolic protein. In this work native (unphosphorylated) and JNK3 phosphorylated Bax proteins are studied on artificial bilayer membranes for pore formation. Phosphorylated Bax formed pore on the bilayer lipid membrane whereas native one does not. In cells undergoing apoptosis the pore formed by the phosphorylated Bax could be important in cytochrome c release from the mitochondrial intermembrane space to the cytosol. The low conductance (1.5 nS) of the open state of the phosphorylated Bax pore corresponds to pore diameter of 0.9 nm which is small to release cytochrome c (∼3.4 nm). We hypothesized that JNK3 phosphorylated Bax protein can form bigger pores after forming complexes with other mitochondrial proteins like VDAC, t-Bid etc. to release cytochrome c.
MDCK cells are widely used to study the differential targeting of membrane transporters to apical and basolateral membrane but its canine origin limited the commercial tools available for the analysis of protein trafficking machinery. Because apical and basolateral membranes are only found in differentiated epithelial cells, genes critical for differential targeting may be specifically up-regulated upon MDCK cell differentiation. To search for these genes, a cross-species screening strategy was used. We first analyzed the human microarray data for protein trafficking-related genes that were up-regulated in colon carcinoma Caco2 cells upon differentiation. The results of mouse 44K gene expression microarray analysis were then used to extract additional candidate genes that showed higher expression in normal colon epithelium compared to primary embryonic fibroblasts. Finally, NCBI genomic sequence information was used to design RT-PCR primers for 13 candidate and 10 negative control genes and used to analyze MDCK cells at 2, 13 and 17 days after seeding. To determine whether the gene up-regulation was specific in epithelial differentiation, we also performed RT-PCR on rat non-differentiating intestinal IEC-6 cells and mouse C2C12 cells, a differentiating myoblast model. Of the 13 candidate genes, 3 genes, SDCBP2, KIF12, KIF27, met all criteria of specific up-regulation in differentiated MDCK cells. In addition, KIF13A showed up-regulation in differentiated MDCK and C2C12 cells but not in IEC-6 cells cultured for the same duration. The functions of these genes need to be analyzed in the future. This cross-species screening strategy may be useful for other non-human, non-rodent cell models.
Voltage-Dependent Anion Channel (VDAC) phosphorylated by c-Jun N-terminal Kinase-3 (JNK3) was incorporated into the bilayer lipid membrane. Single-channel electrophysiological properties of the native and the phosphorylated VDAC were compared. The open probability versus voltage curve of the native VDAC displayed symmetry around the voltage axis, whereas that of the phosphorylated VDAC showed asymmetry. This result indicates that phosphorylation by JNK3 modifies voltage-dependence of VDAC.
Many basic science questions remain regarding protein functions in the pathogen: host interaction, especially in the trunk disease fungi family, the Botryosphaeriaceae, which are a global problem for economically important plants, especially fruiting trees. Proteomics is a highly useful technology for studying protein expression and for discovering novel proteins in unsequenced and poorly annotated organisms. Current fungal proteomics approaches involve 2D SDS-PAGE and extensive, complex, protein extraction methodologies. In this work, a modified Folch extraction was applied to protein extraction to perform both de novo peptide sequencing and peptide fragmentation analysis/protein identification of the plant and human fungal pathogen Lasiodiplodia theobromae. Both bioinformatics approaches yielded novel peptide sequences from proteins produced by L. theobromae in the presence of exogenous triglycerides and glucose. These proteins and the functions they may possess could be targeted for further functional characterization and validation efforts, due to their potential uses in biotechnology and as new paradigms for understanding fungal biochemistry, such as the finding of allergenic enolases, as well as various novel proteases, including zinc metalloproteinases homologous to those found in snake venom. This work contributes to genomic annotation efforts, which, hand in hand with genomic sequencing, will help improve fungal bioinformatics databases for future studies of Botryosphaeriaceae. All data, including raw data, are available via the ProteomeXchange data repository with identifier PXD005283. This is the first study of its kind in Botryosphaeriaceae.
