scholarly journals A New MAP Kinase Protein Involved in Estradiol-Stimulated Reproduction of the Helminth ParasiteTaenia crassiceps

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Galileo Escobedo ◽  
Gloria Soldevila ◽  
Guadalupe Ortega-Pierres ◽  
Jesús Ramsés Chávez-Ríos ◽  
Karen Nava ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Map Kinases ◽  
Host Cells ◽  
Cross Contamination ◽  
Flow Cytometry Analysis ◽  
Cellular Processes ◽  
17Β Estradiol ◽  
Activation Motif

MAP kinases (MAPK) are involved in the regulation of cellular processes such as reproduction and growth. In parasites, the role of MAPK has been scarcely studied. Here, we describe the participation of an ERK-like protein in estrogen-dependent reproduction of the helminth parasiteTaenia crassiceps. Our results show that 17β-estradiol induces a concentration-dependent increase in the bud number of in vitro cultured cysticerci. If parasites are also incubated in presence of an ERK-inhibitor, the stimulatory effect of estrogen is blocked. The expression of ERK-like mRNA and its corresponding protein was detected in the parasite. The ERK-like protein was over-expressed by all treatments. Nevertheless, a strong induction of phosphorylation of this protein was observed only in response to 17β-estradiol. Cross-contamination by host cells was discarded by flow cytometry analysis. Parasite cells expressing the ERK-like protein were exclusively located at the subtegument tissue by confocal microscopy. Finally, the ERK-like protein was separated by bidimensional electrophoresis and then sequenced, showing the conserved TEY activation motif, typical of all known ERK 1/2 proteins. Our results show that an ERK-like protein is involved in the molecular signalling during the interaction between the host andT. crassiceps, and may be considered as target for anti-helminth drugs design.

scholarly journals A20 regulates the therapeutic effect of anti-PD-1 immunotherapy in melanoma

2020 ◽  
Vol 8 (2) ◽  
pp. e001866
Author(s):  
Weinan Guo ◽  
Jinyuan Ma ◽  
Sen Guo ◽  
Huina Wang ◽  
Sijia Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Mouse Model ◽  
Therapeutic Effect ◽  
Immune Checkpoint ◽  
Antitumor Immunity ◽  
Flow Cytometry Analysis ◽  
Melanoma Patients

BackgroundThe therapeutic effect of immune checkpoint blockers, especially the neutralizing antibodies of programmed cell death (PD-1) and its ligand programmed death ligand 1 (PD-L1), has been well verified in melanoma. Nevertheless, the dissatisfactory response rate and the occurrence of resistance significantly hinder the treatment effect. Inflammation-related molecules like A20 are greatly implicated in cancer immune response, but the role of tumorous A20 in antitumor immunity and immunotherapy efficacy remains elusive.MethodsThe association between tumorous A20 expression and the effect of anti-PD-1 immunotherapy was determined by immunoblotting, immunofluorescence staining and flow cytometry analysis of primary tumor specimens from melanoma patients. Preclinical mouse model, in vitro coculture system, immunohistochemical staining and flow cytometry analysis were employed to investigate the role of A20 in regulating the effect of anti-PD-1 immunotherapy. Bioinformatics, mass spectrum analysis and a set of biochemical analyzes were used to figure out the underlying mechanism.ResultsWe first discovered that upregulated A20 was associated with impaired antitumor capacity of CD8+T cells and poor response to anti-PD-1 immunotherapy in melanoma patients. Subsequent functional studies in preclinical mouse model and in vitro coculture system proved that targeting tumorous A20 prominently improved the effect of immunotherapy through the invigoration of infiltrating CD8+T cells via the regulation of PD-L1. Mechanistically, A20 facilitated the ubiquitination and degradation of prohibitin to potentiate STAT3 activation and PD-L1 expression. Moreover, tumorous A20 expression was highly associated with the ratio of Ki-67 percentage in circulating PD-1+CD8+T cells to tumor burden.ConclusionsTogether, our findings uncover a novel crosstalk between inflammatory molecules and antitumor immunity in melanoma, and highlight that A20 can be exploited as a promising target to bring clinical benefit to melanomas refractory to immune checkpoint blockade.

scholarly journals Membrane Binding, Cellular Cholesterol Content and Resealing Capacity Contribute to Epithelial Cell Damage Induced by Suilysin of Streptococcus suis

Pathogens ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 33 ◽  
Author(s):  
Désirée Vötsch ◽  
Maren Willenborg ◽  
Walter M.R. Oelemann ◽  
Graham Brogden ◽  
Peter Valentin-Weigand
Keyword(s):  
Flow Cytometry ◽  
Epithelial Cells ◽  
Cell Damage ◽  
Binding Capacity ◽  
Cholesterol Content ◽  
Economic Losses ◽  
Flow Cytometry Analysis ◽  
Cellular Cholesterol

Streptococcus (S.) suis is a major cause of economic losses in the pig industry worldwide and is an emerging zoonotic pathogen. One important virulence-associated factor is suilysin (SLY), a toxin that belongs to the family of cholesterol-dependent pore-forming cytolysins (CDC). However, the precise role of SLY in host–pathogen interactions is still unclear. Here, we investigated the susceptibility of different respiratory epithelial cells to SLY, including immortalized cell lines (HEp-2 and NPTr cells), which are frequently used in in vitro studies on S. suis virulence mechanisms, as well as primary porcine respiratory cells, which represent the first line of barrier during S. suis infections. SLY-induced cell damage was determined by measuring the release of lactate dehydrogenase after infection with a virulent S. suis serotype 2 strain, its isogenic SLY-deficient mutant strain, or treatment with the recombinant protein. HEp-2 cells were most susceptible, whereas primary epithelial cells were hardly affected by the toxin. This prompted us to study possible explanations for these differences. We first investigated the binding capacity of SLY using flow cytometry analysis. Since binding and pore-formation of CDC is dependent on the membrane composition, we also determined the cellular cholesterol content of the different cell types using TLC and HPLC. Finally, we examined the ability of those cells to reseal SLY-induced pores using flow cytometry analysis. Our results indicated that the amount of membrane-bound SLY, the cholesterol content of the cells, as well as their resealing capacity all affect the susceptibility of the different cells regarding the effects of SLY. These findings underline the differences of in vitro pathogenicity models and may further help to dissect the biological role of SLY during S. suis infections.

scholarly journals Oxidative Stress as a Possible Target in the Treatment of Toxoplasmosis: Perspectives and Ambiguities

2021 ◽  
Vol 22 (11) ◽  
pp. 5705
Author(s):  
Karolina Szewczyk-Golec ◽  
Marta Pawłowska ◽  
Roland Wesołowski ◽  
Marcin Wróblewski ◽  
Celestyna Mila-Kierzenkowska
Keyword(s):  
Oxidative Stress ◽  
Animal Studies ◽  
Treatment Options ◽  
Natural Antioxidants ◽  
Redox Status ◽  
Host Cells ◽  
Common Disease ◽  
Parasite Viability

Toxoplasma gondii is an apicomplexan parasite causing toxoplasmosis, a common disease, which is most typically asymptomatic. However, toxoplasmosis can be severe and even fatal in immunocompromised patients and fetuses. Available treatment options are limited, so there is a strong impetus to develop novel therapeutics. This review focuses on the role of oxidative stress in the pathophysiology and treatment of T. gondii infection. Chemical compounds that modify redox status can reduce the parasite viability and thus be potential anti-Toxoplasma drugs. On the other hand, oxidative stress caused by the activation of the inflammatory response may have some deleterious consequences in host cells. In this respect, the potential use of natural antioxidants is worth considering, including melatonin and some vitamins, as possible novel anti-Toxoplasma therapeutics. Results of in vitro and animal studies are promising. However, supplementation with some antioxidants was found to promote the increase in parasitemia, and the disease was then characterized by a milder course. Undoubtedly, research in this area may have a significant impact on the future prospects of toxoplasmosis therapy.

scholarly journals Lumican Inhibits Osteoclastogenesis and Bone Resorption by Suppressing Akt Activity

2021 ◽  
Vol 22 (9) ◽  
pp. 4717
Author(s):  
Jin-Young Lee ◽  
Da-Ae Kim ◽  
Eun-Young Kim ◽  
Eun-Ju Chang ◽  
So-Jeong Park ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Bone Formation ◽  
Map Kinases ◽  
Osteoclast Differentiation ◽  
Dependent Manner ◽  
Dual Action ◽  
Dose Dependent ◽  
Resorptive Activity

Lumican, a ubiquitously expressed small leucine-rich proteoglycan, has been utilized in diverse biological functions. Recent experiments demonstrated that lumican stimulates preosteoblast viability and differentiation, leading to bone formation. To further understand the role of lumican in bone metabolism, we investigated its effects on osteoclast biology. Lumican inhibited both osteoclast differentiation and in vitro bone resorption in a dose-dependent manner. Consistent with this, lumican markedly decreased the expression of osteoclastogenesis markers. Moreover, the migration and fusion of preosteoclasts and the resorptive activity per osteoclast were significantly reduced in the presence of lumican, indicating that this protein affects most stages of osteoclastogenesis. Among RANKL-dependent pathways, lumican inhibited Akt but not MAP kinases such as JNK, p38, and ERK. Importantly, co-treatment with an Akt activator almost completely reversed the effect of lumican on osteoclast differentiation. Taken together, our findings revealed that lumican inhibits osteoclastogenesis by suppressing Akt activity. Thus, lumican plays an osteoprotective role by simultaneously increasing bone formation and decreasing bone resorption, suggesting that it represents a dual-action therapeutic target for osteoporosis.

scholarly journals Phosphorylation of the mitochondrial triphosphate tunnel metalloenzyme TTM1 regulates programmed cell death in senescence

2020 ◽  
Author(s):  
Purva Karia ◽  
Keiko Yoshioka ◽  
Wolfgang Moeder
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Map Kinases ◽  
Mitochondrial Protein ◽  
Mitochondrial Outer Membrane ◽  
Mitogen Activated Protein ◽  
Animal Growth ◽  
Triphosphate Tunnel Metalloenzyme

ABSTRACTThe role of mitochondria in programmed cell death (PCD) during animal growth and development is well documented, but much less is known for plants. We previously showed that the Arabidopsis thaliana triphosphate tunnel metalloenzyme (TTM) proteins TTM1 and TTM2 are tail-anchored proteins that localize in the mitochondrial outer membrane and participate in PCD during senescence and immunity, respectively. Here, we show that TTM1 is specifically involved in senescence induced by abscisic acid (ABA). Moreover, phosphorylation of TTM1 by multiple mitogen-activated protein kinases (MAPKs) regulates its function and turnover. A combination of proteomics and in vitro kinase assays revealed three major phosphorylation sites of TTM1 (S10, S437, and S490), which are phosphorylated upon perception of senescence cues such as ABA and prolonged darkness. S437 is phosphorylated by the MAP kinases MPK3 and MPK4, and S437 phosphorylation is essential for TTM1 function in senescence. These MPKs, together with three additional MAP kinases (MPK1, MPK7, and MPK6), phosphorylate S10 and S490, marking TTM1 for protein turnover, which likely prevents uncontrolled cell death. Taken together, our results show that multiple MPKs regulate the function and turnover of the mitochondrial protein TTM1 during senescence-related PCD, revealing a novel link between mitochondria and PCD.SummaryEmail addresses: [email protected]

scholarly journals The Role of Monocytes/Macrophages In The Pathogenesis of Immune Associated Diffuse Alveolar Hemorrhage

2021 ◽  
Author(s):  
Qing Wei ◽  
Xun Chen ◽  
Jing Liu ◽  
Yan Li ◽  
Guangmin Nong
Keyword(s):  
Flow Cytometry ◽  
Critical Role ◽  
Lavage Fluid ◽  
Peripheral Blood Monocyte ◽  
Healthy Children ◽  
Group 3 ◽  
Group 2 ◽  
Stimulation Of

Abstract Backgroud The studies in the immnue associated diffuse alveolar hemorrahge (DAH) animal models showed that monocytes/macrophages played an critical role in the pathogenesis.Whether monocytes/macrophages contribute to the pathogenesis of immune associated DAH in human is still unknow. The aim of this study was to explore the role of monocytes/macrophages in the pathogenesis of immune associated DAH in human.Methods This study was conducted in two parts. In the first part, 37 children with immune associated DAH were included (DAH group), and 18 healthy children were recruited as the controls (HC group). Peripheral blood monocyte subtype was analyzed using flow cytometry. In the second part, 24 children with immune associated DAH were included (DAH group), and 13 children with acute airway foreingn body or mild benign airway stenosis were included as the controls (HC group). Bronochoalveolar lavage fluid (BALF) was collected using bronchoscope. Cytokines in the BALF supernatant were detected using cytometric bread array. BALF supertanant was used to stimulated the macrophages in vitro. The mRNA relative expressions of IL-1β, TNFα, IL-6, TGM2, CD163 and MRC1 were detected using quantitative real-time PCR, and the expressions of CD14, CD80, CD86, CD163 and CD206 were detected using flow cytometry. Results 1. The percentage of classical monocyte was significantly increased, whereas the percentages of intermediate and non-classical monocyte were significantly decreased in the DAH group, when compared to those in the HC group. 2. The levels of MCP-1, IL-6 and IL-8 were all significantly higher in the BALF supernatant from the DAH group, when compared to those form the HC group. 3. The mRNA relative expressions of IL-1β and IL-6 as well as the expression of CD86 were significantly higher, whereas the mRNA relative expression of MRC1 as well as the expressions of CD163 and CD206 were significantly lower under the stimulation of BALF supernatant from the DAH group, when compared to that from the HC group. Conclusions Monocytes/macrophages might participate in the pathogenesis of immune associated DAH in human by enhanced M1 polarization.

scholarly journals A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP

2009 ◽  
Vol 206 (9) ◽  
pp. 1899-1911 ◽  
Author(s):  
Sarah M. McWhirter ◽  
Roman Barbalat ◽  
Kathryn M. Monroe ◽  
Mary F. Fontana ◽  
Mamoru Hyodo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Map Kinases ◽  
Second Messenger ◽  
Innate Immune ◽  
Guanosine Monophosphate ◽  
Host Cells ◽  
Type I Interferons ◽  
Interferon Response ◽  
Type I

The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. The cyclic dinucleotide cyclic-di–guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. We provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by c-di-GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, nuclear factor κB, and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid–sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.

Immunomodulation by 17β-estradiol in bivalve hemocytes

2006 ◽  
Vol 291 (3) ◽  
pp. R664-R673 ◽  
Author(s):  
Laura Canesi ◽  
Caterina Ciacci ◽  
Lucia Cecilia Lorusso ◽  
Michele Betti ◽  
Tiziana Guarnieri ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Hydrolytic Enzymes ◽  
Physiological Role ◽  
Estrogen Receptor Α ◽  
Nitrite Accumulation ◽  
No Production ◽  
17Β Estradiol

In mammals, estrogens have dose- and cell-type-specific effects on immune cells and may act as pro- and anti-inflammatory stimuli, depending on the setting. In the bivalve mollusc Mytilus, the natural estrogen 17β-estradiol (E2) has been shown to affect neuroimmune functions. We have investigated the immunomodulatory role of E2 in Mytilus hemocytes, the cells responsible for the innate immune response. E2 at 5–25 nM rapidly stimulated phagocytosis and oxyradical production in vitro; higher concentrations of E2 inhibited phagocytosis. E2-induced oxidative burst was prevented by the nitric oxide (NO) synthase inhibitor NG-monomethyl-l-arginine and superoxide dismutase, indicating involvement of NO and O2−; NO production was confirmed by nitrite accumulation. The effects of E2 were prevented by the antiestrogen tamoxifen and by specific kinase inhibitors, indicating a receptor-mediated mechanism and involvement of p38 MAPK and PKC. E2 induced rapid and transient increases in the phosphorylation state of PKC, as well as of a aCREB-like (cAMP responsive element binding protein) transcription factor, as indicated by Western blot analysis with specific anti-phospho-antibodies. Localization of estrogen receptor-α- and -β-like proteins in hemocytes was investigated by immunofluorescence confocal microscopy. The effects of E2 on immune function were also investigated in vivo at 6 and 24 h in hemocytes of E2-injected mussels. E2 significantly affected hemocyte lysosomal membrane stability, phagocytosis, and extracellular release of hydrolytic enzymes: lower concentrations of E2 resulted in immunostimulation, and higher concentrations were inhibitory. Our data indicate that the physiological role of E2 in immunomodulation is conserved from invertebrates to mammals.

scholarly journals In Vitro Gynogenesis and Flow Cytometry Analysis of the Regenerated Haploids of Black Cumin (Nigella sativa)

HortScience ◽  
2018 ◽  
Vol 53 (5) ◽  
pp. 681-686 ◽  
Author(s):  
Mohammed Elsayed El-Mahrouk ◽  
Mossad K. Maamoun ◽  
Antar Nasr EL-Banna ◽  
Soliman A. Omran ◽  
Yaser Hassan Dewir ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nigella Sativa ◽  
Plant Production ◽  
Haploid Plant ◽  
Naphthalene Acetic Acid ◽  
Ms Medium ◽  
Flow Cytometry Analysis ◽  
Black Cumin ◽  
Haploid Plants

In vitro ovule culture could be used to generate homozygous lines through the production of haploid plants. The present study reports on in vitro regeneration and production of haploid plants through ovule cultures and identification of the regenerated haploids using flow cytometry. The ovules were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kin), 2,4-dichlorophenoxyacetic acid (2,4-D), and naphthalene acetic acid (NAA) at 0, 0.5, 1, and 2 mg·L−1 for their gynogenesis. Among different plant growth regulators (PGRs) tested, 2,4-D at 2 mg·L−1 produced direct gynogenesis. The highest callogenesis percentage (100%) was obtained on MS medium containing 1 mg·L−1 2,4-D and 2 mg·L−1 NAA. Flow cytometry analysis was used to identify the regenerated haploids. It also confirmed gynogenic occurrence at 1 and 2 mg·L−1 2,4-D with percentages of 21.7% and 41%, respectively. Therefore, 2,4-D proved effective for the induction of haploids in black cumin. The regenerated haploids were developed on MS medium without PGRs. The obtained results of in vitro gynogenesis and haploid plant production can tremendously facilitate breeding programs of black cumin.

scholarly journals Functional genetic encoding of sulfotyrosine in mammalian cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xinyuan He ◽  
Yan Chen ◽  
Daisy Guiza Beltran ◽  
Maia Kelly ◽  
Bin Ma ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Biochemical Characterization ◽  
Trna Synthetase ◽  
Functional Verification ◽  
Cellular Processes ◽  
Genetic Encoding ◽  
Efficient Expression

Abstract Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

Sign in / Sign up

Export Citation Format

Share Document