scholarly journals C-type lectins CTL4 and CTLMA2: conserved heterodimeric structure and glycan specificity in Anopheles mosquitoes

2019 ◽  
Author(s):  
Ritika Bishnoi ◽  
Alicia Contet ◽  
Christopher J Day ◽  
David Chun-Feng Hou ◽  
Lauren A. Profitt ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Scattering Data ◽  
Connective Tissues ◽  
Anopheles Mosquitoes ◽  
Molecular Features ◽  
X Ray Scattering ◽  
Specific Variation ◽  
Malaria Mosquitoes

AbstractThe C-type lectins CTL4 and CTLMA2 cooperatively influence Plasmodium infection in the malaria vector Anopheles. Here we report the purification and biochemical characterization of CTL4 and CTLMA2 from An. gambiae and An. albimanus. CTL4 and CTLMA2 are known to form a disulfide-bridged heterodimer via an N-terminal tri-cysteine CXCPC motif. We demonstrate in vitro that CTL4 and CTLMA2 intermolecular disulfide formation is promiscuous within this motif. Furthermore, CTL4 and CTLMA2 exhibit charge complementarity that promotes the formation of higher oligomeric states at physiological pH. Both lectins bind specific sugars, with an apparent preference for glycosaminoglycan motifs comprising β1-3/β1-4 linkages between glucose (Glc), galactose (Gal) and their respective hexosamines. Small-angle x-ray scattering data supports a compact heterodimer between the CTL domains. Recombinant CTL4/CTLMA2 is functional in vivo, reversing the enhancement of phenoloxidase activity in dsCTL4-treated mosquitoes. We propose these molecular features underline a common function for CTL4/CTLMA2 in mosquitoes, with species and strain-specific variation in degrees of activity in response to Plasmodium infection.Author SummaryMosquitoes of the genus Anopheles are vectors for the single-celled parasite Plasmodium, the causative agent of malaria. Mosquitoes, like all insects, utilize the process of melanization for both wound healing and defense against pathogens. CTL4 and CTLMA2 are two proteins found in Anopheles mosquitoes that act as inhibitors of melanization, so understanding their molecular function is important to understanding the immune response of Anopheles mosquitoes to Plasmodium infection. We have purified CTL4 and CTLMA2 from two species of Anopheles and studied their molecular properties with a variety of biochemical and biophysical techniques. We also verified that our purified protein is functional by injecting it into mosquitoes. We learned that CTL4 and CTLMA2 are joined together by a disulfide bond between any one of three cysteine residues near the N-terminus of each protein. The CTL4/CTLMA2 complex is compact, but can associate into larger structures in solution, probably because of a loop in each protein that carries an opposite charge. The proteins cooperatively bind calcium and sugars, specifically glycosaminoglycan sugars, which are typically present in the connective tissues of insects. This information will aid in further investigations of the function of CTL4 and CTLMA2.

scholarly journals Solution structure, glycan specificity and of phenol oxidase inhibitory activity of Anopheles C-type lectins CTL4 and CTLMA2

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ritika Bishnoi ◽  
Gregory L. Sousa ◽  
Alicia Contet ◽  
Christopher J. Day ◽  
Chun-Feng David Hou ◽  
...  
Keyword(s):  
Malaria Vector ◽  
Solution Structure ◽  
Biochemical Characterization ◽  
Phenol Oxidase ◽  
Sub Saharan Africa ◽  
Scattering Data ◽  
Molecular Features ◽  
Sub Saharan

Abstract Malaria, the world’s most devastating parasitic disease, is transmitted between humans by mosquitoes of the Anopheles genus. An. gambiae is the principal malaria vector in Sub-Saharan Africa. The C-type lectins CTL4 and CTLMA2 cooperatively influence Plasmodium infection in the malaria vector Anopheles. Here we report the purification and biochemical characterization of CTL4 and CTLMA2 from An. gambiae and An. albimanus. CTL4 and CTLMA2 are known to form a disulfide-bridged heterodimer via an N-terminal tri-cysteine CXCXC motif. We demonstrate in vitro that CTL4 and CTLMA2 intermolecular disulfide formation is promiscuous within this motif. Furthermore, CTL4 and CTLMA2 form higher oligomeric states at physiological pH. Both lectins bind specific sugars, including glycosaminoglycan motifs with β1-3/β1-4 linkages between glucose, galactose and their respective hexosamines. Small-angle x-ray scattering data supports a compact heterodimer between the CTL domains. Recombinant CTL4/CTLMA2 is found to function in vivo, reversing the enhancement of phenol oxidase activity in dsCTL4-treated mosquitoes. We propose these molecular features underline a common function for CTL4/CTLMA2 in mosquitoes, with species and strain-specific variation in degrees of activity in response to Plasmodium infection.

The in vivo distribution and dynamics of DNA topoisomerase II in Drosophila embryonic nuclei and chromosomes

1993 ◽  
Vol 51 ◽  
pp. 74-75
Author(s):  
Jason R. Swedlow ◽  
Neil Osheroff ◽  
Tim Karr ◽  
John W. Sedat ◽  
David A. Agard
Keyword(s):  
Topoisomerase Ii ◽  
Biochemical Characterization ◽  
Developmental Cycle ◽  
Dna Topoisomerase Ii ◽  
Chromosomal Distribution ◽  
Dna Topoisomerase ◽  
Ideal System ◽  
Dnase Treatment

DNA topoisomerase II is an ATP-dependent double-stranded DNA strand-passing enzyme that is necessary for full condensation of chromosomes and for complete segregation of sister chromatids at mitosis in vivo and in vitro. Biochemical characterization of chromosomes or nuclei after extraction with high-salt or detergents and DNAse treatment showed that topoisomerase II was a major component of this remnant, termed the chromosome scaffold. The scaffold has been hypothesized to be the structural backbone of the chromosome, so the localization of topoisomerase II to die scaffold suggested that the enzyme might play a structural role in the chromosome. However, topoisomerase II has not been studied in nuclei or chromosomes in vivo. We have monitored the chromosomal distribution of topoisomerase II in vivo during mitosis in the Drosophila embryo. This embryo forms a multi-nucleated syncytial blastoderm early in its developmental cycle. During this time, the embryonic nuclei synchronously progress through 13 mitotic cycles, so this is an ideal system to follow nuclear and chromosomal dynamics.

scholarly journals Endothelial-to-Mesenchymal Transition in Human Adipose Tissue Vasculature Alters the Particulate Secretome and Induces Endothelial Dysfunction

2019 ◽  
Vol 39 (10) ◽  
pp. 2168-2191 ◽  
Author(s):  
Bronson A. Haynes ◽  
Li Fang Yang ◽  
Ryan W. Huyck ◽  
Eric J. Lehrer ◽  
Joshua M. Turner ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Endothelial Dysfunction ◽  
Smooth Muscle Actin ◽  
Phenotypic Change ◽  
Alpha Smooth Muscle Actin ◽  
Mesenchymal Transition ◽  
Molecular Features ◽  
Endothelial To Mesenchymal Transition

Objective: Endothelial cells (EC) in obese adipose tissue (AT) are exposed to a chronic proinflammatory environment that may induce a mesenchymal-like phenotype and altered function. The objective of this study was to establish whether endothelial-to-mesenchymal transition (EndoMT) is present in human AT in obesity and to investigate the effect of such transition on endothelial function and the endothelial particulate secretome represented by extracellular vesicles (EV). Approach and Results: We identified EndoMT in obese human AT depots by immunohistochemical co-localization of CD31 or vWF and α-SMA (alpha-smooth muscle actin). We showed that AT EC exposed in vitro to TGF-β (tumor growth factor-β), TNF-α (tumor necrosis factor-α), and IFN-γ (interferon-γ) undergo EndoMT with progressive loss of endothelial markers. The phenotypic change results in failure to maintain a tight barrier in culture, increased migration, and reduced angiogenesis. EndoMT also reduced mitochondrial oxidative phosphorylation and glycolytic capacity of EC. EVs produced by EC that underwent EndoMT dramatically reduced angiogenic capacity of the recipient naïve ECs without affecting their migration or proliferation. Proteomic analysis of EV produced by EC in the proinflammatory conditions showed presence of several pro-inflammatory and immune proteins along with an enrichment in angiogenic receptors. Conclusions: We demonstrated the presence of EndoMT in human AT in obesity. EndoMT in vitro resulted in production of EV that transferred some of the functional and metabolic features to recipient naïve EC. This result suggests that functional and molecular features of EC that underwent EndoMT in vivo can be disseminated in a paracrine or endocrine fashion and may induce endothelial dysfunction in distant vascular beds.

scholarly journals Enzymatic activity in turkey, duck, quail and chicken liver microsomes against four human cytochrome P450 prototype substrates and aflatoxin B1

2011 ◽  
Vol 1 (1) ◽  
pp. 4 ◽  
Author(s):  
Hansen W. Murcia ◽  
Gonzalo J. Díaz ◽  
Sandra Milena Cepeda
Keyword(s):  
Cytochrome P450 ◽  
Aflatoxin B1 ◽  
Enzyme Activities ◽  
High Performance ◽  
Biochemical Characterization ◽  
Liver Microsomes ◽  
Cytochrome P450 Enzymes ◽  
Catalytic Rate

Cytochrome P450 enzymes (CYP) are a group of monooxygenases able to biotransform several kinds of xenobiotics including aflatoxin B1 (AFB1), a highly toxic mycotoxin. These enzymes have been widely studied in humans and others mammals, but there is not enough information in commercial poultry species about their biochemical characteristics or substrate specificity. The aim of the present study was to identify CYPs from avian liver microsomes with the use of prototype substrates specific for human CYP enzymes and AFB1. Biochemical characterization was carried out in vitro and biotransformation products were detected by high-performance liquid chromatography (HPLC). Enzymatic constants were calculated and comparisons between turkey, duck, quail and chicken activities were done. The results demonstrate the presence of four avian ortholog enzyme activities possibly related with a CYP1A1, CYP1A2, CYP2A6 (activity not previously identified) and CYP3A4 poultry orthologs, respectively. Large differences in enzyme kinetics specific for prototype substrates were found among the poultry species studied. Turkey liver microsomes had the highest affinity and catalytic rate for AFB1 whereas chicken enzymes had the lowest affinity and catalytic rate for the same substrate. Quail and duck microsomes showed intermediate values. These results correlate well with the known in vivo sensitivity for AFB1 except for the duck. A high correlation coefficient between 7-ethoxyresorufin-Odeethylase (EROD) and 7-methoxyresorufin- O-deethylase (MROD) activities was found in the four poultry species, suggesting that these two enzymatic activities might be carried out by the same enzyme. The results of the present study indicate that four prototype enzyme activities are present in poultry liver microsomes, possibly related with the presence of three CYP avian orthologs. More studies are needed in order to further characterize these enzymes.

scholarly journals MiR-154-5p-MCP1 Axis Regulates Allergic Inflammation by Mediating Cellular Interactions

2021 ◽  
Vol 12 ◽  
Author(s):  
Misun Kim ◽  
Hyein Jo ◽  
Yoojung Kwon ◽  
Myeong Seon Jeong ◽  
Hyun Suk Jung ◽  
...  
Keyword(s):  
Allergic Inflammation ◽  
Passive Cutaneous Anaphylaxis ◽  
Allergic Reactions ◽  
Dependent Manner ◽  
Cellular Interactions ◽  
Array Analysis ◽  
Molecular Features ◽  
Rat Basophilic Leukemia Cells

In a previous study, we have demonstrated that p62, a selective receptor of autophagy, can regulate allergic inflammation. In the present study, microRNA array analysis showed that miR-154-5p was increased by antigen (DNP-HSA) in a p62-dependent manner in rat basophilic leukemia cells (RBL2H3). NF-kB directly increased the expression of miR-154-5p. miR-154-5p mediated in vivo allergic reactions, including passive cutaneous anaphylaxis and passive systemic anaphylaxis. Cytokine array analysis showed that antigen stimulation increased the expression of MCP1 in RBL2H3 cells in an miR-154-5p-dependent manner. Reactive oxygen species (ROS)-ERK-NF-kB signaling increased the expression of MCP1 in antigen-stimulated RBL2H3 cells. Recombinant MCP1 protein induced molecular features of allergic reactions both in vitro and in vivo. Anaphylaxis-promoted tumorigenic potential has been known to be accompanied by cellular interactions involving mast cells, and macrophages, and cancer cells. Our experiments employing culture medium, co-cultures, and recombinant MCP1 protein showed that miR-154 and MCP1 mediated these cellular interactions. MiR-154-5p and MCP1 were found to be present in exosomes of RBL2H3 cells. Exosomes from PSA-activated BALB/C mouse induced molecular features of passive cutaneous anaphylaxis in an miR-154-5p-dependent manner. Exosomes from antigen-stimulated RBL2H3 cells enhanced both tumorigenic and metastatic potentials of B16F1 melanoma cells in an miR-154-5p-dependent manner. Exosomes regulated both ROS level and ROS mediated cellular interactions during allergic inflammation. Our results indicate that the miR-154-5p-MCP1 axis might serve as a valuable target for the development of anti-allergy therapeutics.

Cell Mechanics Drives Migration Modes

2020 ◽  
Vol 15 (01) ◽  
pp. 1-34 ◽  
Author(s):  
Claudia Tanja Mierke
Keyword(s):  
Extracellular Matrix ◽  
Cell Mechanics ◽  
Single Cells ◽  
Biochemical Properties ◽  
Migration And Invasion ◽  
Environmental Cues ◽  
Connective Tissues ◽  
Migration Of Cells

The classical migration modes, such as mesenchymal or amoeboid migration modes, are essentially determined by molecular, morphological or biochemical properties of the cells. These specific properties facilitate the cell migration and invasion through artificial extracellular matrices mimicking the environmental conditions of connective tissues. However, during the migration of cells through narrow extracellular matrix constrictions, the specific extracellular matrix environments can either support or impair the invasion of cells. Beyond the classical molecular or biochemical properties, the migration and invasion of cells depends on intracellular cell mechanical characteristics and extracellular matrix mechanical features. The switch between cell states, such as epithelial, mesenchymal or amoeboid states, seems to be mainly based on epigenetic changes and environmental cues that induce the reversible transition of cells toward another state and thereby promote a specific migration mode. However, the exact number of migration modes is not yet clear. Moreover, it is also unclear whether every individual cell, independent of the type, can undergo a transition between all different migration modes in general. A newer theory states that the transition from the jamming to unjamming phase of clustered cells enables cells to migrate as single cells through extracellular matrix confinements. This review will highlight the mechanical features of cells and their matrix environment that regulate and subsequently determine individual migration modes. It is discussed whether each migration mode in each cell type is detectable or whether some migration modes are limited to artificially engineered matrices in vitro and can therefore not or only rarely be detected in vivo. It is specifically pointed out how the intracellular architecture and its contribution to cellular stiffness or contractility favors the employment of a distinct migration mode. Finally, this review envisions a connection between mechanical properties of cells and matrices and the choice of a distinct migration mode in confined 3D microenvironments.

scholarly journals A Novel JAK1 Mutant Breast Implant-Associated Anaplastic Large Cell Lymphoma Patient-Derived Xenograft Fostering Pre-Clinical Discoveries

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1603 ◽  
Author(s):  
Danilo Fiore ◽  
Luca Vincenzo Cappelli ◽  
Paul Zumbo ◽  
Jude M. Phillips ◽  
Zhaoqi Liu ◽  
...  
Keyword(s):  
Cell Lymphoma ◽  
Breast Implant ◽  
Large Cell ◽  
Tumor Xenograft ◽  
Sensitivity Threshold ◽  
Primary Lymphoma ◽  
Molecular Features ◽  
Primary Sample

Breast implant-associated lymphoma (BIA-ALCL) has recently been recognized as an independent peripheral T-cell lymphoma (PTCL) entity. In this study, we generated the first BIA-ALCL patient-derived tumor xenograft (PDTX) model (IL89) and a matching continuous cell line (IL89_CL#3488) to discover potential vulnerabilities and druggable targets. We characterized IL89 and IL89_CL#3488, both phenotypically and genotypically, and demonstrated that they closely resemble the matching human primary lymphoma. The tumor content underwent significant enrichment along passages, as confirmed by the increased variant allele frequency (VAF) of mutations. Known aberrations (JAK1 and KMT2C) were identified, together with novel hits, including PDGFB, PDGFRA, and SETBP1. A deep sequencing approach allowed the detection of mutations below the Whole Exome Sequencing (WES) sensitivity threshold, including JAK1G1097D, in the primary sample. RNA sequencing confirmed the expression of a signature of differentially expressed genes in BIA-ALCL. Next, we tested IL89’s sensitivity to the JAK inhibitor ruxolitinib and observed a potent anti-tumor effect, both in vitro and in vivo. We also implemented a high-throughput drug screening approach to identify compounds associated with increased responses in the presence of ruxolitinib. In conclusion, these new IL89 BIA-ALCL models closely recapitulate the primary correspondent lymphoma and represent an informative platform for dissecting the molecular features of BIA-ALCL and performing pre-clinical drug discovery studies, fostering the development of new precision medicine approaches.

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.

Biochemical characterization of bona fide polycystin-1 in vitro and in vivo

2001 ◽  
Vol 38 (6) ◽  
pp. 1421-1429 ◽  
Author(s):  
Alessandra Boletta ◽  
Feng Qian ◽  
Luiz F. Onuchic ◽  
Alessandra Bragonzi ◽  
Marina Cortese ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Bona Fide

scholarly journals Hematopoetic precursors respond to a unique B lymphocyte-derived factor in vivo

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1784-1789 ◽  
Author(s):  
E Niskanen ◽  
J Gorman ◽  
PC Isakson
Keyword(s):  
B Cells ◽  
Colony Formation ◽  
High Performance ◽  
B Lymphocyte ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Plasma Clot ◽  
Lentil Lectin

Abstract In this study we detected a factor that stimulates the proliferation of bone marrow-derived hematopoietic precursors in diffusion chambers implanted in mice. This factor, called diffusible colony-stimulating factor (D-CSF), was found in medium conditioned in the presence of spleen and peripheral blood cells from mice with B cell leukemia (BCL1). After the administration of D-CSF, the number of colonies formed in the plasma clot inside the chamber (CFU-DG) was increased, as were the number of hematopoietic precursors (CFU-MIX, CFU-S, CFU-C, and BFU-E) as judged by a subculture of diffusion chamber contents. Depletion of macrophages and T cells from the spleen cell suspension did not decrease the production of D-CSF, thereby indicating that it was derived from B cells. Neoplastic BCL1 cells appear to be the source because D-CSF could not be detected in medium conditioned with normal B cells. BCL1-conditioned medium (CM) did not enhance CFU-MIX, BFU-E, and CFU-C colony formation in vitro, which suggested that D-CSF is different from multi-CSF, EPA, or CSF. The addition of BCL1 CM to multi- CSF-, erythroid potentiating activity (EPA), and CSF (EL-4CM)- containing cultures had no effect on CFU-MIX, BFU-E, and CFU-C colony formation, thus indicating the absence of a synergistic or inhibitory activity. On the other hand, EL-4 CM, which stimulates CFU-MIX, BFU-E, and CFU-C in vitro, had no effect on CFU-DG in vivo. Biochemical characterization of BCL1 CM revealed that D-CSF is relatively heat stable and loses its bioactivity with protease treatments. It binds to lentil-lectin, according to gel-filtration chromatography has a relative molecular weight of approximately 43,000, and on reverse-phase high-performance liquid chromatography elutes with acetonitrile. These data also indicate that transformed B cells may serve as a source for hematopoietic regulators that act on hematopoietic precursors in vivo.

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