scholarly journals Novel Antimalarial Inhibitors That Specifically Target the Invasion Motor Protein Myosin A in Malaria Parasites

Proceedings ◽  
2019 ◽  
Vol 22 (1) ◽  
pp. 9
Author(s):  
Janna Ehlert ◽  
Arne Alder ◽  
Viola Introini ◽  
Julia Weder ◽  
Pietro Cicuta ◽  
...  
Keyword(s):  
Covalent Binding ◽  
Critical Role ◽  
Resistance Mechanisms ◽  
Myosin Isoforms ◽  
Malaria Parasites ◽  
Wide Range ◽  
Reversible Covalent ◽  
Parasite Motility ◽  
Vitro Analysis

Malaria remains a devastating disease with nearly half a million deaths per year. The WHO reports a stagnating number of new infections every year without a significant decline, as a result of insufficient access to antimalarials in endemic regions as well as complex resistance mechanisms of the parasites against current treatments. Due to its critical role during the parasite’s life cycle, the invasion motor myosin A is a promising target, which has not yet been considered in drug discovery. Myosins appeared to be undruggable since they are ubiquitously expressed and involved in a wide range of cellular processes. In total, the protein superfamily of myosins comprises 35 known subclasses. However, recent studies highlighted the possibility to modulate the myosin motor activity of specific myosin isoforms and classes using small allosteric effector molecules. Exploiting the concept of reversible covalent binding, we show the development of highly potent and specific inhibitors of the key motor myosin A of the glideosome—a sophisticated motor machinery involved in parasite motility and host cell invasion. Combining chemical synthesis with biophysical in vitro analysis confirmed the preferential inhibition of the target protein in the submicromolar range. The developed compounds show significant antiparasitic activities and block efficiently glideosome-associated processes, parasite proliferation, and parasitemia of the malaria parasites. Our findings demonstrate the high potential of our approach using reversible covalent binding to develop new allosteric inhibitors, targeting specifically the key invasion motor as a novel drug target to treat infections caused by malaria parasites.

scholarly journals Stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes by human monocytes

2009 ◽  
Vol 42 (2) ◽  
pp. 103-106 ◽  
Author(s):  
Maria Imaculada Muniz-Junqueira ◽  
Carlos Eduardo Tosta
Keyword(s):  
Plasmodium Falciparum ◽  
Blood Circulation ◽  
Defense Mechanisms ◽  
Healthy Adult ◽  
Critical Role ◽  
Immune Serum ◽  
Human Monocytes ◽  
Malaria Parasites

Monocytes/macrophages play a critical role in the defense mechanisms against malaria parasites, and are the main cells responsible for the elimination of malaria parasites from the blood circulation. We carried out a microscope-aided evaluation of the stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes, by human monocytes. These cells were obtained from healthy adult individuals by means of centrifugation through a cushion of Percoll density medium and were incubated with erythrocytes infected with Plasmodium falciparum that had previously been incubated with a pool of anti-plasmodial immune serum. We described the stages of phagocytosis, starting from adherence of infected erythrocytes to the phagocyte membrane and ending with their destruction within the phagolisosomes of the monocytes. We observed that the different erythrocytic forms of the parasite were ingested by monocytes, and that the process of phagocytosis may be completed in around 30 minutes. Furthermore, we showed that phagocytosis may occur continuously, such that different phases of the process were observed in the same phagocyte.

scholarly journals In vitro analysis of a transcription termination site for RNA polymerase II.

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795 ◽  
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

scholarly journals The Diverse Roles of the Global Transcriptional Regulator PhoP in the Lifecycle of Yersinia pestis

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1039
Author(s):  
Hana S. Fukuto ◽  
Gloria I. Viboud ◽  
Viveka Vadyvaloo
Keyword(s):  
Yersinia Pestis ◽  
Current Knowledge ◽  
Response Regulator ◽  
Expression Profiles ◽  
Critical Role ◽  
Gene Expression Profiles ◽  
Acanthamoeba Castellanii ◽  
Wide Range

Yersinia pestis, the causative agent of plague, has a complex infectious cycle that alternates between mammalian hosts (rodents and humans) and insect vectors (fleas). Consequently, it must adapt to a wide range of host environments to achieve successful propagation. Y. pestis PhoP is a response regulator of the PhoP/PhoQ two-component signal transduction system that plays a critical role in the pathogen’s adaptation to hostile conditions. PhoP is activated in response to various host-associated stress signals detected by the sensor kinase PhoQ and mediates changes in global gene expression profiles that lead to cellular responses. Y. pestis PhoP is required for resistance to antimicrobial peptides, as well as growth under low Mg2+ and other stress conditions, and controls a number of metabolic pathways, including an alternate carbon catabolism. Loss of phoP function in Y. pestis causes severe defects in survival inside mammalian macrophages and neutrophils in vitro, and a mild attenuation in murine plague models in vivo, suggesting its role in pathogenesis. A Y. pestisphoP mutant also exhibits reduced ability to form biofilm and to block fleas in vivo, indicating that the gene is also important for establishing a transmissible infection in this vector. Additionally, phoP promotes the survival of Y. pestis inside the soil-dwelling amoeba Acanthamoeba castellanii, a potential reservoir while the pathogen is quiescent. In this review, we summarize our current knowledge on the mechanisms of PhoP-mediated gene regulation in Y. pestis and examine the significance of the roles played by the PhoP regulon at each stage of the Y. pestis life cycle.

Evaluation of drought resistance-related traits in durum wheat somaclonal lines selected in vitro

2004 ◽  
Vol 44 (1) ◽  
pp. 27 ◽  
Author(s):  
M. Bajji ◽  
P. Bertin ◽  
S. Lutts ◽  
J-M. Kinet
Keyword(s):  
Durum Wheat ◽  
Somaclonal Variation ◽  
Drought Resistance ◽  
In Vitro Selection ◽  
Culture Media ◽  
Field Studies ◽  
Resistance Mechanisms ◽  
Wide Range ◽  
Triticum Durum Desf

Somaclonal variation associated with in vitro selection has been used as a source of variability to improve drought resistance of 3 durum wheat (Triticum durum Desf.) cultivars (Selbera, Sebou, and Kyperounda). In a previous study, R0 plants with improved drought resistance-related characters were regenerated after selection on culture media containing polyethylene glycol (PEG). This improvement was transmitted to the R1 progeny. The present study analysed the behaviour of the selected tissue culture-derived lines in subsequent R2, R3 and R4�generations. Differences in electrolyte leakage, chlorophyll fluorescence (Fv/Fm), stomatal conductance and days to heading were found between the parental cultivars and most of their in vitro-derived lines. The changes may differ from one cultivar to another. Many promising somaclonal lines still presented improvement for at least 3 of the 4�parameters measured comparatively to initial cultivars. Somaclonal variation thus appears to induce a wide range of modifications among individual components of drought-resistance mechanisms. These improved traits could be valuable if shown to be inherited and to give enhanced agronomic performances in future field studies.

Reversible Covalent Binding of Neratinib to Human Serum Albumin In Vitro

2010 ◽  
Vol 4 (4) ◽  
pp. 220-227 ◽  
Author(s):  
Appavu Chandrasekaran ◽  
Li Shen ◽  
Susan Lockhead ◽  
Aram Oganesian ◽  
Jianyao Wang ◽  
...  
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Covalent Binding ◽  
Reversible Covalent

scholarly journals EXP1 is required for organization of the intraerythrocytic malaria parasite vacuole

2019 ◽  
Author(s):  
Timothy Nessel ◽  
John M. Beck ◽  
Shima Rayatpisheh ◽  
Yasaman Jami-Alahmadi ◽  
James A. Wohlschlegel ◽  
...  
Keyword(s):  
Genome Editing ◽  
Translational Control ◽  
Protein Identification ◽  
Parasitophorous Vacuole ◽  
Critical Role ◽  
Transferase Activity ◽  
Glutathione S Transferase ◽  
Malaria Parasites ◽  
Vacuole Membrane

AbstractIntraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) that closely overlays the parasite plasma membrane (PPM) and constitutes the barrier between parasite and host compartments. The PVM is the site of several essential transport activities but the basis for organization of this membrane system is unknown. We utilized the second-generation promiscuous biotin ligase BioID2 fused to EXP2 or HSP101 to probe the content of the PVM, identifying known and novel candidate PVM proteins. Among the best represented hits were members of a group of single-pass integral membrane proteins that constitute a major component of the PVM proteome but whose function remains unclear. We investigated the function of EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 genome editing system to install the TetR-DOZI-aptamers system for conditional translational control. EXP1 knockdown was essential for intraerythrocytic development and accompanied by profound changes in vacuole ultrastructure, including increased separation of the PVM and PPM and formation of abnormal membrane structures in the enlarged vacuole lumen. While previous in vitro studies indicated EXP1 possesses glutathione S-transferase activity, a mutant version of EXP1 lacking a residue important for this activity in vitro still provides substantial rescue of endogenous exp1 knockdown in vivo. Intriguingly, while activity of the Plasmodium translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the translocon pore-forming protein EXP2 was substantially altered. Collectively, our results reveal a novel PVM defect that indicates a critical role for EXP1 in maintaining proper PVM organization.ImportanceLike other obligate intracellular apicomplexans, blood-stage malaria parasites reside within a membrane-bound compartment inside the erythrocyte known as the parasitophorous vacuole. Although the vacuole is the site of several transport activities essential to parasite survival, little is known about its organization. To explore vacuole biology, we adopted recently developed proteomic (BioID2) and genetic (CRISPR/Cpf1) tools for use in Plasmodium falciparum, which allowed us to query the function of the prototypical vacuole membrane protein EXP1.Knockdown of EXP1 showed that a previously reported glutathione S-transferase activity cannot fully account for the essential function(s) of EXP1 and revealed a novel role for this protein in maintaining normal vacuole morphology and PVM protein arrangement. Our results provide new insight into vacuole organization and illustrate the power of BioID2 and Cpf1 (which utilizes a T-rich PAM uniquely suited to the P. falciparum genome) for proximity protein identification and genome editing in P. falciparum.

scholarly journals Enhanced VWF biosynthesis and elevated plasma VWF due to a natural variant in the murine Vwf gene

Blood ◽  
2006 ◽  
Vol 108 (9) ◽  
pp. 3061-3067 ◽  
Author(s):  
Heidi L. Lemmerhirt ◽  
Jordan A. Shavit ◽  
Gallia G. Levy ◽  
Suzanne M. Cole ◽  
Jeffrey C. Long ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Inbred Mice ◽  
Inbred Mouse ◽  
Mouse Strains ◽  
Genetic Modifiers ◽  
Wide Range ◽  
Natural Variant ◽  
Vitro Analysis

Abstract Both genetic and environmental influences contribute to the wide variation in plasma von Willebrand factor (VWF) levels observed in humans. Inbred mouse strains also have highly variable plasma VWF levels, providing a convenient model in which to study genetic modifiers of VWF. Previously, we identified a major modifier of VWF levels in the mouse (Mvwf1) as a regulatory mutation in murine Galgt2. We now report the identification of an additional murine VWF modifier (Mvwf2). Mvwf2 accounts for approximately 16% of the 8-fold plasma VWF variation (or ∼ 25% of the genetic variation) observed between the A/J and CASA/RkJ strains and maps to the murine Vwf gene itself. Twenty SNPs were identified within the coding regions of the A/J and CASA/RkJ Vwf alleles, and in vitro analysis of recombinant VWF demonstrated that a single SNP (+7970G>A) and the associated nonsynonymous amino acid change (R2657Q) confers a significant increase in VWF biosynthesis from the CASA/RkJ Vwf allele. This change appears to represent a unique gain of function that likely explains the mechanism of Mvwf2 in vivo. The identification of a natural Vwf gene variant among inbred mice affecting biosynthesis suggests that similar genetic variation may contribute to the wide range of VWF levels observed in humans.

scholarly journals Polyphosphate blocks tumour metastasis via anti-angiogenic activity

2007 ◽  
Vol 406 (1) ◽  
pp. 49-55 ◽  
Author(s):  
Kyu Yeon Han ◽  
Bok Sil Hong ◽  
Yae Jin Yoon ◽  
Chang Min Yoon ◽  
Yoon-Keun Kim ◽  
...  
Keyword(s):  
Critical Role ◽  
Wide Distribution ◽  
Mitogen Activated Protein Kinase ◽  
Cell Surface Receptor ◽  
Inorganic Polyphosphate ◽  
Angiogenic Activity ◽  
Tumour Metastasis ◽  
Wide Range

PolyP (inorganic polyphosphate) is a linear polymer of many tens or hundreds of orthophosphate residues found in a wide range of organisms, including bacteria, fungi, insects, plants and vertebrates. Despite its wide distribution in mammalian tissues and plasma, the biological functions of polyP on tumour metastasis and angiogenesis have not been previously examined. In the present study, we have shown that polyP effectively blocked in vivo pulmonary metastasis of B16BL6 cells by suppression of neovascularization, whereas it did not affect proliferation or adhesion to extracellular matrix proteins. PolyP not only inhibited bFGF (basic fibroblast growth factor)-induced proliferation and ERK (extracellular-signal-regulated kinase)/p38 MAPK (mitogen-activated protein kinase) activation of human endothelial cells, but also blocked the binding of bFGF to its cognate cell-surface receptor. Furthermore, polyP inhibited bFGF-induced in vitro and in vivo angiogenesis, suggesting that polyP possesses an anti-angiogenic activity. Since neovascularization is essential for tumour metastasis, our present findings clearly indicate that polyP has an in vivo anti-metastatic activity via its anti-angiogenic activity. Taken together with the fact that angiogenesis occurs under various normal and pathological conditions, our observations suggest that endogenous polyP may play a critical role during embryonic development, wound healing and inflammation, as well as in the progress of pathological diseases such as rheumatoid arthritis and cancer.

scholarly journals In vivo experiments do not support the charge zipper model for Tat translocase assembly

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Felicity Alcock ◽  
Merel PM Damen ◽  
Jesper Levring ◽  
Ben C Berks
Keyword(s):  
Structural Model ◽  
Protein Translocation ◽  
Critical Role ◽  
Oligomeric State ◽  
In Vivo Experiments ◽  
Charged Residues ◽  
Twin Arginine Translocase ◽  
Vitro Analysis

The twin-arginine translocase (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat translocation site is formed by substrate-triggered oligomerization of the protein TatA. Walther and co-workers have proposed a structural model for the TatA oligomer in which TatA monomers self-assemble using electrostatic ‘charge zippers’ (Cell (2013) 132: 15945). This model was supported by in vitro analysis of the oligomeric state of TatA variants containing charge-inverting substitutions. Here we have used live cell assays of TatA assembly and function in Escherichia coli to re-assess the roles of the charged residues of TatA. Our results do not support the charge zipper model. Instead, we observe that substitutions of charged residues located in the TatA amphipathic helix lock TatA in an assembled state, suggesting that these charged residues play a critical role in the protein translocation step that follows TatA assembly.

scholarly journals Preparation and In Vitro Characterization of Gels Based on Bromelain, Whey and Quince Extract

Gels ◽  
2021 ◽  
Vol 7 (4) ◽  
pp. 191
Author(s):  
Amalia Mazilu (Moldovan) ◽  
Violeta Popescu ◽  
Codruta Sarosi ◽  
Radu Dumitrescu ◽  
Andrea Maria Chisnoiu ◽  
...  
Keyword(s):  
Tooth Enamel ◽  
Whey Proteins ◽  
Antibacterial Properties ◽  
Blue Dye ◽  
Sem Images ◽  
Sds Page ◽  
Wide Range ◽  
Vitro Analysis ◽  
In Vitro Analysis

The growing interest in the appearance and color of teeth has led to the emergence of a wide range of teeth whitening methods, both in dental offices and in patients’ homes. Concerns about the possible side effects or toxic effects of peroxide-based whitening gels leads to the identification of alternative whitening methods, based on natural compounds with mild action on tooth enamel and remineralizing effect. In this context, this study describes the preparation and in vitro analysis of whitening gels based on natural active agents—bromelain, quince and whey—using organic (polyacrylate, polyethylene glycol) and/or inorganic (silicate) excipients. Five natural products gels were prepared, containing bromelain extract, quince extract and whey, in various proportions. Two supplementary gels, one containing Lubrizol and another containing SiO2, were prepared. All gels were submitted for multiple in vitro analysis such as: SDS-PAGE analysis, UV-vis and FTIR spectroscopy, SEM microscopy, antibacterial activity on Streptococcus mutans ATCC 25175, Porphyromonas gingivalis ATCC 33277, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. The quince extract sample was the only one which completely discolored the blue dye on SDS-PAGE analysis. On the UV-vis spectra, the 303 nm band is assigned to an in situ modified form of bromelain. SEM images of gels containing SiO2 particles show evident marks of these particles, while the rest of the gels containing Lubrizol or whey are more uniform. Regarding antibacterial tests, the SiO2 gel samples did not show inhibition in any strains, but the other tested samples varied in the size of the inhibition diameter depending on the amicrobial strain tested; the protease activity of bromelain modulates the composition of the added whey proteins. Bromelain added as a nanoencapsulated assembly better preserves its integrity. The prepared gels showed antibacterial properties.

Sign in / Sign up

Export Citation Format

Share Document