scholarly journals Biological Activity of a C-Terminal Fragment ofPasteurella multocida Toxin

2001 ◽  
Vol 69 (6) ◽  
pp. 3628-3634 ◽  
Author(s):  
Christian Busch ◽  
Joachim Orth ◽  
Nabil Djouder ◽  
Klaus Aktories
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Essential Role ◽  
Pasteurella Multocida ◽  
Culture Medium ◽  
Inositol Phosphate ◽  
Complete Loss ◽  
Protein Toxin ◽  
Terminal Fragment

ABSTRACT The protein toxin of Pasteurella multocida PMT is a potent mitogen and activator of phospholipase Cβ. In this study different toxin fragments were investigated. A C-terminal fragment encompassing amino acids 581 through 1285 (PMT581C) was constructed, which was inactive toward intact embryonic bovine lung (EBL) cells after addition to culture medium but caused reorganization of the actin cytoskeleton and rounding up of cells when introduced into the cells by electroporation. As the holotoxin, the toxin fragment PMT581C induced an increase in total inositol phosphate levels after introduction into the cell by electroporation. A C-terminal fragment shorter than PMT581C as well as N-terminal fragments were inactive. Exchange of cysteine-1165 for serine in the holotoxin resulted in a complete loss of the ability to increase inositol phosphate levels. Correspondingly, the mutated toxin fragment PMT581C.C1165S was inactive after cell introduction by electroporation, suggesting an essential role of Cys-1165 in the biological activity of the toxin.

scholarly journals Essential role of glucose transporter GLUT3 for post-implantation embryonic development

2008 ◽  
Vol 200 (1) ◽  
pp. 23-33 ◽  
Author(s):  
S Schmidt ◽  
A Hommel ◽  
V Gawlik ◽  
R Augustin ◽  
N Junicke ◽  
...  
Keyword(s):  
Essential Role ◽  
Glucose Transporter ◽  
Growth Arrest ◽  
Complete Loss ◽  
Transporter Gene ◽  
Wild Type ◽  
Post Implantation ◽  
Time Point

Deletion of glucose transporter geneSlc2a3(GLUT3) has previously been reported to result in embryonic lethality. Here, we define the exact time point of growth arrest and subsequent death of the embryo.Slc2a3−/−morulae and blastocysts developed normally, implantedin vivo, and formed egg-cylinder-stage embryos that appeared normal until day 6.0. At day 6.5, apoptosis was detected in the ectodermal cells ofSlc2a3−/−embryos resulting in severe disorganization and growth retardation at day 7.5 and complete loss of embryos at day 12.5. GLUT3 was detected in placental cone, in the visceral ectoderm and in the mesoderm of 7.5-day-old wild-type embryos. Our data indicate that GLUT3 is essential for the development of early post-implanted embryos.

Essential role of amino acids in αD–β4 loop of a Bacillus thuringiensis Cyt2Aa2 toxin in binding and complex formation on lipid membrane

Toxicon ◽  
2013 ◽  
Vol 74 ◽  
pp. 130-137 ◽  
Author(s):  
Kunat Suktham ◽  
Wanwarang Pathaichindachote ◽  
Boonhiang Promdonkoy ◽  
Chartchai Krittanai
Keyword(s):  
Amino Acids ◽  
Bacillus Thuringiensis ◽  
Complex Formation ◽  
Essential Role ◽  
Lipid Membrane

scholarly journals Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

2001 ◽  
Vol 69 (12) ◽  
pp. 7839-7850 ◽  
Author(s):  
Gillian D. Pullinger ◽  
R. Sowdhamini ◽  
Alistair J. Lax
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Pasteurella Multocida ◽  
Transmembrane Domain ◽  
Polyclonal Antibodies ◽  
Full Length ◽  
Cell Binding ◽  
Terminal Fragment ◽  
C Terminus ◽  
N Terminus

ABSTRACT The locations of the catalytic and receptor-binding domains of thePasteurella multocida toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed α/β domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.

scholarly journals The Role of Amino Acids for the In Vitro Culture of Some Species of Forage Legumes I. Trifolium pratense L

2013 ◽  
Vol 70 (1) ◽  
pp. 87-92
Author(s):  
Gabriela Maria VICAȘ ◽  
Mircea SAVATTI
Keyword(s):  
Amino Acids ◽  
In Vitro Culture ◽  
Culture Medium ◽  
Trifolium Pratense ◽  
Basal Medium ◽  
Red Clover ◽  
Forage Legumes ◽  
Trifolium Pratense L

Establishing the effect of the amino acids as additional additives to the culture medium is and will be in the future one of our concerns of interest for the in vitro culture of some plants. The present study examines the effect of the glicocol added to the LS basal medium over the embryos of the Trifolium pratense L specie cultivated in vitro. There were followed: the percentage of plant regeneration of the red clover, its multiplication capacity and the formation of the root system, and also the evolution of the callus obtained on mediums with 2,4D, BA and amino acid.

scholarly journals A Role for the Sendai Virus P Protein Trimer in RNA Synthesis

1998 ◽  
Vol 72 (5) ◽  
pp. 4274-4280 ◽  
Author(s):  
Joseph Curran
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Essential Role ◽  
Rna Synthesis ◽  
Sendai Virus ◽  
Coiled Coil ◽  
The Third ◽  
P Protein ◽  
P Proteins

ABSTRACT The SeV P protein is found as a homotrimer (P3) when it is expressed in mammalian cells, and trimerization is mediated by a predicted coiled-coil motif which maps within amino acids (aa) 344 to 411 (the BoxA region). The bacterially expressed protein also appears to be trimeric, apparently precluding a role for phosphorylation in the association of the P monomers. I have examined the role of P trimerization both in the protein’s interaction with the nucleocapsid (N:RNA) template and in the protein’s function on the template during RNA synthesis. As with the results of earlier experiments (32), I found that both the BoxA and BoxC (aa 479 to 568) regions were required for stable binding of P to the N:RNA. Binding was also observed with P proteins containing less than three BoxC regions, suggesting that trimerization may be required to permit contacts between multiple BoxC regions and the N:RNA. However, these heterologous trimers failed to function in viral RNA synthesis, indicating that the third C-terminal leg of the trimer plays an essential role in P function on the template. We speculate that this function may involve the movement of P (and possibly the polymerase complex) on the template and the maintenance of processivity.

scholarly journals Fate of uptaken host proteins in Taenia solium and Taenia crassiceps cysticerci

2018 ◽  
Vol 38 (4) ◽  
Author(s):  
Jeanette Flores-Bautista ◽  
José Navarrete-Perea ◽  
Gladis Fragoso ◽  
Ana Flisser ◽  
Xavier Soberón ◽  
...  
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Taenia Solium ◽  
Physiological Role ◽  
Essential Amino Acids ◽  
Host Proteins ◽  
Taenia Crassiceps ◽  
The Matrix ◽  
Host Parasite

During the study of host–parasite relationships in taeniid parasite diseases, including cysticercosis and hydatidosis, reports have described the presence of host proteins in the cyst fluid and tissue of metacestodes. However, the fate or role of host elements inside the parasite remains barely explored. After the publication of genomes of four cestode species, it became clear that these organisms possess a limited biosynthetic capability. The initial goal of the present study was to determine if uptaken host proteins could be a source of essential amino acids for cysticerci. To track the utilization of uptaken proteins, we added metabolically labeled IgG-3H and GFP-3H to the culture medium of Taenia crassiceps cysticerci. Incorporation of labeled amino acid was evaluated by fluorography in cysticerci extracts. Our results showed that the use of uptaken proteins by cysticerci as a source of amino acids appeared negligible. Exploring alternative fates for the host proteins, proteomic analysis of the protein matrix in calcareous corpuscles was carried out. Since T. crassiceps does not contain calcareous corpuscles, proteomic analyses were performed in corpuscles of Taenia solium cysticerci. Our results demonstrated that host proteins represented approximately 70% of protein content in the calcareous corpuscles. The presence of the two major uptaken host proteins, namely albumin and IgG, was also demonstrated by Western blot in the matrix of corpuscles. Our findings strongly suggested that the uptake and disposal of host proteins involve calcareous corpuscles, expanding the physiological role of these mineral concretions to a far more important level than previously proposed.

Processing of pro-PTHRP by the prohormone convertase, furin: effect on biological activity

1995 ◽  
Vol 268 (5) ◽  
pp. E832-E838 ◽  
Author(s):  
B. Liu ◽  
D. Goltzman ◽  
S. A. Rabbani
Keyword(s):  
High Pressure ◽  
Biological Activity ◽  
Culture Medium ◽  
Potential Role ◽  
Prohormone Convertase ◽  
Marked Inhibition ◽  
Gel Permeation ◽  
Parathyroid Hormone Related Peptide ◽  
Conditioned Culture Medium

We have examined the conversion of parathyroid hormone-related peptide (PTHRP) from its NH2-terminally extended precursor pro-PTHRP. Within pro-PTHRP, an amino acid sequence exists that can serve as a substrate for the prohormone convertase, furin. To evaluate the potential role of furin in processing of this entity, we expressed pro-PTHRP in COS-7 cells, which normally produce this enzyme. Transiently transfected COS-7 cells secreted high levels of PTHRP into conditioned culture medium. Cotransfection of these cells with antisense furin cDNA resulted in marked inhibition of furin mRNA expression and secretion of an NH2-terminal fragment of pro-PTHRP, which comigrated with synthetic pro-PTHRP-(-12-->+36) on gel-permeation high-pressure liquid chromatography. In an adenylate cyclase bioassay, condition medium containing this fragment and synthetic pro-PTHRP-(-12-->+36) both exhibited lower potency than synthetic PTHRP-(1-36) and conditioned medium containing PTHRP produced by COS-7 cells in the absence of antisense furin. These results demonstrate the capacity of furin to convert pro-PTHRP to a more active product and suggest a role for this enzyme in the normal intracellular processing of this hormone.

scholarly journals Proline-dependent regulation of collagen metabolism

2019 ◽  
Vol 77 (10) ◽  
pp. 1911-1918 ◽  
Author(s):  
Ewa Karna ◽  
Lukasz Szoka ◽  
Thi Yen Ly Huynh ◽  
Jerzy A. Palka
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
De Novo ◽  
Connective Tissue Diseases ◽  
Collagen Molecule ◽  
Collagen Biosynthesis ◽  
Potential Mechanism ◽  
Free Medium ◽  
Cells Cultured

AbstractThis review is focused on recent data on the role of proline (Pro) in collagen biosynthesis and cellular metabolism. It seems obvious that one of the main substrates for collagen biosynthesis Pro is required to form collagen molecule. The question raised in this review is whether the Pro for collagen biosynthesis is synthesized “de novo”, comes directly from degraded proteins or it is converted from other amino acids. Recent data provided evidence that extracellular Pro (added to culture medium) had significant, but relatively little impact on collagen biosynthesis in fibroblasts (the main collagen synthesized cells) cultured in the presence of glutamine (Gln). However, extracellular Pro drastically increased collagen biosynthesis in the cells cultured in Gln-free medium. It suggests that Pro availability determines the rate of collagen biosynthesis and demand for Pro in fibroblasts is predominantly met by conversion from Gln. The potential mechanism of this process as well as possible implication of this knowledge in pharmacotherapy of connective tissue diseases is discussed in this review.

scholarly journals Phospholipase D activity is essential for actin localization and actin-based motility in Dictyostelium

2005 ◽  
Vol 389 (1) ◽  
pp. 207-214 ◽  
Author(s):  
Soha ZOUWAIL ◽  
Trevor R. PETTITT ◽  
Stephen K. DOVE ◽  
Margarita V. CHIBALINA ◽  
Dale J. POWNER ◽  
...  
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Essential Role ◽  
Kinase Activity ◽  
Complete Loss ◽  
Complete Inhibition ◽  
Cellular Processes ◽  
Small Clusters ◽  
Actin Localization

PLD (phospholipase D) activity catalyses the generation of the lipid messenger phosphatidic acid, which has been implicated in a number of cellular processes, particularly the regulation of membrane traffic. In the present study, we report that disruption of PLD signalling causes unexpectedly profound effects on the actin-based motility of Dictyostelium. Cells in which PLD activity is inhibited by butan-1-ol show a complete loss of actin-based structures, accompanied by relocalization of F-actin into small clusters, and eventually the nucleus, without a visible fall in levels of F-actin. Addition of exogenous phosphatidic acid reverses the effects of butan-1-ol, confirming that these effects are caused by inhibition of PLD. Loss of motility correlates with complete inhibition of endocytosis and a reduction in phagocytosis. Inhibition of PLD caused a major decrease in the synthesis of PtdIns(4,5)P2, which could again be reversed by exogenously applied phosphatidic acid. Thus the essential role of PLD signalling in both motility and endocytosis appears to be mediated directly via regulation of PtdIns(4)P kinase activity. This implies that localized PLD-regulated synthesis of PtdIns(4,5)P2 is essential for Dictyostelium actin function.

The Role of Microfilaments in Early Meiotic Maturation of Mouse Oocytes

2005 ◽  
Vol 11 (2) ◽  
pp. 146-153 ◽  
Author(s):  
Patricia G. Calarco
Keyword(s):  
Plasma Membrane ◽  
Confocal Microscopy ◽  
Essential Role ◽  
Culture Medium ◽  
Cytochalasin B ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Meiotic Maturation ◽  
Over Time

Mouse oocyte microfilaments (MF) were perturbed by depolymerization (cytochalasin B) or stabilization (jasplakinolide) and correlated meiotic defects examined by confocal microscopy. MF, microtubules, and mitochondria were vitally stained; centrosomes (γ-tubulin), after fixation. MF depolymerization by cytochalasin in culture medium did not affect central migration of centrosomes, mitochondria, or nuclear breakdown (GVBD); some MF signal was localized around the germinal vesicle (GV). In maturation-blocking medium (containing IBMX), central movement was curtailed and cortical MF aggregations made the plasma membrane wavy. Occasional long MF suggested that not all MF were depolymerized. MF stabilization by jasplakinolide led to MF aggregations throughout the cytoplasm. GVBD occurred (unless IBMX was present) but no spindle formed. Over time, most oocytes constricted creating a dumbbell shape with MF concentrated under one-half of the oocyte cortex and on either side of the constriction. In IBMX medium, the MF-containing half of the dumbbell over time sequestered the GV, MF, mitochondria, and one to two large cortical centrosomes; the non-MF half appeared empty. Cumulus processes contacted the oocyte surface (detected by microtubule content) and mirrored MF distribution. Results demonstrated that MF play an essential role in meiosis, primarily through cortically mediated events, including centrosome localization, spindle (or GV) movement to the periphery, activation of (polar body) constriction, and establishment of oocyte polarity. The presence of a cortical “organizing pole” is hypothesized.

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