scholarly journals A Cytosolic Serine Endopeptidase from Trypanosoma cruzi Is Required for the Generation of Ca2+ Signaling in Mammalian Cells

1997 ◽  
Vol 136 (3) ◽  
pp. 609-620 ◽  
Author(s):  
Barbara A. Burleigh ◽  
Elisabet V. Caler ◽  
Paul Webster ◽  
Norma W. Andrews
Keyword(s):  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Rat Kidney ◽  
Biologically Active ◽  
Early Event ◽  
Peptidase Activity ◽  
Active Peptides ◽  
Oligopeptidase B ◽  
Normal Rat ◽  
Signaling Activity

An early event in the Trypanosoma cruzi cell invasion process, the recruitment of host lysosomes, led us to investigate the involvement of signal transduction. Infective trypomastigotes were found to contain a soluble Ca2+-signaling activity for mammalian cells that is sensitive to protease inhibitors. Inhibitor and substrate utilization profiles were used to purify a candidate peptidase for involvement in this process, from which we isolated a full-length cDNA clone. The sequence revealed a novel enzyme, denominated T. cruzi oligopeptidase B, which is homologous to members of the prolyl oligopeptidase family of serine hydrolases, known to participate in the maturation of biologically active peptides. The T. cruzi oligopeptidase B was expressed as a fully active product in Escherichia coli, and antibodies to the recombinant enzyme inhibited both peptidase activity and Ca2+ signaling induced in normal rat kidney cells by trypomastigote extracts. Our data suggest that the T. cruzi oligopeptidase B participates in processing events in the cytoplasm of the parasites, generating a factor with Ca2+-signaling activity for mammalian cells.

scholarly journals Adhesion-dependent and Contractile Ring-independent Equatorial Furrowing during Cytokinesis in Mammalian Cells

2005 ◽  
Vol 16 (8) ◽  
pp. 3865-3872 ◽  
Author(s):  
Masamitsu Kanada ◽  
Akira Nagasaki ◽  
Taro Q.P. Uyeda
Keyword(s):  
Mammalian Cells ◽  
Rat Kidney ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Cellular Slime Mold ◽  
Animal Cells ◽  
Contractile Ring ◽  
Normal Rat ◽  
Cellular Slime ◽  
Coated Surfaces

Myosin II-dependent contraction of the contractile ring drives equatorial furrowing during cytokinesis in animal cells. Nonetheless, myosin II-null cells of the cellular slime mold Dictyostelium divide efficiently when adhering to substrates by making use of polar traction forces. Here, we show that in the presence of 30 μM blebbistatin, a potent myosin II inhibitor, normal rat kidney (NRK) cells adhering to fibronectin-coated surfaces formed equatorial furrows and divided in a manner strikingly similar to myosin II-null Dictyostelium cells. Such blebbistatin-resistant cytokinesis was absent in partially detached NRK cells and was disrupted in adherent cells if the advance of their polar lamellipodia was disturbed by neighboring cells. Y-27632 (40 μM), which inhibits Rho-kinase, was similar to 30 μM blebbistatin in that it inhibited cytokinesis of partially detached NRK cells but only prolonged furrow ingression in attached cells. In the presence of 100 μM blebbistatin, most NRK cells that initiated anaphase formed tight furrows, although scission never occurred. Adherent HT1080 fibrosarcoma cells also formed equatorial furrows efficiently in the presence of 100 μM blebbistatin. These results provide direct evidence for adhesion-dependent, contractile ring-independent equatorial furrowing in mammalian cells and demonstrate the importance of substrate adhesion for cytokinesis.

scholarly journals Inhibition of Chromosomal Separation Provides Insights into Cleavage Furrow Stimulation in Cultured Epithelial Cells

1998 ◽  
Vol 9 (8) ◽  
pp. 2173-2184 ◽  
Author(s):  
Sally P. Wheatley ◽  
Christopher B. O’Connell ◽  
Yu-li Wang
Keyword(s):  
Epithelial Cells ◽  
Mammalian Cells ◽  
Topoisomerase Ii ◽  
Rat Kidney ◽  
Myosin Ii ◽  
Daughter Cells ◽  
Irregular Shapes ◽  
Anaphase Onset ◽  
Cultured Epithelial Cells ◽  
Normal Rat

While astral microtubules are believed to be primarily responsible for the stimulation of cytokinesis in Echinodermembryos, it has been suggested that a signal emanating from the chromosomal region and mediated by the interzonal microtubules stimulates cytokinesis in cultured mammalian cells. To test this hypothesis, we examined cytokinesis in normal rat kidney cells treated with an inhibitor of topoisomerase II, (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane, which prevents the separation of sister chromatids and the formation of a spindle interzone. The majority of treated cells showed various degrees of abnormality in cytokinesis. Furrows frequently deviated from the equatorial plane, twisting daughter cells into irregular shapes. Some cells developed furrows in regions outside the equator or far away from the spindle. In addition, F-actin and myosin II accumulated at the lateral ingressing margins but did not form a continuous band along the equator as in control cells. Imaging of microinjected 5- (and 6-) carboxymtetramethylrhodamine-tubulin revealed that a unique set of microtubules projected out from the chromosomal vicinity upon anaphase onset. These microtubules emanated toward the lateral cortex, where they delineated sites of microtubule bundle formation, cortical ingression, and F-actin and myosin II accumulation. As centrosome integrity and astral microtubules appeared unperturbed by (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane treatment, the present observations cannot be easily explained by the conventional model involving astral microtubules. We suggest that in cultured epithelial cells the organization of the chromosomes dictates the organization of midzone microtubules, which in turn determines and maintains the cleavage activity.

CORTICOSTEROIDOGENESIS IN ISOLATED ADRENAL CELLS: EFFECT OF ADRENOCORTICOTROPHIC HORMONE, ADENOSINE 3′,5′-MONOPHOSPHATE AND β1–24ADRENOCORTICOTROPHIC HORMONE DIAZOTIZED TO POLYACRYLAMIDE

1972 ◽  
Vol 55 (1) ◽  
pp. 127-139 ◽  
Author(s):  
M. C. RICHARDSON ◽  
D. SCHULSTER
Keyword(s):  
Time Course ◽  
Biologically Active ◽  
Target Cells ◽  
Adrenal Cells ◽  
Active Peptides ◽  
Biologically Active Peptides ◽  
Normal Rat ◽  
Acth Concentration ◽  
A Cell ◽  
Competitive Protein Binding

SUMMARY A cell suspension was prepared from normal rat adrenal tissue by collagenase digestion of decapsulated adrenal glands. Corticosterone produced by these cells was assayed both by competitive protein binding and by radioimmunoassay (range: 0–1 ng; sensitivity: 30 pg). Before incubation, the medium and cells equivalent to one gland contained 27·7 ± 3·2 ng corticosterone which increased to 36·3 ± 7·0 ng corticosterone after 1 h of incubation. Although a normal sigmoid log dose—response curve was obtained using adrenocorticotrophin (ACTH) (ED50:1·2 × 10−4 i.u./ml), it was observed that log (ACTH concentration) was directly proportional to log (corticosterone produced) between 1 × 10−7 and 1 × 10−3 i.u. ACTH/ml. A similar linear relationship was demonstrated for the effect of adenosine 3′,5′-monophosphate on steroidogenesis. Time-course studies revealed that after addition of ACTH there was a definite lag (3 min) before onset of increased steroidogenesis; within 5 min a constant corticosterone production rate was achieved. β1–24 Adrenocorticotrophin was shown to retain its ability to stimulate steroidogenesis when diazotized to beads of polyacrylamide too large to enter cells. Evidence is provided that the activity of the β1–24 ACTH—polyacrylamide beads is not due to biologically active peptides cleaved from the complex. It is concluded that ACTH can stimulate steroidogenesis without entering its target cells.

scholarly journals Role in host cell invasion of Trypanosoma cruzi-induced cytosolic-free Ca2+ transients.

1994 ◽  
Vol 179 (3) ◽  
pp. 1017-1022 ◽  
Author(s):  
I Tardieux ◽  
M H Nathanson ◽  
N W Andrews
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Pertussis Toxin ◽  
Rat Kidney ◽  
Host Cells ◽  
Channel Blockers ◽  
Signaling Mechanisms ◽  
Normal Rat ◽  
G Protein Coupled ◽  
Unique Mechanism

Trypanosoma cruzi enters cells by a unique mechanism, distinct from phagocytosis. Invasion is facilitated by disruption of host cell actin microfilaments, and involves recruitment and fusion of host lysosomes at the site of parasite entry. These findings implied the existence of transmembrane signaling mechanisms triggered by the parasites in the host cells before invasion. Here we show that infective trypomastigotes or their isolated membranes, but not the noninfective epimastigotes, induce repetitive cytosolic-free Ca2+ transients in individual normal rat kidney fibroblasts, in a pertussis toxin-sensitive manner. Parasite entry is inhibited by buffering or depleting host cell cytosolic-free Ca2+, or by pretreatment with Ca2+ channel blockers or pertussis toxin. In contrast, invasion is enhanced by brief exposure of the host cells to cytochalasin D. These results indicate that a trypomastigote membrane factor triggers cytosolic-free Ca2+ transients in host cells through a G-protein-coupled pathway. This signaling event may promote invasion through modulation of the host cell actin cytoskeleton.

scholarly journals Sec6/8 complexes on trans-Golgi network and plasma membrane regulate late stages of exocytosis in mammalian cells

2001 ◽  
Vol 155 (4) ◽  
pp. 593-604 ◽  
Author(s):  
Charles Yeaman ◽  
Kent K. Grindstaff ◽  
Jessica R. Wright ◽  
W. James Nelson
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Rat Kidney ◽  
Brefeldin A ◽  
Intercellular Junctions ◽  
Trans Golgi Network ◽  
Cargo Protein ◽  
Normal Rat ◽  
Golgi Network ◽  
Exocytic Pathway

Sec6/8 complex regulates delivery of exocytic vesicles to plasma membrane docking sites, but how it is recruited to specific sites in the exocytic pathway is poorly understood. We identified an Sec6/8 complex on trans-Golgi network (TGN) and plasma membrane in normal rat kidney (NRK) cells that formed either fibroblast- (NRK-49F) or epithelial-like (NRK-52E) intercellular junctions. At both TGN and plasma membrane, Sec6/8 complex colocalizes with exocytic cargo protein, vesicular stomatitis virus G protein (VSVG)-tsO45. Newly synthesized Sec6/8 complex is simultaneously recruited from the cytosol to both sites. However, brefeldin A treatment inhibits recruitment to the plasma membrane and other treatments that block exocytosis (e.g., expression of kinase-inactive protein kinase D and low temperature incubation) cause accumulation of Sec6/8 on the TGN, indicating that steady-state distribution of Sec6/8 complex depends on continuous exocytic vesicle trafficking. Addition of antibodies specific for TGN- or plasma membrane–bound Sec6/8 complexes to semiintact NRK cells results in cargo accumulation in a perinuclear region or near the plasma membrane, respectively. These results indicate that Sec6/8 complex is required for several steps in exocytic transport of vesicles between TGN and plasma membrane.

scholarly journals High Glycogen Levels in Brains of Rats with Minimal Environmental Stimuli: Implications for Metabolic Contributions of Working Astrocytes

2002 ◽  
Vol 22 (12) ◽  
pp. 1476-1489 ◽  
Author(s):  
Nancy F. Cruz ◽  
Gerald A. Dienel
Keyword(s):  
Rat Brain ◽  
Sensory Stimulation ◽  
Enzyme Inactivation ◽  
Biologically Active ◽  
Metabolic Labeling ◽  
Tissue Sampling ◽  
Normal Rat ◽  
Sampling Procedures ◽  
Very High

The concentration of glycogen, the major brain energy reserve localized mainly in astrocytes, is generally reported as about 2 or 3 μmol/g, but sometimes as high as 3.9 to 8 μmol/g, in normal rat brain. The authors found high but very different glycogen levels in two recent studies in which glycogen was determined by the routine amyloglucosidase procedure in 0.03N HCl digests either of frozen powders (4.8 to 6 μmol/g) or of ethanol-insoluble fractions (8 to 12 μmol/g). To evaluate the basis for these discrepant results, glycogen was assayed in parallel extracts of the same samples. Glycogen levels in ethanol extracts were twice those in 0.03N HCl digests, suggesting incomplete enzyme inactivation even with very careful thawing. The very high glycogen levels were biologically active and responsive to physiologic and pharmacological challenge. Glycogen levels fell after brief sensory stimulation, and metabolic labeling indicated its turnover under resting conditions. About 95% of the glycogen was degraded under in vitro ischemic conditions, and its “carbon equivalents” recovered mainly as glc, glc-P, and lactate. Resting glycogen stores were reduced by about 50% by chronic inhibition of nitric oxide synthase. Because neurotransmitters are known to stimulate glycogenolysis, stress or sensory activation due to animal handling and tissue-sampling procedures may stimulate glycogenolysis during an experiment, and glycogen lability during tissue sampling and extraction can further reduce glycogen levels. The very high glycogen levels in normal rat brain suggest an unrecognized role for astrocytic energy metabolism during brain activation.

Activity of β-Caryophyllene Oxide Derivatives Against Trypanosoma cruzi, Mammalian Cells, and Horseradish Peroxidase

2021 ◽  
Author(s):  
Gisele Bulhões Portapilla ◽  
Luiz Miguel Pereira ◽  
Rafael Augusto Soldi ◽  
Péricles Gama Abreu Filho ◽  
Inara Fernanda Lage Gallo ◽  
...  
Keyword(s):  
Horseradish Peroxidase ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Caryophyllene Oxide
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