Effects of calcium on flagellar movement in the trypanosome Crithidia oncopelti

1976 ◽  
Vol 65 (1) ◽  
pp. 229-242 ◽  
Author(s):  
M. E. Holwill ◽  
J. L. McGregor
Keyword(s):  
Calcium Ion ◽  
High Speed ◽  
Calcium Concentration ◽  
Competitive Inhibition ◽  
Wave Direction ◽  
Wave Shape ◽  
Low Concentrations ◽  
High Calcium ◽  
High Concentrations

1. The effects of calcium on the motility of different preparations of flagella from Crithidia oncopelti were studied using stroboscopic and high-speed cine photographic techniques. 2. By varying the concentration of calcium in suspensions of chemically treated samples of the organism it was found that changes occurred in bend shape, wave direction and frequency. 3. Waves on the flagellum of the organisms in vivo possess the unusual ability to propagate from tip to base, but reverse in direction during an avoiding response. In chemically extracted and reactivated preparations tip to base propagation was observed only at low concentrations (less than 10(−4) mol m-3) of calcium ion; at high concentrations base to tip propagation only was seen. In cells treated with ion across membranes, tip to base propagation was seen only in the presence of EGTA; when calcium was added the majority of organisms propagated waves from base to tip. 4. At certain values (ca. 10(−3) mol m-3) of the calcium concentration the wave shape had meander-like characteristics, whereas at higher and lower concentrations it was more sinsoidal. At high calcium concentrations only one wave appeared on the flagellum whereas at low values two or three were observed. 5. A reduction in frequency at high calcium concentrations was probably due to competitive inhibition of magnesium ions. 6. The results suggest that wave reversal in living Crithidia is induced by the release of calcium ions within the flagellum following stimulation of the membrane. In terms of the sliding filament model of flagellar activity the effects of calcium suggest that the ion is effective in modifying the interaction between the spoke head and central sheath and may control the relative direction of microtubular sliding.

scholarly journals Colchicine-binding protein of the liver. Its characterization and relation to microtubules.

1975 ◽  
Vol 66 (3) ◽  
pp. 609-620 ◽  
Author(s):  
C Patzelt ◽  
A Singh ◽  
Y L Marchand ◽  
L Orci ◽  
B Jeanrenaud
Keyword(s):  
High Speed ◽  
Specific Binding ◽  
Electrophoretic Analysis ◽  
Binding Activity ◽  
Low Concentrations ◽  
Electrophoretic Behavior ◽  
High Affinity Binding Site ◽  
High Concentrations

Colchicine-binding activity of mouse liver high-speed supernate has been investigated. It has been found to be time and temperature dependent. Two binding activities with different affinities for colchicine seem to be present in this high-speed supernate, of which only the high-affinity binding site (half maximal binding at 5 x 10(-6) M colchicine) can be attributed to microtubular protein by comparison with purified tubulin. Vinblastine interacted with this binding activity by precipitating it when used at high concentrations (2 x 10(-3) M), and by stabilizing it at low concentrations (10(-5) M). Lumicolchicine was found not to compete with colchicine. The colchicine-binding activity was purified from liver and compared with that of microtubular protein from brain. The specific binding activity of the resulting preparation, its electrophoretic behavior, and the electron microscope appearance of the paracrystals obtained upon its precipitation with vinblastine permitted its identification as microtubular protein (tubulin). Electrophoretic analysis of the proteins from liver supernate that were precipitated by vinblastine indicated that this drug was not specific for liver tubulin. Preincubation of liver supernate with 5 mM EGTA resulted in a time-dependent decrease of colchicine-binding activity, which was partly reversed by the addition of Ca++. However, an in vitro formation of microtubules upon lowering the Ca++ concentration could not be detected. Finally, a method was developed enabling that portion of microtubular protein which was present as free tubulin to be measured and to be compared with the total amount of this protein in the tissue. This procedure permitted demonstration of the fact that, under normal conditions, only about 40% of the tubulin of the liver was assemled as microtubules. It is suggested that, in the liver, rapid polymerization and depolymerization of microtubules occur and may be an important facet of the functional role of the microtubular system.

Mechanisms of vanadium action: insulin-mimetic or insulin-enhancing agent?

2000 ◽  
Vol 78 (10) ◽  
pp. 829-847 ◽  
Author(s):  
Margaret C Cam ◽  
Roger W Brownsey ◽  
John H McNeill
Keyword(s):  
Lipid Metabolism ◽  
Metabolic Effects ◽  
Isolated Cells ◽  
Insulin Mimetic ◽  
Endogenous Insulin ◽  
Carbohydrate And Lipid Metabolism ◽  
Low Concentrations ◽  
Tissue Sensitivity ◽  
High Concentrations

The demonstration that the trace element vanadium has insulin-like properties in isolated cells and tissues and in vivo has generated considerable enthusiasm for its potential therapeutic value in human diabetes. However, the mechanisms by which vanadium induces its metabolic effects in vivo remain poorly understood, and whether vanadium directly mimics or rather enhances insulin effects is considered in this review. It is clear that vanadium treatment results in the correction of several diabetes-related abnormalities in carbohydrate and lipid metabolism, and in gene expression. However, many of these in vivo insulin-like effects can be ascribed to the reversal of defects that are secondary to hyperglycemia. The observations that the glucose-lowering effect of vanadium depends on the presence of endogenous insulin whereas metabolic homeostasis in control animals appears not to be affected, suggest that vanadium does not act completely independently in vivo, but augments tissue sensitivity to low levels of plasma insulin. Another crucial consideration is one of dose-dependency in that insulin-like effects of vanadium in isolated cells are often demonstrated at high concentrations that are not normally achieved by chronic treatment in vivo and may induce toxic side effects. In addition, vanadium appears to be selective for specific actions of insulin in some tissues while failing to influence others. As the intracellular active forms of vanadium are not precisely defined, the site(s) of action of vanadium in metabolic and signal transduction pathways is still unknown. In this review, we therefore examine the evidence for and against the concept that vanadium is truly an insulin-mimetic agent at low concentrations in vivo. In considering the effects of vanadium on carbohydrate and lipid metabolism, we conclude that vanadium acts not globally, but selectively and by enhancing, rather than by mimicking the effects of insulin in vivo.Key words: vanadium, insulin-mimetic, insulin-like, insulin-enhancing.

scholarly journals Hypogonadism associated withCyp19a1 (Aromatase) post-transcriptional upregulation inCelf1-KO mice

2015 ◽  
pp. MCB.00074-15 ◽  
Author(s):  
Gaella Boulanger ◽  
Marie Cibois ◽  
Justine Viet ◽  
Alexis Fostier ◽  
Stéphane Deschamps ◽  
...  
Keyword(s):  
Rna Binding ◽  
Hypogonadotropic Hypogonadism ◽  
Type I ◽  
Male Gametes ◽  
Low Concentrations ◽  
Reporter Assays ◽  
High Concentrations ◽  
In Vitro Interaction

CELF1 is a multifunctional RNA-binding protein that controls several aspects of RNA fate. The targeted disruption of theCelf1gene in mice causes male infertility due to impaired spermiogenesis, the post-meiotic differentiation of male gametes. Here, we investigated the molecular reasons that underlie this testicular phenotype. By measuring sex hormone levels, we detected low concentrations of testosterone inCelf1-null mice. We investigated the effect ofCelf1disruption on the expression levels of steroidogenic enzyme genes, and we observed thatCyp19a1was upregulated.Cyp19a1encodes aromatase, which transforms testosterone into estradiol. Administration of testosterone or the aromatase inhibitor Letrozole partly rescued the spermiogenesis defects, indicating that a lack of testosterone associated with excessive aromatase contributes to the testicular phenotype. In vivo and in vitro interaction assays demonstrated that CELF1 binds toCyp19a1mRNA, and reporter assays supported the conclusion that CELF1 directly repressesCyp19a1translation. We conclude that CELF1 downregulatesCyp19a1/Aromatasepost-transcriptionally to achieve high concentrations of testosterone compatible with spermiogenesis completion. We discuss the implications of these findings with respect to reproductive defects in men, including patients suffering from isolated hypogonadotropic hypogonadism and myotonic dystrophy type I.

Effects of polymethoxylated flavone metabolites on apoB100 secretion and MTP activity in Huh7.5 cells

2021 ◽  
Vol 18 ◽  
Author(s):  
Danielle R. Gonçalves ◽  
Thais B. Cesar ◽  
John A. Manthey ◽  
Paulo I. Costa
Keyword(s):  
Lipid Synthesis ◽  
Dependent Manner ◽  
Transfer Protein ◽  
Low Concentrations ◽  
Wide Range ◽  
Cell Assays ◽  
Polymethoxylated Flavones ◽  
High Concentrations

Background: Citrus polymethoxylated flavones (PMFs) reduce the synthesis of liver lipoproteins in animal and in vitro cell assays, but few studies have evaluated the direct effects of their metabolites on this highly regulated process. Objective: To investigate the effects of representative metabolites of PMF on the secretion of liver lipoproteins using the mammalian cell Huh7.5. Method: In this study, the influences of three PMFs and five previously isolated PMF metabolites on hepatic apoB-100 secretion and microsomal transfer protein (MTP) activity were evaluated. Tangeretin (TAN), nobiletin (NOB) and 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF), and their glucuronides (TAN-Gluc, NOB-Gluc and HMF-Gluc) and oxidatively demethylated metabolites (TAN-OH, NOB-OH, HMF-OH) were incubated with Huh7.5 cells to measure their inhibitory effects on lipid synthesis. Results: The results showed that TAN, HMF and TAN-OH reduced the secretion of apoB-100 in a dose-dependent manner, while NOB and the other tested metabolites showed no inhibition. MTP activity in the Huh7.5 cells was significantly reduced in the presence of low concentrations of TAN, and in high concentrations of NOB-OH. This study also showed that PMFs and PMF metabolites produced a wide range of effects on apoB-100 secretion and MTP activity. Conclusion: The results suggest that while PMFs and their metabolites control dyslipidemia in vivo, the inhibition of MTP activity cannot be the only pathway influenced by these compounds.

Effects of high calcium levels on the disturbed extrusion of the second polar body during in vitro fertilization in C3H/He mouse substrains

Zygote ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 83-85
Author(s):  
Y. Ohta-Takada ◽  
Y. Nagao ◽  
S. Kito
Keyword(s):  
Calcium Concentration ◽  
Inbred Mice ◽  
Polar Body ◽  
Efficient Production ◽  
Vitro Fertilization ◽  
Practical Applications ◽  
High Calcium ◽  
High Concentrations ◽  
Second Polar Body

SummaryWe previously reported that high concentrations (≥3.42 mM) of calcium during in vitro fertilization (IVF) disturbed the extrusion of the second polar body (PBII) in C3H/He inbred mice. In this study, the substrain specificity of this phenomenon was examined under 1.71–6.84 mM calcium concentration in ova from six C3H/He mouse commercially available substrains in Japan. PBII extrusion in ova from J substrains was not affected by calcium concentrations (<10% at any calcium level), but was grossly disturbed at high calcium levels in the ova of other substrains. This result has practical applications for the efficient production of normal zygotes by IVF, therefore contributing to the reduction in the numbers of donor animals for further zygote or embryo manipulation. Care must be taken in choosing IVF medium for particular strains and substrains.

Stimulation and inhibition of FVIII-specific memory B-cell responses by CpG-B (ODN 1826), a ligand for Toll-like receptor 9

Blood ◽  
2011 ◽  
Vol 117 (1) ◽  
pp. 259-267 ◽  
Author(s):  
Peter Allacher ◽  
Christina K. Baumgartner ◽  
Aniko G. Pordes ◽  
Rafi U. Ahmad ◽  
Hans Peter Schwarz ◽  
...  
Keyword(s):  
B Cells ◽  
Memory B Cells ◽  
Cpg Odn ◽  
Specific Memory ◽  
Cpg Oligodeoxynucleotide ◽  
Low Concentrations ◽  
Essential Components ◽  
High Concentrations

Abstract Factor VIII (FVIII)–specific memory B cells are essential components for regulating anamnestic antibody responses against FVIII in hemophilia A with FVIII inhibitors. We asked how stimulation and inhibition of FVIII-specific memory B cells by low and high concentrations of FVIII, respectively, are affected by concurrent activation of the innate immune system. Using CD138− spleen cells from hemophilic mice treated with FVIII to study restimulation and differentiation of memory B cells in vitro, we tested modulating activities of agonists for Toll-like receptors (TLRs) 2, 3, 4, 5, 7, and 9. Ligands for TLR7 and 9 were most effective. They not only amplified FVIII-specific memory responses in the presence of stimulating concentrations of FVIII, but also countered inhibition in the presence of inhibitory concentrations of FVIII. Notably, CpG oligodeoxynucleotide (CpG-ODN), a ligand for TLR9, expressed biphasic effects. It amplified memory responses at low concentrations and inhibited memory responses at high concentrations, both in vitro and in vivo. Both stimulatory and inhibitory activities of CpG-ODN resulted from specific interactions with TLR9. Despite their strong immunomodulatory effects in the presence of FVIII, ligands for TLR induced negligible restimulation in the absence of FVIII in vitro and no restimulation in the absence of FVIII in vivo.

scholarly journals Inhibition by oxalomalate of rat liver mitochondrial and extramitochondrial aconitate hydratase

1971 ◽  
Vol 125 (2) ◽  
pp. 557-562 ◽  
Author(s):  
A. Adinolfi ◽  
V. Guarriera-Bobyleva ◽  
S. Olezza ◽  
A. Ruffo
Keyword(s):  
Rat Liver ◽  
Initial Rate ◽  
Competitive Inhibition ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Enzyme ◽  
Aconitate Hydratase ◽  
Inhibited Oxidation ◽  
Low Concentrations ◽  
High Concentrations

1. The effect of oxalomalate on the oxidation of citrate and cis-aconitate in rat liver mitochondria, and on the activity of mitochondrial and cytoplasmic aconitate hydratase, has been investigated. 2. Oxalomalate that was added to intact rat liver mitochondria at high concentrations (2mm) produced complete inhibition of citrate and cis-aconitate oxidation, but lower concentrations (0.1–0.25mm) inhibited oxidation of citrate more than that of cis-aconitate. 3. Aconitate hydratase that was either extracted from mitochondria or soluble in the cytoplasm, was strongly inhibited by low concentrations of oxalomalate (0.01–0.2mm), the mitochondrial enzyme being more sensitive than the soluble one. 4. Oxalomalate, when added together with citrate, produced competitive inhibition; the Ki values calculated were 1×10−6m for the mitochondrial and 2.5×10−6m for the cytoplasmic enzyme. 5. With both the enzymic preparations oxalomalate added together with the substrates inhibited the initial rate of the reaction citrate→cis-aconitate more than that of the reaction isocitrate→cis-aconitate. 6. After 2min of preincubation of the inhibitor with either of the enzymic preparations the inhibition increased tenfold and became irreversible; under these conditions both the reactions were inhibited to the same extent. 7. The inhibition by oxalomalate of aconitate hydratase appeared to be similar in many respects to that produced by fluorocitrate on the same enzyme.

Ethanol effects on cardiomyocyte contractility

2000 ◽  
Vol 98 (4) ◽  
pp. 401-407 ◽  
Author(s):  
Leanne M. D. DELBRIDGE ◽  
Pamela J. CONNELL ◽  
Peter J. HARRIS ◽  
Trefor O. MORGAN
Keyword(s):  
High Speed ◽  
Imaging Techniques ◽  
Cardiac Contractility ◽  
Ethanol Exposure ◽  
High Speed Imaging ◽  
Low Concentrations ◽  
Cardiomyocyte Contractility ◽  
Contraction Cycle ◽  
Kinetics Of

Little is known about the direct cardiac effects of socially common sub-intoxication levels of ethanol. Previous studies evaluating the responses of normal cardiomyocytes to short-term ethanol exposure have utilized ethanol concentrations equivalent to extreme intoxication or lethal levels in vivo. The purpose of the present study was to investigate the contractile responses of isolated rat ventricular cardiomyocytes during exposure to relatively low concentrations of ethanol in the range 0.05–0.5% (v/v) (8.6–86 mM) under physiological conditions (3 Hz stimulation; 36 °C; BSA vehicle). High-speed imaging techniques were used to study the kinetics of myocyte contraction, and shortening parameters were calculated for mechanistic evaluation. The concentration–response relationship was not linear and exhibited two plateau phases, suggesting at least two mechanisms of action of ethanol on cardiomyocyte contraction. At 0.05% (8.6 mM), ethanol treatment produced a 14.4% decrease in maximum myocyte shortening. The maximum rates of cell shortening and lengthening were similarly impaired, but there was no effect on contraction cycle timing at this low concentration. At 0.30% (51 mM), ethanol reduced maximum shortening by 40.2%, prolonged excitation–contraction coupling latency and abbreviated the contraction cycle time by 38%. The inotropic modulatory effect of ethanol was exaggerated in the absence of protein in the superfusion buffer. This is the first report which identifies ethanol at 0.05% (v/v) as a modulator of cardiac contractility. Kinetic analyses indicate that the mechanism of action involves disturbance of sarcoplasmic reticulum function, and this may contribute to arrhythmogenic vulnerability – especially in an in vivo context of heightened compensatory sympathetic drive.

Protease-activated receptors 1 and 4 mediate thrombin signaling in endothelial cells

Blood ◽  
2003 ◽  
Vol 102 (9) ◽  
pp. 3224-3231 ◽  
Author(s):  
Hiroshi Kataoka ◽  
Justin R. Hamilton ◽  
David D. McKemy ◽  
Eric Camerer ◽  
Yao-Wu Zheng ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Wild Type ◽  
Protease Activated Receptors ◽  
Erk Phosphorylation ◽  
Low Concentrations ◽  
Extracellular Signal ◽  
High Concentrations ◽  
Mouse Aorta

AbstractDefining the relative importance of protease-activated receptors (PARs) for thrombin signaling in mouse endothelial cells is critical for a basic understanding of thrombin signaling in these cells and for the rational use of knockout mice to probe the roles of thrombin's actions on endothelial cells in vivo. We examined thrombin- and PAR agonist–induced increases in cytoplasmic calcium, phosphoinositide hydrolysis, extracellular signal-regulated kinase (ERK) phosphorylation, and gene expression in endothelial cells from wild-type and PAR-deficient mice. PAR1 and PAR4 agonists triggered responses in wild-type but not in Par1–/– and Par4–/– endothelial cells, respectively. Calcium imaging confirmed that a substantial fraction of individual endothelial cells responded to both agonists. Compared with wild-type cells, Par1–/– endothelial cells showed markedly decreased responses to low concentrations of thrombin, and cells that lacked both PAR1 and PAR4 showed no responses to even high concentrations of thrombin. Similar results were obtained when endothelial-dependent vasorelaxation of freshly isolated mouse aorta was used as an index of signaling in native endothelial cells. Thus PAR1 is the major thrombin receptor in mouse endothelial cells, but PAR4 also contributes. These receptors serve at least partially redundant roles in endothelial cells in vitro and in vivo and together are necessary for the thrombin responses measured.

scholarly journals Involvement of the gp130 cytokine transducer in MtT/S pituitary somatotroph tumour development in an autocrine-paracrine model

2004 ◽  
pp. 595-604 ◽  
Author(s):  
M Graciarena ◽  
A Carbia-Nagashima ◽  
C Onofri ◽  
C Perez-Castro ◽  
D Giacomini ◽  
...  
Keyword(s):  
Cell Line ◽  
Growth Regulation ◽  
Functional Characterization ◽  
Cell Number ◽  
Pituitary Cells ◽  
Tumour Development ◽  
Low Concentrations ◽  
High Concentrations ◽  
And Control

OBJECTIVE: gp130 cytokines are placed as auto-paracrine regulators of pituitary function, since they, as well as their receptors, have been shown to be expressed in and to act in normal and tumoral anterior pituitary cells. The objective of this work was to study their involvement in a model that shows the interaction between different cellular types that participate in a tumorigenic process. DESIGN: The dependence of a pituitary somatotrophic cell line (MtT/S) on a gp130 cytokine-producing folliculostellate (FS) cell line (TtT/GF) for tumorigenesis in vivo has been described. In order to study the participation of gp130 cytokines in the auto-paracrine stimulation of MtT/S growth, we generated MtT/S gp130 sense (gp130-S) and gp130 antisense (gp130-AS) clones stably transfected with pcDNA3/gp130 sense and pcDNA3/gp130 antisense vectors respectively. METHODS AND RESULTS: Functional characterization studies revealed that gp130-AS clones have an inhibited gp130 signalling, and proliferation studies showed that they have an impaired response to gp130 cytokines but respond normally to other independent stimuli. When injected into nude mice, MtT/S clones respond differently depending on cell number; at high concentrations MtT/S clones alone generated tumours equivalent in size to tumours derived from MtT/S plus TtT/GF cells. At low concentrations, MtT/S sense and control clones generated tumours of smaller size than tumours derived from these same clones plus TtT/GF cells, showing a dependence on FS cells. In both cases MtT/S gp130-AS clones had impaired tumour development. Furthermore, vessel density was significantly lower in tumours derived from gp130-AS plus TtT/GF cells. CONCLUSIONS: This study underlines the importance of gp130 cytokines in proliferation and establishes its role in auto-paracrine pituitary growth regulation.

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