scholarly journals Vacuolar proton pyrophosphatase activity and pyrophosphate (PPi) in Toxoplasma gondii as possible chemotherapeutic targets

2000 ◽  
Vol 349 (3) ◽  
pp. 737-745 ◽  
Author(s):  
Claudia O. RODRIGUES ◽  
David A. SCOTT ◽  
Brian N. BAILEY ◽  
Wanderley DE SOUZA ◽  
Marlene BENCHIMOL ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Proton Transport ◽  
Bone Diseases ◽  
31P Nmr Spectroscopy ◽  
Dependent Manner ◽  
Subcellular Compartment ◽  
Culture Cells ◽  
Thiol Reagent ◽  
Intracellular Proliferation ◽  
Dose Dependent

The addition of PPi promoted the acidification of a subcellular compartment in cell homogenates of Toxoplasma gondii tachyzoites, implying the presence of a proton-translocating pyrophosphatase. The proton gradient was collapsed by addition of the K+/H+ antiporter nigericin, and was also inhibited by addition of the PPi analogue aminomethylenediphosphonate (AMDP). Both proton transport and PPi hydrolysis were dependent upon K+, but Na+ caused partial inhibition of these activities. PPi hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, NaF and to the thiol reagent N-ethylmaleimide. This activity was unaffected by common inhibitors of phosphohydrolases, except that NaO3V (sodium orthovanadate) stimulated the activity by 87%. Immunofluorescence microscopy, using antisera raised against conserved peptide sequences of a plant vacuolar pyrophosphatase, suggested that the pyrophosphatase in T. gondii tachyzoites was located in the plasma membrane and intracellular vacuoles of the parasite. High-field 31P-NMR spectroscopy showed that PPi was more abundant than ATP in tachyzoites. Bisphosphonates (PPi analogues), drugs that are used in the treatment of bone diseases, inhibited proton transport and PPi hydrolysis in tachyzoite homogenates, and also inhibited intracellular proliferation of tachyzoites in tissue culture cells.

scholarly journals Metabolism and Selective Toxicity of 6-Nitrobenzylthioinosine in Toxoplasma gondii

1999 ◽  
Vol 43 (10) ◽  
pp. 2437-2443 ◽  
Author(s):  
Mahmoud H. el Kouni ◽  
Vincenzo Guarcello ◽  
Omar N. Al Safarjalani ◽  
Fardos N. M. Naguib
Keyword(s):  
Toxoplasma Gondii ◽  
Human Fibroblasts ◽  
Adenosine Kinase ◽  
Host Cells ◽  
Dependent Manner ◽  
Selective Toxicity ◽  
Link Type ◽  
Infected Cells ◽  
Dose Dependent ◽  
Uninfected Host

ABSTRACT The purine nucleoside analogue NBMPR {nitrobenzylthioinosine or 6-[(4-nitrobenzyl)thio]-9-β-d-ribofuranosylpurine} was selectively phosphorylated to its nucleoside 5′-monophosphate byToxoplasma gondii but not mammalian adenosine kinase (EC2.7.1.20). NBMPR was also cleaved in toxoplasma to its nucleobase, nitrobenzylmercaptopurine. However, nitrobenzylmercaptopurine was not a substrate for either adenosine kinase or hypoxanthine-guanine-xanthine phosphoribosyltransferase (EC 2.4.2.8). Because of this unique and previously unknown metabolism of NBMPR by the parasite, the effect of NBMPR as an antitoxoplasmic agent was tested. NBMPR killed T. gondii grown in human fibroblasts in a dose-dependent manner, with a 50% inhibitory concentration of approximately 10 μM and without apparent toxicity to host cells. Doses of up to 100 μM had no significant toxic effect on uninfected host cells. The promising antitoxoplasmic effect of NBMPR led to the testing of other 6-substituted 9-β-d-ribofuranosylpurines, which were shown to be good ligands of the parasite adenosine kinase (M. H. Iltzsch, S. S. Uber, K. O. Tankersley, and M. H. el Kouni, Biochem. Pharmacol. 49:1501–1512, 1995), as antitoxoplasmic agents. Among the analogues tested, 6-benzylthioinosine,p-nitrobenzyl-6-selenopurine riboside,N 6-(p-azidobenzyl)adenosine, andN 6-(p-nitrobenzyl)adenosine, like NBMPR, were selectively toxic to parasite-infected cells. Thus, it appears that the unique characteristics of purine metabolism inT. gondii render certain 6-substituted 9-β-d-ribofuranosylpurines promising antitoxoplasmic drugs.

scholarly journals Acidocalcisomes and a vacuolar H+-pyrophosphatase in malaria parasites

2000 ◽  
Vol 347 (1) ◽  
pp. 243-253 ◽  
Author(s):  
Norma MARCHESINI ◽  
Shuhong LUO ◽  
Claudia O. RODRIGUES ◽  
Silvia N. J. MORENO ◽  
Roberto DOCAMPO
Keyword(s):  
Dependent Manner ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Acetoxymethyl Ester ◽  
Malaria Parasites ◽  
Subcellular Compartment ◽  
Calcium Indicator ◽  
Thiol Reagent ◽  
Acidic Compartment ◽  
Acidic Organelles

Plasmodium berghei trophozoites were loaded with the fluorescent calcium indicator, fura-2 acetoxymethyl ester, to measure their intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i was increased in the presence of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin. Trophozoites also possess a significant amount of Ca2+ stored in an acidic compartment. This was indicated by: (1) the increase in [Ca2+]i induced by bafilomycin A1, nigericin, monensin, or the weak base, NH4Cl, in the nominal absence of extracellular Ca2+, and (2) the effect of ionomycin, which cannot take Ca2+ out of acidic organelles and was more effective after alkalinization of this compartment by addition of bafilomycin A1, nigericin, monensin, or NH4Cl. Inorganic PPi promoted the acidification of a subcellular compartment in cell homogenates of trophozoites. The proton gradient driven by PPi collapsed by addition of the K+/H+ exchanger, nigericin, and eliminated by the PPi analogue, aminomethylenediphosphonate (AMDP). Both PPi hydrolysis and proton transport were dependent upon K+, and Na+ caused partial inhibition of these activities. PPi hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, sodium fluoride, dicyclohexylcarbodi-imide and to the thiol reagent, N-ethylmaleimide. Immunofluorescence microscopy using antibodies raised against conserved peptide sequences of a plant vacuolar pyrophosphatase (V-H+-PPase) suggested that the proton pyrophosphatase is located in intracellular vacuoles and the plasma membrane of trophozoites. AMDP caused an increase in [Ca2+]i in the nominal absence of extracellular Ca2+. Ionomycin was more effective in releasing Ca2+ from this acidic intracellular compartment after treatment of the cells with AMDP. Taken together, these results suggest the presence in malaria parasites of acidocalcisomes with similar characteristics to those described in trypanosomatids and Toxoplasma gondii, and the colocalization of the V-H+-PPase and V-H+-ATPase in these organelles.

scholarly journals In vitro Anti-parasitic Activity of Pelargonium X. asperum Essential Oil Against Toxoplasma gondii

2021 ◽  
Vol 9 ◽  
Author(s):  
Si-Yang Huang ◽  
Na Yao ◽  
Jia-Kang He ◽  
Ming Pan ◽  
Zhao-Feng Hou ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Essential Oils ◽  
Detrimental Effect ◽  
Dependent Manner ◽  
Cupressus Sempervirens ◽  
Membrane Surfaces ◽  
Limited Effectiveness ◽  
Parasitic Activity ◽  
Dose Dependent

Toxoplasmosis is a global zoonotic disease, and one-third of the human population is chronically infected by Toxoplasma gondii. Due to the limited effectiveness and prominent side effects of the existing drugs, there is a dire need for the discovery of new therapeutic options in the treatment of toxoplasmosis. In this study, five essential oils (EO) were screened for their anti-parasitic activity against T. gondii. The cytotoxicity of essential oils was evaluated using the MTT assay on human foreskin fibroblast cells. The CC50 values of Eucalyptus globulus EO, Cupressus sempervirens EO, Citrus aurantifolia EO, Melaleuca alternifolia EO, and Pelargonium X. asperum (Pa) EO were found to be 22.74, 7.25, 15.01, 6.26, and 4.77 mg/mL, respectively. Only PaEO exhibited anti-parasitic activity, and inhibited the growth of T. gondii in a dose-dependent manner. In addition, treatment with PaEO, was found to reduce the volume of T. gondii tachyzoites and make their membrane surfaces rough. These results showed that PaEO was able to inhibit the growth of T. gondii by reducing invasion, which may be due to its detrimental effect on the ability of tachyzoites to move. These findings suggest that PaEO could be a potential anti-T. gondii drug, which may facilitate the development of new and effective treatments against toxoplasmosis.

scholarly journals Limonoid Triterpene, Obacunone Increases Runt-Related Transcription Factor 2 to Promote Osteoblast Differentiation and Function

2021 ◽  
Vol 22 (5) ◽  
pp. 2483
Author(s):  
Kyung-Ran Park ◽  
SooHyun Kim ◽  
MyoungLae Cho ◽  
Hyung-Mun Yun
Keyword(s):  
Transcription Factor ◽  
Osteoblast Differentiation ◽  
Bone Diseases ◽  
Nuclear Accumulation ◽  
Root Bark ◽  
Dependent Manner ◽  
Matrix Mineralization ◽  
Related Transcription Factor ◽  
Anti Inflammatory ◽  
Dose Dependent

Root bark of Dictamnus dasycarpus Turcz. has been widely used as a traditional medicine and is a well-known anti-inflammatory agent. We isolated limonoid triterpene, obacunone (Obac) from the dried root bark of D. dasycarpus. Obac has been reported to exhibit varieties of biological activities including anti-inflammatory, anti-cancer, and anti-oxidant effects. This study aimed to investigate the beneficial effects and biological mechanisms of Obac in osteoblast differentiation and bone matrix mineralization. In the present study, Obac at concentrations ranging from 1 to 100 μM showed no proliferation effects in MC3T3-E1. The treatment of Obac (1 and 10 μM) increased wound healing and migration rates in a dose-dependent manner. Alkaline phosphatase (ALP) staining and activity showed that Obac (1 and 10 μM) enhanced early osteoblast differentiation in a dose-dependent manner. Obac also increased late osteoblast differentiation in a dose-dependent manner, as indicated by the mineralized nodule formation of ARS staining. The effects of Obac on osteoblast differentiation was validated by the levels of mRNAs encoding the bone differentiation markers, including Alp, bone sialoprotein (Bsp), osteopontin (Opn), and osteocalcin (Ocn). Obac increased the expression of bone morphogenetic protein (BMP), and the phosphorylation of smad1/5/8, and the expression of runt-related transcription factor 2 (RUNX2); Obac also inhibited GSK3β and upregulated the protein level of β-catenin in a dose-dependent manner during osteoblast differentiation. Obac-mediated osteoblast differentiation was attenuated by a BMP2 inhibitor, Noggin and a Wnt/β-catenin inhibitor, Dickkopf-1 (Dkk1) with the abolishment of RUNX2 expression and nuclear accumulation by Obac. Taken together, the findings of this study demonstrate that Obac has pharmacological and biological activates to promote osteoblast differentiation and bone mineralization through BMP2, β-catenin, and RUNX2 pathways, and suggest that Obac might be a therapeutic effect for the treatment and prevention of bone diseases such as osteoporosis and periodontitis.

Down-Regulation of Melanin Synthesis and Transfer by Paeonol and Its Mechanisms

2007 ◽  
Vol 35 (01) ◽  
pp. 139-151 ◽  
Author(s):  
Shi-Hai Xie ◽  
Zhi-Qiang Chen ◽  
Peng-Cheng Ma
Keyword(s):  
Dependent Manner ◽  
Melanin Synthesis ◽  
Down Regulation ◽  
Culture Cells ◽  
Pigmentary Disorders ◽  
Human Melanocytes ◽  
Key Enzyme ◽  
Moutan Cortex ◽  
Direct Inhibitors ◽  
Dose Dependent

Down-regulation of melanin synthesis and\or melanin transfer are\is required for recovery of pigmentary disorders. It is known that direct inhibitors of tyrosinase, the key enzyme in melanin synthesis, such as hydroquinone with a phenol structure, suppress melanin synthesis. We screened some herbal monomers using human melanocytes and found that paeonol, a major phenolic component of Moutan Cortex, down-regulated melanin synthesis. The melanin synthesis and tyrosinase activity were inhibited by paeonol in a dose-dependent manner. The expression levels of tyrosinase mRNA and protein were also reduced by paeonol. We further studied the inhibitory effects of paeonol on melanin transfer in co-culture of melanocytes and keratinocytes. More than 50% of inhibition of melanin transfer was observed at concentration of 200 μM of paeonol and the increased melanin transfer induced by SLIGRL, the PAR-2 activating peptide, was also reduced by paeonol. However, paeonol did not influence the expression level of PAR-2 mRNA in co-culture cells. These results indicate that the depigmenting effect of paeonol might be due to its down-regulation of melanogenesis and melanin transfer.

Effects of Low Molecular Weight Heparin and Unfragmented Heparin on Induction of Osteoporosis in Rats

1990 ◽  
Vol 63 (03) ◽  
pp. 505-509 ◽  
Author(s):  
Thomas Mätzsch ◽  
David Bergqvist ◽  
Ulla Hedner ◽  
Bo Nilsson ◽  
Per Østergaar
Keyword(s):  
Molecular Weight ◽  
Bone Mineral ◽  
Low Molecular Weight Heparin ◽  
Zinc Content ◽  
Low Molecular Weight ◽  
Dependent Manner ◽  
Bone Mineral Mass ◽  
Bone Ash ◽  
Dose Dependent ◽  
Mineral Mass

SummaryA comparison between the effect of low molecular weight heparin (LMWH) and unfragmented heparin (UH) on induction of osteoporosis was made in 60 rats treated with either UH (2 IU/ g b w), LMWH in 2 doses (2 Xal U/g or 0.4 Xal U/g) or placebo (saline) for 34 days. Studied variables were: bone mineral mass in femora; fragility of humera; zinc and calcium levels in serum and bone ash and albumin in plasma. A significant reduction in bone mineral mass was found in all heparin-treated rats. There was no difference between UH and LMWH in this respect. The effect was dose-dependent in LMWH-treated animals. The zinc contents in bone ash were decreased in all heparin-treated rats as compared with controls. No recognizable pattern was seen in alterations of zinc or calcium in serum. The fragility of the humera, tested as breaking strength did not differ between treatment groups and controls. In conclusion, if dosed according to similar factor Xa inhibitory activities, LMWH induces osteoporosis to the same extent as UH and in a dose-dependent manner. The zinc content in bone ash was decreased after heparin treatment, irrespective of type of heparin given.

Effects of NO-Donors on Thrombus Formation and Microcirculation in Cerebral Vessels of the Rat

1996 ◽  
Vol 76 (01) ◽  
pp. 111-117 ◽  
Author(s):  
Yasuto Sasaki ◽  
Junji Seki ◽  
John C Giddings ◽  
Junichiro Yamamoto
Keyword(s):  
Blood Flow ◽  
Thrombus Formation ◽  
Dependent Manner ◽  
Red Cell ◽  
Cell Velocity ◽  
Red Cell Velocity ◽  
The Mean ◽  
Wall Shear ◽  
Dose Dependent

SummarySodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), are known to liberate nitric oxide (NO). In this study the effects of SNP and SIN-1 on thrombus formation in rat cerebral arterioles and venules in vivo were assessed using a helium-neon (He-Ne) laser. SNP infused at doses from 10 Μg/kg/h significantly inhibited thrombus formation in a dose dependent manner. This inhibition of thrombus formation was suppressed by methylene blue. SIN-1 at a dose of 100 Μg/kg/h also demonstrated a significant antithrombotic effect. Moreover, treatment with SNP increased vessel diameter in a dose dependent manner and enhanced the mean red cell velocity measured with a fiber-optic laser-Doppler anemometer microscope (FLDAM). Blood flow, calculated from the mean red cell velocity and vessel diameters was increased significantly during infusion. In contrast, mean wall shear rates in the arterioles and venules were not changed by SNP infusion. The results indicated that SNP and SIN-1 possessed potent antithrombotic activities, whilst SNP increased cerebral blood flow without changing wall shear rate. The findings suggest that the NO released by SNP and SIN-1 may be beneficial for the treatment and protection of cerebral infarction

Melatonin actions in the heart; more than a hormone

2018 ◽  
Vol 1 (1) ◽  
pp. 21-26 ◽  
Author(s):  
Darío Acuña-Castroviejo ◽  
Maria T Noguiera-Navarro ◽  
Russel J Reiter ◽  
Germaine Escames
Keyword(s):  
Cell Membranes ◽  
Myocardial Cell ◽  
Rat Tissues ◽  
Dependent Manner ◽  
Broad Distribution ◽  
Multiple Organs ◽  
High Doses ◽  
Extrapineal Melatonin ◽  
Dose Dependent ◽  
Different Doses

Due to the broad distribution of extrapineal melatonin in multiple organs and tissues, we analyzed the presence and subcellular distribution of the indoleamine in the heart of rats. Groups of sham-operated and pinealectomized rats were sacrificed at different times along the day, and the melatonin content in myocardial cell membranes, cytosol, nuclei and mitochondria, were measured. Other groups of control animals were treated with different doses of melatonin to monitor its intracellular distribution. The results show that melatonin levels in the cell membrane, cytosol, nucleus, and mitochondria vary along the day, without showing a circadian rhythm. Pinealectomized animals trend to show higher values than sham-operated rats. Exogenous administration of melatonin yields its accumulation in a dose-dependent manner in all subcellular compartments analyzed, with maximal concentrations found in cell membranes at doses of 200 mg/kg bw melatonin. Interestingly, at dose of 40 mg/kg b.w, maximal concentration of melatonin was reached in the nucleus and mitochondrion. The results confirm previous data in other rat tissues including liver and brain, and support that melatonin is not uniformly distributed in the cell, whereas high doses of melatonin may be required for therapeutic purposes.

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