scholarly journals Prodigiosins uncouple lysosomal vacuolar-type ATPase through promotion of H+/Cl− symport

1998 ◽  
Vol 334 (3) ◽  
pp. 731-741 ◽  
Author(s):  
Shoji OHKUMA ◽  
Tomohiko SATO ◽  
Masayuki OKAMOTO ◽  
Hidekazu MATSUYA ◽  
Kunizo ARAI ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Dependent Manner ◽  
Lysosomal Ph ◽  
Vacuolar Acidification ◽  
Ic50 Values ◽  
Atp Inhibition ◽  
Glycoprotein Processing ◽  
Type V ◽  
Micro Organisms ◽  
Lysosomal Acidification

We reported previously [Kataoka, Muroi, Ohkuma, Waritani, Magae, Takatsuki, Kondo, Yamasaki and Nagai (1995) FEBS Lett. 359, 53–59] that prodigiosin 25-C (one of the red pigments of the prodigiosin group produced by micro-organisms like Streptomycesand Serratia) uncoupled vacuolar H+-ATPase, inhibited vacuolar acidification and affected glycoprotein processing. In the present study we show that prodigiosin, metacycloprodigiosin and prodigiosin 25-C, all raise intralysosomal pH through inhibition of lysosomal acidification driven by vacuolar-type (V-)ATPase without inhibiting ATP hydrolysis in a dose-dependent manner with IC50 values of 30–120 pmol/mg of protein. The inhibition against lysosomal acidification was quick and reversible, showing kinetics of simple non-competitive (for ATP) inhibition. However, the prodigiosins neither raised the internal pH of isolated lysosomes nor showed ionophoric activity against H+ or K+ at concentrations where they strongly inhibited lysosomal acidification. They required Cl- for their acidification inhibitory activity even when driven in the presence of K+ and valinomycin, suggesting that their target is not anion (chloride) channel(s). In fact, the prodigiosins inhibited acidification of proteoliposomes devoid of anion channels that were reconstituted from lysosomal vacuolar-type (V-)ATPase and Escherichia coli phospholipids. However, they did not inhibit the formation of an inside-positive membrane potential driven by lysosomal V-ATPase. Instead, they caused quick reversal of acidified pH driven by lysosomal V-ATPase and, in acidic buffer, produced quick acidification of lysosomal pH, both only in the presence of Cl-. In addition, they induced swelling of liposomes and erythrocytes in iso-osmotic ammonium salt of chloride but not of gluconate, suggesting the promotion of Cl- entry by prodigiosins. These results suggest that prodigiosins facilitate the symport of H+ with Cl- (or exchange of OH- with Cl-) through lysosomal membranes, resulting in uncoupling of vacuolar H+-ATPase.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

Synthesis and Assessment of Novel Anti-chlamydial Benzylidene Acylhydrazides Derivatives

2018 ◽  
Vol 15 (1) ◽  
pp. 31-36 ◽  
Author(s):  
Xiaofeng Bao ◽  
Ying Xue ◽  
Chao Xia ◽  
Yin Lu ◽  
Ningjing Yang ◽  
...  
Keyword(s):  
Inhibitory Effect ◽  
Dependent Manner ◽  
Iron Starvation ◽  
Hydrochloride Salt ◽  
Obligate Intracellular ◽  
Biphasic Life Cycle ◽  
Ic50 Value ◽  
Ic50 Values ◽  
Dose Dependent ◽  
Better Than

Background: Chlamydiae, characterized by a unique biphasic life cycle, are a group of Gram-negative obligate intracellular bacterial pathogens responsible for diseases in a range of hosts including humans. Benzylidene acylhydrazide CF0001 could inhibit chlamydiae independent of iron starvation and T3SS inhibition. This finding promoted us to design and synthesize more benzylidene acylhydrazides to find novel anti-chlamydial agents. Methods: The carboxylic acids 1a-1d were coupled with Boc-hydrazide inpresence of EDCI and DMAP to obtain the intermediate 2a-2d in 60-62% yields. N-Boc deprotections were performed to obtain hydrazide hydrochloride salt 3a-3d. Nextly, the hydrazides were subjected to condensation with aldehydes to obtain benzylidene acylhydrazides 4a-4g in 30-52% yields in two steps. Results: Compound 4d exhibited best inhibitory effect on the formation and growth of chlamydial inclusions. The IC50 value of compound 4d for infectious progenies was 3.55 µM, better than 7.30 µM of CF0001. Conclusion: To find novel anti-chlamydial agents, we have designed and synthesized benzylidene acylhydrazides 4a-4g. Compounds 4a, 4d, 4g showed inhibitory activity on C. muridarum with the IC50 values from 3.55-12 µM. The 3,5-dibromo-4-hydroxyl substitutes on ring B are critical to keep their anti-chlamydial activity. Compound 4d inhibited C. muridarum in a dose-dependent manner without apparent cytotoxicity.

scholarly journals Targeting BMI-1 with PLGA–PEG nanoparticle-containing PTC209 modulates the behavior of human glioblastoma stem cells and cancer cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Elham Poonaki ◽  
Fatemeh Ariakia ◽  
Mohammad Jalili-Nik ◽  
Mehdi Shafiee Ardestani ◽  
Gholamhossein Tondro ◽  
...  
Keyword(s):  
Drug Loading ◽  
Polycomb Group ◽  
Dependent Manner ◽  
Migration Ability ◽  
Jnk Signaling ◽  
Human Glioblastoma ◽  
Core Member ◽  
Ic50 Values ◽  
Human Gbm ◽  
Dose Dependent

AbstractDespite advances in glioblastoma (GBM) treatments, current approaches have failed to improve the overall survival of patients. The oncogene BMI-1, a core member of the polycomb group proteins, is a potential novel therapeutic target for GBM. To enhance the efficacy and reduce the toxicity, PTC209, a BMI-1 inhibitor, was loaded into a PLGA–PEG nanoparticle conjugated with CD133 antibody (Nano-PTC209) and its effect on the behavior of human GBM stem-like cells (GSCs) and the human glioblastoma cell line (U87MG) was assessed. Nano-PTC209 has a diameter of ~ 75 nm with efficient drug loading and controlled release. The IC50 values of Nano-PTC209 for GSCs and U87MG cells were considerably lower than PTC209. Nano-PTC209 significantly decreased the viability of both GSCs and U87MG cells in a dose-dependent manner and caused a significant enhancement of apoptosis and p53 levels as well as inhibition of AKT and JNK signaling pathways. Furthermore, Nano-PTC209 significantly inhibited the migration ability, decreased the activity of metalloproteinase-2 and -9, and increased the generation of reactive oxygen species in both GSCs and U87MG cells. Our data indicate that PLGA–PEG nanoparticle conjugated with CD133 antibody could be an ideal nanocarrier to deliver PTC209 and effectively target BMI-1 for potential approaches in the treatment of GBM.

Cytotoxic Effects of Ferula Latisecta on Human Glioma U87 Cells

Drug Research ◽  
2019 ◽  
Vol 69 (12) ◽  
pp. 665-670 ◽  
Author(s):  
Mohammad Jalili-Nik ◽  
Hamed Sabri ◽  
Ehsan Zamiri ◽  
Mohammad Soukhtanloo ◽  
Mostafa Karimi Roshan ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Gelatin Zymography ◽  
Annexin V ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Induced Apoptosis ◽  
Natural Herb ◽  
First Time ◽  
U87 Cells

AbstractGlioblastoma multiforme (GBM) is the fatal type of astrocytic tumors with a survival rate of 12 months. The present study, for the first time, evaluated the cytotoxic impacts of Ferula latisecta (F. latisecta) hydroalcoholic extract on U87 GBM cell line. The MTT assay measured the cellular toxicity following 24- and 48 h treatment with various doses of F. latisecta (0–800 μg/mL). Apoptosis was evaluated by an Annexin V/propidium iodide (PI) staining 24 h after treatment by F. latisecta. Moreover, to determine the cellular metastasis of U87 cells, we used a gelatin zymography assay (matrix metalloproteinase [MMP]-2/-9 enzymatic activity). The outcomes showed that F. latisecta mitigated the viability of U87 cells in a concentration- and time-dependent manner with IC50 values of 145.3 and 192.3 μg/mL obtained for 24- and 48 h treatments, respectively. F. latisecta induced apoptosis in a concentration-dependent manner after 24 h. Also, MMP-9 activity was significantly decreased following 24 h after treatment concentration-dependently with no change in MMP-2 enzymatic activity. This study showed that F. latisecta induced cytotoxicity and apoptosis, and mitigated metastasis of U87 GBM cells. Hence, F. latisecta could be beneficial as a promising natural herb against GBM after further studies.

scholarly journals Biochemical and biological activity of arginine deiminase from Streptococcus pyogenes M22

2016 ◽  
Vol 94 (2) ◽  
pp. 129-137 ◽  
Author(s):  
Eleonora A. Starikova ◽  
Alexey V. Sokolov ◽  
Anna Yu. Vlasenko ◽  
Larisa A. Burova ◽  
Irina S. Freidlin ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Streptococcus Pyogenes ◽  
Group A Streptococcus ◽  
Bacterial Pathogen ◽  
Arginine Deiminase ◽  
Dependent Manner ◽  
Group A ◽  
Micro Organisms ◽  
Dose Dependent

Streptococcus pyogenes (group A Streptococcus; GAS) is an important gram-positive extracellular bacterial pathogen responsible for a number of suppurative infections. This micro-organism has developed complex virulence mechanisms to avoid the host’s defenses. We have previously reported that SDSC from GAS type M22 causes endothelial-cell dysfunction, and inhibits cell adhesion, migration, metabolism, and proliferation in a dose-dependent manner, without affecting cell viability. This work aimed to isolate and characterize a component from GAS type M22 supernatant that suppresses the proliferation of endothelial cells (EA.hy926). In the process of isolating a protein possessing antiproliferative activity we identified arginine deiminase (AD). Further study showed that this enzyme is most active at pH 6.8. Calculating Km and Vmax gave the values of 0.67 mmol·L–1 and 42 s−1, respectively. A distinctive feature of AD purified from GAS type M22 is that its optimum activity and the maximal rate of the catalytic process is close to neutral pH by comparison with enzymes from other micro-organisms. AD from GAS type M22 suppressed the proliferative activity of endothelial cells in a dose-dependent mode. At the same time, in the presence of AD, the proportion of cells in G0/G1 phase increased. When l-Arg was added at increasing concentrations to the culture medium containing AD (3 μg·mL–1), the enzyme’s capacity to inhibit cell proliferation became partially depressed. The proportion of cells in phases S/G2 increased concomitantly, although the cells did not fully recover their proliferation activity. This suggests that AD from GAS type M22 has potential for the suppression of excessive cell proliferation.

scholarly journals Anti-Inflammatory Activities of Compounds Isolated from the Rhizome of Anemarrhena asphodeloides

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2631 ◽  
Author(s):  
Zeyuan Wang ◽  
Jianfeng Cai ◽  
Qing Fu ◽  
Lingping Cheng ◽  
Lehao Wu ◽  
...  
Keyword(s):  
Nuclear Factor Κb ◽  
Microglial Cells ◽  
No Production ◽  
Dependent Manner ◽  
Platycodin D ◽  
Protein Levels ◽  
Chemical Structures ◽  
Ic50 Values ◽  
Anti Inflammatory ◽  
Anemarrhena Asphodeloides

Fifteen unreported compounds in Anemarrhena asphodeloides, iriflophene (3), hostaplantagineoside C (7), tuberoside G (8), spicatoside B (9), platycodin D (14), platycoside A (15), platycodin D2 (16), polygalacin D2 (17), platycodin D3 (18), isovitexin (20), vitexin (21), 3,4-dihydroxyallylbenzene-3-O-α-l-rhamnopyranosyl(1→6)-β-d-glucopyranoside (22), iryptophan (24), adenosine (25), α-d-Glucose monoallyl ether (26), together with eleven known compounds (1, 2, 4–6, 10–13, 19 and 23), were isolated from the rhizomes of Anemarrhena asphodeloides. The chemical structures of these compounds were characterized using HRMS and NMR. The anti-inflammatory activities of the compounds were evaluated by investigating their ability to inhibit LPS-induced NO production in N9 microglial cells. Timosaponin BIII (TBIII) and trans-hinokiresinol (t-HL) exhibited significant inhibitory effects on the NO production in a dose-dependent manner with IC50 values of 11.91 and 39.08 μM, respectively. Immunoblotting demonstrated that TBIII and t-HL suppressed NO production by inhibiting the expressions of iNOS in LPS-stimulated N9 microglial cells. Further results revealed that pretreatment of N9 microglial cells with TBIII and t-HL attenuated the LPS-induced expression tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) at mRNAs and protein levels. Moreover, the activation of nuclear factor-κB (NF-κB) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways were inhibited by TBIII and t-HL, respectively. Our findings indicate that the therapeutic implication of TBIII and t-HL for neurogenerative disease associated with neuroinflammation.

scholarly journals ATP-dependent thermostabilization of human P-glycoprotein (ABCB1) is blocked by modulators

2019 ◽  
Vol 476 (24) ◽  
pp. 3737-3750 ◽  
Author(s):  
Sabrina Lusvarghi ◽  
Suresh V. Ambudkar
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Dependent Manner ◽  
Deficient Mutant ◽  
Concentration Dependent Manner ◽  
Glycoprotein P ◽  
Binding Domains ◽  
P Glycoprotein

P-glycoprotein (P-gp), an ATP-binding cassette transporter associated with multidrug resistance in cancer cells, is capable of effluxing a number of xenobiotics as well as anticancer drugs. The transport of molecules through the transmembrane (TM) region of P-gp involves orchestrated conformational changes between inward-open and inward-closed forms, the details of which are still being worked out. Here, we assessed how the binding of transport substrates or modulators in the TM region and the binding of ATP to the nucleotide-binding domains (NBDs) affect the thermostability of P-gp in a membrane environment. P-gp stability after exposure at high temperatures (37–80°C) was assessed by measuring ATPase activity and loss of monomeric P-gp. Our results show that P-gp is significantly thermostabilized (>22°C higher IT50) by the binding of ATP under non-hydrolyzing conditions (in the absence of Mg2+). By using an ATP-binding-deficient mutant (Y401A) and a hydrolysis-deficient mutant (E556Q/E1201Q), we show that thermostabilization of P-gp requires binding of ATP to both NBDs and their dimerization. Additionally, we found that transport substrates do not affect the thermal stability of P-gp either in the absence or presence of ATP; in contrast, inhibitors of P-gp including tariquidar and zosuquidar prevent ATP-dependent thermostabilization in a concentration-dependent manner, by stabilizing the inward-open conformation. Altogether, our data suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing the ATP-dependent dimerization of the NBDs of P-gp.

LUMI-AGGREGOMETER STUDIES OF THE INITIAL ATP-SECRETION FROM COLLAGEN-ADHERENT PLATELETS

1987 ◽  
Author(s):  
R Malmgren
Keyword(s):  
Platelet Adhesion ◽  
Rank Order ◽  
Shape Change ◽  
Dense Granule ◽  
Dependent Manner ◽  
Equal Degree ◽  
Atp Secretion ◽  
Ic50 Values ◽  
Dose Dependent ◽  
Platelet Shape

We have earlier, with the use of a lumi-aggregometer and sub-aggregating doses of collagen (0.2-0.8 ug/ml PRP), been able to detect the initial, aspirin-insensitive secretion of ATP from the collagen-adherent platelets, and to correlate this secretion to the doses of collagen, and onset and degree of subsequent shape change of non-adherent platelets (Malmgren, Thromb Res 4:445, 1986). The present study shows, that 200 ATU of hirudin,which reduced near-maximal aggregation and ATP-secretion induced by high collagen doses (2.5 ug/ml PRP) from 3.35 ± 0.2 uM to 2.85 ± 0.1 uM, did neither reduce the secreted amount of ATP that were 82.5 ± 15 nM in control samples and 90 ± 27.5 nM in hirudin-treated samples, nor reduce platelet shape change when platelets were challenged with 0.31 ug collagen /ml PRP. (200 ATU hirudin completely abolished an equal degree of platelet shape change induced by 0.01 U thrombin). Assuming that 3 % of the platelets in PRP were actually adhering to the collagen fibrils, the secreted amount corresponds to 14.6 ±0.04 pmoles ATP/106adheringplatelets, amounts which closely represented 100 % of their dense granule content. The finding confirms that hirudin does not inhibit platelet adhesion and also indicates, that thrombin-mediated activation of secretory pathways appears not to be involved during the initial phase of platelet-collagen interactions.Dipyridamole (DPA) and dibutyryl cAMP (DBcAMP) inhibited ATP-secretion and platelet aggregation in a dose-dependent manner at high collagen concentrations, but only DBcAMP caused a dose-dependent reduction of ATP secretion (IC50 =10-4 M) induced by sub-aggregating doses of collagen. DPA was devoid of effect in this respect and thus did not inhibit platelet adhesion.Yohimbine, dihydroergotamine and phentolamine reduced ATP-secretion induced by sub-aggregating collagen doses in the mentioned rank order of potency, and with IC50 values in the micromolar range. Ketanserin, ritanserin and propranolol were devoid of effect. The findings suggest that the initial collagen-plate-let interaction involve alfareceptor-mediated mechanisms that may encompass adhesion, while DBcAMP probably interacts with secretory mechanisms connected to phosphatidylinositol turnover.

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