Averrhoa carambola Leaf Extract Induces Apoptosis-Like Death with Increased ROS Generation in Leishmania donovani.

PURPOSE: The parasitic disease leishmaniasis is responsible for high mortality and morbidity rates worldwide. The visceral form is the most severe form of leishmaniasis (or leishmaniosis), which is caused predominantly by Leishmania donovani. Currently, clinically recommended antileishmanial drugs are not convenient because of several medical complications and resistance issues. Phytocompounds are the best candidates in this regard. The present study aimed to evaluate the antileishmanial activity of Averrhoa carambola leaf extract. METHODS: The antipromastigote activity and cytotoxicity were assessed using the MTT assay. Morphological distortions were determined using phase contrast microscopy and scanning electron microscopy (SEM). Reactive oxygen species (ROS) production, nonprotein thiol depletion and apoptotic death in promastigotes were determined via flow cytometry. UV-visible spectroscopy and energy dispersive X-ray (EDX) spectroscopy was performed for elemental analysis. Fourier-transform infrared spectroscopy (FTIR) and liquid chromatography‒mass spectrometry (LCMS) were used to characterize the phytocomponent(s) present in the extract. RESULTS: The chloroform extract of Averrhoa carambola leaf (ACCEX) (IC50 = 50.76 ± 1.7 µg/mL) exhibited the highest activity, followed by the ethyl acetate, hexane, and methanol extracts. ACCEX has also exhibited lower toxicity towards host macrophages. ACCEX also induced morphological distortions in promastigotes, with significant generation of ROS and the concomitant apoptosis initiation followed by a decrease in the nonprotein thiol level. The major phytometabolites present in ACCEX were identified from the National Institute of Standards and Technology (NIST) database and from a literature review. CONCLUSIONS: This study suggested that Averrhoa carambola leaf extracts are rich in some classes of biologically active phytocompounds and exhibit good antileishmanial activity.

Epigallocatechin Gallate with Potent Anti-Helicobacter pylori Activity Binds Efficiently to Its Histone-like DNA Binding Protein.

Helicobacter pylori (H. pylori)-a human gastric pathogen-forms a major risk factor for the development of various gastric pathologies such as chronic inflammatory gastritis, peptic ulcer, lymphomas of mucosa-associated lymphoid tissues, and gastric carcinoma. The complete eradication of infection is the primary objective of treating any H. pylori-associated gastric condition. However, declining eradication efficiencies, off-target effects, and patient noncompliance to prolong and broad-spectrum antibiotic treatments has spurred the clinical interest to search for alternative effective and safer therapeutic options. As natural compounds are safe and privileged with high levels of antibacterial-activity, previous studies have tested and reported a plethora of such compounds with potential in vitro/in vivo anti-H. pylori activity. However, the mode of action of majority of these natural compounds is unclear. The present study has been envisaged to compile the information of various such natural compounds and to evaluate their binding with histone-like DNA-binding proteins of H. pylori (referred here as Hup) using in silico molecular docking-based virtual screening experiments. Hup-being a major nucleoid-associated protein expressed by H. pylori-plays a strategic role in its survival and persistent colonization under hostile stress conditions. The ligand with highest binding energy with Hup-that is, epigallocatechin-(-)gallate (EGCG)-was rationally selected for further computational and experimental testing. The best docking poses of EGCG with Hup were first evaluated for their solution stability using long run molecular dynamics simulations and then using fluorescence and nuclear magnetic resonance titration experiments which demonstrated that the binding of EGCG with Hup is fairly strong (the resultant apparent dissociation constant (k D) values were equal to 2.61 and 3.29 ± 0.42 µM, respectively).

PKCζ-NADPH Oxidase-PKCα Dependent Kv1.5 Phosphorylation by Endothelin-1 Modulates Nav1.5-NCX1-Cav1.2 Axis in Stimulating Ca2+ Level in Caveolae of Pulmonary Artery Smooth Muscle Cells.

Endothelin-1 (ET-1) is a potent endogenously derived vasoconstrictor, which increases pulmonary hypertension via stimulation of [Ca2+]i level in pulmonary artery smooth muscle cells (PASMCs). In this communication, we sought to investigate the mechanism by which ET-1 causes stimulation of Ca2+ concentration in caveolae vesicles of bovine PASMCs (BPASMCs). ET-1 activates PKC-α in the caveolae vesicles by O2.- derived from PKCζ-NADPH oxidase dependent pathway. PKC-α phosphorylates Kv1.5 channels leading to a marked stimulation of Na+ and Ca2+ concentration in the caveolae vesicles. The stimulation of Ca2+ concentration in the caveolae vesicles by ET-1 occurs predominantly via Cav1.2 channels. Additionally, an increase in Na+ concentration by ET-1 due to stimulation of Nav1.5 channels marginally increases Ca2+ level in the caveolae vesicles via reverse-mode Na+/Ca2+ exchanger (NCX-1) and also through "slip-mode conductance" Nav1.5 channels. 4-AP, a well-known inhibitor of Kv channels, also increases Ca2+ concentration in the caveolae vesicles via Cav1.2 channels, reverse-mode NCX-1 and Nav1.5 channels by phosphorylation independent modulation of Kv1.5 channels without the involvement of PKCζ-NADPH oxidase-PKCα signaling axis. Overall, PKCζ-NADPH oxidase-PKCα dependent phosphorylation of Kv1.5 by ET-1 modulates Nav1.5-NCX1-Cav1.2 axis for stimulation of Ca2+ concentration in caveolae vesicles of BPASMCs, which provides a crucial mechanism for better understanding of ET-1-mediated modulation of pulmonary vascular tone.

Bioassay-based Corchorus capsularis L. leaf-derived ß-sitosterol exerts antileishmanial effects against Leishmania donovani by targeting trypanothione reductase.

Leishmaniasis, a major neglected tropical disease, affects millions of individuals worldwide. Among the various clinical forms, visceral leishmaniasis (VL) is the deadliest. Current antileishmanial drugs exhibit toxicity- and resistance-related issues. Therefore, advanced chemotherapeutic alternatives are in demand, and currently, plant sources are considered preferable choices. Our previous report has shown that the chloroform extract of Corchorus capsularis L. leaves exhibits a significant effect against Leishmania donovani promastigotes. In the current study, bioassay-guided fractionation results for Corchorus capsularis L. leaf-derived ß-sitosterol (ß-sitosterolCCL) were observed by spectroscopic analysis (FTIR, 1H NMR, 13C NMR and GC-MS). The inhibitory efficacy of this ß-sitosterolCCL against L. donovani promastigotes was measured (IC50 = 17.7 ± 0.43 µg/ml). ß-SitosterolCCL significantly disrupts the redox balance via intracellular ROS production, which triggers various apoptotic events, such as structural alteration, increased storage of lipid bodies, mitochondrial membrane depolarization, externalization of phosphatidylserine and non-protein thiol depletion, in promastigotes. Additionally, the antileishmanial activity of ß-sitosterolCCL was validated by enzyme inhibition and an in silico study in which ß-sitosterolCCL was found to inhibit Leishmania donovani trypanothione reductase (LdTryR). Overall, ß-sitosterolCCL appears to be a novel inhibitor of LdTryR and might represent a successful approach for treatment of VL in the future.

Curative efficacy of purified serine protease inhibitor PTF3 from potato tuber in experimental visceral leishmaniasis.

To overcome the drug toxicity and frequent resistance of parasites against the conventional drugs for the healing of human visceral leishmaniasis, innovative plant derived antileishmanial components are very imperative. Fuelled by the complications of clinically available antileishmanial drugs, a novel potato serine protease inhibitor was identified with its efficacy on experimental visceral leishmaniasis (VL). The serine protease inhibitors from potato tuber extract (PTEx) bearing molecular mass of 39 kDa (PTF1), 23 kDa (PTF2) and 17 kDa (PTF3) were purified and identified. Among them, PTF3 was selected as the most active inhibitor (IC50 143.5 ± 2.4 µg/ml) regarding its antileishmanial property. Again, intracellular amastigote load was reduced upto 83.1 ± 1.7% in pre-treated parasite and 88.5 ± 0.5% in in vivo model with effective dose of PTF3. Protective immune response by PTF3 was noted with increased production of antimicrobial substances and up-regulation of pro-inflammatory cytokines. Therapeutic potency of PTF3 is also followed by 80% survival in infected hamster. The peptide mass fingerprint (MALDI-TOF) results showed similarity of PTF3 with serine protease inhibitors database. Altogether, these results strongly propose the effectiveness of PTF3 as potent immunomodulatory therapeutics for controlling VL.

Exquisite binding interaction of 18ß-Glycyrrhetinic acid with histone like DNA binding protein of Helicobacter pylori: A computational and experimental study.

The Histone-like DNA binding protein is one of the most abundant nucleoid associated protein expressed by human gastric-pathogen, Helicobacter pylori (H. pylori). The protein -referred here as Hup- has been recognized as a potential drug target for developing therapeutic strategies against H. pylori. However, no attempts have been made, so far, to perturb the functioning of Hup through small molecules. As a first step in this direction, we virtually screened a natural product library containing 56 drug-like bioactive compounds and rationally selected 18ß-Glycyrrhetinic acid (GrA) for further computational and experimental testing of its binding interaction with Hup at the molecular level. The binding modes for GrA-Hup complexes were identified using in silico molecular docking methods and their solution dynamics and stability were evaluated using long run molecular dynamics simulations. Next, we experimentally demonstrated this binding interaction using fluorescence-quenching and ligand based NMR approaches. The fluorescence quenching and NMR titration experiments resulted into apparent dissociation constant (kD) for GrA-Hup binding equal to 87±12 µM and 36.6±1.5 µM, respectively. The various results demonstrate that GrA exhibits an exquisite binding interaction with Hup and would serve as an important molecular scaffold for developing next generation anti-H. pylori agents.

Role of PKCζ-NADPH oxidase signaling axis in PKCα-mediated Giα2 phosphorylation for inhibition of adenylate cyclase activity by angiotensin II in pulmonary artery smooth muscle cells.

We sought to determine the mechanism by which angiotensin II (AngII) inhibits isoproterenol induced increase in adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) production in bovine pulmonary artery smooth muscle cells (BPASMCs). Treatment with AngII stimulates protein kinase C-ζ (PKC-ζ), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and PKC-α activities, and also inhibits isoproterenol induced increase in AC activity and cAMP production in the cells. Pertussis toxin pretreatment eliminates AngII caused inhibition of isoproterenol induced increase in AC activity without a discernible change in PKC-ζ, NADPH oxidase, and PKC-α activities. Treatment of the cells with AngII increases α2 isoform of Gi (Giα2) phosphorylation; while pretreatment with chemical and genetic inhibitors of PKC-ζ and NADPH oxidase attenuate AngII induced increase in PKC-α activity and Giα2 phosphorylation, and also reverse AngII caused inhibition of isoproterenol induced increase in AC activity. Pretreatment of the cells with chemical and genetic inhibitors of PKC-α attenuate AngII induced increase in Giα2 phosphorylation and inhibits isoproterenol induced increase in AC activity without a discernible change in PKC-ζ and NADPH oxidase activities. Overall, PKCζ-NADPH oxidase-PKCα signaling axis plays a crucial role in Giα2 phosphorylation resulting in AngII-mediated inhibition of isoproterenol induced increase in AC activity in BPASMCs.

Role of PLD-PKCζ signaling axis in p47phox phosphorylation for activation of NADPH oxidase by angiotensin II in pulmonary artery smooth muscle cells.

We sought to determine the mechanism by which angiotensin II (ANGII) stimulates NADPH oxidase-mediated superoxide (O2 .- ) production in bovine pulmonary artery smooth muscle cells (BPASMCs). ANGII-induced increase in phospholipase D (PLD) and NADPH oxidase activities were inhibited upon pretreatment of the cells with chemical and genetic inhibitors of PLD2, but not PLD1. Immunoblot study revealed that ANGII treatment of the cells markedly increases protein kinase C-α (PKC-α), -δ, -ε, and -ζ levels in the cell membrane. Pretreatment of the cells with chemical and genetic inhibitors of PKC-ζ, but not PKC-α, -δ, and -ε, attenuated ANGII-induced increase in NADPH oxidase activity without a discernible change in PLD activity. Transfection of the cells with p47phox small interfering RNA inhibited ANGII-induced increase in NADPH oxidase activity without a significant change in PLD activity. Pretreatment of the cells with the chemical and genetic inhibitors of PLD2 and PKC-ζ inhibited ANGII-induced p47phox phosphorylation and subsequently translocation from cytosol to the cell membrane, and also inhibited its association with p22phox (a component of membrane-associated NADPH oxidase). Overall, PLD-PKCζ-p47phox signaling axis plays a crucial role in ANGII-induced increase in NADPH oxidase-mediated O2 .- production in the cells.

White jute (Corchorus capsularis L.) leaf extract has potent leishmanicidal activity against Leishmania donovani.

In pursuit of effective, safe and affordable antileishmanial drugs, the current study was designed to explore Corchorus capsularis L. leaf extract (CCEx) as an effective leishmanicidal substitute against Leishmania donovani. The leaf extract displays potent antileishmanial activity against L. donovani promastigotes with an IC50 value of 79.00 ±â€¯0.3 µg/ml. CCEx also significantly induces intracellular reactive oxygen species (ROS) with a concomitant decrease in the level of non-protein thiols in virulent parasites. Additionally, CCEx treatment induces substantial morphological alterations in parasites. Moreover, reagent-based phytochemical analysis of the extract revealed the presence of various phytochemical constituents. Further study is underway to identify the bioactive component(s) or fraction(s) of CCEx through bioassay-guided fractionation.

Protective role of epigallocatechin-3-gallate in NADPH oxidase-MMP2-Spm-Cer-S1P signalling axis mediated ET-1 induced pulmonary artery smooth muscle cell proliferation.

The signalling pathway involving MMP-2 and sphingosine-1-phosphate (S1P) in endothelin-1 (ET-1) induced pulmonary artery smooth muscle cell (PASMC) proliferation is not clearly known. We, therefore, investigated the role of NADPH oxidase derived O2.--mediated modulation of MMP2-sphingomyeline-ceramide-S1P signalling axis in ET-1 induced increase in proliferation of PASMCs. Additionally, protective role of the tea cathechin, epigallocatechin-3-gallate (EGCG), if any, in this scenario has also been explored. ET-1 markedly increased NADPH oxidase and MMP-2 activities and proliferation of bovine pulmonary artery smooth muscle cells (BPASMCs). ET-1 also caused significant increase in sphingomyelinase (SMase) activity, ERK1/2 and sphingosine kinase (SPHK) phosphorylations, and S1P level in the cells. EGCG inhibited ET-1 induced increase in SMase activity, ERK1/2 and SPHK phosphorylations, S1P level and the SMC proliferation. EGCG also attenuated ET-1 induced activation of MMP-2 by inhibiting NADPH oxidase activity upon inhibiting the association of the NADPH oxidase components, p47phox and p67phox in the cell membrane. Molecular docking study revealed a marked binding affinity of p47phox with the galloyl group of EGCG. Overall, our study suggest that ET-1 induced proliferation of the PASMCs occurs via NADPH oxidase-MMP2- Spm- Cer-S1P signalling axis, and EGCG attenuates ET-1 induced increase in proliferation of the cells by inhibiting NADPH oxidase activity.

Drug Resistance in Protozoan Parasites: An Incessant Wrestle for Survival.

Nowadays, drug resistance in parasites is considered to be one of the foremost concerns in health and disease management. It is interconnected worldwide and undermines the health of millions of people, threatening to grow worse. Unfortunately, it does not receive serious attention from every corner of society. Consequently, drug resistance in parasites is gradually complicating and challenging the treatment of parasitic diseases. In this context, we have dedicated ourselves to review the incidence of drug resistance in the protozoan parasites Plasmodium, Leishmania, Trypanosoma, Entamoeba and Toxoplasma gondii. Moreover, understanding the role of ATP-binding cassette (ABC) transporters in drug resistance is essential in the control of parasitic diseases. Therefore, we also focused on the involvement of ABC transporters in drug resistance, which will be a superior approach to find ways for better regulation of diseases caused by parasitic infections.

Serine protease inhibitors rich Coccinia grandis (L.) Voigt leaf extract induces protective immune responses in murine visceral leishmaniasis.

Leishmaniasis is a parasite-mediated tropical disease affecting millions of individuals worldwide. The available antileishmanial chemotherapeutic modalities exhibit adverse toxicity, exorbitant price and advent of drug-resistant parasites. Hence, plant-derived products are an alternative preference for the emergence of novel and effective antileishmanial agents that rejuvenate the host immunity with limited toxicity. The present work is complementary to our previous report that revealed the in vitro antileishmanial and immunomodulatory activity of Coccinia grandis (L.) Voigt leaf extract (Cg-Ex) rich in serine protease inhibitors. Thus, preliminary objectives of the study were to elucidate the leishmanicidal activity and host effector mechanism in Leishmania donovani infected BALB/c mice treated with Cg-Ex. Oral administration of Cg-Ex significantly reduced the spleen and liver parasite burden at dose-dependently. The parasite elimination was associated with generation of ROS and NO that are interrelated with up-regulation of disease-suppressing Th1 cytokines and down-regulation of disease-promoting Th2 cytokines at both protein and mRNA level. Moreover, Cg-Ex augmented the delayed-type hypersensitivity (DTH) response and serum IgG2a level which are correlated with the diminution of parasite burden with no hepatic and renal toxicity. Additionally, histological analysis of spleen depicted the improvement of structural disorganization of white and red pulp after Cg-Ex treatment. Therefore, our intriguing findings have presented the first indication of in vivo antileishmanial efficacy through activation of pro-inflammatory immune responses of the host by a natural plant leaf extract (Cg-Ex) containing serine protease inhibitors which could have a role as a potential immunomodulator against visceral leishmaniasis.

Functional attribution of LdISP, an endogenous serine protease inhibitor from Leishmania donovani in promoting infection.

Leishmaniasis, a parasitic disease caused by unicellular eukaryotic protozoa of the genus Leishmania, affects more than 12 million people worldwide. Events of leishmaniasis are based on the infection of the mammalian host, precisely macrophages, where both host and parasite derived proteases and endogenous inhibitors are significant. Pathogen derived protease inhibitors have generated considerable interest as they often act as an agent promoting infection and parasitic survivability. An endogenous serine protease inhibitor from Indian strain of Leishmania donovani was previously identified by our group and named as LdISP. It has been found to inhibit neutrophil elastase (NE), responsible for natural inflammation process. However, LdISP's role in progression of infection or the proteomics based structural exposition has not been explored. The present study is aimed to localize and validate the potential role of LdISP in infectivity. We found that LdISP localized endogenously and treatment of infected host cells with LdISP curbs ROS and NO production. Additionally, in silico studies are carried out to predict the putative amino acid residues of LdISP involved in the inhibition process. Taken together, our results demonstrate that LdISP eventually exerts a pronounced role in L. donovani infection.

Role of curcumin in PLD activation by Arf6-cytohesin1 signaling axis in U46619-stimulated pulmonary artery smooth muscle cells.

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to produce phosphatidic acid (PA) which in some cell types play a pivotal role in agonist-induced increase in NADPH oxidase-derived [Formula: see text]production. Involvement of ADP ribosylation factor (Arf) in agonist-induced activation of PLD is known for smooth muscle cells of systemic arteries, but not in pulmonary artery smooth muscle cells (PASMCs). Additionally, role of cytohesin in this scenario is unknown in PASMCs. We, therefore, determined the involvement of Arf and cytohesin in U46619-induced stimulation of PLD in PASMCs, and the probable mechanism by which curcumin, a natural phenolic compound, inhibits the U46619 response. Treatment of PASMCs with U46619 stimulated PLD activity in the cell membrane, which was inhibited upon pretreatment with SQ29548 (Tp receptor antagonist), FIPI (PLD inhibitor), SecinH3 (inhibitor of cytohesins), and curcumin. Transfection of the cells with Tp, Arf-6, and cytohesin-1 siRNA inhibited U46619-induced activation of PLD. Upon treatment of the cells with U46619, Arf-6 and cytohesin-1 were translocated and associated in the cell membrane, which were not inhibited upon pretreatment of the cells with curcumin. Cytohesin-1 appeared to be necessary for in vitro binding of GTPγS with Arf-6; however, addition of curcumin inhibited binding of GTPγS with Arf-6 even in the presence of cytohesin-1. Our computational study suggests that although curcumin to some extent binds with Tp receptor, yet the inhibition of Arf6GDP to Arf6GTP conversion appeared to be an important mechanism by which curcumin inhibits U46619-induced increase in PLD activity in PASMCs.

Leishmania donovani serine protease encapsulated in liposome elicits protective immunity in experimental visceral leishmaniasis.

This study is aimed to evaluate the protective effect of L. donovani intracellular serine protease (SP-Ld) in combination with Freund's adjuvant and liposomal formulations against experimental visceral leishmaniasis (VL). The animals were immunized with SP-Ld in combination with adjuvant and evaluated for its immunogenicity and protective efficacy against Leishmania donovani. The infection was initially assessed by microscopic examination. Immunogenicity of SP-Ld was measured by detecting protease specific-IgG, IgG1 and IgG2a levels by ELISA. Cytokines levels were measured by ELISA and Reverse Transcription Polymerase Chain Reaction (RT-PCR). The vaccine efficacy of SP-Ld was also evaluated by measuring antibody response and survival potency in hamster model. SP-Ld vaccinated Balb/c mice resulted significant reduction of parasite burden with increased levels of IgG2a and decreased levels of IgG1. SP-Ld vaccination also induced Th1 type immune response with the rise of IL-12, IFN-γ and TNF-α with decreased levels of IL-10 and TGF-ß. Importantly, liposomal incorporated SP-Ld exerted better protection rather than in combination with Freund's adjuvant. Additionally, liposome encapsulated SP-Ld vaccinated hamsters continued to survive beyond 8 months against virulent L. donovani post challenge. Overall, these findings demonstrated SP-Ld as an effective immunogen which opens a new perspective for the generation of potential vaccine candidate against leishmaniasis.

Role of catechins on ET-1-induced stimulation of PLD and NADPH oxidase activities in pulmonary smooth muscle cells: determination of the probable mechanism by molecular docking studies.

The treatment of human pulmonary artery smooth muscle cells with ET-1 stimulates the activity of PLD and NADPH oxidase, but this stimulation is inhibited by pretreatment with bosentan (ET-1 receptor antagonist), FIPI (PLD inhibitor), apocynin (NADPH oxidase inhibitor), and EGCG and ECG (catechins having a galloyl group), but not EGC and EC (catechins devoid of a galloyl group). Herein, using molecular docking analyses based on our biochemical studies, we determined the probable mechanism by which the catechins containing a galloyl group inhibit the stimulation of PLD activity induced by ET-1. The ET-1-induced stimulation of PLD activity was inhibited by SecinH3 (inhibitor of cytohesin). Arf6 and cytohesin-1 are associated in the cell membrane, which is not inhibited by the catechins during ET-1 treatment of the cells. However, EGCG and ECG inhibited the binding of GTPγS with Arf6, even in the presence of cytohesin-1. The molecular docking analyses revealed that the catechins containing a galloyl group (EGCG and ECG) with cytohesin-1-Arf6GDP, but not the catechins without a galloyl group (EGC and EC), prevent GDP-GTP exchange in Arf6, which seems to be an important mechanism for inhibiting the activation of PLD induced by ET-1, and subsequently increases the activity of NADPH oxidase.

Role of ADP ribosylation factor6- Cytohesin1-PhospholipaseD signaling axis in U46619 induced activation of NADPH oxidase in pulmonary artery smooth muscle cell membrane.

Treatment of human pulmonary artery smooth muscle cells (HPASMCs) with the thromboxane A2 receptor antagonist, SQ29548 inhibited U46619 stimulation of phospholipase D (PLD) and NADPH oxidase activities in the cell membrane. Pretreatment with apocynin inhibited U46619 induced increase in NADPH oxidase activity. The cell membrane contains predominantly PLD2 along with PLD1 isoforms of PLD. Pretreatment with pharmacological and genetic inhibitors of PLD2, but not PLD1, attenuated U46619 stimulation of NADPH oxidase activity. U46619 stimulation of PLD and NADPH oxidase activities were insensitive to BFA and Clostridium botulinum C3 toxin; however, pretreatment with secinH3 inhibited U46619 induced increase in PLD and NADPH oxidase activities suggesting a major role of cytohesin in U46619-induced increase in PLD and NADPH oxidase activities. Arf-1, Arf-6, cytohesin-1 and cytohesin-2 were observed in the cytosolic fraction, but only Arf-6 and cytohesin-1 were translocated to the cell membrane upon treatment with U46619. Coimmunoprecipitation study showed association of Arf-6 with cytohesin-1 in the cell membrane fraction. In vitro binding of GTPγS with Arf-6 required the presence of cytohesin-1 and that occurs in BFA insensitive manner. Overall, BFA insensitive Arf6-cytohesin1 signaling axis plays a pivotal role in U46619-mediated activation of PLD leading to stimulation of NADPH oxidase activity in HPASMCs.

Coccinia grandis (L.) Voigt Leaf Extract Exhibits Antileishmanial Effect Through Pro-inflammatory Response: An In Vitro Study.

The conventional drugs used for the treatment of human visceral leishmaniasis have concerns about the toxicity and most importantly parasite resistance. To overcome these troubles, more efforts are made for the development of innovative therapeutic agents having effective antileishmanial activity and simultaneously stimulate adaptive immune system of host cells. Hence, search for new leishmanicidal from the natural origin like plants has shown its effectiveness for the treatment of this tropical disease. The aim of this study is to investigate and characterize the antileishmanial efficacy of Coccinia grandis (L.) Voigt leaf extract (Cg-Ex) with its immunomodulatory property against Leishmania donovani in an in vitro experimental model. Cg-Ex significantly reduces the intracellular L. donovani parasite load with IC50 value 193 ± 0.78 µg/ml, but it has lower cytotoxicity on the murine RAW 264.7 macrophage cell line. Interestingly, Cg-Ex induces the generation of potent antimicrobials like reactive oxygen species and nitric oxide dose dependently in infected murine macrophages. Moreover, the increased production of Th1 cytokines (IL-12, TNF-α) with a concurrent decrease of Th2 cytokines (IL-10, TGF-ß) was also observed in Cg-Ex-treated infected host macrophages. Our results thus confirm that serine protease inhibitor(s)-rich Cg-Ex exhibits antileishmanial activity in vitro, and this was mediated through the modulation of pro-inflammatory cytokines. On the whole, the present findings first demonstrate the antileishmanial property of Cg-Ex targeting the Leishmania serine protease resulting protection of host cells with Th1 cytokine expression. Thus, these data indicate that C. grandis leaf extract (Cg-Ex) might be considered as a new lead for designing alternative and novel natural therapeutic against visceral leishmaniasis.

Inhibition of pro-/active MMP-2 by green tea catechins and prediction of their interaction by molecular docking studies.

Matrix metalloproteinases (MMPs) play a crucial role in developing different types of lung diseases, e.g., pulmonary arterial hypertension (PAH). Green tea polyphenolic catechins such as EGCG and ECG have been shown to ameliorate various types of diseases including PAH. Our present study revealed that among the four green tea catechins (EGCG, ECG, EC, and EGC), EGCG and ECG inhibit pro-/active MMP-2 activities in pulmonary artery smooth muscle cell (PASMC) culture supernatant. Based on the above, we investigated the interactions of pro-/active MMP-2 with the green tea catechins by computational methods. In silico analysis revealed a strong interaction of pro-/active MMP-2 with EGCG/ECG, and galloyl group has been observed to be responsible for this interaction. The in silico analysis corroborated our experimental observation that EGCG and ECG are active in preventing both the proMMP-2 and MMP-2 activities. Importantly, these two catechins appeared to be better inhibitors for proMMP-2 in comparison to MMP-2 as revealed by gelatin zymogram and also by molecular docking studies. In many type of cells, activation of proMMP-2 occurs via an increase in the level of MT1-MMP (MMP-14). We, therefore, determined the interactions of MT1-MMP with the green tea catechins by molecular docking analysis. The study revealed a strong interaction of MT1-MMP with EGCG/ECG, and galloyl group has been observed to be responsible for the interaction.

Inhibition of MMP-9 by green tea catechins and prediction of their interaction by molecular docking analysis.

Green tea polyphenolic catechins have been shown to prevent various types of diseases such as pulmonary hypertension (PAH), cancer and cardiac and neurological disorders. Matrix metalloproteinases (MMPs) play an important role in the development of PAH. The present study demonstrated that among the four green tea catechins (EGCG, ECG, EC and EGC), EGCG and ECG inhibit pro-/active MMP-9 activities in pulmonary artery smooth muscle cell culture supernatant. Based on the above, we investigated the interactions of pro-/active MMP-9 with the green tea catechins by computational methods. In silico molecular docking analysis revealed a strong interaction between pro-/active MMP-9 and EGCG/ECG, and galloyl group appears to be responsible for this enhanced interaction. The molecular docking studies corroborate our experimental observation that EGCG and ECG are mainly active in preventing both the proMMP-9 and MMP-9 activities.