scholarly journals Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin N. Nelson ◽  
Savannah G. Beakley ◽  
Sierra Posey ◽  
Brittney Conn ◽  
Emma Maritz ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Cryptococcus Neoformans ◽  
Mammalian Cells ◽  
Cysteine Proteases ◽  
Dependent Manner ◽  
Life Threatening ◽  
Opportunistic Fungal Pathogen ◽  
Dose Dependent ◽  
Lysosomal Proteins

AbstractCryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

scholarly journals Proteolytic Cleavage of Cyclin E Leads to Inactivation of Associated Kinase Activity and Amplification of Apoptosis in Hematopoietic Cells

2002 ◽  
Vol 22 (7) ◽  
pp. 2398-2409 ◽  
Author(s):  
Suparna Mazumder ◽  
Bendi Gong ◽  
Quan Chen ◽  
Judith A. Drazba ◽  
Jeffrey C. Buchsbaum ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Cyclin E ◽  
Genotoxic Stress ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Dose Dependent

ABSTRACT Cyclin E/Cdk2 is a critical regulator of cell cycle progression from G1 to S in mammalian cells and has an established role in oncogenesis. Here we examined the role of deregulated cyclin E expression in apoptosis. The levels of p50-cyclin E initially increased, and this was followed by a decrease starting at 8 h after treatment with genotoxic stress agents, such as ionizing radiation. This pattern was mirrored by the cyclin E-Cdk2-associated kinase activity and a time-dependent expression of a novel p18-cyclin E. p18-cyclin E was induced during apoptosis triggered by multiple genotoxic stress agents in all hematopoietic tumor cell lines we have examined. The p18-cyclin E expression was prevented by Bcl-2 overexpression and by the general caspase and specific caspase 3 pharmacologic inhibitors zVAD-fluoromethyl ketone (zVAD-fmk) and N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), indicating that it was linked to apoptosis. A p18-cyclin E276-395 (where cyclin E276-395 is the cyclin E fragment containing residues 276 to 395) was reconstituted in vitro, with mutagenesis experiments, indicating that the caspase-dependent cleavage was at amino acid residues 272 to 275. Immunoprecipitation analyses of the ectopically expressed cyclin E1-275, cyclin E276-395 deletion mutants, and native p50-cyclin E demonstrated that caspase-mediated cyclin E cleavage eliminated interaction with Cdk2 and therefore inactivated the associated kinase activity. Overexpression of cyclin E276-395, but not of several other cyclin E mutants, specifically induced phosphatidylserine exposure and caspase activation in a dose-dependent manner, which were inhibited in Bcl-2-overexpressing cells or in the presence of zVAD-fmk. Apoptosis and generation of p18-cyclin E were significantly inhibited by overexpressing the cleavage-resistant cyclin E mutant, indicating a functional role for caspase-dependent proteolysis of cyclin E for apoptosis of hematopoietic tumor cells.

scholarly journals Regulation of ATP-induced calcium release in COS-7 cells by calcineurin

2000 ◽  
Vol 348 (1) ◽  
pp. 173-181 ◽  
Author(s):  
Arun BANDYOPADHYAY ◽  
Dong-Wook SHIN ◽  
Do Han KIM
Keyword(s):  
Mammalian Cells ◽  
Calcium Release ◽  
Calcineurin Inhibitors ◽  
Dependent Manner ◽  
Transfected Cells ◽  
High Level ◽  
Dose Dependent ◽  
Constitutively Active

Experiments were conducted to examine the role of calcineurin in regulating Ca2+ fluxes in mammalian cells. In COS-7 cells, increasing concentrations (1-10 μM) of ATP triggered intracellular Ca2+ release in a dose-dependent manner. Treatment of the cells with calcineurin inhibitors such as cyclosporin A (CsA), deltamethrin and FK506 resulted in an enhancement of ATP-induced intracellular Ca2+ release. Measurement of calcineurin-specific phosphatase activity in vitro demonstrated a high level of endogenous calcineurin activities in COS-7 cells, which was effectively inhibited by the addition of deltamethrin or CsA. The expression of constitutively active calcineurin (CnA∆CaMAI) inhibited the ATP-induced increase in intracellular Ca2+ concentration ([Ca2+]i), in both the presence and the absence of extracellular Ca2+. These results suggest that the constitutively active calcineurin prevented Ca2+ release from the intracellular stores. In the calcineurin-transfected cells, treatment with CsA restored the calcineurin-mediated inhibition of intracellular Ca2+ release. Protein kinase C-mediated phosphorylation of Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] was partly inhibited by the extracts prepared from the vector-transfected cells and completely inhibited by those from cells co-transfected with CnA∆CaMAI and calcineurin B. On the addition of 10 μM CsA, the inhibited phosphorylation of Ins(1,4,5)P3R was restored in both the vector-transfected cells and the calcineurin-transfected cells. These results show direct evidence that Ca2+ release through Ins(1,4,5)P3R in COS-7 cells is regulated by calcineurin-mediated dephosphorylation.

scholarly journals Strong Antifungal Activity of SS750, a New Triazole Derivative, Is Based on Its Selective Binding Affinity to Cytochrome P450 of Fungi

2002 ◽  
Vol 46 (2) ◽  
pp. 308-314 ◽  
Author(s):  
Masaru Matsumoto ◽  
Kazuya Ishida ◽  
Akihiro Konagai ◽  
Kazunori Maebashi ◽  
Takemitsu Asaoka
Keyword(s):  
Cytochrome P450 ◽  
Antifungal Activity ◽  
In Vivo Studies ◽  
Dependent Manner ◽  
Strong Affinity ◽  
Dose Dependent ◽  
Potent Inhibitory Activity ◽  
In Vitro Antifungal Activity

ABSTRACT SS750 [(R)-(−)-2-(2,4-difluorophenyl)-1-(ethylsulfonyl)-1,1-difluoro-3-(1H-1,2,4-triazol-1-yl)-2-propanol] is a new triazole, and its potential as an antifungal agent was evaluated by in vitro and in vivo studies. In a comparison of the MICs at which 50% of isolates are inhibited (MIC50s) for all strains of Candida species and Cryptococcus neoformans tested, SS750 was four times or more active than fluconazole and had activity comparable to that of itraconazole. The most important advantage of SS750 was that, when the MIC90s were compared, SS750 had 64 and 32 times greater antifungal activities than fluconazole against Candida krusei and Candida glabrata, respectively, which are intrinsically less susceptible to fluconazole. In cyclophosphamide-immunosuppressed mouse models of systemic and pulmonary candidiasis caused by C. albicans, oral SS750 prolonged the number of days of survival of infected animals in a dose-dependent manner and was 4 and ≥64 times more potent than fluconazole and itraconazole, respectively. In a safety profile, SS750, like fluconazole, had less of an affinity for binding to mammalian cytochrome P450 compared with that of ketoconazole, despite its strong affinity for binding to fungal cytochrome P450. The mechanism for the increased in vitro antifungal activity of SS750 against C. krusei is partially due to the potent inhibitory activity (3.7 times versus that of fluconazole) of C. krusei cytochrome P450 sterol 14α-demethylase; SS750 showed a strong affinity for binding to cytochrome P450 of C. krusei, indicating that SS750 acts by inhibiting the cytochrome P450 sterol 14α-demethylase of fungal cells.

scholarly journals Targeting TMPRSS2 and Cathepsin B/L Together May Be Synergistic Against SARS-CoV-2 Infection

2020 ◽  
Author(s):  
Pranesh Padmanabhan ◽  
Rajat Desikan ◽  
Narendra Dixit
Keyword(s):  
Cathepsin B ◽  
Protein S ◽  
Cysteine Proteases ◽  
Target Cells ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Protease Expression ◽  
Entry Efficiency

<p>The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry <i>in vitro</i>. This blockade may be achieved <i>in vivo</i> through ‘repurposing’ drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics <i>in vitro</i>. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available <i>in vitro</i> data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways <i>in vivo</i> may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry. <br></p>

scholarly journals Targeting TMPRSS2 and Cathepsin B/L Together May Be Synergistic Against SARS-CoV-2 Infection

2020 ◽  
Author(s):  
Pranesh Padmanabhan ◽  
Rajat Desikan ◽  
Narendra Dixit
Keyword(s):  
Cathepsin B ◽  
Protein S ◽  
Cysteine Proteases ◽  
Target Cells ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Protease Expression ◽  
Entry Efficiency

<p>The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry <i>in vitro</i>. This blockade may be achieved <i>in vivo</i> through ‘repurposing’ drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics <i>in vitro</i>. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available <i>in vitro</i> data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways <i>in vivo</i> may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry. <br></p>

scholarly journals Copper Acyl Salicylate Has Potential as an Anti-CryptococcusAntifungal Agent

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Adepemi O. Ogundeji ◽  
Boitumelo F. Porotloane ◽  
Carolina H. Pohl ◽  
Pravin S. Kendrekar ◽  
Olihile M. Sebolai
Keyword(s):  
Antifungal Activity ◽  
Metal Complex ◽  
Growth Inhibition ◽  
Metabolic Activity ◽  
Immune Cells ◽  
Dependent Manner ◽  
Content Type ◽  
Therapeutic Outcomes ◽  
Dose Dependent

ABSTRACTThein vitroantifungal activity of aspirin against cryptococcal cells has been reported. However, the unwanted effects of aspirin may limit its clinical application. Conceivably, a derivative of aspirin could overcome this challenge. Toward this end, this study considered the usage of an aspirinate-metal complex, namely, copper acyl salicylate (CAS), as an anti-Cryptococcusantifungal agent. Additionally, the study examined the effects of this compound on macrophage function. Thein vitrosusceptibility results revealed that cryptococcal cells were vulnerable (in a dose-dependent manner) to CAS, which might have effected growth inhibition by damaging cryptococcal cell membranes. Interestingly, when CAS was used in combination with fluconazole or amphotericin B, synergism was observed. Furthermore, CAS did not negatively affect the growth or metabolic activity of macrophages; rather, it sensitized those immune cells to produce interferon gamma and interleukin 6, which, in turn, might have aided in the phagocytosis of cryptococcal cells. Compared to our aspirin data, CAS was noted to be more effective in killing cryptococcal cells (based on susceptibility results) and less toxic toward macrophages (based on growth inhibition results). Taking these findings together, it is reasonable to conclude that CAS may be a better anti-Cryptococcusdrug that could deliver better therapeutic outcomes, compared to aspirin.

scholarly journals Targeting TMPRSS2 and Cathepsin B/L Together May Be Synergistic Against SARS-CoV-2 Infection

2020 ◽  
Author(s):  
Pranesh Padmanabhan ◽  
Rajat Desikan ◽  
Narendra Dixit
Keyword(s):  
Cathepsin B ◽  
Protein S ◽  
Cysteine Proteases ◽  
Target Cells ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Protease Expression ◽  
Entry Efficiency

<p>The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry <i>in vitro</i>. This blockade may be achieved <i>in vivo</i> through ‘repurposing’ existing drugs and offers a potential treatment option for COVID-19, currently in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics <i>in vitro</i>. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. We showed, analysing our model, that the synergy was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, we found that available <i>in vitro</i> data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Exploiting the synergy may improve the deployability of drug combinations targeting host proteases required for SARS-CoV-2 entry. </p>

scholarly journals Targeting TMPRSS2 and Cathepsin B/L together may be synergistic against SARS-CoV-2 infection

2020 ◽  
Vol 16 (12) ◽  
pp. e1008461
Author(s):  
Pranesh Padmanabhan ◽  
Rajat Desikan ◽  
Narendra M. Dixit
Keyword(s):  
Cathepsin B ◽  
Protein S ◽  
Cysteine Proteases ◽  
Target Cells ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Protease Expression ◽  
Entry Efficiency

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through ‘repurposing’ drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways in vivo may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry.

scholarly journals Cryptococcus neoformans Enters the Endolysosomal Pathway of Dendritic Cells and Is Killed by Lysosomal Components

2008 ◽  
Vol 76 (10) ◽  
pp. 4764-4771 ◽  
Author(s):  
Karen L. Wozniak ◽  
Stuart M. Levitz
Keyword(s):  
Dendritic Cells ◽  
Cryptococcus Neoformans ◽  
Human Monocyte ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Autologous Plasma ◽  
Murine Bone Marrow ◽  
Transmission Electron ◽  
Opportunistic Fungal Pathogen ◽  
Dose Dependent

ABSTRACT Cryptococcus neoformans is an opportunistic fungal pathogen that primarily causes disease in immunocompromised individuals. Dendritic cells (DCs) can phagocytose C. neoformans, present cryptococcal antigen, and kill C. neoformans. However, early events following C. neoformans phagocytosis by DCs are not well defined. We hypothesized that C. neoformans traffics to the endosome and the lysosome following phagocytosis by DCs and is eventually killed in the lysosome. Murine bone marrow-derived DCs (BMDCs) or human monocyte-derived DCs (HDCs) were incubated with live, encapsulated C. neoformans yeast cells and opsonizing antibody. Following incubation, DCs were intracellularly stained with antibodies against EEA1 (endosome) and LAMP-1 (late endosome/lysosome). As assessed by confocal microscopy, C. neoformans trafficked to endosomal compartments of DCs within 10 min and to lysosomal compartments within 30 min postincubation. For HDCs, the studies were repeated using complement-sufficient autologous plasma for the opsonization of C. neoformans. These data showed results similar to those for antibody opsonization, with C. neoformans localized to endosomes within 20 min and to lysosomes within 60 min postincubation. Additionally, the results of live real-time imaging studies demonstrated that C. neoformans entered lysosomal compartments within 20 min following the initiation of phagocytosis. The results of scanning and transmission electron microscopy demonstrated conventional zipper phagocytosis of C. neoformans by DCs. Finally, lysosomal extracts were purified from BMDCs and incubated with C. neoformans to determine their potential to kill C. neoformans. The extracts killed C. neoformans in a dose-dependent manner. This study shows that C. neoformans enters into endosomal and lysosomal pathways following DC phagocytosis and can be killed by lysosomal components.

Oxygenation of 18-hydroxycorticosterone as the final reaction for aldosterone biosynthesis

1984 ◽  
Vol 107 (3) ◽  
pp. 395-400 ◽  
Author(s):  
Itaru Kojima ◽  
Etsuro Ogata ◽  
Hiroshi Inano ◽  
Bun-ichi Tamaoki
Keyword(s):  
Carbon Monoxide ◽  
Hydroxysteroid Dehydrogenase ◽  
Dependent Manner ◽  
In Vitro Production ◽  
Mitochondrial Preparation ◽  
Final Reaction ◽  
Vitro Production ◽  
Dose Dependent ◽  
Cytochrome P 450

Abstract. Incubation of 18-hydroxycorticosterone with the sonicated mitochondrial preparation of bovine adrenal glomerulosa tissue leads to the production of aldosterone, as measured by radioimmunoassay. The in vitro production of aldosterone from 18-hydroxycorticosterone requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide. Cytochrome P-450 inhibitors such as metyrapone, SU 8000. SU 10603, SKF 525A, amphenone B and spironolactone decrease the biosynthesis of aldosterone from 18-hydroxycorticosterone. These results support the conclusion that the final reaction in aldosterone synthesis from 18-hydroxycorticosterone is catalyzed by an oxygenase, but not by 18-hydroxysteroid dehydrogenase. By the same preparation, the production of [3H]aldosterone but not [3H]18-hydroxycorticosterone from [1,2-3H ]corticosterone is decreased in a dose-dependent manner by addition of non-radioactive 18-hydroxycorticosterone.

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