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1.
Bioorg Chem ; 141: 106890, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37783099

RESUMO

Conformational restriction was addressed towards the development of more selective and effective antileishmanial agents than currently used drugs for treatment of Leishmania donovani; the causative parasite of the fatal visceral leishmaniasis. Five types of cyclopentane-based conformationally restricted miltefosine analogs that were previously explored in literature as anticancer AKT-inhibitors were reprepared and repurposed as antileishmanial agents. Amongst, positions-1 and 2 cis-conformationally-restricted compound 1a and positions-2 and 3 trans-conformationally-restricted compound 3b were highly potent eliciting sub-micromolar IC50 values for inhibition of infection and inhibition of parasite number compared with the currently used miltefosine drug that showed low micromolar IC50 values for inhibition of infection and inhibition of parasite number. Compounds 1a and 3b eradicated the parasite without triggering host cells cytotoxicity over more than one log concentration interval which is a superior performance compared to miltefosine. In silico studies suggested that conformational restriction conserved the conformer capable of binding LdAKT-like kinase while it might be possible that it excludes other conformers mediating undesirable effects and/or toxicity of miltefosine. Together, this study presents compounds 1a and 3b as antileishmanial agents with superior performance over the currently used miltefosine drug.


Assuntos
Antiprotozoários , Leishmania donovani , Proteínas Proto-Oncogênicas c-akt , Ciclopentanos/farmacologia , Reposicionamento de Medicamentos , Antiprotozoários/química
2.
Biochem Biophys Res Commun ; 569: 193-198, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34256188

RESUMO

Visceral leishmaniasis (VL) is a fatal infectious disease caused by viscerotropic parasitic species of Leishmania. Current treatment options are often ineffective and toxic, and more importantly, there are no clinically validated drug targets available to develop next generation therapeutics against VL. Topoisomerase IB (TopIB) is an essential enzyme for Leishmania survival. The enzyme is organized as a bi-subunit that is distinct from the monomeric topoisomerase I of human. Based on this unique feature, we synthesized peptides composed of partial amino acid sequences of small subunit of Leishmania donovani (Ld) TopIB to confirm a decrease in catalytic activity by interfering the interaction between the two subunits. One of the synthetic peptides, covering essential amino acids for catalytic activity of LdTopIB, interrupted the enzymatic activity. Next, we examined 151 compounds selected from virtual screening in a functional assay and identified three LRL-TP compounds with a significant decrease in LdTopIB activity (IC50 of LRL-TP-85: 1.3 µM; LRL-TP-94: 2.9 µM; and LRL-TP-101: 35.3 µM) and no effects on Homo sapiens (Hs) TopIB activity. Based on molecular docking, the protonated tertiary amine of inhibitors formed key interactions with S415 of the large subunit. The EC50 values of LRL-TP-85, LRL-TP-94, and LRL-TP-101 were respectively 4.9, 1.4, and 27.8 µM in extracellular promastigote assay and 34.0, 53.7, and 11.4 µM in intracellular amastigote assay. Overall, we validated the protein-protein interaction site of LdTopIB as a potential drug target and identified small molecule inhibitors with anti-leishmanial activity.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , Leishmania donovani/enzimologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Inibidores da Topoisomerase I/farmacologia , Animais , Antiprotozoários/química , Antiprotozoários/farmacologia , Células Cultivadas , DNA/química , DNA/genética , DNA/metabolismo , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , Humanos , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/genética , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/prevenção & controle , Camundongos , Modelos Moleculares , Estrutura Molecular , Conformação de Ácido Nucleico , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Células THP-1 , Inibidores da Topoisomerase I/química
3.
Pathogens ; 9(5)2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32443883

RESUMO

Protozoan parasites of the genus Leishmania are the causative agents of leishmaniasis, a spectrum of a disease that threatens public health worldwide. Although next-generation therapeutics are urgently needed, the early stage of the drug discovery process is hampered by very low hit rates from intracellular Leishmania phenotypic high-throughput screenings. Designing and applying a physiologically relevant in vitro assay is therefore in high demand. In this study, we characterized the infectivity, morphology, and drug susceptibility of different Leishmania and host cell infection combinations. Primary bone marrow-derived macrophage (BMDM) and differentiated human acute monocytic leukemia (THP-1) cells were infected with amastigote or promastigote forms of Leishmania amazonensis and Leishmania donovani. Regardless of host cell types, amastigotes were generally well phagocytosed and showed high infectivity, whereas promastigotes, especially those of L. donovani, had predominantly remained in the extracellular space. In the drug susceptibility test, miltefosine and sodium stibogluconate (SSG) showed varying ranges of activity with 14 and >10-fold differences in susceptibility, depending on the host-parasite pairs, indicating the importance of assay conditions for evaluating antileishmanial activity. Overall, our results suggest that combinations of Leishmania species, infection forms, and host cells must be carefully optimized to evaluate the activity of potential therapeutic compounds against Leishmania.

4.
Cell Chem Biol ; 25(10): 1242-1254.e8, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30057298

RESUMO

Mechanisms underlying cancer cell death caused by inhibitors of subcellular Hsp70 proteins have been elucidated. An inhibitor of Hsp70, apoptozole (Az), is mainly translocated into lysosomes of cancer cells where it induces lysosomal membrane permeabilization, thereby promoting lysosome-mediated apoptosis. Additionally, Az impairs autophagy in cancer cells owing to its ability to disrupt the lysosomal function. However, the Az-triphenylphosphonium conjugate, Az-TPP-O3, localizes mainly to mitochondria of cancer cells where it inhibits the mortalin-p53 interaction and induces mitochondrial outer membrane permeabilization, consequently leading to mitochondria-mediated apoptosis. Unlike Az, Az-TPP-O3 does not have an effect on autophagy in cancer cells. Collectively, the findings indicate that inhibitors of lysosomal Hsp70 and mitochondrial mortalin enhance cancer cell death via distinctively different mechanisms. Additionally, the findings arising from this effort demonstrate that studies aimed at determining subcellular locations and functions of small-molecule modulators provide a deeper understanding of their modes of action in cells.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Lisossomos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Benzamidas/farmacologia , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Imidazóis/farmacologia , Lisossomos/metabolismo , Lisossomos/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Compostos Organofosforados/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia
5.
Sci Rep ; 5: 17642, 2015 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-26631605

RESUMO

The ATPase activities of Hsp70 and Hsc70 are known to be responsible for regulation of various biological processes. However, little is known about the roles of Hsp70 and Hsc70 in modulation of immune responses to antigens. In the present study, we investigated the effect of apoptozole (Az), a small molecule inhibitor of Hsp70 and Hsc70, on immune responses to protein antigens. The results show that mice administered with both protein antigen and Az produce more antibodies than those treated with antigen alone, showing that Az enhances immune responses to administered antigens. Treatment of mice with Az elicits production of antibodies with a high IgG2c/IgG1 ratio and stimulates the release of Th1 and Th2-type cytokines, suggesting that Az activates the Th1 and Th2 immune responses. The observations made in the present study suggest that inhibition of Hsp70 and Hsc70 activities could be a novel strategy designing small molecule-based adjuvants in protein vaccines.


Assuntos
Antígenos/imunologia , Benzamidas/imunologia , Benzamidas/farmacologia , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Imidazóis/imunologia , Imidazóis/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Animais , Benzamidas/química , Citocinas/sangue , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Proteínas de Choque Térmico HSC70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Hemocianinas/imunologia , Imidazóis/química , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia
6.
Chem Biol ; 22(3): 391-403, 2015 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-25772468

RESUMO

The heat shock protein HSP70 plays antiapoptotic and oncogenic roles, and thus its inhibition has been recognized as a potential avenue for anticancer therapy. Here we describe the small molecule, apoptozole (Az), which inhibits the ATPase activity of HSP70 by binding to its ATPase domain and, as a result, induces an array of apoptotic phenotypes in cancer cells. Affinity chromatography provides evidence that Az binds HSP70 but not other types of heat shock proteins including HSP40, HSP60, and HSP90. We also demonstrate that Az induces cancer cell death via caspase-dependent apoptosis by disrupting the interaction of HSP70 with APAF-1. Animal studies indicate that Az treatment retards tumor growth in a xenograft mouse model without affecting mouse viability. These studies suggest that Az will aid the development of new cancer therapies and serve as a chemical probe to gain a better understanding of the diverse functions of HSP70.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Imidazóis/farmacologia , Neoplasias/tratamento farmacológico , Adenosina Trifosfatases/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP70/efeitos dos fármacos , Células HeLa , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias/enzimologia , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Distribuição Aleatória , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Mol Biosyst ; 9(5): 978-86, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23385664

RESUMO

To date, many efforts have been made to detect lectins in cells by using single imaging techniques. However, only a few dual-labeled glycan-based probes, which integrate advantageous features of two imaging methods to enhance the visualization of biological processes associated with lectins in cells, have been reported. Herein we describe the synthesis of dual fluorescence and magnetic resonance imaging agent conjugated neoglycopeptides and their application in the simultaneous imaging of lectins in mammalian cells. The dual-labeled neoglycopeptides bind to lectins on cell surfaces and subsequently enter the cells via lectin-mediated endocytosis. The results of these efforts show that the novel dual-labeled neoglycopeptides are effective fluorescence and MR imaging agents for monitoring biological processes associated with lectins.


Assuntos
Membrana Celular/metabolismo , Endocitose , Glicoproteínas/metabolismo , Lectinas/metabolismo , Receptor de Asialoglicoproteína/química , Receptor de Asialoglicoproteína/metabolismo , Assialoglicoproteínas/química , Assialoglicoproteínas/metabolismo , Corantes Fluorescentes/química , Gadolínio/química , Glicoproteínas/síntese química , Glicoproteínas/química , Células Hep G2 , Compostos Heterocíclicos com 1 Anel/química , Humanos , Lectinas/química , Imageamento por Ressonância Magnética , Microscopia Confocal , Modelos Químicos , Estrutura Molecular , Ligação Proteica , Reprodutibilidade dos Testes , Rodaminas/química , Coloração e Rotulagem/métodos
8.
J Hazard Mater ; 209-210: 92-8, 2012 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-22301079

RESUMO

Although 1,2-dibromoethane (EDB) is a common groundwater contaminant, there is the lack of knowledge surrounding EDB biodegradation, especially under aerobic conditions. We have performed an extensive microcosm study to investigate the biodegradation of EDB under simulated in situ and biostimulated conditions. The materials for soil microcosms were collected from an EDB-contaminated aquifer at the Massachusetts Military Reservation in Cape Cod, MA. This EDB plume has persisted for nearly 40 years in both aerobic and anaerobic EDB zones of the aquifer. Microcosms were constructed under environmentally relevant conditions (field EDB and DO concentrations; incubated at 12°C). The results showed that natural attenuation occurred under anaerobic conditions but not under aerobic conditions, explaining why aerobic EDB contamination is so persistent. EDB degradation rates were greater under biostimulated conditions for both the aerobic and anaerobic microcosms. Particularly for aerobic biostimulation, methane-amended microcosms degraded EDB, on average, at a first order rate eight times faster than unamended microcosms. The best performing replicate achieved an EDB degradation rate of 7.0 yr(-1) (half-life (t(1/2))=0.10 yr). Residual methane concentrations and the emergence of methanotrophic bacteria, measured by culture independent bacterial analysis, provided strong indications that EDB degradation in aerobic methane-amended microcosms occurred via cometabolic degradation. These results indicate the potential for enhanced natural attenuation of EDB and that methane could be considered co-substrate for EDB bioremediation for the EDB-contaminated groundwater in aerobic zone.


Assuntos
Biodegradação Ambiental , Dibrometo de Etileno/metabolismo , Microbiologia da Água , Poluentes Químicos da Água/metabolismo , Aerobiose , Anaerobiose
9.
Chem Soc Rev ; 41(8): 3245-63, 2012 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-22293658

RESUMO

Autophagy or self-eating is a complicated cellular process that is involved in protein and organelle digestion occurring via a lysosome-dependent pathway. This process is of great importance in maintaining normal cellular homeostasis. However, disruption of autophagy is closely associated with various human diseases such as cancer, neurodegenerative disorders, heart disease and pathogen infection. Therefore, small molecules that modulate autophagy can be employed to dissect this complex process and ultimately could have high potential for the treatment of a variety of diseases. This critical review discusses general aspects of autophagy, autophagy-associated diseases and autophagy regulators for biological research and therapeutic applications (207 references).


Assuntos
Autofagia/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Doença , Avaliação Pré-Clínica de Medicamentos , Humanos
10.
J Am Chem Soc ; 133(50): 20267-76, 2011 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-22074182

RESUMO

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cell-surface anion channel that permeates chloride and bicarbonate ions. The most frequent mutation of CFTR that causes cystic fibrosis is the deletion of phenylalanine at position 508 (ΔF508), which leads to defects in protein folding and cellular trafficking to the plasma membrane. The lack of the cell-surface CFTR results in a reduction in the lifespan due to chronic lung infection with progressive deterioration of lung function. Hsc70 plays a crucial role in degradation of mutant CFTR by the ubiquitin-proteasome system. To date, various Hsc70 inhibitors and transcription regulators have been tested to determine whether they correct the defective activity of mutant CFTR. However, they exhibited limited or questionable effects on restoring the chloride channel activity in cystic fibrosis cells. Herein, we show that a small molecule apoptozole (Az) has high cellular potency to promote membrane trafficking of mutant CFTR and its chloride channel activity in cystic fibrosis cells. Results from affinity chromatography and ATPase activity assay indicate that Az inhibits the ATPase activity of Hsc70 by binding to its ATPase domain. In addition, a ligand-directed protein labeling and molecular modeling studies also suggest the binding of Az to an ATPase domain, in particular, an ATP-binding pocket. It is proposed that Az suppresses ubiquitination of ΔF508-CFTR maybe by blocking interaction of the mutant with Hsc70 and CHIP, and, as a consequence, it enhances membrane trafficking of the mutant.


Assuntos
Adenosina Trifosfatases/metabolismo , Benzamidas/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Imidazóis/metabolismo , Mutação , Adenosina Trifosfatases/química , Benzamidas/química , Sítios de Ligação , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Imidazóis/química , Espectroscopia de Ressonância Magnética , Transporte Proteico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ubiquitinação
11.
Chem Asian J ; 6(8): 2107-13, 2011 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21634013

RESUMO

A facile and efficient solid-phase synthesis of linear peptide-based glycoclusters with various valences and different spatial arrangements of the sugar ligands is described. The synthetic strategy includes 1) solid-phase synthesis of fluorophore-labeled, alkyne-containing peptides, 2) coupling of azide-linked, unprotected mono-, di-, and trisaccharides to the alkyne-conjugated peptides on a solid support by click chemistry, and 3) release of the fluorophore-labeled glycoclusters from the solid support. By using this methodology, 32 fluorescent glycoclusters with a valence ranging from 1 to 4 and different spatial arrangements of the sugar ligands were prepared. Lectin-binding properties of the glycoclusters were initially examined by using microarrays immobilized by various lectins. These glycoclusters were then employed to detect the cell-surface carbohydrate-binding proteins in bacteria. Finally, the uptake of glycoclusters by mammalian cells through receptor-mediated endocytosis was evaluated. The results, obtained from the in vitro and in vivo studies, indicate that the binding affinities toward immobilized and cell-surface proteins are highly dependent on the valence and spatial arrangements of the sugar ligands in glycoclusters.


Assuntos
Química Click/métodos , Corantes Fluorescentes/química , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Lectinas/metabolismo , Química Click/economia , Endocitose , Escherichia coli/química , Proteínas de Escherichia coli/análise , Glicopeptídeos/síntese química , Células Hep G2 , Humanos , Lactose/química , Lactose/metabolismo , Análise Serial de Proteínas , Ligação Proteica
12.
Mol Cells ; 31(6): 573-8, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21533547

RESUMO

During orthodontic tooth movement, local hypoxia and enhanced osteoclastogenesis are observed in the compression side of periodontal tissues. The receptor activator of nuclear factor-κB ligand (RANKL) is an osteoblast/stromal cell-derived factor that is essential for osteoclastogenesis. In this study, we examined the effect of hypoxia on RANKL expression in human periodontal ligament fibroblasts (PDLFs) to investigate the relationship between local hypoxia and enhanced osteoclastogenesis in the compression side of periodontal tissues. Hypoxia significantly enhanced the levels of RANKL mRNA and protein as well as hypoxia inducible factor-1α (HIF-1α) protein in PDLFs. Constitutively active HIF-1α alone significantly increased the levels of RANKL expression in PDLFs under normoxic conditions, whereas dominant negative HIF-1α blocked hypoxia-induced RANKL expression. To investigate further whether HIF-1α directly regulates RANKL transcription, a luciferase reporter assay was performed using the reporter vector containing the RANKL promoter sequence. Exposure to hypoxia or overexpression of constitutively active HIF-1α significantly increased RANKL promoter activity, whereas dominant negative HIF-1α blocked hypoxia-induced RANKL promoter activity. Furthermore, mutations of putative HIF-1α binding elements in RANKL promoter prevented hypoxia-induced RANKL promoter activity. The results of chromatin immunoprecipitation showed that hypoxia or constitutively active HIF-1α increased the DNA binding of HIF-1α to RANKL promoter. These results suggest that HIF-1α mediates hypoxia-induced up-regulation of RANKL expression and that in compression side periodontal ligament, hypoxia enhances osteoclastogenesis, at least in part, via an increased RANKL expression in PDLFs.


Assuntos
Fibroblastos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ligamento Periodontal/citologia , Ligante RANK/genética , Hipóxia Celular , Células Cultivadas , Imunoprecipitação da Cromatina , Genes Reporter , Humanos , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Ligamento Periodontal/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Ligante RANK/metabolismo , Transcrição Gênica , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
Biochem Biophys Res Commun ; 408(3): 399-404, 2011 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-21514273

RESUMO

Tumor necrosis factor-α (TNF-α) is known to suppress adipocyte differentiation via a ß-catenin-dependent pathway. However, the mechanisms by which TNF-α induces Wnt/ß-catenin signaling pathway in adipocytes is unclear. Msx2, a homeobox transcription factor, is known to increase osteoblast differentiation through activation of the Wnt/ß-catenin pathway. Therefore, in the present study, we investigated whether TNF-α activates the Wnt/ß-catenin signaling pathway via the induction of Msx2 expression in 3T3-L1 preadipocytes. We found that TNF-α transiently increased Msx2 expression as well as the expression of canonical Wnt signaling molecules, including Wnt3a, Wnt7a, Wnt7b, Wnt10b, low-density lipoprotein receptor-related protein 5 (LRP5) and T-cell factor 1 (TCF1). Furthermore, TNF-α increased ß-catenin/TCF-dependent transcriptional activity. To better understand the role of Msx2 in Wnt signaling, we examined the effects of Msx2 overexpression and knockdown on Wnt/ß-catenin signaling. Msx2 overexpression alone significantly increased the levels of Wnt3a, Wnt7a, Wnt7b, Wnt10b, LRP5 and TCF1 expression, whereas knockdown of Msx2 using small interfering RNA prevented TNF-α-induced expression of Wnt signaling molecules. Taken together, the results of this study indicate that TNF-α enhances the Wnt/ß-catenin signaling pathway by inducing Msx2 expression, which in turn suppresses adipocytic differentiation.


Assuntos
Adipócitos/metabolismo , Adipogenia , Proteínas de Homeodomínio/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
14.
Bone ; 49(2): 242-9, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21514407

RESUMO

Nuclear factor of activated T cell (NFAT) is a key transcription factor for receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation. However, it is unclear whether NFAT plays a role in the expression of RANKL in osteoblasts. High extracellular calcium ([Ca(2+)](o)) increases intracellular calcium, enhances RANKL expression in osteoblasts/stromal cells, and induces osteoclastogenesis in a coculture of osteoblasts and hematopoietic bone marrow cells. Because intracellular calcium signaling activates the calcineurin/NFAT pathway, we examined the role of NFAT activation on high [Ca(2+)](o)-induced RANKL expression in MC3T3-E1 subclone 4 (MC4) cells. Among the family of NFAT transcription factors, expression of NFATc1 and NFATc3, but not NFATc2, NFATc4 or NFAT5, was observed in MC4 cells. High [Ca(2+)](o) increased the expression levels of NFATc1, NFATc3 and RANKL. Cyclosporin A and FK506, inhibitors of calcineurin phosphatase, blocked high [Ca(2+)](o)-induced expression of NFAT and RANKL. Knockdown of NFATc1 and NFATc3 by siRNA prevented high [Ca(2+)](o)-induced RANKL expression, whereas overexpression of NFATc1 and NFATc3 induced RANKL expression. Furthermore, overexpressed NFATc1 upregulated NFATc3 expression, but NFATc1 knockdown decreased NFATc3 expression. Chromatin immunoprecipitation and reporter assay results showed that NFATc3, but not NFATc1, directly binds to the RANKL promoter and stimulates RANKL expression. In summary, these results demonstrate that high [Ca(2+)](o) increases expression of RANKL via activation of the calcineurin/NFAT pathway in osteoblasts. In addition, high [Ca(2+)](o) induces the activation and expression of NFATc1; NFATc3 expression and activity are subsequently increased; and NFATc3 directly binds to the RANKL promoter to increase its expression.


Assuntos
Cálcio/farmacologia , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Animais , Western Blotting , Calcineurina/genética , Calcineurina/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , Camundongos , Mutagênese Sítio-Dirigida , Fatores de Transcrição NFATC/genética , Ligante RANK/genética , Ligante RANK/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
J Microbiol Biotechnol ; 19(4): 339-45, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19420987

RESUMO

We evaluated the activity and abundance of the crude oil- degrading bacterium Nocardia sp. H17-1 during bioremediation of oil-contaminated soil, using real-time PCR. The total petroleum hydrocarbon (TPH) degradation rate constants (k) of the soils treated with and without H17-1 were 0.103 d-1 and 0.028 d-1, respectively. The degradation rate constant was 3.6 times higher in the soil with H17-1 than in the soil without H17-1. In order to detect and quantify the Nocardia sp. H17-1 in soil samples, we quantified the genes encoding 16S ribosomal RNA (16S rRNA), alkane monooxygenase (alkB4), and catechol 2,3-dioxygenase (23CAT) with real-time PCR using SYBR green. The amounts of H17-1 16S rRNA and alkB4 detected increased rapidly up to 1,000-folds for the first 10 days, and then continued to increase only slightly or leveled off. However, the abundance of the 23CAT gene detected in H17-1-treated soil, where H17-1 had neither the 23CAT gene for the degradation of aromatic hydrocarbons nor the catechol 2,3-dioxygenase activity, did not differ significantly from that of the untreated soil (alpha=0.05, p>0.22). These results indicated that H17-1 is a potential candidate for the bioaugmentation of alkane-contaminated soil. Overall, we evaluated the abundance and metabolic activity of the bioremediation strain H17-1 using real-time PCR, independent of cultivation.


Assuntos
Biodegradação Ambiental , Nocardia/crescimento & desenvolvimento , Petróleo/metabolismo , Reação em Cadeia da Polimerase/métodos , Poluentes do Solo/metabolismo , Análise de Variância , Contagem de Colônia Microbiana , Genes Bacterianos , Hidrocarbonetos/metabolismo , Cinética , Modelos Lineares , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Nocardia/enzimologia , Nocardia/genética , Nocardia/metabolismo , RNA Ribossômico 16S/genética
16.
J Microbiol Biotechnol ; 17(1): 67-73, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18051355

RESUMO

The present study compared the microbial diversity and activity during the application of various bioremediation processes to crude oil-contaminated soil. Five different treatments, including natural attenuation (NA), biostimulation (BS), biosurfactant addition (BE), bioaugmentation (BA), and a combined treatment (CT) of biostimulation, biosurfactant addition, and bioaugmentation, were used to analyze the degradation rate and microbial communities. After 120 days, the level of remaining hydrocarbons after all the treatments was similar, however, the highest rate (k) of total petroleum hydrocarbon (TPH) degradatioN was observed with the CT treatment (P < 0.05). The total bacterial counts increased during the first 2 weeks with all the treatments, and then remained stable. The bacterial communities and alkane monooxygenase gene fragment, alkB, were compared by denaturing gradient gel electrophoresis (DGGE). The DGGE analyses of the BA and CT treatments, which included Nocardia sp. H17-1, revealed a simple dominant population structure, compared with the other treatments. The Shannon-Weaver diversity index (H') and Simpson dominance index (D), calculated from the DGGE profiles using 16S rDNA, showed considerable qualitative differences in the community structure before and after the bioremediation treatment as well as between treatment conditions.


Assuntos
Biodegradação Ambiental , Petróleo/metabolismo , Microbiologia do Solo , Poluentes do Solo/metabolismo , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Sequência de Bases , Biodiversidade , Primers do DNA/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Ribossômico/genética , DNA Ribossômico/isolamento & purificação , Poluição Ambiental , Genes Bacterianos , Oxigenases de Função Mista/genética
17.
J Environ Biol ; 27(2 Suppl): 311-6, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-17436516

RESUMO

Phytotoxicity, microbial activity, plant uptake and microbial degradation were examined using Rumex crispus in TNT and/or cadmium contaminated columns (TNT: 100 mg/kg of soil and Cd: 10 mg/kg of soil). The growth of plants was significantly inhibited by TNT, but not by Cd. The microbial activity was highly increased by plant root growth, decreased by Cd, and slightly reduced by TNT. The plant uptake of Cd was relatively well in Cd-contaminated column, but lowered by TNT in TNT+Cd-contaminated column. The microbial degradation of TNT occurred much faster in planted columns than in unplanted columns with minor effect of Cd (less 2-ADNT was produced). Therefore, it may be effective to treat TNT first and then Cd using phytoremediation in the TNT plus Cd contaminated sites.


Assuntos
Cádmio/metabolismo , Recuperação e Remediação Ambiental/métodos , Plantas/metabolismo , Poluentes do Solo/metabolismo , Trinitrotolueno/metabolismo , Cádmio/toxicidade , Desenvolvimento Vegetal , Plantas/efeitos dos fármacos , Microbiologia do Solo , Poluentes do Solo/toxicidade , Trinitrotolueno/toxicidade
18.
Artigo em Inglês | MEDLINE | ID: mdl-15478936

RESUMO

The phytotoxic effects of crude oil and oil components on the growth of red beans (Phaseolus nipponesis OWH1) and corn (Zea mays) was investigated. In addition, the beneficial effects of bioremediation with the oil-degrading microorganism, Nocardia sp. H17-1, on corn and red bean growth in oil-contaminated soil was also determined. It was found that crude oil-contaminated soil (10,000mg/kg) was phytotoxic to corn and red beans. In contrast, obvious phytotoxicity was not observed in soils contaminated with 0-1000 mg/kg of aliphatic hydrocarbons such as decane (C10) and eicosane (C20). Phytotoxicity was observed in soils contaminated with 10-1000mg/kg of the poly aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, and pyrene. It was observed that phytotoxicity increased with the number of aromatic rings, and that corn was more sensitive than red beans to PAH-contaminated soil. Bioremediation with Nocardia sp. H17-1 reduced phytotoxicity more in corn than in red bean, suggesting that this microbial species might degrade PAHs to some degree.


Assuntos
Petróleo/toxicidade , Phaseolus/crescimento & desenvolvimento , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Poluentes do Solo/toxicidade , Zea mays/crescimento & desenvolvimento , Biodegradação Ambiental , Microbiologia do Solo
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