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1.
Annu Rev Biochem ; 79: 445-70, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20370423

RESUMO

Nitric oxide synthases (NOSs) are multidomain metalloproteins first identified in mammals as being responsible for the synthesis of the wide-spread signaling and protective agent nitric oxide (NO). Over the past 10 years, prokaryotic proteins that are homologous to animal NOSs have been identified and characterized, both in terms of enzymology and biological function. Despite some interesting differences in cofactor utilization and redox partners, the bacterial enzymes are in many ways similar to their mammalian NOS (mNOS) counterparts and, as such, have provided insight into the structural and catalytic properties of the NOS family. In particular, spectroscopic studies of thermostable bacterial NOSs have revealed key oxyheme intermediates involved in the oxidation of substrate L-arginine (Arg) to product NO. The biological functions of some bacterial NOSs have only more recently come to light. These studies disclose new roles for NO in biology, such as taking part in toxin biosynthesis, protection against oxidative stress, and regulation of recovery from radiation damage.


Assuntos
Bactérias/metabolismo , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/metabolismo , Animais , Arginina/metabolismo , Bactérias/enzimologia , Óxidos de Nitrogênio/metabolismo
2.
Anal Chem ; 2024 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-38321842

RESUMO

Raman spectroscopy is a popular process analytical technology (PAT) tool that has been increasingly used to monitor and control the monoclonal antibody (mAb) manufacturing process. Although it allows the characterization of a variety of quality attributes by developing chemometric models, a large quantity of representative data is required, and hence, the model development process can be time-consuming. In recent years, the pharmaceutical industry has been expediting new drug development in order to achieve faster delivery of life-changing drugs to patients. The shortened development timelines have impacted the Raman application, as less time is allowed for data collection. To address this problem, an innovative Just-in-Time (JIT) strategy is proposed with the goal of reducing the time needed for Raman model development and ensuring its implementation. To demonstrate its capabilities, a proof-of-concept study was performed by applying the JIT strategy to a biologic continuous process for producing monoclonal antibody products. Raman spectroscopy and online two-dimensional liquid chromatography (2D-LC) were integrated as a PAT analyzer system. Raman models of antibody titer and aggregate percentage were calibrated by chemometric modeling in real-time. The models were also updated in real-time using new data collected during process monitoring. Initial Raman models with adequate performance were established using data collected from two lab-scale cell culture batches and subsequently updated using one scale-up batch. The JIT strategy is capable of accelerating Raman method development to monitor and guide the expedited biologics process development.

3.
Biotechnol Bioeng ; 121(5): 1688-1701, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38393313

RESUMO

Perfusion cell culture has been gaining increasing popularity for biologics manufacturing due to benefits such as smaller footprint, increased productivity, consistent product quality and manufacturing flexibility, cost savings, and so forth. Process Analytics Technologies tools are highly desirable for effective monitoring and control of long-running perfusion processes. Raman has been widely investigated for monitoring and control of traditional fed batch cell culture process. However, implementation of Raman for perfusion cell culture has been very limited mainly due to challenges with high-cell density and long running times during perfusion which cause extremely high fluorescence interference to Raman spectra and consequently it is exceedingly difficult to develop robust chemometrics models. In this work, a platform based on Raman measurement of permeate has been proposed for effective analysis of perfusion process. It has been demonstrated that this platform can effectively circumvent the fluorescence interference issue while providing rich and timely information about perfusion dynamics to enable efficient process monitoring and robust bioreactor feed control. With the highly consistent spectral data from cell-free sample matrix, development of chemometrics models can be greatly facilitated. Based on this platform, Raman models have been developed for good measurement of several analytes including glucose, lactate, glutamine, glutamate, and permeate titer. Performance of Raman models developed this way has been systematically evaluated and the models have shown good robustness against changes in perfusion scale and variations in permeate flowrate; thus models developed from small lab scale can be directly transferred for implementation in much larger scale of perfusion. With demonstrated robustness, this platform provides a reliable approach for automated glucose feed control in perfusion bioreactors. Glucose model developed from small lab scale has been successfully implemented for automated continuous glucose feed control of perfusion cell culture at much larger scale.


Assuntos
Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Cricetinae , Animais , Cricetulus , Células CHO , Perfusão , Glucose/análise , Análise Espectral Raman
4.
Anal Chem ; 95(49): 18130-18138, 2023 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-38015205

RESUMO

Real-time monitoring of biopharmaceutical reactors is becoming increasingly important as the processes become more complex. During the continuous manufacturing of monoclonal antibodies (mAbs), the desired mAb product is continually created and collected over a 30 day process, where there can be changes in quality over that time. Liquid chromatography (LC) is the workhorse instrumentation capable of measuring mAb concentration as well as quality attributes such as aggregation, charge variants, oxidation, etc. However, traditional offline sampling is too infrequent to fully characterize bioprocesses, and the typical time from sample generation to data analysis and reporting can take weeks. To circumvent these limitations, an automated online sampling multidimensional workflow was developed to enable streamlined measurements of mAb concentration, aggregation, and charge variants. This analytical framework also facilitates automated data export for real-time analysis of up to six bioreactors, including feedback-controlling capability using readily available LC technology. This workflow increases the data points per bioreactor, improving the understanding of each experiment while also reducing the data turnaround time from weeks to hours. Examples of effective real-time analyses of mAb critical quality attributes are illustrated, showing substantial throughput improvements and accurate results while minimizing labor and manual intervention.


Assuntos
Produtos Biológicos , Reatores Biológicos , Retroalimentação , Anticorpos Monoclonais/química , Cromatografia Líquida
5.
Anal Chem ; 89(21): 11357-11365, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-28981255

RESUMO

Combining process analytical technology (PAT) with continuous production provides a powerful tool to observe and control monoclonal antibody (mAb) fermentation and purification processes. This work demonstrates on-line liquid chromatography (on-line LC) as a PAT tool for monitoring a continuous biologics process and forced degradation studies. Specifically, this work focused on ion exchange chromatography (IEX), which is a critical separation technique to detect charge variants. Product-related impurities, including charge variants, that impact function are classified as critical quality attributes (CQAs). First, we confirmed no significant differences were observed in the charge heterogeneity profile of a mAb through both at-line and on-line sampling and that the on-line method has the ability to rapidly detect changes in protein quality over time. The robustness and versatility of the PAT methods were tested by sampling from two purification locations in a continuous mAb process. The PAT IEX methods used with on-line LC were a weak cation exchange (WCX) separation and a newly developed shorter strong cation exchange (SCX) assay. Both methods provided similar results with the distribution of percent acidic, main, and basic species remaining unchanged over a 2 week period. Second, a forced degradation study showed an increase in acidic species and a decrease in basic species when sampled on-line over 7 days. These applications further strengthen the use of on-line LC to monitor CQAs of a mAb continuously with various PAT IEX analytical methods. Implementation of on-line IEX will enable faster decision making during process development and could potentially be applied to control in biomanufacturing.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Reatores Biológicos , Cromatografia por Troca Iônica/métodos , Animais , Anticorpos Monoclonais/química , Soluções Tampão , Células CHO , Cromatografia por Troca Iônica/instrumentação , Cricetulus , Concentração de Íons de Hidrogênio
6.
Proc Natl Acad Sci U S A ; 109(52): 21486-91, 2012 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-23236186

RESUMO

Early secretory and endoplasmic reticulum (ER)-localized proteins that are terminally misfolded or misassembled are degraded by a ubiquitin- and proteasome-mediated process known as ER-associated degradation (ERAD). Protozoan pathogens, including the causative agents of malaria, toxoplasmosis, trypanosomiasis, and leishmaniasis, contain a minimal ERAD network relative to higher eukaryotic cells, and, because of this, we observe that the malaria parasite Plasmodium falciparum is highly sensitive to the inhibition of components of this protein quality control system. Inhibitors that specifically target a putative protease component of ERAD, signal peptide peptidase (SPP), have high selectivity and potency for P. falciparum. By using a variety of methodologies, we validate that SPP inhibitors target P. falciparum SPP in parasites, disrupt the protein's ability to facilitate degradation of unstable proteins, and inhibit its proteolytic activity. These compounds also show low nanomolar activity against liver-stage malaria parasites and are also equipotent against a panel of pathogenic protozoan parasites. Collectively, these data suggest ER quality control as a vulnerability of protozoan parasites, and that SPP inhibition may represent a suitable transmission blocking antimalarial strategy and potential pan-protozoan drug target.


Assuntos
Antiparasitários/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Desenho de Fármacos , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Animais , Antiparasitários/química , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Sequência de Bases , Biologia Computacional , Resistência a Medicamentos/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Hep G2 , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/parasitologia , Dados de Sequência Molecular , Parasitos/efeitos dos fármacos , Parasitos/enzimologia , Parasitos/crescimento & desenvolvimento , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Inibidores de Proteases/química , Inibidores de Proteassoma/farmacologia , Proteólise/efeitos dos fármacos , Proteoma/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasma/enzimologia , Toxoplasma/crescimento & desenvolvimento , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimento
7.
Nat Chem Biol ; 8(10): 814-6, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22941045

RESUMO

Thaxtomin phytotoxins produced by plant-pathogenic Streptomyces species contain a nitro group that is essential for phytotoxicity. The N,N'-dimethyldiketopiperazine core of thaxtomins is assembled from L-phenylalanine and L-4-nitrotryptophan by a nonribosomal peptide synthetase, and nitric oxide synthase-generated NO is incorporated into the nitro group, but the biosynthesis of the nonproteinogenic amino acid L-4-nitrotryptophan is unclear. Here we report that TxtE, a unique cytochrome P450, catalyzes L-tryptophan nitration using NO and O(2).


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Indóis/metabolismo , Óxido Nítrico/metabolismo , Piperazinas/metabolismo , Plantas/microbiologia , Streptomyces/metabolismo , Triptofano/metabolismo , Biocatálise
8.
J Pharm Sci ; 112(3): 691-699, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36279953

RESUMO

The use of multi-attribute method (MAM) for identity and purity testing of biopharmaceuticals offers the ability to complement and replace multiple conventional analytical technologies with a single mass spectrometry (MS) method. Phase-appropriate method validation is one major consideration for the implementation of MAM in a current Good Manufacturing Practice (cGMP) environment. We developed a MAM workflow for therapeutic monoclonal antibodies (mAbs) with optimized sample preparation using lysyl endopeptidase (Lys-C) digestion. In this study, we evaluated the assay performances of this platform MAM workflow for identity, product quality attributes (PQAs) monitoring and new peak detection (NPD) for single and coformulated mAbs. An IgG4 mAb-1 and its coformulations were used as model molecules in this study. The assay performance evaluation demonstrated the full potential of the platform MAM approach for its intended use for characterization and quality control of single mAb-1 and mAb-1 in its coformulations. To the best of our knowledge, this is the first performance evaluation of MAM for mAb identity, PQA monitoring, and new peak detection (NPD) in a single assay, featuring 1) the first performance evaluation of MAM for PQA monitoring using Lys-C digestion with a high-resolution MS, 2) a new approach for mAb identity testing capable of distinguishing single mAb from coformulations using MAM, and 3) the performance evaluation of NPD for MAM with Lys-C digestion. The developed platform MAM workflow and the MAM performance evaluation paved the way for its GMP qualification and enabled clinical release of mAb-1 in GMP environment with MAM.


Assuntos
Anticorpos Monoclonais , Produtos Biológicos , Anticorpos Monoclonais/química , Espectrometria de Massas/métodos , Controle de Qualidade , Digestão
9.
Proc Natl Acad Sci U S A ; 106(43): 18183-8, 2009 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-19841256

RESUMO

Deinococcus radiodurans (Dr) withstands desiccation, reactive oxygen species, and doses of radiation that would be lethal to most organisms. Deletion of a gene encoding a homolog of mammalian nitric oxide synthase (NOS) severely compromises the recovery of Dr from ultraviolet (UV) radiation damage. The Deltanos defect can be complemented with recombinant NOS, rescued by exogenous nitric oxide (NO) and mimicked in the wild-type strain with an NO scavenging compound. UV radiation induces both upregulation of the nos gene and cellular NO production on similar time scales. Growth recovery does not depend on NO being present during UV irradiation, but rather can be manifested by NO addition hours after exposure. Surprisingly, nos deletion does not increase sensitivity to oxidative damage, and hydrogen peroxide does not induce nos expression. However, NOS-derived NO upregulates transcription of obgE, a gene involved in bacterial growth proliferation and stress response. Overexpression of the ObgE GTPase in the Deltanos background substantially alleviates the growth defect after radiation damage. Thus, NO acts as a signal for the transcriptional regulation of growth in D. radiodurans.


Assuntos
Deinococcus/metabolismo , Deinococcus/efeitos da radiação , Óxido Nítrico/metabolismo , Raios Ultravioleta , Deinococcus/crescimento & desenvolvimento , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Viabilidade Microbiana/efeitos da radiação , Proteínas Monoméricas de Ligação ao GTP/genética , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Regulação para Cima/efeitos da radiação
10.
Biotechnol Prog ; 37(5): e3187, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34164947

RESUMO

Protein concentration determination is a necessary in-process control for the downstream operations within biomanufacturing. As production transitions from batch mode to an integrated continuous bioprocess paradigm, there is a growing need to move protein concentration quantitation from off-line to in-line analysis. One solution to fulfill this process analytical technology need is an in-line index of refraction (IoR) sensor to measure protein concentration in real time. Here the performance of an IoR sensor is evaluated through a series of experiments to assess linear response, buffer matrix effects, dynamic range, sensor-to-sensor variability, and the limits of detection and quantitation. The performance of the sensor was also tested in two bioprocessing scenarios, ultrafiltration and capture chromatography. The implementation of this in-line IoR sensor for real-time protein concentration analysis and monitoring has the potential to improve continuous bioprocess manufacturing.


Assuntos
Anticorpos Monoclonais/análise , Reatores Biológicos , Proteínas Recombinantes/análise , Refratometria/métodos , Animais , Células CHO , Cromatografia , Cricetinae , Cricetulus , Humanos , Ultrafiltração
11.
Protein Expr Purif ; 73(1): 78-82, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20303407

RESUMO

Over-expression of heme binding proteins in Escherichia coli often results in sub-optimal heme incorporation and the amount of heme-bound protein produced usually varies with the protein of interest. Complete heme incorporation is important for biochemical characterization, spectroscopy, structural studies, and for the production of homogeneous commercial proteins with high activity. We have determined that recombinant proteins expressed in E. coli often contain less than a full complement of heme because they rather are partially incorporated with free-base porphyrin. Porphyrin-incorporated proteins have similar spectral characteristics as the desired heme-loaded targets, and thus are difficult to detect, even in purified samples. We present a straightforward and inexpensive solution to this problem that involves the co-expression of native ferrochelatase with the protein of interest. The method is shown to be effective for proteins that contain either Cys- or His-ligated hemes.


Assuntos
Escherichia coli/genética , Ferroquelatase/biossíntese , Heme/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Ferroquelatase/química , Ferroquelatase/genética , Ferroquelatase/metabolismo , Hemeproteínas/química , Hemeproteínas/genética , Hemeproteínas/metabolismo , Histidina/genética , Histidina/metabolismo , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise Espectral , Análise Espectral Raman
12.
Clin Exp Gastroenterol ; 11: 97-103, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29559804

RESUMO

BACKGROUND/AIMS: As increasing numbers of Crohn's disease (CD) cases are being recognized in India, so the differential diagnosis of CD and gastrointestinal tuberculosis (GITB) is becoming increasingly important. If patients are misdiagnosed with GITB, toxicity may result from unnecessary anti-TB therapy and treatment of the primary disease (ie, CD) gets delayed. We therefore aimed to assess the accuracy of various parameters that can be used to predict GITB diagnosis at index evaluation. MATERIALS AND METHODS: This was a prospective, unicentric, observational study carried out in the gastroenterology department of a tertiary care hospital between August 2011 and January 2013. Patients who presented to our hospital and were suspected of having GITB were included in our study. Patients were then followed up over a 6-month period. STATISTICAL ANALYSIS: Chi-square test was used to analyze the data. RESULTS: Of the 69 patients with GITB, 49 (71.01%) had thickening of the involved part of the colon and 33 (47.83%) had abdominal lymphadenopathy. The ileocecal valve was involved in 58 patients (84.05%) Histological detection of granulomas had 78.95% specificity, 36.23% sensitivity, and 51.40% accuracy. Tuberculosis polymerase chain reaction was found to have 78.95% specificity, 71.01% sensitivity, and 73.83% accuracy. BACTEC-MGIT culture was found to have 100% specificity, 20.29% sensitivity, and 48.60% accuracy. CONCLUSION: Although histology is helpful in ruling out other conditions, TB-specific findings such as caseating granuloma and acid-fast bacilli are rarely seen. Instead, tuberculosis polymerase chain reaction has the highest diagnostic accuracy followed by BACTEC culture.

13.
MAbs ; 10(7): 945-950, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30130442

RESUMO

For many protein therapeutics including monoclonal antibodies, aggregate removal process can be complex and challenging. We evaluated two different process analytical technology (PAT) applications that couple a purification unit performing preparative hydrophobic interaction chromatography (HIC) to a multi-angle light scattering (MALS) system. Using first principle measurements, the MALS detector calculates weight-average molar mass, Mw and can control aggregate levels in purification. The first application uses an in-line MALS to send start/stop fractionation trigger signals directly to the purification unit when preset Mw criteria are met or unmet. This occurs in real-time and eliminates the need for analysis after purification. The second application uses on-line ultra-high performance size-exclusion liquid chromatography to sample from the purification stream, separating the mAb species and confirming their Mw using a µMALS detector. The percent dimer (1.5%) determined by the on-line method is in agreement with the data from the in-line application (Mw increase of approximately 2750 Da). The novel HIC-MALS systems demonstrated here can be used as a powerful tool for real-time aggregate monitoring and control during biologics purification enabling future real time release of biotherapeutics.


Assuntos
Anticorpos Monoclonais/química , Produtos Biológicos/química , Terapia Biológica/métodos , Cromatografia/instrumentação , Difusão Dinâmica da Luz/métodos , Animais , Anticorpos Monoclonais/metabolismo , Produtos Biológicos/metabolismo , Técnicas de Química Analítica , Humanos , Peso Molecular , Agregação Patológica de Proteínas
14.
Int J Biol Macromol ; 73: 160-9, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25449123

RESUMO

The study shows development and optimization of modified release interpenetrating polymer network (IPN) macromolecules (beads) of oxcarbazepine using sodium alginate-egg albumin prepared by ionotropic gelation method and CaCl2 as a cross-linker. Independent variables were identified based on preliminary study of investigation. The effect of amount of both polymers on drug entrapment efficiency (DEE,%), bead size (µm) and cumulative drug release at 8 h (Q8h, %) were optimized using 3(2) factorial design. The DEE, average size and Q8h were found in the range of 65.08-91.02%, 976-1084 µm and 73.50-94.06% respectively. The beads were also characterized by FTIR, DSC, SEM and XRD. The experiential responses were coincided well with predicted values obtained by Design-Expert(®) 8.0.6.1 software. The swelling of beads were influenced by the pH of a release medium. The in vitro drug release from IPN beads exhibited sustained release Hixson-Crowell pattern with anomalous non-Fickian diffusion mechanism concluding that the developed sodium alginate-egg albumin IPN composite beads are suitable for sustained delivery of oxcarbazepine for desired period.


Assuntos
Carbamazepina/análogos & derivados , Microesferas , Polímeros/química , Alginatos/química , Análise de Variância , Carbamazepina/administração & dosagem , Carbamazepina/química , Química Farmacêutica , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Cinética , Ovalbumina/química , Oxcarbazepina , Tamanho da Partícula , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
15.
Int J Biol Macromol ; 65: 72-80, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24418343

RESUMO

Mucilage from the last decades has been found to be very attractive, interesting and useful in development of desired pharmaceutical dosage forms. Various applications of plant based mucilage have a wide potentiality in drug formulations. Lepidium sativum Linn. (family: Brassicaceae) is one of the mucilage containing fast growing, edible annual herb. Its various parts (roots, leaves and seeds) have been used to treat various human ailments. It mainly contains alkaloids, saponins, anthracene glycosides, carbohydrates, proteins, amino acids, flavanoids, and sterols as chief phytochemical constituents. Its seed extracts have been screened for various biological activities like hypotensive, anti-microbial, bronchodilator, hypoglycemic and allelopathic, whereas its seed coat mucilage has been isolated using different methods to make it effective excipient of desired functionality as a part of pharmaceutical applications. Through keen references of reported work on Lepidium sativum Linn., in this review, we have focused on its seed coat mucilage isolation methods, chemical constituents, pharmacological profile and versatile application of Lepidium sativum Linn.


Assuntos
Lepidium sativum/química , Mucilagem Vegetal , Animais , Humanos , Mucilagem Vegetal/química , Mucilagem Vegetal/isolamento & purificação , Mucilagem Vegetal/farmacologia , Mucilagem Vegetal/uso terapêutico , Segurança , Sementes/química
16.
Cell Host Microbe ; 13(1): 15-28, 2013 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-23332153

RESUMO

Following intracellular replication, the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii cause host cell cytolysis to facilitate parasite release and disease progression. Parasite exit from infected cells requires the interplay of parasite-derived proteins and host actin cytoskeletal changes; however, the host proteins underlying these changes remain obscure. We report the identification of a Gα(q)-coupled host-signaling cascade required for the egress of both P. falciparum and T. gondii. Gα(q)-coupled signaling results in protein kinase C (PKC)-mediated loss of the host cytoskeletal protein adducin and weakening of the cellular cytoskeleton. This cytoskeletal compromise induces catastrophic Ca(2+) influx mediated by the mechanosensitive cation channel TRPC6, which activates host calpain that proteolyzes the host cytoskeleton allowing parasite release. Reinforcing the feasibility of targeting host proteins as an antiparasitic strategy, mammalian PKC inhibitors demonstrated activity in murine models of malaria and toxoplasmosis. Importantly, an orally bioavailable PKC inhibitor prolonged survival in an experimental cerebral malaria model.


Assuntos
Autofagia , Interações Hospedeiro-Parasita , Malária Falciparum/metabolismo , Plasmodium falciparum/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Toxoplasma/fisiologia , Toxoplasmose/metabolismo , Animais , Autólise , Cálcio/metabolismo , Linhagem Celular , Citoesqueleto/metabolismo , Humanos , Malária Falciparum/genética , Malária Falciparum/parasitologia , Malária Falciparum/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas G/genética , Toxoplasmose/genética , Toxoplasmose/parasitologia , Toxoplasmose/fisiopatologia
17.
J Mol Cell Biol ; 2(5): 234-6, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20086004

RESUMO

Homologs to mammalian nitric oxide synthases are found in many mostly Gram-positive bacteria. In some genera such as bacilli, and staphylococci, these enzymes produce protects against oxidative damage, this effect has now been shown to provide an advantage against antibiotics that kill by increasing cellular levels of reactive oxygen species.


Assuntos
Antibacterianos/metabolismo , Bactérias/metabolismo , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Espécies Reativas de Oxigênio/metabolismo
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