RESUMO
Our objective is to develop a new therapy for the treatment of stroke. Currently, the only effective therapy for acute ischemic stroke is the thrombolytic agent recombinant tissue plasminogen activator. α1-Antitrypsin (AAT), a serine proteinase inhibitor with potent anti-inflammatory, anti-apoptotic, antimicrobial, and cytoprotective activities, could be beneficial in stroke. The goal of this study is to test whether AAT can improve ischemic stroke outcome in an established rat model. Middle cerebral artery occlusion was induced in male rats via intracranial (i.c.) microinjection of endothelin-1. Five to 10 minutes after stroke induction, rats received either i.c. or intravenous delivery of human AAT. Cylinder and vibrissae tests were used to evaluate sensorimotor function before and 72 hours after middle cerebral artery occlusion. Infarct volumes were examined via either 2,3,5-triphenyltetrazolium chloride assay or magnetic resonance imaging 72 hours after middle cerebral artery occlusion. Despite equivalent initial strokes, at 72 hours, the infarct volumes of the human AAT treatment groups (local and systemic injection) were statistically significantly reduced by 83% and 63% (P < .0001 and P < .05, respectively) compared with control rats. Human AAT significantly limited sensory motor system deficits. Human AAT could be a potential novel therapeutic drug for the protection against neurodegeneration after ischemic stroke, but more studies are needed to investigate the protective mechanisms and efficacy in other animal models.
Assuntos
Encéfalo/efeitos dos fármacos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , alfa 1-Antitripsina/farmacologia , Animais , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Encéfalo/fisiopatologia , Citoproteção , Modelos Animais de Doenças , Endotelina-1 , Humanos , Infarto da Artéria Cerebral Média/induzido quimicamente , Infarto da Artéria Cerebral Média/diagnóstico , Infarto da Artéria Cerebral Média/fisiopatologia , Injeções Intravenosas , Imageamento por Ressonância Magnética , Masculino , Microinjeções , Atividade Motora/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Ratos Sprague-Dawley , Limiar Sensorial/efeitos dos fármacos , Fatores de Tempo , alfa 1-Antitripsina/administração & dosagemRESUMO
Several COVID-19 vaccines, some more efficacious than others, are now available and deployed, including multiple mRNA- and viral vector-based vaccines. With the focus on creating cost-effective solutions that can reach the low- and medium- income world, GreenLight Biosciences has developed an mRNA vaccine candidate, GLB-COV2-043, encoding for the full-length SARS-CoV-2 Wuhan wild-type spike protein. In pre-clinical studies in mice, GLB-COV2-043 induced robust antigen-specific binding and virus-neutralizing antibody responses targeting homologous and heterologous SARS-CoV-2 variants and a TH1-biased immune response. Boosting mice with monovalent or bivalent mRNA-LNPs provided rapid recall and long-lasting neutralizing antibody titers, an increase in antibody avidity and breadth that was held over time and generation of antigen-specific memory B- and T- cells. In hamsters, vaccination with GLB-COV2-043 led to lower viral loads, reduced incidence of SARS-CoV-2-related microscopic findings in lungs, and protection against weight loss after heterologous challenge with Omicron BA.1 live virus. Altogether, these data indicate that GLB-COV2-043 mRNA-LNP vaccine candidate elicits robust protective humoral and cellular immune responses and establishes our mRNA-LNP platform for subsequent clinical evaluations.
Assuntos
COVID-19 , Cricetinae , Animais , Humanos , Camundongos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , SARS-CoV-2/genética , Modelos Animais , RNA Mensageiro/genética , Anticorpos Neutralizantes , Anticorpos Antivirais , Imunogenicidade da VacinaRESUMO
PURPOSE: To develop an efficient and safe strategy to introduce a therapeutic gene into target cells in vivo for cancer therapy. The overall efficiency is based on proper selection of the delivery vector and expressed protein. METHODS: A plasmid coding for a specific cytotoxic fusion peptide, p14ARF-TAT, was evaluated in a xenograft mouse tumor model. The expressed peptide consisted of three domains, a secretory signal, a membrane permeability segment and a cytotoxic fragment. Gene expression was verified in U87-MG cells by Western blot and cytotoxicity confirmed with CyQuant assay. To improve the delivery, a FGF2 targeting peptide, MQLPLATC, was incorporated into the vector, which was evaluated using a luciferase-expressing plasmid. RESULTS: The luciferase activity in vitro was two-fold higher with the targeted formulations, and cytotoxicity was three-fold higher with expression of the p14ARF-TAT protein. A murine xenograph model of human glioma (U87-MG cells) tumors was used to address in vivo activity. FGF2-targeted lipoplexes demonstrated increased tumor volume reduction as compared to non-targeted formulations. RT-PCR and Western blot of tumor homogenizes indicated p14ARF-TAT expression in tumors along with other tissues. CONCLUSION: p14ARF-TAT was cytotoxic and is a promising approach when combined with an efficient targeting.
Assuntos
Fator 2 de Crescimento de Fibroblastos/genética , Produtos do Gene tat/genética , Técnicas de Transferência de Genes , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Proteínas Recombinantes de Fusão/genética , Proteína Supressora de Tumor p14ARF/genética , Animais , Apoptose , Western Blotting , Cátions , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Sobrevivência Celular , Marcação de Genes , Terapia Genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Marcação In Situ das Extremidades Cortadas , Luciferases/genética , Camundongos , Camundongos Nus , Estrutura Molecular , Transplante de Neoplasias , Tamanho da Partícula , Plasmídeos/administração & dosagem , Plasmídeos/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , TransfecçãoRESUMO
Polyethylenimine (PEI) was conjugated to phospholipase A(2) (PLA(2)) in an effort to improve transfection efficiency. PLA(2) was conjugated to PEI using EDC as a coupling reagent. The activity of enzyme in the conjugate was measured. DNA condensation ability of the conjugate to polymer was determined. The resultant nanoparticles were characterized by dynamic and electrophoretic light scattering. Two reporter genes were used to evaluate transfection efficiency in human embryonic kidney (HEK293) and human hepatoma (HepG2) cell lines. Conjugate was shown to retain PLA(2) activity and its ability to condense plasmid DNA, resulting in nanoparticles of a similar size to native PEI. The results demonstrated at N/P ratios of 15 and 20 showed 13- and 8-fold increase in transfection efficiency, respectively, compared to the maximum transfection efficiency of PEI (N/P ratio of 5) in the whole range of N/P ratios tested, from 5 to 60 in HepG2 cells. Toxicity studies in HepG2 cells showed uncomplexed conjugate had similar toxicity as PEI, and when complexed with DNA the conjugate had a significantly reduced toxicity. The results clearly indicate the potential for this approach to improve efficiencies of nonviral gene delivery vectors.
Assuntos
Técnicas de Transferência de Genes , Nanopartículas/química , Fosfolipases A2/química , Polietilenoimina/química , Polímeros/química , Linhagem Celular , Sobrevivência Celular , Células Hep G2 , Humanos , Modelos Teóricos , Fosfolipases A2/metabolismo , Polímeros/síntese química , TransfecçãoRESUMO
PURPOSE: Experiments were conducted to evaluate the utility of a peptide receptor ligand to improve transfection efficiency as part of a polyethylenimine-polyethylene glycol (PEI-PEG) polyplex. The 7-mer peptide (MQLPLAT), targeted toward the fibroblast growth factor 2 (FGF2) receptor, was recently identified using a phage-display library method as possessing a high degree of specificity for the FGF2 receptor without the mutagenicity associated with FGF itself. Two approaches (pre-modification or post-modification) to incorporate the peptide into the PEGylated polyplex were compared in terms of their effect on particle size, surface charge, DNA condensation ability, toxicity, cellular uptake and transfection efficiency. METHODS: The peptide was conjugated to branched PEI (25 kDa) via a PEG spacer either before (pre-modified) or after (post-modified) complexation of PEI with DNA. Polyethyleneimine was conjugated to the PEG spacer (N-hydroxy succinimide (NHS) -PEG-maleimide (Mal)) through the NHS group. The FGF2 peptide was synthesized to contain a cysteine at the carboxyl end (MQLPLATC) and conjugated to the PEG spacer via the Maleimide group. Conjugates were evaluated using (1)H NMR, amino acid analysis, and picrylsulfonic acid assay. DNA condensation was evaluated using agarose gel electrophoresis and cellular toxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular uptake was measured using flow cytometry and transfection efficiency was determined using a luciferase reporter gene assay. RESULTS: Both pre- and post-modification approaches led to a decrease in the zeta potential of the resulting polyplexes but did not alter their size. The pre-modification of PEI did not affect its ability to condense DNA. However, polyplexes formed with the pre-conjugated PEI did not improve cell uptake or transfection efficiency. In contrast, polyplexes that were post-modified with the FGF2 peptide resulted in a 3-fold increase in cell uptake and a 6-fold increase in transfection efficiency. Both pre- and post-modified polyplexes resulted in lower toxicity compared with unmodified PEI. CONCLUSIONS: The results indicate that the FGF2 peptide improves transfection efficiency when used as part of post-modified PEI/PEG polyplex. When used with pre-modified PEI/PEG, the beneficial effect of the peptide on transfection is not evident, probably because, in this case, the peptide ligand is not readily accessible to the FGF receptor.
Assuntos
Peptídeos/genética , Polietilenoglicóis/química , Polietilenoimina/química , Receptores de Fatores de Crescimento de Fibroblastos/genética , Células Cultivadas , DNA/administração & dosagem , DNA/biossíntese , DNA/genética , Eletroquímica , Citometria de Fluxo , Técnicas de Transferência de Genes , Humanos , Indicadores e Reagentes , Ligantes , Espectroscopia de Ressonância Magnética , Tamanho da Partícula , Peptídeos/administração & dosagem , Plasmídeos/genética , Propriedades de Superfície , Sais de Tetrazólio , Tiazóis , TransfecçãoRESUMO
The purpose of this research was to describe the application of lyophilization in the delivery of siRNA using cationic lipids by addressing the long-term formulation/stability issues associated with cationic lipids and to understand the mechanism of lyoprotection. siRNA liposomes complexes were formed in different potential cyro/lyoprotectants and subjected to either lyophilization or freeze thaw cycles. siRNA, liposomes and/or lipoplexes were tested for activity, SYBR Green I binding, cellular uptake and particle size. The lipoplexes when lyophilized in the presence of sugars as lyoprotectants could be lyophilized and reconstituted without loss of transfection efficacy but in ionic solutions they lost 65-75% of their functionality. The mechanism of this loss of activity was further investigated. The lyophilization process did not alter siRNA's intrinsic biological activity as was evident by the ability of lyophilized siRNA to retain functionality and SYBR green I binding ability. While the lipoplex size dramatically increased ( approximately 50-70 times) after lyophilization in the absence of non-ionic lyoprotectants. This increase in size correlated to the decrease in cellular accumulation of siRNA and a decrease in activity. In conclusion, siRNAs can be applied in cationic lipid lyophilized formulations and these complexes represent a potential method of increasing the stability of pre-formed complex.
Assuntos
Carboidratos/química , Crioprotetores/química , Liofilização , Lipídeos/química , Lipossomos , Estabilidade de RNA , RNA Interferente Pequeno/química , Animais , Benzotiazóis , Linhagem Celular Tumoral , Diaminas , Ácidos Graxos Monoinsaturados/química , Corantes Fluorescentes/metabolismo , Congelamento , Glucose/química , Lactose/química , Camundongos , Compostos Orgânicos/metabolismo , Tamanho da Partícula , Fosfatidiletanolaminas/química , Compostos de Amônio Quaternário/química , Quinolinas , RNA Interferente Pequeno/metabolismo , Sacarose/química , Transfecção , Trealose/químicaRESUMO
Small interference RNA (siRNA) is an important research tool, and also has the potential for clinical application. RNA interference (RNAi) approaches allow degradation of selective mRNA coding for pathogenic or disease-related proteins. RNAi pathway can be taken advantage of by the delivery of chemically synthesized siRNA. To fully attain its potential a sufficient siRNA must be delivered to the cell's cytoplasm. Cellular delivery of polyanions such as siRNA, while a challenging problem, may be addressed by the use of cationic macromolecules, the two major classes being lipids and polymers. In this study we compared two model cationic vectors liposomes (lipoplexes) and polyethelyenimine (PEI) (polyplexes). Complexes of the cationic macromolecules and siRNA did not differ in terms of their cellular uptake as determined by flow cytometry. However, it was demonstrated that the lipoplexes decomplexed more easily than the polyplexes. Differences in the biological activity of the siRNA were observed using commercially available siTOX siRNA. Lipoplexes resulted in dose-dependent siRNA activity; to 76.4 +/- 5.9% cell death was seen 48 hours posttransfection using 80 nmol siTOX. In summary, the selection of delivery vector can have a profound impact on biological activity of siRNA molecules. siRNA decomplexation from the cationic vector might be an important factor in the future development of new vectors.
Assuntos
Lipossomos/metabolismo , Polietilenoimina/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transfecção/métodos , Animais , Apoptose , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Marcação de Genes , Camundongos , RNA Interferente Pequeno/genéticaRESUMO
Natural defences in the human body function to protect us from numerous environmental toxins and exposure to potential harmful biological agents. An important frontline defence is antimicrobial peptides. These peptides occur at environmental interfaces and serve to limit bacterial invasion. There has been little work comparing specific peptides as potential antimicrobial compounds. In this study, we evaluated the antimicrobial activity of peptides from the human beta-defensin (HBD) family against four species of Bacillus, chosen as models for Bacillus anthracis, a potential bioweapon. The impact of peptide concentration, sequence and protein binding was evaluated on their biological activity. The results indicated that HBD-3 was the most biologically active against Bacillus subtilis and Bacillus licheniformis, whilst HBD-2 was found to be most active against Bacillus cereus and Bacillus thuringiensis. Moreover, the antimicrobial activity of the peptides was directly related to peptide concentration and indirectly related to albumin concentration (i.e. protein binding).
Assuntos
Anti-Infecciosos/farmacologia , Bacillus/classificação , Bacillus/efeitos dos fármacos , beta-Defensinas/farmacologia , Sequência de Aminoácidos , Anti-Infecciosos/química , Humanos , Testes de Sensibilidade Microbiana/métodos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/farmacologia , beta-Defensinas/químicaRESUMO
INTRODUCTION: Peptide antibiotics as new therapeutic agents are becoming a popular option to investigate due to their broad bacterial target selectivity and limited resistance problems. Although attractive, these new drug candidates have several limitations including low potency and delivery issues which face all peptides/proteins. METHODS: In this study, we designed a plasmid expression system for human beta defensin 3. This sequence was cloned from a human epithelial lung cell into a CMV driven expression cassette. This expression plasmid was then evaluated for its ability to produce human-beta defensin 3 with the use of the non-viral transfection agent, polyethylenimine (PEI). RESULTS: The results indicate the expression cassette was transcriptionally active in HEK 293 cells, as measured by RT-PCR and that a beta defensin peptide was produced by the cells as confirmed by Western blot. The biological activity of the peptide was confirmed against both gram negative E. coli and gram positive Bacillus species using in vitro screening. CONCLUSION: Both the cultured media as well as the transfected cell lysate demonstrated biological activity demonstrating the peptide is also secreted.
Assuntos
Antibacterianos/biossíntese , Antibacterianos/farmacologia , Defensinas/biossíntese , Defensinas/farmacologia , Terapia Genética , Western Blotting , Linhagem Celular , Meios de Cultura , Sistemas de Liberação de Medicamentos , Eletroforese em Gel de Poliacrilamida , Células Epiteliais/metabolismo , Escherichia coli/efeitos dos fármacos , Excipientes , Vetores Genéticos , Humanos , Polietilenoimina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , beta-Defensinas/biossíntese , beta-Defensinas/genéticaRESUMO
Nerve growth factor (NGF) therapy has been proposed to treat cognitive impairments in aged patients including those with Alzheimer's disease. Various viral vectors, including adeno-associated virus serotype 2 (AAV2), have been investigated for their ability to deliver NGF in brain. In this study, hybrid vectors (AAV2/5) consisting of the genome of recombinant AAV2 and the capsid of AAV serotype 5 were evaluated for their ability to deliver NGF and green fluorescent protein (GFP) genes into brain. Compared to AAV2, AAV2/5 consistently led to more septal neurons being transduced with GFP over a wider range of distribution. However, both types of vector provided similar levels of long-term (17 weeks) protection of septal cholinergic neurons from axotomy and led to similar levels of NGF accumulation in this region. These results demonstrate that rAAV-mediated NGF gene delivery is neuroprotective for an extended period of time, but that factors other than transduction efficiency appear to determine transgenic NGF expression in septum.
Assuntos
Axotomia , Proteínas do Capsídeo/genética , Vetores Genéticos , Fatores de Crescimento Neural/genética , Sistema Nervoso Parassimpático/fisiologia , Septo do Cérebro/fisiologia , Animais , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Ensaio de Imunoadsorção Enzimática , Fórnice/fisiologia , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley , Técnicas EstereotáxicasRESUMO
Although NGF gene therapy has been proposed to treat age- or disease-related brain cholinergic decline, little is known about the ectopic expression or function of this trophic factor after transduction in the brain especially over long intervals. The neuron-targeting, recombinant adeno-associated virus serotype 2 (rAAV2) vector was used to express mouse NGF with C-terminal myc-tag in septum using a full-length preproNGF sequence. While the predominant form of endogenous NGF immunoreactivity in septum was 31 kd of proNGF, almost all of the ectopic NGF-immunoreactivity attributable to the rAAV2-mediated transduction in this region was recovered as mature NGF. Transgene expression was found in both cholinergic and GABAergic neurons, with the number of transduced neurons dependent on vector dose. To determine the long-term effects of this NGF-expression on neuron function, fimbria-fornix (FF) lesions were conducted 6 months after NGF gene transfer. NGF gene transfer attenuated the lesion-induced loss of septal cholinergic but not GABAergic neurons, indicating that long-term expression did not eliminate this response, which has been noted over short intervals. The effects and dose dependency of NGF gene delivery on neuroprotection and neurotrophism were also examined. NGF transduction increased cholinergic cell size in the septum, but required a higher vector dose than neuroprotection. These results reveal potential long-term benefits as well as concerns for genetically modifying septal NGF gene expression to preserve neuronal viability and function.
Assuntos
Técnicas de Transferência de Genes , Fator de Crescimento Neural/genética , Neurônios/patologia , Septo do Cérebro/embriologia , Animais , Western Blotting , Sobrevivência Celular , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Proteínas de Fluorescência Verde , Hipocampo/citologia , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Neurônios/metabolismo , Precursores de Proteínas/genética , Ratos , Ratos Sprague-Dawley , Septo do Cérebro/metabolismo , TransgenesRESUMO
Successful gene therapy depends on efficient gene transfer vectors. Viral vectors and non-viral vectors have been investigated extensively. Cationic lipids are non-viral vectors, which resemble traditional pharmaceuticals, display little immunogenicity, and have no potential for viral infection. However, toxicity and low transfection efficiency are two barriers limiting the clinical applications of cationic lipids. Over the last decade, hundreds of cationic lipids have been synthesized to address these problems. In this brief review, we summarized recent research results concerning the structures of DNA/liposomes complexes, some important strategies used to design different classes of cationic lipids, and use of disulfide cationic lipids in plasmid DNA delivery.
Assuntos
Sistemas de Liberação de Medicamentos , Terapia Genética/métodos , Vetores Genéticos/química , Lipídeos/química , Plasmídeos/síntese química , Animais , Cátions , Sistemas de Liberação de Medicamentos/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Lipídeos/administração & dosagem , Plasmídeos/administração & dosagem , Plasmídeos/genéticaRESUMO
UNLABELLED: A cholesteryl ester analog, cholesteryl 1,3-diiopanoate glyceryl ether (C2I), was synthesized and investigated for its potential use for the detection of atherosclerotic lesions in rabbits. METHODS: (125)I-labeled C2I was incorporated into acetylated low-density lipoprotein (AcLDL). The resultant complex, (125)I-C2I-AcLDL, was injected intravenously into 2 groups of rabbits, fed cholesterol and normal chow, at a dose of 555 kBq/kg. Tissue samples were taken 24 h after injection for the biodistribution study. Atherosclerotic lesions and C2I deposition in aortic samples were examined by Sudan IV staining and autoradiography, respectively. RESULTS: The levels of C2I in blood and aortic samples in cholesterol-fed animals were 2- to 3-fold higher than those in the control group (P < 0.05). The autoradiography results correlated well with the Sudan IV staining results, indicating sites of C2I deposition superimposed on lesion sites. CONCLUSION: C2I was preferentially taken up and retained at atherosclerotic lesion sites, suggesting its potential use for the detection of early atherosclerosis.
Assuntos
Arteriosclerose/diagnóstico , Éteres de Glicerila , Radioisótopos do Iodo , Lipoproteínas LDL/metabolismo , Animais , Autorradiografia , Compostos Azo , Masculino , CoelhosRESUMO
Polyethylenimine (PEI) is an effective vehicle for in vivo gene delivery in many tissues including brain. PEI mediates transgene expression in brain neurons and glia. To investigate whether PEI-mediated nerve growth factor (NGF) gene transfer protected axotomized septal cholinergic neurons, we injected linear PEI (in vivo jetPEI, Qbiogene) complexed with a plasmid encoding for mouse NGF (PEI/pNGF-W) into the rat septum. PEI complexed with a plasmid encoding for green fluorescent protein (PEI/pGFP) was used as the control. PEI-mediated gene expression was predominantly neuronal. Fimbria-fornix transections (FFTs), conducted 1 day after rats were injected with control vector, resulted in a 70% loss of septal cholinergic neurons. In contrast, PEI/pNGF-W injection prior to FFTs attenuated the loss of septal cholinergic neurons. This is the first study, to our knowledge, that shows the neuroprotective effects induced by PEI-mediated trophic factor gene transfer in brain.
Assuntos
Técnicas de Transferência de Genes , Fator de Crescimento Neural/genética , Neurônios/patologia , Sistema Nervoso Parassimpático/patologia , Polietilenoimina/farmacologia , Septo do Cérebro/patologia , Animais , Axotomia , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Indicadores e Reagentes , Proteínas Luminescentes , Camundongos , Sistema Nervoso Parassimpático/citologia , Ratos , Ratos Sprague-Dawley , Septo do Cérebro/citologia , Técnicas Estereotáxicas , TransgenesRESUMO
Recombinant adeno-associated virus-2 (rAAV2) under control of the chicken beta actin promoter/truncated CMV enhancer (CBA) was investigated for its ability to transduce primary cultures of rat brain neurons, microglia and astrocytes. This vector was highly effective in all three cell types in heparin-sensitive manners (astrocytes, microglia and neurons transduced by >98%, 75%, and 95%, respectively). However, astrocytes co-cultured with neurons were not transduced. rAAV2/CBA is an important new method for genetic manipulation of brain cells, though this may be modulated by interactions among cell types.
Assuntos
Astrócitos/fisiologia , Dependovirus/genética , Microglia/fisiologia , Transdução Genética/métodos , Animais , Astrócitos/citologia , Astrócitos/virologia , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Humanos , Microglia/citologia , Microglia/virologia , Neurônios/citologia , Neurônios/fisiologia , Neurônios/virologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genéticaRESUMO
Non-viral gene transfer is an alternative to viral vectors for gene transfer. However, non-viral transgene expression remains undesirably low and transient. Matrix attachment regions (MARs) are DNA elements that are defined by their high affinity for the nuclear matrix. MARs may also be related to long-term transgene expression in vitro. The purpose of this research is to evaluate human interferon-beta MARs element in various cell types. This was accomplished by constructing MARs-containing plasmid DNA (pDNA) and comparing their transgene expression with non-MARs-containing pDNA. We found that MARs-containing pDNA increased and prolonged the expression in Chinese hamster ovary (CHO) cells, but not in human neuroblastoma cells (SKnSH) and neuronal cells (primary neuron, astroglia and microglia). From the cotransfection experiment, MARs-containing pDNA had a trans effect on another pDNA expression. A PCR method was used to monitor the intracellular distribution of pDNA after cellular fractionalization. We found that non-MAR containing pDNA demonstrates similar intracellular distribution as non-MARs-containing pDNA.
Assuntos
Regiões de Interação com a Matriz/genética , Ácidos Nucleicos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Transfecção/métodos , Animais , Sítios de Ligação/fisiologia , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , Humanos , Ácidos Nucleicos/genética , RatosRESUMO
BACKGROUND: To date, recombinant tissue plasminogen activator (rtPA) is the only approved drug for ischemic stroke. It is intravenously administered functioning as a thrombolytic agent and is used to obtain reperfusion of the affected area of the brain. Excitotoxicity, inflammation and apoptosis are all involved in delayed neuronal death following stroke and offer multiple opportunities to intervene with neuroprotective agents. Gelsolin (GSN) is an actin- and calcium-binding protein mediating the disassembly of actin filaments and activity of calcium channels. It also functions as a regulator of apoptosis and inflammatory responses. This study tests the hypothesis that increasing the concentration of the form of GSN known as plasma GSN (pGSN) near an infarct will provide neuroprotection following ischemic stroke. METHODS: We induced middle cerebral artery occlusion (MCAO) in male rats via intracranial injection of endothelin-1 (ET-1), a potent vasoconstrictor, and then treated with local delivery of pGSN. Whole brain laser Doppler perfusion imaging was performed through the skull to assess MCAO effectiveness. Cylinder and vibrissae tests evaluated sensorimotor function before and 72 h after MCAO. Infarct volumes were examined 72 h after MCAO via 2, 3, 5-triphenyltetrazolium chloride (TTC) assay. RESULTS: Estimates of relative cerebral perfusion were significantly decreased in all groups receiving MCAO with no differences detected between treatments. Despite equivalent initial strokes, the infarct volume of the pGSN treatment group was significantly reduced compared with the untreated MCAO rats at 72 h. ET-1 induced significant deficits in both cylinder and vibrissae tests while pGSN significantly limited these deficits. CONCLUSION: Gelsolin could be a promising drug for protection against neurodegeneration following ischemic stroke.
RESUMO
Cationic paclitaxel nanoparticles were developed and the possible delivery mechanism was explored by cellular uptake studies. In vitro cytotoxicity of paclitaxel-loaded nanoparticles was evaluated with NIH-3T3 cells and multidrug resistant MDR-3T3 cells (with active P-glycoprotein). The IC(50)s of paclitaxel nanoparticles, liposomal paclitaxel, and Taxol((R)) on NIH-3T3 cells were 0.7 microg/mL, 3.0 microg/mL, and 3.6 microg/mL, respectively, and on MDR-3T3 cells changed to 1.4 microg/mL, 4.4 microg/mL, and 7.3 microg/mL respectively. After addition of verapamil (nonspecific P-glycoprotein inhibition), the IC(50)s on MDR-3T3 cells changed to 0.3 microg/mL, 0.7 microg/mL, and 1.5 microg/mL, respectively. The cellular uptake study of NBD-DOPE labeled nanoparticles by MDR-3T3 cells showed more cellular associated fluorescence than neutral liposomes (EPC/cholesterol). The cellular uptake was not affected by verapamil. Fluorescent nanoparticle-encapsulated 10-nonyl bromide acridine orange also demonstrated an enhanced uptake compared to neutral liposomes. The cellular uptake was increased after verapamil's addition. The cellular uptake of formulations with increased positive charges and the competition of free cationic lipid GL89 demonstrated that the positive charge of the particles enhanced the cellular uptake. In conclusion, although the cationic paclitaxel nanoparticle is susceptible to P-glycoprotein efflux, it is still a promising delivery system for paclitaxel, because of enhanced uptake, which resulted in significantly increased cytotoxicity.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Nanopartículas/química , Paclitaxel/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/farmacocinética , Cátions , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Camundongos , Microscopia Eletrônica de Transmissão , Células NIH 3T3 , Paclitaxel/administração & dosagem , Paclitaxel/farmacocinética , Tamanho da Partícula , Solubilidade , Propriedades de Superfície , Verapamil/farmacologiaRESUMO
Escherichia coli can not synthesize methionine from 5-methylthioribose (MTR) but instead exports this sulfur-containing, energy-rich molecule into the surrounding medium. Transforming E. coli with plasmids that direct expression of the cloned coliphage T3 S-adenosyl-L-methionine (SAM) hydrolase (SAMase) induces the met regulon by cleaving the SAM co-repressor to form 5'-methylthioadenosine, which is then cleaved to produce MTR. To test the effect of in vivo SAMase activity on MTR production and its fate, cultures were incubated in the presence of [35S]methionine and [methyl-3H]methionine. Cells with SAMase activity produced significantly enhanced levels (up to 40-fold in some trials) of extracellular MTR -- the only radiolabeled compound released in significant amounts -- when compared with controls. SAM synthetase (metK) mutants transformed with SAMase expression vectors did not show this increase, verifying the path through SAM as the sole route to MTR production. SAMase expression had little or no effect on intracellular MTR pools, levels of radiolabeled macromolecules, or the transfer of methyl groups to compounds that could be precipitated by trichloroacetic acid. Thus, MTR appears to be a dead-end metabolite in E. coli, begging questions about how this has evolved, the mechanism of MTR export for the cell, and whether the release of MTR is important for some other activity.
Assuntos
Escherichia coli/metabolismo , S-Adenosilmetionina/metabolismo , Tioglicosídeos/metabolismo , Transporte Biológico , Escherichia coli/genética , Hidrolases/genética , Hidrolases/metabolismo , Hidrólise , Metionina/farmacocinética , Plasmídeos/genética , Transformação BacterianaRESUMO
Regulation of methionine biosynthesis in Escherichia coli involves a complex of the MetJ aporepressor protein and S-adenosylmethionine (SAM) repressing expression of most genes in the met regulon. To test the role of SAM in the regulation of met genes directly, SAM pools were depleted by the in vivo expression of the cloned plasmid vector-based coliphage T3 SAM hydrolase (SAMase) gene. Cultures with in vivo SAMase activity were assayed for expression of the metA, B, C, E, F, H, J, K and R genes in cells grown in methionine-rich complete media as well as in defined media with and without l-methionine. In vivo SAMase activity dramatically induced expression between 11- and nearly 1000-fold depending on the gene assayed for all but metJ and metH, and these genes were induced over twofold. metJ : : Tn5 (aporepressor defective) and metK : : Tn5 (SAM synthetase impaired; produces <5 % of wild-type SAM) strains containing in vivo SAMase activity produced even higher met gene activity than that seen in comparably prepared cells with wild-type genes for all but metJ in a MetJ-deficient background. The SAMase-mediated hyperinduction of metH in wild-type cells and of the met genes assayed in metJ : : Tn5 and metK : : Tn5 cells provokes questions about how other elements such as the MetR activator protein or factors beyond the met regulon itself might be involved in the regulation of genes responsible for methionine biosynthesis.