RESUMO
Spermine synthase is an aminopropyltransferase that adds an aminopropyl group to the essential polyamine spermidine to form tetraamine spermine, needed for normal human neural development, plant salt and drought resistance, and yeast CoA biosynthesis. We functionally identify for the first time bacterial spermine synthases, derived from phyla Bacillota, Rhodothermota, Thermodesulfobacteriota, Nitrospirota, Deinococcota, and Pseudomonadota. We also identify bacterial aminopropyltransferases that synthesize the spermine same mass isomer thermospermine, from phyla Cyanobacteriota, Thermodesulfobacteriota, Nitrospirota, Dictyoglomota, Armatimonadota, and Pseudomonadota, including the human opportunistic pathogen Pseudomonas aeruginosa. Most of these bacterial synthases were capable of synthesizing spermine or thermospermine from the diamine putrescine and so possess also spermidine synthase activity. We found that most thermospermine synthases could synthesize tetraamine norspermine from triamine norspermidine, that is, they are potential norspermine synthases. This finding could explain the enigmatic source of norspermine in bacteria. Some of the thermospermine synthases could synthesize norspermidine from diamine 1,3-diaminopropane, demonstrating that they are potential norspermidine synthases. Of 18 bacterial spermidine synthases identified, 17 were able to aminopropylate agmatine to form N1-aminopropylagmatine, including the spermidine synthase of Bacillus subtilis, a species known to be devoid of putrescine. This suggests that the N1-aminopropylagmatine pathway for spermidine biosynthesis, which bypasses putrescine, may be far more widespread than realized and may be the default pathway for spermidine biosynthesis in species encoding L-arginine decarboxylase for agmatine production. Some thermospermine synthases were able to aminopropylate N1-aminopropylagmatine to form N12-guanidinothermospermine. Our study reveals an unsuspected diversification of bacterial polyamine biosynthesis and suggests a more prominent role for agmatine.
Assuntos
Bactérias , Proteínas de Bactérias , Espermidina Sintase , Espermina Sintase , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Espermidina/metabolismo , Espermidina/análogos & derivados , Espermidina/biossíntese , Espermidina Sintase/metabolismo , Espermidina Sintase/genética , Espermina/metabolismo , Espermina/análogos & derivados , Espermina/biossíntese , Espermina Sintase/metabolismo , Espermina Sintase/genética , Poliaminas/metabolismo , Alquil e Aril Transferases/biossíntese , Alquil e Aril Transferases/genética , Agmatina/química , Agmatina/metabolismoRESUMO
Agmatine, an endogenous derivative of arginine, has been found to be effective in treating idiopathic pain, convulsion, stress-mediated behavior, and attenuate the withdrawal symptoms of drugs like morphine. In the early stages of ischemic brain injury in animals, exogenous agmatine treatment was found to be neuroprotective. Agmatine is also considered as a putative neurotransmitter and is still an experimental drug. Chemically, agmatine is called agmatine 1-(4-aminobutyl guanidine). Crystallographic study data show that positively-charged guanidine can bind to the protein containing Gly and Asp residues, and the amino group can interact with the complimentary sites of Glu and Ser. In this study, we blocked the amino end of the agmatine by conjugating it with FITC, but the guanidine end was unchanged. We compared the neuroprotective function of the agmatine and agmatine-FITC by treating them in neurons after excitotoxic stimulation. We found that even the amino end blocked neuronal viability in the excitotoxic condition, by NMDA treatment for 1 h, was increased by agmatine-FITC, which was similar to that of agmatine. We also found that the agmatine-FITC treatment reduced the expression of nitric oxide production in NMDA-treated cells. This study suggests that even if the amino end of agmatine is blocked, it can perform its neuroprotective function.
Assuntos
Agmatina/farmacologia , Neurônios/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Agmatina/química , Animais , Células Cultivadas , Córtex Cerebral/citologia , Feminino , Feto/citologia , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/farmacologia , Camundongos Endogâmicos ICR , N-Metilaspartato/toxicidade , Fármacos Neuroprotetores/química , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
The biogenic polyamines, spermine, spermidine (Spd) and putrescine (Put) are present at micro-millimolar concentrations in eukaryotic and prokaryotic cells (many prokaryotes have no spermine), participating in the regulation of cellular proliferation and differentiation. In mammalian cells Put is formed exclusively from L-ornithine by ornithine decarboxylase (ODC) and many potent ODC inhibitors are known. In bacteria, plants, and fungi Put is synthesized also from agmatine, which is formed from L-arginine by arginine decarboxylase (ADC). Here we demonstrate that the isosteric hydroxylamine analogue of agmatine (AO-Agm) is a new and very potent (IC50 3â¢10-8 M) inhibitor of E. coli ADC. It was almost two orders of magnitude less potent towards E. coli ODC. AO-Agm decreased polyamine pools and inhibited the growth of DU145 prostate cancer cells only at high concentration (1 mM). Growth inhibitory analysis of the Acremonium chrysogenum demonstrated that the wild type (WT) strain synthesized Put only from L-ornithine, while the cephalosporin C high-yielding strain, in which the polyamine pool is increased, could use both ODC and ADC to produce Put. Thus, AO-Agm is an important addition to the set of existing inhibitors of the enzymes of polyamine biosynthesis, and an important instrument for investigating polyamine biochemistry.
Assuntos
Acremonium/química , Agmatina , Carboxiliases , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Agmatina/análogos & derivados , Agmatina/química , Animais , Carboxiliases/antagonistas & inibidores , Carboxiliases/química , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Masculino , CamundongosRESUMO
Bioreducible cationic polymers have gained considerable attention in gene delivery due to their low cytotoxicity and high efficiency. In the present work, we reported a cationic polymer, poly(disulfide-l-lysine)-g-agmatine (denoted as SSL-AG), and evaluated its ability to transfer pEGFP-ZNF580 plasmid (pZNF580) into human umbilical vein endothelial cells (HUVECs). This SSL-AG polymeric carrier efficiently condensed pZNF580 into positively charged particles (<200 nm) through electrostatic interaction. This carrier also exhibited excellent buffering capacity in the physiological environment, good pDNA protection against enzymatic degradation and rapid pDNA release in a highly reducing environment mainly because of the responsive cleavage of disulfide bonds in the polymer backbone. The hemolysis assay and in vitro cytotoxicity assay suggested that the SSL-AG carrier and corresponding gene complexes possessed both good hemocompatibility and great cell viability in HUVECs. The cellular uptake of the SSL-AG/Cy5-oligonucleotide group was 3.6 times that of the poly(l-lysine)/Cy5-oligonucleotide group, and its mean fluorescence intensity value was even higher than that of the PEI 25 kDa/Cy5-oligonucleotide group. Further, the intracellular trafficking results demonstrated that the SSL-AG/Cy5-oligonucleotide complexes exhibited a high nucleus co-localization rate (CLR) value (36.0 ± 2.8%, 3.4 times that of the poly (l-lysine)/Cy5-oligonucleotide group, 1.6 times that of the poly(disulfide-l-lysine)-g-butylenediamine/Cy5-oligonucleotide group) at 24 h, while the endo/lysosomal CLR value was relatively low. This suggested that SSL-AG successfully delivered plasmid into HUVECs with high cellular uptake, rapid endosomal escape and efficient nuclear accumulation owing to the structural advantages of the bioreducible and agmatine groups. In vitro transfection assay also verified the enhanced transfection efficiency in the SSL-AG/pZNF580 group. Furthermore, the results of CCK-8, cell migration and in vitro/vivo angiogenesis assays revealed that pZNF580 delivered by SSL-AG could effectively enhance the proliferation, migration and vascularization of HUVECs. In a word, the SSL-AG polymer has great potential as a safe and efficient gene carrier for gene therapy.
Assuntos
Agmatina/química , Técnicas de Transferência de Genes , Polilisina/química , Agmatina/síntese química , Agmatina/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Masculino , Camundongos , Tamanho da Partícula , Polilisina/síntese química , Polilisina/farmacologia , Propriedades de SuperfícieRESUMO
As a cationic non-viral gene delivery vector, poly(agmatine/ N, N'-cystamine-bis-acrylamide) (AGM-CBA) showed significantly higher plasmid DNA (pDNA) transfection ability than polyethylenimine (PEI) in NIH/3T3 cells. The transfection expression of AGM-CBA/pDNA polyplexes was found to have a non-linear relationship with AGM-CBA/pDNA weight ratios. To further investigate the mechanism involved in the transfection process of poly(AGM-CBA), we used pGL3-control luciferase reporter gene (pLUC) as a reporter pDNA in this study. The distribution of pLUC in NIH/3T3 cells and nuclei after AGM-CBA/pLUC and PEI/pLUC transfection were determined by quantitative polymerase chain reaction (qPCR) analysis. The intracellular trafficking of the polyplexes was evaluated by cellular uptake and nuclei delivery of pLUC, and the intracellular availability was evaluated by the ratio of transfection expression to the numbers of pLUC delivered in nuclei. It was found that pLUC intracellular trafficking did not have any correlation with the transfection expression, while an excellent correlation was found between the nuclei pLUC availability and transfection expression. These results suggested that the intracellular availability of pLUC in nuclei was the rate-limiting step for pLUC transfection expression. Further optimization of the non-viral gene delivery system can be focused on the improvement of gene intracellular availability.
Assuntos
Núcleo Celular/metabolismo , Genes Reporter/genética , Luciferases/genética , Luciferases/metabolismo , Plasmídeos/genética , Transfecção/métodos , Acrilamidas/química , Agmatina/química , Animais , Camundongos , Células NIH 3T3 , Polietilenoimina/químicaRESUMO
In order to balance transfection efficiency and cytotoxicity as well as screen the optimal polymers for gene delivery, a series of amphoteric copolymers (poly(CBA-AGM/GABA)s) composed of different ratios between agmatine (AGM) and γ-aminobutyric acid (GABA) monomers were synthesized. The AGM containing positively charged guanidinium groups was used to improve transfection efficiency, while the GABA containing negatively charged carboxyl groups was used to decrease cytotoxicity. It is hypothesized that the amphoteric poly(CBA-AGM/GABA)s synthesized at the optimal ratio of both components would well balance transfection efficiency and cytotoxicity. By comparing these polymers' essential features in gene delivery, the ideal ratio between AGM and GABA was optimized. AGM80, which contained 80% AGM and 20% GABA, showed favorable properties for gene delivery, including moderate DNA condensation capacity, high cellular uptake, strong nuclear localization ability, high transfection efficiency, and low cytotoxicity, indicating that this polymer is very promising as a potent and nontoxic gene carrier.
Assuntos
Técnicas de Transferência de Genes , Poliaminas/química , Polímeros/química , Transfecção/métodos , Agmatina/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Química Sintética/métodos , Camundongos , Microscopia Confocal , Modelos Químicos , Estrutura Molecular , Células NIH 3T3 , Poliaminas/síntese química , Poliaminas/farmacologia , Polímeros/síntese química , Polímeros/farmacologia , Ácido gama-Aminobutírico/químicaRESUMO
The enzyme 4-guanidinobutyrase (GBase) catalyzes the hydrolysis of 4-guanidinobutyric acid (GB) to 4-aminobutyric acid (GABA) and urea. Here we describe methods to estimate urea and GABA that were suitably adapted from the published literature. The urea is determined by colorimetric assay using modified Archibald's method. However, the low sensitivity of this method often renders it impractical to perform fine kinetic analysis. To overcome this limitation, a high sensitive method for detecting GABA is exploited that can even detect 1 µM of GABA in the assay mixture. The samples are deproteinized by perchloric acid (PCA) and potassium hydroxide treatment prior to HPLC analysis of GABA. The method involves a pre-column derivatization with o-phthalaldehyde (OPA) in combination with the thiol 3-mercaptopropionic acid (MPA). The fluorescent GABA derivative is then detected after reversed phase high performance liquid chromatography (RP-HPLC) using isocratic elution. The protocols described here are broadly applicable to other biological samples involving urea and GABA as metabolites.
Assuntos
Agmatina/metabolismo , Aspergillus niger/metabolismo , Agmatina/química , Bioensaio , Cromatografia Líquida de Alta Pressão , Ureia/química , Ureo-Hidrolases/química , Ureo-Hidrolases/metabolismo , Ácido gama-Aminobutírico/química , Ácido gama-Aminobutírico/metabolismo , o-Ftalaldeído/química , o-Ftalaldeído/metabolismoRESUMO
This paper reports on the development of montmorillonite (MMT)-reinforced hydrogels, based on a peptidomimetic polyamidoamine carrying guanidine pendants (AGMA1), as substrates for the osteo-induction of osteoblast precursor cells. AGMA1 hydrogels of various degrees of crosslinking responded favourably to MMT reinforcement, giving rise to composite hydrogels with shear storage modulus G', when fully swollen in water, up to 200 kPa, i.e. 20 times higher than the virgin hydrogels and of the same order or higher than other hydrogel-based composites proposed for orthopaedic applications. This significant improvement was ascribed to the effective interpenetration between the polymer matrix and the inorganic filler. AGMA1-MMT hydrogels, when evaluated as scaffolds for the osteogenic differentiation of mouse calvaria-derived pre-osteoblastic MC3T3-E1 cells, proved able to support cell adhesion and proliferation and clearly induced differentiation towards the osteoblastic phenotype, as indicated by different markers. In addition, AGMA1-MMT hydrogels proved completely degradable in aqueous media at pH 7.4 and did not provide any evidence of cytotoxicity. The experimental evidence suggests that AGMA1-MMT composites definitely warrant potential as scaffolds for osteoblast culture and bone grafts. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Bentonita/química , Osso e Ossos/metabolismo , Hidrogéis/química , Oligopeptídeos/química , Osteoblastos/metabolismo , Poliaminas/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Agmatina/análogos & derivados , Agmatina/química , Animais , Osso e Ossos/citologia , Linhagem Celular , Teste de Materiais , Camundongos , Osteoblastos/citologiaRESUMO
Agmatine (1-amino-4-guanidinobutane) plays an important role in a range of metabolic functions, in particular in the brain. Agmatinases (AGMs) are enzymes capable of converting agmatine to the polyamine putrescine and urea. AGMs belong to the family of Mn2+-dependent ureahydrolases. However, no AGM from a mammalian source has yet been extracted in catalytically active form. While in human AGM the six amino acid ligands that coordinate the two Mn2+ ions in the active site are conserved, four mutations are observed in the murine enzyme. Here, we demonstrate that similar to its human counterpart murine AGM does not appear to have in vitro catalytic activity, independent of the presence of Mn2+. However, in presence of agmatine both enzymes are very efficient in promoting cell growth of a yeast strain that is deficient in polyamine biosynthesis (Saccharomyces cerevisiae strain TRY104Δspe1). Furthermore, mutations among the putative Mn2+ binding residues had no effect on the ability of murine AGM to promote growth of the yeast culture. It thus appears that mammalian AGMs form a distinct group within the family of ureahydrolases that (i) either fold in a manner distinct from other members in this family, or (ii) require accessory proteins to bind Mn2+ in a mechanism related to that observed for the Ni2+-dependent urease.
Assuntos
Agmatina/metabolismo , Manganês/metabolismo , Ureo-Hidrolases/metabolismo , Agmatina/química , Animais , Sítios de Ligação , Manganês/química , Camundongos , Proteínas Recombinantes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ureo-Hidrolases/química , Ureo-Hidrolases/genéticaRESUMO
Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli.
Assuntos
Sistemas de Transporte de Aminoácidos/química , Antiporters/química , Proteínas de Escherichia coli/química , Agmatina/química , Sistemas de Transporte de Aminoácidos/genética , Antiporters/genética , Arginina/química , Arginina/genética , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Especificidade por SubstratoRESUMO
Commensal and pathogenic enteric bacteria have developed several systems to adapt to proton leakage into the cytoplasm resulting from extreme acidic conditions. One such system involves arginine uptake followed by export of the decarboxylated product agmatine, carried out by the arginine/agmatine antiporter (AdiC), which thus works as a virtual proton pump. Here, using classical and targeted molecular dynamics, we investigated at the atomic level the mechanism of arginine transport through AdiC of E. coli. Overall, our MD simulation data clearly demonstrate that global rearrangements of several transmembrane segments are necessary but not sufficient for achieving transitions between structural states along the arginine translocation pathway. In particular, local structural changes, namely rotameric conversions of two aromatic residues, are needed to regulate access to both the outward- and inward-facing states. Our simulations have also enabled identification of a few residues, overwhelmingly aromatic, which are essential to guiding arginine in the course of its translocation. Most of them belong to gating elements whose coordinated motions contribute to the alternating access mechanism. Their conservation in all known E. coli acid resistance antiporters suggests that the transport mechanisms of these systems share common features. Last but not least, knowledge of the functional properties of AdiC can advance our understanding of the members of the amino acid-carbocation-polyamine superfamily, notably in eukaryotic cells.
Assuntos
Agmatina/química , Sistemas de Transporte de Aminoácidos/química , Antiporters/química , Arginina/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Agmatina/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Antiporters/metabolismo , Arginina/metabolismo , Sítios de Ligação , Transporte Biológico , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Cinética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato , TermodinâmicaRESUMO
Electrospun poly-l-lactic acid (PLLA) nanofiber mats carrying surface amine groups, previously introduced by nitrogen atmospheric pressure nonequilibrium plasma, are embedded into aqueous solutions of oligomeric acrylamide-end capped AGMA1, a biocompatible polyamidoamine with arg-gly-asp (RGD)-reminiscent repeating units. The resultant mixture is finally cured giving PLLA-AGMA1 hydrogel composites that absorb large amounts of water and, in the swollen state, are translucent, soft, and pliable, yet as strong as the parent PLLA mat. They do not split apart from each other when swollen in water and remain highly flexible and resistant, since the hydrogel portion is covalently grafted onto the PLLA nanofibers via the addition reaction of the surface amine groups to a part of the terminal acrylic double bonds of AGMA1 oligomers. Preliminary tested as scaffolds, the composites prove capable of maintaining short-term undifferentiated cultures of human pluripotent stem cells in feeder-free conditions.
Assuntos
Hidrogéis/química , Nanofibras/química , Células-Tronco Pluripotentes/metabolismo , Poliaminas/química , Poliésteres/química , Alicerces Teciduais/química , Agmatina/análogos & derivados , Agmatina/química , Técnicas de Cultura de Células , Humanos , Células-Tronco Pluripotentes/citologiaRESUMO
The development of topical microbicides is a valid approach to protect the genital mucosa from sexually transmitted infections that cannot be contained with effective vaccination, like HSV and HIV infections. A suitable target of microbicides is the interaction between viral proteins and cell surface heparan sulfate proteoglycans (HSPGs). AGMA1 is a prevailingly cationic agmatine-containing polyamidoamine polymer previously shown to inhibit HSPGs dependent viruses, including HSV-1, HSV-2, and HPV-16. The aim of this study was to elucidate the mechanism of action of AGMA1 against HSV infection and assess its antiviral efficacy and biocompatibility in preclinical models. The results show AGMA1 to be a non-toxic inhibitor of HSV infectivity in cell cultures and human cervicovaginal histocultures. Moreover, it significantly reduced the burden of infection of HSV-2 genital infection in mice. The investigation of the mechanism of action revealed that AGMA1 reduces cells susceptibility to virus infection by binding to cell surface HSPGs thereby preventing HSV attachment. This study indicates that AGMA1 is a promising candidate for the development of a topical microbicide to prevent sexually transmitted HSV infections.
Assuntos
Agmatina/análogos & derivados , Antivirais/farmacologia , Colo do Útero/virologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Poliaminas/farmacologia , Agmatina/química , Agmatina/farmacologia , Animais , Anti-Infecciosos Locais/farmacologia , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Colo do Útero/citologia , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Herpes Simples/tratamento farmacológico , Papillomavirus Humano 16/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Poliaminas/química , Células VeroRESUMO
Lately, researchers have made several efforts to improve vaccine production to fight highly contagious respiratory diseases like influenza. One of the most promising options for reducing the impact of this virus is DNA vaccination. However, a large quantity of highly pure plasmid DNA (pDNA) is necessary to attain this goal. The present work describes the production and purification of the plasmid NTC7482-41H-VA2HA expressing influenza virus hemagglutinin using an agmatine monolith. This ligand was chosen to purify supercoiled (sc) pDNA from complex lysates because of its versatile multimodal character. Its natural intervention in several biological systems together with its similarity with the highly studied arginine ligand allowed the development of a simpler and more specific purification process. Agmatine works under two strategies: descending ammonium sulfate gradient and ascending sodium chloride gradient. Furthermore, pH manipulation revealed an important role in pDNA isoforms selectivity. Dynamic binding capacity (DBC) experiments were performed varying different parameters and showed an increase with pDNA concentration, while high flow rate and high pH had the opposite effect. Sc pDNA was purified with high yield and was efficient with respect to cell transfection and cell viability. This monolith showed to be appropriate to purify the plasmid NTC7482-41H-VA2HA, providing a valuable tool for pDNA influenza vaccines preparation.
Assuntos
Agmatina/química , Cromatografia de Afinidade/métodos , Vacinas contra Influenza/isolamento & purificação , Plasmídeos/isolamento & purificação , Vacinas de DNA/isolamento & purificação , Animais , Linhagem Celular , Fibroblastos , Humanos , Vacinas contra Influenza/genética , Plasmídeos/genética , Transfecção , Vacinas de DNA/genéticaRESUMO
A new type of bioreducible poly(amido amine) copolymer is synthesized by the Michael addition polymerization of cystamine bisacrylamide (CBA) with 4-aminobutylguanidine (agmatine, AGM) and 4-aminobutanol (ABOL). Since the positively charged guanidinium groups of AGM and the hydroxybutyl groups of ABOL in the side chains have shown to improve the overall transfection efficiency of poly(amido amine)s, it is hypothesized that poly(CBA-ABOL/AGM) synthesized at the optimal ratio of both components would result in high transfection efficiency and minimal toxicity. In this study, a series of the poly(CBA-ABOL/AGM) copolymers is synthesized as gene carriers. The polymers are characterized and luciferase transfection efficiencies of the polymers in various cell lines are investigated to select the ideal ratio between AGM and ABOL. The poly(CBA-ABOL/AGM) containing 80% AGM and 20% ABOL has shown the best transfection efficiency with the lowest cytotoxicity, indicating that this polymer is very promising as a potent and nontoxic gene carrier.
Assuntos
Agmatina/química , Amino Álcoois/química , Cistamina/análogos & derivados , Lactatos/química , Transfecção/métodos , Agmatina/farmacologia , Amino Álcoois/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cistamina/química , Cistamina/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Lactatos/farmacologia , Luciferases/genética , Luciferases/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Oxirredução , PolimerizaçãoRESUMO
Agmatine-containing bioreducible polymer, poly(cystaminebis(acrylamide)-agmatine) (poly(CBA-AG)) was synthesized for gene delivery systems. It could form 200-300 nm sized and positively charged polyplexes with pDNA, which could release pDNA in reducing the environment due to the internal disulfide bonds cleavage. Poly(CBA-AG) also showed a spontaneous degradation behavior in aqueous condition in contrast to the backbone polymer, poly(cystaminebis(acrylamide)-diaminobutane) (poly(CBA-DAB)) lacking guanidine moieties, probably due to the self-catalyzed hydrolysis of internal amide bonds by guanidine moieties. The cytotoxicity of poly(CBA-AG) was cell-dependent but minimal. Poly(CBA-AG) exhibited highly enhanced transfection efficiency in comparison with poly(CBA-DAB) and even higher transfection efficiency than PEI25k. However, cellular uptake efficiency of the polyplexes did not show positive correlation with the transfection efficiency. Confocal microscopy observation revealed that pDNA delivered by poly(CBA-AG) was strongly accumulated in cell nuclei. These results suggested that high transfection efficiency of poly(CBA-AG) may be derived from the efficient pDNA localization in cell nuclei by guanidine moieties and that the polyplexes dissociation via self-catalyzed hydrolysis as well as disulfide bonds cleavage in cytosol also may facilitate the transfection process. Finally, poly(CBA-AG)/pJDK-apoptin polyplex showed a high anticancer activity induced by apoptosis, demonstrating a potential of poly(CBA-AG) as a gene carrier for cancer gene therapy.
Assuntos
Agmatina/química , Plásticos Biodegradáveis/química , Núcleo Celular/metabolismo , Técnicas de Transferência de Genes , Plasmídeos/química , Núcleo Celular/genética , Células HeLa , HumanosRESUMO
MicroRNA-based therapeutic applications have fostered a growing interest in the development of microRNAs purification processes in order to obtain the final product with high purity degree, good quality and biologically active. The pre-miR-29 deficiency or overexpression has been associated to a number of clinically important diseases, and its therapeutic application can be considered. Monolithic columns emerged as a new class of chromatographic supports used in the plasmid DNA purification platforms, being an interesting alternative to the conventional particle-based columns. Thus, the current work describes, for the first time, a new affinity chromatography method that combines the high selectivity of agmatine ligands with the versatility of monoliths to specifically and efficiently purify pre-miR-29 from other small RNA species and Rhodovulum sulfidophilum impurities. The effect of different flow rates on pre-miR-29 separation was also evaluated. Moreover, breakthrough experiments were designed to study the effect of different RNA concentrations on the modified monolithic support binding capacity, being verified that the dynamic binding capacity for RNA molecules is dependent of the feed concentration. In order to achieve higher efficiency and selectivity, three different binding and elution strategies based on increased sodium chloride (1.75-3M) or arginine (100mM) and decreased ammonium sulfate (2.4-0M) stepwise gradients are described to purify pre-miR-29. As a matter of fact, by employing elution strategies using sodium chloride or arginine, an improvement in the final pre-miR-29 yields (97.33 and 94.88%, respectively) as well as purity (75.21 and 90.11%, respectively) were obtained. Moreover, the quality control analysis revealed that the level of impurities (proteins, endotoxins, sRNA) in the final pre-miR-29 sample was negligible. In fact, this new monolithic support arises as a powerful instrument on the microRNA purification to be used in further clinical applications, providing a more rapid and economical purification platform.
Assuntos
Agmatina/química , Cromatografia de Afinidade/métodos , MicroRNAs/isolamento & purificação , Sulfato de Amônio , Arginina/química , Concentração de Íons de Hidrogênio , Plasmídeos , Cloreto de SódioRESUMO
Poly(amidoamine)s (PAAs) are multifunctional tert-amine polymers endowed with high structural versatility. Here we report on the screening of a minilibrary of PAAs against a panel of viruses. The PAA AGMA1 showed antiviral activity against herpes simplex virus, human cytomegalovirus, human papillomavirus 16, and respiratory syncytial virus but not against human rotavirus and vesicular stomatitis virus. The results suggest the contribution of both a polycationic nature and side guanidine groups in imparting antiviral activity.
Assuntos
Agmatina/química , Antivirais/química , Antivirais/farmacologia , Poliaminas/química , Poliaminas/farmacologia , Rotavirus/efeitos dos fármacos , Citomegalovirus/efeitos dos fármacos , Humanos , Simplexvirus/efeitos dos fármacos , Vesiculovirus/efeitos dos fármacosRESUMO
To exploit the binding affinity for efficient plasmid purification, agmatine and histamine were immobilized on carboxymethylated dextran surface. Their binding strength is evaluated with oligonucleotides and pUC19 (2.69 kbp), pVAX1-LacZ (6.05 kbp) and pcDNA3-myc-FLNa S2152A (14 kbp) isoforms by measuring the KD using SPR-biosensor. The oligonucleotides and plasmid isoforms bind more strongly to histamine than to agmatine. Concerning the oligonucleotides, the highest affinities are found for poly 30, and polyT and polyG series showed lowest affinity with both ligands. These results are corroborating with the hetero-oligonucleotides since the lowest binding affinity is found for GGGTTT, indicating that thymidine and guanosine together decrease the binding strength. The largest plasmid has the highest binding, while pUC19 shows the weaker binding. The linear isoform exhibit the highest binding affinity to both amino acid-derivatives. Decreasing the pH above 6, the interaction strength of linear isoform increases possibly due to positively charged of the surface, which enhances electrostatic interactions. However, no binding response is detected with high salt concentrations (1 M NaCl) and for different buffers. The affinity information accessible with the biosensor offers new insight into nucleic acids-ligand interactions, as well as, new criteria leading the optimization of the novel ligands for preparative plasmid purification.
Assuntos
Agmatina/química , DNA/química , Histamina/química , Ressonância de Plasmônio de Superfície , Plasmídeos/química , Ressonância de Plasmônio de Superfície/métodosRESUMO
In this paper, novel core-shell polymeric affinity beads based on fibrous grafting and functionalization with a salt resistance affinity ligand were developed to separate and deplete serum albumin (SA) from human serum. Poly(hydroxypropyl methacrylate/ethyleneglycole dimethacrylate), p(HPMA/EGDMA), beads were prepared via suspension polymerization, and were grafted with poly(glycidyl methacrylate) (p(GMA)) via surface-initiated atom transfer radical polymerization (SI-ATRP) method. The grafted p(GMA) fibrous chains on the beads were modified with an affinity ligand (i.e., agmatine). The binding capacity of the affinity beads to SA was determined using aqueous solution of SA in a batch system. Batch adsorption studies showed that the amount of adsorbed SA was found to be 156.7 mg/g at 25 °C. The maximum adsorption capacity for affinity beads was observed at around pH 5.5. Adsorption of SA onto affinity beads significantly increased with increasing temperature, and reached a value 177.8 mg/g beads at 35 °C. The equilibrium data were found to be well described by Langmuir model, while the kinetic data were well fitted to the pseudo-second-order kinetic. The degree of the purity of SA was determined by using HPLC. Before and after adsorption, the peak areas of SA were used in the calculation of separated SA.