RESUMO
We synthesized a previously identified ß-tubulin-derived G protein-coupled receptor kinase 2 (GKR2) peptide (GR-11-1; DEMEFTEAESNMN) and its amino-terminal extension (GR-11-1-N; GEGMDEMEFTEAESNMN) and carboxyl-terminal extension (GR-11-1-C; DEMEFTEAESNMNDLVSEYQ) peptides with the aim of finding a high-affinity peptide substrate for GRK2. GR-11-1-C showed high affinity for GRK2, but very low affinity for GKR5. Its specificity and sensitivity for GKR2 were greater than those of GR-11-1 and GR-11-1-N. These findings should be useful in designing tools for probing GKR2-mediated intracellular signaling pathways, as well as GRK2-specific drugs.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Insetos , Fosforilação , Transdução de Sinais/fisiologia , Tubulina (Proteína)/metabolismoRESUMO
In clathrin-mediated endocytosis (CME), specificity and selectivity for cargoes are thought to be tightly regulated by cargo-specific adaptors for distinct cellular functions. Here, we show that the actin-binding protein girdin is a regulator of cargo-selective CME. Girdin interacts with dynamin 2, a GTPase that excises endocytic vesicles from the plasma membrane, and functions as its GTPase-activating protein. Interestingly, girdin depletion leads to the defect in clathrin-coated pit formation in the center of cells. Also, we find that girdin differentially interacts with some cargoes, which competitively prevents girdin from interacting with dynamin 2 and confers the cargo selectivity for CME. Therefore, girdin regulates transferrin and E-cadherin endocytosis in the center of cells and their subsequent polarized intracellular localization, but has no effect on integrin and epidermal growth factor receptor endocytosis that occurs at the cell periphery. Our results reveal that girdin regulates selective CME via a mechanism involving dynamin 2, but not by operating as a cargo-specific adaptor.
Assuntos
Dinamina II/metabolismo , Endocitose , Células Epiteliais/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Linhagem Celular , Membrana Celular/enzimologia , Membrana Celular/metabolismo , HumanosRESUMO
In the course of measuring the intracellular antibacterial activity of antibiotics using a human alveolar epithelial cell line A549, we discovered that the antimicrobial activity of several carbapenems (CPs) decreased in the supernatant of the cells cultured with fetal calf serum (FCS)-free RPMI1640 medium (RPMI). Further investigation revealed A549 culture supernatant inhibited the antibacterial activity of CPs but did not inactivate other types of antibiotics. CE-TOFMS and LC-TOFMS metabolomics analysis of the supernatant revealed the presence of l-cysteine (Cys), which is not an original component in RPMI. Cys is known to hydrolyze and inactivate CPs in a time- and concentration-dependent manner. In this study, the inactivating effects of A549 culture supernatant on the imipenem (IPM) were examined. Antimicrobial activity of 100 µg/mL IPM decreased to 25% with two-fold dilution of A549 supernatant incubated for 3 h. l-Cystine (CS), the Cys oxide, and an original component in RPMI did not inactivate IPM. However, the inactivating effects of A549 supernatant on IPM corresponds with the Cys concentration and depends on the CS content of the culture medium. Addition of FCS to the culture medium decreased the Cys concentration and reduced inactivation of IPM in a dose-dependent manner. Our data suggest that IPM were inactivated by Cys reduced from CS, and this CS-to-Cys conversion must be considered when evaluating the antimicrobial activity of CPs in cell culture. Further studies are needed to understand if the same inactivation occurs around the cells in the human body.
Assuntos
Antibacterianos/metabolismo , Carbapenêmicos/metabolismo , Cisteína/metabolismo , Cistina/metabolismo , Imipenem/metabolismo , Células A549 , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Meios de Cultura Livres de Soro/química , Meios de Cultura Livres de Soro/metabolismo , Relação Dose-Resposta a Droga , Humanos , Imipenem/farmacologia , Inativação Metabólica , Metabolômica , Micrococcus luteus/efeitos dos fármacos , OxirreduçãoRESUMO
A series of amino acid substitutions was made in a previously identified ß-tubulin-derived GRK2 substrate peptide (404DEMEFTEAESNMN416) to examine the role of amino acid residues surrounding the phosphorylation site. Anionic amino acid residues surrounding the phosphorylation site played an important role in the affinity for GRK2. Compared to the original peptide, a modified peptide (Ac-EEMEFSEAEANMN-NH2) exhibited markedly higher affinity for GRK2, but very low affinity for GRK5, suggesting that it can be a sensitive and selective peptide for GRK2.
Assuntos
Substituição de Aminoácidos/genética , Quinase 2 de Receptor Acoplado a Proteína G/genética , Peptídeos/química , Tubulina (Proteína)/química , Sequência de Aminoácidos/genética , Quinase 2 de Receptor Acoplado a Proteína G/química , Humanos , Fosforilação , Especificidade por SubstratoRESUMO
The plant, Cynomorium songaricum Rupr., is used as a traditional medicine in China and Mongolia. In the present study, two new water-soluble polysaccharides isolated from C. songaricum Rupr. were purified by successive Sephadex G-75 and G-50 column chromatographies and then characterized by high resolution NMR and IR spectroscopies. The molecular weights of two polysaccharides were determined by an aqueous GPC to be [Formula: see text] = 3.7 × 10(4) and 1.0 × 10(4), respectively. In addition, it was found that the polysaccharide with the larger molecular weight was an acidic polysaccharide. It was found that the iodine-starch reaction of both isolated polysaccharides was negative and the methylation analysis gave 2, 4, 6-tri-O-methyl alditol acetate as a main product. NMR and IR measurements and sugar analysis revealed that both polysaccharides had a (1 â 3)-α-d-glucopyranosidic main chain with a small number of branches. After sulfation, the sulfated C. songaricum Rupr. polysaccharides were found to have a potent inhibitory effect on HIV infection of MT-4 cells at a 50% effective concentration of 0.3-0.4 µg/ml, a concentration that has almost the same high activity as standard dextran and curdlan sulfates, EC50 = 0.35 and 0.14 µg/ml, respectively. The 50% cytotoxic concentration was low, CC50>1000 µg/ml. In addition, the interaction between the sulfated polysaccharides and poly-l-lysine as a model protein compound was investigated by a surface plasmon resonance to reveal the anti-HIV mechanism.
Assuntos
Fármacos Anti-HIV/isolamento & purificação , Fármacos Anti-HIV/farmacologia , Cynomorium/química , Polissacarídeos/isolamento & purificação , Polissacarídeos/farmacologia , Fármacos Anti-HIV/química , China , Dextranos , Infecções por HIV/tratamento farmacológico , Humanos , Medicina Tradicional Chinesa , Medicina Tradicional da Mongólia , Modelos Biológicos , Estrutura Molecular , Peso Molecular , Ressonância Magnética Nuclear Biomolecular , Caules de Planta/química , Polilisina/química , Polissacarídeos/química , Solubilidade , Espectrofotometria Infravermelho , ÁguaRESUMO
Among synthetic kinesin spindle protein (KSP) inhibitor compounds, KPYB10602, a six-member lactam-fused carbazole derivative was the most potent in vitro against cell growth of human ovarian cancer, A2780. KPYB10602 caused dose-dependent suppression of tumor growth in vivo. Mitotic arrest due to KPYB10602 was confirmed in vitro, and was characterized by inhibition of securin degradation. Apoptosis after mitotic arrest was associated with an increase in the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Increase of reactive oxygen species (ROS) and caspase pathway were also involved. Furthermore, KPYB10602 caused little neurotoxicity in vivo. Therefore, KPYB10602 could be a promising candidate as an anti-tumor agent with reduced adverse events for treating human ovarian cancer.
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Cinesinas/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Ovário/efeitos dos fármacos , Animais , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Cinesinas/metabolismo , Camundongos Endogâmicos BALB C , Mitose/efeitos dos fármacos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Ovário/metabolismo , Ovário/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
We describe a case of central venous catheter-related fungemia caused by Cryptococcus liquefaciens, a non-neoformans and non-gattii Cryptococcus, in a non-HIV patient. A 71-year-old man with diffuse large B-cell lymphoma receiving antineoplastic chemotherapy was febrile approximately 30 weeks after central venous port insertion, and C. liquefaciens was isolated from all three performed blood cultures as well as a central venous catheter tip culture. In vitro antifungal susceptibility tests showed that this yeast isolate was susceptible to low concentrations of amphotericin B, fluconazole, itraconazole and voriconazole yet was resistant to 5-fluorocytosine (MIC: >64 µg/ml), unlike Cryptococcus neoformans. Treatment of the patient with oral and intravenous voriconazole was effective and consistent with the susceptibility tests. Although non-neoformans and non-gattii Cryptococcus spp. are considered non-pathogenic environmental yeast, they may rarely be the causative agents of serious infections in humans, as in the present case.
Assuntos
Infecções Relacionadas a Cateter/microbiologia , Cateteres Venosos Centrais/efeitos adversos , Criptococose/microbiologia , Fungemia/microbiologia , Idoso , Infecções Relacionadas a Cateter/tratamento farmacológico , Cateteres Venosos Centrais/microbiologia , Criptococose/tratamento farmacológico , Fungemia/tratamento farmacológico , Humanos , MasculinoRESUMO
The nationwide surveillance of antibacterial susceptibility to meropenem (MEPM) and other parenteral antibiotics against clinical isolates during 2012 in Japan was conducted. A total of 2985 strains including 955 strains of Gram-positive bacteria, 1782 strains of Gram-negative bacteria, and 248 strains of anaerobic bacteria obtained from 31 medical institutions were examined. The results were as follows; 1. MEPM was more active than the other carbapenem antibiotics tested against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MEPM was also active against most of the species tested in Gram-positive and anaerobic bacteria, except for multi-drug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA). 2. Of all species tested, there were no species, which MIC90 of MEPM was more than 4-fold higher than those in our previous studies in 2009 or 2006. Therefore, the tendency to increase in antimicrobial resistance rates was not observed. 3. MEPM resistance against Pseudomonas aeruginosa was 17.8% (56/315 strains). Compared to our previous results, it was the lowest than that in 2006 and 2009. 4. Carbapenem-resistant Klebsiella pneumoniae, and multi-drug-resistant Acinetobacter species, which emerged in worldwide, were not observed. 5. The proportion of extended-spectrum beta-lactamase (ESBL) strains was 6.2% (59/951 strains) in enterobacteriaceae, which increased compared with that of our previous studies in 2009 or before. Whereas, the proportion of metallo-beta-lactamase strains was 1.6% (5/315 strains) in P. aeruginosa, which was stable. In conclusion, the results from this surveillance suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem for serious infections treatment at present, 17 years passed after available for commercial use in Japan.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Tienamicinas/farmacologia , Farmacorresistência Bacteriana , Humanos , Meropeném , Testes de Sensibilidade MicrobianaRESUMO
Dynamin plays an important role in membrane fission during endocytosis, and its middle domain is involved in the formation of functional oligomers. In this study, we found that replacement of Arg-386 with Gly in the middle domain region of dynamin 1 did not affect the intermolecular interactions of dynamin 1 in the presence of phosphatidylserine-liposomes. But, unexpectedly, this variant showed lower guanosine 5'-triphosphatase activity in the absence of phosphatidylserine-liposomes and enhanced monomer formation from oligomers. Our results indicate that GTPase activity in the absence of lipids is important in the dissociation of oligomer complexes, i.e., reduced basal dynamin 1 GTPase activity is associated with instability of dynamin oligomers.
Assuntos
Substituição de Aminoácidos , Arginina , Dinamina I/química , Dinamina I/metabolismo , Glicina , Multimerização Proteica/genética , Sequência de Aminoácidos , Dinamina I/genética , Endocitose/genética , Estabilidade Enzimática/genética , Glicina/genética , Glicina/metabolismo , Células HeLa , Humanos , Lipossomos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fosfatidilserinas/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Transferrina/metabolismoRESUMO
Boron neutron capture therapy (BNCT) is a type of radiation therapy and a new modality for cancer treatment. The radiation used in BNCT is a very low energy neutron called a "thermal neutron", and unlike other radiation, it has no effect on treating cancer on its own. However, when this neutron collides with boron-10 (10B), which is a stable isotope of boron, fission occurs into a high-energy helium nucleus (α-particle) and a lithium nucleus. Moreover, the effect of this fission reaction is limited to a range of about 10 µm, which corresponds to the approximate size of one cell. Therefore, the basic principle of BNCT is "cell-selective" radiation therapy that only damages cells that have taken up 10B present in the area irradiated with thermal neutrons. For the practical application of BNCT, it is indispensable to generate a boron drug capable of selectively accumulating 10B in cancer cells. We have successfully developed a boron drug for BNCT targeting amino acid transporters. We have obtained manufacturing and marketing approval for the world's first boron drug for BNCT, Steboronine® intravenous drip bag 9000 mg/300 mL (March 25, 2020), for indications of locally unresectable recurrent or advanced unresectable head and neck cancer. This uses Borofalan (10B), which is 10B introduced into l-phenylalanine, as a drug substance. This review describes the progress of drug development and future prospects of boron drugs for BNCT.
Assuntos
Terapia por Captura de Nêutron de Boro/métodos , Boro , Desenvolvimento de Medicamentos/métodos , Neoplasias de Cabeça e Pescoço/radioterapia , Isótopos , Sistemas de Transporte de Aminoácidos , Boro/administração & dosagem , Boro/uso terapêutico , Humanos , Infusões Intravenosas , Isótopos/administração & dosagem , Isótopos/uso terapêutico , Nêutrons , Fissão Nuclear , FenilalaninaRESUMO
The antibacterial activity of meropenem (MEPM) and other parenteral antibiotics against clinical isolates of 2655 strains including 810 strains of Gram-positive bacteria, 1635 strains of Gram-negative bacteria, and 210 strains of anaerobic bacteria obtained from 30 medical institutions during 2009 was examined. The results were as follows; (1) MEPM was more active than the other carbapenem antibiotics tested against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MEPM was also active against most of the species tested in Gram-positive and anaerobic bacteria, except for multidrug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA). (2) MEPM maintained potent and stable antibacterial activity against Pseudomonas aeruginosa. The proportion of MEPM-resistant strains to ciprofloxacin-resistant strains or imipenem-resistant strains were 53.1% and 58.0% respectively. (3) The proportion of extended-spectrum beta-lactamase (ESBL) strains was 3.1% (26 strains) in enterobacteriaceae. And the proportion of metallo-beta-lactamase strains was 2.0% (6 strains) in P. aeruginosa. (4) Of all species tested, there were no species except for Bacteroides fragilis group, which MIC90 of MEPM was more than 4-fold higher than those in our previous study. Therefore, there is almost no significant decrease in susceptibility of clinical isolates to meropenem. In conclusion, the results from this surveillance study suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem for serious infections treatment at present, 14 years passed after available for commercial use in Japan.
Assuntos
Antibacterianos/farmacologia , Bactérias Anaeróbias/efeitos dos fármacos , Bactérias Anaeróbias/isolamento & purificação , Infecções Bacterianas/microbiologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/isolamento & purificação , Tienamicinas/farmacologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Formas de Dosagem , Farmacorresistência Bacteriana , Humanos , Lactente , Recém-Nascido , Japão , Meropeném , Pessoa de Meia-Idade , Sistema Respiratório/microbiologia , Fatores de Tempo , Urina/microbiologia , Adulto JovemRESUMO
Dynamin (Dyn) 1 plays a role in recycling of synaptic vesicles, and thus in nervous system function. We previously showed that sertraline, a selective serotonin reuptake inhibitor (SSRI), is a mixed-type inhibitor of Dyn 1 with respect to both GTP and L-alpha-phosphatidyl-L-serine (PS) in vitro, and we suggested that it may regulate the neurotransmitter transport by modulating synaptic vesicle endocytosis via inhibition of Dyn 1 GTPase. Here, we investigated the effect of sertraline on endocytosis of marker proteins in human neuroblastoma SH-Sy5Y cells and HeLa cells. Sertraline inhibited endocytosis in both cell lines. Western blotting showed that SH-Sy5Y expresses Dyn 1 and Dyn 2, while HeLa expresses only Dyn 2. GTPase assay showed that sertraline inhibited Dyn 2 as well as Dyn 1. Therefore, the effect of sertraline on endocytosis was mediated by Dyn 2, at least in HeLa cells, as well as by Dyn 1 in cell lines that express it. Moreover, the inhibition mechanism of transferrin (Tf) uptake by sertraline differed from that in cells expressing Dyn 1 K44A, a GTP binding-defective variant, and sertraline did not interfere with the interaction between Dyn 1 and PS-liposomes.
Assuntos
Dinamina II/antagonistas & inibidores , Dinamina I/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Sertralina/farmacologia , Proteínas Sanguíneas/química , Linhagem Celular Tumoral , Dinamina I/genética , Dinamina I/metabolismo , Dinamina II/genética , Dinamina II/metabolismo , Células HeLa , Humanos , Neurônios/metabolismo , Fosfatidilserinas/metabolismo , Fosfoproteínas/química , Estrutura Terciária de Proteína/genética , Transferrina/metabolismoRESUMO
BACKGROUND: In order to investigate the physiological role of lignin carbohydrate complex present in Lentinus edodes mycelia extract (LEM), this material was separated into seven fractions. MATERIALS AND METHODS: Three high molecular weight fractions (Frs. I-III) were prepared from the water extract by successive ethanol fractionation, dialysis and lyophilization. Four higher molecular weight fractions were prepared from the NaOH extract of the residue, followed by acid precipitation (Fr. IV) and stepwise ethanol precipitation (Frs. V-VII). RESULTS: All fractions showed higher anti-HIV activity than the water extract. Fr. IV showed the highest anti-HIV activity and most potently inhibited the NO production by LPS-stimulated mouse macrophage-like cells (RAW264.7, J774.1). ESR spectroscopy demonstrated that all fractions scavenged superoxide anion and hydroxyl radical. These properties are similar to those displayed by lignin carbohydrate complex, but not by glucans. HPLC analysis demonstrated the presence of lignin precursors, but not that of tannins, flavonoids and their related compounds. CONCLUSION: These results suggest a significant role of lignin-like substances in the expression of several important biological properties displayed by LEM.
Assuntos
Proteínas Fúngicas/química , Lignina/análise , Lignina/farmacologia , Polissacarídeos/química , Animais , Fármacos Anti-HIV/isolamento & purificação , Fármacos Anti-HIV/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Sequestradores de Radicais Livres/farmacologia , HIV/efeitos dos fármacos , Radical Hidroxila/metabolismo , Lignina/isolamento & purificação , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Peso Molecular , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/metabolismo , Superóxidos/metabolismoRESUMO
Lysophosphatidylcholine (lysoPtdCho) is produced by the phospholipase A2-mediated hydrolysis of phosphatidylcholine and can stimulate proliferation and apoptosis of vascular smooth muscle cells. We examined the influence of fetal bovine serum (FBS) concentration in the culture medium on lysoPtdCho-mediated apoptosis and proliferation of human aortic smooth muscle cells (HASMCs) as well as on the activation of extracellular signal-regulated kinases (ERK)1/2. In the presence of 1% FBS, HASMC viability increased after lysoPtdCho treatment at 1 and 10 µM but decreased at 25 and 50 µM. However, lysoPtdCho increased HASMC viability in a dose-dependent manner in the presence of 10% FBS. The activity of caspase 3/7 in HASMCs was increased by 25 µM lysoPtdCho in the presence of 1% FBS, but not 10% FBS. Furthermore, lysoPtdCho at 1 and 10 µM triggered ERK1/2 phosphorylation in the presence of 1% FBS, but not at 10% FBS. Thus, lysoPtdCho-mediated HASMC apoptosis, proliferation, and ERK1/2 activation are dependent on the concentration of FBS.
Assuntos
Aorta/citologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Lisofosfatidilcolinas/farmacologia , Músculo Liso Vascular/citologia , Soro/fisiologia , Animais , Bovinos , Células Cultivadas , Ativação Enzimática , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismoRESUMO
To elucidate the role of long alkyl group in sulfated poly- and oligosaccharides on anti-HIV activity, the interaction between sulfated 3-O-octadecyl-(1â6)-α-d-glucopyranan with potent anti-HIV activity and liposomes with diameters of 58 ± 20 nm and 107 ± 28 nm as models of HIV were investigated. SPR measurements of sulfated 3-O-octadecyl-(1â6)-α-d-glucopyranans bearing 2.8 mol% of the octadecyl group and the liposome (diameter = 58 ± 20.0 nm and ζ=0 mV) resulted in an apparent association- ka = 6 × 105 1/M, a dissociation-rate kd = 4 × 10-4 1/s, and a dissociation constants KD = 8 × 10-10 M. The particle size of the sulfated 3-O-octadecyl-(1â6)-α-d-glucopyranan (67 ± 14 nm) measured by DLS increased to 104 ± 25 nm, whereas the ζ potential (-29 mV) was unchanged (-33 mV). For dextran sulfate without an alkyl group, no interaction was observed. These results suggest that the long octadecyl group penetrated into the liposome and sulfated glucopyranan was covered on the liposome to increase the anti-HIV activity. The 107 nm liposome exhibited similar results.
Assuntos
Glucanos/análise , Sulfatos/análise , Ressonância de Plasmônio de Superfície , Configuração de Carboidratos , Lipossomos/análise , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Three new spherical sulfated cellobiose-polylysine dendrimers of increasing generations bearing negatively charged sulfate groups were prepared by sulfating the corresponding cellobiose-polylysine dendrimers. The first, second, and third-generation derivatives exhibited potent anti-HIV activity with EC50 values of 3.7, 0.6, and 1.5 µg/mL, respectively, in constant to sulfated oligosaccharides with low anti-HIV activity, while the second-generation sulfated dendrimer was the most active. Surface plasmon resonance measurements with poly-l-lysine bearing positively charged amino acids as a model of the HIV surface glycoprotein gp120, indicated that the second-generation dendrimer had the lowest dissociation constant (KD = 1.86 × 10-12 M). Both the particle size and ζ potential increased in the presence of poly-l-lysine. It was proven that the moderate distance between the terminal sulfated cellobiose units in the second-generation dendrimer favored the high anti-HIV activity, owing to the electrostatic interactions developed due to the cluster effect.
Assuntos
Fármacos Anti-HIV/farmacologia , Celobiose/farmacologia , Dendrímeros/farmacologia , HIV-1/efeitos dos fármacos , Polilisina/farmacologia , Sulfatos/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Linhagem Celular Tumoral , Celobiose/química , Dendrímeros/química , Humanos , Testes de Sensibilidade Microbiana , Conformação Molecular , Polilisina/química , Sulfatos/químicaRESUMO
BACKGROUND: Control of inflammation is essential for the clinical management of many common human diseases. However, there are few generally applicable strategies to convert an abnormal intracellular signal into a gene expression that leads to normalization of the intracellular environment. Recently, we proposed a novel strategy termed D-RECS (i.e. drug or gene delivery system responding to cellular signals) to convert an intracellular signal to transgene expression. In the present study, we applied this concept to inflammatory cells using Ikappa-B kinase as a signal molecule that triggers the gene expression. METHODS: Candidate cationic substrates of Ikappa-B kinase (IKK)beta were synthesized and their reactivity was investigated. Then, polymers grafted with these peptides were prepared by radical polymerization. Polymer/DNA complexes (polyplexes) were prepared by mixing plasmid DNAs with the polymers. The behaviour of these polyplexes by adding IKKbeta was examined. Furthermore, changes of gene expression were evaluated after the microinjection of polyplex into living cells under conditions of nuclear factor (NF)-kappaB activation. RESULTS: Synthetic peptides with additional lysine residues were well phosphorylated by IKKbeta. Both the polymer and the polyplex were also phosphorylated by IKKbeta. The results of gel shift assay showed that the polyplex was disintegrated and free DNA was released in the presence of IKKbeta. The polyplex comprising-green fluorescent protein plasmid DNA and the polymer expressed the transgene in living cells exposed to a pro-inflammatory stimulus. CONCLUSIONS: Our concept of cell-specific gene expression was demonstrated to work in inflammatory cells. This method may provide a unique strategy for gene therapy exclusively in inflammatory cells.
Assuntos
Expressão Gênica , Quinase I-kappa B/metabolismo , Inflamação/metabolismo , Transgenes , Animais , Ativação Enzimática , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Quinase I-kappa B/genética , Camundongos , Estrutura Molecular , Peptídeos/genética , Peptídeos/metabolismoRESUMO
Mastic is a resinous exudate obtained from the stem and the main leaves of Pistacia lentiscus. We have reported the antiplaque effect of mastic-containing chewing gum on the oral cavity. We hypothesize that mastic may be a multifunctional food which has some beneficial pharmaceutical properties. The aim of this study was to assess the biological activity of solid and liquid types of mastic by cytotoxicity against fibroblasts, radical-scavenging activities and inhibitory effect on cell death of oral polymorphonuclear leukocytes (OPMNs). Mastic showed selective antibacterial action against Porphyromonas gingivalis and Prevotella melaninogenica, but no anti-HIV activity. Among a total of thirteen human cell types, promyelocytic leukemia HL-60 was the most sensitive to the cytotoxicity of mastic, followed by myeloblastic leukemia (ML-1, KG-1), erythroleukemia (K-562), oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4), hepatocellular carcinoma (HepG2), glioblastoma (T98G, U87MG) and normal oral cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast, most resistant). Mastic did not induce the differentiation of myelogenous leukemic cells into maturing cells with higher nitroblue tetrazolium-reducing activity, but induced apoptotic cell death, characterized by internucleosomal DNA fragmentation, caspase-3 activation and a decline in the intracellular concentration of putrescine. The cytotoxicity of mastic against leukemic cells did not diminish during its storage. On the other hand, mastic inhibited the spontaneous apoptosis of OPMNs. Mastic showed hydroxyl radical-scavenging activity. The selective antibacterial and apoptosis-modulating activity of mastic suggests its possible beneficial effects on oral health.
Assuntos
Anti-Infecciosos/farmacologia , Apoptose , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias/tratamento farmacológico , Neutrófilos/metabolismo , Resinas Vegetais/farmacologia , Caspase 3/metabolismo , Morte Celular , Diferenciação Celular , Linhagem Celular Tumoral , Fragmentação do DNA , Células HL-60 , Humanos , Células K562 , Resina Mástique , Putrescina/farmacologiaRESUMO
This study aims to quantitatively investigate the interaction between sulfated polysaccharides with potent anti-HIV activity, dextran and curdlan sulfates with negatively charged sulfate groups, and poly-L-lysine as a model protein and oligopeptides from a HIV surface glycoprotein gp120 with positively charged amino acids using surface plasmon resonance (SPR) and dynamic light scattering (DLS) to elucidate the anti-HIV mechanism of sulfated polysaccharides. The apparent association- (ka) and dissociation rate (kd) constants of dextran and curdlan sulfates against poly-L-lysine were kaâ¯=â¯6.92â¯×â¯104-2.17â¯×â¯106â¯1/Ms and kdâ¯=â¯4.29â¯×â¯10-5-2.22â¯×â¯10-4â¯1/s; these kinetic constants were dependent on the molecular weights and degree of sulfation of sulfated polysaccharides. For interaction, the three oligopeptides from the HIV gp120 were peptide A 297TRPNNNTRKRIRIQRGPGRA316 with several lysine (K) and arginine (R) in the V3 loop region, peptide B 493PLGVAPTKAKRRVVQREKR511 with several K and R in the C-terminus region, and oligopeptide C 362KQSSGGDPEIVTHSFNCGG380 with few basic amino acids in the CD4 binding domain. Sulfated polysaccharides exhibited strong interaction against oligopeptides A and B, (kaâ¯=â¯5.48â¯×â¯104-2.96â¯×â¯106â¯1/Ms. and kdâ¯=â¯1.74â¯×â¯10-4-6.24â¯×â¯10-3â¯1/s), no interaction was noted against oligopeptide C. Moreover, the particle size and zeta potential by DLS indicated the interaction between sulfated polysaccharides and oligopeptides A and B, suggesting the anti-HIV mechanism of sulfated polysaccharides to be the electrostatic interaction of negatively charged sulfated polysaccharides and HIV at the positively charged amino acid regions.
Assuntos
Oligopeptídeos/química , Peptídeos/química , Polissacarídeos/química , Sulfatos/química , Dextranos/química , Proteína gp120 do Envelope de HIV/química , Peso Molecular , Polilisina/química , Ressonância de Plasmônio de Superfície/métodos , beta-Glucanas/químicaRESUMO
Vaccines typically contain an antigen, delivery system (vehicle), and adjuvant, all of which contribute to inducing a potent immune response. Consequently, design of new vaccines is difficult, because the contributions and interactions of these components are difficult to distinguish. Here, it is aimed to develop an easy-to-use, non-immunogenic, injectable depot system for sustained antigen release that will be suitable for assessing the efficacy of prolonged antigen exposure per se for inducing an immune response. This should mimic real-life infections. Recombinant elastin-like polypeptides with periodic cysteine residues (cELPs) are selected, which reportedly show little or no immunogenicity, as carriers and tetanus toxoid (Ttd) as an antigen. After subcutaneous injection of the mixture, cELP rapidly forms a disulfide cross-linked hydrogel in situ, within which Ttd is physically incorporated, affording a biodegradable antigen depot. A series of Ttd-containing hydrogels is examined. A single injection induces high levels of tetanus antibody with high avidity for at least 20 weeks in mice. The chain length of cELP proves critical, whereas differences in hydrophobicity has little effect, although hydrophilic cELPs are more rapidly biodegraded. This system's ability to distinguish the contribution of sustained antigen release to antibody induction should be helpful for rational design of next-generation vaccines.