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Mar Drugs ; 11(4): 1113-25, 2013 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-23549283


This study was aimed at developing a sensitive and selective HPLC method with postcolumn fluorescence derivatization for the detection of propylene glycol alginate sodium sulfate (PSS) in rat plasma. Plasma samples were prepared by a simple and fast ultrafiltration method. PSS was extracted from rat plasma with D-glucuronic acid as internal standard. Isocratic chromatographic separation was performed on a TSKgel G2500 PWxL column with the mobile phase of 0.1 M sodium sulfate at a flow rate of 0.5 mL/min. Analyte detection was achieved by fluorescence detection (FLD) at 250 nm (excitation) and 435 nm (emission) using guanidine hydrochloride as postcolumn derivatizing reagent in an alkaline medium at 120 °C. The calibration curve was linear over a concentration range of 1-500 µg/mL, and the lower limit of detection (LLOD) was found to be 250 ng/mL. This validated method was applied successfully to the pharmacokinetic study of PSS and PSS-loaded poly lactic-co-glycolic acid (PLGA) nanoparticles (PSS-NP) in rat plasma after a single intravenous (PSS only) and oral administration (PSS and PSS-NP). Significant differences in the main pharmacokinetic parameters of PSS and PSS-NP were observed. The relative bioavailability of PSS-NP was 190.10% compared with PSS which shows that PSS-NP can improve oral bioavailability.

Alginatos/farmacocinética , Cromatografia Líquida de Alta Pressão/métodos , Ácido Láctico/química , Nanopartículas , Ácido Poliglicólico/química , Administração Oral , Alginatos/análise , Alginatos/química , Animais , Disponibilidade Biológica , Calibragem , Feminino , Fluorescência , Injeções Intravenosas , Limite de Detecção , Masculino , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos , Ratos Wistar , Sulfatos/química
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 30(1): 95-8, 2013 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-23450490


OBJECTIVE: To screen for potential mutations in an ethnic Han Chinese family from Shanxi with hereditary multiple exostoses. METHODS: Polymerase chain reaction and DNA sequencing were used to screen potential mutations in EXT1 and EXT2 genes. RESULTS: For EXT1 gene, two synonymous mutations (P477P and E587E), three intronic mutations (c.1537 -48A>G, c.1721 +203A>G and c.1722 -103C>G) were detected. For EXT2 gene, five intronic mutations (c.-29 -148A>T, c.1080 -18T>A, c.1336 -93C>T, c.1526 -166C>T, and c.1526 -195C>T) were identified. Among these, EXT1 P477P, EXT1 E587E and EXT2 c.1080 -18T>A are polymorphisms listed by Multiple Osteochondroma Mutation Database, whilst the other 7 sites have not been reported. CONCLUSION: No mutations have been found among all exons of the EXT1 and EXT2 genes in this family. Linkage analysis is necessary for identifying the cause of this disease.

Grupo com Ancestrais do Continente Asiático/genética , Exostose Múltipla Hereditária/diagnóstico , Exostose Múltipla Hereditária/genética , Mutação , N-Acetilglucosaminiltransferases/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Criança , Pré-Escolar , China , Éxons , Feminino , Genótipo , Humanos , Íntrons , Masculino , Pessoa de Meia-Idade , Adulto Jovem
Braz. j. pharm. sci ; 49(1): 185-191, Jan.-Mar. 2013. graf, tab
Artigo em Inglês | LILACS | ID: lil-671414


The urine excretion of L-carnitine (LC), acetyl-L-carnitine (ALC) and propionyl-Lcarnitine (PLC) and their relations with the antioxidant activities are presently unknown. Liquid L-carnitine (2.0 g) was administered orally as a single dose in 12 healthy subjects. Urine concentrations of LC, ALC and PLC were detected by HPLC. Superoxide dismutase (SOD), total antioxidative capacity (T-AOC), malondialdehyde (MDA) and nitrogen monoxidum (NO) activities were measured by spectrophotometric methods. The 0~2 h, 2~4 h, 4~8 h, 8~12 h, 12~24 h excretion of LC was 53.13±31.36 µmol, 166.93±76.87 µmol, 219.92±76.30 µmol, 100.48±23.89 µmol, 72.07±25.77 µmol, respectively. The excretion of ALC was 29.70±14.43 µmol, 80.59±32.70 µmol, 109.85±49.21 µmol, 58.65±18.55 µmol, and 80.43±35.44 µmol, respectively. The urine concentration of PLC was 6.63±4.50 µmol, 15.33±12.59 µmol, 15.46±6.26 µmol, 13.41±11.66 µmol and 9.67±7.92 µmol, respectively. The accumulated excretion rate of LC was 6.1% within 24h after its administration. There was also an increase in urine concentrations of SOD and T-AOC, and a decrease in NO and MDA. A positive correlation was found between urine concentrations of LC and SOD (r = 0.8277) or T-AOC (r = 0.9547), and a negative correlation was found between urine LC excretions and NO (r = -0.8575) or MDA (r = 0.7085). In conclusion, a single oral LC administration let to a gradual increase in urine L-carnitine excretion which was associated with an increase in urine antioxidant enzymes and the total antioxidant capacities. These data may be useful in designing therapeutic regimens of LC or its analogues in the future.

A excreção urinária de L-carnitina (LC), acetil-L-carnitina (ALC) e propionil-L-carnitine (PLC) e as suas relações com as atividades antioxidantes são presentemente desconhecidos. Líquido de L-carnitina (2,0 g) foi administrada por via oral como uma dose única em 12 indivíduos saudáveis. As concentrações urinárias de LC, PLC e ALC foram detectados por HPLC. Atividades superóxido dismutase (SOD), a capacidade antioxidante total (T-AOC), malondialdeído (MDA) e óxido nítrico (NO) foram medidas por métodos espectrofotométricos. O 0~2 h, 2~4 h, 4~8 h, 8~12 h, 12~24 h excreção de LC foi 53,13±31.36 µmol, 166,93±76.87 µmol, 219,92±76.30 µmol, 100,48±23.89 µmol, 72,07±25.77 µmol, respectivamente. A excreηão de ALC foi 29,70±14.43 µmol, 80,59±32.70 µmol, 109,85±49.21 µmol, 58,65±18.55 µmol, e 80,43±35.44 µmol, respectivamente. A concentraηão de urina de PLC foi 6,63±4.50 µmol, 15,33±12.59 µmol, 15,46±6.26 µmol, 13,41±11.66 µmol e 9,67±7.92 µmol, respectivamente. A taxa de excreηão acumulada de LC foi de 6,1% 24 horas após sua administração. Houve também um aumento nas concentrações de urina de SOD e T-COA e diminuição de NO e de MDA. Correlação positiva foi encontrada entre as concentrações de urina de LC e SOD (r = 0,8277) ou T-AOC (r = 0,9547) e correlação negativa entre a excreção de LC e NO (r = -0,8575) ou MDA (r = 0,7085). Em conclusão, a administração oral única de LC leva ao aumento gradual na excreção urinária de L-carnitina, que foi associada com o aumento das enzimas antioxidantes na urina e as capacidades antioxidantes totais. Estes dados podem ser úteis no futuro para o planejamento de esquemas terapêuticos de LC ou os seus análogos, no futuro.

Humanos , Acetilcarnitina/farmacocinética , Carnitina/farmacocinética , Antioxidantes/farmacocinética , Cromatografia Líquida de Alta Pressão/métodos
Zhonghua Yi Xue Za Zhi ; 93(46): 3698-702, 2013 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-24534354


OBJECTIVE: To explore the correlations of integrons, gene cassettes and drug resistance phenotypes in 90 multi-drug resistant Gram-negative bacteria. METHODS: Class I/II/III integron and variable region of positive strains of 90 Gram-negative bacteria were amplified by PCR and types of integron variable region gene cassettes analyzed by DNA sequence. And the resistant rates of integron positive and negative strains were tested by drug susceptibility. RESULTS: The detection rate of integron was 81.1% (73/90) in 90 Gram-negative bacteria. The integron types were class I (n = 70), class II (n = 3) and class III (n = 0). Based on the BLAST analysis by GenBank database, in the amplified fragments of Class I integron positive strains variable region gene ranging from 730 to 3300 bp, 8 types of integron structure were identified. And there were aadB (n = 11), aac (6')-II (n = 7), aadA5 (n = 10), dfrA17-aadA5 (n = 14), dfrA12-OrfF-aadA2(n = 1), aacA4-catB8-aadA1(n = 24), aacC1-OrfA-OrfB-aadA1 (n = 3), catB3-aadB-dhfrV-aacA4-nit1-nit2 (n = 1), in which catB3-aadB-dhfrV-aacA4-nit1-nit2 was a new resistance gene cassette; the variable region fragment of class II integron positive strain was 1600 bp, with 3 carrier strains of sat2-aadA1 gene cassette.Susceptibility testing showed that the antimicrobial resistance rate of integron positive strains to aminoglycosides and sulfa were significantly higher than those of integron negative strains and accorded with the results of integration variable region gene cassettes; the positive strains were more sensitive to amikacin with a resistance rate of 32.9% (24/73); and the drug resistance rates of all beta-lactam strains were ≥ 80%. CONCLUSIONS: There is a higher carrier rates of classI integron in Gram-negative bacteria. And the resistant phenotype is related with the types of resistance gene cassettes of integron variable region.

Farmacorresistência Bacteriana Múltipla/genética , Bactérias Gram-Negativas/efeitos dos fármacos , Integrons/genética , Antibacterianos/farmacologia , Sequência de Bases , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética
Fen Zi Xi Bao Sheng Wu Xue Bao ; 40(5): 329-38, 2007 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-18254338


Here reported a 1060 bp cDNA cloning of LeCOP1LIKE gene by EST probing and RT-PCR method. In order to characterize function of this gene, a LeCOP1LIKE antisense expression vector was transformed into Micro-Tom via Agrobacterium-mediated transformation method and 10 independent transgenic lines were obtained. RT-PCR analysis showed that the expression of LeCOP1LIKE gene was evidently repressed in 4 lines of them. The transgenic plants were much shorter than their wild type control, their chlorophyll content was increased but the seed development was obviously suppressed. All these results suggested that the cloned LeCOP1LIKE gene was a negative regulator of photomorphogenesis in tomato.

Carotenoides/genética , DNA Antissenso/genética , DNA Complementar/genética , Lycopersicon esculentum/genética , Plantas Geneticamente Modificadas/metabolismo , Carotenoides/metabolismo , DNA Antissenso/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Vetores Genéticos , Licopeno , Lycopersicon esculentum/crescimento & desenvolvimento , Rhizobium/genética , Transfecção
Fen Zi Xi Bao Sheng Wu Xue Bao ; 39(1): 1-8, 2006 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-16944565


Plant receptor-like kinases (RLKs) play important roles in plant growth, development and responses to environmental stress. RNA interference (RNAi) is a powerful tool to study functions of RLKs. In this study, a 312bp 3'end cDNA fragment of the soybean receptor-like kinase gene rlpk2 was chosen to construct the rlpk2-RNAi expression cassette. The binary vector pART27-R2 harboring rlpk2-RNAi expression cassette was constructed through three times subcloning via mid-clone vector and then transformed into Agrobacterium LBA4404. Three independent transgenic plants were obtained by Agrobactium-mediated soybean cotyledon transformation method. RT-PCR analysis showed that rlpk2 gene was successfully knocked down in all transgenic plants. It was found that photosynthesis activities of rlpk2-RNAi transgenic leaves were much improved, suggesting rlpk2 may function as a negative regulator of soybean leaf functions and/or chloroplast structure.

Vetores Genéticos/genética , Proteínas de Plantas/genética , Interferência de RNA , Soja/genética , Transformação Genética/genética , Regulação da Expressão Gênica de Plantas , Modelos Genéticos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Soja/crescimento & desenvolvimento , Soja/metabolismo
Fen Zi Xi Bao Sheng Wu Xue Bao ; 39(3): 208-16, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16944594


Protein kinases play key roles in transduction of both environmental and developmental signals in higher plants. Here reported full-length cDNA cloning and preliminary structural and functional analysis of a novel soybean receptor-like protein kinase (RLK) gene (GenBank Accession No.AY687390). The deduced protein product of this gene contains a transmembrane region, an intracellular catalytic domain possessing serine/threonine kinase activity, and an extracellular domain which lacks the N-terminus signal peptide. Bioinformatic analyses revealed highly similarity between this soybean gene and a group of the Arabidopsis RLK genes which all lack of N-terminus signal sequence and belong to RLCK subfamily. Thus, it was designated GmRLCK. Some residues of GmRLCK that showed high probability to be phosphorylated were also predicted. RT-PCR analysis revealed higher expression levels of GmRLCK in soybean cotyledons, roots, flowers and young pods; whereas lower expression levels were detected in radicles, stems and mature leaves at vegetative stage. Phylogeny analysis suggested that GmRLCK was more close to and might have a common ancestor with several senescence-associated RLKs.

Proteínas Quinases/genética , Proteínas de Soja/genética , Soja/genética , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis , Clonagem Molecular , DNA de Plantas/análise , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Dados de Sequência Molecular , Fosforilação , Filogenia , Proteínas Quinases/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas de Soja/metabolismo , Soja/classificação , Soja/metabolismo
Plant Mol Biol ; 61(6): 829-44, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16927199


We report here the cloning and characterization of a soybean receptor-like kinase (RLK) gene, designated GmSARK (Glycine max senescence-associated receptor-like kinase), which is involved in regulating leaf senescence. The conceptual protein product of GmSARK contains typical domains of LRR receptor-like kinases: a cytoplasmic domain with all the 11 kinase subdomains, a transmembrane domain and an extracelullar domain containing 9 Leucine-Rich Repeat (LRR) units that may act as a receptor. The expression of GmSARK in soybean leaves was up-regulated in all the three tested senescence systems: senescing cotyledons, dark-induced primary leaf senescence and the natural leaf senescence process after florescence. Furthermore, the RNA interference (RNAi)-mediated knocking-down of GmSARK dramatically retarded soybean leaf senescence. A more complex thylakoid membrane system, higher foliar level of chlorophyll content and a very remarkable delay of senescence-induced disintegration of chloroplast structure were observed in GmSARK-RNAi transgenic leaves. A homolog of maize lethal leaf-spot 1 gene, which has been suggested to encode a key enzyme catalyzing chlorophyll breakdown, was isolated and nominated Gmlls1. The expression level of Gmgtr1 gene, which encodes a key enzyme of chlorophyll synthesis, was also analyzed. It was found that Gmlls1 was up-regulated and Gmgtr1 was down-regulated during senescence in wild-type soybean leaves. However, both of the up-regulation of Gmlls1 and down-regulation of Gmgtr1 were retarded during senescence of GmSARK-RNAi transgenic leaves. In addition, over-expression of the GmSARK gene greatly accelerated the senescence progression of CaMV 35S:GmSARK transgenic plants. Taken together, these results strongly suggested the involvement of this LRR-RLK in regulation of soybean leaf senescence, maybe via regulating chloroplast development and chlorophyll accumulation. Multiple functions of GmSARK besides its regulation of leaf senescence were also discussed.

Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas Quinases/genética , Soja/genética , Sequência de Aminoácidos , Cloroplastos/fisiologia , Cloroplastos/ultraestrutura , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Flores/enzimologia , Flores/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Folhas de Planta/enzimologia , Folhas de Planta/fisiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/metabolismo , Interferência de RNA , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Soja/enzimologia , Soja/fisiologia , Tilacoides/ultraestrutura
Artigo em Chinês | MEDLINE | ID: mdl-16775399


5'-Flanking region of rlpk2 gene was cloned by using PCR-based genomic DNA walking method (Fig.1), and designated P(rlpk2) (GenBank Accession Number: AY870314). Results of sequencing analysis showed that P(rlpk2) contains several putative cis-elements that respond to CTK, ABA, GA or IAA, as well as elements that respond to different environmental stresses, such as dehydration, coldness, wounding and etc. An expression vector containing the P(rlpk2)-GUS fusion gene was constructed (Fig.2) and transferred into soybean seedlings and Micro-Tom cotyledon explants by Agrobacterium-mediated method. A strong transient expression of GUS was observed in both soybean plumule and Micro-Tom cotyledon callus (Fig.3), thus confirming the promoter activity of P(rlpk2).

Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Proteínas Quinases/genética , Soja/genética , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Dados de Sequência Molecular , Reguladores de Crescimento de Planta/farmacologia , Soja/enzimologia