RESUMO
Site-specific recombination has been developed into a genetic engineering tool for higher eukaryotes. The manipulation of newly introduced DNA is now possible in the course of genetic transformation procedures, thus making the process more predictable and reliable. Also, a wide variety of chromosomal rearrangements using site-specific recombination have been documented both in metazoan and plant species. Applying such methods to plants opens new avenues for large-scale chromosome engineering in the future.
Assuntos
Engenharia Genética/métodos , Mutagênese Sítio-Dirigida/genética , Plantas/genética , Recombinação Genética/genética , Cromossomos de Plantas/genética , DNA de Plantas/genética , Técnicas de Transferência de Genes , Células Vegetais , Plantas Geneticamente ModificadasRESUMO
PURPOSE: Peptidomimetic thrombin inhibitors derived from Nalpha-(2-naphthylsulfonyl)-3-amidino-phenylalanine with different basic and acidic substituents were investigated with respect to their intestinal transport behavior. METHODS: Intestinal permeability coefficients were studied using Caco-2 monolayers and a reversed-phase HPLC method for quantitation. RESULTS: Apparent permeability coefficients Papp of compounds with a free amidino group were in general low (<10 x 10(-8) cm/s) and independent of the structure of the amide part (C-terminus). Polarized efflux, however, was strongly affected by substituents in the amide moiety yielding the following efflux ratios (ER): methylpiperidide (1) (ER 45) > piperidine carboxylic acid methylester (ER 6-11) > piperidine carboxylic acids (ER 1.9-2.9) > piperazide (ER -0.17). Efflux of (1) was temperature-dependent, but independent of the enantiomeric configuration, accompanied by an increase in transepithelial electrical resistance (TEER), and could be reduced by P-gp inhibitors (PSC 833, Cremophor EL) but not by indomethacin. Replacement of the amidine group of (1) by aminomethyl-, amino-, and oxamidine- moieties drastically increased absorptive permeability (46-68 fold) with ER < 3.4. In contrast, the oxamidine with a C-terminal nipecotic acid residue (8) displayed also a temperature dependent efflux- without altering TEER (ER 22). This efflux was sensitive to PSC833/Cremophor EL and indomethacin. CONCLUSIONS: Basic and acidic residues of amidino-phenylalanine-derived thrombin inhibitors mediate affinity to intestinal efflux pumps. presumably P-gp and MRP. P-gp mediated efflux was related to a net positive charge and accompanied by an increased TEER. Among the methylpiperide (1) promoieties studied the oxamidino group seems to be very promising in overcoming both transport and efflux problems frequently encountered with peptidomimetics containing amidines.
Assuntos
Amidinas/metabolismo , Hemostáticos/antagonistas & inibidores , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Trombina/antagonistas & inibidores , Amidinas/química , Células CACO-2 , Permeabilidade da Membrana Celular , Fenômenos Químicos , Físico-Química , Cromatografia Líquida de Alta Pressão , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Indicadores e Reagentes , Absorção Intestinal/efeitos dos fármacos , Mimetismo Molecular , Fenilalanina/química , Solubilidade , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Relação Estrutura-Atividade , TemperaturaRESUMO
PURPOSE: Aim of the study was the evaluation of the potential of novel tetanus toxoid (TT) loaded nanoparticles (NP) for electing an immune response in mice against TT. METHODS: Six week-old female Balb/c mice were immunized by oral (p.o.), nasal (i.n.) and intraperitoneal (i.p.) application of TT NP loaded by adsorption. As polymer a novel polyester, sulfobutylated poly(vinyl alcohol)-graft-poly(lactide-co-glycolide), SB(43)-PVAL-g-PLGA was used. Blood samples were collected 4 and 6 weeks after immunization and assayed for serum IgG- as well as IgA antibody titers by ELISA. NP formulations varying in size and loading were compared to alum adsorbates as well as to TT solutions. RESULTS: Both, p.o. and i.n. administration of TT associated NP increased serum titers up to 3 x 10(3) (IgG) and 2 x 10(3) (IgA). While small NP induced significantly higher titers then larger ones after oral administration, intermediate NP induced antibodies after nasal application. Of the mucosal routes investigated, i.n. seems to be more promising compared to p.o. immunization. CONCLUSIONS: Antigen loaded NP prepared from surface modified polyesters combined with CT show considerable potential as a vaccine delivery system for mucosal immunization. The results warrant further experiments to explore in more detail the potential use of NP as mucosal vaccine delivery system.
Assuntos
Ácido Láctico/química , Ácido Poliglicólico/química , Polímeros/química , Álcool de Polivinil/química , Toxoide Tetânico/administração & dosagem , Administração Intranasal , Administração Oral , Animais , Anticorpos Antibacterianos/biossíntese , Fenômenos Químicos , Físico-Química , Sistemas de Liberação de Medicamentos , Ensaio de Imunoadsorção Enzimática , Excipientes , Feminino , Imunidade nas Mucosas , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Microesferas , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Tétano/imunologia , Toxoide Tetânico/imunologiaRESUMO
On-line liquid chromatography-gas chromatography (LC-GC) has been applied to the analysis of steryl esters in cocoa butter. Separation of the steryl esters was achieved after on-line transfer to capillary GC. HPLC removes the large amount of triglycerides and pre-separates the components of interest, thus avoiding time-consuming sample preparation prior to GC analysis. The identities of the compounds were confirmed by GC-MS investigation of the collected HPLC fraction and by comparison of the mass spectra (chemical ionization using ammonia as ionization gas) to those of synthesized reference compounds. Using cholesteryl laurate as internal standard, steryl esters were quantified in commercial cocoa butter samples, the detection limit being 3 mg/kg and the quantification limit 10 mg/kg, respectively. Only slight differences in percentage distributions of steryl esters depending on the geographical origin of the material were observed. The patterns were shown to remain unchanged after deodorization. The method described might be a valuable tool for authenticity assessment of cocoa butter.
Assuntos
Cromatografia Gasosa/métodos , Cromatografia Líquida de Alta Pressão/métodos , Óleos de Plantas/química , Ácidos Esteáricos/análise , Ésteres/análise , Estudos de Avaliação como Assunto , Reprodutibilidade dos Testes , Ácidos Esteáricos/químicaRESUMO
Numerous authors have demonstrated uptake of micro- and nanospheres, consisting of natural or synthetic polymeric materials from the gastrointestinal tract over the past two decades. The exploitation of particulate carrier systems for the delivery of peptides and other hydrophilic macromolecules via the oral route remains a challenging task due to morphological and physiological absorption barriers in the gastrointestinal tract. This review examines recent progress in the field of nanoparticle uptake from this site of administration. Since most studies have been performed with poly(styrene) particles of different sizes relatively little is known about both the effect of physicochemical particle properties critical for absorption after peroral application, and the mechanisms of gastrointestinal particle uptake. Apart from particle size, type and composition of the polymers used for micro- or nanoencapsulation are crucial for an uptake and transport across mucosal barriers. Factors such as particle surface charge and hydrophilic/hydrophobic balance of these polymeric materials have not been investigated systematically since adjustment of these particle properties is almost impossible without synthetic modification of the polymers. The current findings will be reviewed and compared to those obtained with nanoparticles consisting of a novel class of charged comb polyesters, poly(2-sulfobutyl-vinyl alcohol)-graft-poly(D,L-lactic-co-glycolic acid), SB-PVAL-g-PLGA, allowing adjustment of physicochemical nanoparticle properties with a single class of polymers.
Assuntos
Peptídeos/administração & dosagem , Polímeros/farmacologia , Administração Oral , Animais , Biodegradação Ambiental , Portadores de Fármacos , Excipientes , Humanos , Microesferas , Mucosa/efeitos dos fármacos , Mucosa/metabolismoRESUMO
Different absorption enhancing principles for a potent cyclopeptidic alpha(nu)beta(3)-antagonist (EMD 121974) were investigated in monolayers of a human intestinal cell line (Caco-2). Transepithelial transport was quantitated by reversed-phase high-performance liquid chromatography. Cytotoxic effects were characterized by determination of transepithelial electrical resistances (TEERs), propidium iodide (PI)-influx, FITC-phalloidin staining and the release of cytosolic lactate dehydrogenase (LDH). Medium chain fatty acids (MCFAs, NaC10, NaC12) and taurocholate (NaTC) were the most efficient enhancers of cyclopeptide and FITC-dextran 4400 permeability coefficients, displaying different time profiles of activity. Whereas NaTC (15 mM) showed almost a constant permeation enhancing effect from 20 min up to 120 min (ca. 12-fold), MCFA absorption enhancement was markedly dependent on incubation time (NaC10, 20 min: 1.2-fold, 120 min: 17-fold; NaC12, 20 min: 4.3-fold, 120 min: 13-fold). All cytotoxicity assays demonstrated that MCFAs were significantly more cytotoxic than NaTC. Ion pairing with hydrophobic amino acids and heptane sulfonate distinctly increased octanol-buffer partition coefficients of the cationic cyclopeptide but did not enhance its transepithelial permeability. Nanoparticles as well as beta-cyclodextrin neither affected integrity of the cells nor transport properties of the cyclopeptide. In summary, significant absorption enhancement was only observed with NaTC or MCFAs. Increase in permeability coefficients using NaTC occurred rapidly with acceptable cytotoxicities and merits further investigations.
Assuntos
Absorção Intestinal/fisiologia , Peptídeos Cíclicos/farmacocinética , Receptores de Vitronectina/antagonistas & inibidores , Transporte Biológico , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ácidos Decanoicos/farmacologia , Dimiristoilfosfatidilcolina/farmacologia , Condutividade Elétrica , Humanos , L-Lactato Desidrogenase/metabolismo , Ácidos Láuricos/farmacologia , Micelas , Microesferas , Octanóis/química , Venenos de Serpentes , Solubilidade , Ácido Taurocólico/farmacologiaRESUMO
PURPOSE: Different lipophilic derivatives of a potent alphaIIbbeta3-antagonist with benzamidino-oxazolidinone structure were investigated with respect to transport and metabolism properties to evaluate their potential as prodrugs with improved absorption behavior. METHODS: Intestinal transport and metabolism of the compounds were studied in Caco-2 monolayers under in vitro conditions and quantitated by a reversed-phase HPLC- method. Peroral bioavailability in cynomolgus monkeys and inhibition of platelet aggregation (guinea pig) were compared to in vitro permeability coefficients. RESULTS: N-alkoxycarbonyl- and N-benzoyl-derivatization of the benzamidine-parent drug increased the apparent permeabilities across Caco-2 monolayers by a factor of 25-100 fold. Most prodrugs were transported mainly by passive diffusion, whereas the methoxycarbonyl-derivative EMD 122347 displayed directional transport from basolateral (BL) to apical (AP). This polarized efflux was concentration dependent (saturable kinetics with Km = 207 microM, Vmax = 0.275 nmol cm(-2) min(-1)) and could be reduced in the presence of verapamil (300 microM), an inhibitor of p-glycoprotein. Cell mediated cleavage of the prodrugs was low and showed only slight differences to hydrolysis in buffer solution, indicating a predominantly non enzymatic cleavage. Both peroral bioavailability (monkey) and the inhibition of ex-vivo platelet aggregation (guinea pig) gave the same rank order as the permeability coefficients obtained in Caco-2 monolayers. CONCLUSIONS: Alkoxycarbonylamidine- and benzoylamidine promoieties of a RGD mimetic alphaIIbbeta3-antagonist considerably increased both effect bioavailabilities in animal experiments as well as in-vitro permeability in cell monolayers, demonstrating the potential of this approach to enhance transport of peptidomimetic drugs.
Assuntos
Absorção Intestinal/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Animais , Transporte Biológico , Biotransformação , Soluções Tampão , Células CACO-2 , Fenômenos Químicos , Físico-Química , Cromatografia Líquida de Alta Pressão , Eletrofisiologia , Cobaias , Humanos , Técnicas In Vitro , Macaca fascicularis , Octanóis/química , Agregação Plaquetária/efeitos dos fármacos , Solubilidade , Espectrofotometria UltravioletaRESUMO
We report the characterization of a maize Wee1 homologue and its expression in developing endosperm. Using a 0.8-kb cDNA from an expressed sequence tag project, we isolated a 1.6-kb cDNA (ZmWee1), which encodes a protein of 403 aa with a calculated molecular size of 45.6 kDa. The deduced amino acid sequence shows 50% identity to the protein kinase domain of human Wee1. Overexpression of ZmWee1 in Schizosaccharomyces pombe inhibited cell division and caused the cells to enlarge significantly. Recombinant ZmWee1 obtained from Escherichia coli is able to inhibit the activity of p13(suc1)-adsorbed cyclin-dependent kinase from maize. ZmWee1 is encoded by a single gene at a locus on the long arm of chromosome 4. RNA gel blots showed the ZmWee1 transcript is about 2.4 kb in length and that its abundance reaches a maximum 15 days after pollination in endosperm tissue. High levels of expression of ZmWee1 at this stage of endosperm development imply that ZmWee1 plays a role in endoreduplication. Our results show that control of cyclin-dependent kinase activity by Wee1 is conserved among eukaryotes, from fungi to animals and plants.
Assuntos
Proteínas de Ciclo Celular , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Zea mays/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Drosophila , Escherichia coli , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica de Plantas , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Filogenia , Proteínas Tirosina Quinases/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe , Transcrição Gênica , Zea mays/genética , Zea mays/crescimento & desenvolvimentoAssuntos
Infecções Oportunistas Relacionadas com a AIDS/parasitologia , Pneumopatias Parasitárias/parasitologia , Toxoplasmose/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Adulto , Animais , Líquido da Lavagem Broncoalveolar/parasitologia , Humanos , Pneumopatias Parasitárias/diagnóstico , Masculino , Toxoplasma/isolamento & purificaçãoRESUMO
The sensitivity to the major antimicrobial agents of consecutively isolated gram-negative rods in intensive care units in Switzerland was investigated. A majority of strains originated from clinical specimens where infection or colonization could not be distinguished from each other. A total of 1024 isolates from 482 patients were tested by a standardized microtiter method. Of the beta-lactam antibiotics, imipenem (92% of bacteria sensitive), ceftazidime (90%), and aztreonam (85%) showed the greatest activity. 94% of the isolates were sensitive to ciprofloxacin, and 93% and 91% to amikacin and tobramycin respectively. There where considerable differences locally and in time, probably reflecting small outbreaks due to resistant bacteria and because selective pressure is locally dependent on the antimicrobial agents used. 122 patients were observed for an extended period. In 67 (55%) the same gram-negative rods persisted for up to 42 days without development of resistance. In 41 patients (34%) there was a change to other more resistant bacteria, especially to multi-resistant Pseudomonas aeruginosa, Flavobacterium meningo-septicum, Xanthomonas maltophilia or Enterobacter cloacae. In 12 patients (10%) bacteria of the same species with resistance to additional antimicrobial agents were observed.
Assuntos
Antibacterianos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Unidades de Terapia Intensiva , Resistência Microbiana a Medicamentos , Resistência a Múltiplos Medicamentos , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Lactamas , Testes de Sensibilidade Microbiana , Resistência beta-LactâmicaRESUMO
This paper describes how from a situation within the European Community where each Member State had its own individual regulations for materials and articles in contact with food, the Community has developed a harmonized approach. The view is expressed that whilst this harmonization is desirable, the EC legislation as currently written is impractical. Flaws in the principles underlying EC controls are identified and whilst some parts of the harmonized regulations can form the basis for a workable scheme, substantial revision is needed. Proposals for improvement to the current Directives are suggested.
Assuntos
União Europeia , Manipulação de Alimentos/legislação & jurisprudência , Indústria de Processamento de Alimentos/legislação & jurisprudência , Legislação sobre Alimentos , Plásticos/toxicidade , Animais , Contaminação de Alimentos , Humanos , Cooperação Internacional , Camundongos , Testes de Mutagenicidade , RatosRESUMO
Progeny recovered from backcrossed transgenic maize tissue culture regenerants (R0) were analyzed to determine the segregation, expression, and stability of the introduced genes. Transgenic A188 x B73 R0 plants (regenerated from embryogenic suspension culture cells transformed by microprojectile bombardment; see [9]) were pollinated with nontransformed B73 pollen. Inheritance of a selectable marker gene, bar, and a nonselectable marker gene, uidA, was analyzed in progeny (R1) representing four independent transformation events. Activity of the bar gene product, phosphinothricin acetyltransferase (PAT), was assessed in plants comprising the four R1 populations. The number of R1 plants containing PAT activity per total number of R1 plants recovered for each population was 2/7, 19/34, 3/14 and 73/73. Molecular analysis confirmed the segregation of bar in three R1 populations and the lack of segregation in one R1 population. Cosegregation analysis indicated genetic linkage of bar and uidA in all four R1 populations. Analysis of numerous R2 plants derived from crossing transformed R1 plants with nontransformed inbreds revealed 1:1 segregation of PAT activity in three of four lines, including the line that failed to segregate in the R1 generation. Integrated copies of bar in one line appeared to be unstable or poorly transmitted.
Assuntos
Acetiltransferases/genética , Ligação Genética/genética , Marcadores Genéticos/genética , Plantas Geneticamente Modificadas/genética , Zea mays/enzimologia , Acetiltransferases/metabolismo , Aminobutiratos/farmacologia , Southern Blotting , Resistência a Medicamentos/genética , Expressão Gênica , Vetores Genéticos/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/enzimologia , Plasmídeos/genética , Transformação Genética , Zea mays/efeitos dos fármacos , Zea mays/genéticaAssuntos
Bactérias/isolamento & purificação , Hospitais Comunitários , Bactérias/classificação , Líquidos Corporais/microbiologia , Enterobacteriaceae/isolamento & purificação , França , Haemophilus influenzae/isolamento & purificação , Humanos , Estudos Prospectivos , Staphylococcus/isolamento & purificaçãoRESUMO
A reproducible system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed with the bacterial gene bar using microprojectile bombardment. Transformed calli were selected from the suspension cultures using the herbicide bialaphos. Integration of bar and activity of the enzyme phosphinothricin acetyltransferase (PAT) encoded by bar were confirmed in all bialaphos-resistant callus lines. Fertile transformed maize plants (R0) were regenerated, and of 53 progeny (R1) tested, 29 had PAT activity. All PAT-positive progeny analyzed contained bar. Localized application of herbicide to leaves of bar-transformed R0 and R1 plants resulted in no necrosis, confirming functional activity of PAT in the transgenic plants. Cotransformation experiments were performed using a mixture of two plasmids, one encoding PAT and one containing the nonselected gene encoding [beta]-glucuronidase. R0 plants regenerated from co-transformed callus expressed both genes. These results describe and confirm the development of a system for introduction of DNA into maize.
RESUMO
Stable transformed Black Mexican Sweet (BMS) maize callus was recovered from suspension culture cells bombarded with plasmid DNA that conferred resistance to the herbicide bialaphos. Suspension culture cells were bombarded with a mixture of two plasmids. One plasmid contained a selectable marker gene, bar, which encoded phosphinothricin acetyl transferase (PAT), and the other plasmid encoded a screenable marker for ß-glucuronidase (GUS). Bombarded cells were selected on medium containing the herbicide bialaphos, which is cleaved in plant cells to yield phosphinothricin (PPT), an inhibitor of glutamine synthetase. The bialaphos-resistant callus contained the bar gene and expressed PAT as assayed by PPT inactivation. Transformants that expressed high levels of PAT grew more rapidly on increasing concentrations of bialaphos than transformants expressing low levels of PAT. Fifty percent of the bialaphos-resistant transformants tested (8 of 16) expressed the nonselected gene encoding GUS.
RESUMO
A direct antimicrobial susceptibility test and a direct identification of Gram-stained urine specimens positive for Enterobacteriaceae using a new instrument (Cobas-Bact) were compared by means of the conventional Kirby-Bauer agar diffusion disk method and the spot indole test, an in-house set of identification tests or API 20E. Bacteria from 191 cases of monomicrobial bacteriuria due to members of the family Enterobacteriaceae were tested. Direct susceptibility testing was performed in 180 cases (94%), providing 1649 antibiotic-microorganism combinations. A complete agreement was reached in 82% and essential agreement in 92% of cases. Minor discrepancies were found in 163 (9.9%) major ones in 125 (7.6%) and very major ones in 10 (0.6%) of examinations. 72% of all minor and 71% of all major discrepancies were caused by two antibiotics: cephalothin and nitrofurantoin. Of the very major discrepancies, 50% were due to amoxicillin. Of 171 direct identifications obtained, 130 (76%) were "high-confidence" correct identifications (percentage of likelihood p greater than or equal to 80%), 25 (14.6%) "low-confidence" identifications (percentage of likelihood p less than 80%) and 16 (9.4%) misidentifications. On the whole, this direct and rapid Cobas-Bact identification and susceptibility testing procedure provided satisfying information within 5-6 h after collection of urine specimens positive for members of the Enterobacteriaceae family.