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1.
Clin Microbiol Infect ; 14(3): 282-6, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18093230

RESUMO

Diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) requires adhesion of microorganisms to enterocytes. Hence, a promising approach to immunoprophylaxis is to elicit antibodies against colonisation factor antigens (CFAs). Genes encoding the most prevalent ETEC-specific surface antigens were cloned into Vibrio cholerae and Salmonella vaccine strains. Expression of surface antigens was assessed by electron-microscopy. Whereas negative staining was effective in revealing CFA/I and CS3, but not CS6, immunolabelling allowed identification of all surface antigens examined. The V. cholerae vaccine strain CVD103 did not express ETEC-specific colonisation factors, whereas CVD103-HgR expressed CS3 only. However, expression of both CFA/I and CS3 was demonstrated in Salmonella Ty21a.


Assuntos
Antígenos de Bactérias/análise , Antígenos de Superfície/análise , Vacinas Bacterianas/química , Vacinas contra Cólera/química , Escherichia coli Enterotoxigênica/genética , Proteínas de Escherichia coli/análise , Vacinas contra Salmonella/química , Antígenos de Bactérias/genética , Antígenos de Superfície/genética , Vacinas Bacterianas/imunologia , Vacinas contra Cólera/imunologia , Clonagem Molecular , Proteínas de Escherichia coli/genética , Expressão Gênica , Humanos , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Salmonella/química , Salmonella/imunologia , Vacinas contra Salmonella/imunologia , Vibrio cholerae/química , Vibrio cholerae/imunologia
2.
Gene ; 272(1-2): 249-55, 2001 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-11470531

RESUMO

In plants gene knock-outs and targeted mutational analyses are hampered by the inefficiency of homologous recombination. We have developed a strategy to enrich for rare events of homologous recombination in Arabidopsis using combined positive and negative selection. The T-DNA targeting construct contained two flanking regions of the target alcohol dehydrogenase gene as homologous sequences, and neomycin phosphotransferase and cytosine deaminase as positive and negative markers, respectively. A root explant transformation procedure was used to obtain transgenic calli. Among 6250 transformants isolated by positive selection, 39 were found to be resistant to negative selection as well. Of these 39, at least one had undergone homologous recombination correlated with a unidirectional transfer of information. Although the ADH locus was not changed, our data demonstrate that a homologous recombination event can be selected by positive negative selection in plants.


Assuntos
Arabidopsis/genética , Recombinação Genética , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Southern Blotting , Análise Mutacional de DNA/métodos , DNA de Plantas/genética , Fluoruracila/farmacologia , Mutagênese , Transformação Genética
4.
Acta Biotheor ; 48(3-4): 259-72, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11291944

RESUMO

This work presents an hydrodynamical model of heat stroke, which is a physiopathological state of stress, due to an exposure of animals to an ambient temperature of approximatively 40 degrees C during two hours. The evolution of body temperature during this stress process is characterised by three phases. A first phase of increase is followed by a plateau which occurs before a second phase of increase which can be lethal. The model is based on the analogy of a boat progressively caught in a whirlpool. The evolution of the degree of freedom lost by the boat is mathematically analysed and this study leads to the same three phases. The theoretical curves calculated during this study are well in agreement with the experimental curves obtained with animals. This analogy is compared to a previous one which has been made during another experiment with animals constrained by chemical intoxications. It seems that stress can be considered as a vital vorticity and that hydrodynamic models are powerful tools in understanding this physiopathological state.


Assuntos
Regulação da Temperatura Corporal/fisiologia , Modelos Animais de Doenças , Exaustão por Calor/fisiopatologia , Alternativas aos Testes com Animais , Animais , Humanos , Modelos Teóricos , Estresse Fisiológico/fisiopatologia
5.
Infect Immun ; 67(7): 3680-5, 1999 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10377160

RESUMO

The mucosal and systemic immune responses after primary and booster immunizations with two attenuated live oral vaccine strains derived from a noninvasive (Vibrio cholerae) and an invasive (Salmonella typhi) enteric pathogen were comparatively evaluated. Vaccination with S. typhi Ty21a elicited antibody-secreting cell (ASC) responses specific for S. typhi O9, 12 lipopolysaccharide (LPS), as well as significant increases in levels of immunoglobulin G (IgG) and IgA antibodies to the same antigen in serum. A strong systemic CD4(+) T-helper type 1 cell-mediated immune (CMI) response was also induced. In contrast to results with Ty21a, no evidence of a CMI response was obtained after primary immunization with V. cholerae CVD 103-HgR in spite of the good immunogenicity of the vaccine. Volunteers who received a single dose of CVD 103-HgR primarily developed an IgM ASC response against whole vaccine cells and purified V. cholerae Inaba LPS, and seroconversion of serum vibriocidal antibodies occurred in four of five subjects. Serum IgG anti-cholera toxin antibody titers were of lower magnitude. For both live vaccines, the volunteers still presented significant local immunity 14 months after primary immunization, as revealed by the elevated baseline antibody titers at the time of the booster immunization and the lower ASC, serum IgG, and vibriocidal antibody responses after the booster immunization. These results suggest that local immunity may interfere with colonization of the gut by both vaccine strains at least up to 14 months after basis immunization. Interestingly, despite a low secondary ASC response, Ty21a was able to boost both humoral (anti-LPS systemic IgG and IgA) and CMI responses. Evidence of a CMI response was also observed for one of three volunteers given a cholera vaccine booster dose. The direct comparison of results with two attenuated live oral vaccine strains in human volunteers clearly showed that the capacity of the vaccine strain to colonize specific body compartments conditions the pattern of vaccine-induced immune responses.


Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Vacinas contra Cólera/imunologia , Imunidade nas Mucosas , Imunidade , Vibrio cholerae/imunologia , Especificidade de Anticorpos , Humanos , Imunização , Isotipos de Imunoglobulinas
6.
Biologicals ; 27(4): 303-14, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10686057

RESUMO

The NucliSens Extractor in combination with the 2.0 version of the Roche Cobas HCV Amplicor test has been validated by five European blood screening laboratories in a multi-centre study. For testing the performance characteristics of this HCV-NAT method, the European Pharmacopoeia validation guidelines were followed. The CLB VQC reference reagents were used for testing robustness and sensitivity. After a technical improvement in the extraction stations, the NucliSens Extractor appeared to be contamination-free as was proved by testing negative controls alternating with samples containing a high HCV-RNA concentration. The Pelicheck HCV-RNA genotype 1 dilution panel was tested 74 times in the five laboratories and an overall 95% detection limit of 80 genome equivalents (geq)/ml was found. In one laboratory the Pelicheck panel was tested in 25 runs and here a 95% detection limit of 32 geq/ml was achieved. In this laboratory the Pelispy HCV-RNA run control samples of 140 geq/ml were consistently picked up in all extractor stations. In addition the laboratories have tested a WHO HCV-RNA genotype 1 standard dilution series 39 times and a Pelicheck HCV-RNA genotype 3 reference panel in 32 test runs. The limiting dilution analysis enabled us to compare the detection efficiency of the NucliSens-Amplicor method for the genoype 1 and genotype 3 isolates and to calibrate the reference reagents against each other. The combined Nuclisens-Amplicor method was found to detect the genotype 3 isolate in the Pelicheck HCV-RNA panels with 2-3 fold lower efficiency than the genotype 1 standard (assuming that the historical calibration of the genotype 3 against the genotype 1 standard is correct). In this study of a single method 1 IU of the WHO HCV-RNA standard was found to be equivalent to 5.1 geq of the VQC HCV-RNA standard (95% confidence intervals 3.1-9.1 geq). To avoid confusion with the use of the CLB VQC reagents we accept the NIBSC collaborative study in which calibration by a variety of methods showed that the Pelispy 380 geq/ml run control is equivalent to 100 IU/ml of the WHO standard. This multi-centre validation study demonstrates that the 95% detection limit of the NucliSens HCV Amplicor method lies far below the detection limits required by the international regulatory bodies.


Assuntos
Hepacivirus/genética , Hepacivirus/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/sangue , RNA Viral/genética , Europa (Continente) , Estudos de Avaliação como Assunto , Genótipo , Guias como Assunto , Humanos , Plasma/virologia , Padrões de Referência , Reprodutibilidade dos Testes , Organização Mundial da Saúde
7.
Acta Biotheor ; 47(3-4): 173-90, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10855265

RESUMO

This work is a qualitative study of an organism's physiological adaptative response to stress. The experimental data were selected from a previous study leading to the conclusion that stress may be considered as a topological retraction within a vital space that must be more precisely defined. The experimental methodology uses rat poisoning by neurotoxins. The control parameter is the intensity of the toxic doses. Measured parameters are the animals' survival rate and the kinetics of cerebral acetylcholinesterase activity. The results, when expressed as a function of the inverted doses, show a characteristic evolution. The pattern of the curve closely resembles a vortex profile. This analogy is studied more extensively in both the physical and biological domains. These findings help to clarify the concept of biological stress which presents the same vectorial properties as hydrodynamic vorticity. In particular, the dissipation of stress and the dissipation of vorticity seem to obey the same laws. This observation is valid for both diffusion and convection processes. The decompensation phase of stress could be compared with the instability and turbulence in flows. Our approach in this paper is mainly to establish a general and phenomenological description of the stress response fitting experimental observations.


Assuntos
Adaptação Fisiológica/fisiologia , Estresse Fisiológico/fisiopatologia , Acetilcolinesterase/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Relação Dose-Resposta a Droga , Humanos , Modelos Teóricos , Neurotoxinas/toxicidade , Intoxicação/fisiopatologia , Ratos , Estresse Fisiológico/complicações
8.
Mol Membr Biol ; 14(1): 31-3, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9160339

RESUMO

The fluidity of isolated plasma and disc membranes from bovine rod outer segments has been compared by electron-spin resonance spectroscopy using the stearic acid spin labels 5-SASL and 16-SASL. This comparison is of interest, given the fact that the disc membranes arise by invagination from the plasma membrane. Analysis of the order parameter in the hydrophilic part of the membrane, and of rotational correlation time in the hydrophobic part, showed a difference of fluidity between these two types of membranes, with a higher fluidity in the disc membranes in both their hydrophilic and hydrophobic regions. For example, at a temperature of 37 degrees C, plasma and disc membranes exhibited values of 0.64 and 0.61 for the order parameter (S), and 9.1 and 14.5 s-1 for the rotational correlation frequency (v), respectively. These findings are discussed in relation to previously described differences in the biochemical compositions of the membranes.


Assuntos
Membrana Celular/química , Membranas Intracelulares/química , Fluidez de Membrana , Segmento Externo da Célula Bastonete/química , Animais , Bovinos , Espectroscopia de Ressonância de Spin Eletrônica
9.
Plant Cell ; 8(11): 2057-66, 1996 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8953770

RESUMO

The HO endonuclease promotes gene conversion between mating-type alleles in yeast by a DNA double-strand break at the site of conversion (the MAT-Y/Z site). As a first step toward understanding the molecular basis of homologous recombination in higher plants, we demonstrate that expression of HO in Arabidopsis enhances intrachromosomal recombination between inverted repeats of two defective beta-glucuronidase (gus) genes (GUS- test construct). One of these genes has the Y/Z site. The two genes share 2.5 kb of DNA sequence homology around the HO cut site. Somatic recombination between the two repeats was determined by using a histochemical assay of GUS activity. The frequency of Gus+ sectors in leaves of F1 plants from a cross between parents homozygous for the GUS- test construct and HO, respectively, was 10-fold higher than in F1 plants from a cross between the same plant containing the GUS- test construct and a wild-type parent. Polymerase chain reaction analysis showed restoration of the 5' end of the GUS gene in recombinant sectors. The induction of intrachromosomal gene conversion in Arabidopsis by HO reveals the general utility of site-specific DNA endonucleases in producing targeted homologous recombination in plant genomes.


Assuntos
Arabidopsis/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Recombinação Genética , Modelos Químicos , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Proteínas de Saccharomyces cerevisiae
10.
Gene ; 178(1-2): 43-9, 1996 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-8921890

RESUMO

A series of cosmid vectors, termed pSSVI215, pSSVI216-1, pSSVI216-2, pSSVI217, and pSSVI218, were constructed in order to facilitate the downstream processing of large inserts. Each vector has dual cos sites as well as a kanamycin resistance (KmR) gene flanked by recognition sites for the very rare cutter I-SceI meganuclease as well as symmetrical NotI and SwaI sites (SCEKAN cassette). Several unique cloning sites, including BamHI, are present on one side of the cassette between the I-SceI and NotI/SwaI sites. The various cosmids differ from each other by one or more of the following features: origin of replication (ori), size, host range, and conjugal transfer capability. Inserts combined with the SCEKAN cassette can be isolated on a NotI or SwaI fragment from any of these vectors and easily subcloned into the vector of choice by selecting for the adjacent KmR gene which can later be removed by I-SceI restriction and self-ligation. In addition, the SCEKAN cassette can be conveniently excised from plasmid pSSVI214 such that any plasmid can easily be fitted with the present system. The subcloning strategy afforded by the new vectors was successfully applied to an approximately 37-kb fragment from the V. cholerae O139 genome carrying the rfb locus which encodes the O-serotype specificity of this organism.


Assuntos
Clonagem Molecular/métodos , Cosmídeos , Vetores Genéticos , Mapeamento Cromossômico , Resistência a Canamicina/genética , Vibrio cholerae/genética
11.
Infect Immun ; 64(9): 3565-70, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8751900

RESUMO

The rfb region from Vibrio cholerae O139 strain MO45 was cloned from cosmid gene banks established in Escherichia coli HB101, using an immunoblot assay for screening of the correct clones. Immunoblot analysis of lipopolysaccharide (LPS) preparations revealed the presence of two types of positive clones: (i) those expressing only a short core-linked O polysaccharide (SOPS) and (ii) those also expressing a highly polymerized capsular polysaccharide (CPS) not bound to the E. coli K-12 LPS core. In addition, the latter clones appear to contain a locus which may encode a putative regulator of SOPS and CPS chain length. Further characterization in E. coli showed that CPS constitutes a barrier against large particles such as the bacteriophage Ffm but not against bacteriophage lambda or P1. In addition, a portion of the K-12 LPS core may not be substituted with SOPS. Loci associated with the two clonal types were transferred into V. cholerae CH19, an rfbAB deletion mutant of CVD103-HgR deficient in the production of the homologous Inaba O polysaccharide. This resulted in the stable expression of SOPS, alone or together with CPS, that was indistinguishable from that of wild-type V. cholerae O139. Strains CH25 and CH26, which correspond to CH19 bearing the V. cholerae O139 rfb region integrated into the chromosome, were found to be genetically stable and essentially identical to the parent CVD103-HgR with respect to physiological properties such as cell motility, mercury resistance, toxicity, and production of the cholera toxin B subunit. Rabbits immunized with CH25 elicited high titers of anti-O139 SOPS- and CPS-specific serum antibodies. These strains possess characteristics desirable in candidate live oral vaccines against V. cholerae O139.


Assuntos
Cápsulas Bacterianas/imunologia , Proteínas de Bactérias/genética , Vacinas Bacterianas/imunologia , Vibrio cholerae/imunologia , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/genética , Clonagem Molecular , Colífagos/crescimento & desenvolvimento , Escherichia coli , Genes Bacterianos , Coelhos , Proteínas Recombinantes , Vacinas Sintéticas/imunologia
12.
Vaccine ; 14(6): 526-31, 1996 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8782351

RESUMO

Vibrio cholerae CVD103-HgR, the first live attenuated vaccine licensed for human use produced by recombinant DNA technology, was genetically compared to its parent strains 569B and CVD103. The genetic stability for both lyophilized vaccine in final container form and for viable organisms shed from vaccinees was determined. Results obtained lead us to conclude: (i) the genetic composition of the examined genes in CVD103-HgR is identical to that of the parent strains except for the alterations induced; (ii) the level of mercury resistance depends on the orientation of the mer operon within hlyA, with the highest level being observed for the orientation found in CVD103-HgR; (iii) no DNA sequences from plasmids used in construction remain in the genome; (iv) the strain is genetically stable; and (v) both CVD103-HgR and its parent strains contain defective lysogenic prophages. We have further confirmed that a certain amount of restriction fragment length polymorphism (RFLP) exists around the chromosomal ctx locus within V. cholerae strains of the classical biotype (detectable on chromosomal DNA restricted by either HindIII or EcoRI, but not PstI).


Assuntos
Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Vibrio cholerae/genética , Vibrio cholerae/imunologia , Bacteriófagos/isolamento & purificação , Resistência Microbiana a Medicamentos , Estabilidade de Medicamentos , Humanos , Mercúrio/toxicidade , Especificidade da Espécie , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/virologia
13.
Mol Microbiol ; 19(5): 949-63, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8830276

RESUMO

Making use of a newly designed mobilizable suicide vector, the genetic determinants encoding Shigella sonnei lipopolysaccharide (LPS) were stably integrated into the chromosome of the live attenuated Vibrio cholerae vaccine strain CVD103-HgR. Expression studies showed that the production of complete S. sonnei O-polysaccharide (O-PS)-bearing LPS was limited in bivalent recombinant strains that were also proficient in the synthesis of the host-encoded Inaba O-PS. Conversely, high amounts of LPS carrying S. sonnei O-PS are produced in monovalent Inaba-deficient derivatives, even in those strains which do not co-express the compatible R1 LPS core. Thus, the non-enterobacterial V. cholerae LPS core efficiently acts as a receptor for covalent binding of S. sonnei O-PS provided that competition with the host O-PS is avoided. Expression of the R1 core interferes with cell division in recombinant V. cholerae without affecting other physiological properties of vaccine strain CVD103-HgR. Both monovalent and bivalent strains stimulated high serum-antibody titres specific for their respective O-serotype(s) when administered to rabbits. The potential of V. cholerae as an expression carrier for heterologous O-serotypes is discussed.


Assuntos
Proteínas de Fímbrias , Lipopolissacarídeos , Shigella sonnei/genética , Vibrio cholerae/genética , Animais , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Cromossomos Bacterianos , Clonagem Molecular , Genes Bacterianos , Vetores Genéticos/genética , Lipopolissacarídeos/imunologia , Antígenos O/imunologia , Pili Sexual , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Shigella sonnei/metabolismo , Temperatura , Vibrio cholerae/imunologia
14.
Infect Immun ; 64(2): 576-84, 1996 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8550210

RESUMO

Previous experimentation has highlighted a number of difficulties in the development of carrier-based bivalent vaccines (J.-F. Viret and D. Favre, Biologicals 22:361-372, 1994) In an attempt to obviate these carrier strains. Toward this aim, a series of defined rfbInaba deletion (delta rfbInaba) mutants of the cholera vaccine strain V. cholerae CVD103-HgR (O1 Inaba serotype) and derivative bearing the chromosomally integrated locus encoding the S. sonnei O-PS were constructed and characterized. The various mutations disrupt genes thought to be involved in either the synthesis of perosamine, the synthesis of 3-deoxy-L-glycero tetronic acid, or the O-PS transport functions together with synthesis of the perosamine synthetase. Some deletions were obtained only in strains expressing the heterologous lipopolysaccharide (LPS). Viable delta rfbInaba deletions in CVD103-HgR profoundly altered some of its phenotypic properties. The same deletions present in CVD103-HgR derivatives expressing the heterologous LPS affected their phenotypes only to a lesser extent. Only in strains in which perosamine synthesis was specifically abolished could high amounts of core-bound S. sonnei O-PS be synthesized. Two such strains (CH21, which expresses both the R1 core and the S. sonnei O-PS, and CH22, which expresses only the latter antigenic determinant) were further analyzed and were found to be indistinguishable from CVD103-HgR with regard to lack of enterotoxin activity, choleragenoid production, mercury resistance, pilin production, and, for CH22, motility. Mice immunized with CH22 produced high titers of S. sonnei O-PS-specific antibodies.


Assuntos
Vacinas Bacterianas/imunologia , Antígenos O/biossíntese , Shigella sonnei/imunologia , Vacinas Sintéticas/imunologia , Vibrio cholerae/genética , Administração Oral , Animais , Anticorpos Antibacterianos/sangue , Clonagem Molecular , Deleção de Genes , Vetores Genéticos , Imunização , Camundongos
15.
Psychiatry Res ; 59(3): 255-8, 1996 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-8930032

RESUMO

Erythrocyte membrane characteristics were compared in 15 normal women and 15 women with anorexia nervosa; the patients were studied at hospital admission and again after 1 month of refeeding. At admission, physical properties of erythrocyte membranes, studied with electron spin resonance spectrometry, significantly differed between the anorexic patients and the normal volunteers. Fluidity from the hydrophobic part of the erythrocyte membrane, estimated by the correlation frequency, was decreased in the patients. After 1 month of refeeding, fluidity increased. One of the possible mechanisms of the variation of membrane fluidity could be the effect of cholesterol on membrane structure. Increased cholesterol levels in anorexic subjects could reduce fluidity. These alterations in membrane fluidity could explain some of the neurobiological abnormalities observed in anorexia nervosa.


Assuntos
Anorexia Nervosa/sangue , Ingestão de Alimentos , Membrana Eritrocítica/fisiologia , Fluidez de Membrana/fisiologia , Adolescente , Índice de Massa Corporal , Peso Corporal , Colesterol/sangue , Feminino , Humanos
16.
Plant Mol Biol ; 28(6): 1127-32, 1995 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7548830

RESUMO

The coding sequence for FLP recombinase, originally from the 2 mu plasmid of Saccharomyces cerevisiae, was introduced into Arabidopsis behind the cauliflower mosaic virus 35S promoter. FLP activity was monitored by the glucuronidase activity resulting from inversion of an antisense-oriented GUS reporter gene flanked by a pair of FRT target sites in inverted repeat. FLP-dependent Gus activity was observed in both transient assays and transgenic plants. The FLP system will be useful for a variety of in planta genetic manipulations.


Assuntos
Arabidopsis/metabolismo , Inversão Cromossômica , DNA Nucleotidiltransferases/metabolismo , Recombinação Genética , DNA Nucleotidiltransferases/genética , Proteínas Fúngicas/metabolismo , Plantas Geneticamente Modificadas , Saccharomyces cerevisiae/enzimologia
17.
Anal Biochem ; 230(1): 75-84, 1995 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-8585633

RESUMO

In order to specifically modify the fatty acid composition of cell membrane phospholipids, we have developed an original method based on the transfer of pure phospholipid molecular species to membranes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) subclasses containing 18:2n-6 and 22:6n-3 at the sn-2 position were incorporated into human platelet membranes using the endogenous phosphatidylinositol/PC transfer protein (PI/PC-TP) and the phospholipid transfer protein from maize (L-TP), respectively. PI/PC-TP was shown to catalyze a strict exchange of phospholipids between platelet membranes and unilamellar vesicles containing 1,2-diacylglycerophosphocholine (diacyl-GPC; 16:0/18:2-GPC, or 16:0/22:6-GPC). The proportions of 18:2n-6 and 22:6n-3 in diacyl-GPC of platelet membranes were gradually increased from 10.7 to 16.9% and from 0.8 to 10.1%, respectively, whereas the PE and PI fatty acid compositions were not changed. The diacyl-GPC enrichment in 22:6n-3 and 18:2n-6 did not induce changes in membrane fluidity parameters measured by electron-spin resonance of 5- and 16-nitroxy stearic acids. Similarly, 18:2n-6 and 22:6n-3 esterified in 1,2-diacylglycerophosphoethanolamine (diacyl-GPE) have been incorporated in platelet membranes by an apparent exchange process under conditions where donor vesicles had a phospholipid composition equivalent to that of platelet membranes. The proportions of 18:2n-6 and 22:6n-3 were selectively and progressively increased from 6.0 to 21.2% and from 2.2 to 17.2%, respectively, in diacyl-GPE of platelet membranes. Thus, the L-TP- and PI/PC-TP-catalyzed enrichment can be used for studying the modulation of membrane biological activities by defined changes of fatty acid composition of specific phospholipid classes or subclasses.


Assuntos
Proteína de Ligação a Androgênios , Plaquetas/química , Proteínas de Transporte/sangue , Ácidos Graxos Insaturados/sangue , Proteínas de Membrana , Fosfatidilinositóis , Fosfolipídeos/química , Proteínas de Saccharomyces cerevisiae , Membrana Celular/fisiologia , Humanos , Fluidez de Membrana , Fosfatidilcolinas/sangue , Proteína de Ligação a Fosfatidiletanolamina , Fosfatidiletanolaminas/sangue , Proteínas de Transferência de Fosfolipídeos , Zea mays
18.
Biotechnology (N Y) ; 13(7): 683-5, 1995 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9634805

RESUMO

We have compared the in vivo therapeutic potential of anti-tetanus toxin (TT) human Fab antibodies derived from a combinatorial phage display library to established polyclonal and monoclonal reagents. The oligoclonality and fine specificity distribution of the synthetic anti-TT Fab preparations was comparable to the antibody spectrum present in the donor serum and the affinities determined for the synthetic phage-bound Fab (Phab) and soluble Fab were in the same range as their monoclonal and polyclonal counterparts. On a weight basis, the protective capacity of the new oligoclonal preparations in vivo (16.4 IU/100 micrograms Fab) was comparable to those of the best combinations of hybridoma derived human monoclonal antibodies, and far better than those exhibited by the polyclonal serum antibodies of the donor (0.29 IU/100 micrograms IgG) or by a standard commercial human tetanus immunoglobulin preparation. These data suggest that recombinant antibodies may become a safe and effective alternative to human plasma-derived immunoglobulins for passive immunization.


Assuntos
Biblioteca Gênica , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Toxoide Tetânico/imunologia , Tétano/terapia , Animais , Anticorpos Monoclonais/uso terapêutico , Reações Antígeno-Anticorpo , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Camundongos
20.
J Lipid Res ; 36(1): 47-56, 1995 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7706947

RESUMO

The incorporation of albumin-bound docosahexaenoic acid (22:6n-3), but not linoleic acid (18:2n-6), into cellular phospholipids inhibits platelet aggregation induced by the thromboxane analogue U46619. [3H]U46619 specific binding to thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors, as well as specific binding of the antagonist [3H]SQ29548 to these sites were also decreased in these modified cells (P. G., Swann et al. 1990. J. Biol. Chem. 265: 21692-21697). More than 80% of the 22:6n-3 incorporated in these cells was esterified in the various endogenous phospholipid classes and the remaining was found in neutral lipids and in the unesterified fatty acid pool. In this study, we determined whether the effects observed could be attributed to the esterification of 22:6n-3 in phospholipids and whether the 22:6n-3 biological activity might depend on its esterification in specific phospholipid classes. Therefore, pure phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecular species were transferred to platelet membranes, using lipid transfer proteins. PC and PE containing palmitate (16:0) and 22:6n-3 or 16:0 and 18:2n-6 at position sn-1 and sn-2, respectively, were incorporated into membranes only at the expense of the corresponding endogenous phospholipid class, by an apparent exchange process. When such modified membranes were tested for specific binding of U46619 and SQ29548, a significant decrease of the receptor site affinity was only observed in membranes highly enriched with 1-palmitoyl-2-docosahexaenoyl-glycerophosphocholine (16:0/22:6-GPC). Fluidity parameters measured by electron spin resonance of 5- and 16-nitroxy-stearic acids were not significantly different in membranes enriched with 16:0/22:6-GPC relative to those enriched with 16:0/18:2n-6-GPC, arguing against a generalized perturbation of the membrane due to 22:6n-3 incorporation. We conclude that molecular species of PC with 22:6n-3 at the sn-2 position can affect TXA2/PGH2 receptors. The selectivity of the inhibitory effect of PC containing 22:6n-3 is discussed.


Assuntos
Plaquetas/ultraestrutura , Membrana Celular/metabolismo , Lipídeos de Membrana/sangue , Fosfolipídeos/sangue , Receptores de Prostaglandina/metabolismo , Receptores de Tromboxanos/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Plaquetas/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes , Membrana Celular/química , Ácidos Docosa-Hexaenoicos/sangue , Espectroscopia de Ressonância de Spin Eletrônica , Esterificação , Ácidos Graxos Insaturados , Humanos , Hidrazinas/metabolismo , Ácido Linoleico , Ácidos Linoleicos/sangue , Fluidez de Membrana , Fosfatidilcolinas/sangue , Fosfatidiletanolaminas/sangue , Agregação Plaquetária/efeitos dos fármacos , Endoperóxidos Sintéticos de Prostaglandinas/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2 , Tromboxano A2/análogos & derivados , Tromboxano A2/metabolismo
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