RESUMEN
Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I: --> 3)-alpha-L-Rha-(1 --> 2)-alpha-L-Rha-(1 --> 2)-alpha-L-Rha-(1 --> 3)-alpha-L-Rha-(1 -->; II: --> 3)-alpha-L-Rha-(1 --> 3)-alpha-L-Rha-(1 --> 2)-alpha-L-Rha-(1 --> 3)-alpha-L-Rha-(1 -->. The branched polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588 have the same L-rhamnan backbone with repeating units I and II and a lateral chain of (alpha1 --> 4)- or (alpha1 --> 3)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral alpha-D-Fuc3NAc residues were characterized.
Asunto(s)
Antígenos O/química , Pseudomonas syringae/clasificación , Acetilgalactosamina/análogos & derivados , Secuencia de Carbohidratos , Epítopos , Fucosa/análogos & derivados , Datos de Secuencia Molecular , Monosacáridos/inmunología , Antígenos O/inmunología , Pseudomonas syringae/química , Pseudomonas syringae/inmunología , Ramnosa/inmunología , SerotipificaciónRESUMEN
Results of our studies permit reaching the conclusion that oral intake of the mineral water Naftusya as the leading factor with an additional prescription of the course of the hyperbaric oxygenation procedures promote enhancement of functional reserves, stabilize free-radical processes and bodily system of antioxidant defence, favour stabilization of cell membranes, initiate decorporative action, diminish depression of immunity and supply oxygen to those enzymic systems whose activity has been adversely affected by hypoxia.
Asunto(s)
Colonias de Salud , Oxigenoterapia Hiperbárica , Centrales Eléctricas , Traumatismos por Radiación/rehabilitación , Liberación de Radiactividad Peligrosa , Humanos , Aguas Minerales/administración & dosificación , UcraniaRESUMEN
Down syndrome (DS) is the most frequent genetic disorder with mental retardation and caused by trisomy 21. Although the gene dosage effect hypothesis has been proposed to explain the impact of extra chromosome 21 on the pathology of DS, a series of evidence that challenge this hypothesis has been reported. The availability of the complete sequences of genes on chromosome 21 serves now as starting point to find functional information of the gene products, but information on gene products is limited so far. We therefore evaluated expression levels of six proteins whose genes are encoded on chromosome 21 (synaptojanin-1, chromosome 21 open reading frame 2, oligomycin sensitivity confering protein, peptide 19, cystatin B and adenosine deaminase RNA-specific 2) in fetal cerebral cortex from DS and controls at 18-19 weeks of gestational age using Western blot analysis. Synaptojanin-1 and C21orf2 were increased in DS, but others were comparable between DS and controls, suggesting that the DS phenotype cannot be simply explained by gene dosage effects. We are systematically quantifying all proteins whose genes are encoded on chromosome 21 in order to provide a better understanding of the pathobiochemistry of DS at the protein level. These studies are of significance as they show for the first time protein levels that are carrying out specific function in human fetal brain with DS.
Asunto(s)
Encéfalo/metabolismo , Cromosomas Humanos Par 21 , Síndrome de Down/genética , Dosificación de Gen , Western Blotting , Encéfalo/embriología , Estudios de Casos y Controles , Femenino , HumanosRESUMEN
Trisomy 21 (Down syndrome, DS) is the most common genetic cause of mental retardation, resulting from triplication of the whole or distal part of human chromosome 21. Overexpression of genes located on chromosome 21, as a result of extra gene load, has been considered a central hypothesis for the explanation of the DS phenotype. This gene dosage hypothesis has been challenged, however. We have therefore decided to study proteins whose genes are encoded on chromosome 21 in brain of patients with DS and Alzheimer's disease (AD), as all patients with DS from the fourth decade show Alzheimer-related neuropathology. Using immunoblotting we determined Coxsackievirus and adenovirus receptor (CAR), Claudin-8, C21orf2, Chromatin assembly factor 1 p60 subunit (CAF-1 p60) in frontal cortex from DS, AD and control patients. Significant reduction of C21orf2 and CAF-1 p60, but comparable expression of CAR and claudin-8 was observed in DS but all proteins were comparable to controls in AD, even when related to NSE levels to rule out neuronal cell loss or actin to normalise versus a housekeeping protein. Reduced CAF-1 p60 may reflect impaired DNA repair most probably due to oxidative stress found as early as in fetal life continuing into adulthood. The decrease of C21orf2 may represent mitochondrial dysfunction that has been reported repeatedly and also data on CAR and claudin-8 are not supporting the gene-dosage hypothesis at the protein level. As aberrant expression of the four proteins was not found in brains of patients with AD, decreased CAF and C21orf2 can be considered specific for DS.
Asunto(s)
Corteza Cerebral/metabolismo , Proteínas Cromosómicas no Histona/biosíntesis , Cromosomas Humanos Par 21/metabolismo , Proteínas de Unión al ADN/biosíntesis , Síndrome de Down/metabolismo , Biosíntesis de Proteínas , Anciano , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Encéfalo/patología , Corteza Cerebral/patología , Factor 1 de Ensamblaje de la Cromatina , Proteínas Cromosómicas no Histona/genética , Cromosomas Humanos Par 21/genética , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/genética , Síndrome de Down/genética , Síndrome de Down/patología , Regulación hacia Abajo/fisiología , Humanos , Masculino , Persona de Mediana Edad , Proteínas/genética , Estadísticas no ParamétricasRESUMEN
The O-polysaccharide (OPS) was obtained from the lipopolysaccharide of Pseudomonas syringae pv. delphinii NCPPB 1879(T) and studied by sugar and methylation analyses, Smith degradation, and (1)H- and (13)C-NMR spectroscopy. The OPS was found to contain residues of L-rhamnose (L-Rha) and 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc), and the following structure of the major (n = 2) and minor (n = 3) heptasaccharide repeating units of the OPS was established: [carbohydrate structure: see text]. The OPS is distinguished by the presence of oligosaccharide side chains consisting of three D-Fuc3NAc residues that are connected to each other by the (alpha 1-->2)-linkage. The OPS is characterized by a structural heterogeneity due to a different position of substitution of one of the four L-rhamnose residues in the main chain of the repeating unit as well as to the presence of oligosaccharide units with an incomplete side chain.
Asunto(s)
Galactosa/análogos & derivados , Galactosa/análisis , Antígenos O/química , Antígenos O/aislamiento & purificación , Pseudomonas/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Galactosa/química , Lipopolisacáridos/química , Espectroscopía de Resonancia Magnética , Estructura MolecularRESUMEN
In T-cells HIV-1 Nef exerts various functions and interacts with actin. In astrocytes interaction of Nef with cellular proteins is poorly understood. Therefore, human astrocytic cell clones stably transfected with nef-genes derived from HIV-1 Bru and its myristoylation-defective TH-variant were investigated by confocal laser scanning microscopy for expression of Nef and cytoskeleton proteins actin and GFAP, a marker for activated astrocytes. Myristoylated Nef was detected in cytoplasm, Golgi and plasmamembrane, while non-myristoylated Nef was exclusively cytoplasmic. Nef co-localised with GFAP in the perinuclear region of astrocytes. In contrast, Nef did not interact with actin filaments in human astrocytes. Nef/GFAP interaction could contribute to changes in morphology and activation state of astrocytes shown previously which are both critical for development of astrogliosis in HIV-1 infected brain.
Asunto(s)
Astrocitos/metabolismo , Citoesqueleto/metabolismo , Productos del Gen nef/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , VIH-1 , Actinas/análisis , Actinas/biosíntesis , Astrocitos/citología , Línea Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Expresión Génica , Productos del Gen nef/análisis , Productos del Gen nef/genética , Proteína Ácida Fibrilar de la Glía/análisis , Aparato de Golgi/metabolismo , VIH-1/genética , Humanos , Microscopía Confocal , Ácidos Mirísticos/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
DNA immunization permits evaluation of possible antagonistic or synergistic effects between the encoded components. The protein expression capacity in vitro was related to the immunogenicity in vivo of plasmids encoding the HIV-1 regulatory genes tat rev, and nef. Neither Tat nor Rev expression was influenced by co-expression in vitro of all three proteins, while Nef expression was slightly inhibited. With the combination of genes, the T-cellular responses of mice against Rev and Nef were inhibited compared with those when single gene immunization was used. No interference was detected for the Tat T-cell response. Thus, co-immunization with certain genes may result in inhibition of specific immune responses.
Asunto(s)
Vacunas contra el SIDA , Vectores Genéticos , VIH-1 , Linfocitos T/virología , Vacunas de ADN , Vacunas contra el SIDA/inmunología , Animales , Clonación Molecular , Mapeo Epitopo , Regulación Viral de la Expresión Génica , Genes nef/genética , Genes nef/inmunología , Genes rev/genética , Genes rev/inmunología , Genes tat/genética , Genes tat/inmunología , VIH-1/genética , VIH-1/inmunología , Humanos , Resistencia a la Kanamicina/genética , Resistencia a la Kanamicina/inmunología , Ratones , Neomicina , Papillomaviridae/genética , Plásmidos , Poli A/genética , Poli A/inmunología , Linfocitos T/inmunología , Vacunas de ADN/inmunologíaRESUMEN
The O-methylation pattern of the O polysaccharide (OPS) of the lipopolysaccharide of Pseudomonas syringae pv. phaseolicola GSPB 1552 was revealed by methylation (CD3I) analysis, Smith degradation, and NMR spectroscopy. Together with the major O repeats consisting of D-rhamnopyranose (D-Rhap) and D-fucofuranose (D-Fucf), there are minor repeats (approximately 30%) containing 3-O-methyl-D-rhamnose (D-acofriose), which is 2-substituted in the interior repeats and occupies the terminal non-reducing end of the OPS. It was suggested that the methylated O repeats are linked to each other nearby the non-reducing end of the OPS and that the 'biological' O repeat of the OPS has the following structure: [molecular structure: see text].
Asunto(s)
Manosa/análogos & derivados , Antígenos O/química , Pseudomonas/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Manosa/química , Metilación , Datos de Secuencia Molecular , Peso Molecular , Resonancia Magnética Nuclear Biomolecular , Ramnosa/químicaRESUMEN
The O polysaccharide (OPS) moiety of the lipopolysaccharide (LPS) of a phytopathogenic bacterium Pseudomonas syringae pv. ribicola NCPPB 1010 was studied by sugar and methylation analyses, Smith degradation, and 1H- and 13C-NMR spectroscopy, including 2D COSY, TOCSY, NOESY and H-detected 1H,13C HMQC experiments. The OPS structure was elucidated, and shown to be composed of branched pentasaccharide repeating units (O repeats) of two types, major (1) and minor (2), differing in the position of substitution of one of the rhamnose residues. Both O repeats form structurally homogeneous blocks within the same polysaccharide molecule. Although P. syringae pv. ribicola NCPPB 1010 demonstrates genetic relatedness and similarity in the OPS chemical structure to some other P. syringae pathovars, it did not cross-react with any OPS-specific mAbs produced against heterologous P. syringae strains. Therefore, we propose to classify P. syringae pv. ribicola NCPPB 1010 in a new serogroup, O8.
Asunto(s)
Antígenos O/química , Polisacáridos Bacterianos/química , Pseudomonas/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Lipopolisacáridos/química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Monosacáridos/análisis , Antígenos O/inmunología , Pseudomonas/inmunología , Pseudomonas/patogenicidad , SerotipificaciónRESUMEN
The O polysaccharide (OPS) of the lipopolysaccharide (LPS) of Pseudomonas syringae pv. atrofaciens IMV 7836 and some other strains that are classified in serogroup O1 was shown to be a novel linear alpha-D-rhamnan with the tetrasaccharide O repeat -->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-R hap-(1-->2)- alpha-D-Rhap-(1--> (chemotype 1A). The same alpha-D-rhamnan serves as the backbone in branched OPSs with lateral (alpha1-->3)-linked D-Rhap, (beta1-->4)-linked D-GlcpNAc, and (alpha1-->4)-linked D-Fucf residues (chemotypes 1B, 1C, and 1D, respectively). Strains of chemotype 1C demonstrated variations resulting in a decrease of the degree of substitution of the backbone 1A with the lateral D-GlcNAc residue (chemotype 1C-1A), which may be described as branched regular left arrow over right arrow branched irregular --> linear OPS structure alterations (1Cleft arrow over right arrow 1C-1A --> 1A). Based on serological data, chemotype 1D was suggested to undergo a 1D left arrow over right arrow 1D-1A alteration, whereas chemotype 1B showed no alteration. A number of OPS backbone-specific monoclonal antibodies (MAbs), Ps(1-2)a, Ps(1-2)a(1), Ps1a, Ps1a(1), and Ps1a(2), as well as MAbs Ps1b, Ps1c, Ps1c(1), Ps1d, Ps(1-2)d, and Ps(1-2)d(1) specific to epitopes related to the lateral sugar substituents of the OPSs, were produced against P. syringae serogroup O1 strains. By using MAbs, some specific epitopes were inferred, serogroup O1 strains were serotyped in more detail, and thus, the serological classification scheme of P. syringae was improved. Screening with MAbs of about 800 strains representing all 56 known P. syringae pathovars showed that the strains classified in serogroup O1 were found among 15 pathovars and the strains with the linear OPSs of chemotype 1A were found among 9 of the 15 pathovars. A possible role for the LPS of P. syringae and related pseudomonads as a phylogenetic marker is discussed.
Asunto(s)
Antígenos O , Pseudomonas/clasificación , Pseudomonas/inmunología , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Western Blotting , Secuencia de Carbohidratos , Ensayo de Inmunoadsorción Enzimática , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Antígenos O/química , Antígenos O/inmunología , SerotipificaciónRESUMEN
OBJECTIVE: HIV accessory protein Nef is expressed early in the infectious cycle of the virus and has been shown to be an effective immunogen in humoral and cellular immune responses. We have used two different self-replicating pBN vectors and one non-replicating pCGal2 derived (pCG) vector expressing HIV-1 Nef in DNA immunisation of mice in order to determine their efficiency in raising humoral and cellular immune responses. DESIGN AND METHODS: The expression of Nef by the three plasmids was tested by transfections into COS-1 cells. Balb/c mice were immunised with the pBN-NEF and pCGE2-NEF constructs using gold particle bombardment. Immunoblotting and immunocytochemistry were used to detect in vitro expression of Nef. 51Cr release assay, ELISA and immunoblotting were used to detect cellular and humoral immune responses in immunised mice. RESULTS: Efficient in vitro expression of Nef was detected in pBN and pCGE2-NEF transfected cells, in pBN-NEF transfected cells the expression lasting up to three weeks. Anti-Nef antibodies in sera of 13 of 16 pBN-NEF immunised mice were detected within four weeks after the last immunisation, whereas only 2 of 12 pCGE2-NEF immunised mice had very weak anti-Nef antibodies. Twelve of the pBN-NEF immunised mice (75%) and 6 the pCGE2-NEF immunised mice (50%) showed Nef-specific cytotoxic T lymphocyte (CTL) responses within four weeks. CONCLUSIONS: We conclude that the three eukaryotic expression vectors tested are capable of inducing a cell mediated immune response towards HIV-1 Nef and should be considered as part of a genetic HIV vaccine.
Asunto(s)
Vacunas contra el SIDA/inmunología , Productos del Gen nef/inmunología , Vacunas de ADN/inmunología , Animales , Células COS , Femenino , Vectores Genéticos , Anticuerpos Anti-VIH/sangre , Inmunización , Ratones , Ratones Endogámicos BALB C , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The composition and structure of the O-polysaccharide of the lipopolysaccharide of Pseudomonas syringae pathovar garcae ICMP 8047 were studied using methylation analyses, Smith degradation, and 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments. The polysaccharide was found to contain L-rhamnose and 3-acetamido-3, 6-dideoxy-D-galactose (D-Fuc3NAc) in the ratio 4:1 and to consist of two types of pentasaccharide repeating units. The major (1) and minor (2) repeating units differ from each other only in the position of substitution of one of the rhamnose residues in the main chain. Similar structural heterogeneity has been reported formerly in O-polysaccharides of some other P. syringae strains having a similar monosaccharide composition. A Fuc3NAc residue is attached to the main rhamnan chain as a side chain by a (alpha1-->4) glycosidic linkage; this has not hitherto been described in P. syringae: [figure].
Asunto(s)
Antígenos O/química , Pseudomonas/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Espectroscopía de Resonancia Magnética , Datos de Secuencia MolecularRESUMEN
Recombinant HIV-1 Nef protein, but not Tat, gp120, and gp160, provoked leukocyte recruitment into the CNS in a rat model. The strong reduction of bioactivity by heat treatment of Nef, and the blocking effect of the mAb 2H12, which recognizes the carboxy-terminal amino acid (aa) residues 171-190 (but not of mAb 3E6, an anti-Nef Ab of the same isotype, which maps the aa sequence 168-175, as well as a mixture of mAbs to CD4) provided evidence for the specificity of the observed Nef effects. Using a modified Boyden chamber technique, Nef exhibited chemotactic activity on mononuclear cells in vitro. Coadministration of the anti-Nef mAb 2H12, as well as treatment of Nef by heat inhibited Nef-induced chemotaxis. Besides soluble Nef, chemotaxis was also induced by a Nef-expressing human astrocytoma cell line, but not by control cells. These data suggest a direct chemotactic activity of soluble Nef. The detection of elevated levels of IL-6, TNF-alpha, and IFN-gamma in rat cerebrospinal fluid 6 h after intracisternal Nef injection hint at the additional involvement of indirect mechanisms in Nef-induced leukocyte migration into rat CNS. These data propose a mechanism by which HIV-1 Nef protein may be essential for AIDS neuropathogenesis, as a mediator of the recruitment of leukocytes that may serve as vehicles of the virus and perpetrators for disease through their production of neurotoxins.
Asunto(s)
Movimiento Celular/inmunología , Sistema Nervioso Central/inmunología , Productos del Gen nef/inmunología , VIH-1/inmunología , Leucocitos Mononucleares/virología , Neutrófilos/virología , Animales , Sistema Nervioso Central/citología , Sistema Nervioso Central/virología , Líquido Cefalorraquídeo/citología , Líquido Cefalorraquídeo/inmunología , Líquido Cefalorraquídeo/metabolismo , Quimiocina CCL2/líquido cefalorraquídeo , Quimiotaxis de Leucocito/inmunología , Cisterna Magna , Cámaras de Difusión de Cultivos , Relación Dosis-Respuesta Inmunológica , Productos del Gen nef/administración & dosificación , Productos del Gen nef/genética , VIH-1/genética , Humanos , Inyecciones , Interferón gamma/líquido cefalorraquídeo , Interleucina-6/líquido cefalorraquídeo , Recuento de Leucocitos , Leucocitos Mononucleares/inmunología , Masculino , Neutrófilos/inmunología , Ratas , Ratas Wistar , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Factor de Necrosis Tumoral alfa/líquido cefalorraquídeo , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
The AIRE gene (autoimmune regulator), coding for a putative transcriptional regulatory factor, is mutated in autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy (APECED). We have investigated the expression of the AIRE gene by mRNA in situ hybridization and immunohistochemistry in various human tissues. Here we show that AIRE is expressed in distinct cells in thymus medulla, and also in rare cells in lymph node paracortex and medulla, and in spleen and fetal liver, but not in the target organs of autoimmune destruction. Double immunofluorescence studies revealed that in thymus medulla both epithelial (cytokeratin positive) and non-epithelial cells expressed AIRE. Subcellularly, AIRE was localised in nuclear dots in thymus and lymph node and also in transfected cells. The cellular localisation of AIRE and its nuclear localisation, compatible with its predicted protein domains, suggest that AIRE may regulate the mechanisms involved in the induction and maintenance of immune tolerance.
Asunto(s)
Tolerancia Inmunológica , Timo/citología , Factores de Transcripción/metabolismo , Biomarcadores/análisis , Núcleo Celular/química , Colchicina/farmacología , Citocalasina B/farmacología , Citoplasma/química , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Células Dendríticas/química , Células Dendríticas/metabolismo , Células Epiteliales/química , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Inmunohistoquímica , Hibridación in Situ , Hígado/química , Hígado/citología , Hígado/embriología , Hígado/metabolismo , Ganglios Linfáticos/química , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Bazo/química , Bazo/citología , Bazo/metabolismo , Timo/química , Timo/inmunología , Timo/metabolismo , Factores de Transcripción/genética , Transfección , Células U937 , Proteína AIRERESUMEN
I detail the results of adapting a rigorous algorithm, derived for multiple-scattering simulations in photon correlation spectroscopy, for modeling multiple-scattering suppression by a cross-correlation system that employs one laser beam and two slightly tilted detectors. The practical significance of the proposed numerical technique is shown for optimization of an arbitrary design configuration of cross correlation and for prediction of the ideal performance that is possible with that design. It is shown that the behavior of the coherent factor modeled versus the angle between detectors is in agreement with experimental data and analytical investigation. This factor permits mapping of the spatial extent of the single-scattering and the multiple-scattering speckles. The map holds important information about the optimal displacement of detectors for a given measurement setup, and it permits a comprehensive investigation of suppression of the scattering components, even when their magnitudes are small.
RESUMEN
O polysaccharides (OPS) of the lipopolysaccharides (LPS) of Pseudomonas syringae pathovar tomato GSPB 483 and pathovar maculicola IMV 381 were studied by 1H-NMR and 13C-NMR spectroscopy, including two-dimensional COSY, rotating-frame NOE spectroscopy (ROESY), and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments. The OPS from both strains were shown to be chemically identical. Two structurally different types of repeating units (O repeats 1 and 2) were elucidated in each OPS. The minor O repeat is a pentasaccharide having the structure 2. The same structure has been reported earlier for some other OPS of P. syringae. The major O repeat is a hexasaccharide with the novel structure 1 which is distinguished from 2 by the presence of the second lateral residue of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc) and by a different position of substitution of one of the L-rhamnose (Rha) residues in the backbone. [structures: see text] Structural and serological diversity of OPS of strains belonging to P. syringae pv. tomato and pv. maculicola and phylogenetic relatedness of the strains are discussed.
Asunto(s)
Lipopolisacáridos/química , Antígenos O/química , Pseudomonas/inmunología , Conformación de Carbohidratos , Secuencia de Carbohidratos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Antígenos O/inmunología , Oligosacáridos/química , Polisacáridos Bacterianos/química , Pseudomonas/patogenicidad , SerologíaRESUMEN
The O polysaccharide (OPS) of the lipopolysaccharide of Pseudomonas syringae pv. coriandricola GSPB 2028 (NCPPB 3780, W-43) was studied by Smith degradation and 1H-NMR and 13C-NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments. The OPS was shown to consist of pentasaccharide O repeats of two types both containing four L-rhamnose and one 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc) residue. Structure 1 of the major O repeat which had been established earlier [Das, S., Ramm, M., Kochanowski, H. & Basu, S. (1994) J. Bacteriol. 176, 6550-6557], was confirmed by our data, and a new structure 2 was elucidated for the minor O repeat and found to differ from the structure 1 only in the position of substitution of one of the rhamnose residues in the main chain. [structures: see text] A role of structural and immunochemical features of the LPS for defining the taxonomical position of the bacterium studied is discussed.
Asunto(s)
Antígenos O/química , Polisacáridos Bacterianos/química , Pseudomonas/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Antígenos O/inmunología , Polisacáridos Bacterianos/inmunología , Pseudomonas/inmunología , Análisis de Secuencia , SerologíaRESUMEN
A novel protein encoded by the C210RF2 gene in chromosomal locus 21q22.3 was characterized by immunochemistry. This chromosomal region is known to contain genes for human diseases such as non-syndromic autosomal recessive deafness (DFNB8/10) and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Polyclonal murine antisera were produced against the multivalent peptides deduced from the amino acid sequence of the polypeptide. Immunological reactivity of the obtained antisera was tested with primary cells or established cell lines. On western blotting, the polyclonal sera recognized a single protein product of 25 Kd expressed in cell lines of epithelial and lymphoid origin. Subsequent immunochemistry of several human tissues indicated the ubiquitous expression of the protein. Immunofluorescence studies and co-staining with a mitochondrial-specific dye suggest the subcellular localization of the protein to mitochondria. Mitochondrial localization is also predicted by computer analysis of the polypeptide sequence. As deafness is known to be caused in some instances by defects in mitochondrial function, C210RF2 is a plausible candidate gene for DFNB8/10.
Asunto(s)
Cromosomas Humanos Par 21 , Sordera/genética , Genes , Mitocondrias/química , Mitocondrias/genética , Secuencia de Aminoácidos , Técnica del Anticuerpo Fluorescente , Genes/inmunología , Humanos , Sueros Inmunes/química , Inmunohistoquímica , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismo , SíndromeRESUMEN
We describe the characterization of the B-cell epitopes of HIV-1 regulatory proteins Tat and Rev. The prevalence of antibodies to these proteins among human immunodeficiency virus (HIV)-1-infected individuals was examined by enzyme-linked immunosorbent assay (ELISA) and by Western blotting. The Tat and Rev antibody-positive sera were selected for epitope mapping performed with partially overlapping synthetic peptides bound to polyethylene pins. Eighteen and twelve percent of HIV-infected individuals had antibodies against Tat or Rev, respectively. In Tat, four epitopic regions were identified, situated within amino acids 6-10 (PRLEP), 21-37 (ACTNCYCKKCCFHCQVC), 39-58 (ITKALGISYGRKKRRQRRRA) and 74-82 (TSQSRGDPT). The most frequently recognized epitopic regions were located in the middle of the protein. In Rev, the two most frequently recognized epitopic regions were near the amino terminus of the protein within amino acids 12-20 (LIRTVRLIK) and 38-49 (RRNRRRRWRERQ). A third epitope was mapped around amino acids 55-62 (ISERILGT) and a fourth around amino acids 78-83 (LERLTU). To analyze the specificity of Tat and Rev epitopes, soluble synthetic peptides representing the identified epitopes were used in an ELISA assay, and the recognition of most epitopes was shown to be specific for HIV-1-infected individuals. In addition, many of the Tat and Rev epitopes were shown to overlap with regions having functional activity or with regions previously identified as T-cell epitopes.
Asunto(s)
Linfocitos B/virología , Epítopos/análisis , Productos del Gen rev/genética , Productos del Gen tat/genética , Infecciones por VIH/inmunología , Seropositividad para VIH/inmunología , VIH-1/genética , Linfocitos T/virología , Secuencia de Aminoácidos , Linfocitos B/inmunología , Finlandia , Productos del Gen rev/biosíntesis , Productos del Gen rev/química , Productos del Gen tat/biosíntesis , Productos del Gen tat/química , Seronegatividad para VIH/inmunología , VIH-1/inmunología , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Linfocitos T/inmunología , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Murine monoclonal antibodies (MAbs) reacting with Pseudomonas syringae lipopolysaccharide (LPS) O polysaccharides (OPS) composed of tetra- and tri-alpha-D-rhamnose repeats in the backbone [3)D-Rha(alpha1-3)D-Rha(alpha1-2)D-Rha(alpha1-2)D-Rha(alpha1] and [3)D-Rha(alpha1-3)D-Rha(alpha1-2)D-Rha(alpha1] were generated and used for immunochemical analysis and for serological classification of the bacteria. A total of 195 of 358 P. syringae strains tested representing 21 pathovars were shown to share a common epitope, 1a, and were classified into serogroup O1. All strains with pathovars aptata, glycinea, japonica, phaseolicola, and pisi, most of the strains with pathovars atrofaciens and striafaciens, and half of the strains with pathovar syringae were classified into serotypes O1a', O1b, O1c, and O1d within serogroup O1. Serogroup-specific epitope 1a was inferred to be related to the (alpha1-2)D-Rha(alpha1-3) site of the OPS backbone. The serotype-specific epitopes 1b, 1c, 1d, and 1a' were inferred as relating to the immunodominant lateral (alpha1-3)D-Rha, (beta1-4)D-GlcNAc, and (alpha1-4)D-Fuc substituents and backbone-located site (alpha1-3)D-Rha(alpha1-2), respectively, of OPSs that share the common tetra-D-rhamnose repeats in the backbone. A total of 7.3% of the strains studied, all with pathovars morsprunorum and lapsa, were classified as serotypes O2a and O2d within serogroup 02. Serotype-specific epitope 2a was inferred as being related to the backbone-located site D-Rha(alpha1-3)D-Rha and epitope 2d to the immunodominant lateral (alpha1-4)D-Fuc residue of OPS consisting of tri-D-rhamnose repeats in the backbone. Epitope 2d alternated with 2a within the same LPS molecule and did not cross-react with epitope 1d. Serotypes O2a and O2d were observed in some strains correlating with the coexpression of the two chemotypes of OPS by the same strain. The serogroup O1-specific MAb Ps1a reacted weakly but definitely with all strains from serogroup 02. We propose serological formulas for serogroups O1 and 02 as well as for individual strains within these serogroups.