RESUMO
Quantifying signals and uncertainties in climate models is essential for the detection, attribution, prediction and projection of climate change1-3. Although inter-model agreement is high for large-scale temperature signals, dynamical changes in atmospheric circulation are very uncertain4. This leads to low confidence in regional projections, especially for precipitation, over the coming decades5,6. The chaotic nature of the climate system7-9 may also mean that signal uncertainties are largely irreducible. However, climate projections are difficult to verify until further observations become available. Here we assess retrospective climate model predictions of the past six decades and show that decadal variations in North Atlantic winter climate are highly predictable, despite a lack of agreement between individual model simulations and the poor predictive ability of raw model outputs. Crucially, current models underestimate the predictable signal (the predictable fraction of the total variability) of the North Atlantic Oscillation (the leading mode of variability in North Atlantic atmospheric circulation) by an order of magnitude. Consequently, compared to perfect models, 100 times as many ensemble members are needed in current models to extract this signal, and its effects on the climate are underestimated relative to other factors. To address these limitations, we implement a two-stage post-processing technique. We first adjust the variance of the ensemble-mean North Atlantic Oscillation forecast to match the observed variance of the predictable signal. We then select and use only the ensemble members with a North Atlantic Oscillation sufficiently close to the variance-adjusted ensemble-mean forecast North Atlantic Oscillation. This approach greatly improves decadal predictions of winter climate for Europe and eastern North America. Predictions of Atlantic multidecadal variability are also improved, suggesting that the North Atlantic Oscillation is not driven solely by Atlantic multidecadal variability. Our results highlight the need to understand why the signal-to-noise ratio is too small in current climate models10, and the extent to which correcting this model error would reduce uncertainties in regional climate change projections on timescales beyond a decade.
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OBJECTIVES: Although the rotation of lower extremities has gained increasing recognition as a risk factor for anterior cruciate ligament (ACL) injury. This study clarified the influence of lower extremity rotation on the knee during single-leg landing. DESIGN AND SETTING: We recruited 30 students to perform single-leg landing from a height of 30 cm with their lower extremities in neutral, and externally and internally rotated. The knee abduction, flexion angles, and abduction angular velocity were measured. Furthermore, the abduction angle was analyzed at knee flexion angles of 15°, 20°, 25°, and 30° and compared among the three conditions using a repeated measures analysis of variance with Bonferroni post hoc tests. RESULTS: The maximum abduction angle was significantly greater when internally rotated than in the neutral. The maximum abduction angular velocity was significantly greater in the internally rotated compared to in the neutral. Finally, the abduction angle at a knee flexion angle of 30° was significantly greater when internally rotated compared to in the neutral. CONCLUSION: Rotation of the lower extremities affects knee kinematics, and landing on a knee that is internally rotated may increase the risk of ACL injury.
Assuntos
Lesões do Ligamento Cruzado Anterior , Perna (Membro) , Humanos , Fenômenos Biomecânicos , Rotação , Articulação do Joelho , Extremidade InferiorRESUMO
Studies in our laboratory and elsewhere have shown that it is possible to propagate antigen-specific murine T cells in vitro with resultant specific stepwise enrichment of antigen-induced proliferative cells. The proliferative responses of these T cells are antigen specific and dependent upon the presence of antigen-presenting cells (spleen cells) that share the I-A subregion with the proliferating T cell. Using techniques of soft-agar cloning, it has been further possible to isolate clones of antigen-reactive T lymphocytes from such long-term cultures. Data suggesting that these were clones of antigen-reactive T cells were obtained by studying the recognition of antigen in association with antigen-presenting cells with a panel of such clones of antigen-reactive T cells. Proof of clonality was obtained by subcloning. Clones derived from F1-immune mice can be divided into three separate categories: one clone recognizes antigen in association with antigen-presenting determinants of parent A and the F1; the second type recognizes antigen in association with antigen-presenting determinants of parent B and the F1; and the third type recognizes antigen only in association with antigen-presenting determinants of the F1 mouse. Genetic studies on the major histocompatibility complex requirements for antigen presentation to such F1-reactive T cell clones suggests that the hybrid antigen-presenting determinant in this system results from transcomplementation of products of the I-A region of haplotypes a and b. These studies support the concept developed in our laboratory that there exist unique F1 hybrid determinants on (A/J X C57BL/6) F1 cells and suggest that these determinants can be utilized physiologically by hybrid mice in immunocompetent cellular interactions.
Assuntos
Genes MHC da Classe II , Antígenos de Histocompatibilidade Classe II/imunologia , Hibridização Genética , Linfócitos T/imunologia , Animais , Células Clonais , Epitopos , Feminino , Ativação Linfocitária , Complexo Principal de Histocompatibilidade , Masculino , Camundongos , Camundongos Endogâmicos , Recombinação GenéticaRESUMO
Using murine (T,G)-A--L-reactive T cell clones, we have demonstrated the existence of unique homozygous antigen-presenting determinants expressed on C57bl/6 mice, controlled by the I-A subregion of the murine major histocompatibility complex (MHC), which are not expressed on semisyngeneic (C57Bl/6 x A/J)F1 [(B6A)F1] cells. Additionally, we were able to demonstrate that there exist (T,G)-A--L-reactive clones in F1 mice derived between low responder and high responder parents [(B6A)F1] that recognize antigen in association with transcomplementing hybrid I-A subregion determinants expressed uniquely on (B6A)F1 cells not expressed on cells of either of the parental strains. These data suggest that phenotypic high responsiveness exhibited by (higher responder x low responder)F1 mice was not simply controlled by the high responder parental genome, but was controlled at the phenotypic level of expression of antigen-presenting determinants. Such antigen-presenting determinants can be created by complementation using products of the low responder as well as high responder genome. The significance of the existence of such F1 specific hybrid antigen-presenting determinants for T cell specificity and recognition of self was discussed.
Assuntos
Antígenos , Epitopos , Peptídeos/imunologia , Linfócitos T/imunologia , Animais , Células Cultivadas , Células Clonais/imunologia , Cruzamentos Genéticos , Homozigoto , Células Híbridas/imunologia , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Fatores de TempoRESUMO
Mixed isotype A beta dE alpha d class II molecule-restricted antigen-reactive T cell clones were obtained from (BALB/c x B6E alpha d)F1 mice. These T cell clones responded to keyhole limpet hemocyanin in the presence of (BALB/c x B6E alpha d)F1 but not CBF1 APCs. Both anti-A beta d and anti-E alpha mAbs blocked the proliferative responses of these clones. The frequency of such mixed isotype A beta E alpha-restricted T cell clones in (BALB/c x B6E alpha d)F1 mice was estimated to be approximately 10% from our limiting dilution cloning. The existence of such mixed isotype class II molecule-restricted T cells would have important implications for the expansion of the T cell repertoire as well as the induction of autoimmunity.
Assuntos
Antígenos CD4/imunologia , Linfócitos T/imunologia , Animais , Células Clonais , Camundongos , Camundongos TransgênicosRESUMO
Long-term-cultured poly(Tyr, Glu)-poly-D,L,-Ala-poly-Lys [(T,G)-A--L]-reactive T cells and clones derived from (high responder x low responder)F1 [(C57BL/6 x A/J)F1] mice were shown to recognize (T,G)-A--L presented by cells from low responder strain A/J mice. The antigen-presenting determinant(s) that allowed recognition of (T,G)-A--L by such T cell clones was controlled by the I-A subregion of the major histocompatibility complex. These results suggest that there is no functional defect in the ability of low responder Ir gene products (I-A antigens) to associate with (T,G)-A--L for effective recognition by T cells. Although these results might tentatively be interpreted to suggest that Ir gene-controlled low responsiveness is due to the inability of the T cell to recognize the association between (T,G)-A--L and low responder I-A gene products, it is similarly possible that there might be a defect in the functional capabilities of low responder antigen-presenting cells to effectively process (T,G)-A--L into immunodominant epitopes.
Assuntos
Genes MHC da Classe II , Antígenos de Histocompatibilidade Classe II , Linfócitos T/imunologia , Animais , Adesão Celular , Células Cultivadas , Células Clonais/imunologia , Camundongos , Peptídeos/imunologia , Receptores de Antígenos , Baço/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
We established a novel monoclonal antibody, RP/14, that can protect B cells from apoptosis induced by irradiation or dexamethasone. A molecule recognized by RP/14 (the RP antigen) was expressed on B cells with B220bright, IgMdull, and IgDbright. Immunoprecipitation experiments revealed that RP/14 recognized a monomeric protein with an approximate molecular mass of 105 kD. Stimulation of B cells with RP/14 for 48 h induced B cell proliferation and blastogenesis. In contrast to B cells of wild-type mice, X-linked immunodeficient (XID) B cells did not proliferate upon stimulation with RP/14, although the RP antigen was expressed to the same extent as that of wild-type B cells. These results suggest that the RP antigen-mediated signaling pathway is important for rescuing B cells from apoptosis and is deficient in XID B cells.
Assuntos
Anticorpos Monoclonais/imunologia , Apoptose , Linfócitos B/imunologia , Síndromes de Imunodeficiência/imunologia , Ativação Linfocitária , Cromossomo X , Animais , Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos B/fisiologia , Antígenos CD40 , Ligação Genética , Imunoglobulina D/análise , Imunoglobulina M/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteção Radiológica , Ratos , Ratos WistarRESUMO
Protein C is an important regulatory mechanism of blood coagulation. Protein C functions as an anticoagulant when converted to the active serine protease form on the endothelial cell surface. Thrombomodulin (TM), an endothelial cell surface receptor specific for thrombin, has been identified as an essential component for protein C activation. Although protein C can be activated directly by the thrombin-TM complex, the conversion is known as a relatively low-affinity reaction. Therefore, protein C activation has been believed to occur only in microcirculation. On the other hand, we have identified and cloned a novel endothelial cell surface receptor (EPCR) that is capable of high-affinity binding of protein C and activated protein C. In this study, we demonstrate the constitutive, endothelial cell-specific expression of EPCR in vivo. Abundant expression was particularly detected in the aorta and large arteries. In vitro cultured, arterial endothelial cells were also found to express abundant EPCR and were capable of promoting significant levels of protein C activation. EPCR was found to greatly accelerate protein C activation by examining functional activity in transfected cell lines expressing EPCR and/or TM. EPCR decreased the dissociation constant and increased the maximum velocity for protein C activation mediated by the thrombin-TM complex. By these mechanisms, EPCR appears to enable significant levels of protein C activation in large vessels. These results suggest that the protein C anticoagulation pathway is important for the regulation of blood coagulation not only in microvessels but also in large vessels.
Assuntos
Fatores de Coagulação Sanguínea , Endotélio Vascular/metabolismo , Proteína C/metabolismo , Receptores de Superfície Celular/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticoagulantes/farmacologia , Coagulação Sanguínea/fisiologia , Linhagem Celular , Endotélio Vascular/citologia , Ativação Enzimática/fisiologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Cinética , Modelos Biológicos , Serina Endopeptidases/metabolismo , Trombina/fisiologia , Trombomodulina/fisiologia , Transfecção/genéticaRESUMO
Toll-like receptor 4 (TLR4) is a mammalian homologue of Drosophila Toll, a leucine-rich repeat molecule that can trigger innate responses against pathogens. The TLR4 gene has recently been shown to be mutated in C3H/HeJ and C57BL/10ScCr mice, both of which are low responders to lipopolysaccharide (LPS). TLR4 may be a long-sought receptor for LPS. However, transfection of TLR4 does not confer LPS responsiveness on a recipient cell line, suggesting a requirement for an additional molecule. Here, we report that a novel molecule, MD-2, is requisite for LPS signaling of TLR4. MD-2 is physically associated with TLR4 on the cell surface and confers responsiveness to LPS. MD-2 is thus a link between TLR4 and LPS signaling. Identification of this new receptor complex has potential implications for understanding host defense, as well as pathophysiologic, mechanisms.
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Antígenos de Superfície/imunologia , Proteínas de Drosophila , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Animais , Antígenos de Superfície/genética , Sequência de Bases , Linhagem Celular , DNA Complementar/genética , Humanos , Antígeno 96 de Linfócito , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Receptor 4 Toll-Like , Receptores Toll-Like , TransfecçãoRESUMO
The susceptibility to infections induced by Gram-negative bacteria is largely determined by innate immune responses to bacteria cell wall lipopolysaccharide (LPS). The stimulation of B cells by LPS enhances their antigen-presenting capacity and is accompanied by B cell proliferation and secretion of large quantities of LPS-neutralizing antibodies. Similar to macrophages and neutrophils, the LPS-induced activation of B cells is dependent on Toll-like receptor (TLR)4. Here, we demonstrate that the responses of B cells to LPS are also regulated by another TLR protein, RP105, which is predominantly expressed on mature B cells in mice and humans. The analysis of mice homozygous for the null mutation in the RP105 gene revealed impaired proliferative and humoral immune responses of RP105-deficient B cells to LPS. Using originally LPS-unresponsive Ba/F3 cells expressing exogenous TLR4 and RP105, we demonstrate the functional cooperation between TLR4 and RP105 in LPS-induced nuclear factor kappaB activation. These data suggest the existence of the TLR4-RP105 signaling module in the LPS-induced B cell activation.
Assuntos
Antígenos CD , Linfócitos B/imunologia , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/fisiologia , Transdução de Sinais/imunologia , Animais , Antígenos de Superfície/fisiologia , Linfócitos B/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Cruzamentos Genéticos , Éxons , Linfonodos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Sequências Repetitivas de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Baço/imunologia , Linfócitos T/imunologiaRESUMO
Signals through the B cell antigen receptor lead to a variety of cellular events such as activation, anergy, and apoptosis. B cells select these outcomes to establish and maintain self-tolerance, and to mount adequate antibody responses. However, it is not fully understood how one and the same signal causes such different consequences. In the present study, we have studied the effect of activation signals on the outcome of responses to antigen receptor ligation. Two distinct growth-promoting signals were used to activate B cells. Ligation of either RP105, a newly discovered B cell surface molecule, or the CD40 molecule, drove B cells to proliferate. Resultant blastic cells were then exposed to anti-immunoglobulin M (IgM). Blast cells that had been stimulated with anti-RP105 ceased growing and underwent apoptosis after cross-linking of surface IgM. Coligation of the Fc gamma receptor IIB with surface IgM augmented, rather than aborted, this response. In contrast to RP105-activated B cells, blast cells that had been activated by CD40 ligation were unaltered by anti-IgM. On the other hand, CD40-activated B cells became extremely susceptible to Fas-mediated apoptosis, whereas RP105-activated B cells were much less sensitive. Anti-IgM-induced apoptosis in RP105 blasts was independent of Fas, because it was demonstrable with Fas-deficient MRL-lpr/lpr mice. These results demonstrate that the nature of an initial activation signal has a great influence on the fate of activated B cells after (re)engagement of the antigen receptor. RP105, as well as CD40, may be important in this life/death decision.
Assuntos
Antígenos CD , Apoptose , Linfócitos B/citologia , Antígenos CD40/fisiologia , Ativação Linfocitária , Proteínas de Membrana/fisiologia , Receptores de Antígenos de Linfócitos B/fisiologia , Animais , Ciclo Celular , Imunoglobulina M/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Agregação de Receptores , Transdução de Sinais , Receptor fas/fisiologiaRESUMO
Previous reports have demonstrated that accessory cells function to present soluble protein antigens in association with gene products encoded within the I region of the major histocompatibility complex (MHC) to antigen-reactive T helper cells. The biochemical events that occur during antigen presentation are, however, not well-documented primarily because of the difficulties involved in purifying sufficient numbers of homogeneous antigen-presenting cells. In this paper, a number of Ia-positive B lymphocyte tumor lines are shown to be capable of presenting soluble protein antigens to antigen-reactive continuous T cell lines in an MHC-restricted fashion. The characterization of the antigen presentation function of these tumor cells indicates that the tumor cells have many of the functional antigen-presenting characteristics previously thought to be limited to macrophages. These tumor cells should provide a useful model system for determining the biochemical events that occur in antigen uptake and processing as well as for determining the potential interactions between processed antigen and Ia molecules on the plasma membrane of these antigen-presenting cells.
Assuntos
Antígenos de Neoplasias/genética , Linfócitos B/imunologia , Complexo Principal de Histocompatibilidade , Linfócitos T/imunologia , Animais , Epitopos , Antígenos de Histocompatibilidade Classe II , Soros Imunes/farmacologia , Cinética , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Neoplasias Experimentais/imunologiaRESUMO
Physical interaction between human lymphomas and murine bone marrow derived stromal cells were studied. Nalm-6 pre-B cells adhered to BMS2 stromal cells and subsequently migrated beneath them, while Ramos Burkitt lymphoma cells, adhered but did not migrate. Four mAbs were established against Nalm-6 cells, which were able to block initial adhesion of Nalm-6 cells. Two of them were directed against the alpha 4 chain of VLA-4, and other two recognized the beta 1 chain of VLA integrins. Therefore, the initial adhesion of Ramos and Nalm-6 cells to BMS2 was largely mediated by the VLA-4 integrin expressed on lymphocytes. The corresponding ligand on stromal cells appears to be VCAM-1, because antibodies against murine VCAM-1 blocked the adhesion. However, antibodies against the alpha chain of VLA-4 were not capable of blocking subsequent migration beneath stromal cells. In contrast, antibodies against the beta chain of VLA integrins blocked the migration beneath stromal cells as well as the initial adhesion. Because a common beta chain can be shared among integrins, the role of other VLA integrins in Nalm-6 cells migration was investigated. VLA-5 and VLA-6 as well as VLA-4 were expressed on Nalm-6 cells, but not on Ramos cells. Additional blocking experiments revealed that VLA-4 and VLA-5 are likely to work in concert to mediate the migration of Nalm-6 cells beneath stromal cells. Thus, particular VLA integrins appear to be responsible not only for lymphocyte adhesion but also for migration with respect to stromal cells. These findings may have implications for cell-cell interactions and directed migration of lymphocytes in bone marrow and other tissues.
Assuntos
Medula Óssea/fisiologia , Adesão Celular , Receptores de Antígeno muito Tardio/fisiologia , Animais , Anticorpos Monoclonais , Linfócitos B/imunologia , Células da Medula Óssea , Movimento Celular , Células Cultivadas , Fibronectinas/imunologia , Imunofluorescência , Humanos , Linfoma , Camundongos , Receptores de Antígeno muito Tardio/biossíntese , Receptores de Antígeno muito Tardio/imunologia , Células Tumorais CultivadasRESUMO
Two new mAbs (M/K-1 and M/K-2) define an adhesion molecule expressed on stromal cell clones derived from murine bone marrow. The protein is similar in size to a human endothelial cell adhesion molecule known as VCAM-1 or INCAM110. VCAM-1 is expressed on endothelial cells in inflammatory sites and recognized by the integrin VLA-4 expressed on lymphocytes and monocytes. The new stromal cell molecule is a candidate ligand for the VLA-4 expressed on immature B lineage lymphocytes and a possible homologue of human VCAM-1. We now report additional similarities in the distribution, structure, and function of these proteins. The M/K antibodies detected large cells in normal bone marrow, as well as rare cells in other tissues. The antigen was constitutively expressed and functioned as a cell adhesion molecule on cultured murine endothelial cells. It correlated with the presence of mRNA which hybridized to a human VCAM-1 cDNA probe. Partial NH2 terminal amino acid sequencing of the murine protein revealed similarities to VCAM-1 and attachment of human lymphoma cells to murine endothelial cell lines was inhibited by the M/K antibodies. All of these observations suggest that the murine and human cell adhesion proteins may be related. The antibodies selectively interfered with B lymphocyte formation when included in long term bone marrow cultures. Moreover, they caused rapid detachment of lymphocytes from the adherent layer when added to preestablished cultures. The VCAM-like cell adhesion molecule on stromal cells and VLA-4 on lymphocyte precursors may both be important for B lymphocyte formation.
Assuntos
Linfócitos B/metabolismo , Moléculas de Adesão Celular/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Linfócitos B/citologia , Adesão Celular , Moléculas de Adesão Celular/análise , Linhagem Celular , Camundongos , Dados de Sequência Molecular , Receptores de Antígeno muito Tardio/metabolismo , Molécula 1 de Adesão de Célula VascularRESUMO
An evaluation was made of a fully automated index of psoriasis, termed Computer-assisted Area and Severity Index (CASI). This method requires taking digital photographs of the target skin area(s) with a colour reference marker, Casmatch. The CASI evaluates the severity of the psoriasis from the size and redness of the lesion(s). In five patients with mild psoriasis vulgaris mainly observed on their trunk, 18 photographs of the trunk were taken every 2 weeks. Three of the five patients [Psoriasis Area and Severity Index (PASI) of 3.0, 3.6 and 10.1, respectively] were treated with oral cyclosporin 3 mg/kg/day for 4 weeks. The mean +/- SD area of lesion selected by a dermatologist was 2.3 +/- 1.3% of the total skin area. This method achieved extraction performance for psoriasis of 72.1 +/- 19.4% for sensitivity and 97.4 +/- 2.0% for specificity. CASI correlated strongly with PASI (r = 0.92), but not with Skindex16 (r = 0.35). Although only erythema was evaluated, our preliminary results indicate that this method is capable of quantifying psoriasis lesions.
Assuntos
Interpretação de Imagem Assistida por Computador/métodos , Psoríase/patologia , Índice de Gravidade de Doença , Calibragem , Cor , Ciclosporina/uso terapêutico , Fármacos Dermatológicos/uso terapêutico , Humanos , Fotografação/métodos , Psoríase/tratamento farmacológico , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Acral melanocytic nevi have parallel patterns on dermoscopy; however, it is not understood why pigment distributions exhibit such patterns. AIM: To clarify the reason why melanin distributions in acral melanocytic nevi exhibit a parallel pattern on dermoscopy. METHODS: A serial sectioning perpendicular to the skin markings was performed, and each section was stained with modified Fontana-Masson staining to pick out melanin granules. These sections were then reconstructed to three-dimensional images by an image processing software, and these three-dimensional images were analyzed. RESULTS: Melanin columns in the cornified layer were mainly seen in the dermoscopic images of acral melanocytic nevi. In the parallel-furrow pattern melanin columns were arranged vertically, and in the fibrillar pattern they were arranged in a slanting fashion. CONCLUSION: Melanin columns in the cornified layer, not melanin in the basal layer, mainly contribute to the dermoscopic pattern of acral melanocytic nevi.
Assuntos
Dermoscopia , Doenças do Pé/patologia , Mãos/patologia , Imageamento Tridimensional/métodos , Melaninas/análise , Nevo Pigmentado/patologia , Neoplasias Cutâneas/patologia , Humanos , Processamento de Imagem Assistida por Computador , Melaninas/metabolismo , Nitrato de Prata , Pele/metabolismo , Pele/patologiaRESUMO
Chlamydia pneumoniae is a widespread pathogen of humans causing pneumonia and bronchitis. There are many reports of an association between C.PNEUMONIAE: infection and atherosclerosis. We determined the whole genome sequence of C.PNEUMONIAE: strain J138 isolated in Japan in 1994 and compared it with the sequence of strain CWL029 isolated in the USA before 1987. The J138 circular chromosome consists of 1 226 565 nt (40.7% G+C) with 1072 likely protein-coding genes that is 3665 nt shorter than the CWL029 genome. Plasmids, phage- or transposon-like sequences were not identified. The overall genomic organization, gene order and predicted proteomes of the two strains are very similar, suggesting a high level of structural and functional conservation between the two unrelated isolates. The most conspicuous differences in the J138 genome relative to the CWL029 genome are the absence of five DNA segments, ranging in size from 89 to 1649 nt, and the presence of three DNA segments, ranging from 27 to 84 nt. The complex organization of these 'different zones' may be attributable to a unique system of recombination.
Assuntos
Chlamydophila pneumoniae/genética , Genoma Bacteriano , Composição de Bases , Sequência de Bases , Chlamydophila pneumoniae/isolamento & purificação , Cromossomos Bacterianos , DNA Circular/genética , Humanos , Japão , Polimorfismo Genético , Mapeamento por RestriçãoRESUMO
A monoclonal antibody (mAb, A) recognizing the FAD-binding domain of 4-aminobenzoate hydroxylase (4-aminobenzoate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating, decarboxylating), EC 1.14.13.27) from Agaricus bisporus, a common edible mushroom, had been produced (Tsuji, H., Ogawa, T., Bando, N., Kimoto, M. and Sasaoka, K. (1990) J. Biol. Chem. 265, 16064-16067). In the present study, three other mAbs (B1, B2 and B3) against the enzyme have been further prepared in order to facilitate the structural characterization of the enzyme. The three new mAbs immunoblotted the enzyme. The four mAbs, including A, were specific for different epitopes on the enzyme. B1 and B2 immunoprecipitated the apoenzyme and the immunoprecipitation was inhibited in the presence of FAD, whereas B3 failed to immunoprecipitate the apoenzyme in the absence or presence of FAD. B1 and B2 competed with FAD for the binding to the apoenzyme. These findings show that B1 and B2 recognize the FAD-binding domain of the enzyme in analogy with A. The immunoblotting analyses of the peptides obtained from the enzyme by digestion with lysyl endopeptidase (EC 3.4.21.50) provided useful knowledge as to the location of the epitopes to the mAbs on the enzyme, suggesting that the FAD-binding domain of the enzyme can be located and characterized by detailed investigations on the location of the epitopes.
Assuntos
Agaricus/enzimologia , Anticorpos Monoclonais/biossíntese , Oxigenases de Função Mista/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Sítios de Ligação , Epitopos/imunologia , Flavina-Adenina Dinucleotídeo/metabolismo , Hibridomas/imunologia , Immunoblotting , Técnicas de Imunoadsorção , Camundongos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Serina Endopeptidases/metabolismoRESUMO
By intraperitoneal injection of 1-aminoproline, death after severe convulsion was observed in rats (LD50 of 1-amino-L-proline, 26 mg per kg of body weight for young male rats fed a normal diet). The vitamin B-6-deficient rats were more sensitive to this hydrazino acid than the normal rats. The toxic effect was completely prevented by the administration of pyridoxine. 1-Amino-D-proline was less toxic than the L-isomer. By the 1-aminoproline treatment, the most remarkable changes in the free amino acid levels were the striking increases in the concentrations of alpha-aminoadipic acid, citrulline and cystathionine in all the tissues tested, except in brain. Some unidentified ninhydrin-positive substances appeared. These results indicate that 1-aminoproline greatly disturbed the amino acid pattern, i.e. the amino acid metabolism in rats.
Assuntos
Prolina/análogos & derivados , Piridoxina/antagonistas & inibidores , Aminoácidos/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Dose Letal Mediana , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Especificidade de Órgãos , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Prolina/farmacologia , Prolina/toxicidade , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Deficiência de Vitamina B 6/metabolismoRESUMO
A membrane-bound nonspecific triphosphatase of E. coli was solubilized and purified to a homogeneous SDS-acrylamide gel electrophoresis band. It was found to be a single polypeptide of 16 kDa requiring no Mg2+, with an optimal pH at 6.5. The substrate specificity was broad and a nonspecific Mg2+-independent ribonucleoside-triphosphatase (NTPase) activity was expressed together with thiamin-triphosphatase activity. The molecular size and characteristics were clearly different from the known NTPase (EC 3.6.1.15). Using the purified thiamin-triphosphatase II, ATP:thiamin-diphosphate phosphoryl transferase (EC 2.7.4.15) activity was demonstrated with an optimal pH of approx. 5.3. Considering its kinetic parameters and other characteristics, however, the thiamin triphosphate synthesizing activity was not thought to take part in cellular thiamin triphosphate synthesis. The possibility that thiamin-triphosphatase II plays a part in the hydrolysis of thiamin triphosphate to control its cellular level is suggested.