RESUMO
The atomic scale structure of aluminum in amorphous alumina films processed by direct liquid injection chemical vapor deposition from aluminum tri-isopropoxide (ATI) and dimethyl isopropoxide (DMAI) is investigated by solid-state 27Al nuclear magnetic resonance (SSNMR) using a very high magnetic field of 20.0 T. This study is performed as a function of the deposition temperature in the range 300-560 °C, 150-450 °C, and 500-700 °C, for the films processed from ATI, DMAI (+H2O), and DMAI (+O2), respectively. While the majority of the films are composed of stoichiometric aluminum oxide, other samples are partially or fully hydroxylated at low temperature, or contain carbidic carbon when processed from DMAI above 500 °C. The quantitative analysis of the SSNMR experiments reveals that the local structure of these films is built from AlO4, AlO5, AlO6 and Al(O,C)4 units with minor proportions of the 6-fold aluminum coordination and significant amounts of oxycarbides in the films processed from DMAI (+O2). The aluminum coordination distribution as well as the chemical shift distribution indicate that the films processed from DMAI present a higher degree of structural disorder compared to the films processed from ATI. Hydroxylation leads to an increase of the 6-fold coordination resulting from the trend of OH groups to integrate into AlO6 units. The evidence of an additional environment in films processed from DMAI (+O2) by 27Al SSNMR and first-principle NMR calculations on Al4C3 and Al4O4C crystal structures supports that carbon is located in Al(O,C)4 units. The concentration of this coordination environment strongly increases with increasing process temperature from 600 to 700 °C favoring a highly disordered structure and preventing from crystallizing into γ-alumina. The obtained results are a valuable guide to the selection of process conditions for the CVD of amorphous alumina films with regard to targeted applications.
RESUMO
This work reconsiders the GATC motif distribution in a 1.6 Mb segment of the Escherichia coli genome, compared to its distribution in phages and plasmids. At first sight the distribution of GATC words looks random. But when a realistic model of the chromosome (made of average genes having the same codon usage as in the real chromasome), is used as a theoretical reference, strong biasesare observed. GATC pairs such as GATCNNGATC are under-represented while there is a strong positive selection for motifs separated by 10, 19, 70 and 1100 bp. The last class is the only one present in E. coli parasites. It can be ascribed to the triggering sequences of the long-patch mismatch repair system. The 6 bp class overlaps with the consensus of CAP (catabolite activator protein) and FNR (fumarate/nitrate regulator) binding sites, thus accounting for counter-selection. The other classes, which could be targets for a nucleic acid-binding protein, are almost always present inside protein coding sequences, and are members of clusters of GATC motifs. Analysis of the genes containing these motifs suggests that they correspond to a regulatory process monitoring the shift from anaerobic to aerobic growth conditions. In particular this regulation, closing down transcription of a large number of genes involved in intermediary metabolism would be well suited for the cold and oxygen shift from the mammal's gut to the standard environmental conditions. In this process the methylation status of GATC clusters would be very important for tuning transcription, and a DNA binding protein, probably a member of the cold-shock proteins family would be needed for alleviating the effects mediated by slackening of the pace of methylation during the shift.
Assuntos
Bacteriófagos/genética , DNA Bacteriano/genética , Escherichia coli/genética , Oligonucleotídeos/genética , Plasmídeos/genética , Análise de Sequência de DNA , Sequência de Bases , Dados de Sequência MolecularRESUMO
The effect of bacterial flora on cytokine production from resident peritoneal macrophages was investigated in the mouse. The production of IL-1, IL-6 and TNF-alpha was determined in germ-free, and "conventionalized" mice, as well as in monoxenic mice implanted with either the Gram-negative bacterium E. coli, or the Gram-positive organism Bifidobacterium bifidum. Macrophages from the "conventionalized" mice produced significantly more IL-1 and IL-6 in vitro than those of the germ-free mice. IL-1 and IL-6 production from germ-free mice implanted with E. coli was comparable to that from "conventionalized" mice. However, implantation with Bifidobacterium bifidum did not increase production of these two cytokines above levels observed for macrophages from the germ-free mice. A little TNF-alpha was produced by only the macrophages from the "conventionalized" and monoxenic mice implanted with E. coli. Soon after implantation, the bacterial flora stimulated cytokine production by mouse peritoneal macrophages and our results suggest that Gram negative bacteria are the most efficient stimulus for this production.
Assuntos
Citocinas/biossíntese , Intestinos/microbiologia , Macrófagos/imunologia , Animais , Bifidobacterium/imunologia , Escherichia coli/imunologia , Feminino , Vida Livre de Germes , Interleucina-1/biossíntese , Interleucina-6/biossíntese , Intestinos/imunologia , Camundongos , Camundongos Endogâmicos C3H , Cavidade Peritoneal/citologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Besides its role as a barrier against potential pathogens, intestinal flora is presumed to protect the host by priming the immunological defense mechanisms. In this respect, the influence of intestinal flora on macrophage precursors was examined, and its modulating effect was compared on LPS-induced cytokine production by macrophages derived from bone marrow and spleen precursors (BMDM and SDM respectively). The regulation of IL-1, IL-6, TNF-alpha and IL-12 production in macrophages from germ-free and from three groups of flora-associated mice, conventional, conventionalized and E. coli-mono-associated mice, was investigated. The whole flora inhibited IL-1, TNF-alpha and IL-12 secretion by BMDM, whereas it had a stimulatory effect on IL-12 secretion by SDM. Implantation of E. coli alone enhanced cytokine secretion by BMDM but had a more limited effect than whole flora on SDM, enhancing only TNF-alpha and IL-12 secretion. Study of expression of mRNA showed a correlation with protein secretion for IL-6 but not for TNF-alpha and IL-1. IL-12 enhancement in BMDM seemed to be dependent on regulation of p35 mRNA expression while it was correlated to increased p40 mRNA expression in SDM. The results demonstrated that intestinal flora modulated bone marrow and spleen macrophage cytokine production in a differential manner and suggested a role for bacteria other than E. coli among the whole flora. The contrasting effects exerted by the intestinal flora on bone marrow and spleen precursors are an interesting observation in view of the different functions of these organs in immunity. The finding that intestinal flora enhanced IL-12 production in spleen is also potentially important since this cytokine is implicated in the determination of the relative levels of Th1 and Th2 responses and plays a pivotal role in host defense against intracellular microorganisms.
Assuntos
Células da Medula Óssea/metabolismo , Citocinas/biossíntese , Intestinos/microbiologia , Macrófagos/metabolismo , Baço/metabolismo , Animais , Bactérias/isolamento & purificação , Fenômenos Fisiológicos Bacterianos , Sequência de Bases , Contagem de Colônia Microbiana , Citocinas/genética , Primers do DNA , Feminino , Interleucinas/biossíntese , Interleucinas/genética , Camundongos , Camundongos Endogâmicos C3H , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Baço/citologia , Fatores de Crescimento Transformadores/biossíntese , Fatores de Crescimento Transformadores/genéticaRESUMO
Ro52 antigen has recently been identified as TRIM21 protein, but the clinical significance of anti-Ro52/TRIM21 antibodies remains controversial. The aim of this multicentric study was to investigate the significance of anti-Ro52 antibodies without anti-SSA/Ro60 antibodies in various connective diseases. Sera were selected by each laboratory using its own method (ELISA, immunodot or Luminex technology), and then performed with ANA Screen BioPlex™ reagent (BIO-RAD). Among the 247 screened sera, 155/247 (63%) were confirmed as anti-Ro52 positive and anti-SSA/Ro60 negative. These sera were analyzed for the detection of other antibodies in relation with clinical settings. Isolated anti-Ro52 antibodies were detected in 89/155 (57%) sera. For the remaining sera (66/155), the main antibodies associations were Sm/SmRNP or Chromatin (n=38; 57%), Jo1 (n=17; 26%) and CenpB (n=9; 14%). Clinical data from the 155 patients showed high prevalence in autoimmune diseases (73%) including myositis or dermatomyositis (n=30), lupus (n=23); Sjögren and/or sicca syndrome (n=27); CREST or Systemic sclerosis (n=11) and autoimmune hepatitis (n=11). We found that pulmonary manifestations were often associated with the presence of anti-Ro52 antibodies (n=34, 22%), in addition with anti-tRNA synthetases, anti-SRP or anti-Ku antibodies (18/34) or isolated in half of cases (16/34). Separate detection of anti-Ro52 antibodies might be useful in related antisynthetase syndrome diagnosis. The presence of anti-Ro52 antibodies should probably precede development of autoimmune disease and must induce sequential follow-up of positive patients, particularly in interstitial lung disease progression.
Assuntos
Anticorpos/sangue , Doenças Autoimunes/sangue , Pneumopatias/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos/imunologia , Autoanticorpos/sangue , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Feminino , Humanos , Pneumopatias/imunologia , Masculino , Pessoa de Meia-Idade , Ribonucleoproteínas/imunologia , Adulto JovemRESUMO
A program for assembling sequences by using a global approach has been developed. By successive steps, a more and more precise classification of DNA fragments permits the positioning of the sequences on the contig; after having detected the pairs of overlapping sequences, groups are formed such that all sequences in a group overlap. Sequences common to several groups enable the groups to be ordered in a series. Ambiguities in the order of groups can arise at this stage, due to the presence of repeated fragments; different solutions are then proposed. Putting the groups into order leads to a preclassification of sequences. The fragments are then aligned by group, by searching for words common to all sequences in the group, and using an algorithm of dynamic programming. A detailed example on a set of nine sequences accompanies the description of the method.
Assuntos
Análise de Sequência de DNA/métodos , Software , Algoritmos , Sequência de Bases , DNA/genética , DNA Complementar/genética , Técnicas Genéticas , Dados de Sequência Molecular , Alinhamento de Sequência/métodos , Alinhamento de Sequência/estatística & dados numéricos , Análise de Sequência de DNA/estatística & dados numéricosRESUMO
To assess the involvement of bacterial microflora in the development of host defenses, we compared in vitro LPS-induced cytokine production by macrophages in germ-free and E. coli monoxenic mice. E. coli implantation significantly increased IL-1 and IL-6 and, to a lesser extent, TNF activities of peritoneal and bone marrow-derived macrophages. These results suggest that exposure to microflora primes macrophages for an enhanced cytokine production, which may contribute to the activation of the antiinfectious defense. The priming was not restricted to peritoneal macrophages but was associated with a more general effect of the flora since the enhanced response of bone marrow-derived macrophages indicates an effect on macrophage precursors. Furthermore, a higher ability of peritoneal macrophages to produce IL-1 in axenic and monoxenic mice was observed as compared to bone marrow-derived macrophages. In contrast, bone marrow-derived macrophages demonstrated a higher ability to produce IL-6 and TNF but only 3 weeks after bacterial administration.
Assuntos
Medula Óssea/metabolismo , Citocinas/biossíntese , Escherichia coli/fisiologia , Vida Livre de Germes , Macrófagos Peritoneais/metabolismo , Macrófagos/metabolismo , Animais , Células da Medula Óssea , Feminino , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Linfonodos/microbiologia , Mesentério , Camundongos , Camundongos Endogâmicos C3H , Fator de Necrose Tumoral alfa/metabolismoRESUMO
[Cu(salen)Gd(pta)(3)] (1), [Cu(acacen)Gd(pta)(3)] (2), and [Cu(acacen)Gd(hfa)(3)] (3) are three heterobimetallic [Cu(II)Gd(III)] complexes of general formula [Cu(SB)Gd(beta-dik)(3)], in which a N,N',O,O' Schiff base (SB) ligand [acacen = N,N'-ethylenebis(acetylacetoniminate(-)), salen = N,N'-ethylenebis(salicylideneiminate(-))] tetracoordinates Cu(II) and chelates Gd(III) as a tris(beta-diketonate) complex [hfa = 1,1,1,5,5,5-hexafluoropentane-2,4-dionate(-); pta = 1,1,1-trifluoro-5,5-dimethylhexane-2,4-dionate(-)]. They crystallize as a triclinic structure (space group P). The cell parameters are a = 9.8616(10) A, b = 12.1976(13) A, c = 18.4187(22) A, alpha = 90.671(14) degrees, beta = 100.588(13) degrees, gamma = 103.684(12) degrees, V = 2113 A(3), and Z = 2 for 1; a = 9.7560(11) A, b = 12.2924(13) A, c = 18.9368(22) A, alpha = 88.449(14) degrees, beta = 87.269(14) degrees, gamma = 67.629(12) degrees, V = 2098 A(3), and Z = 2 for 2; and a = 12.5726(15) A, b = 15.5985(18) A, c = 18.3724(21) A, alpha = 85.963(13) degrees, beta = 85.411(14) degrees, gamma = 80.766(14) degrees, V = 3539 A(3), and Z = 4 for 3. The Cu(O,O')Gd bridging cores show folding angles about O,O' in the range 139 degrees -147 degrees and intramolecular Cu small middle dot small middle dot small middle dotGd distances of about 3.3 A. In the solid state, the molecules form centrosymmetric pseudodimers [Cu(SB)Gd(beta-dik)(3)](2), through the overlap of the Cu(SB) entities. Resulting intradimer Cu...Cu distances are 5.941(1) A for 1, 4.831(1) A for 2, and 4.511(1) and 3.868(1) A for 3 which comprises two symmetrically independent dimers. The temperature dependence of complexes 1-3 was investigated in the range 1.8-300 K and revealed weak ferromagnetic interactions. Results are discussed in light of the structural features and of available magnetostructural data for other heterobimetallic [Cu(II)Gd(III)] complexes, including [Cu(salen)Gd(hfa)(3)] (4) (Ramade, I.; Kahn, O.; Jeannin, Y.; Robert, F. Inorg. Chem. 1997, 36, 930-936).
RESUMO
The purpose of this study was to evaluate the immunomodulatory activity of a peptide derived from bovine beta-casein (beta-CN), the beta-CN (193-209) peptide, on mouse macrophages that were obtained either from germfree (GF) or from human flora-associated (HF) mice. Macrophages were derived from bone marrow (BMDM) in the presence of recombinant macrophage colony-stimulating factor and exposed to the peptide or lipopolysaccharide (LPS). Membrane marker expression [F4/80, Mac-1, major histocompatibility complex (MHC) class II antigens] and phagocytic activity were assessed by flow cytometry. Production of tumor necrosis factor-alpha and interleukin (IL)-6 was measured by bioassays and production of IL-1alpha, IL-1beta and IL-12 by ELISA. The expression of cytokine mRNA was determined using semi-quantitative reverse transcription-polymerase chain reaction. The beta-CN (193-209) peptide up-regulated MHC class II antigen expression and phagocytic activity of BMDM from GF and HF mice. Its enhancing effect on phagocytosis was greater than that after LPS stimulation (P < 0.01). The peptide induced notable levels of cytokine mRNA in BMDM from GF and HF mice, but it was a significantly weaker inducer of cytokine secretion than LPS. Nevertheless, although flora implantation had no stimulatory influence on basal MHC class II and basal cytokine levels, cells from HF mice were more susceptible than those from GF mice to the peptide effects on these variables. These results indicate that the beta-CN (193-209) peptide could enhance antimicrobial activity of macrophages without proinflammatory effects.
Assuntos
Caseínas/farmacologia , Vida Livre de Germes , Macrófagos/efeitos dos fármacos , Complexo Principal de Histocompatibilidade/efeitos dos fármacos , Fagocitose , Animais , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/imunologia , Caseínas/imunologia , Bovinos , Feminino , Citometria de Fluxo , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C3HRESUMO
To investigate the adjuvant effect of intestinal flora on macrophage-colony-stimulating factor-responsive macrophage progenitors from spleen and bone marrow, we compared progenitor numbers and phenotypic characteristics of in vitro matured macrophages in germ-free and flora-associated mice (conventional, Escherichia coli-monoassociated and conventionalized mice). The data obtained show that the flora affected differentially bone marrow and spleen progenitors. It increased the numbers of progenitors in the spleen but not in the bone marrow. It did not modify the expression of F4/80, Mac-1 and major histocompatibility complex (MHC) class II on bone-marrow-derived macrophages (BMDM), while it clearly up-regulated MHC class II expression on spleen-derived macrophages (SDM). This effect was more pronounced in flora-associated ex germ-free mice than in conventional mice and it was greatly enhanced in the absence of M-CSF. In vitro stimulation by lipopolysaccharide had no effect on marker expression of BMDM, while it decreased F4/80 and enhanced MHC class II molecules on SDM from germ-free and flora-associated mice. However, the expression of MHC class II remained lower in germ-free mice. Enhancement of MHC class II molecule expression on SDM may contribute to the protective role of flora, because successful immune responses are dependent on the expression of these molecules.