Antigen binding properties of purified immunoglobulin A and reconstituted secretory immunoglobulin A antibodies.

The hybridoma cell line ZAC3 expresses Vibrio cholerae lipopolysaccharide (LPS)-specific mouse IgA molecules as a heterogeneous population of monomeric (IgAm), dimeric (IgAd), and polymeric (IgAp) forms. We describe a gentle method combining ultrafiltration, ion-exchange chromatography, and size exclusion chromatography for the simultaneous and qualitative separation of the three molecular forms. Milligram quantities of purified IgA molecules were recovered allowing for direct comparison of the biological properties of the three forms. LPS binding specificity was tested after purification; IgAd and IgAp were found to bind strongly to LPS whereas IgAm did not. Secretory IgA (sIgA) could be reconstituted in vitro by combining recombinant secretory component (rSC) and purified IgAd or IgAp, but not IgAm. Surface plasmon resonance-based binding experiments using LPS monolayers indicated that purified reconstituted sIgA and IgA molecules recognize LPS with identical affinity (KA 1.0 x 10(8)M-1). Thus, this very sensitive assay provides the first evidence that the function of SC in sIgA complex is not to modify the affinity for the antigen. KA falls to 6.6 x 10(5) M-1 when measured by calorimetry using detergent-solubilized LPS and IgA, suggesting that the LPS environment is critical for recognition by the antibody.

The metal ion binding protein Cnnm4 is mutated in rod-cone dystrophy/amelogenesis imperfecta syndrome

Purpose:To identify the gene causing rod-cone dystrophy/amelogenesis imperfecta Methods:Homozygosity mapping was performed using the Affymetrix 50K XbaI array in one family and candidate genes in the linked interval were sequenced with ABI Dye Terminator, vers. 1 in the index patient of 3 families. The identified mutations were screened in normal control individuals. Expression analyses were performed on RNA extracted from the brain, various parts of the eye and teeth; immunostaining was done on mouse eyes and jaw and knock-down experiments were carried out in zebrafish embroys. Results:Sequencing the coding regions of ancient conserved domain protein 4 (CNNM4), a metal ions transporter, revealed a 1-base pair duplication (p.L438fs) in family A, a p.R236Q mutation in family B and a p.L324P in family C. All these mutations were homozygous and involved very conserved amino acids in paralogs and orthologs. Immunostaining and RT-PCR confirmed that CNNM4 was strongly expressed in various parts of the eye and in the teeth. Morpholino experiments in zebrafish showed a loss of ganglion cells at 5 days post fertilization. Conclusions:The rod-cone dystrophy/amelogenesis imperfecta syndrome is caused by mutation in CNNM4 and is due to aberrant metal ion homeostasis.

Esterco líquido de bovinos leiteiros combinado com adubação mineral sobre atributos químicos de um Latossolo Bruno

Este trabalho foi conduzido com o objetivo de avaliar a influência de doses de adubação com esterco líquido de gado leiteiro, combinada com adubação mineral, sobre atributos químicos de um Latossolo Bruno, em sistema plantio direto e rotação de culturas de inverno e verão (sorgo/aveia-preta/milho/azevém/milho/azevém), para produção de silagem, nas camadas de 0-5, 5-10, 10-30, 30-50 e 50-80 cm de profundidade. Os tratamentos foram distribuídos em três blocos casualizados, divididos em 12 parcelas por bloco, em arranjo fatorial 3 x 4, sendo três doses de adubação mineral (0, 50 e 100 % da dose recomendada para as culturas) e quatro doses de adubação orgânica (0, 30, 60 e 90 m³ ha-1 ano-1). O esterco líquido aumentou o pH de forma linear na camada de 0-5 cm e quadrática na de 30-50 cm. A adubação mineral reduziu linearmente os valores de pH nas profundidades de 0-5 e 5-10 cm, havendo efeito quadrático na profundidade de 50-80 cm. A acidez potencial diminuiu na profundidade de 5-10 cm, com comportamento quadrático na profundidade de 10 a 30 cm. Houve incremento do Ca2+ trocável na profundidade de 0-5 cm ao se adubar com esterco, mas não se verificou efeito da adubação mineral sobre este atributo. Observou-se aumento nos níveis Mg2+ trocável nos tratamentos com esterco, até a profundidade de 30 cm; já para o fertilizante mineral os aumentos foram observados em profundidade superior a 30 cm. Houve incremento linear da saturação por bases com o aumento das doses de esterco, até 10 cm de profundidade; a adubação mineral diminuiu a saturação por bases na profundidade de 5-10 cm, havendo efeito quadrático na profundidade de 50-80 cm.

Ubiquitination of the Epstein-Barr virus-encoded latent membrane protein 1 depends on the integrity of the TRAF binding site.

The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus functions as a constitutively activated receptor of the tumor necrosis factor receptor family. LMP1 is a short-lived protein that is ubiquitinated and degraded by the proteasome. We have previously shown that LMP1 recruits the adapter protein tumor necrosis factor receptor-associated factor 3 (TRAF3) to lipid rafts. To test if TRAFs are involved in LMP1's ubiquitination, we have mutated the LMP1 CTAR1 site that has been identified as a TRAF binding site. We show that the CTAR1 mutant (CTAR1(-)) is expressed after transfection at a similar level to wild-type LMP1, and behaves as wild-type LMP1 with respect to membrane localization. However, CTAR1(-) does not bind TRAF3. We demonstrate that ubiquitination of CTAR1(-) is significantly reduced when compared to wild-type LMP1. In addition, the expression of wild-type LMP1 induces the ubiquitination, an effect that is significantly reduced when the CTAR1(-) is expressed. Taken together, our results suggest that TRAF proteins are involved in the ubiquitination of LMP1, and that their binding to LMP1 may facilitate their own ubiquitination.

Penicillin-binding protein gene alterations in Streptococcus uberis isolates presenting decreased susceptibility to penicillin.

Streptococcus uberis is an environmental pathogen commonly causing bovine mastitis, an infection that is generally treated with penicillin G. No field case of true penicillin-resistant S. uberis (MIC > 16 mg/liter) has been described yet, but isolates presenting decreased susceptibility (MIC of 0.25 to 0.5 mg/liter) to this drug are regularly reported to our laboratory. In this study, we demonstrated that S. uberis can readily develop penicillin resistance in laboratory-evolved mutants. The molecular mechanism of resistance (acquisition of mutations in penicillin-binding protein 1A [PBP1A], PBP2B, and PBP2X) was generally similar to that of all other penicillin-resistant streptococci described so far. In addition, it was also specific to S. uberis in that independent resistant mutants carried a unique set of seven consensus mutations, of which only one (Q(554)E in PBP2X) was commonly found in other streptococci. In parallel, independent isolates from bovine mastitis with different geographical origins (France, Holland, and Switzerland) and presenting a decreased susceptibility to penicillin were characterized. No mosaic PBPs were detected, but they all presented mutations identical to the one found in the laboratory-evolved mutants. This indicates that penicillin resistance development in S. uberis might follow a stringent pathway that would explain, in addition to the ecological niche of this pathogen, why naturally occurring resistances are still rare. In addition, this study shows that there is a reservoir of mutated PBPs in animals, which might be exchanged with other streptococci, such as Streptococcus agalactiae, that could potentially be transmitted to humans.

Role of bacterial biomass in the sorption of Ni by biomass-birnessite assemblages

Birnessites precipitated by bacteria are typically poorly crystalline Mn(IV) oxides enmeshed within biofilms to form complex biomass-birnessite assemblages. The strong sorption affinity of bacteriogenic birnessites for environmentally important trace metals is relatively well understood mechanistically, but the role of bacterial cells and extracellular polymeric substances appears to vary among trace metals. To assess the role of biomass definitively, comparison between metal sorption by biomass at high metal loadings in the presence and absence of birnessite is required. We investigated the biomass effect on Ni sorption through laboratory experiments utilizing the birnessite produced by the model bacterium, Pseudomonas putida. Surface excess measurements at pH 6?8 showed that birnessite significantly enhanced Ni sorption at high loadings (up to nearly 4-fold) relative to biomass alone. This apparent large difference in affinity for Ni between the organic and mineral components was confirmed by extended X-ray absorption fine structure spectroscopy, which revealed preferential Ni binding to birnessite cation vacancy sites. At pH >= 7, Ni sorption involved both adsorption and precipitation reactions. Our results thus support the view that the biofilm does not block reactive mineral surface sites; instead, the organic material contributes to metal sorption once high-affinity sites on the mineral are saturated.

In vitro-binding of the natural siderophore enantiomers pyochelin and enantiopyochelin to their AraC-type regulators PchR in Pseudomonas.

The enantiomeric siderophores pyochelin and enantiopyochelin of Pseudomonas aeruginosa and Pseudomonas protegens promote growth under iron limitation and activate transcription of their biosynthesis and uptake genes via the AraC-type regulator PchR. Here we investigated siderophore binding to PchR in vitro using fluorescence spectroscopy. A fusion of the N-terminal domain of P. aeruginosa PchR with maltose binding protein (MBP-PchR'PAO) bound iron-loaded (ferri-) pyochelin with an affinity (Kd) of 41 ± 5 μM. By contrast, no binding occurred with ferri-enantiopyochelin. Stereospecificity of a similar fusion protein of the P. protegens PchR (MBP-PchR'CHA0) was less pronounced. The Kd's of MBP-PchR'CHA0 for ferri-enantiopyochelin and ferri-pyochelin were 24 ± 5 and 40 ± 7 μM, respectively. None of the proteins interacted with the iron-free siderophore enantiomers, suggesting that transcriptional activation by PchR occurs only when the respective siderophore actively procures iron to the cell.

The promoter of the gene encoding 3',5'-cyclic adenosine monophosphate (cAMP) response element binding protein contains cAMP response elements: evidence for positive autoregulation of gene transcription.

The transcriptional transactivational activities of the phosphoprotein cAMP-response element-binding protein (CREB) are activated by the cAMP-dependent protein kinase A signaling pathway. Dimers of CREB bind to the palindromic DNA element 5'-TGACGTCA-3' (or similar motifs) called cAMP-responsive enhancers (CREs) found in the control regions of many genes, and activate transcription in response to phosphorylation of CREB by protein kinase A. Earlier we reported on the cyclical expression of the CREB gene in the Sertoli cells of the rat testis that occurred concomitant with the FSH-induced rise in cellular cAMP levels and suggested that transcription of the CREB gene may be autoregulated by cAMP-dependent transcriptional proteins. We now report the structure of the 5'-flanking sequence of the human CREB gene containing promoter activity. The promoter has a high content of guanosines and cytosines and lacks canonical TATA and CCAAT boxes typically found in the promoters of genes in eukaryotes. Notably, the promoter contains three CREs and transcriptional activities of a promoter-luciferase reporter plasmid transfected to placental JEG-3 cells are increased 3- to 5-fold over basal activities in response to either cAMP or 12-O-tetradecanoyl phorbol-14-acetate, and give 6- to 7-fold responses when both agents are added. The CREs bind recombinant CREB and endogenous CREB or CREB-like proteins contained in placental JEG-3 cells and also confer cAMP-inducible transcriptional activation to a heterologous minimal promoter. Our studies suggest that the expression of the CREB gene is positively autoregulated in trans.

Perdas de nutrientes via subsuperfície em colunas de solo sob fertilização mineral e orgânica

O uso de dejetos de animais na produção agrícola pode ocasionar a contaminação ou poluição das águas por nutrientes, principalmente com aplicações consecutivas na mesma área. Nesse sentido, o objetivo deste trabalho foi avaliar o efeito da aplicação de dejeto líquido de bovinos e fertilizantes minerais na lixiviação de NH4+, NO3-, P e K. Para essa avaliação, foram coletadas colunas indeformadas de solo, num experimento de campo que estava sendo conduzido por sete anos consecutivos e que continha 12 tratamentos, resultantes da combinação de quatro doses de dejeto líquido de bovinos com três doses de fertilizante mineral. O experimento de campo era conduzido num Latossolo Bruno distrófico em sistema de plantio direto com sorgo, milho, aveia-preta e azevém em rotação. Em laboratório, essas colunas, de acordo com os respectivos tratamentos de campo, foram submetidas à aplicação do dejeto líquido de bovinos (0, 30, 60 e 90 m³ ha-¹, aplicados em dose única) e dos fertilizantes minerais (0, 50 e 100 % da dose recomendada para o milho - 120, 60 e 60 kg ha-1 de N, P2O5 e K2O). Imediatamente após a aplicação de dejeto e fertilizante, a água percolada por coluna foi coletada e analisada em cinco turnos contínuos, totalizando 516 mm - aproximadamente três volumes de poros. Constatou-se aumento nas concentrações de N-NH4+, P e K com as doses de dejeto e aumento de N-NO3- e K com as doses de fertilizante mineral. No entanto, com exceção do N-NO3-, as concentrações de N-NH4+ e P estiveram abaixo do limite máximo permitido pela legislação brasileira, indicando a importância da infiltração de água no solo na redução do potencial poluidor dos dejetos.

Success of a general school-based physical activity intervention on bone mineral content depends on pubertal stage but not on gender

Aims: We performed a randomised controlled trial in children of both gender and different pubertal stages to determine whether a school-based physical activity (PA) program during a full schoolyear influences bone mineral content (BMC) and whether there are differences in response for boys and girls before and during puberty. Methods: Twenty-eight 1st and 5th grade classes were cluster randomised to an intervention (INT, 16 classes, n=297) and control (CON; 12 classes, n=205) group. The intervention consisted of a multi-component PA intervention including daily physical education during a full school year. Each lesson was predetermined, included about ten minutes of jumping or strength training exercises of various intensity and was the same for all children. Measurements included anthropometry (height and weight), tanner stages (by self-assessment), PA (by accelerometry) and BMC for total body, femoral neck, total hip and lumbar spine using dualenergy X-ray absorptiometry (DXA). Bone parameters were normalized for gender and tanner stage (pre- vs. puberty). Analyses were performed by a regression model adjusted for gender, baseline height, baseline weight, baseline PA, post-intervention tanner stage, baseline BMC, and cluster. Researchers were blinded to group allocation. Children in the control group did not know about the intervention arm. Results: 217 (57%) of 380 children who initially agreed to have DXA measurements had also post-intervention DXA and PA data. Mean age of prepubertal and pubertal children at baseline was 9.0±2.1 and 11.2±0.6 years, respectively. 47/114 girls and 68/103 boys were prepubertal at the end of the intervention. Compared to CON, children in INT showed statistically significant increases in BMC of total body (adjusted z-score differences: 0.123; 95%>CI 0.035 to 0.212), femoral neck (0.155; 95%>CI 0.007 to 0.302), and lumbar spine (0.127; 95%>CI 0.026 to 0.228). Importantly, there was no gender*group, but a tanner*group interaction consistently favoring prepubertal children. Conclusions: Our findings show that a general, but stringent school-based PA intervention can improve BMC in elementary school children. Pubertal stage, but not gender seems to determine bone sensitivity to physical activity loading.

Fibronectin-binding proteins and clumping factor A in Staphylococcus aureus experimental endocarditis: FnBPA is sufficient to activate human endothelial cells.

Surface molecules of Staphylococcus aureus are involved in the colonization of vascular endothelium which is a crucial primary event in the pathogenesis of infective endocarditis (IE). The ability of these molecules to also launch endothelial procoagulant and proinflammatory responses, which characterize IE, is not known. In the present study we investigated the individual capacities of three prominent S. aureus surface molecules; fibronectin-binding protein A (FnBPA) and B (FnBPB) and clumping factor A (ClfA), to promote bacterial adherence to cultured human endothelial cells (ECs) and to activate phenotypic and functional changes in these ECs. Non-invasive surrogate bacterium Lactococcus lactis, which, by gene transfer, expressed staphylococcal FnBPA, FnBPB or ClfA molecules were used. Infection of ECs increased 50- to 100-fold with FnBPA- or FnBPB-positive recombinant lactococci. This coincided with EC activation, interleukin-8 secretion and surface expression of ICAM-1 and VCAM-1 and concomitant monocyte adhesion. Infection with ClfA-positive lactococci did not activate EC. FnBPA-positive L. lactis also induced a prominent tissue factor-dependent endothelial coagulation response that was intensified by cell-bound monocytes. Thus S. aureus FnBPs, but not ClfA, confer invasiveness and pathogenicity to non-pathogenic L. lactis organisms indicating that bacterium-EC interactions mediated by these adhesins are sufficient to evoke inflammation as well as procoagulant activity at infected endovascular sites.

Comparison of H/Al stoichiometry of mineral and organic soils in Brazil

Exchangeable Al has been used as a criterion for the calculation of lime requirement in several Brazilian States. However, the laboratory method with extraction by a 1 mol L-1 KCl solution followed by indirect alkaline titration is not accurate for some Brazilian soils, mainly in the case of soils with high organic matter content. The objective of this study was therefore to evaluate the stoichiometry of H+/Al3+ in KCl soil extracts. The results suggested that organically complexed Al is the main contributor to exchangeable acidity in soils enriched with organic matter. Liming recommendations for organic soils based exclusively on exchangeable Al determined by the NaOH titration method should therefore be revised.

Estresse salino no crescimento inicial e nutrição mineral de gliricídia (Gliricidia sepium (Jacq.) Kunth ex Steud) em solução nutritiva(1)

A salinidade é um dos fatores que mais limitam o crescimento e desenvolvimento de plantas na região semiárida. A sobrevivência destas em ambientes salinos dependerá de processos adaptativos, que envolvem absorção, transporte e distribuição de íons nos vários órgãos da planta. Com o objetivo de avaliar o crescimento e a nutrição mineral de mudas de gliricídia cultivada em diferentes condições de salinidade, realizou-se um experimento em telado de náilon da Unidade Acadêmica de Engenharia Florestal da Universidade Federal de Campina Grande, Patos - PB. As sementes foram colocadas para germinar em vasos de Leonard, contendo solução nutritiva de Hoagland & Arnon (50 % da concentração original), com as concentrações de NaCl: 0, 100 200 e 400 mmol L-1. Os tratamentos foram distribuídos em delineamento inteiramente casualizado, com quatro repetições, com uma planta por vaso. Aos 60 dias após a emergência, as plantas foram colhidas e avaliadas quanto a altura, matéria seca e teores de N, P, K, Ca, Mg, S e Na na raiz, no caule e nas folhas. O aumento da salinidade promoveu reduções no crescimento e nos teores de macronutrientes, ocorrendo o inverso nos teores de Na, sobretudo na raiz. A gliricídia mostrou-se sensível à salinidade.

Teores de nitrogênio mineral do solo para predição do potencial produtivo de cevada

O teor de N mineral do solo não é usado na recomendação de adubação nitrogenada para cultivos de grãos no Brasil, devido à sua elevada instabilidade por efeito dos fatores ambientais. Seu uso poderia melhorar a eficiência das adubações e reduzir os custos de produção. Foi feita uma análise dos teores de NH4+ e de NO3- do solo, até 15 cm de profundidade, no período da pré-semeadura e do rendimento de grãos em 61 ensaios de cevada, em 20 municípios, em três Regiões Fisiográficas do Rio Grande do Sul, de 2002 a 2005. Os rendimentos foram divididos em classes (muito baixo, baixo, médio, alto e muito alto), que foram relacionadas aos teores de N mineral, usando o modelo de regressão de Gompertz. O ajuste indicou dois marcos de N mineral delimitadores do rendimento relativo: 8 e 18 mg kg-1 de N no solo. Abaixo de 8 mg kg-1 de N houve predomínio de muito baixos rendimentos, evidenciando o efeito da baixa disponibilidade sobre a produtividade. Acima de 18 mg kg-1 de N, este nutriente não foi mais fator decisivo na formação do potencial produtivo, sendo indicativo de potencial produtivo muito alto. Esses valores poderão futuramente ser incorporados como base de um modelo de recomendação de adubação nitrogenada.