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1.
J Struct Biol ; 199(1): 1-11, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28552722

RESUMO

Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind with high affinity and specificity to different antigens. They are small (molecular weight ca. 15kDa) and are usually easy to produce in microorganisms. Here we have evaluated the feasibility of a nanobody binding to HER2 for application in immuno-electron microscopy. To obtain highest labeling efficiency combined with optimal specificity, different labeling conditions were analysed, which included nanobody concentration, fixation and blocking conditions. The obtained optimal protocol was applied for post-embedment labeling of Tokuyasu cryosections and for pre-embedment labeling of HER2 for fluorescence microscopy and both transmission and scanning electron microscopy. We show that formaldehyde fixation after incubation with the anti-HER2 nanobody, improves labeling intensity. Among all tested blocking agents the best results were obtained with a mixture of cold water fish gelatine and acetylated bovine serum albumin, which prevented a-specific interactions causing background labeling while preserving specific interactions at the same time. In conclusion, we have developed a nanobody-based protocol for immuno-gold labeling of HER2 for Tokuyasu cryosections in TEM as well as for pre-embedment gold labeling of cells for both TEM and SEM.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Microscopia Imunoeletrônica/métodos , Receptor ErbB-2/análise , Anticorpos de Domínio Único/imunologia , Fixação de Tecidos/métodos , Animais , Ouro , Humanos , Microscopia Imunoeletrônica/normas , Receptor ErbB-2/imunologia , Projetos de Pesquisa , Coloração e Rotulagem/normas , Fixação de Tecidos/normas
2.
Sci Total Environ ; 553: 340-348, 2016 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-26930307

RESUMO

Woody patches in semiarid environments favor the establishment of other plants. Facilitation may be favored by an increase in soil fertility. Dissolved organic matter (DOM), is the most active fraction of soil organic matter and may contain compounds affecting plant establishment, as allelochemicals, hormone-like substances and metal carriers. However, information on DOM contents and composition in these environments is scarce. In this paper, we study the impact of woody patches on DOM in Stipa tenacissima L. steppes and discuss its implications for community dynamics. DOM under patch- and inter-patch areas, was analyzed for elemental composition, UV-Vis indices and organic acid content. Element concentration and composition in DOM, and organic acid concentration were similar in patch- and inter-patch areas. Yet, soils under patches were richer in DOC, aromatic species and organic acids (particularly fumaric acid) than soils in inter-patch areas. Dominant species affected organic matter concentration and quality in complex ways. Thus, patches dominated by Ephedra fragilis showed higher concentrations of TOC and aromatics than those dominated by other species. Rhamnus lycioides patches showed the highest accumulation of fumaric acid, which may contribute to its successful recruitment rate and expansion in the area. Our results show substantial differences in the amount and composition of DOM and specific compounds affecting soil functionality and plant dynamics. Further studies on the effects of such changes on seedling performance are needed to increase our understanding of plant-plant interactions in semiarid environments.

3.
J Biol Chem ; 275(35): 26885-91, 2000 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-10859310

RESUMO

Most lipases of Gram-negative bacteria require a lipase-specific foldase (Lif) in order to fold in the periplasm into their active, protease-resistant conformation prior to their secretion. The periplasmic domain of the Lif (amino acids 44-353) of Burkholderia glumae was purified as a His-tagged protein, and its function in the folding of lipase was studied in vitro. Refolding of the denatured lipase into its active conformation was dependent on the presence of the Lif. Circular dichroism revealed that the lipase refolded in the absence of Lif into a form with a native-like conformation, which was more stable against heat-induced denaturation than the native form, but was enzymatically inactive. This form of the protein could be activated by adding Lif after several hours, which demonstrates that the function of this chaperone is to help lipase to overcome an energetic barrier in the productive folding pathway rather than to prevent it from entering a non-productive pathway. The Lif was shown to interact with the native lipase in protease-protection experiments as well as by affinity chromatography, consistent with a role of the Lif late in the folding process. These results demonstrate that the Lif functions in a way analogous to the propeptides of many bacterial proteases and indicate that the amino acid sequence of the lipase does not contain all the information required for the protein to adopt its three-dimensional structure.


Assuntos
Burkholderia/enzimologia , Lipase/metabolismo , Chaperonas Moleculares/metabolismo , Dicroísmo Circular , Dissulfetos/química , Histidina/química , Cinética , Chaperonas Moleculares/química , Dobramento de Proteína
4.
Mol Gen Genet ; 261(4-5): 770-6, 1999 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10394914

RESUMO

Folding of lipases that are secreted by Pseudomonads and other gram-negative bacteria via the type II secretion pathway is facilitated by dedicated chaperones, called lipase-specific foldases (Lifs). Lifs are membrane-anchored proteins with a large periplasmic domain. The functional interaction between the Lif and its cognate lipase is specific, since the Pseudomonas aeruginosa Lif was found not to substitute for Lifs from Burkholderia glumae or Acinetobacter calcoaceticus. However, the P. aeruginosa Lif was able to activate the lipase from the closely related species P. alcaligenes. Hybrid proteins constructed from parts of the P. aeruginosa and B. glumae Lifs revealed that the C-terminal 138 amino acids of the B. glumae Lif determine the specificity of the interaction with the cognate lipase. Furthermore, the periplasmic domain of the B. glumae Lif was functional when cloned in frame with a cleavable signal sequence, which demonstrates that the membrane anchor is not essential for Lif function in vivo. However, the recombinant Lif was released into the medium, indicating that the function of the membrane anchor is to prevent secretion of the Lif together with the lipase.


Assuntos
Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Lipase/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Acinetobacter calcoaceticus/genética , Acinetobacter calcoaceticus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Burkholderia/genética , Burkholderia/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Lipase/genética , Plasmídeos , Pseudomonas/genética , Pseudomonas/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Especificidade por Substrato
5.
Biochem Biophys Res Commun ; 229(3): 869-75, 1996 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-8954986

RESUMO

The recent elucidation of the 3-D structure of the outer membrane protein PhoE of Escherichia coli provides an excellent tool for a detailed analysis of the structure-function relationship of this pore-forming protein. For this purpose, a fast and efficient method for the purification of mutant porins is needed. A histidine-tag was engineered between the signal sequence and the N terminus of mature PhoE. The recombinant PhoE protein was normally assembled into the outer membrane and could be purified by immobilized metal affinity chromatography. Part of the total amount of the trimers dissociated into folded monomers during purification. The histidine-tag did not change the electrophysical characteristics of the protein in lipid bilayers. Hence, the method is useful for the fast purification of mutant porins for functional and structural characterization.


Assuntos
Escherichia coli/metabolismo , Porinas , Proteínas de Escherichia coli , Histidina , Porinas/química , Porinas/isolamento & purificação , Porinas/metabolismo , Relação Estrutura-Atividade
6.
Mech Dev ; 52(1): 51-64, 1995 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-7577675

RESUMO

Prx1 and Prx2 (previously called MHox and S8, respectively) are the members of a small subfamily of vertebrate homeobox genes expressed during embryogenesis from gastrulation onwards. We directly compared the expression domains of the Prx genes in detail in mouse and in addition some aspects of these patterns in chicken. In addition to the superficially similar expression patterns of Prx1 and Prx2 in cranial mesenchyme, limb buds, axial mesoderm, and branchial arches and their derivatives, we detect major differences at many sites particularly in heart and brain. Our analysis indicated in several cases a correlation with regions developing into connective tissues. From at least day 8.5, Prx-1 expression was observed in the heart, initially in the endocardial cushions and later in the developing semilunar and atrioventricular valves. Prx2 develops early on a diffuse myocardial expression pattern and is later higher expressed in the ventricular septum and in particular in the ductus arteriosus. Prx2 is never expressed in the brain, whereas Prx1 is expressed, from at least day 9.5 onwards, in a unique distinct domain in the ventral part of the hypothalamus, as well as in a broader region of the telencephalon.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Coração/embriologia , Prosencéfalo/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , Clonagem Molecular , Tecido Conjuntivo/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Especificidade de Órgãos , Alinhamento de Sequência
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