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1.
J Am Soc Mass Spectrom ; 34(3): 394-400, 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36706338

RESUMO

Alzheimer's disease (AD) is a neurodegenerative disorder of increasing concern. It belongs to diseases termed tauopathies which are characterized by inclusions of abnormally hyperphosphorylated and truncated forms of the protein tau. Studies of tauopathies often focus on detection and characterization of these aberrant tau proteoforms, in particular the phosphorylation sites, which represent a significant analytical challenge for example when several phosphosites can be present on the same peptide. Such isomers can even be difficult to fully separate chromatographically. Since recently introduced cyclic ion mobility-mass spectrometry can offer different selectivity, we have investigated the closely positioned phosphorylation sites S214, T212, and T217 of a tryptic peptide from proline rich region of tau-TPSLPTPPTREPK. The conformational heterogeneity of the isomeric peptides in the gas phase hindered their separation due to their overlapping arrival time distributions. Increasing the resolution of the analysis alone is insufficient to distinguish the peptides in a mixture typical of patient samples. We therefore developed a method based on a combination of collision-induced dissociation, isomeric product ions (m/z 677) mobility separation and post-mobility dissociation to aid in analyzing the isomeric phosphopeptides of tau in diseased brain extract. For all three isomers (T212, S214, and T217), the ion mobility signal of the ion at m/z 677 was still observable at the concentration of 0.1 nmol/L. This work not only offers insights into the phosphorylation of tau protein in AD but also provides an analytical workflow for the characterization of challenging pathological protein modifications in neurodegenerative diseases.


Assuntos
Doença de Alzheimer , Humanos , Encéfalo/metabolismo , Espectrometria de Massas/métodos , Fosfopeptídeos/química , Proteínas tau/isolamento & purificação , Proteínas tau/metabolismo
2.
Anal Chem ; 94(36): 12435-12443, 2022 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-36049221

RESUMO

Native mass spectrometry is a powerful tool for the analysis of noncovalent complexes. When coupled with high-resolution ion mobility, this technique can be used to investigate the conformational changes induced in said complexes by different solution or gas-phase conditions. In this study, we describe how a new-generation high-resolution ion mobility instrument equipped with a cyclic ion mobility cell can be utilized for the analysis of large biomolecular systems, including temperature-induced protein aggregates of masses greater than 1.5 MDa, as well as a 63 kDa oligonucleotide complex. The effects of and the interplay between the voltages applied to the different components of the cyclic ion mobility spectrometry system on ion transmission and arrival time distribution were demonstrated using biomolecules covering the m/z range 2000-10,000. These data were used to establish a theoretical framework for achieving the best separation in the cyclic ion mobility system. Finally, the cyclic ion mobility mass spectrometer was coupled with a temperature-controlled electrospray ionization source to investigate high-mass protein aggregation. This analysis showed that it was possible to continuously monitor the change in abundance for several conformations of MDa aggregates with increasing temperature. This work significantly increases the range of biomolecules that can be analyzed by both cyclic ion mobility and temperature-controlled electrospray ionization mass spectrometry, providing new possibilities for high-resolution ion mobility analysis.


Assuntos
Espectrometria de Mobilidade Iônica , Agregados Proteicos , Conformação Molecular , Proteínas/química , Espectrometria de Massas por Ionização por Electrospray/métodos
4.
Anaesthesist ; 70(11): 977-990, 2021 11.
Artigo em Alemão | MEDLINE | ID: mdl-34709412

RESUMO

Endoscopy is most frequently performed in intensive care units (ICU) for gastrointestinal bleeding; however, there are other indications for performing an endoscopy on the ICU. This article shows the indications for this, the background and the peri-interventional and postinterventional management. The endoscopic placement of a postpyloric feeding tube is a well-established procedure. For anastomotic leakage in the esophagus and rectum, the endoscopic vacuum therapy is the treatment of choice. Gastrointestinal motility disorders are a frequent phenomenon in critically ill patients and are associated with increased mortality. With a cecal diameter > 9-12 mm, endoscopic decompression can be performed; however, this is associated with an increased risk of perforation and should only be carried out after the failure of conservative treatment.


Assuntos
Descompressão Cirúrgica , Vértebras Lombares , Cuidados Críticos , Estado Terminal , Endoscopia , Humanos , Unidades de Terapia Intensiva
5.
Front Bioinform ; 1: 774300, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-36303783

RESUMO

In interdisciplinary fields such as systems biology, good communication between experimentalists and theorists is crucial for the success of a project. Theoretical modeling in physiology usually describes complex systems with many interdependencies. On one hand, these models have to be grounded on experimental data. On the other hand, experimenters must be able to understand the interdependent complexities of the theoretical model in order to interpret the model's results in the physiological context. We promote interactive, visual simulations as an engaging way to present theoretical models in physiology and to make complex processes tangible. Based on a requirements analysis, we developed a new model for gas exchange in the human alveolus in combination with an interactive simulation software named Alvin. Alvin exceeds the current standard with its spatio-temporal resolution and a combination of visual and quantitative feedback. In Alvin, the course of the simulation can be traced in a three-dimensional rendering of an alveolus and dynamic plots. The user can interact by configuring essential model parameters. Alvin allows to run and compare multiple simulation instances simultaneously. We exemplified the use of Alvin for research by identifying unknown dependencies in published experimental data. Employing a detailed questionnaire, we showed the benefits of Alvin for education. We postulate that interactive, visual simulation of theoretical models, as we have implemented with Alvin on respiratory processes in the alveolus, can be of great help for communication between specialists and thereby advancing research.

6.
J Am Soc Mass Spectrom ; 31(3): 742-751, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-31971791

RESUMO

Ion mobility spectrometry (IMS) represents a considerable asset for analytics of complex samples as it allows for rapid mass spectrometric separation of compounds. IMS is even more useful for the separation of isobaric compounds when classical separation methods such as liquid chromatography or electrophoresis cannot be used, e.g., during matrix-assisted laser desorption/ionization (MALDI) analyses of biological surfaces. In the present study, we proved the usefulness of IMS for pharmacological applications of MALDI analyses on tissue sections. To illustrate our proof-of-concept, we used the anthelmintic drug mebendazole (MBZ) as a model. Using this exemplary drug, we demonstrated the possibility of using ion mobility to discriminate a drug in tissues from the biological background that masked its signal at low concentrations. In this proof-of-concept, the IMS mode together with the use of a profiling approach for sample preparation enabled quantification of the model drug MBZ from tissue sections in the concentration range 5 to 5,000 ng/g and with a limit of detection of 1 ng/g of tissue, within 2 h. This study highlights the importance of IMS as a separation method for on-surface quantification of drugs in tissue sections.


Assuntos
Anti-Helmínticos/farmacocinética , Mebendazol/farmacocinética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Anti-Helmínticos/análise , Espectrometria de Mobilidade Iônica/economia , Espectrometria de Mobilidade Iônica/métodos , Mebendazol/análise , Camundongos Nus , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/economia , Fatores de Tempo , Distribuição Tecidual
7.
Anal Bioanal Chem ; 411(24): 6275-6285, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30868190

RESUMO

Today, bottom-up protein identification in MALDI-MS is based on employing singly charged peptide ions, which are predominantly formed in the ionization process. However, peptide mass fingerprinting (PMF) with subsequent tandem MS confirmation using these peptide ions is often hampered due to the lower quality of fragment ion mass spectra caused by the higher collision energy necessary for fragmenting singly protonated peptides. Accordingly, peptide ions of higher charge states would be of high interest for analytical purposes, but they are usually not detected in MALDI-MS experiments as they overlap with singly charged matrix clusters and peptide ions. However, when utilizing ion mobility spectrometry (IMS), doubly charged peptide ions can be actively used by separating them from the singly protonated peptides, visualized, and selectively targeted for tandem MS experiments. The generated peptide fragment ion spectra can be used for a more confident protein identification using PMF with tandem MS confirmation, as most doubly protonated peptide ions yield fragment ion mass spectra of higher quality compared to tandem mass spectra of the corresponding singly protonated precursor ions. Mascot protein scores can be increased by approximately 50% when using tandem mass spectra of doubly charged peptide ions, with ion scores up to six times higher compared with ion scores of tandem mass spectra from singly charged precursors.


Assuntos
Espectrometria de Mobilidade Iônica/métodos , Peptídeos/análise , Proteínas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Peptídeos/química
8.
Rapid Commun Mass Spectrom ; 32(24): 2099-2105, 2018 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-30230090

RESUMO

RATIONALE: In-source decay (ISD) matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry with a 1,5-diaminonaphthalene (1,5-DAN) matrix is used for the structural characterisation of peptides. However, MALDI spectra are intrinsically complicated by the presence of matrix ions, which interfere with the peptide fragments. This may cause false-positive results or reduced sequence coverage. This paper reports investigations of ISD processes in an intermediate pressure MALDI ion source and a protocol for the removal of interfering ions using ion mobility separation (IMS). METHODS: An intermediate pressure MALDI source of a Q-IMS-Q-TOF instrument (Synapt G2) has been employed for the ISD of selected peptides using a 1,5-DAN matrix. RESULTS: Successful coupling of the MALDI source tuned for ISD experiments using IMS is demonstrated. The IMS made it possible to remove interfering matrix ions effectively from the spectra and thus to increase the confidence of spectral interpretation. Extensive fragment series corresponding to N-Cα bond cleavages were observed under optimised conditions; on the other hand, weaker series of ions caused by peptide bond cleavages were prevalent for default conditions and/or the α-hydroxycinnamic acid matrix. CONCLUSIONS: Ion mobility has been used for the elimination of matrix ions. The technique has been applied to top-down sequencing of non-tryptic peptides, such as the human palmitoylated analogue of prolactin-releasing peptide used in recent obesity studies, and human and insect antimicrobial peptides.


Assuntos
Espectrometria de Massas/métodos , Peptídeos/química , Animais , Peptídeos Catiônicos Antimicrobianos/química , Humanos , Insetos , Espectrometria de Massas/instrumentação , Hormônio Liberador de Prolactina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
9.
Urol Int ; 94(3): 296-306, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25170747

RESUMO

OBJECTIVE: This study sought to evaluate the clinical outcome after extended sentinel lymph node dissection (eSLND) and radical retropubic prostatectomy (RRP) in patients with clinically localized prostate cancer (PCa). SUBJECTS AND METHODS: From August 2002 until February 2011, a total of 819 patients with clinically localized PCa, confirmed by biopsy, were treated with RRP plus eSLND. Biochemical recurrence-free survival (RFS), cancer-specific survival (CSS), and overall survival (OS) were assessed with Kaplan-Meier curves. Various histopathological parameters were analyzed by univariate and multivariate analysis. RESULTS: The mean follow-up was 5.3 years. Lymph node (LN) metastases occurred in 140 patients. We removed an average of 10.9 LNs via eSLND from patients with pN1 PCa. Postoperatively, 121 pN1 patients temporarily received adjuvant androgen deprivation therapy. The mean survival periods for RFS, RFS after secondary treatment, CSS, and OS were 4.7, 7.0, 8.8, and 8.1 years, respectively. The cancer-specific death rate of the 140 pN1 patients was 13.6%. RFS, CSS, and OS were significantly correlated with pathological margin status, LN density, the total diameter of evident metastases, and membership in the subgroup 'micrometastases only'. CONCLUSION: Despite the presence of LN metastases, patients with a low nodal tumor burden demonstrate a remarkable clinical outcome after undergoing eSLND and RRP, thus suggesting a potential curative therapeutic approach.


Assuntos
Excisão de Linfonodo , Prostatectomia/métodos , Neoplasias da Próstata/cirurgia , Idoso , Biópsia , Intervalo Livre de Doença , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/patologia , Estudos Retrospectivos , Fatores de Risco , Resultado do Tratamento
10.
Plant J ; 73(4): 546-54, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23075038

RESUMO

Despite the ecological and evolutionary importance of nectar, mechanisms controlling its synthesis and secretion remain largely unknown. It is widely believed that nectar is 'secreted phloem sap', but current research reveals a biochemical complexity that is unlikely to stem directly from the phloem. We used the short daily peak in production of extrafloral nectar by Acacia cornigera to investigate metabolic and proteomic dynamics before, during and after 2 h of diurnal secretion. Neither hexoses nor dominating nectar proteins (nectarins) were detected in the phloem before or during nectar secretion, excluding the phloem as the direct source of major nectar components. Enzymes involved in the anabolism of sugars, amino acids, proteins, and nectarins, such as invertase, ß-1,3-glucanase and thaumatin-like protein, accumulated in the nectary directly before secretion and diminished quantitatively after the daily secretion process. The corresponding genes were expressed almost exclusively in nectaries. By contrast, protein catabolic enzymes were mainly present and active after the secretion peak, and may function in termination of the secretion process. Thus the metabolic machinery for extrafloral nectar production is synthesized and active during secretion and degraded thereafter. Knowing the key enzymes involved and the spatio-temporal patterns in their expression will allow elucidation of mechanisms by which plants control nectar quality and quantity.


Assuntos
Acacia/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Néctar de Plantas/metabolismo , Acacia/enzimologia , Glucana 1,3-beta-Glucosidase/genética , Glucana 1,3-beta-Glucosidase/metabolismo , Especificidade de Órgãos , Floema/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteólise , Proteoma/análise , Proteômica , Especificidade da Espécie , Fatores de Tempo , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismo
11.
Onkologie ; 35(10): 556-61, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23038225

RESUMO

BACKGROUND: The admission of patients with malignancies to an intensive care unit (ICU) still remains a matter of substantial controversy. The identification of factors that potentially influence the patient outcome can help ICU professionals make appropriate decisions. PATIENTS AND METHODS: 90 adult patients with hematological malignancy (leukemia 47.8%, high-grade lymphoma 50%) admitted to the ICU were analyzed retrospectively in this single-center study considering numerous variables with regard to their influence on ICU and day-100 mortality. RESULTS: The median simplified acute physiology score (SAPS) II at ICU admission was 55 (ICU survivors 47 vs. 60.5 for non-survivors). The overall ICU mortality rate was 45.6%. With multivariate regression analysis, patients admitted with sepsis and acute respiratory failure had a significantly increased ICU mortality (sepsis odds ratio (OR) 9.12, 95% confidence interval (CI) 1.1- 99.7, p = 0.04; respiratory failure OR 13.72, 95% CI 1.39-136.15, p = 0.025). Additional factors associated with an increased mortality were: high doses of catecholamines (ICU: OR 7.37, p = 0.005; day 100: hazard ratio (HR) 2.96, p < 0.0001), renal replacement therapy (day 100: HR 1.93, p = 0.026), and high SAPS II (ICU: HR 1.05, p = 0.038; day 100: HR 1.2, p = 0.027). CONCLUSION: The decision for or against ICU admission of patients with hematological diseases should become increasingly independent of the underlying malignant disease.


Assuntos
Neoplasias Hematológicas/mortalidade , Neoplasias Hematológicas/terapia , Unidades de Terapia Intensiva/estatística & dados numéricos , Admissão do Paciente/estatística & dados numéricos , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Alemanha/epidemiologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Análise de Sobrevida , Taxa de Sobrevida
12.
J Proteome Res ; 11(10): 4947-60, 2012 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-22905865

RESUMO

Herbivory leads to changes in the allocation of nitrogen among different pools and tissues; however, a detailed quantitative analysis of these changes has been lacking. Here, we demonstrate that a mass spectrometric data-independent acquisition approach known as LC-MS(E), combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and (15)N flux in a high throughput manner. The reliable identification/quantitation of rabbit phosphorylase b protein spiked into leaf protein extract was achieved. The linear dynamic range, reproducibility of technical and biological replicates, and differences between measured and expected (15)N-incorporation into the small (SSU) and large (LSU) subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO) and RuBisCO activase 2 (RCA2) of Nicotiana attenuata plants grown in hydroponic culture at different known concentrations of (15)N-labeled nitrate were used to further evaluate the procedure. The utility of the method for whole-plant studies in ecologically realistic contexts was demonstrated by using (15)N-pulse protocols on plants growing in soil under unknown (15)N-incorporation levels. Additionally, we quantified the amounts of lipoxygenase 2 (LOX2) protein, an enzyme important in antiherbivore defense responses, demonstrating that the approach allows for in-depth quantitative proteomics and (15)N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.


Assuntos
Nicotiana/metabolismo , Nitrogênio/metabolismo , Folhas de Planta/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Algoritmos , Sequência de Aminoácidos , Animais , Teorema de Bayes , Cromatografia Líquida/normas , Herbivoria , Funções Verossimilhança , Lipoxigenase/química , Lipoxigenase/isolamento & purificação , Lipoxigenase/metabolismo , Dados de Sequência Molecular , Isótopos de Nitrogênio/metabolismo , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos/normas , Fosforilase b/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Coelhos , Padrões de Referência , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/normas , Espectrometria de Massas em Tandem/normas , Nicotiana/química
13.
PLoS One ; 6(10): e26676, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22046331

RESUMO

Although the importance of insect saliva in insect-host plant interactions has been acknowledged, there is very limited information on the nature and complexity of the salivary proteome in lepidopteran herbivores. We inspected the labial salivary transcriptome and proteome of Helicoverpa armigera, an important polyphagous pest species. To identify the majority of the salivary proteins we have randomly sequenced 19,389 expressed sequence tags (ESTs) from a normalized cDNA library of salivary glands. In parallel, a non-cytosolic enriched protein fraction was obtained from labial salivary glands and subjected to two-dimensional gel electrophoresis (2-DE) and de novo peptide sequencing. This procedure allowed comparison of peptides and EST sequences and enabled us to identify 65 protein spots from the secreted labial saliva 2DE proteome. The mass spectrometry analysis revealed ecdysone, glucose oxidase, fructosidase, carboxyl/cholinesterase and an uncharacterized protein previously detected in H. armigera midgut proteome. Consistently, their corresponding transcripts are among the most abundant in our cDNA library. We did find redundancy of sequence identification of saliva-secreted proteins suggesting multiple isoforms. As expected, we found several enzymes responsible for digestion and plant offense. In addition, we identified non-digestive proteins such as an arginine kinase and abundant proteins of unknown function. This identification of secreted salivary gland proteins allows a more comprehensive understanding of insect feeding and poses new challenges for the elucidation of protein function.


Assuntos
Proteínas de Insetos/análise , Lepidópteros/química , Proteoma , Proteínas e Peptídeos Salivares/análise , Transcriptoma , Animais , Herbivoria , Proteínas de Insetos/genética , Mariposas , Proteômica/métodos , RNA Mensageiro/análise , Proteínas e Peptídeos Salivares/genética
14.
J Proteome Res ; 10(5): 2633-42, 2011 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-21452889

RESUMO

Apoptosis is of crucial importance in the life of multicellular organisms. In holometabolous insects, particularly in Lepidoptera, apoptosis is essential in biological processes such as metamorphosis and defense against pathogens. Apoptosis is tightly regulated and involves many proteins, among them caspases, which play a central role. In mammals, almost 300 targets of caspases have been described, and the expression of more than a hundred proteins has been shown to be altered in apoptotic cells. To date, the molecular pathways controlling apoptosis are poorly understood in Lepidoptera. Here, we used a comparative approach aiming to identify candidate proteins potentially implicated in these pathways. We examined changes occurring, in the proteome of a Helicoverpa armigera-derived cell line, upon induction by actinomycin D. We identified 13 proteins for which the relative abundance was significantly altered. Among these, the abundance of procaspase-1 decreased in apoptotic cells, reflecting its processing into the active form. We characterized its properties by heterologous expression and correlated the observed substrate specificity with changes in caspase activity in HaAM1 cells after induction. We also identified three chaperones as well as several putative pro- and anti-apoptotic proteins. Altogether, these data suggest that apoptotic pathways in Lepidoptera share similarities with the ones described in mammals.


Assuntos
Apoptose/fisiologia , Mariposas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Animais , Apoptose/genética , Caspase 1/metabolismo , Caspases/metabolismo , Linhagem Celular , Primers do DNA/genética , Dactinomicina , Eletroforese em Gel Bidimensional , Espectrometria de Massas , Mariposas/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Proc Natl Acad Sci U S A ; 108(12): 4897-901, 2011 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-21383196

RESUMO

Chrysomelid leaf beetles use chemical defenses to overcome predatory attack and microbial infestation. Larvae of Chrysomela lapponica that feed on willow sequester plant-derived salicin and other leaf alcohol glucosides, which are modified in their defensive glands to bioactive compounds. Salicin is converted into salicylaldehyde by a consecutive action of a ß-glucosidase and salicyl alcohol oxidase (SAO). The other leaf alcohol glucosides are not oxidized, but are deglucosylated and esterified with isobutyric- and 2-methylbutyric acid. Like some other closely related Chrysomela species, certain populations of C. lapponica shift host plants from willow to salicin-free birch. The only striking difference between willow feeders and birch feeders in terms of chemical defense is the lack of salicylaldehyde formation. To clarify the impact of host plant shifts on SAO activity, we identified and compared this enzyme by cloning, expression, and functional testing in a willow-feeding and birch-feeding population of C. lapponica. Although the birch feeders still demonstrated defensive gland-specific expression, their SAO mRNA levels were 1,000-fold lower, and the SAO enzyme was nonfunctional. Obviously, the loss of catalytic function of the SAO of birch-adapted larvae is fixed at the transcriptional, translational, and enzyme levels, thus avoiding costly expression of a highly abundant protein that is not required in the birch feeders.


Assuntos
Adaptação Fisiológica/fisiologia , Oxirredutases do Álcool/biossíntese , Betula , Besouros/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas de Insetos/biossíntese , Folhas de Planta , Salix , Oxirredutases do Álcool/genética , Animais , Sequência de Bases , Álcoois Benzílicos/metabolismo , Besouros/genética , Comportamento Alimentar/fisiologia , Glucosídeos/metabolismo , Proteínas de Insetos/genética , Dados de Sequência Molecular
16.
Proc Biol Sci ; 278(1722): 3225-32, 2011 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-21429930

RESUMO

Glandular chemical defence relying on the action of salicylaldehyde is characteristic for Chrysomela leaf beetle larvae. The salicylaldehyde precursor salicin, sequestered from salicaceous host plants, is deglucosylated and the aglycon further oxidized by a salicyl alcohol oxidase (SAO) to the respective aldehyde. SAOs, key enzymes in salicin-based glandular chemical defence, were previously identified and shown to be of a single evolutionary origin in Chrysomela species. We here identified and characterized SAO of Phratora vitellinae, the only species outside the genus Chrysomela that produce salicylaldehyde as a defensive compound. Although Chrysomela and Phratora are not closest relatives, their SAOs share glucose-methanol-choline oxidoreductase (GMC) affiliation, a specific GMCi subfamily ancestor, glandular tissue-specific expression and almost identical gene architectures. Together, this strongly supports a single origin of SAOs of both Chrysomela and Phratora. Closely related species of Chrysomela and P. vitellinae use iridoids as defensive compounds, which are like salicylaldehyde synthesized by the consecutive action of glucosidase and oxidase. However, we elucidated SAO-like sequences but no SAO proteins in the glandular secretion of iridoid producers. These findings support a different evolutionary history of SAO, related genes and other oxidases involved in chemical defence in the glandular system of salicylaldehyde and iridoid-producing leaf beetle larvae.


Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Álcoois Benzílicos/metabolismo , Besouros/enzimologia , Besouros/imunologia , Evolução Molecular , Glucosídeos/metabolismo , Aldeídos/química , Aldeídos/metabolismo , Animais , Sequência de Bases , Teorema de Bayes , Western Blotting , Linhagem Celular , Análise por Conglomerados , Besouros/genética , Besouros/metabolismo , Larva/enzimologia , Larva/imunologia , Larva/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Estrutura Molecular , Filogenia , Análise de Sequência de DNA , Especificidade da Espécie , Espectrometria de Massas em Tandem
17.
J Gastrointestin Liver Dis ; 19(4): 399-404, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21188331

RESUMO

BACKGROUND AND AIM: Acute or chronic liver failure is associated with numerous complications and patients may require intensive care treatment, which is complex, time-consuming and often highly resource-intensive. Thus, it is necessary to identify clinical parameters that allow quick risk stratification. METHODS: In 117 patients with acute or chronic liver failure requiring ICU admission, the clinical parameters, risk scores and results of microbiological examinations were documented and correlated with the outcome (survivor vs. non-survivor). RESULTS: Predictors of outcome were: Child-Pugh-Score (p < 0.01), MELD-Score (p < 0.01), SAPS-II-Score (p < 0.05), bilirubin (p < 0.01), Glasgow Coma Scale (GCS) (p < 0.02), urine output (p < 0.01), requirement of catecholamine administration (p <0.004), serum creatinine (p < 0.01). The strongest predictors of outcome were in a multivariate model GCS (p = 0.006) and MELD-score (p = 0.001). CONCLUSIONS: Risk stratification in our patient collective was feasible. Apart from parameters to assess kidney function and circulation, various scoring systems that had previously not been evaluated for this kind of patient collective seem to be the main predictors of outcome.


Assuntos
Cuidados Críticos , Doença Hepática Terminal/diagnóstico , Falência Hepática Aguda/diagnóstico , Adulto , Bilirrubina/sangue , Biomarcadores/sangue , Catecolaminas/uso terapêutico , Distribuição de Qui-Quadrado , Creatinina/sangue , Doença Hepática Terminal/mortalidade , Doença Hepática Terminal/terapia , Feminino , Alemanha , Escala de Coma de Glasgow , Mortalidade Hospitalar , Humanos , Falência Hepática Aguda/mortalidade , Falência Hepática Aguda/terapia , Modelos Logísticos , Masculino , Técnicas Microbiológicas , Pessoa de Meia-Idade , Prognóstico , Medição de Risco , Fatores de Risco , Índice de Gravidade de Doença , Análise de Sobrevida , Taxa de Sobrevida , Fatores de Tempo , Micção
18.
Electrophoresis ; 31(15): 2680-5, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20665926

RESUMO

An on-line microfluidic system for determination of dissociation constants of enzyme/substrate weak interactions by nanoESI-MS is introduced. The microchip was designed to permit the enzyme and the substrate to mix by molecular diffusion in a pressure-driven laminar flow. Introduction of reagent solutions into the chip was an optimized combination of a micropump and an autosampler to enable automation of the measurements. The system performance was tested for monitoring of non-covalent interactions of hen egg white lysozyme with N-acetyl glucosamine oligomers. Dissociation constant (K(D)) of the hen egg white lysozyme-N-acetyl glucosamine oligomer complex was determined by non-linear regression analysis, and the range of K(D) values (39+/-6 x 10(-6) and 19.6+/-8 x 10(-6) M for manual and automated infusions, respectively) confirms the previously reported values. Such miniaturization of a continuous-flow enzyme assay system to a microfluidic format can maximize the capabilities of mass spectrometric detection, reduce the sample size and analysis time required, as well as the associated costs for on-line enzymatic kinetic studies.


Assuntos
Acetilglucosamina/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Muramidase/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Galinhas , Cinética
19.
Plant Physiol ; 152(4): 2232-42, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20190094

RESUMO

Native flower visitors removed less nectar from trypsin proteinase inhibitor (TPI)-silenced Nicotiana attenuata plants (ir-pi) than from wild-type plants in four field seasons of releases, even when the nectar repellent, nicotine, was also silenced. Analysis of floral chemistry revealed no differences in the emission of the floral attractants benzylacetone and benzaldehyde or in the concentrations of nectar sugar and nicotine between wild-type and ir-pi flowers, suggesting that these two lines are equally able to attract insect visitors. TPI activity was found in all wild-type flower parts and was highest in anther heads, while TPI activity was not found in any parts of ir-pi flowers. The nectar of ir-pi flowers contained 3.6-fold more total proteins than the nectar of wild-type flowers. Proteomics analysis and hydrogen peroxide (H2O2) measurements revealed that ir-pi nectar contained more nectarins and nectar germin-like proteins and about 1.5-fold more H2O2 compared with wild-type nectar. Field experiments with wild-type flowers supplemented with a solution containing sugar and glucose oxidase demonstrated a causal association between the accumulation of H2O2 and the reduction in nectar removal. These results showed that silencing TPI expression increases the accumulation of nectar proteins and H2O2 levels, which in turn reduces nectar removal by native insect floral visitors. The effect of silencing TPIs on nectar protein accumulation suggests an endogenous regulatory function for TPIs in N. attenuata flowers. The repellency of H2O2 to floral visitors raises new questions about the qualities of nectar that make it attractive for pollinators.


Assuntos
Inativação Gênica , Glicoproteínas/metabolismo , Peróxido de Hidrogênio/metabolismo , Proteínas de Plantas/metabolismo , Inibidores de Proteases/farmacologia , Superóxido Dismutase/metabolismo
20.
Lab Chip ; 10(3): 320-5, 2010 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-20091003

RESUMO

Silicon nanowire arrays were patterned onto silicon chips by a combination of lithography and chemical vapor deposition using the vapor-liquid-solid growth method. Thus, highly reproducible sample deposition zones were obtained that were used for laser desorption ionization (LDI) mass spectrometric analysis of lipidic species with lithium salts as dopants. Using a conventional UV laser (337 nm), hydrocarbons and numerous lipids (triglycerides, diglycerides, wax esters) could be effectively lithiated yielding [M + Li](+) ions. Upon doping with lithium hydroxide the SiNW arrays yielded high signal-to-noise ratios with low limits of detection (e.g. 750 pg tripalmitin on target with S/N 5) and efficient ionization for a range of fatty acids (FA), mono-, di- and triglycerides and hydrocarbons (HC), in the form of [FA-H + 2Li](+), [mono- or diglyceride-H(2)O + Li](+), or [triglyceride + Li](+) and [HC + Li](+), respectively. It is expected that these chips will find a broad range of applications in the analysis of natural compounds and food control.


Assuntos
Lipídeos/análise , Lipídeos/química , Espectrometria de Massas/métodos , Microfluídica/métodos , Nanotubos/química , Nanotubos/ultraestrutura , Silício/química , Teste de Materiais , Propriedades de Superfície
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