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1.
Anal Chem ; 91(24): 15941-15950, 2019 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-31738517

RESUMO

The design and synthesis of a proline-based reporter isobaric Tandem Mass Tag structure (TMTpro) is presented. An analysis is made of the performance of the new TMTpro tags in comparison with the current commercially available dimethylpiperidine-reporter-based TMT10/11 reagents. The new reporter structure provides a set of 16 tags for use with resolution of 6.3 mDa mass differences in high resolution mass spectrometers and a set of 9 reagents with 1 Da spacing between reporter ions for single dalton analysis using 9 heavy nuclei per tag. We show similar performance in terms of peptide identification rates and quantification between the TMTpro 16-plex and TMT10/11-plex reagents. We also demonstrate the suitability of the TMTpro reagents for phosphopeptide analysis. The ability to pool 16 samples reduces the overall amount of sample required for each channel, and we anticipate that TMTpro reagents will be a useful enhancement for any protocol that benefits from sample pooling and should reduce missing data.

2.
Plant Physiol Biochem ; 143: 232-245, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31521962

RESUMO

Many physiological and molecular responses to salt stress have been investigated after a salt shock. However, salt shock rarely happens in agricultural practice. In the field, salts accumulate gradually due to poor agricultural management. Thus in salinity research, it is more reasonable to investigate plant reaction after stepwise acclimation to salt stress. Previous studies demonstrate that salt shock induces Phase 0, a short-term effect that shows transient water loss and rapid turgor decrease; salt stress after stepwise acclimation avoids Phase 0 effects and induces Phase 1. During Phase 1, plants show maintenance of turgor. In this study, salt shock and stepwise acclimation to salt stress were separated at physiological and transcriptional levels. Four major experiments were conducted: 1) leaf turgor changes were monitored in real time after salt application to separate Phase 0 and Phase 1 effects at the physiological level, 2) RNA-sequence analysis was conducted in Arabidopsis thaliana L. to identify potential marker genes that are involved in plant water relations to distinguish Phase 0 and Phase 1 at transcript level, 3) these selected marker gene candidates were identified in Arabidopsis at different Phase 0 and Phase 1 time points via qRT-PCR, 4) these candidates were further evaluated in Zea mays L. (a model plant for applied research in plant physiology and an important crop plant) via qRT-PCR. In future salinity research, marker genes that are both applicable in Arabidopsis and maize have the potential to differentiate salt shock and stepwise acclimation to salt stress.

3.
ACS Chem Neurosci ; 10(10): 4264-4279, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31464424

RESUMO

Exposure to low levels of environmental contaminants, including pesticides, induces neurodevelopmental toxicity. Environmental and food contaminants can reach the brain of the fetus, affecting brain development and leading to neurological dysfunction. The pesticide endosulfan is a persistent pollutant, and significant levels still remain detectable in the environment although its use is banned in some countries. In rats, endosulfan exposure during brain development alters motor activity, coordination, learning, and memory, even several months after uptake, and does so in a sex-dependent way. However, the molecular mechanisms driving these effects have not been studied in detail. In this work, we performed a multiomics study in cerebellum from rats exposed to endosulfan during embryonic development. Pregnant rats were orally exposed to a low dose (0.5 mg/kg) of endosulfan, daily, from gestational day 7 to postnatal day 21. The progeny was evaluated for cognitive and motor functions at adulthood. Expression of messenger RNA and microRNA genes, as well as protein and metabolite levels, were measured on cerebellar samples from males and females. An integrative analysis was conducted to identify altered processes under endosulfan effect. Effects between males and females were compared. Pathways significantly altered by endosulfan exposure included the phosphatidylinositol signaling system, calcium signaling, the cGMP-PKG pathway, the inflammatory and immune system, protein processing in the endoplasmic reticulum, and GABA and taurine metabolism. Sex-dependent effects of endosulfan in the omics results that matched sex differences in cognitive and motor tests were found. These results shed light on the molecular basis of impaired neurodevelopment and contribute to the identification of new biomarkers of neurotoxicity.

4.
Microscopy (Oxf) ; 68(5): 379-384, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31340024

RESUMO

Novel para-crystalline structures resembling prolamellar bodies in etioplasts were found in the invasion zones of indeterminate root nodules of Vicia faba, which possess persistent meristems and exhibit sequential developmental stages. The para-crystalline structures existed in most cells in the area of the invasion zone and a hexagonal arrangement of tubular membranes was recognized. Extensive membranes, apparently procured from the structures, were often in contact with the bacteria in young infected cells. We propose that the para-crystalline structures serve as a reservoir of membranes for the formation of the numerous symbiosomes that propagate and fill the infected cells, and suggest naming them pro-symbiosome membrane bodies.


Assuntos
Cloroplastos/ultraestrutura , Cristalização , Nódulos Radiculares de Plantas/ultraestrutura , Vicia faba/anatomia & histologia , Membrana Celular/ultraestrutura , Microscopia Eletrônica , Vicia faba/ultraestrutura
5.
Sci Rep ; 9(1): 4478, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30872628

RESUMO

The lack of biomarkers for early diagnosis, clinical stratification and to monitor treatment response has hampered the development of new therapies for amyotrophic lateral sclerosis (ALS), a clinically heterogeneous neurodegenerative disorder with a variable site of disease initiation and rate of progression. To identify new biomarkers and therapeutic targets, two separate proteomic workflows were applied to study the immunological response and the plasma/brain proteome in phenotypic variants of ALS. Conventional multiplex (TMT) proteomic analysis of peripheral blood mononuclear cells (PBMCs) was performed alongside a recently introduced method to profile neuronal-derived proteins in plasma using brain tissue-enhanced isobaric tagging (TMTcalibrator). The combined proteomic analysis allowed the detection of regulated proteins linked to ALS pathogenesis (RNA-binding protein FUS, superoxide dismutase Cu-Zn and neurofilaments light polypeptide) alongside newly identified candidate biomarkers (myosin-9, fructose-bisphosphate aldolase and plectin). In line with the proteomic results, orthogonal immunodetection showed changes in neurofilaments and ApoE in bulbar versus limb onset fast progressing ALS. Functional analysis of significantly regulated features showed enrichment of pathways involved in regulation of the immune response, Rho family GTPases, semaphorin and integrin signalling. Our cross-phenotype investigation of PBMCs and plasma/brain proteins provides a more sensitive biomarker exploratory platform than conventional case-control studies in a single matrix. The reported regulated proteins may represent novel biomarker candidates and potentially druggable targets.

6.
Biochem Biophys Rep ; 14: 168-177, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29872749

RESUMO

Protein aggregation in biofluids is a poorly understood phenomenon. Under normal physiological conditions, fluid-borne aggregates may contain plasma or cell proteins prone to aggregation. Recent observations suggest that neurofilaments (Nf), the building blocks of neurons and a biomarker of neurodegeneration, are included in high molecular weight complexes in circulation. The composition of these Nf-containing hetero-aggregates (NCH) may change in systemic or organ-specific pathologies, providing the basis to develop novel disease biomarkers. We have tested ultracentrifugation (UC) and a commercially available protein aggregate binder, Seprion PAD-Beads (SEP), for the enrichment of NCH from plasma of healthy individuals, and then characterised the Nf content of the aggregate fractions using gel electrophoresis and their proteome by mass spectrometry (MS). Western blot analysis of fractions obtained by UC showed that among Nf isoforms, neurofilament heavy chain (NfH) was found within SDS-stable high molecular weight aggregates. Shotgun proteomics of aggregates obtained with both extraction techniques identified mostly cell structural and to a lesser extent extra-cellular matrix proteins, while functional analysis revealed pathways involved in inflammatory response, phagosome and prion-like protein behaviour. UC aggregates were specifically enriched with proteins involved in endocrine, metabolic and cell-signalling regulation. We describe the proteome of neurofilament-containing aggregates isolated from healthy individuals biofluids using different extraction methods.

7.
PLoS One ; 13(3): e0193958, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29529096

RESUMO

Grass pollen is the main cause of hay fever and allergic asthma in warm temperate climates during summer. The aim of this study was to determine the content of group 5 major allergens in pollen grains of agriculturally important grass species/cultivars. For each cultivar flowering dates and pollen production of cut anthers were observed in the field and in a climate chamber, respectively. An ELISA was used to quantify the group 5 allergens (Phl p5) in pollen extracts which were gained from the grass species Kentucky bluegrass, perennial rye grass, timothy, cocksfoot, annual / Italian rye grass, hybrid rye grass and festulolium. The group 5 allergen content of species varied between 0.01 ng (Kentucky bluegrass) and 0.06 ng (timothy) per pollen grain. On cultivar level the pollen allergenic content differed up to 74-times within the selected grass species. Results from this study might be helpful for the reduction of allergen exposure coming from agriculture grass production e.g. by an adapted grass selection or by the cultivation of grasses with low allergenic content in plant breeding.


Assuntos
Alérgenos , Poaceae , Pólen/imunologia , Rinite Alérgica Sazonal/etiologia , Agricultura , Ensaio de Imunoadsorção Enzimática , Humanos , Poaceae/crescimento & desenvolvimento , Estações do Ano , Temperatura Ambiente , Tempo (Meteorologia)
8.
Plant Physiol Biochem ; 113: 198-207, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28236753

RESUMO

Salt stress affects yield formation of corn (Zea mays L.) at various physiological levels resulting in an overall grain yield decrease. In this study we investigated how salt stress affects kernel development of two corn cultivars (cvs. Pioneer 3906 and Fabregas) at and shortly after pollination. In an earlier study, we found an accumulation of hexoses in the kernel tissue. Therefore, it was hypothesized that hexose uptake into developing endosperm and embryo might be inhibited. Hexoses are transported into the developing endosperm by carriers localized in the plasma membrane (PM). The transport is driven by the pH gradient which is built up by the PM H+-ATPase. It was investigated whether the PM H+-ATPase activity in developing corn kernels was inhibited by salt stress, which would cause a lower pH gradient resulting in impaired hexose import and finally in kernel abortion. Corn grown under control and salt stress conditions was harvested 0 and 2 days after pollination (DAP). Under salt stress sucrose and hexose concentrations in kernel tissue were higher 0 and 2 DAP. Kernel PM H+-ATPase activity was not affected at 0 DAP, but it was reduced at 2 DAP. This is in agreement with the finding, that kernel growth and thus kernel setting was not affected in the salt stress treatment at pollination, but it was reduced 2 days later. It is concluded that inhibition of PM H+-ATPase under salt stress impaired the energization of hexose transporters into the cells, resulting in lower kernel growth and finally in kernel abortion.


Assuntos
ATPases Translocadoras de Prótons/metabolismo , Cloreto de Sódio/farmacologia , Estresse Fisiológico/fisiologia , Zea mays/efeitos dos fármacos , Zea mays/enzimologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Parede Celular/enzimologia , Ativação Enzimática , Ensaios Enzimáticos , Hexoses/metabolismo , Concentração de Íons de Hidrogênio , Proteínas de Plantas/efeitos adversos , Proteínas de Plantas/metabolismo , Sementes/efeitos dos fármacos , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Estresse Fisiológico/efeitos dos fármacos , Sacarose/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo
9.
Sci Total Environ ; 524-525: 427-39, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-25930242

RESUMO

Urban heat islands in the subsurface contain large quantities of energy in the form of elevated groundwater temperatures caused by anthropogenic heat fluxes (AHFS) into the subsurface. The objective of this study is to quantify these AHFS and the heat flow they generate in two German cities, Karlsruhe and Cologne. Thus, statistical and spatial analytical heat flux models were developed for both cities. The models include the spatial representation of various sources of AHFS: (1) elevated ground surface temperatures, (2) basements, (3) sewage systems, (4) sewage leakage, (5) subway tunnels, and (6) district heating networks. The results show that the district heating networks induce the largest AHFS with values greater than 60 W/m(2) and one order of magnitude higher than fluxes from other sources. A covariance analysis indicates that the spatial distribution of the total flux depends mainly on the thermal gradient in the unsaturated zone. On a citywide scale, basements and elevated ground surface temperatures are the dominant sources of heat flow. Overall, 2.1 PJ/a and 1.0 PJ/a of heat are accumulated on average in Karlsruhe and the western part of Cologne, respectively. Extracting this anthropogenically originated energy could sustainably supply significant parts of the urban heating demand. Furthermore, using this heat could also keep groundwater temperatures from rising further.

10.
J Proteome Res ; 14(6): 2500-10, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25939058

RESUMO

We present a novel tandem mass tag solid-phase amino labeling (TMT-SPAL) protocol using reversible immobilization of peptides onto octadecyl-derivatized (C18) solid supports. This method can reduce the number of steps required in complex protocols, saving time and potentially reducing sample loss. In our global phosphopeptide profiling workflow (SysQuant), we can cut 24 h from the protocol while increasing peptide identifications (20%) and reducing side reactions. Solid-phase labeling with TMTs does require some modification to typical labeling conditions, particularly pH. It has been found that complete labeling equivalent to standard basic pH solution-phase labeling for small and large samples can be achieved on C18 resins under slightly acidic buffer conditions. Improved labeling behavior on C18 compared to that with standard basic pH solution-phase labeling is demonstrated. We analyzed our samples for histidine, serine, threonine, and tyrosine labeling to determine the degree of overlabeling and observed higher than expected levels (25% of all peptide spectral matches (PSMs)) of overlabeling at all of these amino acids (predominantly at tyrosine and serine) in our standard solution-phase labeling protocol. Overlabeling at all of these sites is greatly reduced (4-fold, to 7% of all PSMs) by the low-pH conditions used in the TMT-SPAL protocol. Overlabeling seems to represent a so-far overlooked mechanism causing reductions in peptide identification rates with NHS-activated TMT labeling compared to that with label-free methods. Our results also highlight the importance of searching data for overlabeling when labeling methods are used.


Assuntos
Concentração de Íons de Hidrogênio , Fosfopeptídeos/química , Aminas/química , Linhagem Celular Tumoral , Humanos , Espectrometria de Massas em Tandem
11.
PLoS One ; 9(3): e90948, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24670416

RESUMO

OBJECTIVE: LC-MS/MS phospho-proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed a global phospho-proteomic workflow to determine activity of signaling pathways and drug targets in pancreatic cancer tissue for clinical application. METHODS: Peptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal adenocarcinoma and background pancreas (n = 12), were labelled with tandem mass tags (TMT 8-plex), separated by strong cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using IMAC and TiO2, prior to analysis. In-house, commercial and freeware bioinformatic platforms were used to identify relevant biological events from the complex dataset. RESULTS: Of 2,101 proteins identified, 152 demonstrated significant difference in abundance between tumor and non-tumor tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were new candidate markers such as HIPK1 & MLCK. Of the 6,543 unique phosphopeptides identified (6,284 unique phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho guanine nucleotide exchange factors & MRCKα) and formation of focal adhesions. Activator phosphorylation sites on FYN, AKT1, ERK2, HDAC1 and other drug targets were found to be highly modulated (≥2 fold) in different cases highlighting their predictive power. CONCLUSION: Here we provided critical information enabling us to identify the common and unique molecular events likely contributing to cancer in each case. Such information may be used to help predict more bespoke therapy suitable for an individual case.


Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Proteoma/metabolismo , Sequência de Aminoácidos , Biomarcadores/metabolismo , Dano ao DNA , Reparo do DNA , Análise Discriminante , Matriz Extracelular/metabolismo , Adesões Focais/metabolismo , Ontologia Genética , Humanos , Análise dos Mínimos Quadrados , Fosfopeptídeos/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Pseudópodes/metabolismo , Transdução de Sinais , Regulação para Cima
12.
Nat Commun ; 4: 1616, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23511480

RESUMO

Selected groups of peptides, including those that are presented by major histocompatibility complex (MHC) proteins, have been proposed to transmit information to the olfactory system of vertebrates via their ability to stimulate chemosensory neurons. However, the lack of knowledge about such peptides in natural sources accessible for nasal recognition has been a major barrier for this hypothesis. Here we analyse urinary peptides from selected mouse strains with respect to genotype-related individual differences. We discover many abundant peptides with single amino-acid variations corresponding to genomic differences. The polymorphism of major urinary proteins is reflected by variations in prominent urinary peptides. We also demonstrate an MHC-dependent peptide (SIINFEKL) occurring at very low concentrations in mouse urine. Chemoreceptive neurons in the vomeronasal organ detect and discriminate single amino-acid variation peptides as well as SIINFEKL. Hence, urinary peptides represent a real-time sampling of the expressed genome available for chemosensory assessment by other individuals.


Assuntos
Genótipo , Mucosa Nasal/citologia , Peptídeos/urina , Células Receptoras Sensoriais/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Complexo Principal de Histocompatibilidade , Espectrometria de Massas , Camundongos , Peptídeos/química
13.
Hum Mol Genet ; 19(22): 4437-52, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20817635

RESUMO

The mitochondrial chaperone mortalin has been linked to neurodegeneration in Parkinson's disease (PD) based on reduced protein levels in affected brain regions of PD patients and its interaction with the PD-associated protein DJ-1. Recently, two amino acid exchanges in the ATPase domain (R126W) and the substrate-binding domain (P509S) of mortalin were identified in Spanish PD patients. Here, we identified a separate and novel variant (A476T) in the substrate-binding domain of mortalin in German PD patients. To define a potential role as a susceptibility factor in PD, we characterized the functions of all three variants in different cellular models. In vitro import assays revealed normal targeting of all mortalin variants. In neuronal and non-neuronal human cell lines, the disease-associated variants caused a mitochondrial phenotype of increased reactive oxygen species and reduced mitochondrial membrane potential, which were exacerbated upon proteolytic stress. These functional impairments correspond with characteristic alterations of the mitochondrial network in cells overexpressing mutant mortalin compared with wild-type (wt), which were confirmed in fibroblasts from a carrier of the A476T variant. In line with a loss of function hypothesis, knockdown of mortalin in human cells caused impaired mitochondrial function that was rescued by wt mortalin, but not by the variants. Our genetic and functional studies of novel disease-associated variants in the mortalin gene define a loss of mortalin function, which causes impaired mitochondrial function and dynamics. Our results support the role of this mitochondrial chaperone in neurodegeneration and underscore the concept of impaired mitochondrial protein quality control in PD.


Assuntos
Proteínas de Choque Térmico HSP70/genética , Mitocôndrias/fisiologia , Chaperonas Moleculares/genética , Doença de Parkinson/genética , Idoso , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Variação Genética , Humanos , Masculino , Potencial da Membrana Mitocondrial/genética , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Espécies Reativas de Oxigênio/metabolismo
14.
Proteome Sci ; 8: 24, 2010 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-20459704

RESUMO

BACKGROUND: Often high-quality MS/MS spectra of tryptic peptides do not match to any database entry because of only partially sequenced genomes and therefore, protein identification requires de novo peptide sequencing. To achieve protein identification of the economically important but still unsequenced plant pathogenic oomycete Plasmopara halstedii, we first evaluated the performance of three different de novo peptide sequencing algorithms applied to a protein digests of standard proteins using a quadrupole TOF (QStar Pulsar i). RESULTS: The performance order of the algorithms was PEAKS online > PepNovo > CompNovo. In summary, PEAKS online correctly predicted 45% of measured peptides for a protein test data set.All three de novo peptide sequencing algorithms were used to identify MS/MS spectra of tryptic peptides of an unknown 57 kDa protein of P. halstedii. We found ten de novo sequenced peptides that showed homology to a Phytophthora infestans protein, a closely related organism of P. halstedii. Employing a second complementary approach, verification of peptide prediction and protein identification was performed by creation of degenerate primers for RACE-PCR and led to an ORF of 1,589 bp for a hypothetical phosphoenolpyruvate carboxykinase. CONCLUSIONS: Our study demonstrated that identification of proteins within minute amounts of sample material improved significantly by combining sensitive LC-MS methods with different de novo peptide sequencing algorithms. In addition, this is the first study that verified protein prediction from MS data by also employing a second complementary approach, in which RACE-PCR led to identification of a novel elicitor protein in P. halstedii.

15.
J Proteome Res ; 9(5): 2696-704, 2010 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-20201589

RESUMO

Targeted proteomic approaches such as multiple reaction monitoring (MRM) overcome problems associated with classical shotgun mass spectrometry experiments. Developing MRM quantitation assays can be time consuming, because relevant peptide representatives of the proteins must be found and their retention time and the product ions must be determined. Given the transitions, hundreds to thousands of them can be scheduled into one experiment run. However, it is difficult to select which of the transitions should be included into a measurement. We present a novel algorithm that allows the construction of MRM assays from the sequence of the targeted proteins alone. This enables the rapid development of targeted MRM experiments without large libraries of transitions or peptide spectra. The approach relies on combinatorial optimization in combination with machine learning techniques to predict proteotypicity, retention time, and fragmentation of peptides. The resulting potential transitions are scheduled optimally by solving an integer linear program. We demonstrate that fully automated construction of MRM experiments from protein sequences alone is possible and over 80% coverage of the targeted proteins can be achieved without further optimization of the assay.


Assuntos
Algoritmos , Inteligência Artificial , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/análise , Proteômica/métodos , Animais , Benzenossulfonatos/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Modelos Lineares , Cadeias de Markov , Melanoma/metabolismo , Camundongos , Niacinamida/análogos & derivados , Compostos de Fenilureia , Proteoma/análise , Piridinas/farmacologia , Reprodutibilidade dos Testes , Sorafenibe
16.
Genome Res ; 20(6): 837-46, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20237107

RESUMO

Pristionchus pacificus is a nematode model organism whose genome has recently been sequenced. To refine the genome annotation we performed transcriptome and proteome analysis and gathered comprehensive experimental information on gene expression. Transcriptome analysis on a 454 Life Sciences (Roche) FLX platform generated >700,000 expressed sequence tags (ESTs) from two normalized EST libraries, whereas proteome analysis on an LTQ-Orbitrap mass spectrometer detected >27,000 nonredundant peptide sequences from more than 4000 proteins at sub-parts-per-million (ppm) mass accuracy and a false discovery rate of <1%. Retraining of the SNAP gene prediction algorithm using the gene expression data led to a decrease in the number of previously predicted protein-coding genes from 29,000 to 24,000 and refinement of numerous gene models. The P. pacificus proteome contains a high proportion of small proteins with no known homologs in other species ("pioneer" proteins). Some of these proteins appear to be products of highly homologous genes, pointing to their common origin. We show that >50% of all pioneer genes are transcribed under standard culture conditions and that pioneer proteins significantly contribute to a unimodal distribution of predicted protein sizes in P. pacificus, which has an unusually low median size of 240 amino acids (26.8 kDa). In contrast, the predicted proteome of Caenorhabditis elegans follows a distinct bimodal protein size distribution, with significant functional differences between small and large protein populations. Combined, these results provide the first catalog of the expressed genome of P. pacificus, refinement of its genome annotation, and the first comparison of related nematode models at the proteome level.


Assuntos
Genômica , Modelos Biológicos , Nematoides/metabolismo , Proteômica , Algoritmos , Animais , Cromatografia Líquida , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica , Nematoides/genética , Espectrometria de Massas em Tandem
17.
Redox Rep ; 8(6): 384-8, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14980072

RESUMO

Peptide methionine sulfoxide reductases are important enzymes in the defense against cellular oxidative stress as they reduce methionine sulfoxide, the product of methionine oxidation by physiologically relevant reactive oxygen species. Two distinct enzyme classes, MSRA and MSRB, have evolved for selectively reducing the two epimers, methionine-S-sulfoxide and methionine-R-sulfoxide. A new human MSR enzyme (hMSRB2) specifically reducing methionine-R-sulfoxide, which showed a conversion rate for peptide-bound methionine-S-sulfoxide similar to hMSRB1, was characterized with respect to its tissue expression. As previously found for hMSRB1, expression of hMSRB2 mRNA was weak in brain, but strong in heart and skeletal muscle. In contrast to hMSRB1, its expression was high in smooth muscle-containing organs (digestive system, bladder), lung and aorta, while hMSRB1 displayed a higher expression than hMSRB2 in liver and kidney.


Assuntos
Metionina/metabolismo , Oxirredutases/metabolismo , Sulfóxidos/metabolismo , Northern Blotting , Encéfalo/metabolismo , DNA Complementar/metabolismo , Bases de Dados como Assunto , Relação Dose-Resposta a Droga , Humanos , Metionina Sulfóxido Redutases , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta , Oxigênio/metabolismo , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio , Proteínas Recombinantes/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição Tecidual
18.
FEBS Lett ; 527(1-3): 91-4, 2002 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-12220640

RESUMO

Human CBS1 is a methionine sulfoxide reductase of type B (MSRB) as it specifically reduced Met-R-SO in peptides with dithiothreitol or the thioredoxin system as reductants. Mutation C169S in the active site completely abolished enzymatic activity, while mutation W110A only reduced activity and C105S had no effect. Like human MSRA, hCBS1 showed in vivo reducing activity coexpressed with the Drosophila ShC/B potassium channel in oocytes, by accelerating the overall inactivation time course. hCBS1-encoding mRNA is most abundant in muscle tissues, especially in the heart and thereby shows an expression pattern different to the human MSRA.


Assuntos
Oxirredutases/genética , Oxirredutases/metabolismo , Animais , Domínio Catalítico , Células Cultivadas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Humanos , Espectrometria de Massas , Metionina Sulfóxido Redutases , Músculo Esquelético/metabolismo , Mutagênese Sítio-Dirigida , Miocárdio/metabolismo , Oócitos/fisiologia , Oxirredutases/química , Peptídeos/química , Peptídeos/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Superfamília Shaker de Canais de Potássio , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estereoisomerismo , Distribuição Tecidual
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