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1.
Atherosclerosis ; 269: 159-165, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29366988

RESUMO

BACKGROUND AND AIMS: While inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) is known to result in dramatic lowering of LDL-cholesterol (LDL-C), it is poorly understood how it affects other lipid species and their metabolism. The aim of this study was to characterize the alterations in the lipidome of plasma and lipoprotein particles after administration of PCSK9 inhibiting antibody to patients with established coronary heart disease. METHODS: Plasma samples were obtained from patients undergoing a randomized placebo-controlled phase II trial (EQUATOR) for the safe and effective use of RG7652, a fully human monoclonal antibody inhibiting PCSK9 function. Lipoprotein fractions were isolated by sequential density ultracentrifugation, and both plasma and major lipoprotein classes (VLDL-IDL, LDL, HDL) were subjected to mass spectrometric lipidomic profiling. RESULTS: PCSK9 inhibition significantly decreased plasma levels of several lipid classes, including sphingolipids (dihydroceramides, glucosylceramides, sphingomyelins, ceramides), cholesteryl esters and free cholesterol. Previously established ceramide ratios predicting cardiovascular mortality, or inflammation related eicosanoid lipids, were not altered. RG7652 treatment also affected the overall and relative distribution of lipids in lipoprotein classes. An overall decrease of total lipid species was observed in LDL and VLDL + IDL particles, while HDL-associated phospholipids increased. Following the treatment, LDL displayed reduced lipid cargo, whereas relative lipid proportions of the VLDL + IDL particles were mostly unchanged, and there were relatively more lipids carried in the HDL particles. CONCLUSIONS: Administration of PCSK9 antibody significantly alters the lipid composition of plasma and lipoprotein particles. These changes further shed light on the link between anti-PCSK9 therapies and cardiovascular risk.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticolesterolemiantes/uso terapêutico , Doença das Coronárias/tratamento farmacológico , Lipídeos/sangue , Lipoproteínas/sangue , Inibidores de PCSK9 , Idoso , Anticorpos Monoclonais Humanizados , Biomarcadores/sangue , Centrifugação com Gradiente de Concentração , Doença das Coronárias/sangue , Doença das Coronárias/diagnóstico , Feminino , Finlândia , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Pró-Proteína Convertase 9/metabolismo , Fatores de Tempo , Resultado do Tratamento
2.
Eur J Cancer ; 70: 122-132, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27914242

RESUMO

BACKGROUND: Exosomes have recently appeared as a novel source of noninvasive cancer biomarkers, since these nanovesicles contain molecules from cancer cells and can be detected in biofluids. We have here investigated the potential use of lipids in urinary exosomes as prostate cancer biomarkers. METHODS: A high-throughput mass spectrometry quantitative lipidomic analysis was performed to reveal the lipid composition of urinary exosomes in prostate cancer patients and healthy controls. RESULTS: Control samples were first analysed to characterise the lipidome of urinary exosomes and test the reproducibility of the method. In total, 107 lipid species were quantified in urinary exosomes. Several differences, for example, in cholesterol and phosphatidylcholine, were found between urinary exosomes and exosomes derived from cell lines, thus showing the importance of in vivo studies for biomarker analysis. The 36 most abundant lipid species in urinary exosomes were then quantified in 15 prostate cancer patients and 13 healthy controls. Interestingly, the levels of nine lipids species were found to be significantly different when the two groups were compared. The highest significance was shown for phosphatidylserine (PS) 18:1/18:1 and lactosylceramide (d18:1/16:0), the latter also showed the highest patient-to-control ratio. Furthermore, combinations of these lipid species and PS 18:0-18:2 distinguished the two groups with 93% sensitivity and 100% specificity. Finally, in agreement with the reported dysregulation of sphingolipid metabolism in cancer cells, alteration in specific sphingolipid lipid classes were observed. CONCLUSION: This study shows for the first time the potential use of exosomal lipid species in urine as prostate cancer biomarkers.


Assuntos
Biomarcadores Tumorais/urina , Exossomos/química , Lipídeos/análise , Próstata/citologia , Neoplasias da Próstata/urina , Área Sob a Curva , Estudos de Casos e Controles , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Espectrometria de Massas/métodos , Próstata/química , Neoplasias da Próstata/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
3.
Oncotarget ; 7(48): 79885-79900, 2016 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-27829218

RESUMO

2-fluoro-2-deoxy-D-glucose (FDG), labeled with 18F radioisotope, is the most common imaging agent used for positron emission tomography (PET) in oncology. However, little is known about the cellular effects of FDG. Another glucose analogue, 2-deoxy-D-glucose (2DG), has been shown to affect many cellular functions, including intracellular transport and lipid metabolism, and has been found to improve the efficacy of cancer chemotherapeutic agents in vivo. Thus, in the present study, we have investigated cellular effects of FDG with the focus on changes in cellular lipids and intracellular transport. By quantifying more than 200 lipids from 17 different lipid classes in HEp-2 cells and by analyzing glycosphingolipids from MCF-7, HT-29 and HBMEC cells, we have discovered that FDG treatment inhibits glucosylceramide synthesis and thus reduces cellular levels of glycosphingolipids. In addition, in HEp-2 cells the levels and/or species composition of other lipid classes, namely diacylglycerols, phosphatidic acids and phosphatidylinositols, were found to change upon treatment with FDG. Furthermore, we show here that FDG inhibits retrograde Shiga toxin transport and is much more efficient in protecting cells against the toxin than 2DG. In summary, our data reveal novel effects of FDG on cellular transport and glycosphingolipid metabolism, which suggest a potential clinical application of FDG as an adjuvant for cancer chemotherapy.


Assuntos
Fluordesoxiglucose F18/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/efeitos da radiação , Células Cultivadas , Endocitose/efeitos dos fármacos , Endocitose/efeitos da radiação , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos da radiação , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/efeitos da radiação , Células HT29 , Humanos , Metabolismo dos Lipídeos/efeitos da radiação , Células MCF-7 , Metaboloma/efeitos da radiação , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/efeitos da radiação , Toxina Shiga/metabolismo
4.
Biochem J ; 470(1): 23-37, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26251444

RESUMO

2-Deoxy-D-glucose (2DG) is a structural analogue of glucose with well-established applications as an inhibitor of glycolysis and N-glycosylation. Importantly, 2DG has been shown to improve the efficacy of several cancer chemotherapeutic agents in vivo and thus it is in clinical studies in combination with chemotherapy and radiotherapy. However, although 2DG has been demonstrated to modulate many cellular functions, including autophagy, apoptosis and cell cycle control, little is known about the effects of 2DG on intracellular transport, which is of great importance when predicting the effects of 2DG on therapeutic agents. In addition to proteins, lipids play important roles in cellular signalling and in controlling cellular trafficking. We have, in the present study, investigated the effects of 2DG on cellular lipid composition and by use of protein toxins we have studied 2DG-mediated changes in intracellular trafficking. By quantifying more than 200 individual lipid species from 17 different lipid classes, we have found that 2DG treatment changes the levels and/or species composition of several lipids, such as phosphatidylinositol (PI), diacylglycerol (DAG), cholesteryl ester (CE), ceramide (Cer) and lysophospho-lipids. Moreover, 2DG becomes incorporated into the carbohydrate moiety of glycosphingolipids (GSLs). In addition, we have discovered that 2DG protects cells against Shiga toxins (Stxs) and inhibits release of the cytotoxic StxA1 moiety in the endoplasmic reticulum (ER). The data indicate that the 2DG-induced protection against Stx is independent of inhibition of glycolysis or N-glycosylation, but rather mediated via the depletion of Ca(2+) from cellular reservoirs by 2DG. In conclusion, our results reveal novel actions of 2DG on cellular lipids and Stx toxicity.


Assuntos
Citoproteção/efeitos dos fármacos , Desoxiglucose/farmacologia , Lipídeos de Membrana/metabolismo , Toxinas Shiga/toxicidade , Linhagem Celular , Citoproteção/fisiologia , Humanos
5.
Anal Bioanal Chem ; 407(17): 5091-9, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25618760

RESUMO

Lysophospholipids (LPLs) are an essential family of lipids, which serve as bioactive molecules and as precursors and intermediates of the glycerophospholipid and sphingolipid metabolisms. In this work we primarily focused on the subgroup lysoglycerophospholipids that comprise a polar headgroup at the sn-3 position and a fatty acyl group at either the sn-1 or sn-2 position of the glycerol backbone giving rise to the two potential regioisomers 1-acyl-2-LPL and 2-acyl-1-LPL, respectively. We established a quantitative lysophospholipidomics method combining hydrophilic interaction chromatography (HILIC) with the scheduled multiple reaction monitoring (sMRM) algorithm for profiling a vast number of LPLs simultaneously, including the 1-acyl-2-LPL and 2-acyl-1-LPL regioisomers. This approach facilitates baseline separation of monitored lipid classes and regioisomers, including sufficient separation of species having a different degree of unsaturation overcoming the overlapping effect of M + 2 isotopes. The lipid class-based separation improves the quantification of each molecular species as the internal standard elutes together with the endogenous species. The potential of this method is illustrated by analyzing LPLs from human plasma and skin samples. Altogether, 68 molecular lipid species, consisting of 110 regioisomers, were detected in plasma and 43 molecular lipids, consisting of 67 regioisomers, in skin samples. The novel skin LPL profile reveals that most of the lipid species exist as 2-acyl-1-LPL, in comparison to plasma where 1-acyl-2-LPLs are the dominant species.


Assuntos
Cromatografia Líquida/métodos , Lisofosfolipídeos/análise , Lisofosfolipídeos/sangue , Pele/química , Espectrometria de Massas em Tandem/métodos , Acilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Estereoisomerismo
6.
J Biol Chem ; 290(7): 4225-37, 2015 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-25519911

RESUMO

Exosomes are vesicles released by cells after fusion of multivesicular bodies with the plasma membrane. In this study, we have investigated whether ether lipids affect the release of exosomes in PC-3 cells. To increase the cellular levels of ether lipids, the ether lipid precursor hexadecylglycerol was added to cells. Lipidomic analysis showed that this compound was in fact able to double the cellular levels of ether lipids in these cells. Furthermore, increased levels of ether lipids were also found in exosomes released by cells containing high levels of these lipids. Interestingly, as measured by nanoparticle tracking analysis, cells containing high levels of ether lipids released more exosomes than control cells, and these exosomes were similar in size to control exosomes. Moreover, silver staining and Western blot analyses showed that the protein composition of exosomes released in the presence of hexadecylglycerol was changed; the levels of some proteins were increased, and the levels of others were reduced. In conclusion, this study clearly shows that an increase in cellular ether lipids is associated with changes in the release and composition of exosomes.


Assuntos
Exossomos/química , Exossomos/metabolismo , Éteres de Glicerila/farmacologia , Lipídeos/análise , Corpos Multivesiculares/metabolismo , Neoplasias da Próstata/metabolismo , Humanos , Masculino , Neoplasias da Próstata/patologia , Células Tumorais Cultivadas
7.
Cell Mol Life Sci ; 71(21): 4285-300, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24740796

RESUMO

Shiga toxin-producing Escherichia coli bacteria cause hemorrhagic colitis and hemolytic uremic syndrome in humans. Currently, only supportive treatment is available for diagnosed patients. We show here that 24-h pretreatment with an ether lipid precursor, the alkylglycerol sn-1-O-hexadecylglycerol (HG), protects HEp-2 cells against Shiga toxin and Shiga toxin 2. Also the endothelial cell lines HMEC-1 and HBMEC are protected against Shiga toxins after HG pretreatment. In contrast, the corresponding acylglycerol, DL-α-palmitin, has no effect on Shiga toxicity. Although HG treatment provides a strong protection (~30 times higher IC50) against Shiga toxin, only a moderate reduction in toxin binding was observed, suggesting that retrograde transport of the toxin from the plasma membrane to the cytosol is perturbed. Furthermore, endocytosis of Shiga toxin and retrograde sorting from endosomes to the Golgi apparatus remain intact, but transport from the Golgi to the endoplasmic reticulum is inhibited by HG treatment. As previously described, HG reduces the total level of all quantified glycosphingolipids to 50-70% of control, including the Shiga toxin receptor globotriaosylceramide (Gb3), in HEp-2 cells. In accordance with this, we find that interfering with Gb3 biosynthesis by siRNA-mediated knockdown of Gb3 synthase for 24 h causes a similar cytotoxic protection and only a moderate reduction in toxin binding (to 70% of control cells). Alkylglycerols, including HG, have been administered to humans for investigation of therapeutic roles in disorders where ether lipid biosynthesis is deficient, as well as in cancer therapy. Further studies may reveal if HG can also have a therapeutic potential in Shiga toxin-producing E. coli infections.


Assuntos
Glicerol/química , Éteres de Glicerila/química , Toxinas Shiga/química , Transporte Biológico , Biotinilação , Linhagem Celular , Membrana Celular/metabolismo , Citosol/metabolismo , Endocitose , Retículo Endoplasmático/metabolismo , Escherichia coli/metabolismo , Éter/química , Glicoesfingolipídeos/química , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Concentração Inibidora 50 , Lipídeos/química , Ácido Palmítico/química , Toxina Shiga/química , Triexosilceramidas/química
8.
Cell Mol Life Sci ; 71(6): 1097-116, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23921715

RESUMO

Cell density is one of the extrinsic factors to which cells adapt their physiology when grown in culture. However, little is known about the molecular changes which occur during cell growth and how cellular responses are then modulated. In many cases, inhibitors, drugs or growth factors used for in vitro studies change the rate of cell proliferation, resulting in different cell densities in control and treated samples. Therefore, for a comprehensive data analysis, it is essential to understand the implications of cell density on the molecular level. In this study, we have investigated how lipid composition changes during cell growth, and the consequences it has for transport of Shiga toxin. By quantifying 308 individual lipid species from 17 different lipid classes, we have found that the levels and species distribution of several lipids change during cell growth, with the major changes observed for diacylglycerols, phosphatidic acids, cholesterol esters, and lysophosphatidylethanolamines. In addition, there is a reduced binding and retrograde transport of Shiga toxin in high density cells which lead to reduced intoxication by the toxin. In conclusion, our data provide novel information on how lipid composition changes during cell growth in culture, and how these changes can modulate intracellular trafficking.


Assuntos
Contagem de Células , Metabolismo dos Lipídeos , Sintaxina 1/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Colesterol/metabolismo , Diglicerídeos/metabolismo , Globosídeos/metabolismo , Glicoesfingolipídeos/metabolismo , Células HeLa , Células Hep G2 , Humanos , Lisofosfolipídeos/metabolismo , Ácidos Fosfatídicos/metabolismo , Transporte Proteico , Toxina Shiga/metabolismo , Sintaxina 1/genética , Triexosilceramidas/metabolismo
9.
PLoS One ; 8(9): e75904, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24098742

RESUMO

The ether-lipid precursor sn-1-O-hexadecylglycerol (HG) can be used to compensate for early metabolic defects in ether-lipid biosynthesis. To investigate a possible metabolic link between ether-linked phospholipids and the rest of the cellular lipidome, we incubated HEp-2 cells with HG. Mass spectrometry analysis revealed major changes in the lipidome of HG-treated cells compared to that of untreated cells or cells treated with palmitin, a control substance for HG containing an acyl group instead of the ether group. We present quantitative data for a total of 154 species from 17 lipid classes. These species are those constituting more than 2% of their lipid class for most lipid classes, but more than 1% for the ether lipids and glycosphingolipids. In addition to the expected ability of HG to increase the levels of ether-linked glycerophospholipids with 16 carbon atoms in the sn-1 position, this precursor also decreased the amounts of glycosphingolipids and increased the amounts of ceramide, phosphatidylinositol and lysophosphatidylinositol. However, incubation with palmitin, the fatty acyl analogue of HG, also increased the amounts of ceramide and phosphatidylinositols. Thus, changes in these lipid classes were not ether lipid-dependent. No major effects were observed for the other lipid classes, and cellular functions such as growth and endocytosis were unaffected. The data presented clearly demonstrate the importance of performing detailed quantitative lipidomic studies to reveal how the metabolism of ether-linked glycerophospholipids is coupled to that of glycosphingolipids and ester-linked glycerophospholipids, especially phosphatidylinositols.


Assuntos
Glicerol/farmacologia , Glicerofosfolipídeos/biossíntese , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/isolamento & purificação , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Glicerofosfolipídeos/química , Humanos , Estrutura Molecular , Espectrometria de Massas em Tandem
10.
World J Gastroenterol ; 14(28): 4462-72, 2008 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-18680224

RESUMO

AIM: To characterise the effect of energy restriction (ER) on liver lipid and primary metabolite profile by using metabolomic approach. We also investigated whether the effect of energy restriction can be further enhanced by modification of dietary protein source and calcium. METHODS: Liver metabolomic profile of lean and obese C57Bl/6J mice (n = 10/group) were compared with two groups of weight-reduced mice. ER was performed on control diet and whey protein-based high-calcium diet (whey + Ca). The metabolomic analyses were performed using the UPLC/MS based lipidomic platform and the HPLC/MS/MS based primary metabolite platform. RESULTS: ER on both diets significantly reduced hepatic lipid accumulation and lipid droplet size, while only whey + Ca diet significantly decreased blood glucose (P < 0.001) and serum insulin (P < 0.01). In hepatic lipid species the biggest reduction was in the level of triacylglycerols and ceramides while the level of cholesterol esters was significantly increased during ER. Interestingly, diacylglycerol to phospholipid ratio, an indicator of relative amount of diabetogenic diglyceride species, was increased in the control ER group, but decreased in the whey + Ca ER group (P < 0.001, vs obese). ER on whey + Ca diet also totally reversed the obesity induced increase in the relative level of lipotoxic ceramides (P < 0.001, vs obese; P > 0.05, vs lean). These changes were accompanied with up-regulated TCA cycle and pentose phosphate pathway metabolites. CONCLUSION: ER-induced changes on hepatic metabolomic profile can be significantly affected by dietary protein source. The therapeutic potential of whey protein and calcium should be further studied.


Assuntos
Cálcio da Dieta/farmacologia , Proteínas Alimentares/farmacologia , Metabolismo Energético/fisiologia , Fígado Gorduroso/metabolismo , Animais , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Modelos Animais de Doenças , Fígado Gorduroso/fisiopatologia , Insulina/sangue , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
11.
J Chromatogr A ; 1185(1): 139-44, 2008 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-18255080

RESUMO

The neutral sugar composition of acid hydrolyzed extracts of cellulose fiber samples, i.e. oat spelt, wheat straw, thermomechanica pulp (TMP) made of spruce, aspen stemwood, and bleached birch kraft pulp, was determined by a new capillary zone electrophoresis (CZE) method employing an alkaline background electrolyte. The method relies on in-capillary reaction and direct UV detection at wavelength 270 nm. Neutral carbohydrates D-(+)-galactose, D-(+)-glucose, L-rhamnose, D-(+)-mannose, D-(-)-arabinose, and D-(+)-xylose were simultaneously separated. The calibration plots were linear over a range from 10 to 150 mg/L for D-(+)-galactose, L-rhamnose, D-(+)-mannose, and D-(-)-arabinose and from 50 to 400mg/L for D-(+)-glucose and D-(+)-xylose. Relative standard deviations (RSDs) of peak areas during a 5-day analysis period varied from 3.3% for galactose to 11.8% for rhamnose. RSDs of migration times varied between 0.3 and 0.7%. The detection limit (at S/N 3) was 5mg/L for each monosaccharide. The results obtained by CZE agreed well with results obtained by high-performance anion-exchange chromatography. Glucose and xylose were the two predominant monosaccharides in the plants, except in the spruce TMP sample where glucose and mannose dominated.


Assuntos
Eletroforese Capilar/métodos , Monossacarídeos/análise , Plantas/química , Avena/química , Carboidratos/análise , Hidrólise , Picea/química , Triticum/química
12.
FEMS Yeast Res ; 8(1): 140-54, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17425669

RESUMO

Saccharomyces cerevisiae CEN.PK113-1A was grown in glucose-limited chemostat culture with 0%, 0.5%, 1.0%, 2.8% or 20.9% O2 in the inlet gas (D=0.10 h(-1), pH 5, 30 degrees C) to determine the effects of oxygen on 17 metabolites and 69 genes related to central carbon metabolism. The concentrations of tricarboxylic acid cycle (TCA) metabolites and all glycolytic metabolites except 2-phosphoglycerate+3-phosphoglycerate and phosphoenolpyruvate were higher in anaerobic than in fully aerobic conditions. Provision of only 0.5-1% O2 reduced the concentrations of most metabolites, as compared with anaerobic conditions. Transcription of most genes analyzed was reduced in 0%, 0.5% or 1.0% O2 relative to cells grown in 2.8% or 20.9% O2. Ethanol production was observed with 2.8% or less O2. After steady-state analysis in defined oxygen concentrations, the conditions were switched from aerobic to anaerobic. Metabolite and transcript levels were monitored for up to 96 h after the transition, and this showed that more than 30 h was required for the cells to fully adapt to anaerobiosis. Levels of metabolites of upper glycolysis and the TCA cycle increased following the transition to anaerobic conditions, whereas those of metabolites of lower glycolysis generally decreased. Gene regulation was more complex, with some genes showing transient upregulation or downregulation during the adaptation to anaerobic conditions.


Assuntos
Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Aerobiose , Anaerobiose , Carbono , Ciclo do Ácido Cítrico , Meios de Cultura/farmacologia , Metabolismo Energético/efeitos dos fármacos , Glicólise , Redes e Vias Metabólicas , Oxigênio/metabolismo , Oxigênio/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos
13.
Protein Expr Purif ; 51(2): 216-26, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16997574

RESUMO

Heterologous expression of two fungal chitinases, Chit33 and Chit42, from Trichoderma harzianum was tested in the different compartments and on the surface of Escherichia coli cells. Our goal was to find a fast and efficient expression system for protein engineering and directed evolution studies of the two fungal enzymes. Cytoplasmic overexpression resulted in both cases in inclusion body formation, where active enzyme could be recovered after refolding. Periplasmic expression of Chit33, and especially of Chit42, proved to be better suited for mutagenesis purposes. Recombinant chitinases from the periplasmic expression system showed activity profiles similar to those of the native proteins. Both chitinases also degraded a RET (resonance energy transfer) based bifunctionalized chitinpentaose substrate in a similar manner as reported for some putative exochitinases in the glycosyl hydrolase family 18, offering a sensitive way to assay their activities. We further demonstrated that Chit42 can also be displayed on E. coli surface and the enzymatic activity can be measured directly from the whole cells using methylumbelliferyl-chitinbioside as a substrate. The periplasmic expression and the surface display of Chit42, both offer a suitable expression system for protein engineering and activity screening in a microtiter plate scale. As a first mutagenesis approach we verified the essential role of the two carboxylic acid residues E172 (putative proton donor) and D170 (putative stabilizer) in the catalytic mechanism of Chit42, and additionally the role of the carboxylic acid E145 (putative proton donor) in the catalytic mechanism of Chit33.


Assuntos
Quitinases/biossíntese , Quitinases/genética , Trichoderma/enzimologia , Citoplasma/enzimologia , Escherichia coli/metabolismo , Glucosídeos/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Naftalenossulfonatos , Oligossacarídeos/metabolismo , Periplasma/enzimologia , p-Dimetilaminoazobenzeno
14.
J Agric Food Chem ; 53(18): 7279-87, 2005 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-16131143

RESUMO

Twenty-seven barley (Hordeum vulgare L.) samples collected from growing sites in Scandinavia in 2001 and 2002 were examined to study the effect of endosperm structure on malting behavior. Samples were micromalted, and several malt characteristics were measured. Samples were classified as having a mealier or steelier endosperm on the basis of light transflectance (LTm). Because endosperm structure is greatly dependent on protein content, three barley sample pairs with similar protein contents were chosen for further analysis. During malting, the steelier barley samples produced less root mass, but showed higher respiration losses and higher activities of starch-hydrolyzing enzymes. Malts made from steelier barley had a less friable structure, with more urea-soluble D hordein and more free amino nitrogen and soluble protein. The reason for these differences may lie in the structure or localization of the hordeins as well as the possible effects of endosperm packing on water uptake and movement of enzymes.


Assuntos
Hordeum/anatomia & histologia , Sementes/anatomia & histologia , Parede Celular/ultraestrutura , Fenômenos Químicos , Físico-Química , Glutens , Hordeum/química , Nitrogênio/análise , Proteínas de Plantas/análise , Proteínas de Plantas/química , Sementes/química , Sementes/enzimologia , Solubilidade , Amido/análise
15.
Anal Chem ; 77(10): 3038-44, 2005 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-15889891

RESUMO

An antibody-based solid-phase extraction method for filtered 384-well plates was developed for a medical drug candidate having two enantiomeric forms in order to demonstrate the potential of the use of recombinant antibody fragments as specific and efficient immunosorbents. An immobilization method using a six-histidine tag of the antibody fragment and mild oxidation was applied in order to immobilize antibody fragments in an oriented and kinetically stable way that ensured high capacity of the antibody support. Phosphate buffer or plasma spiked with enantiomers were used as samples. Selective solid-phase extraction was followed by liquid chromatography-mass spectrometry analysis. Average recoveries for buffer and plasma samples ranged from 79 to 122% and 80 to 108%, respectively. Good linearity was observed in the concentration range of 30-3000 ng/mL of the enantiomer.


Assuntos
Anticorpos/química , Técnicas de Imunoadsorção , Proteínas Recombinantes/imunologia , Anticorpos/imunologia , Soluções Tampão , Cromatografia Líquida de Alta Pressão/métodos , Histidina/química , Espectrometria de Massas/métodos , Oxirredução , Fosfatos/química , Plasma , Proteínas Recombinantes/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estereoisomerismo
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