Research progress of proteomics in important tapeworm / 中华地方病学杂志

Taeniasis is a kind of zoonotic parasitosis which seriously endangers human health. Drug therapy and vaccine control is one of the hotspots in current research. At present, proteomics has become an important tool for developing vaccines and understanding the pathogenic mechanism. The differences in protein patterns between different developmental stages of parasites may reflect the specific strategies and adaptive mechanisms used in the constantly changing environment and life cycle. Therefore, this article reviews the proteomics research progress of various tapeworms, aiming at providing a reference for prevention and control of Taeniasis.

Plasma and Cell Lysate Proteins Associated With Treatment Outcome in Acute Myeloid Leukaemia

@#Introduction: Drug-resistance is a major hindrance to successful treatment of AML. Current predictive biomarkers are mainly genetic aberrations and insufficient in foretelling treatment outcome in all acute myeloid leukaemia (AML) due to its heterogeneous and aggressive nature. Proteins are stable and reliable. Secreted proteins in AML may have predictive or prognostic values for early intervention. Proteomic studies on AML are few and further investigations will benefit in selection of best markers. The aim of the study was to identify differentially expressed plasma proteins in AML with different treatment outcome. Methods: Two-dimensional electrophoresis (2-DE) technique was utilised to identify proteins differentially expressed in chemo-sensitive/chemo-resistant AML. Plasma and peripheral blood mononuclear cell (PBMC) lysate proteome analysis were performed on six chemo-resistant, four chemo-sensitive and six healthy controls and seven chemo-resistant, three chemo-sensitive and six healthy controls, respectively. Each experiment was conducted in duplicate or triplicate. Images were captured and protein spots detected by software. Differentially expressed protein spots were excised from gel and proteins were identified using LC/MS/MS. Proteins spots that were also detected in healthy controls were excluded. Results: Comparing mean % volume of each spot demonstrated significantly enhanced expression of apoliprotein-E (APO-E) and haptoglobin (HP) (p<0.05) in plasma and HNRNP H1 (p=0.049) in cell lysate of chemo-sensitive group. Serotransferrin (STF) from plasma and DNA-PK from cell lysate (p=0.01) were associated with chemo-resistance. Conclusion: This preliminary study identified several potential predictive biomarkers associated with chemo-resistance/chemo-sensitivity to treatment in AML. Further studies with a larger number of samples are required to validate the results.

Estudio proteómico 2DE-DIGE en plasma sanguíneo de pacientes en etapa infantil con leucemia linfoblástica aguda / 2DE-DIGE proteomic study in blood plasma of patients in childhood stage with acute lymphoblastic leukemia / Estudo proteômico 2DE-DIGE no plasma sanguíneo de pacientes em estágio infantil com leucemia linfoblástica aguda

Resumen En Colombia, durante la última década, la leucemia linfoblástica aguda (LLA) ha causado más del 40% de las muertes por cáncer en menores de edad. Entre los factores que influyen en estas cifras, el diagnóstico tardío es uno de los factores que más afecta el éxito del tratamiento. Por lo anterior, esta investigación se centró en el estudio del proteoma plasmático de niños colombianos diagnosticados con LLA tipo B, en comparación con controles en la búsqueda de proteínas que podrían ser clasificadas como biomarcadores de diagnóstico. En vista de los avances en las herramientas proteómicas y de espectrometría de masas y teniendo en cuenta que son una alternativa para abordar la complejidad molecular de enfermedades como el cáncer, se utilizó una aproximación proteómica basada en una separación por electroforesis bidimensional diferencial (2DE-DIGE) con posterior separación por cromatografía líquida acoplada a espectrometría de masas (LC-MS) en tándem. Se encontraron ocho proteínas con expresión diferencial en plasma de pacientes con LLA-B, entre las cuales resaltan la Serotransferrina, la Alfa-1-antitripsina, la Haptoglobina, la Alfa-2-glicoproteína de zinc y el Complemento C3.

Abstract In Colombia, during the last decade, acute lymphoblastic leukemia (ALL) has caused more than 40% of cancer deaths in children. Among the factors that influence these figures, late diagnosis is one of the factors that affects the treatment success. Therefore, this research focused on the plasma proteome study of Colombian children diagnosed with B-cell ALL, as compared with healthy controls in the search of proteins that could be classified as diagnostic biomarkers. Now, in view of the advances in the proteomics and mass spectrometry tools and taking into account that they are an alternative to address the molecular complexity of diseases such as cancer, a proteomic approach, based on bidimensional difference gel electrophoretic separation (2DE-DIGE) coupled to LC-MS/ MS, was used. We found eight differentially expressed proteins in plasma from B-cell ALL patients as follows: Serotransferrin, Alpha-1-antitrypsin, Haptoglobin, Zinc-alpha-2-glycoprotein, and Complement C3.

Resumo Na Colômbia, durante a última década, a leucemia linfoblastica aguda (LLA) tem sido o câncer com maior incidência, com mais de 40% das mortes por câncer em menores atribuídas a essa doença. Entre os fatores que influenciam esses números, o diagnóstico tardio talvez seja o fator mais sensível que afeta negativamente o sucesso do tratamento. Esta pesquisa enfocou o estudo do proteoma plasmático de crianças colombianas diagnosticadas com LLA tipo B, dada a sua alta incidência, em comparação com controles na busca por proteínas que poderiam ter potencialidade para serem classificadas como biomarcadores diagnósticos. Agora, em vista dos avanços nas ferramentas de proteômica e espectrometria de massa e sabendo que eles são uma alternativa para abordar a complexidade molecular de doenças como o câncer, usamos uma abordagem proteômica baseada em uma separação por eletroforese bidimensional diferencial (2DE-DIGE) com subsequente separação por cromatografia líquida acoplada a espectrometria de massa em tandem. Encontramos 8 proteínas com expressão diferencial no plasma de pacientes com LBA, dentre os quais a Serotransferrina, a Alfa-1-antitripsina, a Haptoglobina, a Glicoproteína alfa-2-zinco e o Complemento C3.

Separation and identification of differential protein in rat's bone with fluorosis and calcium supplementation intervention / 生物工程学报

In order to explore the mechanisms underlying the calcium alleviating fluorosis at protein level, we made an attempt to establish fluorosis and calcium supplementation rat models to isolate and identify bone differential proteins. The bone proteins of different groups were compared by two-dimensional electrophoresis (2-DE) and mass spectrometry (MALDI-TOF MS), and analyzed by gene ontology annotation, pathway enrichment and interaction networks. The 17 proteins were identified in the fluorosis group (F) and the fluorosis calcium supplement group (F+Ca), including type I collagen (Col1a1), actin (Actb), protein glutamine transferase 2 (Tgm2), compared with the control group (C). These differential proteins are enriched in 38 bone metabolic pathways such as focal adhesion, PI3K-Akt signaling pathway, and AMPK signaling pathway. And the functions of these proteins are mainly related to cytoskeleton, energy metabolism, substance transport, ion channel, and apoptosis. Therefore, it is speculated that calcium may alleviate the fluoride-induced bone damage by regulating the focal adhesion, PI3K-Akt, AMPK and other signaling pathway, but the specific mechanism needs further research.

Differential Proteomics Analysis of Endometriosis in Blood Stasis Syndrome / 中国结合医学杂志

OBJECTIVE@#To investigate the innate characters of 3 endometriosis (EMT) syndromes, blood stasis (BS), qi stagnation and blood stasis (QSBS) as well as Shen (Kidney) deficiency and blood stasis (KDBS) in terms of proteomics, lay a molecular biological basis for the differentiation of various blood stasis syndromes of EMT, establish a EMT microscopic syndrome differentiation and diagnosis system in terms of proteomics, discover the evolution principles and therapeutic targets of these EMT syndromes, and search their signifificant molecular markers and genetic intervention targets.@*METHODS@#Six specimens from the ectopic and entopic endometrium tissues of patients with EMT in each syndrome, BS, QSBS as well as KDBS, in the early proliferative phase of the menstrual cycle, and 6 specimens from normal endometrium tissues in the early proliferative phase of the menstrual cycle were obtained. Three groups were formed in each syndrome by mixing two random specimens in equal amount, and then their respective two-dimensional electrophoresis graphs were obtained after total protein extraction. Finally, the detected differences in protein expression were identifified through matrix-assisted laser desorption Ionization-time of flflight mass spectrometry (MALDI-TOF/MS) and protein database.@*RESULTS@#The results of differential proteins expressed in each syndrome were shown as follows: BS syndrome had 2 differential proteins in entopic endometrium and 1 differential protein in ectopic endometrium; KDBS syndrome had 3 in entopic endometrium and 3 in ectopic endometrium; and QSBS syndrome had 3 in entopic endometrium and 4 in ectopic endometrium. It was found out that annexin was highly expressed in both entopic and ectopic endometrium of KDBS syndrome; and myosin light chain 3 was highly expressed in both entopic and ectopic endometrium of QSBS syndrome.@*CONCLUSION@#There are differential protein expressions among the 3 EMT syndromes, which might be the inner origin of syndrome characters, and these differential proteins might be the candidate biomarkers for the pathogenesis of various EMT syndromes.

Separation and identification of differential protein in the insect-resistant and insect-susceptible mungbean / 生物工程学报

In order to find the insect-resistant composition and differentially expressed proteins of mungbean, Jinlv No.7, B20 and Weilv 2117 were used as experimental materials, and the differential proteins and functions of mungbean varieties that are resistant and susceptible to bruchids were compared and analyzed by two dimensional gel electrophoresis (2-DE) and identified by mass spectrometry. Among the samples, 15 protein spots showed a more than 2.5 times reproducible up-regulated, significance 6 of them were successfully identified by the database, and involved three kinds of proteins. They are the alpha and beta subtype 8S globulin, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBis CO) subunits binding protein and the precursor peptide chains for amylase inhibitor and trypsin inhibitor. The protein expressions of B49 (alpha subtype 8S globulin) and B31 (RuBis CO subunits binding protein) of insect-resistant mungbean were 10 000 and 23 times higher than that of insect-susceptible mungbeans. It stunted the growth and even death of the Callosobruchus chinensis L. that alpha and beta subtype 8S globulin and RuBis CO subunits binding protein and precursor peptide chains for amylase inhibitor and trypsin inhibitor of insect-resistant mungbean, the bruchid resistance effect of these three proteins need to further verified in terms of the quantity and the combined effect.

Screening and Identification of Differential Proteins of Serum in Individuals Susceptible to Noise Induced Hearing Loss in Tunnel / 听力学及言语疾病杂志

Objective To explore the differential expression of protein in the serum of individuals susceptible to noise induced hearing loss (NIHL) susceptible individuals working in the military tunnel.Methods A total of 40 soldiers from one tunnel construction troop were divided into the susceptible group and the nonsusceptible group.Twenty soldiers were selected for each group.The average age of the susceptible group was 24.79±2.03 years old and their thresholds of the speech and high frequencies were 22.43±8.31 dB HL and 48.55± 11.54 dB HL,respectively.The average age of the nonsusceptible group was 23.67 ± 3.56 years old and their thresholds of the speech and high frequencies were 13.40±4.13 dB HL and 9.40±2.54 dB HL,respectively.Five microliter peripheral venous blood samples were collected from each individual Two-dimensional electrophoresis (2-DE) and MALDI-TOF-MS were used to separate and identify the differentially expressed proteins.Results Thirty-seven protein spots differentially expressing between the NIHL susceptible and nonsusceptible were found after 2 DE.Compared by mascort score,10 differential proteins were harvested.Among these,5 peptides including proteasome subunit alpha-5,complement C4-A,haptoglobin,apolipoprotein A-I and vitronectin were upregulated,and other 5 ones,including Lysozyme C,beta-2 glycoprotein-1,pigment epithelium derived factor,35 kDa trypsin inhibitor heavy chain H and transthyretin were downregulated in NIHL susceptible individuals.The differences were statistically significant(P＜0.05).Conclusion The differentially expressed proteins were closely related to oxidative stress responses in susceptible individuals,including proteasome subunit alpha-5,complement C4A,haptoglobin,apolipoprotein A-I,beta-2 glycoprotein-1,pigment epithelium derived factor,35 kDa trypsin inhibitor heavy chain H and transthyretin.They might participate in the occurrence of NIHL through this way.The proteins harvested from this study were expected to be specific candidate serum NIHL susceptibility biomarkers in blood to help screen susceptible individuals.

Analysis of molting fluid in silkworm (Bombyx mori) by two-dimensional electrophoresis and mass spectrometry / 生物工程学报

Molting fluid, a liquid between the old epidermis and new epidermis, plays an important role in the process of ecdysis and metamorphosis for insect. In order to explore the function of molting fluid, we used two-dimensional electrophoresis to study the molting fluid during the prepupal stage and pre-eclosion stage. More than 200 protein spots were found in the molting fluid of the 2 stages, which distributed in the 4-9 of pI and 10-180 kDa of molecular weight. We selected 42 spots to be analyzed by the matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF/TOF) from the molting fluid of pre-eclosion stage, of which 34 proteins were identified successfully, including apolipoprotein, protease and protease inhibitors, chitin-binding protein and protein involved in immunity. Some of the proteins demonstrated differential expression between the two stages of metamorphosis. In order to validate the result from proteomics analysis, we studied expression of the apolipoprotein D by Q-PCR from the developmental stages. The results showed that the gene encoding apolipoprotein D had the high expression from the 1st day to the 4th day of the pupa stage, which indicated they could be involved in eclosion due to the abundant accumulation in the late pupa. Our results offered more clues for understanding the mechanism of ecdysis and metamorphosis in insect and could give reference for further study of molting fluid.

Two-dimensional electrophoresis analysis of tuberculosis pleural effusion / 实用医学杂志

Objective To analyze the different expressed protein of pleural effusion caused by tuberculosis and to identify proteins associated with tuberculous pleural effusion for building an economic , rapid, and accurate diagnostic method. Methods Two-dimensional gel electrophoresis technology was applied to separate protein in pleural effusion of 20 cases of tuberculous pleurisy ,19 cases of lung cancer patients and 6 cases of transudate. Analysis of isoelectric point, the range of molecular weight, matching rate and grey value of the protein was carried out by the PDQuest8.0 software.Then the electrophoregram was compared to get the distinct protein. Results There were 13 differential protein spots between tuberculous pleural effusion and the transudateand 9 protein spots were highly expressed for two folds, but 4 protein spots poorly expressed for 0.5 folds in pleural effusion caused by tuberculous pleurisy. There were 11 differential protein spots between tuberculous and malignant pleural effusion and 5 protein spots were highly expressed , but 4 protein spots poorly expressed in pleural effusion caused by tuberculous pleurisy , while 2 protein spots were expressed only in the pleural effusion caused by lung cancer. Conclusion Two-dimensional electrophoresis technology is available to acquire an electrophoretogram of pleural effusion caused by tuberculosis , lung cancer and transudate with well resolution and high repetition rate.In addition, there are different protein spots.

Implementación de una metodología para la separación de proteomas de plasma humano mediante electroforesis bidimensional / Implementation of a methodology to separate human plasma proteomes by two-dimensional electrophoresis / Implementação de uma metodologia para a separação do proteoma do plasma humano por eletroforese bidimensional

En este trabajo se implementaron las condiciones para la separación de proteomas de plasma sanguíneo por electroforesis bidimensional. En muestras de plasma de infante y adulto se evaluaron dos sistemas de pretratamiento de la muestra para reducir el rango dinámico de las proteínas: inmunodepleción de proteínas abundantes y enriquecimiento de proteínas de baja abundancia. Los proteomas se separaron por electroforesis bidimensional y las imágenes se analizaron con el programa Melanie 7.0. Se encontró que ambos métodos de pretratamiento fueron reproducibles y permitieron ver las diferencias en los proteomas de infante y adulto, como muestran los análisis de componentes principales y de clasificación jerárquica tipo heatmap. El porcentaje de recuperación de proteínas fue mayor con la inmunodepleción en comparación con el enriquecimiento proteico. Estos resultados permitieron concluir que con la inmunodepleción, se tiene mayor control de las proteínas eliminadas y por tanto menor pérdida de información, lo que permite su aplicación en estudios exploratorios para la identificación de potenciales biomarcadores de enfermedad.

The conditions to separate blood plasma proteomes by two-dimensional electrophoresis were implemented. In plasma samples from infant and adult two sample pretreatment systems to reduce the proteins dynamic range were evaluated: Immunodepletion of abundant proteins and protein-enrichment of low abundance proteins. Proteomes were separated by two-dimensional electrophoresis and the images were analyzed using Melanie 7.0. It was found that both pretreatment methods were reproducible and allowed to see the differences in the proteomes of infant and adult, as evidenced by the principal component analysis and heatmap, a type of hierarchic classification. The percent recovery of proteins was greater with the immunodepletion method, compared to the protein enrichment system. With these results, we conclude that reducing the complexity of plasma by immunoaffinity showed better control of unrecovered proteins and therefore less loss of information, which allows its application on exploratory studies to identify potential biomarkers of disease.

O objetivo deste trabalho foi otimizar as condições para a separação de proteomas do plasma sanguíneo por eletroforese bidimensional para sua aplicação na procura de potenciais biomarcadores. Trabalhou-se com amostras de plasma de crianças e adultos, avaliando dois sistemas de pre-tratamento da amostra para diminuir o espectro dinâmico das proteínas, imunodepleção de proteínas abundantes e enriquecimento de proteínas de baixa abundância. Os proteomas foram separados por eletroforese bidimensional e as imagens foram analisadas com o programa Melanie 7.0. Foi encontrado que ambos métodos de pre-tratamento foram reprodutíveis e permitiram observar as diferenças nos proteomas de crianças e adultos, como foram evidenciadas com as análises de componentes principais e de classificação hierárquica tipo heatmap. A porcentagem de recuperação de proteínas foi maior na imunodepleção do que obtido pelo enriquecimento proteico. Estes resultados permitiram concluir que com a imunodepleção há um controle mais eficiente das proteínas eliminadas e assim uma menor perda de informação, isto permite sua aplicação em estudos exploratórios orientados na identificação de potenciais biomarcadores da doença.

Evaluation of different protein extraction methods for banana (Musa spp.) root proteome analysis by two-dimensional electrophoresis.

Four protocols viz., the trichloroacetic acid-acetone (TCA), phenol-ammonium acetate (PAA), phenol/SDS-ammonium acetate (PSA) and trisbase-acetone (TBA) were evaluated with modifications for protein extraction from banana (Grand Naine) roots, considered as recalcitrant tissues for proteomic analysis. The two-dimensional electrophoresis (2-DE) separated proteins were compared based on protein yield, number of resolved proteins, sum of spot quantity, average spot intensity and proteins resolved in 4-7 pI range. The PAA protocol yielded more proteins (0.89 mg/g of tissues) and protein spots (584) in 2-DE gel than TCA and other protocols. Also, the PAA protocol was superior in terms of sum of total spot quantity and average spot intensity than TCA and other protocols, suggesting phenol as extractant and ammonium acetate as precipitant of proteins were the most suitable for banana rooteomics analysis by 2-DE. In addition, 1:3 ratios of root tissue to extraction buffer and overnight protein precipitation were most efficient to obtain maximum protein yield.

A Microfluidic Chip with Titanium Dioxide Membrane Enhanced Pseudo Valve Used for the Two-Dimensional Electrophoresis / 分析化学

Two-dimensional electrophoresis performed on a microfluidic chip could reduce the consumption of sample and reagents, realizing high-throughput analysis in a very short time. During the isoelectric focusing ( IEF) separation, the buffer for the two different separation methods should be kept from being mixed. After the IEF, the protein was transferred from the IEF channel into the second dimension channels. Based on the pseudo valve structure, this paper reported a method to deposit a titanium dioxide membrane at the interface of two dimensional channels, which enhanced the performance of the pseudo valve. The IEF electrophoresis and SDS-polyacrylamide gel electrophoresis ( SDS-PAGE ) procedures were characterized respectively. The total separation took 10-15 min, and the protein's migration rate was linear with the migration time, and inversely proportional to the logarithm of protein molecular weight. Based on the above results, the differential two-dimensional electrophoresis was performed to test the deviation between different measurements.

In-vitro Antibacterial Activity of Herba Taraxaci Extract on Escherichia coli / 广州中医药大学学报

Objective To evaluate the inhibitory activity of Herba Taraxaci extract on Escherichia coli DH5α (E. coli DH5α) and to investigate proteomic response of E. coli. Methods Medicinal powder of Herba Taraxaci was extracted with the solvents of different polarity ( n-hexane, ethyl acetate, distilled water) , and then the obtained 8 different extracts were subjected to thin layer chromatography ( TLC) analysis. Microdilution method was performed to detect the minimum inhibitory concentration ( MIC) of different extracts and the growth curves were described. The protein expression profiles of E . coli treated with the extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel electropheresis ( SDS-PAGE) and two dimensional electrophoresis (2-DE) . Results Water decoction of Herba Taraxaci could obviously suppress the growth of E. coli with a MIC of 1.95 mg/mL. The different extractions exhibited no antibacterial activity except ethyl acetate phase 3 with a MIC of 0.13 mg/mL, which was equal to 19.23 mg/mL of crude drugs. The results of TLC analysis showed that chlorogenic acid was undetectable in n-hexane extract and ethyl acetate phase 1 extract, and ethyl acetate phase 2 and 3 extracts showed obviously increased spots. The results of SDS-PAGE and 2-DE showed that water decoction of Herba Taraxaci had inhibitory effect on the expression of functional protein. The results of 2-DE showed that after treatment with ethyl acetate phase 3 at the concentration of 2 × MIC for 21 hours, the amount of protein spots were 92 less than those of the blank control group, the spots of E. coli DH5α soluble protein with expression amount down-regulated doubly were 24, and those with expression amount up-regulated doubly were 19. Ethyl acetate phase 3 extract had an effect on down-regulating the protein expression of E. coli DH5α soluble protein pH3-10, and water decoction of Herba Taraxaci had inhibitory effect on E. coli DH5αprotein expression. Conclusion Herba Taraxaci has significant antibacterial activity on E. coli DH5α, and the water-soluble fraction of chlorogenic acid and caffeic acid might be the active components. The possible antibacterial mechanism may be related with the regulation of bacterial protein expression.

Avoiding acidic region streaking in two-dimensional gel electrophoresis: Case study with two bacterial whole cell protein extracts.

Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost.

Microproteínas urinarias en diabéticos tipo 2 normoalbuminúricos / Urinary microproteins in normoalbuminuric type 2 diabetic patients / Microproteínas urinárias em diabéticos tipo 2 normoalbuminúricos

En pacientes diabéticos tipo 2 normoalbuminúricos se observa la presencia de Microproteínas Urinarias (MU) en el rango 68-25 kDa. El objetivo del trabajo fue identificar en distintos estadios de la nefropatía diabética si dicho rango corresponde a un marcador de daño tubular. Se estudiaron 119 orinas espontáneas de pacientes diabéticos tipo 2; se les midió la relación albúmina/creatinina urinaria y la creatinina sérica. Se dividieron en 5 grupos: 71 normoalbuminúricos, 28 microalbuminúricos, 12 macroalbuminúricos, 2 urémicos en pre-diálisis y 6 en hemodiálisis. Las MU se detectaron en geles de poliacrilamida en 2 dimensiones para uso clínico y se analizaron con el programa Image J 1.30v. La identificación de las MU se realizó por “immunoblotting” o por espectrometría de masa MALDI-TOF-TOF. El 66% de los normoalbuminúricos presentaron las siguientes MU: orosomucoide, fragmento de 35 kDa de la cadena pesada H4 del inter alfa I inhibidor de tripsina y Beta Trace, las cuales no reflejaron daño tubular debido a que la concentración de las mismas no se incrementó en los pacientes en hemodiálisis, en comparación con los normoalbuminúricos. Dichas proteínas están vinculadas al endotelio vascular y podrían constituir un marcador urinario vascular-tubular renal de utilidad clínica en patologías sistémicas con riesgo cardiovascular y funcionalidad renal conservada.

Urinary excretion of microproteins (MU) was detected in normoalbuminuric type 2 diabetic patients, in the range of 68-25 kDa using SDS-PAGE with silver staining. The purpose of this study was to identify MU in diabetic patients in different grades of diabetic nephropathy, in order to clarify the diagnostic relevance as a marker of renal tubular damage. In the spontaneous urine of 119 type 2 diabetic patients, urinary albumin, urinary creatinine and serum were determined. Five groups were formed: 71 normoalbuminuric, 28 microalbuminuric, 12 macroalbuminuric, 2 in pre-dialysis and 6 in hemodialysis. The MU were separated by two-dimensional polyacrylamid gel electrophoresis for clinical use (2D UC) and were identified by immunoblotting or MALDI-TOF-TOF mass spectrometry and analyzed using Image J version 1.30v. Of the normoalbuminurics patients studied, 66% excreted the following MU: orosomucoid, 35 kDa fragment of inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) and Beta Trace; but their concentrations did not reflect tubular damage because they exhibited a progressive downregulation. These proteins are involved in vascular endothelium, and they could be a marker of renal tubular-microvascular disease that would be useful in systemic diseases with cardiovascular risk and with preserved renal function.

Em pacientes diabéticos tipo 2 “normoalbuminúricos” se observa a presença de microproteínas urinárias (MU) em um intervalo compreendido entre 68 e 25 kDa. O objetivo do trabalho é identificar em distintos estágios da nefropatia diabética se tal intervalo corresponde a um marcador de dano tubular. Foram estudadas 119 amostras de urina espontânea de pacientes diabéticos tipo 2; foi medida a reação albumina/creatinina urinária e a creatinina sérica. Dividiram-se em 5 grupos: 71 normoalbuminúricos, 28 microalbuminúricos, 12 macroalbuminúricos, 2 urêmicos pré-dialise e 6 em hemodiálise. Foram detectadas as MU em géis de poliacrilamida em duas dimensões para o uso clínico e analisadas com o programa Image J versão 1.30v. A identificação das MU foi realizada por “immunoblotting” ou por espectrometria de massa MALDI-TOF-TOF. 66% de normoalbuminúricos apresentaram as seguintes MU: orosomucoide, Fragmento de 35 kDa da cadeia pesada H4 do interalfa inibidor da tripsina e Beta Trace, as quais não mostraram dano tubular devido a que a concentração das mesmas não está aumentada nos pacientes em hemodiálise, em comparação com os normoalbuminúricos. Estas proteínas estão envolvidas com o endotélio vascular e poderiam constituir um marcador urinário vascular-tubular renal de utilidade clínica em patologias sistêmicas com risco cardiovascular e funcionalidade renal conservada.

Proteomic fingerprinting of N-linked glycoproteins involved in hepatocellular carcinoma / 解剖学报

Objective To identify differentially expressed N-linked glycoproteins between hepatocellular carcinoma ( HCC) and adjacent non-tumorous liver tissues .Methods N-linked glycoproteome was extracted by multi-lectin affinity chromatography comprising concanavalin A (ConA), lentil lectin (LCH), and snowdrop lectin (GNA) and subsequently subjected to two-dimensional electrophoresis ( 2DE ) and mass spectrometry ( MS ) for identification of differential glycoproteins between 10 pairs of HCC and adjacent non-cancer tissue .Western blotting was used to verify different expression of human liver carboxylesterase 1 (hCE1), haptoglobin (HP)and cathepsin D (CD).Invasion potential in vitro was examined after si-RNA mediated CD gene scilencing .Results LC-ESI-MS/MS identified a total of 28 differentially expressed glycoproteins (14 up-regulation and 14 down-regulated).Western blotting detected consistent down-regulation of hCE1 and HP, and up-regulation of pro-cathepsin D (pCD) in HCC.Up-regulation of ConA-binding CD (ConA-CD), however , was verified in HCC only after ConA-CD enrichment by ConA chromatography .Down-regulation of CD expression mediated by CD-siRNA markedly inhibited the in vitro invasive potential of SNU449 and SNU473.Conclusion Dysregulation of HP , hCE1 expression and alteration of glycans linked to CD may play crucial roles in pathogenesis of HCC.

Comparative Study on Naotaifang Containing Serum and Plasma Proteomics (Peptide) by Proteomics / 世界科学技术-中医药现代化

Through comparative study on Naotaifang containing serum and plasma proteomics (peptide), this article revealed differential proteins (peptides) in the Naotaifang. The characteristics of differential proteins were identified with mass spectrometry. It provides scientific evidences for the pharmacodynamic material basis and Chinese herbal medicine plasma pharmacological method development in the exploration of Naotaifang. A total of 20 healthy adult SD rats were randomly divided into the control group, Naotaifang treatment group according to their weights. Ten rats in each group. Intragastric administration of medication was given for seven consecutive days. Before surgery, rats were fed with water but without food. One hour after the last drug administration, 10% chloral hydrate was injected for intraperitoneal anesthesia. Blood was taken through the common carotid artery. Serum and plasma samples were made after blood was taken from each rat. Serum and plasma samples of five rats were randomly selected from each group. And the two-dimensional electrophoresis (2-DE) technique was used in the comparative study of serum pro-teomics (peptide). The 300 DPI scanning and PDQuest 7.3.0 were used in the analysis. The ESI-MS/MS was used to identify important differences in proteins and screen characteristic serum and plasma protein. The results showed that 20 differential proteins of 5 plasma samples were identified. There were 15 types of proteins expressing up-regulation and 5 types expressing down-regulation. Comparative analysis on the 2-DE gel pictures of Naotaifang containing serum, 19 differential proteins of 5 plasma samples were identified, among which 15 types of proteins express up-regulation and 4 down-regulation. Comparative analysis on the 2-DE gel pictures of Naotaifang containing serum and Naotaifang containing plasma showed that 24 differential proteins of 5 plasma samples were identified, among which 9 types of proteins express up-regulation and 15 down-regulation. The highly expressed proteins were selected to MALDI-TOF-MS between Naotaifang containing serum and Naotaifang containing plasma. There were six successful-ly identified proteins, which were inter-alpha trypsin inhibitor, heavy chain 3, group-specific component, comple-ment factor B, Receptor Complexed with A Heterodimeric Fc, isoform CRA-d, Transferrin. It was concluded that protein with obvious changes in the Naotaifang containing serum and plasma may be related with fibrinolysis and an-ticoagulant. These proteins are involved in angiogenesis, inflammation and other pathological regulations of physiolog-ical processes. They are of great significance in the study of effective target and its signal transduction pathway of Naotaifang.

Protein expression in silica dust-induced transdifferentiated rats lung fibroblasts / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To analyze the expression of different proteins in free silica-induced transdifferentiated rat lung fibroblasts.</p><p><b>METHODS</b>Rat lung fibroblasts and alveolar macrophages were cultured. A transdifferentiation model of rat lung fibroblasts was established. Free silica was used as a stimulator for rat lung fibroblasts. Changes in α-SMA were detected by immunohistochemistry and Western blot, respectively. Protein of lung fibroblasts was extracted and analyzed by two-dimensional electrophoresis (2-DE).</p><p><b>RESULTS</b>Six protein spots were identified by mass spectrometry, including glyceraldehyde 3-phosphate-dehydrogenase, peroxiredoxin 5, heterogeneous nuclear ribonucleoprotein A2, transgelin 2, keratin K6 and vimentin.</p><p><b>CONCLUSION</b>Some proteins are changed in free silica-induced transdifferentiated rat lung fibroblasts.</p>

Difference analysis of proteome between diabetic cataract and age related cataract / 中华实验眼科杂志

Background With the changes of diet and living style,the diabetes has become the major diseases affecting human health.Diabetic cataract is a common complication of diabetes. Objective The present study was to investigate the difference of lens proteomics between diabetic cataract and age related cataract using two dimensional electrophoresis (2-DE) and mass spectrometry in order to postpone happening of diabetic cataract and offer the effective approach to the prevention and therapy of diabetic cataract. Methods The lenses were obtained from 8 diabetic patients and 12 age-related cataract patients during the surgery to extract the protein by lysis and centrifugation.The lens proteins were separated using immobilized pH gradients 2-DE.Image analysis was carried out using PDQuest Advanced-8.0.1 software package.Significant difference of the crystallines was identified by matrixassisted laser adsorption/ionization time of-flight-mass spectrometry (MALDI-TOF-MS) and peptide mass fingerprint combined with protein database. Results The maps of 2-DE showed that lens proteins of diabetic cataract and age related cataract were in the section of pH 5-9 with the relative molecular weight 14000-97000;while relative molecular weight of more abundant crystalline was localized at 20000-31000.About 3 differential protein spots were detected by image analysis software.Two crystallines,αB and βB1 crystallin,were identified using MALDI-TOF-MS.Conclusions Proteomic analysis of lens can be accomplished and the proteins can be well separated,moreover,differential proteins can be analyzed using 2-DE and mass spectrometry between diabetic cataract and age related cataract.These results indicate that αB and βB1 crystallin proteins accelerate the development of diabetic cataract.This technique offers a new avenue for clarity of lens proteins of diabetic cataract other than age related cataract.

STGC3 inhibits xenograft tumor growth of nasopharyngeal carcinoma cells by altering the expression of proteins associated with apoptosis

STGC3 is a potential tumor suppressor that inhibits the growth of the nasopharyngeal carcinoma cell line CNE2; the expression of this protein is reduced in nasopharyngeal carcinoma compared with normal nasopharyngeal tissue. In this study, we investigated the tumor-suppressing activity of STGC3 in nude mice injected subcutaneously with Tet/pTRE-STGC3/CNE2 cells. STGC3 expression was induced by the intraperitoneal injection of doxycycline (Dox). The volume mean of Tet/pTRE-STGC3/CNE2+Dox xenografts was smaller than that of Tet/pTRE/CNE2+Dox xenografts. In addition, Tet/pTRE-STGC3/CNE2+Dox xenografts showed an increase in the percentage of apoptotic cells, a decrease in Bcl-2 protein expression and an increase in Bax protein expression. A proteomic approach was used to assess the protein expression profile associated with STGC3-mediated apoptosis. Western blotting confirmed the differential up-regulation of prohibitin seen in proteomic analysis. These results indicate that overexpression of STGC3 inhibits xenograft growth in nude mice by enhancing apoptotic cell death through altered expression of apoptosis-related proteins such as Bcl-2, Bax and prohibitin. These data contribute to our understanding of the function of STGC3 in human nasopharyngeal carcinoma and provide new clues for investigating other STGC3-associated tumors.