Proteomic analysis of blood level of proteins before and after operation in patients with esophageal squamous cell carcinoma at high-incidence area in Henan Province

AIM:To characterize the protein files in blood from same patients with esophageal squamous cell carcinoma(ESCC) before and after operation at the high-incidence area for ESCC in Henan Province,China. METHODS:Two-dimensional electrophoresis,silver staining and ImageMaster 2-DE analysis software were applied to the determination of protein files in the blood obtained from normal controls and ESCC patients before and after operation. RESULTS:A total of 655,662 and 677 protein spots were identified,respectively,from the normal controls and ESCC patients before and after operation.No significant difference in Me number of protein spots was observed between Me normal group and ESCC patients.A total of seven protein spots were identified with a dramatic difference among the samples before and after operation.Six protein spots were up-regulated and one protein spot was down-regulated in the group after operation compared with those in normal and before operation.Three protein spots were further characterized by matrix-assisted laser desorption/ionization time of flying mass spectrometry(MALDI-TOF-MS).The proteins from these three spots were identified as serum amyloid A (SAA),amyloid related serum protein and haptoglobin. CONCLUSION:Serum amyloid A,amyloid related serum protein and haptoglobin may be related with ESCC and/or surgery.The significance of these proteins needs to be further characterized.The present study provides informative data for the establishment of serum protein profiles related with ESCC.     

Proteomic analysis of hippocampus in the rat

Objective To analyze the protein expression in the rat hippocampus by the proteomic approach. Methods Proteins from hippocampal tissue homogenates of the rat were separated by two-dimensional gel electrophoresis (2-DE), and stained with colloidal Coomassie blue to produce a high-resolution map of the rat hippocampus proteome. Selected proteins from this map were digested with trypsin, and the resulting tryptic peptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) The mass spectrometric data were used to identify the proteins through searches of the NCBI protein sequence database. Results 37 prominent proteins with various functional characteristics were identified. The identified brain protein classes covered metabolism enzymes, cytoskeleton proteins, heat shock proteins, antioxidant proteins,signalling proteins, proteasome-related proteins, neuron-specific proteins and glial-associated proteins. Furthermore,3 hypothetical proteins, unknown proteins so far only proposed from their nucleic acid structure, were identified.Conclusion This study provides the first unbiased characterization of proteins of the rat hippocampus and will be used for future studies of differential protein expression in rat models of neurological disorders.     

Comparative proteomics analysis of human gastric cancer

AIM： To isolate and identify differentially expressed proteins between cancer and normal tissues of gastric cancer by two-dimensional electrophoresis （2-DE） and matrix-assisted laser desorption/ionization time-of- flight mass spectrometry （MALDI-TOF-MS）. METHODS： Soluble fraction proteins of gastric cancer tissues and paired normal tissues were separated by 2-DE. The differentially expressed proteins were selected and identified by MALDI-TOF-MS and database search. RESULTS： 2-DE profiles with high resolution and reproducibility were obtained. Twenty-three protein spots were excised from sliver staining gel and digested in gel by trypsin, in which fifteen protein spots were identified successfully. Among the identified proteins, there were ten over-expressed and five under-expressed proteins in stomach cancer tissues compared with normal tissues. CONCLUSION： In this study, the well-resolved, reproducible 2-DE patterns of human gastric cancer tissue and paired normal tissue were established and optimized and certain differentially-expressed proteins were identified. The combined use of 2-DE and MS provides an effective approach to screen for potential tumor markers.     

Comparative study of proteome between primary cancer and hepatic metastatic tumor in colorectal cancer

AIM: To identify the differential proteins associated with colorectal cancer genesis and hepatic metastasis.METHODS: Hydrophobic protein samples were extracted from normal colorectal mucosa, primary cancer lesion and hepatic metastatic foci of colorectal cancer. With twodimensional electrophoresis and image analysis,differentially expressed protein spots were detected, and the proteins were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and peptide mass fingerprint analysis.RESULTS: Significant alterations of the proteins in number and expression levels were discovered in primary cancer and hepatic metastatic foci, the expression of a number of proteins was lost in 25-40 ku, but protein spots was increased in 14-21ku, compared with normal mucosa. Nine differentially expressed protein spots were identified. Three proteins expressed in normal mucosa, but lost in primary cancer and hepatic metastasis, were recognized as calmodulin, ribonuclease 6 precursor and mannosidase-α.Proapolipoprotein was expressed progressively from normal mucosa to primary cancer and hepatic metastasis. The differentially expressed protein of beta-globin was found in normal mucosa and hepatic metastatic tumor, but lost in primary cancer lesion. Cdc 42, a GTP-binding protein, was identified in hepatic metastasis. The protein spots of C4 from primary cancer, M7 and M9 from hepatic metastasis had less homology with the proteins in database.CONCLUSION: Variations of hydrophobic protein expression in colorectal cancer initiation and hepatic metastasis are significant and can be observed with two-dimensional electrophoresis. The expression of calmodulin, ribonuclease 6 precursor and mannosidase-α is lost but the expression of proapolipoprotein is enhanced which is associated with colorectal cancer genesis and hepatic metastasis. Cdc 42 and beta-globin are expressed abnormally in hepatic metastasis. Protein C4, M7 and M9 may be associated with colorectal cancer genesis and hepatic metastasis.     

Mutation of cytotoxin-associated gene A affects expressions of antioxidant proteins of Helicobacter pylori

AIM： To determine if disruption of the cagA gene of Helicobacter pylori （H pylori） has an effect on the expression of other proteins at proteome level. METHODS： Construction of a cagA knock out mutant Hp27_△cagA （cagA） via homologous recombination with the wild-type strain Hp27 （cagA＋） as a recipient was performed. The method of sonication-urea-CHAPS-DTT was employed to extract bacterial proteins from both strains. Soluble proteins were analyzed by two-dimensional electrophoresis （2-DE）. Images of 2-DE gels were digitalized and analyzed. Only spots that had a statistical significance in differential expression were selected and analyzed by matrix-assisted laser desorption/ionization- time of flight mass spectrometry （MALDI-TOF-MS）. Biological information was used to search protein database and identify the biological function of proteins. RESULTS： The proteome expressions between wild-type strain and isogenic mutant with the cagA gene knocked-out were compared. Five protein spots with high abundance in bacteria proteins of wild-type strains, down-regulated or absently expressed in bacteria proteins of mutants, were identified and analyzed. From a quantitative point of view, the identified proteins are related to the cagA gene and important antioxidant proteins of Hpylori, including alkyl hydroperoxide reductase （Ahp）, superoxide dismutase （SOD） and modulator of drug activity （Mda66）, respectively, suggesting that cagA is important to maintain the normal activity of antioxidative stress and ensure H pylori persistent colonization in the host. CONCLUSION： cagA gene is relevant to the expressions of antioxidant proteins of H pylori, which may be a novel mechanism involved in H pylori cagA pathogenesis.     

Differentially expressed proteins of gamma-ray irradiated mouse intestinal epithelial cells by two-dimensional electrophoresis and MALDI-TOF mass spectrometry

AIM: To identify the differentially expressed proteins involved in ionizing radiation in mice and to explore new ways for studying radiation-related proteins.METHODS: Bal B/c mice grouped as sham-irradiation, 3h and 72 h irradiation were exposed to 9.0Gy single dose of γ-irradiation. Intestinal epithelia were isolated from mice,and total proteins were extracted with urea containing solution. A series of methods were used, including twodimensional electrophoresis, PDQuest 2-DE software analysis, peptide mass fingerprinting based on matrixassisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and SWISS-PROT database searching, to separate and identify the differential proteins.Western blotting and RT-PCR were used to validate the differentially expressed proteins.RESULTS: Mouse intestine was severely damaged by 9.0 Gy γ-irradiation. Tmage analysis of two-dimensional gels revealed that averages of 638±39, 566±32 and 591±29 protein spots were detected in 3 groups, respectively, and the majority of these protein spots were matched. About 360 protein spots were matched between normal group and 3 h irradiation group, and the correlation coefficient was 0.78 by correlation analysis of gels. Also 312 protein spots matched between normal group and 72 h irradiation group, and 282 protein spots between 3 h and 72 h irradiation groups. Twenty-eight differential protein spots were isolated from gels, digested with trypsin, and measured with MALDI-TOF-MS. A total of 25 spots yielded good spectra, and 19 spots matched known proteins after database searching. These proteins were mainly involved in anti-oxidation, metabolism, signal transduction,and protein post-translational processes. Western-blotting confirmed that enolase was up-regulated by γ-irradiation. Upregulation of peroxiredoxin ! was verified by applying RTPCR technique, but no change occurred in Q8VC72.CONCLUSION: These differentially expressed proteins might play important roles when mouse intestine was severely injured by γ-irradiation. It is suggested that differential proteomic analysis may be a useful tool to study the proteins involved in radiation damage of mouse intestinal epithelia.     

应用蛋白质组学技术筛检肝癌血清差异蛋白质并分离鉴定阿朴脂蛋白AⅡ

Objective To screen differential proteins in serum from hepatocellular carcinoma (HCC) patients by Proteomic Technology and to purify and identify them. Methods Surface enhanced laser desorp-tion Ionization time of flight-mass spectrum (SELDI-TOF-MS) was employed to screen differential proteins in serum from 33 HCC patients and 33 control cases, and then to purify and identify them using isoelectric precipitation, Tricine sodium dodecyl sulphate ployacrylamide gel electrophoresis (Tricine-SDS-PAGE) and high performance liquid chromatography tandem Mass Spectrum (HPLC-MS). Result 65 protein peaks in the range of relative molecular weight from 2000 to 10 000 were found significant difference (P < 0.05) between the patient group and control group. Based on these differential protein peaks, diagnostic model for HCC detection was established and its sensitivity and specificity were 100% and 96.97 % respectively. Proteins with 8706.5 and 8579.2 relative molecular weights (the t value was 2.562 and 2.783 respectively, and P value was 0.013 and 0.015 respectively) out of the 65 differential proteins were purified and identified, and then recognized as Apolipoprotein A Ⅱ (Apo AⅡ). Conclusion Apo A II is probably a differential protein of HCC and maybe related to the pathogenesis of HCC.     

应用蛋白质组学技术筛检肝癌血清差异蛋白质并分离鉴定阿朴脂蛋白AⅡ

Objective To screen differential proteins in serum from hepatocellular carcinoma (HCC) patients by Proteomic Technology and to purify and identify them. Methods Surface enhanced laser desorp-tion Ionization time of flight-mass spectrum (SELDI-TOF-MS) was employed to screen differential proteins in serum from 33 HCC patients and 33 control cases, and then to purify and identify them using isoelectric precipitation, Tricine sodium dodecyl sulphate ployacrylamide gel electrophoresis (Tricine-SDS-PAGE) and high performance liquid chromatography tandem Mass Spectrum (HPLC-MS). Result 65 protein peaks in the range of relative molecular weight from 2000 to 10 000 were found significant difference (P < 0.05) between the patient group and control group. Based on these differential protein peaks, diagnostic model for HCC detection was established and its sensitivity and specificity were 100% and 96.97 % respectively. Proteins with 8706.5 and 8579.2 relative molecular weights (the t value was 2.562 and 2.783 respectively, and P value was 0.013 and 0.015 respectively) out of the 65 differential proteins were purified and identified, and then recognized as Apolipoprotein A Ⅱ (Apo AⅡ). Conclusion Apo A II is probably a differential protein of HCC and maybe related to the pathogenesis of HCC.     

Differences in expression of retinal proteins between diabetic and normal rats

AIM： To compare and identify the differences in expression of retinal proteins between normal and diabetic rats, and to analyze the molecular pathogenetic mechanisms of retinal diseases caused by diabetes.
METHODS： Changes in protein expression of retinal tissues from diabetic and normal rats were observed using 2-dimensional polyacrylamide gel electrophoresis （2-DE）. Some protein spots exhibiting statistically significant variations （P 〈 0.05） were selected randomly and identified by tandem mass spectrometry and analyzed by bioinformatics.
RESULTS： 2-DE showed that the expression was upregulated in 5 retinal proteins, down-regulated in 23retinal proteins, and disappeared in 8 retinal proteins.Eight spots were identified from the 36 spots by tandem mass spectrometry （MS/MS） and analyzed by bioinformatics. Guanylate kinase 1, triosephosphate isomerase 1, ATP synthase subunit d, albumin and dimethylarginine dimethylaminohydrolase 2 played an important role in signal transduction. Triosephosphate isomerase 1, crystallin alpha B, ATP synthase subunit d and peroxiredoxin 6 were involved in energy metabolism of retinal tissues. Guanylate kinase 1 played an important role in photoexcitation of retinal rod photoreceptor cells.Whether crystallin beta A1 plays a role in diabetic retinas is unknown so far.
CONCLUSION： There are differences in expression of retinal proteins between diabetic and normal rats.These proteins may be involved in the mechanisms and prognosis of retinal diseases caused by diabetes.     

Comparative study of proteome between primary cancer and hepatic metastatic tumor in colorectal cancer

AIM:To identify the differential proteins associated withcolorectal cancer genesis and hepatic metastasis.METHODS:Hydrophobic protein samples were extractedfrom normal colorectal mucosa,primary cancer lesion andhepatic metastatic foci of colorectal cancer.With two-dimensional electrophoresis and image analysis,differentially expressed protein spots were detected,andthe proteins were identified by matrix assisted laserdesorption/ionization-time of flight-mass spectrometry andpeptide mass fingerprint analysis.RESULTS:Significant alterations of the proteins in numberand expression levels were discovered in primary cancerand hepatic metastatic foci,the expression of a number ofproteins was lost in 25-40 ku,but protein spots wasincreased in 14-21ku,compared with normal mucosa.Ninedifferentially expressed protein spots were identified.Threeproteins expressed in normal mucosa,but lost in primarycancer and hepatic metastasis,were recognized ascalmodulin,ribonuclease 6 precursor and mannosidase-αProapolipoprotein was expressed progressively from normalmucosa to primary cancer and hepatic metastasis.Thedifferentially expressed protein of beta-globin was found innormal mucosa and hepatic metastatic tumor,but lost inprimary cancer lesion.Cdc 42,a GTP-binding protein,wasidentified in hepatic metastasis.The protein spots of C4from primary cancer,M7 and M9 from hepatic metastasishad less homology with the proteins in database.CONCLUSION:Variations of hydrophobic protein expressionin colorectal cancer initiation and hepatic metastasis aresignificant and can be observed with two-dimensionalelectrophoresis.The expression of calmodulin,ribonuclease6 precursor and mannosidase-α is lost but the expressionof proapolipoprotein is enhanced which is associated withcolorectal cancer genesis and hepatic metastasis.Cdc 42and beta-globin are expressed abnormally in hepaticmetastasis.Protein C4,M7 and M9 may be associated withcolorectal cancer genesis and hepatic metastasis.     

State of knowledge of helminth fauna of freshwater fishes of Poland

A total 89 fish and lamprey species has been recorded from Polish freshwater habitats. Twenty-seven of them (30.3%) have not been surveyed for parasitic helminthes. Some of the latter fishes are either rare or not easily accessible. Other live only in specific habitats in scattered localities. An important obstacle for studying parasite faunas of some fishes may be their status on an endangered species. Among the non-surveyed fishes, are those which have been relatively recently introduced to Poland or migrated there on their own. The present paper attempts to review all hitherto not studied helminthologically fish species, their habitats, localities and current protection status.     

高血压降压治疗目标的再认识

根据传统的高血压水平的定义,1993年WHO高血压治疗指南提出血压控制目标为<140/90mm Hg(1mm Hg=0.133kPa),但是并非所有患者都必须将血压降至同一水平,而应根据患者情况进行个体化治疗。Framingham进行的一项长达10～12年的心血管事件研究发现,第5年后,正常上限血压[收缩压(SBP     

Lack of correlation of degree of villous atrophy with severity of clinical presentation of coeliac disease

BACKGROUND AND AIM: Both the clinical presentation and the degree of mucosal damage in coeliac disease vary greatly. In view of conflicting information as to whether the mode of presentation correlates with the degree of villous atrophy, we reviewed a large cohort of patients with coeliac disease. PATIENTS AND METHODS: We correlated mode of presentation (classical, diarrhoea predominant or atypical/silent) with histology of duodenal biopsies and examined their trends over time. RESULTS: The cohort consisted of 499 adults, mean age 44.1 years, 68% females. The majority had silent coeliac disease (56%) and total villous atrophy (65%). There was no correlation of mode of presentation with the degree of villous atrophy (p=0.25). Sixty-eight percent of females and 58% of males had a severe villous atrophy (p=0.052). There was a significant trend over time for a greater proportion of patients presenting as atypical/silent coeliac disease and having partial villous atrophy, though the majority still had total villous atrophy. CONCLUSIONS: Among our patients the degree of villous atrophy in duodenal biopsies did not correlate with the mode of presentation, indicating that factors other than the degree of villous atrophy must account for diarrhoea in coeliac disease.