Morphological and pathologic changes of experimental chronic atrophic gastritis (CAG)and the regulating mechanism of protein expression in rats

Objective： To study the pathologic change and molecular regulation in cell proliferation and apoptosis of gastric mucosa in rats with chronic atrophic gastritis （CAG）, and evaluate the possible mechanisms. Methods： Rats were administered with 60% alcohol or 2% salicylate sodium, 20 mmol/L deoxycholate sodium and 0.1% ammonia water to establish chronic atrophic gastritis （CAG） models. The gastric specimens were prepared for microscopic view with hematoxylin and eosin （H-E） and alcian blue （A-B） stain. The number of infiltrated inflammatory cells, the thickness of the mucosa gland layer （μm） and the number of gastric glands were calculated. The damage of barrier in mucosa with erosion or ulceration, and the thickness of mucin were examined by scanned electron microscope （SEM）. The levels of PGE2, EGF （epiderminal growth factor） and gastrin in the serum were measured with radioimmunoassay or ELISA method. The immunohistochemistry method was used to observe the number of G cells, the expression of protein of EGFR （EGF receptor）, C-erbB-2, p53, p6 and bcl-2 in gastric tissue. Results： Under SEM observation, the gastric mucosa was diffused erosion or ulceration and the thickness of mucin was decreased. Compared with normal rats, the grade of inflammatory cell infltration in CAG rats was elevated, whereas the thickness and number of gastric gland were significantly lower （P〈0.05）. Compared with normal level of （0.61±0.28） μg/L, EGF in CAG （2.24±0.83） μg/L was significantly higher （P〈0.05）. The levels of PGEz and gastrin in serum were significantly lower in CAG rats than that in normal rats （P〈0.05）. Immunohistochemistry detection showed that the number of G cell in antrum was lower in CAG group （P〈0.05）. Imrauno-stain showed EGFR protein expression in the basal and bilateral membrane, and the cytoplasma in atrophic gastric gland, while negative expression was observed in normal gastric epithelial cells. Positive staining of p53 and p 16 protein was localized in the nucleus of epithelial cells. The former was higher positively expressed in atrophic gland, while the later was higher positively stained in normal gastric tissue, bcl-2 protein was positively stained in the cytoplasma in atrophic gastric gland, while very weakly stained in normal gastric tissue. Conclusion： The pathological findings in gastric gland accorded with the Houston diagnostic criteria of antrum-predominant CAG. CAG in rats was related with the damage of barrier in gastric mucosa and the misbalance of cell proliferation and apoptosis. There was high protein expression of oncogene, while inhibitor of suppressor gene in CAG rats indicated high trend of carcinogenesis.

Association of gene and protein expression and genetic polymorphism of CC chemokine ligand 4 in colorectal cancer

BACKGROUND Leukocytes,such as T cells and macrophages,play an important role in tumorigenesis.CC chemokine ligand(CCL)4,which is produced by lymphocytes and macrophages,has been found to be expressed in the mucosa of the gastrointestinal tract and is a potent chemoattractant for various leukocytes.AIM To examine CCL4 expression and its genetic polymorphism rs10491121 in patients with colorectal cancer(CRC)and evaluate their prognostic significance.METHODS Luminex technology was used to determine CCL4 Levels in CRC tissue(n=98),compared with paired normal tissue,and in plasma from patients with CRC(n=103),compared with healthy controls(n=97).Included patients had undergone surgical resection for primary colorectal adenocarcinomas between 1996 and 2019 at the Department of Surgery,Ryhov County Hospital,Jönköping,Sweden.Reverse transcription quantitative PCR was used to investigate the CCL4 gene expression in CRC tissue(n=101).Paired normal tissue and TaqMan single nucleotide polymorphism assays were used for the CCL4 rs10491121 polymorphism in 610 CRC patients and 409 healthy controls.RESULTS The CCL4 protein and messenger RNA expression levels were higher in CRC tissue than in normal paired tissue(90%,P<0.001 and 45%,P<0.05,respectively).CRC tissue from patients with localized disease had 2.8-fold higher protein expression levels than that from patients with disseminated disease.Low CCL4 protein expression levels in CRC tissue were associated with a 30%lower cancer-specific survival rate in patients(P<0.01).The level of plasma CCL4 was 11%higher in CRC patients than in healthy controls(P<0.05)and was positively correlated(r=0.56,P<0.01)with the CCL4 protein level in CRC tissue.The analysis of CCL4 gene polymorphism rs10491121 showed a difference(P<0.05)between localized disease and disseminated disease in the right colon,with a dominance of allele A in localized disease.Moreover,the rate of the A allele was higher among CRC patients with mucinous cancer than among those with nonmucinous cancer.CONCLUSION The present study indicates that the CRC tissue levels of CCL4 and CCL4 gene polymorphism rs10491121,particularly in the right colon,are associated with clinical outcome in CRC patients.

Loss of fragile histidine triad protein expression in inflammatory bowel disease

AIM： To investigate the expression of fragile histidine triad （FHIT） protein in 64 patients with ulcerative colitis （UC） and Crohn＇s disease （CD）, and its relation with clinicopathological data. METHODS： Rabbit-anti-FHIT antibody was used to detect FHIT protein expression in 64 formalin-fixed, paraffin-embedded tissue specimens of inflammatory bowel disease （IBD） by citrate-microwave-streptavidin （SP）-HRP immunohistochemical method. RESULTS： The positive FHIT protein expression was 22.79% ± 16.16%, 42.14% ± 16.82% in active and remittent phases of UC, 36.07% ± 19.23% in CD, and 57.05% ±8.86% in normal colon mucosa. Statistically significant differences in FHIT protein expression were observed between the active and remittent phases of UC, between the active phase of UC and normal colon mucosa, as well as between the remittent phase of UC and normal colon mucosa, and between CD and normal colon mucosa. CONCLUSION： Our results show that FHIT protein expression is completely absent or reduced in IBD, suggesting that the FHIT gene might be associated with the oncogenesis and progression of IBD, an early event from inflammatory conditions to carcinoma in IBD.

HER2 gene status and the relationship with p21 protein expression in gastric cancer

Objective： We aimed to analysis the HER2 gene status and its relationship with p21 protein expression in gastric carcinoma. Methods： Fluorescence in situ hybridisation （FISH） and immunohistochemistry （IHC） techniques were used to detect HER2 gene status and p53 protein in 59 cases of gastric cancer. Results： FISH detection of HER2 gene amplification rate was 16.9% （10/59）, HER2 gene amplification in 49 cases without copy number gain and gene amplification were a total of 49.2% （29/59）. HER2 protein expression was 42.4% （25/59）, HER2 gene amplification rates in patients with ＋＋＋, ＋＋ HER2 protein expression were 3/3 and 5/8, while in patients with ＋ HER2 protein expression, it was 2/14, there was significant difference （P 0.05）. p21 protein expression rate was 49.2% （29/59）, HER2 gene amplification rates and p21 protein expression had significant difference in tumor invasion depth, lymph node metastasis （P 0.05）; had no statistical significance in histological type, age, gender differences （P 0.05）. Conclusion： HER2 gene amplification rate and gene copy number had positively correlation with p21 protein expression, HER2 gene status and expression of p21 protein combined detection can provide a reference value in gastric cancer metastasis, patient’s condition development and prognosis, it also can guide clinical development of individual treatment.

Oncoprotein expression and inhibition of apoptosis during colorectal tumorigenesis

AIMS To study bcl-2 and P53 protein expression and inhibition of apoptosis during colorectal tumorigenesis. METHODS Expression of bcl -2 and p53 in 45 colorectal ade- nomas and 61 colorectal carcinomas was detected by immunohis- tochemical staining. RESULTS The bcl-2 and P53 protein expression was uniformly negative in normal mucosa,whereas bcl-2 and p53 positive rates were significantly higher in adenoma and carcinoma than in nor- reals(P<0.01 ).The area with strong bcl-2 expression was of- ten the area with severely dysplasia.In colorectal adenoma,ex- pression of p53 increased with the increasing size and dysplasia, in adenomas≥20 mm being higher than adenomas<10 mm(77, 8% vs 35.0%,P<0.05).p53 was relevant to differentiation and Duke's staging.A significant inverse correlation was found between bcl-2 and p53 in immunostaining in the adenomas,but not in the carcinomas.Furthermore,carcinomas with a high per- centage of bcl-2 positive cells were significantly more likely to have low rates of apoptosis. CONCLUSIONS These results suggest that bcl-2 gene appears to be an early event in colorectal tumorigenesis that can inhibit apoptosis,p53 expression plays an important role in the develop- ment and malignant change of colorectal adenoma,bcl-2 and p53 may be used as a good marker relating to cell apoptosis.

Differential protein expression in rat cortical astrocytes following fluid percussion injury Two-dimensional gel electrophoresis and mass-spectrum detection

BACKGROUND： The Glasgow Coma Scale, computer tomography, and nuclear magnetic resonance imaging have been frequently used to diagnose brain injury. However, these methods do not accurately and quantitatively evaluate injury degree. However, proteomics displays some advantages. To date, there are few proteomics studies based on primary astrocyte cultures from a fluid percussion injury model. OBJECTIVE： To detect differential protein expression in rat cerebral cortical astrocytes following fluid percussion injury using two-dimensional gel electrophoresis and mass spectrum and to determine specific biological markers of brain injury. DESIGN, TIME AND SETTING： Complete, randomized grouping and proteomics experiments were performed at the Molecular Pathological Laboratory, Central Laboratory and Tianjin Key Laboratory for Biomarkers of Occupational and Environmental Hazard of Medical College of Chinese People＇s Armed Police Force from October 2007 to May 2008. MATERIALS： Inverted phase-contrast microscope was purchased from Olympus, Japan. PROTEAN IEF Cell isoelectric focusing electrophoresis system and PROTEAN II Xi-Cell vertical electrophoresis system were purchased from Bio-Rad, USA. Autofiex MALDI-TOF mass spectrometer was purchased from Bruker, Germany. METHODS： A total of 90 culture dishes, fully coated with Sprague Dawley rat cortical astrocytes, were randomly divided into control （n = 30） and injury （n = 60） groups. Astrocytes in the injury group were subjected to fluid percussion and subdivided into 4-hour （n = 30） and 48-hour injury （n = 30） groups. MAIN OUTCOME MEASURES： Cell morphology was observed using inverted phase-contrast microscopy. Cell total protein was extracted from each group, followed by two-dimensional gel electrophoresis and silver staining, and the differential protein expression was analyzed using PDQuest 7.0 software. Protein peptide mass fingerprinting of differential protein spots was obtained by matrix assisted laser desorption/ionization-time of flight mass spectrometry. The National Center for Biotechnology Information （NCBI） protein database was retrieved by Mascot to primarily identify protein type, Finally, differential protein expression was detected by Western blot analysis. RESULTS： Following fluid percussion injury, astrocytes displayed obvious swelling and increased intercellular space, with some cell detachment; the number of dead cells was significantly greater than the control group （P 〈 0.05）. Expression intensity of 114 protein spots was significantly greater in the injury group compared with the control group （P〈 0.05）; 9 of the 114 protein spots were identified and peptJde matching scores of 8 spots were 〉 61 （P 〈 0.05）. Protein types were identified and included cellular retinol binding protein, brain fatty acid binding protein 7, $100 calcium binding protein All, 60S acidic ribosomal protein P2, calponin 3, breast carcinoma amplified sequence 2 homolog, eukaryotic translation initiation factor 1A, and hypothetical protein LOC685814. Western blot detection revealed brain fatty acid binding protein 7 expression in cortical astrocytes, which increased with injury time compared with the control group （P 〈 0.05）. CONCLUSION： Results from this study showed morphological and proteomic changes in cortical astrocytes following fluid percussion injury. Brain fatty acid binding protein 7 was expressed in astrocytes and possibly played an important role in injury repair. Mass-spectrum identified differentially expressed proteins that correlated with cell metabolism regulation, signal transduction, and translation initiation, and could serve as specific biological markers of brain injury.

Protein expression of sensory and motor nerves Two-dimensional gel electrophoresis and mass spectrometry

The present study utilized samples from bilateral motor branches of the femoral nerve, as well as saphenous nerves, ventral roots, and dorsal roots of the spinal cord, to detect differential protein expression using two-dimensional gel electrophoresis and nano ultra-high performance liquid chromatography electrospray ionization mass spectrometry tandem mass spectrometry techniques. A mass spectrum was identified using the Mascot search. Results revealed differential expression of 11 proteins, including transgelin, Ig kappa chain precursor, plasma glutathione peroxidase precursor, an unnamed protein product （gil55628）, gfyceraldehyde-3-phosphate dehydrogenase-like protein, lactoylgfutathione lyase, adenyfate kinase isozyme 1, two unnamed proteins products （gil55628 and gi11334163）, and poly（rC）-binding protein 1 in motor and sensory nerves. Results suggested that these proteins played roles in specific nerve regeneration following peripheral nerve injury and served as specific markers for motor and sensory nerves.

THE STUDY ON RELATIONSHIP BETWEEN CIGARETTE SMOKING AND THE p53 PROTEIN AND P21 PROTEIN EXPRESSION IN NON-SMALL LUNG CANCER

This paper discusses the relationship between cigarette smoking and the p53 protein and P21 protein expression by the immunohistochemical analysis in 93 cases with lung cancer in which squamous cell carcinoma accounted for 45 cases, adenocarcinoma 48 cases. The results showed that positive proportion of p53 protein expression was 74.20% (28 of 37 squamous cell carcinoma, 21 of 30 adenocarcinomas) in cigarette smoking group with lung cancers, and 38.46% (3 of 8 squamous cell carcinoma, 7 of 18 adenocarcinomas) in nonsmoking group with lung cancers. The difference was statistically significant. Odds ratio was 4.14 and confidence limits for OR was 1.42-12.52. A dose-related presents in the p53 protein expression for the smoking amount and smoking years. The positive proportion of P21 protein expression was 79.31% (21 of 28 squamous cell carcinoma, 25 of 30 adenocarcinomas) in cigarette smoking group with lung cancers, and 82.75% (10 of 11 squamous, 14 of 18 adenocarcinomas) in nonsmoking group with lung cancers, the difference was not statistically significant. But their positive proportion of P21 protein expression were very high in both groups. It was indicated that no relationship between cigarette smoking and the P21 protein expression. We suggest that the p53 gene could be a common target of tobacco-associated carcinogenesis in lung cancer.

Protein Expression of Goat Follicular Fluid （GFF）

Oocyte maturation process is very complex. Until now synthesis and protein function as well as pathway mechanism in oocyte maturation process are not yet well understood. The mechanism of goat follicular fluid （GFF） in maturation in vitro medium needs to be deeply studied before it is implemented widely. The aim of the research was analyzing the GFF to know protein expression of ERK1, ERK 2, p90rsk, Cyclin-Bl and cdc25A by Western Blotting method. GFF collection by aspiration from small follicle （〈 4 mm） and big follicle （4-8 mm） by using needle with spuit size of 18 G. It was concluded that there was protein expression of ERK2 and p90rsk, at 42 kDa and 22 kDa respectively.

Using the SELDI ProteinChip System to Detect Changes in Protein Expression in Vero Cells after Infection

人的疱疹单一的病毒(HSV-1 ) 1 引起美容，眼睛，并且 encephalitic 疾病并且与潜伏的感染和癌症被联系。这里，我们开发了由使用 SELDI 蛋白质薄片在在 vitro 有教养的 Vero 房间检测蛋白质表示的变化在蛋白质水平学习 HSV-1 感染的致病的一个工具。在有为 12， 24 或 48 h 的 HSV-1 和文化的感染以后，房间被收获并且 lysed。IMAC3 数组被用于 SELDI-TOF-MS 在感染前后检测 proteomic 差别。薄片检测了一系列差别表示蛋白质山峰。有趣地，在 16 912 Da 的山峰和 17 581 Da 与 ISG15 的分子的团精确相应，它可以在感染的过程期间参予抗病毒的活动。因此，我们获得了的结果能用作一个基础学习在病毒和它的主人之间的 HSV-1 和相互作用的致病。另外，他们能为 HSV-1 感染的治疗在新治疗学的目标的发现帮助。关键词 SELDI 蛋白质薄片 - Vero 房间 - HSV-1 - 蛋白质表达式 CLC 数字 R373 基础项目：中国(30540075 ) 的国家自然科学基础；

Temozolomide chemotherapy based on MGMT protein expression for patients with malignant gliomas:a report of 40 cases

Objective This study is to evaluate the efficacy and toxicity of temozolomide (TMZ) chemotherapy based on O 6 -methylguanine-DNA methyltransferase (MGMT) protein expression in patients with malignant gliomas. Methods A total of 40 patients with pathologically confirmed malignant gliomas were enrolled. All patients had pretreated with radiotherapy and had assessable lesions.

Proteomics of the differential protein expressions in human glioma cell line U251 cells infected with human cytomegalovirus,as demonstrated by the surface enhanced laser desorption/ionization(SELDI)protein chip system

The proteomics of the differential protein expressions in human glioma cell line U251 cells infected with human cytomegalovirus （HCMV） was investigated at the protein level by using the surface enhanced laser desorption/ionization （SELDI） protein chip system in order to develop a method of study for the pathogenesis of HCMV infection. In this study, the cultured U251 cells were infected with HCMV in good condition and the supernatants of lysates and the extracellular fluids of the cultivated infected cells were quantitatively defined for the expressed proteins. The proteomics of the differential protein expression in cells before and after infection was analyzed by WCX2 arrays on the protein chip reader. It was demonstrated that the eytopathic effects of infected cells appeared on the 5th day after infection, however, the differential protein expression was evident at 6 h after infection as revealed by RT-PCR and mass spectrometry. The protein peaks captured from different batches of samples, from the same sample detected with different arrays or for the different times were all equivalent. With the molecular weight range from 2000 Da to 3000 Da, chip captured 82 peaks from the intracellular fluids and 11 protein peak from the cellular fluid in which compared with the control group, the protein peaks with molecular weight of 13 536.3 Da, 10 046.1 Da and 17 106.2 Da were close to those of β-amyloid protein, caspase-1 precursor and LPS-induced TNF-α factor respectively, which showed brief up-regulation 4 h after infection, and continued to raise 48 h later. These results infer that these proteins may be related to the apoptosis induced by HCMV infection, thus suggesting that the apoptosis induced by HCMV infection may play a role in the pathogenesis of HCMV infection.

Phosphatase and tensin homology deleted in chromosome 10,hypoxia-inducible factor-1 alpha gene expression in colorectal adenoma and adenocarcinoma and their relation to vascular endothelial growth factor protein expression

To examine phosphatase and tensin homology deleted in chromosome 10 (PTEN),hypoxia-inducible factor-1 alpha (HIF-1 alpha) gene expressions and their relation to vascular endothelial growth factor(VEGF) protein expression in the patients with human colorectal adenomas and adenocarcinomas.Methods The expression of PTEN,HIF-1 alpha gene was detected by using in situ hybridization,and the VEGF expression levels by immunohistochemistry in colorectal adenomas and primary colorectal adenocarcinoma.Results Strong expression of HIF-1 alpha was detectable in the majority of colorectal dadenocarcinoma,particularly surrounding areas of necrosis in adenocarcinoma.PTEN,HIF-1 alpha mRNA and VEGF protein were positive in 51.6%,67.7% and 59.7% respectively in 62 cases of adenocarcinomas,and 77.8%,44.4% and 33.3% respectively in 18 cases of adenomas.The positive rate of VEGF was higher in the patients with colorectal adenocarcinomas than that in those with adenomas,whereas that of PTEN mRNA was contrary.HIF-1 mRNA expression was correlated significantly with lymph node metastasis,liver metastasis,Duke’s stage and recurrence.During colorectal tumor progression,the expression of HIF-1 alpha mRNA was positively correlated with the VEGF protein expression (χ2= 4.751 ,P<0.05),but negatively with the PTEN mRNA expression(χ2=21.84,P<0.01).Conclusion The absence or low expression of PTEN and the increased levels of HIF-1α and VEGF may paly an important role in carcinogenesis and progression of colorectal carcinoma.These results suggest that VEGF upregulated by HIF-1 alpha gene may be involved in angiogenesis of colorectal adenocarcinoma.4 refs,1 tab.

Effect of cluster needling at scalp acupoints on differential protein expression in rat brain tissue after acute focal cerebral ischemia

Objective:To explore the function of cluster needling at scalp points therapy on regulating differential protein's expression at different time points in middle cerebral artery occlusion(MCAO)model rats.Methods:Fifty-four rats were divided into three groups randomly and 18 rats in each group.The groups respectively were the model group(group M,n=18),cluster needling at scalp points group(group C,n=18),false operation group(group F,n=18).Each group was then assigned in three subgroups,including 24-h,7-day,and 14-day subgroups.Six rats in each subgroup.Acupuncture at Baihui(GV20)and 2 points beside Baihui,which was 3 e4 mm away from the midline.Longa score was used to evaluated neurological effects.Proteomics methods were used to identify differentially expression proteins with a standard of fold change greater than 1.5 and P<.05 at different times.Results:1.Nerve function scoring:The nerve function scores at 7 and 14 days decreased in group C,which showed better neural function than group M(P<.05).2.Fold change in proteins:Group M showed932 differentially expressed proteins compared with group F,and among them,414 proteins showed significant changes in expression after acupuncture.The expression levels of Cdc42 and GFAP were increased,and Mag,Shank2,and MBP levels were decreased.In the Gene Ontology analysis,the cellular component consisted of the terms cytoplasm,cytoskeleton,lysosome,and plasma membrane.The main related biological processes were cellecell signaling,protein transport,aging,and cell adhesion.Many synaptic and metabolic pathways were found by KEGG analysis.Conclusion:Cluster needling at scalp acupoints can improve the nerve function score and improve dyskinesia in MCAO model rats.Cluster needling at scalp acupoints can regulate the expression of 414 proteins,including Cdc42,GFAP,Mag,Shank2,and MBP,which are related to cerebral ischemia.The differential proteins are major concentration in cytoplasm,cytoskeleton,lysosomes,and plasma membrane,participate in cellecell signaling,protein transport,aging,and cell adhesion,and act through multiple synaptic and metabolic pathways to exert their biological functions.

Gene cloning,protein expression,and enzymatic characterization of a double-stranded RNA degrading enzyme in Apolygus lucorum

RNA interference(RNAi)is a powerful tool that post-transcriptionally silences target genes in eukaryotic cells.However,silencing efficacy varies greatly among different insect species.Recently,we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA injection.The disappearance of double-stranded RNA(dsRNA)could be a potential factor that restricts RNAi efficiency.Here,we found that dsRNA can be degraded in midgut fluids,and a dsRNase of A.lucorum(AldsRNase)was identified and characterized.Sequence alignment indicated that its 6 key amino acid residues and the Mg2+-binding site were similar to those of other insects’dsRNases.The signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali dsRNase.AldsRNase showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle,with peaks at the 4th instar ecdysis in the whole body.The purified AldsRNase protein obtained by heterologously expressed can rapidly degrade dsRNA.When comparing the substrate specificity of AldsRNase,3 specific substrates(dsRNA,small interfering RNA,and dsDNA)were all degraded,and the most efficient degradation is dsRNA.Subsequently,immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut cells.Through cloning and functional study of AldsRNase,the enzyme activity and substrate specificity of the recombinant protein,as well as the subcellular localization of nuclease,the reason for the disappearance of dsRNA was explained,which was useful in improving RNAi efficiency in A.lucorum and related species.

Expression of non-structural protein NS3 gene of Bombyx mori densovirus (China isolate)

The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 （China isolate）, named BmDNV-3, is a kind of bidensovirus. It is a new type of virus with unique replication mechanisms. To investigate the effects of the NS3 gene during viral DNA replication, a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus （China isolate）. Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe, respectively. The NS3 protein was expressed in Escherichia coli BL21. The pFastBacHTe-NS3 was transformed to E. coli DHIOBac. The recombinant bacmid baculoviruses （rBacmid-EGFP-NS3） isolated from the white colonies were transfected into BmN-4 cells using a transfection reagent. BraN-4 cells were infected with recombinant virus to express fusion proteins. The expression of fusion protein around 30 kDa in E. coli BL21 was identified by SDS-PAGE, Western blotting, and mass spectrometry. The expressed NS3 protein by B. mori nucleopolyhedrovirus bacmid system was confirmed by Western blotting using an anti-NS3 polyclonal antibody. And about 45 kDa protein was found. The expressed fusion protein was smaller than the expected size of EGFP-NS3, 55 kDa. Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected larvae with smaller molecular size.

QUANTITATION OF P53 PROTEIN EXPRESSION IN GASTROINTESTINAL SMOOTH MUSCLE TUMORSCLINICOPATHOLOGICAL CORRELATION AND PROGNOSTIC SIGNIFICANCE

Quantitative analysis of P53 protein expression was performed on paraffin-embedded tissues from 55 smooth muscle tumors of the gastrointestinal tract, using immunofluo-rescence and flow cytometry. No positive expression was found in normal smooth muscle tissues of the gastrointestinal tract. Over-expression of P53 gene was found in a significantly higher proportion in leiomyosarcomas (90%) and potentially malignant smooth muscle tumors (75%) as compared to leiomyomas (14%) (P< 0.005). The quantitation of P53 expression was found to be progressively enhanced in the sequence from leiomyoma through potentially malignant smooth muscle tumor to leiomyosarcoma (P< 0.005). It was markedly over-expressed when the mitotic counts ranged from one to more than one per 10 high power fields (P< 0.005) or the mild cytologic atypia was found (P< 0.005). The five-year survival rate was significantly higher in patients with low-expression of P53 than in those with over-expression of P53 (P< 0.005). It was suggested that P53 over-expression might be associated with the transformation of leiomyoma into leiomyosarcoma and could be used as an objective parameter in distinguishing the malignant from the benign and predicting the prognosis of patients with smooth muscle rumors of the gastrointestinal tract.

An Improved Strategy for Efficient Expression and Purification of Soluble HIV-1 Tat Protein in E.coli

Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.

Proteomics Profiling of Host Cell Response via Protein Expression and Phosphorylation upon Dengue Virus Infection

Dengue virus(DENV)infection is a worldwide public health threat.To date,the knowledge about the pathogenesis and progression of DENV infection is still limited.Combining global profiling based on proteomic analysis together with functional verification analysis is a powerful strategy to investigate the interplay between the virus and host cells.In the present study,quantitative proteomics has been applied to evaluate host responses(as indicated by altered proteins and modifications)in human cells(using K562 cell line)upon DENV-2 infection,as DENV-2 spreads most widely among all DENV serotypes.Comparative analysis was performed to define differentially expressed proteins in the infected cells compared to the mock-control,and it revealed critical pathogen-induced changes covering a broad spectrum of host cellular compartments and processes.We also discovered more dramatic changes(>20%,160 regulated phosphoproteins)in protein phosphorylation compared to protein expression(14%,321 regulated proteins).Most of these proteins/phosphoproteins were involved in transcription regulation,RNA splicing and processing,immune system,cellular response to stimulus,and macromolecule biosynthesis.Western blot analysis was also performed to confirm the proteomic data.Potential roles of these altered proteins were discussed.The present study provides valuable large-scale protein-related information for elucidating the functional emphasis of host cell proteins and their post-translational modifications in virus infection,and also provides insight and protein evidence for understanding the general pathogenesis and pathology of DENV.