Basic & Clinical Medicine

Application of Proteomic Techniques to Ubiquitin-Proteasome System

2007, 27(1): 1-7.

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Protein degradation via ubiquitin-proteasome system(UPS) is involved with a wide variety of signalling pathway, from cell cycle and transcription to development. The mass spectrometry-based proteomics techniques are powerful tools in the research of UPS. The recent advance in the study of UPS by proteomics techniques are briefly reviewed.

Proteomic approaches to post-translational modification by ubiquitin family proteins

2007, 27(1): 8-15.

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Post-translational modification by ubiquitin and ubiquitin-like modifiers(Ubls) is one of the most important mechanisms regulating a wide range of cellular processes in eukaryotes. Previous research shows that, through covalently modification by ubiquitin or ubls, the substrate proteins can be regulated in many different ways like stability, subcellular localization, enzymatic activity, protein-protein interaction, etc.. Therefore, we can imagine, ubiquitin and ubls are playing very important roles in cellular and biological processes by modifying plenty of proteins. To better understand the ubiquitin and ubls system, proteomic approaches have been developed to purify and identify more protein substrates. Large-scale idendification of ubiquitin/ubls - modification sites by mass spectrometry is particularly important for understanding the molecular mechanism and function of ubiquitin/ubls modification. Until now, more and more scientists are getting interested and participating in proteomics research of ubiquitin/Ubl modifications. This review summarizes the rencent works in this field.

Ubiquitin-proteasome system and cardiovascular diseases

2007, 27(1): 16-20.

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The ubiquitin-proteasome system (UPS), containing ubiquitin-activating enzyme (E1), ubuquitin-conjugating enzyme (E2s), ubiquitin-ligase (E3s) and 26S proteasome, is involved in the degradation of most intracellular proteins. One major function of UPS is to prevent accumulation of non-functional, potentially toxic proteins. Moreover, it has become clear that the UPS fulfills an important function in most aspects of eukaryotic biology, such as inflammation, cell proliferation, intracellular signaling, transcriptional control, and apoptosis. Recent studies demonstrated that the UPS regulates the initiation and progression of atherosclerosis, ischemia-reperfusion injury, familial cardiomyopathies, hypertrophic and heart failure. This review briefly summarizes present knowledge about structure, function, regulation of UPS and its pathophysiological role in cardiovascular diseases.

Effect of adrenomedullin on hypoxic pulmonay vascular structural remodeling and proadrenomedullin N-terminal 20-peptide in rats

2007, 27(1): 21-24.

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Objective To investigate the effect of adrenomedullin (ADM) on hypoxic pulmonary vascular structural remodeling and proadrenomedullin N-terminal 20-peptide (PAMP) in rats with hypoxic pulmonary hypertension. Methods Twenty-four male Wistar rats were randomly divided into control group (n=8), hypoxia group (n=8) and hypoxia with ADM group (n=8). ADM was subcutaneously administered into rats of hypoxia with ADM group by mini-osmotic pump (300ng/h). The concentration of plasma PAMP was measured by radioimmunoassay, and the expression of PAMP in pulmonary artery was detected by immunohistochemical assay. Results Mean pulmonary arterial pressure, the ratio of right ventricular mass to left ventricular plus septal mass, and relative medial thickness and relative medial area of pulmonary arteries were significantly increased in hypoxic rats as compared with controls (P<0.01, respectively). Ultrastructural changes were also found in pulmonary arteries of hypoxic rats. Meanwhile, plasma PAMP concentration and the expression of PAMP by pulmonary arteries in hypoxic rats were markedly increased compared with controls. However, ADM obviously ameliorated above changes. Conclusion Intramolecular regulation of ADM may play an important role in the regulation of ADM on hypoxic pulmonary hypertension.

Screening of proteins interacting with neuropeptide CART

2007, 27(1): 25-30.

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Abstract : Objective: The objective was to look　for cocaine-and amphetamine-regulated　transcript peptide(CART) receptors or proteins interacted to CART. Method :A cDNA library from rat brain was constructed. Then the BacterioMatch two-hybrid system was used to screening for proteins interacted with CART by using CART41-102 as bait. Result:The cDNA library and bait pBT- CART41-102 were constructed successfully. The library harvested 3.37×106 individuals, and the reconstruction rate was 98.5%. 5.01x106 cotransformed clones were screened, 93 clones were conformtively identified to interacting with CART and were DNA sequenced. By bioinformation analysis, 6 known proteins interacted to CART were identified. Other unknown interaction proteins that had short exactly similiar motif to 22 membraneproteins or receptors were also identified. Conclusion: CART might interact to proteins about energy balance, cell proliferation and enzymes related to protein processing and degrading .

Cardiotrophin-1(CT-1) promote cardiac myocyte survival via mitogen-activated protein kinase(MAPK)-dependent signaling pathway

Fu Shi-Gan

2007, 27(1): 31-35.

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Objiective: Cardiac muscle cells play a critical role in maintaining the normal function of the heart. Cardiotrophin-1(CT-1), a potent cardiac survival factor, is capable of inhibiting apoptosis or promoting survival in cardiomyocytes. We sought to elucidate the mechanism of CT-1 promoting cardiac myocyte survival in cultured neonatal rat cardiomyocytes. To explore the potential signaling pathway that might be responsible for this effect. Methods: We examined the cardiac myocyte survival effect of CT-1 in cultured neonatal rat cardiomyocytes. The cardiomyocytes were stained [3-（4，5-dimethyl－thiaziazol-2-yl）-2-5-diphenyltetrazolium bromide，MTT] and counted. Results: The survival rate of cardiac myocytes was increased by CT-1 in a dose-dependent manner (10-10～10-7 mol/L) and in a time-dependent manner(1~4d, 10-8 mol/L) in cultured neonatal rat cardiomyocytes. Pretreatment of PD098059 (5×10-5mol/L) ，a MAPK blocker, decreased significantly survival rate of cardiac myocytes by promoted CT-1. The phorbol 12-myristate 13-acetate (PMA) (1×10-5mol/L)，a PKC activator, increased significantly this effect of CT-1,but inhibited significantly by MAPK blocker PD098059. Conclusion: These results indicate that CT-1 is a potent factor of promoting cardiac myocyte survival, and increase significantly survival rate of cardiac myocytes in a dose-dependent and a time-dependent manner in cultured neonatal rat cardiomyocytes. The MAPK signaling pathway mediates CT-1 induced cardiac myocyte survival .PKC signaling molecule may be a upstream signaling transduction pathway which cascades of MAPK in CT-1 induced cardiac myocyte survival .

Expression of ion channel gene after MSCs differentiating into cardiomyocytes

2007, 27(1): 36-39.

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Objective To study the expression of L-type calcium channel gene(α1c) and transient outward potassium channel gene (kv4.3 )during the procession of MSCs differentiating into cardiomyocytes. methods MSCs were isolated cultured,passaged, purified in vitro,and then exposed to 5-aza for 24 hours. we observed the morphology with microscopy. The expression ofα1C and kv4.3 gene was detected by Reverse transcription-polymerase chain reaction.Result:It displayed that MSCs had already expressed α1c and kv4.3.mRNA before 5-aza treatment. The expression of kv4.3 was significantly increased after 5-aza exposure at 24 hours ,4th day ,7th day,14th day,wheras the expression of α1C was impaired. Conlusion:L-type calcium channel and transient outward potassium channel played important roles during procession of MSCs differentiating into cardiomyocytes.

Pathological changes of testis tissue in male SARS patients

2007, 27(1): 40-43.

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Ojective To study the pathological changes of testis tissue in male SARS patients. Methods Tissue specimens were studied by HE staining、TUNEL and immunohistochemistry(IHC). Results All testes from the SARS patients demonstrated that widespead germ cells destruction,few or no spermatozoon in the seminiferous epithelium and the lumen, thickened basement membrane、 peritubular fibrosis、 vascular congestion and leukocytes infiltration obviously. The apoptotic seminiferous cells increased significantly(P<0.05).The numbers of CD3+ T lymphocyte and CD68+ macrophage increased significantly in the interstitial tissue compared with the control group(P<0.05), and both cells were observed infiltration in the tubules. Conclusions Our study implied that SARS caused orchitis.

Involvement of COX-2 in effect of heme oxygenase-1 on cardioprotection from anocia/reoxygenation induced injury

2007, 27(1): 44-48.

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Objective To investigate the protection effect and mechanism of PhaseⅡenzyme inducer CPDT (5,6-dihydrocyclopenta[C]- 1, 2-dithiole-3-thione) against threo-hydroxyaspartate-induced injury of motor neuron in the cultured spinal cord slices of rats. Methods Organotypic spinal cord slices of rat pup was divided into normal control group, model group(THA 100μmol/L) and PhaseⅡenzyme inducer CPDT(5, 15, 30μmol/ L) treated groups. The morphology change of spinal cord slices was observed with inverted microscope. Ventralαmotor neurons' survivals were evaluated by immunohistochemical staining with monoclonal antibody SMI32, a nonphosphorylated neurofilament marker. Lactate dehydrogenase (LDH) and malonaldehyde (MDA) levels in culture medium were also measured. Results The slices of THA group showed that the number of Ventralαmotor neurons significantly decreased, but LDH enzyme activity and MDA level in culture medium increased. After CPDT(15, 30μmol/L) pretreatment, the spinal cord slices in vitro grew well and showed the similar change with the slices of normal control group. The number ofαmotor neurons significantly increased with intervention of CPDT, compared with model group. LDH and MDA level in culture medium also decreased. Conclusion PhaseⅡenzyme inducer, CPDT may inhibit THA-induced motor neuron injury by inhibiting lipid superoxide and scavenging free radical.

Expression of connective tissue growth factor in liver fibrotic tissues of human and rats

2007, 27(1): 49-52.

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Objective To explore the role of connective tissue growth factor（CTGF）in the mechanism of liver fibrosis. Methods Liver fibrosis models of rats were made by subcutaneously injecting with CCl4. On the other hand, 32 human liver cirrhosis and 12 normal liver tissues were collected too. Immunohistochemical staining was used to detect the expression of CTGF in these tissues. Results The expression of CTGF in rats after injection with CCl4 were found chiefly in hepatic stellate cells(HSC) and hepatocytes and all significantly higher than those in normal rats(P<0.01,P<0.05)..Moreover, it present with progressive tendency for the expression of CTGF in rats respectively at 1st,4th and 8th week after injection with CCl4(P<0.01,P<0.05). The same tendency was found in human normal and fibrotic liver tissues(P<0.01) too. Conclusions The overexpression of CTGF may accelerate the procedure of liver fibrosis via enhancing HSC proliferation and the ECM increasing, and play an important role in liver fibrogenesis. , and their liver tissues were used for examining CTGF by immunohistochemistry stain.

Relationship between the functional integrity of human sperm membrane and seminal parameters

2007, 27(1): 53-56.

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Objective To study the relation between the function of sperm membrane and seminal parameters. Methods Five hundred and three semen samples were collected from 149 normal men and 354 infertile men. Sperm concentration，motility，viability，Semen WBC concentration，morphology and sperm tail hypo-osmotic swelling (HOS) were analyzed according to WHO criteria. Results We observed sperm HOS score in infertile group was lower than that in control group（P<0.05）.Comparing with control group, the percentage of the concentration ,motility, viability ,as well as the percentage of normal morphology sperm, were significantly decreased in the normal and the abnormal HOS score infertile group, however, the rate of Head defects sperm and semen WBC concentration were significantly increased in these two groups（P<0.05）.In addition, the rate of Tail defects sperm in normal HOS score infertile group was higher than that in control group（P<0.05）.Comparing with abnormal HOS scores infertile group , sperm motility ,as well as sperm viability, was significantly decreased in normal HOS score infertile group（P<0.05）. Significant positive relationship of sperm HOS score was observed with sperm concentration ,motility and viability（P<0.05）.There was negative relationship between sperm HOS scores and semen WBC concentration（P<0.05）. Conclusion Our study shows that an important relationship can be found between the functional integrity of sperm membrane and semen parameters. Detection of sperm membrane function plays a key role in evaluation of male fertility.

Application of genechip in screening for differetially expressed genes among hepatocellula carcinomas, cirrhotic liver tissue and normal liver

2007, 27(1): 57-62.

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Objective To compare gene expression profiles among hepatocellular carcinomas(HCC), cirrhotic liver tissue and normal liver and analyze their biological information. Methods Total RNA was isolated from hepatocellular carcinomas, cirrhotic liver tissue and normal liver respectively, purified into mRNA by oligotex, and reverse-transcribed to synthetize two kinds of fluorescences (Cy3-dUTP, Cy5-dUTP) labeled cDNA probes. The targets were mixed together and hybridized with the genes on the genechip contained 4096 genes. The following comparisons were performed: hepatocellular carcinomas versus normal liver, cirrhotic liver versus normal liver. Genes that were differentially expressed between these groups were identified based on signal-to-noise ratios by using GenePix Pro3.0 software. Results 141 genes were found have closely relation to HCCs, some of these genes were related to cell cycle and signal transduction, such as MCM7, YWHAH, among these, there are 23 genes whose expression tendency consistent with that of cirrhotic liver's. When compared with normal liver, there were 2 genes belong to metallothionein family downregulated in HCCs but upregulated in cirrhotic liver. Conclusions The inactivation of anticancer genes, the abnormality of metabolism related genes and the activation of cell proliferation cycle related genes may have a vital role in HCC. Research into these genes systematically may help to discover new markers of early diagnosis, to guide treatment proposal and to evaluate prognosis better.

A study on the relationship between the microsatellite polymoerphism in the heme oxygenase-1 gene promoter and expressive levels of induced niteic oxide synthase

2007, 27(1): 63-67.

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This study investigated the relationship between the microsatellite polymorphism in the Heme Oxygenase-1(HO-1)gene promoter and expressive levels of induced nitric oxide synthase(iNOS). The relationship was analyzed by techniques of polymerase chain reaction(PCR) and immunohistochemistry in the lung tissues from 80 cases of the patients with lung cancer ,containing 50 cases of patients with COPD (COPD group) and 30 patients without COPD (control group).The results showed that the stronger positive rate of iNOS expression in COPD group was significant higher than those of the control groups（P ﹤0.01）and that the stronger positive rate of iNOS expression in the subjects with a class L allele of the (GT)n repeats of the HO-1 gene promoter（L/L, L/M and L/S）was statistical higher compared with that of the subjects without a class L allele ( M/M, M/S and S/S)（P ﹤0.01）. This results suggest that there is likely a increase of the expressive levels of iNOS in the subjects of genotypes with ≥ 30 GT repeats in the HO-1 gene promoter region, which may be associated with the susceptibility to COPD.

Experimental study on the effect of cyclooxygenase 2 in the pathogeny of alcoholic liver disease in rat model

2007, 27(1): 68-71.

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Objective To explore the effect of cyclooxygenase 2(COX-2) in the pathogeny of alcoholic liver disease (ALD). Methods A model of ALD was developed with Wistar rats by intragastric alcohol. Liver samples were collected in the end of 4th, 8th, 12th and 16th week respectively. The concentrations of triglyceride(TG), oxygen free radical (ORF), superoxide dismutase (SOD), anti-superoxide anion (ASOA), 6-ketone-prostaglandin F1α( 6-K-PGF1α), thromboxane B2(TXB2) and malondiadehyde ( MDA) in the liver homogenate were measured by biochemistry chromatometry. Pathology of hepatic tissue was observed by hematoxylin eosin，sudan Ⅳand sirius red staining.The protein and mRNA expression of COX-2 in liver tissues were examined by immunohistochemical method and reverse transcription-polymerase chain raction (RT-PCR) respectively. Results In the end of 4th ,8th ,12th and 16th week,the content of COX-2 mRNA in liver tissue of model rats were 1.29,1.70,1.92 and 3.38 times the same as normal rats(P<0.01,except for 4th week),and the expression of cox-2 protein increased also. Pearson correlation analysis showed that the COX-2 mRNA expression was correlated positively with the content of OFR,MDA,TG,and TXB2 (P<0.01),and correlated negatively with the content of ASOA, SOD and 6-K-PGF1αin the liver homogenate(P<0.01). Conclusion There is close correlation between COX-2 and oxidative stress，which plays an important role in the etiopathegenesis of ALD.

Expression changes of DBP RBP and TTR after acute PE in rat model

2007, 27(1): 72-75.

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Objective To study the expression changes and metabolic effects of DBP, RBP and TTR after acute pulmonary embolism(PE) in a rat model. Methods We constructed a rat acute PE model by injecting emboli into the jugular vein. The serum protein levels of DBP, RBP and TTR were validated by western blot methods. The mRNA levels of DBP, RBP and TTR were identified by semi-quantitative RT-PCR. The serum concentrations of 25(OH)D3 and FT4 were determined by means of RIA, and that of Vitamin A were checked by microfluorescence. Results The serum protein levels of DBP, RBP and TTR and their mRNA levels in rat liver all decreased gradually after acute PE. On the contrary, the serum concentrations of 25(OH)D3, Vitamin A and FT4 increased sharply after acute PE. Conclusion The synthesis of DBP, RBP and TTR in rat liver was depressed obviously after acute PE, which resulted in the down-regulation of the serum protein levels of them. The low serum concentrations of DBP, RBP and TTR would lead to the release of their ligands------25(OH)D3, Vitamin A and FT4, which would play important roles in the pathophysiologic changes after acute PE.

Protein kinase C and phosphatidyliositol 3 kinase involved in the mechanism of myocardial remodeling in patients with congestive heart failure

2007, 27(1): 76-80.

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Objective To investigate the involvement of mitogen-activated protein kinase（MAPK）and PI3K(phosphatidylinositol 3 kinase)/ Protein kinase B (Akt) signal transduction pathways in the mechanism of myocardium remodeling in patients with congestive heart failure(CHF) Methods Thirty nine patients of mitral valve disease with CHF were randomly selected and 30 cases of healthy persons were included as controls. Cardiac function parameters were measured by echocardiography. Concentrations of AngⅡ in plasma and myocardial tissues were determined by radio immunoassay. Activity of PKC was determined by using competive prote in binding method, activity of MAPK was detected by the methods of immunoprecitipation. Immunoprecitipation was used to assay the protein expression and phosphorylation of PI3K and Akt(Protein kinase B), protein expression of c-fos and α－skeletal-actin in myocardial tissues. Results Pathological changes of myocardial tissues in CHF with valvular heart disease showed typical myocardial remodeling. The hypertrophy was dominant at early stagy of CHF, while at end stage the characteristics include disordered alignament of the myocytes, the discontinuity and dissolving of cardiomyofibrills, destroyed subcellular organs, and the hyperplasia of interstitial tissue. AngⅡconcentrations in plasm and myocardial tissues in patients with CHF were higher than those in the control group(P<0.01), their levels were positively correlated to the levels of CHF; the activities of PKC and MAPK in myocardial tissues in patients with CHF were higher than those in control group(P<0.01), their levels were positively correlated to the levels of CHF (P<0.01). Compared to control group, phosphorylation of PI3K , Akt were higer in heart function class Ⅱgroup than that of heart function classⅢ and heart function class Ⅳgroups(P<0.01),there were no difference between heart function class Ⅲand Ⅳgroups; protein expression of c-fos was obvious in heart function Ⅱgroup (P<0.01) , but it is higer in heart function class Ⅲ and Ⅳ groups compared with heart function Ⅱgroup(P<0.01). The protein expression of α－skeletal-actin also increased in myocardial tissues in CHF groups, but positively correlated to cardiac function, there were big differences from control group(P<0.01). Conclusion Both PKC/MAPK and PI3K/Akt signal pathways involve in the pathogenesis of myocardial remodeling in CHF patients with valvular heart disease

Primary culture of nontumorigrnic human mammary epithelial

2007, 27(1): 81-84.

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Objective: to investigate the feasibility of primary culture of nontumorigenic human mammary epithelial cells, and the effect of proliferation due to hormone. Method: Normal human mammary tissue was taken from mammary segment ectomy, and the tissue were digested by collagenase type Ⅰ. Epithelial depuration was carried out by integration of burning and enzyme digestion. The cells we acquired were identified by observation of cell morphology by light and Transmission Electron Microscopy (TEM). Result: We found it is feasible to culture nontumorigenic human mammary epithelial cells. Conclusion:β-estradiol and progesterone at 1×10-5g/L can promote the proliferation of the normal human mammary epithelial cells.

Bcl-2 and bax expression and apoptosis in fetal rat myocardial cells of intrahepatic cholestasis of pregnancy

2007, 27(1): 85-86.

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Compared survivin with NMP22in Bladder carcinoma diagnosis value

2007, 27(1): 87-88.

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An observation of electrogastrography in pediaric chronic superficial gastritis cases with different gastroscopic features

2007, 27(1): 89-90.

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Effect of ginkgo biloba enalapril and both of them on transdifferentiation of rats rubular epotheloal cell after unilateral ureteral obstruction

2007, 27(1): 91-93.

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Research progresses on genomics and proteins of Chlamydia spp

2007, 27(1): 94-99.

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Following genomes from various strains of Chlamydia spp being sequenced, it was found that there were only tiny changes in their gene components, the infected host variety and pathogenisis process were very different. At presence, the genomes from Chlamydia trachomatis, C.muridarum, C .pneumoniae, Chlamydophila caviae were studied in detail, which revealed the relationship of chlamydial genomes and multiple members causes. The discovery of polymorphic membrane protein gene family, type III secretion gene and peptidoglycan coding gene showed the great research success in the field. After the complete nucleotide acids of various species and strains of Chlamydia spp were sequenced the many proteins were further knew, involving the outer membrane protein, major outer membrane protein, hot shock protein and polymorphic membrane protein with different functions and structures. Fully understanding the properties of these proteins is in favor of studying mechanisms of pathogenisis and immunity of Chlamydia as well as high-effective gene engineering vaccine against chlamydiosis.

ICAM-1 and renal renal ischmic-reperfusion

2007, 27(1): 100-104.

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Abstract: It has been well-documented that ICAM-1 is implicates in pathophysiology of ischemic-reperfusion injury in the kidney. Renal injury after ischemia appears to be a consequence of not only tissue hypoxia from interrupted blood supply but also from the process of reperfusion which leads to an active inflammatory process. Infiltrating leukocytes are the potential source of reactive oxygen species. Proteolytic enzymes and cytokines, which during reperfusion may play a detrimental role. It has been suggested that ICAM-1 plays a key role during leukocyte adhesion and recruitment to inflamed tissue. The goal of the review is to explore the effect and significance of ICAM-1 as well as its mechanism in renal ischemia-reperfunsion.

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