[Dynamic expression of protein tyrosine phosphatase SHP2 in liver tissue of rats with carbon tetrachloride-induced liver fibrosis].

Objective: To investigate the dynamic expression of protein tyrosine phosphatase SHP2 in liver tissue of rats with carbon tetrachloride (CCl(4))-induced liver fibrosis. Methods: Rat liver fibrosis model was established by intraperitoneal injection of CCl(4). Rat liver tissue histopathological changes were detected by HE and Masson-trichrome staining. Immunohistochemical staining, Western blot and real-time fluorescent quantitative PCR were used to detect SHP2 protein and mRNA expression in rat liver tissue. One-way analysis of variance was used for the comparison of means between multiple groups, and the LSD test was used for further inter-group comparison. Results: CCl(4)-induced rat liver fibrosis model was successfully constructed, and with the extension of modeling time, the degree of liver fibrosis in rats were aggravated gradually. Immunohistochemical staining results showed that SHP2 was mainly expressed in the cytoplasm of rat liver tissues. With the aggravation of liver fibrosis, the number of cells with positive expression of SHP2 was aggravated gradually (P < 0.05). Western blot and real-time fluorescent quantitative PCR results showed that the expressions of SHP2 protein and mRNA in rat fibrotic liver tissues at different times in week 2, 4, 6, and 8 were higher in modeling than control group (P < 0.05), and was aggravated gradually with the liver fibrosis aggravation (P < 0.05). Conclusion: The expression of SHP2 protein and mRNA in the liver tissue of rats with CCl(4)-induced liver fibrosis increased gradually with the degree of liver fibrosis, and the degree of increase was consistent with the degree of liver fibrosis.

Brachial artery flow-mediated dilatation and carotid intima-media thickness in young ED patients with insulin resistance.

The evidence of a close relationship between cardiovascular disease and erectile dysfunction (ED) is well documented. The aim of this study is to investigate whether there is an early asymptomatic impairment of the peripheral vasculature in young ED patients without obvious cardiovascular disease. We studied a total of 261 ED patients (19-40 years old) and 40 age-matched healthy controls. All participants received questionnaires of cardiovascular risk factors and erectile function assessment, were subjected to lab tests of fasting blood sample, and underwent the ultrasonographic examination of brachial artery flow-mediated dilation (FMD) and carotid intima-media thickness (c-IMT). Insulin resistance (IR) was measured by the homeostasis model assessment of insulin resistance (HOMA-IR). Compared with normal human controls, FMD was significantly lower, whereas the average c-IMT was significantly greater in ED patients. An inverse correlation was found between FMD and mean c-IMT. The ED patients had significantly higher levels of fasting glucose, fasting insulin and HOMA-IR index, but showed relatively lower total testosterone and prolactin levels than the controls. Both FMD and c-IMT showed a significant correlation with International Index of Erectile Function-5 questionnaire (IIEF-5) score, age and HOMA-IR. Multivariate stepwise regression analysis demonstrated that age, HOMA-IR and IIEF-5 score were the risk factors associated with FMD and c-IMT. In conclusion, young ED patients in association with IR display diminished FMD and increased c-IMT. Furthermore, ED, HOMA-IR and age are independent predictors of the two subclinical atherosclerotic markers.

Role of selenoprotein S (SEPS1) -105G>A polymorphisms and PI3K/Akt signaling pathway in Kashin-Beck disease.

OBJECTIVE: To investigate the relationship between SEPS1 polymorphism and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in Kashin-Beck disease (KBD) and further explore the pathogenesis of KBD. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to detect SEPS1 -105G>A polymorphism in 232 cases and 331 controls. The protein expressions of PI3K/Akt signaling molecules in whole blood and chondrocytes were detected by Western blot. RESULTS: The frequencies of SEPS1 -105G>A genotype AA (21.1% vs 3.0%) and minor allele A (34.1% vs 16.0%) in KBD are significantly higher than those in controls (OR: 8.020, 95% confidence interval (95% CI) 6.341-10.290, P < 0.0001; OR: 2.470, 95% CI 2.001-4.463, P < 0.0001, respectively). SEPS1 AA genotype was an independent risk factor for KBD (adjusted OR: 9.345, 95% CI 4.254-20.529; P < 0.0001). The expression of Gßγ, PI3Kp110, pAkt and pGSK3ß in KBD group were higher than that in control group (all P < 0.05). Gßγ, pAkt and pGSK3ß protein expression of AA and GA increased than GG (all P < 0.05). Cell apoptosis was increasing and molecule expression of PI3K/Akt signaling pathway were up-regulated in the tert-Butyl hydroperoxide (tBHP)-injured group, the cell apoptosis and expression levels of PI3K/Akt in Na2SeO3 group were decreased. CONCLUSIONS: The SEPS1 -105G>A is associated with an increased risk of KBD and influences the expression of PI3K/Akt signaling pathway in KBD patients. Apoptosis induced by tBHP in chondrocyte might be mediated via up-regulation of PI3K/Akt, Na2SeO3 has an effect of anti-apoptosis by down-regulating of PI3K/Akt signaling pathway.

Insulin gene VNTR genotype associates with frequency and phenotype of the autoimmune response to proinsulin.

Immune responses to autoantigens are in part controlled by deletion of autoreactive cells through genetically regulated selection mechanisms. We have directly analyzed peripheral CD4+ proinsulin (PI) 76-90 (SLQPLALEGSLQKRG)-specific T cells using soluble fluorescent major histocompatibility complex class II tetramers. Subjects with type I diabetes and healthy controls with high levels of peripheral proinsulin-specific T cells were characterized by the presence of a disease-susceptible polymorphism in the insulin variable number of tandem repeats (INS-VNTR) gene. Conversely, subjects with a 'protective' polymorphism in the INS-VNTR gene had nearly undetectable levels of proinsulin tetramer-positive T cells. These results strongly imply a direct relationship between genetic control of autoantigen expression and peripheral autoreactivity, in which proinsulin genotype restricts the quantity and quality of the potential T-cell response. Using a modified tetramer to isolate low-avidity proinsulin-specific T cells from subjects with the susceptible genotype, transcript arrays identified several induced pro-apoptotic genes in the control, but not diabetic subjects, likely representing a second peripheral mechanism for maintenance of tolerance to self antigens.

Incompatibility mutants of IncFII plasmid NR1 and their effect on replication control.

DNA from the replication control region of plasmid NR1 or of the Inc- copy mutant pRR12 was cloned into a pBR322 vector plasmid. These pBR322 derivatives were mutagenized in vitro with hydroxylamine and transformed into Escherichia coli cells that harbored either NR1 or pRR12. After selection for the newly introduced pBR322 derivatives only, those cells which retained the unselected resident NR1 or pRR12 plasmids were examined further. By this process, 134 plasmids with Inc- mutations in the cloned NR1 or pRR12 DNA were obtained. These mutants fell into 11 classes. Two of the classes had plasmids with deletions or insertions in the NR1 DNA and were not examined further. Plasmids with apparent point mutations were classified by examining (i) their ability to reconstitute a functional NR1-derived replicon (Rep+ or Rep-), (ii) the copy numbers of the Rep+ reconstituted replicons, (iii) the cross-reactivity of incompatability among the various mutant classes and parental plasmids, and (iv) the trans effects of the mutants on the copy number and stable inheritance of a coresident plasmid.

Analysis of the individual regulatory components of the IncFII plasmid replication control system.

Replication of the IncFII plasmid NR1 is controlled by regulating the amount of synthesis of the repA1 initiator protein at both the transcriptional and translational levels. We have examined mutations which have altered each of these levels of regulation, resulting in different plasmid copy numbers. The genes which encode each of the individual wild-type or mutant regulatory components from the replication control region of NR1 have been cloned independently into pBR322 vectors, and their effects in trans, either individually or in various combinations, on plasmid incompatibility, stability, copy number, and repA1 gene expression have been defined.

Incompatibility and IncFII plasmid replication control.

The DNA coding for replication control and incompatibility of the plasmid NR1 serves as a template in vivo and in vitro for RNA transcription in both directions. In the rightward direction, RNA synthesis begins from 2 different promoters, one of which is regulated and the other constitutive. In vivo, each of these transcripts is more than 1,000 nucleotides long, terminating near the estimated site for the origin of replication. These transcripts serve as messenger RNA for several proteins. One protein (repA1) is required for replication and another (repA2) serves as the repressor for the regulated rightward promoter. RNA synthesis in the leftward direction is constitutive and produces a single transcript of 91 nucleotides which is complementary in sequence to the rightward transcripts. This small transcript is the incompatibility product which regulates the replication of the plasmid. When the intracellular concentration of the small transcript is experimentally varied, the rate of translation of the rightward transcripts and the rate of initiation of replication (plasmid copy number) vary inversely to its concentration. The mode of action of this inhibitor RNA is likely to be formation of an RNA-RNA duplex with the rightward transcripts, thereby inhibiting the translation which would produce the required replication protein. The probability that a rightward transcript will escape interaction with the small RNA molecules and thus allow replication to initiate can be predicted from the laws of mass action based on base-stacking free energies for the likely sequences of initial contact. The estimated intracellular RNA concentrations, based on quantitative hybridization experiments, are agreement with those predicted from the calculated equilibrium constants for duplex formation.

IncFII plasmid incompatibility product and its target are both RNA transcripts.

The region of DNA coding for incompatibility (inc) and copy number control (cop) of the IncFII plasmid NR1 is transcribed in both the rightward and leftward directions. The rightward transcripts serve as mRNA for the repA1 protein, which is required for replication. A small, 91-base leftward transcript is synthesized from the opposite DNA strand and is complementary to a portion of the rightward mRNA near its 5' end. A 262-base-pair Sau3A restriction fragment that encodes the small leftward transcript, but does not include the rightward transcription promoters, was cloned into the vector pBR322 or pUC8. The same fragment was cloned from an Inc- mutant of NR1 that does not make the small leftward transcript. Transcription through the cloned fragments in these derivatives was under control of the tetracycline resistance gene in pBR322 or the lac promoter-operator in pUC8. In one orientation of the inserted DNA, a hybrid transcript containing rightward NR1 RNA sequences was synthesized. In the other orientation, a hybrid transcript containing leftward NR1 RNA sequences was synthesized. These plasmids were used to vary the intracellular levels of the rightward or leftward NR1 RNA transcripts and to test their effects in trans on various coresident derivatives of NR1. An excess of rightward NR1 RNA in trans stimulated expression of the essential repA1 gene and caused an increase in the copy number of a coresident NR1 plasmid. An excess of leftward NR1 RNA in trans inhibited the expression of the repA1 gene and lowered the coresident NR1 copy number, thereby causing incompatibility. A pBR322 derivative with no transcription through the cloned NR1 DNA had no effect in trans. These results suggest that the small leftward transcript is the incompatibility inhibitor of NR1 and that its target is the complementary portion of the rightward mRNA.