Cellular context- and protein level-dependent interaction of pluripotency factor OCT4A with multiple octamer motifs of the same target gene.

AIMS: To compare how OCT4A proteins interact with and regulate multiple OCT4A-octamer motifs (OMs) in different regions of the FOS gene expressed in somatic cancer cells versus pluripotent stem cells. MATERIALS AND METHODS: Two FOS reporter gene systems harboring predicted OMs or their mutational counterparts were introduced into HeLa and NCCIT cells with varying OCT4A protein levels. The transcription of dsGFP reflecting FOS expression was quantitated by RT-qPCR, the OCT4A-OMs binding and the correlation between OCT4A and FOS transcription was determined by ChIP-PCR and RNA-Seq, respectively. KEY FINDINGS: In NCCIT cells, abundant OCT4A proteins bound to and inhibited OM1 and OM2 at the promoter of the FOS gene. RA-induced OCT4A down-regulation transiently increased FOS transcription. In contrast, in HeLa cells that contain much lower levels of endogenous OCT4A proteins, OCT4A primarily bound to and activate OM1 thereby promoting FOS transcription. OCT4A KO significantly reduced FOS expression. Ectopically introduced OCT4A, at its leaked or induced expression level, promoted FOS transcription by binding to OM2/OM3 or OM1/OM3, respectively. Thus, the interaction of OCT4A proteins with different OMs is cellular context- and protein level-dependent, and such complicated OCT4A binding mode can only be reflected by a dsGFP-based reporter harboring the full-length FOS gene but not by that merely having the FOS promoter. SIGNIFICANCE: Our findings unravel an additional layer of regulatory mechanisms that account for the cellular context- and dose-related versatile functions of OCT4A protein, and further underscore the importance of precise modulation of OCT4A in the regenerative medicine and anticancer therapies.

A Comparison of the Cystotome-Assisted Prechop and Phaco-Chop Phacoemulsification Nucleotomy Techniques.

BACKGROUND The aim of this study was to compare the intra-operative parameters and post-operative outcomes of cataract surgery performed using the cystotome-assisted prechop (CAP) and phaco-chop techniques. MATERIAL AND METHODS Fifty-two eyes with age-related cataract in the CAP group, and 63 eyes in the phaco-chop group were enrolled for analysis in this study, and the surgical outcomes were reported 1 day and/or 1 week, and 1 month post-operatively. RESULTS The CAP technique was associated with statistically significantly lower cumulative dissipated energy compared with the phaco-chop technique (P<0.001). The mean endothelial cell loss in the CAP group was statistically significantly lower than that of the phaco-chop group 1 week (5.6±5.9% versus 8.8±8.7%, P=0.020) and 1 month post-operatively (6.3±6.8% versus 9.8±9.9%, P=0.026). The change in the central corneal thickness between the 2 groups was significantly different at 1 day post-operatively (3.3±3.1% versus 4.9±4.6%, P=0.036). The change in the 8.0 mm central corneal volume between the 2 groups was significantly different at 1 day and 1 week post-operatively (6.5±6.1% versus 10.9±7.9%, P=0.001; 3.2±4.7% versus 5.4±5.7%, P=0.029, respectively). CONCLUSIONS The CAP technique showed lower ultrasound energy consumption and less endothelial damage and corneal edema than the phaco-chop technique. It might therefore prove a cost-effective prechop method for cataract surgery.

Akt­mediated phosphorylation of Oct4 is associated with the proliferation of stem­like cancer cells.

Oct4 protein encoded by POU5F1 plays a pivotal role in maintaining the self­renewal of pluripotent stem cells; however, its presence in cancer cells remains controversial. In the present study, we provided evidence that the transcripts of authentic OCT4 gene (OCT4A) and its multiple pseudogenes were detected in a variety of cancer cell lines. A few major bands were also detected by western blotting using an anti­Oct4A monoclonal antibody. Moreover, an anti­Oct4­pT235 antibody was used to identify a band in the majority of the tested cancer cell lines that coincided with one of the anti­Oct4A bands which was decreasable by a specific shRNA. The Oct4­pT235 signals were also detected in human glioblastoma and liver cancer specimens by immunofluorescence microscopy and immunohistochemistry. U87 glioblastoma cells were cultured in a neural stem cell medium to induce the formation of neurospheres rich in stem­like cancer cells. The levels of Oct4­pT235 in the sphere cells were markedly increased compared to their monolayer parental cells, a result that was accompanied by upregulation of the PI3K­Akt pathway. Akti­1/2, a specific inhibitor of Akt, effectively reduced the level of Oct4­pT235 and attenuated the proliferation of U87 sphere cells. ITE, an agonist of the aryl hydrocarbon receptor, also significantly attenuated the Akt­mediated phosphorylation of Oct4 in glioblastoma and liver cancer cells, and reduced their tumorigenic potential in a xenograft tumor model. Taken together, we concluded that the Akt­mediated phosphorylation of Oct4A or its homolog protein was associated with the proliferation of stem­like cancer cells that may serve as a novel biomarker and drug target for certain types of cancer.

The levels of carbohydrate antigen 19-9 are associated with gender and age in Chinese population.

BACKGROUND: CA 19-9 is one of the most frequently used biomarkers for tumors. METHODS: We analyzed the influential factors of the Carbohydrate Antigen 19-9 (CA 19-9) levels in Chinese general population to better interpret the results of the CA 19-9 screening tests. 36,924 apparently healthy individuals and 1,335 patients with gastrointestinal (GI) tumors were involved in this study. Serum CA 19-9 levels were measured using a Roche Cobas E601. RESULTS: The serum CA 19-9 levels in apparently healthy individuals were correlated with age and gender. Its level was positively correlated with the age of males, while it showed a V-shape curve in female populations. This effect could be due to sex hormones, such as prolactin, which were found to have a negative effect on the level of CA 19-9 in the present study. CONCLUSIONS: Our data indicates that gender and age could affect the CA19-9 serum level. We can set up different cut-off values according to the patient's gender and age, which can help us to get a more individualized representation in different populations.

5,10-Methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) gene polymorphisms and adult meningioma risk.

The causes of meningiomas are not well understood. Folate metabolism gene polymorphisms have been shown to be associated with various human cancers. It is still controversial and ambiguous between the functional polymorphisms of folate metabolism genes 5,10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) and risk of adult meningioma. A population-based case­control study involving 600 meningioma patients (World Health Organization [WHO] Grade I, 391 cases; WHO Grade II, 167 cases; WHO Grade III, 42 cases) and 600 controls was done for the MTHFR C677T and A1298C, MTRR A66G, and MTR A2756G variants in Chinese Han population. The folate metabolism gene polymorphisms were determined by using a polymerase chain reaction­restriction fragment length polymorphism assay. Meningioma cases had a significantly lower frequency of MTHFR 677 TT genotype [odds ratio (OR) = 0.49, 95 % confidence interval (CI) 0.33­0.74; P = 0.001] and T allele (OR = 0.80, 95 % CI 0.67­0.95; P = 0.01) than controls. A significant association between risk of meningioma and MTRR 66 GG (OR = 1.41, 95 % CI 1.02­1.96; P = 0.04) was also observed. When stratifying by the WHO grade of meningioma, no association was found. Our study suggested that MTHFR C677T and MTRR A66G variants may affect the risk of adult meningioma in Chinese Han population.

[Effect of siRNA for mutant p53 gene on biological behavior of gastric cancer cell line SGC7901].

OBJECTIVE: To investigate the effects of silencing the expression of mutant p53 gene with RNA interference technique on biological behavior of gastric cancer SGC7901 cells. METHODS: Recombinant plasmid of mutant p53 gene-targeting siRNA was transfected into gastric cancer SGC7901 cells by Lipofectamine(TM)2000. The expressions of mutant p53 gene mRNA and protein were identified by RT-PCR and Western blot assay. The proliferation of SGC7901 cells and changes of Oxaliplatin (OXA) drug sensitivity were detected by MTT assay. The cell cycle distribution and apoptosis rate were analyzed by flow cytometry. Changes in cell tumorigenicity ability were analyzed by colony formation assay and xenograft tumor formation in nude mice. RESULTS: The expression of mutant p53 in SGC7901 cells was remarkable suppressed by mutant p53-siRNA. The cell growth curve of the transfected group turned to left compared to untransfected group and control group. The cell number of G(0)/G(1) phase of transfected group was decreased by 7.4% and 6.7% respectively, and that of S phase was increased both by 17.2% compared to control group and untransfected group, and the cell apoptosis rate induced by Oxaliplatin was remarkable decreased. The IC(50) of OXA in untransfected group, control group and transfected group were 15.88 µmol/L, 14.32 µmol/L and 20.34 µmol/L respectively. The colony formation rate and tumorigenicity ability in nude mice of transfected group increased remarkably. CONCLUSION: Mutant p53-siRNA can significantly inhibit the expression of mutant p53 in SGC7901 cells, however, the use of RNA interference targeting mutant p53 gene in the treatment of gastric cancer is still uncertain.

Laminin-α1 LG4-5 domain binding to dystroglycan mediates muscle cell survival, growth, and the AP-1 and NF-κB transcription factors but also has adverse effects.

In our previous studies, we showed laminin binds α-dystroglycan in the dystrophin glycoprotein complex and initiates cell signaling pathways. Here, differentiated C2C12 myocytes serve as a model of skeletal muscle. C2C12 cells have a biphasic response to the laminin-α(1) laminin globular (LG) 4-5 domains (1E3) dependent on the concentration used; at low concentrations of 1E3 (<1 µg/ml), myoblast proliferation is increased while higher concentrations (>1 µg/ml) cause apoptosis in myoblasts and differentiated myotubes. This alters the activation of the transcription factors activator protein-1 (AP-1) and NF-κB via laminin-dystrophin glycoprotein complex (DGC)-src-grb2-sos1-Rac1-Pak1-c-jun N-terminal kinase (JNK)p46 and laminin-DGC-Gßγ-phosphatidylinositol 3-kinase (PI3K)-Akt pathways, respectively. A specific antibody against Ser(63) phosphorylated c-jun completely blocks or supershifts the AP-1-DNA binding resulting from laminin binding but only partially blocks or supershifts the AP-1-DNA binding resulting from 1E3. This suggests that AP-1 contains phosphorylated c-jun in the presence of hololaminin but contains a different composition in the presence of 1E3. Nuclear NF-κB was only upregulated by a low concentration of 1E3 and is then diminished by a higher concentration; it also has a biphasic response. Nuclear localization of NF-κB is affected by PI3K/Akt signaling, and DGC associated PI3K activity also shows a biphasic response to 1E3. Furthermore, our data suggest that activation of c-jun N-terminal kinase participates in the cell survival pathway and suggest that NF-κB is involved in both survival and cell death. A model is presented which incorporates these observations.

[Relationship between inflammation and neointimal proliferation after coronary stent implantation in porcine model].

OBJECTIVE: To study the relationship between inflammation and neointimal proliferation after coronary stent implantation in porcine model. METHODS: Twenty normal minipigs were randomly divided into group A (implanted with 316L bare metal stents), group B (implanted with 605L bare metal stents), group C (implanted with PLGA coating 605L stents), and group D (implanted with rapamycin-loaded PLGA coating 605L stents). Each minipig was implanted with two same stents in left anterior descending artery and right coronary artery. Four weeks later, the animals were sacrificed and histomorphometric measurements on the stent-segment coronary arteries were made to calculate the correlation between inflammation area and neointimal area. RESULTS: Group D had the smallest neointimal area [(0.64 +/- 0.38) mm2, P < 0. 001] and inflammation area (median 0.00 mm2, P = 0.009) among all the groups, while there were no statistical differences among group A, B, and C in neointimal area [(2.09 +/- 0.90), (2.11 +/- 1.07), and (1.42 +/- 0.35) mm2 respectively] and in inflammation area (0.22 , 0.21, and 0.09 mm2, respectively). Bivariate correlation analysis showed that the inflammation area was positively correlated with the neointimal area (P < 0.001, correlation coefficient = 0.719). When stent type, mean injury score, and EEL area were adjusted, partial correlations analysis showed that the inflammation area was still positively correlated with the neointimal area (P = 0.01, correlation coefficient = 0.498). CONCLUSION: Inflammation promotes the neointimal proliferation after coronary stent implantation. Sirolimus-eluting stent may reduce the inflammatory response.

[Effects of atenolol and metoprolol on cardiomyocyte apoptosis and related gene expression after acute myocardial infarction in rats].

OBJECTIVE: To compare the beneficial effects of Atenolol and Metoprolol on cardiomyocyte apoptosis and related gene expressions after acute myocardial infarction (AMI) in rats. METHODS: AMI model was established with the ligation of anterior descending coronary artery in 251 randomly selected female SD rats. Twenty-four hours after operation, the 124 survivors were randomly assigned to AMI control group (MI group, n = 43), Atenolol group (group A, 10 mg x kg(-1) d(-1), n = 39), and Metoprolol group (group B, 20 mg x kg(-1) x d(-1), n = 42). Sham operation group (group S, n = 27) was also established. Two subgroup (48 h subgroup and 4 weeks subgroup) was randomly divided in each group according to the time points. Drugs were given to each treatment group by gastric gavage 24 h after ligation. Cardiomyocyte apoptosis was detected with terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and DNA ladder. Bcl-2, bax and caspase-3 genes were detected with immunohistochemistry and Western blot analysis. RESULTS: Compared with AMI control group, myocyte apoptosis rate (MAR) significantly decreased only in infarction area (P < 0.01) in group B. Bcl-2 expression was found to increase in myocytes of infarction, border and non-infarcted areas except for non-infarcted area of group A. Changes of the expressions of bax and caspase-3 was not significant. Four weeks after AMI, MAR was found to decrease significantly in scar, border and non-infarcted areas (P < 0.05, P < 0.01) in both group A and group B. No significant changes of bcl-2, bax and caspase-3 expressions was found except for a significant decrease of bax expression in non-infarcted area of group A. As indicated by Western blot, no significant change of the expressions of caspase-3, bcl-2 and bax were found in myocytes of group A and group B compared with AMI control group; however, bcl-2/bax ratio significantly increased to the same level of sham-operated group (P < 0.05). CONCLUSION: Both Atenolol and Metoprolol treatment can reduce cardiomyocyte apoptosis in infarction/scar, border and non-infarcted areas after AMI, mainly through the increase of bcl-2 expression and bcl-2/bax ratio.

[Beneficial effects of carvedilol on cardiomyocyte apoptosis and bcl-2/bax expression after acute myocardial infarction an experiment with rats].

OBJECTIVE: To investigate the beneficial effects of carvedilol on cardiomyocyte apoptosis and related gene expression after acute myocardial infarction (AMI). METHODS: Eighty-three female SD rats underwent ligation of left anterior descending coronary artery, and were randomly assigned to 2 groups 24 hours later: carvedilol (n = 40, 10 mg.kg(-1).d(-1)was administered via direct gastric gavage 24 hs after the ligation, Group C) and AMI control group (n = 43, normal saline of the same volume was given by gastric gavage, Group MI). Another 27 rats were used as sham operation group (Group S, administered with normal saline too). The rats of each group were killed and their hearts were taken out 48 hours and 4 weeks after observation respectively (MI-48 h, MI-4 week, C-48 h, C-4 week, S-48 h, and S-4 week subgroups). TUNEL and DNA gel electrophoresis were used to detect the cardiomyocyte apoptosis. Immunohistochemistry and Western blotting were used to detect the expression of bcl-2 and bax. RESULTS: The apoptotic indices of the infracted/scar, border and non-infarcted areas at any time-point of Group MI were all significantly higher than those of Group S (all P < 0.05). Only the apoptotic indices of the infracted/scar and border areas of the C-4 week subgroup were significantly lower than those of the MI-4 week subgroup (both P < 0.05), and were close to those of the non-infarcted area. DNA gel electrophoresis showed that the positive rate of Group S at any time-point were both 0, the positive rate of MI-48 h subgroup and C-48 h subgroup were both significantly higher than that of Group S (both P < 0.05) without significant difference between these 2 groups, and the positive rates of the MI-4 week subgroup and C-4 week subgroup were both 0. Immunohistochemistry showed that the bax gene expression was slightly to significantly increased in the infarcted/scar, border, and non-infarcted areas of the MI-48 h and MI-4 week subgroups. The bcl-2 expression was significantly increased only in the infracted area of the MI-48 h subgroup. The bcl-2 expression was slightly increased in the infracted and border areas of the C-48 h subgroup and the bax expression was significantly decreased in the infracted/scar area of the C-4 week subgroup. Western blotting showed that (1) the bcl-2 expression of the S-4 week subgroup was significantly higher than that of the S-48 h subgroup (P < 0.05), (2) the bcl-2 expression and bax expression of the MI-48 h subgroup were significantly higher than that of the S-48 h subgroup (P < 0.05 - 0.01), the bcl-2/bax ratio of the MI-48 h subgroup was significantly lower that that of the S-48 h subgroup, however, there were no significant differences in the bcl-2 and bax expression and bcl-2/bax ratio between the MI-4 week subgroup and S-48 h subgroup (all P > 0.05), and (3) There were no significant difference in the bcl-2 and bax expression between Group A and Group S (all P > 0.05), however, the bcl-2/bax ratios at the 2 time-points of Group C were both significantly higher than those of Group MI. CONCLUSION: Cardiomyocyte apoptosis occurs in the infarction/scar, border and non-infarcted areas after AMI. Prolonged treatment with carvedilol reduces cardiomyocyte apoptosis in the scar and border areas and increases the expression ratio of bcl-2/bax.

Binding of laminin alpha1-chain LG4-5 domain to alpha-dystroglycan causes tyrosine phosphorylation of syntrophin to initiate Rac1 signaling.

Previously, a signaling pathway was described [Oak, Zhou, and Jarrett (2003) J. Biol. Chem. 278, 39287-39295] that links matrix laminin binding on the outside of the sarcolemma to Grb2 binding to syntrophin on the inside surface of the sarcolemma and by way of Grb2-Sos1-Rac1-PAK1-JNK ultimately results in the phosphorylation of c-jun on Ser(65). How this signaling is initiated was investigated. Grb2-binding to syntrophin is increased by the addition of either laminin-1 or the isolated laminin alpha1 globular domain modules LG4-5, a protein referred to as E3. This identifies the LG4-5 sequences as the region of laminin responsible for signaling. Since laminin alpha1 LG4 is known to bind alpha-dystroglycan, this directly implicates alpha-dystroglycan as the laminin-signaling receptor. E3 or laminin-1 increase Grb2-binding and Rac1 activation. In the presence of E3 or laminin-1, syntrophin is phosphorylated on a tyrosine residue, and this increases and alters Grb2 binding. The alpha-dystroglycan antibody, IIH6, which blocks binding of laminins to alpha-dystroglycan, blocks both the laminin-induced Sos1/2 recruitment and syntrophin phosphorylation, showing that it is alpha-dystroglycan binding the LG4-5 region of laminin that is responsible. The C-terminal SH3 domain of Grb2 (C-SH3) binds only to nonphosphorylated syntrophin, and phosphorylation causes the Grb2 SH2 domain to bind and prevents SH3 binding. Syntrophin, tyrosine phosphate, beta-dystroglycan, and Rac1 all co-localize to the sarcolemma of rat muscle sections. A model for how this phosphorylation may initiate downstream events in laminin signaling is presented.

[Comparison of doxycycline, losartan, and their combination in the prevention of post-infarction remodeling in rats].

OBJECTIVE: To compare the effects of doxycycline, losartan, and their combination in the prevention of left ventricular remodeling (LVRM) after acute myocardial infarction (AMI) in rats. METHODS: Twenty-four hours after the induction of AMI, the 254 survival rats were randomly assigned to the following groups and received drug treatment: (1) AMI controls (n=64), (2) doxycycline (30 mg x kg(-1) x d(-1), n = 63), (3) losartan (10 mg x kg(-1) x d(-1), n = 62), and (4) combination doxycycline and losartan (30 and 10 mg x kg(-1) x d(-1) respectively, n = 65) treatment groups. Also, sham operated rats (n = 30) were selected randomly. Each group was further divided into three subgroups of 1, 2, and 4 weeks of treatment. After the completion of treatment, hemodynamic studies were performed. Then, the heart of rat was fixed and analyzed pathologically. RESULTS: Exclusive of the dead rats and the hearts with the myocardial infarction size < 35% or > 50%, complete experimental data were obtained in 157 rats. Besides sham operated rats, there was no significant difference in myocardial infarction sizes among the 12 subgroups of AMI control and drug treatment groups (P> 0.05). Compared with sham operated rats, left ventricular end diastolic pressure (LVEDP) and left ventricular absolute weight and relative weight (LVAW and LVRW) were significantly increased in 1, 2, and 4 week subgroups of AMI controls (P < 0.05, P < 0.01, and P < 0.001, respectively), with LVEDP elevated more significantly in 4 week than in 1 and 2 week subgroups (P < 0.01); whereas the maximum rising and dropping rate of left ventricular pressure (+/-dp/dt) and its corrected value by left ventricular systolic pressure (+/-dp/dt/LVSP) were all significantly decreased only at 4 week subgroup of AMI controls (P < 0.001). Compared with AMI controls group, LVEDP was significantly decreased in all 1, 2, and 4 week subgroups of the three treatment groups (P < 0.05, P < 0.01, and P < 0.001, respectively); LVAW and LVRW were significantly decreased in 2 and 4 week subgroups of losartan and combination groups (P < 0.05, P < 0.01, P < 0.001, respectively), and in only 4 week subgroup in doxycycline (P < 0.05, P < 0.01, and P < 0.001, respectively); whereas the maximum dropping rate of left ventricular pressure and the corrected value of left ventricular pressure rising and dropping rate were significantly increased only in 4 week subgroups of all three treatment groups (P < 0.05, P < 0.01, respectively). There is no significant difference in all indices above among the three treatment groups at all three time points (P > 0.05). CONCLUSION: It is indicated that doxycycline can prevent left ventricular remodeling and improve its systolic and diastolic function after AMI in rats, with the equivalent effect to that of losartan. There seems no additive effect when the two drugs are used in combination.

Acquisition of Fas resistance by Fas receptor mutation in a childhood B-precursor acute lymphoblastic leukemia cell line, MML-1.

Programmed cell death, or apoptosis, is a physiological means of eliminating unwanted cells and maintaining immune homeostasis. One of the primary mechanisms is the Fas (CD95)/Fas ligand system. Its inactivation in normal cells and malignant cells may be involved in malignant trans-formation and refractory clinical course, respectively. We established a Fas resistant clone and evaluated the molecular basis for its mechanism of resistance. The Fas-sensitive leukemia cell line, MML-1, was established from a child with B-precursor acute lymphoblastic leukemia. A Fas resistant clone, MML-1R, was obtained by co-culture selection with anti-Fas antibody CH-11. Flow cytometry analysis showed both cell lines had equivalent expression of cell surface CD13, 15, 19, 22 and Fas receptor. Western blot analysis revealed equal expression of FADD (Fas-associated death domain protein), caspase-3 and -8. MML-1 was quite sensitive to both CH-11 and etoposide-induced apoptotis. By contrast, MML-1R had similar sensitivity to etoposide but no response to CH-11. Fas receptor mutation analysis showed a heterozygous death domain A --> G point mutation at 1009 bp, causing a switch from glutamine to glycine at amino acid 256. Immunoprecipitation assay showed decreased binding of Fas to FADD. We also found that etoposide bypassed Fas-FADD interaction in MML-1R by activating caspase-8 and caspase-3. These results indicate that Fas resistance can result from mutations of the gene encoding the Fas receptor which result in decreased FADD binding, thereby blocking formation of the death inducing signaling complex. Screening for similar Fas mutations in therapy resistant malignancies would lead to a better understanding of tumorigenesis and recurrence.

[Comparison of doxycycline, losartan, and their combination on the expression of matrix metalloproteinase, tissue inhibitor of matrix metalloproteinase, and collagen remodeling in the noninfarcted myocardium after acute myocardial infarction in rats].

OBJECTIVE: To compare the effects of matrix metalloproteinase (MMP) inhibitor doxycycline, losartan, and their combination on the expression of MMP-8, 13, tissue inhibitor of MMP-1, 2 (TIMP-1, 2), and collagen remodeling in the noninfarcted myocardium after acute myocardial infarction (AMI) in rats. METHODS: Two hundred and fifty-four AMI rats, induced by left coronary ligation, were randomly assigned to the following groups: (1) AMI controls group (n = 64); (2) doxycycline group (30 mg x kg(-1) x d(-1), n = 63); (3) losartan group (10 mg x kg(-1) x d(-1), n = 62); (4) concomitant doxycycline and losartan group (30 and 10 mg x kg(-1) x d(-1) respectively, n = 65); and (5) Sham-operated rats (n = 30), which were randomly selected to serve as noninfarction controls. Each group was further divided into three subgroups of 1, 2, and 4 weeks that received treatment. After the completion of treatment, the rats were killed. The mRNA and protein expression of MMPs and TIMPs in the noninfarcted myocardium were quantified by RT-PCR and Western blot, respectively. The type I and type III collagen volume fraction (CVF) of the noninfarced myocardium were assessed immunohistochemically. RESULTS: No significant difference existed in myocardial infarction sizes among the 12 subgroups of AMI controls and the three treatment groups (42%-48%, all P > 0.05). Compared with sham operated rats, the mRNA and protein expression of MMP-8 and 13 significantly increased by 39%-183% in all three subgroups of AMI controls (all P < 0.05), except both of their mRNA expressions in 2-week subgroups; the mRNA and protein levels of TIMP-1 increased only in 1-week subgroup of AMI controls by 104% and 67%, respectively (both P < 0.05); the mRNA of TIMP-2 increased in all 1, 2, and 4-week subgroups by 144%-232% (all P < 0.05), but its protein expression lagged and only enhanced in 2 and 4-week subgroups of AMI controls by 231% and 332%, respectively (both P < 0.05). Meanwhile, both type I and type III CVF of noninfarcted myocardium significantly increased in all three subgroups of AMI controls (type I CVF: 3.01%-5.64% vs 1.53%-1.67%, P < 0.01-0.001; type III CVF: 2.19%-4.42% vs 1.46%-1.59%, P < 0.05-0.001), with type I CVF being higher in 4-week than in 1 and 2-week subgroups (5.64% vs 3.01% and 3.02% respectively, all P < 0.05). Compared with AMI controls, all three kinds of treatment significantly reduced the increased mRNA and protein expressions of MMP-8, 13 and TIMP-1, 2 after AMI by 14%-60% (all P < 0.05), as well as type I/III CVF in their 2 and 4-week subgroups (type I CVF: 1.56%-2.38% vs 3.02%-5.64%, P < 0.05-0.001; type III CVF: 1.92%-2.65% vs 4.19%-4.42%, P < 0.05-0.01), except for doxycycline's effect on type III CVF in any of its three subgroups (all P > 0.05). Among the three treatment groups, significant differences existed in the above mentioned indicators only at some subgroup levels (all P < 0.05). CONCLUSIONS: Like losartan, doxycycline can also suppress the enhanced mRNA and protein expression of MMP-8, 13 and TIMP-1, 2, and reduce type I collagen deposition in the noninfarcted myocardium after AMI in rats. However, it has no effect on type III collagen deposition.

Laminin-alpha1 globular domains 3 and 4 induce heterotrimeric G protein binding to alpha-syntrophin's PDZ domain and alter intracellular Ca2+ in muscle.

Alpha-syntrophin is a component of the dystrophin glycoprotein complex (DGC). It is firmly attached to the dystrophin cytoskeleton via a unique COOH-terminal domain and is associated indirectly with alpha-dystroglycan, which binds to extracellular matrix laminin. Syntrophin contains two pleckstrin homology (PH) domains and one PDZ domain. Because PH domains of other proteins are known to bind the betagamma-subunits of the heterotrimeric G proteins, whether this is also a property of syntrophin was investigated. Isolated syntrophin from rabbit skeletal muscle binds bovine brain Gbetagamma-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, alpha- and beta-dystroglycan, or dystrophin precipitate a complex with Gbetagamma from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of Gbetagamma binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of Gsalpha and Gbetagamma occurs and active Gsalpha, measured as GTP-gamma35S bound, decreases. Because intracellular Ca2+ is elevated in Duchenne muscular dystrophy and Gsalpha is known to activate the dihydropyridine receptor Ca2+ channel, whether laminin also altered intracellular Ca2+ was investigated. Laminin-1 decreases active (GTP-gammaS-bound) Gsalpha, and the Ca2+ channel is inhibited by laminin-1. The laminin alpha1-chain globular domains 4 and 5 region, the region bound by DGC alpha-dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to alpha-dystroglycan inhibits Gbeta binding by syntrophin in C2C12 myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor.

Galectin-1 supports survival of naive T cells without promoting cell proliferation.

Naive T cells do not proliferate but remain alive in vivo. In contrast, naive T cells rapidly die in an in vitro culture, suggesting that some factors that are present at the sites of naive T cell circulation in vivo but missing in the bovine serum-containing culture medium, are necessary for their survival. The present study was designed to search for such factors. By functional screening of the cDNA library from murine lymph node-derived stromal cells (LNS) that effectively support the survival of naive T cells, we found that nascent polypeptide-associated complex (alpha-NAC) promoted T cell survival. A conditioned medium derived from culture supernatant of Cos7 cells transfected with alpha-NAC gene supported T cell survival, indicating that alpha-NAC induced production of soluble factor(s) that were secreted into the medium. By examining the products that were cloned from a functional screening of the cDNA library from alpha-NAC-transfected NIH3T3 cells but were not detected in that from control vector-transfected cells, galectin-1 was found as a soluble factor in the conditioned medium of the LNS. Our study demonstrates the novel role of galectin-1 as a soluble factor that functions to maintain naive T cell survival without inducing cell proliferation.

[Effects of doxycycline on the expression of matrix metalloproteinase and tissue inhibitor of MMP in myocardium after acute myocardial infarction: an animal experiment with rats].

OBJECTIVE: To assess the effects of doxycycline, a matrix metalloproteinase (MMP) inhibitor, on the expression of MMP-8 and -13, and tissue inhibitor of MMP-1 and -2 (TIMP-1 and -2), and on collagen remodeling in the noninfarcted myocardium after acute myocardial infarction (AMI). METHODS: 212 female SD rats underwent ligation of left coronary artery so as to cause AMI. Twenty-four hours after the 127 surviving rats were randomly divided into 2 groups: AMI control group (n = 64) and AMI doxycycline treatment (30 mg.kg(-1).d(-1)) group (n = 63). Then the rats in these 2 groups were re-divided into 3 sub-groups of 18 approximately 24 rats according to the course of treatment: 1-week, 2-week, and 4-week subgroups. Thirty female SD rats underwent sham operation and then were re-divided into 3 groups of 10 rats: 1-week, 2-week, and 4-week subgroups. By the end of each course the rats were killed and their hearts were taken out. A piece of myocardium from the left ventricle was taken and stained with hematoxyline and eosin to examine the area of infarction. The mRNA expressions of MMP and TIMP in the non-infarcted myocardium were detected by RT-PCR. The protein expressions of MMP and TIMP in the non-infarcted myocardium were detected by Western blotting. The type I and type III collagen volume fraction (CVF) in the noninfarcted myocardium was examined by immunohistochemistry. RESULTS: There was no significant difference in the MI size among the 6 subgroups of AMI control and doxycycline groups (42% approximately 48%, all P > 0.05). Compared with sham operated rats, the mRNA expressions of MMP-8 and 13 were significantly increased by 54% approximately 183% in all three subgroups of AMI controls (all P < 0.05), and the mRNA expressions of MMP-8 and 13 in the 1-week and 4-week were significantly increased by 39% approximately 160% in these 3 subgroups (all P < 0.05). The mRNA and protein expressions of TIMP-1 were enhanced only in the 1-week subgroup of AMI controls by 104% and 67% respectively (both P < 0.05). The mRNA expression of TIMP-2 was significantly increased in all 1-, 2-, and 4-week subgroups by 144% approximately 232% (all P < 0.05), however, the protein expression of TIMP-2 was increased only in the 2- and 4-week subgroups of AMI control group by 231% and 332% respectively (both P < 0.05). Compared with the sham operation group, both the type I CVF and type III CVF of the noninfarcted myocardium were significantly increased in all three subgroups of the AMI control group (type I CVF: 3.01% approximately 5.64% vs 1.53% approximately 1.67%, P < 0.01 approximately 0.001; type III CVF: 2.19% approximately 4.42% vs 1.46% approximately 1.59%, P < 0.05 approximately 0.001), with type I CVF being higher in the 4-week subgroup than in the 1 and 2-week subgroups (5.64% vs 3.01% and 3.02% respectively, all P < 0.05). In comparison with the 3 subgroups of the AMI control group, the mRNA and protein expressions of MMP-8 and 13 and TIMP-1 and 2 after AMI in the doxycycline group were significantly lower by 14% approximately 51% (all P < 0.05). In comparison with that in the AMI control group, the type I collagen deposition in the doxycycline group was significantly lower 2 and 4 weeks after AMI (1.94% vs 3.02% and 1.97% vs 5.64% respectively, P < 0.01 approximately 0.001), however, there was no significant difference in type III CVF among the different subgroups (all P > 0.05). CONCLUSION: Doxycycline suppresses the enhanced mRNA and protein expressions of MMP-8 and 13 and TIMP-1 and 2, and type I collagen deposition in the noninfarcted myocardium after AMI, but it has no effect on type III collagen deposition.

[Chronic sheep modal for pulmonary valve implantation with domestic bileaflet mechanical prosthesis].

OBJECTIVE: The aim of this study was to identify the long-term character of the domestic bileaflet mechanical valve in the chronic implanted sheep model and to evaluate the potential value of the modal. METHODS: Six adult sheep underwent implanted mechanical bileaflet valve in pulmonary position under the cardio-pulmonary bypass with beating heart. The chronic implanted sheep model was built up and observed in the respects of a long-term survival, function of prosthesis and pathological specimen. RESULTS: Six adult sheep survived with good condition after operation. The average survival period of six sheep was (221 +/- 208) days. Two sheep were postoperatively sacrificed in 41 and 71 days, respectively. The necropsy revealed normal valve function without thrombosis, periprosthetic leakage and overgrowth of fibrous tissue. One sheep died from dysfunction of prosthetic valve at the postoperative 196 days. The reason was the prosthetic thrombosis with slight overgrowth of fibrous tissue in periprosthesis. The other two sheep died from severe anemia at the postoperative 196 days and 234 days, and the autopsy revealed no abnormal finding else. And one remained to survive with good condition up to now (over 617 days) and was checked by Doppler echocardiogram twice at the postoperative 438 days and 479 days, respectively. The results showed normal function of the bileaflet valve in pulmonary position. CONCLUSION: The long-term good effects would be achieved by using the implanted new domestic bileaflet valve in pulmonary position of sheep.

Skeletal muscle signaling pathway through the dystrophin glycoprotein complex and Rac1.

The dystrophin glycoprotein complex has been proposed to be involved in signal transduction. Here we have shown that laminin binding causes syntrophin to recruit Rac1 from the rabbit skeletal muscle. Laminin-Sepharose and syntrophin-Sepharose bind a protein complex containing Rac1 from the muscle membranes. The presence of heparin, which inhibits laminin interactions, prevents recruitment of Rac1. The dystrophin glycoprotein complex recruits Rac1 via syntrophin through a Grb2.Sos1 complex. A syntrophin antibody also prevents recruitment of Rac1, suggesting that the signaling complex requires syntrophin. PAK1 is in turn bound by Rac1. c-Jun NH2-terminal kinase-p46 is phosphorylated and activated only when laminin is present, and the p54 isoform is activated when laminin is depleted or binding is inhibited with heparin. In the presence of laminin, c-Jun is activated in both skeletal muscle microsomes and in C2C12 myoblasts, and proliferation increases in C2C12 myoblasts. We postulate that this pathway signals muscle homeostasis and hypertrophy.

Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro.

Little is known about the homeostatic mechanisms by which the levels of peripheral lymphocytes are maintained. The survival of naïve T cells in vivo must be maintained by some factors that have not been characterized in an in vitro culture system. In this study, we established a culture system of stromal cells derived from murine lymph nodes and investigated the action of the stromal cells in supporting the survival of resting T cells in vitro. Most of the T cells cocultured with the stromal cells did not die, and the supernatant of cultured stromal cells increase the viability of T cells. This T-cell survival-supporting activity was maintained for more than 7 days. Although interleukin (IL)-4, IL-6, IL-7, and interferon-beta also rescued peripheral T cells from spontaneous cell death, medium-soluble and heat-sensitive factor(s) derived from the stromal cells supported the survival of T cells more effectively and for a longer time than did these cytokines. T cells maintained in the culture system with the stromal cells appeared to remain in a resting G0/G1 state and did not show remarkable DNA synthesis. From these results, it is presumed that some soluble factor(s) other than the tested cytokines that have been identified as supporting T-cell survival are produced from lymph node stromal cells. These factor(s) play an important role in maintenance of resting T cells.