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Background: Kawasaki disease (KD) is an acute systemic vasculitis of unknown etiology. Cyclophilin A (CypA), also known as PPIA, has been identified to play a vital role in the pathogenesis of card...

BackgroundTemozolomide can be used in the treatment of pituitary adenoma. Its efficacy can be evaluated by the expression of 06-methylguanine-DNA methyltransferase (MGMT). However, the previous study population was mainly adult. This study was the first to evaluate the expression of MGMT in children and adolescents with pituitary adenomas.MethodsThe clinical features and biological characteristics of 38 cases of pituitary adenoma in children and adolescents were analyzed retrospectively. The expression of MGMT, Ki-67, p53 was marked by immunohistochemical staining.ResultsIn this cohort, the expression of MGMT protein is 16/38 (42.11%). The low expression rate of MGMT in children and adolescent somatotroph adenomas was only 11.11%, which was much lower than that of adults. Ki-67 expression range of 1% -10%（mean 4.24 ± 2.71%）. The positive rate of p53 was 22/38 (57.89%). The statistical analysis showed that the expression of MGMT was related to the diameter of the tumor (P=0.01), the diameter of the tumor was large, and the expression of MGMT was high. The expression of MGMT was not associated with age, sex, tumor type, invasiveness, texture, apoplexy, recurrence, and expression of p53 and Ki-67.ConclusionSomatotroph adenomas, large-diameter children and adolescent pituitary adenomas may be not suitable for temozolomide treatment. To determine whether temozolomide responds to salvage therapy in children and adolescents with pituitary adenomas, MGMT expression should be assessed in all pituitary adenomas, the time span between specimen collection and temozolomide treatment needs to be considered because of the instability of MGMT protein expression.

Background Kawasaki disease (KD) is an acute febrile illness of early childhood. The exact etiology of the disease remains unknown. At present, research on KD is mostly limited to susceptibility genes, infections, and immunity. However, research on the correlation between gut microbiota and KD is rare. Methods Children with a diagnosis of acute KD and children undergoing physical examination during the same period were included. At the time of admission, the subjects’ peripheral venous blood and feces were collected. Faecal samples were analyzed for bacterial taxonomic content via high-throughput sequencing. The abundance, diversity, composition, and characteristic differences of the gut microbiota in KD and healthy children were compared by alpha diversity, beta diversity, linear discriminant analysis and LDA effect size analysis. Blood samples were used for routine blood examination, biochemical analysis, and immunoglobulin quantitative detection. Results Compared with the control group, the community richness and structure of gut microbiota in the KD group was significantly reduced (Chao1 richness estimator, mean 215.85 in KD vs. mean 725.76 in control, p < 0.01; Shannon diversity index, mean 3.32 in KD vs. mean 5.69 in control, p < 0.05). LEfSe analysis identified two strains of bacteria significantly associated with KD: Bacteroidetes and Dorea. Bacteroidetes were enriched in healthy children (mean 0.16 in KD vs. mean 0.34 in control, p < 0.05). Dorea was also enriched in healthy children but rarely existed in children with KD (mean 0.002 in KD vs. mean 0.016 in control, p < 0.05). Compared with the control, IgA and IgG in the KD group decreased (IgA, median 0.68 g/L in KD vs. median 1.06 g/L in control, p < 0.001; IgG, median 6.67 g/L in KD vs. median 9.71 g/L in control, p < 0.001), and IgE and IgM levels were not significantly changed. Conclusions Dysbiosis of gut microbiota occurs in children with acute KD and may be related to the etiology or pathogenesis of KD. It is worth noting that for the first time, we found that Dorea, a hydrogen-producing bacterium, was significantly reduced in children with acute KD. Overall, our results provide a theoretical basis for the prevention or diagnosis of KD based on intestinal microecology.

Tourette syndrome (TS) is a complex neuropsychiatric disorder with chronic motor and vocal tics. Though the etiology is elusive, strong evidence for a genetic contribution to TS has been established. To date, various chromosomal or genetic alterations have been implicated in its pathogenesis. Recently, the deletion in the arylacetamide deacetylase gene (AADAC) was reported to be associated with TS. To investigate the association between the AADAC gene variants and TS, we conducted genetic analysis of the AADAC gene in 200 Han Chinese patients and 300 ethnicity-matched normal controls. Two variants, including a heterozygous splice-site variant, c.361 + 1G > A (rs762169706), and a missense variant, c.744A > T (p.R248S, rs186388618), were identified in two unrelated patients. The c.361 + 1G > A variant, absent in 300 ethnicity-matched controls, led to the deletion of exon 2 in AADAC mRNA, probably associated with development of TS. The c.744A > T variant, predicted to be damaging, was identified in two normal controls. The findings indicate that the AADAC gene c.361 + 1G > A variant may be a potential candidate factor for TS development, though further investigations are warranted.

Recent studies have found that viral myocarditis (VMC) associated with coxsackievirus B3 (CVB3) causes autophagy activation after infection, but the specific mechanism is not clear. The present study demonstrated that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB)/mammalian target of rapamycin (mTOR) signaling pathway participates in CVB3-induced autophagy. We found that the light chain 3 (LC3)-II/LC3-I ratio was increased and p62 and p-mTOR were altered at different times during CVB3 infection. To further assess the effects of this signaling pathway on CVB3 infection and viral replication, we selected 24 h post-inoculation (h.p.i.) as our research time point to conduct our next study. We inhibited the function of PI3K, Akt1 and mTOR. The outcome showed that inhibition of PI3K with ZSTK474 alleviated autophagy and decreased CVB3 mRNA replication and VP1 expression. Inhibition of mTOR with rapamycin promoted autophagy and viral mRNA replication but did not impact VP1 expression. Inhibition of Akt with MK2206 aggravated autophagy induced by viral infection. In our research, p62 exhibited a decrease at the beginning of infection but then increased as infection time increased. This finding may serve as a clue to elucidate the function of autophagy at different times of infection. However, the details merit further study. In conclusion, our findings suggest that the PI3K/Akt/mTOR signaling pathway participates in the process of autophagy induced by CVB3 infection. This finding may provide a new perspective of CVB3-induced autophagy.

Familial renal glucosuria (FRG) is a rare condition that involves isolated glucosuria despite normal blood glucose levels. Mutations in the solute carrier family 5 member 2 (SLC5A2) gene, which encodes sodium-glucose cotransporter 2 (SGLT2), have been reported to be responsible for the disease. Genetic testing of the SLC5A2 gene was conducted in a Chinese family with FRG. A number of online tools were used to predict the potential effect of the identified mutations on SGLT2 function. Additionally, the SLC5A2 mutations previously reported in PubMed were summarized. A novel compound heterozygous mutation (c.514T>C, p.W172R; c.1540C>T, p.P514S) of the SLC5A2 gene in a Chinese child with FRG was identified. In total, 86 mutations of the SLC5A2 gene have been reported to be associated with FRG. The novel compound heterozygous mutation (c.514T>C, p.W172R; c.1540C>T, p.P514S) of the SLC5A2 gene may be responsible for the onset of FRG. The present study provides a starting point for further investigation of the molecular pathogenesis of the SLC5A2 gene mutation in patients with FRG.

Coxsackievirus B3 (CVB3) infection has been shown to stimulate autophagy. We have demonstrated that the inhibition of phosphoinositide 3‑kinase (PI3K)/protein kinase B/mammalian target of rapamycin complex (mTORC) signaling pathway could affect the autophagic reaction induced by CVB3 infection in our previous study. However, the processes associating autophagy and CVB3 replication remain to be determined. In the present study, CVB3‑induced autophagy and its impact on viral replication were investigated. Rapamycin (inhibitor of mTOR) and ZSTK474 (inhibitor of PI3K) were used to change the autophagic reaction caused by CVB3 in Hela cells at different post‑infection (p.i.) time points (6, 9, 12 and 24 h p.i.), meanwhile, we detected the CVB3 mRNA replication and CVB3 capsid protein VP1 expression following the change of autophagy. Here, it was showed that ZSTK474 and Rapamycin promoted CVB3‑induced autophagy, as well as decreasing CVB3 mRNA replication and CVB3 capsid protein VP1 expression at 6 and 9 h p.i. ZSTK474 also alleviated CVB3‑induced autophagy, and decreased CVB3 mRNA replication and VP1 expression at 12 and 24 h p.i. However, Rapamycin continued to promote CVB3‑induced autophagy and increase CVB3 mRNA replication at 12 and 24 h p.i, as well as increase VP1 expression at 12 h, but not at 24 h, p.i. In the present study, we found Rapamycin and ZSTK474 have differential effects at different p.i. time‑points regarding CVB3 replication and CVB3‑induced autophagy. This indicates that the association between CVB3‑induced autophagy and viral replication depends on the infection time. During the early course of infection, autophagy may help host cells clear the virus, thereby providing protection, whereas when the infection time increases, autophagy may be exploited for viral replication.

Kawasaki disease (KD) is an acute systemic vasculitis that predominantly affects children and can result in coronary artery lesions (CALs). Thrombomodulin (TM) is a critical cofactor in the protein C anticoagulant system. The TM C1418T (rs1042579) polymorphism is associated with a high risk of cardiac-cerebral vascular diseases. But the association of the TM C1418T polymorphism with susceptibility to KD, CAL formation, and intravenous immunoglobulin (IVIG) resistance is still unclear. In our study, we examined the TM C1418T polymorphism in 122 children with KD and 126 healthy children and revealed the correlation between the TM C1418T polymorphism and KD, CAL formation, and IVIG resistance.

Kawasaki disease (KD) is an autoimmune disorder that can induce coronary artery aneurysms, particularly in the case of delayed diagnosis and/or treatment. Early diagnosis is important for treatment and reduces the risk of heart injury. The aim of the present study was to identify differentially expressed genes by comparing the levels of gene expression in human umbilical vein endothelial cells following treatment with plasma from healthy individuals and patients with acute or convalescent KD. Following comparison of the control and acute KD groups, 385 up-regulated and 537 down-regulated genes were identified in the acute KD group. In the convalescent group, 505 and 879 genes were up-regulated and down-regulated, respectively, relative to the control group. Genes involved in the immune system and cell growth factors were up-regulated, while genes functioning in methylation were down-regulated, following treatment with KD plasma. In addition, five potential candidate molecular markers of KD, C-X-C motif chemokine ligand 2 (CXCL2), interleukin (IL) 8, tripartite motif containing 58 (TRIM58), immunoglobulin superfamily member 3 (IGSF3) and runt related transcription factor 1 (RUNX1) were identified by microarray analysis and verified using quantitative polymerase chain reaction. A significant positive correlation was identified between the neutrophil polys and expression levels of four of these candidate genes, including CXCL2, IL8, TRIM58, and IGSF3 (all P

Coxsackievirus B3 (CVB3) is a common human pathogen for acute myocarditis, pancreatitis, non-septic meningitis, and encephalitis; it induces a direct cytopathic effect (CPE) and apoptosis on infected cells. The Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT/PKB)/mammalian target of Rapamycin (mTOR) signaling pathway regulates several cellular processes and it is one of the most important pathways in human networks. However, the effect and mechanism of PI3K/AKT/mTOR signaling pathway in CVB3 infected cells are poorly understood. In this study, we demonstrate that inhibition of PI3K/AKT/mTOR signaling pathway increased CVB3-induced CPE and apoptosis in HeLa cells. The activity of downstream targets of PI3K and mTOR is attenuated after CVB3 infection and inhibitors of PI3K and mTOR made their activity to decrease more significantly. We further show that LY294002 and Rapamycin, the inhibitor of PI3K and mTOR respectively, promote CVB3-induced CPE and apoptosis. Taken together, these data illustrate a new and imperative role for PI3K/AKT/mTOR signaling in CVB3 infection in HeLa cells and suggest an useful approach for the therapy of CVB3 infection.

This work illustrated the mechanism contributing to the process of Phosphatidylinostiol 3-kinase (PI3K)/protein kinase B (PKB)/mammalian target of rapamycin (mTOR) signaling pathway, which has been demonstrated to play an important role in virus-induced apoptosis, which contributes to the Viral Myocarditis (VMC) pathogeneses. We examined the expression of Bax, Bim, caspase-3, caspase-9, and viral replication after Coxsackievirus B3 (CVB3) infection using the mTOR inhibitor and PI3K inhibitor pretreated HeLa cells, respectively. Apoptosis in different groups was determined by flow cytometry. Bax, Bim, caspase-9, and caspase-3 were examined by semiquantitative polymerase chain reaction (PCR) and Western blot analysis. The expression of CVB3 mRNA and viral capsid protein VP1 were analyzed by semiquantitative PCR and Western blot analysis distinctively. We found that rapamycin and LY294002 promote CVB3-induced cytopathic effect (CPE) and apoptosis. CVB3 replication in host cells is mediated in mRNA and protein expression by rapamycin and LY294002. Moreover, comparing with controls, at 12 and 24 h of postinfection (p.i.), Bim and Bax expression increased in cells after treated with rapamycin or LY294002, which also stimulates the activation of procaspase-9, and the CVB3-induced caspase-3 self-cleavage. However, in the meantime, the mRNA expression of caspase-9 and caspase-3 did not have an obvious change. In summary, our results demonstrated that the mTOR-signaling pathway plays an important role in CVB3-induced CPE and apoptosis, which is indispensable in VMC, via regulating Bim, Bax, caspase-9, caspase-3, and viral replication. Our findings may provide a new perspective and a deeper understanding of the mechanism of CVB3-induced apoptosis which, in turn, may help with the development of new therapy for the CVB3 infection.

Background A clinical overlap between essential tremor (ET) and Parkinson's disease (PD) has prompted investigation whether these disorders share common genetic factors. The paired-like homeodomain transcription factor 3 gene (PITX3) has been shown to play an important role for the differentiation and survival of midbrain dopaminergic neurons in the substantia nigra pars compacta. The preferential degeneration of those dopaminergic neurons is the pathological hallmark in PD. Aims of the study The purpose of this study was to evaluate whether PITX3 variants are related to susceptibility of ET in Chinese Han population. Methods Genetic analysis of two variants rs3758549 and rs4919621 of the PITX3 gene was conducted in 200 Chinese Han patients with ET and 426 controls. Results We did not identify any statistically significant difference in either genotypic or allelic frequencies of variants between the ET patients and control cohort (all P > 0.05). Haplotype analysis of two variants in the PITX3 gene showed no potential association between the haplotypes and risk of ET (all P > 0.05). Conclusions Our data suggest that PITX3 variants rs3758549 and rs4919621 are not associated with ET in Chinese Han population.

Eukaryotic initiation factor 3a (eIF3a) has been suggested to play a regulatory role in mRNA translation. Decreased eIF3a expression has been observed in differentiated cells while higher levels have been observed in cancer cells. However, whether eIF3a plays any role in differentiation and development is currently unknown. Here, we investigated eIF3a expression during mouse development and its role in differentiation of colon epithelial cells. We found that eIF3a expression was higher in fetal tissues compared with postnatal ones. Its expression in intestine, stomach, and lung abruptly stopped on the 18th day in gestation but persisted in liver, kidney, and heart throughout the postnatal stage at decreased levels. Similarly, eIF3a expression in colon cancer cell lines, HT-29 and Caco-2, drastically decreased prior to differentiation. Enforced eIF3a expression inhibited while knocking it down using small interference RNA promoted Caco-2 differentiation. Thus, eIF3a may play some roles in development and differentiation and that the decreased eIF3a expression may be a pre-requisite of intestinal epithelial cell differentiation.

Fkbp52 and Fkbp51 are tetratricopeptide repeat proteins found in steroid receptor complexes, and Fkbp51 is an androgen receptor (AR) target gene. Although in vitro studies suggest that Fkbp52 and Fkbp51 regulate hormone binding and/or subcellular trafficking of receptors, the roles of Fkbp52 and Fkbp51 in vivo have not been extensively investigated. Here, we evaluate their physiological roles in Fkbp52-deficient and Fkbp51-deficient mice. Fkbp52-deficient males developed defects in select reproductive organs (e.g. penile hypospadias and prostate dysgenesis but normal testis), pointing to a role for Fkbp52 in AR-mediated signaling and function. Surprisingly, ablation of Fkbp52 did not affect AR hormone binding or nuclear translocation in vivo and in vitro. Molecular studies in mouse embryonic fibroblast cells uncovered that Fkbp52 is critical to AR transcriptional activity. Interestingly, Fkbp51 expression was down-regulated in Fkbp52-deficient males but only in affected tissues, providing further evidence of tissue-specific loss of AR activity and suggesting that Fkbp51 is an AR target gene essential to penile and prostate development. However, Fkbp51-deficient mice were normal, showing no defects in AR-mediated reproductive function. Our work demonstrates that Fkbp52 but not Fkbp51 is essential to AR-mediated signaling and provides evidence for an unprecedented Fkbp52 function, direct control of steroid receptor transcriptional activity.

Autoimmune hepatitis represents a ubiquitous human health problem and has a poor prognosis. Dihydroquercetin (DHQ), a well-known antioxidant, significantly inhibits fulminant hepatitis through anti-oxidant and anti-inflammation mechanisms. In this study, we show that administration of DHQ ameliorated concanavalin A (ConA)-induced mouse liver injury by increasing the survival rate, reducing the serum ALT and AST level, preventing histopathological injuries and decreasing pro-inflammatory cytokine mRNA expression in hepatic tissue. As macrophages/Kupffer cells in oxidative stress and pro-inflammatory mediators play an important role in the pathogenesis of immune-mediated hepatitis, we further exposed mouse RAW264 macrophage cell lines to ConA in vitro and found that DHQ significantly inhibited mRNA expression and secretion of IFN-γ and TNF-α in cell culture supernatant. In addition, DHQ significantly enhanced heme oxygenase-1 (HO-1) expression in a dose- and time-dependent manner via increased Nrf2 expression in cytoplasm and nuclear translocation. Furthermore, DHQ enhanced phosphorylation of three members of the mitogen-activated protein kinase (MAPK) family, and cell treatment with MEK/ERK (PD98059), p38 (SB203580) and JNK (SP600125) inhibitors reduced DHQ-induced HO-1 expression. These results indicate that DHQ possesses hepatoprotective properties against ConA-induced liver injury, which are attributed to its ability to scavenge oxidative stress and to inhibit the release of inflammatory mediators via upregulation of HO-1 activity through the MAPK/Nrf2 signaling pathway in macrophages/Kupffer cells.

FK506-binding protein 52 (FKBP52) is a tetratricopeptide repeat protein that associates with steroid receptors in complexes containing heat shock protein 90. To investigate the role of FKBP52 in steroid-regulated physiology, we generated FKBP52-deficient mice. FKBP52 (-/-) females are sterile due to a complete failure of implantation, a process that requires estrogen (ER) and progesterone receptors (PR). Because the uterus expresses two forms of PR, PR-A and PR-B, we investigated all three receptors as potential targets of FKBP52 action. FKBP52 (-/-) uteri showed a normal growth response to estradiol, and unaltered expression of genes controlled by ER and PR-B. In contrast, FKBP52 (-/-) uteri were neither able to express two PR-A-regulated genes, nor undergo decidualization in response to progesterone, suggesting that FKBP52 specifically regulates PR-A at this organ. Analysis of uterine PR heterocomplexes showed preferential association of FKBP52 with PR-A compared with PR-B. Loss of FKBP52 neither disrupted the PR-A/heat shock protein 90 interaction, nor impaired uterine PR-A hormone-binding function, demonstrating the essential role of FKBP52 in PR-A action to be downstream of the hormone-binding event. Transcription studies in +/+ and -/- mouse embryonic fibroblast cells showed a near-complete loss of PR-A activity at mouse mammary tumor virus and synthetic progesterone response element promoters, although partial reductions of ER and PR-B were also observed. Partial disruptions of ovulation and mammary development were also found in FKBP52 (-/-) females. Taken as a whole, our results show FKBP52 to be an essential regulator of PR-A action in the uterus, while being a nonessential but contributory regulator of steroid receptors in the mammary and ovary. These data may now provide the basis for selective targeting of steroid-regulated physiology through tetratricopeptide repeat proteins.

Background Kawasaki disease (KD) is a childhood systemic vasculitis that exhibits a specific preference for the coronary arteries. The aetiology remains unknown and there are no especially diagnostic tests. microRNAs (miRNAs) are 18 to 23 nucleotides non-coding RNAs that are negative regulator of gene expression and play a crucial role in the regulatory network of the genome. Recently, circulating miRNAs have been found presentation in human plasma and displayed some characteristics of the ideal biomarker. However, few researches explored differentially expressed miRNAs in the plasma of KD patients. Our study is to identify circulating miRNAs in KD plasma which can serve as potential biomarkers of KD diagnosis. Materials and methods The total of five pairs of acute KD and normal plasma samples were analyzed using ABI miRNAs TLDA Assay chip. Differentially expression of miR-125a-5p in plasma were confirmed by quantitative real-time PCR (qRT-PCR) in independent cohort (acute KD = 30, convalescent KD = 30 and healthy control = 32). After bioinformatics prediction, miR-125a-5p vector and inhibitor were transfected into HUVECs respectively, to observe MKK7 expression as a potential target gene. Flow cytometry was used to analyze apoptosis. The mRNA and protein levels of desired genes including MKK7, Caspase-3, Bax and Bcl2 were detected by qRT-PCR and western blotting. Results Eighteen miRNAs were differentially expressed in acute KD’s plasma compared with healthy control. miR-125a-5p was significantly increased in plasma of KD patients (p = 0.000), but no variation between acute and convalescent KD (p = 0.357). Moreover, the results from the gain and loss functions of miR-125a-5p in HUVECs have shown that miR-125a-5p remarkably suppressed MKK7 expression, as a novel target gene. Importantly, miR-125a-5p also induced apoptosis in HUVECs through inhibition MKK7 levels to regulate Bax/Bcl2 pathway resulting to activate Caspase-3. Conclusion Our study indicated that the circulating miR-125a-5p levels in KD’s plasma have remarkably evaluated compared with healthy individuals. miR-125a-5p might play a role in the development of KD by regulating target gene MKK7 to induce apoptosis in vascular endothelial cells. Therefore, our findings have suggested that detected miR-125a-5p levels in plasma could be used as a potential biomarker in early KD diagnosis.

Endothelin-1 (ET-1) and the renin-angiotensin system (RAS) are involved in the pathogenesis of cardiac dysfunction. The Mas receptor is a functional binding site for angiotensin (Ang)‑(1-7), which is now considered a critical component of the RAS and exerts cardioprotective effects. To the best of our knowledge, the present study aimed to examine, for the first time, the effects of ET-1 on Mas expression in cultured human cardiomyocytes. Human cardiomyocytes were treated with ET-1 at different concentrations (1, 5, 10, 20 and 30 nM) for varied time periods (0.5, 1.5, 3, 4.5 or 6 h) with or without the transcription inhibitor actinomycin D, endothelin A (ETA) receptor blocker BQ123 and ETB receptor blocker BQ788, or different kinase inhibitors. ET-1 decreased the Mas mRNA level in a statistically significant dose- and time-dependent manner within 4.5 h, which was reflected in the dose-dependent downregulation of Mas promoter activity, Mas protein levels and Ang-(1-7) binding on the cell membrane. Actinomycin D (1 mg/ml), BQ123 (1 µM), p38 mitogen-activated protein kinase (MAPK) siRNA and inhibitor PD169316 (25 µM), completely eliminated the inhibitory effects of ET-1 on Mas expression in human cardiomyocytes. In conclusion, the present study demonstrated that ET-1 downregulates Mas expression at the transcription level in human cardiomyocytes via the ETA receptor by a p38 MAPK‑dependent mechanism. This study provides novel insights into the function of ET-1 and the Ang‑(1-7)/Mas axis in cardiac pathophysiology.

Hypospadias is a common birth defect in humans, yet its etiology and pattern of onset are largely unknown. Recent studies have shown that male mice with targeted ablation of FK506-binding protein-52 (Fkbp52) develop hypospadias, most likely due to actions of Fkbp52 as a molecular co-chaperone of the androgen receptor (AR). Here, we further dissect the developmental and molecular mechanisms that underlie hypospadias in Fkbp52-deficient mice. Scanning electron microscopy revealed a defect in the elevation of prepucial swelling that led to the onset of the ventral penile cleft. Interestingly, expression of Fkbp52 was highest in the ventral aspect of the developing penis that undergoes fusion of the urethral epithelium. Although in situ hybridization and immunohistochemical analyses suggested that Fkbp52 mutants had a normal urethral epithelium signaling center and epithelial differentiation, a reduced apoptotic cell index at ventral epithelial cells at the site of fusion and a defect of genital mesenchymal cell migration were observed. Supplementation of gestating females with excess testosterone partially rescued the hypospadic phenotype in Fkbp52 mutant males, showing that loss of Fkbp52 desensitizes AR to hormonal activation. Direct measurement of AR activity was performed in mouse embryonic fibroblast cells treated with dihydrotestosterone or synthetic agonist R1881. Reduced AR activity at genes controlling sexual dimorphism and cell growth was found in Fkbp52-deficient mouse embryonic fibroblast cells. However, chromatin immunoprecipitation analysis revealed normal occupancy of AR at gene promoters, suggesting that Fkbp52 exerts downstream effects on the transactivation function of AR. Taken together, our data show Fkbp52 to be an important molecular regulator in the androgen-mediated pathway of urethra morphogenesis.

Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.

Our previous studies have shown that the inhibition of phosphatidylinositol 3-kinase (PI3K) or mTOR complex 1 can obviously promote the Coxsackievirus B3 (CVB3)-induced apoptosis of HeLa cells by regulating the expression of proapoptotic factors. To further illustrate it, Homo sapiens eIF4E-binding protein 1 (4EBP1), p70S6 kinase (p70S6K), Akt1 and Akt2 were transfected to HeLa cells, respectively. And then, we established the stable transfected cell lines. Next, after CVB3 infection, apoptosis in different groups was determined by flow cytometry; the expressions of Bim, Bax, caspase-9 and caspase-3 were examined by real-time fluorescence quantitative PCR and western blot analysis; the expression of CVB3 mRNA and viral capsid protein VP1 were also analyzed by real-time fluorescence quantitative PCR, western blot analysis and immunofluorescence, respectively. At the meantime, CVB3 replication was observed by transmission electron microscope. We found that CVB3-induced cytopathic effect and apoptosis in transfected groups were more obvious than that in controls. Unexpectedly, apoptosis rate in Akt1 group was higher than others at the early stage after viral infection and decreased with the viral-infected time increasing, which was opposite to other groups. Compared with controls, the expression of CVB3 mRNA was increased at 3, 6, 12 and 24 h postinfection (p. i.) in all groups. At the meantime, VP1 expression in 4EBP1 group was higher than control during the process of infection, while the expressions in the other groups were change dynamically. Moreover, overexpression of 4EBP1 did not affect the mRNA expressions of Bim, Bax, caspase-9 and caspase-3; while protein expressions of Bim and Bax were decreased, the self-cleavages of caspase-9 and caspase-3 were stimulated. Meanwhile, overexpression of p70S6K blocked the CVB3-induced Bim, Bax and caspase-9 expressions but promoted the self-cleavage of caspase-9. In the Akt1 group, it is noteworthy that the expressions of Bim protein were higher than controls at 3 and 6 h p. i. but lower at 24 h p. i., and the expression of Bax protein were higher at 6 and 24 h p. i., while their mRNA expressions were all decreased. Furthermore, overexpression of Akt1 stimulated the procaspase-9 and procaspase-3 expression but blocked their self-cleavages. Overexpression of Akt2, however, had little effect on Bim, Bax and caspase-3, while prevented caspase-9 from self-cleavage at the late stage of CVB3 infection. As stated above, our results demonstrated that overexpression of 4EBP1, p70S6K, Akt1 or Akt2 could promote the CVB3-induced apoptosis in diverse degree via different mediating ways in viral replication and proapoptotic factors in BcL-2 and caspase families. As 4EBP1, p70S6K and Akt are the important substrates of PI3K and mammalian target of rapamycin (mTOR), we further illustrated the role of PI3K/Akt/mTOR signaling pathway in the process of CVB3-induced apoptosis. Published version