The effect of angiotensin-converting enzyme polymorphism on hemodynamic response to endotracheal intubation in hypertensive patients.

Endotracheal intubation elicits a hemodynamic response associated with increased heart rate and blood pressure that is influenced by the angiotensin-converting enzyme (ACE) insertion (I)/deletion (D) genetic polymorphism which may be of importance also for the pressure response to anesthesia. A total of 337 patients underwent abdominal surgery in general anesthesia and 16% were D/D-homozygotes, 45% were I/D heterozygotes and 39% of the patients were I/I homozygotes. Before surgery most patients were in treatment for arterial hypertension. Systolic and diastolic pressure, heart rate and concentrations of catecholamines in blood were determined before and after induction of anesthesia and for up to 10 minutes following endotracheal intubation. Anesthesia decreased blood pressure and for patients presenting ID and DD, blood pressure and heart rate reached similar levels but compared to II-homozygotes, D-carriers demonstrated significantly higher levels for systolic pressure and heart rate before and after intubation (p < 0.05). The blood levels of catercholamines were similar in the three genotype groups. The incidence of ECG-determined myocardial ischemia was higher in D-allele carriers compared to I-allele homozygotes (DD 22%, ID 25% vs. II 5%). In response to anesthesia and intubation and independent of sympathetic nervous activity, D-allele carriers for ACE polymorphism increased blood pressure response and higher risk for development of cardiovascular complications compared to patients homozygous for the I-allele.

Association of TNF-α, TGF-ß1, IL-10, IL-6, and IFN-γ gene polymorphism with acute rejection and infection in lung transplant recipients.

Infection and rejection are common complications faced by lung transplant recipients (LTRs) and have become major impediments to long-term survival. Cytokines may play an important role in the development of these complications. In this study, we explored the correlation between TNF-α (-308 A/G), TGF-ß1 (+869 T/C, +915 G/C), IL-10 (-592 C/A, -819 T/C, -1082 G/A), IL-6 (-174 G/C), and IFN-γ (+874 T/A) gene polymorphisms and the incidence of acute rejection and infection. Transplant outcomes were reviewed in a retrospective cohort of 113 LTRs from a single center between December 2004 and November 2012. Cytokine polymorphisms were measured using sequence-specific primer-based PCR. HLA typing was performed for the donors and recipients. We found that the LTRs with the IL-10 -819 CC and -592 CC genotypes had a significantly decreased risk of infection (p = 0.017, OR = 0.177, 95% CI = 0.04-0.85). However, we found no significant association between cytokine polymorphisms and acute rejection. Furthermore, the data revealed that the occurrence of acute rejection was strongly associated with infection episodes (χ(2)  = 8.5256, p < 0.01). These results suggest that LTRs possessing the IL-10 -819 CC and -592 CC genotype may be protected from the occurrence of infection. Our results demonstrated that infection is an important cause of acute rejection for LTRs.

Expression of amyloid-ß protein and amyloid-ß precursor protein after primary brain-stem injury in rats.

Amyloid-ß (Aß) protein and its precursor, amyloid-ß precursor protein (ß-APP), have traditionally been used in the diagnosis of Alzheimer disease. Their use in diagnosis of traumatic brain injury by forensic analysis is becoming more widespread. However, to date, no reliable small animal model exists to evaluate these brain injury indicators. To address this, we have studied primary brain-stem injury in rats to assess the appearance of diffuse axonal injury in brain sections and correlate these findings with appearance of Aß and relative ß-APP mRNA levels. Using an EnVision 2-step immunohistochemical staining method to measure axon diameter, we found that there was significant difference in axon diameters within the medulla oblongata and several time points after brain injury, ranging from 3 to 24 hours. In addition, mRNA expression levels of ß-APP increased following brain injury, peaking 3 hours following injury and decreasing back to baseline levels by 24 hours after injury. These results suggest that using immunohistochemistry and reverse transcription-polymerase chain reaction to detect changes in Aß-associated axonal changes and ß-APP mRNA levels, respectively, can be useful for the diagnosis of diffuse axonal injury during autopsy at early time points following fatal brain injury.

[NF-κ B mediates the effect of glucosylceramide synthase on P-glycoprotein modulation in a drug-resistance leukemia cell line].

OBJECTIVE: To investigate whether transcription factor-kappaB (NF-κ B) is involved in the modulation of P-glycoprotein (P-gp) by glucosylceramide synthase (GCS) in a multidrug resistance leukemia cell line K562/A02 and to explore the relationship between NF-κ B and extracelluar signal-regulated kinase (ERK). METHODS: K562/A02 cells were treated with GCSsiRNA, pyrrolidine dithiocarbamate (PDTC, a NF-κ B specific inhibitor) and U0126 (a MEK1/2 inhibitor), respectively. The expression of GCS and multidrug resistance protein 1 (MDR1) mRNA were analyzed with qRT-PCR. Various proteins of different groups were measured by Western blotting. RESULTS: After transfected with GCSsiRNA for 48 h, GCS mRNA were reduced by 62% (51%-73%) and MDR1 mRNA was reduced by 52% (43%-61%) in the K562/A02 cells. Compared with the negative control, relative expression of NF-κ B p65 in nuclear and P-ERK1/2 were both down-regulated, and P-gp was also inhibited significantly at 72 h after transfected with GCSsiRNA (P< 0.05). In addition, the expression of P-gp was decreased at 24 h with 80 µ mol/L PDTC and 48 h with 20 µ mol/L PDTC. P-ERK1/2 was inhibited significantly when the cells were treated with 20 µ mol/L U0126 for 48 h. The expression of NF-κ B p65 in nuclear and P-gp were also down-regulated. CONCLUSION: NF-κ B can modulate the effect of GCS on P-gp in K562/A02 cells. P-ERK1/2 can activate NF-κ B in above signal transduction pathway.

Quantitative detection of T315I mutations of BCR::ABL1 using digital droplet polymerase chain reaction.

BACKGROUND: T315I mutations of the BCR::ABL1 gene lead to resistance to tyrosine kinase inhibitors (TKIs). This study evaluated the performance of digital droplet polymerase chain reaction (ddPCR) in quantifying T315I mutations and their frequency in Philadelphia chromosome (Ph) positive hematological patients. METHODS: The course of disease and BCR::ABL1 fusion transcripts (e13a2, e14a2 and e1a2) were retrospectively reviewed in 21 patients with acute lymphoblastic leukemia (ALL) and 85 patients with chronic myeloid leukemia (CML). T315I mutation analysis was carried out using ddPCR and the limit of detection was assessed using mutant T315I DNA at varying variant allele fractions. RESULTS: T315I mutations were found in two ALL patients and one CML patient without remission in molecular biology and with mutation burdens of 29.20%, 40.85%, and 3.00%, respectively. The mutation burden of ALL patients was higher than that of CML patients, but there was no significant difference between the two (p-value = 0.0536). The test's limit of detection was 0.02% with a correlation coefficient greater than 0.99 between the expected and actual detection abundances. CONCLUSION: T315I mutations have a high incidence in Ph-positive ALL patients even if the course of disease is short. In molecular biology, T315I mutation detection is indicated for CML patients not in remission.

Simultaneous Extraction of Bone Marrow RNA and DNA from Patients with Hematologic Diseases Using a Combined Magnetic Bead Method within 1 Hour.

BACKGROUND: TRIzolTM is widely used for RNA and DNA extraction. However, this method is laborious and time-consuming. The objective of this study was to validate a time-effective and labor-saving protocol. METHODS: The TRIzol method was used to separate the aqueous phase, protein, and phenol layer of bone marrow samples from 12 patients with hematological diseases. Subsequently, RNA and DNA were extracted from the aqueous layer containing RNA and phenol layer containing DNA, respectively, using magnetic bead extraction kits. The quantity and purity of extracted RNA and DNA were examined using a NanoDrop spectrophotometer. Quantitative fluorescence PCR amplification of the ABL1 gene was performed to verify the effectiveness of the extracted RNA and DNA for downstream experiments. RNA and DNA from another 16 bone marrow samples were extracted to compare the performance of the two methods. RESULTS: Co-extraction of RNA and DNA was completed within 1 h. The data showed that RNA and DNA yield ranged from 13.1 to 204.5 ng/µL and 33.1 to 228.8 ng/µL, respectively. The A260/A280 ratios of RNA and DNA samples ranged from 1.82 to 2.01 and 1.73 to 1.91, respectively. RNA and DNA extracted using this scheme exhibited ideal performance in quantitative fluorescence PCR. The present protocol showed better quality and effectiveness in extracting RNA and DNA compared to the TRIzol method. CONCLUSIONS: This protocol for RNA and DNA co-extraction is fast, labor-saving, and high throughput. It can be adopted for routine molecular biology analyses, particularly for non-reproducible specimens.

Glucocorticoid receptor ß regulates injury-mediated astrocyte activation and contributes to glioma pathogenesis via modulation of ß-catenin/TCF transcriptional activity.

Astrocytes react to central nervous system (CNS) injury and participate in gliotic responses, imparting negative, as well as positive effects on axonal regeneration. Despite the considerable biochemical and morphological changes astrocytes undergo following insult, and the known influence of steroids on glial activation, details surrounding glucocorticoid receptor expression and activity are lacking. Such mechanistic information is essential for advancing and enhancing therapies in the treatment of CNS injuries. Using an in vitro wound-healing assay, we found glucocorticoid receptor ß (GRß), not GRα, is upregulated and acts as a regulator of gliosis after injury. In addition, our results suggest that GRß interacts with ß-catenin and is a necessary component for proliferation and migration in both injured astrocytes and glioma cells. Further analysis indicated GRß/ß-catenin interaction as a key modulator of astrocyte reactivity through sustained Wnt/ß-catenin/TCF signaling in its dominant-negative effect on GRα mediated trans-repression by a GSK-3ß-independent manner. These findings expand our knowledge of the mechanism of GRß action in promoting astrocyte proliferation and migration following injury and in glioma. This information furthers our understanding the function of glucocorticoid receptor in CNS injury and disease, as well as in the basic biochemical responses astrocytes undergo in response to injury and glioma pathogenesis.

Human cytomegalovirus glycoprotein B genotypic distributions and viral load in symptomatic infants.

INTRODUCTION: HCMV infection is widespread in humans. This retrospective study aimed to explore the relationship between human cytomegalovirus (HCMV) glycoprotein B (gB) genotype distribution, viral load, and the demographic and clinical features of symptomatic infants. The detection rate of HCMV in blood and urine samples was also compared. METHODOLOGY: Retrospective data from 265 infants who underwent urine HCMV DNA testing were analyzed. The viral load and gB genotype were detected in 91 HCMV positive infants by quantitative fluorescence polymerase chain reaction (PCR) and DNA sequencing, respectively. RESULTS: The positive rate of HCMV infection was 46.04% (122/265) in all infants, and increased rapidly with age. Among the 91 infants investigated, liver function abnormality was the most common diagnosis (34/91, 37.36%), followed by pneumonia (21/91, 23.07%). Sequence analysis of gB yielded two genetic subtypes: the most prevalent gB3 (47/91, 51.65%), followed by gB1 (44/91, 48.35%). The gB3 HCMV infection was more prevalent in infants aged 0-2 months than in infants aged 3-12 months (χ2 = 4.38, p = 0.0364). The data showed that ALT and AST levels were significantly higher in the anti-HCMV IgM+IgG- group than in the anti-HCMV IgM+IgG+ and IgM-IgG+ groups. In addition, this study showed that the detection rate of HCMV DNA in the blood was significantly lower than that in the urine (χ2 = 6.7131, p = 0.0096). CONCLUSIONS: This study presents the HCMV infection status of infants and its relationship with their demographic characteristics and clinical manifestations. In addition, this study suggests that urinary PCR is the most appropriate assay for detecting HCMV infections.

Genistein attenuated allergic airway inflammation by modulating the transcription factors T-bet, GATA-3 and STAT-6 in a murine model of asthma.

BACKGROUND: Genistein, a flavonoid in legumes and some herbal medicines, has various biological actions. Previous studies have shown that genistein decreased airway inflammation in allergic asthma. However, studies on how genistein affects immunoreactions in asthma are very limited. OBJECTIVE: It was the aim of this study to investigate the effect of genistein on T helper 1 (Th1)/Th2 cytokines in a murine asthma model and to explore its underlying mechanisms. METHODS: The asthma model was set up both in vivo and in vitro: the mice were divided into four groups in vivo, i.e. control group, ovalbumin-sensitized (OVA) group, Gen20 group (20 mg/kg genistein) and Gen40 group (40 mg/kg genistein), and into three groups in vitro, i.e. control group, OVA group, genistein group. Changes in lung histology were observed and concentrations of interleukin-4, interleukin-5 and interferon-γ in bronchoalveolar lavage fluid and serum were measured by enzyme-linked immunosorbent assay. The mRNA expression of GATA binding protein 3 (GATA-3), signal transducer and activator of transcription 6 (STAT-6) and T-box transcription factor (T-bet) in the lungs and CD4+ T cells of each group were detected by real-time PCR and the corresponding proteins were detected by Western blot. RESULTS: The results showed that genistein attenuated OVA-induced airway inflammation, decreased Th2-type cytokines and increased Th1-type cytokines. Additionally, our data suggested that genistein may modulate the Th1/Th2 reaction by inhibiting GATA-3 and STAT-6 production while increasing T-bet production. CONCLUSION: Genistein may modulate the immunomodulatory actions caused by Th1/Th2 cytokines in asthma, at least partially, by the down-regulation of GATA-3 and STAT-6 and the up-regulation of T-bet.

[Effect of glucosylceramide synthase on P-gp expression by ERK signal transduction pathway in leukemia multi-drug resistance cell line].

OBJECTIVE: To investigate the effect of glucosylceramide synthase (GCS) on P-glycoprotein (P-gp) expression via extracellular signal-regulated kinase (ERK) pathways in leukemia K562/A02 cell line. METHODS: K562/A02 multidrug resistance cells were treated with GCS siRNA and U0126, respectively. Expression of multidrug resistance protein 1 (MDR1) mRNA was analyzed with qRT-PCR. Phosphorylated ERK1/2, total ERK1/2 protein and P-gp in different groups were measured with Western blotting. RESULTS: After treated with U0126, P-ERK1/2 was decreased along with the increased U0126 concentration. P-ERK1/2 and P-gp were apparently down-regulated by U0126 at the concentrations of 20 µmol/L, 40 µmol/L and 60 µmol/L. After being transfected with GCS siRNA, GCS mRNA was inhibited by 70.50% (58.00%-76.00%) in K562/A02 cells. Compared with the negative control, both P-ERK1/2 and P-gp were inhibited significantly after RNAi for 72 hours (P<0.01 and P<0.05, respectively. CONCLUSION: GCS may affect the expression of P-gp by ERK signal transduction pathway in leukemia cells.

[Association between suppressors of cytokine signaling mRNA expression and Th1/Th2 balance in children with asthma].

OBJECTIVE: Suppressors of cytokine signaling (SOCS) have been shown to play an important role in regulating cytokines, such as intracellular interferon (IFN) and interleukin (IL), in the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. At present, the association between SOCS and asthma is still under study. The aim of this study is to explore the relationship of SOCS-1 and SOCS-3 mRNA expression in peripheral blood mononuclear cells (PBMCs) with the intracellular IFN-'/IL-4 ratio in CD4+ T cells and specific IgE (sIgE) level in children with asthma. METHODS: BMCs were collected from 44 children with allergic asthma (4-14 years) and 30 healthy children. The intracellular IFN-'/IL-4 ratio in CD4+ T cells was measured by flow cytometry. Total RNAs were extracted from the PBMCs and SOCS-1 and SOCS-3 mRNA expression was measured by SYBR Green I quantitative RT-PCR. RESULTS: Compared with the healthy children, children with allergic asthma showed a lower level of intracellular IFN-' in peripheral blood [(15.7±2.0)% vs (19.1±2.7)%] and IFN-'/IL-4 ratio (3.4±1.5 vs 4.8±2.9) and higher SOCS-1 mRNA expression (-Ct, 11.1±1.9 vs 12.6±2.8). There was a negative relationship between SOCS-1 mRNA expression and the percentage of IFN-'-producing CD4+ T cells in peripheral blood in both asthmatic and healthy children (P<0.05). No correlation was found between SOCS-1 and SOCS-3 expression and sIgE level. CONCLUSIONS: Children with allergic asthma have elevated levels of SOCS-1 mRNA in PBMCs, which is associated with Th2-skewed immune response.

High-resolution melting PCR analysis for genotyping the gene polymorphism of TNF-α, TGF-ß1, IL-10, and IFN-γ in lung transplant recipients.

BACKGROUND: High-resolution melting (HRM) analysis is a genotyping method which has the advantages of simple, rapid, low-cost and closed-tube operation. OBJECTIVES: This study evaluated HRM analysis as an option for detecting the single nucleotide polymorphism (SNP) of cytokine, and profiled the distribution of cytokine gene polymorphism in the lung transplant recipients (LTRs). MATERIAL AND METHODS: High-resolution melting-polymerase chain reaction (HRM-PCR) assays for genotyping tumor necrosis factor alpha (TNF-α) (-308 A/G), tumor growth factor beta 1 (TGF-ß1) (+869 T/C), interleukin 10 (IL-10) (-592 C/A, -819 T/C, -1082 G/A), and interferon gamma (IFN-γ) (+874 T/A) SNPs were developed on the LightCycler® 480. The SNPs of the aforementioned cytokine genes in 322 LTRs and 266 normal controls were detected using HRM-PCR approach. To confirm the accuracy of the HRM-PCR assay, we randomly selected 100 samples from the LTRs and detected the aforementioned SNPs with sequence-specific primer-polymerase chain reaction (SSP-PCR) method, using a commercial kit. RESULTS: The data show that the HRM-PCR assay can distinguish all the cytokine SNPs, and the results of HRM-PCR analysis are in complete concordance to the genotyping results obtained using a commercial kit (κ = 1.0). Our data also show that the allele and genotype frequencies of the abovementioned cytokine are not significantly different between the LTRs and the control groups (p > 0.05). In addition, we found the genotypes of TGF-ß1 +869 associated with high expression phenotype were prevalent in the LTRs. On the contrary, for TNF-α -308, IL-10 and IFN-γ, the genotypes associated with low expression phenotype were most common in the LTRs. CONCLUSIONS: In this study, we described a rapid, low-cost and high-throughput HRM-PCR technology for genotyping cytokine SNPs. Our data may be utilized for future studies examining the associations of cytokine gene polymorphisms with the prognosis of the LTRs.

[Study on the correlation of GCS and MDR1 genes in inducing multidrug resistance in human K562/A02 cell line].

OBJECTIVE: To investigate the correlation of glucosylceramide synthase (GCS) gene and multidrug resistance 1 (MDR1) gene in inducing multidrug resistance in human multidrug-resistant K562/A02 cell line, and search for a novel strategy for reversing multidrug resistance of leukemia cells. METHODS: The expression levels of GCS and MDR1 mRNA in K562 and K562/A02 cells were assayed by RT-PCR. siRNAs targeting the GCS and MDR1 gene were transfected into K562/A02 cells with liposome, respectively. The differential expression of GCS and MDR1 mRNAs, as well as their correlation, were detected by RT-PCR and real time quantitative-PCR(QPCR). RESULTS: The expression level of GCS and MDR1 mRNA was dramatically lower in drug-sensitive K562 cells compared with the K562/A02 cells. The GCS mRNA was inhibited by 73%(59%-82%) and MDR1 mRNA expression was down regulated by 67% (38%-82%) in K562/A02 cells after being transfected with GCS siRNA. The expression level of MDR1 mRNA was inhibited by 81%(63%-91%) and GCS mRNA expression had no apparent change in K562/A02 cells treated with MDR1 small interference RNA(siRNA). CONCLUSION: Positive correlation was detected between the expression of GCS and MDR1 mRNA in K562/A02 cells and MDR1 mRNA expression was down regulated after silencing the GCS gene expression.

Effects of Dexmedetomidine and ACE Genotype on Cardiovascular Response During the Decannulation Period of General Anesthesia in Patients With Essential Hypertension.

PURPOSE: This study investigated the effects of dexmedetomidine on cardiovascular response during the decannulation period of general anesthesia in patients with different genotypes of angiotensin-converting enzyme (ACE) and essential hypertension. METHODS: The present study enrolled patients with essential hypertension and American Society of Anesthesiologists class II or III who were scheduled to undergo abdominal surgery under general anesthesia. Patients were assigned to 1 of 6 groups according to ACE genotype, as detected by polymerase chain reaction-restriction fragment length polymorphism, as follows: DD; ID; II; and DD, ID, and II each with dexmedetomidine (Dex). Dexmedetomidine was intravenously infused at 0.5 µg/kg/h for 30 min before the end of surgery in groups DD (Dex), ID (Dex), and II(Dex). Anesthesia was induced and maintained by the same anesthetics in all patients. Systolic and diastolic blood pressure, heart rate (HR), ECG, and rate-pressure product were recorded before anesthesia induction; at 30 min before the end of surgery; at the end of surgery; and at 0, 1.5, 5, and 10 min after extubation. FINDINGS: A total of 210 patients were enrolled (n = 35 per genotype). After extubation, systolic and diastolic blood pressure, HR, and RPP were increased markedly from baseline in groups DD, ID, and II; the increases were greater in groups DD and ID than in group II. No significant changes in blood pressure, HR, or RPP were found, and proper sedative was achieved in groups DD (Dex), ID (Dex), and II(Dex). The prevalences of cardiac arrhythmia were higher in groups DD and ID than in groups II, DD (Dex), ID (Dex), and II(Dex). IMPLICATIONS: Patients essential hypertension and the ACE D allele had a strong hemodynamic response to tracheal extubation, on which dexmedetomidine was found to have both a prevention and treatment effect.

[Expression of the CDH13 gene and BCR/ABL fusion gene in chronic myeloid leukemia patients and their relationship].

OBJECTIVE: To investigate the expression of the CDH13 gene and BCR/ABL fusion gene in chronic myeloid leukemia(CML) patients at different stages and explore their relationship. METHODS: RNA was isolated from peripheral blood in 30 healthy adults, 22 primary CML patients and 25 blastic crisis of CML patients. We examined the expression of the CDH13 gene and BCR/ABL fusion gene using EVA Green real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: The expression of the BCR/ABL fusion gene in the patients with blastic crisis CML was 4.72 folds as that of the patients with primary CML. The expression of the CDH13 gene in the primary and blastic crisis CML patients was significantly reduced to 36% and 25% of that in healthy controls, respectively. Moreover, negative correlation was found between the expressions of these two genes. CONCLUSION: The study showed that the reduction of the CDH13 expression at different clinical stage of CML may account for the defective cell adhesion in CML, and the expression of the CDH13 gene was probably down-regulated by the BCR/ABL fusion gene.

Bioinformatic analysis identifies potentially key differentially expressed genes in oncogenesis and progression of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is one of the most common and lethal types of cancer within the urinary system. Great efforts have been made to elucidate the pathogeny. However, the molecular mechanism of ccRCC is still not well understood. The aim of this study is to identify key genes in the carcinogenesis and progression of ccRCC. The mRNA microarray dataset GSE53757 was downloaded from the Gene Expression Omnibus database. The GSE53757 dataset contains tumor and matched paracancerous specimens from 72 ccRCC patients with clinical stage I to IV. The linear model of microarray data (limma) package in R language was used to identify differentially expressed genes (DEGs). The protein-protein interaction (PPI) network of the DEGs was constructed using the search tool for the retrieval of interacting genes (STRING). Subsequently, we visualized molecular interaction networks by Cytoscape software and analyzed modules with MCODE. A total of 1,284, 1,416, 1,610 and 1,185 up-regulated genes, and 932, 1,236, 1,006 and 929 down-regulated genes were identified from clinical stage I to IV ccRCC patients, respectively. The overlapping DEGs among the four clinical stages contain 870 up-regulated and 645 down-regulated genes. The enrichment analysis of DEGs in the top module was carried out with DAVID. The results showed the DEGs of the top module were mainly enriched in microtubule-based movement, mitotic cytokinesis and mitotic chromosome condensation. Eleven up-regulated genes and one down-regulated gene were identified as hub genes. Survival analysis showed the high expression of CENPE, KIF20A, KIF4A, MELK, NCAPG, NDC80, NUF2, TOP2A, TPX2 and UBE2C, and low expression of ACADM gene could be involved in the carcinogenesis, invasion or recurrence of ccRCC. Literature retrieval results showed the hub gene NDC80, CENPE and ACADM might be novel targets for the diagnosis, clinical treatment and prognosis of ccRCC. In conclusion, the findings of present study may help us understand the molecular mechanisms underlying the carcinogenesis and progression of ccRCC, and provide potential diagnostic, therapeutic and prognostic biomarkers.

PKCγ promotes axonal remodeling in the cortico-spinal tract via GSK3ß/ß-catenin signaling after traumatic brain injury.

Traumatic brain injury (TBI) is a common cause of death and disability. Enhancing the midline-crossing of the contralateral corticospinal tract (CST) to the denervated side of spinal cord facilitates functional recovery after TBI. Activation of the gamma isoform of PKC (PKCγ) in contralateral CST implicates its roles in promoting CST remodeling after TBI. In this study, we deployed loss and gain of function strategies in N2a cells and primary cortical neurons in vitro, and demonstrated that PKCγ is not only important but necessary for neuronal differentiation, neurite outgrowth and axonal branching but not for axonal extension. Mechanically, through the phosphorylation of GSK3ß, PKCγ stabilizes the expression of cytosolic ß-catenin and increase GAP43 expression, thus promoting axonal outgrowth. Further, rAAV2/9-mediated delivery of constitutive PKCγ in the corticospinal tract after unilateral TBI in vivo additionally showed that specifically delivery of active PKCγ mutant to cortical neuron promotes midline crossing of corticospinal fibers from the uninjured side to the denervated cervical spinal cord. This PKCγ-mediated injury response promoted sensorimotor functional recovery. In conclusion, PKCγ mediates stability of ß-catenin through the phosphorylation of GSK3ß to facilitate neuronal differentiation, neurite outgrowth and axonal branching, and PKCγ maybe a novel therapeutic target for physiological and functional recovery after TBI.

Overexpression of glucosylceramide synthase in associated with multidrug resistance of leukemia cells.

Ceramide, as a second messenger, initiates one of the major signal transduction pathways in tumor apoptosis. Glucosylceramide synthase (GCS) catalyzes glycosylation of ceramide and produces glucosylceramide. Through GCS, ceramide glycosylation allows cellular escape from ceramide-induced programmed cell death. Here we investigated the expression of GCS in human leukemia cells and an association between GCS and multidrug resistance of leukemia cells. Using RT-PCR technique the level of GCS gene was detected in 65 clinical multidrug resistance/non-resistance cases with leukemia, and in K562 and K562/A02 cell lines. AlamarBlue Assay was applied to confirm the multidrug resistant of K562/A02 cells. PPMP, which is a chemical inhibitor for GCS, was used to determine the relationship between GCS and drug-resistance in K562/A02 cells. In addition, multidrug resistance gene (mdr1), Bcl-2 and Bax mRNA was also analyzed by RT-PCR. The expression of GCS and mdr1 mRNA in clinic multidrug resistance samples exhibited significantly increased compared with clinic drug sensitive group (P<0.05). There was the positive correlation both the expression of GCS and mdr1 genes in leukemia samples (P<0.01, gamma=0.7). AlamarBlue Assay showed that the K562/A02 cell line was 115-fold more resistant to adriamycin and 36-fold more resistant to vincristine compared with drug-sensitive K562 cell line. There also was significant expression difference of GCS and mdr1 genes between K562 and K562/A02 cells. Bcl-2 gene exhibited higher expressions whatever in clinic drug-resistance samples or K562/A02 cells, whereas the expressions of Bax gene were higher in drug-sensitive samples and K562 cells. PPMP increased sensitivity to adriamycin toxicity by inhibiting GCS in K562/A02 cells. Therefore, it is suggested that a high level of GCS in leukemia is possible contributed to multidrug resistance of leukemia cells. Abnormally expressions of the genes in associated with cell apoptosis might be one of the main molecular pathology mechanisms of multidrug resistance caused by GCS gene.

Nuclear Smad6 promotes gliomagenesis by negatively regulating PIAS3-mediated STAT3 inhibition.

To date, the molecular mechanism underlying constitutive signal transducer and activator of transcription 3 (STAT3) activation in gliomas is largely unclear. In this study, we report that Smad6 is overexpressed in nuclei of glioma cells, which correlates with poor patient survival and regulates STAT3 activity via negatively regulating the Protein Inhibitors of Activated STAT3 (PIAS3). Mechanically, Smad6 interacts directly with PIAS3, and this interaction is mediated through the Mad homology 2 (MH2) domain of Smad6 and the Ring domain of PIAS3. Smad6 recruits Smurf1 to facilitate PIAS3 ubiquitination and degradation, which also depends on the MH2 domain and the PY motif of Smad6. Consequently, Smad6 reduces PIAS3-mediated STAT3 inhibition and promotes glioma cell growth and stem-like cell initiation. Moreover, the Smad6 MH2 transducible protein restores PIAS3 expression and subsequently reduces gliomagenesis. Collectively, we conclude that nuclear-Smad6 enhances glioma development by inducing PIAS3 degradation and subsequent STAT3 activity upregulation.

Association of leptin, visfatin, apelin, resistin and adiponectin with clear cell renal cell carcinoma.

Although an association between obesity and the occurrence of renal cell carcinoma (RCC) has been identified, the mechanism by which obesity functions to increase this risk of cancer remains unclear. Leptin, visfatin, apelin, resistin and adiponectin are peptide hormones secreted by adipocytes; it is considered that these may affect RCC development by exerting effects on proliferation, cell growth and inflammation. The aim of the present study was to investigate the association between the aforementioned adipokine genes and clear cell RCC (CC-RCC). The GSE6344 dataset was downloaded from the Gene Expression Omnibus database, and the relative expression levels of the adipokine genes were analyzed. To verify the results of the mRNA microarray, 77 paired samples of CC-RCC and corresponding adjacent normal tissue were allocated into two groups. The extraction of total RNA was conducted, and the mRNA expression of adipokine genes was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The data from the GSE6344 dataset indicated that the expression of visfatin and apelin was upregulated (P<0.0001 and P<0.01, respectively), and adiponectin was downregulated (P<0.001) in the CC-RCC tissues compared with the adjacent normal tissues. The data from RT-qPCR demonstrated that visfatin and resistin gene expression was increased (P<0.01 and P<0.05, respectively) in the CC-RCC tissues. Furthermore, the mRNA expression level of leptin and adiponectin in the adjacent normal tissue was higher than those in the cancer tissue (P<0.01). The current study verifies that visfatin and adiponectin are associated with an increased risk of CC-RCC, which presents further insights into the molecular mechanisms of CC-RCC tumorigenesis.