Circulating levels of adipocytokine omentin-1 in patients with renal cell cancer

Renal cell carcinoma (RCC) is the fifth most common cancer worldwide, and becomes one of the leading causes of genitourinary cancer-related death in both males and females. Genetic alternations, alcohol consumption, occupationally harmful exposure and even obesity are well-established risk factors of RCC. Omentin-1 is a plasma adipokine synthesized in visceral adipose tissue, and its circulating serum concentration alters not only in conditions associated with insulin resistance such as Polycystic Ovary Syndrome (PCOS), but also in colorectal cancer and prostate cancer. To our best knowledge, the relationship between omentin-1 and RCC has not been clarified previously. Thus, we evaluated serum omentin-1 levels in RCC patients in the current matched case-control study. Forty-one patients newly diagnosed with RCC and forty-two healthy controls confirmed by the comprehensive medical examination were assessed. The omentin-1 concentrations were determined via utilizing enzyme-linked immunosorbent assays (ELISA) in the paired groups, in which the patients and healthy controls had no statistically significant differences in gender, age, systolic blood pressure (SBP), diastolic blood pressure (DBP), waist-hip ratio (WHR), estimate glomerular filtration rate (eGFR), body-mass index (BMI) and biochemical parameters. The omentin-1 levels in healthy people were 9.86 ± 1.44 ng/mL and the circulating omentin-1 levels were dramatically decreased to 3.62 ± 0.76 ng/mL in RCC patients (p < 0.001). Besides, we revealed a negative correlation between omentin-1 with WHR (r = −0.261, p = 0.017) and BMI (r = −0.310, p = 0.004), further indicating BMI was the main influential factor on omentin-1 levels (p = 0.0091). Follow-up studies would be conducted to establish the concrete mechanisms underlying the altered circulating levels of omentin-1 and elucidate the interaction between “RCC complex system” and adipose tissues, which may together provide promising and novel pharmacological insights for RCC theragnosis in the near future.     

ABO blood group and risk of renal cell cancer

Background: The genetic determinants of sporadic renal cell cancer (RCC) are largely unknown. Previous studies have suggested associations between ABO blood group and risk of various cancers. However, its relationship to RCC remains unclear and no prospective data are available. Methods: We prospectively followed up 77,242 women in the Nurses’ Health Study and 30,071 men in the Health Professionals Follow-Up Study from 1996 to 2008. The information on the ABO blood group was collected from participants’ self-reports in 1996. Incidence of pathology-confirmed RCC was compared using hazard ratios (HRs) and 95% confidence intervals (CIs) derived from Cox proportional hazards models. Results: During 12 years of follow-up, 163 cases of incident RCC were documented in women and 88 cases in men. The multivariate HRs between non-O blood group (combined group of A, AB, and B) vs. blood group O were 1.51 (95% CI 1.09–2.09) in women, 1.08 (95% CI 0.70–1.66) in men, and 1.32 (95% CI 0.95–1.82) in the pooled cohorts. The associations between ABO blood group and RCC were consistent across strata of known risk factors for RCC including age, obesity, smoking, and history of hypertension (Pinteraction ≥ 0.32). Conclusions: We found a suggestive non-significant association between non-O blood group and increased risk of RCC in the pooled cohorts of men and women, and this association was significant in women. Our findings need to be replicated by other prospective studies.     

Rhomboid domain containing 2 (RHBDD2): A novel cancer-related gene over-expressed in breast cancer

In the course of breast cancer global gene expression studies, we identified an uncharacterized gene known as RHBDD2 (Rhomboid domain containing 2) to be markedly over-expressed in primary tumors from patients with recurrent disease. In this study, we identified RHBDD2 mRNA and protein expression significantly elevated in breast carcinomas compared with normal breast samples as analyzed by SAGE (n = 46) and immunohistochemistry (n = 213). Interestingly, specimens displaying RHBDD2 over-expression were predominantly advanced stage III breast carcinomas (p = 0.001). Western-blot, RT-PCR and cDNA sequencing analyses allowed us to identify two RHBDD2 alternatively spliced mRNA isoforms expressed in breast cancer cell lines. We further investigated the occurrence and frequency of gene amplification and over-expression affecting RHBDD2 in 131 breast samples. RHBDD2 gene amplification was detected in 21% of 98 invasive breast carcinomas analyzed. However, no RHBDD2 amplification was detected in normal breast tissues (n = 17) or breast benign lesions (n = 16) (p = 0.014). Interestingly, siRNA-mediated silencing of RHBDD2 expression results in a decrease of MCF7 breast cancer cells proliferation compared with the corresponding controls (p = 0.001). In addition, analysis of publicly available gene expression data showed a strong association between high RHBDD2 expression and decreased overall survival (p = 0.0023), relapse-free survival (p = 0.0013), and metastasis-free interval (p = 0.006) in patients with primary ER-negative breast carcinomas. In conclusion, our findings suggest that RHBDD2 over-expression behaves as an indicator of poor prognosis and may play a role facilitating breast cancer progression.     

FGFR3 mutational status and protein expression in patients with bladder cancer in a Jordanian population

Bladder cancer accounts for nearly 5% of all newly diagnosed cancers in Jordan, with a much higher frequency in males. Recent studies have shown that activating mutations in FGFR3 are the most common findings in non-invasive low grade bladder tumors. In this study, we, retrospectively, investigated a cohort of 121 bladder cancer patients with various grades and stages of the tumor for molecular changes in FGFR3. Overexpression of FGFR3 was observed in 49%, 34%, 15%, and 2% of pTa, pT1, pT2, and pT3 cases, respectively. Further, FGFR3 expression was positive in 45%, 26%, and 30% of G1, G2 and G3 cases, respectively. Mutational analysis of exons 7, 10 and 15 of FGFR3 identified four previously reported mutations, namely R248C (n = 4; 10%), S249C (n = 23; 59%), Y375C (n = 7; 18%), G382R (n = 4; 10%), and one novel mutation, G382E (n = 1; 3%). Our results indicate that both mutations and overexpression of FGFR3 are correlated together, and are more prevalent in early stage (pTa and pT1) and low grade (G1 and G2) bladder tumors. Survival analysis showed no contribution of changes in FGFR3 on the patient's survival. Multivariate Cox proportional hazards model analysis of overall survival for the following variables: age, gender, stage and grade of tumor, and FGFR3 (expression and mutation) revealed that age, stage and grade of tumor are independent predictors of overall survival in patients with bladder cancer. Our work is the first to address the molecular status of FGFR3 in Jordanian patients with bladder cancer, and provides further support for FGFR3 as a key player in the initiation of bladder tumors.     

Luminal (Her2 negative) prognostic index and survival of breast cancer patients

PurposeThe group of luminal (Her2 negative) is distinguished from other subtypes of breast cancer. We aimed to produce a prognostic index specific for luminal (Her2 negative) subtype breast cancer that could assist clinical treatment.MethodsThe test set comprised 406 consecutive luminal (Her2 negative) breast cancer patients. The relationship of 11 clinicopathologic factors including survivin with the 5-year disease-free survival was analyzed.ResultsIn univariate analysis, TNM stage, surgery, tumor size, lymph node involvement, and survivin expression were prognostic factors. In multivariate analysis, tumor size [HR (95% CI): 1.98 (1.12–3.49), p = 0.019], the number of lymph node metastasis [HR (95% CI): 1.75 (1.33–2.29), p < 0.0001] and the expression of progesterone receptor [HR (95% CI): 0.58 (0.36–0.95), p = 0.029] can independently predict prognosis. Prognostic index (PI) was calculated as 0.68 × tumor size + 0.56 × the number of lymph node metastasis − 0.54 × PR. According to the PI, patients were categorized into three groups: low, middle, and high risk group with the 5-year disease-free survival rates of 91.91%, 84.97% and 70.47%, respectively (P < 0.001). In the validation set, the luminal prognostic index (LPI) remained significant.ConclusionThe LPI may be a useful tool for evaluating the outcome of patients with luminal (Her-2 negative) breast cancer.     

Prognostic significance of survivin expression in renal cell cancer and its correlation with radioresistance

Survivin, an important inhibitor of apoptosis, has been found to play an important role in the initiation, progression, and chemoradioresistance of human malignancies. Previously, we have reported that upregulation of survivin in oral squamous cell carcinoma correlates with poor prognosis and chemoresistance. The aim of this study was to assess prognostic significance of survivin protein expression in RCC and analyze its correlation with radiosensitivity of RCC cells. RT-PCR and Western blot assays were performed to detect survivin mRNA and protein expression in normal human kidney epithelial cell line (HKEC) or RCC cell lines. The expression of survivin mRNA in RCC and corresponding nontumor kidney tissues was also detected by RT-PCR. Immunohistochemistry was performed to determine survivin protein expression in 75 cases of RCC tissue samples. Moreover, the association of survivin protein expression with clinicopathogical factors and prognosis of RCC patients was statistically analyzed. Small interfering RNA was used to knockdown the endogenous survivin expression in RCC cell line (ACHN) and evaluate the effects of survivin knockdown on proliferation, apoptosis, and radiosensitivity of RCC cell line. RCC cells showed sufficient expression of survivin mRNA and protein, but the expression of survivin gene was not detected in normal HKEC. Moreover, the expression level of survivin mRNA in RCC tissues was significantly higher than that in corresponding nontumor kidney tissues. The immunostaining of survivin protein was mainly located in cytoplasm of RCC tumor cells. Tumor pathological stage (P = 0.028), grade (P = 0.004), and lymph node metastasis (P = 0.017) of RCC patients were significantly correlated with survivin protein expression. In addition, patients with high survivin levels had a significantly shorter overall survival than those with low levels (P < 0.001), and the expression of survivin protein was an independent prognostic factor for RCC patients (P = 0.008). The expression of survivin gene could be reduced in RCC cell line and survivin knockdown could inhibit growth and enhance in vivo radiosensitivity of RCC cell line by inducing apoptosis enhancement. Taken together, the status of survivin protein expression may be an independent factor for predicting the prognosis of RCC patients and tumor-specific survivin knockdown combined with radiotherapy will be a potential strategy for RCC therapy.     

Thromboxane synthase expression and correlation with VEGF and angiogenesis in non-small cell lung cancer

Background: Thromboxane synthase (TXS) metabolizes prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with angiogenesis and poor outcome. TXS has been identified as a potential therapeutic target in NSCLC. This study examines a link between TXS expression, angiogenesis, and survival in NSCLC. Methods: TXS and VEGF metabolite levels were measured in NSCLC serum samples (n = 46) by EIA. TXB₂ levels were correlated with VEGF. A 204-patient TMA was stained for TXS, VEGF, and CD-31 expression. Expression was correlated with a range of clinical parameters, including overall survival. TXS expression was correlated with VEGF and CD-31. Stable TXS clones were generated and the effect of overexpression on tumor growth and angiogenesis markers was examined in-vitro and in-vivo (xenograft mouse model). Results: Serum TXB₂ levels were correlated with VEGF (p < 0.05). TXS and VEGF were expressed to a varying degree in NSCLC tissue. TXS was associated with VEGF (p < 0.0001) and microvessel density (CD-31; p < 0.05). TXS and VEGF expression levels were higher in adenocarcinoma (p < 0.0001) and female patients (p < 0.05). Stable overexpression of TXS increased VEGF secretion in-vitro. While no significant association with patient survival was observed for either TXS or VEGF in our patient cohort, TXS overexpression significantly (p < 0.05) increased tumor growth in-vivo. TXS overexpression was also associated with higher levels of VEGF, microvessel density, and reduced apoptosis in xenograft tumors. Conclusion: TXS promotes tumor growth in-vivo in NSCLC, an effect which is at least partly mediated through increased tumor angiogenesis.     

Molecular cloning and bioinformatics analysis of a novel spliced variant of survivin from human breast cancer cells.

Survivin gene and its two alternatively spliced variants, survivin-2 B and survivin- Delta Ex3 gene were cloned from human breast cancer cell lines B-cap37 firstly. A new gene designated as survivin-image (SI) was cloned from above cell lines, which has not been reported yet to clone from any cell lines. It was found that the novel gene 507 bp comprises partial survivin gene (345 bp), partial image gene (155 bp) of eye cancer and other insertion of 7 bp by analyzing with a series of recent bioinformatics software at the level of nucleotide and protein deduced. Predicted 3-D structures of the new molecule showed greatly similar to that of survivin in N-terminal containing BIR by homology modeling. These results suggested SI gene (GenBank accession No.AY830084) might be a novel alternatively spliced isoforms of the survivin gene involved in other functional significances related to tumorigenesis.     

Expression of orexin receptors 1 (OX1R) and 2 (OX2R) in the porcine pituitary during the oestrous cycle

Orexin A and B, also termed hypocretin 1 and 2, are associated with the stimulation of food intake and arousal. The biological actions of the hormones are mediated via two distinct G protein-coupled receptors, termed orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). OX1R is selective for orexin A and OX2R binds orexin A and orexin B with similar affinity. The present study analyzed mRNA and protein expressions of OX1R and OX2R in adenohypophysis (AP) and neurohypophysis (NP) of cycling pigs. The tissue samples were harvested on days 2–3, 10–12, 14–16, and 17–19 of the oestrous cycle. Using quantitative real-time PCR higher OX1R gene expression was detected in AP on days 2–3 relative to days 10–12, 14–16 and 17–19 (p < 0.05). In NP the OX1R mRNA level was elevated on days 10–12 compared to the remaining stages (p < 0.05). OX2R gene expression in AP was the lowest on days 10–12 (p < 0.05 compared to days 2–3 and 17–19) and the expression peak occurred on days 17–19 (p < 0.05 vs. the all studied stages). In NP the highest (p < 0.05) expression of OX2R mRNA was noted on days 17–19 in relation to the remaining periods. OX1R protein content in AP was greatest on days 10–12 (p < 0.05), whereas in NP it was greatest on days 2–3 and 14–16 (p < 0.05 vs. days 10–12 and 17–19). In both cases the lowest OX1R protein expression was observed during follicular phase (p < 0.05 in relation to three remaining studied stages). OX2R protein in AP was lower (p < 0.05) on days 2–3 and 14–16 compared to days 10–12 and 17–19. In NP the lowest (p < 0.05) expression of this protein was on days 17–19 and the highest on days 10–12 (p < 0.05 compared to days 2–3 and 17–19). In summary, the present findings provide the first evidence that OX1R and OX2R mRNAs and proteins occur in the pituitary of the pig and indicate the dependence of orexin receptor expression on the endocrine reproductive state.     

Aflatoxin contamination of wheat flour and the risk of esophageal cancer in a high risk area in Iran

Background: Golestan province in northeastern Iran has been known as a high-risk area for esophageal cancer (EC). This study was conducted to assess aflatoxin (AF) contamination of wheat flour (WF) samples in high and low EC-risk areas of Golestan province. Methods: Four WF samples were collected randomly from each of 25 active silos throughout the province in 2009. The levels of AFs were measured using the High-performance liquid chromatography method. Using the data of EC rates obtained from Golestan population-based cancer registry, the province was divided into high and low risk areas for EC. Student t-test and multivariate regression analysis were used to compare the levels of aflatoxins as well as the condition of silos between the two areas. Results: One hundred WF samples were collected. The mean levels of total aflatoxin and aflatoxin B1 was 1.99 and 0.53 ng g^?1, respectively. The levels of total AF (p = 0.03), AFG2 (p = 0.02) and AFB1 (p = 0.003) were significantly higher in samples obtained from high risk area. Multivariate regression analysis showed that humidity of silo was the most important source of difference between silos of the two areas (p = 0.04). Conclusion: We found a positive relationship between AF level of WF samples and the risk of EC. So, AF contamination may be a possible risk factor for EC in our region. We also found that humidity of silos was the most important determinant of AF contamination of WF. Intensive control of silos conditions including humidity and temperature are needed especially in high EC-risk areas.     

Adenovirus-mediated tissue factor pathway inhibitor gene transfer induces apoptosis by blocking the phosphorylation of JAK-2/STAT-3 pathway in vascular smooth muscle cells

ObjectiveIn our previous study, we have demonstrated that tissue factor pathway inhibitor (TFPI) gene could induce vascular smooth muscle cell (VSMC) apoptosis. This study was conducted to investigate whether the overexpression of the TFPI gene can induce VSMC apoptosis by inhibiting JAK-2/STAT-3 pathway phosphorylation and thereby inhibiting the expression of such downstream targets as the apoptotic protein Bcl-2 and cell cycle protein cyclin D1. The effect of TFPI on the expression of survivin, a central molecule in cell survival, was also investigated.MethodsRat VSMCs were infected with recombinant adenovirus containing either the TFPI (Ad-TFPI) or LacZ (Ad-LacZ) gene or DMEM in vitro. TFPI expression was detected by ELISA. TUNEL staining and electron microscope were carried out to determine the apoptosis of VSMCs. The expression levels of JAK-2, p-JAK-2, STAT-3, p-STAT-3, cyclin D1, Bcl-2 and survivin were examined by western blot analysis.ResultsTFPI protein was detected in the TFPI group after gene transfer and the peak expression was at the 3rd day. At the 3rd, 5th and 7th days after gene transfer, the apoptotic rates by TUNEL assay in the TFPI group were 10.91 ± 1.66%, 13.46 ± 1.28% and 17.04 ± 1.95%, respectively, whereas those in the LacZ group were 3.28 ± 0.89%, 4.01 ± 0.72% and 4.89 ± 1.17%, respectively. We observed cell contraction, slight mitochondrial swelling, nuclear pyknosis and apoptotic body formation in TFPI-treated VSMCs using electron microscopy. JAK-2, p-JAK-2, STAT-3, p-STAT-3, cyclin D1 and Bcl-2, which are all involved in the JAK-2/STAT-3 pathway, were detected in the VSMCs on the 3rd, 5th and 7th days after gene transfer, which is consistent with previously demonstrated time points when VSMCs apoptosis occurred. The expression levels of p-JAK-2, p-STAT-3, cyclin D1 and Bcl-2 were significantly decreased over time in the TFPI group (each P < 0.05) but not in the Ad-LacZ and DMEM groups. However, this attenuation of expression was not observed for JAK-2 and STAT-3 in any of the groups at any time points after gene transfer (each P > 0.05). The expression level of survivin in the TFPI group also weakened significantly over time compared with the levels in the Ad-LacZ and DMEM groups (each P < 0.05) at the 3rd, 5th and 7th days after gene transfer.ConclusionThe results demonstrated that TFPI played an apoptosis-inducing role in VSMCs in a manner that involves both the suppression of JAK-2/STAT-3 pathway phosphorylation and the down-regulation of survivin. Our data show for the first time that targeting the JAK-2/STAT-3 pathway and survivin by overexpressing TFPI may be a new avenue for the treatment of restenosis.     

Chitinase 3 like 1 is associated with tumor angiogenesis in cervical cancer

Elevated serum levels of a secreted glycoprotein chitinase 3 like 1 (CHI3L1) are associated with poor prognosis and short survival time of patients with cervical cancer (CxCa). Our previous microarray data showed the increased expression of CHI3L1 in invasive CxCa compared to normal tissue, implicating a potential role of CHI3L1 in CxCa. To establish the pathological role of CHI3L1 in the development of CxCa, this study focused on its expression in CxCa and angiogenic impacts in tumor vessel formation. CHI3L1 activated angiogenesis by promoting endothelial cell migration and tube formation in vitro but failed to protect CxCa cell lines, CaSki and HeLa against apoptosis induced by γ-irradiation. In addition, the capability of CHI3L1 to induce proliferation and migration of CaSki and HeLa cells was cell type specific. In an analysis of 103 specimens from CxCa patients, increased expression levels of CHI3L1 mRNA and protein in invasive CxCa were 4-fold (P < 0.05) and 2-fold (P < 0.01), respectively, stronger than those in normal subjects. The immunostaining of CHI3L1 was positively correlated with VEGF expression (P = 0.0019) and microvessel density (P = 0.0110). Moreover, CHI3L1 expression was also positively associated with cancer metastasis (P = 0.011). The data suggest the crucial role of CHI3L1 by promoting angiogenesis, which may contribute to the development and progression of CxCa. The findings help establish CHI3L1 as a prognostic biomarker and therapeutic target for CxCa patients.     

Proteomics identified nuclear N-myc downstream-regulated gene 1 as a prognostic tissue biomarker candidate in renal cell carcinoma

The aim of this study was to identify proteins with aberrant expression in clear cell renal cell carcinoma (ccRCC), and elucidate their clinical utilities. The protein expression profiles of primary ccRCC tumor tissues and neighboring non-tumor tissues were obtained from 9 patients by two-dimensional difference gel electrophoresis and mass spectrometry. Comparative analysis of 3771 protein spots led to the identification of 73 proteins that were expressed at aberrant levels in tumor tissues compared with non-tumor tissues. Among these 73 proteins, we further focused on N-myc downstream-regulated gene 1 protein (NDRG1). NDRG1 expression is regulated by members of myc family as well as by p53, HIF1A, and SGK1. The biological and clinical significance of NDRG1 is controversial for various malignancies and no detailed studies on NDRG1 have been reported in ccRCC until our study. For the 82 newly enrolled ccRCC patients, immunohistochemical analysis revealed a significant association between nuclear NDRG1 and favorable prognosis (p < 0.05). Multivariate analysis demonstrated the role of NDRG1 as an independent factor of progression-free survival (p = 0.01). Subsequent in vitro gene suppression assay demonstrated that NDRG1 silencing significantly enhanced cell proliferation and invasion of RCC cells. The cytotoxic effects of NDRG1 up-regulation induced by an iron chelator were also confirmed. These findings suggest that nuclear NDRG1 has tumor suppressive effects, and the NDRG1 expression may have clinical values in ccRCC. Nuclear NDRG1 may provide additional insights on molecular backgrounds of ccRCC progression, and contribute to the development of novel therapeutic strategy.     

Correlation between VEGFR-2 receptor kinase domain-containing receptor (KDR) mRNA and angiotensin II receptor type 1 (AT1-R) mRNA in endometrial cancer

PurposeAngiogenesis, a multistep process that results in new blood vessel formation from preexisting vasculature is essential for both the growth of solid tumour and for metastasis. Stimulation of vascular endothelial growth factor receptor (VEGFR), a transmembrane glycoprotein, results in mitogenesis. Within this family of receptors, VEGFR 2/kinase-insert-domain containing receptor appears to be principally upregulated during tumorigenesis. The aim of this study was to determine the expression of VEGFR-2/kinase-insert-domain containing receptor (KDR) and its correlation with angiotensin receptor type 1 (AT1-R) and clinical factors in endometrial carcinoma.MethodsThe expression of KDR and AT1-R was studied in endometrial carcinoma and normal endometrium by Real-time RT-PCR and Western blot analysis in 136 samples. The expression profile was correlated with the clinicopathological characteristics of endometrial adenocarcinoma.ResultsWe noted a significant correlation between the expression of KDR and AT1-R in tumour grade G1, G2 and G3 (R_s = 0.50; p = 0.002, R_s = 0.69; p = 0.0001, R_s = 0.52; p = 0.005, respectively). In stage I and stage II carcinoma, a significant correlation was also found between the expression of KDR and AT1-R (R_s = 0.70, p = 0.0001, R_s = 0.67; p = 0.001, respectively). Moreover significant correlation was observed between both KDR and AT1-R in tissue with different myometrial invasion (R_s = 0.54, p = 0.0001, R_s = 0.68; p = 0.0001; respectively for tumours with invasion into the inner half and invasion into the outer half).ConclusionsBasing on received correlation between AT1-R and KDR expression and previous results we speculate that angiotensin through AT1-R modulates KDR expression and thus have influence on local VEGF level. However, further studies are required to clarify the biological interaction between KDR, AT1-R and other hormonal regulators in endometrial carcinoma.     

Characterization and PCR application of a new high-fidelity DNA polymerase from Thermococcus waiotapuensis

The family B DNA polymerase gene from the euryarchaeon Thermococcus waiotapuensis (Twa) contains an open reading frame of 4404 bases that encodes 1467 amino acid residues. The gene is split by two intein-coding sequences that forms a continuous open reading frame with the three polymerase exteins. Twa DNA polymerase genes with (whole gene) and without (genetically intein-spliced) inteins were expressed in Escherichia coli Rosetta(DE3)pLysS. The inteins of the expressed whole gene were easily spliced during purification. The molecular mass of the purified Twa DNA polymerase was about 90 kDa, as estimated by SDS-PAGE. The optimal pH for Twa DNA polymerase activity was 6.0 and the optimal temperature was 75 °C. The enzyme was activated by magnesium ions. The half-life of the enzyme at 99 °C was about 4 h. The optimal buffer for PCR with Twa DNA polymerase was 50 mM Tris–HCl (pH 8.2), 2.0 mM MgCl₂, 30 mM KCl, 2.0 mM (NH₄)₂SO₄, 0.01% Triton X-100, and 0.005% BSA. The PCR fidelity of Twa DNA polymerase was higher than Pfu, KOD and Vent DNA polymerases. A ratio of 15:1 Taq:Twa DNA polymerase efficiently facilitated long-range PCR.     

The regulated in development and DNA damage response 2 (REDD2) gene mediates human monocyte cell death through a reduction in thioredoxin-1 expression

In a previous study, we identified the regulated in development and DNA damage response 2 (REDD2) gene as a highly expressed gene in human atherosclerotic lesions in comparison to normal artery, as well as in cultured human macrophages, and showed its implication in oxidized low-density lipoprotein (LDL)-induced macrophage death sensitivity. In this article, we attempt to identify the mechanism by which REDD2 induces such a phenomenon. Transient transfection of U-937 monocytic cells with a pCI.CMV.REDD2 expression vector increased by approximately twofold the mRNA levels of REDD2 in comparison to control cells transfected with pCI.CMV.GFP. Reactive oxygen species (ROS) production was significantly induced in REDD2-transfected cells compared with control cells (157 ± 48 and 100 ± 8 arbitrary units/mg cell protein, respectively; p < 0.05). Moreover, a significant increase in parameters known to reflect the oxidative modifications of LDL was observed. Among enzymes involved in ROS production or degradation, we found a specific reduction in thioredoxin-1 (Trx-1) mRNA (～ 52 ± 7% decrease, p < 0.01 vs control cells) and protein (～ 60 ± 4% decrease, p < 0.001 vs control cells) levels in cells overexpressing REDD2 in comparison to control cells. In contrast, transfection of U-937 cells with siRNA against REDD2 decreased the mRNA levels of REDD2 by ～ 60% and increased Trx-1 mRNA and protein levels. Moreover, we observed no or a moderate increase in Bax (proapoptotic) and a significant decrease in Bcl2 (antiapoptotic) gene expression in cells that overexpress REDD2 compared to control cells. In addition, we showed that Trx-1 mRNA and protein levels were increased at low H₂O₂ doses and decreased at higher doses. Interestingly, macrophages isolated from human atherosclerotic lesions differentially express REDD2 and Trx-1. Indeed, in certain patients, levels of REDD2 mRNA were low and those of Trx-1 mRNA were high. In contrast, in other patients, levels of REDD2 were high and levels of Trx-1 mRNA were low.     

Cardioprotective effects of lipoic acid,quercetin and resveratrol on oxidative stress related to thyroid hormone alterations in long-term obesity

This study investigated possible mechanisms for cardioprotective effects of lipoic acid (LA), quercetin (Q) and resveratrol (R) on oxidative stress related to thyroid hormone alterations in long-term obesity. Female C57BL/6 mice were fed on high-fat diet (HFD), HFD + LA, HFD + R, HFD + Q and normal diet for 26 weeks. Body weight, blood pressure, thyroid hormones, oxidative stress markers, angiotensin converting enzyme (ACE), nitric oxide synthase (NOS) and ion pump activities were measured, and expression of cardiac genes was analyzed by real-time polymerase chain reaction. HFD induced marked increase (P < .05) in body weight, blood pressure and oxidative stress, while plasma triidothyronine levels reduced. ACE activity increased (P < .05) in HFD mice (0.69 ± 0.225 U/mg protein) compared with controls (0.28 ± 0.114 U/mg protein), HFD + LA (0.231 ± 0.02 U/mg protein) and HFD + Q (0.182 ± 0.096 U/mg protein) at 26 weeks. Moreover, Na⁺/K⁺-ATPase and Ca^2 +-ATPase activities increased in HFD mice whereas NOS reduced. A 1.5-fold increase in TRα1 and reduction in expression of the deiodinase iodothyronine DIO1, threonine protein kinase and NOS3 as well as up-regulation of AT1α, ACE, ATP1B1, GSK3β and Cja1 genes also occurred in HFD mice. Conversely, LA, Q and R inhibited weight gain; reduced TRα1 expression as well as increased DIO1; reduced ACE activity and AT1α, ATP1B1 and Cja1 gene expression as well as inhibited GSK3β; increased total antioxidant capacity, GSH and catalase activity; and reduced blood pressure. In conclusion, LA, resveratrol and quercetin supplementation reduces obesity thereby restoring plasma thyroid hormone levels and attenuating oxidative stress in the heart and thus may have therapeutic potential in heart diseases.     

Metabolic activation of phenanthrene by human and mouse cytochromes P450 and pharmacokinetics in CYP1A2 knockout mice

In the present study V79 Chinese hamster cells were genetically engineered for stable expression of the cytochromes P450 1A1, 1A2, 1B1, and 2E1 from man and mouse to investigate species-specific differences in the regioselective metabolism and toxicity of phenanthrene (Phe), the simplest polycyclic aromatic hydrocarbon (PAH) forming a bay-region. Phe is present in various environmental samples and serves as a model substrate for PAH exposure in human biomonitoring studies. For this reason we explored metabolite profiles and metabolite-dependent cytotoxic activities in vitro. The total turnover of CYP-mediated transformation of Phe was as follows: human CYP1B1 > CYP1A1 > CYP1A2 ? CYP2E1, and for mouse CYP1A2 ? CYP2E1 > CYP1A1. Striking species differences were seen as mouse CYP1B1 did not activate Phe at all, but human CYP1B1 exhibited a significant metabolic turnover comparable to CYP1A1 and CYP1A2. In vivo studies monitoring the whole blood Phe elimination in CYP1A2 knockout and wild-type mice after oral administration confirmed involvement of CYP1A2 in the bioactivation of Phe, but other processes must contribute also. Our data suggest that in humans not only CYP1A2 expressed solely in the liver plays a crucial role in Phe metabolism, but also constitutively expressed extrahepatic CYP1B1 in tissues such as lung, kidney or intestine. This finding will substantially improve the validity of human biomonitoring studies using individual Phe metabolites for the assessment of PAH exposure.