Quercetin, but not rutin and quercitrin, prevention of H2O2-induced apoptosis via anti-oxidant activity and heme oxygenase 1 gene expression in macrophages

In the present study, we examine the protective mechanism of quercetin (QE) on oxidative stress-induced cytotoxic effect in RAW264.7 macrophages. Results of Western blotting show that QE but not its glycoside rutin (RUT) and quicitrin-induced HO-1 protein expression in a time- and dose-dependent manner, and HO-1 protein induced by QE was blocked by an addition of cycloheximide or actinomycin D. Induction of HO-1 gene expression by QE was accompanied by inducing ERKs, but not JNKs or p38, proteins phosphorylation. Addition of PD98059, but not SB203580 or SP600125, significantly attenuates QE-induced HO-1 protein and mRNA expression associated with blocking the expression of phosphorylated ERKs proteins. H(2)O(2) addition reduces the viability of cells by MTT assay, and appearance of DNA ladders, hypodiploid cells, and an increase in intracellular peroxide level was detected. Addition of QE, but not QI or RUT, significantly reduced the cytotoxic effect induced by H(2)O(2) associated with blocking the production of intracellular peroxide, DNA ladders, and hypodiploid cells. QE protection of cells from H(2)O(2)-induced apoptosis was significantly suppressed by adding HO inhibitor SnPP or ERKs inhibitor PD98059. Additionally, QE protects cells from H(2)O(2)-induced a decrease in the mitochondrial membrane potential and a release of cytochrome c from mitochondria to cytosol by DiOC6 and Western blotting assay, respectively. Activation of apoptotic proteins including the caspase 3, caspase 9, PARP, D4-GDI proteins was identified in H(2)O(2)-treated cells by Western blotting and enzyme activity assay, and that was significantly blocked by an addition of QE, but not RUT and QI. Furthermore, HO-1 catalytic metabolites carbon monoxide (CO), but not Fe(2+), Fe(3+), biliverdin or bilirubin, performed protective effect on cells from H(2)O(2)-induced cell death with an increase in HO-1 protein expression and ERKs protein phosphorylation. These data suggest that induction of HO-1 protein may participate in the protective mechanism of QE on oxidative stress (H(2)O(2))-induced apoptosis, and reduction of intracellular ROS production and mitochondria dysfunction with blocking apoptotic events were involved. Differential anti-apoptotic effect between QE and its glycosides RUT and QI via distinct HO-1 protein induction was also delineated.     

Clofibrate and perfluorodecanoate both upregulate the expression of the pregnane X receptor but oppositely affect its ligand-dependent induction on cytochrome P450 3A23

The pregnane X receptor (PXR) interacts with a vast array of structurally dissimilar chemicals and confers induction of several major types of drug metabolizing enzymes such as cytochrome P450s (CYP). We previously reported that the expression of PXR was markedly increased in rats treated with clofibrate and perfluorodecanoic acid (PFDA). The present study was undertaken to test the hypothesis that induced expression of PXR increases PXR ligand-dependent induction on CYP3A23. Rat hepatocytes were treated with clofibrate or PFDA individually, or along with PXR ligand pregnenolone 16alpha-carbonitrile (PCN), and the levels of PXR and CYP3A23 were determined by Western blots. Both clofibrate and PFDA markedly increased the expression of PXR with PFDA being more potent, and the induction was abolished by actinomycin D, an inhibitor for mRNA synthesis. As expected, PCN alone markedly induced the expression of CYP3A23. Interestingly, co-treatment with clofibrate enhanced the induction, whereas co-treatment with PFDA suppressed it. Clofibrate and PFDA represent multi-classes of chemicals called peroxisome proliferators including many therapeutic agents and industrial pollutants. The opposing effects of clofibrate and PFDA on the PCN-induced expression of CYP3A23 suggest that peroxisome proliferators likely increase the expression of PXR but differentially alter its ligand-dependent induction. The interaction between PXR inducer and ligand provides a novel mechanism on how functionally and structurally distinct chemicals cooperatively regulate the expression of xenobiotic-metabolizing enzymes and transporters.     

Suppressor of cytokine signaling-2: a growth hormone-inducible inhibitor of intestinal epithelial cell proliferation

BACKGROUND & AIMS: Growth hormone (GH) and insulin-like growth factor-I (IGF-I) increase intestinal growth. GH is thought to act indirectly via IGF-I. In several models, including rats given total parenteral nutrition (TPN), IGF-I more potently stimulates mucosal growth than GH, even when GH induces similar circulating IGF-I levels. These studies test the hypothesis that GH induces a suppressor of cytokine signaling (SOCS), which inhibits intestinal epithelial cell (IEC) proliferation. METHODS: Rats on TPN received vehicle, GH, or IGF-I. Jejunal SOCS (SOCS-1, -2, -3, and cytokine-inducible SH2-domain-containing protein [CIS]) and IGF-I messenger RNA (mRNA) were quantified. Caco-2, IEC-6 cells, and SOCS-2 null and wild-type (WT) mice were used to examine the expression and functional role of SOCS-2. RESULTS: As reported previously, IGF-I, but not GH, prevented mucosal atrophy during TPN, although GH elevated plasma IGF-I and increased body weight. GH, but not IGF-I, induced jejunal SOCS-2 mRNA. SOCS-2 mRNA levels in GH and IGF-I-treated rats inversely correlated with mucosal weight. SOCS-2 is expressed in Caco-2 cells, and elevated SOCS-2 expression in postconfluent cells is associated with reduced proliferative rates. SOCS-2 overexpression in Caco-2 cells inhibited cell proliferation and promoted differentiation. In IEC-6 cells, GH induced SOCS-2 and reduced basal or IGF-I-induced proliferation. GH also reduced proliferative activity in isolated crypts from WT but not SOCS-2 null mice, and SOCS-2 null crypts showed enhanced proliferative responses to GH and IGF-I. SOCS-2 null mice have increased intestinal weight and length. CONCLUSIONS: SOCS-2 is a GH-inducible, novel inhibitor of intestinal epithelial cell proliferation and intestinal growth.     

肾癌中P16表达的变化及其意义

目的探讨抑癌基因P16纯合性缺失与肾癌发生发展的关系。方法用RT-PCR等方法检测24例肾细胞癌和10例对照组(非癌症肾组织)P16基因mRNA的表达。结果在24例肾癌中,P16基因表达缺失14例(58.3%),中等阳性表达6例(25%),弱阳性表达4例(16.7%)。而对照组均呈强阳性表达(100%)。P16基因与肾癌病理分级无相关性,与临床分期有显著相关(P<0.05)。结论抑癌基因P16在肾细胞癌的生长、浸润和转移过程中可能起重要作用,对肾脏恶性肿瘤的诊断、治疗和预后的判定,可能具有潜在的临床价值。     

Hantavirus and acute appendicitis—The diagnosis behind the diagnosis?

A 33-year-old man with a history of acute lower abdominal pain was admitted to the emergency room. After laparoscopic appendectomy and pathological confirmed acute appendicitis the patient developed thrombocytopenia and acute renal failure. Serological testing for hantaviruses revealed a positive result for PUUV IgG and IgM. Immunohistochemical work-up detected PUUV antigen in endothelial cells of capillaries and larger vessels. The high percentage of patients with hantavirus infection and severe abdominal pain is remarkable and, up to now, unexplained. To our knowledge this is the first report demonstrating PUUV antigen in the human intestine. Further studies are warranted whether hantaviruses are setting the stage for a secondary bacterial infection or cause an inflammation itself.     

Detection of hepatitis B virus DNA in paraffin-embedded intrahepatic and extrahepatic cholangiocarcinoma tissue in the northern Chinese population

This study explored the importance of hepatitis B virus infection in cholangiocarcinoma pathogenesis in northern China. The clinical data of 66 patients with cholangiocarcinoma were analyzed. The hepatitis B virus gene was amplified using nested polymerase chain reaction, and the hepatitis B virus-related antigen was detected using immunohistochemistry in formalin-fixed, paraffin-embedded tissue from patients with intrahepatic cholangiocarcinoma (n = 23) and extrahepatic cholangiocarcinoma (n = 43). Hepatitis B surface antigen seropositivity was found in 52.2% (12/23) of intrahepatic cholangiocarcinoma cases and 13.9% (6/43) of extrahepatic cholangiocarcinoma cases. Hepatitis B virus DNA (X region) was detectable in 34.8% (8/23) of intrahepatic cholangiocarcinoma cases. Hepatitis B surface antigen and/or hepatitis B core antigen was detectable in 30.4% (7/23) of intrahepatic cholangiocarcinoma cases. All cases with detected viral protein were also positive for hepatitis B virus DNA. In contrast, no hepatitis B virus antigens or hepatitis B virus gene was detected in any of the 43 extrahepatic cholangiocarcinoma cases. Our findings strongly suggest that chronic hepatitis B virus infection is a significant risk factor for intrahepatic cholangiocarcinoma, but not for extrahepatic cholangiocarcinoma, in northern China. Hepatitis B virus infection is potentially independently associated with intrahepatic cholangiocarcinoma.     

The expression and role of c-Myc in mouse hair follicle morphogenesis and cycling

Although the function of c-Myc has been clarified in many tissues, until now its expression and role in hair follicle morphogenesis and the hair cycle remains unknown. In this study we detected c-Myc expression pattern in the process of mouse hair follicle development and normal cycle. We found that during hair follicle morphogenesis, the stage-specific expression of c-Myc was detected in mouse skin and was predominantly localized to the hair follicle epithelium. c-Myc expression was also consistently found in mouse skin throughout the hair follicle cycle. Through the in vivo injection of c-Myc inhibitory peptide and c-Myc expression plasmid, we also investigated the direct effects of c-Myc on the hair follicle structures during the hair follicle cycle. Our results showed that c-Myc inhibitory peptide significantly restrained the development of anagen hair follicles, while the injection of plasmid DNA encoding c-Myc in vivo clearly promoted anagen development. Our data indicate that c-Myc may play an important role in the proliferation and differentiation of the hair follicle keratinocytes during hair follicle development. c-Myc also was shown to participate in the regulation of the mouse hair growth cycle and could promote the proliferation of the hair matrix keratinocytes as well as the differentiation of the inner root sheath.     

Development and evaluation of one-step rRT-PCR and immunohistochemical methods for detection of Rift Valley fever virus in biosafety level 2 diagnostic laboratories

Rift Valley fever virus (RVFV) is a zoonotic insect transmitted virus endemic to Africa and the Arabian Peninsula. Infection causes abortions and high mortality in newborn ruminants. The overall human infection rate is <1%; however, fatality rates in those with severe clinical disease have been reported as high as 29%. The potential of RVFV as a bioterrorism agent and/or being accidentally introduced into North America is widely recognized. Currently, regional veterinary biosafety level 2 (BSL-2) diagnostic laboratories lack safe, modern, validated diagnostic tests to detect RVFV. An existing one-step real-time RT-PCR (rRT-PCR) assay was modified for quick virus inactivation for use in BSL-2 laboratories, evaluated on serum and tissue samples from experimentally infected lambs and calves, and compared to virus isolation. Viremia was detected in all inoculated sheep with titers reaching 10^6.5 plaque forming units/ml, or up to 10¹⁰ viral RNA copies/ml. Viremia in calves was lower and not detected in all inoculated animals; however, all animals became transiently febrile and were infected as determined by rRT-PCR of tissues. Virus was isolated from rRT-PCR-positive liver and/or spleen in 33% of lamb and 41% of calf samples between 2 and 7 days post inoculation. For RVFV antigen detection, reagents are typically produced at BSL-3Ag or BSL-4 conditions and require inactivation and safety testing for use outside of containment. In this study, antiserum against recombinant RVFV-nucleocapsid (N) was produced to develop an immunohistochemical (IHC) assay which was subsequently evaluated on formalin fixed lamb and calf tissues at BSL-2 laboratory conditions. Antigen was detected by IHC in 79% of rRT-PCR-positive sheep and 70% of rRT-PCR-positive calf tissues tested. Once validated and approved by national regulatory agencies, these assays can be safely produced and distributed to regional diagnostic laboratories, providing capacity for early detection of RVFV in suspected ruminant samples.     

Development and validation of a multiplex RT-PCR method for the simultaneous detection of five grapevine viroids

Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd), Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd) are the five viroids known to infect naturally grapevines. We developed a multiplex RT-PCR (mRT-PCR) method for the simultaneous detection of these five viroids and the amplification of the cDNA fragment of a host-derived mRNA (actin mRNA) as an internal positive control. Specific primers for each targeted viroid were designed by taking into account the sequence variability within and between the viroid species and tested in silico. The method was validated by testing 57 grapevine samples from Iran and showed reliability and high sensitivity. The RT-PCR-negative samples were further assayed by Northern-blot hybridization. For this, a method was developed for the simultaneous detection of three different grapevine viroids on a single hybridization membrane. In this survey, HSVd, GYSVd-1, AGVd, and GYSVd-2 were detected in 100, 95, 93, and 65% of the samples tested, respectively, confirming the wide distribution of these viroids in Iran. CEVd was not detected in any of the samples collected. Based on these results, HSVd is proposed as a positive internal control for mRT-PCR in the areas where this viroid is widespread, so as to reduce the time and costs of DNase treatment, which is required when a host-derived internal control is used. The mRT-PCR method has the potential to be used routinely for large-scale surveys and certification programs.     

血小板反应素-1在前列腺癌患者中的表达及意义

摘要：目的探讨血小板反应素1（TsP一1）在前列腺癌患者癌组织及其外周血中的表达及其意义。方法应用免疫组化技术检测TSP-1在前列腺癌组织（前列腺癌组）中的表达;以半定量RTPCR技术检测TSP-lmRNA在患者外周血中的表达,并与前列腺增生患者（前列腺增生组）进行比较。结果前列腺癌组TSP-1阴性表达6例,弱阳性表达16例,阳性表达4例,增生组TSP-1弱阳性表达12例,阳性表达16例,无阴性表达,前列腺癌组TSP-1表达强度较增生组显著降低（P〈O．01）;TSP-1mRNA在前列腺癌组织中较前列腺增生组织中表达明显下调（P〈0．01）。TSPlmRNA在前列腺癌组外周血中的表达较增生组明显下调（P〈o．01）。结论TSP-1基因及蛋白表达水平在前列腺癌组织中明显下调,在前列腺癌患者外周血中表达水平明显下调。