Celastrol induced hepatocellular carcinoma cell death via ROS-mediated DNA damage / 中国药理学通报

Aim To investigate the role of eelastrol in reactive oxygen species ( ROS) accumulation and DNA damage in hepatocellular carcinoma cells, and further investigate its effect on apoptosis induction in cancer cells.Methods Human liver cancer HepG2 and Huh7 cells were cultured with celastrol, then the morphological changes of cells were observed under microscope.MTT assay was employed to detect the proliferation of hepatocellular carcinoma cells, CM-H2DCFDA probe to detect intracellular ROS levels, and immunofluorescence to detect the expression level of -y-H2AX in celastrol-treated cells.Hoechst 33258 staining was used to observe nuclear condensation and fragmentation; Flow cytometry was used to evaluate cell death.J J Western blot was applied to measure the expression levels of -y-H2 AX, caspase-3, PARP and other proteins.Results Celastrol had a significant inhibitory effect on the proliferation of liver cancer cells in dose-dependent manner.Comparer] with the eontrol group, the eell viability was reduced and intracellular ROS level also increased significantly after celastrol treatment in a dose-dependent manner ( P < 0.05 ).With Hoechst staining, typical apoptotic characteristics such as nuclear chromatin condensation and fragmentation were observed in celastrol-treated cells.Western blot results showed that pro-form of caspase-3 significantly decreased, and the cleavage of PARP markedly increased by celastrol.After pretreatment with ROS inhibitor NAC, celastrol-mediated caspase-3 activation and PARP cleavage were significantly reversed ( P < 0.05 ).Conclusions Celastrol can induce apoptosis in hepatocellular carcinoma cells, and its anti-cancer effect is dependent on ROS-mediated DNA damage.

Echinococcus multilocularis infection leads to increased expression of proliferation and anti-apoptosis factors in liver cells in BALB/c mice / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects of Echinococcus multilocularis on host liver cell proliferation in vivo using a BALB/c mouse alveolar hydatid infection model.</p><p><b>METHODS</b>Sixty-five 8-10-week-old female BALB/c mice were randomly divided into an experimental group (n = 40) and a control group (n = 25) and administered an abdominal injection into the left liver lobe of E. multilocularis protoscolices in saline solution or saline solution alone, respectively. At post-injection day 2, 8, 30, 60, and 90, liver samples were collected for analysis of lesions and lesion-adjacent tissue by hematoxylin-eosin staining and differential expression of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin A, and cyclin B1 by immunohistochemical staining. The significance of intergroup differences was assessed by Student's t-test.</p><p><b>RESULTS</b>The control group showed normal liver histology at all time points. The experimental group developed E. multilocularis lesions that showed increased severity of pathological features, such as inflammatory cell invasion, steatosis and fibrous connective tissue hyperplasia, over time. At post-injection days 2 and 8, enlarged, binuclear and apocyte hepatocytes were observed close to the lesions. At post-injection days 30, 60, and 90, the number of hepatocytes expressing PCNA progressively increased in the experimental group, and the numbers were significantly higher than in the control group (7.01 +/- 1.89 vs. 1.03 +/- 0.52, 8.41 +/- 2.80 vs. 0.93 +/- 0.31, and 13.4 +/- 4.43 vs. 1.07 +/- 0.94; all P < 0.05). The same progressively increasing trend was seen in the number of hepatocytes expressing CyclinD1, but was only significantly different from controls at post-injection days 30 and 60 (6.73 +/- 2.52 vs. 0.48 +/- 0.43 and 8.22 +/- 3.09 vs. 0.55 +/- 0.34; both P < 0.05). In contrast, the number of hepatocytes expressing cyclin A was significantly increased at post-injection day 30 and then showed a decreasing trend at days 60 and 90, although the numbers of expressing cells remained significantly higher than control levels at all time points (7.75 +/- 3.05 vs. 0.69 +/- 0.36, 3.42 +/- 1.80 vs. 1.14 +/- 0.42, and 3.03 +/- 1.50 vs. 0.69 +/- 0.31; all P < 0.05). The number of hepatocytes expressing CyclinB1 in the experimental group was less robust than the other cyclins (with a general temporal trend of increase followed by decrease), but the differential expression was not significantly different from the control levels at any time point.</p><p><b>CONCLUSION</b>E. multilocularis infection may promote the expression of host factors related to proliferation and anti-apoptosis in liver. This pathogen-mediated modulation of host cell-survival mechanisms may provide a rationale explanation for the clinical observations of hepatomegaly and the unexpected survival of alveolar echinococcosis patients following major hepatic resection.</p>

Expression of ERK1/2 MAPK signaling transduction pathway in esophageal cancers in Kazakh patients / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression variation and significance of ERK1/2 MAPK signaling transduction pathway in the pathogenesis of esophageal squamous cell carcinoma (ESCC) in Kazakh patients.</p><p><b>METHODS</b>The expression level of p-ERK1/2 after serum starvation and treatment with U0126 inhibitor was detected in esophageal cancer cell line EC9706 by Western blot assay. The mRNA level of total ERK1/2 (t-ERK1/2) and expression level of t-ERK1/2 and p-ERK1/2 proteins of 25 pairs of ESCC and adjacent normal esophageal mucosal tissues of Kazakh patients were examined and identified by real-time quantitative PCR (qRT-PCR) and Western blotting, respectively. The expression of p-ERK1/2 protein was verified by immunohistochemistry in 126 paraffin-embeded specimens, including 19 normal esophageal mucosa, 55 esophageal carcinomas in situ and 52 invasive carcinomas.</p><p><b>RESULTS</b>ERK1/2 MAPK signaling transduction pathway was in an active status in the EC9706 cells. The expression level of p-ERK1/2 in Ec9706 cells reached a peak at 10 min after transient serum stimulation, and p-ERK1/2 expression was totally restrained after the treatment with 50 µmol/L U0126. In the 25 pairs of ESCC and adjacent normal mucosa, the t-ERK1 mRNA level was 1.92 ± 3.49 in the ESCC tissues and 3.67 ± 7.47 in the adjacent normal mucosa. The t-ERK1 mRNA level in ESCC tissues was significantly lower than that in adjacent normal mucosa (P < 0.05), whereas there was no significant difference of t-ERK2 mRNA level between them(P > 0.05). The expression levels of p-ERK1 and p-ERK2 proteins were 0.87 ± 0.14 and 0.79 ± 0.10 in the ESCC tissues, and 1.10 ± 0.13 and 1.32 ± 0.12 in the adjacent normal mucosae. p-ERK1/2 protein in the ESCC tissues was significantly lower than that in the adjacent normal tissue (P < 0.01). However, there was no significant difference between their t-ERK1/2 protein levels (P > 0.05). In the 126 cases of paraffin-embeded specimens, positive expressions of both p-ERK1 and p-ERK2 in esophageal cancer tissues were 7.7% (4/52), significantly lower than those in adjacent normal mucosa (31.6%, 6/19) and carcinoma in situ (85.5%, 47/55, P < 0.05).</p><p><b>CONCLUSIONS</b>ERK1/2 MAPK signaling pathway is in an active status in esophageal cancer and adjacent normal mucosa. Our results imply that the activation of p-ERK1/2 MAPK signaling transduction pathway plays a role in the early pathogenesis of ESCC in Kazakh patients.</p>

Molecular characterization of a signal-regulated kinase homolog from Echinococcus granulosus / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Cystic echinococcosis due to Echinococcus granulosus (E. granulosus) is one of the most important chronic helminthic diseases, especially in sheep/cattle-raising regions. The larval stage of the parasite forms a cyst that grows in the liver, lung, or other organs of the host. To ensure a long life in the host tissues, the parasite establishes complex inter-cellular communication systems between its host to allow its differentiation toward each larval stage. Recent studies have reported that this communication is associated with the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade in helminth parasites, and in particular that these protein kinases might serve as effective targets for a novel chemotherapy for cystic echinococcosis. The aim of the present study investigated the biological function of a novel ERK ortholog from E. granulosus, EgERK.</p><p><b>METHODS</b>DNA encoding EgERK was isolated from protoscolices of E. granulosus and analyzed using the LA Taq polymerase chain reaction (PCR) approach and bioinformatics. Reverse transcription PCR (RT-PCR) was used to determine the transcription level of the gene at two different larval tissues. Western blotting was used to detect levels of EgERK protein. The expression profile of EgERK in protoscolices was examined by immunofluorescence.</p><p><b>RESULTS</b>We cloned the entire Egerk genomic locus from E. granulosus. In addition, two alternatively spliced transcripts of Egerk, Egerk-A, and Egerk-B were identified. Egerk-A was found to constitutively expressed at the transcriptional and protein levels in two different larval tissues (cyst membranes and protoscolices). Egerk-A was expressed in the tegumental structures, hooklets, and suckers and in the tissue surrounding the rostellum of E. granulosus protoscolices.</p><p><b>CONCLUSIONS</b>We have cloned the genomic DNA of a novel ERK ortholog from E. granulosus, EgERK (GenBank ID HQ585923), and found that it is constitutively expressed in cyst membrane and protoscolex. These findings will be useful in further study of the biological functions of the gene in the growth and development of Echinococcus and will contribute to research on novel anti-echinococcosis drug targets.</p>

Isolation and identification of Sclerotinia stem rot causal pathogen in Arabidopsis thaliana / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

A new stem rot disease is found to occur naturally on Arabidopsis plants in greenhouses of Fuzhou, China. In order to identify its pathogen, we conducted a series of fungal isolation and purification, plant reinoculation, and ascus and ascospore induction from the sclerotia. The isolate caused typical water-soaked lesions after reinoculation and produced sclerotia both on Arabidopsis plants and culture medium plates, and the sclerotia could be induced to produce discal apothecia and 8 binucleate ascospores per ascus. These disease symptom and fungal morphology data revealed that the fungus Sclerotinia sclerotiorum (Lib.) de Bary was the pathogen for Arabidopsis stem rot. To confirm this, we further amplified its large subunit ribosomal DNA (LSU rDNA) by polymerase chain reaction (PCR), and compared the sequence with the known LSU rDNA sequences in GenBank. The results show that the sequence shares the highest identities with the LSU rDNAs of different S. sclerotiorum strains. Taking all these data together, we concluded that the fungus that caused the Arabidopsis stem rot is S. sclerotiorum (Lib.) de Bary. This is the first report that Arabidopsis is naturally infected by S. sclerotiorum.

Renal dysfunction in workers exposed to arsenic and cadmium / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To examine the nephrotoxicity induced caused by combined effect of arsenic and cadmium in exposed workers.</p><p><b>METHODS</b>Urinary cadmium and arsenic were used as the exposure biomarkers of cadmium and arsenic. Urinary beta2-microglobulin (Ubeta2-MG), albumin (UALB) and N-acetyl-beta-D-glucosaminidase (UNAG) were measured as the effective biomarkers of tubular and glomerular dysfunction induced by cadmium and arsenic.</p><p><b>RESULTS</b>The combination of cadmium and arsenic induced more severe renal injury than that caused by either of the chemicals given alone. There were positive correlations and significant dose-effect among the concentrations of urinary cadmium, arsenic and levels of Ubeta2-MG, UALB, UNAG (P<0.05, P<0.01).</p><p><b>CONCLUSION</b>Cadmium combined with arsenic may have additive effect on renal dysfunction in workers exposed to arsenic and cadmium.</p>

Optimization of T-dNA insertional mutagenesis and analysis of mutants of Magnaporthe grisea / 生物工程学报

The rice blast fungus Magnaporthe grisea causes one of the most destructive diseases of rice around the world. Significant progresses have been made recently in genomics studies of the fungus, opening new era of the functional genomics which requires to generate a large scale of gene knockout mutants. It has been demonstrated that T-DNA insertional mutagenesis is a powerful tool of functional genomics not only for plants but also for fungi. In this paper, we optimized the conditions for T-DNA insertional mutagenesis of M. grisea using Agrobacterium tumefaciens-mediated transformation (ATMT) approach. We employed the binary vector pBHtl constructed by Dr. S. Kang's laboratory at the Pennsylvania State University, which carries the bacterial hygromycin B phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker to transform the conidia of M. grisea. We optimized the conditions for T-DNA insertional mutagenesis including the medium, dosage of hygromycin B, cefotaxime and carbenicillin to select the transformants and inhibit the growth of A. tumefaciens after co-culturing. The dosage to inhibit non-transformants could vary from 200-600microg/mL among different M. grisea isolates so that the optimal dosage of the antibiotics should be decided according to isolates. Rice polished agar medium was found the best selection medium which would facilitate the mutant sporulation and minimize the contamination chance. In average, about 500 transformants could be obtained when transforming 1 x 10(6) spores at the optimum condition, among which 85% had T-DNA insertion detected by polymerase chain reaction (PCR) and thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). Fifteen out of 1520 transformants showed mutation in colony morphology. Within 58 randomly selected mutants, it was found that there were 4 sporulation-decreased mutants, 8 less germination mutants and 9 appressorium defective mutants. Several virulent mutants to C101LAC(Pi-1)and 75-1-127(Pi-9)were also obtained which would facilitate cloning the corresponding avirulence genes.