Hepatocellular carcinoma with colonic metastasis.

Hepatocellular carcinoma with colonic metastasis is rare. It mainly occurs by direct invasion and presents with bloody stools. We describe a patient with haematogenous metastasis to the rectum who presented with tenesmus. To our knowledge, such an association has not been reported previously. Colonic metastasis should be considered when patients with hepatocellular carcinoma present with bloody stools or tenesmus.

Rubella seroepidemiology and estimations of the catch-up immunisation rate and persistence of antibody titers in pregnant women in Taiwan.

OBJECTIVE: To examine rubella seroepidemiology, and estimate rates of catch-up immunisation and persistence of antibody titers in pregnant women in Taiwan after mass immunisation. DESIGN: A retrospective study. SETTING: Two medical centres and four regional hospitals specialising in obstetric care. SAMPLE: A total of 43,640 prenatal rubella test results for pregnant women from 2001 to 2008. METHODS: Rubella immunoglobulin G (IgG) antibody assay. MAIN OUTCOME MEASURES: Seronegativity, rate of catch-up immunization, and antibody decline. RESULTS: The seronegativity was 10.9% in all pregnant women. Immigrant women had higher seronegativity than indigenous women (OR 2.86; 95% CI 2.65, 3.01). Indigenous women born prior to implementation of the vaccination programmes were more susceptible (20.1%) to rubella infection than were women born thereafter (6.7%). Rates of seropositive conversion were low in both Taiwanese-born and foreign-born women (11.5 and 30.7%, respectively). The rubella antibody titers for vaccinated Taiwanese women in the 1971-1976 and after-1976 birth cohorts declined by 0.6 and 2.3% per year, respectively. CONCLUSIONS: This study demonstrates high seronegativity of older indigenous and immigrant women, a low catch-up immunisation rate, and the persistence of rubella antibodies in Taiwan after mass vaccination. Our study suggests that a single dose of rubella vaccine in teenagers effectively increased rubella seropositivity during their childbearing years. This finding is useful for countries that lack the resources necessary for a two-dose regimen. We recommend free rubella antibody tests to women of childbearing age and free vaccination as required. All postpartum women testing negative for rubella antibodies should be vaccinated before they leave hospital.

Cadmium-induced autophagy and apoptosis are mediated by a calcium signaling pathway.

The cytotoxicity of cadmium (Cd) induced autophagy and apoptosis in MES-13 cells was determined by flow cytometry. Autophagy was also assessed by formation of autophagosomes and processing of LC3. Pharmacological inhibition of autophagy resulted in increased of cell viability, suggesting autophagy plays a role in cell death in Cd-treated mesangial cells. Cd also induced a rapid elevation in cytosolic calcium ([Ca(2+)](i) ), and modulation of [Ca(2+)](i) via treatment with IP (3)R inhibitor or knockdown of calcineurin resulted in a change in the proportion of cell death, suggesting that the release of calcium from the ER plays a crucial role in Cd-induced cell death. Inhibition of Cd-induced ERK activation by PD 98059 suppressed Cd-induced autophagy, and BAPTA-AM eliminated activation of ERK. BAPTA-AM also inhibited Cd-induced mitochondrial depolarization and activation of caspases. These findings demonstrated that Cd induces both autophagy and apoptosis through elevation of [Ca(2+)](i), followed by Ca(2+)-ERK and Ca(2+)-mitochondria-caspase signaling pathways.

Antiplatelet autoantibodies elicited by dengue virus non-structural protein 1 cause thrombocytopenia and mortality in mice.

BACKGROUND: The mechanisms responsible for thrombocytopenia associated with dengue fever (DF) and dengue hemorrhage fever (DHF) remain unclear. OBJECTIVE: In this study, we investigated the pathogenic effects of dengue virus (DENV) non-structural protein 1 (NS1) on the elicitation of platelet cross-reactive antibodies. RESULTS: The results showed that anti-DENV NS1 immunoglobulins (Igs) derived from both patients with DF/DHF and recombinant NS1-immunized rabbits could opsonize normal human platelets and enhance platelet-macrophage engagements in vitro. In addition, treatments with anti-NS1 Igs abnormally activated human platelets and induced thrombocytopenia in mice. These prothrombotic characteristics of anti-NS1 Ig might increase the disease burden of coagulant-aberrant DHF patients. To test this hypothesis, we injected anti-NS1 Igs into C57BL/6J mice that were preconditioned into a hypercoagulable state by warfarin treatments. When given before but not after platelet-lysate pre-adsorption, the anti-NS1 Igs injection treatments significantly increased mortality, fibrin deposition in lung, and plasma D-dimer levels, but significantly decreased anticoagulant proteins C, protein S and antithrombin III. CONCLUSIONS: These results suggest that the platelet-bound antibody fractions of anti-NS1 Ig are prothrombotic, which might exacerbate the severity of disease in hosts with an imbalanced coagulant system.

Cholesterol-3-beta, 5-alpha, 6-beta-triol induced genotoxicity through reactive oxygen species formation.

The mutagenicity of oxysterols, cholesterol-3beta,5alpha,6beta-triol (alpha-Triol), 7-keto-cholesterol (7-Keto) and cholesterol-5alpha,6alpha-epoxide (alpha-Epox) were examined by the Ames method and chromosome aberration test in this study. Only alpha-Triol concentration-dependently caused an increase of bacterial revertants in the absence of metabolic activating enzymes (S9), but not 7-keto and alpha-Epox. The mutagenic effect of alpha-Triol was reduced by the addition of S9. On the other hand, although alpha-Triol significantly induced chromosome aberration in CHO-K1 cells with and without S9. However, the addition of S9 reduced the degree of abnormal structure chromosome compared to without S9 mix. Catalase and superoxide dismutase (SOD) inhibited alpha-Triol induced increase of revertants in Salmonella typhimurium and chromosome aberration frequency in CHO cells, suggesting that reactive oxygen species (ROS) might be involved in the genotoxic effect of alpha-Triol. Treatment with alpha-Triol increased the ROS production in CHO cells, which could be attenuated by catalase and SOD. Results in this study suggested, for the first time that alpha-Triol, causes genotoxic effect in an ROS-dependent manner.

RARRES3 expression positively correlated to tumour differentiation in tissues of colorectal adenocarcinoma.

RARRES3 is a retinoid-inducible class II tumour-suppressor gene. This study analysed the expression of RARRES3 protein in normal, adenoma and carcinoma tissues of the colorectum and its correlation with tumour differentiation. The expression of RARRES3 protein in 151 paraffin-embedded colorectal tissues (11 distal normal mucosa, 20 adenoma and 120 colorectal adenocarcinoma) was determined by immunohistochemistry. RARRES3 protein was expressed in all 11 distal normal, 120 adjacent normal and 20 adenoma tissues. In distal normal tissues, RARRES3 protein was expressed at the highest levels in differentiated mucosal epithelial cells. Among 120 carcinoma tissues, RARRES3 protein was detected in 97.6% (40 out of 41), 79.4% (54 out of 68) and 17.3% (three out of 11) of well-, moderately and poorly differentiated tumours, respectively. The expression of RARRES3 protein was positively correlated to tumour differentiation (test for trend, P<0.0001). Also, levels of RARRES3 protein were found to be higher in the normal tissues adjacent to 14.6% (six out of 41), 51.5% (35 out of 68), and 90.1% (10 out of 11) of well-, moderately and poorly differentiated tumours, respectively. The decreases in tumour differentiation and RARRES3 expression were significantly correlated compared to the adjacent normal tissues (test for trend, P<0.0001). The prognostic implication of RARRES3 protein expression was studied in 107 tumour, and no statistical difference in survival was observed. The expression of RARRES3 protein was positively correlated to cellular differentiation of normal and adenocarcinoma tissues of the colorectum, which supports the role of RARRES3 in normal and malignant epithelial differentiation of colorectum. RARRES3 expression was decreased only in carcinoma tissue, which suggests that altered RARRES3 expression occurs late in colorectal carcinogenesis.

Loss of cyclin D1 and p16 expression correlates with local recurrence in nasopharyngeal carcinoma following radiotherapy.

BACKGROUND: The cyclin D1/p16/Rb pathway plays a critical role in tumorigenesis and each component of this pathway may be affected in various malignancies. The purpose of this study was to investigate the expression and prognostic significance of these proteins in nasopharyngeal carcinoma (NPC). PATIENTS AND METHODS: Sixty-five patients undergoing radiotherapy for NPC were analyzed. The expression of cyclin D1, p16 and pRb was evaluated with immunohistochemical analysis of archived pretreatment tumor materials and expression of these proteins was correlated with clinicopathological parameters. RESULTS: Positive expression of cyclin D1 was observed in 43 of 65 NPCs (66%). p16 and pRb inactivation was identified in 42 of 65 (65%) and four of 65 (6%) tumors, respectively. All but seven tumors (58 of 65, 89%) contained at least one alternation in the cyclin D1/p16/Rb pathway. Loss of cyclin D1 as well as p16 was closely related to local recurrence after radiotherapy for NPC (P = 0.015 and 0.047). No association between pRb expression and clinicopathological outcome was apparent. CONCLUSIONS: The study's results suggest that the cyclin D1/p16/Rb pathway plays an important role in NPC tumorigenesis. We also find that cyclin D1 and p16 protein levels in NPC may be of use clinically as a predictor of local tumor control.

The MinE ring required for proper placement of the division site is a mobile structure that changes its cellular location during the Escherichia coli division cycle.

Placement of the division site at midcell in Escherichia coli requires the MinE protein. MinE acts by imparting topological specificity to the MinCD division inhibitor, preventing the inhibitor from acting at the midcell site while permitting it to block division at other unwanted sites along the length of the cell. It was previously shown that MinE assembled into a ring structure that appeared to be localized near midcell, apparently explaining the ability of MinE to specifically counteract MinCD at midcell. We report here that the MinE ring is not fixed in position near midcell but is a dynamic structure that undergoes a repetitive cycle of movement first to one cell pole and then to the opposite pole. Taken together with studies of the dynamic behavior of the MinD protein, the results suggest that the topological specificity of division site placement may not involve a localized action of MinE to counteract the MinCD division inhibitor at midcell but rather the ability of MinE to move the division inhibitor away from midcell and to the cell poles.

Structural basis for the topological specificity function of MinE.

Correct positioning of the division septum in Escherichia coli depends on the coordinated action of the MinC, MinD and MinE proteins. Topological specificity is conferred on the MinCD division inhibitor by MinE, which counters MinCD activity only in the vicinity of the preferred midcell division site. Here we report the structure of the homodimeric topological specificity domain of Escherichia coli MinE and show that it forms a novel alphabeta sandwich. Structure-directed mutagenesis of conserved surface residues has enabled us to identify a spatially restricted site on the surface of the protein that is critical for the topological specificity function of MinE.

Excellent effect of steroid plus azathioprine in a young woman with pernicious anaemia and systemic lupus erythematosus.

We describe a 29-year-old woman who developed pernicious anaemia 2 years after the diagnosis of systemic lupus erythematosus. This is a rare association despite the relationship between the autoimmune aetiologies of these two conditions. Seven other cases have been described, but our report demonstrates a case with an excellent response to steroid and azathioprine.

Mice immunized with DNA encoding a modified Pseudomonas aeruginosa exotoxin A develop protective immunity against exotoxin intoxication.

A recombinant plasmid, which contains the Pseudomonas aeruginosa exotoxin A (PE) gene with a C-terminal deletion, was inserted into expression vector pSecTag Xpress. The expression of this bacterial exotoxin in an animal cell was first demonstrated in 3T3 cell by transient transfection and western blot assay. Recombinant plasmid DNA was then injected intramuscularly to BALB/c mice, anti-PE specific antibodies were found in all animals vaccinated with plasmid containing the PE gene and with 'detoxicated' recombinant PE protein. Mice vaccinated with DNA were protected from the intoxication of lethal dosage of P. aeruginosa exotoxin A. Our results indicated that mice vaccinated with DNA encoding the PE gene could express PE protein in vivo, induced specific immune response, and provided sufficient protective immunity that safeguarded mice from the injection of lethal dosage of PE toxin.

Gene therapy with tumor vaccine increases the survival of hepatoma-bearing mice.

BACKGROUND: Tumor vaccines combined with cytokine gene therapy and Bacillus Calmette-Guérin (BCG) were tested for prevention and therapeutic effects in the H6 mouse hepatoma model. METHODS: Plasmid DNA of expression vectors carrying cDNA of mouse interleukin (IL)-2 and mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) were used for transfection to obtain H6 mouse hepatoma cells that secreted IL-2 (H6/IL-2) or GM-CSF (H6/GM-CSF). For tumor prevention, groups of mice were immunized twice with irradiated tumor cells with untransduced H6, H6/IL-2, H6/GM-CSF, or an equal mixture of H6/IL-2 and H6/GM-CSF. Three weeks later, these mice were inoculated subcutaneously with live H6 hepatoma cells, and tumor growth was measured. For therapeutic studies, mice first inoculated with live H6 cells were treated three days later with various irradiated tumor cell vaccines alone or in combination with BCG. Subsequent tumor growth was measured. RESULTS: In tumor prevention studies, significant protection from tumor growth has been observed in animals vaccinated with irradiated cytokine-secreting H6 cells compared with those immunized with irradiated parental H6 cells. In tumor therapy studies, subsequent administration of irradiated H6/GM-CSF cells in combination with BCG impeded the tumorigenicity of preinoculated live H6 hepatoma cells. CONCLUSIONS: These results suggest that cytokine-secreting tumor vaccines have a prophylactic effect and BCG, in combination with irradiated H6/GM-CSF cells, shows a synergistic effect on delaying the growth of H6 mouse hepatomas.

Involvement of Fas (CD95/APO-1) and Fas ligand in apoptosis induced by ganciclovir treatment of tumor cells transduced with herpes simplex virus thymidine kinase.

Transduction of cancer cells with herpes simplex virus thymidine kinase gene (HSVtk) followed by prodrug ganciclovir (GCV) treatment has been shown to induce apoptosis. In this study, four murine tumors including B16F10 melanoma, NG4TL4 sarcoma, H6 hepatoma and 1MEA 7R.1 hepatoma were found to vary in sensitivity to this gene therapy strategy in vitro but, at effective doses of GCV, the HSVtk-transduced cells of all four tumors showed similar kinetics of early rise in p53 protein levels, then cell cycle S-/G2-phase arrest and finally signs of apoptosis. Immunoblot analyses revealed that Fas (CD95/APO-1), Fas ligand (FasL) and two downstream mediators, RIP and caspase-3, (CPP32, YAMA, Apopain) were increased in GCV-treated HSVtk-transduced tumor cells the cell cycle arrest and before apoptosis. Increased expression of FasL could also be observed in vivo in HSVtk-transduced tumors induced to regress by GCV treatment. Enzyme measurements using specific substrate showed that the caspase-3 activation followed kinetically the FasL expression. More than half of the HSVtk/GCV-induced cell death could be abrogated by addition to the cell culture medium of a specific antisense oligonucleotide to block FasL synthesis, a recombinant Fas/Fc chimeric protein to compete with Fas receptor for FasL binding, or cell-permeable specific tetrapeptide inhibitors of caspase-3 or caspase-8.

Inhibition of DNA synthesis by downregulation of cyclin A but not Skp 2 overexpression in human hepatocellular carcinoma cells.

Cyclin A is an S and G2/M phase regulatory protein and associates with Skp 2 in many transformed cells. Our previous results showed that 12 (39%) and 17 (55%) out of 31 hepatocellular carcinoma (HCC) patients exhibited higher protein expression levels of cyclin A and Skp 2, respectively, in their tumorous compared to non-tumorous tissues. In the present study, we used Western blot analysis to show that 3 out of 6 HCC cell lines, HA59T, HA22T and HCC36, exhibited overexpression of cyclin A, among which the HCC36 cell line also expressed a higher Skp 2 protein level. Moreover, we used the antisense oligonucleotide phosphorothioates to down regulate the overexpression of cyclin A and Skp 2 proteins to determine whether or not these two proteins are involved in the mitogenesis of HCC36 cells. After treatment with antisense oligonucleotide phosphorothioates, the gene product of cyclin A or Skp 2 was suppressed dose-dependently as revealed by Western blot analyses. By [3H]thymidine incorporation assay, we found that downregulation of cyclin A but not Skp 2 overexpression could inhibit the DNA synthesis ability of HCC36 cells, suggesting that abnormal Skp 2 expression is not directly correlated with the HCC proliferation.

The hexY genes of Erwinia carotovora ssp. carotovora and ssp. atroseptica encode novel proteins that regulate virulence and motility co-ordinately.

Mutations located in a new gene, hexY, in Erwinia carotovora ssp. carotovora (Ecc) and ssp. atroseptica (Eca) cause strong upregulation of production of exoenzyme virulence factors and motility. The hexY gene encodes a novel 14.4 kDa protein with no known homologues. The hexY mRNA transcript has an unusually long (525bp) 5' untranslated region, which may be important for post-transcriptional regulation. An elevated level of transcription of two exoenzyme genes, pelCand celV, was observed in the HexY mutant background. The levels of cellulase and protease in a HexY mutant were independent of the presence of PGA, suggesting a role for HexY in the induction of these enzymes seen upon PGA addition. Electron microscopy revealed that HexY cells were hyperflagellated, perhaps contributing to the hypermotility phenotype of this mutant. The HexY mutant M5 exhibited enhanced maceration capacity on potato tubers. Therefore, the hexY gene and its gene product may define another level of regulation of virulence determinants in Ecc and Eca.

The hexA gene of Erwinia carotovora encodes a LysR homologue and regulates motility and the expression of multiple virulence determinants.

We have identified a gene important for the regulation of exoenzyme virulence factor synthesis in the plant pathogen Erwinia carotovora ssp. carotovora (Ecc) and virulence and motility in Erwinia carotovora ssp. atroseptica (Eca). This gene, hexA (hyperproduction of exoenzymes), is a close relative of the Erwinia chrysanthemi (Echr) gene pecT and encodes a member of the LysR family of transcriptional regulators. hexA mutants in both Ecc and Eca produce abnormally high levels of the exoenzyme virulence factors pectate lyase, cellulase and protease. In addition, Eca hexA mutants show increased expression of the fliA and fliC genes and hypermotility. Consistent with a role as a global regulator, expression of hexA from even a low-copy plasmid can suppress exoenzyme production in Ecc and Eca and motility in Eca. Production of the quorum-sensing pheromone OHHL in Ecc hexA is higher throughout the growth curve compared with the wild-type strain. Overexpression of Ecc hexA also caused widespread effects in several strains of the opportunistic human pathogen, Serratia. Low-copy hexA expression resulted in repression of exoenzyme, pigment and antibiotic production and repression of the spreading phenotype. Finally, mutations in hexA were shown to increase Ecc or Eca virulence in planta.