Mir-138-5p acts as a tumor suppressor by targeting pyruvate dehydrogenase kinase 1 in human retinoblastoma.

OBJECTIVE: MicroRNAs have caught more attention for their role in tumor progression. Retinoblastoma (RB) is one of these ordinary malignant tumors. This study aims to identify whether mir-138-5p can regulate the development of RB, and find out its potential mechanism. MATERIALS AND METHODS: Mir-138-5p expression in RB cells was monitored by RT-qPCR. Besides, the role of mir-138-5p in RB development was explored through function experiments in vitro. The potential mechanism was further explored by RT-qPCR, luciferase assay, and Western blot assay. RESULTS: In our investigation, mir-138-5p was lower-expressed in RB cells than that in retinal pigment epithelial cells. Moreover, overexpression of mir-138-5p repressed cell viability, migration and invasion, and induced apoptosis of RB cells, while downregulated mir-138-5p increased cell viability, migration and invasion, and reduced apoptosis of RB cells. Furthermore, pyruvate dehydrogenase kinase 1 (PDK1) could be downregulated via overexpression of mir-138-5p, while PDK1 was upregulated via knockdown of mir-138-5p. CONCLUSIONS: Our results suggested that mir-138-5p could repress the development of RB via suppressing PDK1, which may offer a new vision for interpreting the mechanism of RB tumorigenesis.

In vitro evaluation of antioxidant activities of aqueous extracts from natural and cultured mycelia of Cordyceps sinensis.

Cordyceps sinensis, one of the best known traditional Chinese medicines and health foods, has been highly valued for the treatment of a wide range of diseases and reported to have antioxidant properties. In the present study, the antioxidant activities of hot-water extracts from natural and cultured mycelia of C. sinensis were investigated and evaluated using six in vitro assays, including inhibition of linoleic acid peroxidation; scavenging abilities on DPPH•, hydroxyl and superoxide anion radicals; the reducing power and the chelating ability on ferrous ions. Among these assays, the extracts showed the best effect on the inhibition of linoleic peroxidation with the lowest IC50 values and with an inhibition rate over 90% at concentration of 0.8-1.6 mg/ml, more stable than that of α-tocopherol, a recognised natural antioxidant. The scavenging activities on superoxide anion and hydroxyl radicals of the two extracts were slightly lower than that of butylated hydroxytoluene. DPPH• scavenging activities of both extracts reached over 80% inhibition at 4-8 mg/ml. Both extracts showed moderate reducing power and ferrous ion chelating activity. The IC50 value of the extract from cultured mycelia in all the tests, except for linoleic acid peroxidation, was significantly lower than that of natural mycelia. There was no evident correlation between the antioxidant activity and the content of protein, polysaccharides and mannitol of extracts from C. sinensis; the antioxidant activity may be due to a combined effect of these or some other compounds. These results suggested that both the extracts from cultured and natural mycelia have direct and potent antioxidant activities and that the cultured mycelia of the fungus could be used for the antioxidant activity to reduce the human demands on the natural resources of the fungus, an endangered species.

A fatal case of hepatic failure possibly induced by nitrosofenfluramine: a case report.

A 42-year-old female developed fulminant hepatic failure after having ingested an undetermined quantity of a herbal product over a period of approximately four months prior to the onset of her illness. Clinically, the cause of liver failure was assessed to be drug-induced and she eventually underwent total hepatectomy, with porto-caval shunting, in anticipation of a living-unrelated liver transplant. Unfortunately, her condition deteriorated and she died less than 48 hours post-operatively, approximately three weeks post-admission. An autopsy showed that the subject was deeply jaundiced and severely obese (BMI: 47.1 kg m(-2)), with evidence of diffuse haemorrhage, including the presence of 1.35 l of blood in the peritoneal cavity. The liver had been removed and was later recovered as a formalin-fixed specimen which was markedly contracted, comprising multiple micronodules interspersed with extensive areas of dense fibrotic tissue. Histologically, there was massive necrosis of the hepatic parenchyma, such that the residual hepatocytes were disposed as nodules displaying variable cellular regeneration and ballooning degeneration, attended by florid ductal proliferation and mixed inflammatory infiltrates. Infective, autoimmune, metabolic, vascular, neoplastic and most other natural causes of massive hepatocellular necrosis were effectively excluded. Analysis of the post-mortem blood samples yielded fluconazole, metronidazole, frusemide, lignocaine and tramadol, (therapeutic agents administered to the patient during her last illness). Subsequent analysis of the residual capsules revealed that they were adulterated by fenfluramine, N-nitrosofenfluramine (1.3-1.6 mg per capsule), nicotinamide (13.3-15.6 mg per capsule) and thyroid extract. None of the herbal ingredients is currently known to be hepatotoxic and much the same applies to fenfluramine, nicotinamide (except when taken in mega-doses) and thyroid extract. However, as nitrosamines are known to be variably hepatotoxic, it would be reasonable to surmise that, in the absence of a more plausible cause of liver damage, N-nitrosofenfluramine was the likely cause of massive hepatocellular necrosis in this instance.

Role of PTCH and p53 genes in early-onset basal cell carcinoma.

Basal cell carcinoma (BCC) is the most common skin cancer in the Western world. Ultraviolet (UV) exposure, race, age, gender, and decreased DNA repair capacity are known risk factors for the development of BCC. Of these, UVB irradiation from sunlight is the most significant risk factor. The incidence of sporadic BCC increases in individuals older than age 55, with the greatest incidence reported in individuals who are older than 70, and is rare in individuals who are younger than 30. In this study, we analyzed 24 BCC samples from individuals who had BCC diagnosed by the age of 30. Fifteen single-stranded conformation polymorphism variants in the PTCH gene were identified in 13 BCC samples. Sequence analysis of these single-stranded conformation polymorphism variants revealed 13 single nucleotide changes, one AT insertion, and one 15-bp deletion. Most of these nucleotide changes (nine of 15) were predicted to result in truncated PTCH proteins. Fifteen p53 mutations were also found in 11 of the 24 BCC samples. Thirty-three percent (five of 15) and 60% (nine of 15) of the nucleotide changes in the PTCH and p53 genes, respectively, were UV-specific C-->T and CC-->TT nucleotide changes. Our data demonstrate that the p53 and PTCH genes are both implicated in the development of early-onset BCC. The identification of UV-specific nucleotide changes in both tumor suppressor genes suggests that UV exposure is an important risk factor in early onset of BCC.

Mutation report: identification of a germline mutation in keratin 17 in a family with pachyonychia congenita type 2.

Pachyonychia congenita type 2 (PC-2), also known as Jackson-Lawler type PC, is an autosomal dominant disorder characterized by hypertrophic nail dystrophy associated with focal keratoderma and multiple pilosebaceous cysts. It has been demonstrated that PC-2 is associated with germline mutations in the keratin 17 (K17) gene and in its expression partner keratin 6b. In this report, we describe a novel germline mutation in K17, M88T, in a family with PC-2.

PTEN/MMAC1 mutations in hepatocellular carcinomas.

Mutations in the PTEN/MMAC1 gene have been identified in several types of human cancers and cancer cell lines, including brain, endometrial, prostate, breast, thyroid, and melanoma. In this study, we screened a total of 96 hepatocellular carcinoma (HCC) samples from Taiwan, where HCC is the leading cancer in males and third leading cancer in females, for mutations in the PTEN/MMAC1 gene. Complete sequence analysis of these samples demonstrated a missense mutation in exon 5 (K144I) and exon 7 (V255A) from HCC samples B6-21 and B6-2, respectively. A putative splice site mutation was also detected in intron 3 from sample B6-2. Both B6-21 and B6-2 were previously shown to contain missense mutations in the coding sequences of the p53 gene. Functional studies with the two missense mutations demonstrated that while mutation V255A in exon 7 resulted in a loss of phosphatase activity, mutation K144I in exon 5 retained its phosphatase activity. Additionally, we identified a silent mutation (P96P) in exon 5 of the PTEN/MMAC1 gene from HCC sample B6-22. These data provide the first evidence that the PTEN/MMAC1 gene is mutated in a subset of HCC samples.

Repression of transactivation of the retinoic acid receptor beta2 promoter in human breast cancer cells.

In previous studies, we have shown that the expression of retinoic acid receptor beta2 (RARbeta2) is altered in certain breast cancer cell lines. To investigate the mechanism responsible for this change, we studied in detail the RARbeta2 promoter in cell lines which demonstrated altered expression and compared these results to cell lines in which RARbeta2 was expressed normally. Direct DNA sequencing failed to identify alterations in the sequences of the known response elements in the cell lines manifesting altered expression patterns. By contrast, electrophoretic mobility shift studies of the proteins binding to these response elements demonstrated striking differences in the cells in which expression was altered, when compared to patterns seen in normal cells. Moreover, transient transfection studies using constructs of the RARbeta2 promoter demonstrated an absence of transactivation in the lines in which the expression of this gene was altered. These data suggest that the mechanism responsible for loss of induction of RARbeta2 in breast tumor cells is, at least in part, transcriptional repression.

Acidic and neutral drugs screen in blood with quantitation using microbore high-performance liquid chromatography-diode array detection and capillary gas chromatography-flame ionization detection.

Blood previously acidified with aqueous saturated ammonium chloride solution was extracted with ethyl acetate. The dried extract was subjected to acetonitrile-hexane partition. The acetonitrile portion was analysed for the presence of acidic and neutral drugs by HPLC-DAD (200 mm x 2.1 mm I.D. microbore ODS-Hypersil column) and GC-FID (25 m narrow-bore x 0.25 mm I.D. HP-5 column with 0.33 micron film thickness). The protocol was found to be suitable for both clinical toxicology (including emergency toxicology) and postmortem toxicology. At least 66 drugs of interest were unequivocally identified by RRTs (HPLC) and UV spectra (DAD) match while another 12 were unequivocally identified by double RRTs match (HPLC and GC). Quantitation was facilitated by incorporating calibration blood standards in each assay batch. The five drugs most commonly encountered in clinical blood specimens (1150 cases) were: paracetamol (47.4% of the cases); chlormezanone (6.6%), theophylline (1.74%), naproxen (1.65%) and mefenamic acid (1.56%). The following drugs were detected in toxicologically significant quantities in postmortem blood specimens (245 cases): phenobarbitone (1.22% of the cases), naproxen (0.82%), chlormezanone (0.82%), theophylline (0.82%), carbamazepine (0.41%) and paracetamol (0.41%).

Analysis of recombinant human tumor necrosis factor beta by capillary electrophoresis.

Capillary electrophoresis was used for the analysis of recombinant human tumor necrosis factor beta (rhTNF-beta). Reproducible separation of the protein could be achieved in Polybrene-coated capillaries. Optimum conditions for separation were achieved with a pH 4.5 sodium acetate buffer. Besides Polybrene-coated capillaries, other commercially available column and column-coating reagents were also examined for the separation of rhTNF-beta. Capillary electrophoresis was found to be a potentially useful method for product analysis and process monitoring in recombinant-DNA technology.