RESUMO
BACKGROUND: Snake venoms are the complex mixtures of different compounds manifesting a wide array of biological activities. The venoms of kraits (genus Bungarus, family Elapidae) induce mainly neurological symptoms; however, these venoms show a cytotoxicity against cancer cells as well. This study was conducted to identify in Bungarus fasciatus venom an active compound(s) exerting cytotoxic effects toward MCF7 human breast cancer cells and A549 human lung cancer cells. METHODS: The crude venom of B. fasciatus was separated by gel-filtration on Superdex HR 75 column and reversed phase HPLC on C18 column. The fractions obtained were screened for cytotoxic effect against MCF7, A549, and HK2 cell lines using colorimetric assay with the tetrazolium dye MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The primary structure of active protein was established by ultra high resolution LC-MS/MS. The molecular mechanism of the isolated protein action on MCF7 cells was elucidated by flow cytometry. RESULTS: MTT cell viability assays of cancer cells incubated with fractions isolated from B. fasciatus venom revealed a protein with molecular mass of about 13 kDa possessing significant cytotoxicity. This protein manifested the dose and time dependent cytotoxicity for MCF7 and A549 cell lines while showed no toxic effect on human normal kidney HK2 cells. In MCF7, flow cytometry analysis revealed a decrease in the proportion of Ki-67 positive cells. As Ki-67 protein is a cellular marker for proliferation, its decline indicates the reduction in the proliferation of MCF7 cells treated with the protein. Flow cytometry analysis of MCF7 cells stained with propidium iodide and Annexin V conjugated with allophycocyanin showed that a probable mechanism of cell death is apoptosis. Mass spectrometric studies showed that the cytotoxic protein was phospholipase A2. The amino acid sequence of this enzyme earlier was deduced from cloned cDNA, and in this work it was isolated from the venom as a protein for the first time. It is also the first krait phospholipase A2 manifesting the cytotoxicity for cancer cells.
RESUMO
OBJECTIVE: to assess the relations between cardiovascular risk factors and psychological or socio-economic status. METHODS: We used a self-questionnaire including data upon usual risk factors, anxiety, depression, behavioural pattern, somatic manifestations, life style, incomes and work-related stress. We gathered 955 people, mainly attending an occupational health examination, filling anonymously the same restricted questionnaire (83 questions). RESULTS: Non-working people represented only 4.4 per cent of the study group and included mainly retired people. They differed significantly from wage earners by a higher level of cardiovascular risk and depression, respectively 15.8 (s.d. 11.3) versus 7.8 (6.7) and 5.6 (3.5) versus 3.9 (2.8). These differences can be related to older age of this group. Among working people, 30 per cent felt job-insecurity; they differed significantly from working people by a higher level of anxiety, 8.9 (3.7) versus 7.5 (3.3). There was no difference concerning the absolute cardiovascular risk or the behavioural pattern. A comprehensive self questionnaire is proposed.
