The structure of synenkephalin (pro-enkephalin 1-73) is dictated by three disulfide bridges

Mass spectrometry of fragments produced by limited proteolytic digestion of pro-enkephalin was used to locate the disulfide bridges in synenkephalin (pro-enkephalin 1-73), a domain which contains sorting information for targeting the pro-neuropeptide to the granules of the regulated secretory pathway in neuroendocrine cells. Mass spectrometric analysis was optimized by using chemicals that gave low interference with the ionization and desorption processes, and computer software which simplified the identification of all possible disulfide-linked peptide fragments. Three disulfide bridges between Cys2-Cys24, Cys6-Cys28, and Cys9-Cys41 were identified. Protein conformational prediction of synenkephalin1-42 shows beta-turns which facilitate the formation of these disulfide bonds.     

Newly established MST-1 tumour cell line and tumour-infiltrating lymphocyte culture from a patient with soft tissue melanoma (clear cell sarcoma) and their potential applications to patient immunotherapy

The establishment and characterisation of paired autologous tumour cell line (MST-1) and tumour-infiltrating lymphocyte (TIL) culture from a tumour mass of a 14-year-old Taiwanese girl with soft tissue melanoma are described. MST-1 cells grown in vitro were heterogeneous in morphology, ranging from floating round cells, loosely attached round/oval or elongated cells with prominent pseudopod-like processes, to well-attached spindle and elongated dendritic cells without obvious pseudopods. Immunostaining revealed that major melanoma-associated antigens, such as S100 protein, HMB-45, melanotransferrin, chondroitin sulphate proteoglycan, and the gangliosides GD2 and GD3, were consistently expressed by the tumour tissue, severe combined immunodeficiency (SCID) mouse xenograft and derived cell lines. Flow cytometric analysis of the tumour DNA content showed an index of 1.8 relative to normal peripheral blood lymphocyte DNA. Chromosome analysis revealed all cells at a hypotetraploid level with several clonal chromosome aberrations, including deletions at 10p and 12q, an addition at 12q, translocations t(1;14) and t(5;6). Electron microscopy showed melanosome structures. This observation and the expression of the major melanoma-associated antigens were all indicative of the melanocytic origin of MST-1 tumour. Interleukin-2 (IL-2) expanded TILs had the predominant CD8+ phenotype and the capacity to lyse cells of the cultured autologous tumour. The availability of the soft tissue melanoma cell line, the SCID mouse xenograft tumour system as well as autologous TILs described herein would provide useful materials for identifying T-cell-defined antigens as well as a model system for devising individualised cancer biotherapeutic strategies. This cell line can also be used for further studies aimed at uncovering the histogenesis of this rare cancer.     

Contragestational effects of dihydroartemisinin and artesunate

In experiments carried out in mice, hamsters, guinea pigs and rabbits both dihydroartemisinin and artesunate showed contragestational effect. In mice and rabbits they caused embryo absorption whereas in hamsters and guinea pigs they induced abortion. The contragestational ED50 of dihydroartemisinin given sc on d 7 of pregnancy in mice and d 5 of pregnancy in hamsters were 32.8(27.7-38.9) mg.kg-1 and 6.1(5.6-6.7) mg.kg-1 respectively. The ED50 of this drug given im on d 18 of pregnancy in guinea pigs was 18.3(13.9-24.2) mg.kg-1. Dihydroartemisinin also showed mid-pregnancy terminating effect in hamsters. The contragestational ED50 of artesunate given sc on d 5 of pregnancy in hamsters and the ED50 of sodium artesunate given sc on d 5-8 of pregnancy in hamsters were 12.2(10.3-14.4) mg.kg-1 and 1.0(0.9-1.2) mg.kg-1 daily respectively. Results of light microscopic examination revealed that dihydroartemisinin was selectively toxic to embryo sac. At dose levels sufficient to induce embryo sac necrosis, dihydroartemisinin did not injure the uterus and ovary of the maternal animals. On the ground of the foregoing observations we consider that dihydroartemisinin, artesunate and their analogous drugs should not be used to treat malaria in pregnant women and there is the possibility to exploit intentional abortion agents from artemisinin derivatives.     

A fatty acid-induced decrease in pyruvate dehydrogenase activity is an important determinant of beta-cell dysfunction in the obese diabetic db/db mouse

We studied the effects of fatty acid oxidation on insulin secretion of db/db mice and underlying molecular mechanisms of these effects. At 2-3 months of age, db/db mice were markedly obese, hyperglycemic, and hyperinsulinemic. Serum free fatty acid (FFA) levels were increased in 2-month-old (1.5 +/- 0.1 vs. 1.1 +/- 0.1 mmol/l, P < 0.05) and 3-month-old (1.9 +/- 0.1 vs. 1.2 +/- 0.1 mmol/l, P < 0.01) mice compared with the age and sex-matched db/+ mice serving as controls. Glucose-induced insulin release from db/db islets was markedly decreased compared with that from db/+ islets and was specifically ameliorated (by 54% in 2-month-old and 38% in 3-month-old mice) by exposure to a carnitine palmitoyltransferase I inhibitor, etomoxir (1 micromol/l). Etomoxir failed to affect the insulin response to alpha-ketoisocaproate. The effect of etomoxir on glucose-induced insulin release was lost after culturing db/db islets in RPMI medium containing 22 mmol/l glucose but no fatty acid. Culture of db/+ islets with 0.125 mmol/l palmitate led to a decrease in glucose-induced insulin secretion, which was partially reversible by etomoxir. Both islet glucose oxidation and the ratio of glucose oxidation to utilization were decreased in db/db islets. Etomoxir significantly enhanced glucose oxidation by 60% and also the ratio of oxidation to glucose utilization (from 27 +/- 2.5 to 37 +/-3.0%, P < 0.05). Pyruvate dehydrogenase (PDH) activity was decreased in islets of db/db mice (75 +/-4.2 vs. 91 +/- 2.9 nU/ng DNA, P < 0.01), whereas PDH kinase activity was increased (rate of PDH inactivation -0.25 +/- 0.02 vs. - 0.11 +/- 0.02/min, P < 0.0 1). These abnormalities were partly but not wholly reversed by a 2-h preexposure to etomoxir. In conclusion, elevated FFA levels in the db/db mouse diminish glucose-induced insulin secretion by a glucose-fatty acid cycle in which fatty acid oxidation inhibits glucose oxidation by decreasing PDH activity and increasing PDH kinase activities.