Identification of Delta5-desaturase from Mortierella alpina by heterologous expression in Bakers' yeast and canola

A DNA fragment with homology to Delta6-desaturases from borage and cyanobacteria was isolated after polymerase chain reaction amplification of Mortierella alpina cDNA with oligonucleotide primers corresponding to the conserved regions of known Delta6-desaturase genes. This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 446 amino acids from a M. alpina library. Expression of this open reading frame from an inducible promoter in Saccharomyces cerevisiae in the presence of various substrates revealed that the recombinant product had Delta5-desaturase activity. The effects of growth and induction conditions as well as host strain on activity of the recombinant Delta5-desaturase in S. cerevisiae were evaluated. Expression of the M. alpina Delta5-desaturase cDNA in transgenic canola seeds resulted in the production of taxoleic acid (Delta5,9-18:2) and pinolenic acid (Delta5,9,12-18:3), which are the Delta5-desaturation products of oleic and linoleic acids, respectively.     

Interleukin 12 augments the liver-associated immunity and reduces liver metastases

BACKGROUND/AIMS: It has been reported recently that in vivo administration of interleukin-12 (IL-12) augments the cytotoxic activity of natural killer (NK)/T cells and shows a powerful anti-tumor activity. In this study, we evaluated that the IL-12 effect on liver-associated immunity and in vivo efficacy on the hepatic metastases in a rat model. MATERIALS AND METHODS: Varying amounts of mouse recombinant IL-12 were injected intraperitoneally for 5 days to adult male Fischer rats and hepatic sinusoidal lymphocytes (HSL) were collected. Purified HSL are spontaneously cytolytic to both conventional NK-sensitive target (YAC-1) and NK-resistant target (RCN-H4) tumor cells. RESULTS: IL-12 was found to increase the number of HSL and the cytolytic activity against these target cells in a dose-dependent fashion. Flow cytometric analysis showed that IL-12 caused an increase of CD8+ subpopulation in HSL and a double staining study revealed that the increased subpopulation was not CD3+8+ (cytotoxic T cell) fraction, but actually CD3-8+ (NK cell) fraction. Experimental liver metastases was markedly reduced in rats treated intraperitoneally with IL-12. CONCLUSION: These results demonstrate that IL-12 augments the cytolytic activity of HSL and suggests this cytokine as an attractive choice for liver metastases therapy.     

Effect of hyperglycemia on brain cell membrane function and energy metabolism during hypoxia-ischemia in newborn piglets

The purpose of this study was to test the hypothesis that hyperglycemia ameliorates changes in brain cell membrane function and preserves cerebral high energy phosphates during hypoxia-ischemia in newborn piglets. A total of 42 ventilated piglets were divided into 4 groups, normoglycemic/normoxic(group 1, n=9), hyperglycemic/normoxic(group 2, n=8), normoglycemic/hypoxic-ischemic(group 3, n=13) and hyperglycemic/hypoxic-ischemic(group 4, n=12) group. Cerebral hypoxia-ischemia was induced by occlusion of bilateral common carotid arteries and simultaneous breathing with 8% oxygen for 30 min. Hyperglycemia (blood glucose 350-400 mg/dl) was maintained for 90 min before and throughout hypoxia-ischemia using modified glucose clamp technique. Changes in cytochrome aa3 were continuously monitored using near infrared spectroscopy. Blood and CSF glucose and lactate were monitored. Na+, K+-ATPase activity, lipid peroxidation products (conjugated dienes), tissue high energy phosphates (ATP and phosphocreatine) levels and brain glucose and lactate levels were determined biochemically in the cerebral cortex. During hypoxia-ischemia, glucose levels in blood and CSF were significantly elevated in hyperglycemic/hypoxic-ischemic group compared with normoglycemic/hypoxic-ischemic group, but lactate levels in blood and CSF were not different between two groups. At the end of hypoxia-ischemia of group 3 and 4, triangle up Cyt aa3, Na+, K+-ATPase activity, ATP and phosphocreatine values in brain were significantly decreased compared with normoxic groups 1 and 2, but were not different between groups 3 and 4. Levels of conjugated dienes and brain lactate were significantly increased in groups 3 and 4 compared with groups 1 and 2, and were significantly elevated in group 4 than in group 3 (0.30+/-0.11 vs. 0.09+/-0.02 micromol g-1 protein, 26.4+/-7.6 vs. 13.1+/-2.6 mmol kg-1, p<0.05). These findings suggest that hyperglycemia does not reduce the changes in brain cell membrane function and does not preserve cerebral high energy phosphates during hypoxia-ischemia in newborn piglets. We speculate that hyperglycemia may be harmful during hypoxia-ischemia due to increased levels of lipid peroxidation in newborn piglet.     

Cytotoxic effects of quinoxaline derivatives on human cancer cell lines

The cytotoxicities of 6,7-modified-5,8-quinoxalinedione derivatives and heterocyclic quinoxaline derivatives containing nitrogen, sulfur, and oxygen on human lung adenocarcinoma cell (PC 14), human gastric adenocarcinoma cell (MKN 45), and human colon adenocarcinoma cell (colon 205) were examined in vitro using MTT assay. Pyrido[1,2-a]imidazo[4,5-g]quinoxaline-6,11-dione (10) was markedly cytotoxic against MKN 45 compared with adriamycin and cis-platin used as anticancer drugs. The IC50 value of compound 10 was 0.073 microM while those of adriamycin and cis-platin were 0.12 microM and 2.67 microM, respectively.