Interleukin-10 reduces natural killer (NK) sensitivity of tumor cells by downregulating NK target structure expression

We examined the effect of exogenous IL-10 on the sensitivity of rat W14 and W31 tumor cells to natural killer (NK) cell-mediated cytotoxicity in relation to previously identified NK target structure (NKTS) expressed on these cells. We also examined the effect of endogenous interleukin-10 (IL-10) on rat IL-10 cDNA-introduced W31 cells (W31T-H, a high-IL-10-producer clone; W31T-L, a low-IL-10-producer clone). Both exogenous and endogenous IL-10 had no effect on the proliferative activity of these cells, but incubation of cells with recombinant human (rh) IL-10 resulted in a dose-dependent decrease in the expression of NKTS recognized by mAb 109. The expression level of NKTS on W31T-H cells was dramatically decreased compared with that on W31T-L cells and parental W31 cells. In addition, treatment of W31 cells with the culture supernatants of W31T-H cells could downregulate the expression of the NKTS. Moreover, NK sensitivity of W31T-H was suppressed down to a level equivalent to that of W31 cells pretreated with exogenous rhIL-10, and cytolysis could no longer be inhibited by mAb 109. We previously demonstrated that IL-10 downregulated MHC class I expression in this model. Nevertheless, NK susceptibility was also decreased. Taken together, these results suggest that the IL-10-mediated decrease in NKTS expression has a larger effect than decreased MHC class I expression on NK sensitivity. Thus, our data reveal a novel mechanism for an IL-10-mediated escape of tumor cells from host immune surveillance by downregulation of NKTS expression.     

Yeast two-hybrid screening using constitutive-active caspase-7 as bait in the identification of PA28gamma as an effector caspase substrate

Caspase-3 and -7 represent executioner/effector caspases that directly cause apoptotic morphological changes by cleaving various death substrates. The substrates for caspases generally interact with active caspases, but not with inactive zymogens of caspase or procaspases. Here, to isolate proteins that interact with caspase-7, we established a yeast two-hybrid screening system using reversed-caspase-7, a constitutive active mutant of caspase-7 as a bait plasmid. Screening of an adult brain cDNA library led to isolation of proteasome activator 28 subunit, PA28gamma. In vitro translates of PA28gamma were cleaved by both recombinant caspase-3 and -7. Mutagenesis of potential cleavage site DGLD80 to EGLE80 completely abolished caspase-mediated cleavage. Moreover, endogenous PA28gamma was cleaved during not only Fas-induced apoptosis of HeLa cells, but also cisplatin-induced cell death of MCF7 cells, which are devoid of caspase-3. These findings indicate that PA28gamma is an endogenous substrate for caspase-3 and -7 and that yeast two-hybrid screening using reversed-caspase is a novel and useful approach to clone substrates for effector caspases.     

TLC-densitometric analysis of artemisinin for the rapid screening of high-producing plantlets of Artemisia annua L

A simple TLC-densitometric technique has been developed for the rapid and accurate analysis of artemisinin in a large number of Artemisia annua plantlets cultured in vitro. This new analytical method is based on the structural conversion of artemisinin on a silica gel layer by ammonia vapour to form 10-azadesoxyartemisinin, a chromophore-containing compound (lambdamax 320 nm) that can be detected by UV-based TLC densitometry. The TLC system was evaluated quantitatively in terms of product stability, precision, accuracy and calibration. Good linearity was obtained in the range of 0.01-0.12 microg artemisinin. The technique appeared to be accurate and sensitive as compared with the complicated pre-column reaction-HPLC technique. Among 90 samples of A. annua plantlets, the artemisinin content in the leaves appeared to be highly variable, ranging from 0.02 to 0.67% w/w dry weight. These results demonstrate that densitometric TLC can be a cheap and simple technique for the accurate screening of high-artemisinin-producing plants.     

Expression of the ubiE gene of Geobacillus stearothermophilus V in Escherichia coli K-12 mediates the evolution of selenium compounds into the headspace of selenite- and selenate-amended cultures

The ubiE gene of Geobacillus stearothermophilus V, with its own promoter, was cloned and introduced into Escherichia coli. The cloned gene complemented the ubiE gene deficiency of E. coli AN70. In addition, the expression of this gene in E. coli JM109 resulted in the evolution of volatile selenium compounds when these cells were grown in selenite- or selenate-amended media. These compounds were dimethyl selenide and dimethyl diselenide.     

Effects of weight loss on liver and erythrocyte polyunsaturated fatty acid pattern and oxidative stress status in obese patients with non-alcoholic fatty liver disease

Our aim was to study the influence of weight loss on the fatty acid (FA) composition of liver and erythrocyte phospholipids and oxidative stress status in obese, non-alcoholic, fatty liver disease (NAFLD) patients. Seven obese NAFLD patients who underwent subtotal gastrectomy with a gastro-jejunal anastomosis in roux and Y were studied immediately and 3 months after surgery. Seven non-obese patients who underwent anti-reflux surgery constituted the control group. Serum F2-isoprostane levels were measured by GS/NICI-MS/MS and FA composition was determined by GC. At the time of surgery, controls and obese patients exhibited a hepatic polyunsaturated fatty acid (PUFA) pattern that correlated with that of erythrocytes. Three months after surgery, NAFLD patients lost 21% of initial body weight; serum F2-isoprostane levels decreased by 76%; total PUFA, long-chain PUFA (LCPUFA), n-3 PUFA, and n-3 LCPUFA increased by 22, 29, 81, and 93%, respectively; n-6/n-3 LCPUFA ratio decreased by 51%; docosahexaenoic acid/docosapentaenoic acid ratio increased by 19-fold; and the n-3 product/precursor ratio (20: 5 + 22: 5 + 22: 6)/18: 3 increased by 164% (p<0.05). It is concluded that weight loss improves the n-3 LCPUFA status of obese patients in association with significant amelioration in the biomarkers of oxidative stress, membrane FA insaturation, and n-3 LCPUFA biosynthesis capacity, thus representing a central therapeutic issue in the improvement of obesity-related metabolic alterations involved in the mechanism of hepatic steatosis.     

Two new isodrimene sesquiterpenes from the fungal culture broth of Polyporus arcularius

Two new isodrimene sesquiterpene derivatives, 2(S)-hydroxyalbicanol (1, =(2S,4aS,8S,8aS)-8-(hydroxymethyl)-4,4,8a-trimethyl-7-methylenedecahydronaphthalen-2-ol) and 2(S)-hydroxyalbicanol 11-acetate (2, =((1S,4aS,7S,8aS)-7-hydroxy-5,5,8a-trimethyl-2-methylenedecahydronaphthalen-1-yl)methyl acetate) were isolated from the culture broth of the fungus Polyporus arcularius, together with two phenylpropanediols, (1S,2S)- and (1R,2S)-1-phenyl-1,2-dihydroxypropane (3, 4). Compound 3 is reported as a naturally occurring compound for the first time. The structures of the compounds were elucidated on the basis of spectroscopic analysis. Compound 1 exhibited growth inhibition of lettuce seedlings with IC₅₀ values of 1.3 mM to hypocotyl and 1.7 mM to radicle.     

Evaluation of the role of the endoplasmic reticulum-Golgi transit in the biogenesis of peroxisomal membrane proteins in wild type and peroxisome biogenesis mutant CHO cells

Peroxisomes are thought to be formed by division of pre-existing peroxisomes after the import of newly synthesized proteins. However, it has been recently suggested that the endoplasmic reticulum (ER) provides an alternative de novo mechanism for peroxisome biogenesis in some cells. To test a possible role of the ER-Golgi transit in peroxisome biogenesis in mammalian cells, we evaluated the biogenesis of three peroxisomal membrane proteins (PMPs): ALDRP (adrenoleukodystrophy related protein), PMP70 and Pex3p in CHO cells. We constructed chimeric genes encoding these PMPs and green fluorescent protein (GFP), and transiently transfected them to wild type and mutant CHO cells, in which normal peroxisomes were replaced by peroxisomal membrane ghosts. The expressed proteins were targeted to peroxisomes and peroxisomal ghosts correctly in the presence or absence of Brefeldin A (BFA), a drug known to block the ER-Golgi transit. Furthermore, low temperature did not disturb the targeting of Pex3p-GFP to peroxisomes. We also constructed two chimeric proteins of PMPs containing an ER retention signal "DEKKMP": GFP-ALDRP-DEKKMP and myc- Pex3p-DEKKMP. These proteins were mostly targeted to peroxisomes. No colocalization with an ER maker was found. These results suggest that the classical ER-Golgi pathway does not play a major role in the biogenesis of mammalian PMPs.