Multilevel structure-activity profiling reveals multiple green tea compound families that each modulate ubiquitin-activating enzyme and ubiquitination by a distinct mechanism.

We developed and implemented a reconstituted system to screen for modulators of the ubiquitination of proliferating cell nuclear antigen, a process that activates pathways of DNA damage tolerance and drug resistance. We identified the primary putatively health-beneficial green tea polyphenol epigallocatechin gallate (EGCG) and certain related small molecules as potent inhibitors of ubiquitination. EGCG directly and reversibly targets the ubiquitin-activating enzyme Uba1, blocking formation of the Uba1~ubiquitin thioester conjugate and thus ubiquitination and in the cell. Structure-activity relationship profiles across multiple biochemical and cellular assays for a battery of EGCG analogues revealed distinct chemical and mechanism-of-action clusters of molecules, with catechin gallates, alkyl gallates, and myricetin potently inhibiting ubiquitination. This study defines a number of related though distinct first-in-class inhibitors of ubiquitination, each series with its own unique activity pattern and mechanistic signature.

The Anti-Inflammatory Role of Mannich Curcuminoids; Special Focus on Colitis.

The incidence of inflammatory bowel disease (IBD) increases gradually in Western countries with high need for novel therapeutic interventions. Mannich curcuminoids, C142 or C150 synthetized in our laboratory, have been tested for anti-inflammatory activity in a rat model of TNBS (2,4,6-trinitrobenzenesulphonic acid) induced colitis. Treatment with C142 or C150 reduced leukocyte infiltration to the submucosa and muscular propria of the inflamed gut. C142 or C150 rescued the loss of body weight and C150 decreased the weight of standard colon preparations proportional with 20% less tissue oedema. Both C142 and C150 curcumin analogues caused 25% decrease in the severity of colonic inflammation and haemorrhagic lesion size. Colonic MPO (myeloperoxidase) enzyme activity as an indicator of intense neutrophil infiltration was 50% decreased either by C142 or C150 Mannich curcuminoids. Lipopolysaccharide (LPS) co-treatment with Mannich curcuminoids inhibited NF-κB (nuclear factor kappa B) activity on a concentration-dependent manner in an NF-κB-driven luciferase expressing reporter cell line. Co-treatment with LPS and curcuminoids, C142 or C150, resulted in NF-κB inhibition with 3.57 µM or 1.6 µM half maximal effective concentration (EC50) values, respectively. C150 exerted a profound inhibition of the expression of inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-4 (IL-4) in human PBMCs (peripheral blood mononuclear cells) upon LPS stimulus. Mannich curcuminoids reported herein possess a powerful anti-inflammatory activity.

Isolated hypercholesterolemia leads to steatosis in the liver without affecting the pancreas.

BACKGROUND: Lipid accumulation in the liver and pancreas is primarily caused by combined hyperlipidemia. However, the effect of isolated hypercholesterolemia without hypertriglyceridemia is not fully described. Therefore, our aim was to investigate whether hypercholesterolemia alone leads to alterations both in hepatic and pancreatic lipid panel and histology in rats. METHODS: Male Wistar rats were fed with 2% cholesterol +0.25% cholate-supplemented diet or standard chow for 12 weeks. Blood was collected at weeks 0, 4, 8 and 12 to measure serum cholesterol and triglyceride levels. At week 12, both the pancreas and the liver were isolated for further histological and biochemical analysis. Hepatic and plasma fatty acid composition was assessed by gas chromatography. Expression of mRNA of major enzymes involved in saturated/unsaturated fatty acid synthesis was analyzed by qPCR. In separate experiments serum enzyme activities and insulin levels were measured at week 9. RESULTS: At week 12, rats fed with 2% cholesterol +0.25% cholate-supplemented diet were characterized by elevated serum cholesterol (4.09 ± 0.20 vs. 2.89 ± 0.22 mmol/L, *p < 0.05) while triglyceride (2.27 ± 0.05 vs. 2.03 ± 0.03 mmol/L) and glucose levels (5.32 ± 0.14 vs. 5.23 ± 0.10 mmol/L) remained unchanged. Isolated hypercholesterolemia increased hepatic lipid accumulation, hepatic cholesterol (5.86 ± 0.22 vs. 1.60 ± 0.15 ng/g tissue, *p < 0.05) and triglyceride contents (19.28 ± 1.42 vs. 6.78 ± 0.71 ng/g tissue, *p < 0.05), and hepatic nitrotyrosine level (4.07 ± 0.52 vs. 2.59 ± 0.31 ng/mg protein, *p < 0.05). The histology and tissue lipid content of the pancreas was not affected. Serum total protein level, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities remained unchanged in response to isolated hypercholesterolemia while serum alkaline phosphatase activity (ALP) significantly increased. Plasma insulin levels did not change in response to isolated hypercholesterolemia suggesting an intact endocrine function of the pancreas. Isolated hypercholesterolemia caused a significantly increased hepatic and serum fatty acid level associated with a marked alteration of fatty acid composition. Hepatic expression of Δ9-desaturase (SCD1) was increased 4.92×, while expression of Δ5-desaturase and Δ6-desaturase were decreased (0.447× and 0.577×, respectively) due to isolated hypercholesterolemia. CONCLUSIONS: Isolated hypercholesterolemia leads to hepatic steatosis and marked alterations in the hepatic lipid profile without affecting the pancreas. Altered fatty acid profile might mediate harmful effects of cholesterol in the liver.

Investigation of the Antiproliferative Properties of Natural Sesquiterpenes from Artemisia asiatica and Onopordum acanthium on HL-60 Cells in Vitro.

Plants and plant extracts play a crucial role in the research into novel antineoplastic agents. Four sesquiterpene lactones, artecanin (1), 3ß-chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guaia-11(13)-en-12,6α-olide (2), iso-seco-tanapartholide 3-O-methyl ether (3) and 4ß,15-dihydro-3-dehydrozaluzanin C (4), were isolated from two traditionally used Asteraceae species (Onopordum acanthium and Artemisia asiatica). When tested for antiproliferative action on HL-60 leukemia cells, these compounds exhibited reasonable IC50 values in the range 3.6-13.5 µM. Treatment with the tested compounds resulted in a cell cycle disturbance characterized by increases in the G1 and G2/M populations, while there was a decrease in the S phase. Additionally, 1-3 elicited increases in the hypodiploid (subG1) population. The compounds elicited concentration-dependent chromatin condensation and disruption of the membrane integrity, as revealed by Hoechst 33258-propidium staining. Treatment for 24 h resulted in significant increases in activity of caspases-3 and -9, indicating that the tested sesquiterpenes induced the mitochondrial pathway of apoptosis. The proapoptotic properties of the sesquiterpene lactones were additionally demonstrated withannexin V staining. Compounds 1 and 2 increased the Bax/Bcl-2 expression and decreased the expressions of CDK1 and cyclin B2, as determined at the mRNA level by means of RT-PCR. These experimental results indicate that sesquiterpene lactones may be regarded as potential starting structures for the development of novel anticancer agents.

The effect of a preparation of minerals, vitamins and trace elements on the cardiac gene expression pattern in male diabetic rats.

BACKGROUND: Diabetic patients have an increased risk of developing cardiovascular diseases, which are the leading cause of death in developed countries. Although multivitamin products are widely used as dietary supplements, the effects of these products have not been investigated in the diabetic heart yet. Therefore, here we investigated if a preparation of different minerals, vitamins, and trace elements (MVT) affects the cardiac gene expression pattern in experimental diabetes. METHODS: Two-day old male Wistar rats were injected with streptozotocin (i.p. 100 mg/kg) or citrate buffer to induce diabetes. From weeks 4 to 12, rats were fed with a vehicle or a MVT preparation. Fasting blood glucose measurement and oral glucose tolerance test were performed at week 12, and then total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 41012 oligonucleotides. RESULTS: Significantly elevated fasting blood glucose concentration and impaired glucose tolerance were markedly improved by MVT-treatment in diabetic rats at week 12. Genes with significantly altered expression due to diabetes include functional clusters related to cardiac hypertrophy (e.g. caspase recruitment domain family, member 9; cytochrome P450, family 26, subfamily B, polypeptide; FXYD domain containing ion transport regulator 3), stress response (e.g. metallothionein 1a; metallothionein 2a; interleukin-6 receptor; heme oxygenase (decycling) 1; and glutathione S-transferase, theta 3), and hormones associated with insulin resistance (e.g. resistin; FK506 binding protein 5; galanin/GMAP prepropeptide). Moreover the expression of some other genes with no definite cardiac function was also changed such as e.g. similar to apolipoprotein L2; brain expressed X-linked 1; prostaglandin b2 synthase (brain). MVT-treatment in diabetic rats showed opposite gene expression changes in the cases of 19 genes associated with diabetic cardiomyopathy. In healthy hearts, MVT-treatment resulted in cardiac gene expression changes mostly related to immune response (e.g. complement factor B; complement component 4a; interferon regulatory factor 7; hepcidin). CONCLUSIONS: MVT-treatment improved diagnostic markers of diabetes. This is the first demonstration that MVT-treatment significantly alters cardiac gene expression profile in both control and diabetic rats. Our results and further studies exploring the mechanistic role of individual genes may contribute to the prevention or diagnosis of cardiac complications in diabetes.

Hypothalamic gene expression in ω-3 PUFA-deficient male rats before, and following, development of hypertension.

Dietary deficiency of ω-3 fatty acids (ω-3 DEF) produces hypertension in later life. This study examined the effect of ω-3 DEF on blood pressure and hypothalamic gene expression in young rats, before the development of hypertension, and in older rats following the onset of hypertension. Animals were fed experimental diets that were deficient in ω-3 fatty acids, sufficient in short-chain ω-3 fatty acids or sufficient in short- and long-chain ω-3 fatty acids, from the prenatal period until 10 or 36 weeks-of-age. There was no difference in blood pressure between groups at 10 weeks-of-age; however, at 36 weeks-of-age ω-3 DEF animals were hypertensive in relation to sufficient groups. At 10 weeks, expression of angiotensin-II(1A) receptors and dopamine D(3) receptors were significantly increased in the hypothalamic tissue of ω-3 DEF animals. In contrast, at 36 weeks, α(2a) and ß(1) adrenergic receptor expression was significantly reduced in the ω-3 DEF group. Brain docosahexaenoic acid was significantly lower in ω-3 DEF group compared with sufficient groups. This study demonstrates that dietary ω-3 DEF causes changes both in the expression of key genes involved in central blood pressure regulation and in blood pressure. The data may indicate that hypertension resulting from ω-3 DEF is mediated by the central adrenergic system.

A cell-microelectronic sensing technique for the screening of cytoprotective compounds.

In recent years, a new cell-based high throughput paradigm has emerged, which seeks to identify novel, pharmacologically active cytoprotective compounds. The essence of this approach is to create experimental models of cell injury relevant for a particular disease by establishing in vitro cell-based models, followed by high-throughput testing of compounds that affect the cellular response in a desired manner. Prior approaches typically used simple end-point analyses. To assess the cytoprotective effects of novel drug candidates in real-time, we have applied a cell-microelectronic sensing technique (RT-CES), which measures changes in the impedance of individual microelectronic wells that correlates linearly with cell index (reflecting cell number, adherence and cell growth), thereby allowing the continuous determination of cell viability during oxidative stress. In vitro cytotoxicity was elicited by hydrogen peroxide in myocytes (H9c2) and hepatocytes (Hep3B). Cells were post-treated at 30 min with various reference molecules and novel cytoprotective compounds. Cytoprotection detected in the RT-CES system correlated well with the results of two classical end-point-based methods (improvement in MTT and reduction of LDH release). The RT-CES method, when used as described in the current report, is suitable for the screening of molecular libraries to identify molecules or molecule combinations that attenuate oxidative stress-induced cell damage.

Nutrigenomic approaches to study the effects of n-3 PUFA diet in the central nervous system.

Deficiencies in essential, mainly omega-3 and omega-6 (n-3, n-6) long chain polyunsaturated fatty acids (LC-PUFA) result in visual and cognitive impairment and disturbances in mental functions in animals and could be the main reason for the increasing incidence of different mental disorders in humans. Traditional approaches cannot give us a detailed picture on how dietary lipids exert their effects, because they focus on only a few genes or biomarkers. Dietary lipids not only influence the biophysical state of the cell membranes but, via direct and indirect routes, they also act on multiple pathways including signalling and gene and protein activities. Therefore, to understand the molecular basis of the effects and roles of n-3 PUFA in the central nervous system global screening techniques such as DNA- or protein microarrays were used to assess the changes, in a global way, at the transcriptome and at the proteome level. With DNA microarrays we found that cholesterol and fish oil (high in PUFA) diets altered the expression of several genes involved in raft formation and membrane protrusions. By using protein microarrays we detected a decreased concentration of protein kinase C beta, gamma, phospholipase C gamma and other changes in the expression level of proteins involved in the signal transduction pathway in the brain in response to high cholesterol diet. Besides the known cellular effects of lipid nutritions (changing eicosanoid make up, effects on membrane fluidity and raft stability) it is now evident that dietary lipids influence gene and protein activity levels, protein modifications and probably play important role in modulating protein aggregation.

Gene expression and molecular composition of phospholipids in rat brain in relation to dietary n-6 to n-3 fatty acid ratio.

Rats were fed from conception till adulthood either with normal rat chow with a linoleic (LA) to linolenic acid (LNA) ratio of 8.2:1 or a rat chow supplemented with a mixture of perilla and soy bean oil giving a ratio of LA to LNA of 4.7:1. Fat content of the feed was 5%. Fatty acid and molecular species composition of ethanolamine phosphoglyceride was determined. Effect of this diet on gene expression was also studied. There was an accumulation of docosahexaenoic (DHA) and arachidonic acids (AA) in brains of the experimental animals. Changes in the ratio sn-1 saturated, sn-2 docosahexaenoic to sn-1 monounsaturated, sn-2 docosahexaenoic were observed. Twenty genes were found overexpressed in response to the 4.7:1 mixture diet and four were found down-regulated compared to normal rat chow. Among them were the genes related to energy household, lipid metabolism and respiration. The degree of up-regulation exceeded that observed with perilla with a ratio of LA to LNA 8.2:1 [Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 2619]. It was concluded that brain sensitively reacts to the fatty acid composition of the diet. It was suggested that alteration in membrane architecture and function coupled with alterations in gene expression profiles may contribute to the observed beneficial impact of n-3 type polyunsaturated fatty acids on cognitive functions.

The role of n-3 polyunsaturated fatty acids in brain: modulation of rat brain gene expression by dietary n-3 fatty acids.

Rats were fed either a high linolenic acid (perilla oil) or high eicosapentaenoic + docosahexaenoic acid (fish oil) diet (8%), and the fatty acid and molecular species composition of ethanolamine phosphoglycerides was determined. Gene expression pattern resulting from the feeding of n-3 fatty acids also was studied. Perilla oil feeding, in contrast to fish oil feeding, was not reflected in total fatty acid composition of ethanolamine phosphoglycerides. Levels of the alkenylacyl subclass of ethanolamine phosphoglycerides increased in response to feeding. Similarly, levels of diacyl phosphatidylethanolamine molecular species containing docosahexaenoic acid (18:0/22:6) were higher in perilla-fed or fish oil-fed rat brains whereas those in ethanolamine plasmalogens remained unchanged. Because plasmalogen levels in the brains of rats fed a n-3 fatty acid-enriched diet increased, it is plausible, however, that docosahexaenoic acid taken up from the food or formed from linolenic acid was deposited in this phospholipid subclass. Using cDNA microarrays, 55 genes were found to be overexpressed and 47 were suppressed relative to controls by both dietary regimens. The altered genes included those controlling synaptic plasticity, cytosceleton and membrane association, signal transduction, ion channel formation, energy metabolism, and regulatory proteins. This effect seems to be independent of the chain length of fatty acids, but the n-3 structure appears to be important. Because n-3 polyunsaturated fatty acids have been shown to play an important role in maintaining normal mental functions and docosahexaenoic acid-containing ethanolamine phosphoglyceride (18:0/22:6) molecular species accumulated in response to n-3 fatty acid feeding, a casual relationship between the two events can be surmised.