Cloning, Expression, and Functional Analysis of the Full-Length cDNA of Acetyl-CoA C-acetyltransferase (AACT) Genes Related to Terpenoid Synthesis in Platycodon grandiflorus.

Platycodon grandiflorus is a well-known and widely distributed traditional herbal medicine and functional food in Asia, with triterpenoids as the main bioactive component in its roots. Acetyl-CoA C-acetyltransferase (AACT) is the initiation enzyme in the mevalonate pathway and plays an important role in the biosynthesis of terpenoids. OBJECTIVE: The objective of this study was to clone and identify the PgAACT function in P. grandiflorus. METHODS: The full-length sequence of PgAACT genes was isolated and cloned from P. grandiflorus by polymerase chain reaction (PCR). The recombinant plasmid was constructed using the pET-32a vector and expressed in E. coli Transetta (DE3) cells. Subcellular localization of AACT was observed in the epidermal cells of N. tabacum. Quantitative reverse transcription-PCR (qRT-PCR) was used to identify the PgAACT gene transcription levels. After MeJA treatment, the changes in AACT gene expression were observed, and UHPLC-Q-Exactive Orbitrap MS/MS was used to detect the changes in P. grandiflorus saponins. RESULTS: In this study, two full-length cDNAs encoding AACT1 (PgAACT1) and AACT2 (PgAACT2) were isolated and cloned from P. grandiflorus. The deduced PgAACT1 and PgAACT2 proteins contain 408 and 416 amino acids, respectively. The recombinant vectors were constructed, and the protein expression was improved by optimizing the reaction conditions. Sodium dodecyl sulphate-polycrylamide gel electrophloresis and western blot analysis showed that the PgAACT genes were successfully expressed, with molecular weights of the recombinant proteins of 61 and 63 kDa, respectively. Subcellular localization showed that the PgAACT genes were localized in the cytoplasm. Tissue specificity analysis of P. grandiflorus from different habitats showed that PgAACT genes were expressed in the roots, stems, and leaves. After MeJA treatment, the expression level of PgAACT genes and the content of total saponins of P. grandiflorus were significantly increased, suggesting that PgAACT genes play an important role in regulating plant defense systems. CONCLUSION: Cloning, expression, and functional analysis of PgAACT1 and PgAACT2 will be helpful in understanding the role of these two genes in terpene biosynthesis.

Cloning, Prokaryotic Expression, and Purification of Acetyl-CoA C-Acetyltransferase from Atractylodes lancea.

BACKGROUND: Cangzhu (Atractylodes lancea), a valuable and common traditional Chinese medicinal herb, is primarily used as an effective medicine with various health-promoting effects. The main pharmacological bioactive ingredients in the rhizome of A. lancea are terpenoids. Acetyl-CoA C-acetyltransferase (AACT) is the first enzyme in the terpenoid synthesis pathway and catalyzes two units of acetyl-CoA into acetoacetyl-CoA. OBJECTIVE: The objective of the present work was to clone and identify function of AlAACT from Atractylodes lancea. METHODS: A full-length cDNA clone of AlAACT was isolated using PCR and expressed in Escherichia coli. The expressed protein was purified using Ni-NTA agarose column using standard protocols. AlAACT was transiently expressed in N. benthamiana leaves to determine their subcellular location. The difference in growth between recombinant bacteria and control bacteria under different stresses was observed using the droplet plate experiment. RESULTS: In this study, a full-length cDNA of AACT (AlAACT) was cloned from A. lancea, which contains a 1,227 bp open reading frame and encodes a protein with 409 amino acids. Bioinformatic and phylogenetic analysis clearly suggested that AlAACT shared high similarity with AACTs from other plants. The recombinant protein pET32a(+)/AlAACT was successfully expressed in Escherichia coli BL21 (DE3) cells induced with 0.4 mM IPTG at 30°C as the optimized condition. The recombinant enzyme pET-32a-AlAACT was purified using the Ni-NTA column based on the His-tag, and the molecular weight was determined to be 62 kDa through SDS-PAGE and Western Blot analysis. The recombinant protein was eluted with 100, 300, and 500 mM imidazole; most of the protein was eluted with 300 mM imidazole. Under mannitol stress, the recombinant pET-32a- AlAACT protein showed a substantial advantage in terms of growth rates compared to the control. However, this phenomenon was directly opposite under NaCl abiotic stress. Subcellular localization showed that AlAACT localizes to the nucleus and cytoplasm. CONCLUSION: The expression and purification of recombinant enzyme pET-32a-AlAACT were successful, and the recombinant strain pET-32a-AlAACT in showed better growth in a drought stress. The expression of AlAACT-EGFP fusion protein revealed its localization in both nuclear and cytoplasm compartments. This study provides an important foundation for further research into the effects of terpenoid biosynthesis in A. lancea.

Mikania micrantha Extract Inhibits HMG-CoA Reductase and ACAT2 and Ameliorates Hypercholesterolemia and Lipid Peroxidation in High Cholesterol-Fed Rats.

The present study aimed to determine the effect of an ethyl acetate extract of Mikania micrantha stems (EAMMS) in hypercholesterolemia-induced rats. Rats were divided into a normal group (NC) and hypercholesterolemia induced groups: hypercholesterolemia control group (PC), simvastatin group (SV) (10 mg/kg) and EAMMS extract groups at different dosages of 50, 100 and 200 mg/kg, respectively. Blood serum and tissues were collected for haematological, biochemical, histopathological, and enzyme analysis. Total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, creatinine, malondialdehyde (MDA) level, as well as enzymes of HMG-CoA reductase (HMGCR) and acetyl-CoA acetyltransferase 2 (ACAT2), were measured. Feeding rats with high cholesterol diet for eight weeks resulted in a significantly (p < 0.05) increased of TC, TG, LDL-C, AST, ALT and MDA levels. Meanwhile, the administration of EAMMS extract (50, 100 and 200 mg/kg) and simvastatin (10 mg/kg) significantly reduced (p < 0.05) the levels of TC, TG, LDL-C and MDA compared to rats in the PC group. Furthermore, all EAMMS and SV-treated groups showed a higher HDL-C level compared to both NC and PC groups. No significant difference was found in the level of ALT, AST, urea and creatinine between the different dosages in EAMMS extracts. Treatment with EAMMS also exhibited the highest inhibition activity of enzyme HMGCR and ACAT2 as compared to the control group. From the histopathological examination, liver tissues in the PC group showed severe steatosis than those fed with EAMMS and normal diet. Treatment with EAMMS extract ameliorated and reduced the pathological changes in the liver. No morphological changes showed in the kidney structure of both control and treated groups. In conclusion, these findings demonstrated that EAMMS extract has anti-hypercholesterolemia properties and could be used as an alternative treatment for this disorder.

The potential of antioxidant-rich Maoberry (Antidesma bunius) extract on fat metabolism in liver tissues of rats fed a high-fat diet.

BACKGOUND: Obesity and dyslipidemia are major risk factors associated with non-alcoholic fatty liver disease (NAFLD). NAFLD refers to the accumulation of fat in more than 5% of the liver without alcohol consumption. NAFLD is the most common liver disease and is rapidly becoming a global public health problem. Maoberry (Antidesma bunius) is a fruit rich in antioxidants, especially phenolic compounds, which are reported to have benefits for patients with NAFLD. METHODS: We evaluated the effect of Maoberry extract on fat metabolism in liver tissues of high fat diet-induced rats. Five (5) groups (n = 12) of male Sprague-Dawley (SD) rats were divided into those given a high fat diet with no treatment (HF), different dosages of Maoberry extracts (0.38 [ML], 0.76 [MM) and 1.52 [MH] g/kg body weight) and 10 mg/kg statin (STAT). The rats were fed a high fat diet for 4 weeks to induce obesity and subsequently continued more for 12 weeks with treatments of Maoberry extracts or STAT. The levels of triglyceride, liver enzymes, oxidative stress and inflammation markers, triglyceride synthesis regulators, and pathology of the liver in high fat diet-induced rats were investigated. RESULTS: Feeding Maoberry extract in MH groups resulted in decreasing levels of serum alanine aminotransferase (ALT), liver triglyceride, liver thiobarbituric acid reactive substances (TBARS) and mRNA expression of tumour necrosis factor (TNF)-α, interleukin (IL)-6, glycerol-3-phosphate acyltransferase (GPAT)-1 and acetyl-coenzyme A carboxylase (ACC) compared with the HF group (P < 0.05). Moreover, histopathological study of the liver showed reduced fat droplets in the Maoberry extract treatment groups, especially in MH groups and STAT treatment groups. CONCLUSIONS: The improvements of fat metabolism in liver tissues of rats fed a high-fat diet were observed in Maoberry extracts treatment groups. The underline mechanism that link to fat metabolism might be through the process accompanied with down-regulated the gene expression of key enzymes of lipid production, antioxidant activity, and anti-inflammation properties of Maoberry extracts which contains high levels of phenolic and flavonoid compounds.

Graviola (Annona muricata) attenuates behavioural alterations and testicular oxidative stress induced by streptozotocin in diabetic rats.

Oxidative stresses intensify the progression of diabetes-related behavioural changes and testicular injuries. Graviola (Annona muricata), a small tree of the Annonaceae family, has been investigated for its protective effects against diabetic complications, oxidative stress, and neuropathies. This study was planned to investigate the effects of graviola on behavioural alterations and testicular oxidative status of streptozotocin (STZ; 65 mg/kg)-induced diabetic rats. Forty adult male Wistar rats were equally allocated into four groups: control (received normal saline 8 ml/kg orally once daily), diabetic (received normal saline orally once daily), graviola (GR; received 100 mg/kg/day; orally once daily), and diabetic with graviola (Diabetic+GR; received 100 mg/kg/day; once daily). Behavioural functions were assessed using standard behavioural paradigms. Also, oxidative statuses of testis were evaluated. Results of behavioural observations showed that diabetes induced depression-like behaviours, reduction of exploratory and locomotor activities, decreased memory performance, and increased stress-linked behaviours. These variations in diabetic rats were happened due to oxidative stress. Interestingly, treatment of diabetic rats with graviola for four weeks alleviated all behavioural changes due to diabetes. Also, rats in graviola-treated groups had greater testicular testosterone and estradiol levels compared with diabetic rats due to significant rise in testicular acetyl-CoA acetyltransferase 2 expression. In the same context, graviola enhanced the antioxidant status of testicular tissues by significantly restoring the testicular glutathione and total superoxide dismutase that fell during diabetes. In addition, Graviola significantly decreased the expression of apoptotic (Bax) and inflammatory (interleukin-1ß) testicular genes. In conclusion, these data propose that both the hypoglycemic and antioxidative potential of graviola are possible mechanisms that improve behavioural alterations and protect testis in diabetic animals. Concomitantly, further clinical studies in human are required to validate the current study.

The effect of bergamot polyphenolic fraction on lipid transfer protein system and vascular oxidative stress in a rat model of hyperlipemia.

BACKGROUND: Experimental and epidemiological studies show that bergamot polyphenolic fraction (BPF) ameliorates the serum lipemic profile, normalizes blood pressure and improves non alcoholic fatty liver disease in patients suffering from metabolic syndrome. Despite this evidence, the molecular mechanisms responsible for these beneficial effects remain unclear. The aim of our study is to clarify the effects of BPF on the lipoprotein assembly and to identify oxidative stress biomarkers correlating hyperlipidaemia and BPF-induced metabolic changes. METHODS: Male Wistar rats (180-200 g) were randomly assigned to receive a standard diet, a hypercholesterolemic diet or a hypercholesterolemic diet+BPF (20 mg/Kg/rat daily, gavage), respectively, for 90 days. Total cholesterol (tChol), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglycerides (TG) and fasting plasma glucose were evaluated at the baseline as well as at the end of the treatment. To assess the effect of BPF on the Lipid Transfer Protein System, detection of ACAT, LCAT, CETP, PON1, Apo A1 and Apo B have also been carried out. Finally, the lipid peroxidation biomarker (TBARS) and oxyLDL were also measured. RESULTS: BPF prevented tChol, LDL-C, TG and fasting plasma glucose enhancement and improved HDL-C. Treatment of hyperlipæmic rats with BPF significantly restored altered the serum concentration of lipemic biomarkers and the activity of ACAT, LCAT, CETP and PON1, an effect accompanied by the concomitant normalization of Apo A1 and APO B levels. In addition, TBARS levels were reduced significantly by the treatment with BPF. CONCLUSIONS: BPF prevents diet-induced alteration of the lipid profile in rats, counteracting oxidative stress and improving the dysregulation of the Lipid Transfer Protein System. These data add new insights into the molecular mechanisms underlying the beneficial role of BPF in the therapy of hyperlipidaemia, thus suggesting a novel approach in the prevention of cardiovascular disease.

Prevention by L-carnitine of DNA damage induced by 3-hydroxy-3-methylglutaric and 3-methylglutaric acids and experimental evidence of lipid and DNA damage in patients with 3-hydroxy-3-methylglutaric aciduria.

3-hydroxy-3-methylglutaric aciduria (HMGA) is an inherited disorder of the leucine catabolic pathway in which occurs a deficiency of the 3-hydroxy-3-methylglutaryl-CoA lyase enzyme. Therefore, the organic acids 3-hydroxy-3-methylglutaric (HMG) and 3-methylglutaric (MGA), mainly, accumulate in tissues of affected patients. Lately, much attention has been focused on free radicals as mediators of tissue damage in human diseases, causing lipid peroxidation, protein oxidation and DNA damage. The treatment of this disease is based in a restricted protein ingest and supplementation with l-carnitine (LC), an antioxidant and detoxifying agent. In the present work, we investigated the in vitro oxidative damage to DNA induced by the accumulation of organic acids and oxidative stress parameters in vivo of patients with 3-HMG, as well as the effect of the recommended therapy. The in vitro DNA damage was analyzed by the alkaline comet assay in leukocytes incubated with HMG and MGA (1 mM, 2.5 mM and 5 mM) and co-incubated with LC (90 µM and 150 µM). The in vivo urinary 15-F2t-isoprostane levels and urinary oxidized guanine species were measured by ELISA kits in patient's urine before and after the treatment with LC. HMG and MGA induced a DNA damage index (DI) significantly higher than that of the control group. The DI was significantly reduced in the presence of LC. It was also verified a significant increase of oxidized guanine species and urinary isoprostane levels, biomarker of oxidative DNA damage and lipid peroxidation respectively, in patients before treatment. After the treatment and supplementation with LC, patients presented significantly lower levels of those biomarkers. Analyzing the data together, we can conclude that HMGA patients present oxidative lipid and DNA damage, which is induced by HMG and MGA, and the antioxidant therapy with LC can prevent that kind of injuries.

Disrupting cholesterol esterification by bitter melon suppresses triple-negative breast cancer cell growth.

Triple negative breast cancer (TNBC) is aggressive with a worse prognosis. We have recently shown that bitter melon extract (BME) treatment was more effective in inhibition of TNBC tumor growth in mouse models as compared to ER positive breast tumor growth. Aberrant dysregulation of lipid metabolism is associated with breast cancer progression, however, anti-cancer mechanism of BME linking lipid metabolism in breast cancer growth remains unexplored. Here, we observed that accumulation of esterified cholesterol was reduced in BME treated TNBC cell lines as compared to control cells. We next evaluated expression levels of acyl-CoA: cholesterol acyltransferase 1 (ACAT-1) in TNBC cells treated with BME. Our results demonstrated that BME treatment inhibited ACAT-1 expression in TNBC cells. Subsequently, we found that sterol regulatory element-binding proteins-1 and -2, and FASN was significantly reduced in BME treated TNBC cell lines. Low-density lipoprotein receptor was also downregulated in BME treated TNBC cells as compared to control cells. We further demonstrated that BME feeding reduced tumor growth in TNBC mammospheres implanted into NSG mice, and inhibits ACAT-1 expression. To our knowledge, this is the first report demonstrating BME suppresses TNBC cell growth through ACAT-1 inhibition, and have potential for additional therapeutic regimen against human breast cancer.

Natural Extracts Abolished Lipid Accumulation in Cells Harbouring non-favourable PNPLA3 genotype.

Background & aims. G-allele of PNPLA3 (rs738409) favours triglycerides accumulation and steatosis. In this study, we examined the effect of quercetin and natural extracts from mushroom and artichoke on reducing lipid accumulation in hepatic cells. MATERIAL AND METHODS: Huh7.5 cells were exposed to oleic acid (OA) and treated with quercetin and extracts to observe the lipid accumulation, the intracellular-TG concentration and the LD size. Sterol regulatory element binding proteins-1 (SREBP-1), peroxisome proliferator-activated receptor (PPARα-γ) and cholesterol acyltransferase (ACAT) gene expression levels were analysed. RESULTS: Quercetin decreased the intracellular lipids, LD size and the levels of intracellular-TG through the down-regulation of SREBP-1c, PPARγ and ACAT1 increasing PPARα. The natural-extracts suppressed OA-induced lipid accumulation and the intracellular-TG. They down-regulate the hepatic lipogenesis through SREBP-1c, besides the activation of lipolysis through the increasing of PPARα expression. CONCLUSIONS: Quercetin and the aqueous extracts decrease intracellular lipid accumulation by down-regulation of lipogenesis and up-regulation of lipolysis.

The association and significance of H3K27me3 and a folate metabolic gene ACat2 in neural tube defects.

AIM: To study the association between the expression of H3K27me3 and ACat2 (a folate metabolic protein), in order to elucidate the protective mechanism of folic acid (FA) in neural tube defects (NTDs). METHODS: Eighteen female SD rats were randomly divided into normal, NTD and FA group. NTD group was induced by all-trans retinoic acid (ATRA) at E10d. FA group was fed with FA supplementation since 2 weeks before pregnancy, followed by ATRA induction. At E15d, FA level in the embryonic neural tube was determined by ELISA. Neural stem cells (NSCs) were isolated. Cell proliferation was compared by CCK-8 assay. The differentiation potency was assessed by immunocytochemical staining. H3K27me3 expression was measured by immunofluorescence method and Western blot. ACat2 mRNA expression was detected by qRT-PCR. RESULTS: Cultured NSCs formed numerous Nestin-positive neurospheres. After 5 days, they differentiated into NSE-positive neurons and GFAP-positive astrocytes. When compared with controls, the FA level in NTD group was significantly lower, the ability of cell proliferation and differentiation was significantly reduced, H3K27me3 expression was increased, and ACat2 mRNA expression was decreased (P <0.05). The intervention of FA notably reversed these changes (P <0.05). H3K27me3 expression was negatively correlated with the FA level (rs = -0.908, P <0.01) and ACat2 level (rs = -0.879, P <0.01) in the neural tube. CONCLUSION: The increased H3K27me3 expression might cause a disorder of folate metabolic pathway by silencing ACat2 expression, leading to reduced proliferation and differentiation of NSCs, and ultimately the occurrence of NTD. FA supplementation may reverse this process.

Cinnamon extract improves the body composition and attenuates lipogenic processes in the liver and adipose tissue of rats.

In models of metabolic disorders, cinnamon improves glucose and lipid metabolism. This study explores the effect of chronic supplementation with aqueous cinnamon extract (CE) on the lipid metabolism of rats. Male adult Wistar rats were separated into a control group (CTR) receiving water and a CE Group receiving aqueous cinnamon extract (400 mg of cinnamon per kg body mass per day) by gavage for 25 consecutive days. Cinnamon supplementation did not change the food intake or the serum lipid profile but promoted the following changes: lower body mass gain (P = 0.008), lower relative mass of white adipose tissue (WAT) compartments (P = 0.045) and higher protein content (percentage of the carcass) (P = 0.049). The CE group showed lower leptin mRNA expression in the WAT (P = 0.0017) and an important tendency for reduced serum leptin levels (P = 0.059). Cinnamon supplementation induced lower mRNA expression of SREBP1c (sterol regulatory element-binding protein 1c) in the WAT (P = 0.001) and liver (P = 0.013) and lower mRNA expression of SREBP2 (P = 0.002), HMGCoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase) (P = 0.0003), ACAT1 (acetyl-CoA acetyltransferase 1) (P = 0.032) and DGAT2 (diacylglycerol O-acyltransferase 2) (P = 0.03) in the liver. These changes could be associated with the reduced esterified cholesterol and triacylglycerol content detected in this tissue. Our results suggest that chronic ingestion of aqueous cinnamon extract attenuates lipogenic processes, regulating the expression of key enzymes and transcriptional factors and their target genes, which are directly involved in lipogenesis. These molecular changes possibly promote adaptations that would prevent an increase in circulating cholesterol and triacylglycerol levels and prevent lipid accumulation in tissues, such as liver and WAT. Therefore, we speculate that cinnamon may also be useful for preventing or retarding the development of lipid disorders.

Anti-apoptotic and Beneficial Metabolic Activities of Resveratrol in Type II Gaucher Disease.

Gaucher disease (GD) is one of the most common lysosomal storage disorders and is caused by an inherited deficiency in glucocerebrosidase. Resveratrol is a phytoalexin that has many beneficial activities, including anti-oxidant, anti-apoptotic, and neuroprotective effects. The aim of this study was to determine if resveratrol has a therapeutic effect on primary fibroblast cells derived from a patient with type II GD. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to determine the effect of resveratrol on cell viability. The expression patterns of apoptosis-inducing factor (AIF), Bcl-2-associated X protein (Bax), caspase-3, acetyl-coenzyme A acetyltransferase 1 (ACAT1), E3-binding protein (E3BP), and citrate synthase (CS) were evaluated by Western blotting to characterize the effect of resveratrol treatment on GD cells. TLC was performed to determine glucosylceramide levels in resveratrol-treated GD cells. Resveratrol increased GD cell viability compared to untreated control cells. Further, resveratrol treatment dose-dependently decreased the apoptotic factors AIF, Bax, and cleaved caspase-3 levels, whereas ACAT1, E3BP, and CS expression dose-dependently increased. TLC analysis showed reduced levels of intracellular glucosylceramides in resveratrol-treated GD cells. These findings demonstrate that resveratrol can reduce cellular stress resulting from glucosylceramide accumulation, and suggest that resveratrol should be studied further as a novel therapeutic agent for GD.

[Cloning and expression analysis of a acetyl-CoA U-acetyltransferase gene (TwAACT) from Tripterygium wilfordii].

In this study, based on the transcriptome data, we cloned the full-length cDNAs of TwAACT gene from Tripterygium wilfordii suspension cells, and then analyzed the bioinformation of the sequence, detected the genetic differential expression after being induced by methyl jasmonate (MeJA) by RT-PCR. The full-length cDNA of the TwAACT was 1 704 bp containing a 1 218 bp open reading frame (ORF) encoding a polypeptide of 405 amino acids (GeneBank accession No. KP297934). The deduced isoelectric point (pI) was 6.10, a calculated molecular weight was about 41.20 kDa, and online prediction showed that TwAACT had two catalytic active sites. After the induction of MeJA, the relative expression level of TwAACT increased rapidly. The expression level of TwAACT was highest at 24 h. TwAACT was cloned firstly, that laid the foundation for identifying thegene and illustrating thebiosynthesis mechanism and its synthetic biology.

[Cloning and gene expression of acetyl-CoA C-acetyl transferase gene (AsAACT) from Aquilaria sinensis].

OBJECTIVE: This study aimed to clone the acetyl-CoA C-acetyl transferase (AACT) gene from Aquilaria sinensis and analyze the bioinformatics and expression of the gene. METHOD: One unique sequence containing partly AACT gene sequence was discovered in our previous transcriptome dataset of A. sinensis. AACT gene was cloned by RT-PCR and RACE strategy with the template of RNA extracted from A. sinensis stem. The bioinformatic analysis of this gene and its corresponding protein was performed. The AsAACT expression in calli was analyzed with GADPH gene as an internal control gene in wounded condition by qRT-PCR technique. RESULT: One unique sequence of AACT, named as AsAACT, was cloned from A. sinensis. The full length of AsAACT cDNA was containing a 1 236 bp ORF that encoded 411 amino acids. The result of qRT-PCR displayed that the highest expression level was at 4 h. which indicated that it was possibly involved in early-stage response to wound. CONCLUSION: Cloning and analyzing AsAACT gene from A. sinensis provided basic information for study the function and expression regulation of AsAACT in terpenoid biosynthesis.

Changes in cholesterol homeostasis modify the response of F1B hamsters to dietary very long chain n-3 and n-6 polyunsaturated fatty acids.

BACKGROUND: The plasma lipoprotein response of F1B Golden-Syrian hamsters fed diets high in very long chain (VLC) n-3 polyunsaturated fatty acids (PUFA) is paradoxical to that observed in humans. This anomaly is attributed, in part, to low lipoprotein lipase activity and is dependent on cholesterol status. To further elucidate the mechanism(s) for these responses, hamsters were fed diets containing supplemental fish oil (VLC n-3 PUFA) or safflower oil (n-6 PUFA) (both 10% [w/w]) and either cholesterol-supplemented (0.1% cholesterol [w/w]) or cholesterol-depleted (0.01% cholesterol [w/w] and 10 days prior to killing fed 0.15% lovastatin+2% cholestyramine [w/w]). RESULTS: Cholesterol-supplemented hamsters fed fish oil, relative to safflower oil, had higher non-high density lipoprotein (HDL) cholesterol and triglyceride concentrations (P < 0.001) which were associated with lower hepatic low density lipoprotein (LDL) receptor, sterol regulatory element binding protein (SREBP)-1c and acyl-CoA: cholesterol acyl transferase-2 (ACAT) mRNA and protein (p < 0.05), and higher hepatic apolipoprotein (apo) B-100 and apo E protein levels. In contrast, cholesterol-depleted hamsters fed fish oil, relative to safflower oil, had lower non-HDL cholesterol and triglyceride concentrations (P < 0.001) which were associated with lower hepatic SREBP-1c (p < 0.05) but not apo B-100, apo E or ACAT-2 mRNA or protein levels. Independent of cholesterol status, fish oil fed hamsters had lower HDL cholesterol concentrations (p < 0.001), which were associated with lower hepatic apoA-I protein levels (p < 0.05). CONCLUSION: These data suggest disturbing cholesterol homeostasis in F1B hamsters alters their response to dietary fatty acids, which is reflected in altered plasma lipoprotein patterns and regulation of genes associated with their metabolism.

[Molecular cloning and SNP analysis of a acetyl-CoA C-acetyltransferase gene (SmAACT) from Salvia miltiorrhiza].

Acetyl-CoA C-acetyltransferase (AACT) is the first enzyme in the terpene synthesis pathway, catalyzed two units of acetyl-CoA to acetoacetyl-CoA. In order to study the tanshinone biosynthesis in Salvia miltiorrhiza, a novel AACT gene, SmAACT, was cloned using cDNA microarray and RACE strategy. The full length cDNA of SmAACT is 1 623 bp (accession No. EF635969), which contained a 1 200 bp open reading frame (ORF) encoding a 399 amino acid protein. Nine introns were found in the genomic sequence. SmAACT was upregulated by YE and Ag+ elicitors both with cDNA microarray and quantitative RT-PCR analyses along with the accumulation of tanshinones. Sequence homology comparison and phylogenetic analysis all suggested that SmAACT belonged to the class of acetyl-CoA C-acetyltransferase. The transcription level of SmAACT was relatively higher in root than that in stem and leaf tissues. SNP analysis revealed that SmAACT was highly variable in the region of 6 to 9 introns with 33 SNPs in the 600 bp region, there are 5 SNPs in the cDNA region while they are all synonymous cSNPs. Some special genotypes were found in Salvia miltiorrhiza from different areas. SmAACT will be an useful gene for further analyze the mechanism of gene regulation among the tanshinones biosynthesis.

Development and validation of a transcreener assay for detection of AMP- and GMP-producing enzymes.

Screening of AMP- and GMP-producing enzymes such as phosphodiesterases (PDEs), ligases, and synthetases would be simplified by the ability to directly detect unmodified nucleoside monophosphates. To address this need, we developed polyclonal and monoclonal antibodies that recognize AMP and GMP with nanomolar sensitivity and high selectivity vs. the corresponding triphosphate and 3',5'-cyclic monophosphate nucleotides that serve as substrates for many enzymes in these classes. One of these antibodies was used to develop a Transcreener AMP/GMP assay with a far red fluorescence polarization (FP) readout. This polyclonal antibody exhibited extremely high selectivity, with IC(50) ratios of 6,000 for ATP/AMP, 3,810 for cAMP/AMP, and 6,970 for cGMP/GMP. Standard curves mimicking enzymatic conversion of cAMP, cGMP, and ATP to the corresponding monophosphates yielded Z' values of >0.85 at 10% conversion. The assay reagents were shown to be stable for 24 h at room temperature, both before and after dispensing. The Transcreener AMP/GMP FP assay was used for enzymatic detection of cGMP- and cAMP-dependent PDEs 4A1A, 3A, and 9A2 and ATP-dependent ligases, acetyl CoA synthetase, and ubiquitin- activating enzyme (UBE1). Shifts of >100 mP were observed in the linear part of the progress curves for all enzymes tested, and the PDE isoforms exhibited the expected substrate and inhibitor selectivity. These studies demonstrate that direct immunodetection of AMP and GMP is a flexible, robust enzyme assay method for diverse AMP- and GMP-producing enzymes. Moreover, it eliminates many of the shortcomings of other methods including the need for fluorescently labeled substrates, the low signal:background inherent in substrate depletion assays, and the potential for interference with coupling enzymes.

Lycopene in association with alpha-tocopherol or tomato lipophilic extracts enhances acyl-platelet-activating factor biosynthesis in endothelial cells during oxidative stress.

Lipophilic compounds contained in tomato can prevent cardiovascular diseases by modulating the atherogenic processes in vascular endothelium mediated by oxidized low-density lipoproteins (LDLs). We investigated the effects of lycopene on the metabolism of platelet-activating factor (PAF) and its much less biologically active acyl analog, acyl-PAF, known to prevent LDL oxidation. Lycopene, or lycopene in association with alpha-tocopherol, or whole tomato lipophilic extracts (containing more than 80% lycopene) were used in experiments in which endothelial cells (ECs) are known to synthesize PAF following H(2)O(2)-induced oxidative stress. The results indicated that in each case H(2)O(2)-stimulated PAF biosynthesis in ECs, which is catalyzed by acetyl-CoA acetyltransferase (AT), appeared strongly inhibited. However, acyl-PAF biosynthesis, which also occurs through the PAF-dependent transacetylase (TA), was significantly increased by lycopene only when it was in association with alpha-tocopherol or with the minor compounds present in the whole lipophilic tomato extract. These findings suggest that alpha-tocopherol or lipophilic compounds present in tomato juice potentiate the effects of lycopene on the modulation of PAF and acyl-PAF biosynthesis in ECs during oxidative stress.

Decreased liver fatty acid binding capacity and altered liver lipid distribution in mice lacking the liver fatty acid-binding protein gene.

Although liver fatty acid-binding protein (L-FABP) is an important binding site for various hydrophobic ligands in hepatocytes, its in vivo significance is not understood. We have therefore created L-FABP null mice and report here their initial analysis, focusing on the impact of this mutation on hepatic fatty acid binding capacity, lipid composition, and expression of other lipid-binding proteins. Gel-filtered cytosol from L-FABP null liver lacked the main fatty acid binding peak in the fraction that normally comprises both L-FABP and sterol carrier protein-2 (SCP-2). The binding capacity for cis-parinaric acid was decreased >80% in this region. Molar ratios of cholesterol/cholesterol ester, cholesteryl ester/triglyceride, and cholesterol/phospholipid were 2- to 3-fold greater, reflecting up to 3-fold absolute increases in specific lipid classes in the order cholesterol > cholesterol esters > phospholipids. In contrast, the liver pool sizes of nonesterified fatty acids and triglycerides were not altered. However, hepatic deposition of a bolus of intravenously injected [14C]oleate was markedly reduced, showing altered lipid pool turnover. An increase of approximately 75% of soluble SCP-2 but little or no change of other soluble (glutathione S-transferase, albumin) and membrane (fatty acid transport protein, CD36, aspartate aminotransferase, caveolin) fatty acid transporters was measured. These results (i) provide for the first time a quantitative assessment of the contribution of L-FABP to cytosolic fatty acid binding capacity, (ii) establish L-FABP as an important determinant of hepatic lipid composition and turnover, and (iii) suggest that SCP-2 contributes to the accumulation of cholesterol in L-FABP null liver.