OBJECTIVETo relatively quantify the gene expression of fatty acid synthase in squamous cell carcinoma, adjacent tissue, and some normal oral tissues by real-time quantitative PCR.

METHODSThe tissues were collected fresh from surgical specimens. The collected tissues were minced. Then the total RNA was extracted. The RNA was reversely transcripted into cDNA with random prime. And then the cDNA was amplified by real-time quantitative PCR to quantify the gene expression of FAS according to an internal control GAPDH. The difference of FAS gene expression was compared between squamous cell carcinoma, adjacent tissue, and some normal oral tissues.

RESULTSThe expression of FAS of squamous cell carcinoma was notably higher than the other two (P < 0.001).

CONCLUSIONReal-time quantitative PCR provides a method for monitoring the expression of fatty acid synthetic activity in squamous cell carcinoma, adjacent and normal tissues.

Carcinoma, Squamous Cell ; Fatty Acid Synthases ; Humans ; Mouth Neoplasms ; Polymerase Chain Reaction

The over expression of fatty acid synthase (FAS), a key enzyme in biosynthesis of fatty acid, can enhance enzyme activity and result in the malignant behavior, special material metabolism and energy metabolism of tumors. The expression of FAS is significantly higher in prostate cancer than in normal prostate tissues, which shows that FAS can be used as a marker in the early diagnosis of prostate cancer. The abnormally increased expression of FAS in prostate cancer may offer a new target for the drug treatment of the disease.
Biomarkers, Tumor ; metabolism ; Early Diagnosis ; Fatty Acid Synthases ; metabolism ; Humans ; Male ; Prostatic Neoplasms ; diagnosis ; enzymology

Three long-chain polyunsaturated fatty acids, docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), are the most biologically active polyunsaturated fatty acids in the body. They are important in developing and maintaining the brain function, and in preventing and treating many diseases such as cardiovascular disease, inflammation and cancer. Although mammals can biosynthesize these long-chain polyunsaturated fatty acids, the efficiency is very low and dietary intake is needed to meet the requirement. In this study, a multiple-genes expression vector carrying mammalian A6/A5 fatty acid desaturases and multiple-genes expression vector carrying mammalian Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases coding genes was used to transfect HEK293T cells, then the overexpression of the target genes was detected. GC-MS analysis shows that the biosynthesis efficiency and level of DHA, EPA and ARA were significantly increased in cells transfected with the multiple-genes expression vector. Particularly, DHA level in these cells was 2.5 times higher than in the control cells. This study indicates mammal possess a certain mechanism for suppression of high level of biosynthesis of long chain polyunsaturated fatty acids, and the overexpression of Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases broke this suppression mechanism so that the level of DHA, EPA and ARA was significantly increased. This study also provides a basis for potential applications of this gene construct in transgenic animal to produce high level of these long-chain polyunsaturated fatty acid.
Acetyltransferases ; genetics ; metabolism ; Arachidonic Acid ; biosynthesis ; Docosahexaenoic Acids ; biosynthesis ; Eicosapentaenoic Acid ; biosynthesis ; Fatty Acid Desaturases ; genetics ; metabolism ; Fatty Acid Synthases ; genetics ; metabolism ; Fatty Acids, Unsaturated ; biosynthesis ; Genetic Vectors ; HEK293 Cells ; Humans ; Transfection

Fatty acid synthase (FAS) catalyses the reaction between acetyl-CoA and malonyl-CoA to produce fatty acids. It is one of the most important enzyme in lipid biosynthesis. FAS of the oleaginous yeast Rhodosporidium toruloides has two acyl carrier protein (ACP) domains and a distinct subunit composition compared with FASs of other species. As ACP is a protein cofactor crucial for fatty acid chain elongation, more ACPs in the FAS may facilitate the reaction. To study the biochemical and structural properties of this novel FAS from R. toruloides, plasmids were constructed and transformed into Escherichia coli BL21 (DE3). The strain ZWE06 harboring plasmids pET22b-FAS1 and pET24b-FAS2 could co-overexpress the two subunits. The recombinant FAS was purified by sequentially using ammonium sulphate precipitation, sucrose density gradient centrifugation and anion exchange chromatography. The specific activity of the recombinant FAS was 548 mU/mg. The purified complex would be used to study enzyme kinetics and protein structure of FAS, and heterogeneous expression and purification will facilitate revealing the mechanism of this novel FAS with double ACPs.
Acyl Carrier Protein ; Basidiomycota ; enzymology ; Chromatography ; Escherichia coli ; metabolism ; Fatty Acid Synthases ; biosynthesis ; genetics ; Fatty Acids ; biosynthesis ; Plasmids ; Recombinant Proteins ; biosynthesis ; genetics

To understand molecular modules related to polyunsaturated fatty acids (PUFA) synthesis and eventually produce PUFA at high efficiency, we developed a protein complex analysis technology in Synechocystis sp. PCC 6803, and applied it to identify possible partner proteins interacting with the key enzymes that catalyze PUFA biosynthesis. We first constructed a recombinant expression of protein of slr1609 encoding the fatty acid activation enzyme, by fusing 3xFLAG tag with the target protein. Then we verified its expression by Western blotting targeting 3xFLAG tag. To maximize purification of Slr1609 protein complex, we optimized the protein expression conditions of Slr1609 in Synechocystis in a 5 L fermenter by monitoring its gene expression using RT-qPCR. The purification of the Slr1609 protein complexes was demonstrated by a Native-PAGE analysis. Finally, LC-MS/MS proteomic analysis allowed identification of the possible partner proteins interacting with Slr1609.
Bacterial Proteins ; chemistry ; Chromatography, Liquid ; Fatty Acid Synthases ; chemistry ; Fatty Acids, Unsaturated ; biosynthesis ; Proteome ; chemistry ; Proteomics ; Synechocystis ; enzymology ; Tandem Mass Spectrometry

OBJECTIVETo investigate the hepatic expression of sterol regulatory element-binding proteins-1c (SREBP-1c) in patients with nonalcoholic fatty liver disease (NAFLD).

METHODSNAFLD patients were confirmed using clinical and pathological methods. The expressions of protein and mRNA of SREBP-1 c in the livers were detected by immunohistochemistry, reverse transcription and polymerase chain reactions. The expression of protein of fatty acid synthases (FAS) controlled by SREBP-1c was observed by immunofluorescence. Levels of triglyceride and cholesterol in serum were measured.

RESULTSThe serum levels of triglyceride and cholesterol were positively correlated with hepatic adipose degeneration (r=0.71 or 0.70, P less than 0.05). The levels of SREBP-1c protein in the livers of NAFLD patients were significantly higher than those of the control group (2.19+/-0.31 vs 1.15+/-0.20, t=11.06, P less than 0.05), and the more severe the fatty degeneration, the stronger their expression (F=24.54, P less than 0.01). The expressions of the SREBP-1C mRNA in the livers showed the same changes (0.69+/-0.02 vs 0.40+/-0.02, t= -14.63, P less than 0.05). The expression of FAS was enhanced significantly in the livers of the NAFLD patients.

CONCLUSIONEnhanced expression of SREBP-1 c and FAS maybe a factor causing hepatic lipopexia in NAFLD patients.

Adult ; Fatty Acid Synthases ; metabolism ; Fatty Liver ; metabolism ; Female ; Humans ; Liver ; metabolism ; Male ; Middle Aged ; Sterol Regulatory Element Binding Protein 1 ; metabolism

We employed the whole-cell patch clamp technique to investigate the effects of arachidonic acid (AA) on barium inward current through the L-type calcium channels (IBa) and on osmotic stretch-induced increase of IBa in guinea-pig antral gastric myocytes. Under isosmotic condition, AA inhibited IBa in a dose-dependent manner to 91.1 +/- 1.4, 72.0 +/- 3.2, 46.0 +/- 1.8, and 20.3 +/- 2.3% at 1, 5, 10, 30 mM, respectively. The inhibitory effect of AA was not affected by 10 micrometer indomethacin, a cyclooxygenase inhibitor. Other unsaturated fatty acids, linoleic acid (LA) and oleic acid (OA) were also found to suppress IBa but stearic acid (SA), a saturated fatty acid, had no inhibitory effect on IBa. The potency sequence of these inhibitory effects was AA (79.7 +/- 2.3%) > LA (43.1 +/- 2.7%) > OA (14.2 +/- 1.1%) at 30 mM. On superfusing the myocyte with hyposmotic solution (214 mOsm) the amplitude of IBa at 0 mV increased (38.0 +/- 5.5%); this increase was completely blocked by pretreatment with 30 mM AA, but not significantly inhibited by lower concentrations of AA (1, 5 and 10 micrometer) (P > 0.05). Unsaturated fatty acids shifted the steady-state inactivation curves of IBa to the left; the extent of shift caused by AA was greater than that caused by LA. The activation curve was not affected by AA or LA. The results suggest that AA and other unsaturated fatty acids directly modulate L-type calcium channels and AA might modulate the hyposmotic stretch-induced increase of L-type calcium channel current in guinea-pig gastric smooth muscle.
Arachidonic Acid* ; Barium ; Calcium Channels* ; Calcium Channels, L-Type ; Calcium* ; Fatty Acids, Unsaturated ; Indomethacin ; Linoleic Acid ; Muscle Cells* ; Muscle, Smooth ; Oleic Acid ; Prostaglandin-Endoperoxide Synthases

OBJECTIVETo study the effects of soybean isoflavone on liver lipid, serum lipid, antioxidant index and hepatic lipid metabolism associated factors in nonalcoholic fatty liver rats.

METHODSThirty-six male rats (SD) were randomly divided into four groups by weight: normal control group, nonalcoholic fatty liver disease (NAFLD) model control group, low-dose isoflavone treatment group (10 mg/kg) and high-dose isoflavone group (20 mg/kg), 9 rats in each group. Normal control rats were fed with D12450B (10% fat energy), model control and isoflavone intervention rats were fed with D12492 (60% fat energy). Twelve weeks later, liver lipid, serum lipid and antioxidant index were observed. Liver sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS) and peroxisome proliferators activated receptor alpha (PPAR alpha) were detected by western blotting.

RESULTSLiver triglyceride (TG) in normal control group, NAFLD model control group, low-dose isoflavone group and high-dose isoflavone group were (8.11 ± 4.13), (57.06 ± 16.95), (31.26 ± 10.48), (31.38 ± 13.25) mmol/mg protein, respectively (F = 22.569, P < 0.01); liver free fatty acid (FFA) were (0.030 ± 0.007), (0.042 ± 0.009), (0.038 ± 0.009), (0.032 ± 0.005) µmol/mg protein, respectively (F = 4.857, P < 0.01); liver superoxide dismutase (SOD) activity were (502.29 ± 23.71), (201.83 ± 16.99), (228.93 ± 21.71), (238.08 ± 15.96) U/mg protein, respectively (F = 9.555, P < 0.01); liver malondialdehyde (MDA) were (1.29 ± 0.29), (2.85 ± 0.73), (2.07 ± 0.49), (2.03 ± 0.37) nmol/mg protein, respectively (F = 13.449, P < 0.01); SREBP-1c protein expression were 0.45 ± 0.16, 1.42 ± 0.30, 1.02 ± 0.31, 0.47 ± 0.27, respectively (F = 24.515, P < 0.01); FAS protein expression were 0.27 ± 0.08, 1.97 ± 0.47, 1.35 ± 0.30, 0.49 ± 0.12, respectively (F = 60.361, P < 0.01); PPARα protein expression were 2.03 ± 0.56, 0.41 ± 0.17, 0.81 ± 0.27, 0.66 ± 0.16, respectively (F = 37.97, P < 0.01).

CONCLUSIONSoy isoflavone can reduce the hepatic lipid deposition and increase antioxidant capacity, the mechanism may be related to inhibition of SREBP-1c and activation of PPARα expression in liver.

Animals ; Fatty Acid Synthases ; metabolism ; Fatty Liver ; metabolism ; Isoflavones ; pharmacology ; Lipid Metabolism ; Liver ; drug effects ; metabolism ; Male ; Non-alcoholic Fatty Liver Disease ; PPAR alpha ; metabolism ; Rats ; Rats, Sprague-Dawley ; Soybeans ; chemistry ; Sterol Regulatory Element Binding Protein 1 ; metabolism

OBJECTIVETo detect the expression of epidermal fatty acid-binding protein (E-FABP) and fatty acid synthase (FAS) in human breast cancer and identify the potential markers and therapeutic targets for breast cancer.

METHODSFAS and E-FABP expressions were detected in 76 patients with infiltrating ductal breast carcinoma using RT-PCR, immunohistochemistry and Western blotting. The possible associations of the expression of the two proteins with the major clinicopathological factors were analyzed.

RESULTSE-FABP and FAS expression levels were significantly decreased (P<0.05) in grade III as compared with grades I and II infiltrating ductal breast carcinoma. There was a positive correlation between E-FABP and FAS expressions, but their expressions were not correlated to the clinicopathological factors of the patients except for the tumor grades. High E-FABP expression level in grades I and II tumors were associated with an early increased responsiveness to FAS.

CONCLUSIONThe variation of the E-FABP and FAS expressions in the lesions is associated with increase of the risk for breast cancer, and the results of this study provide evidence for developing new molecular markers of high-risk lesions and identifying new the targets for breast cancer therapy.

Adult ; Aged ; Biomarkers, Tumor ; biosynthesis ; genetics ; Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; pathology ; Fatty Acid Synthases ; biosynthesis ; genetics ; Fatty Acid-Binding Proteins ; biosynthesis ; genetics ; Female ; Humans ; Immunohistochemistry ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo explore the effects of zinc-alpha2-glycoprotein (ZAG) on body weight and body fat in high-fat-diet (HFD)-induced obesity in mice and the possible mechanism.

METHODSThirty-six male mice were fed with standard food (SF) (n = 9) and HFD (n = 27), respectively. Five weeks later, 9 mice fed with HFD were subjected to ZAG expression plasmid DNA transfection by liposome transfection method, and another 9 mice to negative control plasmid transfection. Two weeks later, serum ZAG level in the mice was assayed by Western blot, and the effects of ZAG over-expression on body weight, body fat, serum biochemical indexes, and adipose tissue of obese mice were evaluated. The mRNA expressions of fatty acid synthase (FAS) and hormone-sensitive lipase (HSL) in liver tissue were determined by reverse transcription-polymerase chain reaction.

RESULTSSerum ZAG level significantly lowered in simple HFD-fed mice in comparison to SF-fed mice (0.51 +/- 0.10 AU vs. 0.75 +/- 0.07 AU, P < 0.01). Further statistical analysis demonstrated that ZAG level was negatively correlated with body weight (r = -0.56, P < 0.001), epididymal fat mass (r = -0.67, P < 0.001), percentage of epididymal fat (r = -0.65, P < 0.001), and increased weight (r = -0.57, P < 0.001) in simple SF- and HFD-fed mice. ZAG over-expression in obese mice reduced body weight and the percentage of epididymal fat. Furthermore, FAS mRNA expression decreased (P < 0.01) and HSL mRNA expression increased (P < 0.001) in the liver in ZAG over-expressing mice.

CONCLUSIONSZAG is closely related to obesity. Serum ZAG level is inversely correlated with body weight and percentage of body fat. The action of ZAG is associated with reduced FAS expression and increased HSL expression in the liver of obese mice.

Adipose Tissue ; metabolism ; Animals ; Fatty Acid Synthases ; genetics ; physiology ; Liver ; enzymology ; Male ; Mice ; Mice, Obese ; Seminal Plasma Proteins ; blood ; physiology ; Sterol Esterase ; genetics ; physiology ; Weight Loss