Expression Patterns Of Enzymes Research Articles

The expression pattern of proteolytic enzymes of cathepsin family in two important porcine skeletal muscles

The cathepsins, that belong to protease enzymes are present in lysosomes of various type of cells. They are considered to play an important role in many metabolic processes. In turn, polymorphisms of CTSD, CTSF and CTSZ genes (A70G, G22C, A37G, respectively) are associated with economically important pig traits. Therefore, the aim of our study was to estimate the expression patterns of CTS genes in two important porcine muscles: ham and loin. Our study was performed on 166 sows of four Polish pigs breeds differing in muscle-mass: Landrace, Large White, Pietrain and Puławska. As an internal control: OAZ1, RPS29 and RPL27 genes were used. In both muscles of interest the highest level of expression was established for CSTB inh gene and the lowest for CTSF gene (P<0.001). In the loin the highest transcript level for all investigated cathepsin genes was detected for Puławska breed (P<0.01), which is used to produce dry-cured ham. Therefore, cathepsin activity should be under control during dry-cured ham production this process. The wide spectrum of cathepsin enzymes make them important in proteolysis, therefore the studies aiming to find genetic markers in these proteins should be further carried out.

Soluble Ecto-5′-nucleotidase (5′-NT), Alkaline Phosphatase, and Adenosine Deaminase (ADA1) Activities in Neonatal Blood Favor Elevated Extracellular Adenosine

Extracellular adenosine, a key regulator of physiology and immune cell function that is found at elevated levels in neonatal blood, is generated by phosphohydrolysis of adenine nucleotides released from cells and catabolized by deamination to inosine. Generation of adenosine monophosphate (AMP) in blood is driven by cell-associated enzymes, whereas conversion of AMP to adenosine is largely mediated by soluble enzymes. The identities of the enzymes responsible for these activities in whole blood of neonates have been defined in this study and contrasted to adult blood. We demonstrate that soluble 5'-nucleotidase (5'-NT) and alkaline phosphatase (AP) mediate conversion of AMP to adenosine, whereas soluble adenosine deaminase (ADA) catabolizes adenosine to inosine. Newborn blood plasma demonstrates substantially higher adenosine-generating 5'-NT and AP activity and lower adenosine-metabolizing ADA activity than adult plasma. In addition to a role in soluble purine metabolism, abundant AP expressed on the surface of circulating neonatal neutrophils is the dominant AMPase on these cells. Plasma samples from infant observational cohorts reveal a relative plasma ADA deficiency at birth, followed by a gradual maturation of plasma ADA through infancy. The robust adenosine-generating capacity of neonates appears functionally relevant because supplementation with AMP inhibited whereas selective pharmacologic inhibition of 5'-NT enhanced Toll-like receptor-mediated TNF-α production in neonatal whole blood. Overall, we have characterized previously unrecognized age-dependent expression patterns of plasma purine-metabolizing enzymes that result in elevated plasma concentrations of anti-inflammatory adenosine in newborns. Targeted manipulation of purine-metabolizing enzymes may benefit this vulnerable population.

Effect of Maturity Stage on the Gene Expression of Antioxidative Enzymes in Cucumber (Cucumis sativus L.) Fruits Under Chilling Stress

The gene expression patterns of antioxidative enzymes in cucumber (Cucumis sativus L.) fruits at four different maturity stages, immature (3-8 d after anthesis (DAA), mature (9-16 DAA), breaker (17-22 DAA), and yellow (35-40 DAA), were determined before and after cold storage at 2°C for 9 d and after subsequent rewarming at 20°C for 2 d. The electrolyte leakage and malondialdehyde content in cucumber fruits were increased after cold storage and subsequent rewarming. Increased expressions of peroxidase, ascorbate peroxidase (APX), and monodehydroascorbate reductase after cold storage played an important role in cucumber fruits to cope with chilling injury. The elevated cyt-superoxide dismutase, catalase, APX and dehydroascorbate reductase after subsequent rewarming in cucumber fruits facilitated the recovery from chilling stress. The highest expression levels of all the seven antioxidative enzyme genes in yellow fruits might be responsible for the enhanced chilling tolerance. Cucumber fruits at earlier developmental stages was more susceptible to chilling stress than those at later stages. The relative higher gene expressions of antioxidative enzymes genes at earlier developmental stages may be the responses to the sever oxidative stress caused by chilling injury.

5-Aminolevulinic acid enhances photosynthetic gas exchange, chlorophyll fluorescence and antioxidant system in oilseed rape under drought stress

This study evaluates the role of exogenous foliar application of 5-aminolevulinic acid (ALA) on water relations, gas exchange, chlorophyll fluorescence, and the activities and gene expression patterns of antioxidant enzymes in leaves of oilseed rape under drought stress and recovery conditions. Seedlings at four-leaf stage were imposed to well-watered condition (80 % of water-holding capacity) or drought stress (40 % of water-holding capacity) and subsequently foliar sprayed with water or ALA (30 mg l−1). Drought suppressed the accumulation of plant biomass and decreased chlorophyll content and leaf water status (relative water content and water potential). The actual quantum yield of photosystem II and electron transport rates were hampered in parallel to net photosynthetic rate. However, drought stress induced the accumulation of malondialdehyde (MDA) and hydrogen peroxide, enhanced the activities of catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR) and superoxide dismutase and up-regulated the expression of APX and GR. After rehydration for 4 days, the growth of drought-treated seedlings was restored to normal level for most of the physiological parameters. Foliar application of ALA maintained relatively higher leaf water status and enhanced chlorophyll content, net photosynthetic rate, actual quantum yield of photosystem II, photochemical quenching, non-photochemical quenching and electron transport rates in stressed leaves. Exogenous ALA also alleviated the accumulation of MDA and hydrogen peroxide, increased the activities of antioxidant enzymes and enhanced the expression of CAT and POD in drought-treated plants. These results indicate that ALA may effectively protect rapeseed seedlings from damage induced by drought stress.

A Genome-Wide Screen Indicates Correlation between Differentiation and Expression of Metabolism Related Genes

Differentiated tissues may be considered as materials with distinct properties. The differentiation program of a given tissue ensures that it acquires material properties commensurate with its function. It may be hypothesized that some of these properties are acquired through production of tissue-specific metabolites synthesized by metabolic enzymes. To establish correlation between metabolism and organogenesis we have carried out a genome-wide expression study of metabolism related genes by RNA in-situ hybridization. 23% of the metabolism related genes studied are expressed in a tissue-restricted but not tissue-exclusive manner. We have conducted the screen on whole mount chicken (Gallus gallus) embryos from four distinct developmental stages to correlate dynamic changes in expression patterns of metabolic enzymes with spatio-temporally unique developmental events. Our data strongly suggests that unique combinations of metabolism related genes, and not specific metabolic pathways, are upregulated during differentiation. Further, expression of metabolism related genes in well established signaling centers that regulate different aspects of morphogenesis indicates developmental roles of some of the metabolism related genes. The database of tissue-restricted expression patterns of metabolism related genes, generated in this study, should serve as a resource for systematic identification of these genes with tissue-specific functions during development. Finally, comprehensive understanding of differentiation is not possible unless the downstream genes of a differentiation cascade are identified. We propose, metabolic enzymes constitute a significant portion of these downstream target genes. Thus our study should help elucidate different aspects of tissue differentiation.

Selective induction of glutathione S-transferases in round spermatids from the Brown-Norway rat by the chemotherapeutic regimen for testicular cancer

Chemotherapeutic drugs can affect DNA in male germ cells, thereby impacting on the integrity of the genome transmitted to offspring. Drug metabolizing enzymes can protect cells from xenobiotic insult. We analyzed the expression pattern of such enzymes in isolated round spermatids from rats exposed to drugs used to treat testicular cancer: bleomycin, etoposide, and cisplatin (BEP). The number of isozymes expressed and the overall relative expression values were highest for the glutathione S-transferases (GSTs). Moreover, BEP treatment significantly increased the expression of 8 GSTs and 3 aldehyde dehydrogenases. Increased expression of GST isozymes was confirmed by qRT-PCR and Western blot analysis. Although Gst genes can be targets for epigenetic modifications, promoter DNA methylation was not affected by BEP treatment. As GSTs are involved in drug resistance mechanisms, we hypothesize that BEP induction of GST expression may lead to the survival of damaged germ cells and the production of abnormal sperm.

Short-Cut Pathway to Synthesize Cellulose of Encysting Acanthamoeba

The mature cyst of Acanthamoeba is highly resistant to various antibiotics and therapeutic agents. Cyst wall of Acanthamoeba are composed of cellulose, acid-resistant proteins, lipids, and unidentified materials. Because cellulose is one of the primary components of the inner cyst wall, cellulose synthesis is essential to the process of cyst formation in Acanthamoeba. In this study, we hypothesized the key and short-step process in synthesis of cellulose from glycogen in encysting Acanthamoeba castellanii, and confirmed it by comparing the expression pattern of enzymes involving glycogenolysis and cellulose synthesis. The genes of 3 enzymes, glycogen phosphorylase, UDP-glucose pyrophosphorylase, and cellulose synthase, which are involved in the cellulose synthesis, were expressed high at the 1st and 2nd day of encystation. However, the phosphoglucomutase that facilitates the interconversion of glucose 1-phosphate and glucose 6-phosphate expressed low during encystation. This report identified the short-cut pathway of cellulose synthesis required for construction of the cyst wall during the encystation process in Acanthamoeba. This study provides important information to understand cyst wall formation in encysting Acanthamoeba.

Lipid mediator metabolic profiling demonstrates differences in eicosanoid patterns in two phenotypically distinct mast cell populations

Mast cells are inflammatory cells that play key roles in health and disease. They are distributed in all tissues and appear in two main phenotypes, connective tissue and mucosal mast cells, with differing capacities to release inflammatory mediators. A metabolic profiling approach was used to obtain a more comprehensive understanding of the ability of mast cell phenotypes to produce eicosanoids and other lipid mediators. A total of 90 lipid mediators (oxylipins) were characterized using liquid chromatography-tandem mass spectrometry (LC-MS/MS), representing the cyclooxygenase (COX), lipoxygenase (LO), and cytochrome P450 (CYP) metabolic pathways. In vitro-derived murine mucosal-like mast cells (MLMC) and connective tissue-like mast cells (CTLMC) exhibited distinct mRNA expression patterns of enzymes involved in oxylipin biosynthesis. Oxylipins produced by 5-LO and COX pathways were the predominant species in both phenotypes, with 5-LO products constituting 90 ± 2% of the CTLMCs compared with 58 ± 8% in the MLMCs. Multivariate analyses demonstrated that CTLMCs and MLMCs secrete differing oxylipin profiles at baseline and following calcium ionophore stimulation, evidencing specificity in both a time- and biosynthetic pathway-dependent manner. In addition to the COX-regulated prostaglandin PGD(2) and 5-LO-regulated cysteinyl-leukotrienes (e.g., LTC(4)), several other mediators evidenced phenotype-specificity, which may have biological implications in mast cell-mediated regulation of inflammatory responses.

Chondroitin Sulfate Is a Crucial Determinant for Skeletal Muscle Development/Regeneration and Improvement of Muscular Dystrophies

Skeletal muscle formation and regeneration require myoblast fusion to form multinucleated myotubes or myofibers, yet their molecular regulation remains incompletely understood. We show here that the levels of extra- and/or pericellular chondroitin sulfate (CS) chains in differentiating C2C12 myoblast culture are dramatically diminished at the stage of extensive syncytial myotube formation. Forced down-regulation of CS, but not of hyaluronan, levels enhanced myogenic differentiation in vitro. This characteristic CS reduction seems to occur through a cell-autonomous mechanism that involves HYAL1, a known catabolic enzyme for hyaluronan and CS. In vivo injection of a bacterial CS-degrading enzyme boosted myofiber regeneration in a mouse cardiotoxin-induced injury model and ameliorated dystrophic pathology in mdx muscles. Our data suggest that the control of CS abundance is a promising new therapeutic approach for the treatment of skeletal muscle injury and progressive muscular dystrophies.

Tungstate Reduces the Expression of Gluconeogenic Enzymes in STZ Rats

AimsOral administration of sodium tungstate has shown hyperglycemia-reducing activity in several animal models of diabetes. We present new insights into the mechanism of action of tungstate.MethodsWe studied protein expression and phosphorylation in the liver of STZ rats, a type I diabetes model, treated with sodium tungstate in the drinking water (2 mg/ml) and in primary cultured-hepatocytes, through Western blot and Real Time PCR analysis.ResultsTungstate treatment reduces the expression of gluconeogenic enzymes (PEPCK, G6Pase, and FBPase) and also regulates transcription factors accountable for the control of hepatic metabolism (c-jun, c-fos and PGC1α). Moreover, ERK, p90rsk and GSK3, upstream kinases regulating the expression of c-jun and c-fos, are phosphorylated in response to tungstate. Interestingly, PKB/Akt phosphorylation is not altered by the treatment. Several of these observations were reproduced in isolated rat hepatocytes cultured in the absence of insulin, thereby indicating that those effects of tungstate are insulin-independent.ConclusionsHere we show that treatment with tungstate restores the phosphorylation state of various signaling proteins and changes the expression pattern of metabolic enzymes.

BMP-2 and BMP-7: differential regulation of chondrogenic differentiation

BMP-2 and BMP-7: differential regulation of chondrogenic differentiation

Differential Expression of Metabolic Genes in Tumor and Stromal Components of Primary and Metastatic Loci in Pancreatic Adenocarcinoma

BackgroundPancreatic cancer is the fourth leading cause of cancer related deaths in the United States with a five-year survival rate of 6%. It is characterized by extremely aggressive tumor growth rate and high incidence of metastasis. One of the most common and profound biochemical phenotypes of animal and human cancer cells is their ability to metabolize glucose at high rates, even under aerobic conditions. However, the contribution of metabolic interrelationships between tumor cells and cells of the surrounding microenvironment to the progression of cancer is not well understood. We evaluated differential expression of metabolic genes and, hence, metabolic pathways in primary tumor and metastases of patients with pancreatic adenocarcinoma.Methods and FindingsWe analyzed the metabolic gene (those involved in glycolysis, tri-carboxylic acid pathway, pentose-phosphate pathway and fatty acid metabolism) expression profiles of primary and metastatic lesions from pancreatic cancer patients by gene expression arrays. We observed two principal results: genes that were upregulated in primary and most of the metastatic lesions; and genes that were upregulated only in specific metastatic lesions in a site-specific manner. Immunohistochemical (IHC) analyses of several metabolic gene products confirmed the gene expression patterns at the protein level. The IHC analyses also revealed differential tumor and stromal expression patterns of metabolic enzymes that were correlated with the metastasis sites.ConclusionsHere, we present the first comprehensive studies that establish differential metabolic status of tumor and stromal components and elevation of aerobic glycolysis gene expression in pancreatic cancer.

Sialome analysis of the cephalochordate Branchiostoma belcheri, a key organism for vertebrate evolution

Sialic acid, a common terminal substitution of glycoconjugates, has been so far consistently identified in all vertebrates as well as in a growing number of bacterial species. It is assumed to be widely distributed among animal species of the deuterostome phylum, based on its identification in few echinoderm and all vertebrate species. However, whole sections of deuterostome, especially those intermediate species between invertebrates and vertebrates including cephalochordates, urochordates and hemichordates, are still unexplored in term of sialylation capacities. The discovery of functional sialic acid machinery in some of these species may shed new light onto the evolution of glycosylation capacities in deuterostome lineage. In a first approach, we investigated the sialylation pattern of a cephalocordate species, Branchiostoma belcheri, which occupies a strategic phylogenetic position to understand the transition of invertebrates toward vertebrates. Structural analysis of B. belcheri glycoconjugates established that this organism synthesizes large quantities of various sialic acids, some of which present rare or novel structures such as methylated sialic acids. These sialic acids were shown to be mainly associated with mono- and disialylated core 1-type O-glycans. Moreover, screening of the animal organs revealed the existence of exquisite tissue specificity in the distribution of sialic acids. Description of sialylation profiles was then correlated with the expression patterns of key enzymes involved in the biosynthesis of major forms of sialic acids, which provides the first complete overview of the sialylation patterns in cephalochordates.

Abstract A46: Glycolysis and oxidative phosphorylation in cervical cancer: Correlation with FDG PET/CT and its prognostic implication

Abstract Purpose: The usefulness of 2-[18F]-fluoro-2-deoxy-D-glucose ([18F]FDG) positron emission tomography/computed tomography (PET/CT) can help predict the presence and the extent of malignant cells. The aims of current study were to investigate the expression patterns of genes or enzymes in glucose metabolism, and to determine whether the FDG PET/CT finding correlates with these proteomic markers in surgical specimens. Patients and Methods: Fifty patients with uterine cervical cancer who underwent [18F]FDG PET/CT before treatment were included in this study. Tumor sections were stained by immunohistochemistry for glucose transporter 1 (GLUT1), glucose transporter 3 (GLUT3), hypoxia-inducible factor-1α (HIF-1α), hexokinase 2 (HK2), glycolysis markers (pyruvate kinase M2 (PKM2), phosphofructokinase (PFK), glycoaldehyde-phosphate dehydrogenase (GAPDH), pyruvate dehydrogenase (PDH)), electron transporter chain marker (β-F1-ATPase), mitochondrial transcription factor A (TFAM), oncogene (c-Myc), and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α). Results: In vivo glucose uptake of primary cervical cancer (R = −0.298, P = 0.036) and metastatic lymph nodes (R = −0.298, P = 0.037), as assessed by FDG uptake inversely correlates with the mitochondrial biogenesis determined by immunohistochemical staining of PGC-1α. Cellular GLUT1 expression was also inversely correlated with the expression of PGC-1α (R = −0.413, P = 0.003). Moreover, the level of mitochondrial oxidative phosphorylation expressed by β–F1-ATPase inversely correlates with that of PFK (r = −0.337, P = 0.021). Expression of PGC-1α was lower in squamous cell carcinoma than that of adenocarcinoma (P &amp;lt; 0.001), however, GLUT1 expression was higher in squamous cell carcinoma (P = 0.003). Of interest, HK2 expression in primary tumor was significantly lower in patients with lymph node metastasis (P = 0.003). Conclusion: The results highlight the relevance of the alteration of the glucose metabolism by aerobic glycolysis in cervical cancer, and affording a mechanistic explanation for FDG PET/CT imaging. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A46.

Genotype-dependent responses of wheat (Triticum aestivum L.) seedlings to drought, UV-B radiation and their combined stresses

Experiments were conducted under controlled conditions to investigate the growth and physiological - biochemical responses of wheat ( Triticum aestivum L.) seedlings to UV-B, drought, and their combined stresses. Both UV-B and drought treatments retarded seedling growth with UV-B having worse impact on wheat plants. Chlorophyll content and relative water content (RWC) in leaves, as stress makers, were significantly affected by UV-B and drought, respectively. The increased rate and amount of H 2 O 2 were stress-different and genotype-dependent. Likely, the temporary expression patterns of antioxidant enzymes (superoxide dismutase, SOD, and catalase, CAT) and compounds (proline, and ascobate acid, AsA) exhibited differences under the tested stressful conditions in the two genotypes, indicating that they play significant roles in plant responses to these stresses. Pre-application of either stress reduced the damage caused by subsequent application of the other stress, and this induced defense was greater by UV-B than by drought. Compared to the stress applied separately, the combined application of drought and UV-B at the same time resulted in more adverse effects on the wheat seedlings of the susceptible variety, and more positive effects on the tolerant wheat genotype. These results provide novel insights into understanding the cellular and molecular mechanisms responsible for plant tolerance to various stresses and their interactions. Key words : Wheat (Triticum aestivum L.), UV-B radiation, drought, combined stress, antioxidant system, genotype.

Expression pattern of enzymes related to juvenile hormone metabolism in the silkworm, Bombyx mori L.

The physiological balance of juvenile hormone (JH) in insects depends on its biosynthesis and degradation pathway. Three key enzymes namely, juvenile hormone esterase (JHE), juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone diol kinase (JHDK) are required for degradation in insects. Our present results showed that JHE and JHEH exhibited expression in almost all the tissues. This indicated that JHE and JHEH might degrade JH simultaneously. In addition, the highest levels of JHDK were observed in the midgut, with trace level being found in the malpighian tubule and haemocytes. Since the midgut is a digestive organ and not a JH target, it was hypothesized that both JHE and JHEH hydrolyzed JH to JH diol (JHd) which was then transported to midgut and hydrolyzed further by JHDK, to be finally excretedout of the body. Also the expression studies on JH degradation enzymes in different tissues and stages indicated that the activities of the three enzymes are specific and coincident with the JH functions in silkworm, Bombyx mori L.

211 Expression of No Synthases and Redox Enzymes in Umbilical Arteries From Newborns Born Small, Appropriate, and Large for Gestational Age

Background: Modified expression of NO synthases and prooxidative and antioxidative enzymes accompany endothelial dysfunction, the first stage of atherosclerosis. Humans born small (SGA) and large (LGA) for gestational age are at higher risk to develop atherosclerosis later in life than humans born appropriate (AGA) for gestational age. We hypothesized that indicators of endothelial dysfunction could be detectable already at birth.Aims: To find out whether the expression patterns of NO synthases (eNOS, iNOS, nNOS), of prooxidative enzymes (components of NADPH oxidases, NOX1, NOX2, p22phox, p47phox), and of antioxidative enzymes (superoxide dismutase 1, 2, and 3, catalase, gluthation peroxidase 1) in umbilical arteries differ between SGA, LGA, and AGA newborns.Methods: Protein expression was determined by Western blotting in homogenisated samples of umbilical arteries of healthy term SGA, LGA, and AGA newborns (n = 12 in each group). Optical densities of proteins of interest were normalized in relation to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a standard.Results: Protein expression of NO synthases, of prooxidative enzymes, and of antioxidative enzymes was similar in the three groups.Conclusions: Protein expression patterns of enzymes involved in endothelial function and redox status did not differ in umbilical arteries of term SGA, LGA, and AGA newborns. Thus, indicators of arterial endothelial dysfunction in terms of aberrant enzyme expression in SGA or LGA newborns vs. AGA newborns were not present at birth.

Food utilization of two pelagic crustaceans in the Greenland Sea: Meganyctiphanes norvegica (Euphausiacea) and Hymenodora glacialis (Decapoda, Caridea)

Large pelagic crustaceans from Greenland Sea waters, the northern krill Meganyc- tiphanes norvegica (Euphausiacea) and the decapod shrimp Hymenodora glacialis (Caridea), were captured in depths down to 1500 m and studied with respect to their physiological food utilization abilities. Both species showed distinct differences in the amount of total lipids (TLs), lipid class and fatty acid (FA) compositions as well as proteolytic enzyme activities. In M. norvegica, the overall amount of TLs and storage lipids was much lower than in H. glacialis, and triacylglycerols formed the major lipid fraction with a mean of 48% TLs. Major FAs comprised the trophic markers 20:1(n-9) and 22:1(n-11), indicating the ingestion of calanid copepods. Additionally, the FAs 22:6(n-3), 18:1(n-9) and 16:0 prevailed. In H. glacialis, TLs (mean = 44% dry mass) were about twice as high as in krill, with wax esters comprising up to 89% TLs. H. glacialis seems to accumulate these lipids as energy reserves to survive periods of food limitation. Moreover, high lipid levels, particularly wax esters, also help to maintain neutral buoyancy. The major FA in H. glacialis was 18:1(n-9); other dominant FAs were 20:1(n-9) and 22:1(n-11), typical of calanid copepods, as well as the diatom trophic marker 16:1(n-7). Both species showed omnivorous feeding behaviour with a strong tendency towards carni- vory. Total proteolytic activities in midgut gland tissue were higher in M. norvegica than in H. glacialis. In M. norvegica, proteinases were dominated by serine proteinases, whereas cysteine proteinases formed the major group in H. glacialis. High proteolytic activity in M. norvegica indicates a high digestive potential for proteins and efficient utilization of prey. The presence of different pro- teinase classes in both species may be due to different group-specific enzyme expression patterns between euphausiids and caridean decapods. Both species follow highly deviating life strategies, as reflected by their specific lipid and enzymatic characteristics.

Expression of Long-chain Fatty Acyl-CoA Synthetase 4 in Breast and Prostate Cancers Is Associated with Sex Steroid Hormone Receptor Negativity

Previous studies have shown that key enzymes involved in lipid metabolic pathways are differentially expressed in normal compared with tumor tissues. However, the precise role played by dysregulated expression of lipid metabolic enzymes and altered lipid homeostasis in carcinogenesis remains to be established. Fatty acid synthase is overexpressed in a variety of cancers, including breast and prostate. The purpose of the present study was to examine the expression patterns of additional lipid metabolic enzymes in human breast and prostate cancers. This was accomplished by analysis of published expression databases, with confirmation by immunoblot assays. Our results indicate that the fatty acid-activating enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4), is differentially expressed in human breast cancer as a function of estrogen receptor alpha (ER) status. In 10 separate studies, ACSL4 messenger RNA (mRNA) was overexpressed in ER-negative breast tumors. Of 50 breast cancer cell lines examined, 17 (89%) of 19 ER-positive lines were negative for ACSL4 mRNA expression and 20 (65%) of 31 ER-negative lines expressed ACSL4 mRNA. The inverse relationship between ER expression and ACSL4 expression was also observed for androgen receptor status in both breast and prostate cancers. Furthermore, loss of steroid hormone sensitivity, such as that observed in Raf1-transfected MCF-7 cells and LNCaP-AI cells, was associated with induction of ACSL4 expression. Ablation of ACSL4 expression inMDA-MB-231 breast cancer cells had no effect on cell proliferation; however, sensitivity to the cytotoxic effects of triacsin C was increased three-fold in the cells lacking ACSL4.

Expression of methylation pathway enzymes in bovine oocytes and preimplantation embryos

The methylation pathway, which consists of two metabolic cycles of nutrients, i.e., the methionine and folate cycles, generates S-adenosylmethionine, the methyl donor for the methylation of DNA and histones. Using reverse transcription-polymerase chain reaction, we examined the gene expression patterns of the methylation pathway enzymes during bovine oocyte maturation and preimplantation embryonic development up to the blastocyst stage. Bovine oocytes were demonstrated to have the mRNA of all methylation pathway enzymes examined, namely, methionine adenosyltransferase 1A (MAT1A), MAT2A, MAT2B, S-adenosylhomocysteine hydrolase (AHCY), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), betaine-homocysteine methyltransferase (BHMT), serine hydroxymethyltransferase 1 (SHMT1), SHMT2, and 5,10-methylenetetrahydrofolate reductase (MTHFR). All the transcripts were consistently expressed throughout all developmental stages, except for MAT1A, which was not detected from the 8-cell stage onward and BHMT, which was not detected in the 8-cell stage. Immunofluorescence analysis of MAT1A protein revealed the relatively higher expression in oocytes and early cleavage stage embryos up to the 8-cell stage compared with the morula and blastocyst stage. Further, to investigate the effects of methylation pathway disruption during the earliest stages of embryonic development, the effects of exogenous homocysteine on preimplantation development and DNA methylation of bovine embryos were investigated in vitro. As results, high concentrations of homocysteine induced hypermethylation of genomic DNA as well as developmental retardation in bovine embryos. These results provide a new insight into nutrient-sensitive epigenetic regulation and perturbation at the earliest stage of our life.