Effects of caffeic acid phenethyl ester (CAPE) on angiogenesis,apoptosis and oxidatıve stress ın various cancer cell lines

ABSTRACT

Cancer is a common cause of death worldwide. Approximately 80% of cancer patients use complementary or alternative medicines for treatment. Caffeic acid phenethyl ester (CAPE), the main active component of propolis, exhibits cytotoxic, antiproliferative and anti-cancer effects. Despite its anticancer effects CAPE exhibits no known harmful effects toward normal cells. We investigated the effects of CAPE on angiogenesis, apoptosis and oxidative stress using MDA MB-231, N2a and COLO 320 cell lines and CAPE treatments at 24 and 48 h. A two dimensional cell culture system was used and the findings were evaluated by an indirect immunohistochemical method and H-scores were calculated. CAPE was effective for all three cancer cell lines. After 24 and 48 h, we found a significant decrease in live cells and increased stress in the cells based on e-NOS and i-NOS levels.     

Activation of neutral-sphingomyelinase,MAPKs, and p75 NTR-mediating caffeic acid phenethyl ester–induced apoptosis in C6 glioma cells

Background

Caffeic acid phenethyl ester (CAPE), a component of propolis, is reported to possess anti-inflammatory, anti-bacterial, anti-viral, and anti-tumor activities. Previously, our laboratory demonstrated the in vitro and in vivo bioactivity of CAPE and addressed the role of p53 and the p38 mitogen-activated protein kinase (MAPK) pathway in regulating CAPE-induced apoptosis in C6 glioma cells.

Results

C6 cancer cell lines were exposed to doses of CAPE; DNA fragmentation and MAPKs and NGF/P75NTR levels were then determined. SMase activity and ceramide content measurement as well as western blotting analyses were performed to clarify molecular changes. The present study showed that CAPE activated neutral sphingomyelinase (N-SMase), which led to the ceramide-mediated activation of MAPKs, including extracellular signal-regulated kinase (ERK), Jun N-terminus kinase (JNK), and p38 MAPK. In addition, CAPE increased the expression of nerve growth factor (NGF) and p75 neurotrophin receptor (p75NTR). The addition of an N-SMase inhibitor, GW4869, established that NGF/p75NTR was the downstream target of N-SMase/ceramide. Pretreatment with MAPK inhibitors demonstrated that MEK/ERK and JNK acted upstream and downstream, respectively, of NGF/p75NTR. Additionally, CAPE-induced caspase 3 activation and poly [ADP-ribose] polymerase cleavage were reduced by pretreatment with MAPK inhibitors, a p75NTR peptide antagonist, or GW4869.

Conclusions

Taken together, N-SMase activation played a pivotal role in CAPE-induced apoptosis by activation of the p38 MAPK pathway and NGF/p75NTR may explain a new role of CAPE induced apoptosis in C6 glioma.     

Short-term parenteral application of α-tocopherol leads to increased concentration in plasma and tissues of the rat

Numerous studies suggest that supplemental vitamin E prior to or during vast surgeries might diminish or even prevent ischemia/reperfusion-induced injuries. In the present placebo-controlled study male Sprague-Dawley rats were supplemented parenterally or orally with α-tocopherol for three consecutive days. The applied amount of α-tocopherol was 2.3 μmol per day for oral and 1.2 μmol per day for parenteral supplementation. The enrichment of vitamin E concentrations in plasma and tissue samples (aortic endothelium, liver, and lung) was determined by HPLC. The vitamin E level was elevated following intravenous supplementation in plasma (21.4±1.9 μmol/L vs. 10.2±1.7 μmol/L in parenteral control group), in aortic endothelium (1.1±0.2 pmol/mm² vs. 0.5±0.1 pmol/mm²) and in liver and lung (41.3±7.5 pmol/mg vs. 22.9±6.5 pmol/mg and 75.6±13.6 pmol/mg vs. 51.7±5.9 pmol/mg, respectively). Oral supplementation for three days also led to an increased level in liver (38.2±7.7 pmol/mg vs. 22.9±6.6 pmol/mg in oral control group) and in lung (67.8±5.7 pmol/mg vs. 51.7±9.3 pmol/mg) but not in aortic endothelium or plasma (0.8±0.3 pmol/mm² vs. 0.6±0.3 pmol/mm² and 12.0±2.2 μmol/L vs. 10.7±2.6 μol/L.)     

Loss of cadherin-binding proteins β-catenin and plakoglobin in the heart leads to gap junction remodeling and arrhythmogenesis

Arrhythmic right ventricular cardiomyopathy (ARVC) is a hereditary heart muscle disease that causes sudden cardiac death (SCD) in young people. Almost half of ARVC patients have a mutation in genes encoding cell adhesion proteins of the desmosome, including plakoglobin (JUP). We previously reported that cardiac tissue-specific plakoglobin (PG) knockout (PG CKO) mice have no apparent conduction abnormality and survive longer than expected. Importantly, the PG homolog, β-catenin (CTNNB1), showed increased association with the gap junction protein connexin43 (Cx43) in PG CKO hearts. To determine whether β-catenin is required to maintain cardiac conduction in the absence of PG, we generated mice lacking both PG and β-catenin specifically in the heart (i.e., double knockout [DKO]). The DKO mice exhibited cardiomyopathy, fibrous tissue replacement, and conduction abnormalities resulting in SCD. Loss of the cadherin linker proteins resulted in dissolution of the intercalated disc (ICD) structure. Moreover, Cx43-containing gap junction plaques were reduced at the ICD, consistent with the arrhythmogenicity of the DKO hearts. Finally, ambulatory electrocardiogram monitoring captured the abrupt onset of spontaneous lethal ventricular arrhythmia in the DKO mice. In conclusion, these studies demonstrate that the N-cadherin-binding partners, PG and β-catenin, are indispensable for maintaining mechanoelectrical coupling in the heart.     

Caffeic acid phenethyl ester and its related compounds limit the functional alterations of the isolated mouse brain and liver mitochondria submitted to in vitro anoxia–reoxygenation: Relationship to their antioxidant activities

It is an important therapeutic strategy to protect mitochondria from oxidative stress, especially during ischemia–reperfusion. In the present study, an attempt has been made to evaluate the protective effects of caffeic acid phenethyl ester (CAPE) and its related phenolic compounds on mouse brain and liver mitochondria injury induced by in vitro anoxia–reoxygenation. Added before anoxia or reoxygenation, CAPE markedly protected coupled respiration with the decrease in state 4 and the increases in state 3, respiratory control ratio (RCR) and ADP/O ratio in a concentration-dependent manner. CAPE effectively protected mitochondria by inhibiting the mitochondrial membranes fluidity decrease, the lipoperoxidation and the protein carbonylation increase, which indicated its protective action against the mitochondrial oxidative damage. Meanwhile, CAPE blocked the enhanced release of cardiolipin (CL) and cytochrome c (Cyt c). The related phenolic compounds like caffeic acid (CA), ferulic acid (FA) and ethyl ferulate (EF) also had different-degree protective effects. CAPE and CA were more potent than FA and EF. Their structural differences played the key role in their activity levels. These results suggest that CAPE and its related phenolic compounds protect mitochondria mainly correlated to their antioxidative activities and may be of interest for the prevention and therapy of ischemia–reperfusion injuries.     

A comparison of the in vitro binding of α-tocopherol to microsomes of lung,liver, heart and brain of the rat

The in vitro binding of α-tocopherol to microsomes of lung, liver, heart and brain of the rat was studied with the insoluble tocopherol ligand presented as a complex with bovine serum albumin. Under these conditions, all microsomes showed nonsaturable binding of α-tocopherol and the amount bound to microsomes was linearly proportional to the concentration of albumin-complexed tocopherol. Increasing the amount of α-tocopherol bound to microsomes in this manner reduced the extent of lipid peroxidation induced by added ferrous iron. The apparent affinities of the microsomes for α-tocopherol, as indicated by the amount bound at a given concentration of albumin-complexed tocopherol, decreased in the order brain > liver ≈ heart > lung. The differences in affinity did not correlate with total fatty acid content (r = − 0.39), total unsaturated fatty acid content (r = − 0.26), or with the content of fatty acids containing two or more double bonds (r = − 0.01). A high positive correlation was found with the content of fatty acids containing three or more double bonds (r = + 0.96). Since lung microsomes contain approx. 6-times the tocopherol levels of liver and brain and about twice that of heart microsomes, these results show that the in vivo levels of microsomal tocopherol do not reflect microsomal affinity for this biological antioxidant.     

Dietary supplementation with (R)-α-lipoic acid reverses the age-related accumulation of iron and depletion of antioxidants in the rat cerebral cortex

Abstract

Accumulation of divalent metal ions (e.g. iron and copper) has been proposed to contribute to heightened oxidative stress evident in aging and neurodegenerative disorders. To understand the extent of iron accumulation and its effect on antioxidant status, we monitored iron content in the cerebral cortex of F344 rats by inductively coupled plasma atomic emission spectrometry (ICP-AES) and found that the cerebral iron levels in 24–28-month-old rats were increased by 80% (p<0.01) relative to 3-month-old rats. Iron accumulation correlated with a decline in glutathione (GSH) and the GSH/GSSG ratio, indicating that iron accumulation altered antioxidant capacity and thiol redox state in aged animals. Because (R)-α-Lipoic acid (LA) is a potent chelator of divalent metal ions in vitro and also regenerates other antioxidants, we monitored whether feeding LA (0.2% [w/w]; 2 weeks) could lower cortical iron and improve antioxidant status. Results show that cerebral iron levels in old LA-fed animals were lower when compared to controls and were similar to levels seen in young rats. Antioxidant status and thiol redox state also improved markedly in old LA-fed rats versus controls. These results thus show that LA supplementation may be a means to modulate the age-related accumulation of cortical iron content, thereby lowering oxidative stress associated with aging.     

Is rat an appropriate animal model to study the involvement of D-serine catabolism in schizophrenia? Insights from characterization of D-amino acid oxidase

D-Amino acid oxidase (DAAO; EC1.4.3.3) has been proposed to play a main role in the degradation of D-serine, an allosteric activator of the N-methyl-D-aspartate-type glutamate receptor in the human brain, and to be associated with the onset of schizophrenia. To prevent excessive D-serine degradation, novel drugs for schizophrenia treatment based on DAAO inhibition were designed and tested on rats. However, the properties of rat DAAO are unknown and various in vivo trials have demonstrated the effects of DAAO inhibitors on d-serine concentration in rats. In the present study, rat DAAO was efficiently expressed in Escherichia coli. The recombinant enzyme was purified as an active, 40 kDa monomeric flavoenzyme showing the basic properties of the dehydrogenase-oxidase class of flavoproteins. Rat DAAO differs significantly from the human counterpart because: (a) it possesses a different substrate specificity; (b) it shows a lower kinetic efficiency, mainly as a result of a low substrate affinity; (c) it differs in affinity for the binding of classical inhibitors; (d) it is a stable monomer in the absence of an active site ligand; and (e) it interacts with the mammalian protein modulator pLG72 yielding a ~100 kDa complex in addition to the ~200 kDa one, as formed by the human DAAO. Furthermore, the concentration of endogenous D-serine in U87 glioblastoma cells was not affected by transfection with rat DAAO, whereas it was significantly decreased when expressing the human homologue. These results raise doubt on the use of the rat as a model system for testing new drugs against schizophrenia and indicate a different physiological function of DAAO in rodents and humans.     

Abscisic acid enhances tolerance to spring freeze stress and regulates the expression of ascorbate–glutathione biosynthesis-related genes and stress-responsive genes in common wheat

Molecular Breeding - Hybrid sterility is a major obstacle to the development of superior inter-subspecific hybrids between indica and japonica subspecies of Asian-cultivated rice. To overcome...     

Modeling the acid–base properties of glutathione in different ionic media, with particular reference to natural waters and biological fluids

The acid-base properties of γ-L-glutamyl-L-cysteinyl-glycine (glutathione, GSH) were determined by potentiometry (ISE-H(+), glass electrode) in pure NaI((aq)) and in NaCl((aq))/MgCl(2(aq)), and NaCl((aq))/CaCl(2(aq)) mixtures, at T = 298.15 K and different ionic strengths (up to I(c) ~ 5.0 mol L(-1)). In addition, the activity coefficients of glutathione were also determined by the distribution method at the same temperature in various ionic media (LiCl((aq)), NaCl((aq)), KCl((aq)), CsCl((aq)), MgCl(2(aq)), CaCl(2(aq)), NaI((aq))). The results obtained were also used to calculate the Specific ion Interaction Theory (SIT) and Pitzer coefficients for the dependence on medium and ionic strength of glutathione species, as well as the formation constants of weak Mg(j)H( i )(GSH)((i+2j-3)) and Ca(j)H(i)(GSH)((i+2j-3)) complexes. Direct calorimetric titrations were also carried out in pure NaCl((aq)) and in NaCl((aq))/CaCl(2(aq)) mixtures at different ionic strengths (0.25 ≤ I (c )/mol L(-1) ≤ 5.0) in order to determine the enthalpy changes for the protonation and complex formation equilibria in these media at T = 298.15 K. Results obtained are useful for the definition of glutathione speciation in any aqueous media containing the main cations of natural waters and biological fluids, such as Na(+), K(+), Mg(2+), and Ca(2+). Finally, this kind of systematic studies, where a series of ionic media (e.g., all alkali metal chlorides) is taken into account in the determination of various thermodynamic parameters, is useful for the definition of some trends in the thermodynamic behavior of glutathione in aqueous solution.     

Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. Formation and properties of an aminoacyl-transfer ribonucleic acid–transferase I complex

1. Transferase I of rat liver binds aminoacyl-tRNA to form a relatively stable complex, which is retained on cellulose nitrate filters. This reaction proceeds at both 0 degrees C and 37 degrees C and is inhibited by GTP. The resulting product is stabilized by GTP and Mg(2+). 2. Only very low quantities of deacylated tRNA are bound by transferase I. 3. Methods are described for the preparative isolation of the transferase I-aminoacyl-tRNA complex from incubation mixtures by using ion-exchange procedures. 4. The transferase I-aminoacyl-tRNA complex becomes readily bound to ribosomes. The presence of Mg(2+) is essential for the binding. GTP stimulates this reaction but is not absolutely required. 5. It is concluded that the formation of the transferase I-aminoacyl-tRNA complex may be the primary reaction in the binding of aminoacyl-tRNA to mammalian ribosomes and that, unlike in bacterial systems, GTP is not absolutely required for this step.     

Disease-associated single amino acid mutation in the calf-1 domain of integrin α3 leads to defects in its processing and cell surface expression

Integrin α3β1, a receptor for laminins, is involved in the structural and functional organization of epithelial organs, including the lung, kidney, and skin. Recently, a missense mutation that causes substitution of Arg628 with Pro (R628P) in the calf-1 domain of human α3 was shown to be associated with disorders of the lung, kidney, and skin. Here, we found that the R628P mutation leads to aberrations in the posttranslational processing of α3. Specifically, α3 with the R628P mutation showed hardly any cleavage at the calf-2 domain, which usually occurs in the Golgi apparatus during the delivery of de novo-synthesized α3. The mutant α3 retained the ability to associate with integrin β1, but not with the tetraspanin CD151, and the bound β1 was a partially glycosylated immature form, the maturation of which also takes place in the Golgi apparatus. Furthermore, the cell surface expression of the mutant protein was markedly reduced. These results suggest that the R628P mutation leads to a deficit in the transport of α3β1 from the ER to the Golgi apparatus. When Arg628 was mutated to Gln or Glu, instead of Pro, the resulting mutants did not display aberrations in processing or CD151 binding, indicating that the presence of Pro, rather than the absence of Arg, at amino acid residue 628 of α3 is important for the abnormalities in the R628P mutant. In support of this notion, a homology modeling analysis of the calf-1 domain of α3 showed that replacement with Pro, but not with Gln or Glu, caused partial disruption of the β-sheet structures. Furthermore, the ER-associated degradation of the R628P mutant was not enhanced compared with that of the wild-type protein, suggesting that the deficits in the posttranslational processing and cell surface expression of the R628P mutant are independent of the ER-associated degradation, but arise from the defect in its export from the ER. We conclude that the calf-1 domain is required for the transport of α3 from the ER to the Golgi apparatus to maintain the integrity of epithelial tissues, and hence the impairment of the calf-1 domain by the R628P mutation leads to severe diseases of the kidneys, lungs, and skin.     

Variation of the by-product spectrum during α-ketoglutaric acid production from raw glycerol by overexpression of fumarase and pyruvate carboxylase genes in Yarrowia lipolytica

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric, isocitric, pyruvic (PA), and α-ketoglutaric (KGA) acids, triggered by growth limitation and excess of carbon source. This is leading to an increased interest in this non-conventional yeast for biotechnological applications. To improve the KGA production by Y. lipolytica for an industrial application, it is necessary to reduce the amounts of by-products, e.g., fumarate (FU) and PA, because production of by-products is a main disadvantage of the KGA production by this yeast. We have examined whether the concentration of secreted organic acids (main product KGA and PA as major by-product and FU, malate (MA), and succinate (SU) as minor by-products) can be influenced by a gene-dose-dependent overexpression of fumarase (FUM) or pyruvate carboxylase (PYC) genes under KGA production conditions. Recombinant Y. lipolytica strains were constructed, which harbor multiple copies of the respective FUM1, PYC1 or FUM1, and PYC1 genes. Overexpression of the genes FUM1 and PYC1 resulted in strongly increased specific enzyme activities during cultivation of these strains on raw glycerol as carbon source in bioreactors. The recombinant Y. lipolytica strains showed different product selectivity of the secreted organic acids KGA, PA, FU, MA, and SU. Concentrations of the by-products FU, MA, SU, and PA decreased significantly at overproduction of FUM and increased at overproduction of PYC and also of FUM and PYC simultaneously. In contrast, the production of KGA with the multicopy strains H355A(FUM1) and H355A(FUM1-PYC1) was comparable with the wild-type strain H355 or slightly lower in case of H355(PYC1). KGA productivity was not changed significantly compared with strain H355 whereas product selectivity of the main product KGA was increased in H355A(FUM1).     

Effects of the dietary protein content and the feeding level on protein and energy metabolism in Iberian pigs growing from 50 to 100 kg body weight

Nutritional requirements of the Iberian pig, a slow-growing, obese porcine breed, are not well defined and seem to differ from those of conventional or high-performing pigs. The effects of the dietary protein content and the feeding level on the utilisation of metabolisable energy (ME) and the rates of gain, protein, and fat deposition were studied with 81 Iberian castrates growing from 50 to 100 kg body weight (BW) by using the comparative slaughter technique. The animals were fed 4 diets providing 145, 120, 95, and 70 g ideal crude protein (CP) per kg dry matter (DM), and containing 13.94, 14.29, 14.56, and 14.83 MJ ME per kg DM, respectively. Three levels of feeding were evaluated: 0.60, 0.80, and 0.95 × ad libitum intake. Growth rate increased (linear and quadratic, P < 0.001) as the dietary ideal CP content decreased. It also increased with the feeding level (linear, P < 0.001; quadratic, P < 0.05). Gain:feed and gain:ME intake improved by decreasing the ideal CP content in the diet (linear, P < 0.001 and P < 0.05, respectively; quadratic P < 0.001 for both variables). Increasing the feeding level improved linearly gain:feed and gain:ME intake ( P < 0.001). Protein deposition (PD):ME intake ranged between 1.23 and 1.44 g/MJ, and it showed a tendency to reach the maximum value when the diet providing 95 g ideal CP per kg DM was fed (quadratic, P = 0.078). When this diet was offered at 0.95 × ad libitum, PD reached a maximum value of 71 g/day. This dietary treatment resulted in average values for average daily gain and retained energy (RE) of 854 g/day and 21.4 MJ/day, respectively. The average rate of gain was 19.93 g/MJ increase in ME intake, equivalent to an energy cost of 50.2 kJ ME per g gain, irrespective of the dietary ideal CP content. Also, the overall marginal efficiency of protein deposition (ΔPD:ΔME; g/MJ) was 1.34. Increasing the feeding level led to increases in PD (linear, P <  0.001) and RE (linear, P <  0.001; quadratic, P <  0.01) irrespective of the dietary ideal CP concentrations. Between 50 and 100 kg BW, the chemical composition of 1 kg gain averaged 78, 592, 28.7, and 284 g for CP, fat, ash, and water respectively. The net efficiency of use of ME for growth ( k_g) and the maintenance energy requirements were 0.606 and 396 kJ/kg BW ^0.75 per day, respectively. The results support earlier findings that the genotype has marked effects on protein and energy metabolism of growing pigs and underline important compositional differences of the Iberian pig compared with conventional or modern porcine genotypes.     

Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems?

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.     

Predominance of IL-10 and TGF-β production from the mouse macrophage cell line, RAW264.7, in response to crude antigens from Clonorchis sinensis

Parasitic helminths are well-known to have the ability to modulate host immune responses. In this study, we investigated the fundamental immunoregulatory mechanism of the liver fluke Clonorchis sinensis (C. sinensis) using a murine macrophage RAW 264.7 (RAW) cell line and mouse bone marrow derived macrophages (BMDMs). We found that C. sinensis crude antigen (CA) is able to differentiate macrophage RAW cells into dendritic-like cells that can be detected by morphological observations. In addition, CA induces prominent secretion of anti-inflammatory cytokines such as IL-10 and TGF-β; however, we did not observe changes in cell surface markers that are involved in antigen recognition, antigen presentation, and T cell activation. Additionally, CA treatment induced ERK and JNK phosphorylation, and unexpectedly, elevated levels of IL-10 and TGF-β were inhibited by the addition of an ERK-specific inhibitor. Taken together, these data demonstrate that CA from C. sinensis may modulate host immune responses by upregulating anti-inflammatory cytokines via the regulation of ERK.     

Determination of fatty acid profiles,cholesterol and D,E, K vitamin’s content in Astacus leptodactylus caught from wild prior to reproduction period

In this study, fatty acids compositions, fat-soluble vitamins (D, E, and K) and cholesterol in the gonads, hepatopancreas, and abdomen muscle of male and female Astacus leptodactylus caught from wild were determined in their prior of reproduction period. The results showed that in female A. leptodactylus, compared with hepatopancreas, the ovaries contained a significantly high level of C16:0, C16:1n-7, C18:1n-9, C22:6n-3, the ratio of n-3 to n-6 (p < 0.05). In females, the values of δ-tocopherol, α-tocopherol, K₂ and D₂ vitamins, and cholesterol obtained in ovaries were significantly higher than those of obtained in abdomen muscle and hepatopancreas (p < 0.05). In male A. leptodactylus δ-tocopherol was found to be 1.91 μg/g in testes, which is significantly higher than that of abdomen muscle, hepatopancreas, and vasa deferentia (p < 0.05). In conclusion, the findings of present study are especially constructive for both crayfish biologists and farmers for preparing broodstock diets for crayfish at reproduction units.     

Dynamic or inert metabolism? Turnover of N‐acetyl aspartate and glutathione from d‐[1‐13C]glucose in the rat brain in vivo

The rate of (13)C-label incorporation into both aspartyl (NAA C3) and acetyl (NAA C6) groups of N-acetyl aspartate (NAA) was simultaneously measured in the rat brain in vivo for up to 19 h of [1-(13)C]glucose infusion (n = 8). Label incorporation was detected in NAA C6 approximately 1.5 h earlier than in NAA C3 because of the delayed labeling of the precursor of NAA C3, aspartate, compared to that of NAA C6, glucose. The time courses of NAA were fitted using a mathematical model assuming synthesis of NAA in one kinetic compartment with the respective precursor pools of aspartate and acetyl coenzyme A (acetyl-CoA). The turnover rates of NAA C6 and C3 were 0.7 +/- 0.1 and 0.6 +/- 0.1 micromol/(g h) with the time constants 14 +/- 2 and 13 +/- 2 h, respectively, with an estimated pool size of 8 micromol/g. The results suggest that complete label turnover of NAA from glucose occurs in approximately 70 h. Several hours after starting the glucose infusion, label incorporation into glutathione (GSH) was also detected. The turnover rate of GSH was 0.06 +/- 0.02 micromol/(g h) with a time constant of 13 +/- 2 h. The estimated pool size of GSH was 0.8 micromol/g, comparable to the cortical glutathione concentration. We conclude that NAA and GSH are completely turned over and that the metabolism is extremely slow (< 0.05% of the glucose metabolic rate).     

Identification of a cyclooxygenase gene from the red alga Gracilaria vermiculophylla and bioconversion of arachidonic acid to PGF(2α) in engineered Escherichia coli

Prostaglandins (PGs) are important local messenger molecules in many tissues and organs of animals including human. For applications in medicine and animal care, PGs are mostly purified from animal tissues or chemically synthesized. To generate a clean, reliable, and inexpensive source for PGs, we have now engineered expression of a suitable cyclooxygenase gene in Escherichia coli and achieved production levels of up to 2.7 mg l⁻¹ PGF_2α. The cyclooxygenase gene cloned from the red alga Gracilaria vermiculophylla appears to be fully functional without any eukaryotic modifications in E. coli. A crude extract of the recombinant E. coli cells is able to convert in vitro the substrate arachidonic acid (AA) to PGF_2α. Furthermore, these E. coli cells produced PGF_2α in a medium supplemented with AA and secreted the PGF_2α product. To our knowledge, this is the first report of the functional expression of a cyclooxygenase gene and concomitant production of PGF_2α in E. coli. The successful microbial synthesis of PGs with reliable yields promises a novel pharmaceutical tool to produce PGF_2α at significantly reduced prices and greater purity.