Porphyrogenic effects and induction of heme oxygenase in vivo by δ-aminolevulinic acid

The effects of single large doses of the porphyrin-heme precursor ?d-aminolevulinic acid on tissue porphyrins and on δ-aminolevulinate synthase and heme oxygenase, the rate-living enzymes of liver heme synthesis and degradation respectively, were studied in the chick embryo in ovo, in the mouse and in the rat. δ-Aminolevulinic acid treatment produced a distinctive pattern characterized by extensive tissue porphyrin accumulation and alterations in these rate-limiting enzymes in the liver. Repression of basal or allylisopropylacetamide-induced liver δ-aminolevulinate synthase was observed and, in the mouse and the rat, induction of liver heme oxygenase after δ-aminolevulinic acid treatment, in a manner similar to the known effects of hemin on these enzymes. In the chick embryo liver in ovo heme oxygenase was substantially higher than in rat and mouse liver, and was not significantly induced by δ-aminolevulinic acid or other compounds, including hemin, CS₂ and CoCl₂. Levulinic acid, an analogue of δ-aminolevulinic acid, did not induce heme oxygenase in mouse liver. δ-Aminolevunilic acid treatment did not impair ferrochelatase activity but was associated with slight and variable decreases in liver cytochrome P-450. Treatment of chick embryos with a small ‘priming’ dose of 1,4-dihydro-3,5-dicarbethoxycollidine, which impairs liver ferrochelatase activity, accentuated porphyrin accumulation after δ-aminolevulinic acid in the liver. These observations indicate that exogenous δ-aminolevulinic acid is metabolized to porphyrins in a number of tissues and, at least in the liver, to a physiologically significant amount of heme, thereby producing an increase in the size of one or more of the heme pools that regulate both heme systhesis and degradation. It is also possible than when δ-aminolevulinic acid is markedly overproduced in vivo it may be transported to many tissues and re-enter the heme pathway and alter porphyrin-heme metabolism in cells and tissues other than those in which its overproduction primarily occurs.     

Constituents of the essential oil of Lavandula latifolia

Thirty components were identified in Lavandula latifolia essential oil (spike oil). One of the compounds, espliegol (δ-terpineol), is a new natural product.     

Evidence of increased synthesis of δ-aminolevulinic acid dehydratase in experimental lead-poisoned rats

δ-Aminolevulinic acid dehydratase (porphobilinogen synthase; 5-aminolevulinate hydro-lyase, EC 4.2.1.24) was purified from rat and rabbit erythrocytes to a homogeneous state. Specific activities were 26.0 and 26.6 units/mg protein for the rat and rabbit enzymes, respectively, and their estimated molecular weight was 280 000, each consisting of 8 subunits of M_r 35 000. In order to quantitate rat δ-aminolevulinic acid dehydratase at several stages of lead-poisoning, a radioimmunoassay technique using goat antiserum against the rat enzyme was developed for the first time. This technique was specific, reproducible and high sensitive allowing determination of 1 ng enzyme. When drinking water containing 25 mM lead acetate was given daily to rats ad lib. the δ-aminolevulinic acid dehydratase activity in the blood, assayed without any pretreatment, decreased to 8% of the control level on the next day. On the contrary, the restored enzyme activity, assayed in the presence of Zn²⁺ and dithiothreitol, was greater than normal by the fourth day of lead administration in bone-marrow cells and by the ninth day in the peripheral blood. The increased activity level stayed the same from the ninth day onward. The enzyme content as determined directly by the radioimmunoassay technique at this stage was about 2-fold above that the control. There was no significant difference in the number of reticulocytes and the distribution profile of different types of reticulocytes between the lead-exposed and non-exposed rats. Therefore, the increase in the amount of δ-aminolevulinic acid dehydratase in erythrocytes of lead-poisoned rats was suggested to be due to an increased rate of synthesis in the bone-marrow cells.     

树轮木质素甲氧基稳定氢同位素比率测定方法研究进展

在森林树木合成的有机化合物中,氢元素(H)主要源于大汽水,经植物光合与生理代谢参与生物地球化学循环。近年来,树轮木质素甲氧基稳定氢同位素比率(δ²H_LM)作为新的古气候和古环境研究的代用指标,重建了多个地区的降水稳定同位素比率及气候变化信息,展现了其特有的优势。本文综述了现有树轮δ²H_LM测定的详细分析方法和基本原理,从树轮木质素含量、单体组成等方面对树轮δ²H_LM测定方法的稳定性和有效性进行评价,阐述了树轮木质素甲氧基稳定同位素指标现有研究成果。中纬度地区森林树轮δ²H_LM在记录气温变化和降水稳定同位素变化等方面有着巨大的潜力。但是树轮δ²H_LM的研究尚处于起步阶段,主要表现为: 1) 研究区局限于北半球中纬度地区,研究对象局限于针叶树种;2) 高分辨率树轮δ²H_LM研究有待开展,以弥补硝化纤维稳定氢同位素记录的局限;3) 树轮δ²H_LM在植物生理和森林生态研究方面的潜力有待开发。     

Atorvastatin prevents peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) downregulation in lipopolysaccharide-stimulated H9c2 cells

Although abnormalities in cardiac fatty acid metabolism are involved in the development of several cardiac pathologies, the mechanisms underlying these changes are not well understood. Given the prominent role played by peroxisome proliferator-activated receptor β/δ (PPARβ/δ in cardiac fatty acid metabolism, the aim of this study was to examine the effects of nuclear factor (NF)-κB activation on the activity of this nuclear receptor. Embryonic rat heart-derived H9c2 cells stimulated with lipopolysaccharide (LPS) showed a reduction (38%, P < 0.05) in the mRNA levels of the PPARβ/δ-target gene pyruvatedehydrogenase kinase 4 (PDK4) that was prevented in the presence of the NF-κB inhibitors parthenolide (10 μM) and atorvastatin (10 μM). Electrophoretic mobility shift assay revealed that both parthenolide and atorvastatin significantly decreased LPS-stimulated NF-κB binding activity in H9c2 cardiac cells. LPS-stimulation of H9c2 cardiac cells also led to a 30% reduction (P < 0.05) in the mRNA levels of PPARγ Coactivator 1 (PGC-1) that was consistent with the reduction in the protein levels of this coactivator. In the presence of either atorvastatin or parthenolide, the reduction in PGC-1 expression was prevented. Co-immunoprecipitation studies showed that LPS-stimulation led to a reduction in the physical interaction between PGC-1 and PPARβ/δ and that this reduction was prevented in the presence of atorvastatin. Finally, electrophoretic mobility shift assay revealed that parthenolide and atorvastatin prevented LPS-mediated reduction in PPARβ/δ binding activity in H9c2 cardiac cells. These results suggest that LPS-mediated NF-κB activation inhibits the expression of genes involved in fatty acid metabolism by a mechanism involving reduced expression of PGC-1, which in turn affects the PPARβ/δ transactivation of target genes involved in cardiac fatty acid oxidation.     

Hydrogen sulfide donor sodium hydrosulfide-improved heat tolerance in maize and involvement of proline

Hydrogen sulfide (H₂S) has long been considered as a phytotoxin, but nowadays as a cell signal molecule involved in growth, development, and the acquisition of stress tolerance in higher plants. In the present study, hydrogen sulfide donor, sodium hydrosulfide (NaHS), pretreatment markedly improved germination percentage of seeds and survival percentage of seedlings of maize under heat stress, and alleviated an increase in electrolyte leakage of roots, a decrease in tissue vitality and an accumulation of malondialdehyde (MDA) in coleoptiles of maize seedlings. In addition, pretreatment of NaHS could improve the activity of Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and lower proline dehydrogenase (ProDH) activity, which in turn induced accumulation of endogenous proline in maize seedlings. Also, application of proline could enhance endogenous proline content, followed by mitigated accumulation of MDA and increased survival percentage of maize seedlings under heat stress. These results suggest that sodium hydrosulfide pretreatment could improve heat tolerance of maize and the acquisition of this heat tolerance may be involved in proline.     

γ-Mangostin from Garcinia Mangostana Pericarps as a Dual Agonist That Activates Both PPARα and PPARδ

We tested the peroxisome proliferator-activated receptor (PPAR)δ agonistic activity of a Garcinia mangostana pericarp extract to develop a treatment for the metabolic syndrome, and demonstrated γ-mangostin to be an active compound on the basis of a luciferase reporter gene assay. γ-Mangostin induced the expression of the uncoupling protein-3 (UCP-3) gene which is related to energy expenditure and fat metabolism in L6 cells. We showed that γ-mangostin is a dual agonist that activates both PPARδ and PPARα. γ-Mangostin also induced the expression of acyl-CoA synthase and carnitine palmitoyl-transferase 1A genes in HepG2 cells. These results suggest the potential of γ-mangostin as a preventive agent of the metabolic syndrome.     

In vitro characterization of the inhibitor resistance of glutamate-1-semialdehyde aminotransferase from the cyanobacterium Synechococcus PCC630l GR6

Glutamate-l-semialdehyde (GSA) aminotransferase catalyses the final step in the C5 pathway converting glutamate to the tetrapyrrole precursor δ-aminolaevulinic acid. This enzyme is sensitive to gabaculine (2,3-dihydro-3-amino benzoic acid) and to 4-amino-5-fluoropentanoic acid (AFPA), which are irreversible, mechanism-based inhibitors of pyridoxal phosphatedependent enzymes. Spontaneous mutants of Synechococcus PCC6301 resistant to these inhibitors contain altered enzyme that displays corresponding resistance to high concentrations of the inhibitor. The enzyme from strain GR6, resistant to both inhibitors, contains a three-amino-acid deletion at positions 5–7 and a Met²⁴⁸ → Ile substitution. The enzyme from strain K40 resistant to AFPA but not to gabaculine, contains a Ser¹⁶³ → Thr substitution. GSA aminotransferases containing either the deletion or the substitution that are characteristic of the GR6 mutant were produced in Escherichia coli using the expression vector pMalc2. These engineered mutant enzymes were characterized in terms of their catalytic parameters and sensitivities to gabaculine and AFPA. Furthermore, maltose binding protein/aminotransferase fusion proteins were characterized spectrophotometrically to monitor the interaction of bound cofactor with diamino- and dioxocompounds related to the substrate and both inhibitors. Results were compared with those for similarly produced recombinant wild-type, K40 and GR6 GSA aminotransferases. The engineered products with either the N-terminal deletion or the Met²⁴⁸ → Ile substitution displayed catalytic efficiencies that were intermediate between the wild-type and GR6 or K40 enzymes. However, with respect to their absorption spectra, sensitivity to inhibitors and the reactivity of bound cofactor, they were essentially wild-type. These in vitro studies demonstrate that both changes in enzyme structure are necessary to obtain the distinctive properties of the GR6 aminotransferase, including resistance to high concentrations of gabaculine and AFPA.     

Relationship between a point mutation S97C in CK1δ protein and its affect on ATP-binding affinity

CK1δ (Casein kinase I isoform delta) is a member of CK1 kinase family protein that mediates neurite outgrowth and the function as brain-specific microtubule-associated protein. ATP binding kinase domain of CK1δ is essential for regulating several key cell cycle signal transduction pathways. Mutation in CK1δ protein is reported to cause cancers and affects normal brain development. S97C mutation in kinase domain of CK1δ protein has been involved to induce breast cancer and ductal carcinoma. We performed molecular docking studies to examine the effect of this mutation on its ATP-binding affinity. Further, we conducted molecular dynamics simulations to understand the structural consequences of S97C mutation over the kinase domain of CK1δ protein. Docking results indicated the loss of ATP-binding affinity of mutant structure, which were further rationalized by molecular dynamics simulations, where a notable loss in 3-D conformation of CK1δ kinase domain was observed in mutant as compared to native. Our results explained the underlying molecular mechanism behind the observed cancer associated phenotype caused by S97C mutation in CK1δ protein.     

Two continuous coupled assays for ornithine-δ-aminotransferase

We have developed two new continuous coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ¹-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of l-glutamate, produced during the regeneration of the cofactor pyridoxal 5’-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red l-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of l-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the l-glutamate oxidase (GluOx) reporter enzyme.