The isolation and identification of 2,4-diketones present in human and animal tissues and in urine.

The chemical isolation of 2,3-diketone fraction from hydrolysates of various mammalian tissues has been accomplished by the use of a modified Girard T procedure. This fraction, which constitutes a new lipid class, has been resolved by gas chromatography into a number of homologous 2,4-diketones, ranging in chain length from C13 to C25. After separation by preparative gas chromatography, the following compounds have been unequivocally identified: 2,4-heptadecanedione, 2,4-nonadecanedione, 2,4-heneicosanedione, 2,4-docosanedione, and delta12-2,4-heneicosenedione. The 2,4-diketones appear to exist in tissues in the free state or in labile combination. They are present also in human urine.     

Bioflavonoids as antiradicals,antioxidants and DNA cleavage protectors

Flavonoids have recently aroused considerable interest because of their broad pharmacological activity. In fact, flavonoids have been reported to have antiviral, antiallergic, antiplatelet, anti-inflammatory and antitumoral activities. The pharmacological properties of bioflavonoids have been ascribed both to the concomitant inhibition of enzymes involved in the production of free radicals and to their free-radical scavenging and iron chelating capacity. However the antioxidant capacity of bioflavonoids due to free-radical scavenging and/or to iron chelating is still controversial. In this study, we have investigated the free-radical scavenging capacity of bioflavonoids (rutin, catechin, and naringin). In addition, the effects of these polyphenols on xanthine oxidase activity, spontaneous lipid peroxidation, and DNA cleavage were investigated. The bioflavonoids under examination showed a dose-dependent free-radical scavenging effect, a significant inhibition of xanthine oxidase activity, and an antilipoperoxidative capacity. In addition, they showed a protective effect on DNA cleavage. This revised version was published online in July 2006 with corrections to the Cover Date.     

Recovery of malondialdehyde in urine as a 2,4-dinitrophenylhydrazine derivative analyzed with high-performance liquid chromatography

Malondialdehyde (MDA) in urine was measured as a 2,4-dinitrophenylhydrazine (DNPH) derivative using high-performance liquid chromatography (HPLC) for the analysis. MDA standard coeluted with a peak obtained from rat urine after i.p. administration of MDA standard. This peak was also the only peak containing 14C after injection of a [14C]MDA standard, and was shown by mass spectrometry to contain 1-(2,4-dinitrophenyl)pyrazole, the derivative formed when MDA is treated with DNPH. Depending on the amount given (0.3-5.5 mumol), the recovery (after 24 h sampling period) in urine was 0.7-2.6%. This apparent non-linear kinetics may relate to several factors, such as dose-dependent metabolism. However, the peak urinary concentration approached the expected plasma concentration and reproducible recovery data were obtained, suggesting that MDA was passively excreted in a reasonably stable form. These data indicate that monitoring MDA excretion in urine can give useful information about lipid peroxidation in vivo.     

Effect of reduced and oxidized glutathione on physico-chemical properties of erythrocyte membranes]

The parameters describing the structural and functional state of membranes depending on the level of reduced glutathione in erythrocytes were studied. It was shown, that the decrease in the concentration of reduced intracellular glutathione in erythrocytes upon metabolic depletion (prolonged incubation of cells at 37 degrees C in the absence of glucose) or a rapid irreversible depletion of glutathione with 1-chloro-2,4-dinitrobenzene enhances lipid peroxidation processes in membranes, inhibits the membrane-bound NAD.H methemoglobin reductase activity and decreases the intensity of 1,6-diphenyl-1,3,5-hexatrien fluorescence. The data obtained suggest that the depletion of reduced intracellular glutathione causes changes in the physicochemical state of the erythrocyte membrane: the accumulation of lipid peroxidation products, changes in the physical state of lipid bilayer and the inhibition of membrane-bound NAD.H-methemoglobin reductase activity.     

Another anti-allergic mechanism: antibody IgE deglycosylation induced by a substance extracted from human urine

Enzymically-deglycosylated antibody IgE lost its allergic activity in mouse systemic anaphylaxis, though the IgE kept its antibody activity. IgE antibody obtained from mice treated with a substance extracted from human urine was deglycosylated. This IgE also lost the allergic activity on the systemic anaphylaxis but kept its antibody activity. These findings strongly suggest that glycosylation of IgE has a close relation to the binding of the Fc receptor and that humans have another antiallergic mechanism: in vivo IgE antibody deglycosylation induced by the substance.     

Inhibition of adenosine-3',5'-cyclic monophosphate phosphodiesterase by potential antiallergic compounds.

The potential antiallergic compounds doxantrazole (3- (5-tetrazolyl)-thioxanthone 10, 10-dioxide) and CTD (3- carboxythioxanthone 10, 10-dioxide) are inhibitors of the phosphodiesterases of human and guinea pig lung and beef heart. Disodium cromoglycate is a weak inhibitor of all these enzymes. It is suggested that the antiallergic activity of doxantrazole and CTD is due, at least in part, to their ability to elevate intracellular cAMP levels by inhibiting phosphodiesterase activity.     

Cabbage cryoprotectin is a member of the nonspecific plant lipid transfer protein gene family

We have recently purified a protein (cryoprotectin) from the leaves of cold-acclimated cabbage (Brassica oleracea) to electrophoretic homogeneity, which protects thylakoids isolated from the leaves of nonacclimated spinach (Spinacia oleracea) from freeze-thaw damage. Sequencing of cryoprotectin showed the presence of at least three isoforms of WAX9 proteins, which belong to the class of nonspecific lipid transfer proteins. Antibodies raised against two synthetic peptides derived from the WAX9 proteins recognized a band of approximately 10 kD in western blots of crude cryoprotectin preparations. This protein and the cryoprotective activity could be precipitated from solution by the antiserum. We show further that cryoprotectin is structurally and functionally different from WAX9 isolated from the surface wax of cabbage leaves. WAX9 has lipid transfer activity for phosphatidylcholine, but no cryoprotective activity. Cryoprotectin, on the other hand, has cryoprotective, but no lipid transfer activity. The cryoprotective activity of cryoprotectin was strictly dependent on Ca(2+) and Mn(2+) and could be inhibited by chelating agents, whereas the lipid transfer activity of WAX9 was higher in the presence of ethylenediaminetetraacetate than in the presence of Ca(2+) and Mn(2+).     

川芎粗多糖抗氧化和细胞毒活性研究

通过川芎粗多糖(LCP)对DPPH离子、超氧阴离子、.OH、总抗氧化能力、螯合能力、还原性和脂质过氧化能力的抗氧化效果和对肝癌细胞HepG2的抑制作用研究。结果显示:LCP具有较强的还原力,对超氧阴离子自由基、脂质过氧化产物有良好的抑制作用,其对肝癌细胞HepG2也具有一定的抑制作用。LCP有望作为天然抗氧化剂及功能性食品得到开发与应用。     

The isolation and characterisation of a putative adipocyte precursor cell type from the white adipose tissue of the chicken (Gallus domesticus)

The conditions of isolation and culture of a chicken adipose stromal-derived cell strain are described and compared with chicken lung fibroblasts in vitro. The stromal cells accumulated intracellular lipid during the post-confluency culture period, this being by contrast with lung fibroblasts. Much higher levels of intracellular lipid were accumulated by the stromal cells when whole chicken serum or chicken plasma lipoproteins were added to the culture medium. Lipoprotein lipase activity emerged in stromal cells maintained post confluency. This activity was absent from pre-confluent stromal cells and pre- and post-confluent fibroblasts. The incorporation of 14C-acetate, 3H-oleic acid and 14C-glucose into lipids by the stromal cells exhibited a pattern compatible with a concerted shift in the metabolism of the cells towards lipid storage, particularly in the form of triacylglycerols derived from exogenous fatty acids. It is proposed that, in common with the mammalian species previously studied, the white adipose tissue of the chicken (Gallus domesticus) contains a cell type with properties which allow its preliminary identification as an adipocyte precursor cell capable of adipose conversion in vitro. The confirmation of this proposition is amenable to further investigation.     

Alkylated derivatives of poly(ethylacrylic acid) can be inserted into preformed liposomes and trigger pH-dependent intracellular delivery of liposomal contents

Poly(ethylacrylic acid) (PEAA) is a pH-sensitive polymer that undergoes a transition from a hydrophilic to a hydrophobic form as the pH is lowered from neutral to acidic values. In this work we show that pH sensitive liposomes capable of intracellular delivery can be constructed by inserting a lipid derivative of PEAA into preformed large unilamellar vesicles (LUV) using a simple one step incubation procedure. The lipid derivatives of PEAA were synthesized by reacting a small proportion (3%) of the carboxylic groups of PEAA with C10 alkylamines to produce C10-PEAA. Incubation of C10-PEAA with preformed LUV resulted in the association of up to 8% by weight of derivatized polymer with the LUV without inducing aggregation. The resulting C10-PEAA-LUV exhibited pH-dependent fusion and leakage of LUV contents on reduction of the external pH below pH 6.0 as demonstrated by lipid mixing and release of calcein encapsulated in the LUV. In addition, C10-PEAA-LUV exhibited pH dependent intracellular delivery properties following uptake into COS-7 cells with appreciable delivery to the cell cytoplasm as evidenced by the appearance of diffuse intracellular calcein fluorescence. It is demonstrated that the cytoplasmic delivery of calcein by C10-PEAA-LUV could be inhibited by agents (bafilomycin or chloroquine) that inhibit acidification of endosomal compartments, indicating that this intracellular delivery resulted from the pH-dependent destabilization of LUV and endosomal membranes by the PEAA component of the C10-PEAA-LUV. It is concluded that C10-PEAA-LUV represents a promising intracellular delivery system for in vitro and in vivo applications.     

2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenases from Burkholderia cepacia 2a and Ralstonia eutropha JMP134

2,4-Dichlorophenoxyacetate (2,4-D)/alpha-ketoglutarate (alpha-KG) dioxygenase has been purified to apparent homogeneity from Burkholderia cepacia strain 2a, which utilizes 2,4-D as sole carbon source. The enzyme required ferrous ions, and was a homodimer composed of subunits having an Mr of approximately 32,000. The reaction catalysed consumed one mol each of 2,4-D, alpha-KG and dioxygen, with the production of one mol each of succinate, 2,4-dichlorophenol and glyoxylate. Maximum activity was exhibited at pH 7.8 and 25 degrees C, and reactivity was enhanced by the presence of ascorbate and cysteine. Mn2+, Zn2+, Cu2+, Fe3+ and Co2+ were inhibitory, and chemical modification of the dioxygenase revealed that thiol groups were essential for activity. The enzyme was active towards other substituted phenoxyacetates, but reacted most rapidly with 2,4-D. The apparent Michaelis constants for 2,4-D and alpha-KG were 109 and 8.9 microM, respectively. The properties of this enzyme are compared with those of the 2,4-D/alpha-KG dioxygenase from Ralstonia eutropha JMP134, which exhibits a differing N-terminal amino-acid sequence, and a different temperature 'optimum', pH optimum, substrate specificity and sensitivity to thiol-binding reagents.     

Antioxidant activity of the halophyte Limonium tetragonum and its major active components

In this study, the antioxidant potentials of crude extracts and solvent-partitioned fractions of Limonium tetragonum were assessed by measuring their ability to scavenge intracellular reactive oxygen species (ROS) generated in HT-1080 cells. Following activity-oriented separation, four flavonol glycosides were isolated as active principles and their chemical structures were determined by 2 D NMR and by comparison with reported spectral data. The isolated compounds (1?C4) were evaluated for their antioxidant capacity using three different activity tests; degree of occurrence of intracellular ROS, lipid peroxidation in HT-1080 cells and the extent of oxidative damage of genomic DNA purified from HT-1080 cells. All compounds exhibited significantly inhibited the generation of intracellular ROS and lipid peroxidation in HT-1080 cells, and significantly inhibited DNA oxidation. In addition, direct free radical scavenging effects of these compounds were investigated using the electron spin resonance (ESR) spin-trap technique.     

Role of a guanine nucleotide regulatory protein in the activation of phospholipase C by different chemoattractants

It is well established that formyl peptide chemoattractants can activate a phospholipase C in leukocytes via a pertussis toxin (PT)-sensitive guanine nucleotide regulatory (G) protein. Whether this pathway is similarly used by chemoattractant receptors as a class has been unclear. We now report that lipid and peptide chemoattractants in direct comparative studies induced similar amounts of initial (less than or equal to 15 sec) inositol trisphosphate (IP3) release in human polymorphonuclear leukocytes, but the response to lipid chemoattractants was more transient. Production of IP3 by all chemotactic factors was inhibited by treatment of the cells with PT, indicating that chemotactic factor receptors as a class are coupled to phospholipase C via a G protein that is a substrate for ADP ribosylation by PT. The peptide and lipid factors had comparable chemotactic activity, which was also inhibitable by PT. However, transient activation of phospholipase C is apparently an insufficient signal for full cellular activation, since the lipid chemotactic factor leukotriene B4 and platelet-activating factor were poor stimuli for O2- production and lysosomal enzyme secretion compared with N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe). Nonetheless, treatment with PT inhibited O2- production and enzyme secretion in response to all chemoattractants, but as previously noted, did not affect Ca2+ ionophores, lectins, or phorbol myristate acetate. Formyl peptide and lipid chemotactic factors induced similar levels of Ca2+ mobilization when monitored by Quin 2 or chlortetracycline (CTC) fluorescence. Although these responses to fMet-Leu-Phe were blocked by PT, the Quin 2 and initial CTC response to the lipid factors were only partially susceptible. Thus, the lipid factors apparently utilize an additional PT-resistant mechanism for redistributing intracellular Ca2+. This latter process requires extracellular Ca2+ and may be independent of the PT-sensitive G protein.     

Platinum complexes of diaminocarboxylic acids and their ethyl ester derivatives: the effect of the chelate ring size on antitumor activity and interactions with GMP and DNA

A number of new Pt(II) complexes is described having the general formula PtCl(2)(LL), where LL is a chelating diamine ligand. Ligands LL were chosen as D,L-2,3-diaminopropionic acid and its ethyl ester, and D,L-2,4-diaminobutyric acid and its ethyl ester. The compounds were characterized using analytical and spectroscopic methods. The influence of the size of the chelate ring and its functionalization on the biological properties was studied. It was demonstrated by circular dichroism (CD) that the effects on the secondary structure of DNA induced by the four complexes are different. The interaction takes place at the N7 position of the purine bases, as shown by NMR studies. The platinum complexes of 2,3-diaminopropionic acid and 2,4-diaminobutyric acid are able to form intrastrand adducts with DNA and to distort the double helix by changing the base stacking. The ethyl ester derivatives uncoil the DNA from the B form to the C form. The interactions with 5'-GMP and DNA were compared with their antitumor activity. The platinum complexes of diaminocarboxylic acids exhibit cytotoxic activity in the A431, HeLa, and HL-60 cell lines in a dose- and time-dependent manner.     

Inhibition of membrane-associated methyltransferases by a cholesterol-based metal chelator

We have designed, synthesized, and characterized a metal chelating compound that is based on the structure of cholesterol and contains the high affinity metal chelating group, lysine nitrilotriacetic acid (Lys-NTA). Using the enzyme isoprenylcysteine carboxylmethyltransferase (Icmt) from yeast as a model integral membrane metalloenzyme, we find that this agent potently inhibits Icmt activity with an IC(50) value between 35 and 75 microM, which is at least 40 times more potent than the best known Icmt metal chelating inhibitor, Zincon. We propose that the rigid hydrophobic cholesterol moiety promotes partitioning into the membrane, enabling the metal-binding NTA group(s) to inactivate the enzyme by metal chelation. Because this compound is based on a naturally occurring membrane lipid and appears to chelate metals buried deeply within water insoluble environments, this agent may also be useful as a general tool for identifying previously unappreciated metal dependencies of other classes of membrane proteins.     

Flavonoid antioxidants: chemistry,metabolism and structure-activity relationships

Flavonoids are a class of secondary plant phenolics with significant antioxidant and chelating properties. In the human diet, they are most concentrated in fruits, vegetables, wines, teas and cocoa. Their cardioprotective effects stem from the ability to inhibit lipid peroxidation, chelate redox-active metals, and attenuate other processes involving reactive oxygen species. Flavonoids occur in foods primarily as glycosides and polymers that are degraded to variable extents in the digestive tract. Although metabolism of these compounds remains elusive, enteric absorption occurs sufficiently to reduce plasma indices of oxidant status. The propensity of a flavonoid to inhibit free-radical mediated events is governed by its chemical structure. Since these compounds are based on the flavan nucleus, the number, positions, and types of substitutions influence radical scavenging and chelating activity. The diversity and multiple mechanisms of flavonoid action, together with the numerous methods of initiation, detection and measurement of oxidative processes in vitro and in vivo offer plausible explanations for existing discrepancies in structure-activity relationships. Despite some inconsistent lines of evidence, several structure-activity relationships are well established in vitro. Multiple hydroxyl groups confer upon the molecule substantial antioxidant, chelating and prooxidant activity. Methoxy groups introduce unfavorable steric effects and increase lipophilicity and membrane partitioning. A double bond and carbonyl function in the heterocycle or polymerization of the nuclear structure increases activity by affording a more stable flavonoid radical through conjugation and electron delocalization. Further investigation of the metabolism of these phytochemicals is justified to extend structure-activity relationships (SAR) to preventive and therapeutic nutritional strategies.     

Sulfation of a polysaccharide produced by a marine filamentous fungus Phoma herbarum YS4108 alters its antioxidant properties in vitro

Free radicals and other reactive oxygen species (ROS) are generated by all aerobic cells and are widely believed to play a significant role in aging as well as a number of degenerative or pathological diseases. This study compared the free radical-scavenging properties and antioxidant activity of YCP, a polysaccharide from the mycelium of a marine filamentous fungus Phoma herbarum YS 4108 and its two chemically sulfated derivatives YCP-S1 and YCP-S2. Sulfation, which masks hydroxyl groups of YCP polysaccharide molecule, could introduce new antioxidant activity, such as superoxide and hydroxyl radicals scavenging activity, metal chelating action, lipid peroxidation and linoleic acid oxidation inhibition capability. Furthermore, sulfated YCP was more potent than YCP at protecting erythrocytes against oxidative damage hemolysis. The current data suggest for the first time that sulfation of polysaccharide significantly increases its antioxidant activity and the chemical modification of polysaccharides may allow the preparation of derivatives with new properties and a variety of applications.     

Selective in vitro antioxidant properties of bisphosphonates

The aim of this study was to investigate the in vitro antioxidant profile of different bisphosphonates. Bisphosphonates were tested for their xanthine oxidase and microsomal lipid peroxidation inhibiting capacity. Furthermore, the effect of these different compounds on DPPH, a stable radical, was investigated. Clodronate, risedronate, and pyrophosphate were further tested for their hydroxyl radical scavenging activity. None of the tested compounds showed xanthine oxidase inhibiting activity or DPPH scavenging activity. All the tested bisphosphonates exhibited inhibiting capacities on the microsomal lipid peroxidation. The hydroxyl radical scavenging activity was dependent on the order of adding the different reagents and was highest for risedronate. Bisphosphonates possess an inhibiting activity on the microsomal lipid peroxidation and the Fenton reaction. In these reactions iron plays an important role suggesting that the selective in vitro antioxidant properties of the bisphosphonates are due to their iron chelating characteristics.     

Identification of a novel chloride channel expressed in the endoplasmic reticulum, golgi apparatus, and nucleus

MID-1 is a Saccharomyces cerevisiae gene encoding a stretch-activated channel. Using MID-1 as a molecular probe, we isolated rat cDNA encoding a protein with four putative transmembrane domains. This gene encoded a protein of 541 amino acids. We also cloned the human homologue, which encoded 551 amino acids. Messenger RNA for this gene was expressed abundantly in the testis and moderately in the spleen, liver, kidney, heart, brain, and lung. In the testis, immunoreactivity of the gene product was detected both in the cytoplasm and the nucleus. When expressed in Chinese hamster ovary cells, the gene product was located in intracellular compartments including endoplasmic reticulum and the Golgi apparatus. When microsome fraction obtained from the transfected cells, but not from mock-transfected cells, was incorporated into the lipid bilayer, an anion channel activity was detected. Unitary conductance was 70 picosiemens in symmetric 150 mm KCl solution. We designated this gene Mid-1-related chloride channel (MCLC). MCLC encodes a new class of chloride channel expressed in intracellular compartments.     

The metabolism of 2-chloro-1-(2′,4′-dichlorophenyl)vinyl diethyl phosphate (Chlorfenvinphos) in the dog and rat

1. A single oral dose of [(14)C]Chlorfenvinphos to rats is quantitatively eliminated in 4 days. Rats do not show a sex difference in the elimination pattern and show only a small degree of biological variation in the total excretion data. Of the label 87.2% is excreted in the urine (67.5% in the first day after dosage), 11.2% in the faeces and 1.4% in the expired gases; less than 0.9% of (14)C is present in the gut and contents after 4 days. 2. After oral administration of [(14)C]Chlorfenvinphos to dogs, 94.0% (91.8-97.6%) of the (14)C is excreted in the urine and faeces during 4 days. Dogs do not show a sex difference in the pattern of elimination, and excretion of radioactivity in the urine is very rapid: 86.0% of (14)C during 0-24hr. 3. Chlorfenvinphos is completely metabolized in rats and dogs: unchanged Chlorfenvinphos is absent from the urine and from the carcass, when elimination is complete. In rats, 2-chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate accounts for 32.3% of a dose of Chlorfenvinphos, [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid]uronic acid for 41.0%, 2,4-dichloromandelic acid for 7.0%, 2,4-dichlorophenylethanediol glucuronide for 2.6% and 2,4-dichlorohippuric acid for 4.3%; in dogs, 2-chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate accounts for 69.6%, [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid] uronic acid for 3.6%, 2,4-dichloromandelic acid for 13.4% and 2,4-dichlorophenylethanediol glucuronide for 2.7%. 4. Dogs and rats show a species difference in the rate of excretion of (14)C in the urine, and in the proportions of the metabolites, with the exception of 2,4-dichlorophenylethanediol glucuronide, that are excreted in the urine. Alternative explanations for the latter species difference are suggested. 5. 2-Chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate and 2,4-dichlorophenacyl chloride probably lie on the main metabolic pathway of Chlorfenvinphos, since, in common with that insecticide, they give rise to [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid]uronic acid and 2,4-dichloromandelic acid as major metabolites in the urine. 6. The proposed scheme for the metabolism of Chlorfenvinphos represents a detoxication mechanism.