Biological role of hepoxilins: upregulation of phospholipid hydroperoxide glutathione peroxidase as a cellular response to oxidative stress?

The 12S-lipoxygenase (12S-LOX) pathway of arachidonic acid (AA) metabolism is bifurcated at 12(S)-hydroperoxy-5Z,8Z,10E (12S-HpETE) in the reduction route to form 12S-hydroxy-eicosatetraenoic acid (12S-HETE) and in 8(S/R)-hydroxy-11(S),12S-trans-epoxyeicosa-5Z,9E,14Z-trienoic acid (HXA3) synthase pathway, previously known as isomerization route, to form hepoxilins. Earlier we showed that the HXA3 formation is restricted to cellular systems devoid of hydroperoxide reducing enzymes, e.g. GPxs, thus causing a persistent oxidative stress situation. Here, we show that HXA3 at as low as 100 nM concentration upregulates phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA and protein expressions, whereas other metabolites of AA metabolism 12S-HpETE and 12S-HETE failed to stimulate the PHGPx. Moreover, the decrease in 12S-HpETE below a threshold value of the hydroperoxide tone causes both suppression of the overall 12S-LOX activity and a shift from HXA3 formation towards 12S-HETE formation. We therefore propose that under persistent oxidative stress the formation of HXA3 and the HXA3-induced upregulation of PHGPx constitute a compensatory defense response to protect the vitality and functionality of the cell.     

Effect of nitrogen concentration on the activity of manganese peroxidase ofPhanerochœte chrysosporium

Manganese peroxidase as an extracellular enzyme is produced by the white rot fungusPhanerochœte chrysosporium under nutrient nitrogen or carbon limitation. The effect of nitrogen concentration on the activity of manganese peroxidase was studied using ammonium nitrate andl-asparagine as nitrogen sources. The highest activity of the enzyme was observed in cultures grown in a medium containing 75 mg/L ammonium nitrate and 0.15 g/Ll-asparagine. Manganese peroxidase was not detectable in cultures grown in the presence 0.5 g/L ammonium nitrate and 1 g/Ll-asparagine.     

Effect of phosphatidylcholine vesicles on the activity of DNA polymerase-α

Summary Phosphatidylcholine vesicles stimulate the activity of the DNA polymerase- from calf thymus. This effect is dependent upon the way of addition of the Mg ions, and the extent of the ³H-dTTP incorporation is closely related to the concentration of the vesicles. A role of phospholipids on the activity of the DNA-related enzymes is suggested.     

Effect of water activity control on the catalytic performance of surfactant–Arthromyces ramosus peroxidase complex in toluene

Arthromyces ramosus peroxidase (ARP) was successfully modified with a synthetic surfactant for one-electron oxidation reaction of a hydrophobic substrate in toluene. Although UV–visible absorption spectrum of surfactant–ARP complex in toluene showed slight red shift of Soret band compared to that in water, the complex can catalyze oxidation reaction of o-phenylenediamine (o-PDA) with hydrogen peroxide. It appeared that thermodynamic water activity in the reaction system has dominant effect on either the catalytic activity or the stability in the catalytic cycle. Steady-state kinetics under the optimal condition revealed that the specific constant (k_cat/K_m) of ARP complex for o-PDA was 2 orders of magnitude lower than that in aqueous media, while only 13-fold lower for hydrogen peroxide. The reduction of catalytic activity caused by altering the reaction media from water to toluene was found to be mainly due to the low specific constant of ARP complex for o-PDA rather than hydrogen peroxide.     

Is phospholipid a required cofactor for the activity of mammalian signal peptidase?

To determine whether phospholipid is required for the activity of mammalian signal peptidase, the enzyme was partially purified from porcine pancreas and then extensively freed of phospholipid by SP-Sephadex C-50 chromatography. The delipidated enzyme showed signal peptidase activity, with a low concentration of detergent. Phospholipid was found to release the enzyme from the inhibition due to excess detergent.     

Biochemical and genetic studies on rabbit hemoglobin. II. Electrophoretic polymorphism of theα-chain

A genetic polymorphism of rabbit (Oryctolagus cuniculus) hemoglobin chain is demonstrated by means of acid starch gel electrophoresis. The polymorphism is not detected by isoelectric focusing and may be based upon neutral for neutral amino acid substitutions in accordance with previous findings by means of amino acid sequencing. Segregation analysis was performed on 15 matings with 49 offspring and confirmed the initial genetic hypothesis of three common codominant alleles at an autosomal locus. The calculated gene frequencies in a random sample of 86 unrelated individuals areHBA*1=0.73,HBA*2=0.22, andHBA*3=0.05.     

Interferon β enhances the natural killer activity of patients with bladder carcinoma

We have investigated the effect of interferon (INFß) on the natural killer (NK) cytotoxic activity of peripheral blood mononuclear cells (PBMC) from patients with superficial and infiltrative transitional-cell carcinoma of the bladder (TCC) against both NK-sensitive and NK-resistant target cells. The normal NK activity found in PBMC from these patients can be significantly enhanced by short-term incubation (18 h) with INFß (P<0.05). The depressed NK cytotoxic activity found in PBMC from patients with infiltrative TCC can also be significantly enhanced, but not normalized, by short-term incubation with INFß (P<0.05). In kinetic studies we found that the maximal levels of the INFß-promoted cytotoxic activity against NK-sensitive and against NK-resistant target cells in PBMC from TCC patients were reached after 18 h of culture. Short-term-INFß-incubated PBMC from patients with TCC of the bladder also showed marked cytotoxic activity against NK-resistant target cells. The effector cells of the INFß-induced cytotoxic activity in PBMC from patients with TCC were CD16⁺ CD3^– NK cells. This cytotoxic inducer effect of INFß synergized with that of interleukin-2. In conclusion, INFß can enhance the NK activity of PMBC from patients with TCC of the bladder.     

α-Tocopherol Improves Microcirculatory Dysfunction on Fructose Fed Hamsters

Fructose, an everyday component of western diet associated to chronic hyperglycemia and enhanced free radical production, impairs endothelial function and supplementation with antioxidants might improve it. In this study we investigated if vitamin E could reverse the microvascular damage elicited by fructose. Male Syrian golden hamsters drank either 10% fructose solution (F) or filtered water (C), combined with three concentrations of vitamin E in their chows [zero, normal (VE) or 5X (5XVE)] during 60 days. Microvascular reactivity in response to topical application of acetylcholine (Ach; endothelium-dependent vasodilator) or sodium nitroprusside (SNP; endothelium-independent vasodilator) and macromolecular permeability increase induced by either 30 min ischemia followed by reperfusion (I/R) or topical application of histamine (5 μM) were assessed using the cheek pouch preparation. Compared to controls (drinking filtered water), fructose-drinking animals showed decreased vasodilatation to acetylcholine in all concentrations tested (-56.2% for 10^-9M, -53.9% for 10^-7M and -43.7% for 10^-5M). On the other hand, vitamin E supplementation resulted in increased responses for both water and fructose drinking groups (177.4% for F vs. F/5XVE and 241.6% for C vs. C/5XVE for 10^-5M Ach). Endothelial-independent vasodilatation explored by topical application of SNP was restored and even enhanced with the supplementation of 5X vitamin E in both groups (80.1% for F vs. F/5XVE; 144.2% for C vs. C/5XVE; 3.4% of difference for C/5XVE vs. F/5XVE on 10^-5M SNP). The number of leaky sites after I/R and histamine stimuli in vitamin E supplemented animals decreased (-25.1% and -15.3% for F vs. F/5XVE; and -21.7% and -16% of leaky sites comparing C vs. C/5XVE, respectively for I/R and histamine stimuli) pointing to tightening of the endothelial barrier for macromolecular permeability. Our results strongly suggest that vitamin E could improve the endothelial function and permeability barrier and also reverse impairments elicited by sugar overload.     

Dimerization of piceatannol by Momordica charantia peroxidase and α-glucosidase inhibitory activity of the biotransformation products

Stilbenes, especially those oligomers, have great potential to be antihyperglycemic agents. In this study, eight stilbene dimers, including five new ones, were obtained by biotransformation of piceatannol using Momordica charantia peroxidase (MCP) for the first time. Their structures were established on the basis of spectroscopic evidences. These piceatannol dimers displayed potential α-glucosidase inhibitory activities, and trans double bond, tetrahydrofuran ring, and free adjacent phenolic dihydroxyls were found to be important for their activities. Enzymatic biotransformation of stilbenes by M.charantia peroxidase (MCP) was showed to be a prominent way to produce oligomeric stilbenes for antihyperglycemic development.     

Deiodination of l-thyroxine and its activity on the oxidation in vitro of reduced nicotinamide–adenine dinucleotide by peroxidase plus hydrogen peroxide

When l-thyroxine activates the oxidation of NADH by peroxidase+H(2)O(2), little removal of phenolic-ring iodine atoms becomes apparent until most of the NADH has been oxidized, after which it increases markedly. This extensive deiodination is accompanied by loss of the ability of thyroxine to catalyse the oxidation of NADH by peroxidase+H(2)O(2). The slight deiodination observed before the appearance of extensive deiodination is somewhat higher when the effect of thyroxine on NADH oxidation is greater, and lower when thyroxine has exerted a slighter effect. ICN (but not I(2) or thyronine) catalyses NADH oxidation, in both the presence and the absence of peroxidase+H(2)O(2): thyroxine+peroxidase+H(2)O(2) are thus comparable with ICN alone in their effects on NADH oxidation. The obvious conclusion from the above observation, namely that the active moiety is the halogen liberated from thyroxine (or ICN) is, however, not directly supported by some of the results obtained by measuring the degree of deiodination of thyroxine in the system. In an attempt to reconcile some apparently contradictory conclusions, it is suggested that, when thyroxine activates oxidation of NADH by peroxidase+H(2)O(2), the diphenyl ether structure is undergoing cyclic deiodination and iodination. This would be accompanied by the maintenance in the reaction medium of an oxidized form of iodine, similar to that liberated by ICN, which would be the actual active moiety, until the NADH concentration becomes so low that the diphenyl ether structure is ruptured oxidatively. An alternative explanation is that thyroxine is oxidized to a form that either oxidizes NADH or loses iodine in competing reactions.     

Effect of dehydroepiandrosterone derivatives on the activity of 5α-reductase isoenzymes and on cancer cell line PC-3

It is well known that testosterone (T) under the influence of 5α-reductase enzyme is converted to dihydrotestosterone (DHT), which causes androgen-dependent diseases. The aim of this study was to synthesize new dehydroepiandrosterone derivatives (3a–e, 4a–i, 6 and 7) having potential inhibitory activity against the 5α-reductase enzyme. This paper also reports the in vivo pharmacological effect of these steroidal molecules. The results from this study showed that all compounds exhibited low inhibitory activity for 5α-reductase type 1 and 2 enzymes and they failed to bind to the androgen receptor. Furthermore, in the in vivo experiment, steroids 3b, 4f, and 4g showed comparable antiandrogenic activity to that of finasteride; only derivatives 4d and 7 produced a considerable decrease in the weight of the prostate gland of gonadectomized hamsters treated with (T). On the other hand, compounds 4a, f and h showed 100% inhibition of the growth of prostate cancer cell line PC-3, with compound 4g having a 98.2% antiproliferative effect at 50 μM. The overall data indicated that these steroidal molecules, having an aromatic ester moiety at C-3 (4f–h), could have anticancer properties.     

Assembly of α-synuclein aggregates on phospholipid bilayers

The spontaneous self-assembly of α-synuclein (α-syn) into aggregates of different morphologies is associated with the development of Parkinson's disease. However, the mechanism behind the spontaneous assembly remains elusive. The current study shows a novel effect of phospholipid bilayers on the assembly of the α-syn aggregates. Using time-lapse atomic force microscopy, it was discovered that α-syn assembles into aggregates on bilayer surfaces, even at the nanomolar concentration range. The efficiency of the aggregation process depends on the membrane composition, with the greatest efficiency observed for of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS). Importantly, assembled aggregates can dissociate from the surface, suggesting that on-surface aggregation is a mechanism by which pathological aggregates may be produced. Computational modeling revealed that dimers of α-syn assembled rapidly, through the membrane-bound monomer on POPS bilayer, due to an aggregation-prone orientation of α-syn. Interaction of α-syn with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) leads to a binding mode that does not induce a fast assembly of the dimer. Based on these findings, we propose a model in which the interaction of α-syn with membranes plays a critical role initiating the formation of α-syn aggregates and the overall aggregation process.     

Fusogenic activity of δ-haemolysin from Staphylococcus aureus in phospholipid vesicles in the liquid-crystalline phase

A study has been conducted of the interaction of the lytic toxin δ-haemolysin with vesicles of phospholipid, using electron microscopy, fluorescence depolarisation and excimer fluorescence. The peptide is shown to be a fusogen towards phosphatidylcholine vesicles in fluid phases. In the presence of gel phase lipid, fusion between fluid and gel phases is not seen. Fluid phase lipid vesicles are fused together to form large multilamellar structures, and initial vesicle size does not appear to be important since small unilamellar vesicles and large unilamellar vesicles are similarly affected. Fusogenic activity of δ-haemolysin is compared to that of melittin. The former is a progressive fusogen for fluid phase lipid, while the latter causes vesicle fusion in a manner related to occurrence of a lipid phase transition.     

Biological activity of pyrazole and imidazole-dehydroepiandrosterone derivatives on the activity of 17β-hydroxysteroid dehydrogenase

The enzyme type 5 17β-hydroxysteroid dehydrogenase 5 (17β-HSD5) catalyzes the transformation of androstenedione (4-dione) to testosterone (T) in the prostate. This metabolic pathway remains active in cancer patients receiving androgen deprivation therapy. Since physicians seek to develop advantageous and better new treatments to increase the average survival of these patients, we synthesized several different dehydroepiandrosterone derivatives. These compounds have a pyrazole or imidazole function at C-17 and an ester moiety at C-3 and were studied as inhibitors of 17β-HSD5. The kinetic parameters of this enzyme were determined for use in inhibition assays. Their pharmacological effect was also determined on gonadectomized hamsters treated with Δ⁴-androstenedione (4-dione) or testosterone (T) and/or the novel compounds. The results indicated that the incorporation of a heterocycle at C-17 induced strong 17β-HSD5 inhibition. These derivatives decreased flank organ diameter and prostate weight in castrated hamsters treated with T or 4-dione. Inhibition of 17β-HSD5 by these compounds could have therapeutic potential for the treatment of prostate cancer and benign prostatic hyperplasia.     

Forced dimerization increases the activity of ΔEGFR/EGFRvIII and enhances its oncogenicity

Delta epidermal growth factor receptor (ΔEGFR), an in-frame deletion mutant of the extracellular ligand-binding domain, which occurs in about 30% of glioblastoma, is a potent oncogene that promotes tumor growth and progression. The signaling of ΔEGFR is ligand-independent and low intensity, allowing it to evade the normal mechanisms of internalization and degradation by the endocytic machinery and hence is persistent. The basis of the oncogenic potential of ΔEGFR remains incompletely understood, including whether dimerization plays an important role in its signal and whether its oncogenic potential is dependent on its relatively low intensity, when compared with the acutely activated wild-type receptor. To examine these two important questions, we have generated a chimeric ΔEGFR that allows forced dimerization via domains derived from variants of the FKBP12 protein that are brought together by FK506 derivatives. Forced dimerization of chimeric ΔEGFR significantly increased the intensity of its signal, as measured by receptor phosphorylation levels, suggesting that the naturally occurring ΔEGFR does not form strong or stable dimers as part of its low level signal. Interestingly, the increased activity of dimerized, chimeric ΔEGFR did not promote receptor internalization, implying that reduced rate of endocytic downregulation of ΔEGFR is an inherent characteristic. Significantly, forced dimerization enhanced the oncogenic signal of the receptor, implying that the ΔEGFR is a potent oncogene despite, not because of its low intensity.     

Influence of glucose on the α-glucoside permease activity of yeast

Summary The change in the -glucoside permease activity of baker's yeast, Saccharomyces cerevisiae, has been followed in the presence of maltose and/or glucose in the medium. Three separate effects of glucose on the permease were distinguished: an immediate effect that apparently involves a conformational transformation of the permease, an inactivation of the permease before the initiation of growth, and a repression and derepression of the synthesis of permease. Conceivable mechanisms for regulation of the glucose effects are briefly discussed.     

Isolation of β-Tocopherol from the Root Bark of the Mulberry Tree

Kluyvera citrophila KY7844 enzymatically catalyzed N-acylation of 7-amino-desacetoxycephalosporanic acid with 1-(1H)-tetrazolylacetate methylester to produce 7-[1-(1H)-tetrazolylacetamido]-desacetoxycephalosporanic acid. The product was purified and characterized.     

Observations on the effect of hurricane “Carla” on insect activity

Observations in the laboratory under conditions of controlled temperature and relative humidity on the effects of low barometric pressures on insect activity resulting fron hurricane Carla, one of the largest and most destructive hurricanes to affect the middle coast of Texas, revealed that stable flies (STOMOXYS CALCITRANS (L.)), house flies (MUSCA DOMESTICA L.), and Mexican fruit flies (ANASTREPHA LUDENS (Loew)), cockroaches (BLATELLA GERMANICA (L.)), PERIPLANETA AMERICANA (L.), and BLABERUS GIGANTEUS (L.) and mosquitoes (AEDES AEGYPTI (L.), exhibited nervous activity concomitant with the reduction in barometric pressure. Field studies throughout the critical storm period also indicated unusual arthropod activity. Following the hurricane, certain insect species emerged in abnormally large numbers, particularly fall armyworms (LAPHYGMA FRUGIPERDA (J.E. Smith)), asps (MEGALOPYGE OPERCULARIS (J. E. Smith)), and southern green stink bugs (NEZARA VIRIDULA (L.)).

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Zusammenfassung WÄhrend des Hurrikans Carla, einer der grössten und verheerendsten Orkane, die je auf die mittlere Küste von Texas eingewirkt haben, wurden im Laboratorium mit kontrollierter Temperatur und Feuchte, die Wirkungen von niedrigem Barometerdruck auf die InsektenaktivitÄt untersucht. Stallfliegen (STOMOXYS CALCITRANS (L.)), Hausfliegen (MUSCA DOMESTICA L.), Mexikanische Fruchtfliegen (ANASTREPHA LUDENS (Loew)), Schaben (BLATELLA GERMANICA L.), PERIPLANETA AMERICANA (L.) und BLABERUS GIGANTEUS (L.) und Mücken (AEDES AEGYPTI (L.)) zeigten wÄhrend der Verminderung des Barometerdruckes eine verstÄrkte AktivitÄt. Bei Studien im Freien wÄhrend der kritischen Orkanperiode wurde ebenfalls eine ungewöhnlich grosse AktivitÄt der Arthopoden beobachtet. Nach dem Orkan tauchten bestimmte Insektenarten in abnorm grosser Zahl auf. besonders (LAPHYGMA FRUGIPERDA (J.E.Schmith)), (MEGALOPYGE OPERCULARIS(J.E.Schmith)) und. (NEZARA VIRIDULA (L.)).

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Résumé L'effet, sur l'activité des insectes, des basses pressions barométriques résultant de l'ouragan Carla, un des plus vastes et des plus destructeurs qui aient affecté la cÔte moyenne du Texas, a été. observé au laboratoire dans des conditions de température et d'humidité relative constantes. Les mouches des étables (STOMOXYS CALCITRANS (L.)), les mouches domestiques (MUSCA DOMESTICA L.), les mouches à fruits mexicaines (ANASTREPHA LUDENS (Loew)), les cafards (BLATELLA GERMANICA (L.)), PERIPLANETA AMERICANA (L.) et BLABERUS GIGANTEUS (L.) et les moustiques (AEDES AEGYPTI (L.)) ont manifesté une activité accrue correspondant à la réduction de la pression barométrique. Des études faites en plein air pendant la période d'ouragan ont également permis d'observer chez les arthropodes une activité inaccoutumée. A la suite de l'ouragan sont apparus en quantités anormales certains insectes tels que le(LAPHYGMA FRUGIPERDA (J.E. Smith)), le (MEGALOPYGE OPERCULARIS (J.E.Smith)) et le (NEZARA VIRIDULA (L.)).