Spectral and enzymatic properties of human recombinant myeloperoxidase: comparison with the mature enzyme.

Human recombinant myeloperoxidase (recMPO), purified from an engineered Chinese hamster ovary (CHO) cell line, has been characterized and compared to the mature enzyme isolated from polymorphonuclear leukocytes. Both molecules appear essentially similar in physicochemical enzymatic terms according to the following observations. 1. The unprocessed recombinant protein displays the characteristic light absorption spectra of ferric mature MPO and exhibits its typical spectral changes in the presence of dithionite or hydrogen peroxide. 2. The addition of 14C-labeled 5-aminolevulinic acid, a heme precursor, to the culture medium of recombinant CHO cells yields labeled recMPO, indicating the presence of a heme-like structure in the molecule. 3. Like mature MPO, recMPO has a peroxidatic activity and catalyzes the oxidation of chloride ions in the presence of hydrogen peroxide, producing hypochlorous acid as measured by the monochlorodimedon assay. For both enzymes, the chlorinating activity optimally occurs around pH 5.0 at about 100 microM of hydrogen peroxide and is strongly inhibited by methimazole. 4. Diethylpyrocarbonate significantly reduces the enzymatic activity of both molecules, suggesting that histidine residues may be of prime importance in the active site of the enzymes. 5. According to infrared spectroscopy data, both enzymes present a very similar secondary structure organization. In conclusion, the data suggest that the processing of the precursor enzyme (recMPO) into the mature form occurs without major structural and functional consequences.     

In vitro evaluation of glutathione peroxidase (GPx)-like activity and antioxidant properties of some Ebselen analogues

Four analogues of Ebselen were synthesized and their glutathione peroxidase activity and antioxidant property evaluated and compared to Ebselen. Among the studied compounds, only diselenide [3] exhibited both glutathione peroxidase activity and radical-scavenging capability. Compounds [3] and [4] showed a strong inhibitory effect (53% and 43%, respectively) on the lipid peroxidation of linoleic acid compared to Ebselen and selenide derivatives ([1] and [2]) which were less active (28%, 26% and 18% inhibition, respectively). A concentration-dependent inhibitory effect was also found in the model of the formation of ABTS*+ radical cation: 65% and 89% inhibition for compound [3] at 10(-4) M and 5 x 10(-5) M, respectively, and 68% and 90% for compound [4], compared to 14% and 52% inhibition for Ebselen and the diselenides [1] and [2] (29%, 46% and 45%, 68%, respectively). By EPR spin trapping technique, the following inhibitory profile of the Ebselen analogues was observed towards the formation of thiyl radicals: Ebselen = [3]>[1]>[2]>[4]. Studies with compound [3] are in progress on oxidative stress cell models.     

Yoyo-bang: a risk-aversion investment strategy by a perennial insect society

In 1978, Oster and Wilson proposed a bang-bang investment strategy for social insects in which colony size at maturity amplifies colony reproduction. In this paper, the investment strategies of the monogyne form of the fire ant, Solenopsis invicta, were compared to the predictions of the bang-bang model. Demographic census data, collected on fire ant mounds excavated every month during the years 1985 and 1988, revealed that colony reproduction was independent of colony size (~50,000 to ~250,000 workers). Why were mature S. invicta colonies up to five times larger than they needed to be to reproduce an annual batch of sexual offspring? To address this question, Oster and Wilson's bang-bang model was modified to a "yoyo-bang" investment strategy for perennial societies. In the yoyo-bang model, excess workers are a disposable reserve - a buffer - that can oscillate up or down depending on resource availability without adversely affecting annual reproductive cycles. The yoyo-bang model links colony size, colony survival and lifetime reproductive fitness.     

Effects of glucocorticoids on the respiratory burst of Chlamydia-primed THP-1 cells

We previously observed that the respiratory burst of human monocytes (THP-1 cell line) triggered by phorbol myristate acetate was strongly enhanced by a priming of the cells by Chlamydia pneumoniae [Biochem. Biophys. Res. Commun. 287 (2001) 781]. We describe here the modifications of the responses of Chlamydia-primed THP-1 cells to hydrocortisone (HCT) and methylprednisolone (MPL). HCT and MPL inhibited the production of the cytokines TNFα and IL-8. But HCT, which inhibited the respiratory burst in LPS-primed monocytes, paradoxically stimulated the phenomenon in Chlamydia-primed cells; MPL exerted no significant effect. Both glucocorticoids did not significantly modify the triggering effect of Chlamydia on NF-κB binding activity. On the expression of p22^phox, a protein subunit of the NADPH oxidase, HCT had an increasing and MPL a decreasing effect. Glucocorticoids thus had unexpected effects on the inflammatory response of Chlamydia-primed monocytes.     

The effect of inhibition of endothelial cell cyclooxygenase on arterial thrombosis

Arterial platelet thrombosis was induced in vivo in a branch of the mesenteric artery of the white Wistar rat by topical superfusion by adenosine diphosphate following local de-endothelialization of the dissected segment by means of a small electrical current. Detection of the thrombotic phenomena was performed by projection of the arterial segment onto a set of light sensitive elements, wich allows the registration of several discriminating parameters. The addition of arachidonic acid to the superfusing mixture results in an increase in thromboformation; this increasing effect can be blocked completely by prior inhibition od the vessel wall cyclooxygenase activity by inhibitors such as flurbiprofen or indomethacin. We therefore concluded that the arachidonate cascade, triggered within the endothelial cells, is involved in platelet-vessel wall interaction leading ultimately to local thrombosis.     

Hyperhydricity of Prunus avium shoots cultured on gelrite: a controlled stress response.

Hyperhydricity is a physiological disorder frequently affecting shoots vegetatively propagated in vitro. Hyperhydric shoots are characterised by a translucent aspect due to a chlorophyll deficiency, a not very developed cell wall and a high water content. Hyperhydricity of Prunus avium shoots was expressed in vitro in one multiplication cycle by replacing the gelling agent agar (normal shoots: NS) by gelrite (hyperhydric shoots: HS). P. avium shoots evolving towards the hyperhydric state produced higher amounts of ethylene, polyamines (PAs) and proline, which are substances considered as stress markers. A higher activity of glutathione peroxidase (GPX; EC 1.11.1.9), involved in organic hydroperoxide elimination, suggested an increased production of these compounds in HS. The unchanged free fatty acid composition indicated no HS membrane damages compared to NS. The ploidy level of HS nuclei was not affected, but the bigger size and the lower percentage of nuclei during the S phase suggested a slowing down of the cell cycle. The results argued for a stress response of the HS, but no signs of oxidative damages of lipid membrane and nucleus were observed. The discussion points out paradoxical results in a classical analysis of stress and suggests an alternative way of defense mechanisms in HS, involving homeostatic regulation and controlled degradation processes to maintain integrity and vital functions of the cell.     

Trypsin-like activity and thromboxane release in adult respiratory distress syndrome

Plasmatic immunoreactive trypsin (IRT), thromboxane and trypsin-like enzymatic activity were measured in 117 patients at risk of developing adult respiratory distress syndrome (ARDS) (53 multiple injury, 30 abdominal surgery, 17 acute pancreatitis, 12 burnt and 5 disseminated intravascular coagulation patients). 69 of these patients developed ARDS.Immunoreactive trypsin and thromboxane were measured by radio-immuno-assay and trypsin-like enzymatic activity by spectrophotometry, using a specific chromogenic substrate.Mean IRT value was 675 ng/ml in ARDS and 265 ng/ml in non ARDS patients (p < 0.05). Mean IRT value was 685 ng/ml in sepatic and 170 ng/ml in non septic patients (p < 0.01). An abnormal trypsin-like enzymatic activity was measured in 26 ARDS patients. In 60 patients (37 ARDS and 23 non ARDS), thromboxane appeared in plasma simultaneously or about 24 hours after the beginning of IRT release. The importance of thromboxane release parallels the intensity of IRT. Originating from pancreas, trypsin can appear in plasma either by absortion from gastrointestinal tract or after pancreatic ischemia.     

Peroxynitrite reacts with biological nitrogen-containing cyclic molecules by a radical pathway, as demonstrated by ultraweak luminescence coupled with ESR technique.

Ultraweak luminescence (uwCL) was coupled with electron spin resonance to study the reactions of 3 heterocyclic compounds (tryptophan, serotonin and imidazole) with peroxynitrite at pH 8.7. Tryptophan and serotonin reacted with emission of a flash peak of light (5 s) followed by a long-living light emission of +/- 80 s. Addition of the spin trap 4-POBN at different intervals, after the beginning of reaction revealed that a short-living free radical was produced in the case of serotonin and imidazole, but that with tryptophan, the initial radical rearranged into a relatively long-living radical, which was still formed when 4-POBN was added after 55 s (decreasing phase of uwCL).