Human endothelin converting enzyme-2 (ECE2): characterization of mRNA species and chromosomal localization.

Generation of the functionally pleiotropic members of the endothelin vasoactive peptide family is critically catalyzed by unique type II metalloproteases, termed endothelin converting enzymes (ECE). Isolation of human ECE-2 (EC 3.4.24.71) cDNAs revealed deduced open reading frames of 787 and 765 amino acids with approximately 60% identity with human ECE-1. Characterization of mRNA variants revealed mRNA structural diversity at the 5'-terminus. Two mRNA species exist containing distinct first and second exons. Furthermore, in one of these species, an in-frame deletion of the intracytoplasmic domain removed 29 amino acids. Because of the previously reported human genetic diseases ascribed to germline mutations of member genes of the endothelin family, ECE2 was localized in human chromosomes with fluorescence in situ hybridization and radiation hybrid mapping to 3q28-q29 and SHGC-20171/D3S1571, respectively.     

Assay of zofenopril and its active metabolite zofenoprilat by liquid chromatography coupled with tandem mass spectrometry

Zofenopril is a pro-drug designed to undergo metabolic hydrolysis yielding the active free sulfhydryl compound zofenoprilat, which is an angiotensin converting enzyme (ACE) inhibitor, endowed also with a marked cardioprotective activity. A simple, highly sensitive specific LC–MS–MS method was developed for the determination of zofenopril and zofenoprilat in human plasma. In order to prevent oxidative degradation of zofenoprilat and its internal standard, their free sulfhydryl groups were protected by treatment with N-ethylmaleimide (NEM), which produced the succinimide derivatives. The compounds and their corresponding fluorine derivatives, used as internal standards, were extracted from plasma with toluene. The reconstituted dried extracts were chromatographed and then monitored by a triple-stage-quadrupole instrument operating in the negative ion spray ionization mode. The method was validated over the concentration range of 1–300 ng/ml for zofenopril and 2–600 ng/ml for zofenoprilat. Inter- and intra-assay precision and accuracy of both zofenopril and zofenoprilat were better than 10%. The limit of quantitation was 1 ng/ml with zofenopril and 2 ng/ml with zofenoprilat. Extraction recovery proved to be on average 84.8% with zofenopril and 70.1% with zofenoprilat. Similar recoveries were shown by the above two internal standards. The method was applied to measure plasma concentrations of zofenopril and zofenoprilat in 18 healthy volunteers treated orally with zofenopril calcium salt at the dose of 60 mg.     

Biodiversity and ecosystem functioning: It is time for dispersal experiments

The experimental study of the relationship between biodiversity and ecosystem function has mainly addressed the effect of species and number of functional groups. In theory, this approach has mainly focused on how extinction affects function, whereas dispersal limitation of ecosystem function has been rarely discussed. A handful of seed introduction experiments, as well as numerous observations of the effects of long‐distance dispersal of alien species, indicate that ecosystem function may be strongly determined by dispersal limitation at the local, regional and/or global scales. We suggest that it is time to replace biodiversity manipulation experiments, based on random draw of species, with those addressing realistic scenarios of either extinction or dispersal. Experiments disentangling the dispersal limitation of ecosystem function should have to take into account the probability of arrival. The latter is defined as the probability that a propagule of a particular species will arrive at a particular community. Arrival probability depends on the dispersal ability and the number of propagules of a species, the distance a species needs to travel, and the permeability of the matrix landscape. Current databases, in particular those in northwestern and central Europe now enable robust estimation of arrival probability in plant communities. We suggest a general hypothesis claiming that dispersal limitation according to arrival probability will have ecosystem‐level effects different from those arising due to random arrival. This hypothesis may be rendered more region‐, landscape‐ or ecosystem‐specific by estimating arrival probabilities for different background conditions.     

Functional Effects of Adiponectin on Endothelial Progenitor Cells

Adiponectin is an adipokine whose plasma levels are inversely correlated to metabolic syndrome components. Adiponectin protects against atherosclerosis and decreases risks in myocardial infarction. Endothelial progenitor cells (EPCs) are a heterogeneous population of circulating cells involved in vascular repair and neovascularization. EPCs number is reduced in patients with cardiovascular disease. We hypothesize that the positive effects of adiponectin against atherosclerosis are explained in part by its interactions with EPCs. Cells were obtained from healthy volunteers' blood by mononuclear cell isolation and plating on collagen‐coated dishes. Three sub‐populations of EPCs were identified and characterized using flow cytometry. EPCs' expression of adiponectin receptors, AdipoR1, and AdipoR2 was evaluated by quantitative PCR. The effects of recombinant adiponectin on EPCs' susceptibility to apoptosis were assessed. Finally, expression of neutrophil elastase by EPCs and activity of this enzyme on adiponectin processing were assessed. Quantitative PCR analysis of EPCs mRNAs showed that AdipoR1 mRNA is expressed at higher levels than AdipoR2. Expression of AdipoR1 protein was confirmed by western blot. Adiponectin significantly increased survival of two sub‐populations of EPCs in conditions of serum deprivation. Such effect could not be demonstrated in the third EPCs sub‐population. We also demonstrated that EPCs, particularly one sub‐population, express neutrophil elastase. Neutrophil elastase activity was confirmed in EPCs' conditioned media. Adiponectin protects some EPCs sub‐populations against apoptosis and therefore could modulate EPCs ability to induce repair of vascular damage. Neutrophil elastase activity of EPCs could locally modulate adiponectin activity by its involvement in the generation of the globular form of adiponectin.     

Evaluation of a radioreceptor assay to assess exogenous estrogen activity in serum of patients with breast cancer.

A radioreceptor assay (RRA) for the determination of total estrogen activity, was set up and used to assess the possible presence of exogenous molecules with estrogen activity in serum; a comparison was made with the specific radioimmunoassay (RIA) for the endogenous estrogen 17-B estradiol (17-B-E2). The assay was first performed on sera from healthy people taking estrogens in the form of oral contraceptives or lotions for local application whose total estrogenic activity in the blood was assumed to be abnormal. The assay was then performed on serum from 98 patients with early breast cancer and 20 patients with metastasis, not undergoing hormone therapy. A higher estrogen activity was found in 2.5% of sera compared to the activity found using the RIA method which is specific for endogenous estrogen 17-B-E2, the RRA/17-B-E2 ratio being higher than 3. Increased estrogen activity was found in 10% serum samples from digoxin treated cardiopathic patients, with an RRA/17-B-E2 ratio ranging from 4.4 to 20. The RRA assay could prove useful for showing up exogenous estrogen activity from various sources (drugs, food) in sera of people in whom estrogen stimulation could be potentially dangerous (i.e. in patients with hormone-sensitive tumors). This exogenous activity could support a certain degree of neoplastic stimulation and, therefore, unfavourably condition the patients' therapeutic response.     

LEFKOVITCH'S ERROR

Abstract — Lefkovitch's formula for the probability of incompatibility between two binary characters can give incorrect results because it redundantly counts some possible compatibilities. The inaccuracy occurs when the characters have the same number of terminals showing the apomorphic state.     

Dormancy in peach (Prunus persica L.) flower buds

Morphological studies were carried out with peach flower buds collected monthly in 1989 and 1990, from two months before leaf fall (7 March) until two to three weeks before bloom (7/8 August). Chilled (2–4°C for 30 days) and unchilled buds were exposed to 20 to 25°C, 100% RH and continuous light. Gibberellin A₃ (3 ng or 30 ng) was applied to some of the non-chilled cuttings at three days intervals. Then, 12, 19, and 26 days after they were planted, the buds were sampled and processed for histological studies. Cultured flower buds (chilled or unchilled) had accelerated anther and gynoecium morphogenesis after 12 days under controlled conditions, compared to buds processed immediately after collection from the field. Chilling treatment augmented the bud culture effect, while Gibberellin A₃ applications to the excised buds retarded bud morphogenesis to a stage comparable to that of buds collected directly from the field. This, suggests that the comparatively high levels of Gibberellin A_1/3 we previously found in mid winter [15, 18] could be at least one of the factors that controls floral bud dormancy by retarding anther and gynoecium development.