Structure of the prolidase from Pyrococcus furiosus

The structure of prolidase from the hyperthermophilic archaeon Pyrococcus furiosus (Pfprol) has been solved and refined at 2.0 A resolution. This is the first structure of a prolidase, i.e., a peptidase specific for dipeptides having proline as the second residue. The asymmetric unit of the crystals contains a homodimer of the enzyme. Each of the two protein subunits has two domains. The C-terminal domain includes the catalytic site, which is centered on a dinuclear metal cluster. In the as-isolated form of Pfprol, the active-site metal atoms are Co(II) [Ghosh, M., et al. (1998) J. Bacteriol. 180, 4781-9]. An unexpected finding is that in the crystalline enzyme the active-site metal atoms are Zn(II), presumably as a result of metal exchange during crystallization. Both of the Zn(II) atoms are five-coordinate. The ligands include a bridging water molecule or hydroxide ion, which is likely to act as a nucleophile in the catalytic reaction. The two-domain polypeptide fold of Pfprol is similar to the folds of two functionally related enzymes, aminopeptidase P (APPro) and creatinase. In addition, the catalytic C-terminal domain of Pfprol has a polypeptide fold resembling that of the sole domain of a fourth enzyme, methionine aminopeptidase (MetAP). The active sites of APPro and MetAP, like that of Pfprol, include a dinuclear metal center. The metal ligands in the three enzymes are homologous. Comparisons with the molecular structures of APPro and MetAP suggest how Pfprol discriminates against oligopeptides and in favor of Xaa-Pro substrates. The crystal structure of Pfprol was solved by multiple-wavelength anomalous dispersion. The crystals yielded diffraction data of relatively high quality and resolution, despite the fact that one of the two protein subunits in the asymmetric unit was found to be significantly disordered. The final R and R(free) values are 0.24 and 0.28, respectively.     

Susceptibility of two strains of Amblyseius andersoni Chant. (Acari: phytoseiidae) to dithiocarbamate fungicides

The effect of five fungicides on adult female mortality, fecundity, egg hatch and on larval mortality was examined in the laboratory using a fungicide-susceptible and a fungicide-resistant strain of the predaceous phytoseiid mite, Amblyseius andersoni Chant. The combined effect of each fungicide was compared using a coefficient of toxicity computed using the above four toxicity factors. The susceptible and resistant strains were most affected by the bidithiocarbommate fungicides mancozeb, metiram and propineb. Ziram and dithianon had little effect on the S strain and ziram had little effect on the R strain. The toxicity of dithianon was not tested using the R strain. A resistance factor (LC₅₀ R strain: LC₅₀ S strain) of 6.13 for mancozeb was estimated for the R strain. Mancozeb resistance was a heritable trait and was primarily associated with the adult female.     

Activation studies with amines and amino acids of the α-carbonic anhydrase from the pathogenic protozoan Trypanosoma cruzi

The activation of a α-class carbonic anhydrase (CAs, EC 4.2.1.1) from Trypanosoma cruzi (TcCA) was investigated with the best known classes of activators, the amino acids and aromatic/heterocyclic amines. The best TcCA activators were l-/d-DOPA and 4-amino-l-phenylalanine, which had activation constants in the range of 0.38–0.83?µM. Low micromolar activators were also l-/d-Trp, l-/d-Tyr, l-Gln, histamine and serotonin (K_As of 1.79–4.92?µM), whereas l-/d-His, l-/d-Phe and l-Asp were less effective activators (K_As of 6.39–18.7?µM). Amines such as dopamine, pyridyl-alkylamines, aminoethyl-piperazine or l-adrenaline, were devoid of activating effects on TcCA. Since the role of autacoids as many of these compounds investigated here is not known for the life cycle of T. cruzi, our work provides new tools for further investigations of factors connected with this protozoan pathogen infection.     

Long-term study (1974-1998) of seasonal changes in the phytoplankton in Lake Geneva: a multi-table approach

The monitoring of Lake Geneva began after one decade of eutrophicationand has provided a uniform set of phytoplankton data. This studyaimed to define the mean annual pattern of the seasonal successionsof phytoplankton species, and to determine whether inter-annualdistortions in this seasonal structure occur. We analysed the25 annual patterns using the STATIS multi-table method. Thephytoplankton successions in the first part of the year fittedwell with the pattern predicted by the Plankton Ecology Group(PEG) model. But the temporal evolution in the summer phytoplanktoncommunity differed from the PEG model, and was subject to between-yearsdifferences. We identified three homogeneous periods for theannual patterns: 1974–1985, 1986–1991 (except 1988)and 1992–1996 (including 1988). During the first period,phytoplankton succession followed the reference annual pattern,the typical autumnal community was missing during the secondperiod, and this autumn community developed earlier during thethird period. This study reflects the good ability of the phytoplanktoncommunity of Lake Geneva to resist both inter-annual fluctuationsand brutal shifts in the annual functioning of the system. Webelieve these shifts resulted from a gradual change in environmentalparameters, including the deepening of the phosphorus-depletedlayer and a change in its timing as a result of the long-termmeteorological changes.     

Chronic Cadmium Treatment Promotes Oxidative Stress and Endothelial Damage in Isolated Rat Aorta

Cadmium is a highly toxic metal that is present in phosphate fertilizers, and the incidence of cadmium poisoning in the general population has increased, mainly due to cigarette smoking. Once absorbed, cadmium accumulates in the tissues, causing harmful effects including high blood pressure, endothelial damage and oxidative stress. Oxidative stress is known to efficiently produce oxidized low-density lipoprotein and consequently atherosclerosis, mainly in the aorta. However, the mechanisms through which endothelial damage is induced by cadmium have not been elucidated. Thus, the aim of this study was to investigate the effects of this metal in the isolated aorta and the possible role of oxidative stress. Rats received 100 mg.L⁻¹ cadmium chloride (CdCl₂) in the drinking water or distilled water alone for four weeks. The pressor effect of cadmium was followed throughout the exposure period by tail plethysmography. At the end of the fourth week, the blood cadmium content was established, and the vascular reactivity of the isolated aorta to phenylephrine, acetylcholine and sodium nitroprusside was analyzed in the context of endothelium denudation and incubation with L-NAME, apocynin, losartan, enalapril, superoxide dismutase (SOD) or catalase. We observed an increased response to phenylephrine in cadmium-treated rats. This increase was abolished by catalase and SOD incubation. Apocynin treatment reduced the phenylephrine response in both treatment groups, but its effect was greater in cadmium-treated rats, and NOX2 expression was greater in the cadmium group. These results suggested that cadmium in blood concentrations similar to those found in occupationally exposed populations is able to stimulate NOX2 expression, contributing to oxidative stress and reducing NO bioavailability, despite enhanced eNOS expression. These findings suggest that cadmium exposure promotes endothelial damage that might contribute to inflammation, vascular injury and the development of atherosclerosis.     

Prolactin and thyrotropin response to thyrotropin-releasing hormone in premenopausal women with fibrocystic disease of the breast

Plasma PRL, TSH, total and free T4, total and free T3, and 17 beta-estradiol were evaluated in 29 premenopausal women with well-documented fibrocystic disease of the breast and in 29 healthy matched controls. Plasma PRL and TSH dynamics after acute TRH injection (200 micrograms i.v.) were also determined. All hormonal measurements were performed in the follicular phase of the menstrual cycle. Neither patients nor controls showed any thyroid function impairment. Basal plasma levels of the examined hormones were in the normal range in both groups. When considering data pertinent to PRL and TSH secretory patterns after TRH stimulation, no difference was recorded between patients and controls for TSH secretion, evaluated in terms of maximum peak, net (delta) and percent (delta %) increase above the baseline level and integrated area of response. On the contrary, the response of PRL was significantly higher in patients than controls (maximum peak at 20 min, mean +/- SE, 119.9 +/- 14.1 vs. 60.8 +/- 5.5 ng/ml, p less than 0.001; integrated area of response, 5,725 +/- 908 vs. 3,243 +/- 266 ng/ml/120 min, p less than 0.01). The results are compatible with the view that, in most patients with fibrocystic disease of the breast, there are abnormalities in the control of PRL secretion, which lead to enhanced release of the hormone after stimulation. In such cases the control of TSH appears to be operating normally.     

Soluble proteins from chemosensory organs of Eurycantha calcarata (Insects, Phasmatodea)

Three related nucleotide sequences, encoding mature proteins of 108-113 amino acids, have been obtained from antennal cDNA of the Phasmid Eurycantha calcarata. Among these, one is also expressed in the tarsi as demonstrated by N-terminal sequence and mass spectrometric analyses of protein samples isolated from both organs. PCR experiments performed with specific primers, showed that this species is also expressed in the mouth organs and in the cuticle, while the other two are antennal specific. All three isoforms are similar to Drosophila OS-D and other proteins reported in several insect orders, but one of them is significantly different from the other two. The best conserved elements are the N-terminal region and the four cysteine residues. Accurate ESMS measurements indicated that all cysteines are involved in two disulphide bonds and ruled out the occurrence of additional post-translational modifications. Polyclonal antibodies, raised against the purified protein, did not react with proteins of the same class expressed in another Phasmid species, Carausius morosus, and in the orthopteran Schistocerca gregaria, nor did antibodies against these proteins recognise those of E. calcarata.     

Ecology and management of exotic and endemic Asian longhorned beetle Anoplophora glabripennis

• 1 The Asian longhorned beetle is native to China and Korea, and was found for the first time outside its native habitat in the U.S.A. in 1996, with subsequent detections being made in Canada and several European countries.
• 2 We review the taxonomy, distribution, basic biology, behaviour, ecology and management of endemic and exotic Anoplophora glabripennis, including information that is available in the extensive Chinese literature.
• 3 This species has caused massive mortality of Populus species in China and models have demonstrated that it could become established in many locations worldwide.
• 4 Anoplophora glabripennis is polyphagous but prefers Acer, Salix and Populus, section Aigeiros.
• 5 Although A. glabripennis adults do not disperse far when surrounded by host trees, they have the potential to fly more than 2000 m in a season.
• 6 Volatile organic compounds from preferred host trees are attractive to A. glabripennis and this attraction is heightened by drought stress. Males and females orientate to a volatile released by female A. glabripennis and males attempt to copulate after contacting a sex pheromone on the female cuticle.
• 7 At present, A. glabripennis is being (or has been) eradicated from areas where it has been introduced. After detection, extensive surveys are conducted and, if breeding populations are detected, at the very least, infested trees are removed and destroyed. Close attention is paid to imported solid wood packaging material to prevent new introductions.
• 8 Standard practice to control A. glabripennis in China is to spray insecticides in tree canopies. In North America, largely as a preventative measure, systemic insecticides are injected into trees. Entomopathogenic fungi have been developed for the control of A. glabripennis, and entomopathogenic nematodes, coleopteran and hymenopteran parasitoids and predatory woodpeckers have been investigated as biocontrol agents.
• 9 Ecological control of A. glabripennis in China involves planting mixtures of preferred and nonpreferred tree species, and this practice can successfully prevent outbreaks.