AtCYS1, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death.

In plants, cysteine protease inhibitors are involved in the regulation of protein turnover and play an important role in resistance against insects and pathogens. AtCYS1 from Arabidopsis thaliana encodes a protein of 102 amino acids that contains the conserved motif of cysteine protease inhibitors belonging to the cystatin superfamily (Gln-Val-Val-Ala-Gly). Recombinant A. thaliana cystatin-1 (AtCYS1) was expressed in Escherichia coli and purified. AtCYS1 inhibits the catalytic activity of papain (Kd = 4.0 x 10-2 micro m, at pH 7.0 and 25 degrees C), generally taken as a molecular model of cysteine proteases. The molecular bases for papain inhibition by AtCYS1 have been analysed taking into account the three-dimensional structure of the papain-stefin B complex. AtCYS1 is constitutively expressed in roots and in developing siliques of A. thaliana. In leaves, AtCYS1 is strongly induced by wounding, by challenge with avirulent pathogens and by nitric oxide (NO). The overexpression of AtCYS1 blocks cell death activated by either avirulent pathogens or by oxidative and nitrosative stress in both A. thaliana suspension cultured cells and in transgenic tobacco plants. The suppression of the NO-mediated cell death in plants overexpressing AtCYS1 provides the evidence that NO is not cytotoxic for the plant, indicating that NO functions as cell death trigger through the stimulation of an active process, in which cysteine proteases and theirs proteinaceous inhibitors appear to play a crucial role.     

Home range dynamics of male roe deerCapreolus capreolus in a mountainous habitat

Male spacing behaviour of roe deer Capreolus capreolus (Linnaeus, 1758) was studied in a wooded mountainous habitat in the Casentinesi Forest National Park, Italy. Data were collected using radio-tracking techniques from March 1997 to February 1998. Annual, seasonal, and bimonthly home ranges were analysed. Different factors may influence male spacing behaviour throughout the year. Winter home range sizes may be dependent on environmental conditions, while social factors could determine a high level of individual variability during the territorial and reproductive period. Prime age males showed great spatial stability, in contrast to the ranging movements of yearlings.     

Forecasting airborne pollen concentrations: Development of local models

People's sensitivity to allergies may representone of the most important health factors of thenext century to which attention must be paid inorder to reduce the incidence of social costsand improve the quality of life.Taking into consideration the earnest requestsof the medical-scientific communityEmilia-Romagna ARPA (Regional Agency for thePrevention of the Environment) moved theattention from the monitoring to a short andmedium term prediction of the concentration ofallergenic pollens in the air in order toachieve a more effective therapeutic action.Our main objectives are to improve seasonalforecasts and to interpret anomalous years.A neural network model for grass pollenforecasting has been implemented. Inputvariables were meteorological situations, i.e.,daily temperature (max., min. and average) andrainfall, in addition to combinations ofindividual variables and their thresholds. Theoutput was daily pollen concentration.The model was able to understand and predictanomalous years. We demonstrate that therelationships between pollen concentrations andmeteorological situations are independent fromsite. This means that such models canunderstand the differences in differentareas.     

Biopsy coupled to quantitative immunofluorescence: a new method to study the human vascular endothelium.

Limited availability of endothelial tissue is a major constraint when investigating the cellular mechanisms of endothelial dysfunction in patients with metabolic and cardiovascular diseases. We propose a novel approach that combines collection of 200-1,000 endothelial cells from a superficial forearm vein or the radial artery, with reliable measurements of protein expression by quantitative immunofluorescence analysis. This method was validated against immunoblot analysis in cultured endothelial cells. Levels of vascular endothelial cell activation, oxidative stress, and nitric oxide synthase expression were measured and compared in five patients with severe chronic heart failure and in four healthy age-matched subjects. In summary, vascular endothelial biopsy coupled with measurement of protein expression by quantitative immunofluorescence analysis provides a novel approach to the study of the vascular endothelium in humans.     

Ganglion cells and topographically related nerves in the vallate papilla/von Ebner gland complex.

Ganglion cells and topographically related nerves in the vallate papilla/von Ebner gland complex were investigated in rat tongue by cytochemical, immunocytochemical, and ultrastructural methods to evaluate the possible presence of different neuronal subpopulations. Immunostaining for neurofilaments and protein gene product 9.5 revealed ganglionic cell bodies and nerve fibers. A large part of the neurons were positive at immunostaining for neuronal nitric oxide synthase (NOS), vesicular acetylcholine transporter (VAChT), or vasoactive intestinal peptide (VIP). A small subset of nerve fibers revealed immunoreactivity for cholecystokinin. Axons traveling under the lingual epithelium were evidenced by their content of calcitonin gene-related peptide (CGRP) or substance P (SP). Cell bodies positive for SP or CGRP were not detected. Using methods of co-localization, three different neuronal classes were detected. The main population was composed of AChE/NADPH-diaphorase (NADPHd)-positive cells. Small groups of acetylcholine esterase (AChE)-positive/NADPHd-negative cells were visible. Isolated neurons were AChE-negative/NADPHd-positive. The results of co-localization experiments for VAChT/NOS were consistent with those obtained by cytochemical co-localization of AChE and NADPHd. Experiments of co-localization for peptidergic and nitrergic structures revealed CGRP- and SP-immunoreactive fibers in the vallate papilla/von Ebner gland ganglion. In conclusion, the results demonstrated in the VP/VEG complex peptidergic, cholinergic, and nitrergic neurons. The presence of different neuronal subclasses suggests that a certain degree of functional specialization may exist.     

Properties of endogenous β-glucosidase of a Pichia anomala strain isolated from Sicilian musts and wines

The AL 112 strain, isolated from 361 yeast strains in Sicilian musts and wines, has been identified by biochemical and molecular methods as belonging to Pichia anomala, and your endogenous β-glucosidase (βG, EC 3.2.1.21) subsequently characterised. This strain not only has extremely high specific productivity of βG, but above all shows arabinosidase (Ara, EC 3.2.1.55) activity, essential for aroma enhancement of wine. βG from Al 112 is activated by ethanol at the concentrations typically found in wine; it is not inhibited by fructose, whilst glucose, a non-competitive inhibitor, despite lowering activity, actually protects the enzyme from factors that could damage it. It has an optimum temperature of 20 °C, compatible with typical cellar conditions, and stability in model must-wine and wine solutions ≥40 days.     

Linkage disequilibrium between GABRB3 gene and nonsyndromic familial cleft lip with or without cleft palate

The malformation of nonsyndromic cleft lip with or without cleft palate (CL/P) is a common congenital disease that affects approximately 1/1000 newborns in Caucasian populations. Genetic studies indicate that CL/P has the characteristics of a complex genetic trait. Linkage analysis and mouse-model knockout studies have suggested several candidate genes mapping in different chromosome regions for CL/P malformation. On these grounds, we have investigated, by linkage disequilibrium (LD) and parametric and nonparametric linkage analyses, five different candidate genes, including those for the beta3 subunit of the gamma-aminobutyric acid receptor (GABRB3), glutamic acid decarboxylase 1 (GAD1), retinoic acid receptor alpha (RARA), transforming growth factor beta3 (TGFB3), and msh ( Drosophila) homeobox homolog 1 (MSX1). Interestingly, a significant LD between GABRB3 and CL/P was obtained ( P-value=0.008 in the allele-wise analysis for multiallelic markers), suggesting that the GABRB3 gene is involved in this congenital disease. This new finding in humans is in agreement with previously reported data obtained with the murine model. Indeed, mouse studies indicate a role for gamma-aminobutyric acid (GABA) and its receptor in normal palate development. Exclusion of the GAD1 gene, which encodes the GABA-producing enzyme, in CL/P pathogenesis was obtained in our study. Moreover, we were unable to confirm the involvement of the MSX1 gene in nonsyndromic CL/P. Modest evidence of LD between marker alleles and CL/P was found at the RARA and TGFB3 loci suggesting a minor role for these genes in our family set of nonsyndromic CL/P.     

Microsomal epoxide hydrolase polymorphisms and lung cancer risk: a quantitative review

To investigate the role of microsomal epoxide hydrolase (mEH) polymorphisms in the aetiology of lung cancer and to assess the interaction between mEH polymorphisms and smoking, we performed a meta-analysis of seven published studies, which included 2078 cases and 3081 controls, and a pooled analysis of eight studies (four published and four unpublished at that time) with a total of 986 cases and 1633 controls. The combined metaanalysis odds ratios (ORs) were 0.98 (95% confidence interval [CI] = 0.72-1.35) for polymorphism at amino acid 113 in exon 3 (His/His versus Tyr/Tyr genotype) and 1.00 (95% CI= 0.71-1.41) for polymorphism at amino acid 139 in exon 4 (Arg/Arg versus His/ His genotype). In the pooled analysis, we observed a significant decrease in lung cancer risk (OR = 0.70, 95% CI = 0.51-0.96) for exon 3 His/His genotype after adjustment for age, sex, smoking and centre. The protective effect of exon 3 polymorphism seems stronger for adenocarcinoma of the lung than for other histological types. The OR for high predicted mEH activity, compared with low activity, was 1.54 (95% CI = 0.77-3.07) in the meta analysis and 1.18 (95% CI = 0.92-1.52) in the pooled analysis. We did not find a consistent modification of the carcinogenic effect of smoking according to mEH polymorphism, although the risk of lung cancer decreased among never smokers with high mEH activity and among heavy smokers with the exon 3 His/His genotype. In conclusion, this study suggests a possible effect of mEH polymorphisms at exon 3 in modulating lung cancer. If present, this effect may vary among different populations, possibly because of interaction with genetic or environmental factors.     

Endosome to Golgi Transport of Ricin Is Independent of Clathrin and of the Rab9- and Rab11-GTPases

The plant toxin ricin is transported to the Golgi and the endoplasmic reticulum before translocation to the cytosol where it inhibits protein synthesis. The toxin can therefore be used to investigate pathways leading to the Golgi apparatus. Except for the Rab9-mediated transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network (TGN), transport routes between endosomes and the Golgi apparatus are still poorly characterized. To investigate endosome to Golgi transport, we have used here a modified ricin molecule containing a tyrosine sulfation site and quantified incorporation of radioactive sulfate, a TGN modification. A tetracycline-inducible mutant Rab9S21N HeLa cell line was constructed and characterized to study whether Rab9 was involved in transport of ricin to the TGN and, if not, to further investigate the route used by ricin. Induced expression of Rab9S21N inhibited Golgi transport of mannose 6-phosphate receptors but did not affect the sulfation of ricin, suggesting that ricin is transported to the TGN via a Rab9-independent pathway. Moreover, because Rab11 is present in the endosomal recycling compartment and the TGN, studies of transient transfections with mutant Rab11 were performed. The results indicated that routing of ricin from endosomes to the TGN occurs by a Rab11-independent pathway. Finally, because clathrin has been implicated in early endosome to TGN transport, ricin transport was investigated in cells with inducible expression of antisense to clathrin heavy chain. Importantly, endosome to TGN transport (sulfation of endocytosed ricin) was unchanged when clathrin function was abolished. In conclusion, ricin is transported from endosomes to the Golgi apparatus by a Rab9-, Rab11-, and clathrin-independent pathway.