Identification of the main ubiquitination site in human erythroid alpha-spectrin

Erythroid spectrin is the main component of the red cell membrane skeleton, which is very important in determining the shape, resistance to mechanical stresses and deformability of red cells. Previously we demonstrated that human erythroid alpha-spectrin is ubiquitinated in vitro and in vivo, and using recombinant peptides we identified on repeat 17 the main ubiquitination site of alpha-spectrin. In order to identify the lysine(s) involved in the ubiquitination process, in the present study we mutated the lysines by site-directed mutagenesis. We found that ubiquitination was dramatically inhibited in peptides carrying the mutation of lysine 27 on repeat 17 (mutants K25,27R and K27R). We also demonstrated that the correct folding of this protein is fundamental for its recognition by the ubiquitin conjugating system. Furthermore, the region flanking lysine 27 showed a 75% similarity with the leucine zipper pattern present in many regulatory proteins. Thus, a new potential ubiquitin recognition motif was identified in alpha-spectrin and may be present in several other proteins.     

Biomanipulation of Lago di Candia (Northern Italy): a three-year experience of aquatic macrophyte management

A long-term experimental aquatic plant management programme has been in progress since 1985 in the eutrophic Lago di Candia (Northern Italy). 7.7 ha of water chestnut (Trapa natans L.) were harvested in 1986, 8.2 ha in 1987 and 11.0 ha in 1988, from August to October, removing 334, 290 and 418 tons of fresh plant material, respectively, from the lake. This annual harvesting of about 50% of the total water chestnut cover resulted in the removal from the lake of about 70 kg yr⁻¹ of phosphorus, a significant portion of the external annual loading originating mainly from run-off and precipitation. In addition, up to 38t yr⁻¹ of organic matter and 1t yr⁻¹ of nitrogen were removed. First results of the effect of the harvesting on Secchi-disc transparency, oxygen, total phosphorus, phytoplankton and zooplankton are discussed also in connection with a study of fish manipulation (Giussaniet al., 1990).     

Identification of genetic loci affecting the establishment and development of Echinococcus multilocularis larvae in mice

Alveolar echinococcosis (AE) is a severe hepatic disorder caused by larval infection by the fox tapeworm Echinococcus multilocularis. The course of parasitic development and host reactions are known to vary significantly among host species, and even among different inbred strains of mice. As reported previously, after oral administration of parasite eggs, DBA/2 (D2) mice showed a higher rate of cyst establishment and more advanced protoscolex development in the liver than C57BL/6 (B6) mice. These findings strongly suggest that the outcome of AE is affected by host genetic factor(s). In the present study, the genetic basis of such strain-specific differences in susceptibility/resistance to AE in murine models was studied by whole-genome scanning for quantitative trait loci (QTLs) using a backcross of (B6 × D2)F₁ and D2 mice with varying susceptibility to E. multilocularis infection. For cyst establishment, genome linkage analysis identified one suggestive and one significant QTL on chromosomes (Chrs.) 9 and 6, respectively, whereas for protoscolex development, two suggestive and one highly significant QTLs were detected on Chrs. 6, 17 and 1, respectively. Our QTL analyses using murine AE models revealed that multiple genetic factors regulated host susceptibility/resistance to E. multilocularis infection. Moreover, our findings show that establishment of the parasite cysts in the liver is affected by QTLs that are distinct from those associated with the subsequent protoscolex development of the parasite, indicating that different host factors are involved in the host–parasite interplay at each developmental stage of the larval parasite. Further identification of responsible genes located on the identified QTLs could lead to the development of effective disease prevention and control strategies, including an intensive screening and clinical follow-up of genetically high-risk groups for AE infection.     

Identification and kinetics analysis of a novel heparin-binding site (KEDK) in human tenascin-C

The interaction between tenascin-C (TN-C), a multi-subunit extracellular matrix protein, and heparin was examined using a surface plasmon resonance-based technique on a Biacore system. The aims of the present study were to examine the affinity of fibronectin type III repeats of TN-C fragments (TNIII) for heparin, to investigate the role of the TNIII4 domains in the binding of TN-C to heparin, and to delineate a sequence of amino acids within the TNIII4 domain, which mediates cooperative heparin binding. At a physiological salt concentration, and pH 7.4, TNIII3-5 binds to heparin with high affinity (K(D) = 30 nm). However, a major heparin-binding site in TNIII5 produces a modest affinity binding at a K(D) near 4 microm, and a second site in TNIII4 enhances the binding by several orders of magnitude, although it was far too weak to produce an observable binding of TNIII4 by itself. Moreover, mutagenesis of the KEDK sequence in the TNIII4 domain resulted in the significant reduction of heparin-binding affinity. In addition, residues in the KEDK sequences are conserved in TN-C throughout mammalian evolution. Thus the structure-based sequence alignment, mutagenesis, and sequence conservation data together reveal a KEDK sequence in TNIII4 suggestive of a minor heparin-binding site. Finally, we demonstrate that TNIII4 contains binding sites for heparin sulfate proteoglycan and enhances the heparin sulfate proteoglycan-dependent human gingival fibroblast adhesion to TNIII5, thus providing the biological significance of heparin-binding site of TNIII4. These results suggest that the heparin-binding sites may traverse TNIII4-5 and thus require KEDK in TNIII4 for optimal heparin-binding.     

Pollen deposition in country villages of Eastern Liguria (Northern Italy)

Summary Recent pollen rain in 14 country villages of Ligurian hinterland was examined, in the framework of pollen deposition studies based upon moss polsters analysis, now in progress. The above mentioned localities are prevailingly situated in the Trebbia, Aveto and Vara valleys, within 300 and 1000 m a.s.l. For each locality a moss sample was examined (two for Torriglia: north and south) consisting of several sub-samples from various zones of the built-up area. All the samples have proved rich in pollen and provided spectra in accordance with qualitative and quantitative features of the urbanized areas vegetation and the spontaneous one of the neighbourhood. Sticking to general considerations, an attempt was made to compare the present results with those of the city area of Genova, previously obtained with the same methods.     

Experimental analysis of some factors affecting parental expenditure and investment inGasterosteus aculeatus (Gasterosteidae)

Synopsis Parental investment is the cost of providing parental care. Parental investment was measured in the paternal stickleback,Gasterosteus aculeatus, by comparing future survival (measured indirectly as energy content of the body) and growth of parental and non-parental males. The time taken by parental males to renest was also measured. Male energy content was unaffected after a single parental cycle and no difference in growth rate was detected. Re-nesting was delayed. The effect of stressing parental males by exposing them to potential predators of their offspring and reducing their ration level, was also investigated. Stressed males had reduced energy contents in comparison with unstressed parental males. The time taken by stressed males to re-nest was unaffected. Males on low rations did not fan significantly less than well-fed males. Males exposed to brood predators did fan significantly less than parental males not exposed to brood predators, but the former did spend nearly 60% of their time attacking the predators when present.     

Identification of the ligand binding site in Fas (CD95) and analysis of Fas-ligand interactions.

Fas (CD95), a member of the tumor necrosis factor receptor superfamily, and its ligand (FasL), a tumor necrosis factor-like protein, are intensely studied because their interaction on the cell surface is critical for the induction of programmed cell death (apoptosis) and the regulation of immune responses. The structure and specificity of the extracellular binding domains of Fas and its ligand were studied, in different laboratories, by combining molecular modeling, mutagenesis, and a variety of binding and functional experiments. Residues critical for the receptor-ligand interaction were identified and, in the absence of experimentally determined structures, binding sites and details of the Fas-ligand interactions were predicted. These studies provide an instructive example for the close combination of prediction and experiment and illustrate how insights into the structure and binding characteristics of Fas and its ligand were gradually refined. Discussed methodological aspects are representative of structure-function studies on extracellular domains of other single-path transmembrane proteins.     

Occurrence of radon in some schools of Parma and Reggio Emilia (Northern Italy)

Summary Forty-seven measurement of radon concentration were made in some schools of Parma, Reggio Emilia, Albinea and Borzano. The method used was that of activated carbon canisters, which were placed in classrooms, laboratories, libraries and headmaster's offices for at least 48 hours in the period November '90–March '91. It was possible to determine the amount of radon in each canister counting the Pb-214 and Bi-214 gamma emitters by means of NaI (Tl) and Ge (I) detectors. The mean radon concentrations were: 20 Bq/m³ in Parma; 24 Bq/m³ in Reggio Emilia; 46 Bq/m³ in Borzano and 52 Bq/m³ in Albinea. The values recorded in schools are similar to the values previously recorded in dwellings of Parma and Reggio Emilia.     

Determinant factors in the seedling establishment of Pasania edulis (Makino) Makino

Pasania edulis (Makino) Makino is one of the dominant Fagucea tree species in evergreen broad-leaved forests in southern Japan, and its regeneration success may have a major impact on the dynamics of evergreen broad-leaved forests. We conducted a field survey on the population process from acorn production to seedling establishment of P. edulis in an evergreen broad-leaved forest in Kagoshima, southern Japan, from 1995 to 2009. The acorn crop varied greatly among the 14 cohorts, with mast cropping being recorded every 3–4 years. The mortality rate of acorns was very high for all 14 cohorts (99.3–100 %). Important mortality factors were failure to mature (empty acorns), attack by Curculio weevil on trees, predation of dropped acorns by Apodemus mice and large or medium-sized mammals (wild boar (Sus scrofa leucomystax), badger (Meles meles anakuma), and raccoon dog (Nyctereutes procyonoides viverrinu)), and attacks on germinated acorns by the acorn borer (Coccotrypes graniceps). Among these factors, predation by Apodemus mice was the greatest contributor to annual fluctuations in total mortality until seedling establishment. Large or medium-sized mammals and the acorn borer also caused severe damage to dropped acorns in some years, but contributed little to annual fluctuations in total mortality. For successful regeneration, mast cropping was essential. However, a small population of Apodemus mice, that is, a low predation pressure, was also required during mast years.     

Identification and analysis of the acyl carrier protein (ACP) docking site on beta-ketoacyl-ACP synthase III

The molecular details that govern the specific interactions between acyl carrier protein (ACP) and the enzymes of fatty acid biosynthesis are unknown. We investigated the mechanism of ACP-protein interactions using a computational analysis to dock the NMR structure of ACP with the crystal structure of beta-ketoacyl-ACP synthase III (FabH) and experimentally tested the model by the biochemical analysis of FabH mutants. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The ACP interaction surface was defined by mutations that compromised FabH activity in the ACP-dependent assay but had no effect in the ACP-independent assay. ACP docked to a positively charged/hydrophobic patch adjacent to the active site tunnel on FabH, which included a conserved arginine (Arg-249) that was required for ACP docking. Kinetic analysis and direct binding studies between FabH and ACP confirmed the identification of Arg-249 as critical for FabH-ACP interaction. Our experiments reveal the significance of the positively charged/hydrophobic patch located adjacent to the active site cavities of the fatty acid biosynthesis enzymes and the high degree of sequence conservation in helix II of ACP across species.     

Root Strength and Root Area Ratio of Forest Species in Lombardy (Northern Italy)

Forest vegetation is known to increase hillslope stability by reinforcing soil shear resistance and by influencing hydrologic conditions of soil. Although the importance of plant root systems for hillslope stability has received considerable attention in recent years, the quantification of such an effect needs more investigation. In this paper, we present a synthesis of the data gathered in the last 5 years for some species in different locations of the Alps and Prealps of Lombardy (Northern Italy) with the aim to increase our knowledge on root tensile strength and on Root Area Ratio distribution within the soil. Concerning root tensile strength we developed tensile strength–diameter relationships for eight species: green alder (Alnus viridis(Chaix) D.C.), beech (Fagus sylvatica L.), red willow (Salix purpurea L.), goat willow (Salix caprea L.), hazel (Corylus avellana L.), European ash (Fraxinus excelsior L.), Norway spruce (Picea abies (L.) Karst.) and European larch (Larix decidua Mill.). Results show a great variability among the different species and also for the same species. In general, however, root strength (in terms of tension) tends to decrease with diameter according to a power law, as observed by other Authors. Comparing the power law fitting curves for the considered species, it can be observed that they fall in a relatively narrow band, with the exception of hazel, which appears the most resistant. Concerning the evaluation of root distribution within the soil we estimated the Root Area Ratio (the ratio between the area occupied by roots in a unit area of soil) according to its depth for five species (beech, Norway spruce, European larch, mixed hazel and ash) in three locations of Lombardy. Results show that there is a great variability of root density for the same species well as for different points at the same locality. The general behaviour of root density, in any case, is to decrease with depth according to a gamma function for all the studied species. The results presented in this paper contribute to expanding the knowledge on root resistance behaviour and on root density distribution within the soil. The studied location have allowed the implementation of soil–root reinforcement models and the evaluation of the vegetation contribution to soil stability.     

The main pollen season of Parietaria in Genoa (Italy): forecast possibilities

The authors propose a statistical model for evaluating the Beginning of the Main Pollen Season (BMPS) of Urticaceae (cf. Parietaria) in Genoa (Italy). For eight consecutive years (1981–1988) the mean daily concentrations of this pollen grain were studied in relation to the two meteorological factors which are most significant in triggering the beginning of maximal pollination, namely, mean daily air temperature and rainfall. From a multiple-regression statistical analysis, a formula has been extrapolated, which, one the basis of these meteorological data for the month of January, can be used to predict the onset of the BMPS to within nine days.     

Seasonal variability in pyrrolizidine alkaloids in Senecio inaequidens from the Val Venosta (Northern Italy)

Senecio inaequidens is a neophyte originating from South Africa that has managed to spread to Europe and colonize large areas. This plant has toxic pyrrolizidine alkaloids (PAs) that represent a health risk to humans and animals. The aim of this work was to measure the content of PAs of these plants in continuous intervals over the entire growing season. The plants were separated into their organs, such as sprouts, stems, leaves and inflorescences. PAs were extracted from the dried plants using an acidic methanol solution, purified and measured by GC/MS. The average PA content of plants over the growing season was 0.33% of dry weight. The highest PA levels were about 1% in the dry weight and were found in the young sprouts and flower heads. There were nine PAs present, of which six could be identified. The main alkaloid was retrorsine followed by senecivernine, senecionine, integerrimine, usaramine and seneciphylline. Due to the high PA content of the inflorescences, the long flowering period and the rapid invasion dynamics, this species presents a high health risk for humans and animals.     

Phytoplankton succession in the euphotic zone of Lake Corlo (Northern Italy) during summer stratification

Abstract

Lake Corlo is a dammed reservoir for hydro-electrical purposes in Northern Italy. Weekly samplings were made from May to August 1985 in the euphotic layer. Two major categories, flagellates and diatoms, alternatively dominated the algal community. Flagellates prevailed with stratified conditions. The strong influence of hydraulic conditions on phytoplakton succession is emphasized.     

Landscape history in the subalpine karst region of Moncodeno (Lombardy Prealps, Northern Italy)

In situ larch stumps have been found above the present forest line in the karst region of Moncodeno, Grigna Settentrionale, northern Italy, an area where very few trees currently grow. Samples from living trees at the treeline and in the forest nearby were collected to develop a reference chronology for cross-dating the stumps. The latter span the period between 1218 and 1900, and together with the living trees, which cover the last 150 years, they have led to the development of a 784-year larch tree-ring chronology (1218–2001). Age and location of the stumps reflect past human activity. Deforestation and grazing have intensified the erosion processes in the area. The soil has thinned out and fragmented allowing karst landforms, previously buried by the soil, to emerge. Presently, the rocky outcrops limit the establishment of young larches so that only few scattered trees are currently found.     

Tick species infesting ruminants in Italy: ecological and bio-climatic factors affecting the different regional distribution

The distribution of tick species able to infest ruminants in Italy is briefly reviewed on the basis of published records and new data obtained in several surveys carried out mainly in northern Italy. The most frequent and abundant tick species found on ruminants throughout Italy are Ixodes ricinus and Dermacentor marginatus, the first gradually replacing its association with sheep by the association with wild ruminants. Rhipicephalus bursa was mainly found on cattle while R. turanicus, which is considered one of the most sheep-associated species, was found only on wild boar. Haemaphysalis punctata, H. sulcata and H. parva are the other tick species quite frequently found on small ruminants when they are reared on pastures mainly in central-southern Italy. However, the land exploitation of these last decades has dramatically reduced the diversity of Italian environment and significantly modified the distribution and the abundance of ticks causing a heavy spread of the tick species characterized by a strong adaptability such as I. ricinus and R. sanguineus. If this trend continues, it is possible to hypothesise that few "resistant" tick species will gradually replace most of the others. This hypothesis seems to be confirmed by the high percentage observed for I. ricinus (57.4%) and R. sanguineus (35.73%) out of 8,712 specimens collected from several different hosts and from the environment.