Thermodynamics and kinetics of the thermal unfolding of plastocyanin

The thermal denaturation of plastocyanin in aqueous solution was investigated by means of DSC, ESR and absorbance techniques, with the aim of determining the thermodynamic stability of the protein and of characterizing the thermally induced conformational changes of its active site. The DSC and absorbance experiments indicated an irreversible and kinetically controlled denaturation path. The extrapolation of the heat capacity and optical data at infinite scan rate made it possible to calculate the kinetic and thermodynamic parameters associated with the denaturation steps. The denaturation pathway proposed, and the parameters found from the calorimetric data, were checked by computer simulation using an equation containing the information necessary to describe the denaturation process in detail. ESR and absorbance measurements have shown that structural changes of the copper environment occur during the protein denaturation. In particular, the geometry of the copper-ligand atoms changes from being tetrahedral to square planar and the disruption of the active site precedes the global protein denaturation. The thermodynamic enthalpic change, the half-width transition temperature, and the value of ΔCp, were used to calculate the thermodynamic stability, ΔG, of the reversible process over the entire temperature range of denaturation. The low thermal stability found for plastocyanin, is discussed in connection with structural factors stabilizing the native state of a protein. Received: 17 July 1997 / Revised version: 22 November 1997 / Accepted: 15 January 1998     

The ant fauna of Castelporziano Presidential Reserve (Rome,Italy) as a model for the analysis of ant community structure in relation to environmental variation in Mediterranean ecosystems

Ants are often used as ecological indicators in environmental monitoring programs, but rarely in Europe, probably because of a lack of baseline knowledge on the relationship between ant community structure and environmental variation. We present this baseline knowledge for Italian ecosystems that are typical of the Mediterranean climate, in order to assess the value of ants as ecological indicators in this biogeographic zone. Sampling areas were selected inside the Castelporziano Natural Reserve (Rome, Italy). The elevated variety of habitats and diverse ecological conditions, as well as the different levels of human impact in the Reserve, make this area a suitable location to develop and test biological indicators of environmental quality. The ant community structure was compared among several Mediterranean ecosystems using pitfall trapping, multidimensional scaling, the indicator species analysis and the Functional Groups Approach. Key factors for differences in ant community structure seem to be the presence/absence of water, the level of light and microhabitat diversity. Our results suggest that Castelporziano is an informative area in order to develop a study model for the Italian ant fauna and the Functional Group Approach is a promising procedure to associate differences in ant community structures with differences in habitat stress and disturbance.     

Membrane lipids and proteins as modulators of urothelial endocytic vesicles pathways

The increased studies on urinary bladder umbrella cells as an important factor for maintaining the permeability barrier have suggested new pathways for the discoidal/fusiform endocytic vesicles which is one of the main features of the umbrella cells. The biological role of these vesicles was defined, for many years, as a membrane reservoir for the umbrella cell apical plasma membrane which are subject to an increased tension during the filling phase of the micturition cycle and, therefore, the vesicles are fused with the apical membrane. Upon voiding, the added membrane is reinserted via a non-clathrin or caveolin-dependant endocytosis thereby restoring the vesicle cytoplasmic pool. However, in the last decade, new evidence appeared indicating alternative pathways of the endocytic vesicles different than the cycling process of exocytosis/endocytosis. The purpose of this review is to analyze the molecular modulators, such as membrane lipids and proteins, in the permeability of endocytic vesicles, the sorting of endocytosed material to lysosomal degradation pathway and recycling of both membrane and fluid phases.     

The identification of globin messenger ribonucleic acid in newt erythropoietic cells.

Polyadenylated [poly(A)+]-RNA isolated from newt (Triturus cristatus) erythropoietic cells contained two main species sedimenting at 9S and 25S, and minor amounts of a 15-20S component. The 9S poly(A)+-RNA fraction induced synthesis of newt haemoglobin and globins in frog oocytes and in an mRNA-dependent rabbit reticulocyte lysate, confirming its identity as newt globin mRNA. Translation of 9S globin mRNA in reticulocyte lysate was concentration-dependent, the patterns of globin synthesis suggesting both preferential utilization and unequal amounts of the different globin mRNA subspecies. Globin mRNA activity was also evident in the 25S poly(A)+-RNA fraction whose localization in polyribosomes excluded its function as a nuclear globin mRNA precursor. Denaturation in formamide and estimation of its relative methyl content indicated that the 25S poly(A)+-RNA fraction contained equimolar amounts of 9S globin mRNA and 26S rRNA. Translation of the 25S fraction in reticulocyte lysate was less efficient than that of comparable amounts of 9S globin mRNA and induced a pattern of globin synthesis similar to that obtained with subsaturating amounts of 9S mRNA. The 25S mRNA-rRNA complex was considered to be a non-physiological aggregate generated by extraction of RNA in the presence of buffers of moderate to high ionic strength.     

Cysteine residues in a synthetic peptide corresponding to human follicle-stimulating hormone beta-subunit receptor-binding domain 81-95 [hFSH-beta-(81-95)] modulate the in vivo effects of hFSH-beta-(81-95) on the mouse estrous cycle.

We have previously reported that synthetic peptide amides corresponding to subdomains of the human FSH 3-subunit, hFSH-beta-(33--53) and hFSH-beta-(81--95), interact with the external domain of the FSH receptor in two in vitro model systems. Consistent with these in vitro observations, we found that intraperitoneal (i.p.) administration of each of these peptides prolonged vaginal estrus in normally cycling mice in vivo. Both hFSH-beta-(33--53) and hFSH-beta-(81--95) contain cysteine (Cys) residues with free sulfhydryl groups of potential significance in receptor interactions. To assess the possible involvement of these groups in the in vivo effects of hFSH-beta-(33--53) and hFSH-beta-(81--95), synthetic peptide analogs were prepared in which all Cys residues were replaced with serine (Ser). In the present study, we demonstrate that the in vivo effect of hFSH-beta-(33--53) on the mouse estrous cycle, extension of vaginal estrus, was not changed by substitution of Cys-51 with Ser. In contrast, mice receiving the Ser-substituted analog of hFSH-beta-(81--95) had normal estrus stages, but were arrested in diestrus. hFSH-beta-(33--53)-(81--95), a linear peptide encompassing both domains, also prolonged vaginal estrus. The Ser-substituted analog of this peptide, however, prolonged vaginal estrus in some of the mice tested and induced cycle arrest at diestrus in others. hFSH-beta-(90--95), the active subdomain at the C-terminus of hFSH-beta-(81--95), extended vaginal estrus, but diestrus stages were of normal duration. Its Ser-substituted analog, however, prolonged the estrus stage of the majority of mice treated, but induced diestrus arrest in some. The differing responses to these peptides are presumably due to interactions of the synthetic peptides with different regions of the FSH receptor. This further suggests that one consequence of ligand interaction with multiple receptor binding domains may be variable effects on ovarian function, and that Cys to Ser analogs may have value in the design of a novel class of synthetic peptides capable of fertility regulation and control.