The disintegrins ADAM10 and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein

We showed previously that PrPc undergoes constitutive and phorbol ester-regulated cleavage inside the 106-126 toxic domain of the protein, leading to the production of a fragment referred to as N1. Here we show by a pharmacological approach that o-phenanthroline, a general zinc-metalloprotease inhibitors, as well as BB3103 and TAPI, the inhibitors of metalloenzymes ADAM10 (A disintegrin and metalloprotease); and TACE, tumor necrosis factor alpha-converting enzyme; ADAM17), respectively, drastically reduce N1 formation. We set up stable human embryonic kidney 293 transfectants overexpressing human ADAM10 and TACE, and we demonstrate that ADAM10 contributes to constitutive N1 production whereas TACE mainly participates in regulated N1 formation. Furthermore, constitutive N1 secretion is drastically reduced in fibroblasts deficient for ADAM10 whereas phorbol 12,13-dibutyrate-regulated N1 production is fully abolished in TACE-deficient cells. Altogether, our data demonstrate for the first time that disintegrins could participate in the catabolism of glycosyl phosphoinositide-anchored proteins such as PrPc. Second, our study identifies ADAM10 and ADAM17 as the protease candidates responsible for normal cleavage of PrPc. Therefore, these disintegrins could be seen as putative cellular targets of a therapeutic strategy aimed at increasing normal PrPc breakdown and thereby depleting cells of the putative 106-126 "toxic" domain of PrPc.     

Innate immune recognition of microbes through Nod1 and Nod2: implications for disease

Nod1 and Nod2 are cytosolic proteins involved in intracellular recognition of microbes and their products. Recently, it was shown that these proteins recognize different moieties of bacterial peptidoglycan (PGN) mediating non-specific pathogen resistance and possibly generating signals for the adaptive immune response. Moreover, mutations in the gene encoding Nod2 are associated with increased susceptibility to chronic inflammatory disorders.     

The production and evaluation of antibodies for enzyme immunoassay of AZTTP

We describe the development of the first enzyme immunoassay for quantifying AZTTP that does not use of radioactive labeling. Anti-AZTTP antibodies were raised in rabbits by immunizing with an AZTTP-kelhoyle limpet hemocyanin (KLH) conjugate. Competitive immunoassays indicated a nanomolar sensitivity to AZTTP. One of the antisera produced was specific for AZTTP.     

Gas Exchange, MRS and NIRS Assessment of Metabolic Transients in Skeletal Muscle

The study of the kinetics of O₂ consumption (o₂) at the onsetand offset of constant-load submaximal exercise (o₂ on- andoff-kinetics) is useful from a practical point of view (a fasteradjustment of oxidative metabolism following an increased metabolicdemand reduces the need for substrate level phosphorylation,with implications on exercise tolerance and muscle fatigue)and can give valuable insights into the regulation of oxidativemetabolism in skeletal muscle. Measurements have been carriedout both in man and in animals, at the tissue and at the wholebody level. At the tissue level, the o₂ on- and off-kineticswere determined: a) Directly, by dynamic solution of the Fickequation throughout the transients; attempts were also madeto obtain similar informations by near-infrared spectroscopy.b) Indirectly, from the kinetics of phosphocreatine hydrolysisand resynthesis, by chemical methods or by ³¹P magnetic resonancespectroscopy. At the whole body level, o₂ on- and off-kineticsare determined from breath-by-breath measurements of pulmonarygas exchange. The o₂ = f(t) function is a complex one, particularlyduring the on-transient. The so-called "phase 2" of the o₂ on-response,as well as the o₂ off-response, yield relevant metabolic informations.In muscle the o₂ on- and off-kinetics are characterized by half-times(t) of 15–20 sec. At the whole-body level, t of the o₂on-kinetics show a wider variability, related to the experimentalprotocol and to other factors. The o₂ off-phase is more constant,and its kinetic parameters appear closer to those obtained atthe tissue level. The study of the o₂ kinetics is valuable fora functional evaluation of skeletal muscle oxidative metabolism.In ordinary conditions muscle o₂ kinetics appears mainly imposedby intrinsic (metabolic) rather than extrinsic (O₂ delivery)factors.     

Day-night variation in aggressive behavior among psychiatric inpatients

Self-directed aggressive behaviors of human beings show a 24h pattern. The aim of this study was to evaluate if violence of psychiatric inpatients against one another and hospital staff varies over the 24h. The clock time occurrence of 334 episodes of assault behaviors by 119 psychiatric inpatients (78 males and 41 females, mean age 34.8 ± 11.3 years) committed during a 5-year span in the psychiatric unit of the university-based hospital of Ferrara, Italy, was evaluated. The clock time of each event was categorized by hour during the 24h and into one of four 6h intervals for analysis of temporal variation by cosinor and χ² tests, respectively. A significant 24h variation, characterized by an early afternoon peak, was detected irrespective of gender and number (single vs. repeated) of episodes committed. Changes during the 24h in ward activity, patient contact, and endogenous circadian rhythms are likely to contribute to the observed 24h pattern, although further study is needed to confirm our findings and to define causal factors. (Chronobiology International, 18(3), 503-511, 2001)     

Population ecology, nonlinear dynamics, and social evolution. I. Associations among nonrelatives

Using an individual-based and genetically explicit simulation model, we explore the evolution of sociality within a population-ecology and nonlinear-dynamics framework. Assuming that individual fitness is a unimodal function of group size and that cooperation may carry a relative fitness cost, we consider the evolution of one-generation breeding associations among nonrelatives. We explore how parameters such as the intrinsic rate of growth and group and global carrying capacities may influence social evolution and how social evolution may, in turn, influence and be influenced by emerging group-level and population-wide dynamics. We find that group living and cooperation evolve under a wide range of parameter values, even when cooperation is costly and the interactions can be defined as altruistic. Greater levels of cooperation, however, did evolve when cooperation carried a low or no relative fitness cost. Larger group carrying capacities allowed the evolution of larger groups but also resulted in lower cooperative tendencies. When the intrinsic rate of growth was not too small and control of the global population size was density dependent, the evolution of large cooperative tendencies resulted in dynamically unstable groups and populations. These results are consistent with the existence and typical group sizes of organisms ranging from the pleometrotic ants to the colonial birds and the global population outbreaks and crashes characteristic of organisms such as the migratory locusts and the tree-killing bark beetles.     

Determination of hammerhead ribozyme kinetic constants at high molar ratio ribozyme-substrate

 Hammerhead ribozymes provide valuable tools in the field of gene therapy due to their cleavage specificity and the broad range of RNA targets. A major prerequisite for the selection of suitable ribozymes for in vivo application is represented by in vitro determination of ribozyme cleavage kinetic constants. From the experimental cleavage data, kinetic constants are usually calculated under the assumption of rapid conversion of the substrate into the ribozyme-substrate complex. However, this condition is often not satisfied for ribozymes carrying additional RNA stretches, due to cloning strategies or necessary for ribozyme expression in the cell. To overcome this problem, we propose a mathematical model which is able to calculate ribozyme kinetic constants in the case of non-rapid conversion of substrate into ribozyme-substrate complex. In addition, our system gives the opportunity to evaluate the nature of the S conversion into ES through the determination of a model parameter. The validity of the proposed model is restricted to the hypothesis of a ribozyme excess over the substrate at the beginning of the cleavage reaction and to the absence of any mass exchange with the external environment. Received: 1 February 2001 / Revised version: 1 September 2001 / Published online: 23 August 2002     

Down-regulation of macrophage CD9 expression by interferon-gamma.

CD9, a member of the tetraspanin family is a cell surface marker expressed on myeloid and nonmyeloid as well as on neoplastic cells. The present study has focused on the role of inflammation and macrophage activation in the regulation of CD9 expression. We report that the expression of CD9 on primary cultures of murine peritoneal macrophages was down regulated by Interferon-gamma, IFN-gamma. This down regulation was concentration-dependent and maximal by 48 h. The changes in surface expression were consistent with similar reductions in CD9 protein and message levels by Western and Northern blot analyses. The mechanism by which IFN-gamma decreases CD9 expression appears to be through the Stat1 signaling pathway as Stat1 knockout mice did not demonstrate any reduction in CD9 expression by IFN-gamma treatment. These results represent the first evidence for the down regulation of CD9 expression with macrophage activation.