Hybridase activity of human ribonuclease-1 revealed by a real-time fluorometric assay

Human ribonuclease-1 (hRNase-1) is an extracellular enzyme found in exocrine pancreas, blood, milk, saliva, urine and seminal plasma, which has been implicated in digestion of dietary RNA and in antiviral host defense. The enzyme is characterized by a high catalytic activity toward both single-stranded and double-stranded RNA. In this study, we explored the possibility that hRNase-1 may also be provided with a ribonuclease H activity, i.e. be able to digest the RNA component of RNA:DNA hybrids. For this purpose, we developed an accurate and sensitive real-time RNase H assay based on a fluorogenic substrate made of a 12 nt 5′-fluorescein-labeled RNA hybridized to a complementary 3′-quencher-modified DNA. Under physiological-like conditions, hRNase-1 was found to cleave the RNA:DNA hybrid very efficiently, as expressed by a k(cat)/K(m) of 330 000 M(−1) s(−1), a value that is over 180-fold higher than that obtained with the homologous bovine RNase A and only 8-fold lower than that measured with Escherichia coli RNase H. The kinetic characterization of hRNase-1 showed that its hybridase activity is maximal at neutral pH, increases with lowering ionic strength and is fully inhibited by the cytosolic RNase inhibitor. Overall, the reported data widen our knowledge of the enzymatic properties of hRNase-1 and provide new elements for the comprehension of its biological function

Molecular Interactions of Human Plasminogen with Fibronectin-binding Protein B (FnBPB), a Fibrinogen/Fibronectin-binding Protein from Staphylococcus aureus

none7siStaphylococcus aureus is a commensal bacterium that has the ability to cause superficial and deep-seated infections. Like several other invasive pathogens, S. aureus can capture plasminogen from the human host where it can be converted to plasmin by host plasminogen activators or by endogenously expressed staphylokinase. This study demonstrates that sortase-anchored cell wall-associated proteins are responsible for capturing the bulk of bound plasminogen. Two cell wall-associated proteins, the fibrinogen- and fibronectin-binding proteins A and B, were found to bind plasminogen, and one of them, FnBPB, was studied in detail. Plasminogen captured on the surface of S. aureus- or Lactococcus lactis-expressing FnBPB could be activated to the potent serine protease plasmin by staphylokinase and tissue plasminogen activator. Plasminogen bound to recombinant FnBPB with a KD of 0.532 μm as determined by surface plasmon resonance. Plasminogen binding did not to occur by the same mechanism through which FnBPB binds to fibrinogen. Indeed, FnBPB could bind both ligands simultaneously indicating that their binding sites do not overlap. The N3 subdomain of FnBPB contains the full plasminogen-binding site, and this includes, at least in part, two conserved patches of surface-located lysine residues that were recognized by kringle 4 of the host protein.openPietrocola, G; Nobile, G; Gianotti, V; Zapotoczna, M; Foster, Tj; Geoghegan, Ja; Speziale, P.Pietrocola, Giampiero; Nobile, Giulia; Gianotti, Valentina; Zapotoczna, M; Foster, Tj; Geoghegan, Ja; Speziale, Pietr

Clinical Effects of an ACT-Group Training in Children and Adolescents with Attention-Deficit/Hyperactivity Disorder

Abstract Objective The aim of the present study is evaluate the effectiveness of an Acceptance and Commitment Therapy (ACT)-based training protocol, in adjunct to token economy and previous parent training, in a sample of children with Attention-Deficit/Hyperactivity Disorder (ADHD). By promoting the reduction of immediate responses to thoughts and feelings, we aimed to reduce the impulsive behaviour of children and to improve their self-regulation. Methods The protocol was centred on awareness of the present moment, defusion and acceptance of feelings and emotions. Behavioural (Conners' Parent Rating Scale -Revised: Long version, CPRS-R:L) and severity measures (Clinical Global Impression -Severity, CGI-S) were assessed before and after treatment in a clinical sample of 31 children aged 8–13 years. Results At the end of the ACT protocol, children showed significant improvement in global functioning and behavioural symptoms. There were significant improvements in the CPRS subscales Cognitive Problems (p = 0.005), Hyperactivity (p = 0.006), Perfectionism (p = 0.017), ADHD Index (p = 0.023), Global Index: Restless–Impulsive (p = 0.023), Global Index: Total (p = 0.036), DSM IV Inattentive (p = 0.029), DSM IV Hyperactive–Impulsive (p = 0.016), and DSM IV Total (p = 0.003). When controlling for the confounding effect of pharmacological therapy, comorbidities and socio-economic status, treatment maintained a significant effect on the CPRS subscales Perfectionism (partial η2 = 0.31, p < 0.01), Global Index: Restless–Impulsive (partial η2 = 0.29, p < 0.01), Global Index: Total (partial η2 = 0.31, p < 0.01), DSM IV Hyperactive–Impulsive (partial η2 = 0.20, p = 0.02). Symptom severity as rated by CGI-S scores decreased in 74.2% of the children. Conclusions This preliminary work on an Acceptance and Commitment Therapy-based child training in children affected by ADHD resulted in significant improvements, measured by a rating scale specific for ADHD

Bacterial Expression of Mouse Argonaute 2 for Functional and Mutational Studies

RNA interference (RNAi) is a post-transcriptional gene-silencing process that occurs in many eukaryotic organisms upon intracellular exposure to double-stranded RNA. Argonaute 2 (Ago2) protein is the catalytic engine of mammalian RNAi. It contains a PIWI domain that is structurally related to RNases H and possibly shares with them a two-metal-ion catalysis mechanism. Here we describe the expression in E. coli of mouse Ago2 and testing of its enzymatic activity in a RISC assay, i.e., for the ability to cleave a target RNA in a single position specified by a complementary small interfering RNA (siRNA). The results show that the enzyme can load the siRNA and cleave the complementary RNA in absence of other cellular factors, as described for human Ago2. It was also found that mutation of Arg669, a residue previously proposed to be involved in substrate and/or B metal ion binding, doesn’t affect the enzymatic activity, suggesting that this residue doesn’t belong to the active site

Latent classes of emotional and behavioural problems in epidemiological and referred samples and their relations to DSM-IV diagnoses

Researchers\u2019 interest have recently moved toward the identification of recurrent psychopathological profiles characterized by concurrent elevations on different behavioural and emotional traits. This new strategy turned to be useful in terms of diagnosis and outcome prediction. We used a person-centred statistical approach to examine whether different groups could be identified in a referred sample and in a general-population sample of children and adolescents, and we investigated their relation to DSM-IV diagnoses. A latent class analysis (LCA) was performed on the Child Behaviour Checklist (CBCL) syndrome scales of the referred sample (N = 1225), of the general-population sample (N = 3418), and of the total sample. Models estimating 1-class through 5-class solutions were compared and agreement in the classification of subjects was evaluated. Chi square analyses, a logistic regression, and a multinomial logistic regression analysis were used to investigate the relations between classes and diagnoses. In the two samples and in the total sample, the best-fitting models were 4-class solutions. The identified classes were Internalizing Problems (15.68%), Severe Dysregulated (7.82%), Attention/Hyperactivity (10.19%), and Low Problems (66.32%). Subsequent analyses indicated a significant relationship between diagnoses and classes as well as a main association between the severe dysregulated class and comorbidity. Our data suggested the presence of four different psychopathological profiles related to different outcomes in terms of psychopathological diagnoses. In particular, our results underline the presence of a profile characterized by severe emotional and behavioural dysregulation that is mostly associated with the presence of multiple diagnosis