Cyclo(His-Pro) up-regulates heme oxygenase 1 via activation of Nrf2-ARE signalling

Paraquat (1,1'-dimethyl-4,4'-bipyridinium), a widely used non-selective herbicide, is a redox cycling agent with adverse effects on dopamine systems. Epidemiological data have shown that exposure to paraquat is one of the several risk factors for Parkinson's disease. We have already shown that cyclo(His-Pro), an endogenous cyclic dipeptide produced by the cleavage of the thyrotropin releasing hormone, has a cytoprotective effect through a mechanism involving Nrf2 activation that decreases production of reactive oxygen species and increases glutathione synthesis. Using primary neuronal cultures and PC12 cells as targets of paraquat neurotoxicity, we addressed whether and how cyclo(His-Pro) causes cellular protective response against paraquat-mediated cell death. We found that cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion by up-regulating Nrf2 gene expression, triggering its nuclear accumulation and activating the expression of heme oxygenase1. These protective effects were abolished by RNA interference-mediated Nrf2 knock down whereas were unaffected by RNA interference-mediated Keap1 knock down. Inhibition of heme oxygenase activity decreased cyclo(His-Pro)-induced neuroprotection. These results suggest that cyclo(His-Pro), acting as a selective activator of the brain modulable Nrf2 pathway, may be a promising candidate as neuroprotective agent that act through induction of phase II genes.     

Identification of a phosphorylated form of phosphoenolpyruvate carboxykinase from the yeast Saccharomyces cerevisiae

A phosphoprotein of 65 kDa, as determined by SDS-gel electrophoresis, has been isolated from yeast crude extracts. This phospho form copurifies with phosphoenolpyruvate carboxykinase in the enzyme purification procedure worked out in our laboratory (Tortora, P., Hanozet, G.M. and Guerritore, A. (1985) Anal. Biochem. 144, 179-185). Moreover, both proteins bind strongly to 5'AMP-Sepharose 4B in the presence of Mn2+, whereas a substantially lower binding occurs if Mn2+ is replaced by Mg2+. This binding pattern is consistent with the well-known Mn2+-dependence of yeast phosphoenolpyruvate carboxykinase. These data suggest that the 65-kDa protein might be a phosphorylation product of the native enzyme. Furthermore, although the phospho form is not immunoprecipitated by anti-phosphoenolpyruvate carboxykinase antibodies, addition of Protein A-Sepharose CL-4B to crude extracts preincubated with the antibodies results in the binding to the resin of the phospho form, thus providing immunological evidence for its identification as a modified form of native enzyme. The same 65-kDa phosphoprotein is detectable in extracts from cells grown in the presence of [32P]Pi, as well as in cell extracts incubated with [gamma-32P]ATP. Moreover, digestion of the phosphoprotein with BrCN or with Staphylococcus aureus V8 proteinase, yields two and three fragments, respectively, which appear parallel to digestion products of phosphoenolpyruvate carboxykinase, again supporting the proposed identification. Finally, analysis of the phosphorylated amino acids in the 65-kDa protein shows that phosphoserine is the only labelled phosphoamino acid.     

The nitrite reductase gene of Pseudomonas aeruginosa: Effect of growth conditions on the expression and construction of a mutant by gene disruption

Abstract The expression of nitrite reductase has been tested in a wild-type strain of Pseudomonas aeruginosa (Pao1) as a function of nitrate concentration under anaerobic and aerobic conditions. Very low levels of basal expression are shown under non-denitrifying conditions (i.e. absence of nitrate, in both aerobic and anaerobic conditions); anaerobiosis is not required for high levels of enzyme production in the presence of nitrate. A Pseudomonas aeruginosa strain, mutated in the nitrite reductase gene, has been obtained by gene replacement. This mutant, the first of this species described up to now, is unable to grow under anaerobic conditions in the presence of nitrate. The anaerobic growth can be restored by complementation with the wild-type gene.     

Studies on glucose-induced inactivation of gluconeogenetic enzymes in adenylate cyclase and cAMP-dependent protein kinase yeast mutants

Glucose-induced inactivation of the gluconeogenetic enzymes fructose-1,6-biphosphatase, cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase was tested in yeast mutants defective in adenylate cyclase (cyr1 mutation) and in the cAMP-binding subunit of cAMP-dependent protein kinase (bcy 1 mutation). In the mutant AM7-11D (cyr1 mutation), glucose-induced cAMP overshoot was absent, and no significant inactivation of the gluconeogenetic enzymes was detected, thus supporting the role of cAMP in the process. Moreover, in the mutant AM9-8B (bcy1 mutation), no cAMP-dependent protein kinase activity was evidenced, and, in addition, a normal inactivation pattern was observed, thus indicating that other mechanisms evoked by glucose might be required in the process. In the double mutant AM7-11DR-4 (cyr1 bcy1 mutations), no inactivating effect was triggered by the sugar: this suggests that cAMP exerts some additional effect on the process, besides the activation of cAMP-dependent protein kinase. Furthermore, in AM7-11D, extracellular cAMP triggered about 50% of inactivation of fructose-1,6-bisphosphatase; this effect was largely reversed in acetate medium plus cycloheximide even after 150 min of incubation. However, an extensive and essentially irreversible inactivation was evidenced in the presence of glucose plus cAMP, whereas glucose alone was only slightly effective. Therefore, the reversible effect of cAMP, which probably corresponds to enzyme phosphorylation, seems to be required for the irreversible, probably proteolytic, glucose-stimulated inactivation of this enzyme. Cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase in AM7-11D were also inactivated by cAMP, and much more by glucose plus cAMP, whereas glucose was practically ineffective. However, reversibility of the effect was not detected, and, in addition, no phosphorylation of phosphoenolpyruvate carboxykinase could be evidenced. Therefore, the sugar quite probably stimulates proteolysis of these enzymes, but the mechanism of cAMP in their degradation has still to be defined.     

Uniparental isodisomy of chromosome 2 causing MRPL44-related multisystem mitochondrial disease

Mutations in nuclear-encoded protein subunits of the mitochondrial ribosome are an increasingly recognised cause of oxidative phosphorylation system (OXPHOS) disorders. Among them, mutations in the MRPL44 gene, encoding a structural protein of the large subunit of the mitochondrial ribosome, have been identified in four patients with OXPHOS defects and early-onset hypertrophic cardiomyopathy with or without additional clinical features. A 23-year-old individual with cardiac and skeletal myopathy, neurological involvement, and combined deficiency of OXPHOS complexes in skeletal muscle was clinically and genetically investigated. Analysis of whole-exome sequencing data revealed a homozygous mutation in MRPL44 (c.467 T?>?G), which was not present in the biological father, and a region of homozygosity involving most of chromosome 2, raising the possibility of uniparental disomy. Short-tandem repeat and genome-wide SNP microarray analyses of the family trio confirmed complete maternal uniparental isodisomy of chromosome 2. Mitochondrial ribosome assembly and mitochondrial translation were assessed in patient derived-fibroblasts. These studies confirmed that c.467 T?>?G affects the stability or assembly of the large subunit of the mitochondrial ribosome, leading to impaired mitochondrial protein synthesis and decreased levels of multiple OXPHOS components. This study provides evidence of complete maternal uniparental isodisomy of chromosome 2 in a patient with MRPL44-related disease, and confirms that MRLP44 mutations cause a mitochondrial translation defect that may present as a multisystem disorder with neurological involvement.

     

Benthic marine flora in the Tuscan Archipelago. A first contribution: Isles of Capraia,Elba, Formiche di Grosseto,Giglio, Scoglio d'Africa,Montecristo and Giannutri

Abstract

Benthic marine flora of Tuscan Archipelago has been studied. Several samples were collected along five islands and some rocks. This paper is a compendium of new and already published data, in which 267 species, varieties, forms and stadia of algae and seagrasses are listed: 5 Bangiophyceae, 182 Florideophyceae, 45 Phaeophyceae, 6 Chlorophyceae, 28 Bryopsidophyceae, 1 Angiospermae.     

Obstructive lung diseases and inhaler treatment: results from a national public pragmatic survey

Background

The opinions held by the general population on obstructive lung disease and inhaler devices could influence asthma and chronic obstructive pulmonary disorder (COPD) management and treatment adherence.The aim of the present public pragmatic survey was to evaluate the opinions, beliefs and perceptions of Italian people with respect to respiratory diseases as well as their perspectives on the use of inhaler devices.

Methods

This survey was conducted on a group of 2,008 individuals forming a representative sample of the Italian population aged 15 years and over. It was based on personal interviews that were administered in the homes of the respondents using a structured questionnaire that took approximately 30 minutes.

Results

Awareness of obstructive lung diseases is poor. Asthma, but not COPD, was perceived as a common and increasingly prevalent disease by the majority of the interviewees. Allergy, pollution and smoking were considered to be responsible for both of these diseases. The rates at which the respondents claimed to be suffering from asthma and COPD were lower than expected (4% and 2%, respectively). Inhaled drugs were recognised as the main treatment by 65% of the respondents. The great majority of respondents attributed positive characteristics to the inhaler device (e.g., safety, reliability, effectiveness, ease of use and practicality). Compared to people who have never used inhaler devices, individuals who suffer from asthma or COPD were more confident in their use and showed a greater belief in their safety, reliability and trustworthiness. People older than 64 years showed less attention to the properties of these devices.

Conclusions

The present results highlight the need for public interventions aimed at improving awareness of obstructive lung disease and reveal various potentialities and critical issues for inhaler device usage. Switching of devices was considered feasible by most of the interviewees, as long as the choice is carefully explained by their physician.