Paola Martelli

Proteomic response to physiological fermentation stresses in a wild-type wine strain of Saccharomyces cerevisiae

We report a study on the adaptive response of a wild-type wine Saccharomyces cerevisiae strain, isolated from natural spontaneous grape must, to mild and progressive physiological stresses due to fermentation. We observed by two-dimensional electrophoresis how the yeast proteome changes during glucose exhaustion, before the cell enters its complete stationary phase. On the basis of their identification, the proteins representing the S. cerevisiae proteomic response to fermentation stresses were divided into three classes: repressed proteins, induced proteins and autoproteolysed proteins. In an overall view, the proteome adaptation of S. cerevisiae at the time of glucose exhaustion seems to be directed mainly against the effects of ethanol, causing both hyperosmolarity and oxidative responses. Stress-induced autoproteolysis is directed mainly towards specific isoforms of glycolytic enzymes. Through the use of a wild-type S. cerevisiae strain and PMSF, a specific inhibitor of vacuolar proteinase B, we could also distinguish the specific contributions of the vacuole and the proteasome to the autoproteolytic process.

Identification of a Novel RalGDS-related Protein as a Candidate Effector for Ras and Rap1

Although Ras and Rap1 share interaction with common candidate effector proteins, Rap1 lacks the transforming activity exhibited by Ras proteins. It has been speculated that Rap antagonizes Ras transformation through the formation of nonproductive complexes with critical Ras effector targets. To understand further the distinct biological functions of these two closely related proteins, we searched for Rap1b-binding proteins by yeast two-hybrid screening. We identified multiple clones that encode the COOH-terminal sequences of a protein that shares sequence identity with RalGDS and RGL, which we have designated RGL2. A 158-amino acid COOH-terminal fragment of RGL2 (RGL2 C-158) bound to Ras superfamily proteins which shared identical effector domain sequences with Rap1 (Ha-Ras, R-Ras, and TC21). RGL2 C-158 binding was impaired by effector domain mutations in Rap1b and Ha-Ras. Furthermore, RGL2 C-158 bound exclusively to the GTP-, but not the GDP-bound form of Ha-Ras. Finally, coexpression of RGL2 C-158 impaired oncogenic Ras activation of transcription from a Ras-responsive promoter element and focus-forming activity in NIH 3T3 cells. We conclude that RGL2 may be an effector for Ras and/or Rap proteins.

GM1 gangliosidosis and Morquio B disease: an update on genetic alterations and clinical findings

GM1 gangliosidosis and Morquio B syndrome, both arising from beta-galactosidase (GLB1) deficiency, are very rare lysosomal storage diseases with an incidence of about 1:100,000-1:200,000 live births worldwide. Here we report the beta-galactosidase gene (GLB1) mutation analysis of 21 unrelated GM1 gangliosidosis patients, and of 4 Morquio B patients, of whom two are brothers. Clinical features of the patients were collected and compared with those in literature. In silico analyses were performed by standard alignments tools and by an improved version of GLB1 three-dimensional models. The analysed cohort includes remarkable cases. One patient with GM1 gangliosidosis had a triple X syndrome. One patient with juvenile GM1 gangliosidosis was homozygous for a mutation previously identified in Morquio type B. A patient with infantile GM1 gangliosidosis carried a complex GLB1 allele harbouring two genetic variants leading to p.R68W and p.R109W amino acid changes, in trans with the known p.R148C mutation. Molecular analysis showed 27 mutations, 9 of which are new: 5 missense, 3 microdeletions and a nonsense mutation. We also identified four new genetic variants with a predicted polymorphic nature that was further investigated by in silico analyses. Three-dimensional structural analysis of GLB1 homology models including the new missense mutations and the p.R68W and p.R109W amino acid changes showed that all the amino acid replacements affected the resulting protein structures in different ways, from changes in polarity to folding alterations. Genetic and clinical associations led us to undertake a critical review of the classifications of late-onset GM1 gangliosidosis and Morquio B disease.

Photosensitizing action of furocoumarins on membrane components and consequent intracellular events.

The photodamage induced in membrane components by furocoumarins is reviewed. The oxygen-dependent photoreactions between furocoumarins and cell membrane constituents lead mainly to lipid peroxidation and the formation of cross-linking in ghost proteins, whereas the oxygen-independent photoreactions lead essentially to a C4 cycloaddition between the furocoumarin and the unsaturated fatty acids. In the latter, cycloadducts are formed between the 3,4 double bond of the furocoumarin and the olefinic double bond of the unsaturated fatty acid. The stereochemical structures of these cycloadducts and the reaction mechanism of the cycloaddition are discussed. Finally, the modulation of several membrane systems by furocoumarins and the consequent intracellular events are reviewed.

Effectiveness and tolerability of perampanel in children and adolescents with refractory epilepsies—An Italian observational multicenter study

PURPOSE To evaluate the efficacy and tolerability of Perampanel (PER) in children and adolescents with refractory epilepsies in daily clinical practice conditions. PATIENTS AND METHODS This Italian multicenter retrospective observational study was performed in 16 paediatric epilepsy centres. Inclusion criteria were: (i) ≤18 years of age, (ii) history of refractory epilepsy, (iii) a follow-up ≥5 months of PER add-on therapy. Exclusion criteria were: (i) a diagnosis of primary idiopathic generalized epilepsy, (ii) variation of concomitant AEDs during the previous 4 weeks. Response was defined as a ≥50% reduction in monthly seizure frequency compared with the baseline. RESULTS 62 patients suffering from various refractory epilepsies were included in this study: 53% were males, the mean age was 14.2 years (range 6-18 years), 8 patients aged <12 years. Mean age at epilepsy onset was 3.4 years and the mean duration of epilepsy was 10.8 years (range 1-16), which ranged from 2 seizures per-month up to several seizures per-day (mean number=96.5). Symptomatic focal epilepsy was reported in 62.9% of cases. Mean number of AEDs used in the past was 7.1; mean number of concomitant AEDs was 2.48, with carbamazepine used in 43.5% of patients. Mean PER daily dose was 7.1mg (2-12mg). After an average of 6.6 months of follow-up (5-13 months), the retention rate was 77.4% (48/62). The response rate was 50%; 16% of patients achieved ≥75% seizure frequency reduction and 5% became completely seizure free. Seizure aggravation was observed in 9.7% of patients. Adverse events were reported in 19 patients (30.6%) and led to PER discontinuation in 4 patients (6.5%). The most common adverse events were behaviour disturbance (irritability and aggressiveness), dizziness, sedation and fatigue. CONCLUSION PER was found to be a safe and effective treatment when used as adjunctive therapy in paediatric patients with uncontrolled epilepsy.

Postgenomics of Neisseria meningitidis for vaccines development.

Meningococcal disease is a global problem. Multivalent (A, C, Y, W135) conjugate vaccines have been developed and licensed; however, an effective vaccine against serogroup B has not yet become available. Outer membrane vesicle (OMV) vaccines have been used to disrupt serogroup B epidemics and outbreaks. Postgenomic technologies have been useful in aiding the discovery of new protein vaccine candidates. Moreover, proteomic technologies enable large-scale identification of membrane and surface-associated proteins, and provide suitable methods to characterize and standardize the antigen composition of OMV-based vaccines.

Adenylyl cyclase activity in roots of Pisum sativum

Abstract An extract exhibiting adenylyl cyclase activity was obtained from roots of pea seedlings and the product of its incubation with ATP and Mg 2+ isolated and examined by tandem mass spectrometry. The data presented constitute the first unambiguous identification of the product of a putative adenylyl cyclase from a higher plant source as adenosine 3′,5′-cyclic monophosphate (cyclic AMP). A plot of reaction velocity against substrate concentration was sigmoidal, indicating allostery; 100 nM GTP activates the enzyme but 100μM inhibits it. Inclusion of an ATP- regenerating system in the enzymic assay stimulated at the higher GTP concentration but not at the lower concentration. Possible effectors, including zeatin, gibberellin A 3 , indolylacetic acid and calmodulin were examined; the results suggest that there is an additional, unidentified component of the enzymic system. The enzyme is associated with particulate fractions.

High performance liquid chromatography analysis of the furanochromones khellin and visnagin in various organs of Ammi visnaga (L.) Lam. at different developmental stages

The content of the furanochromones khellin and visnagin, in the organs of Ammi visnaga (L.) Lam. at different developmental stages, has been examined. Determinations have been performed by means of a high performance liquid chromatography (HPLC) technique which allows both the separation and the quantitative determination of these chromones. Unripe fruits are the richest in both chromones, but the collection of ripe dry fruits--as it occurs in Egyptian folk-medicine--seems more reasonable because they might not undergo degradation processes during desiccation and storage.

Adenylate cyclase in roots of Ricinus communis; stimulation by GTP and Mn 2+

Abstract A ‘sedimentable’ adenylate cyclase has been found in the roots of Ricinus communis plantlets. The enzymatic activity is stimulated by MnCl 2 , NaF and GTP. The results obtained constitute the first report of a higher plant adenylate cyclase requiring GTP.

Inactivation of Helicobacter pylori cagA Gene Affects Motility

Background.  The cytotoxin‐associated protein CagA is a Helicobacter pylori immunodominant antigen whose gene resides in the cag pathogenicity island. Our purpose was to determine if the disruption or deletion of cagA gene could have an effect on the expression of other proteins at the proteome level. We analyzed two H. pylori strains, 328 and G27 wild‐type, bearing the cag pathogenicity island, and their respective isogenic cagA− mutants.