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Biomedical subjects

M Molinari

Publications and source records attributed to M Molinari.

At least 19 recordsLinked to original sources
PubMed1998

The acid activation of Helicobacter pylori toxin VacA: structural and membrane binding studies.

The cell vacuolating activity of the protein toxin VacA, released by Helicobacter pylori, is strongly increased in vitro by exposure to acidic pH followed by neutralization. This short acid exposure does not increase significantly the binding of VacA to cell or to lipid membranes. However, membrane photolabeling with photoactivatable radioactive phospholipids and ANS binding studies show that VacA transiently exposed to pH equal or lower than 5 changes conformation and exposes on its surface hydrophobic segments. Both the 32 and the 58 kDa subunits of the toxin insert in the lipid bilayer and interact with the fatty acid chains of phospholipids. Membrane binding and penetration are enhanced by incubating target cells or liposomes with the toxin at mild acidic pH values, similar to those present around H. pylori on the stomach mucosa. These findings are discussed with respect to the critical step in cell intoxication consisting in the translocation of the active toxin domain into the cell cytosol. We suggest that membrane translocation takes place at the plasma membrane level.

Affinity Labels↗
PubMed1998

Prothrombin G20210A mutant genotype is a risk factor for cerebrovascular ischemic disease in young patients.

The factor II G20210A mutation is a recently identified congenital risk factor for venous thrombosis. Its role in artery disease is still undefined. We investigated 72 patients (35 male and 37 female) with documented ischemic stroke occurred before 50 years of age and without risk factors such as diabetes, hypertension, and hyperlipidemia; 198 thrombosis-free individuals were investigated as the control group. We found 7 heterozygotes (9.7%) and 2 homozygotes (2.7%) for the mutant factor II allele among the patients and 5 heterozygotes (2.5%) among the controls; the mutant factor II allele frequency in the patient group (7.6%, 95% confidence interval [CI], 3.3 to 11.9) was significantly higher than in the controls (1.2%; 95% CI, 0.1 to 2.3; P = .0001). The prevalence of other investigated mutant alleles (factor V G1691A, methylenetetrahydrofolate reductase C677T) did not significantly differ between the two groups. The odds ratio for ischemic stroke associated with the carriership of the mutant factor II allele (both heterozygous and homozygous genotypes) was 5.1 (95% CI, 1.6 to 16.3). Heterozygous genotype was associated with a 3.8-fold increased risk for cerebral ischemia (95% CI, 1.1 to 13.1); in particular, assuming an expected prevalence of homozygotes in the general population of 1.6 to 10,000 according to the Hardy-Weinberg equilibrium, the risk associated with the homozygous genotype was estimated exceedingly high, being increased 208-fold.

Adolescent↗
PubMed1998

Selective inhibition of Ii-dependent antigen presentation by Helicobacter pylori toxin VacA.

A major virulence factor in the stomach chronic infection by Helicobacter pylori is a protein toxin (VacA), which alters cell membrane trafficking of late endosomal/prelysosomal compartments. Its role in the chronic infection established by H. pylori is unknown. To test the possibility that VacA alters antigen processing taking place in prelysosomal compartments, we have used the well-established model of antigen processing and presentation consisting of tetanus toxoid-specific human (CD4(+)) T cells stimulated by autologous antigen-pulsed Epstein-Barr virus-transformed B cells. We found that VacA interferes with proteolytic processing of tetanus toxin and toxoid and specifically inhibits the Ii-dependent pathway of antigen presentation mediated by newly synthesized major histocompatibility complex (MHC) class II, while leaving unaffected the presentation pathway dependent on recycling MHC class II. The results presented here suggest that VacA may contribute to the persistence of H. pylori by interfering with protective immunity and that this toxin is a new useful tool in the study of the different pathways of antigen presentation.

Antigen Presentation↗
PubMed1998

Action site and cellular effects of cytotoxin VacA produced by Helicobacter pylori.

Cells treated with the VacA toxin from Helicobacter pylori develop large membrane-bound vacuoles that originate from the late endocytotic pathway. Using different experimental approaches, we showed that VacA can induce vacuoles by acting within the cell cytosol. Moreover, separation of VacA-induced vacuoles at an early stage of formation, using a novel isopycnic density ultracentrifugation method, allowed us to show that they resemble a hybrid compartment, containing elements of both late endosomes and lysosomes. Functional defects of the endocytotic pathway were also studied before any macroscopic vacuolation is evident. VacA-intoxicated cells degrade extracellular ligands with reduced efficiency and, at the same time, they secrete acidic hydrolases into the extracellular medium, normally sorted to lysosomes. All these findings indicate that VacA translocates into the cell cytosol where it causes a lesion of the late endosomal/lysosomal compartments, such that protein trafficking across this crucial cross-point is altered with consequences that may be relevant to the pathogenesis of gastroduodenal ulcers.

Bacterial Proteins↗
PubMed1997

Vacuoles induced by Helicobacter pylori toxin contain both late endosomal and lysosomal markers.

Intoxication of mammalian cells with the vacuolating toxin (VacA) released by Helicobacter pylori causes the formation of large acidic vacuoles containing the vacuolar ATPase proton pump and Rab7, a late endosome marker. Here, we describe a novel subcellular fractionation procedure, and we show that nanomolar concentrations of VacA induce a clear redistribution of lysosomal membrane glycoproteins among endocytic compartments. This redistribution is an early event in the process of cellular intoxication by VacA and precedes the formation of macroscopic vacuoles. The absence of the cation independent mannose 6-P receptor and the presence of Rab7 and of lysosomal membrane proteins in the newly formed compartment suggest that the vacuolating toxin induces the accumulation of a post-endosomal hybrid compartment presenting both late endosomal and lysosomal features.

Animals↗
PubMed1997

Prospective study of the features, indications, and surgical treatment in 513 consecutive patients affected by Crohn's disease.

BACKGROUND: The aim of this prospective study was to elucidate the features, indications, and surgical treatment in patients affected by complications of Crohn's disease. METHODS: Between January 1985 and July 1996, 513 consecutive patients (248 male, 265 female; mean age, 38 years) were operated on for 542 occurrences of Crohn's disease. Data were collected prospectively. RESULTS: Indications for abdominal surgery were often multiple but included failure of medical management (n = 220), obstruction (n = 94), intestinal fistula (n = 68), mass (n = 56), abdominal abscess (n = 33), hemorrhage (n = 7), and peritonitis (n = 9). Four hundred sixty-four abdominal procedures were performed, necessitating 425 intestinal resections and 97 stricture plasties. The use of stricture plasty was more common in the second half of the study (16.0% versus 7.3%, second half versus first half; p < 0.01). Perioperative complications occurred in 75 of the 464 abdominal operations (16%). There were no deaths. One hundred thirty patients (25%) required operation for perineal complications of Crohn's disease. The presence of Crohn's disease in the rectal mucosa was associated with a higher risk for permanent stomas in patients requiring operation for treatment of perianal Crohn's disease (67% versus 11%; p < 0.001). CONCLUSIONS: Patterns of surgical treatment in Crohn's disease are changing, with more emphasis on nonresectional options. The presence of rectal involvement significantly increases the need for a permanent stoma in patients with perianal Crohn's disease.

Adolescent↗
PubMed1997

Spatial event processing.

The present review advances experimental evidence on the cerebellar involvement in spatial data processing. In particular, data on Morris water maze (MWM) performances of hemicerebellectomized (HCbed) rats indicate a specific cerebellar role within the procedural aspects of spatial functions. In MWM testing, HCbed animals are impaired in developing efficient exploration strategies and display only old and rather ineffective ways for acquiring spatial information, such as peripheral circling around the pool. This behavior is not exhibited if spatial mapping abilities are preoperatively acquired. Thus, MWM experimental data point toward a procedural deficit that specifically impairs the acquisition phase. The characteristics of the cerebellar involvement in affecting the procedures needed for spatial data management are discussed in the light of recent theories on spatial data processing and on cerebellar timing and ordering functions.

Animals↗
PubMed1997

Verbal fluency and agrammatism.

Since the beginning of this century it has been documented that cerebellar lesions induce speech deficits but these were thought to result from lack of motor coordination in the muscular activity needed for phonation. The pure motor nature of the cerebellum has been challenged on different grounds, and cerebellar activation has been documented in language-related tasks independently from motor activity. This chapter reviews the available evidence in favor of a cerebellar contribution to linguistic processing, focusing mainly on clinical observations in patients. The clinical findings are discussed in the light of recent theories on cerebellar functions.

Cerebellar Diseases↗
PubMed1997

Cerebellum and procedural learning: evidence from focal cerebellar lesions.

The aim of the present study was to investigate the influence of focal cerebellar lesions on procedural learning. Eight patients with cerebellar lesions and six control subjects were tested in a serial reaction-time task. A four-choice reaction-time task was employed in which the stimuli followed (or not) a sequence repeated 10 times, with the subjects aware (or not) of the item sequence. Learning was manifested by the reduction in response latency over the sequential blocks. Acquisition of declarative knowledge of the sequence was also tested. Reaction times displayed by patients with cerebellar lesions, even though they tended to be longer than those of control subjects in all testing conditions, significantly differed from control subjects only when the stimuli were presented in sequence. The reaction times in sequential trials were still statistically significant when simple motor response times were taken into account. Cerebellar patients were also significantly impaired in detecting and repeating the sequence. On the other hand, when the sequence was learned before testing, motor performances were significantly improved in all subjects. These data indicate that cerebellar lesions induce specific impairment in the procedural learning of a motor sequence and suggest a role of the cerebellar circuitry in detecting and recognizing event sequences.

Adult↗
PubMed1997

Cerebellar contribution to spatial event processing: right/left discrimination abilities in rats.

Recently, we demonstrated the involvement of cerebellar circuits in the procedural components of spatial information processing by testing hemicerebellectomized (HCbed) rats in classical spatial paradigms, such as the Morris Water Maze and the water T-maze. Since procedural components are strongly present in these tests, an impairment also in processing more abstract spatial information, linked to 'where an object is' rather than to 'how to find it', could be hidden by the severe procedural deficits. On this basis, we investigated the influence of cerebellar lesions on spatial abilities strictly reducing procedural variables by employing an active avoidance task, first without and then with a request for right/left discrimination. In the two-way active avoidance task without spatial requests, controls and cerebellar operated rats developed active avoidance responses which were not statistically different, demonstrating that this kind of associative learning is not significantly affected by hemicerebellectomy (HCb). A second experimental group of cerebellar lesioned rats was tested in a modified version of this basic paradigm in which a right/left discrimination request was added. This group displayed severe deficits, which even in the last testing sessions prevented them from performing comparably to the control animals. Reversal of the rewarded choice, even if it affected the performances of both controls and operated rats in the first inversion trials, elicited the lowest number of correct responses in HCbed rats throughout the entire spatial reversal learning, suggesting a severe deficit in the ability to change an initially learned behaviour. These results demonstrate that, beside having a marked impairment in facing procedural components of spatial processing, cerebellar lesioned rats are severely defective also in right/left discrimination tasks, suggesting a role of cerebellar networks also in the discriminative spatial information processing.

Analysis of Variance↗
PubMed1997

Proteolysis by calpains: a possible contribution to degradation of p53.

p53 is a short-lived transcription factor that is frequently mutated in tumor cells. Work by several laboratories has already shown that the ubiquitin-proteasome pathway can largely account for p53 destruction, at least under specific experimental conditions. We report here that, in vitro, wild-type p53 is a sensitive substrate for milli- and microcalpain, which are abundant and ubiquitous cytoplasmic proteases. Degradation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility to calpains, some of them being largely resistant to degradation and others being more sensitive. This result suggests that the different mutants tested here adopt slightly different conformations to which calpains are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by using the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, activation of calpains by calcium ionophore led to a reduction of p53 in mammalian cells, and the effect was blocked by cell-permeant calpain inhibitors. Cotransfection of p53-null cell lines with p53 and calpastatin expression vectors resulted in an increase in p53-dependent transcription activity. Taken together, these data support the idea that calpains may also contribute to the regulation of wild-type p53 protein levels in vivo.

Animals↗
PubMed1997

Spatial dysgraphia and cerebellar lesion: a case report.

Spatial dysgraphia is a writing disorder that occurs in patients with right hemisphere lesion. We report a patient with cerebellar atrophy and spatial dysgraphia. To explain this finding, we hypothesize a discoordination between planning of the movement and performance due to a lack of the cerebellar modulation between supratentorial (premotor cortex) and peripheral (proprioceptive) afference during the ongoing handwriting movement.

Aged↗
PubMed1996

Interaction with damaged DNA induces selective proteolytic cleavage of p53 to yield 40 kDa and 35 kDa fragments competent for sequence-specific DNA binding.

The p53 protein binds sites of primary DNA damage via its C-terminus. This interaction in some way activates sequence-specific binding (via the central core domain) and transactivation of p53 target genes. We now show that interaction with non-specific DNA, but not specific DNA targets, induces selective proteolysis of p53 to give a 40 kDa fragment, comprising the core plus C-terminus, and a 35 kDa conformationally intact core domain. Proteolytic cleavage was limited and yielded roughly equivalent proportions of full length p53 and the 40 kDa and 35 kDa fragments. Significantly, both 40 kDa and 35 kDa products were activated for sequence-specific DNA binding. Similar p53-related products were induced by exposure of cells to DNA damage. We propose that some functions of p53 can be activated by proteolytic processing and that this may be important in the cellular response to DNA damage.

3T3 Cells↗
PubMed1996

Purification of active calpain by affinity chromatography on an immobilized peptide inhibitor.

Most purification schemes of calpain (CANP) involve a number of chromatographic steps. The final preparations often contain impurities, including degradation fragments. Two peptide-affinity columns were developed, using peptides of 27 amino acids and 30 amino acids, corresponding to the products of exons 1B and 1C, respectively, of the natural inhibitor (calpastatin) gene, coupled to CNBr-activated Sepharose 4B. Crude preparations of calpain, isolated by anion-exchange chromatography on a DEAE-Sepharose column, were incubated with a reversible or an irreversible synthetic inhibitor which blocks the catalytic subunit of the enzyme in the inactive 80-kDa form. The crude preparation was then loaded onto the peptide column in the presence of calcium. Calpain was eluted with an EGTA-containing buffer. Using the two peptide-affinity columns connected in tandem, calpain was isolated with a high degree of purity, suitable for structural and mechanistic studies, i.e. as an 80/30-kDa heterodimer or in the form of dissociated monomers.

Amino Acid Sequence↗
PubMed1996

Development of GABA and calcium binding proteins immunoreactivity in the rat hippocampus following neonatal anoxia.

The consequences of neonatal anoxia (N2 100% for 25 min at 30 h after birth) on the rat hippocampus were studied 7-60 days postnatally with immunocytochemistry for gamma-aminobutyric acid (GABA), parvalbumin (PV) and calbindin-D28k (CB). In both sham-treated and anoxic rats, GABA immunoreactivity presented a mature expression since early stages, while PV and CB immunoreactivity showed a major postnatal development. In anoxic animals, a significant reduction in the number of hippocampal GABA-immunoreactive neurons was observed at all time-points analysed, a transitory effect on PV immunoreactivity was seen at P7 and P21, while no modifications in the number of CB-immunoreactive neurons could be found. Thus, selective vulnerability of GABA-containing neurons and relative resistance of neurons in which PV or CB immunoreactivity is present or is expressed later, occur in the hippocampus after neonatal anoxia. The role of calcium binding proteins (CBP) in nerve cell protection is discussed.

Animals↗
PubMed1996

Prospective study of the incidence, timing and treatment of pouchitis in 104 consecutive patients after restorative proctocolectomy.

OBJECTIVE: To determine the incidence timing and effectiveness of treatment of symptomatic pouchitis following restorative proctocolectomy with ileal J-pouch anal anastomosis. DESIGN: A cohort analytical study. SETTING: University hospitals, a tertiary referral center; all subjects entered into the study followed up for a minimum of 12 months (mean follow-up, 40 months). PATIENTS: One hundred four consecutive patients undergoing restorative proctocolectomy with ileal J-pouch anal anastomosis for either ulcerative colitis (n = 97) or familial adenomatous polyposis (n = 7) between June 1986 and December 1994. INTERVENTIONS: Patients with symptomatic pouchitis were treated with either oral metronidazole or ciprofloxacin. OUTCOMES: Diagnosis of pouchitis was determined by clinical symptoms and confirmed with endoscopy. Response to oral antibodies was determined by resolution of symptoms. RESULTS: Fifty-two patients (50%) experience at lest 1 episode of pouchitis. The first episode of pouchitis occurred within the first 12 months after restoration of intestinal continuity in 56% of the cases. In 2 patients it occurred after 30 months. Response to antibiotic treatment was 96%. Two thirds of patients had multiple episodes. Chronic pouchitis occurred in 6 patients, necessitating pouch removal in 2. CONCLUSIONS: The incidence of pouchitis after ileal J-pouch anal anastomosis is approximately 50% with two thirds of these patients having multiple episodes. Chronic pouchitis occurs in a minority of patients. In chronic pouchitis, the risk of pouch loss is substantial.

Adenomatous Polyposis Coli↗