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M Molinari

Publications and source records attributed to M Molinari.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1996

Trends in the anatomical organization and functional significance of the mammalian thalamus.

The last decade has witnessed major changes in the experimental approach to the study of the thalamus and to the analysis of the anatomical and functional interrelations between thalamic nuclei and cortical areas. The present review focuses on the novel anatomical approaches to thalamo-cortical connections and thalamic functions in the historical framework of the classical studies on the thalamus. In the light of the most recent data it is here discussed that: a) the thalamus can subserve different functions according to functional changes in the cortical and subcortical afferent systems; b) the multifarious thalamic cellular entities play a crucial role in the different functional states.

Animals↗
PubMed1996

Branched connections to the septum and to the entorhinal cortex from the hippocampus, amygdala, and diencephalon in the rat.

Neuronal cell populations giving origin to bifurcating projections to the septum and the entorhinal cortex were studied in the rat by means of double retrograde labeling using the fluorescent tracers Fast Blue and Diamidino Yellow. Double labeled pyramidal neurons were consistently detected in the temporal level of the CA1 area and subiculum of the hippocampal formation, where they represented at least 50% of the cells retrogradely labeled from the entorhinal injections. Double labeled neurons were also detected in the amygdala, where they prevailed in the basal complex. Scattered double labeled neurons were observed in a number of hypothalamic nuclei, with a slight predominance in the preoptic region. Finally, a few double labeled cells were detected in the midline thalamus, and especially in the thalamic paraventricular nucleus. In all these structures, double labeled neurons were located ispilaterally to the injection sites. The present data indicate that the septum and entorhinal cortex are tightly interconnected by axonal bifurcations deriving from a variety of telencephalic and diencephalic sources.

Amygdala↗
PubMed1996

Cerebellar contribution to spatial event processing: Morris water maze and T-maze.

Recently, a cognitive function of cerebellar networks has been challenging the traditional view of the cerebellum as a motor control centre. Among the cognitive abilities reported to be affected by cerebellar deficits is the capacity to solve a spatial problem. We investigated the influence of a cerebellar lesion on spatial abilities by behavioural analysis of rats that had undergone surgical hemicerebellectomy (HCb; HCbed rats). Experiments were performed with a Morris water maze (MWM) and a water T-maze in both cue and place versions (visible or hidden platform respectively). Results indicate a severe impairment in coping with spatial information in all phases of MWM testing as well as in the T-maze paradigm. However, if the MWM cue phase was prolonged, HCbed rats displayed some ability to learn platform position, although at a level significantly different from controls. They succeeded in finding the platform, even in a pure place paradigm, such as finding a hidden platform with the starting points sequentially changed. Retention testing was also performed, demonstrating that HCb affects acquisition but not retention of spatial information. HCbed animals exhibit such disrupted exploration behaviour that they can display only peripheral circling, and they can acquire spatial relations only when proximal cues are available. Furthermore, in all phases of testing, platform finding for HCbed animals is essentially based on place strategies. Thus, a specific pattern of spatial behaviour, markedly different from that displayed following hippocampal or cortical lesions, characterizes cerebellar lesioned rats. These results are discussed taking into account the role in procedural learning recently assigned to cerebellar networks, demonstrating that the cerebellar circuits represent the keystone of the procedural components of spatial event processing.

Analysis of Variance↗
PubMed1995

Subdivisions of macaque monkey auditory cortex revealed by calcium-binding protein immunoreactivity.

The aim of this investigation was to characterize auditory areas of the primate cerebral cortex on the basis of chemoarchitecture. Cortical areas of the supratemporal plane were delineated in Macaca fuscata (M. fuscata) by immunocytochemical staining for parvalbumin, staining for cytochrome oxidase, examination of cyto- and myeloarchitecture, and retrograde tracing of corticocortical connections. Comparative observations were made on Macaca fascicularis (M. fascicularis). Differential staining of fiber plexuses, probably of thalamic origin, identifies a central core zone of dense immunostaining and a surrounding zone of moderate-to-dense immunostaining composed of anteromedial, lateral, and posteromedial fields. Outside the second zone, there is a third anterolateral zone of weaker immunoreactivity, and, outside that zone, there is a fourth zone in which immunoreactivity is virtually absent. Differences in parvalbumin immunostaining in the auditory fields may reflect differences in relative contributions of thalamic inputs from parvalbumin-immunoreactive cells in the medial geniculate complex. The central core zone and the surrounding three fields can be correlated with major auditory fields previously defined by multiunit mapping and thalamocortical connectivity. The core zone contains a large principal field and an anterior extension. The pattern of corticocortical connections between these and adjoining fields suggests that the anteromedial, lateral, and posteromedial fields represent first steps in three streams of connections passing outward from auditory into association cortex. M. fuscata has an unusually large auditory cortex that is more deeply placed in the lateral sulcus in comparison to that of M. fascicularis. A small annectant gyrus provides a guide to the position of the primary auditory area.

Animals↗
PubMed1995

Auditory thalamocortical pathways defined in monkeys by calcium-binding protein immunoreactivity.

This study investigated differentiation of Macaca fuscata auditory thalamus into chemically defined nuclei forming relays to auditory cortical areas. The thalamus was stained immunocytochemically for parvalbumin and 28 kDa calbindin in normals and in brains in which retrogradely transported tracers were injected into middle layers of auditory cortical areas or applied to the cortical surface. Parvalbumin- and calbindin-immunoreactive cells show a complementary distribution in ventral, anterodorsal, posterodorsal, and magnocellular medial geniculate nuclei. The ventral nucleus has a high density of parvalbumin cells and few calbindin cells, and the anterodorsal nucleus has a high density of parvalbumin cells and moderate numbers of calbindin cells. Both nuclei have a dense parvalbumin-immunoreactive neuropil formed by terminations of fibers ascending in the brachium of the inferior colliculus. The posterodorsal nucleus has approximately equal proportions of parvalbumin and calbindin cells; neuropil staining is weak but contains terminations of calbindin-immunoreactive fibers ascending in the midbrain tegmentum. The magnocellular nucleus contains domains of parvalbumin and calbindin cells. Parvalbumin cells in the ventral nucleus project to a central core of auditory cortex with densest parvalbumin immunoreactivity. Those in anterodorsal and posterodorsal nuclei project to surrounding auditory fields with less dense parvalbumin immunoreactivity; those in the magnocellular nucleus project widely to auditory and other fields. Injections of middle cortical layers label a large majority of parvalbumin cells in the ventral, anterodorsal, or posterodorsal nuclei and in the magnocellular nucleus. Superficial deposits label calbindin cells only, usually in more than one nucleus, implying a widespread projection system.

Animals↗
PubMed1995

Patchy and laminar terminations of medial geniculate axons in monkey auditory cortex.

The object of this study was to identify the terminal distributions of thalamocortical axons arising in chemically characterized subdivisions of the medial geniculate complex. Large injections of wheat germ agglutinin-conjugated horseradish peroxidase or small injections of Phaseolus vulgaris leucoagglutinin were made in the medial geniculate complex of Macaca fuscata. The terminal distributions of labeled axons in the cortex were correlated with auditory cortical fields demonstrable by different intensities of immunoreactivity for parvalbumin. Fibers from the ventral nucleus terminated mainly in layer IV and deep portion of layer III (IIIB), with additional terminations in layers I-IIIA and in layer VI. In layers IIIB-IV, a major terminal plexus was formed by a small number of dense patches, 300-500 microns in diameter, surrounded by smaller satellite patches. The patches conformed to a similarly lobulated pattern of parvalbumin fiber immunoreactivity. Terminations of some individually labeled thalamocortical fibers were restricted to a single patch, whereas others innervated more than one patch by collateral branches. Fibers from the dorsal nuclei ending in areas of less dense parvalbumin immunoreactivity surrounding the primary auditory cortex formed much larger terminal patches centered largely in layer IIIB. Fibers from the magnocellular nucleus had relatively few terminal branches but innervated extremely wide areas by collaterals of single axons. Two types of axons arose from the magnocellular nucleus, one terminating preferentially in middle cortical layers and the other exclusively in layer I. These may arise respectively from parvalbumin- and calbindin-immunoreactive cell populations in the magnocellular nucleus.

Animals↗
PubMed1995

Purification of mu-calpain by a novel affinity chromatography approach. New insights into the mechanism of the interaction of the protease with targets.

A calmodulin-binding motif is a common structural feature of a number of calpain substrates (1). Since a calmodulin-like domain has been identified in both subunits of the calpain molecule, the proposal was made that the domain(s) would recognize the calmodulin-binding motifs of the substrates prior to the enzymatic modification by calpain. In keeping with the proposal, a successful attempt to purify mu-calpain from human erythrocytes was made by using an affinity chromatography approach in which the synthetic peptide C49, containing the calmodulin-binding domain of the plasma membrane Ca(2+)-ATPase, was coupled to a Sepharose matrix. The calmodulin-like domain of the catalytic subunit of human mu-calpain expressed in Escherichia coli was also retained by the C49-Sepharose column. Both mu-calpain and the calmodulin-like domain interacted with C49 in a Ca(2+)-dependent way and were eluted from the column by Ca(2+)-chelating agents. The finding confirmed the interaction between the calmodulin-binding domain of the plasma membrane Ca(2+)-ATPase and the calmodulin-like domain of mu-calpain. Experiments were performed to establish whether irreversibly inactivated mu-calpain or its expressed C-terminal portion containing the calmodulin-like domain could activate the hydrolysis of ATP by the plasma membrane Ca2+ pump, in keeping with evident ATPase stimulation of the same pump by calmodulin. A stimulation was observed, but it was much weaker than that induced by calmodulin.

Amino Acid Sequence↗
PubMed1995

p53 in complex with DNA is resistant to ubiquitin-dependent proteolysis in the presence of HPV-16 E6.

The tumour suppressor p53 is a transcription factor with high affinity for specific DNA target sequences. Wild type p53 has a very short half life in normal cells but the protein shows transient accumulation in response to DNA damage, accompanied by up-regulation of target genes such as p21 and induction of growth arrest in G1 of the cell cycle. The rapid turnover of p53 may involve the ubiquitin-dependent proteolytic pathway. In order to investigate p53 turnover we have employed an in vitro system with rabbit reticulocyte lysate, in which ubiquitin-dependent degradation of p53 is mediated by the oncoprotein E6 of human papilloma virus type 16 (HPV-16). Using this system we have previously shown that E6-mediated degradation is preferential for p53 in the 1620+ conformation (reactive with the monoclonal antibody PAb1620). p53-1620+ is a pre-requisite for specific DNA binding and we have now asked if p53 in complex with DNA remains susceptible to ubiquitin-dependent proteolysis in the presence of E6. Our results indicate that p53-DNA complexes are resistant to degradation, whereas the 'free' protein is completely degraded within 20 min. Moreover, E6 did not complex with p53-DNA, possibly due to masking of sites recognised either by E6 or by the E6-associated protein (E6-AP) which facilitates E6-p53 interaction. Preincubation with E6 inhibited the DNA binding capacity of p53 and this effect could be explained, at least in part, by ubiquitination of the p53 protein.

Base Sequence↗
PubMed1995

PEST sequences do not influence substrate susceptibility to calpain proteolysis.

Mutations lowering the PEST score of domains surrounding the calmodulin (CaM)-binding region of the plasma membrane Ca(2+)-ATPase failed to influence the susceptibility of the enzyme to mu-calpain (mu-CANP). Synthetic peptides corresponding to the high PEST score C-terminal sequences A18 and B28 had no effect on the rate of pump proteolysis by mu-CANP, i.e. the peptides did not compete for a putative high PEST score recognition site for mu-CANP in the pump molecule. An accessible CaM-binding region appears to be critical for substrate (i.e. the Ca2+ pump) proteolysis and probably also for its recognition by mu-CANP; phosphorylation of the CaM-binding domain of the pump or its occupation by CaM significantly decreased the rate of proteolysis.

Amino Acid Sequence↗
PubMed1995

Motor cortex changes in a patient with hemicerebellectomy.

To evaluate reorganisation of motor pathways following a cerebellar lesion, we studied motor cortex excitatory responses and inhibitory effects after transcranial stimulation, together with segmental spinal cord excitability, in one patient who had undergone hemicerebellectomy. We compared the results obtained using different forms of stimulation capable of activating the cortico-spinal tract at different sites. Results were compared between sides. We previously reported that the threshold for responses is higher in the motor cortex contralateral to the impaired hemicerebellum and the right/left threshold asymmetry is clearly greater than normal when a circular coil centred over the vertex is used. In the present study, using electrical anodal stimulation, no side difference was observed. Significant interside differences were absent also when the durations of the silent periods or the mean amplitude of the flexor carpi radialis H reflex between the two sides were compared. The outcome is that the interside differences previously observed are mainly due to reduction in the intrinsic excitability properties of the motor cortex functionally related to the impaired hemicerebellum and not to modification of the inhibitory properties of the cortex or to spinal mechanisms.

Cerebellum↗
PubMed1995

Pseudotumor cerebri as presenting syndrome of Addisonian crisis.

In a patient who developed clinical signs of intracranial hypertension, bilateral papilledema and diplopia, in association with mild hypotension, hyponatremia and hyperkalemia, the hypothesis of Addison's disease was raised and confirmed. Substitutional therapy led to complete recovery. The present paper represents the first report of pseudotumor cerebri as the only clinical sign of an Addisonian crisis.

Addison Disease↗
PubMed1995

[Primary echinococcal retroperitoneal cysts. (Observation of a case)].

The Authors, after a careful review of the international literature, report one case of retroperitoneal primitive echinococcus cyst. They describe the anatomo-pathological and clinically specific qualities of this rare condition and emphasize the importance of radical surgical therapy.

Adult↗
PubMed1994

Ca(2+)-activated neutral protease is active in the erythrocyte membrane in its nonautolyzed 80-kDa form.

The aim of this study was to investigate the process leading to Ca(2+)-activated neutral protease (CANP) activation in vivo. The unautolyzed form of CANP has been targeted to the erythrocyte membrane by increasing, in a controlled way, the Ca2+ concentration in the cells; this was achieved by incubating erythrocytes with the Ca2+ ionophore A23187 and fixed Ca2+ concentrations. After isolation of the CANP-bearing erythrocyte membrane, we could observe that CANP remained bound to the membrane in the 80-kDa unautolyzed form in the presence of low Ca2+ concentrations (1.75 microM); under these conditions, the preferred CANP substrates (the Ca(2+)-ATPase and Band 3) were cleaved. That the cleavage was due to CANP was shown by the finding that the two substrates were not degraded in the presence of a membrane-permeable irreversible CANP inhibitor, Cbz-Leu-Leu-Tyr-CHN2, nor when the free Ca2+ concentration was decreased to sub microM levels with EDTA. The findings suggest an activation mechanism of CANP based on its translocation to the membrane rather than on its autolysis. In this mechanism, CANP would become reversibly activated on the membrane and would return to the quiescent state after dissociating from it when the cell Ca2+ concentration has returned to the physiological, submicromolar level.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1994

Chemical compartmentation and relationships between calcium-binding protein immunoreactivity and layer-specific cortical caudate-projecting cells in the anterior intralaminar nuclei of the cat.

Neurons projecting to the parietal cortex or striatum and neurons showing immunoreactivity for the calcium-binding proteins parvalbumin and 28KD-calbindin were examined in the anterior intralaminar nuclei (IL) of the cat. Retrograde tracing from deep or superficial parietal cortical layers or from the caudate nucleus was coupled with immunohistochemistry to determine which of these proteins were expressed in the projection neurons. It was found that IL neurons project to deep as well as to superficial layers of the parietal cortex, that IL-cortical neurons could be differentiated into two populations according to their cortical projection pattern and their soma size, and that IL neurons projecting to the parietal cortex or to the striatum express 28KD calbindin immunoreactivity but not parvalbumin immunoreactivity. The distribution of immunoreactivity to 28KD calbindin and parvalbumin in the neuropil showed a consistent complementary distribution pattern in the IL. The compartments based on differential parvalbumin and 28KD calbindin expression may indicate the presence of functionally segregated units in IL.

Animals↗