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Biomedical subjects

A Gatti

Publications and source records attributed to A Gatti.

At least 37 records · Page 2Linked to original sources
PubMed2000

Phosphorylation of human p53 on Thr-55.

The pleiotropic function of p53 is believed to be greatly influenced by phosphorylation, and several sites on p53 are known to be targets for distinct protein kinases. In this study, we observed that affinity-purified p53 from overexpressing cells was phosphorylated by a co-purified protein kinase in vitro. To identify phosphorylation site(s), the resulting phosphorylated p53 protein was subjected to primary and secondary proteolytic cleavage, and phosphopeptides were fractionated by a two-dimensional peptide gel system. Phosphoamino acid analysis and manual Edman degradation of the isolated phosphopeptides enabled us to unequivocally identify Thr-55 as the major phosphorylation site on p53. Furthermore, comparative phosphopeptide mapping data suggest that DNA-PK is not the kinase responsible for this phosphorylation. Significantly, using a phospho-specific antibody for Thr-55, we have shown that Thr-55 is indeed phosphorylated in vivo. These data define Thr-55 as a novel phosphorylation site and for the first time show threonine phosphorylation of human p53.

DNA-Activated Protein Kinase↗
PubMed2000

Clinical and pharmacological phase I study with accelerated titration design of a daily times five schedule of BBR3464, a novel cationic triplatinum complex.

OBJECTIVES: To define the maximum tolerated dose (MTD), the toxicity and pharmacokinetic profile of BBR3464, a novel triplatinum complex. PATIENTS AND METHODS: Fourteen patients with advanced solid tumors not responsive to previous antitumor treatments received BBR 3464 on a daily x 5 schedule every twenty-eighth day. The drug was given as a one-hour infusion with pre-and post-treatment hydration (500 ml in one hour) and no antiemetic prophylaxis. The starting dose was 0.03 mg/m2/day. A modified accelerated titration escalation design was used. Total and free platinum (Pt) concentrations in plasma and urine were assessed by ICP-MS on days 1 and 5 of the first cycle. RESULTS: Dose was escalated four times up to 0.17 mg/m2/day. Short-lasting neutropenia and diarrhea of late onset were dose-limiting and defined the MTD at 0.12 mg/m2. Nausea and vomiting were rare, neither neuro- nor renal toxic effects were observed. BBR3464 showed a rapid distribution phase of 1 hour and a terminal half-life of several days. At 0.17 mg/m2 plasma Cmax and AUC on day 5 were higher than on day 1, indicating drug accumulation. Approximately 10% of the equivalent dose of BBR3464 (2.2%-13.4%) was recovered in a 24-hour urine collection. CONCLUSIONS: The higher than expected incidence of neutropenia and GI toxicity might be related to the prolonged half-life and accumulation of total and free Pt after daily administrations. Lack of nephrotoxicity and the low urinary excretion support the use of the drug without hydration. The single intermittent schedule has been selected for clinical development.

Adolescent↗
PubMed2000

Familial mastocytosis associated with neurosensory deafness.

Mastocytosis is a disease characterized by excessive accumulation of mast cells in different tissues and symptoms caused by the release of mast cell mediators. The skin is frequently directly involved in mastocytosis. The disease is rarely seen in other members of the subjects' family; only 49 cases of familial mastocytosis have been reported. Familial mastocytosis associated with hearing loss may represent a newly described inherited entity. We describe a brother and sister exhibiting skin mastocytosis and neurosensory deafness, associated with a history of hearing loss in their father's family. The appearance of the mast cell disease in two siblings, who presented with similar clinical features represents a familial form of mastocytosis; the association with an inherited form of deafness may constitute a new syndrome. Our patients show several features similar to some previously reported cases but different insofar that additional congenital defects and mental retardation are absent.

Adolescent↗
PubMed2000

Divergent regulation of HIV-1 replication in PBMC of infected individuals by CC chemokines: suppression by RANTES, MIP-1alpha, and MCP-3, and enhancement by MCP-1.

We investigated the role of different CC chemokines, including regulated upon activation normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-lalpha (MIP-1alpha), monocyte chemotactic protein-1 (MCP-1), and MCP-3 on virus replication in cultures established from CD8+ T cell-depleted peripheral blood mononuclear cells (PBMC) of HIV-infected individuals that were either cocultivated with allogeneic T cell blasts (ATCB) of uninfected individuals or directly stimulated by mitogen plus interleukin-2. RANTES was the only chemokine that showed a clear-cut suppressive effect on HIV replication in both culture systems, although inhibitory effects were frequently also observed with MIP-1alpha, MCP-3, and, occasionally, with MCP-1. In contrast, MCP-1 frequently enhanced HIV production in most patients' cultures or cocultures that were characterized by secreting relatively low levels (<20 ng/mL) of MCP-1. When CD8-depleted PBMC of HIV+ individuals were cocultivated with ATCB of uninfected healthy donors, a positive correlation was observed between MCP-1 concentrations and the enhancement of HIV-1 replication occurring after depletion of CD8+ cells from donors' cells. Depletion of CD14+ cells (monocytes) from ATCB resulted in the down-regulation of virus replication during co-cultivation with CD8-depleted PBMC of infected individuals. Of interest, MCP-1 up-regulated HIV production in these CD14-depleted ATCB cocultures. Altogether these observations suggest that MCP-1 may represent an important factor enhancing HIV spreading, particularly in anatomical sites, such as the brain, where infection of macrophages and microglial cells plays a dominant role.

Adult↗
PubMed1999

Defective nef alleles in a cohort of hemophiliacs with progressing and nonprogressing HIV-1 infection.

Deletion of the nef gene results in viral attenuation and confers protection against challenge with wild-type simian immunodeficiency virus in macaques. Regarding HIV-1 infection, a few long-term nonprogressors (LTNP) with nef deletions have been described. In this study, the nef genes of a group of seven LTNP and eight progressors, all belonging to the same cohort of infected hemophiliacs, were analyzed by cloning and sequencing from both virion RNA and peripheral blood mononuclear cell-associated proviral DNA. Defective nef sequences coexisted with full-length nef open reading frames in five of seven LTNP and two of eight progressors. The proportion of disrupted nef sequences within each individual was significantly higher in LTNP (ranging from 10 to 63%) than in progressors (ranging from 9 to 21%) (P = 0.013). Moreover, in-frame small deletions predicting to encode Nef were found in all RNA- and DNA-derived clones from one LTNP and four progressors. A chimeric virus in which the nef gene of NL4.3 was substituted with the nef allele containing the deletion of two alanines at position 49-50 found in two progressors showed a defective replicative capacity compared to NL4.3 virus. In summary, hemophiliacs with either progressing or nonprogressing HIV-1 infection are characterized by the presence of defective nef variants.

Adolescent↗
PubMed1999

Multisite autophosphorylation of p21-activated protein kinase gamma-PAK as a function of activation.

p21-activated protein kinase (PAK) is a family of serine/threonine kinases whose activity is stimulated by binding to small G-proteins such as Cdc42 and subsequent autophosphorylation. Focusing on the ubiquitous gamma-isoform of PAK in this study, baculovirus-infected insect cells were used to obtain recombinant gamma-PAK, while native gamma-PAK was isolated from rabbit reticulocytes. Two-dimensional gel electrophoresis of gamma-PAK followed by immunoblot analysis revealed a similar profile for native and recombinant gamma-PAK, both consisting of multiple protein spots. Following Cdc42-stimulated autophosphorylation, the two-dimensional profiles of native and recombinant gamma-PAK were characterized by a similar acidic shift, suggesting a common response to Cdc42. To understand the effect of differential phosphorylation on its activation status, gamma-PAK autophosphorylation was conducted in the presence or absence of activators such as Cdc42 and histone II-AS, followed by tryptic digestion and comparative two-dimensional phosphopeptide mapping. The major phosphopeptides were subjected to a combination of manual and automated amino acid sequencing. Overall, eight autophosphorylation sites were identified in Cdc42-activated gamma-PAK, six of which are in common with those previously reported in alpha-PAK, while Ser-19 and Ser-165 appear to be uniquely phosphorylated in the gamma-form. Further, the phosphorylation of Ser-141, Ser-165, and Thr-402 was found to correlate with gamma-PAK activation.

Amino Acid Sequence↗
PubMed1999

A two-dimensional peptide gel electrophoresis system for phosphopeptide mapping and amino acid sequencing.

A novel two-dimensional electrophoresis system to be carried out on polyacrylamide gels under nondenaturing conditions was developed to efficiently fractionate the peptides resulting from endoproteinase digestion of 32P-labeled proteins. In particular, nondenaturing gel isoelectric focusing was combined with alkaline 40% polyacrylamide gel electrophoresis to generate phosphopeptide maps with high reproducibility, thus allowing both protein fingerprinting and comparative analysis of different samples. The potential application of this method for subsequent amino acid sequencing of the isolated phosphopeptides was further demonstrated by successful manual and automated Edman sequencing. Taken together these data show that such a simple and precise approach is suitable for both analytical and preparative aims.

Amino Acid Sequence↗
PubMed1999

New insights into the mechanism of inhibition of p53 by simian virus 40 large T antigen.

Simian virus 40 (SV40) large tumor antigen (T antigen) has been shown to inhibit p53-dependent transcription by preventing p53 from binding to its cognate cis element. Data presented in this report provide the first direct functional evidence that T antigen, under certain conditions, may also repress p53-dependent transcription by a mechanism in which the transactivation domain of p53 is abrogated while DNA binding is unaffected. Specifically, p53 purified as a complex with T antigen from mouse cells was found to bind DNA as a transcriptionally inactive intact complex, while that purified from human cells was found to bind DNA independently of T antigen and could activate p53-dependent transcription. This difference in activity may be dependent on a different interaction of T antigen with mouse and human p53 and, in addition, on the presence of super T, which is found only in transformed rodent cells. These results suggest that subtle yet important differences exist between the inhibition of p53 by T antigen in mouse and human cells. The implications of this finding with respect to SV40-associated malignancies are discussed.

Animals↗
PubMed1999

[Evaluation of the biocompatibility of grafts for bone defects].

BACKGROUND: This paper examines a new material composed of polylactic and polyglycolic acids mixed with destrane easy to handle in bone defect reconstruction in periodontology and implantology: its name is fisiograft. METHODS: According to the European directives, fisiograft was checked for some biological tests selected for its specific use. In particular, tests of cytotoxicity, genotoxicity, sensitization and implantation in rabbit's femurs were carried out. RESULTS: The results show that the composite material is not cytotoxic when it is in direct and indirect contact with murine fibroblasts. It is not genotoxic and not allergenic. The implantation in rabbit, already after 30 days, shows that around the material there is a rapid bone growth and the material is reabsorbing. The degradation is suitable for its clinical use. CONCLUSIONS: Fisiograft showed to be biocompatible, easy to handle and its degradation kinetics in bone is appropriate.

Alveolar Ridge Augmentation↗
PubMed1999

Can protrusion of the tongue stop seizures in Rolandic epilepsy?

We report the case of a child with benign partial epilepsy with centro-temporal spikes (BECT) in whom protrusion of the tongue stopped the interictal abnormalities, and describe the polygraphic EEG recording of a seizure which terminated upon voluntary protrusion of the tongue. We mention the close link between the post-central (somatosensory) cortex and pre-central (motor) cortex, and how the primary sensory area has direct access to the motor cortex. We also examine how a tactile stimulus may provoke the inhibition of an epileptic discharge.

Child↗
PubMed1998

Panoramic coronary angiography.

OBJECTIVES: We describe a new imaging technique for coronary angiography. BACKGROUND: The conventional approach to coronary angiography exploits static perspective imaging over multiple cardiac cycles, using a limited number of empirically selected views. This approach entails both lack and redundancy of information and may result in suboptimal visualization of the individual lesion, contributing to diagnostic inaccuracy. METHODS: We developed a new imaging technique exploiting dynamic perspective, obtained by transverse 180 degree rotation of the C arm of a conventional angiographic unit during standard selective coronary opacification and filming. This technique yields a picture of the coronary tree isocentrically rotating around the longitudinal axis and conveying complete three-dimensional information. RESULTS: A complete diagnostic run for both coronary arteries, including two 25 degree cranial and two 25 degree caudal scans is accomplished with a total cine time of 16 s and 45 ml of contrast medium, about half of that required by conventional angiography. In a series of 129 consecutive patients studied by both the conventional and the new technique with quantitative measurements of the severity of the stenoses, the final diagnosis was identical in 65. In no case was a stenosis detected only by the conventional approach. However, in 31 patients the new technique permitted identification of 34 critical stenoses (79+/-8% [mean +/- SD]) either underestimated (61+/-3% n = 24, p < 0.001) or undetected (21+/-22%, n = 10, p < 0.001) in the standard projections. In a further 28 cases, 33 subcritical lesions (60+/-5%) were visualized in the rotational images but were insignificant (24+/-22% p < 0.001) in the standard projections. In five additional patients, distinct laminar plaques were clearly visualized only by the panoramic approach. CONCLUSIONS: This new technique can be easily implemented on conventional angiographic equipment at no additional cost. It provides complete, operator-independent exploitation of the angiographic information, resulting in enhanced diagnostic accuracy.

Coronary Angiography↗
PubMed1998

Intracranial hypotension syndrome: neuroimaging in five spontaneous cases and etiopathogenetic correlations.

Intracranial hypotension (IH) is essential or, more frequently, secondary. This syndrome is characterized by severe postural headache and low opening cerebrospinal fluid (CSF) pressure; although other symptoms may exist. In this study five patients are investigated. Neuroimaging showed: on computerized tomography scan (CT), poor visualization of the cerebral sulci with small ventricles; on magnetic resonance imaging (MRI), subdural fluid collections with enhancement on the convexity, along the tentorium and in the upper cervix after administration of contrast medium and downward displacement of the brain. Radionuclide cisternography was normal in the two patients who underwent this treatment as well as the meningeal biopsy in another patient. In all patients the opening CSF pressure was low or unmeasurable. The clinical syndrome spontaneously recovered contextually to normalization of neuroradiological findings. The possible pathogenesis (dural border cell layer tear) was discussed and the importance of diagnostic confirmation with MRI and measurement of CSF pressure when IH is thought to be present was underlined.

Adult↗
PubMed1998

Peripheral neuropathy in chronic respiratory insufficiency.

Peripheral neuropathy commonly occurs in patients with chronic obstructive lung disease (COPD). We report the presence of peripheral neuropathy in 19 of our 30 COPD patients (63.3%): 7 patients had clinical signs of a symmetric motor and sensory polyneuropathy, 12 patients had only subclinical evidence of peripheral nervous system involvement. Neurophysiological studies showed low amplitude compound muscle action potentials (CMAP) and sensory action potentials (SAP) with only slight reduction of nerve conduction velocity in affected patients: these data confirm an axonal polyneuropathy. The severity of the peripheral nervous system involvement in COPD patients was correlated with hypercapnia, the degree of disability and thus with the severity of COPD. Hypoxia, age and duration of the disease were not related with the presence of polyneuropathy. Improvement of respiratory function produced slight but progressive improvement of neurological symptoms. Within one year, also neurophysiological studies revealed a progressive and statistically significant improvement in CMAP and SAP amplitude and motor and sensory conduction velocity and, in some cases, normal electromyography.

Action Potentials↗
PubMed1997

Okadaic acid interferes with phorbol-ester-mediated down-regulation of protein kinase C-alpha, C-delta and C-epsilon.

A prolonged cell exposure of all examined cell types to tumour-promoting phorbol esters leads to a substantial inactivation and degradation of protein kinase C (PKC), a phenomenon known as down-regulation. With a combination of one- and two-dimensional immunoblot analyses we have previously shown the existence in PC12 cells of distinct PKC-alpha forms that differentially respond to cell treatment with phorbol ester [Gatti, A. & Robinson, P. J. (1996) J. Biol. Chem. 271, 31 718-31722]. Using the same experimental model, in the present study we investigated a possible relationship between PKC-alpha phosphorylation and its down-regulation. The exposure of PC12 cells to okadaic acid, a potent inhibitor of biologically relevant protein phosphatases, was found to partially protect PKC-alpha against phorbol-ester-mediated down-regulation. Further, a similar protective effect of okadaic acid was observed for PKC-delta and PKC-epsilon, which are also expressed in PC12 cells. These results indicate that the tumour-promoting activity of okadaic acid itself may be due to a sustained phosphorylation of PKC.

Animals↗
PubMed1997

Serologic survey for selected viral pathogens in brown bears from Italy.

Blood samples were collected from six captive bears and nine free-ranging Marsican brown bears (Ursus arctos marsicanus) in the Abruzzo National Park, Italy, between 1991 and 1995. Sera were tested for evidence of exposure to canine distemper virus (CDV), canine adenovirus type 2, canine coronavirus, and canine parvovirus type 2 (CPV-2). Serologic evidence of CDV and CPV-2-exposure was found in both captive and free-ranging bears. This may be the first report of CDV exposure in free-ranging bears.

Adenoviridae Infections↗
PubMed1996

Unique phosphorylation of protein kinase C-alpha in PC12 cells induces resistance to translocation and down-regulation.

Cell exposure to phorbol ester stimulates translocation and activation of protein kinase C (PKC), ultimately followed by its down-regulation. Upon activation, PKC-alpha, the best studied isotype of the PKC family, undergoes changes in its phosphorylation state. With a two-dimensional immunoblot procedure we have previously shown the existence in PC12 cells of several multiply phosphorylated forms of PKC-alpha, whose number increases in response to phorbol esters (Gatti, A., Wang, X., and Robinson, P. J. (1996) Biochim. Biophys. Acta 1313, 111-118). Using the same experimental system, here we report that besides the predominant pool of 80-kDa PKC-alpha forms that respond to phorbol ester by translocating to the cell membranes and down-regulating, there is a small pool of cytosolic 82-kDa PKC-alpha forms that are characterized by a more acidic pI and by an unique resistance to phorbol ester-mediated translocation and down-regulation. The appearance of similarly slower migrating and more acidic PKC-alpha forms is reproduced upon in vitro autophosphorylation in the presence of phosphatidylserine and phorbol ester, but not in the presence of calcium. These results suggest that site-specific transphosphorylation or autophosphorylation of this kinase may regulate its subcellular localization and susceptibility to down-regulation.

Animals↗
PubMed1996

Protein kinase C-alpha is multiply phosphorylated in response to phorbol ester stimulation of PC12 cells.

The protein kinase C (PKC) family consists of a number of closely related isotypes, whose in vivo phosphorylation state is regulated in a dynamic fashion by the enzyme's activators. We have investigated here the changes in PKC phosphorylation in response to phorbol ester. Using a combination of hydroxylapatite chromatography and immunoblot with isotype-specific antibodies, we identified PKC-alpha, -delta, -epsilon, and -zeta as the isotypes expressed in PC12 cells. A two-dimensional immunoblot approach was then developed to measure the changes in the phosphorylation state of PKC-alpha before and after exposure of intact PC12 cells to phorbol ester. We found a pool of four differentially migrating PKC-alpha forms in untreated cells, which undergoes an acidic shift after phorbol ester. Furthermore, a similar shift in the two-dimensional immunoblot profile of PKC-alpha was the result of the enzyme autophosphorylation upon in vitro treatment with a combination of phosphatidylserine and phorbol ester, an effect which was enhanced by co-application of purified bovine lung cGMP-dependent protein kinase-I (PKG-I). These results demonstrate a multiple phosphorylation of PKC-alpha in untreated PC12 cells and suggest that various levels of autophosphorylation and trans-phosphorylation of this isoenzyme may occur in response to phorbol ester.

Animals↗