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Biomedical subjects

M Fiorini

Publications and source records attributed to M Fiorini.

At least 19 recordsLinked to original sources
PubMed2006

Detection of CSF 14-3-3 protein in Guillain-Barré syndrome.

OBJECTIVE: To search for biologic markers in the Guillain-Barré syndrome (GBS), we studied CSF samples from patients with GBS and neuropathy of various etiologies for the presence of 14-3-3 protein. METHODS: CSF samples from patients with GBS, chronic neuropathies, motor neuron disease (MND), definite sporadic Creutzfeldt-Jakob disease (sCJD), and normal control subjects were analyzed by standard immunoblot assay, using a polyclonal anti-14-3-3 antibody. CSF samples were also tested with antibodies recognizing specific isoforms of 14-3-3 proteins, either after one-dimensional or two-dimensional electrophoretic separation. RESULTS: A positive 14-3-3 assay was observed in 29 of 38 patients with GBS and in 4 patients with MND and other neuropathies, including 2 subjects with vasculitic neuropathy (VN). In GBS, 14-3-3 protein was detected as early as 12 to 48 hours after disease onset and showed an isoform pattern different from that encountered in patients with noninflammatory neuropathies, VN, MND, and sCJD. Immunohistochemical studies performed in archival fatal GBS cases disclosed marked 14-3-3 expression by mononuclear inflammatory infiltrates and Schwann cells. CONCLUSION: CSF 14-3-3 assay may represent a useful biologic marker in patients with Guillain-Barré syndrome.

14-3-3 Proteins↗
PubMed2006

Excitation energy transfer in ion pairs of polymethine cyanine dyes: efficiency and dynamics.

The present work deals with singlet excitation energy transfer (EET) occurring in contact ion pairs (CIPs) of several anionic oxonol analogues (acting as EE donors) and cationic cyanines (acting as acceptors) characterized by off resonance individual transitions. Combining conductometric and spectroscopic measurements with decreasing solvent polarity, we were able to observe a progressive ion pairing leading first to solvent-separated ion pairs (SSIPs) and then to CIPs. Analysis of the absorption spectra of three selected salts (A2,C1, A2,C2, and A1,C4) in chloroform-toluene mixtures showed that the transformation of SSIP into CIP involves the appearance of a certain exciton coupling, the extent of which decreases regularly with increasing gap between the local excitation energies. Fluorescence excitation spectra showed that EET occurs in CIP, and EET efficiencies were evaluated with a procedure expressly devised for weakly emitting donors. These were between 0.2 and 0.65 for the examined ion pairs involving anions A1 and A2. The spectroscopic study was complemented by a theoretical investigation aimed at establishing the dynamic regime of the observed EET. From classical MD simulations and local full geometry optimizations, A2,C1 and A2,C2 were found to form rather stable sandwich-type CIP structures with interchromophore distances (R) of about 0.45-0.50 nm. The donor-acceptor electronic coupling was calculated in terms of Coulombic interactions between atomic transition charges. For CIP, the electronic coupling was decidedly beyond the limit of the weak coupling required for an incoherent Förster-type mechanism. Thus, we tried to arrange the EET dynamics within the theory developed by Kimura, Kakitani, and Yamato (J. Phys. Chem. B 2000, 104, 9276) for the intermediate coupling case, which provides analytical expressions of time-dependent occupation probability, EET rate, and coherency in terms of two basic quantities: the electronic coupling and a correlation time related to the Franck-Condon factor. The latter was shown to be primarily modulated by Förster's spectral overlap integral (related in turn to the excitation energy gap). Calculations were carried out for the three sample systems using three values of the electronic coupling roughly corresponding to CIP, 1.0, and 2.0 nm interchromophore distances. At the CIP distance, EET in both A2,C1 and A2,C2 was predicted to occur with a partial exciton mechanism, very short transfer times (about 10 fs), and high degree of coherence. In A1,C4 (having the largest energy gap), EET was found to occur with a hot-transfer mechanism. More or less hot-transfer dynamics appeared to be retained by all three systems at R = 1.0 nm. Fully incoherent EET appeared to become operative only at distances larger than 2.0 nm.

Journal Article↗
PubMed2005

Phosphorylated 14-3-3zeta protein in the CSF of neuroleptic-treated patients.

The authors describe 12 neuroleptic-treated patients with dementia of various etiologies who showed CSF elevation of phosphorylated 14-3-3zeta and normal tau protein levels. This contrasted with elevated amounts of 14-3-3 gamma, epsilon, and unphosphorylated zeta coupled to high tau protein levels in Creutzfeldt-Jakob disease and negative 14-3-3 assay in drug-free patients with dementia. Characterization of CSF 14-3-3 isoforms and determination of tau protein level can help to distinguish different etiologies of dementia.

14-3-3 Proteins↗
PubMed2001

pH-dependent prion protein conformation in classical Creutzfeldt-Jakob disease.

In transmissible spongiform encephalopathies, the cellular prion protein (PrP(C)) undergoes a conformational change from a prevailing alpha-helical structure to a beta-sheet-rich, protease-resistant isoform, termed PrP(Sc). PrP(C) has two characteristics: a high affinity for Cu(2+) and a strong pH-dependent conformation. Lines of evidence indicate that PrP(Sc) conformation is dependent on copper and that acidic conditions facilitate the conversion of PrP(C) --> PrP(Sc). In each species, PrP(Sc) exists in multiple conformations, which are associated with different prion strains. In sporadic Creutzfeldt-Jakob disease (sCJD), different biochemical types of PrP(Sc) have been identified according to the size of the protease-resistant fragments, patterns of glycosylation, and the metal-ion occupancy. Based on the site of cleavage produced by proteinase K, we investigated the conformational stability of PrP(Sc) under acidic, neutral, and basic conditions in 42 sCJD subjects. Our study shows that only one type of sCJD PrP(Sc), associated with the classical form, shows a pH-dependent conformation, whereas two other biochemical PrP(Sc) types, detected in distinct sCJD phenotypes, are unaffected by pH variations. This novel approach demonstrates the presence of three types of PrP(Sc) in sCJD.

Blotting, Western↗
PubMed2001

Negative regulation of receptor tyrosine kinase signals.

In Metazoans a number of cellular functions are controlled by receptor tyrosine kinases (RTKs) during development and in postnatal life. The execution of these programs requires that signals of adequate strength are delivered for the appropriate time within precise spatial boundaries. Several RTK inhibitors have been identified in invertebrate and mammalian organisms. Because they are involved in tuning and termination of receptor signals, negative regulators of RTK activity fulfill a fundamental function in the control of receptor signaling.

Animals↗
PubMed2001

Chemical force microscopy with active enzymes.

The adhesion forces have been measured between an atomic force microscope tip derivatized with an active enzyme, shikimate kinase, and an ATP mimic immobilized on a gold surface. Experiments with competitive binding of other ligands in solution show that the observed adhesion forces arise predominantly from specific interactions between the immobilized enzyme and surface-bound adenine derivative. These experiments represent a step in the development of a screening methodology based upon chemical force microscopy.

Binding, Competitive↗
PubMed2001

Transplantation of chondrocytes seeded on a hyaluronan derivative (hyaff-11) into cartilage defects in rabbits.

Different methods have been used to improve chondrocyte transplantation for the repair of articular cartilage defects. Several groups of biomaterials have been proposed as support for in vitro cell growth and for in vivo implantation. Here. we describe a new approach investigating the healing of rabbit cartilage by means of autologous chondrocytes seeded on a hyaluronan derivative referred to as Hyaff-11. Full thickness defects were created bilaterally in the weight-bearing surface of the medial femoral condyle of both femora of New Zealand male rabbits. The wounds were then repaired using both chondrocytes seeded on the biomaterial and biomaterial alone. Controls were similarly treated but received either no treatment or implants of the delivery substance. Histologic samples from in and around the defect sites were examined 1, 3 and 6 months after surgery and were scored from 0 to 16. Statistically significant differences in the quality of the regenerated tissue were found between the grafts carried out with biomaterial carrying chondrocyte cells compared to the biomaterial alone or controls. This study demonstrates the efficacy of this hyaluronan-based scaffold for autologous chondrocytes transplantation.

Animals↗
PubMed2000

Development of autologous T lymphocytes in two males with X-linked severe combined immune deficiency: molecular and cellular characterization.

We report on two patients affected with severe combined immune deficiency (SCID) with an unusual immunological phenotype and a substantial number of autologous, poorly functioning T cells. Distinct mutations identified at the IL2RG locus in the two patients impaired IL-2-mediated signaling but affected T-cell lymphopoiesis differently, resulting in generation of a polyclonal or oligoclonal T-cell repertoire. These observations add to the growing complexity of the immunological spectrum of SCID in humans and indicate the need for detailed immunological and molecular investigations in atypical cases.

Adolescent↗
PubMed2000

Complete genomic organization of the human JAK3 gene and mutation analysis in severe combined immunodeficiency by single-strand conformation polymorphism.

JAK3 deficiency in humans results in autosomal recessive T-B+ severe combined immunodeficiency disease (SCID), a severe immunodeficiency that can only be cured by bone marrow transplantation. We unraveled the complete organization of the human JAK3 gene, which includes 23 exons. This information allowed us to set up a molecular screening test that enabled us to diagnose JAK3 deficiency in 14 patients from 12 unrelated families with T-B+ SCID. In order to define the mutations, we used a nonradioactive single-strand conformation polymorphism (SSCP)/heteroduplex (HD) assay based on exon-specific polymerase chain reaction (PCR). In this cohort of patients, 15 independent JAK3 gene mutations have been identified, including 7 that have not been described previously. Mutation analysis information was used for genetic counseling and prenatal diagnosis.

Consanguinity↗
PubMed2000

Of genes and phenotypes: the immunological and molecular spectrum of combined immune deficiency. Defects of the gamma(c)-JAK3 signaling pathway as a model.

Cytokines play a major role in lymphoid development. Defects of the common gamma chain (gamma(c)) or of the JAK3 protein in humans have been shown to result in a severe combined immune deficiency (SCID), with a profound defect in T and natural killer (NK)-cell development, whereas B-cell generation is apparently unaffected (T-B+NK-SCID). While extensive molecular and biochemical analysis of these patients has been instrumental in understanding better the biological properties of the gamma(c) and JAK3 protein, an unexpected phenotypic heterogeneity of gamma(c) and JAK3 deficiency has emerged, indicating the need for appropriate and extensive investigations even in patients with atypical presentations. At the same time, characterization of the defects has been instrumental in the development of novel therapeutic approaches, from in utero hematopoietic stem cell transplantation to gene therapy.

Cytokines↗
PubMed2000

Inhibition of ErbB-2 mitogenic and transforming activity by RALT, a mitogen-induced signal transducer which binds to the ErbB-2 kinase domain.

The product of rat gene 33 was identified as an ErbB-2-interacting protein in a two-hybrid screen employing the ErbB-2 juxtamembrane and kinase domains as bait. This interaction was reproduced in vitro with a glutathione S-transferase fusion protein spanning positions 282 to 395 of the 459-residue gene 33 protein. Activation of ErbB-2 catalytic function was required for ErbB-2-gene 33 physical interaction in living cells, whereas ErbB-2 autophosphorylation was dispensable. Expression of gene 33 protein was absent in growth-arrested NIH 3T3 fibroblasts but was induced within 60 to 90 min of serum stimulation or activation of the ErbB-2 kinase and decreased sharply upon entry into S phase. New differentiation factor stimulation of mitogen-deprived mammary epithelial cells also caused accumulation of gene 33 protein, which could be found in a complex with ErbB-2. Overexpression of gene 33 protein in mouse fibroblasts inhibited (i) cell proliferation driven by ErbB-2 but not by serum, (ii) cell transformation induced by ErbB-2 but not by Ras or Src, and (iii) sustained activation of ERK 1 and 2 by ErbB-2 but not by serum. The gene 33 protein may convey inhibitory signals downstream to ErbB-2 by virtue of its association with SH3-containing proteins, including GRB-2, which was found to associate with gene 33 protein in living cells. These data indicate that the gene 33 protein is a feedback inhibitor of ErbB-2 mitogenic function and a suppressor of ErbB-2 oncogenic activity. We propose that the gene 33 protein be renamed with the acronym RALT (receptor-associated late transducer).

3T3 Cells↗
PubMed2000

X-chromosome inactivation and mutation pattern in the Bruton's tyrosine kinase gene in patients with X-linked agammaglobulinemia. Italian XLA Collaborative Group.

BACKGROUND: The diagnosis of X-linked agammaglobulinemia (XLA) is not always clearcut. Not all XLA conform to the classic phenotype and less than 50% of affected boys have a family history of immunodeficiency. Mutations in the gene for Bruton's tyrosine kinase (BTK) are responsible for the majority of agammaglobulinemia cases. However, a certain proportion of patients may have mutations involving other genes, although they show with an XLA phenotype. We performed BTK gene mutation analysis in 37 males with presumed XLA and analyzed the pattern of X-chromosome inactivation (XCI) in 31 mothers to evaluate the relevance of these approaches to diagnosis and genetic counseling. MATERIALS AND METHODS: Twenty affected males with a sporadic occurrence and 17 familial cases belonging to nine families were enrolled within the framework of the Italian Multicenter Clinical Study on XLA. We used non-isotopic RNase cleavage assay (NIRCA), followed by cDNA sequence determination to screen for BTK mutations and X-chromosome inactivation analysis for carrier detection. RESULTS: Using the cDNA-based approach, the identification of BTK gene abnormalities confirmed the clinical diagnosis of XLA in 31 of 37 affected infants. Missense was the most frequent mutational event and the kinase domain was mostly involved. In addition, nine novel mutations were identified. In sporadic cases, BTK gene abnormalities were identified in 9 out of 10 patients whose mothers had a nonrandom pattern of XCI and in 5 out of 6 patients whose mother had a random pattern of XCI. With the exception of one family, all patients with a familial occurrence and born to mothers with a nonrandom pattern of XCI had mutations of the BTK gene. CONCLUSIONS: Our findings indicate that in sporadic cases BTK gene sequencing is the only reliable tool for a definitive diagnosis of XLA and support XCI as the first diagnostic tool in the mothers of affected males in multiple generations. Furthermore, our molecular analysis confirms that 10-20% of BTK-unaltered patients have disorders caused by defects in other genes.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1999

Painless intussusception and altered mental status.

A 7-month-old child presented to the emergency department (ED) with 2 hours of painless, nonprojectile emesis and a normal mental status. Over a 3-hour period in the ED, the child remained pain-free, but developed hematemesis, hematochezia, and lethargy, progressing to unresponsiveness. The patient was evaluated for toxic ingestion, intracranial bleed, sepsis/meningitis, and intraabdominal pathology. The diagnosis was made by an abdominal ultrasound, which demonstrated an ileal-cecal intussusception that ultimately required surgical reduction. This case illustrates an insidious and poorly understood presentation of a common childhood affliction, as well as the utility of abdominal ultrasound in evaluating a hemodynamically stable patient with intussusception.

Consciousness↗
PubMed1999

Prenatal molecular diagnosis of Wiskott-Aldrich syndrome by direct mutation analysis.

We have performed prenatal diagnosis for Wiskott Aldrich syndrome (WAS) in two unrelated families by direct gene analysis. Using a combined non-radioactive analysis of single-strand conformational polymorphism (SSCP) and heteroduplex formation (HD), followed by automated sequencing, we studied DNA from chorionic villus sampling (CVS), allowing the diagnosis of one affected and one healthy male at the 12th week of gestation.

Base Sequence↗
PubMed1999

Prenatal diagnosis of JAK3 deficient SCID.

The JAK3 gene, encoding a tyrosine kinase functionally coupled to cytokine receptors which share the common gamma chain, has been identified as the defective gene for autosomal recessive severe combined immunodeficiency (SCID). Thus, specific mutational diagnosis has become possible. We screened all exons with a combined single strand conformational polymorphism and hetero-duplex formation assay followed by sequence analysis to identify specific mutations in two families. This assay was used on chorionic villus sampling derived DNA in two fetuses from two unrelated families, where we found mutations in both parents. We were able to exclude the mutations in both fetuses by the 12th week of gestation. The described method for first-trimester prenatal diagnosis of autosomal recessive T-B+SCID provides a valid tool to aid in genetic counselling and possibly prenatal therapy in this disease.

Alleles↗
PubMed1998

Monocytes from Wiskott-Aldrich patients display reduced chemotaxis and lack of cell polarization in response to monocyte chemoattractant protein-1 and formyl-methionyl-leucyl-phenylalanine.

Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by trombocytopenia, eczema, and progressive decline of the immune function. In addition, lymphocytes and platelets from WAS patients have morphologic abnormalities. Since chemokines may induce morphologic changes and migration of leukocytes, we investigated the monocyte response to chemoattractants in cells from WAS patients with an identified mutation in the WAS protein gene. Here, we report that monocytes derived from four patients with molecularly defined typical WAS have a severely impaired migration in response to FMLP and to the chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha compared with normal donors. Conversely, neither MCP-1 binding to monocytes nor induction of the respiratory burst by MCP-1 and FMLP is significantly different between WAS patients and normal donors. Within a few minutes of stimulation, monocytes respond to chemokines with increased expression of adhesion molecules and with morphologic changes such as cell polarization. Although up-regulation of CD11b/CD18 expression following stimulation with FMLP or MCP-1 is preserved in WAS patients, cell polarization is dramatically decreased. Staining of F-actin by FITC-phalloidin in monocytes stimulated with chemoattractants shows F-actin to have a rounded shape in WAS patients, as opposed to the polymorphic distribution of F-actin in the polarized monocytes from healthy donors. These results suggest that WAS protein is involved in the monocyte response to the chemokines MCP-1 and macrophage inflammatory protein-1alpha.

Cell Movement↗
PubMed1998

Defective actin polymerization in EBV-transformed B-cell lines from patients with the Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive disorder characterized by eczema, thrombocytopenia, and immunodeficiency. An allelic variant of the disease is characterized by isolated thrombocytopenia (XLT). The gene responsible for WAS/XLT (WASP) encodes for a 502 amino acid protein (WASP) that is possibly involved in actin binding and cytoskeleton organization. The expression of WASP and the distribution of F-actin and alpha-actinin (which binds to and stabilizes actin filaments) have been analysed in lymphoblastoid cell lines from six patients with WAS and one with XLT. Western blot and immunocytochemistry did not reveal WASP expression in four WAS patients, whereas two WAS patients (with a moderate clinical course) expressed trace amounts of mutant WASP. In contrast, the XLT patient expressed normal amounts of WASP. Furthermore, cell lines from WAS and XLT patients also markedly differed in F-actin polymerization and alpha-actinin distribution. In particular, severe defects of cytoplasmic F-actin expression and of F-actin-positive microvillus formation, and impaired capping of alpha-actinin, were observed in all patients who lacked WASP. As a whole, the degree of impairment of WASP protein expression in WAS/XLT seems to correlate with anomalies of cytoskeletal organization, strongly supporting a role for WASP in the regulation of F-actin polymerization.

Actinin↗