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V Barone

Publications and source records attributed to V Barone.

At least 37 records · Page 2Linked to original sources
PubMed1997

Requirement for the ryanodine receptor type 3 for efficient contraction in neonatal skeletal muscles.

The skeletal isoform of Ca2+ release channel, RyR1, plays a central role in activation of skeletal muscle contraction. Another isoform, RyR3, has been observed recently in some mammalian skeletal muscles, but whether it participates in regulating skeletal muscle contraction is not known. The expression of RyR3 in skeletal muscles was studied in mice from late fetal stages to adult life. RyR3 was found to be expressed widely in murine skeletal muscles during the post-natal phase of muscle development, but was not detectable in muscles of adult mice, with the exception of the diaphragm and soleus muscles. RyR3 knockout mice were generated, and it was shown that skeletal muscle contraction in these mice was impaired during the first weeks after birth. In skeletal muscles isolated from newborn RyR3(-/- )mice, but not in those from adult mice, the twitch elicited by electrical stimulation and the contracture induced by caffeine were strongly depressed. These results provide the first evidence that RyR3 has a physiological role in excitation-contraction coupling of neonatal skeletal muscles. The disproportion between the low amount of RyR3 and the large impact of the RyR3 knockout suggests that this isoform contributes to the amplification of Ca2+ released by the existing population of ryanodine receptors (RyR1).

Age Factors↗
PubMed1997

Frequency of RET mutations in long- and short-segment Hirschsprung disease.

Hirschsprung disease, or congenital aganglionic megacolon, is a genetic disorder of neural crest development affecting 1:5,000 newborns. Mutations in the RET proto-oncogene, repeatedly identified in the heterozygous state in both long- and short-segment Hirschsprung patients, lead to loss of both transforming and differentiating capacities of the activated RET through a dominant negative effect when expressed in appropriate cellular systems. The approach of single-strand conformational polymorphism analysis established for all the 20 exons of the RET proto-oncogene, and previously used to screen for point mutations in Hirschsprung patients allowed us to identify seven additional mutations among 39 sporadic and familial cases of Hirschsprung disease (detection rate 18%). This relatively low efficiency in detecting mutations of RET in Hirschsprung patients cannot be accounted by the hypothesis of genetic heterogeneity, which is not supported by the results of linkage analysis in the pedigrees analyzed so far. Almost 74% of the point mutations in our series, as well as in other patient series, were identified among long segment patients, who represented only 25% of our patient population. The finding of a C620R substitution in a patient affected with total colonic aganglionosis confirms the involvement of this mutation in the pathogenesis of different phenotypes (i.e., medullary thyroid carcinoma and Hirschsprung). Finally the R313Q mutation identified for the first time in homozygosity in a child born of consanguineous parents is associated with the most severe Hirschsprung phenotype (total colonic aganglionosis with small bowel involvement).

DNA Mutational Analysis↗
PubMed1996

Exclusion of linkage between RET and neuronal intestinal dysplasia type B.

Neuronal Intestinal Dysplasia type B (NID B) is a complex alteration of the enteric nervous system belonging to the group of intestinal dysganglionoses which may involve rectum, colon, and small intestine. Second only to Hirschsprung disease (HSCR), NID B is one of the most frequent causes of chronic constipation and pseudo-obstructive intestinal dysmotility. Since NID B is often associated with HSCR and point mutations in the RET proto-oncogene have been identified in HSCR patients, we analyzed two NID B pedigrees to investigate if RET mutations might cause also the NID B phenotype. Linkage analysis demonstrated that the NID B locus is not linked to RET in the pedigrees analyzed. Further genetic analyses will possibly improve the understanding of the cause and facilitate diagnostic procedures in NID B.

Drosophila Proteins↗
PubMed1996

Prevalence and parental origin of de novo RET mutations in Hirschsprung's disease.

In contrast with the reported almost exclusive paternal origin of de novo mutations in MEN 2A, FMTC and MEN 2B, de novo mutations in Hirschsprung patients arise both on paternal and maternal chromosomes. This distinctive feature of RET mutations associated with Hirschsprung's disease and of the RET mutations associated with thyroid cancer indicates a basic biological difference between the mutational events leading to the different phenotypes.

DNA Mutational Analysis↗
PubMed1995

Molecular cloning and functional characterization of a GABA/betaine transporter from human kidney.

The human homologue of the canine GABA/betaine transporter (BGT-1) was isolated from a kidney inner medulla cDNA library. The coding sequence predicts a 614 amino acids protein with the typical features of neurotransmitter transporter family. The gene maps to chromosome 12p13 and, in addition to kidney, is also expressed in brain, liver, heart, skeletal muscle, and placenta. Functional studies reveal a Km = 20 microM for GABA transport and a coupling to Na+ and Cl- with a stoichiometry 3 Na+:2 Cl-:1 GABA. At 500 microM the GABA transport was inhibited by various compounds with the following potency order: quinidine > verapamil > phloretin > betaine.

Amino Acid Sequence↗
PubMed1994

Point mutations affecting the tyrosine kinase domain of the RET proto-oncogene in Hirschsprung's disease.

Hirschsprung's disease is a genetic disorder of neural crest development affecting 1 in 5,000 births. It is characterized by the absence of intramural ganglion cells in the hindgut, which often results in partial to complete intestinal obstruction during the first years of life. An autosomal dominant gene causing this disease was recently mapped to chromosome 10q11.2 (refs 1,2), using an interstitial deletion of this region isolated in a cell hybrid. It was subsequently localized to a 250-kilobase interval which contains the RET proto-oncogene. Using flanking intronic sequences as primers to amplify 12 of the 20 exons of RET from genomic DNA of 27 Hirschsprung's disease patients, we have now identified four mutations (one frameshift and three missense) that totally disrupt or partially change the structure of the tyrosine kinase domain of the RET protein (Ret). Mutations in the extracellular cysteine-rich domain of Ret have been identified previously in patients with multiple endocrine neoplasia type 2A, and a targeted mutation in the tyrosine kinase domain of the same gene produces intestinal aganglionosis and kidney agenesis in homozygous transgenic mice. Our results support the hypothesis that RET, in addition to its potential role in tumorigenesis, plays a critical role in the embryogenesis of the mammalian enteric nervous system.

Base Sequence↗
PubMed1994

Identification of the Cys634-->Tyr mutation of the RET proto-oncogene in a pedigree with multiple endocrine neoplasia type 2A and localized cutaneous lichen amyloidosis.

Following the recent identification of specific germline mutations of the RET proto-oncogene in Multiple Endocrine Neoplasia type 2A (MEN2A) patients, we looked for mutations of this gene in a pedigree showing recurrence of MEN2A and localized Cutaneous Lichen Amyloidosis (CLA). Basal calcitonin and/or pentagastrin test performed in all the 10 available members of this pedigree confirmed the clinical diagnosis and allowed the presymptomatic identification of an additional carrier. A cys634-->tyr missense mutation, already reported as causative in MEN2A patients, was identified after SSCP analysis and direct sequencing of exon 11 of the RET protooncogene in one individual affected with both MEN2A and CLA, thus suggesting a common etiology for the two disorders. Taking advantage of the observation of an RsaI restriction site in the sequence surrounding the mutated codon, we could demonstrate that the same mutation is present in three other affected members, in the presymptomatic carrier and in one additional 25 years old healthy member who shows a mildly positive pentagastrin test.

Amyloidosis↗
PubMed1994

Heterogeneity and low detection rate of RET mutations in Hirschsprung disease.

Mutations in some exons of the RET proto-oncogene were recently observed in Hirschsprung patients. Using DNA polymorphisms and single-strand conformation polymorphism analysis for the whole coding sequence of the RET proto-oncogene, 82 unrelated Hirschsprung patients were screened systematically. A total of 4 complete deletions of RET and 12 point mutations were identified, each present in no more than one patient and distributed along the whole gene. De novo mutations could be documented in 4 patients. Southern blot and fluorescence in situ hybridization analysis carried out in a restricted number of patients did not reveal any deletion of RET. The low efficiency in detecting mutations of RET in Hirschsprung patients (20%) may originate mainly from genetic heterogeneity.

Blotting, Southern↗
PubMed1994

DNA polymorphisms and conditions for SSCP analysis of the 20 exons of the ret proto-oncogene.

Recently identified mutations affecting different domains of the RET proto-oncogene are associated with Multiple Endocrine Neoplasia type 2A (MEN 2A) and type 2B (MEN 2B), familial and sporadic Medullary Thyroid Carcinomas (MTC) and Hirschsprung disease (HSCR). In order to facilitate the screening for RET mutations, and to study possible genotype-phenotype correlations, we established exon-intron junctions and extended the intronic sequences flanking the 20 exons of this gene. This made it possible to design primers and to develop PCR conditions useful for SSCP analysis of the whole RET coding sequence. Nine conformational variants were observed which after sequencing turned out to be 8 silent mutations and a conservative amino acid substitution. Restriction analysis performed on DNA samples from unrelated controls confirmed the polymorphic nature of six of these nucleotide changes and made it possible to estimate the frequency of the corresponding alleles.

Base Sequence↗
PubMed1993

Close linkage with the RET protooncogene and boundaries of deletion mutations in autosomal dominant Hirschsprung disease.

Tight linkage with the RET proto-oncogene (Zmax = 3.41 at theta = 0.00), analysis of recombinants and detection of a familial microdeletion in a large pedigree restrict the mapping of the Hirschsprung (HSCR) gene previously localized on proximal 10q. The molecular characterization of the familial microdeletion and of 3 additional cytogenetically visible de novo deletions, isolated in somatic cell hybrids, identify a smallest region of overlap of 250 Kb. This contains the RET proto-oncogene where missense mutations causing multiple endocrine neoplasia type 2A (MEN 2A) phenotype were recently found. The pentagastrin test (which detects preclinical forms of MEN 2A or B) is negative in adult HSCR patients with deletions of the RET gene. This represents a good candidate for the search of mutations causing HSCR.

Cell Line↗
PubMed1991

A forskolin and verapamil sensitive K+ current in human tracheal cells.

A voltage-dependent K+ current has been revealed in whole-cell recordings carried out on immortalized cells obtained from the human tracheal epithelium. At positive membrane potentials the current shows a time dependent inactivation which is accelerated by increasing the depolarizing step. Forskolin, a direct activator of adenylyl cyclase, and verapamil, a Ca2+ channel blocker, induce the K+ current to inactivate more rapidly. Control experiments show that the action of these two compounds is not mediated by cyclic AMP and Ca2+. The application of 1,9-dideoxyforskolin, an analogue which does not stimulate adenylate cyclase, inhibits the current in the same way as forskolin; on the contrary, the dibutyryl analogue of cyclic AMP is ineffective. Furthermore, eliminating extracellular Ca2+ does not affect K+ current kinetics. Tetraethylammonium is an effective blocker of this current with an IC50 of 0.3 mM.

Cell Line↗
PubMed1991

Preferred conformation of peptides from C alpha,alpha- symmetrically disubstituted glycines: aromatic residues.

The conformational preference of C(alpha,alpha-diphenylglycine (D-phi-g) and C(alpha,alpha)-dibenzylglycine (Dbz) residues was assessed in selected derivatives and small peptides by conformational energy computations, ir absorption, 1H-nmr, and x-ray diffraction. Conformational energy computations on the two monopeptides strongly support the view that these C(alpha,alpha)-symmetrically disubstituted glycines are conformationally restricted and that their minimum energy conformation falls in the fully extended (C5) region. The results of the theoretical analyses appear to be in agreement with the solution and crystal-state structural propensities of three derivatives and seven di- and tripeptides.

Amino Acid Sequence↗
PubMed1991

Characterization of chloride and cation channels in cultured human keratinocytes.

Patch-clamp experiments on human cultured keratinocytes revealed the presence of three types of ion channel. The first type was a Cl(-)-selective channel, the current/voltage relationship of which showed outward rectification, the mean conductance at positive and negative membrane potentials being 66 pS and 16 pS respectively. The second type of channel showed almost equal permeability to alkali ions but was impermeable to Cl- and to the large organic cation N-methyl-D-glucamine. Its current/voltage relationship was linear with a mean unitary conductance of 18 pS in symmetrical 140 mmol/l NaCl. Finally, the third type was a large-conductance cation channel, which had in physiological ionic conditions a peculiar rectifying current/voltage relationship, the shape of which was strongly dependent on the concentration of divalent cations on both sides of the membrane. Lowering of Ca2+ and/or Mg2+ on either side of the patch led to a marked increase of the single-channel current. With identical solutions without Ca2+ Mg2+ on both sides of the patch the current/voltage relationship became ohmic and reached a conductance of 150-200 pS. In addition, channel activity was reversibly affected by changes of the external Ca2+ concentration. In particular, open-channel probability strongly increased at negative membrane potentials when the external Ca2+ was lowered from millimolar to micromolar values. Whole-cell experiments confirm the role of the extracellular Ca2+ as a modulator of the cation conductance.

Barium↗
PubMed1989

Structural versatility of peptides containing C alpha, alpha-dialkylated glycines: conformational energy computations, i.r. absorption and 1H n.m.r. analysis of 1-aminocyclopropane-1-carboxylic acid homopeptides.

Conformational energy computations on the 1-aminocyclopropane-1-carboxylic acid mono-, di-, and tripeptide amides, Ac-(Ac3c)n-NHMe (n = 1-3), indicate that this C alpha, alpha-dialkylated, cyclic alpha-amino acid residue is conformally restricted and that type-I(I') beta-bends and distorted 3(10)-helices are particularly stable conformations for the di- and tripeptide amides, respectively. The results of the theoretical analysis are in agreement with those obtained in an i.r. absorption and 1H n.m.r. investigation in chloroform solution of Ac3c-rich tri- and tetrapeptide esters. A comparison is also made with the conclusions extracted from our previous work on peptides rich in Aib (alpha-aminoisobutyric acid), Ac5c (1-aminocyclopentane-1-carboxylic acid), and Ac6c (1-aminocyclohexane-1-carboxylic acid).

Alkylation↗
PubMed1989

Structural versatility of peptides containing C alpha, alpha-dialkylated glycines. An X-ray diffraction study of six 1-aminocyclopropane-1-carboxylic acid rich peptides.

The molecular and crystal structures of six fully blocked, Ac3c-rich peptides to the tetramer level were determined by X-ray diffraction. The peptides are Fmoc-(Ac3c)2-OMe-CH3OH, Ac-(Ac3c)2-OMe, t-Boc-Ac3c-L-Phe-OMe, pBrBz-(Ac3c)3-OMe.H2O, Z-Gly-Ac3c-Gly-OTmb.(CH3)2CO, and t-Boc-(Ac3c)4-OMe.2H2O. Type-I (I') beta-bends and distorted 3(10)-helices were found to be typical of the tri- and tetrapeptides, respectively. In the dipeptides, too short to form beta-bend conformations, other less common structural features may be observed. The average geometry of the cyclopropyl moiety of the Ac3c residue is asymmetric and the N-C alpha-C' bond angle is significantly expanded from the regular tetrahedral value. A comparison with the structural preferences of other extensively investigated C alpha, alpha-dialylated alpha-amino acids is made and the implications for the use of the Ac3c residue in conformational design are examined.

Alkylation↗
PubMed1987

Conformational preferences and self-association modes of two diastereomeric statine derivatives.

The conformational preferences and self-association modes of the two diastereomeric N-acetyl, methylamides of 3-hydroxy, 4-amino, 6-methylheptanoic acid (statine) with (R, S) and (S, S) configurations at the 3-hydroxy and 4-amino carbons, respectively, have been determined in solution as well as in the solid state by infrared absorption, 1H nuclear magnetic resonance, and X-ray diffraction. Conformational energy computations have also been performed in parallel. In the crystal state, the change in chirality of the hydroxyl group induces different intermolecular H-bonding schemes in the (R, S) isomer compared to the two structurally distinct molecules in the asymmetric unit of the (S, S) isomer. Different propensities to self-aggregate are seen in solvents of low polarity. In solvents of high polarity, however, the molecules of both isomers are largely solvated, while still keeping some local conformational restriction. Conformational energy computations indicate that in vacuo the two diastereomers exhibit different flexibility, and a preferred conformation with a different type of intramolecular H-bond.

Amino Acids↗