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R Bruzzone

Publications and source records attributed to R Bruzzone.

At least 37 records · Page 2Linked to original sources
PubMed1999

Functional characteristics of skate connexin35, a member of the gamma subfamily of connexins expressed in the vertebrate retina.

Retinal neurons are coupled by electrical synapses that have been studied extensively in situ and in isolated cell pairs. Although many unique gating properties have been identified, the connexin composition of retinal gap junctions is not well defined. We have functionally characterized connexin35 (Cx35), a recently cloned connexin belonging to the gamma subgroup expressed in the skate retina, and compared its biophysical properties with those obtained from electrically coupled retinal cells. Injection of Cx35 RNA into pairs of Xenopus oocytes induced intercellular conductances that were voltage-gated at transjunctional potentials >/= 60 mV, and that were also closed by intracellular acidification. In contrast, Cx35 was unable to functionally interact with rodent connexins from the alpha or beta subfamilies. Voltage-activated hemichannel currents were also observed in single oocytes expressing Cx35, and superfusing these oocytes with medium containing 100 microm quinine resulted in a 1.8-fold increase in the magnitude of the outward currents, but did not change the threshold of voltage activation (membrane potential = +20 mV). Cx35 intercellular channels between paired oocytes were insensitive to quinine treatment. Both hemichannel activity and its modulation by quinine were seen previously in recordings from isolated skate horizontal cells. Voltage-activated currents of Cx46 hemichannels were also enhanced 1. 6-fold following quinine treatment, whereas Cx43-injected oocytes showed no hemichannel activity in the presence, or absence, of quinine. Although the cellular localization of Cx35 is unknown, the functional characteristics of Cx35 in Xenopus oocytes are consistent with the hemichannel and intercellular channel properties of skate horizontal cells.

Animals↗
PubMed1998

Complex structural involvement of chromosome 7 in primary myelodysplastic syndromes determined by fluorescence in situ hybridization.

Cytogenetic analysis of 72 consecutive de novo myelodysplastic syndrome patients revealed monosomy 7 in 12 cases. In 4 of these cases, the -7 was the only abnormality, whereas the remaining 8 cases showed additional chromosomal aberrations. Fluorescence in situ hybridization (FISH) utilizing chromosome 7 alpha-satellite and painting probes and other specific probes, when necessary, provided evidence of unusual and unsuspected structural rearrangements involving chromosome 7. FISH analysis showed that the small fragment found in one patient and the ring found in each of two other patients were chromosome 7-derived rings. FISH also revealed the insertion of chromosome 7 sequences into autosomes in three other patients and unusual translocations in the remaining two patients. By comparing the results obtained by using banding techniques to those obtained by using the FISH technique, we deduced the involvement of chromosome 7 with partial deletion of the short arm in all eight examined patients. Our study confirms the ability of FISH to detect chromosomal aberrations that would otherwise not be identified and the tendency of chromosome 7 to be involved in many different rearrangements. From a clinical point of view, we confirm that patients affected by myelodysplastic syndromes with complex karyotypes involving chromosome 7 do not respond to treatment and have a poor prognosis.

Anemia, Refractory, with Excess of Blasts↗
PubMed1998

Cloning and expression of two related connexins from the perch retina define a distinct subgroup of the connexin family.

We have cloned cDNAs for two closely related connexins (Cx), Cx35 and Cx34.7, from a perch retinal cDNA library. Sequencing of PCR products from genomic DNA revealed that both connexins have an intron 71 bp after the translation initiation site; in Cx35, the intron is 900 bp in length, whereas in Cx34.7 it is approximately 20 kb. Southern blots of genomic DNA suggest that the two connexins represent independent single copy genes. In Northern blots, Cx35 and Cx34.7 transcripts were detected in retina and brain; Cx34.7 also showed a weak signal in smooth muscle (gut) RNA. Antibodies against Cx35 labeled a 30 kDa band on a Western blot of retinal membranes, and in histological sections, the pattern of antibody recognition was consistent with labeling of bipolar cells and unidentified processes in the inner plexiform and nerve fiber layers. When expressed in Xenopus oocytes, Cx35 and Cx34.7 formed homotypic gap junctions, but the junctional conductance between paired oocytes expressing Cx35 was 10-fold greater than that recorded for gap junctional channels formed by Cx34.7. The homotypic gap-junctional channels were closed in a voltage-dependent manner but with relatively weak voltage sensitivity. Heterotypic gap junctions formed by Cx35 and Cx34.7 displayed junctional conductances similar to those of Cx34.7 homotypic pairs and showed a slightly asymmetric current-voltage relationship; the side expressing Cx35 exhibited a higher sensitivity to transjunctional potentials. An analysis of the sequence and gene structure of the connexin family revealed that perch Cx35 and Cx34.7, skate Cx35, and mouse Cx36 constitute a novel gamma subgroup.

Animals↗
PubMed1998

Connexin32 mutations associated with X-linked Charcot-Marie-Tooth disease show two distinct behaviors: loss of function and altered gating properties.

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is associated with mutations in the gene encoding connexin32 (Cx32), which is expressed in Schwann cells. We have compared the functional properties of 11 Cx32 mutations with those of the wild-type protein by testing their ability to form intercellular channels in the paired oocyte expression system. Although seven mutations were functionally incompetent, four others were able to generate intercellular currents of the same order of magnitude as those induced by wild-type Cx32 (Cx32wt). In homotypic oocyte pairs, CMTX mutations retaining functional activity induced the development of junctional currents that exhibited changes in the sensitivity and kinetics of voltage dependence with respect to that of Cx32wt. The four mutations were also capable of interacting in heterotypic configuration with the wild-type protein, and in one case the result was a marked rectification of junctional currents in response to voltage steps of opposite polarity. In addition, the functional CMTX mutations displayed the same selective pattern of compatibility as Cx32wt, interacting with Cx26, Cx46, and Cx50 but failing to do so with Cx40. Although the functional mutations exhibited sensitivity to cytoplasmic acidification, which induced a >/=80% decrease in junctional currents, both the rate and extent of channel closure were enhanced markedly for two of them. Together, these results indicate that the functional consequences of CMTX mutations of Cx32 are of two drastically distinct kinds. The presence of a functional group of mutations suggests that a selective deficit of Cx32 channels may be sufficient to impair the homeostasis of Schwann cells and lead to the development of CMTX.

Animals↗
PubMed1997

Gap junctions: getting the message through.

Connexin proteins make intercellular channels - gap junctions - which provide a direct pathway for cell-cell signaling in vertebrates. Studies of mice lacking connexin genes have demonstrated the need for intercellular transfer of messenger molecules and are uncovering the specific functions of each connexin.

Animals↗
PubMed1997

Amplified c-MYC sequences localized by fluorescence in-situ hybridization on double minute chromosomes in acute myeloid leukemias.

Double minute chromosomes (dmin) are small acentric fragments frequently observed when karyotyping human tumor cells. They are considered the cytogenetic manifestation of gene amplification. The finding of dmin in leukemia is a rare event usually associated with progression of the disease and unfavorable prognosis. We present four patients affected by myeloid disorders with an abnormal karyotype and a variable number of dmin. In an attempt to clarify the origin of the dmin and the amplified gene, we utilized a fluorescent in-situ hybridization (FISH) technique and a panel of specific probes. The results of the analysis indicate that, although chromosomes 8 are apparently uninvolved, dmin retained c-MYC sequencs in three cases. By observing previously reported cases, we found that the majority of patients with myeloid disorders and dmin showed an amplified c-MYC gene, regardless of the chromosomal abnormalities. The FISH technique proved to be informative in demonstrating gene amplification in both metaphase and interphase cells. Finally, in the one patient carrying a 20q deletion, FISH allowed the detection of a previously unreported translocation between a 16p and the 20q-, confirming the ability of the technique to understand complex karyotypes.

Aged↗
PubMed1997

High frequency of trisomy 8 in acute promyelocytic leukemia: a fluorescence in situ hybridization study.

Correct diagnosis of acute promyelocytic leukemia (APL) requires proof of the translocation (15;17)(q24;q11), which appears to be absolutely specific for this particular type of myeloid disorder. We studied the karyotypes of 29 consecutive APL patients at diagnosis: in 5 of them banding techniques failed to detect the t(15;17). In these seemingly cytogenetically negative cases, fluorescence in situ hybridization (FISH) with a chromosome 17 painting probe detected a high percentage of mitoses with 3 hybridization signals: one derived from the intact chromosome 17, and 2 from the rearranged chromosomes 15 and 17. Trisomy 8 (+8) as a secondary chromosomal abnormality was observed in 8 cases (27.5%), confirming that the t(15;17) favors the acquisition of an extra chromosome 8. One of these 8 cases showed a marker that was interpreted by FISH analysis as der(8) with duplication of a segment of the long arm carrying the c-MYC allele. Clinical features of patients with t(15;17) and +8 were no different from patients with t(15;17) alone. The usefulness of FISH to standard banding techniques in the detection of specific structural and/or numerical chromosomal abnormalities is confirmed in this report.

Adult↗
PubMed1997

Connexins, gap junctions and cell-cell signalling in the nervous system.

Connexins form a multigene family of polytopic membrane proteins that, in vertebrates, are the constitutive subunits of intercellular channels and provide the structural basis for electrical coupling. The appearance of electrical coupling in the nervous system is developmentally regulated and restricted to distinct cell types. Electrical coupling between neurons persists after the establishment of chemical transmission, thus suggesting that this form of cell-cell signalling may be functionally interrelated with, rather than alternative to chemical transmission. Furthermore, evidence for the possible role of gap junctions in human neurological diseases is also mounting, following the discovery that the X-linked form of Charcot-Marie-Tooth syndrome, a demyelinating neuropathy of the peripheral nervous system, is associated with mutations in a connexin gene. These findings raise new questions on the significance of connexin diversity and on their functional role in the nervous system.

Animals↗
PubMed1996

Loss of telomeric sequences in a ring derived from chromosome 8 in refractory anemia with excess of blasts in transformation.

Using the fluorescence in situ hybridization technique, we analyzed a ring chromosome that appeared as a karyotype evolution in a patient affected by refractory anemia with excess of blasts in transformation. Metaphases hybridized with a chromosome-8-specific centromeric probe indicated that the ring retained the centromere of chromosome 8. Successively, utilizing a probe specific for all human telomeres, we observed that the ring lost telomeric sequences. This study demonstrated that the formation of a ring chromosome in hematologic disorders can cause loss of genetic material not revealed by banding techniques and therefore providing further proof of the advantages of molecular cytogenetic techniques.

Aged↗
PubMed1996

Connections with connexins: the molecular basis of direct intercellular signaling.

Adjacent cells share ions, second messengers and small metabolites through intercellular channels which are present in gap junctions. This type of intercellular communication permits coordinated cellular activity, a critical feature for organ homeostasis during development and adult life of multicellular organisms. Intercellular channels are structurally more complex than other ion channels, because a complete cell-to-cell channel spans two plasma membranes and results from the association of two half channels, or connexons, contributed separately by each of the two participating cells. Each connexon, in turn, is a multimeric assembly of protein subunits. The structural proteins comprising these channels, collectively called connexins, are members of a highly related multigene family consisting of at least 13 members. Since the cloning of the first connexin in 1986, considerable progress has been made in our understanding of the complex molecular switches that control the formation and permeability of intercellular channels. Analysis of the mechanisms of channel assembly has revealed the selectivity of inter-connexin interactions and uncovered novel characteristics of the channel permeability and gating behavior. Structure/function studies have begun to provide a molecular understanding of the significance of connexin diversity and demonstrated the unique regulation of connexins by tyrosine kinases and oncogenes. Finally, mutations in two connexin genes have been linked to human diseases. The development of more specific approaches (dominant negative mutants, knockouts, transgenes) to study the functional role of connexins in organ homeostasis is providing a new perception about the significance of connexin diversity and the regulation of intercellular communication.

Amino Acid Sequence↗
PubMed1996

The cellular Internet: on-line with connexins.

Most cells communicate with their immediate neighbors through the exchange of cytosolic molecules such as ions, second messengers and small metabolites. This activity is made possible by clusters of intercellular channels called gap junctions, which connect adjacent cells. In terms of molecular architecture, intercellular channels consist of two channels, called connexons, which interact to span the plasma membranes of two adjacent cells and directly join the cytoplasm of one cell to another. Connexons are made of structural proteins named connexins, which compose a multigene family. Connexin channels participate in the regulation of signaling between developing and differentiated cell types, and recently there have been some unexpected findings. First, unique ionic- and size-selectivities are determined by each connexin; second, the establishment of intercellular communication is defined by the expression of compatible connexins; third, the discovery of connexin mutations associated with human diseases and the study of knockout mice have illustrated the vital role of cell-cell communication in a diverse array of tissue functions.

Amino Acid Sequence↗
PubMed1996

Multiple connexin proteins in single intercellular channels: connexin compatibility and functional consequences.

In vertebrates, the protein subunits of intercellular channels found in gap junctions are encoded by a family of genes called connexins. These channels span two plasma membranes and result from the association of two half channels, or connexons, which are hexameric assemblies of connexins. Physiological analysis of channel formation and gating has revealed unique patterns of connexin-connexin interaction, and uncovered novel functional characteristics of channels containing more than one type of connexin protein. Structure-function studies have further demonstrated that unique domains within connexins participate in the regulation of different functional properties of intercellular channels. Thus, gap junctional channels can contain more than one connexin, and this structural heterogeneity has functional consequences in vitro. Moreover, emerging evidence for the existence of intercellular channels containing multiple connexins in native tissues suggests that the functional diversity generated by connexin-connexin interaction could contribute to complex communication patterns that have been observed in vivo.

Animals↗
PubMed1996

Granulocytes with segmented nucleus retain normal chromosomes 17 in Philadelphia chromosome-positive chronic myeloid leukemia with i(17q) and pseudo-Pelger anomaly. A case report studied with fluorescence in situ hybridization.

Previous reports suggested a correlation between the deletion of the terminal region of the short arm of a chromosome 17 and the appearance of dysgranulopoiesis in myeloproliferative disorders. Using the dual-color fluorescence in situ hybridization technique we analyzed the bone marrow and peripheral blood cells of a Philadelphia chromosome-positive chronic myeloid leukemia (CML) patient showing at the onset of transformation into blastic crisis both metaphases with the i(17q) as well as granulocytes without nuclear segmentation. This phenomenon is defined as pseudo-Pelger-Huët anomaly. Using two probes, one specific for 17p and one for 17q, we determined the presence or absence of the i(17q) in both metaphase and interphase cells. Moreover, we observed that all cells with a polysegmented nucleus typical of mature granulocytes did not have i(17q) but had two normal chromosomes 17. This observation confirmed the correlation between 17p deletion and the appearance of pseudo-Pelger anomaly. This finding may also be useful from a clinical point of view: the appearance of pseudo-Pelger cells in CML indicates that 17p deletion actually occurred. This event implies a negative prognosis.

Blast Crisis↗
PubMed1996

Dominant inhibition of intercellular communication by two chimeric connexins.

1. The physiological significance of communication through gap junction channels has been difficult to assess because channel activity cannot be experimentally modulated in a specific manner. To address this problem we have constructed chimeric connexins that function as dominant-negative inhibitors of intercellular channel activity.

Animals↗
PubMed1995

Intercellular channels in teleosts: functional characterization of two connexins from Atlantic croaker.

Gap junction channels, composed of protein subunits termed connexins, are believed to play a critical role in the process of oocyte differentiation and maturation. We have used the paired Xenopus oocyte assay to characterize functionally two connexin genes, connexin-32.2 and connexin-32.7, recently cloned from the ovary of the Atlantic croaker (Micropogonia undulatus), a species that has emerged as a useful model to study the process of maturation of the ovarian follicle. We have found that, while both connexin proteins were expressed at comparable levels in Xenopus oocytes, only one, connexin-32.2, was functionally competent to induce the formation of intercellular channels. Connexin-32.2 channels exhibited voltage-dependent closure that was similar to, but distinct from that of previously characterized mammalian connexins. In addition, the silent connexin-32.7 was unable to functionally interact with connexin-32.2, either in heterotypic channels or as dominant negative inhibitor. Because connexin-32.2 expression is strikingly regulated during oocyte maturation, these data provide further evidence for a role of intercellular channels in the control of oocyte-follicular cell interactions.

Animals↗
PubMed1995

Fluorescence in situ hybridization provides evidence for two-step rearrangement in a masked Ph chromosome formation.

A chronic myelogenous leukemia (CML) patient with a masked Ph chromosome due to the translocation (9;10;22)(q34;q24;q11) is reported. Banding analysis showed a 9q+ chromosome typical of standard t(9;22)(q34;q11), and fluorescence in situ hybridization studies confirmed the involvement of a chromosome 10 in the masked Ph formation and also the presence of 3' ABL-DNA sequences in the der(22). This complex rearrangement could be explained by two consecutive translocations: the first, a standard t(9;22) (q34;q11), the second, a translocation between a chromosome 10 and the der(22) with a breakpoint in sequences derived from chromosome 9 telomeric to the ABL gene. By reverse transcription polymerase chain reaction (RT-PCR), we studied the BCR/ABL transcript junction: a chimeric m-RNA b3-a2, indicating a breakpoint within the major breakpoint cluster region, was found.

Adult↗