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R Bruzzone

Publications and source records attributed to R Bruzzone.

At least 19 recordsLinked to original sources
PubMed2000

Molecular and functional diversity of neural connexins in the retina.

Electrical synapses (gap junctions) in neuronal circuits have become a major focus in the study of network properties such as synchronization and oscillation (Galarreta and Hestrin, 1999; Gibson et al., 1999). Despite the recent progress made in unraveling the contribution of gap junctions to network behavior, little is known about the molecular composition of the junctional constituents. By cloning gap junction proteins [connexins (Cxs)] from zebrafish retina and through functional expression, we demonstrate that the retina possesses a high degree of connexin diversity, which may account for differential functional properties of electrical synapses. Three new Cxs, designated as zebrafish Cx27.5 (zfCx27.5), zfCx44.1, and zfCx55.5, and the carp ortholog of mammalian Cx43 were cloned. By in situ hybridization and in situ RT-PCR, we demonstrate that the four fish connexin mRNAs show differential localization in the retina. Transient functional expression in paired Xenopus oocytes and in the neuroblastoma N2A cell line indicate an extreme range of electrophysiological properties of these connexins in terms of voltage dependence and unitary conductance. For instance, the new zfCx44.1 exhibited high sensitivity to voltage-induced closure with currents decaying rapidly for transjunctional potentials >10 mV, whereas zfCx55.5 channels showed an opposite voltage dependence in response to voltage steps of either polarity. Moreover, although zfCx44.1 channels showed unitary conductance as high as any previously reported for junctional channels (nearly 300 pS), zfCx55. 5 and zfCx27.5 exhibited much lower unitary conductances (<60 pS).

Animals↗
PubMed2000

Gap junctions: fates worse than death?

The reasons for the molecular heterogeneity of connexin channels in vivo remain unclear. Functional replacement of one connexin gene with another has now revealed unexpected phenotypes and shows that cellular homeostasis depends not simply on cell-cell communication but also on the correct types of connexin.

Animals↗
PubMed2000

Functional properties, developmental regulation, and chromosomal localization of murine connexin36, a gap-junctional protein expressed preferentially in retina and brain.

Retinal neurons of virtually every type are coupled by gap-junctional channels whose pharmacological and gating properties have been studied extensively. We have begun to identify the molecular composition and functional properties of the connexins that form these 'electrical synapses,' and have cloned several that constitute a new subclass (gamma) of the connexin family expressed predominantly in retina and brain. In this paper, we present a series of experiments characterizing connexin36 (Cx36), a member of the gamma subclass that was cloned from a mouse retinal cDNA library. Cx36 has been localized to mouse chromosome 2, in a region syntenic to human chromosome 5, and immunocytochemistry showed strong labeling in the ganglion cell and inner nuclear layers of the mouse retina. Comparison of the developmental time course of Cx36 expression in mouse retina with the genesis of the various classes of retinal cells suggests that the expression of Cx36 occurs primarily after cellular differentiation is complete. Because photic stimulation can affect the gap-junctional coupling between retinal neurons, we determined whether lighting conditions might influence the steady state levels of Cx36 transcript in the mouse retina. Steady-state levels of Cx36 transcript were significantly higher in animals reared under typical cyclic-light conditions; exposure either to constant darkness or to continuous illumination reduced the steady-state level of mRNA approximately 40%. Injection of Cx36 cRNA into pairs of Xenopus oocytes induced intercellular conductances that were relatively insensitive to transjunctional voltage, a property shared with other members of the gamma subclass of connexins. Like skate Cx35, mouse Cx36 was unable to form heterotypic gap-junctional channels when paired with two other rodent connexins. In addition, mouse Cx36 failed to form voltage-activated hemichannels, whereas both skate and perch Cx35 displayed quinine-sensitive hemichannel activity. The conservation of intercellular channel gating contrasts with the failure of Cx36 to make hemichannels, suggesting that the voltage-gating mechanisms of hemichannels may be distinct from those of intact intercellular channels.

Animals↗
PubMed2000

Intercellular communication in the eye: clarifying the need for connexin diversity.

In the vertebrate eye, virtually every cell type is directly coupled to its neighbors by intercellular channels present in gap junctions. Although these structures share the common property of allowing adjacent cells to directly exchange ions, second messengers and small metabolites, intercellular channels in the eye also play a specific role in distinct functions such as neuronal transmission at electrotonic synapses in the retina, and the maintenance of homeostasis in the avascular lens. The structural proteins comprising these channels, the connexins (Cx), are a multigene family of which many members are expressed in the eye, even in the same cell type. This molecular heterogeneity poses the crucial question of whether and how a diversity in gap junctional structural proteins influences intercellular communication in ocular tissues. This review will focus on two recent advances in the understanding of connexin diversity in regard to the eye. First, connexin knockouts have demonstrated that postnatal development and homeostasis in the lens requires multiple connexin proteins. Secondly, functional characterization of new connexins that are abundantly expressed in the retina has revealed biophysical properties that mimic those recorded from retinal neurons.

Animals↗
PubMed2000

Connexin channels in Schwann cells and the development of the X-linked form of Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease comprises a group of genetically heterogenous disorders of the peripheral nervous system. The X-linked form of Charcot-Marie-Tooth (CMTX) is associated with mutations in the gene encoding the gap junction protein connexin32 (Cx32), which is expressed in Schwann cells. Immunocytochemical evidence suggests that Cx32 is localized to the incisures of Schmidt-Lanterman and the paranodes of myelinating Schwann cells, where it appears to form reflexive gap junctions. It is currently thought that this cytoplasmic continuity provides a much shorter diffusion pathway for the transport of ions, metabolites and second messenger molecules through intracellular channels between the adaxonal and peri-nuclear regions of Schwann cells, across the myelin sheath. This review summarizes our current understanding of the role of connexins in Schwann cells and focuses on the lessons for channel function and disease pathophysiology derived from the functional analysis of Cx32 mutations. One of the most intriguing aspects emerging from this work is that several mutations retain functional competence, although the mutated channels exhibit altered gating properties. This suggests that partial and/or selective disruption of the radial communication pathway formed by Cx32 is sufficient to cause a functional deficit and lead to the development of CMTX. The next challenge will be to define, at the molecular level, the sequence of events involved in the disease process. The presence of a group of functional mutations should help understand the cellular basis of CMTX, by allowing the identification of the specific molecules that need to be exchanged through Cx32 channels, but are excluded from the mutated ones.

Animals↗
PubMed2000

Complex chromosome rearrangements may locate the bcr/abl fusion gene sites other than 22q11.

BACKGROUND AND OBJECTIVE: From 5-8% of Philadelphia (Ph) positive patients with chronic myeloid leukemia (CML) show variant translocations in which at least a third chromosome in addition to 9q34 and 22q11 is involved. The formation mechanisms and clinical significance of variant Ph translocations are still unclear. The BCR/ABL chimeric gene encoding for chimeric proteins is always present and maps on the 22q- regardless of the type of translocation. We studied two apparently Ph negative CML patients with unusual karyotypes both showing a typical b3a2 rearrangement. DESIGN AND METHODS: Dual-color fluorescence in situ hybridization (FISH) can visualize BCR and ABL genes and localize the BCR/ABL fusion gene. We used FISH to study the formation mechanisms of variant Ph translocations in two patients. RESULTS: The chimeric BCR/ABL gene was located on a locus other than the expected 22q11 in both patients. In the first case the fusion signal was present on the 9q34 band whereas in the second patient it was detected on chromosome 8, involved in masked Ph formation. INTERPRETATION AND CONCLUSIONS: The location of the hybrid BCR/ABL gene on chromosomes other than 22q- is a rare event which can only be observed using the FISH technique. When these unusual translocations occur the hypothesis most often put forward is that several consecutive cytogenetic events have taken place. The factors which regulate the formation of these breakpoints have yet to be clarified. The FISH technique allows the identification of chromosome rearrangements that could not otherwise be detected by conventional banding procedures. The location of the hybrid BCR/ABL gene on sites other than 22q11 represents a rare type of variant Ph translocation. The real frequency and clinical significance of such rearrangements need to be investigated.

Aged↗
PubMed1999

Different TBX5 interactions in heart and limb defined by Holt-Oram syndrome mutations.

To better understand the role of TBX5, a T-box containing transcription factor in forelimb and heart development, we have studied the clinical features of Holt-Oram syndrome caused by 10 different TBX5 mutations. Defects predicted to create null alleles caused substantial abnormalities both in limb and heart. In contrast, missense mutations produced distinct phenotypes: Gly80Arg caused significant cardiac malformations but only minor skeletal abnormalities; and Arg237Gln and Arg237Trp caused extensive upper limb malformations but less significant cardiac abnormalities. Amino acids altered by missense mutations were located on the three-dimensional structure of a related T-box transcription factor, Xbra, bound to DNA. Residue 80 is highly conserved within T-box sequences that interact with the major groove of target DNA; residue 237 is located in the T-box domain that selectively binds to the minor groove of DNA. These structural data, taken together with the predominant cardiac or skeletal phenotype produced by each missense mutation, suggest that organ-specific gene activation by TBX5 is predicated on biophysical interactions with different target DNA sequences.

Adult↗
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↗