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F Mammano

Publications and source records attributed to F Mammano.

At least 19 recordsLinked to original sources
PubMed2005

Functional analysis of R75Q mutation in the gene coding for Connexin 26 identified in a family with nonsyndromic hearing loss.

Mutations in the gene (GJB2) coding for Connexin 26 (Cx26) are responsible for genetic forms of sensorineural hearing loss. This article describes a family characterized by congenital profound hearing loss, inherited in an autosomal dominant fashion and associated to a R75Q substitution in Cx26. Cell transfection and fluorescence imaging, dye transfer experiments and dual patch clamp recording showed that the mutant completely prevents the formation of functional channels despite assembling into junctional plaques, in communication incompetent HeLa cells. The disease is not associated with palmar and plantar keratosis in any of the family members, suggesting that R75Q substitution is not sufficient for the development of the complete syndromic phenotype. The association of palmar and plantar keratosis with profound hearing loss may be dependent on genetic background, requiring a functional interaction between the mutated Cx26 and other epidermally expressed connexins.

Connexin 26↗
PubMed2005

Polymorphism of the human immunodeficiency virus type 2 (HIV-2) protease gene and selection of drug resistance mutations in HIV-2-infected patients treated with protease inhibitors.

We described the baseline polymorphism of the human immunodeficiency virus type 2 (HIV-2) protease gene from 94 treatment-naive patients and the longitudinal follow-up of 17 protease inhibitor-treated patients. Compared to the HIV-2 consensus sequences, baseline polymorphism involved 47 positions. Substitutions selected under treatment were observed at positions corresponding to HIV-1 resistance mutations as well as at positions of currently unknown impact on HIV-1.

Adult↗
PubMed2004

A novel mechanism for HIV1-mediated bystander CD4+ T-cell death: neighboring dying cells drive the capacity of HIV1 to kill noncycling primary CD4+ T cells.

CD4+ T-cell death is a crucial feature of AIDS pathogenesis, but the mechanisms involved remain unclear. Here, we present in vitro findings that identify a novel process of HIV1 mediated killing of bystander CD4+ T cells, which does not require productive infection of these cells but depends on the presence of neighboring dying cells. X4-tropic HIV1 strains, which use CD4 and CXCR4 as receptors for cell entry, caused death of unstimulated noncycling primary CD4+ T cells only if the viruses were produced by dying, productively infected T cells, but not by living, chronically infected T cells or by living HIV1-transfected HeLa cells. Inducing cell death in HIV1-transfected HeLa cells was sufficient to obtain viruses that caused CD4+ T-cell death. The addition of supernatants from dying control cells, including primary T cells, allowed viruses produced by living HIV1-transfected cells to cause CD4+ T-cell death. CD4+ T-cell killing required HIV1 fusion and/or entry into these cells, but neither HIV1 envelope-mediated CD4 or CXCR4 signaling nor the presence of the HIV1 Nef protein in the viral particles. Supernatants from dying control cells contained CD95 ligand (CD95L), and antibody-mediated neutralization of CD95L prevented these supernatants from complementing HIV1 in inducing CD4+ T-cell death. Our in vitro findings suggest that the very extent of cell death induced in vivo during HIV1 infection by either virus cytopathic effects or immune activation may by itself provide an amplification loop in AIDS pathogenesis. More generally, they provide a paradigm for pathogen-mediated killing processes in which the extent of cell death occurring in the microenvironment might drive the capacity of the pathogen to induce further cell death.

Acquired Immunodeficiency Syndrome↗
PubMed2002

No evidence for calcium electrogenic exchanger in frog semicircular canal hair cells.

We investigated the possibility that, in hair cells mechanically isolated from frog semicircular canals, Ca2+ extrusion occurs via a Na+ : Ca2+ (cardiac type) or a Na+ : Ca2+,K+ (retinal type) exchanger. Cells concurrently imaged during whole-cell patch-clamp recordings using the Ca2+ sensitive fluorescent dye Oregon Green 488 BAPTA-1 (100 micro m) showed no voltage dependence of Ca2+ clearance dynamics following a Ca2+ load through voltage-gated Ca2+ channels. Reverse exchange was probed in hair cells dialyzed with a Ca2+- and K+-free solution, containing a Na+ concentration that saturates the exchanger, after zeroing the contribution to the whole-cell current from Ca2+ and K+ conductances. In these conditions, no reverse exchange current was detected upon switching from a Ca2+-free external solution to a solution containing concentrations of Ca2+ alone, or Ca2+ + K+ that saturated the exchanger. By contrast, the same experimental protocol elicited peak exchange currents exceeding 100 pA in gecko rod photoreceptors, used as positive controls. In both cell types, we also probed the forward mode of the exchanger by rapidly increasing the intracellular Ca2+ concentration using flash photolysis of two novel caged Ca2+ complexes, calcium 2,2'-([1-(2-nitrophenyl)ethane-1,2-diyl]bis(oxy))bis(acetate) and calcium 2,2'-([1-(4,5-dimethoxy-2-nitrophenyl)ethane-1,2-diyl]bis(oxy)) bis(acetate), in the presence of internal K+ and external Na+. No currents were evoked by UV-triggered Ca2+ jumps in hair cells, whereas exchanger conformational currents up to 400 pA, followed by saturating forward exchange currents up to 40 pA, were recorded in rod photoreceptors subjected to the same experimental conditions. We conclude that no functional electrogenic exchanger is present in this hair cell population, which leaves the abundant plasma membrane Ca2+-ATPases as the primary contributors to Ca2+ extrusion.

Animals↗
PubMed2001

Primary and recombinant HIV type 1 strains resistant to protease inhibitors are pathogenic in mature human lymphoid tissues.

Preserved peripheral CD4+ T cell counts despite virologic failure in patients undergoing protease inhibitor (PI)-containing antiviral regimens are a frequent occurrence in human immunodeficiency virus (HIV) disease. One hypothesis to explain the relative sparing of CD4+ T cells is that HIV strains exhibiting PI resistance concomitantly are attenuated in terms of cytopathicity for mature T cells. To test this hypothesis, we used a three-dimensional human tonsil histoculture microenvironment to assess the pathogenic potential of a panel of primary and recombinant HIV-1 strains derived from patients experiencing PI failure. All the viruses tested replicated efficiently in these cultures and, in some cases, better than comparable wild-type viral isolates. Furthermore, the PI-resistant strains depleted CD4+ T cells potently and comparably with wild-type isolates in these ex vivo lymphoid tissues. These results demonstrate that PI-resistant viruses are not inherently less pathogenic for mature T cells. Therefore, the sustained peripheral lymphocyte counts in patients with selective virologic failure may be due to specific defects in viral replication in other cell compartments or to an undefined host adaptation to viral infection during PI therapy.

CD4-Positive T-Lymphocytes↗
PubMed2001

Action of 2,3-butanedione monoxime on capacitance and electromotility of guinea-pig cochlear outer hair cells.

1. Whole-cell patch-clamp recordings were obtained from isolated cochlear outer hair cells (OHCs) while applying 2,3-butanedione monoxime (BDM) by pressure. BDM (5 mM) shifted the range of voltage sensitivity of membrane capacitance and cell length in the hyperpolarised direction by -49.6 +/- 4.0 mV (n = 12; mean +/- S.E.M.), without appreciable effects on membrane conductance. The shift was completely reversible and dose dependent, with a Hill coefficient of 1.8 /- 0.4 and a half-maximal dose of 3.0 +/- 0.8 mM (values +/- S.D). 2. The shift of the capacitance curve was also reproducible in cells whose natural turgor had been removed. BDM had no detectable effect on the capacitance of Deiters' cells, a non-sensory cell type of the organ of Corti. 3. The effect of BDM on membrane capacitance was faster than that of salicylate. At similar saturating concentrations (20 mM), the time constant of the capacitance changes was 1.8 +/- 0.3 s (n = 3) for salicylate and 0.75 +/- 0.06 s (n = 3) for BDM. The recovery periods were 13 +/- 1 s and 1.7 +/- 0.4 s, respectively (means +/- S.E.M.). 4. The effect of BDM, a known inorganic phosphatase, was compared to the effects of okadaic acid, trifluoperazine and W-7, which are commonly used in studies of protein phosphorylation. Incubation of OHCs with okadaic acid (1 microM, 30-60 min) shifted the voltage sensitivity of the membrane capacitance in the hyperpolarised direction. Incubation with trifluoperazine (30 microM) and W-7 (150 microM) shifted it in the opposite, depolarised direction. BDM induced hyperpolarising shifts even in the presence of W-7. 5. Simultaneous measurement of membrane capacitance and intracellular free Ca2+ concentration ([Ca2+]i) showed that BDM action on OHC voltage-dependent capacitance and electromotility is not mediated by changes of [Ca2+]i. 6. Our results suggest that: (a) the effects of BDM are unrelated to its inorganic phosphatase properties, cell turgor conditions or Ca2+ release from intracellular stores; and (b) BDM may target directly the voltage sensor of the OHC membrane motor protein.

Animals↗
PubMed2001

Purinergic control of intercellular communication between Hensen's cells of the guinea-pig cochlea.

1. Hensen's cells in the isolated cochlea were stimulated by extracellular adenosine 5'-triphosphate (ATP) applied to their endolymphatic surface while changes in membrane current and intracellular calcium concentration ([Ca2+]i) were measured simultaneously. The response consisted of (i) an initial rapid inward current accompanied by elevation of the [Ca2+]i, (ii) a more slowly rising inward current accompanied by a rise of the [Ca2+]i and (iii) a slowly developing reduction of input conductance. 2. The slower responses were maintained in the absence of extracellular Ca2+. Similar responses were produced by increasing the [Ca2+]i via UV flash photolysis of intracellular D-myo-inositol 1,4,5-trisphosphate, P4(5)-(1-(2-nitrophenyl)ethyl) ester (caged InsP3) loaded at pipette concentrations of 8-16 microM. 3. The slow inward current, reversing around 0 mV, was blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). 4. Bath application of U-73122 (1 microM), a phospholipase C inhibitor, eliminated the slow Ca2+-release component of the response to ATP. It is proposed that the effects of ATP are mediated by the co-activation of ionotropic P2X and metabotropic P2Y receptors. 5. Immunohistochemistry using light and electron microscopy revealed that inositol 1,4,5-trisphosphate (InsP3) receptors delineate a network within the cells. 6. The coupling ratio (CR) between cell pairs measured in dual patch-clamp recordings was 0.356 +/- 0.024. The coupling reversibly decreased to 51 % of the control within 2 min of applying 100 microM ATP. Flash photolysis of 32 microM intracellular caged InsP3 and 1 mM caged Ca2+ reduced CR to 42 and 62 % of the control, respectively. 7. We propose that endolymphatic ATP via P2X and P2Y receptors can control intercellular communication amongst Hensen's cells by reducing gap junction conductance in a Ca2+- and InsP3-dependent manner.

Adenosine Triphosphate↗
PubMed2001

Can you still see the cochlea for the molecules?

It is now established that the mammalian cochlea uses active amplification of incoming sound to achieve sensitivity. Cellular details are emerging slowly. Recent studies of sensory hair cells have highlighted the possible molecular bases for amplification and the components of sensitivity regulation within the cochlea: a synthesis is likely to depend on effective and physiologically informed modelling.

Animals↗
PubMed2001

Impaired replication of protease inhibitor-resistant HIV-1 in human thymus.

Many HIV-1-infected patients treated with protease inhibitors (PI) develop PI-resistant HIV-1 variants and rebounds in viremia, but their CD4+ T-cell counts often do not fall. We hypothesized that in these patients, T-cell counts remain elevated because PI-resistant virus spares intrathymic T-cell production. To test this, we studied recombinant HIV-1 clones containing wild-type or PI-resistant protease domains, as well as uncloned isolates from patients, in activated peripheral blood mononuclear cells, human thymic organ cultures and human thymus implants in SCID-hu Thy/Liv mice. In most cases, wild-type and PI-resistant HIV-1 isolates replicated to similar degrees in peripheral blood mononuclear cells. However, the replication of PI-resistant but not wild-type HIV-1 isolates was highly impaired in thymocytes. In addition, patients who had PI-resistant HIV-1 had abundant thymus tissue as assessed by computed tomography. We propose that the inability of PI-resistant HIV-1 to replicate efficiently in thymus contributes to the preservation of CD4+ T-cell counts in patients showing virologic rebound on PI therapy.

Adult↗
PubMed2001

Intracellular calcium dynamics and membrane conductance changes evoked by Deiters' cell purinoceptor activation in the organ of Corti.

Deiters' cells function as supporting cells for the sensory-motor outer hair cells of the mammalian cochlea and are interconnected by gap junctions. Here the electrical and Ca2+ responses of Deiters' cells evoked by purinergic stimulation were investigated in the organ of Corti, the auditory sensory epithelium. Adenosine 59-triphosphate (ATP, 50-100 microM) applied focally by pressure increased the intracellular free Ca2+ concentration ([Ca2+]i). At the same time ATP evoked an early inward current that was followed by an outward component, reflecting a sustained Ca2+-dependent reduction of the pre-stimulus offset current. These responses were maintained when Ca2+ was removed from the extracellular medium (0 [Ca2+]o), indicating a contribution to Ca2+ signalling from P2Y metabotropic receptors. UV photolysis of caged inositol 1,4,5-triphosphate (InsP3, 16 microM) produced Ca2+ responses similar to those evoked by exogenous ATP, accompanied by reduction of the offset current. In Deiters' cells uncoupled by octanol (1mM), ATP activated only the early inward current, suggesting that functional gap junctions are required in the late phase of the current responses. Following the delivery of UV flashes to pairs of Deiters' cells loaded with caged InsP3, the electrical coupling ratio (CR), monitored by double patch-clamp recordings, was strongly attenuated. These data support the idea that, by promoting inflow of cations and by controlling gap-junction conductance in a Ca2+-and InsP3-dependent way, ATP might serve a protective role in the cochlea.

Animals↗
PubMed2001

Dynamics of intracellular calcium in hair cells isolated from the semicircular canal of the frog.

Changes in cytosolic free Ca(2+) concentration ([Ca(2+)]i) were monitored optically in hair cells mechanically isolated from frog semicircular canals using the membrane-impermeant form of the Ca(2+)-selective dye Oregon Green 488 BAPTA-1 (OG, 100 microM). Cells stimulated by depolarization under whole-cell voltage clamp conditions revealed Ca(2+) entry at selected sites (hotspots) located mostly in the lower (synaptic) half of the cell body. [Ca(2+)]i at individual hotspots rose with a time constant tau1 approximately 70 ms and decayed with a bi-exponential time-course (tau2 approximately 160, tau3 approximately 2500 ms) following a 160 ms depolarization to -20 mV. With repeated stimulation [Ca(2+)]i underwent independent amplitude changes at distinct hotspots, suggesting that the underlying Ca(2+) channel clusters can be regulated differentially by intracellular signalling pathways. Block by nifedipine indicated that the L-type Ca(2+)channels are distributed at different densities in distinct hotspots. No diffusion barrier other than the nuclear region was found in the cytosol, so that, during a prolonged depolarization (lasting up to 1s), Ca(2+) was able to reach the cell apical ciliated pole. The effective Ca(2+) diffusion constant, measured from the progression of Ca(2+) wavefronts in the cytosol, was approximately 57 microm(2)/s. Our results indicate that in these hair cells, buffered diffusion of Ca(2+) proceeds evenly from the source point to the cell interior and is dominated by the diffusion constant of the endogenous mobile buffers.

Animals↗
PubMed2001

Determination of coreceptor usage of human immunodeficiency virus type 1 from patient plasma samples by using a recombinant phenotypic assay.

We developed a recombinant virus technique to determine the coreceptor usage of human immunodeficiency virus type 1 (HIV-1) from plasma samples, the source expected to represent the most actively replicating virus population in infected subjects. This method is not subject to selective bias associated with virus isolation in culture, a step required for conventional tropism determination procedures. The addition of a simple subcloning step allowed semiquantitative evaluation of virus populations with a different coreceptor (CCR5 or CXCR4) usage specificity present in each plasma sample. This procedure detected mixtures of CCR5- and CXCR4-exclusive virus populations as well as dualtropic viral variants, in variable proportions. Sequence analysis of dualtropic clones indicated that changes in the V3 loop are necessary for the use of CXCR4 as a coreceptor, but the overall context of the V1-V3 region is important to preserve the capacity to use CCR5. This convenient technique can greatly assist the study of virus evolution and compartmentalization in infected individuals.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Changes in human immunodeficiency virus type 1 populations after treatment interruption in patients failing antiretroviral therapy.

Mutations in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and protease that confer resistance to antiretroviral agents are usually accompanied by a reduction in the viral replicative capacity under drug-free conditions. Consequently, when antiretroviral treatment is interrupted in HIV-1-infected patients harboring drug-resistant virus, resistant quasi-species appear to be most often replaced within several weeks by wild-type virus. Using a real-time PCR-based technique for the selective quantification of resistant viral sequences in plasma, we have studied the kinetics of the switch from mutant to wild-type virus and evaluated the extent to which minority populations of resistant viruses not detected by genotyping persist in these individuals. Among 12 patients with viruses expressing the V82A or L90M resistance mutation who had undergone a 3-month interruption of therapy and for whom conventional genotyping had revealed an apparent total reconversion to wild-type virus, minority populations expressing these mutations, representing 0.1 to 21% of total virus, were still detectable in 9 cases. Kinetic studies demonstrated that viruses expressing resistance mutations could be detected for >5 months after the discontinuation of treatment in some patients. Most of the minority resistant genomes detected more than 3 months after the interruption of therapy carried only part of the mutations present in the resistant viruses prior to treatment interruption and appeared to result from the emergence of existing strains selected at earlier stages in the development of drug resistance. Thus, following the interruption of treatment, viral populations containing resistance mutations can persist for several months after the time when conventional genotyping techniques detect only wild-type virus. These populations include viral strains with only some of the resistance mutations initially present, strains that presumably express better fitness under drug-free conditions.

Anti-HIV Agents↗
PubMed2001

Frequency dependence of electrical coupling in Deiters' cells of the guinea pig cochlea.

Immunolabeling with antibodies against connexins 26 and 30 showed that, in the guinea pig cochlea, supporting Deiters' cells are massively interconnected and form an orderly network within the organ of Corti. In paired patch-clamp recordings the coupling ratio (CR) of adjacent Deiters' cells at the apex of the cochlea (approximately 0.31) was 3-fold smaller than in isolated cell pairs due to shunting afforded by multicellular connectivity. With sinusoidal current stimuli the delay in signal propagation between adjacent cells increased with increasing frequency whereas the amplitude did not change significantly up to 200 Hz (corner frequency Fc approximately 220 Hz). Depolarizing voltage commands applied to an outer hair cell (OHC) elicited outward potassium currents in the OHC and inward currents in the abutting Deiters' cells, supplying direct evidence for potassium buffering in the organ of Corti. Computational analysis indicates that electrical signals injected into a Deiters' cell are transmitted across a network segment spanning 8 cell diameters. Thus electrical coupling in the organ of Corti is unlikely to influence the selectivity of frequency filtering performed mechanically by the mammalian cochlea.

Animals↗
PubMed2000

Water permeability of cochlear outer hair cells: characterization and relationship to electromotility.

The distinguishing feature of the mammalian outer hair cells (OHCs) is to elongate and shorten at acoustic frequencies, when their intracellular potential is changed. This "electromotility" or "electromechanics" depends critically on positive intracellular pressure (turgor), maintained by the inflow of water through yet uncharacterized water pathways. We measured the water volume flow, J(v), across the plasma membrane of isolated guinea pig and rat OHCs after osmotic challenges and estimated the osmotic water permeability coefficient, P(f), to be approximately 10(-2) cm/sec. This value is within the range reported for osmotic flow mediated by the water channel proteins, aquaporins. J(v) was inhibited by HgCl(2), which is known to block aquaporin-mediated water transport. P(f) values that were estimated for OHCs from neonatal rats were of the order of approximately 2 x 10(-3) cm/sec, equivalent to that of membranes lacking water channel proteins. In an immunofluorescence assay we showed that an anti-peptide antibody specific for aquaporins labels the lateral plasma membrane of the OHC in the region in which electromotility is generated. Using patch-clamp recording, we found that water influx into the OHC is regulated by intracellular voltage. We also found that the most pronounced increases of the electromotility-associated charge movement and of the expression of OHC water channels occur between postnatal days 8 and 12, preceding the onset of hearing function in the rat. Our data indicate that electromotility and water transport in OHCs may influence each other structurally and functionally.

Aging↗
PubMed2000

Two distinct Ca(2+)-dependent signaling pathways regulate the motor output of cochlear outer hair cells.

The outer hair cells (OHCs) of the cochlea have an electromotility mechanism, based on conformational changes of voltage-sensitive "motor" proteins in the lateral plasma membrane. The translocation of electrical charges across the membrane that accompanies electromotility imparts a voltage dependency to the membrane capacitance. We used capacitance measurements to investigate whether electromotility may be influenced by different manipulations known to affect intracellular Ca(2+) or Ca(2+)-dependent protein phosphorylation. Application of acetylcholine (ACh) to the synaptic pole of isolated OHCs evoked a Ca(2+)-activated apamin-sensitive outward K(+) current. It also enhanced electromotility, probably because of a phosphorylation-dependent decrease of the cell's axial stiffness. However, ACh did not change the voltage-dependent capacitance either in conventional whole-cell experiments or under perforated-patch conditions. The effects produced by the Ca(2+) ionophore ionomycin mimicked those produced by ACh. Hyperpolarizing shifts of the voltage dependence of capacitance and electromotility were induced by okadaic acid, a promoter of protein phosphorylation, whereas trifluoperazine and W-7, antagonists of calmodulin, caused opposite depolarizing shifts. Components of the protein phosphorylation cascade-IP(3) receptors and calmodulin-dependent protein kinase type IV-were immunolocalized to the lateral wall of the OHC. Our results suggest that two different Ca(2+)-dependent pathways may control the OHC motor output. The first pathway modulates cytoskeletal stiffness and can be activated by ACh. The second pathway shifts the voltage sensitivity of the OHC electromotile mechanism and may be activated by the release of Ca(2+) from intracellular stores located in the proximity of the lateral plasma membrane.

Acetylcholine↗
PubMed2000

GABA- and glutamate-mediated network activity in the hippocampus of neonatal and juvenile rats revealed by fast calcium imaging.

In the rat hippocampus, during the first postnatal week, network activity is characterized by GABA-driven giant depolarizing potentials (GDPs) associated with calcium signals that are readily blocked when the GABAA antagonist bicuculline is applied to the bath. Towards the end of the first postnatal week, in concomitance with the shift of GABA responses from the depolarizing to the hyperpolarizing direction, functional glutamatergic connections start appearing. At this developmental stage, application of bicuculline blocks GABAA-mediated inhibition and induces the appearance of interictal epileptiform discharges. In the present experiments, we have used a high spatio-temporal resolution imaging system to compare, on a time scale of tens of ms, the onset and propagation of fast calcium transients generated within a GABAergic or glutamatergic network. We found that, during the first postnatal week, calcium signals associated to evoked GDPs arise from the activation of a local circuitry of neurons spanning the stratum radiatum and the pyramidal layer. Similar activation patterns were elicited by focal application of GABA in the presence of kynurenic acid, a broad spectrum ionotropic glutamatergic antagonist, and were blocked by bicuculline. During the second postnatal week, in the presence of bicuculline, calcium signals associated with interictal discharges evoked by stimulation of glutamatergic fibres propagated along the well-defined three-synaptic pathway from the dentate gyrus to the CA1 hippocampal area.

Action Potentials↗