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F Mammano

Publications and source records attributed to F Mammano.

At least 37 records · Page 2Linked to original sources
PubMed2000

Cholinergic control of membrane conductance and intracellular free Ca2+ in outer hair cells of the guinea pig cochlea.

We have studied the action of cholinergic agonists on outer hair cells, both in situ and isolated from the cochlea of the guinea pig, combining new fast CCD technology for Ca2+ imaging and conventional patch-clamp methods. Carbachol (1 mM) activated a current with a reversal potential near -70 mV and a bell-shaped I-V curve, suggesting that it was a Ca2+ activated K+ current. In a few cells, this current was preceded by a transient inward current, probably owing to an influx of Ca2+ and other cations through the acetylcholine (ACh) receptors. The amplitude of the Ca2+ signal was maximal in a circumscribed region at the basal pole of the cell and decreased steeply towards the apical pole, compatible with Ca2+ influx and/or Ca2+ induced Ca2+ release at the cells base. The time course of the Ca2+ rise was fastest at the base, but it was still slightly slower, and more rounded, than that of the K+ current. In some recordings the K+ current was observed without any measurable change of intracellular Ca2+. The K+ current was potentiated (18%) by caffeine (5 mM), and decreased (19%) by ryanodine (0.1 mM) in the majority of cells tested. The results are discussed in terms of a labile intracellular Ca2+ store located at the base of the cell, close to the Ca2+ permeable ACh receptor channels and Ca2+ activated K+ channels, whose contribution to the Ca2+ rise occurring in the region of the channels is variable, and probably dependent on its ability to refill with Ca2+.

Acetylcholine↗
PubMed2000

The karyophilic properties of human immunodeficiency virus type 1 integrase are not required for nuclear import of proviral DNA.

Integrase (IN) is a key component of the preintegration nucleoprotein complex (PIC), which transports the retroviral genome from the cytoplasm to the nucleus of newly infected cells. Retroviral IN proteins have intrinsic karyophilic properties, which for human immunodeficiency virus type 1 (HIV-1) are currently attributed to regions that display sequence homology to previously characterized nuclear localization signals. We asked here whether the karyophilic properties of HIV-1 IN are involved in the nuclear import of PIC. We mutated three conserved basic regions in the C-terminal domain of IN and analyzed the effects of mutations on subcellular localization of the protein, viral particle composition, IN dimerization within virions, and infectivity. Alteration of two sequences caused the loss of nuclear accumulation of IN and drastically reduced the capacity of the protein to multimerize. Mutation of the most C-terminal sequence had no effect on the subcellular localization and dimerization of IN. Nevertheless, conservation of all three sequences was required for viral infectivity. Despite the perturbation of IN subcellular localization, all mutant viruses displayed normal reverse transcription and nuclear transport of PICs in newly infected cells. The replicative defect was instead at the level of integration, for which all mutants were markedly affected in vivo. Besides reinforcing the association between dimerization of IN and nuclear accumulation of the enzyme, our data demonstrate that subcellular localization of IN alone cannot predict the fate of the PICs.

Amino Acid Sequence↗
PubMed2000

Retracing the evolutionary pathways of human immunodeficiency virus type 1 resistance to protease inhibitors: virus fitness in the absence and in the presence of drug.

Human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors (PI) is a major obstacle to the full success of combined antiretroviral therapy. High-level resistance to these compounds is the consequence of stepwise accumulation of amino acid substitutions in the HIV-1 protease (PR), following pathways that usually differ from one inhibitor to another. The selective advantage conferred by resistance mutations may depend upon several parameters: the impact of the mutation on virus infectivity in the presence or absence of drug, the nature of the drug, and its local concentration. Because drug concentrations in vivo are subject to extensive variation over time and display a markedly uneven tissue distribution, the parameters of selection for HIV-1 resistance to PI in treated patients are complex and poorly understood. In this study, we have reconstructed a large series of HIV-1 mutants that carry single or combined mutations in the PR, retracing the accumulation pathways observed in ritonavir-, indinavir-, and saquinavir-treated patients. We have then measured the phenotypic resistance and the drug-free infectivity of these mutant viruses. A deeper insight into the evolutionary value of HIV-1 PR mutants came from a novel assay system designed to measure the replicative advantage of mutant viruses as a function of drug concentration. By tracing the resultant fitness profiles, we determined the range of drug concentrations for which mutant viruses displayed a replicative advantage over the wild type and the extent of this advantage. Fitness profiles were fully consistent with the order of accumulation of resistance mutations observed in treated patients and further emphasise the key importance of local drug concentration in the patterns of selection of drug-resistant HIV-1 mutants.

Amino Acid Substitution↗
PubMed1999

ATP-Induced Ca(2+) release in cochlear outer hair cells: localization of an inositol triphosphate-gated Ca(2+) store to the base of the sensory hair bundle.

We used a high-performance fluorescence imaging system to visualize rapid changes in intracellular free Ca(2+) concentration ([Ca(2+)](i)) evoked by focal applications of extracellular ATP to the hair bundle of outer hair cells (OHCs): the sensory-motor receptors of the cochlea. Simultaneous recordings of the whole-cell current and Calcium Green-1 fluorescence showed a two-component increase in [Ca(2+)](i). After an initial entry of Ca(2+) through the apical membrane, a second and larger, inositol triphosphate (InsP(3))-gated, [Ca(2+)](i) surge occurred at the base of the hair bundle. Electron microscopy of this intracellular Ca(2+) release site showed that it coincides with the localization of a unique system of endoplasmic reticulum (ER) membranes and mitochondria known as Hensen's body. Using confocal immunofluorescence microscopy, we showed that InsP(3) receptors share this location. Consistent with a Ca(2+)-mobilizing second messenger system linked to ATP-P2 receptors, we also determined that an isoform of G-proteins is present in the stereocilia. Voltage-driven cell shape changes and nonlinear capacitance were monitored before and after ATP application, showing that the ATP-evoked [Ca(2+)](i) rise did not interfere with the OHC electromotility mechanism. This second messenger signaling mechanism bypasses the Ca(2+)-clearance power of the stereocilia and transiently elevates [Ca(2+)](i) at the base of the hair bundle, where it can potentially modulate the action of unconventional myosin isozymes involved in maintaining the hair bundle integrity and potentially influence mechanotransduction.

Adenosine Triphosphate↗
PubMed1999

Imaging neuronal calcium fluorescence at high spatio-temporal resolution.

A rapid fluorescence imaging system was developed and utilised to investigate the time-course of intracellular calcium concentration ([Ca2+]i) gradients generated by action potentials in CA1-CA3 pyramidal cells within brain slices of the rat hippocampus. The system, which is based on a fast commercial CCD camera, can acquire hundreds of 128 x 128 pixel images in sequence, with minimal inter-frame interval of 2.5 ms (400 frames/s) and 12 bit/pixel accuracy. By synchronising patch clamp recordings with image capture, the timing of transmembrane potential variation, ionic Ca2+ current and Ca2+ diffusion were resolved at the limit of the relaxation time for the dye-Ca2+ binding reaction (approximately 5 ms at room temperature). Numerical simulations were used to relate measured fluorescence transients to the spatio-temporal distribution of intracellular Ca2+ gradients. The results obtained indicate that dye reaction-diffusion contributes critically to shaping intracellular ion gradients.

Action Potentials↗
PubMed1999

An optical recording system based on a fast CCD sensor for biological imaging.

This paper presents technical details, hardware and software of a complete imaging system which uses a fast CCD sensor and a 41 Msample/s A/D converter to acquire full-frame 12 bit/pixel digitized images with a time resolution of 1.25 ms/image. This apparatus permits to resolve intracellular Ca2+ gradients in individual cells as well as the spatio-temporal pattern of neural activity of cell assemblies in neural tissue.

Animals↗
PubMed1999

Effects of human immunodeficiency virus type 1 resistance to protease inhibitors on reverse transcriptase processing, activity, and drug sensitivity.

Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors often display a reduced replicative capacity as a result of an impairment of protease function. Such fitness-impaired viruses display Gag precursor maturation defects. Here, we report that some protease inhibitor-resistant viruses also display abnormalities in the processing of reverse transcriptase (RT) by the protease. In three recombinant viruses carrying resistant protease sequences from patient plasma, we observed a marked decrease in the amount of mature RT subunits and of particle-associated RT activity compared to their parental pretherapy counterparts. We investigated the possibility that a decrease in the amount of particle-associated mature RT could affect the sensitivity of the corresponding virus to RT inhibitors. We observed a twofold increase of sensitivity to zidovudine (AZT) when a virus which carried AZT mutations was processed by a resistant protease. Interestingly, the presence of AZT-resistance mutations partially rescued the replication defect associated with the mutated protease. The interplay between resistance to protease inhibitors and to RT inhibitors described here may be relevant to the therapeutic control of HIV-1 infection.

Anti-HIV Agents↗
PubMed1999

Oligomerization within virions and subcellular localization of human immunodeficiency virus type 1 integrase.

Previous biochemical and genetic evidence indicated that the functional form of retroviral integrase protein (IN) is a multimer. A direct demonstration of IN oligomerization during the infectious cycle was, however, missing, due to the absence of a sensitive detection method. We describe here the generation of infectious human immunodeficiency virus type 1 (HIV-1) viral clones carrying IN protein tagged with highly antigenic epitopes. In this setting, we could readily visualize IN both in producer cells and in viral particles. More interestingly, we detected IN oligomers, the formation of which was dependent on disulfide bridges and took place inside virions. Additionally, expression of a tagged HIV-1 IN in the absence of other viral components resulted in almost exclusive nuclear accumulation of the protein. Mutation of a conserved cysteine in the proposed dimer interface determined the loss of viral infectivity, associated with a reduction of IN oligomer formation and the redistribution of the mutated protein in the nucleus and cytoplasm. Epitope tagging of HIV-1 IN expressed alone or in the context of a replication-competent viral clone provides powerful tools to validate debated issues on the implication of this enzyme in different steps of the viral cycle.

Cell Nucleus↗
PubMed1998

How well do we understand the cochlea?

As sensory cells, hair cells within the mammalian inner ear convert sounds into receptor potentials when their projecting stereocilia are deflected. The organ of Corti of the cochlea contains two types of hair cell, inner and outer hair cells, which differ in function. It has been appreciated for over two decades that although inner hair cells act as the primary receptor cell for the auditory system, the outer hair cells can also act as motor cells. Outer hair cells respond to variation in potential, and change length at rates unequalled by other motile cells. The forces generated by outer hair cells are capable of altering the delicate mechanics of the cochlear partition, increasing hearing sensitivity and frequency selectivity. The discovery of such hair-cell motility has modified the view of the cochlea as a simple frequency analyser into one where it is an active non-linear filter that allows only the prominent features of acoustic signals to be transmitted to the acoustic nerve by the inner hair cells. In this view, such frequency selectivity arises through the suppression of adjacent frequencies, a mechanical effect equivalent to lateral inhibition in neural structures. These processes are explained by the interplay between the hydrodynamic interactions among different parts of the cochlear partition and the effective non-linear behaviour of the cell motor.

Animals↗
PubMed1998

Pseudotyping of Moloney leukemia virus-based retroviral vectors with simian immunodeficiency virus envelope leads to targeted infection of human CD4+ lymphoid cells.

In view of our recent findings that a truncated form of the envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) was efficiently incorporated into MoMLV particles, we studied the generation of Moloney murine leukemia virus (MoMLV)/simian immunodeficiency virus (SIV) pseudotypes. Unlike HIV-1, both the wild-type SIV Env and a truncated form, which lacks most of the cytoplasmic domain of the transmembrane glycoprotein, were incorporated into MoMLV particles and generated infectious retroviral vectors which could transduce CD4+ sMAGI macaque cells. The infection depended on target cell CD4 expression, and was neutralized by both soluble CD4 and sera from SIV-infected macaques. We also observed pseudotype-mediated gene transfer of a green fluorescent protein marker into the CD4+ CEMX174 and C8166 lymphoid cell lines. More importantly, primary human lymphocytes were also successfully transduced ex vivo by MoMLV/SIV pseudotypes, albeit at lower efficiency, and gene transfer was specifically restricted to the CD4+ subset. These findings demonstrate that MoMLV/SIV pseudotypes can be used to transduce cells which are susceptible to SIV infection, and thus might be advantageously employed in animal models for direct in vivo delivery of gene therapy-based approaches.

Animals↗
PubMed1998

Loss of viral fitness associated with multiple Gag and Gag-Pol processing defects in human immunodeficiency virus type 1 variants selected for resistance to protease inhibitors in vivo.

We examined the viral replicative capacity and protease-mediated processing of Gag and Gag-Pol precursors of human immunodeficiency virus (HIV) variants selected for resistance to protease inhibitors. We compared recombinant viruses carrying plasma HIV RNA protease sequences obtained from five patients before protease inhibitor therapy and after virus escape from the treatment. Paired pretherapy-postresistance reconstructed viruses were evaluated for HIV infectivity in a quantitative single-cycle titration assay and in a lymphoid cell propagation assay. We found that all reconstructed resistant viruses had a reproducible decrease in their replicative capacity relative to their parental pretherapy counterparts. The extent of this loss of infectivity was pronounced for some viruses and more limited for others, irrespective of the inhibitor used and of the level of resistance. In resistant viruses, the efficiency of Gag and Gag-Pol precursor cleavage by the protease was impaired to different extents, as shown by the accumulation of several cleavage intermediates in purified particle preparations. We conclude that protease inhibitor-resistant HIV variants selected during therapy have an impaired replicative capacity related to multiple defects in the processing of Gag and Gag-Pol polyprotein precursors by the protease.

Cell Line, Transformed↗
PubMed1998

Resistance-associated loss of viral fitness in human immunodeficiency virus type 1: phenotypic analysis of protease and gag coevolution in protease inhibitor-treated patients.

We have studied the phenotypic impact of adaptative Gag cleavage site mutations in patient-derived human immunodeficiency virus type 1 (HIV-1) variants having developed resistance to the protease inhibitor ritonavir or saquinavir. We found that Gag mutations occurred in a minority of resistant viruses, regardless of the duration of the treatment and of the protease mutation profile. Gag mutations exerted only a partial corrective effect on resistance-associated loss of viral fitness. Reconstructed viruses with resistant proteases displayed multiple Gag cleavage defects, and in spite of Gag adaptation, several of these defects remained, explaining the limited corrective effect of cleavage site mutations on fitness. Our data provide clear evidence of the interplay between resistance and fitness in HIV-1 evolution in patients treated with protease inhibitors.

Amino Acid Sequence↗
PubMed1997

Truncation of the human immunodeficiency virus type 1 envelope glycoprotein allows efficient pseudotyping of Moloney murine leukemia virus particles and gene transfer into CD4+ cells.

Human immunodeficiency virus type 1 (HIV-1) can readily accept envelope (Env) glycoproteins from distantly related retroviruses. However, we previously showed that the HIV-1 Env glycoprotein complex is excluded even from particles formed by the Gag proteins of another lentivirus, visna virus, unless the matrix domain of the visna virus Gag polyprotein is replaced by that of HIV-1. We also showed that the integrity of the HIV-1 matrix domain is critical for the incorporation of wild-type HIV-1 Env protein but not for the incorporation of a truncated form which lacks the 144 C-terminal amino acids of the cytoplasmic domain of the transmembrane glycoprotein. We report here that the C-terminal truncation of the transmembrane glycoprotein also allows the efficient incorporation of HIV-1 Env proteins into viral particles formed by the Gag proteins of the widely divergent Moloney murine leukemia virus (Mo-MLV). Additionally, pseudotyping of a Mo-MLV-based vector with the truncated rather than the full-length HIV-1 Env allowed efficient transduction of human CD4+ cells. These results establish that Mo-MLV-based vectors can be used to target cells susceptible to infection by HIV-1.

Animals↗
PubMed1996

Differential expression of outer hair cell potassium currents in the isolated cochlea of the guinea-pig.

1. Whole-cell currents were recorded from outer hair cells (OHCs) in undissociated tissues from the organ of Corti. The experiments allowed ionic currents to be measured in cells with precise localization on the three most apical cochlear turns. 2. Two major potassium currents were expressed in the cells. One current, named IK, was half-activated at -24 mV and was most prominent in the most apical turn, turn 4. A second, named IK.n, was half-activated at -92 mV and was the major contributor to the current-voltage (I-V) curve of cells from the more basal turns, turns 3 and 2, of the cochlea. 3. IK was specifically blocked by 100 microM 4-aminopyridine (4-AP). In contrast, IK.n was reduced by 5 mM external barium. Superfusion with zero calcium produced no effect on currents in the range from -60 to 0 mV, but reduced currents by a maximum of 15% outside this range. 4. The cell input conductance increased systematically from 3.4 nS in turn 4 to 40 nS in turn 2 measured at a holding potential of -70 mV. 5. The mean leak conductance, measured from the slope of the I-V curve at -110 mV, decreased systematically from 5.2 nS in turn 2, to 2.9 nS in turn 3 and 2.2 nS in turn 4. 6. These data show that hair cell properties can be determined in undissociated cells and are likely to provide a good estimate of the properties of the cells in the intact cochlea. Differences with the properties of isolated OHCs are discussed.

4-Aminopyridine↗
PubMed1996

Electrophysiological properties of Hensen's cells investigated in situ.

Tight-seal whole-cell patch clamp recordings were obtained in situ from supporting Hensen's cells within the intact organ of Corti of the adult guinea pig. In normal phosphate buffer solution we estimated 20-50 cells to be coupled by gap junctions to the cell under the patch pipette. In the presence of 1 mM octanol, an uncoupling agent, it was possible to identify an outward current which activated upon depolarization above -20 mV and approached saturation above 70 mV. An inward current was seen with hyperpolarizations below -80 mV. These are broadly similar to the currents of Hensen's cells in vitro. Measured differences of the underlying conductance indicate that the currents are sensitive to the procedure used to isolate cells.

Animals↗
PubMed1996

Biophysics of the cochlea. II: Stationary nonlinear phenomenology.

Nonlinearities affecting cochlear mechanics produce appreciable compression in the basilar membrane (BM) input/output (I/O) functions at the characteristic frequency for sound-pressure levels (SPLs) as low as 20 dB (re: 20 microPa). This is thought to depend upon saturation of the outer hair cell (OHC) mechanoelectrical transducer (MET). This hypothesis was tested by solving a nonlinear integrodifferential equation that describes the BM vibration in an active cochlea. The equation extends a previously developed linear approach [Mammano and Nobili, J. Acoust. Soc. Am. 93, 3320-3332 (1993)], here modified to include saturating MET, with a few corrections mainly concerning tectorial membrane resonance and OHC coupling to the BM. Stationary solutions were computed by iteration in the frequency domain for a wide range of input SPLs, generating BM I/O functions, frequency response envelopes, and two-tone distortion products. Traveling-wave amplitude envelopes were also computed for a fixed suppressor and several suppressed tones in order to evidence the phenomenon of two-tone suppression (frequency masking) at the mechanical level. All results accord nicely with experimental data.

Basilar Membrane↗