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J M Aran

Publications and source records attributed to J M Aran.

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

Fast, slow, and steady-state effects of contralateral acoustic activation of the medial olivocochlear efferent system in awake guinea pigs: action of gentamicin.

The function of the medial olivocochlear efferent system was observed in awake guinea pigs by recording, in the absence of ipsilateral external acoustic stimulation, the ensemble background activity (EBA) of the VIIIth nerve from an electrode chronically implanted on the round window of one ear. The EBA was measured by calculating the power value of the round window signal in the 0.5- to 2.5-kHz band after digital or analog (active) filtering. This EBA was compared with and without the addition of a low-level broadband noise to the opposite ear. The contralateral broadband noise (CLBN, 55 dB SPL) induced, via the efferent system, a decrease (suppression) of this EBA. With the use of noise bursts of different durations, two components in this suppression could be observed. After the onset of a 1-s CLBN, the power value of the EBA decreased rapidly by 38.0 +/- 4.2% (mean +/- SD, n = 3), with a latency of <10 ms and a decay time constant of 13.1 +/- 1.0 ms (fast effect). At the offset of the 1-s CLBN, EBA came back to prestimulation values with a similar latency and a time constant of 15.5 +/- 2.9 ms. During longer CLBN stimulation (>/=1 min), EBA presented, after the fast decrease, an additional, slower decrease of 15.6 +/- 3.1%, with a delay of 9.8 +/- 1.3 s and a decay time constant of 16.1 +/- 5.0 s (n = 12, slow effect), and then remained remarkably constant for as long as observed, i.e., >2 h (steady state). The average global suppression was thus up to 47.8 +/- 5.8% of the basal, pre-CLBN-stimulation EBA value. At the offset of the CLBN, EBA returned to pre-CLBN level with fast and slow phases, with, for the slow phase, no delay and a time constant of 32.1 +/- 8.1 s. Fast and slow changes in EBA power values were observed after a single injection of gentamicin (GM) at different doses (150, 200, and 250 mg/kg). At 150 and 200 mg/kg, GM progressively and reversibly blocked the rapid effect, but the slow component of the efferent medial suppression remained remarkably unchanged. However, at higher doses both the fast and slow suppressions were totally yet still reversibly blocked. These observations indicate that the medial olivocochlear efferent system exerts sustained influences on outer hair cells and that this effect develops in two different steps that may have different basic cellular mechanisms.

Acoustic Stimulation↗
PubMed1997

[Decalcified, lyophilized, sterile heterotopic porcine ossicular xenografts. Experimental evaluation in the guinea pig].

Using the guinea pig middle ear model, we assessed decalcified, lyophylized, sterile heterotopic porcine ossicular xeno-implants based on a histology (optic and electron scan microscope) and immunologic (immunofluorscence) methods. Implants were placed in the middle ear and others in the dorsal subcutaneous area. Allo-implants were compared as controls. Implants were placed in the middle ear in 54 animals and skin implants in 14. Under the influence of BMP, the implant ossified in all cases in the middle ear. Intense immune recruitment was not observed. Inversely, there was a mononuclear infiltration reaction to the skin implants with formation of a fibrous capsule, immunoglobulin and complement influx and consequently sequestration. The allo-implants were partially reossified. These findings confirm the value of decalcification with hydrochloric acid for BMP induction, independent of species and the failure of attempted immune despecification. Implant outcome is not dependent on its antigen load, which is high compared with its weight, but on the site of implantation. The middle ear appears to be a privileged site of implantation.

Animals↗
PubMed1996

Changes in CM and CAP with sedation and temperature in the guinea pig: facts and interpretation.

The influence of xylazine on the amplitude, latency and waveform of VIIIth nerve compound action potential (CAP) and cochlear microphonic (CM) in response to clicks at 95 dB SPL in normal awake preimplanted guinea pigs was investigated. The animals' temperature was monitored but no thermoregulation was exerted, except in one control experiment. Following a 0.2 ml injection of xylazine, CM showed minor variations while CAP audiograms for tone pips between 0.5 and 25 kHz remained normal. However, a progressive decrease in temperature and a strongly correlated increase in CAP amplitude and in N1 and N2 latencies were noticed. For peak N1 the changes were equivalent to linear amplitude and time expansions, and could be reproduced through CAP synthesis with convolution methods using time expanded unit response model and firing density functions. All changes were maximal after 2 h of sedation and recovered within approximately another 2 h. Whereas xylazine is known to induce hypothermia, all the changes disappeared if the animal was thermoregulated. Therefore the changes are interpreted as a result of hypothermia. The mechanism of N1 latency lengthening and increase in amplitude during hypothermia can be understood as a simultaneous increase in spike duration, hair cell/nerve synaptic delay and postsynaptic time constant. This hypothesis yielded a theoretical temperature coefficient for N1 latency (-52 microseconds/degree C) matching that measured experimentally (-55 microseconds/degree C). When compared with peak N1, peak N2 appeared relatively more expanded. Arguments about the origin of N2 are discussed.

Acoustic Stimulation↗
PubMed1996

Complete restoration of glucocerebrosidase deficiency in Gaucher fibroblasts using a bicistronic MDR retrovirus and a new selection strategy.

Retrovirus-mediated gene transfer is currently the most common method for the application of genetic therapy to cancer and many inherited and acquired disorders. Here we report the generation of an amphotropic producer cell line (CA2) that synthesizes viral particles carrying a bicistronic cassette in which the selectable MDR1 cDNA encoding P-glycoprotein (P-gp) a multidrug efflux pump, and the human glucocerebrosidase (GC) gene are transcriptionally fused. Transduction of human Gaucher fibroblasts with this recombinant virus allowed coordinate expression of P-gp and GC. Treatment of the transduced fibroblasts with various cytotoxic substrates of P-gp selected for cells with increased expression of GC, which paralleled the stringency of drug selection. Thus, selection of the genetically modified Gaucher fibroblasts in 1 microgram/ml colchicine raised their GC activity levels from nearly undetectable to those present in WI-38 normal human fibroblasts, correcting the enzyme deficiency present in Gaucher cells. Moreover, by simultaneously inhibiting the P-gp pump, it was possible to use much lower concentrations of colchicine to select for high-level expression of MDR1 and GC. Thus, selection with colchicine at 5 ng/ml in combination with the P-gp inhibitors verapamil or PSC 833 produced a complete correction of the GC deficiency in the CA2-transduced fibroblasts. These combination regimens, already in clinical use for the treatment of multidrug-resistant malignancies, may prove useful in gene therapy trials when utilized for high level selection of a nonselectable gene such as glucocerebrosidase when transcriptionally fused to the MDR1 gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Expression of the human multidrug resistance and glucocerebrosidase cDNAs from adeno-associated vectors: efficient promoter activity of AAV sequences and in vivo delivery via liposomes.

Recombinant adeno-associated viruses (rAAV) are attractive tools for gene therapy. We designed plasmids in which the human multidrug resistance gene (hMDR1) cDNA was placed downstream from portions of the 5' end of AAV including either a 234-bp cassette or the entire AAV p5 promoter. The drug-resistant phenotype conferred by the P-glycoprotein (Pgp) efflux pump encoded by the hMDR1 cDNA was used to select NIH-3T3 cells transfected with these plasmids. The 234-bp region alone showed promoter activity similar in strength to that of the entire p5 promoter or the retroviral Harvey murine sarcoma virus long terminal repeat (LTR); this result demonstrates that the 234-bp cassette might be used as a small and efficient promoter in rAAV designed to express large genes approaching the packaging limit of AAV particles. After transfection of AAV-MDR1 vectors, the integration of MDR1 sequences into the host cell genome was demonstrated by fluorescent in situ hybridization (FISH). In addition, Southern analysis of low-molecular-weight DNA extracted from drug-resistant cells grown under continuous selection pressure indicated the persistence of nonintegrated AAV-MDR1 plasmids. Coordinate expression of Pgp and human glucocerebrosidase (hGC) was observed in drug-selected NIH-3T3 cells transfected with a bicistronic vector in which MDR1 cDNA was linked to hGC cDNA via the encephalomyocarditis internal ribosome entry site sequence. Moreover, following a single intravenous injection of the bicistronic vector complexed to cationic liposomes into recipient mice, delivery of MDR1 and GC cDNAs was achieved in all the organs we tested. Our results demonstrate that the efficiency of liposomes as vehicles for in vitro and in vivo gene delivery, the advantages of AAV-vectors, and the use of MDR1 as a selectable marker might be successfully combined in gene therapy protocols.

3T3 Cells↗
PubMed1996

The efferent-mediated suppression of otoacoustic emissions in awake guinea pigs and its reversible blockage by gentamicin.

The physiology of the medial efferent olivocochlear system involves suppressive interactions of contralateral sounds on ipsilateral sound-evoked responses, but its role is largely unknown to date. Medial efferents act at the level of cochlear outer hair cells via cholinergic synapses and might affect their mechanical activity, thereby modulating auditory sensitivity. The aim of the present work was to obtain noninvasive measurements of distortion-product otoacoustic emissions (DPOEs), which reflect outer hair cell function, in order to establish the characteristics of medial efferent-induced suppression in awake, restrained guinea pigs. A clear suppression of DPOEs was induced by continuous contralateral white noise presented at 20-70 dB SPL, in the absence of any confounding effect of anesthesia, middle-ear muscles, or acoustic cross talk. Recently, acute injection of a high dose of the aminoglycoside antibiotic gentamicin (150 mg/kg) was reported to alter the suppressive effect of contralateral noise on eighth nerve-compound action potentials, presumably by blocking efferent synapses to outer hair cells. This hypothesis was confirmed with DPOEs for which a single injection of gentamicin at the same dose abolished suppression after about 1-2 h, whereas no change in basal levels was observed. Complete recovery was obtained after 48 h. This experiment may provide an easy, noninvasive tool for studying auditory function with and without functioning efferents.

Animals↗
PubMed1996

Construction and characterization of a selectable multidrug resistance-glucocerebrosidase fusion gene.

Gene fusions can be employed to ensure concomitant expression of two different proteins under the same transcriptional control elements. We have synthesized a retroviral expression vector (pHaMG1) containing a human multidrug resistance (MDR1)-glucocerebrosidase (GC) chimeric gene inserted between the long terminal repeats of the Harvey murine sarcoma virus. When introduced into psi-CRE mouse fibroblasts, pHaMG1 conferred the drug-selectable multidrug resistance phenotype, and drug-resistant clones produced active human GC of about 60 kDa. Percoll gradient fractionation of homogenates prepared from transfectants confirmed correct targeting of P-glycoprotein to the plasma membrane and of GC to lysosomes. Although this construction was designed as a translational fusion of the MDR1 gene product, P-glycoprotein, and human GC, no evidence for a fusion protein was found in transfected cells, and an analysis of the RNAs transcribed from the integrated pHaMG1 retroviral vector suggests that either P-glycoprotein and GC are translated from one mRNA and rapidly processed into two proteins or they are translated separately from different mRNAs. These results reveal the feasibility of using fusion genes, which are smaller than alternative constructions with two promoters or with an internal ribosome entry site, for coexpression of selectable and nonselectable cDNAs in retroviral vectors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Surface adenosine deaminase. A novel B-cell marker in chronic lymphocytic leukemia.

Previous studies found that ADA is present on the surface of mononuclear blood cells from healthy patients. Because the expression of this surface antigen depends upon the cell type, the presence of ADA on the plasma membrane of cells from patients with malignant hematologic diseases was studied by flow cytometry. The highest percentage of expression was found in CLL, whereas the lowest was found in T-cell-derived malignancies. The enzyme expression in immortalized cell lines showed a similar pattern, with the highest expression (95% +/- 5%) in the SKW64 B-derived cell line, the lowest (15% +/- 5%) in Jurkat T-lymphoma derived cells, and the intermediate (32% +/- 8%) in K562 cells derived from a chronic myelogenous leukemia. Double labeling ADA/CD5 and ADA/CD19, as well as the correlation of ADA expression with the expression of other surface markers, indicate that surface ADA might be considered a novel marker for CLL.

Adenosine Deaminase↗
PubMed1995

Uptake of amikacin by hair cells of the guinea pig cochlea and vestibule and ototoxicity: comparison with gentamicin.

The distribution of amikacin (AK), an exclusive cochleo-toxic aminoglycosidic antibiotic (AA), and of gentamicin (GM), which is both cochleo- and vestibulo-toxic, has been studied in cochlear and vestibular hair cells. Guinea pigs were treated during six days with one daily injection of AK (450 mg/kg/day) or GM (60 mg/kg/day). AAs were detected, using immunocytochemical technique with scanning laser confocal microscopy, in isolated cells from guinea pigs sacrificed from 2 to 30 days after the end of the treatments. Results demonstrate a rapid uptake (as soon as after 2-day treatment) of both AAs by cochlear and vestibular hair cells and a very slow clearance. Particularly GM and AK are detected in type I and type II hair cells of the utricles and cristae ampullaris. The presence of these two molecules with different toxic potentialities towards cochlear and vestibular hair cells indicates that the selective ototoxicity of aminoglycosides cannot be explained simply on the basis of particular uptake and accumulation in the different sensory hair cells.

Amikacin↗
PubMed1995

Piezoelectric middle ear implant preserving the ossicular chain.

The authors developed a research program on piezoelectric middle ear implants. A piezoelectric vibrator was implanted on guinea pigs, enabling piezoelectric auditory-evoked recording. The shape and sizes of a vibrator for humans were determined by a human temporal bone study. These human-type vibrators showed good performances in vitro. A particular vibrator for humans that can be implanted on the stapes' head, without irreversibly interrupting the ossicular chain, was designed by the authors.

Animals↗
PubMed1995

[Development of a piezo-electrical vibrator for middle ear implant. State of research].

Authors develop a piezoelectric vibrator for middle ear implant. Animal experimentation, on guinea pigs, demonstrated the efficiency and good tolerance of this vibrators. A study on human temporal bones led them to design a vibrator implantable on the stapes for humans. The in vitro study of the performances of this vibrators showed an excellent frequency fidelity between 0.5 and 8 kHz, and output levels over 110 dB SPL. A vibrator for humans, implantable on the head of the stapes, without interruption of the ossicular chain was realized. Its study on human temporal bones in implantation position, has begun, in parallel with a theoretical study with a mathematical model.

Animals↗
PubMed1994

Rapid, reversible elimination of medial olivocochlear efferent function following single injections of gentamicin in the guinea pig.

Measurements were made of the ability of nonsimultaneous, contralaterally presented broadband noise maskers to suppress ipsilaterally-evoked compound action potentials (CAPs) of the auditory nerve to 8-kHz tone pips in guinea pigs. Contralateral acoustic stimuli have been shown to suppress responses to ipsilateral stimuli through the action of the medial olivocochlear efferent bundle. In this study, maximum suppression across subjects ranged from 2-6 dB. The ability of the contralateral noise to suppress ipsilateral CAPs was rapidly reduced beginning within minutes of a single intramuscular injection of the aminoglycoside antibiotic gentamicin (150 mg/kg), and, in general, was completely abolished by 1.5-2 h. Changes in suppression were presumably produced by gentamicin-induced blockade of presynaptic calcium channels on the medial olivocochlear efferents and closely follow the pharmacokinetics of gentamicin in perilymph. Reductions in efferent-mediated suppression were observed in the absence of any measurable change in either CAP thresholds to different frequencies or CAP input/output. Suppression was partially restored in some animals after 24 h, and was fully recovered in all subjects within 96 h postinjection. The present studies suggest that measures of efferent function might form the rational basis for an objective means to assess aminoglycoside insult on the cochlea in the absence of hair cell damage. These results also indicate that single injections of gentamicin offer a reversible, non-invasive model in which medial efferent function can be studied in behavioral animals.

Acoustic Stimulation↗
PubMed1994

Drug-selected coexpression of human glucocerebrosidase and P-glycoprotein using a bicistronic vector.

Bicistronic cassettes under control of a single promoter have recently been suggested as useful tools for coordinate expression of two different foreign proteins in mammalian cells. Using the long 5' untranslated region of encephalomyocarditis virus as translational enhancer of the second gene, a bicistronic unit composed of cDNA for human P-glycoprotein [the product of the multidrug resistance gene, MDR1 (also called PGY1)] as selectable marker and cDNA for human glucocerebrosidase (GC; EC 3.2.1.45) (a membrane-associated lysosomal hydrolase) was constructed. NIH 3T3 cells transfected with a Harvey murine sarcoma virus retroviral vector carrying this bicistronic cassette (pHaMCG) express active P-glycoprotein and GC and expression of both proteins augments coordinately with selection for increased colchicine resistance. Percoll gradient analysis of homogenates showed that GC was targeted to the lysosomal fraction. The ability to select for expression of GC with natural product drugs after introduction of the pHaMCG retroviral vector may be useful in gene therapy strategies for Gaucher disease.

3T3 Cells↗
PubMed1994

Differential sensitivity to rotation measured on potentials evoked by electrical stimulation of the guinea-pig ear.

Responses to electrical stimulation of the ear applied between round-window and vertex electrodes were recorded in awake guinea-pigs from the same electrodes or from separate vertex/mastoid subdermal needle electrodes. They were averaged during opposite phases of sinusoidal rotation or before and after constant velocity rotation. In both cases the responses were subtracted from each other and yielded differential per- or post-rotatory "electrovestibular" responses. For comparison, responses were also recorded in the same animals and conditions of electrical stimulation during silence and during silence and during presentation of a broad-band noise. The difference yielded "electroacoustic" responses. In round-window records, electrovestibular and electroacoustic responses presented typical compound nerve action potential patterns. Electrovestibular responses could be recorded for head angular velocities as low as 3 degrees sec-1 at 0.1 Hz. Response amplitude showed a logarithmic relation to head velocity. Changes in amplitude, as a function of time after rotation, were comparable to those reported for vestibular nerve fibre responses. In vertex/mastoid records, electroacoustic responses presented a sequence of peaks similar to the click-evoked auditory brain-stem responses, and electrovestibular responses presented two peaks, presumably representing contributions of central vestibular structures. Such "electrovestibulography" permits the study of an individual ear and makes available to the investigator a large range of vestibular stimulation conditions.

Animals↗
PubMed1994

Plastic changes in ipsi-contralateral differences of auditory cortex and inferior colliculus evoked potentials after injury to one ear in the adult guinea pig.

In normal adult guinea pigs, evoked potentials recorded at the ipsilateral auditory cortex to monaural high-frequency acoustic stimuli present higher thresholds and lower amplitudes than at the contralateral cortex; in the inferior colliculus, such ipsi-contralateral differences (ICDs) are smaller than in the auditory cortex. Changes in the ICDs were studied after opposite ear injury. Following quasi-complete hair cell destruction induced by sisomicin injection into the contralateral inner ear, threshold ICDs almost disappeared after about two to six days and ipsilateral amplitudes progressively increased in two to three weeks. The occurrence of ICDs at higher auditory centers revealed in this study, indicates peculiar processing of high frequency stimuli in normal guinea pigs. The alteration of ICDs after opposite ear impairment provides a new possibility to study the auditory plasticity in adult animals.

Acoustic Stimulation↗
PubMed1993

Mammalian cochlear outer hair cells density evaluated by means of an optical tweezer.

We report on the first individual measurements of guinea pig's cochlear outer hair cells densities. Cells were isolated in vitro and manipulated with an optical tweezer. They were levitated in an upward laser beam coaxially trapping the cells. Then they were released by switching off the laser and let fall down in upright position. Measuring their speed and using the Stokes' law, we calculated their mean density. In our experimental frame, the results suggest that the density of the cellular body (between the basal nucleus and the apical cuticular plate) remains quasi constant whatever the cells' length. This implies that density variation of the cellular body does not participate in an intrinsic tuning mechanism.

Animals↗