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Biomedical subjects

E Mira

Publications and source records attributed to E Mira.

At least 19 recordsLinked to original sources
PubMed1999

Caloric stimulation of ampullar receptors: a new method to produce mechanically-evoked responses in frog semicircular canals.

A microthermistor positioned close to the exposed posterior semicircular canal in isolated labyrinth preparations of the frog was used to stimulate the sensory organ. Our results indicated that, depending on the position of the heater, the induced endolymphatic convection currents may result in either excitatory or inhibitory cupular deflections and thus in a modulation of ampullar receptor resting activity. Other possible thermal-dependent mechanisms, such as a direct action of the stimulus on vestibular sensors or endolymphatic volume changes, had, in the present experimental conditions, a minor role. Caloric stimulation could therefore represent a novel method to stimulate the semicircular canals 'in situ'.

Animals↗
PubMed1999

The matrix metalloproteinase-9 regulates the insulin-like growth factor-triggered autocrine response in DU-145 carcinoma cells.

The androgen-independent human prostate adenocarcinoma cell line DU-145 proliferates in serum-free medium and produces insulin-like growth factors (IGF)-I, IGF-II, and the IGF type-1 receptor (IGF-1R). They also secrete three IGF-binding proteins (IGFBP), IGFBP-2, -3, and -4. Of these, immunoblot analysis revealed selective proteolysis of IGFBP-3, yielding fragments of 31 and 19 kDa. By using an anti-IGF-I-specific monoclonal antibody (mAb), we detect surface receptor-bound IGF-I on serum-starved DU-145 cells, which activates IGF-1R and triggers a mitogenic signal. Incubation of DU-145 cells with blocking anti-IGF-I, anti-IGF-II, or anti-IGF-I plus anti-IGF-II mAb does not, however, inhibit serum-free growth of DU-145. Conversely, anti-IGF-1R mAb and IGFBP-3 inhibit DNA synthesis. IGFBP-3 also modifies the DU-145 cell cycle, decreases p34(cdc2) levels, and IGF-1R autophosphorylation. The antiproliferative IGFBP-3 activity is not IGF-independent, since des-(1-3)IGF-I, which does not bind to IGFBP-3, reverses its inhibitory effect. DU-145 also secretes the matrix metalloproteinase (MMP)-9, which can be detected in both a soluble and a membrane-bound form. Matrix metalloproteinase inhibitors, but not serpins, abrogate DNA synthesis in DU-145 associated with the blocking of IGFBP-3 proteolysis. Overexpression of an antisense cDNA for MMP-9 inhibits 80% of DU-145 cell proliferation that can be reversed by IGF-I in a dose-dependent manner. Inhibition of MMP-9 expression is also associated with a decrease in IGFBP-3 proteolysis and with reduced signaling through the IGF-1R. Our data indicate an IGF autocrine loop operating in DU-145 cells, specifically modulated by IGFBP-3, whose activity may in turn be regulated by IGFBP-3 proteases such as MMP-9.

3T3 Cells↗
PubMed1999

Concerted activity of tyrosine phosphatase SHP-2 and focal adhesion kinase in regulation of cell motility.

The coordinated interplay of substrate adhesion and deadhesion is necessary for cell motility. Using MCF-7 cells, we found that insulin-like growth factor I (IGF-I) induces the adhesion of MCF-7 to vitronectin and collagen in a dose- and time-dependent manner, suggesting that IGF-I triggers the activation of different integrins. On the other hand, IGF-I promotes the association of insulin receptor substrate 1 with the focal adhesion kinase (FAK), paxillin, and the tyrosine phosphatase SHP-2, resulting in FAK and paxillin dephosphorylation. Abrogation of SHP-2 catalytic activity with a dominant-negative mutant (SHP2-C>S) abolishes IGF-I-induced FAK dephosphorylation, and cells expressing SHP2-C>S show reduced IGF-I-stimulated chemotaxis compared with either mock- or SHP-2 wild-type-transfected cells. This impairment of cell migration is recovered by reintroduction of a catalytically active SHP-2. Interestingly, SHP-2-C>S cells show a larger number of focal adhesion contacts than wild-type cells, suggesting that SHP-2 activity participates in the integrin deactivation process. Although SHP-2 regulates mitogen-activated protein kinase activity, the mitogen-activated protein kinase kinase inhibitor PD-98059 has only a marginal effect on MCF-7 cell migration. The role of SHP-2 as a general regulator of cell chemotaxis induced by other chemotactic agents and integrins is discussed.

Cell Adhesion↗
PubMed1999

Insulin-like growth factor I-triggered cell migration and invasion are mediated by matrix metalloproteinase-9.

MCF-7 cells migrate through vitronectin-coated filters in response to insulin-like growth factor I (IGF-I); migration is inhibited by the matrix metalloproteinase (MMP) inhibitor BB-94, but not by the serine proteinase inhibitor aprotinin. MMP-9 was identified in the conditioned medium of MCF-7 cells; in addition, fluorescence-activated cell sorting analysis revealed its presence on the cell surface, where MMP-9 activity was also found using a specific fluorogenic peptide. Furthermore, the messenger RNA encoding MMP-9 was detected in MCF-7 cells by PCR. The IGF-I concentration leading to maximal MCF-7 invasion produces an increase in cell surface proteolytic activity after short incubation periods. At 18 h, however, preincubation of MCF-7 cells with IGF-I produces at 18 h a dose-dependent decrease in cell-associated MMP-9 activity and an increase in soluble MMP-9. MCF-7 invasion is dependent on the alpha(v)beta5 integrin, a vitronectin receptor. The levels of alpha(v)- and beta5-subunits expressed in MCF-7 cells depend on the IGF-I concentration, which triggers an increase in both of these subunits. Based on these results, we suggest that IGF-I-induced MCF-7 cell migration is mediated by the MMP-9 activity on the cell surface and by alpha(v)beta5 integrin.

Breast Neoplasms↗
PubMed1998

Expression and purification of human stromelysin 1 and 3 from baculovirus-infected insect cells.

Stromelysin 1 (ST1) is a member of the matrix metalloproteinase (MMP) family probably involved in extracellular matrix degradation. Stromelysin 3 (ST3), considered by sequence homology to be a member of the MMP family of proteases, is specifically expressed in the stroma adjacent to the invasive tumoral cells, but its role in cancer progression remains to be elucidated. Genes encoding ST1 and ST3 were expressed in lepidopteran insect cells using the baculovirus expression vector system. Recombinant baculoviruses were obtained after cloning the full-length cDNA of ST1 and ST3 in plasmids pBacPAK1 and pBacPAK9, respectively. Sf9 insect cells infected with the recombinant baculovirus overexpressed the zymogen proST1 (60 kDa) in an insoluble form, a peak of expression being reached from 24 h postinfection. After solubilization in 8 M urea, and further refolding, activation, and purification, 0.3 mg of mature ST1 (30 kDa), purified to 90% homogeneity, was obtained per 5 x 10(8) infected cells. Recombinant ST1 exhibited proteolytic activity on alpha2-macroglobulin, casein, fibronectin, alpha1-antitrypsin, and laminin. The recombinant zymogen proST3 (55 kDa) was expressed as a soluble form in insect cells, maximal expression occurring at 72 h postinfection. After purification to 95% homogeneity, 2.5 mg of proST3 was obtained per 5 x 10(8) infected cells. A number of proteases including plasmin, urokinase, and ST1 were shown to be able to cleave proST3 giving rise to defined bands of 50-30 kDa. The ST3 mature form of 45 kDa (mST3) was also expressed in the baculovirus system and the obtained protein, 2. 5 mg per 5 x 10(8) infected cells purified to 80% homogeneity, was shown to be active on both casein degradation and alpha2-macroglobulin entrapment assays. Our results suggest that the baculovirus system offers a convenient and efficient means to produce ST1 and ST3 in order to carry out further biochemical studies.

Animals↗
PubMed1998

Effects of betahistine on vestibular receptors of the frog.

Betahistine is widely used in the symptomatic treatment of peripheral and central vestibular disorders. However, its remains unknown whether the drug can act directly on inner ear sensory organs. To this end, the effects of betahistine (10(-7)-10(-2) M) were examined on isolated preparations of frog semicircular canal mounted in a double-celled bath which allowed drug administration both in the endolymphatic and in the perilymphatic fluid. The effects of betahistine were evaluated by recording ampullar receptor potentials and nerve firing rate both at rest and during mechanical stimulation of the isolated preparation. The results demonstrated that endolymphatic administration of betahistine had no effect, whereas its perilymphatic administration could reduce greatly ampullar receptor resting discharge but had little effect on mechanically evoked responses. This observation may explain the anti-vertigo effects of betahistine. Vertigo is normally due to uncontrolled changes in vestibular receptor resting discharge. It is therefore probable that any factor able to reduce the resting firing rate of vestibular receptors and, in consequence, its variations, may have an anti-vertigo action.

Animals↗
PubMed1998

[A computerized database for managing otorhinolaryngologic oncology patients].

In recent years the management and interdisciplinary treatment of oncological patients has become extremely complex due to the progress made in diagnosis and therapy. As a result, the knowledge required to treat patients can no longer be simply memorized or manually filed. Computer technology provides the ideal instrument for organizing, saving and analyzing data from head and neck tumor patients. The authors have prepared a computerized database to meet the following needs: ease of use, even for non computer savvy users; minimal ambiguity for data entry; use for both clinical and scientific purposes; possibility to create a network with similar database at other Centers; possibility to expand to include image management. The archive is based on a personal computer with an INTEL 80486 microprocessor, 40 Mb RAM, DOS 6.0. and Windows 3.1. The software includes four main routines: a) formulation and management of tables where oncological data are gathered; b) entry and management of patient-related clinical data; c) statistical processing for epidemiological and oncological research and; d) management of basic computer services. In clinical practice the database allows the following: a) preparation of a monthly chart of check-ups, b) rapid tracking of patients lost to followup, c) printout of a summary of the clinical history of each patient at the time of check-up and rapid updating at the end of the examination, d) automatic production of forms such as discharge letters and reports to be shared with related services (i.e. medical oncology, radiotherapy). In addition, the database is a powerful, versatile research tool which can promptly provide all sorts of oncological data and can automatically prepare tables, diagrams, correlations, survival curves. The system was developed from 1993 to 1995 and has been operative, with a few minor modifications and updates, since 1995. Today the database contains more than 1200 oncological cases and the system is used daily by medical and paramedical personnel alike. Approximately 15 new cases are entered a month and 80 cases updated after follow-up.

Head and Neck Neoplasms↗
PubMed1997

Identification of insulin-like growth factor-binding protein-1 as a potential physiological substrate for human stromelysin-3.

To elucidate the physiological role of human stromelysin-3 (hST-3) in tumor progression and/or wound healing, insulin-like growth factor-binding protein-1 (IGFBP-1) was analyzed as a potential physiological substrate. hST-3 proteolysis generates two fragments of 16 and 9 kDa that react with IGFBP-1 monoclonal antibody, although they do not bind insulin-like growth factor-I (IGF-I) in ligand blot. N-terminal sequencing shows that hST-3 cleaves IGFBP-1 at the His140-Val141 bond located in the IGFBP-1 midregion. We show that IGFBP-1 inhibits IGF-I-induced survival and proliferation of BAF/3 cells, as well as IGF-I-mediated activation of phosphatidylinositol 3-kinase (PI 3-K). Co-incubation of the IGF-I. IGFBP-1 complex with hST-3 restores IGF-I-induced proliferation and PI 3-K kinase activity in these cells. BAF/3 proliferation is significantly increased with the hST-3-treated IGF-I.IGFBP-1 complex compared with that obtained using IGF-I alone. To produce this enhanced proliferation, IGF-I must bind to IGFBP-1 before hST-3 proteolysis, demonstrated using an IGF-I variant that does not bind IGFBP. IGFBP-1 also inhibits IGF-I-induced proliferation of the MCF-7 breast adenocarcinoma, and this inhibition was not seen in hST-3-transfected MCF-7 cells. Such proteolysis may thus play a role in in vivo tumor progression. These results indicate that hST-3 may regulate IGF-I bioavailability by proteolyzing IGFBP, thus favoring cell survival and proliferation.

Humans↗
PubMed1997

Evaluation of fluorometric and zymographic methods as activity assays for stromelysins and gelatinases.

To measure matrix metalloproteinase (MMP) activity in a large number of samples it is advisable to use easily automated methods. We have evaluated and compared the activity of stromelysin-1 (MMP-3), matrilysin (MMP-7), 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) by zymogram analysis and fluorescent substrate degradation assays. FITC-casein and the fluorogenic peptide Dnp-Pro-beta-cyclo-hexyl-Ala-Gly-Cys(Me)-His-Ala-Lys-(N-Me-Abz)-NH 2 were used as fluorescent substrates. FITC-casein was more efficiently degraded than the fluorogenic peptide by all MMPs tested except MMP-9. MMP-2 was not significantly able to degrade the fluorogenic peptide. Gelatin zymography was the most sensitive method to detect the activity of both gelatinases but quantitation problems compromise its use. The degradation of fluorogenic substrates by MMPs could be inhibited by the chelating agent EDTA and by the tissue inhibitor of metalloproteinases 2 (TIMP-2), an MMP-specific inhibitor. Fluorometric methods represent a good alternative for MMP activity measurement, especially when a large number of samples must be processed.

Calcium↗
PubMed1997

[Surfactants of the airways. Critical review and personal research].

The literature proving the presence of a surface tension lowering substance (STLS) on the lining layer of mammalian Eustachian tube (ET) is critically reviewed. A further review of the chemical studies on tubal washings based on chromatographic analysis methods (TLC and HPLC) is performed, and is concluded that ET epithelium is coated by a mixture of phospholipids, similar but not identical to the pulmonary surfactant and with similar but less powerful surface activity. In both cases, and with minor differences between the different mammalian species, phosphatidylcholine (PC), and in particular its disaturated fraction, dipalmitoilphosphatidylcholine (DPPC), is the predominating and the most active compound. ET surfactant is synthesized by ET epithelium and secreted in form of osmiophilic multilamellar bodies into the tubal lumen. The exact function of the ET surfactant is not fully understood: it may play an important role in ET physiology by facilitating the tubal opening to allow for aeration of the middle ear and adequate drainage or could act as a release agent, preventing solid-to-solid adhesion of the tubal walls and contrasting the adhesive action of the glycoproteins of the mucous blanket. On the other hand a phospholipidic surfactant seems to be produced by the mucosa of the other parts of the upper airways, i.e. nose and trachea. In this case a surface active agent could act in preventing the transudation of serum into the lumen, in enhancing the phagocytosis or in facilitating the mucociliary transport. Recent data on humans, suggesting that a relative deficiency or an alterated production of tubal surfactant could play a role in the pathogenesis of secretory otitis media (SOM) or middle ear effusion (MEE), are reviewed. Administration of exogenous surfactant or pharmacological stimulation of the production of tubal surfactant could improve ET function and be of value in some cases of SOM. Personal data, suggesting than ambroxol (a drug stimulating the production of pulmonary surfactant by the alveolar type II pneumocytes) exerts a similar activating effect on the tubotympanal secretory cells, are reported. These data support the results of clinical studies on the treatment of SOM with ambroxol.

Ambroxol↗
PubMed1996

Expression and purification of human matrilysin produced in baculovirus-infected insect cells.

The baculovirus expression system was used to produce recombinant human matrilysin. Expression of promatrilysin reached a peak at 72 h post-infection. Most of the recombinant protein remained in the intracellular fraction in an insoluble form, which after renaturation was purified by S-Sepharose and Green A Dyematrex chromatography in order to remove host proteases. Active recombinant matrilysin degraded casein, type I and type IV collagens and fibronectin. Expression of recombinant human matrilysin using the baculovirus system represents a useful tool for obtaining large amounts of this metalloproteinase in order to carry out further biochemical studies and to screen for inhibitors.

Animals↗
PubMed1996

Chemosensitization and drug accumulation assays as complementary methods for the screening of multidrug resistance reversal agents.

Two methods based on the reversion of adriamycin-resistance o the increase of Rhodamine 123 accumulation in a multidrug resistant (MDR) cell line have been simplified and adapted for the screening of MDR reversal agents. Both methods are carried out in microtiter plates, are highly sensitive and can be easily automated. In both assays verapamil and the cyclosporine derivative PSC 833 could be detected at concentrations lower than 1 and 0.05 microM, respectively. Depending on the MDR cell line used, drugs exhibiting the collateral sensitivity phenomenon can be selected in the cytotoxicity assay, while interferences due to sample toxicity are easily avoided in the dye accumulation assay.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Comparison of mizolastine with loratadine in the treatment of perennial allergic rhinitis.

Mizolastine is a new, non-sedating antihistamine providing satisfactory symptomatic relief in seasonal allergic rhinitis. The purpose of this study has been to compare mizolastine to loratadine in perennial allergic rhinitis. This multicentre, double-blind study has involved 68 patients, randomly allocated, after a one-week placebo run-in, to 10 mg mizolastine or 10 mg loratadine, both given on a once-daily basis, for four weeks. Comparable symptom relief occurs in both groups resulting, respectively for mizolastine and loratadine, in a 66.6% and a 61.3% decrease in total nasal score, to a 74.8% and a 76.4% decrease in total ocular score, and to a 69.0% and a 64.8% decrease in global total score. Safety is satisfactory in both groups. Mizolastine is at least as effective as loratadine in relieving perennial allergic rhinitis symptoms and its safety profile allows its use in the treatment of this disease.

Adult↗
PubMed1996

[High-dose carboplatin superselective intraarterial chemotherapy in advanced head and neck cancer].

Advances in vascular radiology techniques for superselective transfemoral arterial infusion prompted us to evaluate the effects of high-dose rapid regional carboplatin infusion for patients with advanced head and neck squamous cell carcinomas. Twenty untreated patients received three infusions of carboplatin (300-350 mg/m2) every 2 weeks with this method. All the infusions were performed without any complication. Treatment was well tolerated, with moderate (Grade 1-3 WHO) local toxicity (stomatitis, dermatitis and alopecia) and minimal (Grade 1-2 WHO) myelosuppression. The total response index (complete response plus partial response) was 94% for primary tumors and 50% for neck metastases. Neoadjuvant chemotherapy employing superselective rapid infusion of high-dose carboplatin is a feasible, relatively nontoxic, effective technique and may have important applications in multimodality therapy of untreated patients with advanced head and neck cancer.

Adult↗
PubMed1995

General view of vestibular disorders.

The role of the labyrinthine receptors is to transduce the forces associated with head acceleration into a biological signal. Vestibular centres in the brain use this signal, in cooperation with visual and somatosensory signals, to detect the motion of the head and its position relative to gravity vector and to produce motor reflexes to maintain equilibrium and accurate vision during head movements. The evaluation of vestibular function is the mainstay of the general otoneurological examination and relies upon testing for static and dynamic imbalance in the vestibular system, positional testing, eye movement testing and testing for visual-vestibular interaction. The more recent aspects of two conditions, benign paroxysmal positional vertigo and ocular tilt reaction, are discussed. Their pathophysiological mechanisms are reinterpreted and re-evaluated on the basis of present knowledge of vestibulo-oculomotor function.

Eye↗
PubMed1995

Osmolar changes and neural activity in frog vestibular organs.

The effects of hypotonic and hypertonic solutions (the normal value was 240 mOsm) on posterior canal resting and evoked discharge were studied in isolated labyrinth preparations. Hypotonic solutions (60-180 mOsm) were obtained by reducing the perilymphatic NaCl content. Hypertonic solutions (300-420 mOsm) were obtained by adding to normal perilymphatic solutions suitable amounts of NaCl, glucose, sucrose, glycerol, mannitol and urea. The results demonstrated that any kind of receptor activity was inhibited by hypotonic solutions. On the contrary, hypertonic solutions produced different effects on resting and evoked activity. The resting discharge was, with the exception of urea, constantly increased whereas the evoked responses were constantly decreased by all the hypertonic solutions tested. The possible effects of media with changed osmolarity in Meniere's patients is also discussed.

Animals↗