Biomedical subjects

A Felipe

Publications and source records attributed to A Felipe.

At least 37 records · Page 2Linked to original sources

PubMed1997

Cytoskeletal-dependent activation of system A for neutral amino acid transport in osmotically stressed mammalian cells: a role for system A in the intracellular accumulation of osmolytes.

System A activity for neutral amino acid transport is increased after hypertonic shock in NBL-1 (an epithelial cell line) and CHO-K1 cells (a nonepithelial cell line) by a mechanism which is consistent with the synthesis of a regulatory protein that activates preexisting system A carrier proteins (Ruiz-Montasell et al., 1994, Proc. Natl. Acad. Sci. USA, 91,9569-9573). In this study, we have further investigated this biological response by determining the role of cytoskeletal structures in system A regulation by hypertonic stress. Using inhibitors of the microfilament and microtubule networks, we show that the increase in system A activity after hypertonic treatment requires the integrity of both cytoskeletal structures in NBL-1 cells, although the increase in system A activity triggered by amino acid starvation is completely insensitive to any of these drugs. In contrast, the enhancement of system A activity in osmotically stressed CHO-K1 cells is not sensitive to inhibitors of the microtubule network. In both cell types, the results suggest that the inhibitors block the increase of system A activity. System A transport decreases when CHO-K1 cells return to isotonic conditions by a mechanism that is insensitive to inhibitors of protein and mRNA synthesis. The increase in system A transport activity is also followed by the accumulation of neutral amino acids (fourfold for alanine), which is totally blocked by the same agents (cycloheximide and actinomycin D) that prevent the increase in system A activity after hypertonic treatment, thus indicating that system A is crucial for maintaining a high concentration of organic osmolytes inside the cell.

Amino Acids↗

PubMed1997

Implementations of a novel algorithm for colour constancy.

In agreement with the principles of the relativistic model proposed by Creutzfeldt et al., with the photometric rule (lightness anchoring rule) and with the influence of simultaneous contrast in the appearance of a visual scene, we propose a first-stage mechanism yielding substantial colour constancy. We have defined a set of first-stage colour descriptors, and to test their utility, we have performed a simulation using a Machine Vision System (MVS). The statistical stability of the descriptors for Munsell samples under different illuminants is good.

Algorithms↗

PubMed1997

Na+,K(+)-ATPase expression in maleic-acid-induced Fanconi syndrome in rats.

Na+,K(+)-ATPase activity and its alpha 1 subunit protein and mRNA in kidney cortex were monitored in rats developing Fanconi syndrome after the administration of maleate. Na+,K(+)-ATPase activity was significantly lower than in saline-injected controls, although this was partially mediated by a general, non-specific decrease in the cortex protein content. 2. The low activity of the sodium pump correlated with low abundance of alpha 1 subunit mRNA and protein levels. Hsp60 protein levels were also decreased in kidney cortex from maleate-treated rats. 3. Kidney cortex brush-border membrane vesicles from maleate-treated rats showed a marked decrease in Na(+)-dependent alanine and glucose transport, which was not dependent on the Na(+)-transmembrane gradient itself, a finding which is consistent with a more stable effect at the plasma membrane level. 4. The effect of maleate may be partially non-specific and involve a great variety of proteins, but seems to be restricted to selected tissues because alpha 1 subunit Na+,K(+)-ATPase and hsp60 protein amounts were not significantly modified in livers from rats developing Fanconi syndrome. 5. These results show that maleate administration induces a low activity of selected concentrative transport systems and a decrease in Na+,K(+)-ATPase activity and expression. The combination of both effects may explain the increased excretion of most organic solutes present in rats developing Fanconi syndrome.

Alanine↗

PubMed1997

Human contrast sensitivity in coherent Maxwellian view: effect of coherent noise and comparison with speckle.

Lasers have been used in vision for measuring the neural contrast sensitivity function (CSF) by forming interference fringes on the retina. We distinguish among three kinds of illumination with lasers: incoherent (without noise), Maxwellian or coherent (with coherent noise), and diffuse coherent (with speckle). The three have different characteristics and different CSF's. A coherent imaging system is designed to measure the CSF with fully coherent illumination. This is the CSF of the whole visual system, although it is measured with gratings imaged on the retina. It therefore differs from the neural CSF's measured by other authors with partially coherent illumination. However, the neural CSF's are also obtained in this study with and without noise. The effects of coherent noise and speckle on both the visual system and neural sensitivities are studied and compared. Coherent noise differs from speckle in the following ways: (1) It behaves as a high-pass filter, reducing sensitivity in the low-spatial-frequency range, whereas speckle is a low-pass filter; (2) quantitatively, coherent noise reduces neural sensitivity by a factor k(m) with a maximum value between 4 and 6, whereas speckle reduces neural sensitivity by a factor ks with a maximum value of approximately 25 (1.4 log units) for a 3-mm pupil and up to 35 (1.55 log units) for a 1-mm pupil; (3) the masking effect of the coherent noise is affected by changes in luminance but not by changes in pupil diameter; however, the pupil size is the main parameter affecting the masking effect of the speckle.

Contrast Sensitivity↗

PubMed1996

Regulation of Na+,K(+)-ATPase and the Na+/K+/Cl- co-transporter in the renal epithelial cell line NBL-1 under osmotic stress.

The long-term adaptation of the Na+,K(+)-ATPase to hypertonicity was studied using the bovine renal epithelial cell line NBL-1. Na+,K(+)-ATPase activity measured in intact cells as the ouabain-sensitive fraction of Rb+ uptake was stimulated (40% above controls) after incubating the cells in hypertonic medium. This stimulation was not correlated with significant changes in the amount of Na+,K(+)-ATPase alpha 1 subunit protein. Nevertheless, the amount of alpha 1 but not beta 1 subunit mRNA progressively increased after hypertonic shock (3-4-fold above basal values). These results suggest that the alpha 1 subunit gene is modulated by medium osmolarity, although this does not necessarily involve enhanced translation of the mRNA into active alpha 1 protein. Indeed, the increase in the biological activity of the Na+,K(+)-ATPase is abolished when the electrochemical Na+ transmembrane gradient is depleted by monensin, which is consistent with a post-translational effect on the activity of the sodium pump. A furosemide-sensitive component of Rb+ uptake, attributable to Na+/K+/Cl- co-transporter activity, was very low when cells were cultured in a regular medium, but was greatly induced after hypertonic shock. This induction could not be blocked by cycloheximide. Colcemide addition slightly reduced the absolute increase in Na+/K+/Cl- co-transporter activity, while cytochalasin B significantly potentiated the effect triggered by hypertonic shock. It is concluded: (i) that in NBL-1 cells the alpha 1 but not the beta 1 subunit of the Na+,K(+)-ATPase is encoded by an osmotically sensitive gene, and (ii) that the Na+/K+/Cl- co-transporter, although an osmotically sensitive carrier, is induced by a mechanism that is independent of protein synthesis but may rely, in an undetermined manner, on the structure of the cytoskeletal network.

Animals↗

PubMed1996

Nucleoside uptake in rat liver parenchymal cells.

Rat liver parenchymal cells express Na(+)-dependent and Na(+)- independent nucleoside transport activity. The Na(+)-dependent component shows kinetic properties and substrate specificity similar to those reported for plasma membrane vesicles [Ruiz-Montasell, Casado, Felipe and Pastor-Anglada (1992) J. Membr. Biol. 128, 227-233]. This transport activity shows apparent K(m) values for uridine in the range 8-13 microM and a Vmax of 246 pmol of uridine per 3 min per 10(5) cells. Most nucleosides, including the analogue formycin B, cis-inhibit Na(+)-dependent uridine transport, although thymidine and cytidine are poor inhibitors. Inosine and adenosine inhibit Na(+)-dependent uridine uptake in a dose-dependent manner, reaching total inhibition. Guanosine also inhibits Na(+)-dependent uridine uptake, although there is some residual transport activity (35% of the control values) that is resistant to high concentrations of guanosine but may be inhibited by low concentrations of adenosine. The transport activity that is inhibited by high concentrations of thymidine is similar to the guanosine-resistant fraction. These observations are consistent with the presence of at least two Na(+)-dependent transport systems. Na(+)-dependent uridine uptake is sensitive to N-ethylmaleimide treatment, but Na(+)-independent transport is not. Nitrobenzylthioinosine (NBTI) stimulates Na(+)-dependent uridine uptake. The NBTI effect involves a change in Vmax, it is rapid, dose-dependent, does not need preincubation and can be abolished by depleting the Na+ transmembrane electrochemical gradient. Na(+)-independent uridine transport seems to be insensitive to NBTI. Under the same experimental conditions, NBTI effectively blocks most of the Na(+)-independent uridine uptake in hepatoma cells. Thus the stimulatory effect of NBTI on the concentrative nucleoside transporter of liver parenchymal cells cannot be explained by inhibition of nucleoside efflux.

Affinity Labels↗

PubMed1996

Hormonal regulation of concentrative nucleoside transport in liver parenchymal cells.

Na(+)-dependent uridine uptake is stimulated in isolated rat liver parenchymal cells by glucagon. This effect is transient, reaches maximum levels of stimulation 10 min after hormone addition, and is dose-dependent. Glucagon action can be mimicked by agents that are able to hyperpolarize the plasma membrane (e.g. monensin) and by dibutyryl cyclic AMP. The effects triggered by glucagon, monensin and dibutyryl cyclic AMP are not additive, suggesting a common mechanism of action. 8-(4-Chloro-phenylthio)adenosine 3':5'-cyclic monophosphate (PCT), a cyclic AMP analogue but also a nucleoside analogue, markedly stimulates Na(+)-dependent uridine uptake in an additive manner to that triggered by monensin, similarly to the effect described for nitrobenzylthioinosine. Considering the roles reported for nucleosides in liver metabolism, the use of PCT as a cyclic AMP analogue should be precluded. Insulin is also about to up-regulate Na(+)-dependent uridine uptake by a mechanism which involves a stable induction of this transport activity at the plasma-membrane level. This is consistent with a mechanism involving synthesis and insertion of more carriers into the plasma membrane. It is concluded that the recently characterized hepatic concentrative nucleoside transporter is under short-term hormonal regulation by glucagon, through mechanisms which involve membrane hyperpolarization, and under long-term control by insulin. This is the first report showing hormonal modulation of the hepatic concentrative nucleoside transporter.

Animals↗

PubMed1996

Effects of cyclosporine A on Na,K-ATPase expression in the renal epithelial cell line NBL-1.

The bovine renal epithelial cell line NBL-1 has been used to monitor the effects of cyclosporine A (CsA) on Na+,K(+)-ATPase activity and expression. CsA at two single doses (0.6 mg/liter and 2.5 mg/liter) inhibits the ouabain-sensitive component of Rb+ uptake, assumed to be Na+,K(+)-ATPase, but increases the low activity of a furosemide-sensitive component corresponding to a Na+/K+/Cl- cotransporter. CsA addition also induces a slight decrease of alpha 1 subunit mRNA levels, without altering the already low beta 1 subunit mRNA amounts. Hypertonic treatment of NBL-1 cells leads to a significant increase in both Na+,K(+)-ATPase activity and alpha 1 subunit mRNA amounts, but does not modify beta 1 subunit mRNA levels. The differential response of the alpha 1 and beta 1 subunit genes may explain why hypertonic treatment does not result in higher alpha 1 protein expression, and supports the view that increased activity relies upon post-translational events, despite the likely transcriptional activation of the alpha 1 subunit gene. The addition of CsA does not alter the hypertonicity-mediated increase of Na+,K(+)-ATPase activity but blocks the accumulation of alpha 1 subunit mRNA. In conclusion, CsA may compromise the ion handling by renal cells as a result of the inhibition of basal Na+,K(+)-ATPase activity and the stimulation of Na+/K+/Cl- cotransport activity. Moreover, this is the first report showing that CsA may affect the long-term adaptation of the pump by altering its subunit gene expression.

Animals↗

PubMed1996

Differential regulation of Na(+)-K(+)-ATPase in the obese Zucker rat.

Expression of Na(+)-K(+)-adenosinetriphosphatase (ATPase) in tissues from obese and lean Zucker rats was monitored. The phosphatase activity of the sodium pump was increased in liver and intestinal mucosa from obese animals but was unaltered in skeletal muscle, brown adipose tissue, kidney, and heart. Induction of Na(+)-K(+)-ATPase activity was correlated with increased alpha 1-subunit protein amounts in liver and intestinal mucosa, although alpha 1-subunit mRNA levels were increased only in liver tissue. Neither protein nor mRNA amounts for both subunits were significantly altered in the other tissues analyzed. The only exception was a decrease in the amount of beta 1-protein in kidney from obese rats. alpha 2-Subunit protein and alpha 2- and beta 2-mRNA levels were not altered in brown adipose tissue, heart, and soleus. In summary, this study shows that in obese Zucker rats the expression of the sodium pump is enhanced in tissues that are directly involved in nutrient uptake and processing. This adaptation may be related to the ongoing hyperphagia and to tissue hypertrophia but develops in a different manner in each tissue, suggesting differential regulation of alpha 1-subunit expression.

Animals↗

PubMed1995

Induction of the high-affinity Na(+)-dependent glutamate transport system XAG- by hypertonic stress in the renal epithelial cell line NBL-1.

The high-affinity Na(+)-dependent glutamate transport system XAG- is induced (threefold increase in Vmax. with no change in Km) by hypertonicity in the renal epithelial cell line NBL-1. This effect is dependent on protein synthesis and glycosylation and is accompanied by an increase in EAAC1 mRNA levels. Other Na(+)-dependent transport systems in this cell line do not respond to hypertonic stress. In contrast to recent findings [Ruiz-Montasell, Gomez-Angelats, Casado, Felipe, McGivan and Pastor-Anglada (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9569-9573] showing that increased system A activity after hyperosmotic shock results from induction of a regulatory protein, this is the first demonstration that hypertonicity may increase the expression of the gene for an amino acid transport protein itself.

Amino Acid Transport System X-AG↗

PubMed1995

Na+,K(+)-ATPase expression during the early phase of liver growth after partial hepatectomy.

Na+,K(+)-ATPase expression has been studied in the early phase of liver growth after partial hepatectomy to ascertain whether its increased activity is due to stable effects, involving de novo synthesis and insertion of pumps into the plasma membrane. Na+,K(+)-ATPase activity progressively increases after partial hepatectomy, reaching a three-fold induction above basal values 12 h after surgery. mRNA amounts of both alpha 1 and beta 1 subunits are rapidly increased up to two-fold for alpha 1 and nearly three-fold for beta 1, at 9 and 12 h post-hepatectomy, respectively. This correlates with increased abundance of both subunit proteins. The results prove that the increase of Na+,K(+)-ATPase activity correlates with higher expression of both subunit proteins and mRNAs, although the characteristics of the induction suggest that some translational and post-translational events may be equally involved in the increased activity of the pump.

Animals↗

PubMed1995

Effect of protein malnutrition on neutral amino acid transport by rat hepatocytes during development.

Hepatocytes from suckling rats whose mothers were fed a low-protein diet (9% protein) showed a lower capacity for Na(+)-dependent L-alanine uptake [due to a decrease in maximal uptake rate (Vmax) of a low-affinity component of transport] and were not able to respond to insulin or glucagon, whereas those from suckling pups whose mothers were fed the control diet (17% protein) had already developed the ability to upregulate L-alanine transport after hormone treatment. When animals from low-protein-fed mothers were weaned onto a hypoprotein diet, the overall capacity for Na(+)-dependent L-alanine uptake (apparent Vmax) and its responsiveness to pancreatic hormones were restored. Hepatocytes from these animals showed a lower response to glucocorticoid treatment. Amino acid availability was dramatically decreased in suckling and weanling rats fed a low-protein diet. These results support the hypothesis that nutrient supply is an important factor in the proper development of hepatic transport functions during the suckling-weaning transition.

Aging↗

PubMed1995

Starvation-induced increase of hepatic alanine uptake is related to changes in sensitivity to SH-group reagents.

Changes in L-alanine transport in plasma membrane vesicles from livers of control and 24- and 48-h starved adult rats and the sensitivity of alanine uptake to sulfhydryl group reagents [N-ethylmaleimide (NEM) and p-chloromercuribenzenesulfonate (p-CMBS)] were studied. The portal concentration of certain amino acids was measured, and the relationship between L-alanine transport kinetic parameters and amino acid levels was analyzed. Starvation only induced a decrease in portal concentration of these amino acids that are mainly carried by Na(+)-dependent systems (85 and 61% for 24- and 48-h starved rats, respectively). Portal alanine concentration was lower in 24-h starved animals than in control rats (370 vs. 587 microM) and further decreased after 48 h of fasting (228 microM). Starvation induced an increase in maximum velocity (Vmax) values of Na(+)-dependent L-alanine transport (7.19, 8.97, and 12.38 pmol.U 5'-nucleotidase-1.10 s-1 for control and 24- and 48-h starved rats, respectively) with slight, but not significant, changes in the apparent Michaelis constant (Km) values (3.35, 2.63, and 2.20 mM for control and 24- and 48-h starved rats, respectively). Portal alanine showed a directly close correlation with Km values and inverse with Vmax values. The mean affinity constant values for the effects of NEM and p-CMBS on Na(+)-dependent L-alanine transport were lower in 48- (2.57 and 0.13 mM, respectively) and 24-h starved rats (3.59 and 0.32 mM, respectively) than in control rats (8.56 and 0.59 mM, respectively) and showed a directly strong correlation with kinetic characteristics of L-alanine transport and portal alanine concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloromercuribenzenesulfonate↗

PubMed1995

Determination of the susceptibility in vitro of 54 isolates of Mycobacterium fortuitum against three fluoroquinolones using two methods.

The in vitro susceptibility to ofloxacin, norfloxacin and ciprofloxacin or 54 Mycobacterium fortuitum isolates originating from clinical samples (7) of patients attending the Hospital Universitario de Canarias and Hospital del Tórax, and from environmental (47) sources, were determined. For this, two methods were used: dilution in agar with Middlebrook 7H10 Agar as a base medium culture, and broth microdilution, with Mueller-Hinton Broth without supplement. The different isolates under study revealed a uniform susceptibility by both methods against ciprofloxacin. 100% inhibition was obtained from a Minimum Inhibitory Concentration (MIC) of 0.25 microgram/ml, and 2 micrograms/ml of ciprofloxacin, for broth microdilution and dilution in agar, respectively. For ofloxacin and norfloxacin, all the isolates were inhibited at an MIC of 0.5 microgram/ml, by the broth microdilution method, which contrasted sharply with an MIC of 32 micrograms/ml, in the case of dilution in agar. In this study, we have observed the existence of differences in the in vitro susceptibility of the isolates of M. fortuitum against the three fluoroquinolones assayed, mainly for ofloxacin and norfloxacin, by both methods. We, therefore, consider it necessary to establish a standardized, reproducible assay method, for the study of sensitivity to atypical mycobacteria.

Anti-Infective Agents↗

PubMed1995

Determination of the in vitro susceptibility of 220 Mycobacterium fortuitum isolates to ten antimicrobial agents.

The authors investigated the in vitro susceptibility to antimicrobial agents of 220 Mycobacterium fortuitum isolates originating from clinical samples (14) of patients attending the Hospital Universitario de Canarias and Hospital del Tórax, and from environmental sources (206): 3 from sea water, 10 from the water supply and 193 from sewage. The Minimum Inhibitory Concentration (MIC) was calculated using the broth microdilution method with Mueller-Hinton Broth without supplement. Amikacin was the most efficacious antimicrobial agent against all the isolates of M. fortuitum with an MIC which was considerably lower than its critical concentration. The good results achieved with amikacin in vitro are confirmed by those obtained in vivo, with patients infected with M. fortuitum. No significant difference was found in the efficacy of amikacin and ofloxacin against all the isolates assayed.

Anti-Bacterial Agents↗

PubMed1995

Ontogeny of L-alanine uptake in plasma membrane vesicles from rat liver.

Alanine uptake into liver plasma membrane vesicles was studied at different stages of postnatal rat development. Before weaning, alanine hepatic uptake showed lower values for the global KM than after weaning (0.34, 0.77, 1.45, and 1.61 mM for 1-, 15-, and 28-d-old and adult rats, respectively). Alanine uptake capacity increased progressively until reaching maximum values in the adult state (values for Vmax: 0.078, 0.199, 0.317, and 0.613 nmol alanine/mg protein/3 s for 1-, 15-, and 28-d-old and adult rats, respectively). These results seem to point to a prevalence of a high affinity, low capacity alanine transport component (traditionally assumed to be attributable to system A) in newborn and suckling rats, in agreement with our previous results on isolated hepatocytes (Martínez-Mas JV, Casado J, Felipe A, Marin JJG, Pastor-Anglada M: Biochem J 293: 819-824, 1993). The suckling-weaning developmental transition seems to play a role in establishing the pattern of adult hepatic alanine transport characterized by a higher capacity but a lower affinity (because most alanine is taken up by system ASC) inasmuch as KM values show a 100% increase after weaning, although Vmax values continue to increase steadily until the adult age.

Alanine↗

PubMed1995

Phospholipid and fatty acid composition in stored sheep erythrocytes of different densities.

The mammalian erythrocyte is an ideal model for studies of membrane aging under conditions of storage. The present study describes the variations in the membrane lipid composition of three density groups (light, 1.110 < d < 1.125; intermediate, 1.125 < d < 1.130, and dense, 1.130 < d < 1.140) of sheep erythrocytes separated by centrifugation in a discontinuous Ficoll density gradient after storage at 4 degrees C in a nutrient medium for up to 6 days. The only changes in phospholipid composition took place in the erythrocyte light fraction where sphingomyelin (SM) and phosphatidic acid increased (p < 0.05), whereas phosphatidylethanolamine (PE) decreased (p < 0.05). Moreover, polyunsaturated fatty acids (20:4 and 22:6) decreased during storage, whereas lipid fluorescence increased (p < 0.01) after 24 hours of storage in all the fractions separated. These observations suggest a lipid peroxidation process in all three erythrocyte groups during storage.

Animals↗

PubMed1995

[In vitro sensitivity of Mycobacterium chelonae strains to various antimicrobial agents].

The in vitro susceptibility of 32 Mycobacterium chelonae strains to 10 antimicrobial agents was determined. The sources of the different strains were: clinical samples from patients treated at the Hospital Universitario de Canarias and Hospital del Tórax (General and Chest facilities) and from environmental sources (water supply, sewage, swimming pools and the sea). The susceptibility tests were performed by a broth microdilution method (Mueller-Hinton Broth). The results showed amikacin as the most effective antimicrobial agent against M. chelonae isolates, then ofloxacin and cefoxitin. However no statistical difference was detected among them. The least effective was imipenem, followed by ciprofloxacin and norfloxacin.

Amikacin↗