Biomedical subjects

A Felipe

Publications and source records attributed to A Felipe.

At least 19 recordsLinked to original sources

PubMed2006

Muscle wasting in cancer and ageing: cachexia versus sarcopenia.

Muscle wasting during cancer and ageing share many common metabolic pathways and mediators. Due to the size of the population involved, both cancer cachexia and ageing sarcopenia may represent targets for future promising clinical investigations. Cancer cachexia is a syndrome characterized by a marked weight loss, anorexia, asthenia and anemia. In fact, many patients who die with advanced cancer suffer from cachexia. The degree of cachexia is inversely correlated with the survival time of the patient and it always implies a poor prognosis. In recent years, age-related diseases and disabilities have become of major health interest and importance. This holds particularly for muscle wasting, also known as sarcopenia that decreases the quality of life of the geriatric population, increasing morbidity and decreasing life expectancy. The cachectic factors (associated with both depletion of fat stores and muscular tissue) can be divided into two categories: of tumour origin and humoural factors. In conclusion, more research should be devoted to the understanding of muscle wasting mediators, both in cancer and ageing, in particular the identification of common mediators may prove as a good therapeutic strategy for both prevention and treatment of wasting both in disease and during healthy ageing.

Aging↗

PubMed2004

Developing a virtual reality application for training nuclear power plant operators: setting up a database containing dose rates in the refuelling plant.

Operators in Nuclear Power Plants can receive high doses during refuelling operations. A training programme for simulating refuelling operations will be useful in reducing the doses received by workers as well as minimising operation time. With this goal in mind, a virtual reality application is developed within the framework of the CIPRES project. The application requires doses, both instantaneous and accumulated, to be displayed at all times during operator training. Therefore, it is necessary to set up a database containing dose rates at every point in the refuelling plant. This database is based on radiological protection surveillance data measured in the plant during refuelling operations. Some interpolation routines have been used to estimate doses through the refuelling plant. Different assumptions have been adopted in order to perform the interpolation and obtain consistent data. In this paper, the procedures developed to set up the dose database for the virtual reality application are presented and analysed.

Computer Simulation↗

PubMed2002

Different Kv2.1/Kv9.3 heteromer expression during brain and lung post-natal development in the rat.

The Kv2.1/Kv9.3 heteromer generates an O2 sensitive potassium channel and induces a slow deactivation that has important consequences for brain and lung physiology. We examined the developmental regulation of Kv2.1 and Kv9.3 mRNAs in brain and lung. Both genes followed parallel expression patterns in brain, increasing progressively through post-natal life. In lung, however, the expression of the two genes followed opposite trends: Kv2.1 transcripts decreased, while Kv9.3 mRNA increased. The Kv9.3/Kv2.1 ratio shows that while in brain the expression of both genes followed a similar pattern, the relative abundance of Kv9.3 increased steadily through post-natal life in lung. Furthermore, there is selective regulation of gene expression during the suckling-weaning transition. Our results suggest that different Kv9.3/Kv2.1 ratios could have physiological implications in both organs during post-natal development, and that diet composition and selective tissue-specific insulin regulation modulate the expression of Kv2.1 and Kv9.3.

Animals↗

PubMed2002

One-step reverse transcription polymerase chain reaction for semiquantitative analysis of mRNA expression.

Analysis of mRNA expression is one of the main targets of scientific research. However, its quantification can be difficult, especially when dealing with low-expression mRNAs (ionic channels, carriers, receptors, etc.) or when only small samples are available (human biopsies). Here we suggest an easy, rapid and reliable method to assess semiquantitative changes in mRNA that combines several technical improvements: i) one-step reverse transcription polymerase chain reaction (RT-PCR) from total RNA; ii) addition of ethidium bromide to the gel, which provides a more homogeneous binding to DNA; iii) direct capture of the gel image using a charged-coupled device camera and then saving the image on computer before quantification, which increases resolution and thus improves and shortens the analysis; and iv) the use of 18S rRNA as a control, which is especially useful when samples from activation, differentiation and proliferation models are used. The technique was validated by checking the system conditions of image capturing and quantification. This was corroborated by a study of Kvl.3 ion channel expression in the brain. In these conditions, the wide range of PCR cycles and total RNA allows us to correlate relative gene expression and direct input of the target gene.

Animals↗

PubMed2001

Lipopolysaccharide-induced apoptosis of macrophages determines the up-regulation of concentrative nucleoside transporters Cnt1 and Cnt2 through tumor necrosis factor-alpha-dependent and -independent mechanisms.

In murine bone marrow macrophages, lipopolysaccharide (LPS) induces apoptosis through the autocrine production of tumor necrosis factor-alpha (TNF-alpha), as demonstrated by the fact that macrophages from TNF-alpha receptor I knock-out mice did not undergo early apoptosis. In these conditions LPS up-regulated the two concentrative high affinity nucleoside transporters here shown to be expressed in murine bone marrow macrophages, concentrative nucleoside transporter (CNT) 1 and 2, in a rapid manner that is nevertheless consistent with the de novo synthesis of carrier proteins. This effect was not dependent on the presence of macrophage colony-stimulating factor, although LPS blocked the macrophage colony-stimulating factor-mediated up-regulation of the equilibrative nucleoside transport system es. TNF-alpha mimicked the regulatory response of nucleoside transporters triggered by LPS, but macrophages isolated from TNF-alpha receptor I knock-out mice similarly up-regulated nucleoside transport after LPS treatment. Although NO is produced by macrophages after LPS treatment, NO is not involved in these regulatory responses because LPS up-regulated CNT1 and CNT2 transport activity and expression in macrophages from inducible nitric oxide synthase and cationic amino acid transporter (CAT) 2 knock-out mice, both of which lack inducible nitric oxide synthesis. These data indicate that the early proapoptotic responses of macrophages, involving the up-regulation of CNT transporters, follow redundant regulatory pathways in which TNF-alpha-dependent- and -independent mechanisms are involved. These observations also support a role for CNT transporters in determining extracellular nucleoside availability and modulating macrophage apoptosis.

Animals↗

PubMed2001

Developmental regulation of the concentrative nucleoside transporters CNT1 and CNT2 in rat liver.

BACKGROUND/AIMS: The pattern of nucleoside transporter expression in hepatocytes was studied in the developing rat liver. METHODS: Hepatocytes isolated from fetuses, neonates and adult rats were used for uridine uptake measurements and concentrative nucleoside transporter (CNT) expression. RESULTS: Adult hepatocytes showed the highest Na-dependent uridine uptake, but fetal hepatocytes exhibited a significant NBTI-sensitive component of equilibrative Na+-independent transport, which was either negligible or absent in neonatal and adult rat hepatocytes. Low Na+-dependent uridine uptake was associated with low amounts of CNT1 and CNT2 transporter proteins, both with apparent Km values in the low micromolar range. Hepatocyte primary cultures from 20-day-old fetuses showed very low amounts of CNT2 mRNA, and expressed both carrier proteins. Incubation of fetal hepatocytes with dexamethasone and T3 resulted in a significant increase in Na+-dependent uridine uptake and an accumulation of the CNT2 protein and mRNA. CONCLUSIONS: The expression of concentrative nucleoside carriers in hepatocytes from developing rat liver is developmentally regulated. Addition of endocrine factors known to induce differentiation of fetal hepatocytes results in selective up-regulation of CNT2 expression.

Animals↗

PubMed2001

Hepatic transport of gluconeogenic substrates during tumor growth in the rat.

Hepatic gluconeogenic substrates (alanine, lactate, and glycerol) transport have been studied in liver plasma membrane vesicles from rats bearing the ascitic tumor Yoshida AH-130 hepatoma. Hepatic alanine uptake was increased in membrane vesicles from tumor-bearing animals as compared with those isolated from non-tumor-bearing controls. Although no changes were observed in relation with KM (2.19 and 2.10 mM for control and tumor groups, respectively), the presence of the tumor caused a clear increase in Vmax (3.07 and 5.04 nmol alanine/mg protein, respectively). The time course of lactate uptake showed no differences between the tumor-bearing animals and their corresponding controls. Both time course and kinetic experiments showed that liver glycerol uptake was due to passive diffusion and therefore cannot contribute to explain the enhanced utilization of this hepatic gluconeogenic substrate during tumor growth. The results suggest that hepatic alanine uptake may be an important factor accounting for its increased utilization for glucose synthesis in tumor-bearing rats.

Alanine↗

PubMed2001

Macrophages require different nucleoside transport systems for proliferation and activation.

To evaluate the mechanisms involved in macrophage proliferation and activation, we studied the regulation of the nucleoside transport systems. In murine bone marrow-derived macrophages, the nucleosides required for DNA and RNA synthesis are recruited from the extracellular medium. M-CSF induced macrophage proliferation and DNA and RNA synthesis, whereas interferon gamma (IFN-gamma) led to activation, blocked proliferation, and induced only RNA synthesis. Macrophages express at least the concentrative systems N1 and N2 (CNT2 and CNT1 genes, respectively) and the equilibrative systems es and ei (ENT1 and ENT2 genes, respectively). Incubation with M-CSF only up-regulated the equilibrative system es. Inhibition of this transport system blocked M-CSF-dependent proliferation. Treatment with IFN-gamma only induced the concentrative N1 and N2 systems. IFN-gamma also down-regulated the increased expression of the es equilibrative system induced by M-CSF. Thus, macrophage proliferation and activation require selective regulation of nucleoside transporters and may respond to specific requirements for DNA and RNA synthesis. This report also shows that the nucleoside transporters are critical for macrophage proliferation and activation.

Animals↗

PubMed2000

Nitric oxide regulates nucleoside transport in activated B lymphocytes.

Activation of human B lymphocytes by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) results in the differential regulation of nucleoside uptake [Soler, C., Felipe, A., Mata, J. F., Casado, F. J., Celada, A., Pastor-Anglada, M. (1998) J. Biol. Chem. 273, 26939-26945]. Because nitric oxide (NO) is involved in the modulation of the apoptotic response of B cells, the effects of NO on the regulatory responses of these transport systems to phorbol esters has been studied in Raji cells by a combination of approaches that involve arginine depletion, inhibition of nitric oxide synthase, and non-enzymatic production of NO using a donor. Human B lymphocytes express three transport systems involved in nucleoside uptake: N1 and N5, which are concentrative and Na+-dependent, and the nitrobenzylthioinosine-sensitive equilibrative system es. Raji cells do not express significant amounts of iNOS mRNA or protein; thus, NO production is presumably constitutive. The data are consistent with a role of NO in maintaining the basal transport activities of the three systems: N1, N5, and es. However, the up-regulatory effect of PMA on N1 and N5 does not require NO, whereas the inhibition of es transport activity does. In summary, NO differentially modulates nucleoside transport systems in activated human B lymphocytes and thus, NO may also be involved in the regulation of nucleoside (i.e., adenosine) disposal by activated B cells.

Arginine↗

PubMed2000

Nutritional regulation of nucleoside transporter expression in rat small intestine.

BACKGROUND & AIMS: Concentrative nucleoside transporters CNT1 (pyrimidine preferring) and CNT2 (purine preferring) may be involved in the uptake of nucleoside-derived drugs used in antiviral and chemical therapies. The possibility that nucleoside carrier isoform expression is modulated by nutrient availability has been studied. METHODS: CNT1 and CNT2 tissue distribution was determined by Western blot analysis. The effect of 48-hour starvation on CNT expression was then studied. Nucleoside transporter expression and uptake activity were measured in jejunal brush border plasma membrane vesicles from fed and starved rats. The expression of nucleoside transporters was later determined in a second model of nutrient deficiency: rats fed a purified diet with or without nucleotides for 10 days. RESULTS: CNT1 and CNT2 nucleoside transporters were expressed in a wider variety of tissues than expected from messenger RNA distribution analysis. CNT1 was sensitive to nutrient availability in small intestine and, accordingly, jejunal brush border membrane vesicles from 48-hour-fasted rats showed increased expression of CNT1 and enhanced Na(+)-dependent thymidine and gemcitabine uptake. This effect was mimicked by feeding semipurified diets lacking nucleotides. CONCLUSIONS: Substrate availability modulates nucleoside transporter expression (CNT1) in rat jejunum in vivo.

Animal Nutritional Physiological Phenomena↗

PubMed2000

Selective loss of nucleoside carrier expression in rat hepatocarcinomas.

Evidence that hepatoma cell lines show differential expression of concentrative nucleoside transporters (CNT1 and CNT2) prompted us to study the transporter proteins in 2 models of hepatocarcinogenesis, the chemically induced Solt and Farber model and the albumin-SV40 large T antigen (Alb-SV40) transgenic rat. CNT1 expression was lower in tumor biopsy specimens from Alb-SV40 rat livers than in normal tissue. Immunocytochemistry revealed that the CNT1 protein was indeed absent in the tumor lesions. CNT1 was also absent in a cell line, L25, derived from the Alb-SV40 transgenic rat liver tumors, whereas another cell line, L37, derived from the normal-appearing parenchyma, retained the expression of both carrier isoforms. The protein expression correlated with the nucleoside transport properties of these cell lines. Moreover, although CNT2 expression was highly dependent on the growth characteristics of the 2 cell lines, as was CNT1 (albeit to a lower extent) in L37 cells, it was not expressed in L25 cells at any stage of cell growth. In contrast to the transgenic model of hepatocarcinogenesis, in the chemically induced tumors the expression of CNT2 was lower, although still detectable. In summary, these data indicate that hepatocarcinogenesis leads to a selective loss or diminished expression of nucleoside carrier isoforms, a feature that may be relevant to our understanding of the molecular basis of the bioavailability of those drugs that are nucleoside derivatives and may be substrates of these carriers. The transport properties and isoform-expression profile of the L25 and L37 cell lines make them suitable hepatocyte culture models with which to study nucleoside transport processes and drug sensitivity.

Animals↗

PubMed1999

Oxygen sensitivity of cloned voltage-gated K(+) channels expressed in the pulmonary vasculature.

Hypoxic pulmonary vasoconstriction is initiated by inhibiting one or more voltage-gated potassium (Kv) channel in the vascular smooth muscle cells (VSMCs) of the small pulmonary resistance vessels. Although progress has been made in identifying which Kv channel proteins are expressed in pulmonary arterial (PA) VSMCs, there are conflicting reports regarding which channels contribute to the native O(2)-sensitive K(+) current. In this study, we examined the effects of hypoxia on the Kv1.2, Kv1.5, Kv2.1, and Kv9.3 alpha subunits expressed in mouse L cells using the whole-cell patch-clamp technique. Hypoxia (PO(2)= approximately 30 mm Hg) reversibly inhibited Kv1.2 and Kv2.1 currents only at potentials more positive than 30 mV. In contrast, hypoxia did not alter Kv1.5 current. Currents generated by coexpression of Kv2.1 with Kv9.3 alpha subunits were reversibly inhibited by hypoxia in the voltage range of the resting membrane potential (E(M)) of PA VSMCs ( approximately 28% at -40 mV). Coexpression of Kv1.2 and Kv1.5 alpha subunits produced currents that displayed kinetic and pharmacological properties distinct from Kv1.2 and Kv1.5 channels expressed alone. Moreover, hypoxia reversibly inhibited Kv1.2/Kv1.5 current activated at physiologically relevant membrane potentials ( approximately 65% at -40 mV). These results indicate that (1) hypoxia reversibly inhibits Kv1.2 and Kv2.1 but not Kv1.5 homomeric channels, (2) Kv1.2 and 1.5 alpha subunits can assemble to form an O(2)-sensitive heteromeric channel, and (3) only Kv1.2/Kv1.5 and Kv2.1/Kv9.3 heteromeric channels are inhibited by hypoxia in the voltage range of the PA VSMC E(M). Thus, these heteromeric channels are strong candidates for the K(+) channel isoforms initiating hypoxic pulmonary vasoconstriction.

Animals↗

PubMed1999

Anatomicohistological characteristics of the ovary of the coypu (Myocastor coypus).

Using histological, histochemical and macroscopic and microscopic measurement techniques, the macroscopic and microscopic structures of coypu ovaries were studied in sexually mature virgin females. The mature ovaries of the coypu were ovoid or elongated bodies, not encapsulated, covered by a single layer of epithelium. They had a parenchyma formed by follicles at different stages of evolution and true and accessory corpora lutea. The interstitial tissue was a prominent and permanent structure in the ovaries. Some ovaries contained a few rete ovarii in the hilus.

Animals↗

PubMed1999

Lack of effect of clinical doses of cyclosporin A on erythrocyte Na+/K+-ATPase activity.

Cyclosporin A (CsA) may exert its cytotoxic effects by altering the activity of different plasma membrane transport systems. Although CsA may act at the gene level, it has been also suggested that it can directly alter transport processes at the plasma membrane. To examine this possibility in a physiological context, we determined Na(+)/K(+)-ATPase activity in erythrocytes from two groups of subjects receiving CsA treatment: group I consisted of kidney transplant patients, and group II comprised patients with steroid-resistant idiopathic nephrotic syndrome. Group I patients showed a marked decrease (35%) in the activity of the Na(+)/K(+)-ATPase in erythrocytes immediately after surgery, before the initiation of CsA treatment. The activity remained low 2 days after the introduction of CsA, but had recovered to the original (pre-surgery) value 1 month later. Group II patients showed the same pattern of erythrocyte Na(+)/K(+)-ATPase activity as those in group I. When the blood CsA levels from all patients were plotted against the corresponding erythrocyte Na(+)/K(+)-ATPase transport activity, a significant linear correlation was found. Higher levels of CsA in the blood were correlated significantly with increased Na(+)/K(+)-ATPase activities. The blood sodium concentration was also correlated positively with both erythrocyte Na(+)/K(+)-ATPase activity and blood CsA concentration. Thus CsA treatment is not associated with inhibition of the Na(+)/K(+)-ATPase in erythrocytes.

Adult↗

PubMed1998

Regulation of nucleoside transport by lipopolysaccharide, phorbol esters, and tumor necrosis factor-alpha in human B-lymphocytes.

Nucleoside transport systems and their regulation in human B-lymphocytes have been characterized using the cell lines Raji and Bare lymphoma syndrome-1 (BLS-1) as experimental models. These cells express at least three different nucleoside transport systems as follows: a nitrobenzylthioinosine-sensitive equilibrative transport system of the es-type, which appears to be associated with hENT1 expression, and two Na+-dependent transport systems that may correspond to N1 and to the recently characterized N5-type, which is nitrobenzylthioinosine-sensitive and guanosine-preferring. B cell activators such as phorbol 12-myristate 13-acetate and lipopolysaccharide (LPS) up-regulate both concentrative transport systems but down-regulate the equilibrative es-type transporter, which correlates with lower hENT1 mRNA levels. These effects are dependent on protein kinase C activity. Phorbol 12-myristate 13-acetate and LPS also induce an increase in tumor necrosis factor-alpha (TNF-alpha) mRNA levels, which suggest that this cytokine may mediate some of the effects triggered by these agents, since addition of TNF-alpha alone can increase N1 and N5 transport activities by a mechanism that also depends on protein kinase C activation. Interestingly, TNF-alpha down-regulates es activity, but this effect cannot be abolished by inhibiting protein kinase C. This study reveals differential regulation of nucleoside transport systems following activation of human B-lymphocyte cell lines by agents of physiological relevance such as TNF-alpha and LPS. Moreover, it indicates that the recently characterized N5 transport system can also be regulated following B cell activation, which may be relevant to lymphocyte physiology and to the treatment of lymphocyte malignancies.

B-Lymphocytes↗

PubMed1998

Na+-dependent nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells.

Hepatocytes show a Na+-dependent nucleoside transport activity that is kinetically heterogeneous and consistent with the expression of at least two independent concentrative Na+-coupled nucleoside transport systems (Mercader et al. Biochem. J. 317, 835-842, 1996). So far, only a single nucleoside carrier-related cDNA (SPNT) has been isolated from liver cells (Che et al. J. Biol. Chem. 270, 13596-13599, 1995). This cDNA presumably encodes a plasma membrane protein responsible for Na+-dependent purine nucleoside transport activity. Thus, the liver must express, at least, a second nucleoside transporter which should be pyrimidine-preferring. Homology cloning using RT-PCR revealed that a second isoform is indeed present in liver. This second isoform turned out to be identical to the 'epithelial-specific isoform' called cNT1, which shows in fact high specificity for pyrimidine nucleosides. Although cNT1 mRNA is present at lower amounts than SPNT mRNA, the amounts of cNT1 protein, when measured using isoform-specific polyclonal antibodies, were even higher than the SPNT protein levels. Moreover, partially purified basolateral plasma membrane vesicles from liver were enriched in the SPNT but not in the cNT1 protein, which suggests that the subcellular localization of these carrier proteins is different. SPNT and cNT1 protein amounts in crude membrane extracts from 6 h-regenerating rat livers are higher than in the preparations from sham-operated controls (3.5- and 2-fold, respectively). These results suggest that liver parenchymal cells express at least two different isoforms of concentrative nucleoside carriers, the cNT1 and SPNT proteins, which show differential regulation and subcellular localization.

Animals↗

PubMed1998

Differential expression and regulation of nucleoside transport systems in rat liver parenchymal and hepatoma cells.

Primary cultures of rat-liver parenchymal cells show carrier-mediated nucleoside uptake by a mechanism that mainly involves concentrative, Na+-dependent transport activity. In contrast, the hepatoma cell line FAO shows high nucleoside transport activity, although it is mostly accounted for by Na+-independent transport processes. This is associated with a low amount of sodium purine nucleoside transporter (SPNT) mRNA. SPNT encodes a purine-preferring transporter expressed in liver parenchymal cells. To analyze whether SPNT expression is modulated during cell proliferation, SPNT mRNA levels were determined in the early phase of liver growth after partial hepatectomy and in synchronized FAO cells that had been induced to proliferate. SPNT mRNA amounts increased as early as 2 hours after partial hepatectomy. FAO cells induced to proliferate after serum refeeding show an increase in SPNT mRNA levels, which is followed by an increase in Na+-dependent nucleoside uptake and occurs before the peak of 3H-thymidine incorporation into DNA. FAO cells also express significant equilibrative nucleoside transport activity, which may be accounted for by the expression of the nitrobenzylthioinosine (NBTI)-sensitive and -insensitive isoforms, rat equilibrative nucleoside transporter 1 (rENT1) and rENT2, respectively. Interestingly, rENT2 mRNA levels follow a similar pattern to that described for SPNT when FAO cells are induced to proliferate, whereas rENT1 appears to be constitutively expressed. Liver parenchymal cells show low and negligible mRNA levels for rENT1 and rENT2 transporters, respectively, although most of the equilibrative transport activity found in hepatocytes is NBTI-resistant. It is concluded that: 1) SPNT expression is regulated both in vivo and in vitro in a way that appears to be dependent on cell cycle progression; 2) SPNT expression may be a feature of differentiated hepatocytes; and 3) equilibrative transporters are differentially regulated, rENT2 expression being cell cycle-dependent. This is consistent with its putative role as a growth factor-induced delayed early response gene.

Animals↗

PubMed1998

Transport and mode of action of nucleoside derivatives used in chemical and antiviral therapies.

Nucleoside analogues used in cancer and anti-viral therapies interfere with nucleotide metabolism and DNA replication, thus inducing their pharmacological effects. A long-awaited goal in the understanding of the pharmacological properties of these molecules, that is the molecular characterization of nucleoside plasma-membrane transporters, has been achieved very recently. These carrier proteins are encoded by at least two gene families and new isoforms remain to be identified. Direct demonstration of translocation of these drugs by nucleoside transporters has already been provided and most of them can inhibit natural nucleoside transport, probably in a competitive manner. The expression of these genes is clearly tissue-specific and might depend on the differentiated status of a cell. This is relevant because the sensitivity of a cell to a drug can depend on the type of nucleoside carrier expressed, and the drug itself might modulate nucleoside carrier expression. In this article, Marçal Pastor-Anglada, Antonio Felipe and Javier Casado discuss recent studies on the regulation of nucleoside carrier expression and of the molecular determinants of substrate specificity. Better knowledge of these will contribute to an improved design of therapies based on nucleoside derivatives.

Acquired Immunodeficiency Syndrome↗