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J Bertran

Publications and source records attributed to J Bertran.

At least 19 recordsLinked to original sources
PubMed2006

y+ LAT-1 mediates transport of the potent and selective iNOS inhibitor, GW274150, in control J774 macrophages.

This study has characterised the transport mechanism(s) for the novel and selective inhibitor of inducible nitric oxide synthase (iNOS), GW274150, in murine macrophage J774 cells. Transport of GW274150 was saturable (K(m) = 0.24 +/- 0.01 mM and V(max) of 8.5 +/- 0.12 pmol.microg protein(-1) min(-1)), pH-insensitive and largely Na(+)-independent. Transport was also susceptible to trans-stimulation and was significantly inhibited by a 10-fold excess of L-arginine, L-lysine, L-leucine, L-methionine, L-glutamine and 6-diazo-5-oxo-L-norleucine but not by other amino acids or by N-ethylmaleimide. More importantly, the inhibitions caused by the neutral amino acids were critically dependent on Na(+). These results strongly implicate system y(+)L in the transport of GW274150. Northern blot analysis confirmed this by revealing the presence of transcripts for y(+)LAT-1 but not y(+)LAT-2. Thus, taken together, our data show for the first time that J774 macrophages express y(+)LAT-1 transporters and that these carriers mediate transport of GW2741500 at least in these cells.

Amino Acid Transport System y+L↗
PubMed2006

Effects of ionization, metal cationization and protonation on 2'-deoxyguanosine: changes on sugar puckering and stability of the N-glycosidic bond.

The influence of oxidation, protonation, and metal cationization with Cu(+) and Cu(2+) on the strength of the N-glycosidic bond in 2'-deoxyguanosine has been studied by means of quantum chemical calculations. In all cases, the N9-C1' bond distance increases (0.03-0.06 A) upon introducing positive charge in the guanine moiety, the observed variations being more important for the dicationic systems. Binding energies show that the effect of X(n)(+) in guanine hinders the homolytic dissociation, whereas it largely favors the heterolytic process. With respect to the deoxyribose ring, it has been found that metal binding, oxidation, and protonation do not significantly change the values of the phase angle of pseudorotation P. However, the glycosyl torsion angle chi varies considerably (from 242.0 degrees to 189.8 degrees) as a consequence of a stabilizing guanine-sugar (H8-O4') interaction due to the increase of acidity of guanine C8-H8 upon cationization.

Algorithms↗
PubMed2001

Functional analysis of mutations in SLC7A9, and genotype-phenotype correlation in non-Type I cystinuria.

Cystinuria (OMIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in nephrolithiasis of cystine. Mutations in SLC3A1, which encodes rBAT, cause Type I cystinuria, and mutations in SLC7A9, which encodes a putative subunit of rBAT (b(o,+)AT), cause non-Type I cystinuria. Here we describe the genomic structure of SLC7A9 (13 exons) and 28 new mutations in this gene that, together with the seven previously reported, explain 79% of the alleles in 61 non-Type I cystinuria patients. These data demonstrate that SLC7A9 is the main non-Type I cystinuria gene. Mutations G105R, V170M, A182T and R333W are the most frequent SLC7A9 missense mutations found. Among heterozygotes carrying these mutations, A182T heterozygotes showed the lowest urinary excretion values of cystine and dibasic amino acids. Functional analysis of mutation A182T after co-expression with rBAT in HeLa cells revealed significant residual transport activity. In contrast, mutations G105R, V170M and R333W are associated to a complete or almost complete loss of transport activity, leading to a more severe urinary phenotype in heterozygotes. SLC7A9 mutations located in the putative transmembrane domains of b(o,+)AT and affecting conserved amino acid residues with a small side chain generate a severe phenotype, while mutations in non-conserved residues give rise to a mild phenotype. These data provide the first genotype-phenotype correlation in non-Type I cystinuria, and show that a mild urinary phenotype in heterozygotes may associate with mutations with significant residual transport activity.

Amino Acid Sequence↗
PubMed2000

Gas phase intramolecular proton transfer in cationized glycine and chlorine substituted derivatives (M-Gly, M = Na+, Mg2+, Cu+, Ni+, and Cu2+): existence of Zwitterionic structures?

The intramolecular proton transfer in cationized glycine and chlorine substituted derivatives with M = Na+, Mg2+, Ni+, Cu+, and Cu2+ has been studied with the three parameter B3LYP density functional method. The coordination of metal cations to the oxygens of the carboxylic group of glycine stabilizes the zwitterionic structure. For all monocations the intramolecular proton transfer occurs readily with small energy barriers (1-2 kcalmol(-1)). For the dication Mg2+ and Cu2+ systems, the zwitterionic structure becomes very stable. However, whereas for Mg2+, the proton transfer process takes place spontaneously, for Cu2+ the reaction occurs with an important energy barrier. The substitution of the hydrogens of the amino group by chlorine atoms decreases the basicity of nitrogen, which destabilizes the zwitterionic structure. For monosubstituted glycine complexed with Na+, the zwitterionic structure still exists as a minimum, but for disubstituted glycine no minimum appears for this structure. In contrast, for Mg2+ complexed to mono- and disubstituted glycine, the zwitterionic structure remains the only minimum, since the enhanced electrostatic interaction with the dication overcomes the destabilizing effect of the chlorine atoms.

Journal Article↗
PubMed2000

Heteromeric amino acid transporters explain inherited aminoacidurias.

In the past 5 years, the first genes responsible for aminoacidurias caused by defects in renal reabsorption transport mechanisms have been identified. These diseases are type I and non-type I cystinuria and lysinuric protein intolerance. This knowledge came from the molecular characterization of the first heteromeric amino acid transporters in mammals. In 1992, rBAT and 4F2hc (genes SLC3A1 and SLC3A2, respectively, in the nomenclature of the Human Genome Organization) were identified as putative heavy subunits of mammalian amino acid transporters. In 1994, it was demonstrated that mutations in SLC3A1 cause type I cystinuria. Very recently, several light subunits of the heteromeric amino acid transporters have been identified. In 1999, a putative light subunit of rBAT (the SLC7A9 gene; complementary DNA and protein termed amino acid transporter) and a light subunit of 4F2hc (the SLC7A7 gene; cDNA and protein termed y+LAT-1) were shown to be the non-type I cystinuria and lysinuric protein intolerance genes, respectively. In this review, the characteristics of these heteromeric amino acid transporters and their role in these inherited aminoacidurias is described.

Amino Acid Transport Disorders, Inborn↗
PubMed1999

A multifactorial health risk questionnaire.

OBJECTIVE: to assess the relations between cardiovascular risk factors and psychological or socio-economic status. METHODS: We used a self-questionnaire including data upon usual risk factors, anxiety, depression, behavioural pattern, somatic manifestations, life style, incomes and work-related stress. We gathered 955 people, mainly attending an occupational health examination, filling anonymously the same restricted questionnaire (83 questions). RESULTS: Non-working people represented only 4.4 per cent of the study group and included mainly retired people. They differed significantly from wage earners by a higher level of cardiovascular risk and depression, respectively 15.8 (s.d. 11.3) versus 7.8 (6.7) and 5.6 (3.5) versus 3.9 (2.8). These differences can be related to older age of this group. Among working people, 30 per cent felt job-insecurity; they differed significantly from working people by a higher level of anxiety, 8.9 (3.7) versus 7.5 (3.3). There was no difference concerning the absolute cardiovascular risk or the behavioural pattern. A comprehensive self questionnaire is proposed.

Health Status Indicators↗
PubMed1998

Targeted integration of a recombinant globin gene adeno-associated viral vector into human chromosome 19.

Transfer of a globin gene into stem cells along with the regulatory elements required to achieve high level expression in maturing erythroid cells would provide effective gene therapy for Cooley's Anemia. We have explored the use of recombinant adeno-associated viral (rAAV) vectors for this purpose. A vector designated rHS32A gamma*3'RE that contains regulatory elements from the locus control and flanking regions, integrates as a stable head-to-tail concatamer in erythroleukemia cells at a high multiplicity of infection and exhibits high level, regulated gamma globin gene expression. Inducible expression of the non-structural Rep proteins of wild-type AAV in HeLa cells transduced with rAAV vectors does not increase overall integration frequency, but targeted integration of rHS32A gamma*'3'RE into human chromosome 19 was documented.

Chromosome Mapping↗
PubMed1998

Molecular biology of mammalian plasma membrane amino acid transporters.

Molecular biology entered the field of mammalian amino acid transporters in 1990-1991 with the cloning of the first GABA and cationic amino acid transporters. Since then, cDNA have been isolated for more than 20 mammalian amino acid transporters. All of them belong to four protein families. Here we describe the tissue expression, transport characteristics, structure-function relationship, and the putative physiological roles of these transporters. Wherever possible, the ascription of these transporters to known amino acid transport systems is suggested. Significant contributions have been made to the molecular biology of amino acid transport in mammals in the last 3 years, such as the construction of knockouts for the CAT-1 cationic amino acid transporter and the EAAT2 and EAAT3 glutamate transporters, as well as a growing number of studies aimed to elucidate the structure-function relationship of the amino acid transporter. In addition, the first gene (rBAT) responsible for an inherited disease of amino acid transport (cystinuria) has been identified. Identifying the molecular structure of amino acid transport systems of high physiological relevance (e.g., system A, L, N, and x(c)- and of the genes responsible for other aminoacidurias as well as revealing the key molecular mechanisms of the amino acid transporters are the main challenges of the future in this field.

Amino Acid Sequence↗
PubMed1997

Gene transfer into hematopoietic cells.

Transfer of a gene into stem cells with subsequent lineage-specific gene expression is a desired goal with many potential therapeutic applications. Retroviral vectors developed from murine leukemia viruses reproducibly transfer genes into murine stem cells, but are inefficient at gene insertion into stem cells of larger animals or man. A growing knowledge of stem cell biology suggests that this inefficiency reflects the quiescent state of stem cells, even when incubated in the presence of multiple cytokines and low expression of the receptor for amphotropic retroviral vectors. Alternative vector systems are being explored in an effort to overcome these barriers to stem cell-targeted gene transfer. Our work has shown that recombinant adeno-associated virus vectors, which have the potential for transducing quiescent cells, transfer, express and integrate a globin gene linked to its normal regulatory elements in human erythroid cells, but only at very high multiplicities of infection. The integrated genome was stable and the encoded globin gene was expressed at levels equivalent to a normal globin gene.

Animals↗
PubMed1997

Genotypic analysis of cutaneous T-cell lymphoma: a comparative study of Southern blot analysis with polymerase chain reaction amplification of the T-cell receptor-gamma gene.

The diagnosis of early cutaneous T-cell lymphoma (CTCL) is a difficult point in dermatology. Recently, Southern blot analysis (SBA) and polymerase chain reaction (PCR) have been used to detect clonality in initial lesions in which clinical and histological findings are unspecific. Forty-one samples from 25 patients with CTCL were investigated for the presence of T-cell receptor-gamma gene rearrangement using a nested PCR technique and analysed by polyacrylamide gel electrophoresis (PAGE). Conventional SBA was also performed on 28 samples from 20 of these patients. In addition, 20 samples corresponding to patients with large plaque parapsoriasis (LPP), cutaneous B-cell lymphoma (CBCL) and eczema were analysed by PCR in the same way as were the CTCL specimens. Most of the CTCL specimens (81%) showed clonality on PCR analysis. Among patients with mycosis fungoides, 71% of initial patch lesions and 100% of plaques and tumours showed clonal disease. Clonality could be detected in three of four histologically negative post-treatment lesions. Clonal rearrangement was detected in one of three patients with LPP and in three of 10 patients with CBCL. None of the samples corresponding to patients with eczema showed positive results. SBA was significantly less sensitive than PCR in detecting clonality in CTCL patients (42% among early disease and 60% among advanced cases). The results indicate that this PCR/PAGE technique is a reliable and useful method for the detection of clonality in early skin lesions of CTCL patients and probably in the identification of silent extracutaneous involvement.

Adolescent↗
PubMed1996

Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors.

Adeno-associated virus has a broad host range, is nonpathogenic, and integrates into a preferred location on chromosome 19, features that have fostered development of recombinant adeno-associated viruses (rAAV) as gene transfer vectors for therapeutic applications. We have used an rAAV to transfer and express the murine cationic amino acid transporter which functions as the ecotropic retroviral receptor, thereby rendering human cells conditionally susceptible to infection by an ecotropic retroviral vector. The proportion of human HeLa cells expressing the receptor at 60 h varied as a function of the multiplicity of infection (MOI) with the rAAV. Cells expressing the ecotropic receptor were efficiently transduced with an ecotropic retroviral vector encoding a nucleus-localized form of beta-galactosidase. Cells coexpressing the ecotropic receptor and nucleus-localized beta-galactosidase were isolated by fluorescence-activated cell sorting, and cell lines were recovered by cloning at limiting dilution. After growth in culture, all clones contained the retroviral vector genome, but fewer than 10% (3 of 47) contained the rAAV genome and continued to express the ecotropic receptor. The ecotropic receptor coding sequences in the rAAV genome were under the control of a tetracycline-modulated promoter. In the presence of tetracycline, receptor expression was low and the proportion of cells transduced by the ecotropic retroviral vector was decreased. Modulation of receptor expression was achieved with both an episomal and an integrated form of the rAAV genome. These data establish that functional gene expression from an rAAV genome can occur transiently without genome integration.

Amino Acid Transport Systems↗
PubMed1995

CD34+ cell positive selection from mobilized peripheral blood by an indirect immunomagnetic method: effect of the type of mobilization and assessment of tumor depletion ability.

Mobilized peripheral blood has been shown to be a suitable source of hematopoietic progenitor cells for autologous transplantation in oncologic patients. However, tumor cell contamination can potentially occur, although to a lesser extent than in the bone marrow. CD34+ cell positive selection has been developed as a system for the ex vivo purging of remaining tumor cells when used in mobilized peripheral blood. This method has shown a lower purification potential than that obtained with bone marrow or cord blood. The reason for this is not clear, but different groups have tried to improve the purity and yield of the positive selection on mobilized peripheral blood by predepletion of nonlymphoid cell populations, since they can induce nonspecific binding. The present study was designed to test an indirect immunomagnetic CD34+ cell selection method to make it reproducible, feasible, and effective for purging mobilized peripheral blood. Twenty-nine samples from mobilized peripheral blood were tested. The median starting CD34+ percentage was 0.8 (0.3%-4.2%), and the median final purity was 87% (32.7%-99.7%), with a median yield of 44.8% (15%-83.5%). The highest purity was reproducibly achieved when the starting percentage of CD34+ cells was higher than 0.65% (median purity 93.7, range 81%-99.7%, CV 6%) on samples obtained from patients primed with chemotherapy alone or chemotherapy plus recombinant human granulocyte-colony stimulating factor. No relation was found between the content of contaminating nucleated cells and the final CD34+ cell purity. This method showed a depletion capacity, assessed by PCR on samples contaminated with K562 leukemic cells, of about 3 logs. These results indicate that this indirect immunomagnetic method can produce high purity CD34+ cell fractions from mobilized peripheral blood with a high efficiency of tumor depletion.

Antigens, CD34↗
PubMed1995

Presentation of a PCR-nuclease protection strategy for minimal residual disease monitoring in B-ALL.

Methods for detecting residual malignant cells in patients suffering from lymphoid malignancies have neither been sufficiently sensitive nor easy to routinize, hampering a potential prediction of disease outcome. Taking advantage of clone-specific DNA sequences, generated during lymphocyte differentiation and the polymerase chain reaction, some strategies have been developed for several groups. Up to now the most specific and sensitive methodology, which consists of designing leukemia-specific oligonucleotides, requires sequencing of the complementary determining region III-DNA for each particular patient and is too laborious to be applied to each case for routine monitoring in most hospital laboratories. In an attempt to achieve an easy way to detect residual malignant cells in B lymphoproliferative diseases, we have used a new PCR-based approach, named here as PCR-nuclease protection assay, consisting of: (i) amplification of DNA segments corresponding to the complementarity determining region III of the immunoglobulin heavy chain genes from samples at disease diagnosis; (ii) isolation of the disease-specific single-stranded DNA; (iii) labeling of the single-stranded DNA to generate specific probes; (iv) hybridization to amplified DNA from samples corresponding to different disease phases; and (v) digestion with S1-nuclease. Using this approach, we could detect one malignant cell in a background of 10(5) healthy cells. The sensitivity and specificity of this approach compares with those of the above mentioned specific oligonucleotide strategy in detecting residual malignant B cells. Moreover, this strategy is much less tedious and could be used by most hospital laboratories.

Base Sequence↗
PubMed1993

rBAT, related to L-cysteine transport, is localized to the microvilli of proximal straight tubules, and its expression is regulated in kidney by development.

We have recently isolated a renal cDNA clone (rBAT) that induces amino acid transport in oocytes either as a component or as a specific activator of a system bo,(+)-like transporter. (Bertran, J., Werner, A., Moore, M. L., Stange, G., Markovich, D., Biber, J., Testar, X., Zorzano, A., Palacín, M., and Murer, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5601-5605. In order to obtain additional information on the rBAT protein, we have investigated the cellular localization of rBAT and its expression during development in rat kidney. A polyclonal antibody raised against rBAT recognizes a specific protein band of approximately 90 kDa, which is highly enriched in rat renal brush border membranes. Immunofluorescence and immunoelectron microscopy studies demonstrated that rBAT is expressed in the microvilli domain of S3 epithelial cells (i.e. straight tubules). The onset of rBAT mRNA expression in kidney was detected during late fetal life. In keeping with this, induction in oocytes of L-cystine uptake due to fetal rBAT-related mRNA was approximately 10% of the induction obtained with rBAT-related mRNA from adult kidneys. rBAT mRNA levels were low in early postnatal life, and only at the end of lactation did they increase steeply, attaining approximately 50% of adult values after weaning. rBAT protein was undetectable in total membrane preparations of kidneys from fetuses and early neonates, weakly detected during lactation, and represented < 15% of adult values after weaning. The postnatal expression of rBAT and its specific location in the microvilli of epithelial cells from the S3 segment of the proximal tubule coincide with postnatal maturation of cystine resorption and with the site of high affinity resorption of cysteine in kidney. This is consistent with the involvement of rBAT in a b(o,+)-like high-affinity resorption system for cysteine in the proximal straight tubule of the nephron.

Amino Acid Sequence↗
PubMed1993

Expression cloning of a human renal cDNA that induces high affinity transport of L-cystine shared with dibasic amino acids in Xenopus oocytes.

A renal cDNA clone (rBAT) that induces system bo,+-like amino acid transport activity in Xenopus oocytes has recently been isolated (Bertran, J., Werner, A., Moore, M. L., Strange, G., Markovich, D., Biber, J., Testar, X., Zorzano, A., Palacín, and Murer, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5601-5605). Here we show the isolation of a cDNA clone by screening a human kidney cortex cDNA library for expression of sodium-independent transport of L-[3H]arginine in Xenopus oocytes. The cRNA of this clone induces in oocytes, in addition to the uptake of L-arginine, that of L-[35S]cystine and L-[3H]leucine. Expressed uptake of these amino acids is mutually cis-inhibitable by the other 2 amino acids. Expressed uptake of L-cystine is saturable and shows an apparent Km in the micromolar range. All these characteristics resemble induction of system bo,+ related to rBAT in the oocytes. Human rBAT mRNA (approximately 2.5 kilobases) is found in kidney, small intestine (i.e., jejunum), pancreas, and liver. Human kidney poly(A)+ RNA (mRNA) induces sodium-independent uptake of L-cystine, L-arginine, and L-leucine in Xenopus oocytes. Hybrid depletion with an antisense oligonucleotide of the isolated clone greatly prevents (80-97%) human kidney mRNA-dependent induction of the uptake of these amino acids (i.e., L-cystine, L-arginine, and L-leucine). The isolated clone (2304 base pairs in length) contains a poly(A) tail and encodes a predicted 78.8-kDa protein which is 85 and 80% identical to the rabbit and rat rBAT, respectively. This predicted protein corresponds to a membrane glycoprotein, and contains six potential N-glycosylation sites which might be functional in the oocyte: [35S] methionine labeling of oocytes shows a specific band of 94 kDa in crude membranes of these human cRNA-injected oocytes; treatment of these oocytes with tunicamycin shifts the cRNA-specific translation product to approximately 72 kDa. We conclude that we have isolated a functional cDNA corresponding to human rBAT. The isolation of this human cDNA would lead to the study of the possible involvement of rBAT in human hyperaminoacidurias.

Amino Acid Sequence↗
PubMed1993

Two mRNA transcripts (rBAT-1 and rBAT-2) are involved in system b0,(+)-related amino acid transport.

Previously, we isolated a cDNA clone (rBAT-1) of 2.2 kilobase pairs (kb) from a rabbit kidney cortex cDNA library, encoding a protein involved in sodium-independent transport of L-dibasic amino acids, L-cystine, and some neutral amino acids via a system related to b0,(+)-like activity (Bertran, J., Werner, A., Moore, M. L., Stange, G., Markovich, D., Biber, J., Testar, X., Zorzano, A., Palacin, M., and Murer, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5601-5605). In Northern blot hybridization using an rBAT-1 cDNA probe, 2.2- and 3.9-kb mRNA species were observed. Here we describe the isolation of a 3.9-kb cDNA clone (rBAT-2) by expression cloning using Xenopus laevis oocytes corresponding to the 3.9-kb mRNA species. On the basis of sequence analysis, in vitro translation (major protein of approximately 78 kDa), and functional analysis (expression of transport function), we conclude that rBAT-1- and rBAT-2-related proteins are identical: 677 amino acids in length, with most likely only one transmembrane-spanning domain. There are seven differences in the nucleotide composition within a common overlap of 2189 nucleotides, resulting in 2 amino acid replacements. In comparison with rBAT-1, rBAT-2 has 26 additional nucleotides at the 5'-end, an identical location of the first polyadenylation signal, and approximately 1.7 kb of 3'-untranslated sequence (rich in AT(U) motifs) prior to a poly(A) tail of 63 adenines. We conclude that rBAT-1 and rBAT-2 encode the same protein and that the major difference seems to be related to the use of different polyadenylation signals.

Amino Acid Sequence↗
PubMed1993

Effect of benzyl succinate on insulin receptor function and insulin action in skeletal muscle: further evidence for a lack of spare high-affinity insulin receptors.

Benzyl succinate inhibited insulin binding and tyrosine receptor kinase in a concentration-dependent manner in the partially purified insulin receptor preparation from rat skeletal muscle. Benzyl succinate lowered the apparent number of high-affinity insulin binding sites. We have made use of the inhibitory effect of benzyl succinate to investigate the possible presence of spare high-affinity insulin receptors in muscle. Benzyl succinate inhibited the effect of a supramaximal concentration of insulin on 3-O-methylglucose uptake, 2-(methylamino)isobutyric acid uptake and lactate production by the incubated muscle. Furthermore, the inhibitory effect of benzyl succinate on insulin binding in vitro closely correlated with its inhibitory effect on insulin action in vivo. These findings suggest the absence of spare high-affinity insulin receptors in skeletal muscle. In contrast to data obtained in skeletal muscle, benzyl succinate did not affect the maximally insulin-stimulated glucose transport, although it caused a marked decrease in insulin sensitivity in isolated rat adipocytes, for which the existence of spare insulin receptors is well documented.

3-O-Methylglucose↗