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R Valdés

Publications and source records attributed to R Valdés.

At least 19 recordsLinked to original sources
PubMed2006

TGF-beta transcriptionally activates the gene encoding the high-affinity adenosine transporter CNT2 in rat liver parenchymal cells.

The nucleoside transporter CNT2 is the highest-affinity adenosine transporter identified so far. Recent evidence suggests that CNT2 has functions other than salvage (i.e. modulation of purinergic responses). Here we identified TGF-beta1 as a potent inducer of CNT2 protein expression in liver parenchymal cells. By contrast, CNT1, which is a target of multifunctional cytokines involved in liver cell proliferation, does not respond to TGF-beta1 treatment. Cloning of a murine CNT2 gene sequence with promoter-like activity enabled us to demonstrate that this cytokine exerts this effect by transcriptionally activating the CNT2-encoding gene in a JNK-dependent manner. The evidence that CNT2 is not a target of multifunctional cytokines involved in hepatocyte proliferation, but instead, of a cytokine that plays major roles in differentiation and apoptosis, further supports the view that the main physiological role of this transporter protein is not nucleoside salvage.

5' Flanking Region↗
PubMed2004

Production and biochemical characterization of the recombinant Boophilus microplus Bm95 antigen from Pichia pastoris.

The new antigen Bm95 from the cattle tick Boophilus microplus was recently isolated, cloned and expressed in the methylotrophic yeast Pichia pastoris. The recombinant protein has shown to induce protection in cattle against infestations of B. microplus under controlled and production conditions. In this paper we report the production and large-scale purification of the Bm95 protein, following a simple and cost-effective process. The antigen was obtained highly aggregated, forming particles ranging from 26 to 30 nm and with purity higher than 80%. The process yield was 0.55 g of pure Bm95 protein per liter of culture. The 98% of the primary structure of the recombinant protein was verified by mass spectrometry. Three amino acid changes in comparison with the sequence deduced from cDNA were detected by LC-MS/MS. The antigen was also obtained N-glycosylated, as previously reported for heterologous protein expression in P. pastoris.

Amino Acid Sequence↗
PubMed2003

Expanded bed adsorption processing of mammalian cell culture fluid: comparison with packed bed affinity chromatography.

A comparison between expanded bed adsorption and conventional packed bed Protein A Fast Flow to purify the anti-rHBsAg mAbs from feedstock is presented in this work. Direct capture by STREAMLINE expanded bed adsorption chromatography resulted in 92% product recovery and sevenfold more concentrated product with similar purity levels compared to that obtained by the standard packed method. The process time and buffer consumption were reduced in the expanded bed adsorption method not only with the binding-elution conditions but also with the use of NaOH during the cleaning-in-place step. The latter is the most widely accepted agent in downstream processing, being a cost effective technique that provides not only efficient cleaning but also sanitizes complete column systems and destroys pirogens.

Adsorption↗
PubMed2003

EEG and behavioral changes following neurofeedback treatment in learning disabled children.

Neurofeedback (NFB) is an operant conditioning procedure, by which the subject learns to control his/her EEG activity. On one hand, Learning Disabled (LD) children have higher values of theta EEG absolute and relative power than normal children, and on the other hand, it has been shown that minimum alpha absolute power is necessary for adequate performance. Ten LD children were selected with higher than normal ratios of theta to alpha absolute power (theta/alpha). The Test Of Variables of Attention (TOVA) was applied. Children were divided into two groups in order to maintain similar IQ values, TOVA values, socioeconomical status, and gender for each group. In the experimental group, NFB was applied in the region with highest ratio, triggering a sound each time the ratio fell below a threshold value. Noncontingent reinforcement was given to the other group. Twenty half-hour sessions were applied, at a rate of 2 per week. At the end of the 20 sessions, TOVA, WISC and EEG were obtained. There was significant improvement in WISC performance in the experimental group that was not observed in the control group. EEG absolute power decreased in delta, theta, alpha and beta bands in the experimental group. Control children only showed a decrease in relative power in the delta band. All changes observed in the experimental group and not observed in the control group indicate better cognitive performance and the presence of greater EEG maturation in the experimental group, which suggests that changes were due not only to development but also to NFB treatment.

Behavior↗
PubMed2002

Chromatographic removal combined with heat, acid and chaotropic inactivation of four model viruses.

The virus removal of protein A affinity chromatography, inactivation capacity, acid pH and a combination of high temperature with a chaotropic agent was determined in this work. The model viruses studied were sendaivirus, human immunodeficency virus (HIV-IIIb), human poliovirus type-II, human herpesvirus I and canine parvovirus. The protein A affinity chromatography showed a maximum reduction factor of 8 logs in the case of viruses larger than 120 nm size, while for small viruses (18-30 nm) the maximum reduction factor was about 5 logs. Non viral inactivation was observed during the monoclonal antibody elution step. Low pH treatment showed a maximum inactivation factor of 7.1 logs for enveloped viruses. However, a weak inactivation factor (3.4 logs) was obtained for DNA nonenveloped viruses. The combination of high temperature with 3 M KSCN showed a high inactivation factor for all of the viruses studied. The total clearance factor was 23.1, 15.1, 13.6, 20.0 and 16.0 logs for sendaivirus, HIV-IIIb, human poliovirus type-II, human herpesvirus I and canine parvovirus, respectively.

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

Stirrer tank: an appropriate technology to immobilize the CB.Hep-1 monoclonal antibody for immunoaffinity purification.

The CB.Hep-1 monoclonal antibody was coupled to CNBr-activated Sepharose CL 4B at three different immobilization scales for purification of recombinant hepatitis B surface antigen. Standard laboratory apparatus to obtain immunosorbents of 1 l (scale I) and 3 l (scale II) as well as a stirrer tank to prepare 6 l immunosorbents (scale III) were used. The binding capacity at scale III was 2- and 1.5-fold higher with respect to the scales II and I, while a reduction in the ligand leakage of 5- and 2-folds was observed. Immunosorbents from scale II showed a significantly reduced adsorption, and an increased ligand leakage. Differences in the coupling efficiency were not observed. Antigen purity eluted from the immunosorbents was always above 85%.

Antibodies, Monoclonal↗
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

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↗
PubMed2000

[Change in metabolic risk factors in obese patients in treatment].

BACKGROUND: Obesity is a threat to health due to its association with cardiovascular risk factors. AIM: To assess the change in metabolic risk factors after weight reduction in obese patients. PATIENTS AND METHODS: A retrospective analysis of 277 obese patients (198 female) aged 39.5 +/- 12 years old, that completed a six month treatment period, consisting in a calorie restricted diet (800-1200 Kcal/day), physical activity, education and group psychological counseling. At the beginning and at the end of the program, weight, waist circumference, resting energy expenditure (REE), serum lipid profile, plasma glucose and plasma insulin levels were measured. RESULTS: During the treatment period, body mass index decreased from 38 +/- 7 to 33.2 +/- 6.4 kg/m2 with a weight loss of 11.7 kg, waist circumference decreased from 106.6 +/- 14.6 to 93.9 +/- 13.5 cm, REE decreased from 1774 +/- 383 to 1585 +/- 267 Kcal/day, blood glucose fell by 5.8%, plasma insulin fell by 40.4%, total cholesterol fell by 7.5%, LDL cholesterol fell by 8.7%, triglycerides fell by 26.6%, and total cholesterol/HDL ratio fell by 12.2%. CONCLUSIONS: Weight loss was associated with improvement in metabolic cardiovascular risk factors in obese patients.

Adolescent↗
PubMed1999

Comparison of different elution conditions for the immunopurification of recombinant hepatitis B surface antigen.

An immunoaffinity chromatographic method was developed using a mAb immunosorbent to purify recombinant hepatitis B surface antigen (r-HBsAg) from yeast. Elution conditions using a mAb-coated ELISA were improved to select the best conditions to purify r-HBsAg. The optimum results in terms of total quantitative recovery were obtained using 20 mM Tris pH 11.6. An increase in the CB.Hep-1 mAb (anti-HBsAg) useful immunosorbents half-life and in its yield per cycle was obtained when alkaline elution conditions were used. Moreover, the basic conditions do not affect either the antigenic characteristics or the purity or the molecular integrity of r-HBsAg.

Chromatography, Affinity↗
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

Nucleoside transporters and liver cell growth.

Liver parenchymal cells show a wide variety of plasma membrane transporters that are tightly regulated by endocrine and nutritional factors. This review summarizes work performed in our laboratory on these transport systems, particularly nucleoside transporters, which are up-regulated in physiological situations associated with liver cell growth. Rat hepatocytes show a Na+-dependent nucleoside transport activity that is stimulated by pancreatic hormones. Indeed, this biological activity appears to be the result of the co-expression of at least two isoforms of nucleoside carriers, CNT1 and CNT2 (also called SPNT). These two transporters are up-regulated during the early phase of liver growth after partial hepatectomy, although to different extents, suggesting differential regulation of the two isoforms. The recent generation of isoform-specific antibodies allowed us to demonstrate that carrier expression may also have complex post-transcriptional regulation on the basis of the lack of correspondence between mRNA and protein levels. The analysis of nucleoside transport systems in hepatoma cells and the comparison with those in hepatocytes has also provided evidence that the differentiation status of liver parenchymal cells may determine the pattern of nucleoside transporters expressed.

Animals↗
PubMed1996

Epitope mapping, V-region DNA sequence, and neutralizing Fab fragments of two monoclonal antibodies against the HIV-1 V3 loop.

BACKGROUND: Experimental evidence suggests that neutralizing antibodies could constitute an important factor in the control of AIDS progression and that the V3 loop of gp120 constitutes the main target for such purposes. We have previously developed two neutralizing murine monoclonal antibodies (Mabs) against the V3 region of the HIV-1 MN strain. OBJECTIVES: To characterize those Mabs in terms of fine specificity and DNA sequence of their V regions and to study if Fab fragments retain their neutralizing potential in vitro. STUDY DESIGN: A set of 12-mer alanine substituted peptides were employed for epitope mapping using two ELISA procedures: (1) indirect, with each peptide bound to polystyrene plates, and (2) competitive, with co-incubation of peptides and Mabs in solution. The V regions of both Mabs were PCR amplified from cDNA and their nucleotide sequences were determined. Finally, Fab fragments of Mab 10F10 were generated and their neutralizing capacity against the MN isolated was assessed. RESULTS: We first restricted the minimal length of the epitopes recognized by 2C4 and 10F10 to the 12-mer peptide KRIHIGPGRAFY. The core of the epitopes recognized by Mabs 2C4 and 10F10 were IHIGP-R and IHIG-R, respectively. While substitution of proline in position 7 completely abolished the binding of 2C4, it only reduced that of 10F10 by 50%. Finally, Fab fragments of Mab 10F10 were still able to neutralize the HIV-1 MN strain in vitro. CONCLUSION: This subtle distinction in the fine mapping of the epitope recognized by Mabs 2C4 and 10F10 should correspond to three amino acid differences that we found in the heavy chain V-regions. The Fab fragments of Mab 10F10 retained the neutralizing capacity. This indicates that HIV neutralization by anti V3 Mabs is an Fc independent process.

Amino Acid Sequence↗
PubMed1995

[Malignant melanoma in an ovarian dermoid cyst].

A case of patient that during a cesarean section, a dark zone of the Omentum was biopsied, and informated as melanosis. Then appeared a cystic mass in the left ovary, and an ooforectomy was performed, resulting a Primary Malignant melanoma.

Adult↗
PubMed1994

Multiepitope polypeptide of the HIV-1 envelope induces neutralizing monoclonal antibodies against V3 loop.

A gene encoding a multiepitope polypeptide (MEP) has been synthesized. It contains the information for (1) an 11-amino acid (aa) epitope from the C1 region of gp120 of HIV-1 and (2) 3 epitopes of 15 amino acids each, from the central part of the V3 loop of isolates MN, SC, and WMJII. These four segments are linked by the short spacer peptide AGGGA. This gene was cloned in a plasmid vector and expressed in Escherichia coli as a fusion product with a 62-aa fragment of human IL-2. The recombinant protein TAB1 was purified by washed pellet procedures and reversed-phase HPLC. TAB1 was recognized in ELISAs by 25 of 27 sera from seropositive individuals. Mice were immunized and several hybridomas were obtained. Two of them secrete monoclonal antibodies that react with synthetic peptides from isolates MN, WMJI, WMJIII, and SC with an affinity constant in the range of 10(8) M-1. They also recognized peptides from isolates SF2 and WMJII, but at much lower affinity. The results obtained from peptide ELISAs indicate that the putative epitope recognized by these MAbs lies within the sequence IHIGPGRAFYT. Classic neutralization assays demonstrated that MAb 2C4 neutralizes 50% of the MN isolate at 0.6 micrograms/ml but fails to neutralize IIIB and SF2 strains. The presence of antibodies directed against every one of the component peptides in the sera of rabbits immunized with TAB1 was also documented.

Amino Acid Sequence↗