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Biomedical subjects

C Vaquero

Publications and source records attributed to C Vaquero.

At least 19 recordsLinked to original sources
PubMed2006

Lack of correlation between DNA copy number and mRNA expression levels of c-myc in gamma-radiation-induced mouse thymic lymphomas by using quantitative real-time PCR.

BACKGROUND: It is well documented that over-expression of the c-myc proto-oncogene occurs in the vast majority of mouse thymic lymphomas induced by gamma-irradiation, evidencing the importance of this gene in T-cell lymphomagenesis. However, it remains unknown whether elevated levels of c-myc expression are driven by extra c-myc copy numbers. MATERIALS AND METHODS: Here we use a quantitative test on the basis of real-time PCR to determine the cellular copy number of c-myc in a set of 14 g-radiation- induced thymic lymphomas obtained from (C57BL/6J x BALB/cJ) F1 hybrid mice with increased mRNA c-myc expression. RESULTS: Since 5 out of 14 (35.7%) cases had no extra copy numbers of c-myc, gene amplification was obviously not the cause of c-myc over-expression in these tumours. In the remaining 9 tumours, c-myc over-expression was also accompanied with extra DNA copy numbers. Therefore, c-myc amplification might be a consequence of the genomic instability subsequent to the up-regulation of c-myc. However, linear regression analysis showed a lack of correlation between increasing DNA copy numbers and mRNA over expression of c-myc in these tumours (r = 0.029, p = 0.94). CONCLUSION: De-regulation of c-myc does not necessarily imply amplification of this gene in these tumours. This report is, to our knowledge, the first one comparing c-myc amplification with expression in lymphomas of the T-cell lineage.

Animals↗
PubMed2006

Phylogeography and alpha taxonomy of the common dolphin (Delphinus sp.).

The resolution of taxonomic classifications for delphinid cetaceans has been problematic, especially for species in the genera Delphinus, Tursiops and Stenella. The frequent lack of correspondence between morphological and genetic differentiation in these species raises questions about the mechanisms responsible for their evolution. In this study we focus on the genus Delphinus, and use molecular markers to address questions about speciation and the evolution of population structure. Delphinus species have a worldwide distribution and show a high degree of morphological variation. Two distinct morphotypes, long-beaked and short-beaked, have been considered different species named D. capensis and D. delphis, respectively. However, genetic differentiation between these two forms has only been demonstrated in the Pacific. We analysed samples from eight different geographical regions, including two morphologically defined long-beaked form populations, and compared these with the eastern North Pacific populations. We found high differentiation among the populations described as long-beaked instead of the expected monophyly, suggesting that these populations may have evolved from independent events converging on the same morphotype. We observed low genetic differentiation among the short-beaked populations across a large geographical scale. We interpret these phylogeographical patterns in the context of life history and population structure in related species.

Animal Migration↗
PubMed2004

New GABAA receptor alpha5 subunit gene polymorphism that may confound genotyping.

We report the discovery of a new GABAA receptor alpha5 subunit gene polymorphism close to the polymorphism described by Glatt et al. (GT)5GCGTGC(GT)21. This new polymorphism is of great importance, because it means that non-denaturing acrylamide gels used to separate the different alleles of the polymorphism described by Glatt et al. cannot distinguish an allele with the sequence: (GT)4GCGTGC(GT)n from another allele with the sequence: (GT)4(GCGT)4GC(GT)(n-6). These gel fragments are separated by size, which would be the same in these two cases. An alternative would be to use an analysis method that can detect base changes, for instance, single strand conformation polymorphism (SSCP) or denaturing gradient gel electrophoresis (DGGE).

Base Sequence↗
PubMed2004

[Progress towards the use of DNA microarray technology for the study of wild Plasmodium strains].

Plasmodium falciparum is still a major cause of mortality in the world. Due to growing drug resistance in most endemic countries, the use of tools allowing large-scale analysis of P. falciparum biology is increasingly urgent. In addition to gene sequence data, post-genomic methods including microarray-based transcript profiling allow complete Plasmodium gene expression. However, application of this technology has been limited to study of samples presenting large quantities of total RNA (8 microg to 50 microg). Indeed at least two replicas (one technical and one biological) are necessary to ensure the statistical strength of results. This constraint excludes the use of biological materials hardly available and of wild strain samples. Many methods have been developed to facilitate the use of microarray technology with smaller quantities of total RNA. Currently the use of amplification techniques, various fluorochrome markers, and labelled cDNA and/or RNA probes avoiding all amplification steps, to enhance detection sensitivity has enabled reliable assays to be performed with less material. However these enhancement techniques appear to have a biasing effect on results and the use of powerful new markers is still limited for technical and practical reasons. At the present time radioactive labeling is the most reliable technique for assays using small quantities of total RNA. This approach is not only compatible with competitive hybridization but also enables all microarray screening criteria. Findings from our laboratory support the effectiveness of radioactive labeling for microarray-based determinations on P. falciparum.

Animals↗
PubMed2002

Laparoscopic approach to incisional hernia.

BACKGROUND: After more than 8 years of working in the field, we thought it would be interesting to evaluate our experience in the laparoscopic repair of abdominal wall hernias, focusing attention on the lessons learned with time. METHODS: From January 1994 to November of 2000, a total of 270 patients with abdominal wall hernias were treated in our center using the laparoscopic approach. The data collected and analyzed were preoperative evaluation, operative findings, early and long-term complications, and recurrences. RESULTS: The mean follow-up time was 44 months, mean surgical time was 85 min, and mean hospital stay was 1.5 days. The average number of abdominal wall defects was 4.8 per patient. There were 9 (3.3%) small bowel perforations. Conversion to open surgery was required in 1 case (0.3%). Minor early postoperative complications occurred in 38 patients (14.07%). Twenty patients (7.4%) developed persistent postoperative abdominal pain. There was 1 case (0.3%) of small bowel incarceration through the mesh border and another case (0.3%) of small bowel leakage due to ischemia and subsequent peritonitis. The relapse rate was 4.4% (12 cases). CONCLUSION: The laparoscopic approach is a valuable option in the management of abdominal wall hernias, but it requires experience in laparoscopic surgery and there is a specific learning curve for the technique.

Abdominal Pain↗
PubMed2001

Two dimerization domains in the trans-activation response RNA-binding protein (TRBP) individually reverse the protein kinase R inhibition of HIV-1 long terminal repeat expression.

Trans-activation response (TAR) RNA-binding protein (TRBP) is a cellular protein that binds to the human immunodeficiency virus-1 (HIV-1) TAR element RNA. It has two double-stranded RNA binding domains (dsRBDs), but only one is functional for TAR binding. TRBP interacts with the interferon-induced protein kinase R (PKR) and inhibits its activity. We used the yeast two-hybrid assay to map the interaction sites between the two proteins. We show that TRBP and PKR-N (178 first amino acids of PKR) interact with PKR wild type and inhibit the PKR-induced yeast growth defect in this assay. We characterized two independent PKR-binding sites in TRBP. These sites are located in each dsRBD in TRBP, indicating that PKR-TRBP interaction does not require the RNA binding activity present only in dsRBD2. TRBP and its fragments that interact with PKR reverse the PKR-induced suppression of HIV-1 long terminal repeat expression. In addition, TRBP activates the HIV-1 long terminal repeat expression to a larger extent than the addition of each domain. These data suggest that TRBP activates gene expression in PKR-dependent and PKR-independent manners.

Amino Acids↗
PubMed2001

Evidence of a possible epigenetic inactivation mechanism operating on a region of mouse chromosome 19 in gamma-radiation-induced thymic lymphomas.

Loss of heterozygosity (LOH) analysis, performed in 68 gamma-radiation-induced primary thymic lymphomas of F1 hybrid mice, provided evidence of significant LOH on chromosome 19 in a region defined by the D19Mit106 (22 cM) and D19Mit100 (27 cM) markers (Thymic Lymphoma Suppressor Region 8, TLSR8). Cd95 and Pten, two genes mapped at this region, were inactivated in a vast majority of these tumors (85.3% for Cd95 and 61.8% for Pten). Moreover, altered expression of Cd95 and Pten occurred concomitantly in 34 of 68 (50%) thymic lymphomas suggesting a coordinated mechanism of inactivation of these genes. Surprisingly, we also found that Jak2, a proto-oncogene located between Cd95 and Pten, was simultaneously inactivated in a significant fraction of the tumors analysed (24 of 34, 70.6%). Taken together these findings and the lack of mutations in the coding sequences of the mentioned genes clearly suggest a possible regional epigenetic inactivation mechanism on mouse chromosome 19 operating during the development of these tumors.

Animals↗
PubMed2001

Natural antisense transcripts are detected in different cell lines and tissues of cats infected with feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) is a lentivirus inducing an AIDS-like disease in cats, thus providing an interesting model for AIDS study. FIV and HIV-1 possess a similar genomic arrangement of structural and non-structural genes, whose expression is regulated by related genetic mechanisms. On the DNA strand complementary to the HIV-1 envelope messenger, an open reading frame (ORF) has been identified which encompasses the Rev Responsive Element (RRE), a cis-acting element critical to HIV gene expression. This ORF was highly conserved among HIV-1 isolates and had the potential to encode a hydrophobic protein. A corresponding antisense transcript was detected in cells infected with HIV-1, and appeared to encode an antisense protein. In the present study, we have identified a well-conserved antisense ORF, also coincident with the RRE region, in the genome of 21 FIV isolates. The predicted protein, 103 amino acids in length, is highly hydrophobic, as is the case for that of HIV-1. In addition, we have shown that a corresponding transcript, complementary to the transmembrane sequence of the FIV envelope gene, was produced in different cellular and viral contexts, that is, ex vivo and in vivo in FIV infected cell lines and tissues of infected cats, respectively. Expression of antisense transcripts might therefore be of general importance in the natural history of retroviruses.

Amino Acid Sequence↗
PubMed2001

Native and artificial reticuloplasmins co-accumulate in distinct domains of the endoplasmic reticulum and in post-endoplasmic reticulum compartments.

We compared the subcellular distribution of native and artificial reticuloplasmins in endosperm, callus, and leaf tissues of transgenic rice (Oryza sativa) to determine the distribution of these proteins among endoplasmic reticulum (ER) and post-ER compartments. The native reticuloplasmin was calreticulin. The artificial reticuloplasmin was a recombinant single-chain antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER (scFvT84.66-KDEL). We found that both molecules were distributed in the same manner. In endosperm, each accumulated in ER-derived prolamine protein bodies, but also in glutelin protein storage vacuoles, even though glutelins are known to pass through the Golgi apparatus en route to these organelles. This finding may suggest that similar mechanisms are involved in the sorting of reticuloplasmins and rice seed storage proteins. However, the presence of reticuloplasmins in protein storage vacuoles could also be due to simple dispersal into these compartments during protein storage vacuole biogenesis, before glutelin deposition. In callus and leaf mesophyll cells, both reticuloplasmins accumulated in ribosome-coated vesicles probably derived directly from the rough ER.

Calcium-Binding Proteins↗
PubMed2000

Transabdominal surgical approach in the management of renal tumors involving the retrohepatic inferior vena cava.

Multiple reports over the last decade have documented the clear superiority of surgery over other alternative treatments in the management of renal cell carcinoma with extension into the inferior vena cava (IVC). Controversy persists, however, regarding the management of tumor thrombus that extends retrohepatically, but not entering the right atrium. In this report, we retrospectively review our experience with the use of a feasible transabdominal technique without any form of bypass or anticoagulation for safe removal of renal tumor involving the retrohepatic IVC. From 1988 to 1998, 132 patients with renal cell carcinoma underwent radical nephrectomies at the urology service of our hospital. Five patients (3.8%) had retrohepatic venous extension through the renal vein into the IVC. Our transabdominal approach was accomplished by complete mobilization of the liver, control of the hepatocaval connection, total vascular exclusion of the IVC without heparin administration, removal of the tumor thrombus, and primary closure of the longitudinal vena cavotomy. From our results, we found the transabdominal approach with total vascular exclusion of the IVC to be satisfactory, with no early deaths, acceptable morbidity, and a remarkable limitation of blood loss and transfusion requirements.

Adult↗
PubMed2000

A study of selected Plasmodium yoelii messenger RNAs during hepatocyte infection.

We investigated the expression of several mRNAs in exoerythrocytic and erythrocytic stages of Plasmodium yoelii in infected mice, focusing our attention on genes thought to be involved in signal transduction (like pypka and pymap-1, encoding homologues of cAMP-dependent and mitogen-activated protein kinases, respectively) and cell cycle progression (those encoding the cdc2-related kinases Pycrk-1, Pycrk-3 and Pymrk). Messengers coding for enzymes involved in general processes such as DNA replication and RNA transcription (both subunits of the ribonucleotide reductase (Pyrnr1, Pyrnr2) and RNA polymerase II) as well as a messenger coding for Pys21, a sexual stage-specific protein, were also investigated. Total RNA was prepared from livers of infected mice at different times post sporozoite inoculation. In contrast to the pys21 transcript, which was observed only in infected erythrocytes, all messenger species could be detected in the liver by RT-PCR, peaking at 43 h post infection, a time when parasite burden was maximum, and decreasing markedly thereafter to become hardly visible at 168 h. Some transcripts (pypka, pymap-1, pyrnr1 and pyrnr2) could be detected 12 h after infection, while others (pymrk and pyrnapolII) did not become detectable until 24 h. In addition, we characterised all these messengers by Northern blot of total RNAs extracted from infected erythrocytes. Taken together, these data suggest that a similar set of regulatory genes is expressed during both exoerythrocytic and erythrocytic schizogony.

Animals↗
PubMed2000

Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies.

This report describes the stable expression of a medically important antibody in the staple cereal crops rice and wheat. We successfully expressed a single-chain Fv antibody (ScFvT84.66) against carcinoembryonic antigen (CEA), a well characterized tumor-associated marker antigen. scFv constructs were engineered for recombinant antibody targeting to the plant cell apoplast and ER. Up to 30 microg/g of functional recombinant antibody was detected in the leaves and seeds of wheat and rice. We confirmed that transgenic dry seeds could be stored for at least five months at room temperature, without significant loss of the amount or activity of scFvT84.66. Our results represent the first transition from model plant expression systems, such as tobacco and Arabidopsis, to widely cultivated cereal crops, such as rice and wheat, for expression of an antibody molecule that has already shown efficacy in clinical applications. Thus, we have established that molecular pharming in cereals can be a viable production system for such high-value pharmaceutical macromolecules. Our findings provide a strong foundation for exploiting alternative uses of cereal crops both in industrialized and developing countries.

Animals↗
PubMed2000

Analysis of a binding difference between the two dsRNA-binding domains in TRBP reveals the modular function of a KR-helix motif.

Double-stranded RNA-binding proteins constitute a large family with conserved domains called dsRBDs. One of these, TRBP, a protein that binds HIV-1 TAR RNA, has two dsRBDs (dsRBD1 and dsRBD2), as indicated by computer sequence homology. However, a 24-amino-acid deletion in dsRBD2 completely abolishes RNA binding, suggesting that only one domain is functional. To analyse further the similarities and differences between these domains, we expressed them independently and measured their RNA-binding affinities. We found that dsRBD2 has a dissociation constant of 5.9 x 10-8 M, whereas dsRBD1 binds RNA minimally. Binding analysis of 25-amino-acid peptides in TRBP and other related proteins showed that only one peptide in TRBP and one in Drosophila Staufen bind TAR and a GC-rich TAR-mimic RNA. Whereas a 25-mer peptide derived from dsRBD2 (TR5) bound TAR RNA, the equivalent peptide in dsRBD1 (TR6) did not. Molecular modelling indicates that this difference can mainly be ascribed to the replacement of Arg by His residues. Mutational analyses in homologous peptides also show the importance of residues K2 and L3. Analysis of 15-amino-acid peptides revealed that, in addition to TR13 (from TRBP dsRBD2), one peptide in S6 kinase has RNA-binding properties. On the basis of previous and the present results, we can define, in a broader context than that of TRBP, the main outlines of a modular KR-helix motif required for binding TAR. This structural motif exists independently from the dsRBD context and therefore has a modular function.

Amino Acid Sequence↗
PubMed1999

Real-time study of interactions between a composite DNA regulatory region (HIV-1 LTR NRE) and several transcription factors of nuclear extracts.

Here we describe the first real-time study of nuclear protein interaction with a composite DNA regulatory region. We studied the interplay between the three target sites of the negative regulatory element (NRE) of HIV-1 LTR, comprising a noncanonical GATA site overlapping two negative regulatory regions, USF and NFIL-6, and their corresponding transcription factors in nuclear extracts. By bandshift analysis, no GATA binding activity could be detected between LTR NRE and different nuclear extracts, although evidenced by in vitro footprinting. Additionally, the LTR NRE and a USF oligonucleotide showed identical retarded complexes. BIAcore study of these interactions revealed the binding of huGATA-3, as well as USF, to the immobilized LTR NRE oligonucleotide. Competition analyses, performed with GATA, USF, and NFIL-6 oligonucleotides, clearly showed that this regulatory region could bind both huGATA-3 and USF factors. Finally, the presence of USF and huGATA-3 proteins in the complexes formed with LTR NRE was ascertained using specific anti-huGATA-3 and anti-USF2 polyclonal antibodies.

CCAAT-Enhancer-Binding Protein-delta↗
PubMed1999

Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves.

To evaluate the expression of different forms of a tumor-specific antibody in plants, we adapted a recently described Agrobacterium-mediated transient expression system. A recombinant single-chain Fv antibody (scFvT84.66) and a full-size mouse/human chimeric antibody (cT84.66) derived from the parental murine mAb T84. 66 specific for the human carcinoembryonic antigen were engineered into a plant expression vector. Chimeric T84.66 heavy and light chain genes were constructed by exchanging the mouse light and heavy chain constant domain sequences with their human counterparts and cloned into two independent plant expression vectors. In vivo assembly of full-size cT84.66 was achieved by simultaneous expression of the light and heavy chains after vacuum infiltration of tobacco leaves with two populations of recombinant Agrobacterium. Upscaling the transient system permitted purification of functional recombinant antibodies from tobacco leaf extracts within a week. His6-tagged scFvT84.66 was purified by immobilized metal affinity chromatography and cT84.66 by protein A affinity chromatography. Sufficient amounts of recombinant antibodies were recovered for detailed characterization by SDS/PAGE, Western blotting, and ELISA.

Animals↗
PubMed1999

Laparoscopic treatment vs open surgery in the solution of major incisional and abdominal wall hernias with mesh.

BACKGROUND: Despite being one of the most exact indications, laparoscopic treatment of eventrations and ventral hernias is barely known among the array of laparoscopic techniques. METHODS: A total of 60 patients were assigned at random over a 3-year period to two homogeneous groups to be operated on for major ventral hernias with mesh. Half of them were operated upon laparoscopically and the rest with open surgery. Early and longer-term complications were analyzed, as were operative time and postoperative hospital stays. RESULTS: The two groups were homogeneous in terms of demographic and clinical characteristics. The group that was operated on laparoscopically presented a lower rate of postoperative and longer-term complications; similarly, surgery time was significantly lower (p < 0.05). Hospitalization time was also significantly lower than in the group undergoing conventional open surgery (p < 0.05). CONCLUSIONS: Laparoscopic treatment of postoperative eventration and primary ventral hernia reduces complications and relapse rates, eliminates reintervention through mesh infection, reduces operative time, and considerably shortens the hospital stay.

Female↗