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Biomedical subjects

J M Navarro

Publications and source records attributed to J M Navarro.

At least 19 recordsLinked to original sources

Frequent chromosome 8q gains in human small cell lung carcinoma detected by arbitrarily primed-PCR genomic fingerprinting.

The arbitrarily primed-polymerase chain reaction (AP-PCR) genomic fingerprinting method was applied to detect chromosomal numerical imbalances in 13 small cell lung carcinomas (SCLC). Increases and decreases in the intensity of the AP-PCR bands from several chromosomes, representing gains of chromosomes 1, 7, 16, and X, and losses of chromosomes 2, 10, and 22, were recurrent events in independent tumors. In addition, the most common alterations detected were increases in signal intensity of three AP-PCR bands derived from genomic sequences on chromosome 8q (eight of 13 tumors: 62%). To define whether the 8q gains observed in the AP-PCR analysis include the C-MYC gene at chromosome 8q24 or not, we performed targeted AP-PCR analysis of the C-MYC gene. The C-MYC gene was amplified in five of the eight tumors with gains of 8q, but in none of the remaining five tumors in which 8q gains were not detected. These results indicate that chromosome 8q gain occurs frequently in SCLC and gained regions often, but do not always, include the C-MYC gene. Moderate increases in copy number of the C-MYC gene and additional gene(s) in the 8q region appear to be under positive selection during SCLC progression.

Carcinoma, Small Cell↗

The HIV-1 Vpr co-activator induces a conformational change in TFIIB.

Vpr is a HIV-1 virion-associated protein which plays a role in viral replication and in transcription and cell proliferation. We have previously reported that Vpr stimulates transcription of genes lacking a common DNA target sequence likely through its ability to interact with TFIIB. However, the molecular mechanism of the Vpr-mediated transcription remains to be precisely defined. In this in vitro study, we show that the binding site of Vpr in TFIIB overlaps the domain of TFIIB which is engaged in the intramolecular bridge between the N- and C-terminus of TFIIB, highly suggesting that binding of Vpr may induce a change in the conformation of TFIIB. Indeed, with a partial proteolysis assay using V8 protease, we demonstrate that Vpr has the ability to change the conformation of TFIIB. We investigated in this partial proteolysis assay a series of Vpr-mutated proteins previously defined for their transactivation properties. Our data show a correlation between the ability of Vpr-mutated proteins to stimulate transcription and their ability to induce a conformational change in TFIIB, indicating a functional relevance of the Vpr-TFIIB interaction.

Binding Sites↗

A functional chimera of mammalian guanylyl and adenylyl cyclases.

Adenylyl and guanylyl cyclases synthesize second messenger molecules by intramolecular esterification of purine nucleotides, i.e., cAMP from ATP and cGMP from GTP, respectively. Despite their sequence homology, both families of mammalian cyclases show remarkably different regulatory patterns. In an attempt to define the functional domains in adenylyl cyclase responsible for their isotypic-common activation by Galphas or forskolin, dimeric chimeras were constructed from soluble guanylyl cyclase alpha1 subunit and the C-terminal halves of adenylyl cyclases type I, II, or V. The cyclase-hybrid generated cAMP and was inhibited by P-site ligands. The data establish structural equivalence and the ability of functional complement at the catalytic sites in both cyclases. Detailed enzymatic characterization of the chimeric cyclase revealed a crucial role of the N-terminal adenylyl cyclase half for stimulatory actions, and a major importance of the C-terminal part for nucleotide specificity.

Adenylyl Cyclase Inhibitors↗

The use of arbitrarily primed polymerase chain reaction in cancer research.

There is a great need for techniques that detect the genome alterations present in cancer cells. Here, we present a review of the arbitrarily primed polymerase chain reaction (AP-PCR), a genomic mutation detection method with some unique advantages: (i) It can detect most types of mutations that usually occur in tumors (except point mutations). (ii) It is especially useful to detect moderate gains in DNA, which most methods currently in use cannot detect. (iii) It allows detection and cloning of alterations in a single experiment. (iv) It is inexpensive and does not require special equipment. We discuss some characteristics of this method and review some of its achievements in cancer research.

Acrylic Resins↗

DNA repair enzyme uracil DNA glycosylase is specifically incorporated into human immunodeficiency virus type 1 viral particles through a Vpr-independent mechanism.

The Vpr protein, encoded by the human immunodeficiency virus type 1 (HIV-1) genome, is one of the nonstructural proteins packaged in large amounts into viral particles. We have previously reported that Vpr associates with the DNA repair enzyme uracil DNA glycosylase (UDG). In this study, we extended these observations by investigating whether UDG is incorporated into virions and whether this incorporation requires the presence of Vpr. Our results, with highly purified viruses, show that UDG is efficiently incorporated either into wild-type virions or into Vpr-deficient HIV-1 virions, indicating that Vpr is not involved in UDG packaging. Using an in vitro protein-protein binding assay, we reveal a direct interaction between the precursor form of UDG and the viral integrase (IN). Finally, we demonstrate that IN-defective viruses fail to incorporate UDG, indicating that IN is required for packaging of UDG into virions.

Cell Line↗

Ecto-5'-nucleotidase from a human colon adenocarcinoma cell line. Correlation between enzyme activity and levels in intact cells.

Differences on 5'-nucleotidase activity in intact Rugli and BCS-TC2 cells (rat glioblastoma and human colon adenocarcinoma cell lines, respectively) are not due to differences in the characteristics of the ectoenzyme. A membrane-bound 5'-nucleotidase from BCS-TC2 cells has been purified to homogeneity with a high specific activity (130 U/mg), yielding a single 72-kDa band on SDS-PAGE. It is a metalloenzyme and, after inhibition by EDTA, its activity can be partially restored by divalent cations. The hydrolysis of the nucleosides 5'-monophosphate used as substrate follows Michaelis-Menten kinetics; ADP and concanavalin A are competitive and non-competitive inhibitors of the AMPase activity, respectively. This ecto-5'-nucleotidase is a high-mannose glycoprotein; deglycosylation converts the 72-kDa into a 59-kDa protein with a concomitant activity loss. The enzyme purified from BCS-TC2 cells shows similar characteristics from that previously isolated from Rugli cells; differences between them are mainly due to glycosylation. Polyclonal antibodies against 5'-nucleotidase from BCS-TC2 cells also show cross-reactivity with the enzyme from Rugli cells. When the ectoenzyme activity is measured in cells in culture, Rugli cells present a higher activity than BCS-TC2 cells however, they express very low amounts of ecto-5'-nucleotidase. Our results also show a reduction in protein level and enzyme activity associated with a decrease in the differentiation degree and an increase in tumorigenicity of human colon adenocarcinoma BCS-TC2 sublines.

5'-Nucleotidase↗

A role for human immunodeficiency virus type 1 Vpr during infection of peripheral blood mononuclear cells.

Studies analysing human immunodeficiency virus type 1 replication in primary cells have demonstrated that Vpr, although dispensable, plays a role along with the matrix (MA) protein in allowing nuclear localization of viral preintegration complexes in non-dividing monocyte-derived macrophages (MDMs). In the current study, experimental infection conditions to analyse the role of Vpr, independently of MA, during infection of PHA/IL-2-stimulated peripheral blood mononuclear cells (PBMC) were designed. It was shown that the absence of Vpr results in a subtle effect on virus production in long-term infection. PCR analysis of the steps of virus retrotranscription during a single cycle of replication in stimulated PBMC revealed that the absence of Vpr alone correlates with an impairment in the nuclear localization of viral DNA. Our data indicate that Vpr is involved in the virus life-cycle during infection of dividing PBMC, presumably as it is during infection of MDMs.

Cells, Cultured↗

Differentiation of BCS-TC2 human colon adenocarcinoma cells by sodium butyrate: increase in 5'-nucleotidase activity.

We have analysed the major effects of sodium butyrate on the morphology, protein content and induction of epithelial differentiation markers in human colon adenocarcinoma BCS-TC2 cells. Sodium butyrate alters the cell morphology, inducing a larger cellular size, flattening and vacuolization. The protein content per cell increases, whereas the proliferation rate is reduced. Moreover, cell death by apoptosis is also observed. Butyrate-treated cells show higher levels of alkaline phosphatase activity and carcinoembryonic antigen, suggesting that this agent induces the in vitro differentiation of BCS-TC2 cells. These effects are reversible and time and dose dependent. In addition, we have observed that the ectoenzyme 5'-nucleotidase activity also increases during this treatment, suggesting that it could be considered as a new differentiation marker for this type of carcinoma cells. These results contribute to the understanding of the action of sodium butyrate as a potential cancer chemotherapeutic agent.

5'-Nucleotidase↗

Endocarditis caused by nonhemolytic group B streptococcus.

We report a case of bacterial endocarditis caused by nonhemolytic group B streptococcus (GBS) in a 67-year-old man with no predisposing risk factors. Nonhemolytic GBS strains rarely cause illness and are usually detected in perinatal infections. We believe this to be the first reported case of endocarditis caused by a nonhemolytic strain of GBS.

Aged↗

The human immunodeficiency virus type 1 Vpr transactivator: cooperation with promoter-bound activator domains and binding to TFIIB.

Since the first report documenting that HIV-1 Vpr was involved in the stimulation of transactivation of several unrelated promoters, little additional information has been reported. By using transient transfection experiments, we confirmed and extended these previously reported data. Further in vivo experiments showed that Vpr can co-operatively stimulate transactivation activity of a minimal promoter containing one GAL4 DNA-binding site, when it is co-expressed with different heterologous activator domains fused to GAL4 DNA-binding domain. Thus, Vpr could transactivate in concert with an activator domain, but has no effect on the transactivation of a minimal promoter in the absence of activator protein. To investigate whether Vpr can interact with components of the basal transcriptional machinery, in vitro protein-protein binding assays were performed using either translated, radiolabeled Vpr or TFIIB proteins and glutathione S-transferase Vpr or TFIIB chimeric proteins. We demonstrated that the portion of Vpr ranging from amino acids 15 to 77 interacts specifically with the basal transcription factor TFIIB. Also, our data indicated that the N-terminal domain of TFIIB is required for the interaction.

Adenovirus E1A Proteins↗

Chromosomal assignment of human DNA fingerprint sequences by simultaneous hybridization to arbitrarily primed PCR products from human/rodent monochromosome cell hybrids.

We have developed a technique for the simultaneous chromosomal assignment of multiple human DNA sequences from DNA fingerprints obtained by the arbitrarily primed polymerase chain reaction (AP-PCR). Radioactively labeled human AP-PCR products are hybridized to DNA fingerprints generated with the same arbitrary primer from human/rodent monochromosome cell hybrids after electroblotting to a nylon membrane. Human-specific hybridization bands in the human/rodent fingerprints unambiguously determine their chromosome of origin. We named this method simultaneous hybridization of arbitrarily primed PCR DNA fingerprinting products (SHARP). Using this approach, we determined the chromosomal origins of most major bands of human AP-PCR fingerprints obtained with two arbitrary primers. Altogether, the chromosomal localization of near 50 DNA fragments, comprehensive of all human chromosomes except chromosomes 21 and Y, was achieved in this simple manner. Chromosome assignment of fingerprint bands is essential for molecular karyotyping of cancer by AP-PCR DNA fingerprinting. The SHARP method provides a convenient and powerful tool for this purpose.

Animals↗

Human immunodeficiency virus type 1 Vpr protein binds to the uracil DNA glycosylase DNA repair enzyme.

The role of the accessory gene product Vpr during human immunodeficiency virus type 1 infection remains unclear. We have used the yeast two-hybrid system to identify cellular proteins that interact with Vpr and could be involved in its function. A cDNA clone which encodes the human uracil DNA glycosylase (UNG), a DNA repair enzyme involved in removal of uracil in DNA, has been isolated. Interaction between Vpr and UNG has been demonstrated by in vitro protein-protein binding assays using translated, radiolabeled Vpr and UNG recombinant proteins expressed as a glutathione S-transferase fusion protein. Conversely, purified UNG has been demonstrated to interact with Vpr recombinant protein expressed as a glutathione S-transferase fusion protein. Coimmunoprecipitation experiments confirmed that Vpr and UNG are associated within cells expressing Vpr. By using a panel of C- and N-terminally deleted Vpr mutants, we have determined that the core protein of Vpr, spanning amino acids 15 to 77, is involved in the interaction with UNG. We also demonstrate by in vitro experiments that the enzymatic activity of UNG is retained upon interaction with Vpr.

Animals↗

Elements controlling the expression and induction of the skin hyperproliferation-associated keratin K6.

The suprabasal keratin 6 (K6) is remarkable among the keratins as, in addition to being constitutively expressed in different stratified epithelia, it is induced in epidermis under hyperproliferative conditions, such as benign or malignant tumors, psoriasis, and wound healing. In addition, this keratin is also induced in skin treated with 12-O-tetradecanoylphorbol-13-acetate or retinoic acid (RA). These characteristics make the study of K6 regulatory elements an especially interesting issue, in particular because these elements could be useful in designing gene constructs for the therapy of skin diseases. We have analyzed by mobility shift and footprinting experiments the cell type-specific enhancer of the bovine K6 beta gene (Blessing, M., Jorcano, J. L., and Franke, W. W. (1989) EMBO J. 8, 117-126) and have identified an AP-2-like element, two AP-1 elements (one of them composite), and a retinoic acid-responsive element (RARE). Mutagenesis experiments and cotransfections with retinoic acid receptors show that the RARE mediates enhancer activation by RA. Chloramphenicol acetyltransferase assays show that under normal culture conditions, the AP-1 element retains most of the enhancer transcriptional activity, while the RARE and AP-2 are weakly active. However, following RA treatment, the AP-1 element is repressed and the RARE is activated, resulting in an overall stimulation of the enhancer by RA in the BMGE+H cells used in our study. These results explain in part the complex and sometimes contradictory response of keratin 6 to hyperproliferative stimuli.

Animals↗

Analysis of the control of expression and tissue specificity of the keratin 5 gene, characteristic of basal keratinocytes. Fundamental role of an AP-1 element.

The keratin 5 gene presents a complex regulation, since it is expressed at different rates in the basal cells of most, if not all, stratified epithelia. We have analyzed the 5'-upstream region of the bovine keratin 5 (BK5) gene and found that the 5.2 kilobases preceding the gene mimic in vitro the cell type-specific expression of BK5. Most of the transcriptional activity maps to an enhancer located between positions -762 and -1009. The only regulatory element found in this enhancer by electrophoretic mobility shift, competition, and footprinting experiments is a consensus AP-1 site. Mutation of this site abolishes the activity of the enhancer and reduces to 25% the activity of the 5.2-kilobase upstream promoter region. Surprisingly, although the AP-1 presents indistinguishable footprints in all cell types tested, the enhancer is active only in some of them. Even an oligonucleotide containing the AP-1 region protected from DNase I is active in epithelial cells lines but not in NIH 3T3 fibroblasts, suggesting that this region could constitute an epithelium-specific AP-1 element. We also show that BK5 does not respond to phorbol ester induction, which suggests that the regulation of this gene by AP-1 must be complex and probably different from several other suprabasal, AP-1-regulated cellular and viral genes.

3T3 Cells↗

The maedi-visna virus Tat protein induces multiorgan lymphoid hyperplasia in transgenic mice.

Sheep infected with maedi-visna virus experience immunological disorders leading to progressive chronic diseases involving the brain, lung, spleen, and lymph nodes. To study the biological activity of the viral transactivating Tat protein, we generated transgenic mice carrying the tat gene. Analysis of the transgenic mouse tissues for tat mRNA revealed that while low at the messenger level, the expression of the transgene correlated with dramatic follicular lymphoproliferative disorders involving the lung, spleen, lymph nodes, and skin. This finding suggests that the viral protein possesses a high pathological potency. Our findings show that the maedi-visna virus tat gene product contributes to the pathogenesis of multiorgan proliferative disorders associated with maedi-visna virus infection.

Animals↗

[Detection of Brucella with an automatic hemoculture system: Bact/Alert].

BACKGROUND: The ability of in vitro and in vivo detection of Brucella spp. with the Bact/Alert system was studied. METHODS: Three strains of Brucella melitensis and two of Brucella abortus were used. Different dilutions of the five strains were performed in trypticase soy broth (TSB), achieving concentrations of 1 cfu/ml, 5 cfu/ml, 10 cfu/ml and 100 cfu/ml. Ten ml of each dilution and strain were inoculated into 5 aerobic bottles Bact/Alert and 5 biphasic Hemóline bottles. Furthermore, over a 9 month period, 8,216 bottles of Bact/Alert bottles from hospitalized patients and from the emergency department were processed in the authors' laboratory. RESULTS: The mean detection time for Brucella growth was from 2 to 3 days with the Bact/Alert system, and 14 days in the biphasic bottles. Former bottles processed in the authors' laboratory, 11 aerobic bottles belonged to 5 patients in whom brucelosis was confirmed by bloodculture. The Bact/Alert system detected Brucella melitensis in only on bottle at 2.9 days of incubation. In 7 bottles Bact/Alert detected B. melitensis by a blind pass of these bottles at 10 to 20 days of incubation. CONCLUSIONS: These results suggest that the Bact/Alert system does not totally solve the diagnosis of brucellosis. Blind passes of the bloodcultures are required.

Bacteriological Techniques↗