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Biomedical subjects

M Nadal

Publications and source records attributed to M Nadal.

At least 37 records · Page 2Linked to original sources
PubMed1999

Cloning, expression and chromosomal localization of a human testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.

6-Phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2) is a bifunctional enzyme responsible for the synthesis and breakdown of Fru-2,6-P2, a key metabolite in the regulation of glycolysis. Several genes encode distinct PFK-2/FBPase-2 isozymes that differ in their tissue distribution and enzyme regulation. In this paper, we present the isolation of a cDNA from a human testis cDNA library that encodes a PFK-2/FBPase-2 isozyme. Sequencing data show an open reading frame of 1407 nucleotides that codifies for a protein of 469 amino acids. This has a calculated molecular weight of 54kDa and 97% similarity with rat testis PFK-2/FBPase-2, with complete conservation of the amino acid residues involved in the catalytic mechanism. Fluorescence in-situ hybridization (FISH) localized testis PFK-2/FBPase-2 gene (PFKFB4) in human chromosome 3 at bands p21-p22. A Northern blot analysis of different rat tissues showed the presence of a 2.4-kb mRNA expressed specifically in testis. In mammalian COS-1 cells, the human testis cDNA drives expression of an isozyme with a molecular weight of 55kDa. This isozyme shows clear PFK-2 activity. Taken together, these results provide evidence for a new PFK-2/FBPase-2 gene coding for a human testis isozyme.

Amino Acid Sequence↗
PubMed1999

Molecular cloning, characterization, and chromosomal localization of the mouse homologue of CD84, a member of the CD2 family of cell surface molecules.

CD84 is a member of the immunoglobulin gene superfamily (IgSF) with two Ig-like domains expressed primarily on B lymphocytes and macrophages. Here we describe the cloning of the mouse homologue of human CD84. Mouse CD84 cDNA clones were isolated from a macrophage library. The nucleotide sequence of mouse CD84 was shown to include an open reading frame encoding a putative 329 amino acid protein composed of a 21 amino acid leader peptide, two extracellular immunoglobulin (Ig)-like domains, a hydrophobic transmembrane region, and an 87 amino acid cytoplasmic domain. Mouse CD84 shares 57.3% amino acid sequence identity (88.7%, considering conservative amino acid substitutions) with the human homologue. Chromosome localization studies mapped the mouse CD84 gene to distal chromosome 1 adjacent to the gene for Ly-9, placing it close to the region where other members of the CD2 IgSF (CD48 and 2B4) have been mapped. Northern blot analysis revealed that the expression of mouse CD84 was predominantly restricted to hematopoietic tissues. Two species of mRNA of 3.6 kilobases (kb) and 1.5 kb were observed. The finding that the pattern of expression was restricted to the hematopoietic system and the conserved sequence of the mouse CD84 homologue suggests that the function of the CD84 glycoprotein may be similar in humans and mice.

Amino Acid Sequence↗
PubMed1999

Enhanced pancreatic tumor regression by a combination of adenovirus and retrovirus-mediated delivery of the herpes simplex virus thymidine kinase gene.

We have evaluated the effectiveness of combining the different characteristics of retrovirus and adenovirus to apply the herpes simplex virus thymidine kinase gene (HSVtk) and ganciclovir (GCV) treatment for gene therapy of pancreatic cancer. Transduction of NP-18 human pancreatic cells in culture by either the adenoviral vector (ADV/tk) or the retroviral vector (Rv/tk) followed by GCV treatment resulted in a GCV dose-dependent cytotoxic effect. A bystander effect was determined, both in NP-18 cultures and in xenogeneic cell mixtures of NP-18 and PA317 cells. Studies in vivo indicated that the effectiveness of tumor regression after HSVtk gene transfer and GCV treatment was dependent first on the tumor size at the time of viral injection and secondly, in large tumors, on the type of virus administered. The administration of the viral combination (ADV/tk + vector producer cells VPC-Rv/tk) was the best approach tested and resulted in a dramatic reduction in tumor mass after 4 days of GCV treatment which was maintained for the treatment period. Remarkably, two animals presented a complete eradication of the tumor. Thus, the HSVtk/GCV system when administered using a viral combination (ADV/tk + VPC-Rv/tk), may be a promising suicide gene therapy for pancreatic carcinomas.

Adenocarcinoma↗
PubMed1999

Analysis of DNA cleavage by reverse gyrase from Sulfolobus shibatae B12.

Reverse gyrase is a type I-5' topoisomerase, which catalyzes a positive DNA supercoiling reaction in vitro. To ascertain how this reaction takes places, we looked at the DNA sequences recognized by reverse gyrase. We used linear DNA fragments of its preferred substrate, the viral SSV1 DNA, which has been shown to be positively supercoiled in vivo. The Sulfolobus shibatae B12 strain, an SSV1 virus host, was chosen for production of reverse gyrase. This naturally occurring system (SSV1 DNA-S. shibatae reverse gyrase) allowed us to determine which SSV1 DNA sequences are bound and cleaved by the enzyme with particularly high selectivity. We show that the presence of ATP decreases the number of cleaved complexes obtained whereas the non-hydrolyzable ATP analog adenosine 5'-[beta, gamma-imido]triphosphate increases it without changing the sequence specificity.

Adenosine Triphosphate↗
PubMed1999

Cloning, expression, and mapping of PDCD9, the human homolog of Gallus gallus pro-apoptotic protein p52.

We report the sequence, tissue distribution, and chromosome location of a novel gene, PDCD9 (programmed cell death 9). PDCD9 is the mammalian counterpart of the Gallus gallus pro-apoptotic protein p52. The human cDNA has an open reading frame of 1,314 nucleotides and was predicted to encode a protein of 438 amino acids with a calculated mass of 50 kDa. The protein sequences of chicken, mouse, and human PDCD9 are remarkably conserved. PDCD9 mRNA is expressed ubiquitously in adult tissues, displaying a stronger signal in heart, skeletal muscle, kidney, and liver. PDCD9 was mapped to chromosome 5q11.

Amino Acid Sequence↗
PubMed1998

Temperature, template topology, and factor requirements of archaeal transcription.

Although Archaea are prokaryotic and resemble Bacteria morphologically, their transcription apparatus is remarkably similar to those of eukaryotic cell nuclei. Because some Archaea exist in environments with temperatures of around 100 degreesC, they are likely to have evolved unique strategies for transcriptional control. Here, we investigate the effects of temperature and DNA template topology in a thermophilic archaeal transcription system. Significantly, and in marked contrast with characterized eucaryal systems, archaeal DNA template topology has negligible effect on transcription levels at physiological temperatures using highly purified polymerase and recombinant transcription factors. Furthermore, archaeal transcription does not require hydrolysis of the beta-gamma phosphoanhydride bond of ATP. However, at lower temperatures, negatively supercoiled templates are transcribed more highly than those that are positively supercoiled. Notably, the block to transcription on positively supercoiled templates at lowered temperatures is at the level of polymerase binding and promoter opening. These data imply that Archaea do not possess a functional homologue of transcription factor TFIIH, and that for the promoters studied, transcription is mediated by TATA box-binding protein, transcription factor TFB, and RNA polymerase alone. Furthermore, they suggest that the reduction of plasmid linking number by hyperthermophilic Archaea in vivo in response to cold shock is a mechanism to maintain gene expression under these adverse circumstances.

Archaea↗
PubMed1998

Nerve growth factor acutely reduces chemical transmission by means of postsynaptic TrkA-like receptors in squid giant synapse.

Tyrosine phosphorylation has been shown to be an important modulator of synaptic transmission in both vertebrates and invertebrates. Such findings hint toward the existence of extracellular ligands capable of activating this widely represented signaling mechanism at or close to the synapse. Examples of such ligands are the peptide growth factors which, on binding, activate receptor tyrosine kinases. To gain insight into the physiological consequences of receptor tyrosine kinase activation in squid giant synapse, a series of growth factors was tested in this preparation. Electrophysiological, pharmacological, and biochemical analysis demonstrated that nerve growth factor (NGF) triggers an acute and specific reduction of the postsynaptic potential amplitude, without affecting the presynaptic spike generation or presynaptic calcium current. The NGF target is localized at a postsynaptic site and involves a new TrkA-like receptor. The squid receptor crossreacts with antibodies generated against mammalian TrkA, is tyrosine phosphorylated in response to NGF stimulation, and is blocked by specific pharmacological inhibitors. The modulation described emphasizes the important role of growth factors on invertebrate synaptic transmission.

Animals↗
PubMed1998

Cerebral hemodynamic changes during sustained hypocapnia in severe head injury: can hyperventilation cause cerebral ischemia?

Hyperventilation (HV) is routinely used in the management of increased intracranial pressure (ICP) in severe head injury. However, this treatment continues to be controversial because it has been reported that long-lasting reduced cerebral blood flow (CBF) due to profound sustained hypocapnia may contribute to the development or deterioration of ischemic lesions. Our goal in this study was to analyze the effects of sustained hyperventilation on cerebral hemodynamics (CBF, ICP) and metabolism (arterio jugular differences of lactates = AVDL). CO2-reactivity and CBF was estimated using AVDO2 (arteriojugular differences of oxygen content). Global cerebral ischemia and increased anaerobic metabolism were considered according to AVDO2 and AVDL respectively. Thirty-three patients with severe and moderate head injury and increased ICP were included. Within 72 hours after accident, patients were hyperventilated for a period of 4 hours. During this time jugular oxygen saturation (SjO2), arterial oxygen saturation (SaO2), ICP, mean arterial blood pressure (MABP), AVDO2 and AVDL were recorded. In our study, most patients preserved CO2-reactivity (88.2%). In these cases HV was very effective in lowering ICP. Our findings showed that this reduction was due to a CBF decrease. According to basal AVDO2 twenty-five patients (75.7%) were considered as hyperemic and eight (24.2%) as not hyperemic. Global ischemia and increased anaerobic metabolism were detected in one case in the non-hyperemic group. According to AVDO2 and AVDL, no adverse effects were found during four hours of HV in hyperemic patients. Nevertheless, AVDO2 and AVDL are global measurements and might not detect regional ischemia surrounding focal lesions such as contusions and haematomas. We suggest that monitoring of AVDO2 or other haemometabolic variables should be mandatory when sustained HV is used in the management of head injury patients.

Adolescent↗
PubMed1998

A patient with autistic disorder and a 20/22 chromosomal translocation.

The case history of a 3-year-old boy without speech and who met 10 criteria of an autistic condition (DSM-IV) (American Psychiatric Association 1994) is reported. Psychometric evaluation, excluding the verbal scale, resulted in an IQ score of 56. The cytogenetic study showed a 20/22 translocation and an interstitial deletion within the region 22q11: 45,XY, -22, +der(20), t(20;22) (q13.3;q11.2), which was confirmed by fluorescence in situ hybridisation (FISH). Although deletions at 22q11 are responsible for the DiGeorge syndrome; clinical, metabolic, and neurological image studies of the patient were inconsistent with this syndrome. In the clinical examination the patient presented with a mildly dysmorphic facies, pectus excavatum, and a short thumb. A 99mTc HMPAO brain perfusion SPECT showed a hypoperfusion of the left temporoparietal cortex. As there have been no previous reports of autistic patients with abnormalities involving both chromosomes 20 and 22, these findings merit some discussion either as a possible cause of autism or as accompanying factors.

Autistic Disorder↗
PubMed1998

Role of UEV-1, an inactive variant of the E2 ubiquitin-conjugating enzymes, in in vitro differentiation and cell cycle behavior of HT-29-M6 intestinal mucosecretory cells.

By means of differential RNA display, we have isolated a cDNA corresponding to transcripts that are down-regulated upon differentiation of the goblet cell-like HT-29-M6 human colon carcinoma cell line. These transcripts encode proteins originally identified as CROC-1 on the basis of their capacity to activate transcription of c-fos. We show that these proteins are similar in sequence, and in predicted secondary and tertiary structure, to the ubiquitin-conjugating enzymes, also known as E2. Despite the similarities, these proteins lack a critical cysteine residue essential for the catalytic activity of E2 enzymes and, in vitro, they do not conjugate or transfer ubiquitin to protein substrates. These proteins constitute a distinct subfamily within the E2 protein family and are highly conserved in phylogeny from yeasts to mammals. Therefore, we have designated them UEV (ubiquitin-conjugating E2 enzyme variant) proteins, defined as proteins similar in sequence and structure to the E2 ubiquitin-conjugating enzymes but lacking their enzymatic activity (HW/GDB-approved gene symbol, UBE2V). At least two human genes code for UEV proteins, and one of them, located on chromosome 20q13.2, is expressed as at least four isoforms, generated by alternative splicing. All human cell types analyzed expressed at least one of these isoforms. Constitutive expression of exogenous human UEV in HT-29-M6 cells inhibited their capacity to differentiate upon confluence and caused both the entry of a larger proportion of cells in the division cycle and an accumulation in G2-M. This was accompanied with a profound inhibition of the mitotic kinase, cdk1. These results suggest that UEV proteins are involved in the control of differentiation and could exert their effects by altering cell cycle distribution.

Amino Acid Sequence↗
PubMed1998

Molecular cloning, expression, and chromosomal localization of a ubiquitously expressed human 6-phosphofructo-2-kinase/ fructose-2, 6-bisphosphatase gene (PFKFB3).

We report the identification of a human 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase gene (PFKFB3) isolated from a human fetal brain cDNA library. The gene was localized to 10p15-->p14 by fluorescence in situ hybridization. The entire cDNA (4,322 bp) codes for a polypeptide of 520 amino acid residues (molecular weight, 59.571 kDa). Structural analysis showed the presence of a kinase domain located at the amino terminus and a bisphosphatase domain at the carboxy terminus, characteristic of previously described 6-phosphofructo-2-kinase/fructose 2, 6-bisphosphatase isozymes. In addition, a phosphorylation site for cAMP-dependent protein kinase was found at the carboxy terminus. Northern blot analysis showed the presence of a unique 4.8-kb mRNA expressed in the different tissues studied. In mammalian COS-1 cells, this cDNA drives the expression of an active isozyme. Taken together, these results identify the presence of a gene coding for a human 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase isozyme which is ubiquitously expressed.

Animals↗
PubMed1997

High-titer retroviral vectors containing the enhanced green fluorescent protein gene for efficient expression in hematopoietic cells.

Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. We have developed a producer cell line that yields high titers of amphotropic retroviral vectors carrying the enhanced green fluorescent protein (EGFP) gene, a codon humanized, red-shifted variant of the green fluorescent protein (GFP) gene, which can be used as a selectable marker. We have used a hybrid vector that has been shown to efficiently drive gene expression in hematopoietic cells. Virtually all murine and human cell lines and primary human hematopoietic cells tested were transduced with varying efficiency after incubation with vector-containing supernatants. Human CD34(+) cells obtained from cord blood or aphereses products were transduced using a protocol that involves daily addition of vector-containing supernatants for 6 consecutive days. At day 6, up to 16% of the cells expressed EGFP, as assessed by flow cytometry. Sorted EGFP-expressing cells were able to produce fluorescent hematopoietic colonies. EGFP's main advantages are its fast flow cytometry determination and the possibility of cell sorting and simultaneous evaluation of the transduction efficiency along with other phenotypic markers.

3T3 Cells↗
PubMed1997

CD84 leukocyte antigen is a new member of the Ig superfamily.

cDNA isolated from a human B-cell line Raji library was analyzed and shown to encode the full-length cDNA sequence of a novel cell-surface glycoprotein, initially termed HLy9-beta. The predicted mature 307-amino acid protein was composed of two extracellular Ig-like domains, a hydrophobic transmembrane region, and an 83-amino acid cytoplasmic domain. The extracellular Ig-like domains presented structural and sequence homology with a group of members of the Ig superfamily that included CD2, CD48, CD58, and Ly9. Northern blot analysis showed that the expression of HLy9-beta was predominantly restricted to hematopoietic tissues. Chromosome localization studies mapped the HLy9-beta gene to chromosome 1q24, where other members of this Ig superfamily (CD48 and HumLy9) have been mapped. CD84 monoclonal antibodies (MoAbs) were shown to react with cells transfected with the cloned cDNA. These MoAbs were further used to show that CD84 is expressed as a single chain cell-surface glycoprotein of Mr 64,000 to 82,000, which was highly glycosylated. CD84 had a unique pattern of expression, being found predominantly on lymphocytes and monocytes. Thus, the glycoprotein HLy9-beta is recognized by MoAbs previously clustered as CD84 and represents a newly identified member of the Ig superfamily that may play a significant role in leukocyte activation.

Amino Acid Sequence↗
PubMed1997

Hin-mediated inversion on positively supercoiled DNA.

Hin recombinase requires negatively supercoiled DNA for an efficient inversion. We have generated positively supercoiled plasmid DNA using reverse gyrase from Sulfolobus shibatae and subjected it to the Hin-mediated inversion reaction. Both Hin and Fis showed the same DNA binding activity regardless of the superhelical handedness of the substrate plasmid. However, inversion activity on positively supercoiled DNA was less than 1% of negatively supercoiled DNA. Assays designed to probe steps in inversion, showed that on positively supercoiled DNA, Hin was able to cleave the recombination sites with the same efficiency shown on negatively supercoiled DNA but was not able to exchange the cleaved DNA. Based on the theoretical differences between positive and negative supercoiling, our data may suggest that unwinding of the double helix at recombination sites is needed after DNA cleavage for strand exchange to occur.

Binding Sites↗
PubMed1997

Down syndrome: characterisation of a case with partial trisomy of chromosome 21 owing to a paternal balanced translocation (15;21) (q26;q22.1) by FISH.

A patient with a typical Down syndrome (DS) phenotype and a normal karyotype was studied by FISH. Using painting probes, we found that the patient had partial trisomy of chromosome 21 owing to an unbalanced translocation t(15;21) (q26; q22.1) of paternal origin. To correlate genotype with phenotype as accurately as possible, we localised the breakpoint using a contig of YACs from the long arm of chromosome 21 as probes and performed FISH. We ended up with two YACs, the most telomeric giving signal on the der (15) in addition to signal on the normal chromosome 21 and the most centromeric giving signal only on both normal chromosomes 21. From these results we could conclude that the breakpoint must be located within the region encompassing YACs 280B1 and 814C1, most likely near one end of either YAC or between them, since neither YAC814C1 nor 280B1 crossed the breakpoint (most likely between marker D21S304 and marker D21S302) onband 21q22.1. The same study was performed on the chromosomes of the father and of a sister and a brother of the patient; all three carried a balanced translocation between chromosomes 15 and 21 and had a normal phenotype. We also performed a prenatal study using FISH for the sister. The fetus was also a carrier of the balanced translocation.

Adult↗
PubMed1997

Minibrain (MNBH) is a single copy gene mapping to human chromosome 21q22.2.

We have identified a human homologue of the Drosophila mnb gene (MNBH) on chromosome 21, while another study mapped an EST clone (R38268) with similarity to minibrain to human chromosome 1. This report describes the mapping of MNBH to a single locus on human chromosome 21q22.2 by FISH and Southern blotting. Comparison of the similarities between the two sequences and mnb demonstrates that MNBH on chromosome 21 is the true human homologue of Drosophila mnb.

Amino Acid Sequence↗