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Biomedical subjects

D Alexandre

Publications and source records attributed to D Alexandre.

At least 19 recordsLinked to original sources
PubMed2001

Genomic structure, chromosomal localization and expression profile of a novel melanoma differentiation associated (mda-7) gene with cancer specific growth suppressing and apoptosis inducing properties.

Abnormalities in cellular differentiation are frequent occurrences in human cancers. Treatment of human melanoma cells with recombinant fibroblast interferon (IFN-beta) and the protein kinase C activator mezerein (MEZ) results in an irreversible loss in growth potential, suppression of tumorigenic properties and induction of terminal cell differentiation. Subtraction hybridization identified melanoma differentiation associated gene-7 (mda-7), as a gene induced during these physiological changes in human melanoma cells. Ectopic expression of mda-7 by means of a replication defective adenovirus results in growth suppression and induction of apoptosis in a broad spectrum of additional cancers, including melanoma, glioblastoma multiforme, osteosarcoma and carcinomas of the breast, cervix, colon, lung, nasopharynx and prostate. In contrast, no apparent harmful effects occur when mda-7 is expressed in normal epithelial or fibroblast cells. Human clones of mda-7 were isolated and its organization resolved in terms of intron/exon structure and chromosomal localization. Hu-mda-7 encompasses seven exons and six introns and encodes a protein with a predicted size of 23.8 kDa, consisting of 206 amino acids. Hu-mda-7 mRNA is stably expressed in the thymus, spleen and peripheral blood leukocytes. De novo mda-7 mRNA expression is also detected in human melanocytes and expression is inducible in cells of melanocyte/melanoma lineage and in certain normal and cancer cell types following treatment with a combination of IFN-beta plus MEZ. Mda-7 expression is also induced during megakaryocyte differentiation induced in human hematopoietic cells by treatment with TPA (12-O-tetradecanoyl phorbol-13-acetate). In contrast, de novo expression of mda-7 is not detected nor is it inducible by IFN-beta+MEZ in a spectrum of additional normal and cancer cells. No correlation was observed between induction of mda-7 mRNA expression and growth suppression following treatment with IFN-beta+MEZ and induction of endogenous mda-7 mRNA by combination treatment did not result in significant intracellular MDA-7 protein. Radiation hybrid mapping assigned the mda-7 gene to human chromosome 1q, at 1q 32.2 to 1q41, an area containing a cluster of genes associated with the IL-10 family of cytokines. Mda-7 represents a differentiation, growth and apoptosis associated gene with potential utility for the gene-based therapy of diverse human cancers.

Antigens, Neoplasm↗
PubMed2001

Pituitary adenylate cyclase-activating polypeptide and its receptors in amphibians.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide of the secretin/glucagon/vasoactive intestinal polypeptide superfamily, has been initially characterized in mammals in 1989 and, only 2 years later, its counterpart has been isolated in amphibians. A number of studies conducted in the frog Rana ridibunda have demonstrated that PACAP is widely distributed in the central nervous system (particularly in the hypothalamus and the median eminence) and in peripheral organs including the adrenal gland. The cDNAs encoding the PACAP precursor and 3 types of PACAP receptors have been cloned in amphibians and their distribution has been determined by in situ hybridization histochemistry. Ontogenetic studies have revealed that PACAP is expressed early in the brain of tadpoles, soon after hatching. In the frog Rana ridibunda, PACAP exerts a large array of biological effects in the brain, pituitary, adrenal gland, and ovary, suggesting that, in amphibians as in mammals, PACAP may act as neurotrophic factor, a neurotransmitter and a neurohormone.

Adrenal Glands↗
PubMed2001

Pituitary adenylate cyclase-activating polypeptide stimulates secretoneurin release and secretogranin II gene transcription in bovine adrenochromaffin cells through multiple signaling pathways and increased binding of pre-existing activator protein-1-like transcription factors.

Secretoneurin (SN) is a novel bioactive peptide that derives from the neuroendocrine protein secretogranin II (SgII) by proteolytic processing and participates in neuro-immune communication. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP-38) dose-dependently stimulates (EC(50) approximately 3 nM) SN release (up to 4-fold) and SgII gene expression (up to 60-fold) in cultured bovine adrenochromaffin cells. The effect of PACAP on both SN secretion and SgII mRNA levels is rapid and long lasting. We analyzed in this neuroendocrine cell model the transduction pathways involved in both SN secretion and SgII gene transcription in response to PACAP. The cytosolic calcium chelator BAPTA-AM and the nonselective calcium channel antagonist NiCl(2) equally inhibited both secretion of the peptide and transcription of the SgII gene, indicating a major contribution of calcium influx in PACAP-induced SN biosynthesis and release in chromaffin cells. Inhibition of protein kinase A (PKA) or C (PKC) also reduced PACAP-evoked SN release but did not alter the stimulatory effect of PACAP on SgII mRNA levels. Conversely, application of mitogen-activated protein kinase inhibitors suppressed PACAP-induced SgII gene expression. The effect of PACAP on SgII mRNA levels, like the effect of the PKC stimulator 12-O-tetradecanoylphorbol-13-acetate (TPA), was not affected by cycloheximide, whereas the effects of the PKA stimulator forskolin or cell-depolarization by high K(+) were significantly reduced by the protein synthesis inhibitor. PACAP and TPA both increased the binding activity of the SgII cAMP response element to trans-acting factors present in chromaffin cell nuclear extracts, which are recognized by antibodies to activator protein-1-related proteins. These data indicate that SN biosynthesis is regulated by PACAP in chromaffin cells through complex signaling cascades, suggesting that SN may play a function during trans-synaptic stimulation of the adrenal medulla.

Animals↗
PubMed2001

Structural and functional identification of the pituitary adenylate cyclase-activating polypeptide receptor VPAC2 from the frog Rana tigrina rugulosa.

Recently, a frog pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) receptor (fPVR) has been characterized, and interestingly, this receptor exhibits characteristics of both mammalian PACAP type II receptors VPAC(1)R and VPAC(2)R. In order to investigate the receptors responsible for mediating the actions of VIP and PACAP in amphibians, in this report, a frog VPAC(2) receptor (fVPAC(2)R) cDNA was isolated. fVPAC(2)R shares 47.7, 46.9 and 62.5% amino acid sequence identity with fPVR, human VPAC(1)R and human VPAC(2)R respectively. Functionally, fVPAC(2)R, when expressed in CHO cells, was responsive to both frog peptides including VIP, PACAP38 and PACAP27 where the EC(50) values of these peptides in intracellular cAMP production were 0.15, 0.18 and 0.16 microM respectively. The pharmacological profiles of human peptides (VIP, PACAP38 and peptide histidine methionine) to stimulate frog and human VPAC(2)Rs were compared, and it was found that these peptides could only activate the frog receptor at micromolar concentrations. fVPAC(2)R was found to be widely distributed in various peripheral tissues as well as several regions of the brain. The presence of the receptor transcripts suggests the functional roles of the receptor in mediating the actions of PACAP and/or VIP in these tissues. As VIP and particularly PACAP27 are highly conserved peptides in vertebrate evolution, comparative studies of these peptides and their receptors in non-mammalian vertebrates should provide clues to better understand the physiology of these important peptides in human and other vertebrates.

Amino Acid Sequence↗
PubMed2000

RaSH, a rapid subtraction hybridization approach for identifying and cloning differentially expressed genes.

Human melanoma cells growth-arrest irreversibly and terminally differentiate on treatment with a combination of fibroblast interferon and the protein kinase C activator mezerein. This experimental protocol also results in a loss of tumorigenic potential and profound changes in gene expression. Various cloning and cDNA microarray strategies are being used to determine the complete spectrum of gene expression changes underlying these alterations in human melanoma cells. An efficient approach, Rapid Subtraction Hybridization (RaSH), has been developed that is permitting the identification of genes of potential relevance to cancer growth control and terminal cell differentiation. RaSH cDNA libraries are prepared from double-stranded cDNAs that are enzymatically digested into small fragments, ligated to adapters, and PCR amplified followed by incubation of tester and driver PCR fragments. This subtraction hybridization scheme is technically simple and results in the identification of a high proportion of differentially expressed sequences, including known genes and those not described in current DNA databases. The RaSH approach represents an efficient methodology for identifying and cloning genes displaying differential expression that associate with and potentially regulate complex biological processes.

Base Sequence↗
PubMed2000

Structure and distribution of the mRNAs encoding pituitary adenylate cyclase-activating polypeptide and growth hormone-releasing hormone-like peptide in the frog, Rana ridibunda.

The structure of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been characterized in several species including protochordates, fish, amphibians, birds, and mammals. Although PACAP has been shown to stimulate frog pituitary and adrenal cell activity, the structure of the PACAP precursor and the expression of its gene have not yet been reported in any amphibian species. In this study, we have characterized two cDNA variants encoding PACAP of the frog Rana ridibunda, one of which encodes a second peptide exhibiting strong homologies to growth hormone-releasing hormone (GHRH) of fish and mammals. Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that PACAP/GHRH-like peptide mRNAs are predominantly expressed in the brain and spinal cord and, to a lesser extent, in the neurointermediate lobe of the pituitary. Other tissues including the testis and the distal lobe of the pituitary do not express the PACAP precursor gene. The distribution of PACAP/GHRH-like peptide mRNAs in the frog brain has been determined by in situ hybridization histochemistry. High levels of expression were found in the accessory olfactory bulb, the distal pallium, the ventral part of the magnocellular preoptic nucleus, the ventral hypothalamic nucleus, the posterior tuberculum, and the ventral habenular nucleus. These data contribute to the understanding of the evolution of the PACAP and GHRH genes in vertebrates and provide the anatomical bases to elucidate the roles of PACAP and the GHRH-like peptide in amphibians.

Amino Acid Sequence↗
PubMed1999

Somatotopy of the lateral line projection in larval zebrafish.

We examined the topography of the lateral line primary projection in zebrafish larvae by double labeling. The projections of two identified neuromasts of the posterior lateral line are seen as two separate sets of fibers that show reproducible spatial relationships: the projection of the anterior neuromast is always ventrolateral to that of a more posteriorly located neuromast. The same rule applies to the projection of anterior lateral line neuromasts. The position of the neuromasts along the antero posterior axis of the fish therefore is represented in the central projection of the sensory neurons. This somatotopy is similar to, and may be at the origin of, the tonotopic projection of the cochlear hair cells in mammals.

Animals↗
PubMed1999

A cloned frog vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide receptor exhibits pharmacological and tissue distribution characteristics of both VPAC1 and VPAC2 receptors in mammals.

Three receptor subtypes for the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been identified in mammals: the PAC1 receptor (PAC1-R) which is selectively activated by PACAP, and two VPAC receptors (VPAC1-R and VPAC2-R), which are equally stimulated by PACAP and VIP. The structures of PACAP and VIP have been well conserved during evolution, but little is known about VIP/PACAP receptors in nonmammalian species. An amphibian VIP/PACAP receptor complementary DNA (cDNA) has been cloned and characterized from a frog (Rana ridibunda) pituitary cDNA library. The predicted protein contains seven putative transmembrane domains and exhibits the highest sequence identity (65%) with the human VPAC1-R. The cloned cDNA was transiently expressed in LLC-PK1 cells, and its pharmacological profile was determined in comparison with the human VPAC1-R. Both PACAP and VIP stimulated cAMP accumulation through the cloned receptor with an EC50 of about 30 nM. In contrast, secretin, at concentrations that stimulate the human VPAC1-R, had no effect on cAMP production. RT-PCR analysis revealed the widespread distribution of this frog VIP/PACAP receptor in peripheral tissues. In situ hybridization histochemistry using a complementary RNA probe showed that the receptor gene is highly expressed in several hypothalamic and thalamic nuclei and to a lesser extent in the pallium and striatum. In the pituitary, the highest messenger RNA levels were detected in the distal lobe. Taken together, these data show that the cloned frog receptor shares several common features with both the VPAC1-R and VPAC2-R of mammals; the frog receptor exhibits the highest sequence identity with mammalian VPAC1-R, but the lack of effect of secretin and the brain distribution of the receptor are reminiscent of the characteristics of the mammalian VPAC2-R. The sequence of the frog receptor should thus prove useful to decipher the structure-activity relationships of the VIP/PACAP receptor family.

Adenylyl Cyclases↗
PubMed1996

Molecular cloning of frog secretogranin II reveals the occurrence of several highly conserved potential regulatory peptides.

Secretogranin II (SgII) is an acidic secretory protein present in large dense core vesicles of neuronal and endocrine cells. Based on the sequence of a peptide derived from the processing of SgII in the brain of the frog Rana ridibunda, degenerate oligonucleotides were used to clone the cDNA encoding frog SgII from a pituitary cDNA library. This cDNA encodes a 574 amino acid protein which exhibits 46-48% sequence identity with mammalian SgII and contains 11 pairs of basic amino acids. Four potential processing products delimited by pairs of basic residues exhibited a much higher degree of identity (68-82%) with the corresponding mammalian SgII sequences. The frog SgII mRNA is approximately 4 kb in length and is differentially expressed in the brain and endocrine tissues. The present data reveal that several SgII-derived peptides have been highly conserved during evolution, suggesting that these peptides may play important neuroendocrine regulatory functions.

Amino Acid Sequence↗
PubMed1996

Ectopic expression of Hoxa-1 in the zebrafish alters the fate of the mandibular arch neural crest and phenocopies a retinoic acid-induced phenotype.

Considerable evidence has demonstrated that retinoic acid influences the formation of the primary body axis in vertebrates and that this may occur through the regulation of Hox gene expression. In this study, we show that the phenotype induced by exogenous retinoic acid in the zebrafish can also be generated by the overexpression of Hoxa-1 following injection of synthetic RNA into the fertilised egg. The isolation, sequence and expression pattern of the zebrafish Hoxa-1 gene is described. We show that exogenously applied retinoic acid causes the ectopic accumulation of Hoxa-1 message during gastrulation in the hypoblast in the head region. Overexpression of Hoxa-1 following injection of RNA causes abnormal growth of the anterior hindbrain, duplication of Mauthner neurons in rhombomere (r) 2 and fate changes of r2 mesenchymal and neurogenic neural crest. These results are discussed in terms of the role of Hoxa-1 in controlling anterior hindbrain patterning and the relationship between expression of Hoxa-1 and retinoic acid.

Amino Acid Sequence↗
PubMed1995

gamma delta lineage-specific transcription of human T cell receptor gamma genes by a combination of a non-lineage-specific enhancer and silencers.

The expression of the T cell receptor (TcR) gamma genes is restricted to TcR gamma delta + T lymphocytes. Transgenic and somatic cell hybrid experiments had suggested that the expression of a functionally rearranged TcR gamma gene was extinguished in TcR alpha beta + T cells, possibly by putative cis-acting transcriptional silencers. We have identified such negative cis-acting sequences in the 3' non-coding region of the human TcR gamma (TRG) locus, upstream of an enhancer located at 6.5 kb of the TcR C gamma 2 gene (TRGC2). These silencers were capable of repressing the transcription from a minimal heterologous promoter in a position- and orientation-independent fashion. When analyzed individually, the silencers and the enhancer were equally active in the TcR alpha beta + and TcR gamma delta + T cell lines studied. In contrast, the association of the enhancer with either silencer was shown to restrict transcription to the TcR gamma delta + T cell lines.

Base Sequence↗
PubMed1992

The human gamma/delta + and alpha/beta + T cells: a branched pathway of differentiation.

In human peripheral blood, most of the CD3+ cells express the alpha/beta T cell receptor. A smaller fraction of CD3+ T cells express the gamma/delta T cell receptor (from 1 to 10% depending the individuals, with an average of 3-5%). Interestingly, although the alpha/beta + T cells never express the gamma chain at the cell surface, most of them (about 98%) rearrange the gamma locus on both alleles, the remaining 2% alpha/beta + T cells have one rearranged TRG locus. We previously proposed that V-J joinings in the human TRG locus occurred sequentially and we recently demonstrated that two successive rearrangements may occur on the same chromosome [Alexandre et al. (Int. Immunol, 3, 973-982, 1991)]. In this paper, we discuss the implications of these sequential rearrangements on the relatedness of the human gamma/delta + and alpha/beta + T cell lineages.

Cell Differentiation↗
PubMed1991

Reciprocal hybrid joints demonstrate successive V-J rearrangements on the same chromosome in the human TCR gamma locus.

Novel variable (V)--joining (J) gene rearrangements are described in the human T cell receptor gamma locus, in which, on the one hand, the V3 variable gene is joined to the heptamer--nonamer recombination signals of the J1 segment and, on the other hand, the J1 segment is joined to the V3 recombination signals through head-to-head fusion. These recombination products, or hybrid joints, have been originated through an inversion of 47 kb DNA. Interestingly the inverted DNA stretch contains a normal V9-J9 rearrangement. These findings are the first direct demonstration that successive rearrangements occur, on the same chromosome, in the human T cell receptor gamma locus, and suggest that the chronology of the joining events plays a role in the ontogeny of T cells and their differentiation in gamma/delta + and alpha/beta + lineages.

Amino Acid Sequence↗