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Biomedical subjects

R Roubin

Publications and source records attributed to R Roubin.

At least 19 recordsLinked to original sources

let-756, a C. elegans fgf essential for worm development.

In vertebrates, Fibroblast Growth Factors (FGFs) and their receptors are involved in various developmental and pathological processes, including neoplasia. The number of FGFs and their large range of activities have made the understanding of their precise functions difficult. Investigating their biology in other species might be enlightening. A sequence encoding a putative protein presenting 30-40% identity with the conserved core of vertebrate FGFs has been identified by the C. elegans sequencing consortium. We show here that this gene is transcribed and encodes a putative protein of 425 amino acids (aa). The gene is expressed at all stages of development beyond late embryogenesis, peaking at the larval stages. Loss-of-function mutants of the let-756 gene are rescued by the wild type fgf gene in germline transformation experiments. Two partial loss-of-function alleles, s2613 and s2809, have a mutation that replaces aa 317 by a stop. The truncated protein retains the FGF core but lacks a C-termins portion. These worms are small and develop slowly into clear and scrawny, yet viable and fertile adults. A third allele, s2887, is inactivated by an inversion that disrupts the first exon. It causes a developmental arrest early in the larval stages. Thus, in contrast to the other nematode fgf gene egl-17, let-756/fgf is essential for worm development.

Amino Acid Sequence↗

The family of Caenorhabditis elegans tyrosine kinase receptors: similarities and differences with mammalian receptors.

Transmembrane receptors with tyrosine kinase activity (RTK) constitute a superfamily of proteins present in all metazoans that is associated with the control and regulation of cellular processes. They have been the focus of numerous studies and are a good subject for comparative analyses of multigene families in different species aimed at understanding metazoan evolution. The sequence of the genome of the nematode worm Caenorhabditis elegans is available. This offers a good opportunity to study the superfamily of nematode RTKs in its entirety and to compare it with its mammalian counterpart. We show that the C. elegans RTKs constitute various groups with different phylogenetic relationships with mammalian RTKs. A group of four RTKs show structural similarity with the three mammalian receptors for the vascular endothelial growth factors. Another group comprises RTKs with a short extracellular region, a feature not known in mammals; the genes encoding these RTKs are clustered on chromosome II with other gene families, including genes encoding chitinase-like proteins. Most of the C. elegans RTKs have no direct orthologous relationship with any mammalian RTK, providing an illustration of the importance of the separate evolution of the different phyla.

Animals↗

Of worms and men: an evolutionary perspective on the fibroblast growth factor (FGF) and FGF receptor families.

FGFs (fibroblast growth factors) play major roles in a number of developmental processes. Recent studies of several human disorders, and concurrent analysis of gene knock-out and properties of the corresponding recombinant proteins have shown that FGFs and their receptors are prominently involved in the development of the skeletal system in mammals. We have compared the sequences of the nine known mammalian FGFs, FGFs from other vertebrates, and three additional sequences that we extracted from existing databases: two human FGF sequences that we tentatively designated FGF10 and FGF11, and an FGF sequence from Caenorhabditis elegans. Similarly, we have compared the sequences of the four FGF receptor paralogs found in chordates with four non-chordate FGF receptors, including one recently identified in C. elegans. The comparison of FGF and FGF receptor sequences in vertebrates and nonvertebrates shows that the FGF and FGF receptor families have evolved through phases of gene duplications, one of which may have coincided with the emergence of vertebrates, in relation with their new system of body scaffold.

Amino Acid Sequence↗

Structure and developmental expression of mouse Garp, a gene encoding a new leucine-rich repeat-containing protein.

Proteins with leucine-rich repeats (LRR) constitute a large family of molecules playing a role in protein-protein interactions and signal transduction. They are involved in various cellular processes in different species. We characterized the organization and pattern of expression of the mouse Garp gene. It is composed of two coding exons, expressed as a major 4.3 kb mRNA, and encodes a putative LRR transmembrane protein with an extracellular region almost entirely made of 20 repeats, and a short intracytoplasmic region. The mouse GARP deduced amino-acid sequence is highly similar to that of the human protein. The Garp gene is expressed in various areas in the mid-gestation developing embryo, including skin, lens fibre cells, nasal cavity, smooth and skeletal muscles, lung, and megakaryocytes of the fetal liver. In the adult it is expressed in the megakaryocytes of the spleen and in endothelial cells of the placenta. The data suggests that GARP might be involved in platelet-endothelium interactions.

Aging↗

Human beta-globin gene polymorphisms characterized in DNA extracted from ancient bones 12,000 years old.

Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the beta-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for beta-globin frameworks by sequencing through two variable positions and for a polymorphic (AT) chi (T) gamma microsatellite 500 bp upstream of the beta-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible.

Archaeology↗

The basic domain of the lentiviral Tat protein is responsible for damages in mouse brain: involvement of cytokines.

The HIV and visna lentiviruses induce an inflammatory reaction in the central nervous system (CNS) of the infected hosts leading to dysmyelination, demyelination, and neuronal loss. The basic domain of the transactivating Tat protein has been involved in CNS damage. Infusion of basic containing domain Tat peptides in the lateral ventricle (systemic injection) or in the grey matter, i.e., hippocampus and thalamus (local injection), induced an inflammatory process characterized by the formation of an edema and invasion of macrophage accompanied by reactive astrogliosis. Control peptides originating from either lentiviral proteins or irrelevant protein as ovalbumin did not lead to any inflammatory reaction or cell death. The inflammation led to the loss of ependymal cells in the lateral ventricles and neurons in the grey matter. RNA extracted from the Tat-injected hemisphere reacted with TNF-alpha, IL-1 alpha and beta, and IL-6 probes. The macrophage/microglia inducible nitric oxyde synthase was also expressed. Blockade of TNF-alpha by a pentoxifylline treatment led to the decrease of IL-1 and iNOS expression accompanied by a reduction of the volume of the lesions indicating that the Tat-induced lesions might be mediated by TNF production.

Amino Acid Oxidoreductases↗

Transcriptional activation of the NCAM gene during myogenic differentiation.

Earlier work has shown that in vitro myogenesis of muscle cells is accompanied by important changes in the expression of NCAM mRNAs and proteins, consisting mainly in an upregulation of the smaller transcripts and the 120- to 125-kD NCAM isoforms they encode. By measuring run-on transcription rates in nuclei isolated from C2 myoblasts and myotubes, we show in this report that transcriptional control can account for the increase in NCAM mRNA levels during myogenesis. Hence, the quantitative changes in overall abundance of NCAM transcripts and the concomitant qualitative changes in the mRNA pattern are regulated by independent mechanisms. Moreover, as transcriptional activity was stimulated to similar extents in the 5' and 3' region of the gene, we can exclude premature transcription termination as a mechanism involved in the preferential synthesis of shorter transcripts. Further support for transcriptional regulation of the NCAM gene is provided by experiments showing increased NCAM promoter activity in differentiating cells. As this increase was observed with 4.5 kb but not with 426 bp of upstream sequence, it seems to involve one or more upstream regulatory elements.

Animals↗

Evaluation of the effect of suramin on neural cell growth and N-CAM expression.

Suramin, a polysulfonated naphthylurea, is currently under investigation for treatment of advanced malignancy and has been shown to exhibit antiproliferative effects on some cells. We investigated its action on two cell lines of neural origin, one with neuronal (N2A) and the other with glial (C6) phenotype, as well as on brain primary cultures. We showed that suramin completely inhibited astrocytoma proliferation for an optimal dose of 1000 micrograms/ml but had the opposite effect on neuroblastoma cells. For these cells, doses as low as 12.5 micrograms/ml first increased cell proliferation and then led to massive cell death. This cytotoxic effect, which could be compatible with an internalization of the drug by the cells, was also observed for postmitotic neurons in brain primary cultures. In both cell lines, suramin was responsible for an accumulation of the neural cell adhesion molecule at the cell surface. One of the causes was the inhibition by suramin on the liberation processes of the phosphatidylinositol anchored Mr 120,000 isoform. At the mRNA level, suramin (12.5 to 50 micrograms/ml) induced an increase of all neural cell adhesion molecule transcripts in N2A but not in C6 cells. Suramin did not have an overall effect on transcription rates or RNA stability as the levels of transcripts coding for PrPc, another cell surface molecule, and actin were not affected. Our data demonstrated pleiotropic action of suramin. The neurotoxic effect exerted on neurons needs to be considered as possible outcomes for the use of suramin in humans.

Animals↗

Modulation of NCAM expression by transforming growth factor-beta, serum, and autocrine factors.

The expression of NCAM (neural cell adhesion molecule) is precisely regulated in terms of cell type specificity and developmental control. We searched for extracellular factors that may be involved in this regulation using N2A neuroblastoma and NIH 3T3 fibroblastic cells. Factors contained in FBS promoted a two- to threefold increase in NCAM protein and mRNA abundance in both cell lines. This increase in NCAM expression in high serum could be entirely attributed to enhanced levels of the NCAM-140 message. Modulation of NCAM synthesis via an autocrine mechanism is suggested by the observation that medium conditioned by N2A cells stimulated NCAM mRNA expression by 3T3 and N2A cells. Among the pure factors tested, transforming growth factor-beta (TGF beta) was found to act as an inducer of NCAM expression in 3T3 but not in N2A cells. 3T3 cells responded to exposure to TGF beta with a two- to threefold increase in NCAM protein and mRNA. Exposure of early-passage embryonic cells to TGF beta resulted in four- and twofold increases in NCAM protein and mRNA abundance, respectively, suggesting a role for TGF beta in modulating NCAM expression in the embryo. TGF beta seems to act by stimulating the transcriptional activity of the NCAM gene because it did not affect transcript stability and stimulated transcription from a proximal promoter element of the NCAM gene.

Animals↗

Biosynthesis of paf-acether. Paf-acether but not leukotriene C4 production is impaired in cultured macrophages.

After adherence for 24 or 48 h mouse peritoneal macrophages, upon a zymosan challenge, synthesized 114 +/- 55 and 82 +/- 31 pmol of paf-acether (paf)/mg of protein respectively, as compared with 513 +/- 195 pmol of paf/mg of protein in 2 h-adherent macrophages (means +/- S.D., n = 10). By contrast, 24 h- and 48 h-adherent macrophages exposed to zymosan produced more leukotriene C4 (2.7 +/- 1.1 and 1.4 +/- 0.2 nmol/mg of protein respectively, n = 5) than did 2 h-adherent macrophages (0.5 +/- 0.2 nmol/mg of protein, n = 5). Paf production was not altered when 2 h- and 24 h-adherent cells were cultured and/or stimulated in the presence of 5 microM-indomethacin, 10 microM-nordihydroguaiaretic acid or 100 microM-BW755C as compared with untreated cells. These results indirectly exclude the regulation of paf production by arachidonic acid metabolites. We investigated the efficiency of the enzymic steps which govern paf synthesis. We showed that the anabolic process was not impaired since (1) the amounts of alkylacylglycerophosphocholine and lyso-paf were similar in 2 h-, 24 h- and 48 h-adherent macrophages; (2) adding synthetic lyso-paf or acetyl-CoA to intact cells did not increase paf production in zymosan-stimulated 24 h- and 48 h-adherent macrophages; (3) the basal level of acetyltransferase was comparable in 2 h-, 24 h- and 48 h-adherent macrophages and in all cases was increased by 2-3 times upon zymosan challenge. We also showed that impaired paf production in 24 h- and 48 h-cultured macrophages was not due to the nature of the stimulus used to induce its synthesis.

Acetyltransferases↗

Recombinant human tumour necrosis factor (rTNF)2 enhances leukotriene biosynthesis in neutrophils and eosinophils stimulated with the Ca2+ ionophore A23187.

Recombinant human tumour necrosis factor alpha (rTNF) did not cause the release of leukotrienes (LT) by human neutrophils and eosinophils. It did, however, prepare neutrophils and eosinophils for enhanced production of LT after activation with the calcium ionophore A23187. The effect was observed with 10(2) to 10(4) units/ml of rTNF which enhanced LTB4 production by ionophore-stimulated neutrophils, by 40 to 400%, while 10(3) to 10(4) units/ml of rTNF increased LTC4 production by ionophore-stimulated eosinophils by 30 to 120%. The enhancement was observed when granulocytes were incubated for 5 to 10 min with rTNF before stimulation with the ionophore. Increased leukotriene production was attributed to the induction of fatty acid hydrolase and/or 5-lipoxygenase activities on the basis of the increment in the total counts released after 3H-arachidonic acid labelling of neutrophils pretreated with rTNF as compared to control medium.

Calcimycin↗

Biosynthesis of paf-acether: VIII: Impairment of paf-acether production in activated macrophages does not depend upon acetyltransferase activity.

Activated peritoneal macrophages (M phi) from mice injected with Bacilli Calmette-Guérin, trehalose dimycolate, a defined immunostimulant derived from Mycobacterium tuberculosis, or streptococci C74 (St), synthesized two to three times less paf-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in response to a zymosan challenge than did (0.7 +/- 0.2 nmol/mg protein) resident-M phi (R-M phi). To assess at which step the paf-acether biosynthetic pathway was impaired, the content in phospholipid paf-acether precursors was evaluated. The alkyl-acyl-glycerophosphocholine content was comparable in R-M phi (50.4 +/- 18.6 nmol/mg protein) and activated-M phi (40.0 +/- 6.5 to 63.9 +/- 7.7 nmol/mg protein), as well as the lyso paf-acether content (0.85 +/- 0.18 nmol/mg protein for R-M phi vs 0.63 +/- 0.16 to 1.23 +/- 0.21 nmol/mg protein for activated macrophages). The nonlimiting rate of the phospholipid substrate was strengthened by experiments showing that incubation of the various populations with lyso paf-acether did not yield increased amounts of paf-acether. Similarly, incubation of R-M phi or St-M phi with sodium acetate increased paf-acether production to the same extent in both populations, ruling out the acetate substrate deficiency as the cause of the impaired production. The level of acetyltransferase activity, the enzyme that transfers an acetate moiety of acetyl coenzyme A (acetyl-CoA) onto lyso paf-acether, was very low in thioglycolate (TG)-elicited M phi but high in R-M phi and activated ones. In all cases, it was increased by two to three times upon zymosan challenge. This suggested that increased paf-acether catabolism and not impaired anabolism could be responsible for the marked reduced formation noted in activated macrophages. The addition of acetyl-CoA (200 microM) to the various macrophage monolayers restored paf-acether formation by activated cells to R-M phi values but with delayed kinetics as compared with paf-acether formation induced by zymosan. The enhancing effect of acetyl-CoA on paf-acether production was inhibited upon oleyl-CoA addition, suggesting that acetyl-CoA may increase paf-acether production by preventing the reacylation of lyso paf-acether resulting from paf-acether degradation. In conclusion, the paf-acether output in some inflammatory macrophages may be regulated by the level of acetyltransferase activity, because it is observed in TG-M phi. However, we present the first evidence for another mechanism of regulation, most probably related to the deacylation/reacylation of paf-acether precursors and metabolites.

Acetates↗

Formation of prostaglandins, leukotrienes and paf-acether by macrophages.

Prostaglandins (PG) and leukotrienes (LT)--arachidonic acid-dependent metabolites--and paf-acether (platelet-activating factor)--an ether phospholipid--are potent mediators of allergic and inflammatory reactions. Their structures, chemical synthesis and biosynthetic pathways have been recently described. These mediators are produced by various cells with proinflammatory activities including the macrophages upon interaction with a specific secretagogue stimulus (phagocytosis of zymosan particles, immune-complexes); in IgE-dependent hypersensitivity reactions; upon interaction with one of these mediators. Formation of these mediators by macrophages depends upon their local environment. Qualitative and/or quantitative variations in their synthesis are observed depending on the tissue they are derived from (alveole or peritoneum) and on the type of inflammation (immunologic specific or not). Their potent biological activities (increase of vascular permeability, smooth muscle contraction, cardiac and vascular effects and/or chemotactism) suggest a role for these mediators in various pathologies.

Animals↗

Paf-acether generates chemiluminescence in human neutrophils in the absence of cytochalasin B.

In the absence of cytochalasin B, synthetic Paf-acether (0.1-10 microM) induced oxygen radical production in polymorphonuclear neutrophils as measured by the luminol-dependent chemiluminescence ( LDCL ) test. This effect was observed after a lag period of 10 s and was maximal between 5 and 15 min. In the presence of cytochalasin B, the kinetics were shortened, but the lag period was not modified and the same concentrations of the agonist had to be used to induce LDCL . None of the structural analogs tested (2-lyso Paf-acether, Paf-acether enantiomer, 1 ester analog of Paf-acether, lyso-phosphatidylcholine) were active, irrespective of the presence of cytochalasin B. Paf-acether (10 microM) shortened the kinetics of opsonized zymosan (10 micrograms/ml)-induced LDCL and enhanced it by 550% and 250% at 5 min and 10 min respectively, without affecting the peak value. Similar results were obtained using non-opsonized zymosan (100 micrograms/ml). Lower concentrations of Paf-acether (0.1 microM) were also able to increase oxygen radical production induced by low doses of zymosan and opsonized zymosan. The triggering and enhancing effects of Paf-acether on oxygen radical production by resting and stimulated polymorphonuclear neutrophils support the role of Paf-acether in inflammation.

Cytochalasin B↗

PAF-acether-induced plasma exudation in rat skin is independent of platelets and neutrophils.

The ability of Paf-acether to induce increased plasma protein extravasation (IPPE) was assessed in the skin of rats by measuring extravasation of 125I-bovine serum albumin. Paf-acether elicited dose-dependent IPPE with a threshold of 0.04 pmol and a maximum at 4.5 nmol, with an effective concentration (EC50) of 23 pmol. The EC50 of the 1-0-ester Paf-acether analog was 1.5 nmol whereas 2-lyso Paf-acether was ineffective in inducing IPPE. Serotonin and histamine were respectively 100 and 1,00-fold less potent than Paf-acether. High doses of Paf-acether also resulted in a dose related accumulation of III-indium oxine labeled platelets. Ultrastructural and radioisotopic studies showed that Paf-acether-induced IPPE was not dependent upon platelet accumulation. In addition, platelet release products were ruled out in this phenomenon, since a "cocktail" of indomethacin, methysergide and pyrilamine-maleate failed to significantly reduce Paf-acether-induced IPPE. Neutrophils were not involved in Paf-acether -induced IPPE since nitrogen mustard-treated animals presented with IPPE not significantly different from controls. Finally, the specificity of Paf-acether on IPPE formation was demonstrated by its complete prevention by prior intravenous injection of Paf-acether (6 ug/kg), but not of Paf-acether analogs, histamine or serotonin.

Animals↗

The role of immunomodulators in the production of lipid mediators by macrophages (M phi).

There is evidence indicating that bioreactive lipid mediators, PAF-acether (platelet-activating factor: 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and arachidonic acid (AA) metabolites, are formed upon deacylation of ether lipids. M phi obtained from mice treated with the sterile irritant thioglycolate exhibited an impaired formation of PAF-acether, leucotrienes (LT) C4 and prostaglandins (PG). In order to assess whether the impaired formation in lipid mediators is a general feature of M phi found at inflammatory sites, we have compared the capacity of resident (R-M phi) and immunostimulant-activated M phi to release, upon a zymosan challenge, PAF-acether and cyclooxygenase and lipoxygenase products. Activated M phi was obtained from mice injected intraperitoneally with non-viable C74 streptococci (St-M phi), bacilli Calmette-Guerin (BCG-M phi) or trehalose dimycolate (TDM-M phi), a defined immunostimulant isolated from Mycobacterium tuberculosis. All populations were capable of releasing PAF-acether. However, the amount of cell-associated PAF-acether was reduced by 75-90% in activated M phi populations as compared to R-M phi. Although the acetyltransferase level was comparable, acetyl-CoA supplementation restored the formation of PAF-acether by activated M phi to control (R-M phi) level. The amount of 14C-AA metabolites released by St-M phi was much lower compared with TDM-M phi or R-M phi, as was the amount of LTC4 detected as SRS contractile activity after HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Lipid-mediator synthesis in peritoneal macrophages from mice injected with immunostimulants.

The metabolism of bioreactive lipid mediators was studied in two types of activated macrophages (M phi). We compared the capacity of resident and activated M phi to release, upon a zymosan challenge, cyclooxygenase and lipoxygenase products as well as PAF-acether (platelet-activating factor) and its 2-lyso precursor. Activated M phi were obtained from mice injected intraperitoneally either with nonviable C74 streptococci (St-M phi) or with trehalose dimycolate, a defined immunostimulant isolated from Mycobacterium tuberculosis (TDM-M phi). Both activated populations exhibited common features: conversion of endogenous [14C]arachidonic acid into prostaglandin E2 and thromboxane A2 rather than into prostaglandin I2 and low biosynthesis of PAF-acether, probably due to an impairment of the acetylation step. However, contrary to St-M phi, TDM-M phi did not display a marked overall reduction of arachidonate metabolism. In addition, as compared to resident M phi, TDM-M phi presented a ratio of thromboxane B2/6-ketoprostaglandin F1 alpha 30-fold higher, a better conversion of leukotriene C to leukotriene D and a higher capacity to release the PAF-acether they synthesize. These macrophages thus seem to be valuable tools for studying the formation of mediators and for determining specific markers of an activated state.

Adjuvants, Immunologic↗