RÉSUMÉ
Fat mass, adipocyte size and metabolic responsiveness, and preadipocyte differentiation decrease between middle and old age. We show that expression of CCAAT/enhancer binding protein (C/EBP)-alpha, a key regulator of adipogenesis and fat cell function, declined substantially with aging in differentiating preadipocytes cultured under identical conditions from rats of various ages. Overexpression of C/EBP alpha in preadipocytes cultured from old rats restored capacity to differentiate into fat cells, indicating that downstream differentiation-dependent genes maintain responsiveness to regulators of adipogenesis. C/EBP alpha-expression also decreased with age in fat tissue from three different depots and in isolated fat cells. The overall level of C/EBP beta, which modulates C/EBP alpha-expression, did not change with age, but the truncated, dominant-negative C/EBP beta-liver inhibitory protein (LIP) isoform increased in cultured preadipocytes and isolated fat cells. Overexpression of C/EBP beta-LIP in preadipocytes from young rats impaired adipogenesis. C/EBP delta, which acts with full-length C/EBP beta to enhance adipogenesis, decreased with age. Thus processes intrinsic to adipose cells involving changes in C/EBP family members contribute to impaired adipogenesis and altered fat tissue function with aging. These effects are potentially reversible.
Sujet(s)
Tissu adipeux/croissance et développement , Vieillissement/physiologie , Protéines liant les séquences stimulatrices de type CCAAT/génétique , Famille multigénique/génétique , Adipocytes/physiologie , Animaux , Protéine alpha liant les séquences stimulatrices de type CCAAT/génétique , Protéine bêta de liaison aux séquences stimulatrices de type CCAAT/génétique , Cellules cultivées , Rats , Rats de lignée F344 , Cellules souches/physiologie , Facteurs de transcription/génétiqueRÉSUMÉ
The sequencing of Leishmania major Friedlin chromosome 1 (Chr1), Chr3, and Chr4 has been completed. and several other chromosomes are well underway. The complete genome sequence should be available by 2003. Over 1,000 full-length new genes have been identified, with the majority (approximately 75%) having unknown function. Many of these may be Leishmania (or kinetoplastid) specific. Most interestingly, the genes are organized into large (> 100-500 kb) polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a "divergent" manner, i.e., the mRNAs for the two sets of genes are both transcribed towards the telomeres. Nuclear run-on analysis suggests that transcription is initiated in both directions within the "divergent" region. Chr3 and Chr4 contain two "convergent" clusters, with a single "divergent" gene at one telomere of Chr3. Sequence analysis of several genes from the LD1 region of Chr35 indicates a high degree of sequence conservation between L. major and L. donovani/L. infantum within protein-coding open reading frames (ORFs), with a lower degree of conservation within the non-coding regions. Immunization of mice with recombinant antigen from two of these genes, BTI (formerly ORFG) and ORFF, results in significant reduction in parasite burden following Leishmania challenge. Recombinant ORFF antigen shows promise as a serodiagnostic. We have also developed a tetracycline-regulated promoter system, which allows us to modulate gene expression in Leishmania.
Sujet(s)
Génome de protozoaire , Leishmania/génétique , Animaux , Séquence nucléotidique , Cartographie chromosomique , Gènes de protozoaire , Leishmania/classification , Leishmania/physiologieRÉSUMÉ
To determine whether an ongoing response to Leishmania major would affect the response to a non-cross-reacting, non-leishmanial antigen, susceptible BALB/c mice and resistant C3H mice were infected with L. major parasites expressing Escherichia coli beta-galactosidase (beta-GAL); this parasite was designated L. major-betaGAL. BALB/c and C3H mice responded to infection with L. major-betaGAL by mounting a CD4 T-cell response to both parasite antigens and to the reporter antigen, beta-GAL. The phenotypes of these T cells were characterized after generating T-cell lines from infected mice. As expected, BALB/c mice responded to infection with L. major-betaGAL by producing interleukin 4 in response to the parasite and C3H mice produced gamma interferon (IFN-gamma) in response to the parasite and beta-GAL. Interestingly, however, BALB/c mice produced IFN-gamma in response to beta-GAL. Taken together, these results demonstrate that priming of IFN-gamma-producing cells can occur in BALB/c mice despite the fact the animals are simultaneously mounting a potent Th2 response to L. major.
Sujet(s)
Leishmania major/immunologie , Leishmaniose cutanée/immunologie , Lymphocytes auxiliaires Th1/immunologie , beta-Galactosidase/immunologie , Animaux , Anticorps antiprotozoaires/biosynthèse , Lymphocytes T CD4+/immunologie , Interféron gamma/biosynthèse , Interféron gamma/pharmacologie , Lipopolysaccharides/pharmacologie , Activation des macrophages , Souris , Souris de lignée BALB C , Souris de lignée C3H , TransfectionRÉSUMÉ
Transposable elements are useful tools for insertional mutagenesis and have many potential applications in the characterization of complex genomes. Here we describe a system which facilitates the construction of large transposon insertion libraries useful for genome sequencing and functional genomic analysis. We developed two transposons, TyK and TyK'GFP+, which can be introduced into target DNAs by Ty1-mediated transposition in vitro, and several modifications which decrease the frequency of false transposition events and direct the recovery of transpositions into passenger rather than vector DNA. Insertions of TyK'GFP+ additionally may yield fusions to the Aequorea green fluorescent protein (GFP), useful in studies of gene expression and protein targeting. Transposition in vitro was obtained into target DNAs of up to 50 kb in size, restriction mapping showed insertion to be relatively random, and the sequence of 55 insertion sites showed neither strong site nor base compositional preference. Our data suggest that TyK-based artificial transposons will be suitable for a variety of genetic applications in many organisms.
Sujet(s)
Éléments transposables d'ADN , Vecteurs génétiques , Mutagenèse par insertion , Saccharomyces cerevisiae/génétique , ADN fongique/génétique , Escherichia coli/génétique , Transformation génétiqueRÉSUMÉ
OBJECTIVE: To review recent findings about changes with age in the replication and differentiation of preadipocytes, the progenitor cells in fat tissue that are capable of differentiating into fat cells, and to examine possible links between these alterations and age-related changes in fat tissue function. DESIGN: A survey and analysis of recent literature concerning changes in preadipocyte and fat cell function with age. CONCLUSIONS: Intrinsic aging changes in fat cells and preadipocytes as well as in factors extrinsic to fat tissue (such as food intake and absorption and hormonal status) contribute to age-related alterations in fat tissue function and cellularity. Changes with age in preadipocyte number, replicative potential, and capacity for differentiation, which may be linked to aging changes in fat cell size, number, and function, have been identified. The decline in preadipocyte capacity for differentiation and the associated decline in fat cell lipogenic capacity may be particularly important in contributing to the decrease in fat mass and alterations in fat tissue function that occur between middle- and old age. These declines result from blunting of the changes in gene expression that occur during preadipocyte differentiation and may, in turn, be related to altered regulation of particular transcription factors that control the preadipocyte differentiation program and maintenance of fat cell function.
Sujet(s)
Tissu adipeux/cytologie , Vieillissement/anatomopathologie , Absorption , Tissu adipeux/physiologie , Sujet âgé , Vieillissement/génétique , Vieillissement/physiologie , Animaux , Numération cellulaire , Différenciation cellulaire/génétique , Division cellulaire/génétique , Taille de la cellule , Consommation alimentaire , Régulation de l'expression des gènes , Hormones/physiologie , Humains , Métabolisme lipidique , Adulte d'âge moyen , Cellules souches/cytologie , Cellules souches/physiologie , Facteurs de transcription/génétiqueRÉSUMÉ
Differentiation of adipocytes is accompanied by secretion of molecules stimulating angiogenesis in vivo and endothelial cell growth and motility in vitro. We demonstrate that the angiogenic and motility-stimulating activities secreted by adipocytes are separable from the endothelial cell mitogenic activity by fractionation of adipocyte-conditioned medium. The major differentiation-dependent angiogenic molecule was purified and identified by GCMS as 1-butyryl-glycerol (monobutyrin). Monobutyrin levels increase at least 200-fold during adipocyte differentiation and represent a major fraction of the total angiogenic activity. Synthetic monobutyrin shows the same spectrum of biological activities as the adipocyte-derived factor: stimulation of angiogenesis in vivo and microvascular endothelial cell motility in vitro, with no effect on endothelial cell proliferation. Angiogenesis is stimulated at doses as low as 20 pg when tested in the chick chorioallantoic membrane assay. These results strongly suggest that monobutyrin is a key regulatory molecule in an angiogenic process linked to normal cellular and tissue development.
Sujet(s)
Tissu adipeux/cytologie , Agents angiogéniques , Endothélium vasculaire/physiologie , Glycérides/biosynthèse , Substances de croissance , Tissu adipeux/métabolisme , Animaux , Dosage biologique , Différenciation cellulaire , Division cellulaire/effets des médicaments et des substances chimiques , Mouvement cellulaire/effets des médicaments et des substances chimiques , Cellules cultivées , Embryon de poulet , Chromatographie sur couche mince , Endothélium vasculaire/cytologie , Endothélium vasculaire/effets des médicaments et des substances chimiques , Chromatographie gazeuse-spectrométrie de masse , Glycérides/isolement et purification , Glycérides/pharmacologie , Mâle , Néovascularisation pathologique , RatsRÉSUMÉ
We have studied the structure and regulation of glycerophosphate dehydrogenase (GPD) mRNA during mouse adipocyte and muscle cell differentiation. This message has a size of 2.8 kilobases that includes a coding segment of 1050 bases and a large untranslated 3' end of about 1700 bases. There is a high degree of amino acid homology (91%) between the mouse adipocyte and rabbit muscle GPD proteins. GPD mRNA is not detected in myoblasts, but, as in adipogenesis, it is expressed upon differentiation into myotubes. The modulation of GPD mRNA by cyclic AMP analogues and tumor necrosis factor has been examined in both adipocytes and myotubes. Dibutyryl cAMP or 8-bromo-cAMP causes a large reduction of GPD mRNA levels in both cell types, with less than 20% remaining after 18 h of treatment. Tumor necrosis factor effects a dramatic and rapid reduction in GPD mRNA in fat cells and a slower but significant decrease in the level of this mRNA in muscle cells. These results indicate that GPD gene expression is linked to cell differentiation in both fat and muscle cells, and suggest certain similarities in hormonal modulation in both cell types.
Sujet(s)
Tissu adipeux/cytologie , Glycerolphosphate dehydrogenase/génétique , Muscles/cytologie , ARN messager/métabolisme , Tissu adipeux/enzymologie , Séquence d'acides aminés , Animaux , Séquence nucléotidique , Butyrates/pharmacologie , Acide butyrique , Différenciation cellulaire , AMP cyclique/analogues et dérivés , AMP cyclique/pharmacologie , Dexaméthasone/pharmacologie , Souris , Muscles/enzymologie , LapinsRÉSUMÉ
We have isolated the mouse gene encoding adipocyte P2, aP2, the differentiation-dependent adipocyte protein homologous to myelin P2. The aP2 gene is present in a single copy in the mouse and is present in single or few copies in species from human to Drosophila. The entire gene spans 4 kilobases and consists of four exons encoding 25, 57, 34, and 16 amino acids; the overall exon structure is similar to the gene encoding liver fatty acid binding protein. A plasmid vector was constructed containing the entire aP2 gene with flanking sequences, modified by linker insertion. When this gene is stably introduced into 3T3-F442A cells, it is expressed only upon adipose differentiation, with a time course of induction very similar to that of the endogenous aP2 gene. We have compared the DNA sequence of the 5'-flanking region of the aP2 gene to the promoter regions of two other genes activated during adipocyte differentiation, glycerol-3-phosphate dehydrogenase and adipsin, and find a 13-base region of homology (Formula: see text) present in multiple copies in the 5'-flanking region of each gene. An adjacent 15-base sequence is present only in glycerol-3-phosphate dehydrogenase and aP2 genes. Both of these elements share homology with putative viral enhancer core sequences. These results indicate that the aP2 gene contains sequence information necessary for differentiation-dependent expression in fat cells; common elements shared by adipocyte-specific genes may play a role in this process.
Sujet(s)
Tissu adipeux/physiologie , Protéines de transport/génétique , Protéines tumorales , Protéines de tissu nerveux , Tissu adipeux/cytologie , Séquence d'acides aminés , Animaux , Différenciation cellulaire , Clonage moléculaire , Facteur D du complément , Endopeptidases/génétique , Protéine-7 de liaison aux acides gras , Protéines de liaison aux acides gras , Acides gras/métabolisme , Régulation de l'expression des gènes , Gènes , Glycerolphosphate dehydrogenase/génétique , Souris , Régions promotrices (génétique) , Similitude de séquences d'acides nucléiques , Serine endopeptidases , Distribution tissulaireRÉSUMÉ
We have examined the cellular mechanisms by which heparin potentiates the ability of 3T3-adipocytes to stimulate the formation of new blood vessels. Both anticoagulant and non-anticoagulant heparin species enhanced the angiogenic activity of adipocyte-secreted products in the chick chorioallantoic membrane assay, indicating that the angiotropic effect of this glycosaminoglycan is independent of its effect on the coagulation cascade. Heparin alone was unable to produce a neovascular response. The ability of heparin to modulate three endothelial functions in vitro thought to be related to angiogenesis were examined: protease activity, motility, and mitogenesis. Heparin caused a 100% increase in the adipocyte-induced stimulation of endothelial cell plasminogen activator activity and motility, but had no effect on proliferation. The enhancement of plasminogen activator and chemoattractant activities had a similar ED50 (1-2 micrograms/ml) and optimum dose (10-30 micrograms/ml). When we examined the direct effect of heparin on the activity of two distinct plasminogen activator enzymes--urokinase and tissue-type--a dual action of heparin was observed: tissue-type enzyme activity was stimulated 100% by heparin at 10 micrograms/ml, whereas urokinase activity was inhibited by 77% at this dose. These data suggest that heparin potentiates angiogenesis in vivo by stimulating endothelial cell plasminogen activator, motility, or both. Our results further suggest that for adipocyte-induced blood vessel formation, in contrast to other angiogenesis systems, heparin does not appear to affect the mitogenic activity.
Sujet(s)
Tissu adipeux/cytologie , Aorte/effets des médicaments et des substances chimiques , Héparine/pharmacologie , Néovascularisation pathologique/induit chimiquement , Tissu adipeux/physiologie , Animaux , Aorte/cytologie , Phénomènes biomécaniques , Bovins , Chimiotaxie/effets des médicaments et des substances chimiques , Synergie des médicaments , Endothélium/cytologie , Endothélium/effets des médicaments et des substances chimiques , Mitogènes/physiologie , Activateurs du plasminogène/physiologieRÉSUMÉ
The complete sequence of 129 amino acids has been determined for one of three closely related lysozymes c purified from cow stomach mucosa. The sequence differs from those known for 17 other lysozymes c at 39-60 positions, at one of which there has been a deletion of 1 amino acid. The glutamate replacement at position 101 and the deletion of proline at position 102 eliminate the aspartyl-prolyl bond that is present between these positions in all other mammalian lysozymes c tested. This bond appears to be the most acid-sensitive one in such lysozymes at physiological temperature. Of the 40 positions previously found to be invariant among lysozymes c, only one has undergone substitution in the cow lineage. This modest number of changes at novel positions is consistent with the inference, based on tree analysis and antigenic comparisons, that the tempo of evolutionary change in the cow lysozyme lineage has not been radically different from that in other lysozyme c lineages. The mutations responsible for the distinctive catalytic properties and stability of cow lysozyme c could be a minor fraction of the total that have been fixed in the cow lineage.
Sujet(s)
Bovins/métabolisme , Isoenzymes/analyse , Lysozyme/analyse , Estomac/enzymologie , Séquence d'acides aminés , Animaux , Artiodactyla/métabolisme , Poulets , Réactions croisées , Muqueuse gastrique/enzymologie , Humains , Isoenzymes/immunologie , Lysozyme/immunologieRÉSUMÉ
A major regulatory shift affecting the expression of lysozyme c may have been involved in the origin of two groups of mammals whose nutrition depends on foregut bacteria. A survey of 23 mammalian species reveals that the lysozyme c activity per g of stomach mucosa is many times higher for ruminants and a leaf-eating monkey than for animals lacking a foregut. The implication is that stomach lysozyme c functions as a major digestive enzyme in ruminant-like mammals, helping to make those bacterial which enter the stomach from the foregut available for hydrolysis by conventional digestive enzymes. The high level of stomach lysozyme is due to more enzyme molecules rather than to an increase in the activity of each molecule. This was shown for the cow by purifying the three, non-allelic lysozymes c that account for the lysozyme activity in gastric mucosa and measuring their specific activities and for other foregut fermenters by immunological titration. Lysozyme appears in the stomach mucosa before birth and reaches adult levels before weaning. Other tissues tested from cattle lack lysozyme c and may instead have low levels of another lysozyme that could belong to the g class, the first indication that lysozyme g may be present in mammals. The lysozymes of eight ruminants, four Old World monkeys, and 12 other animals were compared as regards the ability to lyse bacterial cells under various conditions and to resist inactivation by pepsin. There are differences among these species in the dependence of the rate of bacterial lysis on time, pH, and ionic strength. Although not every lysozyme was tested in all of these catalytic respects, there were no exceptions to the following generalizations. First, at ionic strengths above 0.1 and pH values above 5, the rate of lysis by ruminant and monkey lysozymes c rose with the time of reaction, whereas the rate was more nearly constant for the other animal lysozymes. Second, the lytic activity at neutral pH is lower than at pH 5 for the ruminant and monkey lysozymes c when the ionic strength is over 0.1; by contrast, for other lysozymes c under these conditions the activity at neutral pH is about as high as at pH 5. This latter property, which may be viewed as an adaptation for functioning as a digestive enzyme in the stomach, can be explained in part by differences in electrostatic interactions between lysozyme and the substrate due to the relatively non-basic nature of ruminant and monkey lysozymes compared to other lysozymes c.(ABSTRACT TRUNCATED AT 400 WORDS)
Sujet(s)
Artiodactyla/métabolisme , Lysozyme/métabolisme , Oligosaccharides , Estomac/enzymologie , Animaux , Muqueuse gastrique/enzymologie , Concentration en ions d'hydrogène , Immunodiffusion , Cinétique , Concentration osmolaire , Pepsine A/métabolisme , Tétroses/pharmacologie , Distribution tissulaireRÉSUMÉ
Gene fusions between the mouse mammary tumor virus long terminal repeat and the E. coli lacZ gene have been shown to exhibit hormone dependent expression of beta-galactosidase activity. These constructions were used in transient expression experiments to assess the effects of specific modifications introduced into the region upstream of the transcription initiation site. 5' deletions demonstrate that sequences sufficient for wild-type promoter function are contained downstream of residue -64 relative to the initiation site. Other deletions define a region of approximately 80 base pairs between -220 and -140 which contains sequences essential for hormonal control. Between this control region and the promoter lie sequences dispensable for both functions.