RESUMO
Substantial evidence supports a functional role for cyclooxygenase- and lipoxygenase-catalyzed arachidonic and linoleic acid metabolism in cancer development. Genetic intervention studies firmly established cause-effect relations for cyclooxygenase-2, but cyclooxygenase-1 may also be involved. In addition, pharmacologic cyclooxygenase inhibition was found to suppress carcinogenesis in both experimental mouse models and several cancers in humans. Arachidonic acid-derived eicosanoid or linoleic acid-derived hydro[peroxy]fatty acid signaling are likely to be involved impacting fundamental biologic phenomena as diverse as cell growth, cell survival, angiogenesis, cell invasion, metastatic potential and immunomodulation. However, long chain unsaturated fatty acid oxidation reactions indicate antipodal functions of distinct lipoxygenase isoforms in carcinogenesis, i.e., the 5- and platelet-type 12-lipoxygenase exhibit procarcinogenic activities, while 15-lipoxygenase-1 and 15-lipoxygenase-2 may suppress carcinogenesis.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Lipoxigenase/metabolismo , Neoplasias/enzimologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Apoptose , Araquidonato Lipoxigenases/metabolismo , Ácido Araquidônico/metabolismo , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Transformação Celular Neoplásica , Ciclo-Oxigenase 2/metabolismo , Ácidos Eicosanoicos/metabolismo , Humanos , Isoenzimas , Ácido Linoleico/metabolismo , Peróxidos Lipídicos/metabolismo , Lipoxigenase/efeitos dos fármacos , Invasividade Neoplásica , Neoplasias/metabolismo , Neovascularização Patológica , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Transdução de SinaisRESUMO
Flat gold nanostructures on inert substrates like glass or graphite were illuminated by single intensive laser pulses with fluences above the gold melting threshold. The liquid structures produced in this way are far from their equilibrium shape, and a dewetting process sets in. On a time scale of a few nanoseconds, the liquid contracted toward a sphere. During this contraction, the center of mass moved upward, which could lead to detachment of droplets from the surface due to inertia. The resulting velocities were on the order of 10 meters per second for droplets with radii in the range of 100 nanometers.
RESUMO
Dendritic cell (DC) differentiation from human CD34(+) hematopoietic progenitor cells (HPCs) can be triggered in vitro by a combination of cytokines consisting of stem cell factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha. The immune response regulatory cytokines, IL-4 and IL-13, promote DC maturation from HPCs, induce monocyte-DC transdifferentiation, and selectively up-regulate 15-lipoxygenase 1 (15-LO-1) in blood monocytes. To gain more insight into cytokine-regulated eicosanoid production in DCs we studied the effects of IL-4/IL-13 on LO expression during DC differentiation. In the absence of IL-4, DCs that had been generated from CD34(+) HPCs in response to stem cell factor/granulocyte-macrophage colonystimulating factor/tumor necrosis factor alpha expressed high levels of 5-LO and 5-LO activating protein. However, a small subpopulation of eosinophil peroxidase(+) (EOS-PX) cells significantly expressed 15-LO-1. Addition of IL-4 to differentiating DCs led to a marked and selective down-regulation of 5-LO but not of 5-LO activating protein in DCs and in EOS-PX(+) cells and, when added at the onset of DC differentiation, also prevented 5-LO up-regulation. Similar effects were observed during IL-4- or IL-13-dependent monocyte-DC transdifferentiation. Down-regulation of 5-LO was accompanied by up-regulation of 15-LO-1, yielding 15-LO-1(+) 5-LO-deficient DCs. However, transforming growth factor beta1 counteracted the IL-4-dependent inhibition of 5-LO but only minimally affected 15-LO-1 up-regulation. Thus, transforming growth factor beta1 plus IL-4 yielded large mature DCs that coexpress both LOs. Localization of 5-LO in the nucleus and of 15-LO-1 in the cytosol was maintained at all cytokine combinations in all DC phenotypes and in EOS-PX(+) cells. In the absence of IL-4, major eicosanoids of CD34(+)-derived DCs were 5S-hydroxyeicosatetraenoic acid (5S-HETE) and leukotriene B(4), whereas the major eicosanoids of IL-4-treated DCs were 15S-HETE and 5S-15S-diHETE. These actions of IL-4/IL-13 reveal a paradigm of eicosanoid formation consisting of the inhibition of one and the stimulation of another LO in a single leukocyte lineage.
Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Células Dendríticas/efeitos dos fármacos , Eicosanoides/biossíntese , Interleucina-4/farmacologia , Antígenos CD34/metabolismo , Araquidonato 15-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/genética , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/enzimologia , Células Dendríticas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Ácidos Hidroxieicosatetraenoicos/biossíntese , Interleucina-13/farmacologia , Microscopia Confocal , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Monócitos/metabolismo , Fator de Células-Tronco/farmacologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/enzimologia , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1 , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The 5-lipoxygenase (5-LO) pathway in human CD34(+) hematopoietic progenitor cells, which were induced to differentiate into dendritic cells (DCs) by cytokines in vitro and in DCs of lymphoid tissues in situ, was examined. Extracts prepared from HPCs contained low levels of 5-LO or 5-LO-activating protein. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plus tumor necrosis factor-alpha (TNF-alpha) promoted DC differentiation and induced a strong rise in 5-LO and FLAP expression. Fluorescence-activated cell sorter (FACS) analyses identified a major DC population coexpressing human leukocyte antigen (HLA)-DR/CD80 and monocytic or Langerhans cell markers. Transforming growth factor-beta1 (TGF-beta-1), added to support DC maturation, strongly promoted the appearance of CD1a(+)/Lag(+) Langerhans-type cells as well as mature CD83(+) DCs. TGF-beta-1 further increased 5-LO and FLAP expression, recruited additional cells into the 5-LO(+) DC population, and promoted production of 5-hydroxyeicosatetraenoic acid and leukotriene B(4) in response to calcium (Ca(++)) ionophore A23187. These in vitro findings were corroborated by 5-LO expression in distinct DC phenotypes in vivo. Scattered 5-LO and FLAP in situ hybridization signals were recorded in cells of paracortical T-lymphocyte-rich areas and germinal centers (GCs) of lymph nodes (LNs) and tonsil and in cells of mucosae overlying the Waldeyer tonsillar ring. 5-LO protein localized to both CD1a(+) immature DCs and to CD83(+) mature interdigitating DCs of T-lymphocyte-rich areas of LNs and tonsil. As DCs have the unique ability to initiate naive lymphocyte activation, our data support the hypothesis that leukotrienes act at proximal steps of adaptive immune responses. (Blood. 2000;96:3857-3865)
Assuntos
Antígenos CD34/análise , Araquidonato 5-Lipoxigenase/metabolismo , Células Dendríticas/enzimologia , Araquidonato 5-Lipoxigenase/biossíntese , Araquidonato 5-Lipoxigenase/genética , Ácido Araquidônico/farmacologia , Calcimicina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Fatores Quimiotáticos/biossíntese , Citocinas/farmacologia , Células Dendríticas/citologia , Sangue Fetal/citologia , Citometria de Fluxo , Antígenos HLA-DR , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Ácidos Hidroxieicosatetraenoicos/biossíntese , Imuno-Histoquímica , Imunofenotipagem , Ionóforos/farmacologia , Leucotrieno B4/biossíntese , Linfonodos/enzimologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Macrófagos/enzimologia , Tonsila Palatina/enzimologiaRESUMO
Xylose reductases catalyse the initial reaction in the xylose utilisation pathway, the NAD(P)H+H+ dependent reduction of xylose to xylitol. In this work, the xylose reductase gene from Candida tenuis CBS 4435 was cloned and successfully expressed in E. coli. From the purified and partially sequenced protein primers were deduced for PCR. The fragment obtained was used for Southern blot analysis and screening of a subgenomic library. The clone containing the open reading frame was sequenced; the gene consisted of 969 nucleotides coding for a 322 amino acids protein with a molecular mass of 36 kDa. Putative regulatory signals were identified with the help of a Saccharomyces cerevisiae regulatory sequence database. In order to express the xylose reductase in E. coli, the gene was placed under positive and negative control. At low temperatures, the xylose reductase was expressed in soluble and active form up to about 10% of the soluble protein; with rising temperatures formation of visible inclusion bodies occurred. In refolding experiments we were able to recover the major portion of xylose reductase activity from the pellet fraction.
Assuntos
Aldeído Redutase/genética , Candida/genética , Xilose/metabolismo , Aldeído Redutase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Candida/enzimologia , Catálise , Clonagem Molecular , DNA Fúngico , Escherichia coli/genética , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
We cloned and successfully expressed the gene for xylitol dehydrogenase from Galactocandida mastotermitis in Escherichia coli. The amino acid sequence revealed that the enzyme belongs to the superfamily of zinc containing, medium-chain alcohol dehydrogenases. The enzyme catalyses the second step in the xylose utilising pathway converting xylose to xylulosephosphate. Xylulose-phosphate is further degraded by the transaldolase and transketolase reactions of the pentose phosphate pathway. The purified xylitol dehydrogenase from G. mastotermitis was subjected to partial amino acid sequence analysis. The resulting amino acid information was then used to construct oligonucleotide probes for PCR amplification. The PCR product was used to screen a genomic library. The identified xdh gene includes one short intron at its 5' end. Putative regulatory signals were identified with the help of Saccharomyces cerevisiae regulatory sequence databases. An intronless xdh transcript, cloned by RT-PCR, was actively expressed in pBTac1 at 37 degrees C to approximately 8% of the soluble E. coli protein. Furthermore, the kinetic parameters were determined and conditions were found to stabilise the soluble and active protein.
Assuntos
Candida/genética , Desidrogenase do Álcool de Açúcar/genética , Xilose/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Candida/enzimologia , Clonagem Molecular , D-Xilulose Redutase , DNA Fúngico , Escherichia coli/genética , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The complete amino acid sequence of the hydantoinase from Arthrobacter aurescens DSM 3745 has been derived by automated Edman degradation. This is the first ever reported amino acid sequence of a non-ATP-dependent hydantoinase, which hydrolyzes 5'-monosubstituted hydantoin derivatives L-selectively. A homology search performed in protein and nucleic acid databases retrieved only distantly related proteins. All of these are members of the recently described protein superfamily of amidohydrolases related to ureases (Holm and Sander, Proteins 28: 72-82, 1997). Phylogenetic analysis revealed that the novel hydantoinase forms a new branch separate from other hydantoin cleaving enzymes like dihydropyrimidinases (EC 3.5.2.2) and allantoinases (EC 3.5.2.5). Our results suggests that the enzymes of this protein superfamily have evolved from a common ancestor and therefore are the product of divergent evolution. We show further that the enclosed gene families developed very early in evolution, probably prior to the formation of the three domains, Archaea, Eukarya and Bacteria. Hydantoinases related to ATP-dependent N-methylhydantoinases (EC 3.5.2.14) or 5-oxoprolinases (EC 3.5.2.9) do not belong to this superfamily.
Assuntos
Amidoidrolases/química , Arthrobacter/enzimologia , Amidoidrolases/isolamento & purificação , Sequência de Aminoácidos , Animais , Dados de Sequência MolecularRESUMO
We studied expression of the 5-lipoxygenase (5-LO) pathway in normal human skin. In situ hybridization revealed a 5-LO mRNA-containing epidermal cell (EC) population that was predominantly located in the midportion of the spinous layer, in outer hair root sheaths, and in the epithelial compartment of sebaceous glands. Examination of skin specimens by immunohistochemistry and of primary ECs by flow cytometry mapped the 5-LO protein exclusively to Langerhans cells (LCs). The LC 5-LO protein was largely found in the nuclear matrix, in nuclear envelopes, and perinuclear regions as indicated by in situ confocal laser scan microscopy. Reverse transcription-PCR and immunoblot analyses of purified primary EC populations further indicated that LCs are major 5-LO expressing cells. Enriched primary LCs were also found to contain 5-LO activating protein (FLAP), leukotriene (LT) C4 synthase, and LTA4 hydrolase. By contrast, 5-LO, FLAP, and LTC4 synthase were undetectable or largely reduced, but LTA4 hydrolase transcripts and protein were identified in ECs depleted of LCs. These data show that naive LCs are major, and possibly the sole, 5-LO pathway expressing cells in the normal human epidermis.
Assuntos
Araquidonato 5-Lipoxigenase/biossíntese , Epiderme/enzimologia , Células de Langerhans/enzimologia , Humanos , Hibridização In Situ , RNA Mensageiro/análiseRESUMO
A cDNA-library has been constructed from Nicotiana plumbaginifolia seedlings, and the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GapN, EC 1.2.1.9) was isolated by plaque hybridization using the cDNA from pea as a heterologous probe. The cDNA comprises the entire GapN coding region. A putative polyadenylation signal is identified. Phylogenetic analysis based on the deduced amino acid sequences revealed that the GapN gene family represents a separate ancient branch within the aldehyde dehydrogenase superfamily. It can be shown that the GapN gene family and other distinct branches of the superfamily have its phylogenetic origin before the separation of primary life-forms. This further demonstrates that already very early in evolution, a broad diversification of the aldehyde dehydrogenases led to the formation of the superfamily.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/genética , Nicotiana/enzimologia , Proteínas de Plantas/genética , Plantas Tóxicas , Aldeído Desidrogenase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Southern Blotting , Clonagem Molecular , Genes de Plantas , Dados de Sequência Molecular , Família Multigênica , Fosforilação , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Streptococcus mutans/genéticaRESUMO
1. The objective of the present study was to determine the effects of dexamethasone on key constituents of prostaglandin and leukotriene biosynthesis, cyclo-oxygenase-2 (COX-2) and 5-lipoxygenase activating protein (FLAP). The human monocytic cell line THP-1 was used as a model system. mRNA and protein levels of COX-2 and FLAP were determined by Northern and Western blot analyses, respectively. 2. Low levels of COX-2 and FLAP mRNA were expressed in undifferentiated THP-1 cells, but were induced upon differentiation of the cells along the monocytic pathway by treatment with phorbol ester (TPA, 5 nM). Maximal expression was observed after two days. 3. Coincubation of the undifferentiated cells with dexamethasone (10(-9) - 10(-6) M) and phorbol ester prevented induction of COX-2 mRNA, but did not affect the induction of FLAP mRNA. 4. Dexamethasone downregulated COX-2 mRNA and protein in differentiated, monocyte-like THP-1 cells. In contrast, FLAP mRNA and protein were upregulated by dexamethasone in differentiated THP-1 cells. After 24 h, FLAP mRNA levels were increased more than 2 fold. Dexamethasone did not change 5-lipoxygenase mRNA expression. 5. Release of prostaglandin E2 (PGE2) and peptidoleukotrienes was determined in cell culture supernatants of differentiated THP-1 cells by ELISA. Calcium ionophore-dependent PGE2 synthesis was associated with COX-2 expression, whereas COX-1 and COX-2 seemed to participate in arachidonic acid-dependent PGE2 synthesis. Very low levels of peptidoleukotrienes were released from differentiated THP-1 cells upon incubation with ionophore. Treatment with dexamethasone did not significantly affect leukotriene release. 6. These data provide evidence that prostaglandin synthesis is consistently downregulated by glucocorticoids. However, the glucocorticoid-mediated induction of FLAP may provide a mechanism to maintain leukotriene biosynthesis through more efficient transfer of arachidonic acid to the 5-lipoxygenase reaction, in spite of inhibitory effects on other enzymes of the biosynthetic pathway.
Assuntos
Proteínas de Transporte/genética , Dexametasona/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana/genética , Monócitos/efeitos dos fármacos , Proteínas Ativadoras de 5-Lipoxigenase , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Ciclo-Oxigenase 2 , Eicosanoides/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Monócitos/metabolismo , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Groups of hospitalized patients with psychotic and affective disorders (N = 60) underwent musculoskeletal structural examination. Psychotic and affective disorders each tend to affect a different portion of the musculoskeletal system, with psychotic patients exhibiting increased musculoskeletal dysfunction in the lower extremities and affective-disorders patients exhibiting increased cervical and thoracic dysfunction. At the clinical level, the structural examination may be used to correlate psychiatric disorders with dysfunctional regions of the musculoskeletal system.
Assuntos
Transtornos do Humor/diagnóstico , Sistema Musculoesquelético/patologia , Medicina Osteopática/métodos , Exame Físico/métodos , Transtornos Psicóticos/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medicina Osteopática/normas , Exame Físico/normas , Reprodutibilidade dos TestesRESUMO
The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase catalyses the irreversible reaction of glyceraldehyde-3-phosphate to 3-phosphoglycerate by the reduction of NADP to NADPH. This is in contrast to the extensively analysed phosphorylating glyceraldehyde-3-phosphate dehydrogenases which catalyse the reversible reaction of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. Sequence analysis revealed that the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is not related to the phosphorylating glyceraldehyde-3-phosphate dehydrogenases but a member of the aldehyde dehydrogenase superfamily. The aldehyde dehydrogenases are of ancient origin and they have already existed in the progenote as indicated by phylogenetic analysis. Thus the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase can be found in all three domains, archaea, bacteria and eukarya. The catalytic mechanism of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase and the other aldehyde dehydrogenases resembles a thioester mechanism involving the universally conserved cysteine 298 (pea GAPN). The cofactor of the aldehyde dehydrogenases is bound in a new mode to a structure described as beta-alpha,beta-fold.
Assuntos
Evolução Molecular , Gliceraldeído-3-Fosfato Desidrogenases , Sequência de Aminoácidos , Animais , Sequência de Bases , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Cinética , Dados de Sequência Molecular , Fosforilação , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
We studied the expression of arachidonate 5-lipoxygenase (5-LO) in a cell line of human keratinocytes (HaCaT) and in normal human skin keratinocytes in tissue culture. In undifferentiated keratinocytes 5-LO gene expression was low or undetectable as determined by 5-LO mRNA, protein, cell-free enzyme activity, and leukotriene production in intact cells. However, after shift to culture conditions that promote conversion of prokeratinocytes into a more differentiated phenotype, 5-LO gene expression was markedly induced in HaCaT cells and, to a lesser extent, in normal keratinocytes. These results show that 5-LO gene expression is an intrinsic property of human skin keratinocytes.
Assuntos
Araquidonato 5-Lipoxigenase/biossíntese , Regulação Enzimológica da Expressão Gênica , Queratinócitos/metabolismo , Pele/metabolismo , Araquidonato 5-Lipoxigenase/genética , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Indução Enzimática , Humanos , Ácidos Hidroxieicosatetraenoicos/análise , Leucotrieno B4/biossíntese , Leucotrieno C4/biossíntese , Masculino , Pênis/citologia , RNA Mensageiro/análise , Pele/citologiaRESUMO
An assay for the quantitative determination of the hydroxylation profile of long-chain fatty acids is described for gas chromatography negative-ion chemical ionization mass spectrometry and stable isotope dilution using [carboxyl-18O2]-labeled internal standards. The assay has been applied to the study of fatty acids isolated from body fluids, tissue, and cultured cells. Examples for the analyses of biological systems expressing 5-, 8-, 12-, or 15-lipoxygenase activity are given and the most important sources of analytical errors are addressed. Increased specificity compared to analysis by negative-ion chemical ionization, at the cost of sensitivity, can be achieved by the use of positive-ion electron impact ionization for the investigation of hydrogenated pentafluorobenzylester/trimethylsilylether derivatives. The method described provides complete, specific, and quantitative profiles of hydroxylated fatty acids originally present in biological samples or generated in vitro by incubation with polyunsaturated fatty acid substrates such as linoleic or arachidonic acid.
Assuntos
Ácidos Linoleicos Conjugados , Lipoxigenase/metabolismo , Animais , Linhagem Celular , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Ácidos Hidroxieicosatetraenoicos/análise , Ácidos Linoleicos/análise , CamundongosRESUMO
Eicosanoid biosynthesis in animal cells either results from agonist-stimulated phospholipase activation (endogenous pathway) or from lipoprotein receptor-mediated uptake and lysosomal lipid hydrolase-dependent release of AA (exogenous pathway) (see Fig. 1 for schematic representation). LDL stimulates eicosanoid formation through delivery of substrate AA to enzymes of oxidative AA metabolism. The classical LDL receptor is a control point of the effects of LDL AA on eicosanoid formation in different tissues: LDL AA metabolism occurs in several cell types of mesenchymal and epithelial origin and generates the formation of distinct eicosanoid patterns in each case. The LDL AA pathway does appear to couple directly to the PGH synthase reaction, whereas it does not couple directly to the 5-lipoxygenase reaction. We expect that a more complete characterization of the LDL unsaturated fatty acid pathway in different tissue will yield additional information on the biochemistry of lipoproteins, AA, and eicosanoids.
Assuntos
Ácidos Araquidônicos/metabolismo , Eicosanoides/metabolismo , Transdução de Sinais , Animais , Ativação Enzimática , Humanos , Modelos Biológicos , Fosfolipases/metabolismo , Receptores de LDL/fisiologia , Sistemas do Segundo MensageiroRESUMO
Our results can be summarized as follows. LDL stimulates eicosanoid formation through delivery of substrate AA to enzymes of oxidative AA metabolism. The classical LDL receptor controls the effect of LDL AA on eicosanoid formation. LDL AA metabolism occurs in several cell types of mesenchymal and epithelial origin and generates the formation of distinct eicosanoid patterns in a tissue-specific way. LDL inhibits the PGH synthase, and the LDL-dependent inhibition of the enzyme resembles the inhibition by unesterified AA. The LDL AA pathway does appear to couple directly to the PGH synthase reaction, but it does not appear to couple directly to the 5-lipoxygenase reaction. We expect that a more complete characterization of the LDL unsaturated fatty acid pathway in different tissues will yield additional information on the biochemistry of both lipoproteins and AA metabolism.
Assuntos
Ácidos Graxos Insaturados/metabolismo , Lipoproteínas/metabolismo , Receptores de LDL/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Eicosanoides/metabolismo , Fibroblastos/metabolismo , Humanos , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacologia , Modelos Biológicos , Monócitos/metabolismoRESUMO
Non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDH, NADP-specific, EC 1.2.1.9) operates in the cytosol of autotrophic eukaryotes where it generates NADPH for biosynthetic processes from photosynthetic glyceraldehyde 3-phosphate exported from the chloroplast by the phosphate translocator. Here we report the first cloning and characterization of cDNAs encoding complete polypeptide chains of nonphosphorylating GAPDH from pea and maize by using oligonucleotide probes derived from amino acid sequences determined for the purified enzyme. Unexpectedly, nonphosphorylating GAPDH cannot be aligned with the well-known sequences of phosphorylating GAPDH, but shares about 30% amino acid identity with various specialized and non-specialized aldehyde dehydrogenases (ALDHs) of eubacteria and eukaryotes. A phylogenetic analysis of this ALDH superfamily reveals a complex evolutionary pattern with numerous major branches carrying genes from eubacteria, eukaryotes, or both, encoding enzymes that are specific or non-specific for particular aldehyde substrates. This topology suggests a concomitant emergence of multiple substrate specificities from non-specialized ALDH during an early evolutionary phase of intense metabolic diversification. Although unrelated at the sequence level, non-phosphorylating aldehyde dehydrogenases and phosphorylating GAPDH resemble one another with respect to catalytic hydride transfer and covalent thiol ester formation. Whether or not this reflects an ancestral relationship can only be decided when crystallographic data for ALDH enzymes have become available.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/química , Plantas/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , Catálise , Clonagem Molecular , DNA Complementar , Fabaceae/enzimologia , Dados de Sequência Molecular , Fosforilação , Plantas Medicinais , Homologia de Sequência de Aminoácidos , Zea mays/enzimologiaRESUMO
We have isolated and sequenced three cDNA clones from early embryonal stages of Xenopus laevis containing highly repetitive sequences. By means of poly A+ RNA hybridization we could show that simple repeats of the type AC/TG are ubiquitously transcribed in the ovary and in early embryonal stages of Xenopus laevis.
Assuntos
DNA , Regulação da Expressão Gênica no Desenvolvimento , Sequências Repetitivas de Ácido Nucleico , Transcrição Gênica , Animais , Sequência de Bases , DNA Complementar , Dados de Sequência Molecular , Xenopus laevisRESUMO
We have isolated a cDNA clone from a Xenopus laevis tadpole cDNA library which probably codes for the large subunit of glutathione synthetase. The corresponding protein comprises 474 amino acids and shows a significant homology with the large subunit of glutathione synthetase of Schizosaccharomyces pombe. RNase protection experiments revealed that the gene is transcribed during oogenesis and that zygotic expression starts after midblastula transition. Transcripts are also detected in various adult tissues suggesting an ubiquitous distribution of the corresponding protein.