RESUMO
BACKGROUND: The Insulin-like growth factor (IGF) pathway plays a role in tumour development and progression. In vivo, IGF1 activity is regulated by the IGF binding proteins (IGFBPs). IGFBP4 inhibits the activity of IGF1 but proteolytic cleavage by pregnancy-associated plasma protein-A (PAPP-A) releases active IGF1. A modified IGFBP4, dBP4, which was resistant to PAPP-A cleavage but retained IGF1 binding capacity, was engineered, expressed in Human Embryonic Kidney (HEK) 293 cells and purified. This study examined the effects of dBP4 on IGF1-induced cell migration, invasion and angiogenesis in vitro. The effect of intra-tumour injections of dBP4 on tumour angiogenesis and metastasis was examined using the 4T1.2luc orthotopic model of breast cancer. METHODS: PAPP-A resistance and IGF binding capacity of dBP4 were characterized by Western blot and surface plasmon resonance, respectively. 4T1.2luc are mouse mammary adenocarcinoma cells transfected with luciferase to allow in vivo imaging. The effect of dBP4 on IGF1-induced Akt activation in 4T1.2luc cells was assessed by Western blot. Cell migration and invasion assays were performed using 4T1.2luc cells. Angiokit™ assays and Matrigel® implants were used to assess the effects of dBP4 on angiogenesis in vitro and in vivo, respectively. An orthotopic breast cancer model - 4T1.2luc cells implanted in the mammary fat pad of BALB/c mice - was used to assess the effect of intra tumour injection of purified dBP4 on tumour angiogenesis and metastasis. Tumour growth and lung metastasis were examined by in vivo imaging and tumour angiogenesis was evaluated by CD31 immunohistochemistry. RESULTS: Our engineered, PAPP-A resistant IGFBP4 (dBP4) retained IGF1 binding capacity and inhibited IGF1 activation of Akt as well as IGF1-induced migration and invasion by 4T1.2 mammary adenocarcinoma cells. dBP4 inhibited IGF1-induced angiogenesis in vitro and in Matrigel implants in vivo. Direct intra-tumour injection of soluble dBP4 reduced angiogenesis in 4T1.2 luc mammary tumours tumour and reduced lung metastasis. CONCLUSION: A PAPP-A resistant IGFBP4, dBP4, inhibits angiogenesis and metastasis in 4T1.2 mammary fat pad tumours. This study highlights the therapeutic potential of dBP4 as an approach to block the tumour-promoting actions of IGF1.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Neovascularização Patológica/metabolismo , Proteína Plasmática A Associada à Gravidez/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Modelos Animais de Doenças , Feminino , Humanos , Imuno-Histoquímica , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Metástase Neoplásica , Fosforilação , Proteólise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas RecombinantesRESUMO
BACKGROUND: Staphylococcus epidermidis is a commensal of the human skin that has been implicated in infective endocarditis and infections involving implanted medical devices. S. epidermidis induces platelet aggregation by an unknown mechanism. The fibrinogen-binding protein serine-aspartate repeat protein G (SdrG) is present in 67-91% of clinical strains. OBJECTIVES: To determine whether SdrG plays a role in platelet activation, and if so to investigate the role of fibrinogen in this mechanism. METHODS: SdrG was expressed in a surrogate host, Lactococcus lactis, in order to investigate its role in the absence of other staphylococcal components. Platelet adhesion and platelet aggregation assays were employed. RESULTS: L. lactis expressing SdrG stimulated platelet aggregation (lag time: 2.9 +/- 0.5 min), whereas the L. lactis control did not. L. lactis SdrG-induced aggregation was inhibited by alpha(IIb)beta3 antagonists and aspirin. Aggregation was dependent on both fibrinogen and IgG, and the platelet IgG receptor FcgammaRIIa. Preincubation of the bacteria with Bbeta-chain fibrinopeptide inhibited aggregation (delaying the lag time six-fold), suggesting that fibrinogen acts as a bridging molecule. Platelets adhered to L. lactis SdrG in the absence of fibrinogen. Adhesion was inhibited by alpha(IIb)beta3 antagonists, suggesting that this direct interaction involves alpha(IIb)beta3. Investigation using purified fragments of SdrG revealed a direct interaction with the B-domains. Adhesion to the A-domain involved both a fibrinogen and an IgG bridge. CONCLUSION: SdrG alone is sufficient to support platelet adhesion and aggregation through both direct and indirect mechanisms.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Transporte/fisiologia , Ativação Plaquetária , Staphylococcus epidermidis/química , Fibrinogênio/metabolismo , Humanos , Lactococcus lactis/genética , Adesividade PlaquetáriaRESUMO
BACKGROUND: Aspirin (acetylsalicylic acid) irreversibly inhibits platelet cyclooxygenase (COX)-1, the enzyme that converts arachidonic acid (AA) to the potent platelet agonist thromboxane (TX) A2. Despite clear benefit from aspirin in patients with cardiovascular disease (CAD), evidence of heterogeneity in the way individuals respond has given rise to the concept of 'aspirin resistance.' AIMS: To evaluate the hypothesis that incomplete suppression of platelet COX as a consequence of variation in the COX-1 gene may affect aspirin response and thus contribute to aspirin resistance. PATIENTS AND METHODS: Aspirin response, determined by serum TXB2 levels and AA-induced platelet aggregation, was prospectively studied in patients (n = 144) with stable CAD taking aspirin (75-300 mg). Patients were genotyped for five single nucleotide polymorphisms in COX-1 [A-842G, C22T (R8W), G128A (Q41Q), C644A (G213G) and C714A (L237M)]. Haplotype frequencies and effect of haplotype on two platelet phenotypes were estimated by maximum likelihood. The four most common haplotypes were considered separately and less common haplotypes pooled. RESULTS: COX-1 haplotype was significantly associated with aspirin response determined by AA-induced platelet aggregation (P = 0.004; 4 d.f.). Serum TXB2 generation was also related to genotype (P = 0.02; 4 d.f.). CONCLUSION: Genetic variability in COX-1 appears to modulate both AA-induced platelet aggregation and thromboxane generation. Heterogeneity in the way patients respond to aspirin may in part reflect variation in COX-1 genotype.
Assuntos
Aspirina/farmacologia , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/fisiologia , Agregação Plaquetária/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único , Adulto , Ácido Araquidônico , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/genética , Inibidores de Ciclo-Oxigenase/farmacologia , Resistência a Medicamentos/genética , Feminino , Haplótipos , Humanos , Funções Verossimilhança , Masculino , Farmacogenética , Agregação Plaquetária/genética , Estudos Prospectivos , Tromboxano A2/sangueRESUMO
Shedding of the ectodomain of angiotensin-converting enzyme (ACE) and numerous other membrane-anchored proteins results from a specific cleavage in the juxtamembrane (JM) stalk, catalyzed by "sheddases" that are commonly activated by phorbol esters and inhibited by peptide hydroxamates such as TAPI. Sheddases require a stalk of minimum length and steric accessibility. However, we recently found that substitution of the ACE stalk with an epidermal growth factor (EGF)-like domain from the low-density lipoprotein receptor (LDL-R) did not abolish shedding; cleavage of the ACE-JMEGF chimera occurred at a Gly-Phe bond in the third disulfide loop of the EGF domain. We have now constructed two additional stalk chimeras, in which the native stalk in ACE was replaced with the EGF domain from factor IX (ACE-JMfIX) and with a cysteine knot motif (ACE-JMmin23). Like the ACE-JMEGF chimera, the ACE-JMfIX and -JMmin23 chimeras were also shed, but mass spectral analysis revealed that the cleavage sites were adjacent to, rather than within, the disulfide-bonded domains. Homology modeling of the LDL-R EGF domain revealed that the third disulfide loop is larger and more flexible than the equivalent loop in the factor IX EGF domain. Similarly, the NMR structure of the Min-23 motif is highly compact. Hence, cleavage within a disulfide-bonded domain appears to require an unhindered loop. Interestingly, unlike wild-type ACE and the ACE-JMEGF and -JMmin23 chimeras, shedding of the ACE-JMfIX chimera was not stimulated by phorbol or inhibited by TAPI, but instead was inhibited by 3,4-dichloroisocoumarin, indicating the activity of an alternative sheddase. In summary, the ACE shedding machinery is highly versatile, but an accessible JM sequence, in the form of a flexible stalk or an exposed loop within or adjacent to a folded domain, appears to be required. Moreover, alternative sheddases are recruited, depending on the nature of the JM sequence.
Assuntos
Dissulfetos/metabolismo , Proteínas de Membrana/metabolismo , Peptidil Dipeptidase A/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Células CHO , Cricetinae , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/genética , Vetores Genéticos/metabolismo , Humanos , Hidrólise , Cinética , Proteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Peptidil Dipeptidase A/genética , Estrutura Terciária de Proteína/genética , Receptores de LDL/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismoRESUMO
Angiotensin-converting enzyme (ACE) is one of a growing number of integral membrane proteins that is shed from the cell surface through proteolytic cleavage by a secretase. To investigate the requirements for ectodomain shedding, we replaced the glycosylphosphatidylinositol addition sequence in membrane dipeptidase (MDP) - a membrane protein that is not shed - with the juxtamembrane stalk, transmembrane (TM) and cytosolic domains of ACE. The resulting construct, MDP-STM(ACE), was targeted to the cell surface in a glycosylated and enzymically active form, and was shed into the medium. The site of cleavage in MDP-STM(ACE) was identified by MS as the Arg(374)-Ser(375) bond, corresponding to the Arg(1203)-Ser(1204) secretase cleavage site in somatic ACE. The release of MDP-STM(ACE) and ACE from the cells was inhibited in an identical manner by batimastat and two other hydroxamic acid-based zinc metallosecretase inhibitors. In contrast, a construct lacking the juxtamembrane stalk, MDP-TM(ACE), although expressed at the cell surface in an enzymically active form, was not shed, implying that the juxtamembrane stalk is the critical determinant of shedding. However, an additional construct, ACEDeltaC, in which the N-terminal domain of somatic ACE was fused to the stalk, TM and cytosolic domains, was also not shed, despite the presence of a cleavable stalk, implying that in contrast with the C-terminal domain, the N-terminal domain lacks a signal required for shedding. These data are discussed in the context of two classes of secretases that differ in their requirements for recognition of substrate proteins.
Assuntos
Membranas Intracelulares/química , Peptidil Dipeptidase A/química , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Arginina/química , Ácido Aspártico Endopeptidases , Western Blotting , Divisão Celular , Citosol/química , DNA Complementar/metabolismo , Dipeptidases/química , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Endopeptidases/química , Inibidores Enzimáticos/farmacologia , Humanos , Espectrometria de Massas , Metaloendopeptidases/química , Microscopia Confocal , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Serina/química , Transfecção , Células Tumorais CultivadasRESUMO
Methylarginines inhibit nitric oxide synthases (NOS). Cellular concentrations of methylarginines are determined in part by the activity of dimethylarginine dimethylaminohydrolase (DDAH; EC 3.5.3. 18). We have cloned human DDAH and identified and expressed a second novel DDAH isoform (DDAH I and II respectively). DDAH I predominates in tissues that express neuronal NOS. DDAH II predominates in tissues expressing endothelial NOS. These results strengthen the hypothesis that methylarginine concentration is actively regulated and identify molecular targets for the tissue and cell-specific regulation of methylarginine concentration.
Assuntos
Amidoidrolases , Hidrolases/metabolismo , Isoenzimas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Humanos , Hidrolases/química , Hidrolases/genética , Isoenzimas/química , Isoenzimas/genética , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The role of juxtamembrane stalk glycosylation in modulating stalk cleavage and shedding of membrane proteins remains unresolved, despite reports that proteins expressed in glycosylation-deficient cells undergo accelerated proteolysis. We have constructed stalk glycosylation mutants of angiotensin-converting enzyme (ACE), a type I ectoprotein that is vigorously shed when expressed in Chinese hamster ovary cells. Surprisingly, stalk glycosylation did not significantly inhibit release. Introduction of an N-linked glycan directly adjacent to the native stalk cleavage site resulted in a 13-residue, proximal displacement of the cleavage site, from the Arg-626/Ser-627 to the Phe-640/Leu-641 bond. Substitution of the wild-type stalk with a Ser-/Thr-rich sequence known to be heavily O-glycosylated produced a mutant (ACE-JGL) in which this chimeric stalk was partially O-glycosylated; incomplete glycosylation may have been due to membrane proximity. Relative to levels of cell-associated ACE-JGL, rates of basal, unstimulated release of ACE-JGL were enhanced compared with wild-type ACE. ACE-JGL was cleaved at an Ala/Thr bond, 14 residues from the membrane. Notably, phorbol ester stimulation and TAPI (a peptide hydroxamate) inhibition of release-universal characteristics of regulated ectodomain shedding-were significantly blunted for ACE-JGL, as was a formerly undescribed transient stimulation of ACE release by 3, 4-dichloroisocoumarin. These data indicate that (1) stalk glycosylation modulates but does not inhibit ectodomain shedding; and (2) a Ser-/Thr-rich, O-glycosylated stalk directs cleavage, at least in part, by an alternative shedding protease, which may resemble an activity recently described in TNF-alpha convertase null cells [Buxbaum, J. D., et al. (1998) J. Biol. Chem. 273, 27765-27767].
Assuntos
Proteínas de Membrana/metabolismo , Peptidil Dipeptidase A/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Carboidratos/análise , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Cricetinae , Glicosilação , Humanos , Cinética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/biossíntese , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de Sequência , SolubilidadeRESUMO
Specialized proteases, referred to as sheddases, secretases, or membrane-protein-solubilizing proteases (MPSPs), solubilize the extracellular domains of diverse membrane proteins by catalyzing a specific cleavage in the juxtamembrane stalk regions of such proteins. A representative MPSP (tumor necrosis factor-alpha convertase) was cloned recently and shown to be a disintegrin metalloprotease that is inhibited by peptide hydroxamates including the compound TAPI. Substrate determinants that specify cleavage by MPSPs remain incompletely characterized, but may include the physicochemical properties of the stalk or unidentified recognition motifs in the stalk or the extracellular domain. We constructed a mutant angiotensin-converting enzyme (ACE) in which the stalk has been replaced with an epidermal growth factor (EGF)-like domain (ACE-JMEGF), to test the hypothesis that MPSP cleavage requires an open, comparatively unfolded or extended stalk. Wild-type ACE is a type I transmembrane (TM) ectoprotein that is efficiently solubilized by a typical MPSP activity. We found that ACE-JMEGF was solubilized inefficiently and accumulated in a cell-associated form on transfected Chinese hamster ovary (CHO) cells; cleavage was stimulated by phorbol ester and inhibited by TAPI, features typical of MPSP activity. Determination of the C-terminus of soluble ACE-JMEGF revealed that, surprisingly, cleavage occurred at a Gly-Phe bond between the fifth and sixth cysteines within the third disulfide loop of the EGF-like domain. Reduction of intact CHO cells with tributylphosphine resulted in the rapid release of ACE-JMEGF (but not wild-type ACE) into the medium, suggesting that a proportion of membrane-bound ACE-JMEGF is cleaved but remains cell-associated via disulfide tethering. The mechanism for the release of ACE-JMEGF in the absence of chemical reduction is unclear. We conclude that the presence of a compact, disulfide-bridged domain does not per se inhibit cleavage by an MPSP activity, but ectodomain release is prevented by disulfide tethering to the TM domain.
Assuntos
Dissulfetos/farmacologia , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/fisiologia , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/fisiologia , Dibutirato de 12,13-Forbol/farmacologia , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Sítios de Ligação/genética , Células CHO , Fracionamento Celular , Cricetinae , Dipeptídeos/farmacologia , Fator de Crescimento Epidérmico/genética , Hidrólise/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Cinética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Peptidil Dipeptidase A/genética , Substâncias Redutoras/farmacologiaRESUMO
MtDNA sequencing was used to assess the phylogeographic structure of four species of Hawaiian freshwater fishes: Lentipes concolor, Stenogobius hawaiiensis, Sicyopterus stimpsoni, and Awaous guamensis. Samples of each species were collected from streams on the northeast side of Kauai, Maui, Molokai, Oahu, and Hawaii. We sequenced segments from both coding and noncoding regions (638-1391 bp) in each species. Sequence analysis uncovered genetic variability in these fishes but no evidence of strong geographic structure among island populations. This result is most readily explained by the fishes' larval marine life stage (amphidromy), which likely facilitates gene flow among island populations. By constraining genetic differentiation among populations, amphidromy may impede speciation in these fishes, possibly explaining why the Hawaiian freshwater fish fauna is depauperate compared to other species-rich Hawaiian faunas. It may also provide them with a kind of evolutionary flexibility atypical of other, more isolated island faunas and allow natural restocking to occur in streams that have been restored to suitable conditions. Comparisons of restriction site and sequence data suggested similar population genetic conclusions for all species except S. stimpsoni, for which the restriction site data is questioned.
Assuntos
DNA Mitocondrial , Peixes/genética , Variação Genética , Animais , Sítios de Ligação , Peixes/classificação , Água Doce , Havaí , Filogenia , Mapeamento por Restrição , Análise de Sequência de DNAAssuntos
Óxido Nítrico Sintase/genética , Animais , Baculoviridae , Western Blotting/métodos , Linhagem Celular , Clonagem Molecular/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Vetores Genéticos , Humanos , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo III , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Spodoptera , Transfecção/métodos , Ensaio de Placa Viral/métodosRESUMO
Diverse membrane proteins are solubilized by a specific proteolytic cleavage in the stalk sequence adjacent to the membrane anchor, with release of the extracellular domain. Examples are the amyloid precursor protein, membrane-bound growth factors and angiotensin-converting enzyme (ACE). The identities and characteristics of the responsible proteases remain elusive. We have studied this process in Chinese hamster ovary (CHO) cells stably expressing wild-type ACE (WT-ACE) or juxtamembrane (stalk) deletion or chimaera mutants. Determination of the C termini (i.e. the cleavage sites) of released, soluble wild-type and mutant ACE by MALDI-TOF mass spectrometry indicated that the membrane-protein-solubilizing protease (MPSP) in CHO cells is not constrained by a particular cleavage site motif or by a specific distance from the membrane, but instead may position itself with respect to the putative proximal, folded extracellular domain adjacent to the stalk. Nevertheless, kinetic analyses of release rates indicated that a minimum distance from the membrane must be preserved. Interestingly, soluble full-length (anchor-plus) WT-ACE incubated with fractions of, or intact, CHO cells was not cleaved. In all cases, release was stimulated by a media change or by the addition of phorbol ester, with rate enhancements of 5- and 50-fold, respectively, for WT-ACE. The phorbol ester effect was abolished by staurosporine, a protein kinase C (PKC) inhibitor. We propose that the CHO cell MPSP that solubilizes ACE: (1) only cleaves proteins embedded in a membrane; (2) requires an accessible stalk and cleaves at a minimum distance from both the membrane and proximal extracellular domain; (3) positions itself primarily with respect to the proximal extracellular domain and (4) is regulated in part by a PKC-dependent mechanism.
Assuntos
Células CHO/metabolismo , Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Peptidil Dipeptidase A/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Células CHO/citologia , Células CHO/enzimologia , Cricetinae , Humanos , Cinética , Metaloproteinases da Matriz Associadas à Membrana , Proteínas de Membrana/química , Proteínas de Membrana/genética , Metaloendopeptidases/química , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Mutação/genética , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizAssuntos
Isoenzimas/biossíntese , Óxido Nítrico Sintase/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Baculoviridae , Encéfalo/enzimologia , Cartilagem/efeitos dos fármacos , Cartilagem/enzimologia , Linhagem Celular , Cromatografia de Afinidade/métodos , Clonagem Molecular/métodos , Técnicas de Cultura/métodos , Primers do DNA , DNA Complementar , Eletroforese em Gel de Poliacrilamida/métodos , Endotélio Vascular/enzimologia , Feminino , Expressão Gênica , Biblioteca Gênica , Humanos , Interleucina-1/farmacologia , Isoenzimas/isolamento & purificação , Neurônios/enzimologia , Óxido Nítrico Sintase/isolamento & purificação , Placenta/enzimologia , Reação em Cadeia da Polimerase/métodos , Gravidez , RNA Mensageiro/biossíntese , RNA Mensageiro/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Mapeamento por Restrição , Spodoptera , Transfecção/métodos , Veias UmbilicaisRESUMO
We have previously demonstrated high expression of rat neuronal nitric oxide synthase (NO synthase) in a baculovirus system [Charles, I. G., Chubb, A., Gill, R., Clare, J., Lowe, P. N., Holmes, L. S., Page, M., Keeling, J. G., Moncada, S. & Riveros-Moreno, V. (1993) Biochem. Biophys. Res. Commun. 196, 1481-1489], where a small proportion of the expressed enzyme was soluble and active, but the majority was insoluble (approximately 15% of the total insoluble proteins). NO synthase is a complex enzyme, requiring several cofactors for full activity. These include tightly bound FAD, FMN, heme and tetrahydrobiopterin, in addition to calmodulin and NADPH. Here, we report that a substantial proportion of the total NO synthase produced becomes soluble following addition of hemin (2.5 micrograms/ml) to the culture medium. However, the enzyme purified under these conditions had very low specific activity, 50 nmol.min-1.mg-1, after ADP-Sepharose affinity purification. Full activity (approximately 800 nmol.min-1.mg-1) could, however, be obtained by including precursors for the cofactors, nicotinic acid, riboflavin, and sepiapterin in the culture medium. We demonstrate that the enzyme activity is exclusively associated with the dimeric form of the enzyme, which had the following molar ratios for the cofactors: heme, 0.92; FAD, 0.57; FMN, 0.34; H4biopterin, 0.32, with a specific activity of 1500 nmol.min-1.mg-1. The provision of substantial quantities of good quality enzyme, as described here, will facilitate the studies on the relationship between enzyme structure and its mechanism of catalysis.
Assuntos
Aminoácido Oxirredutases/isolamento & purificação , Encéfalo/enzimologia , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Animais , Baculoviridae/genética , DNA Complementar/química , Hemina/farmacologia , Concentração de Íons de Hidrogênio , Óxido Nítrico Sintase , Ratos , Proteínas Recombinantes/isolamento & purificação , SolubilidadeRESUMO
Discusses Nottingham's approach to developing a public health role explicitly alongside traditional health visiting. Argues that their approach makes significant improvements to the health status of a selected deprived area.
Assuntos
Enfermagem em Saúde Comunitária/organização & administração , Área Carente de Assistência Médica , Modelos de Enfermagem , Prevenção de Acidentes , Inglaterra , Humanos , Áreas de Pobreza , Desenvolvimento de Programas/métodosRESUMO
Incubation of human articular chondrocytes with interleukin 1 beta results in the time-dependent expression of nitric oxide (NO) synthase. We report here the isolation of a cDNA clone which encodes a protein of 1153 amino acids with a molecular mass of 131,213 Da and a calculated isoelectric point of 7.9. CHO cells transfected with a plasmid harboring this cDNA clone expressed NO synthase activity that was inhibited by some L-arginine analogues. The deduced amino acid sequence of the human chondrocyte inducible NO synthase shows 51% identity and 68% similarity with the endothelial NO synthase and 54% identity and 70% similarity with the neuronal NO synthase. The similarity (88%) between the human chondrocyte NO synthase cDNA sequence and that reported for the murine macrophage suggests that the inducible class of enzyme is conserved between different cell types and across species.
Assuntos
Aminoácido Oxirredutases/genética , Cartilagem/enzimologia , Aminoácido Oxirredutases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Clonagem Molecular , Cricetinae , DNA Complementar/genética , Ativação Enzimática , Genes , Humanos , Interleucina-1/farmacologia , Dados de Sequência Molecular , Óxido Nítrico Sintase , Oligodesoxirribonucleotídeos/química , Proteínas Recombinantes , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
The surface antigen P.69/pertactin of Bordetella pertussis has been expressed using the polyhedron promoter of baculovirus in cultured insect cells. Either full-length or truncated prn DNA was used to express P.69 pertactin. The full-length gene gave rise to low levels of P.93 precursor protein, some of which was processed to P.69. The shortened prn expressed P.69 pertactin directly at levels up to 3.5 mg per litre. P.69 vaccinated animals were protected against aerosol challenge with virulent B. pertussis bacteria.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Baculoviridae/imunologia , Bordetella pertussis/imunologia , Fatores de Virulência de Bordetella , Animais , Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Bordetella pertussis/genética , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Genes Bacterianos/genética , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Vacina contra Coqueluche/imunologiaRESUMO
A DNA sequence encoding rat neuronal NO synthase (nNOS) was isolated and cloned into the baculovirus expression vector pVL1393 to generate pVLRBNOS. Transfection of Spodoptera frugiperda Sf-21 cells with the construct pVLRBNOS resulted in the synthesis of high levels of neuronal NO synthase. Analysis of the expression pattern revealed soluble, enzymatically active NO synthase in the cytoplasm of cell extracts. Active enzyme could also be purified from culture supernatants using 2'-5' ADP sepharose affinity chromatography. This enzyme was recognised by antibodies to the native nNOS and showed a similar degree of inhibition by arginine analogs as the native nNOS. The majority of the NOS synthesised had accumulated as insoluble "inclusion-body" material. The purification of recombinant nNOS from insect cells should facilitate characterisation of neuronal NO synthase.
Assuntos
Aminoácido Oxirredutases/biossíntese , Aminoácido Oxirredutases/genética , Encéfalo/enzimologia , Neurônios/enzimologia , Aminoácido Oxirredutases/isolamento & purificação , Animais , Baculoviridae , Sequência de Bases , Western Blotting , Linhagem Celular , Clonagem Molecular , Primers do DNA , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Biblioteca Gênica , Dados de Sequência Molecular , Mariposas , Óxido Nítrico Sintase , Oligonucleotídeos Antissenso , Reação em Cadeia da Polimerase , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Mapeamento por Restrição , TransfecçãoRESUMO
In humans, the H1 RNA, the RNA subunit of RNase P, is synthesized by RNA polymerase III. We have used block replacement mutagenesis to identify the sequences necessary for in vitro transcription of H1 RNA. We find that multiple cis-acting elements located in the H1 RNA 5'-flanking region are necessary for H1 RNA synthesis; no internal sequences are essential. Required cis-acting elements include sequences resembling proximal sequence element, distal sequence element, and TATA motifs. In this respect, the H1 RNA promoter is similar in structure to the promoters of the genes encoding the U6 snRNA, the 7 SK RNA and the MRP RNA. However, our mutational analysis indicates that the H1 promoter is unexpectedly complex, with several additional cis-acting elements spanning nearly 70 base pairs of the H1 RNA gene 5'-flanking sequence.
Assuntos
Endorribonucleases/genética , Regulação Enzimológica da Expressão Gênica , Regiões Promotoras Genéticas , RNA Polimerase III/metabolismo , RNA Catalítico/genética , RNA/genética , Transcrição Gênica , Sequência de Bases , DNA , Endorribonucleases/metabolismo , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , RNA Catalítico/metabolismo , Ribonuclease P , Moldes GenéticosRESUMO
A combination of the 69-kDa outer membrane protein and filamentous hemagglutinin (FHA), both isolated from lymphocytosis promoting factor (LPF; pertussis toxin) minus mutants of Bordetella pertussis, is protective in the mouse intracerebral challenge potency (Kendrick) test. A combination of the same 69-kDa protein and LPF is approximately 15 times less effective. These data suggest that, surprisingly, the 69-kDa protein in tandem with FHA is the most relevant combination for mouse protection; consequently such a combination may be a more suitable acellular pertussis vaccine candidate than the LPF/FHA combinations, which have never been satisfactorily protective in the mouse test. Preparation of samples of the 69-kDa protein of acceptable protective quality remains difficult. Attempts were made to screen the most suitable batches of the preparations by exploiting some recently discovered properties of the 69-kDa protein: the characteristic chromatofocusing pattern of the protein, the affinity for lymphocytes, and the ability to bind to nicotinamide adenine dinucleotide. None of these tests was able to replace the mouse intracerebral challenge potency test for final quality assessment.