RESUMO
The global malaria agenda has undergone a reorientation from control of clinical cases to entirely eradicating malaria. For that purpose, a key objective is blocking transmission of malaria parasites from humans to mosquito vectors. The new antimalarial drug candidate NITD609 was evaluated for its transmission-reducing potential and compared to a few established antimalarials (lumefantrine, artemether, primaquine), using a suite of in vitro assays. By the use of a microscopic readout, NITD609 was found to inhibit the early and late development of Plasmodium falciparum gametocytes in vitro in a dose-dependent fashion over a range of 5 to 500 nM. In addition, using the standard membrane feeding assay, NITD609 was also found to be a very effective drug in reducing transmission to the Anopheles stephensi mosquito vector. Collectively, our data suggest a strong transmission-reducing effect of NITD609 acting against different P. falciparum transmission stages.
Assuntos
Anopheles/parasitologia , Antimaláricos/farmacologia , Gametogênese/efeitos dos fármacos , Indóis/farmacologia , Insetos Vetores/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/patogenicidade , Compostos de Espiro/farmacologia , AnimaisRESUMO
Our previous study demonstrated that, stanniocalcin-1 (STC1) was a target of histone deacetylase (HDAC) inhibitors and was involved in trichostatin A (TSA) induced apoptosis in the human colon cancer cells, HT29. In this study, we reported that the transcriptional factor, specificity protein 1 (Sp1) in association with retinoblastoma (Rb) repressed STC1 gene transcription in TSA-treated HT29 cells. Our data demonstrated that, a co-treatment of the cells with TSA and Sp1 inhibitor, mithramycin A (MTM) led to a marked synergistic induction of STC1 transcript levels, STC1 promoter (1 kb)-driven luciferase activity and an increase of apoptotic cell population. The knockdown of Sp1 gene expression in TSA treated cells, revealed the repressor role of Sp1 in STC1 transcription. Using a protein phosphatase inhibitor okadaic acid (OKA), an increase of Sp1 hyperphosphorylation and so a reduction of its transcriptional activity, led to a significant induction of STC1 gene expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binding on STC1 proximal promoter in TSA treated cells. The binding of Sp1 to STC1 promoter was abolished by the co-treatment of MTM or OKA in TSA-treated cells. Re-ChIP assay illustrated that Sp1-mediated inhibition of STC1 transcription was associated with the recruitment of another repressor molecule, Rb. Collectively our findings identify STC1 is a downstream target of Sp1.
Assuntos
Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicoproteínas/biossíntese , Ácidos Hidroxâmicos/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Repressoras/metabolismo , Fator de Transcrição Sp1/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/genética , Glicoproteínas/genética , Humanos , Ácido Okadáico/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Plicamicina/análogos & derivados , Plicamicina/farmacologia , Proteínas Repressoras/genética , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Fator de Transcrição Sp1/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
The critical events in clearance or persistence of hepatitis C virus (HCV) infection are unknown but likely to be determined early in acute infection. Type 1 and type 2 cytokine production was assessed by HCV peptide ELISpot and multiplex in vitro cytokine production assays in longitudinally collected samples from 20 untreated participants enrolled in the Australian Trial in Acute Hepatitis C (ATAHC); a prospective cohort of acute HCV infection (77% injecting drug users, IDU). Significantly higher interleukin-10 (IL-10) production (P = 0.048), in the relative absence of interferon-gamma (IFN-γ) and IL-2 production, was present early in HCV infection in those who progressed to chronic infection. In contrast, viral clearance was associated with a greater magnitude and broader specificity of IFN-γ (magnitude P < 0.001, breadth P = 0.004) and IL-2 responses, in the relative absence of IL-10. Early IL-10 production was correlated with higher HCV RNA level at baseline (P = 0.046) and week 12 (P = 0.018), while IFN-γ and IL-2 production was inversely correlated with HCV RNA level at baseline (IFN-γ P = 0.020, IL-2 P = 0.050) and week 48 (IFN-γ P = 0.045, IL-2 P = 0.026). Intracellular staining (ICS) indicated the HCV-specific IFN-γ response was primarily from CD8(+) T cells and NK cells, whereas IL-10 production was predominantly from monocytes, with a subset of IL-10 producing CD8(+) T cells present only in those who progressed to chronic infection. IL-10, an immunoregulatory cytokine, appears to play a key role in progression to chronic HCV infection.
Assuntos
Progressão da Doença , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Interleucina-10/imunologia , RNA Viral/sangue , Adolescente , Adulto , Austrália , Usuários de Drogas , ELISPOT/métodos , Feminino , Hepacivirus/patogenicidade , Hepatite C Crônica/virologia , Humanos , Interferon gama/análise , Interferon gama/imunologia , Interleucina-10/análise , Interleucina-2/análise , Interleucina-2/imunologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto JovemRESUMO
Clathrin-coated vesicles mediate diverse processes such as nutrient uptake, downregulation of hormone receptors, formation of synaptic vesicles, virus entry, and transport of biosynthetic proteins to lysosomes. Cycles of coat assembly and disassembly are integral features of clathrin-mediated vesicular transport (Fig. 1a). Coat assembly involves recruitment of clathrin triskelia, adaptor complexes and other factors that influence coat assembly, cargo sequestration, membrane invagination and scission (Fig. 1a). Coat disassembly is thought to be essential for fusion of vesicles with target membranes and for recycling components of clathrin coats to the cytoplasm for further rounds of vesicle formation. In vitro, cytosolic heat-shock protein 70 (Hsp70) and the J-domain co-chaperone auxilin catalyse coat disassembly. However, a specific function of these factors in uncoating in vivo has not been demonstrated, leaving the physiological mechanism and significance of uncoating unclear. Here we report the identification and characterization of a Saccharomyces cerevisiae J-domain protein, Aux1. Inactivation of Aux1 results in accumulation of clathrin-coated vesicles, impaired cargo delivery, and an increased ratio of vesicle-associated to cytoplasmic clathrin. Our results demonstrate an in vivo uncoating function of a J domain co-chaperone and establish the physiological significance of uncoating in transport mediated by clathrin-coated vesicles.
Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , DNA Fúngico/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Sequência de Aminoácidos , Animais , Transporte Biológico Ativo , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Modelos Biológicos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Fosfoproteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
The function of natural killer T (NKT) cells in the immune system has yet to be determined. There is some evidence that their defect is associated with autoimmunity, but it is still unclear how they play a role in regulating the pathogenesis of T cell-mediated autoimmune diseases. It was originally proposed that NKT cells could control autoimmunity by shifting the cytokine profile of autoimmune T cells toward a protective T helper 2 cell (Th2) type. However, it is now clear that the major function of NKT cells in the immune system is not related to their interleukin (IL)-4 secretion. In fact, NKT cells mainly secrete interferon (IFN)-gamma and, activated in the presence of IL-12, acquire a strong inflammatory phenotype and cytotoxic function.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Interferon gama/biossíntese , Interleucina-12/fisiologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Autoimunidade , Diferenciação Celular , Diabetes Mellitus Tipo 1/genética , Inflamação , Interleucina-12/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Fenótipo , Células Th2/imunologiaRESUMO
BACKGROUND: The Australian Trial in Acute Hepatitis C (ATAHC) is a National Institutes of Health-funded prospective cohort study of the natural history and efficacy of treatment in individuals with recently acquired hepatitis C. Enrollment is open to both human immunodeficiency virus (HIV)-infected and -uninfected individuals. The aim of this article was to evaluate characteristics and virological outcomes among HIV-infected individuals enrolled in ATAHC. METHODS: Eligibility criteria included the first positive result of testing for anti-hepatitis C virus (HCV) antibody within 6 months and either clinical hepatitis diagnosed within the past 12 months or documented anti-HCV seroconversion within the past 24 months. RESULTS: Of the initial 103 patients enrolled, 27 (26%) were HIV infected. HIV-infected patients were more likely to be older, to have HCV genotype 1 infection and high levels of HCV RNA at baseline than were HCV-monoinfected patients. Sexual acquisition accounted for the majority (56%) of HCV infections among HIV-infected patients, compared with only 8% of HCV-monoinfected patients. The median duration from estimated HCV infection to treatment was 30 weeks. Treatment with 24 weeks of pegylated interferon and ribavirin resulted in rates of undetectability of HCV RNA of 95%, 90%, and 80% at weeks 12, 24, and 48, respectively. Undetectability at week 4 was achieved in 44% of patients and yielded positive and negative predictive values for sustained virological response of 100% and 33%, respectively. CONCLUSIONS: Significant differences were demonstrated between HIV-infected and HIV-uninfected individuals enrolled in ATAHC. Treatment responses among HIV-infected individuals with both acute and early chronic infection are encouraging and support regular HCV screening of high-risk individuals and early treatment for recently acquired HCV infection.
Assuntos
Infecções por HIV/complicações , Hepatite C/tratamento farmacológico , Hepatite C/patologia , Adulto , Fatores Etários , Antivirais/uso terapêutico , Austrália , Estudos de Coortes , Feminino , Genótipo , Hepacivirus/classificação , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Humanos , Interferons/uso terapêutico , Estudos Longitudinais , Masculino , Estudos Prospectivos , RNA Viral/sangue , Ribavirina/uso terapêutico , Fatores de Risco , Resultado do Tratamento , Carga ViralRESUMO
Enzymatic ligation of discrete nanoparticle-DNA conjugates creates nanoparticle dimer and trimer structures in which the nanoparticles are linked by single-stranded DNA, rather than by double-stranded DNA as in previous experiments. Ligation was verified by agarose gel and small-angle X-ray scattering. This capability was utilized in two ways: first, to create a new class of multiparticle building blocks for nanoscale self-assembly and, second, to develop a system that can amplify a population of discrete nanoparticle assemblies.
Assuntos
Adutos de DNA/síntese química , DNA Ligases/química , Nanopartículas Metálicas/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Adutos de DNA/química , DNA de Cadeia Simples , Eletroforese em Gel de Poliacrilamida , Ouro/química , Modelos Moleculares , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Chemoresistance and therapeutic selectivity are major obstacles to successful chemotherapy of ovarian cancer. Manganese superoxide disumutase (MnSOD) is an important antioxidant enzyme responsible for the elimination of superoxide radicals. We reported here that MnSOD was significantly elevated in ovarian cancer cells and its overexpression was one of the mechanisms that increased resistance to apoptosis in cancer cells. Knockdown of MnSOD by small-interfering RNA (siRNA) led to an increase in superoxide generation and sensitisation of ovarian cancer cells to the two front-line anti-cancer agents doxorubicin and paclitaxel whose action involved free-radical generation. This synergistic effect was not observed in non-transformed ovarian surface epithelial cells. Furthermore, our results revealed that this combination at the cellular level augmented activation of caspase-3 and caspase-9, but not caspase-8, suggesting involvement of an intrinsic apoptotic pathway. Evaluation of signalling pathways showed that MnSOD siRNA enhanced doxorubicin- and paclitaxel-induced phosphorylation of extracellular signal-regulated kinase 1/2. Akt activation was not affected. These results identify a novel chemoresistance mechanism in ovarian cancer, and show that combination of drugs capable of suppressing MnSOD with conventional chemotherapeutic agents may provide a novel strategy with a superior therapeutic index and advantage for the treatment of refractory ovarian cancer.
Assuntos
Caspase 9/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/enzimologia , Superóxido Dismutase/antagonistas & inibidores , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Caspase 9/genética , Inibidores de Caspase , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Feminino , Humanos , Oligopeptídeos/farmacologia , Neoplasias Ovarianas/genética , Paclitaxel/farmacologia , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , TransfecçãoRESUMO
The Hippo pathway is a central regulator of tissue development and homeostasis, and has been reported to have a role during vascular development. Here we develop a bioluminescence-based biosensor that monitors the activity of the Hippo core component LATS kinase. Using this biosensor and a library of small molecule kinase inhibitors, we perform a screen for kinases modulating LATS activity and identify VEGFR as an upstream regulator of the Hippo pathway. We find that VEGFR activation by VEGF triggers PI3K/MAPK signaling, which subsequently inhibits LATS and activates the Hippo effectors YAP and TAZ. We further show that the Hippo pathway is a critical mediator of VEGF-induced angiogenesis and tumor vasculogenic mimicry. Thus, our work offers a biosensor tool for the study of the Hippo pathway and suggests a role for Hippo signaling in regulating blood vessel formation in physiological and pathological settings.
Assuntos
Técnicas Biossensoriais , Transdução de Sinais/fisiologia , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Western Blotting , Feminino , Células HEK293 , Humanos , Imuno-Histoquímica , Mutagênese Sítio-Dirigida , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
In Saccharomyces cerevisiae, clathrin is necessary for localization of trans-Golgi network (TGN) membrane proteins, a process that involves cycling of TGN proteins between the TGN and endosomes. To characterize further TGN protein localization, we applied a screen for mutations that cause severe growth defects in combination with a temperature-sensitive clathrin heavy chain. This screen yielded a mutant allele of RIC1. Cells carrying a deletion of RIC1 (ric1Delta) mislocalize TGN membrane proteins Kex2p and Vps10p to the vacuole. Delivery to the vacuole occurs in ric1Delta cells also harboring end3Delta to block endocytosis, indicative of a defect in retrieval to the TGN rather than sorting to endosomes. SYS1, originally discovered as a multicopy suppressor of defects caused by the absence of the Rab GTPase YPT6, was identified as a multicopy suppressor of ric1Delta. Further comparison of ric1Delta and ypt6Delta cells demonstrated identical phenotypes. Multicopy plasmids expressing v-SNAREs Gos1p or Ykt6p, but not other v- and t-SNAREs, partially suppressed phenotypes of ric1Delta and ypt6Delta cells. SLY1-20, a dominant activator of the cis-Golgi network t-SNARE Sed5p, also functioned as a multicopy suppressor. Because Gos1p and Ykt6p interact with Sed5p, these results raise the possibility that TGN membrane protein localization requires Ric1p- and Ypt6p-dependent retrieval to the cis-Golgi network.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Proteínas de Transporte Vesicular , Rede trans-Golgi/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/farmacologia , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/farmacologia , Fatores de Troca do Nucleotídeo Guanina , Membranas Intracelulares/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/farmacologia , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Fenótipo , Transporte Proteico/efeitos dos fármacos , Proteínas Qb-SNARE , Proteínas SNARE , Saccharomyces cerevisiae/química , Supressão Genética/efeitos dos fármacos , Fatores de Transcrição/genética , Transformação Genética , Rede trans-Golgi/genética , Rede trans-Golgi/metabolismoRESUMO
Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes in Saccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and alpha-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or alpha-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the beta subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.
Assuntos
Clatrina/genética , Proteínas do Tecido Nervoso/genética , Fosfoproteínas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Carboxipeptidases/metabolismo , Catepsina A , Clatrina/química , Clonagem Molecular , Vesículas Revestidas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Mutação , Proteínas do Tecido Nervoso/química , Fosfoproteínas/química , Testes de Precipitina , Ligação ProteicaRESUMO
A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is reduced in pep12 Delta and vps34 Delta, two mutants that block Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p. Interestingly, a Vps10C(t) Delta p mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.
Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Endossomos/metabolismo , Proteínas Fúngicas/metabolismo , Pró-Proteína Convertases , Receptores de Superfície Celular/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Rede trans-Golgi/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Membrana Celular/metabolismo , Clatrina/genética , Clatrina/metabolismo , Proteínas Fúngicas/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Testes de Precipitina , Transporte Proteico , Proteínas Qa-SNARE , Receptores de Superfície Celular/genética , Subtilisinas/genética , Subtilisinas/metabolismo , Leveduras/genética , Leveduras/metabolismoRESUMO
Malaria control and elimination are threatened by the emergence and spread of resistance to artemisinin-based combination therapies (ACTs). Experimental evidence suggests that when an artemisinin (ART)-sensitive (K13 wild-type) Plasmodium falciparum strain is exposed to ART derivatives such as dihydroartemisinin (DHA), a small population of the early ring-stage parasites can survive drug treatment by entering cell cycle arrest or dormancy. After drug removal, these parasites can resume growth. Dormancy has been hypothesized to be an adaptive physiological mechanism that has been linked to recrudescence of parasites after monotherapy with ART and, possibly contributes to ART resistance. Here, we evaluate the in vitro drug sensitivity profile of normally-developing P. falciparum ring stages and DHA-pretreated dormant rings (DP-rings) using a panel of antimalarial drugs, including the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691. We report that while KDU691 shows no activity against rings, it is highly inhibitory against DP-rings; a drug effect opposite to that of ART. Moreover, we provide evidence that KDU691 also kills DP-rings of P. falciparum ART-resistant strains expressing mutant K13.
Assuntos
Antimaláricos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Pirazinas/farmacologia , Animais , Artemisininas/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/patogenicidadeRESUMO
BACKGROUND: Manganese-binding sites are found in several heme peroxidases, namely manganese peroxidase (MnP), chloroperoxidase, and the cationic isozyme of peanut peroxidase. The Mn-binding site in MnP is of particular interest. Oxidation of Mn(II) to Mn(III) is a key step in the biodegradation of lignin, a complex phenylpropanoid polymer, as well as many aromatic pollutants. Cytochrome c peroxidase (CcP), which is structurally homologous to MnP despite a poor sequence homology, does not bind manganese. Thus, engineering a Mn-binding site into CcP will allow us to elucidate principles behind designing metal-binding sites in proteins, to understand the structure and function of this class of Mn-binding centers, and to prepare novel enzymes that can degrade both lignin and other xenobiotic compounds. RESULTS: Based on a comparison of the crystal structures of CcP and MnP, a site-directed triple mutant (Gly41-->Glu, Val45-->Glu, His181-->Asp) of residues near the putative Mn-binding site in CcP was prepared and purified to homogeneity. Titrating MnSO4 into freshly prepared mutant CcP resulted in electronic absorption spectral changes similar to those observed in MnP. The calculated apparent dissociation constant and the stoichiometry of Mn-binding of CCP were also similar to MnP. Titration with MnSO4 resulted in the disappearance of specific paramagnetically shifted nuclear magnetic resonance spectroscopy signals assigned to residues close to the putative Mn-binding site in the mutant CcP. None of the spectral features were observed in wild-type CcP. In addition, the triple mutant was capable of oxidizing Mn(II) at least five times more efficiently than the native CcP. CONCLUSIONS: A Mn-binding site has been created in CcP and based on our spectroscopic studies the designed Mn-binding site is similar to the Mn-binding site in MnP. The results provide a basis for understanding the structure and function of the Mn-binding site and its role in different heme peroxidases.
Assuntos
Citocromo-c Peroxidase/metabolismo , Manganês/metabolismo , Peroxidases/metabolismo , Sítios de Ligação , Citocromo-c Peroxidase/genética , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Mutagênese Sítio-Dirigida , Peroxidases/genética , Plasmídeos , Espectrofotometria UltravioletaRESUMO
Stanniocalcin 1 (STC1) is a hypocalcemic hormone that is known to play an important role in calcium metabolism in teleost fish. An increase in blood Ca(2) (+) levels stimulates its synthesis and release. The biological action of STC1 inhibits gill Ca(2) (+) transport (GCAT), but we as yet have no clear understanding of how STC1 inhibits GCAT. In the present study, we characterized the binding, signaling, and action of STC1 on gill cells. Treatment of gill cell cultures with the extracts of corpuscles of Stannius or recombinant STC1 proteins (STC1-V5) led to an increase in cytosolic cAMP levels. Using in situ ligand-binding assays, we demonstrated that STC1-V5 binds to both lamellar and inter-lamellar regions of gill sections. The binding sites were significantly increased in gill sections obtained from fish adapted to high-Ca(2) (+) (2 mM) freshwater (FW) as compared with those from fish adapted to low-Ca(2) (+) (0.2 mM) FW. Receptor-binding assays illustrated specific binding of STC1-alkaline phosphatase to plasma membrane (Kd of 0.36 nM), mitochondria (Kd of 0.41 nM), and nuclear (Kd of 0.71 nM) preparations from gill cells. STC1 binding capacity was significantly greater in the plasma membrane preparations of gills obtained from fish adapted to high-Ca(2) (+) FW. Using isolated pavement cells and mitochondria-rich cells in cAMP assays, we obtained results indicating that both cell types responded to STC1. To illustrate the biological action of STC1, we conducted Ca(2) (+) imaging experiments to demonstrate the effects of STC1 on thapsigargin-induced elevation of cytosolic Ca(2) (+). Our results indicated that STC1 exerted its inhibitory action via a cAMP pathway to lower intracellular Ca(2) (+) levels. Intriguingly, we were able to block the action of STC1 using an inhibitor, NS-398, of cyclooxygenase-2 (COX-2), which is known to stimulate the activity of sarcoplasmic and endoplasmic reticulum Ca(2) (+)-ATPase (SERCA). A follow-up experiment in which gill cells were incubated with STC1 revealed a downregulation of the epithelial Ca(2) (+) channel (ecacl) but an upregulation of cox-2 expression. The ECaCl is a gatekeeper for Ca(2) (+) entry, whereas COX-2 mediates an activation of SERCA. Taking these results together, the present study is, to our knowledge, the first to provide evidence of STC1 binding and signaling as well as the first to decipher the mechanism of the effect of STC1 on fish gills.
Assuntos
Enguias/metabolismo , Proteínas de Peixes/metabolismo , Glicoproteínas/metabolismo , Animais , Sinalização do Cálcio , Células Cultivadas , AMP Cíclico/metabolismo , Ciclo-Oxigenase 2/metabolismo , Brânquias/citologia , Células HEK293 , Humanos , Ligação ProteicaRESUMO
1,25-Dihydroxy-16-ene vitamin D3 is a synthetic analog of 1,25-dihydroxyvitamin D3, the most physiologically active metabolite of vitamin D3. The renal metabolism of 1,25-dihydroxy-16-ene vitamin D3 had been studied previously using a perfused rat kidney system [Reddy et al., Bioorg Med Chem Lett 3: 1879-1884, 1993], and its C-24 oxidative metabolic pathway had been found to be different from that of 1,25-dihydroxyvitamin D3 by HPLC. To further delineate the differences between the C-24 oxidative metabolic pathways of 1,25-dihydroxyvitamin D3 and 1,25-dihydroxy-16-ene vitamin D3 in this present study we investigated the C-24 oxidation pathway of 1,25-dihydroxy-16-ene vitamin D3 using a novel detection approach based on on-line capillary liquid chromatography coupled to electrospray tandem mass spectrometry. Two types of tandem mass spectrometric detection were employed to characterize the metabolites in the kidney perfusate: (a) the preliminary screening of metabolites by parent scan, which led to the tentative discovery of the production of 1,23,25-trihydroxy-24-oxo-16-ene vitamin D3, a new metabolite of 1,25-dihydroxy-16-ene vitamin D3, and (b) the pharmacokinetic studies of the substrate, 1,25-dihydroxy-16-ene vitamin D3 and its metabolites by multiple reaction monitoring. In the latter, the mass spectrometric sensitivity for quantification was found to be about 20-fold better than UV detection. The current work concluded that the C-24 oxidative metabolic pathway of 1,25-dihydroxy-16-ene vitamin D3 closely mimicked that of its natural counterpart. Furthermore, the use of mass spectrometry permitted the clearance rate of the starting substrate to be studied at a more physiological level (ng/mL or submicromolar level), which had not been possible previously by HPLC-UV detection.
Assuntos
Calcitriol/análogos & derivados , Cromatografia Líquida de Alta Pressão/métodos , Rim/metabolismo , Espectrometria de Massas/métodos , Animais , Calcitriol/química , Calcitriol/metabolismo , Masculino , Perfusão , Ratos , Ratos Sprague-Dawley , Sensibilidade e Especificidade , Triazóis/químicaRESUMO
Eighty-seven families with early onset scoliosis were evaluated. These were divided into 3 groups: resolving infantile idiopathic scoliosis (15 families), progressive infantile idiopathic scoliosis (21 families), and congenital scoliosis due to vertebral malformations (51 families). The children with congenital scoliosis were subdivided into those who had closed neural arch defects (19 families) and those who did not (32 families). Resolving infantile idiopathic scoliosis was usually associated with plagiocephaly, and both deformations tended to show spontaneous recovery. These children were otherwise normal. Seven (33%) of the children with progressive infantile idiopathic scoliosis were mentally retarded, but only 2 had a congenital malformation. In contrast, 18 (33%) of the children with congenital scoliosis had other malformations, but only 2 were mentally retarded. The recurrence risk for scoliosis was low in each group studied. However, there was an increased risk (4% for sibs) of neural tube defects in the families with congenital scoliosis (with or without neural arch defects). This sib risk was apparent for probands with only a single hemivertebrum in addition to probands with more extensive vertebral defects and would support an etiological relationship between neural tube defects and other vertebral malformations.
Assuntos
Escoliose/genética , Anormalidades Múltiplas/genética , Criança , Pré-Escolar , Feminino , Aconselhamento Genético , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/genética , Masculino , Defeitos do Tubo Neural/genética , Risco , Escoliose/classificação , Escoliose/epidemiologia , Escócia , Espinha Bífida Oculta/genética , Coluna Vertebral/anormalidadesRESUMO
The conversion of methyl carbamate to the corresponding free amine is described for a series of 2-amino-2-deoxy-D-glucosamine derivatives. Cleavage of methoxycarbonyl moiety with MeSiCl(3) and triethylamine in dry THF at 60 degrees C and subsequent aqueous hydrolysis yields the free amine in 54 to 93% yields. The selective cleavage of methyl carbamates with MeSiCl(3) in the presence of a 2,2,2-trichloroethoxycarbonyl group or 2-azido glycosides affords selectively, orthogonal N-deprotected carbohydrates.
Assuntos
Glucosídeos/química , Metilglicosídeos/química , Catálise , HidróliseRESUMO
In the presence of interferon-gamma (IFN-gamma), pancreatic ductal epithelial cells grow continuously, and islets undergo neogenesis. To determine whether these new islets are derived from conventional precursors, we tested whether IFN-gamma can complement the loss of transcription factors known to regulate pancreatic development. We analyzed the effect of a transgene on lethality in mice lacking the transcription factors Pax4, Pax6, or Pdx-1, by intercrossing such mice with transgenic mice whose pancreatic cells make IFN-gamma (ins-IFN-gamma mice). However, IFN-gamma expression did not rescue these mice from the lethal mutations, because no homozygous knockout mice carrying the IFN-gamma transgene survived, despite the survival of all other hemizygous gene combinations. This outcome demonstrates that the pathway for IFN-gamma regeneration requires the participation of Pax4, Pax6, and Pdx-1. We conclude that the striking islet regeneration observed in the ins-IFN-gamma NOD strain is regulated by the same transcription factors that control initial pancreatic development.