RESUMO
On the basis of the crystal structure of bovine ß4Gal-T1 enzyme, mutation of a single amino acid Y289 to L289 (Y289L) changed its donor specificity from Gal to N-acetyl-galactosamine (GalNAc). A chemoenzymatic method that uses GalNAc analogues like GalNAz or 2-keto-Gal as sugar donors with the enzyme Y289L-ß4Gal-T1 has identified hundreds of cytosolic and nuclear proteins that have O-GlcNAc modifications. To avoid potential cytotoxicity at Mn(2+) concentrations required to selectively modify GlcNAc residues on the surface of live cells, we have engineered a Mg(2+)-dependent enzyme. Previously, we found that the mutation of the metal-binding residue Met-344 to His-344 in bovine ß4Gal-T1 enzyme altered its metal-ion specificity in such a way that the M344H-ß4Gal-T1 enzyme exhibits better catalytic activity with Mg(2+) than with Mn(2+). Here, we find that, when these two mutations are combined, the double mutant, Y289L-M344H-ß4Gal-T1, transfers GalNAc and its analogue sugars to the acceptor GlcNAc in the presence of Mg(2+). Using this mutant enzyme, we have detected free GlcNAc residues on the surface glycans of live HeLa cells and platelets. The specific transfer of a synthetic sugar with a chemical handle to the terminal GlcNAc residues on the surface of live cells provides a novel tool for selective modification, detection, and isolation of GlcNAc-ending glycans present on the cellular surface.
Assuntos
Acetilglucosamina/análise , Acetilglucosamina/metabolismo , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Glicoconjugados/metabolismo , Mutação Puntual , Animais , Plaquetas/metabolismo , Bovinos , Galactosiltransferases/química , Expressão Gênica , Glicoconjugados/química , Células HeLa , Humanos , Magnésio/metabolismo , Modelos Moleculares , Engenharia de ProteínasRESUMO
In recent years, sugars with a unique chemical handle have been used to detect and elucidate the function of glycoconjugates. Such chemical handles have generally been part of an N-acetyl moiety of a sugar. We have previously developed several applications using the single mutant Y289L-ß1,4-galactosyltransferase I (Y289L-ß4Gal-T1) and the wild-type polypeptide-α-GalNAc-T enzymes with UDP-C2-keto-Gal. Here, we describe for the first time that the GlcNAc-transferring enzymes-R228K-Y289L-ß4Gal-T1 mutant enzyme, the wild-type human ß1,3-N-acetylglucosaminyltransferase-2 and human Maniac Fringe-can also transfer the GlcNAc analog C2-keto-Glc molecule from UDP-C2-keto-Glc to their respective acceptor substrates. Although the R228K-Y289L-ß4Gal-T1 mutant enzyme transfers the donor sugar substrate GlcNAc or its analog C2-keto-Glc only to its natural acceptor substrate, GlcNAc, it does not transfer to its analog C2-keto-Glc. Thus, these observations suggest that the GlcNAc-transferring glycosyltransferases can generally accommodate a chemical handle in the N-acetyl-binding cavity of the donor sugar substrate, but not in the N-acetyl-binding cavity of the acceptor sugar.
Assuntos
Galactose/análogos & derivados , Galactose/química , Hexosiltransferases/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Membrana/química , N-Acetilglucosaminiltransferases/química , Acetilglucosamina/química , Substituição de Aminoácidos , Sítios de Ligação , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia de Afinidade , Clonagem Molecular , Escherichia coli , Fator VII/química , Glucosiltransferases , Glicosilação , Hexosiltransferases/biossíntese , Hexosiltransferases/genética , Hexosiltransferases/isolamento & purificação , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , N-Acetilglucosaminiltransferases/biossíntese , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/isolamento & purificação , Oligossacarídeos/química , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Propriedades de SuperfícieRESUMO
The Global Virus Network (GVN) was established in 2011 to strengthen research and responses to emerging viral causes of human disease and to prepare against new viral pandemics. There are now 45 GVN Centers of Excellence and 7 Affiliate laboratories in 29 countries. The 10th International GVN meeting was held from November 28-30, 2018 in Veyrier du Lac, France and was co-hosted by the two GVN Centers of Excellence, the Mérieux Foundation and the University of Veterinary Medicine Hannover (TiHo). The theme of this 10th International GVN meeting was "Eradication and control of (re-) emerging viruses". This report highlights the recent accomplishments of GVN researchers in several important areas of medical virology, including strategies for the eradication of smallpox, measles, polio, SARS and vector-borne or zoonotic infections, emergence and intervention strategies for retroviruses and arboviruses, preparedness for outbreaks of Filo- and other hemophilic viruses, pathogenesis, impact and prevention of respiratory viruses, as well as, viruses affecting the central and peripheral nervous system. Also threats in crisis settings like refugee camps were presented.
Assuntos
Doenças Transmissíveis Emergentes/virologia , Congressos como Assunto , Saúde Global , Viroses/prevenção & controle , Vírus , Animais , Doenças Transmissíveis Emergentes/prevenção & controle , França , Humanos , Internacionalidade , Pandemias/prevenção & controle , Zoonoses/prevenção & controle , Zoonoses/virologiaRESUMO
The Global Virus Network (GVN) was established in 2011 to strengthen research and responses to emerging viral causes of human disease and to prepare against new viral pandemics. There are now 52 GVN Centers of Excellence and 9 Affiliate laboratories in 32 countries. The 11th International GVN meeting was held from June 9-11, 2019 in Barcelona, Spain and was jointly organized with the Spanish Society of Virology. A common theme throughout the meeting was globalization and climate change. This report highlights the recent accomplishments of GVN researchers in several important areas of medical virology, including severe virus epidemics, anticipation and preparedness for changing disease dynamics, host-pathogen interactions, zoonotic virus infections, ethical preparedness for epidemics and pandemics, one health and antivirals.
Assuntos
Doenças Transmissíveis Emergentes , Saúde Global , Saúde Única/tendências , Viroses , Animais , Antivirais , Arbovírus/efeitos dos fármacos , Arbovírus/genética , Arbovírus/metabolismo , Mudança Climática , Doenças Transmissíveis Emergentes/tratamento farmacológico , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/transmissão , Coronavirus/efeitos dos fármacos , Coronavirus/genética , Coronavirus/metabolismo , Ebolavirus/efeitos dos fármacos , Ebolavirus/genética , Ebolavirus/metabolismo , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/prevenção & controle , Hepatite B/tratamento farmacológico , Hepatite B/epidemiologia , Hepatite B/prevenção & controle , Interações Hospedeiro-Patógeno , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/epidemiologia , Influenza Humana/prevenção & controle , Internacionalidade , Pandemias , Vacinas Virais , Viroses/tratamento farmacológico , Viroses/epidemiologia , Viroses/transmissão , Vírus/efeitos dos fármacos , Vírus/genética , Vírus/metabolismo , Zoonoses/tratamento farmacológico , Zoonoses/epidemiologia , Zoonoses/transmissãoRESUMO
The Global Virus Network (GVN) was established in 2011 to strengthen research and responses to emerging viral causes of human disease and to prepare against new viral pandemics. There are now 40 GVN Centers of Excellence and 6 Affiliate laboratories in 24 countries. The 2017 meeting was held from September 25-27 in Melbourne, Australia, and was hosted by the Peter Doherty Institute for Infection and Immunity and the Institut Pasteur. This report highlights the recent accomplishments of GVN researchers in several important areas of medical virology, including the recent Zika epidemic, infections by human papillomavirus, influenza, HIV, hepatitis C, HTLV-1, and chikungunya viruses, and new and emerging viruses in the Australasia region. Plans for the 2018 meeting also are noted.
Assuntos
Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/prevenção & controle , Pandemias/prevenção & controle , Viroses/epidemiologia , Viroses/prevenção & controle , Austrália , Pesquisa Biomédica/tendências , Saúde Global , Humanos , Disseminação de Informação , Serviços de InformaçãoRESUMO
The past five years have seen a revolution in the treatment of chronic hepatitis C, as short duration oral regimens of direct-acting antiviral drugs (DAAs), with nearly 100% cure rates for all genotypes, have replaced longer courses of ribavirin and injected interferon. Although initially very expensive, these DAAs are now becoming available in generic equivalents in countries with large numbers of chronically infected people, such as India. However, a number of obstacles may hinder the delivery of these drugs in resource-limited settings, including lack of access to diagnostic testing and the restriction of treatment to a small number of medical specialists. New approaches are therefore needed to make DAAs available to the estimated 71 million infected people, many of whom disproportionately live in low- or middle-income countries. A recent pilot study (ASCEND) of hepatitis C management in a low-income population in Washington, D.C., demonstrated that trained nurse practitioners, primary care physicians and hepatologists were equally successful in diagnosing and treating patients, indicating that such an approach might be successful in resource-limited regions of the world. Members of the Global Virus Network have received funding to carry out a similar training project in a region of India with a high prevalence of hepatitis C. This paper reviews the challenges of delivering DAA therapy in low- and middle-income countries, describes plans for performing and evaluating the effectiveness of a training program in India, and discusses future needs for the eventual elimination of hepatitis C.
Assuntos
Antivirais/uso terapêutico , Recursos em Saúde , Hepatite C Crônica/tratamento farmacológico , Antivirais/administração & dosagem , Países em Desenvolvimento , District of Columbia , Genótipo , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hepatite C Crônica/epidemiologia , Humanos , Índia/epidemiologia , Projetos Piloto , Pobreza , Ribavirina/uso terapêuticoRESUMO
The Global Virus Network (GVN) was established in 2011 in order to strengthen research and responses to current viral causes of human disease and to prepare against new viral pandemic threats. There are now 38 GVN Centers of Excellence and 6 Affiliate laboratories in 24 countries. GVN scientists meet annually to learn about each other's current research, address collaborative priorities and plan future programs. The 2016 meeting was held from October 23-25 in Hokkaido, Japan, in partnership with the Japanese Society for Virology, the National Institute of Infectious Diseases of Japan and the Research Center for Zoonosis Control of Hokkaido University. This report highlights the accomplishments of GVN researchers in many priority areas of medical virology, including the current Zika epidemic, infections by human papillomavirus, influenza, Ebola, Lassa, dengue, HIV, hepatitis C, and chikungunya viruses, and the development of improved diagnostics and new vaccines.
Assuntos
Doenças Transmissíveis/virologia , Cooperação Internacional , Vírus/patogenicidade , Animais , Doenças Transmissíveis/epidemiologia , Doenças Transmissíveis/terapia , Congressos como Assunto , Surtos de Doenças , Monitoramento Epidemiológico , Saúde Global , Humanos , Japão , Pandemias , Pesquisa , ZoonosesRESUMO
In response to the outbreak of Zika virus (ZIKV) infection in the Western Hemisphere and the recognition of a causal association with fetal malformations, the Global Virus Network (GVN) assembled an international taskforce of virologists to promote basic research, recommend public health measures and encourage the rapid development of vaccines, antiviral therapies and new diagnostic tests. In this article, taskforce members and other experts review what has been learned about ZIKV-induced disease in humans, its modes of transmission and the cause and nature of associated congenital manifestations. After describing the make-up of the taskforce, we summarize the emergence of ZIKV in the Americas, Africa and Asia, its spread by mosquitoes, and current control measures. We then review the spectrum of primary ZIKV-induced disease in adults and children, sites of persistent infection and sexual transmission, then examine what has been learned about maternal-fetal transmission and the congenital Zika syndrome, including knowledge obtained from studies in laboratory animals. Subsequent sections focus on vaccine development, antiviral therapeutics and new diagnostic tests. After reviewing current understanding of the mechanisms of emergence of Zika virus, we consider the likely future of the pandemic.
Assuntos
Infecção por Zika virus/epidemiologia , África/epidemiologia , América/epidemiologia , Animais , Ásia/epidemiologia , Controle de Doenças Transmissíveis/métodos , Testes Diagnósticos de Rotina , Transmissão de Doença Infecciosa , Descoberta de Drogas/tendências , Transmissão Vertical de Doenças Infecciosas , Malformações do Sistema Nervoso/epidemiologia , Infecção por Zika virus/complicações , Infecção por Zika virus/transmissãoRESUMO
BACKGROUND: Alpha-lactalbumin (α-LA) is a calcium-bound mammary gland-specific protein that is found in milk. This protein is a modulator of ß1,4-galactosyltransferase enzyme, changing its acceptor specificity from N-acetyl-glucosamine to glucose, to produce lactose, milk's main carbohydrate. When calcium is removed from α-LA, it adopts a molten globule form, and this form, interestingly, when complexed with oleic acid (OA) acquires tumoricidal activity. Such a complex made from human α-LA (hLA) is known as HAMLET (Human A-lactalbumin Made Lethal to Tumor cells), and its tumoricidal activity has been well established. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, we have used site-specific labeling, a technique previously developed in our laboratory, to label HAMLET with biotin, or a fluoroprobe for confocal microscopy studies. In addition to full length hLA, the α-domain of hLA (αD-hLA) alone is also included in the present study. We have engineered these proteins with a 17-amino acid C-terminal extension (hLA-ext and αD-hLA-ext). A single Thr residue in this extension is glycosylated with 2-acetonyl-galactose (C2-keto-galactose) using polypeptide-α-N-acetylgalactosaminyltransferase II (ppGalNAc-T2) and further conjugated with aminooxy-derivatives of fluoroprobe or biotin molecules. CONCLUSIONS/SIGNIFICANCE: We found that the molten globule form of hLA and αD-hLA proteins, with or without C-terminal extension, and with and without the conjugated fluoroprobe or biotin molecule, readily form a complex with OA and exhibits tumoricidal activity similar to HAMLET made with full-length hLA protein. The confocal microscopy studies with fluoroprobe-labeled samples show that these proteins are internalized into the cells and found even in the nucleus only when they are complexed with OA. The HAMLET conjugated with a single biotin molecule will be a useful tool to identify the cellular components that are involved with it in the tumoricidal activity.
Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Biotina/metabolismo , Lactalbumina/metabolismo , Lactalbumina/farmacologia , Ácidos Oleicos/metabolismo , Ácidos Oleicos/farmacologia , Engenharia de Proteínas/métodos , Coloração e Rotulagem/métodos , Sequência de Aminoácidos , Antineoplásicos/química , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Lactalbumina/química , Lactalbumina/genética , Modelos Moleculares , Imagem Molecular , Dados de Sequência Molecular , Ácidos Oleicos/química , Ácidos Oleicos/genética , Dobramento de Proteína , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Pfiesteria spp. are mixotrophic armored dinoflagellates populating the Atlantic coastal waters of the United States. They have been a focus of intense research due to their reported association with several fish mortality events. We have now used a clonal culture of Pfiesteria piscicida and several new environmental isolates to describe growth characteristics, feeding, and factors contributing to the encystment and germination of the organism in both laboratory and environmental samples. We also discuss applied methods of detection of the different morphological forms of Pfiesteria in environmental samples. In summary, Pfiesteria, when grown with its algal prey, Rhodomonas sp., presents a typical growth curve with lag, exponential, and stationary phases, followed by encystment. The doubling time in exponential phase is about 12 h. The profiles of proliferation under a standard light cycle and in the dark were similar, although the peak cell densities were markedly lower when cells were grown in the dark. The addition of urea, chicken manure, and soil extracts did not enhance Pfiesteria proliferation, but crude unfiltered spent aquarium water did. Under conditions of food deprivation or cold (4 degrees C), Pfiesteria readily formed harvestable cysts that were further analyzed by PCR and scanning electron microscopy. The germination of Pfiesteria cysts in environmental sediment was enhanced by the presence of live fish: dinospores could be detected 13 to 15 days earlier and reached 5- to 10-times-higher peak cell densities with live fish than with artificial seawater or f/2 medium alone. The addition of ammonia, urea, nitrate, phosphate, or surprisingly, spent fish aquarium water had no effect.
Assuntos
Aquicultura , Sedimentos Geológicos/parasitologia , Peixes Listrados/crescimento & desenvolvimento , Pfiesteria piscicida/crescimento & desenvolvimento , Animais , DNA de Protozoário/análise , DNA Ribossômico/genética , Escuridão , Eucariotos/crescimento & desenvolvimento , Dosagem de Genes , Peixes Listrados/fisiologia , Luz , Microscopia Eletrônica de Varredura , Pfiesteria piscicida/genética , Pfiesteria piscicida/isolamento & purificação , Pfiesteria piscicida/fisiologia , Reação em Cadeia da Polimerase , Especificidade da Espécie , Esporos de Protozoários/crescimento & desenvolvimentoRESUMO
Advanced glycation end products (AGEs) have been proposed as the pathological mechanisms underlying diabetic chronic complications. They may also play a role in the pathogenesis of diabetic osteopenia, although their mechanisms of action remain unclear. We investigated the protein (immunofluorescence) and gene expression (realtime RT-PCR) of two receptors for AGEs, RAGE and galectin-3, as well as their regulation by AGEs, and the apoptotic effect on osteoblast-like cells (UMR106 and MC3T3E1) in culture. AGEs up-regulated the expression of RAGE and galectin-3 in both cells lines. These effects were accompanied by an increase in the corresponding mRNA in the non-tumoral MC3T3E1 but not in the osteosarcoma UMR106 cells. Finally, we demonstrated that a 24 h exposure to AGEs induced apoptosis in both cell lines. Thus, AGEs-receptors may play important roles in the bone alterations described in aging and diabetic patients.
Assuntos
Apoptose , Produtos Finais de Glicação Avançada/farmacologia , Osteoblastos/efeitos dos fármacos , Receptores Imunológicos/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Imunofluorescência , Galectina 3/genética , Galectina 3/metabolismo , Camundongos , Osteoblastos/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
An increase in the interaction between advanced glycation end-products (AGEs) and their receptor RAGE is believed to contribute to the pathogenesis of chronic complications of Diabetes mellitus, which can include bone alterations such as osteopenia. We have recently found that extracellular AGEs can directly regulate the growth and development of rat osteosarcoma UMR106 cells, and of mouse calvaria-derived MC3T3E1 osteoblasts throughout their successive developmental stages (proliferation, differentiation and mineralisation), possibly by the recognition of AGEs moieties by specific osteoblastic receptors which are present in both cell lines. In the present study we examined the possible expression of RAGE by UMR106 and MC3T3E1 osteoblastic cells, by immunoblot analysis. We also investigated whether short-, medium- or long-term exposure of osteoblasts to extracellular AGEs, could modify their affinity constant and maximal binding for AGEs (by 125I-AGE-BSA binding experiments), their expression of RAGE (by immunoblot analysis) and the activation status of the osteoblastic ERK 1/2 signal transduction mechanism (by immunoblot analysis for ERK and P-ERK). Our results show that both osteoblastic cell lines express readily detectable levels of RAGE. Short-term exposure of phenotypically mature osteoblastic UMR106 cells to AGEs decrease the cellular density of AGE-binding sites while increasing the affinity of these sites for AGEs. This culture condition also dose-dependently increased the expression of RAGE and the activation of ERK. In proliferating MC3T3E1 pre-osteoblasts, 24-72 h exposure to AGEs did not modify expression of RAGE, ERK activation or the cellular density of AGE-binding sites. However, it did change the affinity of these binding sites forAGEs, with both higher- and lower-affinity sites now being apparent. Medium-term ( 1 week) incubation of differentiated MC3T3E1 osteoblasts with AGEs, induced a simultaneous increase in RAGE expression and in the relative amount of P-ERK. Mineralising MC3T3E1 cultures grown for 3 weeks in the presence of extracellular AGEs showed a decrease both in RAGE and P-ERK expression. These results indicate that, in phenotypically mature osteoblastic cells, changes in ERK activation closely follow the AGEs-induced regulation of RAGE expression. Thus, the AGEs-induced biological effects that we have observed previously in osteoblasts, could be mediated by RAGE in the later stages of development, and mediated by other AGE receptors in the earlier pre-osteoblastic stage.
Assuntos
Produtos Finais de Glicação Avançada/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osteoblastos/metabolismo , Células 3T3 , Animais , Sítios de Ligação , Western Blotting , Diferenciação Celular , Divisão Celular , Linhagem Celular Tumoral , Colágeno Tipo I/química , Relação Dose-Resposta a Droga , Ativação Enzimática , Immunoblotting , Cinética , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Fenótipo , Ligação Proteica , Ratos , Fatores de TempoRESUMO
The accumulation of irreversible advanced glycation endproducts (AGEs) on long-lived proteins, and the interaction of AGEs with cellular receptors such as AGE-R3/galectin-3 and RAGE, are considered to be key events in the development of long-term complications of diabetes mellitus, Alzheimer's disease, uremia and ageing. The aim of this study was to investigate the expression and sub-cellular distribution of galectin-3, as well as its possible modulation by AGEs, in MC3T3E1 mouse calvaria-derived osteoblasts and in UMR 106 rat osteosarcoma cells. Both osteoblastic lines were cultured either with control bovine serum albumin (BSA) or with AGEs-BSA for 48 h. Cells were evaluated for galectin-3 expression by fixing and immunofluorescent microscopic analysis; or Western blot analysis of whole cell extracts, sub-cellular fractions and culture media. Both cell lines express 30 kDa (monomeric) galectin-3, although expression was about 15-fold lower in the UMR106 osteosarcoma cells. Dimeric (70 kDa) galectin-3 was additionally observed in the UMR106 cells. Immunofluorescent analysis of galectin-3 distribution showed a diffuse cytoplasmic and strong nuclear pattern in MC3T3E1 osteoblasts, and a patchy cytoplasmic pattern in UMR106 cells. Western blot analysis for both cell lines showed that galectin-3 was mainly found in the cytoplasm and in minor amounts in the microsomal fraction, while considerable amounts were secreted into the culture media. Exposure to 100-200 microg/mL AGEs-BSA increased the cellular content of 30 kDa galectin-3 (20-25% for MC3T3E1 and 35-70% for UMR106 versus control BSA, p < 0.05), and decreased the culture media levels of galectin-3 (10-20% for MC3T3E1 and for UMR106 versus control BSA, p < 0.05). These results confirm the expression of galectin-3 in osteoblastic cells, and suggest different levels and sub-cellular distribution of this protein in transformed versus non-transformed osteoblasts. Osteoblastic exposure to AGEs alters their expression and secretion of galectin-3, which could have significant consequences on osteoblast metabolism and thus on bone turnover.