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1.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33384338

RESUMO

Human adenovirus species D (HAdV-D) types are currently being explored as vaccine vectors for coronavirus disease 2019 (COVID-19) and other severe infectious diseases. The efficacy of such vector-based vaccines depends on functional interactions with receptors on host cells. Adenoviruses of different species are assumed to enter host cells mainly by interactions between the knob domain of the protruding fiber capsid protein and cellular receptors. Using a cell-based receptor-screening assay, we identified CD46 as a receptor for HAdV-D56. The function of CD46 was validated in infection experiments using cells lacking and overexpressing CD46, and by competition infection experiments using soluble CD46. Remarkably, unlike HAdV-B types that engage CD46 through interactions with the knob domain of the fiber protein, HAdV-D types infect host cells through a direct interaction between CD46 and the hexon protein. Soluble hexon proteins (but not fiber knob) inhibited HAdV-D56 infection, and surface plasmon analyses demonstrated that CD46 binds to HAdV-D hexon (but not fiber knob) proteins. Cryoelectron microscopy analysis of the HAdV-D56 virion-CD46 complex confirmed the interaction and showed that CD46 binds to the central cavity of hexon trimers. Finally, soluble CD46 inhibited infection by 16 out of 17 investigated HAdV-D types, suggesting that CD46 is an important receptor for a large group of adenoviruses. In conclusion, this study identifies a noncanonical entry mechanism used by human adenoviruses, which adds to the knowledge of adenovirus biology and can also be useful for development of adenovirus-based vaccine vectors.


Assuntos
Adenovírus Humanos , Proteínas do Capsídeo , Regulação Viral da Expressão Gênica , Internalização do Vírus , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , /metabolismo , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Linhagem Celular , Humanos
2.
J Gen Virol ; 99(1): 135-147, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29154744

RESUMO

The vectorization of rare human adenovirus (HAdV) types will widen our knowledge of this family and their interaction with cells, tissues and organs. In this study we focus on HAdV-56, a member of human Ad species D, and create ease-of-use cloning systems to generate recombinant HAdV-56 vectors carrying foreign genes. We present in vitro transduction profiles for HAdV-56 in direct comparison to the most commonly used HAdV-5-based vector. In vivo characterizations demonstrate that when it is delivered intravenously (i.v.) HAdV-56 mainly targets the spleen and, to a lesser extent, the lungs, whilst largely bypassing liver transduction in mice. HAdV-56 triggered robust inflammatory and cellular immune responses, with higher induction of IFNγ, TNFα, IL5, IL6, IP10, MCP1 and MIG1 compared to HAdV-5 following i.v. administration. We also investigated its potential as a vaccine vector candidate by performing prime immunizations in mice with HAdV-56 encoding luciferase (HAdV-56-Luc). Direct comparisons were made to HAdV-26, a highly potent human vaccine vector currently in phase II clinical trials. HAdV-56-Luc induced luciferase 'antigen'-specific IFNγ-producing cells and anti-HAdV-56 neutralizing antibodies in Balb/c mice, demonstrating a near identical profile to that of HAdV-26. Taken together, the data presented provides further insight into human Ad receptor/co-receptor usage, and the first report on HAdV-56 vectors and their potential for gene therapy and vaccine applications.


Assuntos
Adenovírus Humanos/imunologia , Expressão Gênica/efeitos dos fármacos , Terapia Genética/métodos , Vetores Genéticos/imunologia , Vacinação , Vacinas Virais/biossíntese , Adenovírus Humanos/genética , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Quimiocina CXCL9/genética , Quimiocina CXCL9/imunologia , Feminino , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Injeções Intravenosas , Interferon gama/genética , Interferon gama/imunologia , Interleucina-5/genética , Interleucina-5/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/efeitos dos fármacos , Baço/imunologia , Transgenes , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Vacinas Virais/administração & dosagem
3.
Comput Biol Chem ; 70: 65-88, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28822333

RESUMO

This study focuses on the best possible way forward in utilizing inconclusive molecules of PubChem bioassays AID 1332, AID 434987 and AID 434955, which are related to beta-lactamase inhibitors of Mycobacterium tuberculosis (Mtb). The inadequacy in the experimental methods that were observed during the invitro screening resulted in an inconclusive dataset. This could be due to certain moieties present within the molecules. In order to reconsider such molecules, insilico methods can be suggested in place of invitro methods For instance, datamining and medicinal chemistry methods: have been adopted to prioritise the inconclusive dataset into active or inactive molecules. These include the Random Forest algorithm for dataminning, Lilly MedChem rules for virtually screening out the promiscuity, and Self Organizing Maps (SOM) for clustering the active molecules and enlisting them for repositioning through the use of artificial neural networks. These repositioned molecules could then be prioritized for downstream drug discovery analysis.


Assuntos
Mineração de Dados , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Ensaios Enzimáticos , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/análise , Algoritmos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Inibidores de beta-Lactamases/química , beta-Lactamases/metabolismo
4.
Biologicals ; 49: 23-27, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28734743

RESUMO

Foot and mouth disease is a highly contagious disease affecting cloven footed animals. Vaccination using inactivated virus is followed to control the disease. As the immune response conferred by the inactivated vaccine is short lived, there is a need for an alternate vaccine with increased duration of immunity. Inclusion of adjuvant which enhances B and T cell responses is one of the strategies to increase the duration of immune responses of the vaccine. Interleukin 15 is one such a cytokine which improves the cell mediated immune response and also involved in the maintenance of memory T and B cells. In the present communication, we evaluated the role of bovine IL-15 as an adjuvant to inactivated FMD vaccine in guinea pig model. Animals injected with FMD inactivated vaccine and IL-15 plasmid showed improved levels of neutralizing antibodies which were maintained up to 6 months (as the level of neutralizing antibodies is more >1.5 which is considered to give protection). Increased Th1 and Th2 responses (by measuring the level of IL-4 and IFN- gamma responses) were seen in IL-15 adjuvanted guinea pigs compared to animals injected with inactivated vaccine alone.


Assuntos
Adjuvantes Imunológicos , Linfócitos B/imunologia , Vírus da Febre Aftosa , Imunidade Celular/efeitos dos fármacos , Interleucina-15 , Plasmídeos , Linfócitos T/imunologia , Vacinas Virais , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/farmacologia , Animais , Células CHO , Bovinos , Cricetulus , Vírus da Febre Aftosa/química , Vírus da Febre Aftosa/imunologia , Cobaias , Imunidade Celular/genética , Interleucina-15/genética , Interleucina-15/imunologia , Interleucina-15/farmacologia , Plasmídeos/genética , Plasmídeos/farmacologia , Vacinas de Produtos Inativados/química , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/farmacologia , Vacinas Virais/química , Vacinas Virais/imunologia , Vacinas Virais/farmacologia
5.
Dermatol Ther (Heidelb) ; 5(3): 151-69, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26387031

RESUMO

For decades, no cancer therapy had been shown to improve average survival in metastatic melanoma. Two critical events have occurred, the discovery of melanoma driver mutation subsets and the discovery of immune checkpoint inhibitors, which have allowed for the development of modern, effective therapies. These findings have facilitated a rapid emergence of novel therapeutics for the disease with multiple FDA approvals in the last several years. The drugs vemurafenib, trametinib, and dabrafenib, which inhibit the commonly mutated BRAF pathway, have been approved based on improvements in survival outcomes. Agents that block immune checkpoints on lymphocytes allowing for immune cell activity against melanoma have also been approved based on improved survival outcomes such as ipilimumab and nivolumab. Pembrolizumab, another immune checkpoint inhibitor, has also been approved based on the response rate and duration of response in a phase 1 trial. Further agents and combinations of approved agents are positioned to possibly further increase this tally of approved drugs. This review will discuss recently approved novel agents and select drugs in development in advanced melanoma.

6.
J Clin Diagn Res ; 9(1): ZH02-3, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25738103

RESUMO

Head positions can be oriented in a standardized position when the patient stands upright and focusses his/her eyes into a point in infinity. This is the natural head position. This position offers the maximum reproducibility and correlates well with the clinical picture offered to the diagnostician. This article describes an innovative and user friendly method to record natural head position using the head strap double fluid level device, a design modified from the popular fluid level device by Showfety, Vig and Matteson.

7.
J Interferon Cytokine Res ; 34(6): 437-46, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24905200

RESUMO

Adenosine deaminase acting on RNA1 (ADAR1) catalyzes the C6 deamination of adenosine (A) to produce inosine (I) in regions of RNA with double-stranded (ds) character. This process is known as A-to-I RNA editing. Alternative promoters drive the expression of the Adar1 gene and alternative splicing gives rise to transcripts that encode 2 ADAR1 protein size isoforms. ADAR1 p150 is an interferon (IFN)-inducible dsRNA adenosine deaminase found in the cytoplasm and nucleus, whereas ADAR1 p110 is constitutively expressed and nuclear in localization. Dependent on the duplex structure of the dsRNA substrate, deamination of adenosine by ADAR can be either highly site-selective or nonspecific. A-to-I editing can alter the stability of RNA structures and the coding of RNA as I is read as G instead of A by ribosomes during mRNA translation and by polymerases during RNA replication. A-to-I editing is of broad physiologic significance. Both the production and the action of IFNs, and hence the subsequent interaction of viruses with their hosts, are among the processes affected by A-to-I editing.


Assuntos
Adenosina Desaminase/metabolismo , Edição de RNA , RNA de Cadeia Dupla/metabolismo , Animais , Humanos , Proteínas de Ligação a RNA/metabolismo
8.
Virology ; 454-455: 299-310, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24725957

RESUMO

Measles virus (MV) deficient in C protein (C(ko)) expression efficiently induces both stress granules (SG) and interferon (IFNß), whereas isogenic wild-type (WT) and V mutant (V(ko)) viruses do not. We therefore examined the effect of IFNß pretreatment on SG formation, and the roles played by the IFN-inducible double-stranded (ds) RNA-dependent protein kinase (PKR) and dsRNA adenosine deaminase (ADAR1). SG formation in ADAR1-sufficient cells infected with WT or V(ko) mutant virus was enhanced by IFN treatment and was PKR-dependent. SG formation in C(ko) virus-infected cells was already high without IFN treatment and was not further enhanced by IFN. IFN treatment alone, in the absence of infection, induced SG formation in ADAR1-deficient but not ADAR1-sufficient cells. Type I IFN-induced enhancement in SG formation occurred by a canonical IFN signaling response dependent upon STAT1 and STAT2. These results further establish ADAR1 as a suppressor of the interferon and SG innate immune responses.


Assuntos
Adenosina Desaminase/imunologia , Adenosina Desaminase/metabolismo , Interferon beta/imunologia , Interferon beta/metabolismo , Morbillivirus/imunologia , Animais , Chlorocebus aethiops , Células HeLa , Humanos , Morbillivirus/genética , Biossíntese de Proteínas , Proteínas de Ligação a RNA , Células Vero
9.
Virol J ; 10: 269, 2013 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-23984714

RESUMO

BACKGROUND: Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family. After autoproteolytic cleavage, the CHIKV capsid protein (CP) is involved in RNA binding and assembly of the viral particle. The monomeric CP is approximately 30 kDa in size and is small enough for passive transport through nuclear pores. Some alphaviruses are found to harbor nuclear localization signals (NLS) and transport of these proteins between cellular compartments was shown to be energy dependent. The active nuclear import of cytoplasmic proteins is mediated by karyopherins and their export by exportins. As nuclear and cytoplasmic trafficking may play a role in the life cycle of CHIKV, we have sought to identify nuclear localization and nuclear export signals in CHIKV CP in a virus-free system. METHODS: EGFP-fusion proteins of CHIKV CP and mutants thereof were created and used to monitor their intracellular localization. Binding of cellular proteins was confirmed in pull-down assays with purified CP using co-immuoprecipitation. Nuclear localization was demonstrated in a virus-free system using fluorescence microscopy. RESULTS: Here we show that CHIKV CP is a nuclear-cytoplasmic shuttling protein with an active NLS that binds to karyopherin α (Karα) for its nuclear translocation. We also found that the Karα4 C-terminal NLS binding site is sufficient for this interaction. We further demonstrate that CHIKV CP interacts directly with the export receptor CRM1 to transport this viral protein out of the nucleus via a nuclear export signal (NES). The CHIKV CP NES was mapped between amino acids 143 and 155 of CP. Deduced from in silico analyses we found that the NES has a mode of binding similar to the snurportin-1 CRM1 complex. CONCLUSIONS: We were able to show that in a virus-free system that the CHIKV capsid protein contains both, a NLS and a NES, and that it is actively transported between the cytoplasma and the nucleus. We conclude that CHIKV CP has the ability to shuttle via interaction with karyopherins for its nuclear import and, vice versa, by CRM1-dependent nuclear export.


Assuntos
Transporte Ativo do Núcleo Celular , Proteínas do Capsídeo/genética , Vírus Chikungunya/genética , Sinais Direcionadores de Proteínas , Animais , Proteínas do Capsídeo/metabolismo , Núcleo Celular/química , Vírus Chikungunya/fisiologia , Citoplasma/química , Análise Mutacional de DNA , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Microscopia de Fluorescência , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética
10.
PLoS One ; 8(7): e69741, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23894533

RESUMO

Red lined torpedo barbs (RLTBS) (Cyprinidae: Puntius) endemic to the Western Ghats Hotspot of India, are popular and highly priced freshwater aquarium fishes. Two decades of indiscriminate exploitation for the pet trade, restricted range, fragmented populations and continuing decline in quality of habitats has resulted in their 'Endangered' listing. Here, we tested whether the isolated RLTB populations demonstrated considerable variation qualifying to be considered as distinct conservation targets. Multivariate morphometric analysis using 24 size-adjusted characters delineated all allopatric populations. Similarly, the species-tree highlighted a phylogeny with 12 distinct RLTB lineages corresponding to each of the different riverine populations. However, coalescence-based methods using mitochondrial DNA markers identified only eight evolutionarily distinct lineages. Divergence time analysis points to recent separation of the populations, owing to the geographical isolation, more than 5 million years ago, after the lineages were split into two ancestral stocks in the Paleocene, on north and south of a major geographical gap in the Western Ghats. Our results revealing the existence of eight evolutionarily distinct RLTB lineages calls for the re-determination of conservation targets for these cryptic and endangered taxa.


Assuntos
Conservação dos Recursos Naturais , Cyprinidae/anatomia & histologia , Cyprinidae/genética , Espécies em Perigo de Extinção/estatística & dados numéricos , Evolução Molecular , Animais , Geografia , Índia , Rios , Fatores de Tempo
11.
Proc (Bayl Univ Med Cent) ; 26(1): 11-5, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23382601

RESUMO

A 57-year-old woman, who had undergone Roux-en-Y gastric bypass surgery 9 years earlier, was admitted to the intensive care unit because of pneumonia. Despite antibiotic therapy, she died 40 days later, apparently because of sepsis and organ failure related to the pneumonia. However, the patient's family requested an autopsy, which revealed that her death was due to perforation of the Roux limb of her gastric bypass, which had resulted in severe peritonitis. The perforation was caused by a nasogastric tube inserted for enteral nutrition. We discuss ways nasogastric tubes might be inserted more safely after gastric bypass, the response of Baylor University Medical Center at Dallas to this complication, and the role of autopsy in improving the quality of hospital care.

12.
Aquat Biosyst ; 8(1): 27, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23114277

RESUMO

BACKGROUND: The genus Dunaliella (Class - Chlorophyceae) is widely studied for its tolerance to extreme habitat conditions, physiological aspects and many biotechnological applications, such as a source of carotenoids and many other bioactive compounds. Biochemical and molecular characterization is very much essential to fully explore the properties and possibilities of the new isolates of Dunaliella. In India, hyper saline lakes and salt pans were reported to bloom with Dunaliella spp. However, except for the economically important D. salina, other species are rarely characterized taxonomically from India. Present study was conducted to describe Dunaliella strains from Indian salinas using a combined morphological, physiological and molecular approach with an aim to have a better understanding on the taxonomy and diversity of this genus from India. RESULTS: Comparative phenotypic and genetic studies revealed high level of diversity within the Indian Dunaliella isolates. Species level identification using morphological characteristics clearly delineated two strains of D. salina with considerable ß-carotene content (>20 pg/cell). The variation in 18S rRNA gene size, amplified with MA1-MA2 primers, ranged between ~1800 and ~2650 base pairs, and together with the phylogeny based on ITS gene sequence provided a pattern, forming five different groups within Indian Dunaliella isolates. Superficial congruency was observed between ITS and rbcL gene phylogenetic trees with consistent formation of major clades separating Indian isolates into two distinct clusters, one with D. salina and allied strains, and another one with D. viridis and allied strains. Further in both the trees, few isolates showed high level of genetic divergence than reported previously for Dunaliella spp. This indicates the scope of more numbers of clearly defined/unidentified species/sub-species within Indian Dunaliella isolates. CONCLUSION: Present work illustrates Indian Dunaliella strains phenotypically and genetically, and confirms the presence of not less than five different species (or sub-species) in Indian saline waters, including D. salina and D. viridis. The study emphasizes the need for a combined morphological, physiological and molecular approach in the taxonomic studies of Dunaliella.

13.
Appl Biochem Biotechnol ; 167(5): 1340-50, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22434357

RESUMO

Leishmaniasis is a group of diseases with a spectrum of clinical manifestations ranging from cutaneous ulcers to visceral leishmaniasis, which results from the bite of an infected sandfly to human. Attempts to develop an effective vaccine have been shown to be feasible but no vaccine is in active clinical use. This study adopts a Reverse Vaccinology approach to identify common vaccine candidates from both highly pathogenic species Leishmania major and Leishmania infantum. Total proteome of both species were compared to identify common proteins, which are further taken for sub-cellular localization and transmembrane helices prediction. Plasma membrane proteins having only one transmembrane helix were first identified and analyzed which are non-homologous in human and mouse in order to avoid molecular mimicry with other proteins. Selected proteins were analyzed for their binding efficiency to both major histocompatibility complex (MHC) class I and class II alleles. As a result, 19 potential epitopes are screened in this study using different approaches, which can be further verified through in vivo experiments in MHC compatible animal models. This study demonstrates that Reverse Vaccinology approach has potential in discovering various immunogenic antigens from in silico analysis of pathogen's genome or proteome instead of culturing the whole organism by conventional methods.


Assuntos
Biologia Computacional , Leishmania/imunologia , Vacinas/imunologia , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Vacinas/química , Vacinas/genética
14.
PLoS One ; 6(9): e25378, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21966512

RESUMO

BACKGROUND: Hepatitis E virus (HEV) is a non-enveloped plus-strand RNA virus that causes acute hepatitis. The capsid protein open reading frame 2 (ORF2) is known to induce endoplasmic reticulum stress in ORF2 expressing cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study we found that HEV ORF2 activates the expression of the pro-apoptotic gene C/EBP homologous protein (CHOP). ORF2 stimulates the CHOP promoter mainly through AARE (amino acid response elements) and to a minor extent the ERSE (endoplasmic reticulum stress response elements). Activating transcription factor 4 (ATF4) protein binds and activates the AARE regulatory sites of the CHOP promoter. ORF2 expression also leads to increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) that in turn initiates the translation of ATF4 mRNA. The pro-apoptotic gene CHOP is an important trigger to initiate endoplasmic reticulum stress induced apoptosis. However, the activation of CHOP by ORF2 in this study did not induce apoptosis, nor did BCL2-associated X protein (Bax) translocate to mitochondria. Microarray analysis revealed an ORF2 specific increased expression of chaperones Hsp72, Hsp70B', and co-chaperone Hsp40. Co-immunoprecipitation (Co-IP) and in silico molecular docking analysis suggests that HEV ORF2 interacts with Hsp72. In addition, Hsp72 shows nuclear accumulation in ORF2 expressing cells. CONCLUSIONS/SIGNIFICANCE: These data provide new insight into simultaneously occurring counter-acting effects of HEV ORF2 that may be part of a strategy to prevent host suicide before completion of the viral replication cycle.


Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP72/metabolismo , Vírus da Hepatite E/metabolismo , Fator de Transcrição CHOP/metabolismo , Proteínas Virais/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Western Blotting , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP72/genética , Humanos , Imunoprecipitação , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição CHOP/genética , Proteínas Virais/genética
15.
J Biol Chem ; 286(24): 21779-95, 2011 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-21504896

RESUMO

An elevated level of homocysteine, a thiol amino acid, is associated with various complex disorders. The cellular effects of homocysteine and its precursors S-adenosylhomocysteine (AdoHcy) and S-adenosylmethionine (AdoMet) are, however, poorly understood. We used Saccharomyces cerevisiae as a model to understand the basis of pathogenicity induced by homocysteine and its precursors. Both homocysteine and AdoHcy but not AdoMet inhibited the growth of the str4Δ strain (which lacks the enzyme that converts homocysteine to cystathionine-mimicking vascular cells). Addition of AdoMet abrogated the inhibitory effect of AdoHcy but not that of homocysteine indicating that an increase in the AdoMet/AdoHcy ratio is sufficient to overcome the AdoHcy-mediated growth defect but not that of homocysteine. Also, the transcriptomic profile of AdoHcy and homocysteine showed gross dissimilarity based on gene enrichment analysis. Furthermore, compared with homocysteine, AdoHcy treatment caused a higher level of oxidative stress in the cells. However, unlike a previously reported response in wild type (Kumar, A., John, L., Alam, M. M., Gupta, A., Sharma, G., Pillai, B., and Sengupta, S. (2006) Biochem. J. 396, 61-69), the str4Δ strain did not exhibit an endoplasmic reticulum stress response. This suggests that homocysteine induces varied response depending on the flux of homocysteine metabolism. We also observed altered expression of mitochondrial genes, defective membrane potential, and fragmentation of the mitochondrial network together with the increased expression of fission genes indicating that the imbalance in homocysteine metabolism has a major effect on mitochondrial functions. Furthermore, treatment of cells with homocysteine or AdoHcy resulted in apoptosis as revealed by annexin V staining and TUNEL assay. Cumulatively, our results suggest that elevated levels of homocysteine lead to mitochondrial dysfunction, which could potentially initiate pro-apoptotic pathways, and this could be one of the mechanisms underlying homocysteine-induced pathogenicity.


Assuntos
Regulação Fúngica da Expressão Gênica , Homocisteína/metabolismo , Anexina A5/química , Cromatina/química , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/química , Genes Fúngicos , Homocisteína/química , Marcação In Situ das Extremidades Cortadas , Potenciais da Membrana , Modelos Biológicos , Hibridização de Ácido Nucleico , Estresse Oxidativo , Desnaturação Proteica , Saccharomyces cerevisiae/metabolismo , Transcrição Genética
16.
Virol J ; 7: 327, 2010 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-21087473

RESUMO

BACKGROUND: The alphavirus capsid is multifunctional and plays a key role in the viral life cycle. The nucleocapsid domain is released by the self-cleavage activity of the serine protease domain within the capsid. All alphaviruses analyzed to date show this autocatalytic cleavage. Here we have analyzed the sequence requirements for the cleavage activity of Chikungunya virus capsid protease of genus alphavirus. RESULTS: Amongst alphaviruses, the C-terminal amino acid tryptophan (W261) is conserved and found to be important for the cleavage. Mutating tryptophan to alanine (W261A) completely inactivated the protease. Other amino acids near W261 were not having any effect on the activity of this protease. However, serine protease inhibitor AEBSF did not inhibit the activity. Through error-prone PCR we found that isoleucine 227 is important for the effective activity. The loss of activity was analyzed further by molecular modelling and comparison of WT and mutant structures. It was found that lysine introduced at position 227 is spatially very close to the catalytic triad and may disrupt electrostatic interactions in the catalytic site and thus inactivate the enzyme. We are also examining other sequence requirements for this protease activity. CONCLUSIONS: We analyzed various amino acid sequence requirements for the activity of ChikV capsid protease and found that amino acids outside the catalytic triads are important for the activity.


Assuntos
Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Vírus Chikungunya/enzimologia , Vírus Chikungunya/genética , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Animais , Domínio Catalítico , Análise Mutacional de DNA , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
17.
Biochem J ; 396(1): 61-9, 2006 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-16433631

RESUMO

Intracellular thiols like cysteine, homocysteine and glutathione play a critical role in the regulation of important cellular processes. Alteration of intracellular thiol concentration results in many diseased states; for instance, elevated levels of homocysteine are considered to be an independent risk factor for cardiovascular disease. Yeast has proved to be an excellent model system for studying many human diseases since it carries homologues of nearly 40% of human disease genes and many fundamental pathways are highly conserved between the two organisms. In the present study, we demonstrate that cysteine and homocysteine, but not glutathione, inhibit yeast growth in a concentration-dependent manner. Using deletion strains (str2Delta and str4Delta) we show that cysteine and homocysteine independently inhibit yeast growth. Transcriptional profiling of yeast treated with cysteine and homocysteine revealed that genes coding for antioxidant enzymes like glutathione peroxidase, catalase and superoxide dismutase were down-regulated. Furthermore, transcriptional response to homocysteine did not show any similarity to the response to H2O2. We also failed to detect induction of reactive oxygen species in homocysteine- and cysteine-treated cells, using fluorogenic probes. These results indicate that homocysteine- and cysteine-induced growth defect is not due to the oxidative stress. However, we found an increase in the expression of KAR2 (karyogamy 2) gene, a well-known marker of ER (endoplasmic reticulum) stress and also observed HAC1 cleavage in homocysteine- and cysteinetreated cells, which indicates that homocysteine- and cysteine-mediated growth defect may probably be attributed to ER stress. Transcriptional profiling also revealed that genes involved in one-carbon metabolism, glycolysis and serine biosynthesis were up-regulated on exogenous addition of cysteine and homocysteine, suggesting that cells try to reduce the intracellular concentration of thiols by up-regulating the genes involved in their metabolism.


Assuntos
Cisteína/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Homocisteína/farmacologia , Estresse Oxidativo/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina Básica/biossíntese , Fatores de Transcrição de Zíper de Leucina Básica/genética , Meios de Cultivo Condicionados/química , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Glutationa/farmacologia , Glicólise/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/genética , Metionina/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/análise , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Compostos de Sulfidrila/análise
18.
Biochem Biophys Res Commun ; 338(3): 1578-86, 2005 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-16289044

RESUMO

The response of Saccharomyces cerevisiae cells to an aqueous extract of cigarette smoke was studied. Exposure to cigarette smoke extract inhibits yeast growth and results in global changes in gene expression spanning many functional classes of genes. Genes involved in response to oxidative stress are upregulated after a brief exposure to cigarette smoke extract. The effects of cigarette smoke extract on yeast growth can be reversed by treatment with anti-oxidants. Mutants lacking superoxide dismutase gene were hypersensitive to cigarette smoke exposure. YAP1 is a central transcriptional regulator of oxidative stress in yeast. YAP1 dependent expression of beta-galactosidase was enhanced following exposure to cigarette smoke. The overall agreement between our observations and the recently reported effects of cigarette smoke on gene expression in rodent and human cells suggests that yeast can be used as a model system in toxicogenomics studies for monitoring toxic agents and studying the cellular and molecular consequences of exposure to potentially toxic agents.


Assuntos
Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Fumaça , Tabaco/química , Transportadores de Cassetes de Ligação de ATP/genética , Antioxidantes/farmacologia , Calorimetria , Proliferação de Células/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1
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