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1.
Eur J Dent Educ ; 18(1): 39-43, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24423174

RESUMO

Given the rapidly changing demography of populations worldwide, dental professionals of the future need to be able to meet the challenge posed by the evolving landscape in health care needs. Leading institutions are now embedding teaching and learning in special care dentistry (SCD) within their curricula, to provide students with the knowledge, skills and attitudes to meet the oral health needs of vulnerable groups within their communities. The International Association for Disability and Oral Health (iADH) has initiated the development of undergraduate curriculum guidance in SCD through a consensus process. The curriculum in SCD is defined in statements of learning outcomes with many of the skills being transferable across the undergraduate course. This curriculum includes examples of teaching and assessment, designed to enhance critical thinking in relation to SCD and to promote positive attitudes towards disability and diversity. The learning outcomes are designed to be readily adapted to conform to the generic profiles and competencies, already identified in undergraduate frameworks by global educational associations, as well as meeting the requirements of professional regulatory bodies worldwide. Suggestions for teaching and learning are not intended to be prescriptive; rather, they act as a signpost to possible routes to student learning. Ideally, this will require that students have a sufficiently diverse patient case mix during their undergraduate studies, to achieve the required levels of confidence and competence by the time they graduate. Clinical care competencies in SCD emphasise the need for learners to broaden their theoretical knowledge and understanding through practical experience in providing care for people with special health care needs. It is crucial to the development of equitable dental services for all members of a community, that these learning outcomes are embedded into evolving curricula but most importantly, that they are evaluated and refined in a dynamic way with shared learning for all teachers.


Assuntos
Currículo/tendências , Educação em Odontologia/tendências , Especialidades Odontológicas/educação , Especialidades Odontológicas/tendências , Competência Clínica , Avaliação Educacional , Humanos
2.
Science ; 372(6545): 980-983, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34045354

RESUMO

Climate change and other human activities are causing profound effects on marine ecosystem productivity. We show that the breeding success of seabirds is tracking hemispheric differences in ocean warming and human impacts, with the strongest effects on fish-eating, surface-foraging species in the north. Hemispheric asymmetry suggests the need for ocean management at hemispheric scales. For the north, tactical, climate-based recovery plans for forage fish resources are needed to recover seabird breeding productivity. In the south, lower-magnitude change in seabird productivity presents opportunities for strategic management approaches such as large marine protected areas to sustain food webs and maintain predator productivity. Global monitoring of seabird productivity enables the detection of ecosystem change in remote regions and contributes to our understanding of marine climate impacts on ecosystems.

3.
J Neurosci Res ; 88(8): 1651-63, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20077429

RESUMO

Campylobacteriosis is a frequent antecedent event in Guillain-Barré syndrome (GBS), inducing high-titer serum antibodies for ganglioside antigens in the peripheral nervous system (PNS). Molecular mimicry between the lipooligosaccharide (LOS) component of Campylobacter jejuni and human peripheral nerve gangliosides is believed to play an important role in the pathogenesis of GBS. Conventional treatment strategies for patients with GBS include plasmapheresis, intravenous immunoglobulin (IVIG), and immunosuppression, which are invasive or relatively ineffective. In this study, we used our animal model of GBS, in which Lewis rats were immunized with GD3-like LOS isolated from C.jejuni. The animals developed anti-GD3 ganglioside antibodies and manifested neuromuscular dysfunction. To develop novel therapeutic strategies, we treated the animals by intraperitoneal administration of an anti-GD3 antiidiotype monoclonal antibody (BEC2) that specifically interacts with the pathogenic antibody. The treated animals had a remarkable reduction of anti-GD3 antibody titers and improvement of motor nerve functions. The results suggest that ganglioside mimics, such as antiidiotype antibodies, may be powerful reagents for therapeutic intervention in GBS by neutralizing specific pathogenic antiganglioside antibodies.


Assuntos
Anticorpos Anti-Idiotípicos/uso terapêutico , Gangliosídeos/imunologia , Neurite Autoimune Experimental/terapia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Biotinilação/métodos , Campylobacter jejuni/imunologia , Técnicas de Cocultura/métodos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática/métodos , Canais de Potássio Éter-A-Go-Go/imunologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Canais de Potássio Éter-A-Go-Go/farmacocinética , Feminino , Adjuvante de Freund/imunologia , Lipopolissacarídeos , Neurônios Motores/patologia , Neurônios Motores/ultraestrutura , Músculo Esquelético/fisiologia , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/farmacocinética , Neurite Autoimune Experimental/induzido quimicamente , Neurite Autoimune Experimental/complicações , Neurite Autoimune Experimental/imunologia , Doenças da Junção Neuromuscular/tratamento farmacológico , Doenças da Junção Neuromuscular/etiologia , Doenças da Junção Neuromuscular/metabolismo , Técnicas de Cultura de Órgãos , Ratos , Teste de Desempenho do Rota-Rod/métodos , Nervo Isquiático/patologia , Nervo Isquiático/ultraestrutura , Medula Espinal/fisiologia , Fatores de Tempo
4.
Science ; 233(4761): 341-3, 1986 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-17737623

RESUMO

Three ichthyotoxic peptides, pardaxins P-I to P-3, have been isolated from the defense secretion of the sole Pardachirus pavoninus. Pavoninins, the steroid glycosides with shark-repelling ability, had previously been isolated therefrom. Each pardaxin consists of 33 amino acid residues having a distinctly hydrophilic carboxyl terminal region and a predominantly hydrophobic remainder; the pardaxin is thus strongly surfactant. These peptides show marked physical and pharmacological similarities to melittin, the major active constituent of bee venom, yet they lack sequence homology. They are probably also responsible for the predator-repelling property of the sole.

5.
J Neurol Neurosurg Psychiatry ; 79(4): 448-50, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18032455

RESUMO

Alpha-methylacyl-CoA racemase (AMACR) deficiency is a rare disorder of fatty acid metabolism which has recently been described in three adult cases. We have identified a further patient with clinical features of a relapsing encephalopathy, seizures and cognitive decline over a 40 year period. Biochemical studies revealed grossly elevated plasma pristanic acid levels, and a deficiency of AMACR in skin fibroblasts. Sequence analysis of AMACR cDNA identified a homozygous point mutation (c154T>C). This case adds to the phenotypic variation seen in this peroxisomal disorder and highlights the importance of screening for plasma pristanic acid levels in patients with unexplained relapsing encephalopathies.


Assuntos
Encefalopatias Metabólicas Congênitas/diagnóstico , Encefalopatias Metabólicas Congênitas/genética , Demência/diagnóstico , Demência/genética , Ácidos Graxos/sangue , Transtornos Heredodegenerativos do Sistema Nervoso/diagnóstico , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Peroxissômicos/diagnóstico , Transtornos Peroxissômicos/genética , Racemases e Epimerases/deficiência , Doença de Refsum/diagnóstico , Doença de Refsum/genética , Substituição de Aminoácidos/genética , Encéfalo/patologia , Córtex Cerebral/patologia , Feminino , Regulação Enzimológica da Expressão Gênica/fisiologia , Homozigoto , Humanos , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Exame Neurológico , Testes Neuropsicológicos , Fenótipo , Mutação Puntual/genética , Recidiva , Análise de Sequência de DNA
7.
Meat Sci ; 74(2): 388-95, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22062850

RESUMO

Numerous studies have revealed that pre-slaughter stress, like transport, increases the occurrence of pale, soft, and exudative (PSE) pork meat. The molecular mechanism underlying this phenomenon, however, is poorly defined. In this study, we investigated the effects of pre-slaughter transport and subsequent rest on energy metabolism, AMP-activated protein kinase (AMPK) activation, and glycolysis in postmortem pork loin. Results indicated that pre-slaughter transport accelerated ATP depletion, which led to lower energy status in postmortem muscle immediately post-exsanguination when compared with control. The lower energy status led to AMPK activation within 1h postmortem, subsequently increasing glycolysis, leading to rapid glycolysis and high incidence of PSE meat. Allowing pigs to rest after transport restored energy status in muscle ante-mortem. Higher energy status then prevented premature and rapid AMPK activation in postmortem muscle and lessened the negative effects of pre-slaughter transport on meat quality. AMPK regulated glycolysis in postmortem muscle, at least partially, through phosphorylation and activation of phosphofructose kinase-2, since fructose-2,6-diphosphate content, an allosteric activator of phosphofructose kinase-1, was well correlated with AMPK activation and glycolytic rate. This suggests that AMPK is a potential molecular target for the control of PSE incidence in pork.

8.
Chem Sci ; 7(9): 6232-6237, 2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30034763

RESUMO

A new method is introduced for self-assembling citrate-capped gold nanoparticles into supraparticles with crystallographically aligned building blocks. It consists in confining gld nanoparticles inside a cellulose acetate membrane. The constituent nanoparticles are in close contact in the superstructure, and therefore generate hot spots leading to intense Surface-Enhanced Raman Scattering (SERS) signals. They also generate more plasmonic heat than the nanoparticle building blocks. The supraparticles are internalized by cells and show low cytotoxicity, but can kill cancer cells when irradiated with a laser. This, along with the improved plasmonic properties arising from their assembly, makes the gold supraparticles promising materials for applications in bioimaging and nanomedicine.

9.
Clin Neurophysiol ; 127(1): 117-128, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25920940

RESUMO

OBJECTIVES: The significance of infraslow activity (ISA) in focal epilepsies is largely unknown. Recent work has demonstrated ictal ISA to be more widespread in expression than originally understood. Analysis of ISA by stereoelectroencephalography (SEEG) may help to clarify its localizing value, namely the focal versus widespread expression of ISA. METHODS: The ictal SEEG records for fifteen consecutive adult patients were retrospectively analyzed, using both conventional (1.6-70 Hz) and infraslow (0.01-0.1 Hz) bandpass filters. When justified, seizures were averaged in the infraslow band to clarify their stereotypy. Wavelets were used to quantify the time-frequency characteristics of ISA. RESULTS: All clinical seizures were found to possess ISA, and this was markedly invariant across seizures in a given patient. ISA showed biphasic peaks in power, both at ictal onset and offset, with this most prominent in the anatomical structures implicated by conventional analysis. In addition, ISA demonstrated an association with low voltage fast activity, and possessed a more restricted field than conventional activity. CONCLUSIONS: ISA is both widespread (anatomically distributed) and focal (closed electric field). Seizures possess an infraslow spatiotemporal signature. SIGNIFICANCE: Beyond representing a "focus" of paroxysmal activity, ISA must arise from a network process as a component of wideband ictal dynamics. How this relates to clinical definition of the epileptogenic zone requires further study.


Assuntos
Eletroencefalografia , Convulsões/fisiopatologia , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Convulsões/diagnóstico
10.
Oncogenesis ; 5(6): e238, 2016 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-27348268

RESUMO

Composition of the gut microbiota has profound effects on intestinal carcinogenesis. Diet and host genetics play critical roles in shaping the composition of gut microbiota. Whether diet and host genes interact with each other to bring specific changes in gut microbiota that affect intestinal carcinogenesis is unknown. Ability of dietary fibre to specifically increase beneficial gut microbiota at the expense of pathogenic bacteria in vivo via unknown mechanism is an important process that suppresses intestinal inflammation and carcinogenesis. Free fatty acid receptor 2 (FFAR2 or GPR43) is a receptor for short-chain fatty acids (acetate, propionate and butyrate), metabolites of dietary fibre fermentation by gut microbiota. Here, we show FFAR2 is down modulated in human colon cancers than matched adjacent healthy tissue. Consistent with this, Ffar2(-/-) mice are hypersusceptible to development of intestinal carcinogenesis. Dietary fibre suppressed colon carcinogenesis in an Ffar2-dependent manner. Ffar2 played an essential role in dietary fibre-mediated promotion of beneficial gut microbiota, Bifidobacterium species (spp) and suppression of Helicobacter hepaticus and Prevotellaceae. Moreover, numbers of Bifidobacterium is reduced, whereas those of Prevotellaceae are increased in human colon cancers than matched adjacent normal tissue. Administration of Bifidobacterium mitigated intestinal inflammation and carcinogenesis in Ffar2(-/-) mice. Taken together, these findings suggest that interplay between dietary fibre and Ffar2 play a key role in promoting healthy composition of gut microbiota that stimulates intestinal health.

11.
Biochim Biophys Acta ; 1416(1-2): 145-54, 1999 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-9889355

RESUMO

Glucose and fructose enter mammalian cells via facilitated diffusion, a process regulated by five glucose transporter isoforms (GLUT1-5) at the plasma membrane. The tissue-specific pattern of GLUT isoform expression likely reflects differing needs for glucose transport by various tissues. Myocytes must respond expeditiously to increased metabolic demand. A basal isoform, GLUT1, and the insulin-regulatable glucose transporter, GLUT4, have been demonstrated in human myocytes. GLUT3 has a high affinity for glucose, but its presence in human myocardium has not been clearly established. The purpose of this study was to determine whether GLUT3 protein is present in human cardiac myocytes. We examined rapidly frozen myocardial tissue from the explanted heart of seven patients undergoing cardiac transplantation, from the heart of a young, previously healthy male organ donor, from the heart of a 67-year-old woman without known cardiac disease who had a fatal stroke, and from the heart of six human fetuses. GLUT3 protein was detected by immunoblots and localized by light and electron microscopy immunohistochemistry. The presence of GLUT3 protein was verified in myocardial tissue by both immunoblots and immunohistochemistry. Light and electron microscopy confirmed that GLUT3 was in cardiac myocytes. GLUT3 was also demonstrated as a 48 kDa protein in fetal myocardium, which was present at 10 weeks, increased at 15 weeks, then decreased at 20 weeks of gestation. GLUT3 is present in human adult and fetal myocardium. Human myocardial GLUT3 regulation and its role in myocardial glucose uptake remain to be elucidated.


Assuntos
Proteínas de Transporte de Monossacarídeos/análise , Miocárdio/metabolismo , Proteínas do Tecido Nervoso , Idoso , Criança , Feminino , Feto/metabolismo , Idade Gestacional , Transportador de Glucose Tipo 3 , Humanos , Immunoblotting , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Músculo Esquelético/metabolismo , Pericárdio/metabolismo
12.
Biochim Biophys Acta ; 1446(3): 334-40, 1999 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-10524207

RESUMO

Human CD52 (CAMPATH-1 antigen) is an abundant surface molecule on lymphocytes and a favoured target for lymphoma therapy and immunosuppression. It comprises a small glycosylphosphatidylinositol (GPI) anchored peptide to which a large carbohydrate moiety is attached. Structurally similar proteins include the proposed mouse homologue, B7 antigen (B7-Ag; not to be confused with the CD28 ligand), and human and mouse CD24. Sequence similarities between CD52 and B7-Ag precursors are concentrated over the signal peptides and the sequences cleaved during GPI attachment. While the short mature peptides are not apparently homologous, the N-linked glycosylation site is retained in both. We describe similarities in exon-intron organisation, syntenic chromosome positions (human CD52, 1p36; mouse B7-Ag, chromosome 4, between Dsil and D4Nds16) and sequence homology in the promoter regions which strongly suggests that B7-Ag is the mouse homologue of CD52. The structure of these genes is also similar to that of mouse CD24, suggesting a common ancestor. Promoter activities and transcription start sites were also analysed. These results suggest that human CD52 and mouse B7-Ag gene expressions are controlled by TATA-less promoters.


Assuntos
Antígenos CD/genética , Antígenos de Neoplasias , Glicoproteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Antígeno CD52 , Mapeamento Cromossômico , Éxons , Biblioteca Genômica , Humanos , Hibridização in Situ Fluorescente , Íntrons , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico
13.
Neuropsychologia ; 43(9): 1266-76, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15949511

RESUMO

Little is known about episodic and semantic memory in the early predementia stage of Alzheimer's disease (AD), which is referred to as mild cognitive impairment (MCI). To explore person knowledge, item recognition and spatial associative memory, we designed the Face Place Test (FPT). A total of 75 subjects participated: 22 patients with early AD, 24 with MCI and 29 matched controls. As predicted, AD patients showed significant deficits in person naming, item recognition and recall of spatial location (placing). Surprisingly, subjects with MCI were also impaired on all components. There was no significant difference between AD and MCI except on the placing component. Analysis of the relationship between semantic (naming) and episodic (recognition and placing) components of the FPT revealed a significant association between the two episodic tasks, but not between episodic and semantic performance. Patients with MCI show deficits of episodic and semantic memory. The extent of impairment suggests dysfunction beyond the medial temporal lobe. The FPT might form the basis of a sensitive early indicator of AD.


Assuntos
Transtornos Cognitivos/fisiopatologia , Memória/fisiologia , Reconhecimento Visual de Modelos/fisiologia , Semântica , Idoso , Doença de Alzheimer/fisiopatologia , Estudos de Casos e Controles , Intervalos de Confiança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos/estatística & dados numéricos , Razão de Chances , Estimulação Luminosa/métodos
14.
Neuropsychologia ; 43(9): 1338-50, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15949518

RESUMO

Patients with early stage Alzheimer's disease (AD) show deficits in person knowledge and spatial associative memory. The current investigation examined the ability of impairment in these domains to differentiate AD from other overlapping conditions. In experiment 1, 14 AD patients, 21 vascular dementia (VaD) patients, 11 frontal variant frontotemporal dementia (fvFTD) patients and 41 controls were administered a graded faces test. VaD patients demonstrated a level of impairment comparable to the AD group on both the naming and person identification elements of the task. A mild naming deficit was revealed in the fvFTD group. In experiment 2, 22 AD patients, 23 patients with mild cognitive impairment (MCI), 11 fvFTD patients, 13 semantic dementia (SD) patients, and 23 elderly controls were administered the face-place test, a newly developed task that combines naming of famous faces, item recognition and spatial location. The naming component of the face-place test clearly differentiated SD patients from all dementia groups. All patient groups, except those with fvFTD, showed substantial deficits in the item recognition and spatial components. Consistency analyses indicated a fairly robust association between the two episodic components (item recognition and placing), but not between semantic and episodic elements of the FPT. Person knowledge deficits are, therefore, not specific to AD and the employment of face stimuli may influence the performance of SD patients on tasks of episodic memory.


Assuntos
Doença de Alzheimer/fisiopatologia , Aprendizagem por Associação/fisiologia , Demência Vascular/fisiopatologia , Demência/fisiopatologia , Conhecimento , Percepção Espacial/fisiologia , Idoso , Análise de Variância , Estudos de Casos e Controles , Discriminação Psicológica , Feminino , Humanos , Masculino , Entrevista Psiquiátrica Padronizada , Pessoa de Meia-Idade , Testes Neuropsicológicos/estatística & dados numéricos , Reconhecimento Visual de Modelos/fisiologia , Reconhecimento Psicológico/fisiologia , Semântica
15.
Curr Opin Pharmacol ; 1(1): 78-83, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11712540

RESUMO

Major progress in our understanding of the mechanisms of anaesthesia has been made during the past year. Several key advances in defining very specific sites of action on ligand-gated ion channels have been described. Furthermore, new techniques have become available for addressing the identification of binding sites and transduction mechanisms on these receptors. The discovery that anaesthetics affect a recently identified family of potassium channels could also lead to major new findings in the next few years.


Assuntos
Anestésicos Gerais/farmacologia , Neurobiologia , Álcoois/farmacologia , Anestésicos Intravenosos/farmacologia , Animais , Humanos , Receptores de GABA-A/efeitos dos fármacos , Receptores de Glicina/efeitos dos fármacos
16.
Gene ; 167(1-2): 337-8, 1995 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-8566805

RESUMO

The Aspergillus niger (An) gene srpA, encoding a protein with homology to the signal recognition particle (SRP) 54-kDa protein from Saccharomyces cerevisiae (Sc), has been isolated and the nucleotide sequence determined. The putative An srpA gene is comprised of two exons of 78 and 1527 bp separated by a 49-bp intron, and encodes a protein of 534 amino acids that is 53% identical to Sc SRP54.


Assuntos
Aspergillus niger/genética , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas Fúngicas/genética , Genes Fúngicos , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas/metabolismo
17.
Gene ; 209(1-2): 113-22, 1998 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-9583946

RESUMO

Helicobacter pylori persists in the human stomach where it may encounter a variety of DNA-damaging conditions, including gastric acidity. To determine whether the nucleotide excision repair (NER) pathway contributes to the repair of acid-induced DNA damage, we have cloned the putative H. pylori NER gene, uvrB. Degenerate oligonucleotide primers based on conserved amino acid residues of bacterial UvrB proteins were used in PCR with genomic DNA from H. pylori strain 84-183, and the 1.3-kb PCR product from this reaction was used as a probe to clone uvrB from an H. pylori genomic library. This plasmid clone had a 5.5-kb insert containing a 2.0-kb ORF whose predicted product (658 amino acids; 75.9 kDa) exhibited 69.5% similarity to E. coli UvrB. We constructed an isogenic H. pylori uvrB mutant by inserting a kanamycin-resistance cassette into uvrB and verified its proper placement by Southern hybridization. As with uvrB mutants of other bacteria, the H. pylori uvrB mutant showed a greatly increased sensitivity to the DNA-damaging agents methylmethane sulfonate and ultraviolet radiation. The uvrB mutant also was significantly more sensitive than the wild-type strain to killing by low pH, suggesting that the H. pylori nucleotide excision repair (NER) pathway is involved in the repair of acid-induced DNA damage.


Assuntos
Proteínas de Bactérias/genética , DNA Helicases , Reparo do DNA/genética , Proteínas de Escherichia coli , Helicobacter pylori/genética , Filogenia , Raios Ultravioleta , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Sequência de Bases , Clonagem Molecular , Dano ao DNA , Primers do DNA , Escherichia coli/genética , Evolução Molecular , Biblioteca Genômica , Helicobacter pylori/crescimento & desenvolvimento , Helicobacter pylori/efeitos da radiação , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
18.
Gene ; 125(2): 195-8, 1993 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-8462873

RESUMO

We have cloned and determined the nucleotide sequence of a genomic DNA segment from Aspergillus oryzae which contains pepO, the gene encoding the aspartic proteinase, aspergillopepsin O (PEPO). The organization of pepO is strikingly similar to that of pepA from A. niger var. awamori (previously called A. awamori) in that both are composed of four exons and three introns with virtually identical lengths, and the positions of the introns are exactly conserved. From the deduced amino acid (aa) sequence, it appears that PEPO, like other fungal aspartic proteinases, is synthesized as a zymogen containing a putative N-terminal prepro-region of 77 aa followed by a mature protein of 327 aa. Southern blotting experiments suggest that a single copy of pepO exists in the A. oryzae genome.


Assuntos
Ácido Aspártico Endopeptidases/genética , Aspergillus oryzae/genética , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/metabolismo , Aspergillus oryzae/enzimologia , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA Fúngico , Genes Fúngicos , Dados de Sequência Molecular , Mapeamento por Restrição
19.
Gene ; 86(2): 153-62, 1990 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-2182390

RESUMO

We have cloned genomic pepA sequences encoding the aspartic proteinase aspergillopepsin A (PEPA) from Aspergillus awamori using a synthetic oligodeoxyribonucleotide probe. Nucleotide sequence data from the pepA gene revealed that it is composed of four exons of 320, 278, 249, and 338 bp. Three introns which interrupt the coding sequence are 51, 52, and 59 bp in length. Directly downstream from the putative start codon lies a sequence encoding 69 amino acids (aa) which are not present in mature PEPA. Based on similarities to other aspartic proteinases, this region may represent a 20-aa signal peptide followed by a 49-aa propeptide that is rich in basic aa residues. Northern blots of total cellular RNA extracted from A. awamori cells indicate that pepA is transcribed as a single 1.4-kb mRNA. Mutants of A. awamori lacking the pepA structural gene were derived by the following gene replacement strategy. First, we constructed a plasmid in which a 2.4-kb SalI fragment containing the entire pepA coding region was deleted from a 9-kb Eco RI genomic DNA clone and replaced by a synthetic DNA polylinker. Second, a selectable argB gene was inserted into the polylinker. Third, the EcoRI fragment which contained the argB marker flanked by pepA sequences was excised from the plasmid and used to transform an argB auxotroph of A. awamori. From 16-40% of the resulting prototrophic transformants were found to have a PEPA-deficient phenotype when screened with an immunoassay using antibodies specific for PEPA. Southern hybridization experiments confirmed that these mutants resulted from a gene replacement event at the pepA locus.


Assuntos
Ácido Aspártico Endopeptidases , Aspergillus/genética , Deleção Cromossômica , Clonagem Molecular , Endopeptidases/genética , Genes Fúngicos , Sequência de Aminoácidos , Aspergillus/enzimologia , Sequência de Bases , Códon/genética , Endopeptidases/isolamento & purificação , Vetores Genéticos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Mapeamento por Restrição
20.
Free Radic Biol Med ; 27(11-12): 1208-18, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10641713

RESUMO

The reduced glutathione (GSH)/oxidized glutathione (GSSG) redox state is thought to function in signaling of detoxification gene expression, but also appears to be tightly regulated in cells under normal conditions. Thus it is not clear that the magnitude of change in response to physiologic stimuli is sufficient for a role in redox signaling under nontoxicologic conditions. The purpose of this study was to determine the change in 2GSH/GSSG redox during signaling of differentiation and increased detoxification enzyme activity in HT29 cells. We measured GSH, GSSG, cell volume, and cell pH, and we used the Nernst equation to determine the changes in redox potential Eh of the 2GSH/GSSG pool in response to the differentiating agent, sodium butyrate, and the detoxification enzyme inducer, benzyl isothiocyanate. Sodium butyrate caused a 60-mV oxidation (from -260 to -200 mV), an oxidation sufficient for a 100-fold change in protein dithiols:disulfide ratio. Benzyl isothiocyanate caused a 16-mV oxidation in control cells but a 40-mV oxidation (to -160 mV) in differentiated cells. Changes in GSH and mRNA for glutamate:cysteine ligase did not correlate with Eh; however, correlations were seen between Eh and glutathione S-transferase (GST) and nicotinamide adenine dinucleotide phosphate (NADPH):quinone reductase activities (N:QR). These results show that 2GSH/GSSG redox changes in response to physiologic stimuli such as differentiation and enzyme inducers are of a sufficient magnitude to control the activity of redox-sensitive proteins. This suggests that physiologic modulation of the 2GSH/GSSG redox poise could provide a fundamental parameter for the control of cell phenotype.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Glutationa/metabolismo , Oxirredução , Adenocarcinoma , Butiratos/farmacologia , Tamanho Celular , Neoplasias do Colo , Expressão Gênica/efeitos dos fármacos , Glutamato-Cisteína Ligase/genética , Glutationa Transferase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Isotiocianatos/farmacologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas
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