RESUMO
An extended neural network is known to underlie extinction learning. As yet, comparatively little is known about the possible contribution of the cerebellum and the dorsolateral prefrontal cortex (dlPFC). In the present study, transcranial direct current stimulation (tDCS) was used to provide further evidence that the dlPFC and the cerebellum are involved in extinction-related processes. A total of 100 young and healthy human participants were randomly assigned to one of five stimulation groups: (1) anodal tDCS of the cerebellum, (2) cathodal tDCS of the cerebellum, (3) anodal tDCS of the dlPFC, (4) cathodal tDCS of the dlPFC, and (5) sham stimulation. Participants underwent delay eyeblink conditioning using an A-B-A/B renewal paradigm. Two different colors of background light (orange and blue) were used as contexts. On day 1, acquisition of conditioned eyeblink responses was performed in context A, followed by extinction in context B. tDCS was applied during extinction. On day 2, extinction recall was tested in contexts A and B with higher incidence of conditioned responses in acquisition context A compared to extinction context B indicating renewal effects. All groups showed significant effects of acquisition of conditioned eyeblink responses and significant effects of extinction. There was no significant difference in extinction between stimulation groups. During extinction recall, renewal effects were present in all groups, except the group which had received anodal tDCS of the dlPFC during extinction. In the present study, no direct effects of dlPFC or cerebellar tDCS were demonstrated on extinction. Anodal tDCS of the dlPFC, but not the cerebellum, resulted in delayed effects on context-related processes of extinction, possibly explained by shifting attention away from the context and towards the conditioned stimulus during extinction learning. Anodal tDCS of the dlPFC attenuated context-related recall of learned aversive responses. Effects of tDCS, however, were weak and need to be confirmed in future studies. Lack of cerebellar tDCS effects do not exclude a possible role of the cerebellum in extinction-related processes, and are likely explained by methodological limitations of cerebellar tDCS.
Assuntos
Cerebelo/fisiologia , Condicionamento Palpebral/fisiologia , Extinção Psicológica/fisiologia , Córtex Pré-Frontal/fisiologia , Adulto , Feminino , Humanos , Masculino , Estimulação Transcraniana por Corrente Contínua , Adulto JovemRESUMO
Modern microbial mats are widely recognized as useful analogs for the study of biogeochemical processes relevant to paleoenvironmental reconstruction in the Precambrian. We combined microscopic observations and investigations of biomarker composition to investigate community structure and function in the upper layers of a thick phototrophic microbial mat system from a hypersaline lake on Kiritimati (Christmas Island) in the Northern Line Islands, Republic of Kiribati. In particular, an exploratory incubation experiment with (13)C-labeled bicarbonate was conducted to pinpoint biomarkers from organisms actively fixing carbon. A high relative abundance of the cyanobacterial taxa Aphanocapsa and Aphanothece was revealed by microscopic observation, and cyanobacterial fatty acids and hydrocarbons showed (13)C-uptake in the labeling experiment. Microscopic observations also revealed purple sulfur bacteria (PSB) in the deeper layers. A cyclic C(19:0) fatty acid and farnesol were attributed to this group that was also actively fixing carbon. Background isotopic values indicate Calvin-Benson cycle-based autotrophy for cycC(19:0) and farnesol-producing PSBs. Biomarkers from sulfate-reducing bacteria (SRB) in the top layer of the mat and their (13)C-uptake patterns indicated a close coupling between SRBs and cyanobacteria. Archaeol, possibly from methanogens, was detected in all layers and was especially abundant near the surface where it contained substantial amounts of (13)C-label. Intact glycosidic tetraether lipids detected in the deepest layer indicated other archaea. Large amounts of ornithine and betaine bearing intact polar lipids could be an indicator of a phosphate-limited ecosystem, where organisms that are able to substitute these for phospholipids may have a competitive advantage.
Assuntos
Archaea/metabolismo , Bactérias/metabolismo , Carbono/metabolismo , Sedimentos Geológicos/microbiologia , Archaea/classificação , Archaea/citologia , Bactérias/classificação , Bactérias/citologia , Isótopos de Carbono/metabolismo , Ácidos Graxos/análise , Sedimentos Geológicos/química , Microscopia , Oceano Pacífico , Coloração e Rotulagem/métodosRESUMO
Small HERC proteins are defined by the presence of one RCC1-like domain and a HECT domain. Having evolved out of one common ancestor, the four members of the family exhibit a high degree of homology in genomic organization and amino acid sequence, thus it seems possible that they might accomplish similar functions. Here we show that small HERC proteins interact with each other and localize to the same cellular structures, which we identify as late endosomes and lysosomes. We demonstrate interaction of HERC3 with the ubiquitin-like proteins hPLIC-1 and hPLIC-2 and we establish interaction of HERC5 with the metastasis suppressor Nm23B. While hPLIC proteins are not ubiquitinated by HERC3, HERC5 plays an important role in ubiquitination of Nm23B. In summary, although small HERC proteins are highly homologous showing the same subcellular distribution, they undergo different molecular interactions.
Assuntos
Nucleosídeo NM23 Difosfato Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Relacionadas à Autofagia , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endossomos/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Nucleosídeo NM23 Difosfato Quinases/genética , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
The ability to perform X-ray absorption spectroscopy (XAS) in the 300-1500 eV energy range allows measurements to be made on transition metal compounds. This paper describes a detector and the technique used to perform fluorescent measurements on such materials. A variety of test sample results are shown to illustrate the low energy and energy-resolving capabilities of the detector (based on gas microstrip technology). Two possible applications are also demonstrated. The first shows how the detector can be used to gather X-ray absorption spectra for the L edges of transition metals and K edges of light elements (C, O and N). The other shows how the magnetic immunity of the detector can be exploited to study the magnetic properties of materials.
Assuntos
Absorciometria de Fóton/instrumentação , Metais/química , Espectrometria por Raios X/instrumentação , Transdutores , Elementos de Transição/química , Absorciometria de Fóton/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Estudos de Viabilidade , Gases/química , Miniaturização , Fótons , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria por Raios X/métodosRESUMO
BACKGROUND: To investigate the contribution of inflammation to postangioplasty lumen loss, we used an adenoviral gene therapy approach to inhibit the central inflammatory mediator nuclear factor-kappaB (NF-kappaB) by overexpression of its natural inhibitor, IkappaBalpha. METHODS AND RESULTS: The adenovirus carrying human IkappaBalpha was applied immediately after balloon dilatation by a double-balloon catheter in a rabbit iliac artery restenosis model. Immunohistochemistry of IkappaBalpha revealed that mainly smooth muscle cells of the media but also cells of the adventitia were transduced and expressed the transgene IkappaB alpha for >/= 8 days. At this time point, intercellular adhesion molecule-1 (30%) and monocyte chemotactic protein-1 (50%) expression, as well as recruitment of macrophages into the wounded area (90%), were significantly reduced in IkappaB alpha-treated vessels. In addition, expression of inhibitor of apoptosis proteins was reduced and the percentage of apoptotic cells was increased compared with control-treated contralateral vessels. Animals killed 5 weeks after treatment exhibited a significantly reduced degree of lumen narrowing (P<0.02) on the side treated with adenovirus IkappaBalpha. The lumen gain of approximately 40% was due to positive remodeling. CONCLUSIONS: From these data, we conclude that balloon angioplasty-induced activation of NF-kappaB contributes to lumen loss likely via induction of an inflammatory response and a decrease in the rate of apoptosis. These data show for the first time that inflammation mediated by NF-kappaB is involved in postangioplasty lumen narrowing. Specific and more potent inhibitors of NF-kappaB might therefore be a useful therapeutic measure to improve clinical outcome after balloon dilatation.
Assuntos
Oclusão de Enxerto Vascular/metabolismo , Oclusão de Enxerto Vascular/prevenção & controle , Proteínas I-kappa B , NF-kappa B/metabolismo , Adenoviridae/genética , Angiografia Digital , Angioplastia com Balão/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/farmacologia , Dieta Aterogênica , Modelos Animais de Doenças , Expressão Gênica , Oclusão de Enxerto Vascular/patologia , Humanos , Artéria Ilíaca/diagnóstico por imagem , Artéria Ilíaca/metabolismo , Artéria Ilíaca/patologia , Imuno-Histoquímica , Molécula 1 de Adesão Intercelular/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Infiltração de Neutrófilos/efeitos dos fármacos , Coelhos , Transgenes , Grau de Desobstrução Vascular/efeitos dos fármacosRESUMO
Exposure of endothelial and many other cell types to tumor necrosis factor alpha generates both apoptotic and anti-apoptotic signals. The anti-apoptotic pathway leads to activation of the transcription factor NF-kappaB that regulates the expression of genes such as A20 or members of the IAP gene family that protect cells from tumor necrosis factor alpha-mediated apoptosis. In turn, some anti-apoptotic genes have been shown to modulate NF-kappaB activity. Here we demonstrate that XIAP, a NF-kappaB-dependent member of the IAP gene family, is a strong stimulator of NF-kappaB. Expression of XIAP leads to increased nuclear translocation of the p65 subunit of NF-kappaB via a novel signaling pathway that involves the mitogen-activated protein kinase kinase kinase TAK1. We show that TAK1 physically interacts with NIK and with IKK2, and both XIAP or active TAK1 can stimulate IKK2 kinase activity. Thus, XIAP may be part of a system of regulatory loops that balance a cell's response to environmental stimuli.
Assuntos
Apoptose , Endotélio Vascular/metabolismo , MAP Quinase Quinase Quinases , NF-kappa B/metabolismo , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Linhagem Celular , Endotélio Vascular/patologia , Humanos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
The gene coding for the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was isolated from Plasmodium falciparum. The gene contains 1 intron and the A+T content is characteristic for the codon usage of P. falciparum. The predicted open reading frame codes for 337 amino acids (36651Da) and is 63.5% identical to the human erythrocytic GAPDH. GAPDH sequences from several field isolates of P. falciparum displayed 100% conservation. Phylogenetic analysis supports the hypothesis that dinoflagellates and Plasmodium are closely related. The protein encoded by the pfGAPDH was expressed recombinantly in Escherichia coli and exhibited enzymatic activity with NAD(+) but not with NADP(+) as cofactor. Antiserum raised against the recombinantly expressed enzyme detected specifically all developmental stages of cultured P. falciparum blood-stage parasites.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/genética , Plasmodium falciparum/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Escherichia coli/genética , Imunofluorescência , Regulação Enzimológica da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Filogenia , Plasmodium falciparum/enzimologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
PURPOSE: To identify predictors of oral mucositis and gastrointestinal toxicity after high-dose therapy. PATIENTS AND METHODS: Mucositis and gastrointestinal toxicity were prospectively evaluated in 202 recipients of high-dose therapy and autologous or allogeneic stem-cell rescue. Of 10 outcome variables, three were selected as end points: the peak value for the University of Nebraska Oral Assessment Score (MUCPEAK), the duration of parenteral nutritional support, and the peak daily output of diarrhea. Potential covariates included patient age, sex, diagnosis, treatment protocol, transplantation type, stem-cell source, and rate of neutrophil recovery. The three selected end points were also examined for correlation with blood infections and transplant-related mortality. RESULTS: A diagnosis of leukemia, use of total body irradiation, allogeneic transplantation, and delayed neutrophil recovery were associated with increased oral mucositis and longer parenteral nutritional support. No factors were associated with diarrhea. Also, moderate to severe oral mucositis (MUCPEAK > or = 18 on a scale of 8 to 24) was correlated with blood infections and transplant-related mortality: 60% of patients with MUCPEAK > or = 18 had positive blood cultures versus 30% of patients with MUCPEAK less than 18 (P =.001); 24% of patients with MUCPEAK > or = 8 died during the transplantation procedure versus 4% of patients with MUCPEAK less than 18 (P =.001). CONCLUSION: Gastrointestinal toxicity is a major cause of transplant-related morbidity and mortality, emphasizing the need for corrective strategies. The peak oral mucositis score and the duration of parenteral nutritional support are useful indices of gastrointestinal toxicity because these end points are correlated with clinically significant events, including blood infections and treatment-related mortality.
Assuntos
Antineoplásicos/efeitos adversos , Leucemia/complicações , Leucemia/terapia , Mucosa Bucal/efeitos dos fármacos , Nutrição Parenteral , Transplante de Células-Tronco , Estomatite/etiologia , Adolescente , Adulto , Análise de Variância , Antineoplásicos/uso terapêutico , Criança , Bases de Dados Factuais , Diarreia/etiologia , Feminino , Humanos , Leucemia/mortalidade , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Índice de Gravidade de Doença , Estomatite/induzido quimicamente , Estomatite/classificaçãoRESUMO
The 104 kDa microneme-rhoptry protein (p104) is the only known apical complex organelle-specific protein of Theileria parva. p104 exhibits striking structural similarities to circumsporozoite protein and sporozoite surface protein 2 of Plasmodium yoelii. Their primary sequences contain two hydrophobic segments, located at the amino-and the carboxy-terminus. The p104 amino-terminal hydrophobic region was suggested to be a signal peptide for entry into the endoplasmic reticulum and the extreme carboxy-terminal region to function as a membrane anchor. We have studied the biogenesis of p104 in a cell-free expression system and found that p104 is co-translationally transported into membranes derived from endoplasmic reticulum. The amino-terminal signal peptide is not cleaved off and anchors the protein in the membrane with the carboxy-terminal portion translocated into the lumen. We suggest that in vivo p104 is co-translationally integrated into the membrane of the endoplasmic reticulum, from where it is further transported to the microneme-rhoptry complex. Thus, p104 appears to be a suitable marker to study the development of micronemes and rhoptries in T. parva.
Assuntos
Retículo Endoplasmático/metabolismo , Organelas/metabolismo , Sinais Direcionadores de Proteínas/química , Proteínas de Protozoários/metabolismo , Theileria parva/metabolismo , Animais , Proteínas de Membrana/metabolismo , Plasmídeos/genética , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Theileria parva/genética , Transcrição GênicaRESUMO
By differential screening of tumor necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS)- activated endothelial cells (ECs), we have identified a cDNA clone that turned out to be a member of the inhibitor of apoptosis (iap) gene family. iap genes function to protect cells from undergoing apoptotic death in response to a variety of stimuli. These iap genes, hiap1, hiap2, and xiap were found to be strongly upregulated upon treatment of ECs with the inflammatory cytokines TNF-alpha, interleukin 1beta, and LPS, reagents that lead to activation of the nuclear transcription factor kappaB (NF-kappaB). Indeed, overexpression of IkappaBalpha, an inhibitor of NF-kappaB, suppresses the induced expression of iap genes and sensitizes ECs to TNF-alpha-induced apoptosis. Ectopic expression of one member of the human iap genes, human X-chromosome-linked iap (xiap), using recombinant adenovirus overrules the IkappaBalpha effect and protects ECs from TNF-alpha- induced apoptosis. We conclude that xiap represents one of the NF-kappaB-regulated genes that counteracts the apoptotic signals caused by TNF-alpha and thereby prevents ECs from undergoing apoptosis during inflammation.
Assuntos
Apoptose/fisiologia , Regulação da Expressão Gênica/genética , NF-kappa B/fisiologia , Proteínas de Neoplasias/genética , Fator de Necrose Tumoral alfa/farmacologia , Cromossomo X/genética , Adenoviridae/química , Células Cultivadas , DNA/análise , Fragmentação do DNA/genética , Endotélio Vascular/metabolismo , Citometria de Fluxo , Ligação Genética/genética , RNA Mensageiro/metabolismo , Proteínas Virais/fisiologiaRESUMO
Fifty-nine monoclonal antibodies (mAb) were assigned to the adhesion section of the Second International Swine CD Workshop. They were analysed for their reactivity to selected lymphoid cell populations, as well as to non-lymphoid cell lines. Cell lysate ELISAS and Western Blot analyses were also carried out. As a result, thirteen separate cluster groups emerged (p > 0.95). Workshop assignments for adhesion molecules were made: wCD29/49 for mAbs UCP1D2 (#133) and FW4-101 (#165), and PNK-I (#194) and MUC76A (#025) could be assigned to wCD18. For one cluster (FQ1D7, #161 and 2F4, #069) the cellular distribution and MW were characteristic for MHC Class II, and another cluster comprising several antibodies which appeared to recognise MHC Class I. Other clusters could not be assigned to cell surface structures known to be linked to cellular adhesion, however, two further antibodies, 335-2 (#112) and FG1F6 (#156), could be added to SWC1, and the new SWC8 was defined by MIL3 (#077) and MUC20A (#029), binding a ligand of 29-32 kDa. Clustering for these two antibodies was confirmed by blocking studies. The cellular distribution is known for MIL3, recognising an epitope present on granulocytes, B cells, and a subset of T cells expressing CD8 at high intensity.
Assuntos
Anticorpos Monoclonais/metabolismo , Moléculas de Adesão Celular/imunologia , Suínos/imunologia , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Complexo CD3/imunologia , Antígenos CD4/imunologia , Antígenos CD8/imunologia , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Integrinas/imunologia , Subpopulações de Linfócitos/imunologiaRESUMO
The inhibitor of apoptosis (iap) proteins belong to a gene family that protect certain cell to undergo programmed cell death in response to a variety of stimuli. By differential screening we have identified a cDNA clone, designated piap, in porcine aortic endothelial cells (PAEC) that turned out by sequence comparison to be a porcine member of the iap family. The expression of piap is strongly up-regulated upon treatment of endothelial cells (EC) with inflammatory cytokines TNF-alpha, IL-1 beta, and LPS. In EC these stimuli lead to the activation of nuclear transcription factor kappa B (NF-kappa B) that plays a role in countering TNF-alpha induced apoptosis. We demonstrate that adenovirus mediated overexpression of I kappa B alpha, an inhibitor of NF-kappa B suppresses the expression of piap in response to TNF-alpha suggesting that piap is one of the NF-kappa B regulated genes that operates to prevent programmed cell death of EC in inflammation.
Assuntos
Apoptose/genética , Citocinas/farmacologia , Expressão Gênica , Proteínas I-kappa B , NF-kappa B/farmacologia , Proteínas/genética , Sequência de Aminoácidos , Animais , Aorta/química , Proteínas Reguladoras de Apoptose , Sequência de Bases , Células Cultivadas , Sequência Consenso , Proteínas de Ligação a DNA/farmacologia , Endotélio Vascular/química , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Suínos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Asthma management is changing, and there are many potential new drugs undergoing early and late phase trials. Nonetheless, it is unlikely that any dramatic alterations in therapy will occur within the next 3 years. The asthma treatment paradigm has altered over the past 10 or so years, with the emphasis on symptom relief from short acting beta agonists giving way to preventive treatment of underlying airway inflammation with inhaled corticosteroids. More recently, long acting beta agonists have been demonstrated to reduce the need for increasing doses of inhaled steroids in patients with poorly controlled asthma. This article reviews these trends.
Assuntos
Agonistas Adrenérgicos beta/uso terapêutico , Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Glucocorticoides/uso terapêutico , Administração por Inalação , Agonistas Adrenérgicos beta/farmacologia , Antiasmáticos/farmacologia , Broncodilatadores/farmacologia , Broncodilatadores/uso terapêutico , Glucocorticoides/farmacologia , Humanos , Teofilina/farmacologia , Teofilina/uso terapêuticoRESUMO
The schizont stage of the protozoan parasite Theileria parva induces features characteristic of tumor cells in infected bovine T-cell lines. Most strikingly T. parva-infected cell lines acquire unlimited growth potential in vitro. Their proliferative state is entirely dependent on the presence of a viable parasite within the host cell cytoplasm. It has been postulated that parasite proteins either secreted into the host cell or expressed on the parasite surface membrane are involved in the parasite-host cell interaction. We used an in vitro transcription-translation-membrane translocation system to identify T. parva-derived cDNA clones encoding secretory or membrane proteins. Within 600 clones we found one encoding a 17-kDa protein which is processed by microsomal membranes to a 14-kDa protein (11E), presumably by signal peptidase. The processed form is expressed in the T-cell line TpM803 harboring viable parasites. By immunolocalization we show that the 11E protein mostly resides within the parasite, often in close vicinity to membranous structures, but in addition it appears at the surface membrane. Amino acid sequence comparison suggests that 11E belongs to the glutaredoxin family, but is unique so far in containing a signal sequence for endoplasmic reticulum membrane translocation.
Assuntos
Proteínas de Membrana/biossíntese , Biossíntese de Proteínas , Proteína Dissulfeto Redutase (Glutationa) , Proteínas/química , Theileria parva/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/análise , Antígenos de Protozoários/biossíntese , Bovinos , Linhagem Celular , Transformação Celular Neoplásica , Clonagem Molecular , Sequência Consenso , Biblioteca Gênica , Glutarredoxinas , Membranas Intracelulares/metabolismo , Proteínas de Membrana/química , Microssomos/metabolismo , Dados de Sequência Molecular , Peso Molecular , Oxirredutases/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Linfócitos T , Transcrição GênicaRESUMO
Adult tube feeding formulas vary considerably with respect to composition, administration, and cost. Selecting the best product for patients requires a careful analysis of specific patient requirements and resources.
Assuntos
Nutrição Enteral/enfermagem , Alimentos Formulados/análise , Adulto , Educação Continuada em Enfermagem , Nutrição Enteral/instrumentação , Nutrição Enteral/métodos , Alimentos Formulados/provisão & distribuição , Humanos , Necessidades NutricionaisRESUMO
In the pig-to-primate model of xenotransplantation, graft rejection is initiated by binding of the recipient's xenoreactive natural antibodies (XNA), mainly of the IgM type, to antigens constitutively expressed on donor endothelial cells (EC). As a consequence of XNA binding and complement fixation, the EC become activated, which is considered to be a major mechanism promoting hyperacute as well as later phases of graft rejection. It is not clear whether binding of XNA to activated EC also contributes to delayed rejection. We asked whether EC activation by cytokines results in the expression of other novel surface antigens recognized by XNA which might become relevant in progressive stages of graft rejection. We activated porcine aortic EC and smooth muscle cells with tumor necrosis factor (TNF), interleukin 1, or lipopolysaccharide and studied expression of new XNA-binding antigens. Expression of two glycoproteins, gp65 and gp100, was strongly induced by recombinant human TNF in EC but not in smooth muscle cells. Notably, gp100 expression was specific to TNF activation, whereas gp65 could also be induced by interleukin 1 or lipopolysaccharide. Cell surface labeling indicated that gp65 is expressed on the plasma membrane. Recognition of XNA-binding antigens on resting EC occurs via alpha-galactosyl epitopes. In contrast, gp65 and gp100 were recognized independently of this epitope. Our data show that gp65 and gp100 represent selective cytokine-induced markers on EC that may have importance in a porcine-to-primate model of xenotransplantation. Conceivable functions of gp65 and gp100 are discussed.
Assuntos
Anticorpos Heterófilos/imunologia , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Antígenos Heterófilos/imunologia , Aorta/citologia , Aorta/imunologia , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Humanos , Imunoglobulina M/imunologia , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Polissacarídeos/farmacologia , Proteínas Recombinantes , Suínos , TemperaturaRESUMO
NKG2-C is a member of the recently discovered NKG2 family of genes and proteins, which are preferentially expressed on human natural killer (NK) cells. These potential NK cell receptors belong to a larger class of type II transmembrane proteins with a C-type lectin domain. We show here that NKG2-C is expressed as a 36-kDa glycoprotein by translation in vitro, recombinant expression and immunoprecipitation from a human NK cell clone. Further, a recombinant soluble NKG2-C-receptor binds specifically to K562 cells, which are target cells for NK cell killing, and to RPMI 8866 cells, which are feeder cells for NK cells; several other hematopoietic cell lines tested do not show any binding. The binding structures on the surface of K562 cells disappear, concomitant with a loss in susceptibility to killing when the cells are induced to differentiate with phorbol ester and Ca2+ ionophore. Our data suggest the presence of specific target molecules for NKG2-C on K562 cells, since overall glycosylation, Lewis X and Lewis Y structures, as well as the mucin-like CD43 molecule, do not change following induction of the cells. We propose that NKG2-C mediates a specific interaction of NK cells and their target cells with functional importance for NK cell killing.
Assuntos
Células Matadoras Naturais/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores Imunológicos/fisiologia , Animais , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Citotoxicidade Imunológica , DNA Complementar/genética , Cães , Glicosilação , Humanos , Ionomicina/farmacologia , Células Matadoras Naturais/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Glicoproteínas de Membrana/genética , Subfamília C de Receptores Semelhantes a Lectina de Células NK , Nucleopoliedrovírus/genética , Receptores Imunológicos/genética , Receptores de Células Matadoras Naturais , Proteínas Recombinantes de Fusão/metabolismo , Spodoptera/citologia , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
A Theileria parva specific full-length cDNA clone, T7, which encodes a protein with more than 60% homology to heat shock protein 90 (hsp90) of other organisms, has been identified. T7 appears to be a single copy gene. The gene is expressed as a protein of 87 kDa in both the sporozoite and schizont stages of T. parva. The protein was not found in the piroplasm stage, although the corresponding transcript was detected, suggesting post-transcriptional regulation of the gene. In the schizont stage the T7 protein is upregulated upon heat shock and localized in the cytoplasm.
Assuntos
Proteínas de Choque Térmico HSP90/biossíntese , Theileria parva/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Células Cultivadas , Clonagem Molecular , Citoplasma/metabolismo , DNA Complementar/análise , DNA Complementar/isolamento & purificação , DNA de Protozoário/análise , Imunofluorescência , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Linfócitos/parasitologia , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Theileria parva/genética , Theileria parva/fisiologiaRESUMO
The transformed domain maximum likelihood (TDML) algorithm for image motion estimation is presented. This algorithm finds a solution which maximizes a log-likelihood function using a steepest ascent scheme. Important characteristics of the algorithm are the inclusion of noise in the signal model, the consideration of motion as a nonuniform process, and calculation of convergence parameters by means of a linear analysis. Simulation on real image sequences demonstrate the validity of the motion estimator. The experiments also verify the validity of the equations presented for the calculation of the convergence parameters. Additional experiments performed to determine the noise sensitivity of the TDML show that noise resistance can be obtained using a reduced coefficient transform (RCT) TDML algorithm. An additional benefit of using an RCT with the TDML algorithm is an increase in the speed of the algorithm without significant performance degradation. Two of the common transforms, Haar and Walsh-Hadamard, are shown to have some interesting properties when utilized with the RCT-TDML algorithm.
RESUMO
An iterative scheme for frame-to-frame motion estimation from a pair of noisy images is established. The algorithm is developed by assuming that the Karhunen-Loeve coefficients of the motion vector waveform are zero mean and Gaussian random variables. Following the derivation of the generalized maximum likelihood (GML) algorithm, and invoking the maximum a posteriori (MAP) criterion, an iterative motion estimator is developed. A linear analysis of the algorithm is presented, and the convergence of the algorithm is discussed. Simulation experiments are performed and comparisons are made with the GML algorithm the algorithm reported by A.N. Netravali and J.D. Robbins (1979), and the scheme developed by K.P.G. Horn and G.G. Schunck (1981).