RESUMO
Only thin-film technology can satisfy the requirements of high spatial selectivity at high-channel-count electrode array designs by simultaneously good conformability to the targeted tissue through mechanical flexibility enriching future applications of functional neural stimulation. However, caused by the high impact of the microstructure on the mechanical and electrochemical film properties, varying fabrication processes of the same thin-film makes the difference between acute and chronic long-term stable electrodes. The influence of standard clinical electrical pulsing on flexible polyimide-based thin-film platinum electrodes for neuroprostheses, either sputter deposited or evaporated, and different diameters was assessed and compared. The electrochemical and morphological analysis showed a higher corrosion susceptibility and electrochemical degradation for the sputter deposited platinum electrodes with even total failures of smaller diameters. In contrast, the evaporated thin-films provided itself as more stable and reliable metallization with also smaller electrodes keeping their film integrity intact over the experimental period, -appearing to be the preferable material for improving thin-film electrodes' longevity.
Assuntos
Eletrodos , Neurônios , Platina , Estimulação ElétricaRESUMO
The high complexity of the biological response to implanted materials builds a serious barrier against implanted recording and stimulation electrode arrays to succeed in clinically relevant chronic studies. Some of the cell and molecular interactions and their contribution to inflammation and device failure are still unclear. The interrelated mechanisms leading to tissue damage and electrode array failure during simultaneous faradaic, electrochemical reactions and biological response under electrical stimulation are not understood sufficiently. One variable, with which inflammatory and electrode surface processes can be analyzed and assessed, is the pH change in the immediate environment of the material-tissue interface. Here, the greatest challenges are in the biocompatibility and in-vivo long-term stability of selected sensor materials, the measurement of small transient pH oscillations and positioning of the sensor at a defined and nearest possible distance in the micrometer range, to the site of activity without the pH sensing being affected by the material- issue interactions itself. This work represents the in-situ measurement of local and transient pH changes at apulsed electrode with an embedded in-vivo compatible pH sensor and therein differentiating from current approaches of pH sensing during electrical stimulation.
Assuntos
Estimulação Elétrica , Eletrodos , Técnicas Histológicas , Concentração de Íons de HidrogênioRESUMO
The genetic backgrounds of lupus-prone murine models are a valuable resource for studying the influence of environmental exposure on autoimmune diseases in sensitive populations. Epidemiological studies have shown associations between silica exposure and several autoimmune diseases, including scleroderma and systemic lupus erythematosus. To determine whether silica exposure can exacerbate systemic autoimmunity in genetically predisposed animals, New Zealand mixed mice were intranasally instilled twice with saline or saline suspensions of 1 mg silica or 500 micro g TiO2, a dose equivalent in surface area, and were evaluated with respect to health and immune status. Survival in silica exposed NZM mice was decreased compared to saline and TiO2 exposed mice. Proteinuria levels were elevated in silica exposed mice. Levels of circulating immune complexes, autoantibodies to nuclear antigen (ANA), histone, and double stranded DNA were measured every two weeks by ELISA. Circulating immune complexes showed a trend towards an increased acceleration in levels in the silica exposed mice compared to saline and TiO2 exposed mice. ANA levels were significantly higher in silica exposed animals compared to saline and TiO2 exposed animals (0.237 +/- 0.03 versus 0.140 +/- 0.029 and 0.125 +/- 0.03, P < 0.05) 16 weeks postexposure. Autoantibodies to histone were also significantly elevated after 16 weeks in silica exposed animals compared to saline and TiO2 exposed animals (0.227 +/- 0.03 versus 0.073 +/- 0.015 and 0.05 +/- 0.03, P < 0.05). In contrast, serum IgG levels were decreased in silica exposed NZM mice compared to the saline controls, however, IgM levels were unaffected. Lungs of the silica-exposed mice had increased inflammatory infiltrates as well as fibrotic lesions characterized by excess collagen deposition. Therefore, although NZM mice are susceptible to SLE, silica exposure significantly exacerbated the course of disease.
Assuntos
Doenças Autoimunes/etiologia , Predisposição Genética para Doença , Lúpus Eritematoso Sistêmico/etiologia , Dióxido de Silício/imunologia , Animais , Complexo Antígeno-Anticorpo/sangue , Autoanticorpos/biossíntese , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Feminino , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Glomérulos Renais/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NZB , Proteinúria/induzido quimicamente , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/patologia , Silicose/patologia , Taxa de SobrevidaRESUMO
Some inhaled particles are known to lead to inflammation and lung pathology, whereas others do not appear to have long-term effects. Potential mechanisms to account for these differences are only beginning to be understood. In this article we examine whether silica and PM1648 (a model urban particulate) caused selective deletion of the suppressor human alveolar macrophage (HAM) phenotype (RFD1+/7+), and whether this affected cytokine production in an antigen-presenting cell (APC) assay with autologous T lymphocytes. HAM were exposed to the bioactive particulates, silica and PM1648, for 24 hours, then isolated free of extracellular particulates and nonviable cells; HAM were then cultured with autologous lymphocytes in an 11-day APC assay. Silica exposure up-regulated a TH1 lymphocyte-derived cytokine, interferon gamma (IFN-gamma), and a TH2 lymphocyte-derived cytokine, interleukin-4 (IL-4). PM1648 exposure primarily upregulated IL-4. Neither particle exposure had a significant effect on interleukin-10 (IL-10) production. Control particulate exposures with titanium dioxide (TiO2) and wollastonite (Woll) caused no altered APC activity. Silica and PM1648 demonstrated selective toxicity to suppressor macrophages (RFD1+/7+). We propose that, because of the suppressor macrophage phenotype disabling, the activator macrophage (RFD1+/7-) operates free of the suppressor macrophage's influence, enhancing APC activity with increased lymphocyte-derived proinflammatory cytokine production.
Assuntos
Poluentes Ocupacionais do Ar/toxicidade , Células Apresentadoras de Antígenos/imunologia , Compostos de Cálcio/toxicidade , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Silicatos/toxicidade , Dióxido de Silício/toxicidade , Titânio/toxicidade , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Cultivadas , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Interferon gama/metabolismo , Interleucina-4/metabolismo , Pulmão/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacosRESUMO
Scavenger receptors (SR) are responsible for recognition of ligands as diverse as oxidized LDL (endogenous) to respirable particulates (exogenous). A number of recent studies have suggested that these SR ligands induce apoptosis of macrophages. However, the mechanism by which SR triggers apoptosis is not understood. This study used a murine alveolar macrophage cell line (MH-S) to investigate the role of the SR in caspase activation. The presence of SR on MH-S cells was confirmed by FACS analysis and was similar to the distribution found on murine alveolar macrophages. The activity of caspases 1, 3, and 6 was measured following a 6-h exposure to crystalline silica with and without blockers of the SR. Caspase activities were determined by hydrolysis of specific chromogenic substrates and formation of an active enzymatic form (Western for active caspase 3). Silica stimulated significant caspase activity, apoptosis, and necrosis of MH-S cells, which was attenuated by 2F8 (a blocking antibody) and polyinosinic acid (a nonspecific SR antagonist). The results indicate that the SR are necessary for caspase activation and subsequent apoptosis (as well as necrosis) caused by silica in macrophage cells.
Assuntos
Apoptose/efeitos dos fármacos , Macrófagos Alveolares/citologia , Proteínas de Membrana , Receptores Imunológicos/fisiologia , Receptores de Lipoproteínas , Dióxido de Silício/farmacologia , Animais , Caspase 3 , Caspases/metabolismo , Linhagem Celular , Membrana Celular/química , Fragmentação do DNA , Ativação Enzimática , Citometria de Fluxo , Macrófagos Alveolares/química , Macrófagos Alveolares/efeitos dos fármacos , Camundongos , Necrose , Receptores Imunológicos/análise , Receptores Depuradores , Receptores Depuradores Classe A , Receptores Depuradores Classe BRESUMO
Around the time of birth, male rats express higher levels of progesterone receptors in the medial preoptic nucleus (MPN) than female rats, suggesting that the MPN may be differentially sensitive to maternal hormones in developing males and females. Preliminary evidence suggests that this sex difference depends on the activation of estrogen receptors around birth. To test whether estrogen receptor alpha (ER alpha) is involved, we compared progesterone receptor immunoreactivity (PRir) in the brains of male and female neonatal mice that lacked a functional ER alpha gene or were wild type for the disrupted gene. We demonstrate that males express much higher levels of PRir in the MPN and the ventromedial nucleus of the neonatal mouse brain than females, and that PRir expression is dependent on the expression of ER alpha in these regions. In contrast, PRir levels in neocortex are not altered by ER alpha gene disruption. The results of this study suggest that the induction of PR via ER alpha may render specific regions of the developing male brain more sensitive to progesterone than the developing female brain, and may thereby underlie sexual differentiation of these regions.
Assuntos
Animais Recém-Nascidos/metabolismo , Encéfalo/metabolismo , Receptores de Estrogênio/fisiologia , Receptores de Progesterona/metabolismo , Caracteres Sexuais , Animais , Córtex Cerebral/metabolismo , Receptor alfa de Estrogênio , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Área Pré-Óptica/metabolismo , Núcleo Hipotalâmico Ventromedial/metabolismoRESUMO
BACKGROUND AND AIM OF THE STUDY: Stentless aortic valve bioprostheses have become popular because of their superior hemodynamics and expected increased durability. However, the stentless bioprosthesis differs from stented valves in that glutaraldehyde (GA)-treated tissue is implanted in direct contact with the native aorta. The effect of GA-treated tissue on host tissue has not been reported. METHODS: In order to analyze the effect of GA in the healing process, sheep descending aortic conduits treated with 0.625% GA were inserted in the descending thoracic aorta of 10 adult sheep. The implants were removed after 4, 5, 10, 12, 15, 25, 30, 32, 60 and 120 days. The upstream and downstream junctions were evaluated macro- and microscopically, and by immunohistology for smooth muscle cell alpha-actin and von Willebrand factor. RESULTS: By day 60 of implantation, the GA-treated conduits were calcified. By days 60 and 120, the calcification had spread to the host aorta, and was seen as foci of calcification in the junctional area. Acellular areas were also seen in the host aorta near the anastomosis. A fibrotic layer spanning the abluminal aspect of the junction between the implant and host aorta was present at day 4 and continued through 120 days. This layer was characterized by a progressive increase in collagenous matrix and cellularity, as well as new blood vessel formation. The luminal aspect of the junction had a neointimal layer of variable thickness containing alpha-actin-expressing cells covered by a monolayer of von Willebrand factor-expressing cells, seen at 15-30 days and present through 120 days. CONCLUSION: In our model, implanting GA-fixed tissue in direct contact with living tissues resulted in cell death and calcification of host tissue within 60 days. The integrity of the junction did not appear to be compromised. This may be of interest in light of the increased popularity of the stentless aortic bioprosthesis.
Assuntos
Aorta Torácica/patologia , Valva Aórtica/patologia , Bioprótese , Calcinose/patologia , Próteses Valvulares Cardíacas , Actinas/análise , Animais , Implante de Prótese de Valva Cardíaca , Ovinos , Stents , Fatores de Tempo , Transplante Homólogo/patologia , Fator de von Willebrand/análiseRESUMO
H-NS is an abundant nucleoid-associated protein involved in the maintenance of chromosomal architecture in bacteria. H-NS also has a role in silencing the expression of a variety of environmentally regulated genes during growth under nonpermissive conditions. In this study we demonstrate a role for H-NS in the negative modulation of expression of several genes within the ToxR virulence regulon of Vibrio cholerae. Deletion of hns resulted in high, nearly constitutive levels of expression of the genes encoding cholera toxin, toxin-coregulated pilus, and the ToxT virulence gene regulatory protein. For the cholera toxin- and ToxT-encoding genes, elevated expression in an hns mutant was found to occur in the absence of the cognate activator proteins, suggesting that H-NS functions directly at these promoters to decrease gene expression. Deletion analysis of the region upstream of toxT suggests that an extensive region located far upstream of the transcriptional start site is required for complete H-NS-mediated repression of gene expression. These data indicate that H-NS negatively influences multiple levels of gene expression within the V. cholerae virulence cascade and raise the possibility that the transcriptional activator proteins in the ToxR regulon function to counteract the repressive effects of H-NS at the various promoters as well as to recruit RNA polymerase.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Vibrio cholerae/patogenicidade , Toxina da Cólera/genética , Proteínas de Ligação a DNA/genética , Biblioteca Gênica , Fases de Leitura Aberta , Óperon , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Vibrio cholerae/genéticaRESUMO
Signaling by lipopolysaccharide (LPS) through CD14 involves the activation of protein tyrosine kinases of the src family and leads to cytokine production and activation of arachidonic acid metabolism in macrophages. CD45 protein tyrosine phosphatase (PTPase) might play a role in modulating the response through this pathway. Although a critical role in regulation of T-cell signaling for CD45 has been demonstrated, little is known about its role in macrophages. Monoclonal antibodies to CD45 and F(ab')(2) fragments of the monoclonal antibody enhanced the response of differentiated THP-1 monocytic cells to LPS for the release of radiolabeled arachidonic acid metabolites, prostaglandin E(2), and tumor necrosis factor alpha. The enhancing effect of anti-CD45 mAbs was shown to occur primarily through CD14-dependent signaling by performing the experiments under conditions favoring that pathway. Further, LPS may be able to alter the enzymatic activity of CD45, as shown by Western blots of CD45 immunoprecipitates in which LPS caused a transient change in the phosphorylation state of CD45. We conclude that CD45 appears to play a role in LPS-induced responses through the CD14 pathway, possibly through its PTPase activity.
Assuntos
Ácido Araquidônico/metabolismo , Antígenos Comuns de Leucócito/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Células Cultivadas , Dinoprostona/biossíntese , Antígenos Comuns de Leucócito/metabolismo , Receptores de Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Fosforilação , Receptores Fc/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Aortoenteric fistula represents a communication between aortic and duodenal lumen. It is a very rare complication of aortic aneurysmal disease as well as aortic prosthetic surgery. There are only few surgeons who have seen more than one case of aortoenteric fistula. At the Institute of Surgery one case of aortoenteric fistula was treated. It was well diagnosed at the Institute for Internal Medicine, and then presented to the vascular surgeon who operated immediately. The whole diagnostic and therapeutic procedure was well done, but stenosing carcinoma of ascendent colon was found during the operation. Resection were done leading to the dehiscence of bowel anastomosis and fatal outcome for the patient. This case is instructive in a diagnostic and surgical manner.
Assuntos
Aneurisma da Aorta Abdominal/complicações , Doenças da Aorta/etiologia , Ruptura Aórtica/complicações , Duodenopatias/etiologia , Fístula Intestinal/etiologia , Fístula Vascular/etiologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
ToxR and ToxS are integral membrane proteins that activate the transcription of virulence genes in Vibrio cholerae. ToxR can be separated into three different domains: an N-terminal cytoplasmic DNA binding domain, a central transmembrane domain, and a C-terminal periplasmic domain. ToxS is thought to enhance ToxR-mediated transcriptional activation through a periplasmic interaction. By P22 challenge phage selection for DNA binding, in combination with a screen for cholera toxin gene transcription, 12 toxR and toxS positive control mutants producing variant ToxR proteins from the toxRS operon that bind to the cholera toxin promoter but that fail to activate transcription were isolated. One mutation in toxR specifies an E82K change in the predicted helix-loop-helix DNA binding domain and destroys ToxR-mediated activation. Seven toxR mutations included frameshifts and stop codons introduced into the periplasmic domain, and six of these mutations appeared to produce proteolytically processed shorter forms of ToxR, suggesting that even short periplasmic deletions alter the folding of ToxR in the periplasm. Deletion of toxS did not alter the steady-state level of ToxR, and ToxR was found to be capable of binding to DNA in the absence of ToxS even though it did not activate transcription. However, the ToxS L33S variant rendered ToxR susceptible to proteolysis, suggesting that the natural function of ToxS is to complex with ToxR. Therefore, certain alterations that map to the ToxR cytoplasmic DNA binding domain, to the periplasmic domain, or to ToxS separate DNA binding activity from activator function. These data support a model where proper assembly or stability of the periplasmic domain of ToxR is enhanced by ToxS. This chaperone-like activity of ToxS may be required for the formation of the transcriptional activation complex but not the ToxR-DNA complex.
Assuntos
Toxina da Cólera/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Ativação Transcricional , Alelos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/genética , Seleção Genética , Fatores de Transcrição/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/patogenicidade , Virulência/genéticaRESUMO
Ceramide has been shown to be an important second messenger for signal transduction in cells of myeloid lineage. Studies have suggested that lipopolysaccharide (LPS) may activate signaling pathways by mimicking the action of ceramide. We explored this hypothesis with THP-1 cells in terms of the effects of LPS, C2 ceramide, and sphingomyelinase on arachidonic acid metabolism as measured by the release of radiolabeled eicosanoids. Arachidonic acid metabolism was activated by both LPS and ceramide. However, the ratio of prostaglandin E2 to leukotriene C4 was 10 times higher in cells treated with LPS than with ceramide. Unlike LPS, prior exposure to ceramide did not desensitize the cells to subsequent challenge with either LPS or ceramide, nor could LPS desensitize the cells to challenge with ceramide. The results suggest that, although LPS and ceramide may share signaling components, the signaling pathways are not identical.
Assuntos
Ácido Araquidônico/metabolismo , Lipopolissacarídeos/metabolismo , Monócitos/efeitos dos fármacos , Esfingosina/análogos & derivados , Linhagem Celular , Dinoprostona/metabolismo , Humanos , Leucotrieno C4/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/metabolismo , Esfingomielina Fosfodiesterase/farmacologia , Esfingosina/metabolismo , Esfingosina/farmacologiaRESUMO
BACKGROUND: Healthy Start is a 3-year demonstration and education research project designed to evaluate the effectiveness of a multidimensional cardiovascular (CV) risk reduction intervention in preschool centers over a 3-year period of time. METHODS: Two primary interventions are employed. The first is the preschool food service intervention program designed to reduce the total fat in preschool meals and snacks to less than 30% of calories and reduce the saturated fat to less than 10% of calories. The second major intervention is a comprehensive preschool health education curriculum, focused heavily on nutrition. RESULTS: Effectiveness of the intervention will be determined through evaluation of changes in dietary intake of preschool children at school meals and snacks, especially with respect to intake of total and saturated fat. Evaluation of the education component will include assessment of program implementation by teachers, assessment of changes in nutrition knowledge by preschool children, and assessment of changes in home meals that children consume (total and saturated fat content). Blood cholesterol will be evaluated semiannually to evaluate changes that may be due to modification of dietary intake. Growth and body fatness will also be assessed. CONCLUSIONS: While substantial efforts have targeted CV risk reduction and health education for elementary school children, similar efforts aimed at preschool children have been lacking. The rationale for beginning CV risk reduction programs for preschool children is based upon the premise that risk factors for heart disease are prevalent by 3 years of age and tend to track over time, most commonly hypercholesterolemia and obesity, both related to nutrition. Since the behavioral antecedents for nutritional risk factors begin to be established very early in life, it is important to develop and evaluate new educational initiatives such as Healthy Start, aimed at the primary prevention of cardiovascular risk factors in preschool children. The purpose of this publication is to describe the rationale and methods for the Healthy Start project.
Assuntos
Doenças Cardiovasculares/prevenção & controle , Intervenção Educacional Precoce , Educação em Saúde , Ciências da Nutrição/educação , Doenças Cardiovasculares/etiologia , Pré-Escolar , Dieta com Restrição de Gorduras , Feminino , Seguimentos , Serviços de Alimentação , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Masculino , New York , Avaliação de Programas e Projetos de Saúde , Fatores de RiscoRESUMO
This paper analyzes results of 5-year surgical treatment of patients with ruptured abdominal aorta aneurysms (1991.-1995.) at the Clinic for Vascular and Transplantation Surgery of the Institute of Surgery in Novi Sad. 105 patients with abdominal aorta aneurysm underwent surgery, whereas in 31 patients there was a suspicion of rupture and it was confirmed by US and CT examination. One of basic factors to decrease mortality in these patients is early diagnosis and surgery before hemorrhagic shock occurs. Results in hemodynamic stabile patients with blood pressure over 100 mmHg and regular diuresis are much better with mortality of 20%. In order to estimate the correlation of hemodynamic state and outcome of the operation, patients were divided into three groups--hemodynamic stable with blood pressure over 100 mmHg and regular diuresis at admission: hemodynamic unstable patients with signs of mild or moderate shock and blood pressure under 100 mmHg and without initial diuresis which was regulated at the beginning of therapy and hemodynamic unstable patients in severe shock and unmeasurable blood pressure. The highest survival rate (10% mortality) and the least complications occurred in the first group of patients. The total mortality of patients after surgery was 48.48%. Timely diagnosis, suspicion of rupture and adequate first and with urgent transfer to a competent surgical institution are key factors in treatment of this disease and its outcome.
Assuntos
Ruptura Aórtica/cirurgia , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Program teaches management skills beyond those taught in pharmacy school. Pharmacists benefit not only from educational growth but also from networking opportunities. Alumni report that the program has had a positive impact on their personal and career objectives, their department, and their organization. Involvement of administrators helps pharmacy directors take home solutions to specific problems for immediate implementation.
Assuntos
Educação Continuada em Farmácia , Administração Farmacêutica/educação , Serviço de Farmácia Hospitalar , Currículo , Humanos , Serviço de Farmácia Hospitalar/organização & administração , Avaliação de Programas e Projetos de Saúde , Estados UnidosRESUMO
The transmembrane DNA-binding protein, ToxR, of Vibrio cholerae is a global transcriptional regulator of virulence gene expression. ToxR has been shown to interact with promoter regions upstream of both the ctxAB operon encoding cholera toxin, and the regulatory gene toxT. Deletion analysis has shown that a repeated sequence, TTTTGAT, is required for ToxR binding and activation of the ctxAB promoter. However, this sequence is not found upstream of the toxT promoter. Genetic selections using P22 challenge phages were used to define sites within the promoter for ctxAB which are critical for ToxR-DNA interactions. Single-base-pair changes and deletion mutations that impair ToxR binding cluster within two regions: -57 to -69 within two of three tandem TTTTGAT sequences; and from -39 to -47, between the repeat sequences; and the -35 region of the promoter. ToxR does not bind to a synthetic target that has three tandem repeats which lack a flanking upstream and downstream sequence. These results suggest that the ToxR-binding site lies immediately upstream of the - 35 position of the ctx promoter, and that the affinity of ToxR binding to this site is influenced by the repeat sequences.
Assuntos
Proteínas de Bactérias , Toxina da Cólera/genética , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Vibrio cholerae/genética , Bacteriófago P22/genética , Composição de Bases , Sequência de Bases , Sítios de Ligação , Pegada de DNA , DNA Bacteriano/metabolismo , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico , Deleção de Sequência , Ativação Transcricional , Vibrio cholerae/metabolismoRESUMO
Site-directed mutagenesis was used to construct mutant Trp repressors with each of the 38 possible single amino acid changes of the first 2 amino acid residues (Ile79 and Ala80) in the second "recognition" alpha-helix of the helix-turn-helix DNA-binding motif. Eight of these mutant repressors with Ile79 and Ala80 changes are more active than the wild-type protein when tryptophan is limiting, and are super-aporepressors. Eleven mutant repressors have extended DNA-binding specificies in vivo, and bind operators which the wild-type repressor cannot. One mutant repressor, Lys79, has a classical altered specificity phenotype in vivo, and binds the wild-type trp operator less well than wild-type repressor, yet binds a mutant operator better than wild-type repressor. A site-specific nuclease was derived from Lys79 repressor by constructing a double-mutant protein with Lys79 and a sole cysteine residue, Cys49, and alkylating this cysteine with a 1,10-phenanthroline-copper adduct. This nuclease has an altered specificity of DNA binding in vitro. When activated by the addition of thiol and hydrogen peroxide, the Lys79 nuclease cleaves operator DNA within its new recognition sequence with high efficiency.
Assuntos
Proteínas de Bactérias , DNA/química , DNA/metabolismo , Desoxirribonucleases/metabolismo , Estrutura Secundária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Sequência de Bases , Sítios de Ligação , Desoxirribonucleases/química , Escherichia coli/genética , Escherichia coli/metabolismo , Sequências Hélice-Alça-Hélice , Cinética , Modelos Estruturais , Dados de Sequência Molecular , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Especificidade por SubstratoRESUMO
Threonine is found at the third position of the second alpha-helix in the helix-turn-helix motifs of most bacterial DNA-binding proteins. To investigate the role of this conserved residue in Escherichia coli Trp repressor function, plasmids encoding mutant Trp repressors with each of the 19 amino acid changes of Thr-81 were made by site-directed mutagenesis. All 19 changes decrease the activity of Trp holorepressor, indicating that the Thr-81 side-chain is critical for TrpR function. Three mutant repressors, Ser-81, Lys-81 and Arg-81, retain partial DNA-binding activity and inhibit transcription from the wild-type trp promoter/operator complex; challenge-phage assays show that Ser-81 and Lys-81 holorepressors have altered DNA-binding specificities. The side-chain of Thr-81 may make direct contacts with base pairs 4 and 3 of the trp operator, consistent with the nuclear magnetic resonance solution structures of the holorepressor-operator complex.
Assuntos
Proteínas de Bactérias , DNA Bacteriano/metabolismo , Escherichia coli/genética , Sequências Hélice-Alça-Hélice , Proteínas Repressoras/genética , Treonina/química , Sequência de Aminoácidos , Apoproteínas/metabolismo , Sequência de Bases , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Operadoras Genéticas , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Repressoras/metabolismo , Salmonella typhimurium/metabolismo , Relação Estrutura-AtividadeAssuntos
Autoanticorpos/análise , Doenças Autoimunes/imunologia , Encéfalo/imunologia , Esclerose Múltipla/imunologia , Adulto , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/patologia , Encéfalo/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Seguimentos , Humanos , Doenças do Complexo Imune/diagnóstico , Doenças do Complexo Imune/imunologia , Doenças do Complexo Imune/patologia , Masculino , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/patologia , Exame NeurológicoRESUMO
From 1988 to 1993 thirty liver resections were performed in thirty patients. Eight were operated for suffering from hepatocellular carcinoma, four from metastasis of colon's adenocarcinoma, five for great hemangiomas and the rest for focal liver changes. Right lobectomy was performed in six cases, as well as two trisegmentectomies and two left lobectomies while segmentectomies in the rest. Periopretive mortality occurred in two cases as two patients died because of postoperative thrombosis of vena portae and massive gastrointestinal bleeding. Recidive of malignant tumors was established in five patients after year and in other five till the second year. Benign tumors are without recidives. Later resection is a safe procedure with a considerable selection of patients, with use of latest technical conveniences, application of grafts on branches of vena portae and with routine application of intraoperative ultrasonography. The paper contains diagnostical postulates, surgical techniques and anatomic and morphologic types of resection.