RESUMEN
BACKGROUND: Reduced flexion following knee arthroplasty (TKA) may compromise patient's function and outcome. The timing of manipulation under anaesthesia (MUA) has been controversial. We present our experience in a high volume practice and analyse the impact of timing. METHODS: All TKA patients requiring MUA from February 1996 to June 2015 under the care of a single surgeon were analysed. MUA was offered to patients who had ≤ 75° of flexion post-op, providing that they had 30° more flexion preoperatively. To address the impact of timing from primary surgery to MUA on flexion gain we looked at 3 groups: Group I ≤ 90 days, Group II 91-180 days and Group III > 180 days. RESULTS: Sixty two out of 7,423 (0.84%) underwent MUA. The MUA patients were significantly younger than the overall TKA cohort 61.2 vs 70.5 years (p = < 0.01). The median duration between arthroplasty and MUA was 3.9 months (IQR 3.4, Range 1.6-72.5 months). Overall flexion gained at 6-12 Weeks and 1 year post MUA showed significant improvements of 20.9° (p = <0.01) and 25° respectively (p = < 0.01). The flexion gain in group I (≤ 90 days) was significantly better than group III ( > 180 days) both at 6 weeks and 1 year following MUA but not better than group II (90-180 days). CONCLUSIONS: MUA is an effective treatment for reduced flexion following TKA and should be the first line of management after failed physiotherapy. It can still have benefit beyond 6 months but the gains become less effective with time.
RESUMEN
Umbilical cord blood (CB) has emerged as an effective alternative donor source for hematopoietic stem cell transplantation. Despite this success, the prolonged duration of immune suppression following CB transplantation and the naiveté of CB T cells leave patients susceptible to viral infections. Adoptive transfer of ex vivo-expanded virus-specific T cells from CB is both feasible and safe. However, the manufacturing process of these cells is complicated, lengthy, and labor-intensive. We have now developed a simplified method to manufacture a single culture of polyclonal multivirus-specific cytotoxic T cells in less than 30 days. It eliminates the need for a live virus or transduction with a viral vector, thus making this approach widely available and GMP-applicable to target multiple viruses. The use of overlapping PepMixes as a source of antigen stimulation enable expansion of the repertoire of the T cell product to any virus of interest and make it available as a third party "off the shelf" treatment for viral infections following transplantation.
RESUMEN
AIMS: Our aim was to examine the clinical and radiographic outcomes in 257 consecutive Oxford unicompartmental knee arthroplasties (OUKAs) (238 patients), five years post-operatively. PATIENTS AND METHODS: A retrospective evaluation was undertaken of patients treated between April 2008 and October 2010 in a regional centre by two non-designing surgeons with no previous experience of UKAs. The Oxford Knee Scores (OKSs) were recorded and fluoroscopically aligned radiographs were assessed post-operatively at one and five years. RESULTS: The median age of the 238 patients was 65.0 years (interquartile range (IQR) 59.0 to 73.0), the median body mas index was 30.0 (IQR 27.5 to 33.0) and 51.7% were male. There were no intra-operative complications. There was a significant improvement in the median OKS at six weeks (34, IQR 31.0 to 37.0), one year (38, IQR 29.0 to 43.0) and five years (37, IQR 27.0 to 42.0) when compared with the pre-operative scores (16, IQR 13.0 to 19.0) (all p = < 0.01). No patient had progressive radiolucent lines or loosening. A total of 16 patients had died by five years. The cumulative survival at five years was 98.8% and the mean survival time was 5.8 years (95% confidence interval 5.6 to 5.9). A total of seven OUKAs (2.7%) were revised; three within five years and four thereafter, between 5.1 and 5.7 years post-operatively. Five (1.9%) had re-operations within five years. CONCLUSION: The proportion of patients requiring revision at five years is lower than that generally reported for UKA. These findings add support for the use of the cementless OUKA outside the design centre. Longer follow-up is required. Cite this article: Bone Joint J 2017;99-B:623-31.
Asunto(s)
Artroplastia de Reemplazo de Rodilla/métodos , Prótesis de la Rodilla , Anciano , Cementación , Femenino , Fluoroscopía , Estudios de Seguimiento , Humanos , Estimación de Kaplan-Meier , Articulación de la Rodilla/diagnóstico por imagen , Articulación de la Rodilla/cirugía , Masculino , Persona de Mediana Edad , Osteoartritis de la Rodilla/cirugía , Dimensión del Dolor/métodos , Diseño de Prótesis , Falla de Prótesis , Reoperación/estadística & datos numéricos , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
AIMS: Our aim was to report survivorship data and lessons learned with the Corail/Pinnacle cementless total hip arthroplasty (THA) system. PATIENTS AND METHODS: Between August 2005 and March 2015, a total of 4802 primary cementless Corail/Pinnacle THAs were performed in 4309 patients. In March 2016, we reviewed these hips from a prospectively maintained database. RESULTS: A total of 80 hips (1.67%) have been revised which is equivalent to a cumulative risk of revision of 2.5% at ten years. The rate of revision was not significantly higher in patients aged ≥ 70 years (p = 0.93). The leading indications for revision were instability (n = 22, 0.46%), infection (n = 20, 0.42%), aseptic femoral loosening (n = 15, 0.31%) and femoral fracture (n = 6, 0.12%). There were changes in the surgical technique with respect to the Corail femoral component during the ten-year period involving a change to collared components and a trend towards larger size. These resulted in a decrease in the rate of iatrogenic femoral fracture and a decrease in the rate of aseptic loosening. CONCLUSION: The rate of revision in this series is comparable with the best performing THAs in registry data. Most revisions were not directly related to the implants. Despite extensive previous experience with cemented femoral components, the senior author noted a learning curve requiring increased focus on primary stability. The number of revisions related to the femoral component is reducing. Any new technology has a learning curve that may be independent of surgical experience. Cite this article: Bone Joint J 2016;98-B:1589-96.
Asunto(s)
Artroplastia de Reemplazo de Cadera/normas , Prótesis de Cadera , Curva de Aprendizaje , Falla de Prótesis/etiología , Anciano , Artroplastia de Reemplazo de Cadera/educación , Artroplastia de Reemplazo de Cadera/métodos , Cementos para Huesos , Bases de Datos Factuales , Educación Médica Continua , Femenino , Prótesis de Cadera/efectos adversos , Humanos , Inestabilidad de la Articulación/cirugía , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Fracturas Periprotésicas/cirugía , Diseño de Prótesis , Infecciones Relacionadas con Prótesis/cirugía , Reoperación/estadística & datos numéricos , Estudios Retrospectivos , Factores de RiesgoRESUMEN
PURPOSE: To examine the feasibility of a breast cancer-related lymphoedema (BCRL) screening programme. Additionally, to investigate the efficacy of bioimpedance analysis (BIA) compared to circumferential measurements (CM) in detecting BCRL. METHODS: This was a 12-month prospective feasibility study. Participants were recruited from two diagnostic breast clinics and consented to be screened for BCRL. Pre-surgical assessments were conducted, and participants were followed up at quarterly intervals. BIA and CM measurements were conducted at all time points. An L-Dex score of >10 or a 10-U increase from baseline or a ≥5 % increase in proximal, distal or total percentage volume difference (PVD) from baseline was indicative of BCRL. Information was collected on subjective symptoms, potential risk factors, demographics and medical data. Feasibility was based on uptake and retention. RESULTS: One hundred twenty-six participants were recruited with an attrition rate of 16.2 %. Participants' mean age was 59 years with the majority having stage I (63.9 %), infiltrating ductal carcinoma (87.4 %). 31.6 % were identified as having BCRL, 90.3 % detected by CM and 35.5 % by BIA (p = ≤0.0001). We found no significant correlation between BIA and CM. Participants identified as having BCRL had a higher BMI, a recent injury to their 'at-risk' arm and more lymph nodes excised (p = <0.05). These findings were not evident across all time points. A large percentage of participants had transient BCRL when assessed by a lymphoedema physiotherapist. CONCLUSIONS: BCRL screening is acceptable and valued by breast cancer survivors. Work needs to continue to establish the most effective screening tool and the natural behaviour of BCRL within the first-year post-surgery.
Asunto(s)
Neoplasias de la Mama/patología , Linfedema/etiología , Adulto , Anciano , Anciano de 80 o más Años , Pesos y Medidas Corporales , Neoplasias de la Mama/cirugía , Carcinoma Ductal de Mama/patología , Carcinoma Ductal de Mama/cirugía , Estudios de Factibilidad , Femenino , Humanos , Ganglios Linfáticos/patología , Linfedema/diagnóstico , Persona de Mediana Edad , Pletismografía de Impedancia , Periodo Posoperatorio , Estudios Prospectivos , Factores de Riesgo , SobrevivientesRESUMEN
PURPOSE: This study aims to explore gynecological cancer survivors' perceptions and experiences following participation in a randomised controlled trial (RCT) testing the efficacy of a home-based physical activity behavioral change intervention (Donnelly et al., Gynecol Oncol 122:618-624, 2011). METHODS: All participants completing a two-armed parallel RCT were invited to participate in the study (31/33) (Donnelly et al., Gynecol Oncol 122:618-624, 2011). Sixteen participants took part (16/31; physical activity (PA) group n = 9, contact control (CC) group n = 7). Four qualitative group interviews were conducted (focus group size 3-5). A structured interview guide was followed by an independent moderator. Groups were audio recorded, transcribed verbatim, and analyzed using the framework approach (Ritchie and Spencer 2001), a five-stage qualitative method of analysis. RESULTS: One of the most unanimously perceived benefits of taking part in the programme regarded participants' psychological well-being. Additional benefits included improved physical fitness and functioning. Important programme features included the weekly telephone calls from a physiotherapist, the patient-professional relationship, and goal setting. Participants' own motivation and programme timing were also identified as important factors. Suggestions for improvements include: opportunities for social interaction with other gynecological cancer survivors and greater exercise choice. CONCLUSION: Findings suggest that women diagnosed with gynecological cancer perceive participation in physical activity as important and participation provides benefits in terms of psychological well-being and improved physical functioning. Support for continuation of many of the current features of the home-based programme was provided. Findings provide insight and rationale for the selection of components for future home-based physical activity interventions. Findings also support further research into the development of multidimensional interventions for the gynecological cancer population.
Asunto(s)
Terapia por Ejercicio/métodos , Neoplasias de los Genitales Femeninos/psicología , Neoplasias de los Genitales Femeninos/terapia , Actividad Motora , Sobrevivientes/psicología , Adulto , Anciano , Actitud Frente a la Salud , Terapia por Ejercicio/organización & administración , Fatiga/psicología , Fatiga/terapia , Femenino , Grupos Focales , Humanos , Persona de Mediana Edad , Motivación , Evaluación de Programas y Proyectos de Salud , Resultado del TratamientoRESUMEN
OBJECTIVE: To investigate the exercise barriers, facilitators and preferences of a mixed sample of cancer survivors as well as fatigue levels, quality of life (QoL) and the frequency and intensity of exercise that cancer survivors typically engage in. METHODS: An anonymous, postal questionnaire-survey with a convenience sample of 975 cancer survivors was used. Standardised measures were used to establish fatigue (Multidimensional Fatigue Symptom Inventory-Short Form), QoL (European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-C30), exercise frequency and intensity (Leisure Score Index). RESULTS: A 52.3% response rate (n = 456) was achieved. A total of 76.0% were female, with stage I (18.3%) or stage II (21.0%) breast cancer (64.4%), and 62.3% were ≥ 3 years post treatment. A total of 73.5% reported fatigue with 57.2% experiencing fatigue on a daily basis. A total of 68.1% had never been given any advice on how to manage fatigue. A total of 9.4% reported to engage in strenuous physical activity, 43.5% in moderate physical activity and 65.5% in mild physical activity. Respondents experienced difficulties with emotional, cognitive and social functioning and the symptoms of fatigue, insomnia and pain. Barriers that interfered with exercise 'often/very often' were mainly related to respondents' health and environmental factors. A total of 50.2% were interested in exercise and 52.5% felt able to exercise. Exercise facilitators, preferences and motivators provide some insight into cancer survivors' needs in terms of becoming more physically active. CONCLUSIONS: Although cancer survivors continue to experience fatigue and QoL issues long after treatment completion, over half are willing and feel able to participate in exercise. Exercise barriers were mainly health related or environmental issues, however, the main barriers reported were those that had the potential to be alleviated by exercise.
Asunto(s)
Ejercicio Físico , Fatiga , Neoplasias/psicología , Calidad de Vida , Sobrevivientes/psicología , Adulto , Femenino , Encuestas Epidemiológicas , Humanos , Persona de Mediana Edad , Motivación , Actividad Motora , Neoplasias/fisiopatología , Neoplasias/rehabilitación , Dolor , Prioridad del Paciente , Apoyo Social , Factores Socioeconómicos , Encuestas y Cuestionarios , Sobrevivientes/estadística & datos numéricos , Reino UnidoRESUMEN
OBJECTIVE: To determine the feasibility and efficacy of a physical activity behavioural change intervention in managing cancer-related fatigue among gynaecological cancer survivors during and post anti-cancer treatments. METHODS: A two arm, single blind, randomised controlled trial was conducted within the Northern Ireland regional Cancer Centre. Thirty three sedentary gynaecological cancer survivors (stage I-III; ≤3 years post diagnosis), experiencing cancer-related fatigue (mild-severe) took part. Participants were randomly assigned to a behavioural change, moderate intensity physical activity intervention (n=16) or a Contact Control group (n=17). The primary outcome was fatigue (Multidimensional Fatigue Symptom Inventory-Short Form and Functional Assessment in Chronic Illness Therapy-Fatigue subscale). Secondary outcomes included quality of life, physical functioning, positive and negative affect, depression, body composition, sleep dysfunction and self-reported physical activity. Feasibility was assessed based on the recruitment rate, programme and physical activity adherence and participants' programme evaluation, including optional focus groups (n=16). RESULTS: Twenty five percent of eligible women took part (33/134). Participants were 8.7 (SD=9.1) months post diagnosis, with a mean age of 53 (SD=10.3) years. The majority of the sample had a diagnosis of ovarian (n=12) or endometrial cancer (n=11). Significant differences favouring the intervention group were observed for fatigue at 12 weeks and 6 months follow-up (12 week: mean difference=-11.06; 95% confidence interval (CI)=-21.89 to -0.23; effect size (d)=0.13; p=0.046; 6 month: mean difference=-19.48; 95% CI=-19.67 to -19.15; effect size (d)=0.20; p=0.01). A mean of 10 calls (SD=1.2 calls) were delivered to the Physical Activity Group, and 10 (SD=1.6 calls) to the CC group. The intervention was positively perceived based on exit questionnaire and focus group findings. CONCLUSIONS: A physical activity behavioural change intervention for gynaecological cancer survivors is feasible in terms of participants' programme adherence and evaluation, and the intervention demonstrates improvements in fatigue. However, confirmation in the form of a larger fully powered RCT is warranted.
Asunto(s)
Fatiga/rehabilitación , Neoplasias de los Genitales Femeninos/rehabilitación , Conductas Relacionadas con la Salud , Actividad Motora , Fatiga/etiología , Femenino , Grupos Focales , Neoplasias de los Genitales Femeninos/complicaciones , Humanos , Persona de Mediana Edad , Método Simple Ciego , Sobrevivientes , Resultado del TratamientoRESUMEN
OBJECTIVES: To establish factors that influence and contribute to the death of patients with cancer in acute hospitals in Northern Ireland. DESIGN: Retrospective clinical note review. SETTING: 16 acute hospitals, covering 5 Health and Social Care Trusts across Northern Ireland. PARTICIPANTS: 793 adult patients with cancer who died in an acute hospital between July and December 2007 identified through the Northern Ireland Cancer Registry. Information was available for 695 (88%). RESULTS: Thee main reasons for acute hospital deaths were uncovered. First, 26.3% of patients were diagnosed with cancer during their last hospital admission. These patients were significantly different from the rest of the sample in being older, not partnered, having more comorbidities and fewer hospital admissions in their last year of life (all p<0.001). Second, patients were very ill with 78.7% admitted as an emergency, requiring medical attention as a result of cancer-related (37.4%) and urgent physical symptoms (33.5%). Third, despite 38.3% of patients specifically requesting discharge to their usual residence, hospice or other hospital, this was not achieved. For 76.3%, this was owing to a deterioration in their medical condition. However for 12.4% there was a lack of a suitable bed, a care package was not in place for 4.9% and 3.0% lacked the required family support. In addition, preferred place of death was only recorded for 41% of patients. CONCLUSIONS: Late diagnosis of cancer is a problem which requires further research. Training should be in place to ensure that a patient's preferred place of death is discussed, recorded and made part of routine end of life care. To achieve this, all medical staff should know when a patient is dying. Further research is required to establish what enables patients with cancer to die at home.
Asunto(s)
Muerte , Diagnóstico Tardío/estadística & datos numéricos , Hospitales , Neoplasias/diagnóstico , Prioridad del Paciente , Cuidado Terminal , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Hospitales para Enfermos Terminales , Humanos , Masculino , Persona de Mediana Edad , Irlanda del Norte , Alta del Paciente , Estudios RetrospectivosRESUMEN
Using reverse genetics, it is possible to readily add well-defined attenuating mutations to the genome of wild type or incompletely attenuated dengue (DEN) viruses to generate vaccine candidates that exhibit the desired balance between attenuation and immunogenicity. Here, we describe the identification of eight temperature sensitive missense mutations distributed in four non-structural protein genes that specify a 60- to 10,000-fold range of restricted replication in the suckling mouse brain compared to wild type recombinant DEN4 virus. The usefulness of such mutations in flavivirus vaccine design and development is discussed.
Asunto(s)
Encéfalo/virología , Virus del Dengue/genética , Virus del Dengue/fisiología , Genes Virales/genética , Mutación Missense/genética , Proteínas no Estructurales Virales/genética , Replicación Viral , Animales , Animales Lactantes , Humanos , Ratones , Fenotipo , TemperaturaRESUMEN
A recombinant live attenuated dengue virus type 4 (DEN4) vaccine candidate, 2ADelta30, was found previously to be generally well tolerated in humans, but a rash and an elevation of liver enzymes in the serum occurred in some vaccinees. 2ADelta30, a non-temperature-sensitive (non-ts) virus, contains a 30-nucleotide deletion (Delta30) in the 3' untranslated region (UTR) of the viral genome. In the present study, chemical mutagenesis of DEN4 was utilized to generate attenuating mutations which may be useful in further attenuation of the 2ADelta30 candidate vaccine. Wild-type DEN4 2A virus was grown in Vero cells in the presence of 5-fluorouracil, and a panel of 1,248 clones were isolated. Twenty ts mutant viruses were identified that were ts in both simian Vero and human liver HuH-7 cells (n = 13) or only in HuH-7 cells (n = 7). Each of the 20 ts mutant viruses possessed an attenuation phenotype, as indicated by restricted replication in the brains of 7-day-old mice. The complete nucleotide sequence of the 20 ts mutant viruses identified nucleotide substitutions in structural and nonstructural genes as well as in the 5' and 3' UTRs, with more than one change occurring, in general, per mutant virus. A ts mutation in the NS3 protein (nucleotide position 4995) was introduced into a recombinant DEN4 virus possessing the Delta30 deletion, thereby creating rDEN4Delta30-4995, a recombinant virus which is ts and more attenuated than rDEN4Delta30 virus in the brains of mice. We are assembling a menu of attenuating mutations that should be useful in generating satisfactorily attenuated recombinant dengue vaccine viruses and in increasing our understanding of the pathogenesis of dengue virus.
Asunto(s)
Virus del Dengue/efectos de los fármacos , Dengue/virología , Fluorouracilo/farmacología , Mutágenos/farmacología , Regiones no Traducidas 3'/genética , Regiones no Traducidas 5'/genética , Animales , Animales Lactantes , Encéfalo/virología , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Virus del Dengue/genética , Virus del Dengue/fisiología , Genes Virales , Genoma Viral , Humanos , Ratones , Datos de Secuencia Molecular , ARN Helicasas , Recombinación Genética , Serina Endopeptidasas , Temperatura , Vacunas Atenuadas/genética , Células Vero , Proteínas no Estructurales Virales/genética , Vacunas Virales/genética , Replicación ViralRESUMEN
We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8(+) cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2Kb-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8(+) T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8(+) CTL in HSV vaccine design.
Asunto(s)
Antígenos Virales , Herpesvirus Humano 2/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Virales/genética , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/virología , Epítopos/genética , Antígenos H-2 , Herpes Genital/inmunología , Herpes Genital/prevención & control , Herpes Genital/virología , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/aislamiento & purificación , Antígenos de Histocompatibilidad Clase I , Inmunidad Mucosa , Inmunización , Memoria Inmunológica , Masculino , Ratones , Ratones Endogámicos C57BL , Recombinación Genética , Virus Vaccinia/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Six mutants of human epidermal growth factor (EGF), which carry single point substitutions within a surface patch proposed to juxtapose the bound receptor, were prepared and characterized for receptor affinity and mitogenicity. Receptor affinities relative to EGF are G12Q > H16D > Y13W > Q43A approximately = H16A approximately = EGF >> L15A. Notably, the reduced receptor affinity of mutant L15A indicates that Leu15 probably contributes substantially to receptor binding whereas unaltered receptor affinities observed for analogs H16A and Q43A indicate that neither His16 nor Gln43 contributes significantly to this interaction. On the other hand, the observed enhanced receptor affinities of analogs G12Q, Y13W and H16D highlight surface loci where additional productive receptor-binding contacts can be introduced. Interestingly, at acidic pH analog H16A reveals substantially greater receptor affinity than that of EGF, a property which may offer enhanced therapeutic utility in acidic environments in vivo.
Asunto(s)
Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Sitios de Unión , Factor de Crecimiento Epidérmico/genética , Vectores Genéticos , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Relación Estructura-ActividadRESUMEN
New approaches for combinatorial library design and molecular diversity analysis have been developed by extending previous work from the fields of quantitative structure-activity relationship, computational chemistry, and chemical information. Recent work has begun to address design efficiency and validation of descriptors for combinatorial library design.
Asunto(s)
Química Orgánica , Indicadores y Reactivos , Fenómenos Químicos Orgánicos , Relación Estructura-ActividadRESUMEN
Distance geometry methods have been used extensively to build models of molecules of various sizes, including small molecules, peptides, and proteins. These methods are often overlooked as tools for conformational analysis, even though they often perform as well as other conformational sampling methods. We have implemented two new distance geometry approaches in the DGEOM95 package. In the first new method, the traditional embedding algorithm is replaced with a procedure that generates random 4D coordinates for each atom, followed by refinement of these coordinates into 3D using the distance geometry error function. The conformational sampling produced by this method is comparable to that obtained with partial metrization, and superior to that obtained with the original embedding procedure. In the second method, a molecular dynamics step is included in the refinement stage. Although this method can be applied to any embedding algorithm, substantial improvements in sampling are seen primarily with the original embedding algorithm.
Asunto(s)
Modelos Moleculares , Conformación Molecular , Algoritmos , Alcanos/química , Animales , Aprotinina/química , Bovinos , Gráficos por Computador , ADN/química , Encefalina Metionina/química , Sustancias Intercalantes/química , Conformación de Ácido Nucleico , Pentanos/química , Péptidos/química , Programas Informáticos , Termodinámica , Agua/químicaRESUMEN
Synthesis and screening of combinatorial libraries for pharmaceutical lead discovery is a rapidly expanding field. Oligo-N-substituted glycines (NSGs) were one of the earliest sources of molecular diversity in combinatorial libraries. In one of the first demonstrations of the power of combinatorial chemistry, two NSG trimers, CHIR-2279 and CHIR-4531, were identified as nM ligands for two 7-transmembrane G-protein-coupled receptors. The NMR characterization of these two lead compounds was undertaken to verify covalent connectivity and to determine solution conformations, if any. The sequential chemical shift assignments were performed using a new strategy for assigning 1H and 13C resonances of NSGs. The conformational preferences were then determined in both an aqueous co-solvent system and an organic solvent to probe the effects of hydrophobic collapse. NSGs are expected to be more flexible than peptides due to the tertiary amide, with both cis and trans amide bond conformations being accessible. Solution NMR studies indicate that although CHIR-2279 and CHIR-4531 have identical backbones and termini, and very similar side chains, they do not display the same solution conformational characteristics.
Asunto(s)
Oligopéptidos/química , Dimetilsulfóxido , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Proteica , Soluciones , AguaRESUMEN
The tat gene product of the human immunodeficiency virus type 1 (HIV-1) strongly induces the transcription directed by the viral long terminal repeat (LTR). Tat acts by interacting with a target RNA element located immediately downstream of the initiation site. In addition, the action of Tat appears to be assisted by the upstream DNA enhancer elements, including the binding sites for the NF-kappa B/Rel family of host transcription factors. In the present study, we demonstrate that Tat transactivation of the HIV-1 LTR is markedly inhibited by several cytoplasmic inhibitors of NF-kappa B/Rel, suggesting the critical involvement of these host transcription factors in the function of the viral Tat protein. Furthermore, the various NF-kappa B inhibitors appear to have differential effects on Tat. While I kappa B alpha, I kappa B beta, and p100 potently inhibit Tat-mediated transactivation, p105 fails to inhibit, but even moderately synergizes, the action of Tat. We further demonstrate that the action of these NF-kappa B/Rel inhibitors on Tat correlates with their inhibitory activities on the RelA subunit of NF-kappa B. Finally, we show that a degradation-resistant I kappa B alpha mutant is able to potently inhibit Tat-mediated activation of the HIV-1 LTR in both untreated and tumor necrosis factor alpha-stimulated T cells, thus suggesting that such an I kappa B alpha mutant may serve as a constitutive repressor of HIV-1 LTR.
Asunto(s)
Productos del Gen tat/metabolismo , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción , Activación Transcripcional , Línea Celular , Humanos , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Factor de Transcripción ReIB , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Each of the 22 exons and 140 bp of the 5' untranslated region of the gene encoding the beta-subunit of cGMP-phosphodiesterase (PDE6B) were screened by denaturing gradient gel electrophoresis for mutations in the DNAs of 54 unrelated individuals with autosomal dominant retinitis pigmentosa. Six different sequence variants were found in seven patients. Four of the sequence variants did not segregate with disease in the families of the respective probands and/or were present in control DNAs. The remaining two sequence variants, a Leu228His missense in exon 3 and a G to A transition in the tenth base of the splice acceptor site of intron 8, were both present in the same proband. One or the other of the two sequence variants was present in each affected member of the proband's small family and neither sequence variant was present in the one unaffected member nor in 75 unrelated controls. However, no effect on splicing of mRNA was observed in expression studies of DNA constructs containing the G to A transition. Therefore, mutations in PDE6B could not be shown to be the cause of adRP in this group of patients.
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/genética , Genes Dominantes , Hidrolasas Diéster Fosfóricas , Retinitis Pigmentosa/genética , Secuencia de Bases , Northern Blotting , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6 , Cartilla de ADN , Exones , Expresión Génica , Variación Genética , Humanos , Intrones , Datos de Secuencia Molecular , Mutación , Empalme del ARNRESUMEN
We have studied 24 small families with presumed autosomal recessive inheritance of retinitis pigmentosa by a combination of haplotype analysis and exon screening. Initial analysis of the families was made with a dinucleotide repeat polymorphism adjacent to the gene for rod cGMP-phosphodiesterase (PDE6B). This was followed by denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism electrophoresis (SSCPE) of the 22 exons and a portion of the 5' untranslated region of the PDE6B gene in the probands of each family in which the PDE6B locus could not be ruled out from segregating with disease. Two probands were found with compound heterozygous mutations: Gly576Asp and His620(1-bp del) mutations were present in one proband, and a Lys706X null mutation and an AG to AT splice acceptor site mutation in intron 2 were present in the other. Only the affecteds of each of the two families carried both corresponding mutations.
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/genética , Mutación , Retinitis Pigmentosa/genética , Adulto , Secuencia de Bases , Electroforesis , Femenino , Genes Recesivos , Haplotipos , Heterocigoto , Humanos , Masculino , Datos de Secuencia Molecular , Polimorfismo Conformacional Retorcido-Simple , Retinitis Pigmentosa/etiología , Retinitis Pigmentosa/patología , Análisis de Secuencia de ADNRESUMEN
Screening mixtures of synthetic oligomers or fixed templates (e.g., rings) with varying substituents is increasingly the focus of drug discovery programs. CHORTLES is designed and implemented to facilitate representation, storage, and searching of oligomeric and template-based mixtures of any size. Building upon the CHUCKLES method of representing oligomers as both monomer-based sequences and all-atom structures, CHORTLES compactly represents a mixture without explicitly enumerating individual molecules. This method lends itself to a hierarchy relating mixtures to submixtures and individual compounds, as one finds when deconvoluting mixtures in drug lead discovery programs. In addition, we describe two methods of searching mixtures at the monomer level. We also present a simple pictorial representation for describing all components in a mixture, which becomes essential as the list of monomer names is expanded beyond common names (e.g., amino acids).