RESUMEN
Many family medicine and community health researchers use surveys as an original research methodology. Our purpose is to illustrate how survey research provides an important form of quantitative research that can be effectively combined with qualitative data to form a mixed methods study. We first provide an overview of the key principles in survey research and in mixed methods research. We review the various ways that survey can be used in mixed methods studies, citing options such as beginning a study with a survey, using a survey as the second form of data collection, or combining a survey and a form of qualitative data in a single data collection procedure. Finally, we illustrate in a specific example six steps in conducting a mixed methods study using survey research. In a mixed methods study using a survey, primary care researchers should consider six steps. Step 1. Articulate the rationale for mixed methods study. Step 2. Detail quantitative and qualitative databases. Step 3. Identify a mixed methods design. Step 4. Analyse and report the results of the quantitative and qualitative databases. Step 5. Present and show integration. Step 6. Explicate the value of using mixed methods. The ability to combine and integrate survey research into a mixed methods study provides a more rigorous approach to research than conducting only a survey or conducting just a qualitative interview. While requiring skills beyond traditional survey approaches, surveys in primary care offers an opportunity for a high level of sophistication in research methodology.
RESUMEN
Parathyroid hormone-related protein (PTHrP) contains a nuclear localization signal (NLS) sequence within 87-107. NLS sequences are generally capable of penetrating cellular membranes due to a richness of basic amino acid residues, and thus have been used as cell-penetrating peptides (CPPs) to translocate biologically active peptides/proteins into cells. The NLS sequence of PTHrP is not exception to this finding; however, PTHrP(87-107) contains 2 acidic glutamate residues at 99 and 101 within the basic amino acid stretch, which is not commonly observed in other CPPs such as HIV-1 Tat(48-60). In this study, we indicated structure-function relationship of the PTHrP NLS to understand the effect of acidic glutamate residues on cell permeability and intracellular localization. We chemically synthesized PTHrP(87-107) and its N-terminally truncated analogues. Their intracellular localization pattern was analyzed by microscopy, radioimmunoassay, and fluorescence-activated cell sorting. Although all analogues were translocated into cells, internalization by the cytoplasm and/or nucleus was length-dependent; specifically, PTHrP(97-107), PTHrP(95-107), and PTHrP(93-107) were more frequently localized in the cytoplasm. We assume that reduction in the net positive charge within PTHrP NLS analogues resulted in increased cytoplasm- translocation activity. We propose that PTHrP(97-107) is a useful carrier peptide for delivery and expression of cargo molecules in the cytoplasm.
Asunto(s)
Señales de Localización Nuclear/química , Proteína Relacionada con la Hormona Paratiroidea/química , Secuencia de Aminoácidos , Antígeno Carcinoembrionario/química , Antígeno Carcinoembrionario/metabolismo , Línea Celular Tumoral , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Epítopos/química , Epítopos/metabolismo , Humanos , Datos de Secuencia Molecular , Señales de Localización Nuclear/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Péptidos/química , Péptidos/metabolismo , Transporte de Proteínas , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
Glucagon, a gut hormone, is one of the key regulatory elements in glucose homeostasis, and is clinically used for treatment of hypoglycemia and premedication in peroral endoscopy. Dry powder inhaler (DPI) form of glucagon is believed to be a promising new dosage form, and the present study aimed to develop a novel glucagon-DPI using absorption enhancer for improved pharmacological effects. The cytotoxicity of citric and capric acids, the potential absorption enhancers, at 1 and 10 mM was assessed by monitoring extracellular LDH levels in rat alveolar L2 cells, and a concentration- and time-dependent release of LDH was observed in capric acid, but not in citric acid-treated cells. DPI form of glucagon containing citric acid was prepared with a jet mill, and laser diffraction and cascade impactor analyses of the newly developed glucagon-DPI suggested high dispersion and deposition in the respiratory organs with an emitted dose and fine particle fraction of 99.5 and 25%, respectively. Addition of citric acid in glucagon-DPI improved the dissolution behavior, and did not impair the solid-state stability of glucagon-DPI. Intratracheal administration of glucagon-DPI (50 microg-glucagon/kg body weight of rat) containing citric acid led to 2.9-fold more potent hyperglycemic effect in rats, as compared to inhaled glucagon-DPI without citric acid. Based on these physicochemical and pharmacological characterization, the dry powder inhaler of glucagon with addition of citric acid would be of use as an alternative to injection form.
Asunto(s)
Ácido Cítrico/química , Portadores de Fármacos , Glucagón/administración & dosificación , Hipoglucemiantes/administración & dosificación , Administración por Inhalación , Animales , Transporte Biológico , Glucemia/efectos de los fármacos , Línea Celular , Química Farmacéutica , Ácido Cítrico/toxicidad , Ácidos Decanoicos/química , Relación Dosis-Respuesta a Droga , Composición de Medicamentos , Estabilidad de Medicamentos , Glucagón/química , Glucagón/metabolismo , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Rayos Láser , Masculino , Microscopía Electrónica de Rastreo , Nebulizadores y Vaporizadores , Tamaño de la Partícula , Polvos , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/enzimología , Alveolos Pulmonares/patología , Ratas , Ratas Sprague-Dawley , Solubilidad , Propiedades de Superficie , Tecnología Farmacéutica/métodos , Factores de TiempoRESUMEN
Previously, [R(15,20,21), L(17)]-VIP-GRR (IK312532), a long-acting VIP derivative, was proposed as potential drug candidate for the treatment of asthma/COPD. The present work is aimed to elucidate solution-state stability of IK312532 and to develop further stabilized derivative with equipotent or higher biological functions. A stability study on IK312532 was carried out in solution state, and degradation mechanism was deduced by UPLC-MS and amino acid analyses. Three novel VIP derivatives were designed and chemically synthesized on the basis of stability data, being subjected to physicochemical and pharmacological characterization. Solution-state stability studies revealed the gradual degradation of IK312532, following pseudo-first-order kinetics. Chemical modification of IK312532, mainly position at 24, resulted in marked improvement of stability, although the chemical modification had no influence on the secondary structure, receptor binding, and activation of adenylate cyclase in rat lung cells. Novel derivatives also exhibited more potent neurite outgrowth in rat pheochromocytoma PC12 cells when compared to VIP and IK312532, possibly due to improved stability. Deamination of Asn at position 24 might be responsible for degradation of VIP derivative, and stability and chemical modification studies led us to the successful development of novel VIP derivatives with higher stability and biological functions.
Asunto(s)
Péptido Intestinal Vasoactivo/análogos & derivados , Péptido Intestinal Vasoactivo/química , Secuencia de Aminoácidos , Animales , Células Cultivadas , Fenómenos Químicos , Datos de Secuencia Molecular , Neuritas/efectos de los fármacos , Neuritas/fisiología , Células PC12 , Estabilidad Proteica , Ratas , Péptido Intestinal Vasoactivo/genética , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Thymopentin (TP5) is a synthetic pentapeptide fragment, which corresponds to position 32 - 36 of thymic polypeptide thymopoietin. Thymopoietin and TP5 display a variety of biological functions, including phenotypic differentiation of T cells and the regulation of immune systems. Previous chemical modification experiments suggested that there was an absolute requirement for N-terminal amino acids to maintain the biological activity of TP5. On the basis of this structure-activity relationship, we designed and synthesized the C-terminally 5-carboxyfluorescein-coupled TP5 (TP5-FAM) as a fluorescent probe for thymopoietin receptor. TP5-FAM could bind to the membrane of human lymphoid cell lines, MOLT-4 cells, in which the thymopoietin receptor is expressed. The binding is specific and saturable (K(d) = 33 microM). TP5 and human splenopentin are nearly equipotent inhibitors of TP5-FAM binding to the thymopoietin receptor, but porcine secretin did not show any significant inhibition of TP5-FAM binding to MOLT-4 cells. Thus, TP5-FAM is suggested to be a potent and biologically active ligand that would be useful for studying the binding and functional characteristics of the human thymopoietin receptor.
Asunto(s)
Receptores de Péptidos/análisis , Timopentina , Animales , Línea Celular Tumoral , Fluoresceínas , Colorantes Fluorescentes , Humanos , Ligandos , Fragmentos de Péptidos/farmacología , Receptores de Péptidos/antagonistas & inhibidores , Receptores de Péptidos/química , Secretina/farmacología , Relación Estructura-Actividad , Porcinos , Timopentina/química , Timopentina/farmacología , Timopoyetinas/farmacologíaRESUMEN
Alteromonas sp. strain O-7 secretes several proteins in addition to chitinolytic enzymes in response to chitin induction. In this paper, we report that one of these proteins, designated MprIII, is a metalloprotease involved in the chitin degradation system of the strain. The gene encoding MprIII was cloned in Escherichia coli. The open reading frame of mprIII encoded a protein of 1,225 amino acids with a calculated molecular mass of 137,016 Da. Analysis of the deduced amino acid sequence of MprIII revealed that the enzyme consisted of four domains: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The C-terminal extension (PkdDf) was characterized by four polycystic kidney disease domains and two domains of unknown function. Western and real-time quantitative PCR analyses demonstrated that mprIII was induced in the presence of insoluble polysaccharides, such as chitin and cellulose. Native MprIII was purified to homogeneity from the culture supernatant of Alteromonas sp. strain O-7 and characterized. The molecular mass of mature MprIII was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 115 kDa. The optimum pH and temperature of MprIII were 7.5 and 50 degrees C, respectively, when gelatin was used as a substrate. Pretreatment of native chitin with MprIII significantly promoted chitinase activity. Furthermore, the combination of MprIII and a novel chitin-binding protease (AprIV) remarkably promoted the chitin hydrolysis efficiency of chitinase.