RESUMEN
In most clinical laboratories, hematologists rely on the microscopic analysis of stained blood films to accurately classify cells, aiding in the diagnosis and monitoring of a variety of disorders and conditions. Use of the microscope, although considered the gold standard in performing white blood cell differentials, presents a variety of limitations Digital image technology can facilitate a variety of essential job functions in clinical hematology such as: consulting with colleagues, improving training, referencing an abnormal cell, and utilizing archived images for quality assurance and competency assessment. A questionnaire was developed to survey medical laboratory professionals about their perceptions regarding the benefits and limitations for using digital images in clinical hematology. The questionnaire was sent in March 2012 to an entire list of 81 current CellaVision DM96 (CellaVision AB, Sweden) consumers. A response rate of 46% was obtained. Background information on participants, 5-point Likert scale averages, percentage agreement (strongly agree and agree), and disagreement (strongly disagree and disagree) were calculated and analyzed. The benefits of using the CellaVision DM96 rated the strongest by respondents included: decreased eyestrain, consistency among patient results and advantages in training personnel. Respondents reported notable limitations as being: restrictions with accurately estimating platelets and red cell morphology. Digital image software is currently being utilized in preclinical and clinical hematology and offers potential benefits. With upgrades in slide scanning features and improved capabilities to view platelet and red cell morphology, a transition to digital image technology from the conventional method for performing peripheral blood cell differentials is possible.
Asunto(s)
Pruebas Hematológicas/métodos , Procesamiento de Imagen Asistido por Computador , Programas Informáticos , Femenino , Humanos , Masculino , Personal de Laboratorio Clínico , Microscopía , Encuestas y CuestionariosRESUMEN
Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing particular Vbeta elements of T cell receptor. Here, we report the crystal structure of a MAM mutant K201A in apo form (unliganded) at 2.8-A resolutions. We also partially refined the crystal structures of the MAM wild type and another MAM mutant L50A in apo forms at low resolutions. Unexpectedly, the structures of these apo MAM molecules display a three-dimensional domain-swapped dimer. The entire C-terminal domains of these MAM molecules are involved in the domain swapping. Functional analyses demonstrated that the K201A and L50A mutants do not show altered ability to bind to their host receptors and that they stimulate the activation of T cells as efficiently as does the wild type. Structural comparisons indicated that the "reconstituted" MAM monomer from the domain-swapped dimer displays large differences at the hinge regions from the MAM(wt) molecule in the receptor-bound form. Further comparison indicated that MAM has a flexible N-terminal loop, implying that conformational changes could occur upon receptor binding.
Asunto(s)
Antígenos Bacterianos/química , Apoproteínas/química , Mitógenos/química , Mycoplasma arthritidis/metabolismo , Superantígenos/química , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Apoproteínas/genética , Apoproteínas/metabolismo , Cristalografía por Rayos X , Hibridomas/metabolismo , Interleucina-2/biosíntesis , Activación de Linfocitos , Ratones , Mitógenos/genética , Mitógenos/metabolismo , Modelos Moleculares , Mutación , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores Mitogénicos/química , Soluciones , Superantígenos/genética , Superantígenos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen (SAg) that can activate large fractions of T cells bearing particular TCR Vbeta elements. Here we report the mutagenesis, biochemical and biophysical studies on the dimerization of MAM in solution. Our studies showed that although MAM mainly exists as a monomer in solution, a small percentage of MAM molecules form homodimer at high protein concentration, regardless of the presence of Zn2+. A distinct peak corresponding to a MAM homodimer was detected in the presence of EDTA, using both chemical cross-linking and analytical ultracentrifugation methods. Further mutagenesis studies revealed that single mutation of residues at the interface of the crystallographic dimer of MAM does not significantly affect the dimerization of MAM in solution. Circular dichroism (CD) analysis indicated that addition of Zn2+ does not induce conformational changes of MAM from its apo-state. Thermal denaturation experiments indicated that addition of Zn2+ to MAM solution resulted in a decrease of melting point (Tm), whereas addition of EDTA did not affect the Tm of MAM. These results imply that there is no defined Zn2+-binding site on MAM.
