Assembly Kinetics of Vimentin Tetramers to Unit-Length Filaments: A Stopped-Flow Study.

Intermediate filaments (IFs) are principal components of the cytoskeleton, a dynamic integrated system of structural proteins that provides the functional architecture of metazoan cells. They are major contributors to the elasticity of cells and tissues due to their high mechanical stability and intrinsic flexibility. The basic building block for the assembly of IFs is a rod-like, 60-nm-long tetrameric complex made from two antiparallel, half-staggered coiled coils. In low ionic strength, tetramers form stable complexes that rapidly assemble into filaments upon raising the ionic strength. The first assembly products, "frozen" by instantaneous chemical fixation and viewed by electron microscopy, are 60-nm-long "unit-length" filaments (ULFs) that apparently form by lateral in-register association of tetramers. ULFs are the active elements of IF growth, undergoing longitudinal end-to-end annealing with one another and with growing filaments. Originally, we have employed quantitative time-lapse atomic force and electron microscopy to analyze the kinetics of vimentin-filament assembly starting from a few seconds to several hours. To obtain detailed quantitative insight into the productive reactions that drive ULF formation, we now introduce a "stopped-flow" approach in combination with static light-scattering measurements. Thereby, we determine the basic rate constants for lateral assembly of tetramers to ULFs. Processing of the recorded data by a global fitting procedure enables us to describe the hierarchical steps of IF formation. Specifically, we propose that tetramers are consumed within milliseconds to yield octamers that are obligatory intermediates toward ULF formation. Although the interaction of tetramers is diffusion controlled, it is strongly driven by their geometry to mediate effective subunit targeting. Importantly, our model conclusively reflects the previously described occurrence of polymorphic ULF and mature filaments in terms of their number of tetramers per cross section.

Antivirals interacting with hepatitis B virus core protein and core mutations may misdirect capsid assembly in a similar fashion.

Recently, heteroarylpyrimidines (HAP) have been identified as potent inhibitors of capsid maturation. Here we discuss the HAP mode of action comparing the aggregation phenotype of wild-type and mutant core proteins with the respective phenotype imposed by HAP or other agents interacting with core protein. Pertinent tests include core fusion protein-mediated transactivation in a two-hybrid system and capsid formation. The finding that transactivation appeared to be unaffected by HAP, or by mutations preventing assembly, is surprising and raises the question for the structure of the interacting hybrid core proteins: Are they monomers, dimers or even oligomers? A direct activity of core fusion monomers is not excluded but considered to be highly unlikely due to rapid homodimerisation. A role of core fusion dimers in transactivation would indicate distinct interactions with a differential sensitivity to HAP. Regarding significance of data gained in two-hybrid systems, caution is necessary, since the site of transactivation is the nucleus, whereas the real site of the core protein interactions during replication is the cytoplasm. Apparently, HAP leave the monomer-monomer interface of HBV core protein unaffected but prevent capsid maturation by interacting with a region known to be crucial for dimer multimerisation and formation of stable capsids. It is suggested to use antivirals as tools for the elucidation of early steps in genome replication and capsid assembly. A frame for this could be the hypothesis that the virus uses soluble core protein, namely intracellular maturation intermediates of HbeAg for a core targeted self-restriction of replication.

Estudios indicación-prescripción de antihipertensivos en pacientes con hipertension arterial esencial, con o sin diabetes miellitus, de una obra social universitaria / Studies indication-prescription of antihypertensive drugs in patients with essential hypertension with or without diabetes miellitus, a university social work

La Hipertensión arterial esencial (HTA) y la diabetes mellitus (DM) son factores de riesgo cardiovascular altamente prevalentes. El tratamiento farmacológico de la HTA pueden disminuir su morbimortalidad. El objetivo de esta tesis fue describir las recomendaciones sobre prescripción de medicamentos antihipertensivos a personas con HTA y a personas con HTA Y DM (HTA-DM) incluidas en guías de práctica clínica (GPC) de buena calidad y comparar éstas con el perfil de prescripción de una obra social en 2010. Se realizó una búsqueda sitemática de GPC, se preseleccionaron según indicadores básicos de calidad metodológica y se aplicó la metodología Appraisal of Guidelines Research and Evaluation for Europe (AGREE) para su evaluación y selección definitiva por calidad. Se realizó un estudio indicación-prescripción en personas con diagnostico de HTA e HTA-DM que adquirieron medicamentos antihipertensivos en las farmacias de la obra social.