Erythrocytosis-associated HIF-2alpha mutations demonstrate a critical role for residues C-terminal to the hydroxylacceptor proline.

Abstract A classic physiologic response to hypoxia in humans is the up-regulation of the ERYTHROPOIETIN (EPO) gene, which is the central regulator of red blood cell mass. The EPO gene, in turn, is activated by hypoxia inducible factor (HIF). HIF is a transcription factor consisting of an alpha subunit (HIF-alpha) and a beta subunit (HIF-beta). Under normoxic conditions, prolyl hydroxylase domain protein (PHD, also known as HIF prolyl hydroxylase and egg laying-defective nine protein) site specifically hydroxylates HIF-alpha in a conserved LXXLAP motif (where underlining indicates the hydroxylacceptor proline). This provides a recognition motif for the von Hippel Lindau protein, a component of an E3 ubiquitin ligase complex that targets hydroxylated HIF-alpha for degradation. Under hypoxic conditions, this inherently oxygen-dependent modification is arrested, thereby stabilizing HIF-alpha and allowing it to activate the EPO gene. We previously identified and characterized an erythrocytosis-associated HIF2A mutation, G537W. More recently, we reported two additional erythrocytosis-associated HIF2A mutations, G537R and M535V. Here, we describe the functional characterization of these two mutants as well as a third novel erythrocytosis-associated mutation, P534L. These mutations affect residues C-terminal to the LXXLAP motif. We find that all result in impaired degradation and thus aberrant stabilization of HIF-2alpha. However, each exhibits a distinct profile with respect to their effects on PHD2 binding and von Hippel Lindau interaction. These findings reinforce the importance of HIF-2alpha in human EPO regulation, demonstrate heterogeneity of functional defects arising from these mutations, and point to a critical role for residues C-terminal to the LXXLAP motif in HIF-alpha.

Comparison of a novel spray congealing procedure with emulsion-based methods for the micro-encapsulation of water-soluble drugs in low melting point triglycerides

The particle size characteristics and encapsulation efficiency of microparticles prepared using triglyceride materials and loaded with two model water-soluble drugs were evaluated. Two emulsification procedures based on o/w and w/o/w methodologies were compared to a novel spray congealing procedure. After extensive modification of both emulsification methods, encapsulation efficiencies of 13.04% tetracycline HCl and 11.27% lidocaine HCl were achievable in a Witepsol (R)-based microparticle. This compares to much improved encapsulation efficiencies close to 100% for the spray congealing method, which was shown to produce spherical particles of similar to 58 mu m. Drug release studies from a Witepsol (R) formulation loaded with lidocaine HCl showed a temperature-dependent release mechanism, which displayed diffusion-controlled kinetics at temperatures similar to 25 degrees C, but exhibited almost immediate release when triggered using temperatures close to that of skin. Therefore, such a system may find application in topical semi-solid formulations, where a temperature-induced burst release is preferred.

Clinical phenotype of cystic fibrosis patients with the G551D mutation

Background: Data on whether the phenotype of cystic fibrosis (CF) patients with compound heterozygocity for G551D (Gly551Asp) differs from patients with F508del (Phe508del) homozygous mutations is divergent.

A photospheric bright point model

Aims. A magneto-hydrostatic model is constructed with spectropolarimetric properties close to those of solar photospheric magnetic bright points.
Methods. Results of solar radiative magneto-convection simulations are used to produce the spatial structure of the vertical component of the magnetic field. The horizontal component of magnetic field is reconstructed using the self-similarity condition, while the magneto-hydrostatic equilibrium condition is applied to the standard photospheric model with the magnetic field embedded. Partial ionisation processes are found to be necessary for reconstructing the correct temperature structure of the model.
Results. The structures obtained are in good agreement with observational data. By combining the realistic structure of the magnetic field with the temperature structure of the quiet solar photosphere, the continuum formation level above the equipartition layer can be found. Preliminary results are shown of wave propagation through this magnetic structure. The observational consequences of the oscillations are examined in continuum intensity and in the Fe I 6302 angstrom magnetically sensitive line.