HIF signalling: The eyes have it.

The hypoxia inducible factors (HIFs) promote changes in gene expression in response to hypoxia, and mediate key physiological responses such as angiogenesis. They play important roles in development and normal physiology, as well as in ischaemic and other pathologies. The human eye is a complex organ, with tight regulation of vascularisation and oxygen delivery, with the highly specialised retina containing both highly vascularised and avascular regions. This review, written to honour the significant contribution of Lorenz Poellinger to this field, covers the role of the HIFs in normal development of the eye, specifically the vasculature, as well as their roles in numerous retinal pathologies, including ischaemic retinopathies, and age-related macular degeneration (AMD). The characterisation of the HIFs in the eye has improved our understanding of the development, function, and numerous pathologies of the eye, and should inform future therapeutic approaches.

Iodine-131 monitoring in sewage plant outflow.

Three different hospital sites (Oxford, Sutton and Guildford) have performed sampling of their local sewage plant outflow to determine levels of radioactivity resulting from iodine-131 patients undergoing radionuclide therapies. It was found that a maximum of 20% of activity discharged from the hospitals was present in the sewage plant final effluent channel. This is significantly below the level predicted by mathematical models in current use. The results further show that abatement systems to reduce public exposure are unlikely to be warranted at hospital sites.

Radiation protection in fixed PET/CT facilities--design and operation.

We describe the design of a fixed positron emission tomography (PET)/CT facility and the use of a simulated instantaneous dose-rate plot to visually highlight areas of potentially high radiation exposure. We also illustrate the practical implementation of basic radiation protection principles based on the use of distance and shielding and the minimisation of time spent in hot areas. Staff whole body doses for 4 years are presented with results of an optimisation study analysing the dose arising from the different phases within each study using direct reading dosemeters. The total whole body dose for all staff for each patient fell from 9.5 µSv in the first full year of operation to 4.8 µSv in 2008. The maximum dose to an individual member of staff per patient decreased over the same period from 3.2 to 0.9 µSv. The optimisation study showed that the highest dose was recorded during the injection phase.

The emerging role of hypoxia, HIF-1 and HIF-2 in multiple myeloma.

Hypoxia is an imbalance between oxygen supply and demand, which deprives cells or tissues of sufficient oxygen. It is well-established that hypoxia triggers adaptive responses, which contribute to short- and long-term pathologies such as inflammation, cardiovascular disease and cancer. Induced by both microenvironmental hypoxia and genetic mutations, the elevated expression of the hypoxia-inducible transcription factor-1 (HIF-1) and HIF-2 is a key feature of many human cancers and has been shown to promote cellular processes, which facilitate tumor progression. In this review, we discuss the emerging role of hypoxia and the HIFs in the pathogenesis of multiple myeloma (MM), an incurable hematological malignancy of BM PCs, which reside within the hypoxic BM microenvironment. The need for current and future therapeutic interventions to target HIF-1 and HIF-2 in myeloma will also be discussed.

From polyps to people: a highly familiar response to hypoxia.

Obligate aerobes, by definition, require oxygen in order to sustain life. Therefore, changes in environment or physiology that cause metabolic demand for oxygen to exceed supply (hypoxia) can be highly detrimental. Despite considerable variation in physiology and habitat between species, a majority of metazoa employ homologues of the hypoxia-inducible factor (HIF) transcription factors to adapt to oxygen deprivation. Studies in mammals, Drosophila and C. elegans have shown that regulation of HIF-alpha by prolyl hydroxylase (PHD)-mediated proteasomal degradation is conserved, as are a number of HIF target genes. More recently, analysis of coral and beetle HIFs has revealed that, unlike flies and worms, the C-terminal transactivation domain of HIF-alpha and its regulatory hydroxylase FIH-1 are also preserved. The reasons for variable conservation of this system are unknown. However, discovery of the "intermediary" properties of the beetle HIF pathway may prove a useful tool to better define HIF signaling in both mammals and invertebrates.

Attenuation characteristics of MagnaDense high-density concrete at 6, 10 and 15 mv for use in radiotherapy bunker design.

The attenuation characteristics of MagnaDense high-density concrete are measured using the x-ray beams from two Varian linear accelerators at nominal 6, 10, and 15 megavolt energies. The tenth value layers for the primary beam are evaluated under broad- and narrow-beam conditions. The attenuation of secondary radiation is also investigated. Measured data are compared with existing data used in radiotherapy bunker design, derived from the scaling of published tenth value layers for ordinary concrete according to physical density. Instantaneous dose rates around an existing bunker with MagnaDense concrete walls and a conventional concrete roof are predicted using the various different data and compared to actual dose-rate measurements. Primary beam tenth value layer values derived from the broad-beam measurements are found to represent the attenuation properties of the MagnaDense more accurately than those produced by density-based scaling or from narrow-beam measurements.

Turn me on: regulating HIF transcriptional activity.

The hypoxia-inducible factors (HIFs) are critical for cellular adaptation to limiting oxygen and regulate a wide array of genes when cued by cellular oxygen-sensing mechanisms. HIF is able to direct transcription from either of two transactivation domains, each of which is regulated by distinct mechanisms. The oxygen-dependent asparaginyl hydroxylase factor-inhibiting HIF-1alpha (FIH-1) is a key regulator of the HIF C-terminal transactivation domain, and provides a direct link between oxygen sensation and HIF-mediated transcription. Additionally, there are phosphorylation and nitrosylation events reported to modulate HIF transcriptional activity, as well as numerous transcriptional coactivators and other interacting proteins that together provide cell and tissue specificity of HIF target gene regulation.

The hypoxia-inducible factors: key transcriptional regulators of hypoxic responses.

Oxygen depravation in mammals leads to the transcriptional induction of a host of target genes to metabolically adapt to this deficiency, including erythropoietin and vascular endothelial growth factor. This response is primarily mediated by the hypoxia-inducible factors (HIFs) which are members of the basic-helix-loop-helix/Per-ARNT-Sim (bHLH/PAS) transcription factor family. The HIFs are primarily regulated via a two-step mechanism of HIF post-translational modification, increasing both protein stability and transactivation capacity. This review aims to summarise our current understanding of these processes, and discuss the important role of the HIFs in the pathophysiology of many human diseases.

Quantitative assessment of the radiation hazards and risks in sentinel node procedures.

Sentinel node localization using an injected radiopharmaceutical and a gamma probe is performed in many hospitals. Employers have a duty to give appropriate training to staff who may not have been previously formally trained to work with unsealed radioactive sources. A study was performed to assess hazards and risks at all stages of the localization procedure. Whole body doses and finger doses of imaging, surgery and pathology staff were determined. The activity remaining in the tumour specimen, excised nodes and disposable waste from the operating theatre was measured. Any radioactive contamination of the operating theatre and equipment was also ascertained. All results were then assessed in light of current UK radiation protection legislation for the protection of staff and members of the public. Results showed that radiation doses are low and no additional procedures are required for protection of staff, provided the usual procedures for biohazards are in place. However, an information sheet has been prepared for the reassurance of staff, and theatre swabs may need to be stored temporarily before disposal. Injecting and imaging on the day before surgery is preferred, compared with injecting and imaging before surgery on the same day, since this gives lower radiation doses to staff, lower activity in excised specimens and waste, and provides a higher count rate giving better image quality.

Albumin recovery with centrifugal adsorption technology (CAT).

Centrifugal adsorption technology (CAT) is a new compact, countercurrent technology for efficient adsorption from large liquid streams by using adsorbent particles in the micrometer range. CAT seems particularly suited for the recovery of macromolecules at low concentrations, because the small particle dimensions lead to fast mass transfer rates. In this work, the potential of CAT for protein recovery is studied by model and experiment. A predictive model for the separation performance of CAT is presented, incorporating mass transfer resistance and axial dispersion transport in the liquid and the adsorbent phases. The model calculations were compared to experimental data for the adsorption of bovine serum albumin (BSA) on a standard commercial anion-exchange resin with particle diameter d(p) = 50 microm in a pilot-scale CAT apparatus. The model calculations accurately predicted the separation efficiency of CAT. The experimental set-up is shown to be mass transfer limited for the conducted experiments, which agrees with the model predictions. The model was also used to estimate the dimensions and performance of a CAT apparatus for the large-scale recovery of human serum albumin (HSA) from fermentation broth at the scale of 40 tons per year. The resulting equipment dimensions proved to be very small indeed, making CAT a potentially very attractive technology.

Evaluation of a MOSFET radiation sensor for the measurement of entrance surface dose in diagnostic radiology.

A patient dosimetry system using MOSFET technology (Thomson and Neilson Electronics Ltd, Canada) is evaluated for entrance surface dose measurements in diagnostic radiology. The system sensitivity for the standard MOSFET detector coupled to a high sensitivity bias supply was measured to be 1 mV mGy-1. Response of a new high sensitivity dosemeter was measured to be 3 mV mGy-1. The minimum detectable entrance surface dose at which a single measurement can be made with less than 25% total uncertainty at the 95% confidence level was estimated to be 4 mGy for the standard dosemeter and 1.5 mGy for the new high sensitivity dosemeter. The dosemeters were found to be linear with absorbed dose in air, linear with dose rate and reproducible, although they showed some energy dependence across the diagnostic energy range. The system is also compared with thermoluminescent dosimetry (TLD) as a tool for the measurement of entrance surface dose in diagnostic radiology. MOSFET detectors are considered to have advantages over TLD dosemeters with the instant readout of entrance surface dose. These dosemeters do have the disadvantage that they are visible in radiographs, they have a finite shelf life and can only accumulate absorbed dose up to a limiting value after which the dosemeters can no longer be used.

The LXRs: a new class of oxysterol receptors.

The liver X receptors (LXRs) are a family of transcription factors that were first identified as orphan members of the nuclear receptor superfamily. The identification of a specific class of oxidized derivatives of cholesterol as ligands for the LXRs has been crucial to helping understand the function of these receptors in vivo and first suggested their role in the regulation of lipid metabolism. Confirmation of this role has come from the recent analysis of LXR-deficient mice, which has demonstrated the essential function of one of these receptors in the liver as a major sensor of dietary cholesterol.

Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR alpha.

We demonstrate that mice lacking the oxysterol receptor, LXR alpha, lose their ability to respond normally to dietary cholesterol and are unable to tolerate any amount of cholesterol in excess of that which they synthesize de novo. When fed diets containing cholesterol, LXR alpha (-/-) mice fail to induce transcription of the gene encoding cholesterol 7alpha-hydroxylase (Cyp7a), the rate-limiting enzyme in bile acid synthesis. This defect is associated with a rapid accumulation of large amounts of cholesterol in the liver that eventually leads to impaired hepatic function. The regulation of several other crucial lipid metabolizing genes is also altered in LXR alpha (-/-) mice. These results demonstrate the existence of a physiologically significant feed-forward regulatory pathway for sterol metabolism and establish the role of LXR alpha as the major sensor of dietary cholesterol.

Engineering novel specificities for ligand-activated transcription in the nuclear hormone receptor RXR.

BACKGROUND: The retinoid X receptor (RXR) activates transcription of target genes in response to its natural ligand, 9-cis retinoic acid (9cRA), and a number of RXR-specific synthetic ligands. To discover the potential for engineering nuclear receptors for activation of transcription by novel ligands, we used structure-based mutagenesis to change the ligand specificity of RXR. RESULTS: By making substitutions at only two positions (Phe313 and Leu436) we engineered two new classes of RXR proteins that had altered ligand specificities. The first class exhibits decreased activation by 9cRA and increased activation by synthetic ligands. The second class continues to be activated by 9cRA but no longer responds to synthetic ligands. The magnitude of the change in specificity that can be accomplished is greater than 280-fold. CONCLUSIONS: These results confirm that Phe313 and Leu436 are crucial determinants of ligand specificity for RXR and demonstrate that nuclear receptors are exceptionally promising protein scaffolds for the introduction of novel ligand specificities through structure-based protein engineering.

Hairs from patients with maple syrup urine disease show a structural defect in the fiber cuticle.

Models for the surface of cuticle cells in hair fibers consist of a monolayer of fatty acids covalently bound to the underlying protein membrane by thioester linkages. The most prominent of these fatty acids is 18-methyleicosanoic acid (C21a), the synthesis of which requires the oxidative decarboxylation of isoleucine. Maple syrup urine disease (MSUD) is caused by an inherited deficiency in the enzyme branched chain 2-oxo acid dehydrogenase, which leads to the accumulation of branched chain alpha-keto-acids derived from the amino acids, leucine, isoleucine, and valine. Transmission electron microscopy studies of developing hair fibers show a structural defect in the fiber shaft in hair from patients with MSUD. This defect is confined to the cuticle of the fiber, where the cuticle membrane directly apposes the intercellular material. Thus, the defect indicates that C21a is located exclusively on the upper surface of fiber cuticle cells. Lipid analysis of MSUD hairs has demonstrated significant changes in the relative abundance of the covalently bound fatty acids and an almost complete absence of C21a, whereas there was little difference in the amino acid composition compared with normal hair. These results provide further evidence for the existence of the surface lipid monolayer and its crucial role in cellular adhesion.

A comparative study of covalently-bound fatty acids in keratinized tissues.

1. Covalently-bound fatty acids were characterized in keratinous tissues obtained from a wide range of animals. 2. 18-Methyleicosanoic acid was a major component in all the mammalian fur samples examined except monotreme fur. In monotreme fur 26-carbon fatty acids predominated. 3. Fatty acids from feather keratin and reptile skin had different profiles to the alpha-keratins of mammalian fur. 4. The major forms of covalently-bound fatty acids are very similar in species that diverged up to 125 million years ago.