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D R Mole

Publications and source records attributed to D R Mole.

10 recordsLinked to original sources
PubMed2006

Hypoxia-inducible factors: where, when and why?

Hypoxia-inducible factor (HIF) is a family of transcription factors that regulate the homeostatic response to oxygen deprivation during development, physiological adaptation, and pathological processes such as ischemia and neoplasia. Our understanding of the function of different HIF isoforms is being advanced by understanding the processes that regulate their activity, learning where and when they are expressed and what genes they regulate.

Animals↗
PubMed2001

C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation.

HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF.

2,2'-Dipyridyl↗
PubMed2001

Contrasting effects on HIF-1alpha regulation by disease-causing pVHL mutations correlate with patterns of tumourigenesis in von Hippel-Lindau disease.

The von Hippel-Lindau tumour suppressor gene product (pVHL) associates with the elongin B and C and Cul2 proteins to form a ubiquitin-ligase complex (VCBC). To date, the only VCBC substrates identified are the hypoxia-inducible factor alpha subunits (HIF-1alpha and HIF-2alpha). However, pVHL is thought to have multiple functions and the significance of HIF-1alpha and HIF-2alpha regulation for tumour suppressor activity has not been defined. VHL disease is characterized by distinct clinical subtypes. Thus haemangioblastomas (HABs) and renal cell carcinoma (RCC) but not phaeochromocytoma (PHE) occur in type 1 VHL disease. Type 2 subtypes are characterized by PHE susceptibility but differ with respect to additional tumours (type 2A, PHE+HAB but not RCC; type 2B, PHE+ HAB+RCC; type 2C, PHE only). We investigated in detail the effect of 13 naturally occurring VHL mutations (11 missense), representing each phenotypic subclass, on HIF-alpha subunit regulation. Consistent effects on pVHL function were observed for all mutations within each subclass. Mutations associated with the PHE-only phenotype (type 2C) promoted HIF-alpha ubiquitylation in vitro and demonstrated wild-type binding patterns with pVHL interacting proteins, suggesting that loss of other pVHL functions are necessary for PHE susceptibility. Mutations causing HAB susceptibility (types 1, 2A and 2B) demonstrated variable effects on HIF-alpha subunit and elongin binding, but all resulted in defective HIF-alpha regulation and loss of p220 (fibronectin) binding. All RCC-associated mutations caused complete HIF-alpha dysregulation and loss of p220 (fibronectin) binding. Our findings are consistent with impaired ability to degrade HIF-alpha subunit being required for HAB development and RCC susceptibility.

Adrenal Gland Neoplasms↗
PubMed2001

Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.

Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression by oxygen. In oxygenated and iron replete cells, HIF-alpha subunits are rapidly destroyed by a mechanism that involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. Here we show that the interaction between human pVHL and a specific domain of the HIF-1alpha subunit is regulated through hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme we have termed HIF-alpha prolyl-hydroxylase (HIF-PH). An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly as a cellular oxygen sensor.

Amino Acid Sequence↗
PubMed2001

Regulation of HIF by the von Hippel-Lindau tumour suppressor: implications for cellular oxygen sensing.

Hypoxia-inducible factor (HIF) is central in coordinating many of the transcriptional adaptations to hypoxia. Composed of a heterodimer of alpha and beta subunits, the alpha subunit is rapidly degraded in normoxia, leading to inactivation of the hypoxic response. Many models for a molecular oxygen sensor regulating this system have been proposed, but an important finding has been the ability to mimic hypoxia by chelation or substitution of iron. A key insight has been the recognition that HIF-alpha is targeted for degradation by the ubiquitin-proteasome pathway through binding to the von Hippel-Lindau tumour suppressor protein (pVHL), which forms the recognition component of an E3 ubiquitin ligase complex leading to ubiquitylation of HIF-alpha. Importantly, the classical features of regulation by iron and oxygen availability are reflected in regulation of the HIF-alpha/pVHL interaction. It has recently been shown that HIF-alpha undergoes an iron- and oxygen-dependent modification before it can interact with pVHL, and that this results in hydroxylation of at least one prolyl residue (HIF-1alpha, Pro 564). This modification is catalysed by an enzyme termed HIF-prolyl hydroxylase (HIF-PH), and compatible with all previously described prolyl-4-hydroxylases HIF-PH also requires 2-oxoglutarate as a cosubstrate. The key position of this hydroxylation in the degradation pathway of HIF-alpha, together with its requirement for molecular dioxygen as a co-substrate, provides the potential for HIF-PH to function directly as a cellular oxygen sensor. However, the ability of these enzyme(s) to account for the full range of physiological regulation displayed by the HIF system remains to be defined.

Animals↗
PubMed2001

Renal complications of jejuno-ileal bypass for obesity.

Jejuno-ileal bypass has until recently been an accepted treatment for refractory morbid obesity. Although hyperoxaluria causing renal tract calculi is a well-recognized complication, we describe eight patients who developed significant renal failure attributable to hyperoxaluria resulting from this procedure, three requiring renal replacement therapy. We review the literature, describing 18 other cases with renal failure, the mechanisms of hyperoxaluria and its treatment. Because reversal of the bypass may result in stabilization or partial improvement of renal function, these patients require long-term follow-up of renal function.

Adult↗
PubMed2000

Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein.

The von Hippel-Lindau tumor suppressor protein (pVHL) has emerged as a key factor in cellular responses to oxygen availability, being required for the oxygen-dependent proteolysis of alpha subunits of hypoxia inducible factor-1 (HIF). Mutations in VHL cause a hereditary cancer syndrome associated with dysregulated angiogenesis, and up-regulation of hypoxia inducible genes. Here we investigate the mechanisms underlying these processes and show that extracts from VHL-deficient renal carcinoma cells have a defect in HIF-alpha ubiquitylation activity which is complemented by exogenous pVHL. This defect was specific for HIF-alpha among a range of substrates tested. Furthermore, HIF-alpha subunits were the only pVHL-associated proteasomal substrates identified by comparison of metabolically labeled anti-pVHL immunoprecipitates from proteosomally inhibited cells and normal cells. Analysis of pVHL/HIF-alpha interactions defined short sequences of conserved residues within the internal transactivation domains of HIF-alpha molecules sufficient for recognition by pVHL. In contrast, while full-length pVHL and the p19 variant interact with HIF-alpha, the association was abrogated by further N-terminal and C-terminal truncations. The interaction was also disrupted by tumor-associated mutations in the beta-domain of pVHL and loss of interaction was associated with defective HIF-alpha ubiquitylation and regulation, defining a mechanism by which these mutations generate a constitutively hypoxic pattern of gene expression promoting angiogenesis. The findings indicate that pVHL regulates HIF-alpha proteolysis by acting as the recognition component of a ubiquitin ligase complex, and support a model in which its beta domain interacts with short recognition sequences in HIF-alpha subunits.

Animals↗
PubMed1995

Postprandial alkaline tide: does it exist?

The concept of an alkaline tide which compensates for gastric acid secretion suggests the possibility of indirect measurement of gastric acid secretion. This study was designed to determine whether postprandial changes in renal and respiratory function could be used to assess gastric secretion in healthy adults. Volunteers ate one of three standard (low, medium or high protein) breakfasts on separate days. A fall in urine acid output was observed during 2 h after the high protein meal, but not after the medium or low protein meal. Fasting subjects showed a similar fall in urine acid output over a 2-hour period. Pretreatment with ranitidine 150 mg b.i.d. had no effect on basal or postprandial urine acid output. We conclude that changes in urine acid output are not related to the gastric secretory response to food. In a separate study, treatment with omeprazole 20 mg daily had no effect on postprandial respiratory function (minute ventilation; mixed expired CO2; minute volume of CO2; respiratory exchange ratio; venous blood pH, pCO2 or bicarbonate; and end tidal CO2). Thus we were unable to detect a respiratory alkaline tide after a standard breakfast. These findings suggest that any respiratory or urinary compensation for gastric acid secretion is too small to be of physiological or clinical significance.

Adult↗
PubMed1989

Sites of deposition of aqueous aerosols: a study of efficiency of delivery systems for lung ventilation imaging in man.

A study was made of the deposition of 99Tcm-DTPA aerosol in the components of a jet nebulizer-based aerosol production system. Three impaction devices were compared: a ball-bearing separator, a virtual impactor and a step separator. In addition a comparison was made of two types of tubing which carried aerosol from nebulizer to mouthpiece: corrugated and smooth-walled tubing. The retention of aerosol following inhalation was measured in five normal volunteers using different patterns of breathing. Using an aerosol production system which included a ball-bearing separator only a mean of 11% of the radioactivity loaded into the nebulizer was emitted as an aerosol. Some 18% remained in the ball-bearing separator. The ball-bearing and step separators produced similar total outputs (7% and 6% minimum), the step separator producing marginally higher mean output/min. The virtual impactor produced a lower output than the other two impactors studied, only 1%. A larger proportion of the aerosol output was deposited on corrugated tubing (7%) compared with smooth-walled tubing (1%). The retained fraction of the aerosol inhaled by subjects ranged from 16% to 43%. A higher fraction was retained when subjects inhaled deeply and held their breath for 10 s between each breath. The efficiency of radionuclide deposition from aerosol generator to patient ranged from 1.1% to 6% and was determined more by the retention in the subject than by choice of separator or tubing.

Aerosols↗