Kuopio treatment strategy after deep sternal wound infection.

BACKGROUNDS AND AIMS: Infection of sternotomy wound is a rare potentially fatal complication because of the risk for deep sternal infection. Current treatment comprises antibiotics, debridement, negative pressure wound therapy and sometimes transposition of muscle or omental flaps to fill the anterior mediastinal dead space. MATERIAL AND METHODS: The management of 60 consecutive deep sternotomy wound infections is reviewed. The one stage treatment was mostly chosen. In 5 patients after debridement, negative pressure wound therapy was used before flap reconstruction. Fifty-seven patients were rewired and 3 patients had sternectomy. The choice of the flap was based mainly on anatomic location of a sternal wound defect and also on which grafts been used in cardiac operation. RESULTS: The unilateral turnover split pectoralis major flap was the choice for 50 patients. In 8 patients latissimus dorsi flap was used. Rectus abdominis was used as a standalone flap in 4 patients and in combination with pectoralis major in one. All patients survived after deep sternal wound infection. In only 33 patients the recovery was totally uneventful. In the remaining 27 patients there were one or more complications. Not a single flap was lost completely, but due to partial flap necrosis, a redo reconstruction was needed in 3 patients. Negative pressure wound therapy was used after flap reconstruction in eight patients with incomplete post-flap healing to prepare for wound revision and split thickness skin graft. CONCLUSIONS: A structured approach including both cardiac and plastic surgery in case of deep sternal wound infection is recommended. A single stage surgery with the help of muscle flap reconstruction is our standard treatment. With our protocol, we have been able to keep the mortality low.

Pronounced tumour regression after radiotherapy is associated with negative/weak glucose transporter-1 expression in rectal cancer.

UNLABELLED: The aim of this study was to assess glucose transporter-1 (GLUT-1) expression as a predictor of disease outcome in rectal cancer treated by preoperative radio- or chemoradiotherapy. PATIENTS AND METHODS: Operative samples from 175 rectal cancer patients and 78 preoperative biopsies were analysed for GLUT-1 expression using immunohistochemistry. Forty-six patients received long-course radiotherapy, with/without chemotherapy and tumour regression grade was analysed in these specimens. RESULTS: Negative GLUT-1 expression was seen in 25/78 (32%) of the preoperative biopsies and in 38/78 (49%) of the operative samples. There was no significant correlation of GLUT-1 with common clinicopathological factors. A trend towards longer disease-free survival (DFS) for the long-course radiotherapy group patients was seen with negative/weak GLUT-1 staining intensity (p=0.066) and excellent tumour regression grade (p=0.068) in operative samples. Disease-free survival (p=0.068) and disease-specific survival (p=0.024) of the patients with excellent tumour regression were longer than among the patients with moderate or less regression. CONCLUSION: A trend towards longer DFS among patients in favour of negative/weak GLUT-1 staining in the operative samples after long-course radiotherapy is demonstrated.

Preoperative radiotherapy downregulates the nuclear expression of hypoxia-inducible factor-1alpha in rectal cancer.

OBJECTIVE: To assess the value of hypoxia-inducible factor-1alpha (HIF-1alpha) expression as a predictor of disease outcome in rectal cancer treated by preoperative radio- or chemoradiotherapy. MATERIAL AND METHODS: Operative samples from 168 rectal cancer patients and 79 respective preoperative biopsies were analyzed for nuclear HIF-1alpha protein expression using immunohistochemistry by three approaches: (a) positive/negative, (b) the percentage of HIF-positive cancer cells and (c) staining intensity. The patients had received either short- (n = 75) or long-course radiotherapy with or without chemotherapy (n = 39) or no treatment preoperatively (n = 54). RESULTS: HIF-1alpha staining was positive in 70% of the diagnostic biopsies but negative in most of the post-radiotherapy specimens (60%). HIF-1alpha expression in the biopsies was downregulated in 56% of samples taken after preoperative treatment, while negative HIF-1alpha expression was upregulated in 25% of samples. Patients who had HIF-negative tumours after long-course radiotherapy had significantly (P = 0.001) better disease-specific survival (DSS) in univariate analysis. In the multivariate (Cox) regression model, HIF-1alpha lost its significance and only being in the preoperative treatment group was an independent predictor of disease-free survival. In a similar Cox model, disease recurrence and the number of metastatic lymph nodes were independent predictors of DSS. CONCLUSIONS: HIF-1alpha expression was positive in most of the preoperative biopsies but downregulated in most of the operative samples, implicating that preoperative radiotherapy downregulates HIF-1alpha expression in rectal cancer. Negative HIF expression after preoperative long-course radiotherapy was associated with significantly better DSS.

Unilateral spinal anaesthesia for outpatient surgery: a comparison between hyperbaric bupivacaine and bupivacaine-clonidine combination.

BACKGROUND: Low-dose hyperbaric bupivacaine has been used to produce unilateral spinal anaesthesia for outpatient surgery. Unilateral spinal anaesthesia is associated with reduction of hypotension, faster recovery and increased patient satisfaction. Small doses of clonidine have shown effectiveness in intensifying spinal anaesthesia. We investigated the effect of adding 15 microg of clonidine to 5 mg hyperbaric bupivacaine on unilaterality. METHODS: Sixty patients undergoing outpatient knee arthroscopy were randomly allocated to receive either 1.2 ml (6 mg) of hyperbaric bupivacaine or a 1.2 ml solution containing 1.0 ml (5 mg) hyperbaric bupivacaine, 0.1 ml (75 microg) clonidine and 0.1 ml sterile water. The motor block was assessed by a modified Bromage scale and the sensory block by a pinprick. RESULTS: There was a significant difference in the spread of anaesthesia between the operated and contralateral sides in both groups. Seventy-seven per cent of the blocks were unilateral in group B and 73% in group B-C. There was no significant difference between the groups, in unilaterality. The motor block was prolonged in group B-C but it did not affect home-readiness. Patients receiving clonidine needed more vasopressors. There was a significant difference in blood pressures between the groups, being lower in group B-C after 1 h 45 min. CONCLUSION: Using 5 mg hyperbaric bupivacaine with 15 microg of clonidine, the unilaterality can be achieved and spinal anaesthesia intensified without affecting home-readiness. More vasopressors are needed in the beginning, but after the surgery patients experienced less pain.

Expression of carbonic anhydrase IX suggests poor outcome in rectal cancer.

The aim of the study is to assess the value of carbonic anhydrase isozyme IX (CA IX) expression as a predictor of disease-free survival (DFS) and disease-specific survival (DSS) in rectal cancer treated by preoperative radio- or chemoradiotherapy or surgery only. Archival tumour samples from 166 patients were analysed for CA IX expression by three different evaluations: positive/negative, proportion of positivity and staining intensity. The results of immunohistochemical analysis were confirmed by demonstrating CA IX protein in western blotting analysis. Forty-four percent of the operative samples were CA IX positive, of these 34% had weak and 66% moderate/strong staining intensity. In univariate survival analysis, intensity of CA IX expression was a predictor of DFS (P=0.003) and DSS (P=0.034), both being markedly longer in tumours with negative or weakly positive staining. In multivariate Cox model, number of metastatic lymph nodes and CA IX intensity were the only independent predictors of DFS. Carbonic anhydrase isozyme IX intensity was the only independent predictor of DSS, with HR=9.2 for dying of disease with moderate-intense CA IX expression as compared with CA IX-negative/weak cases. Negative/weak CA IX staining intensity is an independent predictor of longer DFS and DSS in rectal cancer.

Hypoxia-activated autophagy accelerates degradation of SQSTM1/p62.

Sequestosome 1 (SQSTM1/p62) is a multifunctional protein involved in signal transduction, protein degradation and cell transformation. Hypoxia is a common feature of solid tumours that promotes cancer progression. Here, we report that p62 is downregulated in hypoxia in carcinoma cells and that the expression is rapidly restored in response to reoxygenation. The hypoxic p62 downregulation did not occur at the mRNA level and was independent of the hypoxic signal mediators hypoxia-inducible factor (HIF) and von Hippel-Lindau tumour suppressor protein as well as the activity of HIF-prolyl hydroxylases and was not mediated by proteosomal destruction. Autophagy was activated in hypoxia and was required for p62 degradation. The hypoxic degradation of p62 was blocked by autophagy inhibitors as well as by the attenuation of Atg8/LC3 expression. Downregulation of p62 was required for hypoxic extracellular regulated kinase (ERK)-1/2 phosphorylation. Attenuation of p62 in normoxia activated and forced expression of p62 in hypoxia blocked the activation of ERK-1/2. The results demonstrate that hypoxic activation of autophagy induces clearance of p62 protein and implies a role for p62 in the regulation of hypoxic cancer cell survival responses.

p38alpha and p38delta mitogen-activated protein kinase isoforms regulate invasion and growth of head and neck squamous carcinoma cells.

Recent studies indicate that the specificity of p38 mitogen-activated protein kinase (MAPK)-mediated cellular stress responses is determined by the expression pattern of the distinct p38 isoforms. Here, we have analysed the function of distinct p38 isoforms in the growth and invasion of head and neck squamous cell carcinomas (HNSCCs). Activation of p38 MAPK by arsenite resulted in inactivation of the ERK1,2 signaling pathway by dephosphorylation of MEK1,2 in primary human epidermal keratinocytes (HEKs), whereas in HNSCC cells this p38-mediated inhibition of the ERK1,2 pathway was absent. Quantitation of p38 pathway component mRNA expression in HNSCC cell lines (n=42) compared to HEKs (n=8) revealed that p38alpha and p38delta isoforms are predominantly expressed in both cell types and that MKK3 is the primary upstream activator expressed. Inhibition of endogenous p38alpha or p38delta activity by adenoviral delivery of corresponding dominant-negative p38 isoforms potently reduced MMP-13 and MMP-1 expressions, and suppressed the invasion of HNSCC cells through collagen. Dominant-negative p38alpha and p38delta inhibited squamous cell carcinoma (SCC) cell proliferation and inhibition of p38alpha activity also compromised survival of SCC cells. p38alpha and p38delta were predominantly expressed in HNSCCs (n=24) and nonneoplastic epithelium in vivo (n=6), with MKK3 being the primary upstream activator. Activation and expression of p38alpha and p38delta by tumor cells was detected in HNSCCs in vivo (n=16). Adenoviral expression of dominant-negative p38alpha or p38delta in cutaneous SCC cells potently inhibited their implantation in skin of severe combined immunodeficiency mice and growth of xenografts in vivo. Our results indicate that p38alpha and p38delta specifically promote the malignant phenotype of SCC cells by regulating cell survival, proliferation and invasion, suggesting these p38 MAPK isoforms as potential therapeutic targets in HNSCCs.

Different mechanisms of syndecan-1 activation through a fibroblast-growth-factor-inducible response element (FiRE) in mucosal and cutaneous wounds.

Syndecan-1 expression is enhanced in cutaneous and mucosal wounds. We have previously demonstrated that wounding-induced syndecan-1 expression in the skin occurs transcriptionally, through a fibroblast-growth-factor-inducible element (FiRE). Here, we show that FiRE is also activated in mucosal wounds. However, both the expression patterns and the activation mechanisms of FiRE are different from those in the skin. In the mucosa in vivo, the activation starts and ends earlier than in cutaneous wounds. FiRE is first detected at around 12 hours in keratinocytes, and the activation declines by the third day after wounding occurs. The activation is seen on the migrating sheet of epithelial mucosa, as in the case of cutaneous wounding. In contrast to the situation in vivo, organ-cultured mucosal wounds exhibit no FiRE activity, while organ-cultured cutaneous wounds show robust activity. Activation in mucosal wounds is enhanced, however, by the application of epidermal growth factor. This suggests that exogenous growth factor activity is required for activation of syndecan-1 in mucosal wounds but not in cutaneous wounds.

Endovascular treatment of aortic aneurysms in Finland: the first four years' experience.

BACKGROUND AND AIMS: In this study the results of endovascular treatment of aortic aneurysms in Finland are presented and compared to the results of the Eurostar registry. MATERIAL AND METHODS: A total of 229 patients with aortic aneurysm were treated in five different Finnish centres during 1996-2000. The data of these patients were collected prospectively by surgeon or interventional radiologist involved. During the same period of time 2464 patients were registered in the Eurostar registry. RESULTS: The procedure was performed successfully in 97% of patients in Finland, and the 30-day mortality was 0,9%. A graft limb thrombosis was detected in 9% of the patients in Finland. A permanent primary endoleak at the first 30-day control was seen in 23 patients (10%). During the follow-up 17 secondary endoleaks (7%) were detected. A secondary intervention was necessary in 26% of the patients. Three patients (1.3%) had late rupture of the abdominal aortic aneurysm. CONCLUSIONS: According to the Finnish short-time results, endovascular treatment of aortic aneurysms is safe and associated with relatively low morbidity and mortality. The mid-term results are more disappointing with relatively many graft thromboses and endoleaks, and a frequent need of secondary interventions.

CT angiography in the assessment of carotid artery atherosclerosis.

OBJECTIVE: The aim of the study was to evaluate the ability of CT angiography (CTA) to detect atherosclerosis of the carotid arteries in comparison with 3D time-of-flight MR angiography (3D TOF MRA), using contrast angiography and intravascular ultrasound (IVUS) as a reference. MATERIAL AND METHODS: Contrast angiography and CTA were performed in 31 patients (mean age 65 years, range 45-79). The image quality was evaluated, atherosclerotic lesions were registered, and diameter stenosis degree was visually estimated from axial source images of CTA and 3D TOF MRA (21 patients), and results of interpretations were compared. The comparison of quantitative measurements was performed using IVUS as a reference. RESULTS: Contrast angiography detected 51 lesions (mean diameter stenosis 50%, range 10-100%), and CTA detected all same lesions. CTA provided better image quality and consistency of image interpretation than 3D TOF MRA. IVUS verified 29 atherosclerotic lesions with a mean diameter stenosis of 35%, (range 4-40%). CTA yielded a sensitivity of 96% to 100% (< or =10% or < or =20% diameter stenosis regarded as normal) for the detection of lesions with reference to IVUS. CONCLUSION: CTA seems feasible and accurate for the detection of atherosclerosis in carotid arteries.

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.

Results of infrainguinal bypass surgery: an analysis of 263 consecutive operations.

BACKGROUND AND AIMS: The purpose of this clinical study was to assess the success of infrainguinal revascularization in the treatment of lower limb ischaemia. MATERIAL AND METHODS: 226 consecutive patients underwent 263 femoropopliteal (n = 194) or femorodistal (n = 69) bypass operations during 1988-1996 at a university hospital. Records of all patients were reviewed. Late control visits including clinical and colour doppler ultrasound examinations were programmed for 109 patients. Initial success, primary and secondary patencies, limb salvage and survival rates were determined and factors affecting outcome were analysed in various patient categories. RESULTS: Initial success rate was 92% (243/263). The primary and secondary patencies were 70/83% and 52/63% at one and five years, respectively. The corresponding limb salvage rates for patients with chronic critical ischaemia were 82% and 77%. The number of diseased vessels in the treated limb correlated negatively with the primary patency. Advanced age did not affect primary patency or limb salvage rates. Diabetes and the use of distal revascularizations were independent predictors of poorer limb salvage. Diabetes and renal insufficiency proved to shorten life expectancy. CONCLUSIONS: Infrainguinal revascularizations are effective regardless of patient's age. The extent of atherosclerotic changes in the operated limb, diabetes and renal insufficiency are factors affecting outcome.

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.

Proximal promoter of the murine syndecan-1 gene is not sufficient for the developmental pattern of syndecan expression in B lineage cells.

Syndecan-1 (CD138) is a cell membrane proteoglycan that binds extracellular matrix components and various growth factors. The role of syndecan-1 in the control of cell growth and morphology has been illustrated by its altered expression in hematological malignancies such as multiple myeloma as well as some solid tumors. It has been reported that the expression of syndecan-1 in cells of the B lineage is developmentally regulated such that pre-B cells and plasma cells express syndecan-1 while mature B cells do not. Thus, we investigated whether the proximal promoter region of the murine syndecan-1 promoter was able to confer the observed on-off-on expression of syndecan-1 in cells of the B lineage as they develop from pre-B cells to plasma cells. Experiments carried out using deletion mutants of the proximal promoter cloned upstream of the CAT reporter gene transfected into murine cell lines, representing the above stages of B-cell development, such as BA/F3 (pro-B cell), 70Z/3 (pre-B cell), 2PK3 (late mature B cell), and MPC-11 (plasma cell), showed detectable levels of CAT expression. The WEHI-231 (mature B cell) cell lines did not show detectable levels of CAT reporter activity. The strong levels of expression were observed with a fragment of the proximal promoter spanning the region from -365 to -95 (from the translation start point). However, Northern analysis of RNA obtained from the five murine B-cell lines, representing various stages of B-cell development, showed that the 70Z/3, MPC-11 but not BA/F3, and 2PK3 cells expressed detectable levels of syndecan-1 mRNA. By FACS analysis, using a rat anti mouse syndecan-1 antibody, syndecan-1 expression on the cell surface was found to correlate with the observed mRNA expression patterns in these cell lines. Our results indicate that the proximal promoter of the murine syndecan-1 promoter is not sufficient for the observed developmental pattern of syndecan expression in B cells.

FT-IR breath test in the diagnosis and control of treatment of methanol intoxications.

A portable Fourier transform infrared (FT-IR) multicomponent point-of-care analyzer was tested for the diagnosis of methanol intoxications. Breath analysis with FT-IR was fast and easy, and no sample preparation was needed. The analyzer was adequately sensitive and accurate in detecting and quantitating clinically relevant amounts of ethanol and methanol in the breath of seriously ill patients. FT-IR spectrometry was also suitable for nearly on-line monitoring of the exhaled ethanol and methanol during hemodialysis. The breath analysis results correlated well with blood samples. The FT-IR method used also has a traceable calibration to physical properties of the analyte, and the measured spectra can be saved for later analysis.

Proximal promoter-independent activation of the far-upstream FGF-inducible response element of syndecan-1 gene.

Far upstream enhancers are predicted to act by looping and activating general transcription factors on core promoters and to require proximal promoter sequences for appropriate gene activation in time and space. We have previously described an FGF-inducible response element (FiRE) located far upstream on the syndecan-1 gene. The FiRE is activated specifically by members of the fibroblast growth factor (FGF) family in NIH3T3 cells. Here we describe the requirements of syndecan-1 proximal promoter for the activation of FiRE by FGF-2. Transient and stable transfections revealed that neither proximal promoter SP1 sites nor TATA-box are required for the FGF-2 induced activation of FiRE. Notably, the enhancer is activated in both orientations by FGF-2 even in the absence of proximal promoter. Importantly, removal of the promoter did not affect the growth factor specificity of FiRE. Proximal promoter independent activation of syndecan-1 gene by FGF-2 might be required during development when syndecan-1 proximal promoter needs to be largely attenuated but simultaneous transient and rapid FGF-2 induced transcription is required.

Transcriptional targeting of adenoviral gene delivery into migrating wound keratinocytes using FiRE, a growth factor-inducible regulatory element.

Impaired cutaneous wound healing is a common complication in diabetes, ischemia and venous insufficiency of lower extremities, and in long-term treatment with corticosteroids or other immunosuppressive agents. In development of gene therapy for wound repair, expression of therapeutic transgenes should be precisely targeted and controlled. Here, we describe a recombinant adenovirus RAdFiRE-EGFP, in which a growth factor inducible element (FiRE) of the murine syndecan-1 gene controls the expression of enhanced green fluorescent protein (EGFP) reporter gene. Treatment of RAdFiRE-EGFP-transduced murine epidermal keratinocytes in culture with FiRE-activating growth factor markedly enhanced the expression of EGFP. In ex vivo organ culture of wounded murine skin transduced with RAdFiRE-EGFP, the EGFP expression was specifically detected in wound margin keratinocytes, but not in intact skin. Activity of EGFP was first detected 2 days after a single application of RAdFiRE-EGFP and persisted up to 10 days. Similarly, FiRE-driven EGFP expression was detected specifically in epidermal keratinocytes in the edge of incisional wounds in murine skin transduced with RAdFiRE-EGFP. In contrast, adenovirus-mediated lacZ expression driven by CMV promoter was detected scattered in epidermal, dermal and subcutaneous layers in ex vivo and in vivo wounds, as well as in intact skin. These data demonstrate the feasibility of FiRE as a tool for transcriptional targeting of adenovirus-mediated transgene expression to cutaneous wound edge keratinocytes.

Prospective trial of infrapopliteal artery balloon angioplasty for critical limb ischemia: angiographic and clinical results.

PURPOSE: To evaluate the safety and efficacy of infrapopliteal percutaneous transluminal angioplasty (PTA) as a primary treatment of chronic critical limb ischemia in a prospective trial. MATERIALS AND METHODS: Infrapopliteal PTA was performed on 72 limbs of 60 patients (mean age, 72 y; range, 38-92 y) and patients were followed for 12-24 months. RESULTS: The primary angiographic success rate for the stenoses was 84% (102 of 121) and that for the occlusions was 61% (41 of 67) with corresponding restenosis rates of 32% and 52% at follow-up angiography performed a mean of 10 months after primary PTA. The rate of major complications was 2.8% (access site pseudoaneurysms in two patients). The primary clinical success was 63% (45 of 72). A 48% cumulative primary patency rate, a 56% secondary patency rate, and a 80% cumulative limb salvage rate were registered at 18 months, as determined with use of Kaplan-Meier analysis. Lack of angiographic improvement at the site of the most severe ischemia and renal insufficiency (serum creatinine level > 130 micromol/L) were independent predictors of poorer long-term clinical results, as determined with use of Cox multiple regression analysis. CONCLUSIONS: Infrapopliteal PTA is a feasible primary treatment of chronic critical limb ischemia with moderate primary angiographic and clinical success, a low complication rate, and a cumulative limb salvage rate comparable with surgical techniques.

The use of low-resolution FT-IR spectrometry for the analysis of alcohols in breath.

Fast and reliable diagnostic methods are needed for detection or exclusion of industrial solvents as a cause of intoxication. Analyzing human breath reveals the presence of any volatile substance. A portable Fourier transform infrared (FT-IR) multicomponent point-of-care analyzer was developed for exhaled breath. The analyzer proved to be accurate and precise in laboratory tests for simultaneous measurement of methanol and ethanol in water. Ethanol, in addition to normal contents of breath, was simultaneously analyzed in human experiments, and the results correlated well with blood samples. FT-IR method has a traceable calibration to physical properties of the analyte. The measured spectra can also be saved and analyzed later. Breath analysis with FT-IR is fast and easy, and no preparation of the sample is needed.

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.