HIF-1␣ is the regulated subunit of the HIF-1 transcription factor, which induces transcription of a number of genes involved in the cellular response to hypoxia. The HIF-1␣ protein is rapidly degraded in cells supplied with adequate oxygen but is stabilized in hypoxic cells. Using polysome profile analysis, we found that translation of HIF-1␣ mRNA in NIH3T3 cells is spared the general reduction in translation rate that occurs during hypoxia. To assess whether the 5ЈUTR of the HIF-1␣ mRNA contains an internal ribosome entry site (IRES), we constructed a dicistronic reporter with the HIF-1␣ 5ЈUTR inserted between two reporter coding regions. We found that the HIF-1␣ 5ЈUTR promoted translation of the downstream reporter, indicating the presence of an IRES. The IRES had activity comparable to that of the well-characterized c-myc IRES. IRES activity was not affected by hypoxic conditions that caused a reduction in cap-dependent translation, and IRES activity was less affected by serum-starvation than was cap-dependent translation. These data indicate that the presence of an IRES in the HIF-1␣ 5ЈUTR allows translation to be maintained under conditions that are inhibitory to cap-dependent translation.

Transcription factors of the bHLH-PAS protein family are important regulators of developmental processes such as neurogenesis and tracheal development in invertebrates. Recently a bHLH-PAS protein, named trachealess (trl) was identified as a master regulator of tracheogenesis. Hypoxia-inducible factor, HIF-1 alpha, is a vertebrate relative of trl which is likely to be involved in growth of blood vessels by the induction of vascular endothelial growth factor (VEGF) in response to hypoxia. In the present study we describe mRNA cloning and mRNA expression pattern of mouse HIF-related factor (HRF), a novel close relative of HIF-1 alpha which is expressed most prominently in brain capillary endothelial cells and other blood vessels as well as in bronchial epithelium in the embryo and the adult. In addition, smooth muscle cells of the uterus, neurons, brown adipose tissue and various epithelial tissues express HRF mRNA as well. High expression levels of HRF mRNA in embryonic choroid plexus and kidney glomeruli, places where VEGF is highly expressed, suggest a role of this factor in VEGF gene activation similar to that of HIF-1 alpha. Given the similarity between morphogenesis of the tracheal system and the vertebrate vascular system, the expression pattern of HRF in the vasculature and the bronchial tree raises the possibility that this family of transcription factors may be involved in tubulogenesis.

Interactions between Ets family members and a vari-

Background and purposeTourniquet application is still a common practice in total knee arthroplasty (TKA) surgery despite being associated with several adverse effects. We evaluated the effects of tourniquet use on functional and clinical outcome and on knee range of motion (ROM).Patients and methods70 patients who underwent TKA were randomized into a tourniquet group (n = 35) and a non-tourniquet group (n = 35). All operations were performed by the same surgeon and follow-up was for 1 year. Primary outcomes were functional and clinical outcomes, as evaluated by KOOS and knee ROM. Secondary outcomes were intraoperative blood loss, surgical time and visibility, postoperative pain, analgesic consumption, and transfusion requirements.ResultsPatients in the non-tourniquet group showed a better outcome in all KOOS subscores and better early knee ROM from surgery to week 8. No difference was detected at the 6- and 12-month follow-ups. Postoperative pain and analgesic consumption were less when a tourniquet was not used. Surgical time and visibility were similar between groups. Intraoperative blood loss was greater when not using a tourniquet, but no postoperative transfusions were required.InterpretationThis study shows that TKA without the use of a tourniquet results in faster recovery in terms of better functional outcome and improved knee ROM. Furthermore, reduced pain and analgesic use were registered and no intraoperative difficulties were encountered.

This study identifies a set of miRNAs deregulated in CIS/RRMS and reconfirms the previously reported underexpression of hsa-miR-20a-5p in MS. hsa-miR-20a-5p and the other validated miRNAs may represent promising candidates for future evaluation as biomarkers for MS and could be of relevance in the pathophysiology of this disease.

Level I, therapeutic study.

BackgroundGenes of the Raf family encode kinases that are regulated by Ras and mediate cellular responses to growth signals. Recently, it was shown that activating mutations of BRaf are found with high frequency in human melanomas. The Ras family member most often mutated in melanoma is NRas.MethodsThe constitutive activation of the Ras/Raf signaling pathway suggests an impact on the clinical course of the tumor. To address this notion, we analyzed tumor DNA from 114 primary cutaneous melanomas and of 86 metastatic lesions obtained from 174 patients for mutations in BRaf (exons 15 and 11) and NRas (exons 1 and 2) by direct sequencing of PCR products and correlated these results with the clinical course.ResultsIn 57.5% of the tumors either BRaf or NRas were mutated with a higher incidence in metastatic (66.3%) than in primary lesions (50.9%). Although the majority of BRaf mutations affected codon 599, almost 15% of mutations at this position were different from the well-described exchange from valine to glutamic acid. These mutations (V599R and V599K) also displayed increased kinase and transforming activity. Surprisingly, the additional BRaf variants D593V, G465R and G465E showed a complete loss of activity in the in vitro kinase assay; however, cells overexpressing these mutants displayed increased Erk phosphorylation. The correlation of mutational status and clinical course revealed that the presence of BRaf/NRas mutations in primary tumors did not negatively impact progression free or overall survival. In contrast, however, for metastatic lesions the presence of BRAF/NRAS mutations was associated with a significantly poorer prognosis, i.e. a shortened survival.ConclusionWe demonstrate a high – albeit lower than initially anticipated – frequency of activating BRaf mutations in melanoma in the largest series of directly analyzed tumors reported to date. Notably, the clinical course of patients harboring activating BRaf mutations in metastatic melanoma was significantly affected by the presence of a constitutive BRaf activation in these.

The vascular endothelial growth factor (VEGF) receptor-2 (Flk-1) is the first endothelial receptor tyrosine kinase to be expressed in angioblast precursors, and its function is essential for the differentiation of endothelial cells and hematopoietic precursors. We have identified cis-acting regulatory elements of the murineFlk-1 gene that mediate endothelium-specific expression of a LacZ reporter gene in transgenic mice. Sequences within the 5′-flanking region of the Flk-1 gene, in combination with sequences located in the first intron, specifically targeted transgene expression to angioblasts and endothelial cells of transgenic mice. The intronic regulatory sequences functioned as an autonomous endothelium-specific enhancer. Sequences of the 5′-flanking region contributed to a strong, uniform, and reproducible transgene expression and were stimulated by the transcription factor HIF-2. The Flk-1 gene regulatory elements described in this study should allow the elucidation of the molecular mechanisms involved in endothelial cell differentiation and angiogenesis.