Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp, Ctenopharyngodon idellus

BACKGROUND: Hypoxia-inducible factors (HIFs) are involved in adaptive and survival responses to hypoxic stress in mammals. In fish, very little is known about the functions of HIFs. RESULTS: We have cloned and characterized two distinct HIF-alpha cDNAs – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp. The deduced gcHIF-1alpha protein is highly similar to the HIF-1alphas (57–68%) from various vertebrate species, while gcHIF-4alpha is a novel isoform, and shows an equivalent degree of amino acid identity (41–47%) to the HIF-1alpha, HIF-2alpha and HIF-3alpha proteins so far described. Parsimony analysis indicated that gcHIF-4alpha is most closely related to the HIF-3alpha proteins. Northern blot analysis showed that mRNA levels of gcHIF-1alpha and gcHIF-4alpha differ substantially under normoxic and hypoxic conditions, while Western blot studies demonstrated that the endogenous protein levels for both gcHIF-1alpha and gcHIF-4alpha are similarly responsive to hypoxia. Our findings suggest that both gcHIF-1alpha and gcHIF-4alpha are differentially regulated at the transcriptional and translational levels. HRE-luciferase reporter assays show that both proteins function as transcription activators and play distinct roles in modulating the hypoxic response in grass carp. CONCLUSION: There are at least two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – in the hypoxia-tolerant grass carp, which are differentially expressed and regulated in different fish organs in response to hypoxic stress. Overall, the results suggest that unique molecular mechanisms operate through these two HIF-alpha isoforms, which underpin the hypoxic response in the hypoxia-tolerant grass carp

Hypoxia induces telomerase reverse transcriptase (TERT) gene expression in non-tumor fish tissues in vivo: the marine medaka (Oryzias melastigma) model

BACKGROUND: Current understanding on the relationships between hypoxia, hypoxia-inducible factor-1 (HIF-1) and telomerase reverse transcriptase (TERT) gene expression are largely based on in vitro studies in human cancer cells. Although several reports demonstrated HIF-1- mediated upregulation of the human TERT gene under hypoxia, conflicting findings have also been reported. Thus far, it remains uncertain whether these findings can be directly extrapolated to non-tumor tissues in other whole animal systems in vivo. While fish often encounter environmental hypoxia, the in vivo regulation of TERT by hypoxia in non-neoplastic tissues of fish remains virtually unknown. RESULTS: The adult marine medaka (Oryzias melastigma) was employed as a model fish in this study. We have cloned and characterized a 3261-bp full-length TERT cDNA, omTERT, which encodes a protein of 1086 amino acids. It contains all of the functional motifs that are conserved in other vertebrate TERTs. Motif E is the most highly conserved showing 90.9–100% overall identity among the fish TERTs and 63.6% overall identity among vertebrates. Analysis of the 5'-flanking sequence of the omTERT gene identified two HRE (hypoxia-responsive element; nt. – 283 and – 892) cores. Overexpression of the HIF-1α induced omTERT promoter activity as demonstrated using transient transfection assays. The omTERT gene is ubiquitously expressed in fish under normoxia, albeit at varying levels, where highest expression was observed in gonads and the lowest in liver. In vivo expression of omTERT was significantly upregulated in testis and liver in response to hypoxia (at 96 h and 48 h, respectively), where concomitant induction of the omHIF-1α and erythropoietin (omEpo) genes was also observed. In situ hybridization analysis showed that hypoxic induction of omTERT mRNA was clearly evident in hepatocytes in the caudal region of liver and in spermatogonia-containing cysts in testis. CONCLUSION: This study demonstrates for the first time, hypoxic regulation of TERT expression in vivo in a whole fish system. Our findings support the notion that hypoxia upregulates omTERT expression via omHIF-1 in non-neoplastic fish liver and testis in vivo. Overall, the structure and regulation of the TERT gene is highly conserved in vertebrates from fish to human

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp, -1

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp, "</p><p>BMC Molecular Biology 2006;7():15-15.</p><p>Published online 20 Apr 2006</p><p>PMCID:PMC1473195.</p><p></p>man HIF-1α; fHIF2α, HIF-2α; hHIF2α, human HIF-2α; hHIF3α, human HIF-3α; gcHIF4α, grass carp HIF-4α. The number on the left of each row denotes the amino acid position. Identical amino acids shared by most of the HIF-α s are shaded in black while similar amino acids are shaded in grey. Dashes (--) indicate gaps inserted for improved alignment. Domains that are typical characteristic of HIF-α proteins are marked on the alignment according to the amino acid positions in human HIF-1α. The two conserved proline residues within the ODD domain are indicated by open arrows (⇩). The closed arrow () indicates the asparagine residue (Asn-803) in C-TAD which controls HIF-1 binding to CBP/p300

Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp, -0

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp, "</p><p>BMC Molecular Biology 2006;7():15-15.</p><p>Published online 20 Apr 2006</p><p>PMCID:PMC1473195.</p><p></p>ions delineating the different domains are indicated and include: bHLH, basic helix-loop-helix domain; PASA/B, Per-ARNT-Sim A/B domains; PAC, domain C-terminal to PAS motifs; ODD, oxygen dependent degradation domain; N-TAD, N-terminal transactivation domain; and C-TAD, C-terminal transactivation domain

Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp, -2

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp, "</p><p>BMC Molecular Biology 2006;7():15-15.</p><p>Published online 20 Apr 2006</p><p>PMCID:PMC1473195.</p><p></p>quences by maximum parsimony (PROTPARS) using the PHYLIP package version 3.57 c [25]. The bootstrap support (SEQBOOT program, PHYLIP package) for each branch (1000 replications) is shown. The standing of the abbreviations and the GenBank/EMBL/Swissprot accession number of the bHLH-PAS-A/B sequences used are provided in