Identification and characterization of hypoxia-induced genes in Carassius auratus blastulae embryonic cells using suppression subtractive hybridization

Organisms living in water are inevitably exposed to periods of hypoxia. Environmental hypoxia has been an important stressor having manifold effects on aquatic life. Many fish species have evolved behavioral, physiological, biochemical and molecular adaptations that enable them to cope with hypoxia. However, the molecular mechanisms of hypoxia tolerance in fish, remain unknown. in this study, we used suppression subtractive hybridization to examine the differential gene expression in CAB cells (Carassius auratus blastulae embryonic cells) exposed to hypoxia for 24 h. We isolated 2100 clones and identified 211 differentially expressed genes (e-value <= 5e-3; Identity > 45%). Among the genes whose expression is modified in cells, a vast majority involved in metabolism, signal transduction, cell defense, angiogenesis, cell growth and proliferation. Twelve genes encoding for ERO1-L, p53, CPO, HO-1, MKP2, PFK-2, cystatin B, GLUT1, BTG1, TGF beta 1, PGAM1, hypothetical protein F1508, were selected and identified to be hypoxia-induced using semi-quantitive RT-PCR and real-time PCR. Among the identified genes, two open reading frames (ORFs) encoding for CaBTG1 and Cacystatin B were obtained. The deduced amino acid sequence of CaBTG1 had 94.1%, 72.8%, 72.8%, 72.8%, 68.6% identity with that of DrBTG1, HsBTG1, BtBTG1, MmBTG1 and XIBTG1. Comparison of Cacystatin B with known cystatin B, the molecules exhibited 49.5 to 76.0% identity overall. These results may provide significant information for further understanding of the adaptive mechanism by which C. auratus responds to hypoxia. (c) 2008 Elsevier Inc. All rights reserved.

Effects of Naotan Pill on repair of neural cells and cognitive disorders in juvenile rats following hypoxia and ischemia

BACKGROUND: Hypoxia and ischemia induce neuronal damage, decreased neuronal numbers and synaptophysin levels, and deficits in learning and memory functions. Previous studies have shown that lycium barbarum polysaccharide, the most effective component of barbary wolfberry fruit, has protective effects on neural cells in hypoxia-ischemia. OBJECTIVE: To investigate the effects of Naotan Pill on glutamate-treated neural cells and on cognitive function in juvenile rats following hypoxia-ischemia. DESIGN, TIME AND SETTING: The randomized, controlled, in vivo study was performed at the Cell Laboratory of Lanzhou University, Lanzhou Institute of Modern Physics of Chinese Academy of Sciences, and Department of Traditional Chinese Medicine of Gansu Provincial Rehabilitation Center Hospital, China from December 2005 to August 2006. The cellular neurobiology, in vitro experiment was conducted at the Institute of Human Anatomy, Histology, Embryology and Neuroscience, School of Basic Medical Sciences, Lanzhou University, and Department of Traditional Chinese Medicine of Gansu Provincial Rehabilitation Center Hospital, China from March 2007 to January 2008. MATERIALS: Naotan Pill, composed of barbary wolfberry fruit, danshen root, grassleaf sweetflag rhizome, and glossy privet fruit, was prepared by Gansu Provincial Rehabilitation Center, China. Rabbit anti-synaptophysin, choline acetyl transferase polyclonal antibody, streptavidin-biotin complex kit and diaminobenzidine kit (Boster, Wuhan, China), as well as glutamate (Hualian, Shanghai, China) were used in this study. METHODS: Cortical neural cells were isolated from neonatal Wistar rats. Neural cell damage models were induced using glutamate, and administered Naotan Pill prior to and following damage. A total of 54 juvenile Wistar rats were equally and randomly assigned into model, Naotan Pill, and sham operation groups. The left common carotid artery was ligated, and then rat models of hypoxic-ischemic injury were assigned to the model and Naotan Pill groups. At 2 days following model induction, rats in the Naotan Pill group were administered Naotan Pill suspension for 21 days. In the model and sham operation groups, rats received an equal volume of saline. MAIN OUTCOME MEASURES: Neural cell morphology was observed using an inverted phase contrast microscope. Survival rate of neural cells was measured by MTT assay. Synaptophysin and choline acetyl transferase expression was observed in the hippocampal CA1 region of juvenile rats using immunohistochemistry. Cognitive function was tested by the Morris water maze. RESULTS: Pathological changes were detected in glutamate-treated neural cells. Neural cell morphology remained normal after Naotan Pill intervention. Absorbance and survival rate of neural cells were significantly greater following Naotan Pill intervention, compared to glutamate-treated neural cells (P < 0.05). Synaptophysin and choline acetyl transferase expression was lowest in the hippocampal CA1 region in the model group and highest in the sham operation group. Significant differences among groups were observed (P < 0.05). Escape latency and swimming distance were significantly longer in the model group compared to the Naotan Pill group (P < 0.05). CONCLUSION: Naotan Pill exhibited protective and repair effects on glutamate-treated neural cells. Naotan Pill upregulated synaptophysin and choline acetyl transferase expression in the hippocampus and improved cognitive function in rats following hypoxia-ischemia.

Cloning of cytoplasmic heat shock protein 90 (FcHSP90) from Fenneropenaeus chinensis and its expression response to heat shock and hypoxia

Heat shock protein 90 (HSP90) works as a multi-functional chaperone and is involved in the regulation of many essential cellular pathways. In this study, we have identified a full-length complementary DNA (cDNA) of HSP90 (FcHSP90) from Chinese shrimp Fenneropenaeus chinensis. FcHSP90 full-length cDNA comprised 2,552 bp, including a 2,181-bp open reading frame encoding 726 amino acids. Both homology analyses using alignment with previously identified HSP90 and a phylogeny tree indicated that FcHSP90 was a cytoplasmic HSP90. Real-time reverse transcription polymerase chain reaction analysis revealed that FcHSP90 was ubiquitously expressed in all the examined tissues but with highest levels in ovary of F. chinensis. FcHSP90 mRNA levels were sensitively induced by heat shock (from 25A degrees C to 35A degrees C) and reached the maximum at 6 h during heat shock treatment. Under hypoxia conditions, FcHSP90 mRNA levels, in both hemocytes and gill, were induced at 2 h and depressed at 8 h during hypoxia stress. The assessment of FcHSP90 mRNA levels under heat shock and hypoxia stresses indicated that the transcription of FcHSP90 was very sensitive to heat shock and hypoxia, so we deduced that FcHSP90 might play very important roles for shrimp to cope with environmental stress.

Effects of temperature, hypoxia, ammonia and nitrate on the bleaching among three coral species

Coral bleaching, which is defined as the loss of colour in corals due to the loss of their symbiotic algae (commonly called zooxanthellae) or pigments or both, is occurring globally at increasing rates, and its harm becomes more and more serious during these two decades. The significance of these bleaching events to the health of coral reef ecosystems is extreme, as bleached corals exhibited high mortality, reduced fecundity and productivity and increased susceptibility to diseases. This decreased coral fitness is easily to lead to reef degradation and ultimately to the breakdown of the coral reef ecosystems. Recently, the reasons leading to coral bleaching are thought to be as follows: too high or too low temperature, excess ultraviolet exposure, heavy metal pollution, cyanide poison and seasonal cycle. To date there has been little knowledge of whether mariculture can result in coral bleaching and which substance has the worst effect on corals. And no research was conducted on the effect of hypoxia on corals. To address these questions, effects of temperature, hypoxia, ammonia and nitrate on bleaching of three coral species were studied through examination of morphology and the measurement of the number of symbiotic algae of three coral species Acropora nobilis, Palythoa sp. and Alveopora verrilliana. Results showed that increase in temperature and decrease in dissolved oxygen could lead to increasing number of symbiotic algae and more serious bleaching. In addition, the concentration of 0.001 mmol/L ammonia or nitrate could increase significantly the expulsion of the symbiotic algae of the three coral species. Except for Acropora nobilis, the numbers of symbiotic algae of other two corals did not significantly increase with the increasing concentration of ammonia and nitrate. Furthermore, different hosts have different stress susceptibilities on coral bleaching.

Hypoxia-inducible factor 1 alpha cDNA cloning and its mRNA and protein tissue specific expression in domestic yak (Bos grunniens) from Qinghai-Tibetan plateau

Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated a-subunit and a constitutively expressed beta-subunit. How animals living on Qinghai-Tibetan plateau adapt to the extreme hypoxia environment is known indistinctly. In this study, the Qinghai yak which has been living at 3000-5000 m attitude for at least two millions of years was selected as the model of high hypoxia-tolerant adaptation species. The HIF-1 alpha ORFs (open reading frames) encoding for two isoforms of HIF-1 alpha have been cloned from the brain of the domestic yak. Its expression of HIF-1 alpha was analyzed at both mRNA and protein levels in various tissues. Both its HIF-1 alpha mRNA and protein are tissue specific expression. Its HIF-1 alpha protein's high expression in the brain, lung, and kidney showed us that HIF-1 alpha protein may play an important role in the adaptation to hypoxia environment. (c) 2006 Elsevier Inc. All rights reserved.

Effects of hypoxia on contents of essential elements in pika and rat heart

The effects of hypoxia on the levels of essential macroelements and trace elements (K, Na, Ca, Mg, Cu, Zn, Fe, and Mn) in the heart muscles of Wistar rats and plateau pikas (Ochotona curzoniae) were studied by atomic absorption spectrometry. Unlike the rat, the plateau pika is tolerant to hypoxia. The levels of K, Na, and the trace element Mn were not significantly changed in rat or pika hearts after exposure to hypoxia for 1, 10, or 25 d at simulated altitudes of 5000 and 7000 m. Other minerals (Ca, Mg, Cu, Zn, and Fe) were significantly affected by hypoxia and the levels followed different time-courses under different hypoxic regimes in these two animals. There were marked differences between the rat and pika in myocardial accumulation of essential elements such as Ca, which was increased to high levels in the rat but not affected in the pika. The results suggest that hypoxia affects animal physiological mechanisms by regulating the levels of essential elements.

Cloning of hypoxia-inducible factor 1 alpha cDNA from a high hypoxia tolerant mammal - plateau pika (Ochotona curzoniae)

Hypoxia-inducible factor I is a transcription factor composed of HIF-1alpha and HIF-1beta. It plays an important role in the signal transduction of cell response to hypoxia. Plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant and cold adaptation species livin only at 3000-5000m above sea level on the Qinghai-Tibet Plateau. In this study, HIF-1alpha cDNA of plateau pika was cloned and its expression in various tissues was studied. The results indicated that plateau pika HIF-1alpha cDNA was highly identical to those of the human (82%), bovine (89%), mouse (82%), and Norway rat (77%). The deduced amino acid sequence (822 bp) showed 90%, 92%, 86%, and 86% identities with those of the human, bovine, house mouse, and Norway rat, respectively. Northern blot analyses detected two isoforms named pLHIF-1alpha and pSHIF-1alpha. The HIF-1alpha mRNA was highly expressed in the brain and kidney, and much less in the heart, lung, liver, muscle, and spleen, which was quite different from the expression pattern of mouse mRNA. Meanwhile, a new variant of plateau pika HIF-1alpha mRNA was identified by RT-PCR and characterized. The deduced protein, composed of 536 amino acids, lacks a part of the oxygen-dependent degradation domain (ODD), both transactivation domains (TADs), and the nuclear localization signal motif (NLS). Our results suggest that HIF-1alpha may play an important role in the pika's adaptation to hypoxia, especially in brain and kidney, and pika HIF-1alpha function pattern may be different from that of mouse HIF-1alpha. Further-more, for the high ratio of HIF-1alpha homology among the animals, the HIF-1alpha gene may be a good phylogenetic performer in recovering the true phylogenetic relationships among taxa. (C) 2004 Elsevier Inc. All rights reserved.

Natural selection on EPAS1 (HIF2 alpha) associated with low hemoglobin concentration in Tibetan highlanders

By impairing both function and survival, the severe reduction in oxygen availability associated with high-altitude environments is likely to act as an agent of natural selection. We used genomic and candidate gene approaches to search for evidence of such genetic selection. First, a genome-wide allelic differentiation scan (GWADS) comparing indigenous highlanders of the Tibetan Plateau (3,200 3,500 m) with closely related lowland Han revealed a genome-wide significant divergence across eight SNPs located near EPAS1. This gene encodes the transcription factor HIF2 alpha, which stimulates production of red blood cells and thus increases the concentration of hemoglobin in blood. Second, in a separate cohort of Tibetans residing at 4,200 m, we identified 31 EPAS1 SNPs in high linkage disequilibrium that correlated significantly with hemoglobin concentration. The sex-adjusted hemoglobin concentration was, on average, 0.8 g/dL lower in the major allele homozygotes compared with the heterozygotes. These findings were replicated in a third cohort of Tibetans residing at 4,300 m. The alleles associating with lower hemoglobin concentrations were correlated with the signal from the GWADS study and were observed at greatly elevated frequencies in the Tibetan cohorts compared with the Han. High hemoglobin concentrations are a cardinal feature of chronic mountain sickness offering one plausible mechanism for selection. Alternatively, as EPAS1 is pleiotropic in its effects, selection may have operated on some other aspect of the phenotype. Whichever of these explanations is correct, the evidence for genetic selection at the EPAS1 locus from the GWADS study is supported by the replicated studies associating function with the allelic variants.

Toxicological Research on Environmental Endocrine Disruptors

Most evidence in terms of endocrine dsiruptors (EDs) mainly originates from studies on reproductive organs. However, in veterbrates, the ability to attain reproductive and development success relays on the intact organization of a complex endocrine system. Disturbances in the regulation of the key hormones and receptors functioning along this system may cause detrimental effects on reproduction and development. Here we reviewed recent studies of EDs on endocrine system. EDs may act on key hormones and receptors along with the hypothalarnic-pituitary-gonald (HPG) axis and lead to reproductive failure. Thyroid disruption may be caused at different levels, for example, the synthesis, transport, binding and cellular uptake along with the hypothalamic-pituitary-thyroid (HPT) axis. Knowledge of model of action EDs is largely via receptors-mediated pathway and alternatively may affect on steroid hormone synthesis. Aquatic hypoxia can influence fish reproduction and thus it is also an endocrine disruptor. Molecular techniques, such as toxicomics, transgenic fish will be employed as powerful tools for environmental EDs risk assessment, as well as in elucidating mechanisms of model action.

U19/Eaf2 Binds to and Stabilizes von Hippel-Lindau Protein

Studies have firmly established a key regulatory role for the tumor suppressor pVHL in the regulation of the vascular system and normal spermatogenesis. Here, we report that knockout of the newly identified tumor suppressor U19/Eaf2 also caused vascular system abnormalities and aspermatogenesis, suggesting a potential link between U19/Eaf2 and pVHL. Coimmunoprecipitation and in vitro binding assays showed an association between U19/Eaf2 and pVHL, whereas deletion mutagenesis revealed the requirement of the NH2 terminus of U19/Eaf2 and both the alpha and beta domains of pVHL for this binding. U19/Eaf2 stabilizes pVHL, as shown by protein stability and pulse-chase studies. Testes and mouse embryonic fibroblasts (MEF) derived from U19/Eaf2 knockout mice expressed reduced levels of pVHL, indicating that full in vivo expression of pVHL indeed requires U19/Eaf2. As expected, U19/Eaf2 knockout MEF cells exhibited an increased level and activity of hypoxia-inducible factor 1 alpha (HIF1 alpha), a protein typically regulated via a pVHL-mediated degradation pathway. Furthermore, angiogenesis in a Matrigel plug assay was significantly increased in U19/Eaf2 knockout mice. The above observations argue that U19/Eaf2 can modulate HIF1 alpha and angiogenesis, possibly via direct binding and stabilization of pVHL. [Cancer Res 2009;69(6):2599-606]

Inductive transcription and protective role of fish heme oxygenase-1 under hypoxic stress

Heme oxygenase-1 is the rate-limiting enzyme in the degradation of heme into biliverdin, carbon monoxide and free divalent iron. In this study, we cloned heme oxygenase isoform 1 (CaHO-1) from a hypoxia-tolerant teleost fish Carassius auratus. The full-length cDNA of CaHO-1 is 1247 bp and encodes a protein of 272 amino acids. RT-PCR and real-time PCR analysis indicated that CaHO-1 was predominantly transcribed in posterior kidney, head kidney, gill and intestine, and induction of gene transcription was observed predominantly in posterior kidney under hypoxic stress. Moreover, the hypoxia-induced transcription was confirmed in goldfish larvae and in in vitro cultured CAB cells. Fluorescence of the HO-1-GFP fusion protein revealed a cytoplasmic and plasma membrane localization, which was consistent with the putative transmembrane structure. Subsequently, we established a stably transfected CAB/pcDNA3.1-HO-1 cell line and a control CAB/pcDNA3.1 cell line, and found that the number of dead cells was obviously reduced in the pcDNA3.1-HO-1-transfected group following 4 days of hypoxic (1% O-2) treatment in comparison with numerous detached dead cells in the control pcDNA3.1-transfected cells. Furthermore, a significant cell viability difference between the two kinds of transfected cells during hypoxia-reoxygenation was revealed. Therefore, the data suggest that fish HO-1 might play a significant protective role in cells in response to hypoxic stress.

Changes in nutrient structure of river-dominated coastal waters: stoichiometric nutrient balance and its consequences

We present an analysis of extensive nutrient data sets from two river-dominated coastal ecosystems, the northern Adriatic Sea and the northern Gulf of Mexico, demonstrating significant changes in surface nutrient ratios over a period of 30 years. The silicon:nitrogen ratios have decreased, indicating increased potential for silicon limitation. The nitrogen:phosphorus and the silicon:phosphorus ratios have also changed substantially, and the coastal nutrient structures have become more balanced and potentially less limiting for phytoplankton growth. It is likely that net phytoplankton productivity increased under these conditions and was accompanied by increasing bottom water hypoxia and major changes in community species composition. These findings support the hypothesis that increasing coastal eutrophication to date may be associated with stoichiometric nutrient balance, due to increasing potential for silicon limitation and decreasing potential for nitrogen and phosphorus limitation. On a worldwide basis, coastal ecosystems adjacent to rivers influenced by anthropogenic nutrient loads may experience similar alterations.

Comparative proteomic profiles of the hepatopancreas in Fenneropenaeus chinensis response to hypoxic stress

Hypoxia, as one suboptimal environmental condition, can affect the physiological state of shrimp during pond aquaculture. To better understand the mechanism of response to hypoxic stress in Chinese shrimp Fenneropenaeus chinensis, proteome research approach was utilized. Differentially expressed proteins of hepatopancreas in adult Chinese shrimp between the control and hypoxia-stressed groups were screened. By 2-DE analysis, 67 spots showed obvious changes after hypoxia. Using LC-ESI-MS/MS, 51 spots representing 33 proteins were identified including preamylase, arginine kinase, phosphopyruvate hydratase, citrate synthase, ATP synthase alpha subunit, chymotrypsin BI, chitinase, ferritin, C-type lectin receptors, transketolase, formylglutathione hydrolase, formyltetrahydrofolate dehydrogenase, aldehyde dehydrogenase, glutathione peroxidase, cytosolic manganese superoxide dismutase, protein disulfide isomerase, beta-actin, oncoprotem nm23, crustacyanin-Cl and so on. These proteins could be functionally classified into several groups such as proteins related to energy production, metabolism-related proteins, immune-related proteins, antioxidant proteins, chaperones, cytoskeleton proteins and ungrouped proteins. The transcription levels of ten selected genes encode the identified proteins were analyzed by real-time PCR at different sampling times of hypoxia. This study is the first analysis of differentially expressed proteins in the hepatopancreas of shrimp after hypoxia and provides a new insight for further study in hypoxic stress response of shrimp at the protein level.

长江口邻近海域缺氧区营养盐及痕量金属的生物地球化学过程研究

长江口邻近海域夏季底层水体存在世界上面积最大的缺氧带。这为研究河口区氧化还原敏感元素的生物地球化学过程提供了天然的实验场所。为了理解长江河口缺氧带存在的生物地球化学过程，本文以长江口邻近海域缺氧带上覆水体、沉积物及间隙水为研究对象，并对其营养盐及重金属进行研究，目的在于揭示长江口邻近海域缺氧带可能存在的生物地球化学过程，为理解长江河口区的物质循环提供科学证据。通过研究得出如下结论：（1）在水体中盐度是影响营养盐分布的主要因素，在高浊度带存在营养盐释放的现象；（2）底层水存在两种显著不同的生物地球化学过程：当盐度<33 PSU时，营养盐与AOU之间为负相关，影响底层海水营养盐分布的主要因素可能为流场；而当盐度>33 PSU时，营养盐与AOU之间存在正相关，影响营养盐分布的主要因素为颗粒有机质的矿化；（3）溶解态痕量金属存在保守型（Mo、U、Ni）和混合型(Fe、Mn、Cu、Pb、Zn)的分布规律，盐度、浮游生物及溶解氧都是影响溶解态痕量金属分布的重要因素；剔除浊度大于20 NTU的数据，分配系数与浊度为显著正相关；（4）间隙水Fe、Mn剖面表明，长江河口表层沉积物存在剧烈的Fe、Mn还原现象；成岩模型模拟结果显示间隙水Mn的模拟剖面与实际观测剖面吻合较好。间隙水U剖面显示，控制U分布的主要因素与Fe有关，但是其它因素如有机质含量对U、Mo的分布也有影响；（5）间隙水营养盐剖面显示近岸与远岸存在两种不同的生物地球化学过程：远岸海域，间隙水营养盐部面主要受早期成岩的控制；而在近岸海域其它过程如氨化及吸附可能是控制间隙水氮剖面分布的主要因素；模拟剖面与NH4+的实测剖面趋于一致，但是同时暗示表层沉积物受到生物扰动；通量计算结果显示，Si、N、P分别占浮游生物每日所需要营养盐数量的15%、10%及0.1%；（6）对痕量金属的黄铁矿化程度进行了分析，发现长江河口痕量金属的黄铁矿化程度异常低，黄铁矿不是影响痕量金属分布的主要因素；导致低黄铁矿化度的原因与长江河口高沉积速率、贫硫、低有机质有关。

Ocean urea fertilization for carbon credits poses high ecological risks

The proposed plan for enrichment of the Sulu Sea, Philippines, a region of rich marine biodiversity, with thousands of tonnes of urea in order to stimulate algal blooms and sequester carbon is flawed for multiple reasons. Urea is preferentially used as a nitrogen source by some cyanobacteria and dinoflagellates, many of which are neutrally or positively buoyant. Biological pumps to the deep sea are classically leaky, and the inefficient burial of new biomass makes the estimation of a net loss of carbon from the atmosphere questionable at best. The potential for growth of toxic dinoflagellates is also high, as many grow well on urea and some even increase their toxicity when grown on urea. Many toxic dinoflagellates form cysts which can settle to the sediment and germinate in subsequent years, forming new blooms even without further fertilization. If large-scale blooms do occur, it is likely that they will contribute to hypoxia in the bottom waters upon decomposition. Lastly, urea production requires fossil fuel usage, further limiting the potential for net carbon sequestration. The environmental and economic impacts are potentially great and need to be rigorously assessed. (C) 2008 Elsevier Ltd. All rights reserved.