Anoxic Levels Research Articles

Abstract IA007: Targeting the hypoxia-induced DDR to improve radiotherapy response

Abstract Regions of hypoxia (insufficient oxygen) occur to some extent in most if not all solid tumors. Within hypoxic regions the levels of oxygen are heterogeneous and can reach near anoxic levels, often referred to as radiobiological hypoxia. Cells experiencing radiobiological hypoxia are significantly more resistant to radiation-induced DNA damage and negatively impact radiotherapy response. A number of clinical studies have demonstrated that hypoxia is a significant indicator of poor patient response. Radiobiological hypoxia, unlike milder hypoxic conditions, leads to a robust DNA damage response although interestingly, this occurs in the absence of detectable DNA damage. Instead, the hypoxia-induced DDR has been attributed to the accumulation of replication stress. Previously, we demonstrated that hypoxia-induced replication stress originated from reduced nucleotide availability due to the oxygen-dependency of ribonucleotide reductase. More, recently we have implicated the accumulation of R-loops in contributing to not only the hypoxia-induced DDR but also transcriptional stress. Numerous factors play key roles in maintaining R-loops and preventing the accumulation of DNA damage including the DNA/RNA helicase SETX. Using SETX has an exemplar, we have demonstrated that targeting R-loop factors leads to increased DNA damage in hypoxic conditions. Currently, we are testing the hypothesis that hypoxia specific R-loop factors are potential therapeutic targets which offer the possibility of increasing the radiosensitivity of hypoxic cells. Citation Format: Ester Hammond. Targeting the hypoxia-induced DDR to improve radiotherapy response [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA007.

Astronomical calibration of the OAE1b from the Col de Pré-Guittard section (Aptian–Albian), Vocontian Basin, France

A high-resolution spectral analysis was performed on magnetic susceptibility (MS) in the Col de Pré-Guittard section (CPG), Vocontian Basin, France to provide a new astronomical time scale of the Aptian–Albian transition and to explore the link between Milankovitch cycles and the occurrence of four laminated black shales events, namely Jacob, Kilian, Paquier and Leenhardt constituting the OAE 1b. The spectral analysis performed on the CPG section records the imprint of a strong predominance of the precession, and 100-kyr and 405-kyr eccentricity cycles. Based on the number of 405-kyr eccentricity cycles, the duration of the interval encompassing the Jacob to Leenhardt levels is calculated at 4.03 myr. The duration between Jacob, Kilian, Paquier and Leenhardt Events are respectively calculated at 1.55 myr, 1.62 myr, and 0.93 myr. The duration calculated for the occurrence of these events is not related to the Milankovitch cycles. However, these events are recorded near local maxima of the 405-kyr filter. Our results show that the 405-kyr eccentricity impacted the recurrence of anoxic levels in the Vocontian Basin, although additional factors, such as the emplacement of flood basalts or oceanic basaltic plateaus have contributed to the development of larger scale marine anoxia.

Mobility of arsenic and vanadium in waterlogged calcareous soils due to addition of zeolite and manganese oxide amendments.

Addition of manganese(IV) oxides (MnO2 ) and zeolite can affect the mobility of As and V in soils due to geochemical changes that have not been studied well in calcareous, flooded soils. This study evaluated the mobility of As and V in flooded soils surface-amended with MnO2 or zeolite. A simulated summer flooding study was conducted for 8 weeks using intact soil columns from four calcareous soils. Redox potential was measured in soils, whereas pH, major cations, and As and V concentrations were measured biweekly in pore water and floodwater. Aqueous As and V species were modeled at 0, 4, and 8 weeks after flooding (WAF) using Visual MINTEQ modeling software with input parameters of redox potential, temperature, pH, total alkalinity, and concentrations of major cations and anions. Aqueous As concentrations were below the critical thresholds (<100μgL-1 ), whereas aqueous V concentrations exceeded the threshold for sensitive aquatic species (2-80μgL-1 ). MnO2 -amended soils were reduced to sub-oxic levels, whereas zeolite-amended and unamended soils were reduced to anoxic levels by 8 WAF. MnO2 decreased As and V mobilities, whereas zeolite had no effect on As but increased V mobility, compared to unamended soils. Arsenic mobility increased under anoxic conditions, and V mobility increased under oxic and alkaline pH conditions. Conversion of As(V) to As(III) and V(V) to V(IV) was regulated by MnO2 in flooded soils. MnO2 can be used as an amendment in immobilizing As and V, whereas the use of zeolite in flooded calcareous soils should be done cautiously.

Latest Hauterivian foraminiferal and calcareous nannofossil assemblages and bioevents from the Pseudothurmannia beds (Central Western Carpathians, Slovakia): A prelude to the Faraoni event

A diverse and abundant assemblage of deep-water agglutinated foraminifera (DWAF) has been recovered from an uppermost Hauterivian ammonite-rich horizon (Pseudothurmannia ohmi Biozone) from the Central Western Carpathians. The section records the last occurrence of the calcareous nannofossil species Litraphidites bollii (Thierstein) preceding the Faraoni anoxic level. Various morphogroups of agglutinated foraminifera show changes in response to trophic regime shift and decreasing oxygenation of the bottom and pore waters in a relatively short time-series. The DWAF data were compared with calcareous nannofossil assemblages and foraminifera analysed from thin sections (calcareous benthic and planktonic foraminifera). Four ecozones were identified, each with a specific taxonomic composition of foraminiferal assemblages. The zone-to-zone changes include fluctuations in the richness and relative abundance of morphogroups and planktonic foraminifera, and dwarfism in certain groups of foraminifera.

Effects of oxygen on the response of mitochondria to X-irradiation and reactive oxygen species-mediated fibroblast activation

Purpose In living organisms, sensitivity to radiation increases in the presence of oxygen (O2) compared with that under anoxic or hypoxic conditions. Here, we investigated whether O2 concentration affected the response of mitochondria to X-rays radiation, which is associated with tumor microenvironment formation via fibroblast activation in radiation-related tumors. Materials and methods O2 concentrations were controlled at <5% (internal environmental oxygen condition) or anoxic levels during culture of normal human diploid lung fibroblasts TIG-3 and MRC-5. Protein expression associated with the response of mitochondria to radiation was assessed using immunostaining or western blotting. Results Induction of DNA damage (marker: γ-H2A histone family member X) and mitochondrial signaling (AMP-activated protein kinase), suppression of mitochondrial metabolic activity, and generation of reactive oxygen species occurred with radiation in cells cultured under 5% and 20% O2 conditions. However, reducing O2 concentration mitigated the effects of radiation on cell growth, mitochondrial damage (parkin), induction of antioxidant responses (nuclear factor E2-related factor 2), and fibroblast activation (α-smooth muscle actin). Radiation did not affect the markers used in this study in the absence of O2. Conclusion O2 concentration affected the response of mitochondria to radiation and reactive oxygen species-mediated fibroblast activation. Higher O2 concentrations enhanced the effects of radiation on mitochondria in human fibroblasts. In vitro studies may overestimate in vivo radiation effects due to high O2 concentrations.

Tumor Hypoxia as a Barrier in Cancer Therapy: Why Levels Matter.

Simple SummaryHypoxia is a common feature of solid tumors and associated with poor outcome in most cancer types and treatment modalities, including radiotherapy, chemotherapy, surgery and, most likely, immunotherapy. Emerging strategies, such as proton therapy and combination therapies with radiation and hypoxia targeted drugs, provide new opportunities to overcome the hypoxia barrier and improve therapeutic outcome. Hypoxia is heterogeneously distributed both between and within tumors and shows large variations across patients not only in prevalence, but importantly, also in level. To best exploit the emerging strategies, a better understanding of how individual hypoxia levels from mild to severe affect tumor biology is vital. Here, we discuss our current knowledge on this topic and how we should proceed to gain more insight into the field.Hypoxia arises in tumor regions with insufficient oxygen supply and is a major barrier in cancer treatment. The distribution of hypoxia levels is highly heterogeneous, ranging from mild, almost non-hypoxic, to severe and anoxic levels. The individual hypoxia levels induce a variety of biological responses that impair the treatment effect. A stronger focus on hypoxia levels rather than the absence or presence of hypoxia in our investigations will help development of improved strategies to treat patients with hypoxic tumors. Current knowledge on how hypoxia levels are sensed by cancer cells and mediate cellular responses that promote treatment resistance is comprehensive. Recently, it has become evident that hypoxia also has an important, more unexplored role in the interaction between cancer cells, stroma and immune cells, influencing the composition and structure of the tumor microenvironment. Establishment of how such processes depend on the hypoxia level requires more advanced tumor models and methodology. In this review, we describe promising model systems and tools for investigations of hypoxia levels in tumors. We further present current knowledge and emerging research on cellular responses to individual levels, and discuss their impact in novel therapeutic approaches to overcome the hypoxia barrier.

Abstract 2672: Localized delivery of granulocyte-macrophage colony-stimulating factor to alleviate tumor hypoxia and immunosuppression in breast cancer

Abstract The purpose of this study is to investigate localized delivery of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential therapeutic modality in breast cancer to alleviate tumor hypoxia and immunosuppression. A therapeutic that has the potential to target and reverse these factors of the tumor microenvironment (TME) would aid in reducing tumor aggressiveness, immune escape, metastasis, and importantly, could potentially be administered as a pre-treatment to reduce resistance to standard therapies such as chemotherapy and radiation. We have previously reported that 100 ng of intratumorally delivered recombinant GM-CSF 1) altered the macrophage profile from immunosuppressive M2-like to immunosupportive M1-like and 2) inhibited angiogenesis and induced near anoxic levels in the tumor due to the dose-dependent production of soluble VEGFR-1 from tumor macrophages that sequestered VEGF. Our current focus is to determine if a dosage window of GM-CSF exists that may normalize leaky and dysfunctional tumor vasculature and alleviate hypoxia in tumors by lesser sVEGFR-1 production, and simultaneously convert the immune cell profile from immunosuppressive to immunosupportive to promote cytotoxic T cell killing. After intratumoral injections with either GM-CSF or saline 3x per week for 2.5 weeks in an orthotopic PyMT murine breast cancer model, we utilized a soluble multifunctional trityl probe and Electron Paramagnetic Resonance Spectrometry to measure intratumoral oxygen and pH in vivo in real time. Our findings demonstrate that intratumoral injections of 15 ng GM-CSF rescued tumor oxygen by ~16% and raised pH ~0.2 units, while 5 ng enhanced oxygen even further by ~40% and raised pH ~0.4 units. Additionally, tumor-associated macrophages (TAMs) that were isolated from Stage IV MMTV-PyMT tumors and treated with 5 ng/ml GM-CSF 3x per week for 2 weeks demonstrated a reduction in IL-10 expression as determined by intracellular staining and flow cytometry. Further, as a result of these data, we have also formulated and characterized GM-CSF-containing PLGA/PEG-PLGA nanoparticles in vitro with average sizes under 200 nm for the purpose of systemic but localized delivery of dose-optimized recombinant GM-CSF to tumors. Nanoparticles yielded an entrapment efficiency of 87.9% +/- 4.2 S.D. and release of functional GM-CSF in vitro. Overall, we have demonstrated the potential of locally delivered recombinant GM-CSF to alleviate tumor hypoxia and acidity in vivo and reverse immunosuppressive TAM phenotype ex vivo, and we have formulated nanoparticles encapsulating recombinant murine GM-CSF as another modality which will be utilized for localized delivery of GM-CSF to breast tumors to achieve similar effects. Citation Format: Nicole Mihalik, Andrey Bobko, Valery Khramtsov, Benoit Driesschaert, Tim D. Eubank. Localized delivery of granulocyte-macrophage colony-stimulating factor to alleviate tumor hypoxia and immunosuppression in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2672.

Reducing Emissions From Degraded Floodplain Wetlands

Freshwater wetlands are significant carbon sinks, however, altering a wetland’s hydrology can reduce its ability to sequester carbon and may lead to the release of previously stored soil carbon. Rehabilitating a wetland’s water table has the potential to restore the natural process of wetland soil carbon sequestration and storage. Further, little is known about the role of microbial communities that mediate carbon cycling during wetland rehabilitation practices. Here, we examined the carbon emissions and microbial community diversity during a wetland rehabilitation process known as ‘environmental watering’ (rewetting) in an Australian, semi-arid freshwater floodplain wetland. By monitoring carbon dioxide (CO2) and methane (CH4) emissions during dry and wet phases of an environmental watering event, we determined that adding water to a degraded semi-arid floodplain wetland reduces carbon emissions by 28-84%. The watering event increased anoxic levels and plant growth in the aquatic zone of the wetland, which may correlate with lower carbon emissions during and after environmental watering due to lower anaerobic microbial decomposition processes and higher CO2 sequestration by vegetation. During the watering event, areas with higher inundation had lower CO2 emissions (5.15 ± 2.50 g CO2 m-2 d-1) compared to fringe areas surrounding the wetland (11.89 ± 4.25 g CO2 m-2 d-1). CH4 flux was inversely correlated with CO2 emissions during inundation periods, showing a 38% (0.013 ± 0.061 g CO2-e m-2 d-1) increase when water was present in the wetland. During the dry phases of environmental watering, there was CH4 uptake within the fringe and aquatic zones (-0.013 ± 0.063 g CO2-e m-2 d-1). A clear succession of soil microbial community was observed during the dry-wet phases of the environmental watering process. This suggests that wetland hydrology plays a large role in the microbial community structure of these wetland ecosystems, and is consequently linked to CO2 and CH4 emissions. Overall, the total carbon emissions (CO2 + CH4) were reduced within the wetland during and after the environmental watering event, due to increasing vegetative growth and subsequent CO2 sequestration. We recommend environmental watering practices in this degraded arid wetland ecosystem to improve conditions for wetland carbon sequestration and storage.

Challenges to DNA replication in hypoxic conditions.

The term hypoxia refers to any condition where insufficient oxygen is available and therefore encompasses a range of actual oxygen concentrations. The regions of tumours adjacent to necrotic areas are at almost anoxic levels and are known to be extremely therapy resistant (radiobiological hypoxia). The biological response to radiobiological hypoxia includes the rapid accumulation of replication stress and subsequent DNA damage response, including both ATR- and ATM-mediated signalling, despite the absence of detectable DNA damage. The causes and consequences of hypoxia-induced replication stress will be discussed.

Assessing Water Quality of Murum Reservoir Halfway Through Impoundment

This paper reports the water quality of Murum Hydroelectric Reservoir, eight months after impoundment of the dam. A total of nine stations were selected in the reservoir. Temperature, pH, dissolved oxygen (DO), biochemical oxygen demand (BOD5), chlorophyll-a and total suspended solids (TSS) were studied. The results showed that the water was warmer at 0.5 m depth. As the depth increased, temperature in the reservoir decreased in the range of 4.40°C to 7.35°C from 0.5 to 20 m depth. The concentration of DO dropped to zero at 4 - 6 m depth leading to anoxic level. pH at 0.5 m depth ranged from 6.40 to 7.20 and they were significantly higher (p<0.05) than those at 10 and 20 m. Chlorophyll-a at 0.5 m were in the range of 4.61 to 20.50 mg/L, relatively higher compared to 10 m and 20 m depths. The results obtained indicates that impoundment of Murum River generally caused water quality deterioration. In addition, this could be due to anthropogenic activities such as plantation and logging activities that are still going on within the catchment during the impoundment. Therefore, further continuous monitoring and investigation is required to determine the long-term water quality condition and identify factors that influence water quality in Murum Reservoir.

Anoxia-mediated calcium release through the mitochondrial permeability transition pore silences NMDA receptor currents in turtle neurons

Mammalian neurons are anoxia sensitive and rapidly undergo excitotoxic cell death when deprived of oxygen, mediated largely by Ca(2+) entry through over-activation of N-methyl-d-aspartate receptors (NMDARs). This does not occur in neurons of the anoxia-tolerant western painted turtle, where a decrease in NMDAR currents is observed with anoxia. This decrease is dependent on a modest rise in cytosolic [Ca(2+)] ([Ca(2+)]c) that is mediated by release from the mitochondria. The aim of this study was to determine whether the mitochondrial permeability transition pore (mPTP) is involved in NMDAR silencing through release of mitochondrial Ca(2+). Opening the mPTP during normoxia with atractyloside decreased NMDAR currents by releasing mitochondrial Ca(2+), indicated by an increase in Oregon Green fluorescence. Conversely, the mPTP blocker cyclosporin A prevented the anoxia-mediated increase in [Ca(2+)]c and reduction in NMDAR currents. Mitochondrial membrane potential (Ψm) was determined using rhodamine-123 fluorescence and decreased with the onset of anoxia in a time frame that coincided with the increase in [Ca(2+)]c. Activation of mitochondrial ATP-sensitive potassium (mK(+)ATP) channels also releases mitochondrial Ca(2+) and we show that activation of mK(+)ATP channels during normoxia with diazoxide leads to Ψm depolarization and inhibition with 5-hydroxydecanoic acid blocked anoxia-mediated Ψm depolarization. Ψm does not collapse during anoxia but rather reaches a new steady-state level that is maintained via ATP hydrolysis by the F1-F0 ATPase, as inhibition with oligomycin depolarizes Ψm further than the anoxic level. We conclude that anoxia activates mK(+)ATP channels, which leads to matrix depolarization, Ca(2+) release via the mPTP, and ultimately silencing of NMDARs.

Temporal variability of near-bottom dissolved oxygen during upwelling off central Oregon

[1] In the productive central-Oregon coastal upwelling environment, wind-driven upwelling, tides, and topographic effects vary across the shelf, setting the stage for varied biogeochemical responses to physical drivers. Current, temperature, salinity, and dissolved oxygen (DO) measurements from three moorings deployed during the upwelling seasons of 2009–2011 off the central-Oregon coast are analyzed over three time bands (interannual, subtidal, tidal) to explore the relationship between mid (70 m) and inner-shelf (15 m) upwelling dynamics and the associated effect on DO. Topographic effects are observed in each time band due to the Heceta and Stonewall Bank complex. Seasonal cumulative hypoxia (DO < 1.4 mL L−1) calculations identify two regions, a well-ventilated inner shelf and a midshelf vulnerable to hypoxia (98 ± 15 days annually). On tidal timescales, along-shelf diurnal (K1) velocities are intensified over the Bank, 0.08 m s−1 compared with 0.03 m s−1 to the north. Interannual variability in the timing of spring and fall transitions, defined using glider-measured continental slope source water temperature, is observed on the midshelf. Interannual source water DO concentrations vary on the order of 0.1 mL L−1. Each spring and summer, DO decline rates are modulated by physical and biological processes. The net observed decrease is about 30% of the expected draw down due to water-column respiration. Physical processes initiate low-oxygen conditions on the shelf through coastal upwelling and subsequently prevent the system via advection and mixing from reaching the potential anoxic levels anticipated from respiration rates alone.

Purification and Properties of White Muscle Lactate Dehydrogenase from the Anoxia-Tolerant Turtle, the Red-Eared Slider,Trachemys scripta elegans

Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a crucial enzyme involved in energy metabolism in muscle, facilitating the production of ATP via glycolysis during oxygen deprivation by recycling NAD+. The present study investigated purified LDH from the muscle of 20 h anoxic and normoxic T. s. elegans, and LDH from anoxic muscle showed a significantly lower (47%) K m for L-lactate and a higher V max value than the normoxic form. Several lines of evidence indicated that LDH was converted to a low phosphate form under anoxia: (a) stimulation of endogenously present protein phosphatases decreased the K m of L-lactate of control LDH to anoxic levels, whereas (b) stimulation of kinases increased the K m of L-lactate of anoxic LDH to normoxic levels, and (c) dot blot analysis shows significantly less serine (78%) and threonine (58%) phosphorylation in anoxic muscle LDH as compared to normoxic LDH. The physiological consequence of anoxia-induced LDH dephosphorylation appears to be an increase in LDH activity to promote the reduction of pyruvate in muscle tissue, converting the glycolytic end product to lactate to maintain a prolonged glycolytic flux under energy-stressed anoxic conditions.

Gene Expression and Physiological Changes of Different Populations of the Long-Lived Bivalve Arctica islandica under Low Oxygen Conditions

The bivalve Arctica islandica is extremely long lived (>400 years) and can tolerate long periods of hypoxia and anoxia. European populations differ in maximum life spans (MLSP) from 40 years in the Baltic to >400 years around Iceland. Characteristic behavior of A. islandica involves phases of metabolic rate depression (MRD) during which the animals burry into the sediment for several days. During these phases the shell water oxygen concentrations reaches hypoxic to anoxic levels, which possibly support the long life span of some populations. We investigated gene regulation in A. islandica from a long-lived (MLSP 150 years) German Bight population and the short-lived Baltic Sea population, experimentally exposed to different oxygen levels. A new A. islandica transcriptome enabled the identification of genes important during hypoxia/anoxia events and, more generally, gene mining for putative stress response and (anti-) aging genes. Expression changes of a) antioxidant defense: Catalase, Glutathione peroxidase, manganese and copper-zinc Superoxide dismutase; b) oxygen sensing and general stress response: Hypoxia inducible factor alpha, Prolyl hydroxylase and Heat-shock protein 70; and c) anaerobic capacity: Malate dehydrogenase and Octopine dehydrogenase, related transcripts were investigated. Exposed to low oxygen, German Bight individuals suppressed transcription of all investigated genes, whereas Baltic Sea bivalves enhanced gene transcription under anoxic incubation (0 kPa) and, further, decreased these transcription levels again during 6 h of re-oxygenation. Hypoxic and anoxic exposure and subsequent re-oxygenation in Baltic Sea animals did not lead to increased protein oxidation or induction of apoptosis, emphasizing considerable hypoxia/re-oxygenation tolerance in this species. The data suggest that the energy saving effect of MRD may not be an attribute of Baltic Sea A. islandica chronically exposed to high environmental variability (oxygenation, temperature, salinity). Contrary, higher physiological flexibility and stress hardening may predispose these animals to perform a pronounced stress response at the expense of life span.

Refined age of the anoxic level in the Cenomanian-Turonian transition of the Mt. Sel’-Bukhra section (Crimean Mountains, Ukraine): Implications of radiolarian analysis

A representative radiolarian assemblage has been macerated for the first time from beds enriched in organic matter at the time of the Oceanic Anoxic Event (OAE-2). The assemblage includes radiolarians characteristic of the late Cenomanian, and is barren of species typical of the incipient Turonian. Consequently, deposition of beds rich in organic matter terminated in the late Cenomanian.

A multi-proxy record of the Latest Danian Event at Gebel Qreiya, Eastern Desert, Egypt

Abstract. The Latest Danian Event (LDE) is a proposed early Palaeogene transient warming event similar to the Paleocene–Eocene Thermal Maximum, albeit of smaller magnitude. The LDE can be correlated with a carbon isotope excursion (‘CIE-DS1’) at Zumaia, Spain, and the ‘top Chron C27n event’ defined recently from ocean drilling sites in the Atlantic and Pacific, supporting a global extent. Yet, records of environmental change during the LDE (e.g. warming and sea-level fluctuations) are still rare. In this study, we focus on the micropalaeontology (calcareous nannofossils and benthic foraminifera), mineralogy and trace element geochemistry of the LDE in the Qreiya 3 section from the southern Tethyan margin in Egypt. In this section, the LDE is characterized by the occurrence of anomalous beds intercalated within upper Danian shales and marls. The event beds of the LDE are situated above an unconformity on top of a shallowing-upwards sequence deposited in a well-oxygenated outer neritic to upper bathyal marine palaeoenvironment. The lower LDE bed is barren of benthic foraminifera, but contains pyrite and fish remains, and is interpreted as an anoxic level formed during rapid relative sea-level rise. Incursion of a Neoeponides duwi (Nakkady, 1950) benthic assemblage in LDE bed II is interpreted as repopulation of the seafloor after anoxia. The sea-level cycle associated with the LDE is estimated at about 50 m maximum in the Qreiya 3 section. The environmental changes at Qreiya 3 are of supra-regional extent, since a similar sequence of events has been observed at other southern Tethyan locations.

Regulation of tail muscle arginine kinase by reversible phosphorylation in an anoxia-tolerant crayfish

Freshwater crayfish, Orconectes virilis, can experience periodic exposures to hypoxia or anoxia due to low water flow (in summer) or ice cover (in winter) in their natural habitat. Hypoxia/anoxia disrupts energy metabolism and triggers mechanisms that to support ATP levels while often also suppressing ATP use. Arginine kinase (AK) (E.C. 2.7.3.3) is a crucial enzyme involved in energy metabolism in muscle, gating the use of phosphagen stores to buffer ATP levels. The present study investigated AK from tail muscle of O. virilis identifying changes to kinetic properties, phosphorylation state and structural stability between the enzyme from aerobic control and 20h anoxic crayfish. Muscle AK from anoxia-exposed crayfish showed a significantly higher (by 59%) K (m) for L: -arginine and a lower I(50) value for urea than the aerobic form. Several lines of evidence indicated that AK was converted to a high phosphate form under anoxia: (a) aerobic and anoxic forms of AK showed well-separated elution peaks on DEAE ion exchange chromatography, (b) ProQ Diamond phosphoprotein staining showed a 64% higher bound phosphate content on anoxic AK compared with the aerobic form, and (c) treatment of anoxic AK with alkaline phosphatase reduced K (m) L: -arginine to aerobic levels whereas incubation of aerobic AK with protein kinase A catalytic subunit raised the K (m) to anoxic levels. The physiological consequence of anoxia-induced AK phosphorylation may be to suppress AK activity in the phosphagen-synthesizing direction and, together with reduced cellular pH and ATP levels, promote the phosphagen-catabolizing direction under anoxic conditions. This is first time that AK has been shown to be regulated by reversible phosphorylation.

EXAFS speciation and phytoavailability of Pb in a contaminated soil amended with compost and gypsum

Due to unregulated uses of lead pellets for hunting purposes in Japan, soils and sediments in some river basins and wetlands have become highly contaminated with Pb. Deterioration of natural vegetation has occurred sporadically in these areas, and therefore revegetation is needed for ecological restoration. The objectives of the present study were to assess the effects of surface applications of compost and gypsum amendments on Pb availability to a watercress plant ( Nasturtium officinale W.T. Aiton) and molecular-scale speciation of Pb in soil solid phases. The compost and gypsum amendments significantly decreased dissolved Pb and Sb in pore water. The concentration of Pb in aboveground plant tissues was 190 mg kg − 1 in the control soil and was reduced to < 20 mg kg − 1 in the compost and gypsum-amended soils. The concentration of Sb in plants grown in the control soil was 13 mg kg − 1 , whereas that in the soils receiving compost and gypsum decreased below detectable levels. Redox potential was higher in vegetated soils (ave. 349 mV) than in the unvegetated soils (ave. 99 mV) due to oxygen introduced by plant roots. Extended X-ray absorption fine structure (EXAFS) spectroscopy illustrated that Pb occurred as Pb sorbed on birnessite and/or ferrihydrite (Pb–Mn/Fe, ~ 60%) and Pb sorbed on organic matter (Pb-org, ~ 15%), and galena (PbS, ~ 10%) in the vegetated and unvegetated control soils. The compost amendment increased the proportion of Pb-org by 2-fold than in the control soils. The amended soils with plant growth decreased the proportion of Pb–Mn/Fe phases by half of that without plant growth. Galena and anglesite (PbSO 4) were not detected in compost-amended soils and even in gypsum-amended soils since a significant soil reduction to anoxic levels did not occur in the entire soil. The present study indicated that, under flooded conditions, surface applications of compost and gypsum amendments reduced plant Pb uptake from the Pb contaminated soil.

The Effect of Pore Water Chemistry on the Biodegradation of the Exxon Valdez Oil Spill

Knowledge of the oxygen and nutrient concentra- tions in an oil-contaminated Prince William Sound (PWS) beach is important for understanding of the oil persistence over two decades after the Exxon Valdez spill. It was tradi- tionally believed that there was enough oxygen in the conta- minated shorelines to sustain aerobic microbial metabolism of oil and that nutrients were the major factors limiting oil biodegradation. In the present study, we analyzed the oxy- gen and nutrients levels in both clean and oily areas on a PWS beach that was heavily contaminated by the Exxon Valdez oil spill. We found that the level of nitrogen and phosphorous were 0.454 mg-N L −1 and 0.033 mg-P L −1 , respectively, which is not sufficient to fully support micro- bial growth, confirming that nutrient concentration was a major factor limiting oil transformation by biological ways. We also observed that the oxygen level varied from higher than 3 mg L −1 in the clean wells to about 1 mg L −1 (near anoxic level) in the oily wells. In addition, the lowest nitrate levels were observed at the oily wells. Altogether, these re- sults suggest that the low level of efficient electron acceptors (oxygen and nitrate) detected at the oily spot is responsible for slow and potentially inefficient biodegradation of the oil.

Imaging of oxygen gradients in monolayer cultured cells using green fluorescent protein

Gradients of Po(2) between capillary blood and mitochondria are the driving force for diffusional O(2) delivery in tissues. Hypoxic microenvironments in tissues that result from diffusional O(2) gradients are especially relevant in solid tumors because they have been related to a poor prognosis. To address the impact of tissue O(2) gradients, we developed a novel technique that permits imaging of intracellular O(2) levels in cultured cells at a subcellular spatial resolution. This was done, with the sensitivity to O(2) ≤3%, by the O(2)-dependent red shift of green fluorescent protein (AcGFP1) fluorescence. Measurements were carried out in a confluent monolayer of Hep3B cells expressing AcGFP1 in the cytoplasm. To establish a two-dimensional O(2) diffusion model, a thin quartz glass slip was placed onto the monolayer cells to prevent O(2) diffusion from the top surface of the cell layer. The magnitude of the red shift progressively increased as the distance from the gas coverslip interface increased. It reached an anoxic level in cells located at ∼220 μm and ∼690 μm from the gas coverslip boundary at 1% and 3% gas phase O(2), respectively. Thus the average O(2) gradient was 0.03 mmHg/μm in the present tissue model. Abolition of mitochondrial respiration significantly dampened the gradients. Furthermore, intracellular gradients of the red shift in mitochondria-targeted AcGFP1 in single Hep3B cells suggest that the origin of tissue O(2) gradients is intracellular. Findings in the present two-dimensional O(2) diffusion model support the crucial role of tissue O(2) diffusion in defining the O(2) microenvironment in individual cells.