Hypoxic Growth Conditions Research Articles

PNUTS (phosphatase nuclear targeting subunit) inhibits retinoblastoma-directed PP1 activity

Protein phosphatase type 1 catalytic subunit (PP1c) is a serine/threonine phosphatase involved in the dephosphorylation of many proteins in eukaryotic cells. It associates with several known targeting or regulatory subunits that directly regulate PP1c activity toward specific substrates. The recently identified Phosphatase Nuclear Targeting Subunit (PNUTS) binds to PP1c and inhibits PP1 activity toward phosphorylase a. One of the substrates of PP1c has been shown to be the cell cycle regulatory protein, Retinoblastoma (pRb). In this study, we show that PNUTS dissociates from PP1c under mildly hypoxic cell growth conditions that lead to an increase of PP1c activity toward pRb. We developed an assay that measures pRb-directed PP1c activity and show that a GST-PNUTS fusion protein inhibits phosphatase activity toward pRb when using PP1c from cell lysates, GST-PP1c, or purified PP1c. These studies suggest that PNUTS is involved in the regulation of PP1c activity toward pRb.

Impact of root morphology on metabolism and oxygen distribution in roots and rhizosphere from two Central Amazon floodplain tree species.

Adaptation to prolonged flooding was investigated using cuttings of two tree species from the Central Amazon white-water floodplain (Várzea). Morphological features and oxygen distribution patterns were correlated with metabolic changes under hypoxia, such as alterations in alcohol dehydrogenase (ADH) activity and adenylate energy charge (AEC) of root cells. Salix martiana (Leyb.) was able to react to hypoxic growth conditions with formation of adventitious roots rich in lysigenous aerenchyma, which facilitates root aeration by longitudinal oxygen transport and rhizosphere oxidation by radial oxygen loss (ROL). The oxygen concentration on the surface of adventitious roots of S. martiana reached 2-3 mg O2 L-1. The low resistance to gas exchange in Salix roots was reflected by low ADH activities, which ranged between 0.03-0.1 μmol NADH mg -1 min-1, and AEC values of 0.8-1 under hypoxic conditions. Adventitious roots were also formed by Tabernaemontana juruana ([Markgr.] Schumann ex. J.F. Macbride) during growth under low-oxygen conditions, although at a later stage. The gas-space continuum in roots of T. juruana was less pronounced, resulting in a 10-fold lower oxygen concentration in the root cortex under oxygen stress compared with adventitious roots of Salix. The lower oxygen content was reflected in 6-fold higher ADH activities and decreased AEC values. ROL occurred only at the non-suberized root tip, suggesting that the suberized hypodermis functions as a barrier against gas exchange between the root and the rhizosphere. These findings indicate that different strategies of adaptation to low oxygen levels are realized in the two species under investigation that occur naturally in the same ecosystem but inhabit different elevation sites.

Vitreoscilla Hemoglobin: INTRACELLULAR LOCALIZATION AND BINDING TO MEMBRANES

The obligate aerobic bacterium, Vitreoscilla, synthesizes elevated quantities of a homodimeric hemoglobin (VHb) under hypoxic growth conditions. Expression of VHb in heterologous hosts often enhances growth and product formation. A role in facilitating oxygen transfer to the respiratory membranes is one explanation of its cellular function. Immunogold labeling of VHb in both Vitreoscilla and recombinant Escherichia coli bearing the VHb gene clearly indicated that VHb has a cytoplasmic (not periplasmic) localization and is concentrated near the periphery of the cytosolic face of the cell membrane. OmpA signal-peptide VHb fusions were transported into the periplasm in E. coli, but this did not confer any additional growth advantage. The interaction of VHb with respiratory membranes was also studied. The K(d) values for the binding of VHb to Vitreoscilla and E. coli cell membranes were approximately 5-6 microm, a 4-8-fold higher affinity than those of horse myoglobin and hemoglobin for these same membranes. VHb stimulated the ubiquinol-1 oxidase activity of inverted Vitreoscilla membranes by 68%. The inclusion of Vitreoscilla cytochrome bo in proteoliposomes led to 2.4- and 6-fold increases in VHb binding affinity and binding site number, respectively, relative to control liposomes, suggesting a direct interaction between VHb and cytochrome bo.

Regulation of macrophage production of vascular endothelial growth factor (VEGF) by hypoxia and transforming growth factor beta-1.

Breast tumors contain high numbers of infiltrating macrophages. The role and function of these cells within the tumor remain unclear, but a number of studies have found an association between poor prognosis and macrophage content in human breast cancer. Both hypoxia and TGFbeta-1 have been shown to regulate VEGF in other cell types. We hypothesized that breast tumor-associated macrophages produce VEGF and that macrophage production of this factor is regulated by both hypoxia and TGFbeta-1. Paraffin-embedded breast tumor sections were stained immunohistochemically with anti-VEGF, anti-CD68, and anti-cytokeratin. Monocytes were matured for 3 days in 20% autologous plasma and activated with 1000 U/mL interferon-gamma for 24 hours. Supernatants were assayed for VEGF protein by ELISA. Total RNA was isolated from cells and reverse transcribed to cDNA, which was used as a template in PCR reactions for VEGF and beta-actin. Both tumor cells and tumor macrophages produce VEGF in human breast tumors. Hypoxia increases VEGF protein and mRNA levels in monocyte-derived macrophages, whereas TGFbeta-1 increases VEGF protein but not mRNA under hypoxic growth conditions. Breast tumor-associated macrophages may contribute to the angiogenic activity of human breast tumors by producing VEGF. Macrophage production of VEGF is upregulated by hypoxia and TGFbeta-1, both of which occur in the tumor environment. Macrophage production of VEGF is regulated at both the mRNA and protein levels.

Maintenance of 32P uptake and transport in Pinus serotina seedlings under hypoxic growth conditions

Maintenance of 32P uptake and transport in Pinus serotina seedlings under hypoxic growth conditions

Maintenance of 32P uptake and transport in Pinus serotina seedlings under hypoxic growth conditions

In the present study, we examined the effects of long‐ and short‐term hypoxia on net uptake and transport of phosphorus to shoots of pond pine (Pinus serotina Michx.), a moderately flood‐tolerant southern pine, and the influence aerenchyma formation might have in maintenance of P uptake and transport. Seedlings were grown under aerobic (250 μM O2) or hypoxic (≤50 μM O2) solution conditions for 5.3 weeks in continuously flowing solution culture containing 100 μM P. Intact seedlings were then labeled with 32P for up to 24 h to determine how short‐ and long‐term hypoxic solution conditions affected rates of unidirectional influx and the accumulation of 32P in roots and shoots. Seedlings in the long‐term hypoxic treatment were grown for 5.3 weeks in hypoxic solution and also labeled in hypoxic uptake solution. The short‐term hypoxic treatments included a 24‐h hypoxic pretreatment followed by time in labeled hypoxic uptake solution for seedlings grown under aerobic or hypoxic conditions; in the latter case, diffusion of atmospheric O2 entry into stem and root collar lenticels was blocked, thus removing any influence that aerenchyma formation might have had on enhancing O2 concentrations of root tissue. Although unidirectional influx rates of 32P in roots of seedlings grown under long‐term hypoxic conditions were 1.4 times those of aerobically grown seedlings, accumulation of 32P in roots was similar after 24 h in labeled uptake solution. These results suggest that 32P efflux was also higher under hypoxic conditions. Higher shoot/root fresh weight ratios and lower shoot P concentrations in seedlings grown under hypoxic solution conditions suggest that the “shoot P demand” per unit root should be high. Yet accumulation of 32P in shoots was reduced by 50% after 24 h in hypoxic uptake solution. Both short‐term hypoxic treatments decreased accumulation of 32P in roots by more than 50%. Short‐term hypoxia decreased shoot accumulation in seedlings grown under aerobic and hypoxic conditions by 84 and 50%. respectively. Short‐ and long‐term hypoxic conditions increased the percentage of root 32P in the nucleic acid and chelated‐P pools, resulting in a significantly smaller percentage of 32P in the soluble inorganic phosphate (pi) pool, the pool available for transport to the shoot. However, a reduction in pool size or in labeling of the pool available for transport cannot fully account for the large reduction in accumulation of 32P in shoots, particularly in the short‐term hypoxic treatment of aerobically grown seedlings. Our results suggest that both influx and transport of 32P to shoots of pond pine seedlings are O2‐dependent processes, and that the transport of 32P to shoots may be more sensitive to hypoxic solution conditions than influx at the cortical and epidermal plasmalemma, with aerenchyma formation supporting a substantial amount of both 32P uptake and transport.

32P uptake and transport to shoots in Pinuus serotina seedlings under aerobic and hypoxic growth conditions

We examined the effects o long‐term hypoxic growth conditions on net uptake and transport of P to shoots of pond Pine (Pinus serotina Michx.), a moderately flood‐tolerant southern pine. Seedlings were grown under aerobic orhypoxic solution conditions for 4–5 weeks in continuously flowing solution culture containing 100 μM P. Short – and long‐term 32P. experiments were then concluded with intact seedlings to determine rates of 32P influx, efflux and net transport to the shoot. Shoot fresh weight/root fresh weight ratios were significantly higher under hypoxic gorwth conditions, reflecting the larger reduction in root growth than shoot growth, despite extensive aerechyma formation in roots. Estimates for the unidirectional influx of 32P in aerobic and hypoxic seedlings were 1.43 and 3.20 μmol P (gFW root)−1 h−1, respectively. However, 32P accumulation between the two treatments became similar within 8 h, suggesting that efflux was also higer in seedlings from the hypoxic treatment. Indeed in a separate experiment, hypoxic growth conditions increased efflux by over 60%. Transport of 32P to shoots was significantly reduced under hypoxic growth conditions, despite higher root P concentrations and lower shoot P concentrations. After 48 h, 32P accumulation in roots was similar between the two treatments. Yet total accumulation of seedling 32P decrcased by 31% under the hypoxic treatment, largely because of reduced transport of 32p to the shoot. The lower accumulation of 32 by shoots of seedlings in the hypoxic treatment may be the result of a direct inhibition on the transport process in O2‐defident tissues, but could also reflect a slower turnover or labeling of the ool available for transport. Indeed, the percentage of total 32P in. roots present in the soluble P. (or transportable form of P) was about 33% lower in seedlings from the hypoxic treatment, probably reflecting increased assimilation into organic compounds as well as chelation with iron. Our results suggest that P transport to the shoots of acclimated seedlings may be more sensitive to hypoxic solution conditions than influx at the root Plasmalemma.

32P uptake and transport to shoots in Pinus serotina seedlings under aerobic and hypoxic growth conditions

32P uptake and transport to shoots in Pinus serotina seedlings under aerobic and hypoxic growth conditions

Effects of root hypoxia and a low P supply on relative growth, carbon dioxide exchange rates and carbon partitioning in Pinus serotina seedlings

The present study was conducted to determine how 10 weeks of root hypoxia and a low P supply altered relative growth, and carbon acquisition and partitioning in a moderately flood tolerant pine. Pond pine (Pinus serotina Michx.) seedlings were grown in continuously flowing solution culture at 5 or 100 μM P, under aerobic or hypoxic solution conditions. Staggered harvests were used to ascertain changes in biomass allocation and relative growth over time. Carbon dioxide exchange rates (CER) were determined by infrared gas analysis, and needles were analyzed for inorganic P (Pi), sucrose, reducing sugars and starch. Although aeration treatment had no significant effect on shoot dry weight or shoot ontogeny, root dry weight of hypoxic seedlings was significantly lower than that of aerobic seedliings after 8 weeks, regardless of the P treatment. Mean relative growth rates (RGR) of roots in the high P treatment initially decreased under hypoxia, but recovered by the sixth week with the production of adventitious roots. Two weeks of hypoxic growth conditions decreased CER and stomatal conductances of seedlings in the high P treatment by more than 30% relative to their aerobic counterparts. Stomatal closure was not accompanied by a decrease in intracellular CO2, but was accompanied by an increase in starch accumulation. Recoveries of CER, stomatal conductance and carbohydrate metabolism coincided with the recovery of root growth. Low P growth conditions did not significantly affect shoot or root dry weight until the sixth week of treatment. However, differences in seedling RGR, particularly needle RGR, were discernable during the first 2 weeks. Low P treatment effects on CER paralleled changes in needle RGR, with needle RGR more affected than CER. After 6 weeks, CER of aerobically grown seedlings in the low P treatment were only 15% lower than CER of seedlings in the high P treatment, despite a 31% and 75% reduction in needle RGR and Pi concentrations, respectively. Increased starch concentrations of recently expanded needles at this time were probably a result of diminished growth. The inhibitory effect of a low P supply on shoot growth, more specifically on needle expansion and emergence of new fascicular needles, probably limited net carbon fixed per plant more than any direct effect of low P on CER per se.

Carbon and phosphorus partitioning in Pinus serotina seedlings growing under hypoxic and low-phosphorus conditions.

Ten-week-old pond pine (Pinus serotina Michx.) seedlings were grown in solution culture at 5 or 100 microM P and under aerobic or hypoxic solution conditions. After 6 and 10 weeks in the treatments, changes in relative growth rate (RGR), P acquisition and allocation, and carbohydrate partitioning were determined by analyzing tissue for total P, soluble sugars and starch. Six weeks of low-P growth conditions decreased seedling dry weight and the ratio of shoot dry weight to root dry weight (S/R) by 39 and 51%, respectively, in comparison to seedlings from the aerobic, high-P (control) treatment. Mean RGRs of shoots in the low-P treatment were reduced by 33%, whereas root growth was unaffected. After 10 weeks of low-P growth conditions, however, both shoot and root RGRs were significantly reduced, and plants had lower S/R ratios than in any other treatment. Slowed shoot growth was accompanied by starch and nonstructural carbohydrate accumulation in needles, indicating that needle growth was not limited by carbohydrate supply. Six weeks of low-P growth conditions decreased total seedling P by 75%, reflecting a 97% reduction in the net uptake rate (NUR). Shoot NUR as a fraction of seedling NUR was also greatly reduced in the low-P treatment, indicating that low-P growth conditions affected P translocation to the shoot more than P accumulation by roots. In contrast, 6 weeks of hypoxic growth conditions decreased total dry weight of seedlings in the high-P treatment by 41% relative to their aerobic counterparts. Root growth was affected more than shoot growth, however, and S/R ratios increased. After 10 weeks, S/R ratios doubled, primarily because of the reduction in root RGR. Nevertheless, roots of hypoxic seedlings contained a higher percentage of total seedling P than their aerobic counterparts. Net P acquisition per seedling decreased by more than 50% under hypoxic growth conditions, as a result of reductions in both root RGR and seedling NUR. Starch accumulation in shoots of hypoxic seedlings reflected reductions both in root growth and in transport of carbohydrates to nonwoody roots. Carbohydrate availability did not appear to be limiting growth of hypoxic woody roots, which are well-aerated internally, but it may have limited metabolic processes in nonwoody roots of seedlings from the high-P treatment.

Buoyant density of EMT6 fibrosarcoma cells: I. effect of hypoxic growth conditions in vitro

The effect of hypoxic culture conditions in vitro on the growth, survival and buoyant density of cancer cells was examined. EMT6 fibrosarcoma cells in the exponential-phase of growth were incubated at 37°C for different times; they were placed in sealed vacuum chambers that contained oxygen ranging in concentration from < 10 ppm (anoxic) to 2.1 x 10 5 ppm (aerobic). After incubation, the cells were centrifuged under isopyknic conditions in linear gradients of Ficoll to determine their buoyant density. Cells that were grown under hypoxic conditions in vitro became less dense than control cultures that were incubated in air. The actual density differences depended on the time of growth and the level of hypoxia. However, these density changes were reversible if the cells were transferred from hypoxic to aerobic conditions before density analysis.