Metabolic Basis for Injury to Plants from Combinations of O(3) and SO(2): Studies with Modifiers of Pollutant Toxicity

Pisum sativum L. cv Alsweet (garden pea) and Lycopersicon esculentum Mill. flacca (mutant tomato) were chosen to evaluate the metabolic basis for plant injury from combinations of O₃ + SO₂. The plants were exposed under conditions reported to specifically alter O₃ or SO₂ toxicity; light versus dark exposures, and treatment with the fungal metabolite fusicoccin (FC), the O₃ injury inhibitor N-[2-(2-oxo-1-imidazolidiny) ethyl]-N′-phenylurea (EDU), and the SO₂ injury stimulator diethyldithiocarbamate (DDTC). Plants were grown in controlled environment chambers and exposed to combinations of O₃ (0.05-0.2 microliters per liter) and SO₂ (0.1-0.3 microliters per liter) for 2 hours. Peas treated with FC had the same or greater injury (quantified by visual rating) with O₃ + SO₂ exposures compared to plants not treated with FC. For plants with open stomata in the dark as well as light, i.e. FC-treated peas and tomatoes, there was no change or an increase in foliar necrosis with O₃ + SO₂ exposures in the dark versus light. Peas treated with EDU had an almost complete absence of O₃ injury, no change in SO₂ injury, and moderate decreases in injury from combinations of O₃ + SO₂ compared to plants not treated with EDU. Tomatoes treated with DDTC showed the same or less injury compared to plants not treated with DDTC and exposed to O₃ or SO₂. The plant responses to the experimental treatments and O₃ + SO₂ resembled O₃ responses more than SO₂ responses. The evidence for O₃-like responses are: no change or increase in injury in the light versus dark, and EDU-induced decreases in injury. Evidences for SO₂-like responses are: incomplete protection from injury with EDU, and no change or increased injury to FC-treated versus untreated plants. Thus, a metabolic mechanism affected by both pollutants may be associated with the combination injury, e.g. effects the plasma membrane.     

A Photorespiratory Mutant of Chlamydomonas reinhardtii

A mutant strain of Chlamydomonas reinhardtii, designated 18-7F, has been isolated and characterized. 18-7F requires a high CO₂ concentration for photoautrophic growth in spite of the apparent induction of a functional CO₂ concentrating mechanism in air-adapted cells. In 2% O₂ the photosynthetic characteristics of 18-7F and wild type are similar. In 21% O₂, photosynthetic O₂ evolution is severely inhibited in the mutant by preillumination in limiting CO₂, although the apparent photosynthetic affinity for inorganic carbon is similar in preilluminated cells and in cells incubated in the dark prior to O₂ evolution measurements. Net CO₂ uptake is also inhibited when the cells are exposed to air (21% O₂, 0.035% CO₂, balance N₂) for longer than a few minutes. [¹⁴C]Phosphoglycolate accumulates within 5 minutes of photosynthetic ¹⁴CO₂ fixation in cells of 18-7F. Phosphoglycolate does not accumulate in wild type. Phosphoglycolate phosphatase activity in extracts from air-adapted cells of 18-7F is 10 to 20% of that in wild-type Chlamydomonas. The activity of phosphoglycolate phosphatase in heterozygous diploids is intermediate between that of homozygous mutant and wild-type diploids. It was concluded that the high-CO₂ requiring phenotype in 18-7F results from a phosphoglycolate phosphatase deficiency. Genetic analyses indicated that this deficiency results from a single-gene, nuclear mutation. We have named the locus pgp-1.     

Fusicoccin and Air Pollutant Injury to Plants : Evidence for Enhancement of SO(2) but Not O(3) Injury

Garden peas (Pisum sativum L. cv Alsweet) and a tomato mutant (Lycopersicon esculentum Mill. var flacca) were sprayed with fusicoccin, a fungal toxin affecting membrane transport properties, before exposure to SO₂ or O₃. Tomatoes treated with 10 micromolar fusicoccin and exposed to SO₂ (0.6 microliter per liter for 2 hours) exhibited twice as much foliar necrosis as untreated plants exposed to SO₂. Peas treated with fusicoccin and exposed to SO₂ (0.7 to 1.0 microliter per liter for 2 hours) exhibited 2 to 6 times more injury than untreated plants exposed to SO₂. Peas treated with fusicoccin and exposed to O₃ had less injury than untreated plants exposed to O₃ (0.1 to 0.3 microliter per liter for 2 hours). Several lines of evidence suggested that the fusicoccin enhancement of SO₂ injury is not the result of increased gas exchange, i.e. the tomato mutant has permanently open stomata under all conditions, and in peas fusicoccin had no effect on SO₂ or H₂O flux in plants exposed to 0.12 microliter per liter SO₂. However, a 21% greater leaf conductance in fusicoccin treated versus untreated plants indicated the possibility of some differences in gas exchange for peas exposed to 1.0 microliter per liter SO₂.     

Activities of Photosystems I and II in Chlamydomonas segnis Adapted and Adapting to Air and Air-enriched with Carbon Dioxide*

Activities of photosystems I and II were compared at a saturating irradiance in air- and 5% CO₂-adapted and adapting Chlamydomonas segnis at the active phase of photosynthesis during the cell cycle. PSII activity was 200% greater in air- than in 5% CO₂-adapted cells, while PSI activity was similar in both types of cells and matched the level of PSII activity in air-adapted cells. As a result, air- and 5% CO₂-adapted cells were characterized by low and high PSI/PSII ratios, respectively. In air-adapted cells, the greater PSII activity (rate of O₂ evolved) exceeded that of photosynthetic (Ps) O₂ evolution, resulting in a Ps/PSII ratio below unity. This was associated with higher levels of catalase activity, lower l -ascorbate content, and higher dehydro-l -ascorbate content than in 5% CO₂-adapted cells. During adaptation to air or 5% CO₂ for 6 h in light, PSI rather than PSII was sensitive to changes in the concentration of CO₂, and the adapting cells acquired the characteristics of air- and 5% CO₂-adapted cells as indicated by PSI/PSII, Ps/PSII, catalase activity, l -ascorbate and dehydro-l -ascorbate contents. The results are discussed in the light of changes in the molecular organization of the thylakoid membranes and enhanced non-cyclic electron transport coupled with O₂-uptake (Mehler reaction) for the generation of the ATP required for CO₂/HCO^?₃-transport in air-adapted and adapting cells.     

Salicylhydroxamic Acid (SHAM) Inhibition of the Dissolved Inorganic Carbon Concentrating Process in Unicellular Green Algae

Rates of photosynthetic O₂ evolution, for measuring K_0.5(CO₂ + HCO₃⁻) at pH 7, upon addition of 50 micromolar HCO₃⁻ to air-adapted Chlamydomonas, Dunaliella, or Scenedesmus cells, were inhibited up to 90% by the addition of 1.5 to 4.0 millimolar salicylhydroxamic acid (SHAM) to the aqueous medium. The apparent K₁(SHAM) for Chlamydomonas cells was about 2.5 millimolar, but due to low solubility in water effective concentrations would be lower. Salicylhydroxamic acid did not inhibit oxygen evolution or accumulation of bicarbonate by Scenedesmus cells between pH 8 to 11 or by isolated intact chloroplasts from Dunaliella. Thus, salicylhydroxamic acid appears to inhibit CO₂ uptake, whereas previous results indicate that vanadate inhibits bicarbonate uptake. These conclusions were confirmed by three test procedures with three air-adapted algae at pH 7. Salicylhydroxamic acid inhibited the cellular accumulation of dissolved inorganic carbon, the rate of photosynthetic O₂ evolution dependent on low levels of dissolved inorganic carbon (50 micromolar Na-HCO₃), and the rate of ¹⁴CO₂ fixation with 100 micromolar [¹⁴C] HCO₃⁻. Salicylhydroxamic acid inhibition of O₂ evolution and ¹⁴CO₂-fixation was reversed by higher levels of NaHCO₃. Thus, salicylhydroxamic acid inhibition was apparently not affecting steps of photosynthesis other than CO₂ accumulation. Although salicylhydroxamic acid is an inhibitor of alternative respiration in algae, it is not known whether the two processes are related.     

Hypoxic culture enhances the expansion of rat bone marrow-derived mesenchymal stem cells via the regulatory pathways of cell division and apoptosis

This study aimed to examine the proliferative behavior and molecular mechanisms of rat bone marrow-derived MSCs (rBMSCs) cultured under three different oxygen concentrations. Passaged rBMSCs exhibited significantly greater proliferation rates at 1% O₂ and 5% O₂ than those at 18% O₂ and the cells exposed to 1% O₂ showed the highest proliferative potential, which was evidenced by the growth curves, colony-forming efficiencies, and CCK-8 absorbance values. The rBMSCs grown under hypoxic culture conditions (1% O₂ and 5% O₂) had the increased percentage of cells in S?+?G₂/M-phase and the decreased apoptotic index, compared with normoxia (18% O₂). It was revealed for the first time that there were more phosphohistone H3 (PHH3)-positive cells and higher expressions of proliferating cell nuclear antigen (PCNA) in the hypoxic cultures of rBMSCs than in the normoxic culture. Hypoxia upregulated the anti-apoptotic protein Bcl-2 and downregulated the pro-apoptotic proteins Bax and the cleaved caspase-3 in cultured rBMSCs. The levels of hypoxia-inducible factor-1α (HIF-1α) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) were increased in the hypoxic-cultured rBMSCs. Nevertheless, no significant difference was observed in p53 level of rBMSCs between different oxygen concentrations. In conclusion, the hypoxia exerts a promoting effect on the in vitro expansion of rBMSCs via several signaling and molecular pathways involved in the control of cell cycle and apoptosis.     

Effect of dissolved inorganic carbon on oxygen evolution and uptake by Chlamydomonas reinhardtii suspensions adapted to ambient and CO2-enriched air

Mass spectrometric measurements of ¹⁶O₂ and ¹⁸O₂ isotopes were used to compare the rates of gross O₂ evolution (E₀), O₂ uptake (U₀) and net O₂ evolution (NET) in relation to different concentrations of dissolved inorganic carbon (DIC) by Chlamydomonas reinhardtii cells grown in air (air-grown), in air enriched with 5% CO₂ (CO₂-grown) and by cells grown in 5% CO₂ and then adapted to air for 6h (air-adapted).At a photon fluence rate (PFR) saturating for photosynthesis (700 mol photons m^-2 s^-1), pH=7.0 and 28°C, U₀ equalled E₀ at the DIC compensation point which was 10M DIC for CO₂-grown and zero for air-grown cells. Both E₀ and U₀ were strongly dependent on DIC and reached DIC saturation at 480 M and 70 M for CO₂-grown and air-grown algae respectively. U₀ increased from DIC compensation to DIC saturation. The U₀ values were about 40 (CO₂-grown), 165 (air-adapted) and 60 mol O₂ mg Chl^-1 h^-1 (air-grown). Above DIC compensation the U₀/E₀ ratios of air-adapted and air-grown algae were always higher than those of CO₂-grown cells. These differences in O₂ exchange between CO₂- and air-grown algae seem to be inducable since air-adapted algae respond similarly to air-grown cells.For all algae, the rates of dark respiratory O₂ uptake measured 5 min after darkening were considerably lower than the rates of O₂ uptake just before darkening. The contribution of dark respiration, photorespiration and the Mehler reaction to U₀ is discussed and the energy requirement of the inducable CO₂/HCO₃ ^- concentrating mechanism present in air-adapted and air-grown C. reinhardtii cells is considered.Abbreviations DIC dissolved inorganic carbon - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - E₀ rate of photosynthetic gross O₂ evolution - PCO photosynthetic carbon oxidation - PFR photon fluence rate - PS I photosystem I - PS II photosystem II - U₀ rate of O₂ uptake in the light - MS mass spectrometer     

Modulation of the Ca2+-sensing function of parathyroid cells in vitro and in hyperparathyroidism

When raising the extracellular Ca²⁺ concentration stepwise from 0.5 to 3.0 mM, bovine parathyroid cells reacted with initial transient and sustained elevations of the cytoplasmic Ca²⁺ concentration (Ca²⁺_i), as well as more than 50% inhibition of parathyroid hormone (PTH) release. Human parathyroid adenoma cells and bovine cells cultured for 1 day or exposed to a low concentration of a monoclonal antiparathyroid antibody exhibited right-shifted dependencies of PTH release and Ca²⁺_i on extracellular Ca²⁺ and reduced Ca²⁺_i transients. The protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) further right-shifted the dose response relationship for Ca²⁺ regulated Ca²⁺_i of the adenoma cells, whereas the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) tended to normalize it, without affecting Ca²⁺_i of normal bovine cells. In cells from an oxyphil adenoma and a parathyroid carcinoma as well as in bovine cells cultured 4 days or exposed to a high concentration of the antiparathyroid antibody, there were no Ca²⁺_i transients, very small increases in steady-state Ca²⁺_i and nonsuppressible PTH release. The results suggest that reduced availability of a putative Ca²⁺-receptor and increased protein kinase C activity may be important factors in the decreased Ca²⁺ sensitivity of abnormal parathyroid cells.     

Phytotoxicity of Air Pollutants: Evidence for the Photodetoxification of SO(2) but Not O(3)

Pisum sativum L. cv Alsweet (garden pea) and Lycopersicon esculentum flacca Mill. (tomato) were used to evaluate the phytotoxicity of SO₂ and O₃ in the light and dark. Plants were grown in controlled environment chambers and exposed to SO₂ or O₃ in the light or dark at the same environmental conditions at which they were grown. The pea plants were treated with fusicoccin to ensure open stomata in the dark; the stomata of the tomato mutant remained open in the dark. Both species exhibited 64% to 80% less foliar necrosis following exposure to SO₂ (0.5 to 1.0 microliter per liter for 2 hours) in the light than in the dark. The decrease in SO₂ injury for light versus dark exposed plants was greater in fully expanded than expanding leaves. Both species exhibited 30% greater foliar necrosis following exposure to O₃ (0.2 microliter per liter for 2 hours) in the light than dark. The increase in O₃ injury in the light versus dark was similar for leaves at all stages of expansion. Leaf conductance to water vapor was 7% to 11% and 23% higher in the light than dark for fusicoccin-treated peas and tomato plants, respectively, indicating greater foliar uptake of both pollutants in the light than dark. Thus, the decreased SO₂ toxicity in the light was not associated with pollutant uptake, but rather the metabolism of SO₂. In contrast, the increased toxicity of O₃ in the light was at least in part associated with increased uptake or could not be separated from it.     

Uptake of inorganic carbon by isolated chloroplasts from air-adapted dunaliella

Neither Dunaliella cells grown with 5% CO₂ nor their isolated chloroplasts had a CO₂ concentrating mechanism. These cells primarily utilized CO₂ from the medium because the K_(0.5) (HCO₃⁻) increase from 57 micromolar at pH 7.0 to 1489 micromolar at pH 8.5, where as the K_(0.5) CO₂ was about 12 micromolar over the pH range. After air adaptation for 24 hours in light, a CO₂ concentrating mechanism was present that decreased the K_0.5 (CO₂) to about 0.5 micromolar and K_0.5 (HCO₃⁻) to 11 micromolar at pH 8. These K_0.5 values suggest that air-adapted cells preferentially concentrated CO₂ but could also use HCO₃⁻ from the medium. Chloroplasts isolated from air-adapted cells had a K_(0.5) for total inorganic carbon of less than 10 micromolar compared to 130 micromolar for chloroplasts from cells grown on high CO₂. Chloroplasts from air-adapted cells, but not CO₂-grown cells, concentrate inorganic carbon internally to 1 millimolar in 60 seconds from 240 micromolar in the medium. Maximum uptake rates occurred after preillumination of 45 seconds to 3 minutes. The CO₂ concentrating mechanism by chloroplasts from air-adapted cells was light dependent and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or flurocarbonyl-cyamidephenylhydrazone (FCCP). Phenazine-methosulfate at 10 micromolar to provide cyclic phosphorylation partially reversed the inhibition by DCMU but not by FCCP. One to 0.1 millimolar vanadate, an inhibitor of plasma membrane ATPase, inhibited inorganic carbon accumulation by isolated chloroplasts. Vanadate had no effect on CO₂ concentration by whole cells, as it did not readily cross the cell plasmalemma. Addition of external ATP to the isolated chloroplast only slightly stimulated inorganic carbon uptake and did not reverse vanadate inhibition by more than 25%. These results are consistent with a CO₂ concentrating mechanism in Dunaliella cells which consists in part of an inorganic carbon transporter at the chloroplast envelope that is energized by ATP from photosynthetic electron transport.     

Effect of sericin on preimplantation development of bovine embryos cultured individually

The silk protein sericin has been identified as a potent antioxidant in mammalian cells. This study was conducted to examine the effects of sericin on preimplantation development and quality of bovine embryos cultured individually. When two-cell-stage embryos were cultured individually for 7 days in CR1aa medium supplemented with 0, 0.1, 0.5, or 1% sericin, rates of total blastocyst formation and development to expanded blastocysts from embryos cultured with 0.5% sericin were higher (P < 0.05) than those from embryos cultured with 0 or 1% sericin. When embryos were cultured individually for 7 days in the CR1aa medium supplemented with 0 or 0.5% sericin under two oxidative stress conditions (50 or 100 μm H₂O₂), the addition of sericin significantly improved the blastocyst formation rate of embryos exposed to 100 μm H₂O₂. However, the protective effect of sericin was not observed in development of embryos exposed to 50 μm H₂O₂. When embryos were exposed to 100 μm H₂O₂ during culture, the DNA fragmentation index of total blastocysts from embryos cultured with 0.5% sericin was lower than blastocysts derived from embryos cultured without sericin (4.4 vs. 6.8%; P < 0.01). In conclusion, the addition of 0.5% sericin to in vitro culture medium improved preimplantation development and quality of bovine embryos cultured individually by preventing oxidative stress.     

Chronic hypoxia upregulates adenosine 2a receptor expression in chromaffin cells via hypoxia inducible factor-2α: role in modulating secretion

Catecholamine (CAT) release from chromaffin tissue plays an essential role in the fetus which develops in a low O₂ environment (hypoxia). To address molecular mechanisms regulating CAT secretion in low O₂, we exposed a fetal chromaffin-derived cell line (MAH cells) to chronic hypoxia (CHox; 2% O₂, 24 h) and assessed gene expression using microarrays, quantitative RT-PCR, and western blot. CHox caused a dramatic ∼12× upregulation of adenosine A2a receptor (A2aR) mRNA, an effect critically dependent upon hypoxia-inducible factor (HIF)-2α which bound the promoter of the A2aR gene. In amperometric studies, acute hypoxia and high K⁺ (30 mM) evoked quantal CAT secretion that was enhanced after CHox, and further potentiated during simultaneous A2aR activation by adenosine. A2aR activation also enhanced stimulus-induced rise in intracellular Ca²⁺ in control, but not HIF-2α-deficient, MAH cells. Thus, A2aR, adenosine, and HIF-2α are key contributors to the potentiation of CAT secretion in developing chromaffin cells during chronic hypoxia.     

Oxygen-coupled Redox Regulation of the Skeletal Muscle Ryanodine Receptor/Ca2+ Release Channel (RyR1): SITES AND NATURE OF OXIDATIVE MODIFICATION*

In mammalian skeletal muscle, Ca²⁺ release from the sarcoplasmic reticulum (SR) through the ryanodine receptor/Ca²⁺-release channel RyR1 can be enhanced by S-oxidation or S-nitrosylation of separate Cys residues, which are allosterically linked. S-Oxidation of RyR1 is coupled to muscle oxygen tension (pO₂) through O₂-dependent production of hydrogen peroxide by SR-resident NADPH oxidase 4. In isolated SR (SR vesicles), an average of six to eight Cys thiols/RyR1 monomer are reversibly oxidized at high (21% O₂) versus low pO₂ (1% O₂), but their identity among the 100 Cys residues/RyR1 monomer is unknown. Here we use isotope-coded affinity tag labeling and mass spectrometry (yielding 93% coverage of RyR1 Cys residues) to identify 13 Cys residues subject to pO₂-coupled S-oxidation in SR vesicles. Eight additional Cys residues are oxidized at high versus low pO₂ only when NADPH levels are supplemented to enhance NADPH oxidase 4 activity. pO₂-sensitive Cys residues were largely non-overlapping with those identified previously as hyperreactive by administration of exogenous reagents (three of 21) or as S-nitrosylated. Cys residues subject to pO₂-coupled oxidation are distributed widely within the cytoplasmic domain of RyR1 in multiple functional domains implicated in RyR1 activity-regulating interactions with the L-type Ca²⁺ channel (dihydropyridine receptor) and FK506-binding protein 12 as well as in “hot spot” regions containing sites of mutation implicated in malignant hyperthermia and central core disease. pO₂-coupled disulfide formation was identified, whereas neither S-glutathionylated nor sulfenamide-modified Cys residues were observed. Thus, physiological redox regulation of RyR1 by endogenously generated hydrogen peroxide is exerted through dynamic disulfide formation involving multiple Cys residues.     

Recovery of cells in vitro from the effects hypoxia and hyperoxia

The cytotoxic effect of high, as well as low, oxygen tension of proliferation and metabolism of Low line cells in culture is reversible even after several days of exposure provided the cells are returned to 95% air + 5% CO₂ environment. This suggests that the activity of certain mechanisms within the cells may have been altered or in other ways inhibited by the abnormal environments but are quite rapidly regenerated once the adverse condition is removed. The cells tolerate a low O₂ exposure for at least 20 days while continuous exposure to high O₂ atmosphere results in degeneration and death after 7–10 days. Both glucose utilization and lactic acid production are elevated in cultures exposed to either low or high O₂ tensions, although they are markedly higher in the latter condition. When cell so exposed are returned to an air + 5% CO₂ atmosphere, rate of glucose uptake and lactic acid formation soon approaches that found in control cultures.     

Effects of Selenium and Topiramate on Cytosolic Ca2+ Influx and Oxidative Stress in Neuronal PC12 Cells

It has been widely suggested that selenium (Se) deficiency play an important role in the pathophysiology of epilepsy. It has been reported that Se provides protection against the neuronal damage in patients and animals with epilepsy by restoring the antioxidant defense mechanism. The neuroprotective effects of topiramate (TPM) have been reported in several studies but the putative mechanism of action remains elusive. We investigated effects of Se and TPM in neuronal PC12 cell by evaluating Ca²⁺ mobilization, lipid peroxidation and antioxidant levels. PC12 cells were divided into eight groups namely control, TPM, Se, H₂O₂, TPM + H₂O₂, Se + H₂O₂, Se + TPM and Se + TPM + H₂O₂. The toxic doses and times of H₂O₂, TPM and Se were determined by cell viability assay which is used to evaluate cell viability. Cells were incubated with 0.01 mM TPM for 5 h and 500 nM Se for 10 h. Then, the cells were exposed to 0.1 mM H₂O₂ for 10 h before analysis. The cells in all groups except control, TPM and Se were exposed to H₂O₂ for 15 min before analysis. Cytosolic Ca²⁺ release and lipid peroxidation levels were higher in H₂O₂ group than in control, Se and TPM combination groups although their levels were decreased by incubation of Se and TPM combination. However, there is no difference on Ca²⁺ release in TPM group. Glutathione peroxidase activity, reduced glutathione and vitamin C levels in the cells were lower in H₂O₂ group than in control, Se and TPM groups although their values were higher in the cells incubated with Se and TPM groups than in H₂O₂ groups. In conclusion, these results indicate that Se induced protective effects on oxidative stress in PC12 cells by modulating cytosolic Ca²⁺ influx and antioxidant levels. TPM modulated also lipid peroxidation and glutathione and vitamin C concentrations in the cell system.     

The Geoglobus acetivorans Genome: Fe(III) Reduction,Acetate Utilization,Autotrophic Growth,and Degradation of Aromatic Compounds in a Hyperthermophilic Archaeon

Geoglobus acetivorans is a hyperthermophilic anaerobic euryarchaeon of the order Archaeoglobales isolated from deep-sea hydrothermal vents. A unique physiological feature of the members of the genus Geoglobus is their obligate dependence on Fe(III) reduction, which plays an important role in the geochemistry of hydrothermal systems. The features of this organism and its complete 1,860,815-bp genome sequence are described in this report. Genome analysis revealed pathways enabling oxidation of molecular hydrogen, proteinaceous substrates, fatty acids, aromatic compounds, n-alkanes, and organic acids, including acetate, through anaerobic respiration linked to Fe(III) reduction. Consistent with the inability of G. acetivorans to grow on carbohydrates, the modified Embden-Meyerhof pathway encoded by the genome is incomplete. Autotrophic CO₂ fixation is enabled by the Wood-Ljungdahl pathway. Reduction of insoluble poorly crystalline Fe(III) oxide depends on the transfer of electrons from the quinone pool to multiheme c-type cytochromes exposed on the cell surface. Direct contact of the cells and Fe(III) oxide particles could be facilitated by pilus-like appendages. Genome analysis indicated the presence of metabolic pathways for anaerobic degradation of aromatic compounds and n-alkanes, although an ability of G. acetivorans to grow on these substrates was not observed in laboratory experiments. Overall, our results suggest that Geoglobus species could play an important role in microbial communities of deep-sea hydrothermal vents as lithoautotrophic producers. An additional role as decomposers would close the biogeochemical cycle of carbon through complete mineralization of various organic compounds via Fe(III) respiration.     

Effects of shear stress cultivation on cell membrane disruption and intracellular calcium concentration in sonoporation of endothelial cells

Microbubble facilitated ultrasound (US) application can enhance intracellular delivery of drugs and genes in endothelial cells cultured in static condition by transiently disrupting the cell membrane, or sonoporation. However, endothelial cells in vivo that are constantly exposed to blood flow may exhibit different sonoporation characteristics. This study investigates the effects of shear stress cultivation on sonoporation of endothelial cells in terms of membrane disruption and changes in the intracellular calcium concentration ([Ca²⁺]_i). Sonoporation experiments were conducted using murine brain microvascular endothelial (bEnd.3) cells and human umbilical vein endothelial cells (HUVECs) cultured under static or shear stress (5 dyne/cm² for 5 days) condition in a microchannel environment. The cells were exposed to a short US tone burst (1.25 MHz, 8 μs duration, 0.24 MPa) in the presence of Definity^TM microbubbles to facilitate sonoporation. Membrane disruption was assessed by propidium iodide (PI) and changes in [Ca²⁺]_i measured by fura-2AM. Results from this study show that shear stress cultivation significantly reduced the impact of ultrasound-driven microbubbles activities on endothelial cells. Cells cultured under shear stress condition exhibited much lower percentage with membrane disruption and changes in [Ca²⁺]_i compared to statically cultured cells. The maximum increases of PI uptake and [Ca²⁺]_i were also significantly lower in the shear stress cultured cells. In addition, the extent of [Ca²⁺]_i waves in shear cultured HUVECs was reduced compared to the statically cultured cells.     

Activation of Methanogenesis by Cadmium in the Marine Archaeon Methanosarcina acetivorans

Methanosarcina acetivorans was cultured in the presence of CdCl₂ to determine the metal effect on cell growth and biogas production. With methanol as substrate, cell growth and methane synthesis were not altered by cadmium, whereas with acetate, cadmium slightly increased both, growth and methane rate synthesis. In cultures metabolically active, incubations for short-term (minutes) with 10 µM total cadmium increased the methanogenesis rate by 6 and 9 folds in methanol- and acetate-grown cells, respectively. Cobalt and zinc but not copper or iron also activated the methane production rate. Methanogenic carbonic anhydrase and acetate kinase were directly activated by cadmium. Indeed, cells cultured in 100 µM total cadmium removed 41–69% of the heavy metal from the culture and accumulated 231–539 nmol Cd/mg cell protein. This is the first report showing that (i) Cd²⁺ has an activating effect on methanogenesis, a biotechnological relevant process in the bio-fuels field; and (ii) a methanogenic archaea is able to remove a heavy metal from aquatic environments.     

Chronic Hypoxia Increases TRPC6 Expression and Basal Intracellular Ca2+ Concentration in Rat Distal Pulmonary Venous Smooth Muscle

Background

Hypoxia causes remodeling and contractile responses in both pulmonary artery (PA) and pulmonary vein (PV). Here we explore the effect of hypoxia on PV and pulmonary venous smooth muscle cells (PVSMCs).

Methods

Chronic hypoxic pulmonary hypertension (CHPH) model was established by exposing rats to 10% O₂ for 21 days. Rat distal PVSMCs were isolated and cultured for in vitro experiments. The fura-2 based fluorescence calcium imaging was used to measure the basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) and store-operated Ca²⁺ entry (SOCE). Quantitative RT-PCR and western blotting were performed to measure the expression of mRNA and levels of canonical transient receptor potential (TRPC) protein respectively.

Results

Hypoxia increased the basal [Ca²⁺]_i and SOCE in both freshly dissociated and serum cultured distal PVSMCs. Moreover, hypoxia increased TRPC6 expression at mRNA and protein levels in both cultured PVSMCs exposed to prolonged hypoxia (4% O₂, 60 h) and distal PV isolated from CHPH rats. Hypoxia also enhanced proliferation and migration of rat distal PVSMCs.

Conclusions

Hypoxia induces elevation of SOCE in distal PVSMCs, leading to enhancement of basal [Ca²⁺]_i in PVSMCs. This enhancement is potentially correlated with the increased expression of TRPC6. Hypoxia triggered intracellular calcium contributes to promoted proliferation and migration of PVSMCs.