Carrier-mediated choline uptake by Krens II ascites cells

Krebs II ascites cells have a low affinity uptake system for choline (K_m = 36 μM, V_m = 76 nmol/min per 2·10⁸ cells). Choline entered the cells and was rapidly phosphorylated (95% of total intracellular soluble label). Trans acceleration of labeled choline from cells preloaded with radiolabeled choline and postincubated in the presence of unlabeled choline indicates that choline transport in Krebs II ascites cells is carrier mediated. Ethanolamine competed for the choline carrier. The uptake was reduced by hemicholinium-3, iodoacetamide and ouabain. The mechanism of choline transport in Krebs Ii ascites cells is in agreement with a linear transport model.     

Gliclazide may have an antiapoptotic effect related to its antioxidant properties in human normal and cancer cells

Experimental and clinical studies suggest that gliclazide may protect pancreatic β-cells from apoptosis induced by an oxidative stress. However, the precise mechanism(s) of this action are not fully understood and requires further clarification. Therefore, using human normal and cancer cells we examined whether the anti-apoptotic effects of this sulfonylurea is due to its free radical scavenger properties. Hydrogen peroxide (H₂O₂) as a model trigger of oxidative stress was used to induce cell death. Our experiments were performed on human normal cell line (human umbilical vein endothelial cell line, HUVEC-c) and human cancer cell lines (human mammary gland cell line, Hs578T; human pancreatic duct epithelioid carcinoma cell line, PANC-1). To assess the effect of gliclazide the cells were pre-treated with the drug. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay was employed to measure the impact of gliclazide on cell viability. Generation of reactive oxygen species, mitochondrial membrane potential (∆Ψ_m), and intracellular Ca²⁺ concentration [Ca²⁺] were monitored. Furthermore, the morphological changes associated with apoptosis were determined using double staining with Hoechst 33258-propidium iodide (PI). Gliclazide protects the tested cells from H₂O₂-induced cell death most likely throughout the inhibition of ROS production. Moreover, the drug restored loss of ΔΨ_m and diminished intracellular [Ca²⁺] evoked by H₂O₂. Double staining with Hoechst 33258-PI revealed that pre-treatment with gliclazide diminished the number of apoptotic cells. Our findings indicate that gliclazide may protect both normal and cancer human cells against apoptosis induced by H₂O₂. It appears that the anti-apoptotic effect of the drug is most likely associated with reduction of oxidative stress.     

Release of Calcium from Mitochondrial and Nonmitochondrial Intracellular Stores in Mouse Pancreatic Acinar Cells by Hydrogen Peroxide

In the present study we have studied how [Ca²⁺] _i is influenced by H₂O₂ in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mm H₂O₂ caused a slow sustained [Ca²⁺] _i increase, reaching a stable plateau after 10–15 min of perfusion. This increase induced by H₂O₂ was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application of 1 mm H₂O₂ to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration of CCK-8 (1 nm) or thapsigargin (0.5 μm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 μm). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H₂O₂-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and FCCP in the presence of H₂O₂ did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a sustained increase of [Ca²⁺] _i . In addition, H₂O₂ was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca²⁺] _i induced by H₂O₂ was abolished by an addition of 2 mm dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H₂O₂ releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H₂O₂ is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases. Received: 15 May 2000/Revised: 4 October 2000     

Salicylic Acid induces cyanide-resistant respiration in tobacco cell-suspension cultures

Cyanide-resistant, alternative respiration in Nicotiana tabacum L. cv Xanthi-nc was analyzed in liquid suspension cultures using O₂ uptake and calorimetric measurements. In young cultures (4-8 d after transfer), cyanide inhibited O₂ uptake by up to 40% as compared to controls. Application of 20 μm salicylic acid (SA) to young cells increased cyanide-resistant O₂ uptake within 2 h. Development of KCN resistance did not affect total O₂ uptake, but was accompanied by a 60% increase in the rate of heat evolution from cells as measured by calorimetry. This stimulation of heat evolution by SA was not significantly affected by 1 mm cyanide, but was reduced by 10 mm salicylhydroxamic acid (SHAM), an inhibitor of cyanide-resistant respiration. Treatment of SA-induced or uninduced cells with a combination of cyanide and SHAM blocked most of the O₂ consumption and heat evolution. Fifty percent of the applied SA was taken up within 10 min, with most of the intracellular SA metabolized in 2 h. 2,6-Dihydroxybenzoic and 4-hydroxybenzoic acids also induced cyanide-resistant respiration. These data indicate that in tobacco cell-suspension culture, SA induces the activity and the capacity of cyanide-resistant respiration without affecting the capacity of the cytochrome c respiration pathway.     

Oxygen inhibition of photosynthesis and stimulation of photorespiration in soybean leaf cells

The occurrence of photorespiration in soybean (Glycine max [L.] Merr.) leaf cells was demonstrated by the presence of an O₂-dependent CO₂ compensation concentration, a nonlinear time course for photosynthetic ¹⁴CO₂ uptake at low CO₂ and high O₂ concentrations, and an O₂ stimulation of glycine and serine synthesis which was reversed by high CO₂ concentration. The compensation concentration was a linear function of O₂ concentration and increased as temperature increased. At atmospheric CO₂ concentration, 21% O₂ inhibited photosynthesis at 25 C by 27%. Oxygen inhibition of photosynthesis was competitive with respect to CO₂ and increased with increasing temperature. The Km (CO₂) of photosynthesis was also temperature-dependent, increasing from 12 μm CO₂ at 15 C to 38 μm at 35 C. In contrast, the Ki (O₂) was similar at all temperatures. Oxygen inhibition of photosynthesis was independent of irradiance except at 10 mm bicarbonate and 100% O₂, where inhibition decreased with increasing irradiance up to the point of light saturation of photosynthesis. Concomitant with increasing O₂ inhibition of photosynthesis was an increased incorporation of carbon into glycine and serine, intermediates of the photorespiratory pathway, and a decreased incorporation into starch. The effects of CO₂ and O₂ concentration and temperature on soybean cell photosynthesis and photorespiration provide further evidence that these processes are regulated by the kinetic properties of ribulose-1,5-diphosphate carboxylase with respect to CO₂ and O₂.     

A study of the impaired growth of roots of Zea mays seedlings at low oxygen concentrations

Abstract. Seedlings of Zea mays L. were grown in the dark at 27°C. Four-day-old seedlings were then exposed for 3 days to solutions equilibrated with gas mixtures to give O₂ concentrations between 0.02 and 0.25 mol m^?3. Root growth was impaired just as severely at 0.06 as 0.02 mol O₂ m^?3 while growth at 0.16 mol O₂ m^?3 was about the same as in solutions in equilibrium with air (0.25 mol O₂ m^?3). Growth of young seedlings at low O₂ concentrations was inhibited to the same extent in nutrient solution and 0.5 ml m^?3 CaCl₂, showing that the adverse effect of O₂ deficits on growth was not due to less uptake of inorganic nutrients. Furthermore, at low O₂ concentrations neither exposure of the shoots to a relative humidity of 100% (26.0 g H₂O m^?3) nor excision of the entire shoot enhanced root growth relative to that in plants with shoots at a relative humidity of 50% (13.0 g H₂O m^?3). Therefore, for these seedlings growing in the dark, impairment of root growth at low O₂ concentrations was not a consequence of water deficits in the shoot or of other shoot-root interactions. Total soluble sugars and amino acid concentrations were generally greater at low (0.02–0.06 mol O₂m^?3) than at high O₂ concentrations (0.16–0.25 mol O₂ m ^?3). This applied specifically to the root apices (0–2 mm) and expanding (2–15 mm) tissue except in some experiments where sugar concentrations in expanding tissue were slightly greater at high than at low O₂ concentrations. Critical O₂ pressures for respiration of excised root segments were approximately 0.117 and 0.065 mol O₂ m^?3 in the expanding and expanded zones of the roots, respectively. In contrast, the critical O₂ pressure exceeded 0.20 mol O₂ m^?3 in the apex, suggesting that O₂ supply for metabolic processes is most likely to be sub-optimal in this zone. Our results show clearly that the adverse effects of low O₂ concentrations are unlikely to be a consequence of substrate shortage for either respiration or synthesis of macromolecules; low rates of ATP regeneration in growing root tissues are the logical cause for impaired growth in young seedlings while they are being sustained by seed reserves.     

Interactive effect of leaf age and ozone on mesophyll conductance in Siebold's beech

Mesophyll conductance (G_m) is one of the most important factors determining photosynthesis. Tropospheric ozone (O₃) is known to accelerate leaf senescence and causes a decline of photosynthetic activity in leaves. However, the effects of age-related variation of O₃ on G_m have not been well investigated, and we, therefore, analysed leaf gas exchange data in a free-air O₃ exposure experiment on Siebold's beech with two levels (ambient and elevated O₃: 28 and 62 nmol mol⁻¹ as daylight average, respectively). In addition, we examined whether O₃-induced changes on leaf morphology (leaf mass per area, leaf density and leaf thickness) may affect CO₂ diffusion inside leaves. We found that O₃ damaged the photosynthetic biochemistry progressively during the growing season. The G_m was associated with a reduced photosynthesis in O₃-fumigated Siebold's beech in August. The O₃-induced reduction of G_m was negatively correlated with leaf density, which was increased by elevated O₃, suggesting that the reduction of G_m was accompanied by changes in the physical structure of mesophyll cells. On the other hand, in October, the O₃-induced decrease of G_m was diminished because G_m decreased due to leaf senescence regardless of O₃ treatment. The reduction of photosynthesis in senescent leaves after O₃ exposure was mainly due to a decrease of maximum carboxylation rate (V_cmax) and/or maximum electron transport rate (J_max) rather than diffusive limitations to CO₂ transport such as G_m. A leaf age×O₃ interaction of photosynthetic response will be a key for modelling photosynthesis in O₃-polluted environments.     

Oxygen protection of nitrogenase in Frankia sp. HFPArI3

O₂ protection of nitrogenase in a cultured Frankia isolate from Alnus rubra (HFPArI3) was studied in vivo. Evidence for a passive gas diffusion barrier in the vesicles was obtained by kinetic analysis of in vivo O₂ uptake and acetylene reduction rates in response to substrate concentration. O₂ of NH ₄ ⁺ -grown cells showed an apparent K _m O₂ of approximately 1M O₂. In N₂-fixing cultures a second K _m O₂ of about 215 M O₂ was observed. Thus, respiration remained unsaturated by O₂ at air-saturation levels. In vivo, the apparent K _m for acetylene was more than 10-fold greater than reported in vitro values. These data were inter oreted as evidence for a gas diffusion barrier in the vesicles but not vegetative filaments of Frankia sp. HFPArI3.     

18F-Fluorodeoxyglucose Uptake and Tumor Hypoxia: Revisit 18F-Fluorodeoxyglucose in Oncology Application

This study revisited ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) uptake and its relationship to hypoxia in various tumor models. METHODS: We generated peritoneal carcinomatosis and subcutaneous xenografts of colorectal cancer HT29, breast cancer MDA-MB-231, and non–small cell lung cancer A549 cell lines in nude mice. The partial oxygen pressure (pO₂) of ascites fluid was measured. ¹⁸F-FDG accumulation detected by digital autoradiography was related to tumor hypoxia visualized by pimonidazole binding and glucose transporter-1 (GLUT-1) in frozen tumor sections. RESULTS: Ascites pO₂ was 0.90 ± 0.53 mm Hg. Single cancer cells and clusters suspended in ascites fluid as well as submillimeter serosal tumors stained positive for pimonidazole and GLUT-1 and had high ¹⁸F-FDG uptake. In contrast, ¹⁸F-FDG uptake was significantly lower in normoxic portion (little pimonidazole binding or GLUT-1 expression) of larger serosal tumors or subcutaneous xenografts, which was not statistically different from that in the liver. CONCLUSIONS: Glucose demand (¹⁸F-FDG uptake) in severely hypoxic ascites carcinomas and hypoxic portion of larger tumors is significantly higher than in normoxic cancer cells. Warburg effect originally obtained from Ehrlich ascites carcinoma may not apply to normoxic cancer cells. Our findings may benefit the better understanding of ¹⁸F-FDG PET in oncology application.     

Hydrogen Peroxide Inhibits Gap Junctional Coupling and Modulates Intracellular Free Calcium in Cochlear Hensen Cells

The double whole-cell patch-clamp configuration was applied to analyze gap junctional conductance (G _j ) of isolated pairs of cochlear supporting Hensen cells of guinea pig under control conditions and in the presence of hydrogen peroxide (H₂O₂). Under control conditions, the dependence of G _j on transjunctional voltage (V _j ) appeared to vary between different cell pairs with a maximum value of about 40 nS at V _j close to 0 mV. The voltage dependence and the maximum amplitude of G _j stayed constant for at least 2 hr. Addition of H₂O₂ to the bath at concentrations above 0.08 mm caused a significant decrease of G _j , but the membrane potential of about −30 mV was not affected. In parallel, intracellular free calcium ([Ca²⁺]_i) was followed using fura-2. At 0.8 mm H₂O₂, a sustained increase of [Ca²⁺]_i was observed, while 0.08 mm H₂O₂ evoked an oscillating-like behavior of [Ca²⁺]_i. We propose that the H₂O₂-evoked inhibition of gap junctional coupling of Hensen cells is closely related to pathophysiological conditions such as noise- induced hearing loss, aminoglycoside-related ototoxicity and presbycusis, which are known to be associated with production of free radicals. Received: 10 July 2000/Revised: 4 January 2001     

Novel role of TRPV2 in promoting the cytotoxicity of H2O2-mediated oxidative stress in Human hepatoma cells

Oxidative stress is important for the initiation and progression of cancers, which confers the cells with a survival advantage by inducing oxidative adaption and drug resistance. Therefore, developing strategies to promote oxidative stress-induced cytotoxicity could be important for cancer therapy. Herein, we found that H₂O₂-mediated oxidative stress increases TRPV2 expression in human hepatoma (HepG2 and Huh-7) cells. This occurred at the mRNA and protein levels in a dose-dependent manner. The significance of TRPV2 in promoting H₂O₂-induced cell death was demonstrated in gain and loss of function studies with overexpression and knockdown of TRPV2, respectively. Mechanistically, H₂O₂-induced cell death involves inhibition of pro-survival signaling proteins (Akt, Nrf2) and activation of pro-death signaling proteins (p38, JNK1). Overexpression of TRPV2 in H₂O₂-treated hepatoma cells aggravates the inhibition of Akt and Nrf2, while it enhances the activation of p38 and JNK1 at the early stage of cell death. Interestingly, increased expression of TRPV2 in HepG2 cells improved the efficacy of stress-associated chemicals to induce cell death. Our findings suggest that TRPV2 acts as an important enhancer for H₂O₂-induced cytotoxicity. This process occurred by the inhibition of Akt and Nrf2 as well as the early activation of p38 and JNK1. These findings have important implications for inhibition of oxidative adaption and drug resistance.     

Involvement of the oxidative burst in phytoalexin accumulation and the hypersensitive reaction

The role of the oxidative burst, transient production of activated oxygen species such as H₂O₂ and superoxide (O₂⁻) in elicitation of phytoalexins and the hypersensitive reaction (HR) was investigated in white clover (Trifolium repens L.) and tobacco (Nicotiana tabacum L.). H₂O₂ and O₂⁻ production was measured as chemiluminescence (CL) mediated by luminol, which was added to suspension-cultured white clover just before measurement in an out-of-coincidence mode scintillation counter. Maximum CL occurred between 10 and 20 min after addition of 0.4 × 10⁸ colony-forming units/mL of incompatible Pseudomonas corrugata or 158 μm HgCl₂. Autoclaved P. corrugata produced a slightly higher response. Elicitation of cells with 25 μm HgCl₂ did not produce CL. Preincubation of plant cells in superoxide dismutase, which converts O₂⁻ to H₂O₂, for 2 min before addition of bacteria did not significantly increase maximum CL levels (P ≥ 0.05). Preincubation of plant cells with catalase for 2 min before addition of bacteria prevented the increase in CL, confirming that H₂O₂ is the substrate for the luminol reaction. Addition of live bacteria or HgCl₂ (25 and 158 μm) to white clover increased levels of the phytoalexin medicarpin during a 24-h period, but addition of autoclaved bacteria did not elicit formation of medicarpin. Preincubation of plant cells with catalase, which quenched the bacteria-induced oxidative burst, did not decrease phytoalexin accumulation. Live bacteria infiltrated into Havana 44 tobacco leaf panels induced development of the HR, but autoclaved bacteria did not. Incubation of live bacteria with superoxide dismutase and catalase before infiltration into tobacco leaves did not interfere with development of the HR. Tobacco leaf panels infiltrated with up to 158 μm HgCl₂ did not develop an HR. These results suggest that an oxidative burst consisting of H₂O₂ and O₂⁻ does occur during these two plant defense responses, but it may not be a necessary element of the signaling system for HR and phytoalexin formation.     

Cysteine Suppresses Oxidative Stress-Induced Myofibrillar Proteolysis in Chick Myotubes

The effects of cysteine as an antioxidant nutrient on change in protein modification and myofibrillar proteolysis in chick myotubes by induction of oxidative stress by H₂O₂ treatment were investigated. Myotubes were treated for 1 h with H₂O₂ (1 mM). After this treatment, the H₂O₂ was removed and the cells were cultured in cysteine (0.1 and 1 mM) containing serum-free medium for 24 h. Protein carbonyl content as an index of protein modification and N^τ-methylhistidine release as an index of myofibrillar proteolysis were increased at 24 h after H₂O₂ treatment, and the increment was reduced by cysteine. Calpain, proteasome and cathepsin (B+L and D) activities were increased at 24 h after H₂O₂ treatment, and the increment was also reduced by cysteine. These results indicate that cysteine suppresses protein modification by oxidative stress, resulting in a decrease of protease acitivities, finally resulting in a decrease in myofibrillar proteolysis in chick myotubes.     

A Proposal for the Expression of the Foamability of Protein Solutions

Mutants exhibiting high catalase activity were derived from Candida boidinii S2 strain AOU-1, from among mutants resistant to H₂O₂, NaN₃ or 3-amino-1,2,4-triazole (ATA). The catalase activity of an ATA-resistant strain was improved by means of a methanol-limited chemostat culture with H₂O₂ supplementation. The catalase activity increased with increasing H₂O₂ concentration in the feed medium in the range where methanol did not remain. Alcohol oxidase activity increased after adaptation of the cells to H₂O₂. Cells of mutant strain SA051 grown under the optimal culture conditions produced 1200 mm formaldehyde in the reaction mixture.     

Enzyme defense against reactive oxygen derivatives. II. Erythrocytes and tumor cells

Summary The enzymatic destruction of oxidizing products produced during metabolic reduction of oxygen in the cell (such as singlet oxygen, H₂O₂ and OH radical) involves the concerted action of superoxide dismutase-which removes O ₂ ^- and yields H₂O₂-and H₂O₂ removing enzymes such as catalase and glutathione peroxidase. A difference in distribution or ratio of these enzymes in various tissues may result in a different reactivity of oxygen radicals.It was found that in red blood cells superoxide dismutase and catalase are extracted in the same fraction as hemoglobin, while glutathione peroxidase appears to be loosely bound to the cellular structure. This suggests that in red blood cells catalase acts in series with superoxide dismutase against bursts of oxygen radicals formed from oxyhemoglobin, while glutathione & peroxidase may protect the cell membrane against low concentrations of H₂O₂. On the other hand, catalase activity is absent in various types of ascites tumor cells, while glutathione peroxidase and superoxide dismutase are found in the cytoplasm. However, the peroxidase/dismutase ratio is lower than in liver cells, and this may provide an explanation for the higher susceptibility of tumor cells to treatments likely to involve oxygen radicals.     

Ecosystem and macrophyte primary production of the Fort River,Massachusetts

Primary production and ecosystem respiration of the Fort River ecosystem, a medium size (mean discharge 1.4 m³/sec) lowland stream in central Massachusetts, U.S.A., were measured using diurnal oxygen techniques from May 1972 to November 1973. During the summer of 1973, vascular hydrophyte production was measured with a modified cropping technique. Whole ecosystem gross primary production ranged from 0.44 g O₂/m²:day in winter to 6.50 g O₂/m^2.day in summer, and averaged 1.78 g O₂/m^2.day for 12 months. Mean ecosystem respiration was 3.65 g O₂/m^2.day for 12 months. Mean ecosystem respiration was 3.65 g O₂/m^2.day.Macrophyte gross production (59.9 g O₂/m^2.year) constitutes 9.2% of annual ecosystem productivity and 15.2% of summer primary production. Macrophytes were little grazed and entered food webs only after death, as detritus. Decomposition occurred near the site of production at relatively rapid rates, thus transport of dead macrophyte material in stream water was low. Data from this and other stream ecosystems suggest that in general, streams are only moderately productive ecosystems which depend to varying degrees on watershed-derived organic matter inputs.     

Synthesis,properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5′,5′-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH₃-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH₃-analogs and the corresponding analogs containing S instead of BH₃ (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH₃-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m₂^7,2′-OGpp_BH3pG D1 and m₂^7,2′-OGpp_BH3pG D2, respectively, than for in vitro transcribed mRNA capped with m₂^7,3′-OGpppG. Higher expression of cancer antigens would make mRNAs containing m₂^7,2′-OGpp_BH3pG D1 and m₂^7,2′-OGpp_BH3pG D2 favorable for anticancer immunization.     

Retention of calcium by mitochondria isolated from Ehrlich ascites tumor cells

Tightly coupled mitochondria isolated from Ehrlich ascites tumor cells accumulate and retain high concentrations of Ca²⁺ in the presence of ATP for periods up to at least 20 min at 25 °C. The presence of inorganic phosphate up to 20 mm does not prevent such Ca²⁺ retention. The tumor mitochondria accumulate Ca²⁺ in the presence of succinate as an energy source but lose the Ca²⁺ after 1–2 min. Addition of ATP (K_m approx 1 mm) to the incubation medium after Ca²⁺ release, induces reaccumulation of the ion. Thus, the ability of the tumor mitochondria to retain Ca²⁺ differs markedly from that of rat liver mitochondria and is seen as being of potential biological significance to the unique metabolic behavior of the ascites tumor cells.     

Photoreaction of manganese catalyst in photosynthetic oxygen evolution

The formation of active O₂ evolving centers following addition of Mn²⁺ to Mn deficient Anacystis nidulans cells yielded an estimate of 6 to 12 Mn atoms associated with each O₂ evolving reaction center. Restoration of activity upon addition of Mn ions is affected in 3 ways: (1) Stimulation of the uptake of exogenous Mn into the cells—this uptake occurs in darkness, but is enhanced 5 to 10 fold by light; a high concentration of DCMU (1 × 10⁻⁵m) decreases this light enhanced influx no more than 50 to 75%; (2) Photoreactivation of the O₂ evolving centers, after excess Mn has been accumulated in the cells essentially no increase in Hill activity is observed unless the cells are illuminated. This photoreactivation is fully inhibited by 10⁻⁶m DCMU and partially by benzoquinone. The Q₁₀ of photoreactivation proper is close to 1; (3) Photoinhibition of the activation—photoreactivation occurs most effectively in weak intensities (< one-fiftieth photosynthetic saturation in normal cells). Apparently at higher intensities an inhibitory photoprocess is overriding. This inhibition proved reversible. The photoreactivation leads to new stable O₂ evolving centers as evidenced by an increase in the rate at saturating intensity, quantum yield, and the O₂ gush.