Bacterial dissolution of pyrite by Thiobacillus ferrooxidans

The kinetics of the dissolution of pure pyrite (FeS₂) particles by Thiobacillus ferrooxidans were studied both theoretically and experimentally. Adsorption and dissolution experiments were carried out at 30 °C and pH=2, by using a batch reactor. The adsorption process of T. ferrooxidans to pyrite surface was rapid in comparison with the bacterial dissolution process. The experimental results for the adsorption equilibrium were well correlated by the Langmuir type isotherm. The growth rate of adsorbed bacteria was found to be proportional to the product of the number of adsorbed cells and the fraction of solid surface unoccupied by cells. A new kinetic model for the bacterial dissolution was presented, and shown to correlate well with the experimental data for the rate of bacterial dissolution and for the time variation in the number of cells in the liquid phase. The specific growth rate of adsorbed bacteria was also evaluated.List of Symbols f weight fraction of iron in pyrite - K _A m³/cells equilibrium constant for cell adsorption - R _A cells/d m³-mixture growth rate of bacteria adsorbed on solid surface - R _L cells/d m³-mixture growth rate of free bacteria in the liquid phase - t d time - V m³ volume of solid-liquid mixture - W kg weight of pyrite - W ₀ kg initial weight of pyrite - X _A cells/kg-solid number of adsorbed cells on solid surface - X _Am cells/kg-solid maximum adsorption capacity - X _L cells/m³-liquid number of free cells existing in the liquid phase - X _T cells/m³-mixture total number of cells - X _TO cells/m³ initial total number of cells - Y _A cells/kg-FeS₂ growth yield of adsorbed bacteria - Y _L cells/kg-Fe²⁺ growth yield of free bacteria - [Fe] _T kg/m³-liquid concentration of total iron in the liquid phase - fraction of pyrite dissolved - _V fraction of adsorption sites unoccupied by cells - _A d^–1 specific growth rate of adsorbed bacteria - _L d^–1 specific growth rate of free bacteria - volume fraction of solid particles in solid-liquid mixture     

Photoinhibition of colonial and unicellular <Emphasis Type="Italic">Microcystis</Emphasis> cells in a summer bloom in Lake Taihu

To evaluate the photoinhibition of colonial and unicellular cells of Microcystis aeruginosa under natural conditions, the maximum and effective quantum yields of photosystem II were measured from variable chlorophyll a fluorescence in samples from Lake Taihu during a summer bloom from June 19 to 21, 2006. Diurnal changes in the photoinhibition of Microcystis cells incubated immediately below the surface in clear bottles for 30 min and in situ samples under natural conditions were measured. At solar noon during the three days, the mean values of maximum quantum yield (F _v/F _m) and effective quantum yield (ΔF/F _m′) for unicellular cells (F _v/F _m = 0.15, ΔF/F _m′ = 0.10) were lower than those for colonial cells (F _v/F _m = 0.25, ΔF/F _m′ = 0.15). For in situ samples, the values of F _v/F _m and ΔF/F _m′ for colonial cells at solar noon on the three days (F _v/F _m 0.30, 0.25, 0.29; ΔF/F _m′ 0.24, 0.21, 0.22) were also higher than those of unicellular cells (F _v/F _m 0.26, 0.18, 0.25; ΔF/F _m′ 0.15, 0.11, 0.14). The results indicate that colony formation has a protective effect on Microcystis cells by reducing the occurrence of photoinhibition under high light intensities.     

Oxidative stress impairs autophagic flux in prion protein-deficient hippocampal cells

We previously reported that autophagy is upregulated in Prnp-deficient (Prnp^0/0) hippocampal neuronal cells in comparison to cellular prion protein (PrP^C)-expressing (Prnp^+/+) control cells under conditions of serum deprivation. In this study, we determined whether a protective mechanism of PrP^C is associated with autophagy using Prnp^0/0 hippocampal neuronal cells under hydrogen peroxide (H₂O₂)-induced oxidative stress. We found that Prnp^0/0 cells were more susceptible to oxidative stress than Prnp^+/+ cells in a dose- and time-dependent manner. In addition, we observed enhanced autophagy by immunoblotting, which detected the conversion of microtubule-associated protein 1 light chain 3 β (LC3B)-I to LC3B-II, and we observed increased punctate LC3B immunostaining in H₂O₂-treated Prnp^0/0 cells compared with H₂O₂-treated control cells. Interestingly, this enhanced autophagy was due to impaired autophagic flux in the H₂O₂-treated Prnp^0/0 cells, while the H₂O₂-treated Prnp^+/+ cells showed enhanced autophagic flux. Furthermore, caspase-dependent and independent apoptosis was observed when both cell lines were exposed to H₂O₂. Moreover, the inhibition of autophagosome formation by Atg7 siRNA revealed that increased autophagic flux in Prnp^+/+ cells contributes to the prosurvival effect of autophagy against H₂O₂ cytotoxicity. Taken together, our results provide the first experimental evidence that the deficiency of PrP^C may impair autophagic flux via H₂O₂-induced oxidative stress.     

Constitutive and inducible aspects of nitrate-nitrogen uptake by Chlamydomonas reinhardtii

Similarly to higher plant root systems, Chlamydomonas reinhardtii Dangeard (UTEX 90) cells exhibited biphasic NO₃^? uptake kinetics. The uptake pattern was similar in cells cultured in 10 mM NO₃^? (NO₃^?-grown), 0.25 mM NO₃^? (N-limited) or 10 mM NO₃^? followed by an 18-h period of N-deprivation (N-starved). In all cell types there was an apparent phase transition in uptake at 1.1 mM NO₃^?, although there were variations in the uptake V_max of both isotherms. The rate of uptake via isotherm 0 ([NO₃^?]<1.1 mM) in N-limited cells was higher than that of either NO₃^?-grown or N-starved cells. In contrast, NO₃^?-grown and N-limited cells exhibited comparable V_max values when supplied with 1.1 to 1.8 mM NO₃^? (isotherm 1). When supplied with 1.6 mM NO₃^?, both N-limited and N-starved cells exhibited enhanced linear uptake after 60 min of incubation. We ascribed this to an induction phenomenon. This trend was not observed when NO₃^?-grown cells were supplied with 1.6 mM NO₃^?, or when N-limited and N-starved cells were supplied with 0.6 mM NO₃^?. The ‘inducible’ aspect of uptake by N-limited cells was blocked by cycloheximide (10 mg l^?1), but not by actinomycin D (5 mg l^?1), thus indicating the involvement of a translational or post-translational event. To investigate this phenomenon further, we analysed the cell proteins of N-limited cells supplied with either 0.6 or 1.6 mM NO₃^? for 90 min, using two-dimensional gel electrophoresis. Comparison of protein profiles enabled the identification of a single cell membrane-associated polypeptide (21 kDa, pI ca 5.5) and ten soluble fraction polypeptides (17–73 kDa, pI ca 5.0 to 7.1) unique to the high NO₃^? treatment. We propose that the ‘inducible’ portion of NO₃^? uptake may provide the means by which C. reinhardtii cells regulate uptake in accordance with assimilatory capacity.     

Comparison of the surface structures of choline-less Torulopsis pintolopesii grown on defined media with choline or methionine

Summary The chemical composition of cell walls from choline-less Torulopsis pintolopesii grown with choline or with methionine was studied. Methioninegrown cells synthesized a weakened cell wall compared to normal choline-grown yeast. The ethylenediamine fractionation procedure yielded three fractions—A, B, and C—with different solubilities. Glucose and mannose were detected in hydrolysed unfractionated cell walls from yeasts grown under both conditions as well as in all fractions. Glucose content was greater in fractions B and C from methioninegrown cells; the mannose content was about the same. Walls from choline-grown cells (W _c ) had 25% more protein than walls from methionine-grown cells (W _m ). The amino acid composition of the proteins of W _c and W _m was not qualitatively altered. Seventeen amino acids were identified; glutamic and aspartic acids and valine predominated. W _c had 3.5 times more lipid than W _m . The amount of phosphorus was the same. Yeasts grown on methionine synthesized more ergosterol than choline-grown cells. The rate of formation of spheroplasts was higher in methionine-grown cells. Rates of incorporation of adenine, glutamic acid, and uracil were similar in cells grown on methionine or choline; incorporation of phenylalanine and tyrosine was depressed in methionine-grown cells.     

Effect of adherence,cell morphology,and lipopolysaccharide on potassium conductance and passive membrane properties of murine macrophage J774.1 cells

Summary The effects of adherence, cell morphology, and lipopolysaccharide on electrical membrane properties and on the expression of the inwardly rectifying K conductance in J774.1 cells were investigated. Whole-cell inwardly rectifying K currents (K _i), membrane capacitance (C _m), and membrane potential (V _m) were measured using the patch-clamp technique. SpecificK _i conductance (G _K i, whole-cell Ki conductance corrected for leak and normalized to membrane capacitance) was measured as a function of time after adherence, and was found to increase almost twofold one day after plating. Membrane potential (V _m) also increased from –42±4 mV (n=32) to –58±2 mV (n=47) over the same time period.G _K i andV _m were correlated with each other;G _L (leak conductance normalized to membrane capacitance) andV _m were not. The magnitudes ofG _K i andV _m 15 min to 2 hr after adherence were unaffected by the presence of 100 m cycloheximide, but the increase inG _K iandV _m that normally occurred between 2 and 8 hr after adherence was abolished by cycloheximide treatment. Membrane properties were analyzed as a function of cell morphology, by dividing cells into three categories ranging from small round cells to large, extremely spread cells. The capacitance of spread cells increased more than twofold within one day after adherence, which indicates that spread cells inserted new membrane. Spread cells had more negative resting membrane potentials than round cells, butG _K i andG _L were not significantly different. Lipopolysaccharide-(LPS; 1 or 10 g/ml) treated cells showed increasedC _m compared to control cells plated for comparable times. In contrast to the effect of adherence, LPS-treated cells exhibited a significantly lowerG _K i than control cells, indicating that the additional membrane did not have as high a density of functionalG _K i channels. We conclude that both adherence and LPS treatment increase the total surface membrane area of J774 cells and change the density of Ki channels. In addition, this study demonstrates that membrane area and density of Ki channels can vary independently of one another.     

Photorespiratory ammonium assimilation in chloroplasts of Chlamydomonas reinhardtii

The effect of CO₂ concentration on the rate of photorespiratory ammonium excretion and on glutamine synthetase (GS) and carbonic anhydrase (CA) isoenzymes activities has been studied in Chlamydomonas reinhardtii cw-15 mutant (lacking cell wall) and in the high CO₂-requiring double mutant cia-3/cw-15 (lacking cell wall and chloroplastic carbonic anhydrase). In cw-15 cells, both the extracellular (CA_ext) and chloroplastic (CA_chl) CA activities increased after transferring cells from media bubbled with 5% CO₂ in air (v/v, high-C_i cells) to 0.03% CO₂ (low-C_i cells), whereas in cia-3/cw-15 cells only the CA_ext was induced after adaptation to low-C_i conditions and the CA_chl activity was negligible. During adaptation to low-C_i conditions in the presence of 1 mM of l-methionine-D,L-sulfoximine (MSX), a specific inhibitor of GS activity, both mutant strains excreted photorespiratory ammonium into nitrogen free medium. In addition, the ammonium excretion rate by cw-15 in the presence of MSX was lower in cells grown and kept at 5% CO₂ than in high-C_i cells adapted to 0.03% CO₂. The double mutant cia-3/cw-15 excreted photorespiratory ammonium at a higher rate than did cw-15. Total GS activity (GS-1 plus GS-2) increased during adaptation to 0.03% CO₂ in both strains of C. reinhardtii. However, only the activity GS-2, which is located in the chloroplast, increased during the adaptation to low CO₂, whereas the cytosolic GS-1 levels remained similar in high and low-C_i cells. We conclude that: (1) cia-3/cw-15 cells lack chloroplastic CA activity; (2) in C. reinhardtii photorespiratory ammonium is refixed in the chloroplasts through the GS-2/GOGAT cycle; and (3) chloroplastic GS-2 concentration changes in response to the variation of environmental CO₂ concentration.     

Transformation inBacillus subtilis. Initial stages of the transformation process and their energy dependence

NaN₃ was found to inhibit transformation but not the irreversible binding of donor³H-DNA in competent cells of the original low-transformable strainBacillus subtilis 168trp ₂ . Addition of NaN₃ to cells of two mutantsBacillus subtilis HT39 and HT46 with an increased transformability decreased substantially the irreversible binding of the donor DNA to the competent cells. The decreased irreversible binding of DNA is caused by an increased osmotic sensitivity of competent cells of the mutants HT39 and HT46 in the presence of NaN₃, leading preferentially to lysis of the competent cells.     

Phenotypic analysis of the ccp1Δ and ccp1Δ-ccp1 mutant strains of Saccharomyces cerevisiae indicates that cytochrome c peroxidase functions in oxidative-stress signaling

Yeast cytochrome c peroxidase (CCP) efficiently catalyzes the reduction of H₂O₂ to H₂O by ferrocytochrome c in vitro. The physiological function of CCP, a heme peroxidase that is targeted to the mitochondrial intermembrane space of Saccharomyces cerevisiae, is not known. CCP1-null-mutant cells in the W303-1B genetic background (ccp1Δ) grew as well as wild-type cells with glucose, ethanol, glycerol or lactate as carbon sources but with a shorter initial doubling time. Monitoring growth over 10 days demonstrated that CCP1 does not enhance mitochondrial function in unstressed cells. No role for CCP1 was apparent in cells exposed to heat stress under aerobic or anaerobic conditions. However, the detoxification function of CCP protected respiring mitochondria when cells were challenged with H₂O₂. Transformation of ccp1Δ with ccp1^W191F, which encodes the CCP^W191F mutant enzyme lacking CCP activity, significantly increased the sensitivity to H₂O₂ of exponential-phase fermenting cells. In contrast, stationary-phase (7-day) ccp1Δ-ccp1^W191F exhibited wild-type tolerance to H₂O₂, which exceeded that of ccp1Δ. Challenge with H₂O₂ caused increased CCP, superoxide dismutase and catalase antioxidant enzyme activities (but not glutathione reductase activity) in exponentially growing cells and decreased antioxidant activities in stationary-phase cells. Although unstressed stationary-phase ccp1Δ exhibited the highest catalase and glutathione reductase activities, a greater loss of these antioxidant activities was observed on H₂O₂ exposure in ccp1Δ than in ccp1Δ-ccp1^W191F and wild-type cells. The phenotypic differences reported here between the ccp1Δ and ccp1Δ-ccp1^W191F strains lacking CCP activity provide strong evidence that CCP has separate antioxidant and signaling functions in yeast.     

Inhibition of photosynthesis by intracellular carbonic anhydrase in microalgae under excess concentrations of CO2

When cells of Chlorococcum littorale that had been grown in air (air-grown cells) were transferred to extremely high CO₂ concentrations (>20%), active photosynthesis resumed after a lag period which lasted for 1–4 days. In contrast, C. littorale cells which had been grown in 5% CO₂ (5% CO₂-grown cells) could grow in 40% CO₂ without any lag period. When air-grown cells were transferred to 40% CO₂, the quantum efficiency of PS II (Φ_II) decreased greatly, while no decrease in Φ_II was apparent when the 5% CO₂-grown cells were transferred to 40% CO₂. In contrast to air-grown cells, 5% CO₂-grown cells showed neither extracellular nor intracellular carbonic anhydrase (CA) activity. Upon the acclimation of 5% CO₂-grown cells to air, photosynthetic susceptibility to 40% CO₂ was induced. This change was associated with the induction of CA. In addition, neither suppression of photosynthesis nor arrest of growth was apparent when ethoxyzolamide (EZA), a membrane-permeable inhibitor of CA, had been added before transferring air-grown cells of C. littorale to 40% CO₂. The intracellular pH value (pH_i) decreased from 7.0 to 6.4 when air-grown C. littorale cells were exposed to 40% CO₂ for 1–2 h, but no such decrease in pH_i was apparent in the presence of EZA. Both air- and 5% CO₂-grown cells of Chlorella sp. UK001, which was also resistant to extremely high CO₂ concentrations, grew in 40% CO₂ without any lag period. The activity of CA was much lower in air-grown cells of this alga than those in air-grown C. littorale cells. These results prompt us to conclude that intracellular CA caused intracellular acidification and hence inhibition of photosynthetic carbon fixation when air-grown C. littorale cells were exposed to excess concentrations of CO₂. No such harmful effect of intracellular CA was observed in Chlorella sp. UK001 cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Differential effects of nitrogen mustard in vivo on the cancer and host cells in MCa-11 tumours

We analysed the effects of nitrogen mustard (HN₂) on the growth, cell cycle distributions, and ratios of tumour cells to host cells for MCa-11 tumours grown in vivo. Treatment of tumour-bearing BALB/c mice with 3 mg/kg of HN₂ produced a significant slowing of MCa-11 tumour growth. Seventy-two hours after treatment in vivo with either 3 or 4 mg/kg of HN₂, the host cells in the treated tumours showed a significantly decreased G₀/G₁ peak and an increased G₂/M peak (P < 0.01), whereas the cancer cells in the treated tumours showed significant increases in the G₀/G₁ peak coupled with relatively decreased proportions of S and G₂/M tumour cells (P < 0.001). The ratio of the total number of cancer cells to the total number of host cells in the tumours was significantly increased 72 h after HN₂ administration (P<0.01). Thirty-two days after treatment with HN₂, the cell cycle distributions of the host and tumour cells in the treatment and control tumours had returned to being identical, but the ratio of the total number of cancer cells to the total number of host cells remained increased in the treated tumours (P<0.01). These results show that the administration in vivo of HN₂ can lead to entirely different cell cycle effects for the host and cancer cells in the same tumour, and that the partial growth arrest of MCa-11 tumours from HN₂ treatment may be due in part to the preferential destruction of host cells rather than solely to a direct cytotoxic effect on the cancer cells.     

Fine structure of the pyloric region and Malpighian papillae of protura (Insecta Apterygota)

The pyloric region of Eosentomon and Acerentomon (Insecta, Protura) is described. In both species the posterior cells of the midgut carry short microvilli. Beneath the epithelial cells there is a muscular pyloric sphincter for closing the intestinal lumen. Behind the sphincter is a wide pyloric chamber lined by cells with very long microvilli which point anteriorly toward the midgut. These cells regulate the passage of the intestinal contents into the hindgut. Secretions from the Malpighian papillae are emitted into the gut at this level. In Eosentomon three regions (R₁, R₂ and R₃) are visible in the Malpighian papillae, whereas in Acerentomon region R₁ is lacking. The R₁ region contains secretory cells with elaborate glycoprotein-containing granules. The R₂ region is composed of cells somewhat resembling the secretory cells of Malpighian tubules of insects. Presumably R₁ and R₂ cells emit secretions into the central cavity of each papilla. Cells of R₃ form a duct for the secretion. It is suggested that the R₂ region represents a basic excretory region, common to Protura, whereas the R₁ region, in Eosentomon, may be a specialized area performing supplementary excretory functions.     

Continuous production of itaconic acid by Aspergillus terreus immobilized in a porous disk bioreactor

Summary Aspergillus terreus NRRL 1960 was grown on porous disks rotating intermittently in and out of the liquid phase. This immobilized fungal cell bioreactor was used to produce itaconic acid from glucose in a continuous operation. The effect of temperature, pH, disk rotation speed, and feed rate on the itaconic acid concentration and volumetric productivity were studied. The highest itaconic acid concentration and volumetric productivity obtained were 18.2 g/l and 0.73 g/l·h, respectively, under the following conditions: temperature at 36°C, pH 3.0, disk rotation speed at 8 rpm, and feed rate at 60 ml/h. These results are better than those by conventional fermentation or by other immobilized method.Nomenclature F feed rate (l/h) - K _1s saturation constant for immobilized cells (g/l) - K _2s saturation constant for suspended cells (g/l) - M ₁ increased mass of immobilized cells (g) - M ₂ total mass of immobilized cells (g) - P concentration of itaconic acid (g/l) - S substrate concentration in and out of the reactor (g/l) - S ₀ substrate concentration in the feed (g/l) - V liquid volume of the reactor (1) - X concentration of the suspended cells (g/l) - Y ₁ apparent yield of the immobilized cells (g cells/g substrate) - Y ₂ apparent yield of the suspended cells (g cell/g substrate) - Y ₃ apparent yield of itaconic acid (g itaconic acid/g substrate) - m ₁ maintenance and by-products coefficient of the immobilized cells (g substrate/g cell·h) - m ₂ maintenance and by-products coefficient of the suspended cells (g substrate/g cell·h) - µ_1max maximum specific growth rate of the immobilized cells (h^-1) - µ_2max maximum specific growth rate of the suspended cells (h^-1)     

Developmental changes in the calcium currents in embryonic chick ventricular myocytes

Summary Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from totalI _Ca activated from a holding potential (V _h) of –80 mV. L-type current (I _L) was activated by depolarizing steps fromV _h –30 or –40 mV. The difference current (I _T) was obtained by subtractingI _L, fromI _Ca.I _T could also be distinguished pharmacologically fromI _L in these cells.I _T was selectively blocked by 40–160 m Ni²⁺, whereasI _L was suppressed by 1 m D600 or 2 m nifedipine. The Ni²⁺-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those ofI _T andI _L defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells,I _T andI _L could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nonetheless, in mostNS cells,I _Ca was partly blocked by Ni²⁺ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separateI _T andI _L by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis. In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V _1/2) of the Ni²⁺-resistant currentI _L averaged –18 mV. In contrast,V _1/2 of the Ni²⁺-sensitiveI _T was –60 mV in 14-day cells, –52 mV in 7-dayS cells, and –43 mV in 7-day NS cells. The half-activation potential was near –2 mV forI _L at both ages, but that ofI _T was –38 mV in 14-day and –29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components ofI _Ca is a negative shift with, embryonic age in the activation and inactivation relationships ofI _T along the voltage axis.     

Glycosylation Influences Gating and pH Sensitivity of I sK

The KvLQT1 and minK subunits that coassemble to form I _sK channels, contain potential N-glycosylation sites. To examine the role of glycosylation in channel function, a Chinese hamster ovary cell line deficient in glycosylation (Lec-1) and its parental cell line (Pro-5) were transiently transfected with human KvLQT1 (hKvLQT1) cDNA, alone and in combination with the rat (rminK) or human minK (hminK) cDNA. Functional KvLQT1 and I _sK currents were expressed in both cell lines, although amplitudes were larger in Pro-5 than Lec-1 cells transfected with hKvLQT1 and hKvLQT1/hminK. For I _sK , but not KvLQT1, the voltage-dependence of activation was shifted to more positive voltages and the activation kinetics were slower in the Lec-1 compared to the Pro-5 cells. The effect of extracellular acidification on recombinant KvLQT1 and I _sK currents was investigated in Pro-5 and Lec-1 cells. Changing external pH (pH _o ) from 7.4 to 6.0 significantly decreased the amplitude and increased the half-activation voltage (V _1/2) of KvLQT1 currents in Pro-5 and Lec-1 cells. In Pro-5 cells, decreasing pH _o reduced I _sK amplitude without increasing V _1/2, whether rminK or hminK was coexpressed with hKvLQT. In contrast, changing pH _o from 7.4 to 6.0 did not significantly change I _sK amplitude in Lec-1 cells. Thus, oligosaccharides attached to the minK subunit affect not only the gating properties, but also the pH sensitivity of I _sK . Received: 12 November 1999/Revised: 31 May 2000     

EFFECTS OF CO2 CONCENTRATION DURING GROWTH ON THE INTRACELLULAR STRUCTURE OF CHLORELLA AND SCENEDESMUS (CHLOROPHYTA)1

Effects of CO₂ concentration during growth on intracellular structure were studied with ftve species of Chlorella and Scenedesmus obliquus. Cells grown under ordinary air conditions (low-CO₂ cells) had a well developed pyrenoid surrounded by starch, while those grown under high CO₂ conditions (high-CO₂ cells) had a less developed pyrenoid or no detectable pyrenoid. Two mitochondria, one at each side of the neck of the projection of the chloroplast close to the pyrenoid, were found in low CO₂ cells of C. vulgaris 11h. Usually, lamellar stacks extended in parallel in the chloroplast of low-CO₂ cells of C. vulgaris 11h, while a grana-like structure was found in high-CO₂ cells. However, in C. pyrenoidosa, grana like structures were found more commonly in low-CO₂ cells than in high-CO₂ cells. These results suggest that development of pyrenoid starch is generally correlated with growth under low CO₂ conditions, whereas CO₂-effects on lamellar stacking are species dependent.     

The promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice)

C₄ plants have two carboxylases which function in photosynthesis. One, phosphoenolpyruvate carboxylase (PEPC) is localized in mesophyll cells, and the other, ribulose bisphosphate carboxylase (RuBPC) is found in bundle sheath cells. In contrast, C₃ plants have only one photosynthetic carboxylase, RuBPC, which is localized in mesophyll cells. The expression of PEPC in C₃ mesophyll cells is quite low relative to PEPC expression in C₄ mesophyll cells. Two chimeric genes have been constructed consisting of the structural gene encoding β-glucuronidase (GUS) controlled by two promoters from C₄ (maize) photosynthetic genes: (i) the PEPC gene (pepc) and (ii) the small subunit of RuBPC (rbcS). These constructs were introduced into a C₃ cereal, rice. Both chimeric genes were expressed almost exclusively in mesophyll cells in the leaf blades and leaf sheaths at high levels, and no or very little activity was observed in other cells. The expression of both genes was also regulated by light. These observations indicate that the regulation systems which direct cell-specific and light-inducible expression of pepc and rbcS in C₄ plants are also present in C₃ plants. Nevertheless, expression of endogenous pepc in C₃ plants is very low in C₃ mesophyll cells, and the cell specificity of rbcS expression in C₃ plants differs from that in C₄ plants. Rice nuclear extracts were assayed for DNA-binding protein(s) which interact with a cis-regulatory element in the pepc promoter. Gel-retardation assays indicate that a nuclear protein with similar DNA-binding specificity to a maize nuclear protein is present in rice. The possibility that differences in pepc expression in a C₃ plant (rice) and C₄ plant (maize) may be the result of changes in cis-acting elements between pepc in rice and maize is discussed. It also appears that differences in the cellular localization of rbcS expression are probably due to changes in a trans-acting factor(s) required for rbcS expression.     

Influence of substrate transport on the activity of immobilized Papaver somniferum cells

Summary Intraparticle diffusion resistance was studied for Papaver somniferum cells immobilized by Ca alginate gel. In callus tissue, these plant cells convert codeinone to codeine. First, the diffusion rates of substrates in the gel were measured, followed by investigation of the consumption rates of the substrates by free cells. The consumption rate of sucrose was zero order in relation to sucrose concentration, whereas that of codeinone was first order in relation to its concentration. The oxygen consumption rate obeyed Michaelis-Menten type kinetics with respect to dissolved oxygen concentration. Combining the reaction rates and diffusion rates allows calculation of the extent of the effect of diffusion limitation on the overall reaction rates. The analysis showed that the effectiveness factor for each substrate was about unity and that the influence of diffusion resistance was negligible. However, the oxygen concentration decreased considerably inside the particle, and this may affect the activity of the plant cell for repeated use over a long time period. Thus, deactivation proceeds due to the oxygen deficit although the temporal reaction rate is not affected.Abbreviations C _c cell concentration (g/l) - C _cod codeinone concentration (g/l) - c _{O 2} dissolved oxygen concentration (g/l) - K _m constant in Eq. (3) (g/l) - K _cod rate constant in Eq. (1) (l/g of cells per second) - k _suc rate constant in Eq. (2) (g sucrose/g of cells per second) - R radius of particles (mm) - r distance from the centre of the particle (mm) - r _cod consumption rate of codeinone (g codeinone/g of cells per second) - r _{O 2} consumption rate of O₂ (g oxygen/g of cells per second) - r _suc consumption rate of sucrose (g sucrose/g of cells per second) - V _m maximum respiration rate (g oxygen/g of cells per second) T. Nozawa is now with the Department of Agricultural Chemistry, University of TokyoT. Isohara is now with the Nippon Steel Corporation     

Quantum Requirements of Photosynthetic Oxygen Evolution and 77 K Fluorescence Emission Spectra in Unicellular Green Algae Grown Under Low- and High-CO2-Conditions

Quantum requirements of photosynthetic oxygen evolution at 682 nm and fluorescence spectra at liquid nitrogen temperature (77 K), were investigated in Dunaliella tertiolecta, Chlamydomonas reinhardtii C-9, Chlorella vulgaris 11g, Chlorella vulgaris C3, and Chlorella pyrenoidosa 8b grown under low- and high-CO₂ conditions. Dunaliella, Chlamydomonas and C. vulgaris 11g show higher quantum requirements and a higher ratio of F_710–740/F_680–695 fluorescence when grown under low-CO₂ conditions, indicating a change in excitation energy distribution towards PS I. In C. pyrenoidosa the quantum requirement for low-CO₂ grown cells is higher than in high-CO₂ grown cells, but there was practically no change in the fluorescence ratio. In C. vulgaris C3, the quantum requirements of low- and high-CO₂ grown cells are the same, but the fluorescence ratio is higher in high-CO₂ grown cells than in low-CO₂ grown cells. These results indicate that most of the low-CO₂ grown cells require more PS I light than high-CO₂ grown cells. It is possible that this energy is used for cyclic electron flow. In C. vulgaris C 3, a mechanism may exist for excitation energy distribution which leads to the same quantum requirements under low- and high-CO₂ conditions.