KINETICS OF SILICON-LIMITED GROWTH IN THE FRESHWATER DIATOM ASTERIONELLA FORMOSA1,2

Growth rates of two clones of the freshwater planktonic diatom Asterionella formosa Hass. were measured under conditions in which external silicon concentrations controlled growth. Clone AfOH2 from Lake Ohrid, Yugoslavia, had a higher maximum growth rate (μ_max= 1.11 doublings/day) and apparent half-saturation constant (K_si] + Si_o= 1.93 μM Si) than clone L262 from Lake Windermere, England. (μ_max= 0.61 doublings/day; K_si+ Si_o= 1.09 μM Si). K_lim, the silicon concentration at μ= 0.9 μ_max, is 13.8 μM Si for clone AfOH2 and 6.5 μM Si for clone L262. These values agree well with published field observations showing A. formosa populations decreasing below 0.5 mg/l SiO₂ (= 8.4 μM Si). Calculations of yield gave a range of 0.5–1.5 μM Si/10⁶ cells for clone AfOH2 and 0.6–1.9 μM Si/10⁶ cells for clone L262.     

TEMPERATURE EFFECTS ON SILICON- AND PHOSPHORUS-LIMITED GROWTH AND COMPETITIVE INTERACTIONS AMONG THREE DIATOMS1

Three diatom species, Stephanodiscus hantzschii (Ehr.) Grun., Asterionella formosa Hass. and Fragilaria crotonensis Kitt. Hass. were isolated from Lake Maarsseveen where they are dominant and show a successional sequence. The physiological responses of each species to temperature and limitation by silicon and phosphorus were determined over the temperature range of 5° to 20° C using short-term batch culture methods. Stephanodiscus hantzschii had higher maximum growth rates than the other two species at all temperatures, and the maximum growth rates of all species increased with increasing temperature. Temperature affected not only maximum growth rates but also half-saturation constants (K_s) and the minimum cell quotas. S. hantzschii had low silicon requirements for growth under Si-limiting conditions, and A. formosa and F. crotonensis had higher and nearly identical silicon requirements. The K_s values for silicon for S. hantzschii were essentially constant from 5° to 20° C but varied greatly for the other two species. A. formosa had the lowest requirements for growth under phosphorus limitation, F. crotonensis was intermediate and S. hantzschii had the highest growth requirements for phosphorus. The K₁ values for phosphorus were constant over the temperature range for both A. formosa and F. crotonensis and were much higher and variable for S. hantzschii. Nutrient competition experiments were performed in continuous cultures at four temperatures and various Si:P ratios. The results generally, but not always, confirmed the predictions based on the Monod relationships for each species. Results not in agreement with predictions were usually because of similar physiological properties of A. formosa and F. crotonensis or because of decreased loss rates for F. crotonensis due to wall growth. In cultures with all three species phosphorus-limited (Si:P > 75), A. formosa often dominated as predicted, although F. crotonensis was sometimes the most abundant species. As predicted, S. hantzschii never dominated at high Si:P ratios. At intermediate Si:P ratios when A. formosa and F. crotonensis were both Si-limited and S. hantzschii P-limited, all three species coexisted because A. formosa and F. crotonensis have almost identical silicon requirements, although sometimes F. crotonensis was more abundant than predicted. At 10°C the results agreed best with the predictions; A. formosa dominated at high Si:P ratios and S. hantzschii dominated as predicted at low Si:P ratios when all three species were Si-limited.     

OLISTHODISCUS LUTEUS (CHRYSOPHYCEAE). III. UPTAKE AND UTILIZATION OF NITROGEN AND PHOSPHORUS1

Uptake and assimilation of nitrogen and phosphorus were studied in Olisthodiscus luteus Carter. A diel periodicity in nitrate reductase activity was observed in log and stationary phase cultures; there was a 10-fold difference in magnitude between maximum and minimum rates, but other cellular features such as chlorophyll a, carbon, nitrogen, C:N ratio (atoms) · cell^?1 were less variable. K_s values (~2 μM) for uptake of nitrate-N and ammonium-N were observed. Phosphorus assimilated · cell^?1· day^?1 varied with declining external phosphorus concentrations; growth rates <0.5 divisions · day^?1 were common at <0.5 μM PO_4-P. Phosphate uptake rates (K_s= 1.0–1.98 μM) varied with culture age and showed multiphasic kinetic features. Alkaline phosphatase activity was not detected. Comparisons of the nutrient dynamics of O. luteus to other phytoplankton species and the ecological implications as related to the phytoplankton community of Narragansett Bay (Rhode Island) are discussed.     

Characterization of a model cyanobacterium Synechocystis sp. PCC 6803 autotrophic growth in a flat‐panel photobioreactor

We characterized the photoautotrophic growth of glucose‐tolerant Synechocystis sp. PCC 6803 in a flat‐panel photobioreactor running on a semicontinuous regime under various lights, temperatures, and influx carbon dioxide concentrations. The maximum reached growth rate was 0.135 h^?1, which corresponds to a doubling time of 5.13 h—a growth speed never reported for Synechocystis before. Saturating red light intensity for the strain was 220–360 μmol(photons) m^?2 s^?1, and we did not observe any photoinhibition up to 660 μmol(photons) m^?2 s^?1. Synechocystis was able to grow under red light only; however, photons of wavelengths 405–585 and 670–700 nm further improved its growth. Optimal growth temperature was 35°C. Below 32°C, the growth rates decreased linearly with temperature coefficient (Q₁₀) 1.70. Semicontinuous cultivation is known to be efficient for growth characterization and optimization. However, the assumption of correct growth rates calculation—culture exponential growth—is often not fulfilled. The semicontinuous setup in this study was operated as a turbidostat. Accurate online OD measurements with high time‐resolution allowed fast and reliable growth rates determination. Repeating diluting frequencies (up to 18 dilutions per day) were essential for rapid growth stability evaluation. The presented setup provides improvement to previously published semicontinuous characterization strategies by decreasing experimental time requirements and maintaining the culture in exponential growth phase throughout the entire characterization procedure.     

Growth kinetics of top and bottom cultures of Saccharomyces spp. in a chemostat using sugarbeet molasses

Growth kinetics were evaluated for three yeast strains of the genus Saccharomyces. Two topfloating strains, SF 115 and SF 116 and one flocculant yeast SF 104 were analyzed in pure and mixed cultures in 1-liter continuous fermentation experiments in a chemostat. Growth was monitored for 72 h at 30°C in a medium containing sugarbeet molasses and 1.0 g/liter each of NH₄H₂PO₄ and urea. SF 115 and SF 116 were found to have lower μ_max values of 0.290 and 0.296 h^?1, respectively, than SF 104, which had a μ_max of 0.364 h^?1. The two top-floating yeasts (SF 115 and SF 116) demonstrated greater affinity for the substrate and utilized substrates at a greater rate. They have K₈ values of 4.03 × 10^?3 M and 3.798 × 10^?3 M, respectively, compared to 9.06 × 10^?3 M for SF 104. A mixed culture of SF 116 and SF and SF 104 was found to have a μ_max of 0.426 h^?1 with a K_s of 6.924 × 10^?3 M. SF 115 grown in mixed culture with SF 104 exhibited a μ_max of 0.473 h^?1 with a K_s of 7.975 × 10^?3 M. In both cases, the SF 104 was the dominant microbe in mixed culture systems.     

EFFECT OF TEMPERATURE,LIGHT AND pH ON GROWTH,PHOTOSYNTHESIS AND RESPIRATION OF THE DINOFLAGELLATE PERIDINIUM CINCTUM FA. WESTII IN LABORATORY CULTURES1

Optimum light, temperature, and pH conditions for growth, photosynthetic, and respiratory activities of Peridinium cinctum fa. westii (Lemm.) Lef were investigated by using axenic clones in batch cultures. The results are discussed and compared with data from Lake Kinneret (Israel) where it produces heavy blooms in spring. Highest biomass development and growth rates occurred at ca. 23° C and ≥50 μE· m^?2·s¹ of fluorescent light with energy peaks at 440–575 and 665 nm. Photosynthetic oxygen release was more efficient in filtered light of blue (BG 12) and red (RG 2) than in green (VG 9) qualities. Photosynthetic oxygen production occurred at temperatures ranging from 5° to 32° C in white fluorescent light from 10 to 105 μE·m^?2·s^?1 with a gross maximum value of 1500 × 10^?12 g·cell^?1·h^?1 at the highest irradiance. The average respiration amounted to ca. 12% of the gross production and reached a maximum value of ca. 270·10^?12 g·cell^?1·h^?1 at 31° C. A comparison of photosynthetic and respiratory Q₁₀-values showed that in the upper temperature range the increase in gross production was only a third of the corresponding increase in respiration, although the gross production was at maximum. Short intermittent periods of dark (>7 min) before high light exposures from a halogen lamp greatly increased oxygen production. Depending on the physiological status of the alga, light saturation values were reached at 500–1000 μE·m^?2·s^?1 of halogen light with compensation points at 20–40 μE·m^?2·s^?1 and I_k-values at 100–200 μE·m^?2·s^?1. The corresponding values in fluorescent light in which it was cultured and adapted, were 25 to 75% lower indicating the ability of the alga to efficiently utilize varying light conditions, if the adaptation time is sufficient. Carbon fixation was most efficient at ca. pH 7, but the growth rates and biomass development were highest at pH 8.3.     

Light/Dark Cycle and Temperature-Their Impact on Phosphate-Limited Growth of Oscillatoria redekei VAN GOOR in Semicontinuous Culture

Phosphate-limited growth of Oscillatoria redekei in semicontinuous culture has been studied under conditions of continuous illumination at 20 °C as well as in a 12/12 hours light-dark cycle at temperatures between 5 °C and 20 °C. The subsistence quota (q₀) amounted to 0.052 μmol mm⁻³ under all conditions, when the phosphate quota was expressed on the basis of cell volume. The interaction between temperature and phosphate quota and its impact on growth rate are described by the following model: Parameter values are t_opt=24.5 °C, t_min=0.95 °C, μ_max =0.873 d⁻¹. The maximum phosphate quota was found to depend on temperature and to increase along with declining temperature.     

Photosynthesis of Marine Macroalgae from Antarctica: Light and Temperature Requirements

The photosynthetic performance of macroalgae isolated in Antarctica was studied in the laboratory. Species investigated were the brown algae Himantothallus grandifolius, Desmarestia anceps, Ascoseira mirabilis, the red algae Palmaria decipiens, Iridaea cordata, Gigartina skottsbergii, and the green algae Enteromorpha bulbosa, Acrosiphonia arcta, Ulothrix subflaccida and U. implexa. Unialgal cultures of the brown and red algae were maintained at 0°C, the green algae were cultivated at 10°C. I_K values were between 18 and 53 μmol m^?2 s^?1 characteristic or low light adapted algae. Only the two Ulothrix species showed higher I_K values between 70 and 74 μmol m^?2 s^?1. Photosynthesis compensated dark respiration at very low photon fluence rates between 1.6 and 10.6 μmol m^?2 s^?1. Values of α were high: between 0.4 and 1.1 μmol O₂ g^?1 FW h^?1 (μmol m^?2 s^?1)^?1 in the brown and red algae and between 2.1 and 4.9 μmol O₂ g^?1 FW h^?1 (μmol m^?2 s^?1)^?1 in the green algal species. At 0°C P_max values of the brown and red algae ranged from 6.8 to 19.1 μmol O₂ g^?1 FW h^?1 and were similarly high or higher than those of comparable Arctic-cold temperate species. Optimum temperatures for photosynthesis were 5 to 10°C in A. mirabilis, 10°C in H. grandifolius, 15°C in G. skottsbergii and 20°C or higher in D. anceps and I. cordata. P: R ratios strongly decreased in most brown and red algae with increasing temperatures due to different Q₁₀ values for photosynthesis (1.4 to 2.5) and dark respiration (2.5 to 4.1). These features indicate considerable physiological adaptation to the prevailing low light conditions and temperatures of Antarctic waters. In this respect the lower depth distribution limits and the northern distribution boundaries of these species partly depend on the physiological properties described here.     

EFFECTS OF LIGHT AND TEMPERATURE ON GROWTH,NITRATE UPTAKE,AND TOXIN PRODUCTION OF TWO TROPICAL DINOFLAGELLATES: ALEXANDRIUM TAMIYAVANICHII AND ALEXANDRIUM MINUTUM (DINOPHYCEAE)1

The two tropical estuarine dinoflagellates, Alexandrium tamiyavanichii Balech and A. minutum Halim, were used to determine the ecophysiological adaptations in relation to their temperate counterparts. These species are the two main causative organisms responsible for the incidence of paralytic shellfish poisoning (PSP) in Southeast Asia. The effects of light (10, 40, 60, and 100 μmol photons·m^?2·s^?1) and temperature (15, 20, and 25°C) on the growth, nitrate assimilation, and PST production of these species were investigated in clonal batch cultures over the growth cycle. The growth rates of A. tamiyavanichii and A. minutum increased with increasing temperature and irradiance. The growth of A. tamiyavanichii was depressed at lower temperature (20°C) and irradiance (40 μmol photons·m^?2·s^?1). Both species showed no net growth at 10 μmol photons·m^?2·s^?1 and a temperature of 15°C, although cells remained alive. Cellular toxin quotas (Q_t) of A. tamiyavanichii and A. minutum varied in the range of 60–180 and 10–42 fmol PST·cell^?1, respectively. Toxin production rate, R_tox, increased with elevated light at both 20 and 25°C, with a pronounced effect observed at exponential phase in both species (A. tamiyavanichii, r²=0.95; A. minutum, r²=0.96). Toxin production rate also increased significantly with elevated temperature (P<0.05) for both species examined. We suggest that the ecotypic variations in growth adaptations and toxin production of these Malaysian strains may reveal a unique physiological adaptation of tropical Alexandrium species.     

The influence of irradiance,photoperiod and temperature on the growth kinetics of three planktonic diatoms

The influence of irradiance, photoperiod and temperature was determined for the growth kinetics of the diatoms Aulacoseira subarctica, Stephanodiscus astraea and Stephanodiscus hantzschii and the results compared with those of cyanobacteria. Irradiance and photoperiod relationships were qualitatively similar to those for cyanobacteria in that: (1) growth rate (K) was proportionally greater under short photoperiods, with ratios of K under continuous light to K under 3:21 light:dark (LD) cycles of 1·50, 1·80 and 2·96 for A. subarctica, S. astraea and S. hantzschii respectively; (2) at subsaturating irradiances, K was proportional to irradiance and independent of temperature with a negligible predicted maintenance growth rate requirement. Apparent growth efficiencies (GE) at subsaturating irradiances were 0·26±0·03, 0·42±0·03 and 0·50±0·03 divisions mol^-1m² for A. subarctica, S. astraea and S. hantzschii with the values for Stephanodiscus species comparable to values for Oscillatoria species. Under a 3:21 LD cycle at 4 °C, light-saturated growth rates were 0·066±0·004, 0·197±0·033 and 0·285±0·018 divisions day^-1 for A. subarctica, S. astraea and S. hantzschii. S. hantzschii growth rate at 4 °C exceeded maximum Oscillatoria growth rates at 23 °C and the S. astraea growth rate at 4 °C was equivalent to O. agardhii growth rate at 20 °C. Temperature increases above 4 °C gave Q₁₀ values between 4 °C and 12 °C of 3·68, 2·39 and 1·92 for A. subarctica, S. astraea and S. hantzschii, but higher temperatures resulted in minor increases in K. S. astraea growth rate peaked at 16 °C, declining sharply at higher temperatures. February to March in situ growth rates in Lough Neagh, mean temperature 4·3 °C, showed that the A. subarctica in situ K of 0·058 divisions day^-1 was close to the laboratory K at 4 °C, but that S. astraea in situ K of 0·101 divisions day^-1 was lower than the laboratory K at 4 °C.     

Purification and characterization of laccase secreted by Phellinus linteus MTCC-1175 and its role in the selective oxidation of aromatic methyl group

A laccase from the culture filtrate of Phellinus linteus MTCC-1175 has been purified to homogeneity. The method involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on DEAE-cellulose. The SDS-PAGE and native-PAGE gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 70 kDa. Using 2.6-dimethoxyphenol, 2.2′[azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] (ABTS) and 4-hydroxy-3,5-dimethoxybenzaldehyde azine as the substrates, the K _m, k _cat and k _cat/K _m values of the laccase were found to be 160 μM, 6.85 s^?1, 4.28 × 10⁴ M^?1 s^?1, 42 μM, 6.85 s^?1, 16.3 × 10⁴ M^?1 s^?1 and 92 μM, 6.85 s^?1, 7.44 × 10⁴ M^?1 s^?1, respectively. The pH and the temperature optima of the P. linteus MTCC-1175 laccase were 5.0 and 45°C, respectively. The activation energy for thermal denaturation of the enzyme was 38.20 kJ/mole/K. The enzyme was the most stable at pH 5.0 after 1 h reaction. In the presence of ABTS as the mediator, the enzyme transformed toluene, 3-nitrotoluene and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde and 4-chlorobenzaldehyde, respectively.     

The response to ribulose bisphosphate4− (RuBP4−) and RuBP-Mg2− in catalysis by structurally divergent RuBP carboxylase/oxygenases

Free ribulose bisphosphate (RuBP^4?) rather than its magnesium complex (RuBP-Mg^2?) was the apparent substrate for spinach ribulose bisphosphate carboxylase/oxygenase. The apparent K_m for total RuBP (pH 8.0 at 30° C) increased with increasing Mg²⁺ concentrations from 11.6 μM at 13.33 mM Mg²⁺ to 32.6 μM at 40.33 mM Mg²⁺. Similarly the apparent K_m for RuBP-Mg^2? complex increased with increasing Mg²⁺ from 9.4 μM at 13.33 mM Mg²⁺ to 29.7 μM at 40.33 mM Mg²⁺. However, the K_m values for uncomplexed RuBP^4? were independent of the (saturating) concentration of Mg²⁺ (K_m=2.2 μM). The V_max did not vary with the changing concentrations of Mg²⁺. In contrast, the K_m for total RuBP remained constant with varying Mg²⁺ concentrations (K_m=59.5 μM) for the enzyme from R. rubrum. The apparent K_m for the RuBP-Mg^2? complex decreased with increasing Mg²⁺ concentrations from 16.0 μM at 7.5 mM Mg²⁺ to 5.9 μM at 27.5 mM Mg²⁺. The initial velocity for the C. vinosum enzyme was also found to be independent of the (saturating) concentration of Mg²⁺ when total RuBP was varied in the assay. Thus the response to total RuBP by these two bacterial enzymes, which markedly differ in structure, was closely similar.     

NUTRIENT REQUIREMENTS OF THE DINOFLAGELLATE PERIDINIUM GATUNENSE1

Optimum nutrient conditions for growth and photosynthesis of Peridinium gatunense (Nygaard) (Peridinium cinctum fa. westii) were investigated using axenic clones in batch cultures. Selenium (Se) had previously been found to be an indispensable growth factor for P. gatunense. Optimal, suboptimal, and supraoptimal concentrations of HCO₃^?, N, Ca, Cl, Mg, P, K, S, Si, EDTA-Na, Fe, Mo, Zn, Mn, Co, Se, B, Br, I, and various trace element mixtures were determined by measuring biomass development, growth rates, ¹⁴C uptake, and/or oxygen production at various concentration gradients of these elements. The general characteristics of the best formulation, medium-L 16, relative to other media, are its high content of NaHCO₃ (1 meq · L^?1) and Mo (0.2 μM) but low concentrations of NO_3-N (150 μM), PO_4-P (10 μM), and Fe (0.4 μM), in addition to its content of Se. The total content of trace metals, except for Se, may be reduced to one-fourth of that in medium-L 16 without altering the major growth-promoting properties of the medium. Medium-L 16 deviated considerably from Lake Kinneret (Israel) water, being much lower in macroelements except for N and P. The pH (8.1–8.4) was in the same range, but the values of conductivity (140 μS · cm^?1), alkalinity (1 meq · L^?1) and NaCl (200 μM) were > 8, 2, and 30 times higher, respectively, in the lake water. Selenium deficiency may limit the growth of P. gatunense in this lake.     

The response to ribulose bisphosphate4− (RuBP4−) and RuBP-Mg2− in catalysis by structurally divergent RuBP carboxylase/oxygenases

Free ribulose hisphosphate (RuBP^4?) rather than its magnesium complex (RuBP-Mg^2?) was the apparent substrate for spinach ribulose bisphosphate carboxylase/oxygenase. The apparent K_m for total RuBP (pH 8.0 at 30° C) increased with increasing Mg²⁺ concentrations from 11.6 μM at 13.33 mM Mg²⁺ to 32.6 μM at 40.33 mM Mg²⁺. Similarly the apparent K_m for RuBP-Mg^2? complex increased with increasing Mg²⁺ from 9.4 μM at 13.33 mM Mg²⁺ to 29.7 μM at 40.33 mM Mg²⁺. However, the K_m values for uncomplexed RuBP^4? were independent of the (saturating) concentration of Mg²⁺ (K_m=2.2 μM). The V_max did not vary with the changing concentrations of Mg²⁺. In contrast, the K_m for total RuBP remained constant with varying Mg²⁺ concentrations (K_m=59.5 μM) for the enzyme from R. rubrum. The apparent K_m for the RuBP-Mg^2? complex decreased with increasing Mg²⁺ concentrations from 16.0 μM at 7.5 mM Mg²⁺ to 5.9 μM at 27.5 mM Mg²⁺. The initial velocity for the C. vinosum enzyme was also found to be independent of the (saturating) concentration of Mg²⁺ when total RuBP was varied in the assay. Thus the response to total RuBP by these two bacterial enzymes, which markedly differ in structure, was closely similar.     

Growth of the plankton diatom Thalassiosira nordenskioeldii Cleve at low silicate concentrations

The combined effect of low silicate concentration and temperature on the growth of the marine plankton diatom Thalassiosira nordenskioeldii Cleve was investigated by means of batch and semi-continuous cultures. Growth rates were measured in thin cell suspensions (less than 500 cells/ml) to prevent the silicate concentration in the medium from decreasing by more than 25 % during the period of measurement.Half-saturation constants of silicate-limited growth were calculated according to the Michaelis-Menten equation. At 3 °C, the constant was 0.09 μg-at. Si/I and at 10 °C, 0.02μg-at. Si/I. In semicontinuous cultures grown for one month at silicate concentrations of 0.3-0.4μg-at. Si/I, the mean cell division rates were 80–100% of the maximum rates recorded at the respective temperatures.It seems unlikely that decreasing silicate concentrations could influence the course of the spring succession of plankton diatom species in arctic or temperate coastal waters.     

Kinetics of interaction of some α- and β-D-monosaccharides with concanavalin A

The rates of formation and dissociation of concanavalin A with some 4-methylumbelliferyl and p-nitrophenyl derivatives of α- and β-D-mannopyranosides and glucopyranosides were measured by fluorescence and spectral stopped-flow methods. All process examined were uniphasic. The second-order formation rate constants varied only from 6.8 · 10⁴ to 12.8 · 10⁴ M^?. s^?1, whereas the first-order dissociation rate constants ranged from 4.1. to 220 s^?1, all at ph 5.0, I = 0.3 M, and 25°C. Dissociation rates thus controlled the value of binding constant. The effect of temperature on these reactions was examined, from which enthalpies and entropies of activation and of reaction could be calculated. The effects of pH at 25°C on the reaction rates of 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside with concanavalin A were examined. The value of the binding constant K_ap (derived from the kinetics) at any pH could be related to the intrinsic binding constant K by the expression K_ap = K_aK(K_a + [H⁺])^?1. The values of K_a, the ionization constant of the protein segment responsive to sugar binding, were 3 · 10^?4 M and 1 · 10^?4 M for 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside, respectively. The binding constant of p-nitrophenyl α-D-mannopyranoside is surprisingly much less sensitive to a pH change from 5.0 to 2.7. Ionic strength had little effect on the binding characteristics of 4-methylumbelliferyl α-D-mannopyranoside to concanavalin A at pH 5.2 and 25°C.     

DIATOM MINERALIZATION OK SILICIC ACID. I Si(OH)4 TRANSPORT CHARACTERISTICS IN NAVICULA PELLICULOSA

Silicic acid transport was studied in the photosynthetic diatom Navicula pelliculosa (Bréb.) Hilse using [⁶⁸Ge] germanic acid (⁶⁸Ge(OH)₄) as a tracer of silicic acid (Si(OH)₄). The initial uptake rate of Si(OH)₄ was dependent on cell number, pH, temperature, light and was promoted by certain monovalent cations in the medium. Na⁺ was more effective than K⁺, whereas Li⁺ and NH⁺₄ were ineffective at promoting uptake. Uncouplers and inhibitors of oxidative phosphorylation and of photophosphorylation reduced uptake by 40–99% of control values. Uptake was also especially sensitive to the sulfhydryl blocking agents at 10^?5 M and to the ionophorous compound valinomycin (10^?7 M) which inhibited uptake by 82%. The Si(OH)₄ transport system displayed Michaelis-Menten-type saturation kinetics with kinetic parameters of K_S= 4.4 p. mol Si(OH)₄· 1^?1, V_max= 334 pmol Si(OH)₄· 10⁶ cells^?1· min^?1. Calculations of the acid soluble silicic acid pool size based on 60 s uptake at 20 μM Si(OH)₄ suggested that intracellular levels of Si could reach 20 mM and as much as 5 mM could exist as free silicic acid, representing maintenance of a 250-fold concentration gradient compared with the medium. Efflux from preloaded cells was dependent on temperature and the Si(OH)₄ concentration of the external medium. In the presence of 100 μMM “cold” Si(OH)₄, approximately 30% of the Si(OH)₄ in preloaded cells was exchanged in 20 min. The initial uptake rate of Si(OH)₄ in logarithmic phase cells was constant, but the uptake rate increased in a linear fashion for 6 h in stationary phase cells. These results suggest that the first step in silica mineralization by diatoms is the active transmembrane transport of Si(OH)₄ by an energy dependent, saturable, membrane-carrier mechanism which requires the monovalent cations Na⁺ and K⁺ and is sensitive to sulfhydryl blocking agents. Silicic acid transport activity also appears to be regulated during different growth stages of the diatom.     

The responses of photosynthetic gas exchange and chlorophyll a fluorescence to changes of irradiance and temperature in two species of Miscanthus

Photosynthetic CO₂ uptake and chlorophyll (Chl) a fluorescence of C₄ perennial grasses, Miscanthus floridulus (Labill) Warb and M. transmorrisonensis Hayata, from altitudes in central Taiwan of 390 and 2700 m, respectively, were studied at 10 and 25 °C to find if the species differ in their photosynthetic responses to a low temperature, and whether their photosystems 2 become more susceptible to the photoinhibition at low temperatures. For both species, the maximum photosynthetic rate (P_max) was reduced when the leaves were exposed to 10 °C. At irradiances higher than 400 μmol m^-2 s^-1, the values of F_v/F_m were reduced in both species at 10 °C but not at 25 °C, which indicated the photoinhibition at 10 °C. Reductions in P_max and the values of F_v/F_m at 10 °C were lesser in M. transmorrisonensis than in M. floridulus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tissue kallikrein processes small proenkephalin peptides

Tissue kallikrein may play a role in processing precursor polypeptide hormones. We investigated whether hydrolysis of natural enkephalin precursors, peptide F and bovine adrenal medulla docosapeptide (BAM-22P), by hog pancreatic kallikrein is consistent with this concept. Incubation of peptide F with this tissue kallikrein resulted in the release of Met⁵-enkephalin and Met⁵-Lys⁶-enkephalin. Met⁵-Lys⁶-enkephalin was the main peptide released, indicating that the major cleavage site was between two lysine residues. At 37°C and pH 8.5, the K_M values for formation of Met⁵-enkephalin and Met⁵-Lys⁶-enkephalin were 129 and 191 μM, respectively. Corresponding k_cat values were 0.001 and 0.03 s⁻¹ and k_cat/K_M ratios were 8 and 1.6·10² M⁻¹ · s⁻¹, respectively. Cleavage of peptide F at acidic pH (5.5) was negligible. When BAM-22P was used as a substrate, Met⁵-Arg⁶-enkephalin was released, thus indicating cleavage between two arginine residues. At pH 8.5, K_M was 64 μM, k_cat was 4.5 s⁻¹, and the k_cat/K_M ratio was 7 · 10⁴ M⁻¹ · s⁻¹. At 5.5, the pH of the secretory granules, K_M, k_cat and k_cat/K_M were 184 μM, 1.9 s⁻¹ and 10⁴ M⁻¹ · s⁻¹, respectively. It is unlikely that peptide F could be a substrate for kallikrein in vivo; however, tissue kallikrein could aid in processing proenkephalin precursors such as BAM-22P by cleaving Arg-Arg peptide bonds.     

PHYSIOLOGICAL RESPONSES OF ANABAENA VARIABILIS (CYANOPHYCEAE) TO INSTANTANEOUS EXPOSURE TO VARIOUS COMBINATIONS OF LIGHT INTENSITY AND TEMPERATURE1

Light intensity and temperature interactions have a complex effect on the physiological process rates of the filamentous bluegreen alga Anabaena variabilis Kütz. The optimum temperature for photosynthesis increased with increasing light intensity from 10°C at 42 μE·m^?2·s^?1 to 35°C at 562 μE·m^?2·s^?1. The light saturation parameter, I_K, increased with increasing temperatures. The maximum photosynthetic rate (2.0 g C·g dry wt.^?1·d^?1) occurred at 35°C and 564 μE·m^?2·s^?1. At 15°C, the maximum rate was 1.25 g C·g dry wt.^?1·d^?1 at 332 μE·m^?2·s^?1. The dark respiration rate increased exponentially with temperature. Under favorable conditions of light intensity and temperature the percent of extracellular release of dissolved organic carbon was less than 5% of the total C fixed. This release increased to nearly 40% under combinations of low light intensity and high temperature. A mathematical model was developed to simulate the interaction of light intensity and temperature on photosynthetic rate. The interactive effects were represented by making the light-saturation parameters a function of temperature.