Short-term binding of fibroblasts to fibronectin: optical tweezers experiments and probabilistic analysis

The biophysical properties of the interaction between fibronectin and its membrane receptor were inferred from adhesion tests on living cells. Individual fibroblasts were maintained on fibronectin-coated glass for short time periods (1–16 s) using optical tweezers. After contact, the trap was removed quickly, leading to either adhesion or detachment of the fibroblast. Through a stochastic analysis of bond kinetics, we derived equations of adhesion probability versus time, which fit the experimental data well and were used to compute association and dissociation rates (k ₊=0.3–1.4 s⁻¹ and k _off=0.05–0.25 s⁻¹, respectively). The bond distribution is binomial, with an average bond number ≤10 at these time scales. Increasing the fibronectin density (100–3000 molecules/μm²) raised k ₊ in a diffusion-dependent manner, leaving k _off relatively unchanged. Increasing the temperature (23–37 °C) raised both k ₊ and k _off, allowing calculation of the activation energy of the chemical reaction (around 20 k _B T). Increasing the compressive force on the cell during contact (up to 60 pN) raised k ₊ in a logarithmic manner, probably through an increase in the contact area, whereas k _off was unaffected. Finally, by varying the pulling force to detach the cell, we could distinguish between two adhesive regimes, one corresponding to one bond, the other to at least two bonds. This transition occurred at a force around 20 pN, interpreted as the strength of a single bond. Received: 2 November 1999 / Revised version: 6 March 2000 / Accepted: 19 April 2000     

The application of atomic force microscopy to topographical studies and force measurements on the secreted adhesive of the green alga Enteromorpha

Atomic force microscopy (AFM) enables the topographical structure of cells and biological materials to be resolved under natural (physiological) conditions, without fixation and dehydration artefacts associated with imaging methods in vacuo. It also provides a means of measuring interaction forces and the mechanical properties of biomaterials. In the present study, AFM has been applied for the first time to the study of the mechanical properties of a natural adhesive produced by a green plant cell. Swimming spores of the green alga Enteromorpha linza (L.) J. Ag. (7–10 μm) secrete an adhesive glycoprotein which provides firm anchorage to the substratum. Imaging of the adhesive in its hydrated state revealed a swollen gel-like pad, approximately 1 μm thick, surrounding the spore body. Force measurements revealed that freshly released adhesive has an adhesion strength of 173 ± 1.7 mN m⁻¹ (mean ± SE; n=90) with a maximum value for a single adhesion force curve of 458 mN m⁻¹. The adhesive had a compressibility (equivalent to Young's modulus) of 0.54 × 10⁶ ± 0.05 × 10⁶ N m⁻² (mean ± SE; n=30). Within minutes of release the adhesive underwent a progressive `curing' process with a 65% reduction in mean adhesive strength within an hour of settlement, which was also reflected in a reduction in the average length of the adhesive polymer strands (polymer extension) and a 10-fold increase in Young's modulus. Measurements on the spore surface itself revealed considerably lower adhesion-strength values but higher polymer-extension values than the adhesive pad, which may reflect the deposition of different polymers on this surface as a new cell wall is formed. The study demonstrates the value of AFM to the imaging of plant cells in the absence of fixation and dehydration artefacts and to the characterisation of the mechanical properties of plant glycoproteins that have potential utility as adhesives. Received: 22 February 2000 / Accepted: 20 April 2000     

Decrease of phosphoribulokinase activity by antisense RNA in transgenic tobacco: definition of the light environment under which phosphoribulokinase is not in large excess

 To test the hypothesis that the contribution of phosphoribulokinase (PRK) to the control of photosynthesis changes depending on the light environment of the plant, the response of transgenic tobacco (Nicotiana tabacum L.) transformed with antisense PRK constructs to irradiance was determined. In plants grown under low irradiance (330 μmol m⁻² s⁻¹) steady-state photosynthesis was limited in plants with decreased PRK activity upon exposure to higher irradiance, with a control coefficient of PRK for CO₂ assimilation of 0.25 at and above 800 μmol m⁻² s⁻¹. The flux control coefficient of PRK for steady-state CO₂ assimilation was zero, however, at all irradiances in plant material grown at 800 μmol m⁻² s⁻¹ and in plants grown in a glasshouse during mid-summer (alternating shade and sun 300–1600 μmol m⁻² s⁻¹). To explain these differences between plants grown under low and high irradiances, Calvin cycle enzyme activities and metabolite content were determined. Activities of PRK and other non-equilibrium Calvin cycle enzymes fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and ribulose-1,5-bisphosphate carboxylase-oxygenase were twofold higher in plants grown at 800 μmol m⁻² s⁻¹ or in the glasshouse than in plants grown at 330 μmol m⁻² s⁻¹. Activities of equilibrium enzymes transketolase, aldolase, ribulose-5-phosphate epimerase and isomerase were very similar under all growth irradiances. The flux control coefficient of 0.25 in plants grown at 330 μmol m⁻² s⁻¹ can be explained because low ribulose-5-phosphate content in combination with low PRK activity limits the synthesis of ribulose-1,5-bisphosphate. This limitation is overcome in high-light-grown plants because of the large relative increase in activities of sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase under these conditions, which facilitates the synthesis of larger amounts of ribulose-5-phosphate. This potential limitation will have maintained evolutionary selection pressure for high concentrations of PRK within the chloroplast. Received: 15 November 1999 / Accepted: 27 January 2000     

Production of Spirulina sp. in sea water supplemented with anaerobic effluents in outdoor raceways under temperate climatic conditions

The use of untreated sea water supplemented with anaerobic effluents from digested pig waste and sodium bicarbonate was evaluated as a low-cost medium for semi-continuous cultivation of a mixed culture of two Spirulina strains in outdoor raceways under temperate climatic conditions (pond temperature in the range 21–26 °C and light intensity in the range 225–957␣μE m⁻² s⁻¹). The mixed culture had a predominant population (86.6 ± 3.9%) of an atypical Spirulina strain consisting of straight filaments, which appeared spontaneously after the strain with helicoidal trichomes had been subcultured. Morphological studies for the identification of the type and size of trichomes of the two strains (HF and SF) were carried out. The proportions of the two strains were observed to be stable during the monitoring period (30 days). Three different sets of semicontinuous cultures were carried out. Sets 1 and 2 were operated under regime 1 (a single addition of anaerobic effluents at time zero and no pH control) during the same season (June and July) of different years. Set 3 was operated under regime 2 (semi-continuous addition of anaerobic effluents and pH control) during the autumn. A minimum productivity of 3.6 g m⁻² day⁻¹ was obtained at one of the lowest temperatures (22.1 °C) and light intensities (245 μE m⁻² s⁻¹) and a maximum productivity of 10.9 g m⁻² day⁻¹ was observed at the highest temperature (25 °C) and highest average light intensity (618 μE m⁻² s⁻¹) registered for sets 1 and 2. The protein content in the Spirulina biomass harvested from these two sets varied from 17% to 65.6%. In set 3, a maximum productivity of 9.0 g m⁻² day⁻¹ was recorded at an average temperature of 24.4 °C and at an average light intensity of 668 μE m⁻² s⁻¹. The protein content in this set under regime 2 varied within a narrower range than in set 1 and set 2 (from 34.8% to 49.1%), apparently because of a continuous availability of ammonia nitrogen at a level of 30–50 mg l⁻¹. However, in terms of the removal of ammonia nitrogen and chemical oxygen demand, regime 1 was more efficient than regime␣2. Received: 3 September 1996 / Received revision: 19 February 1997 / Accepted: 7 March 1997     

Stress-Induced Changes in Optical Properties,Pigment and Fatty Acid Content of <Emphasis Type="Italic">Nannochloropsis</Emphasis> sp.: Implications for Non-destructive Assay of Total Fatty Acids

In order to develop a practical approach for fast and non-destructive assay of total fatty acid (TFA) and pigments in the biomass of the marine microalga Nannochloropsis sp. changes in TFA, chlorophyll, and carotenoid contents were monitored in parallel with the cell suspension absorbance. The experiments were conducted with the cultures grown under normal (complete nutrient f/2 medium at 75 μmol PAR photons/(m² s)) or stressful (nitrogen-lacking media at 350 μmol PAR photons/(m² s)) conditions. The reliable measurement of the cell suspension absorbance using a spectrophotometer without integrating sphere was achieved by deposition of cells on glass–fiber filters in the chlorophyll content range of 3–13 mg/L. Under stressful conditions, a 30–50% decline in biomass and chlorophyll, retention of carotenoids and a build-up of TFA (15–45 % of dry weight) were recorded. Spectral regions sensitive to widely ranging changes in carotenoid-to-chlorophyll ratio and correlated changes of TFA content were revealed. Employing the tight inter-correlation of stress-induced changes in lipid metabolism and rearrangement of the pigment apparatus, the spectral indices were constructed for non-destructive assessment of carotenoid-to-chlorophyll ratio (range 0.3–0.6; root mean square error (RMSE) = 0.03; r ² = 0.93) as well as TFA content of Nannochloropsis sp. biomass (range 5.0–45%; RMSE = 3.23 %; r ² = 0.89) in the broad band 400–550 nm normalized to that in chlorophyll absorption band (centered at 678 nm). The findings are discussed in the context of real-time monitoring of the TFA accumulation by Nannochloropsis cultures under stressful conditions.     

CO2 exchange of the endolithic lichen Verrucaria baldensis from karst habitats in northern Italy

CO₂ exchange of the endolithic lichen Verrucaria baldensis was measured in the laboratory under different conditions of water content, temperature, light, and CO₂ concentration. The species had low CO₂ exchange rates (maximum net photosynthesis: c. 0.45 μmol CO₂ m⁻² s⁻¹; maximum dark respiration: c. 0.3 μmol CO₂ m⁻² s⁻¹) and a very low light compensation point (7 μmol photons m⁻² s⁻¹ at 8°C). The net photosynthesis/respiration quotient reached a maximum at 9–15°C. Photosynthetic activity was affected only after very severe desiccation, when high resaturation respiratory rates were measured. Microclimatic data were recorded under different weather conditions in an abyss of the Trieste Karst (northeast Italy), where the species was particularly abundant. Low photosynthetically active radiation (normally below 40 μmol photons m⁻² s⁻¹), very high humidities (over 80%), and low, constant temperatures were measured. Thallus water contents sufficient for CO₂ assimilation were often measured in the absence of condensation phenomena. Received: 22 September 1996 / Accepted: 26 April 1997     

Phytoplankton biomass in the sub-Antarctic area of the Straits of Magellan (53°S), Chile during spring-summer 1997/1998

In order to provide a better understanding of the dynamics of phytoplankton in the coastal regions of high latitudes, a study was carried out to estimate the dynamics of carbon biomass of autotrophic and heterotrophic algal groups over the austral spring-summer 1997/1998 period. At a fixed station located in the central basin (Paso Ancho) of the Straits of Magellan (53°S), surface water samples were collected at least once a week from September 1997 (early spring) to March 1998 (late summer). Quantitative analysis of biomass of phytoplankton was estimated from geometric volumes, using non-linear equations, and converted to biomass. The pattern of chlorophyll a showed a strong temporal variability, with maximum values (mean 2.8 mg m⁻³) at the austral spring phytoplankton increase or bloom (October/November) and minimum values during early spring (September: <0.5 mg m⁻³) and summer (January/March: 0.5–1.0 mg m⁻³). During the spring bloom, diatoms made up to 90% of the total phytoplankton carbon (0.01–189 μg l⁻¹), followed by a maximum of thecate dinoflagellates (0.08–34 μg l⁻¹), and sporadic high biomass of phytoflagellates during summer. Heterotrophic algal groups such as Gymnodinium and Gyrodinium spp. dominated (70%, in the 5- to 25-μm size range) shortly before the main diatom bloom, and small peaks were observed within spring and early summer periods (0–0.4 μg l⁻¹). Phytoflagellates dominated earlier (spring) with higher carbon biomass (8 μg l⁻¹) and post-bloom periods (summer) when carbon biomass ranged between 1 and 4 μg l⁻¹. Accepted: 6 September 2000     

Variable optimization for the formation of three-dimensional self-organized heart muscle

Cardiac tissue-engineering research is focused on the development of functional three-dimensional (3D) heart muscle in vitro. These models allow the detailed study of critical events in organogenesis, such as the establishment of cell–cell communication and construction and modification of the extracellular matrix. We have previously described a model for 3D heart muscle, termed cardioids, formed by the spontaneous delamination of a cohesive monolayer of primary cells in the absence of any synthetic scaffolding material. In an earlier publication, we have shown that, upon electrical stimulation, cardioids generate a twitch force in the range of 200–300 μN, generate a specific force (twitch force normalized to total cross-sectional area) of 2–4 kN/m², and can be electrically paced at frequencies of up to 10 Hz without any notable fatigue. We have two objectives for the current study: model development and model optimization. Our model development efforts are focused on providing additional characterization of the cardioid model. In this study, we show for the first time that cardioids show a pattern of gene expression comparable to that of cells cultured in two dimensions on tissue culture plastic and normal mammalian heart muscle. Compared with primary cardiac cells cultured on tissue culture plastic, the expression of α-myosin heavy chain (MHC), β-MHC, SERCA2, and phospholamban was significantly higher in cardioids. Our second objective, model optimization, is focused on evaluating the effect of several cell culture variables on cardioid formation and function. Specifically, we looked at the effect of plating density (1.0–4.0 × 10⁶ cells per cardioid), concentration of two adhesion proteins (laminin at 0.2–2.0 μg/cm² and fibronectin at 1–10 μg/cm²), myocyte purity (using preplating times of 15 and 60 min), and ascorbic acid stimulation (1–100 μl/ml). For our optimization studies, we utilized twitch force in response to electrical stimulation as our endpoint metric. Based on these studies, we found that cardioids formed with a plating density in the range 3–4 × 10⁶ cells per cardioid generated the maximum twitch force, whereas increasing the surface adhesion protein (using either laminin or fibronectin) and increasing the myocyte purity both resulted in a decrease in twitch force. In addition, increasing the ascorbic acid concentration resulted in an increase in the baseline force of cardioids, which was recorded in the absence of electrical stimulation. Based on the model development studies, we have shown that cardioids do indeed exhibit a gene expression pattern similar to normal mammalian heart muscle. This provides further validity for the cardioid model. Based on the model optimization studies, we have identified specific cell culture regimes which support cardioid formation and function. These results are specific to the cardioid model; however, they may be translated and applied to other tissue-engineering models. Collectively, the work described in this study provides insight into the formation of functional 3D heart muscle and the effect of several cell culture variables on tissue formation and function.     

The spring mesozooplankton community at South Georgia: a comparison of shelf and oceanic sites

Mesozooplankton (predominantly 200–2000 μm) were sampled at a shelf and an oceanic station close to South Georgia, South Atlantic, during austral spring (October/November) 1997. Onshelf zooplankton biomass was extremely high at 10–16 g dry mass m⁻² (0–150 m), 70% comprising the small neritic clausocalaniid copepod Drepanopus forcipatus. Large calanoid species, principally Calanoides acutus and Rhincalanus gigas, contributed only 8–10%. At the oceanic station, biomass in the sampled water column (0–1000 m) was ∼6.5 g dry mass m⁻² and 4–6 g dry mass m⁻² in the top 200 m. Here, large calanoids composed 40–50% of the standing stock. Antarctic krill (Euphausia superba) occurred in low abundances at both stations. Vertical profiles obtained with a Longhurst Hardy Plankton Recorder indicated that populations of C. acutus and R. gigas, which overwinter at depth, had completed their spring ascent and were resident in surface waters. Dry mass, carbon and lipid values were lower than found in summer but were consistent with overwintered populations. Phytoplankton concentrations were considerably higher at the oceanic station (2–3 mg chlorophyll a m⁻³) and increased over the time on station. In response to this, egg production of both large calanoid species and growth rates of R. gigas approached those measured in summer. Onshelf phytoplankton concentrations were lower (<1 mg m⁻³), and low egg production rates suggested food limitation. Here phytoplankton rations equivalent to 6% zooplankton body C would have been sufficient to clear primary production whereas at the oceanic station daily carbon fixation was broadly equivalent to zooplankton carbon biomass. Accepted: 25 April 1999     

Structural and functional properties of sympagic communities in the annual sea ice at Terra Nova Bay (Ross Sea, Antarctica)

Studies on the chemical and biological properties of annual pack ice at a coastal station in Terra Nova Bay (74°41.72′S, 164°11.63′E) were carried out during austral spring at 3-day intervals from 5 November to 1 December 1997. Temporal changes of nutrient concentrations, algal biomasses, taxonomic composition, photosynthetic pigment spectra and P–E relationships were studied. Quantity, composition and degradation rates of organic matter in the intact sea ice were also investigated. In addition, microcosm experiments were carried out to evaluate photosynthetic and photo-acclimation processes of the sympagic flora in relation to different light regimes. High concentrations of ammonia were measured in four ice-cores (weighted mean values of the cores ranged from 4.3 ± 1.9 μM to 7.2 ± 3.4 μM), whereas nitrate and phosphate displayed high concentrations (up to 35.9 μM and 7.6 μM, respectively) only in the bottom layer (135–145 cm depth). Particulate carbohydrate and protein concentrations in the intact sea ice ranged from 0.5 to 2.3 mg l⁻¹ and 0.2 to 2.0 mg l⁻¹, respectively, displaying a notable accumulation of organic matter in the bottom colored layer, where bacterial enzymatic activities also reached the highest values. Aminopeptidase activity was extremely high (up to 19.7 μM l⁻¹ h⁻¹ ± 0.05 in the bottom layer), suggesting a rapid turnover rate of nitrogen–enriched organic compounds (e.g. proteins). By contrast, bacterial secondary production was low, suggesting that only a very small fraction of mobilized organic matter was converted into bacterial biomass (<0.01‰). The sympagic autotrophic biomass (in terms of chlorophaeopigments) of the bottom layer was high, increasing during the sampling period from 680 to 2480 μg l⁻¹. Analyses of pigments performed by HPLC, as well as microscope observations, indicated that diatoms dominated bottom communities. The most important species were Amphiprora sp. and Nitschia cfr. stellata. Bottom sympagic communities showed an average P ^B _max of 0.12 mgC mg Chl⁻¹ and low photoadaptation index (E _k=18 μE m⁻² s⁻¹, E _m=65 μE m⁻² s⁻¹). Results of the microcosm experiment also indicated that communities were photo-oxidized when irradiance exceeded 100 μE m⁻² s⁻¹. This result suggests that micro- autotrophs inhabiting sea ice might have a minor role in the pelagic algal blooms. Accepted: 4 August 1999     

Morphogenesis and regeneration from stomatal guard cell complexes of cotton (Gossypium hirsutum L.)

 The development of a regeneration system from cotton stomatal guard cells directly on epidermal strips is described. The most important factors affecting embryogenic callus initiation in both of the varieties tested (Coker 312 and 315) were the source of the epidermal tissue, including plant age (4–5 months old), the developmental stage of the flower (opening flower stage) from which bracts were obtained, the composition of the culture medium and light irradiance. The flower developmental stage was critical for callus formation, which was observed only from bracts obtained from opening flowers. In addition, epidermal strips excised from the bract basal region were more responsive in culture than those obtained from the top region. Improved callus initiation was obtained on epidermal strips which had their cuticle in contact with the culture medium. Light irradiance was a limiting factor for embryogenic callus formation, which was observed only in calluses cultured under the lower light irradiance (15.8 μmol m^–2 s^–1). Somatic embryogenesis was observed on callus cultures subcultured consecutively to a culture medium containing naphthalene acetic acid (10.7 μM) and isopentenyladenine (4.9 μM). Histodifferentiation of somatic embryos was improved on a medium containing naphthaleneacetic acid (8.1 μM)+isopentenyladenine (2.5 μM) and abscisic acid (0.19–0.38 μM). Somatic embryo germination and plantlet development were obtained using established protocols with few modifications. On average, one fully developed plant was obtained from the culture of circa 100 epidermal strips in both cultivars. Received: 19 May 2000 / Revision received: 25 August 2000 / Accepted: 29 August 2000     

Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture

Malaysia is the world’s leading producer of palm oil products that contribute US$ 7.5 billion in export revenues. Like any other agro-based industries, it generates waste that could be utilized as a source of organic nutrients for microalgae culture. Present investigation delves upon Isochrysis sp. culture in POME modified medium and its utilization as a supplement to Nanochloropsis sp. in rotifer cultures. The culture conditions were optimized using a 1 L photobioreactor (Temp: 23°C, illumination: 180 ∼ 200 μmol photons m⁻²s⁻¹, n = 6) and scaled up to 10 L outdoor system (Temp: 26–29°C, illumination: 50 ∼ 180 μmol photons m⁻²s⁻¹, n = 3). Algal growth rate in photobioreactor (μ = 0.0363 h⁻¹) was 55% higher compared to outdoor culture (μ = 0.0163 h⁻¹), but biomass production was 1.3 times higher in outdoor culture (Outdoor = 91.7 mg m⁻²d⁻¹; Photobioreactor = 69 mg m⁻²d⁻¹). Outdoor culture produced 18% higher lipid; while total fatty acids (FA) was not significantly affected by the change in culture systems as both cultures yield almost similar concentrations of fatty acids per gram of sample (photobioreactor = 119.17 mg g⁻¹; outdoor culture = 104.50 mg g⁻¹); however, outdoor cultured Isochrysis sp. had 26% more polyunsaturated fatty acids (PUFAs). Rotifers cultured in Isochrysis sp./ Nanochloropsis sp. (1:1, v/v) mixture gave similar growth rate as 100% Nanochoropsis sp. culture (μ = 0.40 d⁻¹), but had 45% higher counts of rotifers with eggs (t = 7, maximum). The Isochrysis sp. culture successfully lowered the nitrate (46%) and orthophosphate (83%) during outdoor culture.     

Initiation of humoral and cellular immune responses in patients with refractory Hodgkin's disease by treatment with an anti-CD16/CD30 bispecific antibody

Fifteen patients with refractory Hodgkin's disease were treated in a dose-escalation trial with the bispecific monoclonal antibody (bi-mAb) HRS-3/A9, which is directed against the Fcγ receptor III (CD16 antigen) and the Hodgkin's-associated CD30 antigen. Treatment consisted of four cycles of four bi-mAb infusions given over 1 h every 3–4 days at different dose levels ranging from 1 mg/m² to 64 mg/m². Measurable serum levels (above 0.1 μg/ml) of circulating bi-mAb could be detected in patients treated with doses above 4 mg/m², reaching peak levels of 9.5 μg/ml immediately after the end of antibody infusion on the highest dose level. Bi-mAb elimination corresponded to second-order kinetics with a terminal half-life time (t _1/2,β) of 28–32 h. Bi-mAb treatment induced the occurrence of human anti-(mouse Ig) antibodies (HAMA) in 6 out of 13 patients initially testing negative. All 6 patients not only developed anti-isotypic anti-(mouse Ig) but also anti-idiotypic and anti-anti-idiotypic antibodies. While no consistent changes of peripheral blood cell counts, or of any lymphocyte subpopulation including natural killer (NK) cells, has been observed, 4 out of 6 evaluable patients treated with doses of at least 4 mg/m² showed an increase of NK cell activity within 2 weeks after treatment, which lasted for a maximum of 12 weeks. Circulating amounts of soluble CD30 antigen could be detected in the serum of 6 patients. However, like the results and time courses of all the other immunological parameters evaluated, this was not predictive for treatment outcome. Received: 16 September 1999 / Accepted: 6 January 2000     

Physiological differences in the growth of <Emphasis Type="Italic">Sargassum horneri</Emphasis> between the germling and adult stages

The effects of temperature, irradiance, and daylength on Sargassum horneri growth were examined at the germling and adult stages to discern their physiological differences. Temperature–irradiance (10, 15, 20, 25, 30°C × 20, 40, 80 μmol photons m⁻²s⁻¹) and daylength (8, 12, 16, 24 h) experiments were carried out. The germlings and blades of S. horneri grew over a wide range of temperatures (10–25°C), irradiances (20–80 μmol photons m⁻²s⁻¹), and daylengths (8–24 h). At the optimal growth conditions, the relative growth rates (RGR) of the germlings were 21% day⁻¹ (25°C, 20 μmol photons m⁻²s⁻¹) and 13% day⁻¹ (8 h daylength). In contrast, the RGRs of the blade weights were 4% day⁻¹ (15°C, 20 μmol photons m⁻²s⁻¹) and 5% day⁻¹ (12 h daylength). Negative growth rates were found at 20 μmol photons m⁻²s⁻¹ of 20°C and 25°C treatments after 12 days. This phenomenon coincides with the necrosis of S. horneri blades in field populations. In conclusion, we found physiological differences between S. horneri germlings and adults with respect to daylength and temperature optima. The growth of S. horneri germlings could be enhanced at 25°C, 20 μmol photons m⁻²s⁻¹, and 8 h daylength for construction of Sargassum beds and restoration of barren areas.     

Effects of temperature on photosynthesis of two morphologically contrasting plant species along an altitudinal gradient in the tropical high Andes

The effects of temperature on photosynthesis of a rosette plant growing at ground level, Acaena cylindrostachya R. et P., and an herb that grows 20–50 cm above ground level, Senecio formosus H.B.K., were studied along an altitudinal gradient in the Venezuelan Andes. These species were chosen in order to determine – in the field and in the laboratory – how differences in leaf temperature, determined by plant form and microenvironmental conditions, affect their photosynthetic capacity. CO₂ assimilation rates (A) for both species decreased with increasing altitude. For Acaena leaves at 2900 m, A reached maximum values above 9 μmol m⁻² s⁻¹, nearly twice as high as maximum A found at 3550 m (5.2) or at 4200 m (3.9). For Senecio leaves, maximum rates of CO₂ uptake were 7.5, 5.8 and 3.6 μmol m⁻² s⁻¹ for plants at 2900, 3550 and 4200 m, respectively. Net photosynthesis-leaf temperature relations showed differences in optimum temperature for photosynthesis (A _o.t.) for both species along the altitudinal gradient. Acaena showed similar A _o.t. for the two lower altitudes, with 19.1°C at 2900 m and 19.6°C at 3550 m, while it increased to 21.7°C at 4200 m. Maximum A for this species at each altitude was similar, between 5.5 and 6.0 μmol m⁻² s⁻¹. For the taller Senecio, A _o.t. was more closely related to air temperatures and decreased from 21.7°C at 2900 m, to 19.7°C at 3550 m and 15.5°C at 4200 m. In this species, maximum A was lower with increasing altitude (from 6.0 at 2900 m to 3.5 μmol m⁻² s⁻¹ at 4200 m). High temperature compensation points for Acaena were similar at the three altitudes, c. 35°C, but varied in Senecio from 37°C at 2900 m, to 39°C at 3550 m and 28°C at 4200 m. Our results show how photosynthetic characteristics change along the altitudinal gradient for two morphologically contrasting species influenced by soil or air temperatures. Received: 5 July 1997 / Accepted: 25 October 1997     

Energy Budget for the Cultured,Zooxanthellate Octocoral <Emphasis Type="Italic">Sinularia flexibilis</Emphasis>

The zooxanthellate octocoral Sinularia flexibilis is a producer of potential pharmaceutically important metabolites such as antimicrobial and cytotoxic substances. Controlled rearing of the coral, as an alternative for commercial exploitation of these compounds, requires the study of species-specific growth requirements. In this study, phototrophic vs. heterotrophic daily energy demands of S. flexibilis was investigated through light and Artemia feeding trials in the laboratory. Rate of photosynthetic oxygen by zooxanthellae in light (≈200 μmol quanta m⁻² s⁻¹) was measured for the coral colonies with and without feeding on Artemia nauplii. Respiratory oxygen was measured in the dark, again with and without Artemia nauplii. Photosynthesis–irradiance curve at light intensities of 0, 50, 100, 200, and 400 μmol quanta m⁻² s⁻¹ showed an increase in photosynthetic oxygen production up to a light intensity between 100 and 200 μmol quanta m⁻² s⁻¹. The photosynthesis to respiration ratio (P/R > 1) confirmed phototrophy of S. flexibilis. Both fed and non-fed colonies in the light showed high carbon contribution by zooxanthellae to animal (host) respiration values of 111–127%. Carbon energy equivalents allocated to the coral growth averaged 6–12% of total photosynthesis energy (mg C g ⁻ 1 buoyant weight day ⁻ 1) and about 0.02% of the total daily radiant energy. “Light utilization efficiency (ε)” estimated an average ε value of 75% 12 h ⁻ 1 for coral practical energetics. This study shows that besides a fundamental role of phototrophy vs. heterotrophy in daily energy budget of S. flexibilis, an efficient fraction of irradiance is converted to useable energy.     

Production of transgenic tall fescue and red fescue plants by particle bombardment of mature seed-derived highly regenerative tissues

 Highly regenerative tissues of tall fescue and red fescue produced from mature seed-derived embryogenic callus were induced and proliferated on medium containing 2,4-dichlorophenoxyacetic acid (4.5 or 9.0 μM), 6-benzylaminopurine (0, 0.044, 0.44 or 2.2 μM) and cupric sulfate (0.1 or 5.0 μM) under dim-light conditions (10 to 30 μE m^–2 s^–1, 16 h light). Tall fescue tissues were transformed with three plasmids containing the genes for hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA;gus), and red fescue with three plasmids containing hpt, uidA and a synthetic green fluorescent protein gene [sgfp(S65T)]. DNA from T₀ plants of eight independently transformed lines from tall fescue and 11 from red fescue were analyzed by PCR and DNA blot hybridization. The co-expression frequency of all three transgenes [hpt/bar/uidA or hpt/uidA/sgfp(S65T)] in transgenic tall fescue and red fescue plants was 25–27%; for two transgenes [hpt/bar or hpt/uidA for tall fescue and hpt/uidA or hpt/sgfp(S65T) for red fescue], the co-expression frequency was 50–75%. Received: 28 September 1999 / Revision received: 13 March 2000 / Accepted: 16 March 2000     

Multiple shoot regeneration of kenaf (Hibiscus cannabinus L.) from a shoot apex culture system

 In this research, a medium was developed that would stimulate multiple shoot initiation from shoot apex explants of Hibiscus cannabinus L. (kenaf). Adventitious shoot formation on a shoot induction media supplemented with combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.5, 2.3 μmol·l^–1) and thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea; TDZ) (0, 1, 5, 20 μmol·l^–1) was evaluated. Multiple shoot induction medium with 1 μmol·TDZ l^–1 resulted in the highest number of regenerated shoots per explant for all three kenaf cultivars tested (Tainung 1, Tainung 2, and Everglades 71). The 2,4-D did not enhance multiple shoot formation. Additionally, kenaf cultivars 7N and SF459 also produced multiple shoots on the induction medium with 1 μmol·TDZ l^–1. Multiple shoot clumps formed after 2 weeks in culture without callus formation. Shoots elongated and rooted within 2 weeks after subculture on a plant growth regulator-free medium. A histological study demonstrated the de novo regeneration of shoots from the shoot apex. Received: 2 February 2000 / Revision received: 30 March 2000 / Accepted: 22 June 2000     

Metabolism of EDTA and its metal chelates by whole cells and cell-free extracts of strain BNC1

The influence of metal ions on the metabolism of ethylenediaminetetraacetate (EDTA) by whole cells and cell-free extracts of strain BNC1 was investigated. Metal-EDTA chelates with thermodynamic stability constants below 10¹² were readily mineralized by whole cells with maximum specific turnover rates of 15 (MnEDTA) to 20 (Ca-, Mg-, and BaEDTA) μmol g protein⁻¹ min⁻¹. With the exception of ZnEDTA, chelates with stability constants greater than 10¹² were not oxidized at a significant rate. However, it was shown for Fe(III)EDTA that even strong complexes can be degraded after pretreatment by addition of calcium and magnesium salts in the pH range 9–11. The range of EDTA chelates converted by cell-free extracts of strain BNC1 did not depend on their thermodynamic stabilities. The EDTA chelates of Ba²⁺, Co²⁺, Mg²⁺, Mn²⁺, and Zn²⁺ were oxidized whereas Ca-, Cd-, Cu-, Fe-, Pb-, and SnEDTA were not. The first catabolic enzyme appears to be an EDTA monooxygenase since it requires O₂, NADH, and FMN for its activity and yields glyoxylate and ethylenediaminetriacetate as products. The latter is further degraded via N,N′-ethylenediaminediacetate. The maximum specific turnover rate with MgEDTA, the favoured EDTA species, was 50–130 μmol g protein⁻¹ min⁻¹, and the K _m value was 120 μmol/l (K _s for whole cells = 8 μmol/l). Whole cells as well as cell-free extracts of strain BNC1 also converted several structural analogues of EDTA. Received: 4 July 1997 / Received revision: 25 September 1997 / Accepted: 29 September 1997     

Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot tips

 Shoot tips from in vitro-grown, cold-hardened stock plants of white poplar (Populus alba L.) were successfully cryopreserved at –196  °C by one-step vitrification. After preculturing at 5  °C for 2 days on hormone-free MS medium containing different sucrose concentrations, and loading for 20 min with 2 m glycerol and 0.4 m sucrose, shoot tips were treated with the PVS2 vitrification solution and plunged directly into liquid nitrogen. Best survival rate (90%) was obtained when shoot tips were precultured on 0.09 m sucrose, hormone-free MS medium, vitrified by exposure to PVS2 solution for 60 min at 0  °C and, following cryopreservation, rewarmed at 40  °C and washed in 1.2 m sucrose solution for 20 min. Regrowth was improved by plating shoot tips on a gelled MS medium containing 1.5 μm N⁶-benzyladenine plus 0.5 μm gibberellic acid, while shoot rooting was achieved on MS medium containing 3 μm indole-3-butyric acid. Following this procedure, almost 60% rooted shoots were obtained from cryopreserved shoot tips. Received: 1 February 1999 / Revision received: 3 May 1999 · Accepted: 21 May 1999