Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes

Summary In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalves. The outer epithelia of the pallial mantle of the pearl oyster, Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20° C in 5% CO₂:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d after the inoculation, 5-bromo-2′-deoxyuridine was added to the culture at 30 μM. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2′-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus, cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of epithelium in bivalves.     

Developmental changes in carboxylase activities in in vitro cultured coconut zygotic embryos: comparison with corresponding activities in seedlings

Phosphoenolpyruvate Carboxylase (PEPC; EC: 4.1.1.31) and Ribulose 1,5-bisphosphate Carboxylase/Oxygenase (RubisCO; EC: 4.1.1.39) enzyme specific activities were measured during the in vitro development of coconut (Cocos nucifera L.) zygotic mature embryos into plantlets and compared with those of palms produced by conventional seed germination. At the time of initiation of germination, high PEPC and low RubisCO activities were measured in both cultured and conventionally germinated embryos, thus indicating an anaplerotic CO₂ fixation. During both in vitro and in planta development, RubisCO progressively took over and became the main route for inorganic carbon fixation. The in vitro-grown coconut plantlets showed a faster decrease in their PEPC:RubisCO ratio than the seedlings, suggesting that an earlier transition from a heterotrophic to an autotrophic mode of carbon fixation takes place in the in vitro-derived material. Just before acclimatization, the RubisCO activity in in vitro-derived plantlets (2.83 μmol CO₂h⁻¹mg⁻¹ _TSP) was lower than that in seedlings (6.98 μmol CO₂h⁻¹mg⁻¹ _TSP) of the same age. Nevertheless, after acclimatization, RubisCO activities were comparable in both in vitro and in planta germinated material This revised version was published online in June 2006 with corrections to the Cover Date.     

Regulation of sucrose storage by amino acid arginine in sugarcane cell suspension cultures

Sugarcane cell cultures were obtained from callus formed on explants derived from young expanding leaves of two early maturing sugarcane varieties viz “CoJ83” and “CoJ86”. The cell cultures were varied with different arginine concentrations in the culture medium. For each cultivar, sucrose content with 20 μM arginine in the culture medium decreased from 3 to 5 days and then increased to 10 days after subculturing. Higher concentration of arginine in the culture medium (60 μM) decreased the sucrose content at different days after subculturing and thus significantly stimulated sucrose mobilization. The activity of sucrose synthase and sucrose phosphate synthase reached maximum while the activity of acid and neutral invertase was minimal in the culture medium with 20 μM arginine. Thus arginine at low concentration (20 μM) enables the cells to accumulate the higher level of sucrose. The optimum level of amino acids can be utilized to regulate the in vivo activity of sucrose synthase, sucrose phosphate synthase and invertase to achieve maximum sucrose accumulation in sugarcane storage tissue.     

Purification and Characterization of Nitrate Reductase from the Halotolerant Cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

Summary Factors affecting the activity of nitrate reductase (E.C.1.7.7.2) from the halotolerant cyanobacterium Aphanothece halophytica were investigated. Cells grown in nitrate-containing medium exhibited higher nitrate reductase activity than cells grown in medium in which nitrate was replaced by glutamine. When ammonium was present in the medium instead of nitrate, the activity of nitrate reductase was virtually non-detectable, albeit with normal cell growth. The enzyme was localized mainly in the cytoplasm. The enzyme was purified 406-fold with a specific activity of 40.6 μmol/min/mg protein. SDS-PAGE revealed a subunit molecular mass of 58 kDa. Gel filtration experiments revealed a native molecular mass of 61 kDa. The K _m value for nitrate was 0.46 mM. Both methyl viologen and ferredoxin could serve as electron donor with K _m values of 4.3 mM and 5.2 μM, respectively. The enzyme was strongly inhibited by sulfhydryl-reactive agents and cyanide. Nitrite, the product of the enzyme reaction, showed little inhibition. Chlorate, the substrate analog, could moderately inhibit the enzyme activity. NaCl up to 200 mM stimulated the activity of the enzyme whereas enzyme inhibition was observed at ≥300 mM NaCl.     

In vitro clonal propagation of an aromatic medicinal herb Ocimum basilicum L. (sweet basil) by axillary shoot proliferation

Summary An efficient protocol for in vitro propagation of an aromatic and medicinal herb Ocimum basilicum L. (sweet basil) through axillary shoot proliferation from nodal explants, collected from field-grown plants, is described. High frequency bud break and maximum number of axillary shoot formation was induced in the nodal explants on Murashige and Skoog (1962) medium (MS) containing N⁶-benzyladenine (BA). The nodal explants required the presence of BA at a higher concentration (1.0 mg·l⁻¹, 4.4 μM) at the initial stage of bud break; however, further growth and proliferation required transfer to a medium containing BA at a relatively low concentration (0.25 mg·gl⁻¹, 1.1 μM). Gibberellic (GA₃) at 0.4 mg·l⁻¹ (1.2 μM) added to the medium along with BA (1.0 mg·l⁻¹, 4.4 μM) markedly enhanced the frequency of bud break. The shoot clumps that were maintained on the proliferating medium for longer durations, developed inflorescences and flowered in vitro. The shoots formed in vitro were rooted on half-strength MS supplemented with 1.0 mg·l⁻¹ (5.0 μM) indole-3-butyric acid (IBA). Rooted plantlets were successfully acclimated in vermi-compost inside a growth chamber and eventually established in soil. All regenerated plants were identical to the donor plants with respect to vegetative and floral morphology.     

The effect of additional red irradiation on the photosynthetic apparatus of <Emphasis Type="Italic">Pisum sativum</Emphasis>

Pisum sativum (L.) plants were grown under “white” luminescent lamps, W [45 μ mol(quantum) m⁻² s⁻¹] or under the same irradiation supplemented with narrow spectrum red light-emitting diodes (LEDs), RE [λ_max = 660 nm, Δλ = 20 nm, 40 μmol(quantum) m⁻² s⁻¹]. Significant differences in the chlorophyll (Chl) a fluorescence parameters, degree of State 1–State 2 transition, and the pigment-protein contents were found in plants grown under differing spectral composition. Addition of red LEDs to the “white light” resulted in higher effective quantum yield of photosystem 2 (PS2), i.e. F′_v/F′_m, linear electron transport (ϕ_PS2), photochemical quenching (q_P), and lower non-photochemical quenching (q_N as well as NPQ). The RE plants were characterised by higher degree State 1–State 2 transition, i.e. they were more effective in radiant energy utilisation. Judging from the data of “green” electrophoresis of Chl containing pigment-protein complexes of plants grown under various irradiation qualities, the percentage of Chl in photosystem 2 (PS2) reaction centre complexes in RE plants was higher and there was no difference in the total Chl bound with Chl-proteins of light-harvesting complexes (LHC2). Because the ratio between oligomeric and monomeric LHC2 forms was higher in RE plants, we suggest higher LHC2 stability in these ones.     

The effect of different closure types, light, and sucrose concentrations on carbon isotope composition and growth ofGardenia jasminoides plantlets during micropropagation and subsequent acclimationex vitro

The growth ofGardenia jasminoides Ellis plantlets and the development of photoautotrophy during two successive culture stages (shoot multiplication and root induction)in vitro was analyzed. We examined the effects of changes in growth conditions (type of tube closure, light, and sugar levels) on the development of photoautotrophy and growth during micropropagation and sought to establish whether they affected later acclimation to conditionsex vitro. During the two stagesin vitro, plantlets were grown in tubes under two different PPFD (50 and 110 μmol m⁻² s⁻¹), in media with three different sucrose concentrations (0, 1.5, and 3.0%, w/v) and with two different CO₂ levels inside the tubes (controlled by either tightly closed caps or loosely sealed caps, and with an external CO₂ concentration of 750 μmol mol⁻¹). The development of photoautotrophy was assessed by determining the difference between the stable carbon isotope composition (δ¹³C) of sugar cane sucrose used as a heterotrophic carbon source and that of leaflets grownin vitro. Plantlets from the root-induction stage showed a more highly developed photoautotrophy than those from the shoot- multiplication stage. At both stages, utilization of closed caps was the treatment which most stimulated development of photoautotrophy in plantlets. Also, lowering PPFD or sucrose concentration induced a greater degree of photoautotrophic development, the strongest effect being observed in plantlets cultured inside loosely sealed tubes. During acclimationex vitro, plantlets taken from loosely sealed tubesin vitro performed better than those cultured inside tightly sealed tubes. The former, as well as recording a larger increase in fresh weight during this stage, also showed more negative δ¹³C in the newly developed leaves, which would seem to indicate a better water status during acclimation. Present results validate the usefulness of δ¹³C analysis of leaflets as a simple technique in assessing the development of photoautotrophy during culturein vitro. In addition, δ¹³C analysis can be extended to evaluate growth conditions during acclimation toex vitro conditions.     

Involvement of betacyanin in chilling-induced photoinhibition in leaves of <Emphasis Type="Italic">Suaeda salsa</Emphasis>

Seeds of Suaeda salsa were cultured in dark for 3 d and betacyanin accumulation in seedlings was promoted significantly. Then the seedlings with accumulated betacyanin (C+B) were transferred to 14/10 h light/dark and used for chilling treatment 15 d later. Photosystem 2 (PS2) photochemistry, D1 protein content, and xanthophyll cycle during the chilling-induced photoinhibition (exposed to 5 °C at a moderate photon flux density of 500 μmol m⁻² s⁻¹ for 3 h) and the subsequent restoration were compared between the C+B seedlings and the control (C) ones. The maximal efficiency of PS2 photochemistry (F_v/F_m), the efficiency of excitation energy capture by open PS2 centres (F_v′/F_m′), and the yield of PS2 electron transport (Φ_PS2) of the C+B and C leaves both decreased during photoinhibition. However, smaller decreases in F_v/F_m, F_v′/F_m′, and Φ_PS2 were observed in the C+B leaves than in C ones. At the same time, the deepoxidation state of xanthophyll cycle, indicated by (A+Z)/(V+A+Z) ratio, increased rapidly but the D1 protein content decreased considerably during the photoinhibition. The increase in rate of (A+Z)/(V+A+Z) was higher but the D1 protein turnover was slower in C+B than C leaves. After photoinhibition treatment, the plants were transferred to a dim irradiation (10 μmol m⁻² s⁻¹) at 25 °C for restoration. During restoration, the chlorophyll (Chl) fluorescence parameters, D1 protein content, and xanthophyll cycle components relaxed gradually, but the rate and level of restoration in the C+B leaves was greater than those in the C leaves. The addition of betacyanins to the thylakoid solution in vitro resulted in similar changes of F_v/F_m, D1 protein content, and (A+Z)/(V+A+Z) ratio during the chilling process. Therefore, betacyanin accumulation in S. salsa seedlings may result in higher resistance to photoinhibition, larger slowing down of D1 protein turnover, and enhancement of non-radiative energy dissipation associated with xanthophyll cycle, as well as in greater restoration after photoinhibition than in the control when subjected to chilling at moderate irradiance.     

Paradoxal pharmacologic effects observed withβ-blocker agents on cardiac cells in culture

Summary The direct effects of propranolol and metoprolol were studied on cultured neonatal rat ventricular myocytes by recording cellular contraction with a video signal analyzer of cells movements. The experiments were performed on 3-d-old cultures forming a synchronously beating monolayer. The spontaneous beating frequency of preparations depended on cultures and varied from 100 to 330 beats/min with a peak for the interval 140 to 149 beats/min. Propranolol and metoprolol were tested at 1, 5, 10μM and 10, 50, 100μM, respectively. At these doses the twoβ-blockers induced chronotropic and inotropic effects in the same range. The two agents induced rapid and short-lasting negative inotropic effects, which were dose-dependent and delayed negative chronotropic effects even with the lowest doses. In some preparations paradoxal effects were evidenced: an increase of the amplitude or frequency of the contractions was observed. The results obtained with 5μM propranolol or 50μM metoprolol could be separated in two groups depending on the basal beating rate (〈 or 〉 to 150 beats/min). In cultures with a rapid frequency, the drugs had a marked negative chronotropic effect and, surprisingly, a positive inotropic effect was observed. These findings confirm the interdependence of the two parameters frequency and amplitude of contraction on this model, and evidence the interest of taking into account the control parameters before any interpretation.     

Environmental induced variations in leaf dark respiration and net photosynthesis of <Emphasis Type="Italic">Quercus ilex</Emphasis> L.

The relationships between dark respiration rate (R _D) and net photosynthetic rate (P _N) in Quercus ilex L. shrubs growing at the Botanical Garden in Rome were analysed. Correlation analysis of the data sets collected in the year 2006 confirmed the dependence among the considered leaf traits, in particular, R _D was significantly (p<0.05) correlated with P _N (r = 0.40). R _D and P _N increased from March to May [1.40±0.10 and 10.1±1.8 μmol(CO₂) m⁻² s⁻¹ mean values of the period, respectively], when air temperature was in the range 14.8–25.2 °C, underlining the highest metabolic activity in the period of the maximum vegetative activity that favoured biomass accumulation. On the contrary, the highest R _D [1.60±0.02 μmol(CO₂) m⁻² s⁻¹], associated to the lowest P _N rates (44 % of the maximum) and carbon use efficiency (CUE) in July underlined the mobilization of stored material during drought stress by a higher air temperature (32.7 °C).     

Functional responses of Acer species to two simulated forest gap environments: leaf-level properties and photosynthesis

Seedlings of eight forest maple (Acer L.) species were grown outdoors through a full season under two irradiation treatments: (a) “gap edge” with a photosynthetic photon flux density of 30 μmol m-² s-¹ and a red:far-red ratio of 0.55, and (b) “gap centre” with 400 μmol m-² s-¹ and a red:far-red ratio of 1.12. Area-based leaf nitrogen concentration was greater in gap centre-grown seedlings, whereas, except for A. saccharum, area-based chlorophyll (Chl) (a+b) was higher in gap edge-grown plants. There was also a significantly lower Chl a/b ratio in gap edge-grown plants. Maximum photosynthetic rate (P _max ) was 60 % higher in the gap-centre treatment. These results are consistent with the functional expectation that shade-acclimated plants will increase their radiant-energy harvesting capacity as a result of limited photon input while gap-acclimated plants will operate more efficiently under bright irradiance by increasing their carboxylation capacity. This inverse relationship between the capacity of the light-harvesting component and the carboxylation component is, however, only partially supported by Chl fluorescence measurements of intact leaves. Compared to gap centre-grown plants, the lower total fluorescence quenching in gap edge-grown plants indicated a lower carboxylation capacity that was in accord with the observed P _max . However, edge-grown seedlings did not show the expected improvement in light-harvesting efficiency and reduction in electron transport of photosystem 2 inferred from their marginally greater t_1/2 and lower F_v/F_m, respectively. Hence while maples acclimated to different irradiation levels by adjusting leaf N and Chl contents, they showed limited acclimation potential at the photosystem level. Variations in the leaf traits examined had only minor effect on low irradiance photosynthesis and sunfleck utilization. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effects of hydration and divalent cations on lamellar-nonlamellar phase transitions in membranes and total lipid extracts from Acholeplasma laidlawii A-EF22 – a 2H NMR study

Acholeplasma laidlawii strain A-EF22 was grown in a medium supplemented with 75 μm α-deuterated palmitic acid (16:0-d ₂) and 75 μm α-deuterated oleic acid (18:1c-d ₂), or with 150 μm 18:1c-d ₂. The fatty acids were incorporated into the membrane lipids and ²H NMR spectra were recorded from intact membranes, total lipid extracts, and the combined glucolipid and neutral lipid fractions of a total lipid extract. The lipids in intact membranes form a bilayer structure up to at least 70 °C. The same result was obtained with membranes digested with pronase, which removes a large fraction of the membrane proteins. A reversed hexagonal liquid crystalline (H_II) phase was formed below 70 °C by the total lipid extracts hydrated with 20 and 30% (w/w) water; in the presence of 40% (w/w) water only one of the extracts formed an H_II phase below 70 °C. The H_II phase was formed at higher temperatures with an increasing water content. However, only a lamellar liquid crystalline (L _α ) phase was formed up to 70 °C by the total lipid extracts when the water concentrations were 50% (w/w) or higher. The temperature (T _LH) for the L _α to H_II phase transition in the combined glucolipid and neutral lipid fractions was only 2–3 °C lower than for the total lipids, and the phospholipids thus have a very modest influence on the T _LH value. Physiologically relevant concentrations of Ca²⁺ and Mg²⁺ ions did not affect the phase equilibria of total lipid extracts significantly. It is concluded from comparison with published data that the membrane lipids of the cell wall-less bacterium A. laidlawii have a smaller tendency to form reversed nonlamellar phases than the membrane lipids of three bacterial species surrounded by a cell wall. Received: 10 March 1997 / Accepted: 4 July 1997     

Effects of light,temperature and salinity on the growth rate of harmful marine diatoms,Chaetoceros convolutus and C. concavicornis that kill netpen salmon

Chaetoceros convolutus and C. concavicornis have been implicated in the death of salmon in netpens in the Pacific Northwest by damaging the salmon's gills. To better understand how environmental factors affect the distribution of these two species, the interacting effects of light, temperature and salinity on growth rate were examined by growing these species under a range of temperatures (4–18 °C), light (10–175 μmol photon m⁻² s⁻¹) and salinities (10–30‰). For C. convolutus, the growth rate showed a hyperbolic relationship with irradiance at 8, 14 and 18 °C and light saturation occurred at 9, 14 and 20 μmol photon m^t s⁻¹ respectively. At 4 °C for C. convolutus and 8 °C for C. concavicornis, cells grew at μ_max, even at the lowest irradiances tested (10 μmol photon m⁻² s⁻¹). For C. convolutus, the amount of light required to saturate growth rate increased with temperature in an approximately linear fashion. The Q₁₀ was 1.88, calculated by averaging over both species. C. concavicornis was the more euryhaline species growing at salinities as low as 17.5‰, while C. convolutus grew only at 25‰ and above.     

Arctic sediments (Svalbard): pore water and solid phase distributions of C,N, P and Si

Pore water and solid phase distributions of C, N, P and Si in sediments of the Arctic Ocean (Svalbard area) have been investigated. Concentrations of organic carbon (C_org) in the solid phase of the sediment varied from 1.3 to 2.8% (mean 1.9%), with highest concentrations found at shallow stations south/southwest of Svalbard. Relatively low concentrations were obtained at the deeper stations north/northeast of Svalbard. Atomic carbon to nitrogen ratios in the surface sediment ranged from below 8 to above 10. For some stations, high C/N ratios together with high concentrations of C_org suggest that sedimentary organic matter is mainly of terrigenous origin and not from overall biological activity in the water column. Organic matter reactivity (defined as the total sediment oxygen consumption rate normalized to the organic carbon content of the surface sediment) correlated with water depth at all investigated stations. However, the stations could be divided into two separate groups with different reactivity characteristics, representing the two most dominant hydrographic regimes: the region west of Svalbard mainly influenced by the West Spitsbergen Current, and the area east of Svalbard where Arctic polar water set the environmental conditions. Decreasing sediment reactivity with water depth was confirmed by the partitioning between organic and inorganic carbon of the surface sediment. The ratio between organic and inorganic carbon at the sediment-water interface decreased exponentially with water depth: from indefinite values at shallow stations in the central Barents Sea, to approximately 1 at deep stations north of Svalbard. At stations east of Svalbard there was an inverse linear correlation between the organic matter reactivity (as defined above) and concentration of dissolved organic carbon (DOC) in the pore water. The more reactive the sediment, the less DOC existed in the pore water and the more total carbonate (C_t or ΣCO₂) was present. This observation suggests that DOC produced in reactive sediments is easily metabolizable to CO₂. Sediment accumulation rates of opaline silica ranged from 0.35 to 5.7 μmol SiO₂ m⁻²d⁻¹ (mean 1.3 μmol SiO₂ m⁻²d⁻¹), i.e. almost 300 times lower than rates previously reported for the Ross Sea, Antarctica. Concentrations of ammonium and nitrate in the pore water at the sediment-water interface were related to organic matter input and water depth. In shallow regions with highly reactive organic matter, a pool of ammonium was present in the pore water, while nitrate conoentrations were low. In areas where less reactive organic matter was deposited at the sediment surface, the deeper zone of nitrification caused a build-up of nitrate in the pore water while ammonium was almost depleted. Nitrate penetrated from 1.8 to ≥ 5.8 cm into the investigated sediments. Significantly higher concentrations of “total” dissolved nitrogen (defined as the sum of NO₃, NO₂, NH₄ and urea) in sediment pore water were found west compared to east of Svalbard. The differences in organic matter reactivity, as well as in pore water distribution patterns of “total” dissolved nitrogen between the two areas, probably reflect hydrographic factors (such as ice coverage and production/import of particulate organic material) related to the dominant water mass (Atlantic or Arctic Polar) in each of the two areas. The data presented were collected during the European “Polarstern” Study (Arctic EPOS) sponsored by the European Science Foundation     

Effects of brassinosteroid infiltration prior to cold treatment on ion leakage and pigment contents in rape leaves

The effect of 24-epibrassinolide (BR₂₇) on cold resistance of rape seedlings was studied by ion leakage and photosynthetic pigment degradation measurements. Aqueous solutions of BR₂₇ were injected into cotyledons or primary leaves of rape plants and these plants were incubated at 2 °C or 20 °C. Cold treatment (2 °C) without BR₂₇ injection elevated the membrane permeability in both primary leaves and cotyledons significantly. Surprisingly, injection of leaves with water or 0.467 % aqueous ethanol solution led to a massive increase in membrane permeability after cold stress at 2 °C. The synergistic effect of leaf infiltration and cold on permeability was abolished by 0.05 and 1.00 μM of BR₂₇ in primary leaves and by 1.00 μM of BR₂₇ in cotyledons. On the other hand, BR₂₇ solutions strongly elevated the membrane permeability at 20 °C, while water and ethanol solutions brought about only negligible increases. Water or ethanol infiltrations strongly reduced the leaf contents of chlorophyll (Chl) a, Chl b and carotenoids at 2 °C but less markedly at 20 °C. However, in seedlings exposed to 2 °C pigments content was significantly higher in BR₂₇-treated leaves as compared to water/ethanol control. There were no differences between pigment contents of leaves injected with BR₂₇ solutions or only water/ethanol at 20 °C. The above data strongly support the stress protecting effect of BR₂₇.     

Carbon-nanotube-modified electrodes for the direct bioelectrochemistry of pseudoazurin

The bioelectrochemistry of the blue copper protein, pseudoazurin, at glassy carbon and platinum electrodes that were modified with single-wall carbon nanotubes (SWNTs) was investigated by multiple scan rate cyclic voltammetry. The protein showed reversible electrochemical behavior at both bare glassy carbon electrodes (GCEs) and SWNT-modified GCEs (SWNT|GCEs); however, direct electrochemistry was not observed at any of the platinum electrodes. The effect of the carbon nanotubes at the GCE was to amplify the current response 1000-fold (nA at bare GCE to μA at SWNT|GCE), increase the apparent diffusion coefficient D _app of the solution-borne protein by three orders of magnitude, from 1.35 × 10⁻¹¹ at bare GCE to 7.06 × 10⁻⁸ cm² s-1 at SWNT|GCE, and increase the heterogeneous electron transfer rate constant k _s threefold, from 1.7 × 10⁻² cm s⁻¹ at bare GCE to 5.3 × 10⁻² cm s⁻¹ at SWNT|GCE. Pseudoazurin was also found to spontaneously adsorb onto the nanotube-modified GCE surface. Well-resolved voltammograms indicating quasi-reversible faradaic responses were obtained for the adsorbed protein in phosphate buffer, with I _pc and I _pa values now greater than corresponding values for solution-borne pseudoazurin at SWNT|GCEs and with significantly reduced ΔE _p values. The largest electron transfer rate constant of 1.7 × 10⁻¹ cm s⁻¹ was achieved with adsorbed pseudoazurin at the SWNT|GCE surface in deaerated buffer solution consistent with its presumed role in anaerobic respiration of some bacteria.     

Cadmium elevates level of protein, amino acids and alters activity of proteolytic enzymes in germinating rice seeds

When seeds of two rice cvs. Ratna and Jaya were germinated under increasing levels of cadmium nitrate (0, 100 and 500 μM) in the medium, a marked decrease in germination percentage was observed with Cd treatments, as compared to controls. There was more absorbed Cd in embryo axes than in endosperms. More uptake resulted with increasing Cd levels in the growth medium in embryo axes. In both rice cultivars, during a germination period of 0 – 120 h, an increased level of protein as well as free amino acids was noted in Cd treatments. Protease activity in general decreased in both embryo axes as well as endosperms due to Cd treatment. In vitro studies showed an enhancement in protease activity in Cd treatments at low Cd levels (50–100 μM), whereas concentrations above this caused inhibition in enzyme activity. Under 500 μM Cd treatments in vivo there was about 30 to 50 percent decline in leucine aminopeptidase (LAP) activity in endosperms, however, carboxypeptidase activity showed a marked increase in endosperms beyond 24 h under Cd treatments. In embryo axes of germinating seeds there was always a decline in peptidase activities, under the influence of cadmium. The leucine amino peptidase and protease activity were always greater in embryo axes in cv. Ratna than cv. Jaya. However, the carboxypeptidase activity was higher in Jaya when compared to Ratna in endosperms under Cd treatments. The results suggest possible suppression of protease and peptidase activities due to Cd treatments in germinating rice seeds leading to altered levels of protein and amino acids.     

Leaf Photosynthesis of the Mangrove Avicennia Germinans as Affected by NaCl

In leaves of the mangrove species Avicennia germinans (L.) L. grown in salinities from 0 to 40 ‰, fluorescence, gas exchange, and δ¹³C analyses were done. Predawn values of F_v/F_m were about 0.75 in all the treatments suggesting that leaves did not suffer chronic photoinhibition. Conversely, midday F_v/F_m values decreased to about 0.55-0.60 which indicated strong down-regulation of photosynthesis in all treatments. Maximum photosynthetic rate (P _max) was 14.58 ± 0.22 μmol m^-2 s^-1 at 0 ‰ it decreased by 21 and 37 % in plants at salinities of 10 and 40 ‰, respectively. Stomatal conductance (g _s) was profoundly responsive in comparison to P _max which resulted in a high water use efficiency. This was further confirmed by δ¹³C values, which increased with salinity. From day 3, after salt was removed from the soil solution, P _max and g _s increased up to 13 and 30 %, respectively. However, the values were still considerably lower than those measured in plants grown without salt addition. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of explant orientation, pH, solidifying agent and wounding on initiation of soybean somatic embryos

Summary Several methods have been developed to obtain somatic embryos of soybean. We report here a new procedure that results in high frequency somatic embryo initiation in a short period of time. Somatic embryos were induced from immature cotyledons of the cultivars “Jack,” “Thorne,” “Resnik,” and “Chapman.” Immature cotyledons were cultured on a medium containing MS salts, B₅ vitamins, 6% sucrose, and 40 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Culture modifications included: orientation of the explants (adaxial or abaxial side of the cotyledon in contact with the medium), adjustment of medium pH (5.7 or 7.0), wounding of cotyledons with scalpel blades, inclusion of ethylene modulators, and use of Noble agar or Gelrite™ as the solidifying agent. The treatment that resulted in the highest embryo induction across the cultivars consisted of abaxial side of the explant facing the medium, pH 7.0 and 0.2% Gelrite™. “Jack” was the most responsive cultivar showing the first embryos as early as 14 d after culture. After 21 d, an average of 44 embryos per cotyledon was obtained with this cultivar. The inclusion of silver nitrate (AgNO₃) in the culture medium did not enhance the number of primary somatic embryos induced per cotyledon, but the addition of 15 μM AgNO₃ did result in a faster production of secondary embryos using the cultivar “Jack.” Wounding of the explants with a scalpel resulted in an earlier induction of somatic embryos. Embryo initials were first observed after only 7 d. Histological examination of cultured cotyledons indicated that the somatic embryos originated from the subepidermal tissues and were of multicellular origin. This somatic embryo induction procedure could be useful for direct transformation work and permits the production of embryogenic tissue within 2 wk.     

Chromium-induced glucose uptake, superoxide anion production, and phagocytosis in cultured pulmonary alveolar macrophages of weanling pigs

The dose-dependent effects of chromium chloride (CrCl₃) and chromium picolinate (CrPic) were evaluated for their glucose uptake, superoxide anion (O ₂ ⁻ ) production, activity of glucose-6-phosphate dehydrogenase, and phagocytosis of incubated pulmonary alveolar macrophages in medium containing no or 5 × 10⁻⁸ M insulin. Glucose uptake was found to increase in cells treated with 20 μg/L CrCl₃. Incubation with 20 μg/L of CrPic enhanced glucose uptake and O ₂ ⁻ production in an insulin-dependent manner. However, the inclusion of CrPic to 100 μg/L in the medium absent of insulin also increased O ₂ ⁻ production. The activity of glucose-6-phosphate dehydrogenase was not affected by either the addition of Cr or insulin. The phagocytosis of Escherichia coli by macrophages was enhanced significantly (p<0.05) in medium containing 10–100 μg/L CrCl₃ or 20–100 μg/L CrPic in the presence of insulin. These results suggest that the addition of 10–20 μg/L CrCl₃ enhances directly the cellular activity of macrophages, whereas the effect of CrPic requires the cooperative action of insulin in enhancing their glucose uptake and phagocytosis.