Down regulation of enkephalin (δ) receptors Demonstration in membrane-bound and solubilized receptors

The pentapeptide leucine enkephalin induced down-regulation of enkephalin receptors in neuroblastoma-glioma NG108-15 hybrid cells in a reversible fashion, whereas the stable enkephalin analogue, d-Ala²-Met-enkephalinamide (AMEA), and the potent opiate alkaloid, etorphine, had a prolonged effect. The opiate alkaloid, morphine, which has low affinity to δ-type enkephalin receptors of these cells did not induce down-regulation, whereas AMEA decreased the binding of both opiate agonists and antagonists but had no effect on the binding of the α₂-adrenergic ligand, [³H]yohimbine. From several experiments that were designed to remove the tightly bound AMEA, and from experiments with solubilized receptor we ruled out the possibility that the decreased binding capacity of enkephalin-treated cells reflects only receptor masking. The study suggests that down-regulation of enkephalin receptors that may also occur in vivo can account for some of the abnormal physiological responses of subjects treated chromically with opiates. However, since opiates from the morphine type can induce opiate tolerance in vivo, but not down-regulation of enkephalin receptors in the cultured cells, we suggest that down-regulation of δ-type opiate receptors may not be prerequisite for the development of the physiological tolerance/dependence on these alkaloids.     

Effects of gibberellic acid and abscisic acid on α-amylase activity and ultrastructure of aleurone cells ofAvena sativa L.

The effects of GA₃ and/or ABA on the α-amylase activity and the ultrastructure of aleurone cells in halves of seeds without embryos (embryo-less half seeds) of oats (Avena sativa L.) were studied. α-Amylase activity was detected by the starch-agar gel method in the aleurone layers of embryo-less half seeds soaked in 1 μM GA₃ solution or 100 μM GA₃+10 μM ABA solution but not in those of seeds soaked in distilled water, 10 μM ABA solution, or 1 μM GA₃+10 μM ABA solution. Ultrastructural examinations of aleurone cells with α-amylase activity showed a decrease in the number of sphaerosomes, the appearance of flattened saccules pressed to the surface of aleurone grains, and the development and transformations of the rER from a slender form to the one with wide inner spaces. In the aleurone cells in which the enzyme activity was not detected, components of the rER showed only slender profiles. The number of sphaerosomes did not decrease, and no flattened saccules appeared in the aleurone cells treated with 10 μM ABA or 1 μM GA₃+10 μM ABA.     

Mycobiota in poultry feeds and natural occurrence of aflatoxins, fumonisins and zearalenone in the Rio de Janeiro State, Brazil

The intake of mycotoxin-contaminated feeds can lead to nutrient losses and may have adverse effects on animal health and on productivity. The aims of this study were (1) to determine the mycobiota present in poultry feed samples, and (2) to evaluate the natural occurrence of aflatoxin B₁, fumonisin B₁ and zearalenone. Fungal counts were similar between all culture media tested (10³ CFU g⁻¹). The most frequent genus isolated was Penicillium spp. (41.26%) followed by Aspergillus spp. (33.33%) and Fusarium spp. (20.63%). High precision liquid chromatography was applied to quantify aflatoxin B₁ and fumonisin B₁. Thin layer chromatography was used to determine zearalenone levels. Aflatoxin B₁ values ranged between 1.2 and 17.5 μg kg⁻¹. Fumonisin B₁ levels ranged between 1.5 and 5.5 μg g⁻¹. Zearalenone levels ranged between 0.1 and 7 μg g⁻¹. The present study shows the simultaneous occurrence of two carcinogenic mycotoxins, aflatoxin B₁ and fumonisin B₁, together with another Fusarium mycotoxin (zearalenone) in␣feed intended for poultry consumption. Many samples contained AFB₁ levels near the permissible maximum and it could affect young animals. A synergistic toxic response is possible in animals under simultaneous exposure.     

Memantine and the amino-alkyl-cyclohexane MRZ 2/579 are moderate affinity uncompetitive NMDA receptor antagonists –in vitro characterisation

Summary. There is general agreement that moderate affinity uncompetitive NMDA receptor antagonists combine good efficacy and tolerability in animal models of disturbances in glutamatergic transmission. There are several theories on which properties are important for this profile including 1, rapid access to the channel at the start of pathological overactivity 2, rapid, voltage-dependent relief of blockade during physiological synaptic activation and 3, partial untrapping. Merz has developed a series of novel uncompetitive NMDA receptor antagonists based on the cyclohexane structure. In cultured hippocampal neurones MRZ 2/579 (1-amino-1,3,3,5,5-pentamethyl-cyclohexane) shows similar blocking kinetics to memantine (K_on 10.7 ^* 10⁴ M⁻¹ sec⁻¹, K_off 0.20 sec⁻¹ at −70 mV) and binds at the same depth in the NMDA receptor channel (δ = 0.8). The potency of MRZ 2/579 assessed as Kd = K_off/K_on = 1.87 μM agrees well with the IC₅₀ of 1.29 μM against steady-state currents in cultured hippocampal neurones (at −70 mV) and with the Ki in [³H]-MK-801 binding of 0.65 μM. MRZ 2/579 protected cultured cortical neurones against glutamate toxicity with an IC₅₀ of 2.16 μM and was also effective in protecting hippocampal slices against hypoxia / hypoglycaemia-induced reduction of fEPSP amplitude in CA1 with an EC₅₀ of 7.01 μM. MRZ 2/579 has similar potency and bio-availability to memantine in vivo assessed using microdialysis, microiontophoresis and MES-induced seizures. Initial characterization in animal models provides strong support for the assump-tion that MRZ 2/579 could be a useful therapeutic in morphine/alcohol dependence, inhibition of morphine tolerance, chronic pain and as a neuroprotective agent. Received August 31, 1999 Accepted September 20, 1999     

The optimal growth conditions for the biomass production of Isochrysis galbana and the effects that phosphorus, Zn2+, CO2, and light intensity have on the biochemical composition of Isochrysis galbana and the activity of extracellular CA

The effects of phosphorus, Zn²⁺, CO₂, and light intensity on growth, biochemical composition, and the activity of extracellular carbonic anhydrase (CA) in Isochrysis galbana were investigated. A significant change was observed when the concentration of phosphorus in the medium was increased from 5 μmol/L to 1000 μmol/L affecting I. galbana’s cell density, biochemical composition, and the activity of extracellular CA. Phosphorous concentration of 50 μmol/L to 500 μmol/L was optimal for this microalgae. The Zn²⁺ concentration at 10 μmol/L was essential to maintain optimal growth of the cells, but a higher concentration of Zn²⁺ (≥ 1000 μmol/L) inhibited the growth of I. galbana. High CO₂ concentrations (43.75 mL/L) significantly increased the cell densities compared to low CO₂ concentrations (0.35 mL/L). However, the activity of extracellular CA decreased significantly with an increasing concentration of CO₂. The activity of extracellular CA at a CO₂ concentration of 43.75 mL/L was approximately 1/6 of the activity when the CO₂ concentration was at 0.35 mL/L CO₂. Light intensity from 4.0 mW/cm² to 5.6 mW/cm² was beneficial for the growth, biochemical composition and the activity of extracellular CA. The lower and higher light intensity was restrictive for growth and changed its biochemical composition and the activity of extracellular CA. These results indicate that phosphorus, Zn²⁺, CO₂, and light intensity are important factors that impact growth, biochemical composition and the activity of extracellular CA in I. galbana.     

GABA Analogues Derived from 4-Aminocyclopent-1-enecarboxylic Acid

The incorporation of extra binding groups onto known ligands is a powerful tool for the development of more potent and selective agents at target sites such as the GABA receptors. In the present work we have attempted to build on the activity of the know potent GABA_A agonist 4-ACP-3-CA and its cis and trans saturated analogues CACP and TACP. We have investigated reactions to add thiol substituents to the α,β-unsaturated system of 4-ACP-3-CA. The reaction was successful with a limited number of thiols but gave products of mixed stereochemistry. The resultant thioether amino acids were screened for activity at human recombinant α₁β₂ γ_2L GABA_A receptors. The most interesting derivative was the benzylthioether which acted as an antagonist with an IC₅₀ of 42 μM for the inhibition of a GABA EC₅₀ dose (50 μM). This study has shown that GABA analogues derived by thiol addition to 4-aminocyclopent-1-enecarboxylic acid display interesting antagonist activity at the α₁β₂γ_2L GABA_A receptor. Special issue article in honour of Dr. Graham Johnston.     

Dose-related influence of sodium selenite on apoptosis in human thyroid follicles in vitro induced by iodine, EGF, TGF-β, and H2O2

Apoptosis of thyroid follicular cells is induced by high doses of iodide, epidermal growth factor (EGF), transforming growth factor-β (TGF-β), as well as H₂O₂ and might be attenuated by antioxidants. Therefore, we examined the apoptotic index induced by these substances in selenium-treated vs untreated human thyroid follicular cells. Reconstituted human thyroid follicles were incubated with sodium selenite (10 or 100 nM) for 72 h; controls received none. The follicles were then distributed to 24-well plates and incubated with potassium iodide (5, 10, or 20 nM), EGF (5 ng/mL), TGF-β (5 ng/mL), or H₂O₂ (100 μM). Apoptosis was determined by a mitochondrial potential assay and the number of apoptotic cells counted by two independent, experienced technicians and the glutathione peroxidase (GPx) activity was determined. A significant increase of apoptic cells was obtained in control thyroid follicles treated with iodine (5, 10, or 20 μM), thyroid-stimulating hormone (TSH) 1, or 10 mU/mL in combination with 5 and 10 μM iodine, EGF (5 ng/mL) and TGF-β (5 ng/mL), or H₂O₂ (100 μM) (p<0.001). In contrast, in thyroid follicles preincubated with 10 or 100 nM sodium selenite, the apoptototic index was identical to the basal rate. In H₂O₂-treated follicles, the apoptotic index was still significantly elevated but 50% lower compared to control cells. The GPx activity increased from 1.4±0.2 to 2.25±0.4 mU/μg DNA with 10 nM selenite and 2.6+0.4 mU/μg DNA with 100 nM selenite. Sodium selenite might increase the antioxidative potential in human thyroid follicles in vitro and therefore diminish the apoptosis induced by TGF-β, EGF, iodide, and even H₂O₂     

Regulation of Medicago sativa L. somatic embryos regeneration by gibberellin A3 and abscisic acid in relation to starch content and α-amylase activity

Somatic embryo quality is an important factor decisive for the efficiency of somatic embryogenesis. Addition gibberellic acid (GA₃) at a concentration of 1 μM to germination medium improved the regeneration of alfalfa somatic embryos. Inhibitory effect of ancymidol, an inhibitor of gibberellin biosynthesis, on germination and conversion may indicate that those processes require endogenous GAs. Since fluridone, an ABA biosynthetic inhibitor, at a concentration of 1 μM, had a slight stimulatory effect on germination of somatic embryos, it may be presumed that embryos contain a too high level of residual ABA after maturation phase (20 μM ABA is used at that phase). The observed improvement of regeneration of somatic embryos by GA₃ was correlated with acceleration of starch hydrolysis through α-amylase activity enhancement by GA₃. In contrast, the inhibitory effect of ABA on the above processes was probably related to inhibition of α-amylase activity and, in consequence, to delayed starch hydrolysis. It is suggested that α-amylase activity can be considered a good marker of the quality of Medicago sativa L. somatic embryos.     

Nitrite utilization by Chaetoceros muelleri under elevated CO2 concentration

Chaetoceros muelleri (Lemn.) was cultured with nitrite (NO₂⁻) or nitrate (NO₃⁻) as the sole nitrogen source and aerated with air or with CO₂-enriched air. Cells of C. muelleri excreted into the medium nitrite produced by reduction of nitrate when grown with 100 μM NaNO₃ as nitrogen source. Accordingly, NO₂⁻ concentration reached 10.4 μM after 95 h at the low CO₂ condition (aerated with air); while the maximum NO₂⁻ concentration was only around 2.0 μM at the high CO₂ condition (aerated with 5% CO₂ in air), furthermore, after 30 h it decreased to no more than 1.0 μM. NO₂⁻ was almost assimilated in 80 h when C. muelleri was cultured at the high CO₂ condition with 100 μM NaNO₂ as sole nitrogen source. At the high CO₂ condition, after 3 h the activity of nitrite reductase was as much as 50% higher than that at the low CO₂ condition. It was indicated that enriched CO₂ concentration could inhibit nitrite excretion and enhance nitrite assimilation by cells. Therefore, aeration with enriched CO₂ might be an effective way to control nitrite content in aquaculture systems.     

Optimizing environmental factors for large-scale multiplication of chrysanthemum (<Emphasis Type="Italic">Chrysanthemum grandiflorum</Emphasis>) in balloon-type bioreactor culture

Summary We have developed optimum culture conditions for the large-scale propagation of chrysanthemum in balloon-type bioreactors to achieve vigorous growth and quality. The effects of NH ₄ ⁺ /NO ₃ ⁻ ratio, air volume, air temperature, photosynthetic photo flux, and an inoculation density on the growth and quality of plantlets were investigated. The best production conditions were an NH ₄ ⁺ :NO ₃ ⁻ ratio of 20∶40 mM, air exchange of 0.1 vvm min⁻¹, air temperature 25°C, photosynthetic photo flux (PPF) at 100 μmol m⁻² s⁻¹, and an inoculation density of 40 nodes Chrysanthemum grandiflorum. Under each of these conditions, the maximum growth rate reached 279.0, 260,0, 20.0, 23.3, and 94.5 (g-fresh weight per plantlet d⁻¹), respectively, at 12 wk of culture. These results specify the key environmental factors that can be regulated to improve the quality and quantity of flowers and increase yield in large-scale bioreactor cultures of chrysanthemum.     

Photosynthetic CO2 affinity of the aquatic carnivorous plant Utricularia australis (Lentibulariaceae) and its investment in carnivory

Aquatic carnivorous plants usually grow in shallow dystrophic waters poor in inorganic N and P. Utricularia australis was chosen as a model plant for its prolific distribution and great ecological plasticity. The photosynthetic CO₂ compensation point and factors associated with investment in carnivory and capture of prey were measured in 17 U. australis micropopulations in Třeboň basin, Czech Republic, together with water chemistry factors at these sites differing greatly in their trophic level, water hardness, and prey availability. Apical shoot growth rate was estimated at some oligotrophic sites. The micropopulations differed greatly in the proportion of traps with animal prey (2.7–70%, mean 26%), trap proportion to total biomass (1.4–42%, mean 26%), mean trap biomass (0.7–63 μg trap⁻¹, mean 19 μg), and maximum trap size (1–3 mm, mean 2.0 mm). CO₂ compensation points ranged from 0.7 to 6.1 μM (mean 2.6 μM). A weak HCO₃ ⁻ use (compensation point 0.51 mM) was found in plants growing in alkaline water. Trap biomass proportion did not correlate significantly with prey capture and CO₂ compensation points with ambient [CO₂]. A very rapid apical growth (2.5–4.2 new nodes day⁻¹) occurred in sand pits. Thus, HCO₃ ⁻ use in U. australis can be induced by growing at very high pH. CO₂ compensation points resembled those known in other aquatic non-carnivorous plants. They did not reflect carnivory. In spite of very rapid apical shoot growth, the relative growth rate of U. australis can be zero in oligotrophic habitats without prey.     

Effects of Yariv phenylglycosides onRosa cell suspensions: Evidence for the involvement of arabinogalactan-proteins in cell proliferation

Treatment of ‘Paul's Scarlet rose (Rosa sp.) cell suspensions with β-D-glucosyl Yariv phenylglycoside (β-D-Glc)₃, a chromophoric molecule that selectively binds arabinogalactan-proteins (AGPs), caused inhibition of cell growth in a concentration-dependent manner, with complete inhibition of growth occurring at 50 μM (β-D-Glc)₃ in the culture medium. Growth was not inhibited by either α-D-galactosyl or β-D-mannosyl Yariv phenylglycosides which do not bind AGPs. Staining of cells with fluorescein diacetate indicated that (β-D-Glc)₃ did not affect cell viability. Upon transfer of 50 μM (β-D-Glc)₃-treated cells to control conditions, cell growth recovered with a time-course similar to that of control cells. Cell sizes in control and (β-D-Glc)₃-treated cultures were similar, indicating that the mechanism of growth inhibition by (β-D-Glc)₃ involved suppression of cell division. Two different analyses of (β-D-Glc)₃-treated cells both showed that approximately 95% of the bound (β-D-Glc)₃ was in the cell wall. Molecules that bound (β-D-Glc)₃ were extracted from the cell wall and were identified as AGPs, as judged by their carbohydrate and amino acid compositions.     

Sigma1 binding in a human neuroblastoma cell line

Behaviorally, sigma₁ agents modulate opioid analgesia. To examine possible mechanisms responsible for these interactions, we have identified a cell line containing both sigma₁ and opioid receptors. [³H](+)-pentazocine binding in BE(2)-C human neuroblastoma cells is high affinity (K_D 3.4±0.7 nM) and high density (B_max 2.98±0.14 pmol/mg protein). Competition studies reveal a selectivity profile similar to that of sigma₁ sites in guinea pig brain. (+)-Pentazocine has no effect upon either basal or forskolin-stimulated cyclase in the BE(2)-C cells, but cAMP accumulation is inhibited by the morphine, DPDPE and naloxone benzoylhydrazone. (+)-Pentazocine at concentrations as high as 10 μM does not affect this opioid effect, implying that sigma₁/opioid interactions are not mediated at the level of the cell. This suggests that their behavioral interactions result from interacting neural circuits. Although (+)-pentazocine is without effect in the cyclase system, it does block carbachol-stimulated phosphoinositol turnover (IC₅₀ 6.5±1.14 μM). The specificity of the effect is confirmed by the ability of haloperidol (1 μM) to shift the IC₅₀ value of (+)-pentazocine 2-fold to the right. Special issue dedicated to Dr. Eric J. Simon.     

High copper levels in the medium improves shoot bud differentiation and elongation from the cultured cotyledons of <Emphasis Type="Italic">Capsicum annuum</Emphasis> L.

The effect of copper sulphate on differentiation and elongation of shoot buds from cotyledonary explants of Capsicum annuum L. cv X-235 was investigated. Shoot buds were induced on medium supplemented with 22.2 μM BAP and 14.7 μM PAA. Elongation of shoot buds was obtained on MS medium containing 13.3 μM BAP + 0.58 μM GA₃. Both shoot induction and elongation media were supplemented with different levels of CuSO₄ (0–5 μM). The levels of CuSO₄ in the induction as well as elongation medium highly influenced the shoot bud formation and their subsequent elongation. Highest number of shoot buds per explant was obtained when the concentration of CuSO₄ was increased 30 times to the normal MS level. Shoot buds formation frequency i.e., the number of shoots formed per explant was increased two fold as compared to those formed on control. Elongation both in terms of percentage and length of shoots was better than that on control. Healthy elongated shoots were rooted on MS medium supplemented with 5.7 μM IAA. Rooted plantlets were transferred to field conditions.     

Gas exchange and photosynthetic performance of the tropical tree <Emphasis Type="Italic">Acacia nigrescens</Emphasis> when grown in different CO<Subscript>2</Subscript> concentrations

The photosynthetic responses of the tropical tree species Acacia nigrescens Oliv. grown at different atmospheric CO₂ concentrations—from sub-ambient to super-ambient—have been studied. Light-saturated rates of net photosynthesis (A _sat) in A. nigrescens, measured after 120 days exposure, increased significantly from sub-ambient (196 μL L⁻¹) to current ambient (386 μL L⁻¹) CO₂ growth conditions but did not increase any further as [CO₂] became super-ambient (597 μL L⁻¹). Examination of photosynthetic CO₂ response curves, leaf nitrogen content, and leaf thickness showed that this acclimation was most likely caused by reduction in Rubisco activity and a shift towards ribulose-1,5-bisphosphate regeneration-limited photosynthesis, but not a consequence of changes in mesophyll conductance. Also, measurements of the maximum efficiency of PSII and the carotenoid to chlorophyll ratio of leaves indicated that it was unlikely that the pattern of A _sat seen was a consequence of growth [CO₂] induced stress. Many of the photosynthetic responses examined were not linear with respect to the concentration of CO₂ but could be explained by current models of photosynthesis.     

Cytotoxic activity induced by crude extracts of Ganoderma lucidum (W. Curt.: Fr.) P. Karst. on mouse myeloma cancer cell-line

Ganoderma lucidum powder using hot water and methanol extraction methods indicated a twofold more active cytotoxic activity with IC₅₀ of 44 ± 3.8 μg/ml in the latter method. The representative dose-response curves of the G. lucidum crude extracts on J558 cell-lines revealed that there were great similarities between the curves which reflected rapid killing activities. The percentage viability of the J558 cell exposed to these crude extracts was dose dependent only up to 150 μg/ml. After which, there was no significant reduction when the dose was increased to 200 or 400 μg/ml. The morphological alterations induced by the crude extract were examined under the phase contrast, fluorescent and electron microscopy. When J558 cells were treated with doses higher than 50 μg/ml of the crude extract, obvious morphological changes and apoptosis occurred after 72 h. At 400 μg/ml, most of the cells showed necrosis characterized as small fragments with uniformly stained red nuclei. The apoptotic and necrotic cells increased by 16.5 and 29.1%, respectively whereas the viable cells decreased by as much as 45.6. The mode of cell death via apoptosis was 3.6% higher than necrosis. However, these morphological changes were not observed in the case of 3T3 cells. Results obtained from scanning electron microscopy and transmission electron microscopy further confirmed the occurrence of various apoptotic and necrotic features.     

Ultrastructure of leaves in C4 Cyperus iria and C3 Carex siderosticta

The ultrastructural aspects ofCyperus iria leaves showing the C₄ syndrome and the typical C₃ species,Carex siderosticta, in the Cyperaceae family were examined.C. iria exhibited the chlorocyperoid type, showing an unusual Kranz structure with vascular bundles completely surrounded by two bundle sheaths. The cellular components of the inner Kranz bundle sheath cells were similar to those found in the NADP-ME C₄ subtype, having centrifugally arranged chloroplasts with greatly reduced grana and numerous starch grains. Their chloroplasts contained convoluted thyla-koids and a weakly-developed peripheral reticulum, although it was extensive mostly in mesophyll cell chloroplasts. The outer mestome bundle sheath layer was sclerenchymatous and generally devoid of organelles, but had unevenly thickened walls. Suberized lamellae were present on its cell walls, and they became polylamellate when traversed by plasmodesmata. Mesophyll cell chloroplasts showed well-stacked grana with small starch grains. InC. siderosticta, vascular bundles were surrounded by the inner mestome sheath and the outer parenchymatous bundle sheath with intercellular spaces. The mestome sheath cells degraded in their early development and remained in a collapsed state, although the suberized lamellae retained polylamellate features. Plastids with a crystalline structure, sometimes membrane-bounded, were found in the epidermal cells. The close interveinal distance was 35–50 μm inC. iria, whereas it was 157–218 μm inC. siderosticta. These ultrastructural characteristics were discussed in relation to their photosynthetic functions.     

Response of growth and fatty acid compositions of Nannochloropsis sp. to environmental factors under elevated CO2 concentration

Nannochloropsis sp. was grown with different levels of nitrate, phosphate, salinity and temperature with CO₂ at 2,800 μl l⁻¹. Increased levels of NaNO₃ and KH₂PO₄ raised protein and polyunsaturated fatty acids (PUFAs) contents but decreased carbohydrate, total lipid and total fatty acids (TFA) contents. Nannochloropsis sp. grew well at salinities from 22 to 49 g l⁻¹, and lowering salinity enhanced TFA and PUFAs contents. TFA contents increased with the increasing temperature but PUFAs contents decreased. The highest eicosapentaenoic acid (EPA, 20:5ω3) content based on the dry mass was above 3% under low N (150 μM NaNO₃) or high N (3000 μM NaNO₃) condition. Excessive nitrate, low salinity and temperature are thus favorable factors for improving EPA yields in Nannochloropsis sp.     

Singlet oxygen formation and chlorophyll a triplet excited state deactivation in the cytochrome b6f complex from Bryopsis?corticulans

We have attempted to investigate the correlation between the detergent-perturbed structural integrity of the Cyt b ₆ f complex from the marine green alga Bryopsis corticulans and its photo-protective properties, for which the nonionic detergents n-octyl-β-d-glucopyranoside (β-OG) and n-dodecyl-β-d-maltoside (β-DM), respectively, were used for the preparation of Cyt b ₆ f, and the singlet oxygen (¹O₂*) production as well as the triplet excited-state chlorophyll a (³Chl a*) formation and deactivation were examined by spectroscopic means. Near-infrared luminescence of ¹O₂ * (~1,270 nm) on photo-irradiation was detected for the β-OG preparation where the complex is mainly in oligomeric state, but not for the β-DM one in which the complex exists in dimeric form. Under anaerobic condition, photo-excitation of Chl a in the β-DM preparation generated ³Chl a* with a lower quantum yield of Φ_T ~ 0.02 and a longer lifetime of ~600 μs with respect to those as in the case of β-OG preparation, Φ_T ~ 0.12 and 200–300 μs. These results prove that the enzymatically active and intact Cyt b ₆ f complex on photo-excitation tends to produce little ³Chl a* or ¹O₂ *, which implies that the pigment–protein assembly of Cyt b ₆ f complex per se is crucial for photo-protection. F. Ma and X.-B. Chen contributed equally to this work.