The photosystem I supercomplex from a primordial green alga Ostreococcus tauri harbors three light-harvesting complex trimers.

As a ubiquitous picophytoplankton in the ocean and an early-branching green alga, Ostreococcus tauri is a model prasinophyte species for studying the functional evolution of the light-harvesting systems in photosynthesis. Here, we report the structure and function of the O. tauri photosystem I (PSI) supercomplex in low light conditions, where it expands its photon-absorbing capacity by assembling with the light-harvesting complexes I (LHCI) and a prasinophyte-specific light-harvesting complex (Lhcp). The architecture of the supercomplex exhibits hybrid features of the plant-type and the green algal-type PSI supercomplexes, consisting of a PSI core, an Lhca1-Lhca4-Lhca2-Lhca3 belt attached on one side and an Lhca5-Lhca6 heterodimer associated on the other side between PsaG and PsaH. Interestingly, nine Lhcp subunits, including one Lhcp1 monomer with a phosphorylated amino-terminal threonine and eight Lhcp2 monomers, oligomerize into three trimers and associate with PSI on the third side between Lhca6 and PsaK. The Lhcp1 phosphorylation and the light-harvesting capacity of PSI were subjected to reversible photoacclimation, suggesting that the formation of OtPSI-LHCI-Lhcp supercomplex is likely due to a phosphorylation-dependent mechanism induced by changes in light intensity. Notably, this supercomplex did not exhibit far-red peaks in the 77 K fluorescence spectra, which is possibly due to the weak coupling of the chlorophyll a603-a609 pair in OtLhca1-4.

Structural characterization of the photosystems in the green alga Chlorella sorokiniana.

MAIN CONCLUSION: The supramolecular organization of the photosystem supercomplexes in the green alga Chlorella sorokiniana belonging to Trebouxiophyceae are essentially the same as those of Chlamydomonas reinhardtii belonging to Chlorophyceae. The photosynthetic conversion of light energy into chemical energy is performed by photosystems II and I (PSII and PSI) embedded within the thylakoid membranes. In plants and green algae, PSII and PSI comprise the core complex and light-harvesting complexes (LHCII and LHCI), forming PSII-LHCII and PSI-LHCI supercomplexes, respectively. The structural information about photosystem supercomplexes of green algae has been limited to chlorophytic algae. Here, to obtain an insight into the evolution of Chlorophyta, we determined the supramolecular organization of the PSII-LHCII and PSI-LHCI supercomplexes from the freshwater green alga Chlorella sorokiniana, which belongs to Trebouxiophyceae. The obtained results showed that the supramolecular organizations of the photosystem supercomplexes in C. sorokiniana were essentially the same as those of the model green alga C. reinhardtii, which belongs to Chlorophyceae, namely PSII-LHCII supercomplex formed the C2S2M2L2 configuration and PSI-LHCI supercomplex was associated with 10 LHCI subunits.

Multimeric and monomeric photosystem II supercomplexes represent structural adaptations to low- and high-light conditions.

An intriguing molecular architecture called the "semi-crystalline photosystem II (PSII) array" has been observed in the thylakoid membranes in vascular plants. It is an array of PSII-light-harvesting complex II (LHCII) supercomplexes that only appears in low light, but its functional role has not been clarified. Here, we identified PSII-LHCII supercomplexes in their monomeric and multimeric forms in low light-acclimated spinach leaves and prepared them using sucrose-density gradient ultracentrifugation in the presence of amphipol A8-35. When the leaves were acclimated to high light, only the monomeric forms were present, suggesting that the multimeric forms represent a structural adaptation to low light and that disaggregation of the PSII-LHCII supercomplex represents an adaptation to high light. Single-particle EM revealed that the multimeric PSII-LHCII supercomplexes are composed of two ("megacomplex") or three ("arraycomplex") units of PSII-LHCII supercomplexes, which likely constitute a fraction of the semi-crystalline PSII array. Further characterization with fluorescence analysis revealed that multimeric forms have a higher light-harvesting capability but a lower thermal dissipation capability than the monomeric form. These findings suggest that the configurational conversion of PSII-LHCII supercomplexes may serve as a structural basis for acclimation of plants to environmental light.

A novel method produces native light-harvesting complex II aggregates from the photosynthetic membrane revealing their role in nonphotochemical quenching.

Nonphotochemical quenching (NPQ) is a mechanism of regulating light harvesting that protects the photosynthetic apparatus from photodamage by dissipating excess absorbed excitation energy as heat. In higher plants, the major light-harvesting antenna complex (LHCII) of photosystem (PS) II is directly involved in NPQ. The aggregation of LHCII is proposed to be involved in quenching. However, the lack of success in isolating native LHCII aggregates has limited the direct interrogation of this process. The isolation of LHCII in its native state from thylakoid membranes has been problematic because of the use of detergent, which tends to dissociate loosely bound proteins, and the abundance of pigment-protein complexes (e.g. PSI and PSII) embedded in the photosynthetic membrane, which hinders the preparation of aggregated LHCII. Here, we used a novel purification method employing detergent and amphipols to entrap LHCII in its natural states. To enrich the photosynthetic membrane with the major LHCII, we used Arabidopsis thaliana plants lacking the PSII minor antenna complexes (NoM), treated with lincomycin to inhibit the synthesis of PSI and PSII core proteins. Using sucrose density gradients, we succeeded in isolating the trimeric and aggregated forms of LHCII antenna. Violaxanthin- and zeaxanthin-enriched complexes were investigated in dark-adapted, NPQ, and dark recovery states. Zeaxanthin-enriched antenna complexes showed the greatest amount of aggregated LHCII. Notably, the amount of aggregated LHCII decreased upon relaxation of NPQ. Employing this novel preparative method, we obtained a direct evidence for the role of in vivo LHCII aggregation in NPQ.

Structural insight into light harvesting for photosystem II in green algae.

Green algae and plants rely on light-harvesting complex II (LHCII) to collect photon energy for oxygenic photosynthesis. In Chlamydomonas reinhardtii, LHCII molecules associate with photosystem II (PSII) to form various supercomplexes, including the C2S2M2L2 type, which is the largest PSII-LHCII supercomplex in algae and plants that is presently known. Here, we report high-resolution cryo-electron microscopy (cryo-EM) maps and structural models of the C2S2M2L2 and C2S2 supercomplexes from C. reinhardtii. The C2S2 supercomplex contains an LhcbM1-LhcbM2/7-LhcbM3 heterotrimer in the strongly associated LHCII, and the LhcbM1 subunit assembles with CP43 through two interfacial galactolipid molecules. The loosely and moderately associated LHCII trimers interact closely with the minor antenna complex CP29 to form an intricate subcomplex bound to CP47 in the C2S2M2L2 supercomplex. A notable direct pathway is established for energy transfer from the loosely associated LHCII to the PSII reaction centre, as well as several indirect routes. Structure-based computational analysis on the excitation energy transfer within the two supercomplexes provides detailed mechanistic insights into the light-harvesting process in green algae.

Structural determination of the large photosystem II-light-harvesting complex II supercomplex of Chlamydomonas reinhardtii using nonionic amphipol.

In photosynthetic organisms, photosystem II (PSII) is a large membrane protein complex, consisting of a pair of core complexes surrounded by an array of variable numbers of light-harvesting complex (LHC) II proteins. Previously reported structures of the PSII-LHCII supercomplex of the green alga Chlamydomonas reinhardtii exhibit significant structural heterogeneity, but recently improved purification methods employing ionic amphipol A8-35 have enhanced supercomplex stability, providing opportunities for determining a more intact structure. Herein, we present a 5.8 Å cryo-EM map of the C. reinhardtii PSII-LHCII supercomplex containing six LHCII trimers (C2S2M2L2). Utilizing a newly developed nonionic amphipol-based purification and stabilizing method, we purified the largest photosynthetic supercomplex to the highest percentage of the intact configuration reported to date. We found that the interprotein distances within the light-harvesting complex array in the green algal photosystem are larger than those previously observed in higher plants, indicating that the potential route of energy transfer in the PSII-LHCII supercomplex in green algae may be altered. Interestingly, we also observed an asymmetric PSII-LHCII supercomplex structure comprising C2S2M1L1 in the same sample. Moreover, we found a new density adjacent to the PSII core complex, attributable to a single-transmembrane helix. It was previously unreported in the cryo-EM maps of PSII-LHCII supercomplexes from land plants.

pH-Responsive Binding Properties of Light-Harvesting Complexes in a Photosystem II Supercomplex Investigated by Thermodynamic Dissociation Kinetics Analysis.

Reorganization of photosynthetic proteins on the thylakoid membrane is an important regulatory process for photoacclimation in photosynthetic organisms. However, the underlying mechanism has been poorly understood due to the lack of methods to analyze the interactions between membrane proteins. To investigate the mechanism, we demonstrated the binding properties of light-harvesting complex proteins (LHCs) in a photosystem II (PSII) supercomplex regulated by pH conditions, which primarily responded to environmental light conditions, using a thermodynamic dissociation kinetics analysis. The results showed that the strongly bound LHCs (∼60%) were responsive to pH conditions, whereas the moderately and loosely bound LHCs (∼40%) were nonresponsive. This result implies that the pH condition alters the binding properties of LHCs in the PSII supercomplex, inducing the reorganization of protein complexes.

Amphipol-assisted purification method for the highly active and stable photosystem II supercomplex of Chlamydomonas reinhardtii.

Photosystem II (PSII) splits water and drives electron transfer to plastoquinone via photochemical reactions using light energy. It is surrounded by light-harvesting complex II (LHCII) to form the PSII-LHCII supercomplex. Complete characterization of its structure and function has, however, been hampered due to instability of the complex in the presence of detergent. To overcome this problem, we developed a new procedure for purifying the PSII-LHCII supercomplexes of Chlamydomonas reinhardtii employing amphipol A8-35. The obtained supercomplexes showed little LHCII dissociation even 4 days after purification. Oxygen-evolving activity was retained within amphipol if the extrinsic polypeptides were kept associated by betaine. Electron microscopy revealed that this method also improved structural uniformity and that the major organization was C2 S2 M2 L2 .

Ten antenna proteins are associated with the core in the supramolecular organization of the photosystem I supercomplex in Chlamydomonas reinhardtii.

Photosystem I (PSI) is a large pigment-protein complex mediating light-driven charge separation and generating a highly negative redox potential, which is eventually utilized to produce organic matter. In plants and algae, PSI possesses outer antennae, termed light-harvesting complex I (LHCI), which increase the energy flux to the reaction center. The number of outer antennae for PSI in the green alga Chlamydomonas reinhardtii is known to be larger than that of land plants. However, their exact number and location remain to be elucidated. Here, applying a newly established sample purification procedure, we isolated a highly pure PSI-LHCI supercomplex containing all nine LHCA gene products under state 1 conditions. Single-particle cryo-EM revealed the 3D structure of this supercomplex at 6.9 Å resolution, in which the densities near the PsaF and PsaJ subunits were assigned to two layers of LHCI belts containing eight LHCIs, whereas the densities between the PsaG and PsaH subunits on the opposite side of the LHCI belt were assigned to two extra LHCIs. Using single-particle cryo-EM, we also determined the 2D projection map of the lhca2 mutant, which confirmed the assignment of LHCA2 and LHCA9 to the densities between PsaG and PsaH. Spectroscopic measurements of the PSI-LHCI supercomplex suggested that the bound LHCA2 and LHCA9 proteins have the ability to increase the light-harvesting energy for PSI. We conclude that the PSI in C. reinhardtii has a larger and more distinct outer-antenna organization and higher light-harvesting capability than that in land plants.

Pharmacokinetics and pharmacodynamics of FSK0808 and Gran after single intravenous drip administration or single subcutaneous administration: comparative study in healthy Japanese adult male subjects.

FSK0808 is a recombinant human granulocyte colony-stimulating factor developed by Fuji Pharma Co., Ltd and Mochida Pharmaceutical Co., Ltd. as a biosimilar product of Gran®. We verified the pharmacokinetic/pharmacodynamic equivalence of FSK0808 and commercially available Gran® by a randomized crossover study of single intravenous dose (200 µg/m(2)) and single subcutaneous dose (400 µg/m(2)) in healthy Japanese adult male subjects. According to the bioequivalence guidelines, the area under the blood concentration - time curve by 48 hours after administration (AUC0-48) in a single intravenous drip (IVD) study, and AUC0-48 and maximum blood concentration (Cmax) in a single subcutaneous (SC) dose study were used as primary endpoints, and the pharmacodynamic parameters including absolute neutrophil count (ANC) or number of CD34 positive cells (CD34(+) cells) as secondary endpoints. The safety was evaluated based on the characteristics and incidence of adverse reactions. As a result, the 90% confidence interval (CI) of the difference in mean value for AUC0-48 among drugs ranged from log(0.8) to log(1.25), in the IVD study, and those for Cmax and AUC0-48 were within the range of log(0.8)-log(1.25) in the SC study. Those for secondary endpoints were all within the range of log(0.8)-log(1.25). Thus, the pharmacokinetics/pharmacodynamics of both drugs were considered equivalent for all routes of administration, and the profiles of adverse reactions were also very similar.

Case of adrenal incidentaloma in which autonomous cortisol production became clear during a very short term.

A 57-year-old woman was admitted to the hospital for the further evaluation of a left adrenal incidentaloma measuring 45 mm x 33 mm. She had no signs of the clinical manifestation of hypercortisolism. An endocrine evaluation revealed that her ACTH level was normal and cortisol values were almost normal pattern excluding the value at 9 PM slightly rising, however, the cortisol was not completely suppressed by the overnight administration of 1 mg dexamethasone. These findings indicated that subtle abnormalities of the hypothalamo-pituitary-adrenal axis were present in this case. After 3 months, surprisingly, the ACTH was suppressed to low levels. Further hormonal investigations revealed that the cortisol level was normal but had an abnormal diurnal rhythm and was not suppressed completely by a 1 mg or an 8 mg overnight dexamethasone dose. Adrenal scintigraphy revealed positive uptake in the left adrenal tumor with no uptake in the right adrenal gland. The patient underwent a left laparoscopic adrenalectomy. Microscopically, the tumor displayed histopathological features in common with ACTH-independent macronodular adrenocortical hyperplasia, including clear cell predominance, a pattern of small compact nests in clear cell areas, and a cord-like arrangement of small compact cells. An in situ hybridization study demonstrated the hybridization signals for P-450scc, 3beta-HSD, P-450c21, P-45011beta, and P-45017a which were observed in the clear cells as well as compact cells, the compact cells being more intensely stained. This case indicates the ability of autonomous cortisol production to become clear during a very short term and a more detailed and careful short-time follow-up should be recommended in patients with adrenal incidentalomas.

Autopsy of a patient with Cushing's Syndrome who was revealed to have pulmonary tumorlets producing ectopic ACTH.

The patient, a 78-year-old female with a 10-year history of type 2 diabetes mellitus, was admitted to our department for evaluation of leg edema and general fatigue. Biochemical investigations revealed hypokalemia and elevated serum cortisol and plasma ACTH levels, with a loss of diurnal rhythm and failure of suppression at high doses (8 mg) of dexamethasone. No pituitary tumor or parasellar tumor was detected by contrast-enhanced computed tomography (CT) or magnetic resonance image scan of the pituitary. High resolution CT of the lung and bronchoscopic examination revealed no abnormalities. Abdominal and pelvic CT indicated bilateral, slightly diffuse, adrenal gland hyperplasia only. These findings led to a diagnosis of ACTH-dependent hypercortisolism from an undefined source. Ten days after admission the patient had a fever and was diagnosed with disseminated intravascular coagulation. Despite intensive treatment about 1 month after admission the patient died from progressive multiple organ failure. At autopsy, a histological examination of the periphery of the right middle lobe of the lung revealed the presence of tumorlets. Immunohistochemical staining of the tumorlets revealed scattered cells containing ACTH and many cells containing chromogranin A that were positive for Grimelius staining. In addition, multiple microabscesses were present throughout most tissues of the body. The ectopic hormonal production observed in the present case suggests that pulmonary tumorlets should thus be considered in the differential diagnosis of Cushing's syndrome, and medical treatment to inhibit steroidogenesis should be started immediately to reduce the risk of complications from hypercortisolism.

Short term intensive insulin therapy improves insulin secretion significantly in type 2 diabetic patients.

To investigate the effects of short-term (1 week) intensive insulin therapy, on glycemic control, insulin secretion, and insulin sensitivity in type 2 diabetic patients, an open prospective study was conducted in sixteen type 2 diabetic patients receiving diet therapy alone or treatment with oral hypoglycemic agents. Of the study subjects, 8 patients were treated with insulin, the remaining 8 patients served as the control group. The metabolic parameters were evaluated once before treatment and once during one of the following treatments : glycemic control as measured by 1,5-anhydro-D-glucitol (1,5-AG) and area under curve of glucose (AUCglucose), insulin secretion as measured by area under curve of daily serum insulin (AUCinsulin), and insulin sensitivity as measured by the K index of the insulin tolerance test (K(ITT)). Post-treatment plasma glucose (AUCglucose) and 1,5-AG levels in patients who had received intensive insulin therapy were comparable to those of the control group. A statistically significant increase in AUCinsulin occurred after intensive insulin therapy for just 1 week, while no change occurred in the control group. Insulin sensitivity (K(ITT)) did not improve significantly in patients treated with insulin or patients from the control group. These results indicate that intensive insulin therapy for 1 week improves insulin secretion remarkably but has little effect on insulin sensitivity in type 2 diabetic patients. Clinically, this suggests that intensive insulin therapy for one week might be one of the initial treatments of choice for such patients.