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Humoral immunity is vital for host protection, yet aberrant antibody responses can trigger harmful inflammation and immune-related disorders. T follicular helper (Tfh) cells, central to humoral immunity, have garnered significant attention for unraveling immune mechanisms. This study shows the role of B-cell Oct-binding protein 1 (Bob1), a transcriptional coactivator, in Tfh cell regulation. Our investigation, utilizing conditional Bob1-deficient mice, suggests that Bob1 plays a critical role in modulating inducible T-cell costimulator expression and cellular respiration in Tfh cells. This regulation maintains the long-term functionality of Tfh cells, enabling their reactivation from central memory T cells to produce antibodies during recall responses. In a bronchial asthma model induced by house dust mite (HDM) inhalation, Bob1 is observed to enhance HDM-specific antibodies, including IgE, highlighting its pivotal function in Tfh cell regulation. Further exploration of Bob1-dependent mechanisms in Tfh cells holds promise for governing protective immunity and addressing immune-related disorders., (© 2024. The Author(s).)

The relationships between T follicular helper (Tfh) cells and antigen-specific immunoglobulins (sIgs) in patients with allergic respiratory diseases who are receiving antigen immunotherapy (AIT) have not been fully clarified. Therefore, we started to perform house dust mite sublingual immunotherapy (HDM-SLIT) for 20 patients with atopic asthma comorbid with allergic rhinitis (AA+AR) who were already receiving ordinary treatments including inhaled corticosteroid (ICS). We examined percentages of circulating T follicular helper (cTfh) and regulatory (cTfr) cells and percentages of circulating regulatory T (cTreg) and B (cBreg) cells by FACS and we examined levels of Der-p/f sIgs by ELISA. Based on the symptom score (asthma control questionnaire: ACQ) and medication score ((global initiative for asthma: GINA) treatment step score) in patients with AA, the patients were divided into responders and non-responders. The percentage of cTfh2 cells significantly decreased and the percentage of cTfh1 cells significantly increased within the first year. Der-p/f sIgEs decreased after a transient elevation at 3 months in both groups. Notably, the percentage of cTfh2 cells and the ratio of cTfh2/cBreg cells and Der-p/f sIgEs greatly decreased in responders from 6 months to 12 months. The percentages of cTfr and cTreg cells showed significant negative correlations with the percentage of cTfh2 cells. The percentage of IL-4 + cTfh cells were significantly decreased and the percentage of IFN-γ + cTfh cells were increased before treatment to 24 months in 6 patients examined (4 responders and 2 non-responders). We performed multi plelogistic regression analysis based on these results, the ratios of cTfh2/cTfr cells and cTfh2/cBreg cells at the start of therapy were statistically effective biomarkers for predicting the response to HDM-SLIT in patients with AA+AR., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Shigehara, Kamekura, Ikegami, Sakamoto, Yanagi, Kamiya, Kodama, Asai, Miyajima, Nishikiori, Uno, Yamamoto, Takano, Chiba, Ohnishi and Ichimiya.)

Complex interactions between keratinocytes and various cell types, such as inflammatory cells and stromal cells, contribute to the pathogenesis of chronic inflammatory skin lesions. In proinflammatory cytokine‒mediated disease settings, IL-9 plays a pathological role in inflammatory dermatitis. However, IL-9‒related mechanisms remain incompletely understood. In this study, we established tamoxifen-induced keratinocyte-specific IL-9RA-deficient mice (K14 CRE/ERT Il9ra Δ/Δ mice) to examine the role of IL-9 in multicellular interactions under chronic skin inflammatory conditions. Studies using an imiquimod-induced psoriasis-like model showed that K14 CRE/ERT Il9ra Δ/Δ mice exhibited a significantly reduced severity of dermatitis and mast cell infiltration compared with control K14 WT Il9ra fl/fl mice. Transcriptome analyses of psoriasis-like lesions showed that the level of peptide Y-Y (Pyy), a member of the neuropeptide Y family, was markedly downregulated in K14 CRE/ERT Il9ra Δ/Δ epidermis. Pyy blockade suppressed epidermal thickening and mast cell numbers in imiquimod-treated wild-type mice. Together with in vitro studies indicating that Pyy induced IL-9 production and chemotactic activity in bone marrow‒derived mast cells, these findings suggest that Pyy-mediated interplay between keratinocytes and mast cells contributes to psoriasiform inflammation. Further investigation focusing on the IL-9‒Pyy axis may provide valuable information for the development of new treatment modalities for inflammatory dermatitis., (Copyright © 2022. Published by Elsevier Inc.)

T follicular helper (Tfh) cells drive humoral immunity by facilitating B cell responses at the initial and recall phases. Recent studies have indicated the possible involvement of Tfh cells in the process of chronic inflammation. However, the functional role of Tfh cells in persistent immune settings remains unclear. Here, we report that CD4 + CD8 + (double-positive, DP; CD3 + CD4 + CD8 + CXCR5 hi PD-1 hi ) Tfh cells, a subset of germinal-center-type Tfh cells, were abundantly present in the fibroinflammatory lesions of patients with immunoglobulin G4-related disease (IgG4-RD). Transcriptome analyses showed that these DP-Tfh cells in the lesions of IgG4-RD preferentially expressed signature genes characteristic of cytotoxic CD8 + T cells, such as Eomes, CRTAM, GPR56, and granzymes, in addition to CD70. Scatter diagram analyses to examine the relationships between tissue-resident lymphocytes and various clinical parameters revealed that the levels of DP-Tfh cells were inversely correlated to the levels of serum IgG4 and local IgG4-expressing (IgG4 + ) memory B cells (CD19 + CD27 + IgD - ) in patients with IgG4-RD. Cell culture experiments using autologous tonsillar lymphocytes further suggested that DP-Tfh cells possess a poor B-cell helper function and instead regulate memory B cells. Since CD4 + (single positive, SP; CD3 + CD4 + CD8 - CXCR5 hi PD-1 hi ) Tfh cells differentiated into DP-Tfh cells under stimulation with IL-2 and IL-7 as assessed by in vitro experiments, these data imply that SP-Tfh cells are a possible origin of DP-Tfh cells under persistent inflammation. These findings highlight the potential feedback loop mechanism of Tfh cells in immune tolerance under chronic inflammatory conditions. Further studies on DP-Tfh cells may facilitate control of unresolved humoral responses in IgG4-RD pathological inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Murayama, Ikegami, Kamekura, Sakamoto, Yanagi, Kamiya, Sato, Sato, Shigehara, Yamamoto, Takahashi, Takano and Ichimiya.)

Objectives: The aim of this study was to determine pathological features of T peripheral helper (Tph)-like (PD-1 + CXCR5 - CD4 + T) cells in IgG4-related disease (IgG4-RD)., Methods: Tph-like cells in the blood and submandibular glands (SMGs) from IgG4-RD patients were analyzed by flow cytometry. Correlations between level of a Tph-like cell subset and clinical parameters of IgG4-RD were investigated. The cytotoxic capacity of Tph-like cells was also examined. Expression profiles of a molecule related to a Tph-like cell subset in IgG4-RD SMGs were assessed by immunohistochemistry., Results: Tph-like cells from IgG4-RD patients highly expressed a fractalkine receptor, CX3CR1. Percentages of circulating CX3CR1 + Tph-like cells were significantly correlated with clinical parameters including IgG4-RD Responder Index, number of involved organs, and serum level of soluble IL-2 receptor. CX3CR1 + Tph-like cells abundantly possessed cytotoxic T lymphocyte-related molecules such as granzyme A, perforin, and G protein-coupled receptor 56. Functional assays revealed their cytotoxic potential against vascular endothelial cells and ductal epithelial cells. Immunohistochemistry showed that fractalkine was markedly expressed in vascular endothelial cells and ductal epithelial cells in IgG4-RD SMGs., Conclusion: CX3CR1 + Tph-like cells are thought to contribute to persistent tissue injury in IgG4-RD and are a potential clinical marker and/or therapeutic target for inhibiting progression of IgG4-RD.

Background: CXCR5 + T follicular helper (T FH ) cells primarily promote B cells to produce an antigen-specific antibody through germinal centers (GCs). T FH cells exist in circulation, and circulating(c) T FH 2 cells, a subset of cT FH cells, are able to help naïve B cells produce IgE in healthy individuals. Conversely, IL-10-producing regulatory B (Breg) cells inhibit an accelerated immune response., Methods: We investigated the roles of cT FH cells and cBreg cells based on a T H 2 response in patients with atopic asthma (AA). Thirty-two patients with AA and 35 healthy volunteers (HV) were enrolled. We examined cT FH cells including their subsets, their expression of ICOS and PD-1, and cBreg cells by flow cytometry and their associations with clinical biomarkers. Plasma levels of CXCL13, which is a counterpart of CXCR5, were also measured using ELISA., Results: In patients with AA, cT FH 2 cells were increased and cT FH 1 cells were decreased compared with those in HV. The expression levels of ICOS on cT FH and their subset cells were elevated and Breg cells were greatly decreased. The plasma levels of CXCL13 in patients with AA were significantly elevated and correlated well with the cT FH 2/cBreg ratio. These cells were examined in 10 patients AA before and after inhaled corticosteroid (ICS) treatment. Interestingly, the percentages and numbers of T FH 2 and ICOS + cT FH cells declined after ICS treatment together with improvements in symptoms and clinical biomarkers., Conclusions: The percentages and numbers of cT FH 2 and ICOS + cT FH cells might be useful as biomarkers of T H 2 typed airway inflammation in patients with AA., (Copyright © 2019 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

POU domain class 2-associating factor 1 (also called Bob1), which is mainly expressed in B cells, regulates B cell homeostasis and controls humoral immune responses. Although Bob1 is known to function reliably in T cell subsets including follicular helper T cells, Th1 cells and Th2 cells, it is unknown whether Bob1 functions in other T cell subsets. In this study, we found that Bob1 knock out (KO) mice are resistant to experimental autoimmune encephalomyelitis (EAE) induced by MOG 35-55 peptide and that Bob1 KO T cells are defective in Th17 differentiation. Importantly, Bob1 interacts with retinoid acid receptor-related orphan receptor (ROR) gamma t (RORγt), a signature transcription factor for Th17 cells, through the ligand-binding domain of RORγt, thereby enhancing IL-17A transcription activity. IL-17A induction by Bob1 requires the ability for its formation of a DNA-Oct1-Bobl ternary complex. Thus, our findings demonstrate that Bob1 enhances IL-17A expression in vivo and in vitro by interacting with RORγt in Th17 cells, suggesting that Bob1 plays a pivotal role in Th17-mediated autoimmune disease., (Copyright © 2019 Elsevier Inc. All rights reserved.)

IgG4-related disease (IgG4-RD) is a chronic fibroinflammatory disease characterized by elevation of serum IgG4 level as well as infiltration of IgG4 + plasma cells in various affected organs. The etiology of IgG4-RD is still not fully understood. Since IgG4-RD is more prevalent in the elderly, aging in itself is considered to be an important risk factor of IgG4-RD. However, the relationship between the pathogenesis of IgG4-RD and immunosenescence remains unknown. To clarify age-related features underlying IgG4-RD, we focused on T follicular regulatory (Tfr) cells, which share forkhead box P3 with regulatory T cells, since the percentage of Tfr cells is known to depend on age. Studies of blood specimens from patients with IgG4-RD and from healthy volunteers demonstrated a marked elevation of circulating Tfr (cTfr) cells in patients with IgG4-RD. Moreover, the percentage of cTfr cells was significantly correlated with various clinical parameters including the level of serum IgG4 and the number of involved organs in IgG4-RD patients. The percentages of tonsillar and blood Tfr cells were increased with aging in healthy volunteers, whereas the suppressive effect of cTfr cells on B cell function in elderly subjects was impaired in comparison with that in young subjects due to a defect in the production of a regulatory cytokine, IL-10. Given that the number of IL-10-producing cTfr cells in IgG4-RD patients was markedly increased compared with that in healthy elderly subjects, these findings suggest that an abnormal aging process of Tfr cells may be related to the pathogenesis of IgG4-RD., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: The aim was to study the pathological role of lymphocytes with a peripheral T helper-cell-like phenotype (PD-1 + CXCR5 - CD4 + ) in IgG4-related disease (IgG4-RD)., Methods: PD-1 + CXCR5 - CD4 + T cells in the blood of patients with IgG4-RD ( n  = 53), patients with SS ( n  = 16) and healthy volunteers ( n  = 34) as controls were analysed by flow cytometry. Correlations between results obtained by flow cytometry and clinical parameters relevant to IgG4-RD were also analysed., Results: The percentage and absolute number of PD-1 + CXCR5 - cells within total CD4 + T cells in IgG4-RD patients were significantly increased compared with those in healthy volunteers. Further analysis showed that there were marked positive correlations of the percentage of PD-1 + CXCR5 - CD4 + T cells with the serum level of IgG4 and the number of organs involved. Interestingly, granzyme A (GZMA) + cells were enriched in PD-1 + CXCR5 - CD4 + T cells, and the percentage and absolute number of GZMA + PD-1 + CXCR5 - CD4 + T cells were significantly elevated in IgG4-RD patients. Although no obvious change was observed in the percentage of total CD4 + T cells, the percentage and absolute number of PD-1 + CXCR5 - CD4 + T cells decreased in accordance with a reduction of serum IgG4 level after treatment with glucocorticoids., Conclusion: In IgG4-RD, circulating CD4 + T-cell populations were composed of PD-1 + CXCR5 - cells, and the ratios of these cells were correlated with clinical manifestations of IgG4-RD. Further analysis of GZMA + PD-1 + CXCR5 - CD4 + T cells might lead to a deeper understanding of the pathogenesis of ectopic lymphoid follicles and the persistent inflammation in IgG4-RD.

The dynamics of fluorescence decay and charge recombination were studied in the ether-extracted photosystem I reaction center isolated from spinach with picosecond resolution over a wide time range up to 100 ns. At all temperatures from 268 to 77 K, a slow fluorescence decay component with a 30-40 ns lifetime was detected. This component was interpreted as a delayed fluorescence emitted from the singlet excited state of the primary donor P700*, which is repopulated through charge recombination that was increased by the lack of secondary acceptor phylloquinone in the sample. Analysis of the fluorescence kinetics allowed estimation of the standard free-energy difference -DeltaG between P700* and the primary radical pair (P700(+)A0(-)) state over a wide temperature range. The values of -DeltaG were estimated to be 160/36 meV at 268/77 K, indicating its high sensitivity to temperature. A temperature-dependent -DeltaG value was also estimated in the delayed fluorescence of the isolated photosystem I in which the secondary acceptor quinone was partially prereduced by preillumination in the presence of dithionite. The results revealed that the temperature-dependent -DeltaG is a universal phenomenon common with the purple bacterial reaction centers, photosystem II and photosystem I reaction centers.

Chlorella, when heterotrophically cultivated in the dark, is able to grow with Zn2+ at 10-40 mM, which is 10 times the concentration lethal to autotrophically grown cells. However, the lag phase is prolonged with increasing concentrations of Zn2+; for example, in this study, 1 d of the control lag phase was prolonged to about 16 d with Zn2+ at 16.7 mM (x2,000 of the control). Once the cells started to grow, the log phase was finished within 4-6 d regardless of Zn concentration, which was almost the same as that of the control. The photosysystem I reaction center chlorophyll, P700, and the far-red fluorescence were detected only after the late log phase of the growth curve, suggesting that chlorophyll-protein complexes can be organized after cell division has ceased. Interestingly, at more than 16.7 mM of Zn2+, Zn-chlorophyll a was accumulated and finally accounted for about 25% of the total chlorophyll a in the late stationary phase. We found that the Zn-chlorophyll a was present in the thylakoid membranes and not in the soluble fractions of the cells. The rather low fluorescence yield at around 680 nm in the stationary phase suggests that Zn-chlorophyll a can transfer its excitation energy to other chlorophylls. Before accumulation of Zn-chlorophyll a, a marked amount of pheophytin a was temporally accumulated, suggesting that Zn-chlorophyll a could be chemically synthesized via pheophytin a.

Phylloquinone, a substituted 1,4-naphthoquinone with an 18-carbon-saturated phytyl tail, functions as a bound one-electron carrier cofactor at the A1 site of photosystem I (PSI). A Feldmann tag line mutant, no. 2755 (designated as abc4 hereafter), showed pale-green young leaves and white old leaves. The mutated nuclear gene encoded 1,4-dihydroxy-2-naphtoic acid phytyltransferase, an enzyme of phylloquinone biosynthesis, and high-performance liquid chromatography analysis revealed that the abc4 mutant contained no phylloquinone, and only about 3% plastoquinone. Photooxidation of P700 of PSI in the abc4 mutant was not observed, and reduced-versus-oxidized difference spectroscopy indicated that the abc4 mutant had no P700. The maximum quantum yield of photosystem II (PSII) in the abc4 mutant was much decreased, and the electron transfer from PSII to PSI in the abc4 mutant did not occur. For the pale-green leaves of the abc4 mutant plant, the ultrastructure of the chloroplasts was almost the same as that of the wild-type plant. However, the chloroplasts in the albino leaves of the mutant were smaller and had a lot of grana thylakoids and few stroma thylakoids. The amounts of PSI and PSII core subunits in the abc4 mutant were significantly decreased compared with those in the wild type. These results suggested that a deficiency of phylloquinone in PSI caused the abolishment of PSI and a partial defect of PSII due to a significant decrease of plastoquinone, but did not influence the ultrastructure of the chloroplasts in young leaves.

Primary photochemistry in photosystem I (PS I) reaction center complex from Acaryochloris marina that uses chlorophyll d instead of chlorophyll a has been studied with a femtosecond spectroscopy. Upon excitation at 630 nm, almost full excitation equilibration among antenna chlorophylls and 40% of the excitation quenching by the reaction center are completed with time constants of 0.6(+/-0.1) and 4.9(+/-0.6) ps, respectively. The rise and decay of the primary charge-separated state proceed with apparent time constants of 7.2(+/-0.9) and 50(+/-10) ps, suggesting the reduction of the primary electron acceptor chlorophyll (A(0)) and its reoxidation by phylloquinone (A(1)), respectively.

The photosystem (PS) I photosynthetic reaction center was modified thorough the selective extraction and exchange of chlorophylls and quinones. Extraction of lyophilized photosystem I complex with diethyl ether depleted more than 90% chlorophyll (Chl) molecules bound to the complex, preserving the photochemical electron transfer activity from the primary electron donor P700 to the acceptor chlorophyll A(0). The treatment extracted all the carotenoids and the secondary acceptor phylloquinone (A(1)), and produced a PS I reaction center that contains nine molecules of Chls including P700 and A(0), and three Fe-S clusters (F(X), F(A) and F(B)). The ether-extracted PS I complex showed fast electron transfer from P700 to A(0) as it is, and to FeS clusters if phylloquinone or an appropriate artificial quinone was reconstituted as A(1). The ether-extracted PS I enabled accurate detection of the primary photoreactions with little disturbance from the absorbance changes of the bulk pigments. The quinone reconstitution created the new reactions between the artificial cofactors and the intrinsic components with altered energy gaps. We review the studies done in the ether-extracted PS I complex including chlorophyll forms of the core moiety of PS I, fluorescence of P700, reaction rate between A(0) and reconstituted A(1), and the fast electron transfer from P700 to A(0). Natural exchange of chlorophyll a to 710-740 nm absorbing chlorophyll d in PS I of the newly found cyanobacteria-like organism Acaryochloris marina was also reviewed. Based on the results of exchange studies in different systems, designs of photosynthetic reaction centers are discussed.

By the ether treatment of lyophilized PSI pigment-protein complexes, all the carotenoids and the secondary acceptor phylloquinone (A1), and more than 90% of the Chl were removed to yield the PSI complex with 9-11 molecules of Chl per reaction-center unit. The complexes retained the primary electron donor and acceptor (P700 and A0), in addition to three FeS clusters (F(X), F(A) and F(B)), and showed an activity of highly efficient electron transfer when phylloquinone was reconstituted. The methods for the preparation and the characterization of the ether-extracted PSI complexes are reviewed in this article. We also review the studies done with this PSI preparation on (1) the identification of the absorption and fluorescence spectra of P700, (2) the nano- and picosecond reaction of A0 and A1, (3) the energy-gap dependency of the reaction rate between A0 and the artificial quinones reconstituted at the A1 site, (4) the direct excitation of P700 followed by the ultra-fast electron transfer from P700 to A0, and (5) the de- and re-stabilization of the PSI structure by the removal and reconstitution, respectively, of antenna Chl in the presence of certain lipids.

For the purpose of examining the Topoisomerase II (Topo II) activity in human kidney cancer cells, we performed experiments with using DNA unknotting method. This method check relative Topo II activity with its conversion of knotted form P4 phage DNA to unknotted form. Our preliminary results demonstrate remarkable activity of Topo II with specific conversion of knotted form P4 DNA to unknotted form in human kidney cancer cells, YCR and ACHN. Moreover, addition of etoposide to the same experiment suppressed Topo II activity in a dose dependent manner. Our results suggest that kidney cancer has a certain amount of Topo II, a target for etoposide. We believe this method is useful to measure Topo II activity in cancer cells and to estimate chemotherapeutic potential of Topo II inhibitors including etoposide in human kidney cancers.

Treatment of H. chlorum membrane preparations with diethyl ether of high degrees of water saturation raised the bacteriochlorophyll (BChl) g' mole fraction, as determined by HPLC analysis of their acetone extracts, toward a level of 40% of total BChl g or higher. Starting from pure BChl g, the BChl g' mole fraction should never exceed 24.6% which is the equilibrium value in diethyl ether. The existence (and possible functioning) of BChl g' in vivo is thus unequivocally demonstrated.

Low-temperature resonance Raman (RR) spectra have been obtained at resonance with the Soret transition of chlorophyll a in photosystem I particles containing large amounts either of the triplet state of P700 or of its radical cation state. Subtracting these spectra from those of resting reaction centers yielded RR spectra of P700 in its neutral, ground state. These spectra arise from two distinct chlorophyll a molecules differing by the strengths of the bonding interactions assumed by their keto carbonyl groups, the stretching frequencies of which are found at 1655 and 1675 cm-1. The present results rule out previous hypotheses that P700 might have consisted of a single, chemically modified chlorophyll a molecule. Neither of the bonding interactions assumed by the keto carbonyls of the P700 chlorophylls most probably involves chlorophyll-chlorophyll bridging through water molecules, as surmised in the so-called special pair models, but likely consists of H bonds with distinct protein sites. The magnesium atoms of the two P700 chlorophylls are 5-coordinated. Hence, the structural model of P700 provided by the present data is qualitatively the same, in terms of bonding interactions, as that currently accepted for the bacterial primary donor.

The reaction center chlorophyll of Photosystem I in spinach chloroplasts was highly enriched. Preparations having 5-9 chlorophylls per 1 P700 were obtained by treating the Photosystem I particles prepared by digitonin treatment of chloroplasts with wet diethyl ether. All P700 present in the extracted particles was found to be photoactive, undergoind oxidation upon illumination.

The linear dichroism of Photosystem I particles containing 10 chlorophylls per P700 has been investigated at 10 K. The particles were oriented by uniaxial squeezing of polyacrylamide gels. The oxidation state of P700 was altered either by incubation of the gels with redox mediators or by low temperature illumination. The QY transitions of the primary electron donor P700, of the remaining unoxidized chlorophyll in P700(+) and of a chlorophyll molecule absorbing at 686 nm, which presumably corresponds to the primary electron acceptor A0, are all preferentially oriented perpendicular to the gel squeezing direction. The QY transition of the chlorophyll forms absorbing at 670 and 675 nm appear tilted at 40 ± 5° from this orientation axis. This orientation of the various chlorophylls is compared to that previously reported for more native Photosystem I particles.

The light-induced changes in the yield of chlorophyll alpha fluorescence and photooxidation of P-700 in the P-700-enriched particles isolated from spinach chloroplasts were studied. 1. Fluorescence emitted from the particles was found to show light-induced transient changes in the yield. In the presence of ascorbate, illumination induced quenching of fluorescence in parallel to the photooxidation of P-700. The time course of dark reduction of photooxidized P-700 agreed well with that of dark recovery of variable fluorescence yield in the presence of ascorbate. When illuminated in the presence of dithionite, the emission yield increased, whereas no photooxidation of P-700 was observed. 2. The yield of variable fluorescence and redox state of P-700 depended similarly upon the redox potential. 3. At liquid nitrogen temperature, illumination induced a rise of the fluorescence yield and a complete photooxidation of P-700 in the ascorbate-treated sample. When the particles had been preincubated with dithionite in the light before cooling, light-induced rise in the fluorescence yield was accompanied by only a small extent of P-700 photooxidation. It is suggested that both the oxidized form of P-700 and the primary electron acceptor act as quenchers for the variable fluorescence. 4. The emission spectrum for the constant part of fluorescence (F679) has a peak at 679 nm, and that for the variable part of fluorescence (F694) has a peak at 694 nm at room temperature. The emission maxima were slightly shifted and the yield of variable fluorescence was markedly enhanced at liquid nitrogen temperature. 5. Excitation spectra determined show a peak at 672 nm for F679, and a peak at 672 nm and a shoulder at 685 nm for F694. Action spectrum for P-700 photooxidation was similar to the excitation spectrum for F694.

The photoreduction of iron-sulfur centers was studied at low temperature in Photosystem I particles from spinach and the cyanobacterium Synechocystis 6803, which contain various amounts of vitamin K-1 (recently tentatively identified as the acceptor A1). The irreversible charge separation that was progressively induced at low temperature between P-700 and FA (or FB) by successive laser flashes was studied at 15 K. Its maximum amount after a large number of flashes was shown to be fairly independent of the number (0, 1 or 2) of vitamins K-1 per reaction center. Moreover, the first flash yield of this charge separation was diminished by only about 50% when vitamin K-1 was completely absent from the particles by comparison with particles containing one or two vitamin K-1 per reaction center. When FA and FB were prereduced, the iron-sulfur center FX was also reversibly photoreduced at 9 K in the absence of vitamin K-1. The implications of these results for the electron pathways of Photosystem I are discussed and it is proposed that a direct electron transfer from A0- to the iron-sulfur centers is highly efficient at low temperature.