Water oxidation by photosystem II is the primary source of electrons for sustained H2 photoproduction in nutrient-replete green algae

The unicellular green alga Chlamydomonas reinhardtii is capable of photosynthetic H₂ production. H₂ evolution occurs under anaerobic conditions and is difficult to sustain due to 1) competition between [FeFe]-hydrogenase (H₂ase), the key enzyme responsible for H₂ metabolism in algae, and the Calvin–Benson–Bassham (CBB) cycle for photosynthetic reductants and 2) inactivation of H₂ase by O₂ coevolved in photosynthesis. Recently, we achieved sustainable H₂ photoproduction by shifting algae from continuous illumination to a train of short (1 s) light pulses, interrupted by longer (9 s) dark periods. This illumination regime prevents activation of the CBB cycle and redirects photosynthetic electrons to H₂ase. Employing membrane-inlet mass spectrometry and H218O, we now present clear evidence that efficient H₂ photoproduction in pulse-illuminated algae depends primarily on direct water biophotolysis, where water oxidation at the donor side of photosystem II (PSII) provides electrons for the reduction of protons by H₂ase downstream of photosystem I. This occurs exclusively in the absence of CO₂ fixation, while with the activation of the CBB cycle by longer (8 s) light pulses the H₂ photoproduction ceases and instead a slow overall H₂ uptake is observed. We also demonstrate that the loss of PSII activity in DCMU-treated algae or in PSII-deficient mutant cells can be partly compensated for by the indirect (PSII-independent) H₂ photoproduction pathway, but only for a short (<1 h) period. Thus, PSII activity is indispensable for a sustained process, where it is responsible for more than 92% of the final H₂ yield.

Many species of green algae have [FeFe]-hydrogenases (H₂ases) (1) that catalyze the reversible reduction of protons to molecular hydrogen:2H++2e−⇄H2.[1]Since [FeFe]-H₂ases are extremely O₂-sensitive (2), reaction 1 typically proceeds under anoxic conditions. With respect to H₂ metabolism, Chlamydomonas reinhardtii is the most studied alga. This alga possesses two [FeFe]-H₂ases in the chloroplast, HYDA1 and HYDA2 (3, 4). In the light, they accept electrons from photosynthetically reduced ferredoxin (FDX1) (5), while in the dark electrons come from the activity of pyruvate ferredoxin oxidoreductase (PFR1) (6). PFR1 catalyzes the oxidation of pyruvate to acetyl-CoA, and its activity is linked to H₂ase via FDX1 (7). Since [FeFe]-H₂ases interact with the photosynthetic electron transport chain at the level of ferredoxin, they may accept electrons originating both from water oxidation via the photosystem II (PSII)-dependent pathway (“direct water biophotolysis”) and from the degradation of organic substrates via a PSII-independent mechanism (“indirect water biophotolysis” or “indirect pathway”) (8). In the latter case, the reductants are supplied to the plastoquinone (PQ) pool by the type II NADPH dehydrogenase (NDA2), thus bypassing PSII (9, 10).The release of H₂ leads to a loss of metabolic energy. In healthy, actively growing C. reinhardtii cultures, H₂ production is therefore only a temporal phenomenon observed during dark anoxia and upon subsequent onset of illumination (11). In contrast to dark fermentation, H₂ photoproduction is a very efficient process that proceeds for only a short period of time (from a few seconds to a few minutes). Two theories have been developed to explain the short duration. The first is based on the oxygen sensitivity of H₂ases (12, 13). In the light, algae accumulate O₂ that is produced by water oxidation at PSII (14). As a result, H₂ photoproduction may cease over time (14, 15), and the duration of this process is reported to shorten with increased light intensity (16). Because of the negative correlation between the rates of H₂ photoproduction and O₂ evolution, the inhibition of H₂ases by O₂ is frequently quoted as the primary reason for the rapid loss in H₂ photoproduction after the onset of illumination (17).Alternatively, the loss in the H₂ photoproduction efficiency during illumination could be explained by the light-induced induction of competitive pathways, which may drain reducing equivalents away from the [FeFe]-H₂ase enzyme (18, 19). Candidates for this role are the Mehler-like reaction driven by flavodiiron proteins (FDPs) (15, 20, 21) and the Calvin–Benson–Bassham (CBB) cycle (22). Compelling evidence for the competition between these two pathways and H₂ production has been accumulated in recent studies (23–25). As CO₂ fixation provides the strongest sink for photosynthetic reductants, it should play a major role in the cessation of H₂ photoproduction in algae when the CBB cycle is active (19, 22).For preventing competition between the [FeFe]-H₂ases and the CBB cycle, we recently devised a pulse-illumination protocol that allows H₂ production in nutrient-replete algal cultures for up to 3 d (23). To achieve this, we specifically selected the duration of light pulses in the light/dark sequence to avoid activation of the CBB cycle, thus allowing for the redirection of photosynthetic electrons toward the [FeFe]-H₂ases. Typically, a train of 1- to 6-s light pulses interrupted by 9-s dark periods is sufficient for sustained H₂ photoproduction in C. reinhardtii cultures (23, 25). Our protocol thus differs from earlier pulse-illumination approaches that aimed at preventing the accumulation of O₂ in the cultures (26).While we could demonstrate competition of [FeFe]-H₂ase with FDPs (25), the origin of reductants for H₂ photoproduction in the pulse-illuminated algae remained unclear. The relatively high efficiency of the process suggests the involvement of water oxidation by PSII, and consequently the simultaneous production of H₂ and O₂. Although widely proposed in the current literature (8, 24), the presence of the direct water biophotolysis in H₂-producing green algae has not yet been proven by direct experimental data.In the present study, we provide clear evidence for the presence of PSII-dependent oxidation of ¹⁸O-labeled water H218O with concomitant evolution of ¹⁶O₂ and ^16,18O₂ during H₂ photoproduction in the pulse-illuminated green alga C. reinhardtii under anoxic conditions. O₂ evolution is balanced by light-dependent and light-independent respiration that sustains the anoxic condition. We also demonstrate that the loss of PSII activity in algae can be partly compensated by the PSII-independent H₂ photoproduction pathway. Nevertheless, the activity of PSII is indispensable for the sustained process, where it contributes to more than 92% of the final H₂ yield.     

Cetuximab plus FOLFOX6 or FOLFIRI in metastatic colorectal cancer: CECOG trial

AIM: To investigate efficacy and safety of cetuximab combined with two chemotherapy regimens in patients with unresectable metastatic colorectal cancer (mCRC). METHODS: Randomized patients received cetuximab with 5-fluorouracil (5-FU), folinic acid (FA) and oxaliplatin (FOLFOX) 6 (arm A, n = 74) or 5-FU, FA and irinotecan (FOLFIRI) (arm B, n = 77). KRAS mutation status was determined retrospectively in a subset of tumors (n = 117). RESULTS: No significant difference was found between treatment arms A and B ...     

Investigations on the effects of alkyl polyglucosides on development and fertility.

Alkyl polyglucosides are non-ionic surfactants which are mainly used in laundry detergents, hard surface cleaners and personal care products. Two in vitro screening test systems were used to investigate the endocrine modulating potential of alkyl polyglucosides. No indications were observed for any estrogenic or anti-estrogenic effects in an MCF-7 E-Screen assay and a reporter gene assay using luciferase-transfected MCF-7 cells. Concentrations exceeding the effect concentration of estradiol by a factor of 1000 did not reveal any indication for an estrogenic activity. Furthermore, in a combination assay investigating the effects of estradiol in the presence of alkyl polyglucoside, no anti-estrogenic potential was observed. In an one-generation screening assay in rats no effects on the fertility were observed up to the highest dose of 1000 mg/kg bw/day alkyl glucoside. Potential embryo-/fetotoxicity and/or teratogenicity was investigated in a segment II study in rats. No effects were noted up to the limit dose of 1000 mg/kg bw/day. All parameters of the treatment groups were comparable with those of the control group. Skeletal and visceral investigations did not detect any treatment-related malformations. For the embryo/fetotoxicity, the teratogenicity and the maternal toxicity a NOAEL of 1000 mg/kg was deduced.     

Neoadjuvant letrozole in postmenopausal estrogen and/or progesterone receptor positive breast cancer: A phase IIb/III trial to investigate optimal duration of preoperative endocrine therapy

Background  

In recent years, preoperative volume reduction of locally advanced breast cancers, resulting in higher rates of breast-conserving surgery (BCS), has become increasingly important also in postmenopausal women. Clinical interest has come to center on the third-generation nonsteroidal aromatase inhibitors (AIs), including letrozole, for such neoadjuvant endocrine treatment. This usually lasts 3–4 months and has been extended to up to 12 months, but optimal treatment duration has not been fully established.     

The anti-tumor arotinoid RO 40-8757 protects bone marrow from the toxic effects of 5-fluorouracil

Combination therapy with 5-Fluorouracil (5-FU) and the arotinoid Ro 40-8757 (mofarotene) of established chemically induced mammary tumors in rats was examined. The cytotoxic drug was administered weekly and Ro 40-8757 was given daily. The dose of Ro 40-8757 used in this study did not have an effect on tumor burden but, in combination with 5-FU, significantly enhanced the reduction in tumor burden and tumor number. In order to determine if Ro 40-8757 had a protective effect on 5-FU-treated animals, several studies were performed with non-tumor-bearing mice. The 5-FU was given once a week for 3 weeks at a dose that was lethal only after the third administration. When this treatment was combined with Ro 40-8757 given 5 times/week, approximately 50% of the mice survived. Examination of the progenitor cell contents of femura and spleens of treated mice indicated that the protective effect of Ro 40-8757 was manifested at the primitive hemopoietic progenitor cell level. Studies with murine bone marrow cells and human breast-cancer cell lines in vitro demonstrated that there was no interaction between the 2 drugs at the cellular level, indicating that the arotinoid does not enhance the ability of cells to metabolize 5-FU. This protective effect of the arotinoid makes it a useful potential partner for combination therapy with 5-FU. © 1994 Wiley-Liss, Inc.     

CD40-expressing carcinoma cells induce down-regulation of CD40 ligand (CD154) and impair T-cell functions

The interaction of CD40 expressed by immunocompetent cells with its ligand CD154 on the surface of T-helper cells plays a crucial role in the immune response. Recently, the presence of CD40 was also demonstrated on a variety of carcinomas. Whereas the critical relevance of CD40 in cytotoxic T-cell priming via dendritic cells is already established, the biological role of CD40/CD154 interactions in nonhematopoetic cells is still unclear. In the present study we demonstrate that CD154 expression density is down-regulated on activated T cells on interaction with CD40+ tumor cells. Naive T cells cocultured with CD40+carcinoma showed impaired functionality as indicated by a reduced frequency of IFN-gamma secreting cells, reduced interleukin 2 secretion, impaired proliferation, and a lack of CD154 re-expression on restimulation. In distinction, T-cell effector lysing capacity was not impaired by CD40-expressing tumor cell targets. The present results suggest that in marked contrast to antigen-presenting cells, CD40 expression on carcinoma cells suppresses T-cell activation. Our findings support the statement that CD40 functions are context dependent and imply a new function for CD40 expressed on nonantigen-presenting cells.     

Anti-p53 in breast cancer: concordance of different assay procedures and association with p53 antigen expression

OBJECTIVE: Anti-p53 levels detected by different methods were compared in a predefined group of patients with breast cancer and correlated with p53 antigen expression in the corresponding tumors. METHODS: P53 autoantibodies were investigated in 165 patients with primary breast cancer using ELISAs with recombinant or native p53. Immunoblot and indirect immunofluorescence (Huh7) were used for confirmation, p53 antigen expression in the tumor was determined immunohistochemically. RESULTS: Using ELISA, overall 18/165 positives (11%) were detected, with only partly concordant results between the assays. Five positive sera were confirmed by immunoblot, and three also by indirect immunofluorescence. Anti-p53-positive patients detected by more than two assays showed accumulated p53 in the tumor (6/6) and mostly suffered from recurrent tumors (4/6; p = 0.02). In these cases, a trend towards a shortened disease-free interval was found (26 vs. 49 months; n.s.). In patients with a positive or borderline result in only one of the serological methods, there was no increased rate of p53 accumulation compared to anti-p53-negative patients (4/19 versus 35/126). CONCLUSIONS: Lack of assay standardization may partly explain the divergence in reports on anti-p53 and its clinicopathological associations. We speculate that, in different groups of patients, anti-p53 might be induced by different mechanisms.