Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced DNA damage

The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA^−/−, FANCC^−/−, FANCA^−/−C^−/−, FANCD2^−/− and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical γH2AX-staining assay. Although the sensitivity of FANCA^−/−, FANCC^−/− and FANCA^−/−C^−/− cells to formaldehyde was comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2^−/− cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, γH2AX foci in FANCD1mt cells persisted for longer times than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex.     

Epistasis in the multiple locus symmetric viability model

The n-locus two-allele symmetric viability model is considered in terms of the parameters measuring the additive epistasis in fitness. The dynamics is analysed using a simple linear transformation of the gametic frequencies, and then the recurrence equations depend on the epistatic parameters and Geiringer's recombination distribution only. The model exhibits an equilibrium, the central equilibrium, where the 2 ⁿ gametes are equally frequent. The transformation simplifies the stability analysis of the central point, and provides the stability conditions in terms of the existence conditions of other equilibria. For total negative epistasis (all epistatic parameters are negative) the central point is stable for all recombination distributions. For free recombination either a central point (segregating one, two, ... or n loci) or the n-locus fixation states are stable. For no recombination and some epistatic parameters positive the central point is unstable and several boundary equilibria may be locally stable. The sign structure of the additive epistasis is therefore an important determinant of the dynamics of the n-locus symmetric viability model. The non-symmetric multiple locus models previously analysed are dynamically related, and they all have an epistatic sign structure that resembles that of the multiplicative viability model. A non-symmetric model with total negative epistasis which share dynamical properties with the similar symmetric model is suggested.Supported in part by NIH grant GM 28016, and by grant 81-5458 from the Danish Natural Science Research Council     

Infrared spectroscopic study of the metal-coordination structures of calcium-binding proteins

Carboxylate (COO⁻) groups can coordinate to metal ions in of the following four modes: ‘unidentate’, ‘bidentate’, ‘bridging’ and ‘pseudo-bridging’ modes. COO⁻ stretching frequencies provide information about the coordination modes of COO⁻ groups to metal ions. We review the Fourier-transform infrared spectroscopy (FTIR) of side-chain COO⁻ groups of Ca²⁺-binding proteins: pike parvalbumin pI 4.10, bovine calmodulin and Akazara scallop troponin C. FTIR spectroscopy of Akazara scallop troponin C has demonstrated that the coordination structure of Mg²⁺ is distinctly different from that of Ca²⁺ in the Ca²⁺-binding site. The assignments of the COO⁻ antisymmetric stretch have been ensured on the basis of the spectra of calcium-binding peptide analogues. The downshift of the COO⁻ antisymmetric stretching mode from 1565 cm^-1 to 1555-1540 cm⁻¹ upon Ca²⁺ binding is a commonly observed feature of FTIR spectra for EF-hand proteins.     

The maximum nitrate reductase activity of the seagrass Zostera noltii (Hornem.) varies along its vertical distribution

Maximum nitrate reductase (NR) activity was measured in two intertidal morphotypes of Zostera noltii (Hornem.) in Ria Formosa tidal lagoon, southern Portugal. The two morphotypes develop in the upper and lower limits of the intertidal meadows. The NR activity was measured using an in vivo method, without cell disruption. NR activity was 30-40 fold higher in leaves than in roots, which indicates that nitrate reduction is essentially made through the aerial part of the plant. The effects of assay temperature (5 °C steps, from 5 to 45 °C), pH (7, 8 and 9) and elevation (upper and lower intertidal) on leaf NR activity were tested in a factorial design (n=5). Both elevation and assay temperature had a significant effect on NR activity, but not pH. NR activity was always higher in the upper intertidal plants, at all temperatures. Activity peaks for upper and lower plants were, respectively, 6.12 μmol NO₂⁻ g⁻¹ DW 0.5 h⁻¹ at 25 °C, and 3.30 μmol NO₂⁻ g⁻¹ DW 0.5h⁻¹ at 35 °C. Further investigation on environmental factors concerning the intertidal environment must be developed, as they are probably responsible for the significant differences found between the values of NR activity in the upper and lower morphotype.     

Ionic and osmotic regulation capabilities of juvenile Gulf of Mexico sturgeon, Acipenser oxyrinchusde sotoi

The salinity tolerance, and hydromineral regulation capabilities of three size groups (small 110–170 g; medium 230–290 g, large 460–700 g; n=48 for each group) of 13-month-old juvenile Gulf of Mexico sturgeon were investigated. Fish (n=6 for each salinity) were transferred directly from freshwater (FW) to a series of experimental salinity treatments (0, 5, 10, 15, 20, 25, 30, and 35 parts per thousand (ppt)). Fish were also acclimated in brackish water (20 ppt) for 2 weeks and transferred to a salinity of 34 ppt. In this condition juvenile Gulf of Mexico sturgeon adapted to saltwater (SW) and maintained their hydromineral balance. FW adapted sturgeon (n=6) had an average blood hemotocrit of 28.2±0.8%, plasma osmolality of 260.7±1.6 mOsm kg⁻¹ H₂O, and plasma ion concentrations of 135.7±1.2 mM l⁻¹ Na⁺, 106.9±1.9 mEq l⁻¹ Cl⁻, and 2.9±0.1 mM l⁻¹ K⁺. In SW adapted sturgeon (n=8) blood parameters averaged 26.9±0.7% for hematocrit, 294.2±2.3 mOsm kg⁻¹ H₂O for osmolality, 152.0±1.7 mM l⁻¹ Na⁺, 149.2±1.4 mEq l⁻¹ for Cl⁻, and 3.1±0.1 mM l⁻¹ K⁺. The method of transfer (abrupt or slow acclimation) directly affected fish survival and the time they took to achieve ionic and osmotic regulation. This SW adaptation appears to be related to body size, the larger the fish the easier the adaptation process. A threshold size of about 170 g was apparent for the fish to adapt to saltwater after 2 weeks of acclimation. Chloride cells were present in both FW and SW adapted sturgeon with SW and brackish water fish having chloride cells significantly (P<0.05) more numerous (561±53 and 598±45 cells mm⁻²) and larger in size (41.0±3.85 and 34.2±4.49 μm²) than FW adapted sturgeon (10±1.0 cells mm⁻² and 22±2.53 μm²). Few chloride cells were observed in the opercular membrane, however, none were found in the pseudobranch and spiracle.     

The role of SNM1 family nucleases in etoposide-induced apoptosis

DNA double strand breaks (DSBs) induced by etoposide, an inhibitor of DNA topoisomerase II, are repaired mainly by non-homologous end joining (NHEJ). Unexpectedly, it was found that at high doses of etoposide, proteins involved in NHEJ, such as KU70/80, DNA-PKcs and ARTEMIS/SNM1C, trigger apoptosis rather than repair of DSBs. Because ARTEMIS is a member of the SNM1 protein family that includes SNM1A and APOLLO/SNM1B, this study examined whether SNM1A and/or APOLLO are also involved in etoposide-induced apoptosis. Using SNM1A^−/− and APOLLO^−/− cells, it was found that both SNM1A and APOLLO participate in etoposide-induced apoptosis. Although cell viability monitored by MTT assay did not differ between SNM1A^−/−/APOLLO^−/−/ARTEMIS^−/−, SNM1A^−/−/APOLLO^−/−, and single gene knockout cells, DNA fragmentation monitored by TUNEL assay differed between these cells, suggesting that the three SNM1 family nucleases function independently, at least during the induction of apoptotic DNA fragmentation.     

Effect of different concentrations of serotonin, histamine and insulin on the hormone (serotonin and ACTH) production of Tetrahymena in nutrient-free physiological milieu

Cell populations of Tetrahymena pyriformisGL were kept in nutrient-free (Losina) milieu and treated with different (10⁻⁶–10⁻²¹ M) concentrations of serotonin, histamine or insulin for 30 min. Following that the hormone (serotonin and adrenocorticotropin (ACTH) content of the cells were measured by immunocytochemical flow cytometric method. Serotonin reduced histamine when applied in 10⁻¹² and 10⁻¹⁵ M concentrations, while elevated ACTH levels when applied in 10⁻⁶, 10⁻⁹ and 10⁻²¹ M concentrations. Histamine reduced serotonin concentration at 10⁻⁹–10⁻²¹ M concentrations and increased ACTH in 10⁻⁶ M. Insulin elevated both hormones’ content in each concentration except at 10⁻¹² M. The results demonstrate that (1) in nutrient-free conditions the hormonal effects differ from that of nutrient-rich (tryptone + yeast) condition; (2) there is an optimal hormone concentration, which causes the strongest effect and this is different for each hormones; (3) the hormone receptors of Tetrahymena are very sensitive; as they react to zeptomolar concentrations. Such small concentration is even more effective than higher ones. Since hormones must become highly diluted in the natural environment of Tetrahymena, it seems that such low concentrations are the actual physiological concentrations.     

Computational experience with a parallel algorithm for tetrangle inequality bound smoothing

Determining molecular structure from interatomic distances is an important and challenging problem. Given a molecule with n atoms, lower and upper bounds on interatomic distances can usually be obtained only for a small subset of the atom pairs, using NMR. Given the bounds so obtained on the distances between some of the atom pairs, it is often useful to compute tighter bounds on all the pairwise distances. This process is referred to as bound smoothing. The initial lower and upper bounds for the pairwise distances not measured are usually assumed to be 0 and ∞. One method for bound smoothing is to use the limits imposed by the triangle inequality. The distance bounds so obtained can often be tightened further by applying the tetrangle inequality—the limits imposed on the six pairwise distances among a set of four atoms (instead of three for the triangle inequalities). The tetrangle inequality is expressed by the Cayley—Menger determinants. For every quadruple of atoms, each pass of the tetrangle inequality bound smoothing procedure finds upper and lower limits on each of the six distances in the quadruple. Applying the tetrangle inequalities to each of the ( ₄ ⁿ ) quadruples requires O(n ⁴) time. Here, we propose a parallel algorithm for bound smoothing employing the tetrangle inequality. Each pass of our algorithm requires O(n ³ log n) time on a CREW PRAM (Concurrent Read Exclusive Write Parallel Random Access Machine) with processors. An implementation of this parallel algorithm on the Intel Paragon XP/S and its performance are also discussed.     

A prezygotic transmission distorter acting equally in female and male zebra finches Taeniopygia guttata

The two parental alleles at a specific locus are usually inherited with equal probability to the offspring. However, at least three processes can lead to an apparent departure from fair segregation: early viability selection, biased gene conversion and various kinds of segregation distortion. Here, we conduct a genome‐wide scan for transmission distortion in a captive population of zebra finches (Taeniopygia guttata) using 1302 single‐nucleotide polymorphisms (SNPs) followed by confirmatory analyses on independent samples from the same population. In the initial genome‐wide scan, we found significant distortion at three linked loci on chromosome Tgu2 and we were able to replicate this finding in each of two follow‐up data sets [overall transmission ratio = 0.567 (95% CI = 0.536–0.600), based on 1101 informative meioses]. Although the driving allele was preferentially transmitted by both heterozygous females [ratio = 0.560 (95% CI = 0.519–0.603)] and heterozygous males [ratio = 0.575 (95% CI = 0.531–0.623)], we could rule out postzygotic viability selection and biased gene conversion as possible mechanisms. Early postzygotic viability selection is unlikely, because it would result in eggs with no visible embryo and hence no opportunity for genotyping, and we confirmed that both females and males heterozygous for the driving allele did not produce a larger proportion of such eggs than homozygous birds. Biased gene conversion is expected to be rather localized, while we could trace transmission distortion in haplotypes of several megabases in a recombination desert. Thus, we here report the rare case of a prezygotically active transmission distorter operating equally effectively in female and male meioses.     

Isocyanides inhibit [Fe]-hydrogenase with very high affinity

[Fe]-Hydrogenase catalyzes the reversible activation of H₂. CO and CN⁻ inhibit this enzyme with low affinity (K_i ≅ 0.1 mM) by binding to the iron site of the bound iron-guanyrylpyridinol cofactor. We report here that isocyanides, which are formally isoelectronic with CO and CN⁻, strongly inhibit [Fe]-hydrogenase (K_i as low as 1 nM). The [NiFe]- and [FeFe]-hydrogenases tested were not inhibited by isocyanides. UV–Vis and infrared spectra revealed that the isocyanides bind to the iron center of [Fe]-hydrogenase. The inhibition kinetics are in agreement with the proposed catalytic mechanism, including the open/closed conformational change of the enzyme.     

SUMO ligase activity of vertebrate Mms21/Nse2 is required for efficient DNA repair but not for Smc5/6 complex stability

Nse2/Mms21 is an E3 SUMO ligase component of the Smc5/6 complex, which plays multiple roles in maintaining genome stability. To study the functions of the vertebrate Nse2 orthologue, we generated Nse2-deficient chicken DT40 cells. Nse2 was dispensable for DT40 cell viability and required for efficient repair of bulky DNA lesions, although Nse2-deficient cells showed normal sensitivity to ionising radiation-induced DNA damage. Homologous recombination activities were reduced in Nse2^−/−/− cells. Nse2 deficiency destabilised Smc5, but not Smc6. In rescue experiments, we found that the SUMO ligase activity of Nse2 was required for an efficient response to MMS- or cis-platin-induced DNA damage, and for homologous recombination, but not for Smc5 stability. Gel filtration analysis indicated that Smc5 and Nse2 remain associated during the cell cycle and after DNA damage and Smc5/Smc6 association is independent of Nse2. Analysis of Nse2^−/−/−Smc5⁻ clones, which were viable although slow-growing, showed no significant increase in DNA damage sensitivity. We propose that Nse2 determines the activity, but not the assembly, of the Smc5/6 complex in vertebrate cells, and this activity requires the Nse2 SUMO ligase function.     

Pseudo-nitzschia and domoic acid fluxes in Santa Barbara Basin (CA) from 1993 to 2008

Blooms of domoic acid (DA) producing Pseudo-nitzschia, regularly occur off the coast of California. Although it has been hypothesized that these blooms are increasing in frequency, the lack of historical records limits our understanding of potential causal mechanisms. In this study, an 15-year time-series (1993–2008) of sediment trap samples collected from the Santa Barbara Basin (SBB) at 540 m were analyzed for Pseudo-nitzschia (n = 196, microscopy and SEM) and DA (n = 206, LC–MS/MS) concentrations and fluxes. Results suggest that there was an abrupt shift towards greater frequency and higher magnitude Pseudo-nitzschia blooms and toxic DA flux events in the SBB after the year 2000. SEM analysis of sediment trap material indicates that these events were mainly blooms of P. australis, with cell fluxes increasing by an order of magnitude from a maximum of 4.5 × 10⁶ cells m⁻² d⁻¹ pre-2000, to as high as 3.2 × 10⁸ cells m⁻² d⁻¹ thereafter. Similarly, sediment trap DA fluxes increased by an average of 13.4 μg m⁻² d⁻¹, with only one large event (>5 μg m⁻² d⁻¹) from 1993 to 1999 versus 16 large DA events from 2000 to 2008. While the causes of this abrupt shift remain ambiguous, we suggest that this shift may be related to natural climate variability associated with a change in phase of the North Pacific Gyre Oscillation (NPGO) and its potential influence on the composition and magnitude of waters that are upwelled into the SBB.     

Bounds on the total particle number for species governed by reaction-diffusion equations in the infinite spatial domain

Differential inequality methods are developed for establishing upper and lower bounds on the total particle numberN(t)=∫θ(x,t) d³ x associated with solutions to nonlinear reaction-diffusion equations of the form ∂θ/∂t=D∇²θ+fθ-gθ ⁿ⁺¹ , whereD(>0),n(>0),f andg are constant parameters. If finite in a neighborhood oft=0,N(t) is bounded below for allt≥0 by a certain derived function oft for equations withg≥0. An upper bound onN(t) is obtained for equations withn=1,f<0 andg<0. These results provide general preservation and extinction criteria for the total particle number.     

Synthesis and characterization of mixed-ligand ruthenium(III) complexes with oxalate and acetylacetonate ions

Two mononuclear mixed-ligand ruthenium(III) complexes with oxalate dianion (ox²⁻) and acetylacetonate ion (2,4-pentanedionate, acac⁻), K₂[Ru(ox)₂(acac)] (1) and K[Ru(ox)(acac)₂] (2), were prepared as a candidate for a building block. In fact, reaction of complex 2 with manganese(II) sulfate gave a heterometallic tetranuclear complex, TBA[Mn^II{(μ-ox)Ru^III(acac)₂}₃] (5) in the presence of tetrabutylammonium (TBA) bromide. The ¹H NMR, UV-Vis, selected IR and FAB mass spectral data of these complexes are presented. Both mixed-ligand ruthenium(III) complexes gave a Nernstian one-electron reduction step in 0.1 mol dm⁻³ Na₂SO₄ aqueous solution on a mercury electrode at 25 °C. Comparison of observed reversible half-wave potentials with calculated values for a series of [Ru(ox)_n(acac)_3 − n]ⁿ⁻ (n=0-3) complexes by using Lever’s ligand electrochemical parameters is presented.     

A new family of Cayley graph interconnection networks based on wreath product and its topological properties

This paper introduces a new type of Cayley graphs for building large-scale interconnection networks, namely WG_n^2m\mathit{WG}_{n}^{2m}, whose vertex degree is m+3 when n≥3 and is m+2 when n=2. A routing algorithm for the proposed graph is also presented, and the upper bound of the diameter is deduced as ⌊5n/2⌋. Moreover, the embedding properties and maximal fault tolerance are also analyzed. Finally, we compare the proposed networks with some other similar network topologies. It is found that WG_n^2m\mathit{WG}_{n}^{2m} is superior to other interconnection networks because it helps to construct large-scale networks with lower cost.     

Sprouty1 and Sprouty2 limit both the size of the otic placode and hindbrain Wnt8a by antagonizing FGF signaling

Multiple signaling molecules, including Fibroblast Growth Factor (FGF) and Wnt, induce two patches of ectoderm on either side of the hindbrain to form the progenitor cell population for the inner ear, or otic placode. Here we report that in Spry1, Spry2 compound mutant embryos (Spry1^−/−; Spry2^−/− embryos), the otic placode is increased in size. We demonstrate that the otic placode is larger due to the recruitment of cells, normally destined to become cranial epidermis, into the otic domain. The enlargement of the otic placode observed in Spry1^−/−; Spry2^−/− embryos is preceded by an expansion of a Wnt8a expression domain in the adjacent hindbrain. We demonstrate that both the enlargement of the otic placode and the expansion of the Wnt8a expression domain can be rescued in Spry1^−/−; Spry2^−/− embryos by reducing the gene dosage of Fgf10. Our results define a FGF-responsive window during which cells can be continually recruited into the otic domain and uncover SPRY regulation of the size of a putative Wnt inductive center.     

Radiative and non-radiative charge recombination pathways in Photosystem II studied by thermoluminescence and chlorophyll fluorescence in the cyanobacterium Synechocystis 6803

The mechanism of charge recombination was studied in Photosystem II by using flash induced chlorophyll fluorescence and thermoluminescence measurements. The experiments were performed in intact cells of the cyanobacterium Synechocystis 6803 in which the redox properties of the primary pheophytin electron acceptor, Phe, the primary electron donor, P₆₈₀, and the first quinone electron acceptor, Q_A, were modified. In the D1Gln130Glu or D1His198Ala mutants, which shift the free energy of the primary radical pair to more positive values, charge recombination from the S₂Q_A⁻ and S₂Q_B⁻ states was accelerated relative to the wild type as shown by the faster decay of chlorophyll fluorescence yield, and the downshifted peak temperature of the thermoluminescence Q and B bands. The opposite effect, i.e. strong stabilization of charge recombination from both the S₂Q_A⁻ and S₂Q_B⁻ states was observed in the D1Gln130Leu or D1His198Lys mutants, which shift the free energy level of the primary radical pair to more negative values, as shown by the retarded decay of flash induced chlorophyll fluorescence and upshifted thermoluminescence peak temperatures. Importantly, these mutations caused a drastic change in the intensity of thermoluminescence, manifested by 8- and 22-fold increase in the D1Gln130Leu and D1His198Lys mutants, respectively, as well as by a 4- and 2.5-fold decrease in the D1Gln130Glu and D1His198Ala mutants, relative to the wild type, respectively. In the presence of the electron transport inhibitor bromoxynil, which decreases the redox potential of Q_A/Q_A⁻ relative to that observed in the presence of DCMU, charge recombination from the S₂Q_A⁻ state was accelerated in the wild type and all mutant strains. Our data confirm that in PSII the dominant pathway of charge recombination goes through the P₆₈₀⁺Phe⁻ radical pair. This indirect recombination is branched into radiative and non-radiative pathways, which proceed via repopulation of P₆₈₀^* from ¹[P₆₈₀⁺Ph⁻] and direct recombination of the ³[P₆₈₀⁺Ph⁻] and ¹[P₆₈₀⁺Ph⁻] radical states, respectively. An additional non-radiative pathway involves direct recombination of P₆₈₀⁺Q_A⁻. The yield of these charge recombination pathways is affected by the free energy gaps between the Photosystem II electron transfer components in a complex way: Increase of ΔG(P₆₈₀^* ↔ P₆₈₀⁺Phe⁻) decreases the yield of the indirect radiative pathway (in the 22-0.2% range). On the other hand, increase of ΔG(P₆₈₀⁺Phe⁻ ↔ P₆₈₀⁺Q_A⁻) increases the yield of the direct pathway (in the 2-50% range) and decreases the yield of the indirect non-radiative pathway (in the 97-37% range).     

Simulation of ‘hitch-hiking’ genealogies

An ancestral influence graph is derived, an analogue of the coalescent and a composite of Griffiths' (1991) two-locus ancestral graph and Krone and Neuhauser's (1997) ancestral selection graph. This generalizes their use of branching-coalescing random graphs so as to incorporate both selection and recombination into gene genealogies. Qualitative understanding of a ‘hitch-hiking’ effect on genealogies is pursued via diagrammatic representation of the genealogical process in a two-locus, two-allele haploid model. Extending the simulation technique of Griffiths and Tavaré (1996), computational estimation of expected times to the most recent common ancestor of samples of n genes under recombination and selection in two-locus, two-allele haploid and diploid models are presented. Such times are conditional on sample configuration. Monte Carlo simulations show that ‘hitch-hiking’ is a subtle effect that alters the conditional expected depth of the genealogy at the linked neutral locus depending on a mutation-selection-recombination balance. Received: 21 July 2000 / Published online: 5 December 2000     

Novel six-coordinate oxorhenium(V) ‘3+2’ mixed-ligand complexes carrying the SNO/SN donor atom set

Ligand exchange reactions of oxorhenium(V) precursors with bidentate SN and tridentate Schiff bases derived from the condensation of ketones or aldehydes with dithiocarbazic acid methyl ester (H₂NNHC(S)SCH₃) produce novel ‘3+2’ mixed-ligand complexes carrying the SNO/SN donor atom set. Thus, reactions of either [NBu₄][ReOCl₄] or Na[ReO(Gluconate)₂] with SNO ligands (H₂Lⁿ) or a mixture of bidentate SN (HL^m) and tridentate SNO (H₂Lⁿ) in methanol solutions lead, respectively, to the six-coordinated mixed ligand oxorhenium(V) compounds of types [ReO(Lⁿ)(HLⁿ)] and [ReO(Lⁿ)(L^m)], combining one tridentate dianionic S⁻NO⁻ donor Schiff base (L) and one bidentate anionic S⁻N donor ligand (HL). Coordination geometry around rhenium is distorted octahedral with the two SN donor atom sets of each ligand defining the equatorial plane, while apical positions are occupied by the oxo group and the oxygen atom of the tridentate SNO ligand (L), as shown by single-crystal X-ray diffraction structure of [ReO(L¹)(HL¹)] 1.     

Hydrogencarbonate is not a tightly bound constituent of the water-oxidizing complex in photosystem II

Since the end of the 1950s hydrogencarbonate (‘bicarbonate’) is discussed as a possible cofactor of photosynthetic water-splitting, and in a recent X-ray crystallography model of photosystem II (PSII) it was displayed as a ligand of the Mn₄O_xCa cluster. Employing membrane-inlet mass spectrometry (MIMS) and isotope labelling we confirm the release of less than one (≈ 0.3) HCO₃⁻ per PSII upon addition of formate. The same amount of HCO₃⁻ release is observed upon formate addition to Mn-depleted PSII samples. This suggests that formate does not replace HCO₃⁻ from the donor side, but only from the non-heme iron at the acceptor side of PSII. The absence of a firmly bound HCO₃⁻ is corroborated by showing that a reductive destruction of the Mn₄O_xCa cluster inside the MIMS cell by NH₂OH addition does not lead to any CO₂/HCO₃⁻ release. We note that even after an essentially complete HCO₃⁻/CO₂ removal from the sample medium by extensive degassing in the MIMS cell the PSII samples retain ≥ 75% of their initial flash-induced O₂-evolving capacity. We therefore conclude that HCO₃⁻ has only ‘indirect’ effects on water-splitting in PSII, possibly by being part of a proton relay network and/or by participating in assembly and stabilization of the water-oxidizing complex.