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PubMed1979

[Selective effects of lindane (gamma-1,2,3,4,5,6 hexachlorocyclohexane) on photosynthesis, membrane transport of amino acids and protein synthesis in Anacystis nidulans (author's transl)].

Effects of the chlorinated hydrocarbon insecticide Lindane on membrane transport of two neutrale amino acids in the Cyanobacterium Anacystis nidulans (Synechococcus AN) were measured. In white light the L-Leucine incorporation into the protein fraction was inhibited, increasing with time. After 30 minutes the degree of inhibition was the same as the effect of DCMU (5 X 10(-6) M) on L-Leucine incorporation. 14CO2-fixation was also reduced at this time. At 717 nm, which enables PS I activity allone, no inhibition was observed. The light energy dependent membrane transport itself of L-Leucine in presence of CAM and of the non-metabolisable alpha-AIB in white light and in monochromatic light of 630 nm and of 717 nm were not influenced by Lindane. The different sites of Lindane action are discussed. It is assumed that in 30 minutes chiefly photosynthesis (PS II and CO2-fixation) is affected by Lindane, resulting in a suppression of protein synthesis caused by a depletion of intermediates of CO2-fixation.

Amino Acids↗
PubMed2005

Single gene retrieval from thermally degraded DNA.

To simulate single gene retrieval from ancient DNA, several related factors have been investigated. By monitoring a 889 bp polymerase chain reaction (PCR) product and genomic DNA degradation, we find that heat and oxygen (especially heat) are both crucial factors influencing DNA degradation. The heat influence, mainly represented by temperature and heating time, affects the DNA degradation via DNA depurination followed by cleavage of nearby phosphodiesters. The heating time influence is temperature-dependent. By reactive oxygen species (ROS) scavenging and 1,3-diphenyl-isobenzofuran (DPBF) bleaching experiments the influence of oxygen on DNA thermal degradation was shown to occur via a singlet oxygen pathway. A comparative study of the thermal degradation of cellular DNA and isolated DNA showed that cellular lipids can aggravate DNA thermal degradation. These results confirm the possibility of gene amplification from thermally degraded DNA. They can be used to evaluate the feasibility of the retrieval of single gene from ancient remains.

Air↗
PubMed2005

Monolayers of photosystem II on gold electrodes with enhanced sensor response--effect of porosity and protein layer arrangement.

Mass transport of the bulk of the analyte to the electrode and through the bioactive layer can be significantly improved by use of the nanoelectrode array and defined arrangement of protein film. This phenomenon has been studied by (i) atomic-force microscopy, (ii) electrochemical measurements of PSII activity, and (iii) digital simulations for an oriented monolayer of histidine-tagged photosystem II (PSII) immobilized on nitrilotriacetic acid (NTA)-modified gold electrodes. The output signal of the electrochemical biosensor is controlled by (i) mass transport from the bioactive layer to electrode and (ii) mass transport between the bulk of the analyte and the electrode. Mass transport through the bioactive layer was electrochemically studied for PSII self-assembled on gold screen-printed electrodes. A densely packed monolayer of PSII has a significant shielding effect toward the diffusion of redox mediator duroquinone (DQ). Mass transport to the planar electrode surface was improved by co-immobilization of bovine-serum albumin (BSA) as spacer biomolecule in the monolayer of PSII. Correlation between the electrochemical properties and surface arrangement of the resulting protein films was clearly observable and confirmed the improved mass-transport properties of structured enzyme monolayers. On the basis of this observation, the application of a bottom-up approach for improvement of electrode performance was proposed and digitally simulated for an infinite array of electrodes ranging in diameter from 50 nm to 5 microm. The nanoelectrode array, with the optimum time window selected for measurements, enables enhancement of mass transport between the bulk of the analyte and the macroelectrode by a factor of up to 50 in comparison with "classical" planar electrodes. Use of a time window enables minimization of crosstalk between individual electrodes in the array. The measurements require methods which suppress the double-layer capacity.

Benzoquinones↗
PubMed2006

Microbial diversity in natural environments: focusing on fundamental questions.

Interactions with Gijs Kuenen and other Dutch scientists have led my lab to fundamental insights into the composition, structure and function of a hot spring cyanobacterial mat community that should influence our thinking about all microbial communities. By focusing on the distribution of molecular sequence variants of predominant mat phototrophs, we have discovered that small-scale sequence variation can be ecologically meaningful. By applying novel cultivation approaches, we have been able to obtain genetically relevant community members and thus to test the hypothesis that closely related sequence variants arose via adaptive evolutionary radiation. By applying the analytical tools of organic geochemistry we have gained insight into the metabolisms of major phototrophic members of the mat community as well as interactions between phototrophic guilds. These observations challenge traditional paradigms about prokaryotic species and cause us to consider evolutionary ecology theory as we develop genome-based methods for high-resolution analysis of the species-like fundamental units comprising microbial communities, and for investigating how such units coordinate the physiological activities within guilds of the community.

Autotrophic Processes↗
PubMed2005

The antenna system of photosystem II from Thermosynechococcus elongatus at 3.2 A resolution.

The content and type of cofactors harboured in the Photosystem II core complex (PS IIcc) of the cyanobacterium Thermosynechococcus elongatus has been determined by biochemical and spectroscopic methods. 17 +/- 1 chlorophyll a per pheophytin a and 0.25 beta-carotene per chlorophyll a have been found in re-dissolved crystals of dimeric PS IIcc. The X-ray crystal structure of PS IIcc from Thermosynechococcus elongatus at 3.2 A resolution clearly shows chlorophyll a molecules arranged in two layers close to the cytoplasmic and lumenal sides of the thylakoid membrane. Each of the cytoplasmic layers contains 9 chlorophyll a, whose positions and orientations are related by a local twofold rotation pseudo-C2 axis passing through the non-haem Fe2+. These chlorophyll a are arranged comparably to those in the antenna domains of PsaA and PsaB of cyanobacterial Photosystem I affirming an evolutionary relation. The chlorophyll a in the lumenal layer are less well conserved between Photosystems I and II and even between CP43 and CP47 with 4 chlorophyll a in the former and 7 in the latter.

Chromatography, High Pressure Liquid↗
PubMed2006

Consistent sets of spectrophotometric chlorophyll equations for acetone, methanol and ethanol solvents.

A set of equations for determining chlorophyll a (Chl a) and accessory chlorophylls b, c2, c1 + c2 and the special case of Acaryochloris marina, which uses Chl d as its primary photosynthetic pigment and also has Chl a, have been developed for 90% acetone, methanol and ethanol solvents. These equations for different solvents give chlorophyll assays that are consistent with each other. No algorithms for Chl c compounds (c2, c1 + c2) in the presence of Chl a have previously been published for methanol or ethanol. The limits of detection (and inherent error, +/- 95% confidence limit), for chlorophylls in all organisms tested, was generally less than 0.1 microg/ml. The Chl a and b algorithms for green algae and land plants have very small inherent errors (< 0.01 microg/ml). Chl a and d algorithms for Acaryochloris marina are consistent with each other, giving estimates of Chl d/a ratios which are consistent with previously published estimates using HPLC and a rarely used algorithm originally published for diethyl ether in 1955. The statistical error structure of chlorophyll algorithms is discussed. The relative error of measurements of chlorophylls increases hyperbolically in diluted chlorophyll extracts because the inherent errors of the chlorophyll algorithms are constants independent of the magnitude of absorbance readings. For safety reasons, efficient extraction of chlorophylls and the convenience of being able to use polystyrene cuvettes, the algorithms for ethanol are recommended for routine assays of chlorophylls. The methanol algorithms would be convenient for assays associated with HPLC work.

Acetone↗
PubMed2005

RAP, an integrated program for monitoring bioluminescence and analyzing circadian rhythms in real time.

To process the large number of circadian rhythmic bioluminescence data that we generated with specially developed, high-throughput monitoring apparatuses, we developed an integrated rhythm-analyzing program (RAP) with a user-friendly graphical interface. RAP does all of the following in real time: (i) displays the bioluminescence time course, (ii) records time-series data, (iii) analyzes the data, (iv) displays the analyzed results, (v) statistically evaluates the analyzed results, and (vi) provides printouts. Because RAP can import files, it can analyze not only bioluminescence data but also other data such as those obtained by DNA array experiments and by Northern and Western blot analyses. The program is a powerful tool for large-scale investigations of biological rhythmic phenomena in general.

Arabidopsis↗
PubMed2005

Extinction coefficients and critical solubilisation concentrations of photosystems I and II from Thermosynechococcus elongatus.

The absorption properties of chlorophyll a (Chla) in active core complexes of photosystems I (PSI) and II (PSII) isolated in high purity from the thermophilic cyanobacterium Thermosynechococcus elongatus were correlated with those of extracts in 80% acetone to determine effective extinction coefficients of protein-bound Chla and molar extinction coefficients of core complexes and reaction centers (RC). These coefficients allow a quick determination of Chla and protein concentrations from steady-state absorption spectra of intact samples without the need for pigment extraction and protein destruction. In the visible range, epsilon(680)(p) = 57 mM(-1) cm(-1) for trimeric PSI (PSIt) and epsilon(674)(p) = 70 mM(-1) cm(-1) for dimeric (PSIId) and monomeric (PSIIm) PSII (error +/-6%; superscript "p" refers to Chla bound to intact protein, subscripts are the peak maxima in nm). The integral extinction coefficient phi(p) = 2.8 nm microM(-1) cm(-1) for the wavelength interval between 550 and 800 nm and the extinction coefficient epsilon(B)(p) = 14 mM(-1) cm(-1) for the smaller absorption maximum (B = 632 nm for PSI and 627 nm for PSII) were found to be essentially the same for both types of PS. The coefficients of PSIt are shown to remain unaltered when 65% (v/v) of the buffer is replaced with glycerol. Molar extinction coefficients of core complexes were determined using Chla/RC ratios of 96+/-1 for PSI and 35+/-2 for PSII based on X-ray data. In addition, the critical solubilisation concentration of n-dodecyl-beta-d-maltoside (betaDM), necessary to keep the core complexes in solution, was determined by turbidimetric titrations. It was found that at least approximately 500 betaDM molecules per PSIt ( approximately 2 betaDM per Chla) and 190 betaDM molecules per PSIIm ( approximately 5 betaDM per Chla, also for PSIId) in excess of the critical micelle concentration of 0.16 +/- 0.03 mM are necessary for a complete solubilisation of the core complexes.

Photosystem I Protein Complex↗
PubMed2005

Extinction coefficients of cytochromes b559 and c550 of Thermosynechococcus elongatus and Cyt b559/PS II stoichiometry of higher plants.

"Reduced minus oxidized" difference extinction coefficients Deltavarepsilon in the alpha-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1+/-1.0 mM(-1) cm(-1) and 27.0+/-1.0 mM(-1) cm(-1) were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from -250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E(m) values at pH 6.5 of 244+/-11 mV and -94+/-21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the alpha-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q(B) site and the microenvironment of the heme group of Cyt b559 are discussed.

Anaerobiosis↗
PubMed2005

Circadian clocks: translation.

One of the big questions in biological rhythms research is how a stable and precise circa-24 hour oscillation is generated on the molecular level. While increasing complexity seemed to be the key, a recent report suggests that circa-24 hour rhythms can be generated by just four molecules incubated in a test tube.

Animals↗
PubMed2005

The occurrence of rapidly reversible non-photochemical quenching of chlorophyll a fluorescence in cyanobacteria.

Cyanobacteria have previously been considered to differ fundamentally from plants and algae in their regulation of light harvesting. We show here that in fact the ecologically important marine prochlorophyte, Prochlorococcus, is capable of forming rapidly reversible non-photochemical quenching of chlorophyll a fluorescence (NPQf or qE) as are freshwater cyanobacteria when they employ the iron stress induced chlorophyll-based antenna, IsiA. For Prochlorococcus, the capacity for NPQf is greater in high light-adapted strains, except during iron starvation which allows for increased quenching in low light-adapted strains. NPQf formation in freshwater cyanobacteria is accompanied by deep Fo quenching which increases with prolonged iron starvation.

Bacterial Proteins↗
PubMed2006

The cyanobacterial principal sigma factor region 1.1 is involved in DNA-binding in the free form and in transcription activity as holoenzyme.

Cyanobacterial principal sigma factor, sigma(A), includes a specifically conserved cluster of basic amino acids in the amino-terminal extension called region 1.1. We found that the sigma(A) in a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 binds DNA in the absence of the core RNA polymerase and that sigma(A) lacking region 1.1 is not able to bind DNA. This indicates that, in the cyanobacterium, region 1.1 participates in DNA-binding, rather than inhibiting the interaction between free sigma and DNA, as found in other principal sigma factors of eubacteria. The results of in vitro transcription assays with the reconstituted RNA polymerase showed that region 1.1 reduces transcription activity from the cpc promoter.

Amino Acid Sequence↗
PubMed2006

Fate determination of the flavin photoreceptions in the cyanobacterial blue light receptor TePixD (Tll0078).

PixD (Tll0078, Slr1694) is a BLUF (sensor of blue light using FAD)-type blue light receptor protein of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 and the mesophilic cyanobacterium Synechocystis sp. PCC 6803. BLUF protein is known to show light-induced approximately 10 nm red shift of flavin absorption that is coupled with strengthening of the hydrogen bond between the O(4) of the isoalloxazine ring and a certain amino acid residue. According to the 3D structure of TePixD we determined, O(4) of the ring is linked to Gln50 and Asn32. A survey of flavin-interacting residues by site-directed mutagenesis showed that Gln50 but not Asn32 is essential for the normal red-shifting photoreaction. Here, we further studied the role of Gln50 and its close neighbor Tyr8. All the mutated proteins of Gln50 and Tyr8 (Q50A, Q50N, Y8A and Y8F) lost the normal red-shifting photoreaction. Y8A, Y8F and Q50N, instead, showed a light-induced flavin triplet state and a low yield of subsequent flavin reduction that is analogous to the photocycle of the LOV (light-oxygen-voltage-sensing) domain of phototropins, while Q50A did not. Fourier-transform infrared (FT-IR) analysis of N32A showed that O(4) of the ring is hydrogen-bonded to Asn32 both in the light and dark. These results, together with the 3D structure, indicate that the hydrogen bond network of Tyr8-Gln50-O(4)/N(5) (flavin) is critical for the light reaction of the BLUF domain. Based on the structural and functional similarities of the BLUF and the LOV domain of phototropins, we propose that the interaction between apoprotein and N(5) of flavin determines the photoreaction of the flavin-binding sensors.

Amino Acid Substitution↗
PubMed2006

Antioxidant properties of recombinant allophycocyanin expressed in Escherichia coli.

Allophycocyanin (APC) is the main core component of phycobilisome found in blue-green algae. The apo-allophycocyanin and its subunits were expressed in Escherichia coli and their antioxidant properties were evaluated using deoxyribose assay. The result showed that both recombinant allophycocyanin fused with maltose binding protein (MBP) tag and 6 x His-tag and their alpha or beta subunits can scavenge hydroxyl radicals successfully, and the separated alpha or beta subunits had a higher inhibition effect on hydroxyl radicals than that when they combined together. The scavenging effects increased with the increasing concentration. These results clearly suggested that apo-allophycocyanin is involved in the antioxidant and radical scavenging activity of phycocyanin, and the antioxidant activity may be partially responsible to the anti-tumor effect of the recombinant allophycocyanin.

Antineoplastic Agents↗