The PsbQ′ protein affects the redox potential of the Q<Subscript>A</Subscript> in photosystem II

Red alga contains four extrinsic proteins in photosystem II (PSII), which are PsbO, PsbV, PsbU, and PsbQ′. Except for the PsbQ′, the composition is the same in cyanobacterial PSII. Reconstitution analysis of cyanobacterial PSII has shown that oxygen-evolving activity does not depend on the presence of PsbQ′. Recently, the structure of red algal PSII was elucidated. However, the role of PsbQ′ remains unknown. In this study, the function of the acceptor side of PSII was analyzed in PsbQ′-reconstituted PSII by redox titration of Q_A and thermoluminescence. The redox potential of Q_A was positively shifted when PsbQ′ was attached to the PSII. The positive shift of Q_A is thought to cause a decrease in the amount of triplet chlorophyll in PSII. On the basis of these results, we propose that PsbQ′ has a photoprotective function when irradiated with strong light.     

The Skull of <Emphasis Type="Italic">Epidolops ameghinoi</Emphasis> from the Early Eocene Itaboraí Fauna,Southeastern Brazil,and the Affinities of the Extinct Marsupialiform Order Polydolopimorphia

The skull of the polydolopimorphian marsupialiform Epidolops ameghinoi is described in detail for the first time, based on a single well-preserved cranium and associated left and right dentaries plus additional craniodental fragments, all from the early Eocene (53–50 million year old) Itaboraí fauna in southeastern Brazil. Notable craniodental features of E. ameghinoi include absence of a masseteric process, very small maxillopalatine fenestrae, a prominent pterygoid fossa enclosed laterally by a prominent ectopterygoid crest, an absent or tiny transverse canal foramen, a simple, planar glenoid fossa, and a postglenoid foramen that is immediately posterior to the postglenoid process. Most strikingly, the floor of the hypotympanic sinus was apparently unossified, a feature found in several stem marsupials but absent in all known crown marsupials. “Type II” marsupialiform petrosals previously described from Itaboraí plausibly belong to E. ameghinoi; in published phylogenetic analyses, these petrosals fell outside (crown-clade) Marsupialia. “IMG VII” tarsals previously referred to E. ameghinoi do not share obvious synapomorphies with any crown marsupial clade, nor do they resemble those of the only other putative polydolopimorphians represented by tarsal remains, namely the argyrolagids. Most studies have placed Polydolopimorphia within Marsupialia, related to either Paucituberculata, or to Microbiotheria and Diprotodontia. However, diprotodonty almost certainly evolved independently in polydolopimorphians, paucituberculatans and diprotodontians, and Epidolops does not share obvious synapomorphies with any marsupial order. Epidolops is dentally specialized, but several morphological features appear to be more plesiomorphic than any crown marsupial. It seems likely Epidolops that falls outside Marsupialia, as do morphologically similar forms such as Bonapartherium and polydolopids. Argyrolagids differ markedly in their known morphology from Epidolops but share some potential apomorphies with paucituberculatans. It is proposed that Polydolopimorphia as currently recognised is polyphyletic, and that argyrolagids (and possibly other taxa currently included in Argyrolagoidea, such as groeberiids and patagoniids) are members of Paucituberculata. This hypothesis is supported by Bayesian non-clock phylogenetic analyses of a total evidence matrix comprising DNA sequence data from five nuclear protein-coding genes, indels, retroposon insertions, and morphological characters: Epidolops falls outside Marsupialia, whereas argyrolagids form a clade with the paucituberculatans Caenolestes and Palaeothentes, regardless of whether the Type II petrosals and IMG VII tarsals are used to score characters for Epidolops or not. There is no clear evidence for the presence of crown marsupials at Itaboraí, and it is possible that the origin and early evolution of Marsupialia was restricted to the “Austral Kingdom” (southern South America, Antarctica, and Australia).     

The First Bite— Profiling the Predatosome in the Bacterial Pathogen Bdellovibrio

Bdellovibrio bacteriovorus is a Gram-negative bacterium that is a pathogen of other Gram-negative bacteria, including many bacteria which are pathogens of humans, animals and plants. As such Bdellovibrio has potential as a biocontrol agent, or living antibiotic. B. bacteriovorus HD100 has a large genome and it is not yet known which of it encodes the molecular machinery and genetic control of predatory processes. We have tried to fill this knowledge-gap using mixtures of predator and prey mRNAs to monitor changes in Bdellovibrio gene expression at a timepoint of early-stage prey infection and prey killing in comparison to control cultures of predator and prey alone and also in comparison to Bdellovibrio growing axenically (in a prey-or host independent “HI” manner) on artificial media containing peptone and tryptone. From this we have highlighted genes of the early predatosome with predicted roles in prey killing and digestion and have gained insights into possible regulatory mechanisms as Bdellovibrio enter and establish within the prey bdelloplast. Approximately seven percent of all Bdellovibrio genes were significantly up-regulated at 30 minutes of infection- but not in HI growth- implicating the role of these genes in prey digestion. Five percent were down-regulated significantly, implicating their role in free-swimming, attack-phase physiology. This study gives the first post- genomic insight into the predatory process and reveals some of the important genes that Bdellovibrio expresses inside the prey bacterium during the initial attack.     

The Role of the ��DELSEED-loop of ATP Synthase

ATP synthase uses a unique rotational mechanism to convert chemical energy into mechanical energy and back into chemical energy. The helix-turn-helix motif, termed “DELSEED-loop,” in the C-terminal domain of the β subunit was suggested to be involved in coupling between catalysis and rotation. Here, the role of the DELSEED-loop was investigated by functional analysis of mutants of Bacillus PS3 ATP synthase that had 3–7 amino acids within the loop deleted. All mutants were able to catalyze ATP hydrolysis, some at rates several times higher than the wild-type enzyme. In most cases ATP hydrolysis in membrane vesicles generated a transmembrane proton gradient, indicating that hydrolysis occurred via the normal rotational mechanism. Except for two mutants that showed low activity and low abundance in the membrane preparations, the deletion mutants were able to catalyze ATP synthesis. In general, the mutants seemed less well coupled than the wild-type enzyme, to a varying degree. Arrhenius analysis demonstrated that in the mutants fewer bonds had to be rearranged during the rate-limiting catalytic step; the extent of this effect was dependent on the size of the deletion. The results support the idea of a significant involvement of the DELSEED-loop in mechanochemical coupling in ATP synthase. In addition, for two deletion mutants it was possible to prepare an α₃β₃γ subcomplex and measure nucleotide binding to the catalytic sites. Interestingly, both mutants showed a severely reduced affinity for MgATP at the high affinity site.F₁F₀-ATP synthase catalyzes the final step of oxidative phosphorylation and photophosphorylation, the synthesis of ATP from ADP and inorganic phosphate. F₁F₀-ATP synthase consists of the membrane-embedded F₀ subcomplex, with, in most bacteria, a subunit composition of ab₂c₁₀, and the peripheral F₁ subcomplex, with a subunit composition of α₃β₃γδε. The energy necessary for ATP synthesis is derived from an electrochemical transmembrane proton (or, in some organisms, a sodium ion) gradient. Proton flow down the gradient through F₀ is coupled to ATP synthesis on F₁ by a unique rotary mechanism. The protons flow through (half) channels at the interface of the a and c subunits, which drives rotation of the ring of c subunits. The c₁₀ ring, together with F₁ subunits γ and ε, forms the rotor. Rotation of γ leads to conformational changes in the catalytic nucleotide binding sites on the β subunits, where ADP and P_i are bound. The conformational changes result in the formation and release of ATP. Thus, ATP synthase converts electrochemical energy, the proton gradient, into mechanical energy in the form of subunit rotation and back into chemical energy as ATP. In bacteria, under certain physiological conditions, the process runs in reverse. ATP is hydrolyzed to generate a transmembrane proton gradient, which the bacterium requires for such functions as nutrient import and locomotion (for reviews, see Refs. 1–6).F₁ (or F₁-ATPase) has three catalytic nucleotide binding sites located on the β subunits at the interface to the adjacent α subunit. The catalytic sites have pronounced differences in their nucleotide binding affinity. During rotational catalysis, the sites switch their affinities in a synchronized manner; the position of γ determines which catalytic site is the high affinity site (K_d1 in the nanomolar range), which site is the medium affinity site (K_d2 ≈ 1 μm), and which site is the low affinity site (K_d3 ≈ 30–100 μm; see Refs. 7 and 8). In the original crystal structure of bovine mitochondrial F₁ (9), one of the three catalytic sites, was filled with the ATP analog AMP-PNP,² a second was filled with ADP (plus azide) (see Ref. 10), and the third site was empty. Hence, the β subunits are referred to as β_TP, β_DP, and β_E. The occupied β subunits, β_TP and β_DP, were in a closed conformation, and the empty β_E subunit was in an open conformation. The main difference between these two conformations is found in the C-terminal domain. Here, the “DELSEED-loop,” a helix-turn-helix structure containing the conserved DELSEED motif, is in an “up” position when the catalytic site on the respective β subunit is filled with nucleotide and in a “down” position when the site is empty (Fig. 1A). When all three catalytic sites are occupied by nucleotide, the previously open β_E subunit assumes an intermediate, half-closed (β_HC) conformation. It cannot close completely because of steric clashes with γ (11).Open in a separate windowFIGURE 1.The βDELSEED-loop. A, interaction of the β_TP and β_E subunits with theγ subunit.β subunits are shown in yellow andγ in blue. The DELSEED-loop (shown in orange, with the DELSEED motif itself in green)of β_TP interacts with the C-terminal helixγ and the short helix that runs nearly perpendicular to the rotation axis. The DELSEED-loop of β_E makes contact with the convex portion of γ, formed mainly by the N-terminal helix. A nucleotide molecule (shown in stick representation) occupies the catalytic site of β_TP, and the subunit is in the closed conformation. The catalytic site on β_E is empty, and the subunit is in the open conformation. This figure is based on Protein Data Bank file 1e79 (32). B, deletions in the βDELSEED-loop. The loop was “mutated” in silico to represent the PS3 ATP synthase. The 3–4-residue segments that are removed in the deletion mutants are color-coded as follows: ³⁸⁰LQDI³⁸³, pink; ³⁸⁴IAIL³⁸⁷, green; ³⁸⁸GMDE³⁹¹, yellow; ³⁹²LSD³⁹⁴, cyan; ³⁹⁵EDKL³⁹⁸, orange; ³⁹⁹VVHR⁴⁰², blue. Residues that are the most involved in contacts with γ are labeled. All figures were generated using the program PyMOL (DeLano Scientific, San Carlos, CA).The DELSEED-loop of each of the three β subunits makes contact with the γ subunit. In some cases, these contacts consist of hydrogen bonds or salt bridges between the negatively charged residues of the DELSEED motif and positively charged residues on γ. The interactions of the DELSEED-loop with γ, its movement during catalysis, the conservation of the DELSEED motif (see 12–14). Thus, the finding that an AALSAAA mutant in the α₃β₃γ complex of ATP synthase from the thermophilic Bacillus PS3, where several hydrogen bonds/salt bridges to γ are removed simultaneously, could drive rotation of γ with the same torque as the wild-type enzyme (14) came as a surprise. On the other hand, it seems possible that it is the bulk of the DELSEED-loop, more so than individual interactions, that drives rotation of γ. According to a model favored by several authors (6, 15, 16) (see also Refs. 17–19), binding of ATP (or, more precisely, MgATP) to the low affinity catalytic site on β_E and the subsequent closure of this site, accompanied by its conversion into the high affinity site, are responsible for driving the large (80–90°) rotation substep during ATP hydrolysis, with the DELSEED-loop acting as a “pushrod.” A recent molecular dynamics (20) study supports this model and implicates mainly the region around several hydrophobic residues upstream of the DELSEED motif (specifically βI386 and βL387)³ as being responsible for making contact with γ during the large rotation substep.

TABLE 1

Conservation of residues in the DELSEED-loop Amino acids found in selected species in the turn region of the DELSEED-loop. Listed are all positions subjected to deletions in the present study. Residue numbers refer to the PS3 enzyme. Consensus annotation: p, polar residue; s, small residue; h, hydrophobic residue; –, negatively charged residue; +, positively charged residue.Open in a separate windowIn the present study, we investigated the function of the DELSEED-loop using an approach less focused on individual residues, by deleting stretches of 3–7 amino acids between positions β380 and β402 of ATP synthase from the thermophilic Bacillus PS3. We analyzed the functional properties of the deletion mutants after expression in Escherichia coli. The mutants showed ATPase activities, which were in some cases surprisingly high, severalfold higher than the activity of the wild-type control. On the other hand, in all cases where ATP synthesis could be measured, the rates where below or equal to those of the wild-type enzyme. In Arrhenius plots, the hydrolysis rates of the mutants were less temperature-dependent than those of wild-type ATP synthase. In those cases where nucleotide binding to the catalytic sites could be tested, the deletion mutants had a much reduced affinity for MgATP at high affinity site 1. The functional role of the DELSEED-loop will be discussed in light of the new information.     

The Fly Agaric,Soma,Father Christmas and the Vikings

1　ThemushroomTheFlyAgaric (Amanitamuscaria (L :Fr.)Hooker)belongstothegenusAmanitaPers .,agroupwhichhasplayedmajorrolesinhumanaffairsthrough outhistory .Itincludesboththemostlydeadlypoi sonousspeciesandpossiblythemostdeliciousspeciesofalloftheediblewildmushrooms.TheFlyAgaricrepresentsthebestknownofallthewildmushroomsandhadthegreatesteffectuponmankindandworldhistory .Itistobeseeninmanychildren′sbooksasthelarge ,brightredmushroomwiththewhitespots,invariablyassociatedwithfairiesandelves.It…     

Here is the evidence,now what is the hypothesis? The complementary roles of inductive and hypothesis‐driven science in the post‐genomic era

It is considered in some quarters that hypothesis-driven methods are the only valuable, reliable or significant means of scientific advance. Data-driven or 'inductive' advances in scientific knowledge are then seen as marginal, irrelevant, insecure or wrong-headed, while the development of technology--which is not of itself 'hypothesis-led' (beyond the recognition that such tools might be of value)--must be seen as equally irrelevant to the hypothetico-deductive scientific agenda. We argue here that data- and technology-driven programmes are not alternatives to hypothesis-led studies in scientific knowledge discovery but are complementary and iterative partners with them. Many fields are data-rich but hypothesis-poor. Here, computational methods of data analysis, which may be automated, provide the means of generating novel hypotheses, especially in the post-genomic era.     

The < folding problem >: can one predict the structure of proteins?

A protein's three-dimensional structure is encoded in its amino acid sequence. The < folding problem > consists in predicting one based on the other. This classic problem of molecular biology has seen important steps forward in recent years. The raw power of today's computers, along with the mobilization of thousands of internauts, have allowed several small proteins to be literally folded up in a computer, through simulations. Moreover, international programs for structural genomics aim to determine the experimental structures of hundreds of proteins in several organisms, and to model the others by homology to known structures. This will lead to a nearly-complete map of the protein structure universe, shedding light on the past evolution and current functions of today's proteins, and suggesting new targets for therapeutic strategies.     

The effect of salinity on the growth,toxin production,and morphology of <Emphasis Type="BoldItalic">Nodularia spumigena</Emphasis> isolated from the Gulf of Gdańsk,southern Baltic Sea

Culture experiments on the toxic Nodularia spumigena strain NSGG-1 isolated from the Gulf of Gdańsk showed a significant effect of salinity on growth and nodularin production. Growth of the NSGG-1 strain, was optimal between 7 and 18 psu, lower at 3 and 24 psu and was significantly inhibited at the extreme salinities of 0 and 35 psu. Nodularin (NOD) content of N. spumigena, estimated by the NOD/Chla ratios, correlated positively with salinity and increased from 0 to 35 psu. The NOD/Chla ratio on day 10 of growth was high, and, reached the maximum at day 30. A sudden increase in salinity from 7 to 18 and 35 psu resulted in plasmolysis of Nodularia cells. Salinity was also observed to have other effects on NSGG-1; the filaments were longest at 7 psu, while an increased number of akinetes were formed at 35 psu. The number of heterocytes was markedly reduced at the extreme salinities. This latter finding might explain why Nodularia blooms do not occur outside a certain salinity range in nitrogen-deficient waters.     

The redox chemistry of the Alzheimer's disease amyloid β peptide

There is a growing body of evidence to support a role for oxidative stress in Alzheimer's disease (AD), with increased levels of lipid peroxidation, DNA and protein oxidation products (HNE, 8-HO-guanidine and protein carbonyls respectively) in AD brains. The brain is a highly oxidative organ consuming 20% of the body's oxygen despite accounting for only 2% of the total body weight. With normal ageing the brain accumulates metals ions such iron (Fe), zinc (Zn) and copper (Cu). Consequently the brain is abundant in antioxidants to control and prevent the detrimental formation of reactive oxygen species (ROS) generated via Fenton chemistry involving redox active metal ion reduction and activation of molecular oxygen. In AD there is an over accumulation of the Amyloid β peptide (Aβ), this is the result of either an elevated generation from amyloid precursor protein (APP) or inefficient clearance of Aβ from the brain. Aβ can efficiently generate reactive oxygen species in the presence of the transition metals copper and iron in vitro. Under oxidative conditions Aβ will form stable dityrosine cross-linked dimers which are generated from free radical attack on the tyrosine residue at position 10. There are elevated levels of urea and SDS resistant stable linked Aβ oligomers as well as dityrosine cross-linked peptides and proteins in AD brain. Since soluble Aβ levels correlate best with the degree of degeneration [C.A. McLean, R.A. Cherny, F.W. Fraser, S.J. Fuller, M.J. Smith, K. Beyreuther, A.I. Bush, C.L. Masters, Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease, Ann. Neurol. 46 (1999) 860-866] we suggest that the toxic Aβ species corresponds to a soluble dityrosine cross-linked oligomer. Current therapeutic strategies using metal chelators such as clioquinol and desferrioxamine have had some success in altering the progression of AD symptoms. Similarly, natural antioxidants curcumin and ginkgo extract have modest but positive effects in slowing AD development. Therefore, drugs that target the oxidative pathways in AD could have genuine therapeutic efficacy.     

α<Subscript>1</Subscript>-Thymosin, α<Subscript>2</Subscript>-interferon,and the LKEKK syntetic peptide inhibit the binding of the B subunit of the cholera toxin to intestinal epithelial cell membranes

The ¹²⁵I-labeled B-subunit of the cholera toxin ([125I]CT-B, specific activity of 98 Ci/mmol) was prepared. This subunit was shown to be bound to the membranes which were isolated from epithelial cells of a mucous tunic of the rat thin intestine with high affinity (K _d = 3.7 nM). The binding of the labeled protein was inhibited by the unlabeled α₂-interferon (IFN-α₂), α₁-thymosin, (TM-α₁), and the LKEKK synthetic peptide corresponding to the 16–20 sequence of TM-α₁ and the 131–135 sequence of human IFN-α₂ (Ki 1.0, 1.5, and 2.0 nM, respectively), whereas the KKEKL unlabeled synthetic peptide did not inhibit the binding (K _i > 100 μМ). The LKEKK peptide and CT-B were shown to dose-dependently increase an activity of the soluble guanylate cyclase (sGC) in the concentration range from 10 to 1000 nM. Thus, the binding of TM- α₁, IFN-α₂, and the LKEKK peptide to the CT-B receptor on a surface of the epithelial cells of the mucous tunic of the rat thin intestine resulted in an increase in the intracellular level of cGMP.     

The G Protein regulators EGL-10 and EAT-16, the G<Subscript>i</Subscript>α GOA-1 and the G<Subscript>q</Subscript>α EGL-30 modulate the response of the <Emphasis Type="Italic">C. elegans</Emphasis>ASH polymodal nociceptive sensory neurons to repellents

Background  

Polymodal, nociceptive sensory neurons are key cellular elements of the way animals sense aversive and painful stimuli. In Caenorhabditis elegans, the polymodal nociceptive ASH sensory neurons detect aversive stimuli and release glutamate to generate avoidance responses. They are thus useful models for the nociceptive neurons of mammals. While several molecules affecting signal generation and transduction in ASH have been identified, less is known about transmission of the signal from ASH to downstream neurons and about the molecules involved in its modulation.