Oxidation states of the manganese cluster during the flash-induced S-state cycle of the photosynthetic oxygen-evolving complex.

The Mn K-edge x-ray absorption spectra for the pure S states of the tetranuclear Mn cluster of the oxygen-evolving complex of photosystem II during flash-induced S-state cycling have been determined. The relative S-state populations in samples given 0, 1, 2, 3, 4, or 5 flashes were determined from fitting the flash-induced electron paramagnetic resonance (EPR) multiline signal oscillation pattern to the Kok model. The edge spectra of samples given 0, 1, 2, or 3 flashes were combined with EPR information to calculate the pure S-state edge spectra. The edge positions (defined as the zero-crossing of the second derivatives) are 6550.1, 6551.7, 6553.5, and 6553.8 eV for S0, S1, S2, and S3, respectively. In addition to the shift in edge position, the S0--> S1 and S1--> S2 transitions are accompanied by characteristic changes in the shape of the edge, both indicative of Mn oxidation. The edge position shifts very little (0.3 eV) for the S2--> S3 transition, and the edge shape shows only subtle changes. We conclude that probably no direct Mn oxidation is involved in this transition. The proposed Mn oxidation state assignments are as follows: S0 (II, III, IV, IV) or (III, III, III, IV), S1 (III, III, IV, IV), S2 (III, IV, IV, IV), S3 (III, IV, IV, IV).

Genes encoding the same three subunits of respiratory complex II are present in the mitochondrial DNA of two phylogenetically distant eukaryotes.

Although mitochondrial DNA is known to encode a limited number (<20) of the polypeptide components of respiratory complexes I, III, IV, and V, genes for components of complex II [succinate dehydrogenase (ubiquinone); succinate:ubiquinone oxidoreductase, EC 1.3.5.1] are conspicuously lacking in mitochondrial genomes so far characterized. Here we show that the same three subunits of complex II are encoded in the mitochondrial DNA of two phylogenetically distant eukaryotes, Porphyra purpurea (a photosynthetic red alga) and Reclinomonas americana (a heterotrophic zooflagellate). These complex II genes, sdh2, sdh3, and sdh4, are homologs, respectively, of Escherichia coli sdhB, sdhC, and sdhD. In E. coli, sdhB encodes the iron-sulfur subunit of succinate dehydrogenase (SDH), whereas sdhC and sdhD specify, respectively, apocytochrome b558 and a hydrophobic 13-kDa polypeptide, which together anchor SDH to the inner mitochondrial membrane. Amino acid sequence similarities indicate that sdh2, sdh3, and sdh4 were originally encoded in the protomitochondrial genome and have subsequently been transferred to the nuclear genome in most eukaryotes. The data presented here are consistent with the view that mitochondria constitute a monophyletic lineage.

Single neuron control over a complex motor program.

While there are many instances of single neurons that can drive rhythmic stimulus-elicited motor programs, such neurons have seldom been found to be necessary for motor program function. In the isolated central nervous system of the marine mollusc Tritonia diomedea, brief stimulation (1 sec) of a peripheral nerve activates an interneuronal central pattern generator that produces the long-lasting (approximately 30-60 sec) motor program underlying the animal's rhythmic escape swim. Here, we identify a single interneuron, DRI (for dorsal ramp interneuron), that (i) conveys the sensory information from this stimulus to the swim central pattern generator, (ii) elicits the swim motor program when driven with intracellular stimulation, and (iii) blocks the depolarizing "ramp" input to the central pattern generator, and consequently the motor program itself, when hyperpolarized during the nerve stimulus. Because most of the sensory information appears to be funneled through this one neuron as it enters the pattern generator, DRI presents a striking example of single neuron control over a complex motor circuit.

Two genes required for the binding of an essential Saccharomyces cerevisiae kinetochore complex to DNA.

Kinetochores are DNA-protein structures that assemble on centromeric DNA and attach chromosomes to spindle microtubules. Because of their simplicity, the 125-bp centromeres of Saccharomyces cerevisiae are particularly amenable to molecular analysis. Budding yeast centromeres contain three sequence elements of which centromere DNA sequence element III (CDEIII) appears to be particularly important. cis-acting mutations in CDEIII and trans-acting mutations in genes encoding subunits of the CDEIII-binding complex (CBF3) prevent correct chromosome transmission. Using temperature-sensitive mutations in CBF3 subunits, we show a strong correlation between DNA-binding activity measured in vitro and kinetochore activity in vivo. We extend previous findings by Goh and Kilmartin [Goh, P.-Y. & Kilmartin, J.V. (1993) J. Cell Biol. 121, 503-512] to argue that DNA-bound CBF3 may be involved in the operation of a mitotic checkpoint but that functional CBF3 is not required for the assembly of a bipolar spindle.

Sequence analysis of the cis-regulatory regions of the bithorax complex of Drosophila.

The bithorax complex (BX-C) of Drosophila, one of two complexes that act as master regulators of the body plan of the fly, has now been entirely sequenced and comprises approximately 315,000 bp, only 1.4% of which codes for protein. Analysis of this sequence reveals significantly overrepresented DNA motifs of unknown, as well as known, functions in the non-protein-coding portion of the sequence. The following types of motifs in that portion are analyzed: (i) concatamers of mono-, di-, and trinucleotides; (ii) tightly clustered hexanucleotides (spaced < or = 5 bases apart); (iii) direct and reverse repeats longer than 20 bp; and (iv) a number of motifs known from biochemical studies to play a role in the regulation of the BX-C. The hexanucleotide AGATAC is remarkably overrepresented and is surmised to play a role in chromosome pairing. The positions of sites of highly overrepresented motifs are plotted for those that occur at more than five sites in the sequence, when < 0.5 case is expected. Expected values are based on a third-order Markov chain, which is the optimal order for representing the BXCALL sequence.

(Table 1) Occurrences of Epimeria georgiana species complex (Amphipoda) in the Weddel and Scotia Sea

DNA barcoding revealed four well-supported clades among amphipod specimens that keyed out to Epimeria georgiana Schellenberg, 1931, three clades with specimens from the southern Scotia Arc and one clade with specimens from the Weddell Sea. Detailed morphological investigations of sequenced specimens were conducted, through light and scanning electron microscopy. High magnification (500-2,000 fold) revealed features such as comb-scales on the first antenna and trich bearing pits on the fourth coxal plate to be similar for all specimens in the four clades. Consistent microstructure character differences in the Weddell Sea specimens combined with high genetic distances (COI divergence>20%) allowed the description of Epimeria angelikae, a species new to science. Specimens of E. georgiana in the other three COI clades from the Scotia Arc were morphologically indistinguishable. Representative specimens of clade A are also illustrated in detail. Our results on the high genetic divergences in epimeriid amphipods support the theory of the southern Scotia Arc being a centre of Antarctic diversification.

Effector ExoU from the type III secretion system is an important modulator of gene expression in lung epithelial cells in response to Pseudomonas aeruginosa infection

Pseudomonas aeruginosa is an important pathogen in immunocompromised patients and secretes a diverse set of virulence factors that aid colonization and influence host cell defenses. An important early step in the establishment of infection is the production of type III-secreted effectors translocated into host cells by the bacteria. We used cDNA microarrays to compare the transcriptomic response of lung epithelial cells to P. aeruginosa mutants defective in type IV pili, the type III secretion apparatus, or in the production of specific type III-secreted effectors. Of the 18,000 cDNA clones analyzed, 55 were induced or repressed after 4 It of infection and could be classified into four different expression patterns. These include (i) host genes that are induced or repressed in a type III secretion-independent manner (32 clones), (ii) host genes induced specifically by ExoU (20 clones), and (iii) host genes induced in an ExoU-independent but type III secretion dependent manner (3 clones). In particular, ExoU was essential for the expression of immediate-early response genes, including the transcription factor c-Fos. ExoU-dependent gene expression was mediated in part by early and transient activation of the AN transcription factor complex. In conclusion, the present study provides a detailed insight into the response of epithelial cells to infection and indicates the significant role played by the type III virulence mechanism in the initial host response.

Mitochondrial control region diversity of the houbara bustard Chlamydotis undulata complex and genetic structure along the Atlantic seaboard of North Africa

The houbara bustard, Chlamydotis undulata, is a declining cryptic desert bird whose range extends from North Africa to Central Asia. Three subspecies are currently recognized by geographical distribution and morphology: C.u.fuertaventurae, C.u.undulata and C.u.macqueenii. We have sequenced 854 bp of mitochondrial control region from 73 birds to describe their population genetic structure with a particular sampling focus on the connectivity between C.u.fuertaventurae and C.u.undulata along the Atlantic seaboard of North Africa. Nucleotide and haplotypic diversity varied among the subspecies being highest in C.u.undulata, lowest in C.u.fuertaventurae and intermediate in C.u.macqueenii. C.u.fuertaventurae and C.u.undulata are paraphyletic and an average nucleotide divergence of 2.08% splits the later from C.u.macqueenii. We estimate that C.u.fuertaventurae and C.u.undulata split from C.u.macqueenii approximately 430 000 years ago. C.u.fuertaventurae and C.u.undulata are weakly differentiated (F-ST = 0.27, N-m = 1.3), indicative of a recent shared history. Archaeological evidence indicates that houbara bustards have been present on the Canary Islands for 130-170 000 years. However, our genetic data point to a more recent separation of C.u.fuertaventurae and C.u.undulata at around 20-25 000 years. Concordant archaeological, climatic opportunities for colonization and genetic data point to a scenario of: (i) initial colonization of the Canary Islands about 130 000 years ago; (ii) a period of secondary contact 19-30 000 years ago homogenizing any pre-existing genetic structure followed by; (iii) a period of relative isolation that persists today.

The preparation and characterization of seven-coordinate tin(IV) complexes of the 2,6-diacetylpyridine Schiff bases of S-alkyl/aryl-dithiocarbazates and the X-ray crystal structure of the [Sn(dapsme)I-2] complex (dapsme=doubly deprotonated form of the 2,6

New tin(IV) complexes of empirical formula, Sn(SNNNS)I-2 (SNNNS = anionic form of the 2,6-diacetylpyridine Schiff bases of S-methyl- or S-benzyldithiocarbazate) have been prepared and characterized by a variety of physico-chemical techniques. The structure of Sn(dapsme)I-2 has been determined by single crystal X-ray crystallographic structural analysis. The complex has a seven-coordinate distorted pentagonal-bipyramidal geometry with the Schiff base coordinated to the tin(IV) ion as a dinegatively charged pentadentate chelating agent via the pyridine nitrogen atom, the two azomethine nitrogen atoms and the two thiolate sulfur atoms. The ligand occupies the equatorial plane and the iodo ligands are coordinated to the tin(IV) ion at axial positions. The distortion from an ideal pentagonal bipyramidal geometry is attributed to the restricted bite size of the pentadentate ligands. (C) 2004 Elsevier Ltd. All rights reserved.

Inhibition of protein interactions with the beta(2) sliding clamp of Escherichia coli DNA polymerase III by peptides from beta(2)-binding proteins

The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.

Pressure and temperature effects on metal-to-metal charge transfer in cyano-bridged Co-III-Fe-II complexes

The effects of pressure and temperature on the energy (E-op) of the metal-to-metal charge transfer (MMCT, Fe-II --> Co-III) transition of the cyano-bridged complexes trans - [(LCoNCFe)-Co-14(CN)(5)](-) and cis-[(LCoNCFe)-Co-14(CN)(5)](-) (where L-14 = 6-methyl-1,4,8,11-tetraazacyclotetradecan-6-amine) were examined. The changes in the redox potentials of the cobalt and iron metal centres with pressure and temperature were also examined and the results interpreted with Marcus Hush theory. The observed redox reaction volumes can mainly be accounted for in terms of localised electrostriction effects. The shifts in E-op due to both pressure and temperature were found to be less than the shifts in the energy difference (E degrees) between the Co-III-Fe-II and Co-II-Fe-III redox isomers. The pressure and temperature dependence of the reorganisational energy, as well as contributions arising from the different spin states of Co-II, are discussed in order to account for this trend. To study the effect of pressure on Co-III electronic absorption bands, a new cyano-bridged complex, trans - [(LCoNCCo)-Co-14(CN)(5)], was prepared and characterised spectroscopically and structurally. X-Ray crystallography revealed this complex to be isostructural with trans -[(LCoNCFe)-Co-14(CN)(5)] center dot 5H(2)O.

Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K-m) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-Angstrom resolution (final R-free value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to A protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.

Dinuclear cyano-bridged Co-III-Fe-II complexes as precursors for molecular mixed-valence complexes of higher nuclearity

The preparation and characterization of a series of trinuclear mixed-valence cyano-bridged Co-III-Fe-II-Co-III compounds derived from known dinuclear [{LnCoIII(mu-NC)}Fe-II(CN)(5)](-) complexes (L-n = N-5 or N3S2 n-membered pendant amine macrocycle) are presented. All of the new trinuclear complexes were fully characterized spectroscopically (UV-vis, IR, and C-13 NMR). Complexes exhibiting a trans and cis arrangement of the Co-Fe-Co units around the [Fe(CN)(6)](4-) center are described (i.e., cis/trans-[{LnCoIII(mu-NC)}(2)Fe-II(CN)(4)](2+)), and some of their structures are determined by X-ray crystallography. Electrochemical experiments revealed an expected anodic shift of the Fe-III/II redox potential upon addition of a tripositively charged {(CoLn)-L-III} moiety. The Co-III/II redox potentials do not change greatly from the di- to the trinuclear complex, but rather behave in a fully independent and noncooperative way. In this respect, the energies and extinction coefficients of the MMCT bands agree with the formal existence of two mixed-valence Fe-II-CN-Co-III units per molecule. Solvatochromic experiments also indicated that the MMCT band of these compounds behaves as expected for a class II mixed-valence complex. Nevertheless, its extinction coefficient is dramatically increased upon increasing the solvent donor number.

Electronic energy-transfer rate constants for geometrical isomers of a bichromophoric macrocyclic complex

The rate of electronic energy transfer (EET) between a naphthalene donor and an anthracene acceptor in [ZnL3]-(ClO4)(2) and [ZnL4](ClO4)(2) was determined by time-resolved fluorescence measurements, where L 3 and L 4 are the geometrical isomers of 6-[(anthracen-9-ylmethyl)amino]-trans-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-13-amine (L-2), substituted with either a naphthalen-1-ylmethyl or naphthalen-2-ylmethyl donor, respectively. The energy transfer rate constant, k(EET), was determined to be (0.92 +/- 0.02) x 10(9) s(-1) for the naphthalen-1-ylmethyl-substituted isomer, while that for the naphthalen-2-ylmethyl-substituted isomer is somewhat faster, with k(EET) = (1.31 +/- 0.01) x 10(9) s(-1). The solid-state structure of [(ZnLCl)-Cl-3]ClO4 has been determined, and using molecular modeling calculations, the likely distributions of solution conformations in CH3CN have been evaluated for both complexes. The calculated conformational distributions in the common trans-III N-based isomeric form gave Forster EET rate constants that account for the differences observed and are in excellent agreement with the experimental values. It is shown that the full range of conformers must be considered to accurately reproduce the observed EET kinetics.

Isomeric distribution and catalyzed isomerization of cobalt(III) complexes with pentadentate macrocyclic ligands. Importance of hydrogen bonding

We have investigated the isomeric distribution and rearrangement of complexes of the type [CoXLn](2+,3+) (where X = Cl-, OH-, H2O, and L-n represents a pentadentate 13-, 14-, and 15-membered tetraaza or diaza-dithia (N-4 or N2S2) macrocycle bearing a pendant primary amine). The preparative procedures for chloro complexes produced almost exclusively kinetically preferred cis isomers (where the pendant primary amine is cis to the chloro ligand) that can be separated by careful cation-exchange chromatography. For L-13 and L-14 the so-called cis-V isomer is isolated as the kinetic product, and for L-15 the cis-VI form (an N-based diastereomer) is the preferred, while for the L-14(S) complex both cis-V and trans-I forms are obtained. All these complexes rearrange to form stable trans isomers in which the pendent primary amine is trans to the monodentate aqua or hydroxo ligand, depending on pH and the workup procedure. In total 11 different complexes have been studied. From these, two different trans isomers of [CoCIL14S](2+) have been characterized crystallographically for the first time in addition to a new structure of cis-V-[CoCIL14S](2+); all were isolated as their chloride perchlorate salts. Two additional isomers have been identified and characterized by NMR as reaction intermediates. The remaining seven forms correspond to the complexes already known, produced in preparative procedures. The kinetic, thermal, and baric activation parameters for all the isomerization reactions have been determined and involve large activation enthalpies and positive volumes of activation. Activation entropies indicate a very important degree of hydrogen bonding in the reactivity of the complexes, confirmed by density functional theory studies on the stability of the different isomeric forms. The isomerization processes are not simple and even some unstable intermediates have been detected and characterized as part of the above-mentioned 11 forms of the complexes. A common reaction mechanism for the isomerization reactions has been proposed for all the complexes derived from the observed kinetic and solution behavior.