Structural features of the metal binding site and dynamics of gallium putidaredoxin, a diamagnetic derivative of a Cys4Fe2S2ferredoxin

The first reconstitution of an Fe₂S₂ferredoxin with a diamagnetic prosthetic group was recently described[Kazanis et al. (1995) J. Am. Chem. Soc., 117, 6625–6626]. Thereplacement of the iron–sulfur cluster of the bacterial ferredoxinputidaredoxin (Pdx) by gallium (Ga³⁺) renders the proteindiamagnetic and permits the use of high-resolution NMR methods to identifyresonances near the metal binding site. We now describe structural featuresof the metal binding site that are not observable by standard NMR methods innative Pdx due to paramagnetic line broadening. These results provide thefirst example of high-resolution NMR-derived structural data concerning themetal binding domain of an Fe₂S₂ ferredoxin, andthe first structural information of any sort for the metal binding site of aferredoxin from this class, which includes adrenodoxin, placental ferredoxinand terpredoxin. Assignments were obtained by applying multidimensional NMRmethods to a series of selectively and nonselectively ¹⁵N- and¹³C/¹⁵N-labeled GaPdx samples. For mostexperiments, a mutant of Pdx was used in which a nonligatingCys⁸⁵ is replaced by serine. All of the major structuralfeatures that were identified in native Pdx are conserved in GaPdx. Theoverall protein dynamics is considerably faster in GaPdx than in the nativeprotein, as reflected by amide proton exchange rates. The C-terminalresidue, Trp¹⁰⁶, also exhibits considerable mobility, asindicated by ¹⁵N{¹H} NOE and ¹⁵NT₁ values of the C-terminal residue of the protein.     

The solution structure of a gallium-substituted putidaredoxin mutant: GaPdx C85S

The Fe₂S₂ cluster of the ferredoxin putidaredoxin (Pdx) can be replaced by a single gallium ion, giving rise to a colorless, diamagnetic protein in which, apart from the metal binding site, the major structural features of the native ferredoxin are conserved. The solution structure of the C85S variant of gallium putidaredoxin (C85S GaPdx), in which a non-ligand cysteine is replaced by a serine, has been determined via multidimensional NMR methods using uniformly ¹⁵N,¹³C labeled samples of C85S GaPdx. Stereospecific assignments of leucine and valine methyl resonances were made using ¹³C,¹H HSQC spectra obtained with fractionally ¹³C-labeled samples, and backbone dihedral angle restraints were obtained using a combination of two-dimensional J-modulated ¹⁵N,¹H HSQC and three-dimensional (HN)CO(CO)NH experiments. A total of 1117 NOE-derived distance restraints were used in the calculations, including 454 short range ($i - j 3$), 456 long range (i - j 4) interresidue restraints and 207 non-trivial intraresidue restraints. 97 and 55 ₁ angular restraints were also included in the calculation of a family of 20 structures using a combined distance geometry-simulated annealing protocol. Most regions of the protein are well defined in the calculations, with an RMSD of 0.525 Å for backbone atoms excluding the metal binding loop (residues 34–48) and the last three C-terminal residues (residues 103–106). Where comparison is possible, these regions show an increase in dynamic behavior over the native protein, as does the loop containing residues 74–76. Structural and dynamic differences between native Pdx and GaPdx are discussed in relation to charge and packing of the metal binding site.     

Disc1 Variation Leads to Specific Alterations in Adult Neurogenesis

Disrupted in schizophrenia 1 (DISC1) is a risk factor for a spectrum of neuropsychiatric illnesses including schizophrenia, bipolar disorder, and major depressive disorder. Here we use two missense Disc1 mouse mutants, described previously with distinct behavioural phenotypes, to demonstrate that Disc1 variation exerts differing effects on the formation of newly generated neurons in the adult hippocampus. Disc1 mice carrying a homozygous Q31L mutation, and displaying depressive-like phenotypes, have fewer proliferating cells while Disc1 mice with a homozygous L100P mutation that induces schizophrenia-like phenotypes, show changes in the generation, placement and maturation of newly generated neurons in the hippocampal dentate gyrus. Our results demonstrate Disc1 allele specific effects in the adult hippocampus, and suggest that the divergence in behavioural phenotypes may in part stem from changes in specific cell populations in the brain.     

Selective and rapid liquid chromatography/negative-ion electrospray ionization mass spectrometry method for the quantification of valacyclovir and its metabolite in human plasma

A rapid, sensitive and specific method was developed for the quantification of valacyclovir and acyclovir in human plasma. Sample preparation was performed by protein precipitation with acetonitrile followed by filtration. Valacyclovir, acyclovir and ganciclovir (internal standard) were separated isocratically on a reversed-phase porous graphitized carbon analytical column (2.1 mm x 125.0 mm i.d., particle size 5 microm), using a mobile phase of acetonitrile/water with 0.05% (v/v) diethylamine (50:50, v/v) at a flow rate of 0.15 mL min(-1) in 4.0 min. Detection was performed by negative electrospray ionization using the selected ion monitoring mode of the deprotonated molecular ions at m/z 323.0 for valacyclovir, 224.0 for acyclovir and 254.0 for ganciclovir. The assay had linear calibration curves over the range 0.020-0.800 microg mL(-1) for valacyclovir and 0.100-20.00 microg mL(-1) for acyclovir. Accuracy and precision were within the acceptance limit of 15%. The method was successfully applied to the analysis of plasma samples obtained from patients after oral administration of valacyclovir.     

Extracellular matrix and the neural stem cell niche

Basal lamina is present in many stem cell niches, but we still have a poor understanding of the role of this and other extracellular matrix (ECM) components. Here, we review current knowledge regarding ECM expression and function in the neural stem cell niche, focusing on the subependymal zone of the adult CNS. An increasing complexity of ECM molecules has been described, and a number of receptors expressed on the stem cells identified. Experiments perturbing the niche using genetics or cytotoxic ablation of the rapidly dividing precursors, or using explant culture models to examine specific growth factors, have been influential in showing how changes in these ECM receptors might regulate neural stem cell behavior. However the role of changes in the matrix itself remains to be determined. The answers will be important, as they will point to the molecules required to engineer niches ex-vivo so as to provide tools for regenerative neuroscience.     

Induced radioresistance: An aspect of induced repair

Summary Irradiation of Escherichia coli cells with UV or X-rays followed by incubation under conditions in which protein synthesis can occur results in a population of cells that is resistant to X-rays; however, this resistance develops only if the cells are recA ⁺ and lexA ⁺, a fact that associates the phenomenon with induced (S.O.S.) repair. By observing separately the component of a culture that is resistant and the component that retains its normal growth, the fraction of induced and uninduced cells for a dose of UV or X-rays can be estimated. Such estimates show that the dose-response for UV induction of resistant cells agrees with that of the recA gene product. Thus induced radioresistance is considered to be due to the changes in the cell occasioned by the derepression of recA and lexA. These changes are expected to be involved with the synapsis of homologous genomes that is necessary for the use of a second genome to repair damage occurring in both strands of a duplex at the same base, as exemplified by a double-strand break or an interstrand crosslink. This consideration is additionally supported by the increased resistance of cells grown to contain multiple genomes in the same envelope, an increased resistance not found in recA ^- or lexA ^- cells. The condition of a completed chromosome is also resistant, again not in recA ^- or lexA ^- cells. We suggest that cell killing by X-rays is due to the double-strand breaks which are not repaired by molecular synapsis before the arrival of the replication polymerase at the break.     

Long-term post-fire vegetation dynamics in Pinus halepensis forests of Central Greece: A functional group approach

A hierarchical approach for plant functional classification was applied to describe long-term vegetation change in Pinus halepensis burned forests. Plant species were initially grouped according to their growth form and afterwards data on species modes of regeneration, persistence and dispersal, together with some other specific competitive advantages were explored, resulting in the identification of 29 different functional groups, 14 for woody and 15 for herbaceous species. Three types of Pinus halepensis forests were identified, according to the structure of the understorey. For each forest type, a post-fire chronosequence of communities was selected for sampling. Data sampling was performed for at least two consecutive years in each community, so as to reduce the shortcomings of the synchronic approach and to increase the age range of each chronosequence. Even though the vast majority of the functional groups proved to be persistent throughout the post-fire development of vegetation, their species richness and abundance did not remain stable. An increase of annual herb richness and abundance was recorded in the first years after the fire, with the leguminous species forming the dominant functional group. For perennial herbs, the most abundant group was of species with vivid lateral growth, while the group of species with subterranean resource organs included the highest number of species. Finally, as far as the woody species are concerned, the groups that played the most important role in defining vegetation structure were the mono-specific group of the pine, the group of resprouting sclerophyllous tall shrubs and the group of obligate seeder short shrubs (with Cistusspp., among others). A negative relationship between the abundance of woody obligate resprouters and the regeneration of woody obligate seeders was found. The advantage of the proposed functional group approach over classical floristic or structural approaches for the long-term study of communities is discussed, together with the applicability of this approach in studies of vegetation risk assessments due to fire regime alterations.