Reduced Activity of Geranylgeranyl Reductase Leads to Loss of Chlorophyll and Tocopherol and to Partially Geranylgeranylated Chlorophyll in Transgenic Tobacco Plants Expressing Antisense RNA for Geranylgeranyl Reductase

The enzyme geranylgeranyl reductase (CHL P) catalyzes the reduction of geranylgeranyl diphosphate to phytyl diphosphate. We identified a tobacco (Nicotiana tabacum) cDNA sequence encoding a 52-kD precursor protein homologous to the Arabidopsis and bacterial CHL P. The effects of deficient CHL P activity on chlorophyll (Chl) and tocopherol contents were studied in transgenic plants expressing antisense CHL P RNA. Transformants with gradually reduced Chl P expression showed a delayed growth rate and a pale or variegated phenotype. Transformants grown in high (500 μmol m⁻² s⁻¹; HL) and low (70 μmol photon m⁻² s⁻¹; LL) light displayed a similar degree of reduced tocopherol content during leaf development, although growth of wild-type plants in HL conditions led to up to a 2-fold increase in tocopherol content. The total Chl content was more rapidly reduced during HL than LL conditions. Up to 58% of the Chl content was esterified with geranylgeraniol instead of phytol under LL conditions. Our results indicate that CHL P provides phytol for both tocopherol and Chl synthesis. The transformants are a valuable model with which to investigate the adaptation of plants with modified tocopherol levels against deleterious environmental conditions.     

Are dendrites in Drosophila homologous to vertebrate dendrites?

Dendrites represent arborising neurites in both vertebrates and invertebrates. However, in vertebrates, dendrites develop on neuronal cell bodies, whereas in higher invertebrates, they arise from very different neuronal structures, the primary neurites, which also form the axons. Is this anatomical difference paralleled by principal developmental and/or physiological differences? We address this question by focussing on one cellular model, motorneurons of Drosophila and characterise the compartmentalisation of these cells. We find that motorneuronal dendrites of Drosophila share with typical vertebrate dendrites that they lack presynaptic but harbour postsynaptic proteins, display calcium elevation upon excitation, have distinct cytoskeletal features, develop later than axons and are preceded by restricted localisation of Par6-complex proteins. Furthermore, we demonstrate in situ and culture that Drosophila dendrites can be shifted from the primary neurite to their soma, i.e. into vertebrate-like positions. Integrating these different lines of argumentation, we propose that dendrites in vertebrates and higher invertebrates have a common origin, and differences in dendrite location can be explained through translocation of neuronal cell bodies introduced during the evolutionary process by which arthropods and vertebrates diverged from a common urbilaterian ancestor. Implications of these findings for studies of dendrite development, neuronal polarity, transport and evolution are discussed.     

Estrogen therapy affects right hemisphere functioning in postmenopausal women

It has been suggested that hormone therapy (HT) in postmenopausal women differentially affects verbal and visuo-spatial abilities which mainly rely on left hemisphere (LH) and right hemisphere (RH) functioning, respectively. Thus, it seems likely that HT-related effects on cognition are driven by associated hormonal changes and their impact on functional brain organization, and functional cerebral asymmetries (FCAs) in particular. The present study investigated HT-related effects on FCAs in sixty-seven postmenopausal women who received hormone therapy either with estrogen (E) alone (n =  14), an E-gestagen combination (n =  22) or without HT (control group, n =  31). Saliva levels of free E and progesterone (P) were analyzed using chemiluminescence assays. FCAs were measured with the visual half-field (VHF) technique using a word matching and a figural comparison task. In agreement with previous results, a postmenopausal control group showed a left hemisphere (LH) advantage in the verbal task and a right hemisphere (RH) advantage in visuo-spatial processing. In contrast, both HT groups revealed significantly reduced FCAs in the figural comparison task as a result of an E-related decrease in RH performance. The findings suggest that E-therapy in postmenopausal women can affect visuo-spatial abilities by modulating the functional brain organization and RH functioning in particular.     

Solution structure of the two-iron rubredoxin of Pseudomonas oleovorans determined by NMR spectroscopy and solution X-ray scattering and interactions with rubredoxin reductase

Here we provide insights into the molecular structure of the two-iron 19-kDa rubredoxin (AlkG) of Pseudomonas oleovorans using solution-state nuclear magnetic resonance (NMR) and small-angle X-ray scattering studies. Sequence alignment and biochemical studies have suggested that AlkG comprises two rubredoxin folds connected by a linker region of approximately 70 amino acid residues. The C-terminal domain (C-Rb) of this unusual rubredoxin, together with approximately 35 amino acid residues of the predicted linker region, was expressed in Escherichia coli, purified in the one-iron form and the structure of the cadmium-substituted form determined at high-resolution by NMR spectroscopy. The structure shows that the C-Rb domain is similar in fold to the conventional one-iron rubredoxins from other organisms, whereas the linker region does not have any discernible structure. This tandem "flexible-folded" structure of the polypeptide chain derived for the C-Rb protein was confirmed using solution X-ray scattering methods. X-ray scattering studies of AlkG indicated that the 70-amino acid residue linker forms a structured, yet mobile, polypeptide segment connecting the globular N- and C-terminal domains. The X-ray scattering studies also showed that the N-terminal domain (N-Rb) has a molecular conformation similar to that of C-Rb. The restored molecular shape indicates that the folded N-Rb and C-Rb domains of AlkG are noticeably separated, suggesting some domain movement on complex formation with rubredoxin reductase to allow interdomain electron transfer between the metal centers in AlkG. This study demonstrates the advantage of combining X-ray scattering and NMR methods in structural studies of dynamic, multidomain proteins that are not suited to crystallographic analysis. The study forms a structural foundation for functional studies of the interaction and electron-transfer reactions of AlkG with rubredoxin reductase, also reported herein.     

Mutations of the mitochondrial holocytochrome c-type synthase in X-linked dominant microphthalmia with linear skin defects syndrome

The microphthalmia with linear skin defects syndrome (MLS, or MIDAS) is an X-linked dominant male-lethal disorder almost invariably associated with segmental monosomy of the Xp22 region. In two female patients, from two families, with MLS and a normal karyotype, we identified heterozygous de novo point mutations--a missense mutation (p.R217C) and a nonsense mutation (p.R197X)--in the HCCS gene. HCCS encodes the mitochondrial holocytochrome c-type synthase that functions as heme lyase by covalently adding the prosthetic heme group to both apocytochrome c and c(1). We investigated a third family, displaying phenotypic variability, in which the mother and two of her daughters carry an 8.6-kb submicroscopic deletion encompassing part of the HCCS gene. Functional analysis demonstrates that both mutant proteins (R217C and Delta 197-268) were unable to complement a Saccharomyces cerevisiae mutant deficient for the HCCS orthologue Cyc3p, in contrast to wild-type HCCS. Moreover, ectopically expressed HCCS wild-type and the R217C mutant protein are targeted to mitochondria in CHO-K1 cells, whereas the C-terminal-truncated Delta 197-268 mutant failed to be sorted to mitochondria. Cytochrome c, the final product of holocytochrome c-type synthase activity, is implicated in both oxidative phosphorylation (OXPHOS) and apoptosis. We hypothesize that the inability of HCCS-deficient cells to undergo cytochrome c-mediated apoptosis may push cell death toward necrosis that gives rise to severe deterioration of the affected tissues. In summary, we suggest that disturbance of both OXPHOS and the balance between apoptosis and necrosis, as well as the X-inactivation pattern, may contribute to the variable phenotype observed in patients with MLS.     

X-ray solution scattering reveals conformational changes upon iron uptake in lactoferrin, serum and ovo-transferrins.

X-ray solution scattering has been used for studying the structural changes that take place upon uptake and release of iron from serum and chicken ovo-transferrin and human lactoferrin. In the case of chicken ovo-transferrin, data have been obtained for both the intact protein and the isolated N and C-lobes with and without iron. These studies reveal that both lobes undergo a change that is consistent with an opening of the inter-domain cleft when iron is removed from the protein. We suggest that the conformational change of the protein increases the specificity of receptor binding and that the closed configuration of the iron-loaded protein is one, or perhaps the, decisive step in the mechanism for receptor-mediated endocytosis.