Two forms of phycobilisomes in the Antarctic red macroalga Palmaria decipiens (Palmariales, Florideophyceae)

The phycobilisomes (PBS), the light-harvesting antennae, from the endemic Antarctic red macroalga Palmaria decipiens were isolated on discontinuous sucrose gradients in two discrete bands and not in one as expected. To exclude methodical faults, we also isolated PBS from the temperate Palmaria palmata and the unicellular red algae Porphyridium cruentum and Rhodella violacea . In P. palmata the PBS were separated in two discrete bands, whereas the PBS from Porphyridium and Rhodella were found in one band. The double-banded PBS (PBS_up and PBS_low) from P. decipiens were further characterized by absorption and fluorescence spectroscopy, native and SDS-PAGE as well as by negative staining. The phycobiliproteins RIII-phycoerythrin, RI-phycocyanin and allophycocyanin were identified and 3 γ -subunits were described. The PBS_up and PBS_low showed no significant differences in their absorption spectra and phycobiliprotein ratios although the negative stained PBS_low were smaller. Differences were found in their low molecular mass subunit complexes, which are assumed to be r-phycoerythrin. The polypeptide pattern of the PBS_up and PBS_low showed no differences in the molecular masses of their subunits and linker polypeptides, but in their percentage distribution. The results suggest that the PBS_low is a closer packed and PBS_up a little more loosely aggregated hemiellipsiodal PBS form. We discuss the ecophysiological function of two PBS forms in P. decipiens and suggest advantages in the rapid acclimation to changes in environmental light conditions.     

Plasticity of human chromosome 3 during primate evolution

Comparative mapping of more than 100 region-specific clones from human chromosome 3 in Bornean and Sumatran orangutans, siamang gibbon, and Old and New World monkeys allowed us to reconstruct ancestral simian and hominoid chromosomes. A single paracentric inversion derives chromosome 1 of the Old World monkey Presbytis cristata from the simian ancestor. In the New World monkey Callithrix geoffroyi and siamang, the ancestor diverged on multiple chromosomes, through utilizing different breakpoints. One shared and two independent inversions derive Bornean orangutan 2 and human 3, implying that neither Bornean orangutans nor humans have conserved the ancestral chromosome form. The inversions, fissions, and translocations in the five species analyzed involve at least 14 different evolutionary breakpoints along the entire length of human 3; however, particular regions appear to be more susceptible to chromosome reshuffling. The ancestral pericentromeric region has promoted both large-scale and micro-rearrangements. Small segments homologous to human 3q11.2 and 3q21.2 were repositioned intrachromosomally independent of the surrounding markers in the orangutan lineage. Breakage and rearrangement of the human 3p12.3 region were associated with extensive intragenomic duplications at multiple orangutan and gibbon subtelomeric sites. We propose that new chromosomes and genomes arise through large-scale rearrangements of evolutionarily conserved genomic building blocks and additional duplication, amplification, and/or repositioning of inherently unstable smaller DNA segments contained within them.     

The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance

Axon guidance requires coordinated remodeling of actin and microtubule polymers. Using a genetic screen, we identified the microtubule-associated protein Orbit/MAST as a partner of the Abelson (Abl) tyrosine kinase. We find identical axon guidance phenotypes in orbit/MAST and Abl mutants at the midline, where the repellent Slit restricts axon crossing. Genetic interaction and epistasis assays indicate that Orbit/MAST mediates the action of Slit and its receptors, acting downstream of Abl. We find that Orbit/MAST protein localizes to Drosophila growth cones. Higher-resolution imaging of the Orbit/MAST ortholog CLASP in Xenopus growth cones suggests that this family of microtubule plus end tracking proteins identifies a subset of microtubules that probe the actin-rich peripheral growth cone domain, where guidance signals exert their initial influence on cytoskeletal organization. These and other data suggest a model where Abl acts as a central signaling node to coordinate actin and microtubule dynamics downstream of guidance receptors.     

The Synechococcus elongatus P signal transduction protein controls arginine synthesis by complex formation with N-acetyl-L-glutamate kinase

This communication identifies, for the first time, a receptor protein for signal perception from the P(II) signal transduction protein in the cyanobacterium Synechococcus elongatus. P(II), a phosphoprotein that signals the carbon/nitrogen status of the cells, forms a tight complex with the key enzyme of the arginine biosynthetic pathway, N-acetylglutamate (NAG) kinase. In complex with P(II), the catalytic activity of NAG kinase is strongly enhanced. Complex formation does not require the effector molecules of P(II), 2-oxoglutarate and ATP, but it is highly susceptible to modifications at the phosphorylation site of P(II), Ser-49. Stable complexes were only formed with the non-phosphorylated form of P(II) but not with Ser-49 mutants. In accordance with these data, NAG kinase activity in S. elongatus extracts correlated with the phosphorylation state of P(II), with high NAG kinase activities corresponding to non-phosphorylated P(II) (nitrogen-excess conditions) and low activities to increased levels of P(II) phosphorylation (nitrogen-poor conditions), thus subjecting the key enzyme of arginine biosynthesis to global nitrogen control.     

Two minimal Tat translocases in Bacillus

Activity of the Tat machinery for protein transport across the inner membrane of Escherichia coli and the chloroplast thylakoidal membrane requires the presence of three membrane proteins: TatA, TatB and TatC. Here, we show that the Tat machinery of the Gram-positive bacterium Bacillus subtilis is very different because it contains at least two minimal Tat translocases, each composed of one specific TatA and one specific TatC component. A third, TatB-like component is apparently not required. This implies that TatA proteins of B. subtilis perform the functions of both TatA and TatB of E. coli and thylakoids. Notably, the two B. subtilis translocases named TatAdCd and TatAyCy both function as individual, substrate-specific translocases for the twin-arginine preproteins PhoD and YwbN, respectively. Importantly, these minimal TatAC translocases of B. subtilis are representative for the Tat machinery of the vast majority of Gram-positive bacteria, Streptomycetes being the only known exception with TatABC translocases.     

P signalling in unicellular cyanobacteria: analysis of redox-signals and energy charge

This communication presents a short outline of the current knowledge on the molecular basis of P_II signal transduction in unicellular cyanobacteria with respect to the perception of environmental stimuli. First, the general characteristics of the P_II signalling system in unicellular cyanobacteria are presented, the hallmark of which is modification by serine-phosphorylation, as compared to the paradigmatic P_II signal transduction system in proteobacteria, which is based on tyrosyl-uridylylation. Then, the focus is turned on the signals controlling P_II phosphorylation state. Recently, the cellular phosphatase (termed PphA), which specifically dephosphorylates phosphorylated P_II (P_II-P) was identified in Synechocystis sp. strain PCC 6803. With the availability of a PphA-deficient mutant and the purified components for in vitro assay of PphA mediated P_II-P dephosphorylation, novel insights into the signals, to which P_II-P dephosphorylation responds, can be obtained. Here we present an investigation of the response of P_II-P dephosphorylation towards treatments that affect the redox-balance of the cells. Furthermore, a possible role of varying ATP/ADP ratios on P_II-P dephosphorylation was examined. From these studies, together with previous investigations, we conclude that P_II-P dephosphorylation specifically responds to changes in the levels of central metabolites of carbon metabolism, in particular 2-oxoglutarate.     

HPLC-based bioactivity profiling of plant extracts: a kinetic assay for the identification of monoamine oxidase-A inhibitors using human recombinant monoamine oxidase-A

An assay for the HPLC-based search for monoamine oxidase-A (MAO-A) inhibitors in plant extracts was established. It combines human recombinant MAO-A, expressed as GST-fusion protein in yeast, with a kinetic measurement of the conversion of kynuramine to 4-hydroxyquinoline. Substrate selectivity and kinetic parameters of the GST-fusion protein were comparable to the wild-type enzyme. The applicability of the assay to HPLC-based activity profiling was tested with plant extracts spiked with small amounts of known MAO inhibitors.     

Localization and functional analysis of PepI, the immunity peptide of Pep5-producing Staphylococcus epidermidis strain 5

Pep5 is a cationic pore-forming lantibiotic produced by Staphylococcus epidermidis strain 5. The producer strain protects itself from the lethal action of its own bacteriocin through the 69-amino-acid immunity peptide PepI. The N-terminal segment of PepI contains a 20-amino-acid stretch of apolar residues, whereas the C terminus is very hydrophilic, with a net positive charge. We used green fluorescent protein (GFP)-PepI fusions to obtain information on its localization in vivo. PepI was found to occur outside the cytoplasm and to accumulate at the membrane-cell wall interface. The extracellular localization appeared essential for conferring immunity. We analyzed the functional role of the specific segments by constructing various mutant peptides, which were also fused to GFP. When the hydrophobic N-terminal segment of PepI was disrupted by introducing charged amino acids, the export of PepI was blocked and clones expressing such mutant peptides were Pep5 sensitive. When PepI was successively shortened at the C terminus, in contrast, its export properties remained unchanged whereas its ability to confer immunity was gradually reduced. The results show that the N-terminal part is required for the transport of PepI and that the C-terminal part is important for conferring the immunity phenotype. A concept based on target shielding is proposed for the PepI immunity mechanism.     

Differential RNA interference: replacement of endogenous with recombinant low density lipoprotein receptor-related protein (LRP)

The interpretation of experiments involving the overexpression of a recombinant cDNA is often hampered by the interference of mRNA expression from the endogenous gene locus. Unless cell lines from naturally occurring mutations or knockout mice are available, difficult and time-consuming gene targeting techniques are required to inhibit endogenous gene expression. Using a method we refer to as "differential RNA interference" we demonstrate that RNA interference can be used to selectively suppress endogenous gene expression without affecting the expression of a co-transfected recombinant version of the same protein. Functional analyses of recombinant low density lipoprotein receptor-related protein (LRP) to study its involvement in lipid metabolism have been shown to be extremely difficult due to its large cDNA and the unavailability of suitable LRP-deficient cell lines. We constructed an expression vector containing the full-length coding sequence of human LRP fused to EGFP and a vector expressing small hairpin RNA directed against the 3'-untranslated region of the wild-type human LRP mRNA (LRP-shRNA). When overexpressed, EGFP-tagged LRP colocalizes with endogenous LRP and stimulates the uptake of LRP ligands. Overexpression of LRP-shRNA vectors significantly inhibits LRP expression, as judged by quantitative RT-PCR, Western blot and immunofluorescence analysis, and it dramatically decreases receptor-associated protein (RAP) uptake. Finally, co-transfection of EGFP-LRP and LRP-shRNA vectors demonstrates selective inhibition of endogenous LRP expression without affecting simultaneous expression of recombinant LRP protein. Thus, utilization of "differential RNA interference" provides a new experimental approach to selectively study the function of any recombinant protein in any given cell line without interference of endogenous protein expression.     

Gene delivery to respiratory epithelial cells by magnetofection

BACKGROUND: For the topical application of DNA vector complexes to the airways, specific extracellular barriers play a major role. In particular, short contact time of complexes with the cell surface caused by the mucociliary clearance hinders cellular uptake of complexes. The aim of this study was to evaluate the ability of magnetofection, a technique based on the principle of magnetic drug targeting, to overcome these barriers in comparison with conventional nonviral gene transfer methods such as lipofection and polyfection. METHODS: Experiments were carried out on permanent (16HBE14o-) and primary airway epithelial cells (porcine and human), and native porcine airway epithelium ex vivo. Transfection efficiency and dose-response relationship of magnetofection were examined by luciferase reporter gene expression. Sedimentation patterns and uptake of gene transfer complexes were characterized by fluorescence and electron microscopy, respectively. RESULTS: We show that (i) application of a magnetic field allows the magnetofectins to sediment and to enrich at the cell surface within a few minutes, (ii) magnetofection bears an improved dose-response relationship, (iii) magnetofection enhances transfection efficiency in both, permanent and primary airway epithelial cells, and (iv) magnetofection leads to significant transgene expression at very short incubation times in an ex vivo airway epithelium organ model. CONCLUSIONS: Magnetofection provides a potential novel method, which may overcome fundamental limitations of nonviral gene transfer to the airways. Due to the accelerated enrichment at the cell surface it may be of major interest for in vivo applications, where long-term incubation times at the target tissue are hardly achievable.