Different genomic structure of mouse and human Lmp7 genes: characterization of MHC-encoded proteasome genes

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L11045 (human LMP7) and L11145 (mouse Lmp-7).     

Think locally: control of ubiquitin‐dependent protein degradation in neurons

The nervous system coordinates many aspects of body function such as learning, memory, behaviour and locomotion. Therefore, it must develop and maintain an intricate network of differentiated neuronal cells, which communicate efficiently with each other and with non‐neuronal target cells. Unlike most somatic cells, differentiated neurons are post‐mitotic and characterized by a highly polarized morphology that determines the flow of information. Among other post‐translational modifications, the ubiquitination of specific protein substrates was recently shown to have a crucial role in the regulation of neuronal development and differentiation. Here, we review recent findings that illustrate the mechanisms that mediate the temporal and spatial control of neuronal protein turnover by the ubiquitin–proteasome system (UPS), which is crucial for the development and function of the nervous system.     

Population genetic structure after 125 years of stocking in sea trout (<Emphasis Type="Italic">Salmo trutta</Emphasis> L.)

Stocking can be an effective management and conservation tool, but it also carries the danger of eroding natural population structure, introducing non-native strains and reducing genetic diversity. Sea trout, the anadromous form of the brown trout (Salmo trutta), is a highly targeted species that is often managed by stocking. Here, we assess the present-day population genetic structure of sea trout in a backdrop of 125 years of stocking in Northern Germany. The study area is characterized by short distances between the Baltic and North Sea river watersheds, historic use of fish from both watersheds for stocking, and the creation of a potential migration corridor between the Baltic and North Sea with the opening of the Kiel Canal 120 years ago. A survey of 24 river systems with 180 SNPs indicates that moderate but highly significant population genetic structure has persisted both within and between the Baltic and North Sea. This genetic structure is characterized by (i) heterogeneous patterns of admixture between the Baltic and North Sea that do not correlate with distance from the Kiel Canal and are therefore likely due to historic stocking practises, (ii) genetic isolation by distance in the Baltic Sea at a spatial scale of <?200 km that is consistent with the homing behaviour of sea trout, and (iii) at least one genetically distinct Baltic Sea river system. In light of these results, we recommend keeping fish of North Sea and Baltic Sea origin separate for stocking, and restricting Baltic Sea translocations to neighbouring river systems.     

Flavin Mononucleotide-Based Fluorescent Proteins Function in Mammalian Cells without Oxygen Requirement

Usage of the enhanced green fluorescent protein (eGFP) in living mammalian cells is limited to aerobic conditions due to requirement of oxygen during chromophore formation. Since many diseases or disease models are associated with acute or chronic hypoxia, eGFP-labeling of structures of interest in experimental studies might be unreliable leading to biased results. Thus, a chromophore yielding a stable fluorescence under hypoxic conditions is desirable. The fluorescence of flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) does not require molecular oxygen. Recently, the advantages of FbFPs for several bacterial strains and yeasts were described, specifically, their usage as a real time fluorescence marker in bacterial expression studies and their ability of chromophore formation under anaerobic conditions. Our objective was to verify if FbFPs also function in mammalian cells in order to potentially broaden the repertoire of chromophores with ones that can reliably be used in mammalian studies under hypoxic conditions. In the present study, we demonstrate for the first time, that FbFPs can be expressed in different mammalian cells, among them murine neural stem cells during proliferative and differentiated stages. Fluorescence intensities were comparable to eGFP. In contrast to eGFP, the FbFP fluorescence did not decrease when cells were exposed to defined hypoxic conditions neither in proliferating nor in differentiated cells. Thus, FbFPs can be regarded as an alternative to eGFP in studies that target cellular structures which are exposed to hypoxic conditions.     

Microbial Community Structure in Midgut and Hindgut of the Humus-Feeding Larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

The guts of soil-feeding macroinvertebrates contain a complex microbial community that is involved in the transformation of ingested soil organic matter. In a companion paper (T. Lemke, U. Stingl, M. Egert, M. W. Friedrich, and A. Brune, Appl. Environ. Microbiol. 69:6650-6658, 2003), we show that the gut of our model organism, the humivorous larva of the cetoniid beetle Pachnoda ephippiata, is characterized by strong midgut alkalinity, high concentrations of microbial fermentation products, and the presence of a diverse, yet unstudied microbial community. Here, we report on the community structure of bacteria and archaea in the midgut, hindgut, and food soil of P. ephippiata larvae, determined with cultivation-independent techniques. Clone libraries and terminal restriction fragment length polymorphism analysis of 16S rRNA genes revealed that the intestines of P. ephippiata larvae contain a complex gut microbiota that differs markedly between midgut and hindgut and that is clearly distinct from the microbiota in the food soil. The bacterial community is dominated by phylogenetic groups with a fermentative metabolism (Lactobacillales, Clostridiales, Bacillales, and Cytophaga-Flavobacterium-Bacteroides [CFB] phylum), which is corroborated by high lactate and acetate concentrations in the midgut and hindgut and by the large numbers of lactogenic and acetogenic bacteria in both gut compartments reported in the companion paper. Based on 16S rRNA gene frequencies, Actinobacteria dominate the alkaline midgut, while the hindgut is dominated by members of the CFB phylum. The archaeal community, however, is less diverse. 16S rRNA genes affiliated with mesophilic Crenarchaeota, probably stemming from the ingested soil, were most frequent in the midgut, whereas Methanobacteriaceae-related 16S rRNA genes were most frequent in the hindgut. These findings agree with the reported restriction of methanogenesis to the hindgut of Pachnoda larvae.     

Intimin-mediated export of passenger proteins requires maintenance of a translocation-competent conformation

Intimins from pathogenic bacteria promote intimate bacterial adhesion to epithelial cells. Several structurally similar domains form on the bacterial cell surface an extended rigid rod that exposes the carboxy-terminal domain, which interacts with the translocated intimin receptor. We constructed a series of intimin-derived fusion proteins consisting of carboxy-terminally truncated intimin and the immunoglobulin light-chain variable domain REIv, ubiquitin, calmodulin, beta-lactamase inhibitor protein, or beta-lactamase. By systematically investigating the intimin-mediated cell surface exposure of these passenger domains in the presence or absence of compounds that interfere with outer membrane stability or passenger domain folding, we acquired experimental evidence that intimin-mediated protein export across the outer membrane requires, prior to export, the maintenance of a translocation-competent conformation that may be distinct from the final protein structure. We propose that, during export, competition exists between productive translocation and folding of the passenger domain in the periplasm into a stable conformation that is not compatible with translocation through the bacterial outer membrane. These results may expand understanding of the mechanism by which intimins are inserted into the outer membrane and expose extracellular domains on the cell surface.     

Localization of the N-terminal domain in light-harvesting chlorophyll a/b protein by EPR measurements

The conformational distribution of the N-terminal domain of the major light-harvesting chlorophyll a/b protein (LHCIIb) has been characterized by electron-electron double resonance yielding distances between spin labels placed in various domains of the protein. Distance distributions involving residue 3 near the N terminus turned out to be bimodal, revealing that this domain, which is involved in regulatory functions such as balancing the energy flow through photosystems (PS) I and II, exists in at least two conformational states. Models of the conformational sub-ensembles were generated on the basis of experimental distance restraints from measurements on LHCIIb monomers and then checked for consistency with the experimental distance distribution between residues 3 in trimers. Only models where residue 3 is located above the core of the protein and extends into the aqueous phase on the stromal side fit the trimer data. In the other state, which consequently is populated only in monomers, the N-terminal domain extends sideways from the protein core. The two conformational states may correspond to two functional states of LHCIIb, namely trimeric LHCIIb associated with PSII in stacked thylakoid membranes and presumably monomeric LHCIIb associated with PSI in nonstacked thylakoids. The switch between these two is known to be triggered by phosphorylation of Thr-6. A similar phosphorylation-induced conformational change of the N-terminal domain has been observed by others in bovine annexin IV which, due to the conformational switch, also loses its membrane-aggregating property.     

Translocation biosensors to study signal-specific nucleo-cytoplasmic transport, protease activity and protein-protein interactions

Regulated nucleo-cytoplasmic transport is crucial for cellular homeostasis and relies on protein interaction networks. In addition, the spatial division into the nucleus and the cytoplasm marks two intracellular compartments that can easily be distinguished by microscopy. Consequently, combining the rules for regulated nucleo-cytoplasmic transport with autofluorescent proteins, we developed novel cellular biosensors composed of glutathione S-transferase, mutants of green fluorescent protein and rational combinations of nuclear import and export signals. Addition of regulatory sequences resulted in three classes of biosensors applicable for the identification of signal-specific nuclear export and import inhibitors, small molecules that interfere with protease activity and compounds that prevent specific protein-protein interactions in living cells. As a unique feature, our system exploits nuclear accumulation of the cytoplasmic biosensors as the reliable readout for all assays. Efficacy of the biosensors was systematically investigated and also demonstrated by using a fully automated platform for high throughput screening (HTS) microscopy and assay analysis. The introduced modular biosensors not only have the potential to further dissect nucleo-cytoplasmic transport pathways but also to be employed in numerous screening applications for the early stage evaluation of potential drug candidates.     

Description of the karyotype of Rhagomys rufescens Thomas, 1886 (Rodentia, Sigmodontinae) from Southern Brazil Atlantic forest

Rhagomys rufescens (Rodentia: Sigmodontinae) is an endemic species of the Atlantic forest from Southern and Southeastern Brazil. Some authors consider Rhagomys as part of the tribe Thomasomyini; but its phylogenetic relationships remain unclear. Chromosomal studies on eight specimens of Rhagomys rufescens revealed a diploid number of 2n = 36 and a number of autosome arms FN = 50. GTG, CBG and Ag-NOR banding and CMA(3) /DAPI staining were performed on metaphase chromosomes. Eight biarmed and nine acrocentric pairs were found in the karyotype of this species. The X and Y chromosomes were both acrocentric. Most of the autosomes and the sex chromosomes showed positive C-bands in the pericentromeric region. The X chromosome showed an additional heterochromatic block in the proximal region of the long arm. Nucleolus organizer regions (NORs) were located in the pericentromeric region of three biarmed autosomes (pairs 4, 6 and 8) and in the telomeric region of the short arm of three acrocentrics (pairs 10, 12 and 17). CMA (3) /DAPI staining produced fluorescent signals in many autosomes, especially in pairs 4, 6, and 8. This study presents cytogenetic data of Rhagomys rufescens for the first time.