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Background: Cyanobacteria possess several cytochrome P450s, but very little is known about their catalytic functions. CYP110 genes unique to cyanaobacteria are widely distributed in heterocyst-forming cyanobacteria including nitrogen-fixing genera Nostoc and Anabaena. We screened the biocatalytic functions of all P450s from three cyanobacterial strains of genus Nostoc or Anabaena using a series of small molecules that contain flavonoids, sesquiterpenes, low-molecular-weight drugs, and other aromatic compounds.
Results: Escherichia coli cells carrying each P450 gene that was inserted into the pRED vector, containing the RhFRed reductase domain sequence from Rhodococcus sp. NCIMB 9784 P450RhF (CYP116B2), were co-cultured with substrates and products were identified when bioconversion reactions proceeded. Consequently, CYP110E1 of Nostoc sp. strain PCC 7120, located in close proximity to the first branch point in the phylogenetic tree of the CYP110 family, was found to be promiscuous for the substrate range mediating the biotransformation of various small molecules. Naringenin and (hydroxyl) flavanones were respectively converted to apigenin and (hydroxyl) flavones, by functioning as a flavone synthase. Such an activity is reported for the first time in prokaryotic P450s. Additionally, CYP110E1 biotransformed the notable sesquiterpene zerumbone, anti-inflammatory drugs ibuprofen and flurbiprofen (methylester forms), and some aryl compounds such as 1-methoxy and 1-ethoxy naphthalene to produce hydroxylated compounds that are difficult to synthesize chemically, including novel compounds.
Conclusion: We elucidated that the CYP110E1 gene, C-terminally fused to the P450RhF RhFRed reductase domain sequence, is functionally expressed in E. coli to synthesize a robust monooxygenase, which shows promiscuous substrate specificity (affinity) for various small molecules, allowing the biosynthesis of not only flavones (from flavanones) but also a variety of hydroxyl-small molecules that may span pharmaceutical and nutraceutical industries.
Amino acids can induce yeast cell adhesion but how amino acids are sensed and signal the modulation of the FLO adhesion genes is not clear. We discovered that the budding yeast Saccharomyces cerevisiae CEN.PK evolved invasive growth ability under prolonged nitrogen limitation. Such invasive mutants were used to identify amino acid transporters as regulators of FLO11 and invasive growth. One invasive mutant had elevated levels of FLO11 mRNA and a Q320STOP mutation in the SFL1 gene that encodes a protein kinase A pathway regulated repressor of FLO11. Glutamine-transporter genes DIP5 and GNP1 were essential for FLO11 expression, invasive growth and biofilm formation in this mutant. Invasive growth relied on known regulators of FLO11 and the Ssy1-Ptr3-Ssy5 complex that controls DIP5 and GNP1, suggesting that Dip5 and Gnp1 operates downstream of the Ssy1-Ptr3-Ssy5 complex for regulation of FLO11 expression in a protein kinase A dependent manner. The role of Dip5 and Gnp1 appears to be conserved in the S. cerevisiae strain ∑1278b since the dip5 gnp1 ∑1278b mutant showed no invasive phenotype.
Secondly, the amino acid transporter gene GAP1 was found to influence invasive growth through FLO11 as well as other FLO genes. Cells carrying a dominant loss-of-function PTR3647::CWNKNPLSSIN allele had increased transcription of the adhesion genes FLO1, 5, 9, 10, 11 and the amino acid transporter gene GAP1. Deletion of GAP1 caused loss of FLO11 expression and invasive growth. However, deletions of FLO11 and genes encoding components of the mitogen-activated protein kinase pathway or the protein kinase A pathway were not sufficient to abolish invasive growth, suggesting involvement of other FLO genes and alternative pathways. Increased intracellular amino acid pools in the PTR3647::CWNKNPLSSIN-containing strain opens the possibility that Gap1 regulates the FLO genes through alteration of the amino acid pool sizes.
Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing 15N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R2 rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding.
Sponges play a key role in Antarctic marine benthic community structure and dynamics and are often a dominant component of many Southern Ocean benthic communities. Understanding the drivers of sponge distribution in Antarctica enables us to understand many of general benthic biodiversity patterns in the region. The sponges of the Antarctic and neighbouring oceanographic regions were assessed for species richness and biogeographic patterns using over 8,800 distribution records. Species-rich regions include the Antarctic Peninsula, South Shetland Islands, South Georgia, Eastern Weddell Sea, Kerguelen Plateau, Falkland Islands and north New Zealand. Sampling intensity varied greatly within the study area, with sampling hotspots found at the Antarctic Peninsula, South Georgia, north New Zealand and Tierra del Fuego, with limited sampling in the Bellingshausen and Amundsen seas in the Southern Ocean. In contrast to previous studies we found that eurybathy and circumpolar distributions are important but not dominant characteristics in Antarctic sponges. Overall Antarctic sponge species endemism is ~43%, with a higher level for the class Hexactinellida (68%). Endemism levels are lower than previous estimates, but still indicate the importance of the Polar Front in isolating the Southern Ocean fauna. Nineteen distinct sponge distribution patterns were found, ranging from regional endemics to cosmopolitan species. A single, distinct Antarctic demosponge fauna is found to encompass all areas within the Polar Front, and the sub-Antarctic regions of the Kerguelen Plateau and Macquarie Island. Biogeographical analyses indicate stronger faunal links between Antarctica and South America, with little evidence of links between Antarctica and South Africa, Southern Australia or New Zealand. We conclude that the biogeographic and species distribution patterns observed are largely driven by the Antarctic Circumpolar Current and the timing of past continent connectivity.
Temporal variation in the detectability of a species can bias estimates of relative abundance if not handled correctly. For example, when effort varies in space and/or time it becomes necessary to take variation in detectability into account when data are analyzed. We demonstrate the importance of incorporating seasonality into the analysis of data with unequal sample sizes due to lost traps at a particular density of a species. A case study of count data was simulated using a spring-active carabid beetle. Traps were ‘lost’ randomly during high beetle activity in high abundance sites and during low beetle activity in low abundance sites. Five different models were fitted to datasets with different levels of loss. If sample sizes were unequal and a seasonality variable was not included in models that assumed the number of individuals was log-normally distributed, the models severely under- or overestimated the true effect size. Results did not improve when seasonality and number of trapping days were included in these models as offset terms, but only performed well when the response variable was specified as following a negative binomial distribution. Finally, if seasonal variation of a species is unknown, which is often the case, seasonality can be added as a free factor, resulting in well-performing negative binomial models. Based on these results we recommend (a) add sampling effort (number of trapping days in our example) to the models as an offset term, (b) if precise information is available on seasonal variation in detectability of a study object, add seasonality to the models as an offset term; (c) if information on seasonal variation in detectability is inadequate, add seasonality as a free factor; and (d) specify the response variable of count data as following a negative binomial or over-dispersed Poisson distribution.
Mitochondrial dynamics and mitophagy play a key role in ensuring mitochondrial quality control. Impairment thereof was proposed to be causative to neurodegenerative diseases, diabetes, and cancer. Accumulation of mitochondrial dysfunction was further linked to aging. Here we applied a probabilistic modeling approach integrating our current knowledge on mitochondrial biology allowing us to simulate mitochondrial function and quality control during aging in silico. We demonstrate that cycles of fusion and fission and mitophagy indeed are essential for ensuring a high average quality of mitochondria, even under conditions in which random molecular damage is present. Prompted by earlier observations that mitochondrial fission itself can cause a partial drop in mitochondrial membrane potential, we tested the consequences of mitochondrial dynamics being harmful on its own. Next to directly impairing mitochondrial function, pre-existing molecular damage may be propagated and enhanced across the mitochondrial population by content mixing. In this situation, such an infection-like phenomenon impairs mitochondrial quality control progressively. However, when imposing an age-dependent deceleration of cycles of fusion and fission, we observe a delay in the loss of average quality of mitochondria. This provides a rational why fusion and fission rates are reduced during aging and why loss of a mitochondrial fission factor can extend life span in fungi. We propose the ‘mitochondrial infectious damage adaptation’ (MIDA) model according to which a deceleration of fusion–fission cycles reflects a systemic adaptation increasing life span.
Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH.
The long sought molecular function of membrane raft-associated flotillin proteins is slowly becoming resolved, partially owing to the increasing knowledge about their interaction partners. Being ubiquitously expressed and evolutionarily highly conserved, flotillins carry out important cellular functions, one of which is the regulation of signal transduction pathways. This study shows that the signaling adaptor protein fibroblast growth factor receptor substrate 2 (FRS2) directly interacts both in vivo and in vitro with flotillin-1 (flot-1). FRS2 is an important docking protein of many receptor tyrosine kinases. It regulates downstream signaling by forming molecular complexes with other adaptor proteins and tyrosine phosphatases, and seems to be a critical mediator of sustained extracellular signal regulated kinase (ERK) activity. Flot-1 has also been implicated in the regulation of ERK activity upon EGF and FGF stimuli. Furthermore, flot-1 forms signalosomes with EGFR and the downstream components of the MAP kinase pathway. The newly discovered interaction between FRS2 and flot-1 was shown to be mediated by the phosphotyrosine binding (PTB) domain and, to a lesser extent, the C-terminus (CT) of FRS2 and by the C-terminus of flot-1. Flot-1 coprecipitated together with FRS2 from murine tissues and cell lysates, demonstrating that this interaction also takes place in vivo. Interestingly, flot-2, which shows a high homology to flot-1 and forms stable oligomeric complexes with it, does not appear to directly interact with FRS2. Novel insights into the functional role of the interaction between flot-1 and FRS2 were provided by the results showing that depletion of flot-1 affects the cellular localization of FRS2. In hepatocytes stably depleted of flot-1, FRS2 appeared to be more soluble. Furthermore, upon pervanadate stimulation of the cells, a small fraction of FRS2 was recruited into detergent resistant membranes, but the recruitment did not take place in the absence of flot-1. Triggered by the same stimulus, a fraction of FRS2 was translocated to the nucleus independently of flot-1. Overexpression of FRS2 has previously been shown to result in increased ERK activation. However, in cells depleted of flot-1, FRS2 was not able to compensate for the compromised ERK activation after EGF or FGF stimulation. This might imply that FRS2 and flot-1 are functionally interconnected and that FRS2 resides upstream of flot-1. Taken together, the results presented here indicate that this complex may be involved in the control of signaling downstream of receptor tyrosine kinases and is important for ensuring a proper signaling response. In the absence of flot-1, increased Tyr phosphorylation of FRS2 was observed. It is known that Tyr and Thr phosphorylation of FRS2 are reciprocally regulated. Since ERK is a known executor of the FRS2 Thr phosphorylation, and ERK activity was shown to be severely diminished upon flot-1 depletion, the increased Tyr phosphorylation of FRS2 was in agreement with this and might be a direct consequence of a decreased ERK activity upon flot-1 depletion. FRS2 owes its name to the major and the first described function of this protein as a substrate for FGFR. PTB domain of FRS2 was published to constitutively bind the juxtamembrane domain of FGFR. In this study, the PTB domain was mapped to be involved in the constitutive interaction with flot-1 and the competition was shown to exist between flot-1 and FGFR1 for binding to FRS2. Another novel interaction partner of FRS2 was discovered in the present study. Cbl-associated protein (CAP) is an adaptor protein with three SH3 domains and it plays a role during insulin signaling by recruiting the signaling complex to lipid rafts. CAP was previously shown to interact with flot-1 via the SoHo domain, and this interaction was found to be crucial for the lipid raft recruitment of other signaling components. Both the PTB domain and CT of FRS2 were found to mediate the interaction with CAP, whereas in CAP, the SoHo domain, together with the third SH3 domain, seems to bind to FRS2. SH3 domains mediate the assembly of specific protein complexes by binding to proline rich sequences, several of which are present in FRS2. Due to overlapping interaction domains, FRS2 and flot-1 competed for the binding to CAP. However, the interaction with neither CAP nor flot-1 was necessary for the observed nuclear translocation of FRS2. Since CAP is expressed as several tissue- and developmental stage-specific isoforms, a further aim of this study was to analyze the expression of its isoforms in mouse embryonic fibroblasts (MEFs). Many new isoforms were discovered here which have not been described in the literature so far. They all contain the SoHo domain and three SH3 domains, but differ among themselves by the presence and length of a proline-rich region that preceeds the SoHo domain and by a novel 20-amino acid (AA) stretch between the second and the third SH3 domain. The length of the proline-rich region turned out to be an important factor determining the strength of the interaction with FRS2. The interaction was found to be weakened by the increasing length of this region. The new isoforms possessing the 20-AA stretch are specifically expressed in murine muscular tissues, with the highest level in the heart. During adipogenesis, we observed a shift in the abundance of the isoforms, in that only the isoforms without the insertion were shown to be upregulated on mRNA level. However, during myogenesis, preferentially expressed isoforms were those with the insertion. The collected data implicate that isoforms with the 20-AA insertion might be more ubiquitous in nondifferentiated/embryonic cells and that the observed "isoform-switch" might be dependent on the cell fate and differentiation state.
Many hominin species are best physically represented and understood by the sum of their dental morphologies. Generally, taxonomic affinities and evolutionary trends in development (ontogeny) and morphology (phylogeny) can be deduced from dental analyses. More specifically, the study of dental remains can yield a wealth of information on many facets of hominin evolution, life history, physiology and ecological adaptation; in short, the organisms paleobiomics. Functionally, teeth present information about dietary preferences, that is, the dietary niche in ecological context and, in turn, masticatory function. As the amount and types of information that can be gleaned from 2-dimensional tooth measurement exhaust themselves, 3-dimensional microscopic modeling and analysis presents a largely fertile ground for reexamination and reinterpretation of dental characteristics (Bromage et al., 2005). As such, a novel, non-destructive approach has been developed which combines the work of two established technologies (confocal microscopy and 3D modeling) adapted specifically for the purpose of mineralized tissue imaging. Through this method, 3D functional masticatory and therefore occlusal molar microwear is able to be visualized, quantified and comparatively analyzed to assess dietary preference in Javanese Homo erectus. This method differs from other microwear investigative techniques (defining 'pits'- vs- 'scratches', microtexture analysis etc.) in that it defines a molars masticatory microwear functional interactions in 3-dimensions as its baseline dataset for further interpretations and analyses. Due to poor specimen collection techniques employed during the first half of the 20th century, the very complex geologic nature of the Sangiran Dome and disagreements over its chronostratigraphy, only very few scientific works have addressed the Sangiran 7 (S7) Homo erectus molar collection (n=25) (e.g. Grine and Franzen, 1994; Kaifu, 2006). Grine and Franzen's (1994) work was a predominantly qualitative initial assessment of the specimens and identified five specimens that might better be ascribed to a fossil pongid rather than H. erectus. They also noted several molars to which tooth position (M1 or M2) was unable to be ascribed (Grine and Franzen, 1994). Kaifu (2006) comparatively examined crown sizes in several S7 molars.
The Sangiran 7 collection originates from two distinct geologic horizons: ten from the older Sangiran Formation (S7a, ~1.7 to 1.0mya) and fifteen from the younger, overlying Bapang Formation (S7b, ~1.0 to .7mya). During this million year period, Java was connected to the mainland during various glacio-eustatic low-stands in sea level. These mainland connections varied in size, extent, climatic condition and therefore in faunal and floral composition. As the S7 sample may be representative of the earliest Homo erectus migrants into Java and spans long durations of occupation, its investigation yields potential to understand the various influences climatic and ecogeographic fluctuations had on these populations. Since the sample consists only of teeth, an ecodietary approach has been deemed the most logical and appropriate investigative approach. Questions regarding the intra- and inter- S7 sample
relationships will also be addressed.
By comparing various aspects of the H. erectus dentition against that of hunter/ gatherer's (H/G) whose diet is known, functional dietary similarity can be directly correlated. Thus a comparative molar sample consisting of the below historic hunter/ gather's (n=63) has been included in order to assess H. erectus's diet in ecological context: Inuit (n=9), Pacific Northwest Tribes (n=11), Fuegians (n=11), Australian Aborigines (n=12) and Bushman (n=20). Methodologically, this approach produces a 3D facet microwear vector (fmv) signature for each molar which can then be compared for statistical similarity.
Microwear (and, as such, the fmv signatures) was defined by the regular, parallel striations found on specific cusp facets known to arise from patterned, directional masticatory movements. This differs significantly from post-mortem or taphonomic microwear which produces striations at irregular angles on multiple, non-masticatory surfaces (Peuch et al.1985, Teaford, 1988). A 'match value' is produced to determine the similarity of two molars fmv's. The 'match values' are ranked (high to low) and these rankings are used to statistically analyze and infer dietary preference: between Sangiran 7 (as an entire sample) compared against that of the historic hunter/ gatherer H. sapiens whose diet and ecogeography is known; within S7a and S7b and then among the S7 sample (eg. S7a-vs-S7b); whether the purported Pongo molars actually affiliate well with H. erectus, the hunter-gatherer's or if they demonstrate distinctly different fmv signatures altogether; whether fmv signatures are useful in distinguishing molars whose tooth position is in doubt (eg. M1 or M2).
When compared against individual H/G molars, the results show that Sangiran 7 H. erectus most closely correlates with Bushmen across all areas of fmv signature analysis. However, within broader dietary categories (yearly reliant on proteinaceous foods; seasonally reliant on proteinaceous foods; not reliant on proteinaceous foods), it was found that H. erectus most closely allied with the two hunter/ gatherer subpopulations associated with the 'Seasonally reliant on proteinaceous foods' (Australian Aboriginals and Pacific Northwest Tribes). There was also evidence for dietary change or specialization over time. As the environment changed during occupation by the earlier Sangiran to the later Bapang individuals, the dietary preference shifted from a focus on vegetative foods to a diet much more inclusive of proteinaceous resources.
These results are considered logical within the larger ecogeographic and chronostratigraphic context of the Sangiran Dome during the Pleistocene. However, a larger sample would be needed to confirm this. Although general dietary preferences can be drawn from this method, it is not possible at present to define specific foods consumed on a daily basis (eg. tubers or tortoise meat).
Out of the five specimens possibly allied with Pongo, S7-14 matched at the 'high' designation with a hunter/ gatherer, S7-62 matched 'moderately', S7-20 matched 'low' while the remaining two were not able to be matched with any other teeth for various reasons. Although designation to Pongo cannot be ruled on at this time using this method, it does demonstrate that at least two of the teeth correlate well with various hunter/ gatherer's who do not share dietary similarity with Pongo. This suggests their designation as Pongo should be more closely reevaluated. As for the four specimens whose tooth position was unsure, S7-14 matched 'highly' with 1st molars, S7-62 and S7-78 matched 'moderately' with 2nd and 1st molars respectively while S7-20 only matched at the 'low' designation. Although this approach is still exploratory, it adds another analytical tool for use in defining tooth position.
In sum, this method has demonstrated its usefulness in defining and functionally analyzing a novel 3D molar microwear dataset to interpret dietary preference. Future work would include a pan- H. erectus molar sample in order to illuminate broader populational, taxonomic and dietary correlations within and amoung all H. erectus specimens. A larger, more heterogenous historic H/G sample would also be included in order to provide a wider dietary comparative population. This method can be further extended to include and compare any and all hominins as well as any organism which produces micro wear upon it molars. Also, the data obtained and resultant fmv signature diagrams have the potential to be incorporated into 3D VR reconstructions of mandibular movement thus recreating mastication in extinct organisms and leading to more robust anatomical and physiological investigations especially when viewed in the context of larger environmental conditions or changes.
Synaptic plasticity is the basis for information storage, learning and memory and is achieved by modulation of the synaptic transmission. The amount of active AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazol-propionic acid) receptors at the synapse determines the transmission properties, therefore the regulation of AMPA receptor trafficking affects the synaptic strength. The protein GRIP (glutamate receptor interacting protein) binds to AMPA receptors and is one of the important regulators of AMPA receptor stability at the synapse (Dong et al., 1997; Osten et al., 2000). Previous studies have shown that the ablation of ephrinB2 or ephrinB3 in the nervous system leads to severe defects in hippocampal LTP (long term potentiation) and LTD (long term depression) (Grunwald et al., 2004). We found that ephrinB2 ligands play an important role in the stabilization of AMPA receptors at the cellular membrane (Essmann et al., 2008). Treating cultured hippocampal neurons with AMPA resulted in a robust AMPA receptor internalization, which could be inhibited by simultaneous ephrinB2 activation with soluble EphB4-Fc fusion proteins. Conditional hippocampal ephrinB2 knock-out (KO) neurons showed enhanced constitutive internalization of AMPA receptors. Interaction and interference experiments revealed that ephrinB ligands and AMPA receptors are bridged by GRIP. This interaction is regulated by phosphorylation of a single serine residue in close proximity to the C-terminal PDZ protein target site in ephrinB ligands (Essmann et al., 2008). To investigate the in vivo relevance of this previously undescribed feature of ephrinB reverse signaling, we generated ephrinB2 S-9>A knock-in mice, where the serine at position -9 was replaced by an alanine to prevent phosphorylation. The mutated ephrinB2 of this mouse line was expressed and able to form clusters following stimulation with the preclustered receptor EphB4-Fc. Surface ephrinB2 cluster size and cluster number was slightly smaller in comparison to wild type (WT) mice. Analyzing AMPA receptor internalization, we oserved an increased basal GluR2 endocytosis in cultured hippocampal neurons of ephrinB2 S-9>A mice. Dendrite and spine morphology was similar in pyramidal CA1 neurons of brain slices from adult ephrinB2 S-9>A and WT mice, suggesting a redundancy between the different ephrinB familily members.
Apart from regulating AMPA receptor stability at the synapse, GRIP1 also has an important role in the secretory pathway to deliver cargo proteins along microtubules to dendrites and synapses (Setou et al., 2002). Proteins involved in synaptic transmission and plasticity, as well as lipids required for the outgrowth and remodeling of dendrites and axons have to be transported. We showed in our laboratory with a directed proteomic analysis using the tandem affinity purification-mass spectrometry methodology (Angrand et al., 2006) and with immunoprecipitation assays with brain lysates that the small regulatory protein 14-3-3 interacts with GRIP1. Further immunoprecipitation assays with lysates from HeLa cells transfected with various parts and sequence mutants of GRIP1 revealed that threonine 956 in the linker region L2 between PDZ6 and PDZ7 of GRIP1 is necessary for the interaction with 14-3-3. GRIP1 has been postulated to influence dendritic arborization and maintenance in hippocampal neurons in culture due to defective kinesin-dependent transport along microtubules (Hoogenraad et al 2005). In order to address the role of the association of GRIP1 and 14-3-3 in dendritogenesis, we transfected rat hippocampal neurons with GRIP1-WT and GRIP1 mutants and performed Sholl analysis to evaluate dendritic arborization defects. We could observe striking increased formation and growth of dendrites in developing neurons as well as in mature neurons overexpressing GRIP1-WT. However, overexpression of GRIP1-T956A, where the threonine 956 was replaced by an alanine to prevent phosphorylation, did not show enhanced dendritogenesis, indicating a role for threonine 956 phosphorylation in dendrite branching. To investigate the importance of the interaction between GRIP1 and 14-3-3 in vivo, we generated transgenic mouse lines with a GRIP1-T956A transgene or a GRIP1-WT transgene as control. These mice were crossed with heterozygous GRIP1 mice and by further breedings we obtained some surviver mice carrying either the wild type or the mutated GRIP1 transgene in the usually embryonic lethal GRIP1-KO background (Bladt et al., 2002; Takamiya et al., 2004). In embryonic day (E) 14.5 cultured hippocampal GRIP1-KO neurons we could observe reduced dendritic growth. We also showed reduced GluR2 staining on the dendritic surface in cultured hippocampal neurons from GRIP1-KO and GRIP1-KO neurons containing the GRIP1-T956A transgene. GRIP1-KO neurons containing the GRIP1-WT transgene showed a similar surface GluR2 signal intensity as WT neurons. Reduced surface GluR2 staining in GRIP1-KO neurons and GRIP1-KO neurons with the GRIP1-T956A transgene might be a consequence of defective kinesin-dependent transport of GluR2 to dendrites, indicating an important role of threonine 956 phosphorylation of GRIP1 for GluR2 trafficking.
Plastids are complex organelles that fulfil numerous essential cellular functions, such as
photosynthesis, amino acid and fatty acid synthesis. he majority of proteins required for
these functions are encoded in the nuclear genome and synthesised on cytosolic ribosomes as
precursors, which are posttranslationally transported to and imported into the organelle by
concerted actions of translocons in the outer and inner chloroplast membrane. For most
preproteins, targeting to the organelle is ensured by a specific import signal, a so called
transit peptide, which is specifically recognised by receptors at the chloroplastês surface. A transit peptide is generally defined as essential and sufficient for precursor targeting to and
translocation into chloroplasts, (however, an analysis of the ability of transit peptides to drive translocation of tightly folded passenger domain revealed that the transit peptide is not
always sufficient for the translocation event. A critical signal length requirement of amino
acids has been determined in vivo and in vitro. In the case of shorter transit peptide, the
succeeding portion of the mature domain provides an extension of an unfolded polypeptide
stretch required for successful translocation. The analysis of the unfolding mode of a folded
model passenger during translocation links the observed transit peptide length requirement
to the action of an energising unit present in the intermembrane space of chloroplasts.
The likely candidate for this energising unit space is putative imsHsp70, previously hypothesised to function in translocation of precursor proteins across the outer membrane. However, as the identity of this protein has up to now remained unknown, its existence has
been a matter of debate. The present study focuses on the isolation and characterisation of
imsHsp70 at the molecular level. Mass spectrometry analyses and in vivo localisation studies
demonstrate that while no specific imsHsp70 exists, multiple cytosolic Hsp70 isoforms are
targeted to the intermembrane space, but not to the stroma of chloroplasts. Thus, a so far unrecognised mode of dual targeting to chloroplasts and cytosol is most likely to ensure the
allocation of (sp s into the intermembrane space.
Crista junctions (CJs) are tubular invaginations of the inner membrane of mitochondria that connect the inner boundary with the cristae membrane. These architectural elements are critical for mitochondrial function. The yeast inner membrane protein Fcj1, called mitofilin in mammals, was reported to be preferentially located at CJs and crucial for their formation. Here we investigate the functional roles of individual domains of Fcj1. The most conserved part of Fcj1, the C-terminal domain, is essential for Fcj1 function. In its absence, formation of CJ is strongly impaired and irregular, and stacked cristae are present. This domain interacts with full-length Fcj1, suggesting a role in oligomer formation. It also interacts with Tob55 of the translocase of outer membrane β-barrel proteins (TOB)/sorting and assembly machinery (SAM) complex, which is required for the insertion of β-barrel proteins into the outer membrane. The association of the TOB/SAM complex with contact sites depends on the presence of Fcj1. The biogenesis of β-barrel proteins is not significantly affected in the absence of Fcj1. However, down-regulation of the TOB/SAM complex leads to altered cristae morphology and a moderate reduction in the number of CJs. We propose that the C-terminal domain of Fcj1 is critical for the interaction of Fcj1 with the TOB/SAM complex and thereby for stabilizing CJs in close proximity to the outer membrane. These results assign novel functions to both the C-terminal domain of Fcj1 and the TOB/SAM complex.
Organelles are surrounded by membranes with a distinct lipid and protein composition. While it is well established that lipids affect protein functioning and vice versa, it has been only recently suggested that elevated membrane protein concentrations may affect the shape and organization of membranes. We therefore analyzed the effects of high chloroplast envelope protein concentrations on membrane structures using an in vivo approach with protoplasts. Transient expression of outer envelope proteins or protein domains such as CHUP1-TM–GFP, outer envelope protein of 7 kDa–GFP, or outer envelope protein of 24 kDa–GFP at high levels led to the formation of punctate, circular, and tubular membrane protrusions. Expression of inner membrane proteins such as translocase of inner chloroplast membrane 20, isoform II (Tic20-II)–GFP led to membrane protrusions including invaginations. Using increasing amounts of DNA for transfection, we could show that the frequency, size, and intensity of these protrusions increased with protein concentration. The membrane deformations were absent after cycloheximide treatment. Co-expression of CHUP1-TM–Cherry and Tic20-II–GFP led to membrane protrusions of various shapes and sizes including some stromule-like structures, for which several functions have been proposed. Interestingly, some structures seemed to contain both proteins, while others seem to contain one protein exclusively, indicating that outer and inner envelope dynamics might be regulated independently. While it was more difficult to investigate the effects of high expression levels of membrane proteins on mitochondrial membrane shapes using confocal imaging, it was striking that the expression of the outer membrane protein Tom20 led to more elongate mitochondria. We discuss that the effect of protein concentrations on membrane structure is possibly caused by an imbalance in the lipid to protein ratio and may be involved in a signaling pathway regulating membrane biogenesis. Finally, the observed phenomenon provides a valuable experimental approach to investigate the relationship between lipid synthesis and membrane protein expression in future studies.
BACKGROUND:
Horizontal gene transfer through natural transformation of members of the microbiota of the lower gastrointestinal tract (GIT) of mammals has not yet been described. Insufficient DNA sequence similarity for homologous recombination to occur has been identified as the major barrier to interspecies transfer of chromosomal DNA in bacteria. In this study we determined if regions of high DNA similarity between the genomes of the indigenous bacteria in the GIT of rats and feed introduced DNA could lead to homologous recombination and acquisition of antibiotic resistance genes.
RESULTS:
Plasmid DNA with two resistance genes (nptI and aadA) and regions of high DNA similarity to 16S rRNA and 23S rRNA genes present in a broad range of bacterial species present in the GIT, were constructed and added to standard rat feed. Six rats, with a normal microbiota, were fed DNA containing pellets daily over four days before sampling of the microbiota from the different GI compartments (stomach, small intestine, cecum and colon). In addition, two rats were included as negative controls. Antibiotic resistant colonies growing on selective media were screened for recombination with feed introduced DNA by PCR targeting unique sites in the putatively recombined regions. No transformants were identified among 441 tested isolates.
CONCLUSIONS:
The analyses showed that extensive ingestion of DNA (100 μg plasmid) per day did not lead to increased proportions of kanamycin resistant bacteria, nor did it produce detectable transformants among the aerobic microbiota examined for 6 rats (detection limit < 1 transformant per 1,1 × 108 cultured bacteria). The key methodological challenges to HGT detection in animal feedings trials are identified and discussed. This study is consistent with other studies suggesting natural transformation is not detectable in the GIT of mammals.
Background: In mixed sugar fermentations with recombinant Saccharomyces cerevisiae strains able to ferment D-xylose and L-arabinose the pentose sugars are normally only utilized after depletion of D-glucose. This has been attributed to competitive inhibition of pentose uptake by D-glucose as pentose sugars are taken up into yeast cells by individual members of the yeast hexose transporter family. We wanted to investigate whether D-glucose inhibits pentose utilization only by blocking its uptake or also by interfering with its further metabolism.
Results: To distinguish between inhibitory effects of D-glucose on pentose uptake and pentose catabolism, maltose was used as an alternative carbon source in maltose-pentose co-consumption experiments. Maltose is taken up by a specific maltose transport system and hydrolyzed only intracellularly into two D-glucose molecules. Pentose consumption decreased by about 20 - 30% during the simultaneous utilization of maltose indicating that hexose catabolism can impede pentose utilization. To test whether intracellular D-glucose might impair pentose utilization, hexo-/glucokinase deletion mutants were constructed. Those mutants are known to accumulate intracellular D-glucose when incubated with maltose. However, pentose utilization was not effected in the presence of maltose. Addition of increasing concentrations of D-glucose to the hexo-/glucokinase mutants finally completely blocked D-xylose as well as L-arabinose consumption, indicating a pronounced inhibitory effect of D-glucose on pentose uptake. Nevertheless, constitutive overexpression of pentose-transporting hexose transporters like Hxt7 and Gal2 could improve pentose consumption in the presence of D-glucose.
Conclusion: Our results confirm that D-glucose impairs the simultaneous utilization of pentoses mainly due to inhibition of pentose uptake. Whereas intracellular D-glucose does not seem to have an inhibitory effect on pentose utilization, further catabolism of D-glucose can also impede pentose utilization. Nevertheless, the results suggest that co-fermentation of pentoses in the presence of D-glucose can significantly be improved by the overexpression of pentose transporters, especially if they are not inhibited by D-glucose.
Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN.PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN.PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN.PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN.PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains.
The neurexin genes (NRXN1/2/3) encode two families (α and β) of highly polymorphic presynaptic proteins that are involved in excitatory/inhibitory synaptic balance. Recent studies indicate that neuronal activation and memory formation affect NRXN1/2/3α expression and alternative splicing at splice sites 3 and 4 (SS#3/SS#4). Neurons in the biological clock residing in the suprachiasmatic nuclei of the hypothalamus (SCN) act as self-sustained oscillators, generating rhythms in gene expression and electrical activity, to entrain circadian bodily rhythms to the 24 hours day/night cycles. Cell autonomous oscillations in NRXN1/2/3α expression and SS#3/SS#4 exons splicing and their links to rhythms in excitatory/inhibitory synaptic balance in the circadian clock were explored. NRXN1/2/3α expression and SS#3/SS#4 splicing, levels of neurexin-2α and the synaptic scaffolding proteins PSD-95 and gephyrin (representing excitatory and inhibitory synapses, respectively) were studied in mRNA and protein extracts obtained from SCN of C3H/J mice at different times of the 24 hours day/night cycle. Further studies explored the circadian oscillations in these components and causality relationships in immortalized rat SCN2.2 cells. Diurnal rhythms in mNRXN1α and mNRXN2α transcription, SS#3/SS#4 exon-inclusion and PSD-95 gephyrin and neurexin-2α levels were found in the SCN in vivo. No such rhythms were found with mNRXN3α. SCN2.2 cells also exhibited autonomous circadian rhythms in rNRXN1/2 expression SS#3/SS#4 exon inclusion and PSD-95, gephyrin and neurexin-2α levels. rNRXN3α and rNRXN1/2β were not expressed. Causal relationships were demonstrated, by use of specific siRNAs, between rNRXN2α SS#3 exon included transcripts and gephyrin levels in the SCN2.2 cells. These results show for the first time dynamic, cell autonomous, diurnal rhythms in expression and splicing of NRXN1/2 and subsequent effects on the expression of neurexin-2α and postsynaptic scaffolding proteins in SCN across the 24-h cycle. NRXNs gene transcripts may have a role in coupling the circadian clock to diurnal rhythms in excitatory/inhibitory synaptic balance.
Protective ant-plant mutualisms that are exploited by non-defending parasitic ants represent prominent model systems for ecology and evolutionary biology. The mutualist Pseudomyrmex ferrugineus is an obligate plant-ant and fully depends on acacias for nesting space and food. The parasite Pseudomyrmex gracilis facultatively nests on acacias and uses host-derived food rewards but also external food sources. Integrative analyses of genetic microsatellite data, cuticular hydrocarbons and behavioral assays showed that an individual acacia might be inhabited by the workers of several P. gracilis queens, whereas one P. ferrugineus colony monopolizes one or more host trees. Despite these differences in social organization, neither of the species exhibited aggressive behavior among conspecific workers sharing a tree regardless of their relatedness. This lack of aggression corresponds to the high similarity of cuticular hydrocarbon profiles among ants living on the same tree. Host sharing by unrelated colonies, or the presence of several queens in a single colony are discussed as strategies by which parasite colonies could achieve the observed social organization. We argue that in ecological terms, the non-aggressive behavior of non-sibling P. gracilis workers — regardless of the route to achieve this social structure — enables this species to efficiently occupy and exploit a host plant. By contrast, single large and long-lived colonies of the mutualist P. ferrugineus monopolize individual host plants and defend them aggressively against invaders from other trees. Our findings highlight the necessity for using several methods in combination to fully understand how differing life history strategies affect social organization in ants.
Treatment of malignant glioma with chemotherapy is limited mostly because of delivery impediment related to the blood-brain tumor barrier (BTB). B1 receptors (B1R), inducible prototypical G-protein coupled receptors (GPCR) can regulate permeability of vessels including possibly that of brain tumors. Here, we determine the extent of BTB permeability induced by the natural and synthetic peptide B1R agonists, LysdesArg9BK (LDBK) and SarLys[DPhe8]desArg9BK (NG29), in syngeneic F98 glioma-implanted Fischer rats. Ten days after tumor inoculation, we detected the presence of B1R on tumor cells and associated vasculature. NG29 infusion increased brain distribution volume and uptake profiles of paramagnetic probes (Magnevist and Gadomer) at tumoral sites (T1-weighted imaging). These effects were blocked by B1R antagonist and non-selective cyclooxygenase inhibitors, but not by B2R antagonist and non-selective nitric oxide synthase inhibitors. Consistent with MRI data, systemic co-administration of NG29 improved brain tumor delivery of Carboplatin chemotherapy (ICP-Mass spectrometry). We also detected elevated B1R expression in clinical samples of high-grade glioma. Our results documented a novel GPCR-signaling mechanism for promoting transient BTB disruption, involving activation of B1R and ensuing production of COX metabolites. They also underlined the potential value of synthetic biostable B1R agonists as selective BTB modulators for local delivery of different sized-therapeutics at (peri)tumoral sites.
The Alpine Region, constituting the Alps and the Dinaric Alps, has played a major role in the formation of current patterns of biodiversity either as a contact zone of postglacial expanding lineages or as the origin of genetic diversity. In our study, we tested these hypotheses for two widespread, sympatric microgastropod taxa - Carychium minimum O.F. Müller, 1774 and Carychium tridentatum (Risso, 1826) (Gastropoda, Eupulmonata, Carychiidae) - by using COI sequence data and species potential distribution models analyzed in a statistical phylogeographical framework. Additionally, we examined disjunct transatlantic populations of those taxa from the Azores and North America. In general, both Carychium taxa demonstrate a genetic structure composed of several differentiated haplotype lineages most likely resulting from allopatric diversification in isolated refugial areas during the Pleistocene glacial periods. However, the genetic structure of Carychium minimum is more pronounced, which can be attributed to ecological constraints relating to habitat proximity to permanent bodies of water. For most of the Carychium lineages, the broader Alpine Region was identified as the likely origin of genetic diversity. Several lineages are endemic to the broader Alpine Region whereas a single lineage per species underwent a postglacial expansion to (re)colonize previously unsuitable habitats, e.g. in Northern Europe. The source populations of those expanding lineages can be traced back to the Eastern and Western Alps. Consequently, we identify the Alpine Region as a significant 'hot-spot' for the formation of genetic diversity within European Carychium lineages. Passive dispersal via anthropogenic means best explains the presence of transatlantic European Carychium populations on the Azores and in North America. We conclude that passive (anthropogenic) transport could mislead the interpretation of observed phylogeographical patterns in general.