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The botanical exploration of Eastern Asia by European travellers and botanists has for a long time attracted the author's attention, and the greater part of the materials for the present work were brought together, many years ago, from various sources of information, frequently unprinted, some of which were only obtainable in China. ...
The formation of vegetable mould : through the action of worms with observations on their habits
(1881)
Background Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-arabinose, which are present in high amounts in lignocellulosic raw materials. Results We describe the engineering of laboratory and industrial S. cerevisiae strains to co-ferment the pentose sugars D-xylose and L-arabinose. Introduction of a fungal xylose and a bacterial arabinose pathway resulted in strains able to grow on both pentose sugars. Introduction of a xylose pathway into an arabinose-fermenting laboratory strain resulted in nearly complete conversion of arabinose into arabitol due to the L-arabinose reductase activity of the xylose reductase. The industrial strain displayed lower arabitol yield and increased ethanol yield from xylose and arabinose. Conclusion Our work demonstrates simultaneous co-utilization of xylose and arabinose in recombinant strains of S. cerevisiae. In addition, the co-utilization of arabinose together with xylose significantly reduced formation of the by-product xylitol, which contributed to improved ethanol production.
Background: Particle Swarm Optimization (PSO) is an established method for parameter optimization. It represents a population-based adaptive optimization technique that is influenced by several "strategy parameters". Choosing reasonable parameter values for the PSO is crucial for its convergence behavior, and depends on the optimization task. We present a method for parameter meta-optimization based on PSO and its application to neural network training. The concept of the Optimized Particle Swarm Optimization (OPSO) is to optimize the free parameters of the PSO by having swarms within a swarm. We assessed the performance of the OPSO method on a set of five artificial fitness functions and compared it to the performance of two popular PSO implementations. Results: Our results indicate that PSO performance can be improved if meta-optimized parameter sets are applied. In addition, we could improve optimization speed and quality on the other PSO methods in the majority of our experiments. We applied the OPSO method to neural network training with the aim to build a quantitative model for predicting blood-brain barrier permeation of small organic molecules. On average, training time decreased by a factor of four and two in comparison to the other PSO methods, respectively. By applying the OPSO method, a prediction model showing good correlation with training-, test- and validation data was obtained. Conclusion: Optimizing the free parameters of the PSO method can result in performance gain. The OPSO approach yields parameter combinations improving overall optimization performance. Its conceptual simplicity makes implementing the method a straightforward task.
The European Strategy on Invasive Alien Species T-PWS(2002) 8 mandates intensified research by member nations on invasive species. This research will not be restricted solely to the biology and remediation of invasive species, but will also evaluate their adverse health effects and economic impact. Previous studies of these issues have only been carried out in the Unites States of America, or in a limited, regional manner. Consequently, 20 plant and animal species from various problem areas (species which pose a threat to public health; losses to agriculture, fisheries, and forestry; damage to public roads and waterways; costs associated with the protection of native species threatened by non-native species as mandated by Recommendation 77 of the Bern Convention were assessed in Germany nation-wide. The accruing costs were sorted into 3 categories: a) direct economic losses, such as those caused by destructive pest species; b) ecological costs, in the form of extra care and protection of native taxa, biotopes, or ecosystems threatened by invasive species; c) costs of measures to combat invasive species. Because of the nature of available data, as well as the different biology and ecology of the invasive species, each had to be treated individually, and the associated costs vary greatly from species to species. Moreover, not all of the species investigated cause economic losses. Accordingly, a nuanced approach to alien species is essential. Cost assessment of losses deriving from ecological damage was only possible in a few cases. Ongoing, multi-year studies incorporating cost/benefit analysis will be necessary to resolve remaining issues.
Prostaglandin E2 (PGE2) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE2 pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE2 receptors) is essential. We therefore examined the production of PGE2 in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE2 on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE2 receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE2 synthesis was determined by mass spectrometry, cell proliferation by DNA [3H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE2 into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE2-dependent proliferation. Exogenously added PGE2 stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE2 was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE2 release, which stimulates cell proliferation via the EP1 receptor.
Herman P. Schwan [1915–2005] was a distinguished scientist and engineer, and a founding father of the field of biomedical engineering. A man of integrity, Schwan influenced the lives of many, including his wife and children, and his many students and colleagues. Active in science until nearly the end of his life, he will be very much missed by his family and many colleagues.
High-throughput gene trapping is a random approach for inducing insertional mutations across the mouse genome. This approach uses gene trap vectors that simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag for the rapid identification of the disrupted gene. Gene trapping has been used by both public and private institutions to produce libraries of embryonic stem (ES) cells harboring mutations in single genes. Presently,~ 66% of the protein coding genes in the mouse genome have been disrupted by gene trap insertions. Among these, however, genes encoding signal peptides or transmembrane domains (secretory genes) are underrepresented because they are not susceptible to conventional trapping methods. Here, we describe a high-throughput gene trapping strategy that effectively targets secretory genes. We used this strategy to assemble a library of ES cells harboring mutations in 716 unique secretory genes, of which 61% were not trapped by conventional trapping, indicating that the two strategies are complementary. The trapped ES cell lines, which can be ordered from the International Gene Trap Consortium (http://www.genetrap.org), are freely available to the scientific community.
Background: Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV) has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. Results: We inserted the coding sequences for green fluorescent protein (GFP) into the proline-rich region (PRR) of the ecotropic envelope protein (Env) and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP) and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. Conclusions: Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.
We have isolated the human protein SNEV as downregulated in replicatively senescent cells. Sequence homology to the yeast splicing factor Prp19 suggested that SNEV might be the orthologue of Prp19 and therefore might also be involved in pre-mRNA splicing. We have used various approaches including gene complementation studies in yeast using a temperature sensitive mutant with a pleiotropic phenotype and SNEV immunodepletion from human HeLa nuclear extracts to determine its function. A human–yeast chimera was indeed capable of restoring the wild-type phenotype of the yeast mutant strain. In addition, immunodepletion of SNEV from human nuclear extracts resulted in a decrease of in vitro pre-mRNA splicing efficiency. Furthermore, as part of our analysis of protein–protein interactions within the CDC5L complex, we found that SNEV interacts with itself. The self-interaction domain was mapped to amino acids 56–74 in the protein's sequence and synthetic peptides derived from this region inhibit in vitro splicing by surprisingly interfering with spliceosome formation and stability. These results indicate that SNEV is the human orthologue of yeast PRP19, functions in splicing and that homo-oligomerization of SNEV in HeLa nuclear extract is essential for spliceosome assembly and that it might also be important for spliceosome stability.
In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase a (pol alpha) and Klenow fragment (exo-) of DNA polymerase I (Escherichia coli ). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electronwithdrawing (trifluoromethyl and dinitro). Both pol a and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol a and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol a and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol {alpha} and Klenow fragment.
Lesion of the rat entorhinal cortex denervates the outer molecular layer of the fascia dentata followed by layer-specific axonal sprouting of uninjured fibers in the denervated zone. One of the candidate molecules regulating the laminar-specific sprouting response in the outer molecular layer is the transmembrane chondroitin sulfate proteoglycan NG2. NG2 is found in glial scars and has been suggested to impede axonal regeneration following injury of the spinal cord. The present study adressed the question whether NG2 could also regulate axonal growth in denervated areas of the brain. Therefore, (1) changes in NG2 mRNA and NG2 protein levels, (2) the cellular and the extracellular localisation of the molecule, (3) the identity of NG2 expressing cells, and (4) the generation of NG2-positive cells were studied in the rat fascia dentata before and following entorhinal deafferentation. Laser microdissection was employed to selectively harvest the denervated molecular layer and combined with quantitative reverse transcription-PCR to measure changes in NG2 mRNA amount (6h, 12h, 2d, 4d, 7d post lesion). The study revealed increases of NG2 mRNA at day 2 (2.5-fold) and day 4 (2-fold) post lesion. Immunocytochemistry was used to detect changes in NG2 protein distribution (1d, 4d, 7d, 10d, 14d, 30d, 6 months post lesion). NG2 staining was increased in the denervated outer molecular layer at 1 day post lesion, reached a maximum at 10 days post lesion, and returned to control levels within 6 month. Interestingly, the accumulation of NG2 protein was strongly restricted to the denervated outer molecular layer forming a border to the unaffected inner molecular layer. Using electron microscopy, NG2-immunoprecipitate was localized not only on glial surfaces and in the extracellular matrix but also in the vicinity of neuronal profiles indicating that NG2 is secreted following denervation. Double-labelings of NG2-immunopositive cells with markers for astrocytes, microglia/macrophages, and oligodendrocytes suggested that NG2-cells are a distinct glial subpopulation before and after entorhinal deafferentation. Bromodeoxyuridine-labeling revealed that some of the NG2-positive cells are postlesional generated. Taken together, the data revealed a layer-specific upregulation of NG2 in the denervated outer molecular layer of the fascia dentata that coincides with the sprouting response of uninjured fibers. This suggests that NG2 could regulate lesion-induced axonal growth in denervated areas of the brain.
Mitochondial NADH:ubiquinone oxidoreductase (complex I) the largest multiprotein enzyme of the respiratory chain, catalyses the transfer of two electrons from NADH to ubiquinone, coupled to the translocation of four protons across the membrane. In addition to the 14 strictly conserved central subunits it contains a variable number of accessory subunits. At present, the best characterized enzyme is complex I from bovine heart with a molecular mass of about 980 kDa and 32 accessory proteins. In this study, the subunit composition of mitochondrial complex I from the aerobic yeast Y. lipolytica has been analysed by a combination of proteomic and genomic approaches. The sequences of 37 complex I subunits were identified. The sum of their individual molecular masses (about 930 kDa) was consistent with the native molecular weight of approximately 900 kDa for Y. lipolytica complex I obtained by BN-PAGE. A genomic analysis with Y. lipolytica and other eukaryotic databases to search for homologues of complex I subunits revealed 31 conserved proteins among the examined species. A novel protein named “X” was found in purified Y. lipolytica complex I by MALDI-MS. This protein exhibits homology to the thiosulfate sulfurtransferase enzyme referred to as rhodanese. The finding of a rhodanese-like protein in isolated complex I of Y. lipolytica allows to assume a special regulatory mechanism of complex I activity through control of the status of its iron-sulfur clusters. The second part of this study was aimed at investigating the possible role of one of these extra subunits, 39 kDa (NUEM) subunit which is related to the SDRs-enzyme family. The members of this family function in different redox and isomerization reactions and contain a conserved NAD(P)H-binding site. It was proposed that the 39 kDa subunit may be involved in a biosynthetic pathway, but the role of this subunit in complex I is unknown. In contrast to the situation in N. crassa, deletion of the 39 kDa encoding gene in Y. lipolytica led to the absence of fully assembled complex I. This result might indicate a different pathway of complex I assembly in both organisms. Several site-directed mutations were generated in the nucleotide binding motif. These had either no effect on enzyme activity and NADPH binding, or prevented complex I assembly. Mutations of arginine-65 that is located at the end of the second b-strand and responsible for selective interaction with the 2’-phosphate group of NADPH retained complex I activity in mitochondrial membranes but the affinity for the cofactor was markedly decreased. Purification of complex I from mutants resulted in decrease or loss of ubiquinone reductase activity. It is very likely that replacement of R65 not only led to a decrease in affinity for NADPH but also caused instability of the enzyme due to steric changes in the 39 kDa subunit. These data indicate that NADPH bound to the 39 kDa subunit (NUEM) is not essential for complex I activity, but probably involved in complex I assembly in Y. lipolytica.
The thesis entitled „Investigations on the significance of nucleo-cytoplasmic transport for the biological function of cellular proteins" aimed to unreveal molecular mechanisms in order to improve our understanding of the impact of nucleo-cytoplasmic transport on cellular functions. Within the scope of this work, it could be shown that regulated nucleo-cytoplasmic transport of a subfamily of homeobox transcription factors controlled their intra- and intercellular transport, and thereby influencing also their transcriptional activity. This study describes a novel regulatory mechanism, which could in general play an important role for the ordered differentiation of complex organisms. Besides cis-active transport Signals, also post-translational modifications can influence the localization and biological activity of proteins in trans. In addition to the known impact of phosphorylation on the transport and activity of STAT1, experimental evidence was provided demonstrating that acetylation affected the interaction of STAT1 with NF-kB p65, and subsequently modulated the expression of apoptosis-inducing NF-kB target genes. The impact of nucleo-cytoplasmic transport on the regulation of apoptosis was underlined by showing that the evolutionary conservation of a NES within the anti-apoptotic protein survivin plays an essential role for its dual function in the inhibition of apoptosis and ordered cell division. Since survivin is considered a bona fide cancer therapy target, these results strongly encourage future work to identify molecular decoys that specifically inhibit the nuclear export of survivin as novel therapeutics. In order to further dissect the regulation of nuclear transport and to efficiently identify transport inhibitors, cell-based assays are urgently required. Therefore, the cellular assay Systems developed in this work may not only serve to identify synthetic nuclear export and Import inhibitors but may also be applied in systematic RNAi-screening approaches to identify novel components of the transport machinery. In addition, the translocation based protease- and protein-interaction biosensors can be applied in various biological Systems, in particular to identify protein-protein interaction inhibitors of cancer relevant proteins. In summary, this work does not only underline the general significance of nucleo-cytoplasmic transport for cell biology, but also demonstrates its potential for the development of novel therapies against diseases like cancer and viral infections.
Calcium-activated potassium channels are fundamental regulators of neuron excitability. SK channels are activated by an intracellular increase of Ca++ (such as occurs during an action potential). They have a small single channel conductance (less than 20pS) and show no voltage dependence of activation. To date, there are only a few examples of high-resolution structures of eukaryotic membrane proteins. All of them were purified from natural sources. Since no abundant natural sources of eukaryotic K+ channels are available we overexpressed rSK2 in order to produce the quantities necessary for structural analysis. Unfortunately the Pichia pastoris expression system did not yield sufficient amount of pure protein, mainly because most of the protein was retained by in the ER and was only partially soluble. Subsequently, two constructs were expressed: SK2-FCYENE (containing a specific sequence that promotes surface expression), and SK2-q-CaM a concatamer of SK2 and calmodulin. Although these proved an improvement in terms of solubilisation, little improvement was found in terms of amounts of purified material obtained. For this reason we tested the Semliki Forest virus expression system, since the protein is expressed in a mammalian system where we hoped that it would be trafficked in the same way as in vivo. Using this system it was possible to express rSK2 and solubilise it with several detergents and to achieve much better purification. However, the levels were still not sufficient for high-resolution structural studies, although sufficient for single particle electron microscopy analysis.
DCD – a novel plant specific domain in proteins involved in development and programmed cell death
(2005)
Background: Recognition of microbial pathogens by plants triggers the hypersensitive reaction, a common form of programmed cell death in plants. These dying cells generate signals that activate the plant immune system and alarm the neighboring cells as well as the whole plant to activate defense responses to limit the spread of the pathogen. The molecular mechanisms behind the hypersensitive reaction are largely unknown except for the recognition process of pathogens. We delineate the NRP-gene in soybean, which is specifically induced during this programmed cell death and contains a novel protein domain, which is commonly found in different plant proteins.
Results: The sequence analysis of the protein, encoded by the NRP-gene from soybean, led to the identification of a novel domain, which we named DCD, because it is found in plant proteins involved in d evelopment and c ell d eath. The domain is shared by several proteins in the Arabidopsis and the rice genomes, which otherwise show a different protein architecture. Biological studies indicate a role of these proteins in phytohormone response, embryo development and programmed cell by pathogens or ozone.
Conclusion: It is tempting to speculate, that the DCD domain mediates signaling in plant development and programmed cell death and could thus be used to identify interacting proteins to gain further molecular insights into these processes.
Prion diseases, also called transmissible spongiform encephalopathies, are a group of fatal neurodegenerative conditions that affect humans and a wide variety of animals. To date there is no therapeutic or prophylactic approach against prion diseases available. The causative infectious agent is the prion, also termed PrPSc, which is a pathological conformer of a cellular protein named prion protein PrPc. Prions are thought to multiply upon conversion of PrPc to PrPSc in a self-propagating manner. Immunotherapeutic strategies directed against PrPc represent a possible approach in preventing or curing prion diseases. Accordingly, it was already shown in animal models, that passive immunization delays the onset of prion diseases. The present thesis aimed at the development of a candidate vaccine towards the active immunization against prion diseases, an immune response, which has to be accompanied by the circumvention of host tolerance to the self-antigen PrPc. The vaccine development was approached using virus-like particles (retroparticles) derived from either the murine leukemia (MLV) or the human immunodeficiency virus (HIV). The display of PrP on the surface of such particles was addressed for both the cellular and the pathogenic form of PrP. The display of PrPc was achieved by either fusion to the transmembrane domain of the platelet derived growth factor receptor (PDGFR) or to the N-terminal part of the viral envelope protein (Env). In both cases, the corresponding PrPD- and PrPE-retroparticles were successfully produced and analyzed via immune fluorescence, Western Blot analysis, immunogold electron microscopy as well as by ELISA methods. Both, PrPD- and PrPE-retroparticles showed effective incorporation of N-terminally truncated forms of PrPc but not for the complete protein. PrPc at this revealed the typical glycosylation pattern, which was specifically removed by a glycosidase enzyme. Upon display of PrPc on retroparticles the protein remained detectable by PrP-specific antibodies under native conditions. Electron microscopy analysis of PrPc-variants revealed no alteration of the characteristic retroviral morphology of the generated particles. MLV-derived PrPD-retroparticles were successfully used in immunization studies. Contrary to approaches using bacterially expressed PrPc, the immunization of mice resulted in a specific antibody response. The display of the pathogenic isoform was aimed by two different strategies. The first one was directed at the conversion of the proteinase K (PK) sensitive from of PrP on the surface of PrPD-retroparticles into the PK resistant form. Albeit specific adaption of the PK digestion assay detecting resistant PrP, no PrP conversion was observed for PrPD-retroparticles. The second approach utilized a replication competent variant of the ecotropic MLV displaying PrPc on the viral Env protein. This MLV variant was stable in cell culture for six passages but did not replicate on scrapie-infected, PrPSc-propagating neuroblastoma cells. Thus, besides PrPc-displaying virus-like particles a replication competent MLV variant was obtained, which stably incorporated PrPc at the N-terminus of the viral Env protein. The incorporation of the cell-surface located PrPc into particles was expected from previously obtained data on protein display in the context of retrovirus-derived particles. Thus, the lack of incorporation observed for the complete PrPc sequence was rather unexpected and was found to be inhibited at both, fusion to PDGFR and the viral Env. In contrast to N-terminally truncated PrPc, the complete PrPc was shown to exhibit increased cell surface internalization rates and half-life times eventually contributing to the observed results. The PrP-vaccination approach described in this work represents the first successful system inducing PrP-specific antibody responses against the prion protein in wt mice. Explanations at this are based on the induction of specific T cell help or effects of the innate immunity, respectively. MLV-and HIV-derived particles bearing the PrP-coding sequence or being replication competent variants generated during this thesis might help to further improve the PrP-specific immune response.