Biomedical subjects

Klaus Richter

Publications and source records attributed to Klaus Richter.

At least 19 recordsLinked to original sources

PubMed2007

Expression profiling of vitamin D treated primary human keratinocytes.

Vitamin D has attracted much attention by its ability to stop cell proliferation and induce differentiation, which became of particular interest for the treatment of cancer and psoriasis. We performed an expression profile of 12 hours and 24 hours 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) treated primary human keratinocytes, to determine the changes in gene expression induced by the steroid in order to improve our understanding of the biological activity of 1alpha,25(OH)(2)D(3). This we expect to be useful for establishing a test system for vitamin D analogs or might open new therapeutic targets or uses for the hormone. For the filter array experiments a non-redundant set of 2135 sequence verified EST clones was used. The normalized raw data of 2 filters per time point were combined and subjected to SAM analysis to further increase the statistical significance. 86 positive and 50 negative genes were identified after 12 h. The numbers went down to 43 positive and 1 negative gene after 24 h of treatment. Fifteen genes are up-regulated over a longer period of time (12 h and 24 h). Results were verified by real-time PCR and/or Northern blots. Targets identified are involved in intracellular signaling, transcription, cell cycle, metabolism, cellular growth, constitution of the extracellular matrix or the cytoskeleton and apoptosis, immune responses, and DNA repair, respectively. Expression profiles showed an initial stop of proliferation and induction of differentiation, and resumed proliferation after prolonged incubation, most likely due to degradation of the hormone.

Base Sequence↗

PubMed2006

Statistical description of eigenfunctions in chaotic and weakly disordered systems beyond universality.

We present a semiclassical approach to eigenfunction statistics in chaotic and weakly disordered quantum systems which goes beyond random matrix theory, supersymmetry techniques, and existing semiclassical methods. The approach is based on a generalization of Berry's random wave model, combined with a consistent semiclassical representation of spatial two-point correlations. We derive closed expressions for arbitrary wave-function averages in terms of universal coefficients and sums over classical paths, which contain, besides the supersymmetry results, novel oscillatory contributions. Their physical relevance is demonstrated in the context of Coulomb blockade physics.

Journal Article↗

PubMed2006

hsp90: twist and fold.

Molecular chaperones are cellular machines that facilitate protein folding. The crystal structures of HtpG, the Escherichia coli homolog of hsp90, reported in this issue (Shiau et al., 2006) together with the recently published structures of an hsp90-cochaperone complex (Ali et al., 2006) and an hsp90-client protein complex (Vaughan et al., 2006), reveal exciting insights into the hsp90 reaction cycle.

Adenine Nucleotides↗

PubMed2006

Dynamical symmetry breaking in transport through molecules.

We analyze the interplay between vibrational and electronic degrees of freedom in charge transport across a molecular single-electron transistor. We focus on the wide class of molecules which possess quasidegenerate vibrational eigenstates, while no degeneracy occurs for their anionic configuration. We show that the combined effect of a thermal environment and coupling to leads, involving tunneling events charging and discharging the molecule, leads to a dynamical symmetry breaking where quasidegenerate eigenstates acquire different occupations. This imbalance gives rise to a characteristic asymmetry of the current versus an applied gate voltage.

Journal Article↗

PubMed2006

NADP-dependent mannitol dehydrogenase, a major allergen of Cladosporium herbarum.

Cladosporium herbarum is an important allergenic fungal species that has been reported to cause allergic diseases in nearly all climatic zones. 5-30% of the allergic population displays IgE antibodies against molds. Sensitization to Cladosporium has often been associated with severe asthma and less frequently with chronic urticaria and atopic eczema. However, no dominant major allergen of this species has been found so far. We present cloning, production, and characterization of NADP-dependent mannitol dehydrogenase of C. herbarum (Cla h 8) and show that this protein is a major allergen that is recognized by IgE antibodies of approximately 57% of all Cladosporium allergic patients. This is the highest percentage of patients reacting with any Cladosporium allergen characterized so far. Cla h 8 was purified to homogeneity by standard chromatographic methods, and both N-terminal and internal amino acid sequences of protein fragments were determined. Enzymatic analysis of the purified natural protein revealed that this allergen represents a NADP-dependent mannitol dehydrogenase that interconverts mannitol and d-fructose. It is a soluble, non-glycosylated cytoplasmic protein. Two-dimensional protein analysis indicated that mannitol dehydrogenase is present as a single isoform. The cDNA encoding Cla h 8 was cloned from a lambda-ZAP library constructed from hyphae and spores. The recombinant non-fusion protein was expressed in Escherichia coli and purified to homogeneity. Its immunological and biochemical identity with the natural protein was shown by enzyme activity tests, CD spectroscopy, IgE immunoblots with sera of patients, and by skin prick testing of Cladosporium allergic patients. This protein therefore is a new major allergen of C. herbarum.

Alcohol Oxidoreductases↗

PubMed2006

Expression profiling of aging in the human skin.

During the last years it was shown that the aging process is controlled by specific genes in a large number of organisms (C. elegans, Drosophila, mouse or humans). To investigate genes involved in the natural aging process of the human skin we applied cDNA microarray analysis of naturally aged human foreskin samples. For the array experiments a non-redundant set of 2135 pre-selected EST clones was used. These arrays were used to probe the patterns of gene expression in naturally aged human skin of five young (3-4 years of age) and five old (68-72 years of age) healthy persons. We found that in total 105 genes change their expression over 1.7-fold during the aging process in the human skin. Of these 43 genes were shown to be down-regulated in contrast to 62 up-regulated genes. Expression of regulated genes was confirmed by real-time PCR. These results suggest that the aging process in the human skin is connected with the deregulation of various cellular processes, like cell cycle control, cytoskeletal changes, inflammatory response, signaling and metabolism.

Aged↗

PubMed2006

Intrinsic inhibition of the Hsp90 ATPase activity.

The molecular chaperone Hsp90 is required for the folding and activation of a large number of substrate proteins. These are involved in essential cellular processes ranging from signal transduction to viral replication. For the activation of its substrates, Hsp90 binds and hydrolyzes ATP, which is the key driving force for conformational conversions within the dimeric chaperone. Dimerization of Hsp90 is mediated by a C-terminal dimerization site. In addition, there is a transient ATP-induced dimerization of the two N-terminal ATP-binding domains. The resulting ring-like structure is thought to be the ATPase-active conformation. Hsp90 is a slow ATPase with a turnover number of 1 ATP/min for the yeast protein. A key question for understanding the molecular mechanism of Hsp90 is how ATP hydrolysis is regulated and linked to conformational changes. In this study, we analyzed the activation process structurally and biochemically with a view to identify the conformational limitations of the ATPase reaction cycle. We showed that the first 24 amino acids stabilize the N-terminal domain in a rigid state. Their removal confers flexibility specifically to the region between amino acids 98 and 120. Most surprisingly, the deletion of this structure results in the complete loss of ATPase activity and in increased N-terminal dimerization. Complementation assays using heterodimeric Hsp90 show that this rigid lid acts as an intrinsic kinetic inhibitor of the Hsp90 ATPase cycle preventing N-terminal dimerization in the ground state. On the other hand, this structure acts, in concert with the 24 N-terminal amino acids of the other N-terminal domain, to form an activated ATPase and thus regulates the turnover number of Hsp90.

Adenosine Triphosphatases↗

PubMed2006

Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library.

The clinical presentation of non-pollen related allergy to hazelnut can be severe and systemic. So far, only a limited number of non-pollen related hazelnut allergens have been identified and characterized. The aim of this study was to identify and clone new hazelnut allergens. A lambda ZAP cDNA library of hazelnut was constructed. The library was screened with serum of six hazelnut allergic patients displaying different IgE-binding patterns on hazelnut immunoblot. Rapid amplification of cDNA ends (RACE) protocols were applied to obtain full-length clones. Expression experiments were carried out in Eschericchia coli. Expression was monitored by SDS-PAGE, protein staining and immunoblotting. A hazelnut cDNA library was constructed. IgE screening resulted in the cloning of two isoforms of a novel putative hazelnut allergen. The clones were identified as oleosins, with theoretical molecular masses of 16.7 and 14.7 kDa and pI of 10.5 and 10.0, respectively. The isoforms demonstrated only 37% amino acid sequence identity but contained the typical hydrophobic stretch in the middle of the protein (53% identity) with the characteristic oleosin proline knot region (11/12 amino acids identical). Expression in E. coli of the longer isoform resulted in a clear band on SDS-PAGE. The expressed protein was recognized on an immunodot blot by IgE from serum that was used for screening the cDNA library. Hazelnut contains multiple isoforms of oleosin. IgE binding of a hazelnut-allergic patient to a recombinant version suggest that hazelnut oleosin is an allergen, as has been described for peanut and sesame.

Amino Acid Sequence↗

PubMed2006

An improved mRFP1 adds red to bimolecular fluorescence complementation.

Protein-protein interactions are fundamental to virtually every aspect of cellular functions. Blue, green and yellow bimolecular fluorescence complementation (BiFC) systems based on GFP and its variants allow the investigation of protein-protein interactions in vivo. We have developed the first red BiFC system based on an improved monomeric red fluorescent protein (mRFP1-Q66T), expanding the range of possible applications for BiFC.

Gene Expression Profiling↗

PubMed2005

Fibrinogen substrate recognition by staphylocoagulase.(pro)thrombin complexes.

Thrombin generation and fibrinogen (Fbg) clotting are the ultimate proteolytic reactions in the blood coagulation pathway. Staphylocoagulase (SC), a protein secreted by the human pathogen Staphylococcus aureus, activates prothrombin (ProT) without proteolysis. The SC.(pro)thrombin complex recognizes Fbg as a specific substrate, converting it directly into fibrin. The crystal structure of a fully active SC fragment containing residues 1-325 (SC-(1-325)) bound to human prethrombin 2 showed previously that SC inserts its Ile(1)-Val(2) N terminus into the Ile(16) pocket of prethrombin 2, inducing a functional active site in the cognate zymogen conformationally. Exosite I of alpha-thrombin, the Fbg recognition site, and proexosite I on ProT are blocked by domain 2 of SC-(1-325). In the present studies, active site-labeled fluorescent ProT analogs were used to quantitate Fbg binding to the SC-(1-325).ProT complex. Fbg binding and cleavage are mediated by expression of a new Fbg-binding exosite on the SC-(1-325).ProT complex, resulting in formation of an (SC-(1-325).ProT)(2).Fbg pentameric complex with a dissociation constant of 8-34 nm. In both crystal structures, the SC-(1-325).(pre)thrombin complexes form dimers, with both proteinases/zymogens facing each other over a large U-shaped cleft, through which the Fbg substrate could thread. On this basis, a molecular model of the pentameric (SC-(1-325).thrombin)(2).Fbg encounter complex was generated, which explains the coagulant properties and efficient Fbg conversion. The results provide new insight into the mechanism that mediates high affinity Fbg binding and cleavage as a substrate of SC.(pro)thrombin complexes, a process that is central to the molecular pathology of S. aureus endocarditis.

Amino Acid Sequence↗

PubMed2005

The activation mechanism of Hsp26 does not require dissociation of the oligomer.

Small heat shock proteins (sHsps) are molecular chaperones that specifically bind non-native proteins and prevent them from irreversible aggregation. A key trait of sHsps is their existence as dynamic oligomers. Hsp26 from Saccharomyces cerevisiae assembles into a 24mer, which becomes activated under heat shock conditions and forms large, stable substrate complexes. This activation coincides with the destabilization of the oligomer and the appearance of dimers. This and results from other groups led to the generally accepted notion that dissociation might be a requirement for the chaperone mechanism of sHsps. To understand the chaperone mechanism of sHsps it is crucial to analyze the relationship between chaperone activity and stability of the oligomer. We generated an Hsp26 variant, in which a serine residue of the N-terminal domain was replaced by cysteine. This allowed us to covalently crosslink neighboring subunits by disulfide bonds. We show that under reducing conditions the structure and function of this variant are indistinguishable from that of the wild-type protein. However, when the cysteine residues are oxidized, the dissociation into dimers at higher temperatures is no longer observed, yet the chaperone activity remains unaffected. Furthermore, we show that the exchange of subunits between Hsp26 oligomers is significantly slower than substrate aggregation and even inhibited in the presence of disulfide bonds. This demonstrates that the rearrangements necessary for shifting Hsp26 from a low to a high affinity state for binding non-native proteins occur without dissolving the oligomer.

Amino Acid Substitution↗

PubMed2005

A central role of Arabidopsis thaliana ovate family proteins in networking and subcellular localization of 3-aa loop extension homeodomain proteins.

The organization of living cells is based on networks of interacting molecules. Systematic analysis of protein interactions of 3-aa loop extension (TALE) homeodomain proteins, fundamental regulators of plant meristem function and leaf development, revealed a highly connected, complex network. The network includes nine members of Arabidopsis thaliana ovate family proteins (AtOFPs), a plant-specific protein family, indicating a close functional connection to TALE homeodomain proteins. Evidence is provided that AtOFP1 is an essential pleiotropic developmental regulator. AtOFP1 and AtOFP5 are shown to associate with the cytoskeleton and to regulate subcellular localization of TALE homeodomain proteins, suggesting a previously unrecognized control mechanism in plant development.

Agrobacterium tumefaciens↗

PubMed2005

Role of orbital dynamics in spin relaxation and weak antilocalization in quantum dots.

We develop a semiclassical theory for spin-dependent quantum transport to describe weak (anti)localization in quantum dots with spin-orbit coupling. This allows us to distinguish different types of spin relaxation in systems with chaotic, regular, and diffusive orbital classical dynamics. We find, in particular, that for typical Rashba spin-orbit coupling strengths, integrable ballistic systems can exhibit weak localization, while corresponding chaotic systems show weak antilocalization. We further calculate the magnetoconductance and analyze how the weak antilocalization is suppressed with decreasing quantum dot size and increasing additional in-plane magnetic field.

Journal Article↗

PubMed2005

Nonlinear resonant transport of Bose-Einstein condensates.

The coherent flow of a Bose-Einstein condensate through a quantum dot in a magnetic waveguide is studied. By the numerical integration of the time-dependent Gross-Pitaevskii equation in the presence of a source term, we simulate the propagation process of the condensate through a double barrier potential in the waveguide. We find that resonant transport is suppressed in interaction-induced regimes of bistability, where multiple scattering states exist at the same chemical potential and the same incident current. We demonstrate, however, that a temporal control of the external potential can be used to circumvent this limitation and to obtain enhanced transmission near the resonance on experimentally realistic time scales.

Journal Article↗

PubMed2005

Semiclassical form factor for spectral and matrix element fluctuations of multidimensional chaotic systems.

We present a semiclassical calculation of the generalized form factor Kab(tau) which characterizes the fluctuations of matrix elements of the operators a and b in the eigenbasis of the Hamiltonian of a chaotic system. Our approach is based on some recently developed techniques for the spectral form factor of systems with hyperbolic and ergodic underlying classical dynamics and f = 2 degrees of freedom, that allow us to go beyond the diagonal approximation. First we extend these techniques to systems with f > 2. Then we use these results to calculate Kab(tau). We show that the dependence on the rescaled time tau (time in units of the Heisenberg time) is universal for both the spectral and the generalized form factor. Furthermore, we derive a relation between Kab(tau) and the classical time-correlation function of the Weyl symbols of a and b.

Journal Article↗

PubMed2004

Optimized RNA amplification using T7-RNA-polymerase based in vitro transcription.

The use of expression profiling to explore a cell's transcriptional landscape has exploded in recent years. In many cases, however, the very limited amount of starting material poses a major problem, making the amplification of the isolated RNA obligatory. The most prominent amplification method used was developed by the Eberwine lab in 1990: cDNA synthesis is started with an oligo(dT) primer containing a T7 RNA polymerase promoter. After second-strand synthesis RNA is transcribed in vitro using T7 RNA polymerase. It has been demonstrated that antisense RNA amplification not only preserves the fidelity of RNA-based microarray analysis but even improves the sensitivity. In our aim to improve the yield of in vitro transcription reactions and to facilitate the use of amplified RNA for the construction of cDNA libraries we tested a series of T7 primers with different 3' flanking sequences containing restriction sites. In addition we tested the impact of different DNA polymerases used for synthesizing the templates on the efficiency of the in vitro transcription reaction. A total of 28 different oligo(dT)-T7 promoter primers were tested. Two of them showed a dramatically increased yield of RNA from the in vitro transcription reaction. The combination of the improved second-strand synthesis with the new T7 primer increased the RNA yield 60-fold compared to the yield of standard procedures.

3' Flanking Region↗

PubMed2004

The Co-chaperone Sba1 connects the ATPase reaction of Hsp90 to the progression of the chaperone cycle.

The molecular chaperone Hsp90 mediates the ATP-dependent activation of a large number of proteins involved in signal transduction. During this process, Hsp90 was found to associate transiently with several accessory factors, such as p23/Sba1, Hop/Sti1, and prolyl isomerases. It has been shown that ATP hydrolysis triggers conformational changes within Hsp90, which in turn are thought to mediate conformational changes in the substrate proteins, thereby causing their activation. The specific role of the partner proteins in this process is unknown. Using proteins from Saccharomyces cerevisiae, we characterized the interaction of Hsp90 with its partner protein p23/Sba1. Our results show that the nucleotide-dependent N-terminal dimerization of Hsp90 is necessary for the binding of Sba1 to Hsp90 with an affinity in the nanomolar range. Two Sba1 molecules were found to bind per Hsp90 dimer. Sba1 binding to Hsp90 resulted in a decreased ATPase activity, presumably by trapping the hydrolysis state of Hsp90ATP. Ternary complexes of Hsp90Sba1 could be formed with the prolyl isomerase Cpr6, but not with Sti1. Based on these findings, we propose a model that correlates the ordered assembly of the Hsp90 co-chaperones with distinct steps of the ATP hydrolysis reaction during the chaperone cycle.

Adenosine Triphosphatases↗

PubMed2004

Semiclassical construction of random wave functions for confined systems.

We develop a statistical description of chaotic wave functions in closed systems obeying arbitrary boundary conditions by combining a semiclassical expression for the spatial two-point correlation function with a treatment of eigenfunctions as Gaussian random fields. Thereby we generalize Berry's isotropic random wave model by incorporating confinement effects through classical paths reflected at the boundaries. Our approach allows one to explicitly calculate highly nontrivial statistics, such as intensity distributions, in terms of usually few short orbits, depending on the energy window considered. We compare with numerical quantum results for the Africa billiard and derive nonisotropic random wave models for other prominent confinement geometries.

Journal Article↗