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Biomedical subjects

Dirk Schneider

Publications and source records attributed to Dirk Schneider.

At least 19 recordsLinked to original sources
PubMed2006

PetG and PetN, but not PetL, are essential subunits of the cytochrome b6f complex from Synechocystis PCC 6803.

The cytochrome b(6)f complex consists of four large core subunits and an additional four low molecular weight subunits, the function of which is elusive thus far. Here we sought to determine whether small subunits PetG, PetL, and PetN are essential for a cyanobacterial cytochrome b(6)f complex. We found that only PetL is dispensable, whereas PetG and PetN appear to be essential. Possible roles of the small cytochrome b(6)f complex subunits are discussed, and observations from our study are compared with previous findings.

Bacterial Proteins↗
PubMed2006

Ssr2998 of Synechocystis sp. PCC 6803 is involved in regulation of cyanobacterial electron transport and associated with the cytochrome b6f complex.

To analyze the function of a protein encoded by the open reading frame ssr2998 in Synechocystis sp. PCC 6803, the corresponding gene was disrupted, and the generated mutant strain was analyzed. Loss of the 7.2-kDa protein severely reduced the growth of Synechocystis, especially under high light conditions, and appeared to impair the function of the cytochrome b6 f complex. This resulted in slower electron donation to cytochrome f and photosystem 1 and, concomitantly, over-reduction of the plastoquinone pool, which in turn had an impact on the photosystem 1 to photosystem 2 stoichiometry and state transition. Furthermore, a 7.2-kDa protein, encoded by the open reading frame ssr2998, was co-isolated with the cytochrome b6 f complex from the cyanobacterium Synechocystis sp. PCC 6803. ssr2998 seems to be structurally and functionally associated with the cytochrome b6 f complex from Synechocystis, and the protein could be involved in regulation of electron transfer processes in Synechocystis sp. PCC 6803.

Amino Acid Sequence↗
PubMed2006

High level expression of a protein precursor for functional studies.

In vitro analyses of type I signal peptidase activities require protein precursors as substrates. Usually, these pre-proteins are expressed in vitro and cleavage of the signal sequence is followed by SDS polyacrylamide gel electrophoresis coupled with autoradiography. Radioactive amino acids have to be incorporated in the expressed protein, since the amount of the in vitro expressed protein is usually very low and processing of the signal peptide cannot be followed by SDS polyacrylamide gel electrophoresis alone. Here we describe a rapid and simple method to express large amounts of a protein precursor in E. coli. We have analyzed the effect of ionophors as well as of azide on the accumulation of expressed protein precursors. Azide blocks the function of SecA and the ionophors dissipate the electrochemical gradient across the cytoplasmic membrane of E. coli. Addition of azide ions resulted in the formation of inclusion bodies, highly enriched with pre-apo-plastocyanine. Plastocyanine is a soluble copper protein, which can be found in the periplasmic space of cyanobacteria as well as in the thylakoid lumen of cyanobacteria and chloroplasts, and the pre-protein contains a cleavable signal sequence at its N-terminus. After purification of cyanobacterial preapo-plastocyanine, its signal sequence can be cleaved off by the E. coli signal peptidase, and protein processing was followed on Coomassie stained SDS polyacrylamide gels. We are optimistic that the presented method can be further developed and applied.

Amino Acid Sequence↗
PubMed2006

Assembly of a transmembrane b-type cytochrome is mainly driven by transmembrane helix interactions.

Folding, assembly and stability of alpha-helical membrane proteins is still not very well understood. Several of these membrane proteins contain cofactors, which are essential for their function and which can be involved in protein assembly and/or stabilization. The effect of heme binding on the assembly and stability of the transmembrane b-type cytochrome b'559 was studied by fluorescence resonance energy transfer. Cytochrome b'559 consists of two monomers of a 44 amino acid long polypeptide, which contains one transmembrane domain. The synthesis of two variants of the b'559 monomer, each carrying a specific fluorescent dye, allowed monitoring helix-helix interactions in micelles by resonance energy transfer. The measurements demonstrate that the transmembrane peptides dimerize in detergent in the absence and presence of the heme cofactor. Cofactor binding only marginally enhances dimerization and, apparently, the redox state of the heme group has no effect on dimerization.

Amino Acid Sequence↗
PubMed2006

The stability of transmembrane helix interactions measured in a biological membrane.

Despite some promising progress in the understanding of membrane protein folding and assembly, there is little experimental information regarding the thermodynamic stability of transmembrane helix interactions and even less on the stability of transmembrane helix-helix interactions in a biological membrane. Here we describe an approach that allows quantitative measurement of transmembrane helix interactions in a biological membrane, and calculation of changes in the interaction free energy resulting from substitution of single amino acids. Dimerization of several variants of the glycophorin A transmembrane domain are characterized and compared to the wild-type (wt) glycophorin A transmembrane helix dimerization. The calculated DeltaDeltaG(app) values are further compared with values found in the literature. In addition, we compare interactions between the wt glycophorin A transmembrane domain and helices in which critical glycine residues are replaced by alanine or serine, respectively. The data demonstrate that replacement of the glycine residues by serine is less destabilizing than replacement by alanine with a DeltaDeltaG(app) value of about 0.4 kcal/mol. Our study comprises the first measurement of a transmembrane helix interaction in a biological membrane, and we are optimistic that it can be further developed and applied.

Amino Acid Sequence↗
PubMed2006

[Post-marketing surveillance on treatment of 5,665 patients with atopic dermatitis using the calcineurin inhibitor pimecrolimus: positive effects on major symptoms of atopic dermatitis and on quality of life].

BACKGROUND: Topical application of the calcineurin inhibitors pimecrolimus and tacrolimus is a current major advance in the therapy of atopic dermatitis. The aims of this post-marketing surveillance were: a) to acquire data on the efficacy and tolerability of pimecrolimus ointment (Elidel) on a very large cohort of patients from outpatient clinics, and b) to assess changes in their quality of life, a parameter not often considered in previous studies. PATIENTS AND METHODS: Included were 5,665 patients with atopic dermatitis. During the observation period, data on efficacy and tolerability were obtained at the beginning of the study, 3 to 10 days after initiation of therapy and after 4 to 6 weeks. Evaluation of symptoms as well as assessment of efficacy and tolerability were based on linear scales and on the percentage of the body surface area (% BSA) involved. Quality of life was assessed by the German version of the "Dermatology Life Quality Index (DLQI)" or the "Children's Dermatology Life Quality Index (CDLQI)". RESULTS: In this largest post-marketing surveillance hitherto performed in Germany, the efficacy of pimecrolimus was judged as "good" by 79.3 % of physicians and by 76.5 % of patients. Tolerability was assessed as "good" by 87.2 % of physicians and by 83.1 % of patients. Major symptoms of atopic dermatitis such as pruritus, erythema or lichenification showed marked reduction after just 3 to 10 days, signalling general improvement of the skin disease. In addition, application of pimecrolimus resulted in a significant improvement of the quality of life scores in both children and adults. CONCLUSION: The present study demonstrates that the good efficacy and tolerability of pimecrolimus ointment which had been shown in controlled trials: i) could also be demonstrated on a very large cohort of patients with atopic dermatitis when used in the outpatient setting, and ii) were paralleled by a significant improvement in the quality of life.

Adolescent↗
PubMed2006

Benefits from the use of a pimecrolimus-based treatment in the management of atopic dermatitis in clinical practice. Analysis of a Swiss cohort.

BACKGROUND: Controlled studies established the efficacy and good tolerability of pimecrolimus cream 1% for the treatment of atopic dermatitis but they may not reflect real-life use. OBJECTIVE: To evaluate the efficacy, tolerability and cosmetic acceptance of a pimecrolimus-based regimen in daily practice in Switzerland. METHODS: This was a 6-month, open-label, multicentre study in 109 patients (55% > or = 18 years) with atopic dermatitis. Pimecrolimus cream 1% was incorporated into patients' standard treatment protocols. RESULTS: The pimecrolimus-based treatment was well tolerated and produced disease improvement in 65.7% of patients. It was particularly effective on the face (improvement rate: 75.0%). Mean pimecrolimus consumption decreased from 6.4 g/day (months 1-3) to 4.0 g/day (months 3-6) as disease improved. Most patients (74.1%) rated their disease control as 'complete' or 'good' and 90% were highly satisfied with the cream formulation. CONCLUSION: The use of a pimecrolimus-based regimen in everyday practice was effective, well tolerated and well accepted by patients.

Administration, Cutaneous↗
PubMed2006

Safety, efficacy, and dosage of 1% pimecrolimus cream for the treatment of atopic dermatitis in daily practice.

INTRODUCTION: Although several controlled clinical trials have demonstrated the efficacy and good tolerability of 1% pimecrolimus cream for the treatment of atopic dermatitis, the results of these trials may not apply to real-life usage. The objective of this study was to evaluate the safety and efficacy of a pimecrolimus-based regimen in daily practice. METHODS: This was a 6-month, open-label, multicenter study in 947 patients aged >or=3 months with atopic dermatitis of all severities. The investigators incorporated 1% pimecrolimus cream into patients' standard treatment protocols on the basis of their clinical diagnosis. Use of topical corticosteroids was allowed at the discretion of the physician. Safety and tolerability were evaluated by monitoring adverse events. Efficacy was evaluated by recording changes in the Investigators' Global Assessment scores and pruritus scores at each visit. RESULTS: No clinically unexpected adverse events were reported. The discontinuation rate for adverse events was 2.3%. The disease improvement rate was 53.7% at week 1 and 66.9% at week 24. The pimecrolimus-based regimen was particularly effective for the treatment of lesions involving the face (improvement rate: 61.9% at week 1 and 76.7% at week 24). The greatest therapeutic response was experienced by pediatric patients with mild or moderate disease. Nonetheless, 64% and 65% of infants and children, respectively, with severe/very severe facial disease at baseline were clear/almost clear of signs of atopic dermatitis on their face at week 24. In patients aged <18 years, most of the improvement occurred within the first week of treatment, while in adults a progressive improvement was observed over the entire study period. Worsening of disease by the end of the study occurred in 9.5% of patients and was most frequent in adults (12.6%). The discontinuation rate for unsatisfactory therapeutic effect was 4.8%. The mean number of treatment days was 135.6 (SD 53.2). The mean drug consumption (non-US centers only) was 4.2 g per treatment day. Drug consumption decreased over time as disease improved. In total, 47% of patients who completed the study never used topical corticosteroids over 6 months. CONCLUSION: In daily practice, incorporation of 1% pimecrolimus cream into patients' standard treatment regimen is well tolerated and improves atopic dermatitis in approximately two-thirds of patients. Disease improvement is particularly evident on the face. The greatest therapeutic response is experienced by pediatric patients with mild or moderate disease. In these patients, most of the improvement is observed within 1 week from the start of treatment.

Administration, Topical↗
PubMed2005

Multiple Rieske proteins in prokaryotes: where and why?

Many microbial genomes have been sequenced in the recent years. Multiple genes encoding Rieske iron-sulfur proteins, which are subunits of cytochrome bc-type complexes or oxygenases, have been detected in many pro- and eukaryotic genomes. The diversity of substrates, co-substrates and reactions offers obvious explanations for the diversity of the low potential Rieske proteins associated with oxygenases, but the physiological significance of the multiple genes encoding high potential Rieske proteins associated with the cytochrome bc-type complexes remains elusive. For some organisms, investigations into the function of the later group of genes have been initiated. Here, we summarize recent finding on the characteristics and physiological functions of multiple high potential Rieske proteins in prokaryotes. We suggest that the existence of multiple high potential Rieske proteins in prokaryotes could be one way of allowing an organism to adapt their electron transfer chains to changing environmental conditions.

Animals↗
PubMed2005

Defining the structural basis for assembly of a transmembrane cytochrome.

To define the structural basis for cofactor binding to membrane proteins, we introduce a manageable model system, which allows us, for the first time, to study the influence of individual transmembrane helices and of single amino acid residues on the assembly of a transmembrane cytochrome. In vivo as well as in vitro analyses indicate central roles of single amino acid residues for either interaction of the transmembrane helices or for binding of the cofactor. The results clearly show that interaction of the PsbF transmembrane helix is independent from binding of the heme cofactor. On the other hand, binding of the cofactor highly depends on helix-helix interactions. By site-directed mutagenesis critical amino acid residues were identified, which are involved in the assembly of a functional transmembrane cytochrome. Especially, a highly conserved glycine residue is critical for interaction of the transmembrane helices and assembly of the cytochrome. Based on the two-stage-model of alpha-helical membrane protein folding, the presented results clearly indicate a third stage of membrane protein folding, in which a cofactor binds to a pre-assembled transmembrane protein.

Amino Acid Sequence↗
PubMed2005

Functional implications of pigments bound to a cyanobacterial cytochrome b6f complex.

A highly purified cytochrome b(6)f complex from the cyanobacterium Synechocystis sp. PCC 6803 selectively binds one chlorophyll a and one carotenoid in analogy to the recent published structure from two other b(6)f complexes. The unknown function of these pigments was elucidated by spectroscopy and site-directed mutagenesis. Low-temperature redox difference spectroscopy showed red shifts in the chlorophyll and carotenoid spectra upon reduction of cytochrome b(6), which indicates coupling of these pigments with the heme groups and thereby with the electron transport. This is supported by the correlated kinetics of these redox reactions and also by the distinct orientation of the chlorophyll molecule with respect to the heme cofactors as shown by linear dichroism spectroscopy. The specific role of the carotenoid echinenone for the cytochrome b(6)f complex of Synechocystis 6803 was elucidated by a mutant lacking the last step of echinenone biosynthesis. The isolated mutant complex preferentially contained a carotenoid with 0, 1 or 2 hydroxyl groups (most likely 9-cis isomers of beta-carotene, a monohydroxy carotenoid and zeaxanthin, respectively) instead. This indicates a substantial role of the carotenoid - possibly for strucure and assembly - and a specificity of its binding site which is different from those in most other oxygenic photosynthetic organisms. In summary, both pigments are probably involved in the structure, but may also contribute to the dynamics of the cytochrome b(6)f complex.

Carotenoids↗
PubMed2004

Rendezvous in a membrane: close packing, hydrogen bonding, and the formation of transmembrane helix oligomers.

The interaction of transmembrane alpha-helices is promoted by a detailed fit between two helical surfaces, which results in close packing and van der Waals interactions of amino acid side chains between two helices. Recent studies additionally indicate an important role of hydrogen bonding for mediating and stabilizing transmembrane helix-helix interactions. The interplay between close packing and electrostatic interactions in influencing the specificity of helix-helix interactions on the one hand and the stability of an existing interaction on the other hand is still unknown. Here, we suggest that close packing mainly determines the specificity of a helix-helix interaction, whereas hydrogen bonding is important for stabilization of a preformed helix dimer.

Biopolymers↗
PubMed2004

Motifs of two small residues can assist but are not sufficient to mediate transmembrane helix interactions.

Both experimental and statistical searches for specific motifs that mediate transmembrane helix-helix interactions showed that two glycine residues separated by three intervening residues (GxxxG) provide a framework for specific interactions. Further work suggested that other motifs of small residues can mediate the interaction of transmembrane domains, so that the AxxxA-motif could also drive strong interactions of alpha-helices in soluble proteins. Thus, all these data indicate that a motif of two small residues in a distance of four might be enough to provide a framework for transmembrane helix-helix interaction. To test whether GxxxG is equivalent to (small)xxx(small), we investigated the effect of a substitution of either of the two Gly residues in the glycophorin A GxxxG-motif by Ala or Ser using the recently developed GALLEX system. The results of this mutational study demonstrate that, while a replacement of either of the two Gly by Ala strongly disrupts GpA homo-dimerization, the mutation to Ser partly stabilizes a dimeric structure. We suggest that the Ser residue can form a hydrogen bond with a backbone carbonyl group of the adjacent helix stabilizing a preformed homo-dimer. While (small)xxx(small) serves as a useful clue, the context of adjacent side-chains is essential for stable helix interaction, so each case must be tested.

Amino Acid Motifs↗
PubMed2004

PetC1 is the major Rieske iron-sulfur protein in the cytochrome b6f complex of Synechocystis sp. PCC 6803.

Many of the completely sequenced cyanobacterial genomes contain a gene family that encodes for putative Rieske iron-sulfur proteins. The Rieske protein is one of the large subunits of the cytochrome bc-type complexes involved in respiratory and photosynthetic electron transfer. In contrast to all other subunits of this complex that are encoded by single genes, the genome of the cyanobacterium Synechocystis PCC 6803 contains three petC genes, all encoding potential Rieske subunits. Most interestingly, any of the petC genes can be deleted individually without altering the Synechocystis phenotype dramatically. In contrast, double deletion experiments revealed that petC1 and petC2 cannot be deleted in combination, whereas petC3 can be deleted together with any of the other two petC genes. Further results suggest a different physiological function for each of the Rieske proteins. Whereas PetC2 can partly replace the dominating Rieske isoform PetC1, PetC3 is unable to functionally replace either PetC1 or PetC2 and may have a special function involving a special donor with a lower redox potential than plastoquinone. A predominant role of PetC1, which is (partly) different from PetC2, is suggested by the mutational analysis and a detailed characterization of the electron transfer reactions in the mutant strains.

Chlorophyll↗
PubMed2004

Long-term efficacy and safety of pimecrolimus cream 1% in adults with moderate atopic dermatitis.

BACKGROUND: Pimecrolimus cream 1% is a non-steroid, selective inflammatory cytokine inhibitor indicated for atopic dermatitis (AD). OBJECTIVE: To compare the safety and efficacy of pimecrolimus cream 1%-based treatment versus conventional therapy in adults with moderate AD. METHODS: Patients were randomized to receive pimecrolimus cream 1% (n = 62) or vehicle (n = 68) at the first signs/symptoms of AD, for 24 weeks as required. A moderately potent topical corticosteroid (prednicarbate 0.25% cream) was allowed in both groups to treat flares. RESULTS: Corticosteroids were required on fewer days in the pimecrolimus group, compared with the vehicle group (9.7 vs. 37.8%, p < 0.001). Furthermore, 59.7% of pimecrolimus-treated patients experienced no flares during the study period, compared with 22.1% of vehicle-treated patients (p < 0.001). Pimecrolimus cream 1% was well tolerated throughout the study. CONCLUSION: For adults with moderate AD, pimecrolimus cream 1% is well tolerated, reduces the incidence of flares, reduces/eliminates corticosteroid use, improves long-term disease control and enhances the patients' quality of life.

Administration, Cutaneous↗
PubMed2003

Involvement of transmembrane domain interactions in signal transduction by alpha/beta integrins.

The alpha and beta subunits of alpha/beta heterodimeric integrins function together to bind ligands in the extracellular region and transduce signals across cellular membranes. A possible function for the transmembrane regions in integrin signaling has been proposed from structural and computational data. We have analyzed the capacity of the integrin alpha(2), alpha(IIb), alpha(4), beta(1), beta(3), and beta(7) transmembrane domains to form homodimers and/or heterodimers. Our data suggest that the integrin transmembrane helices can help to stabilize heterodimeric integrins but that the interactions do not specifically associate particular pairs of alpha and beta subunits; rather, the alpha/beta subunit interaction constrains the extramembranous domains, facilitating signal transduction by a promiscuous transmembrane helix-helix association.

Amino Acid Motifs↗
PubMed2002

Thermostability of membrane protein helix-helix interaction elucidated by statistical analysis.

A prerequisite for the survival of (micro)organisms at high temperatures is an adaptation of protein stability to extreme environmental conditions. In contrast to soluble proteins, where many factors have already been identified, the mechanisms by which the thermostability of membrane proteins is enhanced are almost unknown. The hydrophobic membrane environment constrains possible stabilizing factors for transmembrane domains, so that a difference might be expected between soluble and membrane proteins. Here we present sequence analysis of predicted transmembrane helices of the genomes from eight thermophilic and 12 mesophilic organisms. A comparison of the amino acid compositions indicates that more polar residues can be found in the transmembrane helices of thermophilic organisms. Particularly, the amino acids aspartic acid and glutamic acid replace the corresponding amides. Cysteine residues are found to be significantly decreased by about 70% in thermophilic membrane domains suggesting a non-specific function of most cysteine residues in transmembrane domains of mesophilic organisms. By a pair-motif analysis of the two sets of transmembrane helices, we found that the small residues glycine and serine contribute more to transmembrane helix-helix interactions in thermophilic organisms. This may result in a tighter packing of the helices allowing more hydrogen bond formation.

Archaeal Proteins↗