[Herpes zoster in Germany. A retrospective analyse of SHL data]. / Herpes zoster in Deutschland. Eine retrospektive Analyse von GKV-Daten.

The incidence of herpes zoster in the elderly (50 years and older) 2004 in Germany was determined by retrospectively analysing representative treatment data of the statutory health insurance sample of AOK Hesse/KV Hesse. The overall observed incidence rate of herpes zoster was 9.4 cases per 1,000 person-years (PY). 10.1% of herpes-zoster-patients suffered at least 1 month from pain, the so called postherpetic neuralgia (PHN1), 6.9% had at least 3 months pain (PHN3). Incidence rate of herpes zoster rose markedly with age: from 6.8 per 1,000 PY in 50 to 54 year-olds to 12.4 PY in persons 80 years and older. Incidence rate in the immunocompromised was higher (11.6 per 1,000 PY) than in the immunocompetent (9.1 per 1,000 PY). According to a standardized extrapolation of the sample to the German population, about 300,000 persons 50 years and older suffered from acute herpes zoster on the year 2004 in Germany.

Activation of the redox-regulated molecular chaperone Hsp33--a two-step mechanism.

BACKGROUND: Hsp33 is a novel redox-regulated molecular chaperone. Hsp33 is present in the reducing environment of the cytosol and is, under normal conditions, inactive. The four highly conserved cysteines found in Hsp33 constitute a novel zinc binding motif. Upon exposure to oxidative stress, Hsp33's chaperone activity is turned on. This activation process is initiated by the formation of two intramolecular disulfide bonds. Recently, the 2.2 A crystal structure of Hsp33 has been solved, revealing that Hsp33 is present as a dimer in the structure (Vijayalakshmi et al., this issue, 367-375 [1]). RESULTS: We show here that oxidized, highly active Hsp33 is a dimer in solution. In contrast, reduced and inactive Hsp33 is monomeric. The incubation of reduced Hsp33 in H(2)O(2) leads to the simultaneous formation of two intramolecular disulfide bonds and the concomitant release of zinc. This concentration-independent step is followed by a concentration-dependent association reaction. The dimerization of Hsp33 requires highly temperature-sensitive structural rearrangements. This allows Hsp33's activation process to be greatly accelerated at heat shock temperatures. CONCLUSIONS: The regulation of Hsp33's chaperone function is highly sophisticated. On a transcriptional level, Hsp33 is under heat shock control. This increases the concentration of Hsp33 under heat and oxidative stress, a process that favors dimerization, a critical step in Hsp33's activation reaction. On a posttranslational level, Hsp33 is redox regulated. Dimerization of disulfide-bonded Hsp33 monomers leads to the formation of two extended, putative substrate binding sites. These sites might explain Hsp33's high and promiscuous affinity for unstructured protein folding intermediates.

Association of antibody chains at different stages of folding: prolyl isomerization occurs after formation of quaternary structure.

The folding pathways of multi-domain proteins are still poorly understood due to the complexity of the reaction involving domain folding, association and, in many cases, prolyl cis/trans isomerization. Here, we have established a kinetic model for the folding of the Fab fragment of the antibody MAK 33 with intact disulfide bonds. Folding of the hetero-dimeric protein from the completely denatured, oxidized state comprises the pairwise association of the two domains of each chain with those of the partner protein. Both the reactivation of the Fab fragment in which the two constituent polypeptide chains were covalently linked via a cystine bond (Fab) and that of a mutant lacking this covalent linkage (Fab/-cys) were monitored by ELISA. Folding of the Fab fragment is a slow process, which can be described by a single exponential term. The kinetic phase reflects a folding step after the association of the two chains. The same reaction was detected in the folding of Fab/-cys but an additional rate-limiting step is involved that is due to a unimolecular step in the folding of the isolated light chain. This implies that, during Fab reactivation, Fd associates with the light chain at the stage of an earlier folding intermediate, thus eliminating the additional slow folding step of the light chain observed with Fab/-cys. Both in Fab and Fab/-cys renaturation, the folding reaction after association is determined by prolyl isomerization. Therefore, at least four different association-competent folding intermediates have to be postulated according to the folding stage of light chain and the configuration of at least one prolyl-peptide bond. Using the different substrate specificities of cyclophilin and FK506 binding protein, we have obtained evidence that Pro159 within the Fd fragment may be responsible for the observed slow folding phase after association, although three other proline residues adopt a cis configuration in the native protein. Furthermore, the data suggest that in the case of the Fab fragment, association is a prerequisite for cis/trans isomerization of prolyl peptide bonds, implying that the quaternary but not the tertiary structure determines the cis-configuration of the prolyl residue in Fd involved in the rate-limiting folding reaction.

Dynamics of the GroEL-protein complex: effects of nucleotides and folding mutants.

Chaperonins are a ubiquitous class of ring-shaped oligomeric protein complexes that are of crucial importance for protein folding in vivo. Analysis of the underlying functional principles had relied mainly on model proteins the (un)folding of which is dominated by irreversible side-reactions. We used maltose-binding protein (MBP) as a substrate protein for GroEL, since the refolding of this protein is completely reversible and thus allows a detailed analysis of the molecular parameters that determine the interaction of GroEL with non-native protein. We show that MBP folding intermediates are effectively trapped by GroEL in a diffusion-controlled reaction. This complex is stabilized via unspecific hydrophobic interactions. Stabilization energies for wild-type MBP increasing linearly with ionic strength from 50 kJ/mol to 60 kJ/mol. Depending on the intrinsic folding rate and the hydrophobicity of the substrate protein, the interaction of GroEL with MBP folding intermediates leads to a dramatically decreased apparent refolding rate of MBP (wild-type) or a complete suppression of folding (MBP folding mutant Y283D). On the basis of our data, a quantitative kinetic model of the GroEL-mediated folding cycle is proposed, which allows simulation of the partial reactions of the binding and release cycles under all conditions tested. In the presence of ATP and non-hydrolysable analogues, MBP is effectively released from GroEL, since the overall dissociation constant is reduced by three orders of magnitude. Interestingly, binding of nucleotide does not change the off rate by more than a factor of 3. However the on-rate is decreased by at least two orders of magnitude. Therefore, the rebinding reaction is prevented and folding occurs in solution.

Biochemical characterization of the structure-specific DNA-binding protein Cmb1 from Schizosaccharomyces pombe.

Cmb1, a novel HMG box protein from Schizosaccharomyces pombe, has been characterized biochemically using glutaraldehyde cross-linking, gel-filtration and analytical ultracentrifugation. It was identified as a monomeric, non-spherical protein, with a tendency to aggregate in solution. Limited proteolysis with trypsin and chymotrypsin showed that the C-terminal HMG box was a compact, proteolytically stable domain and the N-terminal region of Cmb1 was relatively unstructured and more easily digested. As Cmb1 was previously identified as a potential mismatch-binding protein, the binding constants and stoichiometry for both homoduplex and heteroduplex DNA were determined using an IASys resonant mirror biosensor. Cmb1 indeed demonstrated a tighter association with mismatched DNA, especially with the C/Delta-mismatch. Expression constructs of Cmb1 were made to study the sections of the protein involved in DNA binding. Constructs with the N-terminal region absent revealed that the C-terminal HMG box was the primary DNA-binding region. The presence of the N-terminal region did, however, facilitate tighter binding to both homoduplex and heteroduplex DNA. The amino acid residues isoleucine 14 and leucine 39 were located as putative intercalating residues using structure guided homology modelling. The model templates were derived from two distinct HMG:DNA complexes: HMG-D bound to homoduplex DNA and HMG 1 bound to cisplatin DNA. Binding studies using the Cmb1 HMG box with point mutations in these residues showed that isoleucine 14 was important for the binding of Cmb1 to homoduplex DNA, but affected binding to mismatches to a lesser extent. In contrast, leucine 39 appeared to have a more significant function in binding to mismatched DNA.

Folding and association of the antibody domain CH3: prolyl isomerization preceeds dimerization.

The simplest naturally occurring model system for studying immunoglobulin folding and assembly is the non-covalent homodimer formed by the C-terminal domains (CH3) of the heavy chains of IgG. Here, we describe the structure of recombinant CH3 dimer as determined by X-ray crystallography and an analysis of the folding pathway of this protein. Under conditions where prolyl isomerization does not contribute to the folding kinetics, formation of the beta-sandwich structure is the rate-limiting step. beta-Sheet formation of CH3 is a slow process, even compared to other antibody domains, while the subsequent association of the folded monomers is fast. After long-time denaturation, the majority of the unfolded CH3 molecules reaches the native state in two serial reactions, involving the re-isomerization of the Pro35-peptide bond to the cis configuration. The species with the wrong isomer accumulate as a monomeric intermediate. Importantly, the isomerization to the correct cis configuration is the prerequisite for dimerization of the CH3 domain. In contrast, in the Fab fragment of the same antibody, prolyl isomerization occurs after dimerization demonstrating that within one protein, comprised of highly homologous domains, both the kinetics of beta-sandwich formation and the stage at which prolyl isomerization occurs during the folding process can be completely different.

Pro-sequence assisted folding and disulfide bond formation of human nerve growth factor.

Nerve growth factor (NGF) is a member of the neurotrophin family. These growth factors support neuronal survival and differentiation. Neurotrophins are synthesized as pre-pro-proteins. Whereas the pre-sequences mediate secretion, the function of the pro-peptides is largely unknown. To test the role of the pro-sequence as a folding enhancer, recombinant human pro-NGF (rh-pro-NGF) was produced in Escherichia coli. The oxidative refolding of rh-pro-NGF and rh-NGF was studied using electrospray mass spectrometry (ESIMS) time-course analysis. This analysis permitted both the identification and quantification of intermediates present during the process. The disulfide bonds formed at different times of the refolding processes were characterized by proteolytic digestion followed by matrix assisted laser desorption ionization mass spectrometry (MALDIMS) analysis. Folding yields and kinetics of rh-pro-NGF were significantly enhanced when compared to the in vitro refolding of mature rh-NGF. These results suggest that the pro-sequence of NGF promotes folding of the mature part.

Novel molecular architecture of the multimeric archaeal PEP-synthase homologue (MAPS) from Staphylothermus marinus.

The phosphoenolpyruvate (PEP)-synthases belong to the family of structurally and functionally related PEP-utilizing enzymes. The only archaeal member of this family characterized thus far is the Multimeric Archaeal PEP-Synthase homologue from Staphylothermus marinus (MAPS). This protein complex differs from the bacterial and eukaryotic representatives characterized to date in its homomultimeric, as opposed to dimeric or tetrameric, structure. We have probed the molecular architecture of MAPS using limited proteolytic digestion in conjunction with electron microscopic, biochemical, and biophysical techniques. The 2.2 MDa particle was found to be organized in a concentric fashion. The 93.7 kDa monomers possess a pronounced tripartite domain structure and are arranged such that the N-terminal domains form an outer shell, the intermediate domains form an inner shell, and the C-terminal domains form a core structure responsible for the assembly into a multimeric complex. The core domain was shown to be capable of assembling into the native multimer by recombinant expression in Escherichia coli. Deletion mutants as well as a synthetic peptide were investigated for their state of oligomerization using native polyacrylamide gel electrophoresis, molecular sieve chromatography, analytical ultracentrifugation, circular dichroism (CD) spectroscopy, and chemical cross-linking. Our data confirmed the existence of a short C-terminal, alpha-helical oligomerization motif that had been suggested by multiple sequence alignments and secondary structure predictions. We propose that this motif bundles the monomers into six groups of four. An additional formation of 12 dimers between globular domains from different bundles leads to the multimeric assembly. According to our model, each of the six bundles of globular domains is positioned at the corners of an imaginary octahedron, and the helical C-terminal segments are oriented towards the centre of the particle. The edges of the octahedron represent the dimeric contacts. Phylogenetic analysis suggests that the ancient predecessor of this family of enzymes contained the C-terminal oligomerization motif as a feature that was preserved in some hyperthermophiles.

Advances in refolding of proteins produced in E. coli.

Inclusion body production is a common theme in recombinant protein technology. Hence, renaturation of these inclusion body proteins is a field of increasing interest for gaining large amounts of proteins. Recent developments of renaturation procedures include the inhibition of aggregation during refolding by the application of low molecular weight additives and matrix-bound renaturation techniques.

Coupling of antibodies via protein Z on modified polyoma virus-like particles.

Therapeutic application of virus-based delivery systems often implies a change of the tropism of these vectors. This can be achieved by insertion of polypeptides (e.g., antibody fragments) in viral coat proteins. Such fusion proteins have only been used in viral vectors so far and, as part of a virus, they have not been available for a detailed biophysical characterization. We analyzed a fusion protein called VP1-Z, which is based on the polyoma virus coat protein VP1 and protein Z. Protein Z is an engineered antibody-binding domain derived from protein A from Staphylococcus aureus. The fusion VP1-Z was constructed by insertion of protein Z in the HI-loop of VP1. As wild-type VP1, VP1-Z formed pentameric capsomers and assembled to VLPs in vitro. The stability of these particles was very similar compared to that of VLPs of wild-type VP1. Protein Z was fully structured in the fusion protein and was still capable of binding antibodies on the surface of VLPs of VP1-Z. Using this fusion protein, we could change the tropism of polyoma VLPs toward cells presenting on their surface the antigen of the coupled antibody.

Characterization of a quaternary-structured folding intermediate of an antibody Fab-fragment.

Antibody folding is a complex process comprising folding and association reactions. Although it is usually difficult to characterize kinetic folding intermediates, in the case of the antibody Fab fragment, domain-domain interactions lead to a rate-limiting step of folding, thus accumulating folding intermediates at a late step of folding. Here, we analyzed a late folding intermediate of the Fab fragment of the monoclonal antibody MAK 33 from mouse (kappa/IgG1). As a strategy for accumulation of this intermediate we used partial denaturation of the native Fab by guanidinium chloride. This denaturation intermediate, which can be populated to about 90%, is indistinguishable from a late-folding intermediate with respect to denaturation and renaturation kinetics. The spectroscopic analysis reveals a native-like secondary structure of this intermediate with aromatic side chains only slightly more solvent exposed than in the native state. The respective partner domains are weekly associated. From these data we conclude that the intramolecular association of the two chains during folding, with all domains in a native-like structure, follows a two-step mechanism. In this mechanism, presumably hydrophobic interactions are followed by rearrangements leading to the exact complementarity of the contact sites of the respective domains.

Changing the surface of a virus shell fusion of an enzyme to polyoma VP1.

Recent developments on virus-like particles have demonstrated their potential in transfecting eucaryotic cells. In the case of particles based on the major coat protein VP1 of polyoma virus, transfection occurs via binding of VP1 to sialic acids. Since sialic acid is present on almost every eucaryotic cell line, this results in an unspecific cell targeting. Generation of a cell-type specificity of this system would imply the presentation of a new function on the surface of VP1. To analyze whether a new functional protein can be placed on VP1, we inserted dihydrofolate reductase from Escherichia coli as a model protein. The effect of such an insertion on both VP1 and the inserted protein was investigated, respectively. The function of VP1, like the formation of pentameric capsomers and its ability to assemble into capsids, was not influenced by the insertion. The inserted dihydrofolate reductase showed major changes when compared to the wild-type form. The thermal stability of the enzyme was dramatically reduced in the fusion protein; nevertheless, the dihydrofolate reductase proved to be a fully active enzyme with only slightly increased K(M) values for its substrates. This model system provides the basis for further modifications of the VP1 protein to achieve an altered surface of VP1 with new properties.

Prolyl isomerases catalyze antibody folding in vitro.

Some slow-folding phases in the in vitro refolding of proteins originate from the isomerization of prolyl-peptide bonds, which can be accelerated by a class of enzymes called prolyl isomerases (PPIs). We used the in vitro folding of an antibody Fab fragment as a model system to study the effect of PPI on a folding reaction that is only partially reversible. We show here that members of both subclasses of PPIs, cyclophilin and FK 506 binding protein (FKBP), accelerate the refolding process and increase the yield of correctly folded molecules. An acceleration of folding was not observed in the presence of the specific inhibitor cyclosporin A, but still the yield of correctly folded molecules was increased. Bovine serum albumin (BSA) increased the yield comparable to cyclophilin but, in contrast, did not influence the rate of reactivation. These effects were observed only when cyclophilin or BSA were present during the first few seconds of refolding. However, the rate-limiting reactivation reaction is still accelerated when PPI is added several minutes after starting refolding. In contrast, the prokaryotic chaperone GroEL influences the refolding yield when added several minutes after initiating refolding. The results show that PPIs influence the folding of Fab in two different ways. (1) They act as true catalysts of protein folding by accelerating the rate-limiting isomerization of Xaa-Pro peptide bonds. Proline isomerization is obviously a late folding step and has no influence on the formation of aggregates within the first seconds of the refolding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Folding of the Fab fragment within the intact antibody.

At present it is not clear to which extent the Fab fragment and the Fc part of an antibody interact in the intact immunoglobulin structure. To determine such potential interactions the unfolding and refolding of an isolated Fab fragment and the respective antibody MAK 33 (kappa/IgG1) are compared. It could be shown that the proline independent renaturation kinetics of both an unfolding intermediate and the fully denatured form of both proteins are identical. Upon denaturation, the loss of antigen binding activity occurs with the same rate for both the Fab fragment and the intact antibody. However, the complete structural unfolding of the Fab part of the antibody is significantly slower than that of the isolated Fab fragment. These kinetic data suggest that the structure of the Fab fragment within the intact antibody is stabilized by interactions, presumably with the Fc part, missing in the isolated Fab.

Domain interactions stabilize the alternatively folded state of an antibody Fab fragment.

The structure of the Fab fragment of the monoclonal antibody MAK 33 (kappa/IgG1) at pH 2 was characterized. Spectroscopic and kinetic analysis revealed a molten globule-like state, characterized by elements of secondary structure but less defined tertiary contacts than in the native state. However, some aromatic side chains are in an asymmetrical environment. This structure was not detected using the isolated light chain or a Fab fragment lacking the covalent linkage of the light chain and Fd via the C-terminal disulfide bond. Therefore, interactions between the two chains, stabilized by the interchain disulfide within the Fab fragment, are essential for formation of the alternatively folded state.

Folding of a synthetic parallel beta-roll protein.

Recently, the design of beta-sheet proteins and concomitant folding studies have attracted increasing attention. A unique natural all-beta domain occurs in a family of cytolytic bacterial toxins, the so-called RTX toxins. This domain consists of a variable number (about 6-45) of tandem repeats of a glycine-rich nine-residue motif with the consensus sequence GGXGXDX(L/I/F)X. The analysis of the three-dimensional structure of alkaline protease from Pseudomonas aeruginosa which possesses six of these repeats revealed that they fold into a novel 'parallel beta-roll' where calcium is bound within the turns connecting the beta-strands. A 75-mer peptide of the sequence NH(2)-WLS-[GGSGNDNLS](8)-COOH was chemically synthesised. Circular dichroism spectroscopy showed that this polypeptide folds in the presence of Ca(2+) and polyethylene glycol into a beta-structure which is presumably identical with the parallel beta-roll. This synthetic beta-roll behaves similarly to the isolated beta-roll domains from Escherichia coli haemolysin or Bordetella pertussis cyclolysin in terms of calcium binding and polymerisation behaviour.

Fibrin encapsulated liposomes as protein delivery system. Studies on the in vitro release behavior.

The efficacy of biologically active proteins in medical therapy depends on the development of suitable drug delivery systems. These delivery systems need to overcome the severe problems connected with the use of proteins such as their usually short half lives in body fluids and their susceptibility to proteolysis and denaturation. Our delivery system combines two widespread devices by encapsulating liposomes containing the model protein horseradish peroxidase (HRP) inside the biopolymer fibrin. The liposomes enable the protein to remain in its preferred aqueous environment and protect it during the polymerization process. Further encapsulation of the liposomes inside fibrin was carried out in order to achieve a depot system with sustained protein release. In vitro experiments showed that the protein filled liposomes were absolutely stable within the fibrin network. In contrast to 'free' HRP, enzyme entrapped in liposomes was completely retained by the fibrin network and wasn't released from the device unless the fibrin was degraded by plasmin.

The role of antivirals in the management of neuropathic pain in the older patient with herpes zoster.

Herpes zoster has been known since ancient times. It is a ubiquitous disease, occurring sporadically without any seasonal preference and is caused by the varicella-zoster virus. It may be defined as an endogenous relapse of the primary infection varicella. Herpes zoster is characterised by typical efflorescences in the innervation region of a cranial or spinal nerve and starts and ends with pain of varying intensity. Currently, several antiviral drugs are approved and many studies have shown that antiviral therapy, started early in the course of disease, can significantly reduce the risk and the duration of postherpetic neuralgia in elderly patients. The effects of all antivirals discussed in this article, given either orally or intravenously, are comparable with regards to the resolution of virus replication, prevention of dissemination of skin lesions and reduction of acute herpes zoster pain. Valaciclovir (valacyclovir), famciclovir and brivudine (brivudin) are comparably effective in the reduction of the incidence and/or prevention of zoster-associated pain and postherpetic neuralgia. Brivudine 125mg once daily is as effective as famciclovir 250mg three times daily in reducing the prevalence and the duration of zoster-associated pain and postherpetic neuralgia, especially if therapy is combined with a structured-pain therapy. The intensity of the therapy for pain should depend on the intensity of the pain that it is treating. Famciclovir and brivudine offer an advantage over other antivirals because they are administered less frequently; this is particularly relevant for elderly patients who may already be taking a number of medications for other diseases. Therefore, antiviral therapy in combination with adequate pain management should be given to all elderly patients as soon as herpes zoster is diagnosed.

Synthesis and biological evaluation of first N-alkyl syn dimeric 4-aryl-1,4-dihydropyridines as competitive HIV-1 protease inhibitors.

A first series of novel N-alkyl substituted syn dimeric 4-aryl-1,4-dihydropyridines 12--17 have been synthesised and evaluated as HIV-1 protease inhibitors in in vitro assays. While the N-methyl derivatives 12 and 13 were almost inactive, with IC(50)-values of about 225 microM, the N-benzyl compounds with varied ester groups all exhibited stronger activities, with IC(50)-values of 11--12 microM for the presently best compounds 16 and 17 with ethyl ester functions. The type of HIV-1 protease inhibition of the novel inhibitors was characterised as competitive. With the increase of observed activity from N-methyl derivatives to N-benzyl compounds the binding mode may correspond to that of cyclic ureas with hydrophobic interactions of the four aromatic residues to the S1/S1' and S2/S2' regions of HIV-1 protease.

[Confidentiality and the refusal to give evidence]. / Schweigepflicht und Zeugnisverweigerungsrecht.

The physician's obligation to give evidence as a witness in a preliminary investigation is in an area of conflict between the duty to tell the truth and preservation of professional discretion. These have to be weighed in the individual case. In opposition to the Anglo-American law, where a possible right of the witness to refuse to give evidence is limited by the principle of "finding the truth", the German law of criminal procedure contains far-reaching rights of a physician and so-called professional assistants witness to refuse to give evidence in sections 53, 53a Code of Criminal Procedure in order to protect the professional secrecy. This privilege refers to all facts, that have become known to the doctor or his staff and therefore it goes beyond the area of the medical discretion in section 203 GCC, that only contains secrets, which were confined to the doctor or which have become known to him. The witness can decide whether he either uses his right to refuse to give evidence or gives evidence without being released from medical confidentiality. In the second case, he risks being punished under section 203 GCC. If a physician is considered as witness in a procedure, the medical files are protected from attachment in section 97 subsection 1 numbers 2 and 3 Code of Criminal Procedure. In cases, where the physician is defendant himself, he cannot refer to this protection.