[Medical confidentiality]. / Ärztliche Schweigepflicht.

Medical confidentiality is a fundamental prerequisite in the patient-physician relationship based on trust and goes back to the Hippocratic oath. It is clearly defined in the German Constitution as well as the medical professional code of conduct. A breach of confidentiality can result in criminal sanctions and professional consequences as well as civil claims for damages and compensation by the affected patients. In routine clinical practice situations repeatedly occur which lead to uncertainty regarding the limits of confidentiality, the right to silence and the obligation to disclosure. The purpose of this article is to explain the legal foundations of medical confidentiality, the right to silence and the obligation to disclosure and to provide practical support for critical questions in routine clinical practice.

[Fractures of the patella]. / Patellafrakturen.

With 1% of all skeletal fractures patellar fractures are rare. The majority of cases are caused by a direct trauma to the flexed knee. The diagnosis is made via the injury mechanism as well as the physical and radiological findings. In conventional x­ray imaging the extent of injury often is underrated, which mostly leads to the necessity of a computed tomography (CT) scan. The aim of the treatment is the reconstruction of the extensor mechanism and the anatomical reconstruction of the articular surface. The type of treatment depends on the fracture type. Tension band wiring still is the most frequently practiced technique. Complications, such as secondary dislocation or migration of the K­wires resulting in revision surgery have been described in up to 30% of the cases. Studies could already show a higher biomechanical stability of osteosynthesis via cannulated screws. Especially in cases of comminuted fractures, osteosynthesis via a locking plate seems to have several advantages but long-term results are not yet available.

[Plate osteosynthesis of patellar fractures]. / Plattenosteosynthese bei Patellafrakturen.

OBJECTIVE: Anatomic reconstruction of the retropatellar articular surface and repair of the extensor mechanism of the knee joint. The osteosynthesis should allow immediate mobilization as part of an early functional postoperative rehabilitation protocol. INDICATIONS: Displaced fractures of the patella, especially multifragment and comminuted fractures with a retropatellar incongruity or dislocation of >2 mm. CONTRAINDICATIONS: Critical local soft tissue because of the risk of postoperative infection. SURGICAL TECHNIQUE: Median skin incision. For simple (transverse) fractures, preservation of the soft tissue and reduction control via the index finger. For complex fractures, lateral arthrotomy and eversion of the patella. Reconstruction of the articular surface from the joint side with optimal visibility. Temporary fixation with Kirschner wires, osteosynthesis with the fixed angle plate. If necessary, additional screws or wires. POSTOPERATIVE MANAGEMENT: Immediate mobilization with full weightbearing in full extension with a knee brace. Extension/flexion 0/0/60° for 4 weeks, then 0/0/90° until the 7th week. Active extension after 6 weeks. Climbing stairs after 12 weeks. RESULTS: Good functional results in combination with a low rate of complications and revisions.

Molecular and physiological characterisation of a 3-phytase from soil bacterium Klebsiella sp. ASR1.

Klebsiella sp. strain ASR1 isolated from an Indonesian rice field is able to hydrolyse myo-inositol hexakis phosphate (phytate). The phytase protein was purified and characterised as a 42 kDa protein accepting phytate, NADP and sugar phosphates as substrates. The corresponding gene (phyK) was cloned from chromosomal DNA using a combined approach of protein and genome analysis, and expressed in Escherichia coli. The recombinant enzyme was identified as a 3-phytase yielding myo-inositol monophosphate, Ins(2)P, as the final product of enzymatic phytate hydrolysis. Based on its amino acid sequence, PhyK appears to be a member of a hitherto unknown subfamily of histidine acid phytate-degrading enzymes with the active site RHGXRXP and HD sequence motifs, and is different from other general phosphatases and phytases. Due to its ability to degrade sodium phytate to the mono phosphate ester, the phyK gene product is an interesting candidate for industrial and agricultural applications to make phytate phosphorous available for plant and animal nutrition.

CNS synaptogenesis promoted by glia-derived cholesterol.

The molecular mechanisms controlling synaptogenesis in the central nervous system (CNS) are poorly understood. Previous reports showed that a glia-derived factor strongly promotes synapse development in cultures of purified CNS neurons. Here, we identify this factor as cholesterol complexed to apolipoprotein E-containing lipoproteins. CNS neurons produce enough cholesterol to survive and grow, but the formation of numerous mature synapses demands additional amounts that must be provided by glia. Thus, the availability of cholesterol appears to limit synapse development. This may explain the delayed onset of CNS synaptogenesis after glia differentiation and neurobehavioral manifestations of defects in cholesterol or lipoprotein homeostasis.

Deactivation of regulatory proteins hnRNP A1 and A2 during SC-1 induced apoptosis.

Phosphorylation and activation of caspases play an important role in the induction of apoptosis. During tumor specific apoptosis, induced by the human monoclonal antibody SC-1, tyrosine phosphorylation and serine dephosphorylation of several proteins is observed. In this paper we describe the identification of two dephosphorylated proteins as heterogeneous nuclear ribonucleoproteins A1 and A2 (hnRNP A1, hnRNP A2). The dephosphorylation of these proteins is important for apoptosis since the amount of apoptotic cell death can be decreased by the specific serine/threonine phosphatase inhibitor okadaic acid. We also investigated the effect of serine kinase inhibitor H7 on SC-1 induced apoptosis, which leads to a dose dependent increase in apoptosis. We could also show that 24 hours after the induction of apoptosis the hnRNP A1 protein is cleaved into different cleavage products. Further, we found a decreased expression of caspase-2 in early apoptosis signalling and an overexpression 24 hours after induction of apoptosis. Our results show that the phosphorylation status of the hnRNP A1 and A2 plays a significant role in early SC-1 induced apoptosis signalling and further indicate the role of caspase activation during the apoptotic process.

Identification of acidic, low molecular mass proteins of Mycobacterium tuberculosis strain H37Rv by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry.

Matrix-assisted laser desorption/ionization-mass spectrometry peptide mass mapping and nano-electrospray ionization tandem mass spectrometry were used to identify acidic, low molecular mass proteins of Mycobacterium tuberculosis strain H37Rv. Proteins were extracted from whole cell lysates of mycobacteria, separated by high resolution two-dimensional electrophoresis (2-DE) and analysed by mass spectrometry (MS). Silver-stained 2-DE patterns resolved about 1800 distinct protein species, 190 of which had an observed isoelectric point and molecular mass in the range of pH 4 to 6 and 6 to 15 kDa, respectively. Seventy-six spots from this range were excised from Coomassie Brilliant Blue G250-stained gels and analysed by MS, from which 72 were identified. These spots were shown to represent products of as many as 50 different protein-coding genes. Ten genes gave rise to more than one protein species. Eleven spots contained more than one protein. The present study led to the identification of 15 mycobacterial proteins with assigned putative functions, 28 conserved hypothetical proteins and one unknown protein. Most proteins of the latter two groups had previously been predicted at the DNA level only. Six additional spots were shown to comprise proteins encoded by open reading frames that have not been predicted for M. tuberculosis H37Rv by genomic investigations.

Tyrosine phosphorylation patterns and size modification of the Helicobacter pylori CagA protein after translocation into gastric epithelial cells.

Helicobacter pylori is one of the most common bacterial pathogens that causes a variety of gastric diseases. During infection, the immuno-dominant H. pylori CagA protein is translocated and tyrosine-phosphorylated in gastric epithelial cells. We compared tyrosine phosphorylation patterns of five CagA variants by two-dimensional electrophoresis (2-DE) and immunoblotting studies. Tyrosine-phosphorylated CagA was detected as two distinct protein species in strains P12, P227, G27 and 26695 suggesting that two tyrosine residues of CagA can be phosphorylated both separately and simultaneously. Prediction programs revealed the presence of three putative tyrosine phosphorylation motifs in the sequences of CagA. Mutations in these motifs were identified suggesting that only two putative phosphorylation-relevant tyrosines are present in each CagA variant. CagA of strain J99 was found to be unique because essential codons were mutated in each of the three motifs and, consequently, revealed no tyrosine phosphorylation signals at all. These findings support the view that CagA from different H. pylori strains can be tyrosine-phosphorylated at one or two out of three predicted positions. Additionally, truncated CagA protein species of about 100-105 kDa (p100CagA) have been detected after infection with some of the H. pylori strains. The isoelectric point determined by both 2-DE and sequence analysis suggested that p100CagA represents the amino (N)-terminal part of the protein. Translocation, tyrosine phosphorylation and size modification of CagA might be involved in host signal transduction and development of gastric disease.

Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains.

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.

Protein kinase C regulates the phosphorylation and cellular localization of occludin.

Occludin is an integral membrane phosphoprotein specifically associated with tight junctions, contributing to the structure and function of this intercellular seal. Occludin function is thought to be regulated by phosphorylation, but no information is available on the molecular pathways involved. In the present study, the involvement of the protein kinase C pathway in the regulation of the phosphorylation and cellular distribution of occludin has been investigated. Phorbol 12-myristate 13-acetate and 1,2-dioctanoylglycerol induced the rapid phosphorylation of occludin in Madin-Darby canine kidney cells cultured in low extracellular calcium medium with a concomitant translocation of occludin to the regions of cell-cell contact. The extent of occludin phosphorylation as well as its incorporation into tight junctions induced by protein kinase C activators or calcium switch were markedly decreased by the protein kinase C inhibitor GF-109203X. In addition, in vitro experiments showed that the recombinant COOH-terminal domain of murine occludin could be phosphorylated by purified protein kinase C. Ser(338) of occludin was identified as an in vitro protein kinase C phosphorylation site using peptide mass fingerprint analysis and electrospray ionization tandem mass spectroscopy. These findings indicate that protein kinase C is involved in the regulation of occludin function at tight junctions.

Proteomics reveals open reading frames in Mycobacterium tuberculosis H37Rv not predicted by genomics.

Genomics revealed the sequence of 3924 genes of the H37Rv strain of Mycobacterium tuberculosis. Proteomics complements genomics in showing which genes are really expressed, and here we show the expression of six genes not predicted by genomics, as proved by two-dimensional electrophoresis and matrix-assisted laser desorption ionization and nano-electrospray mass spectrometry.

Multiple pathways contribute to nuclear import of core histones.

Nuclear import of the four core histones H2A, H2B, H3 and H4 is one of the main nuclear import activities during S-phase of the cell cycle. However, the molecular machinery facilitating nuclear import of core histones has not been elucidated. Here, we investigated the pathways by which histone import can occur. First, we show that core histone import can be competed by the BIB (beta-like import receptor binding) domain of ribosomal protein L23a suggesting that histone import is an importin mediated process. Secondly, affinity chromatography on immobilized core histones revealed that several members of the importin beta family of transport receptors are able to interact with core histones. Finally, we demonstrate that at least four known and one novel importin, importin 9, can mediate nuclear import of core histones into the nuclei of permeabilized cells. Our results suggest that multiple pathways of import exist to provide efficient nuclear uptake of these abundant, essential proteins.

Characterization and crystallization of a novel Sarcocystis muris lectin, SML-2.

A novel lectin (SML-2) consisting of 138 amino acids was isolated from cyst merozoites of Sarcocystis muris and sequenced by Edman degradation and mass spectrometry. All 12 cysteinyl residues are involved in disulfide bridges, four of which are attributed to a characteristic pattern of cysteines as found in the so-called PAN-module superfamily. Crystals of SML-2 diffracting to 2.1 A resolution at a synchrotron were grown by the hanging-drop vapour-diffusion technique. They belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 53.6, b = 128.8, c = 158.2 A and eight molecules in the asymmetric unit. SML-2 cocrystallized with Au galactose results in two different crystal forms. The first form is isomorphous with the native crystals and the second form adopts space group C222(1), with unit-cell parameters a = 74.7, b = 82.0, c = 131.0 A, and diffracts to 2.4 A at a rotating-anode X-ray generator.

Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450scc. Mass spectrometry and Edman degradation of complexes of the steroidogenic hydroxylase system.

NADPH-dependent adrenodoxin reductase, adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, adrenodoxin transfers electrons from the FAD of adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from adrenodoxin reductase to P450. Here, we characterize new covalent, zero-length crosslinks mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide between bovine adrenodoxin and adrenodoxin reductase, and between adrenodoxin and P450scc, respectively, which allow to discriminate between the electron transfer models. Using Edman degradation, mass spectrometry and X-ray crystallography a crosslink between adrenodoxin reductase Lys27 and adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of adrenodoxin. However, in a covalent complex of adrenodoxin and P450scc, adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an adrenodoxin -P450scc complex/adrenodoxin reductase test system. Because the acidic residues Asp76 and Asp79 belong to the binding site of adrenodoxin to adrenodoxin reductase, as well as to the P450scc, the covalent bond within the adrenodoxin-P450scc complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system.

Human monoclonal rheumatoid synovial B lymphocyte hybridoma with a new disease-related specificity for cartilage oligomeric matrix protein.

Joint-specific self-Ags are considered to play an important role in the induction of synovial T and B cell expansion in human rheumatoid arthritis (RA). However, the nature of these autoantigens is still enigmatic. In this study a somatically mutated IgG2 lambda B cell hybridoma was established from the synovial membrane of an RA patient and analyzed for its Ag specificity. A heptameric peptide of cartilage oligomeric matrix protein (COMP) could be characterized as the target structure recognized by the human synovial B cell hybridoma. The clonotypic V(H) sequences of the COMP-specific hybridoma could also be detected in synovectomy material derived from five different RA patients but in none of the investigated osteoarthritis cases (n = 5), indicating a preferential usage of V(H) genes closely related to those coding for a COMP-specific Ag receptor in RA synovial B cells. Moreover, the COMP heptamer was preferentially recognized by circulating IgG in RA (n = 22) compared with osteoarthritis patients (n = 24) or age-matched healthy controls (n = 20; both p < 0.0001). Hence, the COMP-specific serum IgG is likely to reflect local immune responses toward a cartilage- and tendon-restricted Ag that might be crucial to the induction of tissue damage in RA.

TNF-alpha-mediated neutrophil apoptosis involves Ly-GDI, a Rho GTPase regulator.

We investigated intracellular signaling events involved in fibronectin-accelerated TNF-alpha-mediated PMN apoptosis by means of 2-D gel electrophoresis and western blotting. Proteins were sequenced with electrospray ionization mass spectrometry. Apoptosis was quantitated by flow cytometry. We detected a cluster of acidic, high molecular-weight proteins that were only tyrosine phosphorylated when TNF-alpha-treated PMN interacted with fibronectin. Sequence analysis revealed that one of these proteins was Ly-GDI, a regulator of Rho GTPases. Fibronectin increased the TNF-alpha-induced Ly-GDI cleavage, yielding a 23-kD fragment. At 8 h, intact Ly-GDI was decreased to 33% on fibronectin, compared with 69% on PolyHema (P<0.05). Inhibition of tyrosine phosphorylation prevented phosphorylation of Ly-GDI, fibronectin-accelerated Ly-GDI cleavage, and fibronectin-accelerated apoptosis in TNF-alpha-treated PMN. We found that Ly-GDI cleavage was dependent on caspase-3 activation and that caspase-3 inhibition decreased apoptosis. We conclude that tyrosine phosphorylation of Ly-GDI, followed by increased caspase-3-mediated Ly-GDI cleavage, is a signaling event associated with accelerated TNF-alpha-mediated apoptosis on fibronectin.

Protein kinase C alpha-dependent phosphorylation of Golgi proteins.

Golgi-enriched membranes were phosphorylated in order to understand the mechanism for protein kinase C (PKC) regulation of exocytic vesicle formation at the trans-Golgi network. Two of the main PKC substrates were identified as MARCKS and Mac-MARCKS by two-dimensional electrophoresis (2-DE) and mass spectrometric sequencing. Annexin IV and profilin I, two other Golgi-associated proteins--although known as in vitro PKC substrates--were not phosphorylated in the Golgi-bound state.

GDP dissociation inhibitor D4-GDI (Rho-GDI 2), but not the homologous rho-GDI 1, is cleaved by caspase-3 during drug-induced apoptosis.

Different cytotoxic drugs induce cell death by activating the apoptotic programme; a family of cysteinyl aspartate proteases named caspases has been shown to be involved in the initiation as well as the execution of this kind of cell death. In the present study, cleavage of D4-GDI (Rho-GDI 2), an abundant haemopoietic-cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, was demonstrated after treatment of BJAB Burkitt-like lymphoma cells with taxol or epirubicin. The cleavage of D4-GDI occurred simultaneously with the activation of caspase-3 but preceded DNA fragmentation and the morphological changes associated with apoptotic cell death. By using high-resolution two-dimensional gel electrophoresis it was shown that this cleavage is specific: whereas the level of the homologous protein Rho-GDI 1 was not significantly altered during drug-induced apoptosis and in cytochrome c/dATP-activated cellular extracts, D4-GDI disappeared owing to proteolytic cleavage. Inhibitor experiments with Z-DEVD-fmk (in which Z stands for benzyloxycarbonyl and fmk for fluoromethyl ketone) and microsequencing of the D4-GDI fragment revealed that this occurs at the caspase-3 cleavage site. Our results strongly suggest the differential regulation of the homologous GDP dissociation inhibitors Rho-GDI 1 and D4-GDI during drug-induced apoptosis by proteolysis mediated by caspase-3 but not by caspase-1. Owing to their crucial role as modulators of Rho GTPases, this might in turn have a significant impact on the mechanisms that induce the cytoskeletal and morphological changes in apoptotic cells.

Biochemical analysis of proteasomes from mouse microglia: induction of immunoproteasomes by interferon-gamma and lipopolysaccharide.

The 20S proteasome is a multicatalytic threonine protease and serves to process peptides that are subsequently presented as antigenic epitopes by MHC class I molecules. In the brain, microglial cells are the major antigen presenting cells and they respond sensitive to pathologic events. We used cultured mouse microglia and a microglial cell line, the BV-2 line, as a model to study the correlation between microglial activation parameters and structural plasticity of the 20S/26S proteasome. Lipopolysaccharide (LPS)- or interferon-gamma (IFN-gamma)-stimulated microglia or BV-2 cells exhibit properties of activated microglia such as high levels of TNFalpha and IL-6 release. In response to IFN-gamma or LPS, three constitutive beta subunits (beta1/Delta, beta2/MC14, beta5/MB1) were replaced by the immunoproteasome subunits ibeta1/LMP2, ibeta2/MECL-1, and ibeta5/LMP7, indicating that activated microglia adapts its proteasomal subunit composition to the requirements of an optimized MHC class I epitope processing. Induction of immunoproteasomes in BV-2 cells was solely provoked by IFN-gamma, but not by LPS. Moreover, LPS (but not IFN-gamma) triggered the expression of a novel protein of approximately 50 kD as part of the proteasome activator PA700, that is the substrate-recognizing and unfolding unit of the 26S proteasome. These results indicate that both the 20S core protease as well as the proteasome activator PA700 are targets of modulatory subunit replacements or transient association of regulatory components in the course of microglial activation.

Proteomics in human disease: cancer, heart and infectious diseases.

In recent years, genomics has increased the understanding of many diseases. Proteomics is a rapidly growing research area that encompasses both genetic and environmental factors. The protein composition represents the functional status of a biological compartment. The five approaches presented here resulted in the detection of disease-associated proteins. Calgranulin B was upregulated in colorectal cancer, and hepatoma-derived aldose reductase-like protein was reexpressed in a rat model during hepatocarcinogenesis. In these two investigations, attention was focused on one protein, obviously differing in amount, directly after two-dimensional electrophoresis (2-DE). Additional methods, such as enzyme activity measurements and immunohistochemistry, confirmed the disease association of the two candidates resulting from 2-DE subtractive analysis. The following three investigations take advantage of the holistic potential of the 2-DE approach. The comparison of 2-DE patterns from dilated cardiomyopathy patients with those of controls revealed 25 statistically significant intensity differences, from which 12 were identified by amino acid analysis, Edman degradation or matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). A human myocardial 2-DE database was constructed, containing 3300 protein spots and 150 identified protein species. The number of identified proteins was limited by the capacity of our group, rather than by the principle of feasibility. Another field where proteomics proves to be a valuable tool in identifying proteins of importance for diagnosis is proteome analysis of pathogenic microorganisms such as Borrelia burgdorferi (Lyme disease) and Toxoplasma gondii (toxoplasmosis). Sera from patients with early or late symptoms of Lyme borreliosis contained antibodies of various classes against about 80 antigens each, containing the already described antigens OspA, B and C, flagellin, p83/100, and p39. Similarly, antibody reactivity to seven different marker antigens of T. gondii allowed differentiation between acute and latent toxoplasmosis, an important diagnostic tool in both pregnancy and immunosuppressed patients.