The future of aortic surgery in Europe

At least every ten years, each specialty should reflect upon its past, its present and its future, in order to be able to reconfirm the direction in which it is headed, to adopt suggestions from inside and outside and, consequently, to improve. As such, the aim of this manuscript is to provide the interested reader with an overview of how aortic surgery and (perhaps more accurately) aortic medicine has evolved in Europe, and its present standing; also to provide a glimpse into the future, trying to disseminate the thoughts of a group of people actively involved in the development of aortic medicine in Europe, namely the Vascular Domain of the European Association of Cardio-Thoracic Surgery (EACTS).

Genetic and functional characterization of the NanA sialidase from Clostridium chauvoei

ABSTRACT: Clostridium chauvoei is the causative agent of blackleg, a wide spread serious infection of cattle and sheep with high mortality. In this study we have analyzed the sialidase activity of the NanA protein of C. chauvoei and cloned the sialidase gene nanA. Sialidase is encoded as a precursor protein of 722 amino acids with a 26 amino acid signal peptide. The mature sialidase has a calculated molecular mass of 81 kDa and contains the carbohydrate binding module 32 (CBM32, or F5/8 type C domain), the sialic acid binding module CBM40 and the enzymatically active sialidase domain found in all pro- and eukaryotic sialidases. Sialidase activity does not require the CBM32 domain. The NanA protein is secreted by C. chauvoei as a dimer. The nanA gene was found to be conserved and sialidase activity was found in C. chauvoei strains isolated over a period of 50 years from various geographical locations. Antiserum directed against a recombinant 40 kDa peptide containing CBM40 and part of the enzymatically active domain of NanA neutralized the secreted sialidase activity of all C. chauvoei strains tested.

Two domains of the V protein of virulent canine distemper virus selectively inhibit STAT1 and STAT2 nuclear import

Canine distemper virus (CDV) causes in dogs a severe systemic infection, with a high frequency of demyelinating encephalitis. Among the six genes transcribed by CDV, the P gene encodes the polymerase cofactor protein (P) as well as two additional nonstructural proteins, C and V; of these V was shown to act as a virulence factor. We investigated the molecular mechanisms by which the P gene products of the neurovirulent CDV A75/17 strain disrupt type I interferon (IFN-alpha/beta)-induced signaling that results in the establishment of the antiviral state. Using recombinant knockout A75/17 viruses, the V protein was identified as the main antagonist of IFN-alpha/beta-mediated signaling. Importantly, immunofluorescence analysis illustrated that the inhibition of IFN-alpha/beta-mediated signaling correlated with impaired STAT1/STAT2 nuclear import, whereas the phosphorylation state of these proteins was not affected. Coimmunoprecipitation assays identified the N-terminal region of V (VNT) responsible for STAT1 targeting, which correlated with its ability to inhibit the activity of the IFN-alpha/beta-mediated antiviral state. Conversely, while the C-terminal domain of V (VCT) could not function autonomously, when fused to VNT it optimally interacted with STAT2 and subsequently efficiently suppressed the IFN-alpha/beta-mediated signaling pathway. The latter result was further supported by a single mutation at position 110 within the VNT domain of CDV V protein, resulting in a mutant that lost STAT1 binding while retaining a partial STAT2 association. Taken together, our results identified the CDV VNT and VCT as two essential modules that complement each other to interfere with the antiviral state induced by IFN-alpha/beta-mediated signaling. Hence, our experiments reveal a novel mechanism of IFN-alpha/beta evasion among the morbilliviruses.

Collagen XII interacts with avian tenascin-X through its NC3 domain

Large oligomeric proteins often contain several binding sites for different molecules and can therefore induce formation of larger protein complexes. Collagen XII, a multidomain protein with a small collagenous region, interacts with fibrillar collagens through its C-terminal region. However, no interactions to other extracellular proteins have been identified involving the non-collagenous N-terminal NC3 domain. To further elucidate the components of protein complexes present close to collagen fibrils, different extracellular matrix proteins were tested for interaction in a solid phase assay. Binding to the NC3 domain of collagen XII was found for the avian homologue of tenascin-X that in humans is linked to Ehlers-Danlos disease. The binding was further characterized by surface plasmon resonance spectroscopy and supported by immunohistochemical co-localization in chick and mouse tissue. On the ultrastructural level, detection of collagen XII and tenascin-X by immunogold labeling confirmed this finding.

Posttranscriptional regulation of Fas (CD95) ligand killing activity by lipid rafts

Fas (CD95/Apo-1) ligand-mediated apoptosis induction of target cells is one of the major effector mechanisms by which cytotoxic lymphocytes (T cells and natural killer cells) kill their target cells. In T cells, Fas ligand expression is tightly regulated at a transcriptional level through the activation of a distinct set of transcription factors. Increasing evidence, however, supports an important role for posttranscriptional regulation of Fas ligand expression and activity. Lipid rafts are cholesterol- and sphingolipid-rich membrane microdomains, critically involved in the regulation of membrane receptor signaling complexes through the clustering and concentration of signaling molecules. Here, we now provide evidence that Fas ligand is constitutively localized in lipid rafts of FasL transfectants and primary T cells. Importantly, disruption of lipid rafts strongly reduces the apoptosis-inducing activity of Fas ligand. Localization to lipid rafts appears to be predominantly mediated by the characteristic cytoplasmic proline-rich domain of Fas ligand because mutations of this domain result in reduced recruitment to lipid rafts and attenuated Fas ligand killing activity. We conclude that Fas ligand clustering in lipid rafts represents an important control mechanism in the regulation of T cell-mediated cytotoxicity.

Caveolin-1 is required for signaling and membrane targeting of EphB1 receptor tyrosine kinase

Eph receptor tyrosine kinases are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Caveolae are flask-shaped invaginations of the cell membrane; their major structural protein, caveolin-1, has been shown to regulate signaling molecules localized in these micro-domains. The interaction of caveolin-1 with several of these proteins is mediated by the binding of its scaffolding domain to a region containing hydrophobic amino acids within these proteins. The presence of such a motif within the EphB1 kinase domain prompted us to investigate the caveolar localization and regulation of EphB1 by caveolin-1. We report that EphB1 receptors are localized in caveolae, and directly interact with caveolin-1 upon ligand stimulation. This interaction, as well as EphB1-mediated activation of extracellular-signal-regulated kinase (ERK), was abrogated by overexpression of a caveolin-1 mutant lacking a functional scaffolding domain. Interaction between Ephs and caveolin-1 is not restricted to the B-subclass of receptors, since we show that EphA2 also interacts with caveolin-1. Furthermore, we demonstrate that the caveolin-binding motif within the kinase domain of EphB1 is primordial for its correct membrane targeting. Taken together, our findings establish caveolin-1 as an important regulator of downstream signaling and membrane targeting of EphB1.

Neuropsychological problems after paediatric stroke: two year follow-up of Swiss children

AIM: The aim of this study was to obtain information about neurological and cognitive outcome for a population-based group of children after paediatric ischaemic stroke. METHODS: Data from the Swiss neuropaediatric stroke registry (SNPSR), from 1.1.2000 to 1.7.2002, including children (AIS 1) and neonates (AIS 2). At 18-24 months after a stroke, a follow-up examination was performed including a history, neurological and neuropsychological assessment. RESULTS: 33/48 children (22 AIS 1, 11 AIS 2) participated in the study. Neurological outcome was good in 16/33. After childhood stroke mean IQ levels were normal (94), but 6 children had IQ < 85 (50-82) and neuropsychological problems were present in 75%. Performance IQ (93) was reduced compared to verbal IQ (101, p = 0.121) due to problems in the domain of processing speed (89.5); auditory short-term memory was especially affected. Effects on school career were common. Outcome was worse in children after right-sided infarction. Children suffering from stroke in mid-childhood had the best prognosis. There was no clear relationship between outcome and localisation of the lesion. After neonatal stroke 7/11 children showed normal development and epilepsy indicated a worse prognosis in the remaining 4. CONCLUSION: After paediatric stroke neuropsychological problems are present in about 75% of children. Younger age at stroke as well as an emergence of epilepsy were predictors for worse prognosis.

Signal peptide and helical bundle domains of virulent canine distemper virus fusion protein restrict fusogenicity

Persistence in canine distemper virus (CDV) infection is correlated with very limited cell-cell fusion and lack of cytolysis induced by the neurovirulent A75/17-CDV compared to that of the cytolytic Onderstepoort vaccine strain. We have previously shown that this difference was at least in part due to the amino acid sequence of the fusion (F) protein (P. Plattet, J. P. Rivals, B. Zuber, J. M. Brunner, A. Zurbriggen, and R. Wittek, Virology 337:312-326, 2005). Here, we investigated the molecular mechanisms of the neurovirulent CDV F protein underlying limited membrane fusion activity. By exchanging the signal peptide between both F CDV strains or replacing it with an exogenous signal peptide, we demonstrated that this domain controlled intracellular and consequently cell surface protein expression, thus indirectly modulating fusogenicity. In addition, by serially passaging a poorly fusogenic virus and selecting a syncytium-forming variant, we identified the mutation L372W as being responsible for this change of phenotype. Intriguingly, residue L372 potentially is located in the helical bundle domain of the F(1) subunit. We showed that this mutation drastically increased fusion activity of F proteins of both CDV strains in a signal peptide-independent manner. Due to its unique structure even among morbilliviruses, our findings with respect to the signal peptide are likely to be specifically relevant to CDV, whereas the results related to the helical bundle add new insights to our growing understanding of this class of F proteins. We conclude that different mechanisms involving multiple domains of the neurovirulent A75/17-CDV F protein act in concert to limit fusion activity, preventing lysis of infected cells, which ultimately may favor viral persistence.

Modulation of human CYP19A1 activity by mutant NADPH P450 oxidoreductase

Mutations in NADPH P450 oxidoreductase (POR) cause a broad spectrum of human disease with abnormalities in steroidogenesis. We have studied the impact of P450 reductase mutations on the activity of CYP19A1. POR supported CYP19A1 activity with a calculated Km of 126 nm for androstenedione and a Vmax of 1.7 pmol/min. Mutations R457H and V492E located in the FAD domain of POR that disrupt electron transfer caused a complete loss of CYP19A1 activity. The A287P mutation of POR decreased the activities of CYP17A1 by 60-80% but had normal CYP19A1 activity. Molecular modeling and protein docking studies suggested that A287P is involved in the interaction of POR:CYP17A1 but not in the POR:CYP19A1 interaction. Mutations C569Y and V608F in the NADPH binding domain of POR had 49 and 28% of activity of CYP19A1 compared with normal reductase and were more sensitive to the amount of NADPH available for supporting CYP19A1 activity. Substitution of NADH for NADPH had a higher impact on C569Y and V608F mutants of POR. Similar effects were obtained at low/high (5.5/8.5) pH, but using octanol to limit the flux of electrons from POR to CYP19A1 inhibited activity supported by all variants. High molar ratios of KCl also reduced the CYP19A1 supporting activities of C569Y and V608F mutants of POR to a greater extent compared to normal POR and A287P mutant. Because POR supports many P450s involved in steroidogenesis, bone formation, and drug metabolism, variations in the effects of POR mutations on specific enzyme activities may explain the broad clinical spectrum of POR deficiency.

Effects of point mutations in the cytidine deaminase domains of APOBEC3B on replication and hypermutation of hepatitis B virus in vitro

APOBEC3 cytidine deaminases hypermutate hepatitis B virus (HBV) and inhibit its replication in vitro. Whether this inhibition is due to the generation of hypermutations or to an alternative mechanism is controversial. A series of APOBEC3B (A3B) point mutants was analysed in vitro for hypermutational activity on HBV DNA and for inhibitory effects on HBV replication. Point mutations inactivating the carboxy-terminal deaminase domain abolished the hypermutational activity and reduced the inhibitory activity on HBV replication to approximately 40 %. In contrast, the point mutation H66R, inactivating the amino-terminal deaminase domain, did not affect hypermutations, but reduced the inhibition activity to 63 %, whilst the mutant C97S had no effect in either assay. Thus, only the carboxy-terminal deaminase domain of A3B catalyses cytidine deaminations leading to HBV hypermutations, but induction of hypermutations is not sufficient for full inhibition of HBV replication, for which both domains of A3B must be intact.

Characterization of the first FGFRL1 mutation identified in a craniosynostosis patient

Fibroblast growth factor receptor-like 1 (FGFRL1) is a recently discovered transmembrane protein whose functions remain unclear. Since mutations in the related receptors FGFR1-3 cause skeletal malformations, DNA samples from 55 patients suffering from congenital skeletal malformations and 109 controls were searched for mutations in FGFRL1. One patient was identified harboring a frameshift mutation in the intracellular domain of this novel receptor. The patient showed craniosynostosis, radio-ulnar synostosis and genital abnormalities and had previously been diagnosed with Antley-Bixler syndrome. The effect of the FGFRL1 mutation was studied in vitro. In a reporter gene assay, the wild-type as well as the mutant receptor inhibited FGF signaling. However, the mutant protein differed from the wild-type protein in its subcellular localization. Mutant FGFRL1 was mainly found at the plasma membrane where it interacted with FGF ligands, while the wild-type protein was preferentially located in vesicular structures and the Golgi complex. Two motifs from the intracellular domain of FGFRL1 appeared to be responsible for this differential distribution, a tandem tyrosine based motif and a histidine-rich sequence. Deletion of either one led to the preferential redistribution of FGFRL1 to the plasma membrane. It is therefore likely that mutant FGFRL1 contributes to the skeletal malformations of the patient.

Selective pharmacological targeting of a DEAD box RNA helicase

RNA helicases represent a large family of proteins implicated in many biological processes including ribosome biogenesis, splicing, translation and mRNA degradation. However, these proteins have little substrate specificity, making inhibition of selected helicases a challenging problem. The prototypical DEAD box RNA helicase, eIF4A, works in conjunction with other translation factors to prepare mRNA templates for ribosome recruitment during translation initiation. Herein, we provide insight into the selectivity of a small molecule inhibitor of eIF4A, hippuristanol. This coral-derived natural product binds to amino acids adjacent to, and overlapping with, two conserved motifs present in the carboxy-terminal domain of eIF4A. Mutagenesis of amino acids within this region allowed us to alter the hippuristanol-sensitivity of eIF4A and undertake structure/function studies. Our results provide an understanding into how selective targeting of RNA helicases for pharmacological intervention can be achieved.

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

The CopA copper ATPase of Enterococcus hirae belongs to the family of heavy metal pumping CPx-type ATPases and shares 43% sequence similarity with the human Menkes and Wilson copper ATPases. Due to a lack of suitable protein crystals, only partial three-dimensional structures have so far been obtained for this family of ion pumps. We present a structural model of CopA derived by combining topological information obtained by intramolecular cross-linking with molecular modeling. Purified CopA was cross-linked with different bivalent reagents, followed by tryptic digestion and identification of cross-linked peptides by mass spectrometry. The structural proximity of tryptic fragments provided information about the structural arrangement of the hydrophilic protein domains, which was integrated into a three-dimensional model of CopA. Comparative modeling of CopA was guided by the sequence similarity to the calcium ATPase of the sarcoplasmic reticulum, Serca1, for which detailed structures are available. In addition, known partial structures of CPx-ATPase homologous to CopA were used as modeling templates. A docking approach was used to predict the orientation of the heavy metal binding domain of CopA relative to the core structure, which was verified by distance constraints derived from cross-links. The overall structural model of CopA resembles the Serca1 structure, but reveals distinctive features of CPx-type ATPases. A prominent feature is the positioning of the heavy metal binding domain. It features an orientation of the Cu binding ligands which is appropriate for the interaction with Cu-loaded metallochaperones in solution. Moreover, a novel model of the architecture of the intramembranous Cu binding sites could be derived.

The copper-responsive repressor CopR of Lactococcus lactis is a 'winged helix' protein

CopR of Lactococcus lactis is a copper-responsive repressor involved in copper homoeostasis. It controls the expression of a total of 11 genes, the CopR regulon, in a copper-dependent manner. In the absence of copper, CopR binds to the promoters of the CopR regulon. Copper releases CopR from the promoters, allowing transcription of the downstream genes to proceed. CopR binds through its N-terminal domain to a 'cop box' of consensus TACANNTGTA, which is conserved in Firmicutes. We have solved the NMR solution structure of the N-terminal DNA-binding domain of CopR. The protein fold has a winged helix structure resembling that of the BlaI repressor which regulates antibiotic resistance in Bacillus licheniformis. CopR differs from other copper-responsive repressors, and the present structure represents a novel family of copper regulators, which we propose to call the CopY family.

Intracellular localization of the BCL-2 family member BOK and functional implications

The pro-apoptotic BCL-2 family member BOK is widely expressed and resembles the multi-BH domain proteins BAX and BAK based on its amino acid sequence. The genomic region encoding BOK was reported to be frequently deleted in human cancer and it has therefore been hypothesized that BOK functions as a tumor suppressor. However, little is known about the molecular functions of BOK. We show that enforced expression of BOK activates the intrinsic (mitochondrial) apoptotic pathway in BAX/BAK-proficient cells but fails to kill cells lacking both BAX and BAK or sensitize them to cytotoxic insults. Interestingly, major portions of endogenous BOK are localized to and partially inserted into the membranes of the Golgi apparatus as well as the endoplasmic reticulum (ER) and associated membranes. The C-terminal transmembrane domain of BOK thereby constitutes a 'tail-anchor' specific for targeting to the Golgi and ER. Overexpression of full-length BOK causes early fragmentation of ER and Golgi compartments. A role for BOK on the Golgi apparatus and the ER is supported by an abnormal response of Bok-deficient cells to the Golgi/ER stressor brefeldin A. Based on these results, we propose that major functions of BOK are exerted at the Golgi and ER membranes and that BOK induces apoptosis in a manner dependent on BAX and BAK.