Mutation G1629E Increases von Willebrand Factor Cleavage via a Cooperative Destabilization Mechanism

The large multimeric glycoprotein von Willebrand Factor (VWF) plays a pivotal adhesive role during primary hemostasis. VWF is cleaved by the protease ADAMTS13 as a down-regulatory mechanism to prevent excessive VWF-mediated platelet aggregation. For each VWF monomer, the ADAMTS13 cleavage site is located deeply buried inside the VWF A2 domain. External forces in vivo or denaturants in vitro trigger the unfolding of this domain, thereby leaving the cleavage site solvent-exposed and ready for cleavage. Mutations in the VWF A2 domain, facilitating the cleavage process, cause a distinct form of von Willebrand disease (VWD), VWD type 2A. In particular, the VWD type 2A Gly1629Glu mutation drastically accelerates the proteolytic cleavage activity, even in the absence of forces or denaturants. However, the effect of this mutation has not yet been quantified, in terms of kinetics or thermodynamics, nor has the underlying molecular mechanism been revealed. In this study, we addressed these questions by using fluorescence correlation spectroscopy, molecular dynamics simulations, and free energy calculations. The measured enzyme kinetics revealed a 20-fold increase in the cleavage rate for the Gly1629Glu mutant compared with the wild-type VWF. Cleavage was found cooperative with a cooperativity coefficient n = 2.3, suggesting that the mutant VWF gives access to multiple cleavage sites of the VWF multimer at the same time. According to our simulations and free energy calculations, the Gly1629Glu mutation causes structural perturbation in the A2 domain and thereby destabilizes the domain by ～10 kJ/mol, promoting its unfolding. Taken together, the enhanced proteolytic activity of Gly1629Glu can be readily explained by an increased availability of the ADAMTS13 cleavage site through A2-domain-fold thermodynamic destabilization. Our study puts forward the Gly1629Glu mutant as a very efficient enzyme substrate for ADAMTS13 activity assays.     

Relative shortening and functional tethering of spinal cord in adolescent scoliosis – Result of asynchronous neuro-osseous growth, summary of an electronic focus group debate of the IBSE

There is no generally accepted scientific theory for the causes of adolescent idiopathic scoliosis (AIS). As part of its mission to widen understanding of scoliosis etiology, the International Federated Body on Scoliosis Etiology (IBSE) introduced the electronic focus group (EFG) as a means of increasing debate on knowledge of important topics. This has been designated as an on-line Delphi discussion. The Statement for this debate was written by Dr WCW Chu and colleagues who examine the spinal cord to vertebral growth interaction during adolescence in scoliosis. Using the multi-planar reconstruction technique of magnetic resonance imaging they investigated the relative length of spinal cord to vertebral column including ratios in 28 girls with AIS (mainly thoracic or double major curves) and 14 age-matched normal girls. Also evaluated were cerebellar tonsillar position, somatosensory evoked potentials (SSEPs), and clinical neurological examination. In severe AIS compared with normal controls, the vertebral column is significantly longer without detectable spinal cord lengthening. They speculate that anterior spinal column overgrowth relative to a normal length spinal cord exerts a stretching tethering force between the two ends, cranially and caudally leading to the initiation and progression of thoracic AIS. They support and develop the Roth-Porter concept of uncoupled neuro-osseous growth in the pathogenesis of AIS which now they prefer to term ' asynchronous neuro-osseous growth'. Morphological evidence about the curve apex suggests that the spinal cord is also affected, and a 'double pathology' is suggested. AIS is viewed as a disorder with a wide spectrum and a common neuroanatomical abnormality namely, a spinal cord of normal length but short relative to an abnormally lengthened anterior vertebral column. Neuroanatomical changes and/or abnormal neural function may be expressed only in severe cases. This asynchronous neuro-osseous growth concept is regarded as one component of a larger concept. The other component relates to the brain and cranium of AIS subjects because abnormalities have been found in brain (infratentorial and supratentorial) and skull (vault and base). The possible relevance of systemic melatonin-signaling pathway dysfunction, platelet calmodulin levels and putative vertebral vascular biology to the asynchronous neuro-osseous growth concept is discussed. A biomechanical model to test the spinal component of the concept is in hand. There is no published research on the biomechanical properties of the spinal cord for scoliosis specimens. Such research on normal spinal cords includes movements (kinematics), stress-strain responses to uniaxial loading, and anterior forces created by the stretched cord in forward flexion that may alter sagittal spinal shape during adolescent growth. The asynchronous neuro-osseous growth concept for the spine evokes controversy. Dr Chu and colleagues respond to five other concepts of pathogenesis for AIS and suggest that relative anterior spinal overgrowth and biomechanical growth modulation may also contribute to AIS pathogenesis.     

European guidelines for quality assurance in cervical cancer screening: recommendations for clinical management of abnormal cervical cytology, part 1

The current paper presents the first part of Chapter 6 of the second edition of the European Guidelines for Quality Assurance in Cervical Cancer Screening. It provides guidance on how to manage women with abnormal cervical cytology. Throughout this article the Bethesda system is used for cervical cytology terminology, as the European guidelines have recommended that all systems should at least be translated into that terminology while cervical intraepithelial neoplasia (CIN) is used for histological biopsies (Cytopathology 2007; 18 :213–9). A woman with a high‐grade cytological lesion, a repeated low‐grade lesion or with an equivocal cytology result and a positive human papillomavirus (HPV) test should be referred for colposcopy. The role of the colposcopist is to identify the source of the abnormal cells and to make an informed decision as to whether or not any treatment is required. If a patient requires treatment the colposcopist will decide which is the most appropriate method of treatment for each individual woman. The colposcopist should also organize appropriate follow‐up for each woman seen. Reflex testing for high‐risk HPV types of women with atypical squamous cells (ASC) of undetermined significance with referral for colposcopy of women who test positive is a first option. Repeat cytology is a second possibility. Direct referral to a gynaecologist should be restricted to special circumstances. Follow‐up of low‐grade squamous intraepithelial lesion is more difficult because currently there is no evidence to support any method of management as being optimal; repeat cytology and colposcopy are options, but HPV testing is not sufficiently selective, unless for older women. Women with high‐grade squamous intraepithelial lesion (HSIL) or atypical squamous cells, cannot exclude HSIL (ASC‐H) should be referred without triage. Women with glandular lesions require particular attention. In a subsequent issue of Cytopathology, the second part of Chapter 6 will be presented, with recommendations for management and treatment of histologically confirmed intraepithelial neoplasia and guidance for follow‐up of special cases such as women who are pregnant, postmenopausal or immunocompromised.     

Lateral transfer of an EF-1alpha gene: origin and evolution of the large subunit of ATP sulfurylase in eubacteria

It is generally accepted that new genes arise via duplication and functional divergence of existing genes, in accordance with Ohno's model, now called "Mutation During Redundancy," or MDR. In this model, one of the two gene copies is free to acquire novel (although likely related) activities through mutation, since only one copy is required for its original function. However, duplication within a genome is not the only process that might give rise to this situation: acquisition of a functionally redundant gene by lateral gene transfer (LGT) could also initiate the MDR process. Here we describe a probable instance, involving LGT of an archaeal or eukaryotic elongation factor 1alpha (EF-1alpha) gene. The large subunit of ATP sulfurylase (CysN or the N-terminal portion of NodQ), found mainly in proteobacteria, is clearly related to translation elongation factors. However, our analyses show that cysN arose from an EF-1alpha gene initially acquired by LGT, not from a within-genome duplication of the resident EF-Tu gene. To our knowledge, this is the first unequivocal case of LGT followed by functional modification to be described; this mechanism could be a potentially important force in establishing genes with novel functions in genomes.     

The Protein Tyrosine Phosphatase Rptpζ Suppresses Osteosarcoma Development in Trp53-Heterozygous Mice

Osteosarcoma (OS), a highly aggressive primary bone tumor, belongs to the most common solid tumors in growing children. Since specific molecular targets for OS treatment remain to be identified, surgical resection combined with multimodal (neo-)adjuvant chemotherapy is still the only way to help respective individuals. We have previously identified the protein tyrosine phosphatase Rptpζ as a marker of terminally differentiated osteoblasts, which negatively regulates their proliferation in vitro. Here we have addressed the question if Rptpζ can function as a tumor suppressor protein inhibiting OS development in vivo. We therefore analyzed the skeletal phenotype of mice lacking Ptprz1, the gene encoding Rptpζ on a tumor-prone genetic background, i.e. Trp53-heterozygosity. By screening a large number of 52 week old Trp53-heterozygous mice by contact radiography we found that Ptprz1-deficiency significantly enhanced OS development with 19% of the mice being affected. The tumors in Ptprz1-deficient Trp53-heterozygous mice were present in different locations (spine, long bones, ribs), and their OS nature was confirmed by undecalcified histology. Likewise, cell lines derived from the tumors were able to undergo osteogenic differentiation ex vivo. A comparison between Ptprz1-heterozygous and Ptprz1-deficient cultures further revealed that the latter ones displayed increased proliferation, a higher abundance of tyrosine-phosphorylated proteins and resistance towards the influence of the growth factor Midkine. Our findings underscore the relevance of Rptpζ as an attenuator of proliferation in differentiated osteoblasts and raise the possibility that activating Rptpζ-dependent signaling could specifically target osteoblastic tumor cells.     

Complement-Opsonized HIV-1 Overcomes Restriction in Dendritic Cells

DCs express intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. Thus, DCs are productively infected only at very low levels with HIV-1, and this non-permissiveness of DCs is suggested to go along with viral evasion. We now illustrate that complement-opsonized HIV-1 (HIV-C) efficiently bypasses SAMHD1 restriction and productively infects DCs including BDCA-1 DCs. Efficient DC infection by HIV-C was also observed using single-cycle HIV-C, and correlated with a remarkable elevated SAMHD1 T592 phosphorylation but not SAMHD1 degradation. If SAMHD1 phosphorylation was blocked using a CDK2-inhibitor HIV-C-induced DC infection was also significantly abrogated. Additionally, we found a higher maturation and co-stimulatory potential, aberrant type I interferon expression and signaling as well as a stronger induction of cellular immune responses in HIV-C-treated DCs. Collectively, our data highlight a novel protective mechanism mediated by complement opsonization of HIV to effectively promote DC immune functions, which might be in the future exploited to tackle HIV infection.     

Impact of olfactory non-host predator cues on aggregation behaviour and activity in <Emphasis Type="Italic">Polymorphus minutus</Emphasis> infected <Emphasis Type="Italic">Gammarus pulex</Emphasis>

Parasites with a complex life cycle are supposed to influence the behaviour of their intermediate host in such a way that the transmission to the final host is enhanced, but reduced to non-hosts. Here, we examined whether the trophically transmitted bird parasite Polymorphus minutus increases the antipredator response of its intermediate host, the freshwater amphipod Gammarus pulex to fish cues, i.e. non-host cues (‘increased host abilities hypothesis’). Aggregation behaviour and reduced activity are assumed to decrease the predation risk of gammarids by fishes. Uninfected G. pulex are known to aggregate in the presence of a fish predator. In the present study, gammarids were allowed to choose either to join a group of conspecifics or to stay solitary (experiment 1) or between two groups differing in infection status (experiment 2), both in the presence or absence of fish odour. The perception of the groups was limited to mainly olfactory cues. Contrary to the ‘increased host abilities hypothesis’, in infected gammarids of experiment 1, fish cues induced similar aggregation behaviour as in their uninfected conspecifics. In experiment 2, uninfected as well as infected gammarids did not significantly discriminate between infected and uninfected groups. Although only uninfected gammarids reduced their activity in the presence of predator cues, infected G. pulex were generally less active than uninfected conspecifics. This might suggest that P. minutus manipulates rather the general anti-predator behaviour than the plastic response to predation risk.     

Theoretical prediction of the basic helix types in alpha,beta-hybrid peptides

This study provides a complete overview on all possible helical- folding patterns, their stabilities, and their detailed molecular structure in the novel foldamer class of alpha,beta-hybrid peptides on the basis of ab initio molecular orbital (MO) theory. The results indicate a considerable intrinsic potential of backbone folding. As found for other peptide foldamers, representatives of mixed or beta-helices are most stable in more apolar media, whereas polar environments favor the helices with the hydrogen bonds pointing in only one direction. The theoretical results confirm the hydrogen-bonding patterns found in the first experimental studies on these hybrid peptides. Selecting special backbone substitution patterns, the secondary structure potential of the alpha,beta-hybrid peptides could be of great importance for a rational peptide and protein design.     

Evolution of dark-spored Myxomycetes (slime-molds): molecules versus morphology

The Myxomycetes are a major component of soil amoebae, displaying a complex life cycle that terminates in the formation of often macroscopic fruiting bodies. The classification of Myxomycetes is controversial and strongly depends on the weight given by different authors to morphological and developmental characters. We used a molecular approach to establish the phylogenetic relationships in the dark-spored orders Stemonitales and Physarales. Twenty-five small subunit ribosomal RNA gene sequences were obtained, with focus on two Stemonitales genera, Lamproderma and Comatricha. Unexpectedly, our results show that Stemonitales are paraphyletic with Physarales arising from within a Lamproderma clade. The genus Lamproderma itself is polyphyletic and can be divided into two distinct clades. Additionally, we found that Comatricha nigricapillitia comprises two cryptic species, both related to Enerthenema. Our study allows the reappraisal of morphological and developmental characters in the light of molecular data and sets foundations for a new classification of Myxomycetes.