Structural and functional relationships in the virulence-associated cathepsin L proteases of the parasitic liver fluke, Fasciola hepatica

The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and biochemically characterized. By using site-directed mutagenesis, we show that residues at position 67 and 205, which lie within the S2 pocket of the active site, are critical in determining the substrate and inhibitor specificity. FheCL1 exhibits a broader specificity and a higher substrate turnover rate compared with FheCL2. However, FheCL2 can efficiently cleave substrates with a Pro in the P2 position and degrade collagen within the triple helices at physiological pH, an activity that among cysteine proteases has only been reported for human cathepsin K. The 1.4-A three-dimensional structure of the FheCL1 was determined by x-ray crystallography, and the three-dimensional structure of FheCL2 was constructed via homology-based modeling. Analysis and comparison of these structures and our biochemical data with those of human cathepsins L and K provided an interpretation of the substrate-recognition mechanisms of these major parasite proteases. Furthermore, our studies suggest that a configuration involving residue 67 and the "gatekeeper" residues 157 and 158 situated at the entrance of the active site pocket create a topology that endows FheCL2 with its unusual collagenolytic activity. The emergence of a specialized collagenolytic function in Fasciola likely contributes to the success of this tissue-invasive parasite.

Cytohesin Binder and Regulator (Cybr) is Not Essential for T- and Dendritic-Cell Activation and Differentiation

Cybr (also known as Cytip, CASP, and PSCDBP) is an interleukin-12-induced gene expressed exclusively in hematopoietic cells and tissues that associates with Arf guanine nucleotide exchange factors known as cytohesins. Cybr levels are dynamically regulated during T-cell development in the thymus and upon activation of peripheral T cells. In addition, Cybr is induced in activated dendritic cells and has been reported to regulate dendritic cell (DC)-T-cell adhesion. Here we report the generation and characterization of Cybr-deficient mice. Despite the selective expression in hematopoietic cells, there was no intrinsic defect in T- or B-cell development or function in Cybr-deficient mice. The adoptive transfer of Cybr-deficient DCs showed that they migrated efficiently and stimulated proliferation and cytokine production by T cells in vivo. However, competitive stem cell repopulation experiments showed a defect in the abilities of Cybr-deficient T cells to develop in the presence of wild-type precursors. These data suggest that Cybr is not absolutely required for hematopoietic cell development or function, but stem cells lacking Cybr are at a developmental disadvantage compared to wild-type cells. Collectively, these data demonstrate that despite its selective expression in hematopoietic cells, the role of Cybr is limited or largely redundant. Previous in vitro studies using overexpression or short interfering RNA inhibition of the levels of Cybr protein appear to have overestimated its immunological role.

Three-dimensional cultures of normal human osteoblasts: proliferation and differentiation potential in vitro and upon ectopic implantation in nude mice.

This paper is novel andreports on the in vitro establishment of 3-D cultures of human osteoblasts. These were evaluated for protein markers of bone cells. Sequentially alkaline phosphatase, calcium incorporation for matrix mineralisation and then finally osteocalcin expression were detected in cultures. The extracellular matrix was composed of type 1 collagen and as it mineralised, needle shaped crystals were often associated with matrix vesicles initiating mineralisation. In vivo implantation in nude mice showed progression of mineralisation from the inner region outward with peripheral cells in a non-mineralised matrix. Host vessels invaded the implanted cell area. The research has relevance to musculoskeletal tissue engineering.

Species boundaries and phylogenetic relationships within the green algal genus Codium (Bryopsidales) based on plastid DNA sequences

Despite the potential model role of the green algal genus Codium for studies of marine speciation and evolution, there have been difficulties with species delimitation and a molecular phylogenetic framework was lacking. In the present study, 74 evolutionarily significant units (ESUs) are delimited using 227 rbcL exon 1 sequences obtained from specimens collected throughout the genus' range. Several morpho-species were shown to be poorly defined, with some clearly in need of lumping and others containing pseudo-cryptic diversity. A phylogenetic hypothesis of 72 Codium ESUs is inferred from rbcL exon 1 and rps3-rp/16 sequence data using a conventional nucleotide substitution model (GTR + Gamma + I), a codon position model and a covariotide (covarion) model, and the fit of a multitude of substitution models and alignment partitioning strategies to the sequence data is reported. Molecular clock tree rooting was carried out because out-group rooting was probably affected by phylogenetic bias. Several aspects of the evolution of morphological features of Codium are discussed and the inferred phylogenetic hypothesis is used as a framework to study the biogeography of the genus, both at a global scale and within the Indian Ocean. (c) 2007 Elsevier Inc. All rights reserved.

School Ethos and Governor Relationships

This paper argues that an understanding of ethos is essential for appreciating the process of policy implementation in schools. Whilst educational literature consistently refers to the importance of ethos as a means of understanding the distinctive nature of schools, there is little exploration of school ethos as an effective mediator of policy. Drawing on qualitative data collected in three primary schools it will be demonstrated how ethos can act as a bridge or a barrier to the successful construction of the model of collaborative relationships between school governors and heads, as set out by the education reform legislation of the late 1980s

Runx2, p53, and pRB Status as Diagnostic Parameters for Deregulation of Osteoblast Growth and Differentiation in a New Pre-Chemotherapeutic Osteosarcoma Cell Line (OS1)

Osteosarcomas are the most prevalent primary bone tumors found in pediatric patients. To understand their molecular etiology, cell culture models are used to define disease mechanisms under controlled conditions. Many osteosarcoma cell lines (e.g., SAOS-2, U2OS, MG63) are derived from Caucasian patients. However, patients exhibit individual and ethnic differences in their responsiveness to irradiation and chemotherapy. This motivated the establishment of osteosarcoma cell lines (OS1, OS2, OS3) from three ethnically Chinese patients. OS1 cells, derived from a pre-chemotherapeutic tumor in the femur of a 6-year-old female, were examined for molecular markers characteristic for osteoblasts, stem cells, and cell cycle control by immunohistochemistry, reverse transcriptase-PCR, Western blotting and flow cytometry. OS I have aberrant G-banded karyotypes, possibly reflecting chromosomal abnormalities related to p53 deficiency. OS I had ossification profiles similar to human fetal osteoblasts rather than SAOS-2 which ossifies ab initio, (P

The Impact of Shame on the Therapeutic Alliance and Intimate Relationships

Objectives


This study examined the role of shame coping styles and state shame in predicting the therapeutic alliance and intimate relationship functioning in individuals with mental health problems.


Method


A sample of 50 treatment-receiving adults aged 21 to 67 years with a mix of common mental health difficulties was recruited from a clinical psychology service. Participants were given questionnaire measures of shame states, shame coping styles, intimate relationship functioning, and the therapeutic alliance.


Results


Regression analyses indicated that the shame coping strategy of physical and psychological withdrawal was the primary risk factor for development of a less effective therapeutic alliance. Both withdrawal and attack self coping styles were significant predictors of impaired intimate relationship functioning.


Conclusions


These findings have implications for the theoretical role of shame in mental health presentations as well as the potential for internalizing shame coping styles (i.e., withdrawal, attack self) to act as a barrier to successful therapy and interpersonal relationships. The inclusion of shame-focused assessments and interventions in the initial stages of treatment with clients exhibiting these strategies could improve prognosis.

Contact and context: sharing education and building relationships in a divided society

One approach to tackling problems of division in society has been to promote collaboration and engagement between schools separated on ethno-religious lines. Based on some variant of contact theory, the received wisdom is that inter-group encounters can contribute to prejudice reduction and promote more harmonious relationships. Evidence to support this analysis is convincing; however, relatively little is known about the environmental factors that impede or enhance the potential for contact in different contexts. The importance of understanding such factors is underscored in divided jurisdictions, where separate education has been linked to the perpetuation of division and hostility. This paper adopts a qualitative approach to exploring the impact of two inter-school initiatives in Northern Ireland. The projects are located in contrasting socio-political and demographic environments, and research findings point to very different contact outcomes for participants in each. Seemingly relevant factors include the degree of congruence between school and community norms and values, the opportunity to develop relationships outside the school context, the relationships developed between the schools and local communities and the historical, political and social referents used by individuals to navigate the contact experience. The paper concludes with some reflections on factors that may help foster social harmony and on potential policy implications of the findings.

Complex trauma and intimate relationships: The impact of shame, guilt and dissociation

Background: This study examined dissociation, shame, guilt and intimate relationship difficulties in those with chronic and complex PTSD. Little is known about how these symptom clusters interplay within the complex PTSD constellation. Dissociation was examined as a principle organizing construct
within complex PTSD. In addition, the impact of shame, guilt and dissociation on relationship difficulties was explored.
Methods: Sixty five treatment-receiving adults attending a Northern Irish service for conflict-related trauma were assessed on measures of dissociation, state and trait shame, behavioral responses to shame, state and trait guilt, complex PTSD symptom severity and relationship difficulties.
Results: Ninety five percent (n=62) of participants scored above cut-off for complex PTSD. Those with clinical levels of dissociation (n=27) were significantly higher on complex PTSD symptom severity, state and trait shame, state guilt, withdrawal in response to shame and relationship preoccupation than subclinical dissociators (n=38). Dissociation and state and trait shame predicted complex PTSD. Fear of relationships was predicted by dissociation, complex PTSD and avoidance in response to shame, while complex PTSD predicted relationship anxiety and relationship depression.
Limitations: The study was limited to a relatively homogeneous sample of individuals with chronic and complex PTSD drawn from a single service.
Conclusions: Complex PTSD has significant consequences for intimate relationships, and dissociation makes an independent contribution to these difficulties. Dissociation also has an organizing effect on
complex PTSD symptoms.

Sequencing and its consequences:Path dependence and the relationships between genetics and medicalization

Both advocacy for and critiques of the Human Genome Project assume a self-sustaining relationship between genetics and. medicalization. However, this assumption ignores the ways in which the meanings of genetic research are conditional on its position in sequences of events. Based, on analyses of three conditions for which at least one putative gene or genetic marker has been identified, this article argues that critical junctures in the institutional stabilization of phenotypes and the mechanisms that sustain such classifications over time configure the practices and meanings of genetic research. Path dependence is critical to understanding the lack of consistent fit between genetics and medlcalization.