A proline-rich protein, verprolin, involved in cytoskeletal organization and cellular growth in the yeast Saccharomyces cerevisiae

A gene (VRP1) encoding a novel proline-rich protein (verprolin) has been isolated from the yeast Saccharomyces cerevisiae as a result of its hybridization to a chick vinculin cDNA probe. The deduced protein sequence contains 24% proline residues present as proline-rich motifs throughout the verprolin sequence. Several of these motifs resemble recently identified sequences shown to bind Src homology 3 (SH3) domains in vitro. Replacement of the wild-type VRP1 allele with a mutant allele results in strains that grow slower than wild-type strains and are temperature sensitive. The vrp1 mutants are impaired in both cell shape and size and display aberrant chitin and actin localization. We propose that verprolin is involved in the maintenance of the yeast actin cytoskeleton, through interactions with other proteins, possibly containing SH3 domains.     

Biochemical and molecular characterization of a thermostable chitosanase produced by the strain Paenibacillus sp. 1794 newly isolated from compost

Chitosan raises a great interest among biotechnologists due to its potential for applications in biomedical or environmental fields. Enzymatic hydrolysis of chitosan is a recognized method allowing control of its molecular size, making possible its optimization for a given application. During the industrial hydrolysis process of chitosan, viscosity is a major problem; which can be circumvented by raising the temperature of the chitosan solution. A thermostable chitosanase is compatible with enzymatic hydrolysis at higher temperatures thus allowing chitosan to be dissolved at higher concentrations. Following an extensive micro-plate screening of microbial isolates from various batches of shrimp shells compost, the strain 1794 was characterized and shown to produce a thermostable chitosanase. The isolate was identified as a novel member of the genus Paenibacillus, based on partial 16S rDNA and rpoB gene sequences. Using the chitosanase (Csn1794) produced by this strain, a linear time course of chitosan hydrolysis has been observed for at least 6 h at 70 °C. Csn1794 was purified and its molecular weight was estimated at 40 kDa by SDS-PAGE. Optimum pH was about 4.8, the apparent K _m and the catalytic constant k_cat were 0.042 mg/ml and 7,588 min^?1, respectively. The half-life of Csn1794 at 70 °C in the presence of chitosan substrate was >20 h. The activity of chitosanase 1794 varied little with the degree of N-acetylation of chitosan. The enzyme also hydrolyzed carboxymethylcellulose but not chitin. Chitosan or cellulose-derived hexasaccharides were cleaved preferentially in a symmetrical way (“3?+?3”) but hydrolysis rate was much faster for (GlcN)₆ than (Glc)₆. Gene cloning and sequencing revealed that Csn1794 belongs to family 8 of glycoside hydrolases. The enzyme should be useful in biotechnological applications of chitosan hydrolysis, dealing with concentrated chitosan solutions at high temperatures.     

Nutrient concentration-stream discharge relationships during storm events in a first-order stream

The relationship between nutrient element concentration and stream discharge during storm events was studied in a nutrient-rich first order stream. Stream concentrations of NO₃-N, phosphate-P, Ca, and Mg were determined during the course of and following thunderstorms. Nutrient element trajectories were constructed for the elements monitored and some recognizable and reproducible patterns in nutrient concentrations emerged. NO₃-N and phosphate-P generally increased in concentration during the early stages of increasing stream discharge resulting in a general clockwise trajectory. Ca and Mg consistently showed decreases in concentration during rising water, but the pattern of the trajectories was less constant. The patterns seen for NO₃-N and phosphate-P suggest surface run-off as their origin while the patterns for Ca and Mg reflect their primary origin in groundwater. The ability to detect these differences from the analysis of nutrient trajectories suggests the use of this technique for determining the source of other elements in streams.     

5-Androstene-3β,7β,17β-triol (β-AET) slows thermal injury induced osteopenia in mice: relation to aging and osteoporosis

5-Androstene-3β,7β,17β-triol (β-AET), an active metabolite of dehydroepiandrosterone (DHEA), reversed glucocorticoid (GC)-induced suppression of IL-6, IL-8 and osteoprotegerin production by human osteoblast-like MG-63 cells and promoted osteoblast differentiation of human mesenchymal stem cells (MSCs). In a murine thermal injury model that includes glucocorticoid-induced osteopenia, β-AET significantly (p<0.05) preserved bone mineral content, restored whole body bone mineral content and endochondral growth, suggesting reversal of GC-mediated decreases in chondrocyte proliferation, maturation and osteogenesis in the growth plate. In men and women, levels of β-AET decline with age, consistent with a role for β-AET relevant to diseases associated with aging. β-AET, related compounds or synthetic derivatives may be part of effective therapeutic strategies to accelerate tissue regeneration and prevent or treat diseases associated with aging such as osteoporosis.     

Evaluation of miglustat as maintenance therapy after enzyme therapy in adults with stable type 1 Gaucher disease: a prospective,open-label non-inferiority study

Background

Previous studies have provided equivocal data on the use of miglustat as maintenance therapy in Gaucher disease type 1. We report findings from a clinical trial evaluating the effects of miglustat treatment in patients with stable type 1 Gaucher disease after enzyme therapy.

Methods

Adult type 1 Gaucher disease patients stabilized during at least 3 years of previous enzyme therapy were included in this 2-year, prospective, open-label non-inferiority study. The primary endpoint was percent change from baseline in liver volume. Secondary endpoints included changes in spleen volume, hemoglobin concentration and platelet count.

Results

Forty-two patients were enrolled (mean±SD age, 45.1±12.7 years; previous enzyme therapy duration 9.5±4.0 years). Median (range) exposure to miglustat 100 mg t.i.d. was 658 (3–765) days. Twenty-one patients discontinued treatment prematurely; 13 due to adverse events, principally gastrointestinal. The upper 95% confidence limit of mean percent change in liver volume from baseline to end of treatment was below the non-inferiority margin of 10% (–1.1%; 95%CI ?6.0, 3.9%). Mean (95%CI) changes in spleen volume, hemoglobin concentration and platelet count were 102 (24,180) mL, –0.95 (?1.38, –0.53) g/dL and ?44.1 (–57.6, –30.7) ×10⁹/L, respectively.

Conclusions

The primary efficacy endpoint was met; overall there was no change in liver volume during 24 months of miglustat therapy. Several patients showed a gradual deterioration in some disease manifestations, suggesting that miglustat could maintain clinical stability, but not in all patients. Miglustat demonstrated a predictable profile of safety and tolerability that was consistent with that reported in previous clinical trials and experience in clinical practice.

Trial registration

Clinicaltrials.gov identifier NCT00319046

     

Mast cell restricted mouse and human tryptase·heparin complexes hinder thrombin-induced coagulation of plasma and the generation of fibrin by proteolytically destroying fibrinogen

The mouse and human TPSB2 and TPSAB1 genes encode tetramer-forming tryptases stored in the secretory granules of mast cells (MCs) ionically bound to heparin-containing serglycin proteoglycans. In mice these genes encode mouse MC protease-6 (mMCP-6) and mMCP-7. The corresponding human genes encode a family of serine proteases that collectively are called hTryptase-β. We previously showed that the α chain of fibrinogen is a preferred substrate of mMCP-7. We now show that this plasma protein also is highly susceptible to degradation by hTryptase-β· and mMCP-6·heparin complexes and that Lys(575) is a preferred cleavage site in the protein α chain. Because cutaneous mouse MCs store substantial amounts of mMCP-6·heparin complexes in their secretory granules, the passive cutaneous anaphylaxis reaction was induced in the skin of mMCP-6(+)/mMCP-7(-) and mMCP-6(-)/mMCP-7(-) C57BL/6 mice. In support of the in vitro data, fibrin deposits were markedly increased in the skin of the double-deficient mice 6 h after IgE-sensitized animals were given the relevant antigen. Fibrinogen is a major constituent of the edema fluid that accumulates in tissues when MCs degranulate. Our discovery that mouse and human tetramer-forming tryptases destroy fibrinogen before this circulating protein can be converted to fibrin changes the paradigm of how MCs hinder fibrin deposition and blood coagulation internally. Because of the adverse consequences of fibrin deposits in tissues, our data explain why mice and humans lack a circulating protease inhibitor that rapidly inactivates MC tryptases and why mammals have two genes that encode tetramer-forming serine proteases that preferentially degrade fibrinogen.     

Dual equipoise shared decision making: definitions for decision and behaviour support interventions

Background

In the experience of health professionals, it appears that interacting with peers in the workplace fosters learning and information sharing. Informal groups and networks present good opportunities for information exchange. Communities of practice (CoPs), which have been described by Wenger and others as a type of informal learning organization, have received increasing attention in the health care sector; however, the lack of uniform operating definitions of CoPs has resulted in considerable variation in the structure and function of these groups, making it difficult to evaluate their effectiveness.

Objective

To critique the evolution of the CoP concept as based on the germinal work by Wenger and colleagues published between 1991 and 2002.

Discussion

CoP was originally developed to provide a template for examining the learning that happens among practitioners in a social environment, but over the years there have been important divergences in the focus of the concept. Lave and Wenger's earliest publication (1991) centred on the interactions between novices and experts, and the process by which newcomers create a professional identity. In the 1998 book, the focus had shifted to personal growth and the trajectory of individuals' participation within a group (i.e., peripheral versus core participation). The focus then changed again in 2002 when CoP was applied as a managerial tool for improving an organization's competitiveness.

Summary

The different interpretations of CoP make it challenging to apply the concept or to take full advantage of the benefits that CoP groups may offer. The tension between satisfying individuals' needs for personal growth and empowerment versus an organization's bottom line is perhaps the most contentious of the issues that make CoPs difficult to cultivate. Since CoP is still an evolving concept, we recommend focusing on optimizing specific characteristics of the concept, such as support for members interacting with each other, sharing knowledge, and building a sense of belonging within networks/teams/groups. Interventions that facilitate relationship building among members and that promote knowledge exchange may be useful for optimizing the function of these groups.     

Adjuncts or adversaries to shared decision-making? Applying the Integrative Model of behavior to the role and design of decision support interventions in healthcare interactions

Background

Clinical practice guidelines have been a popular tool for the improvement of health care through the implementation of evidence from systematic research. Yet, it is increasingly clear that knowledge alone is insufficient to change practice. The social, cultural, and material contexts within which practice occurs may invite or reject innovation, complement or inhibit the activities required for success, and sustain or alter adherence to entrenched practices. However, knowledge translation (KT) models are limited in providing insight about how and why contextual contingencies interact, the causal mechanisms linking structural aspects of context and individual agency, and how these mechanisms influence KT. Another limitation of KT models is the neglect of methods to engage potential adopters of the innovation in critical reflection about aspects of context that influence practice, the relevance and meaning of innovation in the context of practice, and the identification of strategies for bringing about meaningful change.

Discussion

This paper presents a KT model, the Critical Realism and the Arts Research Utilization Model (CRARUM), that combines critical realism and arts-based methodologies. Critical realism facilitates understanding of clinical settings by providing insight into the interrelationship between its structures and potentials, and individual action. The arts nurture empathy, and can foster reflection on the ways in which contextual factors influence and shape clinical practice, and how they may facilitate or impede change. The combination of critical realism and the arts within the CRARUM model promotes the successful embedding of interventions, and greater impact and sustainability.

Conclusion

CRARUM has the potential to strengthen the science of implementation research by addressing the complexities of practice settings, and engaging potential adopters to critically reflect on existing and proposed practices and strategies for sustaining change.     

The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of beta-carotene accumulation

Despite recent progress in our understanding of carotenogenesis in plants, the mechanisms that govern overall carotenoid accumulation remain largely unknown. The Orange (Or) gene mutation in cauliflower (Brassica oleracea var botrytis) confers the accumulation of high levels of beta-carotene in various tissues normally devoid of carotenoids. Using positional cloning, we isolated the gene representing Or and verified it by functional complementation in wild-type cauliflower. Or encodes a plastid-associated protein containing a DnaJ Cys-rich domain. The Or gene mutation is due to the insertion of a long terminal repeat retrotransposon in the Or allele. Or appears to be plant specific and is highly conserved among divergent plant species. Analyses of the gene, the gene product, and the cytological effects of the Or transgene suggest that the functional role of Or is associated with a cellular process that triggers the differentiation of proplastids or other noncolored plastids into chromoplasts for carotenoid accumulation. Moreover, we demonstrate that Or can be used as a novel genetic tool to induce carotenoid accumulation in a major staple food crop. We show here that controlling the formation of chromoplasts is an important mechanism by which carotenoid accumulation is regulated in plants.