Conversion of the enzyme guanylate kinase into a mitotic-spindle orienting protein by a single mutation that inhibits GMP-induced closing

New protein functions can require complex sequence changes, but the minimal path is not well understood. The guanylate kinase enzyme (GK(enz)), which catalyzes phosphotransfer from ATP to GMP, evolved into the GK domain (GK(dom)), a protein-binding domain found in membrane associate guanylate kinases that function in mitotic spindle orientation and cell adhesion. Using an induced polarity assay for GK(dom) function, we show that a single serine to proline mutation is sufficient to switch extant GK(enz) into a functional GK(dom). The mutation blocks catalysis (GK(enz) function) but allows protein binding and spindle orientation (GK(dom) function). Furthermore, whereas the GK(enz) undergoes a large closing motion upon GMP binding, fluorescence quenching and NMR demonstrate that the S?→?P mutation inhibits GMP-induced GK movements. Disrupting GK closing with a mutation at a different position also leads to GK(dom) function, suggesting that blocking the GK(enz) closing motion is sufficient for functional conversion of GK(enz) to GK(dom). Although subtle changes in protein function can require complex sequence paths, our work shows that entirely new functions can arise from single mutations that alter protein dynamics.     

Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases

Malaria causes worldwide morbidity and mortality, and while chemotherapy remains an excellent means of malaria control, drug-resistant parasites necessitate the discovery of new antimalarials. Peptidases are a promising class of drug targets and perform several important roles during the Plasmodium falciparum erythrocytic life cycle. Herein, we report a multidisciplinary effort combining activity-based protein profiling, biochemical, and peptidomic approaches to functionally analyze two genetically essential P. falciparum metallo-aminopeptidases (MAPs), PfA-M1 and Pf-LAP. Through the synthesis of a suite of activity-based probes (ABPs) based on the general MAP inhibitor scaffold, bestatin, we generated specific ABPs for these two enzymes. Specific inhibition of PfA-M1 caused swelling of the parasite digestive vacuole and prevented proteolysis of hemoglobin (Hb)-derived oligopeptides, likely starving the parasite resulting in death. In contrast, inhibition of Pf-LAP was lethal to parasites early in the life cycle, prior to the onset of Hb degradation suggesting that Pf-LAP has an essential role outside of Hb digestion.     

Efalizumab,a Human Monoclonal Anti-CD11a Antibody,in the Treatment of Moderate to Severe Crohn’s Disease: An Open-Label Pilot Study

Background and Aims  

Efalizumab is a monoclonal antibody targeting CD11a, an adhesion molecule involved in the activation and trafficking of T-lymphocytes. This agent has proven efficacy in the treatment of psoriasis. We performed an open-label study to evaluate the efficacy and safety of efalizumab in Crohn’s disease (CD).     

The H4 histamine receptor agonist, clobenpropit, suppresses human cholangiocarcinoma progression by disruption of epithelial mesenchymal transition and tumor metastasis

Cholangiocarcinoma (CCA) is a biliary cancer arising from damaged bile ducts. Epithelial-mesenchymal transition (EMT) occurs as epithelial cells begin to resemble mesenchymal cells leading to increased invasion potential as the extracellular matrix (ECM) degrades. Histamine exerts its effects by way of four receptors (H1-H4 HRs). Clobenpropit, a potent H4HR agonist, inhibits mammary adenocarcinoma growth. We have shown that (1) cholangiocytes and CCA cells express H1-H4 HRs and (2) the H3HR decreases CCA proliferation. We evaluated the effects of clobenpropit on CCA proliferation, invasion, EMT phenotypes, and ECM degradation. In vitro, we used CCA cell lines to study proliferation, signaling pathways, and the morphological invasive potential. Gene and protein expression of the hepatobiliary epithelial markers CK-7, CK-8, and CK-19, the focal contact protein paxillin, and the mesenchymal markers fibronectin, s100A4, and vimentin were evaluated. Cell invasion across an ECM layer was quantitated and matrix metalloproteinase-1, -2, -3, -9, and -11 gene and protein expression was examined. Evaluation of the specific role of H4HR was performed by genetic knockdown of the H3HR and overexpression of H4HR. Proliferation was evaluated by proliferating cellular nuclear antigen immunoblotting. In vivo, xenograft tumors were treated with either vehicle or clobenpropit for 39 days. Tumor volume was recorded every other day. Clobenpropit significantly decreased CCA proliferation by way of a Ca(2+) -dependent pathway and altered morphological development and invasion. Loss of H3HR expression or overexpression of H4HR significantly decreased CCA proliferation. In vivo, clobenpropit inhibited xenograft tumor growth compared with controls. CONCLUSION: Modulation of H4HR by clobenpropit disrupts EMT processes, ECM breakdown, and invasion potential and decreases tumor growth. Interruption of tumorigenesis and invasion by histamine may add to therapeutic advances for CCAs.     

PKC-θ function at the immunological synapse: prospects for therapeutic targeting

Protein kinase C (PKC)-θ regulates conventional effector T (Teff) cell function. Since this initial finding, it has become clear that the role of PKC-θ in T cells is complex. PKC-θ plays a central role in Teff cell activation and survival, and negatively regulates stability of the immunological synapse (IS). Recent studies demonstrated that PKC-θ is required for the development of natural CD4(+)Foxp3(+) regulatory T (Treg) cells, and mediates negative regulation of Treg cell function. Here, we examine the role of PKC-θ in the IS, evidence for its distinct localization in Treg cells and the therapeutic implications of inhibiting PKC-θ in Teff cells, to reduce effector function, and in Treg cells, to increase suppressor function, for the prevention and treatment of autoimmune and alloimmune disease states.     

Safety, efficacy, and performance of implanted recycled cardiac rhythm management (CRM) devices in underprivileged patients

Introduction: Patients in underdeveloped nations have limited access to life‐saving medical technology including cardiac rhythm management (CRM) devices. We evaluated alternative means to provide such technology to this patient population while assessing the safety and efficacy of such a practice. Methods: Patients in the United States with clinical indications for extraction of CRM devices were consented. Antemortem CRM devices were cleaned and sterilized following a protocol established at our institution. Surveillance in vitro cultures were performed for quality assurance. The functional status of pulse generators was tested with a pacing system analyzer to confirm at least 70% battery life. Most generators were transported, in person, to an implanting institution in Nicaragua. Recipients with a Class I indication for CRM implantation, and meeting economical criteria set forth, were consented for implantation of a recycled device. Results: Between 2003 and 2009, implantation was performed in 17 patients with an average age of 42.1 ± 20.3 years. Of the 17 patients, nine were male and eight were female. Mean follow‐up was 68 ± 38 months. Device evaluation occurred prior to discharge, 4 weeks post implantation, and every 6 months thereafter. There were three deaths during the follow‐up period secondary to myocardial infarction, stroke, and heart failure. Hematoma formation occurred in one patient. No infections, early battery depletion, or device malfunction were identified during follow‐up. Conclusions: Our case series is the longest follow‐up of recipients of recycled antemortem CRM devices. Our findings support the feasibility and safety of this alternative acquisition of life‐saving technology. (PACE 2011; 653–658)     

Continuing professional development for interprofessional teams supporting patients in healthcare decision making

Healthcare professionals and organizations, policy makers, and the public are calling for safe and effective care that is centered on patients' needs, values, and preferences. The goals of interprofessional shared decision making and decision support are to help patients and professionals agree on choices that are effective, health promoting, realistic, and consonant with patients' and professionals' values and preferences. This requires collaboration among professionals and with patients and their family caregivers. Continuing professional development is urgently needed to help healthcare professionals acquire the knowledge, skills, and attitudes necessary to create and sustain a culture of collaboration. We describe a model that can be used to design, implement, and evaluate continuing education curricula in interprofessional shared decision making and decision support. This model aligns curricular goals, objectives, educational strategies, and evaluation instruments and strategies with desired learning and organizational outcomes. Educational leaders and researchers can institutionalize such curricula by linking them with quality improvement and patient safety initiatives.