Coupling of Cdc20 inhibition and activation by BubR1

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1–Cdk1 independently inhibits APC/C–Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.     

G-quadruplex-forming aptamer enhances the peroxidase activity of myoglobin against luminol

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin''s peroxidase activity was discovered. Through our strategy—in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.     

Role of cardiac renin-angiotensin system in sarcoplasmic reticulum function and gene expression in the ischemic-reperfused heart

The aim of this study was to explore the possible participation of cardiac renin-angiotensin system (RAS) in the ischemia-reperfusion induced changes in heart function as well as Ca²⁺-handling activities and gene expression of cardiac sarcoplasmic reticulum (SR) proteins. The isolated rat hearts, treated for 10 min without and with 30 M captopril or 100 M losartan, were subjected to 30 min ischemia followed by reperfusion for 60 min and processed for the measurement of SR function and gene expression. Attenuated recovery of the left ventricular developed pressure (LVDP) upon reperfusion of the ischemic heart was accompanied by a marked reduction in SR Ca²⁺-pump ATPase, Ca²⁺-uptake and Ca²⁺-release activities. Northern blot analysis revealed that mRNA levels for SR Ca²⁺-handling proteins such as Ca²⁺-pump ATPase (SERCA2a), ryanodine receptor, calsequestrin and phospholamban were decreased in the ischemia-reperfused heart as compared with the non-ischemic control. Treatment with captopril improved the recovery of LVDP as well as SR Ca²⁺-pump ATPase and Ca²⁺-uptake activities in the postischemic hearts but had no effect on changes in Ca²⁺-release activity due to ischemic-reperfusion. Losartan neither affected the changes in contractile function nor modified alterations in SR Ca²⁺-handling activities. The ischemia-reperfusion induced decrease in mRNA levels for SR Ca²⁺-handling proteins were not affected by treatment with captopril or losartan. The results suggest that the improvement of cardiac function in the ischemic-reperfused heart by captopril is associated with the preservation of SR Ca²⁺-pump activities; however, it is unlikely that this action of captopril is mediated through the modification of cardiac RAS. Furthermore, cardiac RAS does not appear to contribute towards the ischemia-reperfusion induced changes in gene expression for SR Ca²⁺-handling proteins.     

A highly conserved pocket on PP2A‐B56 is required for hSgo1 binding and cohesion protection during mitosis

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A‐B56 phosphatase through a coiled‐coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A‐B56‐hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A‐B56 substrates containing a canonical B56 binding motif. We find that PP2A‐B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis.     

Haematopoietic stem cell gene therapy to treat autoimmune disease

Autoimmune diseases affect approximately 6% of the population and are characterised by a pathogenic immune response that targets self-antigens. Well known diseases of this nature include type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis. Treatment is often restricted to replacement therapy or immunosuppressive regimes and to date there are no cures. The strategy of utilising autologous or allogeneic haematopoietic stem cell transplantation to treat autoimmunity and induce immunological tolerance has been trailed with various levels of success. A major issue is disease relapse as the autoimmune response is reinitiated. Cells of the immune system originate from bone marrow and have a central role in the induction of immunological tolerance. The ability to isolate and genetically manipulate bone marrow haematopoietic stem cells therefore makes these cells a suitable vehicle for driving ectopic expression of defined autoantigens and induction of immunological tolerance.     

Changes in fatty acid compositions of myocardial lipids in rats with heart failure following myocardial infarction

Changes in fatty acid composition of myocardial lipids were examined in rats with heart failure following myocardial infarction. Left ventricular systolic pressure (LVSP) was decreased and left ventricular end-diastolic pressure (LVEDP) was elevated 24 h, 1 and 12 weeks after left coronary artery ligation (CAL), suggesting the development of heart failure at these periods in this model. Hearts were isolated 24 h, 1 week and 12 weeks after the operation. Myocardial lipids in the infarcted scar tissue, non-infarcted remaining left ventricle including interseptum and right ventricle were separated into phospholipid (PL), triacylglycerol (TG), diacylglycerol (DAG) and free fatty acid (FFA) fractions. In the scar tissue PL content markedly decreased whereas TG, DAG and FFA contents increased 24 h after CAL. Despite a marked decrease in constituted fatty acids of PL fraction in the scar tissue the percentage of arachidonic acid in PL was elevated 12 weeks after CAL, suggesting that release of arachidonic acid during PL degradation was suppressed. In the non-infarcted viable left ventricle PL content remained unchanged throughout the experiment whereas TG, DAG and FFA contents were elevated 24 h after CAL. Despite no changes in PL and other lipid contents in the non-infarcted tissue the percentage of linoleic acid in PL was reduced and that of docosahexaenoic acid in PL was elevated 12 weeks after CAL. Our findings showed that myocardial lipid composition of the non-infarcted left ventricle was altered only in an early stage of the development of heart failure and fatty acid compositions of PL was exchanged in a late stage of the development of heart failure. The exchange may be related to cardiac dysfunction or myocardial remodelling in the rat with heart failure.     

In vitro ovine articular chondrocyte proliferation: experiments and modelling

This study focuses on analysis of in vitro cultures of chondrocytes from ovine articular cartilage. Isolated cells were seeded in Petri dishes, then expanded to confluence and phenotypically characterized by flow cytometry. The sigmoidal temporal profile of total counts was obtained by classic haemocytometry and corresponding cell size distributions were measured electronically using a Coulter Counter. A mathematical model recently proposed ( 1 ) was adopted for quantitative interpretation of these experimental data. The model is based on a 1‐D (that is, mass‐structured), single‐staged population balance approach capable of taking into account contact inhibition at confluence. The model’s parameters were determined by fitting measured total cell counts and size distributions. Model reliability was verified by predicting cell proliferation counts and corresponding size distributions at culture times longer than those used when tuning the model’s parameters. It was found that adoption of cell mass as the intrinsic characteristic of a growing chondrocyte population enables sigmoidal temporal profiles of total counts in the Petri dish, as well as cell size distributions at ‘balanced growth’, to be adequately predicted.     

Transplantation of bone marrow genetically engineered to express proinsulin II protects against autoimmune insulitis in NOD mice

BACKGROUND: Type 1 diabetes (T1D) is a T-cell-dependent autoimmune disease resulting from destructive inflammation (insulitis) of the insulin-producing pancreatic beta-cells. Transgenic expression of proinsulin II by a MHC class II promoter or transfer of bone marrow from these transgenic mice protects NOD mice from insulitis and diabetes. We assessed the feasibility of gene therapy in the NOD mouse as an approach to treat T1D by ex vivo genetic manipulation of normal hematopoietic stem cells (HSCs) with proinsulin II followed by transfer to recipient mice. METHODS: HSCs were isolated from 6-8-week-old NOD female mice and transduced in vitro with retrovirus encoding enhanced green fluorescent protein (EGFP) and either proinsulin II or control autoantigen. Additional control groups included mice transferred with non-manipulated bone marrow and mice which did not receive bone marrow transfer. EGFP-sorted or non-sorted HSCs were transferred into pre-conditioned 3-4-week-old female NOD mice and insulitis was assessed 8 weeks post-transfer. RESULTS: Chimerism was established in all major lymphoid tissues, ranging from 5-15% in non-sorted bone marrow transplants to 20-45% in EGFP-sorted bone marrow transplants. The incidence and degree of insulitis was significantly reduced in mice receiving proinsulin II bone marrow compared to controls. However, the incidence of sialitis in mice receiving proinsulin II bone marrow and control mice was not altered, indicating protection from insulitis was antigen specific. CONCLUSIONS: We show for the first time that ex vivo genetic manipulation of HSCs to express proinsulin II followed by transplantation to NOD mice can establish molecular chimerism and protect from destructive insulitis in an antigen-specific manner.     

Mast cell protease 5 mediates ischemia-reperfusion injury of mouse skeletal muscle

Ischemia with subsequent reperfusion (IR) injury is a significant clinical problem that occurs after physical and surgical trauma, myocardial infarction, and organ transplantation. IR injury of mouse skeletal muscle depends on the presence of both natural IgM and an intact C pathway. Disruption of the skeletal muscle architecture and permeability also requires mast cell (MC) participation, as revealed by the fact that IR injury is markedly reduced in c-kit defective, MC-deficient mouse strains. In this study, we sought to identify the pathobiologic MC products expressed in IR injury using transgenic mouse strains with normal MC development, except for the lack of a particular MC-derived mediator. Histologic analysis of skeletal muscle from BALB/c and C57BL/6 mice revealed a strong positive correlation (R(2) = 0.85) between the extent of IR injury and the level of MC degranulation. Linkage between C activation and MC degranulation was demonstrated in mice lacking C4, in which only limited MC degranulation and muscle injury were apparent. No reduction in injury was observed in transgenic mice lacking leukotriene C(4) synthase, hemopoietic PGD(2) synthase, N-deacetylase/N-sulfotransferase-2 (enzyme involved in heparin biosynthesis), or mouse MC protease (mMCP) 1. In contrast, muscle injury was significantly attenuated in mMCP-5-null mice. The MCs that reside in skeletal muscle contain abundant amounts of mMCP-5 which is the serine protease that is most similar in sequence to human MC chymase. We now report a cytotoxic activity associated with a MC-specific protease and demonstrate that mMCP-5 is critical for irreversible IR injury of skeletal muscle.