Equality to equals and unequals: a revisit of the equivalence and nonequivalence criteria in object-oriented software testing

published_or_final_versio

Structure-function study of ubiquitin c-terminal hydrolase L1 (UCH-L1) by NMR spectroscopy - insights into UCH-L1 mutation's association with the risk of Parkinson's disease

Poster Presentation: P72Protein ubiquitination and deubiquitination, play important roles in many aspects of cellular mechanisms. Its defective regulation results in diseases that range from developmental abnormalities to neurodegenerative diseases and cancer. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) is a protein of 223 amino acids, which is highly abundant in brain, constituting up to 2% of total brain proteins. Although it was originally characterized as a deubiquitinating enzyme, recent studies indicate that it also functions as a ubiquitin ligase and a mono-Ub stabilizer. Down-regulation and extensive oxidative modifications of UCH-L1 have been observed in the brains of Alzheimer’s disease and Parkinson’s disease (PD) patients. Of importance, I93M and S18Y point mutations in the UCH-L1 gene have been reported to be linked to susceptibility to and protection from PD respectively. Hence, the structure of UCH-L1 and the effects of disease associated mutations on the structure and function are of considerable interest. Our circular dichroism studies suggest that the S18Y point mutation only slightly perturbs the structure while a significant decrease in the α-helical content is observed in the I93M mutant. We have determined the solution structure of S18Y and mapping its interaction with ubiquitin by chemical shift perturbation approach. The electrostatic surface potential analysis reveals that the interaction between ubiquitin and UCH-L1-S18Y is primarily electrostatic in nature, with negatively charged residues on the surface of UCH-L1-S18Y interacting with the positively charged residues on the basic face of ubiquitin. Although the active site and the L8 loop in UCH-L1-S18Y adopts conformations similar to that observed in the crystal structure of UCH-L1-WT, both the altered hydrogen bond network and surface charge distributions have demonstrated that the S18Y substitution could lead to profound structural changes. In particular, the difference in the dimeric interfaces of the wild-type and the S18Y mutant has shown that mutation can significantly affect the distribution of the surface-exposed residues involved in the dimeric interface. Such observed difference might weaken the stability of the UCH-L1 dimer and hence may explain the reduced dimerization-dependent ligase activity of UCH-L1-S18Y in comparison to UCH-L1-WT.postprin

Transformation of UML interaction diagrams into contract specifications for object-oriented testing

Testing is an important means to ensure the quality of software systems. Contract specification can be used to formally specify the cluster level of object-oriented software, which can then be tested using TACCLE, an advanced methodology for object-oriented testing. The use of formal specifications as a testing base has many advantages. However, such specifications are not easily understood and therefore not widely used in the software industry. On the other hand, UML, a semi-formal modeling language, is becoming increasingly popular and widely accepted. In particular, UML interaction diagrams specify the dynamic, interacting behavior among the objects of an object-oriented system. If the transformation of UML interaction diagrams into Contract specifications can be automated, the TACCLE methodology can be applied directly to test object-oriented software at the cluster level. In this paper, a method to transform UML interaction diagrams into Contract specifications is proposed based on the UML meta-model. A prototype has been developed. © 2007 IEEE.published_or_final_versio

Discoveries from a phenanthroline-based dynamic combinatorial library: catenane from a copper(I) or copper(II) template?

We report here a DCL study of a phenanthroline-based building block focusing on catenane formation with copper templates. Two [2]catenanes have been amplified by using Cu+ as a template from the DCLs that contain no interlocked compounds in the absence of copper. In addition, an unexpected Cu2+ template effect on the [2]catenane formation was discovered. The observed Cu2+ template effect was found to originate from the in situ reduction of the divalent metal to Cu+.postprin

Isolated heart models for studying cardiac electrophysiology: a historical perspective and recent advances

Experimental models used in cardiovascular research range from cellular to whole heart preparations. Isolated whole hearts show higher levels of structural and functional integration than lower level models such as tissues or cellular fragments. Cardiovascular diseases are multi-factorial problems that are dependent on highly organized structures rather than on molecular or cellular components alone. This article first provides a general introduction on the animal models of cardiovascular diseases. It is followed by a detailed overview and a historical perspective of the different isolated heart systems with a particular focus on the Langendorff perfusion method for the study of cardiac arrhythmias. The choice of species, perfusion method, and perfusate composition are discussed in further detail with particular considerations of the theoretical and practical aspects of experimental settings

Multiple Ca2+ signaling pathways regulate intracellular Ca 2+ activity in human cardiac fibroblasts

Ca2+ signaling pathways are well studied in cardiac myocytes, but not in cardiac fibroblasts. The aim of the present study is to characterize Ca2+ signaling pathways in cultured human cardiac fibroblasts using confocal scanning microscope and RT-PCR techniques. It was found that spontaneous intracellular Ca2+ (Cai 2+) oscillations were present in about 29% of human cardiac fibroblasts, and the number of cells with Cai 2+ oscillations was increased to 57.3% by application of 3% fetal bovine serum. Cai 2+ oscillations were dependent on Ca2+ entry. Cai2+ oscillations were abolished by the store-operated Ca2+ (SOC) entry channel blocker La3+, the phospholipase C inhibitor U-73122, and the inositol trisphosphate receptors (IP3Rs) inhibitor 2-aminoethoxydiphenyl borate, but not by ryanodine. The IP3R agonist thimerosal enhanced Ca2+ i oscillations. Inhibition of plasma membrane Ca2+ pump (PMCA) and Na+-Ca2+ exchanger (NCX) also suppressed Ca i 2+ oscillations. In addition, the frequency of Ca i 2+ oscillations was reduced by nifedipine, and increased by Bay K8644 in cells with spontaneous Cai 2+ oscillations. RT-PCR revealed that mRNAs for IP3R1-3, SERCA1-3, CaV1.2, NCX3, PMCA1,3,4, TRPC1,3,4,6, STIM1, and Orai1-3, were readily detectable, but not RyRs. Our results demonstrate for the first time that spontaneous Cai 2+ oscillations are present in cultured human cardiac fibroblasts and are regulated by multiple Ca2+ pathways, which are not identical to those of the well-studied contractile cardiomyocytes. This study provides a base for future investigations into how Ca2+ signals regulate biological activity in human cardiac fibroblasts and cardiac remodeling under pathological conditions. © 2009 Wiley-Liss, Inc.postprin

Cyclic ADP ribose is a novel regulator of intracellular Ca 2+ oscillations in human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine. However, the cellular biology of these cells is not fully understood. The present study characterizes the cyclic ADP-ribose (cADPR)-mediated Ca 2+ signals in human MSCs and finds that externally applied cADPR can increase the frequency of spontaneous intracellular Ca 2+ (Ca 2+ i) oscillations. The increase was abrogated by a specific cADPR antagonist or an inositol trisphosphate receptor (IP3R) inhibitor, but not by ryanodine. In addition, the cADPR-induced increase of Ca 2+ i oscillation frequency was prevented by inhibitors of nucleoside transporter or by inhibitors of the transient receptor potential cation melastatin-2 (TRPM2) channel. RT-PCR revealed mRNAs for the nucleoside transporters, concentrative nucleoside transporters 1/2 and equilibrative nucleoside transporters 1/3, IP3R1/2/3 and the TRPM2 channel, but not those for ryanodine receptors and CD38 in human MSCs. Knockdown of the TRPM2 channel by specific short interference RNA abolished the effect of cADPR on the Ca 2+ i oscillation frequency, and prevented the stimulation of proliferation by cADPR. Moreover, cADPR remarkably increased phosphorylated extracellular-signal-regulated kinases 1/2 (ERK1/2), but not Akt or p38 mitogen-activated protein kinase (MAPK). However, cADPR had no effect on adipogenesis or osteogenesis in human MSCs. Our results indicate that cADPR is a novel regulator of Ca 2+ i oscillations in human MSCs. It permeates the cell membrane through the nucleoside transporters and increases Ca 2+ oscillationviaactivation of the TRPM2 channel, resulting in enhanced phosphorylation of ERK1/2 and, thereby, stimulation of human MSC proliferation. This study delineates an alternate signalling pathway of cADPR that is distinct from its well-established role of serving as a Ca 2+ messenger for mobilizing the internal Ca 2+ stores. Whether cADPR can be used clinically for stimulating marrow function in patients with marrow disorders remains to be further studied. © 2011 The Authors © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.postprin

Regulation of human cardiac KCNQ1/KCNE1 channel by epidermal growth factor receptor kinase

The aim of the present study was to investigate whether/how the recombinant human cardiac I Ks could be regulated by epidermal growth factor receptor kinase in HEK 293 cells stably expressing hKCNQ1/hKCNE1 genes using the approaches of perforated patch clamp technique, immunoprecipitation and Western blot analysis. It was found that the broad spectrum isoflavone tyrosine kinase inhibitor genistein and the selective epidermal growth factor receptor kinase inhibitor tyrphostin AG556 suppressed the recombinant I Ks, and their inhibition was countered by the protein tyrosine phosphatase inhibitor orthovanadate. The Src-family kinase inhibitor PP2 reduced the current, but the effect was not antagonized by orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hKCNQ1 protein was decreased by genistein or AG556, but not by PP2. These results provide the novel information that epidermal growth factor receptor kinase, but not Src-family kinases, regulates the recombinant cardiac I Ks stably expressed in HEK 293 cells via phosphorylating KCNQ1 protein of the channel. © 2009 Elsevier B.V. All rights reserved.postprin

Human ether-à-go-go gene potassium channels are regulated by EGFR tyrosine kinase

Human ether á-go-go gene potassium channels (hEAG1 or Kv10.1) are expressed in brain and various human cancers and play a role in neuronal excitement and tumor progression. However, the functional regulation of hEAG channels by signal transduction is not fully understood. The present study was therefore designed to investigate whether hEAG1 channels are regulated by protein tyrosine kinases (PTKs) in HEK 293 cells stably expressing hEAG1 gene using whole-cell patch voltage-clamp, immunoprecipitation, Western blot, and mutagenesis approaches. We found that the selective epidermal growth factor receptor (EGFR) kinase inhibitor AG556 (10μM), but not the platelet growth factor receptor (PDGFR) kinase inhibitor AG1295 (10μM) or the Src-family inhibitor PP2 (10μM), can inhibit hEAG1 current, and the inhibitory effect can be reversed by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hEAG1 channels was reduced by AG556, and the reduction was significantly countered by orthovanadate. The hEAG1 mutants Y90A, Y344A and Y485A, but not Y376A and Y479A, exhibited reduced response to AG556. Interestingly, the inhibition effect of AG556 was lost in triple mutant hEAG1 channels at Y90, Y344, and Y485 with alanine. These results demonstrate for the first time that hEAG1 channel activity is regulated by EGFR kinase at the tyrosine residues Tyr 90, Try 344, and Try 485. This effect is likely involved in regulating neuronal activity and/or tumor growth. © 2011 Elsevier B.V.postprin

Tensor-based morphometry and stereology reveal brain pathology in the complexin1 knockout mouse.

Complexins (Cplxs) are small, soluble, regulatory proteins that bind reversibly to the SNARE complex and modulate synaptic vesicle release. Cplx1 knockout mice (Cplx1(-/-)) have the earliest known onset of ataxia seen in a mouse model, although hitherto no histopathology has been described in these mice. Nevertheless, the profound neurological phenotype displayed by Cplx1(-/-) mutants suggests that significant functional abnormalities must be present in these animals. In this study, MRI was used to automatically detect regions where structural differences were not obvious when using a traditional histological approach. Tensor-based morphometry of Cplx1(-/-) mouse brains showed selective volume loss from the thalamus and cerebellum. Stereological analysis of Cplx1(-/-) and Cplx1(+/+) mice brain slices confirmed the volume loss in the thalamus as well as loss in some lobules of the cerebellum. Finally, stereology was used to show that there was loss of cerebellar granule cells in Cplx1(-/-) mice when compared to Cplx1(+/+) animals. Our study is the first to describe pathological changes in Cplx1(-/-) mouse brain. We suggest that the ataxia in Cplx1(-/-) mice is likely to be due to pathological changes in both cerebellum and thalamus. Reduced levels of Cplx proteins have been reported in brains of patients with neurodegenerative diseases. Therefore, understanding the effects of Cplx depletion in brains from Cplx1(-/-) mice may also shed light on the mechanisms underlying pathophysiology in disorders in which loss of Cplx1 occurs