bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular imaging of homodimeric protein–protein interactions in living subjects
Authors: Massoud Tarik F. , Paulmurugan Ramasamy , Gambhir Sanjiv S. 
Published: May 2004

ABSTRACTHomodimeric protein interactions are potent regulators of cellular functions, but are particularly challenging to study in vivo. We used a split synthetic renilla luciferase (hRLUC) complementation‐based bioluminescence assay to study homodimerization of herpes simplex virus type 1 thymidine kinase (TK) in mammalian cells and in living mice. We quantified and imaged homodimerization of TK chimeras containing N‐terminal (N‐hRLUC) or C‐terminal (C‐ hRLUC) fragments of hRLUC in the upstream and downstream positions, respectively (tail‐to‐ head homodimer). This was monitored using luminometry (68‐fold increase, and was significantly [P<0.01] above background light emission) and by CCD camera imaging of living mice implanted with ex vivo transfected 293T cells (2.7‐fold increase, and is significantly [P<0.01] above background light emission). We also made a mutant‐TK to generate N‐hRLUC mutant TK and mutant TK‐C‐hRLUC by changing a single amino acid at position 318 from arginine to cysteine, a key site that has previously been reported to be essential for TK homo‐ dimerization, to support the specificity of the hRLUC complementation signal from TK homodimerization. Ex vivo substrate (8‐3H Penciclovir) accumulation assays in 293T cells expressing the TK protein chimeras showed active TK enzyme. We also devised an experimental strategy by constructing variant TK chimeras (possessing extra N‐hRLUC or C‐hRLUC ‘spacers’) to monitor incremental lack of association of the tail‐to‐head TK homodimer. Application of this potentially generalizable assay to screen for molecules that promote or disrupt ubiquitous homodimeric protein–protein interactions could serve not only as an invaluable tool to understand biological networks but could also be applied to drug discovery and validation in living subjects.
Nitric oxide, a key signaling molecule in the murine early embryonic heart
Authors: Malan Daniela , Ji Guanju , Schmidt Annette , Addicks Klaus , Hescheler Jürgen , Levi Renzo C. , Bloch Wilhelm , Fleischmann Bernd K. 
Published: May 2004

ABSTRACTNitric oxide (NO) is thought to play an important role as a signaling molecule in embryonic and adult cardiomyocytes; however, its involvement in muscarinic signaling is still unclear. The aim of the present work was to analyze the muscarinic modulation of the L‐type Ca2+ current (ICa) in early‐ and late‐stage embryonic ventricular cardiomyocytes. Muscarinic stimulation depressed basal ICa by 30.1 ± 3.2% (n=27) in early‐stage cardiomyocytes. Pharmacological evidence suggested that the muscarinic modulation was mediated through generation of NO, activation of cGMP‐dependent phosphodiesterase (PDE) 2, and ensuing lowering of cyclic AMP/protein kinase A (cAMP/PKA) levels. Conversely, in late‐stage cardiomyocytes, muscarinic regulation of ICa occurred in a NO‐independent manner via inhibition of prestimulated adenylyl cyclase (AC). To unequivocally prove the involvement of NO and to identify the nitric oxide synthase (NOS) isoform(s), we analyzed muscarinic signaling in embryonic ventricular cardiomyocytes of NOS2 (–/–) and NOS3 (–/–) mice. The early‐stage NOS3 (–/–) cardiomyocytes lacked muscarinic modulation, whereas it was preserved in NOS2 (–/–) cells. Moreover, at the late embryonic stage, muscarinic modulation of ICa was intact in both strains. Thus, NO is the key regulator of muscarinic signaling in the early embryonic ventricle, whereas at later stages, signaling occurs through a NO‐independent pathway.
VEGF‐A promotes tissue repair‐associated lymphatic vessel formation via VEGFR‐2 and the α1β1 and α2β1 integrins
Authors: Hong Young‐Kwon , Lange‐Asschenfeldt Bernhard , Velasco Paula , Hirakawa Satoshi , Kunstfeld Rainer , Brown Lawrence F. , Bohlen Peter , Senger Donald R. , Detmar Michael 
Published: May 2004

ABSTRACTVascular endothelial growth factor‐A (VEGF‐A) is strongly up‐regulated in wounded cutaneous tissue and promotes repair‐associated angiogenesis. However, little is known about its role in lymphatic regeneration of the healing skin. We studied wound healing in transgenic mice that overexpress VEGF‐A specifically in the epidermis and in wild‐type mice in the absence or presence of inhibitors of VEGF‐A signaling. Surprisingly, transgenic overexpression of VEGF‐A in the skin promoted lymphangiogenesis at the wound healing site, whereas systemic blockade of VEGFR‐2 prevented lymphatic vessel formation. Studies in cultured lymphatic endothelial cells revealed that VEGF‐A induced expression of the α1 and α2 integrins, which promoted their in vitro tube formation and their haptotactic migration toward type I collagen. VEGF‐A‐induced lymphatic endothelial cord formation and haptotactic migration were suppressed by anti‐α1 and anti‐α2 integrin blocking antibodies, and systemic blockade of the α1 and α2 integrins inhibited VEGF‐A‐driven lymphangiogenesis in vivo. We propose that VEGF‐A promotes lymphatic vasculature formation via activation of VEGFR‐2 and that lineage‐specific differences of integrin receptor expression contribute to the distinct dynamics of wound‐associated angiogenesis and lymphangiogenesis.
Neuroprotective effects of spermine following hypoxia‐ischemia‐induced brain damage: A mechanistic study
Authors: Clarkson Andrew N. , Liu Hanzhong , Pearson Lachlan , Kapoor Mohit , Harrison Joanne C. , Sammut Ivan A. , Jackson David M. , Appleton Ian 
Published: May 2004

ABSTRACTThe polyamines (spermine, putrescine, and spermidine) can have neurotoxic or neuroprotective properties in models of neurodegeneration. However, assessment in a model of hypoxia– ischemia (HI) has not been defined. Furthermore, the putative mechanisms of neuroprotection have not been elucidated. Therefore, the present study examined the effects of the polyamines in a rat pup model of HI and determined effects on key enzymes involved in inflammation, namely, nitric oxide synthase (NOS) and arginase. In addition, effects on mitochondrial function were investigated. The polyamines or saline were administered i.p. at 10mg/kg/day for 6 days post‐HI. Histological assessment 7 days post‐HI revealed that only spermine significantly (P<0.01) reduced infarct size from 46.14 ± 10.4mm3 (HI + saline) to 4.9 ± 2.7 mm3. NOS activity was significantly increased following spermine treatment in the left (ligated) hemisphere compared with nonintervention controls (P<0.01) and HI + saline (P<0.05). In contrast, spermine decreased arginase activity compared with HI + saline but was still significantly elevated in comparison to nonintervention controls (P<0.01). Assessment of mitochondrial function in the HI + saline group, revealed significant and extensive damage to complex‐I (P<0.01) and IV (P<0.001) and loss of citrate synthase activity (P<0.05). No effect on complex II‐III was observed. Spermine treatment significantly prevented all these effects. This study has therefore confirmed the neuroprotective effects of spermine in vivo. However, for the first time, we have shown that this effect may, in part, be due to increased NOS activity and preservation of mitochondrial function.
Toll‐like receptor 4 functions intracellularly in human coronary artery endothelial cells: roles of LBP and sCD14 in mediating LPS‐responses
Authors: Dunzendorfer Stefan , Lee Hyun‐Ku , Soldau Katrin , Tobias Peter S. 
Published: May 2004

ABSTRACTEndothelial cells are activated by microbial agonists through Toll‐like receptors (TLRs) to express inflammatory mediators; this is of significance in acute as well as chronic inflammatory states such as septic shock and atherosclerosis, respectively. We investigated mechanisms of lipopolysaccharide (LPS)‐induced cell activation in human coronary artery endothelial cells (HCAEC) using a combination of FACS, confocal microscopy, RT‐PCR, and functional assays. We found that TLR4, in contrast to TLR2, is not only located intracellularly but also functions intracellularly. That being the case, internalization of LPS is required for activation. We further characterized the HCAEC LPS uptake system and found that HCAEC exhibit an effective LPS uptake only in the presence of LPS binding protein (LBP). In addition to its function as a catalyst for LPS‐CD14 complex formation, LBP enables HCAEC activation at low LPS concentrations by facilitating the uptake, and therefore delivery, of LPS‐CD14 complexes to intracellular TLR4‐MD‐2. LBP‐dependent uptake involves a scavenger receptor pathway. Our findings may be of pathophysiological relevance in the initial response of the organism to infection. Results further suggest that LBP levels, which vary as LBP is an acute phase reactant, could be relevant to initiating inflammatory responses in the vasculature in response to chronic or recurring low LPS.
Apoptotic adaptations from exercise training in skeletal and cardiac muscles
Authors: Siu Parco M. , Bryner Randall W. , Martyn Julie K. , Alway Stephen E. 
Published: May 2004

ABSTRACTThe effect of exercise on apoptosis in postmitotic tissues is not known. In this study, we investigated the effect of regular moderate physical activity (i.e., exercise training) on the extent of apoptosis in rat skeletal and cardiac muscles. Adult Sprague Dawley rats were trained (TR) 5 days weekly for 8 wk on treadmill. Sedentary rats served as controls (CON). An ELISA was used to detect mono‐ and oligonucleosome fragmentation as an indicator of apoptosis. Bcl‐2, Bax, Apaf‐1, AIF, cleaved PARP, cleaved caspase‐3, cleaved/active caspase‐9, heat shock protein (HSP)70, Cu/Zn‐SOD, and Mn‐SOD protein levels were determined by Western analyses. Bcl‐2 and Bax transcript contents were estimated by RT‐PCR. A spectrofluorometric assay was used to determine caspase‐3 activity. DNA fragmentation in ventricles of the TR group decreased by 15% whereas that in soleus of the TR group tended to decrease (P=0.058) when compared with CON group. Protein contents of Bcl‐2, HSP70, and Mn‐SOD increased in both soleus and ventricle muscles of TR animals when compared with CON animals. Apaf‐1 protein content in the soleus of TR animals was lower than that of CON animals. Bcl‐2 mRNA levels increased in both ventricle and soleus muscles of TR animals, and Bax mRNA levels decreased in the soleus of TR animals when compared with CON animals. Furthermore, HSP70 protein content was negatively correlated to Bax mRNA content and was positively correlated to Bcl‐2 protein and mRNA contents. Mn‐SOD protein content was negatively correlated to the apoptotic index, and caspase‐3 activity and was positively correlated to Bcl‐2 transcript content and HSP70 protein content. These data suggest that exercise training attenuates the extent of apoptosis in cardiac and skeletal muscles.
Role of myoglobin in the antioxidant defense of the heart
Authors: Flögel Ulrich , Gödecke Axel , Klotz Lars‐Oliver , Schrader Jürgen 
Published: May 2004

ABSTRACTAlthough the primary function of myoglobin (Mb) has been considered to be cellular O2 storage and supply, recent studies have shown that Mb in addition can act as NO oxidase. Here we report that Mb also significantly contributes to the attenuation of oxidative stress in cardiac muscle. In support of this hypothesis, we found that in isolated perfused hearts of Mb‐deficient (myo‒/‒) mice oxidative challenge by intracoronary infused H2O2 (1‐300 µM) or superoxide formed by 2,3‐dimethoxy‐1,4‐naphtoquinone (0.1‐30 µM), respectively, depressed cardiac contractility to a greater extent than in wild‐type (WT) hearts, e.g., up to [H2O2] = 10 µM there was a significant left ventricular developed pressure (LVDP) decrease in myo‒/‒ hearts only (90.4±4.2 vs. 98.1±0.7% of control, n=6, P<0.05). Likewise in an ischemia/reperfusion protocol, myo‒/‒ hearts showed a delayed recovery of postischemic function as compared with WT controls (e.g., LVDP was 35.6±7.5 vs. 22.4±5.3 mmHg, respectively, after 10 min of reperfusion, P<0.05, n=8), which correlated well with an enhanced release of reactive oxygen species in myo‒/‒ hearts as measured by online lucigenin‐enhanced chemiluminescence [e.g. 465±87 relative light units (RLU) in myo‒/‒ vs. 287±73 RLU in WT after 2.5 min of reperfusion, P<0.05, n=8]. 31P NMR spectroscopy revealed concomitantly a more pronounced phosphocreatine overshoot during reperfusion in the knockout but only minute alterations in ATP and pHi. Our data show that lack of Mb leads to increased vulnerability of cardiac function to oxidative challenge either pharmacologically induced or endogenously generated. We propose that Mb is a key element influencing redox pathways in cardiac muscle to functionally and metabolically protect the heart from oxidative damage.
Cross‐linking of ubiquitin, HSP27, parkin and α‐synuclein by γ‐glutamyl‐ε‐lysine bonds in Alzheimer's neurofibrillary tangles
Authors: Nemes Zoltán , Devreese Bart , Steinert Peter M. , Beeumen Jozef Van , Fésüs László 
Published: May 2004

ABSTRACTThe accumulation of misfolded proteins in intracellular inclusions is a generic feature of neurodegenerative disorders. Although heavily ubiquitylated, the aggregated proteins are not degraded by the proteasomes. A possible reason for this phenomenon may be a modification of deposited proteins by transglutaminases forming γ‐glutamyl‐ε‐lysine (GGEL) cross‐links between distinct proteins. Here, we show that the frequency of GGEL cross‐links is an order of magnitude higher in Alzheimer's brain cortex than in age‐matched or younger controls. This difference is due to the accumulation of GGEL cross‐links in ubiquitin‐immunopositive protein particles present in both Alzheimer's brains and those from aged individuals. The highly cross‐ linked protein aggregates show immunoreactivity to antibodies against tau and neurofilament proteins, and partially also to α‐synuclein, indicating that these structures are inherent in Alzheimer's neurofibrillary tangles and Lewy bodies. Using mass sequence analysis, we identified the same six pairs of peptide sequences cross‐linked in both senile and Alzheimer's specimens: Gln31 and Gln190 of HSP27 protein are cross‐linked with Lys29 and Lys48 of ubiquitin and HSP27 therefore may cross‐link two (poly)ubiquitin chains. One lysine residue of parkin and one of α‐synuclein were also found to be cross‐linked. The data suggest that cross‐linking of (poly)ubiquitin moieties via HSP27 may have a role in the stabilization of the intraneuronal protein aggregates by interference with the proteasomal elimination of unfolded proteins.
An essential role of Cav1.2 L‐type calcium channel for urinary bladder function
Authors: Wegener Jörg W. , Schulla Verena , Lee Tae‐Seong , Koller Angela , Feil Susanne , Feil Robert , Kleppisch Thomas , Klugbauer Norbert , Moosmang Sven , Welling Andrea , Hofmann Franz 
Published: May 2004

ABSTRACTMice deficient in the smooth muscle Cav1.2 calcium channel (SMACKO, smooth muscle α1c‐ subunit calcium channel knockout) have a severely reduced micturition and an increased bladder mass. L‐type calcium current, protein, and spontaneous contractile activity were absent in the bladder of SMACKO mice. K+ and carbachol (CCh)‐induced contractions were reduced to 10‐ fold in detrusor muscles from SMACKO mice. The dihydropyridine isradipine inhibited K+‐ and CCh‐induced contractions of muscles from CTR but had no effect in muscles from SMACKO mice. CCh‐induced contraction was blocked by removing extracellular Ca2+ but was unaffected by the PLC inhibitor U73122 or depletion of intracellular Ca2+ stores by thapsigargin. In muscles from CTR and SMACKO mice, CCh‐induced contraction was partially inhibited by the Rho‐ kinase inhibitor Y27632. These results show that the Cav1.2 Ca2+ channel is essential for normal bladder function. The Rho‐kinase and Ca2+‐release pathways cannot compensate the lack of the L‐type Ca2+ channel.
Expression of Concern: Glycogen synthase kinase 3β (GSK3β) mediates 6‐ hydroxydopamine‐induced neuronal death
Authors: Chen Gang , Bower Kimberly A. , Ma Cuiling , Fang Shengyun , Thiele Carol J. , Luo Jia 
Published: May 2004

ABSTRACTThe causes of sporadic Parkinson's disease (PD) are poorly understood. 6‐Hydroxydopamine (6‐ OHDA), a PD mimetic, is widely used to model this neurodegenerative disorder in vitro and in vivo; however, the underlying mechanisms remain incompletely elucidated. We demonstrate here that 6‐ OHDA evoked endoplasmic reticulum (ER) stress, which was characterized by an up‐regulation in the expression of GRP78 and GADD153 (Chop), cleavage of procaspase‐12, and phosphorylation of eukaryotic initiation factor‐2 a in a human dopaminergic neuronal cell line (SH‐SY5Y) and cultured rat cerebellar granule neurons (CGNs). Glycogen synthase kinase‐3 β (GSK3β) responds to ER stress, and its activity is regulated by phosphorylation. 6‐OHDA significantly inhibited phosphorylation of GSK3β at Ser9, whereas it induced hyperphosphorylation of Tyr216 with little effect on GSK3β expression in SH‐SY5Y cells and PC12 cells (a rat dopamine cell line), as well as CGNs. Furthermore, 6‐OHDA decreased the expression of cyclin D1, a substrate of GSK3β, and dephosphorylated Akt, the upstream signaling component of GSK3β. Protein phosphatase 2A (PP2A), an ER stress‐responsive phosphatase, was involved in 6‐OHDA‐induced GSK3β dephosphorylation (Ser9). Blocking GSK3β activity by selective inhibitors (lithium, TDZD‐8, and L803‐mts) prevented 6‐OHDA‐induced cleavage of caspase‐3 and poly(ADP‐ribose) polymerase (PARP), DNA fragmentations and cell death. With a tetracycline (Tet)‐controlled TrkB inducible system, we demonstrated that activation of TrkB in SH‐ SY5Y cells alleviated 6‐OHDA‐induced GSK3β dephosphorylation (Ser9) and ameliorated 6‐OHDA neurotoxicity. TrkB activation also protected CGNs against 6‐OHDA‐induced damage. Although antioxidants also offered neuroprotection, they had little effect on 6‐OHDA‐induced GSK3β activation. These results suggest that GSK3β is a critical intermediate in pro‐apoptotic signaling cascades that are associated with neurodegenerative diseases, thus providing a potential target site amenable to pharmacological intervention.
Realistic protein–protein association rates from a simple diffusional model neglecting long‐range interactions, free energy barriers, and landscape ruggedness
Authors: Schlosshauer Maximilian , Baker David 
Published: May 2004

AbstractWe develop a simple but rigorous model of protein–protein association kinetics based on diffusional association on free energy landscapes obtained by sampling configurations within and surrounding the native complex binding funnels. Guided by results obtained on exactly solvable model problems, we transform the problem of diffusion in a potential into free diffusion in the presence of an absorbing zone spanning the entrance to the binding funnel. The free diffusion problem is solved using a recently derived analytic expression for the rate of association of asymmetrically oriented molecules. Despite the required high steric specificity and the absence of long‐range attractive interactions, the computed rates are typically on the order of 104–106 M−1 sec−1, several orders of magnitude higher than rates obtained using a purely probabilistic model in which the association rate for free diffusion of uniformly reactive molecules is multiplied by the probability of a correct alignment of the two partners in a random collision. As the association rates of many protein–protein complexes are also in the 105–106 M−1 sec−1 range, our results suggest that free energy barriers arising from desolvation and/or side‐chain freezing during complex formation or increased ruggedness within the binding funnel, which are completely neglected in our simple diffusional model, do not contribute significantly to the dynamics of protein–protein association. The transparent physical interpretation of our approach that computes association rates directly from the size and geometry of protein–protein binding funnels makes it a useful complement to Brownian dynamics simulations.
Activation and inhibition of cyclin‐dependent kinase‐2 by phosphorylation; a molecular dynamics study reveals the functional importance of the glycine‐rich loop
Authors: Bártová Iveta , Otyepka Michal , Kříž Zdeněk , Koča Jaroslav 
Published: May 2004

AbstractNanoseconds long molecular dynamics (MD) trajectories of differently active complexes of human cyclin‐dependent kinase 2 (inactive CDK2/ATP, semiactive CDK2/Cyclin A/ATP, fully active pT160‐CDK2/Cyclin A/ATP, inhibited pT14‐; pY15‐; and pT14,pY15,pT160‐CDK2/Cyclin A/ATP) were compared. The MD simulations results of CDK2 inhibition by phosphorylation at T14 and/or Y15 sites provide insight into the structural aspects of CDK2 deactivation. The inhibitory sites are localized in the glycine‐rich loop (G‐loop) positioned opposite the activation T‐loop. Phosphorylation of T14 and both inhibitory sites T14 and Y15 together causes ATP misalignment for phosphorylation and G‐loop conformational change. This conformational change leads to the opening of the CDK2 substrate binding box. The phosphorylated Y15 residue negatively affects substrate binding or its correct alignment for ATP terminal phospho‐group transfer to the CDK2 substrate. The MD simulations of the CDK2 activation process provide results in agreement with previous X‐ray data.
Identification of the catalytic motif of the microbial ribosome inactivating cytotoxin colicin E3
Authors: Walker Daniel , Lancaster Lorna , James Richard , Kleanthous Colin 
Published: May 2004

AbstractColicin E3 is a cytotoxic ribonuclease that specifically cleaves 16S rRNA at the ribosomal A‐site to abolish protein synthesis in sensitive Escherichia coli cells. We have performed extensive mutagenesis of the 96‐residue colicin E3 cytotoxic domain (E3 rRNase), assayed mutant colicins for in vivo cytotoxicity, and tested the corresponding E3 rRNase domains for their ability to inactivate ribosome function in vitro. From 21 alanine mutants, we identified five positions where mutation resulted in a colicin with no measurable cytotoxicity (Y52, D55, H58, E62, and Y64) and four positions (R40, R42, E60, and R90) where mutation caused a significant reduction in cytotoxicity. Mutations that were found to have large in vivo and in vitro effects were tested for structural integrity through circular dichroism and fluorescence spectroscopy using purified rRNase domains. Our data indicate that H58 and E62 likely act as the acid–base pair during catalysis with other residues likely involved in transition state stabilization. Both the Y52 and Y64 mutants were found to be highly destabilized and this is the likely origin of the loss of their cytotoxicity. The identification of important active site residues and sequence alignments of known rRNase homologs has allowed us to identify other proteins containing the putative rRNase active site motif. Proteins that contained this active site motif included three hemagglutinin‐type adhesins and we speculate that these have evolved to deliver a cytotoxic rRNase into eukaryotic cells during pathogenesis.