Domain deletions outside of the catalytic domain of drosophila tolloid result in loss of protease activity

Introduction The tolloid (TLD)‐related zinc metalloproteinases serve multiple functions in development, being involved in both extracellular matrix assembly and TGF‐β signalling. Signalling by bone morphogenetic proteins BMPs, which form a subgroup of the TGF‐β superfamily, is a highly regulated process which involves cleavage of extracellular BMP antagonists by TLD‐like (TLL) proteases. In vertebrates, BMP‐1 and mTLL‐1 cleave the BMP antagonist chordin to release free BMPs, while in invertebrates, TLD cleaves the BMP antagonist short gastrulation (SOG) to release decapentaplegic (DPP). Mammalian TLD (mTLD), TLL‐1 and TLL‐2 share the same domain structure as drosophila TLD, while BMP‐1 is a shorter splice variant of mTLD which lacks the EGF‐2, CUB‐4 and CUB‐5 domains. Material and methods Site‐directed mutagenesis was used to produce TLD constructs lacking various domains. These were subcloned into the vector pAc5.1V5His, along with wild‐type TLD and full‐length SOG. Transient transfections in drosophila S2 cells were used to produce recombinant proteins, and aliquots of medium containing the appropriate recombinant proteins were dialysed or desalted as required. In vitro digests of SOG by TLD and the TLD mutants were performed overnight at 25°C in the presence of recombinant DPP (R & D Systems). Digests were analysed by SDS Page and Western blotting, and full‐length SOG and SOG fragments were detected using an anti‐V5 antibody (Invitrogen). Results Wild‐type TLD was able to cleave SOG at three distinct sites as previously reported ( Marques et al. 1997 ). A ‘BMP1‐like’ TLD construct, truncated after the CUB3 domain, was efficiently secreted by S2 cells but was unable to cleave SOG. Wild‐type TLD was also found to have enhanced activity in the presence of calcium ions, indicating that it is a calcium‐dependent protease. Removal of the putative calcium‐binding domains from TLD (EGF1 and EGF2) resulted in a decreased amount of enzyme present in the medium of the transfected S2 cells and less (ΔEGF1) or abolished (ΔEGF2 and double deletion) protease activity. Discussion These preliminary findings indicate that the EGF‐2, CUB‐4 and CUB‐5 domains are required for cleavage of SOG by TLD. This is in direct contrast to the cleavage of chordin which is actually cleaved most efficiently by the short‐splice variant BMP‐1 ( Scott et al. 1999 ). In vitro cleavage of SOG also requires the presence of DPP, whereas chordin is cleaved in the absence of exogenous BMPs. It is possible that these domains could be involved in interactions with the substrate or alternatively with DPP itself. The putative calcium‐binding domains, EGF1 and EGF2, appear to be required both for secretion and stability of the protein as well as for protease activity. The mechanism whereby calcium augments the protease activity of TLD is unknown, but it could aid the binding of enzyme to substrate or create specific conformational changes in the enzyme to increase activity.     

Identification of a mutation causing deficient BMP1/mTLD proteolytic activity in autosomal recessive osteogenesis imperfecta

Herein, we have studied a consanguineous Egyptian family with two children diagnosed with severe autosomal recessive osteogenesis imperfecta (AR-OI) and a large umbilical hernia. Homozygosity mapping in this family showed lack of linkage to any of the previously known AR-OI genes, but revealed a 10.27 MB homozygous region on chromosome 8p in the two affected sibs, which comprised the procollagen I C-terminal propeptide (PICP) endopeptidase gene BMP1. Mutation analysis identified both patients with a Phe249Leu homozygous missense change within the BMP1 protease domain involving a residue, which is conserved in all members of the astacin group of metalloproteases. Type I procollagen analysis in supernatants from cultured fibroblasts demonstrated abnormal PICP processing in patient-derived cells consistent with the mutation causing decreased BMP1 function. This was further confirmed by overexpressing wild type and mutant BMP1 longer isoform (mammalian Tolloid protein [mTLD]) in NIH3T3 fibroblasts and human primary fibroblasts. While overproduction of normal mTLD resulted in a large proportion of proα1(I) in the culture media being C-terminally processed, proα1(I) cleavage was not enhanced by an excess of the mutant protein, proving that the Phe249Leu mutation leads to a BMP1/mTLD protein with deficient PICP proteolytic activity. We conclude that BMP1 is an additional gene mutated in AR-OI.     

Enzymatic regulation of pattern: BMP4 binds CUB domains of Tolloids and inhibits proteinase activity

In Xenopus embryos, a dorsal–ventral patterning gradient is generated by diffusing Chordin/bone morphogenetic protein (BMP) complexes cleaved by BMP1/Tolloid metalloproteinases in the ventral side. We developed a new BMP1/Tolloid assay using a fluorogenic Chordin peptide substrate and identified an unexpected negative feedback loop for BMP4, in which BMP4 inhibits Tolloid enzyme activity noncompetitively. BMP4 binds directly to the CUB (Complement 1r/s, Uegf [a sea urchin embryonic protein] and BMP1) domains of BMP1 and Drosophila Tolloid with high affinity. Binding to CUB domains inhibits BMP4 signaling. These findings provide a molecular explanation for a long-standing genetical puzzle in which antimorphic Drosophila tolloid mutant alleles displayed anti-BMP effects. The extensive Drosophila genetics available supports the relevance of the interaction described here at endogenous physiological levels. Many extracellular proteins contain CUB domains; the binding of CUB domains to BMP4 suggests a possible general function in binding transforming growth factor-β (TGF-β) superfamily members. Mathematical modeling indicates that feedback inhibition by BMP ligands acts on the ventral side, while on the dorsal side the main regulator of BMP1/Tolloid enzymatic activity is the binding to its substrate, Chordin.     

Scube regulates synovial angiogenesis-related signaling

Angiogenesis is particularly driven in the synovial microenvironment of Rheumatoid arthritis (RA), and considered as the fundamental cause for the persistent injury and chronic damage. Therefore, exploring the pathomechanism of synovial angiogenesis may provide promising prospects for vascular-targeting treatment of RA. The noval family of Scube proteins is confirmed to overlap significantly in structure characterized by epidermal growth factor (EGF)-like domains and CUB (complement subcomponents C1r/C1s, Uegf, bone morphogenetic protein-1) domain. As secreted glycoprotein and peripheral membrane protein, Scube increases its serum level in response to stimuli of inflammation and hypoxia. In rheumatoid angiogenesis-related signaling system defined by hedgehog (Hh), transforming growth factor (TGF)β and bone morphogenetic protein 2 (BMP2), Scube1 and 2 antagonize BMP2 signaling, suppressing BMP2-induced phospho-Smad1/5/8 level in vivo. Scube3 functions as an endogenous TGFβ receptor ligand, increasing Smad2/3 phosphorylation, and thus upregulates target genes involved in angiogenesis. Via obligate assistance of Scube1 and 3, Scube2 plays a center role to recruit dually lipid-modified Hh transferred from Dispatched A (DispA), increasing Hh secretion by promoting its solubility. These findings support the hypothesis that Scube may regulate synovial angiogenesis may be the ideal vascular targets for anti-rheumatic treatment of RA.     

The Human Complement Component C1R Gene: The Exon-intron Structure and the Molecular Basis of Allelic Diversity

Human C1r is a component of the complement system, which is a major mediator of innate immunity. In this study we investigated the exon‐intron organization of the human C1R gene, which spans 11 kb from the initiation codon to the stop codon, and is very similar in exon‐intron structure to the C1S gene. Six common and rare alleles, C1R*1, C1R*2, C1R*5, C1R*8, C1R*9, and C1R*13, were characterized by five mutations at amino acid positions 114, 135, 146, 167 and 244, in exons 4, 5 and 7 where the CUB1, EGF and CUB2 domains are encoded, respectively. A comparison with the cDNA of the mouse C1r gene showed that C1R*2is likely to be an ancestral allele. In addition, nine nucleotide substitutions and one length polymorphism were found in introns 2, 3, 4, 8 and 10.     

Missense mutations of conserved glycine residues in fibrillin‐1 highlight a potential subtype of cb‐EGF‐like domains

In six index cases/families referred for Marfan syndrome (MFS) molecular diagnosis, we identified six novel mutations in the FBN1 gene: c.1753G>C (p.Gly585Arg), c.2456G>A (p.Gly819Glu), c.4981G>A (p.Gly1661Arg), c.5339G>A (p.Gly1780Glu), c.6418G>A (p.Gly2140Arg) and c.6419G>A (p.Gly2140Glu). These variants, predicted to result in Glycine substitutions are located at the third position of a 4 amino acids loop‐region of calcium‐binding Epidermal Growth Factor‐like (cb‐EGF) fibrillin‐1 domains 5, 9, 24, 25 and 32. Familial segregation studies showing cosegregation with MFS manifestations or de novo inheritance in addition to in silico analyses (conservation, 3D modeling) suggest evidence for a crucial role of the respective Glycine positions. Extending these analyses to all Glycine residue at position 3 of this 4 residues loop in fibrillin‐1 cb‐EGF with the UMD predictor tool and alignment of 2038 available related sequences strongly support a steric strain that only allows Glycine or even Alanine residues for domain structure maintenance and for the fibrillin functions. Our data compared with those of the literature strongly suggest the existence of a cb‐EGF domain subtype with implications for related diseases. © 2009 Wiley‐Liss, Inc.     

The ins and outs of extracellular matrix assembly

Introduction To a rough approximation, the adult human comprises 2–5 kg of cells. The remaining mass originates from the extracellular matrix (ECM). The ECM is highly organized, which is surprising considering the paucity of cells and the fact that the ECM comprises some of the largest and most insoluble macromolecules encoded by the genome. The high level of supramolecular order of the ECM is particularly evident in tendon in which millimetre‐long collagen fibrils lie parallel to each other and to the tendon‐long axis ( Canty & Kadler 2002 ). The work in our laboratory is focused on understanding the molecular and cellular basis of collagen fibrillogenesis. The work is directly relevant to understanding the secretion and assembly of large proteins, tissue homeostasis and embryonic development, as well as understanding the aetiology of heritable and acquired diseases of connective tissue including fibrosis. Materials and methods A multidisciplinary approach is being used including protein biochemistry, recombinant protein expression, immunofluorescence, immunoEM and serial section reconstruction from electron micrographs. Results Using pulse‐chase in organ cultures of embryonic chick tendon we show that approximately 50% of newly synthesized type‐I procollagen is secreted to the ECM where it is cleaved to type‐I collagen, and approximately 50% of the procollagen is cleaved to collagen inside the cells. ImmunoEM shows collagen in specialized secretory vesicles, which bud from the trans‐face of the Golgi apparatus. Serial section reconstruction of E13 chick metatarsal tendon and E15.5 mouse tail tendon shows cross‐striated nascent collagen fibrils, which are approximately 1 µm in length and identical to collagen early collagen fibrils ( Graham et al. 2000 ), enclosed within tube‐like secretory vesicles. The nascent fibrils are secreted via nozzles (plasma membrane protrusions) into tunnel‐shaped spaces between the cells. Bone morphogenetic protein‐1 (ProBMP‐1) is cleaved to active BMP‐1 in the TGN by dibasic convertases ( Graham et al. 2000 ) (e.g. furin) and thereby initiates the assembly of collagen fibrils. Further studies have identified the minimal structure of procollagen C‐proteinase activity of BMP‐1 ( Leighton & Kadler 2003 ). Discussion In vitro studies in the 1980s established that collagen fibrillogenesis resembles a crystallization process, in that it proceeds by distinct nucleation and propagation phases. However, there was no obvious mechanism for how the two phases occurred separately in vivo. Our recent studies show that the nucleation phase occurs in specialized secretory vesicles. The early fibrils are deposited in the ECM via nozzles which are long protrusions of the plasma membranes. The propagation phase for fibril assembly occurs in the ECM. Our studies have direct relevance to the assembly of other matrix polymers, e.g. fibrillin and type‐VI collagen, as well as understanding the molecular basis of ECM assembly in diseases such as fibrosis and wound healing.     

Prediction of the Repeat Domain Structures and Impact of Parkinsonism‐Associated Variations on Structure and Function of all Functional Domains of Leucine‐Rich Repeat Kinase 2 (LRRK2)

Genetic variations of leucine‐rich repeat kinase 2 (LRRK2) are the major cause of dominantly inherited Parkinson disease (PD). LRRK2 protein contains seven predicted domains: a tandem Ras‐like GTPase (ROC) domain and C‐terminal of Roc (COR) domain, a protein kinase domain, and four repeat domains. PD‐causative variations arise in all domains, suggesting that aberrant functioning of any domain can contribute to neurotoxic mechanisms of LRRK2. Determination of the three‐dimensional structure of LRRK2 is one of the best avenues to decipher its neurotoxic mechanism. However, with the exception of the Roc domain, the three‐dimensional structures of the functional domains of LRRK2 have yet to be determined. Based on the known three‐dimensional structures of repeat domains of other proteins, the tandem Roc–COR domains of the Chlorobium tepidum Rab family protein, and the kinase domain of the Dictyostelium discoideum Roco4 protein, we predicted (1) the motifs essential for protein–protein interactions in all domains, (2) the motifs critical for catalysis and substrate recognition in the tandem Roc–COR and kinase domains, and (3) the effects of some PD‐associated missense variations on the neurotoxic action of LRRK2. Results of our analysis provide a conceptual framework for future investigation into the regulation and the neurotoxic mechanism of LRRK2.     

Three alternatively spliced variants of the gene coding for the human bone morphogenetic protein-1

 The human bone morphogenetic protein-1 was originally identified as a protein with the capacity to stimulate bone and cartilage growth in vitro. Its gene sequence identified it as an alternatively spliced human homolog of the Drosophila dorsal-ventral patterning tolloid gene and suggested that it activates transforming growth factor-β-like molecules by proteolytic cleavage. Its expression pattern and its recently identified activity as a procollagen C proteinase, however, suggest that it has a more general function in the early stages of embryogenesis. This view is strengthened by the previous observation of a third alternatively spliced isoform of the gene, called bone morphogenetic protein 1/His. We now show that the gene is expressed in three additional variants, leading to shorter and slightly modified C-termini. The three variants are preferentially expressed in placenta but show individual differences in their expression profiles in other soft tissues. Received: 27 January 1997 / Accepted: 14 October 1997     

Type I Procollagen C‐Propeptide Defects: Study of Genotype–Phenotype Correlation and Predictive Role of Crystal Structure

The type I procollagen carboxyterminal(C‐)propeptides are crucial in directing correct assembly of the procollagen heterotrimers. Defects in these domains have anecdotally been reported in patients with Osteogenesis Imperfecta (OI) and few genotype–phenotype correlations have been described. To gain insight in the functional consequences of C‐propeptide defects, we performed a systematic review of clinical, molecular, and biochemical findings in all patients in whom we identified a type I procollagen C‐propeptide defect, and compared this with literature data. We report 30 unique type I procollagen C‐propeptide variants, 24 of which are novel. The outcome of COL1A1 nonsense and frameshift variants depends on the location of the premature termination codon. Those located prior to 50–55 nucleotides upstream of the most 3’ exon–exon junction lead to nonsense‐mediated mRNA decay (NMD) and cause mild OI. Those located beyond this boundary escape NMD, generally lead to production of stable, overmodified procollagen chains, which may partly be retained intracellularly, and are usually associated with severe‐to‐lethal OI. Proα1(I)‐C‐propeptide defects that permit chain association result in more severe phenotypes than those inhibiting chain association. We demonstrate that the crystal structure of the proα1(III)‐C‐propeptide is a reliable tool to predict phenotypic severity for most COL1A1‐C‐propeptide missense variants, whereas for COL1A2‐C‐propeptide variants, the phenotypic outcome is milder than predicted.     

Skeletal muscle expression of bone morphogenetic protein-1 and tolloid-like-1 extracellular proteases in different fiber types and in response to unloading,food deprivation and differentiation

Members of the bone morphogenetic protein-1/mammalian tolloid (BMP-1/mTLD) family of proteases cleave diverse extracellular proteins, including the growth inhibitor myostatin. The purpose of this work was to examine the expression of BMP-1/mTLD, tolloid-like-1 and -2 (TLL1 and TLL2) in hindlimb muscles of the mouse in vivo and in C₂C₁₂ muscle cells in vitro. Quantitative real-time polymerase chain reaction revealed that neither BMP-1/mTLD nor TLL1 mRNA levels differed between the predominantly fast-twitch tibialis anterior (TA) and gastrocnemius (GAST) muscles and the more slow-twitch soleus (SOL) muscle; TLL2 mRNA levels were not detectable in any of the muscles examined. Interestingly, however, immunohistochemical analysis revealed that BMP-1 protein was expressed in type I and IIa but not in IIb fibers. TLL1 mRNA levels significantly increased in the TA but not the SOL with 3 days of hindlimb suspension and significantly decreased in both TA and SOL in response to 2 days of food deprivation. In contrast, BMP-1/mTLD mRNA levels were unaffected in either muscle by either condition. In addition, BMP-1/mTLD and TLL1 mRNA levels significantly decreased during C₂C₁₂ myoblast differentiation in vitro, and activity of a 1,200-bp mouse TLL1 promoter construct was significantly decreased in C₂C₁₂ myotubes by differentiation, by mutation of an nuclear factor kappa-beta (NF-kappaB) site, or deletion of a sma/mothers against decapentaplegic (SMAD) site. Together, these data demonstrate that TLL1 mRNA levels are altered by loading, energy status, and differentiation, and thus its expression may be regulated so as to modulate activity of myostatin or other extracellular substrates during these adaptive states.     

Identification of 9 novel FBN1 mutations in German patients with Marfan syndrome

We report 9 new mutations in German patients presenting with classical Marfan syndrome. All mutations occur in exons with calcium‐binding (cb) epidermal growth factor‐like (EGF) domains. Five mutations are missense involving exons 12, 27, 30, 44, and 52 with the resultant substitution of cysteine by phenylalanine (C504F), cysteine by tyrosine (C1129Y), tyrosine by cysteine (Y1261C), cysteine by serine (C1833S), and cysteine by tyrosine (C2142Y), respectively. The other four mutations are single base deletions in exons 39, 43, 48, and 58, at nucleotide A4826, C5311, T6018, and A7291, respectively, each resulting in frameshift with premature termination. Four mutations were detected in sporadic cases and are likely to be de novo. Hum Mutat 14:181, 1999. © 1999 Wiley‐Liss, Inc.     

PRG‐4/SZP N‐ and C‐terminal domains: cloning,expression and characterization

Introduction Proteoglycan‐4 (PRG‐4), also known as superficial zone protein/proteoglycan (SZP), is an approximately 345‐kDa mucinous proteoglycan that has been detected in a variety of tissues including cartilage, tendon, bone, heart and liver ( Ikegawa et al. 2000 ). In the synovial joint, PRG‐4 is specifically synthesized by chondrocytes located in the superficial zone of articular cartilage and by some surface‐lining cells of the synovium ( Schumacher et al. 1994 ). Sequence analyses have shown that the N‐ and C‐terminal vitronectin‐like domains of PRG‐4 may impart interesting functions relevant to synovial joint metabolism ( Merberg et al. 1993 ; Flannery et al. 1999 ). The objective of this study was to investigate these potential functions, facilitated by the production of PRG‐4 N‐ and C‐terminal domains as recombinant proteins. Methods cDNAs for the human N‐terminal (exons 2–5) and bovine C‐terminal (exons 7–12) domains of PRG‐4 were obtained by RT‐PCR and cloned into the expression vector pMT‐BiP for inducible, secreted expression in Drosophila S2 cells. Proteins were purified using FLAG‐M2 antibody affinity chromatography and visualized by SDS‐PAGE and Western blotting with PRG‐4‐specific antibodies. The heparin‐binding properties of recombinant proteins were investigated using heparin affinity chromatography. The interactions of recombinant PRG‐4 domains with human plasminogen activator‐inhibitor (PAI)‐1 and bovine type‐II collagen were assayed using standard ELISA techniques. Results Stable cell lines have been generated that express human N‐terminal and bovine C‐terminal PRG‐4 domains. In both cases, two proteins have been purified, possibly due to a splice mechanism by the expression system. N‐terminal sequence data and Western blotting indicate that the two species in each case could represent full‐length and truncated proteins. Analyses of the two PRG‐4 N‐terminal domain species have confirmed the presence of a predicted heparin‐binding domain and indicate that the molecule can bind to PAI‐1, with binding activity localized towards its two somatomedin B domains. The somatomedin B domain of vitronectin is known to bind PAI‐1 ( Seiffert 1997 ). Analyses of the two PRG‐4 C‐terminal species have demonstrated self‐association under nonreducing conditions and binding to heparin and PAI‐1. Discussion The exact role of PRG‐4 in the synovial joint is yet to be elucidated. However, these results point towards the interaction of the N‐ and C‐terminal domains of PRG‐4 with structural molecules such as type‐II collagen and heparin, and functional molecules such as PAI‐1, a serpin that is involved in the fibrinolytic cascade and cell adhesion. These properties are in addition to the well‐documented boundary lubricating activity of the central mucinous region of PRG‐4.     

Apa is a trimeric autotransporter adhesin of Actinobacillus pleuropneumoniae responsible for autoagglutination and host cell adherence

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, and adherence to host cells is a key step in the pathogenic process. Although trimeric autotransporter adhesins (TAAs) were identified in many pathogenic bacteria in recent years, none in A. pleuropneumoniae have been characterized. In this study, we identified a TAA from A. pleuropneumoniae, Apa, and characterized the contribution of its amino acid residues to the adhesion process. Sequence analysis of the C‐terminal amino acid residues of Apa revealed the presence of a putative translocator domain and six conserved HsfBD1‐like or HsfBD2‐like binding domains. Western blot analysis revealed that the 126 C‐terminal amino acids of Apa could form trimeric molecules. By confocal laser scanning microscopy, one of these six domains (ApaBD3) was determined to mediate adherence to epithelial cells. Adherence assays and adherence inhibition assays using a recombinant E. coli‐ ApaBD3 strain which expressed ApaBD3 on the surface of E. coli confirmed that this domain was responsible for the adhesion activity. Moreover, cellular enzyme‐linked immunosorbent assays demonstrated that ApaBD3 mediated high‐level adherence to epithelial cell lines. Intriguingly, autoagglutination was observed with the E. coli‐ ApaBD3 strain, and this phenomenon was dependent upon the association of the expressed ApaBD3 with the C‐terminal translocator domain. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Roles of the third Ig‐like domain of Necl‐5/PVR and the fifth Ig‐like domain of the PDGF receptor in its signaling

The immunoglobulin (Ig)‐like cell adhesion molecule nectin‐like molecule (Necl)‐5/poliovirus receptor is up‐regulated in many types of cancer cells and implicated in their abnormally enhanced cell proliferation and movement. We previously showed that Necl‐5 cis‐interacts with the platelet‐derived growth factor (PDGF) receptor β through the extracellular region and enhances its signaling. Although this cis‐interaction does not affect the PDGF‐induced tyrosine phosphorylation of the receptor, the interaction of the cytoplasmic region of Necl‐5 with sprouty2 and the regulation of its activity are required for the enhancement of the PDGF receptor β signaling by Necl‐5. We investigated here the more detailed mechanism for this cis‐interaction of Necl‐5 with the PDGF receptor β. Necl‐5 contains three Ig‐like domains and the PDGF receptor β contains five Ig‐like domains at their extracellular regions. We showed here that the third Ig‐like domain of Necl‐5 cis‐interacted with the fifth Ig‐like domain of the PDGF receptor β. The recombinant protein of the third Ig‐like domain of Necl‐5 inhibited the cis‐interaction of full‐length Necl‐5 with the PDGF receptor β and the PDGF‐induced activation of the ERK signaling pathway that was enhanced by Necl‐5. These results revealed the novel roles of the third Ig‐like domain of Necl‐5 and the fifth Ig‐like domain of the PDGF receptor β in its signaling.     

Stunning and myocardial contractile autoregulation studied on the isolated isovolumic blood‐perfused dog heart

Aim: To study, for the first time, the effects of stunning on homeometric and heterometric autoregulation. Methods and results: Ischaemia (15 min)/reperfusion (30 min) was induced in the isovolumic blood‐perfused dog heart preparation. Heart rate elevations (n = 9) from 60 to 200 beats min^?1, in steps of 20 beats min^?1, promoted the same inotropic stimulation in control (C) and stunning (S), indicating that ischaemia/reperfusion does not affect the changes in calcium kinetics elicited by the Bowditch effect. Sudden ventricular dilation (VD) (n = 10) evoked an instantaneous increase in developed pressure (Δ₁DP) followed by a continuous slow performance increase (Δ₂DP) in C and S. Δ₁DP (C: 35 ± 2.2 mmHg; S: 27 ± 2.1 mmHg; P = 0.002) and Δ₂DP (C: 20 ± 1.6 mmHg; S: 14 ± 1.3 mmHg; P = 0.002) decreased proportionally, while Δ₂/Δ₁DP (C: 0.57 ± 0.13; S: 0.58 ± 0.14) and slow response time course (T/2) were unchanged (C: 55 ± 6.6 s; S: 57 ± 7.7 s) after ischaemia/reperfusion. The reduction of Δ₁DP can be understood as a decline of the myofilaments calcium responsiveness, the main pathophysiological effect of stunning. The reason for the weakening of Δ₂DP, due to intracellular calcium gain, was not determined but it was supposed that its complete manifestation could be restricted by cyclic adenosine monophosphate (cAMP) myocardial content reduction. As reported by others, Δ₂DP depends on myocardial cAMP, and it has been shown that myocardial cAMP is decreased after ischaemia/reperfusion. Conclusions: Contractile depression due to stunning has no effect on the inotropic stimulation generated by the Bowditch phenomenon. Immediate and time‐dependent enhancements of contraction evoked by sudden VD are proportionally reduced and the slow response time course is unaffected in the stunned myocardium.     

Dynamic analysis of BMP‐responsive smad activity in live zebrafish embryos

Bone morphogenetic proteins (BMPs) are critical players in development and disease, regulating such diverse processes as dorsoventral patterning, palate formation, and ossification. These ligands are classically considered to signal via BMP receptor‐specific Smad proteins 1, 5, and 8. To determine the spatiotemporal pattern of Smad1/5/8 activity and thus canonical BMP signaling in the developing zebrafish embryo, we generated a transgenic line expressing EGFP under the control of a BMP‐responsive element. EGFP is expressed in many established BMP signaling domains and is responsive to alterations in BMP type I receptor activity and smad1 and smad5 expression. This transgenic Smad1/5/8 reporter line will be useful for determining ligand and receptor requirements for specific domains of BMP activity, as well as for genetic and pharmacological screens aimed at identifying enhancers or suppressors of canonical BMP signaling. Developmental Dynamics 240:682–694, 2011. © 2011 Wiley‐Liss, Inc.     

Efficient Autoproteolytic Processing of the MHV-A59 3C-like Proteinase from the Flanking Hydrophobic Domains Requires Membranes

The replicase gene of the coronavirus MHV-A59 encodes a serine-like proteinase similar to the 3C proteinases of picornaviruses. This proteinase domain is flanked on both sides by hydrophobic, potentially membrane-spanning, regions. Cell-free expression of a plasmid encoding only the 3C-like proteinase (3CLpro) resulted in the synthesis of a 29-kDa protein that was specifically recognized by an antibody directed against the carboxy-terminal region of the proteinase. A protein of identical mobility was detected in MHV-A59-infected cell lysates.In vitroexpression of a plasmid encoding the 3CLpro and portions of the two flanking hydrophobic regions resulted in inefficient processing of the 29-kDa protein. However, the efficiency of this processing event was enhanced by the addition of canine pancreatic microsomes to the translation reaction, or removal of one of the flanking hydrophobic domains. Proteolysis was inhibited in the presence ofN-ethylmaleimide (NEM) or by mutagenesis of the catalytic cysteine residue of the proteinase, indicating that the 3CLpro is responsible for its autoproteolytic cleavage from the flanking domains. Microsomal membranes were unable to enhance thetransprocessing of a precursor containing the inactive proteinase domain and both hydrophobic regions by a recombinant 3CLpro expressed fromEscherichia coli.Membrane association assays demonstrated that the 29-kDa 3CLpro was present in the soluble fraction of the reticulocyte lysates, while polypeptides containing the hydrophobic domains associated with the membrane pellets. With the help of a viral epitope tag, we identified a 22-kDa membrane-associated polypeptide as the proteolytic product containing the amino-terminal hydrophobic domain.