Academic literature on the topic 'Expressed Protein Ligation (EPL)'

Over the past few years, synthetic methods for the preparation of complex glycopeptides have been drastically improved. The need for homogenous glycopeptides and glycoproteins with defined chemical structures to study diverse biological phenomena further enhances the development of methodologies. Selected recent advances in synthesis and applications, in which glycopeptides or glycoproteins serve as tools for biological studies, are reviewed. The importance of specific antibodies directed to the glycan part, as well as the peptide backbone has been realized during the development of synthetic glycopeptide-based anti-tumor vaccines. The fine-tuning of native chemical ligation (NCL), expressed protein ligation (EPL), and chemoenzymatic glycosylation techniques have all together enabled the synthesis of functional glycoproteins. The synthesis of structurally defined, complex glycopeptides or glyco-clusters presented on natural peptide backbones, or mimics thereof, offer further possibilities to study protein-binding events.

Abstract Posttranslational modifications (PTMs) of histones represent a crucial regulatory mechanism of nucleosome and chromatin dynamics in various of DNA-based cellular processes, such as replication, transcription and DNA damage repair. Lysine succinylation (Ksucc) is a newly identified histone PTM, but its regulation and function in chromatin remain poorly understood. Here, we utilized an expressed protein ligation (EPL) strategy to synthesize histone H4 with site-specific succinylation at K77 residue (H4K77succ), an evolutionarily conserved succinylation site at the nucleosomal DNA-histone interface. We then assembled mononucleosomes with the semisynthetic H4K77succ in vitro. We demonstrated that this succinylation impacts nucleosome dynamics and promotes DNA unwrapping from the histone surface, which allows proteins such as transcription factors to rapidly access buried regions of the nucleosomal DNA. In budding yeast, a lysine-to-glutamic acid mutation, which mimics Ksucc, at the H4K77 site reduced nucleosome stability and led to defects in DNA damage repair and telomere silencing in vivo. Our findings revealed this uncharacterized histone modification has important roles in nucleosome and chromatin dynamics.

Abstract Prostaglandin E2 (PGE2) is a lipid mediator that acts by ligating 4 distinct G protein–coupled receptors, E prostanoid (EP) 1 to 4. Previous studies identified the importance of PGE2 in regulating macrophage functions, but little is known about its effect on macrophage maturation. Macrophage maturation was studied in vitro in bone marrow cell cultures, and in vivo in a model of peritonitis. EP2 was the most abundant PGE2 receptor expressed by bone marrow cells, and its expression further increased during macrophage maturation. EP2-deficient (EP2−/−) macrophages exhibited enhanced in vitro maturation compared with wild-type cells, as evidenced by higher F4/80 expression. An EP2 antagonist also increased maturation. In the peritonitis model, EP2−/− mice exhibited a higher percentage of F4/80high/CD11bhigh cells and greater expression of macrophage colony-stimulating factor receptor (M-CSFR) in both the blood and the peritoneal cavity. Subcutaneous injection of the PGE2 analog misoprostol decreased M-CSFR expression in bone marrow cells and reduced the number of peritoneal macrophages in wild-type mice but not EP2−/− mice. The suppressive effect of EP2 ligation on in vitro macrophage maturation was mimicked by a selective protein kinase A agonist. Our findings reveal a novel role for PGE2/EP2/protein kinase A signaling in the suppression of macrophage maturation.

Abstrak Beberapa gen yang terekspresi spesfik di epididimis diduga terlibat dalam proses pematangan sperma. Ekspresi gen spesifik di epididimis dipengaruhi oleh androgen, faktor testikuler, dan terekspresi pada masa pubertas. Famili gen yang cukup banyak ditemukan terekspresi di epididimis adalah beta-defensin, salah satunya yaitu beta-defensin 2 (Defb2). Penelitian ini bertujuan untuk mengkarakterisasi gen Defb2 terkait dengan perannya pada proses pematangan sperma. Analisis bioinformatika digunakan pada penelitian ini untuk mendapatkan informasi mengenai struktur gen, signal peptide, dan domain fungsional pada gen Defb2. Analisis quantitative reverse transcriptase-Polymerase Chain Reaction (qRT-PCR) untuk mengetahui ekspresi relatif gen Defb2. Hasil yang diperoleh yaitu Defb2 merupakan protein sekretori karena memiliki signal peptide. Defb2 memiliki domain fungsional berupa N-myristoylation dan protein kinase-C. Gen Defb2 terekspresi spesifik di epididimis. Ekspresi Defb2 dipengaruhi oleh androgen dan faktor testikuler terbukti setelah perlakuan gonadektomi dan efferent duct ligation (EDL) terjadi penurunan ekspresi Defb2. Adapun pada analisis postnatal development terlihat ekspresi gen Defb2 mulai terdeteksi pada hari ke-15 yang merupakan masa pubertas mencit jantan. Kata kunci: androgen, beta-defensin 2 (Defb2), epididimis, pematangan sperma Abstract Several specific genes expressed in the epididymis are thought to be involved in the sperm maturation process. Their specific expression in the epididymis are influenced by androgens, testicular factors, and increased expression at puberty. A family of genes that are commonly found expressed in the epididymis is beta-defensin, one of which is beta-defensin 2 (Defb2). This study was aimed to characterize the Defb2 gene related to its role in the sperm maturation process. Bioinformatics analysis was used in this study to obtain information about gene structure, signal peptides, and functional domains in the Defb2 gene. The quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis was used to determine relative expression of the Defb2 gene. The results showed Defb2 was a secretory protein because it has a signal peptide. Defb2 has a functional domain in the form of N-myristoylation and protein kinase-C. Defb2 gene was specifically expressed in the epididymis. Defb2 expression was influenced by androgens and testicular factors that were proven by post gonadectomy and efferent duct ligation (EDL) decreases in Defb2 expression. As for the postnatal development analysis, the expression of Defb2 gene was initially detected on day 15 which is the puberty of male mice. Keywords: androgen, beta-defensin 2 (Defb2), epididymis, sperm maturation

Antigene e anticorpo sono i due reagenti chiave di un saggio immunodiagnostico. L’indagine di nuove tecniche e il miglioramento di processi quali la purificazione e la marcatura sito-specifica di antigeni e anticorpi possono promuovere lo sviluppo di nuovi reagenti più efficienti capaci di migliorare la performance dei saggi immunodiagnostici. La prima parte di questa tesi è stata focalizzata sull’esplorazione di tecniche biotecnologiche innovative nella produzione di antigeni al fine di migliorare i saggi per la rilevazione degli anticorpi IgM e IgG specifici per il virus Epstein-Barr. Il virus EBV è causa della mononucleosi infettiva ed è associato ad un crescente numero di tumori; per questa motivo è importante sviluppare saggi diagnostici per la rilevazione di EBV ad alta specificità e sensibilità. La proteina VCA p18 è uno degli antigeni più importanti per la diagnosi di EBV. I saggi attuali Diasorin LIAISON EBV VCA IgM and IgG si basano su un singolo antigene corrispondente alla regione C-terminale immunodominante della proteina p18, immobilizzata su fase solida. I vari metodi esplorati in questa tesi hanno permesso di ottenere diverse varianti dell’antigene p18 con lo scopo di migliorare le prestazioni dei saggi EBV VCA IgM e IgG a diversi livelli: 1_produzione dell’antigene p18; 2_immobilizzazione dell’antigene p18 su fase solida; 3_formato di saggio. 1_La lunghezza della regione C-terminale della proteina p18 (57aa), risulta essere considerevole per il processo sintetico ma, allo stesso tempo, troppo piccola per essere prodotta in modo efficiente per via ricombinante. Per superare questo problema, abbiamo esplorato il sistema Elastin Like Polypeptides (ELP)-Inteina basato sull’uso di una proteina capace di effettuare auto-cleavage (inteina) e un tag responsivo alla temperatura (ELP). Questa tecnica si è rivelata un eccellente sistema per la produzione del peptide p18. 2_Lo sviluppo di diverse varianti dell’antigene p18 ha permesso di esplorare varie tecniche per l’immobilizzazione dello stesso antigene su fase solida: coating covalente diretto, attraverso il complesso streptavidina-biotina e attraverso l’uso dei peptidi leucine zipper (o velcro). L’immobilizzazione del peptide p18 su fase solida attraverso questi vari metodi è avvenuta con successo e l’attività immunochimica dell’antigene, immobilizzato con queste tecniche innovative, è comparabile o migliore rispetto a quella del peptide sintetico utilizzato nei saggi attuali. 3_Nonostante il saggio Diasorin LIAISON EBV VCA IgM abbia una buona performance analitica, al fine di ottenere un aumento di specificità, è stato esplorato un nuovo tipo di formato di saggio. Sfortunatamente i risultati indicano che questo diverso tipo di formato raggiunge un livello di specificità minore rispetto a quello del saggio attuale. La seconda parte di questa tesi è stata focalizzata sull’esplorazione di un metodo innovativo per la marcatura sito specifica degli anticorpi. Uno degli approcci più promettenti è basato sulla generazione di gruppi tiolo liberi attraverso la riduzione parziale e selettiva dei ponti disolfuro inter-catena presenti a livello della “hinge region” e la loro reazione con molecole marcanti caratterizzate dal possedere gruppi funzionali reattivi verso i gruppi sulfidrilici. Questa tecnologia è stata utilizzata per la biotinilazione di due diversi anticorpi usati attualmente per il rilevamento della proteina virale p24 di HIV e per l’antigene FGF23. I risultati suggeriscono che la biotinilazione sito-specifica rispetto a quella classica random promuove un miglioramento dell'attività immunochimica degli anticorpi con una conseguente ottimizzazione della performance dei saggi immunodiagnostici.
Antigen and antibody are the two key reagents for an immunodiagnostic assay. Investigation of new techniques and improvement of processes such as purification and site-specific labeling of antigen and antibody molecules can promote the development of new more powerful bioreagents able of improving the performance of immunodiagnostic assays. The first part of this thesis aimed to explore innovative biotechnology techniques in antigen production for the improvement of immunoassays that allow the detection of antibodies directed against the Epstein-Barr virus. EBV virus is the causative agent of infectious mononucleosis and it is considered to be associated with a still increasing number of tumors; for this reason it is important to develop diagnostic assays for EBV detection with high specificity and sensitivity. The viral capsid protein VCA p18 is one of the most important antigens for the diagnosis of EBV. The current Diasorin LIAISON EBV VCA IgM and IgG assays rely on a single antigen, consisting in a synthetic peptide corresponding to the immunodominant C-terminal portion of the p18 protein, which is immobilized on solid phase (indirect format). The several methods explored in this thesis have allowed to obtain different variants of the p18 antigen with the aim to improve the performance of DiaSorin LIAISON EBV VCA IgM and IgG assays at different levels: 1_production of p18 antigen; 2_immobilization of p18 antigen on solid phase; 3_immunoassay format. 1_ The length of the immunodominant C-terminal portion of the p18 protein (57aa) appears to be considerable for the synthetic route but, at the same time, too small to be effectively produced in a recombinant fashion. To overcome this problem, we explored the Elastin Like Polypeptides (ELP)-Intein system, a method based on the use of a self-cleavable protein (the intein) and a temperature responsive tag (ELP). This technique has proved to be an excellent system for the preparation of the p18 peptide. 2_The development of different variants of p18 antigen has enabled to explore different techniques for the immobilization of the same antigen on solid phase: direct covalent coating, through streptavidin-biotin complex and through an innovative procedure based on the use of leucine zipper (or “velcro”) peptides. The immobilization of the p18 peptide on solid phase through these different methods occurred successfully and the immunochemical activity of the antigen, immobilized with these innovative techniques, is comparable or better than that of the synthetic peptide used in the current immunoassays. 3_Despite the DiaSorin LIAISON EBV VCA IgM immonoassay has a good analytical performance, in order to obtain an increase of specificity, a new assay format was explored. Unfortunately, the results indicate that this different type of format reaches a lower level of specificity than that of current assay. The second part of this thesis aimed to explore an innovative method for the site-specific labeling of antibodies. One the most promising approach is based on the generation of free thiol groups by selective partial reduction of the interchain disulfide bridges present at the level of the “hinge region” and their reaction to labels carrying sulfhydryl-reactive chemical groups. This technology was used for the biotinylation of two different antibodies currently used in immunoassays for the HIV p24 viral protein and for FGF23 antigen detection. The results suggest that the site-specific biotinylation compared to the random traditional biotinylation promotes a great improvement of antibodies immunochemical activity with a consequent optimization of immunodiagnostic assays performance.

Abstract:The 90-kb androgen receptor (AR) gene is located at locus Xq11-12 and has eight exons that encode a protein of about 919 amino acids. Exon 1 encodes the transactivational domain, exons 2 and 3 encode the DNA-binding domain, and exons 4 through 8 encode the ligand-binding domain. The AR protein, a nuclear receptor that belongs to the steroid/thyroid hormone receptor family, functions primarily as a DNA-binding transcription factor to regulate gene expression. The AR also has a role in the development of the male phenotype: in fact, cytoplasmic AR is activated by binding testosterone or dihydrotestosterone hormone before translocating to the nucleus. Spinal and Bulbar Muscular Atrophy (SBMA), also known as Kennedy's disease, is an X-linked recessive neurodegenerative disease. The cause of SBMA is expansion of the CAG trinucleotide repeat, which codes for glutamine (Gln, Q), in Exon 1 of the AR gene. Therefore SBMA is considered to be a polyglutamine (polyGln) expansion disorder. In unaffected men, 9-33 CAG repeats coding for Gln are found, while males who inherit more than 37 CAG repeats will develop SBMA. The toxic gain of AR function that is characteristic of SBMA results in a slow progressive muscle weakness, atrophy of the tongue, fasciculations of bulbar, facial and limb muscles, and muscle cramping. A loss of AR function is related to breast enlargement and reduced fertility.The ubiquitin-proteasome system (UPS), which functions in protein degradation, plays an important role in neural cells from SBMA patients. UPS components are found in polyGln-expanded protein aggregates seen in these cells. Therefore previous studies have suggested that the UPS is impaired in SBMA. Previous work suggests that 0QAR and 20QAR can be degraded via the UPS, however, the 50QAR appeared to inhibit the degradation of specific proteins by the UPS. It is also hypothesized that these SBMA-inducing mutant ARs can accumulate in the cell and cause disease by evading UPS degradation. The primary goal of this research was to develop a system for ubiquitinating AR by using an expressed protein ligation (EPL) system. To prove this hypothesis, several intermediate goals have been achieved. PCR-amplified fragments of AR (0Q, 20Q, and 50Q) were cloned into the pTWIN2 vector and contructs were analyzed using restriction enzyme digests and gel electrophoresis. Furthermore, 0QAR, 20QAR, 50QAR and human ubiquitin (HUB) fusion proteins were purified using chitin beads and cleaved using 2-mercapto-ethanesulfonic acid (MESNA). Finally, the 0QAR, 20QAR, 50QAR peptides were ligated with an HA-tagged H2B peptide using EPL. Thus, we successfully developed tools which can be used to explore ubiquitinatination of the AR using EPL systems in vitro. Future work aims to ligate the HUB protein to a peptide to using the EPL method, and then ligating the ubiquitinated peptide to the three different AR peptides. These will be added to active proteasomes to explore the involvement of the UPS in the pathogenesis of SBMA.
Abrégé:Le gène de 90-kb du récepteur androgène (AR) est localisé au locus Xq11-12 et possède huit exons codant pour une protéine d'environ 919 acides aminés. L'exon 1 code pour le domaine transactivationnel, les exons 2 et 3 codent pour le domaine de liaison à l'ADN, et les exons 4 à 8 codent pour le domaine de liaison au ligand. La protéine du AR, un récepteur nucléaire appartenant à la famille des récepteurs hormonaux stéroïdiens/thyroïdiens, agit primordialement en tant que facteur transcriptionnel se liant à l'ADN pour réguler l'expression génique. Le AR possède également un rôle dans le développement du phénotype masculin: en effet, le AR cytoplasmique est activé en se liant aux hormones testostérone ou dihydrotestostérone avant de se translocaliser au noyau.L'amyotrophie bulbo-spinale (SBMA-Spinal and Bulbar Muscular Atrophy), aussi connue sous le nom de maladie de Kennedy, est une maladie neurodégénérative liée au chromosome X récessif. La cause de la SBMA est une expansion du triplet CAG répété, lequel code pour une glutamine (Gln,Q), dans l'exon 1 du gène AR. Ainsi, la SBMA est considérée comme étant un désordre d'expansion polyglutaminique (poly-Gln). Chez l'homme non-affecté, on retrouve 9-33 répétitions CAG codant pour la Gln, alors que l'homme qui hérite plus de 37 répétitions CAG développera la SBMA. Le gain de fonction toxique du AR caractérisant la SBMA se traduit par: une faiblesse musculaire à progression lente, une atrophie de la langue, des fasciculations des muscles bulbaires, faciaux et limbiques et enfin par des crampes musculaires. Une perte de fonction du AR est liée à l'élargissement de la poitrine et à la fertilité réduite.Le système ubiquitine-protéasome (UPS), responsable dans la dégradation protéique, joue un rôle important dans les cellules neurales chez les patients atteints de la SBMA. Les composantes de l'UPS se trouvent dans les aggrégats protéiques de l'expansion poly-Gln dans ces cellules. C'est la raison pour laquelle des études précédentes ont suggéré que l'UPS est affaibli dans la SBMA. D'autres études antérieures suggèrent que les peptides 0QAR et 20QAR peuvent être dégradés par l'UPS, cependant le peptide 50QAR semble inhiber la dégradation par lUPS de certaines protéines spécifiques. L'hypothèse veut que ces mutants AR responsables de la SBMA peuvent s'accumuler dans la cellule et causer la maladie en s'évadant de la dégradation UPS. Le but premier de ce projet était de développer un système d'ubiquitination du AR en appliquant le système de ligation des protéines exprimées (expressed protein ligation-EPL). Afin de prouver cette hypothèse, quelques buts intermédiaires ont été atteints. Les fragments du AR (0Q, 20Q, et 50Q), amplifiés par PCR, ont été clonés dans le vecteur pTWIN2 et les clones ont été analysés sur gels d'électrophorèse suite aux digestions par enzymes de restriction. De plus, les fragments 0QAR, 20QAR et 50QAR ainsi que les protéines de fusion de l'ubiquitine humaine (HUB) ont été purifiés à l'aide de billes de chitine et clivés par l'acide mercapto-2-éthanesulfonique (MESNA).Les peptides 0QAR, 20QAR et 50QAR ont été unis par ligation au peptide de fusion H2B doté d'un tag HA en se servant du système EPL. Ainsi, nous avons développé avec succès des outils permettant d'explorer l'ubiquitination du AR en utilisant les systèmes EPL in vitro. Des expériences à l'avenir viseront à lier par ligation la protéine HUB à un peptide, en utilisant la méthode EPL et ensuite faire la ligation du peptide ubiquitiné aux trois différents peptides AR. Ces derniers seront ajoutés aux protéasomes actifs pour explorer l'implication de l'UPS dans la pathogénèse de la SBMA.

Das fibrillen-bildende beta2-Mikroglobulin (b2M) und das CRF1-Mimetikum mit verzweigter Rückgratstruktur können als „schwierige“ Proteine betrachtet werden, deren Darstellung sich eignet, gegenwärtige Möglichkeiten und Grenzen der Proteinsynthese zu ermitteln. Die Proteine sollen zu spektroskopischen Untersuchungen von Proteinfaltung bzw. Ligand-Rezeptor- Wechselwirkungen eingesetzt werden. Versuche zur Chemosynthese von b2M über drei Segmente führten per NCL zwar zu linearen Produkten mit korrekter Primärstruktur, aber wiederholt wurden zwei, mittels HPLC trennbare Proteine erhalten, deren enzymatische Spaltung zu identischen Fragmenten führte. Eine Isomerisierung (wie z.B. Epimerisierung) als Ursache für die Bildung der zwei Produkte konnte ausgeschlossen werden. Mittels CD- und FTIR-Spektroskopie wurden für beide Produkte beta- Faltblatt-Strukturen ermittelt, die sich sowohl untereinander als auch vom rekombinanten Protein unterschieden. Die „fehlgefalteten“ Syntheseprodukte konnten nicht entfaltet und anschließend in die „korrekte“ Struktur des rekombinanten b2M überführt werden. Es ist denkbar, dass die beobachtete „Fehlfaltung“, deren Ursache nicht geklärt werden konnte, für vom b2M ausgelöste Amyloidosen verantwortlich ist. Das CRF1-Modell, das aus drei zyklischen Peptiden und einem Protein mit Disulfidbrücken besteht, welche auf einem linearen Peptid-Templat verankert sind, wurde durch ein zyklisches Templat zur strukturellen Einschränkung modifiziert. Durch das zyklische Templat ergaben sich keine Syntheseprobleme, aber interessanterweise führte die Zyklisierung des Templats zu einer signifikant höheren Affinität für den Agonisten Urocortin-I im funktionellen Assay. Darüber hinaus wurde gezeigt, dass ein zyklisches Rezeptor-Loop-Peptid mittels EPL im mg-Maßstab erhalten werden kann, was künftig die Synthese isotopenmarkierter Analoga für Struktur-Untersuchungen ermöglicht.
The fibril forming beta2-microglobulin (b2m) and the CRF1 mimic with branched peptide backbone could be considered as “difficult” proteins, whose synthesis is suited for determining present possibilities and limits of protein synthesis. The proteins shall be used for spectroscopic analysis of protein misfolding or ligand-receptor-interaction, respectively. Efforts of the chemosynthesis of b2m over three segments may lead via NCL to linear products with correct primary structure, but two, via HPLC isolatable proteins were repetitively susbstained, whose enzymatic digest lead to identical fragments. An isomerization (such as e. g. epimerization) as reason for the formation of the two products could be excluded. By means of CD and FTIR spectroscopy for both products beta-sheet structure were determined, which differ among themselves as well as from the recombinant protein. The “misfolded” synthetic product could not be unfolded und subsequently converted into the “correct” structure of the recombinant b2m. It is possible that the observed “misfolding”, whose cause could not be clarified, is reasonable for the amyloidosis induced by b2m. The CRF1 model that consists of three cyclic peptides and one protein with disulfid bridges coupled to a linear peptide template, was modified for structural constraints by a cyclic template. In consequence of the cyclic template no synthetic problems aroused, although the cyclisation of the template leads interestingly to a significant higher affinity for the antagonist urocortin-I in the functional assay. Furthermore, it was shown that a cyclic receptor loop peptide could be received via EPL in mg scale, what in future enables the synthesis of isotopically labeled analogs for structure investigations.

A hybrid between human tissue-type plasminogen activator (t-PA) and human single chain urokinase-type plasminogen activator (scu-PA) was obtained by ligation of cDNA fragments encoding the NH2-terminal amino acids 1 to 67 of t-PA and the COOH-ter-minal amino acids 136 to 411, of scu-PA. Both this chimaeric cDNA and cDNA encoding scu-PA were expressed in a mammalian system (HAK-cells) using bovine papilloma virus (BPV) derived vectors. Two stable cell lines were obtained which secreted the recombinant hybrid and the scu-PA at 1 μg/ml and 2 μg/ml u-PA related antigen respectively into the culture medium. Following purification by Zinc chelate Sepharose, immunoadsorption chromatography, benzamidine-Sepharose and Ultrogel AcA44 gel filtration, highly purified proteins were obtained with a yield of about 200 μg/1. SDS gel electrophoresis under reducing conditions showed single bands with M 43,000 and M 50,000 respectively. Following conversion to urokinase with plasmin, both proteins had a specific amidolytic activity comparable to that of natural scu-PA. Both proteins activated plasminogen directly with km1.4 and 0.5 μM and k2 0.0034 s and 0.0027 s . Neither protein bound specifically to fibrin.Thus the fusion of the finger-like domain of t-PA to the COOH-terminal part of scu-PA does not confer fibrin affinity of t-PA to this chimaeric protein. However, peptide material can be fused to the COOH-terminal part of scu-PA without perturbing its enzymatic properties.

Cellular therapy for myocardial injury has improved ventricular function in both animal and clinical studies, though the mechanism of benefit is unclear. This study was undertaken to examine the effects of cellular injection after infarction on myocardial elasticity. Coronary artery ligation of Lewis rats was followed by direct injection of human mesenchymal stem cells (MSC) into the acutely ischemic myocardium. Two weeks post-infarct, myocardial elasticity was mapped by atomic force microscopy. MSC-injected hearts near the infarct region were two-fold stiffer than myocardium from non-infarcted animals but softer than myocardium from vehicle-treated infarcted animals. After eight weeks, the following variables were evaluated: MSC engraftment and left ventricular geometry by histologic methods; cardiac function with a pressure-volume conductance catheter; myocardial fibrosis by Masson trichrome staining; vascularity by immunohistochemistry; and apoptosis by TUNEL assay. The human cells engrafted and expressed a cardiomyocyte protein but stopped short of full differentiation and did not stimulate significant angiogenesis. MSC-injected hearts showed significantly less fibrosis than controls, as well as less left ventricular dilation, reduced apoptosis, increased myocardial thickness, and preservation of systolic and diastolic cardiac function. In summary, MSC injection after myocardial infarction did not regenerate contracting cardiomyocytes but reduced the stiffness of the subsequent scar and attenuated post-infarction remodeling, preserving some cardiac function. Improving scarred heart muscle compliance could be a functional benefit of cellular cardiomyoplasty.

A hybrid human cDNA was constructed by ligation of a cDNA fragment of tissue-type plasminogen activator (t-PA), encoding 5∲-untranslated, the pre-pro region and amino acids Ser 1 through Thr 263, with a cDNA fragment of urokinase-type plasminogen activator (u-PA), encoding amino acids Leu 144 through Leu 411. The hybrid cDNA was expressed in Chinese Hamster Ovary Cells and the translation product purified from the conditioned cell culture media in the presence of aprotinin. On SDS-gel electrophoresis under reducing conditions, the protein migrated as a single band with approximate Mr 70,000 and on immunoblot-ting, it reacted with rabbit antisera raised against human t-PA and against human u-PA. The urokinase-like amidolytic activity (S-2444) of the protein’ was 320 IU/mg but increased to 43,000 IU/mg after treatment with plasmin, which resulted in conversion of the single chain molecule (t-PA/scu-PA) to a two-chain molecule (t-PA/tcu-PA).Both proteins activated plasminogen directly with Michaelis constant (Km) 1.5 μM and catalytic rate constant (km2) 0.0058 s-1 for t-PA/scu-PA and with K = 80 μM and = 5.6 s-1 for t-PA/tcu-PA. CBNr-digested fibrinogen stimulated the activation rate of plasminogen with t-PA/tcu-PA (increase of k2/Km of 88-fold).Both t-PA/scu-PA and t-PA/tcu-PA bound specifically to fibrin albeit ^np$re weakly than t-PA. In an in vitro system composed of a human I-fibrin labeled plasma clot immersed in human plasma, the t-PA/tcu-PA hybrid has a higher fibrin-selectivity of clot lysis than tcu-PA, but this difference was not evident between t-PA/scu-PA and scu-PA. The stability of the t-PA/scu-PA hybrid in plasma was much higher than that of the t-PA/tcu-PA hybrid, a difference comparable to that between scu-PA and tcu-PA.It is concluded that these t-PA/u-PA hybrid proteins combine fibrin-affinity of t-PA with the enzymatic properties of u-PA (either scu-PA or tcu-PA), resulting in improved fibrin-mediated plasminogen activation.