High throughput screening of combinatorial chemical libraries holds tremendous promise for accelerating the process of drug discovery. 1 To increase the screen throughput, a number of strategies have been developed to encode synthetic molecules with DNA or RNA tags so that the whole library can be selected for target binding in one pool and the structures of the selected compounds can be quickly identified by sequencing the DNA or RNA tags. 2 In another strategy, the DNA "bar code" is not physically linked to the encoded small molecule but is embedded within the phage genomic DNA and encapsulated inside the phage capsid proteins to which the small molecules are attached. 3 The phage displayed small molecule library can then be selected for target binding, and upon amplification of the selected phages, the corresponding phagemid is sequenced to give the identity of the selected molecule. Using the phage platform for small molecule encoding has the advantages that the DNA bar code is shielded by the phage capsid proteins, which eliminates the interference of DNA secondary structures with the binding of small molecules to the target proteins, and at the same time the DNA bar code is protected from degradation so that the phage displayed small molecule libraries can be selected for binding with in vivo targets. 4 Also, since the amplification of the phage is spontaneous and highly efficient, the enrichment of the selected phages provides a sensitive readout for library screen. 3 The main drawback of the current method for small molecule phage display is that tens to hundreds of molecules are attached to each phage particle due to the nonspecific nature of the conjugation reaction. The high density of the small molecule ligands on a single phage particle may lead to high avidity of the phages with the target protein due to the multivalent binding of the small molecules carried by the same phage. This may give high background binding of low affinity ligands with the target protein during selection. Moreover, phages displaying high affinity ligands with the target protein may be impossible to elute due to the multivalent binding and thus cannot be amplified and identified during the selection process.To circumvent these problems, here we report a strategy for attaching small molecules site specifically to the peptidyl carrier protein (PCP) monovalently displayed on M13 phage in order to achieve one-phage-one-compound display of the small molecules ( Figure 1). PCPs are 10 kDa stably folded domains excised from nonribosomal peptide synthetases (NRPSs). 5 Sfp phosphopantetheinyl transferase 6a was used to covalently link the small molecules to a specific serine residue of the PCP through a phosphopantetheinyl group using various coenzyme A (CoA)-small molecule conjugates as substrates (1-5). 7 We have shown that the selection of the small molecules monovalently displayed on the phage is highly efficient; typically, a single round of selection gives more than 2000-fold enrichment of the ligands with nM dissociation c...

scriptomic and proteomic analysis of global ischemia and cardioprotection in the rabbit heart. Physiol Genomics 38: 125-137, 2009. First published May 19, 2009 doi:10.1152/physiolgenomics.00033.2009Cardioplegia is used to partially alleviate the effects of surgically induced global ischemia injury; however, the molecular mechanisms involved in this cardioprotection remain to be elucidated. To improve the understanding of the molecular processes modulating the effects of global ischemia and the cardioprotection afforded by cardioplegia, we constructed rabbit heart cDNA libraries and isolated, sequenced, and identified a compendium of nonredundant cDNAs for use in transcriptomic and proteomic analyses. New Zealand White rabbits were used to compare the effects of global ischemia and cardioplegia compared with control (nonischemic) hearts. The effects of RNA and protein synthesis on the cardioprotection afforded by cardioplegia were investigated separately by preperfusion with either ␣-amanitin or cycloheximide. Our results demonstrate that cardioplegia partially ameliorates the effects of global ischemia and that the cardioprotection is modulated by RNA-and protein-dependent mechanisms. Transcriptomic and proteomic enrichment analyses indicated that global ischemia downregulated genes/proteins associated with mitochondrial function and energy production, cofactor catabolism, and the generation of precursor metabolites of energy. In contrast, cardioplegia significantly increased differentially expressed genes/proteins associated with the mitochondrion and mitochondrial function and significantly upregulated the biological processes of muscle contraction, involuntary muscle contraction, carboxylic acid and fatty acid catabolic processes, fatty acid ␤-oxidation, and fatty acid metabolic processes. mitochondrion; ischemia-reperfusion MYOCARDIAL ISCHEMIA-REPERFUSION INJURY occurs as the result of the attenuation or cessation of coronary blood flow such that oxygen delivery to the myocardium is insufficient to meet energy demands. The cessation of myocardial coronary blood flow induces a cascade of cellular events that rapidly alter myocardial cellular homeostasis, leading to cellular dysfunction and/or death and postischemic functional impairment (23).To alleviate the effects of surgically induced ischemia/ reperfusion injury, surgeons use cardioplegia (CP) solutions that allow for the rapid electromechanical arrest of the myocardium through the alteration of cellular electrochemical gradients (40). In a series of studies, we have shown that magnesium-supplemented potassium CP with the addition of diazoxide significantly decreases myocardial cell death and significantly enhances postischemic functional recovery (22,40,43).The mechanisms through which CP affords cardioprotection are complex and cannot be investigated as a single entity; rather, they must be investigated as a system. In previous reports, we and others have used a variety of methods to identify the RNAs and proteins associated with global ischemia (GI) a...

Recently we have shown that the cardioprotection afforded by cardioplegia is modulated by age and gender and is significantly decreased in the aged female. In this report we use microarray and proteomic analyses to identify transcriptomic and proteomic alterations affecting cardioprotection using cold blood cardioplegia in the mature and aged male and female heart. Mature and aged male and female New Zealand White rabbits were used for in situ blood perfused cardiopulmonary bypass. Control hearts received 30 min sham ischemia and 120 min sham reperfusion. Global ischemia (GI) hearts received 30 min of GI achieved by cross-clamping of the aorta. Cardioplegia (CP) hearts received cold blood cardioplegia prior to GI. Following 30 min of GI the hearts were reperfused for 120 min and then used for RNA and protein isolation. Microarray and proteomic analyses were performed. Functional enrichment analysis showed that mitochondrial dysfunction, oxidative phosphorylation and calcium signaling pathways were significantly enriched in all experimental groups. Glycolysis/gluconeogenesis and the pentose phosphate pathway were significantly changed in the aged male only (P < 0.05), while glyoxylate/dicarboxylate metabolism was significant in the aged female only (P < 0.05). Our data show that specific pathways associated with the mitochondrion modulate cardioprotection with CP in the aged and specifically in the aged female. The alteration of these pathways significantly contributes to decreased myocardial functional recovery and myonecrosis following ischemia and may be modulated to allow for enhanced cardioprotection in the aged and specifically in the aged female.

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and left ventricular (LV) myocardium differ in their pathophysiological response to pressure-overload hypertrophy. In this report we use microarray and proteomic analyses to identify pathways modulated by LV-aortic banding (AOB) and RV-pulmonary artery banding (PAB) in the immature heart. Newborn New Zealand White rabbits underwent banding of the descending thoracic aorta [LV-AOB; n ϭ 6]. RV-PAB was achieved by banding the pulmonary artery (n ϭ 6). Controls (n ϭ 6 each) were sham-manipulated. After 4 (LV-AOB) and 6 (RV-PAB) wk recovery, the hearts were removed and matched RNA and proteins samples were isolated for microarray and proteomic analysis. Microarray and proteomic data demonstrate that in LV-AOB there is increased transcript expression levels for oxidative phosphorylation, mitochondria energy pathways, actin, ILK, hypoxia, calcium, and protein kinase-A signaling and increased protein expression levels of proteins for cellular macromolecular complex assembly and oxidative phosphorylation. In RV-PAB there is also an increased transcript expression levels for cardiac oxidative phosphorylation but increased protein expression levels for structural constituents of muscle, cardiac muscle tissue development, and calcium handling. These results identify divergent transcript and protein expression profiles in LV-AOB and RV-PAB and provide new insight into the biological basis of ventricular specific hypertrophy. The identification of these pathways should allow for the development of specific therapeutic interventions for targeted treatment and amelioration of LV-AOB and RV-PAB to ameliorate morbidity and mortality.heart; hypertrophy; microarray; proteomics VENTRICULAR OUTFLOW TRACT obstruction in congenital heart defects such as interrupted aortic arch, coarctation of the aorta on the left side, and pulmonary artery stenosis on the right side, causes progressive cardiac hypertrophy and, if unrelieved, leads to ventricular dilatation with contractile dysfunction and ultimately irreversible heart failure. Initially, the myocardium compensates to elevated pressure-loading by increasing wall thickness to normalize wall stress. These changes allow for maintenance of contractile function in left ventricular pressureoverload hypertrophy [LV-aortic banding (AOB)], whereas in right ventricular pressure-overload hypertrophy [RV-pulmonary artery banding (PAB)] these changes are less effective and progression to failure occurs more rapidly (2, 4, 15).Our previous studies using the rabbit model of LV-AOB and RV-PAB have demonstrated that disease progression involves both cellular and extracellular components of the myocardium. We have demonstrated that changes in cardiomyocyte viability, lack of adaptive capillary growth, fibrosis, and response to metabolic stress are differentially regulated in LV-AOB and RV-PAB (11,13,16,20). However, the mechanisms involved in the compensated phase of LV-AOB and RV-PAB have yet to be clearly elucidated. In this report we present microarray and proteomic analyses of the LV and RV during t...