Fragmentation and Matching of Human MicroRNA Sequences in 3'utr.

AIMS: Definition of sense and antisense microRNA matches in 3'utr. BACKGROUND: Matches of mature microRNAs (m-miRs) in human 3'utr could be traced to mutations producing fragments of original m-miR sequences without physical separation. (The m-miR matches in 5'utr and cds should be by far fewer, but could follow similar patterns). OBJECTIVE: To ascertain if the sense and antisense m-miR fragments in 3'utr occur at similar or different levels. METHODS: Frequency of sense and antisense m-miR matches in 3'utr was examined in the range of 7-22 nucleotides. RESULTS: The fragmentation occurs at gene level by mutation within one of the paired m-miRs, which upon transcription results in increased interactive capability for both former pre-micro (premir) RNA stem partners. The non-mutated stem partner can persist in 3'utr sequences, as is apparent from significant presence of miR-619-5p and miR-5096 and some conservation of 20 other simian- specific m-miR sequences. However, most of m-mir sequences in 3'utr are extensively fragmented, with low preservation of long matches. In flanks of individual m-miR embeds the mutated pre-mir positions are to a degree defined specifically. CONCLUSION: The m-mir matches of various sizes in 3'utr apparently reflect accumulation, on a phylogenetic time scale, of in-sequence point mutations. Across human 3'utr this fragmentation is significantly less for evolutionarily recent human m-miRs that originate in simians compared to human m-miRs first appearing in lower primates, and especially to human m-miRs introduced in nonprimates.

Phosphodiesterase 4B knockout prevents skeletal muscle atrophy in rats with burn injury.

The phosphodiesterase 4 (PDE4)-cAMP pathway plays a predominant role in mediating skeletal muscle proteolysis in burn injury. The present investigations to determine the PDE4 isoform(s) involved in this action revealed that burn injury increased the expression of rat skeletal muscle PDE4B mRNA by sixfold but had little or no effect on expression of other PDE4 isoforms. These observations led us to study the effects of burn in PDE4B knockout (KO) rats. As reported by us previously, burn injury significantly increased extensor digitorum longus (EDL) muscle total and myofibrillar proteolysis in wild-type (WT) rats, but there were no significant effects on either total or myofibrillar protein breakdown in EDL muscle of PDE4B KO rats with burn injury. Moreover, burn injury increased PDE4 activity in the skeletal muscle of WT rats, but this was reduced by >80% in PDE4B KO rats. Also, burn injury decreased skeletal muscle cAMP concentration in WT rats but had no significant effects in the muscles of PDE4B KO rats. Incubation of the EDL muscle of burn-PDE4B KO rats with an inhibitor of the exchange factor directly activated by cAMP, but not with a protein kinase A inhibitor, eliminated the protective effects of PDE4B KO on EDL muscle proteolysis and increased muscle proteolysis to the same extent as in the EDL of burn-WT rats. These novel findings confirm a major role for PDE4B in skeletal muscle proteolysis in burn injury and suggest that an innovative therapy based on PDE4B-selective inhibitors could be developed to treat skeletal muscle cachexia in burn injury without the fear of causing emesis, which is associated with PDE4D inhibition.

The Expansion Segments of 28S Ribosomal RNA Extensively Match Human Messenger RNAs.

Eukaryote ribosomal RNAs (rRNAs) have expanded in the course of phylogeny by addition of nucleotides in specific insertion areas, the expansion segments. These number about 40 in the larger (25-28S) rRNA (up to 2,400 nucleotides), and about 12 in the smaller (18S) rRNA (<700 nucleotides). Expansion of the larger rRNA shows a clear phylogenetic increase, with a dramatic rise in mammals and especially in hominids. Substantial portions of expansion segments in this RNA are not bound to ribosomal proteins, and may engage extraneous interactants, including messenger RNAs (mRNAs). Studies on the ribosome-mRNA interaction have focused on proteins of the smaller ribosomal subunit, with some examination of 18S rRNA. However, the expansion segments of human 28S rRNA show much higher density and numbers of mRNA matches than those of 18S rRNA, and also a higher density and match numbers than its own core parts. We have studied that with frequent and potentially stable matches containing 7-15 nucleotides. The expansion segments of 28S rRNA average more than 50 matches per mRNA even assuming only 5% of their sequence as available for such interaction. Large expansion segments 7, 15, and 27 of 28S rRNA also have copious long (≥10-nucleotide) matches to most human mRNAs, with frequencies much higher than in other 28S rRNA parts. Expansion segments 7 and 27 and especially segment 15 of 28S rRNA show large size increase in mammals compared to other metazoans, which could reflect a gain of function related to interaction with non-ribosomal partners. The 28S rRNA expansion segment 15 shows very high increments in size, guanosine, and cytidine nucleotide content and mRNA matching in mammals, and especially in hominids. With these segments (but not with other 28S rRNA or any 18S rRNA expansion segments) the density and number of matches are much higher in 5'-terminal than in 3'-terminal untranslated mRNA regions, which may relate to mRNA mobilization via 5' termini. Matches in the expansion segments 7, 15, and 27 of human 28S rRNA appear as candidates for general interaction with mRNAs, especially those associated with intracellular matrices such as the endoplasmic reticulum.

The Expansion Segments of Human 28S rRNA Match MicroRNAs Much Above 18S rRNA or Core Segments.

BACKGROUND: The size of eukaryotic 25-28S rRNAs shows a progressive phylogenetically linked increase which is pronounced in mammals, and especially in hominids. The increase is confined to specific expansion segments, inserted at points that are highly conserved from yeast to man. These segments also show a progressive increase in nucleotide bias, mostly the GC bias. Substantial parts of the large expansion segments 7, 15 and 27 of 28S rRNA are known to be exposed at the ribosome surface, with no clear association with ribosomal proteins. These segments could bind extraneous RNAs and proteins to support regulatory events. METHODS: This study examined the possible canonical matching of human 28S rRNA and 18S rRNA segments with 2586 human microRNAs. This was compared with matching of the microRNAs to sectors of 18810 human mRNAs. RESULTS: The overall matching was rather similar across 18S rRNA segments and core segments of 28S rRNA. However, the expansion segments of 28S rRNA (abbreviated ESL) collectively have a much higher (up to two-fold) capacity for the canonical association with microRNAs. This is pronounced in large ESL, and is found to strongly relate to the GC content of microRNAs. CONCLUSION: Oligonucleotides and microRNAs of high GC content through a strong canonical hydrogen bonding could have large activity in regulation of subcellular RNAs. In view of the considerable abundance of ribosomal RNAs in many mammalian tissues, ESL could constitute an important component of microRNA balance, possibly serving to lower the availability of GC-rich microRNAs (and thereby help conservation of GC-rich mRNAs).

Canonical microRNA Matching Differs Greatly Across Groups of G-protein Coupled Receptor mRNAs.

BACKGROUND: Heptahelical G protein coupled receptors (GPCRs) support numerous sensory and metabolic functions and differ considerably in levels of expression. GPCR protein levels should link to regulation of GPCR mRNAs by microRNAs (miRs), which might significantly depend on numbers, size and GC content of the canonical antisense matches in mRNAs. These parameters of GPCR mRNAs have not been studied in detail. METHODS: Canonical matching profiles of human GPCR mRNAs and miRs were examined using segments of 7-15 nucleotides in windows shifted by one position over the entire microRNA sequence. RESULTS: Human GPCRs mRNAs within larger function-related groups have a quite homogenous matching with miRs. Both the GC content and the melting temperature (and hence also the binding energy) are appreciably higher in 5'utr compared to 3'utr matches of the same length. Increase in the GC content correlates significantly with length in the ubiquitous matches of 7-12 nucleotides. However, several GPCR groups strongly differ in overall match numbers and density. The untranslated regions of sensory receptor mRNAs, especially the olfactory and Taste-2 mRNAs, have the lowest match numbers and density and the fewest miR partners. The glucagon and frizzled families show the highest canonical matching. CONCLUSION: Partnership of GPCR mRNAs and miRs could significantly relate to the type of function of the receptor proteins, with mRNAs of the sensory receptors having the lowest and those of metabotropic GPCRs the highest targeting. This could be of interest regarding GPCR regulation by exogenous miRs.

Neuropeptide FF receptors as novel targets for limbic seizure attenuation.

Neuropeptide Y (NPY) is a well established anticonvulsant and first-in-class antiepileptic neuropeptide. In this study, the controversial role of NPY1 receptors in epilepsy was reassessed by testing two highly selective NPY1 receptor ligands and a mixed NPY1/NPFF receptor antagonist BIBP3226 in a rat model for limbic seizures. While BIBP3226 significantly attenuated the pilocarpine-induced seizures, neither of the highly selective NPY1 receptor ligands altered the seizure severity. Administration of the NPFF1/NPFF2 receptor antagonist RF9 also significantly attenuated limbic seizure activity. To further prove the involvement of NPFF receptors in these seizure-modulating effects, low and high affinity antagonists for the NPFF receptors were tested. We observed that the low affinity ligand failed to exhibit anticonvulsant properties while the two high affinity ligands significantly attenuated the seizures. Continuous NPFF1 receptor agonist administration also inhibited limbic seizures whereas bolus administration of the NPFF1 receptor agonist was without effect. This suggests that continuous agonist perfusion could result in NPFF1 receptor desensitization and mimic NPFF1 receptor antagonist administration. Our data unveil for the first time the involvement of the NPFF system in the management of limbic seizures.

Des-acyl-ghrelin (DAG) normalizes hyperlactacidemia and improves survival in a lethal rat model of burn trauma.

Critical illness, including burn injury, results in elevated plasma lactate levels. Dysregulation of PI3K/Akt signaling has been shown to play a predominant role in the inactivation of skeletal muscle PDC and, hence, in hyperlactacidemia in rat models of sepsis and endotoxemia. This observation, and our previous finding that DAG can reverse burn-induced skeletal muscle proteolysis through the activation of PI3K/Akt pathway, led us to hypothesize that DAG may also attenuate hyperlactacidemia in burn injury. Our investigations revealed that burn injury significantly elevated both skeletal muscle lactate production and plasma lactate levels. Moreover, this was accompanied in skeletal muscle by a 5-7 fold increase in mRNA expression of pyruvate dehydrogenase kinases (PDK) 2 and 4, and a ∼30% reduction in PDC activity. DAG treatment of burn rats completely normalized not only the mRNA expression of the PDKs and PDC activity, but also hyperlactacidemia within 24h of burn injury. DAG also normalized epinephrine-induced lactate production by isolated skeletal muscles from normal rats. Moreover, DAG also improved survival in a lethal rat model of burn trauma. These findings with DAG may have clinical implications because chances of survival for critically ill patients are greatly improved if plasma lactate levels are normalized within 24h of injury.

Phosphodiesterase (PDE) inhibitor torbafylline (HWA 448) attenuates burn-induced rat skeletal muscle proteolysis through the PDE4/cAMP/EPAC/PI3K/Akt pathway.

Treatment of rats after burn-injury with the cyclic AMP phosphodiesterase (PDE) inhibitor, torbafylline (also known as HWA 448) significantly reversed changes in rat skeletal muscle proteolysis, PDE4 activity, cAMP concentrations and mRNA expression of TNFα, IL-6, ubiquitin and E3 ligases. Torbafylline also attenuated muscle proteolysis during in vitro incubation, and this effect was blocked by the inhibitor Rp-cAMPS. Moreover, torbafylline significantly increased phospho-Akt levels, and normalized downregulated phospho-FOXO1 and phospho-4E-BP1 in muscle of burn rats. Similarly, torbafylline also normalized phosphorylation levels of Akt and its downstream elements in TNFα+IFNγ treated C2C12 myotubes. Torbafylline enhanced protein levels of exchange protein directly activated by cAMP (Epac) both in skeletal muscle of burn rats and in TNFα+IFNγ treated C2C12 myotubes. Pretreatment with a specific antagonist of PI3K or Epac significantly reversed the inhibitory effects of torbafylline on TNFα+IFNγ-induced MAFbx mRNA expression and protein breakdown in C2C12 myotubes. Torbafylline inhibits burn-induced muscle proteolysis by activating multiple pathways through PDE4/cAMP/Epac/PI3K/Akt.

Dimers of G-protein coupled receptors as versatile storage and response units.

The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs) are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αßγ subunit heterotrimer. With neuropeptide Y (NPY) receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY) receptors and could apply to many receptors that use large peptidic agonists.

On the expansion of ribosomal proteins and RNAs in eukaryotes.

While the ribosome constitution is similar in all biota, there is a considerable increase in size of both ribosomal proteins (RPs) and RNAs in eukaryotes as compared to archaea and bacteria. This is pronounced in the large (60S) ribosomal subunit (LSU). In addition to enlargement (apparently maximized already in lower eukarya), the RP changes include increases in fraction, segregation and clustering of basic residues, and decrease in hydrophobicity. The acidic fraction is lower in eukaryote as compared to prokaryote RPs. In all eukaryote groups tested, the LSU RPs have significantly higher content of basic residues and homobasic segments than the SSU RPs. The vertebrate LSU RPs have much higher sequestration of basic residues than those of bacteria, archaea and even of the lower eukarya. The basic clusters are highly aligned in the vertebrate, but less in the lower eukarya, and only within families in archaea and bacteria. Increase in the basicity of RPs, besides helping transport to the nucleus, should promote stability of the assembled ribosome as well as the association with translocons and other intracellular matrix proteins. The size and GC nucleotide bias of the expansion segments of large LSU rRNAs also culminate in the vertebrate, and should support ribosome association with the endoplasmic reticulum and other intracellular networks. However, the expansion and nucleotide bias of eukaryote LSU rRNAs do not clearly correlate with changes in ionic parameters of LSU ribosomal proteins.

On the segregation of protein ionic residues by charge type.

Based on ubiquitous presence of large ionic motifs and clusters in proteins involved in gene transcription and protein synthesis, we analyzed the distribution of ionizable sidechains in a broad selection of proteins with regulatory, metabolic, structural and adhesive functions, in agonist, antagonist, toxin and antimicrobial peptides, and in self-excising inteins and intron-derived proteins and sequence constructs. All tested groups, regardless of taxa or sequence size, show considerable segregation of ionizable sidechains into same type charge (homoionic) tracts. These segments in most cases exceed half of the sequence length and comprise more than two-thirds of all ionizable sidechains. This distribution of ionic residues apparently reflects a fundamental advantage of sorted electrostatic contacts in association of sequence elements within and between polypeptides, as well as in interaction with polynucleotides. While large ionic densities are encountered in highly interactive proteins, the average ionic density in most sets does not change appreciably with size of the homoionic segments, which supports the segregation as a modular feature favoring association.

Neuropeptide Y increases in vivo hippocampal extracellular glutamate levels through Y1 receptor activation.

Neuropeptide Y's (NPY) anticonvulsant effect is generally attributed to its inhibitory effect on glutamate release from presynaptic nerve terminals, which is nicely demonstrated in in vitro settings. To date no study has attempted to investigate the effect of NPY in vivo on extracellular (EC) glutamate levels thus, via intracerebral microdialysis, we determined NPY's effect on hippocampal glutamate concentrations in vivo, and consequently the involvement of Y(1) receptors to this effect. NPY or the Y(1) agonist D-His26-NPY was intrahippocampally administered in rats for 2h, during which the hippocampal glutamate dialysate levels were monitored. Pilocarpine was subsequently co-administered with NPY or D-His26-NPY to determine their effect on pilocarpine-induced limbic seizures. Unexpectedly we noted that intrahippocampal administration of NPY or D-His26-NPY increased glutamate dialysate levels in a reproducible manner. NPY attenuated pilocarpine induced seizures, whereas D-His26-NPY did not. To clarify the role of Y(1) receptors in NPY's glutamatergic effect, NPY was co-administered with the selective Y(1) antagonist BVD10. Hippocampal Y(1) receptor blockade prevented the NPY-induced increase in hippocampal glutamate, proving that this induced glutamate increase is clearly Y(1) receptor mediated. This is the first evidence that NPY enhances hippocampal EC glutamate overflow in vivo via hippocampal Y(1) receptors without interfering with or contributing to NPY's anticonvulsant effect. Whilst this finding contrasts with the supposed glutamatergic hypothesis for NPY in the hippocampus, it is of significance to further assist in deciphering NPY's mechanisms of action in in vivo settings.

Des-acyl ghrelin exhibits pro-anabolic and anti-catabolic effects on C2C12 myotubes exposed to cytokines and reduces burn-induced muscle proteolysis in rats.

Although ghrelin and GHRP-2 have been shown to inhibit skeletal muscle proteolysis in rats with burn injury, the effects of des-acyl ghrelin (DAG) have not been reported. In this paper, we demonstrate that continuous 24h administration of DAG attenuated burn-induced EDL muscle proteolysis, and normalized elevated TNFα mRNA. Combined treatment of cultured C2C12 myotubes with TNFα and IFN-γ (TNF+IFN) inhibited protein synthesis and increased protein breakdown; DAG abolished both effects. PI3 kinase inhibition by LY294002 and mTOR inhibition by rapamycin blocked the reversal of the anti-anabolic effects of TNF+IFN-treated myotubes by DAG. DAG also reversed or attenuated the TNF+IFN-induced reduction in phosphorylation of Akt, FOXO1, 4E-BP-1, and GSK-3ß in myotubes. Furthermore, DAG attenuated the atrophy signal, phospho-NF-κB, and the mRNA expression of MAFbx and MuRF1, upregulated by TNF+IFN in C2C12 myotubes. We conclude that DAG reduces muscle cachexia produced by injury and proinflammatory cytokines, and that DAG or DAG-based compounds may be useful in treating wasting disorders.

HMGA-targeted phosphorothioate DNA aptamers increase sensitivity to gemcitabine chemotherapy in human pancreatic cancer cell lines.

Elevated high mobility group A (HMGA) protein expression in pancreatic cancer cells is correlated with resistance to the chemotherapy agent gemcitabine. Here, we demonstrate use of HMGA-targeted AT-rich phosphorothioate DNA (AT-sDNA) aptamers to suppress HMGA carcinogenic activity. Cell growth of human pancreatic cancer cells (AsPC-1 and Miapaca-2) transfected with AT-sDNA were monitored after treatment with gemcitabine. Significant increases in cell death in AT-sDNA transfected cells compared to non-AT-rich sDNA treated cells were observed in both cell lines. The data indicate the potential use of HMGA targeted DNA aptamers to enhance chemotherapy efficacy in pancreatic cancer treatment.

Metabolic Profiling Comparison of Human Pancreatic Ductal Epithelial Cells and Three Pancreatic Cancer Cell Lines using NMR Based Metabonomics.

Metabolic profiles of hydrophilic and lipophilic cell extracts from three cancer cell lines, Miapaca-2, Panc-1 and AsPC-1, and a non-cancerous pancreatic ductal epithelial cell line, H6C7, were examined by proton nuclear magnetic resonance spectroscopy. Over twenty five hydrophilic metabolites were identified by principal component and statistical significance analyses as distinguishing the four cell types. Fifteen metabolites were identified with significantly altered concentrations in all cancer cells, e.g. absence of phosphatidylgrycerol and phosphatidylcholine, and increased phosphatidylethanolamine and cholesterols. Altered concentrations of metabolites involved in glycerophospholipid metabolism, lipopolysaccharide and fatty acid biosynthesis indicated differences in cellular membrane composition between non-cancerous and cancer cells. In addition to cancer specific metabolites, several metabolite changes were unique to each cancer cell line. Increased N-acetyl groups in AsPC-1, octanoic acids in Panc-1, and UDP species in Miapaca-2 indicated differences in cellular membrane composition between the cancer cell lines. Induced glutamine metabolism and protein synthesis in cancer cells were indicated by absence of glutamine other metabolites such as acetate, lactate, serine, branched amino acids, and succinate. Knowledge of the specifically altered metabolic pathways identified in these pancreatic cancer cell lines may be useful for identifying new therapeutic targets and studying the effects of potential new therapeutic drugs.

Maintenance of Y receptor dimers in epithelial cells depends on interaction with G-protein heterotrimers.

Treatment of CHO cells expressing human Y receptors (Y(1), Y(2) or Y4 subtype) with pertussis toxin results in a large decrease in functional receptors, with a preferential loss of heteropentameric assemblies of receptor dimers and G-protein trimers. This occurs in parallel to inactivation of the nucleotide site of Gi α subunits, with a half period of about 4 h. The loss could be mainly due to proteolysis at the level of recycling/perinuclear endosomes, and of receptor completion in the ER, since it is reduced by co-treatment with ammonium chloride, an inhibitor of particulate proteinases. Antagonists do not strongly decrease the heteropentameric fraction. These findings indicate that the upkeep of Y receptor dimers in epithelial cell lines depends on the association of receptor oligomers with functional Gi α subunits. This interaction could use the juxtamembrane helix 8 in the fourth intracellular domain, and could also be supported by the C-terminal helix of the third intracellular loop, as outlined in the companion review (Parker et al., Amino Acids, doi: 10.1007/s00726-010-0616-1 , 2010).

Regulatory effects of Y4 receptor agonist (BVD-74D) on food intake.

The objective of this study was to clarify the role of a novel agonist with high selectivity and affinity for Y4 receptors in the regulation of food intake. The Y4 receptor agonist BVD-74D was administered to C57BL/6J mice that were fed with a normal or high-fat diet, and to fatty liver Shionogi (FLS)-ob/ob mice; the food intake, water intake, and body weight gain were measured in these mice. In the mice fed with a normal diet, the cumulative food intake significantly decreased at 20 min and 1 h after the administration of 1 mg/kg of BVD-74D and at 1, 2, 4, 8, and 24 h after the administration of 10 mg/kg of BVD-74D. Moreover, the cumulative water intake and body weight gain significantly decreased in these mice. Among the mice that were fed with a high-fat diet, the cumulative food intake and water intake significantly decreased 1, 2, and 4 h after BVD-74D (10 mg/kg) administration. Furthermore, the cumulative food intake significantly decreased 2 and 4 h after BVD-74D (10 mg/kg) administration in the FLS-ob/ob mice. Thus, we propose that the novel Y4 receptor agonist BVD-74D has suppressive effects on food intake, water intake, and weight gain in normal mice fed with normal diets and on food intake in normal mice fed with high-fat diets and in FLS-ob/ob mice. These findings indicate that the Y4 receptor and its agonist would be promising targets for obesity.

Neuropeptide Y Y5 receptor promotes cell growth through extracellular signal-regulated kinase signaling and cyclic AMP inhibition in a human breast cancer cell line.

Overexpression of neuropeptide Y (NPY) and its receptor system has been reported in various types of cancers. NPY Y5 receptor (Y5R) has been implicated in cell growth and angiogenesis. However, the role of Y5R in breast cancer is unknown. To identify the role of Y5R in breast cancer, we screened several breast cancer cell lines to examine the expression of Y5R and its function in breast cancer. All screened cell lines express both Y1 receptor and Y5R except BT-549, which expresses mainly Y5R. Binding studies showed that NPY, Y5R-selective agonist peptide, and Y5R-selective antagonist (CGP71683A) displaced (125)I-PYY binding in BT-549 cell membranes in a dose-dependent manner. The displacement studies revealed the presence of two binding sites in Y5R with IC(50) values of 29 pmol/L and 531 nmol/L. NPY inhibited forskolin-stimulated cyclic AMP accumulation with an IC(50) value of 52 pmol/L. NPY treatment of BT-549 cells induced extracellular signal-regulated kinase phosphorylation but did not alter intracellular calcium. Y5R activation stimulates BT-549 cell growth, which is inhibited by CGP71683A, pertussis toxin, and extracellular signal-regulated kinase blockade. CGP71683A alone induced cell death in a time- and dose-dependent manner in Y5R-expressing cells. The stimulation of MDA MB-231 cell migration by NPY is inhibited by CGP71683A. Together, our results suggest that Y5R plays an important role in cancer cell growth and migration and could be a novel therapeutic target for breast cancer.

Combining neuropeptide Y and mesenchymal stem cells reverses remodeling after myocardial infarction.

Neuropeptide Y (NPY) induced reentry of differentiated rat neonatal and adult cardiomyocytes into the cell cycle. NPY also induced differentiation of bone marrow-derived mesenchymal stem cells (MSC) into cardiomyocytes following transplantation into infarcted myocardium. Rat neonatal and adult cardiomyocytes were treated in vitro with vehicle, NPY, fibroblast growth factor (FGF; 100 ng/ml), or FGF plus NPY. DNA synthesis, mitosis, and cytokinesis were determined by immunocytochemistry. NPY-induced MSC gene expression, cell migration, tube formation, and endothelial cell differentiation were analyzed. Male rat green fluorescent protein-MSC (2 x 10(6)), pretreated with either vehicle or NPY (10(-8) M) for 72 h, were injected into the border zone of the female myocardium following left anterior descending artery ligation. On day 30, heart function was assessed, and hearts were harvested for histological and immunohistochemical analyses. NPY increased 5-bromo-2'-deoxy-uridine incorporation and promoted both cytokinesis and mitosis in rat neonatal and adult myocytes. NPY also upregulated several genes required for mitosis in MSC, including aurora B kinase, FGF-2, cycline A2, eukaryotic initiation factor 4 E, and stromal cell-derived factor-1alpha. NPY directly induced neonatal and adult cardiomyocyte cell-cycle reentry and enhanced the number of differentiated cardiomyocytes from MSC in the infarcted myocardium, which corresponded to improved cardiac function, reduced fibrosis, ventricular remodeling, and increased angiomyogenesis. It is concluded that a combined treatment of NPY with MSC is a novel approach for cardiac repair.

Ghrelin receptor agonist, GHRP-2, attenuates burn injury-induced MuRF-1 and MAFbx expression and muscle proteolysis in rats.

Thermal injury results in hypermetabolism, loss of body weight, and skeletal muscle wasting in mice and rats. Our earlier studies have demonstrated that ghrelin injection stimulates food intake and growth hormone release and inhibits skeletal muscle proteolysis in rats with thermal injury. We sought to develop a lower molecular weight, stable and longer acting peptide to combat the catabolic responses caused by thermal injury. Towards this goal, we examined the role of the hexapeptide mimetic of ghrelin, growth hormone-releasing peptide-2 (GHRP-2), on expression of E3 ubiquitin ligases and breakdown of muscle protein in rats with thermal injury. Slow in vivo release of GHRP-2 through minipump for 24h attenuated the thermal injury-induced increase in mRNA expression of IL-6 and of the E3 ubiquitin ligases, MuRF-1 and MAFbx, in rat skeletal muscle. Furthermore, burn-induced increases in total and myofibrillar protein breakdown from rat EDL muscle were attenuated by GHRP-2. These findings suggest that catabolic responses resulting from thermal injury can be attenuated by GHRP-2.