Catestatin Improves Post-Ischemic Left Ventricular Function and Decreases Ischemia/Reperfusion Injury in Heart

The Chromogranin A (CgA)-derived anti-hypertensive peptide catestatin (CST) antagonizes catecholamine secretion, and is a negative myocardial inotrope acting via a nitric oxide-dependent mechanism. It is not known whether CST contributes to ischemia/reperfusion injury or is a component of a cardioprotective response to limit injury. Here, we tested whether CST by virtue of its negative inotropic activity improves post-ischemic cardiac function and cardiomyocyte survival. Three groups of isolated perfused hearts from adult Wistar rats underwent 30-min ischemia and 120-min reperfusion (I/R, Group 1), or were post-conditioned by brief ischemic episodes (PostC, 5-cycles of 10-s I/R at the beginning of 120-min reperfusion, Group 2), or with exogenous CST (75 nM for 20 min, CST-Post, Group-3) at the onset of reperfusion. Perfusion pressure and left ventricular pressure (LVP) were monitored. Infarct size was evaluated with nitroblue-tetrazolium staining. The CST (5 nM) effects were also tested in simulated ischemia/reperfusion experiments on cardiomyocytes isolated from young-adult rats, evaluating cell survival with propidium iodide labeling. Infarct size was 61 ± 6% of risk area in hearts subjected to I/R only. PostC reduced infarct size to 34 ± 5%. Infarct size in CST-Post was 36 ± 3% of risk area (P < 0.05 respect to I/R). CST-Post reduced post-ischemic rise of diastolic LVP, an index of contracture, and significantly improved post-ischemic recovery of developed LVP. In isolated cardiomyocytes, CST increased the cell viability rate by about 65% after simulated ischemia/reperfusion. These results suggest a novel cardioprotective role for CST, which appears mainly due to a direct reduction of post-ischemic myocardial damages and dysfunction, rather than to an involvement of adrenergic terminals and/or endothelium.     

Catestatin (CgA344-364) stimulates rat mast cell release of histamine in a manner comparable to mastoparan and other cationic charged neuropeptides

Catestatin (bovine CgA(344-364)) is a cationic peptide, which besides reducing catecholamine secretion from chromaffin cells in vitro also acts a potent vasodilator in the rat in vivo. The alleged histamine releasing effect of catestatin was tested in vitro in rat mast cells. The most active domain of catestatin (bovine CgA(344-358): RSMRLSFRARGYGFR) caused concentration-dependent (0.01-5 microM) release of histamine from peritoneal and pleural mast cells. The potency and efficacy of catestatin was higher than for the wasp venom peptide, mastoparan. Only in the pleural cells was neurotensin (NT) more potent than catestatin, mastoparan and substance P (SP), consistent with a receptor-mediated histamine release by neurotensin. Amongst these cationic peptides, substance P was least effective. The acidic CgA peptide (WE-14, bovine CgA (324-337)) neither stimulated nor modulated histamine release by the cationic peptides. The catestatin and neurotensin evoked histamine release were suppressed by pertussis toxin (PTX), suggesting involvement of a G(i) subunit. Electron micrographs of rat pleural mast cells responding to catestatin revealed a concentration-dependent discharge of granular material. We propose that catestatin activates histamine release from rat mast cells by a mechanism analogous to that already established for mastoparan and other amphiphilic cationic neuropeptides (the peptidergic pathway) and distinct from the mechanism of inhibition of catecholamine release from chromaffin cells.     

Primary sequence characterization of catestatin intermediates and peptides defines proteolytic cleavage sites utilized for converting chromogranin a into active catestatin secreted from neuroendocrine chromaffin cells

Catestatin is an active 21-residue peptide derived from the chromogranin A (CgA) precursor, and catestatin is secreted from neuroendocrine chromaffin cells as an autocrine regulator of nicotine-stimulated catecholamine release. The goal of this study was to characterize the primary sequences of high molecular mass catestatin intermediates and peptides to define the proteolytic cleavage sites within CgA that are utilized in the biosynthesis of catestatin. Catestatin-containing polypeptides, demonstrated by anti-catestatin western blots, of 54-56, 50, 32, and 17 kDa contained NH(2)-terminal peptide sequences that indicated proteolytic cleavages of the CgA precursor at KK downward arrow, KR downward arrow, R downward arrow, and KR downward arrow basic residue sites, respectively. The COOH termini of these catestatin intermediates were defined by the presence of the COOH-terminal tryptic peptide of the CgA precursor, corresponding to residues 421-430, which was identified by MALDI-TOF mass spectrometry. Results also demonstrated the presence of 54-56 and 50 kDa catestatin intermediates that contain the NH(2) terminus of CgA. Secretion of catestatin intermediates from chromaffin cells was accompanied by the cosecretion of catestatin (CgA(344)(-)(364)) and variant peptide forms (CgA(343)(-)(368) and CgA(332)(-)(361)). These determined cleavage sites predicted that production of high molecular mass catestatin intermediates requires cleavage at the COOH-terminal sides of paired basic residues, which is compatible with the cleavage specificities of PC1 and PC2 prohormone convertases. However, it is notable that production of catestatin itself (CgA(344)(-)(364)) utilizes more unusual cleavage sites at the NH(2)-terminal sides of downward arrow R and downward arrow RR basic residue sites, consistent with the cleavage specificities of the chromaffin granule cysteine protease "PTP" that participates in proenkephalin processing. These findings demonstrate that production of catestatin involves cleavage of CgA at paired basic and monobasic residues, necessary steps for catestatin peptide regulation of nicotinic cholinergic-induced catecholamine release.     

High affinity DNA-microtubule interactions: evidence for a conserved DNA-MAP interaction involving unusual high CsCl density repetitious DNA families

We have examined high affinity interactions of chick brain microtubule proteins with ³⁵S labelled tracer DNAs from chick, mouse and D. melanogaster under equilibrium conditions by the nitrocellulose filter binding technique. Ternary reaction mixtures of the above two components and a third component, an excess of unlabelled competitor DNA from either E. coli., mouse, D. melanogaster or chick, were used to measure small fractions of DNA in each case (1–4%) bound to microtubule protein under high stringency- large competitor DNA concentration and 0.5 M NaCl. As seen in part previously (Marx, K.A. and Denial, T. (1985) in The Molecular Basis of Cancer, 172B, 65–75 (Rein, ed), A. Liss, N.Y.) the measured order of competitor DNA strengths was identical for all three tracer DNAs. That is: chick > mouse > D. melanogaster > E. coli competitor DNA. Since the homologous interaction, chick competitor DNA with chick brain microtubule protein, is always the strongest interaction measured, we interpret this as evidence for a conserved protein-DNA sequence interaction. ³⁵S chick DNA tracer sequences, isolated from nitrocellulose filters following the stringent binding in the presence of 0.9 mM^–1 E. coli. competitor DNA, was used in driven reassociation reactions with total chick driver DNA. This fraction was found to be significantly enriched in repetitive chick DNA sequences. Since we have observed a similar phenomenon in mouse, we then compared the stringent binding mouse sequences and showed that the bulk of these sequences did not cross-hybridize with total chick DNA. Finally, all three ³⁵S tracer DNAs binding to nitrocellulose were isolated and sedimented to equilibrium on CsCl density gradients. The CsCl density distributions from all three DNAs showed significant (100-fold) enrichment in classical satellite DNAs as well as higher enrichment in two very unusual high CsCl density families of DNA (1.720–1.740 g/cm³; 1.750–1.765 g/cm³). These families are never observed as distinct bands in total DNA CsCl gradients, nor could we isolate them in purified tubulin control binding experiments. This apparently general phenomena may be identifying some of the sequence families involved in the high affinity microtubule interaction, which appears to be conserved in evolution.     

Interaction of the catecholamine release-inhibitory peptide catestatin (human chromogranin A(352-372)) with the chromaffin cell surface and Torpedo electroplax: implications for nicotinic cholinergic antagonism

The catecholamine release-inhibitory chromogranin A fragment catestatin (chromogranin A(344-364)) exhibits non-competitive antagonism of nicotinic cholinergic signaling in chromaffin cells. A previous homology model of catestatin's likely structure suggested a mode of interaction of the peptide with the nicotinic receptor, but direct evidence has been lacking. Here we found that [125I]-catestatin binds to the surface of intact PC12 and bovine chromaffin cells with high affinity (K(D)=15.2+/-1.53 nM) and specificity (lack of displacement by another [N-terminal] fragment of chromogranin A). Nicotinic agonist (carbamylcholine) did not displace [125I]-catestatin from chromaffin cells, nor did catestatin displace the nicotinic agonist [3H]-epibatidine; these observations indicate a catestatin binding site separate from the agonist binding pocket on the nicotinic receptor, a finding consistent with catestatin's non-competitive nicotinic mechanism. [125I]-catestatin could be displaced from chromaffin cells by substance P (IC(50) approximately 5 microM), though at far lower potency than displacement by catestatin itself (IC(50) approximately 350-380 nM), suggesting that catestatin and substance P occupy an identical or overlapping non-competitive site on the nicotinic receptor, at different affinities (catestatin > substance P). Small, non-peptide non-competitive nicotinic antagonists (hexamethonium or clonidine) did not diminish [125I]-catestatin binding, suggesting distinct non-competitive binding sites on the nicotinic receptor for peptide and non-peptide antagonists. Similar binding and inhibitory profiles for [125I]-catestatin were observed on chromaffin cells as well as nicotinic receptor-enriched Torpedo membranes. Covalent cross-linking of [125I]-catestatin to Torpedo membranes suggested specific contacts of [125I]-catestatin with the delta, gamma, and beta subunits of the nicotinic receptor, a finding consistent with prior homology modeling of the interaction of catestatin with the extracellular face of the nicotinic heteropentamer. We conclude that catestatin occludes the nicotinic cation pore by interacting with multiple nicotinic subunits at the pore vestibule. Such binding provides a physical explanation for non-competitive antagonism of the peptide at the nicotinic receptor.     

Correction of translational defects in patient-derived mutant mitochondria by complex-mediated import of a cytoplasmic tRNA

A variety of clinical disorders result from mutations in mitochondrial tRNA genes, leading to translational defects. We show here that a protein complex from the kinetoplastid protozoon Leishmania induces specific, ATP-dependent import of human cytoplasmic tRNA(1)(Lys) into human mitochondria in vitro. The imported tRNA undergoes efficient aminoacylation within the organelle and supports organellar protein synthesis. Moreover, translation in mitochondria from patients with myclonic epilepsy with ragged red fibers (MERRF) and Kearns-Sayre syndrome (KSS), containing mutant tRNA(Lys) genes, is stimulated to near-wild-type levels and the formation of aberrant polypeptides suppressed by complex-mediated import. These results suggest a novel way to introduce RNAs for the modulation of mitochondrial gene expression.     

Minocycline suppresses oxidative stress and attenuates fetal cardiac myocyte apoptosis triggered by in utero cocaine exposure

This study investigates the molecular mechanisms by which minocycline, a second generation tetracycline, prevents cardiac myocyte death induced by in utero cocaine exposure. Timed mated pregnant Sprague-Dawley (SD) rats received one of the following treatments twice daily from embryonic (E) day 15–21 (E15–E21): (i) intraperitoneal (IP) injections of saline (control); (ii) IP injections of cocaine (15 mg/kg BW); and (iii) IP injections of cocaine + oral administration of 25 mg/kg BW of minocycline. Pups were killed on postnatal day 15 (P15). Additional pregnant dams received twice daily IP injections of cocaine (from E15–E21) + oral administration of a relatively higher (37.5 mg/kg BW) dose of minocycline. Minocycline treatment continued from E15 until the pups were sacrificed on P15. In utero cocaine exposure resulted in an increase in oxidative stress and fetal cardiac myocyte apoptosis through activation of c-Jun-NH₂-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK)-mediated mitochondria-dependent apoptotic pathway. Continued minocycline treatment from E15 through P15 significantly prevented oxidative stress, kinase activation, perturbation of BAX/BCL-2 ratio, cytochrome c release, caspase activation, and attenuated fetal cardiac myocyte apoptosis after prenatal cocaine exposure. These results demonstrate in vivo cardioprotective effects of minocycline in preventing fetal cardiac myocyte death after prenatal cocaine exposure. Given its proven clinical safety and ability to cross the placental barrier and enter into the fetal circulation, minocycline may be an effective therapy for preventing cardiac consequences of in utero cocaine exposure.     

Glucose Directly Promotes Antifungal Resistance in the Fungal Pathogen,Candida spp.

Effects of glucose on the susceptibility of antifungal agents were investigated against Candida spp. Increasing the concentration of glucose decreased the activity of antifungal agents; voriconazole was the most affected drugs followed by amphotericin B. No significant change has been observed for anidulafungin. Biophysical interactions between antifungal agents with glucose molecules were investigated using isothermal titration calorimetry, Fourier transform infrared, and ¹H NMR. Glucose has a higher affinity to bind with voriconazole by hydrogen bonding and decrease the susceptibility of antifungal agents during chemotherapy. In addition to confirming the results observed in vitro, theoretical docking studies demonstrated that voriconazole presented three important hydrogen bonds and amphotericin B presented two hydrogen bonds that stabilized the glucose. In vivo results also suggest that the physiologically relevant higher glucose level in the bloodstream of diabetes mellitus mice might interact with the available selective agents during antifungal therapy, thus decreasing glucose activity by complex formation. Thus, proper selection of drugs for diabetes mellitus patients is important to control infectious diseases.