Post-translational modification by acetylation regulates the mitochondrial carnitine/acylcarnitine transport protein

The carnitine/acylcarnitine transporter (CACT; SLC25A20) mediates an antiport reaction allowing entry of acyl moieties in the form of acylcarnitines into the mitochondrial matrix and exit of free carnitine. The transport function of CACT is crucial for the β-oxidation pathway. In this work, it has been found that CACT is partially acetylated in rat liver mitochondria as demonstrated by anti-acetyl-lys antibody immunostaining. Acetylation was reversed by the deacetylase Sirtuin 3 in the presence of NAD⁺. After treatment of the mitochondrial extract with the deacetylase, the CACT activity, assayed in proteoliposomes, increased. The half-saturation constant of the CACT was not influenced, while the V _max was increased by deacetylation. Sirtuin 3 was not able to deacetylate the CACT when incubation was performed in intact mitoplasts, indicating that the acetylation sites are located in the mitochondrial matrix. Prediction on the localization of acetylated residues by bioinformatics correlates well with the experimental data. Recombinant CACT treated with acetyl-CoA was partially acetylated by non-enzymatic mechanism with a corresponding decrease of transport activity. The experimental data indicate that acetylation of CACT inhibits its transport activity, and thus may contribute to the regulation of the mitochondrial β-oxidation pathway.     

Determination of key metabolites during biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine with Rhodococcus sp. strain DN22.

Rhodococcus sp. strain DN22 can convert hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to nitrite, but information on degradation products or the fate of carbon is not known. The present study describes aerobic biodegradation of RDX (175 microM) when used as an N source for strain DN22. RDX was converted to nitrite (NO(2)(-)) (30%), nitrous oxide (N(2)O) (3.2%), ammonia (10%), and formaldehyde (HCHO) (27%), which later converted to carbon dioxide. In experiments with ring-labeled [(15)N]-RDX, gas chromatographic/mass spectrophotometric (GC/MS) analysis revealed N(2)O with two molecular mass ions: one at 44 Da, corresponding to (14)N(14)NO, and the second at 45 Da, corresponding to (15)N(14)NO. The nonlabeled N(2)O could be formed only from -NO(2), whereas the (15)N-labeled one was presumed to originate from a nitramine group ((15)N-(14)NO(2)) in RDX. Liquid chromatographic (LC)-MS electrospray analyses indicated the formation of a dead end product with a deprotonated molecular mass ion [M-H] at 118 Da. High-resolution MS indicated a molecular formula of C(2)H(5)N(3)O(3). When the experiment was repeated with ring-labeled [(15)N]-RDX, the [M-H] appeared at 120 Da, indicating that two of the three N atoms in the metabolite originated from the ring in RDX. When [U-(14)C]-RDX was used in the experiment, 64% of the original radioactivity in RDX incorporated into the metabolite with a molecular weight (MW) of 119 (high-pressure LC/radioactivity) and 30% in (14)CO(2) (mineralization) after 4 days of incubation, suggesting that one of the carbon atoms in RDX was converted to CO(2) and the other two were incorporated in the ring cleavage product with an MW of 119. Based on the above stoichiometry, we propose a degradation pathway for RDX based on initial denitration followed by ring cleavage to formaldehyde and the dead end product with an MW of 119.     

Bioactive peptides: Conformational study of a cystinyl cycloheptapeptide in its free and calcium complexed forms

The disulphide bridged heptapeptide has been synthesized by classical solution methods. An ion binding study showed the peptide's ability to complex calcium ions with definite stoichiometry. The solution conformation of the peptide in its free and calcium-complexed form has been investigated by CD and nmr. The model structure derived from nmr data has been energy minimized and the resulting structure investigated by molecular dynamics simulation in water. The structure of the equimolar peptide/Ca^2? complex in acetonitrile at room temperature shows the presence of two transannular hydrogen bonds, with the formation of two ring structures of the C₁₀ (type VIa) and C₁₄ type. One peptide unit (Pro-Pro) is cis, all others are trans. © 1993 John Wiley & Sons, Inc.     

Interaction of beta-lactam antibiotics with the mitochondrial carnitine/acylcarnitine transporter

The interaction of beta-lactams with the purified mitochondrial carnitine/acylcarnitine transporter reconstituted in liposomes has been studied. Cefonicid, cefazolin, cephalothin, ampicillin, piperacillin externally added to the proteoliposomes, inhibited the carnitine/carnitine antiport catalysed by the reconstituted transporter. The most effective inhibitors were cefonicid and ampicillin with IC50 of 6.8 and 7.6mM, respectively. The other inhibitors exhibited IC50 values above 36 mM. Kinetic analysis performed with cefonicid and ampicillin revealed that the inhibition is completely competitive, i.e., the inhibitors interact with the substrate binding site. The Ki of the transporter is 4.9 mM for cefonicid and 9.9 mM for ampicillin. Cefonicid inhibited the transporter also on its internal side. The IC50 was 12.9 mM indicating that the inhibition was less pronounced than on the external side. Ampicillin and the other inhibitors were much less effective on the internal side. The beta-lactams were not transported by the carnitine/acylcarnitine transporter. Cephalosporins, and at much lower extent penicillins, caused irreversible inhibition of the transporter after prolonged time of incubation. The most effective among the tested antibiotics was cefonicid with IC50 of 0.12 mM after 60 h of incubation. The possible in vivo implications of the interaction of the beta-lactam antibiotics with the transporter are discussed.     

Occurrence of different inter-varietal and inter-organ defence strategies towards supra-optimal zinc concentrations in two cultivars of Triticum aestivum L.

Two bread wheat (Triticum aestivum L.) cultivars (Albimonte, traditional cultivar very important in Italy since long time; and Manital, more recent, evincing better productive performances) were grown for 10 d in presence of 0.7 (control), 70 or 350 μM ZnSO₄, to verify whether Zn excess was differently managed at inter-varietal and at inter-organ level. Roots were found to be the main site of Zn accumulation, although a moderate metal translocation to leaves occurred in both cultivars. Despite only slight differences in internal Zn concentrations between cultivars, Albimonte seemed to be more sensitive to Zn excess in terms of growth reduction and H₂O₂ accumulation, suggesting that the diversities in responses to Zn stress should be ascribed here to inter-varietal metabolic differences. In both cultivars, increased NAD(P)H oxidation rate by pH-dependent peroxidases, and reduced detoxification activity by catalase and peroxidases, were responsible for Zn-induced H₂O₂ accumulation, while total superoxide dismutase content and activity seemed in general to not change or even depress. Moreover, differences in the content of thiol-peptide compounds (glutathione and phytochelatins) were detected, suggesting indeed the setting up of differential response mechanisms to Zn excess at an inter-varietal and inter-organ level.     

Determination of Endogenous Acetylcholine Release in Freely Moving Rats by Transstriatal Dialysis Coupled to a Radioenzymatic Assay: Effect of Drugs

The technique of intracerebral dialysis in combination with a sensitive and specific radioenzymatic method was used for recovery and quantification of endogenous extracellular acetylcholine from the striata of freely moving rats. A thin dialysis tube was inserted transversally through the caudate nuclei, and the tube was perfused with Ringer solution, pH 6.1, at a constant rate of 2 microliter min-1. The perfusates were collected at 10-min intervals. In the presence of 1 and 10 microM physostigmine, acetylcholine release was 4.5 +/- 0.02 and 7.3 +/- 0.3 pmol/10 min, respectively (not corrected for recovery). The latter concentration of the acetylcholinesterase inhibitor was used in all experiments. Under basal conditions, acetylcholine output was stable over at least 4 h. A depolarizing K+ concentration produced a sharp, reversible 87% increase in acetylcholine output. Both the basal and K+-stimulated release were Ca2+ dependent. The choline uptake inhibitor hemicholinium-3 (20 micrograms intracerebroventricularly) reduced striatal acetylcholine output to 35% of the basal value within 90 min. Scopolamine (0.34 mg/kg s.c.) provoked a sharp enhancement of acetylcholine release of approximately 63% over basal values, whereas oxotremorine (0.53 mg/kg i.p.) transiently reduced acetylcholine release by 54%. These results indicate the physiological and pharmacological suitability of transstriatal dialysis for monitoring endogenous acetylcholine release.     

A metabolomics-based approach for non-invasive diagnosis of chromosomal anomalies

Introduction

Chromosomal anomalies (CA) are the most frequent fetal anomalies.

Objective

To evaluate the diagnostic performance of a machine learning ensemble model based on the maternal serum metabolomic fingerprint of fetal aneuploidies during the second trimester .

Methods

This is a case-control pilot study. Metabolomic profiles have been obtained on serum of 328 mothers (220 controls and 108 cases), using gas chromatography coupled to mass spectrometry. Eight machines learning and classification models were built and optimized. An ensemble model was built using a voting scheme. All samples were randomly divided into two sets. One was used as training set, the other one for diagnostic performance assessment.

Results

Ensemble machine learning model correctly classified all cases and controls. The accuracy was the same for trisomy 21 and 18; also, the other CA were correctly detected. Elaidic, stearic, linolenic, myristic, benzoic, citric and glyceric acid, mannose, 2-hydroxy butyrate, phenylalanine, proline, alanine and 3-methyl histidine were selected as the most relevant metabolites in class separation.

Conclusion

The proposed model, based on the maternal serum metabolomic fingerprint of fetal aneuploidies during the second trimester, correctly identifies all the cases of chromosomal abnormalities. Overall, this preliminary analysis appeared suggestive of a metabolic environment conductive to increased oxidative stress and a disturbance in the fetal central nervous system development. Maternal serum metabolomics can be a promising tool in the screening of chromosomal defects. Moreover, metabolomics allows to extend our knowledge about biochemical alterations caused by aneuploidies and responsible for the observed phenotypes.

     

Cardiac pacemaker battery discharge after external electrical cardioversion for broad QRS Complex Tachycardia

External electrical cardioversion or defibrillation may be necessary in patients with implanted cardiac pacemaker (PM) or implantable cardioverter defibrillator (ICD). Sudden discharge of high electrical energy employed in direct current (DC) transthoracic countershock may damage the PM/ICD system resulting in a series of possible device malfunctions. For this reason, when defibrillation or cardioversion must be attempted in a patient with a PM or ICD, some precautions should be taken, particularly in PM dependent patients, in order to prevent damage to the device. We report the case of a 76-year-old woman with a dual chamber PM implanted in the right subclavicular region, who received two consecutive transthoracic DC shocks to treat haemodynamically unstable broad QRS complex tachycardia after cardiac surgery performed with a standard sternotomic approach. Because of the sternal wound and thoracic drainage tubes together with the severe clinical compromise, the anterior paddle was positioned near the pulse generator. At the following PM test, a complete battery discharge was detected.     

Acylpeptide hydrolase inhibition as targeted strategy to induce proteasomal down-regulation

Acylpeptide hydrolase (APEH), one of the four members of the prolyl oligopeptidase class, catalyses the removal of N-acylated amino acids from acetylated peptides and it has been postulated to play a key role in protein degradation machinery. Disruption of protein turnover has been established as an effective strategy to down-regulate the ubiquitin-proteasome system (UPS) and as a promising approach in anticancer therapy.Here, we illustrate a new pathway modulating UPS and proteasome activity through inhibition of APEH. To find novel molecules able to down-regulate APEH activity, we screened a set of synthetic peptides, reproducing the reactive-site loop of a known archaeal inhibitor of APEH (SsCEI), and the conjugated linoleic acid (CLA) isomers. A 12-mer SsCEI peptide and the trans10-cis12 isomer of CLA, were identified as specific APEH inhibitors and their effects on cell-based assays were paralleled by a dose-dependent reduction of proteasome activity and the activation of the pro-apoptotic caspase cascade. Moreover, cell treatment with the individual compounds increased the cytoplasm levels of several classic hallmarks of proteasome inhibition, such as NFkappaB, p21, and misfolded or polyubiquitinylated proteins, and additive effects were observed in cells exposed to a combination of both inhibitors without any cytotoxicity. Remarkably, transfection of human bronchial epithelial cells with APEH siRNA, promoted a marked accumulation of a mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), herein used as a model of misfolded protein typically degraded by UPS. Finally, molecular modeling studies, to gain insights into the APEH inhibition by the trans10-cis12 CLA isomer, were performed.Our study supports a previously unrecognized role of APEH as a negative effector of proteasome activity by an unknown mechanism and opens new perspectives for the development of strategies aimed at modulation of cancer progression.