[Long-term outcomes and factors for predicting ventricular arrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia].

OBJECTIVE: To evaluate factors for predicting ventricular arrhythmia, the clinical effect of drugs on patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), and their long-term outcomes. METHODS: Six patients diagnosed with CPVT underwent a series of electrocardiograms and 24-hour Holter monitoring. ß-blockers were recommended for all patients, while some patients were also prescribed propafenone and 1 patient underwent catheter-based renal sympathetic denervation (RDN). The characteristics of electrocardiogram, arrhythmia and long-term outcomes were monitored. RESULTS: Syncope episodes did not occur any longer in 1 patient on ß-blocker, but recurred in 3 other patients and 2 patients died (one due to his cessation of metoprolol for 3 months). Inverted and/or bifid T waves and abnormal U wave were observed in the precordial leads. T wave alternans was observed in 4 patients in the precordial leads. These abnormal electrocardiogram features disappeared or diminished with ß-blocker treatment. All spontaneous episodes of ventricular tachycardia occurred prior to sinus tachycardia and frequent polymorphic premature ventricular contractions. CONCLUSIONS: Bifid and/or inverted T waves, T wave alternans and abnormal U waves together with sinus tachycardia and frequent premature ventricular contractions are indicator for predicting ventricular arrhythmia and assessing the effect of ß-blockers. Compliance with ß-blocker treatment is a strong indicator of outcome.

Serum miR-210 and miR-30a expressions tend to revert to fetal levels in Chinese adult patients with chronic heart failure.

BACKGROUND: MicroRNAs (miRNAs) are widely involved in the process of chronic heart failure (HF), which is characterized by reactivation of the fetal gene program. Here, we examined whether the serum expression levels of some HF-related miRNAs in adult HF patients would tend to revert to fetal levels. METHODS AND RESULTS: Serum was obtained from the peripheral venous blood of 22 HF patients, 18 asymptomatic controls, and the umbilical venous blood of 9 fetuses from 9 independent parturitions. Serum pools of the three groups were initially screened against 40 known HF-associated miRNAs via quantitative reverse transcriptase polymerase chain reaction. Twenty-seven miRNAs were stably expressed in the serum pools. Nine miRNAs showed similar expression levels in the HF and fetus groups compared to the controls, two of which (miR-210, miR-30a) were significantly up-regulated in both groups. These miRNAs showed high diagnostic accuracy and correlations with blood N-terminal prohormone of brain natriuretic peptide, identifying them as potential biomarkers for HF. Putative targets of the miRNAs were predicted with online software programs, and the Kyoto Encyclopedia of Genes and Genomes pathway analysis was employed to identify miRNA-regulated functional modules. In particular, miR-210 seemed to be more closely related than miR-30a to the pathological mechanisms of HF, including the calcium signaling, vascular smooth muscle contraction, transforming growth factor-ß signaling, and aldosterone-regulated sodium reabsorption pathways. CONCLUSION: The serum expression levels of some HF-related miRNAs in HF patients tended towards fetal levels. Among them, miR-210 and miR-30a were elevated in the HF and fetus groups.

Structural analysis of bengamide derivatives as inhibitors of methionine aminopeptidases.

Natural-product-derived bengamides possess potent antiproliferative activity and target human methionine aminopeptidases (MetAPs) for their cellular effects. Several derivatives were designed, synthesized, and evaluated as MetAP inhibitors. Here, we present four new X-ray structures of human MetAP1 in complex with the inhibitors. Together with the previous structures of bengamide derivatives with human MetAP2 and tubercular MtMetAP1c, analysis of the interactions of these inhibitors at the active site provides structural basis for further modification of these bengamide inhibitors for improved potency and selectivity as anticancer and antibacterial therapeutics.

[Clinical features of six patients with catecholaminergic polymorphic ventricular tachycardia].

OBJECTIVE: To describe the clinical features of 6 patients with catecholaminergic polymorphic ventricular tachycardia. METHODS: Clinical data including signs and symptoms, electrocardiograms, Holter monitoring electrocardiograms and echocardiography was analyzed. Definite diagnosis was made based on the mutations of RYR2 and CASQ2. RESULTS: From July 2002 to March 2010, 6 consecutive patients referred to our center because of syncope [4 males, mean age (13.0 ± 4.2) years] were diagnosed with CPVT by clinical evaluation and genetic testing. Their electrocardiograms showed T waves with notch or bimodal and tall U waves in right chest leads. There was no J wave, no ST-segment deviation, no prolongation or shortening of QT interval. We captured the so-called "bidirectional and(or) polymorphic ventricular tachycardia (bVT and pVT)" in 2 out of 6 patients by ECG, in 5 out of 6 patients by 24-hours Holter monitor, in 3 out of 6 patients by exercise test. All patients received ß blockers and no syncope occurred during the 3 months follow-up after discharge from hospital. CONCLUSIONS: CPVT is an inherited cardiac channelopathy characterized by syncope and(or) sudden death relatived to motion. The ECG shows T wave alteration and tall U wave in right chest leads. The mode of its onset is bVT and(or) pVT, and can be captured by Holter easily. ß blocker is a safe and effective remedy for suppressing its attack.

Structural analysis of inhibition of Mycobacterium tuberculosis methionine aminopeptidase by bengamide derivatives.

Natural product-derived bengamides possess potent antiproliferative activity and target human methionine aminopeptidases for their cellular effects. Using bengamides as a template, several derivatives were designed and synthesized as inhibitors of methionine aminopeptidases of Mycobacterium tuberculosis, and initial antitubercular activity were observed. Here, we present three new X-ray structures of the tubercular enzyme MtMetAP1c in complex with the inhibitors in the Mn(II) form and in the Ni(II) form. All amide moieties of the bengamide derivatives bind to the unique shallow cavity and interact with a flat surface created by His-212 of MtMetAP1c in the Mn(II) form. However, the active site metal has significant influence on the binding mode, because the amide takes a different conformation in the Ni(II) form. The interactions of these inhibitors at the active site provide the structural basis for further modification of these bengamide inhibitors for improved potency and selectivity.

Inhibition of Mycobacterium tuberculosis methionine aminopeptidases by bengamide derivatives.

Methionine aminopeptidase (MetAP) carries out an essential function of protein N-terminal processing in many bacteria and is a promising target for the development of novel antitubercular agents. Natural bengamides potently inhibit the proliferation of mammalian cells by targeting MetAP enzymes, and the X-ray crystal structure of human type 2 MetAP in complex with a bengamide derivative reveals the key interactions at the active site. By preserving the interactions with the conserved residues inside the binding pocket while exploring the differences between bacterial and human MetAPs around the binding pocket, seven bengamide derivatives were synthesized and evaluated for inhibition of MtMetAP1a and MtMetAP1c in different metalloforms, inhibition of M. tuberculosis growth in replicating and non-replicating states, and inhibition of human K562 cell growth. Potent inhibition of MtMetAP1a and MtMetAP1c and modest growth inhibition of M. tuberculosis were observed for some of these derivatives. Crystal structures of MtMetAP1c in complex with two of the derivatives provided valuable structural information for improvement of these inhibitors for potency and selectivity.

Atorvastatin protects against angiotensin II-induced injury and dysfunction in human umbilical vein endothelial cells through bradykinin 2 receptors.

The morphological and functional integrity of the endothelial cell (EC) is compromised in many cardiovascular diseases such as atherosclerosis, hypertension, and diabetes. Angiotensin II (Ang II) plays important roles in the initiation and progression of these diseases. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) may have cholesterol-independent pleiotropic effects on preventing the EC injury and dysfunction that occurs in these diseases, and the protective effects may relate to bradykinin 2 receptors (B2Rs). Our study was designed to test the hypothesis that atorvastatin, via B2Rs, protects the viability and function of EC exposed to Ang II independent of hemodynamics. The experimental results showed that the cytotoxic effects of Ang II on human umbilical vein endothelial cells were significantly ameliorated by atorvastatin pretreatment (LDH tests, MTT assay, and propdium iodide (PI)/Annexin V-stating analysis), and atorvastatin treatment simultaneously enhanced expression of endothelial nitric oxide synthase and yielded of nitric oxide (NO) and cyclic guanosine monophosphate, but both effects were attenuated by the B2Rs antagonist HOE-140. This study proves the hypothesis and may be pertinent to the complex mechanism of action of statins explaining their long-term beneficial effects in maintaining the morphological and functional integrity of vascular ECs.

Expression and characterization of Mycobacterium tuberculosis methionine aminopeptidase type 1a.

Methionine aminopeptidase (MetAP) carries out the cotranslational N-terminal methionine excision and is essential for bacterial survival. Mycobacterium tuberculosis expresses two MetAPs, MtMetAP1a and MtMetAP1c, at different levels in growing and stationary phases, and both are potential targets to develop novel antitubercular therapeutics. Recombinant MtMetAP1a was purified as an apoenzyme, and metal binding and activation were characterized with an activity assay using a fluorogenic substrate. Ni(II), Co(II) and Fe(II) bound tightly at micromolar concentrations, and Ni(II) was the most efficient activator for the MetAP-catalyzed substrate hydrolysis. Although the characteristics of metal binding and activation are similar to MtMetAP1c we characterized before, MtMetAP1a was significantly more active, and more importantly, a set of inhibitors displayed completely different inhibitory profiles on the two mycobacterial MetAPs in both potency and metalloform selectivity. The differences in catalysis and inhibition predicted the significant differences in active site structure.

Catalysis and inhibition of Mycobacterium tuberculosis methionine aminopeptidase.

Methionine aminopeptidase (MetAP) carries out an important cotranslational N-terminal methionine excision of nascent proteins and represents a potential target to develop antibacterial and antitubercular drugs. We cloned one of the two MetAPs in Mycobacterium tuberculosis (MtMetAP1c from the mapB gene) and purified it to homogeneity as an apoenzyme. Its activity required a divalent metal ion, and Co(II), Ni(II), Mn(II), and Fe(II) were among activators of the enzyme. Co(II) and Fe(II) had the tightest binding, while Ni(II) was the most efficient cofactor for the catalysis. MtMetAP1c was also functional in E. coli cells because a plasmid-expressed MtMetAP1c complemented the essential function of MetAP in E. coli and supported the cell growth. A set of potent MtMetAP1c inhibitors were identified, and they showed high selectivity toward the Fe(II)-form, the Mn(II)-form, or the Co(II) and Ni(II) forms of the enzyme, respectively. These metalloform selective inhibitors were used to assign the metalloform of the cellular MtMetAP1c. The fact that only the Fe(II)-form selective inhibitors inhibited the cellular MtMetAP1c activity and inhibited the MtMetAP1c-complemented cell growth suggests that Fe(II) is the native metal used by MtMetAP1c in an E. coli cellular environment. Finally, X-ray structures of MtMetAP1c in complex with three metalloform-selective inhibitors were analyzed, which showed different binding modes and different interactions with metal ions and active site residues.

Determination of binding affinity of metal cofactor to the active site of methionine aminopeptidase based on quantitation of functional enzyme.

Determination of metal affinity to the active site of metalloenzymes constitutes an integral part in the understanding of enzyme catalysis and regulation. Nonlinear curve fitting of metal titration curves using the multiple independent binding sites (MIBS) model was adapted to determine K(D) values based on functional enzyme concentrations. This approach provides a more accurate evaluation of K(D) compared with existing methods that are based on total protein concentrations. We applied this concept to methionine aminopeptidase from Mycobacterium tuberculosis and showed that it is a monometalated enzyme with a K(D) of 0.13 microM for Co(2+).

Metalloform-selective inhibitors of escherichia coli methionine aminopeptidase and X-ray structure of a Mn(II)-form enzyme complexed with an inhibitor.

Methionine aminopeptidase (MetAP) enzymes require a divalent metal ion such as Mn(II), Fe(II), Co(II), Ni(II), or Zn(II) for its removal of the N-terminal methionine from newly synthesized proteins, but it is not certain which of these ions is most important in vivo. Metalloform-selective MetAP inhibitors could be valuable for defining which metals are physiologically relevant for MetAP activation and could serve as leads for development of new therapeutic agents. We have screened a library of 43 736 small drug-like molecules against Escherichia coli MetAP and identified two groups of potent and highly metalloform-selective inhibitors of the Co(II)-form, and of the Mn(II)-form, of this enzyme. Compound 1 is 790-fold more selective for the Co(II)-form, while compound 4 is over 640-fold more potent toward the Mn(II)-form. The X-ray structure of a di-Mn(II) form of E. coli MetAP complexed with the Mn(II)-form-selective compound 4 was obtained, and it shows that the inhibitor interacts with both Mn(II) ions through the two oxygen atoms of its free carboxylate group. The preferential coordination of the hard (oxygen) donors to Mn(II) may contribute to its superb selectivity toward the Mn(II)-form.