J point elevation in high precordial leads associated with risk of ventricular fibrillation

Introduction

The significance of high precordial electrocardiograms in idiopathic ventricular fibrillation (IVF) is unknown.

Method

This study included 50 consecutive patients (48 men; age, 42 ± 18 years) who had spontaneous ventricular fibrillation not linked to structural heart disease and received implantable cardiac defibrillator therapy. IVF was diagnosed in 35 patients and Brugada syndrome was diagnosed in other 15 patients. Electrocardiograms in high intercostal space were compared between 35 patients with IVF and 105 age- and sex-matched healthy controls (patient: control ratio, 1:3).

Results

The frequency of J point elevation ≥ 0.1 mV in the 4th intercostal spaces was similar between patients with IVF (14%) and healthy controls (7%). However, the frequency of J point elevation ≥ 0.1 mV in the 3rd intercostal space was higher in patients with IVF (40%) than controls (11%) (p < .01). J point elevation was present only in the 3rd intercostal space but not in the 4th intercostal space in 30% of patients with IVF but only in 6% of controls (p < .01). During follow-up, the recurrence of ventricular fibrillation was higher in patients with IVF who had J point elevation in the 3rd intercostal space (36%) and Brugada syndrome(40%) than those with IVF who did not have J point elevation in the 3rd intercostal space(11%) (p < .05 for both).

Conclusion

J point elevation in the 3rd intercostal space was associated with IVF and recurrences of ventricular fibrillation. Electrocardiogram recordings in the high intercostal space may be useful to identify risk of sudden death.

     

Effect of cardiac resynchronization therapy in isolated ventricular noncompaction in adults: follow-up of four cases

Background: An isolated ventricular noncompaction (IVNC) is an unclassified cardiomyopathy and, despite the increasing awareness of and interest in this disorder, the role of cardiac resynchronization therapy (CRT) remains obscure. Objective: The purpose of this study was to clarify the long‐term effect of CRT on IVNC in adult patients. Methods: Four cases of IVNC were included in this study. Before the CRT device was implanted, all four patients (54 ± 16‐year‐old, 4 males) presented with symptomatic congestive heart failure. Echocardiography revealed their systolic dysfunction and their left ventricular ejection fraction (LVEF) was 21 ± 8%. There was also mechanical dyssynchrony observed between the LV septum and free wall area. The QRS duration was “narrow” (112 and 120 ms) in two patients. One patient had been resuscitated from ventricular fibrillation (VF) and two had nonsustained ventricular tachycardia (VT). A CRT defibrillator (CRT‐D) was implanted in three patients with VT/VF and a CRT pacemaker (CRT‐P) in a patient without VT/VF. The LV lead was positioned in a lateral branch of the coronary sinus where a thickened noncompacted wall existed. Results: During the follow‐up period (28 ± 23 months), their congestive heart failure had improved in terms of the cardiothoracic ratio on the chest X‐ray, B‐type natriuretic peptide level, LV systolic dimension, and LVEF. No episodes of defibrillation shocks were observed. Conclusion: CRT may improve the prognosis and quality‐of‐life in patients with an IVNC with mechanical dyssynchrony.     

Long‐term outcome of patients with acquired chronic pure red cell aplasia (PRCA) following immunosuppressive therapy: a final report of the nationwide cohort study in 2004/2006 by the Japan PRCA collaborative study group

Immunosuppressive therapy has been employed as the initial treatment for acquired chronic pure red cell aplasia (PRCA), such as idiopathic, thymoma‐associated, or large granular lymphocyte (LGL) leukaemia‐associated PRCA, which is thought to be immune‐mediated. To explore the overall long‐term outcome following immunosuppression and to identify the risk factors for death in these disorders, we conducted nationwide surveys in Japan 2004 and 2006, and identified a total of 185 patients with acquired chronic PRCA, including 72 idiopathic, 41 thymoma‐associated and 14 LGL leukaemia‐associated cases of PRCA for whom data was available. The present study evaluated 127 patients with these three subsets of PRCA. The median overall survival has not yet been reached in idiopathic PRCA. The estimated median overall survival times in patients with thymoma‐associated and LGL leukaemia‐associated PRCA were 142·1 and 147·8 months, respectively. Twenty‐two deaths were reported, and the response to induction therapy and relapse of anaemia were found to be associated with death. The major causes of death were infection in seven patients and organ failure in another seven patients. The results suggest that maintenance therapy and the management of infectious complications are crucial for improving the prognosis of chronic PRCA.     

Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis

The molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of guanosine 5′-triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC (molybdenum cofactor biosynthesis protein A and C, respectively). Conventionally, MoaA was considered to catalyze the majority of this transformation, with MoaC playing little or no role in the pyranopterin formation. Recently, this view was challenged by the isolation of 3′,8-cyclo-7,8-dihydro-guanosine 5′-triphosphate (3′,8-cH₂GTP) as the product of in vitro MoaA reactions. To elucidate the mechanism of formation of Moco pyranopterin backbone, we performed biochemical characterization of 3′,8-cH₂GTP and functional and X-ray crystallographic characterizations of MoaC. These studies revealed that 3′,8-cH₂GTP is the only product of MoaA that can be converted to cPMP by MoaC. Our structural studies captured the specific binding of 3′,8-cH₂GTP in the active site of MoaC. These observations provided strong evidence that the physiological function of MoaA is the conversion of GTP to 3′,8-cH₂GTP (GTP 3′,8-cyclase), and that of MoaC is to catalyze the rearrangement of 3′,8-cH₂GTP into cPMP (cPMP synthase). Furthermore, our structure-guided studies suggest that MoaC catalysis involves the dynamic motions of enzyme active-site loops as a way to control the timing of interaction between the reaction intermediates and catalytically essential amino acid residues. Thus, these results reveal the previously unidentified mechanism behind Moco biosynthesis and provide mechanistic and structural insights into how enzymes catalyze complex rearrangement reactions.Moco is an essential enzyme cofactor that mediates redox reactions in the active sites of enzymes. Moco-dependent enzymes play central roles in purine and sulfur catabolism in mammals, anaerobic respiration in bacteria, and nitrate assimilation in plants (1, 2). Importantly, Moco must be synthesized de novo in cells because it is chemically unstable, particularly under aerobic conditions, and cannot be taken up as a nutrient (1, 2).During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of GTP into cPMP (3). This unusual transformation involves the insertion of the guanine C-8 between C-2′ and C-3′ of ribose (Fig. 1A) (4). Although this conversion has been shown to require two proteins, MoaA and MoaC (molybdenum cofactor biosynthesis protein A and C, respectively) (4–6), their individual contributions have remained elusive and are the subject of the current study. MoaA belongs to the radical S-adenosyl-l-methionine (SAM) superfamily, of which members catalyze unique free-radical reactions (7). By contrast, MoaC shows no significant sequence or structural similarities to any functionally characterized enzyme. Therefore, the predominant view of the field has been that MoaA catalyzes the majority or all of the rearrangement of GTP to form the pterin structure, with MoaC playing little to no catalytic role in this process (2). In line with this view, studies identifying a putative MoaA product after chemical derivatization suggested that MoaA catalyzed the transformation of GTP to pyranopterin triphosphate (Fig. 1B) (8, 9). This conventional view was challenged by a recent in vitro characterization of MoaA, where formation of 3′,8-cyclic-7,8-dihydro-GTP (3′,8-cH₂GTP; Fig. 1B) rather than pyranopterin triphosphate was proposed (10). This finding suggested the possibility of a novel mechanism in which MoaC, and not MoaA, may ultimately be responsible for pyranopterin ring formation. However, it was also proposed that 3′,8-cH₂GTP could simply be a transient intermediate of MoaA (8, 9), leaving ambiguity about the functions of MoaA and MoaC.Open in a separate windowFig. 1.In vivo and in vitro functional characterization of MoaC. (A) Moco biosynthesis. Symbols indicate the fate of each atom (4). cPMP may also be in a hydrate form in solution (14). (B) Previously proposed structures for the MoaA product. (C) In situ ¹³C NMR characterization of the MoaA product. Shown are the ¹³C NMR spectra of [U-¹³C]GTP (Top), purified [U-¹³C]3′,8-cH₂GTP (Middle), and the MoaA (0.4 mM) reaction using [U-¹³C]GTP (1 mM) as the substrate (Bottom). Numbers are the signal assignments for atoms labeled in A and B. Signals highlighted by * and # are derived from toluensulfonate and glycerol, respectively. (D) Timecourse of the formation of the biosynthetically relevant MoaA product based on the quantitation of 3′,8-cH₂GTP before (open circles) or after (filled circles) its conversion to cPMP. The assay solution contained 65 μM WT-MoaA, 0.2 mM GTP, and 1 mM SAM. (E) In vitro activity of WT and variants of MoaC determined by a coupled assay with MoaA. (F) Steady-state kinetic parameters for WT and variants of MoaC. The assays were performed in the absence of MoaA by using purified 3′,8-cH₂GTP as a substrate. a, only the upper limit (1.0 μM) was determined because the reaction rate became impractically low below this substrate concentration. (G) Moco production in E. coli ΔmoaC expressing WT or variants of MoaC based on anaerobic growth rates (black bars) or NR activity (gray). All data in D–G are average of at least three replicates, and the errors are based on SDs.Most of these previous studies have focused on the characterization of MoaA; the contribution of MoaC has been largely unexplored. Here, we report a comprehensive functional and structural study that clarifies these issues and provides strong evidence that MoaC is the enzyme responsible for pyranopterin backbone formation and that MoaA provides 3′,8-cH₂GTP as the MoaC substrate. Further structural and functional studies revealed that MoaC catalyzes the complex rearrangement of 3′,8-cH₂GTP into cPMP via a unique mechanism involving dynamic conformational changes of substrate and enzyme.     

Homologous trans-editing factors with broad tRNA specificity prevent mistranslation caused by serine/threonine misactivation

Aminoacyl-tRNA synthetases (ARSs) establish the rules of the genetic code, whereby each amino acid is attached to a cognate tRNA. Errors in this process lead to mistranslation, which can be toxic to cells. The selective forces exerted by species-specific requirements and environmental conditions potentially shape quality-control mechanisms that serve to prevent mistranslation. A family of editing factors that are homologous to the editing domain of bacterial prolyl-tRNA synthetase includes the previously characterized trans-editing factors ProXp-ala and YbaK, which clear Ala-tRNA^Pro and Cys-tRNA^Pro, respectively, and three additional homologs of unknown function, ProXp-x, ProXp-y, and ProXp-z. We performed an in vivo screen of 230 conditions in which an Escherichia coli proXp-y deletion strain was grown in the presence of elevated levels of amino acids and specific ARSs. This screen, together with the results of in vitro deacylation assays, revealed Ser- and Thr-tRNA deacylase function for this homolog. A similar activity was demonstrated for Bordetella parapertussis ProXp-z in vitro. These proteins, now renamed “ProXp-ST1” and “ProXp-ST2,” respectively, recognize multiple tRNAs as substrates. Taken together, our data suggest that these free-standing editing domains have the ability to prevent mistranslation errors caused by a number of ARSs, including lysyl-tRNA synthetase, threonyl-tRNA synthetase, seryl-tRNA synthetase, and alanyl-tRNA synthetase. The expression of these multifunctional enzymes is likely to provide a selective growth advantage to organisms subjected to environmental stresses and other conditions that alter the amino acid pool.A high level of accuracy in protein synthesis is essential for normal cell function and proliferation. A critical step in this process is pairing the correct amino acid with the cognate tRNA species by aminoacyl-tRNA synthetases (ARSs). ARSs catalyze the aminoacyl-tRNA (aa-tRNA) formation in two steps involving amino acid activation (step 1) and transfer of the activated amino acid to tRNAs (step 2). Although ARSs have evolved to exhibit specific tRNA-recognition capabilities with an estimated error frequency of 10⁻⁶, a greater number of mistakes arise from the lack of discrimination of near-cognate amino acids, with an estimated error rate of 10⁻⁴ to 10⁻⁵ at this step (1). Misincorporation of amino acids into proteins can be harmful to both eukaryotic and prokaryotic cells (2, 3).Quality control of aa-tRNA formation is achieved by hydrolysis of the aminoacyl-adenylate (“pre-transfer editing”) or deacylation of the mischarged aa-tRNA (“post-transfer editing”) (4, 5). Editing mechanisms are used by both classes of ARSs, and 7 of 22 ARSs possess posttransfer editing sites that are distinct from the aminoacylation active site. The connective polypeptide 1 editing domain is found in class I isoleucyl-tRNA synthetase (IleRS) (6), leucyl-tRNA synthetase (LeuRS) (7, 8), and valyl-tRNA synthetase (ValRS) (9). Editing domains of class II ARSs are more diverse and include the N-terminal domain of threonyl-tRNA synthetase (ThrRS) (10), the related editing domain of alanyl-tRNA synthetase (AlaRS) (11), the β3/ β4 domain of phenylalanine-tRNA synthetase (12), and the insertion domain (INS) of prolyl-tRNA synthetase (ProRS) (13, 14). Mutations in editing domains can have detrimental effects on cells. For example, a mutation in the editing site of AlaRS, which results in only a twofold increase in misacylation in vitro, results in a severe neurodegeneration phenotype in mice (15). Cellular degradation and apoptosis caused by a mutation in the editing domain of ValRS have been reported in murine cells (16). In addition, editing defects in bacteria often result in slower growth rates, delayed growth, or even death (17–22).In addition to the cis-editing domain appended to ARSs, free-standing homologs of editing domains are distributed throughout organisms in all three kingdoms of life as additional checkpoints to maintain translational fidelity in trans. Autonomous trans-editing factors that are evolutionarily related to three class II ARSs, namely, AlaRS, ThrRS and ProRS, have been identified. A homolog of the AlaRS editing domain, AlaXp, is widely distributed and is shown to hydrolyze seryl-tRNA synthetase (Ser-tRNA^Ala) (23, 24). ThrRS-ed is an autonomous editing domain of Ser-tRNA^Thr in crenarchaeal genomes where thrS genes are truncated (25). Bacterial ProRS INS domain homologs include five proteins previously named “YbaK,” “ProXp-ala,” “ProXp-x,” “ProXp-y” (annotated YeaK), and “ProXp-z” (annotated PA2301) (Fig. 1) (26). These domains together with the INS domain of ProRS are collectively known as the “INS superfamily.” Although the INS domain edits mischarged Ala-tRNA^Pro (13, 14), YbaK deacylates Cys-tRNA^Pro (27), which is formed in the active site of ProRS because of the similar size of Cys and Pro (28). ProXp-ala is capable of clearing Ala-tRNA^Pro and compensates for the lack of an INS posttransfer editing domain in many bacteria (23, 26). Functions for ProXp-x, -y, and -z have not yet been reported. Here, for the first time to our knowledge, we explore the in vivo and in vitro substrate specificities of ProXp-y and ProXp-z. These two domains are phylogenetically distinct (26) and differ in length and in the identity of several highly conserved residues. Most notably, proXp-y encodes a functionally critical Lys residue that is present in all other INS superfamily members with the exception of proXp-z, which instead contains a strictly conserved Asn (26).Open in a separate windowFig. 1.Domain structure of E. coli and B. parapertussis ProRS and the INS superfamily. Conserved motifs 1, 2, and 3 (M1–M3, purple), the anticodon-binding domain (ABD, green), and the editing domain (INS domain, blue) are shown. Single-domain INS-like proteins YbaK, ProXp-ala, ProXp-x, ProXp-y, and ProXp-z encoded in the indicated species are shown together with the INS and a truncated mini-INS present in the corresponding ProRS. C. crescentus is Caulobacter crescentus, and R. palustris is Rhodopseudomonas palustris. The known activities of INS, YbaK, and ProXp-ala are color coded as follows: blue, Ala deacylation; red, Cys deacylation. The domains investigated in this work are in orange, and domains of unknown function are in gray.     

Altered intracellular region of MUC1 and disrupted correlation of polarity‐related molecules in breast cancer subtypes

MUC1 glycoprotein is overexpressed and its intracellular localization altered during breast carcinoma tumorigenesis. The present study aimed to clarify the relationship of cytoplasmic localization of MUC1 with the breast cancer subtype and the correlation of 10 molecules associated with cell polarity in breast cancer subtypes. We immunostained 131 formalin-fixed and paraffin-embedded breast cancer specimens with an anti-MUC1 antibody (MUC1/CORE). For 48 of the 131 tumor specimens, laser-assisted microdissection and real-time quantitative RT-PCR were performed to analyze mRNA levels of MUC1 and 10 molecules, β-catenin, E-cadherin, claudin 3, claudin 4, claudin 7, RhoA, cdc42, Rac1, Par3 and Par6. Localization of MUC1 protein varied among breast cancer subtypes, that is, both the apical domain and cytoplasm in luminal A-like tumors (P < 0.01) and both the cytoplasm and cell membrane in luminal B-like (growth factor receptor 2 [HER2]+) tumors (P < 0.05), and no expression was found in triple negative tumors (P < 0.001). Estrogen receptor (ER)+ breast cancers showed higher MUC1 mRNA levels than ER− breast cancers (P < 0.01). The incidence of mutual correlations of expression levels between two of the 10 molecules (55 combinations) was 54.5% in normal breast tissue and 38.2% in luminal A-like specimens, 16.4% in luminal B-like (HER2+), 3.6% in HER2 and 18.2% in triple negative specimens. In conclusion, each breast cancer subtype has characteristic cytoplasmic localization patterns of MUC1 and different degrees of disrupted correlation of the expression levels between the 10 examined molecules in comparison with normal breast tissue.     

Inflammatory features of pancreatic cancer highlighted by monocytes/macrophages and CD4+ T cells with clinical impact

Pancreatic ductal adenocarcinoma (PDAC) is among the most fatal of malignancies with an extremely poor prognosis. The objectives of this study were to provide a detailed understanding of PDAC pathophysiology in view of the host immune response. We examined the PDAC tissues, sera, and peripheral blood cells of PDAC patients using immunohistochemical staining, the measurement of cytokine/chemokine concentrations, gene expression analysis, and flow cytometry. The PDAC tissues were infiltrated by macrophages, especially CD33+CD163+ M2 macrophages and CD4+ T cells that concomitantly express programmed cell death‐1 (PD‐1). Concentrations of interleukin (IL)‐6, IL‐7, IL‐15, monocyte chemotactic protein‐1, and interferon‐γ‐inducible protein‐1 in the sera of PDAC patients were significantly elevated. The gene expression profile of CD14+ monocytes and CD4+ T cells was discernible between PDAC patients and healthy volunteers, and the differentially expressed genes were related to activated inflammation. Intriguingly, PD‐1 was significantly upregulated in the peripheral blood CD4+ T cells of PDAC patients. Correspondingly, the frequency of CD4+PD‐1+ T cells was increased in the peripheral blood cells of PDAC patients, and this increase correlated to chemotherapy resistance. In conclusion, inflammatory conditions in both PDAC tissue and peripheral blood cells in PDAC patients were prominent, highlighting monocytes/macrophages as well as CD4+ T cells with influence of the clinical prognosis.