[Long-term outcome of patients with hypertrophic obstructive cardiomyopathy post percutaneous transluminal septal ablation].

Objective: To evaluate the long-term outcome of patients with hypertrophic obstructive cardiomyopathy(HOCM) after percutaneous transluminal septal ablation(PTSMA). Methods: HOCM patients who underwent PTSMA and surgical myectomy at the Chest Hospital of Shanghai Jiao Tong University from April 2001 to February 2019 were included in this retrospective analysis. Patients were divided into PTSMA group and surgical myectomy group. In addition, patients undergoing PTSMA were further divided into HOCM-PTSMA non-survivor group and HOCM-PTSMA survivor group. The general clinical information, procedural/surgical information and complications during hospitalization were compared between groups. Multivariate Cox regression model was used to analyze the independent risk factors for all-cause death in HOCM patients after PTSMA. Results: A total of 104 patients with HOCM who underwent PTSMA were enrolled. Mean age of the patients was (54±15) years old, including 41 females (38.7%). The follow-up time was 37.5(14.3, 76.8) months. At the last follow-up, 12 patients died (HOCM-PTSMA non-survivor group) and 92 were alive(HOCM-PTSMA survivor group). The proportion of patients with NYHA function class Ⅲ/Ⅳ was higher(P=0.036), and the posterior wall of the left ventricle was thicker(P=0.006) in the HOCM-PTSMA non-survivor group than in the HOCM-PTSMA survivor group. The immediate success rate of PTSMA in this cohort was 66%(70/104). The amount of absolute alcohol during the operation in the HOCM-PTSMA non-survivor group was (2.9±0.8) ml, which tended to be higher as compared to that in the HOCM-PTSMA survivor group((2.4±1.0)ml, P=0.056). Kaplan-Meier survival curve analysis showed that patients with HOCM who underwent PTSMA had an all-cause mortality-free survival rate of 90.1%, 78.3%, and 56.9% at 5, 10 and 15 years, and a HOCM-free survival rate of 91.3%, 79.4% and 57.7% at 5, 10 and 15 years, respectively. Multivariate Cox regression analysis showed that age≥ 65 years was an independent risk factor for all-cause death after PTSMA in patients with HOCM (HR=2.697, 95%CI 1.292-18.977, P=0.020). There were 32 patients in the surgical myectomy group. The proportion of patients with NYHA function class Ⅲ/Ⅳ was higher than that in the PTSMA group(P<0.001), while age, gender, and major comorbidities(atrial fibrillation, coronary heart disease, hypertension, and diabetes) as well as the left atrium dimension were all similar between the two groups(all P>0.05). Patients in the surgical myectomy group were followed up for 38.0(17.6, 64.2)months, and no deaths occurred during the follow-up period. Kaplan-Meier survival curve analysis showed that there were no statistically significant differences in all-cause-free and HOCM-free survival rates between patients in PTSMA group and surgical myectomy group(P=0.089 and 0.110, respectively). Conclusion: PTSMA is safe and effective for the treatment of patients with HOCM, and the long-term survival rate of patients after PTSMA is similar as patients undergoing classical surgical myectomy surgery.

[A novel HAND1 mutation associated with sporadic dilated cardiomyopathy].

Objective: To investigate a novel mutation in the HAND1 gene associated with sporadic dilated cardiomyopathy (DCM). Methods: From February 2013 to February 2017, the clinical data and peripheral venous blood samples were collected from 120 patients with sporadic DCM and 200 healthy controls, who were both from the Fifth People's Hospital of Shanghai, Fudan University and Shanghai Chest Hospital, Shanghai Jiaotong University.The genomic DNA was extracted from the study participants.The coding exons of HAND1 were amplified from the study subjects by polymerase chain reaction, and were sequenced for a potential HAND1 mutation.The online computer programs MUSCLE and Mutation Taster were used to analyze the conversation of an altered amino acid and to predict the disease-causing potential of an identified mutation, respectively.The wild-type HAND1 was cloned and the mutant was generated by site-directed mutagenesis.The Dual-luciferase reporter assay kits were used to explore the functional characteristics of the mutant HAND1. Results: A novel heterozygous mutation, a substitution of thymine for guanine at nucleotide 346 (c.346G>T), predicting the conversion of a glutamic acid-encoding codon into a stop codon at codon 116 (p.E116X), was detected in a patient with sporadic DCM.The nonsense mutation was absent in the 200 control individuals.The altered glutamic acid at amino acid position 116 was highly conserved evolutionarily, and the mutation was predicted to be pathogenic.Biological analyses revealed that the mutant HAND1 lost the ability to transcriptionally activate a target gene. Conclusion: Loss-of-function mutation in HAND1 is likely to be an uncommon cause responsible for sporadic DCM.

The diversity of the DnaJ/Hsp40 family, the crucial partners for Hsp70 chaperones.

DnaJ/Hsp40 (heat shock protein 40) proteins have been preserved throughout evolution and are important for protein translation, folding, unfolding, translocation, and degradation, primarily by stimulating the ATPase activity of chaperone proteins, Hsp70s. Because the ATP hydrolysis is essential for the activity of Hsp70s, DnaJ/Hsp40 proteins actually determine the activity of Hsp70s by stabilizing their interaction with substrate proteins. DnaJ/Hsp40 proteins all contain the J domain through which they bind to Hsp70s and can be categorized into three groups, depending on the presence of other domains. Six DnaJ homologs have been identified in Escherichia coli and 22 in Saccharomyces cerevisiae. Genome-wide analysis has revealed 41 DnaJ/Hsp40 family members (or putative members) in humans. While 34 contain the typical J domains, 7 bear partially conserved J-like domains, but are still suggested to function as DnaJ/ Hsp40 proteins. DnaJA2b, DnaJB1b, DnaJC2, DnaJC20, and DnaJC21 are named for the first time in this review; all other human DnaJ proteins were dubbed according to their gene names, e.g. DnaJA1 is the human protein named after its gene DNAJA1. This review highlights the progress in studying the domains in DnaJ/Hsp40 proteins, introduces the mechanisms by which they interact with Hsp70s, and stresses their functional diversity.

An eukaryotic RuvB-like protein (RUVBL1) essential for growth.

A human protein (RUVBL1), consisting of 456 amino acids (50 kDa) and highly homologous to RuvB, was identified by using the 14-kDa subunit of replication protein A (hsRPA3) as bait in a yeast two-hybrid system. RuvB is a bacterial protein involved in genetic recombination that bears structural similarity to subunits of the RF-C clamp loader family of proteins. Fluorescence in situ hybridization analysis demonstrated that the RUVBL1 gene is located at 3q21, a region with frequent rearrangements in different types of leukemia and solid tumors. RUVBL1 co-immunoprecipitated with at least three other unidentified cellular proteins and was detected in the RNA polymerase II holoenzyme complex purified over multiple chromatographic steps. In addition, two yeast homologs, scRUVBL1 and scRUVBL2 with 70 and 42% identity to RUVBL1, respectively, were revealed by screening the complete Saccharomyces cerevisiae genome sequence. Yeast with a null mutation in scRUVBL1 was nonviable. Thus RUVBL1 is an eukaryotic member of the RuvB/clamp loader family of structurally related proteins from bacteria and eukaryotes that is essential for viability of yeast.

Anticancer quinones induce pRb-preventable G2/M cell cycle arrest and apoptosis.

Reactive oxygen species generated during the metabolism of the antitumor quinone 3,6-diaziridinyl-1,4-benzoquinone (DZQ) in human colonic carcinoma HCT116 cells lead to the induction of p21 (WAF1, Cip1, or sdi1), an upstream regulator of the retinoblastoma gene product pRb involved G1 cell cycle control. We here demonstrate that the cell cycle was arrested in G2/M phase following supplementation with DZQ of human osteosarcoma Saos-2 cells (lacking both p53 and pRb) and HCT116 cells. DZQ also induced p21 and apoptosis in Saos-2 cells. The transfection of the Rb gene into Saos-2 cells did not alter the level of p21 induction, but changed cell cycle arrest into G1 phase and prevented apoptosis. These findings suggest that quinones may lead to a p53-independent and pRb-preventable G2/M arrest and apoptosis, which correlate with p21 induction.

The role of NAD(P)H:quinone oxidoreductase in quinone-mediated p21 induction in human colon carcinoma cells.

This study examines the role of NAD(P)H:quinone acceptor oxidoreductase (NQOR) (EC 1.6.99.2) in the metabolism of aziridinylbenzoquinones and the ensuing formation of reactive oxygen species in the induction of the cell cycle inhibitor p21 (WAF1, Cip1, or sdi1) in human colon carcinoma cells. The aziridinylbenzoquinones used were 2,5-diaziridinyl-1,4-benzoquinone (DZQ) and 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone (AZQ). The cell lines used in this study, BE and HT29 human colon carcinoma cell lines, are devoid of and overexpress NQOR activity, respectively. The rate of reduction of the above quinones in BE cells proceeded at similar rates (approximately 170 nmol/min/ mg protein) and, expectedly, it was not affected by the NQOR inhibitor, dicumarol. The metabolism of DZQ in HT29 cells was largely accomplished by NQOR (approximately 94%), whereas that of AZQ was accomplished by dicumarol-insensitive reductases. The metabolism of DZQ in HT29 cells was accompanied by H2O2 formation, which was approximately 10-fold higher than that ensuing from the activation of AZQ. In agreement with these data, the production of H2O2 during the activation of DZQ by purified NQOR was approximately 10-fold higher than that of AZQ. The formation of H2O2 during the metabolism of aziridinylbenzoquinones in BE cells was 24- to 57-fold lower than that in HT29 cells. At variance with HT29 cells, H2O2 formation by BE cells was insensitive to the catalase inhibitor sodium azide. The bioactivation of AZQ and DZQ in BE cells yielded O2.- and HO. as detected by spin trapping/EPR, the intensity of the former adduct being approximately 2-fold higher than that of the latter. These signals were insensitive to dicumarol. The metabolism of DZQ in HT29 cells yielded mainly HO. and a modest contribution of O2.- (ratio HO./O2.- approximately 10), whereas that of AZQ yielded a HO./O2.- approximately 2. The effect of dicumarol on the free radical pattern obtained during DZQ metabolism resulted in a strong inhibition (80%) of HO. production and a substantial increase of O2.- generation. The metabolism of DZQ and AZQ in BE cells was associated with a significant increase of p21 mRNA levels; the former quinone was approximately 2-fold more efficient than the latter. DZQ metabolism in HT29 cells led to an increase of p21 mRNA levels 15-fold higher than that observed with AZQ activation. Dicumarol did not inhibit p21 induction associated with the metabolism of DZQ in the NQOR-deficient BE cells, whereas the inhibitor decreased p21 induction in HT29 cells by approximately 30%. This modest inhibition is likely due to the low concentration of dicumarol used, which did not affect p21 constitutive levels in control experiments carried out in the absence of the quinone. p21 induction in HT29 cells was also inhibited by DTPA, a metal chelator, and N-acetylcysteine, a potent cellular anti-oxidant, suggesting that HO. may serve as an ultimate mediator for the induction. It may be surmised that the higher efficiency of DZQ in p21 induction may be related to its efficient metabolism by NQOR in HT29 cells and the associated high level of reactive oxygen species. The role of reactive oxygen species in p21 induction was further assessed upon supplementation of cells with H2O2:p21 induction in BE cells was 4-fold higher than that in HT29 cells. These findings suggest that assessment of the role of NQOR and reactive oxygen species in p21 induction requires careful consideration of the cell genotype.