Loading...

Current Issue

, Volume 48 Issue 1 Previous Issue    Next Issue
Frontiers in structural biology
YANG Fan,YANG Wei
J Zhejiang Univ (Med Sci), 2019, 48(1): 1-4.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.01
Abstract( 409 )   HTML( 83 )     PDF(459KB)( 419 )
Extraction and purification of NUDT9 homology domain of human transient receptor potential melastatin 2 channel
YE Peiwu,YU Xiafei,MA Cheng,YANG Wei
J Zhejiang Univ (Med Sci), 2019, 48(1): 5-11.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.02
Abstract( 680 )   HTML( 38 )     PDF(1227KB)( 258 )
Objective

To develop methods of extraction and purification of C-terminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel.

Methods

After sonication and centrifuge of Escherichia coli strain Rosetta (DE3) which was induced by isopropylthio-β-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected.

Results

The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl-β-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h.

Conclusion

Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.

I1363T mutation induces the defects in fast inactivation of human skeletal muscle voltage-gated sodium channel
TANG Siyang,YE Jia,LI Yuezhou
J Zhejiang Univ (Med Sci), 2019, 48(1): 12-18.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.03
Abstract( 503 )   HTML( 23 )     PDF(1055KB)( 150 )
Objective

To investigate the mechanism of congenital paramyotonia caused by human skeletal muscle voltage-gated sodium channel hNav1.4 mutant I1363T.

Methods

The conservation of the mutant site were detecled by using amino acid sequence alignment; the C-terminal mCherry fusion hNav1.4 was constructed, and the expression and distribution of wild type and hNav1.4 mutant I1363T were determined by confocal microscopy; the steady-state activation, fast inactivation and window current of wild type and hNav1.4 mutant I1363T were examined by whole-cell patch clamp.

Results

Alignment of the amino acid sequences revealed that Ile1363 is highly conserved in human sodium channels. There was no significant difference in expression level and distribution between wild type and I1363T. Although no significant differences were observed between I1363T mutant and wild type in the activation upon channel gating, the V0.5 of voltage-dependence of fast inactivation of I1363T mutant [(-59.01±0.26) mV] shifted 9 mV towards depolarization as compared with wild type [(-68.03±0.34) mV], and the slope factor of voltage-dependence curve increased to (5.24±0.23) mV, compared with (4.55±0.21) mV of the wild type. Moreover, I1363T showed the larger window current than that of the wild type.

Conclusions

I1363T causes the defect in fast inactivation of hNav1.4, which may increase the excitability of muscle cells and be responsible for myotonia. The increased window current of I1363T may result in an increase of inward Na+ current, could subsequently inactivate the channels and lead to loss of excitability and paralysis.

Structural modeling of selectivity filter in transient receptor pontential melastatin 8 ion channel
XU Lizhen,YANG Fan
J Zhejiang Univ (Med Sci), 2019, 48(1): 19-24.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.04
Abstract( 512 )   HTML( 21 )     PDF(4528KB)( 201 )
Objective

To construct a three-dimensional structural model for the selectivity filter in the transient receptor pontential melastatin 8 (TRPM8) ion channel.

Methods

In the Rosetta computational structural biology suite, multiple rounds of de novo modeling with the kinematic loop closure algorithm were performed.

Results

After nine rounds of computational modeling, we obtained the models of the selectivity filter within the TRPM8 channel with the lowest energy and high convergence. The model showed that the sidechain of D918 points were away from the central ion permeation pathway, while the sidechains of Q914, D920 and T923 pointed towards it.The glycosylation site N934 was located outside the pore region and its side chain directed to the extracellular water environment.

Conclusion

A three-dimensional structural model for the selectivity filter in the TRPM8 ion channel was constructed, which provides reliable structural information for exploring the mechanism of ion selectivity.

Progress on structural biology of voltage-gated ion channels
SONG Fangjun,GUO Hongtao
J Zhejiang Univ (Med Sci), 2019, 48(1): 25-33.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.05
Abstract( 1251 )   HTML( 23 )     PDF(1097KB)( 1534 )

Ion channels mediate ion transport across membranes, and play vital roles in processes of matter exchange, energy transfer and signal transduction in living organisms. Recently, structural studies of ion channels have greatly advanced our understanding of their ion selectivity and gating mechanisms. Structural studies of voltage-gated potassium channels elucidate the structural basis for potassium selectivity and voltage-gating mechanism; structural studies of voltage-gated sodium channels reveal their slow and fast inactivation mechanisms; and structural studies of transient receptor potential (TRP) channels provide complex and diverse structures of TRP channels, and their ligand gating mechanisms. In the article we summarize recent progress on ion channel structural biology, and outlook the prospect of ion channel structural biology in the future.

Application of mechanosensitive channels in sonogenetics
HONG Feifan,LI Yuezhou
J Zhejiang Univ (Med Sci), 2019, 48(1): 34-38.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.06
Abstract( 1077 )   HTML( 28 )     PDF(725KB)( 678 )

As a non-invasive approach, sonogenetics is applied to control neuronal activity. The mechanosensitive channel(MSC), which has low threshold of responding to ultrasound, may be the alternative solution. Sonogenetics is the technique that activates the MSC expressed in targeted neurons by low intensity ultrasound, thus achieve the neuromodulation. In this review, we introduce the mechanosensitive channel of large conductance, transient receptor potential, channels of the two-pore-domain potassium family, Piezo and the recent progress on their application in sonogenetics.

Single-particle cryo-electron microscopy opens new avenues in structural biology of G protein-coupled receptor
LI Chuntao,ZHANG Huibing,ZHANG Yan
J Zhejiang Univ (Med Sci), 2019, 48(1): 39-43.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.07
Abstract( 751 )   HTML( 22 )     PDF(1360KB)( 414 )

G protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors, mediating wide range of cellular and physiological processes through their transducers, G proteins and the-arrestins participate in almost all pathological processes. Recent technological advances are revolutionizing the utility of cryo-electron microscopy (cryo-EM), leading to a tremendous progress in the structural studies of biological macromolecules and cryo-EM has played a leading role in the structural biology of GPCR signaling complex. New discoveries of high-resolution three-dimensional structures of GPCR signaling complexes based on cryo-EM have emerged vigorously, which depict the common structural characteristics of intermolecular interaction between GPCR and G protein complex-the conformational changes of the transmembrane helix 6 of receptors, and also demonstrate the structural basis of G protein subtype selectivity. Single-particle cryo-EM becomes an efficient tool for identifying the molecular mechanism of receptor-ligand interaction, providing important information for understanding GPCR signaling and the structure-based drug design.

New inhibitors targeting bacterial RNA polymerase
SHI Jing,FENG Jue
J Zhejiang Univ (Med Sci), 2019, 48(1): 44-49.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.08
Abstract( 751 )   HTML( 22 )     PDF(1296KB)( 341 )

Rifamycins, a group of bacterial RNA polymerase inhibitors, are the first-line antimicrobial drugs to treat tuberculosis. In light of the emergence of rifamycin-resistant bacteria, development of new RNA polymerase inhibitors that kill rifamycin-resistant bacteria with high bioavailability is urgent. Structural analysis of bacterial RNA polymerase in complex with inhibitors by crystallography and cryo-EM indicates that RNA polymerase inhibitors function through five distinct molecular mechanisms: inhibition of the extension of short RNA; competition with substrates; inhibition of the conformational change of the ‘bridge helix’; inhibition of clamp opening; inhibition of clamp closure. This article reviews the research progress of these five groups of RNA polymerase inhibitors to provide references for the modification of existing RNA polymerase inhibitors and the discovery of new RNA polymerase inhibitors.

Expression of WT1 gene and its prognostic value in patients with acute myeloid leukemia
DU Dongfen,ZHU Lixia,WANG Yungui,YE XiujinG
J Zhejiang Univ (Med Sci), 2019, 48(1): 50-57.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.09
Abstract( 530 )   HTML( 15 )     PDF(842KB)( 205 )
Objective

To investigate the expression of Wilms’ tumor 1 (WT1) gene in patients with acute myeloid leukemia (AML), and to explore its application in predicting prognosis of AML in patients with wild or mutated nucleophosmin 1(NPM1) and Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD).

Methods

One hundred and sixty-seven newly diagnosed AML patients(exclued M3 type)were enrolled in this study. The survival of patients were analyzed with Kaplan-Meier method. The clinical data, laboratory findings and the survival of patients were analyzed and compared between patients with high WT1 expression (high-WT1 group) and those with low WT1 expression (low-WT1 group), as well as among the patients with NPM1 or FLT3-ITD wild type and mutants.

Results

The overall response rates (ORR) in high-WT1 and low-WT1 groups were 65.9% (83/126) and 95.1% (39/41), respectively (P<0.01). Compared with the low-WT1 group, the high-WT1 group had lower 2-y overall survival (OS) rate (46.1% vs. 75.2%,P<0.05) and 2-y disease free survival (DFS) rate (43.5% vs. 68.5%,P<0.05). After induction chemotherapy, the patients with decreased WT1 gene expression ≥1log was associated with higher ORR and 2-y OS rate (all P<0.05), but the advantage of 2-y DFS rate was not shown (P>0.05). In patients with NPM1 wild type, the high-WT1 group had inferior ORR and 2-y OS rate (all P<0.05), while in the patients with FLT3-ITD wild type, the high-WT1 group had inferior ORR, 2-y OS rate and 2-y DFS rate (all P<0.05). In patients with NPM1 or FLT3-ITD mutations, the WT1 expression had no significantly predicting values in treatment efficacy and survival (all P>0.05).

Conclusions

WT1 gene overexpression indicated poor prognosis of AML patients; the patients with decreased WT1 gene expression ≥1 log after the first induction therapy show better prognosis than those with<1 log. The WT1 gene expression level at diagnosis can be used as an unfavorable prognostic factor for AML patients with NPM1 or FLT3-ITD wild types.

Intrauterine infection affects early growth and neurobehavioral development in neonatal rats
SHEN Ying,SUN Yi,GU Weizhong,YU Huimin,YUAN Tianming
J Zhejiang Univ (Med Sci), 2019, 48(1): 58-64.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.10
Abstract( 918 )   HTML( 6 )     PDF(4367KB)( 155 )
Objective

To explore the effects of intrauterine infection on early growth and neurobehavioral development in neonatal rats.

Methods

Escherichia coli (E. coli) was inoculated into uterine cervix of pregnant rats with gestation of 15 d to establish the intrauterine infection model, and the effect on the delivery of pregnant rats was observed. The neonatal rat brain tissue was stained with Hematoxylin-Eosin and the cerebral white matter damage was assessed. Immunohistochemical staining and Western blot analysis were performed to evaluate the expression of glial fibrillary acidic protein (GFAP), 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and neurofilament (NF) in pup brains. Birth weight and early growth development indices were monitored, and neurobehavioral tests were performed to access the change of neurobehavioral development in neonatal rats.

Results

The white blood cell count increased significantly in the uterus and placenta of the pregnant rats after intrauterine E. coli infection and no significant impact was observed on the delivery of pregnant rats. Weak staining and focal rarefaction of cerebral white matter from rats at P7 in intrauterine infection group were observed. The expression of GFAP markedly increased (P<0.05) in infection group, while the level of CNPase and NF in pup brains at P7 significantly decreased (P<0.05 or P<0.01). Compared with control group, the neonatal rats in infection group had lower birth weight and slower weight gain during the suckling period (P<0.05 or P<0.01), and the completion times of ear opening, eye opening, surface righting, negative geotaxis, acoustic startle and swimming test in infection group were significantly delayed (P<0.05 or P<0.01). Conclussion: Intrauterine infection in pregnant rats can induce cerebral white matter damage and retardation of early growth and neurobehavioral development in neonatal rats.

Rictor regulates mitochondrial calcium signaling in mouse embryo stem cell-derived cardiomyocytes
SHAO Ying,WANG Jiadan,ZHU Danyan
J Zhejiang Univ (Med Sci), 2019, 48(1): 65-74.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.11
Abstract( 854 )   HTML( 19 )     PDF(8324KB)( 140 )

Objective: To explore the expression, localization and regulatory effect on mitochondrial calcium signaling of Rictor in embryonic stem cell-derived cardiomyocytes (ESC-CMs). Methods: Classical embryonic stem cell cardiomyogenesis model was used for differentiation of mouse embryonic stem cells into cardiomyocytes. The location of Rictor in ESC-CMs was investigated by immunofluorescence and Western blot. The expression of Rictor in mouse embryonic stem cells was interfered with lentiviral technology, then the superposition of mitochondria and endoplasmic reticulum (ER) in ESC-CMs was detected with immunofluorescence method; the cellular ultrastructure of ESC-CMs was observed by transmission electron microscope; the mitochondrial calcium transients of ESC-CMs was detected by living cell workstation; immunoprecipitation was used to detect the interaction between 1, 5, 5-trisphosphate receptor (IP3 receptor, IP3R), glucose-regulated protein 75 (Grp75) and voltage-dependent anion channel 1 (VDAC1) in mitochondrial outer membrane; the expression of mitochondrial fusion protein (mitonusin-2, Mfn2) was detected by Western blot. Results: Rictor was mainly localized in the endoplasmic reticulum and mitochondrial-endoplasmic reticulum membrane (MAM) in ESC-CMs. Immunofluorescence results showed that Rictor was highly overlapped with ER and mitochondria in ESC-CMs. After mitochondrial and ER were labeled with Mito-Tracker Red and ER-Tracker Green, it was demonstrated that the mitochondria of the myocardial cells in the Rictor group were scattered, and the superimposition rate of mitochondria and ER was lower than that of the negative control group (P < 0.01). The MAM structures were decreased in ESC-CMs after knockdown of Rictor. The results of the living cell workstation showed that the amplitude of mitochondrial calcium transients by ATP stimulation in ESC-CMs was decreased after knockdown of Rictor (P < 0.01). The results of co-immunoprecipitation showed that the interaction between IP3R, Grp75 and VDAC1 in the MAM structure of the cardiomyocytes in the Rictor group was significantly attenuated (P < 0.01); the results of Western blot showed that the expression of Mfn2 protein was significantly decreased (P < 0.01). Conclusion: Using lentiviral technology to interfere Rictor expression in mouse embryonic stem cells, the release of calcium from the endoplasmic reticulum to mitochondria in ESC-CMs decreases, which may be affected by reducing the interaction of IP3R, Grp75, VDAC1 and decreasing the expression of Mfn2, leading to the damage of MAM structure.

Relationship between temperament, parenting style and resilience of children aged 3-5 years
NIU Yubai,ZHANG Lingyan,HAO Zesheng,JI Yuzhu
J Zhejiang Univ (Med Sci), 2019, 48(1): 75-82.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.12
Abstract( 814 )   HTML( 18 )     PDF(642KB)( 441 )
Objective

To investigate the developmental characteristics of resilience in children aged 3-5, and to explore the relationship between temperament, parenting style and resilience.

Methods

A total of 570 preschoolers aged 3-5 years in Hangzhou participated in this study. The children’s teachers completed the assessment of the resilience scale of DECA-P2 (Devereux Early Childhood Assessment for Preschoolers Second Edition); the children’s parents completed assessment of temperament questionnaire CBQ (Children’s Behavior Questionnaire) and parenting style questionnaire PSDQ (Parenting Styles and Dimensions Questionnaire).

Results

Totally 432 valid questionnaires were retrieved with a recovery rate of 75.79%. The levels of initiative and self-regulation of 5-y children were higher than those of children aged 3 or 4 (all P<0.01); the level of attachment/relationship of 5-y children was higher than that of children aged 4 (P<0.01); the levels of initiative and self-regulation of girls were higher than those of boys (P<0.05 or P<0.01). The negative affect dimension of temperament was negatively correlated with resilience (all P<0.05), while the effortful control and authoritative parenting styles were positively correlated with resilience (all P<0.05). The negative affect and effortful control were able to partially predict resilience of children through authoritative parenting style (mediating effect were -0.0143 and 0.0363).

Conclusion

Preschoolers aged 3-5 years with different age and gender show differences in resilience, and parenting styles may play a mediating effect between temperament and resilience.

Risk factors of death in newborns with congenital diaphragmatic hernia
CHEN Dong,HU Yuanjun,WU Yurui,LI Xiaoying
J Zhejiang Univ (Med Sci), 2019, 48(1): 83-88.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.13
Abstract( 622 )   HTML( 16 )     PDF(598KB)( 265 )
Objective

To investigate risk factors of death in newborns with congenital diaphragmatic hernia (CDH).

Method

A total of 126 newborns with CDH from June 2012 to September 2018 were enrolled. Concomitant malformations were recorded by descriptive analysis. Newborns received surgical treatment (n=120) for CDH were divided into survival group and fatal group. The risk factors of death were analyzed by univariate and multivariate logistic regression and the ROC curve with generated with relevant variables.

Result

There were 55 CDH newborns with concomitant malformations (43.7%), including 20 cases (15.9%) with multi-malformation. Logistic regression analysis showed that premature rupture of membranes (PROM), postoperative atelectasis, long duration of postoperative mechanical ventilation, postoperative high oxygenation index (OI) were related to death (all P<0.05), and the delayed surgery was a protective factor (P<0.05). In ROC analysis of postoperative OI in predicting death, the area under the curve (AUC) was 0.841, with the cutoff value of 5.74, the sensibility and specificity of OI was 81.0% and 75.0%, respectively(P<0.01).

Conclusions

Newborns with CDH have a high rate of malformations. The risk factors of death were PROM, postoperative atelectasis, postoperative long duration of mechanical ventilation and higher postoperative OI, and delayed surgery may reduce mortality.

Mechanisms of herpes simplex virus latency and reactivation
SUN Boqiang,WANG Qiongyan,PAN Dongli
J Zhejiang Univ (Med Sci), 2019, 48(1): 89-101.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.14
Abstract( 1106 )   HTML( 29 )     PDF(1265KB)( 504 )

Herpes simplex virus (HSV), including HSV-1 and HSV-2, is an important pathogen that can cause many diseases. Usually these diseases are recurrent and incurable. After lytic infection on the surface of peripheral mucosa, HSV can enter sensory neurons and establish latent infection during which viral replication ceases. Moreover, latent virus can re-enter the replication cycle by reactivation and return to peripheral tissues to start recurrent infection. This ability to escape host immune surveillance during latent infection and to spread during reactivation is a viral survival strategy and the fundamental reason why no drug can completely eradicate the virus at present. Although there are many studies on latency and reactivation of HSV, and much progress has been made, many specific mechanisms of the process remain obscure or even controversial due to the complexity of this process and the limitations of research models. This paper reviews the major results of research on HSV latency and reactivation, and discusses future research directions in this field.

Advances in molecular mechanism of vascular remodeling in pulmonary arterial hypertension
XIAO Li,TONG Xiaoyong
J Zhejiang Univ (Med Sci), 2019, 48(1): 102-110.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.15
Abstract( 858 )   HTML( 16 )     PDF(881KB)( 702 )

Pulmonary arterial hypertension (PAH) is a clinical hemodynamic syndrome characterized by elevated pulmonary arterial pressure and pulmonary vascular resistance leading to right heart failure and death. Vascular remodeling is the most prominent histopathological feature of PAH, which is regulated by many factors. Endoplasmic reticulum stress, calcium disorder and mitochondrial dysfunction are involved in the vascular cell proliferation and apoptosis by regulating intracellular calcium homeostasis and cellular metabolism. Epigenetic phenomenon such as DNA damage and abnormal expression of miRNA are also involved in the regulation of abnormal proliferation of vascular cells. Vascular cell phenotype switching including endothelial-mesenchymal transition and smooth muscle cell phenotype switching play an important role in abnormal proliferation of vascular cells. Vascular remodeling is produced by a variety of cells and molecular pathways, and aiming at multiple targets which is expected to find a new breakthrough in the treatment of PAH, and to improve abnormal vascular remodeling, delay or even reverse the progression of PAH.

Progress on correlation between cell senescence and idiopathic pulmonary fibrosis
ZHAO Shihao,ZHANG Xue,KE Yuehai
J Zhejiang Univ (Med Sci), 2019, 48(1): 111-115.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.16
Abstract( 568 )   HTML( 15 )     PDF(490KB)( 315 )

Cellular senescence is a key factor driving age-related diseases. Recent studies have revealed that senescence-associated secretory phenotype, telomere attrition, epigenetic changes, and mitochondrial autophagy damage may mediate the pathogenesis of senescence-related idiopathic pulmonary fibrosis (IPF). Reducing the level of cellular senescence or clearing senescent cells can down-regulate the expression of fibrosis factors and alleviate the symptoms of IPF. In this review, we outlined the role and mechanism of cellular senescence in IPF.

Research advances on the role of exosomes in chemotherapy resistance of ovarian cancer
SHEN Xiameng,LYU Weiguo
J Zhejiang Univ (Med Sci), 2019, 48(1): 116-120.   https://doi.org/10.3785/j.issn.1008-9292.2019.02.17
Abstract( 578 )   HTML( 18 )     PDF(514KB)( 298 )

Chemotherapy resistance is one of the biggest challenges in treatment of ovarian cancer. Mounting evidence shows that the exosomes shedding from tumor cells are considered to be involved in chemotherapy resistance of ovarian cancer by enhanced exosomal export of drugs, transferring RNAs or proteins and interfering with the bioactivity of therapeutic anti-tumor antibodies. In this review, we display the correlation between exosomes and chemotherapy resistance of ovarian cancer, the mechanism of exosomes involved in chemotherapy resistance of ovarian cancer, and discuss the potential clinical values of exosomes in chemotherapy resistance of ovarian cancer.

17 articles