Publications by authors named "Aizhen Zhang"

29 Publications

  • Page 1 of 1

Piccolo is essential for the maintenance of mouse retina but not cochlear hair cell function.

Aging (Albany NY) 2021 Apr 21;13(8):11678-11695. Epub 2021 Apr 21.

School of Life Science and Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University, Jinan 250100, China.

Piccolo is a presynaptic protein with high conservation among different species, and the expression of Piccolo is extensive in vertebrates. Recently, a small fragment of Piccolo (Piccolino), arising due to the incomplete splicing of intron 5/6, was found to be present in the synapses of retinas and cochleae. However, the comprehensive function of Piccolo in the retina and cochlea remains unclear. In this study, we generated knockout mice using CRISPR-Cas9 technology to explore the function of Piccolo. Unexpectedly, whereas no abnormalities were found in the cochlear hair cells of the mutant mice, significant differences were found in the retinas, in which two layers (the outer nuclear layer and the outer plexiform layer) were absent. Additionally, the amplitudes of electroretinograms were significantly reduced and pigmentation was observed in the fundoscopy of the mutant mouse retinas. The expression levels of Bassoon, a homolog of Piccolo, as well as synapse-associated proteins CtBP1, CtBP2, Kif3A, and Rim1 were down-regulated. The numbers of ribbon synapses in the retinas of the mutant mice were also reduced. Altogether, the phenotype of -/- mice resembled the symptoms of retinitis pigmentosa (RP) in humans, suggesting might be a candidate gene of RP and indicates knockout mice are a good model for elucidating the molecular mechanisms of RP.
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http://dx.doi.org/10.18632/aging.202861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8109093PMC
April 2021

Deficiency for Lcn8 causes epididymal sperm maturation defects in mice.

Biochem Biophys Res Commun 2021 Apr 22;548:7-13. Epub 2021 Feb 22.

School of Life Science and Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University, Jinan, 250100, PR China. Electronic address:

Lipocalin family members, LCN8 and LCN9, are specifically expressed in the initial segment of mouse caput epididymis. However, the biological functions of the molecules in vivo are yet to be clarified. In this study, CRISPR/Cas9 technology was used to generate Lcn8 and Lcn9 knockout mice, respectively. Lcn8 and Lcn9 male mice showed normal spermatogenesis and fertility. In the cauda epididymis of Lcn8 male mice, morphologically abnormal sperm was increased significantly, the proportion of progressive motility sperm was decreased, the proportion of immobilized sperm was elevated, and the sperm spontaneous acrosome reaction (AR) frequency was increased. Conversely, the knockout of Lcn9 did not have any effect on the ratio of morphologically abnormal sperm, sperm motility, and sperm spontaneous AR frequencies. These results demonstrated the role of LCN8 in maintaining the sperm quality in the epididymis, and suggested that the deficiency of LCN8 leads to epididymal sperm maturation defects.
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http://dx.doi.org/10.1016/j.bbrc.2021.02.052DOI Listing
April 2021

Cdc14a has a role in spermatogenesis, sperm maturation and male fertility.

Exp Cell Res 2020 10 15;395(1):112178. Epub 2020 Jul 15.

School of Life Science and Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University, Jinan, 250100, PR China. Electronic address:

Cdc14a is an evolutionarily conserved dual-specific protein phosphatase, and it plays different roles in different organisms. Cdc14a mutations in human have been reported to cause male infertility, while the specific role of Cdc14a in regulation of the male reproductive system remains elusive. In the present study, we established a knockout mouse model to study the function of Cdc14a in male reproductive system. Cdc14a male mice were subfertile and they could only produce very few offspring. The number of sperm was decreased, the sperm motility was impaired, and the proportion of sperm with abnormal morphology was elevated in Cdc14a mice. When we mated Cdc14a male mice with wild-type (WT) female mice, fertilized eggs could be found in female fallopian tubes, however, the majority of these embryos died during development. Some empty spaces were observed in seminiferous tubule of Cdc14a testes. Compared with WT male mice, the proportions of pachytene spermatocytes were increased and germ cells stained with γH2ax were decreased in Cdc14a male mice, indicating that knockout of Cdc14a inhibited meiotic initiation. Subsequently, we analyzed the expression levels of some substrate proteins of Cdc14a, including Cdc25a, Wee1, and PR-Set7, and compared those with WT testes, in which the expression levels of these proteins were significantly increased in Cdc14a testes. Our results revealed that Cdc14a male mice are highly subfertile, and Cdc14a is essential for normal spermatogenesis and sperm function.
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http://dx.doi.org/10.1016/j.yexcr.2020.112178DOI Listing
October 2020

Simultaneous Study of Anti-Ferroptosis and Antioxidant Mechanisms of Butein and ()-Butin.

Molecules 2020 Feb 5;25(3). Epub 2020 Feb 5.

School of Basic Medical Science, Guangzhou University of Chinese Medicine, Guangzhou 510006, China.

To elucidate the mechanism of anti-ferroptosis and examine structural optimization in natural phenolics, cellular and chemical assays were performed with 2'-hydroxy chalcone butein and dihydroflavone ()-butin. C11-BODIPY staining and flow cytometric assays suggest that butein more effectively inhibits ferroptosis in erastin-treated bone marrow-derived mesenchymal stem cells than ()-butin. Butein also exhibited higher antioxidant percentages than ()-butin in five antioxidant assays: linoleic acid emulsion assay, Fe-reducing antioxidant power assay, Cu-reducing antioxidant power assay, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO)-trapping assay, and α,α-diphenyl-β-picrylhydrazyl radical (DPPH)-trapping assay. Their reaction products with DPPH were further analyzed using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS). Butein and ()-butin produced a butein 5,5-dimer ( 542, 271, 253, 225, 135, and 91) and a ()-butin 5',5'-dimer ( 542, 389, 269, 253, and 151), respectively. Interestingly, butein forms a cross dimer with ()-butin ( 542, 523, 433, 419, 415, 406, and 375). Therefore, we conclude that butein and (S)-butin exert anti-ferroptotic action via an antioxidant pathway (especially the hydrogen atom transfer pathway). Following this pathway, butein and (S)-butin yield both self-dimers and cross dimers. Butein displays superior antioxidant or anti-ferroptosis action to (S)-butin. This can be attributed the decrease in π-π conjugation in butein due to saturation of its α,β-double bond and loss of its 2'-hydroxy group upon biocatalytical isomerization.
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http://dx.doi.org/10.3390/molecules25030674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036861PMC
February 2020

Role of CCR7 on dendritic cell‑mediated immune tolerance in the airways of allergy‑induced asthmatic rats.

Mol Med Rep 2019 Nov 20;20(5):4425-4432. Epub 2019 Sep 20.

Department of Respiration Medicine, People's Hospital of Shanxi Province, Taiyuan, Shanxi 030001, P.R. China.

Dendritic cells (DCs) have an important role in initiating and maintaining the immune inflammatory response in allergic asthma, and CC chemokine receptor 7 (CCR7) is directly involved in the pathogenesis of DC‑ and T cell‑mediated allergic asthma. The present study aimed to investigate the effects of CCR7 on DC‑mediated immune tolerance in allergic asthma. In the present study, bone marrow‑derived DCs were transfected with an adenovirus encoding the rat CCR7 gene or a short hairpin RNA targeting CCR7 (sh‑CCR7). Rats injected with DCs overexpressing CCR7 or presenting CCR7 knockdown were examined. After the rats were injected with DCs via the tail vein, bronchoalveolar lavage fluid was collected to assess its cellular composition. The protein expression levels of CCR7 in DCs were determined using immunohistochemistry and western blot analysis. The protein expression levels of interferon‑γ (IFN‑γ), interleukin‑4 (IL‑4), IL‑10, IL‑12, transforming growth factor‑β (TGF‑β) and immunoglobulin E (IgE) were determined by ELISA. Compared with the control group, the protein expression level of CCR7 was significantly higher in the CCR7 overexpression group and significantly lower in sh‑CCR7 group. Similarly, the number of DCs was higher in the CCR7 overexpression group and lower in the sh‑CCR7 group. The protein expression levels of IL‑10 and TGF‑β were significantly lower in the CCR7 overexpression group and higher in the sh‑CCR7 group. In addition, the expression levels of IL‑4, IL‑12, IFN‑γ and IgE were higher in the CCR7 overexpression group and lower in the sh‑CCR7 group. The present results suggested that the role of cytokines and IgE in immune inflammation and immune tolerance in allergic asthma may be associated with the expression level of CCR7 in DCs, suggesting that CCR7 may serve a role in DC‑mediated immune tolerance in allergic asthma.
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http://dx.doi.org/10.3892/mmr.2019.10694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797982PMC
November 2019

A knock-in mouse model of Pendred syndrome with Slc26a4 L236P mutation.

Biochem Biophys Res Commun 2019 07 30;515(2):359-365. Epub 2019 May 30.

Institute of Developmental Biology, School of Life Science, Shandong University, Jinan, Shandong, China. Electronic address:

SLC26A4 gene mutations lead to Pendred syndrome and non-syndromic hearing loss (DFNB4). The mouse model is well used to study the pathology of Pendred syndrome, however, mice with different Slc26a4 mutations exhibit different phenotypes, and these mice have severe deafness and inner ear malformations that are not imitated less severely Human phenotype. In this study, we generated a knock-in mouse model of Pendred syndrome with Slc26a4 L236P mutation to mimic the most common mutation found in human. Some L236P mice were observed to have significant vestibular dysfunction including torticollis and circling, the giant otoconia and destruction of the otoconial membrane was observed in L236P mice. Unlike other profoundly deafness in Slc26a4 mouse model, L236P mice present mild to profound hearing loss, consistent with the hearing threshold, inner ear hair cells also lost from slight to significant. Together, these data demonstrate that the L236P mouse phenotype is more similar to the human phenotype and should be used as a tool for further research into the human Pendred syndrome.
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http://dx.doi.org/10.1016/j.bbrc.2019.05.157DOI Listing
July 2019

Ufm1-Specific Ligase Ufl1 Regulates Endoplasmic Reticulum Homeostasis and Protects Against Heart Failure.

Circ Heart Fail 2018 10;11(10):e004917

Vascular Biology Center (J.L., G.Y., W.M., A.Z., J.Z., H.S.), Medical College of Georgia, Augusta University.

Background: Defects in protein homeostasis are sufficient to provoke cardiac remodeling and dysfunction. Although posttranslational modifications by ubiquitin and ubiquitin-like proteins are emerging as an important regulatory mechanism of protein function, the role of Ufm1 (ubiquitin-fold modifier 1)-a novel ubiquitin-like protein-has not been explored in either the normal or stressed heart.

Methods And Results: Western blotting revealed that Ufl1 (Ufm1-specific E3 ligase 1)-an enzyme essential for Ufm1 modification-was increased in hypertrophic mouse hearts but reduced in the failing hearts of patients with dilated cardiomyopathy. To determine the functional role of Ufl1 in the heart, we generated a cardiac-specific knockout mouse and showed that Ufl1-deficient mice developed age-dependent cardiomyopathy and heart failure, as indicated by elevated cardiac fetal gene expression, increased fibrosis, and impaired cardiac contractility. When challenged with pressure overload, Ufl1-deficient hearts exhibited remarkably greater hypertrophy, exacerbated fibrosis, and worsened cardiac contractility compared with control counterparts. Transcriptome analysis identified that genes associated with the endoplasmic reticulum (ER) function were dysregulated in Ufl1-deficient hearts. Biochemical analysis revealed that excessive ER stress preceded and deteriorated along with the development of cardiomyopathy in Ufl1-deficient hearts. Mechanistically, Ufl1 depletion impaired (PKR-like ER-resident kinase) signaling and aggravated cardiomyocyte cell death after ER stress. Administration of the chemical ER chaperone tauroursodeoxycholic acid to Ufl1-deficient mice alleviated ER stress and attenuated pressure overload-induced cardiac dysfunction.

Conclusions: Our results advance a novel concept that the Ufm1 system is essential for cardiac homeostasis through regulation of ER function and that upregulation of myocardial Ufl1 could be protective against heart failure.
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http://dx.doi.org/10.1161/CIRCHEARTFAILURE.118.004917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205741PMC
October 2018

Tuberous sclerosis complex-mediated mTORC1 overactivation promotes age-related hearing loss.

J Clin Invest 2018 11 24;128(11):4938-4955. Epub 2018 Sep 24.

School of Life Science and Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University, Jinan, China.

The underlying molecular mechanisms of age-related hearing loss (ARHL) in humans and many strains of mice have not been fully characterized. This common age-related disorder is assumed to be closely associated with oxidative stress. Here, we demonstrate that mTORC1 signaling is highly and specifically activated in the cochlear neurosensory epithelium (NSE) in aging mice, and rapamycin injection prevents ARHL. To further examine the specific role of mTORC1 signaling in ARHL, we generated murine models with NSE-specific deletions of Raptor or Tsc1, regulators of mTORC1 signaling. Raptor-cKO mice developed hearing loss considerably more slowly than WT littermates. Conversely, Tsc1 loss led to the early-onset death of cochlear hair cells and consequently accelerated hearing loss. Tsc1-cKO cochleae showed features of oxidative stress and impaired antioxidant defenses. Treatment with rapamycin and the antioxidant N-acetylcysteine rescued Tsc1-cKO hair cells from injury in vivo. In addition, we identified the peroxisome as the initial signaling organelle involved in the regulation of mTORC1 signaling in cochlear hair cells. In summary, our findings identify overactive mTORC1 signaling as one of the critical causes of ARHL and suggest that reduction of mTORC1 activity in cochlear hair cells may be a potential strategy to prevent ARHL.
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http://dx.doi.org/10.1172/JCI98058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205401PMC
November 2018

Tprn is essential for the integrity of stereociliary rootlet in cochlear hair cells in mice.

Front Med 2019 Dec 30;13(6):690-704. Epub 2018 Aug 30.

School of Life Science, Shandong University, Jinan, 250100, China.

Tprn encodes the taperin protein, which is concentrated in the tapered region of hair cell stereocilia in the inner ear. In humans, TPRN mutations cause autosomal recessive nonsyndromic deafness (DFNB79) by an unknown mechanism. To determine the role of Tprn in hearing, we generated Tprn-null mice by clustered regularly interspaced short palindromic repeat/Cas9 genome-editing technology from a CBA/CaJ background. We observed significant hearing loss and progressive degeneration of stereocilia in the outer hair cells of Tprn-null mice starting from postnatal day 30. Transmission electron microscopy images of stereociliary bundles in the mutant mice showed some stereociliary rootlets with curved shafts. The central cores of the stereociliary rootlets possessed hollow structures with surrounding loose peripheral dense rings. Radixin, a protein expressed at stereocilia tapering, was abnormally dispersed along the stereocilia shafts in Tprn-null mice. The expression levels of radixin and β-actin significantly decreased.We propose that Tprn is critical to the retention of the integrity of the stereociliary rootlet. Loss of Tprn in Tprn-null mice caused the disruption of the stereociliary rootlet, which resulted in damage to stereociliary bundles and hearing impairments. The generated Tprn-null mice are ideal models of human hereditary deafness DFNB79.
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http://dx.doi.org/10.1007/s11684-018-0638-8DOI Listing
December 2019

Knock-In Mice with Myo3a Y137C Mutation Displayed Progressive Hearing Loss and Hair Cell Degeneration in the Inner Ear.

Neural Plast 2018 5;2018:4372913. Epub 2018 Jul 5.

School of Life Science, Shandong University, Jinan 250100, China.

Myo3a is expressed in cochlear hair cells and retinal cells and is responsible for human recessive hereditary nonsyndromic deafness (DFNB30). To investigate the mechanism of DFNB30-type deafness, we established a mouse model of Myo3a kinase domain Y137C mutation by using CRISPR/Cas9 system. No difference in hearing between 2-month-old Myo3a mutant mice and wild-type mice was observed. The hearing threshold of the ≥6-month-old mutant mice was significantly elevated compared with that of the wild-type mice. We observed degeneration in the inner ear hair cells of 6-month-old Myo3a mutant mice, and the degeneration became more severe at the age of 12 months. We also found structural abnormality in the cochlear hair cell stereocilia. Our results showed that Myo3a is essential for normal hearing by maintaining the intact structure of hair cell stereocilia, and the kinase domain plays a critical role in the normal functions of Myo3a. This mouse line is an excellent model for studying DFNB30-type deafness in humans.
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http://dx.doi.org/10.1155/2018/4372913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6079384PMC
November 2018

Oocyte-specific deletion of Gα induces oxidative stress and deteriorates oocyte quality in mice.

Exp Cell Res 2018 09 17;370(2):579-590. Epub 2018 Jul 17.

School of Life Science and Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University, Jinan 250100, PR China. Electronic address:

The stimulatory heterotrimeric G protein alpha subunit (Gα) is a ubiquitous guanine nucleotide-binding protein that regulates the intracellular cAMP signaling pathway and consequently participates in a wide range of biological events. In the reproductive system, despite Gα being associated with oocyte meiotic arrest in vitro, the exact role of Gα in female fertility in vivo remains largely unknown. Here, we generated oocyte-specific Gα knockout mice by using the Cre/LoxP system. We observed that the deletion of Gα caused complete female infertility. Exclusion of post-implantation abnormalities, oogenesis, fertilization, and early embryo development was subsequently monitored; meiosis in Gα-deficient oocytes precociously resumed in only 43% of antral follicles from mutant mice, indicating that alteration of meiotic pause was not the key factor in infertility. Ovulation process and number were normal, but the rate of morphological abnormal oocytes was apparently increased; spindle organization, fertilization, and early embryo development were impaired. Furthermore, the level of ROS (reactive oxygen species) and the mitochondrial aggregation increased, and antioxidant glutathione (GSH) content, ATP level, mtDNA copy number, and mitochondrial membrane potential decreased in Gα-deficient oocytes. GV oocytes from mutant mice showed early-stage apoptosis. Meanwhile, the Gα knockout-induced decline in oocyte quality and low developmental potential was partially rescued by antioxidant supplementation. To sum up, our results are the first to reveal that the profile of Gα oocyte-specific deletion caused female infertility in vivo, and oxidative stress plays an important role in this event.
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http://dx.doi.org/10.1016/j.yexcr.2018.07.023DOI Listing
September 2018

Protein kinase Cα stimulates hypoxia‑induced pulmonary artery smooth muscle cell proliferation in rats through activating the extracellular signal‑regulated kinase 1/2 pathway.

Mol Med Rep 2017 Nov 12;16(5):6814-6820. Epub 2017 Sep 12.

Department of Respiratory and Critical Care Medicine, Shanxi Provincial People's Hospital Affiliated to Shanxi Medical University, Taiyuan, Shanxi 030012, P.R. China.

Hypoxic pulmonary hypertension (HPH) may contribute to vascular remodeling, and pulmonary artery smooth muscle cell (PASMC) proliferation has an important role in this process. However, no relevant information concerning the role and mechanism of protein kinase C (PKC)α in hypoxia‑induced rat PASMC proliferation has been elucidated. The present study aimed to further investigate this by comparison of rat PASMC proliferation among normoxia for 72 h (21% O2), hypoxia for 72 h (3% O2), hypoxia + promoter 12‑myristate 13‑acetate control, hypoxia + safingol control, hypoxia + PD98059 control and hypoxia + U0126 control groups. The present study demonstrated that protein expression levels of PKCα in rat PASMCs were elevated. In conclusion, through activating the extracellular signal‑regulated 1/2 signaling pathway, PKCα is involved in and initiates PASMC proliferation, thus bringing about pulmonary artery hypertension. These results add to the understanding of the mechanism PKCα in PH formation and lays a theoretical basis for prevention as well as treatment of HPH.
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http://dx.doi.org/10.3892/mmr.2017.7478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5865839PMC
November 2017

Loss of Myh14 Increases Susceptibility to Noise-Induced Hearing Loss in CBA/CaJ Mice.

Neural Plast 2016 22;2016:6720420. Epub 2016 Dec 22.

School of Life Science and Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University, Jinan 250100, China.

MYH14 is a member of the myosin family, which has been implicated in many motile processes such as ion-channel gating, organelle translocation, and the cytoskeleton rearrangement. Mutations in MYH14 lead to a DFNA4-type hearing impairment. Further evidence also shows that MYH14 is a candidate noise-induced hearing loss (NIHL) susceptible gene. However, the specific roles of MYH14 in auditory function and NIHL are not fully understood. In the present study, we used CRISPR/Cas9 technology to establish a Myh14 knockout mice line in CBA/CaJ background (now referred to as Myh14 mice) and clarify the role of MYH14 in the cochlea and NIHL. We found that Myh14 mice did not exhibit significant hearing loss until five months of age. In addition, Myh14 mice were more vulnerable to high intensity noise compared to control mice. More significant outer hair cell loss was observed in Myh14 mice than in wild type controls after acoustic trauma. Our findings suggest that Myh14 may play a beneficial role in the protection of the cochlea after acoustic overstimulation in CBA/CaJ mice.
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http://dx.doi.org/10.1155/2016/6720420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215640PMC
October 2017

Loss of liver kinase B1 causes planar polarity defects in cochlear hair cells in mice.

Front Med 2016 Dec 23;10(4):481-489. Epub 2016 Dec 23.

School of Life Science and Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University, Jinan, 250100, China.

The tumor suppressor gene liver kinase B1 (LKB1), also called STK11, encodes a serine/threonine kinase. LKB1 plays crucial roles in cell differentiation, proliferation, and polarity. In this study, LKB1 conditional knockout mice (LKB1 CKO mice) were generated using Pax2-Cre mice to investigate the function of LKB1 in inner ear hair cells during early embryonic period. LKB1 CKO mice died perinatally. Immunofluorescence and scanning electron microscopy revealed that stereociliary bundles in LKB1 CKO mice were clustered and misoriented, respectively. Moreover, ectopic distribution of kinocilium bundles resulting from abnormal migration of kinocilium was observed in the mutant mice. The orientation of stereociliary bundles and the migration of kinocilia are critical indicators of planar cell polarity (PCP) of hair cells. LKB1 deficiency in LKB1 CKO mice thus disrupted hair cell planar polarity during embryonic development. Our results suggest that LKB1 is required in PCP formation in cochlear hair cells in mice.
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http://dx.doi.org/10.1007/s11684-016-0494-3DOI Listing
December 2016

Loss of Lysyl Oxidase-like 3 Attenuates Embryonic Lung Development in Mice.

Sci Rep 2016 09 20;6:33856. Epub 2016 Sep 20.

School of Life Science and Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University, Jinan 250100, China.

Lysyl oxidase-like 3 (LOXL3), a human disease gene candidate, is a member of the lysyl oxidase (LOX) family and is indispensable for mouse palatogenesis and vertebral column development. Our previous study showed that the loss of LOXL3 resulted in a severe cleft palate and spinal deformity. In this study, we investigated a possible role for LOXL3 in mouse embryonic lung development. LOXL3-deficient mice displayed reduced lung volumes and weights, diminished saccular spaces, and deformed and smaller thoracic cavities. Excess elastic fibres were detected in LOXL3-deficient lungs, which might be related to the increased LOXL4 expression. Increased transforming growth factor β1 (TGFβ1) expression might be involved in the up-regulation of LOXL4 in LOXL3-deficient lungs. We concluded that the loss of LOXL3 attenuates mouse embryonic lung development.
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http://dx.doi.org/10.1038/srep33856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5029289PMC
September 2016

Abnormal mRNA splicing but normal auditory brainstem response (ABR) in mice with the prestin (SLC26A5) IVS2-2A>G mutation.

Mutat Res 2016 08 12;790:1-7. Epub 2016 May 12.

School of Life Science and Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University, Jinan 250100, China. Electronic address:

Prestin is critical to OHC somatic motility and hearing sensitivity in mammals. Several mutations of the human SLC26A5 gene have been associated with deafness. However, whether the IVS2-2A>G mutation in the human SLC26A5 gene causes deafness remains controversial. In this study, we created a mouse model in which the IVS2-2A>G mutation was introduced into the mouse Slc26a5 gene by gene targeting. The homozygous Slc26a5 mutant mice were viable and fertile and displayed normal hearing sensitivity by ABR threshold analysis. Whole-mount immunostaining using prestin antibody demonstrated that prestin was correctly targeted to the lateral wall of OHCs, and no obvious hair cell loss occurred in mutant mice. No significant difference in the amount of prestin protein was observed between mutants and controls using western blot analysis. In OHCs isolated from mutants, the NLC was also normal. However, we observed a splicing abnormality in the Slc26a5 mRNA of the mutant mice. Eleven nucleotides were missing from the 5' end of exon 3 in Slc26a5 mRNA, but the normal ATG start codon in exon 3 was still detected. Thus, the IVS2-2A>G mutation in the Slc26a5 gene is insufficient to cause hearing loss in mice.
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http://dx.doi.org/10.1016/j.mrfmmm.2016.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345126PMC
August 2016

LKB1 Regulates Cerebellar Development by Controlling Sonic Hedgehog-mediated Granule Cell Precursor Proliferation and Granule Cell Migration.

Sci Rep 2015 Nov 9;5:16232. Epub 2015 Nov 9.

School of Life Science and Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University, Jinan 250100, China.

The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1(Atoh1) CKO) to investigate the function of LKB1 in cerebellar development. The LKB1(Atoh1) CKO mice displayed motor dysfunction. In the LKB1(Atoh1) CKO cerebellum, the overall structure had a larger volume and more lobules. LKB1 inactivation led to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1(Atoh1) CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development.
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http://dx.doi.org/10.1038/srep16232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4637891PMC
November 2015

1α,25-Dihydroxyvitamin D3 prevents the differentiation of human lung fibroblasts via microRNA-27b targeting the vitamin D receptor.

Int J Mol Med 2015 Oct 20;36(4):967-74. Epub 2015 Aug 20.

Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China.

Pulmonary fibroblasts have key roles in the formation and maintenance of lung structure and function, and are involved in tissue repair and remodeling. Transforming growth factor‑β1 (TGF‑β1) induces differentiation of fibroblasts into myofibroblasts, the key effector cells in fibrotic states, which are characterized by the expression of α‑smooth muscle actin (α‑SMA) markers. 1α,25‑Dihydroxyvitamin D3 [1,25(OH)2D3] has been implicated in regulating differentiation, and the vitamin D receptor (VDR) may be a regulator of TGF‑β signaling. In addition, there is presently only limited information regarding microRNA (miRNA) regulation of lung fibroblast differentiation. To determine the role of 1,25(OH)2D3 in regulating the differentiation of fibroblasts induced by TGF‑β1 and the functional importance of miR‑27b, cell culture systems, cell transfection and the 3' untranslated region (3'UTR) luciferase assay were employed. 1,25(OH)2D3 inhibited differentiation and downregulated miR‑27b expression in human lung fibroblasts induced by TGF‑β1. In addition, human lung fibroblasts were transfected with miR‑27b mimic or miR‑27b inhibitor, and demonstrated that the overexpression of miR‑27b decreased the VDR protein expression and increased the expression of α‑SMA, while reducing levels of miR‑27b had opposing effects. Finally, the luciferase reporter assays were performed to confirm that miR‑27b directly targeted VDR 3'UTR. Taken together, these results suggest that 1,25(OH)2D3 inhibits lung fibroblast differentiation induced by TGF‑β1 via miR‑27b targeting VDR 3'UTR, which may be used as a novel treatment strategy in differentiation pathways.
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http://dx.doi.org/10.3892/ijmm.2015.2318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564074PMC
October 2015

Loss of lysyl oxidase-like 3 causes cleft palate and spinal deformity in mice.

Hum Mol Genet 2015 Nov 24;24(21):6174-85. Epub 2015 Aug 24.

Institute of Developmental Biology, School of Life Science, Shandong University, 27 Shanda Nanlu, Jinan 250100, China

In mammals, embryonic development are highly regulated morphogenetic processes that are tightly controlled by genetic elements. Failure of any one of these processes can result in embryonic malformation. The lysyl oxidase (LOX) family genes are closely related to human diseases. In this study, we investigated the essential role of lysyl oxidase-like 3 (LOXL3), a member of the LOX family, in embryonic development. Mice lacking LOXL3 exhibited perinatal lethality, and the deletion of the Loxl3 gene led to impaired development of the palate shelves, abnormalities in the cartilage primordia of the thoracic vertebrae and mild alveolar shrinkage. We found that the obvious decrease of collagen cross-links in palate and spine that was induced by the lack of LOXL3 resulted in cleft palate and spinal deformity. Thus, we provide critical in vivo evidence that LOXL3 is indispensable for mouse palatogenesis and vertebral column development. The Loxl3 gene may be a candidate disease gene resulting in cleft palate and spinal deformity.
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http://dx.doi.org/10.1093/hmg/ddv333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599675PMC
November 2015

LKB1 Is Required for the Development and Maintenance of Stereocilia in Inner Ear Hair Cells in Mice.

PLoS One 2015 14;10(8):e0135841. Epub 2015 Aug 14.

Institute of Developmental Biology, School of Life Science, Shandong University, Jinan, Shandong, China.

The LKB1 gene, which encodes a serine/threonine kinase, was discovered to play crucial roles in cell differentiation, proliferation, and the establishment of cell polarity. In our study, LKB1 conditional knockout mice (Atoh1-LKB1-/- mice) were generated to investigate LKB1 function in the inner ear. Tests of auditory brainstem response and distortion product otoacoustic emissions revealed significant decreases in the hearing sensitivities of the Atoh1-LKB1-/- mice. In Atoh1-LKB1-/- mice, malformations of hair cell stereocilliary bundles were present as early as postnatal day 1 (P1), a time long before the maturation of the hair cell bundles. In addition, we also observed outer hair cell (OHC) loss starting at P14. The impaired stereocilliary bundles occurred long before the presence of hair cell loss. Stereociliary cytoskeletal structure depends on the core actin-based cytoskeleton and several actin-binding proteins. By Western blot, we examined actin-binding proteins, specifically ERM (ezrin/radixin/moesin) proteins involved in the regulation of the actin cytoskeleton of hair cell stereocilia. Our results revealed that the phosphorylation of ERM proteins (pERM) was significantly decreased in mutant mice. Thus, we propose that the decreased pERM may be a key factor for the impaired stereocillia function, and the damaged stereocillia may induce hair cell loss and hearing impairments. Taken together, our data indicates that LKB1 is required for the development and maintenance of stereocilia in the inner ear.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135841PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537123PMC
May 2016

Accelerated hepatocellular carcinoma development in CUL4B transgenic mice.

Oncotarget 2015 Jun;6(17):15209-21

Key Laboratory of Experimental Teratology, Ministry of Education, Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, China.

Cullin 4B (CUL4B) is a component of the Cullin 4B-Ring E3 ligase (CRL4B) complex that functions in proteolysis and in epigenetic regulation. CUL4B possesses tumor-promoting properties and is markedly upregulated in many types of human cancers. To determine the role of CUL4B in liver tumorigenesis, we generated transgenic mice that expressed human CUL4B in livers and other tissues and evaluated the development of spontaneous and chemically-induced hepatocellular carcinomas. We observed that CUL4B transgenic mice spontaneously developed liver tumors at a high incidence at old ages and exhibited enhanced DEN-induced hepatocarcinogenesis. There was a high proliferation rate in the livers of CUL4B transgenic mice that was accompanied by increased levels of Cdk1, Cdk4 and cyclin D1 and decreased level of p16. The transgenic mice also exhibited increased compensatory proliferation after DEN-induced liver injury, which was accompanied by activation of Akt, Erk, p38 and NF-κB. We also found that Prdx3 was downregulated and that DEN induced a higher level of reactive oxygen species in the livers of transgenic mice. Together, our results demonstrate a critical role of CUL4B in hepatocarcinogenesis in mice.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4558146PMC
http://dx.doi.org/10.18632/oncotarget.3829DOI Listing
June 2015

Trio gene is required for mouse learning ability.

Brain Res 2015 May 26;1608:82-90. Epub 2015 Feb 26.

Key Laboratory of the Ministry of Education for Experimental Teratology and School of Life Science, Shandong University, Jinan 250100, China. Electronic address:

Trio is a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Trio regulates cytoskeleton dynamics and actin remodeling and is involved in cell migration and axonal guidance in neuronal development. The null allele of the Trio gene led to embryonic lethality, and Trio null embryos displayed aberrant organization in several regions of the brain at E18.5, including hippocampus. Nestin-Trio-/- mice, in which the Trio gene was deleted specifically in the neuronal system by the Nestin-Cre system, displayed severe phenotypes, including low survival rate, ataxia and multiple developmental defects of the cerebellum. All Nestin-Trio-/- mice died before reaching adulthood, which hinders research on Trio gene function in adult mice. Thus, we generated EMX1-Trio-/- mice by crossing Trio-floxed mice with EMX1-Cre mice in which Cre is expressed in the brain cortex and hippocampus. EMX1-Trio-/- mice can survive to adulthood. Trio gene deletion results in smaller brains, an abnormal hippocampus and disordered granule cells in the dentate gyrus (DG) and cornu ammonis (CA). Behavior tests showed that Trio deletion interfered with the hippocampal-dependent spatial learning in the mice, suggesting that Trio plays critical roles in the learning ability of adult mice. We conclude that the Trio gene regulates the neuronal development of the hippocampus and that it affects the intelligence of adult mice.
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http://dx.doi.org/10.1016/j.brainres.2015.02.040DOI Listing
May 2015

Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice.

Dev Biol 2014 Nov 19;395(1):167-81. Epub 2014 Jul 19.

Key Laboratory of the Ministry of Education for Experimental Teratology and School of Life Science, Shandong University, Jinan 250100, China. Electronic address:

Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs.
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http://dx.doi.org/10.1016/j.ydbio.2014.07.007DOI Listing
November 2014

Comprehensive evaluation and clinical implementation of commercially available Monte Carlo dose calculation algorithm.

J Appl Clin Med Phys 2013 Mar 4;14(2):4062. Epub 2013 Mar 4.

Department of Radiation Oncology, Wayne State University School of Medicine, Detroit, MI, USA.

A commercial electron Monte Carlo (eMC) dose calculation algorithm has become available in Eclipse treatment planning system. The purpose of this work was to evaluate the eMC algorithm and investigate the clinical implementation of this system. The beam modeling of the eMC algorithm was performed for beam energies of 6, 9, 12, 16, and 20 MeV for a Varian Trilogy and all available applicator sizes in the Eclipse treatment planning system. The accuracy of the eMC algorithm was evaluated in a homogeneous water phantom, solid water phantoms containing lung and bone materials, and an anthropomorphic phantom. In addition, dose calculation accuracy was compared between pencil beam (PB) and eMC algorithms in the same treatment planning system for heterogeneous phantoms. The overall agreement between eMC calculations and measurements was within 3%/2 mm, while the PB algorithm had large errors (up to 25%) in predicting dose distributions in the presence of inhomogeneities such as bone and lung. The clinical implementation of the eMC algorithm was investigated by performing treatment planning for 15 patients with lesions in the head and neck, breast, chest wall, and sternum. The dose distributions were calculated using PB and eMC algorithms with no smoothing and all three levels of 3D Gaussian smoothing for comparison. Based on a routine electron beam therapy prescription method, the number of eMC calculated monitor units (MUs) was found to increase with increased 3D Gaussian smoothing levels. 3D Gaussian smoothing greatly improved the visual usability of dose distributions and produced better target coverage. Differences of calculated MUs and dose distributions between eMC and PB algorithms could be significant when oblique beam incidence, surface irregularities, and heterogeneous tissues were present in the treatment plans. In our patient cases, monitor unit differences of up to 7% were observed between PB and eMC algorithms. Monitor unit calculations were also preformed based on point-dose prescription. The eMC algorithm calculation was characterized by deeper penetration in the low-density regions, such as lung and air cavities. As a result, the mean dose in the low-density regions was underestimated using PB algorithm. The eMC computation time ranged from 5 min to 66 min on a single 2.66 GHz desktop, which is comparable with PB algorithm calculation time for the same resolution level.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5714370PMC
http://dx.doi.org/10.1120/jacmp.v14i2.4062DOI Listing
March 2013

Increased plasma n-3 polyunsaturated fatty acid is associated with improved insulin sensitivity in type 2 diabetes in China.

Mol Nutr Food Res 2010 May;54 Suppl 1:S112-9

Department of Food Science and Nutrition, Zhejiang University, Hangzhou, P. R. China.

Increased tissue n-3 polyunsaturated fatty acid (PUFA) is associated with improved insulin sensitivity in type 2 diabetes. However, this relationship among Chinese is not clear. To investigate the relationship between plasma phospholipids (PL) fatty acid composition and insulin resistance (IR) in type 2 diabetes mellitus, 186 type 2 diabetes and 180 healthy subjects were studied in this case-control study. In the sex, age and BMI controlled partial correlation, homeostasis model assessment (HOMA)-IR and blood glucose was significantly negatively correlated with plasma PL n-3 PUFA, 20:5n-3 and ratio of n-3:n-6 (p<0.01), and positively correlated with n-6 PUFA (p<0.001) and saturated fatty acid (p<0.05) in the diabetes patients. PL 22:6n-3 was also significantly negatively correlated with HOMA-IR (p<0.01), but not with blood glucose. Fasting insulin was significantly negatively correlated with plasma PL n-3 PUFA, 20:5n-3, 22:6n-3 and ratio of n-3:n-6 (p<0.01). The 18:3n-3 was not associated with HOMA-IR and fasting insulin. The results suggested that increased plasma PL n-3 PUFA, 20:5n-3, 22:6n-3 and ratio of n-3:n-6 PUFA was associated with decreased HOMA-IR in type 2 diabetes. Increased plasma PL n-3 PUFA improves insulin sensitivity in type 2 diabetes.
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http://dx.doi.org/10.1002/mnfr.200900189DOI Listing
May 2010

Diacylglycerol-induced improvement of whole-body insulin sensitivity in type 2 diabetes mellitus: a long-term randomized, double-blind controlled study.

Clin Nutr 2008 Apr 7;27(2):203-11. Epub 2008 Mar 7.

Department of Food Science and Nutrition, Zhejiang University, 268 Kaixuan Road, Hangzhou, Zhejiang 310029, China.

Background & Aims: Diacylglycerol oil has been shown to lower postprandial and fasting serum triacylglycerol levels and reduce body fat. The aim of this study was to investigate the effect of diacylglycerol oil on risk factors of type 2 diabetes mellitus (DM) and cardiovascular disease in type 2 DM patients.

Methods: This was a double-blind controlled parallel study with 127 type 2 DM patients (aged 40-65) recruited in Hangzhou, China. All subjects consumed triacylglycerol oil in the lead-in period (14 days), then they were randomly divided into two groups and consumed diacylglycerol or triacylglycerol oil with a similar fatty acid composition (25 g/day) for 120 days. Blood samples were collected on days 0, 60 and 120 and risk factors of type 2 DM and cardiovascular disease and biochemical parameters were measured by standard methods.

Results: There were a total of 112 subjects who completed the study. Diet intake did not differ significantly between groups. Body weight, BMI, waist circumference, HOMA-IR, serum insulin and leptin levels were significantly reduced from baseline in the diacylglycerol oil group but not in the triacylglycerol oil group. Serum glucose was also significantly improved in patients with higher glucose levels at baseline (>7.00 mmol/L) in the diacylglycerol oil group. Parameters of liver and kidney functions and essential fatty acids in serum phospholipids did not differ between groups.

Conclusions: Diacylglycerol oil consumption improved biomarkers and anthropometric parameters of type 2 DM compared with triacylglycerol oil. No adverse reactions were observed with diacylglycerol oil consumption for type 2 DM patients. Diacylglycerol oil has an equivalent bioavailability as triacylglycerol oil in relation to providing essential fatty acids.
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http://dx.doi.org/10.1016/j.clnu.2008.01.009DOI Listing
April 2008

Cuisine: the concept and its health and nutrition implications--a Hangzhou perspective.

Asia Pac J Clin Nutr 2004 ;13(2):136-40

Medical Nutrition and Food Hygiene Institute, Zhejiang University, Hangzhou, Zhejiang, China, 310006.

Cuisine is an activity that meets human physical and psychological needs. With the development of civilization, cuisine is an important component of culture and includes the dietary profession. However, each nation or each area has its own characteristic cuisine. There are eight major styles of Chinese cuisine, Hangzhou style is an important part of Zhe style. It was divided into two branches named 'Lake branch' and 'Town branch'. An ideal Chinese dish should satisfy in terms of colour, aroma, taste, shape, texture and sustenance. But nowadays, people pay more attention to other aspects of dishes than sustenance. It is estimated that food and beverages will cost up to 570 billion RMB (about 69 billion US dollars) in China this year. The incidence of chronic diseases also increases year after year. There are 40 million diabetic and 70 million obese persons in China. Hence it is important to make efforts to promote in-depth knowledge of cooking and nutrition.
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September 2004

Cuisine: the concept and its health and nutrition implications--global.

Asia Pac J Clin Nutr 2004 ;13(2):131-5

Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, Brisbane, Qld 4029, Australia.

Cuisine, broadly food culture, has evolved greatly in the past ten thousand years, following the domestication of plants and animals which greatly increased the food supply and led to villages, cities and civilizations. Major factors in the evolution of cuisines have been the existing biota, soils, fuel for cooking and climates, followed by new technologies, exploration and trade. These provide the context of the world's amazing variety of cuisines, but not the understanding of why cuisines developed as they have, in particular why China has the world's greatest cuisine. There is evidence that the diet of older women in Zhejiang province meets the recent WHO guidelines for the prevention of chronic disease, consistent with reported longevity in the province. But current changes with the industrialization and globalization of cuisines are associated with increases in chronic diseases, and point to much greater increases in the future.
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September 2004

[Microsomal epoxide hydrolase gene polymorphism and susceptibility to chronic obstructive pulmonary disease in Han nationality of North China].

Zhonghua Nei Ke Za Zhi 2002 Jan;41(1):11-4

Department of Respiratory Medicine, People's Hospital, Peking University, Beijing 100044, China.

Objective: We investigated whether polymorphism in gene for microsomal epoxide hydrolase (mEH) has any bearing on individual susceptibility to the development of chronic obstructive pulmonary disease.

Method: The genotypes of 55 patients with COPD and 52 healthy smoking control subjects were tested with polymerase chain reaction followed by restriction fragment length polymorphism for mEH gene.

Result: The frequency of polymorphic genotypes of mEH showed no difference between the COPD group and the control group. In COPD group mEH exon 3 homozygous wild-type, heterozygote and homozygous mutant was 27.3%, 27.3% and 45.5% respectively and exon 4 homozygous wild-type, heterozygote and homozygous mutant was 72.7%, 18.2% and 9.1% respectively.

Conclusion: Genetic polymorphism in mEH is not associated with development of COPD in Han nationality of North China.
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January 2002