OBJECTIVETo identify the mutation of Cu/Zn superoxide dismutase(SOD1) gene in an amyotrophic lateral sclerosis (ALS) family with unique phenotype.

METHODSFive exons of SOD1 gene were amplified by PCR. The differences of these products were analyzed by PCR-single strand conformation polymorphism and visualized by silver staining.

RESULTSAbnormal bands were found in exons 2 and 5 of SOD1 gene in several familial members. DNA sequence analysis verified that a base pair insertion occurred in the codon area of exon 2 and in the intron area of exon 5. And the insertion mutation of exon 2 led to a frameshift mutation and premature stop. It is a new type of SOD1 mutation which may be associated with familial amyotrophic lateral sclerosis.

CONCLUSIONInsertion mutation of exon 2 may be responsible for the disease of an ALS family in Chongqing.

Adult ; Amyotrophic Lateral Sclerosis ; genetics ; Humans ; Mutation ; Polymorphism, Single-Stranded Conformational ; Superoxide Dismutase ; genetics ; Superoxide Dismutase-1

OBJECTIVE: To explore the genetic basis for a Chinese patient with amyotrophic lateral sclerosis (ALS). METHODS: Peripheral blood samples were collected from the patient and his parents for the extraction of genomic DNA. Genetic variant was identified by whole exome sequencing. Candidate variant was verified by Sanger sequencing of his parents and healthy controls. RESULTS: The patient was found to harbor a heterozygous c.420C>G (p.Asn140Lys) variant of the SOD1 gene. The same variant was not detected in his parents and 100 healthy controls. The variant has not been included in HGMD, dbSNP and other databases. CONCLUSION The c.420C>G variant of the SOD1 gene may underlie the ALS in this patient. Above finding has enriched the spectrum of SOD1 gene variants.
Amyotrophic Lateral Sclerosis/genetics* ; China ; Heterozygote ; Humans ; Superoxide Dismutase-1/genetics* ; Whole Exome Sequencing

BACKGROUNDRNA interference (RNAi) is a potential cure for amyotrophic lateral sclerosis (ALS) caused by dominant, gain-of-function superoxide dismutase 1 (SOD1) mutations. The success of such therapy relies on the functional small interfering RNAs (siRNAs) that can effectively deliver RNAi. This study aimed to design the functional siRNAs targeting ALS-associated mutant alleles.

METHODSA modified dual luciferase system containing human SOD1 mRNA target was established to quantify siRNA efficacy. Coupled with validated siRNAs identified in the literature, we analyzed the rationale of siRNA design and subsequently developed an asymmetry rule-based strategy for designing siRNA. We then further tested the effectiveness of this design strategy in converting a naturally symmetric siRNA into functional siRNAs with favorable asymmetry for gene silencing of SOD1 alleles.

RESULTSThe efficacies of siRNAs could vary tremendously by one base-pair position change. Functional siRNAs could target the whole span of SOD1 mRNA coding sequence as well as non-coding region. While there is no distinguishable pattern of the distribution of nucleobases in these validated siRNAs, the high percent of GC count at the last two positions of siRNAs (P18 and P19) indicated a strong effect of asymmetry rule. Introducing a mismatch at position 1 of the 5' of antisense strand of siRNA successfully converted the inactive siRNA into functional siRNAs that silence SOD1 with desired efficacy.

CONCLUSIONSAsymmetry rule-based strategy that incorporates a mismatch into siRNA most consistently enhances RNAi efficacy and guarantees producing functional siRNAs that successfully silence ALS-associated SOD1 mutant alleles regardless target positions. This strategy could also be useful to design siRNAs for silencing other disease-associated dominant, gain-of-function mutant genes.

Amyotrophic Lateral Sclerosis ; genetics ; Cell Line ; Humans ; RNA Interference ; physiology ; RNA, Small Interfering ; genetics ; physiology ; Superoxide Dismutase ; genetics ; Superoxide Dismutase-1

BACKGROUND: Investigations of the pathogenic mechanisms in motor neurons (MNs) derived from amyotrophic lateral sclerosis (ALS) disease-specific induced pluripotent stem (iPS) cell lines could improve understanding of the issues affecting MNs. Therefore, in this study we explored mutant superoxide dismutase 1 (SOD1) protein expression in MNs derived from the iPS cell lines of ALS patients carrying different SOD1 mutations. METHODS: We generated induced pluripotent stem cell (iPSC) lines from two familial ALS (FALS) patients with SOD1-V14M and SOD1-C111Y mutations, and then differentiated them into MNs. We investigated levels of the SOD1 protein in iPSCs and MNs, the intracellular Ca2+ levels in MNs, and the lactate dehydrogenase (LDH) activity in the process of differentiation into the MNs derived from the controls and ALS patients' iPSCs. RESULTS: The iPSCs from the two FALS patients were capable of differentiation into MNs carrying different SOD1 mutations and differentially expressed MN markers. We detected high SOD1 protein expression and high intracellular calcium levels in both the MN and iPSCs that were derived from the two SOD1 mutant patients. However, at no time did we observe stronger LDH activity in the patient lines compared with the control lines. CONCLUSIONS MNs derived from patient-specific iPSC lines can recapitulate key aspects of ALS pathogenesis, providing a cell-based disease model to further elucidate disease pathogenesis and explore gene repair coupled with cell-replacement therapy. Incremental mutant expressions of SOD1 in MNs may have disrupted MN function, either causing or contributing to the intracellular calcium disturbances, which could lead to the occurrence and development of the disease.
Amyotrophic Lateral Sclerosis/genetics* ; Humans ; Induced Pluripotent Stem Cells ; Motor Neurons ; Mutation/genetics* ; Superoxide Dismutase-1/genetics*

BACKGROUNDSleep/wake disturbances in patients with amyotrophic lateral sclerosis (ALS) are well-documented, however, no animal or mechanistic studies on these disturbances exist. Orexin is a crucial neurotransmitter in promoting wakefulness in sleep/wake regulation, and may play an important role in sleep disturbances in ALS. In this study, we used SOD1-G93A transgenic mice as an ALS mouse model to investigate the sleep/wake disturbances and their possible mechanisms in ALS.

METHODSElectroencephalogram/electromyogram recordings were performed in SOD1-G93A transgenic mice and their littermate control mice at the ages of 90 and 120 days, and the samples obtained from these groups were subjected to quantitative reverse transcriptase-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay.

RESULTSFor the first time in SOD1-G93A transgenic mice, we observed significantly increased wakefulness, reduced sleep time, and up-regulated orexins (prepro-orexin, orexin A and B) at both 90 and 120 days. Correlation analysis confirmed moderate to high correlations between sleep/wake time (total sleep time, wakefulness time, rapid eye movement [REM] sleep time, non-REM sleep time, and deep sleep time) and increase in orexins (prepro-orexin, orexin A and B).

CONCLUSIONSleep/wake disturbances occur before disease onset in this ALS mouse model. Increased orexins may promote wakefulness and result in these disturbances before and after disease onset, thus making them potential therapeutic targets for amelioration of sleep disturbances in ALS. Further studies are required to elucidate the underlying mechanisms in the future.

Amyotrophic Lateral Sclerosis ; genetics ; metabolism ; Animals ; Female ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Male ; Mice ; Mice, Transgenic ; Neuropeptides ; genetics ; metabolism ; Orexins ; Reverse Transcriptase Polymerase Chain Reaction ; Sleep ; physiology ; Superoxide Dismutase ; genetics ; metabolism ; Superoxide Dismutase-1 ; Wakefulness ; physiology

OBJECTIVETo observe the association between SNPs in SOD1 (rs1041740, rs2070424, rs10432782 and rs4998557) and noise-induced hearing loss in Chinese Han population.

METHODSA case-control study was used to study the effects of environmental risk factors on the susceptibility to noise induced hearing loss (NIHL) in 201 sensitive workers and 202 resistant workers. A questionnaire was designed to carry out an investigation, and an occupational health survey was used to identify the occupational risk factors. Genomic DNA was extracted from peripheral blood cells using standard procedures of Takara kit, and 5 ml blood was from each subject. SNPs were detected using standard procedures of TaqMan probe allele identification method.

RESULTSIn SOD1 gene, the A allele of rs2070424 was a protective factor of NIHL, compared with the G allele (OR = 0.67, 95%CI: 0.50 approximately 0.88). The risk with NIHL in subjects with AA genotype was significantly lower than that in subjects with GG genotype (OR = 0.48, 95%CI: 0.26 approximately 0.79). After adjusting the confusion factors, OR was 0.44 and 95%CI was 0.25 approximately 0.78.

CONCLUSIONIn Chinese Han population, the SNP of rs2070424 in SOD1 may be associated with the susceptibility to NIHL.

Adolescent ; Adult ; Alleles ; Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; Female ; Genetic Predisposition to Disease ; Genotype ; Haplotypes ; Hearing Loss, Noise-Induced ; epidemiology ; genetics ; Humans ; Male ; Noise, Occupational ; Polymorphism, Single Nucleotide ; Risk Factors ; Superoxide Dismutase ; genetics ; Superoxide Dismutase-1 ; Young Adult

OBJECTIVETo explore the effects of noise exposure level and cumulative noise exposure (CNE) on the relationship between rs2070424 and rs10432782 SNPs in SOD1 and the susceptibility to noise-induced hearing loss (NIHL).

METHODSA case-control study was performed for investigating the effects of environmental risk factors on the susceptibility to NIHL in 201 sensitive workers and 202 resistant workers.A questionnaire was utilized to investigate the occupational health and to identify the occupational risk factors. The noise exposure levels were detected according to the Chinese standard Measurement of noise in the workplace (GBZ/T 189.8-2007). The peripheral blood samples (5 ml blood for each sample) were from sensitive workers and resistant workers. Genomic DNA was extracted on the basis of the standard procedures of Takara kit. SNPs were detected using standard procedures of TaqMan probe allele identification method.

RESULTSIn group exposed to 85 - 92 dB noise (A), the risk of NIHL in the subjects with the AA genotype of rs2070424 was lower than that in the subjects with the GG genotype, OR = 0.37 (95%CI: 0.17∼ 0.80). In group exposed to > 82 dB CNE (A), the AA genotype of rs2070424 is a protective factor of NIHL, as compared with the GG genotype, OR = 0.25 (95%CI: 0.09 ∼ 0.70). In group exposed to 85 - 92 dB noise (A), the risk of NIHL in the subjects with the GG genotype of rs10432782 was compared with the risk of NIHL in the subjects with the TT genotype, OR = 3.17 (95%CI: 1.16 ∼ 6.89). The GT genotype was compared with TT genotype, OR = 2.39 (95%CI: 1.16 ∼ 4.97). In group exposed to 75 ∼ 82 dB CNE (A), the risk of NIHL in the subjects with the GG genotype was compared with the risk of NIHL in the subjects with the TT genotype, OR = 2.35 (95%CI: 0.96 ∼ 5.72), P = 0.06. The GG genotype may bea risk factor of NIHJ.

CONCLUSIONThe noise exposure level and CNE may influence the relationship between rs2070424, rs10432782 SNPs in SOD1 and noise-induced hearing loss.

Adult ; Case-Control Studies ; Female ; Genetic Predisposition to Disease ; Genotype ; Hearing Loss, Noise-Induced ; etiology ; genetics ; Humans ; Male ; Noise, Occupational ; adverse effects ; Polymorphism, Single Nucleotide ; Superoxide Dismutase ; genetics ; Superoxide Dismutase-1 ; Surveys and Questionnaires

Antioxidant enzymes fused with cell-penetrating peptides could enter cells and protect cells from irradiation damage. However, the unselective transmembrane ability of cell-penetrating peptide may also bring antioxidant enzymes into tumor cells, thus protecting tumor cells and consequently reducing the efficacy of radiotherapy. There are active matrix metalloproteinase (MMP)-2 or MMP-9 in most tumor cellular microenvironments. Therefore, a fusion protein containing an MMP-2/9 cleavable substrate peptide X, a cell-penetrating peptide R9, a glutathione S-transferase (GST), and a human Cu, Zn superoxide dismutase (SOD1), was designed and named GST-SOD1-X-R9. In the tumor microenvironment, GST-SOD1-X-R9 would lose its cell-penetrating peptide and could not enter tumor cells due to the cleavage of substrate X by active MMP-2/9, thereby achieving selected entering normal cells. The complete nucleotide sequence of SOD1-X-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1. The pGEX4T-1-SOD1-X-R9 recombinant plasmid was obtained, and soluble expression of the fusion protein was achieved. GST-SOD1-X-R9 was purified by ammonium sulfate precipitation and GST affinity chromatography. The molecular weight of the fusion protein was approximately 47 kDa, consistent with the theoretical value. The SOD and GST activities were 2 954 U/mg and 328 U/mg, respectively. Stability test suggested that almost no change in either SOD activity or GST activity of GST-SOD1-X-R9 was observed under physiological conditions. The fusion protein could be partially digested by collagenase Ⅳ in solution. Subsequently, the effect of MMP-2/9 activity on transmembrane ability of the fusion protein was tested using 2D and 3D cultured HepG2 cells. Little extracellular MMP-2 activity of HepG2 cells was observed under 2D culture condition. While under the 3D culture model, the size and the MMP-2 activity of the HepG2 tumor spheroid increased daily. GST-SOD1-R9 proteins showed the same transmembrane efficiency in 2D cultured HepG2 cells, but the transmembrane efficiency of GST-SOD1-X-R9 in 3D cultured HepG2 spheres was reduced remarkably. This study provided a basis for further investigating the selectively protective effect of GST-SOD1-X-R9 against oxidative damage in normal cells.
Ammonium Sulfate ; Antioxidants ; Cell-Penetrating Peptides/pharmacology* ; Endopeptidases ; Glutathione Transferase/metabolism* ; Humans ; Matrix Metalloproteinase 2/genetics* ; Matrix Metalloproteinase 9/genetics* ; Recombinant Fusion Proteins ; Recombinant Proteins ; Superoxide Dismutase/metabolism* ; Superoxide Dismutase-1

OBJECTIVE: To determine the protective mechanism of preconditioning to the lung injury induced by ischemia-reperfusion. METHODS: Twelve pigs were randomly divided into 2 groups: control group ( Group C) and ischemic preconditioning group ( Group IP). The concentration of superoxide distrautase (SOD) and malondialdehyde (MDA) in circuit were checked before and after the perfusion to reflect the lipid peroxidation in the lungs. Left lung biopsies were performed immediately after the perfusion and 1 hour postperfusion for histologic examination. The ICAM-1 expression was assessed by immunohistochemical analysis with Envision method and the mRNA expression of ICAM-1 was analyzed by RT-PCR. RESULTS: SOD in Group IP was much higher than that in Group C (P < 0.01 ). MDA in Group IP was much lower than that in Group C ( P < 0.01 ). The lung histologic examination showed that Group C was significantly more serious than Group IP in pulmonary edema, inflammatory cell infiltration, mild focal hemorrhage, and alveolar disruption. The expression of ICAM-1 of lung tissue obviously decreased in Group IP than that in Group C (P <0.01 ). The expression of ICAM-1 mRNA of lung tissue was significantly lower in Group IP than that in Group C (P < 0.01 ). CONCLUSION Lung ischemic preconditioning can reduce the lung injury. The mechanisms of the protective effects of the IP may be related to the increase of SOD and the decrease of MDA. The preconditioning down-regulated the ICAM-1 expression is one of the mechanisms in reducing the lung injury.
Animals ; Female ; Intercellular Adhesion Molecule-1 ; biosynthesis ; genetics ; Ischemia ; Ischemic Preconditioning ; Lung ; blood supply ; Male ; RNA, Messenger ; blood ; Random Allocation ; Reperfusion Injury ; prevention & control ; Superoxide Dismutase ; blood ; Swine

This study aims to investigate the effect of Chaihu Shugan Powder(CHSG) on liver injury in rats with intrahepatic cholestasis by regulating farnesoid X receptor(FXR)/nuclear factor erythroid-2-related factor(Nrf2)/antioxidant response element(ARE) pathway. Eighty-four SD rats were classified into normal group, model group, CHSG-L group(0.5 g·kg~(-1)), CHSG-H group(2.5 g·kg~(-1)), ursodeoxycholic acid group(UDCA group, 100 mg·kg~(-1)), CHSG-H+sh-NC group(2.5 g·kg~(-1) CHSG+subcutaneous injection of sh-NC lentivirus), CHSG-H+sh-FXR group(2.5 g·kg~(-1) CHSG+subcutaneous injection of sh-FXR lentivirus), with 12 rats in each group. Rats were treated with corresponding drugs except for the normal group and the model group, once a day, for 7 days. On 5 th day, rats, except the normal group, were given α-naphthalene isothiocyanate(ANIT) at a dose of 100 mg·kg~(-1), once a day for 3 days to induce intrahepatic cholestasis, and the normal group was given the same amount of normal saline. Rats were anesthetized 1 h after the last administration and the 2 h bile flow was measured. Aeroset chemistry analyzer was employed to detect the levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), total bilirubin(TBIL), and total bile acid(TBA) in rat serum. Based on hematoxylin and eosin(HE) staining, the pathological changes of rat liver tissue were observed. Glutathione peroxidase(GSH-Px), superoxide dismutase(SOD), and malondialdehyde(MDA) in rat liver tissue homogenate were monitored with corresponding kits. Western blot was used to detect the expression of FXR, Nrf2, and heme oxygenase-1(HO-1) proteins in rat liver tissue. Compared with the normal group, the model group showed many spots or concentrated necrotic areas in the liver tissue, infiltration of a large number of inflammatory cells, swelling liver cells with nuclear shrinkage. The 2 h bile flow, levels of GSH-Px and SOD, and relative expression of FXR, Nrf2, and HO-1 proteins were significantly lower, and the levels of ALT, AST, TBIL, TBA and MDA were significantly higher in the model group than in the normal group. Compared with the model group, CHSG-L group, CHSG-H group, and UDCA group demonstrated significant alleviation of pathological damage of the liver tissue, significantly high 2 h bile flow, levels of GSH-Px and SOD, and expression of FXR, Nrf2 and HO-1 proteins, and significantly low levels of ALT, AST, TBIL, TBA and MDA. Compared with the CHSG-H group, the CHSG-H+sh-FXR group had worse liver pathological damage, significantly low levels of 2 h bile flow, levels of GSH-Px and SOD, and expression of FXR, Nrf2, and HO-1 proteins, and significantly high levels of ALT, AST, TBIL, TBA, and MDA. CHSG may protect against liver injury in rats with intrahepatic cholestasis by activating the FXR/Nrf2/ARE pathway.
Rats ; Animals ; 1-Naphthylisothiocyanate/toxicity* ; Powders ; NF-E2-Related Factor 2/genetics* ; Rats, Sprague-Dawley ; Cholestasis, Intrahepatic/drug therapy* ; Liver ; Superoxide Dismutase ; Oxidative Stress