N-glycosylation of proteins is an important post-translation modifying process in organism.The reactions of the protein N-glycosylation pathway occur along the secretory pathway and most of the entail enzymes localized in the endoplasmic reticulum and the Golgi body.The α-mannosidase is a key enzyme of N-glycan synthesis and metabolism,which functions on removing the mannose reside of glycan.This review will focus on the classification of this enzyme and its function on maturation and degradation of N-glycan,the relative disease resulted from the it's mutation.
Mannosidase
Endoplasmic-reticulum-associated protein degradation
Fraxinellone, a furanoid, is one of the bioactive and potentially hepatotoxic constituents from Dictamnus dasycarpus Turcz, which is extensively spread throughout Asian countries. This herb was reported to cause liver injury in clinical application. However, the mechanism behind is still not fully understood. This study mainly focused on the hepatotoxicity of fraxinellone and the underlying mechanism. The current study demonstrated that fraxinellone resulted in a significant elevation of serum alanine aminotransferase and aspartate aminotransferase in a dose-dependent manner in mice after oral administration. Pretreatment with ketoconazole for three successive days could significantly alleviate the hepatotoxicity of fraxinellone. Considering that fraxinellone has a structural alert of furan ring, it is believed that the hepatotoxicity caused by fraxinellone required cytochrome P450-mediated bioactivation. Bioactivation studies were subsequently carried out in vitro and in vivo. Fraxinellone was metabolized into cis-enedial intermediate, an electrophile that was prone to react with glutathione or N-acetyl-lysine through 1,2- or 1,4-addition to form stable conjugates. Ketoconazole significantly inhibited the formation of the glutathione conjugates (M1 and M2) in microsomal incubation and similar finding was obtained in vivo. Phenotyping study indicated that CYP3A4 was the principal enzyme responsible for the bioactivation of fraxinellone. This study suggested that CYP3A4-mediated bioactivation plays an indispensable role in fraxinellone-induced hepatotoxicity. The work performed herein enables us to better understand the hepatotoxicity of fraxinellone as well as the mechanism behind.
To study the effect of human alpha-mannosidase Man2c1 transgene on tumor growth and metastasis in mice.Hepatoma cell H22 or squamous epithelial carcinoma cell S180 was subcutaneously inoculated into the right armpit of mice (wild type mice and 28#, 35#, and 54# transgenic mice). Tumor size was measured every week. Mice were sacrificed on day 9 or 10 and then the tumors were exercised and weighted. Tumors and lungs were fixed in formaldehyde and sectioned. The sections were stained with hematoxylin/eosin and examined under microscope. The red blood cells in spleen were destroyed by Tris-NH4Cl. Natural killer (NK) cell activity was detected with Yac-1 cell as target.H22 and S180 tumors grew faster in all the three transgenic mice (28#, 35#, and 54#) than in wild type mice. The average size and weight of tumors between the transgenic mice and wild type mice were significantly different (P<0.05). Most tumors in the transgenic mice invaded the surrounding tissues. In contrast, nearly all the tumors in wild type mice were capsulized. Three of 10 28# transgenic mice, 5 of 10 35# transgenic mice, 3 of 10 54# transgenic mice, and 1 of 10 wild type mice showed lung metastasis of H22 tumor. Two of 6 28# transgenic mice, 3 of 6 35# transgenic mice, 1 of 6 54# transgenic mice, and 0 of 6 wild type mice showed lung metastasis of S180 tumor. No difference of NK activity in spleen cells was observed between the transgenic mice and wild type mice.hMan2c1 transgene promotes growth, invasion, and metastasis of transplanted H22 and S180 tumors in mice. hMan2cl transgene does not affect NK activity in splenocytes.
Secreted Wnt glycoproteins are one of the major families of cell signaling molecules and have a dual role in embryogenesis and carcinogenesis.Wnt proteins signals act through canonical pathway and non-canonical pathway.Wnt ligands bind to two distinct families of cell-surface receptors: the Frizzled(Fz) receptor family and the LDL-receptor related protein(LRP) family.Wnt-mediated signals are modulated extracellularly by Wnt antagonists.Wnt antagonists are asecreted proteins that bind to Wnt proteins and inhibit their activities.They can be divided into two groups: one is sFRP family,WIF-1 and Cerberus,another is DKK family.Wnt signaling has clearly emerged as a critical pathway in carcinogenesis.It deserves further study on the components of the Wnt pathway to provide valuable targets for potential anti-cancer therapeutic agents.
The object of this study was to develop a simple, rapid, specific, and highly sensitive method to detect HCV core antigen. A nucleic acid aptamer was designed with the high specificity and sensitivity in a nucleic acid lateral flow strip to compete with HCV core antigen and DNA probes. The lower detection limit of the test strip was calculated to be 10 pg/mL with the scanner and 100 pg/mL with naked eyes. Results showed that there were no cross-interactions with other proteins such as HCV NS3, E1/E2 antigens, HIV p24 antigens, or BSA proteins (HCV unrelated protein). When the viral load exceeded 10(4) copies/mL, the positive coincidence rates of ELISA and strip detection, when compared with the HCV RNA assay, were 98.44% and 97.28%, respectively. The results indicated that the ELISA detection and strip assay were in good agreement with the measured value. The results indicated that a nucleic acid lateral flow strip was a simple, rapid, specific, highly sensitive, and cost-effective field-based method for detecting HCV core antigen. The strip assay is an acceptable alternative to diagnose HCV core antigen and to investigate its epidemiology in clinical laboratories lacking specialized equipment and skills.
Objective To investigate the in vivo effect of silenced actin-associated protein Transgelin on the growth of human pancreatic carcinoma xenograft in nude mice.Methods Human pancreatic cancer cell line BxPC3 were transfected with small hairpin RNA (shRNA) eukaryotic expression vector targeting Transgelin gene.RT-PCR and Western blot were used to analyze Transgelin expression after transfection.24 animal models were randomly divided into three groups with 8 in each:Experimental group (transplanted BxPC3/Transgelin shRNA),negative control group (transplanted BxPC3/Neo) and untreated group (transplanted BxPC3).Tumor size was measured weekly.All mice were sacrificed after 28 days.Tumor volume was calculated,inhibitory effect was analyzed.Immunohistochemical staining of paraffin sections for Transgelin and proliferating cell nuclear antigen (PCNA) proteins were performed.Results Tumors varied in sizes among 3 groups (all P ＜ 0.05).On day 21 and 28 tumor was significantly smaller in experimental group than those in control groups.Tumor weighed(0.74 ±0.21) g in experimental group,lower than that in negative control group(1.42 ± 0.28) g and untreated group(1.59 ± 0.24) g (all P ＜ 0.05).The inhibitory effect was 53.5％ in experimental group.The PCNA index was significantly lower in experimental group than those in control groups (all P ＜ 0.05).Conclusions Deletion of Transgelin gene can significantly inhibit the proliferation and tumor growth of BxPC3 cells in nude mice.
Key words:
Pancreatic neoplasms ; RNA, small interfering; Transgelin
Objective To generate a transgenic mouse line expressing human APP695K595N/M596L(Swedish mutation) and establish a transgenic Alzheimer disease model.Methods The transgenic plasmid was constructed by inserting the mutated APP695K595N/M596L gene into the downstream of mouse prion protein promoter.The transgenic mice were produced by microinjection and the genotype was detected by PCR.The gene expression levels were determined by Western blotting.The senile plaques were detected by thioflavin-S staining and visualized directly by fluorescence microscopy.The behavioral changes was examined by Morris water maze test.Results Transgenic C57BL/6J mice were generated with the expression of the APP695K595N/M596L in the brain tissue.The transgenic mice showed significant learning and memory impairments in the Morris water maze at 5 months of age and the extent of the impairments was developed at 7,9 and 11 months of age,comparing with that of age-matched wild type mice(P0.05).Senile plaques were visualized in the CA1 area of hippocampus at 9 and increased at 12 months of age.Conclusions The transgenic mice show a progressive deficits of learning and memory and a progressive formation of senile plaques in the hippocampus,suggesting that this APP transgenic mouse is an useful animal model of Alzheimer disease.
Myocardial infarction (MI) is a major disease burden. Wild-type p53-induced phosphatase 1 (Wip1) has been studied extensively in the context of cancer and the regulation of different types of stem cells, but the role of Wip1 in cardiac adaptation to MI is unknown. We investigated the significance of Wip1 in a mouse model of MI.The study began in June 2014 and was completed in July 2016. We compared Wip1-knockout (Wip1-KO) mice and wild-type (WT) mice to determine changes in cardiac function and survival in response to MI. The heart weight/body weight (HW/BW) ratio and cardiac function were measured before MI. Mouse MI was established by ligating the left anterior descending (LAD) coronary artery under 1.5% isoflurane anesthesia. After MI, survival of the mice was observed for 4 weeks. Cardiac function was examined by echocardiography. The HW/BW ratio was analyzed, and cardiac hypertrophy was measured by wheat germ agglutinin staining. Hematoxylin and eosin (H&E) staining was used to determine the infarct size. Gene expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) was assessed by quantitative real-time polymerase chain reaction (qPCR), and the levels of signal transducers and activators of transcription 3 (stat3) and phosphor-stat3 (p-stat3) were also analyzed by Western blotting. Kaplan-Meier survival analysis, log-rank test, unpaired t-test, and one-way analysis of variance (ANOVA) were used for statistical analyses.Wip1-KO mice had a marginally increased HW/BW ratio and slightly impaired cardiac function before LAD ligation. After MI, Wip1-deficient mice exhibited increased mortality (57.14% vs. 29.17%; n = 24 [WT], n = 35 [Wip1-KO], P< 0.05), increased cardiac hypertrophy (HW/BW ratio: 7 days: 7.25 ± 0.36 vs. 5.84 ± 0.18, n = 10, P< 0.01, and 4 weeks: 6.05 ± 0.17 vs. 5.87 ± 0.24, n = 10, P > 0.05; cross-sectional area: 7 days: 311.80 ± 8.29 vs. 268.90 ± 11.15, n = 6, P< 0.05, and 4 weeks: 308.80 ± 11.26 vs. 317.00 ± 13.55, n = 6, P > 0.05), and reduced cardiac function (ejection fraction: 7 days: 29.37 ± 1.38 vs. 34.72 ± 1.81, P< 0.05, and 4 weeks: 19.06 ± 2.07 vs. 26.37 ± 2.95, P< 0.05; fractional shortening: 7 days: 13.72 ± 0.71 vs. 16.50 ± 0.94, P< 0.05, and 4 weeks: 8.79 ± 1.00 vs. 12.48 ± 1.48, P< 0.05; n = 10 [WT], n = 15 [Wip1-KO]). H&E staining revealed a larger infarct size in Wip1-KO mice than in WT mice (34.79% ± 2.44% vs. 19.55% ± 1.48%, n = 6, P< 0.01). The expression of IL-6 and p-stat3 was downregulated in Wip1-KO mice (IL-6: 1.71 ± 0.27 vs. 4.46 ± 0.79, n = 6, P< 0.01; and p-stat3/stat3: 1.15 ± 0.15 vs. 1.97 ± 0.23, n = 6, P< 0.05).The results suggest that Wip1 could protect the heart from MI-induced ischemic injury.
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