Author Correction: Endothelial IGFBP6 suppresses vascular inflammation and atherosclerosis.
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Published Erratum↗
Biomedical subjects
Zhihua Wang
Publications and source records attributed to Zhihua Wang.
EZH2 variants derived from cryptic splice sites govern distinct epigenetic patterns during embryonic development.
EZH2 catalyzes H3K27me3 and is essential for embryonic development. Although multiple EZH2 variants have been identified, the functional implications and physiological significance of its heterogeneity remain unclear. Here, we revealed that conserved cryptic splice sites generated two EZH2 variants with (EZH2A) or without (EZH2B) a 27-nt region, coding for a 9-aa segment. Structural modeling showed that splice-in or splice-off of the 9-aa segment caused a topological change in EZH2 structure. The 9-aa surplus in EZH2A strengthened its interaction with other PRC2 components, particularly in PRC2.2 holocomplex. We developed point-mutation mouse lines specifically depleting EZH2A or EZH2B (Ezh2amut or Ezh2bmut). Biallelic deletion of Ezh2a caused developmental defects and embryonic lethality between E12.5 and E15.5, while the Ezh2bmut mice were fertile and developed normally. Combined RNA-seq and CUT&Tag analyses in mouse embryonic fibroblasts revealed that EZH2A and EZH2B bound to different genomic loci and affected H3K27me3 deposition in different subsets of genes related to development or the innate immune system, respectively. EZH2A depletion specifically suppressed the expression of genes involved in the development-related Hippo-Yap1 pathway, which might be attributable to a compensatory process mediated by JARID2. Our findings demonstrate that EZH2 heterogeneity from the 9-aa splicing event plays a crucial role in development.
Animals↗
Construction, expression and in vitro biological behaviors of Ig scFv fragment in patients with chronic B cell leukemia.
The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDS-PAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD= 0.9921 ku), which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of Id-HSP complex vaccine for B-CLL.
Cell Proliferation↗
Study on the interactions between CuL(2) and Morin with DNA.
Absorption, fluorescence spectral and viscometric studies have been carried out on the interaction of Morin (2',3,4',5,7-pentahydroxyflavone, ) and its Cu complex, CuL(2) x 2H(2)O [L=Morin (2'-OH group deprotonated), ] with calf thymus DNA. CuL(2) shows different spectral characteristics from that of Morin in the presence of DNA. Increasing fluorescence is seen for CuL(2) with DNA addition whereas decreased fluorescence is observed for Morin. Quenching fluorescence is observed for the DNA-EB system when CuL(2) is added whereas slightly quenched fluorescence is seen for the DNA-EB system with Morin addition. The relative viscosity of DNA and the DNA-EB system increases with the addition of CuL(2.) Hypochromism and a smaller shift are observed in the UV-visible spectra of CuL(2) in the presence of DNA and the denatured temperature of DNA is decreased in the presence of CuL(2). The above results suggested that Morin and CuL(2) can both bind to DNA, but the binding mode is different. The complex binds to DNA mainly by intercalation, while Morin binds in a nonintercalating mode.
Animals↗
MARS: optimistic therapy method in fulminant hepatic failure secondary to cytotoxic mushroom poisoning--a case report.
BACKGROUND: Poisoning by cytotoxic mushrooms (Amanita phalloides and related species) is associated with severe morbidity and a high mortality rate (lethality > 20% in adults and > 50% in children). The main causes of this intoxication are the amatoxines, which inhibit DNA-dependent RNA Polymerase II or B. This interaction leads to a tight complex, and the inhibition is of a non-competitive type (1); in addition to those tight binding inhibitors of adenosine kinase, papain, cathepsin L, cathepsin B, cysteine proteinase and bromelain (2), inhibit the synthesis of messenger RNA in the hepatocytes, decrease the formation of coagulation factors and of immunoglobulins and effect a vasoconstriction. They also have an influence on the transcription and lesions that are seen in cells with rapid protein synthesis, particularly in liver and renal cells, with the cellular changes causing the fragmentation and segregation of all nuclear components, even at low toxin concentrations (3). Phallotoxin, which is the other toxin isolated from death cap, binds with a high affinity to microfilamentous structures - in particular, to F-actin, which stimulates the polymerization of G-actin, stabilizes the F-actin filaments, irreversibly polymerizes actin filaments and causes cholestasis (4). Liver is recognized as the target organ for Amanita phalloides toxins; it is presented by fatty degeneration, acute toxic dystrophy and centrilobular necroses (5). Therapeutic options employed to treat mushroom intoxication, such as hemodiaperfusion on activated charcoal, high dosages of penicillin G, oral charcoal, etc., very often failed to act properly and liver transplantation (when a graft is available) appeared to be the only solution. The most polarized debate concerns the value of extracorporeal elimination. Plasmapheresis and peritoneal dialysis proved much less useful for this purpose; neither haemodialysis (HD) nor haemoperfusion (HP) contributed to the clearance of amatoxin (6, 7). Recently, Stange et al. (8). introduced a new detoxication method (referred to as MARS) for protein-bound substances in patients with liver failure and grade III and IV hepatic encephalopathy. MARS was performed with an albumin-containing dialysate, which is recycled in a closed loop that contains a charcoal cartridge, an anion exchanger resin adsorber and a conventional haemodialyser. With dialysis using an albumin-containing dialysate, protein-bound substances, which are usually not sufficiently dialysable, can be eliminated. The treatments increase the rate of toxin elimination to the extent that the toxic exposure of highly susceptible cells, such as hepatocytes, is minimized. This leads to the surprise recovery of the poisoning patient, despite her severe condition, even as late as up to a week after mushroom ingestion.
Adolescent↗
[Effect of Tumor Vaccine of EL-4 Cell Transduced with IL-12 Gene in C57BL/6 Mice]
In order to study the vaccine potency of gene-modified tumor cells, IL-12 express vector was constructed by using retrovirus. The vector was transfected into EL-4 thymic lymphoma cells and the effect of transfectant on anti-tumor immunity was investigated. The tumorigenicity of EL-4/IL-12 transfectant in syngeneic C57BL/6 mice was decreased significantly after implanted with EL-4/IL-12 transfectant compared with EL-4/Wt or EL-4/Neo groups (P < 0.001). The systemic protective immunity was induced against the challenge with EL-4/Wt (in 8/10 mice) after the rejection of EL-4/IL-12 in vivo experiment, a stronger CTL activity against EL-4/Wt cells and a weak killer activity against syngeneic Lewis lung carcinoma (LLC) cells were obtained in (51)Cr release assay. In vivo depletion analysis suggested that the decreased tumorigenicity mainly depended on CD4(+), CD8(+) and NK cells. Therapeutic vaccines with EL-4/IL-12 cells could retard the growth of established EL-4/Wt tumors significantly compared with those of EL-4/Neo (P < 0.005). These studies suggested that immunotherapy and gene therapy using IL-12 is effective in hematopoietic malignancy and IL-12 has prospects of application in human cancer treatment in the near future.
Journal Article↗
Molecular species analysis of phosphatidylcholine by reversed-phase ion-pair high-performance liquid chromatography.
The retention behavior of molecular species of phosphatidylcholine (PC) is studied by reversed-phase (RP) ion-pair high-performance liquid chromatography (HPLC). Mobile phases contain tetraalkyl ammonium phosphates (TAAPs) in methano-acetonitrile-water. The stationary phase is alkyl-bonded silica. Competitive interactions of TAAPs, analyte solutes, and an RP-HPLC column result in reduced retention of PC molecular species. PC molecular species are eluted at longer retention times with a larger size of TAAP in the mobile phase, and an increase in the TAAP concentration invariably causes a decrease in PC molecular species retention times. There is a linear correlation between the logarithmic retention factors (k) of PC molecular species and the total number of carbon atoms of TAAP, and the logarithm of k values of PC molecular species can be approximated as a linear function of the logarithm of the counter-ion concentration. There is found to be no distinct dependence between k values of PC molecular species and the mobile phase pH.
Chromatography, High Pressure Liquid↗