LIN28 coordinately promotes nucleolar/ribosomal functions and represses the 2C-like transcriptional program in pluripotent stem cells

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28's role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.

Bortezomib and obatoclax for dual blockade of protein degradation pathways show synergistic anti-tumor effect in human acute T lymphoblastic leukemia cells / 南方医科大学学报

OBJECTIVE@#To explore whether bortezomib and a Bcl-2 inhibitor exhibit synergistic anti-tumor effect in human acute T lymphoblastic leukemia cells.@*METHODS@#MTT assay was used to determine the cytotoxicity of bortezomib in the absence or presence of Bcl-2 inhibitors (obatoclax, AT-101 and ABT-199) in Jurkat cells. The effects of drug treatment on the expression of Bcl-2 family proteins, LC3B, p62, ubiquitin, BiP/Grp78, p-JNK, p-p38 and CHOP proteins were examined by Western blotting. Flow cytometry was used to determine the effects of bortezomib and Bcl-2 inhibitors (obatoclax, AT-101 and ABT-199) on cell apoptosis. Quantitative real-time PCR was used to measure the mRNA expression levels of the key regulatory factors of unfolded protein reaction (UPR). A zebrafish xenograft model was used to study the anti-tumor effect of bortezomib, obatoclax and their combination in vivo.@*RESULTS@#Bortezomib or Bcl-2 inhibitors alone inhibited the cell viability of Jurkat cells, but only obatoclax and bortezomib showed synergistic cytotoxicity and pro-apoptotic effect. Obatoclax, rather than AT-101 and ABT- 199, blocked autophagic flux in the cells evidenced by concomitant accumulation of LC3B-Ⅱ and p62. Both bortezomib and obatoclax alone caused accumulation of polyubiquinated proteins, and their combination showed a synergistic effect, which was consistent with their synergistic cytotoxicity. The dual blockade of proteasome and autophagy by the combination of bortezomib and obatoclax triggered unfolded protein response followed by cell apoptosis. Preventing UPS dysfunction by tauroursodeoxycholic acid (TUDCA) significantly attenuated the cytotoxicity and pro-apoptotic effect of bortezomib in combination with obatoclax. In zebrafish xenograft models, bortezomib combined with obatoclax significantly decreased tumor foci formation.@*CONCLUSIONS@#Bortezomib and obatoclax for dual blockade of protein degradation pathways show synergistic anti-tumor effect in human acute T lymphoblastic leukemia cells.

Effect of Ginkgo biloba on liver injury of arsenic poisoning rats caused by corn flour baked by high-arsenic coal / 中华地方病学杂志

Objective To explore the effects and the possible mechanism of Gingko biloba on liver injury due to arsenic poisoning in rats,and to provide experimental evidence for prevention and treatment of arsenic poisoning.Methods The corn powder baked by high arsenic coal was served as the main raw material to make feed containing arsenic.Forty healthy Wistar rats were randomly divided into 5 groups according to their body weights,including control group A,arsenic poisoning group,control group B,natural recovery group and Ginkgo biloba treatment group,eight rats in each group,half male and half female.The control group A rats were fed with normal diet ad libitum for 3.0 months;the arsenic poisoning group rats were freely given feed containing arsenic (100 mg/kg) for 3.0 months;the control group B rats were fed with normal diet ad libitum for 4.5 months;the natural recovery group rats were freely given arsenic (100 mg/kg) feed for 3.0 months,and then given a normal diet for 1.5 months;Ginkgo biloba treatment rats ingested arsenic feed for 3.0 months,and then give Ginkgo biloba solution (25 mg/kg) orally,6 d/week for 1.5 months,then back to normal diet.The content of arsenic in urine,liver,as well as the liver function indices [alanine aminotransferase (ALT),aspartate transaminase (AST),total bile acids (TBA),gamma glutamyl aminopeptidase (GGT),glutathione S-transferase (GSTs)] and the oxidative stress indexes [superoxide dismutase (SOD),glutathione peroxidase (GPx),thiol (-SH),malondialdehyde （MDA)] of liver homogenate,were measured.Results The arsenic content of urine and liver (geometric mean) of the rats in arsenic poisoning group (2 991.24 μg/g Cr,4.29 μg/g) were significantly higher than those in control group A (91.59 μg/g Cr,1.00 μg/g).Urinary arsenic and liver arsenic levels of rats in natural recovery and Ginkgo biloba treatment groups (467.39,334.48 μg/g Cr;,3.15,1.88 μg/g) were higher than those in control group B (99.54 μg/g Cr,0.85 μg/g).The arsenic contents of urine of the rats in natural recovery group,the arsenic contents of urine and liver of rats of Ginkgo biloba treatment group were all lower than those in arsenic poisoning group.The differences were significant (all P ＜ 0.05).The activity/contents of AST,TBA,GGT,GSTs of rats in arsenic poisoning group [(212.88 ± 29.76) U/L,(19.19 ± 4.33) μmol/L,(1.73 ± 0.50) U/L,(196.21 ± 47.38) U/L] were all significantly higher than those in control group A [(142.63 ± 24.20) U/L,(6.23 ± 2.95) μmol/L,(0.77 ± 0.32) U/L,(142.86 ± 28.58) U/L].The activity/contents of TBA,GGT,GSTs in natural recovery group were (17.07 ± 3.92) μ,mol/L,(1.47 ± 0.57) U/L and (178.06 ± 27.37) U/L;and the contents of TBA in Ginkgo biloba treatment group were (13.60 ± 3.00) μmol/L;which were all higher than those in control group B [(7.55 ± 2.45) μmol/L,(0.74 ± 0.51) U/L,(145.17 ± 28.59) U/L].The activity of AST in natural recovery group [(137.44 ± 23.20) U/L],the activity/contents of AST,TBA,GGT and GSTs in Ginkgo biloba treatment group[(129.63 ± 31.25) U/L,(13.60 ± 3.00) μmol/L,(1.15 ± 0.48) U/L,(155.64 ± 20.79) U/L,respectively] were all lower than those in arsenic poisoning group.The content of TBA in Ginkgo biloba treatment group was lower than that of natural recovery group.The differences of those indexes were all significant (all P ＜ 0.05).The activity/contents of SOD,GPx and-SH in arsenic poisoning group [(46.34 ± 11.39),(275.16 ± 92.00) U/mg prot and (0.08 ± 0.02) μmol/mg prot] were all significantly lower than those in control group A [(75.52 ± 8.72),(1 351.01 ± 395.96) U/mg prot,(0.13 ± 0.01) μmol/mg prot].The activity of SOD and GPx in natural recovery group [(42.44 ± 9.58),(694.87 ± 187.01) U/mg prot] were all lower than those in control group B [(68.17 ± 11.11),(1 342.80 ± 185.04) U/mg prot].The activity of GPx in natural recovery group,the activity/contents of SOD,GPx,-SH in Ginkgo biloba treatment group [(63.90 ± 10.44),(1 283.28 ± 373.87) U/mg prot,(0.12-± 0.02) μmol/mg prot] were all higher than those in arsenic poisoning group.The contents of SOD,GPx,-SH in Ginkgo biloba treatment group were higher than those of natural recovery group.The content of MDA in arsenic poisoning group [(3.05 ± 0.94) nmol/mg prot] was higher than that in control group A [(1.67 ± 0.55) nmol/mg prot].The content of MDA of rats in natural recovery and Ginkgo biloba treatment groups were (2.22 ± 0.93),(1.77 ± 0.37) nmol/mg prot,which were lower than those in the arsenic poisoning group.The differences of the above indexes were all significant (all P ＜ 0.05).Conclusion Ginkgo biloba can reduce the accumulation of arsenic in the liver and ameliorate lipid peroxidation,relieve liver injury effectively in rats caused by coal-burning arsenic.

Intervention effects of curcumin on hepatic oxidative stress injury in water arsenic-exposed rats / 中华地方病学杂志

Objective To observe the effects of curcumin on hepatic oxidant stress in water arsenic-exposed rats and to study its mechanism,which can offer references for curcumin used in antioxidant therapy of arsenic poisoning.Methods Thirty-two SD rats were divided into 4 groups according to body weight by random number table,half male and half female.Including control group (lavaged 135 days with deionized water),arsenic poisoning group (lavaged 45 days with deionized water after lavaging 90 days with 10 mg/kg sodium arsenite),pure curcumin group (lavaged 135 days with 1 000 mg/kg curcumin solution) and curcumin treatment group (lavaged 45 days with 1 000 mg/kg curcumin solution after lavaging 90 days with 10 mg/kg sodium arsenite),8 rats in each group.The arsenic contents of urine (urine creatinine corrected) and liver were detected by hydride generation inductively coupled plasma optical emission spectrometer (HG-ICP-OES);the activity of Cu/Zn-superoxide dismutase (SOD1) and catalase (CAT),the contents of malondialdehyde (MDA) in serum and liver homogenate by colorimetric method;the protein expression of liver antioxidant enzyme (SOD 1 and CAT) was assayed by Western blotting.Results The arsenic contents of urine and liver in arsenic poisoning group [(5.83 ± 0.29)μg/g Cr,(15.76 ± 1.65)μg/g] and the arsenic contents of urine in curcumin treatment group [(1.07 ± 0.14)μg/g Cr] were obviously higher than those of control group [(0.40 ± 0.14)μg/g Cr,(4.56 ± 1.05)μg/g,all P ＜ 0.05];compared to arsenic poisoning group,the arsenic contents of urine and liver in curcumin treatment group [(1.07 ± 0.14)μg/g Cr,(5.42 ± 1.76)μg/g] were obviously lower (all P ＜ 0.05).The contents of serum and liver SOD1,CAT and MDA in control group respectively were (102.46 ± 5.03),(29.33 ± 8.13)U/ml,(3.11 ± 0.49)μ mol/L and (204.05 ± 18.33),(126.26 ± 13.19)U/mg prot,(1.62 ± 0.42) μmol/g prot.Compared to the control,the activity of serum and liver SOD1 and CAT in arsenic poisoning group [(60.97 ± 7.94),(13.56 ± 5.14)U/ml and (133.66 ± 11.51),(74.01 ± 13.30)U/mg prot] were lower,the contents of MDA [(7.26 ± 0.54)μmol/L and (2.61 ± 0.52)μmol/g prot] were higher (all P ＜ 0.05).Compared to arsenic poisoning group,the activity of serum and liver SOD1 and CAT in curcumin treatment group [(87.39 ± 9.38),(20.45 ± 6.49) U/ml and (178.27 ± 9.32),(93.70 ± 20.35)U/mg prot] were higher,the contents of MDA [(4.34 ± 0.79)μmol/L and (1.92 ± 0.18)μmol/g prot] were lower (all P ＜ 0.05).The protein expressions of SOD1 and CAT in control group respectively were 0.64 ± 0.32 and 0.72 ± 0.31.Compared to the control group,the protein expressions of SOD1 and CAT in pure curcumin group (1.03 ± 0.23,1.02 ± 0.20) were significantly higher (all P ＜ 0.05) and in arsenic poisoning group (0.34 ± 0.12,0.39 ± 0.11) were lower (all P ＜ 0.05);Compared with the arsenic poisoning group,the protein expressions of SOD1 and CAT in curcumin treatment group (0.58 ± 0.09,0.68 ± 0.29) were significantly higher (all P ＜ 0.05).The arsenic content of urine in rats were positively related with arsenic content of liver and the content of MDA [correlation coefficient (r) =0.952,0.732,all P ＜ 0.05],but negativity related with the activity of SOD1 and CAT in liver (r =-0.874,-0.679,all P ＜ 0.05);the activity of SOD1 and CAT and the content of MDA in serum and liver were positively related (r =0.796,0.484,0.607,all P ＜ 0.05),the activity and protein expression of SOD1 and CAT in liver were positively related (r =0.748,0.424,all P ＜ 0.05).Conclusion The curcumin may improve the activity of hepatic antioxidant enzyme in water arsenic-exposed rats and effectively decrease lipid poroxidation damage caused by arsenic via promoting the excretion of arsenic and the protein expression of hepatic antioxidant enzyme.

Intervention effects of SOD-enriched cili juice on immune function in arseniasis rats caused by coal burning / 中国药理学与毒理学杂志

OBJECTIVE To explore the influence of superoxidase (SOD)-enriched cili juice (CJSOD)on immune function of arseniasis rats,in order to provide a experi mental basis for the search of drugs of immune regulation of the ende mic arsenic poisoning.METHODS Wistar rats were rando mly divided into arsenic 25,50 and 100mg·g-1 groups to be fed 164.67 pp marsenic conta minated grain at levels of 15%,30% and 60%,experi mental period was 90d,CJSOD intervention group was fed with feed contained arsenic 100mg·kg-1 for 90d,then CJSOD was ad ministered at 10mL·kg-1 (6d /week) for 45d.The urine arsenic contents,blood T-ly mphocyte subsets (CD3 +,CD4 +,CD8 + T cell),seru mimmunoglobulin (IgG,IgM,IgA)and complement (C3,C4)were analyzed.RESULTS Co mpared with the normal control group,As 50 and 100mg·kg-1 group had significantly lower CD3 +(46.21 ± 21 .62,31 .31 ±7.73),CD4 +(30.36 ±9.97,25.94 ±12.94)and the ratio of CD4 +/CD8 +(1 .12 ±0.41 , 1 .12 ±0.41 ).As 100mg·kg-1 group showed clearly high levels of C4(81 .18 ±13.23)(P <0.05). Co mpared with the As 100mg·kg-1 group,after intervention with CJSOD,the positive rate of CD3 +(54.06 ±25.77),CD4 +(42.50 ±17.01 ),and the ratio of CD4 +/CD8 +(1 .80 ±0.71 )were significantly increased,while the result of C4 (68.70 ±10.30)and the urine arsenic contents 〔253.82 (158.21 ~494. 11 )〕was significantly decreased(P <0.05).The levels of urine arsenic in rat and CD3 +,CD4 +and CD4 +/CD8 + were negatively correlated,while positive correlation existed between the level of urine arsenic and C4(P <0.01 ).CONCLUSION Arsenic inhibit the immune function in rats.Arsenic load levels were positively correlated with the degree of inhibition.CJSOD preparation can i mprove the immune function of arseniasis rats.

Biomarkers in rats for kidney damage characteristics of arsenism due to coal burning and benchmark dose analysis / 中国药理学与毒理学杂志

OBJECTIVE Study the kidney toxic effects caused by burning coal endemic arsenism in rats,application bench mark dose (BMD) method to investigate the bench mark dose of urinary arsenic (UAs)and the changes in bio markers of renal function.METHODS Wistar rats were fed for 90 d with arsenic 0,25,50,100 mg·kg -1 conta minated feed.Urinary arsenic,kidney arsenic and renal function indicators were determined,and routine pathological and fibrosis of kidney were exa mined.UAs as the exposure bio marker,Uβ2-MG,UNAG and UALB for the effect bio markers,application bench mark dose method to calculate the BMD and BMDL of UAs for each effect bio markers.RESULTS UAs,KAs, Uβ2-MG,UNAG,UALB levels of rats in arsenic 100 mg·kg -1 group were increased than normal group (P <0.05);In light microscope,the results of HE staining of rat kidney in all arsenic dose groups showed infla mmatory cell infiltration,renal tubular epithelial cell swelling,renal interstitial capillary dila-tion,congestion and other varying degrees pathological changes,and the results of masson staining showed varying degrees of tubulointerstitial fibrosis;UAs as the exposure bio marker,Uβ2-MG,UNAG, UALB for the effects of mark,the BMD and BMDL of UAs for Uβ2-MG,UNAG,UALB were calculated, the BMD values were 998.9,1213.5,1386.9 μg·g -1 Cr,the BMDL values were 660.5,803.6 and 909. 4 μg·g -1 Cr,respectively.CONCLUSION Burning coal arsenic pollution can cause kidney da mage in rats,mini mal change nephropathy may be the pri mary pathological in the coal arsenic conta mination of kidney da mage.The BMD and BMDL of UAs were 998.9,660.5 μg·g -1 Cr,the early changes of renal function of burning coal arsenism in rats;it is reco mmended to use the more sensitive bio markers Uβ2-MG to calculate the biological exposure li mits on renal injury caused by arsenic.

Clinical Distribution of Pathogens and Antimicrobial Resistance in Respiratory Ward 2005-2007 / 中华医院感染学杂志

OBJECTIVE To analyze the pathogenic distribution and antimicrobial resistance in respiratory ward and provide the rational information to use antibiotics reasonably. METHODS All pathogens isolated from patients in a respiratory ward from 2005 to 2007 and drug susceptibility results were retrospectively analyzed. RESULTS Totally 264 strains of pathogenic bacteria were isolated,in which 68 strains of Gram-positive bacteria,165 strains of Gram-negative bacteria and 31 strains of fungi.MRSA prevalence was 77.1% and showed a trend of increase.No vancomycin-resistant Staphylococcus aureus or Enterococcus was detected.The resistance rate of Streptoccocus pneumoniae to penicillin,erythromycin and levofloxacin was 44.4-66.7%.Enterobacter and Acinetobacter baumannii showed stable susceptibility to imipenem.Pseudomonas aeruginosa strains were relatively susceptible to cefoperazone /sulbactam,amikacin,gentamicin,piperacillin/tazobactam,ceftazidine,cefepime,cefoperazone and imipenem. CONCLUSIONS The changes in pathogens and antibiotic resistance in the respiratory ward are consistent with the surveillance data in this country,Gram-negative bacteria are still the most common pathogens and the serious degree of bacterial drug resistance is increasing.Our data are useful for the guidance of rational use of antibiotics.