[Deubiquitinating enzyme MINDY1 is an independent risk factor for the maintenance of stemness and poor prognosis in liver cancer cells].

Objective: To explore the key deubiquitinating enzymes that maintain the stemness of liver cancer stem cells and provide new ideas for targeted liver cancer therapy. Methods: The high-throughput CRISPR screening technology was used to screen the deubiquitinating enzymes that maintain the stemness of liver cancer stem cells. RT-qPCR and Western blot were used to analyze gene expression levels. Stemness of liver cancer cells was detected by spheroid-formation and soft agar colony formation assays. Tumor growth in nude mice was detected by subcutaneous tumor-bearing experiments. Bioinformatics and clinical samples were examined for the clinical significance of target genes. Results: MINDY1 was highly expressed in liver cancer stem cells. The expression of stem markers, the self-renewal ability of cells, and the growth of transplanted tumors were significantly reduced and inhibited after knocking out MINDY1, and its mechanism of action may be related to the regulation of the Wnt signaling pathway. The expression level of MINDY1 was higher in liver cancer tissues than that in adjacent tumors, which was closely related to tumor progression, and its high expression was an independent risk factor for a poor prognosis of liver cancer. Conclusion: The deubiquitinating enzyme MINDY1 promotes stemness in liver cancer cells and is one of the independent predictors of poor prognosis in liver cancer.

[Determination of ammonia and potassium in tobacco by ion chromatography].

A new ion chromatographic method was developed for the quantitative determination of ammonia and potassium in tobacco. The tobacco samples were treated with dilute nitric acid and K+, NH4+ were extracted. The method is fast and convenient. The relationships between peak heights and initial concentrations of K+ and NH4+ were linear. The detection limits were 0.05 mg/L for K+ and 0.03 mg/L for NH4+. Recoveries of K+ and NH4+ were 96.7%-106.7% and 96.5%-109.1% respectively. The whole procedure of determination can be completed within 5 min.

[Inhibition of ATP-activated current by met-Enk in isolated DRG neurons of the rat].

The present study aimed to explore modulation of ATP-activated currents (I(ATP)) by met-Enk in rat DRG neurons. I(ATP) was recorded using the whole-cell patch clamp technique. The majority of the neurons examined responded to ATP (90.0 percent;, 45/50) with inward currents. In the 45 ATP sensitive neurons three kinds of responses to application of met-Enk were distinguished: (l) inward currents (29/45), (2) outward currents (9/45), and (3) no effect (7/45). Pretreatment with met-Enk (10(-9)~10(-5) mol/L) suppressed I(ATP) (10(-4) mol/L) in 29 neurons responding to met-Enk with inward currents. The inhibition by met-Enk of I(ATP) could be blocked by naloxone (10(-7) mol/L) in a concentration-dependent manner. Met-Enk of 10(-9), 10(-8), 10(-7), 10(-6) and 10(-5) mol/L suppressed I(ATP) by l3.2+/-5.4 percent; (n=5); 39.2+/-8.6 percent; (n=8), 54.l+/-8.6 percent; (n=8),43.3+/-7.9 percent; (n=7) and 43.l+/-7.9 percent; (n=7) (mean+/-MSE), respectively. A comparison of concentration - response relations of ATP with and without preapplication of met-Enk indicated that after pretreatment with met-Enk (10(-7) mol/L) the curve shifted downward markedly with a decrease of 25 percent; of the maximum value of I(ATP) and unchanged K(d) value. The suppression of I(ATP) by met-Enk was reversed as evidenced by intracellular dialysis of H-9 by using the repatch technique. Taken together, it is suggested that the inhibition by met-Enk of I(ATP) is caused by activation of opiate receptor, which eventually results in phosphorylation of ATP receptor, mediated by modulation of G protein coupling and intracellular signal transduction.

[grp75 protects cells from injuries caused by glucose deprivation].

To elucidate the functions of grp75, CHL cells over-expressing grp75 are cultured in glucose-free medium in order to simulate energy metabolic stress. Their susceptibilities to injuries caused by glucose deprivation are assessed by trypan blue exclusion, LDH leakage measurement and cytometry analysis. Data shows a stronger resistance to glucose deprivation among cells over-expressing grp75 than among cells constitutively expressing grp75. The outcome suggests that grp75 can protect cells from injuries caused by glucose deprivation.

[Effects of injection of L-glutamate into the locus coeruleus complex area on the respiration].

Experiments were performed on 55 urethane anesthetized and flaxedil immobilized rabbits under artificial respiration. Injection of L-Glutamate into the locus coeruleus area led to a marked augmentation of respiration. This effect of L-Glutamate could be significantly attenuated or reversed by a previous injection of prazosin, yohimbine and propranolol into the NTS area. These results indicate that the neurons in the locus coeruleus are involved in the regulation of the respiration as a result of mediation by alpha and beta receptors in the NTS area.

Effects of microinjection of L-glutamate into locus coeruleus complex area on respiration.

The experiments were performed on 55 rabbits vagotomied, anesthetized with urethane and immobilized with flaxedil. Injection of L-glutamate (L-glu) into the locus coeruleus complex (Lc-Sc) area led to a marked increase in respiratory frequency (RF) and phrenic nerve discharge rate (phr.d) (16 +/- 3.5% and 6.5 +/- 2.5%; P < 0.01 and P < 0.05, respectively), a decrease in inspiratory and expiratory duration, and no obvious change in blood pressure (Bp) was observed. While prazosin (0.5 micrograms/microliters), yohimbin (4 micrograms/microliters) and propranolol (2 micrograms/microliters) were preinjected into the nuclei tractus solitraii (NTS) respectively, the above-mentioned effects of L-glu on respiration was blocked. These results indicate that the excitation of neurons in the Lc-Sc can induce an increase in RF and phr. d and the excitative effect of L-glu on RF was mediated by alpha 1, alpha 2 and beta-receptors in the NTS, while the effect on phr.d was mediated differently.