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An Escherichia coli ATP-dependent two-component protease, ClpYQ(HslUV), targets the SulA molecule, an SOS induced protein. ClpY recognizes, unfolds and translocates the substrates into the proteolytic site of ClpQ for degradation. ClpY is divided into three domains N, I and C. The N domain is an ATPase; the C domain allows for oligomerization, while the I domain coordinates substrate binding. In the ClpYQ complex, two layer pore sites, pore I and II, are in the center of its hexameric rings. However, the actual roles of two outer-loop (130~159 aa, L1 and 175~209 aa, L2) of the ClpY-I domain for the degradation of SulA are unclear. In this study, with ATP, the MBP-SulA molecule was bound to ClpY oligomer(s). ClpYΔL1 (ClpY deleted of loop 1) oligomers revealed an excessive SulA-binding activity. With ClpQ, it showed increased proteolytic activity for SulA degradation. Yet, ClpYΔL2 formed fewer oligomers that retained less proteolytic activity, but still had increased SulA-binding activity. In contrast, ClpYΔpore I had a lower SulA-binding activity. ClpYΔ pore I ΔL2 showed the lowest SulA-binding activity. In addition, ClpY (Q198L, Q200L), with a double point mutation in loop 2, formed stable oligomers. It also had a subtle increase in SulA-binding activity, but displayed less proteolytic activity. As a result, loop 2 has an effect on ClpY oligomerization, substrate binding and delivery. Loop 1 has a role as a gate, to prevent excessive substrate binding. Thus, accordingly, ClpY permits the formation of SulA-ClpY (6x) , with ATP(s), and this complex then docks through ClpQ (6x) for ultimate proteolytic degradation.

Methionine is critical for variety of metabolic processes in biological organisms, acting as a precursor or intermediate for many final products. The last step for the synthesis of methionine is the methylation of homocysteine, which is catalyzed by MetE. Here, we use Salmonella enterica serovar Typhimurium LT2 to study the regulation of the metE + gene by an anaerobically induced small non-coding RNA-FnrS, the expression of which is strictly dependent on the anaerobic regulator-FNR. The MetE-HA protein was expressed at an increased level in the fnrS - and hfq - deficient strains under anaerobic conditions. The Hfq protein is predicted to stabilize the binding between small RNA(s) and their target mRNA(s). A transcriptional (op) and translational (pr) metE::lacZ fusion gene were separately constructed, with the metE + -promoter fused to a lacZ reporter gene. In an anaerobic environment, the metE::lacZ (pr) fusion gene and reverse transcription-PCR identified that FnrS and/or FNR negatively regulate metE + mRNA levels in the rich media. Analysis of FnrS revealed a sequence complementary to the 5' mRNA translational initiation region (TIR) of the metE + gene. Mutation(s) predicted to disrupt base pairing between FnrS and metE + TIR were constructed in fnrS, and most of those resulted in the loss of repressive activity. When compensatory mutation(s) were made in metE + 5' TIR to restore base pairing with FnrS, the repressive regulation was completely restored. Therefore, in this study, we identified that in anaerobic phase, there is a repression of metE + gene expression by FnrS and that base-paring, between both expressive transcripts, plays an important role for this negative regulation., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Objective: To investigate the effect of Pteris semipinnata L. (PsL) extract Ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F)-on HepG2 cells and explore its potential mechanism., Methods: Cytotoxicity of 5F was studied in HepG2 cells treated with different doses of 5F (0 - 80 mg/L) for 24 h and cell viability was determined by MTT assay. To analyze apoptosis qualitatively, the Hoechst/PI assay was used. The level of Bax in mitochondria fraction of 5F-treated HepG2 cells was determined by western blotting. The levels of cyto-c and AIF in the cytosol were analyzed by western blotting., Results: The cytotoxicity of 5F on HepG2 cells was elevated with the increasing of 5F concentrations, as evidenced by the cell viability assay. The apoptotic cells characterized by condensed neclei were observed after the exposure of HepG2 cells to 5F. The level of Bax in mitochondria fraction of 5F-treated HepG2 cells increased. The levels of cyto-c and AIF in the cytosol of 5F-treated HepG2 cells increased., Conclusion: 5F mediated apoptosis involves mitochondria-dependent pathway and 5F might have a therapeutic value against human cancer cell lines and especially on hepatocellular carcinoma (HCC) cells.

Objective: To establish the quality standard of PsL injections containing mainly 5F (ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid)., Method: The identification of PsL was performed by thin-layer chromatography, and the content was determined by HPLC. The column was Hypersil C18 (4.6 mm x 250 mm, 5 microm), the mobile phase was the mixture of methane-water-acitic acid (55:45: 0.045) with a flow rate of 1.0 mL x min(-1), the detective wavelength was 254 nm, and the column temperature was maintained at 35 degrees C. The pH value and K+ content of the three batchs injection were determined with pH meter and flame photometric meter, and the contents of tannin, protein, oxalic acid salt and heavy metals were detected by deferent methods., Result: The TLC method was suitable for the identification of PsL5F. The linearity for 5F was obtained over the range of 30-240 microg x mL(-1) (r = 0.999 8), the average recovery of 5F was 99.8%. The injections were of pH value range from 7.80 to 8.20, K+ contents less than 10 mmol x L(-1), and the contents of tannin, protein, oxalic acid salt and heavy metals were qualified with the Chinese pharmacopoeia, respectively., Conclusion: It's sensitive and reliable that can be used as quality control methods of PsL5F injections.

Objective: To investigate the possible mechanisms of nobiletin for anticancer by studying the inhibition effects of nobiletin on tubulin polymerization., Method: In vitro nobiletin was added into the tubulin polymerization-depolymerization system and the absorption values were recorded at 350 nm under 37 degrees C., Result: As compared with controls, the absorption values in reaction system decreased significantly in nobiletin treatment groups. When nobiletin final concentrations in reaction system were 5.0, 7.5, 10.0 and 12.5 micromol x L(-1), the maximum absorption values were 0 130, 0.109, 0.086 and 0.071 with 16.7%, 30.1%, 44.9% and 54.5% of inhibition rate, respectively. The results suggested that nobiletin could inhibit tubulin polymerization., Conclusion: The inhibition effect of nobiletin on tubulin polymerization is the possible mechanism for anticancer.

Objective: To observe the effect of Armillaria mellea polysaccharide on mice bone marrow cells damage caused by Cyclophosphamide., Method: Kunming purebred mice were used and stochastic divided into 5 groups: normal control group, positive control group (rhG-CSF 20 microg x kg(-1) x d(-1)), damage group of Cyclophosphamide (150 mg x kg(-1) x d(-1)), the protective group with A. mellea polysaccharide, low dose (25.0 mg x kg(-1) x d(-1)) and high dose (50.0 mg x kg(-1) x d(-1)). Positive control was s.c. rhGCSF 6 d and i.p. Cyclophosphamide 3 d. A. mellea polysaccharide was i.p. 8 d. and Cyclophosphamide i.p. 3 d. WBC, RBC, PLT, BMNC were counted in peripheral blood and bone marrow cells. The myelogram were analyzed in bone marrow., Result: The WBC, RBC, PLT, BMNC of protective group and positive control group were higher than damage group (P < 0.01) significantly high dose group increaser than low dose group in protective group the numbed of Promyelocytic and lobulation nuclear of marrow., Conclusion: A. mellea polysaccharide has preferably protective effect on damaging mice bone marrow cell caused by Cyclophosphamide.