Ectopic expression of archaeal TRAM-encoding genes in rice improves its drought-tolerance / 生物工程学报

Drought stress affects the growth and development of rice, resulting in severe loss in yield and quality. Ectopic expression of the bacterial RNA chaperone, cold shock protein (Csp), can improve rice drought tolerance. Archaeal TRAM (TRM2 and MiaB) proteins have similar structure and biochemical functions as bacterial Csp. Moreover, DNA replication, transcription and translation of archaea are more similar to those in eukaryotes. To test if archaeal RNA chaperones could confer plant drought tolerance, we selected two TRAM proteins, Mpsy_3066 and Mpsy_0643, from a cold-adaptive methanogenic archaea Methanolobus psychrophilus R15 to study. We overexpressed the TRAM proteins in rice and performed drought treatment at seedling and adult stage. The results showed that overexpression both TRAM proteins could significantly improve the tolerance of rice to drought stress. We further demonstrated in rice protoplasts that the TRAMs could abolish misfolded RNA secondary structure and improve translation efficiency, which might explain how TRAMs improve drought tolerance transgenic rice. Our work supports that ectopic expression of archaeal TRAMs effectively improve drought tolerance in rice.

Molecular markers of autophagy / 药学学报

Autophagy is a physiological process which delivers the mutant cytoplasmic proteins and dysfunctional subcellular organs into lysosomes for degradation to generate fuel in the deficiency conditions. It is mainly classified into macroautophagy, microautophagy and chaperon-mediated autophagy (CMA), as well as the selective autophagy such as mitophagy and aggrephagy. This review mainly introduces the key molecular markers of macroautophagy, CMA and mitophagy.

Role of copper transporter protein in copper accumulation in C6 cells induced by lead exposure / 中国职业医学

OBJECTIVE: To investigate the role of copper transporter protein and copper chaperones in copper accumulation in glioma cell line C6 cells induced by lead acetate exposure. METHODS: i) CCK-8 assay was used to determine the proper lead acetate dose by treating the cells with lead acetate at the final concentration of 0-50 μmol / L for 24. 0 hours. ii) C6 cells were divided into control group and lead-exposure group,treated with 0 and 10 μmol / L lead acetate respectively for24. 0 hours,and then cultured in 2 μmol / L copper chloride for 0. 0,0. 5,1. 0,2. 0,4. 0 and 8. 0 hours; inductively coupled plasma mass spectrometry was used to detect the levels of copper and lead in the cells. Real-time polymerase chain reaction was used to detect the mRNA expression of copper transporter 1( CTR1),divalent metal transporter 1( DMT1),copper-transporting ATPase α polypeptide / β polypeptide( ATP7 A and ATP7B), antioxidant 1 copper chaperone( ATOX1),cytochrome c oxidase copper chaperone( COX17),and copper-chaperone-for-superoxide dismutase( CCS).Laser con-focal microscopy was applied to detect the protein expression of CTR1 and ATP7 A in cells. RESULTS: i) CCK-8assay proved that the 10 μmol / L lead acetate treatment did not affect C6 cells proliferation( P > 0. 05). Thus the final concentration of 10 μmol / L lead acetate was chosen as the treatment dose in later experiments. ii) After 10 μmol / L lead acetate exposure for 24. 0 hours,the lead and copper levels of C6 cells in lead-exposure group were higher than those in the control group( P < 0. 01),but there was no statistical significant difference in the C6 cell survival rate between these two groups( P > 0. 05). After cells were treated with copper,the C6 cell survival rate of lead-exposure group was lower than that in the control group( P < 0. 01). The interactive effect of copper level showed statistical significance between lead exposure and cooper treatment time( P < 0. 01). At the 5 time points from 0. 5-8. 0 hours after exposure to copper,the copper levels in lead-exposure group were higher than those of control group( P < 0. 05). The copper levels in the control group reached a peak after exposure to copper for 2. 0 hours,and maintained at a stable level till the time point of 8. 0hours. The copper levels of lead-exposed groups increased with the increasing time of copper exposure and there was a time-effect relationship,and they reached to the peak at the time point of 8. 0 hours. After 10 μmol / L lead acetate exposure for 24. 0 hours,compared with control group,the CTR1 and DMT1 mRNA relative expression levels in leadexposed group increased by 113. 00% and 36. 00% respectively( P < 0. 01),and the ATP7 A mRNA relative expression level decreased by 25. 00%( P < 0. 01). The protein expression of CTR1 increased by 76. 04%( P < 0. 01),and the protein expression of ATP7 A decreased by 16. 0%( P < 0. 01). There was no significant difference in the mRNA relative expression levels of ATP7 B,ATOX1,COX17 and CCS between the two groups( P > 0. 05). CONCLUSION: Lead acetate exposure can lead to increase accumulation of copper in C6 cells with increasing exposure time showing a time-effect relationship. The increased protein expression of CTR1 and decreased protein expression of ATP7 A might be one of the mechanisms of inducing copper accumulation in cells after the lead acetate exposure.

Heterologous expression, chaperone mediated solubilization and purification of parasitic nematode-specific growth factor-like protein of Setaria digitata

OBJECTIVE@#To clone, express and purify a putative parasitic nematode specific protein of Setaria digitata (S. digitata), filarial nematode that infects livestock and cause significant economic losses in Far East and Asia to be used for structural and functional analyses.@*METHODS@#To characterize uncharacterized gene of S. digitata (SDUG), the herterologous expression of SDUG was carried out in the pET [cloned into pET45b(+)] expression system initially and co-expression of SDUG using chaperone plasmids pG-KJE8, pGro 7, pKJE7, pG-Tf2 and pTf16 containing chaperone proteins of dnaK-dnaJ-grpE-groES-gro-E, groES-groEL, dnaK-dnaJ-grpE, groES-groEL-tig, and tig respectively, was carried out subsequently.@*RESULTS@#Expression of SDUG was seen when Escherichia coli strain BL21(DE3) is used, while concentrating protein largely into the insoluble fraction. The co-expression of SDUG using chaperone plasmid mediated system indicated a significant increase of the protein in the soluble fraction. Of the chaperon plasmid sets, the highest amount of recombinant SDUP in the soluble fraction was seen when pGro7 was used in the presence of 2 mg/mL L-arabinose and 0.6M IPTG concentration in the culture medium and for 3 h of incubation at the temperature of 28 °C. Recombinant SDUG was purified both from soluble and insoluble fractions using Ni affinity chromatography. SDS-PAGE and western blot analyses of these proteins revealed a single band having expected size of ∼24 kDa.@*CONCLUSIONS@#SDUG seems to be more aggregate-prone and hydrophobic in nature and such protein can make soluble by correct selecting the inducer concentrations and induction temperature and its duration.

The application of Chaperon guiding catheter system in endovascular treatment of intracranial aneurysms / 介入放射学杂志

Objective To discuss the application of Chaperon guiding catheter system in endovascular treatment of intracranial aneurysms. Methods A total of 20 patients with intracranial aneurysms were enrolled in this study. The patients hadⅡorⅢtype of aortic arch (n=11) or sclerotic plague at the orifice of internal carotid or vertebral artery (n = 9). Endovascular embolization of the intracranial aneurysm was carried out in all patients. By using Cordis guiding catheter system the catheter was placed into the target artery. Chaperon guiding catheter system was used during the procedure in order to determine whether the Chaperon guiding catheter could be smoothly placed into the target artery or not. Results When the Chaperon guiding catheter system was employed in the endovascular procedure, the difficulties of catheterization caused by the distortion of the aorta or by the plagues on the walls of arteries could be basically overcome. The Guiding catheter could be smoothly placed into the target arteries. Conclusion The Chaperon guiding catheter system can be successfully used in the endovascular treatment for the intracranial aneurysms, especially when the patient has tortuous aorta or there is sclerotic plague on the artery wall. (J Intervent Radiol, 2014, 23:281-283).

Changes of endoplasmic reticulum molecular chaperon GRP78 expression in spinal cord of ischemia/reperfusion injuried rat / 基础医学与临床

Objective To evaluate the changes of GRP78 expression in the Spinal Cord of Ischemia/Reperfusion Injuried Rat.Methods Fifty five adult Wistar rats(250～300 g)were randomly divided into 2 groups as control group(n=5)and operation group(n=50).The spinal cord SCII model was established.The expression of GRP78 was detected in spinal cord tissue through immunohistochemistry(IHC)and Western blot analysis,and the neuronal apoptosis was detected through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)methods.Results The GRP78 expression was increased at 30 min of reperfusion,and peaked at 7 h,but began to decline at 11 h post-reperfusion,and reduced significantly at 23 h.The number of GRP78 positive neurons at 0.5,3,7,11 and 23 h groups was(19.4?1.34),(42.6?2.30),(82.4?2.07),(40.0?1.58)and(18.8?0.83),respectively and significantly higher than that of control group(P

Nuclear localization and the heat shock proteins.

The highly conserved heat shock proteins (HSP) belong to a subset of cellular proteins that localize to the nucleus. HSPs are atypical nuclear proteins in that they localize to the nucleus selectively, rather than invariably. Nuclear localization of HSPs is associated with cell stress and cell growth. This aspect of HSPs is highly conserved with nuclear localization occurring in response to a wide variety of cell stresses. Nuclear localization is likely important for at least some of the heat shock proteins' protective functions; little is known about the function of the heat shock proteins in the nucleus. Nuclear localization is signalled by the presence of a basic nuclear localization sequence (NLS) within a protein. Though most is known about HSP 72’s nuclear localization, the NLS(s) has not been definitively identified for any of the heat shock proteins. Likely more is involved than presence of a NLS; since the heat shock proteins only localize to the nucleus under selective conditions, nuclear localization must be regulated. HSPs also function as chaperons of nuclear transport, facilitating the movement of other macromolecules across the nuclear membrane. The mechanisms involved in chaperoning of proteins by HSPs into the nucleus are still being identified.