Structure-based design and characterization of Parkin-activating mutations.

Autosomal recessive mutations in the Parkin gene cause Parkinson's disease. Parkin encodes an ubiquitin E3 ligase that functions together with the kinase PINK1 in a mitochondrial quality control pathway. Parkin exists in an inactive conformation mediated by autoinhibitory domain interfaces. Thus, Parkin has become a target for the development of therapeutics that activate its ligase activity. Yet, the extent to which different regions of Parkin can be targeted for activation remained unknown. Here, we have used a rational structure-based approach to design new activating mutations in both human and rat Parkin across interdomain interfaces. Out of 31 mutations tested, we identified 11 activating mutations that all cluster near the RING0:RING2 or REP:RING1 interfaces. The activity of these mutants correlates with reduced thermal stability. Furthermore, three mutations V393D, A401D, and W403A rescue a Parkin S65A mutant, defective in mitophagy, in cell-based studies. Overall our data extend previous analysis of Parkin activation mutants and suggests that small molecules that would mimic RING0:RING2 or REP:RING1 destabilisation offer therapeutic potential for Parkinson's disease patients harbouring select Parkin mutations.

Cordycepin improved the cognitive function through regulating adenosine A2A receptors in MPTP induced Parkinson's disease mice model.

BACKGROUND: Parkinson's disease (PD), the most common neurodegenerative disorder, primarily affects dopaminergic neurons in the substantia nigra (SN). In addition to severe motor dysfunction, PD patients appear apparent cognitive impairments in the late stage. Cognitive dysfunction is accompanied by synaptic transmission damage in the hippocampus. Cordycepin has been reported to alleviate cognitive impairments in neurodegenerative diseases. PURPOSE: The study aimed to estimate the protection roles of cordycepin on cognitive dysfunction in PD model and explore the potential mechanisms. METHODS: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to establish the PD model in vivo and in vitro experiments. In the in vivo experiments, the C57BL / 6 mice were intraperitoneally injected with MPTP and intragastric administration with cordycepin. Open field test (OFT) was used to estimate the exercise ability. Spontaneous alternation behavioral (SAB) and morris water maze (MWM) tests were used to evaluate the learning and memory abilities. The hippocampal slices from C57BL / 6 and Kunming mice in the in vitro experiments were used to record field excitatory postsynaptic potential (fEPSP) by electrophysiological methods. Western blotting was used to examine the level of tyrosine hydroxylase (TH) in the in vivo experiments and the levels of adenosine A1 and A2A receptors (A1R and A2AR) in the in vitro experiments, respectively. The drugs of MPTP, cordycepin, DPCPX and SCH58261 were perfused through dissolving in artificial cerebrospinal fluid. RESULTS: Cordycepin could significantly reduce the impairments on motor, exploration, spatial learning and memory induce by MPTP. MPTP reduced the amplitude of LTP in hippocampal CA1 area but cordycepin could improve LTP amplitudes. Cordycepin at dosage of 20 mg/kg also increased the TH level in SN. In the in vitro experiments, MPTP inhibited synaptic transmission in hippocampal Schaffer-CA1 pathway with a dose-dependent relationship, while cordycepin could reverse the inhibition of synaptic transmission. Furthermore, the roles of cordycepin on synaptic transmission could been attenuated in the presence of the antagonists of A1R and A2AR, DPCPX and SCH58261, respectively. Interestingly, the level of A2AR rather than A1R in hippocampus was significantly decreased in the cordycepin group as compared to the control. CONCLUSION: The present study has showed that cordycepin could improve cognitive function in the PD model induced by MPTP through regulating the adenosine A2A receptors. These findings were helpful to provide a new strategy for the dementia caused by Parkinson's disease.

Molecular dynamics study of Cl- permeation through cystic fibrosis transmembrane conductance regulator (CFTR).

The recent elucidation of atomistic structures of Cl- channel CFTR provides opportunities for understanding the molecular basis of cystic fibrosis. Despite having been activated through phosphorylation and provided with ATP ligands, several near-atomistic cryo-EM structures of CFTR are in a closed state, as inferred from the lack of a continuous passage through a hydrophobic bottleneck region located in the extracellular portion of the pore. Here, we present repeated, microsecond-long molecular dynamics simulations of human CFTR solvated in a lipid bilayer and aqueous NaCl. At equilibrium, Cl- ions enter the channel through a lateral intracellular portal and bind to two distinct cationic sites inside the channel pore but do not traverse the narrow, de-wetted bottleneck. Simulations conducted in the presence of a strong hyperpolarizing electric field led to spontaneous Cl- translocation events through the bottleneck region of the channel, suggesting that the protein relaxed to a functionally open state. Conformational changes of small magnitude involving transmembrane helices 1 and 6 preceded ion permeation through diverging exit routes at the extracellular end of the pore. The pore bottleneck undergoes wetting prior to Cl- translocation, suggesting that it acts as a hydrophobic gate. Although permeating Cl- ions remain mostly hydrated, partial dehydration occurs at the binding sites and in the bottleneck. The observed Cl- pathway is largely consistent with the loci of mutations that alter channel conductance, anion binding, and ion selectivity, supporting the model of the open state of CFTR obtained in the present study.

Nano-MgO composites containing plasmid DNA to silence SNCA gene displays neuroprotective effects in Parkinson's rats induced by 6-hydroxydopamine.

Parkinson's disease (PD) always causes dyskinesia and cognitive impairments. The alpha-synuclein (α-syn) accumulation, one of the main pathological characteristics of PD, may impair synaptic structural and synaptic functions. Nano-MgO composites has been reported to interfere α-syn expression. The present study is aim to investigate the roles of nano-MgO composites on cognitive impairments in PD rats. PD rats were formed by 6-hydroxydopamine (6-OH DA) and α-syn expression were evaluated by Western blot. Hippocampal dendritic morphology was examined by Golgi staining. Morris water maze (MWM) test was applied to evaluate learning and memory abilities and population spike was recorded by electrophysiological records in vivo. The results showed that: 6-OH DA-treated up-regulated α-syn levels in striatum and hippocampus and increased the rotational times by APO, but nano-MgO composites could down-regulated α-syn levels. The overall length of dendritic and the total number of intersections were reduced by 6-OH DA, accompanied by the decrease of the dendritic spine density in hippocampal CA1, CA3 and DG regions. Interestingly, nano-MgO composites could alleviate the morphological damages of dendrites. In the MWM test, the escape latencies and the swimming distances in PD rats were increased as compared to the sham group, and nano-MgO composites could reduce the escapes latencies and the swimming distances. Furthermore, 6-OH DA reduced the amplitudes of long-term potentiation (LTP) in hippocampal CA1 region, and 6 mg/kg nano-MgO composites could improve LTP amplitudes. In conclusion, the current findings would be helpful to explore the roles of nano-MgO composites on neuroprotection in PD.

Identification of binding sites for ivacaftor on the cystic fibrosis transmembrane conductance regulator.

Ivacaftor (VX-770) was the first cystic fibrosis transmembrane conductance regulator (CFTR) modulatory drug approved for the treatment of patients with cystic fibrosis. Electron cryomicroscopy (cryo-EM) studies of detergent-solubilized CFTR indicated that VX-770 bound to a site at the interface between solvent and a hinge region in the CFTR protein conferred by transmembrane (tm) helices: tm4, tm5, and tm8. We re-evaluated VX-770 binding to CFTR in biological membranes using photoactivatable VX-770 probes. One such probe covalently labeled CFTR at two sites as determined following trypsin digestion and analysis by tandem-mass spectrometry. One labeled peptide resides in the cytosolic loop 4 of CFTR and the other is located in tm8, proximal to the site identified by cryo-EM. Complementary data from functional and molecular dynamic simulation studies support a model, where VX-770 mediates potentiation via multiple sites in the CFTR protein.

[Effect of electroacupuncture with different frequencies on hindlimb locomotor function and expression of LC3, Beclin 1 and cleaved Caspase-3 proteins in spinal cord injury rats].

OBJECTIVE: To investigate the effect of different frequencies of electroacupuncture (EA) on motor function and expression of autophagy-related proteins microtubule-associated protein light chain 3 (LC3), etc. in spinal cord injury (SCI) rats, so as to reveal its underlying mechanisms and provide a reference for clinical application. METHODS: A total of 100 male SD rats were randomly divided into sham control, model, 2 Hz-EA, 100 Hz-EA and 2 Hz/100 Hz-EA groups(n=20 in each group). EA (2 Hz, 100 Hz or 2 Hz/100 Hz, 0.4-0.5-0.6 mA increased by 0.1 mA every10 min) was applied to "HuatuoJiaji" (EX-HN1, T9 and T11) for 30 min, once a day for 7 days. The rat's hindlimb motor function was assessed by using Basso, Beattie and Bresnahan(BBB) locomotor rating scale, inclined plane test and plantar imprinting test, separately. The histological changes and neuronal apoptosis of the spinal cord tissue were observed by Fluoro-Jade B (FJB) and Nissl staining, respectively. The expression of LC3, Beclin 1 and cleaved Caspase-3 proteins in the spinal cord was detected by Western blot. RESULTS: After modeling, the BBB scores and the angles of inclined plane on day 1, 3 and 7 were significantly decreased (P<0.001), and the number of FJB positive cells, and the expression levels of Beclin 1 and cleaved Caspase-3 proteins were considerably increased in the model group compared with the sham control group (P<0.001, P<0.05). After EA intervention, the BBB score and the angles of inclined plate on day 3 in the 2 Hz/100 Hz-EA group (rather than in the 2 Hz- and 100 Hz-EA groups), the BBB score and the angles of inclined plate on day 7 in both 2 Hz/100 Hz and 100 Hz-EA groups(rather than in the 2 Hz-EA group), and the expression levels of Beclin 1 and LC3-Ⅱ/Ⅰ in both 2 Hz/100 Hz and 100 Hz-EA groups (rather than in the 2 Hz-EA group) on day 7 were obviously increased (P<0.001, P<0.01, P<0.05), while the number of FJB-positive neurons in the 3 EA groups, and the expression of cleaved Caspase-3 protein in both 2 Hz/100 Hz and 100 Hz-EA groups and the LC3-Ⅱ/Ⅰ in the 2 Hz-EA group were obviously decreased relevant to the model group (P<0.001, P<0.05). The expression of Beclin 1 in the 100 Hz-EA group was obviously decreased relevant to the 2 Hz/100 Hz-EA group (P<0.05) .Nissl staining displayed appearance of cavities and fuzzy shape of Nissl bodies with light coloration in the injured spinal cord of model group, which was milder in both 2 Hz/100 Hz-EA and 100 Hz-EA groups. Plantar imprinting tests showed dragging gait prints in the model group due to disability in movement, and relatively distinct foot imprints in both 2 Hz/100 Hz and 100 Hz-EA groups. CONCLUSION: Both 100 Hz and 2 Hz/100 Hz-EA can effectively promote the recovery of hindlimb locomotor function of SCI rats, which may be related to its function in promoting autophagy of damaged nerve cells and in reducing neuronal apoptosis.

Astragalus polysaccharide ameliorates lipopolysaccharide-induced cell injury in ATDC5 cells via miR-92a/KLF4 mediation.

BACKGROUND: Astragalus polysaccharide (APS) is a traditional Chinese herbal medicine with anti-inflammatory and anti-aging activities. OBJECTIVE: This study aimed to explore the effect and associated mechanisms of APS on LPS-induced injury in ATDC5 cells, to evaluate the potential of APS for use as an adjuvant therapy for osteoarthritis (OA). MATERIALS AND METHODS: ATDC5 cells were pre-treated with APS and stimulated with lipopolysaccharide (LPS). Cell viability, ROS generation as well as the expression of IL-6, TNF-α, iNOS and Cox-2 were evaluated by performing CCK8 assay, ROS detection, ELISA, western blot and qRT-PCR. The expression of NF-κB and p38MAPK signal pathways related proteins and KLF4 was measured through western blot. RESULTS: LPS increased the expression of IL-6 and TNF-α, elevated the expression of Cox-2, iNOS and increased ROS generation. APS treatment significantly alleviated LPS-induced damage in ATDC5 cells. Besides, miR-92a was down-regulated while KLF4 was up-regulated by APS. At the same time, the targeting relationship between miR-92a and KLF4 was demonstrated. The inhibitory effects of APS on LPS-induced injury in ATDC5 cells were attenuated by the combination of KLF4 siRNA. In addition, LPS induced NF-κB and p38MAPK signal pathways were decreased by APS treatment. Also, the inhibitory effect of APS on NF-κB and p38MAPK signal pathways was reversed by KLF4 siRNA. CONCLUSIONS: The present study reveals that APS protects ATDC5 cells against LPS induced-injury by regulation of miR-92a/KLF4 axis and suppressing NF-κB and p38MAPK signal pathways.

Comprehensive Adsorption Studies of Doxycycline and Ciprofloxacin Antibiotics by Biochars Prepared at Different Temperatures.

This paper comparatively investigated the removal efficiency and mechanisms of rice straw biochars prepared under three pyrolytic temperatures for two kinds of tetracycline and quinolone antibiotics (doxycycline and ciprofloxacin). The influencing factors of antibiotic adsorption (including biochar dosage, pH, background electrolytes, humic acid, initial antibiotics concentration, contact time, and temperature) were comprehensively studied. The results suggest that biochars produced at high-temperature [i.e., 700°C (BC700)], have higher adsorption capacity for the two antibiotics than low-temperature (i.e., 300-500°C) biochars (BC300 and BC500). Higher surface area gives rise to greater volume of micropores and mesopores, and higher graphitic surfaces of the BC700 contributed to its higher functionality. The maximum adsorption capacity was found to be in the following order: DOX > CIP. The π-π EDA interaction and hydrogen bonding might be the predominant adsorption mechanisms. Findings in this study highlight the important roles of high-temperature biochars in controlling the contamination of tetracycline and quinolone antibiotics in the environment.

[Correlative study between angiotensin-converting enzyme gene polymorphism and hepatorenal syndrome].

OBJECTIVE: To investigate the relationship between insertion/deletion (I/D) polymorphism of angiotensin-converting enzyme (ACE) gene and uncompensated cirrhosis of liver with hepatorenal syndrome (HRS). METHODS: ACE I/D polymorphism was detected by polymerase chain reaction amplification of DNA fragment in 56 patients of uncompensated liver cirrhosis with HRS, and 60 healthy individuals served as the controls. At the same time, alanine aminotransferase, aspartate transaminase, serum creatinine (SCr), blood urea nitrogen (BUN) and glomerular filtration rate (GFR) etc. were measured in all the subjects, and the difference between these variables among different genotypes was noted. RESULTS: There was no significant difference in genotypes and allele frequency between the HRS group and controls(all P>0.05). The I allele frequency was higher than the D allele in all the subjects (all P<0.01). But in the control group, there was no significant difference in the genotype frequency among three genomic groups, while the II genotype frequency was higher than the one of ID and DD (all P<0.05). SCr and BUN of the II genotype were higher in the HRS group than that of ID and DD(both P<0.05) and GFR of the II genotype was lower than the one of ID and DD in the HRS group(P<0.05). CONCLUSION: There is relationship between ACE gene polymorphism and the incidence of uncompensated liver cirrhosis with HRS. II genotype may be the genetic factor of vulnerability to HRS patients with uncompensated cirrhosis of liver. The degree of kidney failure in II genotype population is more serious than in ID and DD individuals with uncompensated liver cirrhosis complicated by HRS.