OBJECTIVE:To investigate the possibility of constructing anti-cancer drug in-situ hydrogel with self-assembling peptide RAD16-Ⅰ. METHODS:The rheological parameters as storage modulus(G′),loss modulus(G″)and phase angle(Δ)of 0.1%,0.2% and 0.5% RAD16-Ⅰ solution containing paclitaxel or not were determined by rheometer before and after mixing with isometric phosphate buffer solution (PBS);RAD16-Ⅰ solution containing paclitaxel or not were mixed with breast cancer MDA-MB-435S cells culture medium to obtain hydrogel,the status and effect of which on cell morphology were observed by in-verted microscope (compared with paclitaxel solution). RESULTS:In RAD16-Ⅰ solution containing paclitaxel or not,G′was close to or slightly higher than G″,and G′and G″had changed slightly as the concentration of peptide increased. Compared with not mixed with PBS,G′increased significantly in concentration-dependent manner after mixed with PBS,and G″also increased but was slighter than G′;Δ decreased significantly. In cell culture media,RAD16-Ⅰ solutions containing paclitaxel could form hy-drogel and maintain their gel form,cancer cells kept same morphology after treated with hydrogel and same concentration of pacli-taxel solution for same time. CONCLUSIONS:RAD16-Ⅰ solutions containing paclitaxel can form hydrogel under simulated physi-ological conditions,which can maintain their gel form and have anti-cancer effect of paclitaxel.

This study is to investigate the amelioration effect of glucocorticoid receptor (GR) antagonist mifepristone on the changes of learning and memory abilities in rat model of depression. In the present study, a 35-day rat chronic unpredictable stress (CUS) model was used to observe both depression-like behaviors with sucrose preference test and open-field test and learning and memory-associated behaviors with Morris water maze test. A total of 45 male adult Sprague-Dawley rats were randomly assigned to three groups of equal size: control group (CON); CUS group (CUS); CUS + mifepristone group (CM). Animals in CM group were first exposed to CUS for 14 days, and then were administered with 50 mg x kg(-1) x d(-1) of mifepristone with continued CUS procedure. Corticosterone EIA Kit was used to detect the concentration of plasma corticosterone (CORT). Nissl staining was used to observe the structure of hippocampus. The results demonstrated that CUS exposure induced both depressive-like and learning and memory-associated behaviors and these deficits were reversed by mifepristone. Compared to CON group, the concentration of plasma CORT increased significantly in CUS group. CUS exposure damaged the structure of hippocampus, whereas mifepristone had an amelioration effect. Together, the structural deficits of hippocampus resulting from long-term stress exposure, which could contribute to the impairment of learning and memory in depression, are reversed by the GR receptor antagonist mifepristone.

Aim To construct the cell model targeted on the damage by α-synuclein for screening anti-Parkinson’s Disease (PD)compounds.Methods The cDNA fragment of α-synucle-in gene was obtained by PCR methods and inserted into the re-combinant prokaryotic plasmid by molecular cloning technique. The recombinant plasmid was transformed into Escherichia coli, and subsequently induced to express α-synuclein protein.The recombinant α-synuclein was purified and identified by affinity chromatography,immunoblotting and mass spectrometry.The cells damage by α-synuclein was evaluated through cell viability measured by 3-(4,5-dimethyl-2-thiazolyl )-2,5-diphenyl-2-H-tetrazolium bromide.Results The obtained cDNA fragment ofα-synuclein in accordance with its theoretic molecular weight was cloned into pET30a plasmid and verified by sequencing.The re-combinant plasmid was transformed into bacteria E.Coli.BL21 (DE3)and induced to express α-synuclein by isopropyl β-D-1 -thiogalactopyranoside (IPTG).The expression condition was op-timized according to the culture temperature,the concentration of IPTG and the proliferation state of bacteria.The purified α-synu-clein was proved to be a 1 5.3 ku molecule weight protein,and could be immunoblotted with anti-α-synuclein antibody.The pu-rified α-synuclein could decrease the viability of PC1 2 cells and primary neurons significantly,and its effect was in a concentra-tion-dependent manner.Conclusion We have succeeded in constructing the cell model targeted on the damage by α-synucle-in.

Aim To study the effect of synapsinⅠon synaptic transmission in rat dentate gyrus induced by(-) clausenamide.Methods The basal synaptic transmission experiment was conducted through electrophysiological recordings.The effect of(-) clausenamide on synapsinⅠ phosphorylation was measured by western blot and confocal microscopy.Results(-)Clausenamide increased the population spike(PS) of hippocampal dentate gyrus.The phosphorylation of synapsinⅠ was increased both in cortex and hippocampus,the maximum effect was observed at 5 min in hippocampus and at 15 min in cortex.Furthermore,(-)clausenamide promoted the phosphorylation of synapsinⅠat a dose-denpendent manner in PC12 cells.The phosphorylation of synapsinⅠ in PC12 cells and synaptosomes incubated with(-)clausenamide was increased and reached maximum at 1~2 min.However,H89,PKA inhibitor,blocked the effect of(-)clausenamide on synapsinⅠ phosphorylation.Conclusion(-)Clausenamide activated synapsinⅠ via PKA signal pathway,which may contribute to the effect of(-)clausenamide on potentiating basal synaptic transmission.

Aim To investigate the role of chemokine-like factor 1 ( CKLF1 ) in SH-SY5 Y cell migration and its molecular regulatory mechanism. Methods SH-SY5Y cells were stimulated with CKLF1 for 0. 5 h, 2 h, 8 h and 24 h, respectively. The migration distance and the percentage of migration cells were recorded by CELLocate analysis. The phosphorylation of focal ad-hesion kinase ( FAK) at Tyr-397 site was detected by Western blot analysis. By chemotaxis assays, we con-firmed the chemotaxis of CKLF1. Furthermore, FAK inhibitor PF-573228 and PLCγ inhibitor U73122 were used for the research of molecular regulatory mecha-nisms involved. Results CKLF1 promoted cell migra-tion and induced a strong increase in the phosphoryla-tion level of FAK-pY397 , which were significantly at-tenuated by the presence of U73122 ( a specific inhibi-tor for PLCγ) . In addition, the chemotaxis of CKLF1 was obviously blocked by the FAK inhibitor PF-573228 . Conclusion CKLF1 induces SH-SY5 Y cell migration via PLCγ/FAK signaling pathway.

Abstract:Aim To study the effect of rotenone on α-synuclein in rat midbrain of Parkinson's disease(PD).Methods Rats were subcutaneouly injected with chronic low dose rotenone(1.0 mg·kg~(-1)·d~(-1)).Movements within 5 minutes were evaluated in open field test.Tyrosine hydroxylase(TH)-positive neuronsin the midbrain were measured with immunofluorescence staining.α-SYN protein level in the midbrain was demonstrated with immunohistochemical staining and Western blot.Results Compared to the control group,latency time,crossing,rearing and rearing time were changed significantly(P<0.05,0.01)in the rotenone group,TH-positive neurons were reduced(P<0.05)and α-SYN protein level in the midbrain was increased(P<0.05).Conclusion Injection with rotenone can induce PD symptom,which may be correlated to α-SYN protein level in the midbrain.

Aim To study the screening of the nucleo-tide sequences might be affected by α-syn in vitro. Methods The nucleotide sequences were synthesized according to the feature of base composition, and then mixed with the α-syn-GFP. The CD was used to ana-lyse the changes of the peak. Result The peak of the CD changed greatly when the α-syn-GFP mixed with the GC-box like sequence. Conclusion The α-syn-GFP might affect the GC-box like sequence after trans-located into the nuclei. Then, it plays a role in physio-logical and pathological conditions by affecting the reg-ulation of gene expression.

Objective To report the clinical and genetic features in a family with Mohr-Tranebjaerg syndrome (MTS).Methods After collecting clinical data of the family,visual and auditory evoked potentials,pure tone audiometry,transient evoked otoacoustic emissions and distortion product otoacoustic emissions were carried out in proband (Ⅲ5).The translocase of inner mitochondrial membrane 8 homolog A (TIMM8A) gene was sequenced in proband,Ⅰ2,Ⅱ 4 and Ⅱ 6.Results Ⅲ5,a 15-year-old boy presented with deafness,slurred speech,difficulty in finger extension and dystonia gradually since 2 years old.Ⅲ1,Ⅲ 2 and Ⅲ 3 presented with deafness at the age of 2.Ⅰ 2,Ⅱ 2,Ⅱ 4 and Ⅱ 6 showed mild decreased visual acuity at the age of 12.Visual evoked potentials revealed prolonged P100 latency in both eyes.Pure tone audiometry revealed severe sensorineural hearing loss.The auditory evoked potentials showed no wave in the bilateral ears.Otoacoustic emissions were not elicited bilaterally.A novel c.133-2delA mutation in TIMM8A gene was identified in Ⅲ 5,Ⅰ2,Ⅱ 4 and Ⅱ 6.Conclusions We confirm the MTS caused by a novel c.133-2delA mutation in TIMM8A gene.Except for the deafness,the development of other symptoms of the disease vary obviously from case to case in the same family.The female carriers with mild lesion of optic nerves.

Aim To study of the expression and distribution of four α-synuclein truncations in three cells.Method Four α-synuclein gene truncations were obtained by PCR method,followed by subcloning into the pEGFP-N1 eukaryotic expression vector.Four obtained recombination plasmids were transfected into MN9D cells,PC12 cells and SH-SY5Y cells using Lipofectamine 2000 respectively.The expression and distribution of four α-synuclein truncations were observed by Confocal.Results Distribution of four α-synuclein truncations was discrepant obviously,the truncations,with more C terminal remained,were prone to emerging in nuclei.Conclusion Localization of α-synuclein protein in cells may be related to the C terminal,and the whole C terminal plays an important role in distribution of α-synuclein into nuclei.

BACKGROUNDHearing impairment has been reported to be common in patients with mitochondrial disorders, a group of diseases characterized by pleiomorphic clinical manifestations due to defects in oxidative phosphorylation of mitochondria. This study aimed to investigate the audiological characteristics in a large cohort of patients with mitochondrial disease.

METHODSComprehensive audiological evaluations, including pure tone audiometry, tympanometry, speech audiometry, otoacoustic emissions, electrocochleography and auditory brainstem evoked potentials, were performed in 73 Chinese patients with mitochondrial encephalomyopathy and with confirmed mitochondrial DNA (mtDNA) defects.

RESULTSAmong the patients, 71% had hearing impairment. However, the incidence rate and severity of hearing impairment were much less in the chronic progressive external ophthalmoplegia (CPEO) subtype than in the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonic epilepsy with ragged red fibers (MERRF) and Kearns-Sayre syndrome (KSS) subtypes. While most of our patients had a predominantly cochlea origin for the hearing deficit, five patients had an auditory neuropathy spectrum disorder and three patients had impairment of both cochlea and auditory cortex.

CONCLUSIONSVarious portions of the auditory system could be involved in patients with mitochondrial diseases, including cochlea, auditory nerve, auditory pathway and cortex. Hearing loss was more associated with multisystem involvement. Genotype, mutant load of mtDNA and other unknown factors could contribute to heterogeneity of hearing impairment in mitochondrial disease.

Adolescent ; Adult ; Aged ; Child ; Female ; Hearing Loss ; physiopathology ; Hearing Loss, Central ; physiopathology ; Humans ; Male ; Middle Aged ; Mitochondrial Encephalomyopathies ; physiopathology ; Young Adult