BACKGROUND: Lot of researches has been made on investigating the influence of neurturin(NTN) on dopaminergic neurons,motor neurons,sensory neurons and sympathetic neurons in respect of its enhancing survival and neuroprotective potential. But reports about its influence on cholinergic neurons still remained less.OBJECTIVE: To study the influence of NTN combined with nerve growth factor(NGF) on the survival of NGF receptor positive neurons in vitro.DESIGN: A repeated measurement and experimental study. based on cell model.SETTING: Department of anatomy in a university.MATERIALS: This study was carried out at the Human Anatomical Department of Guangzhou Medical College between September 2001 and June2002. Neonatal SD rats were adopted,DMEM/F12 culture medium supplemented with 20 g/L additive without serum were filtered and sterilized for cell culture.METHODS: Rats were divided into four groups: NGF group,NTN group,NGF + NTN group and control group. Cells of the former three groups were cultured with NGF,NTN and NGF + NTN of 100 μg/L respectively,which were replaced by culture medium in control group. Cholinergic neurons derived from neonatal rat basal forebrain were primary cultured for observing the influence of NTN,NGF on their survival by using immunohistochemical combined imaging analysis method.MAIN OUTCOME MEASURES: The survival rate and growing state of NGFR positive neurons derived from forebrain of neonatal rats when cultured for 4 days and 8 days in vitro.RESULTS: In 4-day culture,indexes of NTN group were significantly different from control group( P ＜ 0. 05 ),and indexes of NGF + NTN group were better than those of NGF group or NTN group( P ＜ 0. 05); in 8-day culture,all indexes except the number of neuronal processes in NTN group were similar to control group( P ＞ 0.05),and indexes of NGF + NTN group were still better than that of NGF group or NTN group( P ＜ 0. 05); but the number of NGF-R positive neuronal processes in NTN group was more that that in NGF group ( P ＜ 0. 05).CONCLUSION: NTN has neurotrophic effect on NGF-R positive neurons of neonatal rat basal forebrain in vitro by relatively shorter acting time,and the number of neuronal processes induced by NTN is stronger than NGF; Moreover NTN combined with NGF exerts better effect than NTN or NGF alone in vitro.

BACKGROUND:Measurement of cardiac troponin plays an important role in diagnosis of myocardial infarction.OBJECTIVE:To label the rabbit cardiac troponin C(cTnC) by a fluorescent probe 5-iodoaccetamidofluorescein(5-IAF),and to observe whether the 5-IAF can be used to study the interaction between cTnC and other contractile regulatory proteins.DESIGN,TIME AND SETTING:A randomized control experiment was performed at Department of Human Anatomy,Guangzhou Medical College,from January 2002 to December 2005.MATERIAL:Adult rabbits were provided by Experimental Animal Center of Guangzhou Medical College.METHODS:The rabbit cTnC DNA fragment was prepared with RT-PCR method.This gene fragment was cloned to pET expression vector by gene recombination technology.The site-directed mutagenesis were used to produce a mutant containing single cysteine at position 84 by replacing Cys35 with Ser,cTnC(C35S).The cTnC(C35S) was labeled by 5-IAF and 2-(4'-(iodoacetamido) anilino) naphthalene-6sulfonic acid(IAANS),Respectively.And then,the fluorescence emission(steady-state and time-resolved) was performed.MAIN OUTCOME MEASURE:The fluorescence properties of 5-IAF-labeled cTnC(C35S) and IAANS-labeled cTnC(C35S).RESULTS:The excitation of apo-cTnC(C35S)IAF was performed at 491 nm,and the emission peak was at 520 nm.Saturation of cTnC(C35S)IAF with Mg led to a 35% decrease in fluorescence intensity.Another 35% decrease with a 3 nm-blue shift was seen as the protein was saturated with Ca.The two-phase transitions of fluorescence emission from IAANS-labeled cTnC in response to Mg and Ca did not appear in fluorescence emission of 5-IAF-labeled cTnC.However,the Ca-induced conformational change in cTnC remained unchanged no matter which probe was used.Ca titration experiments showed that binding parameters derived from the fluorescence emission of the two probes were comparable.CONCLUSION:5-IAF is an appropriate probe that can be used to study the interaction between cardiac troponin C and other contractile regulatory proteins.