Aim To study the effects of L-borneol on the chloride channel and cell volume of human umbili-cal vein endothelial cells (HUVECs). Methods Whole-cell patch-clamp technique was used to record chloride currents. The expression of ClC-3 protein was down-regulated by siRNA interference technique. The cell volume was measured by dynamic image analysis. Results 20 nmol·L-1L-borneol significantly activa-ted chloride current in HUVEC (79.59 ± 4.90) pA/pF, which could be inhibited by chloride channel blockers,NPPB and DIDS. The outward current inhib-itory rate of NPPB was (95.57 ± 2.57)%, while that of DIDS was (97.28 ± 6.36)%. The chloride current activated by L-borneol significantly decreased after the silence of ClC-3 (27.03 ± 3.89) pA/pF. Cell volume was markedly reduced by L-borneol (14.38 ± 1.58)%,which was inhibited after NPPB appliance. Conclusion L-borneol can activate ClC-3 chloride channel in HUVECs, which induces Cl- outflow then cell volume decrease.

OBJECTIVETo investigate epidemiological, clinical and genetic features of the first Chinese case of Creutzfeldt-Jakob disease (CJD ) with mutation of E200K in PRNP.

METHODSThe general epidemiological and clinical data were collected; CSF 14-3-3 protein was analyzed by Western blot; The PRNP was amplified by PCR and analyzed.

RESULTSA missense mutation in codon 200 (E200K) of the PRNP was identified in this patient; CSF 14-3-3 protein was positive; sleep disturbance was the initial sign and the other symptoms gradually appeared, including memory loss, dizziness and ataxia.

CONCLUSIONThe CJD patient who was first reported in China has a missense mutation in codon 200 (E200K) of the PRNP, and the codon 129 is a methionine homozygous genotype.

Asian Continental Ancestry Group ; China ; Creutzfeldt-Jakob Syndrome ; genetics ; Humans ; Male ; Middle Aged ; Mutation, Missense ; Prion Proteins ; Prions ; genetics

OBJECTIVETo create transgenic mice expressing hamster- and human-PRNP as a model for understanding the physiological function and pathology of prion protein (PrP), as well as the mechanism of cross-species transmission of transmissible spongiform encephalopathies (TSEs).

METHODSHamster and human-PRNP transgenic mice were established by conventional methods. The copy number of integrated PRNP in various mouse lines was mapped by real-time PCR. PRNP mRNA and protein levels were determined by semi-quantitative RT-PCR, real-time RT-PCR, and western blot analysis. Histological analyses of transgenic mice were performed by hematoxylin and eosin (H & E) staining and immunohistochemical (IHC) methods.

RESULTSIntegrated PRNP copy number in various mouse lines was 53 (Tg-haPrP1), 18 (Tg-huPrP1), 3 (Tg-huPrP2), and 16 (Tg-huPrP5), respectively. Exogenous PrPs were expressed at both the transcriptional and translational level. Histological assays did not detect any abnormalities in brain or other organs.

CONCLUSIONWe have established one hamster-PRNP transgenic mouse line and three human-PRNP transgenic mouse lines. These four transgenic mouse lines provide ideal models for additional research.

Animals ; Blotting, Western ; Cricetinae ; DNA ; genetics ; Disease Models, Animal ; Humans ; Immunohistochemistry ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Organ Specificity ; Plasmids ; Prion Diseases ; genetics ; Prion Proteins ; Prions ; genetics ; Real-Time Polymerase Chain Reaction ; Transcription, Genetic

OBJECTIVETo establish a prion disease PrP(Sc) panel from the brain tissues of experimental hamsters and to address the stability of the panel conserved under the specific condition, for evaluating the diagnostic techniques of human and animal's prion diseases.

METHODS30 brain tissues of hamsters infected with scrapie strain 263K intracerebrally and 30 ones of normal hamsters were enrolled in this panel. Each brain sample was prepared to 10%, 1% and 0.5% homogenates and aliquoted into stocks. The presences of PrP(Sc) in each brain sample were evaluated with PrP-specific Western Blots and partially with immunohistochemistry, and the stability of PrP(Sc) signals in each sample were repeatedly assessed half a year later and 3 years later.

RESULTSPrP(Sc) signals were detected in all stocks of 10% brain homogenates from infected hamsters, 26 out of 30 stocks of 1% homogenates and 19 out of 30 stocks of 0.5% homogenates. The assessments of PrP(Sc) signals in all samples half-year and three years later demonstrated almost unchanged. All homogenates of brain tissues of normal hamsters were PrP(Sc) negative.

CONCLUSIONA prion disease PrP(Sc) panel of the brain tissues, which includes 90 PrP(Sc) positive stocks and PrP(Sc) negative ones, was successfully established, with a reliable stability of PrP(Sc) signals.

Animals ; Brain ; metabolism ; Disease Models, Animal ; Humans ; Male ; PrPC Proteins ; pharmacokinetics ; PrPSc Proteins ; Prion Diseases ; metabolism ; Scrapie ; metabolism ; Tissue Distribution

OBJECTIVETo investigate whether gliosis in the brain tissues of the hamsters infected with various amounts of scrapie strain 263K is correlated with the inoculation doses or the incubation times.

METHODSThe total values of glial fibrillary acidic protein (GFAP) in brains were evaluated by Western Blots and the GFAP-stained cells were detected by immunohistochemistry (IHC). The characteristics of GFAP distributions among various groups were defined by quantitive and statistic analyses.

RESULTSCompared with the brain tissues of normal hamsters, remarkably higher total GFAP levels and more GFAP-stained cells were observed in the brain tissues of infected ones, howbeit, no significant difference was addressed among the infected groups.

CONCLUSIONInoculations of various amounts of scrapie strain 263K into experimental hamsters intracerebrally induced the similar patterns of gliosis in the brains at the clinically terminal stage, regardless of infectious doses and incubation times.

Animals ; Brain ; metabolism ; pathology ; Cricetinae ; Gene Expression ; Glial Fibrillary Acidic Protein ; genetics ; metabolism ; Gliosis ; metabolism ; pathology ; Humans ; PrPSc Proteins ; metabolism ; Prion Diseases ; metabolism ; pathology

Objective To describe the epidemiological and clinical characteristics of Creutzfeldt-Jakob disease (CJD) in China. Methods Clinical and epidemical data on patients from China CJD surveillance network was analyzed. Blood and cerebral spinal fluid (CSF) specimens from these patients were collected. Western blot assay was used to detect 14-3-3 protein in CSF, PCR and sequencing assay were used for analyzing the polymorphism of 129 amino acid and mutation of PRNP gene. Results A total number of 31 probable and 11 possible sporadic CJD patients were identified. Additionally, one patient with Gerstmann-Straussler-Scheinker syndrome (GSS) and 2 familial CJD cases were identified. No geographic- or occupational-related events were observed among these cases. The mean age of onset on the probable or possible CJD patients were 56.7 and 57.4 years old, with sex ratios of the probable CJD patients as 8:9 and the possible one as 5:6 respectively. Rapid progressive dementia was the main foremost symptom, presenting in 33.3% of the CJD patients. Probable CJD patients showed more clinical manifestations than those possible ones. Conclusion Geography distribution, occupation, ratio of gender and the mean onset age of the CJD eases in 2008 were consistent with the characteristics of the sporadic CJD.

AIM:To study the effect of ClC-3 gene over-expression on thyroid structure and function in mice. METHODS:Three-months-old FVB mice were used to study the difference of thyroid structure and function between wild-type(WT)mouse and ClC-3 transgene mice.The expression and distribution of ClC-3 in the thyroid of mice were deter-mined by the methods of qPCR,Western blot and immunofluorescence.Behavioral monitoring was performed on the daily activities of mice.Serum concentrations of total triiodothyronine(TT3), total thyroxine(TT4)and thyrotropin(TSH) were measured by ELISA.RESULTS:Compared with the WT group,the expression of ClC-3 in the thyroid of ClC-3 trans-gene group was significantly increased(P<0.05).The thyroid gland showed obvious hyperplasia and the folliculi glandu-lae thyreoideae was significantly bigger in ClC-3 transgene mice(P<0.05).The weight loss was increased in ClC-3 trans-gene mice(P<0.05).The expression of TT3 and TT4 were significantly higher than that of WT group(P<0.05),but the change of TSH was not obvious.CONCLUSION:ClC-3 over-expression results in thyroid hyperplasia and thyroid hor-mone secretion.This study suggests that ClC-3 is likely to be involved in the synthesis of thyroid hormones.

OBJECTIVE: To study the association of copy number of SMN1 and SMN2 with clinical phenotypes in children with spinal muscular atrophy (SMA). METHODS: A total of 45 children with SMA were enrolled. Multiplex ligation-dependent probe amplification was used to measure the gene copy numbers of SMN1 and SMN2. The association of copy number of SMN1 and SMN2 with clinical phenotypes was analyzed. RESULTS: Of the 45 children with SMA, 42 (93%) had a homozygous deletion of SMN1 exons 7 and 8, and 3 (7%) had a deletion of SMN1 exon 7 alone. No association was found between SMA clinical types and the deletion types of SMN1 exons 7 and 8 (P>0.05). There was a significant difference in the distribution of SMN2 gene copy numbers between the children with SMA and the healthy children (P<0.05). The children with SMA usually had two or three copies of SMN2 gene, while the healthy children usually had one or two copies of SMN2 gene. There was a significant difference in the distribution of SMN2 copy numbers among the children with different SMA clinical types (P<0.05). The children with two copies of SMN2 gene had a significantly lower age of onset than those with three or four copies. Most of the children with type I SMA had two or three copies of SMN2 gene. Most of the children with type II SMA had three copies of SMN2 gene. Most of the children with type III SMA had three or four copies of SMN2 gene. Children with a higher copy number of SMN2 gene tended to have an older age of onset and better motor function and clinical outcome, and there was a significant association between SMN2 gene copy number and clinical outcome (P<0.05). CONCLUSIONS The SMN2 gene can reduce the severity of SMA via the dosage compensation effect. SMN2 copy number is associated with the phenotype of SMA, and therefore, it can be used to predict disease severity.
Child ; Humans ; Muscular Atrophy, Spinal ; genetics ; Phenotype ; Survival of Motor Neuron 1 Protein ; genetics ; Survival of Motor Neuron 2 Protein ; genetics

In human prion diseases, phosphorylated-tau deposition has been described in a rare genetic form, Gerstmann-Straussler-Scheinker disease, but is not considered part of the neuropathological picture of Creutzfeldt-Jakob disease. To investigate the possible changes of tau and phosphorylated tau (Ser396/Ser404) in transmissible spongiform encephalopathies (TSEs), the expressions and transcriptions of above biological factors in the brain tissues of 263K- and 139A-infected hamsters were evaluated by Western blots and Real Time PCR, respectively, followed by quantitative analyses of immunoblot images and relative transcriptional levels compared with normal animals. The contents of total tau increased, but phosphorylated tau at Ser396 and Ser404 decreased, regardless of the types of scrapie agents and clinical incubations. Transcriptions of two tau isoforms were also markedly increased. These findings suggested that dephosphorylation of tau at Ser396/Ser404 was a illness-correlative phenomenon in TSEs. Alterations of tau and phosphorylated tau (Ser396/Ser404) were either intermediate or consequent events in TSE pathogenesis and proposed the potential linkage of these bioactive proteins with the pathogenesis of prion diseases.
Animals ; Blotting, Western ; Brain ; metabolism ; Cricetinae ; Gene Expression Regulation ; physiology ; Phosphorylation ; Polymerase Chain Reaction ; PrPSc Proteins ; pathogenicity ; Prion Diseases ; metabolism ; tau Proteins ; metabolism

OBJECTIVETo establish a stable PrP(Sc) panel from brain tissues of experimental hamsters infected with scrapie agent 263K for evaluating diagnostic techniques of human and animals' prion diseases.

METHODSThirty brain tissue samples from hamsters intracerebrally infected with scrapie strain 263K and another 30 samples from normal hamsters were selected to prepare 10%, 1%, and 0.5% brain homogenates, which were aliquoted into stocks. PrP(Sc) in each brain homogenate was determined by proteinase K digestions followed by Western blot assay and partially by immunohistochemistry. Stability and glycoforms of PrP(Sc) were repeatedly detected by PrP(Sc)-specific Western blots in half a year and 3 years later.

RESULTSPrP(Sc) signals were observed in all 10% brain homogenates of infected hamsters. Twenty out of 30 stocks and 19 out of 30 stocks were PrP(Sc) positive in 1% and 0.5% brain homogenatesof infected hamsters, respectively. Twenty-seven out of 30 stocks presented three positive bands in 10% brain homogenates, whereas none of 1% and 0.5% homogenates contained 3 bands. The detection of PrP(Sc)-specific signals stored in half a year and 3 years later demonstrated that the ratio of PrP(Sc) positive samples and glycoforms was almost unchanged. All normal hamsters' brain homogenates were PrP(Sc) negative.

CONCLUSIONA PrP(Sc) panel of prion disease can be established, which displays reliably stable PrP(Sc)-specific signals and glycoforms.

Animals ; Brain ; Cricetinae ; Immunohistochemistry ; Male ; PrPSc Proteins ; classification ; Scrapie