Intracellular ASIC1a regulates mitochondrial permeability transition-dependent neuronal death.

Acid-sensing ion channel 1a (ASIC1a) is the key proton receptor in nervous systems, mediating acidosis-induced neuronal injury in many neurological disorders, such as ischemic stroke. Up to now, functional ASIC1a has been found exclusively on the plasma membrane. Here, we show that ASIC1a proteins are also present in mitochondria of mouse cortical neurons where they are physically associated with adenine nucleotide translocase. Moreover, purified mitochondria from ASIC1a(-/-) mice exhibit significantly enhanced Ca(2+) retention capacity and accelerated Ca(2+) uptake rate. When challenged with hydrogen peroxide (H2O2), ASIC1a(-/-) neurons are resistant to cytochrome c release and inner mitochondrial membrane depolarization, suggesting an impairment of mitochondrial permeability transition (MPT) due to ASIC1a deletion. Consistently, H2O2-induced neuronal death, which is MPT dependent, is reduced in ASIC1a(-/-) neurons. Additionally, significant increases in mitochondrial size and oxidative stress levels are detected in ASIC1a(-/-) mouse brain, which also displays marked changes (>2-fold) in the expression of mitochondrial proteins closely related to reactive oxygen species signal pathways, as revealed by two-dimensional difference gel electrophoresis followed by mass spectrometry analysis. Our data suggest that mitochondrial ASIC1a may serve as an important regulator of MPT pores, which contributes to oxidative neuronal cell death.

Molecular interaction of α-synuclein with tubulin influences on the polymerization of microtubule in vitro and structure of microtubule in cells.

Microtubule dynamics is essential for many vital cellular processes such as in intracellular transport, metabolism, and cell division. Evidences demonstrate that α-synuclein may associate with microtubular cytoskeleton and its major component, tubulin. In the present study, the molecular interaction between α-synuclein and tubulin was confirmed by GST pull-down assay and co-immunoprecipitation. The interacting regions within α-synuclein with tubulin were mapped at the residues 60-100 of α-synuclein that is critical for the binding activity with tubulin. Microtubule assembly assays and sedimentation tests demonstrated that α-synuclein influenced the polymerization of tubulin in vitro, revealing an interacting region-dependent feature. Confocal microscopy detected that exposures of α-synuclein proteins inhibited microtubule formation in the cultured cells, with a length-dependent phenomenon. Our data highlight a potential role of α-synuclein in regulating the microtubule dynamics in neurons. The association of α-synuclein with tubulin may further provide insight into the biological and pathophysiological function of synuclein.

Transient expressions of doppel and its structural analog prionDelta32-121 in SH-SY5Y cells caused cytotoxicity possibly by triggering similar apoptosis pathway.

Doppel (Dpl) is a recently identified prion (PrP)-like protein due to the structural and biochemical similarities, however, its natural function and pathogenic role in neurodegenerative diseases remains unclear. To investigate the possible pathogenic pathway of Dpl and its structural analog for cell apoptosis, mammalian expressing recombinant plasmids containing human PRND gene encoding the full-length Dpl and a truncated human PRNP gene deleting the sequences encoding the peptide from aa 32 to 121 (PrPDelta32-121) were generated. MTT assays showed the cell viabilities of the human neuroblastoma cell line SH-SY5Y receiving Dpl and PrPDelta32-121 expressing plasmids were remarkably lower. Obvious apoptosis phenomena were observed to be associated with the cells transient expressing Dpl and PrPDelta32-121, including reduced mitochondrial transmembrane potential (psim), decreased pro-caspase-3 quantity, more numbers of annexin V- and annexin V/PI-double-stained cells and depressed Bcl-2 level. Moreover, we also found that the Dpl- and PrPDelta32-121-induced cytotoxicities and relevant apoptotic events in SH-SY5Y cells could be fully antagonized by co-expression of the human full-length PrP. These data highly indicate that cytotoxicity induced by the expression of Dpl and truncated PrP in neural derived cells are closely related with the apoptosis process, probably triggering the mitochondrial pathway. It also implies that the cell-benefit activity of the full-length PrP may result from its anti-apoptosis capacity.

Creutzfeldt-Jakob disease in a Chinese patient with a novel seven extra-repeat insertion in PRNP.

Familial transmissible spongiform encephalopathies comprise about 14% of all cases of transmissible spongiform encephalopathy in humans. We report on a patient with a definite diagnosis of familial Creutzfeldt-Jakob disease with an insertional mutation consisting of seven extra octapeptide repeats between codons 51 and 91 in the PRNP gene, associated with a genotype homozygotic for methionine at codon 129 and a novel coding change of the inserted octapeptide region.

Sensitive detection of PrPSc by Western blot assay based on streptomycin sulphate precipitation.

Transmissible spongiform encephalopathies, also termed prion diseases, are fatal neurodegenerative disorders that affect both humans and animals, which are characterized by presences of protease-resistance disease-associated prion protein (PrP(Sc)) in brains. In the present study, we optimized the Western blot assay for PrP(Sc) with a precipitation procedure of streptomycin sulphate. After incubated with suitable amount of streptomycin sulphate, the detective sensitivity for PrP(Sc) was remarkably improved. The precipitation of PrP(Sc) was obviously influenced by pH value in the solution. Employs of PrP(Sc) stock sample into various mimic specimens, including normal hamster brain homogenate, human cerebrospinal fluid and urine, demonstrated that streptomycin precipitation markedly increased the detective sensitivity of PrP(Sc), regardless in low concentration or in large volume. In addition, the PrP(Sc) from a human brain tissue of familiar Creutzfeldt-Jakob disease (fCJD) was efficiently precipitated with streptomycin sulphate. As a sensitive, specific, rapid and flexible protocol for PrP(Sc), the protocol in this study has the potential, alone or combined with other techniques, to detect low levels of PrP(Sc) in the specimens not only from central nerve system, but also from peripheral organs or fluids.

Influence of guanidine on proteinase K resistance in vitro and infectivity of scrapie prion protein PrP(Sc).

As the scrapie prion protein PrP(Sc) is rich in beta-sheets it aggregates into prion rods, which show infectivity and proteinase K (PK) resistance. Consequently, dissociation of prion rods and breakdown of beta-sheets in PrP(Sc) by denaturation results in loss of both infectivity and PK-sensitivity. In this study, the effects of guanidine (Gdn), which solubilizes and denatures proteins by breaking down their higher structure, on the solubility, the PK-resistance in vitro and the infectivity of PrP(Sc) of scrapie strain 263K was examined. The infectivity was assayed by intracerebral inoculation into hamsters. Brain tissues of scrapie-infected hamsters were used for preparation of homogenates and crude extracts of PrP(Sc). A treatment of PrP(Sc) with Gdn enhanced its PK-sensitivity in a dose-dependent manner. The PK-resistance in vitro of PrP(Sc) denatured with lower concentrations of Gdn (<2.5 mol/l) could partially resume by renaturation. Gdn markedly reduced or, at higher concentrations, even destroyed the infectivity of PrP(Sc). On the other hand, the infectivity of PrP(Sc) inactivated by denaturation could be partially restored by renaturation. These results confirmed our assumption that all the alternations in the PK-resistance and the infectivity of PrP(Sc) caused by Gdn resulted from changes in its higher structure. However, it should be emphasized that a complete loss of PK-resistance of PrP(Sc) may not necessarily mean its full non-infectivity.

Comparative study of the effects of several chemical and physical treatments on the activity of protease resistance and infectivity of scrapie strain 263K.

To study the influences of chemical and physical factors on the protease resistant activity in vitro and the infectivity in vivo of scrapie strain 263K, PrPSc from the hamsters infected intracerebrally with scrapie strain 263K were treated with several commonly used disinfection methods, including sodium hydroxide (NaOH), sodium hypochlorite (NaOCl), heating or autoclaving at 80, 100, 121 and 134 degrees C in the solutions with or without 3% sodium dodecyl sulphate (SDS). The protease resistance of PrPSc was analysed by a proteinase K (PK) digesting Western blot and the infectivity of PrPSc was analysed by intracerebral (i.c.) inoculation into experimental hamsters. The results showed that PrPSc signals were removed in the preparations treated with NaOH higher than 0.05 mol/l, NaOCl higher than 0.1%, autoclaved over 121 degrees C, or heated over 80 degrees C in the presence of 3% SDS. Animal challenges revealed that mixing with 2 mol/l NaOH or 2% NaOCl, autoclaving at 134 degrees C, as well as heating at 100 degrees C or autoclaving at 121 degrees C in the solutions with 3% SDS completely blocks the transmission of scrapie 263K in this experimental situation. It is obvious that the removal of PK resistance of PrPSc happened at relatively lower concentration chemicals or lower temperature, while elimination of the infectivity needs more vigorous conditions. Our data provide the useful evidences for several commonly used methods to inactivate TSEs agent and suggest that it is inappropriate to use PrPSc as a surrogate for TSEs infectivity in inactivation experiments.

Detection of epidermodysplasia verruciformis-associated human papillomavirus DNA in nongenital seborrhoeic keratosis.

BACKGROUND: DNA of epidermodysplasia verruciformis (EV)-associated human papillomaviruses (HPVs) has been widely detected in lesions of malignant skin tumours, benign tumours and other proliferative diseases of epithelial origin. OBJECTIVES: To investigate the presence of EV-associated HPV DNA in nongenital seborrhoeic keratosis (SK) and to elucidate the prevalence of distinct HPV genotypes. METHODS: We investigated HPV DNA in 55 nongenital SK biopsies, which were compared with 48 normal skin biopsies (healthy controls) using a nested polymerase chain reaction (PCR) using consensus primers CP65/CP70 and CP66/CP69. The positive PCR products were retracted and used to prepare recombination clones with T-vector. Distinct clones were analysed with endonucleases, and HPV genotypes were identified by direct sequencing. RESULTS: EV-associated HPV DNA was detected in 42 of 55 (76%) nongenital SK biopsies vs. only 13 of 48 (27%) healthy controls (chi2 = 22.087; P < 0.005). The prevalence was higher in patients with more than five lesions than in those with only one lesion (P < 0.05). Ten distinct HPV genotypes were detected in the nongenital SK biopsies: HPV 20, 23, 5, renal transplant recipient (RTR) X7, HPV 17, 37, 17b, RTRX4, RTRX4b and strain SK3. HPV 20 was found in 26 of 42 (62%) positive specimens, followed by HPV 23 (11 of 42, 26%) and HPV 5 (six of 42, 14%). Existence of multiple HPV genotypes was observed in 12 of 42 (29%) positive specimens. In healthy controls, five genotypes of EV-associated HPV (HPV 20, 23, 5, 17 and RTRX4) were detected, with the same predominant genotype of HPV 20 (five of 13, 38%). Several distinct HPV genotypes were found to coexist in four of 13 (31%) positive specimens. CONCLUSIONS: This study provides some evidence that EV-associated HPVs might play a part in the pathogenesis of nongenital SK.

[Diagnosis of bovine spongiform encephalopathy and scrapie by western blot].

The rabbits were immuned with bovine prion protein (BoPrPC) which was expressed in E. coli and anti-PrPC antibody (T1) was obtained. According to pathological prion protein (PrPSC) was resistant to protease treatment, extracts of brain tissue were digested with proteinase K and detected by western blot with T1 antibody. The results showed that protease-resistant pathological PrPSC was existed in golden hamster brain tissue which was inoculated with scrapie strain 263 K, but no protein existed in normal golden hamster brain homogenates which was detected with T1 antibody. Several bovines and sheep from Beijing were used for diagnosis of Bovine spongiform encephalopathy (BSE) and scrapie, their brain tissue were freshly collected and homogenated. The homogenates were separated on SDS-PAGE and detected by western blot with T1 antibody. The results indicated no protease-resistant protein(PrPSC) existed, this suggested they were not infected by BSE and scrapie. The same results were obtained with 1A8 antibody from England. These results indicated we could detect BSE and scrapie with T1 antibody.

N-methyl-D-aspartate enhancement of the glycine response in the rat sacral dorsal commissural neurons.

The effect of N-methyl-D-aspartate (NMDA) on the glycine (Gly) response was examined in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. The application of 100 microM NMDA to SDCN neurons reversibly potentiated Gly-activated Cl- currents (IGly) without affecting the Gly binding affinity and the reversal potential of IGly. A selective NMDA receptor antagonist, APV (100 microM), blocked the NMDA-induced potentiation of IGly, whereas 50 microM CNQX, a non-NMDA receptor antagonist, did not. The potentiation effect was reduced when NMDA was applied in a Ca2+-free extracellular solution or in the presence of BAPTA AM, and was independent of the activation of voltage-dependent Ca2+ channels. Pretreatment with KN-62, a selective Ca2+-calmodulin-dependent protein kinase II (CaMKII) inhibitor, abolished the NMDA action. Inhibition of calcineurin (CaN) further enhanced the NMDA-induced potentiation of IGly. In addition, the GABAA receptor-mediated currents were suppressed by NMDA receptor activation in the SDCN neurons. The present results show that Ca2+ entry through NMDA receptors modulates the Gly receptor function via coactivation of CaMKII and CaN in the rat SDCN neurons. This interaction may represent one of the important regulatory mechanisms of spinal nociception. The results also suggest that GABAA and Gly receptors may be subject to different intracellular modulatory pathways.

Functional significance of sequence variation in the E2 gene and the long control region of human papillomavirus type 16.

The long control region (LCR) and the E2 protein of human papillomaviruses (HPV) are the most important viral factors regulating transcription of the viral oncogenes E6 and E7. Sequence variation within these genomic regions may have an impact on the oncogenic potential of the virus. Sequence variation in the LCR and in the E2 gene of human papillomavirus type 16 (HPV-16) isolates originating from cervical cancer patients from East Hungary was studied. In 30 samples, sequencing and/or single-strand conformation polymorphism analysis revealed variants belonging to the European variant lineage of HPV-16. These variants differed from the reference European clone only slightly in their E2 and LCR sequences. Three samples represented variants belonging to the Asian-American group. These differed from the published reference sequence at several positions in the LCR and E2 regions. Compared to the reference clone, the LCR clones of the European isolates showed very similar transcriptional activities, while that of an Asian-American isolate had approximately 1.7-fold increased activity. Most of the increased activity of the Asian-American isolate could be ascribed to nucleotide changes found at the 3' end of the LCR (nt 7660-7890). The transcriptional transactivation potentials of the HPV-16 E2 isolates differed only slightly from each other, and the differences seemed to be independent of the taxonomic position of the isolates.

Point mutations in SP1 motifs in the upstream regulatory region of human papillomavirus type 18 isolates from cervical cancers increase promoter activity.

Evidence of the functional significance of two naturally occurring mutations at nt 40 or 41 in the Sp1 motif in the promoter proximal segment of the upstream regulatory region (URR) of human papillomavirus (HPV) type 18 is presented. In electrophoretic mobility shift assays, Sp1 protein bound more efficiently to the Sp1 mutant motifs than to the prototype; while in both HeLa and HT3 cells, luciferase activity controlled by the mutant URRs was upregulated 2- and 3-fold, or 4- and 6-fold, in comparison with the prototype URR or HeLa cell-derived URR respectively. The HeLa URR represents a more appropriate baseline for promoter activity, containing a series of point mutations representative of most HPV-18 cancer isolates, including one in the Yin Yang 1 (YY1) site at the P105 promoter. The effect of the Sp1 mutations was found to be largely maintained in the context of the HeLa URR containing the prototype YY1 site.

[Chemical studies of Epimedium acuminatum Franch].

Three flavonol glycosides isolated from the aerial parts of Epimedium acuminatum were identified as baohuoside II, epimedoside A and icariin on the basis of spectral data (UV, IR, MS, and 1HNMR) and chemical properties. Baohuoside II was isolated from the species for the first time.

Prevalence of deletions of YY1-binding sites in episomal HPV 16 DNA from cervical cancers.

Expression of the oncogenes E6 and E7 of human papillomavirus 16 (HPV 16) appears enhanced in pre-malignant and malignant genital tumors. We recently identified a transcriptional silencer upstream of the oncogene promoter P97, comprising 4 binding sites for the cellular YY1 protein. The analysis of the long transcriptional control regions (LCR) of episomal HPV 16 DNAs from primary tumors and lymph-node metastases of 6 patients with cervical cancer revealed deletions and point mutations of YY1 binding sites in 4 cases. To test for the activity of the P97 promoter, the mutated LCRs were cloned in a luciferase reporter gene vector. A point mutation in YY1-recognition site 4, which prevents DNA-protein interaction, did not affect promoter activity, probably due to compensation by the overlapping YY1-binding site 3. However, 5.5- to 6.5-fold increased luciferase expression was obtained under the control of 3 shortened LCRs lacking 2 to 4 YY1-binding sites. A point mutation in YY1-recognition site 2, which was previously shown to stimulate P97 3.5-fold, could be detected in the HPV 16 LCRs from both primary tumor and metastasis, indicating that the mutation is a stable characteristic of HPV 16 DNA associated with the individual cancer. These findings suggest that deletions or mutations of YY1-binding sites play a significant role in over-expression of viral oncogenes and tumor progression.

The E6/E7 promoter of extrachromosomal HPV16 DNA in cervical cancers escapes from cellular repression by mutation of target sequences for YY1.

Human papillomavirus type 16 (HPV16) induces squamous intraepithelial lesions of the cervical mucosa which may develop into invasive cancer. The expression of viral oncogenes in advanced neoplasias appears increased relative to the proliferating cell layers of low grade lesions raising questions about molecular mechanisms of deregulation of transcription. In a lymph node metastasis of a cervical cancer, we observed full-length HPV16 plasmids and molecules with a small deletion, which was mapped to the long control region (LCR). Both wild type and shortened LCR were amplified by PCR, cloned into the promoter test plasmid pBLCAT6 and sequenced to identify a 107 bp deletion from position 7794 to 7901 in the short LCR. CAT expression in cervical cancer-derived HT3, SiHa and CaSki cells appeared 5- to 6-fold increased under the control of the short LCR. This could be traced back to elevated levels of mRNA initiated at the viral oncogene promoter. A slight further increase in CAT expression was noted in the presence of the HPV16 E2 protein which is probably due to the deletion of one E2 binding site and consequent relief from E2 repression. Computer-assisted sequence analysis and band-shift experiments with purified YY1 protein and wild type or mutated oligonucleotides identified four binding sites for this cellular transcriptional repressor within the promoter-proximal segment of the HPV16 LCR, three of which were removed by the deletion. A LCR fragment comprising these YY1 binding sites was cloned in front of the heterologous thymidine kinase gene promoter and suppressed CAT expression 3- to 4-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Kmeriol and other aromatic constituents of Kmeria duperreana.

The CHCl3-soluble fraction of a crude extract of Kmeria duperreana exhibited cytotoxic activity when tested in both KB and P388 tumor-cell cultures. Bioassay-directed fractionation led to the isolation of a cytotoxic alkaloid, liriodenine (1). Other constituents obtained from the extract included scopoletin (2), (-)-3,4,5-trimethoxyphenyl beta-D-glucopyranoside (3), (+)-syringaresinol beta-D-glucopyranoside (4), and a new phenylpropanol, kmeriol (5), whose chemical structure was established through spectroscopic analysis.