去甲基药、化疗药及TRAIL联合诱导神经母细胞瘤细胞凋亡的研究

目的探讨去甲基药5-氮杂胞苷、肿瘤坏死因子相关凋亡诱导配体（TRAIL）及化疗药联合诱导神经母细胞瘤细胞株SH-SYSY（SYSY）凋亡的作用及其发生机制。方法应用逆转录-聚合酶链反应（RT-PCR）方法检测5-氮杂胞苷作用前后SYSY细胞caspase-8的表达；应用四甲基偶氮唑蓝（MTT）比色法及流式细胞仪检测TRAIL、5-氮杂胞苷＋TRAIL、5-氮杂胞苷＋caspase-8抑制剂zIETD-FMK＋TRAIL及5-氮杂胞苷＋化疗药＋TRAIL对SYSY细胞生长及凋亡的影响。结果SYSY细胞不表达caspase-8，5-氮杂胞苷作用1、3、5、7d的SY5Y细胞caspase-8表达逐渐增加。SY5Y细胞对TRAIL不敏感，经5-氮杂胞苷诱导表达caspase-8的SY5Y细胞对TRAIL敏感；化疗药可增强SYSY细胞对TRAIL的敏感性。结论5-氮杂胞苷可通过上调SYSY细胞caspase-8表达而逆转SYSY细胞对TRAIL诱导凋亡的耐受，化疗药可进一步增强TRAIL对SYSY细胞的诱导凋亡作用。     

α-synuclein budding yeast model

Parkinson's disease (PD) is a common neurodegenerative disorder that results from the selective loss of midbrain dopaminergic neurons. Misfolding and aggregation of the protein α-synuclein, oxidative damage, and proteasomal impairment are all hypotheses for the molecular cause of this selective neurotoxicity. Here, we describe a Saccharomyces cerevisiae model to evaluate the misfolding, aggregation, and toxicity-inducing ability of wild-type α-synuclein and three mutants (A30P, A53T, and A30P/A53T), and we compare regulation of these properties by dysfunctional proteasomes and by oxidative stress. We found prominent localization of wild-type and A53T α-synuclein near the plasma membrane, supporting known in vitro lipid-binding ability. In contrast, A30P was mostly cytoplasmic, whereas A30P/A53T displayed both types of fluorescence. Surprisingly, α-synuclein was not toxic to several yeast strains tested. When yeast mutants for the proteasomal barrel (doa3-1) were evaluated, delayed α-synuclein synthesis and membrane association were observed; yeast mutant for the proteasomal cap (sen3-1) exhibited increased accumulation and aggregation of α-synuclein. Both sen3-1 and doa3-1 mutants exhibited synthetic lethality with α-synuclein. When yeasts were challenged with an oxidant (hydrogen peroxide), α-synuclein was extremely lethal to cells that lacked managanese superoxide dismutase Mn-SOD (sod2Δ) but not to cells that lacked copper, zinc superoxide dismutase Cu,Zn-SOD (sod1Δ). Despite the toxicity, sod2Δ cells never displayed intracellular aggregates of α-synuclein. We suggest that the toxic α-synuclein species in yeast are smaller than the visible aggregates, and toxicity might involve α-synuclein membrane association. Thus, yeasts have emerged effective organisms for characterizing factors and mechanisms that regulate α-synuclein toxicity. Author to whom all correspondence and requests should be addressed.     

肿瘤坏死因子相关凋亡诱导配体诱导胶质瘤细胞凋亡机制研究

目的 探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导胶质瘤细胞凋亡的机制.方法 以人重组可溶性TRAIL蛋白(rsTRAIL)处理人胶质瘤细胞U87;AnnexinV-FITC/PI双染色流式细胞术检测细胞凋亡;DiOC6荧光染色流式细胞术检测细胞线粒体跨膜电位(△ψm);比色法测定细胞caspase-3、8、9活性的变化;ELISA法检测胞浆cytC浓度;观察caspase-8阻断剂(Z-IETD-fmk)对U87细胞凋亡、△ψm和caspase-3、8、9活性变化的影响. 结果 rsTRAIL以时间依赖方式诱导U87细胞凋亡.同时导致U87细胞△ψm进行性下降,caspase-3、8、9活性及胞浆内cyt C浓度升高:caspase-8阻断剂可明显减弱rsTRAIL对U87细胞的上述生物学效应. 结论 TRAU通过激活caspase-8间接启动线粒体凋亡途径诱导恶性胶质瘤细胞U87凋亡.     

TRAIL诱发人脑H4神经胶质瘤细胞凋亡的研究

目的研究TRAIL对H4神经胶质瘤细胞的凋亡诱导作用，并探讨其可能机制。方法利用显微镜、透射电镜、流式细胞仪，观察和检测TRAIL对传代培养的肿瘤细胞的凋亡诱导作用，用MTT法检测easpase-8抑制剂zIETD—fmk对TRAIL凋亡诱导作用的抑制情况。结果TRAIL作用后镜下可见到H4细胞凋亡的典型表现；在流式细胞仪检测PI荧光直方图上，出现典型的亚二倍体峰，随着作用时间的延长，H4细胞凋亡率明显增加；zIETD—fmk能显著抑制TRAIL对H4细胞的凋亡诱导作用。结论TRAIL可有效的诱导H4神经胶质瘤细胞凋亡；easpase-8在H4瘤细胞的凋亡过程发挥着重要作用，TRAIL可能是通过激活caspase系统而诱发H4瘤细胞凋亡的。     

Caspase-3 activation is required for reovirus-induced encephalitis <Emphasis Type="Italic">in vivo</Emphasis>

Reovirus infection of neonatal mice provides a classic experimental system for understanding the molecular pathogenesis of central nervous system (CNS) viral infection. CNS tissue injury, caused by many human neurotropic viruses, including herpes viruses and West Nile virus, is associated with caspase-dependent apoptotic neuronal cell death. We have previously shown that reovirus-induced CNS tissue injury results from apoptosis and is associated with activation of both death-receptor and mitochondrial apoptotic pathways culminating in the activation of the downstream effector caspase, caspase-3. In order to directly investigate the role of caspase-3 in virus-induced neuronal death and CNS tissue injury during encephalitis, we have compared the pathogenesis of reovirus CNS infection in mice lacking the caspase-3 gene (caspase-3 (−/−)) to syngeneic wild-type mice. Prior studies of antiapoptotic treatments for reovirus-infected mice have indicated that protection from reovirus-induced neuronal injury can occur without altering the viral titer in the brains of infected mice. We now show that reovirus infection of caspase-3 (−/−) mice was associated with dramatic reduction in severity of CNS tissue injury, decreased viral antigen and titer in the brain, and enhanced survival of infected mice. Following intracerebral inoculation, the authors also show that virus spread from the brain to the eyes in reovirus-infected caspase-3 (−/−) mice, indicating that viral spread was intact in these mice. Examination of brains of long-term survivors of reovirus infection among caspase-3 (−/−) mice showed that these mice eventually clear their CNS viral infection, and do not manifest residual or delayed CNS tissue injury.     

Increased neuronal cell death after intermittent hypercapnic hypoxia in the developing piglet brainstem

We examined the immunohistochemical expression of caspase-3 (CASP3), active caspase-3 and TUNEL in the normal piglet brainstem at 13-14 days of age and evaluated the effects of exposure to 2 vs. 4 days of intermittent hypercapnic hypoxia (IHH) on their expression. Eight nuclei from the level of the caudal medulla were studied. In control piglets, CASP3 was present in approximately 45% of neurons while active caspase-3 and TUNEL were present in approximately 5%, indicating that approximately half the neuronal population of the piglet medulla express caspase-3 in a latent state and that only approximately 5% undergo 'normal' programmed cell death. After 2 days of IHH, CASP3 increased in the nucleus of the solitary tract (NTS), gracile and cuneate nuclei (P<0.05 for all). Active caspase-3 increased in the dorsal motor nucleus of the vagus (DMNV) (P<0.05) but decreased in the lateral reticular nucleus (LRt) (P<0.05), while TUNEL increased in both the DMNV and LRt (P<0.05 for both). After 4 days of IHH, CASP3 remained elevated in the cuneate nucleus (P<0.01) but decreased in the hypoglossal and DMNV (P<0.05) when compared to controls. Active caspase-3 levels were not changed, whereas TUNEL was increased in the DMNV, LRt, and inferior olivary nucleus (P<0.05 for all). These results show that IHH induces neuronal cell death within certain nuclei in the piglet caudal medulla that are functionally important in cardiorespiratory, sleep and arousal control. This could have important implications for clinical conditions including obstructive apnea and prone sleeping as a risk for SIDS.     

TRAIL-induced apoptosis in U-1242 MG glioma cells

ABSTRACT: Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induces apoptosis in U-1242 MG cells. To investigate the molecular events involved in this process, we studied the effects of TRAIL on the localization within membrane fractions of molecules critical to the extrinsic apoptotic pathway. We report here that death receptor-5 (DR5), tumor necrosis factor receptor-1 (TNF-R1), and Fas receptor (FasR) are all located in the caveolin-1-enriched membrane fractions, and TRAIL caused the translocation of DR5, FasR, and TNF-R1 to the caveolar fractions. Caspase-8 is mainly located outside of caveolae, but TRAIL caused it to redistribute to the caveolin-1-enriched fractions where it was cleaved. Within 6 hours, the cleaved caspase-8 appeared in the high-density, noncaveolin fractions. Using confocal microscopy, we found that DR5, caspase-8, and caveolin-1 became progressively concentrated in blebs of plasmalemma as they formed in response to TRAIL. Our results provide the first evidence for the caveolar localization of TNF-R1 and DR5 and the coordinated redistribution among membrane fractions of several death receptors in response to TRAIL. We propose that the coordinated movement of these molecules among membrane compartments is probably an important component of the mechanisms regulating and initiating the extrinsic apoptotic pathway in human glioma cells.     

Catecholamine neuron groups in rat brain slices differ in their susceptibility to excitatory amino acid induced dendritic degeneration

We investigated whether specific types of catecholamine neurons were differentially vulnerable to damage induced by excitatory amino acids (EAAs)in vitro in a rat brain slice preparation. Brain slices, 300 μm thick, were cut horizontally, exposed to either N-methyl-D-aspartate (NMDA) or kainic acid (KA) for 2 h, fixed and then cut into thin (30 μm) sections in the same (horizontal) plane as the slice. The sections were immunolabelled for tyrosine hydroxylase to identify different groups of catecholamine neurons (substantia nigra (SN), paranigral (PN), interfascicular (IF) and hypothalamic A11, A13 and A14) which exhibited prominent dendritic projections in the horizontal plane. Loss of dendrites was used as a sensitive index of damage that precedes the loss of the cell body. Catecholamine neurons differed strikingly in their vulnerability to EAA-induced dendrite degeneration. The most vulnerable were those in the dorsal tier of the SN, whereas the most resistant were those in the hypothalamic All group. For example, in the dorsal tier of SN, NMDA (50 μM) reduced the proportion of neurons with dendrites from 64% (±8% SEM) in controls to 13% (±7%) whereas the majority of All neurons (69±10%) retained their dendrites compared to controls (89%±8%). The other groups of catecholamine neurons exhibited intermediate vulnerability. An essentially similar pattern of differential vulnerability was observed with KA. An understanding of the cellular mechanisms that underlie the particular vulnerability of SN neurons in the slice will aid the discovery of pharmacological therapies to prevent or slow the pathological process in neurodegenerative diseases which involve these neurons.     

NMDA-sensitive neurons profoundly influence delayed staurosporine-induced apoptosis in rat mixed cortical neuronal cultures

We have investigated cell killing in cultured rat embryonic cortical neurons exposed to the protein kinase inhibitor staurosporine, the excitatory amino acid N-methyl- -aspartate (NMDA), or a combination thereof. Our data indicate that there are several populations of neurons that differ in their response to these agents. Cultures exposed to NMDA undergo cell death typified by lactate dehydrogenase (LDH) leakage which is likely primarily necrotic in that little caspase-3 activation or oligonucleosome formation is observed even when followed for 48 h. Cells exposed to staurosporine (STS) exhibit rapid, extensive activation of caspase-3 with coincident LDH leakage, oligonucleosome formation and TUNEL staining. Both LDH leakage and oligonucleosome content were significantly more elevated at 48 h than at 20 h with STS treatment while caspase-3 activity peaked early (8–20 h) and declined markedly by 48 h. Deletion of NMDA-responsive neurons by pre-treatment of the cultures with NMDA for 4 days prevented the late phase (20–48 h) increases in LDH leakage and oligonucleosomes in the remaining neuronal population. Caspase-3 activity was also completely abolished by NMDA pre-treatment. These results indicate that cells susceptible to acute NMDA-induced toxicity can be killed by non-apoptotic means when exposed to NMDA; however, they undergo a delayed, apoptotic death when exposed to STS. Interestingly, removal of NMDA-responsive cells prevents the processing of procaspase-3; thus, STS-induced apoptosis in cells resistant to NMDA-mediated killing proceeds independent of caspase-3 activation. The data indicate that nearly all neurons in these mixed cultures can undergo apoptosis in response to appropriate stimuli such as STS but that the temporal nature, and the pathways activated in response to STS, vary amongst the subpopulations of neurons. These findings may help to explain the simultaneous appearance of features of both apoptosis and necrosis observed in vivo following cerebral ischemia.     

Cytoarchitectural alterations are widespread in cerebral cortex in tuberous sclerosis complex

Tubers are cerebral cortical developmental malformations associated with epilepsy and autism in tuberous sclerosis complex (TSC). The disparity between tuber number and severity of neurological impairment often observed in TSC led us to hypothesize that microscopic structural abnormalities distinct from tubers may occur in TSC. Serial frontal to occipital lobe sections were prepared from five postmortem TSC brain specimens. Sections were probed with cresyl violet stain or NeuN antibodies to define cytoarchitectural abnormalities and phospho-S6 (Ser235/236) antibodies to define mammalian target of rapamycin complex 1 (mTORC1) pathway activation. Tubers identified in all specimens (mean, 5 tubers per brain specimen) were defined by abnormal cortical lamination, dysmorphic neurons, and giant cells (GCs) and exhibited robust phospho-S6 immunolabeling. Histopathological analysis of non-tuber cortices demonstrated that 32% of the sections exhibited microscopic cytoarchitectural alterations, whereas 68% of the sections did not. Four types of morphological abnormalities were defined including: (1) focal dyslamination, (2) heterotopic neurons, (3) small collections of giant cells (GCs) and neurons we termed “microtubers”, (4) isolated GCs we termed “sentinel” cells. When compared with control cortex, phospho-S6 labeling was enhanced in microtubers and sentinel cells and in some but not all areas of dyslamination. There are microscopic cytoarchitectural abnormalities identified in postmortem TSC brain specimens that are distinct from tubers. mTORC1 cascade activation in these areas supports a widespread effect of TSC1 or TSC2 mutations on brain development. Tubers may represent the most dramatic developmental abnormality in TSC; however, more regionally pervasive yet subtle abnormalities may contribute to neurological disability in TSC.     

Hypoxia-ischemia upregulates TRAIL and TRAIL receptors in the immature rat brain

The immature brain is susceptible to inflammatory injury induced by hypoxia-ischemia (HI) or infection, which causes serious neurodevelopmental disabilities in the survivors of preterm births. Recently, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (death receptor DR4/5 and decoy receptor DcR1/2) were reported to mediate various neuroinflammatory responses. However, little information is available regarding the role of TRAIL and its receptors in the immature brain after HI. The purpose of this study was to evaluate the expression of TRAIL and its receptors in the immature brain after HI and relate this expression to neurological function. We performed right common carotid artery ligation followed by hypoxia (6% O(2), 37°C) for 2.5 h to induce HI in postnatal day 3 rats. The distribution of TRAIL and its receptors, caspase-3 and CD68-labeled microglia/macrophages was evaluated 24 h after HI by immunostaining. The protein and mRNA expression of TRAIL and DR5 was measured by Western blot and real-time PCR, respectively. Delayed neuronal loss was evaluated by NeuN and Nissl staining 7 days after HI. Furthermore, neurological deficits were evaluated by a righting reflex test, time of eye opening and T-maze test. The expression of TRAIL, DR5 and DcR1/2 receptors and caspase-3 was more pronounced in the ipsilateral hemisphere compared with the contralateral part and the control group 24 h after HI. DR5/active caspase-3 double-positive cells were observed at 24 h after HI in the ipsilateral hemisphere but not in the contralateral hemisphere. The TRAIL and CD68 double-labeled cells were more pronounced in the ipsilateral cortical regions compared with the corresponding regions of the contralateral part. HI also resulted in a significant increase in TRAIL and DR5 protein and mRNA expression at 24 h, which corresponded to neuronal cell loss 7 days after HI. Furthermore, the HI group displayed impaired neurobehavioral development compared with the control group (p < 0.05). Altogether our results show that the TNF-α superfamily ligand TRAIL is induced on CD68+ microglia/macrophages after perinatal HI and that one of its receptors, DR5, is induced on neocortical neurons and glial cells. That many DR5+ cells were also caspase-3+ strongly supports the conclusion that these signaling molecules are involved in the delayed loss of neurons in the neocortex and in the neurobehavioral deficits that are often seen after perinatal HI.     

Neuronal death in brain infarcts in man

The mechanism of neuronal death in brain ischaemia remains unclear. Morphology, terminal transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) and immunohistochemistry for the pro-apoptotic enzyme caspase-3 (CASP3), for its substrates poly(ADP-ribose) polymerase (PARP) and the DNA-dependent protein kinase catalytic subunit (DNA-PKCS) and for poly(ADP-ribose) (PAR), an end-product of PARP activity, were used to investigate neuronal death in brain infarcts from 15 men and 20 women, aged 46-95 years. The infarcts varied in age from 18 h to several months. Neuronal death was characterized morphologically by cell shrinkage, cytoplasmic hypereosinophilia and moderate nuclear pyknosis with later chromatin dispersal and disintegration, but not features of apoptosis. Occasional apoptotic bodies were seen but these appeared to be related to inflammatory cells, endothelial cells and occasional glia, including satellite cells. Neurones within infarcts showed strong nuclear and cytoplasmic labelling for CASP3 during the first 2 days after infarction. Neuronal DNA-PKCS, PARP and poly(ADP-ribose) immunoreactivity was demonstrable in scattered neurones in and adjacent to infarcts for 18-24 h but thereafter declined to below detectable levels in most cases. TUNEL labelled cells towards the edge of the infarcts, particularly at 2-4 days, but most of the labelling could be prevented by preincubation of the sections in diethyl pyrocarbonate to inactivate endogenous nucleases. Between 3 days and 3 weeks, CASP3 and DNA-PKCS were detected in proliferating capillaries and CASP3, PARP and poly(ADP-ribose) in infiltrating macrophages. Our findings indicate that neuronal death in human brain infarcts has some of the early biochemical features of programmed cell death, with upregulation of CASP3 and rapid disappearance of DNA-PKCS and PARP. However, the morphological changes are not those of apoptosis, the DNA cleavage occurs relatively late, and some of the TUNEL is probably mediated by the release of endogenous endonucleases during protease or microwave pretreatment of the damaged tissue.     

Differential induction of apoptosis by antidepressants in glioma and neuroblastoma cell lines

Several antidepressants, mainly selective serotonin-reuptake inhibitors (SSRIs) and some tricyclic antidepressants (TCAs), have been shown to possess potent apoptotic activity in different cell lines. Our aim was to screen and select those agents with significant activity and elucidate the molecular pathway underlying this process in rat glioma and human neuroblastoma cell lines. We studied the effect of different antidepressants on apoptotic markers, including: cell viability, DNA fragmentation, cytochrome c (Cyt c) release from mitochondria, and caspase-3-like activity. In addition, the involvement of MAPK genes, c-Jun, and ERK was determined. Paroxetine and fluoxetine, SSRIs, clomipramine, a TCA, but not imipramine or mianserin (an atypical antidepressant), caused apoptosis in both cell lines, as assessed by flow cytometry of propidium iodide-stained C6 cells and typical fluorescence microscopy in glioma cells. These apoptotic changes were preceded by rapid increase in p-c-Jun levels, Cyt c release from mitochondria, and increased caspase-3-like activity. Assessment of paroxetine cytotoxicity in primary mouse brain and neuronal cultures showed significantly lower sensitivity to the drug’s proapoptotic activity. These results strongly suggest that selected antidepressants induce apoptosis in neuronal and glial cell lines. Activation of p-c-Jun and subsequent increased Cyt c mitochondrial release participate in the apoptotic mechanism of the antidepressant. The high sensitivity to these drugs of the cancer cell, compared with primary brain tissue, suggests the potential use of these agents in the treatment of brain-derived tumors.     

Association between migraine and a functional polymorphism at the dopamine β-hydroxylase locus

Migraine is a common neurological disorder with a significant genetic component. Although a number of linkage and association studies have been undertaken, the number and identity of all migraine susceptibility genes has yet to be defined. The existence of dopaminergic hypersensitivity in migraine has been recognised on a pharmacological basis and some studies have reported genetic association between migraine and dopamine-related gene variants. Our laboratory has previously reported association of migraine with a promoter STR marker in the dopamine beta hydroxylase (DBH) gene. In the present study, we analysed two additional DBH markers in two independent migraine case–control cohorts. These two markers are putative functional SNPs, one within the promoter (−1021C→T) and another SNP (+1603C→T) in exon 11 of the DBH gene. The results showed a significant association for allelic and genotypic frequency distribution between the DBH marker in the promoter and migraine in the first (P = 0.004 and P = 0.012, respectively) and the second (P = 0.013 and P = 0.031, respectively) tested cohorts. There was no association observed between either genotype and/or allelic frequencies for the DBH marker located in exon 11 and migraine (P ≥ 0.05). The promoter DBH marker, reported associated with migraine in this study, has been shown to affect up to 52% of plasma DBH activity. Varying DBH activity levels have been postulated to be involved in migraine process with an increase of dopamine, resulting from a lower DBH activity shown positively correlated with migraine severity. It is plausible that the functional promoter variant of DBH may play a role in the migraine disorder.     

Studies of Anandamide Accumulation Inhibitors in Cerebellar Granule Neurons: Comparison to Inhibition of Fatty Acid Amide Hydrolase

The endocannabinoid, N-arachidonylethanolamine (AEA) is accumulated by neurons via a process that has been characterized biochemically but not molecularly. Inhibitors of AEA accumulation have been characterized individually but have not been compared in a single study. Our purpose was to compare the potency of five previously described compounds (AM404, AM1172, VDM11, OMDM-2, and UCM707) both as inhibitors of AEA and N-palmitoylethanolamine (PEA) accumulation by cerebellar granule neurons and as inhibitors of AEA hydrolysis. The compounds all inhibited AEA accumulation; AM404, VDM11 and OMDM-2 with IC₅₀ values of approximately 5 μM, whereas AM1172 and UCM707 exhibited IC₅₀ values of 24 and 30 μM, respectively. The compounds also inhibited PEA accumulation; AM404 being the most potent with an IC₅₀ of 6 μM, whereas the other compounds had IC₅₀ values in the range of 30–70 μM. All of the compounds potently inhibited AEA hydrolysis by brain membranes; the K _I values for AM404, VDM11, and UCM707 were less than 1 μM; AM1172 and OMDM-2 exhibited K _I values of 3 and 10 μM, respectively. The IC₅₀ values for inhibition of AEA accumulation were compared to the IC₅₀ values for PEA accumulation and AEA hydrolysis using linear regression. None of the regressions were significant. These data indicate that inhibition of AEA accumulation by neurons is not a result of the inhibition of endocannabinoid hydrolysis and is a process different from the accumulation of PEA. These studies support the hypothesis that the cellular AEA accumulation beyond simple equilibrium between intracellular and extracellular concentrations occurs because AEA binds to an intracellular protein that is not FAAH but that also recognizes the AEA uptake inhibitors.     

Nuclear β-catenin accumulation associates with epithelial morphogenesis in craniopharyngiomas

Activation of the Wnt/wingless signalling cascade is a key mechanism in developmental morphogenesis, whereas aberrant nuclear accumulation of β-catenin in adult tissues seems to be associated with neoplastic transformation and tumour progression. Adamantinomatous craniopharyngiomas carry activating mutations in exon 3 of the β-catenin gene, which results in a distinct pattern of nuclear β-catenin accumulation in up to 95% of respective tumour specimens. To better characterise the impact of nuclear β-catenin aggregation in these neoplasms, we systematically examined epithelial differentiation and cell cycle-associated molecules in accumulating compared to non-accumulating tumour cell clusters using a cohort of 65 adamantinomatous craniopharyngiomas. Monoclonal antibodies directed against cytokeratins 5/6 (CK5/6) were utilised to differentiate squamous from simple epithelium, the latter being identified by immunoreactivity for cytokeratins 8 and 18 (CK8/CK18). Intriguingly, nuclear β-catenin accumulation in whorl-like tumour cell clusters was always associated with a distinct CK8 and CK18 immunoreactivity, whereas surrounding non-accumulating tumour cells showed exclusively squamous differentiation indicated by CK5/6 expression. In addition, a low proliferation activity combined with an increased expression of p21^WAF1/CIP1, a key control protein of the cell cycle, was observed in β-catenin accumulating cells. Our data support an impact of nuclear β-catenin on different cytoarchitectural and epithelial differentiation patterns in adamantinomatous craniopharyngiomas. This work is supported by the Johannes and Frieda Marohn Foundation (to RB and BH).     

Further validation of the Child and Adolescent Scale of Participation (CASP)

Primary objective: To further validate the Child and Adolescent Scale of Participation (CASP) for children and youth with acquired brain injuries and other disabling conditions.

Research design and methods: A cross-sectional design was used. Data were collected on 313 children and youth, aged 3–22, with and without disabling conditions. Children with acquired brain injuries (ABI) were the largest group (56%). Cronbach's alpha, factor analyses and Rasch analyses were used to examine internal scale consistency and structure. Correlation analyses were conducted to examine associations between CASP scores and scores that reflect extent of impairment and impact of environmental barriers. Independent t-tests or analyses of variance were used to examine mean differences in CASP scores in relation to sex, age and disability groups.

Main outcomes and results: Children without disabilities had significantly greater extent of participation (higher CASP scores) than children with disabilities. Greater extent of participation was associated with lesser extent of impairment and environmental barriers. Evidence of internal consistency and internal structure validity was demonstrated. Factor analyses showed that items from similar domains loaded onto one of three factors and a large (63%) proportion of variance was explained. Rasch analyses revealed essentially one unidimensional construct. The item difficulty order closely matched the expected pattern of life situations that children would find more to less challenging to participate in.

Conclusions: Findings were similar to prior research and suggested that the CASP is a promising new measure of participation. However, study design features limit generalizations and definitive conclusions that can be made. Future research is needed to assess the ability of the CASP to detect change over time and to include a larger and more diverse sample.     

Coordinated expression of pro- and antiapoptotic proteins in the hippocampus of neonatal rats

We studied the contents of the apoptotic proteins Bcl-XL, Bax, and intact and active forms of caspase-3 using the immunoblot method in the hippocampus and brainstem of 3-day-old rat pups. The expression of pro- and antiapoptotic proteins in the regions of the developing brain demonstrates individual variability with a variation coefficient ranging from 10 to 40%. In the brainstem, a structure with a low intensity of apoptosis at this period of ontogeny, correlations between the expression levels of these proteins were not significant. However, in the hippocampus we observed a significant positive correlation between the levels of expression of the proapoptotic proteins Bax and the active form of caspase-3 (r = +0.653). The levels of these proteins significantly negatively correlated with the content of the antiapoptotic protein Bcl-XL (r = −0.651 and −0.740, respectively). Our data suggest coordinated expression of the apoptotic cascade proteins in the hippocampus, a brain structure with a high intensity of elimination of excessive cells during early postnatal ontogeny.     

Ghrelin Antagonized 1-Methyl-4-Phenylpyridinium (MPP<Superscript>+</Superscript>)-Induced Apoptosis in MES23.5 Cells

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) acting to stimulate growth hormone release. In the previous study, we have observed the neuroprotective effects of ghrelin on dopaminergic neurons in vivo in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine -treated Parkinson’s disease mice. In order to illustrate the underlying mechanisms, in the present study, we conducted our experiment in vitro in 1-methyl-4-phenylpyridinium (MPP⁺)-treated MES23.5 cells that could express GHS-R1a. Ten- to 1,000-μmol/L MPP⁺ treatment caused decreased cell viability, with increased lactate dehydrogenase leakage. A 200-μmol/L MPP⁺ treatment was chosen to do the further experiments. MES23.5 cells treated with 200 μmol/L MPP⁺ showed decreased mitochondrial transmembrane potential, an elevated level of reactive oxidative species production and activation of caspase-3. Additionally, these cells also showed apoptotic morphological changes. Pretreatment with different doses of ghrelin (10⁻¹²–10⁻⁷ mol/L) could abolish the MPP⁺-induced apoptotic changes in a dose-dependent manner. These results suggested that ghrelin could antagonize MPP⁺-induced apoptosis in MES23.5 cells. The protective effects of ghrelin involved the restoration of mitochondria function. Juanjuan Dong and Ning Song contributed equally to this work.