不同剂量的He—Ne激光对Raji细胞表面电荷的影响

本文以离体培养的Ｒａｊｉ细胞为材料,采用细胞电泳技术检测了不同剂量的Ｈｅ－Ｎｅ激光对Ｒａｊｉ细胞表面电荷的影响,发现低于或者等于０．５Ｊ／ｃｍ＾２的Ｈｅ－Ｎｅ激光能量对膜表面电荷无明显影响（Ｐ〉０．０５）;大于此剂量的Ｈｅ－Ｎｅ激光可使膜表面电荷（绝对值）下降,即负电荷数减小,ＥＰＭ下降（Ｐ〈０．０５）。     

tBu-D-Gly5]NPS, a pure and potent antagonist of the neuropeptide S receptor: in vitro and in vivo studies

Neuropeptide S (NPS) regulates various biological functions by selectively activating the NPS receptor (NPSR). Recently, the NPSR ligand [(t)Bu-D-Gly(5)]NPS was generated and in vitro characterized as a pure antagonist at the mouse NPSR. In the present study the pharmacological profile of [(t)Bu-D-Gly(5)]NPS has been investigated. [(t)Bu-D-Gly(5)]NPS activity was evaluated in vitro in the calcium mobilization assay at the rat NPSR and in vivo in the locomotor activity and righting reflex tests in mice and in the elevated plus maze and defensive burying assays in rats. In vitro, [(t)Bu-D-Gly(5)]NPS was inactive per se while it inhibited the calcium mobilization induced by 30 nM NPS (pK(B) 7.42). In Schild analysis experiments [(t)Bu-D-Gly(5)]NPS (0.1-10 μM) produced a concentration-dependent rightward shift of the concentration-response curve to NPS, showing a pA(2) value of 7.17. In mouse locomotor activity experiments, supraspinal injection of [(t)Bu-D-Gly(5)]NPS (1-10 nmol) dose dependently counteracted NPS (0.1 nmol) stimulant effects. In the mouse righting reflex assay [(t)Bu-D-Gly(5)]NPS (0.1-10 nmol) fully prevented the arousal-promoting action of the natural peptide (0.1 nmol). Finally, [(t)Bu-D-Gly(5)]NPS (3-30 nmol) was able to completely block NPS (1 nmol) anxiolytic-like actions in rat elevated plus maze and defensive burying assays. Collectively, the present results demonstrated that [(t)Bu-D-Gly(5)]NPS behaves both in vitro and in vivo as a pure and potent NPSR antagonist. This compound represents a novel and useful tool for investigating the pharmacology and neurobiology of the NPS/NPSR system.     

Synthetic studies of centromere-associated protein-E (CENP-E) inhibitors: 1.Exploration of fused bicyclic core scaffolds using electrostatic potential map

Centromere-associated protein-E (CENP-E), a mitotic kinesin that plays an important role in mitotic progression, is an attractive target for cancer therapeutic drugs. For the purpose of developing novel CENP-E inhibitors as cancer therapeutics, we investigated a fused bicyclic compound identified by high throughput screening, 4-oxo-4,5-dihydrothieno[3,4-c]pyridine-6-carboxamide 1a. Based on this scaffold, we designed inhibitors for efficient binding at the L5 site in CENP-E utilizing homology modeling as well as electrostatic potential map (EPM) analysis to enhance CENP-E inhibitory activity. This resulted in a new lead, 5-bromoimidazo[1,2-a]pyridine 7, which showed potent CENP-E enzyme inhibition (IC₅₀: 50 nM) and cellular activity with accumulation of phosphorylated histone H3 in HeLa cells. Our homology model and EPM analysis proved to be useful tools for the rational design of CENP-E inhibitors.     

Intracellular aggregation of human stefin B: confocal and electron microscopy study

Background. Protein aggregation is a major contributor to the pathogenic mechanisms of human neurodegenerative diseases. Mutations in the CSTB (cystatin B) gene [StB (stefin B)] cause EPM1 (progressive myoclonus epilepsy of type 1), an epilepsy syndrome with features of neurodegeneration and increased oxidative stress. Oligomerization and aggregation of StB in mammalian cells have recently been reported. It has also been observed that StB is overexpressed after seizures and in certain neurodegenerative conditions, which could potentially lead to its aggregation. Human StB proved to be a good model system to study amyloid fibril formation in vitro and, as we show here, to study protein aggregation in cells. Results. Endogenous human StB formed smaller, occasional cytoplasmic aggregates and chemical inhibition of the UPS (ubiquitin–proteasome system) led to an increase in the amount of the endogenous protein and also increased its aggregation. Further, we characterized both the untagged and T‐Sapphire‐tagged StB on overexpression in mammalian cells. Compared with wild‐type StB, the EPM1 missense mutant (G4R), the aggregate‐prone EPM1 mutant (R68X) and the Y31 StB variant (both tagged and untagged) formed larger cytosolic and often perinuclear aggregates accompanied by cytoskeletal reorganization. Non‐homogeneous morphology of these large aggregates was revealed using TEM (transmission electron microscopy) with StB detected by immunogold labelling. StB‐positive cytoplasmic aggregates were partially co‐localized with ubiquitin, proteasome subunits S20 and S26 and components of microfilament and microtubular cytoskeleton using confocal microscopy. StB aggregates also co‐localized with LC3 and the protein adaptor p62, markers of autophagy. Flow cytometry showed that protein aggregation was associated with reduced cell viability. Conclusions. We have shown that endogenous StB aggregates within cells, and that aggregation is increased upon protein overexpression or proteasome inhibition. From confocal and TEM analyses, we conclude that aggregates of StB show some of the molecular characteristics of aggresomes and may be eliminated from the cell by autophagy. Intracellular StB aggregation shows a negative correlation with cell survival.     

Corticosterone does not change open elevated plus maze-induced antinociception in mice

It has been demonstrated that the exposure of rodents to the standard elevated plus-maze (sEPM: 2 open and 2 enclosed arms) elicits defensive behavioral reactions and antinociception and also activates the hypothalamo-pituitary-adrenal (HPA) axis. We have recently reported that EPM-induced antinociception is particularly observed when rats and mice are exposed to a totally open EPM (oEPM: 4 open arms). Given that the oEPM seems to be a more aversive situation than the sEPM, we hypothesized that oEPM exposure would induce higher plasma levels of corticosterone than sEPM exposure in mice. In this study, we investigated the influence of exposure to eEPM (enclosed EPM: 4 enclosed arms), sEPM or oEPM on plasma corticosterone levels in mice, with or without prior nociceptive stimulation (2.5% formalin injection into the right hind paw). We also tested whether the nociceptive response in the formalin test and oEPM-induced antinociception are altered by adrenalectomy. Results showed that oEPM-exposed mice spent less time licking the injected paw than sEPM- and eEPM-exposed animals. All three types of EPM exposure increased plasma corticosterone when compared to the basal group, but sEPM- and oEPM-exposed mice showed higher corticosterone levels than eEPM-exposed mice. Prior nociceptive stimulation (formalin injection) did not enhance the plasma corticosterone response induced by the three types of EPM exposure. Indeed, formalin injection appeared to provoke a ceiling effect on plasma corticosterone concentration. Furthermore, neither the nociceptive response in the formalin test nor oEPM-induced antinociception was changed by adrenalectomy. Present results suggest that oEPM antinociception does not depend on corticosterone release in mice.     

Nuclear localization of cystatin B, the cathepsin inhibitor implicated in myoclonus epilepsy (EPM1)

Cystatin B is an anti-protease implicated in myoclonus epilepsy, a degenerative disease of the central nervous system. In vitro, cystatin B interacts with and inhibits proteases of the cathepsin family. Confocal microscopy analysis of the subcellular localization of cystatin B and cathepsin B shows that, in vivo, the two proteins are concentrated in different cell compartments. In fact, cystatin B is found mainly in the nucleus of proliferating cells and both in the nucleus and in the cytoplasm of differentiated cells, while cathepsin B, in either case, is essentially cytoplasmic. However, colocalization of cystatin and cathepsin B is observed in the isolated cell matrix and in the nuclear scaffold of differentiated neuroblastoma cells but not of proliferating cells. This suggests that at least a fraction of cystatin B is bound to the protease in differentiated cells. The electron microscopy analysis of the cell matrix confirms the observation made with confocal microscopy. The cellular activity of cathepsin B was analyzed with a fluorogenic cytochemical assay. A fluorescent signal is observed in the cytoplasm of proliferating cells but is undetectable in the cytoplasm of differentiated cells, suggesting that cathepsin B is active mainly during the cell cycle. This result is consistent with the separate compartimentalization of cystatin B and cathepsin B that we have observed in growing cells.     

HCN channel activity-dependent modulation of inhibitory synaptic transmission in the rat basolateral amygdala

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in the central nervous system and play a regulatory role in neuronal excitability. In the present study, we examined a physiological role of HCN channels in the rat basolateral amygdala (BLA). In vitro electrophysiological studies showed that ZD7288 decreased spontaneous inhibitory postsynaptic current (sIPSC) without changing miniature IPSC (mIPSC). HCN channel blockade also attenuated feedback inhibitions in BLA principal neurons. However, blockade of HCN channel had little effects on spontaneous excitatory postsynaptic current (sEPSC) and mEPSC. Therefore, HCN channel appeared to decrease BLA excitability by increasing the action potential-dependent inhibitory control over the BLA principal neurons. Anxiety is reported to be influenced by neuronal excitability in the BLA and inhibitory synaptic transmission is thought to play a pivotal role in regulating overall excitability of the amygdala. As expected, blockade of HCN channels by targeted injection of ZD7288 to the BLA increased anxiety-like behavior under elevated plus maze test. Our results suggest that HCN channel activity can modulate the GABAergic synaptic transmission in the BLA, which in turn control the amygdala-related emotional behaviors such as anxiety.     

An alternate pathway for androgen regulation of brain function: activation of estrogen receptor beta by the metabolite of dihydrotestosterone, 5alpha-androstane-3beta,17beta-diol

The complexity of gonadal steroid hormone actions is reflected in their broad and diverse effects on a host of integrated systems including reproductive physiology, sexual behavior, stress responses, immune function, cognition, and neural protection. Understanding the specific contributions of androgens and estrogens in neurons that mediate these important biological processes is central to the study of neuroendocrinology. Of particular interest in recent years has been the biological role of androgen metabolites. The goal of this review is to highlight recent data delineating the specific brain targets for the dihydrotestosterone metabolite, 5alpha-androstane, 3beta,17beta-diol (3beta-Diol). Studies using both in vitro and in vivo approaches provide compelling evidence that 3beta-Diol is an important modulator of the stress response mediated by the hypothalmo-pituitary-adrenal axis. Furthermore, the actions of 3beta-Diol are mediated by estrogen receptors, and not androgen receptors, often through a canonical estrogen response element in the promoter of a given target gene. These novel findings compel us to re-evaluate the interpretation of past studies and the design of future experiments aimed at elucidating the specific effects of androgen receptor signaling pathways.     

Identification of a novel protein interacting with laforin,the EPM2a progressive myoclonus epilepsy gene product

We have identified an interacting partner protein (encoded by the human EPM2AIP1 gene (approved symbol)) for laforin, the product of the EPM2A gene, which is mutated in an autosomal recessive form of adolescent progressive myoclonus epilepsy. The EPM2AIP1 gene was identified in a screen for laforin-interacting proteins with a human brain cDNA library using the yeast two-hybrid system. The specificity of the interaction was confirmed by coimmunoprecipitation of in vivo-transfected protein and by using EPM2A deletion constructs. Subcellular colocalization of laforin and EPM2AIP1 protein was also demonstrated. The human EPM2AIP1 gene, corresponding to the KIAA0766 cDNA clone in the databases, was characterized and shown, like EPM2A, to be ubiquitously expressed. The gene, which comprises one large exon 1824 nucleotides in length and has alternative 3' untranslated regions, maps to human chromosome 3p22.1. The function is currently not known and extensive analyses do not reveal any homology to other proteins or any obvious structural motifs. Because genetic heterogeneity in Lafora disease has been described, mutational analysis of the EPM2AIP1 gene was performed on non-EPM2A patients, but no mutations were found. The identification of this first binding partner for laforin promises to be an important step toward unraveling the underlying pathogenesis of this severest form of teenage-onset epilepsy.