淫羊藿苷对淀粉样β蛋白片段25-35所致大鼠学习记忆障碍的改善作用

目的观察淫羊藿苷对淀粉样β蛋白片段25-35(Aβ25-35)所致阿尔茨海默病(AD)模型大鼠学习记忆能力的保护作用,并探讨其可能的作用机制。方法Wistar雄性大鼠,右侧海马内注射Aβ25-3510μg制备AD模型,次日起淫羊藿苷30,60和120mg.kg-1灌胃给药,连续14d,d15～19Morris水迷宫检测大鼠空间辨别学习记忆能力;d20检测海马组织中谷胱甘肽过氧化物酶(GSH-PX)、超氧化物歧化酶(SOD)及一氧化氮合酶(NOS)的活性,免疫组化法检测海马内乙酰胆碱酯酶(AChE)和胆碱乙酰转移酶(ChAT)的表达。结果与模型对照组比较,淫羊藿苷给药14d明显改善大鼠学习记忆能力;海马组织中SOD和GSH-PX活性升高,NOS活性降低;海马内AChE及ChAT的表达增加。结论淫羊藿苷可以改善Aβ25-35海马内注射所致AD模型大鼠的学习记忆能力,其作用可能与其增加AChE和ChAT表达,增强SOD和GSH-PX等自由基清除酶活性,降低NOS活性,减少NO释放等多种机制,促进胆碱能递质系统功能的恢复有关。     

精神分裂症与神经调节素1、G72及G-蛋白信号转导调节子4基因的关联研究

目的探索神经调节素1(NRG1)、G72、G-蛋白信号转导调节子4(RGS4)基因变异的独立或联合作用与精神分裂症的关联。方法采用酶切或测序方法,对315例精神分裂症患者和347例正常对照者NRG1、G72、RGS4基因的13个单核苷酸多态性(SNPs)位点进行基因型检测和遗传关联分析。应用Lo-gistic回归分析和风险或保护基因型分层后卡方检验评估三基因的交互作用。结果单位点关联分析发现,NRG1的5个SNPs、G72的1个SNP、RGS4的2个SNPs与精神分裂症关联(P<0.05)。Logistic回归分析构建了三个基因交互作用模型,各自变量的OR分别为:nrg1=3.58,rgs4=1.24,g72_rs947267=1.45,nrg1*rgs4=3.03,nrg1*g72_rs947267=1.06。应用基因型分层后卡方检验表明,三基因存在协同风险效应(OR=2.35～4.05;P<0.05)。结论NRG1、G72及RGS4基因的单独或交互作用与精神分裂症关联。     

Roles of G-protein signaling 5 in tumors

G-protein signaling 5 ( RGS5) belongs to RGS family, which can negatively regulate the conduction of this signaling pathway. RGS5 mainly expresses in vascular pericyte, and is closely related to the occurrence, development and maturation of the blood vessels. Loss of RGS5 results in pericyte maturation, tumor vascular normalization, and these changes can improve the curative effect of combined chemotherapy and immunotherapy, indicating that RGS5 may become a new target of anti-tumor treatment. In addition, RGS5 involves in tumor metastasis and apoptosis, which can improve antineoplastic effect by inducing tumor cells apoptosis.     

FAAH?/? Mice Display Differential Tolerance,Dependence, and Cannabinoid Receptor Adaptation After Δ9-Tetrahydrocannabinol and Anandamide Administration

Repeated administration of Δ⁹-tetrahydrocannabinol (THC), the primary psychoactive constituent of Cannabis sativa, induces profound tolerance that correlates with desensitization and downregulation of CB₁ cannabinoid receptors in the CNS. However, the consequences of repeated administration of the endocannabinoid N-arachidonoyl ethanolamine (anandamide, AEA) on cannabinoid receptor regulation are unclear because of its rapid metabolism by fatty acid amide hydrolase (FAAH). FAAH^−/− mice dosed subchronically with equi-active maximally effective doses of AEA or THC displayed greater rightward shifts in THC dose–effect curves for antinociception, catalepsy, and hypothermia than in AEA dose–effect curves. Subchronic THC significantly attenuated agonist-stimulated [³⁵S]GTPγS binding in brain and spinal cord, and reduced [³H]WIN55,212-2 binding in brain. Interestingly, AEA-treated FAAH^−/− mice showed less CB₁ receptor downregulation and desensitization than THC-treated mice. Experiments examining tolerance and cross-tolerance indicated that the behavioral effects of THC, a low efficacy CB₁ receptor agonist, were more sensitive to receptor loss than those of AEA, a higher efficacy agonist, suggesting that the expression of tolerance was more affected by the intrinsic activity of the ligand at testing than during subchronic treatment. In addition, the CB₁ receptor antagonist, rimonabant, precipitated a markedly reduced magnitude of withdrawal in FAAH^−/− mice treated subchronically with AEA compared with mice treated repeatedly with THC. The findings that repeated AEA administration produces lesser adaptive changes at the CB₁ receptor and has reduced dependence liability compared with THC suggest that pharmacotherapies targeting endocannabinoid catabolic enzymes are less likely to promote tolerance and dependence than direct acting CB₁ receptor agonists.     

Common null variant, Arg192Stop, in a G-protein coupled receptor, olfactory receptor 1B1, associated with decreased serum cholinesterase activity

Aim: Non-functioning single nucleotide polymorphisms (nSNPs) that result in premature termination codons, that is null-alleles of the respective genes, may have phenotypic effects on clinical parameters. We conducted association studies involving several G-protein coupled receptors (GPCRs) that harbor nSNPs, using clinical parameters of liver function in a general population consisting of 2969 Japanese adults. Methods: SNP typings were performed with TaqMan and Invader assays. Quantitative associations between genotypes and clinical parameters were analyzed by analysis of variance. Linkage disequilibrium (LD) was tested by Haploview Version 3.3. Haplotype-based association was performed using the haplo.stats program. Results: A significant correlation (P = 0.0057) was identified between serum cholinesterase activity (CHE) and an nSNP (Arg192Stop) in the olfactory receptor (OR) 1B1 gene, a member of the GPCR gene family. This nSNP was associated with decreased serum CHE (P = 0.0013). LD analysis based on eight selected SNPs at the locus revealed three LD blocks. The Arg192Stop nSNP was located on the second LD block, which covered one-third of the 3'-portion of the gene. Conclusion: These results suggested that the null-allele of OR1B1 might affect metabolism of serum cholinesterase in carriers of this nSNP.     

The neuropeptide Y Y1 receptor is a somatic receptor on dorsal root ganglion neurons and a postsynaptic receptor on somatostatin dorsal horn neurons.

Using indirect immunofluorescence, neuropeptide Y Y1 receptor (Y1 receptor)-like immunoreactivity (LI) was localized close to the plasmalemma of small neurons in lumbar dorsal root ganglia (DRGs) and neurons in the inner lamina II of the lumbar spinal cord of the rat. Using confocal microscopy, colocalization of Y1 receptor-LI and transferrin receptor-LI, a marker for endosomes and coated vesicles, was observed in dot-like structures along the plasmalemma. Under the electron microscope, Y1 receptor-LI was localized in coated vesicles and endosomes, in the membrane of tubular cisternae, sometimes connected to multivesicular bodies, and in the plasmalemma. These complex distribution patterns may reflect receptor turnover and internalization processes. In the lamina II of the spinal dorsal horn, Y1 receptor-LI was localized in the plasmalemma of neurons without any apparent association with paramembrane structures, as described above for the DRG neurons. Many dendrites were Y1 receptor-positive, and some of them made synaptic contacts with unstained axonal terminals. In general, Y1 receptor-LI was localized in the membrane outside the postsynaptic density. Double-immunofluorescence staining showed that most Y1 receptor-immunoreactive neurons in lamina II contained somatostatin-LI. Both in DRG and dorsal horn neurons, the Y1 receptor thus seems to represent a postjunctional/postsynaptic receptor.     

The human antimicrobial peptide dermcidin activates normal human keratinocytes

Background  The skin has evolved an epithelial defence mechanism which is characterized by antimicrobial peptides that inactivate various microorganisms and exhibit stimulatory activities bridging innate and adaptive immunity. Dermcidin (DCD) is a newly isolated antimicrobial peptide produced by the eccrine sweat glands in the skin. Recently, the DCD peptides DCD-1 and DCD-1L have been shown to display in vitro microbicidal activities against bacteria and viruses.
Objectives  Because some skin-derived antimicrobial peptides activate keratinocytes, we investigated whether DCD-1L would also trigger keratinocyte activation.
Methods  Normal human keratinocytes were used in this study. The ability of DCD-1L to induce the production of cytokines/chemokines by keratinocytes was determined by enzyme-linked immunosorbent assay, and various inhibitors were used to investigate the stimulatory mechanism of DCD-1L. Mitogen-activated protein kinase (MAPK) phosphorylation and NF-κB activation were analysed by Western blotting.
Results  DCD-1L stimulated keratinocytes to generate cytokines and chemokines including tumour necrosis factor-α, interleukin-8 (CXCL8), interferon-inducible protein 10 (CXCL10) and macrophage inflammatory protein-3α (CCL20). To determine the molecular mechanism involved, we showed that DCD-1L-mediated cytokine/chemokine production was controlled by both G-protein and MAPK pathways, as evidenced by the inhibitory effects of pertussis toxin and specific inhibitors for p38 and ERK, but not for JNK, on DCD-1L-induced keratinocyte activation. Furthermore, we confirmed that DCD-1L could induce phosphorylation of p38 and ERK, and noticeably upregulated NF-κB activation.
Conclusions  Taken together, the new activity of DCD-1L to stimulate the production of cytokines/chemokines by keratinocytes provides novel evidence for the implication of DCD, beyond its microbicidal ability, in skin immunity.     

Membrane-impermeable estrogen is involved in regulation of calbindin-D9k expression via non-genomic pathways in a rat pituitary cell line,GH3 cells

Estrogen (E2) has been shown to regulate various functions for many pituitary hormones. Recently, the potential roles of non-genomic pathways in E2-induced actions have been proposed in the previous studies, however, the effects of E2 remain to be elucidated in regard to non-genomic induction of cytosolic protein calbindin-D9k (CaBP-9k). To gain a better understanding of the molecular events underlying E2-induced expression of CaBP-9k, rat pituitary tumor cells (GH3 cells) were treated with E-BSA (membrane impermeable E2-conjugated with BSA). Non-genomic induction of CaBP-9k by E-BSA was determined using RT-PCR and western blot analysis. The significant increase in CaBP-9k mRNA level was observed as early as 15 min following treatment with a high concentration of E-BSA (10⁻⁶ M), whereas rapid and significant induction of CaBP-9k protein was noted at 5, 15 and 30 min after E-BSA exposure (p < 0.05). In order to determine the potential involvement of different signaling pathways, several inhibitors were employed, i.e., ICI 182,780 for the estrogen receptor (ER) pathway, pertussis toxin (PTX) for the G-protein-coupled signaling pathway, U0126 (U) for the ERK (extracellular regulated kinase) and wortmannin (W) for the Akt (protein kinase B). Co-treatment with ICI 182, 780 and PTX reversed an E-BSA-induced increase in CaBP-9k mRNA and protein. Although neither U nor W alone attenuated E-BSA-induced effects, these inhibitors together abolished E-BSA-induced CaBP-9k expression, suggesting their involvement in its regulation. Taken together, these results demonstrate the involvement of various signaling pathways in E2-induced regulation of CaBP-9k. In addition, ER and G-protein-coupled signaling pathways may play central roles in the non-genomic activities of E2 and that downstream signaling via ERK and Akt are required to evoke ER-mediated induction of CaBP-9k in vitro.     

Site-directed mutagenesis of the putative human muscarinic M2 receptor binding site.

Experimental probing of the model of the muscarinic M₂ receptor binding site proposed by Hibert et al. [Hibert, M.F., Trumpp-Kallmeyer, S., Bruinsvels, A., Hoflak, K., 1991. Three-dimensional models of neurotransmitter G-binding protein-coupled receptors. Mol. Pharmacol. 40, 8–15.] was achieved by mutating each amino-acid proposed to interact with muscarinic ligands. Pharmacological analysis of the different mutant receptors transiently expressed in human embryonic kidney (HEK/293) cells was performed with a variety of agonists and antagonists. D103A, Y403A and N404A mutations prevented binding of [³H] N-methylscopolamine and [³H] quinuclidinyl benzilate with a reduction in affinity greater than 100-fold, indicating essential contributions of these residues to the binding site for the radioligands. W400A and W155A mutations had very large effects on the binding of [³H] N-methylscopolamine (150-fold, 960-fold) but modest effects on the binding of [³H] quinuclidinyl benzilate (4-fold, 17-fold). In addition, binding of oxotremorine-M, oxotremorine, arecoline and pilocarpine to W155A resulted in a greater than 100-fold decrease in affinity. Threonine mutations (T187A and T190A) alter binding of most agonists but not of antagonists. W99 makes little contribution (<10-fold) to the binding site of the M₂ receptor. D103, W155, W400, Y403 and N404 are likely to be part of the binding site for N-methylscopolamine and also to contribute to the binding site for quinuclidinyl benzilate. Some of the predicted residues do not seem to be part of the M₂ receptor binding site but W155 is important for proper ligand binding on the muscarinic M₂ receptor, as predicted by the proposed model.