Disruption of nNOS-PSD95 protein–protein interaction inhibits acute thermal hyperalgesia and chronic mechanical allodynia in rodents

Background and purpose:

Post-synaptic density protein 95 (PSD95) contains three PSD95/Dosophilia disc large/ZO-1 homology domains and links neuronal nitric oxide synthase (nNOS) with the N-methyl-D-aspartic acid (NMDA) receptor. This report assesses the effects of disruption of the protein–protein interaction between nNOS and PSD95 on pain sensitivity in rodent models of hyperalgesia and neuropathic pain.

Experimental approach:

We generated two molecules that interfered with the nNOS–PSD95 interaction: IC87201, a small molecule inhibitor; and tat-nNOS (residues 1–299), a cell permeable fusion protein containing the PSD95 binding domain of nNOS. We then characterized these inhibitors using in vitro and in vivo models of acute hyperalgesia and chronic allodynia, both of which are thought to require nNOS activation.

Key results:

IC87201 and tat-nNOS (1–299) inhibited the in vitro binding of nNOS with PSD95, without inhibiting nNOS catalytic activity. Both inhibitors also blocked NMDA-induced 3′,5′-cyclic guanosine monophosphate (cGMP) production in primary hippocampal cultures. Intrathecal administration of either inhibitor potently reversed NMDA-induced thermal hyperalgesia in mice. At anti-hyperalgesic doses, there was no effect on acute pain thresholds or motor coordination. Intrathecal administration of IC87201 and tat-nNOS also reversed mechanical allodynia induced by chronic constriction of the sciatic nerve.

Conclusions and implications:

nNOS–PSD95 interaction is important in maintaining hypersensitivity in acute and chronic pain. Disruption of the nNOS–PSD95 interaction provides a novel approach to obtain selective anti-hyperalgesic compounds.     

强啡肽A(1-17)在脊髓的致瘫作用及其与神经毒作用的相关性

在以行为学为观察指标(甩尾镇痛和斜板实验)的基础上,用组织学方法探讨大剂量强啡肽A在脊髓水平的致瘫作用与其神经毒作用的关系。结果表明,给大鼠蛛网膜下腔(it)注射强啡肽A(1-17)20nmol·L^-1,共10μl,给药后5～10min即引起大鼠后肢不可逆性瘫痪、甩尾反射被抑制长达40h以上。大鼠脊髓组织学检查发现,腰、骶段脊髓前角运动神经元大量坏死或严重变性、以腰段损伤最为显著(运动神经元减少87.2%),其次是骶段(减少69.6%),胸段损伤不明显(减少8.2%)。     

Loss of response to epidermal growth factor and retinoic acid accompanies the transformation of human prostatic epithelial cells to tumorigenicity with v-Ki-ras

Growth factor-independent proliferation and loss of response to differentiation factors are believed to be critical elements in carcinogenesis. We have developed an in vitro model of human prostatic carcinogenesis by the introduction of SV40 DNA into normal prostatic epithelial cells to create a transformed, immortal cell line, pRNS-1-1. This non-tumorigenic cell line responded similarly to normal prostatic epithelial cells to most growth- and differentiation-regulatory factors, with the notable exception of loss of response to the inhibitory factor 1,25-dihydroxyvitamin D3. In this study, we describe the introduction of the ras oncogene into pRNS-1-1 cells to create a tumorigenic cell line, pRNS-1-1/ras. In addition to an attenuated response to 1,25-dihydroxyvitamin D3, these cells also became unresponsive to retinoic acid and gained the ability to undergo clonal proliferation in the absence of epidermal growth factor (EGF). EGF- independent growth could not be linked to the production of autocrine transforming growth factor-alpha, but instead was likely due to sustained signaling by the ras oncogene, bypassing ligand-activation of the EGF receptor. Ligand-independent proliferation, coupled with the loss of response to the growth-inhibitory and differentiation agent retinoic acid, may be important elements in the conversion of human prostatic epithelial cells to tumorigenicity.      

榆耳发酵液中新倍半萜——榆耳三醇的结构

从榆耳(Gloeostereum incarnatum S.Ito et Imai)的液体发酵物中经层析分离得一新倍半萜榆耳三醇(gloeosteretriol),根据红外光谱、核磁共振谱和质谱数据推定其结构为Ⅰ,并用X-射线晶体衍射进一步确证其结构和立体构型。     

The combination of cyclophosphomide, thalidomide and dexamethasone is an effective alternative to cyclophosphamide - vincristine - doxorubicin - methylprednisolone as induction chemotherapy prior to autologous transplantation for multiple myeloma: a case-matched analysis

A retrospective case-matched study was conducted to compare the oral regimen CTD (cyclophosphamide - thalidomide - dexamethasone) and infusional CVAMP (cyclophosphamide - vincristine - doxorubicin - methylprednisolone) as induction therapy followed by autologous peripheral blood stem-cell transplantation (PBSCT) for newly diagnosed multiple myeloma patients. The response rate after three cycles of treatment was statistically higher with CTD (n = 27) compared to CVAMP (n = 27) (89% vs. 56%, P = 0.016). Toxicity studies showed more neutropenia (grade 3/4) (4% vs. 60%, P = 0.0002) with CVAMP and more thrombotic episodes with CTD (11% vs. 4%). CTD may emerge as the superior induction regimen prior to PBSCT, in terms of high efficacy and better tolerability.     

Structural determinants in the second intracellular loop of the human cannabinoid CB1 receptor mediate selective coupling to Gs and Gi

BACKGROUND AND PURPOSE

The cannabinoid CB₁ receptor is primarily thought to be functionally coupled to the G_i form of G proteins, through which it negatively regulates cAMP accumulation. Here, we investigated the dual coupling properties of CB₁ receptors and characterized the structural determinants that mediate selective coupling to G_s and G_i.

EXPERIMENTAL APPROACH

A cAMP-response element reporter gene system was employed to quantitatively analyze cAMP change. CB₁/CB₂ receptor chimeras and site-directed mutagenesis combined with functional assays and computer modelling were used to determine the structural determinants mediating selective coupling to G_s and G_i.

KEY RESULTS

CB₁ receptors could couple to both G_s-mediated cAMP accumulation and G_i-induced activation of ERK1/2 and Ca²⁺ mobilization, whereas CB₂ receptors selectively coupled to G_i and inhibited cAMP production. Using CB₁/CB₂ chimeric receptors, the second intracellular loop (ICL2) of the CB₁ receptor was identified as primarily responsible for mediating G_s and G_i coupling specificity. Furthermore, mutation of Leu-222 in ICL2 to either Ala or Pro switched G protein coupling from G_s to G_i, while to Ile or Val led to balanced coupling of the mutant receptor with G_s and G_i.

CONCLUSIONS AND IMPLICATIONS

The ICL2 of CB₁ receptors and in particular Leu-222, which resides within a highly conserved DRY(X)₅PL motif, played a critical role in G_s and G_i protein coupling and specificity. Our studies provide new insight into the mechanisms governing the coupling of CB₁ receptors to G proteins and cannabinoid-induced tolerance.     

1－（2，6－二甲基苯氧基）－2－（3，4－二甲氧基苯乙氨基）丙烷盐酸盐对兔血小板胞浆游离钙浓度的影响

１－（２，６－二甲基苯氧基）－２－（３，４－二甲氧基苯乙氨基）丙烷盐酸盐对兔血小板胞浆游离钙浓度的影响夏敬生，罗湘，曾繁典（同济医科大学临床药理研究室，武汉４３００３０）１－（２，６－二甲基苯氧基）－２－（３，４－二甲氧基苯乙氨基）丙烷盐酸盐［１－（...     

固相法合成心肌兴奋肽及其类似物

用Boc-和Tos-基团分别保护氨基和侧链胍基,以1%交联度聚苯乙烯二苯甲氨基树脂为载体,用DCC固相法合成肽,HF断裂肽树脂键和去除侧链保护基团,粗产物经高效液相层析纯化,合成了心肌兴奋肽Phe-Met-Arg-Phe-NH₂及其类似物Phe-Pro-Arg-Phe-NH₂,并观察了此二种肽对大鼠血压和心率的影响。     

苦木双碱甲和苦木双碱乙的结构

自苦木科(Simaroubaceae)植物苦木(Picrasma quassioides(D.Don.)Benn.)的木质部的乙醇提取物中经硅胶和氧化铝柱层析分离得到两个新β-咔巴啉二聚体生物碱,分别命名为苦木双碱甲(kumujansine)和苦木双碱乙(kumujantine),经光谱(UV,IR,¹H-NMR,^{13C-NMR,2D-NMR和MS)分析确定结构分别为式Ⅰ和Ⅱ。}