Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons

The adipocytokine apelin and its G protein-coupled APJ receptor were initially isolated from a bovine stomach and have been detected in the brain and cardiovascular system. Recent studies suggest that apelin can protect cardiomyocytes from ischemic injury. Here, we investigated the effect of apelin on apoptosis in mouse primary cultures of cortical neurons. Exposure of the cortical cultures to a serum-free medium for 24 h induced nuclear fragmentation and apoptotic death; apelin-13 (1.0-5.0 nM) markedly prevented the neuronal apoptosis. Apelin neuroprotective effects were mediated by multiple mechanisms. Apelin-13 reduced serum deprivation (SD)-induced ROS generation, mitochondria depolarization, cytochrome c release and activation of caspase-3. Apelin-13 prevented SD-induced changes in phosphorylation status of Akt and ERK1/2. In addition, apelin-13 attenuated NMDA-induced intracellular Ca²⁺ accumulation. These results indicate that apelin is an endogenous neuroprotective adipocytokine that may block apoptosis and excitotoxic death via cellular and molecular mechanisms. It is suggested that apelins may be further explored as a potential neuroprotective reagent for ischemia-induced brain damage.     

Structural requirements of anthocyanins in relation to inhibition of endothelial injury induced by oxidized low-density lipoprotein and correlation with radical scavenging activity

Anthocyanins may play an important role in atherosclerosis prevention. However, the structure-function relationships are not well understood. The objective of this study was to compare the inhibitory effect of 21 anthocyanins against oxidized low-density lipoprotein-induced endothelial injury to understand the relationship between anthocyanin chemical structure and the endothelial protective properties, measured as cell viability, MDA production and NO release. Additionally, the intracellular anti-radical activity of the selected anthocyanins was investigated to identify the correlation with endothelial protection. Our results provide evidence that the number of -OH in total or in B-ring, 3′,4′-ortho-dihydroxyl and 3-hydroxyl are the main structural requirements of anthocyanins in suppressing oxidative stress-induced endothelial injury and such inhibitory effect was significantly correlated with the intracellular radical scavenging activity.     

Nω-硝基-L-精氨酸甲酯抑制吗啡镇痛耐受大鼠中脑和脊髓一氧化氮合酶/N-甲基-D-天冬氨酸受体表达上调

采用热甩尾测痛法观察全身应用非特异性一氧化氮合酶（nitric oxide synthase,NOS）抑制剂——N^ω-硝基-L-精氨酸甲酯（L-NAME）对吗啡镇痛耐受形成的影响,并通过观察脊髓和中脑神经元型NOS（nNOS）和N-甲基-D-天冬氨酸（NMDA）受体亚单位表达的变化来阐释NO／NMDA受体在吗啡镇痛耐受形成中的作用。将36只健康成年Sprague-Dawley大鼠平均分为6组（每组6只）：1组为对照组,皮下注射生理盐水1ml;2、3、4、5和6组为处理组,分别皮下注射L-NAME10mg／kg、L-NAME20mg／kg、吗啡10mg／kg、L-NAME10mg／kg＋吗啡10mg／kg、L-NAME20mg／kg＋吗啡10mg／kg,每天2次。在注射前测量大鼠的热甩尾潜伏期（tail-flick latency,TFL）基础值,随后每天第一次给药50min后测量其TFL。第8天最后一次给药80min后（除2组和5组之外）断头取脊髓和中脑,采用RT-PCR技术测量nNOS以及NMDA受体1A（NR1A）和2A（NR2A）亚单位的表达。结果显示,2组大鼠第1天至第7天的TFL与基础值相比无显著差异;3组第7天时的TFL仍显著高于基础值;4组的TFL在第1天时最高,第2至第6天期间逐渐降低,第6天时与基础值相比无显著差异：5组的TFL在实验过程中呈下降趋势,虽然第7天时较第1天有所降低,但是仍然显著高于基础值;6组的TFL变化趋势与5组相同。PT—PCR分析结果显示,与1组相比,3组脊髓和中脑的nNOS mRNA表达显著降低,但NR1A mRNA和NR2A mRNA表达无显著改变;4组的nNOS mRNA、NR1A mRNA和NR2A mRNA表达均显著高于1组。与4组相比,6组的nNOS mRNA、NR1A mRNA和NR2A mRNA表达均显著降低。结果提示,吗啡镇痛耐受大鼠脊髓和中脑的nNOS和NMDA受体表达增加,联合应用L—NAME可抑制长期应用吗啡所致的nNOS表达增加和NMDA受体上调,延缓吗啡镇痛耐受的形成。本研究结果提示,脊髓和中脑的NO／NMDA受体与吗啡镇痛耐受形成密切相关。     

Competitive study of monoclonal antibodies against the HIV-1 Gp41 core structure

Monoclonal antibodies (MAbs) 50.69, 98.6, and T26 bind specifically to the core structure of the human immunodeficiency virus type 1 (HIV-1) envelope transmembrane glycoprotein (gp41). To clarify the specificity of the anti-core structure MAbs, we performed competitive assays using the MAbs to the H9 human T cell line infected with the IIIB strain of HIV-1 (H9/IIIB). Bound MAb 50.69 inhibited MAb 98.6 binding unidirectionally. The reason for the unidirectional cross competition between MAbs 50.69 and 98.6 is not clear, but these results help to define the antigenic structure of gp41 on the surface of infected cells.     

Myostatin induces cachexia by activating the ubiquitin proteolytic system through an NF-kappaB-independent, FoxO1-dependent mechanism

Myostatin, a transforming growth factor-beta (TGF-beta) super-family member, has been well characterized as a negative regulator of muscle growth and development. Myostatin has been implicated in several forms of muscle wasting including the severe cachexia observed as a result of conditions such as AIDS and liver cirrhosis. Here we show that Myostatin induces cachexia by a mechanism independent of NF-kappaB. Myostatin treatment resulted in a reduction in both myotube number and size in vitro, as well as a loss in body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 was reduced, while NF-kappaB (the p65 subunit) localization and expression remained unchanged. In addition, promoter analysis has confirmed Myostatin inhibition of myoD and pax3. An increase in the expression of genes involved in ubiquitin-mediated proteolysis is observed during many forms of muscle wasting. Hence we analyzed the effect of Myostatin treatment on proteolytic gene expression. The ubiquitin associated genes atrogin-1, MuRF-1, and E214k were upregulated following Myostatin treatment. We analyzed how Myostatin may be signaling to induce cachexia. Myostatin signaling reversed the IGF-1/PI3K/AKT hypertrophy pathway by inhibiting AKT phosphorylation thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. Therefore, our results suggest that Myostatin induces cachexia through an NF-kappaB-independent mechanism. Furthermore, increased Myostatin levels appear to antagonize hypertrophy signaling through regulation of the AKT-FoxO1 pathway.     

Evaluation of the stress-inducible production of choline oxidase in transgenic rice as a strategy for producing the stress-protectant glycine betaine

Glycine betaine (GB) is a compatible solute that is also capable of stabilizing the structure and function of macromolecules. Several GB-producing transgenic rice lines were generated in which the Arthrobacter pascens choline oxidase (COX) gene, fused to a chloroplast targeting sequence (TP) was expressed under the control of an ABA-inducible promoter (SIP; stress-inducible promoter) or a ubiquitin (UBI) gene promoter that is considered to be constitutive. This comparison led to interesting observations that suggest complex regulation with respect to GB synthesis and plant growth response under stress. In spite of the use of the well-studied stress-inducible promoter, the highest level of GB accumulation (up to 2.60 micromol g(-1) DW) in the SIP lines grown under saline conditions was not as high as in the UBI lines (up to 3.12 micromol g(-1) DW). Therefore, the use of an ABA-inducible promoter was not more beneficial for de novo production of GB. Interestingly, saline growth conditions enhanced GB accumulation by up to 89% in the SIP lines, whereas up to 44% increase was seen in a UBI line. In all these cases the GB levels were many-fold below the range reported for plant species that produce GB naturally. In spite of lower GB concentrations, statistically greater levels of stress tolerance were found in SIP lines than in UBI lines, suggesting that the stress protection observed in SIP plants cannot be totally explained by the increase in the GB content.     

Nuclear magnetic resonance structure-based epitope mapping and modulation of dust mite group 13 allergen as a hypoallergen

IgE-mediated allergic response involves cross-linking of IgE bound on mast cells by specific surface epitopes of allergens. Structural studies on IgE epitopes of allergens are essential in understanding the characteristics of an allergen and for development of specific allergen immunotherapy. We have determined the structure of a group 13 dust mite allergen from Dermatophagoides farinae, Der f 13, using nuclear magnetic resonance. Sequence comparison of Der f 13 with homologous human fatty acid-binding proteins revealed unique surface charged residues on Der f 13 that may be involved in IgE binding and allergenicity. Site-directed mutagenesis and IgE binding assays have confirmed four surface charged residues on opposite sides of the protein that are involved in IgE binding. A triple mutant of Der f 13 (E41A_K63A_K91A) has been generated and found to have significantly reduced IgE binding and histamine release in skin prick tests on patients allergenic to group 13 dust mite allergens. The triple mutant is also able to induce PBMC proliferation in allergic patients with indices similar to those of wild-type Der f 13 and shift the secretion of cytokines from a Th2 to a Th1 pattern. Mouse IgG serum raised using the triple mutant is capable to block the binding of IgE from allergic patients to wild-type Der f 13, indicating potential for the triple mutant as a hypoallergen for specific immunotherapy. Findings in this study imply the importance of surface charged residues on IgE binding and allergenicity of an allergen, as was also demonstrated in other major allergens studied.