蝎毒多肽提取物对白血病细胞株K562/A02荷瘤鼠耐药性的影响

目的 探讨蝎毒多肽（peptide extract from scorpion venom,PESV）逆转白血病干细胞（leukemiastem cells, LSC）在体内多药耐药（multidrug resistance, MDR）的分子机制。方法 以多药耐药的K562/A02细胞株成模白血病BALB/c裸鼠为例,成模鼠随机分为6组：模型对照组、阿霉素（ADM）组、PESV组、ADM+PESV（H）组、ADM+PESV（M）组、ADM+PESV（L）组。模型对照组给予等体积0.9%氯化钠溶液腹腔注射,其余各组予相应剂量ADM和（或）PESV腹腔注射,连续给药14天。第21天观察各组裸鼠移植瘤生长情况,分别检测瘤块中LSC：细胞膜上P-gp的表达,细胞质中ALDH、PI3K的变化及细胞核中MDR1、NF-κB的活性。结果 K562/A02细胞经免疫磁珠分选前后的CD34+CD38-细胞比率和IC50值分别为31.5%、（60.33±10.68）μg/ml和92.8%、（58.33±9.72）μg/ml,分选后细胞干性显著提高,而耐药性无差异性损失;各组造模裸鼠成瘤率100%。瘤体中LSC：流式细胞仪检测细胞膜上P-gp表达结果：检测对照组89.8%、ADM组91.9%、PESV组88.4%、ADM+PESV（H）组53.9%、ADM+PESV（M）组78.0%、ADM+PESV（L）组78.7%;半定量RT-PCR检测MDR1 mRNA的表达：PESV组＞ADM+PESV（L）组＞ADM+PESV（M）组＞ADM+PESV（H）组＞ADM组;免疫组织化学检测ALDH,显示灰度值ADM组＞PESV组＞ADM+PESV（H）组＞ADM+PESV（M）组＞ADM+PESV（L）组;Western blot检测PI3K分子与Elisa检测NF-κB因子结果一致,在ADM组、PESV组表达上调,在ADM+PESV组中表达下调,下调强度与PESV剂量呈正相关。结论 PESV具有下调白血病干细胞膜上P-gp,细胞质内ALDH、PI3K及细胞核中MDR1、NF-κB的表达水平,增强了白血病K562/A02干细胞在体内对ADM的敏感度,逆转其多药耐药特性。     

人类表皮生长因子受体-2高亲和力模拟肽对卵巢癌细胞SKOV3生长的抑制作用

 目的　研究人类表皮生长因子受体-2（Her-2）高亲和力模拟肽（YCFPDEEGACY）对卵巢癌细胞株SKOV3增殖的影响。方法　化学合成模拟多肽,用MTT法检测合成多肽对SKOV3细胞株体外生长的抑制作用。结果　含模拟肽成分组与阴性对照组比较（t≥2.896,P＜0.05）;单纯EGF组与相应EGF和模拟肽共同作用组比较（t≥3.355,P＜0.05）,显示模拟肽对Her-2高表达的卵巢癌细胞株SKOV3的体外生长有明显抑制作用,对Her-2低表达的乳腺癌细胞株MDA-MB-231无明显抑制作用。结论　Her-2高亲和力模拟多肽可与Her-2高表达的卵巢癌细胞特异结合并能明显抑制其在体外的生长,为卵巢癌靶向生物治疗提供依据。     

The stability and dissolution properties of solid glucagon/γ-cyclodextrin powder

In the present study, the solid-state stability and the dissolution of glucagon/γ-cyclodextrin and glucagon/lactose powders were evaluated. Freeze-dried powders were stored at an increased temperature and/or humidity for up to 39 weeks. Pre-weighed samples were withdrawn at pre-determined intervals and analyzed with HPLC-UV (HPLC = high performance liquid chromatography, UV = ultraviolet), HPLC-ESI-MS (ESI-MS = electrospray ionization mass spectrometry), SEC (size-exclusion chromatography), turbidity measurements and solid-state FTIR (Fourier Transform Infrared Spectroscopy). Dissolution of glucagon was evaluated at pH 2.5, 5.0 and 7.0. In addition, before storage, proton rotating-frame relaxation experiments of solid glucagon/γ-cyclodextrin powder were conducted with CPMAS (¹³C cross-polarization magic-angle spinning) NMR (nuclear magnetic resonance) spectroscopy. In the solid state, glucagon was degraded via oxidation and aggregation and in the presence of lactose via the Maillard reaction. The solid-state stability of glucagon/γ-cyclodextrin powder was better than that of glucagon/lactose powder. In addition, γ-cyclodextrin improved the dissolution of glucagon at pH 5.0 and 7.0 and delayed the aggregation of glucagon after its dissolution at pH 2.5, 5.0 and 7.0. There was no marked difference between the proton rotating-frame relaxation times of pure glucagon and γ-cyclodextrin, and thus, the presence of inclusion complexes in the solid state could not be ascertained by CPMAS NMR. In conclusion, when compared to glucagon/lactose powder, glucagon/γ-cyclodextrin powder exhibited better solid-state stability and more favorable dissolution properties.     

Ghrelin reactive autoantibodies in restrictive anorexia nervosa

Objective

Subjects with restrictive anorexia nervosa (AN) display increased basal plasma levels of ghrelin that normalize after refeeding. The mechanism responsible for increased ghrelin levels in AN is unknown. We studied if changes of ghrelin reactive autoantibodies (autoAbs) could explain elevated plasma ghrelin in AN.

Methods

Plasma levels of autoAbs reactive with ghrelin and des-acyl ghrelin were measured by enzyme-linked immunosorbent assay in subjects with AN before and 1 mo after hospitalization (refeeding) and compared with healthy controls and with plasma levels of ghrelin peptides.

Results

Decreased levels of immunoglobulin (Ig) G, IgM, and IgA classes of autoAbs reacting with acyl ghrelin were found in patients with AN. Addition of des-acyl ghrelin but not of acyl ghrelin peptides at 10⁻⁸ M to plasma before enzyme-linked immunosorbent assay showed in patients with AN but not in controls high levels of IgG autoAbs reacting with des-acyl ghrelin as a result of dissociation of des-acyl ghrelin autoAbs in immune complexes. Plasma levels of acyl and des-acyl ghrelin peptides correlated negatively with des-acyl ghrelin IgG autoAbs. Body mass index, which improved after refeeding, correlated with an increase of acyl ghrelin IgM autoAbs.

Conclusion

These results show that in patients with AN, ghrelin IgG autoAbs exist mainly as immune complexes with des-acyl ghrelin accompanied by a decrease of a free fraction of these autoAbs binding acylated and des-acyl ghrelin. This decrease of bioavailable ghrelin autoAbs may underlie a long-term elevation of plasma ghrelin levels and the resulting phenomenon of ghrelin resistance in malnourished patients with AN.     

Nanostructured Probes for RNA Detection in Living Cells

The ability to visualize in real-time the expression level and localization of specific RNAs in living cells can offer tremendous opportunities for biological and disease studies. Here we review the recent development of nanostructured oligonucleotide probes for living cell RNA detection, and discuss the biological and engineering issues and challenges of quantifying gene expression in vivo. In particular, we describe methods that use dual FRET (fluorescence resonance energy transfer) or single molecular beacons in combination with peptide-based or membrane-permeabilization-based delivery, to image the relative level, localization, and dynamics of RNA in live cells. Examples of detecting endogenous mRNAs, as well as imaging their subcellular localization and colocalization are given to illustrate the biological applications, and issues in molecular beacon design, probe delivery, and target accessibility are discussed. The nanostructured probes promise to open new and exciting opportunities in sensitive gene detection for a wide range of biological and medical applications.     

Inhibition of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infectivity by peptides analogous to the viral spike protein

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is the cause of an atypical pneumonia that affected Asia, North America and Europe in 2002–2003. The viral spike (S) glycoprotein is responsible for mediating receptor binding and membrane fusion. Recent studies have proposed that the carboxyl terminal portion (S2 subunit) of the S protein is a class I viral fusion protein. The Wimley and White interfacial hydrophobicity scale was used to identify regions within the CoV S2 subunit that may preferentially associate with lipid membranes with the premise that peptides analogous to these regions may function as inhibitors of viral infectivity. Five regions of high interfacial hydrophobicity spanning the length of the S2 subunit of SARS-CoV and murine hepatitis virus (MHV) were identified. Peptides analogous to regions of the N-terminus or the pre-transmembrane domain of the S2 subunit inhibited SARS-CoV plaque formation by 40–70% at concentrations of 15–30 μM. Interestingly, peptides analogous to the SARS-CoV or MHV loop region inhibited viral plaque formation by >80% at similar concentrations. The observed effects were dose-dependent (IC50 values of 2–4 μM) and not a result of peptide-mediated cell cytotoxicity. The antiviral activity of the CoV peptides tested provides an attractive basis for the development of new fusion peptide inhibitors corresponding to regions outside the fusion protein heptad repeat regions.     

Inhibition of HIV-1 virus replication using small soluble Tat peptides

Although the introduction of highly active antiretroviral therapy (HAART) has led to a significant reduction in AIDS-related morbidity and mortality, unfortunately, many patients discontinue their initial HAART regimen, resulting in development of viral resistance. During HIV infection, the viral activator Tat is needed for viral progeny formation, and the basic and core domains of Tat are the most conserved parts of the protein. Here, we show that a Tat 41/44 peptide from the core domain can inhibit HIV-1 gene expression and replication. The peptides are not toxic to cells and target the Cdk2/Cyclin E complex, inhibiting the phosphorylation of serine 5 of RNAPII. Using the Cdk2 X-ray crystallography structure, we found that the low-energy wild-type peptides could bind to the ATP binding pocket, whereas the mutant peptide bound to the Cdk2 interface. Finally, we show that these peptides do not allow loading of the catalytic domain of the cdk/cyclin complex onto the HIV-1 promoter in vivo.     

血浆corin、NEP、BNP与心功能衰竭及左室收缩功能的相关性

目的:探讨血浆corin、脑啡肽酶（NEP）、B型利钠肽（BNP）在慢性心功能衰竭患者病情评估中的临床价值及其与左心室收缩功能的相关性。方法:选取2016年1月-2018年1月在我院心内科就诊的患者为研究对象,根据患者心衰评分及EF值分组:无心衰正常EF组（nEF对照组）,无心衰收缩功能减低组（rEF+nHF,rEF≤50%）,和收缩功能减低心衰组（rEF+dHF,rEF≤50%）,每组32例。采用酶联免疫吸附法检测各组血浆corin、NEP、BNP浓度,并比较分析其变化情况。结果:一般临床资料组间比较无差别（P>0.05）。EF减低组血清corin浓度均明显低于nEF对照组（P <0.05）,而血清NEP、BNP浓度均明显高于nEF对照组（P <0.05）。血浆corin和NEP浓度呈负相关关系。多元线性回归分析血浆corin水平是LVEF的独立风险因素。结论:血浆corin、NEP、BNP水平与心衰患者心脏收缩功能变化有关,监测血浆corin水平有望为心衰的治疗提供新的思路。     

Dissection and identification of regions required to form pseudoparticles by the interaction between the nucleocapsid (N) and membrane (M) proteins of SARS coronavirus

When expressed in mammalian cells, the nucleocapsid (N) and membrane (M) proteins of the severe acute respiratory syndrome coronavirus (SARS-CoV) are sufficient to form pseudoparticles. To identify region(s) of the N molecule required for pseudoparticle formation, we performed biochemical analysis of the interaction of N mutants and M in HEK293 cells. Using a peptide library derived from N, we found that amino acids 101-115 constituted a novel binding site for M. We examined the ability of N mutants to interact with M and form pseudoparticles, and our observations indicated that M bound to NΔ(101-115), N1-150, N151-300, and N301-422, but not to N1-150Δ(101-115). However, pseudoparticles were formed when NΔ(101-115) or N301-422, but not N1-150 or N151-300, were expressed with M in HEK293 cells. These results indicated that the minimum portion of N required for the interaction with M and pseudoparticle formation consists of amino acids 301-422.     

8DSS peptide induced effective dentinal tubule occlusion in vitro

Objective

Eight repetitive nucleotide sequences of aspartate–serine–serine (8DSS) derived from dentin phosphoprotein (DPP) has been proved to be a good remineralization agency. In this study, 8DSS peptide was employed to induce dentinal tubule occlusion.