Small envelope protein E of SARS：cloning,expression, purification, CD determination, and bioinformatics analysis

AIM:To obtain the pure sample of SARS small envelope E protein (SARS E protein), study its properties and analyze its possible functions. METHODS: The plasmid of SARS E protein was constructed by the polymerase chain reaction (PCR), and the protein was expressed in the E coli strain. The secondary structure feature of the protein was determined by circular dichroism (CD) technique. The possible functions of this protein were annotated by bioinformatics methods, and its possible three-dimensional model was constructed by molecular modeling. RESULTS: The pure sample of SARS E protein was obtained. The secondary structure feature derived from CD determination is similar to that from the secondary structure prediction. Bioinformatics analysis indicated that the key residues of SARS E protein were much conserved compared to the E proteins of other coronaviruses. In particular, the primary amino acid sequence of SARS E protien is much more similar to that of murine hepatitis virus(MHV) and other mammal coronaviruses. The transmembrane (TM) segment of the SARS E protein is relatively more conserved in the whole protein than other regions. CONCLUSION: The success of expressing the SARS E protein is a good starting point for investigating the structure and functions of this protein and SARS coronavirus itself as well. The SARS E protein may fold in water solution in a similar way as it in membrane-water mixed environment. It is possible that β-sheet I of the SARS E protein interacts with the membrane surface via hydrogen bonding, this β-sheet may uncoil to a random structure in water solution.     

Rat neuronal nicotinic acetylcholine receptors containing α7 subunit： pharmacological properties of ligand binding and function

Aim： To compare pharmacological properties of heterologously expressed homomeric α7 nicotinic acetylcholine receptors （α7 nAChRs） with those of native nAChRs containing α7 subunit （α7＊ nAChRs） in rat hippocampus and cerebral cortex. Methods： We established a stably transfected HEK-293 cell line that expresses homomeric rat α7 nAChRs. We studies ligand binding profiles and functional properties of nAChRs expressed in this cell line and native rat α7＊ nAChRs in rat hippocampus and cerebral cortex. We used [125Ⅰ]-α-bungarotoxin to compare ligand binding profiles in these cells with those in rat hippocampus and cerebral cortex. The functional properties of the α7 nAChRs expressed in this cell line were studied using whole-cell current recording. Results： The newly established cell line, KXα7R1, expresses homomeric α7 nAChRs that bind [125Ⅰ]-α-bungarotoxin with a Kd value of 0.38±0.06 nmol/L, similar to Kd values of native rat α7＊nAChRs from hippocampus （Kd=0.28±0.03 nmol/L） and cerebral cortex （Kd=0.33±0.05 nmol/L）. Using whole-cell current recording, the homomeric α7 nAChRs expressed in the cells were activated by acetylcholine and （-）-nicotine with EC50 values of 280±19μmol/L and 180±40μmol/L, respectively. The acetylcholine activated currents were potently blocked by two selective antagonists of α7 nAChRs, a-bungarotoxin （IC50=19±2 nmol/L） and methyllycaconitine （IC50=100±10 pmol/L）. A comparative study of ligand binding profiles, using 13 nicotinic ligands, showed many similarities between the homomeric α7 nAChRs and native α7＊receptors in rat brain, but it also revealed several notable differences. Conclusion： This newly established stable cell line should be very useful for studying the properties of homomeric α7 nAChRs and comparing these properties to native α7＊ nAChRs.     

Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors

Cysteine-rich peptides from the venom of cone snails （Conus） target a wide variety of different ion channels. One family of conopeptides, the α-conotoxins, specifically target different isoforms of nicotinic acetylcholine receptors （nAChRs） found both in the neuromuscular junction and central nervous system. This family is further divided into subfamilies based on the number of amino acids between cysteine residues. The exquisite subtype selectivity of certain α-conotoxins has been key to the characterization of native nAChR isoforms involved in modulation of neurotransmitter release, the pathophysiology of Parkinson＇s disease and nociception. Structure/function characterization of α-conotoxins has led to the development of analogs with improved potency and/or subtype selectivity. Cyclization of the backbone structure and addition of lipophilic moieties has led to improved stability and bioavailability of α-conotoxins, thus paving the way for orally available therapeutics. The recent advances in phylogeny, exogenomics and molecular modeling promises the discovery of an even greater number of α-conotoxins and analogs with improved selectivity for specific subtypes of nAChRs.     

Loss of C-terminal α-helix decreased SDF-1α-mediated signaling and chemotaxis without influencing CXCR4 internalization

AIM: To investigate the possibility that a novel s-helix-defective mutant of stromal cell-derived factor-1α(SDF-1α) (SDF-1/54R) acts as an antagonist of CXC chemokine receptor 4 (CXCR4). METHODS: According to the genetic sequence of natural SDF-1α, a recombinant α-helix-defective mutant of SDF-1α was designed and some biologic characteristics of this mutant were demonstrated. The migration of Jurkat cells was assessed with chemotactic assay. ERK phosphorylation was analyzed by Western blot with a specific anti-phospho-ERK1/2 antibody.Intracellular calcium influx was examined by flow cytometer with a calcium indicator dye Fluo-3AM. The CXCR4 on the cell surface was detected by flow cytometer with a PE conjoined anti-human CXCR4 antibody. RESULTS：Compared with native SDF-1α, SDF-1/54R displayed apparent decrease in chemotactic ability, ERK1/2 activation,and intracellular calcium influx in Jurkat cells. However, the binding to CXCR4 and inducing CXCR4 internalization of SDF-1/54R did not change outstandingly. Moreover, a competitive inhibitory effect of SDF-1/54R on the migration of Jurkat cells induced by native SDF-1α was confirmed. CONCLUSION: α-helix-defective mutant of SDF-1α, SDF-1/54R that remained both the N-terminus and the central β-sheet region, decreased SDF-1α-mediated signaling and chemotaxis but did not influence CXCR4 internalization, which suggested that SDF-1/54R might be developed as an anti-CHIV inhibitor with high biological potency and low side-effect.     

Identification of probable genomic packaging signal sequence from SARS—CoV genome by bioinformatics analysis

AIM:To predict the probable genomic packaging signal of SARS-CoV by bioinformatics analysis. The derived packaging signal may be used to design antisense RNA and RNA interfere (RANi) drugs treating SARS. methods: Based on the studies about the genomic packaging signals of MHV and BCoV, especially the information about primary and secondary structures, the putative genomic packaging signal of SARS_CoV were analyzed by using bioinformatic tools. Multi-alignment for the genomic sequences was performed among SARS-CoV,MHV,BCoV, PEDV and HCoV 229E. Secondary structures of RNA sequences were also predicted for the identification fo the possible genomic packaging signals. Meanwhile, the N and M proteins of all five viruses were analyzed to study the evolutionary relationship with genomic packaging signals. RESULTS: The putative genomic packaging signal of SARS-CoV locates at the 3′ end of ORF1b near that of MHV and BCoV, where is the most variable region of this gene. The RNA secondary structure of SARS-CoV genomic packaging signal is very similar to that of MHV and BCoV. The same result was also obtained in studying the genomic packaging signals of PEDV and HCoV 229E. Further more, the genomic sequence multi-alignment indicated that the locations of packaging signals of SARS-CoV, PEDV, and HCoV overlaped each other. It seems that the mutation rate of packaging signal sequences is much higher than the N protein, while only subtle variations for the M protein. CONCLUSIONS: The probable genomic packaging signal of SARS-CoV is analogous to that of MHV and BCoV, with the corresponding secondary RNA structure locating at the similar region of ORF1b. The positions where genomic packaging signals exist have suffered rounds of mutations, which may influence the primary structures of the N and M proteins consequently.     

Synthesis and bioassay of β-（1,4）-D-mannans as potential agents against Alzheimer＇s disease

Aim： Oligomannurarate 971 derived from a marine plant has shown neuroprotective effects. In this study we synthesized a series of truncated derivatives of the oligosaccharide, and investigated the effect of these derivatives against Aβ peptide toxicity in vitro. Methods： The sulfoxide method was applied to synthesize the derivatives. SH-SY5Y human neuroblastoma cells were treated with Aβ1-40 （2 pmol/L）, and the cell viability was detected using a CCK8 assay. Results： A series of β-（1,4）-D-mannosyl oligosaccharide, ranging from the disaccharide to the hexasaccharide, were synthesized. Addition of 10 pmol/L β-（1,4）-D-mannobiose 6, β-（1,4）-D-mannotriose 9 or β-（1,4）-D-mannotetraose 12 in SH-SY5Y cells significantly attenuated Aβ1-40-induced toxicity. The efficacies were similar to those caused by 10 pmol/L oligomannurarate 971 or alzhemed. Other oligosaccharides including oligomaltoses and oligocelluloses were less active. Conclusion： Synthetic homogeneous short chain β-（1,4）-D-mannans shows neuroprotective effect against Aβ peptide toxicity similar to that of heterogeneous oligomannurarate 971 and alzhemed.     

Phase Ⅱ metabolites of etofesalamide in filamentous fungi

Aim: To study phase Ⅱ metabolites of etofesalamide in filamentous fungi. Methods: Seven fungi were screened to transform etofesalamide. The metabolites of etofesalamide were assayed using liquid chromatography coupled to mass spectrometry. The major metabolite was subject to enzymatic hydrolysis to confirm its structure. Results: Etofesalamide was converted into two phase Ⅱ metabolites: glucoside and riboside conjugates. Glucoside conjugate was the major product with a yield greater than 90%; no phase Ⅰ metabolites were detected. Conclusion: Glucoside and riboside conjugations of etofesalamide in filamentous fungi differ from the phase Ⅱ metabolism of glucuronidation in mammals.     

Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

包裹在脂质体中胰岛素的二级结构

目的　以单纯胰岛素为对照 ,采用傅立叶红外转换光谱 (FourierTransformInfraredFTIR)研究包裹在脂质体内部胰岛素二级结构的变化。方法　分别对单纯胰岛素、胰岛素与空白脂质体混合物 (样品I)及包裹胰岛素的脂质体 (样品II)样品进行FTIR测定。结果　与单纯胰岛素相比 ,样品Ⅰ和Ⅱ与胰岛素的FTIR谱图形状基本一致 ,仅其中的α -螺旋略有下降 (由36 %分别到 32 %～ 31% ) ,β 折叠略有增加 (由 4 8%分别到 5 3%～ 5 1% )。样品I同样品II相比 ,胰岛素的二级结构无明显差别(α 螺旋分别为 32 %～ 31% ,β 折叠分别为 5 3%～ 5 1% )。说明包裹在脂质体内的胰岛素未与脂质体的膜发生插膜作用 ,所产生与单纯胰岛素二级结构之间微小差别的原因是由于部分胰岛素铺展在脂质体膜表面所致的。结论　FTIR测定被载体包裹的蛋白多肽药物的二级结构 ,具有快速直接和非破坏性的特点。经FTIR测定 ,包裹在脂质体内的胰岛素与单纯胰岛素的二级结构相比无明显的变化 ,仍保持原胰岛素的二级结构     

口服胰岛素脂质体吸收的细胞学机制

目的:在细胞水平研究胰岛素脂质体的口服吸收机制.方法:以Caco-2模型为小肠上皮细胞模型.在不同浓度下进行通透实验,研究动力学机制;用ELISA方法评价小肠黏膜胞内途径吸收药物的能力;用EDTA、维拉帕米处理细胞,研究胞旁途径,以及P-糖蛋白的外排作用.结果:脂质体可以促进胰岛素的吸收;药物的吸收机制不是简单的被动扩散;药物可以从胞内途径、胞外途径通透;维拉帕米对药物的外排作用不显著;药物作用、通透作用对Caco-2细胞单层完整性的改变是暂时的.结论:脂质体包裹的胰岛素可以通过胞内途径、胞外途径吸收.     

正常大鼠静脉注射PEG包裹胰岛素脂质体的降血糖作用

目的以胰岛素为模型药物,评价PEG包裹胰岛素脂质体的降血糖药效学作用.方法采用逆相蒸发法制备PEG包裹的胰岛素脂质体.正常Wistar大鼠分别静脉注射给予胰岛素溶液、胰岛素脂质体及PEG包裹的胰岛素脂质体.采用葡萄糖还原酶法测定血清中的血糖浓度.以梯形法计算血糖-时间曲线上面积(AAC),采用胰岛素溶液的AAC为对照,分别计算胰岛素脂质体和PEG包裹的胰岛素脂质体的药理相对生物利用度.结果 PEG包裹的胰岛素脂质体的包封率为18.33%,平均粒径为58.4 nm.静脉注射给予胰岛素溶液、胰岛素脂质体和PEG包裹的胰岛素脂质体后,其血糖降低的最低百分率(Cmin%)分别为:25.26±5.75%,33.92±12.42% 和42.39±10.5%;达到最低百分率的时间(Tmin)分别为:0.7±0.3,1.2±0.4和2.3±0.7 h.胰岛素脂质体和PEG包裹的胰岛素脂质体的药理相对生物利用度分别为:98.03%和99.70%.结论 PEG包裹的胰岛素脂质体具有相对的缓释作用,降血糖作用更为明显.     

包裹在脂质体内部胰岛素的二级结构

目的以单纯胰岛素为对照，采用傅立叶红外光谱（Fourier transform infrared， FTIR）研究包裹在脂质体内部胰岛素二级结构的变化。方法分别对单纯胰岛素、胰岛素与空白脂质体混合物（样品I）及包裹胰岛素的脂质体（II）样品进行测定。结果样品I和样品II中胰岛素的FTIR谱图形状与单纯胰岛素相比基本一致，仅α-螺旋百分比略有下降（由36.09%分别下降到31.68%和31.45%），β-折叠百分比略有增加（由47.83%分别增加到53.29%和51.36%）。样品I和样品II中胰岛素的二级结构无明显差别（α-螺旋百分比为31.68%和31.45%，β-折叠百分比为53.29%和51.36%）。结论包裹在脂质体内部胰岛素的二级结构与单纯胰岛素相比无明显变化。     

纳米脂质体包裹胰岛素经Caco-2细胞转运的研究

目的　研究脂质体包裹胰岛素、壳聚糖包覆脂质体胰岛素对胰岛素透过Caco 2细胞的促进作用。方法　用培养于Transwell上的Caco 2细胞作为通透模型研究胰岛素的通透促进作用;用ELISA方法检测细胞裂解液中胰岛素的含量;用荧光标记的方法研究药物的通透途径。结果　脂质体,特别是壳聚糖均可以增加胰岛素的通透量。脂质体和壳聚糖可打开细胞间连接,但是生物大分子主要通过跨细胞转运进行。结论　壳聚糖包覆脂质体胰岛素促进胰岛素的跨细胞转运主要通过胞内途径进行。     

胰岛素脂质体的制备及在电致孔下的经皮渗透

采用反相蒸发法制备了平均粒径122.7nm的胰岛素脂质体,并考察了电致孔经皮转运情况.结果表明,在电致孔条件下,胰岛素脂质体2h累积渗透量是载药脂质体被动扩散的1倍;是原药及其与空白脂质体混合物的2～3倍.     

Effect of freeze-drying,cryoprotectants and storage conditions on the stability of secondary structure of insulin-loaded solid lipid nanoparticles

This study aims to monitor the secondary structure behaviour of insulin when it is encapsulated into solid lipid nanoparticles (SLN), under the influence of several critical processing parameters. Insulin was used as a therapeutic protein model. Physicochemical properties of insulin-loaded SLN (Ins-SLN) were assessed, with special focus on the insulin secondary structure after its encapsulation into SLN and after freeze-drying using different cryoprotectants (glucose, fructose and sorbitol). Additionally, a 6-month stability study was performed to evaluate the maintenance of insulin secondary structure over time at different storage conditions (4 °C/60% RH, 25 °C/60% RH, 40 °C/75% RH).     

胰岛素与脂质体的相互作用

目的研究胰岛素与脂质体的相互作用。方法用荧光扫描、荧光淬灭、HPLC测定及微量量热方法,对胰岛素脂质体相互作用样品和超速离心、凝胶柱分离样品进行研究。结果胰岛素与脂质体相互作用后,胰岛素的荧光发射峰未发生蓝移,仅强度有所增加,荧光淬灭实验结果与胰岛素溶液基本相同,K_sv之比分别为0.9及0.81,微量量热实验表明为非共价键结合。分离后的样品经HPLC测定,胰岛素与脂质体的结合率只有0.2%,说明胰岛素与脂质体作用属弱吸附方式,未发生插膜(镶嵌);结合量小且强度低。结论胰岛素与脂质体相互作用的数量及程度均较弱,属于弱吸附的范畴。     

Conformational mapping of the N‐terminal segment of surfactant protein B in lipid using 13C‐enhanced Fourier transform infrared spectroscopy

Abstract: Synthetic peptides based on the N‐terminal domain of human surfactant protein B (SP‐B_1?25; 25 amino acid residues; NH₂‐FPIPLPYCWLCRALIKRIQAMIPKG) retain important lung activities of the full‐length, 79‐residue protein. Here, we used physical techniques to examine the secondary conformation of SP‐B_1?25 in aqueous, lipid and structure‐promoting environments. Circular dichroism and conventional, ¹²C‐Fourier transform infrared (FTIR) spectroscopy each indicated a predominateα‐helical conformation for SP‐B_1?25 in phosphate‐buffered saline, liposomes of 1‐palmitoyl‐2‐oleoyl phosphatidylglycerol and the structure‐promoting solvent hexafluoroisopropanol; FTIR spectra also showed significant β‐ and random conformations for peptide in these three environments. In further experiments designed to map secondary structure to specific residues, isotope‐enhanced FTIR spectroscopy was performed with 1‐palmitoyl‐2‐oleoyl phosphatidylglycerol liposomes and a suite of SP‐B_1?25 peptides labeled with ¹³C‐carbonyl groups at either single or multiple sites. Combining these ¹³C‐enhanced FTIR results with energy minimizations and molecular simulations indicated the following model for SP‐B_1?25 in 1‐palmitoyl‐2‐oleoyl phosphatidylglycerol: β‐sheet (residues 1–6), α‐helix (residues 8–22) and random (residues 23–25) conformations. Analogous structural motifs are observed in the corresponding homologous N‐terminal regions of several proteins that also share the ‘saposin‐like’ (i.e. 5‐helix bundle) folding pattern of full‐length, human SP‐B. In future studies, ¹³C‐enhanced FTIR spectroscopy and energy minimizations may be of general use in defining backbone conformations at amino acid resolution, particularly for peptides or proteins in membrane
environments.     

Secondary structure prediction of 11 mammalian growth hormones

The secondary structure of 11 mammalian growth hormones has been predicted by combining five different methods. Three long helical regions located around residues 20, 120, and 170 constitute the most prominent common feature in the species studied. The strong amphiphilic character of these helices suggests that they can play an important role in protein folding or stability.