Isomerization and Racemization of Menthols

Menthols were converted to Δ³-menthone enol acetate (VII) via menthones having only one asymmetric carbon atom. It was shown that the optical rotation of menthone enol acetate was proportional to the optical purity of starting menthols. Optical purity of original menthol could, therefore, be determined by optical rotation of menthone enol acetate derived from.     

Isomerization and Racemization of Menthols

The isomerization and racemization of menthol isomers have been investigated with copper chromite and Raney nickel catalyst or sodium mentholates. The equilibrium concentration has been found to correspond to 55~56% of menthol with the catalytic isomerization, whereas to 74~75% of menthol with sodium mentholates. From these data, the free energy difference between equatorial and axial group in menthols is calculated to be about 0.5~0.6 Kcal./mole for hydroxyl and 1.4~1.5 Kcal./mole for methyl at 200°C.     

重组猪乳铁蛋白N端的高效表达及抑菌活性检测

为获得表达猪乳铁蛋白基因的重组菌株,并检测其表达的重组猪乳铁蛋白抑菌活性,应用RT-PCR方法从泌乳3d后母猪乳腺组织中扩增了猪乳铁蛋白N端1077bp的PLF-N基因片段,与GenBank上发表的4株猪乳铁蛋白基因序列相比,核苷酸同源性均达到99%以上。为了得到高表达量的PLF-N基因,以扩增的PLF-N片段为参考模板,经过密码子优化,全基因合成了编码猪乳铁蛋白N端的基因PLF-NS。将其定向插入到原核表达载体pET-30b中,转化大肠杆菌BL21,获得了表达PLF-NS的重组菌pET-PLF-NS/BL21;经IPTG诱导,并对表达条件进行优化,以及通过SDS-PAGE和Western blotting分析均表明猪乳铁蛋白得到了正确表达,其产物分子量约为42kDa,最优表达条件下蛋白表达量占菌体总蛋白的32%,表达产物以包涵体形式存在。包涵体经裂解、纯化、复性处理后纯度达到98%。用琼脂孔穴扩散抑菌法检测表明重组猪乳铁蛋白具有明显的抑菌作用。表明通过基因优化对表达量低的基因进行改造使之高效表达,是一种提高表达效率的有效手段。     

江蓠内生真菌NSS1蛋白活性的初步研究

为促进江蓠内生真菌NSS1抗菌蛋白的应用研究,利用硫酸铵沉淀法制备抗菌蛋白,最佳硫酸铵饱和度为65%。采用滤纸片法检测其对细菌指示菌的抑菌活性,抗菌粗蛋白均能抑制五种细菌指示菌的生长,当浓度达到750 μg/mL时,抑制效果最好。采用邻苯三酚自氧化法测定蛋白对超氧阴离子自由基(O-2)的清除作用,MTT法检测蛋白对肿瘤细胞的影响。抗菌蛋白在100℃以下,pH中性时抑菌活性稳定,对紫外线照射不敏感,丙三醇、甲醇和胰蛋白酶对抗菌蛋白的抑菌活性没有影响。不同浓度的蛋白液对超氧阴离子均有清除效果。当蛋白浓度达到360 μg/mL时对肿瘤细胞的抑瘤作用最强。上述结果显示抗菌蛋白具有较强的抑菌活性、抗氧化活性和抗肿瘤活性。     

β-防御素130在大肠杆菌中的串联表达、纯化及生物活性分析

为了研究抗菌肽β-防御素130的生物学活性和实现大规模制备,通过改良其分子结构,构建表达载体pET28a-3×β-defensin130,利用大肠杆菌BL21 (DE3)作为宿主细胞诱导表达后为水溶性蛋白。对纯化后抗菌肽进行抑菌实验、稳定性实验、MTT实验和溶血性实验确定其生物活性。最终成功制备出25 kDa的重组蛋白,对金黄色葡萄球菌(ATCC25923)(45μg/mL)和单增李斯特菌(ATCC221633)(80μg/mL)等革兰氏阴性和阳性菌都表现出极强的抗菌活性,且其抗菌活性不受温度、pH值和蛋白酶消化等影响,MTT细胞毒性实验显示其对HEK293细胞无毒性且对兔源红细胞具有极低的溶血性。这将为新型抗菌肽的开发提供理论基础并推动抗生素替代产业快速发展。     

A non-synonymous mutation in a conserved site of the MTTP gene is strongly associated with protein activity and fatty acid profile in pigs

Despite the economic interest of the fatty acid profile in pigs, no gene has been convincingly associated with this trait so far. Here, the porcine microsomal triglyceride transfer protein ( MTTP ) gene, which plays a crucial role in the assembly of nascent lipoproteins, has been analysed as a positional candidate gene for a QTL affecting the fatty acid composition that was previously identified on chromosome 8 in an Iberian by Landrace F₂ cross. By resequencing a panel of different breeds, a non-synonymous polymorphism in a conserved residue of the lipid transfer domain of MTTP was identified. Association analyses with this polymorphism showed a strong association with the fatty acid composition of porcine fat, much stronger than the QTL effect, in the F₂ cross and in a synthetic Sino-European line. In addition, in vitro activity assays in liver protein extracts have shown that this mutation is also associated with the lipid transfer activity of the MTTP protein ( P  <   0.1). These results suggest that the detected polymorphism is a potential causal factor of the fatty acid composition QTL. There appears to be an interaction between the porcine MTTP genotype and the type of fat source in the pig diet, which would agree with the previous results on the biology of MTTP biology.     

Synthesis of (±)-Muscone from Ethyl 6-Methyl-8-oxopentadecanedioate

(±)-Muscone (3-methylcyclopentadecanone) (8) was synthesized from ethyl 6-methyl-8-oxopentadecanedioate (1) in a 31.9% over-all yield. Ethylene ketal (2) of 1 was cyclized to the acyloin mixture (3) by the acyloin condensation. Reduction of 3 gave 9,9-ethylenedioxy-7-methylcyclopentadecane-1,2-diol (4) which afforded 1,2-ditosyloxy derivative (5). By detosylation according to the Tipson-Cohen procedure, 5 was converted to 9,9-ethylenedioxy-7-methylcyclopentadec-1-ene (6) which was hydrogenated to 8.     

重组猪肺表面活性蛋白A在体外可抑制PRRSV感染宿主细胞

【目的】研究重组猪肺表面活性蛋白A(SP-A)在体外对猪繁殖与呼吸综合征病毒(PRRSV)感染的抑制作用。【方法】采用PCR方法从含有猪SP-A基因的质粒中扩增SP-A基因,并将其插入到含有人CD5信号肽序列的真核表达载体pcDNA3.1A-CD5中,构建成SP-A基因的真核分泌型表达载体pcDNA-CD5-SPA/MH。将重组表达载体通过磷酸钙介导转染HEK293T细胞进行瞬时表达,通过Western blot方法鉴定表达产物,采用Ni-NTA琼脂糖凝胶亲和层析法从培养基中分离和纯化重组SP-A蛋白,通过ELISA方法检测SP-A蛋白与PRRSV的结合活性。将SP-A蛋白与PRRSV孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,感染72 h后测定病毒滴度,分析重组SP-A蛋白对PRRSV感染的抑制作用。【结果】结果表明构建的真核表达载体能够介导SP-A基因在HEK293T细胞中进行分泌表达;表达的重组猪SP-A蛋白能够与PRRSV进行剂量依赖性结合;用重组猪SP-A蛋白与PRRSV进行孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,结果显示SP-A处理的PRRSV感染细胞后的病变程度明显低于对照组。感染72 h后,SP-A处理组的PRRSV在MARC-145细胞和猪肺泡巨噬细胞的滴度明显低于SP-A非处理组。【结论】重组猪SP-A在体外对PRRSV的感染有明显的抑制作用,揭示SP-A具有抗PRRSV的活性。     

Photochemical Isomerization of Leaf Aldehyde

Isopimpinellin, bergapten, xanthotoxin, kokusagine, evoxine and japonin were isolated and identified in the leaves of Orixa japonica (Rutaceae) as feeding inhibitors against Spodoptera litura F. (Noctuidae). Isopimpinellin exhibited the most potent activity among above substances at the feeding inhibitory test. The isolated evoxine was the optical antipode of the previously reported one.     

骆驼蓬(Peganum harmala)蛋白质的分布、提取及活性研究

采用不同溶剂分级提取骆驼蓬（Peganum harmala L．）根、茎、叶及种子中的蛋白质,结果表明总蛋白以种子中的蛋白含量最高,而营养器官以叶中的蛋白含量较高。当年种子总蛋白含量显著高于贮藏种子。不同溶剂分级提取的当年生种子、根、茎和叶中各组分,以碱提组分蛋白含量最高。用含不同盐离子浓度的缓冲液和不同pH值的广泛缓冲液提取骆驼蓬种子的蛋白质,结果表明优化后的缓冲液条件为含0．2mol／LNaCl,pH：7．0～8．0的5mmol／LPBS缓冲液。硫酸铵沉淀法获得的骆驼蓬种子中的蛋白粗提物（1．2mg／mL）对供试真菌交格链孢菌（Alternaria alternata）、指状青霉菌（Penicillium degitatum）、灰霉病菌（Botrytis cinerea）、稻瘟病菌（Magnaporthe grisea）和意大利青霉菌（Penicillium italicum）等5种植物病原真菌均有抑菌作用,其中对意大利青霉菌和交格链孢菌表现出较好的抑菌活性,抑菌环直径分别为19．50和18．50mm。对供试细菌表皮葡萄球菌（Staphylococcus epidermidis）、粪肠球菌（Enterococcus faecalis）、臭鼻克雷伯菌（Klebsiella penumoniae）、福氏志贺氏菌（Shigella flexneri）、金黄色葡萄球菌（Staphylococcus aureua）和鲍曼不动杆菌（Acinetobacter baumannii）等6种病原细菌也有抑菌作用,其中对臭鼻克雷伯菌、福氏志贺氏菌、表皮葡萄球菌等病原细菌等有较好的抑制作用,抑菌圈直径分别10．20、10．10和9．30mm。     

Isolation of the lipid-transporting protein Ns-LTP1 from seeds of the garden fennel flower (<Emphasis Type="Italic">Nigella sativa</Emphasis>)

A novel lipid-transporting protein (Ns-LTP1) has been isolated from seeds of the garden fennel flower Nigella sativa. The molecular mass, N-terminal amino acid sequence, and amino acid composition of the protein have been determined. Ns-LTP1 has a molecular mass of 9602 Da and contains eight cysteine residues which form four disulfide bridges. The protein is capable of suppressing the development of some phytopathogenic fungi and oomycetes.     

猪I型与II型干扰素的克隆、表达及抗病毒活性比较

干扰素(IFN)是由多种细胞受病毒感染或其他生物诱导剂刺激而产生的天然蛋白质,主要功能为抗病毒增殖、调节免疫反应和激活免疫细胞等。本研究克隆并测序了猪干扰素(PoIFN)α、γ、αγ及ω基因。构建原核表达载体pET-His/PoIFN-α、pET-His/PoIFN-γ、pET-His/PoIFN-αγ和pET-His/PoIFN-ω,转化大肠杆菌Rosetta(DE3)进行表达,经纯化、复性得到具有生物学活性的蛋白。用细胞病变抑制法在Marc-145/PRRSV、Marc-145/VSV、PK-15/VSV、Vero/VSV、MDBK/VSV系统上进行抗病毒活性测定,结果表明猪α和αγ融合干扰素有较为显著的抗病毒活性,抗PRRSV活性高达108U/mg;猪γ干扰素活性效价约为α干扰素的1/2到1/3;猪ω干扰素几乎未检测到抗病毒活性,需进一步验证。本研究对干扰素在抗病毒、提高机体免疫方面的应用提供了理论依据。     

猪白细胞介素18基因的克隆、表达及生物学活性检测

通过RT-PCR方法直接从猪脾脏淋巴细胞中扩增出猪白细胞介素18(pIL-18)成熟蛋白基因的cDNA, 克隆到pGEM-T载体, 构建重组质粒pGEM-T-IL18, 转化E.coli JM109感受态细胞, 取PCR和酶切鉴定为阳性的重组质粒进行序列测定。测序结果表明, pIL-18成熟蛋白基因核苷酸长度为474 bp, 编码157个氨基酸。将其克隆到表达载体pGEX6P-1中, 构建重组质粒pGEX-IL18, 转化E.coli BL21感受态细胞, 用IPTG诱导表达。重组菌菌体裂解物SDS-PAGE可检测到相对分子质量为45 kD的重组蛋白, 占菌体总蛋白的28%左右, 以包涵体形式存在。对包涵体进行洗涤, 用MTT法测定表明, 重组蛋白能明显刺激猪脾脏T淋巴细胞增殖反应的活性。     

β-发卡抗菌肽的全新设计及其生物学活性

通过缬氨酸和精氨酸的交替连接形成β-发卡结构的两条侧链,D-脯氨酸和甘氨酸形成β-转角单元以及侧链末端的两个半胱氨酸连接形成一个二硫键,来设计得到全新的由16残基构成的β-发卡抗菌肽VR。对设计得到的抗菌肽VR的生物学活性进行了检测,主要测定了新型β-发卡抗菌肽VR的最小杀菌浓度、对红细胞的溶血活性、杀菌动力学和盐敏感性。结果发现,VR和蜂毒素具有相似的杀菌活性,而溶血活性远低于蜂毒素,这表明VR比蜂毒素具有更高的细胞选择性。在NaCl的浓度低于100 mmol/L时,VR的杀菌活性没有受到影响;在NaCl的浓度为100 mmol/L时,VR具有50%的杀菌活性。综上可见,VR具有较优异的生物学活性,拥有成为抗生素替代物的发展潜力。     

Racemization of Individual Aspartate Residues in Human Myelin Basic Protein

Human myelin basic protein (MBP), a long-lived brain protein, undergoes gradual racemization of its amino acids, primarily aspartic acid and serine. Purified protein was treated at neutral pH with trypsin to yield peptides that were separated by HPLC using a C18 column. Twenty-nine peptides were isolated and analyzed for amino acid composition and aspartate racemization. Each aspartate and asparagine in the protein was racemized to a different extent, ranging from 2.2 to 17.1% D isomer. When the racemization was examined in terms of the beta-structure model of MBP, a correlation was observed in which six aspartate/asparagine residues assumed to be associated with myelin membrane lipids showed little racemization (2.2-4.9% D isomer), whereas five other aspartate residues were more highly racemized (9.9-17.1% D isomer). Although the observed aspartate racemization may be related to steric hindrance by neighboring residues and/or the protein secondary structure, interaction of aspartates with membrane lipids may also be a major factor. The data are compatible with a model in which each MBP molecule interacts with adjacent cytoplasmic layers of myelin membrane through a beta-sheet on one surface and loops and helices on the other surface, thereby stabilizing the myelin multilamellar structure.     

In vitro protein digestion kinetics of protein sources for pigs

In current feed evaluation systems, the nutritional value of protein sources in diets for pigs is based on the ileal digestibility of protein and amino acids, which does not account for the kinetics of protein digestion along the gastrointestinal tract. The objective of the present study was to determine the in vitro protein digestion kinetics of different protein sources (soya bean meal (SBM), wheat gluten (WG), rapeseed meal (RSM), whey powder (WP), dried porcine plasma protein, yellow meal worm larvae and black soldier fly larvae (BSF)). Protein sources were incubated with pepsin at pH 3.5 for 0 to 90 min and subsequently with pancreatin at pH 6.8 for 0 to 210 min at 39°C. The in vitro protein digestion kinetics were described as the kinetics of nitrogen (N) solubilisation and the release of low molecular weight peptides (LMW) (<500 Da). The N solubilisation rate ranged from 0.025 min⁻¹ for BSF to 0.685 min⁻¹ for WP during the incubation with pepsin, and from 0.027 min⁻¹ for RSM to 0.343 min⁻¹ for WP during the incubation with pancreatin. The release rate of LMW peptides ranged from 0.027 min⁻¹ for WG to 0.093 min⁻¹ for WP during the incubation with pepsin, and from 0.029 min⁻¹ for SBM to 0.385 min⁻¹ for WP. Black soldier fly larvae showed a similar release rate of LMW peptides as WP during the incubation with pancreatin. At the end of the sequential incubation with pepsin (90 min) and pancreatin (210 min), WG and WP showed the highest percentage of N present in LMW peptides relative to total N (78% and 79%, respectively), whereas SBM showed the lowest (35%). In conclusion, protein sources for pig diets show substantial differences in in vitro protein digestion kinetics as measured by the kinetics of N solubilisation and the release of LMW peptides. The rate of release of LMW peptides was not correlated to the rate of N solubilisation for each of the protein sources evaluated.     

猪白细胞介素18基因的克隆、表达及生物学活性检测

通过RT-PCR方法直接从猪脾脏淋巴细胞中扩增出猪白细胞介素18(pIL-18)成熟蛋白基因的cDNA, 克隆到pGEM-T载体, 构建重组质粒pGEM-T-IL18, 转化E.coli JM109感受态细胞, 取PCR和酶切鉴定为阳性的重组质粒进行序列测定。测序结果表明, pIL-18成熟蛋白基因核苷酸长度为474 bp, 编码157个氨基酸。将其克隆到表达载体pGEX6P-1中, 构建重组质粒pGEX-IL18, 转化E.coli BL21感受态细胞, 用IPTG诱导表达。重组菌菌体裂解物SDS-PAGE可检测到相对分子质量为45 kD的重组蛋白, 占菌体总蛋白的28%左右, 以包涵体形式存在。对包涵体进行洗涤, 用MTT法测定表明, 重组蛋白能明显刺激猪脾脏T淋巴细胞增殖反应的活性。     

Production of recombinant bovine lactoferrin N-lobe in insect cells and its antimicrobial activity

Lactoferrin is a multifunctional, iron-binding glycoprotein found in physiological fluids of mammals. In the present study, a gene encoding the N-terminal half (N-lobe) of bovine lactoferrin was cloned and expressed in cultured insect cells using a baculovirus expression system. One mutation was found in the lactoferrin N-lobe gene, but it resulted in no amino acid substitution. The recombinant lactoferrin N-lobe was secreted into the culture medium and partially purified by means of an immobilized heparin column. The recombinant lactoferrin N-lobe secreted was not glycosylated, but it possessed antimicrobial activity toward Escherichia coli O111. The recombinant product synthesized and accumulated in the host cells exhibited greater electrophoretic mobility on SDS-PAGE than the secreted product and showed no potency to inhibit the growth of bacteria. It is thought that the product accumulated intracellularly lacks antimicrobial ability due to its degradation in the host cells or due to disruption of the active conformation.     

Antimicrobial activity of extracts of the lichens <Emphasis Type="Italic">Cladonia furcata,Parmelia caperata,Parmelia pertusa,Hypogymnia physodes</Emphasis> and <Emphasis Type="Italic">Umbilicaria polyphylla</Emphasis>

Antimicrobial features of acetone, methanol and aqueous extracts of lichens of Cladonia furcata, Parmelia caperata, Parmelia pertusa, Hypogymnia physodes and Umbilicaria polyphylla were investigated by two different methods at the same time. Testing of antimicrobial activities of extracts from five species of lichens was performed by disc diffusion test in relation to Gram-positive and Gram-negative bacteria and fungal organisms, and through determination of minimal inhibitory concentration (MIC) by Broth Tube Dilution method. The obtained results indicated that acetone and methanol extracts of all investigated lichens in different concentrations manifested selective antibacterial and antifungal activity. That activity was more evident in relation to Gram-positive, than Gram-negative bacteria and fungal organisms. Acetone and methanol extracts of lichens Parmelia pertusa, Hypogymnia physodes and Umbilicaria polyphylla inhibited the growth of all tested microorganisms, most of all of lichens Cladonia furcata and Parmelia caperata. Although, the methanol extracts were generally the most active against the test organisms, the lowest MIC value was measured for acetone extract of species Cladonia furcata 0.39 mg/mL in relation to bacterium Bacillus subtilis. Aqueous extracts of investigated lichens were inactive against all tested organisms.