利用内标为基础的苹果锈果类病毒RT-PCR检测技术

为开发准确、灵敏的苹果锈果类病毒(Apple scar skin viroid,ASSVd)RT-PCR检测方法,以田间染病苹果组织为试材,提取高质量总RNA为模板,在常规RT-PCR检测体系中引入苹果线粒体nad5基因作为内标基因,对RT-PCR反应体系和程序进行优化,并测定其灵敏度和稳定性,最后利用优化的检测体系确定苹果果实着色期最佳检测部位。结果表明:以RNA提取改良法提取总RNA为模板合成cDNA后,进行PCR扩增,优化后的PCR体系(25μL)中cDNA模板2μL、ASSVd(20pmol/μL)正反向引物各1.0μL,nad5(20pmol/μL)正反向引物各0.1μL;扩增程序中退火温度为60.9℃,循环次数为35次;检测灵敏度为37.5ng新鲜样本;果实着色期,染病植株的幼嫩叶片、1a生枝的韧皮部和木质部、果实及部分种子样本均能检测到病毒,最佳检测部位为幼嫩韧皮部。研究结果可以为ASSVd高效、快速、准确的检测提供可靠方法,并为田间病害诊断及无毒苗木繁育提供依据和借鉴。     

Fast curing: A method to improve postharvest quality of onions in hot climate harvest

Most of the onions (Allium cepa cv. Orlando) grown in southern Israel are treated with maleic hydrazide before storage, and are cold-stored for up to 8 months with minimal losses to rots or sprouting. Nevertheless, in most cases the complete dry outer skin (tunic) cracks and loosens, and tends to fall off during storage. To improve onion postharvest quality, bulbs were harvested at 80–100% green leaf drop (top-down), leaving about 10 cm of neck above the bulb. The early harvest reduced skin cracks in 93% of the bulbs, resulting in a sturdy tunic beneath the muddy outer skin. We applied fast curing (FC) at 30 °C and 98% RH for up to 9 days postharvest: the onion neck became 52% narrower after 6 days, similar to the effect of 5 months of cold storage. FC also changed the color of the treated onion bulbs’ outer skin to a darker reddish brown. FC of onions harvested with a long neck and stored for 290 days reduced weight loss and rot by 30% and 80%, respectively, as compared to non-FC onions. The better onion quality induced by FC was accompanied by an increased number of onion skin layers (from an average of 1.8 to 4) and a higher force needed to tear the tunic (average 4.8 N as compared to 3.5 N in the control). FC compressed the effects of 5 months of cold storage into a few days, since most of the bulb neck and tunic changes measured during FC occurred only after long cold storage without FC. Although emission of the lachrymatory factor was not affected by FC, it markedly increased after 5 months of storage. Histological observation showed that FC keeps the onion tunic and inner fleshy scales intact and ensures postharvest quality, even after 8 months of cold storage. The high temperature used for FC can be reached in hot-climate storage areas with minimal energy investment.     

Antibacterial Activity of Long-Chain Polyunsaturated Fatty Acids against Propionibacterium acnes and Staphylococcus aureus

New compounds are needed to treat acne and superficial infections caused by Propionibacterium acnes and Staphylococcus aureus due to the reduced effectiveness of agents used at present. Long-chain polyunsaturated fatty acids (LC-PUFAs) are attracting attention as potential new topical treatments for Gram-positive infections due to their antimicrobial potency and anti-inflammatory properties. This present study aimed to investigate the antimicrobial effects of six LC-PUFAs against P. acnes and S. aureus to evaluate their potential to treat infections caused by these pathogens. Minimum inhibitory concentrations were determined against P. acnes and S. aureus, and the LC-PUFAs were found to inhibit bacterial growth at 32–1024 mg/L. Generally, P. acnes was more susceptible to the growth inhibitory actions of LC-PUFAs, but these compounds were bactericidal only for S. aureus. This is the first report of antibacterial activity attributed to 15-hydroxyeicosapentaenoic acid (15-OHEPA) and 15-hydroxyeicosatrienoic acid (HETrE), while the anti-P. acnes effects of the six LC-PUFAs used herein are novel observations. During exposure to the LC-PUFAs, S. aureus cells were killed within 15–30 min. Checkerboard assays demonstrated that the LC-PUFAs did not antagonise the antimicrobial potency of clinical agents used presently against P. acnes and S. aureus. However, importantly, synergistic interactions against S. aureus were detected for combinations of benzoyl peroxide with 15-OHEPA, dihomo-γ-linolenic acid (DGLA) and HETrE; and neomycin with 15-OHEPA, DGLA, eicosapentaenoic acid, γ-linolenic acid and HETrE. In conclusion, LC-PUFAs warrant further evaluation as possible new agents to treat skin infections caused by P. acnes and S. aureus, especially in synergistic combinations with antimicrobial agents already used clinically.     

激光诱变对黄皮洋葱须根的生物学效应研究

[目的]对激光诱变黄皮洋葱须根的生物学效应进行研究。[方法]采用CO2激光和He-Ne激光对黄皮洋葱2个品种的湿种子进行辐射,通过随机区组设计,利用生物统计学和生理生化的方法,从个体及生化水平上考查激光诱变黄皮洋葱L1代须根的生物学效应。[结果]经激光诱变处理的黄皮洋葱在须根长度、数目、须根重以及根系活力等方面,表现为不同处理引起的生物学效应差异显著,其中,激光种类不同引起须根长度的变异达到5%显著差异;洋葱品种不同,激光辐照引起的须根变异明显,但各处理间差异均未达到5%显著水平;激光剂量不同,引起须根数的变异达5%显著水平;激光辐射对洋葱根系活力表现为刺激效应。[结论]该研究结果为激光诱变黄皮洋葱育种提供了参考依据。     

The effect of culture on morphology, lipid and fatty acid composition, and polyunsaturated fatty acid metabolism of rainbow trout (Oncorhynchus mykiss) skin cells

Rainbow trout (Oncorhynchus mykiss) skin cell cultures were obtained by trypsinization of the tissue and grown in Leibovitz L-15 medium. Lipid class compositions, and fatty acid profiles of total lipids and individual phospholipid classes were determined at different times of culture. The metabolism of polyunsaturated fatty acids (PUFA) was investigated by incubating primary cultures after 7 and 14 days with [1-¹⁴C]18:2n-6 and [1-¹⁴C-]18:3n-3. The change in morphology between epithelial-like primary cultures and fibroblastic-like secondary subcultures was accompanied by alterations in the lipid composition. Polar lipids became predominant by 14 days in culture. The relative proportions of phosphatidylcholine (PC), the most abundant phospholipid, phosphatidylinositol and cholesterol increased significantly, while sphingomyelin decreased. Saturated fatty acids, 18:1n-9, n-6 and n-9PUFA were more abundant in total lipid in cultures at 14 days and 4 months than in cells initially isolated which contained higher percentages of longer chain monoenes and n-3PUFA. The changes in fatty acid composition with time in culture were observed in all the major phospholipid classes. Rainbow trout skin cells in culture desaturated and elongated both 18:2n-6 and 18:3n-3, with 20:4n-6 and 20:5n-3 being the most abundant products, respectively. PC presented the highest incorporation of radioactivity, especially following incubation with 18:3n-3. Lipid metabolism in general increased with the age of primary cultures, with both the amount of C18 PUFA incorporated and metabolized by desaturation/elongation significantly increased in 14 day cultures compared to 7 day cultures. Product/precursor ratios calculated for both n-6 and n-3 fatty acids showed that, while 6 desaturase activity was increased significantly with cell age, 5 desaturase activity was more affected by the fatty acid series, with 18:3n-3 being more readily transformed to 20:5n-3 than 18:2n-6 to 20:4n-6. Further desaturation of 20:5n-3 to hexaenes was low. Overall, the data suggested that the trout skin cell cultures were more similar to mammalian skin fibroblasts than mammalian epidermal/keratinocyte cultures.     

中国美利奴超细型与哈萨克羊毛囊兴盛期皮肤组织消减cDNA文库的构建

【目的】筛选影响绵羊羊毛性状的重要候选基因,为研究羊毛性状形成的分子机制奠定基础。【方法】利用抑制性消减杂交技术构建中国美利奴超细型和哈萨克羊兴盛期皮肤组织间正、反向消减cDNA文库,并对差异基因进行测序和生物信息学分析;应用半定量RT-PCR和实时荧光定量RT-PCR对部分差异基因进行鉴定。【结果】①从所构建绵羊皮肤组织的正、反向消减cDNA文库中分别获得153和143个ESTs,其中分别包括5和4个未知功能的ESTs,推测可能是新基因;对部分已知功能ESTs进行了GO聚类分析、通路分析和蛋白互作分析,结果显示粗、细毛羊之间存在一定差异;②文库中的3个差异基因KRTAP8-2、Trichohyalin和KRTAP3-2均能在皮肤中特异性地高表达,其中KRTAP8-2和Trichohyalin基因在中国美利奴超细型羊皮肤组织中的表达丰度分别是哈萨克羊的3.45和2.07倍,而KRTAP3-2基因在哈萨克羊皮肤组织表达丰度是中国美利奴超细型羊的1.87倍。【结论】成功构建了中国美利奴超细型和哈萨克羊皮肤组织间抑制性消减cDNA文库,初步筛选出一批可能影响羊毛性状的差异表达ESTs。     

杨树皮类脂对感染鸡传染性贫血病毒鸡外周血T细胞亚群含量的影响

[目的]从细胞免疫学角度探讨杨树皮类脂(PBL)对改善感染鸡传染性贫血病毒(CIAV)鸡的免疫器官的细胞免疫水平的影响。[方法]以人工感染与未感染CIAV的1日龄SPF鸡作为试验模型,分别连续饲喂PBL 7 d,在第7、27、47天采用流式细胞术检测外周血中CD4+和CD8+的含量,并计算CD4+/CD8+。[结果]PBL能够增加健康SPF雏鸡外周血的CD4+T淋巴细胞的数量,提高非特异性细胞免疫功能和体液免疫功能。[结论]杨树皮类脂对CIAV诱导的免疫抑制雏鸡外周血的CD4+和CD8+T细胞水平的降低具有一定抑制作用。     

Characterization of Collagen from Different Discarded Fish Species of the West Coast of the Iberian Peninsula

ABSTRACT

Skin collagen of six discarded fish species was analyzed and compared. Acid soluble collagen (ASC) was extracted; a characteristic sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profile for type I collagen was obtained, except for Chimaera mostrosa. Contents of collagen calculated from HPro (31.85% average) were higher than those determined from ASC extracts (17.75% average), with Galeus spp. being the species with the higher percentage. Amino acid analysis revealed the typical composition of collagen, with very few differences among species. Specific profiles were obtained after protease digestion. Denaturation temperature of ASC correlated well with imino and hydroxyproline contents.

Results demonstrate the feasibility of using the obtained collagens in different industrial applications.     

Spongiotic vesicular dermatitis as a cutaneous reaction pattern in seven horses

Over a 6-year period seven adult horses of different breeds and genders developed multifocal, exudative, oozing dermatitis characterized histologically by epidermal spongiotic vesicles and perivascular eosinophilic, neutrophilic and mixed mononuclear inflammation. Three horses were pruritic. Systemic disease was not noted. Two horses had a history of recurrent urticaria (hives) and one horse had nodules or welt-type lesions that progressed to exudative, oozing lesions. Interepithelial immunoglobulin (Ig)G was detected by avidin-biotin complex-peroxidase staining, but the pattern of staining was more consistent with epithelial oedema than specific IgG deposition associated with pemphigus. The exudative oozing lesions developed under circumstances suggesting that dermal oedema progressed to intracellular and intercellular epidermal oedema, which in turn progressed to the spongiotic vesicular epidermal lesions.     

桑籽皮多酚的提取工艺研究

通过单因素试验,分析了不同浸提剂、浸提剂浓度、料液比、浸提时间、浸提温度5个因素对桑籽皮多酚提取量的影响,通过正交试验,确定了桑籽皮多酚的最佳提取条件为:浸提剂(丙酮)体积分数为70%,料液比1∶35,回流温度45℃,回流时间1.5h。在此提取条件下,提取量达到1.332mg/g干籽皮,为桑籽皮多酚的工业化提取提供科学依据。