Lipidome and proteome of lipid droplets from the methylotrophic yeast Pichia pastoris

Lipid droplets (LD) are the main depot of non-polar lipids in all eukaryotic cells. In the present study we describe isolation and characterization of LD from the industrial yeast Pichia pastoris. We designed and adapted an isolation procedure which allowed us to obtain this subcellular fraction at high purity as judged by quality control using appropriate marker proteins. Components of P. pastoris LD were characterized by conventional biochemical methods of lipid and protein analysis, but also by a lipidome and proteome approach. Our results show several distinct features of LD from P. pastoris especially in comparison to Saccharomyces cerevisiae. P. pastoris LD are characterized by their high preponderance of triacylglycerols over steryl esters in the core of the organelle, the high degree of fatty acid (poly)unsaturation and the high amount of ergosterol precursors. The high phosphatidylinositol to phosphatidylserine of ~ 7.5 ratio on the surface membrane of LD is noteworthy. Proteome analysis revealed equipment of the organelle with a small but typical set of proteins which includes enzymes of sterol biosynthesis, fatty acid activation, phosphatidic acid synthesis and non-polar lipid hydrolysis. These results are the basis for a better understanding of P. pastoris lipid metabolism and lipid storage and may be helpful for manipulating cell biological and/or biotechnological processes in this yeast.     

Intestinal caveolin-1 is important for dietary fatty acid absorption

How dietary fatty acids are absorbed into the enterocyte and transported to the ER is not established. We tested the possibility that caveolin-1 containing lipid rafts and endocytic vesicles were involved. Apical brush border membranes took up 15% of albumin bound ³H-oleate whereas brush border membranes from caveolin-1 KO mice took up only 1%. In brush border membranes, the ³H-oleate was in the detergent resistant fraction of an OptiPrep gradient. On OptiPrep gradients of intestinal cytosol, we also found the ³H-oleate in the detergent resistant fraction, separate from OptiPrep gradients spiked with ³H-oleate or ³H-triacylglycerol. Caveolin-1 immuno-depletion of cytosol removed 91% of absorbed ³H-oleate whereas immuno-depletion using IgG, or anti-caveolin-2 or -3 or anti-clathrin antibodies removed 20%. Electron microscopy showed the presence of caveolin-1 containing vesicles in WT mouse cytosol that were 4 fold increased by feeding intestinal sacs 1 mM oleate. No vesicles were seen in caveolin-1 KO mouse cytosol. Caveolin-1 KO mice gained less weight on a 23% fat diet and had increased fat in their stool compared to WT mice. We conclude that dietary fatty acids are absorbed by caveolae in enterocyte brush border membranes, are endocytosed, and transported in cytosol in caveolin-1 containing endocytic vesicles.     

中国野生葡萄mybA转录因子SNP特征分析

本研究以中国野生葡萄14个种为材料,对控制花色苷合成的mybA转录因子进行克隆和序列分析,获得VvmybA1和VlmybA2两个转录因子的全长基因序列,共检测到121个SNP,表现出丰富的遗传多样性。3种中性检测方法比较序列变异模式,结果表明,中国野生种葡萄VvmybA1和VlmybA2基因没有偏离中性模型,反映出基因漂移和选择性中性突变之间的平衡。不同野生种材料的mybA 基因结构存在很高的同源相似性。但是在启动子区、内含子区以及第三个外显子区存在不同程度碱基的缺失、插入和替换,而且野生种葡萄mybA基因存在一些特有序列或突变,这些突变可以作为分子标记区分不同的野生种材料。通过基因结构比对和系统进化树分析,可将野生种葡萄细分为5个类群。初步推测桦叶葡萄和变叶葡萄进化地位较为原始。     

Seminal roots of wild and cultivated barley differentially respond to osmotic stress in gene expression,suberization, and hydraulic conductivity

Wild barley, Hordeum vulgare spp. spontaneum, has a wider genetic diversity than its cultivated progeny, Hordeum vulgare spp. vulgare. Osmotic stress leads to a series of different responses in wild barley seminal roots, ranging from no changes in suberization to enhanced endodermal suberization of certain zones and the formation of a suberized exodermis, which was not observed in the modern cultivars studied so far. Further, as a response to osmotic stress, the hydraulic conductivity of roots was not affected in wild barley, but it was 2.5-fold reduced in cultivated barley. In both subspecies, osmotic adjustment by increasing proline concentration and decreasing osmotic potential in roots was observed. RNA-sequencing indicated that the regulation of suberin biosynthesis and water transport via aquaporins were different between wild and cultivated barley. These results indicate that wild barley uses different strategies to cope with osmotic stress compared with cultivated barley. Thus, it seems that wild barley is better adapted to cope with osmotic stress by maintaining a significantly higher hydraulic conductivity of roots during water deficit.     

土壤中高产蛋白酶菌株产酶条件及酶学性质

【背景】微生物蛋白酶已经成为工业用蛋白酶的主要来源,筛选具有特殊环境适应性的微生物成为生物酶资源的开发热点。【目的】通过对青藏高原土壤微生物产蛋白酶菌株的筛选、优化及相关特性研究,寻找新的蛋白酶资源,为高原菌种资源利用提供科学依据。【方法】采用形态学和分子生物学对筛选菌株进行菌种鉴定,利用单因素试验和正交试验对菌株进行发酵条件优化及酶学性质的探究。【结果】筛选出一株高产蛋白酶菌株XC2,经鉴定菌株XC2为枯草芽孢杆菌(Bacillus subtilis)。XC2最优产酶条件:可溶性淀粉4.0%,牛肉膏1.0%,K~+0.6%,培养温度34°C、初始pH 7.0、接种量2.0%的条件下200 r/min振荡培养13 h,所产蛋白酶活力最高为638.5 U/mL。XC2所产蛋白酶最适反应温度60°C,最适pH9.0;40-50°C、pH8.0-10.0条件下酶活稳定性较高;Mn~(2+)对酶活力有明显激活作用,而Zn~(2+)、Cu~(2+)、Fe~(2+)、Fe3+对酶活力有明显抑制作用。【结论】枯草芽孢杆菌XC2有较强的产碱性蛋白酶的能力,具有较好的应用前景。     

Opportunistic Predation of Wild Turkey Nests by Wild Pigs

Wild pigs (Sus scrofa; i.e., feral hogs, feral swine) are considered an invasive species in the United States. Where they occur, they damage agricultural crops and wildlife habitat. Wild pigs also depredate native wildlife, particularly ground-nesting bird species during nesting season. In areas inhabited by wild turkeys (Meleagris gallopavo), nest destruction caused by wild pigs may affect recruitment. There is debate whether wild pigs actively seek ground-nesting bird nests or depredate them opportunistically. To address this debate, in 2016 we examined the movements of wild pigs relative to artificial wild turkey nests (i.e., control [no artificial nests], moderate density [12.5–25 nests/km²], and high density [25–50 nests/km²]) throughout the nesting season (i.e., early, peak, and late) in south-central Texas, USA. We found no evidence that wild pigs learned to seek and depredate wild turkey nests relative to nest density or nesting periods. Despite wild pigs being important nest predators, depredation was not a functional response to a pulsed food resource and can only be associated with overlapping densities of wild pigs and nests. Protecting reproductive success of wild turkeys will require reducing wild pig densities in nesting habitat prior to nesting season. © 2019 The Wildlife Society.     

Diet and Prey Selection of Dholes in Evergreen and Deciduous Forests of Southeast Asia

Endangered dholes (Cuon alpinus) are restricted to small and declining populations in Southeast Asia, and little is known about how their ecology differs within the region. We used DNA-confirmed scats and prey surveys to determine the seasonal diet and prey selection of dholes in 2 different landscapes that dominate Southeast Asia: closed evergreen forests in hilly terrain in northern Laos, and open deciduous forests in relatively flat terrain in eastern Cambodia. On both sites, muntjac (Muntiacus spp.; 20–28 kg) was the dominant prey item and was selectively consumed over other ungulates in all seasons. Our findings differ from previous conclusions, based largely on studies from India, that the preferred prey weight range of dholes was either 40–60 kg or 130–190 kg. Other important prey were sambar (Rusa unicolor) in Laos, and wild pig (Sus scrofa) and banteng (Bos javanicus) in Cambodia. Seasonal differences in overall diet occurred in Laos, but not Cambodia, primarily because of an increase in livestock consumption. The mean number of dhole scats in group defecation sites was higher in Cambodia (5.9 ± 0.5 [SE]) than Laos (2.4 ± 0.2), suggesting pack sizes were larger in Cambodia. Our results suggest that regardless of land cover type, prey diversity, or pack size, the management of muntjac will be important for conserving dhole populations in Southeast Asia. In Laos, we recommend that local villagers remove livestock from the protected area during the hot-dry season to reduce livestock predation by dholes. © 2020 The Authors. The Journal of Wildlife Management published by Wiley Periodicals LLC on behalf of The Wildlife Society.     

藏猪小肠形态、消化酶及微生物多样性研究

【目的】肠道是动物的主要消化器官,同时也是机体抵抗外源病原菌的重要屏障,已有研究表明,动物的品种、饲养方式、生长阶段均会影响动物的肠道菌群结构,但对舍饲和放牧饲养条件下藏猪的肠道菌群结构,以及藏猪和长白、约克与杜洛克三元杂交猪(DLY猪)的肠道菌群结构是否有差异,尚未见报道。【方法】本研究选取6–7月龄的放牧藏猪、舍饲藏猪和DLY猪的小肠组织,分别采用组织切片法测定各试验猪的肠道形态、酶活性测定试剂盒测定肠道内容物的消化酶活性,高通量测序法测定肠道微生物。【结果】DLY猪小肠的肌层厚度和绒毛高度高于藏猪,而隐窝深度低于藏猪;舍饲藏猪和放牧藏猪的小肠形态没有显著变化。DLY猪小肠的胰蛋白酶活性高于藏猪,而淀粉酶活性低于藏猪。三组猪小肠微生物的优势菌门均为Proteobacteria、Firmicutes和Bacteroidetes;藏猪的优势菌属为Ralstonia和Escherichia,而DLY猪的优势菌属为Ralstonia和Bradyrhizobium,但含量却存在显著性差异。舍饲藏猪与放牧藏猪肠道菌群结构相似度较高,而藏猪与DLY猪肠道菌群结构相似度较低。【结论】放牧藏猪、舍饲藏猪和DLY猪的小肠形态、消化酶活性和肠道菌群结构均存在显著性差异。