GM1 and GM3 gangliosides highlight distinct lipid microdomains within the apical domain of epithelial cells

The apical domain of epithelial cells is composed of distinct subdomains such as microvilli, primary cilia and a non-protruding region. Using the cholesterol-binding protein prominin-1 as a specific marker of plasma membrane protrusions we have previously proposed the co-existence of different cholesterol-based lipid microdomains (lipid rafts) within the apical domain [R?per, K., Corbeil, D. and Huttner, W.B. (2000), Retention of prominin in microvilli reveals distinct cholesterol-based lipid microdomains in the apical plasma membrane. Nat. Cell Biol. 2, 582-592]. To substantiate the hypothesis that the microvillar plasma membrane subdomains contain a distinct set of lipids compared to the planar portion we have investigated the distribution of prominin-1 and two raft-associated gangliosides GM(1) and GM(3) by fluorescence microscopy. GM(1) was found to co-localize with prominin-1 on microvilli whereas GM(3) was segregated from there suggesting its localization in the planar region. Regarding the primary cilium, overlapping fluorescent signals of GM(1) or GM(3) and prominin-1 were observed. Thus, our data demonstrate that specific ganglioside-enriched rafts are found in different apical subdomains and reveal that two plasma membrane protrusions with different structural bases (actin for the microvillus and tubulin for the cilium) are composed of distinct types of lipid.     

Chemiluminescence assay for kanamycin based on target recycling strategy

A chemiluminescence (CL) sensing strategy for kanamycin residue detection in fish samples was established based on luminol-functionalized gold nanoparticles as CL nanoprobe materials combined with DNA hairpin structure and carboxyl-modified magnetic beads. Relying on nucleic acid amplification technology, the system can successfully realize the recycling of kanamycin, so that the biosensor can release a large number of luminol-functionalized gold nanoparticles with excellent CL performance even at a low residual levels of kanamycin. The biosensor strategy showed a good linear relationship with kanamycin in the range 0.09–130 nM, the detection limit was as low as 0.04 nM. This method proves the excellent performance of the sensing strategy and provides a low-cost and high-sensitivity CL analysis strategy for the detection of kanamycin and even other antibiotics.     

表施和混施污泥对团花根系生长的影响

林地利用被认为是污泥资源化利用的重要方式,但施用污泥对林木根系生长的影响报道较少。本研究通过根箱试验,分析表施和混施10%污泥对速生树种团花不同土层根系形态、土壤pH值和电导率动态变化及根系重金属含量的影响,并拟合土壤pH值、电导率和根系重金属含量与根长的关系。结果表明: 与不施污泥(对照)相比,混施污泥显著抑制了团花根长、根表面积和根体积增长,混施污泥120和240 d后,0～20 cm土层总根长分别为不施污泥的76.9%和67.4%;表施污泥对团花根长和根表面积的影响不显著,但显著提高了根体积。混施污泥显著提高了土壤pH值和电导率及根系重金属含量,混施污泥0～20和20～40 cm土层根系镉含量分别是不施污泥的11.5和10.0倍。线性回归拟合分析显示,不同处理0～20 cm土层的电导率与根长均呈显著负相关;表施和混施污泥根系镉含量与根长呈极显著负相关。上述结果表明,混施污泥抑制了团花根系生长,这可能是由于混施污泥提高了土壤电导率和根系镉含量所致,而表施污泥对团花根系生长的作用不明显。     

一种强力霉素诱导型白念珠菌基因敲除与筛选标记再循环工具包

【目的】在白念珠菌中建立一个快捷方便经济的基因敲除与筛选标记再循环的DNA操作系统。【方法】通过ExoIII介导的不依赖于连接酶的克隆策略,在异源筛选标记基因CmLEU2、CdHIS1和CdARG4基因的两侧分别插入了loxP位点,成为筛选标记基因盒扩增的模板。全基因合成了经过白念珠菌密码子优化的rTetR元件,并组装成Tet-on启动子。将密码子优化的重组酶Cre基因置于该启动子控制下。然后将他们插入筛选标记基因CdHIS1和CdARG4的CDS区域,形成筛选标记基因再循环载体。【结果】构建了3个用于白念珠菌基因敲除的侧翼含有loxP位点的筛选标记基因载体,以及2个含有Tet-on启动子控制的Cre酶的载体用于筛选标记基因的再循环。【结论】成功构建了一个白念珠菌中可诱导的基因敲除和筛选标记再循环的载体系统并成功应用于多个基因缺失株构建。这个系统有助于快速构建白念珠菌的单基因和多基因敲除菌株。     

Rational design and characterization of bioplastics from Hermetia illucens prepupae proteins

In this study proteins extracted from prepupae of Hermetia illucens, also known as black soldier fly, are investigated as promising base for a new type of bioplastics for agricultural purposes. Design of experiments techniques are employed to perform a rational study on the effects of different combination of glycerol as plasticizer, citric acid as cross-linking agent and distilled water as solvent on the capability of proteins to form a free-standing film through casting technique, keeping as fixed the quantity of proteins. Glycerol shows interesting properties as plasticizer contributing to the formation of homogenous and free-standing film. Moreover, mechanical and thermal characterizations are performed to estimate the effect of increasing amounts of proteins on the final properties and thickness of the specimens. Proteins derived from H. illucens can be successfully employed as base for bioplastics to be employed for agricultural purposes.     

Enhancing cofactor recycling in the bioconversion of racemic alcohols to chiral amines with alcohol dehydrogenase and amine dehydrogenase by coupling cells and cell-free system

Alcohol dehydrogenase (ADH) and amine dehydrogenase (AmDH)-catalyzed one-pot cascade conversion of an alcohol to an amine provides a simple preparation of chiral amines. To enhance the cofactor recycling in this reaction, we report a new concept of coupling whole-cells with the cell-free system to enable separated intracellular and extracellular cofactor regeneration and recycling. This was demonstrated by the respective biotransformation of racemic 4-phenyl-2-butanol 1a and 1-phenyl-2-propanol 1b to (R)-4-phenylbutan-2-amine 3a and (R)-1-phenylpropan-2-amine 3b . Escherichia coli cells expressing S-enantioselective CpsADH, R-enantioselective PfODH, and NADH oxidase (NOX) was developed to oxidize racemic alcohols 1a–b to ketones 2a–b with full conversion via intracellular NAD⁺ recycling. AmDH and glucose dehydrogenase (GDH) were used to convert ketones 2a–b to amines (R)- 3a–b with 89–94% conversion and 891–943 times recycling of NADH. Combining the cells and enzymes for the cascade transformation of racemic alcohols 1a–b gave 70% and 48% conversion to the amines (R)- 3a and (R)-3 b in 99% ee, with a total turnover number (TTN) of 350 and 240 for NADH recycling, respectively. Improved results were obtained by using the E. coli cells with immobilized AmDH and GDH: (R)- 3a was produced in 99% ee with 71–84% conversion and a TTN of 1410-1260 for NADH recycling, the highest value so far for the ADH–AmDH-catalyzed cascade conversion of alcohols to amines. The concept might be generally applicable to this type of reactions.     

Amino acid starvation‐induced LDLR trafficking accelerates lipoprotein endocytosis and LDL clearance

Mammalian cells utilize Akt‐dependent signaling to deploy intracellular Glut4 toward cell surface to facilitate glucose uptake. Low‐density lipoprotein receptor (LDLR) is the cargo receptor mediating endocytosis of apolipoprotein B‐containing lipoproteins. However, signaling‐controlled regulation of intracellular LDLR trafficking remains elusive. Here, we describe a unique amino acid stress response, which directs the deployment of intracellular LDLRs, causing enhanced LDL endocytosis, likely via Ca²⁺ and calcium/calmodulin‐dependent protein kinase II‐mediated signalings. This response is independent of induction of autophagy. Amino acid stress‐induced increase in LDL uptake in vitro is comparable to that by pravastatin. In vivo, acute AAS challenge for up to 72 h enhanced the rate of hepatic LDL uptake without changing the total expression level of LDLR. Reducing dietary amino acids by 50% for 2 to 4 weeks ameliorated high fat diet‐induced hypercholesterolemia in heterozygous LDLR‐deficient mice, with reductions in both LDL and VLDL fractions. We suggest that identification of signaling‐controlled regulation of intracellular LDLR trafficking has advanced our understanding of the LDLR biology, and may benefit future development of additional therapeutic strategies for treating hypercholesterolemia.     

Bicaudal‐D binds clathrin heavy chain to promote its transport and augments synaptic vesicle recycling

Cargo transport by microtubule‐based motors is essential for cell organisation and function. The Bicaudal‐D (BicD) protein participates in the transport of a subset of cargoes by the minus‐end‐directed motor dynein, although the full extent of its functions is unclear. In this study, we report that in Drosophila zygotic BicD function is only obligatory in the nervous system. Clathrin heavy chain (Chc), a major constituent of coated pits and vesicles, is the most abundant protein co‐precipitated with BicD from head extracts. BicD binds Chc directly and interacts genetically with components of the pathway for clathrin‐mediated membrane trafficking. Directed transport and subcellular localisation of Chc is strongly perturbed in BicD mutant presynaptic boutons. Functional assays show that BicD and dynein are essential for the maintenance of normal levels of neurotransmission specifically during high‐frequency electrical stimulation and that this is associated with a reduced rate of recycling of internalised synaptic membrane. Our results implicate BicD as a new player in clathrin‐associated trafficking processes and show a novel requirement for microtubule‐based motor transport in the synaptic vesicle cycle.     

Fjx1, the murine homologue of the Drosophila four-jointed gene, codes for a putative secreted protein expressed in restricted domains of the developing and adult brain

The Drosophila gene four jointed (fj) codes for a secreted or cell surface protein important for growth and differentiation of legs and wings and for proper development of the eyes. Here we report the cloning of the mouse four-jointed gene (fjx1) and its pattern of expression in the brain during embryogenesis and in the adult. In the neural plate, fjx1 is expressed in the presumptive forebrain and midbrain, and in rhombomere 4, however a small rostral/medial area of the forebrain primordium is devoid of expression. Expression of fjx1 in the neural tube can be divided into three phases. (1) In the embryonic brain fjx1 is expressed in two patches of neuroepithelium: in the midbrain tectum and the telencephalic vesicles. (2) In fetal and early postnatal brain fjx1 is expressed mainly by the primordia of layered telencephalic structures: cortex (ventricular layer and cortical plate), olfactory bulb (subependymal layer and in the mitral cell layer). In addition expression is observed in the superior colliculus. (3) In the adult, fjx1 is expressed by neurons evenly distributed in the telencephalon (isocortex, striatum, hippocampus, olfactory bulb, piriform cortex), in the Purkinje cell layer of the cerebellum, and numerous medullary nuclei. In the embryo, strong expression can further be seen in the apical ectodermal ridge of fore- and hindlimbs and in the ectoderm of the branchial arches.