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Jan Arnold Julia Mahamid Vladan Lucic Alex de?Marco Jose-Jesus Fernandez Tim Laugks Tobias Mayer Anthony?A. Hyman Wolfgang Baumeister Jürgen?M. Plitzko 《Biophysical journal》2016,110(4):860-869
The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. In this study, we present the development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures and describe the incorporation of the new hardware into existing workflows for cellular sample preparation by cryo-FIB. Introduction of fiducial markers and subsequent computation of three-dimensional coordinate transformations provide correlation between light microscopy and scanning electron microscopy/FIB. The correlative approach is employed to guide the FIB milling process of vitrified cellular samples and to capture specific structures, namely fluorescently labeled lipid droplets, in lamellas that are 300 nm thick. The correlation procedure is then applied to localize the fluorescently labeled structures in the transmission electron microscopy image of the lamella. This approach can be employed to navigate the acquisition of cryo-ET data within FIB-lamellas at specific locations, unambiguously identified by fluorescence microscopy. 相似文献
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Lucic D Huang ZH Gu de S Altenburg MK Maeda N Mazzone T 《Journal of lipid research》2007,48(2):366-372
Factors that regulate apolipoprotein E (apoE) secretion by macrophages will have important effects on vessel wall lipid flux and atherosclerosis. Macrophages express the LDL receptor, which binds apoE with high affinity and could thereby affect the net secretion of apoE from macrophages. In these studies, we demonstrate that treatment of J774 macrophages transfected to constitutively express a human apoE3 cDNA with simvastatin, to increase LDL receptor activity, reduces the secretion of apoE. To further examine the relationship between LDL receptor expression and apoE secretion from macrophages, mouse peritoneal macrophages (MPMs) were isolated from mice with constitutively high expression of human LDL receptor to increase overall LDL receptor expression by 2- to 3-fold. Cells with increased LDL receptor expression also showed reduced apoE secretion compared with MPMs with basal LDL receptor expression. The effect of changes in LDL receptor expression on apoE secretion was isoform-specific, with greater reduction of apoE4 compared with apoE3 secretion and no reduction of apoE2 secretion, paralleling the known affinity of each isoform for LDL receptor binding. The effect of the LDL receptor on apoE secretion for each isoform was further reflected in LDL receptor-dependent changes in apoE-mediated cholesterol efflux. These results establish a regulatory interaction between two branches of macrophage sterol homeostatic pathways that could facilitate a rapid response to changes in macrophage sterol content relative to need. 相似文献
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Focussing on the blood-feeding reduviid Rhodnius prolixus, we investigated the structure and function of the hypopharynx in (1) conducting the saliva towards the mouthparts and (2)
bringing together the salivary pump and the stylets to ensure the difficult task of supplying the two closed antidromic streams
of blood and saliva, while allowing the mouthparts to be moved forth and back during the feeding process. The distal apex
of the hypopharynx forms a needle-like structure that is X-shaped in cross section. It arranges the interlocking of the maxillae
in a manner resembling the fixed slider of a zip-lock. Further proximal, the hypopharynx extends into the maxillary food channel
as a wide tongue. The salivary pump possesses two separate efferent ducts. The dorsal duct originates in the retrograde angle
of the cupula (part of the salivarium) and conducts saliva directly into the maxillary salivary channel. The ventral duct
originates at the distal opening of the cupula. It extends into a bag, the distal opening of which can be closed by a ventral
bolster-like cuticle and opened by muscles. We show for the first time for heteropteran mouthparts that the saliva is not
exclusively discharged into the maxillary salivary channel (via the dorsal efferent duct of the salivary pump), but that a
large amount of saliva directly flows into the tube of the labium (via the ventral efferent duct of the salivary pump), which
encloses the piercing stylets. However, within a short section, saliva may also pass from the ventral salivary duct into the
maxillary salivary channel. Similar double salivary efferent ducts are present in the reduviids Triatoma dimidiata, T. infestans, Dipetalogaster maxima, Panstrongylus megistus, in the pyrrhocorid Pyrrhocoris apterus, and in the pentatomid Troilus luridus. It might thus be a more common feature of the Heteroptera. 相似文献
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A gene repertoire for nitrogen transporters in Laccaria bicolor 总被引:1,自引:0,他引:1
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Gelatinous invertebrate zooplankton of the South Adriatic: species composition and vertical distribution 总被引:1,自引:0,他引:1
Batistic Mirna; Krsinic Frano; Jasprica Nenad; Caric Marina; Vilicic Damir; Lucic Davor 《Journal of plankton research》2004,26(4):459-474
The species composition and vertical distribution of gelatinousinvertebrate zooplankton were investigated from April 1993 toJune 1994 in the 01000 m water column at a deep-sea stationin the northern part of the South Adriatic Pit. Fifty-sevenspecies were identified: 11 hydromedusae, 13 calycophores, 3ctenophores, 3 heteropods, 10 pteropods, 8 polychaetes and 9chaetognaths. The pteropod Desmopterus papilio and the heteropodProtatlanta mediterranea were recorded for the first time inthe Adriatic Sea. Data from this study differed from those ofprevious investigations in the South Adriatic as regards thenumerically dominant polychaete and pteropod species. All investigatedgroups generally were more abundant in the upper 100 m and decreasedwith depth. Different vertical distributions of life stageswere observed for those species that occupy a wide depth range:Persa incolorata, Solmissus albescens, Limacina inflata, Cymbuliaperoni, Pelagobia longicirrata, Sagitta lyra and Sagitta decipiens. 相似文献
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Stojcevic D Zivicnjak T Marinculic A Marucci G Andelko G Brstilo M Pavo L Pozio E 《The Journal of parasitology》2004,90(3):666-670
Whether the brown rat (Rattus norvegicus) is a reservoir of Trichinella spp. infection or merely an accidental host, which may be vector of Trichinella spp., continues to be debated. We estimated the prevalence of Trichinella sp. infection in brown rat populations and in domestic pigs in 2 villages in Croatia, where Trichinella sp. infection in pigs has been endemic in the past 10 yr. Trichinella spiralis larvae, identified by a multiplex polymerase chain reaction analyses, were the only species detected in both rats and pigs. In 2001 and 2002, 2,287 rats were collected on 60 farms with different levels of sanitation and with, or without, T. spiralis-infected pigs. The prevalence of infection in rats ranged from 0.2 to 10.7%. Infected rats were detected only on farms with T. spiralis-positive pigs and low sanitation or formerly with low sanitation (P = 0.007, Fisher's exact test), yet no infected rat was detected on farms with T. spiralis-negative pigs. The finding that no infected rat was found on farms with T. spiralis-negative pigs suggests that, in the investigated area, the brown rat is not a reservoir but only a victim of improper pig slaughtering. 相似文献