Isolation and Identification of Cellular Phospholipids

1997 ◽  
Author(s):  
Donald J. Hanahan
Keyword(s):  
Venous Blood ◽  
Volume Ratio ◽  
Trisodium Citrate ◽  
Human Platelets ◽  
Convincing Evidence ◽  
Model Systems ◽  
Membrane Phospholipids ◽  
Isolation And Identification ◽  
Cellular Events ◽  
Plastic Syringe

In this chapter, some of the types of methodologies currently in use for isolation and analysis of cellular phospholipids will be outlined. Such techniques can be readily applied to experiments designed to explore the involvement of phospholipids in cellular events, such as stimulus-induced activation. Primary attention will be paid to the human platelet. If you need to justify the choice of human platelets as the cell of choice, a number of highly creditable reasons can be cited. Only three need to be considered at this point. First, the circulating platelet is of paramount importance in hemostasis, and there is convincing evidence that its membrane phospholipids are intimately involved in this process. Second, these cells can serve as excellent targets or model systems for stimulus-induced activation in which the membrane phospholipids play an important role. Third, human platelets can be isolated from whole blood by a simple, convenient centrifugal approach. Human donors are available at a very reasonable cost, and the platelets obtained from a varied spectrum of donors show remarkable consistency. Thus, one can undertake their isolation using the following method and have them available for immediate experimentation. Nonfasting venous blood is drawn (with informed consent) from male or female subjects between the ages of 20 and 40 years, who are considered to be normal and healthy and had not ingested platelet-active medication for at least 10 days prior. Blood is obtained by insertion of a butterfly infusion set (12-in. tubing from Abbott Hospitals, Inc., North Chicago, IL) with a 1-in. x 19-gauge needle into the antecubital vein. A few milliliters of blood is allowed to flow before a 60-ml plastic syringe containing 7.5 ml of an ACD [10.8% citric acid, 2.2% trisodium citrate, and 2% dextrose (w/v)] solution was attached. Four syringes were filled to the 50-ml mark and inverted gently, and the contents were transferred carefully into 50-ml plastic tubes that are then capped. The blood-to-ACD volume ratio is 6:1 (v/v). The tubes are centrifuged at 2000 rpm (830g) in a Sorvall RT 6000 centrifuge at 24°C for 15 min.

Phospholipid(s) Associations in Cellular Structures

1997 ◽  
Author(s):  
Donald J. Hanahan
Keyword(s):  
Signal Transduction ◽  
Signal Transduction Pathway ◽  
Human Platelets ◽  
Biologically Active ◽  
Membrane Phospholipids ◽  
Stimulus Response ◽  
Cellular Events ◽  
Close Relationship ◽  
A Cell ◽  
In Cells

In the preceding chapter, the intent was to provide the reader with a broad perspective on the chemical characteristics of cellular phospholipids. At the same time, emphasis was placed on the potential usefulness of this information in dissecting the importance of phospholipids in cellular events, such as signal transduction. There is no doubt that the large number of observations reported on the close relationship of phospholipids to the transduction process has stimulated a widespread (and gratifying) interest in these compounds. Certainly it is very clear now that stimulus-induced activation of cells leads to the turnover of specific membrane phospholipids. The following diagram reemphasizes several, but not all, possible reaction pathways that can be invoked during an agonist (stimulus)–induced activation of a cell and gives the possible sequelae: In each of the above reactions, the substrates phosphatidylcholine and phosphatidylinositol bisphosphate normally are considered biologically inactive in membranes. Then, subsequent to activation of cellular phospholipases by a stimulus, biologically active products are formed from these compounds. Thus, inositol bisphosphate triggers the release of calcium ions from intracellular stores, diacylglycerol is implicated in the translocation and activation of protein kinase C, arachidonic acid can be converted to biologically active prostaglandins, and phosphatidic acid can be an agonist in its own right. The major point to be stressed here is that phospholipid turnover is intimately associated with the signal transduction pathway in cells. Hence an understanding of the chemistry of these phospholipids is of major relevance to delineating the complicated process of signal transduction. While investigation of the behavior of phospholipids in this pathway in platelets has been a consuming interest of this author, the main thrust in this book will be simply to acquaint the reader with the chemistry of phospholipids of major importance in signal transduction and also to discuss other phospholipids found in mammalian membranes. Inasmuch as most investigations on stimulus response in cells utilize quite small numbers of cells—for example, a typical experiment on human platelets might use 1 x 109 cells, which would yield ∼50 μg of total lipid—this poses a challenge to an investigator to be able to isolate and identify these lipids.

scholarly journals Solenodon genome reveals convergent evolution of venom in eulipotyphlan mammals

2019 ◽  
Vol 116 (51) ◽  
pp. 25745-25755 ◽  
Author(s):  
Nicholas R. Casewell ◽  
Daniel Petras ◽  
Daren C. Card ◽  
Vivek Suranse ◽  
Alexis M. Mychajliw ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Prey Capture ◽  
Convincing Evidence ◽  
Model Systems ◽  
Ancestral State ◽  
Origin And Evolution ◽  
Physiological Processes ◽  
A Genome ◽  
Taxonomic Groups

Venom systems are key adaptations that have evolved throughout the tree of life and typically facilitate predation or defense. Despite venoms being model systems for studying a variety of evolutionary and physiological processes, many taxonomic groups remain understudied, including venomous mammals. Within the order Eulipotyphla, multiple shrew species and solenodons have oral venom systems. Despite morphological variation of their delivery systems, it remains unclear whether venom represents the ancestral state in this group or is the result of multiple independent origins. We investigated the origin and evolution of venom in eulipotyphlans by characterizing the venom system of the endangered Hispaniolan solenodon (Solenodon paradoxus). We constructed a genome to underpin proteomic identifications of solenodon venom toxins, before undertaking evolutionary analyses of those constituents, and functional assessments of the secreted venom. Our findings show that solenodon venom consists of multiple paralogous kallikrein 1 (KLK1) serine proteases, which cause hypotensive effects in vivo, and seem likely to have evolved to facilitate vertebrate prey capture. Comparative analyses provide convincing evidence that the oral venom systems of solenodons and shrews have evolved convergently, with the 4 independent origins of venom in eulipotyphlans outnumbering all other venom origins in mammals. We find thatKLK1s have been independently coopted into the venom of shrews and solenodons following their divergence during the late Cretaceous, suggesting that evolutionary constraints may be acting on these genes. Consequently, our findings represent a striking example of convergent molecular evolution and demonstrate that distinct structural backgrounds can yield equivalent functions.

Isolation and Identification of Aldosterone from Adrenal Venous Blood

1954 ◽  
Vol 87 (1) ◽  
pp. 141-143 ◽  
Author(s):  
G. L. Farrell ◽  
P. C. Royce ◽  
E. W. Rauschkilb ◽  
H. Hirschmann
Keyword(s):  
Venous Blood ◽  
Isolation And Identification

scholarly journals Quantifying cerebral hypoxia by near-infrared spectroscopy tissue oximetry: the role of arterial-to-venous blood volume ratio

2017 ◽  
Vol 22 (2) ◽  
pp. 025001 ◽  
Author(s):  
Martin B. Rasmussen ◽  
Vibeke R. Eriksen ◽  
Bjørn Andresen ◽  
Simon Hyttel-Sørensen ◽  
Gorm Greisen
Keyword(s):  
Infrared Spectroscopy ◽  
Blood Volume ◽  
Near Infrared Spectroscopy ◽  
Near Infrared ◽  
Venous Blood ◽  
Volume Ratio ◽  
Cerebral Hypoxia ◽  
Venous Blood Volume ◽  
Tissue Oximetry

scholarly journals Dietary supplementation of fructooligosaccharides alleviates enterotoxigenic E. coli-induced disruption of intestinal epithelium in a weaned piglet model

2021 ◽  
pp. 1-27
Author(s):  
Yuheng Luo ◽  
Lei Liu ◽  
Daiwen Chen ◽  
Bing Yu ◽  
Ping Zheng ◽  
...  
Keyword(s):  
Protective Effect ◽  
Intestinal Mucosa ◽  
Intestinal Epithelium ◽  
Venous Blood ◽  
Intestinal Barrier ◽  
Convincing Evidence ◽  
Human Infants ◽  
Sterile Culture ◽  
E Coli ◽  
Weaned Piglet

Abstract Diarrhea caused by pathogens such as enterotoxigenic E. coli (ETEC) is a serious threat to the health of young animals and human infants. Here, we investigated the protective effect of fructooligosaccharides (FOS) on the intestinal epithelium with ETEC-challenge in a weaned piglet model. Twenty-four weaned piglets were randomly divided into three groups: (1) non-ETEC-challenged control (CON), (2) ETEC-challenged control (ECON), and (3) ETEC challenge + 2.5 g/kg FOS (EFOS). On day 19, the CON pigs were orally infused with sterile culture, while the ECON and EFOS pigs were orally infused with active ETEC (2.5 × 109 colony-forming units). On day 21, pigs were slaughtered to collect venous blood and small intestine. Result showed that the pre-treatment of FOS improved the antioxidant capacity and the integrity of intestinal barrier in the ETEC-challenged pigs without affecting their growth performance. Specifically, comparing with ECON pigs, the level of GSH-Px (glutathione peroxidase) and CAT (catalase) in the plasma and intestinal mucosa of EFOS pigs was increased (P<0.05), and the intestinal barrier marked by ZO-1 and plasmatic DAO was also improved in EFOS pigs. A lower level (P<0.05) of inflammatory cytokines in the intestinal mucosa of EFOS pigs might be involved in the inhibition of TLR4/MYD88/NF-κB pathway. The apoptosis of jejunal cells in EFOS pigs was also lower than that in ECON pigs (P<0.05). Our findings provide convincing evidence of possible prebiotic and protective effect of FOS on the maintenance of intestinal epithelial function under the attack of pathogens.

scholarly journals Automated Optical Counting of Blood Platelets

Blood ◽  
1971 ◽  
Vol 38 (4) ◽  
pp. 422-430 ◽  
Author(s):  
GEOFFREY M. BRITTIN ◽  
SHIRLEY A. DEW ◽  
ELVI K. FEWELL
Keyword(s):  
Whole Blood ◽  
Blood Platelets ◽  
Venous Blood ◽  
Human Platelets ◽  
Blood Samples ◽  
Platelet Counts ◽  
Large Numbers ◽  
Significant Difference ◽  
Electronic Method ◽  
Electronic Counting

Abstract We have evaluated the use of an optical particle counter to perform automated platelet counts on whole blood. The erythrocytes were lysed by dilution of whole blood with 2 M urea and the remaining platelets and leukocytes were enumerated by a darkfield microscope optical system that detects light diffracted by them. A suspension of fixed human platelets available commercially was highly satisfactory for standardization. The method gave accurate and reproducible platelet counts, comparable with those of electronic particle counting on venous blood and substantially more reliable platelet counts on thrombocytopenic and finger-puncture blood samples. We believe that errors resulting from the electronic method were caused by technical difficulties of sample handling and not to an intrinsic error in electronic counting. By using the automated optical method we found no significant difference between the platelet counts of capillary and venous blood, although capillary platelet counts had twice the variability of venous counts. The optical technique has important advantages over electronic platelet counting, and its superiority appears to be due to the ability to count platelets in diluted whole blood rather than in plasma. It should prove especially useful in performing the large numbers of platelet counts on thrombocytopenic and finger-puncture blood samples that are increasingly important for management of patients receiving chemotherapy.

DIRECT STIMULATIONOF CA2+-ACTIVATED HUMAN PLATELET PHOSPHOLIPASE A2 BY DIACYLGLYCEROL

1987 ◽  
Author(s):  
D Deykin ◽  
R M Karmer
Keyword(s):  
Enzymatic Activity ◽  
Phospholipase A2 ◽  
Conformational Changes ◽  
Cellular Membrane ◽  
Human Platelets ◽  
Stable Form ◽  
Membrane Phospholipids ◽  
Kinase C ◽  
Snake Venom Phospholipase ◽  
Phospholipase A2 Activity

These studies examined the effect of diacylglycerol on Ca2+-dependent phospholipase A2 from human platelets. Phospholipase A2 was solubilized and partially purified to a stable form in the presence of octylglucoside and its enzymatic activity determined using sonicated arachidonoyl phosphatidylcholine (PC) as substrate. (Kramer RM, et al: BBA 878:394, 1986) Phospholipase A2 activity was increased when dioleoylglycerc_ was incorporated into the substrate arachidonoyl-PC. In the presence of 1 uM (29 mol %) sn-1,2-dioleoylglycerol the enzymatic activity was stimulated 4.1-fold. Exogenously added sn-l-oleoyl-2-acetoylglycerol also enhanced phospholipase A2 activity, producing a maximal stimulation of 1.6-fold at a concentration of 25 uM. Comparative studies conducted with pancreatic, bee-venom and snake venom phospholipase A2 showed that the activity of these extracellular phospholipases towards the arachidonoyl-PC substrate was also increased by diacylglycerol, but the stimulation was less than observed for platelet phospholipase A2. We conclude that in stimulated platelets Ca2+-activated phospholipase A2 may be regulated by newly generated diacylglycerols, not only via protein kinase C-mediated events, but also directly through conformational changes imposed by the diglycerides on cellular membrane phospholipids.

scholarly journals Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids

2018 ◽  
Vol 115 (33) ◽  
pp. E7720-E7727 ◽  
Author(s):  
Spencer T. Glantz ◽  
Erin E. Berlew ◽  
Zaynab Jaber ◽  
Benjamin S. Schuster ◽  
Kevin H. Gardner ◽  
...  
Keyword(s):  
Electrostatic Interaction ◽  
Signal Transmission ◽  
Model Systems ◽  
Membrane Localization ◽  
Diffusion Limit ◽  
Dependent Manner ◽  
Membrane Phospholipids ◽  
Anionic Phospholipids ◽  
Domain Of Unknown Function

We report natural light–oxygen–voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10−7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure–function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit.

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