scholarly journals The induction of secondary cytolytic T lymphocytes by solubilized membrane proteins.

1978 ◽  
Vol 147 (3) ◽  
pp. 946-951 ◽  
Author(s):  
M Mescher ◽  
L Sherman ◽  
F Lemonnier ◽  
S Burakoff
Keyword(s):  
Plasma Membranes ◽  
Active Form ◽  
Protein A ◽  
Gel Filtration ◽  
Biologically Active ◽  
Cytolytic T Lymphocytes ◽  
Histocompatibility Complex ◽  
Cell Plasma ◽  
Immunological Specificity ◽  
Membrane Bound

Membrane-bound antigens responsible for induction of a secondary allogeneic murine cytolytic T-cell (CTL) response have been obtained in a soluble, biologically active form by deoxycholate solubilization of tumor cell plasma membranes. The active proteins are soluble by the criteria of both ultracentrifugation and gel filtration. The immunological specificity of the induced CTL and removal of the activity from solution by treatment with B6 anti-P815 (anti-H-2d) antiserum and Protein A-Sepharose demonstrate that the CTL-inducing activity is dependent upon solubilized major histocompatibility complex antigens.

scholarly journals Characterization of the binding of plasminogen activators to plasma membranes from human liver

1992 ◽  
Vol 287 (3) ◽  
pp. 911-915 ◽  
Author(s):  
G Nguyen ◽  
S J Self ◽  
C Camani ◽  
E K O Kruithof
Keyword(s):  
Human Liver ◽  
Plasma Membranes ◽  
Gel Filtration ◽  
Mannose Receptor ◽  
Hepatoma Cells ◽  
Tissue Type ◽  
High Affinity ◽  
Liver Membranes ◽  
Membrane Bound ◽  
Pai 1

The binding of tissue-type plasminogen activator (t-PA) to membranes prepared from human liver was investigated, and a specific, saturable, high-affinity binding site (Kd = 3.4 nM) was identified. The binding of t-PA to liver membranes was not affected by an excess of D-mannose or D-galactose, or by active urokinase (u-PA), whereas binding of t-PA to membranes prepared from human HepG2 hepatoma cells was inhibited by u-PA. HepG2-membrane-bound t-PA was fully complexed to PA inhibitor 1 (PAI-1), whereas liver-membrane-bound t-PA was not complexed. Gel filtration on Sephacryl S300 of membrane proteins solubilized in deoxycholate revealed that high-affinity t-PA binding activity elutes at an apparent molecular mass of 40 kDa. Monoclonal antibodies specific for the growth factor and the kringle 2 domains inhibited the binding of t-PA to liver membranes and the catabolism of t-PA by rat hepatoma cells. Human liver membranes also bound u-PA; binding was inhibited by pro-u-PA, the N-terminal fragment of u-PA, but not by the 33 kDa form of u-PA or by t-PA. Our results show that human liver membranes contain a specific 40 kDa binding protein for t-PA that is different from the PAI-1-dependent receptor described on HepG2 cells and the mannose receptor isolated from human liver.

scholarly journals Membrane isolation on polylysine-coated beads. Plasma membrane from HeLa cells.

1977 ◽  
Vol 75 (1) ◽  
pp. 119-134 ◽  
Author(s):  
C M Cohen ◽  
D I Kalish ◽  
B S Jacobson ◽  
D Branton
Keyword(s):  
Adenosine Triphosphatase ◽  
Wheat Germ ◽  
Plasma Membranes ◽  
Cell Attachment ◽  
Isolation Procedure ◽  
Chemical Analyses ◽  
Isolation Techniques ◽  
Cell Plasma ◽  
Membrane Bound ◽  
Membrane Isolation

HeLa cell plasma membranes have been purified after binding cells to polylysine-coated polyacrylamide beads. Cell attachment to beads and membrane recovery were maximal in a sucrose-acetate buffer, pH 5.0, at 25 degrees C. Measurements of ouabain-sensitive NaK-adenosine triphosphatase, membrane-bound 125I-wheat germ agglutinin, and chemical analyses showed that membranes on beads were of comparable or greater purity than membranes isolated by conventional methods. Because the isolation procedure is rapid (approximately 2.5 h), and produces membranes whose protoplasmic surfaces are fully exposed, it should be a useful supplement to standard isolation techniques.

scholarly journals Stimulation of Toll-like receptor 3 and 4 induces interleukin-1β maturation by caspase-8

2008 ◽  
Vol 205 (9) ◽  
pp. 1967-1973 ◽  
Author(s):  
Jonathan Maelfait ◽  
Elisabeth Vercammen ◽  
Sophie Janssens ◽  
Peter Schotte ◽  
Mira Haegman ◽  
...  
Keyword(s):  
Active Form ◽  
Biologically Active ◽  
Poly I:C ◽  
Caspase 8 ◽  
Caspase 1 ◽  
Polyinosinic:Polycytidylic Acid ◽  
Membrane Bound ◽  
Acute And Chronic Inflammation ◽  
Stimulation Of

The cytokine interleukin (IL)-1β is a key mediator of the inflammatory response and has been implicated in the pathophysiology of acute and chronic inflammation. IL-1β is synthesized in response to many stimuli as an inactive pro–IL-1β precursor protein that is further processed by caspase-1 into mature IL-1β, which is the secreted biologically active form of the cytokine. Although stimulation of membrane-bound Toll-like receptors (TLRs) up-regulates pro–IL-1β expression, activation of caspase-1 is believed to be mainly initiated by cytosolic Nod-like receptors. In this study, we show that polyinosinic:polycytidylic acid (poly[I:C]) and lipopolysaccharide stimulation of macrophages induces pro–IL-1β processing via a Toll/IL-1R domain–containing adaptor-inducing interferon-β–dependent signaling pathway that is initiated by TLR3 and TLR4, respectively. Ribonucleic acid interference (RNAi)–mediated knockdown of the intracellular receptors NALP3 or MDA5 did not affect poly(I:C)-induced pro–IL-1β processing. Surprisingly, poly(I:C)- and LPS-induced pro–IL-1β processing still occurred in caspase-1–deficient cells. In contrast, pro–IL-1β processing was inhibited by caspase-8 peptide inhibitors, CrmA or vFLIP expression, and caspase-8 knockdown via RNAi, indicating an essential role for caspase-8. Moreover, recombinant caspase-8 was able to cleave pro–IL-1β in vitro at exactly the same site as caspase-1. These results implicate a novel role for caspase-8 in the production of biologically active IL-1β in response to TLR3 and TLR4 stimulation.

scholarly journals The biologically active form of the sea urchin egg receptor for sperm is a disulfide-bonded homo-multimer.

1994 ◽  
Vol 125 (4) ◽  
pp. 817-824 ◽  
Author(s):  
K Ohlendieck ◽  
J S Partin ◽  
W J Lennarz
Keyword(s):  
Sea Urchin ◽  
Disulfide Bonds ◽  
Plasma Membranes ◽  
Active Form ◽  
Electrophoretic Analysis ◽  
Biologically Active ◽  
Major Protein ◽  
Dependent Manner ◽  
Surface Receptors ◽  
Sperm Receptor

Since many cell surface receptors exist in their active form as oligomeric complexes, we have investigated the subunit composition of the biologically active sperm receptor in egg plasma membranes from Strongylocentrotus purpuratus. Electrophoretic analysis of the receptor without prior reduction of disulfide bonds revealed that the surface receptor exists in the form of a disulfide-bonded multimer, estimated to be a tetramer. These findings are in excellent agreement with the fact that the NH2-terminus of the extracellular domain of the sperm receptor is rich in cysteine residues. Studies with cross-linking agents of various length and hydrophobicity suggest that no other major protein is tightly associated with the receptor. Given the multimeric structure of the receptor, we investigated the effect of disulfide bond reduction on its biological activity. Because in quantitative bioassays fertilization was found to be inhibited by treatment of eggs with 5 mM dithiothreitol, we undertook more direct studies of the effect of reduction on properties of the receptor. First, we studied the effect of addition of isolated, pure receptor on fertilization. Whereas the non-reduced, native receptor complex inhibited fertilization in a dose-dependent manner, the reduced and alkylated receptor was inactive. Second, we tested the ability of the isolated receptor to mediate binding of acrosome-reacted sperm to polystyrene beads. Whereas beads coated with native receptor bound sperm, those containing reduced and alkylated receptor did not. Thus, these results demonstrate that the biologically active form of the sea urchin sperm receptor consists only of 350 kD subunits and that these must be linked as a multimer via disulfide bonds to produce a complex that is functional in sperm recognition and binding.

scholarly journals Purification and characterization of protein disulphide-isomerase from the unicellular green alga Chlamydomonas reinhardii. A 120 kDa dimer antigenically distinct from the vertebrate enzyme

1990 ◽  
Vol 268 (1) ◽  
pp. 63-68 ◽  
Author(s):  
D D Kaska ◽  
K I Kivirikko ◽  
R Myllylä
Keyword(s):  
Green Alga ◽  
Polyclonal Antibodies ◽  
Active Form ◽  
Protein A ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Chlamydomonas Reinhardii ◽  
Protein Disulphide Isomerase ◽  
Unicellular Green Alga ◽  
A Subunit

Protein disulphide-isomerase (PDI) has been isolated from the unicellular green alga Chlamydomonas reinhardii and purified by (NH4)2SO4 precipitation, gel filtration and DEAE-Sephacel, hydroxyapatite and f.p.l.c. chromatography. The active algal enzyme is a 120 kDa dimer with a subunit molecular mass of 60 kDa when determined by SDS/PAGE. Although similar in size to the previously isolated vertebrate PDIs, the algal enzyme is antigenically distinct, polyclonal antibodies against the algal PDI showing no cross-reactivity with the vertebrate enzyme on immunoblots, and vice versa. The anti-(algal PDI) antiserum did not inhibit algal PDI activity, and C. reinhardii PDI could be immobilized on anti-PDI-Protein A-Sepharose in active form. In contrast with the situation in vertebrates, where PDI functions as a subunit of prolyl 4-hydroxylase, the C. reinhardii PDI is not associated with the algal prolyl 4-hydroxylase.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Processing and Characterization of Recombinant von Willebrand Factor Expressed in Different Cell Types Using a Vaccinia Virus Vector

1992 ◽  
Vol 67 (01) ◽  
pp. 154-160 ◽  
Author(s):  
P Meulien ◽  
M Nishino ◽  
C Mazurier ◽  
K Dott ◽  
G Piétu ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Von Willebrand Factor ◽  
Human Fibroblasts ◽  
Active Form ◽  
Cell Types ◽  
Biologically Active ◽  
L Cells ◽  
Von Willebrand ◽  
Different Cell Types ◽  
Willebrand Factor

SummaryThe cloning of the cDNA encoding von Willebrand factor (vWF) has revealed that it is synthesized as a large precursor (pre-pro-vWF) molecule and it is now clear that the prosequence or vWAgll is responsible for the intracellular multimerization of vWF. We have cloned the complete vWF cDNA and expressed it using a recombinant vaccinia virus as vector. We have characterized the structure and function of the recombinant vWF (rvWF) secreted from five different cell types: baby hamster kidney (BHK), Chinese hamster ovary (CHO), human fibroblasts (143B), mouse fibroblasts (L) and primary embryonic chicken cells. Forty-eight hours after infection, the quantity of vWF antigen found in the cell supernatant varied from 3 to 12 U/dl depending on the cell type. By SDS-agarose gel electrophoresis, the percentage of high molecular weight forms of vWF varied from 39 to 49% relative to normal plasma for BHK, CHO, 143B and chicken cells but was less than 10% for L cells. In all cell types, the two anodic subbands of each multimer were missing. The two cathodic subbands were easily detected only in BHK and L cells. By SDS-PAGE of reduced samples, pro-vWF was present in similar quantity to the fully processed vWF subunit in L cells, present in moderate amounts in BHK and CHO and in very low amounts in 143B and chicken cells. rvWF from all cells bound to collagen and to platelets in the presence of ristocetin, the latter showing a high correlation between binding efficiency and degree of multimerization. rvWF from all cells was also shown to bind to purified FVIII and in this case binding appeared to be independent of the degree of multimerization. We conclude that whereas vWF is naturally synthesized only by endothelial cells and megakaryocytes, it can be expressed in a biologically active form from various other cell types.

Innovative approaches to the correction of chronotrope status of pregnant and lactating women

2016 ◽  
pp. 37-40
Author(s):  
S.I. Zhuk ◽  
◽  
K.K. Bondarenko ◽  
Keyword(s):  
Folic Acid ◽  
Neural Tube ◽  
Active Form ◽  
Biologically Active ◽  
Lactating Women ◽  
Pathological Conditions ◽  
Prevention And Treatment ◽  
The Impact ◽  
High Bioavailability ◽  
Key Words Mthfr

Most recent studies show the impact of violations in the metabolism of folate and metin period in the pathogenesis of neural tube defects (NTD) of the fetus. Metafolin has a number of advantages, which primarily includes direct intake of substances in biologically active form and the optimum effect, even in the case when the patient homozygote and/or heterozygote genotype 677С T polymorphism in MTHFR. With the aim of prevention and treatment of various pathological conditions related to folate deficiency during pregnancy, it is advisable to apply vitamin-mineral complexes, containing metafolin - active form of folate with high bioavailability. Key words: MTHFR, metafolin, folic acid, pregnancy.

scholarly journals Folate in Red Rhapsody Strawberry—Content and Storage Stability.

Proceedings ◽  
2020 ◽  
Vol 70 (1) ◽  
pp. 47
Author(s):  
Julius Rami ◽  
Caroline Dumler ◽  
Nadine Weber ◽  
Michael Rychlik ◽  
Gabriele Netzel ◽  
...  
Keyword(s):  
Storage Stability ◽  
Active Form ◽  
Food Composition ◽  
Biologically Active ◽  
Stable Isotope Dilution ◽  
Isotope Dilution Assay ◽  
C Storage ◽  
Vitamin B9 ◽  
Adults And Children ◽  
And Storage

Folate (Vitamin B9) is critical for a range of biological functions in adults and children, including DNA, protein and neurotransmitter synthesis. It is also essential for the healthy development of the fetus in early pregnancy and for the prevention of neural tube defects, such as spina bifida. Strawberries are considered a tasty and healthy fruit consumed all over the world and may potentially be an important dietary source of natural folates. However, the relative importance of strawberry as a dietary source will depend on the total folate concentration, vitamer profile, storage stability and bioavailability to humans. Red Rhapsody, an important commercial strawberry cultivar in Australia, was screened for its folate content and storage stability by stable isotope dilution assay (SIDA). Total folate content ranged from 90–118 μg/100 g fresh weight (fw), which was well above the value in the Australian Food Composition Database (39 μg/100 g fw). 5-Methyltetrahydrofolate, the biologically active form in humans, was the principal vitamer present. Furthermore, folate remained relatively stable during refrigerated (4 °C) storage (loss of only 28% after 14 days of storage). This information is relevant for consumers since the inherent perishability of strawberry fruit makes refrigerated storage common practice in Australian households.

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