scholarly journals Studies on freezing and thawing of flat ice on the water surface.

1993 ◽  
Vol 55 (3) ◽  
pp. 179-182
Author(s):  
Shigeru NAKATSUKA ◽  
Masao ANDO
Keyword(s):  
Water Surface ◽  
Freezing And Thawing

Immunoferritin localization of intracellular antigens in positively stained frozen sections

1978 ◽  
Vol 36 (2) ◽  
pp. 168-169
Author(s):  
K. T. Tokuyasu
Keyword(s):  
Surface Tension ◽  
Water Surface ◽  
Thin Layers ◽  
The Other ◽  
Positive Staining ◽  
Frozen Sections ◽  
The Past ◽  
Ultrathin Frozen Sections ◽  
Definition Of

During the past investigations of immunoferritin localization of intracellular antigens in ultrathin frozen sections, we found that the degree of negative staining required to delineate u1trastructural details was often too dense for the recognition of ferritin particles. The quality of positive staining of ultrathin frozen sections, on the other hand, has generally been far inferior to that attainable in conventional plastic embedded sections, particularly in the definition of membranes. As we discussed before, a main cause of this difficulty seemed to be the vulnerability of frozen sections to the damaging effects of air-water surface tension at the time of drying of the sections.Indeed, we found that the quality of positive staining is greatly improved when positively stained frozen sections are protected against the effects of surface tension by embedding them in thin layers of mechanically stable materials at the time of drying (unpublished).

Three-Dimensional Structure of Cloned T3 Connector Protein at 1.6nm Resolution

1990 ◽  
Vol 48 (1) ◽  
pp. 282-283
Author(s):  
José L. Carrascosa ◽  
José M. Valpuesta ◽  
Hisao Fujisawa
Keyword(s):  
Dna Sequences ◽  
Expression Vector ◽  
Three Dimensional ◽  
Deae Cellulose ◽  
Dna Packaging ◽  
Dimensional Structure ◽  
Two Dimensional ◽  
Freezing And Thawing ◽  
Three Dimensional Structure ◽  
Dimensional Reconstruction

The head to tail connector of bacteriophages plays a fundamental role in the assembly of viral heads and DNA packaging. In spite of the absence of sequence homology, the structure of connectors from different viruses (T4, Ø29, T3, P22, etc) share common morphological features, that are most clearly revealed in their three-dimensional structure. We have studied the three-dimensional reconstruction of the connector protein from phage T3 (gp 8) from tilted view of two dimensional crystals obtained from this protein after cloning and purification.DNA sequences including gene 8 from phage T3 were cloned, into Bam Hl-Eco Rl sites down stream of lambda promotor PL, in the expression vector pNT45 under the control of cI857. E R204 (pNT89) cells were incubated at 42°C for 2h, harvested and resuspended in 20 mM Tris HC1 (pH 7.4), 7mM 2 mercaptoethanol, ImM EDTA. The cells were lysed by freezing and thawing in the presence of lysozyme (lmg/ml) and ligthly sonicated. The low speed supernatant was precipitated by ammonium sulfate (60% saturated) and dissolved in the original buffer to be subjected to gel nitration through Sepharose 6B, followed by phosphocellulose colum (Pll) and DEAE cellulose colum (DE52). Purified gp8 appeared at 0.3M NaCl and formed crystals when its concentration increased above 1.5 mg/ml.

Factor VIII-Related Activities in Normal, Haemophilic and von Willebrand’s Disease Platelet Fractions

1978 ◽  
Vol 40 (02) ◽  
pp. 288-301 ◽  
Author(s):  
P Meucci ◽  
I R Peake ◽  
A L Bloom
Keyword(s):  
Factor Viii ◽  
Electrophoretic Mobility ◽  
Concanavalin A ◽  
Freezing And Thawing ◽  
Von Willebrand's Disease ◽  
Factor Viii Related Antigen ◽  
Von Willebrand’S Disease ◽  
Normal Plasma ◽  
Significant Difference ◽  
Ristocetin Cofactor

SummaryFactor VIII-related activities have been studied in platelet fractions in order to try to reconcile the conflicting findings of other workers, and to extend the studies. In platelets from 16 normal subjects procoagulant factor VIII was not detected. The amount of factor VIII-related antigen (FVIIIR: AG) in the cytosol per mg of protein was about twice that in the membrane fraction and about ten times that in the debris fraction. There was no significant difference between the amount of FVIIIR: AG and ristocetin cofactor (RistCof) activity in each fraction. The findings in haemophilic platelets were similar. In von Willebrand’s disease (vWd) one serverely affected patient had no detectable factor VIII related activities in any platelet fraction. In 5 patients with intermediate vWd results were normal. In a further 5, with more prolonged bleeding times, no FVIIIR: RistCof was detected in platelets, despite a normal amount of FVIIIR: AG in the cytosol and debris. The electrophoretic mobility of cytosol FVIIIR: AG was increased in all normals and patients, while that in the membrane and debris fractions had normal mobility. Cytosol FVIIIR: AG eluted later than normal FVIIIR: AG on gel filtration on Sepharose 2B, and also showed reduced antibody binding in an immunoradiometric assay. Precipitation of FVIIIR: AG by concanavalin A was incomplete in all platelet fractions from normals, and even more reduced in vWd platelet fractions. The results suggest the possibility of two types of platelet FVIIIR: AG.A factor VIII-related antigen was shown to be associated with normal washed platelets by immunofluorescence techniques (Bloom et al. 1973). Since then, several studies have been reported on the localisation of factor VIII related antigen (FVIIIR: AG), factor VIII procoagulant activity (FVIII: C) and factor VIII related ristocetin cofactor activity (FVIIIR: RistCof) within the platelets. Initially, Howard et al. (1974) indicated that FVIIIR: AG was firmly bound to the platelet membrane, and noted that in lysed platelets the level of FVIIIR: AG as measured by electroimmunodiffusion was higher than that in whole platelet suspensions. However, further studies by Nachman and Jaffe (1975) showed that FVIIIR: AG was also present to a considerable extent in the granules, and they detected none in the platelet cytosol. Bouma and colleagues (1975) were, however, able to find FVIIIR: AG and FVIIIR: RistCof in the cytosol upon freezing and thawing platelets. This FVIIIR: AG had an electrophoretic mobility comparable to that of normal plasma. They also noted that platelets which were air dried apparently had a granular FVIIIR:AG localisation by immunfluorescence; however, intact platelets in suspension did not stain by this method.Recently Ruggeri et al. (1977) and Sultan et al. (1977) have also found FVIIIR: AG in the cytosol, and the former authors reported it to have increased electrophoretic mobility when compared to normal plasma FVIIIR:AG. Results concerning the localisation of FVIIIR: AG in normal platelets have thus been conflicting. Similarly, in the few reports available concerning platelet FVIIIR: AG in von Willibrand’s disease variable results have also been obtained (Ruggeri et al. 1977, Howard et al. 1974, Shearn et al. 1974 and Bouma et al. 1975).In this study we report on the localisation of factor VIII-related activities in normal, haemophilic and von Willebrand’s disease platelets using available standard techniques as well as precipitation of FVIIIR: AG with the plant lectin concanavalin A, a procedure which has been shown to detect abnormal forms of FVIIIR:AG in certain types of von Willebrand’s disease (Peake and Bloom 1977).

Daunorubicin and Platelet Function

1981 ◽  
Vol 45 (01) ◽  
pp. 038-042 ◽  
Author(s):  
M E Pogliani ◽  
R Fantasia ◽  
G Lambertenghi-Deliliers ◽  
E Cofrancesco
Keyword(s):  
Electron Microscope ◽  
Cellular Membrane ◽  
Hemorrhagic Diathesis ◽  
Clot Retraction ◽  
Freezing And Thawing ◽  
Platelet Factor ◽  
High Doses ◽  
Very High ◽  
Platelet Functions

SummaryThe influence of Daunorubicin on some platelet functions in vitro was investigated, using different concentrations of the drug (0.01-0.02-0.04 μg/ml). Daunorubicin was shown to inhibit Collagen and Thrombin induced platelet aggregation and the intensity of inhibition depended on both drug concentration and the time of preincubation.Daunorubicin was also shown to inhibit the release reaction, the platelet prostaglandin pathway and the availability platelet factor 3; the drug at concentrations for clinical use does not damage the platelet membrane, as is the case with the freezing and thawing test, in platelet uptake of 14C-serotonin and as confirmed by the electron microscope. When very high doses (0.16 mg) of Daunorubicin are used, lysis of the platelets can be observed and this is confirmed under the electron microscope by the presence of empty platelets with fractures at the level of the cytoplasmic membrane.Finally, Daunorubicin causes irreversible inhibition of reptilase clot-retraction, even if this is less severe than with Vincristine. Working with gel-filtered platelets, it would appear that the inhibition exercised by the drug on platelet reactions is not caused through modifications in Ca++ metabolism.The authors suggest that Daunorubicin, at the dosages used clinically, induces in vitro thrombocytopathy without damaging the cellular membrane as confirmed by the electron microscope.This impairment of platelet functions could play a part in hemorrhagic diathesis observed during Daunorubicin therapy.

Assessment of Damage to Human Platelets after Aggregation and Other Injuries by Microscopic Observation and Estimation of Serotonin Uptake

1970 ◽  
Vol 23 (02) ◽  
pp. 261-275 ◽  
Author(s):  
G Zbinden ◽  
J. N Mehrishi ◽  
S Tomlin
Keyword(s):  
Platelet Aggregation ◽  
Electrophoretic Mobility ◽  
Human Platelets ◽  
Freezing And Thawing ◽  
Low Degree ◽  
Microscopic Evaluation ◽  
Negative Surface ◽  
Negative Surface Charge ◽  
Charged Macromolecules ◽  
5 Ht Uptake

SummaryThe severity of platelet damage induced by hyper- and hypotonic NaCl solutions and freezing and thawing was assessed by microscopic evaluation and measuring inhibition of 5-HT uptake. The same techniques were used to quantitate the effects of aggregating agents. The positively charged macromolecules PS, Poly-L und Poly-O reduced the net negative surface charge as determined by microelectrophoresis, caused platelet aggregation and inhibited 5-HT uptake. The damaging effects of Poly-L and Poly-O were more severe and more closely related to concentration than that of PS. The negatively charged macromolecules Poly-IC and NaPS increased the anodic electrophoretic mobility. Poly-IC and heparin caused a low degree of platelet clumping and no inhibition of 5-HT uptake. NaPS produced severe platelet damage with extensive clumping and complete inhibition of 5-HT uptake. Na laurate had the same effect, but did not alter electrophoretic mobility. ADP caused concentration-dependent platelet aggregation and inhibition of 5-HT uptake. The effects of ADP and NaPS were compared in agitated and non-agitated platelet samples containing identical concentrations of the 2 compounds. Agitation was found to increase the degree of platelet clumping and to reduce 5-HT uptake.

Bovine Platelet Proteins II. Purification of Platelet Fibrinogen

1969 ◽  
Vol 21 (03) ◽  
pp. 419-427 ◽  
Author(s):  
N. O Solum ◽  
S Łopaciuk
Keyword(s):  
Analytical Ultracentrifugation ◽  
Insoluble Fraction ◽  
Disc Electrophoresis ◽  
Plasma Fibrinogen ◽  
Starch Gel Electrophoresis ◽  
Freezing And Thawing ◽  
Insoluble Material ◽  
High Resolution Power ◽  
Starch Gel ◽  
Platelet Proteins

Summary1. Platelet fibrinogen has been purified from washed bovine platelets. The procedure was based on the methods for purification of plasma fibrinogen by fractionated precipitations and extractions with ethanol and glycine below 0°, and precipitation of proteins by dimethylformamide at 0°.2. The platelet extract obtained by freezing and thawing of the cells, freed from insoluble material by centrifugation at 23,000 x g for 30 min, contained 0.22 ±0.003mg fibrinogen per 109 platelets. Total protein of this fraction was 0.77 ±0.08 mg per 109 platelets whereas that of the insoluble fraction was 0.79 ±0.09 mg per 109 platelets.3. The most purified platelet fibrinogen fraction contained 91-98% of the protein in a thrombin-clottable state. The yield was approx. 20%. It showed homogeneity in analytical ultracentrifugation, in immunoelectrophoresis using an antiserum produced by immunization of rabbits against platelet extract, and in starch gel electrophoresis using a discontinuous system of Tris HCl and borate buffers offering a high resolution power towards the platelet proteins. Polyacrylamide disc electrophoresis revealed two to three faint lines behind the main fibrinogen line. At least one such line was also observed with purified plasma fibrinogen.

Antithrombin III Antigen in Human Platelets

1985 ◽  
Vol 54 (03) ◽  
pp. 599-602 ◽  
Author(s):  
M Léon Alhenc-Gelas ◽  
M Aiach ◽  
A Gorenflot ◽  
J P Andreux
Keyword(s):  
Arachidonic Acid ◽  
Enzyme Immunoassay ◽  
Antithrombin Iii ◽  
Human Platelets ◽  
Mean Value ◽  
Normal Blood ◽  
Freezing And Thawing ◽  
Healthy Donors ◽  
At Iii ◽  
Storage Granules

SummaryImmunoreactive AT III was found in human platelets. AT III antigen was quantified in platelets taken from each of 17 healthy donors by a specific competitive enzyme immunoassay using purified AT III and AT III antibodies. AT III antigen levels in extracts of washed platelets disrupted by freezing and thawing ranged from 32 to 140 ng per 109 platelets with a mean value of 70.3 ± 27.3. When stimulated by arachidonic acid, the platelets released AT III antigen together with immunoreactive fibrinogen. These results show that AT III is present in platelets at a level corresponding to approximately 0.01% of total antithrombin in normal blood, and suggest that platelet AT III, like fibrinogen, is contained in the storage granules.

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