Reconstitution of rubella hemagglutinin on liposomes

1980 ◽  
Vol 26 (8) ◽  
pp. 899-904 ◽  
Author(s):  
Michel Trudel ◽  
Madeleine Ravaoarinoro ◽  
Pierre Payment
Keyword(s):  
Density Gradient ◽  
Immunoelectron Microscopy ◽  
Sedimentation Rate ◽  
Rubella Virus ◽  
Tween 80 ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Differential Centrifugation ◽  
Pure Preparation ◽  
Dicetyl Phosphate

The hemagglutinin of rubella virus has been purified by differential centrifugation through a sucrose density gradient after disruption of purified virus with Tween 80 – ether. The purified isolated hemagglutinin was then adsorbed on liposomes which had been prepared by mixing lecithin and dicetyl phosphate in a 3.5:1 molar ratio. The complex of hemagglutinin adsorbed on the virosomes had a higher sedimentation rate, enabling their separation from free hemagglutinin. It was thus possible to obtain a pure preparation of virosomes by rate zonal centrifugation in a sucrose density gradient containing 0.5 M NaCl. Immunoelectron microscopy showed aggregation of these virosomes with a rubella immune antiserum; this would suggest that the HA subunits are oriented in the same way as on the whole virus.

scholarly journals THE SEDIMENTATION PROPERTIES OF THE SKIN-SENSITIZING ANTIBODIES OF RAGWEED-SENSITIVE PATIENTS

1964 ◽  
Vol 120 (1) ◽  
pp. 31-43 ◽  
Author(s):  
Burton R. Andersen ◽  
Wilton E. Vannier
Keyword(s):  
Density Gradient ◽  
Sedimentation Rate ◽  
Gradient Method ◽  
Sucrose Density Gradient ◽  
Major Portion ◽  
Density Gradient Ultracentrifugation ◽  
Cell Method ◽  
Average Value ◽  
Γ Globulins

The sedimentation coefficients of the skin-sensitizing antibodies to ragweed were evaluated by the moving partition cell method and the sucrose density gradient method. The most reliable results were obtained by sucrose density gradient ultracentrifugation which showed that the major portion of skin-sensitizing antibodies to ragweed sediment with an average value of 7.7S (7.4 to 7.9S). This is about one S unit faster than γ-globulins (6.8S). The data from the moving partition cell method are in agreement with these results. Our studies failed to demonstrate heterogeneity of the skin-sensitizing antibodies with regard to sedimentation rate.

scholarly journals Some Properties of Rose Mosaic Virus from South Australia

1970 ◽  
Vol 23 (5) ◽  
pp. 1197 ◽  
Author(s):  
AA Basit ◽  
RIB Francki
Keyword(s):  
North America ◽  
Physical Properties ◽  
Mosaic Virus ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
South Australia ◽  
Sucrose Density Gradient ◽  
Differential Centrifugation ◽  
Sucrose Density Gradient Centrifugation

Isolates of rose mosaic virus (RMV) from South Australia were purified by differential centrifugation of cucumber extracts clarified by emulsification with ether, followed by sucrose density-gradient centrifugation. The virus was shown to be serologically similar to and to have many physical properties in common with RMV from North America. However, the Australian isolates studied appear to hlwe narrower host ranges.

scholarly journals Solid-phase radioimmunoassay of rubella virus immunoglobulin M antibodies: comparison with sucrose density gradient centrifugation test

1977 ◽  
Vol 5 (3) ◽  
pp. 257-262
Author(s):  
O H Meurman ◽  
M K Viljanen ◽  
K Granfors
Keyword(s):  
Density Gradient ◽  
Rubella Virus ◽  
False Positive ◽  
Solid Phase ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Igm Antibodies ◽  
Solid Phase Radioimmunoassay

The solid-phase radioimmunoassay (RIA) method developed in our laboratory for demonstrating rubella virus-specific immunoglobulin G (IgG) antibodies (Kalimo et al., 1976) was further developed for demonstrating IgM antibodies. A total of 188 serum specimens were tested. The statistical probability of obtaining a false-positive IgM result, based on determinations of 100 rubella-negative sera, was below 0.001. Nonspecific inhibitors and IgM antibodies against other viruses tested did not interfere in the assay. In 2 out of 20 (10%) serum specimens with rheumatoid factor, a false-positive IgM result was obtained. The new RIA method was compared with sucrose density gradient centrifugation, followed by hemagglutination inhibition testing of the separated immunoglobulins with respect to demonstrating IgM antibodies. In patients with acute rubella infection, IgM antibodies were demonstrated by RIA in 9 out of 20 acute-phase sera and in all 20 early-convalescent-phase sera, compared with 7 out of 20 acute-phase sera and 19 out of 20 early-convalescent-phase sera by sucrose density gradient centrifugation. The results obtained indicate that the RIA method is reliable and sensitive and suitable for routine diagnostic use.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

Characterization and Partial Purification of Heterodisperse Polysomal RNAs in Normal and Neoplastic Cells

1972 ◽  
Vol 58 (2) ◽  
pp. 71-94
Author(s):  
Ada Sacchi ◽  
Gianni Chinali ◽  
Susetta Pons ◽  
Michela Galdieri ◽  
Piero Cammarano
Keyword(s):  
Size Distribution ◽  
Density Gradient ◽  
Rat Liver ◽  
Sucrose Density Gradient ◽  
Partial Purification ◽  
Messenger Rnas ◽  
Alkaline Ph ◽  
Sedimentation Profile ◽  
Molecular Weights ◽  
Phenol Extraction

The size distribution of cytoplasmic messenger RNAs (m-RNA) has been studied in rat liver and in monodifferentiated cells (mouse reticulocytes and myelomas). It has been found that the RNA which exhibits a « rapid turnover » and a polydisperse profile of radioactivity is refractory to phenol extraction. This property has been exploited to selectively isolate m–RNA from the phenol residue by means of an extraction at an alkaline pH. The sucrose density gradient profiles of m–RNA isolated from monodifferentiated cells show monodisperse peaks having the sedimentation coefficients expected on the basis of the molecular weights of monocistronic messages for α and β chains of hemoglobin (reticulocytes) and L and H chains of immunoglobulin (myelomas). The sedimentation profile of cytoplasmic m–RNA associated with rat liver polysomes shows a much broader distribution, with sedimentation coefficients ranging from 8 S to 28 S.

scholarly journals Ribonucleic Acid Biosynthesis in Human Leukocytes

Blood ◽  
1966 ◽  
Vol 28 (2) ◽  
pp. 188-200 ◽  
Author(s):  
MARTIN J. CLINE
Keyword(s):  
Density Gradient ◽  
Ribonucleic Acid ◽  
Turnover Rate ◽  
Rna Synthesis ◽  
Gradient Analysis ◽  
Sucrose Density Gradient ◽  
Acid Biosynthesis ◽  
Total Load ◽  
Human Leukocytes ◽  
Particle Ingestion

Abstract Phagocytosis has profound effects on several aspects of the RNA metabolism of human leukocytes. The major changes induced by particle ingestion appear to be (1) an increased uptake of pyrimidine precursors from the suspending medium, (2) a contraction in the size of the nucleotide pool, (3) an accelerated rate of destruction of preexisting RNA, and (4) an increased rate of RNA synthesis. Sucrose density gradient analysis of the newly synthesized RNA suggests that several classes of RNA are involved in this process. The increased turnover rate of the nucleotide pool and of the cellular RNA of the leukocyte is proportional, within limits, to the total load of ingested particles.

scholarly journals Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

ANALYSIS OF RIBOSOMES AND POLYSOMES DERIVED FROM RABBIT MAMMARY GLANDS DURING PREGNANCY AND LACTATION

1971 ◽  
Vol 49 (4) ◽  
pp. 667-NP ◽  
Author(s):  
I. D. HERRIMAN ◽  
G. D. BAIRD ◽  
JUDY M. BRUCE
Keyword(s):  
Electron Microscopy ◽  
Density Gradient ◽  
Gradient Analysis ◽  
Late Pregnancy ◽  
Sucrose Density Gradient ◽  
Mammary Glands ◽  
Pregnancy And Lactation

SUMMARY Whole-ribosome and polysome-enriched fractions were prepared from the mammary glands of rabbits during late pregnancy and lactation. The composition of the fractions was determined by sucrose density gradient analysis and electron microscopy. The range of size of polysomal aggregates was similar in the late-pregnant and lactating gland, with aggregates containing five to nine ribosomal units predominating. However, the amount of polysomes relative to monosomes was invariably found to increase after parturition. The greater portion of this increase was accounted for by the increased abundance of aggregates containing five to nine units.

Methods in coccidiosis research: separation of oocysts from faeces

Parasitology ◽  
1976 ◽  
Vol 73 (3) ◽  
pp. 311-326 ◽  
Author(s):  
J. F. Ryley ◽  
R. Meade ◽  
Judith Hazelhurst ◽  
Thelma E. Robinson
Keyword(s):  
Glass Bead ◽  
Large Scale ◽  
Potassium Dichromate ◽  
Tween 80 ◽  
Sucrose Density Gradient ◽  
Potassium Dichromate Solution ◽  
Physical Effort ◽  
Faecal Material ◽  
Collection Medium ◽  
Forced Aeration

Factors which may be important in the large-scale extraction of coccidial oocysts from faeces have been investigated with Eimeria tenella. Age of bird, inoculum, feeding status at the time of inoculation, period of collection, feeding status during collection, collection medium, homogenization and sieving, flotation, washing, sporulation and further purification have all been considered. The aim has been to establish a method to produce the maximum number of oocysts of a required degree of purity and viability, with the expenditure of the minimum amount of physical effort, time, animals and chemicals. In our method, groups of chickens 3–4 weeks of age are inoculated with 5000 oocysts of E. tenella and food is supplied ad lib. Over the period 5–8 days after inoculation, faeces are collected in trays containing 2% (w/v) potassium dichromate solution, while food intake is restricted. The faecal material is homogenized, passed once through 40 and 100 mesh sieves, centrifuged and the oocysts recovered from the sediment by 3 flotations in saturated salt solution. Following washing, oocysts are sporulated by forced aeration at 30°C and may be further purified by hypochlorite treatment, or passage in 5% Tween 80 solution through a glass bead column followed by sucrose density gradient centri-fugation. Routine passages along these lines over a 5 year period have given a recovery of 46% of the oocysts excreted by over 7000 birds.

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