scholarly journals Conditions for an in vitro culture of murine mixed hematopoietic colonies and their putative cellular origin

Blood ◽  
1982 ◽  
Vol 60 (1) ◽  
pp. 99-107
Author(s):  
BA Miller ◽  
DE Siedler ◽  
CD Dunn ◽  
AT Huang
Keyword(s):  
Gel Filtration ◽  
Colony Growth ◽  
Antigenic Determinants ◽  
Anion Exchange Column ◽  
Erythroid Progenitor ◽  
Cellular Origin ◽  
Mouse Liver Cell ◽  
Fetal Mouse Liver

The supernatant fluid of stimulated spleen cells (PHA-SCM) supported in vitro colony growth of murine marrow. In the absence of exogenous erythropoietin, it stimulated the growth of (1) myeloid colonies and (2) distinct mixed colonies containing erythroid cells, granulocytes, macrophages, and infrequently megakaryocytes in a setting structurally resembling biopsied marrow. The cells that form mixed colonies reside in a density range of 1.058–1.068 g/ml in a discontinuous albumin gradient. Active supernatant was produced by T cells in combination with a macrophage factor. DNA synthesis correlated with activity. PHA- SCM differed from erythropoietin (EPO) when chromatographed on lectin columns and did not contain EPO activity as demonstrated by the fetal mouse liver cell (FMLC) assay. The activity for mixed colony growth could be eluted from an anion exchange column with 0.07 M NaCl and eluted in a gel filtration column at a distance corresponding to a molecular weight of 39,000. Mixed colony-forming cells responsive to PHA-SCM were found to be Ia-H-2+. BFU-Es, CFU-Cs, and progenitors for myeloid colonies responsive to PHA-SCM were also H-2+ but showed significant sensitivity to anti-Ia antisera reflecting variable antigenic density. The mixed colony-forming cell appeared less differentiated than myeloid or erythroid progenitor cells examined, and its antigenic determinants are consistent with those observed for the pluripotent stem cell assayed in vivo (CFU-S).

scholarly journals Conditions for an in vitro culture of murine mixed hematopoietic colonies and their putative cellular origin

Blood ◽  
1982 ◽  
Vol 60 (1) ◽  
pp. 99-107 ◽  
Author(s):  
BA Miller ◽  
DE Siedler ◽  
CD Dunn ◽  
AT Huang
Keyword(s):  
Gel Filtration ◽  
Colony Growth ◽  
Antigenic Determinants ◽  
Anion Exchange Column ◽  
Erythroid Progenitor ◽  
Cellular Origin ◽  
Mouse Liver Cell ◽  
Fetal Mouse Liver

Abstract The supernatant fluid of stimulated spleen cells (PHA-SCM) supported in vitro colony growth of murine marrow. In the absence of exogenous erythropoietin, it stimulated the growth of (1) myeloid colonies and (2) distinct mixed colonies containing erythroid cells, granulocytes, macrophages, and infrequently megakaryocytes in a setting structurally resembling biopsied marrow. The cells that form mixed colonies reside in a density range of 1.058–1.068 g/ml in a discontinuous albumin gradient. Active supernatant was produced by T cells in combination with a macrophage factor. DNA synthesis correlated with activity. PHA- SCM differed from erythropoietin (EPO) when chromatographed on lectin columns and did not contain EPO activity as demonstrated by the fetal mouse liver cell (FMLC) assay. The activity for mixed colony growth could be eluted from an anion exchange column with 0.07 M NaCl and eluted in a gel filtration column at a distance corresponding to a molecular weight of 39,000. Mixed colony-forming cells responsive to PHA-SCM were found to be Ia-H-2+. BFU-Es, CFU-Cs, and progenitors for myeloid colonies responsive to PHA-SCM were also H-2+ but showed significant sensitivity to anti-Ia antisera reflecting variable antigenic density. The mixed colony-forming cell appeared less differentiated than myeloid or erythroid progenitor cells examined, and its antigenic determinants are consistent with those observed for the pluripotent stem cell assayed in vivo (CFU-S).

scholarly journals Erythroid regulatory factors: correlation of detectable titers in vivo and in vitro

Blood ◽  
1978 ◽  
Vol 52 (6) ◽  
pp. 1238-1242
Author(s):  
CD Dunn ◽  
RD Lange
Keyword(s):  
Liver Cell ◽  
Mouse Liver ◽  
Fetal Mouse ◽  
Regulatory Factors ◽  
Mouse Liver Cell ◽  
Fetal Mouse Liver

Titers of erythroid regulatory factors (ERF), as measured in vivo with a standard erythrocythemic mouse technique, showed a highly significant correlation with those obtained in vitro with a fetal mouse liver cell (FMLC) assay. The results from the assay in vitro were somewhat higher than would be predicted from the assay in vivo. Nevertheless, the FMLC technique has been found to be a valid and useful technique particularly in the study of serum ERF titers.

scholarly journals Erythroid regulatory factors: correlation of detectable titers in vivo and in vitro

Blood ◽  
1978 ◽  
Vol 52 (6) ◽  
pp. 1238-1242 ◽  
Author(s):  
CD Dunn ◽  
RD Lange
Keyword(s):  
Liver Cell ◽  
Mouse Liver ◽  
Fetal Mouse ◽  
Regulatory Factors ◽  
Mouse Liver Cell ◽  
Fetal Mouse Liver

Abstract Titers of erythroid regulatory factors (ERF), as measured in vivo with a standard erythrocythemic mouse technique, showed a highly significant correlation with those obtained in vitro with a fetal mouse liver cell (FMLC) assay. The results from the assay in vitro were somewhat higher than would be predicted from the assay in vivo. Nevertheless, the FMLC technique has been found to be a valid and useful technique particularly in the study of serum ERF titers.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

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.

Biochemical and pharmacokinetic characterisation of two PEGylated variants of dipetarudin

2009 ◽  
Vol 102 (09) ◽  
pp. 454-459 ◽  
Author(s):  
Anne Koehler ◽  
Goetz Nowak ◽  
Mercedes López
Keyword(s):  
Gel Filtration ◽  
Pharmacokinetic Profile ◽  
Peripheral Compartment ◽  
Therapeutic Application ◽  
Similar Activity ◽  
Elimination Half ◽  
K 12 ◽  
Extravascular Compartment

SummaryDipetarudin was coupled to polyethylene glycol (PEG)-5000 residues in order to improve its pharmacokinetic profile and to enhance its anticoagulant efficacy. The resulting compounds, mono-and di-PEGylated dipetarudin were purified by gel filtration. Mono-PEGylated dipetarudin exhibited similar activity like its non-conjugated equivalent both in vitro and in vivo. However, di-PEGylated dipetarudin showed longer distribution and elimination half-lives and higher area under the time-concentration curve in comparison with the unmodified inhibitor which may be attributed to decreased renal clearance. Futhermore, ratio k 12/k 21 decreased when the number of PEG chains coupled to dipetarudin increased. It means that the intercompartment transfer of dipetarudin, characterised by a fast distribution and a high retention in the peripheral compartment, is reverted by coupling to PEG. Thus, the transfer of mono-PEGylated dipetarudin between these compartments is similar in both senses and the transfer of di-PEGylated dipetarudin is slower from vascular to extravascular compartment than vice versa. Our results show that di-PEGylated dipetarudin produces a better and longer anticoagulant effect than unmodified dipetarudin which is a desirable attribute for future therapeutic application.

scholarly journals Fitness of Isolates of Phytophthora capsici Resistant to Mefenoxam from Squash and Pepper Fields in North Carolina

Plant Disease ◽  
2008 ◽  
Vol 92 (10) ◽  
pp. 1439-1443 ◽  
Author(s):  
Adalberto C. Café-Filho ◽  
Jean Beagle Ristaino
Keyword(s):  
North Carolina ◽  
Phytophthora Capsici ◽  
Colony Growth ◽  
Relative Fitness ◽  
In Planta ◽  
Phytophthora Blight ◽  
In Vivo Experiments ◽  
First Time

Despite the wide adoption of mefenoxam (Ridomil Gold EC) for vegetables in North Carolina, the incidence of Phytophthora blight on pepper (Capsicum annuum) and squash (Cucurbita pepo) is high. Seventy-five isolates of Phytophthora capsici were collected in five pepper and one squash field in order to assess mefenoxam sensitivity. The relative fitness of resistant and sensitive isolates was contrasted in vitro by their respective rates of colony growth and their ability to produce sporangia in unamended V8 juice agar medium. In in vivo experiments, the aggressiveness of isolates on pepper was evaluated. The frequency of resistant isolates in North Carolina populations was 63%, considerably higher than resistance levels in areas where mefenoxam is not widely adopted. Resistant isolates grew on amended media at rates >80 to 90% and >100% of the nonamended control at 100 μg ml-1 and 5 μg ml-1, respectively. Sensitive isolates did not growth at 5 or 100 μg ml-1. All isolates from three fields, including two pepper and a squash field, were resistant to mefenoxam. Populations from other fields were composed of either mixes of sensitive and resistant isolates or only sensitive isolates. Response to mefenoxam remained stable during the course of in vitro and in planta experiments. Occurrence of a mefenoxam-resistant population of P. capsici on squash is reported here for the first time in North Carolina. When measured by rate of colony growth, sporulation in vitro, or aggressiveness in planta, fitness of resistant isolates was not reduced. Mefenoxam-resistant isolates from squash were as aggressive on pepper as sensitive or resistant pepper isolates. These results suggest that mefenoxam-resistant populations of P. capsici are as virulent and fit as sensitive populations.

Age-dependent changes in volume and haemoglobin content of erythrocytes in the carp (Cyprinus carpio L.)

1989 ◽  
Vol 141 (1) ◽  
pp. 133-149 ◽  
Author(s):  
W. Speckner ◽  
J. F. Schindler ◽  
C. Albers
Keyword(s):  
Gel Filtration ◽  
Linear Increase ◽  
Human Erythrocytes ◽  
Main Peak ◽  
Red Cell ◽  
Human Haemoglobin ◽  
Cell Fractions ◽  
Haemoglobin Content

Carp erythrocytes were fractionated by angle-head centrifugation which yielded fractions with a linear increase in density. Haematological examinations revealed that the heavier red blood cells of carp had greater volumes (MCV), more haemoglobin (MCH) and higher haemoglobin concentrations (MCHC) than light ones. The same experiments with human red cell fractions yielded a decrease in MCV, constant MCH and an increase in MCHC. Haemoglobin content in individual erythrocytes was also determined by scanning stage absorbance cytophotometry to establish the frequency distribution of the cellular haemoglobin contents. In carp, the distribution was symmetrical with the means increasing with density. No such change with cell density was found in human erythrocytes. Both carp and human erythrocytes incorporated [2-14C]glycine in vitro. After gel filtration, radioactivity was detected in carp, but not in human, haemoglobin fractions. 14C was found in all three haemoglobin fractions, obtained by isoelectric focusing, and was present in the haem and in the globin. [2-14C]glycine-labelled erythrocytes were reinjected into chronically cannulated carp and followed in vivo for several months. With time, the main peak of scintillation counts shifted from red cell fractions of low to high density. This is considered as evidence that density and age of red cells in carp are positively correlated and that erythrocytes can synthesize haemoglobin while circulating in the peripheral blood.

HEMODIALYSIS OF THE RAT

1966 ◽  
Vol 44 (5) ◽  
pp. 849-859 ◽  
Author(s):  
Sumner M. Robinson ◽  
David A. Hurwitz ◽  
Robert Louis-Ferdinand ◽  
William F. Blatt
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Low Molecular Weight

A technique is described for hemodialysis of either anesthetized or non-restrained rats. In the apparatus the dialysis plates of an autoanalyzer system are used with only minor modification. The efficiency of this method has been evaluated with regard to the clearance of saccharides, both in vitro and in vivo, as well as the extraction of nitrogenous low molecular weight moieties from circulating blood. Approximately 50% of the dialyzable material was obtained in a 1-hour dialysis. Further fractionation of the dialyzate was accomplished by gel filtration (Sephadex G-25).

scholarly journals Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

1998 ◽  
Vol 66 (9) ◽  
pp. 4374-4381 ◽  
Author(s):  
John C. McMichael ◽  
Michael J. Fiske ◽  
Ross A. Fredenburg ◽  
Deb N. Chakravarti ◽  
Karl R. VanDerMeid ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bacterial Attachment ◽  
Vaccine Candidates ◽  
Isolation And Characterization ◽  
Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

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