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Biomedical subjects

G R Johnson

Publications and source records attributed to G R Johnson.

At least 181 records · Page 10Linked to original sources
PubMed1985

Erythroid progenitor cells and stimulating factors during murine embryonic and fetal development.

Murine embryonic and fetal yolk-sacs, peripheral blood, and livers were assayed for hemopoietic multipotential and progenitor cell content between days 6 and 13 of gestation. Multipotential cells (Mix-CFC), erythroid-committed progenitor cells (BFU-E), and nonerythroid progenitor cells (predominantly GM-CFC) were assayed by their ability to form hemopoietic colonies in vitro when stimulated by pokeweed-mitogen-stimulated spleen-cell-conditioned media (as a source of Multi-CSF) and either human or murine erythropoietin. Late erythroid progenitor cells (CFU-E) were stimulated to form colonies by erythropoietin. Mix-CFC, BFU-E, and nonerythroid cells were first detected on day 8 in yolk-sacs, day 9 in peripheral blood, and day 11 in liver. Maximum absolute numbers of yolk-sac Mix-CFC (182), BFU-E (331), and non-erythroid CFC (1358) occurred at 11 days of gestation. The maximum frequency of peripheral blood mix-CFC (24/10(5) cells) and BFU-E (55/10(5) cells) occurred at ten days of gestation. The absolute numbers of hepatic Mix-CFC, BFU-E, nonerythroid CFC, and CFU-E increased exponentially from 11 to 13 days' gestation. CFU-E were first detected at nine days in peripheral blood, at ten days in yolk-sac, and 11 days in liver and at all ages were equally responsive to erythropoietin. The maximum frequency (151/10(5) cells) of CFU-E in the peripheral blood and the maximum number per yolk-sac (1699) both occurred on day 11 of gestation. In confirmation of previous studies, yolk-sac fluid was found to contain a macrophage colony-stimulating activity. In addition, an activity capable of stimulating fetal liver CFU-E was also detected in yolk-sac fluid. However, no activity (Multi-CSF) capable of stimulating Mix-CFC or BFU-E was detected in either yolk-sac fluid or fetal plasma.

Animals↗
PubMed1985

Preparation of human erythropoietin for tissue culture.

A simple two-step procedure has been developed for the production of human urinary erythropoietin suitable for tissue culture use. Lyophilized human urinary protein was fractionated by phenyl-Sepharose chromatography, the erythropoietin binding in 2 M lithium chloride and being eluted by an ascending linear gradient to 6 M guanidine hydrochloride. The erythropoietin was further purified by ethanol (75% vol/vol) precipitation. This two-step procedure resulted in a 25-fold purification of erythropoietin with a 50% recovery of the starting activity. The erythropoietin preparation was suitable for use in murine or human cultures, being free of protease activity, inhibitory factors, colony-stimulating factors, and erythroid-enhancing activities.

Chromatography, Gel↗
PubMed1985

Characterization of murine erythropoietin.

Although erythropoietins from human and sheep sources have been purified, a mouse erythropoietin remains essentially uncharacterized despite the fact that the mouse is most commonly used to assay for erythropoietin. Because other murine growth factors may have erythropoietic activity it will be essential to compare these with erythropoietin from murine sources rather than from other species. Here we compare the physicochemical properties and some of the bioactivities of erythropoietins from anemic mouse serum and human urine in parallel fractionations. Both molecules showed similar molecular weights (40-45,000) by gel filtration and similar retention characteristics on reverse-phase high-performance columns. However, they differed in their relative solubility in ammonium sulfate, hydrophobicity on phenyl-Sepharose, and in their ability to bind to a phenyl-boronate agarose column. Despite these differences the two molecules stimulated the same number of CFU-E in the mouse fetal liver assay and did so with similar dose-response relationships. The data indicate that putative murine erythropoietic stimuli must be compared with murine erythropoietin and not human urine erythropoietin before concluding, on the basis of fractionation results, that a factor is different from erythropoietin.

Ammonium Sulfate↗
PubMed1984

The use of discontinuous Percoll gradients to separate populations of cells from human bone marrow and peripheral blood.

Cells from human bone marrow samples were separated on discontinuous Percoll gradients and the effects of variations in osmolality were studied. Yield of committed progenitor cells as determined by CFU-c and BFU-E assays was improved when a gradient of discontinuous osmolality (360-320 mosM) was compared with gradients of uniform osmolality (320 mosM and 290 mosM). The CFU-c and BFU-E containing fraction recovered from this gradient also contained fewer T cells. When used for separation of peripheral blood samples, the same gradient yielded populations of mononuclear cells, neutrophils and platelets of more than 90% purity.

Adolescent↗
PubMed1984

Characterization of two populations of CFU-S fractionated from mouse fetal liver by fluorescence-activated cell sorting.

The properties of spleen colony forming units (CFU-S) contained in two murine fetal liver cell populations (pre-colony-forming cell (CFC) and CFC fractions) obtained after fluorescence-activated cell sorting fractionation on the basis of pokeweed mitogen binding have been analyzed. Individual spleen colonies generated by both populations were examined to determine cellularity, differential cell morphology, as well as CFU-S and in vitro CFC content. Spleen colonies from both populations contained approximately equal numbers of cells 7 days after transplantation; at 12 days, however, CFU-S from the pre-CFC fraction produced four times as many cells per colony than colonies from the CFC fraction. Although the frequency of spleen colonies containing CFU-S was similar, in comparison to the CFC fraction, spleen colonies obtained from the pre-CFC fraction contained higher absolute numbers of CFU-S. This was most evident with CFU-S assayed 12 days after transplantation of 12-day spleen colony cells (pre-CFC fraction, 84% of colonies contained CFU-S, mean of 527 per colony; CFC fraction, 77% of colonies contained CFU-S, mean of 41 per colony). Similarly, the in vitro CFC content of 7- or 12-day spleen colonies was higher in colonies produced by pre-CFC fraction CFU-S. No significant differences were observed in either the proportion of CFU-S in S-phase or the morphology of spleen colonies produced by either cell population.

Animals↗
PubMed1984

Factors affecting the generation of mast cells from multipotential colony-forming cells.

The cells responsible for the long-term in vitro generation of murine mast cells have been examined. Sequential analysis of all colony types obtained from cultures of spleen or bone marrow cells showed that only colonies derived from multipotential cells (mixed-erythroid colonies) or mast cell progenitors, contained cells responsible for mast cell generation in liquid cultures. Primary colony growth and subsequent maintenance of mast cells in liquid cultures was dependent upon pokeweed mitogen-stimulated spleen cell-conditioned medium (SCM). Mixed-erythroid colonies from 14-day cultures of spleen cells had the greatest capacity for mast cell generation. Analysis by clone splitting and transfer to high (20%) and low (2.5%) concentrations of SCM showed that the concentration of SCM used in either the primary colony culture or subsequent liquid culture phase altered both the proliferative capacity of the mast cells generated and the frequency of mast cell progenitors within individual mixed-erythroid colonies. Thus, mixed-erythroid colonies stimulated with 2.5% SCM contained the highest proportion of mast cell progenitors (34% of colonies) and when stimulated with 20% SCM, approximately fourfold higher numbers of mast cells were produced at weekly intervals from liquid cultures maintained in 2.5% SCM compared to parallel liquid cultures containing 20% SCM. These studies confirm the hemopoietic origin of mast cells and demonstrate that a factor(s) in SCM is able to modulate their proliferative potential.

Animals↗
PubMed1984

A method for determining the mechanical characteristics of orthotic knee joints.

All orthotic equipment must operate at the highest possible standards of safety and, should structural failure of a loaded component occur, there must be a minimal possibility of injury to the user. Because of the lack of definitive data on the in-service loading of lower limb orthoses it is not possible to base a test procedure on "real" loading conditions. In this paper a method of destructive testing, based on the assumption that the predominant loading consists of bending about the medio-lateral and anterior-posterior axes, is described. This method makes it possible to measure the bending strength of a knee joint side member assembly and to define the brittleness of the failure. It is suggested that the latter definition makes it possible to predict the potential safety of a particular knee joint should in-service failure occur. Some laboratory failures are described and recommendations, based on the test programme, are made for new joint designs.

Elasticity↗
PubMed1983

Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor.

A naturally occurring inducer of terminal differentiation in a murine myelomonocytic leukemia cell line (WEHI-3B) was purified to apparent homogeneity from medium conditioned by lungs from mice injected with bacterial endotoxin. The factor was purified over 400,000-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Bio-Gel P-60 in 1 M acetic acid, reverse-phase high performance liquid chromatography on a phenyl-silica column, and high performance liquid chromatography on a gel filtration column. During the first two steps, the differentiation-inducing factor was separated completely from a known proliferative regulator for normal myeloid cells, granulocyte-macrophage colony-stimulating factor, but it co-purified through all remaining steps with a distinct granulocyte-specific colony-stimulating factor. The purified factor showed a single protein band of Mr = 24,000-25,000 on sodium dodecyl sulfate-polyacrylamide gels coincident with both differentiation-inducing and granulocyte colony-stimulating activity. The granulocyte-specific colony-stimulating factor was active on WEHI-3B cells and normal granulocytic progenitor cells in vitro at the same half-maximally active concentration of 3 X 10(-12) M.

Animals↗
PubMed1983

Immunotherapy of murine leukemia. IX. The requirement for the Fc portion of antibody for successful passive serum therapy of Friend leukemia virus-induced disease.

The possible role of an antibody-dependent cellular cytotoxicity (ADCC)-type mechanism in the passive serum therapy of Friend leukemia virus (FLV)-induced erythroleukemia has been investigated by determining whether successful serum protection requires an intact Fc portion on the administered antibody. F(ab')2 fragments of IgG extracted from chimpanzee anti-FLV and goat anti-FLV gp71 antisera were prepared and compared with whole serum and uncleaved IgG for their capacity to protect DBA/2 mice against challenge with leukemogenic dose of FLV. Despite demonstrating in vitro virus neutralizing activity equivalent to that seen with antiviral serum or IgG, the virus-specific F(ab')2 preparations were devoid of protective activity. Given that passively administered F(ab')2 of goat origin have been reported to persist at stable levels in the mouse circulation, the failure of these F(ab')2 preparations to protect against virus challenge cannot be ascribed to rapid clearance from the treated animals. These results indicate that the passive serum therapy of FLV-induced disease is Fc dependent, consistent with the involvement of an ADCC-type mechanism, as well as confirming the previous suggestion that virus neutralization does not represent the sole mechanism of serum protection in this system.

Animals↗
PubMed1983

Hemopoiesis in spleen and bone marrow cultures.

Long-term cultures established from spleen cells were compared to those established from bone marrow cells for their ability to maintain hemopoiesis as measured by the presence of hemopoietic progenitor cells (in vitro CFU) and multipotent stem cells (CFU-S). The frequency of both in vitro CFU and CFU-S increased dramatically during the first 2 weeks in the spleen cultures. Following this early peak of activity, the number of progenitors and stem cells declined to undetectable levels by week 6 of culture. During this short phase of hemopoiesis, large amounts of GM-CSF could be detected in the supernatant of the spleen cultures. In contrast, bone marrow cultures did not share this early peak of hemopoiesis; however, they maintained activity for much longer periods of time than did the spleen cultures. When spleen stem cells were seeded onto functional bone marrow adherent cells, spleen-derived in vitro CFU were maintained well beyond week 6 of culture. Spleen cultures established from athymic nu/nu mice showed a greatly reduced ability to support hemopoiesis while those from S1/S1d mice maintained GM-CFU as well as cultures from normal mice.

Animals↗
PubMed1983

Factors altering the ability of multipotential hemopoietic colony-forming cells to self-generate or form progenitor cells.

Individual in vitro colonies grown from multipotential cells have been analysed for their content of colony-forming cells including BFU-E, Mix-CFC and non-erythroid CFC. The CFC content of mixed colonies was higher in colonies grown from spleen cells than in colonies grown from bone marrow cells and was unaffected by the addition of thymocytes or postendotoxin serum. When the incubation period was extended from 7 to 14 days, CFC content of mixed colonies increased. However, plate mapping and reculturing of individual mixed colonies demonstrated that different subsets of mixed colonies were present in day 7 and day 14 cultures. Although the number of mixed colonies developing decreased when stimulated by decreasing concentrations of pokeweed mitogen-stimulated spleen cell conditioned medium (SCM) the mean number of cells per colony remained unaltered and the content of CFC in such colonies increased. With the SCM concentration range tested, as the SCM concentration was decreased, the absolute number of mixed colonies containing CFC rose from 0.6 per culture to 2.4 per culture. Over this same SCM concentration range the mean number of CFC per mixed colony increased from 1.3 to 185.2. These data suggest that amplification within mixed colonies of the number of cells capable of colony formation may occur at the expense of the production of differentiating cells.

Animals↗
PubMed1983

Collagen synthesis in cultured human skin fibroblasts: effect of ascorbic acid and its analogs.

In confluent human skin fibroblasts maintained in 0.5% serum-supplemented medium. L-ascorbate specifically stimulated the rate of incorporation of labeled proline into total collagenase-sensitive protein, without changing the specific activity of the intracellular free proline. This influence of ascorbate reached a maximum at 30 microM and continued for at least 4 days, resulting in a 4-fold increase. The ascorbate effect occurred in cells at both confluent and subconfluent densities and was evident at all serum concentrations from 0.5-20%. The effect was independent of duration of the radioactive pulse between 2-6 h. D-Ascorbate, D-isoascorbate, and L-dehydroascorbate also stimulated collagen synthesis but at considerably higher concentrations, i.e., 250-300 microM. The stimulation of collagen synthesis by ascorbate and its analogs was accompanied by a decline in prolyl hydroxylase activity and a rise in lysyl hydroxylase activity; again L-ascorbate was found to be most effective. Dimethyltetrahydropterine and L-lactate failed to produce these effects.

Ascorbic Acid↗
PubMed1983

Hemopoietic progenitors, stimulating factors, and hemoglobin switching.

Studies with murine hematopoietic cells have characterized three separate molecular species acting at some point on the erythropoietic lineage (GM-CSF, BPA, Epo). These studies have been performed mainly with adult tissues, and during the embryonic phase of murine development only macrophage colony-stimulating factor and an activity capable of stimulating CFU-E can be detected in yolk sac fluid. Multipotential or erythroid-committed progenitor cells (BFU-E) obtained from 9-day murine embryos produce only adult hemoglobins when stimulated with an adult source of BPA. Studies with human cord blood erythroid progenitor cells have failed to detect a human equivalent of murine BPA. Clone transfer experiments suggest that erythropoietin alone is required to stimulate human cord blood BFU-E. Experiments of similar design are required to determine the role of various hemopoietic regulatory factors in adult human erythropoiesis.

Animals↗