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Biomedical subjects

L K Johnson

Publications and source records attributed to L K Johnson.

At least 91 records · Page 5Linked to original sources
PubMed1980

Nuclear accumulation of epidermal growth factor in cultured rat pituitary cells.

Epidermal growth factor (EGF) is a mitogen for epidermal cells in vivo and for a wide variety of cells in culture. Recently, we and others have reported that EGF can also regulate the cellular levels of various hormones and fibronectin at concentrations which only minimally influence cell division. In addition, EGF treatment of GH3 cells affects chromatin structure such that isolated nuclei from treated cells have an increased capacity to bind bacterial RNA polymerase in initiation site complexes. Thus, the data suggest that various nuclear functions are modulated by EGF in GH3 cells despite its failure to affect DNA synthesis or cell proliferation. Recently, Yanker and Shooter have reported on the nuclear accumulation of nerve growth factor (NGF) in PC12 cells in which NGF does not promote cell division but does influence RNA and protein synthesis while inducing overt differentiation (neurite outgrowth). The similarities between the two systems and the various theories regarding the mechanism by which mitogens exert their growth-promoting and other effects led us to investigate whether an interaction between EGF and the cell nucleus can be demonstrated after surface binding and internalization of EGF in GH3 cells. We report here that when its lysosomal degradation is inhibited by chloroquine, EGF accumulates in the nucleus.

Animals↗
PubMed1980

Epidermal growth factor and expression of specific genes: effects on cultured rat pituitary cells are dissociable from the mitogenic response.

Cultured rat pituitary tumor cells, GH3/D6, which synthesize both growth hormone and prolactin, have cell-surface epidermal growth factor (EGF) receptor sites (34,000 per cell) that bind 125I-labeled EGF with a high affinity (Kd approximately 1 nM). Prolonged treatment of the cells with EGF did not stimulate cell division but did inhibit thyroid hormone-stimulated cell growth. In addition, EGF altered the morphology of the cells from a rounded to an elongated conformation. EGF also induced a perturbation of chromatin structure in GH3 cell nuclei that was detected by an increase (40%) in the number of rifampicin-resistant initiation sites for bacterial RNA polymerase. This was accompanied by an increased synthesis of prolactin and an inhibition of synthesis of growth hormone. In the presence of EGF, the synthesis of growth hormone was no longer inducible by thyroid hormone, but it remained responsive to glucocorticoids. The results demonstrate that EGF can elicit major effects on the cellular phenotype and expression of specific genes in the absence of a proliferative response. This suggests that EGF can also regulate differentiated cellular functions.

Animals↗
PubMed1980

Processing of the precursor to corticotropin and beta-lipotropin in humans.

The biosynthesis of human corticotropin (ACTH) was studied in organ culture of pituitary adenomas and by translating mRNA from an ectopic ACTH-producing tumor in a cell-free system. Peptides similar to human ACTH, beta-lipotropin, and the amino-terminal glycopeptide are cleaved from a common precursor with an apparent molecular weight of 35,000 on sodium dodecyl sulfate/polyacrylamide gels. The precursors synthesized in pituitary and ectopic ACTH-producing tissues are indistinguishable. The cleavage sites of the peptide chain appear to be similar to those previously deduced for murine and bovine ACTH. Immunoprecipitation studies suggest that the primary structure of the precursor peptide is similar in all three species. However, glycosylation is different in the human and murine precursors: the precursor to human ACTH appears to be glycosylated only in the amino-terminal fragment, not in the ACTH or beta-lipotropin sequences. Studies with an autopsied normal human pituitary suggest that neither normal nor adenomatous pituitary tissue glycosylates the ACTH sequence.

Adrenocorticotropic Hormone↗
PubMed1980

Early events after infection of Escherichia coli by bacteriophage T5. III. Inhibition of uracil-DNA glycosylase activity.

The activity of uracil-DNA glycosylase in Escherichia coli decreases dramatically to less than 10% of its original level after infection of the cells by phage T5. Phage-induced protein synthesis is required for this inhibition to occur, and the inhibition is induced by a mutant capable of injecting only the first 8% of its DNA. The inhibitor activity in extracts of infected cells is heat labile and nondialyzable, and will inhibit enzyme activity present in extracts of uninfected cells.

Cell-Free System↗
PubMed1980

Kinetics of 51CrEDTA in the rat.

The kinetics of 51Cr EDTA in the rat has been analysed. A single bolus injection after an equilibration period of 30 min, resulted in a steep monoexponential plasma clearance (T 1/2 = 40.8 +/- 4.3 min). To facilitate estimation of the EDTA space, renal clearance was eliminated by renal vascular ligation. The extrarenal clearance of EDTA was very slow (T 1/2 approximately 800 min) according to a linear function. The EDTA space was 25.7 +/- 1.8 ml/100 g body weight. The EDTA space is considered to measure the extracellular fluid volume.

Animals↗
PubMed1980

Utility of serum ferritin as a measure of iron deficiency in normal males undergoing repetitive phlebotomy.

Hematologic indices and iron balance data were obtained on 22 normal male volunteers who were subjected to a mean +/- SD phlebotomy of 164 +/- 34 ml whole blood/mo while living in a controlled environment. Over an average stay of 5 mo, volunteers did not develop anemia, but did display a reduction in iron stores that was quantitated by measurement of serum ferritin and iron balance. The percent saturation of transferrin and the usual erythrocyte parameters did not reflect changes in iron status. Loss of iron, which was calculated from quantitative phlebotomy and iron balance data, showed that a decrease of 1 ng of serum ferritin represented a loss of 4.5 +/- 5.3 mg of iron in 10 men whose initial serum ferritins were greater than 25 ng/ml, and 25.3 +/- 58.8 mg of iron in 7 men whose initial serum ferritins were less than 25 ng/ml. The period required for 3 volunteers who consumed a self-selected mixed diet at home to replace their depleted iron stores to prephlebotomy levels was about 4.5 mo. The sensitivity of serum ferritin as an index of iron stores was affirmed. In addition it was found that normal men who were consuming a mixed diet containing about 15 mg of iron daily and losing blood at a rate of 164 +/- 34 ml/mo did not increase their iron absorption sufficiently to compensate for the iron loss.

Adult↗
PubMed1980

Accuracy of predictions of long-term energy needs.

Energy needs as estimated by various methods were compared restrospectively with actual energy needs of eleven subjects who participated in long-term metabolic studies. Eight methods were compared with actual energy needs. The most satisfactory were the Harris and Benedict formula, the Kleiber formula, and the Mayo Foundation Nomogram, with an additional percentage allowed for activity energy. Interviewing methods were the least satisfactory for estimating energy needs. None of the methods was satisfactory for long-term use with individuals, indicating the necessity of follow-up and adjustment when energy intake is externally controlled.

Adolescent↗
PubMed1979

Stimulation and inhibition of cellular functions by glucocorticoids. Correlations with rapid influences on chromatin structure.

The effect of media conditions on the glucocorticoid response has been examined in three types of cultured cells. In rat pituitary tumor cells (GC cells) growth hormone production was stimulated by glucocorticoids provided fresh culture media was present (enriched media conditions). In contrast, dexamethasone either failed to induce or deinduce growth hormone synthesis if added to cultures which had not received fresh media for 3 days (depleted media condition). With human skin fibroblasts, cortisol stimulated [3H]thymidine incorporation in the enriched condition but inhibited this response in the depleted condition. In mouse lymphoma (S49) cells the enriched media conditions significantly delayed the killing response to glucocorticoids (20% killing after 24 h versus 90% killing after 24 h for the depleted condition). Thus, the magnitude and in some cases, the direction of the glucocorticoid response are sensitive to the conditions to which the cells are exposed. In all three cell types the steroid also rapidly (detectable by 15 min, maximal by 2 h) altered chromatin structure as detected by a change in the number of initiation sites for Escherichia coli RNA polymerase assayed under cell-free conditions. This early nuclear response could be in a positive or negative direction and was also affected by the culture conditions; enriched media favored a positive or less negative effect on the initiation sites by the steroid, while depleted media favored a steroid-induced inhibition of this chromatin function. In S49 and GC cells the kinetics and magnitude of the change in chromatin closely followed receptor . glucocorticoid complex binding to nuclei while removal of dexamethasone from the culture media resulted in a rapid (t 1/2 = approximately 20 min) disappearance of the effect which paralleled loss of bound hormone from the nucleus. The glucocorticoid effect on chromatin was not observed in two lines of glucocorticoid-resistant mutant S49 cells. One line (R-) lacks detectable glucocorticoid receptors; the other line (Nti) has receptors that bind the hormone normally, but the receptor . glucocorticoid complexes bind more avidly to the nucleus. These results suggest that the receptor is involved in both the stimulatory and the inhibitory effects on chromatin. The findings in the Nti cells and of a slight lag between nuclear binding of receptors and initiation site alteration implies that some receptor property, in addition to nuclear binding per se, is responsible for the influence on chromatin. These results are discussed in terms of a model in which steroid hormones initiate their actions by influencing a reaction that modifies chromatin structure. The direction and magnitude of the reaction, and its effect on the expression of specific genes, are dictated by the metabolic state and differentiation of the cell.

Animals↗
PubMed1979

Inhibition of low density lipoprotein uptake in confluent endothelial cell monolayers correlates with a restricted surface receptor redistribution.

Binding of either low density lipoprotein (LDL) or Concanavalin A (ConA) to actively growing vascular endothelial cells is associated with a redistrubution of the appropriate cell surface receptor sites which form patches and caps. This receptor lateral mobility is greatly restricted when endothelial cells reach confluence and adopt the configuration of a cell monolayer composed of closely apposed and non-overlapping cells. In this case, although the cells still exhibit specific LDL binding to the appropriate cell surface receptor sites, neither the binding of LDL nor of ConA induces a receptor redistribution. The lack of LDL receptor redistribution correlates with a marked decrease in the rate of LDL internalization. In contrast, no such density-dependent changes are observed in cell types which grow on top of each other and form multiple cell layers at confluence. Thus, neither LDL nor ConA induced cap formation in either sparse or confluent smooth muscle cell cultures and the same rate of LDL internalization is observed at both cell densities. Similarly, adsorptive endocytosis of cationized LDL (which enters the cells independently of the LDL receptor sites) was not correlated with a detectable receptor redistribution, nor was it significantly affected by changes in cell density and spatial organization. The formation of a confluent cell monolayer resting on an underlying basement membrane might therefore provide, via a change in membrane dynamics, a mechanism whereby the endothelium of large blood vessels can function as a protective barrier against the high circulating levels of LDL in plasma.

Cell Division↗
PubMed1979

Loss of hybridizable ribosomal DNA from human post-mitotic tissues during aging: I. Age-dependent loss in human myocardium.

DNA obtained from 29 male humans at autopsy was hybridized in liquid phase with tritium-labelled 18 and 28S ribosomal RNA (rRNA) in order to determine whether a change in the dosage in rDNA, which codes for rRNA, occurs during human aging. The individuals ranged in age from 3 months to 76 years. It was found that the amount of rDNA hybridizable per 260 nm absorption unit by DNA decreases by about 0.5% per year with a regression coefficient of about -0.83. These findings confirm earlier ones from this laboratory and indicate either a loss of these key genes during aging of humans or decreased hybridizability due to some other factor or factors. In any event, this degree of loss or inactivation of genes involving an all protein synthesis would seem to impair function of post-mitotic cells in response to maximal stress to about the same degree that function is lost in various human organ systems during aging, as defined by Shock and others.

Adolescent↗
PubMed1979

Developmental restrictions on transcription: determinants of the developmental program and their role in aging.

A developing plant system, the soybean hypocotyl has been used to investigate early transcription events which restrict auxin induced cellular proliferation to the appropriate developmental stage. Auxin treatment of 4-day old seedlings resulted in an early (6 hour) activation of chromatin-bound RNA polymerase activity wihich approached 200% of control values by 18 hours. This occurred without a detectable alteration in chromatin template capacity (assayed with exogenous RNA polymerase) and resulted in the synthesis of "induced RNA transcripts" as determined in vitro by nearest neighbor analysis. In contrast, auxin treatment of unresponsive 8-day old seedlings did not alter the chromatin-bound RNA polymerase activity. Hormonal activation did, however, result in the exposure of "induced template" regions in chromatin which could only be transcribed in vitro if exogenous RNA polymerase was included in the transcription reaction. Isoelectric focusing of the endogenous chromatin-bound and soluble RNA polymerase enzymes from successive developmental stages revealed that the chromatin-bound enzymes at the 2-day stage were first released from the chromatin complex and could be recovered in the soluble pool (4-day stage). This was followed by a gradual disappearance of these subspecies (6-day stage) until only a limited ensemble of RNA polymerase subspecies remained bound to chromatin and free in the soluble pool (8-day stage). Similar analyses of both the bound and free enzymes at the 4 and 8-day stages following auxin treatment revealed that the 4-day soluble enzymes could be induced to rebind to the chromatin complex in a defined sequence after hormone treatment while those of the 8-day hypocotyl were unable to do so. These developmental events indicate that the select loss of certain RNA polymerase subspecies serves to restrict the hormone responsivness of this tissue to the early developmental stages. Such restrictions could thus commit the constituent hypocotyl cells to their terminal post-mitotic phase of development.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1979

Regulation of activity of chromatin receptors for thyroid hormone: possible involvement of histone-like proteins.

Thyroid hormone receptors lose their capability for high-affinity binding of the biologically active triiodothyronine after solubilization and separation from other chromatin proteins. The high-affinity triiodothyronine-binding capacity can be reconstituted by addition of a histone-containing extract of chromatin of purified core histones (H2A, H2B, H3, and H4); a number of other acidic or basic proteins tested were ineffective. The data support a model of the receptor in which a "core" receptor subunit that contains a thyroid hormone-binding site interacts with a regulatory subunit, which is possibly a histone or histone-like species. This interaction with the "core" subunit enables the resulting "holo" receptor to bind biologically active hormones. These data also suggest that histones or related proteins can modulate the activity of nonhistone chromosomal proteins that are involved in regulating the expression of specific genes.

Animals↗
PubMed1979

Appearance in confluent vascular endothelial cell monolayers of a specific cell surface protein (CSP-60) not detected in actively growing endothelial cells or in cell types growing in multiple layers.

The formation of a highly organized vascular and corneal endothelial cell monolayer is associated with the appearance of a 60,000-dalton cell surface protein (CSP-60) (30,000 daltons after reduction with dithiothreitol) which is not detectable in rapidly growing endothelial cells and in subconfluent cultures that do not yet exhibit the strict morphology of a confluent monolayer. It is also absent from vascular smooth muscle cells and from endothelial cultures that are maintained in the absence of fibroblast growth factor and grow on top of each other at confluence. After disorganization of cells in a confluent endothelial monolayer by urea, EDTA, or trypsin, CPS-60 is no longer exposed on the cell surface, but it reappears as soon as the cells readopt their characteristic two-dimensional configuration. This reorganization can be achieved in the presence of cycloheximide and despite removal of fibronectin by urea, EDTA, or trypsin. Maximal amounts of fibronectin and no CSP-60 are detected in subconfluent, but not yet organized, endothelial cultures or in endothelial cells that no longer form a monolayer of nonoverlapping cells at confluence. Likewise, cultures of vascular smooth muscle cells contain fibronectin but no CSP-60. These results suggest that CSP-60, rather than fibronectin, could be involved in the adoption of a monolayer configuration by confluent endothelial cells.

Aorta↗
PubMed1979

Vascular endothelial cells maintained in the absence of fibroblast growth factor undergo structural and functional alterations that are incompatible with their in vivo differentiated properties.

Vascular endothelial cells cultured in the presence of fibroblast growth factor (FGF) adopt at confluence a morphological appearance similar to that of the vascular endothelium in vivo. Similarly, their apical cell surface is, as in vivo, nonthrombogenic. In contrast, when the cultures are maintained in the absence of FGF, the cells undergo within two to three passages structural and functional alterations that are incompatible with their in vivo morphological appearance and physiological function. Cultures maintained in the absence of FGF no longer adopt, upon reaching confluence, the configuration of a monolayer composed of small closely apposed and nonoverlapping, cuboidal cells. Instead, confluent cultures deprived of FGF consist of large, overlapping cells which have lost the polarity of cell surface characteristic of the vascular endothelium. The apical cell surface becomes thrombogenic, as reflected by its ability to bind platelets, whereas fibronectin, which at confluence is normally associated only with the basal cell surface, can be found both on top of and underneath the cell layer. Among other changes, both sparse and confluent cultures maintained in the absence of FGF showed a greatly increased production of fibronectin. CSP-60, a cell surface protein whose appearance is correlative with the adoption of a cell monolayer configuration, can no longer be detected in cultures maintained in the absence of FGF. Overlapping endothelial cells maintained in the absence of FGF can also no longer function as a protective barrier against the uptake of ligands such as low density lipoprotein. Exposure of the culture to FGF induces a restoration of the normal endothelial characteristics concomitant with the adoption of a flattened cell monolayer morphology. These results demonstrate that, in addition to being a mitogen. FGF is involved in controlling the differentiation and phenotypic expression of the vascular endothelium. This is reflected by its effect on the morphological appearance, polarity of cell surfaces, platelet binding capacity, and barrier function of the vascular endothelium.

Animals↗