The incubation of Treponema pallidum with rabbit testicular cells, HEP-2 cells, human foreskin cells, rat cardiac cells, and rat skeletal muscle cells caused morphological disruption of these cultured cells. Control preparations of heat-inactivated treponemes, a high-speed supernatant in which treponemes had been pelleted, and culture medium failed to damage the tissue cells, as did viable treponemes when the cells were incubated in inverted Sykes-Moore chambers. Thus, cellular disruption is not associated with soluble treponemal, soluble inflammatory, or soluble testicular constituents but is mediated by the specific attachment of T pallidum. This organism apparently elaborates some type of toxic activity that lyses membranes: this may explain some of the histopathology of syphilitic disease.
Terminal differentiation of mouse epidermal cells in primary culture was found to be regulated by 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3), the hormonal form of vitamin D3 produced by sequential hydroxylations in the liver and kidney. Epidermal differentiation was stimulated dose dependently by 1 alpha,25(OH)2D3 at concentrations of 0.12 nM or more. In the presence of the vitamin, stratified foci increased in number and size and contiguous foci coalesced. Basal cells in the treated cultures decreased sharply and underwent differentiation into squamous and enucleated cells which sloughed off into the medium during cultivation. The size and density of the cells became larger and lighter during the course of differentiation. 1 alpha,25(OH)2D3 markedly stimulated formation of a cornified envelope, a structure with chemically stable cross-links formed beneath the plasma membrane. Of several derivatives of vitamin D3 examined, 1 alpha,25(OH)2D3 was the most potent in inducing epidermal differentiation. Stimulation of epidermal differentiation was also observed in low calcium medium. DNA synthesis was inhibited dose dependently by 1 alpha,25(OH)2D3. A specific receptor for 1 alpha,25(OH)2D3 was found in the cytosol fraction of the epidermal cells. Scatchard plot analysis revealed that the receptor has an apparent dissociation constant (Kd value) of 54 pM and maximum binding value (Nmax) of 43 fmol/mg protein. The specificity of the receptor was demonstrated by analog competition in the following order: 1 alpha,25(OH)2D3 much greater than 25-hydroxyvitamin D3 greater than 1 alpha-hydroxyvitamin D3 greater than 24R,25-dihydroxyvitamin D3.
The platelet-derived growth factor (PDGF) binds specifically to high-affinity receptors on the surface of bovine aortic smooth muscle cells and 3T3 cells. Conditioned medium from cultured bovine aortic endothelial cells (EC) prevents PDGF binding to these receptors in a dose-dependent manner at 4 degrees C. The I-labeled PDGF that is displaced by the conditioned medium shows no increase in trichloroacetic acid solubility or decrease in binding capability to fresh cells. The competitor activity was identified as a protein by ammonium sulfate precipitability and sensitivity to trypsin. The competitor protein also is found in the serumfree conditioned media from porcine aortic EC and human umbilical vein EC but not in media from bovine aortic smooth muscle cells, human neonatal foreskin fibroblasts, or the interleukin-producing thyoma cell line EL-4. The competitor protein, like PDGF, has no effect on the specific 4 degrees C binding of either I-labeled insulin to 3T3 cells or I-labeled epidermal growth factor to human epidermoid A431 cells. Saturation curves of PDGF binding to smooth muscle cells that had been preincubated in the presence and absence of competitor indicate that the concentration for half-maximal binding of I-labeled PDGF to its receptor (≈ 30 pM) is unchanged by the competitor, whereas the apparent number of available receptor sites or maximal level of binding is greatly diminished. The competitor activity produced by cultured human umbilical vein EC is completely inhibited by antiserum against pure human PDGF, whereas the same PDGF antiserum only partially inhibits the mitogenic activity of the conditioned media. In addition, ≈ 7-fold more crude endothelium-derived growth factor is required for half-maximal inhibition of I-labeled PDGF binding as is required for half-maximal stimulation of DNA synthesis. These results suggest that EC secrete a PDGF-like protein that is biochemically distinct from the majority of EC-derived mitogenic activity.
The possibilities that the growth-promoting effect of the extracellular matrix (ECM) produced by cultured bovine corneal endothelial (BCE) cells could be due to: (1) adsorbed cellular factors released during the cell lysis process leading to the denudation of the ECM; (2) adsorbed serum or plasma factors: or (3) adsorbed exogenous growth factors have been examined. Exposure of confluent BCE cultures to 2 M urea in medium supplemented with 0.5% calf serum denudes the ECM without cell lysis. The ECM prepared by this procedure supports cell growth just as well as ECM prepared by denudation involving cell lysis. Thus, it is unlikely that the growth-promoting properties of ECM are due to adsorbed cellular factors. When the ECM produced by BCE cells grown in defined medium supplemented with high-density lipoprotein, transferrin, and insulin was compared to the ECMs produced by cells grown in the presence of serum- or plasma-supplemented medium, all were found to be equally potent in stimulating cell growth. It is therefore unlikely that the growth-promoting ability of the ECM is due to adsorbed plasma or serum components. When fibroblast growth factor (FGF)-coated and ECM-coated plastic dishes were submitted to a heat treatment (70 degrees C, 30 min) which results in the inactivation of FGF, the growth-supporting ability of FGF-coated dishes was lost, while the comparable ability of ECM-coated dishes was not affected significantly. This observation tends to demonstrate that the active factor present in the ECM is not FGF. Nor is it platelet-derived growth factor (PDGF), since treatment known to destroy the activity of PDGF, such as exposure to dithiothreitol (0.1 M, 30 min, 22 degrees C) or to beta-mercaptoethanol (10%) in the presence or absence of 6 M urea for 30 min at 22 degrees C, does not affect the growth-promoting activity of ECM. It is therefore unlikely that the growth-promoting effect of ECM is due to cellular growth-promoting agents or to plasma or serum factors adsorbed onto the ECM.
The effect of abscisic acid (ABA) on the cold hardiness of cell suspension was investigated. Cell suspension cultures of winter wheat (Triticum aestivum L. cv Norstar), winter rye (Secale cereale L. cv Cougar), and bromegrass Bromo inermis Leyss treated with 7.5 × 10 molar ABA for 4 days at 20°C could tolerate -30°C, whereas the control cultures tolerated only -7 to -8°C. The optimum concentration for increasing the cold hardiness of the cultures was 7.5 × 10 molar. The degree of cold hardiness and the rate of hardening obtained by ABA treatment was significantly higher than that induced by low temperature alone. Of ten species tested, ABA was only effective on those cultures which were capable of cold hardening upon exposure to low temperatures. The results suggest that ABA bypasses the cold requirement for hardening and also suggests that ABA triggers the genetic system(s) responsible for inducing the hardening process.
Recombinant plasmids in which the sequence encoding the bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 22.214.171.124) has been placed under the control of Drosophila heat shock protein 70 (hsp 70) or copia promoters have been introduced into cultured cells of two Drosophila species (Schneider II line of Drosophila malanogaster and D. immigrans) as calcium-phosphate complexes. Within 1-2 days after transfection functional CAT enzyme was detected in cells exposed to either CAT recombinant. The expression of the bacterial information depends on the activity of the Drosophila promoters because plasmids in which the Drosophila DNA fragments were fused to the CAT coding sequence in inverted orientation did not support the synthesis of CAT enzyme activity. Low levels of CAT activity and of hybrid mRNA were detected in cells transformed with hsp-cat recombinants when the cells were maintained at room temperature, and both mRNA levels and CAT activity increased substantially after a brief exposure to 37 degrees C. hsp-cat mRNA has the same 5 terminus as authentic Drosophila hsp 70 messenger. These experiments document a practical system for the introduction and expression of isolated genes in cultured cells of Drosophila.
125I]Insulin binding has been studied with cultured cells from an insulin-resistant patient with the Rabson-Mendenhall syndrome. Previously, this patient has been demonstrated to have decreased insulin binding to circulating blood cells. Similarly, 125I]insulin binding to cultured lymphocytes and cultured fibroblasts was markedly decreased. Whereas the decrease in 125I]insulin binding to cultured lymphocytes appeared to result from a decrease in receptor number in cultured lymphocytes, there appeared to be a reduction in the affinity of 125I]insulin binding to cultured fibroblasts. This discrepancy between the observations with the two cell types is a general phenomenon which has been described in patients with a variety of syndromes of extreme insulin resistance. It is possible that the interpretation of the studies with the fibroblasts is complicated by the confounding influence of receptors for insulin-like growth factors on the surface of those cells. If the number of insulin receptors is sufficiently reduced on fibroblasts from the patient with extreme insulin resistance, then the low level of residual 125I]insulin binding may involve primarily receptors for insulin-like growth factors, which bind insulin with relatively low affinity.
Synthetic ovine corticotropin-releasing factor (CRF) is a 41-residue peptide with high potency and intrinsic activity to stimulate the secretion of ACTH and beta-endorphin-like immunoactivity (beta-End-LI) by cultured adenohypophysial corticotropic cells. The action of CRF in vitro can be potentiated by the weaker secretagogues, vasopressin, oxytocin, epinephrine, norepinephrine, and angiotensin II. CRF-mediated secretion of ACTH and beta-End-LI is noncompetitively inhibited by pretreatment of cells with glucocorticoids. Long term exposure of adenohypophysial cells to CRF results in an increase in total medium plus cell ACTH in the cultures, suggesting that CRF can enhance rates of ACTH synthesis as well as release. CRF also stimulates the secretion of beta-End-LI by corticotropic cells cultured from the neurointermediate lobe. Higher concentrations of CRF are required to stimulate secretion by this cell type than by anterior lobe corticotropic cells. These in vitro results are consistent with CRF playing a major physiological role in the neuroregulation of secretion by anterior lobe corticotropic cells, where the peptide may interact with other modulators.
Modulation of ion channels is of increasing interest as it is an important step in the regulation of cellular functions. We have analysed the effect of 8-bromocyclic AMP on Ca2+ channels in cultured cardiac cells by the patch-clamp method and report here that there was a large increase in the probability of opening of the channels. On the basis of a recently proposed kinetic reaction scheme we suggest that cyclic AMP-dependent phosphorylation of Ca2+ channels primarily promotes the forward rate constants which lead to the open state of a Ca2+ channel during depolarization.