Activin/EDF as an inhibitor of neural differentiation

Activin/EDG, a stimulator of the secretion of follicle stimulating hormone (FSH) from pituitary gland and an inducer of erythroid differentiation for Friend leukemia cells, has since been implicated in a variety of biological roles. Here, we show some novel effects of activin on murine embryonal carcinoma cells (EC cells). First, activin acts as a growth factor on undifferentiated P19 cells, a well characterized EC cell line for the study of mammalian development. Second, activin inhibits the retinoic acid (RA) induced differentiation of P19 cells to neurons and glial cells. The inhibitory effect of activin on neural differentiation, which has yet to be proved in other physiological peptides, is confirmed also on the differentiation of various neuroblastoma cell lines. Our results suggest a possible role of activin as a negative regulator of neural differentiation in mammalian development.     

Cyclic AMP is an inhibitor of stalk cell differentiation in Dictyostelium discoideum

Cyclic AMP and DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) together induce stalk cell differentiation in vitro in Dictyostelium discoideum strain V12M2. The induction can proceed in two stages: in the first, cyclic AMP brings cells to a DIF-responsive state; in the second, DIF-1 alone can induce stalk cell formation. We report here that during the DIF-1-dependent stage, cyclic AMP is a potent inhibitor of stalk cell differentiation. Addition of cyclic AMP at this stage to V12M2 cells appreciably delays, but does not prevent, stalk cell formation. In contrast, stalk cell differentiation in the more common strain NC4 is completely suppressed by the continued presence of cyclic AMP. This fact explains earlier failures to induce stalk cells in vitro in NC4. We now consistently obtain efficient stalk cell induction in NC4 by removing cyclic AMP in the DIF-1-dependent stage. Cyclic AMP also inhibits the production of a stalk-specific protein (ST310) in both NC4 and a V12M2 derivative. Adenosine, a known antagonist of cyclic AMP action, does not relieve this inhibition by cyclic AMP and does not itself promote stalk cell formation. Finally, stalk cell differentiation of NC4 cells at low density appears to require factors in addition to cyclic AMP and DIF-1, but their nature is not yet known. The inhibition of stalk cell differentiation by cyclic AMP may be important in establishing the prestalk/prespore pattern during normal development, and in preventing the maturation of prestalk into stalk cells until culmination.     

Oestrogen as an inhibitor of human NK cell cytolysis

Natural killer (NK) cells are large granular lymphocytes attributed with the ability to lyse certain tumour cells. Previous studies on NK cells have demonstrated only an in vivo suppression of NK cell activity by 17 beta-oestradiol. The suppressive action of oestrogen on other peroxidase-containing leukocytes by virtue of its redox potential has already been documented. In the present study oestrogen suppressed NK cell cytolysis in vitro (determined by the release of [51Cr]chromate from radiolabelled cells) in a dose-dependent manner (p less than 0.01). Parallel experiments demonstrated a similar reduction in NK cell luminol chemiluminescence during activation by K562 tumour cells. Therefore, it would appear that there may be an association between NK cell lysis and their peroxidase/oxygenase activity.     

Identification of an endothelial cell product as an inhibitor of tissue factor activity

Summary A endothelial cell glycoprotein that inhibits the initiation of the coagulation process promoted by tissue factor has been isolated by heparin-sepharose, hydroxyapatite and gel filtration chromatography. As is the case for several other species synthesized by this cell type, the N-linked carbohydrate moiety is sulfated. The identification of this inhibitor hints at the possible existence of a functional balance, between the actvvator of the coagulation cascade and its inhibitor, which may become perturbed in various pathophysiological conditions. This work was supported by grants from NHLB Institute, RJR Nabisco and the Council for Tobacco Research. Editor's statement This paper describes a heparin-binding factor capable of inhibiting coagulation. The work contributes toward understanding the processes of cellular hemostasis, and also may represent the first report of a protein that could find widespread therapeutic use.     

The role of a cell surface inhibitor in early signal transduction associated with the regulation of cell division and differentiation

Serum stimulation of quiescent human fibroblast cultures resulted in a hyperphosphorylation of the nuclear retinoblastoma gene susceptibility product (RB). However, serum stimulation in the presence of 9 x 10(-8) M of a purified bovine sialoglycopeptide (SGP) cell surface inhibitor abrogated the hyperphosphorylation of the RB protein and the subsequent progression of cells through the mitotic cycle. The experimental results suggest that the SGP mediated its cell cycle arrest at a site in the cell cycle that was at the time of RB phosphorylation or somewhat upstream of the modification of this regulatory protein of cell division. Both cells serum-deprived and serum stimulated in the presence of the SGP displayed only a hypophosphorylated RB protein, consistent with the SGP-mediated cell cycle arrest point being near the G1/S interface.     

Albumin functions as an inhibitor of T cell adhesion in vitro

Jurkat T cells were found to adhere to a tissue culture flask or cover glass when 10% fetal bovine serum (FBS) was withdrawn. However, the cells adhered to extracellular matrix, especially fibronectin, regardless of the presence of FBS. We hypothesized that a substance in FBS inhibits T cells' adherence. Through a purification and identification procedure performed on the substance, bovine serum albumin (BSA) was found to inhibit T cell adhesion. BSA, furthermore, inhibited the adhesion of human primary cultured T cells. These results suggest a novel function for albumin as a T cell adhesion inhibitor.     

Osmotic behavior of red blood cell as seen with an ultrasonic method

We have measured the density and ultrasonic velocity (usv) of swine red blood cell (RBC) suspensions in the wide osmolarity range from 300 mOsm to 1400 mOsm in saline solution. The cellular density and compressibility of RBC at each osmolarity were obtained using the fact that the density and the compressibility are additive by volume. The osmolarity dependence of hematocrit was also measured at a constant number concentration of RBC in the range of 300 mOsm to 1700 mOsm. The cellular density and the cellular compressibility of RBC as well as the inverse of hematocrit were expressed well into one unique exponential type equation f (pi) = a [1 - b exp (-c pi)] with a common value for the coefficient c = 0.0025 against the osmolarity pi. The results were analyzed with a simple consideration based only upon the contribution of free water inside the erythrocyte through the volume concentration phi of the free water in it. According to this theoretical analysis, the density and the compressibility of the free water were found to be 0.990 g/cm3 and 4.59 x 10(-11) cm2/dyne which agree closely with 0.998 g/cm3 and 4.59 x 10(-11) cm2/dyn of pure water at 20 degrees C within the experimental error.     

Cyclic AMP as an inducer of the cell differentiation of Trypanosoma cruzi

The addition to epimastigotes cultures of T. cruzi, of either cAMP, monobutyryl-cAMP, dibutyryl-cAMP, 8-Br-cAMP (at 2 mM each), or the cAMP-phosphodiesterase inhibitor, papaverine (0.2 mM), promoted the in vitro differentiation of these parasite forms into metacyclics. This effect of cAMP may also be exerted in vivo in the insect vector, since cAMP was detected in the urine and in the Malpighi secretion fluids of Rodnius prolixus.     

Reversible cross-linking and CO treatment as an approach in red cell stabilization

We explored the use of the reversible cross-linking reagent dimethyl 3,3-dithiobispropionimidate (DTBP) in combination with CO treatment as an approach to stabilizing erythrocyte structure and function. Erythrocytes were cross-linked with different concentrations of DTBP for different times. DTBP increased erythrocyte osmotic stability, blocked lysolecithin-induced echinocytosis, and decreased erythrocyte deformability in a concentration- and time-dependent manner. Reversal of the cross-linking with the reducing agent dithioerythritol (DTE) restored osmotic fragility and response to lysolecithin as well as deformability. Complete reversal, however, is a function of the DTBP concentration and the time of cross-linking. The effects of cross-linking with 5 mM DTBP for 1 h were completely reversible after treatment with 10 mM DTE for 20 min. Longer incubation times or higher concentrations of DTBP resulted in partial reversal by DTE of the effects produced by DTBP. Cross-linking and reversal only slightly reduced the ATP content. The hemoglobin contained in the cross-linked and reversed cells could still undergo reversible oxygenation and deoxygenation. Erythrocytes were pretreated with CO, cross-linked with 5 mM DTBP for 1 or 3 h, loaded with a solution containing 500 mM glucose for 24 h, and freeze-dried in a medium containing 15% (w/v) albumin. Rehydration followed in distilled water. Complete recovery, measured as the percentage of free hemoglobin, was achieved for cells cross-linked with 5 mM DTBP for 3 h and freeze-dried to a final water content of 10-15%. Non-cross-linked cells lysed 100% on rehydration in distilled water. No methemoglobin (MetHb) formation as a result of freeze-drying was detected in CO-treated cells. In non-CO-treated cells 20% of the Hb was converted to MetHb.     

A protein complex expressed during terminal differentiation of monomyelocytic cells is an inhibitor of cell growth

A protein complex (PC) composed of the MRP8 and MRP14 proteins has previously been shown to be a specific inhibitor of casein kinase I and II. This PC is expressed during the late stages of terminal differentiation induced in human promyelocytic HL-60 leukemia cells by 1 alpha,25-dihydroxyvitamin D3 and in human monocytic THP-1 leukemia cells by phorbol 12-myristate 13-acetate. This expression is associated with terminal cell differentiation because incubation of HL-60 cells with an agent or condition that causes suppression of growth but not induction of differentiation does not result in expression of the PC. At concentrations of 5-15 nM, the purified PC inhibited the growth of HL-60 cells and THP-1 cells, as well as other cell types belonging to different cell lineages. This growth inhibition was preceded by a reduction in [32P]phosphate incorporation and, at the higher PC concentrations, was associated with a reduction in [3H]thymidine, [3H]uridine, and [32S]methionine incorporation. The specific expression pattern and growth-inhibitory character of the PC suggests that the complex may have a role in suppressing cell growth during monomyelocytic terminal differentiation induced by specific chemical stimuli and during physiological and pathological events associated with monomyelocytic cell functions.     

Elevation in phosphatidylethanolamine is an early but not essential event for cardiac cell differentiation

The biosynthesis of phosphatidylethanolamine was examined during differentiation of P19 teratocarcinoma cells into cardiac myocytes. P19 cells were induced to undergo differentiation into cardiac myocytes by the addition of dimethyl sulfoxide to the medium. Immunofluorescence labeling confirmed the expression of striated myosin 10 days postinduction of differentiation. The content of phosphatidylethanolamine increased significantly within the first 2 days of differentiation. [1,3-(3)H]Glycerol incorporation into phosphatidylethanolamine was increased 7.2-fold during differentiation, indicating an elevation in de novo synthesis from 1, 2-diacyl-sn-glycerol. The mechanism for the increase in phosphatidylethanolamine levels during cardiac cell differentiation was a 2.8-fold increase in the activity of ethanolaminephosphotransferase, the 1,2-diacyl-sn-glycerol utilizing reaction of the cytidine 5'-diphosphate-ethanolamine pathway of phosphatidylethanolamine biosynthesis. Incubation of P19 cells with the phosphatidylethanolamine biosynthesis inhibitor 8-(4-chlorophenylthio)-cAMP inhibited the differentiation-induced elevation in phosphatidylethanolamine levels but did not affect the expression of striated myosin. The results suggest that elevation in phosphatidylethanolamine is an early event of P19 cell differentiation into cardiac myocytes, but is not essential for differentiation to proceed.     

Decreased intracellular Na+ concentration is an early event in murine erythroleukemic cell differentiation

A decrease in Na+/K+-pump activity is an early event of Friend murine erythroleukemic (MEL) cell differentiation along the erythroid pathway. This decreased Na+/K+-pump activity has been proposed to be an essential step in differentiation which would cause a rise in intracellular Na+ concentration and then, by means of Na+/Ca2+ exchange, an increase in intracellular Ca2+. An increase in intracellular Ca2+ has been proposed to be essential for induction of differentiation. A critical prediction of this Na+-Ca2+ hypothesis is the rise in intracellular Na+. To test this prediction we have measured intracellular Na+ using a novel triple isotope method involving 3H2O, [14C]sucrose, and 22Na to measure total water, extracellular fluid, and Na+, respectively. 22Na equilibration occurred in less than 10 min. In uninduced cells, intracellular Na+ was 15.2 +/- 2.2 mM (S.D., n = 22); after induction for 14-16 h with dimethyl sulfoxide, intracellular Na+ decreased significantly (p less than 0.0001) to 8.4 +/- 1.4 mM (n = 21). The time course of the decline in intracellular Na+ paralleled that of the decrease in the Na+/K+-pump activity. These results are in direct contradiction to the Na+-Ca2+ hypothesis and suggest that observed changes in Na+/K+-pump activity can be explained solely on the basis of changes in intracellular Na+. The drop in intracellular Na+ is due to a decrease in Na+ influx. We suggest, however, that the decrease in the Na+ influx is not itself an essential event of differentiation, but may be induced by a change in the flux of another ion coupled to Na+.     

A metalloproteinase inhibitor as an inhibitor of neovascularization.

Metalloproteinases and their endogenous inhibitors are key components of an enzyme system which is important in a number of fundamental biochemical and cellular processes. Our recent work has focused on the role of a particular metalloproteinase, collagenase, and the role of an endogenous inhibitor of this enzyme in the control of neovascularization. The proteolytic degradation of extracellular matrix components by capillary endothelial cells (EC) has been shown to be one of the key prerequisites of the angiogenic process. As part of a study of the effect(s) of the inhibition of collagenase on neovascularization, we have recently reported the purification, characterization and partial NH2-terminal sequence of a cartilage-derived inhibitor (CDI) of angiogenesis in vivo and in vitro. Evidence is presented which suggests that one means of controlling deregulated vascular growth characteristic of a number of "angiogenic diseases" may be at the level of the control of metalloproteinase activity.