Mitogenic Substance Research Articles

Separation of the hot water extracts of sclerotia of Sclerotinia sclerotiorum IFO 9395 into mitogenic and antitumor-active subfractions.

The hot water extract of sclerotia of Sclerotinia sclerotiorum IFO 9395 (TSHW) was divided into representative fractions by ammonium sulfate and ethanol precipitations, and (1----3)-beta-D-glucanase treatment. The ammonium sulfate and ethanol precipitations gave a (1----3)-beta-D-glucan fraction (TSG) and a mannan fraction (TSM). After the degradation of (1----3)-beta-D-glucan in TSHW by (1----3)-beta-D-glucanase treatment, a water-insoluble protein fraction (EDP) and supernatant (EDS) were obtained. Among these fractions, the mitogenic and antitumor activities were mainly observed in EDP and TSG, respectively. On the other hand, the stimulatory effect on the reticuloendothelial system was mainly found in EDP and EDS, and a weak effect was observed in TSG. These findings suggest that the mitogenic and antitumor activities of TSHW were mainly due to the protein and (1----3)-beta-D-glucan, respectively, and that the mitogenic substance (EDP) is tightly bound to (1----3)-beta-D-glucan (TSG) in TSHW, accounting for its solubility in aqueous solution.

A form of basic fibroblast growth factor is secreted into the adluminal fluid of the rat coagulating gland.

Rat coagulating gland and its secreted fluid contain a mitogenic substance that, by numerous criteria, appears to be similar to basic fibroblast growth factor (basic FGF). It is a potent competence factor in the BALB/c3T3 mouse embryo fibroblast assay, elutes from heparin-agarose at concentrations of NaCl greater than 1.0 M, and has an isoelectric point (pI) between 8 and 9. In coagulating gland fluid, its molecular weight was 20,000 +/- 5,000, as indicated by gel filtration on Sephadex G-200. Western blot analysis of purified factor from coagulating gland cytosol and fluid, respectively, revealed immunoreactive bands of 16,000 and 17,000 relative molecular weight similar to that of purified bovine basic FGF.

The role of adrenal nerves in the regulation of adrenocortical functions.

There is now convincing evidence for the distribution of several nerve plexuses in the outer zone of the adrenal cortex. At the ultrastructural level, the close proximity of nerve boutons to cortical cells establishes the anatomical substrate for a direct neural effect on adrenal cortical cell functions. Of those neurotransmitters and neuropeptides identified to date, catecholamine, VIP, and NPY appear to be most prevalent. Importantly, the amounts of morphologically identifiable catecholamine, VIP and NPY are differentially sensitive to alteration of several physiological conditions. Furthermore, the VIP plexus appears to be intrinsic to the adrenal while the catecholamine and NPY nerve fibers enter the adrenal along blood vessels. Together, these results suggest that these multiple nerve plexuses might exert control on several adrenocortical cellular processes in addition to the regulation of adrenal blood flow. Compensatory adrenal growth, a rapid proliferative response to unilateral adrenalectomy, was previously shown to be neurally mediated. The role of the catecholamine innervation in the mediation of this process has now been demonstrated. The elimination of the sympathetic nervous system by neonatal sympathectomy inhibited the proliferative response as measured by DNA synthesis. In vivo administration of beta-adrenergic receptor blockers did not inhibit the compensatory growth response. Furthermore, the beta-adrenergic agonist isoproterenol, inhibited the rate of DNA synthesis both in vivo and in vitro. The direct action of the beta-adrenergic agonist on the adrenocortical cell DNA synthesis rate suggests that the catecholaminergic nerves tonically inhibit cell proliferation associated with compensatory growth and that the release from the beta-adrenergic inhibition is necessary for compensatory growth. Whether inhibition of the beta-adrenergic innervation is the trigger for compensatory growth or whether it is permissive to the action of a still unidentified mitogenic substance, is not yet known. The direct role of VIP and catecholamines in the regulation of steroidogenesis has been investigated in vitro using the perifused capsule-glomerulosa preparation which is representative of a normal outer zone of the adrenal and is the site of the neural plexuses and identified receptors. Both VIP and isoproterenol stimulate steroidogenesis and specifically cause a greater increase in secretion of aldosterone than corticosterone. Although the concentrations of VIP and isoproterenol required to stimulate steroidogenesis are greater than reported circulating levels, release from resident nerves could provide high local concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of B cell stimulatory factor 1/interleukin 4 in clonal proliferation of B cells.

B cell stimulatory factor 1 (BSF-1)/interleukin 4 (IL-4) has striking effects on colony formation in soft agar by small resting B lymphocytes. BSF-1 alone induces colony formation in this cell population, presumably in costimulation with a mitogenic substance present in bacto-agar. In costimulation with anti-IgM antibodies, BSF-1 caused initial proliferation of 8 to 10% of B cells, resulting in a large number of cell clusters (10 to 40 cells/clone) after 3 to 4 days of incubation. However, substantial number of colonies (greater than 40 cells/clone) developed only from these clusters when IL-1 was added to the cultures. Using a modified immunoperoxidase staining technique for the determination of IgM allotype, evidence was obtained that B cell colonies stimulated with BSF-1 are derived from a single progenitor cell. Neutralization of BSF-1 with 11B11 after a culture period of 1 to 4 days inhibits further proliferation of B cell colonies, indicating that the action of BSF-1 extends for several cell generations beyond initial stimulation from the resting state. Furthermore, it is demonstrated that the synergistic action of IL-1 with BSF-1 is confined to the late culture period, indicating a growth-promoting effect by IL-1 for activated B cells.

Ascaris suum: Human lymphocyte mitogenic factor content

Ascaris suum extract was found to contain a mitogenic factor which stimulates human lymphocytes. The extract (100 μg protein/ml) induced an increase in [ 3H]thymidine incorporation into human lymphocytes at a level similar to that obtained with pokeweed mitogen (11 μg/ml). Stimulation of mitosis appeared to be more effective with T lymphocytes than non-T lymphocytes. The mitogenic activity of the extract was reduced by only 27% when treated at 56 C for 30 min or when immersed into boiling water for 1 min. The extract was fractionated into four protein peaks by Sephacryl S-200 column chromatography. Lymphocyte mitogenic activity was observed in the first half of the first protein peak. Allergenic activity, assessed by the passive cutaneous anaphylaxis test in rats, was observed in the latter half of the same peak. These results suggest that A. suum contains both an allergen and a mitogenic substance.

Biochemical and physicochemical characterization of a mitogen obtained from an oriental crude drug, Tohki (Angelica actiloba Kitagawa).

Chemical composition and physicochemical properties of an immunomodulator, which is a non-dialyzable and acetone precipitable material(s) extracted with hot water from Angelica actiloba KITAGAWA (Yamato Tohki) (AIP), were investigated. AIP was composed of about 90% sugar and 10% protein. The major polysaccharide was identified as pectic substance(s) because its main component sugars were found to be arabinose, glucose, and galacturonic acid by gas liquid chromatographic analysis. The pectic substance(s) was not concerned with the mitogenicity of AIP since the activity was similar before and after pectinase(endo-polygalacturonase) treatment. More than half of the mitogenicity was destroyed by acid or alkali treatment. With pronase treatment, the activity was not affected, but the molecular weight of the mitogen was lowered. In addition, the mitogenic substance was partially purified from AIP by pectinase treatment and Westphal's phenol/water fractionation. The partially purified mitogenic substance(s) was rich in protein. These facts suggest that the mitogenicity of AIP was carried by a heat stable and protease resistant protein.

A Micro-Culture System for Cloning Human T Lymphocytes in Agar

A simple and reproducible single-layer micro-agar culture system for cloning of human T lymphocytes has been described. The system consists of an agar layer, in which mononuclear cells from peripheral blood were suspended, and a liquid overlayer containing the mitogenic substance. The advantages of the described method are a low incubation volume (0.5 ml) and the liquid overlayer. The addition of different test substances to the liquid overlayer is simple and easily controllable. Depending on the agar concentration a different number of formed colonies can be found floating in the liquid phase. The morphological, cytochemical and immunological characteristics of the cells from those aggregates could be easily studied. The T-cell characteristics of formed clusters and colonies was confirmed by immunofluorescence and E rosette formation. The effects of agar, serum and cell concentrations, as well as the mitogenic activation caused by three lectins on the development and number of colonies were studied on day 7, 10 and 14 of incubation.

Isolation and characterization of a mitogenic substance for murine and human B lymphocytes from Ulex europeus seeds.

A mitogenic substance, which stimulates murine and human B lymphocytes, has been isolated from the seeds of Ulex europeus. The m.w. of this mitogen was estimated to be approximately 100,000 by sodium dodecyl sulfate disc gel electrophoresis. Under physiologic conditions Ulex mitogen was in the form of a polymer. The isolated mitogen was found to contain 80% carbohydrate, in which glucose, galactose, and rhamnose were the predominant sugars. The carbohydrate portion of the mitogen could be responsible for the mitogenic activity, since the activity was destroyed by periodate treatment but not by protease digestion. It is unlikely that the mitogenic activity is due to the contamination by lipopolysaccharide (LPS), because Ulex mitogen strongly activated spleen cells from C3H/HeJ mice and did not contain a detectable amount of fatty acid. Spleen cells from nude mice, T-depleted spleen cells from conventional mice, or T-depleted spleen cells from conventional mice, or T-depleted lymphocytes from human peripheral blood responded as strongly as lymphocytes (T + B) from normal origin as measured by 3H-thymidine incorporation. It seems that the mitogenic effect is primarily on B cells. Ulex mitogen also induced the increase of immunoglobulin synthesis in murine and human B cells.

Mitogenic and Antigenic Properties of Group A Streptococcal M Protein Preparations

The separation of a mitogenic substance in M protein preparations of Streptococcus pyogenes, type 1 and type 12, is described. The isolation was achieved by gel chromatography on Biogel A 0.5 m, and by immunochromatography on immobilized type specific antibodies. In the delayed cutaneous hypersensitivity test the immunochromatography purified M 1 protein caused erythema but no unspecific mitogenicity could be found by lymphocyte transformation test. In neutralization experiments the mitogenic activity of M protein preparations was specifically inhibited by anti-erythrogenic toxin antisera.

Proliferative response of human diploid fibroblasts to intermittent light exposure.

Three- to four-hour exposure to fluorescent light, one to three times weekly, reproducibly enhanced the proliferation rate of human diploid fibroblasts. This enhancement was observed in WI-38 and a line from whole embryo mince at late population doubling level (PDL) as well as in a line from adult skin at early PDL. Single or multiple exposures of short duration stimulated proliferation, whereas exposures of long duration were cytotoxic. This proliferative response is reversible, and is mediated through the culture medium, Dulbecco Vogt's supplemented with 10% fetal bovine serum. Apparently light produces some mitogenic substance(s) in the culture medium that accumulates in the cells and is toxic or growth-stimulatory depending on its concentration per cell. Another possibility is that light produces in the medium both cytotoxic and growth-stimulatory substances.

Enhancement of [3H-Methyl]Thymidine Incorporation and Replication of Rat Chondrocytes Grown in Tissue Culture by Plasma, Tissue Extracts and Vasopressin1

A pituitary mitogenic peptide, which stimulates cellular replication of a variety of cells maintained in tissue culture, has been identified by other investigators. To study this mitogenic substance, we developed an assay to measure mitogenic substances utilizing fetal rat chondrocytes grown in monolayer culture. Mitogenic activity of added test substances was determined by [3H-methyl]thymidine incorporation into trichloroacetic acid insoluble cell products and increase in total cell number after 24 h exposure. Extracts of whole pituitary glands were more potent in stimulating these cellular indices than either those of liver or muscle, confirming that the chondrocytes are sensitive to the described mitogen. Identically prepared extracts of either anterior or posterior pituitary lobes were mitogenic indicating the presence of two or more mitogenic substances in crude pituitary extracts. Synthetic lysine vasopressin and a beef pitressin concentrate stimulated thymidine incorporation into chondrocytes in the absence of calf serum and this effect was additive to that of calf serum, suggesting that the mitogenic substance of posterior pituitary extracts was vasopressin. The maximum effective dose of vasopressin leading to an increase in either thymidine incorporation or total cell number was between 100 to 500 pg/ml, and as little as 50 pg/ml of hormone elicited an increase in total cell number. The mitogenic effect of both vasopressin and calf serum on chondrocytes was partially inhibited by 1 X 10(-4)M N, O'dibutryl cyclic adenosine 3',5' monophosphate suggesting that cell division of chrondrocytes may be under tonic control by the andenylyl cyclase system. We conclude that vasopressin is a potent mitogen for chondrocytes maintained in tissue culture and its presence must be rigorously excluded in evaluating mitogenic activity of pituitary or serum concentrates.

Persistence of anti-immunoglobulin on the lymphocyte surface.

IT is well established that addition of anti-immunoglobulin (anti-Ig) to lymphocytes expressing Ig receptors produces ‘capping’ followed by endocytosis or ‘sloughing’ of the membrane Ig, the expression of which is greatly diminished until resynthesis occurs1. Another property of anti-Ig in some species, notably the rabbit, is blastogenic activation of lymphocytes2. It is known that for the activation of lymphocytes by plant mitogens, the mitogenic substance must persist on the cell membrane for several hours before irreversible activation of the cell occurs3. Similarly, the activation of rabbit lymphocytes by antiserum to allotypic Ig determinants is largely inhibited if the Ig expressing the target allotype is added within a few hours4. Thus there is an apparent conflict between these two phenomena, one of which centres around the rapid removal of membrane Ig, the other requiring the persistence of anti-Ig for longer periods. Accordingly, experiments were designed to test the degree of retention of anti-Ig and the re-expression of membrane Ig on the surface of lymphocytes treated with anti-Ig sera and retained in tissue culture for several days.

Isolation by isoelectric focusing of a mitogenic substance released by stimulated human lymphocytes

Supernatants of human lymphocytes incubated for 4 hr with extracellular products of group A streptococci (streptococcal filtrate) and then washed and reincubated had a mitogenic activity for homologous lymphocytes. Fractionation by isoelectric focusing of the lymphocyte supernatants and of the streptococcal filtrates showed mitogenic activity in fractions with different isoelectric points. Moreover, human lymphocytes stimulated either with a mitogenic fraction of the lymphocyte supernatant or with a mitogenic fraction of the streptococcal filtrate, after treatment with bromodeoxyuridine (BUdR) and reciprocal restimulation, were responsive only to the heterologous stimulant.

Effects of Bacterial Products on Lymphocytes and Macrophages: Their Possible Role in Natural Resistance to Listeria Infection in Mice

Abstract Fluids from Listeria monocytogenes cultures contain products that possess a number of biologic activities. The fluids contain a mitogenic substance for B lymphocytes. Thus, their injection into mice produces striking hyperplasia of the B cell zones of the spleen. Also, lymphocytes in vitro are stimulated to proliferate, regardless of whether they are first depleted of T lymphocytes by exposure to anti-θ serum plus complement. These fluids injected intraperitoneally activate macrophages as judged by morphologic criteria. Mice that receive the products develop a transitory state of nonspecific resistance to infection by live Listeria. The relationships between B cell mitogenicity and macrophage activation—and protection—were not established. This kind of reaction of lymphoid cells to Listeria products may represent an early nonspecific line of defense against bacterial invasion.

Hormonal Regulation of Breast Development and Activity

The prepubertal mammary gland consists of simple ducts or tubes whose walls are composed of epithelial cells. The process of differentiation seems to include cellular proliferation at irregular sites along the wall of these ducts. The proliferating regions ultimately give rise to the alveoli, the site of milk secretion. Which hormone or hormones serve as the primary mitogenic agent or agents is not certain. However, under in vivo conditions, prolactin seems to be an especially potent mitogenic substance. Under in vitro conditions, insulin stimulates cell division for a time. During the proliferative phase, an increase in DNA indicates increased activity of the replicative process. Increased RNA synthesis also indicates an enhancement of the transcriptional process. Cortisol stimulates synthesis of the rough endoplasmic reticulum and ribosomes and stabilizes the rough endoplasmic reticulum-ribosome complex. There is evidence that prolactin also stimulates secretory protein formation. Several hormones are involved in the complete differentiation process of the secretory epithelial cell. It is not clear whether a given hormone acts as a single focus or at several foci. There is evidence that several hormones do affect equivalent events in the differentiation process.

Fluorescent staining of human chromosomes.

In the mid-1950's, Tjio and Levan applied the technics of plant cytology to the study of human cells and were able to demonstrate for the first time that 46 chromosomes were present in the normal human cell. This technical feat was followed in 1959 by the discovery that Down's syndrome was the result of an extra chromosome. The next technical advance resulted from the observation, in 1960, that kidney-bean extract ("phytohemagglutinin") contained a mitogenic substance that could stimulate peripheral leukocytes to divide. (The name phytohemagglutinin emphasizes the accidental origin of the extract's use as a mitogenic agent; the bean extract . . .