Intracellular Transactivation of the Insulin-like Growth Factor I Receptor by an Epidermal Growth Factor Receptor

Growth factor receptors may be transactivated not only by homologous receptors, but also by heterologous receptors. We have investigated this possibility, using for this purpose R⁻/EGFR cells, which are mouse embryo cells devoid of IGF-I receptors, but overexpressing the EGF receptor. At variance with mouse embryo cells with a wild-type number of IGF-I receptors and overexpressing the EGF receptor, R⁻/EGFR cells cannot grow in EGF only, nor can they form colonies in soft agar. However, if a wild type human IGF-I receptor is stably transfected into R⁻/EGFR cells, growth in EGF and colony formation in soft agar are restored. To determine a possible interaction between the two receptors, we transfected into R⁻/EGFR cells a number of IGF-I receptor mutants with different impaired functions. The only IGF-I receptor that cannot reverse the growth phenotype of R⁻/EGFR cells is a receptor with a point mutation at the ATP-binding site. All other mutant receptors, even when incapable of responding to IGF-I with a mitogenic signal, made R⁻/EGFR cells fully capable of responding with growth to EGF stimulation. IGF-I receptor mutants that are mitogenic but not transforming made R⁻/EGFR cells grow in EGF only, but were incapable of inducing the transformed phenotype. The mutant IGF-I receptors are activated (tyrosyl phosphorylation of IRS-1) in response to EGF. These experiments indicate that certain IGF-I receptor mutants with loss of function can be reactivated intracellularly by an overexpressed EGF receptor and confirm that the C-terminus of the IGF-IR is required for its transforming activity.     

Regulation of cap formation by concanavalin A and the differentiation of myeloid leukemic cells : Relationship to free and anchored surface receptors

There are three types of myeloid leukemic cells. One type (Fc⁺C3⁺D⁺) can be induced by a protein in serum from mice injected with bacterial endotoxin to form rosettes for Fc and C3 receptors, migrate in agar, attach to the surface of a Petri dish and differentiate to mature macrophages and granulocytes. A second type (Fc⁺C3⁺D⁻) can be induced by this protein to form Fc and C3 rosettes, but not to migrate, attach or form mature cells and the third type of cell (Fc⁻C3⁻D⁻), could not even be induced to form rosettes. Fc⁺C3⁺D⁺, Fc⁺C3⁺D⁻ and Fc⁻C3⁻D⁻ cells before induction, showed 50%, 5% and 0% cells with a concanavalin A (ConA)-induced cap, respectively. Treatment with vinblastine or colchicine, but not with lumicolchicine, increased the frequency of cap formation to 100% in Fc⁺C3⁺D⁺, 95% in Fc⁺C3⁺D⁻ but only to 50% in Fc⁻C3⁻D⁻ cells. Of the properties that can be induced, the induction of C3 rosettes, cell migration and cell attachment can be determined 24 h after induction. The increased ability to form a cap produced by vinblastine, did not change the inducibility of cells for these properties. The results indicate that although free surface receptors for ConA and receptors anchored to tubulin can form a cap on myeloid leukemic cells, there are also receptors that may be anchored to structures other than tubulin, that did not form a cap. It is suggested that the ability of myeloid leukemic cells to differentiate is associated with the frequency of ConA surface receptors that are free or have specific types of anchorage.     

Kinetic evidence for a common mechanism of capping on lymphocytes

1. Differences in the rates at which ligands cap various receptors on the same cells, and their sensitivity to various drugs, have been interpreted as evidence that there are distinct mechanisms for `fast' and `slow' cap formation. We have examined the factors which determine the rate of cap formation of three receptors on mouse splenic lymphocytes or thymocytes, and compared the effects of cytochalasin B or colchicine under conditions where the different receptors cap at similar rates. 2. When surface immunoglobulin, concanavalin A receptors, or θ antigen are induced to cap at their maximal rates by appropriate concentrations of one or more cross-linking ligands, the half-time for maximal capping of each receptor population is between 1.5 and 3.0min at 37°C. Slower rates of cap formation are obtained by using non-optimal concentrations of the cross-linking ligands. 3. When the three receptors were induced to cap at similar rates (either maximal or slower), 10μm-cytochalasin B caused a similar decrease in the rate of cap formation for each receptor, without affecting the eventual extent of capping. At comparable capping rates on control cells, colchicine (10μm) increased the rate of cap formation for surface immunoglobulin and concanavalin A receptors to a similar extent, without affecting the eventual extent of cap formation. In contrast, colchicine had no detectable effect on the capping of θ antigen. 4. From these results, we conclude that there are no intrinsic differences in the rates at which different receptors can be induced to cap that can be used to diagnose differences in their mechanisms of cap formation. The observation that ligand concentration and the drugs acting on the cytoskeleton generally affect the rate but not the extent of cap formation accounts for the wide variation in reported effects of the drugs on cap formation measured at fixed times. The receptor-specific effect of colchicine on surface immunoglobulin and concanavalin A receptors, but not θ antigen, is not readily compatible with models of cap formation which depend on lipid or membrane flow.     

Effect of dexamethasone and cytochrome P 450 inhibitors on the formation of 7α-hydroxydehydroepiandrosterone by human adipose stromal cells

7α-Hydroxydehydroepiandrosterone (7α-OHDHA) is a major metabolite of dehydroepiandrosterone (DHA) using adipose stromal cells. To gain a better understanding of the factors regulating DHA metabolism, we examined the effect of dexamethasone and cytochrome P 450 inhibitors on the formation of 7α-OHDHA. Dexamethasone (10⁻⁹ to 10⁻⁷ M) stimulated 7α-OHDHA formation in a dose-dependent manner with a 2- to 5-fold stimulation at 10⁻⁷ M. The dexamethasone stimulated 7α-OHDHA formation was inhibited by RU486 in a dose-dependent manner with suppression to basal levels at 10⁻⁶ M. Progesterone (10⁻⁷ M) had no effect on 7α-OHDHA formation suggesting that the dexamethasone stimulation was acting through the glucocorticoid receptor. Conversion of DHA to 7α-OHDHA was inhibited by ketoconazole and metyrapone. An inhibition of 70–80% was obtained with ketoconazole and 25–60% with metyrapone at concentrations of 10⁻⁵ M. Aminoglutethimide phosphate was less effective than either ketoconazole or metyrapone in inhibiting 7α-OHDHA formation with <30% inhibition at 10⁻⁵ M. These studies indicate that 7-hydroxylation provides an alternative pathway for the metabolism of DHA in peripheral tissues. This pathway, which is regulated by glucocorticoids, may influence the amount of DHA available for conversion to androstenedione and its subsequent aromatization to estrone. The biological role of the 7-oxygenated metabolites and their effects on other steroidogenic pathways have not been established.     

Protection against a Lethal Avian Influenza A Virus in a Mammalian System

The question of how best to protect the human population against a potential influenza pandemic has been raised by the recent outbreak caused by an avian H5N1 virus in Hong Kong. The likely strategy would be to vaccinate with a less virulent, laboratory-adapted H5N1 strain isolated previously from birds. Little attention has been given, however, to dissecting the consequences of sequential exposure to serologically related influenza A viruses using contemporary immunology techniques. Such experiments with the H5N1 viruses are limited by the potential risk to humans. An extremely virulent H3N8 avian influenza A virus has been used to infect both immunoglobulin-expressing (Ig^+/+) and Ig^−/− mice primed previously with a laboratory-adapted H3N2 virus. The cross-reactive antibody response was very protective, while the recall of CD8⁺ T-cell memory in the Ig^−/− mice provided some small measure of resistance to a low-dose H3N8 challenge. The H3N8 virus also replicated in the respiratory tracts of the H3N2-primed Ig^+/+ mice, generating secondary CD8⁺ and CD4⁺ T-cell responses that may contribute to recovery. The results indicate that the various components of immune memory operate together to provide optimal protection, and they support the idea that related viruses of nonhuman origin can be used as vaccines.     

A mesocosm study of Phaeocystis globosa population dynamics: I. Regulatory role of viruses in bloom control

The regulatory role of viruses on population dynamics of the prymnesiophyte Phaeocystis globosa was studied during a mesocosm experiment in relation to growth and loss by microzooplankton grazing and cell lysis. The mesocosms were conducted under varying light conditions (20 and 150 μmol photons m⁻² s⁻¹) and nutrient regime (inorganic nitrogen to phosphorus ratios of 4, 16 and 44). Overall, viruses infecting P. globosa (PgV) were found to be an important cause of cell lysis (30–100% of total lysis) and a significant loss factor (7–67% of total loss). We demonstrate that the morphology of P. globosa cells (solitary versus colonial) differently regulated viral control of P. globosa bloom formation. Reduced irradiance (20 μmol photons m⁻² s⁻¹) was provided for 11 days to select for the solitary cell morphotype. Viruses were able to restrict P. globosa bloom formation even after irradiance became saturating again (150 μmol photons m⁻² s⁻¹). Saturating light conditions from the start of the experiment allowed colony formation and because the colony-morphotype acted as a mechanism reducing viral infection bloom formation succeeded. Nutrient depletion, however, affected specifically the colonies that disintegrated while releasing single cells. Virus infection of these solitary cells resulted in the termination of the bloom. The nature of phytoplankton growth-limiting nutrient (nitrate and/or orthophosphate) did not seem to noticeably affect the level of viral control.     

Effect of microtubule-disrupting agents on superoxide production in human polymorphonuclear leukocytes

We explored the effects of compounds known or proposed to affect microtubule functions on superoxide (O₂⁻) production in human polymorphonuclear leukocytes stimulated by N-formyl-methionyl-phenylalanine (f-Met-Phe), calcium ionophore A23187 and phorbol myristate acetate. F-Met-Phe-induced O₂⁻ production was markedly potentiated not only by microtubule-disrupting agents, including colchicine, vincristine, vinblastine, nocodazole, podophyllotoxin and griseofulvin, but also deuterium oxide (²H₂O), which is proposed to stabilize microtubules, and not affected by lumicolchicine. Ionophore A23187-induced O₂⁻ production was not influenced by colchicine, and markedly enhanced by ²H₂O, whereas phorbol myristate acetate-induced O₂⁻ production was not influenced by colchicine, and slightly inhibited by ²H₂O. ²H₂O did not counteract the effects of colchicine and vice versa. Dibutyryl cyclic AMP and prostaglandin E₁ inhibited O₂⁻ production stimulated by f-Met-Phe and ionophore A23187, whereas phorbol myristate acetate-induced O₂⁻ production was strongly resistant to the inhibitory effect of these agents. The enhancing effect of colchicine and ²H₂O on f-Met-Phe-induced O₂⁻ production was abolished by dibutyryl cyclic AMP. Colchicine promoted concanavalin A cap formation, and ²H₂O produced cancanavalin A patch formation, whereas dibutyryl cyclic AMP did not affect the distribution of concanavalin A receptors. In addition, ²H₂O and dibutyryl cyclic AMP did not interfere with the colchicine-induced concanavalin A cap formation. These findings suggest that f-Met-Phe, ionophore A23187 and phorbol myristate acetate may activate the oxidative metabolism of human polymorphonuclear leukocytes through different mechanisms, and that microtubule-disrupting agents, ²H₂O and cyclic AMP agonists may affect the different steps of the activating system of NAD(P)H oxidase.     

Cell surface mobility of a bacterial R-form glycolipid, mR595, after binding to rat cells, and its effect on the mobility of concanavalin A receptors

Binding of small amounts of glycolipid mR595 to rat cells, followed by sequential incubation of cells at 37 °C with rabbit anti-glycolipid mR595 and fluorescein-conjugated sheep anti-rabbit γ-globulin antisera results in the localization of fluorescence at one pole of the cell surface (capping). Binding of higher amounts of glycolipid mR595 to cells not only inhibits formation of glycolipid caps but those of the ConA receptor-fluorescent ConA complex as well. Glycolipid mR595 binding does not alter [³H]ConA binding to cells but cell agglutination by ConA is inhibited in a competitive fashion. Binding of small amounts of ConA to cells does not affect glycolipid capping. Colchicine and cytochalasin B (CB) treatment of cells inhibits glycolipid cap formation.     

Scn3b knockout mice exhibit abnormal ventricular electrophysiological properties

We report for the first time abnormalities in cardiac ventricular electrophysiology in a genetically modified murine model lacking the Scn3b gene (Scn3b^−/−). Scn3b^−/− mice were created by homologous recombination in embryonic stem (ES) cells. RT-PCR analysis confirmed that Scn3b mRNA was expressed in the ventricles of wild-type (WT) hearts but was absent in the Scn3b^−/− hearts. These hearts also showed increased expression levels of Scn1b mRNA in both ventricles and Scn5a mRNA in the right ventricles compared to findings in WT hearts. Scn1b and Scn5a mRNA was expressed at higher levels in the left than in the right ventricles of both Scn3b^−/− and WT hearts. Bipolar electrogram and monophasic action potential recordings from the ventricles of Langendorff-perfused Scn3b^−/− hearts demonstrated significantly shorter ventricular effective refractory periods (VERPs), larger ratios of electrogram duration obtained at the shortest and longest S₁–S₂ intervals, and ventricular tachycardias (VTs) induced by programmed electrical stimulation. Such arrhythmogenesis took the form of either monomorphic or polymorphic VT. Despite shorter action potential durations (APDs) in both the endocardium and epicardium, Scn3b^−/− hearts showed ΔAPD₉₀ values that remained similar to those shown in WT hearts. The whole-cell patch-clamp technique applied to ventricular myocytes isolated from Scn3b^−/− hearts demonstrated reduced peak Na⁺ current densities and inactivation curves that were shifted in the negative direction, relative to those shown in WT myocytes. Together, these findings associate the lack of the Scn3b gene with arrhythmic tendencies in intact perfused hearts and electrophysiological features similar to those in Scn5a^+/− hearts.     

Effect of zinc-chelating dipeptides on osteoblastic MC3T3-E1 cells: Activation of aminoacyl-tRNA synthetase

The effect of zinc-chelating dipeptides on osteoblastic MC3T3-E1 cells was investigated. As zinc compounds, we used zinc sulfate, AHZ, di(N-acetyl-β-alanyl-l-histidinato)zinc (AAHZ), and di(histidino)zinc (HZ). Cells were cultured for 72 h in the presence of zinc compounds (10⁻⁸–10⁻⁵M). The effect of AHZ (10⁻⁷ and 10⁻⁶M) to increase protein and deoxyribonucleic acid (DNA) contents in the cells was the greatest in comparison with those of other zinc compounds. Zinc sulfate and HZ at 10⁻⁷M did not have an effect on the cellular protein content. AHZ (10⁻⁶M) had a potent effect on cell proliferation, although zinc sulfate (10⁻⁶M) had no effect. β-Alanyl-l-histidine (10⁻⁶ and 10⁻⁵M) did not have an appreciable effect on the cells. Those effects of AHZ (10⁻⁶M) on osteoblastic cells were completely abolished by the presence of cycloheximide (10⁻⁶M). AHZ (10⁻⁸–10⁻⁵M) directly activated [³H]leucyl-tRNA synthetase in the cell homogenate, whereas the effect of zinc sulfate was seen at 10⁻⁶ and 10⁻⁵M. The present study suggests that the chemical form of zinc-chelating β-alanyl-l-histidine (AHZ) can reveal a potent anabolic effect on osteoblastic cells, and that AHZ directly stimulates protein synthesis.     

Control of Epithelial Ion Transport by Cl<Superscript>−</Superscript> and PDZ Proteins

Inhibition of epithelial Na⁺ channels (ENaC) by the cystic fibrosis transmembrane conductance regulator (CFTR) has been demonstrated previously. Recent studies suggested a role of cytosolic Cl⁻ for the interaction of CFTR with ENaC, when studied in Xenopus oocytes. In the present study we demonstrate that the Na⁺/H⁺-exchanger regulator factor (NHERF) controls expression of CFTR in mouse collecting duct cells. Inhibition of NHERF largely attenuates CFTR expression, which is paralleled by enhanced Ca²⁺-dependent Cl⁻ secretion and augmented Na⁺ absorption by the ENaC. It is further demonstrated that epithelial Na⁺ absorption and ENaC are inhibited by cytosolic Cl⁻ and that stimulation by secretagogues enhances the intracellular Cl⁻ concentration. Thus, the data provide a clue to the question, how epithelial cells can operate as both absorptive and secretory units: Increase in intracellular Cl⁻ during activation of secretion will inhibit ENaC and switch epithelial transport from salt absorption to Cl⁻ secretion.This revised version was published online in August 2005 with a corrected cover date.     

Rabbit lymphocyte subpopulations. II. Separation and characterization of two Ig+ cell subpopulations.

Rabbit lymph node cells (Ig⁺Ig^?) were separated into Ig⁺ and Ig^? populations by rosette formation with anti-Ig antibody-coated erythrocytes and centrifugation on Ficoll-Hypaque. Subpopulations of Ig⁺ cells were obtained by treating rosetted cells with autologous serum which dissociated approximately half of the rosettes. The stable rosetted cells (Ig⁺ S) were separated from the labile unrosetted cells (Ig⁺L) by centrifugation on Ficoll-Hypaque. The Ig⁺S population contained most of the Ig-secreting cells and responded poorly to mitogens. The Ig⁺L population contained few Ig-secreting cells and responded well to mitogens. Approximately 50% of Ig⁺L cells became Ig⁺S when cultured with Ig^? cells but this transition did not occur if Ig⁺S cells were added to the culture at the start of the incubation period. Purified Ig⁺ L cells lost their ability to form rosettes when cultured by themselves but retained their ability to form rosettes when cultured wih Ig^? cells. The data indicate that the Ig⁺S and Ig⁺L populations are at different stages in the differentiation of Ig⁺ cells (B cells) and that the Ig⁺L cells are subject to the regulatory influences of both Ig^? and Ig⁺S cells.     

Inhibition of mitochondria-dependent apoptosis by 635-nm irradiation in sodium nitroprusside-treated SH-SY5Y cells

Nitric oxide (NO) is a major factor contributing to the loss of neurons in ischemic stroke, demyelinating diseases, and other neurodegenerative disorders. NO not only functions as a direct neurotoxin, but also combines with superoxide (O₂⁻) by a diffusion-controlled reaction to form peroxynitrite (ONOO⁻), a species that contributes to oxidative signaling and cellular apoptosis. However, the mechanism by which ONOO⁻ induces apoptosis remains unclear, although subsequent formation of reactive oxygen species (ROS) has been suggested. The aim of this study was to further investigate the triggers of the apoptotic pathway using O₂⁻ scavenging with light irradiation to block ONOO⁻ production. Antiapoptotic effects of light irradiation in sodium nitroprusside (SNP)-treated SH-SY5Y cells were assayed by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, DNA fragmentation, flow cytometry, Western blot, and caspase activity assays. In addition, NO, total ROS, O₂⁻, and ONOO⁻ levels were measured to observe changes in NO and its possible involvement in radical induction. Cell survival was reduced to approximately 40% of control levels by SNP treatment, and this reduction was increased to 60% by low-level light irradiation. Apoptotic cells were observed in the SNP-treated group, but the frequency of these was reduced in the irradiation group. NO, O₂⁻, total ROS, and ONOO⁻ levels were increased after SNP treatment, but O₂⁻, total ROS, and ONOO⁻ levels were decreased after irradiation, despite the high NO concentration induced by SNP treatment. Cytochrome c was released from mitochondria of SNP-treated SH-SY5Y cells, but not of irradiated cells, resulting in a decrease in caspase-3 and -9 activity in SNP-treated cells. Finally, these results show that 635-nm irradiation, by promoting the scavenging of O₂⁻, protected against neuronal death through blocking the mitochondrial apoptotic pathway induced by ONOO⁻ synthesis.     

Characteristics of fed-batch cultures of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> containing human-like collagen cDNA at different specific growth rates

Fed-batch cultures of recombinant Escherichia coli BL21 for producing human-like collagen were performed at different specific growth rates (0.1~0.25 h⁻¹) before induction and at a constant value of 0.05 h⁻¹ after induction by the method of pseudo-exponential feeding. Although the final biomass (around 69 g l⁻¹) was almost the same in all fed-batch cultures, the highest product concentration (13.6 g l⁻¹) was achieved at the specific growth rate of 0.15 h⁻¹ and the lowest (9.6 g l⁻¹) at 0.25 h⁻¹. The mean productivity of human-like collagen was the highest at 0.15 h⁻¹ (0.57 g l⁻¹ h⁻¹) and the lowest at 0.1 h⁻¹ (0.35 g l⁻¹ h⁻¹). In the phase before induction, the cell yield coefficient (Y_X/S) decreased when the specific growth rate increased, while the formation of acetic acid increased upto 2.5 g l⁻¹ at 0.25 h⁻¹. The mean product yield coefficient (Y_P/S) also decreased with specific growth rate increasing. The respiration quotient (RQ) increased slightly with specific growth rate increasing before induction, and the mean value of RQ was around 72%. The optimum growth rate for human-like collagen production was 0.15~0.2 h⁻¹.     

De novo purine and pyrimidine biosynthesis during thymocyte concanavalin A stimulation

Measurement of the incorporation of [¹⁴C]formate and [¹⁴C]HCO₃⁻ enable us to characterize the activity of the synthetic pathways of purine and pyrimidine nucleotides in vitro throughout the process of the conA stimulation of rat thymocyte populations enriched for immunocompetent cells by isopycnic centrifugation. Our results show that de novo synthesis compensates for the total absence of exogenous nitrogenous bases and nucleosides in the culture medium. The magnitude of the proliferative response in media supplemented with dialysed fetal calf serum (FCS) was found to be the same as that observed when complete FCS was used in the culture medium. The induction of de novo synthesis (1) contributes to the expansion of the free nucleotide pool (the quantity of ATP measured in the perchloric acid (PCA)-soluble material of a same number of cells is increased by a factor of 10); (2) supplies the nucleotides necessary for nucleic acid synthesis (the total number of cells is increased by a factor of 3 after 4 days culture period). The activity of the metabolic pathways involved appears to be solicited by the dynamic requirements for thymocyte stimulation. For each step in the cellular activation a steady state of adenylic nucleotide synthesis and condensation into nucleic acids is established.     

Sulfalene concentrations in plasma and blood cells of Plasmodium falciparum malaria cases after treatment with metakelfin using high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method using acetonitrile–methanol–1 M perchloric acid–water (25:9:0.8:95, v/v/v) at a flow-rate of 1.0 ml min⁻¹ on LiChrospher 100 RP 18 column (250×4 mm; 5 μm) with UV (254 nm) detection has been developed for the determination of sulfalene in plasma and blood cells after oral administration of the antimalarial drug metakelfin. Calibration curves were linear in the range 0.5–100 μg ml⁻¹. The limit of quantification was 50 ng ml⁻¹. Within-day and day-to-day coefficients of variation averaged 3.84 and 5.31%, respectively. Mean extraction recoveries of sulfalene from plasma and blood cells were 87.21 and 84.65%, respectively. Mean concentrations of sulfalene in plasma of P. falciparum cases on days 2, 7 and 15 were 44.58, 14.90 and 1.70 μg ml⁻¹, respectively; in blood cells concentrations of sulfalene were 7.77, 3.25 and 0.75 μg ml⁻¹, respectively, after oral treatment with two tablets (1000 mg) of metakelfin. Significant difference was recorded on day 2 for sulfalene concentration in blood cells of healthy and P. falciparum cases (t=9.49; P<0.001).     

Influence of humic, fulvic and hydrophilic acids on the growth, photosynthesis and respiration of the dinoflagellate Prorocentrum minimum (Pavillard) Schiller

Blooms of the dinoflagellate Prorocentrum minimum often occur in coastal regions characterized by variable salinity and elevated concentrations of terrestrially derived dissolved organic carbon (DOC). Humic, fulvic and hydrophilic acid fractions of DOC were isolated from runoff entering lower Narragansett Bay immediately after a rainfall event and the influence of these fractions upon P. minimum growth, cell yield, photosynthesis and respiration was examined. All organic fractions stimulated growth rates and cell yields compared with controls (no organic additions), but the extent of stimulation varied with the fraction and its molecular weight. Greatest stimulations were observed with humic and fulvic acids additions; cell yields were more than 2.5 and 3.5 times higher than with hydrophilic acid additions while growth rates were 21 and 44% higher, respectively. Responses to additions of different molecular weight fractions of each DOC fraction suggest that growth rate effects were attributable to specific molecular weight fractions: the >10,000 fraction of humic acids, both the >10,000 and <500 fractions of fulvic acids and the <10,000 fraction of hydrophilic acids. The form and concentration of nitrogen (as NO₃⁻ or NH₄⁺) present also influenced P. minimum response to DOC; 10–20 μg ml⁻¹ additions of fulvic acid had no effect upon growth rates in the presence of NH₄⁺ but significantly increased growth rates in the presence of NO₃⁻, a relationship probably related to fulvic acid effects upon trace metal bioavailability and subsequent regulation of the biosynthesis of enzymes required for NO₃⁻ assimilation. The influence of DOC additions on P. minimum respiration and production rates also varied with the organic fraction and its concentration. Production rates ranged from 1.1 to 3.4 pg O₂ cell⁻¹ h⁻¹, with highest rates observed upon exposure to fulvic and hydrophilic acid concentrations of >10 μm ml⁻¹. Low concentrations (5–10 μg ml⁻¹) of humic acid had no statistically significant effect upon production, but exposure to concentrations >25 μg ml⁻¹ resulted in a 30% decrease in O₂ evolution, probably due to light attenuation by the highly colored humic acid fraction. Respiration rates ranged from 1.2 to 2.7 pg O₂ cell⁻¹ h⁻¹ and were elevated upon exposure to both fulvic and hydrophilic acids, but not to humic acid. These results demonstrate that terrestrially derived DOC fractions play an active role in stimulation of P. minimum growth via direct effects upon growth, yield and photosynthesis as well as via indirect influences such as interactions with nitrogen and effects upon light attenuation.     

Biological effects of bovine glia maturation factor on glial cells in culture

Optimal bioassay conditions for bovine glia maturation factor (GMF) were determined among glial cells from normal glioblasts to glioma cells. Rat glioblasts 4–8 days after subculture show the highest response to GMF with regard to morphological transformation and mitogenic activity. Bovine GMF enhances DNA synthesis of rat glioblasts at 12 hr after stimulation; maximum incorporation of [methyl-³H]thymidine was detected at 18 hr. GMF increases twofold the saturation density of rat glioblasts but does not alter that of C6 astrocytoma cells. The apparent inhibition of mitogenic activity of high doses of GMF is seen in both normal and malignant glial cells.     

Stimulation of lymphocyte receptor capping by the ionophore monensin

Summary The carboxylic ionophore monensin has a biphasic effect on antibody-induced Thy-1 cap formation. At higher concentrations, 5×10^–6–5×10^–5 m monensin causes a significant inhibition of receptor capping similar to that previously found with the Ca²⁺ selective ionophore A23187. At lower concentrations, 5×10^–8–5×10^–7 m capping is stimulated. It is concluded that capping at lower ionophore concentrations is a secific response to the ability of monensin to induce a rise in intracellular Na⁺, which indirectly elevates intracellular Ca²⁺ activity. This in turn activates the contractile machinery required for the aggregation of surface receptors into capped structures. At higher concentrations monensin acts as a nonspecific detergent, which causes detrimental structural alterations in some of the membrane components involved in the capping process.     

The effect of progesterone on the release of arachidonic acid from human endometrial cells stimulated by histamine

Progesterone at concentrations of 10⁻⁷M and 10⁻⁸M inhibits release of [³H]-arachidonic acid from stimulated, perfused, endometrial cells. The effect is independent of the mechanism of stimulation. Cortisol (10⁻⁵M but not 10⁻⁷M) has a similar effect in this system but estradiol (10⁻⁷M) is without effect. There was a positive correlation (p<0.05) between the magnitude of inhibition by progesterone and the day of cycle. The inhibitory action of progesterone on the release of arachidonic acid was greater in endometrial cells than in decidual cells and was apparent after fifteen minutes. The activities of commercial and endometrial cell-free preparations of phospholipase A₂ and phospholipase C were unaffected by the presence of progesterone. We conclude that progesterone modulates release of [³H]-arachidonic acid from endometrial cells by a rapid, indirect action on phospholipase activity.