Terminal differentiation of murine erythroleukemia cells: physical stabilization of end-stage cells

An important limitation in the use of the murine erythroleukenia (MEL) cell system as an in vitro system for the study of terminal erythroid differentiation has been the inability to produce significant numbers of cells which represent the end-point of the pathway in vitro. We show here that a major reason for the failure to observe end-stage cells in vitro is that such cells are physically unstable under the standard culture conditions used for MEL cell differentiation. Modification of these culture conditions by the addition of either bovine serum albumin or Ficoll leads to physical stabilization of end-stage cells. Under such culture conditions, uniform cultures of terminally differentiated MEL cells with morphological characteristics similar to those of normal mouse orthochromatophilic erythroblasts and reticulocytes are observed. Examination of physical and biochemical parameters of these cell populations give values which are similar to values characteristic of mouse reticulocytes. A physically stabilized MEL cell shows a narrow cell volume distribution with an average value of approximately 100 mum(3), similar to the cell volume distribution observed for mouse reticulocytes, while a typical MEL cell culture treated with DMSO but without a stabilizing agent exhibits a broader, more heterogeneous cell volume distribution with an average value of approximately 500 mum(3). Globin mRNA levels and levels of globin synthesis reach values almost equal to those in mouse reticulocytes in cultures of physically stabilized MEL cells while differentiating cultures not treated with a stabilizing agent reach substantially lower values for these parameters. We suggest that the ability to produce populations of MEL cells which undergo complete terminal erythroid differentiation in vitro will allow the analysis of the molecular mechanisms which control the terminal stages of the erythroid differentiation process.     

Nuclear muscarinic acetylcholine receptors in corneal cells from rabbit

PURPOSE: Previous studies have indicated that muscarinic acetylcholine receptors (mAChR) may be present in an unexpected, unique location and play a singular role in cellular growth regulation of rabbit corneal epithelium that may be of general physiologic significance if found in other cells. The purpose of this study was to examine rabbit corneas and corneal cells in culture to determine mAChR location and tissue distribution. METHODS: Using [3H]-propylbenzilylcholine mustard ([3H]PrBChM), which binds covalently to the active site of mAChR, rabbit corneal cross-sections, cultured corneal keratocytes, epithelial and endothelial cells, as well as nuclei isolated from these cultured corneal cells were labeled, stained, and autoradiographed. Nuclei labeled with [3H]PrBChM were further analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. RESULTS: Direct visual confirmation of the localization of mAChRs was obtained. MAChR were found in epithelial and endothelial layers of fresh-frozen corneal cross-sections, in cultured rabbit epithelial and endothelial cells, and on isolated rabbit epithelial and endothelial cell nuclei. mAChR were not detectable in keratocytes with these techniques. When [3H]PrBChM-labeled nuclei from cultured corneal cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, epithelial and endothelial samples showed specific mAChR binding, whereas binding to keratocyte nuclei was not detectable. CONCLUSIONS: As a result of these findings, a revised hypothesis is suggested for the locations and possible functions of mAChR in regulation of growth in corneal and other cells.     

Role of peroxide in AC electrical field exposure effects on friend murine erythroleukemia cells during dielectrophoretic manipulations

Anal fissures, proctalgia fugax, levator ani syndrome, and pruritus ani are common causes of anorectal pain and irritation. The clinician who obtains a thorough history and performs a complete examination can accurately diagnose these disorders. Ancillary tests seldom are helpful and rarely are necessary. Most patients suffering from these conditions readily respond to conservative therapy provided in the primary care practitioner's office.     

The dendritic cell differentiation and antigen-presenting function of the erythroleukemia cells induced by GM-CSF

OBJECTIVE: To investigate the generation of dendritic cells and the change of antigen-presenting function during the differentiation of FBL-3 erythroleukemia cells induced by GM-CSF. METHODS: The effects of GM-CSF on the phenotype, ultrastructure and antigen-presenting function of FBL-3 erythroleukemia cells were observed by FACS, electromicroscopy and 51Cr-release assay. RESULTS: After treatment with 100 ng/ml GM-CSF for 3 days, the expressions of 33 D1 and NLDC 145 which are the specific markers on dendritic cells were increased significantly; MHC-II B7-1, B7-2, ICAM-1, VCAM-1 and CD40 were also upregulated. The membrane of FBL-3 cells was changed into villous surface with dendritic projections. There were plenty of mitochondria in cytoplasm, the nucleus became lobulated. The GM-CSF-treated FBL-3 cells could apparently stimulate the proliferation of allogeneic T lymphocytes, induce the production of IL-2 and improve the specific cytotoxic activity of CTL on FBL-3 cells. CONCLUSION: Erythroleukemia cells were induced to differentiate into the dendritic cells by GM-CSF and obtained the antigen-presenting function.     

The effect of differentiation inducers on the integrin expression of K562 erythroleukemia cells

The DNA sequences of the recA gene from 25 strains of bacteria are known. The evolution of these recA gene sequences, and of the derived RecA protein sequences, is examined, with special reference to the effect of variations in genomic G + C content. From the aligned RecA protein sequences, phylogenetic trees have been drawn using both distance matrix and maximum parsimony methods. There is a broad concordance between these trees and those derived from other data (largely 16S ribosomal RNA sequences). There is a fair degree of certainty in the relationships among the "Purple" or Proteobacteria, but the branching pattern between higher taxa within the eubacteria cannot be reliably resolved with these data.     

Protein kinases C-gamma and -delta are involved in insulin-like growth factor I-induced migration of colonic epithelial cells

BACKGROUND & AIMS: The mechanisms by which epithelial cells migrate during the repair of damaged colonic mucosa are poorly understood. This study investigated the insulin-like growth factor I (IGF-I) signaling pathway leading to HT29-D4 human colonic epithelial cell line migration. METHODS: IGF-stimulated cell migration was determined using a wound model in the presence or absence of kinase inhibitors. Activation of protein kinase C (PKC) was determined by immunodetection. RESULTS: IGF-I and insulin induce cell migration without affecting cell proliferation through their cognate receptors. Des(1-3)-IGF-I, a truncated analogue of IGF-I, was more potent than IGF-I, suggesting that IGF-binding proteins secreted in the medium modulated IGF-I-induced cell migration. Inhibition of phosphatidylinositol 3-kinase, PKC, and mitogen-activated protein kinases eliminated cell restitution. Long-term exposure of cells to phorbol myristate acetate caused the depletion of PKC-delta and -gamma and prevented also IGF-I-induced cell motility. IGF-I also induced activation of PKC-delta and -gamma only. CONCLUSIONS: IGF-I stimulates colonic restitution through the activation of multiple signaling pathways including activation of phosphatidylinositol 3-kinase, PKC-delta and -gamma, and mitogen-activated protein kinases.     

Nuclear but not cytoplasmic phospholipase C beta 1 inhibits differentiation of erythroleukemia cells

A body of evidence has shown the existence of a nuclear phosphoinositide cycle in different cell types. The cycle is endowed with kinases as well as phosphatases and phospholipase C (PLC). Among the PLC isozymes, the beta family is characterized by a long COOH-terminal tail that contains a cluster of lysine residues responsible for nuclear localization. Indeed, PLC beta 1 is the major isoform that has been detected in the nucleus of several cells. This isoform is activated by insulin-like growth factor I, and when this isoform is lacking, as a result of gene ablation, the onset of DNA synthesis induced by this hormone is abolished. On the contrary, PLC beta 1 is down-regulated during the erythroid differentiation of Friend erythroleukemia cells. A key question is how PLC beta 1 signaling at the nucleus fits into the erythroid differentiation program of Friend erythroleukemia cells, and whether PLC beta 1 signaling activity is directly responsible for the maintenance of the undifferentiated state of erythroleukemia cells. Here we present evidence that nuclear PLC beta 1 but not the isoform located at the plasma membrane is directly involved in maintaining the undifferentiated state of Friend erythroleukemia cells. Indeed, when wild-type PLC beta 1 is overexpressed in these cells, differentiation in response to DMSO is inhibited in that the expression of beta-globin is almost completely abolished, whereas when a mutant lacking the ability to localize to the nucleus is expressed, the cells differentiate, and the expression of beta-globin is the same as in wild-type cells.     

D2 receptors in the ventrolateral striatum are involved in feeding behavior in rats

The alpha1-adrenoceptor subtypes mediating contraction of rabbit aorta and urethra were pharmacologically characterized using an isolated organ bath technique. Although aorta was as sensitive as urethra to the contractile action of methoxamine, phenylephrine was about 10 times more potent as a contractile agonist on aorta than on urethra. In aorta, the rank order of agonist sensitivity was norepinephrine > phenylephrine > clonidine > methoxamine whereas the rank order in urethra was clonidine > methoxamine > or = phenylephrine > norepinephrine. A lack of significant correlation between the potency of different alpha1-adrenoceptor antagonists tested against the phenylephrine-induced contraction in aorta and in urethra indicated that different alpha1-adrenoceptor subtypes mediated the contractile response in the two preparations. The potency of different alpha1-adrenoceptor antagonists tested in rabbit urethra was significantly correlated with their affinity for the cloned human alpha1c-, but not alpha1a- or alpha1b-, adrenoceptor subtype. Such a clear correlation with the potency of different alpha1-adrenoceptor antagonists tested in rabbit aorta and their affinity for one subtype of cloned human alpha1-adrenoceptor was not found. Chlorethylclonidine, which produced a 10 000-fold rightward shift in the phenylephrine concentration-response curve for rat aorta, had a weak inhibitory effect in rabbit aorta and urethra as well as in other rabbit tissues (spleen, fundus, renal artery, saphenous artery). The results indicate that significant heterogeneity exists among alpha1-adrenoceptor in rabbit aorta and urethra (alpha1c-adrenoceptor). However, chlorethylclonidine does not seem to be a suitable tool for the differentiation of alpha1-adrenoceptor subtypes in the rabbit.     

N-methyl-D-aspartate receptors in the nucleus accumbens are involved in detection of spatial novelty in mice

Toxic milk mutant (tx) mice accumulate excess copper (Cu) in liver with age and develop symptoms similar to those seen in human Wilson disease. Because metallothionein (MT) is the major Cu-binding protein in tx mouse liver and Cu-MT can enhance lipid peroxidation initiated by an organic hydroperoxide, the potential genotoxicity of Cu-MT in tx mice was assessed in male tx mice (11 to 12 months old) and in age- and sex-matched control wild-type (DL) mice. Toxic milk mutant mice, but not control DL mice, developed regenerative liver nodules (tx-N) with normal histologic appearance. Residual, non-nodular tx mouse liver (tx-R) was microscopically abnormal with large, atypical hepatocytes. The levels of Cu, zinc (Zn), and MT, and the numbers of apoptotic cells (APC) in tx-N, tx-R, and DL livers were measured by atomic absorption spectrophotometry, 109cadmium-heme assay, and the TUNEL method, respectively. Significantly higher levels of MT, Cu, and Zn, as well as increased numbers of APC were found in both tx-N and tx-R compared with DL mouse livers. Intense nuclear and cytoplasmic immunohistochemical staining for MT was observed in both normal and atypical hepatocytes of the tx mouse, whereas only cytoplasmic staining for MT was detected in DL mouse liver tissue. Accumulated Cu could be detected in tx-R and tx-N liver by rhodanine staining but was not detected in other tx mouse organs, or in mouse liver or other organs of DL. The number of APC and level of MT were significantly higher in tx-R liver compared with both tx-N and DL liver. These results suggest that: (a) aged tx mouse accumulate excess Cu in liver accompanied by striking morphologic changes, and (b) although MT binds to Cu in tx mouse liver, the presence of high Cu-MT and Cu in the nucleus can be genotoxic and may lead to enhanced apoptosis.     

Expression of the protein tyrosine phosphatase beta2 gene in mouse erythroleukemia cells induces terminal erythroid differentiation

We have cloned cDNA for protein tyrosine phosphatase beta2, which had been implicated in erythroid differentiation of mouse erythroleukemia cells. Expression of cDNA constructs, in which beta2 cDNA is placed under the control of mouse metallothionein-I promoter, by ZnCl2 converted a significant portion (20 to 38%) of the cells to erythroid-like cells, which is 25-50% of the erythroid differentiation efficiency observed by conventional erythroid-inducing agents. Furthermore, introduction and expression of altered protein tyrosine phosphatase beta2 cDNA constructs designed to produce the enzyme lacking the phosphatase activity inhibited erythroid differentiation by 100-20%, depending upon the concentration of erythroid-inducing agents employed. These results strongly suggest that protein tyrosine phosphatase beta2 is involved in triggering erythroid differentiation in mouse erythroleukemia cells.     

Alpha1-adrenergic and dopaminergic receptors are involved in the anoretic effect of corticotropin-releasing factor in goldfish

This study investigates the noradrenergic and/or dopaminergic receptors subtypes involved in the anoretic action of CRF in goldfish. Agonists and antagonists of alpha1- and alpha2-adrenoceptors, and D1- and D2-dopaminergic receptors were intracerebroventricularly (i.c.v.) administered alone or in combination with CRF in the case of antagonists. Food intake and hypothalamic content of catecholamines and their metabolites were measured at 2 h postinjection. On one hand, alpha1-adrenergic receptor antagonist, but not alpha2, blocked the anoretic effect of CRF. Moreover, we found a blockade of CRF-induced anoretic action by pretreatment with specific D1- and D2-dopaminergic antagonists. On the other hand, the i.c.v. administration of CRF reduced hypothalamic norepinephrine (NE) and dopamine (DA) content, without modifications in their metabolism. Thus, our results suggest that the anoretic effect of CRF appears to be mediated by alpha1-adrenergic and dopaminergic receptors in fish. Second, the reduction in hypothalamic NE and DA synthesis could be due to a direct effect of CRF treatment and/or a secondary effect of food intake reduction.     

Changes in G protein pattern and in G protein-dependent signaling during erythropoietin- and dimethylsulfoxide-induced differentiation of murine erythroleukemia cells

We have studied the expression of G protein subtypes and the role of G protein-dependent signaling in two subclones of RED-1 cells, an erythropoetin(Epo)-sensitive, murine erythroleukemia cell line. Clone 6C8 showed terminal erythroid differentiation in response to a combined treatment with Epo and dimethylsulfoxide. Clone G3 was resistant to these inducers, but responded to Epo with enhanced proliferation. We measured G protein alpha subunit levels by toxin-catalyzed adenosine diphosphate (ADP)-ribosylation with [32P]-nicotinamide adenine dinucleotide (NAD) and by semiquantitative immunoblotting with specific antisera. Native RED-1 cells expressed G alpha i2, alpha i3, alpha s, and alpha q/11, but not alpha i1 and alpha o. Terminal differentiation was associated with a selective loss (approximately 80%) of G alpha i3 and an increase in a truncated cytosolic form of G alpha i2, while the membrane levels of alpha i2, alpha q/11, and alpha s did not change significantly. Treatment of G3 cells with the inducers was without effect on G protein abundance. However, except for alpha s, G3 cells contained significantly higher levels of the different G protein alpha subunits tested. Stimulation of G protein-coupled receptors by thrombin and ADP caused a pertussis toxin (PTX)-inhibitable transient increase in intracellular Ca2+ that was markedly reduced in differentiated cells. In G3 cells, but not in 6C8 cells, thrombin also caused a PTX-sensitive inhibition of isoprenaline-stimulated cyclic 3',5'-adenosine monophosphate (cAMP) formation. Our results show that specific alterations in G protein expression and function are associated with erythroid differentiation of erythroleukemia cells but do not prove a causal relationship. The loss of G alpha i3 may affect cellular responses that are mediated via P2T purine or thrombin receptors.     

Different domains of the ORL1 and kappa-opioid receptors are involved in recognition of nociceptin and dynorphin A

In order to gain further insight into the functional architecture of structurally related G protein-coupled receptors, the ORL1 (nociceptin) and opioid receptors, we have constructed chimeras of ORL1 and mu-, delta- and kappa-opioid receptors, and compared their binding and functional properties with those of the parent receptors. We find in particular that a ORL1-kappa-opioid (O-K) hybrid construct has retained high affinity for non-type-selective opiate ligands, and has acquired the ability to bind and respond to enkephalins and mu- and/or delta-opioid receptor-selective enkephalins analogs, thus behaving like a 'universal' opioid receptor. Most significantly however, whilst the ORL1 and kappa-opioid receptors display high binding preference (KD 0.1 vs. 100 nM) for their respective endogenous ligands, nociceptin and dynorphin A, the O-K chimeric receptor binds both nociceptin and dynorphin A, with high affinity (KD < 1 nM). Together, these data (i) add weight to the hypothesis that the extracellular loops of opioid receptors act as a filter for ligand selection, and (ii) demonstrate that different domains of the ORL1 and kappa-opioid receptors are involved in recognition of their endogenous peptide ligands.     

An activating mutation in the kit receptor abolishes the stroma requirement for growth of ELM erythroleukemia cells, but does not prevent their differentiation in response to erythropoietin

We have previously shown that murine ELM erythroleukemia cells can only be grown in vitro in the presence of a stromal feeder layer, or alternatively stem cell factor (SCF), without which they differentiate. When grown in the presence of SCF, ELM cells can still differentiate in response to erythropoietin (Epo), but growth on stroma prevents this. We previously isolated a stroma-independent ELM variant, ELM-I-1, that is also defective in Epo-induced differentiation. We show here that this variant has an activating mutation in the Kit receptor, converting aspartic acid 814 to histidine. Expression of the mutant receptor in stroma-dependent ELM-D cells causes growth factor-independent proliferation and also gives the cells a selective advantage, in terms of proliferation rate and clonegenicity, compared with ELM-D cells grown in optimal amounts of SCF. Expression of the mutant receptor in ELM-D cells also prevents spontaneous differentiation, but not differentiation induced by Epo. Analysis of mitogenic signaling pathways in these cells shows that the mutant receptor induces constitutive activation of p42/p44 mitogen-activated protein kinases. It also selectively inhibits the expression of p66Shc but not the p46/p52 Shc isoforms (as did treatment of ELM cells with SCF), which is of interest, because p66Shc is known to play an inhibitory role in growth factor signaling.     

Metabotropic glutamate receptors are involved in calcium-induced LTP of AMPA and NMDA receptor-mediated responses in the rat hippocampus

INTRODUCTION: Previous studies have demonstrated a high prevalence of "white coat" hypertension (20%), but it is still controversial if it implies an increase in cardiovascular risk. PATIENTS: Between 1992 and 95 we prospectively studied 175 untreated hypertensive patients aged over 18 years (V Joint National Committee's stage I-II), and 91 controls. DESIGN AND METHODS: The subjects were submitted to clinical evaluation, ambulatory blood pressure monitoring, 24-hour Holter monitoring, signal-averaged ECG, echocardiography/Doppler and ergometry. "White coat" hypertension was defined as mean daytime (6.00-22.00 H) ambulatory blood pressure < 136/87 mm Hg (males) and < 131/86 mm Hg (females). RESULTS: "White coat" hypertension was present in 29 patients (18%). "White coat" hypertension patients had an identical prevalence of smoking, family history of cardiovascular disease, abnormal ECG and retinopathy (> Keith-Wagener II) as patients with daytime hypertension. Ambulatory blood pressure values (24 hour, 6.00-22.00 h, 22.00-6.00 h, sleep, blood pressure load, heart rate) were all significantly different from controls (p < 0.03 to 0.0007). In patients with daytime hypertension, only 24 hour and daytime diastolic ambulatory blood pressure (p < 0.005) were different from "white coat" hypertension patients. Exercise testing blood pressure values (6 min exercise, maximal, 3 min recovery) were significantly different between "white coat" hypertension patients and the control group (n = 70) (p varying from 0.05 to 0.005) but not between "white coat" hypertension and daytime hypertension (n = 33) patients. Diastolic function was studied only in 39 daytime hypertension patients, 10 individuals with "white coat" hypertension and 34 controls (for technical reasons and because we only analyzed individuals younger than 55 years). E velocity and E/A ratio were similar in "white coat" hypertension and daytime hypertension, but only in daytime hypertension patients they reached a significant difference from controls (p = 0.04; p = 0.01), probably due to the small number of patients. CONCLUSIONS: These data (clinical, ambulatory blood pressure, ergometric, diastolic function) suggest that "white coat" hypertension might not be a benign entity.     

Somatodendritic 5-HT1A receptors are critically involved in the anxiolytic effects of 8-OH-DPAT

A simple technique for the fast sampling of biological tissues for electron paramagnetic resonance (EPR) spectroscopy is described. By using tube-like stainless steel cutter, a cylindrical piece of tissue is dissected free; while holding the sample and cutter on a silicone rubber cutting base, the sample is transferred directly into the EPR test tube by pushing the tube into the instrument. The main features of the technique are its simplicity, speed, elimination of the necessity to manipulate frozen tissue (e.g., breaking, chopping, etc.), precise information on the sample volume, the ability to adjust sample size according to need, and avoidance of the use any foreign compounds (such as spin traps). The technique has been successfully applied to the EPR detection of free radicals in rabbit spinal cord exposed to ischemia. It can be easily modified for manipulation with samples in an inert atmosphere.