Anticancer Res, 1993 Nov-Dec, 13(6A), 1925 - 37 Efficacy of pyridoxal treatment in controlling the growth of melanomas in cell culture and an animal pilot study; Maksymowych AB et al.; We have demonstrated, using confocal laser scanning microscopy, that pyridoxal treatment of B16C3 murine melanoma cells inhibits triamcinolone acetonide induced translocation of the glucocorticoid receptor to the nucleus of intact cells . In addition to inhibiting glucocorticoid receptor nuclear translocation, pyridoxal kills B16C3 murine melanoma cells and WM983A human melanoma cells in culture . Cortexolone, a glucocorticoid antagonist, also kills cells in culture . This mechanism, however, appears to initiate in the glucocorticoid receptor signal transducing cascade at a point prior to the impact of pyridoxal treatment alone . The glucocorticoid antagonist RU486 has no detrimental effect on melanoma cell viability, however, in combination with pyridoxal, RU486 extends cell viability . Since pyridoxal kills melanoma cells in culture, a pilot study was carried out examining the efficacy of topical application of a pyridoxal cream to inhibit the growth and/or cause regression of (B16C3) xenograft melanoma tumors in an immunocompetent (Hairless Rhino-J3) and an immunocompromised (Crl: nu/nu (CD1)BR) murine animal model . The results of the study with immunocompetent animals are encouraging . While tumors are brought under control by pyridoxal treatment, further work is needed to determine the most efficacious treatment regimen and to establish formal concentrations for pyridoxal in topical ointments . Trials using immunocompromised animals indicated that although some qualitative differences may be detected between the control and experimental animals, tumor growth in these animals is so aggressive that multiple applications or higher concentrations of pyridoxal may be needed to obtain useful data. Glia, 1993 Nov, 9(3), 176 - 87 Rat ciliary neurotrophic factor (CNTF): gene structure and regulation of mRNA levels in glial cell cultures; Carroll P et al.; The structure of the rat ciliary neurotrophic factor (CNTF) gene and the regulation of CNTF mRNA levels in cultured glial cells were investigated . The rat mRNA is encoded by a simple two-exon transcription unit . Sequence analysis of the region upstream of the transcription start-site did not reveal a typical TATA-box consensus sequence . Low levels of CNTF mRNA were detected in cultured Schwann cells, and CNTF mRNA was not increased by a variety of treatments . Three-week-old astrocyte-enriched cell cultures from new-born rat brain contained easily detectable CNTF mRNA . In astrocyte-enriched cultures, upregulation of CNTF mRNA levels was observed after treatment with IFN-gamma . CNTF mRNA levels were down-regulated in these cells by treatments that elevate intracellular cyclic AMP and by members of the fibroblast growth factor (FGF) family . The implications of these results for potential in vivo functions of CNTF are discussed. Radiats Biol Radioecol, 1993 Nov-Dec, 33(6), 900 - 1 {Effect of chronic neutron irradiation on fibroblast cell culture in Chinese hamster}; Smirnova EN et al.; The effect of monoenergy (30 Gev) neutron radiation on Chinese hamster fibroblasts has been studied by micronuclear test . It has been shown that in the range of doses up to 60 cGy RBE is equal to unity. Acta Otolaryngol, 1993 Nov, 113(6), 772 - 6 Nasal epithelial cell culture as a tool in evaluating ciliary dysfunction; Gilain L et al.; Cultures of respiratory epithelial cells were obtained from nasal polyps collected in patients with and without primary ciliary defect . The ciliary beating frequency and the ciliary beating heterogeneity were determined on native and cultured tissues . We observed a significantly higher (p < 0.01) ciliary beating frequency of cultured ciliated cells, when compared with ciliated cells from the native tissue . The ciliary beating frequency of the cultured ciliated cells from the patient with primary defect (7.9 +/- 2.1 Hz) was significantly lower when compared with the beating frequency of the ciliated cells from the control subject (12.4 +/- 2.0 Hz) . In addition, the percentage of ciliated cells characterized by a beating frequency lower than 8 Hz was 90.7% in the native tissue and 47.5% in the cultured tissue from the patient with ciliary primary defect . In the patient without ciliary primary defect, 90% of the cultured ciliated cells had a homogeneous ciliary beating, whereas in the patient with primary ciliary defect, only 47% of the ciliated cells had a homogeneous ciliary beating . These results suggest that the culture of respiratory cells associated with the functional activity measurement of the ciliated cells represent another way of precisely determining the extent of the primary ciliary dyskinesia defect. Pharm Res, 1993 Nov, 10(11), 1620 - 6 Utilization of a human intestinal epithelial cell culture system (Caco-2) for evaluating cytoprotective agents; Tang AS et al.; Human intestinal epithelial cells (Caco-2) were cultured as confluent monolayers on polycarbonate membranes in Transwells for investigating their applicability in evaluating the cytoprotective activity of sucralfate . The control experiments established a reproducible chemical method (using 0.5 mM indomethacin in Hanks' balanced salt solution) for inducing damage to the Caco-2 cell monolayers . Damage was determined by measuring changes in transepithelial electrical resistance (TEER) . Twenty-day-old Caco-2 cell monolayers were significantly and reproducibly damaged (compared to buffer alone) (P < 0.001) by application of 0.5 mM indomethacin to the apical side for 1 hr . While sucralfate, at a 0.5, 2, or 5 mg/mL concentration in the buffer, was shown not to reverse (treat) the damage caused by indomethacin in this cellular model, it was able to protect (prevent) the cells from indomethacin-induced damage (P < 0.001) . We observed that indomethacin-induced damage to the Caco-2 cell monolayers greatly affected the paracellular pathway since the percentage transport of {3H}methoxyinulin was significantly elevated . In contrast, protection of the Caco-2 cells with 5 mg/mL sucralfate in the presence of the damaging agent resulted in transport of the paracellular marker similar to that in the control (HBSS-treated) cell monolayers . This direct cytoprotective effect was thus independent of vascular factors at neutral pH and was observed to be dose dependent (0.5 to 5 mg/mL) when sucralfate was applied to the cells in the presence of the damaging agent.(ABSTRACT TRUNCATED AT 250 WORDS) Antimicrob Agents Chemother, 1993 Nov, 37(11), 2496 - 9 Use of human renal proximal tubule cell cultures for studying foscarnet-induced nephrotoxicity in vitro; Trifillis AL et al.; Foscarnet is an antiviral agent used for the treatment of cytomegalovirus retinitis and acyclovir-resistant herpes simplex virus infections in AIDS patients . Renal impairment has been reported for many patients treated with foscarnet . We have studied the effects of foscarnet on the viability (estimated by neutral red inclusion) and ultrastructure of cultures of human renal proximal tubule cells (HRPTC) isolated from the kidneys of five cadavers and cultured . The degree of foscarnet-induced toxicity was dose dependent and varied among the HRPTC cultures . The data obtained by using the in vitro system of HRPTC mimic the data of the clinical trials in that there is a dose-dependent individual variation among human cases in response to foscarnet treatment . Thus, these cultures are extremely well-suited to investigations of the mechanism of toxicity at the subcellular level. ORL J Otorhinolaryngol Relat Spec, 1993 Nov-Dec, 55(6), 347 - 51 Synthesis of human cartilage using organotypic cell culture; Bujia J et al.; The limited supply of fresh autologous cartilage tissue for use in reconstructive surgery necessitates the use of vital banked allografts . A feasible in vitro production of cartilage tissue composed of living cells requires the use of modern tissue culture techniques retaining the phenotypic characteristics of chondrocytes . With this purpose in mind, human chondrocytes were isolated and cultured using different culture procedures: monolayer, suspension and agar gel . The differentiation state of chondrocytes as well as proteoglycan and collagen syntheses were assessed by histochemical and immunohistochemical methods . Whereas chondrocytes in monolayer displayed an unstable phenotype and tended to dedifferentiate, in three-dimensional culture the chondrocytes remained morphologically, phenotypically and functionally differentiated . Furthermore, an accumulation of matrix products pericellularly was observed in the agar gel . The results suggest that three-dimensional cultures in agar gel may allow the in vitro production of bioartificial cartilage for transplantation. J Clin Microbiol, 1993 Nov, 31(11), 3046 - 9 Relative frequencies of G (VP7) and P (VP4) serotypes determined by polymerase chain reaction assays among Japanese bovine rotaviruses isolated in cell culture; Suzuki Y et al.; The relative frequencies of both the G (VP7) and P (VP4) serotypes of 40 bovine rotaviruses isolated in cell culture from diarrheic calves in Japan between January 1983 and February 1991 were determined by recently developed polymerase chain reaction assays . Isolates with G serotype 6 and P serotype 5 (UK-like strains) were most frequently found (42.5%) followed by isolates with G6P11 (17.5%), G6P1 (10%), or G10P5 (10%) . Isolates with G10P11 (B223-like strains) were least frequently found (7.5%) . The presence of various combinations of G and P serotypes suggests frequent reassortment in nature among bovine rotaviruses. Plant Mol Biol, 1993 Nov, 23(4), 737 - 47 Metabolic regulation of alpha-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.); Huang N et al.; Expression of two genes in the alpha-amylase gene family is controlled by metabolic regulation in rice cultured cells . The levels of RAmy3D and RAmy3E mRNAs in rice cultured cells are inversely related to the concentration of sugar in the culture medium . Other genes in the rice alpha-amylase gene family have little or no expression in cultured cells; these expression levels are not controlled by metabolic regulation . A RAmy3D promoter/GUS gene fusion was metabolically regulated in the transgenic rice cell line 3DG, just as the endogenous RAmy3D gene is regulated . An assay of GUS enzyme activity in 3DG cells demonstrated that RAmy3D/GUS expression is repressed when sugar is present in the culture medium and induced when sugar is removed from the medium . The 942 bp fragment of the RAmy3D promoter that was linked to the coding region of the GUS reporter gene thus contains all of the regulatory sequences necessary for metabolic regulation of the gene. J Steroid Biochem Mol Biol, 1993 Nov, 46(5), 579 - 83 Comparative effects of short term treatment with norethisterone and sex steroids on gonadotropin secretion in rat pituitary cell cultures; Mendoza ME et al.; The short term effects of norethisterone (NET), progesterone (P), estradiol (E2) and dihydrotestosterone (DHT) on the gonadotropin secretion of pituitary cells, from both male and female rats, in primary culture primed with E2 were studied . In female cells, NET only increased the GnRH-induced secretion of LH, while P increased both LH and FSH . Male pituitary cells showed an increased response to GnRH after P pretreatment only if the E2 concentration was augmented . However with the same E2 conditions pretreatment with NET decreased the stimulated LH, but not FSH secretion . Pretreatment with E2 inhibited LH stimulated secretion from pituitary cells of male but not female rats . Furthermore DHT treatment diminished the GnRH response for both LH and FSH in pituitary cells from both sexes . Androgen pretreatment increased basal gonadotropin secretion in male but not in female cells . Basal FSH secretion was increased by NET pretreatment in male cells . This suggests that NET is metabolized by cultured pituitary cells to A-ring reduced compounds during the 4 h incubation period . The formation of NET metabolites, particularly the 3 beta, 5 alpha and 5 alpha-NET might be responsible for the estrogenic and androgenic effects observed when NET was administered to the cultured pituitary cells. Am J Physiol, 1993 Nov, 265(5 Pt 1), C1266 - 70 Microelectrode measurements of pericellular PO2 in erythropoietin-producing human hepatoma cell cultures; Wolff M et al.; On the basis of Fick's law of gas diffusion, it has been proposed that cells in conventional monolayer cultures may be severely hypoxic . Because knowledge of the cellular O2 availability is important for the interpretation of biochemical and toxicological cell culture work, microelectrode measurements of the pericellular PO2 were carried out using the erythropoietin (Epo)-producing human hepatoma cell lines Hep G2 and Hep 3B as an in vitro model . In confluent hepatoma cultures grown in polystyrene dishes and incubated in air with 5% CO2, the pericellular steady-state PO2 was < 1 mmHg . The rates of the production of immunoreactive Epo and lactate were high due to a misproportion between O2 supply and O2 requirements . Epo production decreased when shaken instead of static cultures were studied, or when the O2 concentration in the gas atmosphere was increased gradually up to 95% . In cultures grown on gas-permeable supports, pericellular and gas PO2 values were very similar, with increased Epo production at lowered PO2 . In agreement with mathematical models, our experimental data make PO2 measurements desirable for studies of O2-dependent biological functions in cell cultures. J Clin Invest, 1993 Nov, 92(5), 2553 - 9 Hypertensive sodium-proton exchanger phenotype persists in immortalized lymphoblasts from essential hypertensive patients . A cell culture model for human hypertension; Rosskopf D et al.; An enhancement of sodium-proton exchange activity is a frequently observed ion transport abnormality in essential hypertension . The cellular basis for this has not yet been elucidated . Due to the lack of a specific cell culture system it has been impossible to distinguish between intrinsic cellular abnormalities and influences exerted by the hypertensive neurohumoral milieu . Using Epstein-Barr virus we have immortalized lymphocytes from controls and from patients with essential hypertension that exhibited enhanced sodium-proton exchanger activity . Sodium-proton exchanger activity was determined in cells loaded with the fluorescent cytosolic pH indicator 2'7'-biscarboxyethyl-5,6-carboxyfluorescein acetoxymethylester (BCECF) after pretreatment with 250 nM of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate for 10 min . Cell lines from hypertensive patients displayed higher Vmax values of sodium-proton exchange than those from normotensive controls (129.6 +/- 30.0 vs . 77.1 +/- 13.2 mmol H+/min.; P < 0.001) . Hill coefficients for H+ were distinctly lower in hypertension compared to normotension (1.12 +/- 0.12 vs . 1.50 +/- 0.14; P < 0.0001) . The enhanced antiporter activity in cell lines from hypertensive patients was not accompanied by a corresponding increase in steady-state NHE-1 mRNA transcript levels, which argues against overexpression of antiporter protein in hypertension . The cells from hypertensive patients with high sodium-proton exchange activity proliferated distinctly faster than those from normotensive controls . These human cell lines represent a novel model to study the mutual interaction between sodium-proton exchange and cell proliferation, and may provide insights into the alterations in ion transport observed in a group of patients with essential hypertension. Khirurgiia (Mosk), 1993 Nov, (11), 26 - 30 {Use of cell cultures in the local treatment of burn wounds}; Glushchenko EV et al.; The article deals with the results of using cultivated allofibroblasts in topical treatment of burn wounds . Allofibroblasts obtained from the derma of burnt individuals were transplanted to the burn wounds of 28 patients . In 12 among 15 patients (80%) with IIIa-IIIb degree burns transplantation induced epithelialization of the burns in 12.1 +/- 2.4 days . Thirteen patients had deep burns, only in 3 of them with a circumscribed burn area transplantation proved effective . Complications of transplantation were encountered in 5 patients--rejection of the transplant in 2 and its suppuration in 3 patients . Allofibroblast transplantation may be applied as an independent method of treatment in IIIa, b degree burns. Dementia, 1993 Nov-Dec, 4(6), 301 - 7 APP expression in primary neuronal cell cultures from P6 mice during in vitro differentiation; Dichgans M et al.; Primary neuronal cell cultures from P6 mice were investigated in order to study amyloid protein precursor (APP) gene expression in differentiating neurons . Cerebellar granule cells which strongly express APP 695 allowed the identification of three distinct isoforms of neuronal APP 695 . The high-molecular-weight form of APP 695 is sialylated . The expression pattern of neuronal APP 695 changes during in vitro differentiation . Sialylated forms become more abundant upon longer cultivation time . The secreted forms of sialylated, neuronal APP 695 are shown to comigrate with APP isolated from cerebrospinal fluid . We suggest that the different sialylation states of APP 695 may reflect the modulation of cell-cell and cell-substrate interactions during in vitro differentiation and regeneration. Alcohol, 1993 Nov-Dec, 10(6), 477 - 80 Pathogenesis of IgA nephropathy in ethanol consumption: animal model and cell culture studies; Smith SM et al.; Using the intragastric ethanol infusion model of IgA nephropathy, we investigated the hypothesis that in this model mesangial changes commence prior to the deposition of IgA . We studied the two cellular components of the glomerular mesangium: the mononuclear phagocyte and the contractile mesangial cell . In the in vivo model, we observed a mononuclear phagocyte influx in the mesangium of alcoholic rats before the deposition of IgA . Using molecular techniques on cultured contractile mesangial cells, we demonstrated a threefold increase in interleukin-6 mRNA expression in contractile cells incubated with ethanol . These mesangial changes in the cellular composition, and in the autocrine cytokine system, suggest a direct role for ethanol in the pathogenesis of IgA nephropathy. Cell Biol Int, 1993 Nov, 17(11), 979 - 83 Differential expression of adult type MHC in satellite cell cultures from regenerating fast and slow rat muscles; Cantini M et al.; The local anaesthetic (Bupivacaine (1-n-butyl-DL-piperidine-2-carboxylic acid-2, 6-dimethyl anilide hydrochloride) has been used to induce myofiber damage (and thus satellite cells proliferation) and thereby represents a tool for increasing the yield of myoblasts from adult muscles . Replicating satellite cells were isolated by enzymatic dissociation from soleus (slow type) and tibialis anterior (fast type) muscles of adult rats, and categorized by the isoform (embryonic, fast and slow) of myosin heavy chain (MHC) expressed following myotube formation in a similar in vitro environment . According to light microscopic criteria, no morphological differences exist between the satellite cell cultures obtained from adult fast and slow muscles after Bupivacaine injection . On the other hand the derived myotubes express, beside the embryonic type, the peculiar myosin heavy chains which characterize the myosin pattern of the donor muscles. J Reprod Fertil, 1993 Nov, 99(2), 519 - 27 Stimulation of rat placental lactogen-II (rPL-II) secretion by cultured trophoblasts by insulin: development of a rat placental cell culture system and effects of peptide hormones on rPL-II secretion in vitro; Kishi K et al.; The purpose of this study was to develop a primary culture system using serum-free medium for rat placental trophoblast cells and to investigate the factors that control rat placental lactogen-II (rPL-II) secretion in vitro . The placentae of day 13 pregnant rats were dissociated in Medium 199 containing 0.1% collagenase and 0.002% DNAase . Dissociated cells were fractionated into five segments by centrifugation through a 40% Percoll density gradient and incubated on rat tail collagen bed in medium SFM-101 for up to 7 days . Fraction B at the Percoll gradient density of 1.05 g ml-1 was enriched with rPL-II-producing cells and the time course of rPL-II secretion was characterized by a rapid increase in the first 2 days, remaining at high values (mean: 14-16 ng micrograms-1 DNA) for the following 2-3 days and decreasing thereafter . The rPL-II-producing cells from faction B identified by immunocytochemical examination accounted for approximately 69% of total cultured cells and consisted of a few giant cells and polygonal cells . Growth factors (bovine insulin, 0.1-20 micrograms ml-1; recombinant human insulin-like growth factor (IGF)-I, IGF-II, 0.1-1.0 micrograms ml-1; murine epidermal growth factor (EGF), 0.001-10 micrograms ml-1), rat pituitary hormones (rat growth hormone, rat prolactin, 0.1-10 micrograms ml-1) and hypothalamic hormones (human growth hormone-releasing hormone (GHRH), corticotrophin-releasing hormone (CRH), LHRH, 0.1-10 micrograms ml-1) were individually added to the culture medium to investigate the putative factors that directly control rPL-II secretion by the trophoblast cells.(ABSTRACT TRUNCATED AT 250 WORDS) Biotechnol Prog, 1993 Nov-Dec, 9(6), 615 - 24 Growth, nutrient consumption, and end-product accumulation in Sf-9 and BTI-EAA insect cell cultures: insights into growth limitation and metabolism; Bedard C et al.; Growth, nutrient consumption, and end-product accumulation were quantitated in shake-flask cultures of two insect cell lines, Sf-9 and BTI-EAA, in three different serum-supplemented media . Per cell consumption or production rates were calculated for most medium components analyzed . Glucose was growth-limiting in TNM-FH medium and was the most important single source of organic-C for the cells in all cultures . Cells utilized fructose and maltose but not sucrose . alpha-Ketoglutarate and malate contributed significantly to the carbon budget of cells in TNM-FH . Lactate generally did not accumulate during growth . Most of the amino acids were consumed by the cells, with the exception of alanine which was produced . Most of the amino acids appeared to be present in adequate supply in the cultures . Glutamate was generally the most rapidly consumed of the amino acids, followed closely by glutamine . Alanine accumulation was correlated with glucose consumption . In Sf-9 cultures, ammonia accumulated only slightly or not at all as long as glucose was present in the medium, and uric acid was detectable at the end of growth and in the stationary phase . Added ammonia up to a concentration of 10 mM did not affect the growth of either cell line . Ammonia and lactate may be of less importance in limiting growth in insect cell cultures than in mammalian cell cultures . A hypothetical outline of the major metabolic pathways of the cultured insect cells is presented on the basis of information obtained here and in the literature. Am J Respir Cell Mol Biol, 1993 Nov, 9(5), 547 - 56 Reverse transcription-polymerase chain reaction (RT-PCR) phenotypic analysis of cell cultures of human tracheal epithelium, tracheobronchial glands, and lung carcinomas; Finkbeiner WE et al.; In order to identify expression of RNA transcripts for a number of important tracheobronchial cell products and molecules, we developed simple reverse transcription-polymerase chain reaction (RT-PCR) assays . Assays included the RNA for two apomucins (MUC1 and MUC2), secretory component, secretory leukocyte inhibitor protein, lysozyme, lactoferrin, 15-lipoxygenase, and the cystic fibrosis transmembrane conductance regulator . We tested RNA of normal and neoplastic origin . Sources of normal tissue included human tracheal surface epithelial cells and tracheobronchial submucosal tissues, acutely isolated human tracheal surface epithelial and tracheobronchial gland acini, and confluent cultures of human tracheal epithelial and tracheobronchial gland cells . Sources of neoplastic tissue included cell lines of non-small cell carcinomas of the lung . RNA expression was correlated with protein expression as assessed by immunocytochemistry . Tracheal surface epithelial tissues, isolated cells and cultures, and tracheobronchial submucosal tissues expressed RNA transcripts for all of the RNA transcripts assayed . Isolated gland acini and cultured gland cells expressed all RNA transcripts except 15-lipoxygenase . Expression of RNA transcripts by non-small cell lung carcinomas was heterogeneous and not necessarily influenced by histopathologic type . In most instances, RNA expression predicted expression of immunocytochemically detectable protein . These RT-PCR assays are useful for characterizing the molecular phenotype of cell cultures derived from normal or neoplastic airway epithelium and for establishing the potential of cultured cells for functional studies. J Immunol, 1993 Nov 1, 151(9), 5062 - 72 Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture; Petzelbauer P et al.; Microvascular endothelial cells (EC) recruit circulating leukocytes at sites of inflammation, in part through cytokine-regulated expression of endothelial-leukocyte adhesion molecules . Adhesion molecule expression varies among vascular beds and among EC within microvessels of a particular vascular bed . In the present study, we have examined the patterns of antigen expression and cytokine responsiveness of dermal microvascular endothelial cells (DMEC) in a skin organ culture model and, for comparison, in cell culture . Within the superficial vascular plexus (SVP) of normal skin, CD36 molecule expression is undetectable on capillary loops and is expressed on DMEC in only 20% of the larger, horizontal vessels . CD36 expression is not modulated by cytokines . Endothelial-leukocyte adhesion molecule-1 (ELAM-1) expression induced at 6 and 24 h by TNF or IL-1, is restricted to the venular side of the capillary loop and to the venules proper . Vascular cell adhesion molecule-1 (VCAM-1) expression is not inducible on EC of the SVP in normal skin by TNF, IL-1, or IL-4, alone or in combination at either time point . When inflamed skin is examined in organ culture, SVP EC are cytokine responsive regarding VCAM-1 expression . Within the deep vascular plexus (DVP) . CD36 molecules are expressed on EC in all capillaries and small vessels . Both ELAM-1 and, to a lesser extent, VCAM-1 expression are inducible by TNF, IL-1, and/or IL-4 on capillaries and larger microvessels at 6 and 24 h . The larger vessels at the dermal-subcutaneous border were found to be CD36-/ELAM-1+/VCAM-1+ after cytokine treatment . CD36 expression of DMEC in cell culture varies from 47 to 98% of cells (mean 75%) in seven separate isolates and is not modified by cytokines . Upon TNF or IL-1 activation, 50 to 90% of DMEC express ELAM-1 molecules at 6 h and expression persists at high levels for 24 h . VCAM-1 expression is negligible at both times . These results with DMEC differ from human umbilical vein EC analyzed in parallel, which are completely CD36- and show transient ELAM-1 and sustained VCAM-1 expression in response to TNF and IL-1 . In summary, we have demonstrated that DMEC comprise a heterogeneous population that differ from umbilical vein EC. J Immunol, 1993 Nov 1, 151(9), 4950 - 63 The effects of stem cell factor on the ultrastructure of Fc epsilon RI+ cells developing in IL-3-dependent murine bone marrow-derived cell cultures; Rottem M et al.; Stem cell factor (SCF) is known to alter the proteoglycans, proteases, and cytokines synthesized by mast cells and to activate basophils . To determine whether SCF could also effect the ultrastructural characteristics of basophils and mast cells, we examined the ultrastructure of these Fc epsilon RI+ cells over 42 days in IL-3-dependent murine bone marrow-derived cell cultures in the presence or absence of SCF . Initial experiments revealed that the addition of SCF to IL-3-dependent cells enhanced their proliferative rate without influencing the percentage of Fc epsilon RI+ or metachromatic cells . We next isolated the Fc epsilon RI+ cells using flow cytometry . Light microscopy of these cells revealed mixed cultures of both immature and mature mast cells and basophils with mature mast cells predominating by 3 wk . One hundred to 150 Fc epsilon RI+ cells were then photographed by electron microscopy at 3, 10, 21, and in some cases, 42 days of culture, and the ultrastructure of each cells was evaluated by morphometry . Each cell was scored as a mast cell or basophil using standard criteria . Analysis of this data revealed that SCF in the presence of IL-3 promoted the development of mast cells, although a significant number of basophils were noted at day 21 but were absent by day 42 . When bone marrow cells cultured in IL-3 + SCF were compared with cells cultured in IL-3 alone, a significant decrease in cell and nuclear size and granule number and size was noted in both mast cells and basophils cultured in IL-3 + SCF, and basophils and mast cells under these conditions most resemble their in vivo counterparts . Thus, SCF in the presence of IL-3 increases the ratio of mast cells to basophils and alters the ultrastructural characteristics of mast cells and basophils toward a more mature phenotype. J Reprod Fertil, 1993 Nov, 99(2), 571 - 5 Effects of hormones, cyclic AMP analogues and growth factors on steel factor (SF) production in mouse Sertoli cell cultures; Tajima Y et al.; Regulation of steel factor (SF) production in Sertoli cells from postnatal mouse testes was studied in a mast cell-Sertoli cell coculture system . Treatment of Sertoli cells with dibutyryl cAMP (50-1000 mumol l-1), forskolin (1-25 mmol l-1), and cholera toxin (10 micrograms ml-1) increased SF production, whereas FSH and theophylline had no significant effect . Furthermore, growth factors and testosterone, which would play some roles in spermatogenesis, were also tested, but none of these stimulated SF production . The constitutive production of SF may, rather, reflect the physiological condition of Sertoli cells in vivo. Eur J Nucl Med, 1993 Nov, 20(11), 1084 - 8 Fluorodeoxyglucose cell incorporation as an index of cell proliferation: evaluation of accuracy in cell culture; Slosman DO et al.; The use of fluorodeoxyglucose (FDG) and positron emission tomography (PET) is recognized as an accurate tool for the specific diagnosis and staging of cancer . It has also been proposed for the monitoring of anticancer therapy . FDG cell incorporation reflects glycolytic activity whereas inhibition of cell proliferation corresponds to an efficient cancer treatment . The relationship between FDG incorporation and cell proliferation has yet to be demonstrated . Therefore, we aimed to correlate the effects of the toxic agents bleomycin and unlabelled meta-iodobenzylguanidine (mIBG) on cellular metabolism and proliferation . We determined the in vitro metabolic and cytotoxic effects of bleomycin and mIBG by measuring the incorporation of fluorine-18 FDG (%UFDG) and hydrogen-3 thymidine (%UTHY) in cells of the human premonocytic line U937 in the presence of increasing concentrations of these agents . Proliferation rate of these cells was studied by means of limiting dilution analysis . %UTHY appeared more sensitive to bleomycin or mIBG-mediated cell injury than %UFDG . After 1 h of exposure to 0.5 microM bleomycin, %UTHY was significantly reduced to 62.0% +/- 10.4% of control value whereas %UFDG remained unchanged (91.6% +/- 5.3%) . Similar results were obtained after 1 h of exposure to increasing concentrations of mIBG (1 microM to 1 mM) . After 20 h of exposure to bleomycin, %UTHY and %UFDG were significantly reduced as a function of concentration . After 20 h of exposure to mIBG, a transient increase in %UFDG up to 149.3% +/- 11.2% with 50 microM mIBG was further followed by a reduction to 20.1% +/- 6.7% with 0.5 mM (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) Brain Res, 1993 Oct 22, 625(2), 337 - 41 Inhibition of nitric oxide formation does not protect murine cortical cell cultures from N-methyl-D-aspartate neurotoxicity; Hewett SJ et al.; We examined the role of nitric oxide in N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity in rat and mouse primary cortical cell cultures . In rat and mouse cultures, the NO synthase inhibitor, NG-Nitro-L-arginine, blocked cGMP formation but not neuronal cell death following a 5-10 min exposure to 300-500 microM NMDA . NG-Monomethyl-L-arginine was also unable to prevent neuronal death . In contrast, the non-competitive NMDA receptor antagonist, dextrorphan, prevented both cGMP formation and cell death . While other data suggest that the synthesis of nitric oxide can mediate NMDA receptor-mediated neurotoxicity, present results suggest that such synthesis is not necessarily required. FEMS Microbiol Lett, 1993 Oct 15, 113(2), 175 - 82 Presumptive identification of common adenovirus serotypes by the development of differential cytopathic effects in the human lung carcinoma (A549) cell culture; Lipson SM et al.; The neutralization test is commonly used in clinical virology laboratories for the identification by serotype of adenovirus isolates . In an effort to conserve reagents and reduce the amount of time in the performance of this assay, we evaluated the significance of differential cytopathic effects for the presumptive identification of lower-numbered adenovirus serotypes that are commonly encountered in the clinical setting . Utilizing the human lung carcinoma (A549) cell culture as our indicator system, two viral induced monolayer degenerations (i.e., cytopathic effects or CPEs) were recognized . Among our wild and the laboratory adapted (i.e., ATCC) adenovirus isolates tested in this study, serotypes 1, 2, 4, 5, 6, 8, 11, 19, 21, 27, and 31 were expectedly characterized by the typically enlarged, rounded, and refractile cells, which eventually aggregated into irregular 'grape-like' clusters . Adenovirus types 3 and 7, however, were characterized by the development of distinct intranuclear inclusions, a flattening and then a web or net-like monolayer degeneration . Differences in the intensity of intranuclear granulation were related by electron microscopy to differences in the quantity of viral crystalline aggregates within the host cell nucleus . A presumptive identification of the commonly encountered adenovirus serotypes 3 and 7 prior to the performance of the neutralization test would result in a conservation of type-specific antiserum, a decreased use of cell cultures and medium, and lastly, reduced medical technologist workload. J Immunol Methods, 1993 Oct 15, 165(2), 193 - 206 A simple and inexpensive high density dialysis tubing cell culture system for the in vitro production of monoclonal antibodies in high concentration; Falkenberg FW et al.; This paper describes the construction and application of a low-cost roller bottle-like culture appliance in which hybridoma cells can be cultivated in high density in dialysis tubing . The appliance facilitates the simultaneous culture of up to four cell lines yielding 50 ml culture volume of each . Samples for follow-up analysis of the cultures can easily be taken when needed through sample ports . In order to obtain high cell densities (at least 10(7) cells/ml), high cell viability (at least 50%) and high antibody yield (at least 1.0 mg/ml) the bottle is rolled at a speed of 4-6 rpm and is gassed continuously by a micropump driven by rechargeable NiCd batteries fixed to the culture flask . Depending on the individual properties of the hybridoma lines tested, the cells may be cultured for 1-2 weeks, and cell densities of up to 30 x 10(6) cells/ml with viabilities of approximately 50% and monoclonal antibodies in concentrations of up to 2.8 mg/ml may be obtained . In their properties the monoclonal antibodies produced by this in vitro procedure are indistinguishable from those prepared in the form of conventional stationary culture supernatant or of ascitic fluid . Specific antibody content is within the same range as in ascitic fluid . Consequently, the monoclonal antibodies can be purified in one step, e.g., by ion exchange chromatography from the culture supernatant . Therefore, the newly developed culture device and the culture method described is a useful alternative to ascites production in live mice. Biochem Biophys Res Commun, 1993 Oct 15, 196(1), 409 - 415 Dermatofibrosarcoma protuberans: increased growth response to platelet-derived growth factor BB in cell culture; Kikuchi K et al.; Dermatofibrosarcoma protuberans (DFSP) is a malignant tumor originating in the dermis . Although it is locally aggressive and recurs unless completely excised, it only rarely metastasizes . In the present study, we established 4 cultured DFSP cell strains, which were almost identical to normal skin fibroblasts when observed under a phase-contrast-microscope, and we observed their responses to various growth factors . DFSP cells showed significantly greater response to platelet-derived growth factor BB(PDGF BB) and transforming growth factor beta 1(TGF beta 1) than normal fibroblasts . We also determined upregulation of PDGF beta receptors in DFSP cells by both 125I PDGF-BB binding assay and immunoblotting analysis . These findings suggest that the interaction between the PDGF-B chain and the overexpression of PDGF beta receptors might play a role in the development of DFSP tumors. Brain Res, 1993 Oct 8, 624(1-2), 75 - 84 Neuronal vs . glial somatostatin in the hypothalamus: a cell culture study of the ontogenesis of cellular location, content and release; Davidson K et al.; Somatostatin (SRIH) is an ubiquitous peptide subserving functions as a hypothalamic-hypophysial regulatory peptide and as a neuromodulator in the CNS, as well as various roles in the periphery . Based on our earlier observations that the ability of primary cultures of foetal rat hypothalamic cells to secrete SRIH developed distinct differences depending on whether they were maintained in a serum-supplemented medium (SSM) or a defined, serum free medium (DM), coupled with recent reports that glia in several brain regions may express neuropeptides, we have investigated the cellular location of SRIH in these cultures . Using immunocytochemical techniques (ICC) for the simultaneous location of SRIH and glial fibrillary acidic protein (GFAP) we found that SRIH-like immunoreactivity (SRIH-LI) was present in a significant number of hypothalamic glial cells around the time of birth . In the presence of serum, this expression persists and even by day 9 in culture a dense and intense immunofluorescent SRIH-LI signal was observed in GFAP positive cells . However, in DM glial expression is switched off rapidly (although GFAP positive cells are still present) until SRIH-LI eventually occurs at a much reduced abundance in GFAP negative neuronal cell types . In addition, the SRIH content of SSM cultures was three-fold greater than that from SSM cultures . Thus our results indicate that the mechanisms for SRIH-LI release from astrocytes appears to be distinctly different to that from neuronal-type cells . We propose that glial cell expression of SRIH-LI may represent a developmentally important phenomenon in the hypothalamus, e.g . serving a trophic role during critical times of development, and that its expression in glia may be stimulated by factors present in serum . Consequently, an understanding of factors which regulate SRIH expression and release from glia is likely to be of importance to our understanding of brain development, injury, repair and diseases of the CNS. J Neurosci, 1993 Oct, 13(10), 4281 - 92 Signal transduction events mediated by the BDNF receptor gp 145trkB in primary hippocampal pyramidal cell culture; Marsh HN et al.; The trkB gene encodes a tyrosine kinase receptor, gp145trkB, for brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) . To understand the role of gp145trkB in the nervous system, we have investigated its expression in embryonic rat hippocampal pyramidal cell cultures and examined the effects of BDNF on signal transduction in the primary neurons . The expression of trkB transcripts was established by PCR analysis and in situ hybridization . In addition to gp145trkB, the pyramidal neuronal cultures expressed transcripts specific for the NT-3 receptor gp145trkC, but not for the high-affinity NGF receptor gp140trk or for p75LNGFR, a low-affinity receptor for all known members of the NGF family of neurotrophins including the gp145trkB ligands, BDNF and NT-4 . The presence of gp145trkB receptors in the primary neuronal cultures was confirmed by immunocytochemical analysis in which > 90% of the cells stained with affinity-purified polyclonal antibodies to gp145trkB . Immunoblots using this antibody revealed a single approximately 140 kDa protein in both adult hippocampus and pyramidal cultures . Addition of recombinant BDNF to these cultures induced the tyrosine phosphorylation of gp145trkB, as determined by antiphosphotyrosine staining of gp145trkB immunoprecipitates . Moreover, BDNF treatment activated the microtubule-associated protein (MAP) kinases, as determined by an increase in MAP2 phosphorylation in vitro . Both the 41 and 44 kDa forms of MAP kinase were activated by BDNF . BDNF also increased c-fos expression in over 90% of the cells . These results indicate that gp145trkB does not require p75LNGFR to form a functional receptor for BDNF in hippocampal pyramidal neurons. J Invest Dermatol, 1993 Oct, 101(4), 634 - 8 Human hair follicle germinative epidermal cell culture; Reynolds AJ et al.; Isolated human hair follicle germinative epidermal cells were observed in vitro for the first time . When cultured alone, this small, round, novel cell type did not grow, divide, take on an outer root sheath-type appearance, or display any obvious signs of epidermal differentiation . We have previously described comparable cells from rat vibrissa follicles . However, in combination with human hair follicle dermal papilla populations, the germinative epidermal cells were stimulated into proliferative and complex interactive behaviors . This included the formation of composite organotypic structures containing not only impressively intact basement membrane, but also the hair-specific form, glassy membrane. J Clin Endocrinol Metab, 1993 Oct, 77(4), 925 - 31 Regulation of inhibin secretion in human placental cell culture by epidermal growth factor, transforming growth factors, and activin; Qu J et al.; In this study, the effects of epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), TGF beta 1, and activin-A on inhibin secretion were investigated in primary culture of human placental cells . Immunoreactive inhibin in the culture medium was measured by immunoenzymatic assay . EGF stimulated testosterone-induced inhibin secretion in placental cells . Although testosterone alone induced only a slight enhancement of inhibin release in the culture, treatment of trophoblast cells with EGF and testosterone caused a significant increase in inhibin secretion, with immunoreactive inhibin levels much higher than those of testosterone or EGF alone . TGF alpha combined with human placental lactogen (hPL) had a stimulatory effect on inhibin secretion in placental cell culture . Simple addition of either TGF alpha or hPL to the culture did not show any effect on inhibin secretion in placental cells . A remarkable augmentation of inhibin secretion was obtained after the trophoblasts were exposed to both TGF alpha and hPL simultaneously . TGF beta 1 and activin-A showed synergistic effects to suppress inhibin secretion in placental cells . TGF beta 1 alone did not show any action on inhibin secretion, and activin-A alone induced a small decrease in inhibin release in the culture . In the presence of activin-A, addition of TGF beta 1 to the culture induced a profound decrease in immunoreactive inhibin levels in the medium . Activin-A could also suppress hCG-induced inhibin secretion in placental cells . Addition of hCG alone resulted in a small, but not significant, increase in inhibin release in the cultured cells, whereas the presence of activin-A combined with hCG in the culture conversely decreased inhibin secretion in the culture, with immunoreactive inhibin levels significantly lower than those in the presence of hCG or activin-A alone . These findings suggest that EGF and TGF alpha, alone or in combination with other hormones, may be stimulators, and TGF beta and activin may act as suppressors of inhibin secretion in human placental cells. J Virol, 1993 Oct, 67(10), 5976 - 88 Characterization of chimeric full-length molecular clones of Aleutian mink disease parvovirus (ADV): identification of a determinant governing replication of ADV in cell culture; Bloom ME et al.; The ADV-G strain of Aleutian mink disease parvovirus (ADV) is nonpathogenic for mink but replicates permissively in cell culture, whereas the ADV-Utah 1 strain is highly pathogenic for mink but replicates poorly in cell culture . In order to relate these phenotypic differences to primary genomic features, we constructed a series of chimeric plasmids between a full-length replication-competent molecular clone of ADV-G and subgenomic clones of ADV-Utah 1 representing map units (MU) 15 to 88 . After transfection of the plasmids into cell culture and serial passage of cell lysates, we determined that substitution of several segments of the ADV-Utah 1 genome (MU 15 to 54 and 65 to 73) within an infectious ADV-G plasmid did not impair the ability of these constructs to yield infectious virus in vitro . Like ADV-G, the viruses derived from these replication-competent clones caused neither detectable viremia 10 days after inoculation nor any evidence of Aleutian disease in adult mink . On the other hand, other chimeric plasmids were incapable of yielding infectious virus and were therefore replication defective in vitro . The MU 54 to 65 EcoRI-EcoRV fragment of ADV-Utah 1 was the minimal segment capable of rendering ADV-G replication defective . Substitution of the ADV-G EcoRI-EcoRV fragment into a replication-defective clone restored replication competence, indicating that this 0.53-kb portion of the genome, wholly located within shared coding sequences for the capsid proteins VP1 and VP2, contained a determinant that governs replication in cell culture . When cultures of cells were studied 5 days after transfection with replication-defective clones, rescue of dimeric replicative form DNA and single-stranded progeny DNA could not be demonstrated . This defect could not be complemented by cotransfection with a replication-competent construction. J Neurochem, 1993 Oct, 61(4), 1470 - 8 Toxic and protective effects of L-dopa on mesencephalic cell cultures; Mytilineou C et al.; The autoxidation of L-DOPA or dopamine (DA) and the metabolism of DA by monoamine oxidase generate a spectrum of toxic species, namely, hydrogen peroxide, oxy radicals, semiquinones, and quinones . When primary dissociated cultures of rat mesencephalon were incubated with L-DOPA (200 microM) for 48 h, the number of tyrosine hydroxylase-positive neurons (DA neurons) was reduced to 69.7% of control values, accompanied by a decrease in {3H}DA uptake to 42.3% of control values; the remaining DA neurons exhibited reduced neurite length and overall deterioration . Lack of simultaneous change in the number of neurons stained with neuron-specific enolase indicated that toxicity was relatively specific for DA neurons . At the same time, the level of GSH, a major cellular antioxidant, rose to 125.2% of control values . Thus, exposure of mesencephalic cultures to L-DOPA results in both damaging and antioxidant actions . Ascorbate (200 microM), an antioxidant, prevented the rise in GSH . The effect of ascorbate on GSH points to an oxidative signal to initiate the rise in GSH content . On the other hand, neither inhibition of monoamine oxidase with pargyline nor addition of superoxide dismutase or catalase to the culture medium prevented the rise in GSH level or the loss in {3H}DA uptake . The latter results tend to exclude the products of monoamine oxidase activity or the presence of hydrogen peroxide or superoxide in the medium as responsible agents for the rise in GSH or neuronal toxicity . In cultures treated with L-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, L-DOPA prevented cell death by L-BSO. Epithelial Cell Biol, 1993 Oct, 2(4), 163 - 9 Differences in secretory profiles of epithelial cell cultures derived from human tracheal and bronchial mucosa and submucosal glands; Taylor GW et al.; The respiratory tract contains macromolecules produced by various epithelia including tracheal and bronchial mucosa and submucosal glands . The objectives of this study were to elucidate and compare the growth and secretory profiles of epithelial cell cultures derived from the human tracheal (TC) and bronchial mucosa (BC) and submucosal glands (GC) . Most experiments were done on third to fourth passage cultures . Secretory glycoconjugates were characterized by a combination of gel filtration and anion-exchange chromatography after enzymic digestion with hyaluronidase of {3H}glucosamine and {35S}sulphate incorporated glycoconjugates secreted into the culture medium . Intracellular mucin-like glycoproteins were characterized by immunohistochemical staining with a human monoclonal respiratory mucin antibody . Results showed that the three cell types exhibited variable growth rates and secretory profiles . Doubling times of GC, BC and TC were 53, 75 and 80 h respectively . Immunocytochemical staining with the mucin antibody demonstrated positive reaction in GC and BC; TC showed no significant reaction . Mucin-like glycoproteins were detected in the spent media of GC and BC whereas TC, under the same conditions, did not produce any detectable amount of the glycoconjugates . Further, the mucin-like materials produced by GC and BC differed in their relative glycosylation and sulphation levels . The production of mucin was independent of substrate and vitamin A as the cultures were propagated on the plastic surfaces and the culture medium lacked vitamin A. J Virol Methods, 1993 Oct, 44(2-3), 329 - 38 An in situ hybridization technique for the study of B19 human parvovirus replication in bone marrow cell cultures; Vassias I et al.; An in situ hybridization technique using digoxigenin labelling was developed to study B19 infection . By using appropriate DNA probes, transcription of structural and non-structural genes was detected in bone marrow cell cultures . Such a simple system is useful to the study of B19-cell interactions in non-permissive cell lines. Toxicol Appl Pharmacol, 1993 Oct, 122(2), 265 - 72 The toxic effects of formate in dissociated primary mouse neural cell cultures; Dorman DC et al.; Primary dissociated mouse cerebrocortical cell cultures containing both neurons and glial cells were used as an experimental model to study the neurotoxic effects of formate, the putative toxic metabolite of methanol . Neural cells were isolated and prepared from the cerebral cortex of fetal CD-1 mice on Gestational Day 15 . Mature 7- to 15-day-old monolayer cultures were exposed to formate (0 to 240 mM) for 8 hr at 37 degrees C over a range of extracellular pH (6.0 to 7.6) . Cytotoxicity was evaluated by histopathology, changes in membrane integrity (lactate dehydrogenase release, LDH; {14C}adenine nucleotide leakage), and mitochondrial metabolic activity {reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT} . Similar quantitative estimates of cell injury were obtained by LDH release or {14C}adenine nucleotide leakage from prelabeled cells . Exposure of neural cells produced time- and concentration-dependent toxic responses . The concentration of formate that resulted in 50% LDH leakage after an 8-hr incubation was estimated to be 45 mM . As determined by light microscopy, formate (20 to 60 mM) was specifically neuronotoxic, primarily affecting large polygonal neurons . Higher concentrations of formate (> or = 120 mM) induced nonspecific cytotoxicity . MTT reduction appeared to be a more sensitive endpoint by showing significant toxic effects at 20 mM (8-hr incubation), while significant leakage of LDH occurred only at formate concentrations > or = 60 mM . Total intracellular ATP concentration was significantly decreased following a 20 or 40 mM formate exposure for 8 hr . These results are consistent with the hypothesis that formate may inhibit mitochondrial function resulting in decreased intracellular ATP and formate-induced neurotoxicity. Br Med Bull, 1993 Oct, 49(4), 860 - 72 Scrapie associated PrP accumulation and its prevention: insights from cell culture; Caughey B; Transmissible spongiform encephalopathies (TSEs), Alzheimer's disease and other amyloidoses result in the accumulation of abnormally stable, potentially amyloidogenic proteins that appear to play central roles in disease pathogenesis . Scrapie-infected tissue culture cells have become well-developed models for studying how the TSE-specific protein, protease-resistant PrP, is made from its apparently normal precursor . The conversion of PrP to the protease-resistant state occurs on the plasma membrane or along an endocytic pathway to the lysosomes . The protease-resistant PrP has a much longer half-life than normal PrP and its accumulation in lysosomes may feature in TSE pathogenesis . Congo red and certain sulfated glycans potently inhibit protease-resistant PrP formation or stabilization in cell culture . These and other observations suggest that an interaction of PrP with glycosaminoglycans is critical in protease-resistant PrP accumulation and raises the possibility that therapeutic strategies for TSEs and other amyloidoses could be based on blocking (pre)amyloid-glycosaminoglycan interactions. J Biotechnol, 1993 Oct, 31(1), 1 - 15 Anticarsia gemmatalis nuclear polyhedrosis virus replication in serum-free and serum-reduced insect cell cultures; Claus JD et al.; In order to develop a financially feasible process to produce Anticarsia gemmatalis Nuclear Polyhedrosis virus in cell culture, we developed a lipidic supplement to replace fetal calf serum in insect cell culture media . The supplement, prepared with an extract of lipids from hen egg yolk, allowed us to reduce the contents of serum in the culture medium from 10% to 1% . IPLB-Sf-21 cells could be kept along consecutive passages in serum-reduced medium . The replication of AgNPV in HEYLE-supplemented cultures was evaluated . Extracellular virions production was the same as in FCS-supplemented-cultures, but the production level of polyhedral inclusion bodies was significantly lowered in HEYLE-supplemented cultures . The reduced production of PIBs is related to a premature releasing of non-occluded particles as well as to a reduced synthesis of polyhedrin protein. Biomaterials, 1993 Oct, 14(12), 917 - 24 Biological evaluation of an ionomeric bone cement by osteoblast cell culture methods; Meyer U et al.; Periosteal derived bovine osteoblast-like cells migrated in culture onto an ionomeric cement . Cell cultures were maintained for 4 weeks and used to study the in vitro behaviour of cells on the ionomeric bone cement (IC) . The cells produced bone matrix proteins (osteocalcin, bone sialoprotein II) and were osteoblast-like . The osteoblast-like cells colonized the substrate in monolayers and produced an extracellular matrix as seen by light and scanning electron microscopy . Morphological comparison between cells growing on the ionomeric bone cement and cortical bone revealed no significant difference in phenotypic expression . Staining for aluminium in osteoblasts growing on the IC showed an uptake and storage of aluminium in the cells . Energy dispersive X-ray microanalysis revealed high concentrations of aluminium and silicon in the periosteal tissue . Despite the known toxic effect of aluminium in vivo and in vitro on osteoblasts, no signs of toxicity were apparent on light and scanning electron microscopy analysis. Proc Natl Acad Sci U S A, 1993 Sep 15, 90(18), 8424 - 8 Derivation of completely cell culture-derived mice from early-passage embryonic stem cells; Nagy A et al.; Several newly generated mouse embryonic stem (ES) cell lines were tested for their ability to produce completely ES cell-derived mice at early passage numbers by ES cell <==> tetraploid embryo aggregation . One line, designated R1, produced live offspring which were completely ES cell-derived as judged by isoenzyme analysis and coat color . These cell culture-derived animals were normal, viable, and fertile . However, prolonged in vitro culture negatively affected this initial totipotency of R1, and after passage 14, ES cell-derived newborns died at birth . However, one of the five subclones (R1-S3) derived from single cells at passage 12 retained the original totipotency and gave rise to viable, completely ES cell-derived animals . The total in vitro culture time of the sublines at the time of testing was equivalent to passage 24 of the original line . Fully potent early passage R1 cells and the R1-S3 subclone should be very useful not only for ES cell-based genetic manipulations but also in defining optimal in vitro culture conditions for retaining the initial totipotency of ES cells. Med Klin (Munich), 1993 Sep 15, 88(9), 520 - 4 {In vitro studies of the beat frequency of ciliary cell cultures after short-term exposure to SO2 and NO2}; Kienast K et al.; Mucociliary transport is an important defense mechanism of the respiratory tract . The aim of this study was to investigate the effect of SO2 and NO2 at different concentrations on ciliary beat frequency (ZSF) . Single ciliated cells were obtained from 25 volunteers by nose brush . ZSF was quantified using video-interference-microscopy . The cells were placed on a polycarbonate membrane, which was in contact with the surface of a reservoir filled with RPMI medium (bicarbonate buffered) or electrolyte solution (Ringer), allowing the cells to be supplied by capillarity . In an exposure chamber the cells were exposed for 30 to 120 min to SO2 2.5 to 15.0 ppm at 37 degrees C . SO2 induced a dose dependent decrease in ZSF of the cells, supported by Ringer solution . 2.5 ppm SO2 caused a 42.8%, 12.5 ppm a nearly 100% decrease (8.10 +/- 0.24 vs . 0.28 +/- 0.20 Hz) . ZSF of cells cultured in RPMI medium was reduced moderately after 12.5 ppm SO2 exposure (7.90 +/- 0.26 vs . 6.66 +/- 0.31 Hz) . In Ringer solution we observed a decrease of pH after 30 min SO2 exposure with 12.5 ppm to a minimum value of 3.6 . In marked contrast, the pH of RPMI medium remained constant at 7.5 under identical conditions . After adding RPMI medium to Ringer solution, ZSF increased in parallel to the pH (5.0 ppm: 2.77 +/- 0.37 to 7.97 +/- 0.49 Hz) . After an initial increase in ZSF, 120 min NO2 exposure to 15.0 ppm yielded a decrease in ZSF of 23.3% under conditions of constant pH.(ABSTRACT TRUNCATED AT 250 WORDS) Int J Cancer, 1993 Sep 9, 55(2), 256 - 61 Retinoic-acid-induced augmentation of molecular species carrying sialosyl Lewis(a) antigen in colorectal-carcinoma cell cultures; Liepkalns VA et al.; In order to study the effects of vitamin-A metabolites on long-term carcinoma-antigen secretion, colorectal-carcinoma cells SW1116 were cultured on membrane filters in totally synthetic media with 0 to 2.6 microM retinoic acid (RA) . RA altered cell division, cell size and soluble-sialosyl Le(a) (S-Le(a) secretion and S-Le(a) accumulation within cells and apical-membrane domains . Cultures treated with RA for 10-12 days grew to lower cell densities (60% of controls) and contained more protein per cell (140% of controls) . RA treated cells also had 5-fold higher levels of S-Le(a) in cells and secreted 9-fold more S-Le(a) into culture media assayed per 24 hr by (ELISA) 19-9 monoclonal antibody binding . As total media S-Le(a) increased, polarity of non-lipid S-Le(a) antigen secretion increased toward the interior (apical) media . High-performance thin-layer immunobinding showed that ganglioside S-Le(a) was higher in RA-fed cells, but could not be detected in apical media of RA-fed or control cells after 24 hr . Western blots indicated that non-lipid sialosyl Lewis(a) was bound to 150- to 180-kDA molecular species principally in cells, but 210- to 300-kDa molecular species appeared in the non-lipid extract of media . Thus, the above RA alterations, monitored by 3 immunochemical techniques, include up to 9-fold stimulation of "constitutive" 150- to 300-kDa sialosyl-Lewis(a) secretion, but ganglioside Lewis(a) is sorted differently and retained by apical membranes. Neurosurgery, 1993 Sep, 33(3), 485 - 8; discussion 488 Determination of the lethal dose of dexamethasone for early passage in vitro human glioblastoma cell cultures; Maciunas RJ et al.; Previous investigators have supported the idea that glucocorticoids may be oncolytic . In this study, the percentage of cell death in two human glioblastoma cell cultures was related to the concentration of dexamethasone that was administered . It was determined that for Cell line 1, the median lethal dose was approximately 500-800 micrograms/ml and the completely lethal dose was about 900-1000 micrograms/ml; the 3H-thymidine uptake to approximate the mitotic rate was 16,607 cpm, and the dexamethasone receptor activity was 228 fmol/mg protein . The median lethal dose and completely lethal dose for Cell line 2 was approximately 500-600 micrograms/ml and 700-1000 micrograms/ml, respectively; the 3H-thymidine uptake was 8402 cpm, and the dexamethasone receptor activity was 137 fmol/mg protein . These lethal concentrations of dexamethasone are probably higher than can be tolerated by systemic delivery . However, it remains to be seen whether the interstitial administration of dexamethasone could achieve local concentrations resulting in the oncolysis of malignant gliomas . The clinical significance of these findings will depend on the local tolerance of normal brain parenchyma to very high doses of dexamethasone . A review of some of the literature is included. J Surg Res, 1993 Sep, 55(3), 307 - 13 An eosinophil chemotactic lymphokine in the supernatant of lymphokine-activated killer (LAK) cell culture: relationship to interleukin-5; Ishimitsu T et al.; The administration of interleukin-2 (IL-2) systemically or locally to cancer patients, either alone or in combination with lymphokine-activated killer (LAK) cells, frequently results in eosinophilia . To investigate the mechanism of such eosinophil accumulation, we measured in vitro eosinophil chemotactic activity (ECA) in the supernatant of LAK cell cultures by a modified Boyden's chamber method . Consequently, a potent and specific ECA was found in the cell-free supernatant of LAK cell cultures . On the other hand, no ECA was found in the supernatant of normal lymphocyte cultures, nor in IL-2 itself . This activity plateaued when 3 x 10(6) lymphocytes were incubated with 1000 U/ml IL-2 for more than 3 days . Sephadex G-100 gel chromatography indicated that the molecular weight of this eosinophil chemotactic factor (ECF) was approximately 45,000 Da . The ECF was stable when heated to 56 degrees C for 30 min but was inactivated by trypsin, indicating that the ECF is a lymphokine which has very similar characteristics to those of IL-5 . Using ELISA for hIL-5, the IL-5 level in the supernatant of LAK cell cultures increased dose dependently with increasing IL-2 concentrations . These results suggest that IL-5 may be responsible for the local recruitment of eosinophils in IL-2/LAK therapy. Cell Tissue Res, 1993 Sep, 273(3), 571 - 5 Collagen fibrillogenesis in a three-dimensional fibroblast cell culture system; Contard P et al.; The purpose of this study was to follow collagen fibril formation in a newly developed three dimensional cell culture system . Human neonatal foreskin fibroblasts were grown on a nylon mesh in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal calf serum and antibiotics . Fibrillogenesis was initiated by the addition of 50 micrograms/ml ascorbate to confluent cultures . Sample meshes were processed for electron microscopy or immuno-electron microscopy . Fibrils approximately 20-30 nm in diameter, with 67 nm periodicity, were first detected five days after the addition of ascorbate . As cultures progressed, cells organized into parallel layers between which collagen fibers continued to form and increase in diameter . By day 50, fiber diameter ranged from 30 to 80 nm and large bundles were seen . No collagen fibril formation occurred in control cultures to which no ascorbate was added . However, large amounts of microfibrils were observed . Antibodies against the aminopropeptide of type I procollagen were found to bind to fibrils with diameters less than 34 nm while antibodies against the aminopropeptide of type III collagen bound primarily to fibers which ranged from 35-54 nm in diameter . We believe that this system, which morphologically resembles a normal dermis, will serve as an excellent model for the study of collagen fibrillogenesis. Nippon Sanka Fujinka Gakkai Zasshi, 1993 Sep, 45(9), 987 - 93 {The effect of steroid hormone on oxytocin receptor expression in primary human uterine myometrial cell culture}; Adachi S et al.; In the present study, we have examined the steroid hormone effect on oxytocin receptor (OTR) expression in human myometrial monolayer culture . The myometrial cells were cultured for 24-72 hours in serum-free media with various concentrations of steroid hormones . The plasma membrane of the cultured cells was then collected . The 125I-oxytocin(OT) was employed for a binding assay and the mean binding and dissociation constant were evaluated by Scatchard analysis . 1 . The OT binding values in the presence of estradiol free, 10(-8) M, and 10(-7) M at 72 hours were 19.85, 28.12, and 36.20pM/mg protein, respectively . 2 . The OT binding values in the presence of estradiol (10(-7) M) at 24, 48, and 72 hours were 17.85, 23.72, and 36.20pM/mg protein, respectively . 3 . Dehydroepiandrosterone sulfate (10(-5) M) exerted almost no effect on the OTR expression by estradiol (10(-7) M) . Cortisol (10(-5) M) decreased the amount of OTR expressed by estradiol . 4 . The changes in OTR expression caused by estradiol (10(-7) M) were inhibited according to the concentration by progesterone . The concentration of progesterone at which OT binding was reduced 50% was 2.7 x 10(-6) M (E/P ratio = 0.037) . These results suggested that estradiol increased the amount of OTR discovered in human myometrial cells in a time and concentration dependent manner, while progesterone inhibited the changes in OTR expression by estradiol in a concentration dependent manner . Thus, it was indicated that the changes in the E/P ratio during pregnancy which regulate the OTR expression control the myometrial contraction. Biochem J, 1993 Sep 1, 294 ( Pt 2), 427 - 33 Use of 18O-labelled leucine and phenylalanine to measure protein turnover in muscle cell cultures and possible futile cycling during aminoacylation; Fuller JC Jr et al.; Amino acids labelled with 18(O) on both carboxy oxygen atoms have the potential for use as non-recyclable tracers to measure protein turnover . During protein synthesis one of the labelled oxygen atoms is removed, and thus release of the mono-labelled amino acid could be used to determine proteolysis . Primary cultures of embryonic-chick skeletal-muscle cells were used to test the use of 18(O2)-labelled Leu to measure proteolysis . For 9-day cultures, prelabelled on days 2-8 with medium containing one-half the Leu as {18O2}Leu and one-half as {2H3}Leu, release of {18(O)}Leu was less than 50% that of {2H}Leu over 24 h, suggesting a loss of the 18O label by a mechanism other than protein synthesis . Medium containing {18(O2)}Leu, {2H3}Leu, {18O2}Phe and {13C}Phe was then incubated with 9-day cultures to compare the rate of loss of the 18(O)-label from Leu and Phe with the rate of uptake of the non-carboxy-oxygen-labelled amino acids . Results for Leu demonstrated an 81% loss of the 18(O) label compared with a 33% decrease in {2H}Leu over 12 h . Loss of the 18(O) label was four times as great for Leu as for Phe . Loss of the 18(O) label was not decreased by addition of cycloheximide or by addition of a 3-fold excess of Ile, Val and Tyr; thus the loss of label was not due to protein synthesis alone or to misbinding to incorrect tRNAs . Infusion of the isotopes into pigs showed that the 18(O) label of Leu was not lost during transamination to alpha-ketoisocaproate (alpha-oxoisohexanoate) . The most probable explanation is that the 18(O) label is lost as a result of the enzymic deacylation of tRNA, that this process is substantially faster for Leu than for Phe, and that this represents a potentially costly futile cycle for Leu. Nippon Sanka Fujinka Gakkai Zasshi, 1993 Sep, 45(9), 994 - 1000 {The effect of oxytocin on production of free fatty acid in primary human uterine myometrial cell culture}; Otsuki T et al.; Oxytocin(OT) is considered to have several activities besides strongly inducing myometrial contraction by activating phosphatidilinositol-specific phospholipase C(PI-PLC) . These include reconstructing the phospholipid constituents of the cell membrane and activating a variety of fatty acid producing systems . On the other hand, pregnancy-related steroid hormones which are produced by the fetus, placenta and mother are considered to be closely involved in the maintenance of pregnancy and the initiation of labor . In the present study with cultured myometrial cells, we examined what effect these steroid hormones might exert on the intramyometrial production of fatty acid by OT . Our results confirmed bi-phasic production of arachidonic acid(AA), linoleic acid(LA), palmitic acid(PA), and stearic acid(SA) by OT . Phase 1 was an increasing but transient phenomenon having its peak at 30 sec . It is considered to be derived from phosphatidylinositol bis-phosphate . Phase 2 was a persistent and increasing phenomenon which was initiated after 120 sec . It is considered to be mediated by Ca-dependent phospholipase . We also studied the effect of steroid hormones on the production of fatty acid . For AA, LA, and PA, we confirmed that dehydroepiandrosterone sulfate(DHAS) shortened the time taken in reaching the peak of Phase 1 to half of that of the control, and progesterone(P) extended the time 2-3 fold . These findings suggest that DHAS, P and F might modify the human myometrial construction mechanism as a factor which regulates the quantity and velocity of fatty acid production. Dev Biol, 1993 Sep, 159(1), 223 - 31 Basic FGF maintains some characteristics of the progress zone of chick limb bud in cell culture; Watanabe A et al.; The apical region of chick limb buds (progress zone, PZ) is known to be essential for limb pattern formation . In the PZ, the cells are in an uncommitted state and change their positional values under the influence of the apical ectodermal ridge (AER) . Using a culture system of chick PZ cells, we have examined the factor(s) which helps to maintain some of the characteristics of PZ cells . It was found that basic FGF (bFGF) stimulated the expression of AV-1 and Msx 1 mRNA, which are rich in PZ cells at early stages, in the cultured PZ cells . These two molecules were rapidly lost in the control culture without the bFGF . Further, bFGF promoted growth and inhibited chondrogenesis of the cultured PZ cells . When quail PZ cells were cultured in FGF medium, harvested, and grafted to the apical region of chick limb buds, in about half of the grafts, the cells distributed to form a long band along the proximodistal axis and participated in normal host cartilage pattern formation as did intact PZ tissues . In contrast, those cultured in the medium without bFGF distributed as a cluster and did not participate in host cartilage pattern formation . The culture with conditioned medium of BRL cells increased the percentage of the grafts participating in normal pattern formation . These results indicate that bFGF helps to maintain some of the characteristics of PZ cells in culture and suggest that bFGF may be a PZ maintenance factor from the AER. J Cereb Blood Flow Metab, 1993 Sep, 13(5), 803 - 10 Secobarbital attenuates excitotoxicity but potentiates oxygen-glucose deprivation neuronal injury in cortical cell culture; Giffard RG et al.; We examined the effects of secobarbital and other sedative-hypnotic barbiturates on the neuronal death induced by exposure to excitatory amino acids or deprivation of oxygen or glucose in mouse cortical cell cultures . N-Methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionate, and kainate toxicities were attenuated in a concentration-dependent fashion by high concentrations of secobarbital or thiopental . Antagonism of NMDA toxicity was not overcome by increasing NMDA concentration and not mimicked by gamma-aminobutyrate . Despite these antiexcitotoxic actions, secobarbital exacerbated the neuronal death induced by deprivation of either glucose alone or oxygen and glucose together; death induced by oxygen deprivation alone was little affected . Thiopental and methohexital also increased oxygen-glucose deprivation injury . A possible explanation for this injury potentiation was provided by the observation that secobarbital enhanced the cellular ATP depletion induced by combined oxygen-glucose deprivation . Deleterious effects on ATP production may counterbalance the protective effects of barbiturates under some conditions. Sci China B, 1993 Sep, 36(9), 1110 - 6 The effects of glutamate and GABA (gamma-amino-butyric-acid) on spontaneous acetylcholine release at the neuromuscular junction in Xenopus laevis embryo cell cultures; Xie ZP et al.; The miniature endplate currents (MEPC's) were recorded at the neuromuscular junction of Xenopus laevis embryo neuron-muscle co-cultured cells . These MEPC's were due to the spontaneous release of acetylcholine from the nerve terminal . After perfusion with glutamate (10 mumol/L), both frequency and amplitude of the MEPC's increased . After washing away of glutamate, this effect persisted . We named this phenomena "Long-Term Facilitation" . GABA (20 mumol/L) on the other hand had an inhibitory effect on both frequency and amplitude of the MEPC's . After washing away of GABA, the MEPC frequency and amplitude increased . We named this effect "Post-Potentiation" . Local perfusion experiments furthermore indicated that the effect of glutamate was restricted to the neuromuscular junction, the effect of GABA was restricted to the soma. Hum Reprod, 1993 Sep, 8(9), 1380 - 6 Development of a human granulosa cell culture model with follicle stimulating hormone responsiveness; Schipper I et al.; In order to study the effects of follicle-stimulating hormone (FSH) on differentiation of granulosa cells, a well-defined and validated in-vitro culture system is indispensable . In this study, pooled follicular aspirates were stimulated in vitro with FSH and luteinizing hormone (LH) for 2, 4 and 6 days, either immediately after plating or after 7 days of preincubation . Cultures were assayed for progesterone and oestradiol production . Fresh cells displayed very high basal progesterone production which could be stimulated with LH but not FSH . After preincubation, addition of LH and FSH resulted in dose-dependent increases of progesterone and oestradiol . When cultured on human fibronectin-coated wells, similar basal but higher progesterone concentrations after stimulation were observed . In comparison with serum-free media, addition of Serum-Plus resulted in higher basal and stimulated progesterone concentration, possibly due to the presence of serum factors . This study demonstrates firstly that after 7 days preincubation, cultures gained responsiveness to FSH but remained responsive to LH during 4 days of stimulation . This suggests a persisting differentiated cell population in vitro . Secondly, the use of human fibronectin extracellular matrix and serum promotes steroid production, either due to factors promoting cell growth and function or to availability of steroid precursors . Therefore one has to be cautious with interpretation of data obtained from this widely used culture system, employing highly differentiated cells obtained after ovarian stimulation for in-vitro fertilization for study of local regulation of granulosa cell function. Cell Biol Int, 1993 Sep, 17(9), 839 - 45 Changes on protein expression associated with salinity tolerance in Brassica cell cultures; Martin JP et al.; The synthesis of proteins from salt-tolerant Brassica oleracea L . var . botrytis L . subvar . cauliflora (Gars.) DC . (cauliflower) cell cultures is modified in relation to controls in several features . There are nine newly induced polypeptides in tolerant cultures (absent in control conditions) . Some of them are only present under low salt levels (85 mM NaCl) . Another group seems to be representative of moderate and high salt levels (170 and 255 mM NaCl), and a third group is present in all the salt conditions tested . On the other hand, the synthesis of most of the polypeptides present in control conditions is modified in salt-tolerant cultures by increasing, decreasing or stopping their synthesis in any of the tested conditions . The relationship between these changes in Brassica and other plant systems is discussed. Appl Environ Microbiol, 1993 Sep, 59(9), 3145 - 6 Evaluation of radioactive and nonradioactive gene probes and cell culture for detection of poliovirus in water samples; Moore NJ et al.; Five nonradioactive probe assays were evaluated by using chemiluminescent and colormetric signals, along with two isotopic assays and cell culture, for the detection of poliovirus in concentrated water samples . In environmental samples, a 100% correlation existed between digoxigenin and single-stranded {32P}RNA probes . All probe assays detected more positive samples than the cell culture did. Biotechnol Prog, 1993 Sep-Oct, 9(5), 510 - 9 Complex coacervate microcapsules for mammalian cell culture and artificial organ development; Matthew HW et al.; A number of combinations of anionic and cationic polymers, the majority being polysaccharides, were screened to determine their suitability for the development of alternative microcapsule formulations capable of supporting cells . The capsules were taken through a limited optimization and then evaluated on the bases of rupture strength, permeability to albumin, and ability of their components to promote the attachment, aggregation, and function of encapsulated rabbit hepatocytes . The widely used alginate-polylysine capsules were employed as a comparative standard in all tests . A number of the new formulations compared favorably with the standard, and some exhibited superior performance in specific areas . Hepatocyte function, as evaluated by the rate of urea synthesis, showed no significant differences between formulations over a 24-h test period . One formulation, composed of the polysaccharides (carboxymethyl)cellulose, chondroitin sulfate A, chitosan, and polygalacturonate, was found to be superior to alginate-polylysine capsules in the areas investigated and supported the long-term survival and growth of liver endothelial cells. Biotechnology (N Y), 1993 Sep, 11(9), 1037 - 41 High level, stable production of recombinant proteins in mammalian cell culture using the herpesvirus VP16 transactivator; Hippenmeyer P et al.; We have engineered mammalian cell lines to produce high levels of heterologous proteins by constructing a cell line that expresses the herpesvirus transactivator, VP16 . Subsequent stable transfection with a gene of interest under control of a herpesvirus immediate early promoter led to a rapid isolation of cell lines producing between 1 and 20 micrograms of protein/million cells/24 hours . This high level expression is stable for at least five months. Biotechniques, 1993 Sep, 15(3), 444 - 7 Tartrate-resistant acid phosphatase gene expression as a facile reporter gene for screening transfection efficiency in mammalian cell cultures; Reddy SV et al.; The efficiency of DNA transfection into mammalian cell cultures has been monitored using a variety of reporter assays . However, the common procedures are expensive, time-consuming and usually cannot identify the transfected cell population directly . In the present communication we describe a simple, inexpensive and efficient method to directly identify DNA transfection in mammalian cells using tartrate-resistant acid phosphatase (TRAP) gene expression . The method involves the transfection of a plasmid (pCT3), which contains TRAP cDNA driven by a CMV promoter, into mammalian cells . The cells can then be stained for TRAP activity, and the transfection efficiency can be determined by simply counting the positively transfected cells in a defined area with a microscope . This method permits screening of mammalian cells for transfection efficiency in multi-well plates . After waiting 30-40 minutes to allow the TRAP assay to saturate, wells can be scored in 1-2 minutes with little difficulty in detecting the transfected cells. FEBS Lett, 1993 Aug 23, 329(1-2), 43 - 6 Metribuzin resistance in photoautotrophic Chenopodium rubrum cell cultures . Characterization of double and triple mutations in the psbA gene; Schwenger-Erger C et al.; Sequence analysis of eight metribuzin-resistant mutants of photoautotrophic Chenopodium rubrum cell cultures revealed new mutations in the psbA gene coding for the 32 kDa herbicide binding protein . Mutants were found to possess either two or three changes in the amino acid sequence of the D1-protein between positions 219 and 272. Eur J Pharmacol, 1993 Aug 15, 246(3), 261 - 7 Cytoprotective effect of NMDA receptor antagonists on prion protein (PrionSc)-induced toxicity in rat cortical cell cultures; Muller WE et al.; Rat cortical cells were incubated with the Scrapie prion protein, PrionSc . At concentrations of 3 ng/ml of PrionSc and higher, the viability of the cells decreased significantly after a 12-h incubation period . Simultaneously, the degree of DNA fragmentation increased . In control experiments with antibodies against PrionSc, PrionSc lost its deleterious effect on neurons . PrionSc did not affect the viability of astrocytes . Drugs known to block NMDA receptor channels, such as memantine (1-amino-3,5-dimethyl-adamantane) (Mem), its analogue 1-N-methylamino-3,5-dimethyl-adamantane as well as (+)-5-methyl-10,11-dihydro-5H-dibenzo{a,d}cyclohepten-5,10-imine maleate (MK-801) prevented the effect of PrionSc . Production of PrionSc in the Scrapie prion-infected subclone of N2 a cells (ScN2 a cells) was not affected by memantine . We conclude that antagonists of the NMDA receptor-channel complex (i) abolish the PrionSc-induced neuronal injury in vitro, and (ii) display no influence on the synthesis and/or the processing of PrionSc. Brain Res, 1993 Aug 13, 619(1-2), 255 - 62 Degradation fragments of L1 antigen enhance tyrosine hydroxylase-immunoreactive neurite outgrowth in mesencephalic cell culture; Poltorak M et al.; The L1 antigen has been implicated in adhesion events controlling axonal elongation during formation of major fiber tracts, and promotes neurite outgrowth in culture . It is possible that injury of brain tissue causes neuronal surface molecules such as L1 antigen to be shed, and degradation fragments may therefore be present adjacent to the damage . These L1 fragments might then influence regeneration or injury-induced growth . We have evaluated neurite outgrowth from tyrosine hydroxylase-positive (TH +) E13 mesencephalic neurons grown in vitro on a substrate of mouse L1 antigen and on L1 degradation fragments separated by molecular weight . Mouse myelin-associated glycoprotein (MAG), laminin, poly-D-lysine, and fetal calf serum served as control substrates . L1 antibodies were added to one set of cultures (experimental), and compared to control cultures containing normal rabbit serum . After 3 days in vitro, the cultures were stained using an antibody against TH, and the length of the TH + neurites was measured by computer-assisted image analysis in a double-blind fashion . TH+ neurites were significantly longer when grown on L1 antigen, as well as on L1 degradation fragments, as compared to the control substrates . As compared to control normal rabbit serum, L1 antibodies eliminated the neurite-promoting effect of both the L1 substrate and the L1 degradation products . In addition, L1 substrates promoted clustering of TH + cells and the formation of loose bundles of TH + neurites . It is suggested that the L1 substrates influence TH-immunoreactive neurite outgrowth, at least partially, through indirect effects on glial cells.(ABSTRACT TRUNCATED AT 250 WORDS) Strahlenther Onkol, 1993 Aug, 169(8), 500 - 7 {Electron microscopic studies of the effect of x-rays and L-3,4-dihydroxyphenylalanine (L-Dopa)--alone and in combination-- on Harding-Passey melanoma cells in monolayer cell culture}; Proske H et al.; Monolayer cultures of Harding-Passey melanoma cells in exponential growth phase were exposed to 8 or 16 Gy by X-ray treatment . The 8 Gy treated cells revealed little ultrastructural changes, while the 16 Gy exposed cells showed increased damage as segregates, swollen mitochondria and vacuoles . Sole treatment with L-3,4-dihydroxyphenylalanine (2 x 10(-4) M L-Dopa) resulted in insignificant electronmicroscopically tangible cell alterations . Combined treatment--starting with 8 Gy irradiation followed by a six-day incubation in the presence of 2 x 10(-4) M L-Dopa--revealed more pronounced cell damage with final cell disintegration; the cytoplasm contained an increased number of vacuoles and segregates, a strongly decreased endoplasmic reticulum as well as swollen mitochondria and less pinocytosis vesicles; the cell surface showed less microvilli . Melanin containing organelles increased after the combination treatment . The growth inhibitory and cell destructive influence of L-Dopa on X-ray pretreated melanogenic melanoma cells was explained with the formation of cytotoxic oxidation products of L-Dopa. J Gen Virol, 1993 Aug, 74 ( Pt 8), 1599 - 609 Replication of Cydia pomonella granulosis virus in cell cultures; Winstanley D et al.; Several primary cell lines that support the complete replication of Cydia pomonella granulosis virus have been established from one culture of C . pomonella embryonic cells . Virus passaged three times in cells and once in larvae showed no change in restriction enzyme fragment patterns . Stages in virus replication observed by electron microscopy resembled those from in vivo studies . Cell lines that were maintained at or below 21 degrees C retained susceptibility to virus over a period of 4 years whereas the same cell lines maintained at 27 degrees C gradually lost their susceptibility and eventually could not be infected at all. Mol Cell Biol, 1993 Aug, 13(8), 4928 - 38 Activation of an imprinted Igf 2 gene in mouse somatic cell cultures; Eversole-Cire P et al.; The mouse insulin-like growth factor II gene (Igf 2), located on distal chromosome 7, is parentally imprinted such that the paternal allele is expressed while the maternal allele is transcriptionally silent . We derived a cell line from a mouse embryo maternally disomic and paternally deficient for distal chromosome 7 (MatDi7) to determine the stability of gene repression in culture . MatDi7 cells maintained Igf2 in a repressed state even after immortalization, except for one randomly picked clone which spontaneously expressed the gene . Igf 2 was expressed in a cell culture derived from a normal littermate; this expression was growth regulated, with Igf 2 mRNA levels increasing in the stationary phase of growth . Analysis of the methylation status of 28 sites distributed over 10 kb of the gene did not show consistent differences associated with expression level in the normal and MatDi7 cell lines, and the CpG island in the Igf 2 promoter remained unmethylated in all of the cell lines . Only with an oncogenically transformed cell line did the promoter become extensively methylated . We attempted to derepress the imprinted gene in MatDi7 cells by treatments known to alter gene expression . Expression of the Igf 2 allele in MatDi7 cells was increased in a dose-dependent manner by treatment with 5-aza-2'-deoxycytidine or bromodeoxyuridine, agents known to change DNA methylation patterns or chromatin conformation . Treatment of the cells with 1-beta-D-arabinofuranosylcytosine, 2'-deoxycytidine, calcium ionophore, heat shock, cold shock, or sodium butyrate did not result in increases in the levels of Igf 2 expression . It seems likely that the mechanism of the Igf 2 imprint involves subtle changes in the methylation or chromatin conformation of the gene which are affected by 5-aza-2'-deoxycytidine and bromodeoxyuridine. Cancer Immunol Immunother, 1993 Aug, 37(3), 169 - 74 Cytokine production in whole blood cell cultures of patients undergoing therapy with biological response modifiers or 5-fluorouracil; Elsasser-Beile U et al.; We have measured the levels of interferon gamma (IFN gamma), tumor necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha), IL-1 beta, and IL-2 in the whole blood cell culture supernatants of 43 tumor patients undergoing a treatment with biological response modifiers or a conventional therapy with 5-fluorouracil and leucovorin . In the blood cell cultures of the 16 patients who received 5-fluorouracil and leucovorin IFN gamma levels decreased (P < or = 0.01) and TNF alpha levels rose (P < or = 0.05) during each therapy cycle . However, in the blood samples a declining number of total leukocytes and lymphocytes was measured (P < or = 0.05) . Progressive disease could be correlated to a tendency towards lower IFN gamma levels in the pretherapeutic cultures of these patients . The second group analyzed consisted of 8 patients receiving a low-dose IL-1 beta therapy . In this group we found either an unchanged or an augmented IFN gamma production of the blood cells during treatment . In the group of 13 patients receiving low-dose recombinant IL-2 (< or = 4.5 x 10(6) IU m-2 day-1) significantly increasing IFN gamma levels were seen in the blood cell cultures during the therapy (P < or = 0.05), although total leukocyte counts decreased . In this group, 4 had stable disease for at least 2 months and 9 patients had tumor progression under therapy . In the cultures of the latter a tendency towards lower IFN gamma values was found . Finally, the cytokine production in the blood cell cultures of 6 patients receiving a combination therapy of IFN alpha and high-dose IL-2 was studied . During this therapy a dramatically reduced production not only of IFN gamma but also of all other measured cytokines was found . In this group all patients had tumor progression under therapy . It is concluded that the measurements of cytokine production in a reproducible whole blood culture system may be useful for monitoring immunological therapies and may help us to find out which doses of biological response modifiers have enhancing or suppressive effects on the functions of the immune cells. Zentralbl Veterinarmed B, 1993 Aug, 40(6), 391 - 6 Oxygen concentration and asexual development of Eimeria tenella in cell cultures; Wrede D et al.; Primary chicken kidney cells in Flexiperm cultures were either inoculated with Eimeria tenella sporozoites or incubated as noninoculated controls . Oxygen concentration was reduced (10 or 15 vol% O2, 5 vol% CO2) or increased (25 or 30 vol% O2, 5 vol% CO2) in a triple gas incubator (Heraeus B 5061 EK/O2) and retained in a CO2-air incubator (20 vol% O2, 5 vol% CO2) 24 hours post inoculation (hpi) . Mature second generation schizonts (mS2) were counted microscopically at 120 hpi and numbers were compared either as mS2 or mS2/cm2 confluent cells . Asexual development of Eimeria tenella was neither stimulated nor inhibited by different oxygen concentrations, indicating that higher numbers of schizonts in cultures under reducing conditions reported earlier are probably a result of increased invasion rates of sporozoites. Trop Anim Health Prod, 1993 Aug, 25(3), 144 - 50 Antibodies to Cowdria ruminantium in Mozambican goats and cattle detected by immunofluorescence using endothelial cell culture antigen; Asselbergs M et al.; Endothelial cell cultures, established from bovine umbilical cord arteries and subsequently infected with Cowdria ruminantium, were used as antigen in the indirect fluorescent antibody test . Bovine sera (374) and caprine sera (388) collected in 6 provinces of Mozambique were tested . Overall, 30.4% of goat sera had antibodies to Cowdria, and 43% of sera collected from cattle . North of the River Save, where the tick Amblyomma variegatum is highly prevalent, overall percentages of positive sera were low, 10% in goats and 20% in cattle . However, south of the river where the tick Amblyomma hebraeum is abundant percentages were much higher, 63.5% in goats and 59.4% in cattle . These results are discussed in relation to field observations that clinical disease is rare or absent in the north with enzootic instability in goats and Friesian calves in the south. J Pediatr Gastroenterol Nutr, 1993 Aug, 17(2), 153 - 60 Newborn rabbit gastric smooth muscle cell culture: EGF and TGF-alpha are potent mitogens; Yuan QX et al.; Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are two in a family of growth-promoting peptides for many gastrointestinal epithelia . This study was designed to assess their mitogenic effect on cultured gastric myocytes and to characterize specific EGF receptors on these cells . Single myocytes were isolated from newborn rabbit gastric fundus and placed into tissue culture . The composition of the culture at confluence as assessed by immunostaining with smooth muscle actin-specific monoclonal antibody (CGA7) was > 95% myocytes . To assess the effect of putative growth factors, freshly isolated myocytes were incubated in Dulbecco's modified Eagle's (DME) medium containing 1% fetal bovine serum in the presence or absence of growth factors . After 6 days, cells were incubated in serum-free medium with {3H}thymidine (1 microCi/ml) in the continued presence or absence of growth factors . After 24 h, EGF and TGF-alpha but not insulin-like growth factor I (IGF-I) induced a dose-dependent increase in {3H}thymidine incorporation . Ten nanomolar EGF or TGF-alpha increased {3H}thymidine incorporation more than sixfold over control . EGF was more potent than was TGF-alpha, with apparent median effective dose (ED50) values of 64 +/- 14 pM and 166 +/- 62 pM (p < 0.05), respectively . EGF bound to cultured myocytes with Kd = 7.6 +/- 1.8 nM and Bmax = 27 +/- 11 pmol/mg DNA or 440,000 receptors/cell . TGF-alpha competed for binding at these receptors . Although IGF-I did not stimulate thymidine incorporation, specific high-affinity receptors for IGF-I were detected on gastric myocytes.(ABSTRACT TRUNCATED AT 250 WORDS) Eur J Pediatr Surg, 1993 Aug, 3(4), 213 - 6 Biliary atresia--bile acids and prostaglandin E2 in cell cultures of bile duct epithelia; Schier F et al.; In a cell culture model of bile duct epithelial cells, the effect of prostaglandin E2, lithocholic acid and deoxycholic acid was studied . Bile acids and prostaglandin are administered postoperatively in biliary atresia empirically as choleretics . Prostaglandin E2 and the bile acids all had inhibitory effects on bile duct epithelial cells in culture . There is no clinical study proving the efficacy of either bile acids or prostaglandin E2 in biliary atresia . The negative results with these substances in cell cultures warrants reserve in their routine clinical use in biliary atresia. Steroids, 1993 Aug, 58(8), 384 - 6 Stimulation of aldosterone production by hemin in calf adrenal glomerulosa cell cultures; Cozza EN et al.; Aldosterone production from 11-deoxycorticosterone was stimulated by hemin in primary cultures and homogenates of calf adrenal zona glomerulosa, in a time- and dose-dependent fashion . The ferrochelatase inhibitor 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) blocked the stimulation of aldosterone mediated by adrenocorticotropin (ACTH) . Addition of hemin after treatment with DDC partially restored ACTH action . These results suggest that hemin may play a role in regulation of aldosterone production. J Neurosci, 1993 Aug, 13(8), 3510 - 24 Combined oxygen and glucose deprivation in cortical cell culture: calcium-dependent and calcium-independent mechanisms of neuronal injury; Goldberg MP et al.; Murine neocortical cell cultures were transiently deprived of both oxygen and glucose, producing widespread neuronal swelling in less than 60 min, followed by neuronal degeneration over the ensuing several hours, despite return to normal medium . Cultured glia (> 95% astrocytes) were irreversibly injured only by oxygen-glucose deprivation exposures exceeding 4-6 hr . Replacing either Na+ or Cl- with impermeant ions blocked acute neuronal swelling but did not prevent delayed neuronal degeneration . While neuronal swelling and death were increased by removing Ca2+ from the exposure medium, combined removal of extracellular Ca2+ together with Na+ or Cl- substitution blocked both acute and delayed injury . If acute swelling was limited by a hyperosmolar medium, then neuronal loss depended on extracellular {Ca2+} . Oxygen-glucose deprivation was associated with a large increase in extracellular glutamate concentration . Both early swelling and later neuronal degeneration were blocked by addition of NMDA receptor antagonists to the exposure medium but not by the AMPA/kainate receptor antagonist 6-cyano-7-dinitroquinoxaline-2,3-dione (CNQX), dihydropyridines nifedipine or nimodipine, or TTX . Oxygen-glucose deprivation induced substantial neuronal uptake of tracer 45Ca2+ from the exposure medium that was reduced by NMDA receptor antagonists and closely paralleled the degree of subsequent neuronal loss . These observations suggest the presence of two distinct components of hypoxic injury, each involving NMDA receptor activation and each capable of leading to neuronal death . Acute swelling is mediated by influx of Na+, Cl-, and water, and is enhanced by removal of extracellular Ca2+ . Delayed neuronal degeneration depends on the presence of extracellular Ca2+ and correlates closely with cellular uptake of 45Ca2+. Ecotoxicol Environ Saf, 1993 Aug, 26(1), 113 - 26 Effects of a heterogeneous set of xenobiotics on growth and plasma membranes of mammalian and fungal cell cultures; Cascorbi I et al.; A comparison of the toxicity of 45 selected, heterogenous substances on two test organisms of different taxonomic levels, the yeast Saccharomyces cerevisiae and Chinese hamster ovary (CHO) cells, was made . In addition, effects on the yeast plasma membrane-integrated H(+)-ATPase and on the CHO adenosine uptake system were investigated . For all test systems, log EC50 values highly correlated with log EC20 values . Good correlations were obtained between CHO proliferation rate and yeast growth rate (r = 0.80) . However, CHO cells were about four times more sensitive than yeast . A good accordance was also found between effects on yeast cell growth and on the H(+)-ATPase, indicating a plasma membrane impairment as a major cause of cytotoxicity . These findings were supported by correlations of log EC20 values with the log Pow as a measure for lipophilicity . Although the test systems demonstrated different dependencies, the main trend reflected an increasing toxicity with increasing lipophilicity . Comparisons with data from in vivo test systems suggest that these in vitro test systems could be implemented for initial estimation of basic toxicity and the detection of outliers thereby reducing the number of tests with higher animals. J Comp Neurol, 1993 Jul 22, 333(4), 567 - 77 Hippocampal regulation of the survival and morphological development of locus coeruleus neurons in dissociated cell culture; Robinson LJ et al.; The influence of target structures on neural development, originally described for the peripheral nervous system, has more recently been investigated in the central nervous system . We sought to determine whether targets regulate the development of the locus coeruleus, with its diffuse and complex projections in marked contrast to the simpler neural circuits of the peripheral nervous systems . Dissociated locus coeruleus cells were grown alone or with the hippocampus in serum-free, chemically defined medium that minimized non-neuronal growth . Coculture with the hippocampus resulted in a significant increase in locus coeruleus tyrosine hydroxylase activity . Elevated tyrosine hydroxylase activity was accompanied by a commensurate increase in the number of tyrosine hydroxylase-immunoreactive cells, suggesting hippocampal enhancement of locus coeruleus survival . Furthermore, when hippocampal cells were added to locus coeruleus dissociates at zero time, or after two days, tyrosine hydroxylase-positive cell number was increased only by hippocampal cells added initially, suggesting that the target does indeed foster survival . The apparent target enhancement of locus coeruleus survival seems to be selective since total protein and total neuron number, estimated with neuron-specific enolase immunocytochemistry, were not affected by the hippocampus . Coculture with the hippocampus also increased the length and complexity of locus coeruleus cell processes . Neither the increase in tyrosine hydroxylase cell number nor the changes in morphology could be attributed to nonspecific effects of the increased cell density in cocultures . Our observations thus suggest that the target hippocampus influences the survival and neurite elaboration of afferent locus coeruleus neurons. Brain Res Dev Brain Res, 1993 Jul 16, 74(1), 146 - 50 Synapse formation in dissociated cell cultures of embryonic chick cerebral neurons; Tokioka R et al.; The development of synapses was confirmed in the primary cultures of dissociated cerebral cortex neurons from chick embryos . Whole-cell patch clamp recording applied to dissociated neurons from 6- to 12-day-old embryos revealed that these neurons form functional synapses . In these cultures, both excitatory and inhibitory postsynaptic responses were observed . Synaptogenesis in our cultures seemed to be in proportion to the embryonic equivalent days, which are the sum of the days in incubation and culture. J Histochem Cytochem, 1993 Jul, 41(7), 1023 - 30 Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections; Gold R et al.; Since DNA fragmentation is a key feature of programmed cell death (PCD) and also occurs in certain stages of necrosis, we have adapted the methodology of in situ nick-translation (ISNT) to detect DNA fragmentation on a single-cell level . We first established the technique for cell preparations . Apoptosis was induced by gamma-irradiation on freshly isolated rat thymocytes . After fixation procedures, ISNT was performed by overnight incubation either with fluorescein-12-dUTP or with digoxigenin-labeled 11-dUTP and DNA polymerase I . The enzymatic incorporation of labeled nucleotides at sites of DNA fragmentation was detected by flow cytometry either directly or indirectly with fluorescein-conjugated anti-digoxigenin . The quantitative results demonstrated close correlation with morphological essays for apoptosis, DNA gel electrophoresis, and ISNT . Proliferating cells determined by bromodeoxyuridine immunofluorescence were not labeled by ISNT . Immunocytochemistry for cell surface antigens in combination with ISNT allowed the identification of specific cell types undergoing PCD . Furthermore, the simultaneous application of photolabeling techniques with ethidium monoazide and ISNT led to the identification of DNA fragmentation in cells with still intact membranes . Extending ISNT to tissue sections of paraformaldehyde-fixed, paraffin-embedded material reliably revealed labeling of cells with typical morphological features of apoptosis . However, this technique was not useful in detecting early stages of necrotic cell death. Matrix, 1993 Jul, 13(4), 323 - 30 A mouse 3T6 fibroblast cell culture model for the study of normal and protein-engineered collagen synthesis and deposition into the extracellular matrix; Lamande SR et al.; Mouse 3T6 fibroblasts deposited an organized collagenous extracellular matrix during long-term culture in the presence of ascorbic acid . The matrix produced by the cells had a similar distribution of collagen types as the mouse dermal matrix, comprising predominantly type I with smaller amounts of types III and V collagens . By day 8 of culture more than 70% of the collagen in the 3T6 matrix was involved in covalent crosslinkages and required pepsin digestion for extraction . Incorporation of NaB3H4 into reducible crosslinks and aldehydes directly demonstrated the involvement of the alpha 1 (I)CB6 and alpha 2(I)CB3.5 in crosslinks . The pattern of reducible crosslinks in the in vitro 3T6 matrix was similar to that in mouse skin suggesting a comparable fibril organization . Processing of procollagen to collagen occurred efficiently throughout the culture period and the rate of collagen production was unaltered during 15 days of culture, indicating that the development of a collagenous matrix does not directly play a role in procollagen processing or biosynthetic regulation . The existence of a preformed matrix did however, increase the efficiency with which newly synthesised collagen was incorporated into the pericellular matrix . At day 0, when there was no measurable matrix present, 29% of the collagen synthesised was deposited, while by day 15, 88% of the collagen was laid down in the matrix . The development of this 3T6 culture system, where collagen is efficiently incorporated into an organized extracellular matrix, will facilitate detailed studies on matrix organization and regulation and provide a system in which protein-engineered mutant collagens can be expressed to determine their effects on the production of a functional extracellular matrix. Horm Metab Res, 1993 Jul, 25(7), 356 - 9 Assessment of bioactivity of WHO/NIH research standard for inhibin (code 86/690) using two separate pituitary cell-culture systems; Parte PP et al.; The Research Standard for inhibin, porcine (No . 86/690) distributed by the National Institute for Biological Standards and Control, U.K . for the bioassay of inhibin was tested for its bioactivity in vitro in two rat pituitary cell culture systems . The system A was obtained from pituitaries of 12 day old immature rats while system B was obtained from pituitaries of mature male rats . The inhibin preparation failed to inhibit basal secretion of FSH in the system A . Instead it stimulated the release of both LH and FSH . 1 ng LHRH induced release of LH was inhibited by 32, 80 and 43% by 0.1, 1 and 10 IU inhibin, respectively . 10 ng LHRH induced release of FSH was inhibited in a none-dose related manner and the maximum inhibition was by 10 IU inhibin (25%) . The same dose of 10 IU inhibin stimulated LHRH-induced release of LH by 35% . In system B, 1, 10, 50, 100 and 200 IU inhibin suppressed basal secretion of FSH by 0, 44, 72, 70 and 48%, respectively, while LH was suppressed by 13, 24, 46, 22 and 21% respectively . The pattern of inhibition of 10 ng LHRH induced release of LH and FSH by inhibin was similar to its effect on basal secretion . A specific dose-related inhibition of FSH was not observed . Our data question the utility of the inhibin standard (86/690). Alcohol, 1993 Jul-Aug, 10(4), 285 - 90 Cocaethylene toxicity in rat primary myocardial cell cultures; Welder AA et al.; Cocaethylene is a unique cocaine metabolite formed in the presence of ethanol by the liver . Neither acute nor chronic cardiotoxic effects of this metabolite have been investigated . The purpose of this study was to establish a time- and dose-dependent toxicity profile for cocaethylene in primary myocardial cell cultures established from 3-5-day-old Sprague-Dawley rats . Alterations in lactate dehydrogenase (LDH) release, lysosomal neutral red (NR) retention, thiobarbituric acid-reactive substances (TBARS), morphology, and beating activity were evaluated after treatment of cultures with cocaethylene doses ranging from 1.0 x 10(-3) to 1.0 x 10(-9) M from 1 to 24 h . LDH release was significantly elevated after 24 h only with those cultures exposed to the highest dose of cocaethylene (1.0 x 10(-3) M) . The highest dose of cocaethylene also significantly depressed NR retention . While all doses of cocaethylene depressed contractile activity and altered cellular morphology by 24 h, there were no TBARS formed up to 15 h . Thus, both low and high doses of cocaethylene are injurious to the cellular integrity and contractility of myocardial cell cultures . Future studies are warranted to determine mechanisms of cocaethylene toxicity in this in vitro model of spontaneously contracting myocardial cells. Neuroscience, 1993 Jul, 55(2), 597 - 605 Aluminum silicate toxicity in cell cultures; Murphy EJ et al.; To assess the cytotoxicity of four clays containing an aluminum silicate--montmorillonite, bentonite, kaolinite and erionite--we used human umbilical vein endothelial, N1E-115 neuroblastoma, and ROC-1 oligodendroglial cells . Morphological examination, lactate dehydrogenase release and fatty acid release were used as indices of trauma . The clays were added in suspension to the cell cultures at concentrations of 0.1, 0.03 and 0.01 mg/ml of medium and the cells were incubated for 1, 6 and 24 h . The clays did not lyse ROC-1 and N1E-115 cells and did not cause a dose-dependent increase in fatty acid levels at 24 h . There were no significant increases in lactate dehydrogenase activity in N1E-115 neuroblastoma or ROC-1 oligodendroglial cells . In human umbilical vein endothelial cells, montmorillonite, kaolinite and bentonite caused a dose-dependent increase in fatty acids at 24 h . All three clays caused cell lysis . We postulate that the cytotoxicity of the clays containing an aluminum silicate towards endothelial cells may disrupt the blood-brain barrier in the affected areas, allowing the entry of the clay particle into the brain . Aluminum silicate clays caused a dose-dependent release of fatty acids in human umbilical vein endothelial cells . The clays also caused lysis of these cells . ROC-1 oligodendroglia and N1E-115 neuroblastoma cells were not lysed by the clays, suggesting that this is not a general phenomenon. Virology, 1993 Jul, 195(1), 140 - 8 Marek's disease virus protein kinase gene identified within the short unique region of the viral genome is not essential for viral replication in cell culture and vaccine-induced immunity in chickens; Sakaguchi M et al.; The open reading frame (ORF) of 1206 bp within the short unique region (Us) of Marek's disease virus type 1 (MDV1) shows significant homology with the herpes simplex virus type 1 US3 gene encoding protein kinase (PK) . The lacZ gene of Escherichia coli was inserted within the ORF, designated MDV1-US3, of MDV1 K544 strain DNA by homologous recombination . The plaque-purified recombinant MDV1 stably expressed the beta-galactosidase encoded by the inserted lacZ gene in infected cells and replicated well as the parental K544 strain . Antibodies against both MDV1 antigen and beta-galactosidase were detected in the sera of ch