Studies on phospholipid biosynthesis in hepatocytes from alcoholic rats by using radiolabeled exogenous precursors

We apply the continuous delayed feedback method of Pyragas to control chaos in the enzymatic Peroxidase-Oxidase (PO) reaction, using the electric current as the control parameter. At each data point in the time series, a time delayed feedback function applies a small amplitude perturbation to inert platinum electrodes, which causes redox processes on the surface of the electrodes. These perturbations are calculated as the difference between the previous (time delayed) signal and the actual signal. Unstable periodic P1, 1(1), and 1(2) orbits (UPOs) were stabilized in the CSTR (continuous stirred tank reactor) experiments. The stabilization is demonstrated by at least three conditions: A minimum in the experimental dispersion function, the equality of the delay time with the period of the stabilized attractor and the embedment of the stabilized periodic attractor in the chaotic attractor.     

Generation of purified neural precursors from embryonic stem cells by lineage selection

Mouse embryonic stem (ES) cells are non-transformed cell lines derived directly from the pluripotent founder tissue in the mouse embryo, the epiblast [1-3]. Aggregation of ES cells triggers the generation of a diverse array of cell types, including neuronal cells [4-7]. This capacity for multilineage differentiation is retained during genetic manipulation and clonal expansion [8]. In principle, therefore, ES cells provide an attractive system for the molecular and genetic dissection of developmental pathways in vitro. They are also a potential source of cells for transplantation studies. These prospects have been frustrated, however, by the disorganised and heterogeneous nature of development in culture. We have therefore developed a strategy for genetic selection of lineage-restricted precursors from differentiating populations. Here, we report that application of such lineage selection enables efficient purification of neuroepithelial progenitor cells that subsequently differentiate efficiently into neuronal networks in the absence of other cell types.     

Intestinal epithelial cell line induction of T cell differentiation from bone marrow precursors

The mechanism whereby the intestinal microenvironment promotes T cell development in the absence of the thymus is unknown. We show that the murine intestine-derived epithelial cell line, MODE-K, can induce T cell differentiation marker expression in vitro on bone marrow (BM) T cell precursors. Three-color flow cytometry analysis of T-cell-depleted C3H BM mononuclear cells (MNC) after 4 days of coculture on monolayers of MODE-K indicated that approximately 25% of MNC expressed CD3 and TCR alpha beta. Of these CD3+ cells, 36% were CD3loCD4-CD8- double negative (DN), 34% were CD3loCD4+CD8 alpha beta+ double positive (DP), and the remainder were CD3hiCD4+CD8- or CD3hiCD4-CD8 alpha beta+ single positive (SP). In addition, the T cells which developed in coculture with MODE-K expressed the early T cell differentiation marker CD24 (heat-stable antigen). These T cells subsets did not develop when BM was cocultured with the LTA fibroblast cell line or in medium alone. Interestingly, preventing cell contact between MODE-K and BM by culturing in Transwell plates did not interfere with the development of T cells expressing the DN, DP, or SP phenotypes. Double-positive T cells did not develop if splenic MNC were cocultured with MODE-K. These results suggest that the intestinal epithelial environment can induce and support the T cell development from bone marrow precursors.     

Three-dimensional culture of hepatocytes in a continuously flowing medium

Rat hepatocytes were maintained on three-dimensional cultures on sponge discs kept in Spinner Baskets (New Brunswick Scientific Co., New Brunswick, NJ, USA) with continuously circulating serum-free hepatocyte growth medium (HGM) containing hepatocyte growth factor (HGF) and epidermal growth factor (EGF). Hepatocytes were embedded in polyester sponge discs with a collagen gel at the concentration of 5 million cells/ml. Atmospheric gas containing 7% CO2 was directly bubbled into the medium. Agitation by the impeller created a continuous medium-flow through the packed hepatocytes. Comparison between identically prepared perfused and stationery cultures showed that hepatocytes in the perfused cultures maintain higher levels of DNA synthesis. These results demonstrate the value of perfusion systems and also show that hepatocytes can proliferate and maintain differentiation in three-dimensional culture environments.     

Synthesis of secretory component by rat hepatocytes in culture

Normal rat hepatocytes were isolated and cultivated in vitro. Synthesis of secretory component was demonstrated by its accumulation in the culture medium, as measured by radioimmunoassay; by incorporation of 14C-leucine in the protein specifically precipitated with anti-secretory component antiserum; and by a positive precipitin reaction of concentrated culture medium with the same antiserum. The results explain the high levels of secretory component found in rat bile and render plausible a mechanism of hepatic IgA transfer involving secretory component as the hepatocyte membrane receptor for polymeric IgA.     

Cyclosporin A induces apoptosis in rat hepatocytes in culture

Salmonella typhimurium phage type (PT) or definitive type (DT) 104 is a virulent pathogen for humans and animals, particularly cattle. It has been isolated increasingly from humans and animals in the United Kingdom and several other European countries and, more recently, in the United States and Canada. Humans may acquire the infection from foods of animal origin contaminated with the infective organism. Farm families are particularly at risk of acquiring the infection by contact with infected animals or by drinking unpasteurized milk. The symptoms in cattle are watery to bloody diarrhea, a drop in milk production, pyrexia, anorexia, dehydration and depression. Infection may result in septicemic salmonellosis and, upon necropsy, a fibrinonecrotic enterocolitis may be observed. The infection occurs more commonly in the calving season than at other times. Feedlot cattle and pigs may also be affected. Prolonged carriage and shedding of the pathogen may occur. Symptoms in humans consist of diarrhea, fever, headache, nausea, abdominal pain, vomiting, and, less frequently, blood in the stool. Salmonella typhimurium DT104 strains are commonly resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline.     

Spinal cord neuronal precursors generate multiple neuronal phenotypes in culture

Neuronal restricted precursors (NRPs) () can generate multiple neurotransmitter phenotypes during maturation in culture. Undifferentiated E-NCAM+ (embryonic neural cell adhesion molecule) immunoreactive NRPs are mitotically active and electrically immature, and they express only a subset of neuronal markers. Fully mature cells are postmitotic, process-bearing cells that are neurofilament-M and synaptophysin immunoreactive, and they synthesize and respond to different subsets of neurotransmitter molecules. Mature neurons that synthesize and respond to glycine, glutamate, GABA, dopamine, and acetylcholine can be identified by immunocytochemistry, RT-PCR, and calcium imaging in mass cultures. Individual NRPs also generate heterogeneous progeny as assessed by neurotransmitter response and synthesis, demonstrating the multipotent nature of the precursor cells. Differentiation can be modulated by sonic hedgehog (Shh) and bone morphogenetic protein (BMP)-2/4 molecules. Shh acts as a mitogen and inhibits differentiation (including cholinergic differentiation). BMP-2 and BMP-4, in contrast, inhibit cell division and promote differentiation (including cholinergic differentiation). Thus, a single neuronal precursor cell can differentiate into multiple classes of neurons, and this differentiation can be modulated by environmental signals.     

Modulation of biochemical activity of hepatocytes in culture by matrix substratum

If critical care nurses and advanced practice nurses could identify patients with an increased risk of an unsuccessful outcome from cardiac arrest resuscitation in the hospital, such patients could be monitored with a heightened vigilance. This is the first nursing study to examine pre-existing variables and outcome of cardiac arrest resuscitation in hospitalized patients. The investigators found that heart rate and respiratory rate increased significantly 8 hours before the cardiac arrest in patients with an unsuccessful outcome of resuscitation.     

Endothelial cell culture from human cerebral cavernous malformations

BACKGROUND AND PURPOSE: The cerebral cavernous malformation (CCM) is a common and frequently unrecognized cause of stroke and epilepsy. It consists of blood-filled caverns lined by endothelial cells (EC) and devoid of mature vessel wall structure. Cultured EC obtained from CCM may express phenotypic and genotypic alterations contributing to CCM pathogenesis. We report the first successful isolation and growth in vitro of primary EC lines from human CCM lesions. METHODS: We developed a procedure for the isolation and growth of EC from human CCM, confirmed their EC origin by a panel of molecular markers, and determined by immunocytochemistry the basic expression patterns of 6 transmembrane receptor protein kinases comparing brain, skin, and CCM primary EC lines grown identically. RESULTS: Several CCM EC lines were established from 2 patients after we treated the excised specimens with 0.3% trypsin/1% EDTA, selective cloning, and growth in MCDB107 containing 0.3 g/L heparin, 0.15 g/L endothelial cell growth supplement, and 15% FBS. The CCM EC showed contact inhibition and a rounded cobblestone appearance. The cells expressed CD31, CD105, von Willebrand factor, and binding sites for Ulex europaeus agglutinin, type 1 and acetylated LDL. They showed low levels of Flt-1, Flk-1, transforming growth factor (TGF)-beta RI, and TGF-beta RII expression but stained strongly with antibodies against Tie-1 and Tie-2. CONCLUSIONS: Cultured CCM EC retained their endothelial phenotype. Brain, skin, and CCM EC lines did not significantly differ in their staining patterns with antibodies against Flt-1, Flk-1, TGF-beta RI, TGF-beta RII, Tie-1, and Tie-2. These cell lines will assist in defining molecular phenotype and genotype alterations in association with CCM.     

Kinetics of erythroid cell precursors in the newborn rat

The nuclear organization of a particular step of cricket late spermiogenesis was examined by an electron microscope study of spermatids dispersed by air-liquid surface tension. Cell spreading techniques facilitate a display of condensing spermatid nuclei sufficient to allow interpretation of chromatin packaging processes. Results indicate that nuclei of late developing cricket spermatids are integrated by multiple packaging units as the result of an orderly aggregation of individual chromatin fibers. Each packaging unit consists of a thick fasicle, formed by the alignment of smooth chromatin fibers, which frays out into tassels of looped fibers.     

Generation of cell polarity in yeast

Yeast cells constitute an excellent system for studying cell polarity. They polarize by means of internally programmed patterns of cell division; they polarize chemotropically towards a partner during mating; and they utilize polarity to segregate cell-fate determinants during division. In the past year, considerable progress has been made towards increasing our understanding of the molecular mechanisms underlying each of these processes.     

Enhanced proliferation of fetal rat hepatocytes in primary culture induced by ritodrine

OBJECTIVE: Although ritodrine crosses the placenta, its direct effect on fetal cell proliferation has not been reported. We hypothesized that beta 2-adrenergic receptor stimulation could promote fetal liver growth. STUDY DESIGN: Ritodrine was added to serum- and hormone-free primary cultures of fetal, neonatal, or adult rat hepatocytes. We measured both tritiated thymidine incorporation into deoxyribonucleic acid and nucleus number. The effect of ritodrine on cell cycle was also analyzed with flow cytometry. RESULTS: Ritodrine enhanced the proliferation of fetal rat hepatocytes. Ritodrine remarkably stimulated deoxyribonucleic acid synthesis of fetal and neonatal but not adult hepatocytes. The effect was dose dependent and was antagonized by propranolol. Analysis of the nuclear deoxyribonucleic acid content derived from flow cytometry revealed that cells stimulated by ritodrine entered S phase. CONCLUSION: These results indicate that ritodrine may promote the proliferation of fetal hepatocytes through the stimulation of beta 2-adrenergic receptors, followed by induction of deoxyribonucleic acid synthesis.     

Isolation and long-term maintenance of differentiated adult chicken hepatocytes in primary culture

Adult chicken hepatocytes were obtained by an adaptation of the two step in situ collagenase perfusion. Usually 0.5 to 1 x 10(9) cells were obtained, with 75 to 95% viability. Hepatocytes attached within 2 h when plated on plastic cell culture dishes and spread in 4 h, surviving for several months in a specific serum-free medium. These cells retained a typical parenchymal cell morphology and the ability to produce a specific protein (albumin) throughout the culture period. We hereby provide a suitable model for studying hepatic metabolism in birds.     

Incorporation of exogenous precursors into neutral lipids and phospholipids in rat hepatocytes: effect of ethanol in vitro

We studied the incorporation of different radioactively labeled exogenous substrates into the lipids of rat hepatocytes previously incubated with ethanol. Glycerol, oleate, and serine were all incorporated into neutral lipids to a significantly greater degree in the presence of ethanol, the increase in radioactivity in the triacylglycerol fraction being quite substantial. A similar ethanol-induced increase was found in the incorporation of these substrates into the various phospholipids. This lipogenic activity did not occur when the metabolism of ethanol was blocked by 4-methylpyrazole, an inhibitor of hepatic ADH (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) activity, thus demonstrating that one of the initial effects of ethanol on lipid biosynthesis was mediated by some products of its metabolism in the liver. The only alteration that persisted in the presence of 4-methylpyrazole was an inhibitory effect on the esterification of free cholesterol from oleate, suggesting that ethanol specifically inhibits hepatic ACAT (acyl CoA:cholesterol O-acyltransferase, EC 2.3.1.26) activity.     

Dexamethasone induces chondrogenesis in organoid culture of cell mixtures from mouse embryos

Previous studies have shown that verbal processing in both normal individuals and a split-brain patient can be strongly affected by the semantic category of an unattended word presented to the left visual field (LVF). The effect was interpreted in terms of inhibition, since responses were slower when the unattended LVF word belonged to the same category as the target word. The present experiment discriminated between two alternative explanations for this finding. Subjects were presented with two letter strings, one in central vision and one to the left or right of centre. Subjects made a speeded lexical decision to the central string, and were instructed to ignore the lateral string. When a word was presented to the LVF subjects responded more slowly when it was semantically related to the central word. When an unattended word was presented to the RVF, its semantic relationship to the central word had no effect on decision latency. This finding is discussed in relation to views of performance laterality and selective attention.     

Defective alpha-granule production in megakaryocytes from gray platelet syndrome: ultrastructural studies of bone marrow cells and megakaryocytes growing in culture from blood precursors

The ultrastructure and cytochemistry of megakaryocytes from two patients with a familial gray platelet syndrome are described. Although the Golgi zones appeared normally developed, the megakaryocytes lacked alpha-granules. Catalase-containing particles were normal in number. In immature megakaryocytes, granules measuring from 0.05-0.1 mu and having an electron-dense core occurred in the Golgi area. These granules, which are considered as the precursors of alpha-granules in normal megakaryocytes, appeared unable to mature, and their number decreased with the megakaryocyte maturation. The presence of dense material in distended demarcation membranes and/or vacuoles suggested that their content was discharged. It is suggested that myelofibrosis present in the bone marrow from these two patients may be related to the possible excretion of a polypeptide growth factor normally contained in the alpha-granules. Megakaryocytes grown by the plasma clot procedure from blood precursors isolated from the two patients also did not exhibit alpha-granules, which were replaced by vacuoles. Our findings suggest that the lack of alpha-granules in gray platelets may be related to a defective megakaryocyte-committed cell, and evidence is presented which suggests that the precursors of alpha-granules are produced but that their contents are then lost.