Fibronectin synthesis by human tubular epithelial cells in culture: effects of PDGF and TGF-beta on synthesis and splicing

Mutations of p53 tumor suppressor gene are the most common genetic alterations in a variety of human carcinomas. The sites of p53 mutations, however, vary in different cancers. The present study was designed to characterize p53 mutations in 40 primary human renal cancer specimens using hot-start-PCR-single-strand conformation polymorphism (SSCP) analysis, sequencing of PCR product and immunohistochemistry. DNA extracted from microdissected paraffin-embedded sections was amplified by hot-start-PCR using oligonucleotide primers specific for exons 4-9 of p53. The mutations were analyzed by PCR-SSCP technique and the generated fragments were denatured and analyzed by 6% polyacrylamide gel electrophoresis. The samples showing a band shift were denatured and sequenced using the Sequenase Version 2.0 DNA Sequencing Kit (US Biochemical, Cleveland, Ohio). Genomic DNA from control samples containing wild-type p53 alleles was sequenced in parallel for confirming mutations in samples that were positive for p53 in the PCR-SSCP analysis. The results of these experiments demonstrate that: (1) there were mutations in p53 exon 5 and 8 in 35% (14 out of 40 samples) of human renal cancer tissues as revealed by PCR-SSCP analysis; (2) DNA sequencing of samples showing frame-shift have hot spot of p53 mutation on exon 8 at codon 244 (GGC-->TGC) and exon 5 at codon 132 [AAG (Lys)-->AGG (Arg)]. This mutation in p53 exon 5 at codon 132 is novel and has not yet been reported; (3) immunohistochemical staining of p53 in renal cancer tissue using mouse anti-human p53 monoclonal antibody, clone PAb 1801, correlated with the p53 mutation assessed by PCR-SSCP. No correlation was found between p53 mutations and tumor stage and grade of renal cancer.     

Optimum amino acid complement for protein synthesis by rat mammary cells in tissue culture

The amino acid requirement of rat mammary cells for milk protein synthesis was investigated in dispersed cell culture. A three-dimensional central composite design utilizing three variables (X1 = lysine; X2= methionine, valine, and arginine; X3 = isoleucine, tryptophan, threonine, phenylalanine, and histidine) at five concentrations each, was duplicated twice with mammary cells from lactating Sprague-Dawley rats. The optimum combination of amino acids for maximum milk protein synthesis from multiple regression models was X1 15.0-, X2 4.5-, and X3 1.5-fold their quantities in Eagle's minimal essential medium with leucine, tyrosine, cystine, and glutamine at the base 1-fold in the medium.     

Receptor-mediated gene transfer to airway epithelial cells in primary culture

The reproducibility of tooth tapping frequencies was measured in young and elderly dentate subjects. Six rates of tapping, i.e. 40, 60, 90, 120, 160 and 200 times per min, were practised to the accompaniment of a metronome for 15 s before recording. After a 15-s break, subjects were asked to reproduce the same rate of tapping without metronome accompaniment, and these movements were recorded. It was determined that the young subjects regulated tooth tapping frequencies by controlling velocity of mandibular movement. On the other hand, the elderly subjects regulated tooth tapping frequency by controlling opening width.     

Metabolism of caffeine to nucleic acid precursors in mammalian cells

The mission of this study was to determine whether or not arteriovenous connections, indicative of a "closed" type of circulation, existed in the human spleen. Spleens from four patients requiring therapeutic splenectomy were the basis for this report. Scanning electron microscopy of plastic corrosion casts, prepared from these four spleens, revealed direct vascular conduits between splenic pulp arteries or arterial capillaries and the venous sinuses in the red pulp. Also demonstrated were a few arteriovenous shunts between pulp arteries or arterial capillaries and pulp or trabecular veins. Inclusion of sized microspheres in low-viscosity perfusion plastic illustrated that some diameters of the connecting shunts were 7-10 mum, with other shunts even smaller. Not only do arteriovenous connections exist in human spleens, but their frequency, as revealed by methods accentuating three-dimensional aspects of the splenic microcirculation, justify future reconsiderations of the functional significance of this closed type of circulation. Examination of samples of the same intact spleens, prepared by freeze-fracture and conventional critical-point drying, also revealed an "open" type circulation structure, namely, pore-patterned sinus walls that could facilitate blood cell movement from pulp cords into venous sinuses. Scanning electron microscopy thus has provided direct evidence that human spleens have both "open" and "closed" circulatory pathways in their microvasculature.     

Stimulation of the adherence of Haemophilus influenzae to human lung epithelial cells by antimicrobial neutrophil defensins

Patients with chronic obstructive pulmonary disease (COPD) frequently have recurrent lower respiratory tract infections with nonencapsulated Haemophilus influenzae. The infected mucosa of these patients is infiltrated with neutrophils, which upon activation may release antimicrobial peptides, including defensins. It was shown that defensins isolated from neutrophils or from sputum samples of COPD patients did not kill H. influenzae from these patients, but they did stimulate its adherence to human bronchial epithelial cells in a time- and dose-dependent manner. Maximal stimulation was observed after 3 h in the presence of > or = 10 micrograms/mL defensins, resulting in 65 +/- 36 cfu/cell (61-fold increase). The enhanced adherence was not solely due to charge effects and was specifically blocked by alpha 1-proteinase inhibitor. Because adherence is the first step in the onset of respiratory tract infections, our findings indicate that neutrophil defensins likely contribute to the pathogenesis of H. influenzae infection in the lower respiratory tract.     

Occurrence of minute ring-shaped nucleoprotein particles in culture media conditioned by mammalian cells

Conditioned media of 21 mammalian cell lines have been examined by electron microscopic and biochemical techniques for the presence of a minute ring-shaped nucleoprotein particle (RSP). Electron microscopy showed the presence of RSP in 20 of the 21 cell lines tested. Comparative analyses of sex cell cultures for radioactive RSP, following 3H-thymidine incorporation, suggested that quantitative differences exist in the amount of RSP detectable in conditioned media of normal and pathologic cells. The cell lines tested included cells of different morphologies, origin and function     

The role of pulmonary epithelial cells in lung cellular reaction to asbestos exposure

BACKGROUND: Standard therapy for febrile neutropenia after chemotherapy has consisted of intravenous antibiotic until resolution of both fever and neutropenia. We attempted to shorten the hospital stay by discontinuing intravenous antibiotics in blood culture negative patients who remained clinically stable and afebrile for 48 hours. PATIENTS AND METHODS: Febrile neutropenic admissions of non-leukemic patients were reviewed. They were divided by three consecutive six month intervals into Group 1 (prior to initiation of the policy), Group 2 (after the policy was instituted), and Group 3 (to monitor the implementation of the policy after the initial six months). RESULTS: There were 134 admissions for neutropenic fever. Median duration of intravenous antibiotic for Group 1 was 7 days (95% Confidence Interval 6-9). It was significantly decreased to 5 days (4-6) and 4 days (3-5) for Groups 2 and 3 respectively (p = 0.004 and p < 0.001). Median duration of hospital stay for Group 1 was 10 days (7-13). It was also significantly decreased to 7 (5-8) and 6 days (5-7) for Groups 2 and 3 respectively (p = 0.04 and p = 0.002). CONCLUSION: Early discontinuation of intravenous antibiotics in patients with negative blood culture who remain afebrile and clinically stable for 48 hours results in shorter duration of hospital stay with potential for reduction in hospital costs.     

Uptake of diphtheria toxin and its fragment A moiety by mammalian cells in culture

Evidence suggesting that diphtheria toxin reaches the cytoplasm of susceptible mammalian cells by two independent mechanisms is presented. A schematic model describing the two processes of toxin entry into the cell is developed. One process of toxin uptake considered to by physiologically significant is passage of the protein toxin through the plasma membrane. This most likely happens by binding of fragment B to receptors on the membrane and by subsequent toxin-membrane interaction so that ultimately fragment A, the enzymatically active moiety, is transported tothe cell interior. This process, which ultimately leads to cessation of protein synthesis and cell death, involves a comparatively small number of toxin molecules. A second mechanism of toxin uptake is by classical pinocytosis. The majority of toxin taken into the cell is accomplished by this process. The fate of toxin taken into HEp-2 cells via pinocytosis is proteolysis by lysosomal enzymes. Thus, such vesicle-bound toxin is ordinarily not expressed biologically. Evidence suggesting that ammonium chloride provides total protection to diphtheria toxin-susceptible cells by preventing entry of toxin by the specific receptor-associated process is also provided; data showing that the ammonium salt immobilizes bound toxin on the plasma membrane of HEp-2 cells are presented. Finally, it is suggested that actively endocytic cells such as guinea pig macrophages interact with toxin in a significantly different manner than do nonphagocytic cells.     

Effect of mimosine on DNA synthesis in mammalian cells

We have designed a general protocol to assess the rate of replicon initiation in mammalian cells in the presence of inhibitors of DNA synthesis. It is based on cross-linking DNA in vivo with trioxsalen, which effectively blocks the movement of the replication forks along DNA, while having little effect on initiation of replication. We applied this protocol to study the effect of the plant amino acid mimosine on the rate of replicon initiation in exponentially growing murine erythroleukemia F4N cells. We found out that during the first 2 h after application of 25-400 microM mimosine, the initiation step was inhibited more efficiently than the overall DNA synthesis. In this respect, the effect of mimosine was similar to that of gamma-ray irradiation and differed from that of hydroxyurea and aphidicolin. The results suggest that in addition to inhibiting the elongation step of DNA synthesis, mimosine inhibits the initiation of DNA replication as well.     

Cell culture for the study of epithelial cells

Keratinocytes depend on the support of fibroblasts or fibroblast products to grow from single cells into colonies. The essentials of a human stratified squamous epithelium can be constructed from single human epidermal keratinocytes and lethally irradiated fibroblasts. Established lines of mouse keratinocytes obtained from teratomas have many of the same properties. In this way it is possible to study many aspects of this epithelial tissue or organ under essentially cell culture conditions.     

Malaria circumsporozoite protein inhibits protein synthesis in mammalian cells

Native Plasmodium circumsporozoite (CS) protein, translocated by sporozoites into the cytosol of host cells, as well as recombinant CS constructs introduced into the cytoplasm by liposome fusion or transient transfection, all lead to inhibition of protein synthesis in mammalian cells. The following findings suggest that this inhibition of translation is caused by a binding of the CS protein to ribosomes. (i) The distribution of native CS protein translocated by sporozoites into the cytoplasm as well as microinjected recombinant CS protein suggests association with ribosomes. (ii) Recombinant CS protein binds to RNase-sensitive sites on rough microsomes. (iii) Synthetic peptides representing the conserved regions I and II-plus of the P.falciparum CS protein displace recombinant CS protein from rough microsomes with dissociation constants in the nanomolar range. (iv) Synthetic peptides representing region I from the P.falciparum CS protein and region II-plus from the P.falciparum, P.berghei or P.vivax CS protein inhibit in vitro translation. We propose that Plasmodium manipulates hepatocyte protein synthesis to meet the requirements of a rapidly developing schizont. Since macrophages appear to be particularly sensitive to the presence of CS protein in the cytosol, inhibition of translation may represent a novel immune evasion mechanism of Plasmodium.     

Evidence for synthesis and release of catecholamines by human amniotic epithelial cells

The present study investigated the presence, possible synthesis and release of catecholamines (CA) by human amniotic epithelial cells (HAEC) using HPLC with electrochemical detection. The presence of CA was indicated by the detection of norepinephrine (NE), dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in extracts of cultured HAEC. Incubation of HAE cells in medium supplemented with 1-tyrosine (CA precursor) and tetrahydrobiopterin (tyrosine hydroxylase cofactor) significantly increased the production of catecholamines, suggesting CA synthesis by HAEC. In contrast, pharmacological inhibition of tyrosine hydroxylase by alpha-methyl-p-tyrosine (MPT) significantly reduced CA production, further confirming CA synthesis by HAEC. Catecholamines were also detected in the cell incubation media, demonstrating the ability of HAEC to spontaneously secrete CA. Moreover, incubation of cells with 50 mM K+ for 10 min increased the amount of CA released into the medium. Additionally, the detection of DOPAC, a primary metabolite of DA, in HAEC strongly indicates that these cells contain DA metabolizing enzymes. The present results suggest that HAEC synthesize and release CA. These cells may be a possible candidate for transplantation therapy of neurodegenerative diseases such as Parkinson's disease and also may serve as a model to study the aspects of catecholaminergic activity.     

Amino acid metabolism of myeloma cells in culture

The growth of myeloma cells in Leibovitz medium supplemented with 20% serum was limited by the depletion of glutamine. A simple modification of the Leibovitz medium by increasing the concentrations of glutamine, lysine, isoleucine, leucine, sodium pyruvate, galactose, and vitamins resulted in over 100% increase in cell growth yield. The total myeloma protein produced by the cells was increased by approximately 90% in modified Leibovitz media. Analysis of spent culture media for 19 amino acids showed that the concentrations of 8 amino acids were reduced; those of 5 amino acids were increased and the other 6 did not change significantly.     

Stimulation of retinal pigment epithelial cell growth by neuropeptides in vitro

PURPOSE: The proliferation of many cell types are regulated by cytokines and neuropeptides by autocrine and paracrine mechanisms. Retinal pigment epithelial (RPE) cells are also regulated by cytokines. But RPE cells are very close to the neural retina which has some neuropeptides. The present study was to investigate the effects of neuropeptides on the growth of RPE cells. METHODS: RPE cells were obtained from the eyes of 11 day old chick embryos and cultured in Dulbecco's modified Eagle's culture medium containing 10% fetal calf serum. The growth of RPE cells was evaluated by [3H]-thymidine uptake. RESULTS: Substance P, beta-endorphin and calcitonin gene-related peptide markedly stimulated the growth of RPE cells. The effects of methionine-enkephalin, somatostatin and vasoactive intestinal peptide were intermediate. The strongest effects of substance P, beta-endorphin and calcitonin gene-related peptide were observed at 10(-6) to 10(-7) M. The stimulation of RPE cells with beta-endorphin was inhibited by naloxone, suggesting that the stimulation with beta-endorphin is mediated by an opioid receptor. beta-endorphin and substance P induced RPE cell growth stimulating activity. Leucine-enkephalin and neuropeptide Y did not affect the growth of RPE cells. CONCLUSIONS: These results suggest that neuropeptides play an important role in the regulation of RPE cell growth.     

Morphology and function of rooster efferent ductule epithelial cells in culture

Regulation of the excurrent ducts of the testis is not well understood, particularly in avian species. To investigate the role of steroid hormones in the male reproductive tract, we developed a primary cell culture of epithelia isolated from rooster ductuli efferentes (efferent ductules). Efferent ductules of the avian testis comprise 77% of the epididymal region and form a mass of tubules containing a heavily folded epithelium enmeshed in connective tissue. The epididymal region was separated by microdissection and small epithelial plaques isolated by serial digestion with collagenase, elastase and repeated pipetting. Isolated cell plaques were cultured in a bicameral chamber on Millicell-CM inserts coated with two layers of basement membrane matrix, consisting primarily of laminin and Types I and IV collagen. Active ciliary beat was observed before plating and this activity was maintained for 14 days in culture. Cell plaques attached within 24 h and outgrowths formed a confluent monolayer by 5-6 days. The epithelial nature of cultured cells was demonstrated by immunocytochemical staining for cytokeratin. Light and electron microscopy confirmed that morphology and polarity of the original epithelial cells were maintained in culture. Cultured efferent ductal epithelium was cuboidal in shape and maintained many of the cytoplasmic organelles typical of these cells in vivo. The uptake of cationic ferritin indicated the endocytotic activity of these cultured cells was maintained. Estrogen receptor mRNA expression was maintained in cultured cells. These data demonstrate avian efferent ductal epithelium can be isolated and grown in defined culture medium for the purpose of determining the role of hormones and other factors in regulating the function of the epididymal region in the bird.     

Stimulation of DNA synthesis by ouabain and concanavalin A in cultures of embryonic neural retina cells

Ouabain and concanavalin A, agents which bind to specific sites in the cell membrane, stimulate DNA synthesis and cell replication in monolayer cultures of neural retina cells from late chick embryos. The results suggest a relationship between control of retina cell replication and properties of the cell membrane. The experiments involved measurements of 3H-thymidine incorporation in primary monolayer cultures (24-48h) of retina cells from embryos of different ages. Stimulation by ouabain was greatest in cells from 14-day embryos, and its magnitude was similar to that elicited in these cell cultures by concanavalin A. Simultaneous treatment of 14-day retina cells with both agents resulted in a greater than additive stimulation of DNA synthesis. Our results demonstrated that, although during normal embryogenesis cell replication in the neural retina has virtually ceased by day 14 of development, some cells retained a capacity for mitogenesis when exposed to conditions such as provided in these experiments. By autoradiography the responding cells were identified as large epithelioid retina cells (LER cells). Under optimal conditions of simultaneous treatment with ouabain and Con A about 20% of the LER cells showed stimulation of DNA synthesis. The nature of LER cells and other aspects of our findings are discussed.     

Binding of fatty acid ethyl esters to albumin for transport to cells in culture

Fatty acid ethyl esters (FAEE) are non-oxidative products of ethanol metabolism that have been proposed to mediate pathological changes in various organs and tissues resulting from excessive ethanol consumption. Evidence supporting this proposal is scant, however, mainly because of the lack of adequate methods with which to solubilize the highly hydrophobic FAEE in aqueous medium for testing under physiological conditions. In this report we describe a simple and practical method for solubilizing FAEE in aqueous medium by binding them to albumin. We also report that the albumin-bound FAEE are readily taken up by rat alveolar macrophages in culture. The availability of FAEE bound to albumin, their main physiological carrier in vivo, will facilitate the investigation of the role that these metabolites may have in mediating pathological changes associated with excess ethanol consumption.