Isolation and long-term survival of adult human sensory neurons in vitro

OBJECTIVE: To determine whether adult human dorsal root ganglion neurons can be isolated and maintained in long-term tissue culture, where they would extend processes. METHODS: Dorsal root ganglia were removed from adult human organ donors within 2 hours of clamping the aorta. They were then treated with enzymes for one hour, triturated to dissociate the neurons and their satellite cells, and the individual neurons were then plated in tissue culture dishes in medium containing serum. RESULTS: Isolated adult human dorsal root ganglion neurons survive in vitro for more than 2 1/2 months, in the absence of exogenously supplied neurotrophins. where they remain electrically excitable and extend processes, CONCLUSIONS: Isolated adult human dorsal root ganglion neurons survive in culture for more than 2 1/2 months, extend processes, and remain electrically excitable, without exogenous neurotrophins. These results suggest that, adult human sensory neurons do not require exogenous neurotrophins for survival and process outgrowth, or that sufficient factors were provided by the small number of satellite cells in the cultures. In addition, the neurons survive well in spite of an initial period of up to 14 hours of hypoxia, between the time the aorta was clamped and when the plated neurons were placed in an incubator with the appropriate O2/CO2 environment.     

Modifications of proteoglycans secreted into the growth medium by young and senescent human skin fibroblasts

The properties of proteoglycans (PGs) secreted into the growth medium by normal young and senescent human skin fibroblasts (HFs) were investigated. In both cases, the incorporation per cell of radioactive precursors into total PGs was similar. The polysaccharide chains of PGs from young and senescent HFs were mainly represented by galactosaminoglycuronans and showed a similar range of size distribution. However, galactosaminoglycuronans of PGs secreted by senescent HFs had a lower content of unsulphated disaccharides and a lower proportion of D-glucuronosyl residues. Moreover, senescent HFs released into the growth medium higher relative amounts of small PGs with chondroitin sulphate, dermatan sulphate chains, such as decorin.     

Binding of low density lipoproteins by proteoglycans synthesized by proliferating and quiescent human arterial smooth muscle cells

Chondroitin sulfate-rich proteoglycans secreted by arterial intima smooth muscle cells appear involved in low density lipoprotein entrapment and modification. Hypothetically, such a process may contribute to atherogenesis. We compared composition and size of those proteoglycans synthesized by proliferating and resting human arterial smooth muscle cells for which low density lipoprotein had affinity. Lipoprotein-binding proteoglycans secreted by proliferating cells were larger than those of resting cells (M(r) = 1.1 x 10(6) versus 0.8 x 10(6). This was primarily caused by increased M(r) of the chondroitin sulfate chains (6 x 10(4) versus 3.5 x 10(4)). The glycosaminoglycan chains of the proteoglycans from both cells were made of more than 90% chondroitin 6-sulfate and chondroitin 4-sulfate in a 6:4 ratio. Affinity chromatography indicated that low density lipoprotein had a higher affinity with the proteoglycans synthesized by proliferating cells than those from resting cells. Measured with gel mobility shift assay, the apparent affinity constant of low density lipoproteins for proteoglycans from proliferating cells was 3-fold higher than that for proteoglycans from resting cells. This increased affinity appeared related to the higher relative proportion of proteoglycans with longer glycosaminoglycan chains secreted by the proliferating cells than those secreted by the resting cells.     

Interferon-gamma inhibits 35S incorporation in heparan sulfate synthesized by human skin fibroblasts

Glycosaminoglycans synthesized by human skin fibroblasts were simultaneously radiolabelled with D-[1-(3H)]glucosamine and Na2(35)SO4. Considering 3H incorporation, we found that IFNgamma increased the production of glycosaminoglycan synthesis, including hyaluronic acid, heparan and chondroitin/dermatan sulfate. In contrast, the production of heparan and chondroitin/dermatan sulfate was slightly decreased on the basis of the 35S signal. Furthermore, when heparan sulfate was treated with nitrous acid, the release of free 35S was greater in control than in treated cells, although the 3H patterns of depolymerization with this agent were similar. These data demonstrate that IFNgamma inhibits the incorporation of sulfate from extracellular medium into heparan sulfate.     

Isolation and characterization of the human X-arrestin gene

In previous publications we have discussed the stabilization mechanism of hydration forces as applied to the development of latex agglutination tests. We describe here how we have obtained stable and reactive IgG-latex conjugates in a high-ionic-strength reaction buffer. To this end we have made agglutination tests with polystyrene beads sensitized with IgG, measuring the immunoaggregation reaction with human C-reactive protein in a stopped-flow nephelometer. The results are compared to those obtained with a F(ab')2-latex conjugate with similar antibody molecule coverage. Adsorption isotherms of F(ab')2 and IgG on latex at pH 7.2 were obtained to study the affinity of these antibodies for the surface. The results of the electrokinetic characterization of the antibody-latex conjugates agree satisfactorily with those obtained from stability studies. This research throws light upon the use of hydration forces as a new approach to stabilizing immunoassay reagents that are colloidally unstable in physiological reaction buffers.     

Isolation and characterization of the human prosaposin promoter

Male C57BL/6N mice were chosen to determine Fos production during acquisition of context-dependent fear and after re-exposure to the conditioning context. Fear-conditioning was induced by a single exposure of mice to a context followed by an electric shock. Control groups consisted of mice exposed to context only (Context group) or to an immediate electric shock. When contextual retention was measured 24 h after conditioning (retention test 1), significant contextual generalization was observed. However, when animals were exposed to a different context from days 2-5 after conditioning and then tested for retention on day 6 (retention test 2), generalization was markedly reduced. After the training, the fear-conditioned mice produced higher Fos levels than mice exposed to an immediate shock in the hippocampus, medial amygdaloid nucleus and parietal somatosensory cortex. Both shock groups produced significantly more Fos than the Context group in the central nucleus of the amygdala. After retention test 1, fear-conditioned mice generated more Fos in the hippocampus and central amygdaloid nucleus than the two control groups. However, all groups exhibited similarly low Fos production after retention test 2. The results demonstrated that simultaneous Fos production in the hippocampus, central and medial nuclei of amygdala and somatosensory parietal cortex closely paralleled the ability of mice to acquire conditioned fear. In contrast, Fos production after the retention tests did not correlate with the expression of conditioned fear.     

Phenotypic and functional characterization in vitro of a multipotent epithelial cell present in the normal adult human breast

The developmental relationships between the different mammary epithelial cell lineages in the human mammary gland are not well defined. To characterize human breast epithelial cells (HBEC) with progenitor activity, we used flow cytometry and single cell sorting to analyze the distribution of cellular phenotypes in primary cultures of reduction mammoplasties and their associated ability to generate colonies in 2-dimensional (D) and 3-D (collagen gel) culture systems. This approach allowed two distinct types of HBEC progenitor populations to be distinguished on the basis of their differential expression of the MUC-1 glycoprotein, CALLA/CD10 and epithelial-specific antigen (ESA). The first type of progenitor, which is enriched in the MUC-1+/CAL-LA-/ESA+ subpopulation, generated colonies of tightly arranged cells in 2-D cultures and small alveolar-like colonies with a central lumen when cultured in a collagen matrix. The cells produced in the colonies and derived from these MUC-1+/CALLA-/ESA+ progenitors were found to express typical luminal epitopes (keratin 8/18, keratin 19, MUC-1, ESA) and showed low levels of expression of myoepithelial epitopes (keratin 14 and CD44v6). The second type of progenitor, which is present in the MUC-1-to +/-/CALLA +/- to +/ESA+ subpopulation, generated mixed colonies of both luminal and myoepithelial cells when seeded in 2-D and 3-D cultures. In 2-D cultures, the centrally located cells exhibited a luminal morphology and expressed ESA, but were heterogeneous in their expression of MUC-1. Radiating from the periphery of these ESA+ HBEC were highly refractile ESA- teardrop-shaped myoepithelial-like cells. When cultured in a collagen matrix, these bipotent progenitors generated large branched colonies composed of a heterogeneous population of cells, with some of the progeny cells expressing luminal epitopes (keratin 8/18, keratin 19 and MUC-1) and others expressing myoepithelial epitopes (keratin 14 and CD44v6). A third type of progenitor, which became apparent is passaged HBEC cultures and was enriched in the MUC-1-/CALLA+/ESA- subpopulation, was found to generate colonies of cells with an exclusively myoepithelial phenotype. These results provide definitive evidence for the existence of multilineage HBEC progenitors in normal adult human mammary tissue. The phenotypic profile of these cells suggest that these multilineage progenitors are a relatively undifferentiated cell since they express low levels of MUC-1 and that they have a luminal location within the mammary epithelium since they are ESA+. Furthermore, we suggest that the MUC-1+/CALLA-/ESA+ and the MUC-1- to +/-/CALLA +/- to +/ESA+ progenitors we have identified and characterized are candidate in vivo alveolar and ductal progenitors, respectively.     

Study of intracellular localization and traffic of newly synthesized ceruloplasmin receptor in cultured human fibroblasts

Ceruloplasmin (Cp) receptor in cells of non-hepatocyte lineage (human HT-1080 fibroblasts) is synthesized by membrane-bound polyribosomes and then becomes a resident of the plasma membrane. The intracellular traffic of [14C]Cp receptor was followed in pulse-chase experiments using specific antibodies. It was shown that pulse-labeled Cp receptor, after reaching the place of its residence in the plasma membrane, is retained there for 90 min and then appears in the cytosol. Immunoactive 20-kD fragments of Cp receptor were found in the culture medium 1.5 h later. The intracellular traffic of 125I-labeled Cp bound to the fibroblast cell surface was traced in parallel chase experiments. It was shown that the internalized Cp receptor was recovered from the floating fraction of the cytosol. Comparison of the dynamics of the retention of internalized [14C]Cp receptor and 125I-labeled Cp in the subcellular compartments demonstrated that the traffic of both proteins within the fibroblasts is coordinated in time and proceeds via a common route. The role of Cp receptor in copper uptake by non-hepatocyte cells is discussed.     

Isolation and characterization of chondroitin sulfate proteoglycans from embryonic quail that influence neural crest cell behavior

The movement of neural crest cells is controlled in part by extracellular matrix. Aggrecan, the chondroitin sulfate proteoglycan from adult cartilage, curtails the ability of neural crest cells to adhere, spread, and move across otherwise favorable matrix substrates in vitro. Our aim was to isolate, characterize, and compare the structure and effect on neural crest cells of aggrecan and proteoglycans purified from the tissues through which neural crest cells migrate. We metabolically radiolabeled proteoglycans in E2.5 quail embryos and isolated and characterized proteoglycans from E3.3 quail trunk and limb bud. The major labeled proteoglycan was highly negatively charged, similar in hydrodynamic size to chick limb bud versican/PG-M, smaller than adult cartilage aggrecan but larger than reported for embryonic sternal cartilage aggrecan. The molecular weight of the iodinated core protein was about 400 kDa, which is more than reported for aggrecan but less than that of chick versican/PG-M. The proteoglycan bore chondroitin sulfate glycosaminoglycan chains of 45 kDa, which is larger than those of aggrecan. It lacked dermatan sulfate, heparan sulfate, or keratan sulfate chains. It bound to collagen type I, like aggrecan, but not to fibronectin (unlike versican/PG-M), collagen type IV, or laminin-1 in solid-phase assays and it bound to hyaluronate in gel-shift assays. When added at concentrations between 10 and 30 microg/ml to substrates of fibronectin, trunk proteoglycan inhibited neural crest cell spreading and migration. Attenuation of cell spreading was shown to be the most sensitive and titratable measure of the effect on neural crest cells. This effect was sensitive to digestion with chondroitinase ABC. Similar cell behavior was also produced by aggrecan and the small dermatan sulfate proteoglycan decorin; however, 30-fold more aggrecan was required to produce an effect of similar magnitude. When added in solution to neural crest cells which were already spread and migrating on fibronectin, the embryonic proteoglycan rapidly and reversibly caused complete rounding of the cells, being at least 30-fold more potent than aggrecan in this activity.     

Synthesis of corneal keratan sulfate proteoglycans by bovine keratocytes in vitro

Keratan sulfate proteoglycans (KSPGs) are the major proteoglycans of the cornea and are secreted by keratocytes in the corneal stroma. Previous studies have been able to show only transient secretion of KSPG in cell culture. In this study, cultures of bovine keratocytes were found to secrete the three previously characterized KSPG proteins into culture medium. Reactivity with monoclonal antibody I22 demonstrated substitution of these proteins with keratan sulfate chains. KSPG constituted 15% of the proteoglycan metabolically labeled with [35S]sulfate in keratocyte culture medium. This labeled KSPG contained keratan sulfate chains of 4700 Da compared to 21,000 Da for bovine corneal keratan sulfate. Labeled keratan sulfate from cultures contained nonsulfated, monosulfated, and disulfated disaccharides that were released by digestion with endo-beta-galactosidase or keratanase II. Nonsulfated disaccharides were relatively more abundant in keratan sulfate from culture than in corneal keratan sulfate. These results show that cultured bovine keratocytes maintain the ability to express all three of the known KSPG proteins, modified with keratan sulfate chains and sulfated on both N-acetylglucosamine and galactose moieties. KSPG made in vitro differs from that found in vivo in the length and sulfation of its keratan sulfate chains. The availability of cell cultures secreting corneal keratan sulfate proteoglycans provides an opportunity to examine biosynthesis and control of this important class of molecules.     

Isolation and identification of the iron-binding organic compounds synthesized by Klebsiella aerogenes

A simple, accurate method for demonstrating very small areas of endothelium is described. Some possible fields for further investigation are suggested. An outline of the possible normal mechanism of endothelial regeneration is described.     

Isolation of a gene product expressed by a subpopulation of human lung fibroblasts by differential display

Fibroblasts are the major cell type responsible for synthesizing matrix constituents in lung and other connective tissues. Evidence indicates that fibroblasts are heterogeneous, and that subpopulations with some distinct properties are clonally selected and expanded in fibrotic diseases. However, few distinct markers capable of demonstrating the presence of fibroblast subpopulations in tissues have been isolated so far. With the objective of identifying proteins that could detect fibroblast subpopulations, we compared the messenger RNA (mRNA) expression of two cultured human lung fibroblast subpopulations by differential display. Total RNA was obtained, complementary DNA (cDNA) was synthesized, and the polymerase chain reaction (PCR) products obtained with several primer pairs were compared. One 724-bp product, which was strongly expressed by one human lung fibroblast subpopulation, was identified and cloned. This product was poorly expressed by the other lung fibroblast subpopulation. The mRNA for the gene encoding this product was not detectable in human smooth-muscle cells, endothelial cells, or epithelial cells, although it was present in dermal fibroblasts. The mRNA was detected in normal and fibrotic human lungs. Search of the National Center for Biotechnology (NCBI) GenBank DNA database with the sequence obtained from this clone revealed no significant matches. However, a search of the NCBI database of expressed sequence tags (dBEST) revealed five different human expressed sequence tag (EST) clones corresponding to the LR8 cDNA sequence. Six additional mouse and one pig EST clones were identified that showed significant similarity to the human fibroblast cDNA. Composites of the entire coding sequences for the human fibroblast gene product and the mouse homologue were assembled from the respective overlapping EST sequences. The open reading frame identified for each composite sequence predicted protein products of 270 and 263 amino acids for the human and mouse sequences, respectively, which were 52% identical, with three gaps. At the amino acid level, no significant sequence similarity was detected with any other sequences in exhaustive searches of the NCBI DNA and protein databases or the Blocks databases. A PCR product with predicted length and sequence was obtained by using a sense primer upstream to LR8 and an antisense primer within LR8. Our results indicate that this differentially displayed product represents a previously undescribed protein that could be useful for distinguishing fibroblasts, and possibly fibroblast subpopulations, from other cell types in lungs and other tissues.     

Isolation and partial characterization of mutagen-sensitive and DNA repair mutants of Chinese hamster fibroblasts

Through a new approach, we have sought to isolate ultraviolet light (UV)-sensitive and DNA repair mutant Chinese hamster fibroblasts. The procedure consisted of 1) mutation induction by 5-bromodeoxyuridine (Brd U)-blacklight and UV treatments; 2) incorporation of 3H-thymidine in repair-proficient cells at high temperature (38.5 degrees C) following UV damage; 3) cold holding (4.0 degrees C) of these cells to induce tritium killing; and 4) recovery and testing of repair-deficient and UV-sensitive cells which have survived and formed colonies at low temperature (34.0 degrees C). In our initial attempt at this protocol, we isolated 72 surviving colonies from 2 x 10(7) cells plated for selection. Of the 72 colonies, 20 demonstrated potential interest and four were selected for extensive study. One, identified as UVs-7, is slightly more sensitive to UV, but not sensitive to X rays or N-acetoxy-2-acetylaminofluorene (NAc-AAF). The mutant exhibits a highly reduced level of unscheduled DNA synthesis (UDS), as compared to the parental line. Two additional lines, UVs-40 UVs-44, are sensitive to UV, X ray, N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and NAc-AAF, but exhibit normal UDS. A fourth line, UVr-23, has enhanced UDS, is resistant to UV, but exhibits no difference in sensitivity to x ray or NAc-AAf. These mutants are all stable, and should be useful for the study of mammalians DNA repair processes and mechanisms of mutagenesis.     

Expression of heat shock protein 27 in adult human third molar dental pulp

Atherosclerosis is a complex progressive process with high morbidity and frequent dramatic mortality. The experience from the developed countries justifies the effectiveness of atherosclerosis prevention. The combination of nonpharmacologic, antiaggregatory and antihyperlipemic prevention reaches currently the effectiveness of surgical intervention, with the exception of sudden events. On the other hand the surgical intervention does not restore the process of atherosclerosis and requires the same secondary prevention if the long term prognosis is to be improved. The review presents the guidelines on nonpharmacologic, antihyperlipemic (up to the combination of statin with fibrates) and the antiaggregatory prevention with the initial dose of ASA being 200 mg and a long term dose being > or = 30 mg of ASA/d treatment. (Tab. 4, Fig. 3, Ref. 25.)     

In vitro wound repair by human gastric fibroblasts: implications for ulcer healing

Fibroblasts modulate epithelial biological activities and play a key role in the ulcer healing process. There is no information regarding the biological response of human gastric fibroblasts to regulatory compounds. The aim of this study was to assess the effects of growth factors and prostaglandins on an in vitro model of human gastric fibroblast wound repair. Subconfluent fibroblast cultures were used to study proliferative responses, determined by [3H]thymidine incorporation into DNA. In vitro wound repair was determined in confluent fibroblast monolayers after mechanical denudation. The presence of putative growth factors secreted by fibroblasts was studied in conditioned medium by heparin-affinity chromatography and immunodetection with specific antibodies. Serum and platelet-derived growth factor (PDGF) -BB induced a dramatic increase in both gastric fibroblast proliferation and closure of wounded cell monolayers, whereas these activities were inhibited by both transforming growth factor (TGF) -beta1 and prostaglandin E1. Basal activities in unstimulated gastric fibroblasts were lower than those obtained in skin fibroblasts. Conditioned medium stimulated fibroblast proliferation and wound repair activity, which was inhibited by the addition of suramin, and was partially dependent on the presence of PDGF-like factor. PDGF is a major, autocrine promotor of human gastric fibroblast-dependent wound repair activities, which are inhibited by prostaglandins and TGF-beta. These findings might be important for future therapeutic ulcer healing approaches.     

Amylose is synthesized in vitro by extension of and cleavage from amylopectin

Amylose synthesis was obtained in vitro from purified Chlamydomonas reinhardtii starch granules. Labeling experiments clearly indicate that initially the major granule-bound starch synthase extends glucans available on amylopectin. Amylose synthesis occurs thereafter at rates approaching or exceeding those of net polysaccharide synthesis. Although these results suggested that amylose originates from cleavage of a pre-existing external amylopectin chain, such transfer of chains from amylopectin to amylose was directly evidenced from pulse-chase experiments. The structure of the in vitro synthesized amylose could not be distinguished from in vivo synthesized amylose by a variety of methods. Moreover high molecular mass branched amylose synthesis preceded that of the low molecular mass, suggesting that chain termination occurs consequently to glucan cleavage. Short pulses of synthesis followed by incubation in buffer with or without ADP-Glc prove that transfer requires the presence of the glucosyl-nucleotide. Taken together, these observations make a compelling case for amylopectin acting as the in vivo primer for amylose synthesis. They further prove that extension is followed by cleavage. A model is presented that can explain the major features of amylose synthesis in plants. The consequences of intensive amylose synthesis on the crystal organization of amylopectin are reported through wide angle x-ray analysis of the in vitro synthesized polysaccharides.     

Surface topography and serum concentration affect the appearance of tenascin in human gingival fibroblasts in vitro

This work tests the hypothesis that chronic alcohol intoxication suppresses the microbicidal activity of Kupffer cells by modulating the expression of cell surface receptors associated with respiratory burst and the release of potent microbicidal agents [i.e., reactive oxygen species (ROS)]. Because alcohol is also a potential risk factor in human immunodeficiency virus-1 (HIV-1) infection, this study examines the effect of HIV-1 glycoprotein 120 (gp120)-induced ROS release by isolated Kupffer cells. After 16 weeks of ethanol feeding, Kupffer cells from male Sprague-Dawley rats were isolated and assayed for HIV-1 gp120-induced superoxide release. Fluorescein isothiocyanate (FITC)-HIV-1 gp120 binding, NADPH oxidase, and protein kinase C activity in Kupffer cells were measured. Results show that HIV-1 gp120 induced the release of superoxide anion in a dose-dependent manner in normal rats. Mannosylated-bovine serum albumin inhibited FITC-HIV-1 gp120-mediated superoxide release in normal Kupffer cells by 85%. Moreover, 83 +/- 6% of Kupffer cells were FITC-HIV 1 gp120-positive, whereas <30% were CD4-positive. In alcohol-fed rats, HIV-1 gp120-induced ROS release was reduced by 70% and FITC-HIV-1 gp120 binding (in terms of fluorescence intensity per 10[6] Kupffer cells) by 44% in Kupffer cells, without any change in percent positive cells for this ligand. Concomitantly, HIV-1 gp120-induced translocation of NADPH oxidase to the plasma membranes of Kupffer cells in alcohol-fed rats was suppressed by 60%. In contrast, alcohol consumption significantly increased total protein kinase C activity and phorbol ester-induced superoxide release by Kupffer cells. These studies demonstrate that Kupffer cells are likely targets of HIV-1 whose binding sites on macrophages could also include mannose-specific receptors. These observations further suggest that suppression of HIV-1 gp120-mediated ROS production in chronic alcoholics is due to altered cell surface receptor expression for gp120, and defective postreceptor signaling mechanisms, which in turn could lead to attenuated microbicidal activity of hepatic macrophages.