Biology of a novel organic solute and steroid transporter, OSTalpha-OSTbeta

Using a comparative approach, recent studies have identified and functionally characterized a new type of organic solute and steroid transporter (OST) from skate, mouse, rat, and human genomes. In contrast to all other organic anion transporters identified to date, transport activity requires the coexpression of two distinct gene products, a predicted 340-amino acid, seven-transmembrane (TM) domain protein (OSTalpha) and a putative 128-amino acid, single-TM domain ancillary polypeptide (OSTbeta). When OSTalpha and OSTbeta are coexpressed in Xenopus oocytes, they are able to mediate transport of estrone 3-sulfate, dehydroepiandrosterone 3- sulfate, taurocholate, digoxin, and prostaglandin E2, indicating a role in the disposition of key cellular metabolites or signaling molecules. OSTalpha and OSTbeta are expressed at relatively high levels in intestine, kidney, and liver, but they are also expressed at lower levels in many human tissues. Indirect immunofluorescence microscopy revealed that intestinal OSTalpha and OSTbeta proteins are localized to the baso-lateral membrane of mouse enterocytes. In MDCK cells, mouse Ostalpha-Ostbeta mediated the vectorial movement of taurocholate from the apical to the basolateral membrane, but not in the opposite direction, indicating basolateral efflux of bile acids. Overall, these findings indicate that OSTalpha-OSTbeta is a heteromeric transporter that is localized to the basolateral membrane of specific epithelial tissues and serves to regulate the export and disposition of bile acids and structurally related compounds from the cell. If confirmed, this model would have important implications for the body's handling of various steroid-derived molecules and may provide a new pharmacologic target for altering sterol homeostasis.     

Retinoic acid-induced modifications in the growth and cell surface components of a human carcinoma (HeLa) cell line

The addition of retinoic acid to cultures of HeLa-S3 cells caused a reduction in cell proliferation rate which became apparent after 72 h and was linearly dependent on retinoic acid concentration in the range 10⁻⁹–10⁻⁵ M. After 72 h of exposure to retinoic acid, the cells assumed a flattened appearance and no longer formed multilayers. These changes were reversed within 48 h after removal of retinoic acid from the medium. Structural analogs of retinoic acid with a free ---COOH group at C-15 were usually more potent in growth inhibition than compounds with an alcohol, aldehyde, ether or ester group. A cellular retinoic acid-binding protein was detected in cell homogenates, and the binding of [³H]retinoic acid to the binding protein was inhibited by most, but not all, analogs possessing a free terminal ---COOH group. For example, the 4-oxo analog of retinoic acid, while capable of inhibiting cellular proliferation, failed to bind to the retinoic acid-binding protein. Analysis of cell surface and cellular glycoproteins by lactoperoxidase-catalysed ¹²⁵I iodination and by metabolic labeling with [³H]glucosamine revealed that a 190000 D glycoprotein which was labeled by both methods and a 230000 D glycoprotein which was labeled only with [³H]glucosamine were labeled more intensely in retinoic acid-treated cells compared with untreated cells. The electrophoretic mobility of the 230000 D glycoprotein could be modified by treatment of intact cells with either neuraminidase or proteolytic enzymes, suggesting that this glycoprotein is also exposed on the cell surface. The cell surface alterations were detected much earlier than the onset of growth inhibition and appeared as early as 24 h after exposure to retinoic acid. The possible relationship between retinoic acid-induced changes in cell membrane structure, cell morphology, and cell proliferation is discussed.     

Stable expression of bioactive recombinant pleurocidin in a fish cell line

Pleurocidin (Ple), a linear cationic peptide of 25 amino acids, is a member of a larger family of antimicrobial peptides present in flatfish. Previous studies have shown that Ple displays a strong antimicrobial activity against a broad spectrum of bacteria and appears to play a role in innate host defence. In this work, the genomic sequence encoding the Ple prepropeptide has been isolated from Limanda limanda and cloned in a vector under the control of a non-viral promoter (the carp β-actin promoter). By using this construction, expression of bioactive Ple was demonstrated in transformed fish cell lines continuously growing for more than 2 years. Furthermore, the study of Ple processing, maturation and secretion (by using fusion with green fluorescence protein) and the high bactericidal activity of the secreted recombinant Ple (detectable in cell supernatants without any concentration) are all reported here, as no other recombinant Ple or fish antimicrobial peptide have been expressed before to that extent. Such an overexpression of recombinant Ple or any other related antimicrobial peptide might improve the chances to develop new antibiotic agents, as well as to provide essential information about the mechanism of action, range of activity and the role in the innate immune response of antibiotic peptides.     

Parathyroid hormone-related protein (PTHrP) in cartilaginous and bony fish tissues.

Tissues from a range of fish were examined for the presence of parathyroid hormone-related protein (PTHrP) to investigate PTHrP protein distribution and PTHrP gene expression in jawless fish, cartilaginous fish, and bony fish. Immunoreactive PTHrP was localized using antisera to N-terminal and mid-molecule regions of human PTHrP and PTHrP gene expression examined using a digoxigenin labeled riboprobe to a conserved region of the mammalian PTHrP gene. In all of the fish studied, PTHrP protein and messenger RNA (mRNA) were localized to the skin, kidney, and skeletal muscle, following the pattern seen in higher vertebrates. Additional sites of localization for both protein and mRNA included gill, nerve cord, and pituitary, as well as developing dermal denticles and rectal gland in the elasmobranch species. The sites of PTHrP distribution indicate that PTHrP may have roles in ionoregulation as well as growth and differentiation in fish, as has been suggested in higher vertebrates. The results imply that the distribution of PTHrP is widespread in fish and that there is homology between the PTHrP molecules found in humans and fish. The conservation of localization and possible similarity of the PTHrP molecules between tetrapods and fish suggests that PTHrP has a number of fundamental roles in vertebrates. J. Exp. Zool. 284:541-548, 1999.     

Transport of organic cations by a renal epithelial cell line (OK)

The goal of this study was to determine the mechanisms involved in the transport of the organic cation, tetraethylammonium (TEA), across the apical membrane of OK cells. [14C]TEA accumulated in OK cell monolayers reaching equilibrium in 2 h. The uptake of [14C]TEA at equilibrium was dependent upon temperature and was inhibited by sodium azide and by various organic cations, including N1-methylnicotinamide (NMN), mepiperphenidol, and cimetidine but not by the organic anion, p-aminohippuric acid. The initial uptake of [14C]TEA was characterized by a saturable process. The mean +/- S.D. Km was 27.8 +/- 2.6 microM and the Vmax was 414 +/- 26.5 pmol/mg protein/min. Both an accelerated efflux and influx of [14C]TEA in the presence of a trans-gradient of unlabeled TEA and NMN was observed, whereas a deaccelerated influx and efflux was observed in the presence of a trans-gradient of mepiperphenidol. The mechanism of interaction between NMN and TEA was examined. NMN significantly increased the apparent Km (mean +/- S.D.) of TEA to 82.8 +/- 16.4 microM (p less than 0.001), whereas the Vmax (mean +/- S.D.) was only slightly affected (478 +/- 72 pmol/mg protein/min) suggesting a competitive inhibition. The stimulatory effect of trans-gradients of NMN on TEA transport was due to an increase in the Vmax of TEA suggesting that NMN trans-stimulates TEA transport by increasing the turnover rate of the exchanger. In the presence of an inwardly directed proton gradient, the efflux at 30 s of [14C]TEA from the OK cell monolayers was significantly accelerated (p less than 0.05). Studies with the pH-sensitive fluorescent probe, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, suggested that TEA could drive the countertransport of protons. In apical membrane vesicles prepared from OK cells, the uptake of [3H]NMN exhibited an apparent "overshoot phenomenon" in the presence of an initial outwardly directed proton gradient. Protons competitively inhibited TEA uptake suggesting that the proton/organic cation and the organic cation/organic cation self exchange mechanism are the same mechanism. This is the first report describing both TEA self-exchange and proton/TEA exchange in the apical membrane of a continuous cell line. OK cells are an excellent model for the study of organic cation transport across the apical membrane.     

Transport of choline and its relationship to the expression of the organic cation transporters in a rat brain microvessel endothelial cell line (RBE4)

The present study was undertaken to elucidate the functional characteristics of choline uptake and deduce the relationship between choline uptake and the expression of organic cation transporters in the rat brain microvessel endothelial cell line RBE4. Confluent RBE4 cells were found to express a high affinity choline uptake system. The system is Na(+)-independent and shows a Michaelis-Menten constant of approx. 20 microM for choline. The choline analogue hemicholinium-3 inhibits choline uptake in these cells with an inhibition constant of approx. 50 microM. The uptake system is also susceptible for inhibition by various organic cations, including 1-methyl-4-phenylpyridinium, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, clonidine, procainamide, and tetramethylammonium. The prototypical organic cation tetraethylammonium shows very little affinity for the choline uptake system in these cells. The inhibition of choline uptake by hemicholinium-3 is competitive. Northern analysis and RT-PCR show that these cells do not express the organic cation transporters OCT2 and OCT3. These cells do express, however, low levels of OCT1, but the functional characteristics of choline uptake in these cells are very different from the known properties of choline uptake via OCT1. The Na(+)-coupled high affinity choline transporter CHT1 is not expressed in these cells as evidenced by RT-PCR. This corroborates the Na(+)-independent nature of choline uptake in these cells. It is concluded that RBE4 cells express an organic cation transporter that is responsible for choline uptake in these cells and that this transporter is not identical to any of the organic cation transporters thus far identified at the molecular level in mammalian cells.     

Arachidonic acid-induced hypersensitive cell death as an assay of downy mildew resistance in pearl millet

Arachidonic acid (AA) induces hypersensitive response (HR) on coleoptile/root regions of two-day-old pearl millet seedlings. The response is comparable to the HR induced by the downy mildew pathogen, Sclerospora graminicola. A time gap in the appearance of cell necrosis among genotypes of pearl millet was related to the degree of resistance to downy mildew. Based on the time required for the development of necrotic spots induced by AA, the pearl millet genotypes were categorised as highly resistant/resistant (HR in 3–6 h), susceptible (HR in 7–12 h) and highly susceptible (HR in 13 h and above). The percentage disease incidence in each genotype was compared with the time required for the development of AA-induced HR. The appearance of hypersensitive cell necrosis was rapid in genotypes having high resistance to downy mildew and was slow in genotypes with high susceptibility. This simple method of screening various pearl millet genotypes in the absence of the pathogen aids in identifying the downy mildew resistant/susceptible host cultivars without the risk of introducing the virulent race of the pathogen.     

Use of an organic buffer (hepes) in human lymphocytoid cell line cultures

Summary N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) added to bicarbonate buffer in cell culture medium was found to promote continuous logarithmic growth of human lymphocytoid cell lines. Consistent high yields of viable cells were obtained in the combined buffer system. Storage of lymphocytoid cells in the combined buffer system at room temperature allowed successful reinitiation of growth of the cultures.     

Some properties of an established fish cell line fromXiphophorus helleri (red swordtail)

Summary A cell line (SWT) was established from embryonic tissue of the red swordtailXiphophorus helleri. The SWT cells grew optimally, at 26°C to 30°C in Eagle's basal medium plus 10% fetal calf serum but failed to grow at 16°C and 37°C. After 50 subcultivations, the cells remained contact-inhibited and were pseudodiploid with a chromosomal modal number of 46. Virological studies demonstrated that SWT cells supported replication of the following viruses at the indicated temperatures: IPN virus (22°C), FV-3 (30°C), and VSV (33°C). The following mammalian, viruses failed to replicate at 33°C: vaccinia, poliovirus 2, herpes simplex, and reovirus 2. Although not replicating, reovirus induced interferon in there cells. This work was supported in part by a grant from the University Research Council. A part of these results was presented at the 22nd Annual Meeting of the Tissue Culture Association, Lake Placid, N. Y. 1971.     

Vimentin and desmin of a cartilaginous fish, the shark Scyliorhinus stellaris: sequence, expression patterns and in vitro assembly

In the shark Scyliorhinus stellaris we have biochemically identified and cDNA-cloned orthologs of human vimentin and desmin, SstV and SstD, as deduced from immunoblotting and sequence alignment with teleost, frog and human vimentin and desmin, respectively. This allowed us to further clarify the relationship of previously identified lower vertebrate intermediate filament proteins to mammalian vimentin and desmin. Immunofluorescence microscopy with antibodies H5 and VIM13.2 showed vimentin expression in shark eye and brain and absence in epithelia, which resembles the situation in higher vertebrates. In addition, SstV is expressed in many mesenchymal cell types which corresponds to the case in terrestrial vertebrates but strongly differs from teleosts. Surprisingly, shark interstitial cells, including fibroblasts, express neither SstV nor keratins but other as yet unidentified intermediate filament proteins as deduced from their reactivity with antibody IFA. In vitro assembly studies of recombinant SstV revealed a temperature optimum for uncompromised filament assembly of 15 degrees C. At 18 degrees C, but more pronounced at 21 degrees C and 24 degrees C, which is notably above the animal's inherent preferred environmental temperature, both, SstV and SstD assemble into thick and inflexible fibers. Thus, environmental temperature apparently is, as a general principle, a driving force for the fine tuning of protein primary structure and eventually 3D structure.     

Not all fish are equal: functional biodiversity of cartilaginous fishes (Elasmobranchii and Holocephali) in Chile

A review of the primary literature on the cartilaginous fishes (sharks, skates, rays and chimaeras), together with new information suggests that 106 species occur in Chilean waters, comprising 58 sharks, 30 skates, 13 rays and five chimaeras. The presence of 93 species was confirmed, although 30 species were encountered rarely, through validated catch records and sightings made in artisanal and commercial fisheries and on specific research cruises. Overall, only 63 species appear to have a range distribution that normally includes Chilean waters. Actual reliable records of occurrence are lacking for 13 species. Chile has a cartilaginous fish fauna that is relatively impoverished compared with the global species inventory, but conservative compared with countries in South America with warm‐temperate waters. The region of highest species richness occurs in the mid‐Chilean latitudes of c. 30–40° S. This region represents a transition zone with a mix of species related to both the warm‐temperate Peruvian province to the north and cold‐temperate Magellan province to the south. This study provides clarification of species occurrence and the functional biodiversity of Chile's cartilaginous fish fauna.     

Immunohistochemical localization of delta sleep-inducing peptide (DSIP) in the brain and pituitary of the cartilaginous fish Scyliorhinus canicula.

The distribution of delta sleep-inducing peptide (DSIP) in the brain and pituitary of the cartilaginous fish Scyliorhinus canicula was investigated using the indirect immunofluorescence technique. Delta sleep-inducing peptide-like immunoreactive cell bodies were mainly observed in the nucleus lateralis tuberis of the hypothalamus. Immunolabeled perikarya were also distributed in the nucleus lobi lateralis hypothalami and in the dorso-lateral wall of the recessus posterioris. Most of these cells, located in the subependymal layers of the infundibulum and lateral lobes, had the typical aspect of cerebrospinal fluid-contacting elements. The DSIP-like immunoreactive fibers were localized in the basal telencephalon, within the regions of the nucleus interstitialis commissurae anterioris and the nucleus entopeduncularis. A dense network of DSIP-positive fibers was seen throughout the midcaudal hypothalamus, the lateral lobes, and the posterior lobe. In the pituitary, numerous DSIP-like immunoreactive cells were detected in the median lobe of the pars distalis. In particular, a high concentration of cells was seen in the dorsal wall of the median lobe, an area which is known to contain melanin-concentrating hormone (MCH)-producing cells. Comparison of the distribution of DSIP- and MCH-like immunoreactive cells revealed that the two neuropeptides are stored in the same cells of the median lobe of the pituitary. These findings provide the first evidence for the presence of a DSIP-related peptide in fish. The distribution of the immunoreactive material supports the view that DSIP may act as a neuromodulator and/or a hypophysiotropic factor. Moreover, the presence of DSIP-like immunoreactive cells in the pars distalis suggests that this peptide may exert autocrine or paracrine effect in the pituitary.