Development of a fluorescence-based assay to screen antiviral drugs against Kaposi's sarcoma associated herpesvirus

Tumors associated with Kaposi's sarcoma–associated herpesvirus infection include Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Virtually all of the tumor cells in these cancers are latently infected and dependent on the virus for survival. Latent viral proteins maintain the viral genome and are required for tumorigenesis. Current prevention and treatment strategies are limited because they fail to specifically target the latent form of the virus, which can persist for the lifetime of the host. Thus, targeting latent viral proteins may prove to be an important therapeutic modality for existing tumors as well as in tumor prevention by reducing latent virus load. Here, we describe a novel fluorescence-based screening assay to monitor the maintenance of the Kaposi's sarcoma–associated herpesvirus genome in B lymphocyte cell lines and to identify compounds that induce its loss, resulting in tumor cell death

Cyclic AMP-stimulated fluid transport in the thyroid: Influence of thyroid stimulators, amiloride and acetazolamide on the dynamics of domes in monolayer cultures of porcine thyroid cells

Confluent monolayer cultures of porcine thyroid cells form dome-shaped elevations by local separation from the plastic culture dish. Formation of domes by epithelial cells in culture is generally considered to be evidence of fluid transport. A computer-controlled data acquisition system was developed to quantitate fluid transport in thyroid cultures by serial measurements of dome elevation. Thyrotrophin (10 mU/ml), prostaglandin E2 (PGE2; 0.01-1 mumol/l), forskolin (1 mumol/l), 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (0.5 mmol/l) and 3-isobutyl-1-methyl-xanthine (0.5 mmol/l) promoted increases in dome height over 5-120 min. Dome growth in the presence of PGE2 (1 mumol/l) was inhibited by amiloride (0.1-100 mumol/l), ouabain (200 mumol/l), or by removal of bicarbonate and glucose from the medium. In media of reduced bicarbonate concentration (1 mmol/l compared with the control concentration of 10 mmol/l), dome growth was inhibited by acetazolamide (0.01-1 mmol/l). These data are consistent with cyclic AMP-stimulated transport of fluid from apical to basal pole of the cells, dependent on sodium entry through the apical pole by an Na+/H+ exchanger

Epidermal Growth-Factor (Egf) Inhibits the Secretomotor Response of the Thyroid - Effects of Egf On Radioiodine Turnover and Fluid Transport in Cultured Porcine Thyroid-Cells

Thyrotrophin (4-256-mu-U/ml) promoted an increase in the rate of release of radioiodine from the organic iodine pool of cultured porcine thyroid cells in follicular formations. This action of TSH was antagonized by low concentrations of epidermal growth factor (EGF; 0.1-5 nmol/1). The maximal effect of EGF was reached by 0.5 nmol/1. EGF (0.5-5 nmol/1) also inhibited the stimulatory effect of 8-chloro cyclic AMP (0.06-1.0 nmol/1) on radioiodine turnover. Exposure of thyroid cultures to media with a calcium concentration of 17.7-mu-mol/1 (1% of normal) resulted in a very marked increase in the rate of release of radioiodine. The effect of TSH in low-calcium media was to inhibit the increased release of radioiodine, and EGF (0.5 nmol/1) antagonized this inhibitory effect of TSH. The calcium ionophore, A23187, stimulated radioiodine release in a dose-dependent fashion, and EGF (1.7 nmol/1) inhibited this response. Fluid transport in thyroid monolayers was stimulated by prostaglandin E2 (PGE2; 1-mu-mol/1). EGF (5 nmol/1) also stimulated fluid transport, but antagonized the effect of PGE2 added subsequently. It was concluded that EGF exerted acute antagonistic effects on thyroid cell responses in vitro to cyclic AMP and agents promoting accumulation of cyclic AMP in time-frames too short for these inhibitory effects to be attributable to the dedifferentiative effect of the growth factor

Bidirectional ion transport in thyroid: Secretion of anions by monolayer cultures that absorb sodium

Cultured porcine thyroid cell monolayers transport Na in an apical-to-basal direction, resulting in the development of a basal-positive transepithelial potential difference (TEP) and the formation of domes (fluid-filled elevations of the cell layer above the culture dish substrate). Stimulation by prostaglandin E (PGE) increases the magnitude of the TEP, the short-circuit current (I(sc)) measured in Transwell Ussing chambers, and the height of domes in cultures grown on impermeable substrates. A phenamil-resistant, PGE-stimulated component of the I(sc) in Transwells and of the TEP in monolayers in conventional culture dishes was inhibitable by bumetanide, a diuretic drug that blocks NaKCl symporters, mediating active transport of Cl. The rate of decrease in height of domes in cultures after addition of phenamil, presumably indicative of transport of fluid in a basal-to-apical direction, was also reduced by bumetanide. Studies with Transwells in Cl-free, HCO-free or Cl- and HCO-free media indicated that thyroid cells transported HCO as well as Cl in a basal-to-apical direction. It was concluded that the thyroid epithelium is both sodium absorbing and anion secreting

Stereochemical challenges in characterizing nitrogenous spiro-axane sesquiterpenes from the Indo-Pacific sponges Amorphinopsis and Axinyssa

An investigation was conducted to identify the structures and bioactive properties of five compounds isolated from the Halichondrida sponges Amorphinopsis foetida and Axinyssa aplysinoides. All compounds possessed the spiro-axane sesquiterpene core and all were substituted at C-2 with nitrogen containing functionality. The stereochernistry of one known compound has been revised to (2R,5R, 10S)-2-formamido-6-axene (3). It exhibited mild selective solid tumor and mild antibacterial activity and was found from Axinyssa. A second known substance whose stereochemistry has also been revised, (2R,5R, 10S)-2-isothiocyanato-6-axene (4) plus its undescribed diastereomer (5) were isolated from Amorphinopsis. Both sponges were the source of two new N-phenethyl-2-formamido-6-axene diastereomeric compounds 6 and 7. No solid tumor or antibacterial activity was found for 4-7. (c) 2006 Elsevier Ltd. All rights reserved

Inhibitory effects of amiloride and its analogues on prostaglandin E2-stimulated fluid transport by cultured porcine thyroid cells: Evidence for apical membrane Na+ channels

Confluent monolayers of cultured porcine thyroid cells transport fluid from the apical to the basal surface, forming circumscribed zones of detachment (domes) from the culture dish substrate. Fluid transport, as measured by increase in dome height, was stimulated by prostaglandin E (PGE; 1μmol/l) and inhibited by amiloride (0.1-100 μmol/l). Values of the inhibition constant [K(i)] with 95% confidence limits for each of a series of amiloride analogues were: 3',4'-dichlorobenzamil (DCB), 0.090(0.045-0.18) μmol/l; 2',4'-dimethylbenzamil (DMB), 0.14 (0.074-0.27) μmol/l; amiloride, 0.72 (0.33-1.8) μmol/l; 5-(N,N-hexamethylene)amiloride (HMA), 71 (5.9-43) μmol/l; 5-(N-ethyl-N-isopropyl)amiloride (EIPA), 33 (15-17) μmol/l; and 2-guanidinobenzimidazole, 243 (110-570) μmol/l. Triaminopyrimidine was ineffective at concentrations up to 1 mmol/l. Since DCB and DMB are known to have a higher affinity for Na channels, while HMA and EIPA show higher affinity for Na/H antiports, it was concluded that PGE-stimulated fluid transport involved an apical membrane Na channel

Electrophysiological correlates of fluid transport in cultured porcine thyroid cells

Confluent monolayers of cultured porcine thyroid cells transport fluid from the apical to the basal surface, forming circumscribed zones of detachment rom the culture dish substrate (domes). The transepithelial potential (TEP), positive on the basal side, was 12.9 ± 0.4 (S.E.M.) mV (n = 93) under control conditions, increasing to 38.9 ± 0.3 mV (n = 281) when fluid transport was stimulated by prostaglandin E (PGE; 1 μmol/l). Forskolin (1 μmol/l) and 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate (0.5 mmol/l) were also effective in increasing TEP. Addition of amiloride in concentrations sufficient to block fluid transport (100 μmol/l) reduced the TEP to 5.8 ± 0.3 mV (n = 76). Substitution of N-methyl-D-glucamine for sodium in the medium reduced the PGE-stimulated TEP to 13.4 ± 0.8 mV (n = 32). Substitution of gluconate for chloride increased the TEP to 40.3 ± 0.4 mV (n = 160). Removal of bicarbonate or potassium from the medium, or addition of ouabain (200 μmol/l) were also effective in reducing the TEP. In media of low bicarbonate concentration (1 mmol NaHCO/l), acetazolamide (1 mmol/l) reduced the TEP. Fluid transport by the monolayer as measured by the change in height of domes was increased by PGE (1 μmol/l). PGE-stimulated fluid transport was inhibited by sodium or chloride ion substitution, bicarbonate removal or the addition of ouabain (200 μmol/l) or amiloride (100 μmol/l). It was concluded that fluid transport in thyroid monolayers is mediated by rheogenic sodium transport with chloride transport being passive, electrogenically coupled to sodium transport. Sodium entry to the apical pole of the cells occurs by an amiloride-sensitive mechanism, and sodium extrusion at the basal pole depends on the Na/K ATPase

The Hoiamides, Structurally Intriguing Neurotoxic Lipopeptides from Papua New Guinea Marine Cyanobacteria

Two related peptide metabolites, one a cyclic depsipeptide, hoiamide B , and the other a linear lipopeptide, hoiamide C, were isolated from two different collections of marine cyanobacteria obtained in Papua New Guinea. Their structures were elucidated by combining various techniques in spectroscopy, chromatography and synthetic chemistry. Both metabolites belong to the unique hoiamide structural class, characterized by possessing an acetate extended and S-adenosyl methionine modified isoleucine unit, a central triheterocyclic system comprised of two -methylated thiazolines and one thiazole, as well as a highly oxygenated and methylated C-15 polyketide unit. The cyclic depsipeptide 2 stimulated sodium influx and suppressed spontaneous Ca2+ oscillations with EC50 values of 3.92 μM and 79.8 nM, respectively, while 3 had no significant effects in these assays