Human Embryonic Kidney Cultures Research Articles

Korean Red Ginseng aqueous extract improves markers of mucociliary clearance by stimulating chloride secretion

BackgroundAbnormal chloride (Cl−) transport has a detrimental impact on mucociliary clearance in both cystic fibrosis (CF) and non-CF chronic rhinosinusitis. Ginseng is a medicinal plant noted to have anti-inflammatory and antimicrobial properties. The present study aims to assess the capability of red ginseng aqueous extract (RGAE) to promote transepithelial Cl− secretion in nasal epithelium. MethodsPrimary murine nasal septal epithelial (MNSE) [wild-type (WT) and transgenic CFTR−/−], fisher-rat-thyroid (FRT) cells expressing human WT CFTR, and TMEM16A-expressing human embryonic kidney cultures were utilized for the present experiments. Ciliary beat frequency (CBF) and airway surface liquid (ASL) depth measurements were performed using micro–optical coherence tomography (μOCT). Mechanisms underlying transepithelial Cl− transport were determined using pharmacologic manipulation in Ussing chambers and whole-cell patch clamp analysis. ResultsRGAE (at 30μg/mL of ginsenosides) significantly increased Cl− transport [measured as change in short-circuit current (ΔISC = μA/cm2)] when compared with control in WT and CFTR−/− MNSE (WT vs control = 49.8±2.6 vs 0.1+/−0.2, CFTR−/- = 33.5±1.5 vs 0.2±0.3, p < 0.0001). In FRT cells, the CFTR-mediated ΔISC attributed to RGAE was small (6.8 ± 2.5 vs control, 0.03 ± 0.01, p < 0.05). In patch clamp, TMEM16A-mediated currents were markedly improved with co-administration of RGAE and uridine 5-triphosphate (8406.3 +/− 807.7 pA) over uridine 5-triphosphate (3524.1 +/− 292.4 pA) or RGAE alone (465.2 +/− 90.7 pA) (p < 0.0001). ASL and CBF were significantly greater with RGAE (6.2+/−0.3 μm vs control, 3.9+/−0.09 μm; 10.4+/−0.3 Hz vs control, 7.3 ± 0.2 Hz; p < 0.0001) in MNSE. ConclusionRGAE augments ASL depth and CBF by stimulating Cl− secretion through CaCC, which suggests therapeutic potential in both CF and non-CF chronic rhinosinusitis.

Modulation of gene expression by adenovirus transformation.

Human adenoviruses (Ad) cause a productive infection in human cells, while in rodent cells in general they bring about only an abortive infection, which occasionally can lead to morphological transformation. In the transformed cells, viral DNA is integrated into the genome and at least the genes of the viral E1 region are expressed. This is consistent with the observation that the transforming activity of the virus is localized in region E1. Rodent cells, e.g., from rat, mouse, or hamster, can be transformed efficiently by Ad DNA, but human cells, including fibroblasts and epithelial cells, are extremely refractory to transformation. Only a few transformed cell lines have been isolated from human embryonic kidney cultures, in spite of considerable efforts (Graham et al. 1977; Byrd et al. 1982). In contrast, human embryonic retina cells can be transformed reproducibly with Ad DNA (Whittaker et al. 1984), although less efficiently than rodent embryo or kidney cultures. The E1 region consists of two transcriptional units, E1A and E1B. For complete morphological transformation both regions are needed, but the E1A region by itself can immortalize rodent cells (Houweling et al. 1980).

Immunological characterization of multiple weight forms of human cell plasminogen activators.

Several molecular weight forms of plasminogen activator (PA) activity have been observed in serum-free conditioned media from human cells in culture. An antibody inhibition technique is described which combines inhibition of enzyme activity by anti-urokinase IgG with sodium dodecyl sulfate-gel electrophoresis to determine whether different molecular weight forms of human cell PA's are immunologically related to urokinase. Plasminogen activator forms with molecular weights of 85,000 to 95,000, 50,000 to 60,000, and 36,000 were inhibited by anti-urokinase IgG. In contrast, PA forms with molecular weights in the 73,000 range from three different types of human cells were not inhibited by comparable concentrations of the antibody. Human embryonic kidney cultures contain only anti-urokinase IgG-inhibitable PA forms, while melanoma-derived Malme-3M cultures contain only anti-urokinase IgG-resistant forms. Cultures of tumorigenic Detroit 562 cells and nontumorigenic IMR-90 cells contain a mixture of "antibody-sensitive" and "antibody-resistant" PA forms. The antibody-resistant 73,000-dalton PA form may be a precursor of the smaller antibody-sensitive, urokinase-related forms, or it may be the product of a second plasminogen activator gene which codes for a protein immunologically and structurally different from urokinase.

Nuclear acidic protein changes in adenovirus-infected human embryo kidney cultures

Adenovirus infection of human embryonic kidney cultures alters the metabolism of the nuclear nonhistone acidic proteins. Analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis shows that infection inhibits the labeling with radioactive amino acids of some nuclear acidic proteins, while the rate of synthesis of other acidic proteins seems to be specifically stimulated. Because not all of the polypeptides having an enhanced rate of synthesis show the same electrophoretic mobilities as the acidic adenoviral structural polypeptides, there appears to be an induction of synthesis of acidic proteins which are nonvirus components.

Transient stimulation of deoxyribonucleic acid-dependent ribonucleic acid polymerase and histone acetylation in human embryonic kidney cultures infected with adenovirus 2 or 12: apparent induction of host ribonucleic acid synthesis.

The synthesis of cell-specific ribonucleic acid (RNA) appeared to be stimulated in human embryonic kidney (HEK) cultures infected with adenovirus 2 or 12. Deoxyribonucleic acid (DNA)-RNA hybridization experiments revealed that by 44 to 70 hr after infection with either virus, the relative amount of pulse-labeled RNA capable of hybridizing with HEK cell DNA increased considerably; such RNA was detected in both nuclear and cytoplasmic fractions. The main increase in apparent host RNA synthesis was preceded by (i) a relatively early transient stimulation of the DNA-dependent RNA polymerase activity in isolated nuclei, and (ii) a small but consistently observed increase in the rate of acetylation of lysine-rich and arginine-rich histone fractions. The Mn(2+)-(NH(4))(2)SO(4) and Mg(2+)-activated RNA polymerase reactions measured in nuclei isolated from cells infected with adenovirus 2 or 12 were stimulated at about the same time; a rapid loss of polymerase activity followed. The augmentation of the two RNA polymerase reactions found in adenovirus 12-infected cells was independent of protein synthesis. After the initial increase, the acetylation rate of histones of cells infected with adenovirus 2 or 12 declined, until late in infection it was approximately 40 to 70% of the control cell rate.

Kinetics of nucleic acid synthesis in human embryonic kidney cultures infected with adenovirus 2 or 12: inhibition of cellular deoxyribonucleic acid synthesis.

Infection of human embryonic kidney (HEK) cell cultures with adenovirus types 2 or 12 resulted in an initial drop in the rate of incorporation of (3)H-thymidine into deoxyribonucleic acid (DNA) during the early latent period of virus growth, followed by a marked rise in label uptake. It was shown by cesium chloride isopycnic centrifugation that, after adenovirus 2 infection, there was a decrease in the rate of incorporation of thymidine into cellular DNA. Moreover, DNA-DNA hybridization experiments revealed that, by 28 to 32 hr after infection with either adenovirus 2 or 12, the amount of isolated pulse-labeled DNA capable of hybridizing with HEK cell DNA was reduced by approximately 60 to 70%. Autoradiographic measurements showed that the inhibition of cellular DNA synthesis was due to a decrease in the ability of an infected cell to synthesize DNA. The adenovirus-induced inhibition of host cell DNA synthesis was not due to degradation of cellular DNA. (3)H-thymidine incorporated into cellular DNA at the time of infection remained acid-precipitable, and labeled material was not incorporated into viral DNA. Furthermore, when zone sedimentation through neutral or alkaline sucrose density gradients was employed, no detectable change was observed in the sedimentation rate of this cellular DNA at various times after infection with adenovirus 2 or 12. In addition, there was no increase in deoxyribonuclease activity in cells infected with either virus. Cultures infected for 38 hr with adenovirus 2 or 12 incorporated three to four times as much (3)H-uridine into ribonucleic acid (RNA) as did non-infected cultures. Furthermore, the net RNA synthesized by infected cultures substantially exceeded that of control cultures. The activity of thymidine kinase was induced, but there was no stimulation of uridine kinase.

Enhanced deoxyribonucleic acid polymerase activity in human embryonic kidney cultures infected with adenovirus 2 or 12.

Deoxyribonucleic acid (DNA) polymerase activity was induced at approximately 18 to 20 hr after infection of secondary cultures of human embryonic kidney cells with adenovirus type 2 or type 12, and, at 30 to 50 hr after infection, the activity of this enzyme increased two- to threefold. The activity of thymidine kinase was also induced, but the activity of deoxycytidylic deaminase was not. The DNA content per cell at 71 hr after infection was 1.6-fold greater in adenovirus 2-infected cultures, and approximately 2.4-fold greater in adenovirus 12-infected cultures, than in the noninfected cultures. Several properties of DNA polymerase were studied. The enzymes in normal and adenovirus 2- or 12-infected cell extracts were saturated by approximately the same concentration of heat-denatured salmon sperm DNA primer (160 mug/ml); the enzyme activities had a similar broad pH optimum between 7.5 and 9. Extracts prepared from cells infected by either adenovirus did not activate DNA polymerase from noninfected cells, nor did the noninfected cell extracts inhibit enzyme activity of infected cell extracts. DNA polymerase in both normal and adenovirus 2- or 12-infected cells was located predominantly in the nucleus. In each case, the cytoplasm had only 30% of the enzyme activity of the nucleus. At 40 hr after infection with adenovirus 2 or 12, the activities of the enzyme in the nuclear and cytoplasmic fractions increased two- to threefold. Puromycin, an inhibitor of protein synthesis, prevented DNA polymerase induction when added to cultures during the 18- to 30-hr postinfection period, and it arrested the additional increase in enzyme activity when added after enzyme induction began. However, the increases in both DNA polymerase and thymidine kinase activities took place after treatment of infected cultures with 1-beta-d-arabinofuranosylcytosine, an inhibitor of DNA synthesis and adenovirus growth.