Pig Kidney Cell Line Research Articles

Neutralizing secretory IgA and IgG do not inhibit attachment of transmissible gastroenteritis virus.

Secretory IgA (sIgA) and IgG from porcine milk and serum, respectively, [3H]uridine-labelled virus, swine testis and pig kidney cell lines were used to examine the neutralized virus-cell interaction. Transmissible gastroenteritis virus (TGEV), 99.99% neutralized by immunoglobulin, was able to attach to the cells. Moreover, sIgA enhanced virus attachment. However, the neutralized virus was unable to enter cells, as demonstrated by the action of proteinase K which removed it from the cell surface. It was also found that pre-attached virus was still neutralizable and that IgG and sIgA had similar TGEV-neutralizing capacities.

Regulation of 1,25-dihydroxyvitamin D3 receptors by vitamin D analogs in cultured mammalian cells.

The pig kidney cell line (LLC-PK1) has been shown to possess 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors and to exhibit functional responses to vitamin D metabolites. Here we report that these receptors appear to undergo homologous up-regulation by 1,25-(OH)2D3 and other vitamin D analogs. This phenomenon was also observed in other cell lines, including human skin fibroblasts and human mammary cancer cells (MCF-7). Treatment with active hormone or vitamin D analogs results in a substantial increase (200-400%) in the number of 1,25-(OH)2D3 receptors without altering the affinity of receptor for hormone. The up-regulated receptor, like the basal receptor, has an apparent Kd of about 0.04 nM and sediments at 3.3S on hypertonic sucrose gradients. In addition, approximately 50% of the total receptors from both control and treated cells bind to DNA-cellulose and elute at 0.18 m KCl. These results indicate that the up-regulated receptor is similar to the classical 1,25-(OH)2D3 receptor. While the time necessary to achieve the maximal receptor increment is 16-20 h, there is a rapid component in the rise observed within 5 min. The maximal effect persists for 4-6 h after hormone removal. The increased binding is not a result of differential receptor localization or extractability. 1,25-(OH)2D3, 1,24,25-trihydroxyvitamin D3, 24,25-(OH)2D3, and 25-hydroxyvitamin D3 all increase receptor binding to similar levels, and the dose required closely reflects the affinities of the various metabolites for the receptor. Treatment of cells with the RNA synthesis inhibitor actinomycin D indicates that the increase in receptors is partially dependent on RNA synthesis. Mutant skin fibroblasts from patients with vitamin D-dependent rickets type II, containing nonresponsive 1,25-(OH)2D3 receptors, failed to exhibit the characteristic up-regulation observed in normal cells. Taken together, these results indicate that vitamin D metabolites regulate the number of 1,25-(OH)2D3 receptors in part by receptor occupancy and, more importantly, by a receptor-mediated induction mechanism.

Production of C-24- and C-23-oxidized metabolites of 1,25-dihydroxycholecalciferol by cultured kidney cells (LLC PK1) and their presence in kidney in vivo.

1,25-Dihydroxy[3H]cholecalciferol was converted into several more-polar metabolites by a cultured pig kidney cell line (LLC PK1). The production of metabolites was stimulated by pretreating the cells with unlabelled 1,25-dihydroxycholecalciferol. A similar profile of metabolites was observed on high-pressure-liquid-chromatographic analysis of an extract from the kidneys of rats dosed intravenously with 1,25-dihydroxy[3H]cholecalciferol. Among the metabolites detected were 1,24,25-trihydroxycholecalciferol, 1,25-dihydroxy-24-oxocholecalciferol, 1,23,25-trihydroxy-24-oxocholecalciferol and 1,25-dihydroxycholecalciferol-26,23-lactone. The results are in accord with data reported for intestinal 1,25-dihydroxycholecalciferol metabolism [Napoli, Pramanik, Royal, Reinhardt & Horst (1983) J. Biol. Chem. 258, 9100-9107]. These data indicate that C-23- and C-24-oxidation of 1,25-dihydroxycholecalciferol are phenomena common to calciferol target tissues, and that regulation of 1,25-dihydroxycholecalciferol homoeostasis is dependent on the rate of its metabolism in addition to the rate of its synthesis.

Cytocidal effect of interferons on porcine renal cells.

Treatment of low-passaged pig kidney cell cultures with interferon (IFN) at greater than or equal to 80 times the limit antiviral dose resulted in death of the whole sheet within 15-30 h. This cytocidal effect was specific for both IFN and target cells. It was induced by type I IFNs from three mammalian species (human, bovine, and porcine), including electrophoretically pure HuIFN-alpha. For each IFN preparation, cytocidal and antiviral titers were closely correlated. The processus was protein synthesis dependent and was inhibited by antibodies specific for IFN. Concerning the cell counterpart, the cytocidal effect was observed on 14 different cell strains, isolated from foetal, neonate or adult pig kidneys. There was two absolute requirements for cytocidal effect to appear: (i) the cells must be of epithelioid phenotype, and (ii) the cells must be in a state of confluent monolayer. Under usual conditions of subpassaging, susceptibility to cytocidal effect was lost after 20-25 passages post-explanation, at which stage the cells acquired properties known for established pig kidney cell lines, i.e., the absence of stringent contact inhibition and a weaker sensibility to antiviral effect. An intriguing feature was that cytocidal effect developed as microplaques of dead cells rapidly enlarging to surrounding cells. Several lines of evidence render highly improbable a virus involvement in this IFN-induced plaque effect. Moreover no contaminant, like virus or mycoplasma, was found in the cell strains studied. Such an IFN-induced cytocidal activity on epithelial renal cells from porcine species provide a so far unique example of short-term lethal effect of IFNs on a normal, non lymphoid cell system.

The development of γ-glutamyltransferase in a pig renal-epithelial-cell line in vitro. Relationship to amino acid transport

The pig kidney-cell line, LLC-PK1, possesses gamma-glutamyltransferase with properties similar to those of the purified renal enzyme. gamma-Glutamyltransferase activity increases after cells are seeded at low density to reach values comparable with those found in kidney cortex when the cells are fully confluent. This increase is paralleled by an increase in alpha-methyl D-glucoside uptake. In contrast, the uptakes of L-leucine and L-alanine decrease during this time. These results suggest that gamma-glutamyltransferase is not important as a mediator of the renal transport of neutral amino acids.

Strain-Specific Synthesis of Mycophenolic Acid by Penicillium roqueforti in Blue-Veined Cheese

Twenty of 80 strains of Penicillium roqueforti were able to produce up to 600 mg of mycophenolic acid (MPA) liter in 2% yeast extract-5% sucrose broth. Sixty-two of these strains had been isolated from the main blue-veined cheese varieties of western Europe or from starter cultures. Of these 62 dairy strains, only 7 had MPA-producing potential in vitro. These seven strains had all been isolated during the period 1975 to 1981 from the blue cheese of one individual factory. In cheese from the market, MPA (up to 5 mg kg) was only found in samples of this same factory. With MPA-producing and -nonproducing strains for the experimental manufacture of blue cheese, MPA synthesis in cheese was only detected with strains which form MPA in yeast extract-sucrose broth. The maximum MPA level at 4 mg kg was similar to that in commercial cheese. Toxicity of MPA was tested with two established human cell lines (Detroit 98 and Girardi Heart) and one established pig kidney cell line (AmII).

Ouabain-sensitive 86Rb(K) influx is linked to transepithelial Na transport in pig kidney cell line

The pig kidney cell line, LLC-PK 1, exhibits rheogenic d-glucose coupled transepithelial Na + transport that is inhibited by phlorizin. By measuring the difference in initial rates of influx of 86Rb + with and without coupled Na + transport, we can demonstrate an 86Rb + uptake linked to Na + transport. The simultaneous determination of phlorizin-inhibited Na coupled d-[ 3H]glucose uptake and 86Rb + influx allows calculation of an Na +/Rb + stoichiometry that is consistent with an electrogenic Na + for Rb + exchange.

1,25-Dihydroxyvitamin D3 receptors and functions in cultured pig kidney cells (LLC PK1). Regulation of 24,25-dihydroxyvitamin D3 production.

Although 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) regulates the renal metabolism of 25-hydroxyvitamin D3 (25-OH-D3), the mechanism is not well understood. The established pig kidney cell line, LLC PK1, was used to study this feedback regulation. These cells possess a receptor for 1,25-(OH)2D3 with a sedimentation coefficient of 3.2 S. Scatchard analysis of 1,25-(OH)2D3 binding to cell cytosol yielded an apparent Kd of 0.12 nM and an Nmax of 26 fmol/mg of cytosol protein. LLC PK1 cells respond to 1,25-(OH)2D3 by changes in the metabolism of 25-OH-D3. When incubated with 25-OH-[3H]D3, homogenates of untreated cells did not produce detectable 1,25-(OH)2[3H]D3 or 24,25-(OH)2[3H]D3. However, after treatment of cell monolayers with 1,25-(OH)2D3 for 8 h, homogenates converted substantial 25-OH-[3H]D2 substrate to 24,25-(OH)2[3H]D3. The appearance of this 24-hydroxylase activity in response to 1,25-(OH)2D3 was time- and dose-dependent. Half-maximal levels of enzyme activity were achieved with 0.13 nM 1,25-(OH)2D3, a concentration almost identical to the Kd of the 1,25-(OH)2D3 receptor. The stimulation of 24-hydroxylase activity was shown to be an induction event; treatment of monolayers with 13 nM 1,25-(OH)2D3 for a 4-h pulse was sufficient to induce maximal activity assayed at h. The presence of the transcriptional inhibitor, actinomycin D, during the 4-h pulse abolished the induction of 24-hydroxylase activity. These results demonstrate for the first time the presence of both 1,25-(OH)2D3 receptors and stimulation of 24-hydroxylase activity within the same established mammalian kidney cell line.

Characterization of (8-lysine) vasopressin binding sites on a pig kidney cell line (LLC-PK1). Evidence for hormone-induced receptor transition.

Characterization of (8-lysine) vasopressin binding sites on a pig kidney cell line (LLC-PK1). Evidence for hormone-induced receptor transition.

Relationship of (8-lysine) vasopressin receptor transition to receptor functional properties in a pig kidney cell line (LLC-PK1).

In intact LLC-PK1 cells, occupancy of vasopressin receptors (Roy, C., and Ausiello, D. A. (1981) J. Biol. Chem. 256, 3415-3522) correlated with cell cAMP production. This relationship was observed as a function of hormone dose, incubation time, and changes in receptor affinity. However, the rate of cAMP production diminished with time in intact cells exposed to high hormone concentrations, even in the presence of a phosphodiesterase inhibitor. A rapid desensitization of adenylate cyclase activity was observed in minutes upon treatment of intact cells with high hormonal concentrations. Desensitization was dose- and time-dependent. Hypertonic sodium chloride, which increased hormonal binding and cell cAMP production, prevented desensitization. The acute decrease in hormone-stimulated adenylate cyclase activity correlated with increased occupancy of low affinity binding sites. EDTA-suspended cells, which have a homogeneous population of binding sites, did not demonstrate desensitization. A proposal is made as to the consequences of this phenomenon at physiological concentrations of vasopressin.

Introduction

Pig kidney cell line LLC-PK1 cultured on a collagen-coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by circumferential occluding junctions. In the presence of 5.5 mM D-glucose, a potential difference (PD) of 2.8 mV, apical bath negative, short-circuit current Isc of 13.2 microA . cm-2, and transepithelial resistance of 211 omega . cm2 were recorded. Isc and PD were reduced by phlorizin added to the apical bath but were unaffected when phlorizin was placed in the basolateral bath. Ouabain or the replacement of Na by tris-(hydroxymethyl)aminomethane or choline abolished the Isc. The sugar concentrations required to produce the half-maximal Isc were 0.13 mM beta-methyl-D-glucoside, 0.28 mM D-glucose, 0.65 mM alpha-methyl-D-glucoside, 0.77 mM 6-deoxy-D-glucose, 4.8 mM D-galactose, and 29 mM 3-O-methylglucose. When [Na] was reduced, the D-glucose required for half-maximal SCC increased. Isotopically 3H- and 14C-labeled D-glucose were used to determine simultaneous bidirectional fluxes; a resultant net apical-to-basolateral flux was present and could be abolished by phlorizin. The transported isotope cochromatographed with labeled D-glucose, indicating negligible metabolism. The cell culture model provides advantages for investigation of mechanisms of transepithelial glucose transport.

Transepithelial glucose transport in cell culture.

Pig kidney cell line LLC-PK1 cultured on a collagen-coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by circumferential occluding junctions. In the presence of 5.5 mM D-glucose, a potential difference (PD) of 2.8 mV, apical bath negative, short-circuit current Isc of 13.2 microA . cm-2, and transepithelial resistance of 211 omega . cm2 were recorded. Isc and PD were reduced by phlorizin added to the apical bath but were unaffected when phlorizin was placed in the basolateral bath. Ouabain or the replacement of Na by tris-(hydroxymethyl)aminomethane or choline abolished the Isc. The sugar concentrations required to produce the half-maximal Isc were 0.13 mM beta-methyl-D-glucoside, 0.28 mM D-glucose, 0.65 mM alpha-methyl-D-glucoside, 0.77 mM 6-deoxy-D-glucose, 4.8 mM D-galactose, and 29 mM 3-O-methylglucose. When [Na] was reduced, the D-glucose required for half-maximal SCC increased. Isotopically 3H- and 14C-labeled D-glucose were used to determine simultaneous bidirectional fluxes; a resultant net apical-to-basolateral flux was present and could be abolished by phlorizin. The transported isotope cochromatographed with labeled D-glucose, indicating negligible metabolism. The cell culture model provides advantages for investigation of mechanisms of transepithelial glucose transport.

Transepithelial transport in cell culture: D-glucose transport by a pig kidney cell line (LLC-PK1).

The pig kidney cell line LLC-PK1 cultured on a collagen coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by occluding junctions. A transepithelial electrical potential (PD) and short-circuit current (SCC) were dependent on the presence of Na and sugar in the apical bathing solution. In the presence of 5.5 mM D-glucose, a PD of 2.8 mV. apical surface negative a SCC of 13 microA cm-2 and transepithelial resistance of 211 ohm.cm2 were recorded. The SCC was promptly reduced by the addition of phlorizin to the apical bath but unaffected when placed in the basolateral bath. The effect on SCC of various sugars was compared by the concentrations required for half-maximal SCC: 0.13 mM beta-methyl-D-glucoside, 0.28 mM D-glucose, 0.65 mM alpha-methyl-D-glucoside, 0.77 mM 6-deoxy-D-glucose, 4.8 mM D-galactose, and 29 mM 3-O-methyl-glucose. When [Na] was reduced, the concentration of D-glucose required for half-maximal SCC increase. Isotopically labeled 3H and 14C D-glucose were used to simultaneously determine bidirectional fluxes; a resultant net apical-to-basolateral transport was present and abolished by phlorizin. The transported isotope cochromatographed with labeled D-glucose, indicating negligible metabolism of transported glucose. The pig kidney cell line, LLC-PK1, provides a cell culture model for the investigation of mechanisms of transepithelial glucose transport.

The white pock mutants of rabbit poxvirus II. The early white pock (μ) host range (hr) mutants of rabbit poxvirus uncouple transcription and translation in nonpermissive cells

A biochemical examination of the white pock (μ) host range (hr) mutants of rabbit poxvirus (RP μhr mutants) on the prototypic nonpermissive pig kidney (PK) cell line allows them to be divided into two groups, “early” or “late,” based on their ability to synthesize viral DNA in these cells. We have studied two early mutants (those unable to synthesize viral DNA) and found that by 8–10 hr after infection of PK cells all translation ceases; however, viral RNA synthesis continues unabated. These data provide evidence for the active control of translation during poxvirus infection. Even though the two mutants both inhibit translation under nonpermissive conditions, they clearly differ biochemically from one another in their total transcriptional capacity. An exmaination of the RNA made by these two RP μhr mutants under nonpermissive conditions further reveals that at times, which would be equivalent to “late” in a normal infection, one mutant has transcribed only 62% and the other 84% of the RNA sequences synthesized in a wild-type infection. Furthermore, transcription continues long after translation has stopped. Both mutants have also been found to be more refractile to uncoating in nonpermissive cells than wild-type virus. We find that the rate of the host-dependent formation of subviral cores is impaired as is the subsequent step of viral-dependent uncoating of the cores. A defective uncoating process might be related to the reduced complexity of transcription observed with these mutants. Despite their similarities, our results suggest that the two mutants are interrupted at different stages within the early phase of infection and that both poxvirus transcription and translation are actively regulated prior to the onset of viral DNA synthesis.

Studies on plasminogen activator from pig kidney cell cultures. III. Purification and characterization

Plasminogen activator in spent tissue culture medium from a continuous line of pig kidney cells was purified on a 25 liter scale by a combination of adsorption on hydroxyapatite, ammonium sulfate precipitation, DEAE-and CM-cellulose chromatography, and gel filtration. The final product had a specific activity of 70,000 to 120,000 units/mg and consisted of numerous immunologically similar species of different size and charge. Two major fractions were obtained by gel filtration. The principal fraction included enzymes of molecular weights >45,000. The other fraction included 30,000 and 24,000 dalton forms. The large forms could be degraded to smaller forms. The isolectric points of the different species were in the range pH 6.8 to 7.2. Active site titrations gave 1.4 ± 0.1 × 10 13 units/mole. The enzyme was similar to, but not identical with, human urokinase in specificity, inhibitor sensitivity, and fibrinolytic activity. The two enzymes were immunologically different.

EQUINE HERPESVIRUSES: ON THE DIFFERENTIATION OF RESPIRATORY FROM FOETAL STRAINS OF TYPE 1

SUMMARY Confirmation of the occurrence of equine herpesvirus type 1 (EHV1) abortion in both epizootic and sporadic form epizootic in Australia for the first time in 1977 against a background in which a reasonably diligent search for such viruses during the preceeding 11 years failed to associate EHV1 with abortion, provided a special opportunity to compare the properties of the newly isolated foetal (F) strains with a collection of endemic respiratory (R) strains that had been recovered at fairly regular intervals since 1967. Using plaque size and host cell range all of 7 R strains tested were clearly distinguishable from 7 of 11 F isolates. The remaining 4 F strains had plaque diameters of R strains but 3 of the 4 viruses conformed in their host cell range with F strains. Only one F isolate (from Tasmania) had both plaque morphology and host cell range of R strain viruses.The mean diameter of plaques produced by R strains in equine foetal kidney (EFK) cells after 4 days under a methyl cellulose overlay was 1.52 mm (range 1.30–1.84 mm) while the mean diameter of small plaques produced by F strains was 0.82 mm (range 0.68–0.91 mm).In addition to EFK cells all R and F strains grew in an equine dermal (EDerm) cell line and all but two of 19 isolates grew in a pig kidney (PK) cell line. None of the low passage R strains grew in bovine embryo tracheal (EBTr) or feline embryo (FEmb) cells whereas all but one of 11 F isolates grew in EBTr cells. 8/11F isolates also grew in FEmb cell line.Growth of viruses at 33° and 40.5°cf. a usual growth temperature of 37° was of no detectable value in differentiating R and F strains of EHV1.In a limited geographic and time frame the criteria of plaque size in EFK cells and growth in EBTr cells unambiguously distinguished between R and F isolates and represent simple markers worthy of additional study.

Selection of foot-and-mouth disease viruses by passage in bovine and swine kidney cell cultures

Two uncloned populations of foot-and-mouth disease virus, one pathogenic for adult mice and the other nonpathogenic, were passaged in cultures of primary bovine kidney (BK) cells and a line of pig kidney (MVPK) cells. Within 10 passages in MVPK cells, the nonpathogenic virus became pathogenic for adult mice, but similar passages of this virus in BK cells did not affect its pathogenicity. In contrast, passage of the pathogenic virus in MVPK cells resulted in a decrease in pathogenicity, but again passage in BK cells had no effect on this characteristics. Neither of the viruses changed in pathogenicity for infant mice during the four passages that were tested. The nonpathogenic virus passaged in BK cells was more infectious for BK than for MVPK cells, but after passage in MVPK cells, this virus was about equally infectious for the two types of cells. Infectivity of the pathogenic virus was relatively unchanged by passage in either type of cell. The parent nonpathogenic virus and the 10th BK cell passage of this virus were much more resistant to adsorption with homogenized mouse kidney than were the MVPK cell passages of nonpathogenic virus. The parent and passaged pathogenic viruses were readily adsorbed. The results demonstrated that passage of the two viruses in MVPK cells had a pronounced selective effect.

The flaviviruses (group B arboviruses): a cross-neutralization study.

SUMMARY Cross-neutralization studies on 42 flaviviruses and their respective antisera were performed by a plaque assay in a line of pig kidney cells. Six tick-borne viruses fell into one subgroup; seven viruses associated with bats and small rodents and a further tick-borne virus fell into a second subgroup. Twenty-two mosquito-borne viruses fell into one major and four minor subgroups. Four mosquito-borne, one tick-borne and one bat virus showed no relationship to any other flavivirus.

Characterization of a type C virus released from the porcine cell line PK(15)

The PK(15) pig kidney cell line produces type C viral particles which are not infectious for a variety of mammalian cell lines. The PK(15) virions contain reverse transcriptase with a molecular weight of 70,000 and a “gs protein” with a molecular weight of 30,000. The reverse transcriptase and gs protein are antigenically related to the homologous proteins of previously studied mammalian type C viruses; however, RNA-DNA hybridization shows that the PK(15) RNA genome is essentially unrelated to known murine, feline, rat, hamster, and primate type C viruses. Normal pig liver contains DNA which is homologous to the PK(15) viral genome, indicating that pigs have endogenous type C viral genetic information.

Biological studies and plaquing of Mokola virus (rabies serogroup) in porcine kidney (PS) cells.

The continuous pig kidney cell line (PS) is very sensitive to the propagation of Mokola virus. No prior adaptation of the virus to the cells was necessary before the demonstration of high virus titers. Virus activity in the cells can be easily quantified in terms of cytopathic effect, titer of complement-fixing antigen in the culture fluid, and plaque formation. There was no inclusion body formation. The plaques were quite clear, measuring 1–2 mm, and were countable from the 4th day. The results of the plaque neutralization test as well as the neutralization test in baby mice showed no significant cross-reaction between Mokola virus and rabies virus.