Primary Kidney Cell Cultures Research Articles

Kidney lipids in galactosylceramide synthase-deficient mice: absence of galactosylsulfatide and compensatory increase in more polar sulfoglycolipids

UDP-galactose:ceramide galactosyltransferase (CGT) catalyzes the final step in the synthesis of galactosylceramide (GalCer). It has previously been shown that CGT-deficient mice do not synthesize GalCer and its sulfated derivative GalCer I(3)-sulfate (galactosylsulfatide, SM4s) but form myelin containing glucosylceramide (GlcCer) and sphingomyelin with 2-hydroxy fatty acids. Because relatively high concentrations of GalCer and SM4s are present also in mammalian kidney, we analyzed the composition of lipids in the kidney of Cgt(-/-) and, as a control, Cgt(+/-) and wild-type mice. The homozygous mutant mice lacked GalCer, galabiaosylceramide (Ga(2)Cer), and SM4s. Yet, they did not show any major morphological or functional defects in the kidney. A slight increase in GlcCer containing 4-hydroxysphinganine was evident among neutral glycolipids. Intriguingly, more polar sulfoglycolipids, that is, lactosylceramide II(3)-sulfate (SM3) and gangliotetraosylceramide II(3),IV(3)-bis-sulfate (SB1a), were expressed at 2 to 3 times the normal levels in Cgt(-/-) mice, indicating upregulation of biosynthesis of SB1a from GlcCer via SM3. Given that SM4s is a major polar glycolipid constituting renal tubular membrane, the increase in SM3 and SB1a in the mice deficient in CGT and thus SM4s appears to be a compensatory process, which could partly restore kidney function in the knockout mice.

Disruption of LT-antigen/p53 complex by heat treatment correlates with inhibition of DNA synthesis during transforming infection with SV40

Transforming infection of Go/G1-arrested primary mouse kidney cell cultures with simian virus 40 (SV40) induces cells to re-enter the S-phase of the cell cycle. In Go-arrested cells, no p53 is detected, whereas in cells induced to proliferate by infection, a gradual accumulation of p53 complexed to SV40 large T-antigen is observed in the nucleus. Heat treatment of actively proliferating SV40-infected cells leads to inhibition of DNA synthesis and growth arrest. To determine the fate of p53 after heat treatment, proliferating infected cells were exposed to mild heat (42.5°C) for increasing lengths of time. The results presented here show that after ninety minutes of treatment, the arrest of DNA synthesis by heat correlates with the disruption of the p53/LT-antigen complex. Longer treatments induce, in addition, a reduction in the solubility of p53, which was recovered tightly associated with the nuclear fraction. This contrasted with large T-antigen, whose solubility remained unaffected by heat treatment. Although the total amount of p53 in the nucleus remained constant, as shown by immunoblot analyses, p53 was no longer detectable after immunoprecipitation or by immunofluorescent staining techniques. These results suggest that heat treatment had either induced conformational changes in its antigenic sites, or had sequestered the sites through aggregation or binding to insoluble nuclear components.Key words: p53, heat shock, LT-antigen/p53 complex, S-phase.

The hydropericardium syndrome and inclusion body hepatitis in domestic fowl.

Hydropericardium syndrome, an emerging disease of poultry, has recently been detected in some countries of Asia and America, particularly in broiler birds aged 3-6 weeks. The disease is characterized by its sudden occurrence with high mortality of up to 80% in broilers and low mortality of under 10% in layers, associated with hydropericardium. Its course is of 7-15 days under natural conditions. The causative agent is probably fowl adenovirus serotype 4, belonging to group I aviadenovirus genus of the family adenoviridae, which can be cultivated in primary cell cultures of chicken kidney and embryo liver cells. The transmission of disease occurs laterally by the oral-faecal route. The livers of affected birds show necrotic foci, and basophilic intranuclear inclusion bodies fill the entire enlarged nucleus of some of the hepatocytes. The disease can be diagnosed from its gross lesions, histopathological changes in the liver and by serological tests, such as agar gel diffusion, counter immunoelectrophoresis, indirect haemagglutination and ELISA. It has been brought under control by inactivated liver organ vaccines (0.25 ml/bird) or inactivated cell culture vaccines (10(3.5) LD50/bird) given by the subcutaneous route at 10-15 days of age. The vaccine is effective in the face of an outbreak and significantly reduces the mortality.

Disruption of LT-antigen/p53 complex by heat treatment correlates with inhibition of DNA synthesis during transforming infection with SV40

Transforming infection of Go/G1-arrested primary mouse kidney cell cultures with simian virus 40 (SV40) induces cells to re-enter the S-phase of the cell cycle. In Go-arrested cells, no p53 is detected, whereas in cells induced to proliferate by infection, a gradual accumulation of p53 complexed to SV40 large T-antigen is observed in the nucleus. Heat treatment of actively proliferating SV40-infected cells leads to inhibition of DNA synthesis and growth arrest. To determine the fate of p53 after heat treatment, proliferating infected cells were exposed to mild heat (42.5 degrees C) for increasing lengths of time. The results presented here show that after ninety minutes of treatment, the arrest of DNA synthesis by heat correlates with the disruption of the p53/LT-antigen complex. Longer treatments induce, in addition, a reduction in the solubility of p53, which was recovered tightly associated with the nuclear fraction. This contrasted with large T-antigen, whose solubility remained unaffected by heat treatment. Although the total amount of p53 in the nucleus remained constant, as shown by immunoblot analyses, p53 was no longer detectable after immunoprecipitation or by immunofluorescent staining techniques. These results suggest that heat treatment had either induced conformational changes in its antigenic sites, or had sequestered the sites through aggregation or binding to insoluble nuclear components.

Correlation between Induction of DNA Fragmentation and Micronuclei Formation in Kidney Cells from Rats and Humans and Tissue-Specific Carcinogenic Activity

Five chemicals, known to induce kidney tumors in rats, were assayed for their ability to induce DNA damage and formation of micronuclei in primary cultures of rat and human kidney cells and in the kidney of intact rats. Significant dose-dependent increases of DNA fragmentation, as measured by the Comet assay, and of micronuclei frequency were obtained in primary kidney cells from both rats and humans with the following concentrations of the five test compounds: lead acetate (not tested for micronuclei induction) and potassium bromate from 0.56 to 1.8 mM, phenacetin from 1 to 3.2 mM, and 1,4-dichlorobenzene and nitrilotriacetic acid from 1.8 to 5.6 mM. In terms of DNA-damaging potency all the five chemicals were more active in rat than in human kidney cells, whereas the potencies in inducing micronuclei formation were similar in the two species with the exception of 1,4-dichlorobenzene, which was slightly more potent in human than in rat cells. Consistently with the results observed in vitro, statistically significant increases in the average frequency of both DNA breaks and micronucleated cells were detected in the kidney of rats given po a single (12 LD50) or three successive daily doses (13 LD50) of the five test compounds. 4,4′-Methylenedianiline, a carcinogen which does not induce kidney tumors in rats, gave negative responses in both in vitro and in vivo assays. These findings give evidence that kidney carcinogens may be identified by short-term genotoxicity assays using as target kidney cells and show that the five chemicals tested produce in primary cultures of kidney cells from human donors effects similar to those observed in rats.

Genomic cloning, structure, and regulatory elements of the 1α,25(OH) 2D 3 down-regulated gene for cyclic AMP-dependent protein kinase inhibitor

The cyclic AMP-dependent protein kinase inhibitor (PKI) mRNA and protein are negatively and tissue-specifically regulated in the kidney by 1α,25(OH) 2D 3. A 17-kb PKI clone, isolated from a chick genomic library, revealed that the PKI gene consists of two exons separated by a 4.5-kb intron. A 411-bp upstream region (constituting 93 bp upstream and 318 bp downstream from the transcriptional start site) containing a putative negative VDRE (nVDRE) fused to the luciferase gene was used for transient transfections of primary cultures of chick kidney cells. Luciferase activity was significantly down-regulated in response to 1α,25(OH) 2D 3. This result suggests that the promoter region containing the putative nVDRE plays a pivotal role in the negative regulation of PKI gene transcription.

DNA vaccine against oncogenic hamster cells transformed by HPV16 E6/E7 oncogenes and the activated ras oncogene.

The capability of DNA to elicit anti-tumour immunity was studied using human papillomavirus type 16 (HPV16)-transformed Syrian hamster cells denoted K3/II. These cells had been derived after cotransfection of primary kidney cell cultures with p16HHMo plasmid containing E6/E7 oncogenes of HPV16 and pEJ6.6 plasmid containing the activated human H-ras oncogene; they express both the HPV16 and activated H-ras genes. As a DNA vaccine, the p16HHMo plasmid was used. Three doses of the plasmid (either 100 microg or 10-15 microg per dose) were administered intramuscularly at 3-week intervals. The animals were challenged with four different doses (10(3)-10(6) per animal) of K3/II cells 10 days after the last plasmid injection. In one experiment the lower dose of plasmid DNA was also given in a mixture with the cationic lipid DOTAP. In another experiment, the pEJ6.6 plasmid (100 microg per dose) was used either alone or in combination with p16HHMo. In all experiments animals inoculated with the same doses of pBR322 plasmid served as controls. A moderate protective effect was observed in animals inoculated with the 100-microg doses of p16HHMo, but not in those inoculated with 10-15 microg of the same plasmid, whether given with or without DOTAP. A protective effect was also observed after administration of the pEJ6. 6 plasmid. At the time of challenge a portion of the p16HHMo-immunized, but not the pBR322-treated, animals possessed antibodies reactive in ELISA with peptides derived from the N-terminal portion of HPV16 E7 protein and with one peptide derived from E6 protein, while two other E6 peptides exhibited non-specific reactivity.

Cisplatin effects on F-actin and matrix proteins precede renal tubular cell detachment and apoptosis in vitro.

In primary cultures of porcine proximal tubular kidney cells and LLC-PK1 cells cisplatin (5 - 50 microM) caused apoptosis and cell detachment; in both systems cell detachment occurred, preceded by a loss of cytoskeletal F-actin stress fibers within 4 - 6 h, and a reduction of mRNA encoding for fibronectin, collagen a2 type (IV) and laminin B2 within 17 - 41 h. Prevention of F-actin damage by phalloidin prevented nuclear fragmentation, suggesting a relation between F-actin damage and apoptosis. Overexpression of Bcl-2 also prevented apoptosis, but did not prevent damage to the F-actin skeleton or the reduction of mRNA expression of the matrix proteins. These results suggest that Bcl-2 overexpression interferes with apoptotic signals downstream of F-actin. The relevance of these results for cell detachment in kidney toxicity is discussed.

The major late promoter and bipartite leader sequence of fowl adenovirus

The region of the fowl adenovirus serotype 10 (FAV-10) genome containing the major late promoter (MLP) and leader sequences was determined and appropriate genomic fragments were cloned and sequenced. A TATA box was identified and the location of the putative transcription start site was determined. By using synthetic primers from the transcription start site in conjunction with oligonucleotides from the coding regions of the penton base and hexon genes, cDNA was produced from late mRNA isolated from cell cultures infected with FAV-10 at 24 h post-infection. The resulting cDNA was cloned and sequenced and the leader sequences thus identified. It was found that the FAV-10 MLP utilized only two leader sequences (a bipartite leader). By comparison with human adenoviruses (HAVs) it appeared that the second leader in HAVs was absent from the FAV-10. The second leader sequences of FAV-10 was larger than either the second or third leaders of HAVs, but was 29 basepairs shorter than the combined size of the leader sequences 2 and 3 from HAV-2. To confirm the transcription start site and leader sequences, single stranded cDNA was produced from mRNA using the primers from within the coding sequence for the penton base or hexon. A tail of dGTP's was added and cDNA synthesis was completed using an oligonucleotide from within the hexon or penton base coding sequence and a second poly-dCTP oligonucleotide. Sequencing of the resultant G-tailed DNA confirmed the location of the transcription start site as an adenosine residue 24 basepairs upstream from the 3-prime (3') end of the TATA box. Sequencing 5' of the TATA box failed to reveal any sequence similarity with the human adenovirus upstream stimulatory factor (USF). Various plasmids were constructed which placed the determined sequences of the MLP, leader, and the region upstream of the TATA box linked to the co-acetyl acid transferase (CAT) gene. These expression plasmids in transient expression assays of CAT activity in primary chicken kidney cell culture with or without FAV-10 co-infection were determined. These experiments showed that the cassette containing sequences 5' of the TATA box expressed CAT to a much greater level than cassettes not containing this upstream region and that the presence of virus significantly increased the activity of the promoter following the onset of viral DNA replication. Without the 5' region, cassettes failed to express above background levels. These results suggest that the basic structure of the fowl adenovirus MLP is similar to that of the human adenovirus although it utilizes a bipartite rather than a tripartite leader sequence.

In Vitro Methods of Assessing Renal Damage

Freshly isolated and primary cultures of rat kidney cells derived from specific nephron segments can be useful in vitro models for studying processes such as drug metabolism, membrane transport, and biochemical mechanisms of chemically induced toxicity. Proximal tubular (PT) and distal tubular (DT) cells were isolated from rat renal cortex by collagenase perfusion and Percoll density-gradient centrifugation. Oxidants produced glutathione (GSH) oxidation and lipid peroxidation and were markedly more cytotoxic to DT cells than to PT cells. Similarly, alkylating agents that target soft nucleophiles such as GSH and protein sulfhydryls were more toxic to DT cells than to PT cells, whereas an alkylating agent that targets hard nucleophiles was equally cytotoxic in the 2 cell types. DT cells were also more sensitive to brief periods of oxygen deprivation and were markedly more susceptible to ATP depletion by treatment with iodoacetate and cyanide than were PT cells. Serum-free, hormonally defined conditions have been optimized for primary culture of rat renal PT and DT cells to maintain differentiated function for up to 9 days. Primary cultures exhibited similar susceptibilities as freshly isolated cells to acute injury from chemical toxicants and the cultures express several isoforms of cytochrome P-450. These studies show that freshly isolated and primary cultures of rat renal PT and DT cells can be used to study both short-term and long-term responses to toxic chemicals.

Tumor necrosis factor α augments amyloid β protein (25–35) induced apoptosis in human cells

No information is yet available on the effect of tumor necrosis factor α (TNF α) on amyloid β protein (A β)-induced cytotoxicity in human cells. For this reason the induction of apoptosis by TNF α and A β (25–35) was studied in primary cultures of human thyroid and kidney cells as well as in the neuroblastoma line SK-N-SH and in DU-145 cells. Apoptosis occurred in all cell types after A β (25–35) treatment, but was markedly enhanced when TNF α was additionally present. This effect was less pronounced in transformed cell lines than in primary cultures, in which TNF α on its own was not cytotoxic. Apoptosis was still more prevalent under serum free culture conditions. The results demonstrate that TNF α may support the occurrence of A β-mediated cell death and thus contribute to the development of pathological changes in Alzheimer's disease (AD).

Primary kidney cells.

Hormonally defined serum-free media have been developed for growth and functional studies with kidney epithelial cell cultures. Not only can several established kidney epithelial cell lines (MDCK and LLC-PK1) be grown in a serum-free environment (1,2), but in addition, primary cultures of kidney epithelial cells can also be grown serum free (1-4). Investigations with primary kidney cell cultures are advantageous for several reasons. First of all, kidney cells can be grown in vitro from the animal of choice. Thus, the results of tissue culture studies can be more closely correlated with animal studies. Secondly, new tissue culture systems can be developed that more closely resemble kidney cells in vivo than those obtained with established kidney cell lines.

DNA damage induced by seven N-nitroso compounds in primary cultures of human and rat kidney cells

Seven N-nitroso compounds (NOC), known to induce kidney tumors in rats, were assayed for DNA-damaging activity in primary cultures of human and rat kidney cells. DNA fragmentation was measured by the alkaline elution technique. Positive responses were obtained in cells of both species with N-nitrosodimethylamine (32 mM), N-nitrosodiethylamine (32 mN), N-nitrosodi- n-propylamine (10 mM), N-ethyl- N-hydroxyethylnitrosamine (18 mM), and streptozotocin (1 mM). N-nitrosodiethanolamine and N-nitrosomorpholine were inactive at the highest concentration tested (32 mM). The responses of human kidney cells were qualitatively similar to those of rat kidney cells, but statistically significant differences between the two species in the DNA-damaging potencies were observed with N-ethyl- N-hydroxyethylnitrosamine and streptozocin, both more genotoxic in rat cells. Taken as a whole, the results suggest on the one hand that the five active NOC might be carcinogenic for the kidney in humans, and on the other hand that the rat kidney cell/DNA damage assay is a valid model for predicting the genotoxic potential of NOC in human kidney cells.

In vitro characterization of renal reabsorption and secretion of folate using primary cultures of human kidney cells

Proximal tubule cells in the kidney play an important role in the homeostasis of folate. Mechanistic studies on folate reabsorption and secretion across the proximal tubular epithelium have been limited. The present studies were aimed at assessing the suitability of human proximal tubule (HPT) cells cultured on membrane inserts as an in vitro model of renal folate processing by examining aspects of folate transport from the apical to basolateral (A-B, reabsorptive) and from the basolateral to apical (B-A, secretory) directions. The data demonstrate that apical membrane binding and cellular uptake of folate in HPT cells were mediated via specific high affinity mechanisms. Apical binding and cellular uptake of folate occurred in a greater proportion from the A-B direction than from the B-A direction, suggesting that folate reabsorption may be the dominant process in HPT cells. However, cellular uptake did occur readily from the B-A direction, in amounts greater than could be explained by leakage through the cell barrier to the apical media, followed by uptake from the A-B direction. These data suggest the presence of specific folate secretory processes in HPT cells. Folate was transferred across the cell layer into the opposite media compartments from both directions, but apparently by nonspecific processes. The present studies suggest that primary cultures of HPT cells grown on microporous membrane inserts may serve as a valuable in vitro model to study the bidirectional transport of folate by kidney cells.

Occurrence and role of an early antigen and evidence for transforming ability of porcine circovirus.

By means of indirect immunofluorescence assay (IFA) using natural swine immune serum and hyperimmune serum from rabbits infected with porcine circovirus (PCV), a PCV antigen was detected present prior to the onset of viral and cellular DNA synthesis in nucleoli of cells of synchronized and growth stimulated infected PS cell cultures grown for more than 12 h in the presence of hydroxyurea. The number of cells containing specifically fluorescing nucleoli increased with increasing time of growth in the presence of hydroxyurea. The concomitant increase in the number of cells containing virus structural (VS) antigen in the nuclei and the increase in the amount of replicative (RF) DNA and accompanying 5 S DNA after release from the hydroxyurea block suggest that EA is involved in induction of PCV DNA replication. Primary pig kidney cell cultures persistently infected with PCV survived mock-infected control cultures for 16 passages. They had lost contact inhibition and formed cell colonies in soft agar at a ratio of 0.1 to 0.4%. Cell lines derived from agar colonies showed properties of transformed cells e.g. low requirement for serum growth factors, ability to overgrow a continuous cell layer, anchorage independence of growth. In transformed cells stimulated to growth and grown in the presence of hydroxyurea, non-structural viral antigen visible by IFA in nucleoli and VS antigen located in the cytoplasm were expressed. Contrary to virus bound nuclear VS antigen in productive infection, accumulation of cytoplasmatic VS antigen was independent of DNA synthesis and caused cell destruction, thus limiting growth of cell layers and colonies in soft agar.

Identification of a New Acute Phase Protein

We have previously reported mouse SIP24 protein as a secreted inducible protein produced by quiescent Balb/c 3T3 cells. SIP24 can be produced in response to many factors, including serum, basic fibroblast growth factor, prostaglandin F2 alpha, phorbol ester, and dexamethasone. Here we present evidence to show that SIP24 is the product of mouse 24P3 mRNA. The 24P3 cDNA was originally cloned from an SV40-transformed quiescent mouse primary kidney cell culture, and it has been classified as a new member of the lipocalin protein family. We show that the SIP24/24P3 protein and mRNA increase dramatically in mouse serum and liver during the acute phase response induced by turpentine injection. Injection of mice with dexamethasone caused a modest increase of SIP24/24P3 mRNA in the liver. Tissue distribution studies revealed that SIP24/24P3 is mainly expressed in liver during the acute phase response. SIP24/24P3 was also detected in the brain and the uterus. In mouse BNL (Balb/c normal liver) cells, the production of SIP24/24P3 is stimulated by tumor necrosis factor alpha, which is a major regulator of the expression of other acute phase proteins. From its pattern of regulation, we conclude that SIP24/24P3 is a new type 1 acute phase protein.

Estrogen metabolism in microsomal, cell, and tissue preparations of kidney and liver from Syrian hamsters.

The estrogen-treated golden Syrian hamster has been used as an experimental model for estrogen-induced and estrogen-dependent cancers, but pathways to neoplastic transformation remain unknown in this animal. Metabolism of estrogens to activated or reactive compounds, followed by subsequent oxidative damage to the target tissue, remains a potential step in the tumorigenic process. In this study, the extent of estrogen metabolism is compared in three different in vitro preparations from untreated and estrogen-treated Syrian hamsters, primary kidney cell cultures, microsomal preparations, and freshly prepared tissue kidney slices. In primary kidney cell cultures, the amount of catechol estrogens decreased upon increasing estrogen (DES) treatment period, and completely disappeared after about 6 months treatment. This decrease is not a result of formation of less amounts of catechol estrogens, but rather reflects the presence of the enzyme systems to further metabolize any formed catechol estrogens, since the amount of catechol estrogens formed, as detected by 3H2O release, is unchanged. The polar metabolites a, b and c increased with estrogen treatment, and metabolite c appeared only after DES treatment. The appearance of polar metabolite c only in kidney preparations from DES-treated animals implies that it may serve as a marker of cellular transformation. Estriol and estrone were detected, but were not affected by DES treatment, while no methoxyestrogens were isolated. Studies of estradiol metabolism in microsomal preparations showed a very low rate of metabolism, compared to the primary kidney cell cultures. In contrast, estrogen metabolism was extensive in kidney slices from untreated hamsters, with only approx. 30% of the substrate estradiol remaining unmetabolized after 6 h of incubation. While no catechol estrogens were detected, a small quantity of estriol, and a large amount of estrone and methoxyestrogens were isolated. The polar metabolite a was the main polar metabolite detected, with very little of metabolite b and no metabolite c. In kidney slices from 4 month DES-treated hamsters, a much higher amount of polar metabolites was detected, and metabolite c appeared after 6 h incubation. Mass spectrometric analysis and HPLC data of metabolite c indicate that this metabolite is 15 alpha-hydroxyesteradiol. This metabolite may serve as a biomarker for changes occurring in the hamster kidney cells under continuous estrogen exposure. Finally, formation of water soluble conjugates was demonstrated in both kidney slices and liver slices from Syrian hamsters, with glucuronide, sulfate and thioether conjugates of estrone and estradiol and glucuronides of catechol estrogens detected.(ABSTRACT TRUNCATED AT 400 WORDS)

A heterogeneous set of FMR1 proteins is widely distributed in mouse tissues and is modulated in cell culture

The fragile X syndrome is an X-linked inherited disease and is the result of transcriptional inactivation of the FMR1 gene and the absence of its encoded FMR protein (FMRP). Using a specific monoclonal antibody directed against human FMRP, we have studied the steady-state levels of its murine homolog in several tissues and organs of adult and young mice. In immunoblot analyses, the antibody recognizes a heterogeneous subset of proteins with apparent molecular weights ranging from 80 to 70 kDa. These proteins are detected in all the 27 tissues tested; however, the relative proportion of each polypeptide recognized varies between tissues, and a significantly higher expression is observed in young animals. Northern blot analysis of RNA extracted from selected tissues from adult mouse shows that these tissues express the major 4.8 kb mRNA, although at different levels, and contain several additional shorter transcripts, particularly in muscular tissues. We also report that expression of the FMR1 gene is modulated in proliferating and quiescent primary mouse kidney cell cultures with an inverse relationship between levels of FMR1 mRNA and of its encoded proteins. This suggests that FMRPs are highly stable in quiescent cells and that FMR1 expression is likely post-transcriptionally controlled. Our results document the widespread expression of the FMR1 gene, and suggest that it is controlled by different mechanisms implicated in cell growth and differentiation.

A Live Attenuated Dengue-L Vaccine Candidate (45AZ5) Passaged In Primary Dog Kidney Cell Culture Is Attenuated And Immunogenic For Humans

A dengue-1 candidate vaccine (45AZ5), previously found to be underattenuated in 2 volunteers, was further attenuated by passage in primary dog kidney (PDK) cell cultures. New candidate vaccines prepared from three levels of PDK-passaged virus, PDK-10, PDK-20, and PDK-27, were each injected into 9 or 10 volunteers. There was a significant, progressive decline in viremia, clinical illness, and hematologic changes from low to high PDK cell passage level. PDK-20 infected all 10 vaccinees and induced viremia in 5, transient fever in 3, symptoms that resulted in curtailed activities for < or = 1 day in 4, and neutralizing antibody in all 10, which persisted for > or = 1 year in 5 of 8 vaccinees tested. Progressive passage in PDK cell culture progressively attenuates vaccine candidate strain 45AZ5 for humans. Because passage level PDK-20 may be suitable for healthy adults at high risk of dengue fever, additional clinical trials of this strain are warranted.

Oscillatory behavior of control-systems of calcium homeostasis in chickens.

Computer simulation of calcium homeostasis in chicks predicted an oscillatory behavior of bone calcium flow and kidney 25-hydroxyvitamin D3-1-hydroxylase with a periodicity of 56 h and a 9 h phase difference between the two signals. In growing chickens subjected to a light: dark cycle of 22:2 h, and intravenously dosed with 45Ca, the temporal changes in plasma 45Ca could be described by an exponential decline with superimposed diurnal oscillations. The activity of the renal 25-hydroxyvitamin D3-1-hydroxylase in chicks subjected to a 12:12 h light: dark cycle ALSO followed diurnal oscillations, with a nadir at the beginning of the light period and a peak 12 h later. The production of 1,25-dihydroxyvitamin D3 by primary cultures of chicken kidney cells oscillated with a periodicity of 5.6 h or shorter. It is suggested that despite the differences in phase and periodicity between the simulation predictions and actual results, the oscillations in both 1-hydroxylase and bone calcium flow could be coupled through the hormonal systems involved in regulation of plasma calcium.