Uptake and utilization of lipids by Paramecium tetraurelia.

Uptake rates of a variety of 14C-labeled fatty acids and complex lipids by Paramecium tetraurelia during 48 h of log-phase growth varied. Fatty acid uptake was maximal during lag phase of growth when phagosome (food vacuole) formation was minimal. Food vacuole formation was shown to be suppressed by the presence of exogenous lipids and by starvation. The rates of uptake of lipids were significantly greater than those of small organic compounds such as amino acids, cyclitols, fatty acid precursors and metabolic intermediates. Significant amounts of radioactivity from 14C-labeled fatty acids were metabolized to 14CO2. The uptake rates of different saturated, straight-chain fatty acids of even carbon numbers were different and were not correlated with chain length, results suggesting that the primary mechanism for uptake of these compounds was neither by bulk transport nor simple diffusion and that carrier-mediated processes could possibly be involved.

Uptake and utilization of small organic molecules by Paramecium tetraurelia.

Several methods for the metabolic radiolabeling of proteins in axenically grown Paramecium tetraurelia were examined. Less than 5% of the initial radioactivity from exogenously supplied radiolabeled compounds was incorporated into cellular proteins under all conditions tested. The relatively low uptake rates of 14C-labeled amino acids and other small organic compounds by the cells and the subsequent metabolism of these molecules producing radioactive CO2 under conditions of rapid culture growth suggested that the transport of most or all of these compounds may be limited to bulk transport in solution and possibly accounted for by the volume transported by food vacuoles. Active transport of these molecules against a concentration gradient via carrier-mediated mechanisms was not apparent. The low rate of amino acid uptake explains the inefficient in vivo radiolabeling of cellular proteins by radioactive amino acids.

Mouse dioxin-inducible cytosolic aldehyde dehydrogenase-3: AHD4 cDNA sequence, genetic mapping, and differences in mRNA levels.

We have cloned and sequenced the murine AHD4 cDNA encoding the 'Class 3' cytosolic aldehyde dehydrogenase (ALDH-3c). The cDNA is 1722 bp in length, excluding the poly(A+) tail, and has 5' and 3' nontranslated regions of 174 bp and 186 bp, respectively. AHD4 encodes a protein of 453 amino acids, including the first methionine (M(r) = 50,466). The murine AHD4 protein is 91% and 80% similar to the rat and human ALDH3c proteins, respectively, 64% identical to the rat microsomal ALDH3 protein, and < 28% similar to ALDH 'Class 1' and 'Class 2' proteins. Surprisingly, in contrast to the rat gene that is expressed in both cell cultures and the intact liver, the murine Ahd-4 gene is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) or benzo[a]pyrene in cell cultures but not in liver of the intact adult or newborn mouse. Southern hybridization analysis of mouse DNA probed with the full-length cDNA reveals that the Ahd-4 gene is likely to span less than a total of 15 kb, and was mapped to chromosome (Chr) 11 between the Mgat-1 and Shbg loci by analysis of two multilocus crosses. AHD4 mRNA levels are strikingly elevated in the untreated mouse hepatoma Hepa-1c1c7 mutant line c37 lacking CYP1A1 (aryl hydrocarbon hydroxylase) activity and in the untreated 14CoS/14CoS mouse cell line having a homozygous deletion of about 1.2 cM on Chr 7. Our data suggest that the Ahd-4 gene in murine cell cultures is regulated by three distinct mechanisms: Ah receptor-mediated induction by TCDD or benzo[a]pyrene, CYP1A1 metabolism-dependent repression, and Chr 7-mediated putative derepression.

Mouse dioxin-inducible NAD(P)H: menadione oxidoreductase: NMO1 cDNA sequence and genetic differences in mRNA levels.

We have cloned and sequenced the mouse NMO1 cDNA, which encodes the NAD(P)H:menadione oxidoreductase [also called NAD(P)H:(quinone acceptor) oxidoreductase; quinone reductase; azo dye reductase; DT diaphorase; EC 1.6.99.2]. The cDNA is 1528 bp in length excluding the poly(A+) tail, and has 5' and 3' nontranslated regions of 108 bp and 595 bp, respectively. The deduced protein contains 274 amino acids, including the first methionine (M(r) = 30,959). The mouse NMO1 protein is: 94% similar to the rat NMO1 and 86.5% to the human NMO1 proteins; 49.3% identical to the human NQO2 protein; and < 20% similar to several dozen other proteins in the quinone oxidoreductase superfamily. Southern hybridization analysis of mouse DNA reveals that the Nmo1 gene is likely to span less than a total of 20 kb. The Nmo1 gene is highly inducible by 2,3,7,8,-tetrachlorodibenzo-p-dioxin (dioxin; TCDD) in mouse liver and mouse cell cultures. TCDD inducibility of NMO1 is detectable at 12 and 18 days of gestation, but markedly elevated at 1-3 weeks post partum as compared with the 6- and 12-week-old mouse. NMO1 mRNA levels are strikingly elevated in the untreated mouse hepatoma Hepa-1c1c7 mutant line c37 lacking CYP1A1 (aryl hydrocarbon hydroxylase) activity, and in the untreated 14CoS/14CoS mouse cell line having an 'oxidative stress response' caused by homozygous deletion of about 3800 kb on chromosome 7. Previous work and the data in this report show that the murine Nmo1 gene is regulated by three distinct mechanisms: CYP1A1 metabolism-dependent repression, Ah receptor-mediated induction by TCDD, and activation by the chromosome 7-mediated oxidative stress response.

Mouse cytosolic class 3 aldehyde dehydrogenase (Aldh3a1): gene structure and regulation of constitutive and dioxin-inducible expression.

The mouse cytosolic aldehyde dehydrogenase ALDH3A1 (encoded by the Aldh3a1 gene) has previously been shown in cell culture to be markedly inducible by 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD; dioxin), downregulated by the metabolism of functional CYP1A1/1A2 enzymes, and upregulated by a gene on Chr 7 that leads to endogenous oxidative stress. In order to study the regulation of Aldh3a1 gene expression, we isolated two overlapping genomic sequences from a B6/CBA mouse genomic library that included the entire Aldh3a1 gene, along with considerable 5' and 3' flanking sequences. The Aldh3a1 gene was shown to span approximately 10 kb and comprise 11 exons including a noncoding first exon. The sequence of 3.18 kb upstream of exon 1 reveals numerous consensus transcription factor-binding sites, some of which were shown to be important in the positive and negative control of Aldh3a1 gene expression; these include seven aromatic hydrocarbon response elements (AHREs), an electrophile response element (EPRE), and AP-1, C/EBP beta, c/EBP alpha, NF-kappaB, Sp1, and NF-1 putative binding sites. Deletion fusion constructs containing regions of the Aldh3a1 gene 5' flanking sequence, ligated to chloramphenicol experiments suggested that the 5' flanking region of the gene contains a strong promoter, at least four functional AHREs appear to act cooperatively in causing dioxin-mediated upregulation, and a putative negative regulatory element (NRE) controls basal gene expression independent of dioxin inducibility. The dioxin-mediated upregulation of Aldh3a1 expression in mouse hepatoma Hepa-1c1c7 cell cultures was shown to depend exclusively on the aromatic hydrocarbon receptor. acetyltransferase (CAT) or luciferase (LUC) reporter genes, were studied. Transient transfection experiments suggested that the 5' flanking region of the gene contains a strong promoter, at least four functional AHREs appear to act cooperatively in causing dioxin-mediated upregulation, and a putative negative regulatory element (NRE) controls basal gene expression independent of dioxin inducibility. The dioxin-mediated upregulation of Aldh3a1 expression in mouse hepatoma Hepa-1c1c7 cell cultures was shown to depend exclusively on the aromatic hydrocarbon receptor.

Extrahepatic expression of NAD(P)H:menadione oxidoreductase, UDP glucuronosyltransferase-1A6, microsomal aldehyde dehydrogenase, and hepatic nuclear factor-1 alpha mRNAs in ch/ch and 14CoS/14CoS mice.

Oxidative stress-induced gene expression in liver of the untreated newborn c14CoS/c14CoS mouse, as compared with that in the cch/cch wild-type mouse, appears to be caused by homozygous loss of the fumarylacetoacetate hydrolase (Fah) gene on Chr 7 and absence of the FAH enzyme, which leads to increased levels of endogenous reactive oxygenated metabolites (ROMs) formed in the tyrosine degradative pathway. In these mice almost all studies to date have been carried out in liver. We have examined the extrahepatic expression of four genes. Two genes are members of the [Ah] battery and induced by ROM-mediated oxidative stress: NAD(P)H:menadione oxidoreductase (Nmo1) and UDP glucuronosyltransferase-1A6 (Ugt1a6). The other two genes are decreased in the livers of 14CoS/ 14CoS mice as compared with that in ch/ch mice: microsomal aldehyde dehydrogenase (Ahd3) and hepatocyte-specific nuclear factor-1 alpha HNF-1 alpha (Hnf1 alpha). In liver plus nine extrahepatic tissues of untreated newborn 14CoS/14CoS mutant and ch/ch wild-type mice, we compared NMO1, UGT1A6, AHD3 and HNF-1 alpha mRNA levels. Our results show a wide variation in extrahepatic tissue-specific expression of all four transcripts and indicate that numerous differences exist in the extrahepatic expression of these genes between 14CoS/14CoS and ch/ch mice.

The kinetics of fatty acid uptake by Paramecium tetraurelia.

The kinetics of radiolabeled fatty acid uptake by the ciliate Paramecium tetraurelia was examined on a homologous series of saturated, straight chain fatty acids of even carbon numbers. Uptake rates increased with chain length from acetate to palmitate. Saturation kinetics was demonstrated for most fatty acids examined, thus ruling out simple diffusion as the major mechanism for fatty acid transport and implicating carrier-mediated, facilitated transport as the major mechanism. Data from most competitive inhibition experiments were too scattered to determine the number of transporter systems present. Cholesterol uptake also exhibited saturation kinetics and hence other sterols, which can satisfy this nutritional requirement, may also be transported by a carrier-mediated mechanism. The uptake of the essential fatty acid oleate was faster than those observed for the saturated acids and could not be explained by only one transport mechanism. Therefore, fatty acid transport also occurs via other kinetically significant routes.

Sustained food vacuole formation by axenic Paramecium tetraurelia and the inhibition of membrane recycling by Alcian Blue.

It is believed that the uptake mechanism of some nutrients by Paramecium tetraurelia primarily involves transport through the cell surface, whereas the uptake of other compounds appears to be restricted to bulk transport during food vacuole (phagosome) formation. In this study, we established that, in axenically grown cells, food vacuole formation occurred at continuous rates over long periods. This information allows quantitation of the volume of media taken up by bulk transport. India ink and latex beads were shown to be inert food vacuole markers and carmine was found to have an initial stimulatory effect on phagosome formation rates. Cultures grown for 3.5 h or longer with the glycocalyx stain Alcian Blue, contained only three phagosomes/cell, whereas cells cultured with the other markers contained 15 phagosomes/cell. Electron microscopy of fecal material that accumulated at the bottom of Alcian Blue-grown cells demonstrated the presence of membranes, suggesting that the vacuolar membrane was eliminated during defecation. Neither cell lysis nor the formation of autophagous vacuoles was detected in Alcian Blue-grown cells, indicating that the stain was not cytotoxic at the concentrations used. Thus it appeared that the binding of Alcian Blue to the digestive vacuole membrane resulted in a loss of the vacuole membranes from the cell which reduced the amount of membranes retrieved and recycled and hence eventually reduced the rate of phagosome formation. Alcian Blue-treated cultures exhibited decreased rate of growth and final density, which is consistent with a decrease in bulk transport of nutrients resulting from reduced membranes of digestive cycle organelles available in the cell.