Abstract: Overexpression of Cu/Zn superoxide dismutase 1 (SOD1) in monocytes blocks reactive oxygen species-induced inhibition of cell growth and apoptosis and renders cells resistant to the toxic effect of tumor necrosis factor (TNF)-alpha, suggesting that TNF-alpha represses the SOD1 gene in these cells. We herein show that TNF-alpha decreases SOD1 mRNA, protein, and promoter activity in U937 cells. Electrophoretic mobility-shift assays (EMSA) show that TNF-alpha decreased binding of three different complexes. Ectopic Sp1 overexpression markedly increased SOD1-basal promoter activity and partially antagonized the TNF-alpha inhibitory effect. In contrast, ectopic c-Jun overexpression mimics TNF-alpha inhibitory effects and antagonizes Sp1 stimulatory effects. In agreement with these findings, EMSA shows a TNF-alpha-induced increase in AP-1 and a decrease in Sp1 DNA binding. Disruption of the C/EBP site decreases, whereas mutation in the Sp1/Egr-1 site completely abolishes DNA-binding and promoter activity. A JNK inhibitor antagonized the negative effects of TNF-alpha on SOD1 promoter activity, suggesting that JNK signaling through c-Jun protein activation is critical for the TNF-alpha-dependent SOD1 repression. A greater understanding of the mechanisms of TNF-alpha-induced SOD1 repression could facilitate the design and development of novel therapeutic drugs for inflammatory conditions.

Abstract: Recent reports in patients with PFIC1 have indicated that a gene defect in ATP8B1 could cause deregulations in bile salt transporters through decreased expression and/or activity of FXR. This study aimed to: (1) define ATP8B1 expression in human hepatobiliary cell types, and (2) determine whether ATP8B1 defect affects gene expressions related to bile secretion in these cells. ATP8B1 expression was detected by RT-PCR in hepatocytes and cholangiocytes isolated from normal human liver and gallbladder. ATP8B1 mRNA levels were 20- and 200-fold higher in bile duct and gallbladder epithelial cells, respectively, than in hepatocytes. RT-PCR analyses of the liver from two patients with PFIC1, one with PFIC2, one with biliary atresia, showed that, compared to normal liver, hepatic expressions of FXR, SHP, CYP7A1, ASBT were decreased at least by 90% in all cholestatic disorders. In contrast, NTCP transcripts were less decreased (by < or = 30% vs. 97%) in PFIC1 as compared with other cholestatic disorders, while BSEP transcripts, in agreement with BSEP immunohistochemical signals, were normal or less decreased (by 50% vs. 97%). CFTR hepatic expression was decreased (by 80%), exclusively in PFIC1, while bile duct mass was not reduced, as ascertained by cytokeratin-19 immunolabeling. In Mz-ChA-2 human biliary epithelial cells, a significant decrease in CFTR expression was associated with ATP8B1 invalidation by siRNA. In conclusion, cholangiocytes are a major site ofATP8B1 hepatobiliary expression. A defect of ATP8B1 along with CFTR downregulation can impair the contribution of these cells to bile secretion, and potentially explain the extrahepatic cystic fibrosis-like manifestations that occur in PFIC1.

Abstract: The role of thyroid hormone [L-3,5,3'-triiodothyronine (T3)] and the thyroid hormone receptor (TR) in regulating growth, development, and metabolic homeostasis is well established. It is also emerging that T3 is associated with oxidative stress through the regulation of the activity of superoxide dismutase-1 (SOD-1), a key enzyme in the metabolism of oxygen free radicals. We found that T3 reverses the activation of the SOD-1 promoter caused by the free radical generators paraquat and phorbol 12-myristate 13-acetate through the direct repression of the SOD-1 promoter by liganded TR. Conversely, the SOD-1 promoter is significantly stimulated by unliganded TRs. This regulation requires the DNA-binding domain of the TR, which is recruited to an inhibitory element between -157 and +17 of the SOD-1 promoter. TR mutations, which abolish recruitment of coactivator proteins, block repression of the SOD-1 promoter. Conversely, a mutation that inhibits corepressor binding to the TR prevents activation. Together, our findings suggest a mechanism of negative regulation in which TR binds to the SOD-1 promoter but coactivator and corepressor binding surfaces have an inverted function. This effect may be important in T3 induction of oxidative stress in thyroid hormone excess.

Abstract: BACKGROUND: There is a substantial clinical overlap between chronic renal failure (CRF) and hypothyroidism, suggesting the presence of hypothyroidism in uremic patients. Although CRF patients have low T3 and T4 levels with normal thyroid-stimulating hormone (TSH), they show a higher prevalence of goiter and evidence for blunted tissue responsiveness to T3 action. However, there are no studies examining whether thyroid hormone receptors (TRs) play a role in thyroid hormone dysfunction in CRF patients. To evaluate the effects of an uremic environment on TR function, we investigated the effect of uremic plasma on TRbeta1 binding to DNA as heterodimers with the retinoid X receptor alpha (RXRalpha) and on T3-dependent transcriptional activity. RESULTS: We demonstrated that uremic plasma collected prior to hemodialysis (Pre-HD) significantly reduced TRbeta1-RXRalpha binding to DNA. Such inhibition was also observed with a vitamin D receptor (VDR) but not with a peroxisome proliferator-activated receptor gamma (PPARgamma). A cell-based assay confirmed this effect where uremic pre-HD ultrafiltrate inhibited the transcriptional activation induced by T3 in U937 cells. In both cases, the inhibitory effects were reversed when the uremic plasma and the uremic ultrafiltrate were collected and used after hemodialysis (Post-HD). CONCLUSION: These results suggest that dialyzable toxins in uremic plasma selectively block the binding of TRbeta1-RXRalpha to DNA and impair T3 transcriptional activity. These findings may explain some features of hypothyroidism and thyroid hormone resistance observed in CRF patients.

Abstract: AIMS: To now, there are no studies reporting whether thyroid hormones (THs) transport play a role in thyroid hormone dysfunction observed in chronic renal failure (CRF). Therefore, the aim of this study was to investigate the transport of THs in erythrocytes from patients with CRF on hemodialysis (HD). METHODS: [125I]-L-triiodothyronine ([125I]T3) and [125I]-L-thyroxine ([125I]T4) erythrocytes uptake was measured at 1 min and 5 min. To study L-triiodothyronine (LT3) and L-thyroxine (LT4) efflux from erythrocytes, we preloaded the cells during 180 min with [125I]T3 or [125I]T4 and measured their [125I]T3 or [125I]T4 efflux during 60 min. RESULTS: [125I]T3 uptake in erythrocytes from uremic patients pre-HD was higher than control subjects by 50% at 1 min and by 55% at 5 min. However, [125I]T4 uptake in erythrocytes from uremic patients was significantly lower at 1min (88%) and at 5 min (63%). LT3 efflux rate was lower and LT4 efflux was significantly higher than in control subjects. After 60-min of efflux, LT3 remained in erythrocytes was 80% higher and LT4 was 57% lower than in normal individuals. Neither [125I]T3 and [125I]T4 uptake, nor efflux rates were changed by hemodialysis. CONCLUSION: Despite the fact that uremic patients on hemodialysis show low serum levels of LT3, changes in LT3 influx and efflux could act as a compensatory mechanism that neutralize thyroid hormone dysfunction in order to maintain the euthyroid state.

Abstract: BACKGROUND: During transport-associated adenosine triphosphate hydrolysis, P-glycoprotein (Pgp) undergoes conformation transitions detected by UIC2, a functional anti-Pgp monoclonal antibody. A newly developed UIC2 shift assay is based on increased UIC2 reactivity in the presence of Pgp substrates. All peripheral blood leukocytes express low Pgp levels. The existing antibody-based detection methods are limited in their sensitivity and require additional techniques to simultaneously analyze Pgp expression and efflux, making it difficult to ascertain the physiologic role of Pgp-mediated transport. METHODS: We validated the UIC2 shift assay against UIC2 immunostaining and DiOC(2) efflux. The UIC2 shift assay was then used to characterize Pgp functional expression and its physiologic substrates in peripheral blood leukocytes. RESULTS: A strong correlation was observed between the UIC2 shift assay versus immunostaining and dye efflux tests. The UIC2 shift assay showed improved sensitivity (compared with conventional UIC2 staining) and allowed for simultaneous detection of Pgp expression and function. Using this assay, we identified several new Pgp substrates, including monensin and retinol, and confirmed that interleukin-2 and interferon-gamma can be transported by Pgp. CONCLUSIONS: Our findings validate the use of the UIC2 shift assay in MDR1 detection and support the idea that Pgp plays a physiologic role in immunoregulation.

Abstract: We characterized T3 efflux in primary cultures of cells derived from human placenta, neonatal rat cardiac myocytes, and rat inner medullary collecting ducts (IMCD). The T3 efflux rate was highest in placenta cells, followed by ventriculocytes, atriocytes, and IMCD cells. Verapamil reversibly blocked [125I]T3 efflux in these cells in a manner that correlated with their T3 efflux rate. Thus, verapamil inhibition of [125I]T3 efflux in placenta cells led to a 432% increase in the [125I]T3 content compared with 33% increase in IMCD cells. Several unlabeled iodothyronines, but not TRIAC, differentially blocked [125I]T3 efflux such as (T4 > T3 > rT3 = D-T3 > D-T4) in placenta cells and (T4 > rT3 = D-T4 = T3 > D-T3) in ventriculocytes, suggesting tissue-specific differences in the carriers/transporters responsible for T3 efflux. This hypothesis draws further support from the fact that D-T3 inhibited [125I]T3 efflux in placenta cells, but not in ventriculocytes. TRIAC did not affect T3 efflux in ventriculocytes or placenta cells, but it greatly inhibited [125I]T3 uptake in these cells, suggesting that [125I]T3 uptake and efflux mechanisms are distinct and appear to be mediated by distinct carrier/transporter proteins. Collectively, these data suggest that differences in thyroid hormone transport in target cells may provide an important mechanism for regulating hormone action in a tissue-specific fashion.

Abstract: Estrogens used in hormone replacement therapy regimens may increase the risk of developing breast cancer. Paradoxically, high consumption of plant-derived phytoestrogens, particularly soybean isoflavones, is associated with a low incidence of breast cancer. To explore the molecular basis for these potential different clinical outcomes, we investigated whether soybean isoflavones elicit distinct transcriptional actions from estrogens. Our results demonstrate that the estrogen 17beta-estradiol effectively triggers the transcriptional activation and repression pathways with both estrogen receptors (ERs) ERalpha and ERbeta. In contrast, soybean isoflavones (genistein, daidzein, and biochanin A) are ERbeta-selective agonists of transcriptional repression and activation at physiological levels. The molecular mechanism for ERbeta selectivity by isoflavones involves their capacity to create an activation function-2 surface of ERbeta that has a greater affinity for coregulators than ERalpha. Phytoestrogens may act as natural selective estrogen receptor modulators that elicit distinct clinical effects from estrogens used for hormone replacement by selectively recruiting coregulatory proteins to ERbeta that trigger transcriptional pathways.

Abstract: The mechanisms responsible for regulating epithelial ATP permeability and purinergic signaling are not well defined. Based on the observations that members of the ATP-binding cassette (ABC)1 family of proteins may contribute to ATP release, the purpose of these studies was to assess whether multidrug resistance-1 (MDR1) proteins are involved in ATP release from HTC hepatoma cells. Using a bioluminescence assay to detect extracellular ATP, increases in cell volume increased ATP release approximately 3-fold. The MDR1 inhibitors cyclosporine A (10 microm) and verapramil (10 microm) inhibited ATP release by 69% and 62%, respectively (p < 0.001). Similarly, in whole-cell patch-clamp recordings, intracellular dialysis with C219 antibodies to inhibit MDR1 decreased ATP-dependent volume-sensitive Cl- current density from -33.1 +/- 12.5 pA/pF to -2.0 +/- 0.3 pA/pF (-80 mV, p < or = 0.02). In contrast, overexpression of MDR1 in NIH 3T3 cells increased ATP release rates. Inhibition of ATP release by Gd3+ had no effect on transport of the MDR1 substrate rhodamine-123; and alteration of MDR1-substrate selectivity by mutation of G185 to V185 had no effect on ATP release. Since the effects of P-glycoproteins on ATP release can be dissociated from P-glycoprotein substrate transport, MDR1 is not likely to function as an ATP channel, but instead serves as a potent regulator of other cellular ATP transport pathways.

Abstract: Export of L-T3 out of the cell is one factor governing the cellular T3 content and response. We previously observed in liver-derived cells that T3 export was inhibited by verapamil, suggesting that it is due to either ATP-binding cassette/multidrug resistance (MDR1/mdr1b) or multidrug resistance-related (MRP1/mrp1) proteins. To test this hypothesis we measured T3 export in FRTL-5, NIH-3T3, and rat hepatoma (HTC) cells that varied in expression of these proteins. FRTL-5 and NIH-3T3 cells were found to contain a T3 efflux mechanism that is verapamil inhibitable, saturable, and stereospecific. By contrast, T3 efflux in HTC cells was slow and unaffected by verapamil. Neither FRTL-5 nor NIH-3T3 cells express mdrlb, but all three cell types express mrpl, as assessed by immunoblotting. Overexpression of MDR1 in NIH-3T3 cells did not enhance verapamil-inhibitable T3 efflux. Photoaffinity labeling of FRTL-5 and NIH-3T3 cells with [125I]L-T3 revealed a labeled 90- to 100-kDa protein that was not present in HTC cells. Verapamil and excess nonradioactive L-T3, but not D-T3, inhibited labeling of this protein. The lack of correlation between T3 efflux and MDR1 and mrpl expression and the finding of a photoaffinity-labeled putative transport protein smaller than MDR1 or mrp1 protein (approximately 170 kDa) suggest that a novel protein is involved in the transport of T3 out of cells.

Abstract: BACKGROUND: Liver regeneration occurs promptly after partial hepatectomy, although the factors regulating this response have not been fully clarified. Molecular events in the regenerative response have been widely characterized after 70% hepatectomy which represents a model of "normal" liver regeneration in rats. More extensive resection results in hepatic failure which has been attributed to a critical loss of hepatic mass. It is not known whether the pattern of genes expressed early in regeneration remains intact after lethal hepatectomy. We hypothesize that the increased expression of selected early response genes remains intact after massive hepatectomy. The aim of this study was to compare the expression of selected genes after 70 and 85% hepatectomy. MATERIALS AND METHODS: One hundred ten Wistar rats were divided into three groups: control group (sham laparotomy) (n = 30), 70% hepatectomy group (n = 40), and 85% hepatectomy group (n = 40). Animals were sacrificed at intervals. Livers were excised and divided into four equal specimens, snap frozen, and stored at -70 degrees C. RNA was extracted by standard methods and preparations were probed for protooncogenes, c-myc, c-fos, and for hepatocyte growth factor, and its receptor, c-met. After overnight exposure of autoradiographs, quantification was accomplished by densitometry of RNA slot blots. RESULTS: After 70% hepatectomy, peaks of maximal expression for both c-myc and c-met were observed after 1 and 12 h. For c-fos, peak of maximal expression was observed at 6 h. For HGF, peak was observed between 12 h and Day 2. After 85% hepatectomy, rats demonstrated similar patterns including peak expression of c-myc at 1 h, but altered peak at 12 h. For c-met, the same pattern was observed between 1 and 12 h. For HGF, two peaks were noted: a first peak at 1 h, and a peak similar to the peak observed after 70% hepatectomy at 12 h. CONCLUSIONS: These results suggest that early molecular events which are part of the regenerative response are largely intact after 85% lethal hepatectomy. We propose that liver dysfunction and the failure of regeneration observed after 85% hepatectomy is not due to alteration of early signaling. Further study will be required to define failure of the regeneration program in this model.

Abstract: Cholestasis, or impaired bile flow, is an important but poorly understood manifestation of liver disease. Two clinically distinct forms of inherited cholestasis, benign recurrent intrahepatic cholestasis (BRIC) and progressive familial intrahepatic cholestasis type 1 (PFIC1), were previously mapped to 18q21. Haplotype analysis narrowed the candidate region for both diseases to the same interval of less than 1 cM, in which we identified a gene mutated in BRIC and PFIC1 patients. This gene (called FIC1) is the first identified human member of a recently described subfamily of P-type ATPases; ATP-dependent aminophospholipid transport is the previously described function of members of this subfamily. FIC1 is expressed in several epithelial tissues and, surprisingly, more strongly in small intestine than in liver. Its protein product is likely to play an essential role in enterohepatic circulation of bile acids; further characterization of FIC1 will facilitate understanding of normal bile formation and cholestasis.

Abstract: BACKGROUND & AIMS: Canalicular secretion is rate limiting in overall blood-to-bile transport of bile acids. Studies using transfected cells have implicated the canalicular ecto-adenosine triphosphatase (ecto-ATPase) in adenosine triphosphate (ATP)-dependent bile acid transport. However, the structural features of this ecto-ATPase are not those anticipated for an in-to-out ATP-dependent transporter. The aim of this study was to explore the possible existence of an ATP-dependent bile acid transport mechanism distinct from ecto-ATPase. METHODS: Bile acid transport activity and ecto-ATPase expression were analyzed in primary rat hepatocytes, rat hepatoma HTC cells, and specially adapted HTC (HTC-R) cells using plasma membrane vesicles and Northern blot, slot blot, ribonuclease protection assay, and Western blot analyses. RESULTS: Plasma membranes isolated from HTC-R cells exhibited ATP-dependent taurocholate transport, which was many-fold greater than that in HTC cells. Hepatocytes showed the highest transport rates. Protein and RNA analyses showed very low expression of ecto-ATPase in HTC and HTC-R cells compared with hepatocytes. There was no difference between the two cell types at both the RNA and protein level. CONCLUSIONS: These findings show the presence in HTC-R cells and, apparently in hepatocytes, of one or more proteins other than the ecto-ATPase that mediate ATP-dependent transport of bile acids.

Abstract: In a model liver cell line, recovery from swelling is mediated by a sensitive autocrine pathway involving conductive release of ATP, P2 receptor stimulation, and opening of membrane Cl- channels (Wang, Y., Roman, R. M., Lidofsky, S. D., and Fitz, J. G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12020-12025). However, the mechanisms coupling changes in cell volume to ATP release are not known. Based on evidence that certain ATP-binding cassette (ABC) proteins may function as ATP channels or channel regulators, we evaluated the potential role of ABC proteins by comparing ATP release and volume regulation in rat HTC and HTC-R hepatoma cells, the latter of which overexpress Mdr proteins. In both cell types, Cl- current activation (ICl-swell) and volume recovery following swelling were dependent on conductive ATP efflux. The rate of volume recovery was approximately 6-fold faster in HTC-R cells compared with HTC cells. This effect is likely due to enhanced ABC protein-dependent ATP release since (i) ICl-swell and cell volume recovery were eliminated by inhibition of P-glycoprotein transport (20 microM verapamil and 15 microM cyclosporin A); (ii) swelling-induced Cl- current density was similar in both cell types (approximately -50 pA/pF; not significant); and (iii) ATP conductance measured by whole-cell techniques was increased approximately 3-fold in HTC-R cells compared with HTC cells. Moreover, HTC-R cells exhibited enhanced survival during hypotonic stress. By modulating ATP release, hepatic ABC proteins may play a key role in the cellular pathways coupling changes in cell volume to ion permeability and secretion.

Abstract:

Abstract: Actions of thyroid hormones (THs) are determined by intracellular free hormone concentration. Here we report that enhanced TH extrusion via a saturable, cold-sensitive mechanism lowers intracellular TH and causes TH resistance in hepatoma cells. Since these cells overexpress multidrug resistance P-glycoproteins and TH extrusion and resistance are blunted by verapamil, P-glycoproteins may mediate this resistance. Verapamil-inhibitable TH efflux was also found in primary hepatocytes, cardiocytes, and fibroblasts. These findings demonstrate that TH extrusion can modulate TH availability and action in mammalian cells.

Abstract: The interest of mammalian biologists in ATP-binding cassette (ABC) proteins is relatively recent. However, ABC proteins are widespread in distribution and have long been known to play an important transport role in prokaryotes. The review includes a brief overview of the structure, regulation, and varied functions of ABC proteins in different cell types as well as a synopsis of the emerging role of ABC proteins in human biology and disease. The review then focuses on the established (canalicular secretion of organic cations by the multidrug resistance, or MDR 1, gene product; ductular secretion of fluid and electrolytes mediated by CFTR), probable (biliary phospholipid secretion by the MDR 2 gene product; secretion of non-bile acid organic anions by the multidrug resistance protein, or MRP), and possible (bile acid secretion; biliary secretion of the signaling molecule, ATP; hormone transport) roles of known and novel ABC proteins in hepatobiliary secretion.

Abstract: Secretion of anionic endo- and xenobiotics is essential for the survival of animal and plant cells; however, the underlying molecular mechanisms remain uncertain. To better understand one such model system--i.e., secretion of bile acids by the liver--we utilized a strategy analogous to that employed to identify the multidrug resistance (mdr) genes. We synthesized the methyl ester of glycocholic acid (GCE), which readily enters cells, where it is hydrolyzed to yield glycocholic acid, a naturally occurring bile acid. The rat hepatoma-derived HTC cell line gradually acquired resistance to GCE concentrations 20-fold higher than those which inhibited growth of naive cells, yet intracellular accumulation of radiolabel in resistant cells exposed to [14C]GCE averaged approximately 25% of that in nonresistant cells. As compared with nonresistant cells, resistant cells also exhibited (i) cross-resistance to colchicine, a known mdr substrate, but not to other noxious substances transported by hepatocytes; (ii) increased abundance on Northern blot of mRNA species up to 7-10 kb recognized by a probe for highly conserved nucleotide-binding domain (NBD) sequences of ATP-binding cassette (ABC) proteins; (iii) increased abundance, as measured by RNase protection assay, of mRNA fragments homologous to a NBD cRNA probe; and (iv) dramatic overexpression, as measured by Western blotting and immunofluorescence, of a group of 150- to 200-kDa plasma membrane proteins recognized by a monoclonal antibody against a region flanking the highly conserved NBD of mdr/P-glycoproteins. Finally, Xenopus laevis oocytes injected with mRNA from resistant cells and incubated with [14C]GCE secreted radiolabel more rapidly than did control oocytes. Enhanced secretion of glycocholic acid in this cell line is associated with overexpression of ABC/mdr-related proteins, some of which are apparently novel and are likely to include a bile acid transport protein.

Abstract:

Abstract: The amino acid sequence of pig liver flavin-containing monooxygenase (PgLFMO) was determined by nucleotide analysis of cDNA clones isolated using synthetic oligonucleotide probes. Most of the clones isolated were missing the 5'-noncoding region and the first seven codons including the putative initiation codon ATG. A full-length cDNA clone, PgLFMO1, was obtained by extending the 5'-end sequence to include an ATG initiation codon and the codons corresponding to the 5'-end using the polymerase chain reaction. Restriction sites for EcoRI and SalI were incorporated at the 5'- and 3'-ends of the cDNA, respectively, by polymerase chain reaction to provide a full-length cDNA clone, pTrcPgLFMO1. Escherichia coli strain NM522 was transformed with pTrcPgLFMO1 and the PgLFMO was expressed under the control of the Trc promoter. Recombinant PgLFMO isolated from the bacterial lysates was purified to homogeneity and found to have N- and S-oxygenation kinetic properties similar to those of the native enzyme. Stereoselective S-oxygenation of (+)- and (-)-4-bromophenyl-1,3-oxathiolane or 2-methyl-1,3-benzodithiole by the expressed PgLFMO showed some differences from that of the native enzyme, however. These data indicate that active PgLFMO was expressed in E. coli but that the enzyme action was distinct from the native enzyme.

Abstract: The cDNA for the adult human liver flavin-containing monooxygenase (form 3) (FMO3) was cloned, sequenced, and expressed in Escherichia coli. The cDNA-expressed FMO3 was used to investigate the regio- and stereoselective N- and S-oxygenation of a number of tertiary amines and sulfides, respectively. For comparison, the N- and S-oxygenation of the same chemicals and drugs were examined with adult human liver microsomes from a normal healthy female donor and FMO1 from pig liver and FMO2 from rabbit lung. Both cDNA-expressed FMO3 and adult human liver microsomes N-oxygenated trifluoperazine or 10-(N,N-dimethylaminoalkyl)-phenothiazines with similar substrate specificities. The substrate specificity for FMO3 differed, however, from that of pig liver FMO1. Nucleophilic sulfur-containing compounds [i.e., thiobenzamide, (4-bromophenyl)-1,3-oxathiolane, and 2-methyl-1,3-benzodithiole] were efficiently S-oxygenated by cDNA-expressed FMO3 and adult human liver microsomes. Stereoselective S-oxygenation of (+)- and (-)-(4-bromophenyl)-1,3-oxathiolane and 2-methyl-1,3-benzodithiole was therefore investigated. In general, the stereoselectivity observed for S-oxygenation in the presence of FMO3 was similar to that observed in the presence of adult human liver microsomes. In most cases examined, however, the stereoselectivity for S-oxygenation was quite distinct from that observed for pig liver FMO1. We conclude that FMO3 is the major form of FMO active in adult human liver. Because the stereoselectivity for X-oxygenation and the substrate specificity for tertiary amine N-oxygenation by cDNA-expressed FMO3 are distinct from those of pig liver FMO1, we conclude that the binding channel for each isoform is quite different.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: The cDNA for a major component of the family of flavin-containing monooxygenases (FMOs) present in adult human liver (i.e., HLFMO-D) has been cloned and expressed in a prokaryotic system. Escherichia coli strain NM522 was transformed with pTrcHLFMO-D, and the HLFMO-D cDNA was expressed under the control of the Trc promoter. A variety of tertiary amine substrates [i.e., chlorpromazine and 10-[(N,N-dimethylamino)alkyl]- 2-(trifluoromethyl)phenothiazines] were efficiently oxygenated by HLFMO-D cDNA expressed in E. coli or by adult human liver microsomes. Approximate dimensions of the substrate binding channel for both adult human liver microsomal FMO and cDNA-expressed HLFMO-D were apparent from an examination of the N-oxygenation of a series of 10-[(N,N-dimethylamino)alkyl]-2-(trifluoromethyl)phenothiazines. The substrate regioselectivity studies suggest that adult human liver FMO form D possesses a distinct substrate specificity compared with form A FMO from animal hepatic sources. It is likely that the substrate specificity observed for cDNA-expressed adult human liver FMO-D may have consequences for the metabolism and distribution of tertiary amines and phosphorus- and sulfur-containing drugs in humans and may provide insight into the physiologic substrate(s) for adult human liver FMO.

Abstract: Complementary DNA (cDNA) clones encoding the adult human liver flavin-containing monooxygenase (FMO; dimethylaniline N-oxidase, EC 1.14.13.8) were isolated from lambda gt10 and lambda gt11 libraries. The cDNA libraries were screened with three synthetic 36-mer oligonucleotide probes derived from the nucleic acid sequence of the pig liver FMO cDNA. The deduced amino acid sequence for the adult human liver FMO was quite distinct from the pig liver FMO, and adult human liver FMO was designated form II (HLFMO II). The full-length cDNA sequence of HLFMO II [2119 base pairs (bp)] had an open reading frame of 1599 nucleotides, which encoded a 533-amino acid protein of Mr 59,179, a 5'-noncoding region of 136 nucleotides and a 3'-noncoding region of 369 nucleotides excluding the poly(A) tail. The deduced amino acid sequence of HLFMO II had 80% similarity with the rabbit liver FMO II but only a 52%, 55%, and 53% amino acid similarity with the rabbit liver (form I), the pig liver (form I), and fetal human liver (form I) FMOs, respectively. RNA analysis of adult human liver RNA showed that there was one HLFMO II mRNA species. Analysis of genomic DNA indicated that HLFMO II was the product of a single gene. These results indicated that the deduced amino acid sequence for HLFMO II contained highly conserved residues and suggested that FMO enzymes were closely related and, undoubtedly, derived from the same ancestral gene.

Abstract: The cellular distribution of calbindin-D 28k mRNA in the rat vestibular ganglion was examined by in situ hybridization. Using a [35S]cDNA probe a neuronal subpopulation expressing calbindin-D 28k mRNA with a strong intensity has been identified. These findings confirm the presence of a subclass of calbindin-immunoreactive neurons in the rat vestibular ganglion.

Abstract: The Calbindins-D (CaBP9K and 28K), like calmodulin, belong to a group of intracellular proteins that bind calcium with high affinity. Each protein is encoded by a separate gene and there is no direct filiation between the two genes. We have demonstrated the tissue-specific expression and regulation of CaBP9K gene. This gene is expressed in the intestine, placenta and uterus of the rat as a single 0.5kb long transcript. Exogenous 1,25(OH)2D3 triggers the rapid synthesis of CaBP9K mRNA and accumulation of translatable CaBP9K mRNA in the duodenum of vitamin D-deficient rats. Calcium also stimulates CaBP9K gene expression in this tissue. In contrast 1,25(OH)2D3 does not change the uterine concentration of CaBP9K but estrogen stimulates the transcription of the CaBP9K gene in the uterus. The promoter region of rat CaBP9K gene contains 1 TATA box and 4 CAAT box-type sequences and several steroid hormone regulatory elements. The CaBP9K gene is therefore a suitable model for studying the tissue-specific regulation of gene expression by steroid hormones.

Abstract:

Abstract: A quantitative study of calbindin-D28K (calcium-binding protein) was carried out on the developing hippocampal formation in normal, hypothyroid, hyperthyroid, and underfed rats. In normal animals, the calbindin-D28K content increased after birth in agreement with the distribution of the protein previously reported by immunocytochemistry. Calbindin-D28K was strikingly spared, compared to the other proteins, from the effects of hypothyroidism. On the contrary, the calbindin-D28K:protein ratio was transiently reduced by hyperthyroidism. Corrective doses of thyroxine to hypothyroid rats increased the calbindin-D28K content whatever the period of the hormonal treatment, but they also had a marked effect on the hippocampal weight and the protein content, especially when the hormone was given on days 2-3. With this latter replacement therapy schedule, the calbindin-D28K:protein ratio dropped from the high value of the hypothyroid animal to normal. Taken together, the results obtained in hypothyroidism, hyperthyroidism and replacement therapy are consistent with a pronounced action of thyroid hormone on hippocampal structures other than those containing calbindin-D28K. Undernutrition, which, like hypo- or hyperthyroidism, also markedly impairs hippocampal growth, affected the calbindin-D28K content per hippocampus but not the calbindin-D28K:protein ratio. This emphasizes the unique influence of thyroid hormone on brain development. The relative preservation of calbindin-D28K in the hippocampal formation of animals lacking thyroid hormone suggests that calbindin-D28K function may be crucial in this brain region.

Abstract: The vitamin D-dependent calcium-binding protein (CaBP), calbindin-D28K (CaBP28K), is present in the central nervous system (CNS), the sensory system, and kidneys of mammals and birds. Recent studies have indicated that several other CaBPs of very similar Mrs are also present in the CNS. This study was carried out to establish the relationship between CaBP28K and other CaBP, particularly spot 35, to provide a basis for further studies on the tissue-specific regulation and distribution of CaBP28K. A cloned pC28 cDNA was isolated from a rat brain expression library using synthetic oligodeoxyribonucleotides (oligos) complementary to rat spot-35 mRNA. This pC28 cDNA had an open reading frame (ORF) of 783 nucleotides (nt) coding for a 261-aa, 30-kDa protein. There was 100% homology between the pC28 sequence and that of the CaBP28K isolated from rat brain cDNA library using a chicken intestinal CaBP28K probe (Hunziker and Schrickel, 1988). Thus the aa and nt sequences of rat CaBP28K and spot 35 are identical. Primer extension studies and Northern analyses show that the major species of CaBP28K mRNA contains a 5'-untranslated region of 132 nt, a coding region of 261 codons and a 3'-untranslated region of 804 nt without the poly(A) tail. The rat CaBP28K probe hybridizes to one major RNA species (1.9 kb) and two minor ones (2.8 and 3.2 kb) in the cerebellum, hippocampus, retina and kidney. This distribution correlates well with the distribution of CaBP28K itself in these organs. Comparison of the genomic organization of the CaBP28K gene with that of other members of the 'EF-hand' CaBP family emphasizes that the CaBP28K gene diverged from the others at the first duplication of the gene encoding one CaBP domain. All the members of the 'EF-hand' gene CaBP family evolved by exon shuffling and specific genomic rearrangements.

Abstract:

Abstract: The evolutionary history of the intracellular calcium-binding protein superfamily is well documented. The members of this gene family are all believed to be derived from a common ancestor, which, itself, was the product of two successive gene duplications. In this study, we have compared and analyzed the structures of the recently described genes coding for these proteins. We propose a series of evolutionary events, which include exon shuffling and intron insertion, that could account for the evolutionary origin of all the members of this superfamily. According to this hypothesis, the ancestral gene, a product of two successive duplications, consisted of at least four exons. Each exon coding for a peptide (a calcium-binding domain) was separated by an intron that had mediated the duplication. Each distinct lineage evolved from this ancestor by genomic rearrangement, with insertion of introns being a prominent feature.

Abstract: The structural organization of the entire rat vitamin-D-dependent calcium-binding protein (9-kDa CaBP) gene was determined by analysis of overlapping genomic clones isolated from a rat genomic library using the rat 9-kDa CaBP cDNA [Desplan C., Heidmann O., Lillie J., Auffray C. and Thomasset M. (1983) J. Biol. Chem. 258, 13502-13505]. These clones together span 30 kbp of rat genomic DNA, with the rat 9-kDa CaBP gene lying in the middle. The 9-kDa CaBP gene is 2.5 kbp long and contains three exons interrupted by two introns. The first exon contains almost the entire 5' untranslated region. The second exon codes for the calcium-binding site I, the third exon codes for site II and the 3' untranslated region. Therefore each of the calcium-binding domains is encoded by single, separate exons. The transcription initiation site was identified by S1 nuclease mapping and primer extension. A consensus sequence TATAAA is localized 31 bp upstream from the cap site and the 'CCAAT-box' lies upstream from the transcription start. Single (AC)25 and (AG)23 repeats are present in the second intron together with an Alu-like sequence. Repetitive elements are present 5 kbp upstream from the cap site and in the 3' flanking region. Comparison of the known rat CaBP sequences (9-kDa CaBP, 28-kDa CaBP, S100 protein) shows that the 9-kDa CaBP is more closely related to the S100 protein than to the 28-kDa CaBP. There is no evidence to indicate that 9-kDa CaBP has arisen from the 28-kDa CaBP.

Abstract: Regulation of the expression of vitamin D-dependent calcium-binding protein (Mr 9000 CaBP) gene by 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was studied in the rat duodenum. In vivo stimulation of Mr 9000 CaBP synthesis was analyzed using a complementary DNA probe and by measuring the rate of Mr 9000 CaBP gene transcription in isolated nuclei (run-on assay). A single 1,25-(OH)2D3 injection (650 pmol/100 g of body weight) induced a 2-fold increase in Mr 9000 CaBP gene transcription within 15 min in the duodenum of vitamin D-deficient rats. RNA synthesis was maximal at 1 h, then decreased until 16 h of postinjection. There was an initial transient accumulation of Mr 9000 CaBP mRNA (from 7 to 15 min), which was followed by a second, significant increase, by 3 h which remained elevated until 16 h. The magnitude and time course of the Mr 9000 CaBP increase was similar to that of its mRNA as early as 1 h after 1,25-(OH)2D3 administration. Mr 9000 CaBP gene transcription was not significantly induced by 1,25-(OH)2D3 in vitamin D-replete rats and no transient accumulation of Mr 9000 CaBP mRNA was observed. Thus, 1,25-(OH)2D3 modulates Mr 9000 CaBP gene expression in at least two ways, a rapid transcriptional stimulation and a post-transcriptional effect preventing degradation of Mr 9000 CaBP transcripts and accounting for their accumulation several hours after the hormone treatment.

Abstract: We investigated the effect of 1 alpha,25-dihydroxyvitamin D3 (1,25 (OH)2 vit D3) on the 3H-thymidine uptake by Balb/c 3T3 cells and by human skin fibroblasts stimulated by normal human serum or by purified PDGF. We found an inhibitory effect of 1,25 (OH)2 vit D3 on the DNA synthesis of Balb/c 3T3 cells grown in the presence of human serum as well as in the presence of PDGF. At 5% human serum this effect is minimal at 10(-12) M 1,25 (OH)2 vit D3 and is maximal at 10(-9) M. On the DNA synthesis of human fibroblasts stimulated by human serum or by PDGF a modulatory effect of 1,25 (OH)2 vit D3 was shown. On these cells the vitamin had a stimulatory effect between 10(-11) and 10(-9) M and an inhibitory effect at very high concentrations (10(-7) M). Our results suggested that the effect of 1,25 (OH)2 vit D3 on fibroblast DNA synthesis could be mediated by interactions with its specific intracellular receptor. 1,25 (OH)2 vit D3 had no any action on the growth of human fibroblasts stimulated by fibroblast growth factor.