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J A Silverman

Publications and source records attributed to J A Silverman.

At least 37 records · Page 2Linked to original sources
PubMed1997

Hepatic canalicular membrane 4: expression of the multidrug resistance genes in the liver.

The liver is presented with a diverse set of nutrients, endogenous metabolites, and xenobiotics it must process for movement to their correct physiologic destinations. These compounds are transported by specific mechanisms that move their substrates, often against a concentration gradient. Several hepatic transporters have been identified such as the multispecific anion transporter, cMOAT, bile acid transporters, ion-motive ATPases, glutathione transporters, purine transporters, and the multidrug resistance-related protein, MRP. This review focuses on the hepatic regulation of the multidrug resistance genes that encode the P-glycoprotein transporters. P-glycoproteins are ATP-dependent integral membrane proteins that have diverse functions such as conferring resistance toward chemotherapeutic drugs and phospholipid movement. The expression of the multidrug resistance genes is regulated in a tissue-specific pattern and can be induced by exposure to chemotherapeutic drugs as well as cytotoxic xenobiotics. The specific molecular mechanisms that govern expression of these genes in normal and neoplastic cells are currently being unraveled.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1997

Characterization of anti-Toxoplasma activity of SDZ 215-918, a cyclosporin derivative lacking immunosuppressive and peptidyl-prolyl-isomerase-inhibiting activity: possible role of a P glycoprotein in Toxoplasma physiology.

The immunosuppressive agent cyclosporin A (CsA) also possesses broad-spectrum antimicrobial activity. Previous investigators have reported that the obligate intracellular protozoan Toxoplasma gondii is sensitive to CsA. We have measured the sensitivity of Toxoplasma to 26 CsA derivatives that maintain only a subset of the parent compound's activity. We identified one compound, SDZ 215-918, that is a particularly potent inhibitor of parasite invasion and replication, with a 50% inhibitory concentration of 0.45 microg/ml, which is 10-fold lower than that of CsA. Kinetic studies demonstrate that activity has a rapid onset (half-life, < or = 20 min) and is initially reversible, although long-term exposure (> 24 h) to 5 microg/ml is lethal; in contrast, this concentration had no effect on host cell protein synthesis or cell division. SDZ 215-918 acts directly on the parasite, as demonstrated by inhibition of macromolecular synthesis in host-free extracellular parasites. Inhibition of invasion is due to a reduction in parasite motility. SDZ 215-918 does not bind to cyclophilins, the ubiquitous cyclosporin-binding proteins, but is a potent inhibitor of the mammalian P glycoprotein, a member of the ATP binding cassette transporter superfamily and the pump responsible for multidrug resistance in cancer and parasite cell lines. SDZ 215-918 blocks the efflux of rhodamine 123 from extracellular parasites, consistent with inhibition of a P glycoprotein-like pump. We suggest that a P glycoprotein or a related transporter plays a crucial role in the biology of Toxoplasma and may be a novel target for antiparasitic compounds. Preliminary studies with animals indicate that SDZ 215-918 inhibits parasite growth in vivo; its relationship to CsA may make it suitable for clinical development.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Prolactin increases ATP-dependent taurocholate transport in canalicular plasma membrane from rat liver.

The taurocholate (TC) maximal secretory rate (SRm) in the isolated perfused liver is increased in postpartum rats and ovariectomized rats treated with ovine prolactin (oPRL). The present studies were designed to characterize the mechanism(s) by which oPRL increases TC transport in the liver. oPRL (300 micrograms/day i.v. for 7 days) increased the SRm 1.6-fold from 185 to 364 nmol.min-1.mg protein-1 in the perfused rat liver and the maximal rate of transport for ATP-dependent transport 1.7-fold from 66 to 109 nmol.min-1.mg protein-1 in canalicular liver plasma membrane (cLPM) vesicles without changing the Michaelis constant (5-6 microM). The oPRL-mediated increases in biliary excretion in the perfused liver and ATP-dependent TC transport in cLPM vesicles were significantly inhibited by cycloheximide treatment (2 mg/kg). oPRL (300 micrograms/day iv for 7 days) increased expression of Ca(2+)-Mg(2+)-ecto-adenosinetriphosphatase mRNA sixfold and increased protein expression two- to threefold, but had no effect on the expression of P-glycoprotein (mdr1b and mdr2) mRNA. Thus the increase in ATP-dependent transport in cLPM vesicles due to oPRL treatment accounts for the increased TC SRm in the perfused liver. The oPRL-mediated increased TC transport may be associated with increased expression of proteins related to bile acid transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Multidrug resistance gene expression in rodents and rodent hepatocytes treated with mitoxantrone.

Overexpression of P-glycoprotein in tumor cells can represent a severe drawback for cancer chemotherapy. P-glycoprotein acts as an efflux transporter for a variety of chemotherapeutic agents. It is encoded by multidrug resistance (mdr) genes of the subfamily 1 in humans (MDR1) and rodents (mdr1a and 1b). Because mdr1 gene expression is inducible in cultured rat hepatocytes and in rat liver with chemical carcinogens such as 2-acetylaminofluorene or aflatoxin B1, which form DNA-binding electrophiles during their metabolism, we investigated whether the DNA-damaging chemotherapeutic drug mitoxantrone may induce multidrug resistance in rodents and in hepatocytes in primary culture. In H4IIE rat hepatoma cells stably transfected with a luciferase construct containing the rat mdr1b promoter, mitoxantrone caused a concentration-dependent increase in promoter activity. Mdr1 gene expression in cultured rat hepatocytes was enhanced at mitoxantrone concentrations greater than or equal to 0.1 microM and in mouse hepatocytes at 5 microM. In hepatocytes from both species, a correlation was found between mdr1 induction and the inhibition of protein synthesis. In vivo, mitoxantrone was a very powerful inducer of mdr1 gene expression in rat liver and small intestine. In rat kidney, induction of mRNA was lower, and a marginal effect was seen in lung. In contrast with rats, no significant induction of mdr1 gene expression was obtained in mouse liver. Probably as a consequence of inhibition of protein synthesis, mitoxantrone did not lead to a pronounced elevation of P-glycoprotein levels in rat liver and kidney.

Animals↗
PubMed1996

Characterization of the rat mdr2 promoter and its regulation by the transcription factor Sp1.

The mdr2 gene encodes a P-glycoprotein that transports phospholipids across the canalicular membrane in hepatocytes. In this report we describe the isolation, sequencing and first functional characterization of the promoter of mdr2. Analysis of 1.6 kb of DNA upstream of the initiation of translation revealed that this sequence has a high GC content, lacks a TATA element and contains a number of putative transcription factor binding sites. We observed that transcription initiates at several sites between -290 and -463 and that this region was critical for promoter activity. Gel mobility shift assays indicated that Sp1 protein binds to a Sp1 consensus site located at -263. Co-expression of Sp1 protein with a reporter construct containing the -263 GC box demonstrated that Sp1 regulates transcription of this promoter. Expression of a non-functional Sp1 protein did not increase transcription from the mdr2 promoter. Mutation of the -263 GC box diminished the response of the promoter to Sp1 protein. Mutation of this site also decreased expression of this promoter in cells which normally express this gene. These data show that Spl has a role in the regulation of mdr2 expression.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1996

Experimental Mallory body formation is accompanied by modulation of the expression of multidrug-resistance genes and their products.

Mallory bodies (MBs) are characteristic morphological features of alcoholic hepatitis and are also found in other chronic liver disorders and hepatocellular neoplasms. MBs can be produced in mouse liver by chronic administration of the porphyrinogenic drugs griseofulvin (GF) and 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC). The mechanisms causing the formation of MBs are poorly understood, and the significance of MB formation during the course of liver disease remains unclear. We investigated the relationship between the mechanisms underlying the formation of MBs and the regulation of multidrug resistance (mdr) genes and their products, the P-glycoproteins (Pgp). Immunofluorescence microscopy using the monoclonal antibody C219 revealed an increase of Pgp expression in almost all hepatocytes after 3 to 8 days of feeding mice DDC- and GF-containing diets. However, after approximately 4 weeks of DDC and approximately 8 weeks of GF feeding, when the first small MBs appeared and loosening and diminution of keratin intermediate filament (KIF) cytoskeleton occurred in some hepatocytes, a decrease or loss of Pgp staining in affected hepatocytes was observed. After feeding mice DDC for 6 weeks and GF for 12 weeks, many hepatocytes contained MBs and displayed a disruption of the immunohistochemically demonstrable KIF meshwork. Double immunofluorescence microscopy with the keratin polyclonal antibody and the mab C219 at this time point revealed a complete loss of Pgp staining in affected cells, although remaining hepatocytes with unaltered KIF meshwork showed a strong reaction with the C219 antibody. Northern blot analyses revealed a significant increase of mdr2 mRNA and, to a lesser extent, of mdr1a mRNA in the livers of DDC- and GF-fed animals.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Regulation of mdr1b gene expression in Fischer, Wistar and Sprague-Dawley rats in vivo and in vitro.

Administration of 2-acetylaminofluorene (2-AAF) to rats increased mdr1b expression in Fischer, Wistar and Sprague-Dawley rat livers; however, the response was much smaller in Sprague-Dawley livers. To investigate the basis of this difference we further examined the regulation of the mdr1b gene in hepatocytes isolated from Fischer, Sprague-Dawley and Wistar rats. A time-dependent increase in basal expression of mdr1b but not mdr2 was observed in hepatocytes isolated from all three strains of rats. After 4 days in culture, a larger increase in mdr1b mRNA levels was observed in Fischer and Wistar rat hepatocytes (3.5- and 4.6-fold respectively) than Sprague-Dawley hepatocytes (2-fold). Treatment of primary hepatocytes with 2-AAF caused an induction of mdr1b expression that varied among the three strains. Notably, Sprague-Dawley hepatocytes were not responsive to 2-AAF. In contrast to the parent compound, the electrophilic metabolites N-hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene caused a dose-dependent induction of mdr1b expression in both Fischer and Sprague-Dawley hepatocytes, indicating that differences in the metabolic activation of 2-AAF between the strains may account for the differences in mdr1b by 2-AAF. Hepatocytes isolated from all three strains of rats showed an equivalent induction of mdr1b after treatment with cycloheximide. Nuclear run-on assays demonstrated that the increases in mdr1b expression with time in culture and after xenobiotic treatment were due to increased transcription.

2-Acetylaminofluorene↗
PubMed1996

PCO2 affects tracheal tone during apnea in anesthetized dogs.

We hypothesized that CO2, like hypoxia and withdrawal of pulmonary slowly adapting receptor input, would cause tracheal constriction during neural apnea (absence of phrenic activity). In seven anesthetized paralyzed dogs ventilated to neural apnea, we increased arterial PCO2 (PaCO2) in steps by adding CO2 to the inspirate while keeping ventilation constant. Increases in PaCO2 caused tracheal constriction during neural apnea in all dogs; 69 +/- 26 (SD)% of the change in tracheal diameter occurred during neural apnea. Average sensitivity of tracheal diameter to CO2 was 0.44 mm/Torr PaCO2. Our data suggest that central chemoreceptor inputs to brain stem neurons controlling smooth muscle of the extrathoracic airway bypass central mechanisms generating inspiration.

Animals↗
PubMed1995

Characterization of the basal and carcinogen regulatory elements of the rat mdr1b promoter.

In this report we characterized the transcriptional regulation of the rat mdr1b gene by xenobiotics. The expression of this gene was increased in primary rat hepatocytes and in the H4-II-E hepatoma cell line by exposure to carcinogens such as aflatoxin B1, N-acetoxy-2-acetylaminofluorene, and methyl methanesulfonate. Nuclear run-on experiments indicated that the higher steady-state levels of mdr1b mRNA were due to an increase in transcription. The 5'-flanking region of the mdr1b gene was isolated, sequenced, and functionally characterized in transient and stable transfection assays. A single transcription start site was identified for this gene; no alternate start sites were used after induction with aflatoxin B1. Deletion analysis of this promoter demonstrated that the sequence between nt -214 and -178 was critical for basal promoter activity. This region did not contain any consensus-binding sites for previously identified transcription factors. A negative regulatory region was also identified between nt -940 and -250. No specific carcinogen-responsive element was identified; the xenobiotic response required a large part of the promoter. These data suggest that the carcinogen induction of mdr1b expression is mediated through sequences that overlap or that are identical to the basal promoter element.

Animals↗
PubMed1995

Dynamic measurement of tracheal diameter in dogs.

We describe and validate a new minimally invasive method for continuous measurement of tracheal diameter in anesthetized dogs. The method is based on measuring displacement of water into and out of a modified endotracheal tube cuff placed in the trachea. The system was calibrated to allow tracheal diameter to be calculated from known cuff volume. The resolution of the method in measuring changes in tracheal diameter is 0.1 mm over a range of approximately 10-25 mm. The apparatus was tested in five dogs by observing the response of the trachea to four stimuli previously shown to alter tracheal tone: stimulation of nasal mucosa, hyperinflation of the lungs, induction of hypocapnea, and infusion of atropine. The observed changes in tracheal diameter were generally consistent with those of previous studies. The direction and extent of changes in tracheal diameter in response to the test conditions were confirmed by fluoroscopy. We conclude that continuous measurement of volume changes in the cuff reflects corresponding changes in tracheal diameter.

Animals↗
PubMed1995

Divergent regulation of the class II P-glycoprotein gene in primary cultures of hepatocytes versus H35 hepatoma by glucocorticoids.

We investigated whether the glucocorticoid-mediated mechanisms controlling P-glycoprotein (pgp2 or mdr1b) are similar in normal hepatocytes compared with the H35 hepatoma cell line. In primary rat hepatocytes, dexamethasone (DEX) caused a dose- and time-dependent decrease in the amount of the pgp2 mRNA, which correlated with functional pgp2 expression (intracellular accumulation of [3H]vincristine). The suppression of pgp2 mRNA was specific for glucocorticoids because a representative estrogen and progestin were without effect, and DEX suppression of pgp2 mRNA could be reversed by cotreatment with an anti-glucocorticoid. DEX suppression of pgp2 mRNA appears to be posttranscriptional because following actinomycin D inhibition of new RNA synthesis, the pgp2 transcript disappeared at a faster rate in DEX treated versus untreated hepatocytes. Moreover, transcriptional activity of chloramphenicol acetyltransferase plasmids containing the pgp2 promoter in primary rat hepatocytes was unaffected by DEX treatment. Thus, suppression of pgp2 mRNA by glucocorticoids in primary hepatocytes is due to a decrease in pgp2 mRNA stability. In contrast, in the H35 hepatoma cell line, DEX dose dependently increased pgp protein and pgp2 mRNA, effects which parallel transcriptional activation of the pgp2 promoter. Activation of the pgp2 promoter was specific for glucocorticoids since a representative estrogen had no significant effect on transcription of the pgp2 promoter and RU486 blocked DEX activation of pgp2 transcription. Transcriptional activation of the pgp2 promoter was not due to a global up-regulation of basal transcription factors because DEX treatment did not activate either a herpes simplex virus thymidine kinase promoter or the SV40 early gene promoter. Further studies with a panel of pgp2 5' sequence deletion plasmids revealed that the minimal promoter (-66 bp) was not activated by DEX. In contrast, inclusion of sequences up to -177 bp restored DEX-dependent transcriptional activation. These are the first studies to demonstrate that glucocorticoids regulate pgp2 by different mechanisms in normal rat hepatocytes compared to the H35 hepatoma cell line.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Regulation and function of the multidrug resistance genes in liver.

The P-glycoproteins are integral membrane proteins that function as ATP-dependent transporters. The multidrug resistance genes which encode P-gp comprise a small gene family, with 2 members in humans and 3 in rodents. The P-gp encoded by the mdr1 gene functions as a drug efflux pump to remove drugs from cells and may serve as a barrier to protect cells from cytotoxic agents. In normal tissues, P-gp is localized on the luminal surface of transporting epithelia in the liver, kidney, small intestine, testes, and blood-brain barrier. Transient exposure to drugs transcriptionally increases the level of expression of the mdr1 genes, however, the cellular pathways critical to this regulation are yet unknown. This observation may have some implications on the level of expression in tumors and response to chemotherapy. Examination of the basal level of MDR expression in tumors may not be a reliable predictor of the effect of P-gp on chemotherapy. Induction of MDR transcription by drugs may further impede the effectiveness of anti-cancer agents. This is most obvious for drugs which are substrates for P-gp transport, however, it also applies significantly to compounds which are not themselves substrates but affect the response to other drugs simultaneously or subsequently administered. A clear understanding of the mechanisms that regulate basal and drug-induced mdr transcription will facilitate development of novel agents which circumvent this obstacle or permit targeted modification of mdr expression. Expressed on the bile canalicular surface of the liver, P-gps represent the first ATP-dependent biliary transporters to be characterized. The P-gp encoded by mdr2 is the major form of P-gp expressed in normal liver and transports phospholipids into bile. A defect in this protein leads to severe liver disease caused by chronic inflammation of the biliary system that results from high concentrations of free bile salts. The cellular origin and molecular basis of the ensuing liver tumors in these mice are unclear. It is possible that the chronic damage to the biliary ductules causes an increased growth rate of the surrounding cells, including putative stem cells in the liver. Thus, these mice may serve as a model for carcinogenesis in which the liver is under constant promotion placing the proliferating cells at increased risk to further genetic alterations or expansion of preexisting, but normally quiescent, mutations. Mdr2-deficient animals may also provide a model for human chronic inflammatory liver disease. Clearly, these exciting results indicate that further characterization of the P-gps as normal physiologic canalicular membrane transporters is necessary.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Mutational analysis of the helical hairpin region of diphtheria toxin transmembrane domain.

Entry of the catalytic domain of diphtheria toxin into the cytoplasma of eukaryotic cells depends on insertion of the T (transmembrane) domain into the endosomal membrane, a process triggered by low pH. To probe the mechanism of insertion, we mutated ionizable residues within the helical hairpin region of the T domain. Only three mutations caused significant effects on cytotoxicity, D295K, E349K, and D352K. Each of these represents a substitution of a basic for an acidic residue at the tip of a helical hairpin. Substitution of Lys for Glu349 or Asp352, in the TH8/9 hairpin, reduced toxicity for Vero cells > 100-fold, whereas a Lys substitution for Asp295, one of 3 acidic residues in the TH5/6/7 hairpin, caused a less marked reduction. All three mutations also altered the pH-dependent formation, and/or ion conductance, of channels formed by the toxin in artificial bilayers or the plasma membrane. E349K or D352K did not alter the pH dependence of conformational changes in the toxin occurring near pH 5. Our findings support the hypothesis that the TH8/9 hairpin inserts into the endosomal membrane after low pH-mediated partial unfolding of the T domain. A positive residue at the tip of this hairpin apparently inhibits insertion and blocks toxin action. The ion-conducting properties of channels formed by selected mutants, described elsewhere, are consistent with this model. The status of the TH5/6/7 hairpin in the integral membrane form of the T domain remains uncertain.

Amino Acid Sequence↗
PubMed1994

Structure-function relationships in diphtheria toxin channels: I. Determining a minimal channel-forming domain.

Diphtheria Toxin (DT) is a 535 amino acid exotoxin, whose active form consists of two polypeptide chains linked by an interchain disulphide bond. DT's N-terminal A fragment kills cells by enzymatically inactivating their protein synthetic machinery; its C-terminal B chain is required for the binding of toxin to sensitive cells and for the translocation of the A fragment into the cytosol. This B fragment, consisting of its N-terminal T domain (amino acids 191-386) and its C-terminal R domain (amino acids 387-535) is responsible for the ion-conducting channels formed by DT in lipid bilayers and cellular plasma membranes. To further delineate the channel-forming region of DT, we studied channels formed by deletion mutants of DT in lipid bilayer membranes under several pH conditions. Channels formed by mutants containing only the T domain (i.e., lacking the A fragment and/or the R domain), as well as those formed by mutants replacing the R domain with Interleukin-2 (IL-2), have single channel conductances and selectivities essentially identical to those of channels formed by wild-type DT. Furthermore, deleting the N-terminal 118 amino acids of the T domain also has minimal effect on the single channel conductance and selectivity of the mutant channels. Together, these data identify a 61 amino acid stretch of the T domain, corresponding to the region which includes alpha-helices TH8 and TH9 in the crystal structure of DT, as the channel-forming region of the toxin.

Amino Acid Sequence↗
PubMed1994

Structure function relationships in diphtheria toxin channels: II. A residue responsible for the channel's dependence on trans pH.

Ion-conducting channels formed in lipid bilayers by diphtheria toxin are highly pH dependent. Among other properties, the channel's single channel conductance and selectivity depend on proton concentrations on either side of the membrane. We have previously shown that a 61 amino acid fragment of DT is sufficient to form a channel having the same pH-dependent single channel properties as that of the intact toxin. This region corresponds to an alpha-helical hairpin in the recently published crystal structure of DT in solution; the hairpin contains two alpha-helices, each long enough to span a membrane, connected by a loop of about nine residues. This paper reports on the single channel effects of mutations which alter the two negatively charged residues in this loop. Changing Glutamate 349 to neutral glutamine or to positive lysine has no effect on the DT channel's single channel conductance or selectivity. In contrast, mutations of Aspartate 352 to neutral asparagine (DT-D352N) or positive lysine (DT-D352K) cause progressive reductions in single channel conductance at pH 5.3 cis/7.2 trans (in 1 M KCl), consistent with this group interacting electrostatically with ions in the channel. The cation selectivity of these mutant channels is also reduced from that of wild-type channels, a direction consistent with residue 352 influencing permeant ions via electrostatic forces. When both sides of the membrane are at pH 4, the conductance difference between wild-type and DT-D352N channels is minimal, suggesting that Asp 352 (in the wild type) is neutral at this pH. Differences observed between wild-type and DT-D352N channels at pH 4.0 cis/7.2 trans (with a high concentration of permeant buffer in the cis compartment) imply that residue 352 is on or near the trans side of the membrane. Comparing the conductances of wild-type and DT-D352K channels at large (cis) positive voltages supports this conclusion. The trans location of position 352 severely constrains the number of possible membrane topologies for this region.

Diphtheria Toxin↗