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Biomedical subjects

J A Silverman

Publications and source records attributed to J A Silverman.

At least 19 recordsLinked to original sources
PubMed2002

The development of marked elevation in white blood cell count does not predict inferior outcome in chronic lymphocytic leukemia.

Although elevation of the white blood cell (WBC) count at diagnosis of chronic lymphocytic leukemia (CLL) appears to predict shortened survival, its significance later in the course of the disease remains unclear. We reviewed all cases of CLL seen in our center between 1980 and 1999 to evaluate the frequency and clinical significance of WBC elevation > 100 x 10(9)/L. CLL was confirmed according to standard diagnostic criteria and data was collected from diagnosis, occurrence of WBC > 100 x 10(9)/L, and last follow-up. 235 consecutive patients with CLL were identified; 94 were excluded. 141 included patients had a median age of 61 years and median WBC 19.7 x 10(9)/L at diagnosis. Median follow-up for all patients was 56 months, and median survival was 104 months. 41 patients (29%) had > or = 1 episode of WBC > 100 x 10(9)1/L, occurring at a median of 38 months from diagnosis. Compared to controls matched for modified Rai stage, development of a WBC > 100 x 10(9)/L did not predict inferior survival (median 107 vs. 101 months, p = 0.72). We conclude that the occurrence of a WBC count > 100 x 10(9)/L in patients with CLL does not shorten the survival, and patients require therapy only if other indications for treatment are present.

Actuarial Analysis↗
PubMed2001

Reverse engineering the (beta/alpha )8 barrel fold.

The (beta/alpha)(8) barrel is the most commonly occurring fold among protein catalysts. To lay a groundwork for engineering novel barrel proteins, we investigated the amino acid sequence restrictions at 182 structural positions of the prototypical (beta/alpha)(8) barrel enzyme triosephosphate isomerase. Using combinatorial mutagenesis and functional selection, we find that turn sequences, alpha-helix capping and stop motifs, and residues that pack the interface between beta-strands and alpha-helices are highly mutable. Conversely, any mutation of residues in the central core of the beta-barrel, beta-strand stop motifs, and a single buried salt bridge between amino acids R189 and D227 substantially reduces catalytic activity. Four positions are effectively immutable: conservative single substitutions at these four positions prevent the mutant protein from complementing a triosephosphate isomerase knockout in Escherichia coli. At 142 of the 182 positions, mutation to at least one amino acid of a seven-letter amino acid alphabet produces a triosephosphate isomerase with wild-type activity. Consequently, it seems likely that (beta/alpha)(8) barrel structures can be encoded with a subset of the 20 amino acids. Such simplification would greatly decrease the computational burden of (beta/alpha)(8) barrel design.

Conserved Sequence↗
PubMed2001

Resistance studies with daptomycin.

We studied the in vitro emergence of resistance to daptomycin using three methods: spontaneous resistance incidence, serial passage in the presence of increasing drug concentrations, and chemical mutagenesis. No spontaneously resistant mutants were obtained for any organism tested (<10(-10) for Staphylococcus aureus, <10(-9) for Staphylococcus epidermidis, <10(-9) for Enterococcus faecalis, <10(-9) for Enterococcus faecium, <10(-8) for Streptococcus pneumoniae). Population analysis demonstrated that bacterial susceptibility to daptomycin is heterogeneous. Assay results were sensitive to calcium concentration and culture density, both of which can affect apparent resistance rates. Stable S. aureus mutants were isolated by both serial passage in liquid media and chemical mutagenesis. The daptomycin MICs for these isolates were 8- to 32-fold higher than for the parental strain. Many mutants with high MICs (>12.5 microg/ml) had significant growth defects but did not display phenotypes typical of S. aureus small colony variants. The voltage component (Delta psi) of the bacterial membrane potential was increased in three independent resistant isolates. In vivo data showed that some daptomycin-resistant mutants had lost significant virulence. For other mutants, the degree of in vitro resistance was greater than the change in in vivo susceptibility. These results suggest that infection with some daptomycin-resistant organisms may still be easily treatable.

Animals↗
PubMed2000

Reduced growth rate accompanied by aberrant epidermal growth factor signaling in drug resistant human breast cancer cells.

We examined transforming growth factor (TGF) alpha, epidermal growth factor (EGF) and EGF receptor (EGFR) expression and signaling in three drug resistant MCF-7 human breast cancer sublines and asked whether these pathways contribute to the drug resistance phenotype. In the resistant sublines, upregulation of both TGFalpha and EGFR mRNA was observed. In an apparent contrast with upregulated growth factor and receptor gene expression, the drug resistant sublines displayed a reduced growth rate. Defects in the EGFR signaling pathway cascade were found in all examined drug resistant sublines, including altered EGF-induced Shc, Raf-1, or mitogen-activated protein kinase phosphorylation. Induction of c-fos mRNA expression by EGF was impaired in the sublines compared to parental MCF-7 cells. In contrast, the induction of the stress-activated protein kinase activity was similar in both parental and drug resistant cells. Evaluating the link between the reduced growth rate and drug resistance, serum starvation experiments were performed. These studies demonstrated that a reduced proliferative activity resulted in a marked reduction in sensitivity to cytotoxic agents in the parental MCF-7 cells. We propose that the altered EGFR levels frequently observed in drug resistant breast cancer cells are associated with perturbations in the signaling pathway that mediate a reduced proliferative rate and thereby contribute to drug resistance.

Anisomycin↗
PubMed2000

Drug target validation: lethal infection blocked by inducible peptide.

Genome projects are generating large numbers of potential new targets for drug discovery. One challenge is target validation, proving the usefulness of a specific target in an animal model. In this paper, we demonstrate a new approach to validation and assay development. We selected in vitro specific peptide binders to a potential pathogen target. By inducing the expression of a selected peptide in pathogen cells causing a lethal infection in mice, the animals were rescued. Thus, by combining in vitro selection methods for peptide binders with inducible expression in animals, the target's validity was rigorously tested and demonstrated. This approach to validation can be generalized and has the potential to become a valuable tool in the drug discovery process.

Amino Acid Sequence↗
PubMed2000

Tracheal constrictor drive above the apneic threshold in anesthetized dogs.

We have previously shown that raising arterial PCO(2) (Pa(CO(2))) by small increments in dogs ventilated below the apneic threshold (AT) results in almost complete tracheal constriction before the return of phrenic activity (Dickstein JA, Greenberg A, Kruger J, Robicsek A, Silverman J, Sommer L, Sommer D, Volgyesi G, Iscoe S, and Fisher JA. J Appl Physiol 81: 1844-1849, 1996). We hypothesized that, if increasing chemical drive above the AT mediates increasing constrictor drive to tracheal smooth muscle, then pulmonary slowly adapting receptor input should elicit more tracheal dilation below the AT than above. In six methohexital sodium-anesthetized, paralyzed, and ventilated dogs, we measured changes in tracheal diameter in response to step increases in tidal volume (VT) or respiratory frequency (f) below and above the AT at constant Pa(CO(2)) ( approximately 40 and 67 Torr, respectively). Increases in VT (400-1,200 ml) caused significantly more (P = 0.005) tracheal dilation below than above AT (7.0 +/- 2.2 vs. 2.8 +/- 1.0 mm, respectively). In contrast, increases in f (14-22 breaths/min) caused similar (P = 0.93) tracheal dilations below and above (1.0 +/- 1.3 and 1.0 +/- 0.8 mm, respectively) AT. The greater effectiveness of dilator stimuli below compared with above the AT is consistent with the hypothesis that drive to tracheal smooth muscle increases even after attainment of maximal constriction. Our results emphasize the importance of controlling PCO(2) with respect to the AT when tracheal smooth muscle tone is experimentally altered.

Animals↗
PubMed1999

Intestinal MDR transport proteins and P-450 enzymes as barriers to oral drug delivery.

Cytochrome P-450 3A4 (CYP3A4), the major phase I drug metabolizing enzyme in humans, and the multidrug efflux pump, MDR or P-glycoprotein (P-gp), are present at high levels in the villus tip enterocytes of the small intestine, the primary site of absorption for orally administered drugs. These proteins are induced or inhibited by many of the same compounds and demonstrate a broad overlap in substrate and inhibitor specificities, suggesting that they act as a concerted barrier to drug absorption. A series of studies from our laboratory of cyclosporine and tacrolimus in humans and a novel cysteine protease inhibitor in rats, dosed concomitantly with inhibitors and inducers of CYP3A4 and P-gp, suggest that gut extraction can be modeled using measures of intestinal metabolism and absorption rate, the latter reflecting changes in P-gp. Results evaluating a preliminary model applied to the CYP3A substrate drugs midazolam, indinavir, saquinavir, and rifabutin suggest that the model may be useful for predicting in vivo intestinal metabolism from in vitro data.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Multidrug-resistance transporters.

P-glycoprotein was initially isolated due to its role in multidrug resistance to cancer chemotherapeutics. Recent work, however, makes it increasingly apparent that this transporter is also involved in the pharmacokinetics of many drugs. P-gp is strategically expressed in the luminal epithelial cells of organs often associated with drug absorption and disposition, for example, hepatocyte canalicular membrane, renal proximal tubules, and the intestinal mucosa. P-gp is also expressed in the endothelial cells comprising the blood-brain barrier. This localization clearly suggests the potential for this protein to serve as a protective mechanism against entry of toxic xenobiotics and also suggests that P-gp is well situated to participate in the removal of therapeutic agents. Numerous investigations with drugs such as digoxin, etoposide, cyclosporine, vinblastine, Taxol, loperamide, dom-peridone, and ondansteron demonstrate that P-gp has an important role in determining the pharmacokinetics of substrate drugs. Pharmacological modulation of P-gp function to increase drug bioavailability, both on a organismal and a cellular level, is one approach currently being explored to enhance therapeutic effectiveness. This approach is not without potential collateral consequences given the wide tissue distribution of P-gp. While animals deficient in P-gp are viable and without obvious abnormalities, the pharmacokinetics and toxic consequences of several compounds are significantly altered in these animals. Thus blockade of the protective P-gp barrier in humans may have adverse effects on substrate drugs. In particular, this situation may arise when several compounds which may be substrates compete for P-gp-mediated transport. Additional multidrug transporters, notably MRP and family members, have been identified and may also determine the fate of pharmaceuticals. Further understanding the physiological role of each of the multidrug transporters is critical for determining their role in pharmacokinetics and for evaluating the consequences of modification of their activities. Such information is also important in the development of novel drugs which may be substrates for these transporters.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

Grapefruit juice activates P-glycoprotein-mediated drug transport.

PURPOSE: Grapefruit juice (GJ) is known to increase the oral bioavailability of many CYP3A-substrates by inhibiting intestinal phase-I metabolism. However, the magnitude of AUC increase is often insignificant and highly variable. Since we earlier suggested that CYP3A and P-glycoprotein (P-gp) form a concerted barrier to drug absorption, we investigated the role of P-gp in GJ-drug interactions. METHODS: The transcellular bidirectional flux of drugs that are (i) CYP3A-and/or P-gp substrates (Vinblastine, Cyclosporine, Digoxin, Fexofenadine, Losartan) or that are (ii) primary CYP3A-substrates (Felodipine, Nifedipine) was evaluated across MDCK-MDR1 cell monolayers with or without GJ, verifying monolayer integrity at all times. RESULTS: While both apical-to-basal (A-B) and basal-to-apical (B-A) fluxes of all CYP3A/P-gp substrates tested were increased in the presence of GJ, the resulting net efflux (B-A/A-B) was in all cases significantly greater with GJ than control (Vin, 28.0 vs. 5.1; CsA, 9.9 vs. 2.8; Dig, 22. 9 vs. 14.7, Fex, 22.3 vs. 11.1, Los, 39.6 vs. 26). In contrast, no such GJ flux effect was observed with Fel and Nif, substrates of CYP3A only (2 vs. 1.7 and 1.2 vs. 1.3). CONCLUSIONS: GJ significantly activates P-gp-mediated efflux of drugs that are substrates of P-gp, potentially partially counteracting the CYP3A-inhibitory effects of GJ.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Inhibition of P-glycoprotein by D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS).

PURPOSE: To investigate whether d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) functions as an inhibitor of P-glycoprotein (P-gp), the multidrug resistance transporter. METHODS: Two assays were used to measure the function of TPGS on P-gp function. First, we examined the ability of TPGS to modulate the cytotoxicity of established, cytotoxic, P-glycoprotein substrates. Parental NIH 3T3 cells and NIH 3T3 cells transfected with the human MDR1 cDNA (G185) were exposed to doxorubicin, paclitaxel, colchicine, vinblastine and 5-fluorouracil (5FU) in the presence or absence of TPGS. Cytotoxicity was assessed with the MTT assay. Second, polarized transport of the P-gp substrates rhodamine 123 (R123), paclitaxel and vinblastine was measured using the human intestinal HCT-8 and Caco-2 cell lines grown in Transwell dishes. Drug flux was measured by liquid scintillation counting or fluorescence spectroscopy of the media. RESULTS: G185 cells were 27-135 fold more resistant to the cytotoxic drugs doxorubicin, vinblastine, colchicine and paclitaxel than the parental NIH 3T3 cells. In contrast 5FU, which is not a P-gp substrate, is equally cytotoxic to parental and G185 cells. Co-administration of TPGS enhanced the cytotoxicity of doxorubicin, vinblastine, paclitaxel, and colchicine in the G185 cells to levels comparable to the parental cells. TPGS did not increase the cytotoxicity of 5FU in the G185 cells. Using a polarized epithelial cell transport assay, TPGS blocked P-gp mediated transport of R123 and paclitaxel in a dose responsive manner. CONCLUSIONS: These data demonstrate that TPGS acts as a reversal agent for P-glycoprotein mediated multidrug resistance and inhibits P-gp mediated drug transport. These results suggest that enhanced oral bioavailability of drugs co-administered with TPGS may, in part, be due to inhibition of P-glycoprotein in the intestine.

3T3 Cells↗
PubMed1998

Induced activation of the Toxoplasma gondii nucleoside triphosphate hydrolase leads to depletion of host cell ATP levels and rapid exit of intracellular parasites from infected cells.

The nucleoside triphosphate hydrolase of Toxoplasma gondii is a potent apyrase. The protein is synthesized in large amounts and transported through the secretory pathway of the parasite and into the vacuolar space in an oxidized and thereby enzymatically inactive form. Complete activation of the purified enzyme is known to require dithiols (e.g. DTT); subcellular fractionation demonstrates that little if any (<5%) of the enzyme in the vacuolar space is active in the absence of DTT. Both native and epitope-tagged nucleoside triphosphate hydrolase (NTPase) were partially activated during immunoprecipitation, precluding precise assessment of enzyme activity in the vacuolar space but suggesting that protein-protein interactions may trigger activation. When infected cells were treated with DTT, the NTPase was activated in a dose-response fashion, as assessed by migration on SDS-polyacrylamide gel electrophoresis and by an increase in enzymatic activity. After activation, enzyme activity decreased with time in the presence of DTT; this inactivation was slowed by the presence of excess ATP. A rapid fall in host cell ATP was accompanied by an abrupt exit of parasites from cells. These results demonstrate that the oxidation/reduction status of the NTPase, the only parasite dense granule protein that contains disulfide bonds, is tightly controlled within the vacuolar space and may influence parasite exit from cells.

Acid Anhydride Hydrolases↗
PubMed1998

Role of P-glycoprotein and cytochrome P450 3A in limiting oral absorption of peptides and peptidomimetics.

Cytochrome P450 3A4 (CYP3A4), the major phase I drug metabolizing enzyme in humans, and the MDR1 gene product P-glycoprotein (P-gp) are present at high concentrations in villus tip enterocytes of the small intestine and share a significant overlap in substrate specificity. A large body of research both in vitro and in vivo has established metabolism by intestinal CYP3A4 as a major determinant of the systemic bioavailability of orally administered drugs. More recently it has been recognized that drug extrusion by intestinal P-gp can both reduce drug absorption and modulate the effects of inhibitors and inducers of CYP3A-mediated metabolism. There is relatively little data regarding the effects of CYP3A and P-gp on peptide drugs; however, studies with the cyclic peptide immunosuppresant cyclosporine as well as peptidomimetics such as the HIV-protease inhibitor saquinavir (Invirase) and a new cysteine protease inhibitor K02 (Morpholine-Urea-Phe-Hphe-Vinyl sulfone; Axys Pharmaceuticals) provide some insight into the impact of these systems on the oral absorption of peptides.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Saquinavir, an HIV protease inhibitor, is transported by P-glycoprotein.

Saquinavir, a peptidomimetic HIV protease inhibitor, has been shown to be effective in reducing patient viral load and reducing mortality. In this report we investigated whether saquinavir is a substrate for the multidrug resistance transporter P-glycoprotein (P-gp), which may reduce the effective intracellular concentration of the drug. G185 cells, which highly express P-gp, are resistant to saquinavir-mediated cytotoxicity, and co-administration of cyclosporine reversed this resistance. Saquinavir and saquinavir mesylate inhibited basolateral to apical transport of the fluorescent dye rhodamine 123 in a polarized epithelial transport assay, a result that suggests competition of these drugs for the P-gp transporter. Finally, we measured specific, directional transport of saquinavir and saquinavir mesylate in an epithelial monolayer model. Transport in the basolateral to apical direction was 3-fold greater than apical to basolateral flux for both saquinavir and saquinavir mesylate and was blocked by co-incubation with the established P-gp reversal agents cyclosporine and verapamil. These data provide evidence that saquinavir is a substrate for the P-gp transporter and suggest that this protein may affect intracellular accumulation of the drug and contribute to its poor oral bioavailability.

3T3 Cells↗
PubMed1997

Functional characterization of the rat mdr1b encoded P-glycoprotein: not all inducing agents are substrates.

The multidrug resistance (mdr) genes encode P-glycoproteins, integral membrane proteins which function as drug efflux transporters. Exposure of animals in vivo and cells in vitro to a variety of xenobiotics leads to increased mdr1 gene expression and higher levels of P-glycoprotein. This response may protect cells from the cytotoxic effects of these compounds. In this investigation we functionally expressed the rat mdr1b gene in NIH 3T3 cells and assessed the ability of the encoded P-glycoprotein to protect these cells from the cytotoxicity of xenobiotics known to induce mdr1b expression. In long-term colony survival assays, stably expressed mdr1b conferred resistance to cytotoxic drugs such as colchicine, vinblastine and doxorubicin, but not to 5-fluorouracil nor to the carcinogens aflatoxin B1 and N-hydroxy-acetylaminofluorene. The mdr reversal agent verapamil restored cytotoxicity of colchicine, doxorubicin, actinomycin D, vinblastine and taxol, but had no effect on the sensitivity of these cells to 5-fluorouracil, aflatoxin B1 or N-hydroxy-acetylaminofluorene. In a competitive transport assay, verapamil and, to a lesser extent, colchicine blocked the increased efflux of the fluorescent dye rhodamine 123 from mdr1b-transfected cells, whereas aflatoxin B1 did not compete for this export. These data demonstrate that expression of the rat mdr1b encoded P-glycoprotein can protect cells from a diverse group of compounds previously identified to be mdr substrates, however, other effective inducers of mdr expression, such as aflatoxin B1 and N-hydroxy-acetylaminofluorene, remain potent cytotoxins despite high levels of P-glycoprotein. The fact that compounds which are not themselves substrates can induce P-glycoprotein expression may have implications for pharmacokinetic interactions and chemotherapy.

3T3 Cells↗
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↗