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Eric J Sorscher

Publications and source records attributed to Eric J Sorscher.

14 recordsLinked to original sources
PubMed2024

Relationships between genomic dissipation and de novo SNP evolution.

Patterns of single nucleotide polymorphisms (SNPs) in eukaryotic DNA are traditionally attributed to selective pressure, drift, identity descent, or related factors-without accounting for ways in which bias during de novo SNP formation, itself, might contribute. A functional and phenotypic analysis based on evolutionary resilience of DNA points to decreased numbers of non-synonymous SNPs in human and other genomes, with a predominant component of SNP depletion in the human gene pool caused by robust preferences during de novo SNP formation (rather than selective constraint). Ramifications of these findings are broad, belie a number of concepts regarding human evolution, and point to a novel interpretation of evolving DNA across diverse species.

Polymorphism, Single Nucleotide↗
PubMed2003

Membrane transplantation to correct integral membrane protein defects.

In this report we show that the tendency of certain viruses to carry host membrane proteins in their envelopes can be harnessed for transplantation of small patches of plasma membrane, including fully functional, polytopic ion channel proteins and their regulatory binding partners. As a stringent model we tested the topologically complex epithelial ion channel CFTR. Initially an attenuated vaccinia virus was found capable of transferring CFTR in a properly folded, functional and regulatable form to CFTR negative cells. Next we generated viruslike particles (VLPs) composed of retroviral structural proteins that assemble and bud at the host cell plasma membrane. These particles were also shown to mediate functional ion channel transfer. By testing the capacity of complex membrane proteins to incorporate into viral envelopes these experiments provide new insight into the permissiveness of viral envelopment, including the ability of incorporated proteins to retain function and repair defects at the cell surface, and serve as a platform for studies of ion channel and membrane protein biochemistry.

Animals↗
PubMed2003

Adenosine receptors and phosphodiesterase inhibitors stimulate Cl- secretion in Calu-3 cells.

We investigated cystic fibrosis transmembrane conductance regulator (CFTR) activation by clinically used phosphodiesterase inhibitors (PDEis) in Calu-3 cell monolayers alone and in combination with A2B adenosine receptor stimulation. This receptor pathway has previously been shown to activate wild-type and mutant CFTR molecules. Several PDEis, including milrinone, cilostazol (Pletal), papaverine, rolipram, and sildenafil (Viagra), produced a short circuit current (Isc) that was glibenclamide-sensitive, achieving 20-85% of forskolin-stimulated Isc. Papaverine, cilostazol, and rolipram also augmented both the magnitude and the duration of Isc following low dose stimulation of adenosine receptors with Ado (0.1-1.0 microM, P < 0.01). Subsequent studies demonstrated that very low concentrations of cilostazol or papaverine (approximately 1/2 peak serum concentrations) were sufficient to activate Isc, and both agents markedly augmented Ado-stimulated Isc (1 microM, P < 0.01). Our results provide evidence that select PDEis, at concentrations achieved as part of systemic therapies, can activate CFTR-dependent Isc in Calu-3 cell monolayers. These studies also indicate that PDEis have the capacity to augment an endogenous CFTR-activating pathway in an "in vivo"-like model system, and supports future investigations of these agents relevant to cystic fibrosis.

Adenosine Deaminase↗
PubMed2003

Antitumor activity of 2-fluoro-2'-deoxyadenosine against tumors that express Escherichia coli purine nucleoside phosphorylase.

The selective expression of Escherichia coli purine nucleoside phosphorylase (PNP) in solid tumors has been successfully used to activate two purine nucleoside analogs [9-(2-deoxy-beta-D-ribofuranosyl)-6-methylpurine (MeP-dR) and 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-araA)] resulting in lasting tumor regressions and cures. E. coli PNP also cleaves 2-fluoro-2'-deoxyadenosine (F-dAdo) to 2-F-adenine, which is the toxic purine analog liberated from F-araA that has high bystander activity and is active against nonproliferating tumor cells. As F-dAdo is 3000 times better than F-araA as a substrate for E. coli PNP, we have evaluated its antitumor activity against D54 gliomas that express E. coli PNP and have characterized its in vivo metabolism in order to better understand its mechanism of action with respect to the other two agents. Like MeP-dR and F-araA-5'-monophosphate (F-araAMP, a prodrug of F-araA), treatment of mice bearing D54 tumors that express E. coli PNP with F-dAdo resulted in excellent antitumor activity. Although F-dAdo was as active as MeP-dR and better than F-araAMP, it was not dramatically better than either compound because of its short plasma half-life and the limited activation of F-adenine to toxic metabolites. Regardless, these results indicated that F-dAdo was also an excellent prodrug for use with gene vectors that deliver E. coli PNP to tumor cells.

Animals↗
PubMed2003

A long-acting suicide gene toxin, 6-methylpurine, inhibits slow growing tumors after a single administration.

We have demonstrated antitumor activity against refractory human glioma and pancreatic tumors with 6-methylpurine (MeP) using either a suicide gene therapy strategy to selectively release 6-methylpurine in tumor cells or direct intratumoral injection of 6-methylpurine itself. A single i.p. injection in mice of the prodrug 9-beta-D-[2-deoxyribofuranosyl]-6-methylpurine (MeP-dR; 134 mg/kg) caused sustained regression lasting over 70 days of D54 (human glioma) tumors transduced with the Escherichia coli purine nucleoside phosphorylase (PNP), and a single intratumoral injection of 6-methylpurine (5-10 mg/kg) elicited prolonged delays of the growth of D54 tumors and CFPAC human pancreatic carcinoma. Because the D54 tumor doubling time is >15 days, the experiments indicate that prodrug activation by E. coli PNP engenders destruction of both dividing and nondividing tumor compartments in vivo and, therefore, address a fundamental barrier that has limited the development of suicide gene strategies in the past. A prolonged retention time of 6-methylpurine metabolites in tumors was noted in vivo (T(1/2) >24 h compared with a serum half-life of <1 h). By high-pressure liquid chromatography, metabolites of [(3)H]MeP-dR were 5- to 6-fold higher in tumors expressing E. coli PNP. These experiments point to new endpoints for monitoring E. coli PNP suicide gene therapy, including intratumoral enzymatic activity, in situ (intratumoral) prodrug conversion, and tumor regressions after direct injection of a suicide gene toxin. The findings also help explain the strong in vivo bystander killing mechanism ascribed by several laboratories to E. coli PNP in the past.

Animals↗
PubMed2002

A macromolecular complex of beta 2 adrenergic receptor, CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA.

It has been demonstrated previously that both the cystic fibrosis transmembrane conductance regulator (CFTR) and beta(2) adrenergic receptor (beta(2)AR) can bind ezrinradixinmoesin-binding phosphoprotein 50 (EBP50, also referred to as NHERF) through their PDZ motifs. Here, we show that beta(2) is the major adrenergic receptor isoform expressed in airway epithelia and that it colocalizes with CFTR at the apical membrane. beta(2)AR stimulation increases CFTR activity, in airway epithelial cells, that is glybenclamide sensitive. Deletion of the PDZ motif from CFTR uncouples the channel from the receptor both physically and functionally. This uncoupling is specific to the beta(2)AR receptor and does not affect CFTR coupling to other receptors (e.g., adenosine receptor pathway). Biochemical studies demonstrate the existence of a macromolecular complex involving CFTR-EBP50-beta(2)AR through PDZ-based interactions. Assembly of the complex is regulated by PKA-dependent phosphorylation. Deleting the regulatory domain of CFTR abolishes PKA regulation of complex assembly. This report summarizes a macromolecular signaling complex involving CFTR, the implications of which may be relevant to CFTR-dysfunction diseases.

Animals↗
PubMed2002

Ablation of internalization signals in the carboxyl-terminal tail of the cystic fibrosis transmembrane conductance regulator enhances cell surface expression.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that undergoes endocytosis through clathrin-coated pits. Previously, we demonstrated that Y1424A is important for CFTR endocytosis (Prince, L. S., Peter, K., Hatton, S. R., Zaliauskiene, L., Cotlin, L. F., Clancy, J. P., Marchase, R. B., and Collawn, J. F. (1999) J. Biol. Chem. 274, 3602-3609). Here we show that a second substitution in the carboxyl-terminal tail of CFTR, I1427A, on Y1424A background more than doubles CFTR surface expression as monitored by surface biotinylation. Internalization assays indicate that enhanced surface expression of Y1424A,I1427A CFTR is caused by a 76% inhibition of endocytosis. Patch clamp recording of chloride channel activity revealed that there was a corresponding increase in chloride channel activity of Y1424A,I1427A CFTR, consistent with the elevated surface expression, and no change in CFTR channel properties. Y14124A showed an intermediate phenotype compared with the double mutation, both in terms of surface expression and chloride channel activity. Metabolic pulse-chase experiments demonstrated that the two mutations did not affect maturation efficiency or protein half-life. Taken together, our data show that there is an internalization signal in the COOH terminus of CFTR that consists of Tyr(1424)-X-X-Ile(1427) where both the tyrosine and the isoleucine are essential residues. This signal regulates CFTR surface expression but not CFTR biogenesis, degradation, or chloride channel function.

Animals↗
PubMed2002

Role for phospholipid interactions in the trafficking defect of Delta F508-CFTR.

Cystic fibrosis commonly occurs as a consequence of the DeltaF508 mutation in the first nucleotide binding fold domain (NBF-1) of CFTR. The mutation causes retention of the mutant CFTR molecule in the endoplasmic reticulum, and this aberrant trafficking event is believed to be due to defective interactions between the mutant NBF-1 domain and other cellular factors in the endoplasmic reticulum. Since the NBF-1 domain has been shown to interact with membranes, we wanted to investigate whether NBF-1 and CFTR interactions with specific phospholipid chaperones might play a role in trafficking. We have found that the recombinant wild-type NBF-1 interacts selectively with phosphatidylserine (PS) rather than phosphatidylcholine (PC). By contrast, NBF-1 carrying the DeltaF508 mutation loses the ability to discriminate between these two phospholipids. In cells expressing DeltaF508-CFTR, replacement of PC by noncharged analogues results in an absolute increase in CFTR expression. In addition, we detected progressive expression of higher molecular weight CFTR forms. Thus, phospholipid chaperones may be important for CFTR trafficking, and contribute to the pathology of cystic fibrosis.

Amino Alcohols↗
PubMed2002

Reactive oxygen nitrogen species decrease cystic fibrosis transmembrane conductance regulator expression and cAMP-mediated Cl- secretion in airway epithelia.

We investigated putative mechanisms by which nitric oxide modulates cystic fibrosis transmembrane conductance regulator (CFTR) expression and function in epithelial cells. Immunoprecipitation followed by Western blotting, as well as immunocytochemical and cell surface biotinylation measurements, showed that incubation of both stably transduced (HeLa) and endogenous CFTR expressing (16HBE14o-, Calu-3, and mouse tracheal epithelial) cells with 100 microm diethylenetriamine NONOate (DETA NONOate) for 24-96 h decreased both intracellular and apical CFTR levels. Calu-3 and mouse tracheal epithelial cells, incubated with DETA NONOate but not with 100 microm 8-bromo-cGMP for 96 h, exhibited reduced cAMP-activated short circuit currents when mounted in Ussing chambers. Exposure of Calu-3 cells to nitric oxide donors resulted in the nitration of a number of proteins including CFTR. Nitration was augmented by proteasome inhibition, suggesting a role for the proteasome in the degradation of nitrated proteins. Our studies demonstrate that levels of nitric oxide that are likely to be encountered in the vicinity of airway cells during inflammation may nitrate CFTR resulting in enhanced degradation and decreased function. Decreased levels and function of normal CFTR may account for some of the cystic fibrosis-like symptoms that occur in chronic inflammatory lung diseases associated with increased NO production.

Animals↗
PubMed2002

Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Since approximately 5% of all mutant CF alleles are stop mutations, it can be calculated that approximately 10% of CF patients carry a premature stop mutation in at least one copy of the CFTR gene. Certain ethnic groups, such as the Ashkenazi Jewish population, carry a much higher percentage of CF stop mutations. Consequently, a therapeutic strategy aimed at suppressing this class of mutation would be highly desirable for the treatment of this common genetic disease. We have shown previously that aminoglycoside antibiotics can suppress premature stop mutations in the CFTR gene in a bronchial epithelial cell line [Nat Med (1997) 3:1280]. To address whether aminoglycosides can suppress a CFTR premature stop mutation in an animal model, we constructed a transgenic mouse with a null mutation in the endogenous CFTR locus (Cftr-/-) that also expressed a human CFTR-G542X cDNA under control of the intestinal fatty acid binding protein promoter. We then investigated whether the daily administration of the aminoglycoside antibiotics gentamicin or tobramycin could restore the expression of a detectable level of CFTR protein. Immunofluorescence staining of intestinal tissues from Cftr-/- hCFTR-G542X mice revealed that gentamicin treatment resulted in the appearance of hCFTR protein at the apical surface of the glands of treated mice. Weaker staining was also observed in the intestinal glands following tobramycin treatment. Short-circuit current measurements made on intestinal tissues from these mice demonstrated that a significant number of positive cAMP-stimulated transepithelial chloride current measurements could be observed following gentamicin treatment (P=0.008) and a near significant number following tobramycin treatment (P=0.052). When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional hCFTR protein by suppressing the hCFTR-G542X premature stop mutation in vivo.

Action Potentials↗
PubMed2002

Elevation of hepatic sulphotransferase activities in mice with resistance to cystic fibrosis.

The severity of intestinal disease in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) (-/-) mice has been reported to co-segregate with gene loci which contain the genes for hydroxysteroid sulphotransferase (SULT). Because of the potential involvement of steroid hormones in CF, we investigated levels of steroid SULT activity in the livers of CFTR mice to determine whether the levels of SULT activity correlate with the occurrence or severity of CF. To elucidate the possible role of SULT activity in ameliorating the deleterious effects of CF in CFTR (-/-) mice, we determined the levels of phenol SULT (PST), hydroxysteroid SULT [dehydroepiandrosterone (DHEA)-ST] and oestrogen SULT (EST) activity in control CFTR (+/+), heterozygous CFTR (+/-) and homozygous CFTR (-/-) mice, which survive to adulthood. The level of PST activity was not significantly different between any of the groups of mice, regardless of sex or genotype. Although DHEA-ST activity was significantly higher in female mice than in male mice, there was no difference in DHEA-ST activity that could be correlated with genotype. In contrast with PST and DHEA-ST activities, we found that some male and all female adult CFTR (-/-) mice had elevated, dramatically different levels of EST from both CFTR (+/+) and CFTR (+/-) mice. Results from these SULT activity experiments were confirmed by Northern-blot analysis of mouse-liver RNA. Subsequent studies with preweanling mice revealed no differences in the levels of EST that could be correlated with genotype. Thus this study indicates that EST is elevated significantly in CFTR (-/-) mice which survive to adulthood and provides important biochemical information that EST levels may be protective in CF.

Adjuvants, Immunologic↗
PubMed2002

Effects of megestrol acetate on weight gain, body composition, and pulmonary function in patients with cystic fibrosis.

OBJECTIVES: Malnutrition is a negative prognostic indicator in patients with cystic fibrosis (CF) and may accentuate pulmonary decline. We tested whether megestrol acetate would have beneficial effects on growth in patients with CF and pancreatic insufficiency. STUDY DESIGN: We performed a randomized, double-blind, placebo controlled study. All patients were taking replacement enzymes to compensate for pancreatic insufficiency. Patients (n = 17) were randomly assigned to receive either megestrol acetate or placebo. RESULTS: The treatment group had a significant increase in weight-for-age z scores compared with placebo and reached 100% of their ideal body weight within 3 months of initiating therapy. Weight gain included both fat and fat-free mass. Improved pulmonary function (forced vital capacity and forced expiratory volume in 1 second) was noted in the treatment group compared with placebo (P <.04). Reversible adrenal suppression was observed in the majority of patients who received megestrol acetate. CONCLUSIONS: Short-term use of megestrol acetate results in significant weight gain and improved pulmonary function in malnourished subjects with CF. Our study provides a controlled basis for this intervention, identifies important side effects, and provides the foundation for multiyear, longitudinal trials in a larger number of patients with CF.

Adolescent↗