Expression and polarized targeting of a rab3 isoform in epithelial cells.

Pathways of polarized membrane traffic in epithelial tissues serve a variety of functions, including the generation of epithelial polarity and the regulation of vectorial transport. We have identified a candidate regulator of polarized membrane traffic in epithelial cells (i.e., rab3B), which is a member of the rab family of membrane traffic regulators. Rab3B is highly homologous to a brain-specific rab3 isoform (rab3A) that targets in a polarized fashion to the presynaptic nerve terminal, where it probably regulates exocytosis. The coding region for human rab3B was cloned from epithelial mRNA using a reverse-transcription polymerase chain reaction strategy. This cDNA clone hybridized to a single mRNA species in Northern blots of poly(A)+ RNA isolated from epithelial cell lines. A rab3B-specific antibody that was raised against recombinant fusion protein recognized a 25-kD band in immunoblots of cell lysates prepared from cultured epithelial cells (e.g., T84 and HT29-CL19A), but not from a variety of nonepithelial cells (e.g., PC12 neuroendocrine cells). Immunofluorescence analysis confirmed that rab3B protein is preferentially expressed in cultured epithelial cells as well as in a number of native epithelial tissues, including liver, small intestine, colon, and distal nephron. Rab3B localized to the apical pole very near the tight junctions between adjacent epithelial cells within all of these cell lines and native epithelial tissues, as determined by immunofluorescence and immunoelectron microscopic analysis. Moreover, this pattern of intracellular targeting was regulated by cell contact; namely, rab3B was reversibly retrieved from the cell periphery as epithelial cell contact was inhibited by reducing the extracellular Ca2+ concentration. Our results indicate that neurons and epithelial cells express homologous rab3 isoforms that target in a polarized fashion within their respective tissues. The pattern and regulation of rab3B targeting in epithelial cells implicates this monomeric GTPase as a candidate regulator of apical and/or junctional protein traffic in epithelial tissues.

Regulation of plasma membrane recycling by CFTR.

The gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) is defective in patients with cystic fibrosis. Although the protein product of the CFTR gene has been proposed to function as a chloride ion channel, certain aspects of its function remain unclear. The role of CFTR in the adenosine 3',5'-monophosphate (cAMP)-dependent regulation of plasma membrane recycling was examined. Adenosine 3',5'-monophosphate is known to regulate endocytosis and exocytosis in chloride-secreting epithelial cells that express CFTR. However, mutant epithelial cells derived from a patient with cystic fibrosis exhibited no cAMP-dependent regulation of endocytosis and exocytosis until they were transfected with complementary DNA encoding wild-type CFTR. Thus, CFTR is critical for cAMP-dependent regulation of membrane recycling in epithelial tissues, and this function of CFTR could explain in part the pleiotropic nature of cystic fibrosis.

The biogenesis, traffic, and function of the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride channel that is encoded by the gene that is defective in cystic fibrosis. This ion channel resides at the luminal surfaces and in endosomes of epithelial cells that line the airways, intestine, and a variety of exocrine glands. In this article we discuss current hypotheses regarding how CFTR functions as a regulated ion channel and how CF mutations lead to disease. We also evaluate the emerging notion that CFTR is a multifunctional protein that is capable of regulating epithelial physiology at several levels, including the modulation of other ion channels and the regulation of intracellular membrane traffic. Elucidating the various functions of CFTR should contribute to our understanding of the pathology in cystic fibrosis, the most common lethal genetic disorder among Caucasians.

Prevention of nitric oxide synthase induction in vascular smooth muscle cells by microtubule depolymerizing agents.

We investigated the role of microtubules in the induction of nitric oxide synthase in cultured vascular smooth muscle cells. We found that like interleukin-1 alpha, lipopolysaccharide elicited a time and concentration-dependent accumulation of cyclic GMP via induction of nitric oxide synthase. Nocodazole and colchicine, two chemically distinct microtubule depolymerizing agents, completely prevented lipopolysaccharide- and interleukin-induced (and nitric oxide-mediated) cyclic GMP generation. In contrast to lipopolysaccharide and interleukin-1 alpha, cyclic GMP accumulation in response to sodium nitroprusside, an exogenous nitrovasodilator, was not altered by either nocodazole or colchicine. Our findings demonstrate that microtubule depolymerizing agents inhibit nitric oxide synthase induction and suggest a prominent role for microtubules in mediating the activation of the inducible nitric oxide pathway in smooth muscle cells.

Cytoskeleton-dependent activation of the inducible nitric oxide synthase in cultured aortic smooth muscle cells.

1. Vascular endothelial and smooth muscle cells generate nitric oxide (NO) via different nitric oxide synthase (NOS) isozymes. Activation of the endothelial constitutive NOS (ecNOS) contributes to the maintenance of cardiovascular homeostasis, whereas expression of the endotoxin- and cytokine-inducible pathway (iNOS) within the vascular smooth muscle is thought to be responsible for the cardiovascular collapse which occurs during septic shock and antitumour therapy with cytokines. Since the cytoskeleton is involved in the activation of certain genes and in some effects of endotoxin in macrophages, we investigated the role of microtubules and microfilaments in the activation of the NO pathway in cultured vascular cells. 2. Depolymerization of microtubules by either nocodazole or colchicine prevented lipopolysaccharide (LPS)- and interleukin-1 beta-induction of NO-dependent cyclic GMP accumulation. Steady state levels of iNOS mRNA, assessed by Northern blot and RT-PCR, and iNOS protein, assessed by Western blotting, were also decreased by either colchicine or nocodazole treatment. 3. Taxol enhanced microtubule polymerization alone, and prevented microtubule depolymerization elicited by nocodazole and colchicine. Associated with its effect on microtubule assembly, taxol prevented the inhibitory effects of nocodazole and colchicine on cyclic GMP accumulation and iNOS mRNA levels. 4. Disruption of microfilaments by cytochalasins had no inhibitory effect on the activation of the inducible NO pathway. 5. In contrast to cytokine-stimulated smooth muscle cells, modulation of either microtubule or microfilament assembly did not affect the constitutive NO pathway in endothelial cells, as endothelial cell- and NO-dependent cyclic GMP accumulation in endothelial-smooth muscle co-cultures remained unchanged. 6. Our findings demonstrate that microtubules play a prominent role in the activation of the inducible NO pathway in response to inflammatory mediators in smooth muscle cells but not of the constitutive synthesis of NO in endothelial cells.

The role of polyunsaturated fatty acid supplementation in intestinal inflammation and neonatal necrotizing enterocolitis.

Dietary polyunsaturated fatty acid (PUFA) supplementation has been shown to reduce the incidence of necrotizing enterocolitis (NEC) in a recent randomized, controlled trial. These compounds are known to modulate the inflammatory cascade and to influence intestinal health in a variety of ways. Although the pathophysiology of NEC is not well understood, recent evidence suggests that platelet-activating factor (PAF) is a key endogenous mediator of intestinal necrosis in animals. Using a neonatal rat model of NEC that includes the key risk factors of asphyxia and formula feeding, we investigated the role of dietary PUFA supplementation on the incidence and pathophysiology of NEC. Our findings suggest that PUFA reduce the incidence of NEC by modulating PAF metabolism and endotoxin translocation.

OS054. Differential strain sensitivities to ischemia/reperfusion in two mouse models of fetal growth restriction.

INTRODUCTION: Uterine ischemia/reperfusion (I/R) in rodents is a common model for the study of fetal growth restriction (FGR). It has been observed that different strains of mice vary in their response to uterine I/R. OBJECTIVES: Our objective was to characterize fetal and placental growth, and uterine and placental inflammation, in pregnant mice from 2 strains (C3H/HeOuJ and C57BL/6J), in response to different uterine I/R modalities. METHODS: Timed-pregnant mice (6-8 in each experimental group) were subjected to unilateral uterine I/R by either total flow restriction (TFR,right ovarian and uterine arterial clamping,5 or 30min) or partial flow restriction (PFR,right ovarian artery clamping,30min), or to sham-operation, on the 14th (for C57 mice) or 15th (for C3H mice) day of gestation. Four days later, fetal and placental weights and fetal loss were evaluated, and myeloperoxidase (MPO) activity was assayed in uterine and placental tissues. Data were analyzed by ANOVA with p<0.05 considered significant. RESULTS: In C3H/HeOuJ mice, TFR/30 min induced significantly (p<0.05) lower fetal and placental weights, and higher MPO activity in both uterus and placenta, compared to sham-operated controls. PFR/30min produced fetal but not placental growth restriction in the C3H mice. In contrast, C57BL/6J mice exhibited much greater sensitivity to uterine I/R: TFR/5 min was adequate to induce FGR in the C57 mice, which was equivalent in proportion to FGR caused by TFR/30min in C3H. C57 mice also exhibited significantly greater fetal loss and higher uterine, but not placental, MPO activity in response to I/R compared to sham-operated controls. CONCLUSION: Reliable FGR can be induced in different strains of mice by uterine I/R through adjustment of intensity, duration, and gestation day of the challenge. Mice of different strains have different tolerance to uterine I/R. These strain disparities would lend themselves to identifying the role of mouse strain-specific genetic determinants which form the bases for the observed differences in sensitivity to I/R-induced FGR.

Cyclic AMP and chloride-dependent regulation of the apical constitutive secretory pathway in colonic epithelial cells.

Epithelial cells of the colonic crypt engage in cAMP-mediated fluid and electrolyte secretion. In addition to participating in electrolyte transport, colonic crypt cells also synthesize and secrete a number of proteins and peptides that play a crucial role in mucosal homeostasis. In the present study we show that cAMP regulates not only electrolyte secretion but also polarized protein secretion in a tissue culture model of colonic crypt cells. We found that apical but not basolateral protein secretion was stimulated by a physiological activator of the cAMP pathway, vasoactive intestinal peptide, as well as by a cell-permeant analogue of cAMP (8-(4-chlorophenylthio)cAMP) at concentrations as low as 12.5 microM. Based on several criteria, we determined that the regulation of protein secretion by cAMP in HT29-CL19A cells occurs via stimulation of constitutive membrane traffic from the trans-Golgi network (TGN) to the apical cell surface. In addition, the regulation of apical protein secretion by cAMP was Cl--dependent with cAMP inhibiting rather than stimulating secretion in Cl--depleted cells. The locus of cAMP action on the secretory pathway is at least in part at the level of the TGN, where it stimulates the sialylation of alpha1-antitrypsin (i.e. one of the identified secretory proteins) in addition to the traffic of secretory proteins from the TGN to the apical cell surface. We propose that a cyclic AMP and Cl--dependent regulation of TGN acidification could modulate both sialylation and secretory vesicle budding at the TGN.

New concepts in necrotizing enterocolitis.

Necrotizing enterocolitis is an overwhelming gastrointestinal emergency that primarily afflicts premature infants born weighing less than 1500 g. Despite years of investigation, the etiology remains unclear, and accepted prevention and treatment strategies are lacking. Studies published over the last year have provided new insight into several aspects of this complex disease. In this review, novel information is presented on (1) the epidemiology; (2) methods of early diagnosis, such as abdominal magnetic resonance imaging; (3) the importance of risk factors, including assessment of feeding strategies and role of bacterial colonization; (4) the pathophysiology, highlighting experimental and clinical trials evaluating the role of inflammatory mediators and growth factors on the disease; (5) preventive strategies, such as anaerobic bacterial supplementation; and (6) surgical interventions, including peritoneal drainage. Understanding some of these important aspects of necrotizing enterocolitis may help improve the outlook of patients with this dreaded disease. Although the incidence of neonatal necrotizing enterocolitis (NEC) and the mortality stemming from this disease have not significantly improved over the last 30 years, there is exciting new information that may significantly improve the outlook of patients with this overwhelming intestinal emergency in the near future.

Intercellular adhesion molecule-1 gene expression by glial cells. Differential mechanisms of inhibition by IL-10 and IL-6.

The central nervous system contains two major glial cell types, astrocytes and microglia, which function as immune effector cells within the central nervous system. We have been studying the ability of glial cells to express gene products involved in immune responsiveness, with an emphasis on expression of ICAM-1. We demonstrated previously that three proinflammatory cytokines, TNF-alpha, IL-1 beta, and IFN-gamma, as well as IFN-gamma plus LPS, can enhance ICAM-1 expression by primary rat astrocytes. In this study, we examined ICAM-1 expression by primary rat microglia and found that only IFN-gamma and IFN-gamma/LPS enhance ICAM-1 gene expression. These data indicate that ICAM-1 expression by astrocytes and microglia is enhanced differentially by various proinflammatory cytokines. We next examined the effect of two cytokines, IL-10 and IL-6, on ICAM-1 expression. IL-10 alone has no effect on ICAM-1 expression, but it inhibits the enhancement of ICAM-1 intracellular and membrane protein expression in both cell types, although it has no influence on ICAM-1 steady-state mRNA levels. These results suggest that IL-10 affects ICAM-1 expression at the translational and/or post-translational level. IL-6 alone also had no effect on ICAM-1 expression in either astrocytes or microglia, but it inhibited induction of both ICAM-1 mRNA and protein expression in these cells. Inhibition of ICAM-1 mRNA steady-state levels by IL-6 was not the result of degradation of the ICAM-1 message, suggesting an effect at the transcriptional level. Thus, both IL-10 and IL-6 can inhibit ICAM-1 expression by glial cells, although they do so by contrasting mechanisms.

cAMP-regulated trafficking of epitope-tagged CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated chloride channel responsible for cAMP-induced Cl secretion across the apical membranes of epithelial cells. To optimize its detection in membrane localization studies, we tagged CFTR with epitope sequences at the carboxy terminus or in the fourth external loop. The function of six different tagged-CFTRs was tested in two different physiological assays. CFTRs containing the M2 epitope responded to cAMP, whereas cells expressing CFTR with the hemagglutinin HA tag showed little or no cAMP response. Using CFTR tagged in the fourth external loop, we demonstrate that cAMP activation using forskolin results in an increase in CFTR in the plasma membrane of HeLa cells. Forskolin inhibited CFTR endocytosis, and this contributes to the increase in cell surface CFTR expression. Our results indicate that regulation of cell surface CFTR contributes to the increase in plasma membrane Cl conductance evoked by cAMP stimulation.

Distinct functional properties of Rab3A and Rab3B in PC12 neuroendocrine cells.

Rab3A and Rab3B are highly homologous monomeric GTPases that are putative regulators of exocytosis in those tissues in which they are expressed. We have characterized and directly compared the targeting and functional properties of these isoforms in PC12 neuroendocrine cells. Rab3A and Rab3B both targeted to norepinephrine (NE)-containing large dense core vesicles (LDCVs) when stably expressed in PC12 cells, as determined by immunofluorescence and membrane fractionation. Both Rab3 isoforms also bound to recombinant rabphilin-3A in a GTP-dependent manner. The membrane association of rabphilin-3A was modestly enhanced in Rab3B-expressing PC12 cells relative to Rab3A-overexpressing cells. In addition, overexpression of Rab3A modestly inhibited Ca2+-evoked NE release, whereas Rab3B and a GTP binding mutant (Rab3B N135I) markedly stimulated the efficiency of [3H]NE secretion by PC12 cells (i.e. secretion normalized to total cell radioactivity). Expression of Rab3B and Rab3B N135I increased not only the efficiency of NE secretion but also the accumulation of [3H]NE into LDCVs (i.e. the secretory cargo available for secretion). Neither of these effects was attributable to changes in the numbers of LDCVs nor the docking of LDCVs at the plasma membrane. Our results indicate that Rab3A and Rab3B have similar membrane targeting properties and are capable of interacting with the same putative downstream effector; i.e. rabphilin-3A. However, these isoforms are functionally distinct monomeric GTPases with Rab3B stimulating a late step in Ca2+-evoked secretion when expressed in PC12 cells.

Regulation of fluid-phase endocytosis in alveolar macrophages.

We investigated whether fluid-phase endocytosis in rabbit alveolar macrophages (AM) was regulated by alterations in intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Suspensions of freshly isolated AM were incubated with anionic dextrans (mol mass = 10 kDa), coupled to fluorescein isothiocyanate (FITC), at either 37 or 4 degrees C. There was a rapid increase in AM-associated fluorescence, quantified by laser flow-cytometry and video microscopy during the first hour of incubation at 37 degrees C, which was directly proportional to the amount of tracer present in the medium. In contrast, at 4 degrees C, AM fluorescence was similar to autofluorescence. Incubation of AM with forskolin (50 microM) or 3-isobutyl-1-methyl xanthine (IBMX; 0.1 mM) increased their cAMP content by 67 +/- 2 and 52 +/- 5% (mean +/- SE; n = 4) and decreased FITC-dextran uptake by 29 +/- 4 and 31 +/- 4% (n = 3). On the other hand, incubation of AM with 0.5 mM IBMX inhibited FITC-dextran uptake by 62 +/- 4% (n = 3), without any further increase in cAMP. Incubation of AM with 0.4 mM 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP), a cell-permeable analogue of cAMP, decreased FITC-dextran uptake by 48 +/- 5% (n = 6). Pulse-chase experiments showed that the rate of FITC-dextran exocytosis was not affected by cAMP. We concluded that fluid-phase endocytosis in rabbit AM is regulated by cAMP and by an additional, cAMP-independent mechanism of IBMX.

Nitric oxide inhibits heterologous CFTR expression in polarized epithelial cells.

Nitric oxide (. NO) has been implicated in a wide range of autocrine and paracrine signaling mechanisms. Herein, we assessed the role of exogenous. NO in the modulation of heterologous gene expression in polarized kidney epithelial cells (LLC-PK(1)) that were stably transduced with a cDNA encoding human wild-type cystic fibrosis transmembrane conductance regulator (CFTR) under the control of a heavy metal-sensitive metallothionein promoter (LLC-PK(1)-WTCFTR). Exposure of these cells to 125 microM DETA NONOate at 37 degrees C for 24 h (a chemical. NO donor) diminished Zn(2+)-induced and uninduced CFTR protein levels by 43.3 +/- 5.1 and 34.4 +/- 17.1% from their corresponding control values, respectively. These changes did not occur if red blood cells, effective scavengers of. NO, were added to the medium. Exposure to. NO did not alter lactate dehydrogenase release in the medium or the extent of apoptosis. Coculturing LLC-PK(1)-WTCFTR cells with murine fibroblasts that were stably transduced with the human inducible. NO synthase cDNA gene also inhibited CFTR protein expression in a manner that was antagonized by 1 mM N(G)-monomethyl-L-arginine in the medium. Pretreatment of LLC-PK(1)-WTCFTR with ODQ, an inhibitor of guanylyl cyclase, did not affect the ability of. NO to inhibit heterologous CFTR expression; furthermore, 8-bromo-cGMP had no effect on heterologous CFTR expression. These data indicate that. NO impairs the heterologous expression of CFTR in epithelial cells at the protein level via cGMP-independent mechanisms.

Effect of polyunsaturated fatty acid (PUFA) supplementation on intestinal inflammation and necrotizing enterocolitis (NEC) in a neonatal rat model.

Inasmuch as long-chain polyunsaturated fatty acids (PUFA, metabolites of the essential n-3 and n-6 fatty acids) are known to modulate inflammation, we hypothesized that supplementation of formula with these compounds would prevent necrotizing enterocolitis (NEC) and intestinal inflammation in our neonatal rat model. Newborn rats were stressed with asphyxia and formula feeding, and randomly assigned to control formula, control with PUFA supplementation, and PUFA with nucleotides. Animals were followed for 72--96 h and assessed for death, gross and histologic NEC, intestinal apoptosis, endotoxemia, and intestinal mRNA synthesis of phospholipase A(2)-II (rate-limiting enzyme for platelet activating factor production), platelet activating factor receptor, and inducible nitric oxide synthase. We found that PUFA reduced the incidence of death and NEC compared with the other groups (NEC 8 of 24 versus 17 of 24 control and 13 of 23 PUFA + nucleotides, p < 0.05). Furthermore, PUFA reduced plasma endotoxemia at 48 h (25 +/- 4 EU/mL versus 276 +/- 39 EU/mL in control and 170 +/- 28 EU/mL in PUFA + nucleotide), intestinal phospholipase A(2)-II expression at 24 h, and platelet activating factor receptor expression at 48 h. Formula supplementation had no effect on apoptosis of intestinal epithelium or intestinal inducible nitric oxide synthase expression. Addition of nucleotides with PUFA abrogated the beneficial effects of PUFA on intestinal inflammation. We conclude that PUFA reduces the incidence of NEC and intestinal inflammation in a neonatal rat model.

Regulated endocytosis in a chloride secretory epithelial cell line.

The colonic epithelial cell line T84 has been shown to be a good model to investigate the regulation of Cl- secretion by the adenosine 3',5'-cyclic monophosphate (cAMP)-mediated second messenger cascade. Regulated exocytic insertion and endocytic retrieval of transport proteins, or proteins that regulate transport proteins, is one mechanism proposed to regulate plasma membrane solute permeabilities. The aims of our studies were to characterize endocytic processes in T84 cells and to investigate their regulation by known activators of Cl- secretion that are mediated by the cAMP second messenger cascade. Forskolin, an activator of adenylate cyclase, caused a marked inhibition of endocytic uptake of the fluid-phase marker horseradish peroxidase (HRP) and the adsorptive marker wheat germ agglutinin conjugated to HRP. Similar inhibition was obtained with vasoactive intestinal peptide, a secretagogue whose receptor is coupled to adenylate cyclase, and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, a membrane-permeable cAMP analogue. 1,9-Dideoxy-forskolin, a forskolin analogue that fails to activate adenylate cyclase, was without effect on endocytosis. Our data show that the net rate of endocytosis, as measured by fluid-phase uptake, is decreased by a cAMP-mediated mechanism. Because the number of Cl- channels or associated regulatory proteins in the plasma membrane reflects a balance between their exocytic insertion and endocytic retrieval, we propose that the cAMP-mediated decrease in endocytosis could contribute to the concomitant increase in plasma membrane Cl- permeability.

Quantitation of alveolar distribution of liposome-entrapped antioxidant enzymes.

Liposome-encapsulated Cu,Zn superoxide dismutase (Cu,Zn SOD) and catalase (CAT) were instilled intratracheally in rabbits, and the temporal and spatial distribution of Cu,Zn SOD and CAT within the lung was assessed at the organ and cellular levels. Specific activities of Cu,Zn SOD and CAT were increased in both lung homogenates and isolated alveolar type II pneumocytes. Peak Cu,Zn SOD activities in lung homogenates and alveolar type II cells were observed 4 h after liposome instillation and returned to control levels by 24 h, whereas CAT activities remained significantly above controls. There were no significant differences in liposome distribution or antioxidant enzyme uptake among lung lobes. The distribution of fluorescently labeled Cu,Zn SOD and CAT was assessed with the use of epifluorescence microscopy and digital image processing to determine patterns of cellular incorporation of liposome-entrapped Cu,Zn SOD and CAT within the lung. Although the mean fluorescence intensity of alveoli from rabbits instilled with liposomes containing labeled Cu,Zn SOD and CAT was greater than autofluorescence observed with either no liposome or empty liposome instillation, fluorescence intensity varied between adjacent alveoli. Both fluorescently labeled Cu,Zn SOD and CAT were located cytosolically, and uptake was not limited to alveolar type II pneumocytes. These results demonstrate that a single intratracheal instillation of liposomes can effect increases in Cu,Zn SOD and CAT activities in distal lung cells, including alveolar type I and type II cells and macrophages.

One-step affinity isolation of recombinant protein using the baculovirus/insect cell expression system.

We have developed two baculovirus transfer vectors which allow single-step affinity isolation of recombinant proteins after expression in insect cells. Using these vectors, recombinant proteins are synthesized as fusions with glutathione-S-transferase and are amenable to enrichment from a crude insect cell lysate using glutathione affinity agarose. After affinity isolation, glutathione-S-transferase can be cleaved from the recombinant polypeptides of interest at an engineered thrombin cleavage site. We used this approach to successfully isolate glutathione-S-transferase, the human low density lipoprotein receptor, two large polypeptides containing cytoplasmic domains of the cystic fibrosis transmembrane conductance regulator (CFTR), and the full-length CFTR. The approach has potential advantages over prokaryotic overexpression of foreign polypeptides, including: (i) eukaryotic post-translational modification of expressed protein, (ii) increased solubility of recombinant fusion proteins synthesized in insect cells leading to increased affinity yield under mild conditions, and (iii) production of large and/or complex polypeptides which might be difficult to purify from prokaryotic cells. The method also allows enrichment of recombinant protein representing a small fraction (less than 5%) of total insect cell protein produced and provides a general method for eukaryotic protein synthesis and isolation which is independent of the particular protein being expressed.

Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells.

We have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropyl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment (3.9 or 1.3 microM) for 24 hr virtually abolished Cl- transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br- uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatment, but not after treatment for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl- transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl- permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl- permeability.

Activation of DeltaF508 CFTR in an epithelial monolayer.

The DeltaF508 mutation leads to retention of cystic fibrosis transmembrane conductance regulator (CFTR) in the endoplasmic reticulum and rapid degradation by the proteasome and other proteolytic systems. In stably transfected LLC-PK1 (porcine kidney) epithelial cells, DeltaF508 CFTR conforms to this paradigm and is not present at the plasma membrane. When LLC-PK1 cells or human nasal polyp cells derived from a DeltaF508 homozygous patient are grown on plastic dishes and treated with an epithelial differentiating agent (DMSO, 2% for 4 days) or when LLC-PK1 cells are grown as polarized monolayers on permeable supports, plasma membrane DeltaF508 CFTR is significantly increased. Moreover, when confluent LLC-PK1 cells expressing DeltaF508 CFTR were treated with DMSO and mounted in an Ussing chamber, a further increase in cAMP-activated short-circuit current (i.e., approximately 7 microA/cm2; P < 0.00025 compared with untreated controls) was observed. No plasma membrane CFTR was detected after DMSO treatment in nonepithelial cells (mouse L cells) expressing DeltaF508 CFTR. The experiments describe a way to augment DeltaF508 CFTR maturation in epithelial cells that appears to act through a novel mechanism and allows insertion of functional DeltaF508 CFTR in the plasma membranes of transporting cell monolayers. The results raise the possibility that increased epithelial differentiation might increase the delivery of DeltaF508 CFTR from the endoplasmic reticulum to the Golgi, where the DeltaF508 protein is shielded from degradative pathways such as the proteasome and allowed to mature.