Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two ATP-binding domains are found in members of the family of ATP-dependent transport proteins, which includes P-glycoprotein and cystic fibrosis transmembrane conductance regulator. To investigate the involvement of the two ATP-binding domains in the ATPase activity of P-glycoprotein, full-length and the 5'-half of human MDR1 cDNA, which encodes P-glycoprotein, were fused with the Escherichia coli lacZ gene and expressed in NIH3T3 cells. Immunoprecipitated full-length P-glycoprotein beta-galactosidase showed ATPase activity with apparent specific activity of 180 nmol/mg/min, a value higher than previously reported, in the presence of phospholipids, suggesting that stabilization of the transmembrane domains is necessary for ATP hydrolysis. N-terminal half P-glycoprotein-beta-galactosidase also showed ability to hydrolyze ATP but with slightly lower specific activity. Both ATPase activities showed similar characteristics when the effect of several inhibitors was analyzed, indicating that the N-terminal ATP-binding domain contains all residues necessary to hydrolyze ATP without interacting with the C-terminal ATP-binding domain.
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PMID:P-glycoprotein. ATP hydrolysis by the N-terminal nucleotide-binding domain. 134 41

In preparation for human trials of gene therapy for cystic fibrosis (CF), we performed a preclinical study of gene transfer into the lungs of baboons. Recombinant adenovirus vectors containing expression cassettes for human cystic fibrosis transmembrane conductance regulator (CFTR) and Escherichia coli beta-galactosidase (lacZ) were instilled through a bronchoscope into limited regions of lung in 14 baboons. A detailed accounting of the extent, distribution, and duration of gene expression is contained in a companion article (Engelhardt et al., 1993b). In this article, we report the results of toxicity studies in which clinical laboratory tests, chest radiographs, and necropsy studies were used to detect adverse effects. The only adverse effect noted was a mononuclear cell inflammatory response within the alveolar compartment of animals receiving doses of virus that were required to induce detectable gene expression. Minimal inflammation was seen at 10(7) and 10(8) pfu/ml, but at 10(9) and more prominently at 10(10) pfu/ml, a perivascular lymphocytic and histiocytic infiltrate was seen. The intensity of inflammation increased between 4 and 21 days. At its greatest intensity, there was diffuse alveolar wall damage with intra-alveolar edema. Airways were relatively spared, despite the intensity of alveolar inflammation. Clinical tests did not accurately reflect the presence of lung inflammation, with the exception of chest radiographs which revealed alveolar infiltrates, but only in regions of lung having the greatest intensity inflammation. We conclude that adenovirus-mediated gene transfer into the lungs of baboons is associated with development of alveolar inflammation at high doses of virus.
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PMID:Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: toxicity study. 751 46

The requirements for efficient translation termination are incompletely understood. Since the local context surrounding stop codons can influence the efficiency of translation termination, premature termination codons introduced by random mutation may not always terminate at the optimal efficiencies expected of naturally occurring stop codons. To investigate whether this could result in physiologically significant levels of read through, we examined the suppression of premature translation termination mutations within a sequence motif of the yeast Ste6 protein (Ste6p) that is highly conserved among members of the ATP-binding cassette (ABC) transporter family. The human cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in individuals with the disease cystic fibrosis, is also a member of this protein family. The mutations examined in Ste6p were chosen because a premature termination codon at the corresponding residue of CFTR has previously been reported to cause less severe pulmonary involvement than some missense mutations, suggesting that low level suppression of this stop codon could be occurring. Our results indicate that these premature stop codons in Ste6p can be suppressed at frequencies as high as 10%. Characterization of this phenomenon using a beta-galactosidase read through assay system showed that a limited sequence context surrounding this site contained information that was sufficient to cause suppression of translation termination. Amino acid sequence analysis of the full-length translation products produced by read through of an amber codon demonstrated that termination suppression was mediated by near-cognate tRNA mispairing that resulted in the insertion of tyrosine, lysine, or tryptophan.
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PMID:Premature translation termination mutations are efficiently suppressed in a highly conserved region of yeast Ste6p, a member of the ATP-binding cassette (ABC) transporter family. 751 33

Adenovirus vectors are a promising vehicle to deliver cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to airway epithelia. However, the value of adenovirus vectors will depend on the efficiency with which the vector can correct the defective fluid transport that is though to underlie the pathogenesis of the disease. To address the efficiency of gene transfer, we applied adenovirus vectors expressing CFTR (Ad2/CFTR-1) or beta-galactosidase to the mucosal surface of primary cultures of airway epithelial cells grown as polarized epithelial monolayers on permeable filter supports. These conditions provide a model that reproduces the physiology of the airways in vivo. We found that after adding 1 moi Ad2/CFTR-1 to the mucosal surface, cAMP agonists stimulated fluid secretion that was within the range observed in epithelia from normal subjects. When we measured electrolyte transport, we found that as little as 0.1 moi partially restored cAMP-stimulated Cl- secretion, and at 10 moi Cl- secretion was in the normal range. A related vector encoding beta-galactosidase generated activity in approximately 20% of cells at an moi of 1 and 90% of cells at an moi of 10. These data suggest that Ad2/CFTR-1 is very efficient at restoring normal fluid and electrolyte transport to CF airway epithelia. Thus, they suggest that relatively low input doses could be used for gene transfer to CF airway epithelia.
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PMID:Correction of cAMP-stimulated fluid secretion in cystic fibrosis airway epithelia: efficiency of adenovirus-mediated gene transfer in vitro. 751 84

Recombinant human adenovirus (Ad) vectors are leading candidates for human gene therapy for cystic fibrosis (CF) based on demonstration of efficient transfer of exogenous genes to rodent respiratory epithelium in vivo and human respiratory cells in vitro. The safety of Ad-mediated gene transfer to the respiratory epithelium and acute (up to 21 days) clinical responses to airway delivery of a replication-deficient recombinant, E1-, E3- Ad type 5-based vector containing the human cystic fibrosis transmembrane conductance regulator cDNA (AdCFTR) were evaluated in rhesus monkeys. Airway delivery of an Ad vector with the lacZ marker gene demonstrated beta-galactosidase expression in epithelial cells. Animals administered intratracheal AdCFTR demonstrated human CFTR cDNA expression in airway epithelial cells. Animals administered AdCFTR intranasal, and 24 hr later, intrabronchial [2 x 10(7) to 5 x 10(10) plaque-forming units (pfu), n = 12], in a fashion similar to a proposed human protocol, or only intrabronchial (10(11) pfu, n = 3), had no significant changes in clinical parameters compared to vehicle controls (n = 6). Microscopic analysis of the lung by necropsy or bronchoalveolar lavage demonstrated a dose-dependent increase in inflammatory cells, primarily lymphocytes, in the area where AdCFTR was delivered, which persisted for at least 2 months in some animals. Serum anti-Ad type 5 neutralizing antibody titers did not rise and shed Ad was not detected. The presence of AdCFTR DNA, analyzed by the polymerase chain reaction (PCR), was not detected in organs outside the lung. These data demonstrate that AdCFTR is well tolerated in non-human primates, although there is dose-dependent inflammation in the lung not clinically apparent.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Acute responses of non-human primates to airway delivery of an adenovirus vector containing the human cystic fibrosis transmembrane conductance regulator cDNA. 752 1

Human fetal lung rudiments (8-12 weeks of development) undergo considerable growth upon microsurgical ectopic implantation in the xenograft-tolerant SCID mouse, and differentiate into a lung-like tissue that includes: (i) bronchial structures lined with pseudostratified, secretory, ciliated epithelium surrounded by smooth muscle and cartilage rings, (ii) submucosal glands, and (iii) alveolar sacs. Normal expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein was detected by immunostaining in those grafts, and similar differentiation was observed from either normal or cystic fibrosis (CF) fetal lung rudiments. Upon microinjection into human CF or normal lung grafts in SCID mice, beta-galactosidase-adenovirus gene constructs were efficiently transduced into epithelial and glandular cells. Such an in vivo replica of the human respiratory tissue may be a useful experimental model to study normal and pathologic lung development, and to assay candidate therapeutic gene constructs preclinically.
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PMID:Gene transfer to human fetal pulmonary tissue developed in immunodeficient SCID mice. 753 Apr 95

We have raised mouse monoclonal antibodies to eight synthetic peptides corresponding to different regions of the human cystic fibrosis transmembrane conductance regulator (CFTR) and rabbit polyclonal antisera to beta-galactosidase fusion proteins which encompass three different regions of CFTR. Immunoblot, immunoprecipitation, immunofluorescence and immunocytochemical experiments demonstrate that, in addition to recognising CFTR, these antibodies recognise one or more immunologically related proteins with a similar molecular mass, calcium responsiveness and tissue distribution to CFTR.
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PMID:Production and characterisation of monoclonal and polyclonal antibodies to different regions of the cystic fibrosis transmembrane conductance regulator (CFTR): detection of immunologically related proteins. 754 88

A new adenovirus-based vector (Ad2/CFTR-1) has been constructed in which the cDNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis (CF) gene product, replaces the early region 1 coding sequences, E1a and E1b. The virus retains the E3 region. Ad2/CFTR-1 and a related construct encoding beta-galactosidase replicate in human 293 cells which provide E1 gene functions in trans. Replication of these recombinant viruses was not detected in a variety of other cells, although very limited viral DNA synthesis and transcription from the E4 and L5 regions could be measured. These E1-deletion vectors were also deficient in cellular transformation, shut-off of host cell protein synthesis, and production of cytopathic effects, even at high multiplicities of infection. Ad2/CFTR-1 produced CFTR protein in a variety of cells including airway epithelia from CF patients. Expression of functional CFTR protein in a CF airway epithelial monolayer was detected by correction of the Cl- transport defect characteristic of CF. Surprisingly low multiplicities of infection (0.1 moi) were sufficient to generate CFTR Cl- current across a CF epithelial monolayer in vitro. These data, together with the lack of obvious toxicity, suggest that Ad2/CFTR-1 should be suitable for CF gene therapy.
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PMID:Development and analysis of recombinant adenoviruses for gene therapy of cystic fibrosis. 769 Nov 87

Previous studies have established that gene transfer into myocardial cells in vivo is detectable after direct injection of plasmid DNA. Recently, adenovirus vectors have been shown to provide an efficient method for gene transfer into a wide range of tissues. Therefore, this study sought to assess the efficiency and stability of adenovirus-mediated gene transfer into myocardium and to compare this method with that using plasmid-based gene transfer techniques. Adult rats underwent myocardial injection via a subdiaphragmatic approach. Gene transfer efficiency was compared using direct injection of an adenovirus vector encoding for the marker gene beta-galactosidase (beta-gal), a control adenovirus vector encoding for the cystic fibrosis transmembrane conductance regulator gene, a plasmid encoding for beta-gal, or a control plasmid. Hearts infected with an adenovirus vector containing the beta-gal gene showed significantly increased beta-gal enzymatic activity compared with hearts injected with beta-gal plasmid. Histological examination revealed that cardiac myocytes were the target of adenovirus-mediated gene transfer. A time course of gene expression showed that beta-gal enzymatic activity peaked during the first week following injection. Adenovirus vectors provide an efficient but transient method for in vivo gene expression in myocardium.
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PMID:Efficient gene transfer into myocardium by direct injection of adenovirus vectors. 822 91

The Sf9 insect Spodoptora frugiperda cell line was used for heterologous expression of the cloned human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, or the cloned beta-galactosidase gene, using the baculovirus Autographa califonica as the infection vector. Using application of the patch-clamp technique, evidence for functional expression of CFTR was obtained according to the following three criteria. Firstly, whole-cell currents recorded 2 days after infection with CFTR revealed a statistically significant increase of membrane conductance, approximately 25 times above that of mock-infected control cells, with the reversal potential of the major current component being governed by the chloride equilibrium potential (ECl). Secondly, in contrast to uninfected cells and cells infected with beta-galactosidase, the membrane conductance to chloride of CFTR-injected cells was stimulated by cytosolic adenosine 3',5'-cyclic monophosphate (cAMP), which was raised by exposing the cells to 10 microM forskolin. Thirdly, recordings of currents through single channels in excised outside-out membrane patches of CFTR-infected cells revealed channels which were clearly different from the native insect chloride channel. Excised outside-out patches of CFTR-infected and forskolin-stimulated cells exhibited wave-like gating kinetics of well-resolved current transitions. All-point Gaussian distributions revealed contributions from several (five to nine) identical channels. Such channels, in excised outside-out patches, studied with a pipette [Cl-] = 40 mM and a bath [Cl-] = 150 mM, rectified the current in agreement with simple electrodiffusion and with a single-channel Goldman-Hodgkin-Katz permeability, PCl = 1. 34 x 10(-14) +/- 0.23 x 10(-14 )cm3/s (n = 5), corresponding to a physiological single-channel conductance of 2.8 +/- 0.5 pS (VM = ECl) and a limiting conductance, gamma150/150, = 7.7 +/- 1.3 pS ([Cl-]Bath = [Cl-]Cell = 150 mM). Currents recorded from multichannel excised outside-out patches could shift from the above mode of resolvable unitary conductance transitions to one which was too fast to reveal the dwell-times of closed and open states. During periods characterized by noisy currents, the variance (sigma2) of current fluctuations about their stationary mean value depicted a U-shaped function of membrane potential, with a minimum value at a pipette potential where the chloride current was shown to be zero. Thus, it can be concluded that the current fluctuations are caused by fast gating of channels specific for chloride ions. Switching back and forth between the two gating modes of clusters of chloride channels occurred from moment to moment in excised patches when the membrane potential was held at a constant value indicating cooperative gating as a result of interaction between neighbouring chloride channels.
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PMID:Clusters of Cl- channels in CFTR-expressing Sf9 cells switch spontaneously between slow and fast gating modes. 876 14


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