Biomedical subjects

R J Gregory

Publications and source records attributed to R J Gregory.

At least 73 records · Page 4Linked to original sources

PubMed1992

Human cystic fibrosis transmembrane conductance regulator directed to respiratory epithelial cells of transgenic mice.

Human cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in transgenic mice under the control of transcriptional elements derived from the human surfactant protein C (SP-C) gene. The hCFTR mRNA was expressed in lungs and testes: in the lung, we found hCFTR mRNA in bronchiolar and alveolar epithelial cells, and CFTR protein in respiratory epithelial cells. While the level of expression of hCFTR mRNA varied, hCFTR mRNA and protein were detected in pulmonary epithelial cells of several lines. Lung weight, morphology, somatic growth and reproductive capacity were not altered by expression hCFTR in lung and testes of the transgenics. Our findings suggest that hCFTR can be safely transferred to lung epithelial cells for CF therapy.

Animals↗

PubMed1992

External fixation of lower limb fractures in children.

Thirty-four tibial and femoral shaft fractures in 32 children between the ages of 3 and 15 years were treated by external fixation over a 5-year period. The indications were fractures occurring in association with other major injuries and failure of conservative treatment to maintain satisfactory reduction. There was one case of delayed union and one early refracture. The overall pin track infection rate was 6%, but the rate for the tibial pins (2.1%) was much lower than for the femoral pins (10.3%). Union was achieved at an average of 11.7 weeks in the femoral fractures and 10.0 weeks in the tibial fractures. The use of external fixation is recommended for childhood femoral and tibial fractures, particularly in children with multiple injuries.

Adolescent↗

PubMed1991

Nucleoside triphosphates are required to open the CFTR chloride channel.

The CFTR Cl- channel contains two predicted nucleotide-binding domains (NBD1 and NBD2); therefore, we examined the effect of ATP on channel activity. Once phosphorylated by cAMP-dependent protein kinase (PKA), channels required cytosolic ATP to open. Activation occurred by a PKA-independent mechanism. ATP gamma S substituted for ATP in PKA phosphorylation, but it did not open the channel. Several hydrolyzable nucleotides (ATP greater than GTP greater than ITP approximately UTP greater than CTP) reversibly activated phosphorylated channels, but nonhydrolyzable analogs and Mg(2+)-free ATP did not. Studies of CFTR mutants indicated that ATP controls channel activity independent of the R domain and suggested that hydrolysis of ATP by NBD1 may be sufficient for channel opening. The finding that nucleoside triphosphates regulate CFTR begins to explain why CF-associated mutations in the NBDs block Cl- channel function.

Adenosine Triphosphate↗

PubMed1991

Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel.

CFTR, the protein associated with cystic fibrosis, is phosphorylated on serine residues in response to cAMP agonists. Serines 660, 737, 795, and 813 were identified as in vivo targets for phosphorylation by protein kinase A. The SPQ fluorescence assay revealed that mutagenesis of any one of these sites did not affect Cl- channel activity. Indeed, concomitant mutagenesis of three of the four sites still resulted in cAMP-responsive Cl- channel activity. However, mutagenesis of all four sites abolished the response. One interpretation of these results is that the CFTR Cl- channel is blocked by the R domain and that phosphorylation on serines by protein kinase A electrostatically repels the domain, allowing passage of Cl-. The four phosphorylation events appear to be degenerate: no one site is essential for channel activity, and, at least in the case of serine 660, phosphorylation at one site alone is sufficient for regulation of Cl- channel activity.

Chloride Channels↗

PubMed1991

Demonstration that CFTR is a chloride channel by alteration of its anion selectivity.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) generates adenosine 3',5'-monophosphate (cAMP)-regulated chloride channels, indicating that CFTR is either a chloride channel or a chloride channel regulator. To distinguish between these possibilities, basic amino acids in the putative transmembrane domains were mutated. The sequence of anion selectivity of cAMP-regulated channels in cells containing either endogenous or recombinant CFTR was bromide greater than chloride greater than iodide greater than fluoride. Mutation of the lysines at positions 95 or 335 to acidic amino acids converted the selectivity sequence to iodide greater than bromide greater than chloride greater than fluoride. These data indicate that CFTR is a cAMP-regulated chloride channel and that lysines 95 and 335 determine anion selectivity.

Amino Acid Sequence↗

PubMed1991

Effect of deleting the R domain on CFTR-generated chloride channels.

The cystic fibrosis transmembrane conductance regulator (CFTR), which forms adenosine 3',5'-monophosphate (cAMP)-regulated chloride channels, is defective in patients with cystic fibrosis. This protein contains two putative nucleotide binding domains (NBD1 and NBD2) and an R domain. CFTR in which the R domain was deleted (CFTR delta R) conducted chloride independently of the presence of cAMP. However, sites within CFTR other than those deleted also respond to cAMP, because the chloride current of CFTR delta R increased further in response to cAMP stimulation. In addition, deletion of the R domain suppressed the inactivating effect of a mutation in NBD2 (but not NBD1), a result which suggests that NBD2 interacts with the channel through the R domain.

Binding Sites↗

PubMed1991

Generation of cAMP-activated chloride currents by expression of CFTR.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. In order to evaluate its function, CFTR was expressed in HeLa, Chinese hamster ovary (CHO), and NIH 3T3 fibroblast cells, and anion permeability was assessed with a fluorescence microscopic assay and the whole-cell patch-clamp technique. Adenosine 3',5'-monophosphate (cAMP) increased anion permeability and chloride currents in cells expressing CFTR, but not in cells expressing a mutant CFTR (delta F508) or in nontransfected cells. The simplest interpretation of these observations is that CFTR is itself a cAMP-activated chloride channel. The alternative interpretation, that CFTR directly or indirectly regulates chloride channels, requires that these cells have endogenous cryptic, chloride channels that are stimulated by cAMP only in the presence of CFTR.

Animals↗

PubMed1991

Comparative evaluation of enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of dourine.

The detection of antibodies against Trypanosoma equiperdum in 689 equid sera was compared by enzyme-linked immunosorbent assay (ELISA), the complement fixation test (CFT) and an indirect immunofluorescent test (IIF). CFT was the least sensitive technique, susceptible to anti-complementary factors and the most technically demanding. IIF was more sensitive, but was only suitable for testing limited numbers of samples. In this study, ELISA was the most sensitive test, the least labour intensive and lends itself to a considerable degree of automation. It is suggested that ELISA would be relatively easy to standardise between laboratories and an ELISA protocol could be adopted as the internationally approved test for equine health certification.

Animals↗

PubMed1991

Maturation and function of cystic fibrosis transmembrane conductance regulator variants bearing mutations in putative nucleotide-binding domains 1 and 2.

One feature of the mutations thus far found to be associated with the disease cystic fibrosis (CF) is that many of them are clustered within the first nucleotide-binding domain (NBD) of the CF transmembrane conductance regulator (CFTR). We sought to discover the molecular basis for this clustering by introducing into the two NBDs of CFTR mutations either mimicking amino acid changes associated with CF or altering residues within highly conserved motifs. Synthesis and maturation of the mutant CFTR were studied by transient expression in COS cells. The ability of the altered proteins to generate cyclic AMP-stimulated anion efflux was assessed by using 6-methoxy-N-(sulfopropyl) quinolinium (SPQ) fluorescence measurements in HeLa cells expressing mutated plasmids. The results show that (i) all CF-associated mutants, with one exception, lack functional activity as measured in the SPQ assay, (ii) mutations in NBD1 are more sensitive to the effects of the same amino acid change than are the corresponding mutations in NBD2, (iii) cells transfected with plasmids bearing CF-associated mutations commonly but not exclusively lack mature CFTR, (iv) NBD mutants lacking mature CFTR fail to activate Cl- channels, and (v) the glycosylation of CFTR, per se, is not required for CFTR function. We reason that the structure of NBD1 itself or of the surrounding domains renders it particularly sensitive to mutational changes. As a result, most NBD1 mutants, but only a few NBD2 mutants, fail to mature or lack functional activity. These findings are consistent with the observed uneven distribution of CFTR missense mutations between NBD1 and NBD2 of CF patients.

Amino Acid Sequence↗

PubMed1991

Identification and regulation of the cystic fibrosis transmembrane conductance regulator-generated chloride channel.

Cystic fibrosis transmembrane conductance regulator (CFTR) generates cAMP-regulated Cl- channels; mutations in CFTR cause defective Cl- channel function in cystic fibrosis epithelia. We used the patch-clamp technique to determine the single channel properties of Cl- channels in cell expressing recombinant CFTR. In cell-attached patches, an increase in cellular cAMP reversibly activated low conductance Cl- channels. cAMP-dependent regulation is due to phosphorylation, because the catalytic subunit of cAMP-dependent protein kinase plus ATP reversibly activated the channel in excised, cell-free patches of membrane. In symmetrical Cl- solutions, the channel had a channel conductance of 10.4 +/- 0.2 (n = 7) pS and a linear current-voltage relation. The channel was more permeable to Cl- than to I- and showed no appreciable time-dependent voltage effects. These biophysical properties are consistent with macroscopic studies of Cl- channels in single cells expressing CFTR and in the apical membrane of secretory epithelia. Identification of the single channel characteristics of CFTR-generated channels allows further studies of their regulation and the mechanism of ion permeation.

Adenosine Triphosphate↗

PubMed1991

Dislocation after primary arthroplasty for subcapital fracture of the hip. Wide range of movement is a risk factor.

Dislocation is the most frequent serious complication following total hip replacement for subcapital femoral fracture. We report a prospective study, using matched groups, which compared the range of hip movement following hip replacement for arthritis and for fracture. The range of movement was significantly greater in the fracture group. We suggest that this is a predisposing factor for dislocation.

Aged↗

PubMed1990

Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.

The gene associated with cystic fibrosis (CF) encodes a membrane-associated, N-linked glycoprotein called CFTR. Mutations were introduced into CFTR at residues known to be altered in CF chromosomes and in residues believed to play a role in its function. Examination of the various mutant proteins in COS-7 cells indicated that mature, fully glycosylated CFTR was absent from cells containing delta F508, delta 1507, K464M, F508R, and S5491 cDNA plasmids. Instead, an incompletely glycosylated version of the protein was detected. We propose that the mutant versions of CFTR are recognized as abnormal and remain incompletely processed in the endoplasmic reticulum where they are subsequently degraded. Since mutations with this phenotype represent at least 70% of known CF chromosomes, we argue that the molecular basis of most cystic fibrosis is the absence of mature CFTR at the correct cellular location.

Animals↗

PubMed1990

Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in cultured cystic fibrosis airway epithelial cells and Cl- channel activation assessed in single cells using a fluorescence microscopic assay and the patch-clamp technique. Expression of CFTR, but not of a mutant form of CFTR (delta F508), corrected the Cl- channel defect. Correction of the phenotypic defect demonstrates a causal relationship between mutations in the CFTR gene and defective Cl- transport which is the hallmark of the disease.

Cell Membrane Permeability↗

PubMed1990

Expression and characterization of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is a common lethal genetic disease that manifests itself in airway and other epithelial cells as defective chloride ion absorption and secretion, resulting at least in part from a defect in a cyclic AMP-regulated, outwardly-rectifying Cl- channel in the apical surface. The gene responsible for CF has been identified and predicted to encode a membrane protein termed the CF transmembrane conductance regulator (CFTR). Identification of a cryptic bacterial promoter within the CFTR coding sequence led us to construct a complementary DNA in a low-copy-number plasmid, thereby avoiding the deleterious effects of CFTR expression on Escherischia coli. We have used this cDNA to express CFTR in vitro and in vivo. Here we demonstrate that CFTR is a membrane-associated glycoprotein that can be phosporylated in vitro by cAMP-dependent protein kinase. Polyclonal and monoclonal antibodies directed against distinct domains of the protein immunoprecipitated recombinant CFTR as well as the endogenous CFTR in nonrecombinant T84 cells. Partial proteolysis fingerprinting showed that the recombinant and non-recombinant proteins are indistinguishable. These data, which establish several characteristics of the protein responsible for CF, will now enable CFTR function to be studied and will provide a basis for diagnosis and therapy.

Chloramphenicol O-Acetyltransferase↗

PubMed1989

Embryonic expression of a Drosophila src gene: alternate forms of the protein are expressed in segmental stripes and in the nervous system.

Expression of the Drosophila src-related gene, Dsrc28C has been investigated at the protein level using monoclonal antibodies. This analysis has revealed that the Dsrc28C gene encodes two protein forms: a 66-kD doublet predicted from the sequence of a cDNA clone and an additional 55-kD form. The 66-kD protein doublet is observed first at the cellular blastoderm stage and is not detectable in embryos after 12 hr of development. Expression of the 55-kD protein lags behind that of the 66-kD doublet and then persists throughout embryogenesis. Indirect immunofluorescence microscopy reveals that Dsrc28C protein is localized to the cell periphery during cellular blastoderm and gastrulation. The cell periphery-associated staining is then resolved into ectodermal stripes along the fully extended germ band. After the stripes fade, cytoplasmic staining of the majority of cells within the central and peripheral nervous system is observed. The 66-kD protein was shown to represent the cell periphery-associated form of the protein through antibody staining of larval salivary glands expressing a heat shock promoter-driven, full-length Dsrc28C cDNA. Staining of embryos with a monoclonal antibody specific for the 66-kD protein indicates that the 55-kD protein is the nervous system form. Thus, the 66- and 55-kD proteins are products of the Dsrc28C gene, which exhibit different temporal and spatial patterns of expression in the embryo.

Amino Acid Sequence↗

PubMed1988

Interaction of Escherichia coli ribosomal protein S8 with its binding sites in ribosomal RNA and messenger RNA.

The ability of ribosomal protein S8 from Escherichia coli to interact with 12 variants of its 16 S rRNA binding site, as well as with a regulatory sequence within spc operon mRNA, has been assessed. Single-site alterations were introduced into the appropriate segment of the E. coli 16 S rRNA gene by mutagenesis in vitro. Their effects on S8-rRNA interaction were measured via a filter-binding assay, utilizing S8 binding sites transcribed in vitro from the altered 16 S rRNA gene fragments. Of the 12 rRNA mutants, six were unable to bind S8. Significantly, five of these occur within a small, phylogenetically conserved internal loop, defined by nucleotides 596-597 and 641-643, suggesting that this structure plays a major role in S8-16 S rRNA recognition. The reduced affinity of S8 for its binding site in these cases was closely correlated with growth defects that resulted from expression of the same mutations in vivo. Alterations at other positions in the S8 binding site had little influence on complex formation or cell growth, as long as they did not disrupt rRNA secondary structure. The specific interaction of S8 with a segment of the spc operon mRNA containing a putative site of translational feedback regulation was demonstrated using appropriate in vitro transcripts in conjunction with the filter-binding assay. The apparent association constant for the S8-mRNA interaction was determined to be approximately 5 x 10(6) M-1, about five times lower than for the interaction of S8 with wild-type 16 S rRNA. The structure of the regulatory binding site, determined by sequence analysis of spc operon mRNA protected by S8 from RNase digestion, was found to contain all of the characteristic features of the 16 S rRNA binding site, demonstrating that the protein associates with structurally similar domains in both RNAs.

Base Sequence↗

PubMed1988

Mutations within the decoding site of Escherichia coli 16S rRNA: growth rate impairment, lethality and intragenic suppression.

Several C----U transitions and small deletions were introduced into the conserved region centered on base C1400 in Escherichia coli 16S rRNA by in vitro mutagenesis. The mutations were placed within rrnB operons on multicopy plasmids under the transcriptional regulation of either the normal rrnB P1P2 promoters or the temperature-inducible PL promoter from bacteriophage lambda and introduced into E. coli hosts. When expressed from the P1P2 promoters, several of the mutant 16S rRNAs impaired cell growth while others, including one in which U replaced C at position 1400 within the ribosomal decoding site, had little or no effect on cell doubling time. However, C----U transitions at positions 1395 and 1407, as well as the deletion of C1400, appeared to render their hosts inviable. Cells in which these mutations were expressed from the lambdaPL promoter died within four generations after induction. Unexpectedly, the lethal phenotype was suppressed intragenically by replacement of G1505 with A, C or U. Suppression may alleviate a functional defect in 30S subunits containing the U1395, U1407 or deltaC1400 mutations.

Base Sequence↗

PubMed1987

Depression in patients referred for psychiatric consultation. A need for a new diagnosis.

The authors analyzed 329 referrals for psychiatric consultation from medical and surgical wards. They found depression to be the most prevalent diagnosis (34%), with Major Depression being the most common DSM-III subtype (49%). Depression was predominant in the elderly (p less than 0.05), in women (p less than 0.05), and in patients with a high degree of psychosocial stressors (p less than 0.01). There were significant differences among the DSM-III subtypes of depression in some of these correlates. The authors emphasize the importance of DSM-III in differentiating among the subtypes of depression in referred patients but they suggest the need for a new diagnosis for depression in the physically ill.

Adjustment Disorders↗