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R J Gregory

Publications and source records attributed to R J Gregory.

At least 19 recordsLinked to original sources
PubMed1994

The cystic fibrosis transmembrane conductance regulator is a dual ATP and chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to a superfamily of proteins implicated in the transport of ions, proteins, and hydrophobic substances. Recent studies have demonstrated that CFTR is a protein kinase A-sensitive anion channel regulated by ATP. In the present study, patch-clamp techniques were used to assess the role of CFTR in the transport of Cl- and ATP. The stable transfection of mouse mammary carcinoma cells, C127i, with the cDNA for human CFTR resulted in the appearance of a diphenylamine-2-carboxylate-inhibitable Cl- channel, which was activated by cAMP under whole-cell and cell-attached conditions and by protein kinase A plus ATP under excised, inside-out conditions. CFTR expression was also associated with the electrodiffusional movement of ATP as indicated by the cAMP activation of ATP currents measured under whole-cell conditions. In excised, inside-out patches, it was demonstrated that ATP currents were mediated by ATP-conductive channels, which were also activated by protein kinase A and blocked by the Cl- channel blocker diphenylamine-2-carboxylate under excised, inside-out conditions. Single-channel currents observed in the presence of asymmetrical Cl-/ATP concentrations indicated that the same conductive pathway was responsible for both ATP and Cl- movement. Thus, CFTR is a multifunctional protein with more than one anion transport capability and may modify signal transduction pathways for Cl- or other secretory processes by the selective delivery of nucleotides to the extracellular domain.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

External ATP and its analogs activate the cystic fibrosis transmembrane conductance regulator by a cyclic AMP-independent mechanism.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel activated by protein kinase A and regulated by ATP in a complex manner. We have applied patch-clamp techniques to C127i mouse mammary carcinoma cells transfected with human CFTR to assess the role of external ATP in the modulation of CFTR function. Extracellular ATP was sufficient to activate non-rectifying, Cl(-)-selective whole-cell currents in CFTR-transfected, but not mock-transfected cells. The ATP-mediated activation of CFTR was independent of protein kinase A since channel activation by ATP was preserved in cells that were (a) depleted of intracellular ATP, (b) incubated with the cAMP antagonist Rp-cAMPS, or (c) exposed to the protein kinase A inhibitor, 5-24 amide. In each of these conditions, 8-Br-cAMP was no longer capable of activating CFTR. The possibility that the extracellular ATP activation of Cl- currents in CFTR-expressing C127i cells was mediated by a P2-type purinergic receptor was supported by studies in which the effect of external ATP on the Cl- currents was mimicked by the ATP analogs, ATP gamma S and beta,gamma-methylene ATP, but not the uridine nucleotide, UTP. Single-channel analysis of ATP-activated Cl -currents under both cell-attached and excised, inside-out patch-clamp configurations indicated that this channel is only present in CFTR-transfected cells and indistinguishable from CFTR. External ATP also activated ATP currents in CFTR-transfected cells, a novel function of CFTR. These findings are consistent with the presence of a purinergic receptor signal transduction mechanism in C127i cells whose activation by external ATP is linked to the activation of CFTR in a cAMP-independent manner. The data provide additional support for the use of ATP and its analogs as alternative therapies in cystic fibrosis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Safety and efficacy of repetitive adenovirus-mediated transfer of CFTR cDNA to airway epithelia of primates and cotton rats.

Gene therapy for cystic fibrosis (CF) will require the safe transfer of CFTR cDNA to airway epithelia in vivo. We showed previously that a recombinant adenovirus, Ad2/CFTR-1, expresses CFTR in vitro. As adenovirus rarely integrates, treatment will require repeated vector administration. We applied Ad2/CFTR-1 to intrapulmonary airway epithelia of cotton rats and nasal epithelia of Rhesus monkeys. In both species we detected CFTR mRNA and protein after repeated administration and in monkeys, protein was detected six weeks after repeat administration. The vector did not replicate and was rapidly cleared. Despite an antibody response, there was no evidence of a local or systemic inflammatory response after repeat administration. These data indicate that repetitive administration of Ad2/CFTR-1 is both safe and efficacious.

Adenoviridae↗
PubMed1994

Grief and loss among Eskimos attempting suicide in western Alaska.

Data were collected prospectively from psychiatric evaluations performed on 53 consecutive Eskimos in the Bering Strait region who attempted suicide. Depressive diagnoses were common (N = 49). Thirty-seven (70%) of the attempts were preceded by a recent interpersonal loss. Sixty percent of the patients had lost a parent during childhood. Poor affective relatedness, especially around issues of loss, was noted in most of the patients. Thus, both multiple losses and limited grieving mechanisms may be important risk factors for attempted suicide in this population.

Adult↗
PubMed1994

The Zung Self-Rating Depression Scale as a potential screening tool for use with Eskimos.

OBJECTIVE: The study assessed the utility of a modified Zung Self-Rated Depression Scale (SDS) in detecting major depression among Eskimo patients in the Bering Straits region of Alaska. METHODS: The modified SDS was administered to Eskimo patients referred for psychiatric evaluation at the community mental health center in Nome. Diagnoses were assigned by a staff psychiatrist based on DSM-III-R criteria. The SDS scores of 25 consecutive patients with a diagnosis of major depression were compared with the scores of 25 consecutive patients assigned other psychiatric diagnoses. RESULTS: The modified SDS was readily understood by patients and took between three and four minutes to complete. The mean SDS score of patients with a diagnosis of major depression was significantly different from that of patients with other diagnoses. The SDS had a sensitivity of 80 percent and specificity of 88 percent for detecting patients with major depression. CONCLUSIONS: The SDS is easy to administer and can differentiate between patients with major depression and those with other psychiatric diagnoses. It can be modified to fit local idiom in an Eskimo community. The SDS shows promise as a clinically relevant screening tool for major depression in populations with limited access to psychiatric care.

Adolescent↗
PubMed1994

After traumatic brain injury: a discussion of consequences.

Traumatic brain injury is followed by many changes in cognition, emotion, social interaction and functioning, and communication. These changes may range from severe to subtle. They create complex difficulties for the individual and his or her network of support. Presented here is a review of the literature on the changes and their impact. The review comprises Part I, on brain injury and its effects, and Part II, on social interaction research.

Adolescent↗
PubMed1993

Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis.

To evaluate the potential of direct transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA for the treatment of cystic fibrosis (CF), we administered an E1-deficient adenovirus, encoding CFTR, to a defined area of nasal airway epithelium of three individuals with CF. This treatment corrected the Cl- transport defect that is characteristic of CF-affected epithelia. After treatment, there was a decrease in the elevated basal transepithelial voltage, and the normal response to a cAMP agonist was restored. We found no evidence of viral replication or virus-associated adverse effects, even at the highest dose tested (25 MOI). These data represent a small step in achieving long-term improvement of CF lung function by gene therapy.

Adenoviridae↗
PubMed1993

Mutations in CFTR associated with mild-disease-form Cl- channels with altered pore properties.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-regulated Cl- channel located in the apical membrane of epithelia. Although cystic fibrosis (CF) is caused by mutations in a single gene encoding CFTR, the disease has a variable clinical phenotype. The most common mutation associated with cystic fibrosis, deletion of a phenylalanine at position 508 (frequency, 67%), is associated with severe disease. But some missense mutations, for example ones in which arginine is replaced by histidine at residue at 117 (R117H; 0.8%), tryptophan at 334 (0.4%), or proline at 347 (0.5%), are associated with milder disease. These missense mutations affect basic residues located at the external end of the second (M2) and in the sixth (M6) putative membrane-spanning sequences. Here we report that, when expressed in heterologous epithelial cells, all three mutants were correctly processed and generated cyclic AMP-regulated apical Cl- currents. Although the macroscopic current properties were normal, the amount of current was reduced. Patch-clamp analysis revealed that all three mutants had reduced single-channel conductances. In addition, R117H showed altered sensitivity to external pH and had altered single-channel kinetics. These results explain the quantitative decrease in macroscopic Cl- current, and suggest that R117, R334 and R347 contribute to the pore of the CFTR Cl- channel. Our results also suggest why R117H, R334W and R347P produce less severe clinical disease and have implications for our understanding of cystic fibrosis.

Animals↗
PubMed1993

Development and analysis of recombinant adenoviruses for gene therapy of cystic fibrosis.

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.

Adenoviruses, Human↗
PubMed1993

Effect of deletion mutations on the function of CFTR chloride channels.

Mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a unique R domain; CFTR functions as a Cl- channel regulated by phosphorylation and by nucleoside triphosphates. To study the domains of CFTR involved in Cl- channel function, we expressed mutants lacking various domains and assayed cAMP-stimulated Cl- channel activity using the halide-sensitive fluorophore, 6-methoxy-N-(3'-sulfopropyl)-quinolinium. We previously reported that deletion of part of the R domain (residues 708-835) produced Cl- channels that were constitutively open. Here we show that more extensive deletions within the R domain failed to generate functional CFTR Cl- channels; the portion of protein that could be deleted without destroying function corresponds to sequences that are not conserved in related proteins. In contrast, when we deleted the two NBDs (either alone, together, or in combination with the R domain), we did not observe functional Cl- channels. CFTR has a unique carboxyl terminus that is conserved across species. However, truncation of the carboxyl terminus (up to, but not including, NBD2) produced a regulated anion permeability similar to that of wild-type CFTR, suggesting that this region is not essential for channel function. Expression of two CF-associated nonsense mutants (G542X and W1316X) also failed to generate functional CFTR Cl- channels. These results help define structure:function relationships for CFTR and identify the domains that are required for Cl- channel function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Pharmacotherapy of depression in the medically ill: directions for future research.

A common problem facing the psychiatric consultant and the medical practitioner is evaluating depression in patients with concurrent medical illnesses. Depression is difficult to recognize in the medically ill, often presenting with "masked" symptoms and organized into unique syndromes. A primary concern of the clinician is identifying those patients who are likely to benefit from an antidepressant trial. Although antidepressants have been shown to sometimes be of benefit in medical populations, the symptoms predicting antidepressant response remain poorly defined. Important directions for future research include 1) evaluating the safety and efficacy of newer antidepressants in the medically ill and 2) identifying those depressive syndromes that may be responsive to pharmacotherapy.

Adult↗
PubMed1992

Treatment of displaced subcapital femoral fractures with total hip replacement.

A series of 46 patients with 46 displaced subcapital femoral fractures were treated with total hip replacements. Indications for treatment were age between 65 and 79 years, a mental test score of greater than 7/13 and mobility sufficient to allow the patient to leave the house at least once a week. The main complication was dislocation (9 per cent), which tended to occur early, and was associated with inexperience of the surgeon. The 6-month mortality was 9 per cent. The mean Harris score of 33 patients reviewed at 3 years was 83. Preoperative mobility was well maintained and there was no evidence of deep sepsis or loosening. We recommend that this method of treatment should be studied further in a prospective comparative fashion.

Aged↗
PubMed1992

Production of cystic fibrosis transmembrane conductance regulator in the milk of transgenic mice.

Here we describe the production of cystic fibrosis transmembrane conductance regulator (CFTR), the product of the gene associated with cystic fibrosis, in the milk of transgenic mice. Mammary specific expression was achieved by placing the CFTR cDNA under the control of the goat beta-casein gene promoter. By fractionation, CFTR was shown to be associated with the membranes that envelop milk fat globules as they are discharged from the apical surface of the mammary epithelia. Since milk fat globules may comprise up to 10% of whole milk, this represents a novel, inexpensive and efficient approach to produce CFTR and possibly other membrane-associated proteins. The availability of large quantities of CFTR could have important implications for the development of new therapies for cystic fibrosis.

Animals↗