Biomedical subjects

Kevin Davis

Publications and source records attributed to Kevin Davis.

6 recordsLinked to original sources

PubMed2005

A constitutively active GPCR retains its G protein specificity and the ability to form dimers.

G protein-coupled receptors (GPCRs) are cell surface proteins which help to regulate the physiology of all the major organ systems within higher eukaryotes. They are stimulated by multiple ligands and activate a range of effector molecules to bring about changes in cell behaviour. The use of constitutively active mutants (CAMs) of GPCRs has enabled a better understanding of receptor activation as CAMs exhibit ligand-independent signalling negating the use of ligands. Here we introduce the fission yeast Schizosaccharomyces pombe as a host for producing CAMs, by describing the isolation and characterization of constitutive mutants of the P-factor receptor (Mam2). One mutant Mam2[P261L] contained a single-amino-acid substitution (Pro261 to Leu) within a region of high homology in GPCRs. Substitution of this proline leads to an 18-fold increase in ligand-independent signalling. We utilized Mam2[P261L] to investigate CAM activity by demonstrating that Mam2[P261L] is efficiently trafficked to the cell surface where it can form fully functional oligomeric complexes with the native receptor. Mam2[P261L] also retains the G protein specificity (RG-profile) of the native receptor and only induces constitutive signalling in the same G proteins. Finally, evidence is provided to indicate that CAM activity results from a reduction in the kinetics of G protein binding. This is the first time that S. pombe has been utilized for isolating and characterizing CAMs and the techniques employed will complement the current systems available for studying these important receptors.

Amino Acid Sequence↗

PubMed2004

Regional anesthesia as an alternative to general anesthesia for abdominal surgery in patients with severe pulmonary impairment.

BACKGROUND: It is known that smokers and patients with chronic obstructive pulmonary disease (COPD) experience a higher rate of pulmonary-related complications following abdominal surgery. The impact of anesthetic technique (regional [RA] versus general [GA] versus combination of both) on the complication rate has not been established. This study examined the outcomes of abdominal surgery performed using RA (epidural or continuous spinal) as the sole anesthetic technique in patients with severe pulmonary impairment (SPI). METHODS: We reviewed a series of 8 general surgery cases performed using RA alone (T4-T6 sensory level) in patients with SPI, as evidenced by an forced expiratory volume in 1 second (FEV(1)) less than 50% predicted and/or home oxygen requirement. One patient also received postoperative epidural analgesia. FEV(1) ranged from 0.3 to 1.84 L; 3 patients required home oxygen therapy, and 5 of the 8 were American Society of Anesthesiology (ASA) class 4. Operations included segmental colectomy (n = 2), open cholecystectomy (n = 1), incisional herniorrhaphy (n = 1), and laparoscopic herniorrhaphy (n = 4). RESULTS: Intraoperative conditions were adequate with RA alone for successful completion of the procedure in all cases. All patients recovered uneventfully except for 1 who developed postoperative pneumonia that resolved with standard therapy. Length of stay was less than 24 hours for 5 of 8 patients. Mortality was 0%. CONCLUSIONS: Abdominal surgery can be safely performed using RA alone in selected high-risk patients, making this option an attractive alternative to GA for those with severe pulmonary impairment.

Abdomen↗

PubMed2004

An efficient bipartite PCR technique to introduce specific changes in large plasmids.

Amplifying an entire double-stranded plasmid by an inverse polymerase chain reaction (PCR) using a pair of tail-to-tail primers is a particularly efficient approach for introducing changes into DNA sequences. However, the approach generally works best for plasmids less than 5 Kb and it can be difficult to amplify the large multicomponent vectors that are used for protein expression in various eukaryotic cells. We have therefore adopted an alternative approach in which two smaller PCR products are generated and then ligated to produce the complete plasmid. A mutagenic primer is used to introduce the desired change and each reaction includes one of a pair of tail-to-tail primers from within an antibiotic resistance gene contained on the plasmid so that the two PCR products contain complementing parts of the complete gene. Ligating the two products generates various combinations but only the correctly ligated molecules recreate the antibiotic resistance gene and are able to replicate in Escherichia coli. When combined with methods to minimize the carryover of template plasmid, this can be an efficient way of introducing mutations into large plasmids.

Mutagenesis, Site-Directed↗

PubMed2003

ACTH and adrenocortical gap junctions.

Since the initial identification of gap junctions in the adrenal gland, it has been proposed that a system involving direct cell-cell communication might be involved in adrenal cortical functions. Gap junction channels do, in fact, provide pathways for direct intercellular exchange of small molecules (<1,000 Da), many of which have the potential to influence a wide range of cellular activities. Gap junctions are composed of proteins called connexin which, in the adrenal cortex, have proven to be remarkably consistent in both type and zonal distribution with connexin 43 (Cx43) as the predominant component in mammalian adrenal glands thus far evaluated. Only the inner two zones of the cortex (zonae fasciculata and reticularis) exhibit significant amounts of Cx43 and functional coupling. Adrenocorticotropin (ACTH) has been shown to increase Cx43 protein in vivo and in vitro, and a strong correlation has been noted between the presence of gap junctions and certain adrenal cortical functions, especially steroidogenic capacity and cell proliferation. This review summarizes evidence of the Cx43 expression in adrenal cortical cells and the likely role of Cx43 in steroidogenesis and cell proliferation. It is concluded that control of gap junction expression in the adrenal gland is hormonally dependent and is functionally linked to adrenal gland zonation.

Adrenal Cortex↗

PubMed2003

Modified yeast cells to investigate the coupling of G protein-coupled receptors to specific G proteins.

G protein-coupled receptors (GPCRs) help to regulate the physiology of all the major organ systems. They respond to a multitude of ligands and activate a range of effector proteins to bring about the appropriate cellular response. The choice of effector is largely determined by the interaction of individual GPCRs with different G proteins. Several factors influence this interaction, and a better understanding of the process may enable a more rational approach to identifying compounds that affect particular signalling pathways. A number of systems have been developed for the analysis of GPCRs. All provide useful information, but the genetic amenability and relative simplicity of yeast makes them a particularly attractive option for ligand identification and pharmaceutical screening. Many, but not all, GPCRs are functional in the budding yeast Saccharomyces cerevisiae, and we have developed reporter strains of the fission yeast Schizosaccharomyces pombe as an alternative host. To provide a more generic system for investigating GPCRs, we created a series of yeast-human Galpha-transplants, in which the last five residues at the C-terminus of the yeast Galpha-subunit are replaced with the corresponding residues from different human G proteins. These enable GPCRs to be coupled to the Sz. pombe signalling machinery so that stimulation with an appropriate ligand induces the expression of a signal-dependent lacZ reporter gene. We demonstrate the specificity of the system using corticotropin releasing factor (CRF) and CRF-related peptides on two CRF receptors. We find that different combinations of ligand and receptor activate different Galpha-transplants, and the specificity of the coupling is similar to that in mammalian systems. Thus, CRF signalled through the Gs- and Gi-transplants, consistent with its regulation of adenylate cyclase, and was more active against the CRF-R1A receptor than against the CRF-R2B receptor. In contrast, urocortin II and urocortin III were selective for the CRF-R2B receptors. Furthermore, urocortin, but not CRF, induced signalling through the CRF-R1A receptor and the Gq-transplant. This is the first time that human GPCRs have been coupled to the signalling pathway in Sz. pombe, and the strains described in this study will complement the other systems available for studying this important family of receptors.

Corticotropin-Releasing Hormone↗

PubMed2002

Identifying regulators of pheromone signalling in the fission yeast Schizosaccharomyces pombe.

The rate and extent of a cell's response to an extracellular stimulus is influenced by regulators that act on the intracellular signalling machinery. Although not directly involved in propagating the intracellular signal, regulators control the activity of the proteins that transmit the signals. To understand this aspect of cell signalling, we studied the pheromone-response pathway in the fission yeast Schizosaccharomyces pombe, a relatively simple signalling system in a genetically tractable organism. Here, we describe the development of yeast strains containing ura4 and lacZ reporter genes under the control of the pheromone-regulated sxa2 promoter and the use of these strains to isolate mutants defective in their ability to regulate signalling. Several different types of mutant were identified. Some mutants were defective in proteins already known to regulate the pheromone-signalling pathway (Rgs1, Map1, Map2). Our approach also identified the MAP kinase phosphatase Pmp1 as a regulator of the pheromone-response pathway. Although previously shown to regulate other MAP kinase pathways in Sz. pombe, this is the first demonstration of a role for Pmp1 in pheromone signalling.

Cell Cycle↗