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Biomedical subjects

C F Higgins

Publications and source records attributed to C F Higgins.

At least 19 recordsLinked to original sources
PubMed2000

The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism.

The bacterial LmrA protein and the mammalian multidrug resistance P-glycoprotein are closely related ATP-binding cassette (ABC) transporters that confer multidrug resistance on cells by mediating the extrusion of drugs at the expense of ATP hydrolysis. The mechanisms by which transport is mediated, and by which ATP hydrolysis is coupled to drug transport, are not known. Based on equilibrium binding experiments, photoaffinity labeling and drug transport assays, we conclude that homodimeric LmrA mediates drug transport by an alternating two-site transport (two-cylinder engine) mechanism. The transporter possesses two drug-binding sites: a transport-competent site on the inner membrane surface and a drug-release site on the outer membrane surface. The interconversion of these two sites, driven by the hydrolysis of ATP, occurs via a catalytic transition state intermediate in which the drug transport site is occluded. The mechanism proposed for LmrA may also be relevant for P-glycoprotein and other ABC transporters.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking.

Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy with serious consequences for the mother and fetus. Two pedigrees have been reported with ICP in the mothers of children with a subtype of autosomal recessive progressive familial intrahepatic cholestasis (PFIC) with raised serum gamma-glutamyl transpeptidase (gamma-GT). Affected children have homozygous mutations in the MDR3 gene (also called ABCB4 ), and heterozygous mothers have ICP. More frequently, however, ICP occurs in women with no known family history of PFIC and the genetic basis of this disorder is unknown. We investigated eight women with ICP and raised serum gamma-GT, but with no known family history of PFIC. DNA sequence analysis revealed a C to A transversion in codon 546 in exon 14 of MDR3 in one patient, which results in the missense substitution of the wild-type alanine with an aspartic acid. We performed functional studies of this mutation introduced into MDR1, a closely related homologue of MDR3. Fluorescence activated cell sorting (FACS) and western analysis indicated that this missense mutation causes disruption of protein trafficking with a subsequent lack of functional protein at the cell surface. The demonstration of a heterozygous missense mutation in the MDR3 gene in a patient with ICP with no known family history of PFIC, analysed by functional studies, is a novel finding. This shows that MDR3 mutations are responsible for the additional phenotype of ICP in a subgroup of women with raised gamma-GT.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2000

The downstream regulatory element of the proU operon of Salmonella typhimurium inhibits open complex formation by RNA polymerase at a distance.

The intracellular concentration of K(+)-glutamate, chromatin-associated proteins, and a downstream regulatory element (DRE) overlapping with the coding sequence, have been implicated in the regulation of the proU operon of Salmonella typhimurium. The basal expression of the proU operon is low, but it is rapidly induced when the bacteria are grown in media of high osmolarity (e.g. 0.3 M NaCl). It has previously been suggested that increased intracellular concentrations of K(+)-glutamate activate the proU promoter in response to increased extracellular osmolarity. We show here that the activation of the proU promoter by K(+)-glutamate in vitro is nonspecific, and the in vivo regulation cannot simply be mimicked in vitro. In vivo specificity requires both the chromatin-associated protein H-NS and the DRE; they are both needed to maintain repression of proU expression at low osmolarity. How H-NS and the DRE repress the proU promoter in vivo has so far been unclear. We show that, in vivo, the DRE acts at a distance to inhibit open complex formation at the proU promoter.

Bacterial Proteins↗
PubMed2000

Repeat administration of DNA/liposomes to the nasal epithelium of patients with cystic fibrosis.

The major cause of mortality in patients with cystic fibrosis (CF) is lung disease. Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene product in the airways is a potential treatment. Clinical studies in which the CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in modest, transient gene expression. It seems likely that repeated administration of the gene transfer vector will be required for long-term gene expression. We have undertaken a double-blinded study in which multiple doses of a DNA/liposome formulation were delivered to the nasal epithelium of CF patients. Ten subjects received plasmid DNA expressing the CFTR cDNA complexed with DC-Chol/DOPE cationic liposomes, whilst two subjects received placebo. Each subject received three doses, administered 4 weeks apart. There was no evidence of inflammation, toxicity or an immune response towards the DNA/liposomes or the expressed CFTR. Nasal epithelial cells were collected 4 days after each dose for a series of efficacy assays including quantitation of vector-specific DNA and mRNA, immunohistochemistry of CFTR protein, bacterial adherence, and detection of halide efflux ex vivo. Airway ion transport was also assessed in vivo by repeated nasal potential difference (PD) measurements. On average, six of the treated subjects were positive for CFTR gene transfer after each dose. All subjects positive for CFTR function were also positive for plasmid DNA, plasmid-derived mRNA and CFTR protein. The efficacy measures suggest that unlike high doses of recombinant adenoviral vectors, DNA/liposomes can be successfully re-administered without apparent loss of efficacy.

Adolescent↗
PubMed2000

Oligomerization of the chromatin-structuring protein H-NS.

H-NS is a major component of the bacterial nucleoid, involved in condensing and packaging DNA and modulating gene expression. The mechanism by which this is achieved remains unclear. Genetic data show that the biological properties of H-NS are influenced by its oligomerization properties. We have applied a variety of biophysical techniques to study the structural basis of oligomerization of the H-NS protein from Salmonella typhimurium. The N-terminal 89 amino acids are responsible for oligomerization. The first 64 residues form a trimer dominated by an alpha-helix, likely to be in coiled-coil conformation. Extending this polypeptide to 89 amino acids generated higher order, heterodisperse oligomers. Similarly, in the full-length protein no single, defined oligomeric state is adopted. The C-terminal 48 residues do not participate in oligomerization and form a monomeric, DNA-binding domain. These N- and C-terminal domains are joined via a flexible linker which enables them to function independently within the context of the full-length protein. This novel mode of oligomerization may account for the unusual binding properties of H-NS.

Amino Acid Sequence↗
PubMed1999

Cysteine-scanning mutagenesis provides no evidence for the extracellular accessibility of the nucleotide-binding domains of the multidrug resistance transporter P-glycoprotein.

Multidrug resistance of cancer cells is, at least in part, conferred by overexpression of P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of active transporters. P-gp actively extrudes chemotherapeutic drugs from cells, thus reducing their efficacy. As a typical ABC transporter, P-gp has four domains: two transmembrane domains, which form a pathway through the membrane through which substrates are transported, and two hydrophilic nucleotide-binding domains (NBDs), located on the cytoplasmic side of the membrane, which couple the energy of ATP hydrolysis to substrate translocation. It has been proposed that the NBDs of ABC transporters, including the histidine permease of Salmonella typhimurium and the cystic fibrosis transmembrane conductance regulator, are accessible from the extracellular surface of the cell, spanning the membrane directly or potentially contributing to the transmembrane pore. Such organization would have significant implications for the transport mechanism. We determined to establish whether the NBDs of P-gp are exposed extracellularly and which amino acids are accessible, using cysteine-scanning mutagenesis and limited proteolysis. In contrast to other transporters, the data provided no evidence that the P-gp NBDs are exposed to the cell surface. The implications for the structure and mechanism of P-gp and other ABC transporters are discussed.

3T3 Cells↗
PubMed1999

Polyadenylation promotes degradation of 3'-structured RNA by the Escherichia coli mRNA degradosome in vitro.

Polyadenylation contributes to the destabilization of bacterial mRNA. We have investigated the role of polyadenylation in the degradation of RNA by the purified Escherichia coli degradosome in vitro. RNA molecules with 3'-ends incorporated into a stable stem-loop structure could not readily be degraded by purified polynucleotide phosphorylase or by the degradosome, even though the degradosome contains active RhlB helicase which normally facilitates degradation of structured RNA. The exoribonucleolytic activity of the degradosome was due to polynucleotide phosphorylase, rather than the recently reported exonucleolytic activity exhibited by a purified fragment of RNase E (Huang, H., Liao, J., and Cohen, S. N. (1998) Nature 391, 99-102). Addition of a 3'-poly(A) tail stimulated degradation by the degradosome. As few as 5 adenosine residues were sufficient to achieve this stimulation, and generic sequences were equally effective. The data show that the degradosome requires a single-stranded "toehold" 3' to a secondary structure to recognize and degrade the RNA molecule efficiently; polyadenylation can provide this single-stranded 3'-end. Significantly, oligo(G) and oligo(U) tails were unable to stimulate degradation; for oligo(G), at least, this is probably due to the formation of a G quartet structure which makes the 3'-end inaccessible. The inaccessibility of 3'-oligo(U) sequences is likely to have a role in stabilization of RNA molecules generated by Rho-independent terminators.

ATP-Binding Cassette Transporters↗
PubMed1999

The equilibrium and kinetic drug binding properties of the mouse P-gp1a and P-gp1b P-glycoproteins are similar.

The gene encoding the multidrug resistance P-glycoprotein (P-gp) is duplicated in rodent species and the functional basis for this remains unresolved. Despite a high sequence similarity, the mouse P-gp1a and P-gp1b isoforms show distinct patterns of tissue distribution which suggest a specific role of the P-gp1b isoform in steroid transport. In the present study possible biochemical differences between the isoforms were directly investigated at the level of drug interaction. There was no detectable difference in the affinity or binding capacity of the two isoforms towards [3H]vinblastine at equilibrium. Similarly, the rate at which [3H]vinblastine associates with P-gp was indistinguishable between the two isoforms. Some modest differences were observed in the relative abilities of the multidrug-resistant (MDR) reversing agents CP100-356, nicardipine and verapamil to displace equilibrium [3H]vinblastine binding to P-gp1a and P-gp1b. The steroid hormone progesterone displayed a low affinity (Ki = 1.2 +/- 0.2 microM for P-gp1a and 3.5 +/- 0.5 microM for P-gp1b), suggesting an unlikely role as a physiological substrate. Thus the mouse isoforms do not appear to exhibit functional differences at the level of initial substrate interaction with protein.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

Environmental influences on DNA curvature.

DNA curvature plays an important role in many biological processes. To study environmental influences on DNA curvature we compared the anomalous migration on polyacrylamide gels of ligation ladders of 11 specifically-designed oligonucleotides. At low temperatures (25 degrees C and below) most of the sequences exhibited a degree of anomalous migration. Increased temperature had a significant effect on the anomalous migration (curvature) of some sequences but limited effects on others; at 50 degrees C only 1 sequence migrated anomalously. Mg2+ had a strong influence on the migration of certain sequences, whilst spermine enhanced the anomalous migration of a different set of sequences. Sequences with a GGC motif exhibited greater curvature than predicted by the presently-used angles for the nearest-neighbour wedge model and are especially sensitive to Mg2+. The data have implications for models for DNA curvature and for environmentally-sensitive DNA conformations in the regulation of gene expression.

DNA↗
PubMed1998

Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome.

The Escherichia coli RNA degradosome is the prototype of a recently discovered family of multiprotein machines involved in the processing and degradation of RNA. The interactions between the various protein components of the RNA degradosome were investigated by Far Western blotting, the yeast two-hybrid assay, and coimmunopurification experiments. Our results demonstrate that the carboxy-terminal half (CTH) of ribonuclease E (RNase E) contains the binding sites for the three other major degradosomal components, the DEAD-box RNA helicase RhlB, enolase, and polynucleotide phosphorylase (PNPase). The CTH of RNase E acts as the scaffold of the complex upon which the other degradosomal components are assembled. Regions for oligomerization were detected in the amino-terminal and central regions of RNase E. Furthermore, polypeptides derived from the highly charged region of RNase E, containing the RhlB binding site, stimulate RhlB activity at least 15-fold, saturating at one polypeptide per RhlB molecule. A model for the regulation of the RhlB RNA helicase activity is presented. The description of RNase E now emerging is that of a remarkably complex multidomain protein containing an amino-terminal catalytic domain, a central RNA-binding domain, and carboxy-terminal binding sites for the other major components of the RNA degradosome.

Base Sequence↗
PubMed1998

Protein kinase C phosphorylation disengages human and mouse-1a P-glycoproteins from influencing the rate of activation of swelling-activated chloride currents.

1. Whole-cell, swelling-activated Cl- currents, ICl(swell), were characterized in Chinese hamster ovary (CHO) cells and found to exhibit time-dependent inactivation at depolarizing potentials, tamoxifen and dideoxyforskolin sensitivity, and an anion permeability sequence: SCN- > I- > Br- > Cl- > F- > gluconate-. 2. CHO cells permanently transfected with either the human MDR1 or mouse mdr1a cDNAs demonstrated an increased rate of activation of ICl(swell) compared with parental cells or those permanently transfected with the mouse mdr1b cDNA. However, no differences in the magnitude of the currents were observed at steady state. 3. Pretreatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) did not affect ICl(swell) in MDR1 or mdr1a permanently transfected CHO cells. In contrast, pretreatment with TPA reduced ICl(swell) in MDR1(G185V)-expressing transfected NIH3T3 fibroblasts. Subsequently, the CHO cell lines were shown to contain significantly reduced levels of protein kinase C (PKC), suggesting that PKC concentrations might be limiting in these cell lines, at least under whole-cell patch clamp conditions. 4. Addition of purified PKC to the pipette solution, followed by a pretreatment with TPA, reduced the rate of ICl(swell) activation in human Pgp- and mouse Pgp1a-expressing CHO cells to the levels observed in parental and mouse Pgp1b-expressing cells. This confirms that PKC is limiting in these cells under whole-cell, patch clamp conditions. Furthermore, these results suggest that PKC-mediated phosphorylation of human Pgp and mouse Pgp1a disengages the influence which these Pgps have on ICl(swell). 5. These studies also demonstrate a functional distinction between the two mouse homologues, Pgp1a and Pgp1b. Although both can function as drug efflux pumps, only Pgp1a can act like human Pgp to influence ICl(swell).

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

A bacterial antibiotic-resistance gene that complements the human multidrug-resistance P-glycoprotein gene.

Bacteria have developed many fascinating antibiotic-resistance mechanisms. A protein in Lactococcus lactis, LmrA, mediates antibiotic resistance by extruding amphiphilic compounds from the inner leaflet of the cytoplasmic membrane. Unlike other known bacterial multidrug-resistance proteins, LmrA is an ATP-binding cassette (ABC) transporter. The human multidrug-resistance P-glycoprotein, encoded by the MDR1 gene, is also an ABC transporter, overexpression of which is one of the principal causes of resistance of human cancers to chemotherapy. We expressed lmrA in human lung fibroblast cells. Surprisingly, LmrA was targeted to the plasma membrane and conferred typical multidrug resistance on these human cells. The pharmacological characteristics of LmrA and P-glycoprotein-expressing lung fibroblasts were very similar, and the affinities of both proteins for vinblastine and magnesium-ATP were indistinguishable. Blockers of P-glycoprotein-mediated multidrug resistance also inhibited LmrA-dependent drug resistance. Kinetic analysis of drug dissociation from LmrA expressed in plasma membranes of insect cells revealed the presence of two allosterically linked drug-binding sites indistinguishable from those of P-glycoprotein. These findings have implications for the reversal of antibiotic resistance in pathogenic microorganisms. Taken together, they demonstrate that bacterial LmrA and human P-glycoprotein are functionally interchangeable and that this type of multidrug-resistance efflux pump is conserved from bacteria to man.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

The Escherichia coli ATP-binding cassette (ABC) proteins.

The recent completion of the Escherichia coli genome sequence (Blattner et al., 1997) has permitted an analysis of the complement of genomically encoded ATP-binding cassette (ABC) proteins. A total of 79 ABC proteins makes this the largest paralogous family of proteins in E. coli. These 79 proteins include 97 ABC domains (as some proteins include more than one ABC domain) and are components of 69 independent functional systems (as many systems involve more than one ABC domain). The ABC domains are often, but not exclusively, the energy-generating domains of multicomponent membrane-bound transporters. Thus, 57 of the 69 systems are ABC transporters, of which 44 are periplasmic-binding protein-dependent uptake systems and 13 are presumed exporters. The genes encoding these ABC transporters occupy almost 5% of the genome. Of the 12 systems that are not obviously transport related, the function of only one, the excision repair protein UvrA, is known. A phylogenetic analysis suggests that the majority of ABC proteins can be assigned to 10 subfamilies. Together with statistical and, importantly, biological evidence, this analysis provides insight into the evolution and function of the ABC proteins.

ATP-Binding Cassette Transporters↗
PubMed1998

Tissue-specific in vivo transcription start sites of the human and murine cystic fibrosis genes.

The in vivo transcription start sites of the human cystic fibrosis transmembrane conductance regulator gene ( CFTR ) and its murine homologue ( Cftr ) have been mapped in a range of tissues using the technique of 5' rapid amplification of cDNA ends (5' RACE). These are the first in vivo transcription start sites for CFTR or Cftr to be reported. Distinct, tissue-specific patterns of CFTR start site usage were identified in both mouse and human. In particular, striking variation in the position of the murine Cftr transcription start site was seen along the length of the intestinal tract; different start sites being utilized in ileum and in duodenum. In humans, distinct transcription start sites are utilized in adult and foetal lungs. In addition, a novel 5'-untranslated exon of murine Cftr , denoted exon -1, was identified and shown to be expressed exclusively in mouse testis. Expression of exon -1-containing Cftr transcripts was shown by mRNA in situ hybridization to be confined to the germ cells and to be regulated during spermatogenesis.

Adult↗
PubMed1998

A divergent CFTR homologue: highly regulated salt transport in the euryhaline teleost F. heteroclitus.

The killifish, Fundulus heteroclitus, is a euryhaline teleost fish capable of adapting rapidly to transfer from freshwater (FW) to four times seawater (SW). To investigate osmoregulation at a molecular level, a 5.7-kilobase cDNA homologous to human cystic fibrosis transmembrane conductance regulator (hCFTR) was isolated from a gill cDNA library from SW-adapted killifish. This cDNA encodes a protein product (kfCFTR) that is 59% identical to hCFTR, the most divergent form of CFTR characterized to date. Expression of kfCFTR in Xenopus oocytes generated adenosine 3',5'-cyclic monophosphate-activated, Cl(-)-selective currents similar to those generated by hCFTR. In SW-adapted killifish, kfCFTR was expressed at high levels in the gill, opercular epithelium, and intestine. After abrupt exposure of FW-adapted killifish to SW, kfCFTR expression in the gill increased severalfold, suggesting a role for kfCFTR in salinity adaptation. Under similar conditions, plasma Na+ levels rose significantly after 8 h and then fell, although it is not known whether these changes are directly responsible for the changes in kfCFTR expression. The killifish provides a unique opportunity to understand teleost osmoregulation and the role of CFTR.

Adaptation, Physiological↗
PubMed1998

Restoration by intratracheal gene transfer of bicarbonate secretion in cystic fibrosis mouse gallbladder.

Gallbladders from cystic fibrosis (CF) mice (Cftrtm1Cam and Cftrtm2Cam) were examined with the short-circuit current technique. The tissues failed to show any electrogenic anion transport in response to forskolin (cAMP stimulus) but responded to the Ca2+ ionophore ionomycin. Administration of the plasmid pTrial10-CFTR2 complexed with cationic liposomes (3beta-[N-(dimethylaminoethane)-carbamoyl]cholesterol and L-alpha-phosphatidylethanolamine dioleolyl) to the airways restored the phenotype of CF gallbladders to that of the wild type, but did not do so when given orally. Formation of human CFTR mRNA in gallbladders of transfected CF null mice was demonstrated. Using the reporter genes pCMV-luc and pCMV-LacZ, we showed that 1) the intratracheal route was more effective than the oral,intravenous, intramuscular, subcutaneous, or intraperitoneal routes in expressing luciferase activity in the gallbladder and 2) beta-galactosidase staining after pCMV-LacZ was confined to the columnar epithelium lining the gallbladder without any discernible activity in it smooth muscle. The discovery of an unusual route for gene transfer to the biliary system may give useful insight into counteracting the consequences of biliary fibrosis in human CF patients.

Animals↗