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Biomedical subjects

C F Higgins

Publications and source records attributed to C F Higgins.

At least 91 records · Page 5Linked to original sources
PubMed1994

mRNA degradation in Escherichia coli: a novel factor which impedes the exoribonucleolytic activity of PNPase at stem-loop structures.

Stem-loop structures can protect upstream mRNA from degradation by impeding the processive activities of 3'-5' exoribonucleases. The ability of such structures to impede exonuclease activity in vitro is insufficient to account for the stability they can confer on mRNA in vivo. In this study we identify a factor from Escherichia coli which specifically impedes the processive activity of the 3'-5' exonuclease PNPase at stem-loop structures in vitro. This factor can, potentially, reconcile the apparent discrepancy between the ability of 3' stem-loop structures to stabilize upstream mRNA in vitro and in vivo. Its mechanism of action, and possible role in regulating mRNA degradation, is discussed.

Bacterial Proteins↗
PubMed1994

Use of transcriptional fusions to monitor gene expression: a cautionary tale.

Gene fusions are frequently used to facilitate studies of gene expression and promoter activity. We have found that certain reporter genes can, themselves, influence promoter activity. For example, the commonly used luxAB reporter genes can activate or repress transcription from a subset of promoters, generating data apparently at odds with those obtained with other reporter genes. These effects are probably related to an intrinsically curved DNA segment in the 5' coding sequence of the luxA gene. Thus, caution must be observed when one is interpreting results obtained with a single reporter gene system such as luxAB.

Artifacts↗
PubMed1994

Tamoxifen blocks chloride channels. A possible mechanism for cataract formation.

Tamoxifen is an antiestrogen frequently used in the treatment of breast cancer and is currently being assessed as a prophylactic for those at high risk of developing tumors. We have found that tamoxifen and its derivatives are high-affinity blockers of specific chloride channels. This blockade appears to be independent of the interaction of tamoxifen with the estrogen receptor and therefore reflects an alternative cellular target. One of the clinical side effects of tamoxifen is impaired vision and cataract. Chloride channels in the lens of the eye were shown to be essential for maintaining normal lens hydration and transmittance. These channels were blocked by tamoxifen and, in organ culture, tamoxifen led to lens opacity associated with cataracts at clinically relevant concentrations. These data suggest a molecular mechanism by which tamoxifen can cause cataract formation and have implications for the clinical use of tamoxifen and related antiestrogens.

3T3 Cells↗
PubMed1994

Drug efflux mediated by the human multidrug resistance P-glycoprotein is inhibited by cell swelling.

P-glycoprotein (P-gp), the product of the human multidrug resistance (MDR1) gene, confers multidrug resistance on cells by acting as an ATP-dependent drug transporter. A method using confocal microscopy was developed to measure the transport activity of P-gp from the rate of movement of doxorubicin, a fluorescent substrate of P-gp, across the membrane of a single cell. Recent work has shown that expression of P-gp enhances the activation of chloride channels in response to cell swelling, suggesting that membrane stretch might switch P-gp from a drug-transporting mode to a mode in which it activates chloride channels. In agreement with this idea, we find that cell swelling inhibits drug efflux in cells expressing P-gp but is without effect on the slower background efflux in cells not expressing P-gp and in cells transiently transfected with a mutated MDR1 in which the ATP hydrolysis sites had been inactivated. The identification of a novel means for inhibiting P-gp-mediated drug transport may have implications for the reversal of multidrug resistance during chemotherapy.

3T3 Cells↗
PubMed1994

Regulation of volume activated chloride channels by protein kinase C-mediated phosphorylation of P-glycoprotein.

The multidrug resistance P-glycoprotein (Pgp) transports hydrophobic drugs out of cells and has been recently associated with volume-activated chloride channels. Activation of these channels by hypotonic swelling was seen to be prevented by protein kinase C (PKC) in cells expressing high levels of Pgp by transfection. HeLa cells possess equivalent chloride currents yet they are not regulated by PKC. HeLa cells do not express Pgp as assessed by Western blotting. Following transfection of HeLa cells with cDNA encoding for Pgp, PKC-dependent suppression of volume activated chloride currents was observed. PKC regulation in transiently transfected HeLa cells was abolished by alanine replacement of the serine/threonine residues in the consensus phosphorylation sites of the linker region of Pgp. Replacement of these residues with glutamate, to mimic the effect of phosphorylation, mimicked the effects of PKC on channel activation. These results indicate that overexpression of Pgp confers PKC-regulation of endogenous volume-activated chloride channels. More generally they favour a model in which Pgp acts as a regulator of volume-activated chloride channels.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Correction of the ion transport defect in cystic fibrosis transgenic mice by gene therapy.

Cystic fibrosis (CF) is a lethal inherited disorder affecting about 1 in 2,000 Caucasians. The major cause of morbidity is permanent lung damage resulting from ion transport abnormalities in airway epithelia that lead to mucus accumulation and bacterial colonization. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a cyclic-AMP-regulated chloride channel. Cyclic-AMP-regulated chloride conductances are altered in airway epithelia from CF patients, suggesting that the functional expression of CFTR in the airways of CF patients may be a strategy for treatment. Transgenic mice with a disrupted cftr gene are appropriate for testing gene therapy protocols. Here we report the use of liposomes to deliver a CFTR expression plasmid to epithelia of the airway and to alveoli deep in the lung, leading to the correction of the ion conductance defects found in the trachea of transgenic (cf/cf) mice. These studies illustrate the feasibility of gene therapy for the pulmonary aspects of CF in humans.

Animals↗
PubMed1993

Volume-activated chloride channels in HeLa cells are blocked by verapamil and dideoxyforskolin.

The possible role of Cl- currents in regulatory volume decrease processes has been explored in HeLa cells using the whole-cell recording mode of the patch-clamp technique. Cells showed very small currents in voltage-clamp experiments performed with Cl(-)-rich, permeant-cation-free (N-methyl-D-glucamine replacement) intracellular and bathing solutions. Exposure of the cells to hypotonic solutions visibly swelled the cells and activated, reversibly, an outward rectifying Cl- current, which decayed at the most depolarised voltages used. Replacement of extracellular Cl- by a series of halide anions, SCN- and gluconate was consistent with an anion selectivity sequence: SCN- > I- > Br- > Cl- > F- > gluconate. The volume-regulated Cl- current was effectively inhibited by 100 microM 5-nitro-2-(3-phenyl-propylamino)-benzoic acid and by 100 microM 4,4'-diisothiocyanotostilbene-2,2-disulphonic acid, substances known to block Cl- channels in a variety of cells. Chloride current activation by hypotonicity was dependent on the presence of ATP in the intracellular solution and this requirement could be replaced by the non-hydrolysable analogue ATP[gamma S] and Mg(2+)-free ATP. The data suggest that the channels responsible for the current described are involved in the regulatory volume decrease in HeLa cells. The characteristics of this Cl- current are similar to those of the current associated with expression of multidrug resistance P-glycoprotein. Furthermore, the currents in HeLa cells were inhibited rapidly and reversibly by verapamil and 1,9-dideoxyforskolin, which are known to inhibit P-glycoprotein function.

Adenosine Triphosphate↗
PubMed1993

The multidrug resistance P-glycoprotein.

P-glycoprotein plays an important role in the resistance of cancers to chemotherapy. Thus, an understanding of the mechanism by which it functions, and its 'normal' physiological role, is of clinical relevance as well as intrinsic interest. Considerable progress towards this goal has been made in the last year or so. In addition, the finding that P-glycoprotein is associated with both a channel and a transporter activity has, potentially, far-reaching implications for an understanding of the nature of channels and transporters.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Chromosomal domains of supercoiling in Salmonella typhimurium.

The chromosomes of enteric bacteria are divided into about 50 independently supercoiled domains. It is not known whether the net level of DNA supercoiling is similar in each domain, or whether the domains are differentially supercoiled. We have addressed this question genetically, using a supercoiling-sensitive promoter to probe the relative levels of supercoiling at defined points around the Salmonella typhimurium chromosome. We conclude that, within the limits of resolution of this approach, the level of supercoiling does not differ significantly between chromosomal domains, and that each domain responds in a similar fashion to factors that perturb supercoiling. These findings have implications for the organization of the bacterial genome.

Amino Acid Transport Systems↗
PubMed1993

A sequence-specific function for the N-terminal signal-like sequence of the TonB protein.

TonB is a proline-rich protein which provides a functional link between the inner and outer membranes of Gram-negative bacteria. TonB is anchored to the inner membrane via an N-terminal signal-like sequence and spans the periplasm, interacting with transport receptors in the outer membrane. We have investigated the role of the N-terminal signal-like peptide in TonB function. Replacement of the N-terminal sequence with heterologous sequences indicates that it has at least three distinct roles in TonB function: (i) to facilitate translocation of TonB across the cytoplasmic membrane; (ii) to anchor TonB to the cytoplasmic membrane; (iii) a sequence-specific functional interaction with the ExbBD proteins.

Amino Acid Sequence↗
PubMed1993

ExbB acts as a chaperone-like protein to stabilize TonB in the cytoplasm.

The TonB protein is required to transduce energy from the cytoplasmic membrane to outer membrane transport proteins of Gram-negative bacteria. Two accessory proteins, ExbB and ExbD, are required for TonB function and it has been suggested that TonB and ExbBD form a complex in the membrane. In this paper we demonstrate that there are two spatially distinct, functional interactions between ExbBD and TonB. First, there is an interaction between ExbBD and the N-terminal signal-like peptide of TonB, probably the formation of a stable complex in the membrane. Second, ExbB interacts with TonB in the cytoplasm. This interaction involves the domain of TonB that is normally periplasmic. Thus, this is a transient interaction which occurs during the synthesis and/or localization of TonB, implying a chaperone-like role for ExbB. The transmembrane topology of ExbB was shown to be consistent with this role.

Amino Acid Sequence↗
PubMed1993

Escherichia coli endoribonuclease RNase E: autoregulation of expression and site-specific cleavage of mRNA.

Mutations in the Escherichia coli rne (ams) gene have a general effect on the rate of mRNA decay in vivo. Using antibodies we have shown that the product of the rne gene is a polypeptide of relative mobility 180 kDa. However, proteolytic fragments as small as 70 kDa, which can arise during purification, also exhibit RNase E activity. In vitro studies demonstrate that the rne gene product, RNase E, is an endoribonuclease that cleaves mRNA at specific sites. RNase E cleaves rne mRNA and autoregulates the expression of the rne gene. In addition we demonstrate RNase E-dependent endonucleolytic cleavage of ompA mRNA, at a site known to be rate-determining for degradation and reported to be cleaved by RNase K. Our data are consistent with RNase K being a proteolytic fragment of RNase E.

Antibodies, Bacterial↗
PubMed1993

Specific inhibitors distinguish the chloride channel and drug transporter functions associated with the human multidrug resistance P-glycoprotein.

Expression of the human multidrug resistance P-glycoprotein is associated with two activities, active drug transport and a volume-regulated chloride channel. In this study we define four classes of compound, based on their differential effects on these two activities. Class I compounds are substrates transported by P-glycoprotein. They also prevent channel activation when added to the cytoplasmic face of the membrane. Class II compounds include reversers of multidrug resistance such as verapamil. These compounds inhibit drug transport and block the chloride channel when added to the outer face of the membrane. Class III compounds include conventional channel blockers which block the chloride channel but do not influence drug transport. Class IV compounds, for example cyclosporin A, appear to inhibit drug transport but do not affect chloride channel activity. These findings have implications for the relationship between the channel and transporter functions associated with P-glycoprotein expression, and for the development of clinical agents which reverse multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

The chromatin-associated protein H-NS interacts with curved DNA to influence DNA topology and gene expression.

H-NS is an abundant structural component of bacterial chromatin and influences many cellular processes, including recombination, transposition, and transcription. We have studied the mechanism of action of H-NS at the osmotically regulated proU promoter. The interaction of H-NS with a curved DNA element located downstream of the proU promoter is required for normal regulation of expression. Heterologous curved sequences can replace the regulatory role of the proU curve. Hence, the luxAB and lacZ reporter genes, which differ in the presence or absence of a curve, can indicate very different patterns of transcription. H-NS interacts preferentially with these curved DNA elements in vitro. Furthermore, in vivo the interaction of H-NS with curved DNA participates in the control of plasmid linking number. The data suggest that H-NS-dependent changes in DNA topology play a role in the osmoregulation of proU expression.

Bacterial Proteins↗
PubMed1992

Separation of drug transport and chloride channel functions of the human multidrug resistance P-glycoprotein.

The human multidrug resistance P-glycoprotein is an active transporter that pumps cytotoxic drugs out of cells. Expression of P-glycoprotein is also associated with a volume-activated chloride channel. Here we address the relationship between these two functions. Drug transport requires ATP hydrolysis while, in contrast, ATP binding is sufficient to enable activation of the chloride channel. The chloride channel and drug transport activities of P-glycoprotein appear to reflect two distinct functional states of the protein that can be interconverted by changes in tonicity. Transportable drugs prevent channel activation but have no effect on channel activity once it has been preactivated by hypotonicity. The transport and channel functions of P-glycoprotein have been separated by directed mutations in the nucleotide-binding domains of the protein. These data provide further evidence that P-glycoprotein is bifunctional with both transport and channel activities. Implications for the design of chemotherapeutic drugs and for the function of the related cystic fibrosis gene product, CFTR, are discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Volume-regulated chloride channels associated with the human multidrug-resistance P-glycoprotein.

Expression of P-glycoprotein, the product of the MDR1 gene, confers multidrug resistance on cell lines and human tumours (reviewed in refs 1,2). P-glycoprotein (relative molecular mass 170,000) is an ATP-dependent, active transporter which pumps hydrophobic drugs out of cells, but its normal physiological role is unknown. It is a member of the ABC (ATP-binding cassette) superfamily of transporters, which includes many bacterial transport systems, the putative peptide transporter from the major histocompatibility locus, and the product of the cystic fibrosis gene (the cystic fibrosis transmembrane regulator, CFTR). CFTR is located in the apical membranes of many secretory epithelia and is associated with a cyclic AMP-regulated chloride channel. At least two other chloride channels are present in epithelial cells, regulated by cell volume and by intracellular Ca2+, respectively. Because of the structural and sequence similarities between P-glycoprotein and CFTR, and because P-glycoprotein is abundant in many secretory epithelia, we examined whether P-glycoprotein might be associated with one or other of these channels. We report here that expression of P-glycoprotein generates volume-regulated, ATP-dependent, chloride-selective channels, with properties similar to channels characterized previously in epithelial cells.

3T3 Cells↗
PubMed1992

The multidrug resistance and cystic fibrosis genes have complementary patterns of epithelial expression.

The cystic fibrosis gene product, CFTR, and the multidrug resistance P-glycoprotein (encoded by the MDR1 gene) are structurally related proteins and both are associated with epithelial chloride channel activities. We have compared their cell-specific expression in the rat by in situ hybridization. In all tissues examined the two genes were found to have complementary patterns of expression, demonstrating exquisite regulation in both cell-specific and temporal fashions. Additionally, a switch in expression from one gene to the other was observed in certain tissues. For example, expression in the intestine switches from CFTR to MDR1 as the cells migrate across the crypt-villus boundary. A switch from CFTR to MDR1 expression was also observed in the uterine epithelium upon pregnancy. These data suggest that CFTR and P-glycoprotein serve analogous roles in epithelial cells and provide additional evidence that P-glycoprotein has a physiological role in regulating epithelial cell volume. The patterns of expression suggest that the regulation of these two genes is coordinately controlled.

ATP Binding Cassette Transporter, Subfamily B, Mem↗