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Biomedical subjects

R E Hancock

Publications and source records attributed to R E Hancock.

At least 109 records · Page 6Linked to original sources
PubMed1996

Antibacterial in vitro activity of fourth generation cephalosporins.

Cefpirome, cefepime and cefaclidine are distinguished by having a positively charged quaternary ammonium at carbon 3 of the dihydrothiazone ring. This confers the distinctive advantages of higher permeability across the outer membrane and low affinity for chromosomal cephalosporinases compared to the third generation cephalosporins which lack this quaternary ammonium moiety. These properties result in a marked advantage against resistant mutants of several species containing either derepressed class C chromosomal beta-lactamases or variant class A beta-lactamase. These unique properties have led to the suggestion that these compounds represent a "fourth generation" of cephalosporins.

Cell Membrane Permeability↗
PubMed1995

Antiviral screening of British Columbian medicinal plants.

One hundred methanolic plant extracts were screened for antiviral activity against seven viruses. Twelve extracts were found to have antiviral activity at the non-cytotoxic concentrations tested. The extracts of Rosa nutkana and Amelanchier alnifolia, both members of the Rosaceae, were very active against an enteric coronavirus. A root extract of another member of the Rosaceae, Potentilla arguta, completely inhibited respiratory syncytial virus. A Sambucus racemosa branch tip extract was also very active against respiratory syncytial virus while the inner bark extract of Oplopanax horridus partially inhibited this virus. An extract of Ipomopsis aggregata demonstrated very good activity against parainfluenza virus type 3. A Lomatium dissectum root extract completely inhibited the cytopathic effects of rotavirus. In addition to these, extracts prepared from the following plants exhibited antiviral activity against herpesvirus type 1: Cardamine angulata, Conocephalum conicum, Lysichiton americanum, Polypodium glycyrrhiza and Verbascum thapsus.

Animals↗
PubMed1995

Pseudomonas aeruginosa outer membrane protein OprF as an expression vector for foreign epitopes: the effects of positioning and length on the antigenicity of the epitope.

OprF, the major outer membrane (OM) protein of Pseudomonas aeruginosa, has been proposed to be comprised of a series of beta-strands separated by periplasmic or surface-exposed loop regions. In this study, a simple malarial epitope was used to demonstrate that OprF can be used as an expression vector to present foreign peptide sequences, namely, the 4-amino-acid (aa) repeating epitope (Asn-Ala-Asn-Pro = NANP) of the circumsporozoite protein of the human malarial parasite Plasmodium falciparum. Eight permissive sites, that allowed the expression and surface exposure of the malarial epitope, were identified throughout OprF. Using a monoclonal antibody (mAb) specific for the malarial epitope, we investigated the effects of positioning and length of the epitope on its antigenicity in the OprF expression vector system. It was demonstrated that the malarial epitope inserted at aa26 was significantly more reactive with the epitope-specific mAb (i.e., more antigenic) when assayed in the context of whole cells whereas those at aa213 and aa290 were more antigenic when assayed in the OM. The malarial epitope inserted at aa188 and aa196 was moderately antigenic, while this epitope inserted at aa215 and aa310 showed low antigenicity with the same mAb in both whole cell and OM assays. For two insertion sites, aa26 and aa213, we demonstrated that the insertion of multiple copies of the epitope enhanced reactivity with the malarial epitope-specific mAb. These data are discussed with respect to the local OprF sequences into which the epitope was inserted.

Amino Acid Sequence↗
PubMed1995

Membrane topology and site-specific mutagenesis of Pseudomonas aeruginosa porin OprD.

Pseudomonas aeruginosa OprD is a 420-amino-acid protein that facilitates the uptake of basic amino acids, imipenem and gluconate across the outer membrane. OprD was the first specific porin that could be aligned with members of the non-specific porin super-family. Utilizing multiple alignments in conjugation with structure predictions and amphipathicity calculations, an OprD-topology model was proposed. Sixteen beta-strands were predicted, connected by short loops at the periplasmic side. The eight external loops were of variable length but tended to be much longer than the periplasmic ones. Polymerase chain reaction (PCR)-based site-specific mutagenesis was performed to delete separately short stretches (4-8 amino acid residues) from each of the predicted external loops. The mutants with deletions in the predicted external loops L1, L2, L5, L6, L7 and L8 were tolerated in both Escherichia coli and P. aeruginosa. The expressed mutant proteins maintained substantial resistance to trypsin treatment in the context of isolated outer membranes. Proteins with deletions in loops L1, L5, L6, L7 and L8 reconstituted similar imipenem supersusceptibility in a P. aeruginosa OprD:: omega background. The L2-deletion mutant only partially reconstituted super-susceptibility, suggesting that loop L2 is involved in imipenem binding. These data were generally consistent with the topology model.

Amino Acid Sequence↗
PubMed1995

Development of multiple-antibiotic-resistant (Mar) mutants of Pseudomonas aeruginosa after serial exposure to fluoroquinolones.

Laboratory-derived fluoroquinolone-resistant mutants were created by serially passaging wild-type Pseudomonas aeruginosa on fluoroquinolone-containing agar to obtain high-level fluoroquinolone resistance (e.g., ciprofloxacin MIC of 1,024 micrograms/ml). With increases of 4- to 32-fold in MICs of fluoroquinolones, these organisms demonstrated (relative to wild-type) normal morphology, resistance to fluoroquinolones only, no change in fluoroquinolone uptake, and no change in lipopolysaccharide profiles or outer membrane protein profiles. Complementation with wild-type Escherichia coli gyrA restored fluoroquinolone susceptibility, suggesting that these were gyrA mutants. After 4- to 32-fold increases in fluoroquinolone MICs (with continued passage on fluoroquinolone-containing agar) isolates demonstrated altered morphology, a multiple-antibiotic-resistant (Mar) phenotype (including cross-resistance to beta-lactams, chloramphenicol, and tetracycline), reduced fluoroquinolone uptake and altered outer membrane proteins (reductions in the 25- and 38-kDa bands as well as several bands in the 43- to 66-kDa region). Complementation with wild-type E. coli gyrA partially reduced the level of fluoroquinolone resistance by approximately 8- to 32-fold, suggesting that these mutants displayed both gyrA and non-gyrA mutations.

Anti-Infective Agents↗
PubMed1995

Cloning and characterization of the Pseudomonas aeruginosa pbpB gene encoding penicillin-binding protein 3.

Clones containing the pbpB gene which encodes penicillin-binding protein (PBP) 3 of Pseudomonas aeruginosa were detected by hybridization by PCR amplification with primers based on the conserved sequences of high-molecular-weight PBPs. The translated amino acid sequence demonstrated 45% identity and had a total of 66% conserved amino acids relative to the Escherichia coli PBP3. The pbpB gene was located upstream of a gene homologous to the E. coli murE gene, which encodes uridine diphosphate-N-acetyl muramic acid-tripeptide synthetase. The overexpressed pbpB gene product reacted with 3H-penicillin G and had an apparent molecular weight of 60,000.

Amino Acid Sequence↗
PubMed1995

Epitope mapping of the Pseudomonas aeruginosa major outer membrane porin protein OprF.

The Pseudomonas aeruginosa major outer membrane protein OprF has been proposed for use as a vaccine and as a target for immunotherapeutic and diagnostic monoclonal antibodies. The well-conserved epitopes for 10 surface-reactive, OprF-specific monoclonal antibodies were localized by both overlapping peptide analysis and immunodetection of OprF peptides generated by cyanogen bromide and the protease papain. Three of the monoclonal antibodies bound to specific overlapping octapeptides, which had been synthesized on 160 pins to cover the entire 326 amino acids of OprF. The highest reactivities were as follows: MA7-1 to the pin with attached peptide GTYETGNK (amino acids 55 to 62), MA7-2 to NLADFMKQ (amino acids 237 to 244), and MA5-8 to TAEGRAIN (amino acids 307 to 314). The other monoclonal antibodies showed no reactivity, indicating that they do not recognize linear epitopes. Two polyclonal sera were also tested and demonstrated weak reactivity with discrete regions of OprF, suggesting that the majority of antibodies produced might recognize conformational epitopes. Utilizing defined peptides generated with cyanogen bromide and papain, the conformational epitopes recognized by the seven monoclonal antibodies were localized to regions that were 42 to 90 amino acids long. These regions were located on two adjacent loops in the middle of an amended structural model of OprF.

Amino Acid Sequence↗
PubMed1995

Isolation and characterization of a family of porin proteins from Helicobacter pylori.

Two-dimensional gel electrophoresis was used to identify heat-modifiable outer membrane proteins, which were candidates for porins, from Helicobacter pylori membrane preparations. Four such proteins with apparent molecular masses of 48, 49, 50, and 67 kDa were isolated. The four proteins copurified together after selective detergent solubilizations followed by anion-exchange chromatography, and each protein was ultimately purified to homogeneity by gel purification. These proteins were then tested for pore-forming ability with a planar lipid bilayer model membrane system. All four proteins appeared to be present as monomers, and they formed pores with low single-channel conductances in 1.0 M KCl of 0.36, 0.36, 0.30, and 0.25 nS, respectively, for the 48-, 49-, 50-, and 67-kDa proteins which we propose to designate HopA, HopB, HopC, and HopD. N-terminal amino acid sequence analyses showed a high degree of homology among all four proteins, and it appears that these proteins constitute a family of related porins in H. pylori.

Amino Acid Sequence↗
PubMed1995

A conserved Aeromonas salmonicida porin provides protective immunity to rainbow trout.

A protein with an apparent M(r) of 28,000 was isolated from outer membrane preparations of Aeromonas salmonicida A440. The protein was tested for the ability to form pores, using a planar lipid bilayer model membrane system. The protein appeared to be a monomer with a single-channel conductance in 1.0 M KCl of 1.96 nS and a cation/anion permeability ratio of 2.91 +/- 0.68. These data show that the porin channel is comparable in size to OmpC and OmpF of Escherichia coli and is relatively nonselective, having some preference for cations over anions. The porin was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a polyclonal antibody was raised. Immunoblot analysis showed that an immunologically cross-reactive protein was present in other Aeromonas strains but not in strains of Vibrio or Yersinia. The N-terminal amino acid sequence of the porin was determined and was found to show some homology to an Aeromonas hydrophila outer membrane protein. This is the second porin species of A. salmonicida to be described, and it differs from the other in subunit molecular weight, aggregation properties, peptidoglycan association, pore size, and antigenicity. Rainbow trout (Oncorhynchus mykiss) immunized intraperitoneally with the purified porin protein were significantly protected from experimental A. salmonicida challenge. This is the first report of successful vaccination against A. salmonicida with a purified outer membrane component.

Aeromonas↗
PubMed1995

The rare outer membrane protein, OmpL1, of pathogenic Leptospira species is a heat-modifiable porin.

The outer membranes of invasive spirochetes contain unusually small amounts of transmembrane proteins. Pathogenic Leptospira species produce a rare 31-kDa surface protein, OmpL1, which has a deduced amino acid sequence predictive of multiple transmembrane beta-strands. Studies were conducted to characterize the structure and function of this protein. Alkali, high-salt, and urea fractionation of leptospiral membranes demonstrated that OmpL1 is an integral membrane protein. The electrophoretic mobility of monomeric OmpL1 was modifiable by heat and reduction; complete denaturation of OmpL1 required prolonged boiling in sodium dodecyl sulfate (SDS), 8 M urea, and 2-mercaptoethanol. When solubilized in SDS at low temperature, a small proportion of OmpL1 exhibited an apparent molecular mass of approximately 90 kDa, indicating the existence of an SDS-unstable oligomer. OmpL1 dimers and trimers were demonstrated by nearest neighbor chemical cross-linking. In order to generate purified protein for functional studies, the ompL1 gene was ligated into the pMMB66 expression plasmid under control of the tac promoter. Although expression in Escherichia coli was toxic, most of the OmpL1 produced was found in the outer membrane, as determined by subcellular fractionation. Purified recombinant OmpL1 was reconstituted into planar lipid bilayers, demonstrating an average single channel conductance of 1.1 nS, similar to the major porin activity of native leptospiral membranes. These findings indicate that OmpL1 spans the leptospiral outer membrane and functions as a porin.

Bacterial Outer Membrane Proteins↗
PubMed1995

Porin activity and sequence analysis of a 31-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp1).

We have recently reported the isolation and purification of the Treponema pallidum outer membrane and the identification of its rare protein constituents, including a 31-kDa protein markedly enriched in the outer membrane preparation (D.R. Blanco, K. Reimann, J. Skare, C.I. Champion, D. Foley, M. M. Exner, R. E. W. Hancock, J. N. Miller, and M. A. Lovett, J. Bacteriol. 176:6088-6099, 1994). In this study, we report the cloning, sequencing, and expression of the structural gene which encodes the 31-kDa outer membrane protein, designated Tromp1. The deduced amino acid sequence from the tromp1 gene sequence encodes a 318-amino-acid polypeptide with a putative 40-amino-acid signal peptide. Processing of Tromp1 results in a mature protein with a predicted molecular mass of 30,415 Da and a calculated pI of 6.6. Secondary-structure predictions identified repeated stretches of amphipathic beta-sheets typical of outer membrane protein membrane-spanning sequences. A topological model of Tromp1 containing 14 transmembrane segments is proposed. Specific antiserum against a recombinant Tromp1 fusion protein was generated and was used to identify native Tromp1 in cellular fractionation. Upon Triton X-114 extraction and phase separation of T. pallidum, the 31-kDa Tromp1 protein was detected in the detergent-phase fraction but not in the protoplasmic cylinder or aqueousphase fractions, consistent with a hydrophobic outer membrane protein. Anti-Tromp1 antiserum was also used to identify native Tromp1 purified from whole T. pallidum by Triton X-100 solubilization followed by nondenaturing isoelectric focusing. Reconstitution of purified Tromp1 into planar lipid bilayers showed porin activity based on the measured single channel conductanes of 0.15 and 0.7 nS in 1 M KCl. These findings demonstrate that Tromp1 is a transmembrane outer membrane porin protein of T. pallidum.

Amino Acid Sequence↗
PubMed1995

Insertion mutagenesis of the Pseudomonas aeruginosa phosphate-specific porin OprP.

The gene encoding the Pseudomonas aeruginosa phosphate-specific porin OprP was subjected to both linker and epitope insertion mutageneses. Nine of the 13 linker mutant genes expressed protein at levels comparable to those obtained with the wild-type gene. These mutant proteins were shown, by indirect immunofluorescence with an OprP-specific antiserum, to be properly exposed at the cell surface. Four of the linker mutant genes expressed protein at reduced levels which were not detectable at the cell surface. A foreign epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum was cloned into the linker sites of 12 of the 13 mutant genes. Seven of the resultant epitope insertion mutant genes expressed surface-exposed protein. Two of these mutant genes presented the foreign epitope at surface-accessible regions as assessed by indirect immunofluorescence with a malarial epitope-specific monoclonal antibody. The data from these experiments were used to create a topological model of the OprP monomer.

Amino Acid Sequence↗
PubMed1995

Isolation and characterization of a conserved porin protein from Helicobacter pylori.

Helicobacter pylori is a causative agent of gastritis in humans and is correlated with gastric ulcer formation. Infections with this bacterium have proven difficult to treat with antimicrobial agents. To better understand how this bacterium transports compounds such as antimicrobial agents across its outer membrane, identification of porin proteins is important. We have recently identified a family of H. pylori porins (HopA to HopD) (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995). Here, we report on an unrelated porin species (HopE) from this bacterium. This protein had a apparent molecular mass of 31 kDa and was seen to form 50- and 90-kDa aggregates that were designated putative dimeric and trimeric forms, respectively. The protein was purified to homogeneity and, with a model planar lipid membrane system, was shown to act as a nonselective pore with a single channel conductance in 1.0 M KCl of 1.5 nS, similarly to other bacterial nonspecific porins. An internal peptide sequence of HopE shared homology with the P2 porin of Haemophilus influenzae. HopE was also shown to be antigenic in vivo as assessed by sera taken from H. pylori-infected individuals and was immunologically conserved with both patient sera and specific monoclonal antibodies. From these data, it appears that HopE is a major nonselective porin of H. pylori. The implications of these findings are discussed.

Amino Acid Sequence↗
PubMed1994

Antifungal screening of medicinal plants of British Columbian native peoples.

One hundred methanolic plant extracts were screened for antifungal activity against 9 fungal species. Eighty-one were found to have some antifungal activity and 30 extracts showed activity against 4 or more of the fungi assayed. The extracts with the greatest fungal inhibition were prepared from Alnus rubra catkins, Artemisia ludoviciana aerial parts, Artemisia tridentata aerial parts, Geum macrophyllum roots, Mahonia aquifolium roots and Moneses uniflora aerial parts. In addition to these, extracts prepared from the following plants also exhibited antifungal activity against all 9 fungi: Asarum caudatum whole plant, Balsamorhiza sagittata roots, Empetrum nigrum branches, Fragaria chiloensis leaves, Gilia aggregata aerial parts and roots, Glehnia littoralis roots, Heracleum lanatum roots, Heuchera cylindrica roots and Rhus glabra branches.

Antifungal Agents↗
PubMed1994

Antimicrobial constituents of Rhus glabra.

The antimicrobial activity of the methanol extract and isolated constituents of Rhus glabra (Anacardiaceae), a species used in folk medicine by North American native people, was evaluated against 11 microorganisms, including gram-positive and gram-negative bacteria. The extract was subsequently fractionated and monitored by bioassays leading to the isolation of three antibacterial compounds, the methyl ester of 3,4,5-trihydroxybenzoic acid (methyl gallate) (minimal inhibition concentration (MIC) 12.5 micrograms/ml), 4-methoxy-3,5-dihydroxybenzoic acid (MIC 25 micrograms/ml) and gallic acid (MIC > 1000 micrograms/ml). The first two compounds are reported here for the first time from Rhus glabra. Their structures were established using spectroscopic and chemical methods.

Anti-Bacterial Agents↗
PubMed1994

The interaction of a recombinant cecropin/melittin hybrid peptide with the outer membrane of Pseudomonas aeruginosa.

A cecropin/melittin hybrid peptide (CEME) produced by recombinant DNA procedures was tested for its ability to interact with the outer membrane of Pseudomonas aeruginosa and found to have identical biological properties to that of chemically synthesized CEME. CEME was shown to kill P. aeruginosa and permeabilize its outer membrane to lysozyme and 1-N-phenylnaphthlyamine, in some cases better than other antimicrobial agents and permeabilizers. CEME demonstrated a high-binding affinity to purified P. aeruginosa lipopolysaccharide (LPS) and LPS in whole-cell environments. These data provide information on the molecular mechanism of CEME antimicrobial activity and strongly suggest that it is taken up across the outer membrane by the self-promoted uptake pathway.

1-Naphthylamine↗