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Biomedical subjects

R Benz

Publications and source records attributed to R Benz.

At least 109 records · Page 6Linked to original sources
PubMed1994

Interaction of Serratia marcescens hemolysin (ShlA) with artificial and erythrocyte membranes. Demonstration of the formation of aqueous multistate channels.

Pore formation by hemolysin (ShlA) of Serratia marcescens was studied in erythrocytes and in artificial lipid bilayer membranes. The results with erythrocytes demonstrated that hemolysin pores varied in size. In erythrocyte membranes with reduced fluidity (0 degrees C), the toxin formed small pores with diameter 1-1.5 nm. In fluid membranes (above 20 degrees C), hemolysin pores with larger diameters (approximately 2.5-3.0 nm) were observed, which may be caused by association of ShlA monomers into oligomers. Comparison of the channels formed by Staphylococcus aureus alpha-toxin with channels formed by ShlA indicated a slightly smaller pore diameter of ShlA pores. Analysis of ShlA in artificial lipid bilayers showed the formation of pores with a broad distribution of single channel conductances, suggesting variable sizes of the ShlA pore. The lower limit for the pore diameter was approximately 1.0 nm. The ShlA pores did not exhibit pronounced ion selectivity nor voltage dependence, supporting the presence of a large water-filled pore.

Bacterial Toxins↗
PubMed1994

Interaction of Clostridium botulinum C2 toxin with lipid bilayer membranes. Formation of cation-selective channels and inhibition of channel function by chloroquine.

Lipid bilayer experiments were performed with the C2-II binding component of the ADP-ribosylating C2 toxin from Clostridium botulinum. The trypsin-activated but not the nonactivated form of the protein was able to increase the specific conductance of artificial lipid bilayer membranes by the formation of ion-permeable channels. The channels had on average a single-channel conductance of 55 pS in 0.1 M KCl and were found to be cation-selective and voltage-dependent. The single-channel conductance was only moderately dependent on the bulk aqueous KCl concentration, which indicated point charge effects on the channel properties. Incubation of the activated C2-II binding component with antibodies against C2-II or with C2-I toxin inhibited channel formation to a large extent. Addition of chloroquine, a known inhibitor of endocytosis in cells, led to a dose-dependent decrease of the C2-II-induced membrane conductance. This result suggested that the activated C2-II component contains a binding site for chloroquine inside the channel. It is discussed that the channels formed by C2-II component are involved in the translocation of C2-I toxin across the target cell membrane.

Alkanes↗
PubMed1994

Energy-coupled transport through the outer membrane of Escherichia coli small deletions in the gating loop convert the FhuA transport protein into a diffusion channel.

Active transport of Fe3+ as ferrichrome complex through the outer membrane of Escherichia coli is mediated by the FhuA outer membrane protein and the TonB-ExbB-ExbD protein complex in the cytoplasmic membrane. The required energy is provided by the electrochemical potential of the cytoplasmic membrane which is assumed to induce a conformation of the TonB protein that causes a conformational change in FhuA so that bound ferrichrome is released into the periplasmic space located between the outer and the cytoplasmic membrane. Excision of segments as small as 12 amino acids in the largest surface loop of FhuA converted FhuA into an open channel through which ferrichrome and antibiotics diffused independent of TonB-ExbB-ExbD. It is proposed that FhuA forms a closed channel which is opened by movement of the gating loop through a kind of allosteric interaction with TonB. The gating loop is also involved in binding of all FhuA ligands which in addition to ferrichrome are the phages T1, T5, phi 80, colicin M and the antibiotic albomycin.

Amino Acid Sequence↗
PubMed1994

Pore formation in artificial membranes by the secreted hemolysins of Proteus vulgaris and Morganella morganii.

Lipid-bilayer experiments were performed with the related hemolysins from Proteus vulgaris and Morganella morganii (HlyA). The addition of the toxins to the aqueous phase bathing lipid-bilayer membranes composed of different lipids resulted in the formation of transient ion-permeable channels. Membranes formed of pure lipids were rather inactive targets for the hemolysins as compared with lipid mixtures such as asolectin. The channels had several different substrates. The major open state had single-channel conductances of 500 pS in 0.15 M KCl at small transmembrane voltages. Experiments with different salts suggested that the hemolysin-induced channels of P. vulgaris and M. morganii were exclusively cation selective at neutral pH, caused by negative charges localized at the channel mouth. The mobility sequence of the cations within the channels was similar if not identical to their mobility sequence in the aqueous phase. The single-channel data were consistent with wide, water-filled channels with estimated minimal diameters of about 1 nm since the large organic cation Tris+ can permeate the channels without any detectable interaction with its interior. Pore-forming properties of these hemolysins were compared with those of HlyA of Escherichia coli. All these toxins share common features, oligomerize probably to form pores in lipid-bilayer membranes and form channels with similar properties which suggests that their structures are more or less identical.

Bacterial Proteins↗
PubMed1994

The mode of action of SA-FF22, a lantibiotic isolated from Streptococcus pyogenes strain FF22.

SA-FF22 is a lanthionine-containing bacteriocidal peptide isolated from Streptococcus pyogenes strain FF22. The peptide interacts closely with non-energised artificial phospholipid vesicles, as evidenced by a 'blue shift' in the fluorescent emissions associated with a tryptophan residue within the peptide sequence. Furthermore, SA-FF22 induced efflux of radiolabelled amino acids from artificially energised cytoplasmic membrane vesicles and arrested uptake of amino acids by intact cells. By measuring the decrease in membrane potential of both starved and energised SA-FF22-treated cells, and through the use of artificial planar membranes, a potential of approximately 100 mV was deduced as the minimum required to induce pore formation by SA-FF22. This threshold potential is independent of the orientation of the applied voltage (i.e. trans or cis orientations are equally effective). Single channel conductance measurements suggested that the pores formed by SA-FF22 are relatively unstable, short-lived and approximately 0.5-0.6 nm in diameter. This is somewhat smaller than those of the previously described, pore-forming lantibiotics and should not allow significant efflux of large molecules such as ATP. Thus, death of affected cells seems to result from membrane-potential disruption and subsequent exhaustion of the cells.

Alanine↗
PubMed1994

Contribution of electrogenic ion transport to impedance of the algae Valonia utricularis and artificial membranes.

The cell membrane of Valonia utricularis contains an electrogenic carrier system for chloride (Wang et al., Biophys J. 59:235-248 (1991)). The electrical impedance of V. utricularis was measured in the frequency range between 1 Hz and 50 kHz. The analysis of the impedance spectra from V. utricularis and its comparison with equivalent circuit models showed that the transport system created a characteristic contribution to the impedance in the frequency range between 10 Hz and 5 kHz. The fit of the impedance spectra with the formalism derived from the theory of carrier-mediated transport allowed the determination of the kinetic parameters of chloride transport through the cell membrane of V. utricularis, and its passive electrical properties. Simultaneous measurements of the kinetic parameters with the charge pulse method demonstrated the equivalence of both experimental approaches with respect to the evaluation of the translocation rate constants of the free and the charged carriers and the total density of carriers within the membrane. Moreover, the impedance spectra of the protonophor-mediated proton transport by FCCP (carbonylcyanide p-trifluoromethoxyphenyl-hydrazone) were measured in model membranes. The carrier system made a substantial contribution to the impedance of the artificial membranes. The analysis of the spectra in terms of a simple carrier system (Benz and McLaughlin, 1983, Biophys. J. 41:381-398) allowed the evaluation of the kinetic and equilibrium parameters of the FCCP-mediated proton transport. The possible application of the measurement of impedance spectra for the study of biological transport systems is discussed.

Biological Transport↗
PubMed1994

Noise analysis of ion current through the open and the sugar-induced closed state of the LamB channel of Escherichia coli outer membrane: evaluation of the sugar binding kinetics to the channel interior.

LamB, a sugar-specific channel of Escherichia coli outer membrane was reconstituted into lipid bilayer membranes and the current noise was investigated using fast Fourier transformation. The current noise through the open channels had a rather small spectral density, which was a function of the inverse frequency up to about 100 Hz. The spectral density of the noise of the open LamB channels was a quadratic function of the applied voltage. Its magnitude was not correlated to the number of channels in the lipid bilayer membrane. Upon addition of sugars to the aqueous phase the current decreased in a dose-dependent manner. Simultaneously, the spectral density of the current noise increased drastically, which indicated interaction of the sugars with the binding site inside the channel. The frequency dependence of the spectral density was of Lorentzian type, although the power of its frequency dependence was not identical to -2. Analysis of the power density spectra using a previously proposed simple model (Benz, R., A. Schmid, and G. H. Vos-Scheperkeuter. 1987. J. Membr. Biol. 100: 12-29), allowed the evaluation of the on- and the off-rate constants for the maltopentaose binding to the binding site inside the LamB channels. This means also that the maltopentaose flux through the LamB channel could be estimated by assuming a simple one-site, two-barrier model for the sugar transport from the results of the noise analysis.

Bacterial Outer Membrane Proteins↗
PubMed1994

Permeability of the cell wall of Mycobacterium smegmatis.

The cell wall of Mycobacterium smegmatis mc2155 was shown to be an effective permeability barrier to hydrophilic compounds. Permeability coefficients to beta-lactams ranged from 10 x 10(-7) to 0.5 x 10(-7) cms-1. Cell wall proteins were solubilized with EDTA and Genapol and were tested for channel-forming activity by reconstitution into lipid bilayers. Proteins were able to induce a voltage-gated cation-selective channel. The mycobacterial porin channel appeared to be water-filled since the single-channel conductance followed the mobility sequence of hydrated ions in the aqueous phase. On the basis of the Renkin equation and the single-channel conductance, the channel diameter was estimated to be around 3 nm. Model calculations showed that cation selectivity may be caused by four negative point-charges at the channel mouth. The permeability properties of the cell wall of intact cells were in good agreement with those of the reconstituted channel. Negatively charged cephalosporins, cefamandole and cephalothin, diffused at a 10- to 20-fold lower rate than the zwitterionic cephaloridine. The mycobacterial porin represents a major hydrophilic pathway of the cell wall of M. smegmatis.

Cations↗
PubMed1994

Role of androgens in the regulation of the human menstrual cycle.

Although previous investigations have examined the importance of androgens in the regulation of the human menstrual cycle, no consensus has been reached, due to conflicting results. We have therefore used the non-steroidal anti-androgen flutamide as a pharmacological probe to evaluate the role of androgens in the control of gonadotropin secretion in normally cycling women. Eight women were studied during control and treatment cycles, during which either placebo (as control) or flutamide (750 mg orally) was given daily. Blood was sampled every other day during the follicular and luteal phases and daily around the expected midcycles for determination of luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol, progesterone and androgens (testosterone, androstenedione, dehydroepiandrosterone sulfate) by radioimmunoassay and immunoradiometric assay. To establish unstimulated and gonadotropin releasing hormone (GnRH)-stimulated gonadotropin profiles, blood samples were frequently collected (every 10 min for 8 h, GnRH 25 micrograms i.v. after 7 h on day 10 in both the control and treatment cycles. Compared to control conditions, the durations of both the follicular and luteal phases did not change considerably during flutamide treatments. Serum androgen levels (testosterone, androstenedione, dehydroepiandrosterone sulfate) were significantly (p < 0.01) reduced during androgen antagonism. Daily gonadotropin and estradiol levels did not differ between control and flutamide cycles, while progesterone secretion tended to be attenuated (p = 0.2) during the luteal phases of the flutamide cycles. The LH and FSH secretory profiles and the GnRH-stimulated gonadotropin responses remained virtually unchanged during androgen antagonism.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1993

Abscisic acid-lipid interactions: a phospholipid monolayer study.

Lipid monolayer studies were performed on a Langmuir trough in the absence and in the presence of the plant hormone abscisic acid (ABA). The ABA-induced effects on the lipid monolayers can be summarized as follows: (i) ABA as the free acid (pH below 5.3) increased the molecular area and slightly decreased the surface pressure in the collapse points of monolayers made of saturated, unsaturated and of mixed lipids; ABA as the anion showed only minor effects. (ii) The ABA-induced area increase of the lipid monolayers decreased when the surface pressure increased, but some ABA remained in the monolayers made of unsaturated phospholipids even at collapse pressure. (iii) The incorporation of ABA into the monolayers could be inhibited by adding the plant sterol beta-sitosterol to the monolayer forming phospholipids. (iv) There was no substantial difference of ABA action on plant phospholipids as compared with other phospholipids. (v) ABA had a much stronger influence on unsaturated phospholipids than on saturated ones. (vi) ABA decreased the phase-transition temperature of saturated phospholipids. These results, which agree with those obtained from phospholipid vesicle studies, indicate that the physical state of the lipid is important for the ability of ABA penetrating into the lipid monolayer. Finally, a possible relevance of these results is discussed in terms of the action of ABA on guard cell membranes of plants.

Abscisic Acid↗
PubMed1993

Anion transport through the phosphate-specific OprP-channel of the Pseudomonas aeruginosa outer membrane: effects of phosphate, di- and tribasic anions and of negatively-charged lipids.

The mechanism of anion transport through the phosphate-starvation inducible OprP-channel of Pseudomonas aeruginosa outer membrane was studied in planar lipid bilayer membranes. The single-channel conductance of OprP was 160 pS in 100 mM chloride solution. Addition of other anions, in particular of phosphate, di and tribasic anions lead to a strong decrease of the chloride conductance. The decrease was used to calculate the stability constants for the binding of these ions to the binding site of the channel on the basis of a one-site two-barrier model. The stability constant of the binding of phosphate to the site was 11,000 M-1 at neutral pH. Surprisingly, di- and tribasic anions, such as sulfate and citrate had a much lower affinity to the binding site inside the channel. Although the single-channel conductance was dependent on the external pH, the stability constants for phosphate binding decrease only slightly for increasing the pH. The use of negatively-charged lipids instead of neutral ones in the planar lipid bilayers had no influence on the single-channel conductance of the OprP-channel, suggesting that the channel is shielded from the influence of surrounding molecules. Its permeability properties are probably not influenced by negatively-charged lipopolysaccharide molecules.

Anions↗
PubMed1993

Characterization of the chloride carrier in the plasmalemma of the alga Valonia utricularis: the inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid.

The effect of the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on the Cl(-)-transport system located in the plasmalemma of cells of the giant marine alga Valonia utricularis was studied by using the charge pulse relaxation technique. Analysis of the biphasic relaxation patterns in terms of the kinetic model published previously (Wang, J., Wehner, G., Benz, R. and Zimmermann, U. (1991) Biophys. J. 59, 235-248) demonstrated that extracellular DIDS dramatically reduced the translocation rate, KAS, of the Cl(-)-carrier complex (maximal inhibition 79%). The translocation rate of the free carrier molecules, KS, as well as the total surface concentration of the carrier, No, were not affected. A Hill-plot of DIDS inhibition on KAS yielded an half-maximal inhibition concentration (IC50) of 3.9 x 10(-5) M and Hill-coefficient of 1.61, suggesting a co-operative binding of the inhibitors to the Cl(-)-carrier. The maximal inhibition of DIDS was dependent on the extracellular Cl(-)-concentration. This inhibition was not competitive to chloride, since it increased and did not decrease with increasing chloride concentration. The DIDS effect decreased with increasing pH-value (investigated pH range between 6.5 and 10). Intravascular DIDS or SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid) had no effect on the biphasic voltage relaxation pattern. These results showed that the binding sites of DIDS must be located on the outer surface of the plasmalemma of V. utricularis and, in turn, supported previous conclusions that the Cl(-)-carrier (which is assumed to be part of the turgor-pressure-sensing mechanism) is only located in the outer membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Use of irreversible electrical breakdown of lipid bilayers for the study of interaction of membranes with surface active molecules.

Lipid bilayers were formed in the presence of different macromolecules and high electrical fields were used to induce mechanical rupture of the membranes. The kinetics of pore formation during irreversible breakdown was studied as a function of the macromolecules. We observed that macromolecules having a strong binding affinity to the membrane alter the time course of pore formation significantly. We propose this method as a simple test for adsorption of macromolecules to membranes.

Adsorption↗
PubMed1993

Characterization of the channel formed by the mycobacterial porin in lipid bilayer membranes. Demonstration of voltage gating and of negative point charges at the channel mouth.

We studied the channel formed by the mycobacterial porin from the cell wall of Mycobacterium chelonae (Trias, J., Jarlier, V., and Benz, R. (1992) Science 258, 1479-1481) by reconstituting the mycobacterial porin and cell wall extracts in lipid bilayer membranes. The channel exhibited two different states in lipid bilayer membranes at 10 mV of applied voltage. One was characterized by a steplike appearance while the other showed a fast, voltage-dependent, flickering behavior between a closed and an open state. The channel was voltage-gated, and starting at 40 mV of applied voltage the mycobacterial porin channel switched to a closed configuration in an asymmetric fashion. The channel was cation-selective and had 2.5-point negative charges at both sides of the channel. Identical channels were observed when membranes were reconstituted with cell wall extracts, suggesting that there is only one porin species in the mycobacterial cell wall.

Bacterial Outer Membrane Proteins↗
PubMed1993

Crystallization and preliminary X-ray diffraction analysis of ScrY, a specific bacterial outer membrane porin.

The sucrose-specific outer membrane porin ScrY of Salmonella typhimurium was isolated from Escherichia coli K-12 strain KS 26 containing the plasmid pPSO112. The protein was purified to homogeneity by differential extraction of the cell envelope in the presence of the detergents sodium dodecyl sulfate and lauryl (dimethyl)-amine oxide (LDAO). The porin had apparent molecular weights of 58 kDa and 120 kDa for the monomer and for the trimer, respectively, on SDS/PAGE. The purified trimers were crystallized using poly(ethylene glycol) 2000 and the detergents octylglucoside (OG) and hexyl-(dimethyl)-amine oxide (C6DAO). X-ray diffraction of the crystals showed reflections to 2.3 A. The space group of the crystals was R3 and the lattice constants of the hexagonal axes were a = b = 112.85 A and c = 149.9 A. The crystal volume per unit of protein molecular weight was 3.47 A3/Da.

Bacterial Outer Membrane Proteins↗
PubMed1993

Conversion of the FhuA transport protein into a diffusion channel through the outer membrane of Escherichia coli.

The FhuA receptor protein is involved in energy-coupled transport of Fe3+ via ferrichrome through the outer membrane of Escherichia coli. Since no energy source is known in the outer membrane it is assumed that energy is provided through the action of the TonB, ExbB and ExbD proteins, which are anchored to the cytoplasmic membrane. By deleting 34 amino acid residues of a putative cell surface exposed loop, FhuA was converted from a ligand specific transport protein into a TonB independent and nonspecific diffusion channel. The FhuA deletion derivative FhuA delta 322-355 formed stable channels in black lipid membranes, in contrast to wild-type FhuA which did not increase membrane conductance. The single-channel conductance of the FhuA mutant channels was at least three times larger than that of the general diffusion porins of E. coli outer membrane. It is proposed that the basic structure of FhuA in the outer membrane is a channel formed by beta-barrels. Since the loop extending from residue 316 to 356 is part of the active site of FhuA, it probably controls the permeability of the channel. The transport-active conformation of FhuA is mediated by a TonB-induced conformational change in response to the energized cytoplasmic membrane. The ferrichrome transport rate into cells expressing FhuA delta 322-355 increased linearly with increasing substrate concentration (from 0.5 to 20 microM), in contrast to FhuA wild-type cells, which displayed saturation at 5 microM. This implies that in wild-type cells ferrichrome transport through the outer membrane is the rate-limiting step and that TonB, ExbB and ExbD are only required for outer membrane transport.

Amino Acid Sequence↗
PubMed1993

Oligomerization of Escherichia coli haemolysin (HlyA) is involved in pore formation.

Coexpression of pairs of nonhaemolytic HlyA mutants in the recombination-deficient (recA) strain Escherichia coli HB101 resulted in a partial reconstitution of haemolytic activity, indicating that the mutation in one HlyA molecule can be complemented by the corresponding wild-type sequence in the other mutant HlyA molecule and vice versa. This suggests that two or more HlyA molecules aggregate prior to pore formation. Partial reconstitution of the haemolytic activity was obtained by the combined expression of a nonhaemolytic HlyA derivative containing a deletion of five repeat units in the repeat domain and several nonhaemolytic HlyA mutants affected in the pore-forming hydrophobic region. The simultaneous expression of two inactive mutant HlyA proteins affected in the region at which HlyA is covalently modified by HlyC and the repeat domain, respectively, resulted in a haemolytic phenotype on blood agar plates comparable to that of wild-type haemolysin. However, complementation was not possible between pairs of HlyA molecules containing site-directed mutations in the hydrophobic region and the modification region, respectively. In addition, no complementation was observed between HlyA mutants with specific mutations at different sites of the same functional domain, i.e. within the hydrophobic region, the modification region or the repeat domain. The aggregation of the HlyA molecules appears to take place after secretion, since no extracellular haemolytic activity was detected when a truncated but active HlyA lacking the C-terminal secretion sequence was expressed together with a nonhaemolytic but transport-competent HlyA mutant containing a deletion in the repeat domain.

Bacterial Proteins↗