Biomedical subjects

R Benz

Publications and source records attributed to R Benz.

At least 127 records · Page 7Linked to original sources

PubMed1993

Kinetics of the iodine- and bromine-mediated transport of halide ions: demonstration of an interfacial complexation mechanism.

Stationary and kinetic experiments were performed on lipid bilayer membranes to study the mechanism of iodine- and bromine-mediated halide transport in detail. The stationary conductance data suggested that four different 1:1 complexes between I2 and Br2 and the halides I- and Br- were responsible for the observed conductance increase by iodine and bromine (I3-, I2Br-, Br2I-, and Br3-). Charge pulse experiments allowed the further elucidation of the transport mechanism. Only two of three exponential voltage relaxations predicted by the Läuger model could be resolved under all experimental conditions. This means that either the heterogeneous complexation reactions kR (association) and kD (dissociation) were too fast to be resolved or that the neutral carriers were always in equilibrium within the membrane. Experiments at different carrier and halide concentrations suggested that the translocation of the neutral carrier is much faster than the other processes involved in carrier-mediated ion transport. The model was modified accordingly. From the charge pulse data at different halide concentrations, the translocation rate constant of the complexed carriers, kAS, the dissociation constant, kD, and the total surface concentration of charged carriers, NAS, could be evaluated from one single charge pulse experiment. The association rate of the complex, kR, could be obtained in some cases from the plot of the stationary conductance data as a function of the halide concentration in the aqueous phase. The translocation rate constant, kAS, of the different complexes is a function of the image force and of the Born charging energy. It increases 5000-fold from Br3- to I3- because of an enlarged ion radius.

Biological Transport↗

PubMed1993

Kinetics of pore size during irreversible electrical breakdown of lipid bilayer membranes.

The kinetics of pore formation followed by mechanical rupture of lipid bilayer membranes were investigated in detail by using the charge-pulse method. Membranes of various compositions were charged to a sufficiently high voltage to induce mechanical breakdown. The subsequent decrease of membrane voltage was used to calculate the conductance. During mechanical breakdown, which was probably caused by the widening of one single pore, the membrane conductance was a linear and not exponential function of time after the initial starting process. In a large number of experiments using various lipids and electrolytes, the characteristic opening process of the pore turned out to be independent of the actual membrane potential and electrolyte concentration. Our theoretical analysis of the pore formation suggested that the voltage-induced irreversible breakdown is due to a decrease in edge energy when the pore had formed. After initiation of the pore, the electrical contribution to surface tension is negligible. The time course of the increase of pore size shows that our model of the irreversible breakdown is in good agreement with mechanical properties of membranes reported elsewhere.

Biophysical Phenomena↗

PubMed1993

TolC of Escherichia coli functions as an outer membrane channel.

Reconstitution experiments were performed with TolC from Escherichia coli outer membrane by using the lipid bilayer membrane technique. TolC was purified by elution of the oligomeric and the monomeric forms out of preparative SDS-PAGE. The oligomeric but not the monomeric form of the protein was able to increase the specific conductance of artificial lipid bilayer membranes. Investigation of the membrane activity in single-channel experiments suggested that TolC formed ion-permeable channels. The channels of 80 pS in 1 M KCl had a much smaller single-channel conductance than the general diffusion pores of E. coli outer membrane (1500 pS). The single-channel conductance was only moderately dependent on the bulk aqueous KCl concentration which indicated either ion binding or charge effects. Titration of TolC-induced membrane conductance with peptides lead to a dose-dependent decrease of the conductance. This result suggested that TolC contained a binding site for peptides. A dissociation constant of 20 mM was calculated for the binding of the tripeptide H-Gly-Gly-Leu-OH to the binding site. The results are consistent with the assumption that TolC acts as an outer membrane channel for peptides.

Amino Acid Sequence↗

PubMed1993

Topology of PhoE porin: the 'eyelet' region.

A model for the topology of the PhoE porin has been proposed according to which the polypeptide traverses the outer membrane sixteen times mostly as amphipathic beta-sheets, thereby exposing eight loops at the cell surface. Until now, no evidence has been obtained for the surface exposure of the third loop. Recently, the structure of porin of Rhodobacter capsulatus has been determined. The proposed model of PhoE is very similar to the structure of the R. capsulatus porin, which has an 'eyelet' region, extending into the interior of the pore. The proposed third external loop of PhoE might form a similar 'eyelet' region. To determine the location of the predicted third external loop of PhoE, multiple copies of an oligonucleotide linker encoding an antigenic determinant of VP1 protein of foot-and-mouth disease virus (FMDV) were inserted. All hybrid proteins were properly inserted in the outer membrane. The monoclonal antibody MA11, directed against the linear FMDV epitope, was able to bind only to intact cells expressing a hybrid PhoE protein with at least three copies of the FMDV epitope present. Antibiotic sensitivity tests and single-channel conductance measurements revealed that the insertions influenced the channel size. These results are consistent with a location of the third loop of PhoE within the pore channel.

Amino Acid Sequence↗

PubMed1993

Chronobiology of prolactin secretion in women: diurnal and sleep-related variations in the pituitary lactotroph sensitivity.

While a nocturnal rise accounts for the marked circadian variability of prolactin (PRL) secretion in humans, the mechanisms subserving this neuroendocrine manifestation are still obscure. Since gonadotropin-releasing hormone (GnRH) stimulates PRL under physiological conditions, we questioned whether changes in the pituitary lactotroph sensitivity to GnRH during the 24-hour cycle may contribute to the expression of circadian PRL rhythmicity. Accordingly, 8 women were studied in the early follicular phase of their cycles (days 2-5) on 6 occasions in random order: during daytime between 10.00 and 14.00 h ('day' studies), at night between 22.00 and 02.00 h, when the women were awake ('night' studies), and finally, during the identical night hours, when the women were asleep ('sleep' studies). On all occasions, blood was collected at 10-min intervals for 4 h, while either GnRH (25 micrograms i.v. bolus) or saline (as control) was injected twice within 2 h. As assessed by the net PRL increments (differences between unstimulated nadir and stimulated peak) and the areas under the PRL response curves, the PRL secretion was not substantially altered following GnRH stimulations during the day studies. In contrast, PRL release was markedly enhanced (p < 0.05 or less vs. day studies), when GnRH was administered during the night studies. This GnRH-stimulated PRL release was even further increased (p < 0.01 vs. day, p < 0.05 vs. night or saline studies), when GnRH had been given during sleep.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1993

The somatotropin-somatomedin axis in adult patients with Turner syndrome: measurement of stimulated GH, GH-BP, IGF-I, IGF-II and IGFBP-3 in 25 patients.

So far, few studies have addressed the regulation of GH and GH-dependent growth factors in adult patients with Turner syndrome. We therefore studied a group of 25 genetically proven patients with Turner syndrome (age 20-50 years) and 10 control women (25-48 years). Turner patients were significantly shorter (148.7 +/- 1.1 cm vs. 169.1 +/- 2.3 cm; mean +/- SE; p < 0.0001) and more overweight [body mass index (BMI)] 25.6 +/- 1.3 vs. 21.4 +/- 0.6 in controls; p < 0.01). No significant differences were present when the integrated GH response to stimulation with arginine and the serum levels of GH-binding protein (GH-BP), IGF-I, IGF-II and binding protein 3 for IGFs (IGFBP-3) were compared between the two groups. However, more detailed analysis revealed significant abnormalities of the somatotropic axis in Turner patients. Pituitary GH secretion was negatively and serum GH-BP positively related to the degree of overweight in normal patients. In Turner patients, no such relationship was present, while IGF-II significantly increased with BMI. IGFBP-3 was positively related to adult height in normal women but not in Turner patients. While serum testosterone values did not affect any of the somatotropic parameters measured, there was a previously unreported, inverse relation between serum estradiol and GH-BP in controls but not in Turner patients. While adult patients with Turner syndrome do not display endocrine features of GH insufficiency, a detailed analysis reveals several abnormalities of the interrelation between anthropometric parameters, sex steroids and the pituitary-somatomedin axis.

Adult↗

PubMed1993

Diurnal variation in the elimination rate of human growth hormone (GH): the half-life of serum GH is prolonged in the evening, and affected by the source of the hormone, as well as by body size and serum estradiol.

The half-lives of endogenous and exogenous (biosynthetic monomeric) GH were compared in the morning and evening in healthy young men (n = 10). In group A, a bolus of GHRH was injected either at 0800 or at 2000 h, whereas in group B hGH was injected iv after suppression of endogenous GH by somatostatin. GH was sampled every 10 min and the t1/2 for GH was determined by deconvolution analysis (two compartments). The GH elimination half-life was shorter in the morning: for endogenous GH, t1/2 was 23 +/- 1.1 min (mean +/- SE) in the morning compared to 26 +/- 1.7 min in the evening (P < 0.02). T1/2 correlated negatively with estradiol (r = -0.78; P < 0.01) and positively with sex hormone-binding globulin (r = 0.71; P < 0.03). The half-life of exogenous 22-kilodalton GH was shorter compared to endogenous GH (P < 0.002), and diurnal variation was even more pronounced: t1/2 was 14 +/- 1.0 min in the morning and 19 +/- 1.0 min in the evening (P < 0.01). These effects were not due to differences in GH distribution volumes. The half-life of exogenous GH was significantly affected by weight (r = -0.8; P < 0.01) and height (r = 0.67; P < 0.05). We conclude that in young males, the rate of GH disappearance from the circulation depends on both diurnal mechanisms as well as the source or structural composition of the hormone. Body size and sex steroids contribute to the variability of GH clearance in healthy man.

Adult↗

PubMed1993

Human chorionic gonadotropin secretion from the early human placenta: in vitro regulation by progesterone and its antagonist.

Local actions of placental neurohormones and sex steroids have been proposed to play a role in the regulation of human chorionic gonadotropin (hCG) release from the human placenta. Accordingly, we utilized an in vitro perifusion system and cultures of placental explants to investigate short- and long-term effects of progesterone and its respective antagonist on hCG secretion from the human first-trimester placenta. Tissue slices (100 mg) obtained from legal pregnancy terminations of 9-12 weeks of gestation were continuously perifused and the effluent collected in fractions of 2-20 min. After initial perifusion periods of 30-140 min, either progesterone, a progesterone antagonist (ZK 98-299), or both were added to the perifusion medium at final concentrations of 10(-4)-10(-9) mol/l. Administration was either continuous or intermittent in 10-min pulses. Furthermore, 50-mg pieces of placental explants were cultured in multiwell tissue culture plates for up to 5 days. During the perifusion studies, hCG (determined by enzyme immunoassay) was released in a pulsatile fashion. This hCG pulsatility was decreased in response to both progesterone and progesterone antagonist at all concentrations tested. In contrast, intermittent administration of either treatment increased the hCG secretion. Secretion of hCG was not affected when progesterone and its antagonist were co-administered at equimolar concentrations. These observations demonstrate the diverging effects of progesterone and its antagonist on hCG secretion from the human first-trimester placenta in vitro, depending on the experimental conditions. Thus, progesterone-modulated hCG secretion appears to be regulated in a complex manner, its fine tuning involving other, as yet uninvestigated intraplacental factors.

Abortion, Induced↗

PubMed1992

Identification of two general diffusion channels in the outer membrane of pea mitochondria.

Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent solubilized mitochondrial membranes of pea seedlings (Pisum sativum). The addition of the detergent-solubilized material to the membranes resulted in a strong increase of the membrane conductance. To identify the proteins responsible for membrane activity the detergent extracts were applied to a hydroxyapatite (HTP) column and the fractions were tested for channel formation. The eluate of the column contained a protein which migrated as a single band with an apparent molecular mass of 30 kDa on SDS-PAGE. This channel was identified as the porin of pea mitochondria since it formed voltage-dependent channels with single-channel conductances of 1.5 and 3.7 nS in 1 M KCl and an estimated effective diameter of about 1.7 nm. Further elution of the column with KCl containing solutions yielded fractions which resulted in the formation of transient channels in lipid bilayer membranes. These channels had a single-channel conductance of 2.2 nS in 1 M KCl and had also the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. Zero-current membrane potential measurements suggested that pea porin was anion-selective in the open state. The selectivity of the second channel was investigated by the measurement of the reversal potential. It was also slightly anion-selective. Its possible role in the metabolism of mitochondria is discussed.

Anions↗

PubMed1992

Porins in the cell wall of mycobacteria.

The cell wall of mycobacteria is an efficient permeability barrier that makes mycobacteria naturally resistant to most antibiotics. Liposome swelling assays and planar bilayer experiments were used to investigate the diffusion process of hydrophilic molecules through the cell wall of Mycobacterium chelonae and identify the main hydrophilic pathway. A 59-kilodalton cell wall protein formed a water-filled channel with a diameter of 2.2 nanometers and an average single-channel conductance equal to 2.7 nanosiemens in 1 M potassium chloride. These results suggest that porins can be found in the cell wall of a Gram-positive bacterium. A better knowledge of the hydrophilic pathways should help in the design of more effective antimycobacterial agents.

Bacterial Proteins↗

PubMed1992

Purification, functional characterization, and cDNA sequencing of mitochondrial porin from Dictyostelium discoideum.

Porin of Dictyostelium discoideum was extracted from mitochondria with Genapol X-80 and was purified by hydroxyapatite and CM-cellulose chromatography. The purified protein displayed a single band of 30 kDa in SDS-polyacrylamide gel electrophoresis. The formation of channels in artificial lipid bilayer membranes defined its function as a channel-forming component. Its average single-channel conductance was 3.9 nanosiemens in 1 M KCl, which suggested that the effective diameter of the channel is approximately 1.7 nm at small transmembrane potentials. The channel displayed a characteristic voltage dependence for potentials higher than 20 mV. It switched to substates of smaller conductance and a selectivity different to that of the open state. The closed state was stabilized at low ionic strength. The cDNA sequence of mitochondrial porin from D. discoideum was determined. It showed little sequence similarities to other known mitochondrial porins. The functional similarity, however, was striking. Localization of the porin in the mitochondrial outer membrane was confirmed by immunogold labeling of cryosections of fixed cells.

Amino Acid Sequence↗

PubMed1992

Investigation of the selectivity of maltoporin channels using mutant LamB proteins: mutations changing the maltodextrin binding site.

Wild-type and seven mutant maltoporins were purified and their channel-forming activities studied after reconstitution into black lipid membranes. The proteins were assayed for alterations at the maltodextrin binding site by measuring the sugar-dependent blockage of ion flux through these channels. Some substitutions (R8H, W74R) caused reduced channel affinity for all maltodextrins without changing single channel conductivities. The channel with a GlySer insertion after residue 9 was also poorly blocked by sugars but unique to this protein, the channel showed a striking, almost exponential increase of affinity with increasing maltodextrin chain length. In mutants with AspPro insertions after residues 79 and 183, there was an increase in affinity for glucose and maltose but not longer maltodextrins. The additional negative charge in the AspPro insertion mutants increased the cation selectivity of maltoporin channels, as did the decrease in positive charge resulting from the R8H substitution. A mutant with a W120C substitution also showed an increased affinity for glucose and maltose but reduced affinity for longer maltosaccharides. In contrast, a Y118F substitution resulted in an 8-fold increase in maltotriose affinity, but lesser improvements for other sugars. These results are interpreted to reflect changes in subsites contributing to an extended binding site within the channel, which in turn determines the overall sugar affinity of maltoporin.

Amino Acid Sequence↗

PubMed1992

Reconstitution of vacuolar ion channels into planar lipid bilayers.

Vacuolar ion channels were characterized after reconstitution into planar lipid bilayers. (1) Channel activity was observed after incorporation of tonoplast-enriched microsomal membranes, purified tonoplast membranes or of solubilized tonoplast proteins. (2) Channels of varying single-channel conductances were detected after reconstitution. In symmetrical 100 mmol l-1 KCl, conductances between 1 and 110 pS were frequently measured; the largest number of independent reconstitution events was seen for single-channel conductances of 16-25 pS (28 experiments), 30-42 pS (26), 49-56 pS (15) and 64-81 pS (15). Channel current usually increased linearly with voltage. (3) In asymmetrical solutions, cation-, non-selective and, for the first time for the tonoplast, anion-selective channels were detected. Ca(2+)-dependent regulation of channel opening was not observed in our reconstitution system. (4) Permeability was also observed for Cl-, NO3-, SO4(2-) and phosphate. (5) After fractionation of tonoplast proteins by size exclusion chromatography, ion channel activity was recovered in specific fractions. (6) Some of these fractions catalyzed sulfate transport after reconstitution into liposomes. The results suggest that different channels are active at the tonoplast membrane at a larger number than has been concluded from previous work.

Conductometry↗

PubMed1992

Regulation of adenylyl cyclase from Paramecium by an intrinsic potassium conductance.

Hyperpolarization of the cell membrane of Paramecium stimulates adenosine 3',5'-monophosphate (cAMP) formation. Manipulations of the K+ resting conductance of the ciliate by adaptation in different buffers affected excitability of the cAMP generating system. Blockade of K+ channels inhibited hyperpolarization-stimulated cAMP formation. A mutant of Paramecium that is unable to control its K+ resting conductance had a defect in cAMP formation. Purified adenylyl cyclase, when incorporated into an artificial lipid bilayer membrane, revealed properties of a voltage-independent K+ channel. This indicates that the adenylyl cyclase of Paramecium has a secondary function as carrier of the K+ resting conductance. A hyperpolarization-activated K+ efflux appears to directly regulate adenylyl cyclase activity in vivo.

Adenylyl Cyclases↗

PubMed1992

Partial purification of a potassium channel with low permeability for sodium from tonoplast membranes of Hordeum vulgare cv. Gerbel.

A potassium-specific tonoplast channel was identified by reconstitution of tonoplast polypeptides into planar lipid bilayer membranes. Highly purified tonoplast membranes were solubilized in Triton X-100-containing buffer and fractionated by size-exclusion chromatography. The protein fractions were assayed for ion channel activity in a planar bilayer system, and the potassium channel was routinely recovered in specific fractions corresponding to an apparent molecular mass of 80 kDa. In symmetrical electrolyte solutions of 100 mM potassium chloride, the potassium channel had a single-channel conductance of 72 pS. Substates of the channel with conductances of 17, 33 and 52 pS were frequently observed. After identification of the channel in low or high KCl, addition of sodium acetate or sodium chloride caused only insignificant conductance changes. This result suggested that the channel was not or little permeable for sodium or chloride, whereas it had similar single-channel conductance for rubidium and caesium ions as compared with potassium ions. The channel is presumably responsible for the equilibration of potassium between the vacuole and the cytosol. The role of the channel in the physiology of the barley cell under salt stress is discussed.

Child, Preschool↗

PubMed1992

The cation-selective substate of the mitochondrial outer membrane pore: single-channel conductance and influence on intermembrane and peripheral kinases.

The polyanion-induced substate of the outer mitochondrial membrane was studied in vivo and in vitro. Study of the substate in artificial bilayers showed that it is highly cation selective. The induction of the substate in intact mitochondria leads to a complete inhibition of the intermembrane kinases, such as creatine kinase and adenylate kinase, which were excluded from the external ATP pool. Peripheral kinases, such as hexokinase, were blocked when they utilized internal ATP. The results with intact mitochondria suggested the existence of two regions of the outer membrane containing channels of different states, which may be involved in the regulation of intermembrane and peripheral kinases.

Adenosine Triphosphate↗

PubMed1992

Role of frequency and amplitude of repetitive HCG stimulations for sustained progesterone secretion from the bovine corpus luteum in vitro.

This investigation aimed at evaluating a role for frequencies and amplitudes of repeated HCG stimulations for the optimal maintenance of progesterone (P4) secretion from the bovine corpus luteum in vitro. Slices (100-120 mg) of midluteal bovine corpora lutea were perifused with medium M199 (0.05% BSA, pH 7.2, 38.5 degrees C) and the perifusion effluent collected at 15 minute intervals for 20-29 hours. Unstimulated P4 release (n = 5) was distinctly pulsatile (by Pulsar pulse algorithm), with pulses occurring every 90 +/- 6 minutes (mean +/- SEM) and pulse amplitudes of 14.4 +/- 1.1 ng. Conversely, no pulses were detected in two control perifusions. Unstimulated P4 release increased during the first 5 perifusion hours (from 39.3 +/- 4.6 to 50.3 +/- 5.6 ng/15 min, p less than 0.01), but then appeared to decline (to 29.3 +/- 1.3 ng/15 min, p less than 0.05) towards the end of the perifusion periods. Hourly pulses of HCG (6.7 mM) did not change the P4 pulse amplitudes (16.6 +/- 2.0 ng), the pulse periodicities (105 +/- 15 min) and overall release rates (34.7 +/- 5.7 ng/15 min), nor did they prevent the decline in P4 secretion towards the end of perifusions (n = 5). In contrast, 2-hourly HCG stimulations maintained stable P4 release rates throughout the perifusion periods (34.7 +/- 6.8 ng/15 min), with P4 pulses of similar amplitudes (14.7 +/- 1.7 ng), but of lower periodicities (135 +/- 2 min, p less than 0.05) than during unstimulated conditions (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Haemolysin-derived synthetic peptides with pore-forming and haemolytic activity.

Escherichia coli haemolysin (Hlya) is a pore-forming protein which belongs to the family of 'Repeat-toxins' (RTX) (Lo et al., 1987; Lally et al., 1989; Kraig et al., 1990). A model for the pore-forming structure of HlyA has been proposed (Ludwig et al., 1991) which consists of eight transmembrane segments all present in this hydrophobic region of HlyA. We report here that two synthetic peptides of 10 and 8 amino acids in length (Pep1 and Pep2, respectively), which are derived from transmembrane segment V, are able to form pores in an artificial lipid bilayer. In addition, Pep1 exhibits strong haemolytic activity when tested on human red blood cells (HRBCs). The haemolytic activity of Pep1 and of E. coli haemolysin is completely inhibited by antibodies raised against Pep1.

Amino Acid Sequence↗