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Biomedical subjects

G Müller

Publications and source records attributed to G Müller.

At least 271 records · Page 15Linked to original sources
PubMed1995

Characterization of non-inducible Tet repressor mutants suggests conformational changes necessary for induction.

Non-inducible tetracycline repressor (TetR) mutants were grouped in three structurally distinct classes. We quantitated in vivo operator binding, inducibility, and in vitro tetracycline binding of mutants from each class. Mutation of residues close to tetracycline (class 1) leads to reduced affinity for the drug. Mutation of residues located at the connection of the DNA-reading head with the protein core (class 2) and at the dimerization interface (class 3) bind inducer with the same affinity as wild-type TetR. These mutations interfere with the induced, but not the operator-binding conformation of TetR. The affinity of some class 1 mutants for tetracycline is less affected than their inducibility, suggesting that the mutated residues are important for triggering those conformational changes necessary for induction.

Amino Acid Sequence↗
PubMed1995

Optical properties of ocular fundus tissues--an in vitro study using the double-integrating-sphere technique and inverse Monte Carlo simulation.

Various models have been published calculating the light transport at the ocular fundus either for interpretation of in vivo reflectance measurements or for the prediction of photocoagulation effects. All these models took the absorption spectra of the pigments located at the ocular fundus, melanin, haemoglobin, xanthophyll, and the photoreceptor pigments, into account. However, light scattering inside the single fundus layers has not been investigated in detail and was, therefore, neglected in the calculations or only considered by very rough approximations. This paper presents measurements on specimens of retina, retinal pigment epithelium, choroid, and sclera using the double-integrating-sphere technique. Absorption coefficients, scattering coefficients, and anisotropy of scattering were calculated by an inverse Monte Carlo simulation from the measured collimated and diffuse transmittance and diffuse reflectance. Conclusions are drawn for the interpretation of fundus reflectance measurements, which are a useful tool in diagnostics and photocoagulation dosimetry.

Animals↗
PubMed1995

Direct stimulation of myocardial glucose transport and glucose transporter-1 (GLUT1) and GLUT4 protein expression by the sulfonylurea glimepiride.

Freshly isolated and primary cultured cardiac myocytes from adult rats were used to elucidate acute and chronic effects of the sulfonylurea drug glimepiride on basal and insulin-stimulated glucose transport and on expression of the transporter isoforms glucose transporter-1 (GLUT1) and GLUT4. A 30-min incubation with glimepiride (100 microM) was unable to modify the initial rates of 3-O-methylglucose transport in freshly isolated cardiocytes, both in the absence or presence of insulin (10(-7) M). Cells were then kept in serum-free culture for 20 h in the presence of glimepiride (10 microM) and a physiological insulin dose. Under these conditions, the sulfonylurea induced an increase in 2-deoxyglucose uptake to 186% of control. This drug effect was dose dependent and could also be demonstrated in the absence of insulin during the culture period. The acute action of insulin on glucose transport was additive to the effect of glimepiride, and the insulin responsiveness of glucose transport remained unaltered in sulfonylurea-treated cultures. Western blot analysis of crude membrane fractions obtained from cultured cardiocytes showed that glimepiride increased the expression of both GLUT1 and GLUT4 to 164% +/- 21% and 148% +/- 5% of control, respectively. We concluded that glimepiride increases cardiac glucose uptake by an insulin-independent pathway, probably involving an increased protein expression of GLUT1 and GLUT4. The increased expression of GLUT4 may have a therapeutic impact on the treatment of insulin-resistant states.

3-O-Methylglucose↗
PubMed1995

[In vitro studies of the specifications for methodological and technical equipment requirements of endoscopic infrared diaphanoscopy].

Endoscopic infrared diaphanoscopy is a method of improving orientation in the nasal cavity during endoscopy-controlled endonasal surgery. The transillumination is expected to facilitate the localisation of the natural passageways between the paranasal sinuses and the nasal cavity during endoscopic procedures. The present study specifies the necessary conditions on the basis of in vitro examinations using an animal model (sheep) and an anatomical preparation of half of the human head. The questions as to what paranasal sinus is at all accessible to diaphanoscopy, whether the method actually enables the connecting pathways between an individual sinus and the nasal cavity to be identified, and what technical demands need to be met, are considered and answered.

Animals↗
PubMed1995

[Revision of the stapes foot plate with various laser systems. 1: Continuous laser irradiation].

During stapedotomy, the small dimensions and highly sensitive anatomic structures present require the highest degree of precision and safety. The risk of damage to middle and inner ear structures through manipulation with conventional instruments can be reduced by non-contact perforation of the footplate with the laser beam. The present study was devised to clarify which of the presently available laser systems was best suited for use in stapes surgery and thus represent a significant alternative to conventional stapedotomy. Isolated human stapes and bovine compact-bone platelets were used to investigate the connections between the parameters of various laser systems and their effects on bone tissue. The aim was to optimize the laser parameters required to achieve a perforation of 500 microns to 600 microns in diameter. In addition, the thermal effects of laser irradiation were assessed. Three different systems were employed: the argon and CO2 lasers in continuous wave (cw) modes and the CO2 laser in superpulse mode. The lasers investigated offered the advantage of achieving an adequately large perforation with one or a few juxtaposed applications but caused in part extensive thermal side effects at the stapes footplate. To reduce thermal effects, footplate perforation was best performed by several juxtaposed single shots at low power, short pulse duration and small beam diameter. The suitability of the argon laser for stapedotomy was rendered doubtful in view of the lower absorption coefficient of the stapes for the argon laser beam and the considerable influence exerted by the degree of pigmentation of the irradiated tissue sites with a resultant poor reproducibility of the perforation diameter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

[Modifying the stapes footplate with various laser systems. II. The pulsed laser].

The object of the present study was to examine the tissue ablation capacity of various pulsed lasers at the stapes footplate. Isolated human stapes and bovine compact-bone platelets (thickness 90 microns) were used to determine effective laser parameters for achieving a perforation measuring 500 microns to 600 microns in diameter. Apart from achieving the perforation diameters, particular attention was given to the form and quality of the perforations, reproducibility of the perforation effect and the thermally altered border zones occurring at the footplate. Four pulsed laser systems were used: excimer, Ho:YAG, Er:YSGG and CO2 lasers. An adequately large perforation generally could only be achieved by several repeated shots at the same application site, since only a small amount of tissue was ablated per application. The mechanism of the photoablation caused the extent of the thermal side effects to be markedly lower than with the continuous wave (cw) and superpulse systems. For this reason and because of their highly reproducible perforation effect, they were basically better suited for stapedotomy than the cw systems. It was possible, however, that as a result of the longer application time and the need to repeatedly irradiate the same application site, the higher pulse counts could prove to be disadvantageous in clinical practice. Among the pulsed laser systems, the Er:YSGG laser had the highest ablation rate at the stapes and was thus the most effective laser for interventions at the footplate. Although somewhat less effective than the Er:YSGG laser in our studies, the Ho:YAG and pulsed CO2 lasers also appear to be suitable for stapes surgery. On the other hand, we did not consider the excimer laser (308 nm) to be particularly effective at the footplate because of its low ablation rates.

Animals↗
PubMed1995

[Thermal stress of the inner ear during laser stapedotomy. I: Continuous-wave laser].

As a consequence of perforating the footplate during laser stapedotomy, direct radiation to the inner ear will warm perilymph and adjacent structures. To determine the possible thermal dangers to cochlear structures from different laser parameters, heat transport mechanisms, temperature increases and temperature fields were investigated in a model system approximating caloric and physiologic changes in the inner ear. The temperature-time course of local cochlear warming showed a rapid convection-dependent increase that reached a peak at about the end of the laser impulse. An increase in power density caused an elevation of the temperature in all laser systems used. Maximum temperatures varied widely for CO2 lasers at the same wave-length, but a different beam-time behavior was found at a distance of 2 mm behind the perforation by using low-power densities. Heat values were lowest at a pulse duration of 50 ms in the superpulse (< 5 degrees C) and continuous wave (cw) modes (< 9 degrees C), while the highest value was found in the pulser mode (to 21 degrees C). After argon laser irradiation at high-power densities, temperatures were nearly independent of location (5.5-13 degrees C). When considering risks of possible inner ear damage from thermal stress during laser stapedotomy, application of the CO2 superpulse and cw laser appears to be safe over a large power-density range. Low energies using a small-beam diameter and short pulse durations (50-100 ms) are recommended. In contrast, use of the CO2 laser in a pulser mode may result in inner-ear damage because of the high temperatures produced. Structures located at a greater distance can be endangered by direct irradiation with the argon laser.

Body Temperature Regulation↗
PubMed1994

Membrane association of lipoprotein lipase and a cAMP-binding ectoprotein in rat adipocytes.

cAMP-binding ectoprotein (Gce1) and lipoprotein lipase (LPL) are anchored to plasma membranes of rat adipocytes by glycosylphosphatidylinositol (GPI) moieties as demonstrated by cleavage by bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), reactivity with anti-crossreacting determinant antibodies (anti-CRD), and metabolic labeling with radiolabeled palmitic acid and myo-inositol. Quantitative release from the membrane of LPL and Gce1 requires both lipolytic removal of their GPI anchors and the presence of either 2 M NaCl or 1 mM inositol 1,2-cyclic monophosphate or inositol 1-monophosphate. PI-PLC-cleaved and released LPL or Gce1 reassociates with isolated plasma membranes of rat adipocytes and, less efficiently, with membranes of 3T3 fibroblasts. The specificity of the reassociation is demonstrated (i) by its inhibition after pretreatment of the membranes with trypsin, (ii) by its competition with inositol 1,2-cyclic monophosphate and inositol 1-monophosphate in a concentration-dependent manner, and (iii) by the limited number of binding sites. Enzymic or chemical removal as well as masking with anti-CRD antibodies of the terminal inositol (cyclic) monophosphate moiety of hydrophilic Gce1 and LPL significantly impairs the reassociation. These data suggest that in rat adipocytes GPI-proteins are not readily released from the cell surface upon lipolytic cleavage, but remain associated through a receptor which specifically recognizes the terminal inositol (cyclic) monophosphate epitope of the (G)PI-PLC-cleaved GPI moiety. This interaction may have implications for the regulated membrane release of GPI-proteins and for their possible internalization.

3T3 Cells↗
PubMed1994

Amino acid requirements of the nucleocapsid protein of HIV-1 for increasing catalytic activity of a Ki-ras ribozyme in vitro.

The nucleocapsid protein NCp7 of HIV-1 is a single-stranded nucleic acid binding protein with several functions such as specific recognition, dimerization and packaging of viral RNA, tRNA annealing to viral RNA and protection against nucleases. Since some of these functions involve annealing and double-stranded RNA-melting activity we applied the nucleocapsid protein to a hammerhead ribozyme specific for the activated Ki-ras mRNA in vitro, which carries at its mutated codon 12 a GUU site. A synthetic ribozyme containing 2'-O-allyl-modified nucleotides and alternatively in vitro transcribed ribozymes were used. At a one to one molar ratio of substrate to ribozyme almost no cleavage is observed at 37 degrees C. Presence of a synthetic nucleocapsid protein significantly increases the catalytic activity of the ribozyme. Kinetic analyses by means of single and multiple turnover reactions performed at various substrate to ribozyme ratios lead to only a slight stimulation of the rate constants for single turnover reactions. The rate constants in multiple turnover reactions, however, are stimulated up to 17-fold by the presence of the nucleocapsid protein. The activating region of the nucleocapsid protein was characterized by a number of mutants. The mutants demonstrate that activation requires both basic amino acid clusters as evidenced by point mutations. Deletion mutants indicate that the second zinc finger is totally dispensable and that replacement of the first zinc finger by a glycine-glycine spacer only slightly reduces the enhancing effect of the nucleocapsid protein on the ribozyme.

Amino Acid Sequence↗
PubMed1994

Stimulation of glucose utilization in 3T3 adipocytes and rat diaphragm in vitro by the sulphonylureas, glimepiride and glibenclamide, is correlated with modulations of the cAMP regulatory cascade.

The long-term hypoglycemic activity of sulphonylurea drugs has been attributed, in part at least, to the stimulation of glucose utilization in extra-pancreatic tissues. The novel sulphonylurea, glimepiride, gives rise to a longer lasting reduction in the blood sugar level in dogs and rabbits compared to glibenclamide (Geisen K, Drug Res 38: 1120-1130, 1988). This cannot be explained adequately by elevated plasma insulin levels. This study investigated whether this prolonged hypoglycemic phase was based on the drug's abilities to stimulate glucose utilization and affect the underlying regulatory mechanisms in insulin-sensitive cells in vitro. It was found that in the absence of added insulin, glimepiride and glibenclamide (1-50 microM) stimulated lipogenesis (3T3 adipocytes) and glycogenesis (isolated rat diaphragm) approximately 4.5- and 2.5-fold, respectively, and reduced the isoproterenol-stimulated lipolysis (rat adipocytes) up to 40-60%. The increased glucose utilization was correlated with a 3-4-fold higher 2-deoxyglucose transport rate and amount of GLUT4 at the plasma membrane, as well as with increased activities of key metabolic enzymes (glycerol-3-phosphate acyltransferase, glycogen synthase) within the same concentration range. Furthermore, the low Km cAMP-specific phosphodiesterase was activated 1.8-fold, whereas the cytosolic cAMP level and protein kinase A activity ratios were significantly lowered after incubation of isoproterenol-stimulated rat adipocytes with the sulphonylureas. In many of the aspects studied the novel sulphonylurea, glimepride, exhibited slightly lower ED50-values than glibenclamide. This study demonstrates correlations existing between drug-induced stimulation of glucose transport/metabolism and cAMP degradation/protein kinase A inhibition as well as between the relative efficiencies of glimepiride and glibenclamide in inducing these extra-pancreatic processes. Therefore, it is suggested that the stimulation of glucose utilization by sulphonylureas is mediated by a decrease of cAMP-dependent phosphorylation of GLUT4 and glucose metabolizing enzymes. The therapeutic relevance of extra-pancreatic effects of sulphonylureas, in general, and of the differences between glimepiride and glibenclamide as observed in vitro in this work, in particular, remain to be elucidated.

3T3 Cells↗
PubMed1994

Selective recognition of cyclic RGD peptides of NMR defined conformation by alpha IIb beta 3, alpha V beta 3, and alpha 5 beta 1 integrins.

The binding of purified fibrinogen receptor alpha IIb beta 3, vitronectin receptor alpha V beta 3, and fibronectin receptor alpha 5 beta 1 to their corresponding ligands in solid-phase binding assays was used to examine the inhibitory activity of various linear and cyclic penta- and hexapeptides of different conformation containing RGD or RAD sequences. Cyclic peptides with different defined backbone conformations were designed by introducing a single D-amino acid or a proline at different positions in the ring. The data were calibrated for alpha IIb beta 3 integrin incorporated into a planar lipid bilayer by a physical method (total internal reflection fluorescence microscopy) which yielded KD = 1.7 microM for a linear RGD peptide and KD = 0.03 microM for fibrinogen. With this integrin, three cyclic hexapeptides ([GRGDFL], [ARGDFV], [GRGDFV]) were 2-4-fold more inhibitory than the linear GRGDS peptide in solid-phase assays and showed similar inhibition as the fibrinogen ligand. Six peptides had the same or a 2-fold lower activity as the linear reference peptide, and three peptides were up to 7-fold less active. Replacement of Arg or Asp by their stereoisomers or Gly by Ala resulted in a 100-1000-fold reduction in activity. With the two other integrins, a single cyclic pentapeptide [RGDFV] was 10-fold more active (alpha V beta 3) or equal in activity (alpha 5 beta 1) to linear GRGDS, while all of the other cyclic peptides were moderately or distinctly less active. Changes in the RGD sequence caused a less dramatic reduction in binding strength for alpha V beta 3 and alpha 5 beta 1 than for alpha II beta 3. Inhibitory activity was compared with the distance between the C beta atoms of Arg and Asp residues as determined by NMR and indicated that the optimum distance is in the range of 0.75-0.85 nm for alpha IIb beta 3 and at or below 0.67 nm for alpha V beta 3 and alpha 5 beta 1. This indicates that alpha IIb beta 3 less sensitive to variations in the RGD backbone structure and can accommodate a larger distance than the integrins alpha V beta 3 and alpha 5 beta 1.

Amino Acid Sequence↗
PubMed1994

Differential interaction of glimepiride and glibenclamide with the beta-cell sulfonylurea receptor. I. Binding characteristics.

Glimepiride is a novel sulfonylurea drug for treatment of non-insulin-dependent diabetes mellitus with higher blood sugar lowering efficacy in diabetic patients than glibenclamide raising the question whether this characteristics is in line with different binding of glimepiride and glibenclamide to the beta-cell sulfonylurea receptor. Scatchard plot analysis of [3H]sulfonylurea binding to membranes isolated from rat beta-cell tumors and (RINm5F) insulinoma cells and to RINm5F cells demonstrated that glimepiride has a 2.5-3-fold lower affinity than glibenclamide. This corresponded well to the 8-9-fold higher koff and 2.5-3-fold higher kon rates of glimepiride compared to glibenclamide as revealed by the dissociation and association kinetics of [3H]sulfonylurea binding and the Kd values calculated thereof. In agreement, the concentrations required for half-maximal displacement of [3H]sulfonylurea bound to beta-cell membranes were significantly higher for glimepiride compared to glibenclamide. However, the binding affinity of glimepiride measured by both equilibrium binding and kinetic binding studies upon solubilization of beta-cell tumor membranes and RINm5F cell membranes increased up to the value for glibenclamide. This was primarily based on a drastic decrease of the dissociation rate constant of glimepiride whereas the kinetics of glibenclamide binding remained largely unaffected upon solubilization. These data suggest that the Kd value alone is not sufficient for characterization of a sulfonylurea drug, since the kinetic binding parameters may also determine its acute blood sugar lowering efficacy.

ATP-Binding Cassette Transporters↗
PubMed1994

Differential interaction of glimepiride and glibenclamide with the beta-cell sulfonylurea receptor. II. Photoaffinity labeling of a 65 kDa protein by [3H]glimepiride.

Glimepiride is a novel sulfonylurea for the treatment of type II-diabetic patients exhibiting different receptor binding kinetics to beta-cell membranes with 8-9-fold higher koff rate and 2.5-3-fold higher kon rate compared to glibenclamide (see accompanying paper (Müller, G. et al. (1994) Biochim. Biophys. Acta 1191, 267-277)). To elucidate the molecular basis for this differential behaviour of glimepiride and glibenclamide, direct photoaffinity labeling studies using beta-cell tumor membranes were performed. [3H]Glimepiride was specifically incorporated into a membrane polypeptide of M(r) = 65,000 under conditions, which led to predominant labeling of a 140 kDa protein by [3H]glibenclamide (Kramer, W. et al. (1988) FEBS Lett. 229, 355-359). Labeling of the 140 kDa protein by [3H]glibenclamide was inhibited by unlabeled glimepiride and, vice versa, glibenclamide inhibited labeling of the 65 kDa protein by [3H]glimepiride. The 65 kDa protein was also specifically photolabeled by the sulfonylurea [125I]35623, whereas an 4-azidobenzoyl derivative of glibenclamide, N3-[3H]33055, exclusively labeled a 33 kDa protein. Competitive Scatchard analysis of [3H]glimepiride-binding and [3H]glibenclamide-binding to RINm5F cell membranes using glibenclamide and glimepiride, respectively, as heterologous displacing compounds yielded non-linear plots. These findings may be explained by cooperative interactions between the 140 and 65 kDa sulfonylurea-binding proteins. The possibility that sulfonylureas of different structure have different access to the 140 and 65 kDa receptor proteins due to the beta-cell membrane barrier was investigated by photoaffinity labeling of solubilized beta-cell membrane proteins. Interestingly, solubilization of beta-cell tumor membranes led to a shift of specific [3H]glibenclamide binding from the 140 kDa to the 65 kDa binding protein, exclusively, and to an increased labeling of the 65 kDa protein by [3H]glimepiride. The labeling of a unique protein is in agreement with similar Kd values measured for both sulfonylureas upon solubilization of beta-cell tumor and RINm5F cell membranes (see accompanying paper). Furthermore, competitive Scatchard plots of [3H]glimepiride binding to solubilized RINm5F cell membrane proteins in the presence of glibenclamide and vice versa approximate linearity suggesting loss of cooperativity between the 140 kDa glibenclamide-binding and 65 kDa glimepiride-binding proteins upon solubilization. The physiological significance of the differential interaction of glimepiride and glibenclamide with different binding proteins was also substantiated by photoaffinity labeling of RINm5F cells leading to labeling of a 140 kDa protein by [3H]glibenclamide and of a 65 kDa protein by [3H]glimepiride.(ABSTRACT TRUNCATED AT 400 WORDS)

ATP-Binding Cassette Transporters↗