Lipid-protein interactions in the membrane: studies with model peptides

We have used fluorescence quenching of tryptophan-containing trans-membrane peptides by bromine-containing phospholipids to study the specificity of peptide-lipid interactions. We have synthesized peptides Ac-K2GLm WLnK2A-amide where m = 7 and n = 9 (L16) and m = 10 and n = 12 (L22). Binding constants of L22 for dioleoylphosphatidylserine [di(C18 : 1)PS] or dioleoylphosphatidic acid [di(C18 : 1)PA] relative to dieoleoylphosphatidylcholine [di(C18 : 1)PC] were close to 1. However, for L16, whilst the bulk of the di(C18 : 1)PA molecules bound with a binding constant relative to di(C18 : 1)PC close to 1, a small number of di(C18 : 1)PA molecules bound much more strongly. Assuming just one high affinity binding site on L16 for anionic lipid, the affinity of the site for di(C18 : 1)PS was calculated to be ca. 8 times that for di (C18 : 1)PC. The relative binding constant was little affected by ionic strength and close contact between the anionic headgroup of di(C18 : 1)PS and a lysine residue on the peptide was suggested. The relative binding constant for di(C18 : 1)PS at this high affinity site was less than for di(C18 : 1)PA. Cholesterol interacts with L22 with an affinity about 0.7 of that of di(C18 : 1)PC. The structure of the peptide itself is important. The peptide Ac-KKGYL6WL8YKKA-amide (Y2L14) incorporated into bilayers of dinervonylphosphatidylcholine [di(C24 : 1)PC] whereas L16 did not incorporate into this lipid. It is suggested that thinning of a lipid bilayer around a peptide to give optimal hydrophobic matching is less energetically unfavourable when a Tyr residue is located in the lipid/water interfacial region.     

Multiple cation binding by bola-amphiphilic crown ethers: Assessment by fast atom bombardment mass spectrometry

Abstract

Bola-amphiphiles having two aza-18-crown-6 ether rings attached by a covalent spacer (O—O) and an analogous tris (macrocycle) (O—O—O) have been prepared and their cation complexation behavior has been assessed by fast atom bombardment and collisionally activated dissociation tandem mass spectrometry; the key finding is that two cations may simultaneously be complexed by a two- or three-crown system and that this complexation may also involve an anion.     

Studies on drug metabolism by fungi colonizing decomposing human cadavers. Part I: DNA sequence-based identification of fungi isolated from postmortem material

Cadavers can be colonized by a wide variety of bacteria and fungi. Some of these microbes could change the concentration or the metabolic pattern of drugs present in postmortem samples. The purpose of this study was to identify fungi from human postmortem material and to further assess their potential role in the metabolism of drugs. Aliquots of 252 postmortem samples (heart blood, liver, kidney, and lung) taken from 105 moderately to severely decomposed bodies were streaked on Sabouraud agar for isolation of fungal species. One part of the samples was worked up immediately after autopsy (group I). The second part had previously been stored at ?20 °C for at least 1 year (group II). Identification of the isolates was achieved morphologically by microscopy and molecularly by polymerase chain reaction amplification and sequencing of markers allowing species identification of the respective genera. Depending on the genus, different gene fragments were used: calmodulin for Aspergillus, β-tubulin for Penicillium, translation elongation factor 1α for Fusarium, and the internal transcribed spacer region of the ribosomal DNA for all remaining genera. A total of 156 fungal strains were isolated from 62 % of the postmortem materials. By using these primers, 98 % of the isolates could be identified to the species level. The most common genera were Candida (60.0 %—six species), Penicillium (10.3 %—two species), Rhodotorula (7.1 %—one species), Mucor (6.4 %—four species), Aspergillus (3.2 %—four species), Trichosporon (3.2 %—one species), and Geotrichum (3.2 %—one species). Group I samples contained 53 % more fungal species than stored samples suggesting some fungi did not survive the freezing process. The isolated fungi might be characteristic for decomposed bodies. The proposed methodology proved to be appropriate for the identification of fungi in this type of material.     

Formulation and In Vitro Evaluation of Sustained Release Microspheres of Diltiazem Hydrochloride Prepared by Emulsification-Internal Gelation Method

The present work describes the formulation of alginate microspheres containing diltiazem hydrochloride by the emulsification-internal gelation method with the use of barium carbonate as a cross-linking agent. The effect of various factors (the concentration of alginate and barium chloride) on the drug loading efficiency and in vitro release were investigated. Fourier transform infrared microscopy (FTIR) and differential scanninig calorimetry (DSC) analysis confirmed the absence of any drug polymer interaction. X-ray diffraction (XRD) pattern showed that there is a decrease crystallinity of the drug. The in vitro drug release profile could be altered significantly by changing various processing parameters to give a controlled release of drug from microcapsules. The stability studies of drug-loaded microcapsules showed that the drug was stable at different storage conditions.     

A thermal-mechanical fatigue crack-growth testing system

An automated system for crack-growth studies under thermal-mechanical loading conditions utilizing a compact tension specimen has been developed. The system uses quartz lamps and a multiple-zone control system to accurately control the specimen temperature according to a predetermined profile. Further, a cyclic mechanical loading can be applied along with cyclic temperature profile in a prescribed manner.     

Fatigue crack growth analysis of adhesively repaired panel using perfectly and imperfectly composite patches

Fatigue crack behavior of cracked aluminum panel repaired with the imperfectly bonded composite patch is analyzed. The imperfection is in the form of debond which could result during the bonding of patch or the service life of the repaired structure. Debonds, of various sizes and at different locations with respect to the crack front, are investigated. An analytical procedure, involving two-dimensional finite element method having three layers to model cracked plate, adhesive and composite patch, is used to compute the stress intensity factors of test coupons. From the computed stress intensity factors, the crack growth rates are obtained analytically by assuming that the relationship between the stress intensity factor and the crack growth rate after repair is the same as the fatigue crack growth relationship for cracked panel material. The fatigue crack growth rates obtained experimentally and analytically are in good agreement with each other and they vary linearly with crack length inside the patch. The experimental results are bounded between its analytical counterparts at the mid-plane and free edge surfaces of the cracked panel. The present analytical procedure can, thus, be used to characterize the effects of imperfectly and perfectly bonded composite patch repairs on the durability and damage tolerance of the repaired structure.     

Structural analysis and molecular modelling of the Cu/Zn-SOD from fungal strain Humicola lutea 103

The native form of Cu/Zn-superoxide dismutase, isolated from fungal strain Humicola lutea 103 is a homodimer that coordinates one Cu(2+) and one Zn(2+) per monomer. Cu(2+) and Zn(2+) ions play crucial roles in enzyme activity and structural stability, respectively. It was established that HLSOD shows high pH and temperature stability. Thermostability of the glycosylated enzyme Cu/Zn-SOD, isolated from fungal strain H. lutea 103, was determined by CD spectroscopy. Determination of reversibility toward thermal denaturation for HLSOD allowed several thermodynamic parameters to be calculated. In this communication we report the conditions under which reversible denaturation of HLSOD exists. The narrow range over which the system is reversible has been determined using the strongest test of two important thermodynamic independent variables (T and pH). Combining both these variables, the "phase diagram" was determined, as a result of which the real thermodynamic parameters (ΔC(p), ΔH(exp)°, and ΔG(exp)°) was established. Because very narrow pH-interval of transitions we assume they are as result of overlapping of two simple transitions. It was found that ΔH(o) is independent from pH with a value of 1.3 kcal/mol and 2.8 kcal/mol for the first and the second transition, respectively. ΔG(o) was pH-dependent in all studied pH-interval. This means that the transitions are entropically driven, these. Based on this, these processes can be described as hydrophobic rearrangement of the quaternary structure. It was also found that glycosylation does not influence the stability of the enzyme because the carbohydrate chain is exposed on the surface of the molecule.     

Characterization of mode II fracture behavior in fiber-reinforced ceramic composite utilizing laser interferometry

A test technique to characterize the mode II fracture behavior in fiber-reinforced ceramic composites utilizing laser interferometry was developed. This was demonstrated by measuring the mode II critical strain energy release rate at room temperature. The present study used the siliconcarbide-fiber/glass-ceramic matrix composite system. Paper was presented at the 1991 SEM Spring Conference on Experimental Mechanics held in Milwaukee, WI on June 9–13.     

Dynamic-finite-element and dynamic-photoelastic analyses of two fracturing homalite-100 plates

A dynamic-finite-element code, HONDO, was used to analyze two single-edged-notch fracturing Homalite-100 plates which had been previously studied by dynamic photoelasticity. A single-edged crack in the finite-element model was advanced in incremental jumps such that the time-averaged crack velocity matched the measured crack velocity in the Homalite-100 plate. Dynamic-energy-release rates were computed for a constant-velocity crack and a crack which arrested after a somewhat constant deceleration. These results were compared with the corresponding dynamic-energy-release rates, which were computed from the dynamic-stress-intensity factors determined by dynamic photoelasticity, and with static-strain energyrelease rates. Despite the crude modeling of the running crack, the coarseness of the finite-element-grid breakdown and the differences in the modeled and actual grip conditions, the computed and measured dynamic-energy-release rates, except for occasional large differences, generally agreed within 10 percent of each other.