Showing papers by "University of Basel published in 1991"

Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast.

Abstract: FK506 and rapamycin are related immunosuppressive compounds that block helper T cell activation by interfering with signal transduction. In vitro, both drugs bind and inhibit the FK506-binding protein (FKBP) proline rotamase. Saccharomyces cerevisiae cells treated with rapamycin irreversibly arrested in the G1 phase of the cell cycle. An FKBP-rapamycin complex is concluded to be the toxic agent because (i) strains that lack FKBP proline rotamase, encoded by FPR1, were viable and fully resistant to rapamycin and (ii) FK506 antagonized rapamycin toxicity in vivo. Mutations that conferred rapamycin resistance altered conserved residues in FKBP that are critical for drug binding. Two genes other than FPR1, named TOR1 and TOR2, that participate in rapamycin toxicity were identified. Nonallelic noncomplementation between FPR1, TOR1, and TOR2 alleles suggests that the products of these genes may interact as subunits of a protein complex. Such a complex may mediate nuclear entry of signals required for progression through the cell cycle.

A review of otoacoustic emissions

Abstract: Otoacoustic emissions measured in the external ear canal describe responses that the cochlea generates in the form of acoustic energy. For the convenience of discussing their principal features, emitted responses can be classified into several categories according to the type of stimulation used to evoke them. On this basis, four distinct but interrelated classes can be distinguished including spontaneous, transiently evoked, stimulus-frequency, and distortion-product otoacoustic emissions. The present review details the findings that have been described for each emission type according to this classification schema. Additionally, the known features of emitted responses are discussed for both normally hearing and hearing-impaired humans and experimental animals, and with respect to their potential clinical applications. The findings reviewed here clearly indicate that future studies of otoacoustic emissions will significantly increase our understanding of the basic mechanisms of cochlear function while, at the same time, provide a new and important clinical tool.

Import of proteins into mitochondria.

Abstract: The biogenesis of mitochondria involves a close interaction between the nucleocytoplasmic and the mitochondrial genetic system. The majority of the mitochondrial proteins are synthesized on cytoplasmic ribosomes and transported into the organelle (1). The mechanism of this protein import has been under debate for many years (e.g., (2,3)). Recent experiments with yeast cells have shown that several cytoplasmic- ally made mitochondrial proteins are initially made as larger precursors, both in vitro as well as in pulse-labeled yeast spheroplasts (4,5). So far, larger precursors have been detected for the following cytoplasmically made mitochondrial proteins: the three largest subunits of the mitochondrial F1-ATPase (i.e., the α, β, and γ subunit, see (4)) and two subunits of the mitochondrial cytochrome bc1 complex (cytochrome c1 and subunit V; (5) and C. Cote, unpublished). Import of in vitro synthesized precursors into isolated mitochondria was demonstrated for the three largest F1-ATPase subunits; as an operational measure of import, we checked whether any of the polypeptides added to the mitochondria became resistant to externally added proteases (4).

Carbon isotope discrimination by plants follows latitudinal and altitudinal trends.

Abstract: In an earlier paper we provided evidence that carbon isotope discrimination during photosynthesis of terrestrial C3 plants decreases with altitude, and it was found that this was associated with greater carboxylation efficiency at high altitudes. Changing partial pressures of CO2 and O2 and changing temperature are possible explanations, since influences of moisture and light were reduced to a minimum by selective sampling. Here we analyse plants sampled using the same criteria, but from high and low altitudes along latitudinal gradients from the equator to the polar ends of plant distribution. These data should permit separation of the pressure and temperature components (Fig. 1). Only leaves of fully sunlit, non-water-stressed, herbaceous C3 plants are compared. The survey covers pressure differences of 400 mbar (ca. 5000 m) and 78 degrees of latitude (ca 25 K of mean temperature of growth period). When habitats of similar low temperature (i.e. high altitude at low latitude and low altitude at polar latitude) are compared, discrimination increases towards the pole (with decreasing altitude and thus increasing atmospheric pressure). Latitudinally decreasing temperature at almost constant atmospheric pressure (samples from low altitude) is associated with a decrease in discrimination. So, polar low-altitude plants have δ13C values half way between humid tropical lowland and tropical alpine plants. It is unlikely that latitudinal changes of the light regime had an effect, since low and high altitude plants show contrasting latitudinal trends in δ13C although local altitudinal differences in overall light consumption were small. These results suggest that both temperature and atmospheric pressure are responsible for the altitudinal trends in 13C discrimination. Temperature effects may partly be related to increased leaf thickness (within the same leaf type) in cold environments. Theoretical considerations and laboratory experiments suggest that it is the oxygen partial pressure that is responsible for the pressure related change in discrimination. The study also provided results of practical significance for the use of carbon isotope data. Within a community of C3 plants, discrimination in species of similar life form, exposed to similar light, water and ambient CO2 conditions ranges over 4‰, with standard deviations for 10–30 species of ±0.6 to 1.2‰. This natural variation has to be taken into account by using a sufficient sample size and standardization of sampling in any attempt at ecological site characterization using carbon isotope data. Evidence of a pronounced genotypic component of this variation in 13C discrimination in wild C3 plant species is provided. Correlations with dry matter partitioning, mesophyll thickness and nitrogen content are also present.

C2‐Symmetric 4,4′,5,5′‐Tetrahydrobi(oxazoles) and 4,4′,5,5′‐Tetrahydro‐2,2′‐methylenebis[oxazoles] as Chiral Ligands for Enantioselective Catalysis Preliminary Communication

Abstract: The synthesis of a series of enantiomerically pure, C2-symmetric 4,4′,5,5′-tetrahydro-2,2′-methylenebis[oxazoles] and 4,4′,5,5′-tetrahydro-2,2′-bi(oxazoles) is reported. Copper complexes with anionic tetrahydromethylenebis[oxazole] ligands are efficient catalysts for the enantioselective cyclopropane formation from olefins and diazo compounds (up to 96% ee in the reaction of styrene with menthyl diazoacetate). Tetrahydrobi(oxazole)iridium(I) complexes were found to catalyze transfer hydrogenations of aryl alkyl ketones with i-PrOH (up to 91% ee). Tetrahydrobi(oxazole)palladium complexes can be used as enantioselective catalysts for allylic nucleophilic substitution (up to 77% ee in the reaction of PhCHCHCH(OAc)Ph with NaHC(COOMe)2).

Molecular mechanism of slow acetylation of drugs and carcinogens in humans.

Abstract: The acetylation polymorphism is one of the most common genetic variations in the transformation of drugs and chemicals. More than 50% of individuals in Caucasian populations are homozygous for a recessive trait and are of the "slow acetylator" phenotype. They are less efficient than "rapid acetylators" in the metabolism of numerous drugs and environmental and industrial chemicals. The acetylation polymorphism is associated with an increased risk of drug toxicity and with an increased frequency of certain cancers. We report the identification of the primary mutations in two alleles of the gene for the N-acetyltransferase (NAT; acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) isozyme NAT2 associated with slow acetylation. These alleles, M1 and M2, account for more than 90% of slow acetylator alleles in the European population we have studied. M1 and M2 were identified by restriction fragment length polymorphisms with Kpn I and Msp I and subsequently cloned and sequenced. M1 and M2 each are characterized by a combination of two different point mutations, one causing an amino acid substitution (Ile-113----Thr in M1, Arg-197----Gln in M2), the other being silent (C 481----T in M1, C 282----T in M2). Functional expression of M1 and M2 and of chimeric gene constructs between mutant and wild-type NAT2 in COS-1 cells suggests that M1 causes a decrease of NAT2 protein in the liver by defective translation, whereas M2 produces an unstable enzyme. On the basis of the mutations described here and a rare mutant allele (M3) reported recently, we have developed a simple DNA amplification assay that allows the predictive genotyping of more than 95% of slow and rapid acetylator alleles and the identification of individuals at risk.

A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole

Abstract: Tobacco contains different isoforms of chitinase (EC 3.2.1.14), a hydrolase thought to be involved in the defense against pathogens. Deduced amino acid sequences for putatively vacuolar, basic chitinases differ from the homologous extracellular, acidic isoforms by the presence of a C-terminal extension. To examine the role of this C-terminal extension in protein sorting, Nicotiana silvestris plants were stably transformed with chimeric genes coding for tobacco basic chitinase A with and without the seven C-terminal amino acids. In plants expressing unmodified chitinase A, the enzyme activity was low in the intercellular wash fluid but high in protoplasts and isolated vacuoles. In contrast, in plants expressing mutant chitinase lacking the C terminus, the activity was high in the intercellular wash fluid but low in protoplasts. N. silvestris plants were also transformed with similar constructions coding for a structurally unrelated, extracellular cucumber chitinase. In plants expressing unmodified cucumber chitinase, its activity was present in the intercellular wash fluid and absent from protoplasts. In plants expressing cucumber chitinase with the C-terminal extension from tobacco chitinase A, activity was low in intercellular wash fluids but high in protoplasts and vacuoles. These results demonstrate that the C-terminal extension of tobacco chitinase A is necessary and sufficient for the vacuolar localization of chitinases and, therefore, that it comprises a targeting signal for plant vacuoles.

Electrostatic effects in proteins: comparison of dielectric and charge models.

Abstract: Two approaches for calculating electrostatic effects in proteins are compared and ana analysis is presented of the dependence of calculated properties on the model used to define the charge distribution. Changes in electrostatic free energy have been calculated using a screened Coulomb potential (SCP) with a distance-dependent effective dielectric permittivity to model bulk solvent effects and a finite difference approach to solve the Poisson-Boltzmann (FDPB) equation. The properties calculated include shifts in dissociation constants of ionizable groups, the effect of annihilating surface charges on the binding of metals, and shifts in redox potentials due to changes in the charge of ionizable groups. In the proteins considered the charged sites are separated by 3.5-12 A. It is shown that for the systems studied in this distance range the SCP yields calculated values which are at least as accurate as those obtained from solution of the FDPB equation. In addition, in the distance range 3-5 A the SCP gives substantially better results than the FDPB equation. Possible sources of this difference between the two methods are discussed. Shifts in binding constants and redox potentials were calculated with several standard charge sets, and the resulting values show a variation of 20-40% between the 'best' and 'worst' cases. From this study it is concluded that in most applications, changes in electrostatic free energies can be calculated economically and reliably using an SCP approach with a single functional form of the screening function.

Mitochondrial proteins essential for viability mediate protein import into yeast mitochondria.

Abstract: Only five mitochondrial proteins are known to be essential for viability of the yeast Saccharomyces cerevisiae; all of them are key components of the mitochondrial protein import system. Other components of this system are not essential for life; they include functionally redundant import receptors on the mitochondrial surface and enzymes acting upon only a few precursor proteins.

FK 506-binding protein proline rotamase is a target for the immunosuppressive agent FK 506 in Saccharomyces cerevisiae.

Abstract: FK 506 and cyclosporin A are potent immunosuppressive compounds that inhibit T-cell activation by interfering with signal transduction. In vitro, FK 506 binds and inhibits the activity of FK 506-binding protein (FKBP), a peptidylprolyl rotamase (cis-trans isomerase). Cyclosporin A acts similarly on a different proline rotamase, cyclophilin. Experiments described here demonstrate genetically that FKBP is a target for FK 506 in vivo. We have isolated the gene encoding the FKBP proline rotamase (FPR1) from Saccharomyces cerevisiae. The encoded yeast protein is highly homologous with bovine and human FKBP and shares no homology with cyclophilin. Disruption of FPR1 and CPR1 (encoding cyclophilin) individually or in combination is not lethal; thus, either enzymatic proline rotamerization is not essential for life or an unknown proline rotamase can substitute for the missing enzymes. Overexpression or disruption of FPR1 confers resistance to growth inhibition by FK 506, suggesting that FKBP is a target for FK 506 in yeast. However, FKBP is only one of at least two targets because strains lacking FKBP are only partially resistant to FK 506.

Monomorphic and polymorphic human arylamine N-acetyltransferases: a comparison of liver isozymes and expressed products of two cloned genes.

Abstract: A genetic polymorphism of human liver arylamine N-acetyltransferase (NAT; EC 2.3.1.5) enzyme activity divides populations into distinguishable "slow acetylator" and "rapid acetylator" phenotypes. Two human genes, NAT1 and NAT2, encoding NAT proteins [DNA Cell Biol. 9:193-203 (1990)] were transiently expressed in cultured monkey kidney COS-1 cells, and the resulting recombinant NAT1 and NAT2 proteins were compared with N-acetyltransferase activities in human liver cytosol with respect to their stability, chromatographic behavior on anion exchange columns, electrophoretic mobility, and arylamine acceptor substrate specificity. NAT1 was far less stable in vitro than NAT2. Under conditions designed to optimize enzyme stability, anion exchange chromatography experiments revealed that enzymes corresponding to both recombinant NAT1 and NAT2 were expressed in human liver. Recombinant and human liver NAT1 enzymes showed the same characteristic selectivity (low apparent Km, high Vmax) for the "monomorphic" substrates p-aminosalicylic acid and p-aminobenzoic acid. Such substrates fail to discriminate between the acetylator phenotypes in vivo. The same criteria established that recombinant NAT2 was indistinguishable from one of two previously observed N-acetyltransferases (NAT2A and NAT2B) whose liver contents correlate with acetylator phenotype in human populations. Recombinant NAT2 and the liver NAT2 isoforms NAT2A and NAT2B selectivity N-acetylated the "polymorphic" substrates sulfamethazine and procainamide, whose disposition in vivo is affected by the acetylation polymorphism. Interestingly, the carcinogen 2-aminofluorene was very efficiently metabolized by both NAT1 and NAT2. Independent regulation of NAT1 and NAT2 genes was suggested by a lack of correlation of NAT1 and NAT2 enzyme activities in cytosols from 39 human livers. The results provide strong evidence that the NAT2 locus is the site of the human acetylation polymorphism. In addition, the use of recombinant NAT1 and NAT2 will allow us to predict whether any given arylamine will be polymorphically acetylated in humans.

A new petrogenetic grid for low‐grade metabasites

Abstract: We have used internally-consistent thermodynamic data to present calculated phase equilibria for the system Na2O-CaO-MgO-Al2O3-SiO2-H2O (NCMASH), in the range 0–500° C and 0.1–10 kbar, involving the phases anorthite, glaucophane, grossular, heulandite, jadeite, laumontite, lawsonite, paragonite, prehnite, pumpellyite, stilbite, tremolite, wairakite, zoisite with excess albite, clinochlore, quartz and pure water. Average activity terms derived from published mineral chemical data were included for clinochlore, glaucophane, prehnite, pumpellyite, tremolite, and zoisite. The new petrogenetic grid delineates stability fields and parageneses of common index minerals in zeolite, prehniteactinolite, prehnite-pumpellyite, pumpellyite-actinolite, blueschist and greenschist facies metabasites. The stability fields of mineral assemblages containing prehnite, pumpellyite, epidote, actinolite (+ albite + chlorite + quartz) were analysed in some detail, using activity data calculated from five specific samples. For example, the prehnite-actinolite facies covers a P-T field ranging from about 220 to 320° C at pressures below 4.5 kbar. The transition from the prehnite-actinolite and pumpellyite-actinolite to greenschist facies occurs at about 250–300° C at 1–3 kbar and at about 250–350° C at 3–8 kbar. P-T fields of individual facies overlap considerably due to variations in chemical composition.

Debrisoquine/sparteine hydroxylation genotype and phenotype: analysis of common mutations and alleles of CYP2D6 in a European population.

Abstract: Four different mutations of the cytochrome P450 CYP2D6 gene associated with the poor metabolizer phenotype (PM) of the debrisoquine/sparteine polymorphism were analyzed by Xba I restriction fragment length polymorphism (RFLP) analysis and a polymerase chain reaction (PCR)-based DNA amplification method in DNA of 394 healthy European subjects; 341 of these were phenotyped by sparteine or debrisoquine administration and urinary metabolic ratios (MR). Our study demonstrates the efficiency of the PCR-test for phenotype prediction; 96.4% of individuals were correctly predicted, i.e., 100% of the extensive metabolizers (EMs) and 86.0% of the poor metabolizers (PMs). In contrast, Xba I RFLP analysis was far less informative, predicting the phenotype in only 26.8% of PMs. By combining both DNA tests, the prediction rate of the PM phenotype increased to 90.6%. A point mutation at a splice-site consensus sequence termed D6-B represented the most common mutant CYP2D6 gene and accounted for more than 75% of ...

Plasma Antioxidant Vitamins and Subsequent Cancer Mortality in the 12-Year Follow-up of the Prospective Basel Study

Abstract: Plasma antioxidant vitamins A, C, and E and carotene were measured in a group of 2,974 men participating in the third examination of the prospective Basel Study in 1971-1973. In 1985, the vital status and mortality of all participants were assessed. A total of 204 men had died from cancer, including 68 with bronchus cancer and 37 with gastrointestinal cancer (20 with stomach cancer and 17 with large bowel cancer excluding cancer of the rectum). Overall mortality from cancer was associated with low mean plasma levels of carotene adjusted for cholesterol (p less than 0.01) and of vitamin C (p less than 0.01). Bronchus and stomach cancers were associated with a low mean plasma carotene level (p less than 0.01). Subjects with subsequent stomach cancer also had lower mean vitamin C and lipid-adjusted vitamin A levels than did survivors (p less than 0.05). After calculation of the relative risk using the Cox model with exclusion of mortality during the first 2 years of follow-up, low plasma carotene (below quartile 1) was associated with a significantly increased risk for bronchus cancer (relative risk (RR) = 1.8, p less than 0.05), low plasma levels of carotene and vitamin A with all cancers (RR = 2.47, p less than 0.01), and low plasma retinol in older subjects (greater than age 60 years) with lung cancer (RR = 2.17, p less than 0.05). Low levels of vitamin C increased the risk of stomach cancer (RR = 2.38) and gastrointestinal cancer (RR = 2.46) in older subjects, but only significantly with the inclusion of the first 2 years. The authors conclude that low plasma levels of antioxidant vitamins are associated with an increased risk of subsequent cancer mortality. This effect was stronger in men above age 60 years at blood sampling, and the effect seems to be site-specific.

Order-parameter saturation and low-temperature extension of Landau theory

Abstract: The temperature evolution of the structural order parameter in displacive phase transitions with high transition temperatures (e.g. quartz, As2O5, LaAlO3, CaCO3, NaNO3, Pb3(PO4)2 etc.) follows a Landau-type behaviour over large temperature intevals. Below a saturation temperatureT s , however, the order parameter tends to become temperature independent. A quantitative analysis shows the correlationk B T s =ħΩ/4, where Ω is the frequency of the relevant local excitation atT s . A general Landau-type expression for the Gibbs energy is given which includes the effects of order-parameter saturation.

Synthesis of Optically Active Bis(2-oxazolines): Crystal Structure of a 1,2-Bis(2-oxazolinyl)benzene ZnCl2 Complex

Abstract: Dinitriles (5–7, 12, 13) react with enantiomerically pure β-amino alcohols (8 – 11,17) under zinc chloride catalysis to give optically active C2-symmetric bis(oxazolines). 1,2-Bis(2-oxazolin-2-yl)benzenes 1a – e are obtained under mild reaction conditions. 1H-NMR spectroscopy indicates the formation of 1:1 complexes 23 of these compounds with ZnCl2. The energy required for a conformational interconversion of zinc dichloride complex 23e was determined by variable-temperature 1H-NMR studies. An X-ray structure analysis was performed with the substituted [1,2-bis(2-oxazolinyl)benzene]zinc dichloride complex 23a.

Determinants for glycophospholipid anchoring of the Saccharomyces cerevisiae GAS1 protein to the plasma membrane.

Abstract: A 125-kDa glycoprotein exposed on the surface of Saccharomyces cerevisiae cells belongs to a class of eucaryotic membrane proteins anchored to the lipid bilayer by covalent linkage to an inositol-containing glycophospholipid. We have cloned the gene (GAS1) encoding the 125-kDa protein (Gas1p) and found that the function of Gas1p is not essential for cell viability. The nucleotide sequence of GAS1 predicts a 60-kDa polypeptide with a cleavable N-terminal signal sequence, potential sites for N- and O-linked glycosylation, and a C-terminal hydrophobic domain. Determination of the anchor attachment site revealed that the C-terminal hydrophobic domain of Gas1p is removed during anchor addition. However, this domain is essential for addition of the glycophospholipid anchor, since a truncated form of the protein failed to become attached to the membrane. Anchor addition was also abolished by a point mutation affecting the hydrophobic character of the C-terminal sequence. We conclude that glycophospholipid anchoring of Gas1p depends on the integrity of the C-terminal hydrophobic domain that is removed during anchor attachment.

Sequential action of mitochondrial chaperones in protein import into the matrix.

Abstract: Translocation and folding of proteins imported into mitochondria are mediated by two matrix-localized chaperones, mhsp70 and hsp60. In order to investigate whether these chaperones act sequentially or in parallel, we studied their interaction with newly imported precursor proteins in isolated yeast mitochondria by coimmunoprecipitation. All precursors bound transiently to mhsp70. Release from mhsp70 required hydrolysis of ATP and did not immediately generate a tightly folded protein. For example, after imported mouse dihydrofolate reductase (a soluble monomeric enzyme) had been released from mhsp70, folding to a protease resistant conformation occurred only after a lag and was much slower than the release. Under standard import conditions, no significant association of DHFR with hsp60 could be detected. Similarly, newly imported hsp60 subunit was released from mhsp70 as an incompletely folded, unassembled intermediate which accumulated at low temperature and assembled to hsp60 14-mer at higher temperature in an ATP-dependent manner. Mas2p (the larger subunit of the MAS-encoded processing protease) first bound to mhsp70, then to hsp60, and only then assembled with its partner subunit, Mas1p. We propose that ATP-dependent release from mhsp70 is insufficient to cause folding of imported proteins and that assembly of hsp60 and Mas2p requires sequential, ATP-dependent interactions with mhsp70 and hsp60.

The effects of phenotypic plasticity on genetic correlations

Abstract: Recent theory suggests that genetic correlations should help to predict the simultaneous response to selection of two or more traits, and much recent research has been directed towards understanding the sources of variation in genetic correlations. Genetic correlations can change from sample to sample, from species to species, from population to population, during the course of development and — within a population, at a fixed stage of development — from one environment to another. These are changes not only in magnitude but also in sign. Theory suggests that genetic correlations should not change sign when the two traits are tightly integrated by physiology or development. Patterns of change of genetic correlations are caused by differences in development and physiology, an understanding of which appears to be necessary to predict the response to selection in natural, heterogeneous environments.

Cleavage and polyadenylation factor CPF specifically interacts with the pre-mRNA 3' processing signal AAUAAA.

Abstract: Cleavage and polyadenylation factor (CPF) is required for the cleavage as well as for the subsequent polyadenylation reaction during 3' processing of messenger RNA precursors. Here, we have investigated the interaction of CPF and poly(A) polymerase with short RNA substrates. CPF activates poly(A) polymerase to elongate RNA primers carrying the canonical hexamer recognition signal AAUAAA. CPF specifically binds to such RNA as shown by gel mobility shift assays and competition experiments. Upon binding of CPF, two polypeptides of 35 kDa and 160 kDa can be covalently crosslinked to the RNA by irradiation with UV light. These polypeptides may correspond to the smallest and the largest subunit contained in purified CPF fractions. In addition, chemical modification-exclusion experiments demonstrate that CPF interacts directly with the AAUAAA recognition signal in the RNA. The entire hexamer signal is involved in binding of CPF since modification of any of its bases interferes with complex formation.

Molecular-resolution images of Langmuir–Blodgett films using atomic force microscopy

Abstract: THE ability to prepare thin films of amphiphilic molecules (Langmuir–Blodgett (LB) films) is valuable to many areas of research. In biology they provide models for ideal membranes; the two-dimensional behaviour and structural phase transitions are of fundamental interest in surface physics; and their tribological characteristics suggest potential engineering applications. For determining the structure of these films, the common techniques such as X-ray and neutron scattering are limited to thick ( ≳200 A) multilayers. Thinner films can be studied by transmission electron microscopy and low-energy electron diffraction1,2, but these electron-beam techniques tend to damage thin films. More recently, the scanning tunnelling microscope3 has provided a non-destructive means of investigating the structures of LB films4–6, but as the films are insulating, the interpretation of such images has been controversial. The atomic force microscope7 is not plagued with these ambiguities, as it does not require a conductive sample. Here we present images, with molecular resolution, of LB films of cadmium arachidate deposited on an amorphous silicate substrate. Despite the disorder in the substrate, the films display a periodic structure over large distances (several hundreds of angstroms). This suggests that the adsorbed molecules near the interface are driven to self-assemble primarily, if not solely, by intermolecular forces rather than by dependence on substrate periodicity.

Biological Applications of Scanning Probe Microscopes

Abstract: PERSPECTIVES AND OVERVIEW...... ...... . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. ......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 INSTRUMENTATION AND THEORETICAL BACKGROUND 8 1 Resolution . . . ..... . . . . . .... .. ......... . . . . . . . . . . . . . . . ... . . . . . . . . ...... . . . . . . . . . . 8 1 Servo System, Scan Devices, and Scan Modes ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... . . . . 83 Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Scanning Tunneling Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .. . . . . . . . . . . . . . . 86 Scanning Force Microscopy . . . . ....... . . . . . . . . . . . . . . ... . . . . ... . . . . ..... . . . . 87 SP M for Biological Applications . . . . . . .. . . . . . . . . . . . . . . ....... . . . .. . . 87 SAMPLE PREPARATION FOR SPM . ........ . . . . ... . . . . . . . . . . ....... . . . . . . . . . . . . . . . . . . . 88 Protein Structure ... .... . ....... ... ...... . . . . . . . . ........ . . . . . . . . . . .. . ........ . . . . . . . . . . . . 88 Preparation Artifacts During Immobilization and Dehydration .. . . . . . . ........ 88 Specimen Supports . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . ..... 89 APPLICATIONS .... . . . . ........ . . . . . . . ......... . . . . . . . . . . . . . . . . ......... . . . . . . . . . . ........ . . . . . . 90 Scanning Tunneling Microscopy ... . . . . ......... .. ...... . . . ......... . . . . . . . . . . . . . . . 90 Scanning Force Microscopy . . . . . . . . ........ . . . . . . . . . ...... . . . . . . 100 CONCLUSIONS AND PROSPECTS . . . . . . . . . . . . . ..... . . . . . . . . . . . .... . . . . . .. . . . . ... . . . 102

Nonclassical hydrophobic effect in membrane binding equilibria.

Abstract: The enthalpy of transfer of four different amphiphilic molecules from the aqueous phase to the lipid membrane was determined by titration calorimetry. The four molecules investigated were the potential-sensitive dye 2-(p-toluidinyl)naphthalene-6-sulfonate (TNS), the membrane conductivity inducing anion tetraphenylborate (TPB), the Ca2+ channel blocker amlodipine [Bauerle, H. D., & Seelig, J. (1991) Biochemistry 30, 7203-7211], and the positively charged local anesthetic dibucaine. All four amphiphiles penetrate into the hydrophobic part of the membrane, and their binding constants, after correcting for electrostatic effects, range between 600 M-1 for dibucaine and 60,000 M-1 for tetraphenylborate. The corresponding changes in free energy were about -6 to -9 kcal/mol. Binding of the amphiphiles to membrane vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was accompanied by exothermic heats of reaction for all four molecules. For TNS, TPB, and amlodipine, the enthalpies of transfer were almost identical and corresponded to delta H approximately -9 kcal/mol, essentially accounting for the total free energy change. Thus, the binding of these charged amphiphiles to the hydrophobic membrane was driven by enthalpy. This is in contrast to the classical hydrophobic effect, where the transfer is considered to be entropy driven. For dibucaine, the enthalpy of transfer was smaller with delta H approximately -2 kcal/mol but was still about one-third of the total free energy change. All enthalpies of transfer exhibited a distinct temperature dependence with molar heat capacities delta Cp of -30 to -100 cal mol-1K-1 for the transfer from water to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)