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Adsorbed proteins that promote cell adhesion mediate the response of cells to biomaterials and scaffolds. As proteins undergo conformational changes upon surface adsorption, their functional display may be significantly affected by surface chemistry or solution conditions during the adsorption process. A high-resolution localization microscopy technique is extended here to probe the conformation of individual fibronectin (Fn) molecules at the glass–water interface under physiological buffer conditions. To map distances, four available cysteines located on the modules FnIII7and FnIII15of dimeric Fn were site-specifically labeled with Cy3B, and their relative positions were determined by stepwise photobleaching with nanometer precision. The four labels on single Fn molecules did not show a uniform or linear arrangement. The distances between label positions were distributed asymmetrically around 33 nm with a tail towards higher distances. Exposure of Fn to denaturing solution conditions during adsorption increased the average distances up to 43 nm for 4 M guanidinium HCl, while changing the solution conditions after the adsorption had no effect, indicating that the observed intra-molecular distances are locked-in during the adsorption process. Also surface coatings of different hydrophobicity altered the conformational distribution, shifting label distances from a median of 24 nm on hydrophilic to 49 nm on hydrophobic surfaces. These results further highlight that the conformation of macromolecules at interfaces depends on the adsorption history. While illustrated here for surface adsorbed Fn, the power of localization-based microscopy extends the repertoire of techniques to characterize biomolecules at interfaces.

Bij een behandeling in een forensisch kader van plegers van seksueel geweld is zicht op risico van herhaling van het probleemgedrag een must. In het verlengde daarvan is onderzoek naar beschermende factoren een interessant en relatief nieuw gebied. Retrospectief werd validatie-onderzoek gedaan naar de psychometrische kwaliteiten van de SAPROF, een instrument dat zich richt op beschermende factoren ten aanzien van toekomstig (seksueel) geweld. De onderzoeksgroep betrof 97 TBS-patiënten die één of meerdere seksuele delicten hadden gepleegd en hiervoor waren behandeld in een TBS-kliniek. Twee groepen werden gedefinieerd: een groep die aan het einde van de behandelperiode wegens onvoldoende behandelresultaat werd overgeplaatst naar een ander forensisch psychiatrisch centrum (Herselectie); en een groep die aan het einde van de behandelperiode uit de TBS-kliniek werd ontslagen vanwege een als succesvol ingeschatte behandeling (Uitstroom). Beide groepen werden getaxeerd aan zowel het begin als het eind van de behandeling. De interbeoordelaarsbetrouwbaarheid was goed. Somscores van de SAPROF bleken aan het eind van de behandeling significant hoger voor de Uitstroom groep ten opzichte van de Herselectie groep. Er werd ten tijde van de behandeling voor de gehele groep een toename van beschermende factoren waargenomen. Deze toename was echter significant groter voor de Uitstroom groep dan voor de Herselectie groep. Binnen de Uitstroom groep werd vervolgens een vergelijking gemaakt tussen zij die wel en zij die niet recidiveerden met een nieuw (seksueel) geweldsdelict na de behandeling. Niet-recidivisten bleken significant meer beschermende factoren te bezitten en zich op dit gebied significant meer te hebben ontwikkeld ten tijde van de behandeling dan Recidivisten. Dit onderzoek bevestigt de waarde voor GZ-psychologen van de SAPROF als taxatie-instrument voor de beoordeling van beschermende factoren voor gewelddadig gedrag en als klinisch evaluatie instrument voor behandelsucces. De SAPROF blijkt in staat met het gemeten behandelresultaat te differentiëren tussen seksuele delinquenten die wel en niet zullen recidiveren na behandeling.

The application of supported lipid bilayer systems as molecular sensors, diagnostic devices, and medical implants is limited by their lack of stability. In an effort to enhance the stability of supported lipid bilayers, three pairs of phosphatidylcholine lipids were designed to cross-link at the termini of their 2-position acyl chain upon the formation of lipid bilayers. The cross-linked lipids span the lipid bilayer, resembling naturally occurring bolaamphiphiles that stabilize archaebacterial membranes against high temperatures. The three reactions investigated here include the acyl chain cross-linking between thiol and bromine groups, thiol and acryloyl groups, and cyclopentadiene and acryloyl groups. All three reactive lipid pairs were found to cross-link in liposomal membranes, as determined by thin-layer chromatography, ion-spray mass spectrometry, and ¹H NMR. The monolayer film properties of the reactive amphiphiles were characterized by surface pressure−area isotherms and showed that stable monolayers formed at the air−water interface with limiting molecular areas comparable to that of pure saturated phosphatidylcholine lipids. Langmuir−Blodgett bilayers of dimyristoylphosphatidylcholine incorporating 15 mol % of the reactive thiol and acryloyl lipids had diffusion coefficients comparable with pure dimyristoylphosphatidylcholine, while bilayers with more than 25 mol % of the reactive lipids were immobile, suggesting that interleaflet cross-linking of the lipids inhibited membrane diffusion. Our results show that the reactive lipids can cross-link within a lipid bilayer and are suitable for assembling supported lipid bilayers using Langmuir−Blodgett deposition. By using terminally reactive amphiphiles to build up supported lipid bilayers with cross-linked leaflets, bolaamphiphiles can be incorporated into asymmetric solid supported membranes to increase their stability in biosensor and medical implant applications.

Cells regulate active transport of intracellular cargo using motor proteins. Recent nanobiotechnology efforts aim to adapt motor proteins to power the movement and assembly of synthetic materials. A motor-protein-based nanoscale transport system (molecular shuttle) requires that the motion of the shuttles be guided along tracks. This study investigates the principles by which microtubules, serving as shuttle units, are guided along micrometer-scale kinesin-coated chemical and topographical tracks, where the efficiency of guidance is determined by events at the track boundary. Thus, we measure the probability of guiding as microtubules reach the track boundary of (1) a chemical edge between kinesin-coated and kinesin-free surfaces, (2) a topography-only wall coated completely with kinesin, and (3) a kinesin-free wall next to a kinesin-coated bottom surface (topography and chemistry combined). We present a guiding mechanism for each surface type that takes into account the physical properties of microtubule filaments and the surface properties (geometry, chemistry), and elucidate the contributions of surface topography and chemistry. Our experimental and theoretical results show that track edges that combine both topography and chemistry guide microtubules most frequently (approximately 90% of all events). By applying the principles of microtubule guidance by microfabricated surfaces, one may design and build motor-protein-powered devices optimized for transport.

To investigate protein and cell surface interactions, robust and increasingly complex model surfaces need to be developed to mimic specific aspects of the cell membrane structure. Polymer-supported lipid monolayers and bilayers present an opportunity to develop rugged cell mimetic surfaces that may be stable in a wide range of in vivo and in vitro applications and characterization techniques. We have investigated the stability of dimyristoylphosphatidylethanolamine (DMPE) monolayers grafted to poly(hydroxyethyl methacrylate) (pHEMA) via 1,1‘-carbonyldiimidazole (CDI) chemistry using X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and fluorescence microscopy. The results illustrate that it is possible to covalently couple the amine-containing lipid headgroup to the carboxyl and hydroxyl groups of the pHEMA and retain a proportion of the lipids at the surface after the samples are sonicated in ethanol. Most importantly, the cross-linking efficiency and retention of the resulting lipid layer is higher if the lipid is transferred to CDI-activated HEMA by Langmuir−Blodgett deposition rather than by adsorption of the lipid directly from solution. Both factors are critical if these monolayers are to form a stable and reproducible model system.

Motor proteins such as kinesin are used in cells to transport intracellular cargo along defined filament paths. The constituents of the transport system (i.e., motor proteins and filaments) can be reconstructed on synthetic surfaces to serve as molecular shuttles that actively transport cargo. Microfabricated open channels have been used in the past to guide microtubules propelled by kinesin adsorbed to the surface. In an effort to better understand the mechanism by which guidance occurs, we present a quantitative measure of the transport efficiency of microtubules along straight polyurethane channels 1 μm deep. Our analysis shows that the probability of a microtubule remaining in a channel drops exponentially as a function of its travel distance and that this characteristic decay distance varies with channel width. We identify microtubule−sidewall collisions as the key event for microtubule escape from a channel and thus the determinant of guiding efficiency. To better understand the relationship between collisions and travel distance, we determine the effect of channel width on (1) the microtubule approach angle to the sidewall, (2) the collision outcome (guided or escaped), and (3) the distance traveled between collisions. Also discussed is how the physical properties of microtubules or actin filaments (i.e., length, bending stiffness, persistence length) and changes in channel geometry (width, height) can describe measured parameters.

The two-dimensional crystallization of the protein streptavidin under biotinylated lipids has provided a convenient model system for studying the molecular mechanisms underlying protein crystallization. At neutral pH, streptavidin forms crystals with C222 symmetry and an X-shaped morphology that arises from asymmetric growth rates along perpendicular axes. The presence or absence of biotin association in the subunits has been connected to the growth asymmetry, but the molecular mechanism coupling biotin binding and growth kinetics has remained uncertain. Here we show evidence that a cooperative interaction between the Asn 23 hydrogen bond to biotin and a protein−protein hydrogen-bonding interaction at Tyr 22−Thr 20 across the crystal contact interface is acting as the structural/energetic connection between biotin binding and the asymmetric crystal growth kinetics. Brewster's angle microscopy and atomic force microscopy studies of a Thr20Ala site-directed mutant revealed that it crystallizes as symmetric squares, while maintaining the molecular C222 arrangement of wild-type protein crystals. These results suggest that the T20−Y22 hydrogen-bond interaction across the protein−protein contact interface is the crystallization contact that is altered by biotin binding to give growth asymmetry in wild-type crystals.

A new approach to image microscopic surface properties is introduced. Information about surface properties such as topography is obtained by repeated acquisition of an optical signal from a large number of microscopic, self-propelled probes moving on random paths across a surface. These self-propelled probes sample the surface in a statistical process in contrast to the deterministic, linear sampling performed by a scanning probe microscope. This method is experimentally demonstrated using microtubules as probes, which are moved by the motor protein kinesin.

Molecular shuttles have been built from motor proteins capable of moving cargo along engineered paths. We illustrate alternative methods of controlling the direction of motion of microtubules on engineered kinesin tracks, how to load cargo covalently to microtubules, and how to exploit UV-induced release of caged ATP combined with enzymatic ATP degradation by hexokinase to turn the shuttles on and off sequentially. These are the first steps in the development of a tool kit to utilize molecular motors for the construction of nanoscale assembly lines.

Light scattering microscopy reveals previously undetected topographic instabilities in phospholipid monolayers at the air/water interface far below the collapse pressure. Following compression through the fluid → condensed phase transition in monolayers of dipalmitoyl phosphatidylcholine, after the disappearance of the fluid phase, the contact regions between condensed domains acquire static roughness as indicated by enhanced light scattering. With further compression, a nanoscale budding process occurs within the roughened regions, while the interiors of the condensed domains remain flat and retain their domain shapes. At monolayer collapse, the buds proliferate across the entire interface, suggesting that the buds detected at lower pressures represent spatially confined fluctuations into the collapse phase. The confinement of static roughness formation and budding to domain contact regions indicates that these topographic instabilities originate from packing defects created where adjacent domain edges with conflicting molecular orientations grow together during the fluid → condensed phase transition.

The carboxyl- and amino-terminal ends of streptavidin are near the site of protein−protein contacts in two-dimensional streptavidin crystals. The role of these C- and N-terminal residues in determining the pH-dependent phase behavior of crystallization has been investigated with site-directed truncation mutants. Commercial streptavidin (consisting primarily of amino acids 14−136) and two recombinant streptavidin forms, spanning residues 13−136 and 13−139, have been crystallized at pH 4−7. The commercial 14−136 protein crystallizes in three distinct lattice symmetries, P1, P2, and C222, respectively, depending on pH. The 13−136 mutant also crystallizes in three distinct lattices, but with a shifted pH profile that is attributed to the N-terminal residue. The presence of amino acids 137−139 inhibits the growth of crystals with P1 symmetry at low pH. In addition, we observe a solid−solid phase transition in situ from the P2 to the P1 crystal forms for the 13−136 recombinant protein at pH 5.2. We also demonstrate the ability of Brewster angle microscopy to distinguish between different crystal forms if protein monolayer densities are sufficiently different.

The two-dimensional crystallization of streptavidin at biotinylated interfaces provides a model system for elucidating the role of interfacial binding dynamics in determining crystal morphologies and phases. In this study, a library of eight well-characterized site-directed mutants with increased biotin dissociation rates has been compared to core streptavidin (dissociation half-life of 59 h). The W79F, W120F, Y43F, and S27A mutants, with half-lives of 9.8 h, 38 min, 20 min, and 10 min, respectively, all displayed the “X”-shaped crystal morphology that is characteristic of core streptavidin. A sharp change in morphology is observed with the N23E (7.2 min) and N23A (4.8 min) mutants. The N23E mutant crystallizes in rectangular shapes, and the N23A displays square crystal morphology. The D128A mutant (1.7 min) crystallizes in elongated needles, but this is the one mutant which displays significant three-dimensional structural alterations. The W120A (<1 min) mutant did not display significant interfacial binding and did not crystallize. Quantitative Brewster angle microscopy was used to characterize the crystallization process. The noncrystalline background remained in equilibrium with the crystalline regions, and thus all the mutants crystallized as a first-order phase transition. The critical surface concentration for crystallization remained constant until the half-life reached 10.4 min for S27A. The square-shaped N23A crystal displayed the lowest critical surface concentration, which was equivalent to the values observed previously for square-shaped crystals obtained via metal binding. Fourier analysis of transmission electron microscopy images demonstrated that all of the mutants crystallized with the same C222 space group characteristic of core streptavidin. The change in crystal shape over a sharp range of dissociation rates is consistent with a change in a rate-limiting microscopic kinetic step that underlies macroscopic morphology. Alternatively, the altered shapes observed with the Asn 23 mutants could be the result of changing structural and energetic coupling between biotin binding and the directly adjacent Thr 20 and Tyr 22 residues which hydrogen bond to each other across the protein−protein crystal contacts.

Luminescence microspectroscopy (LMS) is described here as an in situ approach that combines the spatial resolution and imaging capabilities of optical microscopy with the structural sensitivity of rare earth ion spectroscopy. In this work, LMS, laser-induced luminescence spectroscopy, and conventional Raman spectroscopy were used to analyze the effect of Eu³⁺ admixtures on calcium oxalate crystallization. This new combination of techniques was used to establish a link between crystal morphology, phase, and impurity ion bonding environments for single crystals in contact with their aqueous growth solution. Distinct, spatially resolved Eu³⁺ luminescence spectra were measured for 1−20 μm crystals separated by less than 10 μm. Results show that micromolar quantities of Eu³⁺ significantly inhibit calcium oxalate nucleation in static, low ionic strength (~2 mM) supersaturated solutions at room temperature. Eu³⁺ stabilizes and incorporates into the dihydrate phase of calcium oxalate and the bipyramidal morphology associated with this phase is maintained. Eu³⁺ is believed to occupy Ca²⁺ lattice sites in the dihydrate crystals. Eu³⁺ also associates with calcium oxalate monohydrate during crystallization, yielding several ill-formed crystal morphologies. Two classes of Eu³⁺ bonding environments are identified in the monohydrate, including one with unusual luminescence band energies. The luminescence spectra are interpreted in terms of the local structure of the calcium oxalate crystal lattices. These experiments demonstrate LMS to be a useful approach for a spatially controlled analysis of inorganic crystal growth at surfaces in real time. Potential applications include analysis of template-directed crystallization, biomineralization, phosphors, and ceramic coatings.

The two-dimensional crystallization of streptavidin at functionalized lipid interfaces is one of the best studied model systems for investigating molecular self-assembly processes. This system also provides an opportunity to elucidate the relationship between protein−protein molecular recognition, crystallization solution conditions, and crystal properties such as coherence, space group symmetry, and morphology. A better understanding of these relationships may aid in the design of rational strategies for promoting high-quality protein crystallization and for controlling protein assembly at interfaces in the biomaterials and nanotechnology fields. Here we show that two-dimensional streptavidin crystallization is kinetically controlled and that formation of a single electrostatic interaction at the crystal contact interfaces is a key energetic determinant of the kinetic barriers controlling crystal morphology. Our results also demonstrate that this electrostatic interaction at the streptavidin protein−protein interfaces is responsible for the ionic strength dependence of streptavidin crystallization. Molecular modeling studies of the wild-type crystal that displays C222 symmetry suggested that the side-chain amines of lysine 132 from adjacent proteins interact with each other across the dyad-related crystal contacts. Leucine was substituted at this position (K132L) to remove the need for bridging counterions. Unlike wild-type streptavidin, the K132L mutant crystallizes with rectangular morphology on buffer or on a pure water subphase and analysis of the electron micrographs demonstrates that the crystal retains C222 symmetry in the presence or absence of salt. The kinetic barriers associated with formation of this electrostatic interaction thus underlie the wild-type butterfly crystal morphology.

Brewster angle microscopy (BAM) is extended to probe protein adsorption and aggregation processes at interfaces quantitatively. Since no p-polarized light is reflected from the plain air−water interface under Brewster's angle, the grayscales of BAM images solely depend on the optical thicknesses of interfacial layers; photolabeling is not required. Effective refractive indices are calculated from the grayscales via Fresnel's equations and are finally converted to relative protein surface densities by the Maxwell−Garnett theory. It is shown here that the Maxwell−Garnett theory provides an accurate framework for the description of a protein−water slab. Streptavidin binding to biotinylated monolayers at the air−water interface was chosen as a model system for an in situ study of the formation of two-dimensional streptavidin crystals. H-shaped aggregates are seen with BAM. We identify these as two-dimensional crystals since they are equal in size and shape to those typically observed in fluorescence microscopy which have been characterized by electron diffraction. The difference in protein surface density between the streptavidin crystals and the noncrystalline surrounding is sufficient to provide for a rich contrast without the use of a fluorescence probe. A critical protein surface density equal to 75% of the crystal density is found to be required for the crystals to form, a value that is independent of the protein bulk concentration. We have studied further the compressibility of the two phases. Whereas the density of the crystalline phase remains constant during compression, the noncrystalline phase can be compressed to a surface density which exceeds that of the crystalline phase without initiating further crystal growth. This leads to an inverted contrast in BAM; dark crystals are seen on a bright background. The protein density of the noncrystalline phase can also exceed that of the crystalline phase upon the much slower process of protein adsorption from sufficiently concentrated bulk solutions. The nature of this two-dimensional phase transition is discussed.

The non-respiratory vascular system of T. mossambica gill filaments was studied in serial longitudinal and cross sections. Comparatively few scattered vascular communications occur between the afferent filament artery and the central venous sinus (AVAaff). The efferent filament artery, however, is connected by regularly arranged anastomoses (AVAeff), directly, and sometimes indirectly via nutritive vessels, to the central sinus. These AVAeff are about as numerous as lamellae counted on one side of each filament, although they diminish slightly in number towards the filament base. The relation AVAeff to AVAaff was 17.6:1 in the distal and 17.8:1 in the basal filamental region, while in the tip region of 7 filaments 126 AVAeff but only 1 AVAaff were encountered. No direct connection between the lamellar lacunae and the central sinus was detected. According to these results, non-respiratory intrafilamental blood shunting appears unlikely. AVAeff are assumed to be the main route for blood entering the central venous sinus which would consequently flow into the branchial veins.

Arterio-venous anastomoses (AVA) in gill filaments of Tilapia mossambica exhibit a distinct polarity. Two different types of highly specialized endothelial cells, both of epitheloid shape, line these vessels: Type I cells, contacting the arterial lumen, are elongated and about two to three times as large as type II endothelial cells. Their surface is increased by tentacular protrusions which reach far into the arterial lumen. Filament whorls forming tubelike structures with centrally located glycogen granules are abundant in these cells. Type II endothelial cells are located proximal to the central venous sinus (CVS). Their less abundant and more electron dense cytoplasm is free of filament whorls. There are also intermediate cell forms at approximately the middle of each anastomosis. Short cell processes protrude from all endothelial cell types into the AVA lumen. Outside the indistinct vascular basement lamina, a layer of cover cells tightly envelopes the AVA. These cells are, however, absent around the part of the AVA adjacent to the CVS. Here the endothelial cells are in immediate contact with the interstitium. Endothelial cells sheathed by cover cells reach the interstitium through basal foot processes. Nerve fibre bundles regularly come into close contact with the AVAs. Possible functions of the AVAs, including osmoreception are discussed.

The origin of arterio-venous anastomoses, connecting the efferent filament artery (EFA) with the central venous sinus (CVS) of gill filaments can be well discerned by scanning electron microscopy in the rainbow trout. Corresponding vessels between the afferent filament artery and the CVS could not be detected with the techniques applied. AVA-specific endothelial cells are characterized by their bulky shape and their microvillous surface. The general morphology of AVA's in Salmo gairdneri is very similar to that of AVA's in Tilapia mossambica (Vogel et al., 1974) but they are much longer in the trout. No filament whorls have been encountered in AVA endothelia of Salmo gairdneri.

A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented. This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface. This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals. Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket. The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids.

The primary structures of two neuropeptides, Tabanus atratus adipokinetic hormone (Taa-AKH) and Tabanus atratus hypotrehalosemic hormone (Taa-HoTH), from the corpora cardiaca of horse flies (Diptera: Tabanidae) have been determined. Amino acid sequences of Taa-AKH (less than Glu-Leu-Thr-Phe-Thr-Pro-Gly-Trp-NH2) and Taa-HoTH (less than Glu-Leu-Thr-Phe-Thr-Pro-Gly-Trp-Gly-Tyr-NH2) (where less than Glu = pyroglutamic acid) were determined by automated gas-phase Edman degradation of the peptides deblocked by pyroglutamate aminopeptidase and by fast atom bombardment mass spectrometry. The hormones were synthesized, and the natural and synthetic peptides exhibited identical chromatographic, spectroscopic, and biological properties. When assayed in adult face fly males, Taa-AKH and Taa-HoTH demonstrated hyperlipemic activity, in addition, Taa-HoTH also demonstrated a significant hypotrehalosemic activity.

Charged organic−aqueous interface electrostatics are assessed in order to clarify potential electrostatic contributions to oriented mineral nucleation at planar organic surfaces. Fatty acid and acidic phospholipid monolayers contacting aqueous solutions of sparingly soluble salts serve as model systems. Monolayer electrostatic parameters and ion distributions in the interfacial aqueous solution are calculated using a Gouy−Chapman−Stern electric double-layer model that incorporates the multiple, multivalent cations and anions found in mineralizing solutions. The calculations provide insight into the relationship between surface-directed crystallization and factors such as ion binding, surface charge density, and interfacial ion concentrations. Variations in crystal nucleation, phase, and morphology observed at charged monolayers are interpreted in terms of calculated departures from lattice ion stoichiometry and pH lowering within the interfacial aqueous solution. Lattice matching is addressed in terms of Stern layer calculations of Ca²⁺ ion binding at carboxylic acid monolayers. Arguments are presented suggesting that near negatively charged surfaces, soluble ionic species concentrations are enhanced relative to free anion concentrations, and a link between interfacial anion speciation and oriented mineral nucleation is proposed. Implications for biomineralization and template-directed crystallization at planar surfaces are discussed.

1.Rhinopoma is able to live without drinking water in a dry environment at a temperature of 35° C, when fed on 1 g mealworms/day (observed during 17 days).2.Urine of normal Rhinopoma is on the average less concentrated (2042 mOsm/ kg H2O) than that of normal Rhinolophus (2467 mOsm/kg H2O). The mean urine concentration of thirsting Rhinopoma however is higher (3722 mOsm/kg H2O) than that of thirsting Rhinolophus (2980 mOsm/kg H2O).3.The serum of dehydrated Rhinopoma is slightly more concentrated (401 mOsm/ kg H2O) than that of normal ones (377 mOsm/kg H2O). This is mainly due to a significant increase of the sodium concentration. Neither potassium nor urea concentrations increased significantly during dehydration.Serum of thirsting Rhinolophus on the other hand was considerably more concentrated (546 mOsm/kg H2O) than that of normal ones (413 mOsm/kg H2O). The concentrations of urea as well as sodium had then increased respectively from 208 to 403 mg-% and from 173 to 227 mVal/l.4.The serum of normal Rhinolophus is more concentrated than that of normal Rhinopoma. This is due to a higher sodium concentration. Serum urea concentrations of normal Rhinopoma and normal Rhinolophus do not differ significantly. Mean serum concentration of dehydrated Rhinolophus is much higher than that of dehydrated Rhinopoma — urea as well as sodium being then more concentrated in Rhinolophus serum.5.Changes in urine potassium and urea concentrations, not however in urine sodium concentrations of both animals, correlate with the corresponding urine osmolality.6.There is no difference between the U/Posm ratio of Rhinopoma and that of Rhinolophus under normal conditions. During dehydration however, this ratio is higher in Rhinopoma than in Rhinolophus. U/Posm ratios of thirsting Rhinopoma were much higher than those of normal ones (p\s<0.001). A comparison of thirsting Rhinolophus with those held under normal conditions indicates that the U/Posm ratio of the normal animals tends to be higher than that of thirsting ones. The difference however is not significant (0.2\s> p\s>0.1)..

In fresh water, Fundulus heteroclitus produces copious hypotonic urine.

A forcemeter capable of detecting piconewton forces was assembled from nanoscale building blocks, using a cantilevered microtubule as a beam of known stiffness, which is loaded by a second microtubule transported by kinesin motor proteins adsorbed to the surface and connected to the cantilevered microtubule by the link whose strength is to be determined. Due to the loading rate of less than 1 pN/s, this forcemeter is ideally suited to study the strength of biological receptor/ligand pairs, such as streptavidin/biotin.

The catalytic effect of metmyoglobin on the back thermal cis → trans isomerization reaction of stilbazolium betaine M is reported. This reaction shows substantial acceleration in the presence of metmyoglobin in comparison to the same reaction without the protein or in the presence of metmyoglobin cyanide. It is suggested that the observed thermal reaction acceleration may arise from the coordination of the protonated stilbazolium betaine molecule to the sixth ligand position of the heme iron and subsequent chromophore deprotonation or from the low polarity of the heme pocket microenvironment.