Structural insights into light-driven anion pumping in cyanobacteria.

Transmembrane ion transport is a key process in living cells. Active transport of ions is carried out by various ion transporters including microbial rhodopsins (MRs). MRs perform diverse functions such as active and passive ion transport, photo-sensing, and others. In particular, MRs can pump various monovalent ions like Na+, K+, Cl-, I-, NO3-. The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp. PCC 7509 and is named Synechocystis halorhodopsin (SyHR). The structural study of SyHR may help to understand what makes an MR pump divalent ions. Here we present the crystal structure of SyHR in the ground state, the structure of its sulfate-bound form as well as two photoreaction intermediates, the K and O states. These data reveal the molecular origin of the unique properties of the protein (exceptionally strong chloride binding and proposed pumping of divalent anions) and sheds light on the mechanism of anion release and uptake in cyanobacterial halorhodopsins. The unique properties of SyHR highlight its potential as an optogenetics tool and may help engineer different types of anion pumps with applications in optogenetics.

High-resolution structural insights into the heliorhodopsin family.

Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), has recently been discovered. Unlike in the known rhodopsins, in HeRs the N termini face the cytoplasm. The function of HeRs remains unknown. We present the structures of the bacterial HeR-48C12 in two states at the resolution of 1.5 Å, which highlight its remarkable difference from all known rhodopsins. The interior of HeR's extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (Schiff base cavity [SBC]) surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH, a planar triangular molecule (acetate) is present in the SBC. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs, suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement in fundamental redox biological processes.

[Development and Evaluation of a Health Promotion Programme for Low-Qualified Workers with Special Attention to Transfer]. / Entwicklung und Evaluation eines Gesundheitsförderungsprogramms für Un- und Angelernte unter besonderer Berücksichtigung der Transfersicherung.

A health promotion programme for low-qualified workers was developed with focus on resource and stress management (ReSuM) and with special attention to transfer. ReSuM is a multiplier concept and combines a team intervention for low-qualified workers with an intervention for their supervisors. The effectiveness and efficiency were successfully evaluated within a comprehensive evaluation strategy.

Effects of environmental parameters and irrigation on the turgor pressure of banana plants measured using the non-invasive, online monitoring leaf patch clamp pressure probe.

Turgor pressure provides a sensitive indicator for irrigation scheduling. Leaf turgor pressure of Musa acuminate was measured by using the so-called leaf patch clamp pressure probe, i.e. by application of an external, magnetically generated and constantly retained clamp pressure to a leaf patch and determination of the attenuated output pressure P(p) that is highly correlated with the turgor pressure. Real-time recording of P(p) values was made using wireless telemetric transmitters, which send the data to a receiver base station where data are logged and transferred to a GPRS modem linked to an Internet server. Probes functioned over several months under field and laboratory conditions without damage to the leaf patch. Measurements showed that the magnetic-based probe could monitor very sensitively changes in turgor pressure induced by changes in microclimate (temperature, relative humidity, irradiation and wind) and irrigation. Irrigation effects could clearly be distinguished from environmental effects. Interestingly, oscillations in stomatal aperture, which occurred frequently below turgor pressures of 100 kPa towards noon at high transpiration or at high wind speed, were reflected in the P(p) values. The period of pressure oscillations was comparable with the period of oscillations in transpiration and photosynthesis. Multiple probe readings on individual leaves and/or on several leaves over the entire height of the plants further emphasised the great impact of this non-invasive turgor pressure sensor system for elucidating the dynamics of short- and long-distance water transport in higher plants.

A non-invasive probe for online-monitoring of turgor pressure changes under field conditions.

An advanced non-invasive, field-suitable and inexpensive leaf patch clamp pressure probe for online-monitoring of the water relations of intact leaves is described. The probe measures the attenuated output patch clamp pressure, P(p), of a clamped leaf in response to an externally applied input pressure, P(clamp). P(clamp) is generated magnetically. P(p) is sensed by a pressure sensor integrated into the magnetic clamp. The magnitude of P(p) depends on the transfer function, T(f), of the leaf cells. T(f) consists of a turgor pressure-independent (related to the compression of the cuticle, cell walls and other structural elements) and a turgor pressure-dependent term. T(f) is dimensionless and assumes values between 0 and 1. Theory shows that T(f) is a power function of cell turgor pressure P(c). Concomitant P(p) and P(c) measurements on grapevines confirmed the relationship between T(f) and P(c). P(p) peaked if P(c) approached zero and assumed low values if P(c) reached maximum values. The novel probe was successfully tested on leaves of irrigated and non-irrigated grapevines under field conditions. Data show that slight changes in the microclimate and/or water supply (by irrigation or rain) are reflected very sensitively in P(p).

Evidence for discontinuous water columns in the xylem conduit of tall birch trees.

The continuity of the xylem water columns was studied on 17- to 23-m tall birch trees (trunk diameter about 23 cm; first branching above 10 m) all year round. Fifty-one trees were felled, and 5-cm thick slices or 2-m long boles were taken at regular, relatively short intervals over the entire height of the trees. The filling status of the vessels was determined by (i) xylem sap extraction from trunk and branch pieces (using the gas bubble-based jet-discharge method and centrifugation) and from trunk boles (using gravity discharge); (ii) (1)H nuclear magnetic resonance imaging of slice pieces; (iii) infusion experiments (dye, (86)Rb(+), D(2)O) on intact trees and cut branches; and (iv) xylem pressure measurements. This broad array of techniques disclosed no evidence for continuous water-filled columns, as postulated by the Cohesion-Tension theory, for root to apex directed mass transport. Except in early spring (during the xylem refilling phase) and after extremely heavy rainfall during the vegetation period, cohesive/mobile water was found predominantly at intermediate heights of the trunks but not at the base or towards the top of the tree. Similar results were obtained for branches. Furthermore, upper branches generally contained more cohesive/mobile water than lower branches. The results suggest that water lifting occurs by short-distance (capillary, osmotic and/or transpiration-bound) tension gradients as well as by mobilisation of water in the parenchymatic tissues and the heartwood, and by moisture uptake through lenticels.

The mechanisms of refilling of xylem conduits and bleeding of tall birch during spring.

Seasonal variations in osmolality and components of xylem sap in tall birch trees were determined using several techniques. Xylem sap was extracted from branch and trunk sections of 58 trees using the very rapid gas bubble-based jet-discharge method. The 5-cm long wood pieces were taken at short intervals over the entire tree height. The data show that large biphasic osmolality gradients temporarily exist within the conducting xylem conduits during leaf emergence (up to 272 mosmol x kg(-1) at the apex). These gradients (arising mainly from glucose and fructose) were clearly held within the xylem conduit as demonstrated by (1)H NMR imaging of intact twigs. Refilling experiments with benzene, sucrose infusion, electron and light microscopy, as well as (1)H NMR chemical shift microimaging provided evidence that the xylem of birch represents a compartment confined by solute-reflecting barriers (radial: lipid linings/lipid bodies; axial: presumably air-filled spaces). These features allow transformation of osmolality gradients into osmotic pressure gradients. Refilling of the xylem occurs by a dual mechanism: from the base (by root pressure) and from the top (by hydrostatic pressure generated by xylem-bound osmotic pressure). The generation of osmotic pressure gradients was accompanied by bleeding. Bleeding could be observed at a height of up to 21 m. Bleeding rates measured at a given height decreased exponentially with time. Evidence is presented that the driving force for bleeding is the weight of the static water columns above the bleeding point. The pressure exerted by the water columns and the bleeding volume depend on the water-filling status of (communicating) vessels.

A novel, non-invasive, online-monitoring, versatile and easy plant-based probe for measuring leaf water status.

A high-precision pressure probe is described which allows non-invasive online-monitoring of the water relations of intact leaves. Real-time recording of the leaf water status occurred by data transfer to an Internet server. The leaf patch clamp pressure probe measures the attenuated pressure, P(p), of a leaf patch in response to a constant clamp pressure, P(clamp). P(p) is sensed by a miniaturized silicone pressure sensor integrated into the device. The magnitude of P(p) is dictated by the transfer function of the leaf, T(f), which is a function of leaf patch volume and ultimately of cell turgor pressure, P(c), as shown theoretically. The power function T(f)=f(P(c)) theoretically derived was experimentally confirmed by concomitant P(p) and P(c) measurements on intact leaflets of the liana Tetrastigma voinierianum under greenhouse conditions. Simultaneous P(p) recordings on leaflets up to 10 m height above ground demonstrated that changes in T(f) induced by P(c) changes due to changes of microclimate and/or of the irrigation regime were sensitively reflected in corresponding changes of P(p). Analysis of the data show that transpirational water loss during the morning hours was associated with a transient rise in turgor pressure gradients within the leaflets. Subsequent recovery of turgescence during the afternoon was much faster than the preceding transpiration-induced water loss if the plants were well irrigated. Our data show the enormous potential of the leaf patch clamp pressure probe for leaf water studies including unravelling of the hydraulic communication between neighbouring leaves and over long distances within tall plants (trees).

Effects on capacitance by overexpression of membrane proteins.

Functional Channelrhodopsin-2 (ChR2) overexpression of about 10(4)channels/mum(2) in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C(m)). The C(m) increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C(m) of 1.15+/-0.08 microF/cm(2) in ChR2-expressing cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (0.79+/-0.02 microF/cm(2)). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C(m) (0.85+/-0.07 microF/cm(2)) as compared to non-expressing control cells (0.70+/-0.03 microF/cm(2)). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins.

A combined patch-clamp and electrorotation study of the voltage- and frequency-dependent membrane capacitance caused by structurally dissimilar lipophilic anions.

Interactions of structurally dissimilar anionic compounds with the plasma membrane of HEK293 cells were analyzed by patch clamp and electrorotation. The combined approach provides complementary information on the lipophilicity, preferential affinity of the anions to the inner/outer membrane leaflet, adsorption depth and transmembrane mobility. The anionic species studied here included the well-known lipophilic anions dipicrylamine (DPA(-)), tetraphenylborate (TPB(-)) and [W(2)(CO)(10)(S(2)CH)](-), the putative lipophilic anion B(CF(3))(4)(-) and three new heterocyclic W(CO)(5) derivatives. All tested anions partitioned strongly into the cell membrane, as indicated by the capacitance increase in patch-clamped cells. The capacitance increment exhibited a bell-shaped dependence on membrane voltage. The midpoint potentials of the maximum capacitance increment were negative, indicating the exclusion of lipophilic anions from the outer membrane leaflet. The adsorption depth of the large organic anions DPA(-), TPB(-) and B(CF(3))(4)(-) increased and that of W(CO)(5) derivatives decreased with increasing concentration of mobile charges. In agreement with the patch-clamp data, electrorotation of cells treated with DPA(-) and W(CO)(5) derivatives revealed a large dispersion of membrane capacitance in the kilohertz to megahertz range due to the translocation of mobile charges. In contrast, in the presence of TPB(-) and B(CF(3))(4)(-) no mobile charges could be detected by electrorotation, despite their strong membrane adsorption. Our data suggest that the presence of oxygen atoms in the outer molecular shell is an important factor for the fast translocation ability of lipophilic anions.

Mechanisms of electrically mediated cytosolic Ca2+ transients in aequorin-transformed tobacco cells.

Cytosolic Ca(2+) changes induced by electric field pulses of 50-micros duration and 200-800 V/cm strength were monitored by measuring chemiluminescence in aequorin-transformed BY-2 tobacco cells. In Ca(2+)-substituted media, electropulsing led to a very fast initial increase of the cytosolic Ca(2+) concentration reaching a peak value within <100-200 ms. Peaking of [Ca(2+)](cyt) was followed by a biphasic decay due to removal of Ca(2+) (e.g., by binding and/or sequestration in the cytosol). The decay had fast and slow components, characterized by time constants of approximately 0.5 and 3-5 s, respectively. Experiments with various external Ca(2+) concentrations and conductivities showed that the fast decay arises from Ca(2+) fluxes through the plasmalemma, whereas the slow decay must be assigned to Ca(2+) fluxes through the tonoplast. The amplitude of the [Ca(2+)](cyt) transients increased with increasing field strength, whereas the time constants of the decay kinetics remained invariant. Breakdown of the plasmalemma was achieved at a critical field strength of approximately 450 V/cm, whereas breakdown of the tonoplast required approximately 580 V/cm. The above findings could be explained by the transient potential profiles generated across the two membranes in response to an exponentially decaying field pulse. The dielectric data required for calculation of the tonoplast and plasmalemma potentials were derived from electrorotation experiments on isolated vacuolated and evacuolated BY-2 protoplasts. The electrorotation response of vacuolated protoplasts could be described in terms of a three-shell model (i.e., by assuming that the capacitances of tonoplast and plasmalemma are arranged in series). Among other things, the theoretical analysis together with the experimental data show that genetic manipulations of plant cells by electrotransfection or electrofusion must be performed in low-conductivity media to minimize release of vacuolar Ca(2+) and presumably other vacuolar ingredients.

Foliar water supply of tall trees: evidence for mucilage-facilitated moisture uptake from the atmosphere and the impact on pressure bomb measurements.

The water supply to leaves of 25 to 60 m tall trees (including high-salinity-tolerant ones) was studied. The filling status of the xylem vessels was determined by xylem sap extraction (using jet-discharge, gravity-discharge, and centrifugation) and by (1)H nuclear magnetic resonance imaging of wood pieces. Simultaneously, pressure bomb experiments were performed along the entire trunk of the trees up to a height of 57 m. Clear-cut evidence was found that the balancing pressure (P(b)) values of leafy twigs were dictated by the ambient relative humidity rather than by height. Refilling of xylem vessels of apical leaves (branches) obviously mainly occurred via moisture uptake from the atmosphere. These findings could be traced back to the hydration and rehydration of mucilage layers on the leaf surfaces and/or of epistomatal mucilage plugs. Xylem vessels also contained mucilage. Mucilage formation was apparently enforced by water stress. The observed mucilage-based foliar water uptake and humidity dependency of the P(b) values are at variance with the cohesion-tension theory and with the hypothesis that P(b) measurements yield information about the relationships between xylem pressure gradients and height.

Biophysical characterisation of electrofused giant HEK293-cells as a novel electrophysiological expression system.

Giant HEK293 cells of 30-65 microm in diameter were produced by three-dimensional multi-cell electrofusion in 75 mOsm sorbitol media. These strong hypotonic conditions facilitated fusion because of the spherical shape and smooth membrane surface of the swollen cells. A regulatory volume decrease (RVD), as observed at higher osmolalities, did not occur at 75 mOsm. In contrast to field-treated, but unfused cells, the increase in volume induced by hypotonic shock was only partly reversible in the case of fused giant cells after their transfer into isotonic medium. The large size of the electrofused cells allowed the study of their electrophysiological properties by application of both whole-cell and giant excised patch-clamp techniques. Recordings on giant cells yielded a value of 1.1+/-0.1 microF/cm2 for the area-specific membrane capacitance. This value was consistent with that of the parental cells. The area-specific conductivity of giant cells (diameter > 50 microm) was found to be between 12.8 and 16.1 microS/cm2, which is in the range of that of the parental cells. Measurements with patch-pipettes containing fluorescein showed uniform dye uptake in the whole-cell configuration, but not in the cell-attached configuration. The diffusion-controlled uniform uptake of the dye into the cell interior excludes internal compartmentalisation. The finding of a homogeneous fusion was also supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein ChR2-YFP) in giant cells since no plasma-membrane bound YFP-mediated fluorescence was detected in the interior of the electrofused cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) yielded similar results as for parental cells. Most importantly, the giant cells exhibited a comparable expression density of the channel protein in the plasma membrane as observed in parental cells. This demonstrates that electrofused cells can be used as a heterologous expression system.

Swelling-activated pathways in human T-lymphocytes studied by cell volumetry and electrorotation.

Small organic solutes, including sugar derivatives, amino acids, etc., contribute significantly to the osmoregulation of mammalian cells. The present study explores the mechanisms of swelling-activated membrane permeability for electrolytes and neutral carbohydrates in Jurkat cells. Electrorotation was used to analyze the relationship between the hypotonically induced changes in the electrically accessible surface area of the plasma membrane (probed by the capacitance) and its permeability to the monomeric sugar alcohol sorbitol, the disaccharide trehalose, and electrolyte. Time-resolved capacitance and volumetric measurements were performed in parallel using media of different osmolalities containing either sorbitol or trehalose as the major solute. Under mild hypotonic stress in 200 mOsm sorbitol or trehalose solutions, the cells accomplished regulatory volume decrease by releasing cytosolic electrolytes presumably through pathways activated by the swelling-mediated retraction of microvilli. This is suggested by a rapid decrease of the area-specific membrane capacitance C(m) (microF/cm2). The cell membrane was impermeable to both carbohydrates in 200 mOsm media. Whereas trehalose permeability remained also very poor in 100 mOsm medium, extreme swelling of cells in a strongly hypotonic solution (100 mOsm) led to a dramatic increase in sorbitol permeability as evidenced by regulatory volume decrease inhibition. The different osmotic thresholds for activation of electrolyte release and sorbitol influx suggest the involvement of separate swelling-activated pathways. Whereas the electrolyte efflux seemed to utilize pathways preexisting in the plasma membrane, putative sorbitol channels might be inserted into the membrane from cytosolic vesicles via swelling-mediated exocytosis, as indicated by a substantial increase in the whole-cell capacitance C(C) (pF) in strongly hypotonic solutions.

Kinetics of charge translocation in the passive downhill uptake mode of the Na+/H+ antiporter NhaA of Escherichia coli.

The Na+/H+ antiporter NhaA is the main Na+ extrusion system in E. coli. Using direct current measurements combined with a solid supported membrane (SSM), we obtained electrical data of the function of NhaA purified and reconstituted in liposomes. These measurements demonstrate NhaA's electrogenicity, its specificity for Li+ and Na+ and its pronounced pH dependence in the range pH 6.5-8.5. The mutant G338S, in contrast, presents a pH independent profile, as reported previously. A complete right-side-out orientation of the NhaA antiporter within the proteoliposomal membrane was determined using a NhaA-specific antibody based ELISA assay. This allowed for the first time the investigation of NhaA in the passive downhill uptake mode corresponding to the transport of Na+ from the periplasmic to the cytoplasmic side of the membrane. In this mode, the transporter has kinetic properties differing significantly from those of the previously investigated efflux mode. The apparent Km values were 11 mM for Na+ and 7.3 mM for Li+ at basic pH and 180 mM for Na+ and 50 mM for Li+ at neutral pH. The data demonstrate that in the passive downhill uptake mode pH regulation of the carrier affects both apparent Km as well as turnover (Vmax).

Channelrhodopsins: directly light-gated cation channels.

Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial type chromophores, i.e. all-trans-retinal in the ground state. The green alga Chlamydomonas reinhardtii was recently completely sequenced and the EST (expressed sequence tag) database was made public. We and others detected overlapping partial cDNA sequences that encode two proteins which we termed channelopsins (Chops). The N-terminal half of chop1 (approximately 300 of 712 amino acids) comprises hypothetical seven-transmembrane segments with sequence similarity to the proton pump bacteriorhodopsin and the chloride pump halorhodopsin. Even though the overall sequence homology is low, several amino acids are conserved that define the retinal-binding site and the H+-transporting network in BR (bacteriorhodopsin). Expression of Chop1, or only the hydrophobic core, in Xenopus laevis oocytes, enriched with retinal, produced a light-gated conductance (maximum at approx. 500 nm), which shows characteristics of a channel [ChR1 (channelrhodopsin-1)] that is selectively permeable for protons. Also ChR2 (737 amino acids) is an ion channel that is switched directly by light and also here the hydrophobic N-terminal half of the protein is sufficient to enable light-sensitive channel activity. The action spectrum is blue-shifted (maximum at approx. 460 nm) with respect to ChR1. In addition to protons, ChR2 is permeable to univalent and bivalent cations. We suggest that ChRs are involved in phototaxis of green algae. We show that heterologous expression of ChR2 is useful to manipulate intracellular pCa or membrane potential of animal cells, simply by illumination.

Real-time 3-D dark-field microscopy for the validation of the cross-linking process of alginate microcapsules.

Alginate-based microencapsulation is a promising method for long-term maintenance of cellular and membrane function of the cells and tissue fragments required for in vitro and in vivo biosensors, for tissue engineering and particularly for immunoisolation of non-autologous transplants. Microcapsules of high mechanical strength and optimum permeability can be produced by injection of BaCl2 crystals into alginate droplets before they come into contact with external Ba2+. A key requirement is that the system parameters (number of crystals, speed of the crystal stream etc.) are properly adjusted according to the mannuronic and guluronic acid ratio and the average molecular mass of the alginate as well as to the diameter of the microcapsules. Robust, reliable, rapid and low-cost validation tools are, therefore, needed for assurance of the microcapsule quality. Here, we describe a novel three-dimensional (3-D) dark-field microscopy that allows the real-time measurement of the number and spatial distribution of the injected Ba2+ ions throughout the microcapsules after treatment with sulphate. This novel method requires only a conventional microscope equipped with three polarising filters and a double aperture stop. In contrast to confocal laser scanning microscopy images, peripherally attached BaSO4 precipitates can clearly be distinguished from internal ones. The data also demonstrate that several steps of the alginate gelling process must be improved before such immunoisolation can be used in patients.

Surviving high-intensity field pulses: strategies for improving robustness and performance of electrotransfection and electrofusion.

Electrotransfection and electrofusion, both widely used in research and medical applications, still have to face a range of problems, including the existence of electroporation-resistant cell types, cell mortality and also great batch-to-batch variations of the transfection and fusion yields. In the present study, a systematic analysis of the parameters critical for the efficiency and robustness of electromanipulation protocols was performed on five mammalian cell types. Factors examined included the sugar composition of hypotonic pulse media (trehalose, sorbitol or inositol), the kinetics of cell volume changes prior to electropulsing, as well as the growth medium additives used for post-pulse cell cultivation. Whereas the disaccharide trehalose generally allowed regulatory volume decrease (RVD), the monomeric sugar alcohols sorbitol and inositol inhibited RVD or even induced secondary swelling. The different volume responses could be explained by the sugar selectivity of volume-sensitive channels (VSC) in the plasma membrane of all tested cell types. Based on the volumetric data, highest transfection and fusion yields were mostly achieved when the target cells were exposed to hypotonicity for about 2 min prior to electropulsing. Longer hypotonic treatment (10-20 min) decreased the yields of viable transfected and hybrid cells due to (1) the cell size reduction upon RVD (trehalose) or (2) the excessive losses of cytosolic electrolytes through VSC (inositol/sorbitol). Doping the plasma membrane with lipophilic anions prevented both cell shrinkage and ion losses (probably due to VSC inhibition), which in turn resulted in increased transfection and fusion efficiencies.

Effect of testosterone and oestradiol-17beta on canine hair follicle culture.

Skin biopsies were taken from four body sites (head, thorax, flank and perineum) of three male entire Beagles and the primary hair follicles were isolated. Culture conditions were established to keep the hair follicles growing for up to 7 days. Additionally, hair follicles were incubated in supplemented medium (containing insulin, transferrin, glutamine and sodium selenite) with or without the addition of testosterone (T) (1, 10 or 100 ng/ml) or oestradiol-17beta (E2beta) (0.01, 0.1 or 1 ng/ml), respectively and the daily growth of hair follicles was measured. In vitro daily growth of hair follicles from the thorax was stimulated by the low concentration of both hormones, but the growth of those from the flank was inhibited by the high concentration of both hormones. Hair follicles from the head were positively influenced by the lowest concentration of T and the medium concentration of E2beta. The daily growth of hair follicles from the perineum was not significantly influenced by either hormone.

Excretion of corticosteroid metabolites in urine and faeces of rats.

Stress enhances the production of corticosteroids by the adrenal cortex, resulting in the increased excretion of their metabolites in urine and faeces. An intraperitoneal injection of radioactive corticosterone was applied to adult, male Sprague-Dawley rats to monitor the route and delay of excreted metabolites in urine and faeces. Peak concentrations appeared in urine after 3.2 +/- 1.9 h and in faeces after 16.7 +/- 4.3 h. Altogether about 20% of the recovered metabolites were found in urine and about 80% in faeces. Using high-performance liquid chromatography (HPLC), several peaks of radioactive metabolites were found. Some metabolites were detected by enzyme immunoassay (EIA) using two different antibodies (corticosterone, 11beta-OH-aetiocholanolone). There was a marked diurnal variation with low levels of faecal corticosterone metabolites in the evening and higher values in the morning. This diurnal variation was influenced neither by the intraperitoneal injection of isotonic saline nor by ACTH. However, the administration of dexamethasone eliminated the morning peak for 2 days.