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Iloprost (IP) stimulation (1 microM, 2 h) of Flag-epitope-tagged human IP prostanoid receptor (FhIPR) expressed in HEK293 cells resulted in specific decrease of endogenous G(s)alpha protein in detergent-insensitive, caveolin-enriched, membrane domains (DIMs). Receptor protein FhIPR, caveolin, G(i)alpha and GPI-linked, domain markers CD55 and CD59 were unchanged. The same result was obtained in HEK293 cells expressing FhIPR-G(s)alpha fusion protein. The endogenous G(s)alpha decreased, but the level of Flag-hIPR-G(s)alpha protein did not change. The specific depletion of domain-bound pool of G(s)alpha as consequence of iloprost stimulation was also demonstrated in membrane domains prepared according to alkaline treatment plus sonication protocol (detergent-free procedure of Song et al.). Our data further indicated that in control, quiescent cells only a very small amount of IP prostanoid receptor was present in DIMs together with large amount of its cognate G(s)alpha protein. Expressed in quantitative terms, DIMs contained 30-40% of the total cellular amount of G proteins whereas the content of IP prostanoid receptors was 1-3%. The dominant portion (>95%) of FhIPR as well as FhIPR-G(s)alpha was localised in high-density area of the gradient containing detergent-solubilised proteins. FhIPR and FhIPR-G(s)alpha distribution was similar to that of transmembrane plasma membrane (PM) markers (CD147, MHCI, CD29, Tapa1, the alpha subunit of Na,K-ATPase, transmembrane form of CD58 and CD44). All these proteins are known to be fully solubilised by detergent and thus unable to float in density gradient. Our data indicate that (i) long-term agonist stimulation of IP prostanoid receptor is associated with preferential decrease of its cognate G protein G(s)alpha from membrane domains; receptor level is unchanged. (ii) Very small fraction (1-3%) of total cellular amount of receptors is recovered in DIMs together with roughly 40% of G proteins. These data suggest a "supra-stoichiometric" arrangement of G proteins and corresponding receptors in DIMs.

Many extracellular signals are at the cell surface received by specific receptors, which upon activation transduce information to the appropriate cellular effector molecules via trimeric G proteins. The G protein-mediated cascades ultimately lead to the highly refined regulation of systems such as sensory perception, cell growth, and hormonal regulation. Transmembrane signaling may be seriously deranged in various pathophysiological conditions. Over the last two decades the major experimental effort of our group has been devoted to better understanding the molecular mechanisms underlying transmembrane signaling regulated by G proteins and to the closely related process of desensitization of hormone response. This review provides general information about the basic principles of G protein-regulated transmembrane signaling as well as about our contribution to the current progress in the field.

Using x-ray magnetic circular dichroism, we have detected the very interfacial spins that are responsible for the horizontal loop shift in three different exchange bias sandwiches, chosen because of their potential for device applications. The "pinned" uncompensated interfacial spins constitute only a fraction of a monolayer and do not rotate in an external magnetic field since they are tightly locked to the antiferromagnetic lattice. A simple extension of the Meiklejohn and Bean model is proposed to account quantitatively for the exchange bias fields in the three studied systems from the experimentally determined number of pinned moments and their sizes.

Using x-ray absorption spectromicroscopy we have imaged the uncompensated spins induced at the surface of antiferromagnetic (AFM) NiO(100) by deposition of ferromagnetic (FM) Co. These spins align parallel to the AFM spins in NiO(100) and align the FM spins in Co. The uncompensated interfacial spins arise from an ultrathin CoNiOx layer that is formed upon Co deposition through reduction of the NiO surface. The interfacial Ni spins are discussed in terms of the "uncompensated spins" at AFM/FM interfaces long held responsible for coercivity increases and exchange bias. We find a direct correlation between their number and the size of the coercivity.

The present paper investigated the differences in passive avoidance learning between Sprague--Dawley and Lewis rats. After initial habituation (experimental Part 1), measured as latencies to enter the dark, preferable compartment, the effect of treatment with amphetamine (8 mg kg(-1)b.w.), the retention performance compared with controls (saline) was tested in both rat strains in Parts 2--4. The intervals between Parts 2--4 were 24 or 49 days. Each experimental part consisted of testing lasting 6 days. On the 7th day the rats received drug treatment 1 h before the application of foot shock. The differences between rat strains were already detectable at the beginning of the study. During the repeated exposures of rats in Part 1, only Lewis rats, in contrast to Sprague--Dawley rats, exhibited the habituation. The repeated testing of rats in Parts 2--4, due to previous experience with an aversive stimulus, was considered as the retention test. In Parts 2--3 we observed only minor differences in the responses of both rat strains tested. Also no significant differences were observed between rat strains after amphetamine treatment that induced an amnesia-like effect in all retention trials. However, data shown in Part 4 revealed the largest differences between both strains. Control Lewis rats exhibited significantly higher retention responses than Sprague--Dawley rats. In the latter strain we observed no differences in avoidance latencies between controls and amphetamine treated rats. In Lewis rats the difference in avoidance performance between controls and amphetamine treated animals was highly significant due to their enhanced retention performance. In conclusion, the results presented in this study extend the known behavioural differences in tested rat strains to the passive avoidance procedure that, in addition, was performed for a total period of 4 months. Due to a known deficiency of hypothalamo-pituitary-adrenal axis activity in Lewis rats it can be hypothesized that the behavioural dissociation of this strain from Sprague--Dawley rats could be related to the different activity of this regulatory axis in the rat strains tested., (Copyright 2001 Academic Press.)

The technique used to align liquid crystals-rubbing the surface of a substrate on which a liquid crystal is subsequently deposited-has been perfected by the multibillion-dollar liquid-crystal display industry. However, it is widely recognized that a non-contact alignment technique would be highly desirable for future generations of large, high-resolution liquid-crystal displays. A number of alternative alignment techniques have been reported, but none of these have so far been implemented in large-scale manufacturing. Here, we report a non-contact alignment process, which uses low-energy ion beams impinging at a glancing angle on amorphous inorganic films, such as diamond-like carbon. Using this approach, we have produced both laptop and desktop displays in pilot-line manufacturing, and found that displays of higher quality and reliability could be made at a lower cost than the rubbing technique. The mechanism of alignment is explained by adopting a random network model of atomic arrangement in the inorganic films. Order is induced by exposure to an ion beam because unfavourably oriented rings of atoms are selectively destroyed. The planes of the remaining rings are predominantly parallel to the direction of the ion beam.

Polarization dependent x-ray photoemission electron microscopy was used to investigate the spin structure near the surface of an antiferromagnetic NiO(001) single crystal in response to the deposition of a thin ferromagnetic Co film. For the cleaved NiO surface we observe only a subset of bulklike antiferromagnetic domains which is attributed to minimization of dipolar energies. Upon Co deposition a spin reorientation near the NiO interface occurs, with the antiferromagnetic spins rotating in plane, parallel to the spins of the Co layer. Our results demonstrate that the spin configuration in an antiferromagnet near its interface with a ferromagnet may significantly deviate from that in the bulk antiferromagnet.

Antiferromagnetic layers are a scientifically challenging component in magnetoelectronic devices, such as magnetic sensors in hard-disk heads, or magnetic random-access memory (RAM) elements. In this paper, it is shown that photoelectron emission microscopy (PEEM) is capable of determining the magnetic structure at the interface of ferromagnets and antiferromagnets with high spatial resolution (down to 20 nm). Dichroism effects at the L edges of the magnetic 3d transition metals, using circularly or linearly polarized soft X-rays from a synchrotron source, give rise to a magnetic image contrast. Images, acquired with the PEEM2 experiment at the Advanced Light Source, show magnetic contrast for antiferromagnetic LaFeO3, microscopically resolving the magnetic domain structure in an antiferromagnetically ordered thin film for the first time. Magnetic coupling between LaFeO3 and an adjacent Co layer results in a complete correlation of their magnetic domain structures. From field-dependent measurements, a unidirectional anisotropy resulting in a local exchange bias of up to 30 Oe in single domains could be deduced. The elemental specificity and the quantitative magnetic sensitivity render PEEM a perfect tool to study magnetic coupling effects in multilayered thin-film samples.

Monocytes are recruited as the principal inflammatory cells into the atherosclerotic lesion. In a previous study we demonstrated that a low HDL-cholesterol and the apo E4 allele are associated with an increased proportion of blood monocytes that are characterized by a high expression of Fcgamma-RIIIa (CD16), a dim expression of the lipopolysaccharide (LPS) receptor (CD14) and a high expression of beta1- and beta2-integrins (Rothe et al. Arterioscler Thromb Vasc Cell Biol 1996;16:1437-1447). In this study, 79 hypercholesterolemic patients were treated either with the HMG CoA reductase inhibitor fluvastatin in combination with diet or with placebo and diet in a double-blind and randomized multicenter study, and monitored for the potential effects on the phenotype of peripheral blood monocytes. At baseline, in the whole group of hypercholesterolemic patients the population size of these more mature monocytes (CD14dimCD16+) was positively correlated to triglyceride (P = 0.003) and total serum cholesterol levels (P = 0.012) confirming our previous study. Fluvastatin treatment for 52 weeks was associated with a 24.2% reduction in LDL-cholesterol (P < 0.001) as well as a 40.7% decrease in the expression density of CD14 on all monocytes (P = 0.027). A 24.5% decrease (P < 0.001) of the population of less differentiated CD14brightCD16- monocytes and an 83.1% increase (P = 0.029) of the population of more differentiated CD14dimCD16+ monocytes further confirmed this modification of the phenotype of peripheral blood monocytes. The positive pre-study correlation of the CD14dimCD16+ monocyte subset to the serum cholesterol concentration, but inverse changes of both parameters under fluvastatin therapy, in conclusion indicate that fluvastatin exerts an as yet uncharacterized immunomodulatory effect on either monocyte maturation and differentiation, or extravasation which may also depend on the endothelial phenotype that is independent of the change in serum lipids.

Several papers have indicated the participation of cyclic AMP as a second messenger for the release of neurohypophysial hormones. Since very little is known about the effects of stress and drugs of abuse on this process, we studied the activity of adenylyl cyclase in the neurohypophyses after immobilization stress and chronic amphetamine treatment. Our findings indicate the involvement of cyclic AMP in the regulation of neurohypophysis as well as the increase in total adenylyl cyclase both after application of immobilization stress combined with water immersion and after chronic amphetamine treatment.

We recently reported a positive correlation of the pool size of lipopolysaccharide receptor (CD14)dim and Fc gamma receptor IIIa (CD16a)+ monocytes in peripheral blood to the apolipoprotein E4 (apoE4) phenotype and a negative correlation to high density lipoprotein (HDL) cholesterol levels (Arterioscler Thromb Vasc Biol. 1996;16:1437-1447). In this study, the in vitro differentiation of mononuclear phagocytes derived from healthy blood donors homozygous for the E3/3 or the E4/4 phenotype was analyzed during 7 days of culture in serum-free medium supplemented with macrophage colony-stimulating factor (M-CSF). The CD16a expression, which indicates Fc receptor-dependent phagocytic activity, increased to a significantly higher level in apoE4/4 monocytes than in apoE3/3 cells. The costimulatory molecule CD40, which indicates antigen-presenting capacity, was upregulated more strongly in apoE3/3 monocytes compared with E4/4 cells, but the difference did not reach a significant level. The expression of differentiation-associated surface proteins (CD14, CD33, CD45) and adhesion molecules (CD11a, CD11b, CD11c, CD49d) was not significantly different between apoE3/3 and apoE4/4 monocytes. However, a significantly decreased intracellular apoE concentration and a reduced amount of secreted apoE were found in apoE4/4 monocytes during in vitro differentiation. No differences were found in the surface expression of the low density lipoprotein receptor-related protein (CD91) and the uptake of fluorescence labeled low density lipoprotein between apoE3/3 and apoE4/4 monocytes. These data indicate that the apoE4/4 phenotype significantly influences the M-CSF-dependent differentiation of monocytes toward a more CD16a-positive phagocytic phenotype.

X-ray photoelectron emission microscopy (X-PEEM) was used to study the surface orientation of stylized and rubbed polyimide thin films. Using soft X-rays produced by a synchrotron light source, this technique combines high spatial resolution imaging with near-edge X-ray absorption fine structure (NEXAFS) spectroscopy to yield information on the surface orientation of the films. Stylizing is an ideal model of the rubbing process since the local stress acting on the polyimide to orient the molecules can be calculated. The minimum normal stress necessary to orient the surface of BPDA-PDA films was found to be 45 MPa much lower than the bulk yield stress of 200-300 MPa. Studies of the polyimide films oriented by the conventional rubbing method showed lateral inhomogeneities in the orientation of the polymer at the surface.

Multiparameter flow cytometry reveals a complex heterogeneity of mononuclear phagocyte differentiation within the peripheral blood compartment. In this study, the relation of abnormal cellular lipid metabolism to the phenotype of peripheral blood mononuclear phagocytes, which finally may be related to atherogenesis, was analyzed using recently characterized autosomal recessive defects of lysosomal acid lipase (LAL) expression as model system. The reduction of LAL activity in nine heterozygote, disease free carriers of mutations from two cholesteryl ester storage disease (CESD) pedigrees and the family of a patient with Wolman disease was associated with an increased fraction of monocytes which expressed CD56 (N-CAM) (4.1 +/- 2.7% of monocytes, compared to 2.2 +/- 0.5% in ten controls, P < 0.05), an antigen characteristic of immature myeloid cells, suggesting an increased turnover of monocytes. Furthermore, a trend was observed towards an enhanced blood pool of more mature mononuclear phagocytes which show decreased expression of the 55 kD lipopolysaccharide receptor (CD14) together with either expression of the Fc-gamma-receptor III (CD16) or a high expression of CD33. A similar phenotype of peripheral mononuclear phagocytes was observed in the two CESD patients analyzed. In conclusion, our data suggest that these monogenetic defects of lysosomal lipoprotein metabolism are associated with complex alterations of mononuclear phagocyte differentiation and extravasation.

A trichloroacetic-acid-soluble 14.5-kDa protein (p14.5) has been isolated from human mononuclear phagocytes (MNP) by a combination of trichloroacetic acid extraction, preparative electrophoresis and hydrophobic affinity chromatography; five tryptic peptides were subjected to protein sequencing. The full-length cDNA of the protein was cloned and sequenced from a lambda gt11 human liver library. The cDNA showed a remarkable similarity to a rat protein preferentially expressed in hepatocytes and renal tubular epithelial cells. The encoded protein is 137 amino acids long and similar to members of a new hypothetical family of small proteins with presently unknown function, named YER057c/YJGF. Human recombinant p14.5 inhibits in vitro protein synthesis in a rabbit reticulocyte lysate system. Unlike other inhibitors of protein synthesis, p14.5 is not phosphorylated despite the presence of putative phosphorylation sites. The p14.5 mRNA is weakly expressed in freshly isolated monocytes but is significantly upregulated when these monocytes are subjected to differentiation. This is also reflected by a differentiation-dependent increase in the protein concentration as demonstrated by immunoblots from cytosolic fractions and fluorescence-activated flow cytometry of permeabilized cells. A differentiation-dependent mRNA and protein expression of p14.5 is further suggested by the observation of a low expression in a variety of liver and kidney tumor cells and a high expression in fully differentiated cells as assessed by immunohistochemistry and northern blots. The highest mRNA expression was found in hepatocytes and renal distal tubular epithelial cells and only weak expression was found in other human tissues as evaluated by northern blot analysis. The preferential localization of the immunoreaction product seemed to be cytoplasmatic but, in less differentiated cells, nuclear labeling was occasionally visible. Immunoblotting of subcellular fractions confirmed these data. The high degree of evolutionary conservation of p14.5, the considerable upregulation during cellular differentiation and its potential role as a translational inhibitor may reflect an involvement in basic cellular mechanisms, e.g. a differentiation-dependent regulation of protein synthesis in hepatocytes, renal tubular epithelial cells, smooth muscle cells and MNP.

Mononuclear phagocytes play a major role in the development of vascular lesions in atherogenesis. The goal of our study was to characterize circulating blood monocyte subpopulations as potential cellular markers of systemic immunological abnormalities in hypercholesterolemia. In normal subjects, three-parameter immunophenotyping of whole blood revealed that 61.3 +/- 6.0% of monocytes showed "bright" expression of the lipopolysaccharide receptor (LPSR: CD14) and Fc gamma receptor I (RI: CD64) without expression of Fc gamma-RIII (CD16). Other monocyte subsets (populations 2, 3, 4, and 5) were characterized by the simultaneous expression of both Fc gamma-R's (25.6 +/- 5.0%), isolated expression of Fc gamma-RIII (9.4 +/- 1.7%), or high expression of CD33 (3.7 +/- 1.1%) with only dim expression of CD14, respectively. The smallest subset of monocytes (population 5: 2.1 +/- 0.8%) differed from the predominant population of CD14brightCD64+CD16- monocytes by additional expression of neural cell adhesion molecule (N-CAM: CD56). In a group of hypercholesterolemic patients (n = 19), high density lipoprotein cholesterol levels were negatively correlated to the population size of CD64-CD16+ monocytes. In both healthy subjects (n = 55) and hypercholesterolemic patients, the rare apolipoprotein E3/E4 and E4/E4 phenotypes were associated with a tendency toward a larger population of CD64-CD16+ monocytes. Expression of the variant activation antigen CD45RA by peripheral blood mononuclear phagocytes showed a positive correlation to plasma levels of the atherogenic lipoproteins low density lipoprotein and lipoprotein(a). These data suggest that systemic abnormalities in mononuclear phagocyte subpopulations may play a role in the pathogenesis of atherosclerosis.

Absorption measurements above the K edges of the low-Z atoms, C, N, O, and F, are discussed, which utilize 250-1000-eV synchrotron radiation. The paper emphasizes experimental procedures of soft x-ray absorption and extended absorption fine structure (EXAFS) measurements as well as applications and future prospects of such studies. In particular, we discuss such applications as surface EXAFS, surface absorption fine structure, and ion yield EXAFS measurements.

Single glucose doses of 0.5 g/kg body weight or 1.0 g/kg were intravenously applied at ambient temperatures between 25 degrees C and 30 degrees C pigs aged between zero and 20 hours two and three, five and seven, ten and 14, 20 and 30 days, and eight weeks. With reference to excess glucose curves in the blood and plasma, the half-life of glucose elimination was graphically determined in a semi-logarithmic system. The result obtained was used to calculate the coefficients of assimilation and distribution, as well as the readily exchangeable pool and the transfer rate of glucose. On the average the half-life of glucose elimination in the five groups of the piglets was identical (0.47 hour); in contrast it was faster in weaners (0.17 hour) than in piglets. With reference to the plasma values, the volume of the glucose distribution decreased continuously with growing age. Average glucose transfer amounted to 1.02 mg/g. hr in weaners and thus was twice as much as that recorded for the five groups of the piglets. Reduction of ambient temperatures between 15 degrees C and 18 degrees C had no bearing on the parameters of 30-day piglets. These findings are likely to suggest that there is no increased glucose turnover within the thermal neutral zone in piglets over the first days of age and, consequently, cannot contribute to the development of hypoglycaemia.

Fructose tolerance tests were applied to 54 piglets, aged between zero and 20 hours, two and three, five and seven, as well as 20 and 30 days. Fructose injections per kilogram of body weight were 0.5 g and 1.0 g. Galactose tolerance tests were applied to 33 piglets, aged between zero and 20 hours as well as between five and seven or 20 and 35 days. The galactose dose per kilogram of body weight was 0.5 g. Very slow or no fructose elimination at all was recordable from the circulation of animals, aged between zero and 20 hours, yet, with elimination rates being accelerated along with growing age. Piglets, aged between 20 and 30 days, eliminated fructose from the blood just as fast as glucose (th = 0.49 +/- 0.11 hr). Fructose depressed the glucose levels in the blood in animals, aged between five and seven days, but it failed to do so in animals, aged between 20 and 30 days, Galactose was found to be eliminated speedily from the circulation, that is immediately after birth, a maximum being reached at an age between five and seven days (th = 0.11 +/- 0.02 hr). Fructose space in piglets, aged between five and seven or between 20 and 30 days, was found to be significantly below that recorded from the two younger age groups. Galactose space in piglets, aged between five and seven days, was significantly below that of the other two groups.