Orientation-dependent work function of in situ annealed strontium titanate.

We have used energy-filtered x-ray photoelectron emission microscopy (XPEEM) and synchrotron radiation to measure the grain orientation dependence of the work function of a sintered niobium-doped strontium titanate ceramic. A significant spread in work function values is found. Grain orientation and surface reducing/oxidizing conditions are the main factors in determining the work function. Energy-filtered XPEEM looks ideally suited for analysis of other technologically interesting polycrystalline samples.

Abnormalities in neuromuscular junction structure and skeletal muscle function in mice lacking the P2X2 nucleotide receptor.

ATP is co-released in significant quantities with acetylcholine from motor neurons at skeletal neuromuscular junctions (NMJ). However, the role of this neurotransmitter in muscle function remains unclear. The P2X2 ion channel receptor subunit is expressed during development of the skeletal NMJ, but not in adult muscle fibers, although it is re-expressed during muscle fiber regeneration. Using mice deficient for the P2X2 receptor subunit for ATP (P2X2(-/-)), we demonstrate a role for purinergic signaling in NMJ development. Whereas control NMJs were characterized by precise apposition of pre-synaptic motor nerve terminals and post-synaptic junctional folds rich in acetylcholine receptors (AChRs), NMJs in P2X2(-/-) mice were disorganized: misapposition of nerve terminals and post-synaptic AChR expression localization was common; the density of post-synaptic junctional folds was reduced; and there was increased end-plate fragmentation. These changes in NMJ structure were associated with muscle fiber atrophy. In addition there was an increase in the proportion of fast type muscle fibers. These findings demonstrate a role for P2X2 receptor-mediated signaling in NMJ formation and suggest that purinergic signaling may play an as yet largely unrecognized part in synapse formation.

Real time observation of GeSi/Si(001) island shrinkage due to surface alloying during Si capping.

The Si capping of Ge/Si(001) islands was observed by in situ time-resolved transmission electron microscopy. During the initial stages of the Si deposition, islands were observed not only to flatten but also to shrink in volume. This unexpected shrinkage is explained by taking into account the intermixing of the deposited Si with the wetting layer and a consequently induced diffusion of Ge from the islands into the wetting layer. A model of the capping process which takes into account Ge diffusion is presented which is in good agreement with the experimental data.

Simulated electron energy loss spectra from a C70 crystal.

Electron energy loss spectra are simulated for a C70 crystalline structure. It is found that the simulated spectrum is similar to the unoccupied density of states of a C70 molecule, indicating that the crystalline structure has only a small effect on the spectrum. Unlike the case of a single molecule, however, the main contribution to the second peak in the spectrum cannot be ascribed as being due to the equatorial atoms.

Decreased sensory receptors P2X3 and TRPV1 in suburothelial nerve fibers following intradetrusor injections of botulinum toxin for human detrusor overactivity.

PURPOSE: Botulinum neurotoxin type A (BoNT/A) is effective in the treatment of intractable detrusor overactivity (DO). In addition to its known inhibitory effect on presynaptic release of acetylcholine by motor terminals, there is increasing evidence that BoNT/A may affect sensory fibers. We investigated a possible effect of BoNT/A on human bladder afferent mechanisms by studying the sensory receptors P2X3 and TRPV1 in biopsies from patients with neurogenic or idiopathic DO. MATERIALS AND METHODS: A total of 38 patients (22 with neurogenic DO, 16 with idiopathic DO) with intractable DO were treated with intradetrusor BoNT/A, and bladder biopsies were taken at 4 and 16 weeks. Urodynamics and voiding diary were also recorded. Specimens were studied immunohistochemically for P2X3, TRPV1 and the pan-neuronal marker PGP9.5, in comparison with controls. RESULTS: P2X3-immunoreactive and TRPV1-immunoreactive (-IR) fibers were decreased at 4 weeks after BoNT/A, and more significantly at 16 weeks (paired t test p=0.0004 and p=0.0008, respectively), when significant improvements were observed in clinical and urodynamic parameters. P2X3-IR fiber decrease was significantly correlated with reduction of urgency episodes at 4 and 16 weeks (p=0.0013 at 4 weeks and p=0.02 at 16 weeks), but not maximum cystometric capacity or detrusor pressures. TRPV1-IR fiber decrease showed a similar trend. PGP9.5-IR suburothelial fibers remained unchanged after treatment at both followups (p=0.85 and p=0.21 at 4 and 16 weeks, respectively). Urothelial cell P2X3-IR and TRPV1-IR also appeared unchanged. CONCLUSIONS: Decreased levels of sensory receptors P2X3 and/or TRPV1 may contribute to the clinical effect of BoNT/A in detrusor overactivity.

Self-assembled periodical polycrystalline-ZnO/a-C nanolayers on Zn nanowire.

Zn nanowires with an epitaxial thin surface layer of zinc oxide were dispersed onto amorphous carbon films and stored at room temperature. After 1500 h, a self-organized equal-spaced zinc oxide (approximately 2 nm)/carbon (approximately 2.5 nm) multilayer structure was found to form outside the Zn nanowire, taking the place of the original ZnO surface layer. We carried a systematic study to clarify the self-formation mechanism of the periodical multilayers outside the Zn nanowire and found out that such a configuration originated from a chemical reaction between Zn and CO2 and were formed via a gas phase diffusion-interfacial chemical reaction-phase separation process.

[001] zone-axis bright-field diffraction contrast from coherent Ge(Si) islands on Si(001).

Coherent Ge(Si)/Si(001) quantum dot islands grown by solid source molecular beam epitaxy at a growth temperature of 700 degrees C were investigated using transmission electron microscopy working at 300kV. The [001] zone-axis bright-field diffraction contrast images of the islands show strong periodicity with the change of the TEM sample substrate thickness and the period is equal to the effective extinction distance of the transmitted beam. Simulated images based on finite element models of the displacement field and using multi-beam dynamical diffraction theory show a high degree of agreement. Studies for a range of electron energies show the power of the technique for investigating composition segregation in quantum dot islands.

Bladder and cutaneous sensory neurons of the rat express different functional P2X receptors.

The expression and functional responses of P2X receptors in bladder and cutaneous sensory neurons of adult rats and mice have been studied using immunohistochemistry and patch clamp techniques. Cell bodies of bladder pelvic afferents were identified in L6 and S1 dorsal root ganglia (DRG), following Fast Blue injection into the muscle wall of the urinary bladder. Similarly, cutaneous sensory neurons were identified in L3 and L4 DRG, following Fast Blue injection into the saphenous nerve innervating the skin. Bladder sensory neurons contained only weak to moderate P2X(3)-immunoreactivity (IR), in contrast to strong P2X(3)-IR observed in a sub-population of cutaneous afferents. Whole-cell patch-clamp recordings revealed that approximately 90% of bladder afferent neurons responded to alpha beta-methylene ATP (alpha beta meATP) and ATP (30 microM) with persistent currents, which were inhibited by 2',3'-O-trinitrophenyl-ATP (TNP-ATP) (0.3 microM) to 6.4+/-1.9% and 8.0+/-2.6% of control, respectively (n=8). The remaining bladder sensory neurons demonstrated biphasic, transient or no response to P2X agonists. In contrast, only 24% of cutaneous afferent neurons gave persistent currents to alpha beta meATP (30 microM), with 66% of cells giving transient or biphasic currents and the remaining 10% being non-responsive. Our results suggest that, in contrast to DRG neurons in general, bladder sensory neurons projecting via pelvic nerves express predominantly P2X(2/3) heteromeric receptors, which are likely to mediate the important roles of ATP as a signaling molecule of urinary bladder filling and nociception.

Electron diffraction from nanovolumes of amorphous material using coherent convergent illumination.

Using a conventional transmission electron microscope that incorporates a field emission gun it is possible to focus an electron beam to form a small probe (<1nm full-width at half-maximum). Such a probe can then be used to perform high spatial resolution diffraction experiments. The high spatial resolution allows technologically interesting amorphous volumes, such as those found in glassy intergranular phases or in semiconductor implantations, to be investigated directly. In order to achieve the probe characteristics necessary to investigate nanovolumes of material the probe must be highly convergent which results in it being highly coherent. In this paper we examine the effect of coherent convergent illumination on electron diffraction data taken from nanovolumes of amorphous material. It is shown that, for amorphous volumes as small as 1.2nm in diameter, the additional interference effects induced in the diffraction data by the use of coherent convergent illumination are largely suppressed by the lack of order in amorphous materials. This allows the use of deconvolution techniques, developed for the correction of broadening of the diffraction pattern in the case of incoherent illumination, and the subsequent application of reduced density function (G(r)) analysis, to also be used for coherent illumination.

Deconvolution of electron diffraction patterns of amorphous materials formed with convergent beam.

To perform reduced density function (G(r)) analysis on electron diffraction patterns of amorphous materials formed with convergent beams, the effects of convergence must be removed from the diffraction data. Assuming electrons incident upon the sample in different directions are incoherent, this can be done using deconvolution (Ultramicroscopy 76 (1999) 115). In this letter we show that the combination of an energy filtering transmission electron microscope with an image plate, increases the accuracy with which diffraction data can be measured and, subsequently, the accuracy of the deconvolution.

Limitations on the s-state approach to the interpretation of sub-angstrom resolution electron microscope images and microanalysis.

The s-state approach is useful for analysing transmission electron microscope images of a thin crystalline foil consisting of well-separated atomic columns. It assumes that the signal collected (e.g., the annular dark-field image, the EELS spectrum) can be attributed to only the lowest-energy bound eigenstate of the two-dimensional projected potential of a single column. When, however, columns are close, the form of the bound states depends on more than one column, which implies that interpretation of the signal may not be so simple. For closely spaced columns we show that the simple s-state approach fails for the case of a sub-Angstrom probe initially centred on one column of a pair, because two bound states are excited. The energy in the probe is almost completely transferred to the neighbouring column after it has propagated some tens of nanometres through the foil and then is transferred back. Signals which relate directly to the local probe intensity (e.g. annular dark-field formed by thermal diffuse scattering, EELS) must be analysed in terms of the two bound states. Accurate calculations of bound states of pairs of columns are more demanding than for a single column but sufficient accuracy can be achieved from knowledge of the 1s-states of isolated columns. We provide formulae for the bound states of a column pair. These can be used to determine if image analysis requires the extension to the s-state approach described in this paper.

Cyclic ADP-ribose production by CD38 regulates intracellular calcium release, extracellular calcium influx and chemotaxis in neutrophils and is required for bacterial clearance in vivo.

Cyclic ADP-ribose is believed to be an important calcium-mobilizing second messenger in invertebrate, mammalian and plant cells. CD38, the best-characterized mammalian ADP-ribosyl cyclase, is postulated to be an important source of cyclic ADP-ribose in vivo. Using CD38-deficient mice, we demonstrate that the loss of CD38 renders mice susceptible to bacterial infections due to an inability of CD38-deficient neutrophils to directionally migrate to the site of infection. Furthermore, we show that cyclic ADP-ribose can directly induce intracellular Ca++ release in neutrophils and is required for sustained extracellular Ca++ influx in neutrophils that have been stimulated by the bacterial chemoattractant, formyl-methionyl-leucyl-phenylalanine (fMLP). Finally, we demonstrate that neutrophil chemotaxis to fMLP is dependent on Ca++ mobilization mediated by cyclic ADP-ribose. Thus, CD38 controls neutrophil chemotaxis to bacterial chemoattractants through its production of cyclic ADP-ribose, and acts as a critical regulator of inflammation and innate immune responses.

P2X3 knock-out mice reveal a major sensory role for urothelially released ATP.

The present study explores the possible involvement of a purinergic mechanism in mechanosensory transduction in the bladder using P2X(3) receptor knock-out (P2X(3)-/-) and wild-type control (P2X(3)+/+) mice. Immunohistochemistry revealed abundant nerve fibers in a suburothelial plexus in the mouse bladder that are immunoreactive to anti-P2X(3). P2X(3)-positive staining was completely absent in the subepithelial plexus of the P2X(3)-/- mice, whereas staining for calcitonin gene-related peptide and vanilloid receptor 1 receptors remained. Using a novel superfused mouse bladder-pelvic nerve preparation, we detected a release of ATP proportional to the extent of bladder distension in both P2X(3)+/+ and P2X(3)-/- mice, although P2X(3)-/- bladder had an increased capacity compared with that of the P2X(3)+/+ bladder. The activity of multifiber pelvic nerve afferents increased progressively during gradual bladder distension (at a rate of 0.1 ml/min). However, the bladder afferents from P2X(3)-/- mice showed an attenuated response to bladder distension. Mouse bladder afferents of P2X(3)+/+, but not P2X(3)-/-, were rapidly activated by intravesical injections of P2X agonists (ATP or alpha,beta-methylene ATP) and subsequently showed an augmented response to bladder distension. By contrast, P2X antagonists [2',3'-O-(2,4,6-trinitrophenyl)-ATP and pyridoxal 5-phosphate 6-azophenyl-2',4'-disulfonic acid] and capsaicin attenuated distension-induced discharges in bladder afferents. These data strongly suggest a major sensory role for urothelially released ATP acting via P2X(3) receptors on a subpopulation of pelvic afferent fibers.

Signals that initiate somatic hypermutation of B cells in vitro.

Somatic hypermutation is initiated as B lymphocytes proliferate in germinal centers. The signals that switch on the mutation process are unknown. We have derived an in vitro system to define signals that will initiate mutation in normal, naive splenic B cells. We find that three signals are required to allow detection of somatic mutation in vitro; these are anti-Ig, anti-CD40, and anti-CD38. If any one of these is omitted, mutation remains off. We show that CD40 is obligatory in vivo, as CD40 knockout mice exhibit no Ag-driven mutation. In contrast, CD38 is not, as CD38 knockout mice mutate normally. We believe that, in vitro, CD38, in combination with other stimuli, drives extensive cell division, allowing the detection of mutated sequences. However, in germinal centers in vivo, proliferative activity is instigated by a different molecule. This is the first demonstration of the initiation of hypermutation in vitro with normal splenic B cells using defined stimuli.

Changes in P2X receptor responses of sensory neurons from P2X3-deficient mice.

Dorsal root ganglion (DRG) neurons respond to ATP with transient, persistent or biphasic inward currents. In contrast, the ATP responses in nodose neurons are persistent. These sustained currents are also heterogeneous, with one component being accounted for by P2X2/3 receptors, and the residual response probably mediated by P2X2 receptors, although the direct evidence for this has been lacking. In the present study, we examined the P2X receptors on DRG and nodose neurons from P2X3-deficient (P2X3-/-) mice, using whole cell voltage-clamp recording and immunohistochemistry. We found that all P2X3-/- DRG neurons lacked rapidly desensitizing response to ATP, and both DRG and nodose neurons from P2X3-null mutant mice no longer responded to alpha,beta-methylene ATP (alphabetameATP). In contrast, ATP evoked persistent inward current in 12% of DRG neurons and 84% of nodose neurons from P2X3-/- mice. This retained persistent response to ATP on nodose neurons had an EC50 for ATP of 77 microm, was antagonized by Cibacron blue and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid, potentiated by Zn2+ and acidification, but not enhanced by ivermectin or diinosine pentaphosphate. 2',3'-O-Trinitrophenyl-ATP antagonized this response with an IC50 of 8 microm. All these properties are consistent with those of recombinant P2X2 homomeric receptors. Furthermore, specific P2X2 receptor immunoreactivity detected in wild-type sensory neurons was unaltered in null mutant mice. Therefore, the alphabetameATP-insensitive persistent responses on nodose neurons are likely to be mediated by P2X2 homomers, which contribute to 60% of currents evoked by 100 microm ATP in the wild type.

Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice.

Extracellular ATP is implicated in numerous sensory processes ranging from the response to pain to the regulation of motility in visceral organs. The ATP receptor P2X3 is selectively expressed on small diameter sensory neurons, supporting this hypothesis. Here we show that mice deficient in P2X3 lose the rapidly desensitizing ATP-induced currents in dorsal root ganglion neurons. P2X3 deficiency also causes a reduction in the sustained ATP-induced currents in nodose ganglion neurons. P2X3-null mice have reduced pain-related behaviour in response to injection of ATP and formalin. Significantly, P2X3-null mice exhibit a marked urinary bladder hyporeflexia, characterized by decreased voiding frequency and increased bladder capacity, but normal bladder pressures. Immunohistochemical studies localize P2X3 to nerve fibres innervating the urinary bladder of wild-type mice, and show that loss of P2X3 does not alter sensory neuron innervation density. Thus, P2X3 is critical for peripheral pain responses and afferent pathways controlling urinary bladder volume reflexes. Antagonists to P2X3 may therefore have therapeutic potential in the treatment of disorders of urine storage and voiding such as overactive bladder.

Mice deficient for the ecto-nicotinamide adenine dinucleotide glycohydrolase CD38 exhibit altered humoral immune responses.

CD38 is a membrane-associated ecto-nicotinamide adenine dinucleotide (NAD+) glycohydrolase that is expressed on multiple hematopoietic cells. The extracellular domain of CD38 can mediate the catalysis of NAD+ to cyclic adenosine diphosphoribose (cADPR), a Ca2+-mobilizing second messenger, adenosine diphosphoribose (ADPR), and nicotinamide. In addition to its enzymatic properties, murine CD38 has been shown to act as a B-cell coreceptor capable of modulating signals through the B-cell antigen receptor. To investigate the in vivo physiological function(s) of this novel class of ectoenzyme we generated mice carrying a null mutation in the CD38 gene. CD38-/- mice showed a complete loss of tissue-associated NAD+ glycohydrolase activity, showing that the classical NAD+ glycohydrolases and CD38 are likely identical. Although murine CD38 is expressed on hematopoietic stem cells as well as on committed progenitors, we show that CD38 is not required for hematopoiesis or lymphopoiesis. However, CD38-/- mice did exhibit marked deficiencies in antibody responses to T-cell-dependent protein antigens and augmented antibody responses to at least one T-cell-independent type 2 polysaccharide antigen. These data suggest that CD38 may play an important role in vivo in regulating humoral immune responses.

CD38: a new paradigm in lymphocyte activation and signal transduction.

CD38 is a type II transmembrane glycoprotein that is extensively expressed on cells of hematopoietic and non-hematopoietic lineage. Although the intracellular domain of CD38 is not homologous to any known proteins, the extracellular domain of CD38 is structurally related to enzymes in the ADP-ribosyl cyclase family. The structural homology between CD38 and the cyclase family members extends to functional homology, as the extracellular domain of CD38 can mediate the catalysis of beta-NAD+ into nicotinamide, ADP-ribose (ADPR) and, to a lesser extent, into cyclic ADPR-ribose (cADPR). Extensive investigation in other systems has shown that cADPR is an important regulator of intracellular Ca2+ release. Since engagement of CD38 on hematopoietic cells with anti-CD38 Abs has been shown to have potent effects on a number of in vitro cellular responses, we have speculated that cADPR might control CD38-mediated signal transduction. However, it has been difficult to understand how a mediator which is typically an intracellular signaling molecule could potentiate its effects from an extracellular location, thus posing a dilemma which pertains to all ecto-enzymes and the mechanisms by which they regulate signal transduction and cellular processes. This review describes the biologic properties of murine CD38, its role in humoral immunity, and its signal transduction properties in B lymphocytes. We suggest that signaling through CD38 represents a new paradigm in lymphocyte signal transduction and is predicated upon extracellular, rather than intracellular, crosstalk.

IL-13 protects mice from lipopolysaccharide-induced lethal endotoxemia: correlation with down-modulation of TNF-alpha, IFN-gamma, and IL-12 production.

IL-13 is a potent down-modulator of macrophage proinflammatory activity in vitro, similar in this context to the anti-inflammatory cytokines IL-4 and IL-10. Since IL-10 effectively confers protection to mice from LPS-induced lethal endotoxemia through inhibition of proinflammatory cytokine production, we investigated whether IL-13 may also be capable of providing protection in this experimental model of endotoxic shock. A single injection of recombinant murine IL-13 (rmIL-13; 0.5-10 microg) significantly increased survival in a dose-dependent manner when a lethal i.p. injection of endotoxin was administered to BALB/c mice. This effect appeared to be IL-13 specific, since survival was not affected in mice that received heat-inactivated rmIL-13. rmIL-13 provided significant protection to mice even when given 30 min after LPS injection; however, this protection decreased in a time-dependent manner as the administration of rmIL-13 was delayed by 1, 2, and 5 h following LPS injection. The protective effect of IL-13 was correlated with significant decreases in the production of the inflammatory mediators TNF-alpha, IFN-gamma, and IL-12 as well as a decrease in the anti-inflammatory mediator IL-10. Our data suggest that IL-13 provides protection from LPS-induced lethal endotoxemia in a manner that is similar to but independent from that of IL-10, and therefore can be added to the list of cytokine immunomodulators that might be beneficial in the treatment of septic shock.

X-ray microanalysis of cell elements in normal and cystic fibrosis jejunum: evidence for chloride secretion in villi.

BACKGROUND & AIMS: Cystic fibrosis transmembrane conductance regulator (CFTR) is an adenosine 3',5'-cyclic monophosphate-dependent chloride channel that is defective in cystic fibrosis. The aims of this study were to determine if defective apical chloride secretion in the intestine of patients with cystic fibrosis alters the intracellular electrolyte milieu and to examine the geographical localization of CFTR in the normal intestine. METHODS: The content of intracellular elements was assessed in cryosections using energy-dispersive x-ray microanalysis, and CFTR was identified by immunocytochemistry using commercially available antibodies. RESULTS: Cystic fibrosis jejunum had a significantly lower Na+ content, higher K+ and Cl- content, and higher potassium/phosphorus ratio in both villus and crypt regions. Incubation of normal jejunum with the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (300 mumol/L) resulted in decreased K+ and Cl- content in both crypt and villus regions, indicative of Cl- secretion. CFTR was identified on the surface of normal villus and crypt enterocytes but not in cystic fibrosis samples. CONCLUSIONS: Defective apical chloride channels in cystic fibrosis result in alterations in the intracellular electrolyte milieu. The microanalysis observations and immunocytochemical studies imply a role for villus enterocytes in human intestinal chloride secretion.