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Calcium ion concentration ([Ca(2+)]) in the systemic extracellular fluid, ECF-[Ca(2+)], is maintained around a genetically predetermined set-point, which combines the operational level of the kidney/ and bone/ECF interfaces. The ECF-[Ca(2+)] is maintained within a narrow oscillation range by the regulatory action of Parathyroid Hormone (PTH), Calcitonin, FGF-23, and 1,25(OH)(2)D(3). This model implies two correction mechanisms, i.e. tubular Ca(2+) reabsorption and osteoclast Ca(2+) resorption. Although their alterations have an effect on the ECF-[Ca(2+)] maintenance, they cannot fully account for rapid correction of the continuing perturbations of plasma [Ca(2+)], which occur daily in life. The existence of Ca(2+) fluxes at quiescent bone surfaces fulfills the role of a short-term error correction mechanism in Ca(2+) homeostasis. To explore the hypothesis that PTH regulates the cell system responsible for the fast Ca(2+) fluxes at the bone/ECF interface, we have performed direct real-time measurements of Ca(2+) fluxes at the surface of ex-vivo metatarsal bones maintained in physiological conditions mimicking ECF, and exposed to PTH. To further characterize whether the PTH receptor on osteocytes is a critical component of the minute-to-minute ECF-[Ca(2+)] regulation, metatarsal bones from mice lacking the PTH receptor in these cells were tested ex vivo for rapid Ca(2+) exchange. We performed direct real-time measurements of Ca(2+) fluxes and concentration gradients by a scanning ion-selective electrode technique (SIET). To validate ex vivo measurements, we also evaluated acute calcemic response to PTH in vivo in mice lacking PTH receptors in osteocytes vs littermate controls. Our data demonstrated that Ca(2+) fluxes at the bone-ECF interface in excised bones as well as acute calcemic response in the short-term were unaffected by PTH exposure and its signaling through its receptor in osteocytes. Rapid minute-to-minute regulation of the ECF-[Ca(2+)] was found to be independent of PTH actions on osteocytes. Similarly, mice lacking PTH receptor in osteocytes, responded to PTH challenge with similar calcemic increases.

Causal factors leading to epizootic shell disease (ESD) lesions in American lobster, Homarus americanus H. Milne-Edwards, 1837, are not well understood. We explore the structural and physiological bases for development of ESD from preclinical stages invisible to unaided eye to early visible stages. We present a lobster shell model, which develops structural functional vulnerability and suggests plausible routes to ESD. Medallions of carapace cuticle were obtained from carapace fixed with protocols to minimize movement of mineral and macromolecular components. Rapid processing of medallions was used to encourage large sample sizes compatible with environmental surveys. One- and two-dimensional analytic maps of polished sections of the cuticle, obtained with an electron microprobe, described the composite mineral and polymeric structures. Micro-Raman spectroscopy was used to identify bond properties of phosphates and carbonates, as well as signatures of organic structures. The frequency and properties of structures identified can be monitored through the lobster molting cycle using a high throughput application of micro-computed tomography (μCT). We observed density differences in the calcite layer, exocuticle, and endocuticle, and the frequency and structure of CaCO3 structures in the endocuticle and membranous layer of carapace cuticle during chosen stages of the molting cycle. The correlative microscopy and μCT of shell structures provides improved understanding of the lobster cuticle structure. Detailed structural differences quantified through development and under different environmental conditions can provide insight into causes and vulnerabilities associated with ESD.

A total inventory of density defined objects of American lobster cuticle was obtained by using high resolution 3D Xray tomography, micro-computed-tomography. Through this relatively unbiased sampling approach several new objects were discovered in intermolt cuticle of the lobster carapace. Using free and open-source software the outlines of density-defined objects were obtained and their locations in 3D space calculated allowing population parameters about these objects to be determined. Nearest neighbor distances between objects allowed interpretations of structural relationships of and between objects. Several organule types are recognized by their structural outlines and density signatures. A hierarchy of organule types and distribution suggests they develop during sequential molts. New objects (stalactites, Bouligand spirals, and basal granules) are described as mineral structures with well defined morphological character, allowing them to be recognized by their descriptive names and distributions. The three new cuticular objects appear to represent forms of calcium carbonate deposition in the sequential layers of exocuticle, endocuticle and membranous layer and may provide signatures of underlying epidermal cells.

The elasmoid scales of fish represent a significant internal reservoir of calcium ions (Ca2+), but little is known about the contribution of these scales to the short-term regulation of Ca2+ homeostasis in the extracellular fluid (ECF). This gap in our knowledge is partly due to the technical challenges involved in measuring small Ca2+ fluxes around the scales of live fish in real time. Here, we describe a technique for exfoliating, mounting, and culturing intact living zebrafish Danio rerio scales, then subjecting them to examination using an extracellular, non-invasive, surface-scanning ion-selective electrode technique (SIET). In a Ca2+-sensitive configuration, the SIET can resolve Ca2+ flux values in the low-to-sub picomole/square centimeter/second range, with a spatial resolution of approximately 5 μm. We quantified the Ca2+ fluxes into and out of scales under different extracellular calcemic challenges set to mimic a variety of Ca2+ concentrations in the ECF and showed that the results were similar to those previously reported from isolated mouse metatarsal bone. Our new data extend our current understanding of the role played by fish scales in the short-term homeostatic regulation of Ca2+ concentration in the ECF. They also support the suggestion that scales might provide an inexpensive and complementary model for studying the fundamentals of bone-mediated homeostatic Ca2+ regulation and the diseases that result from its dysregulation.

An insect oocyte with a disc-shaped sink of current on its ventral surface during a vitellogenic phase of development is used to demonstrate modeling of expected currents arising from a mathematically defined source. A simple model with an analytical solution for the expected currents is compared to a more complex model in which expectations are computed by mathematical integration. The currents about two oocytes of different pattern type are interpreted in light of the two models.

Legume plants secrete signaling molecules called flavonoids into the rhizosphere. These molecules activate the transcription of rhizobial nod genes, which encode proteins involved in the synthesis of signaling compounds named Nod factors (NFs). NFs, in turn, trigger changes in plant gene expression, cortical cell dedifferentiation and mitosis, depolarization of the root hair cell membrane potential and rearrangement of the actin cytoskeleton. Actin polymerization plays an important role in apical growth in hyphae and pollen tubes. Using sublethal concentrations of fluorescently labeled cytochalasin D (Cyt-Fl), we visualized the distribution of filamentous actin (F-actin) plus ends in living Phaseolus vulgaris and Arabidopsis root hairs during apical growth. We demonstrated that Cyt-Fl specifically labeled the newly available plus ends of actin microfilaments, which probably represent sites of polymerization. The addition of unlabeled competing cytochalasin reduced the signal, suggesting that the labeled and unlabeled forms of the drug bind to the same site on F-actin. Exposure to Rhizobium etli NFs resulted in a rapid increase in the number of F-actin plus ends in P. vulgaris root hairs and in the re-localization of F-actin plus ends to infection thread initiation sites. These data suggest that NFs promote the formation of F-actin plus ends, which results in actin cytoskeleton rearrangements that facilitate infection thread formation.

Bottom-up modeling of American lobster (Homarus americanus) cuticle explains architecture and function ab initio, from first principles, starting with synthesis of component polymers and progressively building composite structure that should explain observed properties. A top-down perspective decomposes the lobster cuticle starting at the top level of structural complexity and function aiming to descend to the finest detail. Both approaches aim to ultimately model the same cuticle structure. Current bottom-up models of the cuticle do not succeed in explaining key structural and functional detail identified by top-down approaches. Top-down identified structures and associated functions are valuable as bases for potential vulnerabilities to microbial attack. An immediate objective is to inform the bottom-up approach of top-down identified model components critical to cuticle function. Top-down features include detail of protein expression and mineral heterogeneity and their function in observed structures. This function-directed approach provides a better understanding of the distribution and roles of minerals in relation to their immediate cuticle environment. The top-down identified features can hopefully be included in ab initio models to improve our understanding of cuticle design.

This work describes the fabrication and characterization of a Niobium on polyimide flex cable suitable for sub-Kelvin temperatures. The processing used can be extended to high density interconnects and allows for direct integration with printed circuit boards. Several key parameters such as RRR, T c , current carrying capability at 4 K and thermal conductivity in the range from 0.15 to 10 K have been measured. The average T c was found to be 8.9 K, with a minimum of 8.3 K. Several samples allowed for more than 50 mA current at 4 K while remaining in the superconducting state. The thermal conductivity for this flex design is dominated by the polyimide, in our case Pyralin PI-2611, 2 and is in good agreement with published thermal conductivity data for a polyimide called Upilex R. 3

Various carbonate apatite formulae contribute to discrete cuticle structures participating in protective functions of the American lobster, Homarus americanus, integument. Canal walls use their lowest Calcium : Phosphate (Ca : P) ratios to protect exposed surfaces of gland and neuronal canals. The linings insulate more soluble calcium carbonate from attack by acid secreting micro-organisms. A trabecular bone-like structure, called here ‘trabeculae’ in analogy to vertebrates, utilizes high Ca : P in the inner exocuticle demonstrating an efficient use of environmentally scarce phosphate and provides the hardness layer of the cuticle that protects the superficial calcite layer from flexure. Strength is derived from phosphatic trabeculae being embedded in a phenolicly hardened inner exocuticle layer. A third location and use of carbonate apatite is at the interface of the calcite and inner exocuticle where it may cap calcite layer development. A fourth phosphatic localization is seen in cuticular nip...

A major role of lobster integument is protection from microbes. Calcite and amorphous calcium carbonate are the most abundant and most acid vulnerable of the cuticle minerals. We propose that calcite is invested in neutralizing an acidifying environment modulated by the epicuticle. A minor cuticle component is carbonate apatite (CAP), proposed to play critical roles in the integument's structural protective function. The CAP of lobster exhibits a flexible composition; its least soluble forms line the cuticular canals most exposed to the environment. A trabecular CAP structure illustrates efficient use of a sparse phosphate resource, cooperating in the hardness of the inner exocuticle. A schematic model of the cuticle emphasizes structural and chemical diversity. A thin outer calcite layer provides a dense microbial barrier that dissolves slowly through the epicuticle, providing an external, alkaline, unstirred layer that would be inhibitory to bacterial movement and metabolism. Injury to the epic...

3D-stacking of Ultra Thin Chip Packages (UTCP’s) – one of the emerging technologies in the field of high density integration - is presented here. This technology is developed to increase the functionality of the electronic devices and to raise the comfort zone by reducing the overall size and weight of the package. This has a bright future in the area of mobile communication, medical equipments like hearing aids, implants, patient monitoring. This paper describes an approach to produce miniaturized modular packages by using Ultrathin Chip Package Technology, stacking and through-hole (TH) interconnection technology. UTCP technology is a board level packaging concept, based on embedding of ultra-thin chips (∼20 μm) within two 20 μm thick spin-on polyimide layers resulting in a thin flexible chip package of thickness ∼50 μm. Stacking of the 4 layers of UTCPs by using 25 μm thick layers of adhesive films and vacuum lamination processes results in stacked UTCP modules of total thickness ∼300 μm. The connection to the different layers of the UTCPs is realized by drilling THs on the outer contact pads on the stacked packages, followed by metallization of these THs by Cu electroless deposition, electroplating and finally metal structuring. After processing and testing, the stacks can be easily mounted within functional demonstrators (e.g, Hearing Aid device, etc) replacing single silicon die of almost same size, but with 4 times output.

Uridine-cytidine kinase (UCK) 2, an enzyme normally expressed in human placenta and testis and highly overexpressed in many neoplasias of blood and solid tissues, catalyzes monophosphorylation of pyrimidine ribonucleosides with efficiency 15- to 20-fold higher than that of ubiquitously expressed isozyme UCK1. In this paper, we report the synthesis of 3'-(E)-(2-iodovinyl)uridine (IV-14) and its preclinical evaluation as a new radiotracer derived from a UCK2-selective antitumor agent, 3'-(ethynyl)uridine.Radioiodinated IV-14 was prepared from the respective stannyl precursor. (131)I-IV-14 was studied in cellular uptake assays and tested for stability in serum as well as for stability to thymidine phosphorylase, liver-, and mucosa-specific murine uridine phosphorylases. UCK1 and UCK2 expression levels in different tumor cell lines were determined by Western blot. Cellular distribution of (131)I-IV-14 was determined in HL60 cells. Biodistribution studies and gamma-camera scintigraphy were performed on an HL60-xenografted severe combined immunodeficiency (SCID) mouse model.(131)I-IV-14 demonstrated excellent stability in serum. It was stable to human thymidine phosphorylase and to liver- and mucosa-specific murine uridine phosphorylases. Cellular uptake after 24 h of incubation with (131)I-IV-14 was 4.27 +/- 0.21, 3.66 +/- 0.13, 2.69 +/- 0.07, 2.24 +/- 0.18, and 3.26 +/- 0.18 percentage injected dose per 5 x 10(5) Mia-PaCa-2, CX-1, HL60, Capan-1, and Panc-1 cells, respectively. Uptake and retention of IV-14 were regulated by 2 factors: UCK2 expression level and intracellular transport mediated partially via human equilibrating nucleoside transporter 1. A biodistribution study of (131)I-IV-14 in an HL60-xenografted SCID mouse model showed that at 4 h after injection the greatest amount of retained radioactivity was in tumor. The tissue-to-tumor ratio 4 h after injection was 1.0 +/- 0.24 for tumor, 0.40 +/- 0.18 for spleen, 0.25 +/- 0.12 for colon, 0.14 +/- 0.07 for small intestine, and less than 0.1 for other sites. Scintigraphy with (123)I-IV-14 4 h after injection showed the tumor well. In addition, high accumulation of radioiodide in the stomach content was observed and was presumably due to metabolic degradation of IV-14.IV-14 is a UCK2-specific marker, allowing for in vivo addressing of tumors with high RNA synthesis independent of proliferation rate.

We examined exocytosis during oscillatory growth in lily (Lilium formosanum and Lilium longiflorum) and tobacco (Nicotiana tabacum) pollen tubes using three markers: (1) changes in cell wall thickness by Nomarski differential interference contrast (DIC), (2) changes in apical cell wall fluorescence in cells stained with propidium iodide (PI), and (3) changes in apical wall fluorescence in cells expressing tobacco pectin methyl esterase fused to green fluorescent protein (PME-GFP). Using PI fluorescence, we quantified oscillatory changes in the amount of wall material from both lily and tobacco pollen tubes. Measurement of wall thickness by DIC was only possible with lily due to limitations of microscope resolution. PME-GFP, a direct marker for exocytosis, only provides information in tobacco because its expression in lily causes growth inhibition and cell death. We show that exocytosis in pollen tubes oscillates and leads the increase in growth rate; the mean phase difference between exocytosis and growth is –98° ± 3° in lily and –124° ± 4° in tobacco. Statistical analyses reveal that the anticipatory increase in wall material predicts, to a high degree, the rate and extent of the subsequent growth surge. Exocytosis emerges as a prime candidate for the initiation and regulation of oscillatory pollen tube growth.

Purpose: Auger electron emitting radiopharmaceuticals are attractive for targeted nanoirradiation therapy, provided that DNA of malignant cells is selectively addressed. Here, we examine 5-[123/125/131I]iodo-4′-thio-2′-deoxyuridine (ITdU) for targeting DNA in tumor cells in a HL60 xenograft severe combined immunodeficient mouse model. Experimental Design: Thymidine kinase and phosphorylase assays were done to determine phosphorylation and glycosidic bond cleavage of ITdU, respectively. The biodistribution and DNA incorporation of ITdU were determined in severe combined immunodeficient mice bearing HL60 xenografts receiving pretreatment with 5-fluoro-2′-deoxyuridine (FdUrd). Organ tissues were dissected 0.5, 4, and 24 h after radioinjection and uptake of [131I]ITdU (%ID/g tissue) was determined. Cellular distribution of [125I]ITdU was imaged by microautoradiography. Apoptosis and expression of the proliferation marker Ki-67 were determined by immunohistologic staining using corresponding paraffin tissue sections. Results: ITdU is phosphorylated by thymidine kinase 1 and stable toward thymidylate phosphatase-mediated glycosidic bond cleavage. Thymidylate synthase-mediated deiodination of [123/125/131I]ITdU was inhibited with FdUrd. Pretreatment with FdUrd increased preferentially tumor uptake of ITdU resulting in favorable tumor-to-normal tissue ratios and tumor selectivity. ITdU was exclusively localized within the nucleus and incorporated into DNA. In FdUrd-pretreated animals, we found in more than 90% of tumor cells apoptosis induction 24 h postinjection of ITdU, indicating a highly radiotoxic effect in tumor cells but not in cells of major proliferating tissues. Conclusion: ITdU preferentially targets DNA in proliferating tumor cells and leads to apoptosis provided that the thymidylate synthase is inhibited.

Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). In this study, we evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Stable transfectants of COS-1 cells expressing FcgammaRIIA or both FcgammaRIIA/hCERK expression vectors were created. Cell fractionation studies demonstrated that hCERK and the transient receptor potential channel (TRPC-1) were enriched in caveolae fractions. Our data establish that both CERK and TRPC-1 localize to the caveolar microdomains during phagocytosis and that CERK also colocalizes with EIgG in FcgammaRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcgammaRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcgammaRIIA under identical conditions displayed little periphagosomal Ca2+ signaling. The enhanced Ca2+ signals were accompanied by enhanced phagolysosome formation. However, the addition of pharmacological reagents that inhibit store-operated channels (SOCs) reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. The higher Ca2+ signal observed in hCERK transfected cells as well as the fact that CERK colocalized with EIgG during phagocytosis support our hypothesis that Ca2+ signaling is an important factor for increasing phagocytosis and is regulated by CERK in a manner that involves SOCs/TRPCs.

Because of environmental concerns CFC-containing pressurised metered dose inhalers (pMDI) had to be replaced by dry powder inhalers (DPI). The Novolizer, a novel DPI has previously been shown to be as effective as the Turbuhaler in delivering budesonide. The objective of this study was to show non-inferiority of inhaled formoterol therapy delivered through the Novolizer compared to formoterol delivered through the Aerolizer in patients suffering from moderate to severe asthma.In this double-blind, double-dummy, multicentre study 392 patients were randomised and received a dose of 12 microg formoterol twice daily for 4 weeks either through the Aerolizer or the Novolizer. FEV1 after 4 weeks of treatment was the primary variable. Secondary variables were FVC, PEF, consumption of short-acting; 2 adrenoceptor agonists, asthma symptoms, tolerability and safety.After 4 weeks of treatment, the mean trough FEV1 (95% CI) was 2.34 L (2.24-2.45) for the Novolizer and 2.31 L (2.21-2.41) for the Aerolizer. Non-inferiority was proven (p0.0001, pre-defined; of 0.25 L). All secondary variables (incl. PEF) confirmed these findings. Treatment with both devices was safe and well tolerated.Inhalation of 12 microg formoterol twice daily via Novolizer was shown to be equally therapeutically effective compared to the inhalation via Aerolizer in the treatment of moderate to severe persistent asthma. Treatment via both inhalers was safe and well tolerated.

We have examined the arrangement and movement of three major compartments, the endoplasmic reticulum (ER), mitochondria, and the vacuole during oscillatory, polarized growth in lily pollen tubes. These movements are dependent on the actin cytoskeleton, because they are strongly perturbed by the anti-microfilament drug, latrunculin-B, and unaffected by the anti-microtubule agent, oryzalin. The ER, which has been labeled with mGFP5-HDEL or cytochalasin D tetramethylrhodamine, displays an oscillatory motion in the pollen tube apex. First it moves apically in the cortical region, presumably along the cortical actin fringe, and then periodically folds inward creating a platform that transects the apical domain in a plate-like structure. Finally, the ER reverses its direction and moves basipetally through the central core of the pollen tube. When subjected to cross-correlation analysis, the formation of the platform precedes maximal growth rates by an average of 3 s (35-40 degrees ). Mitochondria, labeled with Mitotracker Green, are enriched in the subapical region, and their movement closely resembles that of the ER. The vacuole, labeled with carboxy-dichlorofluorescein diacetate, consists of thin tubules arranged longitudinally in a reticulate network, which undergoes active motion. In contrast to the mitochondria and ER, the vacuole is located back from the apex, and never extends into the apical clear zone. We have not been able to decipher an oscillatory pattern in vacuole motion. Because this motion is dependent on actin and not tubulin, we think this is due to a different myosin from that which drives the ER and mitochondria.

The location and changes in NAD(P)H have been monitored during oscillatory growth in pollen tubes of lily (Lilium formosanum) using the endogenous fluorescence of the reduced coenzyme (excitation, 360 nm; emission,400 nm). The strongest signal resides 20 to 40 microm behind the apex where mitochondria (stained with Mitotracker Green) accumulate. Measurements at 3-s intervals reveal that NAD(P)H-dependent fluorescence oscillates during oscillatory growth. Cross-correlation analysis indicates that the peaks follow growth maxima by 7 to 11 s or 77 degrees to 116 degrees, whereas the troughs anticipate growth maxima by 5 to 10 s or 54 degrees to 107 degrees. We have focused on the troughs because they anticipate growth and are as strongly correlated with growth as the peaks. Analysis of the signal in 10-microm increments along the length of the tube indicates that the troughs are most advanced in the extreme apex. However, this signal moves basipetally as a wave, being in phase with growth rate oscillations at 50 to 60 microm from the apex. We suggest that the changes in fluorescence are due to an oscillation between the reduced (peaks) and oxidized (troughs) states of the coenzyme and that an increase in the oxidized state [NAD(P)(+)] may be coupled to the synthesis of ATP. We also show that diphenyleneiodonium, an inhibitor of NAD(P)H dehydrogenases, causes an increase in fluorescence and a decrease in tube growth. Finally, staining with 5-(and-6)-chloromethyl-2',7'-dichlorohydrofluorescein acetate indicates that reactive oxygen species are most abundant in the region where mitochondria accumulate and where NAD(P)H fluorescence is maximal.

Lily (Lilium formosanum or Lilium longiflorum) pollen tubes, microinjected with a low concentration of the pH-sensitive dye bis-carboxyethyl carboxyfluorescein dextran, show oscillating pH changes in their apical domain relative to growth. An increase in pH in the apex precedes the fastest growth velocities, whereas a decline follows growth, suggesting a possible relationship between alkalinity and cell extension. A target for pH may be the actin cytoskeleton, because the apical cortical actin fringe resides in the same region as the alkaline band in lily pollen tubes and elongation requires actin polymerization. A pH-sensitive actin binding protein, actin-depolymerizing factor (ADF), together with actin-interacting protein (AIP) localize to the cortical actin fringe region. Modifying intracellular pH leads to reorganization of the actin cytoskeleton, especially in the apical domain. Acidification causes actin filament destabilization and inhibits growth by 80%. Upon complete growth inhibition, the actin fringe is the first actin cytoskeleton component to disappear. We propose that during normal growth, the pH increase in the alkaline band stimulates the fragmenting activity of ADF/AIP, which in turn generates more sites for actin polymerization. Increased actin polymerization supports faster growth rates and a proton influx, which inactivates ADF/AIP, decreases actin polymerization, and retards growth. As pH stabilizes and increases, the activity of ADF/AIP again increases, repeating the cycle of events.

Given the importance of the actin cytoskeleton to pollen tube growth, we have attempted to decipher its structure, organization and dynamic changes in living, growing pollen tubes of Nicotiana tabacum and Lilium formosanum, using three different GFP-labeled actin-binding domains. Because the intricate structure of the actin cytoskeleton in rapidly frozen pollen tubes was recently resolved, we now have a clear standard against which to compare the quality of labeling produced by these GFP-labeled probes. While GFP-talin, GFP-ADF and GFP-fimbrin show various aspects of the actin cytoskeleton structure, each marker produces a characteristic pattern of labeling, and none reveals the entire spectrum of actin. Whereas GFP-ADF, and to a lesser extent GFP-talin, label the fringe of actin in the apex, no similar structure is observed with GFP-fimbrin. Further, GFP-ADF only occasionally labels actin cables in the shank of the pollen tube, whereas GFP-fimbrin labels an abundance of fine filaments in this region, and GFP-talin bundles actin into a central cable in the core of the pollen tube surrounded by a few finer elements. High levels of expression of GFP-talin and GFP-fimbrin frequently cause structural rearrangements of the actin cytoskeleton of pollen tubes, and inhibit tip growth in a dose dependent manner. Most notably, GFP-talin results in thick cortical hoops of actin, transverse to the axis of growth, and GFP-fimbrin causes actin filaments to aggregate. Aberrations are seldom seen in pollen tubes expressing GFP-ADF. Although these markers are valuable tools to study the structure of the actin cytoskeleton of growing pollen tubes, given their ability to cause aberrations and to block pollen tube growth, we urge caution in their use.

The mammalian kidney excretes its metabolic acid load through the proton-transporting cells, intercalated cells, in the distal nephron and collecting duct. Fish excrete acid through external organs, gill, or skin; however, the cellular function is still controversial. In this study, molecular and electrophysiological approaches were used to identify a novel cell type secreting acid in skin of zebrafish ( Danio rerio) larvae. Among keratinocytes covering the larval surface, novel proton-secreting ionocytes, proton pump (H+-ATPase)-rich cells, were identified to generate strong outward H+flux. The present work demonstrates for the first time, with a noninvasive technique, H+-secreting cells in an intact animal model, the zebrafish, showing it to be a suitable model in which to study the functions of vertebrate transporting epithelia in vivo.

Multivariate analysis of the morphological variation in six populations of the Rice Stem Borer, Chilo suppressalis, was performed using 15 and 10 landmarks selected from the fore- and hindwings respectively. The raw planar coordinate data were aligned using geometric and mathematical calculations in Kendall’s shape space. Multivariate analysis was performed following transfer of the data to a linear Euclidean space, i.e. tangent space. 311 and 319 images of fore- and hindwings respectively were made using their geometric morphometric characters (26 in the forewings and 16 characters in the hindwings). The analysis showed a significant difference between the sexes and between the populations of Guilan and Mazandran provinces. A direct correlation was observed between morphological and geographic distance.

Background Recent studies have shown that the neurotrophins NGF and BDNF are produced by eosinophils. The influence of neurotrophins in allergic diseases including asthma has been described. The regulation by pharmacological substance remains unclear. Objectives The aim of this study was to assess whether approved pharmacological substances in the treatment of asthma such as corticosteroids or theophylline regulate neurotrophins on a cellular level. Methods Eosinophils were purified by negative immunoselection from allergics and non-allergic donors. Eosinophils were incubated with dexamethasone and theophylline and supernatants were collected for measurement of neurotrophic factors. The content of neurotrophins in eosinophil lysates was determined by ELISA. Regulation of stored NGF and BDNF was demonstrated by Western-blotting and flow cytometry while influence on transcription level was demonstrated by RT-PCR. Results Eosinophils produce and release the neurotrophins NGF and BDNF at different levels in allergics and non-allergics. Dexamethason lead to a significant downregulation of NGF in eosinophils of allergics. The levels of BDNF were not significantly reduced. Theophylline did not influence the levels of NGF nor BDNF significantly. Conclusions The production of the neurotrophin NGF was downregulated by an established substance such as dexamethasone. This might further contribute to the pharmacological potential of corticosteroids in allergic asthma.

We report on the unique effects and benefits of autologous stem cell transplantation in childhood systemic lupus erythematosus (SLE) and describe this procedure in two young girls with severe and refractory disease. The patients' stem cells were mobilized with granulocyte colony-stimulating factor (G-CSF) and collected by CS-3000 Blood Cell Separator (Baxter Healthcare, Round Lake, Ill., USA), and the CliniMACS CD34+ cell selection device (Miltenyi Biotech, Bergisch Gladbach, Germany) was used to obtain CD34+ cells. A total of 1.7x10(6) and 1.0x10(6)/kg CD34+ cells were obtained, with 2.0x10(5) and 1.0x10(4)/kg of CD3+ cells remaining, respectively. The conditioning regimen consisted of cyclophosphamide (50 mg/kg per day for 4 days) plus antithymocyte globulin (ATG-Fresenius, 5 mg/kg per day for 3 days). Neutrophil counts recovered within 9 days in both cases. Within 15 days, the platelet counts recovered and were sustained over 100x10(9)/l. Cushingoid features disappeared completely 3 months after transplantation because of the removal of corticosteroid medication. One 13-year-old child increased her height by 5 cm in 6 months after stopping steroids. She had not increased her height in her previous 7 years of disease. As of the time of this report, the first patient remains in clinical and laboratory remission for nearly 4 years, while the second suffered a relapse of thrombocytopenia 9 months post-transplantation. One residual effect of their treatment is that their CD4+ cell counts remained in the lower range after one year of transplant. The effect of this conditioning regimen plus CD34+ autologous stem cell transplantation on these two children with refractory SLE was beneficial, but long-term follow-up data and additional experience with this procedure are required. Autologous stem cell transplantation may limit the long-term toxicity of therapy in childhood SLE.

Two in vitro assays for the detection of allergen-specific IgE (sIgE) were compared reestimating sera with sIgE to common inhalants (birch t3, timothy g6, mugwort w6, cat dander el, dog dander e5, housdust mite D. pter. dl) and food allergens (FA) (hen's egg fl, cow's milk f2, wheat flour f4, soy bean f14, hazelnut f17, apple f49). Quantitative values (kU/l) of both systems were associated to different degrees depending on the allergen: allergen/n t3/48, g6/47, w6/39, e1/40, e5/42, d1/39. Spearman's rho 0.95***, 0.9***, 0.82***, 0.9***, 0.82***, 0.85***, allergen/n f1/37 f2/38 f4/68 f14/52f17/40 f49/40. Spearman's rho 0.84***, 0.6***, 0.6***, 0.31*, 0.68***, 0.82***, *** p < 0.001; * p < 0.05. Values to inhalants (t3, g6, el, e5) were scattered moderately around an ideal regression line (diagonal between x-and y-axis). In contrast, sIgE-levels to FA (f2, f4, f14, fl7) showed stronger discrepancies between both systems. Similarly, different degrees of association and spreading, depending on the individual allergen, were found comparing semiquantitative classes (0 - 6) of both systems. Taken together, values of sIgE to inhalants detected by different in vitro methods are stronger associated than sIgE to FA; IgE-values to allergens with one dominant major allergen (i.e. t3, el) seem to be more concordant than complex allergen mixtures (i.e. dl); identical units (kU/l) can not garantee equivalent IgE results. The data presented do not allow estimates of the real IgE concentrations or judgement on superiority of one of the tests over the other. The study is restricted to compare both methods analytically, since the diagnostic concordance could not be calculated due to a lack of corresponding clinical data. Despite these limitations, comparative sIgE studies are necessary and helpful to define the concordance of IgE values detected by different immunoassays. They also set the basis for subsequent improvement of allergens used in test systems detecting specific IgE.

Elevated serum levels of antigen-specific immunoglobulin (Ig)E are often associated with allergic respiratory diseases. This parallel-group, randomised, double-blind, placebo-controlled trial was designed to study the influence of omalizumab on the early nasal response to allergen challenge reflected by symptom score and inflammatory marker levels in nasal lavage fluid (NAL). A total of 23 patients with allergic rhinitis took part in the study, 11 were given placebo and omalizumab was administered subcutaneously in 12. Omalizumab or placebo were given at 2- or 4-week intervals based on a patient's body weight and IgE levels to a total dose of 0.016 mg x kg(-1) x IgE(-1) (IU x mL(-1)) every 4 weeks. Compared to placebo, 16 weeks of treatment with omalizumab significantly inhibited allergen challenge-induced nasal symptoms (median symptom score 7.0-0.5 versus 7.0-7.0) and inhibited the increase of human serum albumin (median 15.3-0.12 mg x mL(-1) versus 8.2-19.7 mg x mL(-1)) in the NAL after allergen challenge. Treatment with omalizumab induced a significant decrease in tumour necrosis factor-alpha levels in basal NAL, but no change was seen for histamine. These results indicate that subcutaneously administered monoclonal anti-immunoglobulin-E antibody, omalizumab, inhibits the nasal responses to allergen challenge of patients with allergic rhinitis. Omalizumab may provide a new strategy for the treatment of allergic rhinitis.

Kennedy's disease is a degenerative disorder of motor neurons caused by the expansion of a glutamine tract near the amino terminus of the androgen receptor (AR). Ligand binding to the receptor is associated with several post-translational modifications, but it is poorly understood whether these affect the toxicity of the mutant protein. Our studies now demonstrate that mutation of lysine residues in wild-type AR that are normally acetylated in a ligand-dependent manner mimics the effects of the expanded glutamine tract on receptor trafficking, misfolding, and aggregation. Mutation of lysines 630 or 632 and 633 to alanine markedly delays ligand-dependent nuclear translocation. The K632A/K633A mutant also undergoes ligand-dependent misfolding and aggregation similar to the expanded glutamine tract AR. This acetylation site mutant exhibits ligand-dependent 1C2 immunoreactivity, forms aggregates that co-localize with Hsp40, Hsp70, and the ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase carboxyl terminus of Hsc70-interacting protein (CHIP), and inhibits proteasome function. Ligand-dependent nuclear translocation of the wild-type receptor and misfolding and aggregation of the K632A/K633A mutant are blocked by radicicol, an Hsp90 inhibitor. These data identify a novel role for the acetylation site as a regulator of androgen receptor subcellular distribution and folding and indicate that ligand-dependent aggregation is dependent upon intact Hsp90 function.

Juvenile American shad Alosa sapidissima were subjected to isothermal transfers into sea water (salinity 24) 'early' (1 September; 24° C) and 'late' (10 November; 10° C) in the autumn migratory season. Early acclimation resulted in a modest osmotic perturbation that recovered rapidly. Haematocrit declined by 14% at 24 h, recovering within 48 h. Plasma osmolality increased by 6% at 4 h, recovering within 8 h. Early acclimation caused a two-fold increase in gill Na + , K + -ATPase activity by 24h and a four-fold increase by 4 days. The number of chloride cells on the primary gill filament increased two-fold by 4 days. Chloride cells on the secondary lamellae rapidly decreased from 22 cells mm -1 to

Influx of eosinophils in airway mucosa and airway lumen is a hallmark of bronchial asthma. In-vitro data and animal studies indicate that the T-helper (Th) type-2 cell cytokine, interleukin (IL)-5, plays an important role in eosinophil maturation, differentiation, recruitment, and survival. The objective of this study was to determine whether intravenous treatment with monoclonal anti-IL-5 would affect the number of peripheral blood eosinophils, their activation status, T-cell activation or the pattern of Th1 and Th2 cytokine production. Over a period of 6 months, 19 asthmatics were investigated in a double-blind, placebo-controlled, multicentre study with mepolizumab (SB 240563) anti-IL-5 antibody administered three times. Before each infusion and 12 weeks after the last infusion, peripheral blood leukocytes were examined, qualitative and quantitative distribution of eosinophils and lymphocyte subpopulations, frequencies of IL-2, -3, -4, -5, -10, -13, interferon-gamma-producing CD4 T-cells and serum eosinophil cationic protein (ECP) levels were determined. Treatment with mepolizumab resulted in a marked, rapid and sustained decrease of eosinophil numbers (median values from 300 to 45 per microL) paralleled by decreased levels of serum ECP (median values from 15 to 5 microg x L(-1)). Distribution of T-cell subsets and T-cell cytokine production were not altered during antibody treatment. In conclusion, administration of mepolizumab to asthmatic patients markedly reduces peripheral blood eosinophils without altering the distribution and activation status of lymphocytes.

Summary Background Recent studies have shown that neurotrophins are produced by and can act on several immune-inflammatory cells. The origin of circulating as well as local neurotrophins is unknown. Objectives The aim of this study was to assess whether eosinophils of allergic and non-allergic donors produce, store and release the neurotrophic factors NGF, BDNF and NT-3. Methods Eosinophils were purified by negative immunoselection (purity > 96%) from allergic asthmatics and non-allergic donors (25 to 53 years). The presence of mRNA for neurotrophic factors was evaluated by reverse transcription PCR. Specificity was demonstrated by cloning products and sequencing. Stored NGF, BDNF and NT-3 was demonstrated by Western-blotting and flow cytometry. Eosinophils were incubated and supernatants were collected for measurement of neurotrophic factors after cell stimulation with PAF. Neurotrophin content in eosinophil lysates was determined by ELISA. Results Eosinophils demonstrate mRNA for neurotrophins. Proteins were detectable by Western blot and FACS analysis. Neurotrophins were found in the eosinophil lysates at different amounts comparing allergic and non-allergic donors. Cell stimulation with PAF (10-8–10-5 M) after priming with GM-CSF leads to a dose-dependant release of NGF and BDNF. Conclusions Eosinophils store, produce and release NGF, BDNF and NT-3. They are a possible source of elevated neurotrophin levels found in allergy and asthma.

Asthma bronchiale ist gekennzeichnet durch eine Entzundung der Atemwege mit reversibler Atemwegsobstruktion und erhohter Reagibilitat auf unspezifische Reize. Histologisch besteht ein entzundliches Zellinfiltrat, das durch Lymphozyten und eosinophile Granulozyten charakterisiert ist. Die immunologischen und zellularen Veranderungen beim Asthma bronchiale stehen in engem Zusammenhang mit der neuronalen Innervation und Funktion der Atemwege. Die neuronale Versorgung der Atemwege gliedert sich in einen efferenten sympathischen und parasympathischen Anteil sowie einen afferenten sensiblen Anteil, aus dem uber einen Axon-Reflex atemwirksame Mediatoren freigesetzt werden. Die aus den Neuronen freigesetzten Mediatoren beteiligen sich an der Regulation der Atemwege. In umgekehrter Weise werden Mediatoren aus Zellen, insbesondere Immunzellen (Mastzellen, eosinophile Granulozyten, Lymphozyten), freigesetzt, die wiederum sympathische, parasympathische und sensible Neurone beeinflussen. So werden aus sensiblen Neuronen proinflammatorische Tachykinine wie Substanz P (SP) und Neurokinin A (NKA) freigesetzt, deren Effekte als „neurogene Entzundung“ bezeichnet werden. Die Homoostase diese Systems wird durch degradierende Enzyme wie z. B. die neutrale Endopeptidase (NEP) und die Synthese fordernde Neurotrophine wie z. B. Nerve Growth Factor (NGF) aufrechterhalten.

Background/Objectives: The mechanical aerosol generator, MAGhaler®, is a new chlorofluorocarbon-free inhalation device. The objective of this trial was to show equivalent efficacy and safety of beclomethasone dipropionate (BDP) delivered via the MAGhaler® and the metered-dose inhaler (MDI) in patients with mild to moderate bronchial asthma. Moreover, user-friendliness and acceptance of the two devices were compared. Methods: This was a double-blind, reference-controlled, 12-week trial in 171 patients with asthma receiving BDP (1,000 µg/day) delivered via either the MAGhaler® or the conventional MDI. Respiratory function parameters, clinical symptoms, concomitant intake of salbutamol or fenoterol, adverse events (AEs), laboratory values, and concomitant medications and diseases were recorded. The primary efficacy parameter was mean forced expiratory volume in 1 s (FEV1), measured after 4, 8, and 12 weeks of therapy. Results: The equivalence of the two devices was confirmed (p = 0.003) on the basis of the ratios of the mean FEV1 in weeks 4 to 12. Mean (± SD) FEV1 (MAGhaler®) was 2.24 ± 0.60 l (baseline), 2.61 ± 0.90 litres (week 4), and 2.62 ± 0.87 litres (weeks 4–12). Mean FEV1 (MDI) was 2.28 ± 0.59 litres (baseline), 2.53 ± 0.82 litres (week 4), and 2.56 ± 0.77 litres (weeks 4–12). In total, 33 AEs occurred in 26 (30.2%) patients (MAGhaler®) and 51 AEs in 36 (42.4%) patients (MDI). Most of the AEs were of mild or moderate intensity. The relationship to treatment could not be excluded for 11 AEs in 11 patients (MAGhaler®) and 23 AEs in 18 patients (MDI). Three serious AEs, all unrelated to treatment, occurred in 3 patients (MAGhaler®: 2, MDI: 1). There were no clinically relevant changes in other safety parameters. Most patients either preferred the MAGhaler® or rated the two devices as equally acceptable. Conclusion: The new MAGhaler® was equivalent to the standard MDI in terms of the safety and efficacy of BDP. The improved user-friendliness and acceptance of the MAGhaler® over the conventional MDI represent an important advance in the clinical management of bronchial asthma.

Calcium ions (Ca(2+)), protons (H(+)), and borate (B(OH)(4)(-)) are essential ions in the control of tip growth of pollen tubes. All three ions may interact with pectins, a major component of the expanding pollen tube cell wall. Ca(2+ )is thought to bind acidic residues, and cross-link adjacent pectin chains, thereby strengthening the cell wall. Protons are loosening agents; in pollen tube walls they may act through the enzyme pectin methylesterase (PME), and either reduce demethylation or stimulate hydrolysis of pectin. Finally, borate cross-links monomers of rhamnogalacturonan II (RG-II), and thus stiffens the cell wall. It is demonstrated here that changing the extracellular concentrations of Ca(2+), H(+) and borate affect not only the average growth rate of lily pollen tubes, but also influence the period of growth rate oscillations. The most dramatic effects are observed with increasing concentrations of Ca(2+) and borate, both of which markedly reduce the rate of growth of oscillating pollen tubes. Protons are less active, except at pH 7.0 where growth is inhibited. It is noteworthy, especially with borate, that the faster growing tubes exhibit the shorter periods of oscillation. The results are consistent with the idea that binding of Ca(2+) and borate to the cell wall may act at a similar level to alter the mechanical properties of the apical cell wall, with optimal concentrations being high enough to impart sufficient rigidity to the wall so as to prevent bursting in the face of cell turgor, but low enough to allow the wall to stretch quickly during periods of accelerating growth.

Summary Background Recent studies have shown that nerve growth factor (NGF) can act on several immune cells as well as residential cells. But little is known about their role in modulating eosinophil function via activation of high-affinity receptors. Objectives The aim of this study was to assess whether eosinophils express functional receptors and if their function is influenced by NGF, brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). Methods Eosinophils were purified by negative immunoselection (purity > 96%). High-affinity neurotrophin receptors were demonstrated by reverse transcription polymerase chain reaction, western blotting and flow-cytometry analysis. Functionality of receptors was demonstrated by receptor phosphorylation after ligand binding. Eosinophils were incubated with NGF, BDNF and NT-3, and cells and supernatants were collected for measurement of the mediators IL-4, IL-5, IL-8, transforming growth factor (TGF)-β1, eosinophil cationic protein (ECP), eosinophil protein X (EPX) as well as eosinophil viability. Results Eosinophils expressed mRNA for neurotrophin receptors. Proteins were detectable by western blot and fluorescent-activated cell sorter analysis. The receptors were phosphorylated after stimulation with neurotrophins. After NGF stimulation, a significant increase in IL-4 was detectable. BDNF and NT-3 stimulation led to a significant increase in EPX. Eosinophil viability was not influenced. Conclusions Eosinophils express the functionally active receptors TrkA, TrkB and TrkC. Receptor activation stimulates eosinophils. This might be an additional pathway regulating inflammatory responses in allergic reactions.

Background πβ-endorphin is a derivative of pro-opiomelanocortin. Cells of the immune system can also synthesize and secrete β-endorphin. Its concentration is increased during the allergic reaction and during stress. Increased reactivity during psychological stress of allergic subjects is also well known. Objective Is β-endorphin one physiological link between stress and an exacerbation of the allergic reaction? Methods First, intranasal β-endorphin challenges with subsequent lavages to determine histamine and albumin levels and measurements of nasal flow and resistance in dose-response and time course experiments were performed. Secondly, we examined whether β-endorphin pre-treatment increased the antigen-induced release of histamine and albumin in nasal lavages and the clinical symptoms. Results Exogenous β-endorphin (100 pM−10 µM/mL) induced a dose-dependent increase in nasal symptoms in asymptomatic allergic subjects with rhinitis (n = 14) as well as in non-allergic controls (n = 10), but did not release any mediators into nasal secretion. However, comparing the antigen-evoked release of mediators into nasal secretions with that of a β-endorphin pre-treated antigen challenge we could note a significant enhancement of human serum albumin influx (P

Background The neurotrophins Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF) and Neurotrophin (NT)-3 are produced, stored and released by various immunological cells. The influence of NTs upon the function of these cells is described. Elevated plasma levels were found in inflammatory, autoimmune and allergic diseases with the highest levels in allergic asthma. A connection between bronchial hyper-responsiveness and serum levels has been reported. Objective Little is known about the influence of treatment with inhaled corticosteroids (ICS) on serum NT levels and their influence on the asthmatic state. Methods Eighty-seven volunteers were studied. Thirty-eight were stable allergic asthmatics with constant ICS doses, 29 were asthmatics not receiving anti-asthmatic treatment and 20 were age- and sex-matched healthy controls. Demographic and lung function data were evaluated. NT serum levels were determined by ELISA. Results NGF and BDNF levels were significantly increased in untreated asthmatics compared to the control and the treated group, while NT-3 demonstrated significantly higher levels in treated asthmatics compared to healthy controls. After stabilization of untreated subjects with ICS, the NT levels decreased significantly. Conclusions These results suggest that NTs participate in allergic inflammation and asthma. Effective treatment leads to a decrease of circulating neurotrophic factors.

Self-incompatibility (SI) is a genetically con- trolled system used by many flowering plants to prevent self-pollination. We established, using calcium imaging, that the SI response in Papaver rhoeasL. (poppy) pollen involves a Ca 2+ -mediated intracellular signalling path- way. Here we review what is known about the signalling components and cascades implicated in the SI response in poppy pollen. We present some studies using calcium green (CG-1) that show SI-induced alterations in CG-1 fluorescence and localization. We have begun to exam- ine potential sources of Ca 2+ involved in the responses induced by SI. This work presents preliminary data showing that influx of extracellular Ca 2+ at the "shank" of the pollen tube is possible. This is the first evidence suggesting that influx at this localization may play a role in the SI response. We also describe preliminary studies that begin to investigate whether the phosphoinositide signalling pathway is implicated in the SI response.

Neutral endopeptidase (NEP) is described in airways as the major degrading enzyme of tachykinins such as neurokinin A (NKA) and substance P (SP). Due to its localization and mode of action NEP may play a role in the pathophysiology of bronchial reactivity (BR) especially under the aspect of neurogenic inflammation. Serum NEP concentrations were measured by ELISA to investigate if there is a correlation between serum NEP and the degree of bronchial reactivity expressed by PC20-FEV1 histamine(.). PC20-FEV1 histamine was determined in 31 asthmatic patients [age 31.9+/-1.3 years (mean+/-SEM) FEV1=92.1+/-2.4% (mean+/-SEM) 16 females/15 males]. Prior to the histamine challenge blood samples were obtained and stored at -70 degrees C until determination using ELISA. A significant correlation between serum NEP and the PC20-FEV1 (n=31, r=0.49, P0.01) was found. The results suggest that serum NEP is modulating neuropeptide-induced effects in the pathophysiology of airway responsiveness.

Summary The occurrence of oscillatory behaviours in living cells can be viewed as a visible consequence of stable, regulatory homeostatic cycles. Therefore, they may be used as experimental windows on the underlying physiological mechanisms. Recent studies show that growing pollen tubes are an excellent biological model for these purposes. They unite experimental simplicity with clear oscillatory patterns of both structural and temporal features, most being measurable during real-time in live cells. There is evidence that these cellular oscillators involve an integrated input of plasma membrane ion fluxes, and a cytosolic choreography of protons, calcium and, most likely, potassium and chloride. In turn, these can create positive feedback regulation loops that are able to generate and self-sustain a number of spatial and temporal patterns. Other features, including cell wall assembly and rheology, turgor, and the cytoskeleton, play important roles and are targets or modulators of ion dynamics. Many of these features have similarities with other cell types, notably with apical-growing cells. Pollen tubes may thus serve as a powerful model for exploring the basis of cell growth and morphogenesis. BioEssays 23:86‐94, 2001. fl 2001 John Wiley & Sons, Inc.