Different Groups Of Cells Research Articles

Na+ transport by small airway surface epithelia

BackgroundMaintaining an adequate level of Airway Surface Liquid (ASL) is crucial for the normal functioning of the immune system of the lungs. A balance between the secretion and the absorption of ions and fluid by the airways surface epithelia (ASE) ensures proper amount of ASL for mucociliar clearance. ASE expresses Epithelial Na+ Channel (ENaC) and the Cl‐ channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Currently, there are two opposing hypotheses to explain how the interplay between CFTR and ENaC activity regulate ASL height. One theory proposes that each surface epithelial cell expresses both CFTR and ENaC and is able to secrete or reabsorb fluid and ions. A recent hypothesis proposes that CFTR and ENaC are expressed in different groups of cells. Cells within the pleats of ASE express CFTR and are mainly secretory while the cells of the folds express ENaC and are principally absorptive.QuestionAre the cells of ASE able to transport ions in both directions?MethodsTo detect transepithelial Na+ flux we used Scanning Ion Selective Techniquethe, which measures ion concentration gradient generated by the epithelia using ion selective microelectrodes.We investigated the ion transport in the folds of the swine distal airways under normal Kreb's solution and in the presence of CFTR and ENaC modulators.ResultsASE showed spontaneous basal Na+ transport from the lumen into the tissue. Adding the sodium azide blocked the basal secretion. Stimulation with forskolin or thapsigargin triggered rapid and reversal Na+ transport and was blocked by treatment with the CFTR blocker.ConclusionsThe epithelial cells of the fold regions of ASE are able to secret and absorb Na+. Our results allow us to reject the hypothesis of the monodirectional Na+ transportation in the surface epithelia.

Adaptive developmental plasticity: compartmentalized responses to environmental cues and to corresponding internal signals provide phenotypic flexibility.

BackgroundThe environmental regulation of development can result in the production of distinct phenotypes from the same genotype and provide the means for organisms to cope with environmental heterogeneity. The effect of the environment on developmental outcomes is typically mediated by hormonal signals which convey information about external cues to the developing tissues. While such plasticity is a wide-spread property of development, not all developing tissues are equally plastic. To understand how organisms integrate environmental input into coherent adult phenotypes, we must know how different body parts respond, independently or in concert, to external cues and to the corresponding internal signals.ResultsWe quantified the effect of temperature and ecdysone hormone manipulations on post-growth tissue patterning in an experimental model of adaptive developmental plasticity, the butterfly Bicyclus anynana. Following a suite of traits evolving by natural or sexual selection, we found that different groups of cells within the same tissue have sensitivities and patterns of response that are surprisingly distinct for the external environmental cue and for the internal hormonal signal. All but those wing traits presumably involved in mate choice responded to developmental temperature and, of those, all but the wing traits not exposed to predators responded to hormone manipulations. On the other hand, while patterns of significant response to temperature contrasted traits on autonomously-developing wings, significant response to hormone manipulations contrasted neighboring groups of cells with distinct color fates. We also showed that the spatial compartmentalization of these responses cannot be explained by the spatial or temporal compartmentalization of the hormone receptor protein.ConclusionsOur results unravel the integration of different aspects of the adult phenotype into developmental and functional units which both reflect and impact evolutionary change. Importantly, our findings underscore the complexity of the interactions between environment and physiology in shaping the development of different body parts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0097-x) contains supplementary material, which is available to authorized users.

Impact of the structure of Mo(S,Se)2 interfacial region in electrodeposited CuIn(S,Se)2 solar cells

In this study, we investigate the formation of Mo(S,Se)2 layers in the interface region between the absorber and the back contact in electrodeposited CuIn(S,Se)2 (CISSe) solar cells, and its influence on the resulting devices. Mo(S,Se)2 layers are formed spontaneously in the Mo/CI(S,Se) interface during the annealing of the absorber, but have a serious impact on the solar cell performance, being responsible of the conductivity at this interface. In this paper, we demonstrate that the crystal orientation of the Mo(S,Se)2 layer is the relevant parameter that governs the electrical properties of the interface by the generation of an energy barrier. For this, we have analysed two different groups of cells, with high and low Voc, to determine the origin of the differences. C–V analysis suggested the presence of an energy barrier at the back contact that was related to the existence of a Mo(S,Se)2 layer with c-axis oriented perpendicular to the substrate. Simulations taking into account this feature reproduced the behaviour of the low performing cells. Finally, high-resolution transmission electron microscopy observations were performed to clarify the orientation of the Mo(S,Se)2 layers at the CISSe/Mo(S,Se)2 interface, which is determining for the thickness of the Mo(S,Se)2 layer.

Structure and anterior regeneration of musculature and nervous system in Cirratulus cf. cirratus (Cirratulidae, Annelida).

Annelids provide suitable models for studying regeneration. By now, comprehensive information is restricted to only a few taxa. For many other annelids, comparative data are scarce or even missing. Here, we describe the regeneration of a member of the Cirratulus cirratus species complex. Using phalloidin-labeling and antibody-stainings combined with subsequent confocal laser scanning microscopy, we provide data about the organization of body wall musculature and nervous system of intact specimens, as well as about anteriorly regenerating specimens. Our analyses show that C. cf. cirratus exhibits a prominent longitudinal muscle layer forming a dorsal muscle plate, two ventral muscle strands and a ventral-median muscle fiber. The circular musculature forms closed rings which are interrupted in the area of parapodia. The nervous system of C. cf. cirratus shows a typical rope-ladder like arrangement and the circumesophageal connectives exhibit two separate roots leading to the brain. During regeneration, the nervous system redevelops remarkably earlier than the musculature, first constituting a tripartite loop-like structure which later become the circumesophageal connectives. Regeneration of longitudinal musculature starts with diffuse ingrowth and subsequent structuring into the blastema. In contrast, circular musculature develops independently inside the blastema. Our findings constitute the first analysis of regeneration for a member of the Cirratuliformia on a structural level. Summarizing the regeneration process in C. cf. cirratus, five main phases can be subdivided: 1) wound closure, 2) blastema formation, 3) blastema differentiation, 4) resegmentation, and 5) growth, respectively elongation. Additionally, the described tripartite loop-like structure of the regenerating nervous system has not been reported for any other annelid taxon. In contrast, the regeneration of circular and longitudinal musculature originating from different groups of cells seems to be a general pattern in annelid regeneration.

Ras-dva1 small GTPase regulates telencephalon development in Xenopus laevis embryos by controlling Fgf8 and Agr signaling at the anterior border of the neural plate

ABSTRACTWe previously found that the small GTPase Ras-dva1 is essential for the telencephalic development in Xenopus laevis because Ras-dva1 controls the Fgf8-mediated induction of FoxG1 expression, a key telencephalic regulator. In this report, we show, however, that Ras-dva1 and FoxG1 are expressed in different groups of cells; whereas Ras-dva1 is expressed in the outer layer of the anterior neural fold, FoxG1 and Fgf8 are activated in the inner layer from which the telencephalon is derived. We resolve this paradox by demonstrating that Ras-dva1 is involved in the transduction of Fgf8 signal received by cells in the outer layer, which in turn send a feedback signal that stimulates FoxG1 expression in the inner layer. We show that this feedback signal is transmitted by secreted Agr proteins, the expression of which is activated in the outer layer by mediation of Ras-dva1 and the homeodomain transcription factor Otx2. In turn, Agrs are essential for maintaining Fgf8 and FoxG1 expression in cells at the anterior neural plate border. Our finding reveals a novel feedback loop mechanism based on the exchange of Fgf8 and Agr signaling between neural and non-neural compartments at the anterior margin of the neural plate and demonstrates a key role of Ras-dva1 in this mechanism.

Adaptive developmental plasticity: Compartmentalized responses to environmental cues and to corresponding internal signals provide phenotypic flexibility

The environmental regulation of development can result in the production of distinct phenotypes from the same genotype and provide the means for organisms to cope with environmental heterogeneity. The effect of the environment on developmental outcomes is typically mediated by hormonal signals which convey information about external cues to the developing tissues. While such plasticity is a wide-spread property of development, not all developing tissues are equally plastic. To understand how organisms integrate environmental input into coherent adult phenotypes, we must know how different body parts respond, independently or in concert, to external cues and to the corresponding internal signals. We quantified the effect of temperature and ecdysone hormone manipulations on post-growth tissue patterning in an experimental model of adaptive developmental plasticity, the butterfly Bicyclus anynana. Following a suite of traits evolving by natural or sexual selection, we found that different groups of cells within the same tissue have sensitivities and patterns of response that are surprisingly distinct for the external environmental cue and for the internal hormonal signal. All but those wing traits presumably involved in mate choice responded to developmental temperature and, of those, all but the wing traits not exposed to predators responded to hormone manipulations. On the other hand, while patterns of significant response to temperature contrasted traits on autonomously-developing wings, significant response to hormone manipulations contrasted neighboring groups of cells with distinct color fates. We also showed that the spatial compartmentalization of these responses cannot be explained by the spatial or temporal compartmentalization of the hormone receptor protein. Our results unravel the integration of different aspects of the adult phenotype into developmental and functional units which both reflect and impact evolutionary change. Importantly, our findings underscore the complexity of the interactions between environment and physiology in shaping the development of different body parts.

Differential Activity of Drosophila Hox Genes Induces Myosin Expression and Can Maintain Compartment Boundaries

Compartments are units of cell lineage that subdivide territories with different developmental potential. In Drosophila, the wing and haltere discs are subdivided into anterior and posterior (A/P) compartments, which require the activity of Hedgehog, and into dorsal and ventral (D/V) compartments, needing Notch signaling. There is enrichment in actomyosin proteins at the compartment boundaries, suggesting a role for these proteins in their maintenance. Compartments also develop in the mouse hindbrain rhombomeres, which are characterized by the expression of different Hox genes, a group of genes specifying different structures along their main axis of bilaterians. We show here that the Drosophila Hox gene Ultrabithorax can maintain the A/P and D/V compartment boundaries when Hedgehog or Notch signaling is compromised, and that the interaction of cells with and without Ultrabithorax expression induces high levels of non-muscle myosin II. In the absence of Ultrabithorax there is occasional mixing of cells from different segments. We also show a similar role in cell segregation for the Abdominal-B Hox gene. Our results suggest that the juxtaposition of cells with different Hox gene expression leads to their sorting out, probably through the accumulation of non-muscle myosin II at the boundary of the different cell territories. The increase in myosin expression seems to be a general mechanism used by Hox genes or signaling pathways to maintain the segregation of different groups of cells.

Apoptosis in Glioma Cells Treated with PDT

Despite significant advances in neurosurgical techniques, the median survival time of patients with glioblastoma has improved little over the past 50 years and remains less than one year. Photodynamic therapy (PDT) is presently established as a widely accepted modality for the treatment of a variety of solid tumors. This study evaluated the effect of PDT-Photogem(®) on five glioma cell lines (U87, U138, U251, U343, and T98G). The experiments were carried out in 25-cm(3) flasks with different groups of cells seeded at a density of 1 × 10(5) cells per flask. After 3 h, the medium was removed, and the cells were incubated for 4 h with Photogem (5 μg/mL). After the incubation time, the photosensitizer-containing medium was removed and the cells were irradiated with LED (630 nm, 25 mW/cm(2), 25 J/cm(2)) devices for 17 min. For the final steps of the PDT, the cells were returned to the incubator and kept at 37°C with 5% CO(2) for 24 h, the cell viability assay was assessed using the trypan blue method, and the expression of Caspase 3 mRNA levels was assessed by real-time quantitative PCR. Upon PDT-Photogem(®) treatment, viable cells, as evaluated by the trypan blue dye-exclusion method, decreased in two cell lines (U87 and U138) but not in the other three. Apoptosis, as assessed by the expression of caspase-3 mRNA levels, was at least partly involved in the death mechanism of the cell lines. Collectively, our results indicated that PDT-Photogem(®) can act in glioma cells, thus encouraging new experiments in this field.

An Optimal Transportation Approach for Nuclear Structure-Based Pathology

Nuclear morphology and structure as visualized from histopathology microscopy images can yield important diagnostic clues in some benign and malignant tissue lesions. Precise quantitative information about nuclear structure and morphology, however, is currently not available for many diagnostic challenges. This is due, in part, to the lack of methods to quantify these differences from image data. We describe a method to characterize and contrast the distribution of nuclear structure in different tissue classes (normal, benign, cancer, etc.). The approach is based on quantifying chromatin morphology in different groups of cells using the optimal transportation (Kantorovich-Wasserstein) metric in combination with the Fisher discriminant analysis and multidimensional scaling techniques. We show that the optimal transportation metric is able to measure relevant biological information as it enables automatic determination of the class (e.g., normal versus cancer) of a set of nuclei. We show that the classification accuracies obtained using this metric are, on average, as good or better than those obtained utilizing a set of previously described numerical features. We apply our methods to two diagnostic challenges for surgical pathology: one in the liver and one in the thyroid. Results automatically computed using this technique show potentially biologically relevant differences in nuclear structure in liver and thyroid cancers.

Effect of PKC signalling pathway and aldose reductase on expression of fibronectin induced by transforming growth factor-β1 in human mesangial cells

To study the effect of PKC signalling pathway and aldose reductase (AR) on the expression of fibronectin (FN) induced by transforming growth factor-β1 (TGF-β1). Human mesangial cells (HMCs) were cultured and transfected with pcDNA3-AR, and subject to AR gene silencing with small interfering RNA (siRNA) and then the cell was treated with recombinant human TGF-β1. The AR mRNA expression in the HMCs was examined using real time RT-PCR and protein expression of AR and FN was detected by Western blotting. The cultured HMC treated with TGF-β1 showed increased expression of AR and FN, the normal HMC showed not reduced expression of FN after incubation with single inhibitors of AR.Pre-incubation of cells with inhibitors of AR and PKC, then the different groups of cells were treated with TGF-β1, and the induction effect on FN expression was suppressed (34%) in HMC. HMCs transfected with AR showed a strong protein expression of FN, which was increased by 3.6-fold after treatment with TGF-β1 (P < 0.05), and the induction effect on FN expression was suppressed by GÖ6983 (42%) in HMCs (P < 0.05). The HMC with AR gene knock-down by siRNA showed a decreased expression of AR and 90% decrease of FN protein in HMCs (P < 0.01), and TGF-β1-induced up-regulation of FN was significantly suppressed by siRNA (12%) in HMCs (P < 0.01). AR is capable of regulating FN expression only in the presence of TGF-β1, and this reaction is possibly accomplished through the activation of PKC signalling pathway.

Effects of Intercellular Communication and Stochasticity in Gene Expression On TH1/TH2 Differentiation.

Abstract 2673Poster Board II-649Cytokines provide a main regulatory mechanism of a developing immune response, which allows for intercellular communication. This sharing of information enables cells to make collective decisions based on the state of the system rather than that of a single cell. In our research we focus on several examples of cytokine mediated regulatory mechanisms that function in helper T-cells (Th), whose role is to direct the immune response in the desired course by affecting responses of other cells. To analyze the relative importance of the different components affecting the behavior of the cells we employ mathematical modeling and numerical simulations of specific experimental settings. Quantitative experiments are performed to evaluate unknown parameters and to verify model predictions. We focus on two examples of cytokine mediated regulatory mechanisms that function in the helper T-cell (Th) differentiation. IL-12 and IL-4 direct differentiation into the main fates of Th cells, Th1 and Th2, respectively. By exposing cells to varied combinations of the two cytokines we experimentally map the “decision space” of the differentiation process. We measured, at the single cell level, eight key parameters relating to the state of the cells, from which we can deduce properties of the network describing the system. Our data indicate that under certain mixed conditions, with cells exposed to both IL-12 and IL-4, a new class of Th cells is observed, which secret both IL-4 and IFN-γ upon restimulation. This is preceded by co-expression of the two lineage specific transcription factors T-bet and GATA3 at earlier time points. We incorporate our experimental results and other known data into a cellular automata based simulation. Each cell is simulated as a node on a dynamic network, whose links describe the propagation of intercellular cytokine signals. The intracellular protein network for each node is realized as an automaton with cytokines levels as inputs and their secretion rates as output. By varying model parameters and comparing to experimental results we can gain insight into the significance of the various components driving the differentiation process. We also analyze the roles of heterogeneity in expression levels of cytokine receptors in the differentiation decision. To that end, naïve mice CD4 cells were sorted into separate groups according to their expression level of the IL-4 receptor. The different groups of cells were cultured separately, under “controlled milieu” conditions, at different IL-4 or IL-12 concentrations. Expression levels of key proteins involved in the differentiation process were measured at the single cell level every 24h. This was compared to expression levels of those proteins in a whole population of cells growing together. Our results reveal differences in the differentiation abilities between the sorted, controlled milieu, groups. Additionally, the weighted average of expression levels of the controlled milieu populations is different than that of the full heterogeneous population of cells growing together. These differences are caused by intercellular interactions, as cells secrete cytokines which are involved in and drive the differentiation process (e.g. IL-4, IFN-γ). Thus, these experiments provide an opportunity to evaluate the importance and roles played by intercellular communication in this process, and to asses what are the effects of stochastic levels of a protein (IL-4R in this case) on the following differentiation process. Disclosures:No relevant conflicts of interest to declare.

Identification and functional analysis of a novel TRPC6 mutation associated with late onset familial focal segmental glomerulosclerosis in Chinese patients

Objective Mutations in the TRPC6 gene are responsible for a late onset form of familial focal segmental glomerulosclerosis (FSGS). However, the role of TRPC6 variation in Chinese patients with late onset familial FSGS remains unclear. Here, we screened 31 Chinese pedigrees with late onset familial FSGS for changes in TRPC6 by DNA sequence analysis. Methods Genomic DNA was extracted from peripheral leukocytes. We PCR-amplified each of 13 exons of TRPC6 for sequence analysis. When a novel nucleotide change seemed likely to cause FSGS, we carried out an in vitro research to determine the effects of the mutation on TRPC6 function. HEK 293 cells were transfected stably with vectors containing mutant or wild type TRPC6 cDNA. We then compared the expression of mutant TRPC6 to wild type TRPC6 using Western blot. For the observation of the function of mutant TRPC6 channel compared with wild type TRPC6 channel, Intracellular Ca 2+ concentration was detected using fluorescent indicator Fluo-3 among different groups of cells upon stimulation with 1-oleoyl-2-acetyl sn-glycerol (OAG). Results All the 31 pedigrees with late onset familial FSGS were collected in our department from September 1997 to October 2007. A novel TRPC6 mutation (cytosine 2664 adenine resulting in Glutamine 889 Lysine substitution, Q889K) was identified in one of these pedigrees. Mutant TRPC6 (TRPC6 Q889K) or wild type TRPC6 was stably expressed in HEK293 cells by Western blot. The mutant TRPC6 expression was a little increased without significant difference compared with wild type TRPC6 expression, whereas the intracellular Ca 2+ level in cells expressing mutant TRPC6 was significantly increased compared with that in the cells expressing wild TRPC6 upon stimulation. Conclusion We identified a novel TRPC6 mutation Q889K associated with late onset FFSGS in Chinese pedigrees and this mutation was demonstrated to be “gain of function” by an in vitro functional research.

A microfluidic cell array with individually addressable culture chambers

Microfluidic arrays of living cells have raised a lot of interests recently due to their potential for high throughput screening of cell-based assays. This report presents a microfluidic cell array with individually addressable chambers controlled by pneumatic valves for cell culture and treatment. There are two modes for the cell array to be operated. In the first mode, different groups of cells are directed into designated chambers for culturing and observation. We demonstrate the delivery and culture of enhanced green fluorescent protein (EGFP) expressing and nonfluorescent Chinese hamster ovary (CHO) cells into specific chambers in the array. In the second mode, the chambers are first seeded with the same cell type and different reagents are delivered to specific chambers for cell treatment. We treat cells in designated chambers with Calcein AM and CellTrace calcein red-orange AM to demonstrate the principle. We envision that this microfluidic cell array technology will pave the way to automated high-throughput screening of biomolecules and drugs based on observing cellular phenotypes and responses.

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

Different Gene Expressions on the Left and the Right: A Genotype/Phenotype Mismatch in Need of Attention

Discordance in monozygotic twins has traditionally been explained in terms of environmental influences. A recent investigation has found a difference in epigenetic markers in older but not in younger twins. However, phenotypic differences that depend on an individual's postnatal life style do not address the problem of discordance in congenital malformations, or the reason why malformations are frequently unilateral, often with a preference for one or the other side. One such condition, cleft lip with or without cleft palate, which is preferentially expressed on the left, is a multifactorial condition, that is caused by a failure of the critical timing necessary for different groups of cells to meet and develop into a normal face. This process is dependent on cell proliferation and migration, which are energy-dependent, while the additional requirement for apoptosis to allow cell fusion suggests the involvement of mitochondria. Recent progress in two separate areas of research could lead to a better understanding of the problem of facial clefts: (1) the recognition of an interaction between gene products and mitochondria in the aetiology of neurodegenerative diseases and (2) the discovery of an increasing number of genes, including transcription factors, growth factors and members of the TGF-beta signalling family, that are differentially expressed on the left and right side, thus pointing to a difference in their micro-environment. These findings emphasize the importance of investigating the activity of candidate genes for complex developmental processes separately on the left and right sides. Data presented in this review suggest that differential growth rates may lead to an inversion of laterality. A method is described to test for a possible mitochondrial difference between left and right sides, using a mouse model with cleft lip.

Differential cortical activity for precision and whole‐hand visually guided grasping in humans

Effective grasping involves the remarkable ability to implement multiple grasp configurations such as precision grip (PG; opposition between the index finger and thumb) and whole-hand grasp (WHG), depending on the properties of the object grasped (e.g. size, shape and weight). In the monkey brain, different groups of cells in the anterior-lateral bank of the intraparietal sulcus (area AIP) are differentially active for various hand configurations during grasping of differently shaped objects. Visually guided grasping studies in humans suggest the anterior intraparietal sulcus (aIPS) as the homologue of macaque area AIP, but leave unresolved the question of whether activity in human aIPS reflects the relationship between object size and grasp configuration, as in macaques. To address this issue, a human fMRI study was conducted in which objects were grasped with the right hand while object size was varied. The results indicated that the left aIPS was active when the subjects naturally adopted a PG to grasp the small object but showed a much weaker response when subjects naturally adopted a WHG to grasp the large object. The primary motor cortex and somatosensory cortices were active for both PG and WHG. Our results suggest that, in humans, the aIPS is centrally involved in determining the type of grasp.

Separate Processing of Different Global-Motion Structures in Visual Cortex Is Revealed by fMRI

The visual system has the remarkable ability to extract several types of meaningful global-motion signals, such as radial motion, translation motion, and rotation, for different visual functions and actions. In the monkey brain, different groups of cells in MST respond best to different types of global motion [1, 2] whereas in lower cortical areas including MT, no such differential responses have been found. Here, we show that an area (or areas) lower than MST in the human brain [3] responds to different types of global motion. A series of human functional magnetic resonance imaging (fMRI) experiments, in which attention was controlled for, indicated that the center of radial motion activates the corresponding location in the V3A representation, whereas translation motion activates mainly in a more peripheral representation of V3A. These results suggest that in the human brain, V3A is an area that differentially responds according to the type of global motion.

Unraveling the Molecular Details Involved in the Intimate Link between the Immune and Neuroendocrine Systems

During systemic infections, the immune system can signal the brain and act on different neuronal circuits via soluble molecules, such as proinflammatory cytokines, that act on the cells forming the blood-brain barrier and the circumventricular organs. These activated cells release prostaglandin of the E(2) type (PGE(2)), which is the endogenous ligand that triggers the pathways involved in the control of autonomic functions necessary to restore homeostasis and provide inhibitory feedback to innate immunity. Among these neurophysiological functions, activation of the circuits that control the plasma release of glucocorticoids is probably the most critical to the survival of the host in the presence of pathogens. This review revisits this issue and describes in depth the molecular details (including the emerging role of Toll-like receptors during inflammation) underlying the influence of circulating inflammatory molecules on the cerebral tissue, focusing on their contribution in the synthesis and action PGE(2) in the brain. We also provide an innovative view supporting the concept of "fast and delayed response" involving the same ligands but different groups of cells, signal transduction pathways, and target genes.

Applications of an efficient method for comparing immunogold labelling patterns in the same sets of compartments in different groups of cells.

Quantitative immunoelectron microscopy often involves determining the distributions of gold label in different intracellular compartments and then drawing comparisons between compartments in the same sample of cells or between experimental groups of cells. In the case of within-group comparisons, recent developments in the estimation of relative labelling index and labelling density make it possible to test whether or not particular compartments are preferentially labelled. These methods are ideally suited to analysing gold label restricted to volume (organelle) or surface (membrane) compartments but may be modified to analyse label localised in mixtures of both. Here, a simple and efficient approach to drawing between-group comparisons for label associated with organelles and/or membranes is presented. The method relies on multistage random sampling of specimens (via blocks and microscopic fields) followed by simply counting gold particles associated with different compartments. The distributions of raw gold counts in different groups are then compared by contingency table analysis with statistical degrees of freedom for chi-squared values being determined by the number of compartments and the number of experimental groups of cells. Compartmental chi-squared values making substantial contributions to the total chi-squared values then identify where the main between-group differences reside. The method requires no information about compartment size (for example, organelle profile area or membrane trace length) and does not even depend critically on standardising between-group magnification. Its application is illustrated using datasets from immunolabelling studies designed to localise the KDEL receptor, phosphatidyl-inositol 4,5-bisphosphate, GLUT4 and rab4 at the electron microscopic level.

Estrogen modulates xanthine dehydrogenase/xanthine oxidase activity by a receptor-independent mechanism.

Hypoxia causes up-regulation and activation of xanthine dehydrogenase/xanthine oxidase (XDH/XO) in vitro and in the lungs in vivo. This up-regulation, and the likely corresponding production of reactive oxygen species, may underlie the pathogenesis of an array of disorders. Thus, compounds that prevent hypoxia-induced increase in XDH/XO activity may provide a therapeutic strategy in such disorders. The antioxidant properties of estrogens have been demonstrated in several studies. However, the effect of these compounds on XDH/XO has not been explored previously. The aim of this study was to investigate the effects of estrogen on hypoxia-induced increase in XDH/XO activity. Rat pulmonary artery microvascular endothelial cells were exposed to normoxia or hypoxia in the presence or absence of 17beta- or 17alpha-estradiol. The XDH/XO enzyme and gene promoter activities were measured in different groups of cells. Hypoxia caused a twofold increase in XDH/XO enzymatic and promoter activity. Either of the estradiol stereoisomers prevented the hypoxia-induced increase in XDH/XO enzymatic activity, but not the promoter activity. ICI 182,780, an antagonist of the estrogen receptor, failed to block the inhibitory effect of estradiol on XDH/XO. In conclusion, 17alpha- and 17beta-estradiol modulate the hypoxia-induced regulation of XDH/XO activity at a posttranscriptional level by a receptor-independent mechanism.