[Diagnosis and therapy of attention deficit-/hyperkinetic disorder in adulthood].
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Adolescent↗
Biomedical subjects
K H Krause
Publications and source records attributed to K H Krause.
A novel H(+) conductance in eosinophils: unique characteristics and absence in chronic granulomatous disease.
Efficient mechanisms of H(+) ion extrusion are crucial for normal NADPH oxidase function. However, whether the NADPH oxidase-in analogy with mitochondrial cytochromes-has an inherent H(+) channel activity remains uncertain: electrophysiological studies did not find altered H(+) currents in cells from patients with chronic granulomatous disease (CGD), challenging earlier reports in intact cells. In this study, we describe the presence of two different types of H(+) currents in human eosinophils. The "classical" H(+) current had properties similar to previously described H(+) conductances and was present in CGD cells. In contrast, the "novel" type of H(+) current had not been described previously and displayed unique properties: (a) it was absent in cells from gp91- or p47-deficient CGD patients; (b) it was only observed under experimental conditions that allowed NADPH oxidase activation; (c) because of its low threshold of voltage activation, it allowed proton influx and cytosolic acidification; (d) it activated faster and deactivated with slower and distinct kinetics than the classical H(+) currents; and (e) it was approximately 20-fold more sensitive to Zn(2+) and was blocked by the histidine-reactive agent, diethylpyrocarbonate (DEPC). In summary, our results demonstrate that the NADPH oxidase or a closely associated protein provides a novel type of H(+) conductance during phagocyte activation. The unique properties of this conductance suggest that its physiological function is not restricted to H(+) extrusion and repolarization, but might include depolarization, pH-dependent signal termination, and determination of the phagosomal pH set point.
Adult↗
Calreticulin is essential for cardiac development.
Calreticulin is a ubiquitous Ca2+ binding protein, located in the endoplasmic reticulum lumen, which has been implicated in many diverse functions including: regulation of intracellular Ca2+ homeostasis, chaperone activity, steroid-mediated gene regulation, and cell adhesion. To understand the physiological function of calreticulin we used gene targeting to create a knockout mouse for calreticulin. Mice homozygous for the calreticulin gene disruption developed omphalocele (failure of absorption of the umbilical hernia) and showed a marked decrease in ventricular wall thickness and deep intertrabecular recesses in the ventricular walls. Transgenic mice expressing a green fluorescent protein reporter gene under the control of the calreticulin promoter were used to show that the calreticulin gene is highly activated in the cardiovascular system during the early stages of cardiac development. Calreticulin protein is also highly expressed in the developing heart, but it is only a minor component of the mature heart. Bradykinin-induced Ca2+ release by the InsP3-dependent pathway was inhibited in crt-/- cells, suggesting that calreticulin plays a role in Ca2+ homeostasis. Calreticulin-deficient cells also exhibited impaired nuclear import of nuclear factor of activated T cell (NF-AT3) transcription factor indicating that calreticulin plays a role in cardiac development as a component of the Ca2+/calcineurin/NF-AT/GATA-4 transcription pathway.
Animals↗
Ca2+ regulation of interactions between endoplasmic reticulum chaperones.
Casade Blue (CB), a fluorescent dye, was used to investigate the dynamics of interactions between endoplasmic reticulum (ER) lumenal chaperones including calreticulin, protein disulfide isomerase (PDI), and ERp57. PDI and ERp57 were labeled with CB, and subsequently, we show that the fluorescence intensity of the CB-conjugated proteins changes upon exposure to microenvironments of a different polarity. CD analysis of the purified proteins revealed that changes in the fluorescence intensity of CB-ERp57 and CB-PDI correspond to conformational changes in the proteins. Using this technique we demonstrate that PDI interacts with calreticulin at low Ca2+ concentration (below 100 microM), whereas the protein complex dissociates at >400 microM Ca2+. These are the Ca2+ concentrations reminiscent of Ca2+ levels found in empty or full ER Ca2+ stores. The N-domain of calreticulin interacts with PDI, but Ca2+ binding to the C-domain of the protein is responsible for Ca2+ sensitivity of the interaction. ERp57 also interacts with calreticulin through the N-domain of the protein. Initial interaction between these proteins is Ca2+-independent, but it is modulated by Ca2+ binding to the C-domain of calreticulin. We conclude that changes in ER lumenal Ca2+ concentration may be responsible for the regulation of protein-protein interactions. Calreticulin may play a role of Ca2+ "sensor" for ER chaperones via regulation of Ca2+-dependent formation and maintenance of structural and functional complexes between different proteins involved in a variety of steps during protein synthesis, folding, and post-translational modification.
Animals↗
Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin alpha5beta1.
The ability of Staphylococcus aureus to invade mammalian cells may explain its capacity to colonize mucosa and to persist in tissues after bacteraemia. To date, the underlying molecular mechanisms of cellular invasion by S. aureus are unknown, despite its high prevalence and difficulties in treatment. Here, we show cellular invasion as a novel function for an S. aureus adhesin, previously implicated solely in attachment. S. aureus, but not S. epidermidis, invaded epithelial 293 cells in a temperature- and F-actin-dependent manner. Formaldehyde-fixed and live bacteria were equally invasive, suggesting that no active bacterial process was involved. All clinical S. aureus isolates analysed, but only a subset of laboratory strains, were invasive. Fibronectin-binding proteins (FnBPs) acted as S. aureus invasins, because: (i) FnBP deletion mutants of invasive laboratory strains lost invasiveness; (ii) expression of FnBPs in noninvasive strains conferred invasiveness; and (iii) the soluble isolated fibronectin-binding domain of FnBP (D1-D4) completely blocked invasion. Integrin alpha5beta1 served as host cell receptor, which interacted with staphylococcal FnBPs through cellular or soluble fibronectin. FnBP-deficient mutants lost invasiveness for epithelial cells, endothelial cells and fibroblasts. Thus, fibronectin-dependent bridging between S. aureus FnBPs and host cell integrin alpha5beta1 is a conserved mechanism for S. aureus invasion of human cells. This may prove useful in developing new therapeutic and vaccine strategies for S. aureus infections.
Adhesins, Bacterial↗
Localization of intracellular Ca2+ stores in HeLa cells during infection with Chlamydia trachomatis.
Chlamydia trachomatis elementary bodies (EBs) enter epithelial cells within membrane-bound endosomes that aggregate with each other in a calcium-regulated process, but avoid fusion with lysosomes. Annexin III but not I translocates to chlamydial aggregates and inclusions. In this study, we localize the intracellular Ca2+ stores during the course of infection by analyzing the distribution of three intracellular Ca2+ store proteins: calreticulin, type-1 inositol-1,4, 5-trisphosphate receptor (IP3-R), and Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPase type 2 (SERCA2) in HeLa cells infected with C. trachomatis serovar L2. In uninfected cells, immunofluorescence staining of the proteins showed a fine granular distributed pattern for all three proteins. After infection with C. trachomatis, calreticulin was found at the periphery of chlamydial aggregates and inclusions from 3 to 48 hours post-infection. In infected cells, SERCA2 was intimately associated with chlamydial inclusions after 3 and 24 hours, but not after 48 hours. Moreover, IP3-R was translocated to and colocalized with EB aggregates and chlamydial inclusions and had a distribution very similar to that of SERCA 2. After 24 hours incubation with chlamydiae, there was a local accumulation of [Ca2+]i (105+/-17 nM) in the proximity of chlamydial inclusions, compared to 50+/-13 nM in other parts of the cell cytoplasm. In the absence of extracellular Ca2+, this local accumulation of Ca2+ increased to 295+/-50 nM after adding 50 microM ATP, and to a similar extent after adding 100 nM thapsigargin (Tg). These data indicate that during infection of HeLa cells with chlamydiae, intracellular Ca2+ stores are redistributed, causing local accumulation of Ca2+ in the vicinity of chlamydial inclusions. These changes may trigger the association of certain proteins such as annexins with chlamydia-containing vesicles, and thereby regulation of membrane-membrane interaction during endosome aggregation and inclusion formation.
Adenosine Triphosphate↗
Ca2+-induced exocytosis in individual human neutrophils: high- and low-affinity granule populations and submaximal responses.
We have investigated Ca2+-induced exocytosis from human neutrophils using the whole cell patch-clamp capacitance technique. Microperfusion of Ca2+ buffer solutions (<30 nM to 5 mM free Ca2+) through the patch-clamp pipette revealed a biphasic activation of exocytosis by Ca2+. The first phase was characterized by high affinity (1.5-5 microM) and low apparent cooperativity (<=2) for Ca2+, and the second phase by low affinity (approximately 100 microM) and high cooperativity (>6). Only the second phase was accompanied by loss of myeloperoxidase, suggesting that the low-affinity exocytosis reflected release of peroxidase-positive (primary) granules, while the high-affinity exocytosis reflected release of peroxidase-negative (secondary and tertiary) granules. At submaximal Ca2+ concentrations, only a fraction of a given granule population was released. This submaximal release cannot simply be explained by Ca2+ modulation of the rate of exocytosis, and it suggests that the secretory response of individual cells is adjusted to the strength of the stimulus. The Ca2+ dependence of the high- and low-affinity phases of neutrophil exocytosis bears a resemblance to endocrine and neuronal exocytosis, respectively. The occurrence of such high- and low-affinity exocytosis in the same cell is novel, and suggests that the Ca2+ sensitivity of secretion is granule-, rather than cell-specific.
Calcium↗
Aerolysin induces G-protein activation and Ca2+ release from intracellular stores in human granulocytes.
Aerolysin is a pore-forming toxin that plays a key role in the pathogenesis of Aeromonas hydrophila infections. In this study, we have analyzed the effect of aerolysin on human granulocytes (HL-60 cells). Proaerolysin could bind to these cells, was processed into active aerolysin, and led to membrane depolarization, indicating that granulocytes are potential targets for this toxin. Fura-2 measurements were used to analyze the effect of aerolysin on cytosolic [Ca2+] homeostasis. As expected for a pore-forming toxin, aerolysin addition led to Ca2+ influx across the plasma membrane. In addition, the toxin triggered Ca2+ release from agonist and thapsigargin-sensitive intracellular Ca2+ stores. This Ca2+ release was independent of the aerolysin-induced Ca2+ influx and occurred in two kinetically distinct phases: an initial rapid and transient phase and a second, more sustained, phase. The first, but not the second phase was sensitive to pertussis toxin. Activation of pertussis toxin-sensitive G-proteins appeared to be a consequence of pore formation, rather than receptor activation through aerolysin-binding, as it: (i) was not observed with a binding competent, insertion-incompetent aerolysin mutant, (ii) had a marked lag time, and (iii) was also observed in response to other bacterial pore-forming toxins (staphylococcal alpha-toxin, streptolysin O) which are thought to bind to different receptors. G-protein activation through pore-forming toxins stimulated cellular functions, as evidenced by pertussis toxin-sensitive chemotaxis. Our results demonstrate that granulocytes are potential target cells for aerolysin and that in these cells, Ca2+ signaling in response to a pore-forming toxin involves G-protein-dependent cell activation and Ca2+ release from intracellular stores.
Bacterial Proteins↗
Electron currents generated by the human phagocyte NADPH oxidase.
Electron transport across biological membranes is a well-known feature of bacteria, mitochondria and chloroplasts, where it provides motive forces for vectorial transport processes. In contrast, electron transport is generally not found in the plasma membrane of eukaryotic cells, possibly because it would interfere with electric processes at the plasma membrane. An exception is provided by the phagocyte NADPH oxidase, which generates superoxide (O2.-) through electron transfer from cytosolic NADPH to extracellular oxygen. The enzyme is essential for host defence, and patients with chronic granulomatous disease, who lack the functional enzyme, suffer from severe infections. It has been suggested that electron transfer by the NADPH oxidase might be electrogenic. Here we demonstrate, using the whole-cell patch-clamp technique, the generation of electron currents by the NADPH oxidase in human eosinophil granulocytes. The currents were absent in granulocytes of sufferers of chronic granulomatous disease and under conditions of low oxygen. Generation of electron currents across the plasma membrane of eukaryotic cells has not been observed previously and might be-independently of the generation of superoxide-a physiologically relevant function of the phagocyte NADPH oxidase.
Adult↗
[Smoking and hyperkinetic syndrome].
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Adult↗
[Hyperkinetic syndrome (attention deficit-/hyperactivity disorder) in adulthood].
The clinical picture of adult hyperkinetic syndrome (HKS) or attention deficit/hyperactivity disorder is nearly unknown in Germany. It can be estimated, that approximately one third of affected children also show symptoms as adults. In the combined type of the syndrome symptoms of inattention as well as of hyperactivity and impulsivity are present, a predominantly inattentive or hyperactive-impulsive type is possible. Retrospective diagnosis of HKS in childhood can be difficult. Disorganization, emotional disturbances and stress intolerance are common in adults with HKS as well as residual symptoms of learning disorders like dyslexia, dyscalculia and dysgraphia. In differential diagnosis especially affective, anxiety and antisocial personality disorders have to be considered, for which on the other side a frequent comorbidity with HKS is known. There is strong evidence for genetic transmission. Neurobiological findings revealed dysregulation of neurotransmitters. For treatment stimulants as pemoline and methamphetamin are effective, in addition tricyclic antidepressants or beta blockers; positive effects are probable for moclobemide, bupropion, fluoxetine and venlafaxine.
Adolescent↗
Store-operated Ca2+ influx and stimulation of exocytosis in HL-60 granulocytes.
This study addresses the role of store-operated Ca2+ influx in the regulation of exocytosis in inflammatory cells. In HL-60 granulocytes, which do not possess voltage-operated Ca2+ channels, the chemotactic peptide fMet-Leu-Phe (fMLP) was able to stimulate store-operated Ca2+ influx and to trigger exocytosis of primary granules. An efficient triggering of exocytosis by fMLP required the presence of extracellular Ca2+ and was inhibited by blockers of store-operated Ca2+ influx. However, receptor-independent activation of store-operated Ca2+ influx through thapsigargin did not trigger exocytosis. fMLP was unable to stimulate exocytosis in the absence of cytosolic free Ca2+ concentration [Ca2+]c elevations. However, a second signal generated by fMLP synergized with store-operated Ca2+ influx to trigger exocytosis and led to a left shift of the exocytosis/[Ca2+]c relationship in ionomycin-stimulated cells. The synergistic fMLP-generated signaling cascade was long-lasting, involved a pertussis toxin-sensitive G protein and a phosphatidylinositol 3-kinase. In summary, store-operated Ca2+ influx is crucial for the efficient triggering of exocytosis in HL-60 granulocytes, but, as opposed to Ca2+ influx through voltage-operated Ca2+ channels in neurons, it is not a sufficient stimulus by itself and requires synergistic receptor-generated signals.
Androstadienes↗
Nef-mediated clathrin-coated pit formation.
The sequence of events leading to clathrin-coated pit (CCP) nucleation on the cell surface and to the incorporation of receptors into these endocytic structures is still imperfectly understood. In particular, the question remains as to whether receptor tails initiate the assembly of the coat proteins or whether receptors migrate into preformed CCP. This question was approached through a dissection of the mechanisms implemented by Nef, an early protein of human and simian immunodeficiency virus (HIV and SIV, respectively), to accelerate the endocytosis of cluster of differentiation antigen type 4 (CD4), the major receptor for these viruses. Results collected showed that: (a) Nef promotes CD4 internalization via an increased association of CD4 with CCP; (b) the Nef-mediated increase of CD4 association with CCP is related to a doubling of the plasma membrane area occupied by clathrin-coated structures; (c) this increased CCP number at the plasma membrane has functional consequences preferentially on CD4 uptake and does not significantly affect transferrin receptor internalization or fluid-phase endocytosis; (d) the presence of a CD4 cytoplasmic tail including a critical dileucine motif is required to induce CCP formation via Nef; and (e) when directly anchored to the cytoplasmic side of the plasma membrane, Nef itself can promote CCP formation. Taken together, these observations lead us to propose that CD4 can promote CCP generation via the connector molecule Nef. In this model, Nef interacts on one side with CD4 through a dileucine-based motif present on CD4 cytoplasmic tail and on the other side with components of clathrin-coated surface domain (i.e., adaptins). These Nef-generated complexes would then initiate the nucleation of CCP.
CD4 Antigens↗
Chemoattractant-induced respiratory burst: increases in cytosolic Ca2+ concentrations are essential and synergize with a kinetically distinct second signal.
The role of the cytosolic free Ca2+ concentration ([Ca2+]c) and its relationship to other second messengers in the signalling between chemoattractant [e.g. N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP)] receptors and the NADPH oxidase is still poorly understood. In this study, we have used thapsigargin, an inhibitor of the Ca2+-ATPase of intracellular stores, as a tool to selectively manipulate Ca2+ release from intracellular stores and Ca2+ influx across the plasma membrane. We thereby temporarily separated the Ca2+ signal from other signals generated by fMLP and analysed the consequences on the respiratory burst. Under all conditions investigated, the extent of fMLP-induced respiratory burst activation was critically determined by [Ca2+]c elevation. fMLP was unable to activate the respiratory burst without [Ca2+]c elevation. Thapsigargin-induced Ca2+ influx activated the respiratory burst in the absence of fMLP, but only to approx. 20% of the values observed in the presence of fMLP. The second signal generated by fMLP did not activate the respiratory burst by itself, but acted in synergy with [Ca2+]c elevation. The second signal was long lasting (>15 min) provided that there was no rise in [Ca2+]c and that the receptor was continuously occupied. The second signal was inactivated by high [Ca2+]c elevation. Our results demonstrate that [Ca2+]c elevations are an essential step in the signalling between the fMLP receptor and NADPH oxidase. They also provide novel information about the properties of the second Ca2+-independent signal that activates the respiratory burst in synergy with [Ca2+]c.
Calcium↗
European workshop on the cell biology of phagocytes.
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Cell Degranulation↗
Second-messenger regulation of receptor association with clathrin-coated pits: a novel and selective mechanism in the control of CD4 endocytosis.
CD4, a member of the immunoglobulin superfamily, is not only expressed in T4 helper lymphocytes but also in myeloid cells. Receptor-mediated endocytosis plays a crucial role in the regulation of surface expression of adhesion molecules such as CD4. In T lymphocytes p56lck, a CD4-associated tyrosine kinase, prevents CD4 internalization, but in myeloid cells p56lck is not expressed and CD4 is constitutively internalized. In this study, we have investigated the role of cyclic AMP (cAMP) in the regulation of CD4 endocytosis in the myeloid cell line HL-60. Elevations of cellular cAMP were elicited by 1) cholera toxin, 2) pertussis toxin, 3) forskolin and IBMX, 4) NaF, or 5) the physiological receptor agonist prostaglandin E1. All five interventions led to an inhibition of CD4 internalization. Increased cAMP levels did not inhibit endocytosis per se, because internalization of insulin receptors and transferrin receptors and fluid phase endocytosis were either unchanged or slightly enhanced. The mechanism of cAMP inhibition was further analyzed at the ultrastructural level. CD4 internalization, followed either by quantitative electron microscopy autoradiography or by immunogold labeling, showed a rapid and temperature-dependent association of CD4 with clathrin-coated pits in control cells. This association was markedly inhibited in cells with elevated cAMP levels. Thus these findings suggest a second-messenger regulation of CD4 internalization through an inhibition of CD4 association with clathrin-coated pits in p56lck-negative cells.
CD4 Antigens↗
Effect of biotin depletion on spike-wave seizures in the tottering mutant mouse.
Influence of depletion of biotin on spontaneously occurring spike-wave seizures in the tottering mutant mouse was tested by electrocorticographic recordings over three hours in five mice fed a biotin deficient diet. Frequency of seizures as well as percent of time with epileptic discharges were clearly lowered in the biotin deficient animals compared with eight mice of the same age under normal food (p < 0.01 in Wilcoxon's U-test).
Animals↗