Biomedical subjects

T Haller

Publications and source records attributed to T Haller.

At least 19 recordsLinked to original sources

PubMed2006

A fluorescent microplate assay for exocytosis in alveolar type II cells.

The authors describe a simple, reliable, and quantitative assay to monitor exocytotic fusion of lamellar bodies (LBs) in adherent rat alveolar type II (AT II) cells. The assay is based on fluorescence measurements of LB-plasma membrane (PM) fusions modified for the use in multiwell culture plates to obtain a high-sample throughput. In particular, it is based on the presence of a highly light-absorbing dye in the cell supernatants to increase the specificity of fluorescence signals and to yield pseudo-confocal information from the cells. When the assay was tested with agonist-(ATP) and phorbolester-induced stimulation of LB-PM fusions, the authors found a good correlation with direct microscopic investigations based on single cell recordings. To further validate the assay, they used Curosurf at 10 mg/ml. However, it influenced neither the basal nor the ATP-stimulated rate of LB-PM fusions. This was corroborated by the fact that Curosurf had no effect on resting Ca (2+) levels nor the ATP induced Ca (2+) signals. The results cast new light on previous findings that surfactant phospholipids decrease the rate of secretion in AT II cells in a dose-dependent way. The authors conclude that the inhibitory effect exerted by phospholipids might be due to action on a later step in exocytosis, probably associated with exocytotic fusion pore expansion and content release out of fused vesicles.

Adenosine Triphosphate↗

PubMed2005

Optical measurement of surface tension in a miniaturized air-liquid interface and its application in lung physiology.

We have previously shown that lamellar body-like particles, the form in which pulmonary surfactant is secreted, spontaneously disintegrate when they contact an air-liquid interface, eventually creating an interfacial film. Here, we combined these studies with a new technique enabling the simultaneous and non-invasive measurement of surface tension (gamma). This method is a refinement of the pendant-drop principle. A sapphire cone with a 300-microm aperture keeps the experimental fluid by virtue of surface coherence in a fixed and nearly planar position above the objective of an inverted microscope. The radius of curvature of the fluid meniscus is related to gamma and determines the pattern of light back-reflection upon epi-illumination. This method, which we name "inverted interface", has several novel aspects, in particular its microscopic dimensions. When using lamellar body-like particles freshly released by alveolar type II cells, we found that their conversion at the interface resulted in gamma-reduction close to 30 mN/m. After a fast initial decay, gamma-decrease proceeded slowly and in proportion to single particle conversions. These conversions ceased with time whereas gamma decreased further, probably due to reorganization of the already deposited material. The present investigation indicates that surface film formation by adsorption of large surfactant aggregates is an important mechanism by which gamma is reduced in the lung.

Animals↗

PubMed2004

Tracing surfactant transformation from cellular release to insertion into an air-liquid interface.

Pulmonary surfactant is secreted by alveolar type II cells as lipid-rich, densely packed lamellar body-like particles (LBPs). The particulate nature of released LBPs might be the result of structural and/or thermodynamic forces. Thus mechanisms must exist that promote their transformation into functional units. To further define these mechanisms, we developed methods to follow LBPs from their release by cultured cells to insertion in an air-liquid interface. When released, LBPs underwent structural transformation, but did not disperse, and typically preserved a spherical appearance for days. Nevertheless, they were able to modify surface tension and exhibited high surface activity when measured with a capillary surfactometer. When LBPs inserted in an air-liquid interface were analyzed by fluorescence imaging microscopy, they showed remarkable structural transformations. These events were instantaneous but came to a halt when the interface was already occupied by previously transformed material or when surface tension was already low. These results suggest that the driving force for LBP transformation is determined by cohesive and tensile forces acting on these particles. They further suggest that transformation of LBPs is a self-regulated interfacial process that most likely does not require structural intermediates or enzymatic activation.

Air↗

PubMed2004

[Indentification of institutional factors affecting the well-being of sedentary and nomadic populations living in the Waza-Logone flood plain along the border between Cameroon and Chad].

This article describes an ongoing research project designed to compile a database to promote pastoral development in the lowlands along the Logone and Chari Rivers in Cameroon. A number of sedentary and nomadic populations depend on these flood plains south of Lake Chad for their livelihood. However the natural resources of the area undergo sharp seasonal variations and sometimes become the property of sedentary groups. As a result nomadic communities experience difficulty not only in gaining access to grazing lands and water but also to quality health care (hospital centers, effective medication). The purpose of this study was to define institutional requirements necessary to ensure access to health care resources for both nomadic and sedentary groups. The main problem for the nomadic population is that, unlike the now defunct pre-colonial structures, today's institutions are not compatible with the subsistence strategies of rural populations. These findings suggest that new institutional frameworks for natural resource management could indirectly improve the health status of nomadic pastoralist.

Cameroon↗

PubMed2001

Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells.

In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579-1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Fick's law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation.

Adenosine Triphosphate↗

PubMed2001

A subpopulation of mitochondria prevents cytosolic calcium overload in endothelial cells after cold ischemia/reperfusion.

BACKGROUND: Calcium represents a key mediator of cold ischemia/reperfusion (CIR) injury presumably by affecting mitochondrial function. In this study, we investigated cellular and mitochondrial changes of calcium homeostasis in sublethally damaged human endothelial cells. METHODS: Changes in cellular and mitochondrial calcium concentrations were studied after cold ischemia in University of Wisconsin solution for 12 hr and reperfusion in ringer solution. Cytosolic-free calcium concentration ([Ca2+]c) and mitochondrial-free calcium content ([Ca2+]m) were analyzed by fura-2 and rhod-2 fluorescence, respectively. Pretreatment of cells with ruthenium red (RR) or a H+-ionophore was used to inhibit mitochondrial calcium uptake. Mitochondrial membrane potential (DeltaPsim) was measured by 5,5',6,6'-tetrachloro- 1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide and 3,3'-dihexyloxacarbocyanine iodide fluorescence. RESULTS: Twelve-hr cold ischemia did not induce apoptosis in endothelial cells. In such sublethally damaged cells, [Ca2+]c rose from approximately 20 nmol/L after cold ischemia to approximately 120 nmol/L during reperfusion. Pretreatment with RR leads to an approximately 5-fold rise in [Ca2+]c. Image analysis revealed a significant increase of [Ca2+]m in a subpopulation of mitochondria during reperfusion. This was not the case in RR-pretreated cells. DeltaPsim decreased significantly during cold ischemia and was sustained during reperfusion. The loss of DeltaPsim can be related to a reduced portion of mitochondria exhibiting high DeltaPsim. CONCLUSIONS: Our results suggest that cytosolic calcium influx during CIR is buffered by a selective portion of mitochondria in human umbilical vein endothelial cells. These mitochondria protect cells against cytosolic calcium overload and probably against subsequent cell injury.

Calcium↗

PubMed2001

Inhibition of ATP-induced surfactant exocytosis by dihydropyridine (DHP) derivatives: a non-stereospecific, photoactivated effect and independent of L-type Ca2+ channels.

Purinergic stimulation of surfactant secretion via exocytosis of lamellar bodies is mediated by an elevation of the intracellular Ca2+ concentration ([Ca2+](i)). We tested the dihydropyridine (DHP) analogues isradipine (+/-enantiomers), nifedipine and Bay K 8644 (racemic forms) on ATP-induced surfactant secretion and [Ca2+](i) in single type II cells, using FM1-43 and fura-2 fluorescence. None of the DHPs (2 microM) had an effect on ATP-induced surfactant secretion in the dark. They did, however, inhibit secretion in a concentration-dependent manner during illumination, particularly with UV light. This effect was not stereospecific, because it was mimicked by (-)-isradipine. In addition, (+)- or (-)-isradipine, but not nifedipine or Bay K 8644, elicited a slow increase of [Ca2+](i) during illumination with UV light, which was reversible by exposure to dark. None of the DHPs inhibited the ATP-induced Ca2+ signal. In perforated patch clamp experiments, depolarizing voltage steps did not induce L-type Ca2+ (Sr(2+)) currents, even in the presence of the agonist Bay K 8644 (1 microM). We conclude that impairment of ATP-induced surfactant secretion by all tested DHPs and alterations of Ca2+ homeostasis by isradipine are photoactivated effects, independent of L-type Ca2+ channels.

Adenosine Triphosphate↗

PubMed2001

The conception of fusion pores as rate-limiting structures for surfactant secretion.

It is well established that the release of surfactant phospholipids into the alveolar lumen proceeds by the exocytosis of lamellar bodies (LBs), the characteristic storage organelles of surfactant in alveolar type II cells. Consequently, the fusion of LBs with the plasma membrane and the formation of exocytotic fusion pores are key steps linking cellular synthesis of surfactant with its delivery into the alveolar space. Considering the unique structural organization of LBs or LB-associated aggregates which are found in lung lavages, and the roughly 1-microm-sized dimensions of these particles, we speculated whether the fusion pore diameter of fused LBs might be a specific hindrance for surfactant secretion, delaying or even impeding full release. In this mini-review, we have compiled published data shedding light on a possibly important role of fusion pores during the secretory process in alveolar type II cells.

Animals↗

PubMed2001

Signal transduction pathways in directed migration of human monocytes induced by human growth hormone in vitro.

The human growth hormone (GH) was shown to modulate leukocyte functions such as stimulating directed migration of human monocytes in vitro. Dimerisation of GH-receptors leads to the activation of various signalling mechanisms. As transduction of GH signals to monocytes is unknown, we investigated GH signalling mechanisms in monocyte migration using a modified Boyden chamber chemotaxis assay. Inhibition of tyrosyl phosphorylation of GH receptor-associated tyrosine kinase by tyrphostin-23 or staurosporine blocked GH-stimulated monocyte migration down to random levels. Furthermore, pre-incubation with effective concentrations of 4B-phorbol-12-myristate-13-acetate (PMA), staurosporine and bisindolylmaleimide I, inhibitors of protein kinase C, significantly decreased GH-induced migration, suggesting that PKC is involved in the signalling cascade. Additionally, phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) activation seems to be required. This study revealed signalling pathways in monocyte movement toward GH in vitro.

Calcium↗

PubMed2001

The crotonase superfamily: divergently related enzymes that catalyze different reactions involving acyl coenzyme a thioesters.

Synergistic investigations of the reactions catalyzed by several members of an enzyme superfamily provide a more complete understanding of the relationships between structure and function than is possible from focused studies of a single enzyme alone. The crotonase (or enoyl-CoA hydratase) superfamily is such an example whereby members catalyze a wide range of metabolic reactions but share a common structural solution to a mechanistic problem. Some enzymes in the superfamily have been shown to display dehalogenase, hydratase, and isomerase activities. Others have been implicated in carbon-carbon bond formation and cleavage as well as the hydrolysis of thioesters. While seemingly unrelated mechanistically, the common theme in this superfamily is the need to stabilize an enolate anion intermediate derived from an acyl-CoA substrate. This apparently is accomplished by two structurally conserved peptidic NH groups that provide hydrogen bonds to the carbonyl moieties of the acyl-CoA substrates and form an "oxyanion hole".

Acyl Coenzyme A↗

PubMed2001

Mechanisms of surfactant exocytosis in alveolar type II cells in vitro and in vivo.

Surfactant secretion must be regulated to maintain a low surface tension in the lung during various conditions such as exercise. In vitro studies reveal a slow, unique exocytotic process at the interface of stimulated and constitutive exocytosis. The exocytotic mechanisms and sites of regulation in vivo, however, are still poorly understood.

Animals↗

PubMed2001

Secretion in alveolar type II cells at the interface of constitutive and regulated exocytosis.

Long-term, simultaneous, measurements of cytoplasmic free Ca(2+) concentrations and single exocytotic fusion events in surfactant-secreting type II cells were performed. All fusion (constitutive, phorbol ester-induced, and agonist-induced) was Ca(2+)-dependent. Kinetic analysis revealed that agonist (adenosine triphosphate [ATP])-induced fusion exhibited a kinetic pattern that correlated well with the Ca(2+) signal. The effects of Ca(2+) release from intracellular stores (early) and Ca(2+) entry (late) could be demonstrated for the first time by dissecting the slow (10-to-15-min) fusion response to ATP into these two components. Bath Ba(2+) or Sr(2+) could replace Ca(2+) to elicit a fusion response in thapsigargin-pretreated cells lacking ATP-induced Ca(2+) release from stores. Although the late response was partially inhibited by interrupting the phospholipase D-protein kinase C axis, a high Ca(2+) dependence of the entire secretory course was demonstrated by a significant correlation between the integrated Ca(2+) signal and the fusion response. There was also a highly significant correlation between constitutive and ATP-stimulated fusion activity in individual cells. We propose a common mechanistic model for all types of fusion in this slow secretory cell, in which constitutive and regulated forms of exocytosis are subject to the same principles of regulation.

Adenosine Triphosphate↗

PubMed2001

[Formaldehyde fixation in the third millennium].

This review deals with the general principles and problems of formaldehyde fixation. After a short description of 1) formaldehyde methods of production, 2) chemical properties of formaldehyde solution, and 3) kinetic of formaldehyde binding in tissue, formaldehyde reactivity with the tissue biopolymers, proteins and cucleic acids mainly, are described. How formaldehyde fixation of tissues adversely affects the reactivity of cellular proteins with their respective specific antibody and the ways the most commonly used retrieval techniques in immunohistochemistry act are, thereafter, discussed. Finally, concerns that need to be dealt with when formalin-fixed specimens are used for genomic analysis and studies of DNA expression are highlighted.

DNA↗

PubMed2001

Aciclovir selects for ganciclovir-cross-resistance of human cytomegalovirus in vitro that is only in part explained by known mutations in the UL97 protein.

Phenotypically, ganciclovir-resistant human cytomegalovirus strains could be selected by aciclovir as effectively as by ganciclovir in vitro. Three clinical human cytomegalovirus isolates with different sensitivities against ganciclovir, aciclovir, foscarnet, and cidofovir, but without any mutation in the viral UL97 protein known to confer ganciclovir resistance, were propagated each in duplicate in the presence of ganciclovir or aciclovir. After drug selection, all 12 strains were less susceptible to ganciclovir (increase of 50% focus reduction dose between 2.1- and 31.5-fold) but were still sensitive to foscarnet and cidofovir; 7/12 exhibited a ganciclovir-resistant phenotype with a 50% focus reduction dose >30 microM, and in 6 out of these typical mutations in the UL97 coding region could be found by genotyping. All four strains selected from one isolate carried the identical UL97 mutation at amino acid position 460 (methionine to valine). The decreased sensitivity to ganciclovir and aciclovir in the other strains could neither be attributed to known UL97 mutations nor to mutations in the viral polymerase (UL54), which have been reported to induce resistance.

Acyclovir↗

PubMed2000

Discovering new enzymes and metabolic pathways: conversion of succinate to propionate by Escherichia coli.

The Escherichia coli genome encodes seven paralogues of the crotonase (enoyl CoA hydratase) superfamily. Four of these have unknown or uncertain functions; their existence was unknown prior to the completion of the E. coli genome sequencing project. The gene encoding one of these, YgfG, is located in a four-gene operon that encodes homologues of methylmalonyl CoA mutases (Sbm) and acyl CoA transferases (YgfH) as well as a putative protein kinase (YgfD/ArgK). We have determined that YgfG is methylmalonyl CoA decarboxylase, YgfH is propionyl CoA:succinate CoA transferase, and Sbm is methylmalonyl CoA mutase. These reactions are sufficient to form a metabolic cycle by which E. coli can catalyze the decarboxylation of succinate to propionate, although the metabolic context of this cycle is unknown. The identification of YgfG as methylmalonyl CoA decarboxylase expands the range of reactions catalyzed by members of the crotonase superfamily.

Amino Acid Sequence↗

PubMed2000

New reactions in the crotonase superfamily: structure of methylmalonyl CoA decarboxylase from Escherichia coli.

The molecular structure of methylmalonyl CoA decarboxylase (MMCD), a newly defined member of the crotonase superfamily encoded by the Escherichia coli genome, has been solved by X-ray crystallographic analyses to a resolution of 1.85 A for the unliganded form and to a resolution of 2.7 A for a complex with an inert thioether analogue of methylmalonyl CoA. Like two other structurally characterized members of the crotonase superfamily (crotonase and dienoyl CoA isomerase), MMCD is a hexamer (dimer of trimers) with each polypeptide chain composed of two structural motifs. The larger N-terminal domain contains the active site while the smaller C-terminal motif is alpha-helical and involved primarily in trimerization. Unlike the other members of the crotonase superfamily, however, the C-terminal motif is folded back onto the N-terminal domain such that each active site is wholly contained within a single subunit. The carboxylate group of the thioether analogue of methylmalonyl CoA is hydrogen bonded to the peptidic NH group of Gly 110 and the imidazole ring of His 66. From modeling studies, it appears that Tyr 140 is positioned within the active site to participate in the decarboxylation reaction by orienting the carboxylate group of methylmalonyl CoA so that it is orthogonal to the plane of the thioester carbonyl group. Surprisingly, while the active site of MMCD contains Glu 113, which is homologous to the general acid/base Glu 144 in the active site of crotonase, its carboxylate side chain is hydrogen bonded to Arg 86, suggesting that it is not directly involved in catalysis. The new constellation of putative functional groups observed in the active site of MMCD underscores the diversity of function in this superfamily.

Acyl Coenzyme A↗

PubMed2000

Persistent fusion pores but transient fusion in alveolar type II cells.

The release of vesicle contents following exocytotic fusion is limited by various factors including the size of the fusion pore. Fusion pores are channel-like, narrow structures after formation and proceed through semi-stable states ('fusion pore flickering'), unless they fully expand (full fusion) or close again (transient fusion). Partial release of vesicle contents may occur during transient fusion, which was described to last between milliseconds and seconds, depending on the size of the vesicle. We studied fusion pores in a slow-secreting lung epithelial cell (type II cell) using fluorescence staining of vesicle contents (surfactant) and fluorescence recovery after photobleaching (FRAP). Surfactant is a lipidic material, which is secreted into the alveolar lumen to reduce the surface tension in the lung. We found release of surfactant to be a slow process, which can last for hours. Accordingly, fusion pores in these cells are stable structures, which appear to be a barrier for release. FRAP measurements suggest that transient fusions occasionally take place in these long-lasting fusion pores, resulting in partial release of surfactant into the extracellular space. These data suggest that postfusion mechanisms may regulate the amount of secreted surfactant.

Animals↗

PubMed2000

Mitochondrial calcium overload is restricted to a few mitochondria in endothelial cells after cold ischemia/reperfusion.

Changes in cytosolic and mitochondrial calcium content were studied in an endothelial cell model after simulating cold ischemia reperfusion injury. Image analysis demonstrated that only a subpopulation of mitochondria in endothelial cells accumulate calcium. Observations led to the hypothesis that mitochondria which are in close contact with the plasma membrane are mainly affected by the Ca2+ efflux across that membrane, while those in other parts of the cell remain unaffected.

Adenosine↗