Genetic susceptibility to temporomandibular joint involvement in juvenile idiopathic arthritis.

BACKGROUND: Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic condition of childhood. Temporomandibular joint (TMJ) is among the most commonly affected joints in JIA patients. When JIA involves the TMJ, it may affect condylar growth in the joint; therefore, JIA patients are at risk of unfavourable long-term outcomes from associated joint damage. If undetected, TMJ involvement can lead to various functional disabilities such as reduced mandibular mobility and disorders of the mastication muscles. Limitations in sagittal and vertical mandibular growth can result in micrognathia and anterior open bite with aesthetic and functional restrictions. OBJECTIVE: Genetic factors may play a role in determining which individuals are more prone to develop TMJ disorders or in predicting the severity of the disease process. Therefore, we applied a GWAS approach to identify loci associated with TMJ involvement in a sample of Estonian patients with JIA. Our aim was to address the potential role of genetic susceptibility factors in TMJ-JIA, a condition not previously studied in this context. METHODS: The case group consisted of 55 JIA patients with TMJ involvement and 208 patients without TMJ involvement comprised the control group. The entire cohort was genotyped using the Illumina HumanOmniExpress BeadChip arrays. Imputation was performed using a nationwide reference panel obtained of 2240 individuals whose data were obtained from the Estonian Biobank. RESULTS: We identified six loci as being associated with the risk of TMJ-JIA in Estonian JIA patients. The strongest associations were identified at CD6 rs3019551 (P = 3.80 × 10-6), SLC26A8/MAPK14 rs9470191 (P = 6.15 × 10-6), NLRP3 rs2056795 (P = 8.91 × 10-6) and MAP2K4 rs7225328 (P = 1.64 × 10-5). CONCLUSION: This study provides first insights into the risk-associated loci between JIA and its manifestation in the TMJ. The reported loci are involved in molecular pathways of immunological relevance and likely represent genomic regions that render the TMJ susceptible to involvement by JIA in Estonian patients.

30-day morbidity and mortality after transoral robotic surgery for human papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma: A retrospective analysis of two prospective adjuvant de-escalation trials (MC1273 & MC1675).

OBJECTIVE: Dose de-escalation of adjuvant therapy (DART) in patients with HPV(+)OPSCC was investigated in two prospective Phase II and III clinical trials (MC1273 and MC1675). We report the 30-day morbidity and mortality associated with primary TORS resection in patients enrolled in these trials. MATERIALS AND METHODS: Patients with HPV(+)OPSCC, who underwent TORS resection between 2013 and 2020 were considered in this analysis. The severity of postoperative transoral bleeding was graded using both the Hinni Grade (HG) transoral surgery bleeding scale and the Common Terminology for Adverse Events (CTCAE) v5.0. Post-surgical complications within 30 days of surgery, as well as rates of tracheostomy, PEG and nasogastric tube placement. RESULTS: 219 patients were included. A total of 7 (3.2 %) patients had a tracheostomy placed at the time of surgery, and all were decannulated within 26 days (median: 5, range: 2-26). There were 33 (15.1 %) returns to the emergency department (ED) with 10 (4.6 %) patients requiring readmission. Using the HG scale, 10 (4.6 %) patients experienced ≥ Grade 3 bleeding with no Grade 5 or 6 bleeds. In contrast, using the CTCAE scale, 15 patients (6.8 %) experienced ≥ Grade 3 bleeding with no Grade 5 bleeds. There was one post-operative death in a patient withdrawn from the trial, and no deaths related to hemorrhage. CONCLUSION AND RELEVANCE: TORS for HPV(+)OPSCC in carefully selected patients at a high volume center was associated with low morbidity and mortality.

Genome-wide analysis of nuclear magnetic resonance metabolites revealed parent-of-origin effect on triglycerides in medium very low-density lipoprotein in PTPRD gene.

AIM: The aim of the study was to explore the parent-of-origin effects (POEs) on a range of human nuclear magnetic resonance metabolites. MATERIALS & METHODS: We search for POEs in 14,815 unrelated individuals from Estonian and Finnish cohorts using POE method for the genotype data imputed with 1000 G reference panel and 82 nuclear magnetic resonance metabolites. RESULTS: Meta-analysis revealed the evidence of POE for the variant rs1412727 in PTPRD gene for the metabolite: triglycerides in medium very low-density lipoprotein. No POEs were detected for genetic variants that were previously known to have main effect on circulating metabolites. CONCLUSION: We demonstrated possibility to detect POEs for human metabolites, but the POEs are weak, and therefore it is hard to detect those using currently available sample sizes.

Conflicts, security and marginalisation: institutional change of the pastoral commons in a 'glocal' world.

This paper argues that pastoral commons are under increasing pressure not just from overuse by pastoralists themselves, but from land management policies. Since colonial times, these have been based on a persistent misconception of the nature of pastoral economies and combined with increasing land alienation and fragmentation through government policies and covert privatisation of pastures. The paper focuses especially on pastoral populations in African drylands and is based on long-term research by independent researchers summarising some of their experiences in western, eastern and southern Africa. Most of them are organised in the African Drylands Dialogue, trying to shed some light on the developments in these areas. Before discussing the actual situation of African pastoralists, the authors focus on basic institutional features of the political and economic management of common grazing lands. This is followed by an overview of land alienation processes in colonial times, which serves as a basis for understanding the current land alienation constellations. The paper then moves on to explain how and why pastoralists are framed by the national discourses as the 'other' and the 'troublemaker', even being labelled as terrorists in nation state contexts. This goes hand in hand with a new wave of land alienation in the form of large-scale land acquisitions or 'land grabbing' (including water grabbing and 'green grabbing' processes). The paper then outlines different coping and adaptation strategies adopted by pastoral groups in a context in which a range of different global and local political, economic and ecological situations interrelate ('glocal'). Finally, the paper discusses the way in which pastoralism could be reframed in a participatory way in the future.

Les auteurs de cet article soutiennent que la pression foncière croissante exercée sur les terres collectives pastorales n'est pas seulement imputable à la surexploitation par les pasteurs eux-mêmes mais résulte surtout des politiques de gestion des terres. Depuis le temps des colonies, ces politiques ont reposé sur une perception erronée et tenace de la nature même des économies pastorales, à laquelle se sont greffées l'aliénation croissante des terres et leur fragmentation impulsée par les politiques gouvernementales et par la privatisation dissimulée des prairies. Les auteurs s'intéressent particulièrement aux populations pastorales des régions arides d'Afrique et exposent les conclusions d'une étude conduite sur une longue durée par une équipe indépendante de chercheurs, résumant l'essentiel de leurs observations en Afrique de l'Ouest, de l'Est et australe. La plupart d'entre eux oeuvrent sous les auspices d'African Drylands Dialogue et tentent de faire la lumière sur les évolutions constatées dans ces régions. Avant de se pencher sur la situation des pasteurs africains aujourd'hui, les auteurs décrivent les principales caractéristiques institutionnelles de la gestion politique et économique des terres collectives dédiées au pâturage. Ils retracent ensuite les processus d'aliénation des terres opérés durant l'époque coloniale, qui servent de grille de lecture pour mieux comprendre les constellations actuelles de terres aliénées. Puis les auteurs expliquent comment et pourquoi les discours nationaux désignent les pasteurs comme « l'autre ¼ et le « fauteur de troubles ¼, quand ils ne les dépeignent pas comme des terroristes dans les contextes d'étatsnations. Ces accusations sont indissociables d'une nouvelle vague d'aliénation des terres, qui prend la forme d'acquisitions à grande échelle ou de réquisitions (y compris les processus d'appropriation des cours d'eau ou d'écosystèmes [green grabbing]). Les auteurs détaillent les stratégies mises en oeuvre par les groupes pastoraux pour faire face à cette évolution et s'y adapter, dans un contexte de forte interaction entre de nombreuses situations politiques, économiques et écologiques de portée tant mondiale que locale (niveau dit « glocal ¼). Enfin, les auteurs examinent les perspectives d'avenir du pastoralisme à travers un nouveau cadre de type participatif.

Los autores postulan que el patrimonio pastoral común se encuentra sometido a presiones crecientes, no solo a resultas de su explotación excesiva por parte de los propios pastores, sino también a consecuencia de las políticas de ordenación del territorio. Desde los tiempos coloniales, estas se basan en un equívoco pertinaz acerca del carácter de las economías pastorales, a lo que se suma un nivel creciente de enajenación y fragmentación de las tierras a resultas de las políticas públicas y la privatización encubierta de los pastos. Los autores prestan especial atención a las poblaciones pastorales de las tierras áridas africanas, basándose en investigaciones de larga duración realizadas por investigadores independientes y resumiendo parte de su experiencia en el África occidental, oriental y meridional. La mayoría de ellos están adscritos al African Drylands Dialogue [diálogo sobre las tierras áridas africanas] y tratan por esta vía de arrojar luz sobre la evolución de esas zonas. Antes de presentar la situación real de las sociedades de pastores africanas, los autores se detienen en una serie de rasgos institucionales básicos de la gestión política y económica de los pastizales de propiedad común. A continuación exponen a grandes líneas los procesos de enajenación de las tierras en la época colonial, que encierran elementos básicos para comprender la actual constelación de tierras enajenadas. Después pasan a explicar cómo y por qué en el discurso de ciertos países las sociedades de pastores han acabado representando la alteridad, percibida además como «agitadora¼, hasta llegar a ser etiquetadas de «terroristas¼ en algunos estados-nación, paralelamente a una nueva oleada de enajenación de tierras en forma de adquisiciones a gran escala o «acaparamiento de tierras¼ (lo que incluye procesos de acaparamiento del agua y «acaparamiento ecológico¼). Tras exponer diferentes estrategias de respuesta y adaptación adoptadas por los grupos pastorales en un contexto marcado por la imbricación entre diversas realidades políticas, económicas y ecológicas («glocal¼), los autores concluyen reflexionando sobre el modo en que en el futuro sería posible reestructurar el pastoreo pasando por métodos participativos.

Sleeping beauty mutase (sbm) is expressed and interacts with ygfd in Escherichia coli.

In Escherichia coli, a four-gene operon, sbm-ygfD-ygfG-ygfH, has been shown to encode a putative cobalamin-dependent pathway with the ability to produce propionate from succinate in vitro [Haller T, Buckel T, Retey J, Gerlt JA. Discovering new enzymes and metabolic pathways: conversion of succinate to propionate by Escherichia coli. Biochemistry 2000;39:4622-4629]. However, the operon was thought to be silent in vivo, illustrated by the eponym describing its first gene, "sleeping beauty mutase" (methylmalonyl-CoA mutase, MCM). Of the four genes described, only ygfD could not be assigned a function. In this study, we have evaluated the functional integrity of YgfD and Sbm and show that, indeed, both proteins are expressed in E. coli and that YgfD has GTPase activity. We show that YgfD and Sbm can be co-immunoprecipitated from E. coli extracts using antibody to either protein, demonstrating in vivo interaction, a result confirmed using a strain deleted for ygfD. We show further that, in vitro, purified His-tagged YgfD and Sbm behave as a monomer and dimer, respectively, and that they form a multi-subunit complex that is dependent on pre-incubation of YgfD with non-hydrolysable GTP, an outcome that was not affected by the state of Sbm, as holo- or apoenzyme. These studies reinforce a role for the in vivo interaction of YgfD and Sbm.

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.

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.

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.

[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]. / A la recherche des déterminants institutionnels du bien-être des populations sédentaires et nomades dans la plaine du Waza-Logone de la frontière camerounaise et tchadienne.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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".

[Formaldehyde fixation in the third millennium]. / La fixation formolée à l'aube du troisième millénaire.

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.

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.