The ΔF508-CFTR mutation inhibits wild-type CFTR processing and function when co-expressed in human airway epithelia and in mouse nasal mucosa

Background

Rescue or correction of CFTR function in native epithelia is the ultimate goal of CF therapeutics development. Wild-type (WT) CFTR introduction and replacement is also of particular interest. Such therapies may be complicated by possible CFTR self-assembly into an oligomer or multimer.

Results

Surprisingly, functional CFTR assays in native airway epithelia showed that the most common CFTR mutant, ??F508-CFTR (??F-CFTR), inhibits WT-CFTR when both forms are co-expressed. To examine more mechanistically, both forms of CFTR were transfected transiently in varying amounts into IB3-1 CF human airway epithelial cells and HEK-293 human embryonic kidney cells null for endogenous CFTR protein expression. Increasing amounts of ??F-CFTR inhibited WT-CFTR protein processing and function in CF human airway epithelial cells but not in heterologous HEK-293 cells. Stably expressed ??F-CFTR in clones of the non-CF human airway epithelial cell line, CALU-3, also showed reduction in cAMP-stimulated anion secretion and in WT-CFTR processing. An ultimate test of this dominant negative-like effect of ??F-CFTR on WT-CFTR was the parallel study of two different CF mouse models: the ??F-CFTR mouse and the bitransgenic CFTR mouse corrected in the gut but null in the lung and airways. WT/??F heterozygotes had an intermediate phenotype with regard to CFTR agonist responses in in vivo nasal potential difference (NPD) recordings and in Ussing chamber recordings of short-circuit current (ISC) in vitro on primary tracheal epithelial cells isolated from the same mice. In contrast, CFTR bitransgenic +/? heterozygotes had no difference in their responses versus +/+ wild-type mice.

Conclusions

Taken altogether, these data suggest that ??F-CFTR and WT-CFTR co-assemble into an oligomeric macromolecular complex in native epithelia and share protein processing machinery and regulation at the level of the endoplasmic reticulum (ER). As a consequence, ??F-CFTR slows WT-CFTR protein processing and limits its expression and function in the apical membrane of native airway epithelia. Implications of these data for the relative health of CF heterozygous carriers, for CFTR protein processing in native airway epithelia, and for the relative efficacy of different CF therapeutic approaches is significant and is discussed.     

Pathological characteristics of esophago-respiratory fistulas of esophageal tumor origin

Esophago-respiratory fistulas, evolving as a result of esophageal tumors, are serious and lethal complications on account of the constant respiratory contamination and the inability to swallow. They can develop either as the complication of the end stage disease or sometimes even in the first stage of the malignancy. The objective was to reveal the characteristics of the disease. In a prospective single-center study in the period between 1984 and 2004, 243 fistulas were diagnosed. Their data were analyzed using multivariate analysis. The mean age of patients with fistula was 56.9 years, the male-to-female ratio was 4.3:1. The average time of the complaints was 5.2 months, while the time of manifestation of the fistula was 7.5 months. Dysphagia was diagnosed in 97.5% of the patients, fever in 36.9%, and cachexia in 59.5%, respectively. The average loss of weight was 10.4 kg and the average size of the tumor was 7.7 cm. Endoscopic intubation was performed in 176 cases. The average survival was 3.4 months. Patients with fistula were divided into two groups, where the characteristics of the disease were significantly different. Only in 66.3% was the fistula a late complication. In the other 33.7% of the cases the fistula was diagnosed in younger patients at the early stage of the disease, with a more aggressive, less differentiated histology. In these patients the weight loss, the grade of dysphagia and the size of the tumor were smaller, the possibilities of treatment were fewer, and survival were shorter.     

Study of career motivators affecting the emotional labour of health care professionals in oncology

In the course of their everyday work health care professionals (HCPs) often have to change their true feelings. The literature labels this performance as emotional labor. This article is presenting data on the characteristics of HCPs' most endangered by the negative consequences of emotional labor. Our simple choice question survey was conducted at Debrecen University Medical Healthcare Center with the help of 50 oncology HCPs volunteers. Nearly 90% of the HCPs examined change their true feelings in the course of work. It is very difficult to classify those threatened by the negative upshot of this emotional labor. Due to our research we found appalling differences of work motivation that were tightly interconnected with the respondents' emotional labor and their perceived role/emotional expectations. We succeeded in establishing three clusters and defining each cluster's characteristics. Figures suggest that only somewhat more than the half of the HCPs is authentic professional helper, and 45% of them does not or only slightly perceive the patients' demands concerning their work. Therefore, it is important that the work environment does not only assist the work of HCPs by professional means, but along emotional dimensions as well.     

Measurement of instantaneous velocity vectors of organelle transport: mitochondrial transport and bioenergetics in hippocampal neurons

Impaired transport of mitochondria, in dendrites and axons of neurons, and bioenergetic deficit are increasingly recognized to be of pathological importance in neurodegenerative diseases. To study the relationship between transport and bioenergetics, we have developed what to our knowledge is a novel technique to quantify organelle velocity in cultured cells. The aim was to combine measurement of motion and bioenergetic parameters while minimizing photodynamic oxidative artifacts evoked by fluorescence excitation. Velocity determination from sequential fluorescence images is not trivial, and here we describe an application of “optical flow”, the flow of gray values in grayscale images, to this problem. Based on the principles of photon shot noise occurring in low light level fluorescence microscopy, we describe and validate here an optical flow-based, robust method to measure velocity vectors for organelles expressing fluorescent proteins. This method features instantaneous velocity determination from a pair of images by detecting motion of edges, with no assumptions about the separation or shapes of the objects in the image. Optical flow was used in combination with single mitochondrion assay of mitochondrial thiol redox status by mitochondrially targeted redox-sensitive green fluorescent protein and measurement of mitochondrial membrane potential by tetramethylrhodamine methyl ester. Mitochondrial populations of resting cultured hippocampal neurons were analyzed. It was found that mitochondria with more oxidized thiol redox status have lower membrane potentials and are smaller in size. These mitochondria are more motile than the average; however, mitochondrial motility is only slightly dependent on the observed bioenergetic parameters and is correlated the best to the size of the mitochondria.     

The oxidation state of phospholipids controls the oxidative burst in neutrophil granulocytes

The activation of neutrophil granulocytes has to be carefully controlled to balance desired activity against invading pathogens while avoiding overwhelming activation leading to host tissue damage. We now show that phospholipids are potential key players in this process by either enhancing or dampening the production of reactive oxygen species (ROS) during the oxidative burst. Unoxidized phospholipids induce the production of ROS, and they also work synergistically with FMLP in potentiating the oxidative burst in neutrophil granulocytes. Oxidation of these phospholipids, however, turns them into potent inhibitors of the oxidative burst. OxPls specifically inhibit ROS production by inhibiting the assembly of the phagocyte oxidase complex but do not alter neutrophil viability, nor do they interfere with MAPK activation. Furthermore, up-regulation of the activation marker Mac-1 and phagocytosis of bacteria is not affected. Therefore, phospholipids may act as sensors of oxidative stress in tissues and either positively or negatively regulate neutrophil ROS production according to their oxidation state.     

Why do dusk-active cockchafers detect polarization in the green? The polarization vision in Melolontha melolontha is tuned to the high polarized intensity of downwelling light under canopies during sunset

In the retina of dusk-active European cockchafers, Melolontha melolontha, the linear polarization of downwelling light (skylight or light from the tree canopy) is detected by photoreceptors in upward-pointing ommatidia with maximal sensitivity at 520 nm in the green portion of the spectrum. To date no attempt has been made to answer the question of why these beetles detect polarization in the green. Here we present an atmospheric optical and receptor-physiological explanation of why longer wavelengths are advantageous for the perception of polarization of downwelling light under canopies illuminated by the setting sun. Our explanation focuses on illumination situations during sunset in canopied optical environments, because cockchafers are active at sunset and fly predominantly under canopies during their swarming, feeding, and mating periods. Using three simple atmospheric optical models, we computed the degree of linear polarization, the linearly polarized intensity of downwelling light, the quantum catch, and quantum catch difference between polarization detectors with orthogonal microvilli under canopies illuminated by the setting sun as functions of wavelength and solar zenith angle. Based upon these computations, we show that the green sensitivity of polarization detectors in M. melolontha is tuned to the high polarized intensity of downwelling light in the green under canopies during sunset, an optimal compromise between simultaneous maximization of the quantum catch and the quantum catch difference. We also briefly discuss how green-sensitive polarization detectors can function efficiently enough during the pre-feeding and egg-laying flights of cockchafers, which always occur prior to sunset and under the sky.     

Modelling the fission yeast cell cycle.

The molecular networks regulating basic physiological processes in a cell can be converted into mathematical equations (eg differential equations) and solved by a computer. The division cycle of eukaryotic cells is an important example of such a control system, and fission yeast is an excellent test organism for the computational modelling approach. The mathematical model is tested by simulating wild-type cells and many known cell cycle mutants. This paper describes an example where this approach is useful in understanding multiple rounds of DNA synthesis (endoreplication) in fission yeast cells that lack the main (B-type) mitotic cyclin, Cdc13. It is proposed that the key physiological variable driving progression through the cell cycle during balanced growth and division is the mass/DNA ratio, rather than the mass/nucleus ratio.     

Nitric oxide-induced mitochondrial fission is regulated by dynamin-related GTPases in neurons

Mitochondria are present as tubular organelles in neuronal projections. Here, we report that mitochondria undergo profound fission in response to nitric oxide (NO) in cortical neurons of primary cultures. Mitochondrial fission by NO occurs long before neurite injury and neuronal cell death. Furthermore, fission is accompanied by ultrastructural damage of mitochondria, autophagy, ATP decline and generation of free radicals. Fission is occasionally asymmetric and can be reversible. Strikingly, mitochondrial fission is also an early event in ischemic stroke in vivo. Mitofusin 1 (Mfn1) or dominant-negative Dynamin related protein 1 (Drp1(K38A)) inhibits mitochondrial fission induced by NO, rotenone and Amyloid-beta peptide. Conversely, overexpression of Drp1 or Fis1 elicits fission and increases neuronal loss. Importantly, NO-induced neuronal cell death was mitigated by Mfn1 and Drp1(K38A). Thus, persistent mitochondrial fission may play a causal role in NO-mediated neurotoxicity.     

Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells

The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ΔψM) in pancreatic β-cells. This built on our previously introduced calculation of the absolute magnitude of ΔψM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of tetramethylrhodamine methyl ester and a bis-oxonol plasma membrane potential (ΔψP) indicator. ΔψM is a central mediator of glucose-stimulated insulin secretion in pancreatic β-cells. ΔψM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ΔψM and ΔψP. Glucose addition caused hyperpolarization of ΔψM and depolarization of ΔψP. The hyperpolarization was a monophasic step increase, even in cells where the ΔψP depolarization was biphasic. The biphasic response of ΔψP was associated with a larger hyperpolarization of ΔψM than the monophasic response. Analysis of the relationships between ΔψP and ΔψM revealed that primary dispersed β-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with β-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ΔψM and ΔψP. Finally, we demonstrate a significant problem with using an alternative ΔψM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited β-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusions.