Disparate Effects of p24α and p24δ on Secretory Protein Transport and Processing

Contains fulltext : 34883.pdf ( ) (Open Access)BACKGROUND: The p24 family is thought to be somehow involved in endoplasmic reticulum (ER)-to-Golgi protein transport. A subset of the p24 proteins (p24alpha(3), -beta(1), -gamma(3) and -delta(2)) is upregulated when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated to produce vast amounts of their major secretory cargo, the prohormone proopiomelanocortin (POMC). METHODOLOGY/PRINCIPAL FINDINGS: Here we find that transgene expression of p24alpha(3 )or p24delta(2) specifically in the Xenopus melanotrope cells in both cases causes an effective displacement of the endogenous p24 proteins, resulting in severely distorted p24 systems and disparate melanotrope cell phenotypes. Transgene expression of p24alpha(3) greatly reduces POMC transport and leads to accumulation of the prohormone in large, ER-localized electron-dense structures, whereas p24delta(2)-transgenesis does not influence the overall ultrastructure of the cells nor POMC transport and cleavage, but affects the Golgi-based processes of POMC glycomaturation and sulfation. CONCLUSIONS/SIGNIFICANCE: Transgenic expression of two distinct p24 family members has disparate effects on secretory pathway functioning, illustrating the specificity and non-redundancy of our transgenic approach. We conclude that members of the p24 family furnish subcompartments of the secretory pathway with specific sets of machinery cargo to provide the proper microenvironments for efficient and correct secretory protein transport and processing

Oxidative and nitrosative stress in the diaphragm of patients with COPD

COPD is associated with an increased load on the diaphragm. Since chronic muscle loading results in changes in antioxidant capacity and formation of reactive oxygen and reactive nitrogen species, we hypothesized that COPD has a similar effect on the diaphragm, which is related to the severity of COPD. Catalase activity was determined spectrophotometrically. Levels of 4-hydroxy-2-nonenal (HNE)-protein adducts and 3-nitrotyrosine (NT) formation were measured using western blotting. Levels of malondialdehyde (MDA) were assessed by high-performance liquid chromatography. We found that catalase activity was ~89% higher in the diaphragm of severe COPD patients (FEV1 37 ± 5% predicted) compared with non-COPD patients. MDA levels, a marker for lipid peroxidation, were significantly lower in the diaphragm of COPD patients compared with non-COPD patients, whereas the level of HNE-protein adducts was equal in both groups. NT formation was not different between groups. However, increasing hyperinflation and NT formation were inversely correlated. These results indicate that in COPD the diaphragm adapts to a higher work load by increasing catalase activity, resulting in a reduction in oxidative damage to lipids and tyrosine nitration of proteins

Image Thresholding Technique Based On Fuzzy Partition And Entropy Maximization

Thresholding is a commonly used technique in image segmentation because of its fast and easy application. For this reason threshold selection is an important issue. There are two general approaches to threshold selection. One approach is based on the histogram of the image while the other is based on the gray scale information located in the local small areas. The histogram of an image contains some statistical data of the grayscale or color ingredients. In this thesis, an adaptive logical thresholding method is proposed for the binarization of blueprint images first. The new method exploits the geometric features of blueprint images. This is implemented by utilizing a robust windows operation, which is based on the assumption that the objects have "e;C"e; shape in a small area. We make use of multiple window sizes in the windows operation. This not only reduces computation time but also separates effectively thin lines from wide lines. Our method can automatically determine the threshold of images. Experiments show that our method is effective for blueprint images and achieves good results over a wide range of images. Second, the fuzzy set theory, along with probability partition and maximum entropy theory, is explored to compute the threshold based on the histogram of the image. Fuzzy set theory has been widely used in many fields where the ambiguous phenomena exist since it was proposed by Zadeh in 1965. And many thresholding methods have also been developed by using this theory. The concept we are using here is called fuzzy partition. Fuzzy partition means that a histogram is parted into several groups by some fuzzy sets which represent the fuzzy membership of each group because our method is based on histogram of the image . Probability partition is associated with fuzzy partition. The probability distribution of each group is derived from the fuzzy partition. Entropy which originates from thermodynamic theory is introduced into communications theory as a commonly used criteria to measure the information transmitted through a channel. It is adopted by image processing as a measurement of the information contained in the processed images. Thus it is applied in our method as a criterion for selecting the optimal fuzzy sets which partition the histogram. To find the threshold, the histogram of the image is partitioned by fuzzy sets which satisfy a certain entropy restriction. The search for the best possible fuzzy sets becomes an important issue. There is no efficient method for the searching procedure. Therefore, expansion to multiple level thresholding with fuzzy partition becomes extremely time consuming or even impossible. In this thesis, the relationship between a probability partition (PP) and a fuzzy C-partition (FP) is studied. This relationship and the entropy approach are used to derive a thresholding technique to select the optimal fuzzy C-partition. The measure of the selection quality is the entropy function defined by the PP and FP. A necessary condition of the entropy function arriving at a maximum is derived. Based on this condition, an efficient search procedure for two-level thresholding is derived, which makes the search so efficient that extension to multilevel thresholding becomes possible. A novel fuzzy membership function is proposed in three-level thresholding which produces a better result because a new relationship among the fuzzy membership functions is presented. This new relationship gives more flexibility in the search for the optimal fuzzy sets, although it also increases the complication in the search for the fuzzy sets in multi-level thresholding. This complication is solved by a new method called the "e;Onion-Peeling"e; method. Because the relationship between the fuzzy membership functions is so complicated it is impossible to obtain the membership functions all at once. The search procedure is decomposed into several layers of three-level partitions except for the last layer which may be a two-level one. So the big problem is simplified to three-level partitions such that we can obtain the two outmost membership functions without worrying too much about the complicated intersections among the membership functions. The method is further revised for images with a dominant area of background or an object which affects the appearance of the histogram of the image. The histogram is the basis of our method as well as of many other methods. A "e;bad"e; shape of the histogram will result in a bad thresholded image. A quadtree scheme is adopted to decompose the image into homogeneous areas and heterogeneous areas. And a multi-resolution thresholding method based on quadtree and fuzzy partition is then devised to deal with these images. Extension of fuzzy partition methods to color images is also examined. An adaptive thresholding method for color images based on fuzzy partition is proposed which can determine the number of thresholding levels automatically. This thesis concludes that the "e;C"e; shape assumption and varying sizes of windows for windows operation contribute to a better segmentation of the blueprint images. The efficient search procedure for the optimal fuzzy sets in the fuzzy-2 partition of the histogram of the image accelerates the process so much that it enables the extension of it to multilevel thresholding. In three-level fuzzy partition the new relationship presentation among the three fuzzy membership functions makes more sense than the conventional assumption and, as a result, performs better. A novel method, the "e;Onion-Peeling"e; method, is devised for dealing with the complexity at the intersection among the multiple membership functions in the multilevel fuzzy partition. It decomposes the multilevel partition into the fuzzy-3 partitions and the fuzzy-2 partitions by transposing the partition space in the histogram. Thus it is efficient in multilevel thresholding. A multi-resolution method which applies the quadtree scheme to distinguish the heterogeneous areas from the homogeneous areas is designed for the images with large homogeneous areas which usually distorts the histogram of the image. The new histogram based on only the heterogeneous area is adopted for partition and outperforms the old one. While validity checks filter out the fragmented points which are only a small portion of the whole image. Thus it gives good thresholded images for human face images

Spectral analysis and resolving spatial ambiguities in human sound localization

This dissertation provides an overview of my research over the last five years into the spectral analysis involved in human sound localization. The work involved conducting psychophysical tests of human auditory localization performance and then applying analytical techniques to analyze and explain the data. It is a fundamental thesis of this work that human auditory localization response directions are primarily driven by the auditory localization cues associated with the acoustic filtering properties of the external auditory periphery, i.e., the head, torso, shoulder, neck, and external ears. This work can be considered as composed of three parts. In the first part of this work, I compared the auditory localization performance of a human subject and a time-delay neural network model under three sound conditions: broadband, high-pass, and low-pass. A “black-box” modeling paradigm was applied. The modeling results indicated that training the network to localize sounds of varying center-frequency and bandwidth could degrade localization performance results in a manner demonstrating some similarity to human auditory localization performance. As the data collected during the network modeling showed that humans demonstrate striking localization errors when tested using bandlimited sound stimuli, the second part of this work focused on human sound localization of bandpass filtered noise stimuli. Localization data was collected from 5 subjects and for 7 sound conditions: 300 Hz to 5 kHz, 300 Hz to 7 kHz, 300 Hz to 10 kHz, 300 Hz to 14 kHz, 3 to 8 kHz, 4 to 9 kHz, and 7 to 14 kHz. The localization results were analyzed using the method of cue similarity indices developed by Middlebrooks (1992). The data indicated that the energy level in relatively wide frequency bands could be driving the localization response directions, just as in Butler’s covert peak area model (see Butler and Musicant, 1993). The question was then raised as to whether the energy levels in the various frequency bands, as described above, are most likely analyzed by the human auditory localization system on a monaural or an interaural basis. In the third part of this work, an experiment was conducted using virtual auditory space sound stimuli in which the monaural spectral cues for auditory localization were disrupted, but the interaural spectral difference cue was preserved. The results from this work showed that the human auditory localization system relies primarily on a monaural analysis of spectral shape information for its discrimination of directions on the cone of confusion. The work described in the three parts lead to the suggestion that a spectral contrast model based on overlapping frequency bands of varying bandwidth and perhaps multiple frequency scales can provide a reasonable algorithm for explaining much of the current psychophysical and neurophysiological data related to human auditory localization

V-ATPase-Mediated Granular Acidification Is Regulated by the V-ATPase Accessory Subunit Ac45 in POMC-Producing Cells

The regulation of the V-ATPase, the proton pump mediating intraorganellar acidification, is still elusive. We find that excess of the neuroendocrine V-ATPase accessory subunit Ac45 reduces the intragranular pH and consequently disturbs prohormone convertase activation and prohormone processing. Thus, Ac45 represents the first V-ATPase regulator

Isolation of intact elastin fibers devoid of microfibrils.

Contains fulltext : 48622.pdf (publisher's version ) (Open Access)Purification protocols for elastin generally result in greatly damaged elastin fibers and this likely influences the biological response. We here describe a novel protocol for the isolation of elastin whereby the fibers stay intact, and introduce the term "elastin fiber" for intact elastic fibers with elastin as their sole component. As opposed to elastic fibers, elastin fibers do not contain any microfibrils or associated molecules. Elastin fibers were isolated from equine elastic ligaments according to various protocols and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, amino acid quantification, immunofluorescence assay, transmission/scanning electron microscopy, and cellular reactivity in vivo. The optimal protocol comprised several extraction steps and trypsin digestion. Elastin fibers were free of contaminants and had a smooth, regular appearance. The cellular response to purified, intact elastin fibers was different in comparison with purified, but affected, fibers and to contaminated fibers. Intact fibers consisting only of elastin may be important for both fundamental and applied research, for example, tissue engineering, which need well-defined preparations to study the cellular biological effect of individual components

A biomaterial composed of collagen and solubilized elastin enhances angiogenesis and elastic fiber formation without calcification.

Contains fulltext : 70670.pdf (publisher's version ) (Open Access)Elastin is the prime protein in elastic tissues that contributes to elasticity of, for example, lung, aorta, and skin. Upon injury, elastic fibers are not readily replaced, which hampers tissue regeneration. Incorporation of solubilized elastin (hydrolyzed insoluble elastin fibers or elastin peptides) in biomaterials may improve regeneration, because solubilized elastin is able to promote proliferation as well as elastin synthesis. Porous biomaterials composed of highly purified collagen without and without elastin fibers or solubilized elastin were prepared by freezing and lyophilization. Solubilized elastin formed spherical structures that were incorporated in the collagenous part of the scaffolds and that persisted after chemical crosslinking of the scaffolds. Crosslinked scaffolds were subcutaneously implanted in young Sprague Dawley rats. Collagen-solubilized elastin and collagen scaffolds showed no calcification in this sensitive calcification model, in contrast to scaffolds containing elastin fibers. Collagen-solubilized elastin scaffolds also induced angiogenesis, as revealed by type IV collagen staining, and promoted elastic fiber synthesis, as shown with antibodies against rat elastin and fibrillin-1. It is concluded that scaffolds produced from collagen and solubilized elastin present a non-calcifying biomaterial with a capacity for soft-tissue regeneration, especially in relation to elastic fiber synthesis

Generation of <i>Xenopus</i> with transgene expression of p24α₃ or p24δ₂ specifically in the melanotrope cells.

<p>(A and B) Schematic representation of the linear injection fragments pPOMC-p24α₃-GFP (A) and pPOMC-p24δ₂-GFP (B) containing a <i>Xenopus</i> POMC gene promoter fragment (pPOMC) and the protein-coding sequence of p24α₃-GFP (transgenic lines #605, #55 and #602) or p24δ₂-GFP (lines #125, #115, #124 and #224); pPOMC drives transgene expression specifically to the melanotrope cells. (C) Pituitary-specific GFP-fluorescence (arrows) in living tadpoles transgenic for p24α₃ (line #55) or p24δ₂ (line #224); G, gut; E, eye; N, nose. (D) Fluorescence in the intermediate lobe (IL) and not in the anterior lobe (AL) of the pituitary of adult frogs transgenic for p24α₃ (#55) or p24δ₂ (#224).</p