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

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

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

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

Electron microscopy analysis of wild-type and transgenic melanotrope cells.

<p>(A–C) Wild-type (wt; A1 and A2) intermediate pituitary cells showed a well-developed rough endoplasmic reticulum and extensive Golgi-ribbons. The p24α₃-transgenic cells (#55; B1 and B2) contained Golgi mini-stacks and large electron-dense structures (EDS). The p24δ₂-transgenic cells (#224; C1 and C2) showed an ultrastructure similar to that of wild-type cells. The dotted lines highlight the outline of the Golgi. (D–F) Immuno-electron microscopy analysis of intermediate pituitary melanotrope cells from wt frogs (D), and frogs transgenic for p24α₃ (#55; E) or p24δ₂ (#224; F) using an anti-POMC antiserum. Immunoreactivity was found in dense-core secretory granules (filled arrowheads) in wt and p24α₃- and p24δ₂-transgenic cells and occasionally in newly forming secretion granules still attached to the <i>trans</i>-Golgi network (open arrowheads). In addition, in the p24α₃-transgenic cells a strong POMC-immunolabeling was observed in the EDS, which were localized to the ER lumen (arrow) and occasionally in EDS newly forming within the ER lumen (open arrow). G, Golgi; L, lysosome; M, mitochondrion; N, nucleus; RER, rough endoplasmic reticulum; PM, plasma membrane; sg, immature secretory granules. Bars equal 1 µm (A–C); 500 nm (D–F).</p

Steady-state levels of secretory cargo proteins in wild-type and transgenic <i>Xenopus</i> intermediate pituitary cells.

<p>Western blot analysis of neurointermediate lobe (NIL) lysates from wild-type frogs (wt) and frogs transgenic for p24α₃ (#55) or p24δ₂ (#224) using antibodies directed against the soluble cargo proteins proopiomelanocortin (POMC) and prohormone convertase 2 (PC2), and the transmembrane cargo amyloid-β precursor protein (APP). Tubulin was used as a control for equal loading.</p

Newly synthesized 18K and 18K* POMC differ in N-glycosylation.

<p>Neurointermediate lobes (NILs) from wild-type frogs (wt) and frogs transgenic for p24α₃ (#55) or p24δ₂ (#224) were pulse labeled with [³⁵S]-Met/Cys for 30 min and subsequently chased for 3 hrs. Newly synthesized proteins extracted from the NILs were deglycosylated with PNGaseF (F) or control-treated (C), resolved by 20% SDS-PAGE and visualized by autoradiography; the #55 lanes were exposed three times longer than the other lanes.</p

The effect of p24α₃- or p24δ₂-transgene expression on POMC biosynthesis and processing in <i>Xenopus</i> melanotropes.

<p>(A–C) Neurointermediate lobes (NILs) from wild-type frogs (wt) and frogs transgenic for p24α₃ (#55) or p24δ₂ (#224) were pulse labeled with [³⁵S]-Met/Cys for 30 min and subsequently chased for 3 hrs. Newly synthesized proteins extracted from the NILs (Cells; 5% of extract) and secreted into the incubation medium (Media; 20%) were resolved by 15% SDS-PAGE and visualized by autoradiography. (A) The analysis was performed in six independent experiments and a representative autoradiogram is shown. (B) The amount of newly synthesized 37K POMC in wild-type (n = 16) and the p24α₃-transgenic (n = 10) and p24δ₂-transgenic (n = 6) cells was quantified and is shown relative to the wild-type cells. (C) The amounts of newly synthesized 18K and 18K* POMC in wild-type (n = 12) and the p24α₃-transgenic (n = 5) and p24δ₂-transgenic (n = 6) cells were quantified and are shown relative to wild-type 18K POMC. Indicated are the 18K/18K* ratios and their statistical evaluations. Data are shown as means +/− SEM. n.s., not significant; **, p<0.01; ***, p<0.001.</p