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200 Quart Offshore Pro Series™ Marine Cooler
Item# 3000002237
$399.99 $349.99
Additional Information
Get the ultimate in long-term cold retention while you're out on the water when you bring a Coleman® 200 Quart Offshore Pro Series™ Marine Cooler. Ultimate Xtreme® technology uses extra thick insulation in the wall and lid to keep your items cold for up to 7 days. Large enough to hold 327 cans, you'll have plenty of space to store refreshments or your catch for an entire fishing expedition. Two storage dividers and a dry storage tray also let you organize your items and keep some things out of the ice. Built to withstand the extra demands of a marine environment, this cooler's UVGuard™ material offers protection from the sun's damaging rays, and its stainless steel hardware resists rusting. Whether you're headed on an extended-length boat trip or heading to your favorite out-of-the-way fishing hole, the comfort-grip swing handles make getting to your destination easy and comfortable. You can even secure extra gear to the lid with the integrated tie-down slots. Once you arrive, the Have-A-Seat™ lid will ensure you always have a place to sit and relax, and the cup holders molded into the lid will keep your cold drink close while you enjoy the day. When the fun is done, the leak-resistant channel drain makes it easy to empty any excess water without tilting the cooler. Meanwhile the antimicrobial liner resists odor, mold, mildew and fungus so your cooler stays clean until your next fun adventure on the water.
• Antimicrobial liner resists odor, mold, mildew and fungus
• UVGuard™ material helps protect your cooler from the sun's damaging rays
• Keeps the Ice!™ up to 7 days at temperatures up to 90 °F
• Ultimate Xtreme® technology uses extra thick insulation in the walls and lid for long-term ice retention
• Holds 327 cans
• Dry storage tray gives you a place to store items out of the ice
• 2 storage dividers lets you organize your cooler contents
• Leak-resistant channel drain for easy draining without tilting the cooler
• Have-A-Seat™ Lid supports up to 250 lbs. for a place to sit and rest
• Comfort-grip swing handles for easy carrying
• Rust-resistant, stainless steel hardware
• Rust-resistant, stainless steel spring lid strap locks lid in place when open to prevent accidental closings
• Cup holders molded into the lid keep drinks close
• Integrated tie-down slots let you secure gear to the lid with bungees (sold separately)
• Integrated fish ruler lets you easily size up your catch
• Low CO2 insulation for reduced carbon emissions from foam manufacturing
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100 Quart Xtreme® 5 Cooler
Keep your food and drinks cold for an extended time, and make the fun last longer, when you bring a Coleman® 100 Quart Xtreme® 5 Cooler. …


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subset: commoncrawl


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Apparently, Otto Porter Jr. is waiting for someone to tell him that he is super-awesome or something. Porter, who nearly everyone considers a top draft pick, has not officially announced his entry into the upcoming draft. Meaning, the Georgetown Hoyas can still hold on to the slightest of slight hopes that Porter will be returning to the program next season. ...
What Is Georgetown Hoyas Star Otto Porter Jr. Waiting For?
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What is better in the winter than a good easy recipe for chili? From super bowl parties to chili cook-offs… chili is woven into the fabric of American winter culture. And for good reason; it's AWESOME and there are so many variations.
Oh – and in case you're interested, I'd LOVE to send you a FREE Meal Plan! 10 super easy meals prepared in about 45 minutes for under $80! Sign up right below:
Ok back to the recipe at hand…
And if you've been searching for a good "standard" type chili that everyone will like, look no further! This is it. And it's EASY! My mom cut this recipe out of a newspaper in the early 90's, and it's been our 'family chili' recipe ever since. There might be a few ingredients you are not used to seeing in chili, like cinnamon and chocolate chips (a Cincinnati chili influence), but trust me – they work!
And like I said – this is easy.
Chopping the onions is probably the hardest part.
One of the most important steps is to cook the onions as the ground beef cooks. Add a bit of salt at this step to soften the onions.
After the onions are soft and the beef is cooked, add the garlic and cook for another couple minutes. Drain the beef, and then add everything else except for the kidney beans and simmer for an hour or so. And yeah – even add those chocolate chips and cinnamon (you will thank me).
Then add the kidney beans and cook for another half hour. Top with cheese, serve corn bread or whatever you'd like!
I have so many other great recipes, so make sure that you check them out here.
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The transcription factor NFAT plays essential roles in both immune and non-immune cells. In T cells, store- operated Ca2+ influx through the STIM-ORAI pathway triggers the phosphatase calcineurin, which dephos- phorylates NFAT and promotes its nuclear translocation. The NFAT kinases CK1, GSK3, and DYRK counter the action of calcineurin and maintain NFAT in a phosphorylated, inactive state in the cytoplasm. In this application, we focus on the role of a long intergenic non-coding RNA (lincRNA) named NRON (non- coding repressor of NFAT) in regulation of the Ca2+-calcineurin-NFAT signalling pathway in T cells. NRON was previously identified as a negative regulator of NFAT, and shown to bind the scaffold protein IQGAP1 and nuclear import proteins. We extended these findings by showing that NRON, NFAT, IQGAP1 and NFAT kinases form a high-molecular-weight complex in the cell cytoplasm. siRNAs against NRON, IQGAP1 or both enhanced NFAT activation in response to low-level stimulation, and dramatically increased production of the NFAT-dependent cytokine Interleukin-2 (IL-2) by Jurkat T cells. Likewise, mouse T cells bearing a disruption of the Iqgap1 gene produced considerably more interferon-gamma (IFN?) than wild type T cells. Here we will investigate the role of the lincRNA NRON, RNA-binding proteins (RBPs), and novel modulators of NFAT signalling identified in a genome-wide RNAi screen, in regulating the activity of the Ca2+-calcineurin- NFAT pathway in T cells.
In Aim 1, we will define the biochemical features of the scaffold complex containing NFAT, NRON, IQGAP1, calmodulin, NFAT kinases and importins in resting and activated T cells, asking if components of the complex are involved in recruiting calcineurin in the cytoplasm and if they have a potential function in the nucleus. We will also identify protein partners for NRON and RNA partners for the calcineurin regulator RCAN1.
In Aim 2, we will examine the in vivo role of NRON in thymocytes and T cells by conditional gene disruption in mice. The functions of small non-coding RNAs, particularly microRNAs, have been extensively studied, but the roles of long noncoding lincRNAs in biological processes are just beginning to be elucidated. LincRNAs can target transcription factors to their binding sites in DNA, and can also function as critical components of RNA-protein complexes that regulate gene expression. Our proposed studies should illuminate these possibilities for the important Ca2+-calcineurin-NFAT signaling pathway in immune cells. We have developed a large number of innovative reagents and techniques to study this pathway, and thus are uniquely positioned to elucidate the molecular mechanisms underlying NFAT activation by Ca2+-calcineurin and the NRON scaffold complex.
Calcium is a universal second messenger that is used by all cells, including those of the brain, heart, muscle and immune system, to control a huge diversity of cellular functions. When calcium enters cells, a protein known as NFAT turns on genes important for the proper functioning of the cell. In the previous project period, we showed that NFAT existed in a large complex that contains both RNA and other proteins. Here we will use cutting-edge approaches, including proteomics and next-generation sequencing, to investigate how NFAT travels to the nucleus in response to calcium entry. We expect to identify new regulatory proteins that will provide novel therapies for autoimmunity and other important diseases.
|Pereira, Renata M; Martinez, Gustavo J; Engel, Isaac et al. (2014) Jarid2 is induced by TCR signalling and controls iNKT cell maturation. Nat Commun 5:4540
|Carlson, Thaddeus J; Pellerin, Alex; Djuretic, Ivana M et al. (2014) Halofuginone-induced amino acid starvation regulates Stat3-dependent Th17 effector function and reduces established autoimmune inflammation. J Immunol 192:2167-76
|Fogli, Laura K; Sundrud, Mark S; Goel, Swati et al. (2013) T cell-derived IL-17 mediates epithelial changes in the airway and drives pulmonary neutrophilia. J Immunol 191:3100-11
|Zhou, Yubin; Srinivasan, Prasanna; Razavi, Shiva et al. (2013) Initial activation of STIM1, the regulator of store-operated calcium entry. Nat Struct Mol Biol 20:973-81
|Trifari, Sara; Pipkin, Matthew E; Bandukwala, Hozefa S et al. (2013) MicroRNA-directed program of cytotoxic CD8+ T-cell differentiation. Proc Natl Acad Sci U S A 110:18608-13
|Gudlur, Aparna; Zhou, Yubin; Hogan, Patrick G (2013) STIM-ORAI interactions that control the CRAC channel. Curr Top Membr 71:33-58
|Ko, Myunggon; An, Jungeun; Bandukwala, Hozefa S et al. (2013) Modulation of TET2 expression and 5-methylcytosine oxidation by the CXXC domain protein IDAX. Nature 497:122-6
|Sharma, Sonia; Quintana, Ariel; Findlay, Gregory M et al. (2013) An siRNA screen for NFAT activation identifies septins as coordinators of store-operated Ca2+ entry. Nature 499:238-42
|Vijayanand, Pandurangan; Seumois, Gregory; Simpson, Laura J et al. (2012) Interleukin-4 production by follicular helper T cells requires the conserved Il4 enhancer hypersensitivity site V. Immunity 36:175-87
|Keller, Tracy L; Zocco, Davide; Sundrud, Mark S et al. (2012) Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase. Nat Chem Biol 8:311-7
Showing the most recent 10 out of 50 publications


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10 easy meals for crazy-busy weeknights.
10 easy meals for crazy-busy weeknights.
Easy meals for those crazy-busy weeknights.
PIzza burgers, pasta, and more recipes that are easy to throw together for the whole family to enjoy
Easy meals for those crazy-busy weeknights.
He may seem tough on screen, but this family man has a soft spot for his wife's cooking and talented kid cooks.
Easy recipes to make tonight.
This easy recipe just might turn cauliflower into your favorite veggie.
One month's worth of weeknight dinners to whip up in less than half an hour.
10 easy recipes for crazy-busy nights.
Indulge your guests with these delicious holiday eats.
Now that you've read up on the risk factors, knock 'em out with 6 tailor-made meals.
Dark greens are full of iron, calcium, and vitamins A, C and K. Here are five ways to integrate them into your next meal.
Take your family on a culinary adventure without leaving the dinner table.
Inexpensive ways to put a delicious meal on the table.
Eat your greens! Salads aren't just a lunch thing-here are six dinner-worthy ideas.
How 'Bout These Apples?
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|Publication number
|Publication type
|Application number
|Publication date
|Jun 6, 1995
|Filing date
|Nov 12, 1993
|Priority date
|Nov 12, 1993
|Fee status
|Also published as
|Publication number
|Vito Quaranta, Marketta Hormia
|Original Assignee
|Desmos, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (3), Non-Patent Citations (46), Referenced by (21), Classifications (41), Legal Events (9)
|External Links: USPTO, USPTO Assignment, Espacenet
This research was supported by National Institutes of Health Grants GM 46902 and DE 10063. The government may have certain rights in the invention.
When organs of the body are formed, they develop in neatly organized arrays. Often, cell groups of one kind are separated from cells of another kind by flat strips of connective tissue called basement membranes. In skin, for instance, the superficial layer of epidermal cells adheres to the underlying basement membrane. This skin basement membrane acts as a barrier between the epidermal cells on the outside, and the dermal cells underneath. A similar arrangement of cells occurs in the lining of the gut.
Basement membranes have been implicated in the growth, attachment, migration, repair, and differentiation of their overlying cell populations. Three layers have been defined in basement membranes: a) the lamina lucida, an electron microscope-clear region that resides in close approximation to the overlying cells; b) the lamina densa, an electron dense region of 20-300 nm in width; and c) the sublamina densa that contains anchoring fibrils, microfibrillar bundles and collagen fibers.
Many different types of compounds have now been localized to the basement membrane. Some of these compounds are laminin, collagen IV and heparin sulfate proteoglycans (Verrando et al. Exp. Cell Res. (1987); 170: 116-128). In addition, specific basement membranes have been found to possess other compounds, such as nidogen and entactin.
The principal cell adhesion receptor that epidermal cells use to attach to the basement membrane is called α6 β4. This transmembrane receptor is formed by a combination of two protein moieties α6 and β4. The α6 and β4 proteins are derived from different genes that have been found to be part of the integrin family.
Integrins are versatile family cell adhesion receptors. So far, approximately twenty members have been discovered in the integrin family. These molecules are involved in many types of cell adhesion phenomena in the body. Integrins are signalling molecules that can translate environmental cues into cellular instructions. Further, integrins can also transmit signals in the reverse direction, from the cell interior to the exterior. This has been illustrated in non-adherent cells, such as lymphocytes.
Stimulation of the T-cell antigen receptor, or of the CD3 complex, augments the affinity of certain integrins for their respective ligands. Unfortunately, in adherent cells, changes in the affinities of integrins have been more difficult to demonstrate. However, affinity modulation of one integrin in differentiating epidermal keratinocytes has been described by Adams et al. (Cell (1990); 63: 425-435). For this reason, modifications of cell status initiated by activation or differentiation of other receptors may influence integrin affinity, and ultimately, the adhesive behavior of cells. Further, as a consequence of adhering to a surface, an integrin may actively contribute to modifying cell shape or migration.
Many epithelial cells interact with the underlying extracellular matrix via a junction called the hemidesmosome (Staehelin, (1974) Structure and Function of Intercellular Junctions, Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colo., 191-283). Over the last few years there has been considerable progress in the biochemical characterization of this junction (Schwarz et al., (1990) Annu. Rev. Cell Biol., 6:461-491). The hemidesmosome, with its associated structures such as intermediate filaments and anchoring fibrils, forms an adhesion complex. Disruptions of the epithelial-connective tissue interaction is often accompanied by a disruption of the hemidesmosome complex. For example, in certain blistering skin diseases such as junctional epidermolysis bullosa where epithelial cell-connective tissue interactions is abnormal, it has been proposed that there is a biochemical modification in or loss of a basement membrane zone-associated component of the hemidesmosome.
Two high molecular weight intracellular components of the hemidesmosome have been identified and characterized with the aid of antisera from patients suffering from bullous pemphigoid. This autoimmune disease results in a disruption of the interactions between epithelial cells and connective tissue simultaneously with loss of hemidesmosome integrity (Chapman et al. Br. J. Dermatol (1990); 123: 137-144). Accordingly, it was discovered that bullous pemphigoid patients were producing antibodies against hemidesmosome components. Two hemidesmosome related bullous pemphigoid (BP) antigens have been previously described (Klatte et al., (1989) J. Cell Biol., 109:3377-3390).
One BP antigen is a 230 kD polypeptide that may act as an anchor for cytoskeleton elements in the hemidesmosomal plaque (Jones and Green, (1991) Curr. Opin. Cell Biol., 3:127-132). A second BP antigen is a type II membrane protein that possesses a collagen-like extracellular domain (Giudice et al., (1991) J. Clin. Invest., 87:734-738). (Hopkinson et al., (1992) J. Invest. Dermatol., 3:264-270). In addition, it has been demonstrated that the interaction of the hemidesmosome with the underlying connective tissue involves the α6 β4 integrin heterodimer (Steppet al., (1990), Proc. Natl. Acad. Sci. USA, 87:8970-8974; Jones et al., (1991) Cell Regulation, 2:427-438; Sonnenberg et al., (1991) J. Cell Biol., 113:907-917; Kurpakus et al., (1991) J. Cell Biol., 115:1737-1750). The α6 β4 heterodimer has been localized to hemidesmosomes along the basal surfaces of the rat bladder carcinoma cell line 804 G (Jones et al. Cell Regulation (1991); 2: 427-438). These results suggested that integrins (e.g. α6 β4) may play an important role in the assembly and adhesive functions of hemidesmosomes.
Various prior art efforts have focused on purifying adhesion-facilitating proteins found in basement membranes. For example, Burgeson, et al., Patent Cooperation Treaty Application No. WO92/17498, disclose a protein which they call kalinin. Kalinin is said to facilitate cell adhesion to substrates; however, this material is apparently inactive with respect to hemidesmosome formation. See also, Marinkovich, et al., J. Cell Biol. (1992); 119:695-703 (k-laminin); Rouselie, et al., J. Cell. Biol. (1991); 114:567-576 (kalinin); and Marinkovich, et al., J. Biol. Chem. (1992); 267:17900-17906 (kalinin).
Similarly, a basement glycoprotein of about 600 kD made up of polypeptides in the range of 93.5 kD to 150 kD has been identified, and is known as GB3 or nicein. See, e.g., Verrando, et al., Biochim. Biophys. Acta (1988); 942:45-56; and Hsi, et al., Placenta (1987); 8:209-217. None of these materials have been effective in generating formation of hemidesmosomes, either in vitro or in vivo.
When cultured on tissue culture plastic in vitro, most epithelial cells do not assemble bona fide hemidesmosomes despite the fact that they appear to express all of the hemidesmosomal plaque and transmembrane components mentioned above. Indeed, it is only recently that cell lines such as 804G were discovered to have the ability to readily assemble hemidesmosomes in vitro under regular culture conditions (Riddelle et al., (1991) J. Cell Biol., 112:159-168; Hieda et al., (1992) J. Cell Biol., 116:1497-1506). Such cells are at last allowing detailed cell and biochemical analysis of the dynamics of hemidesmosome assembly.
For instance, it has been reported that substratum-associated staining by anti-hemidesmosome antibodies is greatly diminished in 804G cell cultures that enter in vitro wound sites (Riddelle et al., J. Cell Sci. (1992); 103: 475-490). However, as closure of the wound became complete, anti-hemidesmosome staining along the substratum- attached surface was evident in the cells.
There are, however, many epithelial cells that do not attach to tissue culture dishes in a normal fashion, even after treatment with various growth factors. These cells do not produce normal hemidesmosomes or grow to resemble their in vivo phenotype. It would provide a tremendous advantage to have a system that was capable of maintaining epithelial cell growth in vitro wherein the cells maintained their normal phenotype.
One aspect of the present invention provides a method for inducing hemidesmosome formation in epithelial cells in vitro by the following steps:
growing a first sample of epithelial cells in media under conditions that promote secretion of hemidesmosome-formation facilitating soluble factors, whereby the first cells secrete the factors into the media;
removing the media from the epithelial cells; and
growing a second sample of epithelial cells of a different type in contact with the media whereby the second epithelial cells are induced to attach to a substrate and produce hemidesmosomes.
Preferably, the second epithelial cells are human and the medium is Modified Eagles Medium. The invention also includes culturing the 804G cells in Dulbecco's Modified Eagles Medium OPTI-MEM medium in a 1:1 ratio in the presence of 1% fetal calf serum. Another embodiment provides the 804GMHcell line produced upon culturing in low serum medium. Preferably, the first epithelial cells are either 804G or NBT-II rat bladder carcinoma cells.
Another embodiment of the present invention is a method for growing epithelial cells in vitro by the following steps:
growing 804G rat bladder carcinoma cells in media under conditions that promote the secretion of soluble factors, whereby the 804G cells secrete the factors;
removing the media from the 804G cells; and contacting the media with epithelial cells, whereby the contact promotes hemidesmosome formation in the epithelial cells.
Preferably, the epithelial cells are mammalian; most preferably, they are human skin cells.
The present invention also includes a biocompatible shaped article adapted for use in vivo in a mammal, and a hemidesmosome formation-facilitating protein composition on the shaped article. Preferably, the protein composition is secreted by a tumor cell line of epithelial origin and the tumor cell line is either the rat bladder carcinoma cell line 804G or NBT-II.
Still another embodiment of the present invention is a composition for use in growing mammalian cells consisting of media preconditioned by growth of 804G or 804GMH cells, the media having the property of promoting hemidesmosome formation in cells contacting the media.
The present invention includes the discovery that a soluble factor secreted into the growth media by certain cell lines can stimulate cellular adhesion and hemidesmosome assembly in epithelial cells. One type of cell with this ability is the rat bladder carcinoma cell line 804G. This cell line has been described by Izumi, et al., Cancer Res. (1981); 41:405-409, and is maintained in permanent collection in the laboratory of inventor Jonathan C. R. Jones, from whom the cell line is readily available. This cell line is also available from Ryoichi Oyasu, Department of Pathology, Northwestern University Medical School, Chicago, Ill. The 804G cell line is also maintained as a Budapest Treaty patent deposit by the American Type Culture Collection (ATCC), Rockville, Md., under accession number ATCC CRL 11555 made Feb. 24, 1994. Furthermore, the purification of these soluble factors is greatly facilitated by culturing the 804G cells under low serum conditions due to the virtual absence of contaminating serum proteins. Although the 804G cells were cultured in Dulbecco's Modified Eagles Medium:OPTI-MEM (1:1) in the presence of 1 % fetal calf serum, the use of other media and other concentrations of fetal calf serum, preferably from about 0.1% to about 5%, is also contemplated.
Ultrastructural data have been developed demonstrating that the 804G soluble factor can induce a number of different cell types to develop mature hemidesmosomes and attach to their growth substrate. A solution can now be prepared, having factors secreted by cells such as 804G cells, that can modulate the organization of hemidesmosomal antigens in unrelated cells. This effect appears specific to hemidesmosomal elements since adhesion plaque components do not obviously change their localization in cells treated with the 804G soluble factor.
To demonstrate our new discovery, we provide evidence that the murine 804G factor was capable of inducing assembly of "mature" hemidesmosomes in human HaCaT cells. It can be appreciated that it is uncommon to find compounds from murine cells that have such a profound effect on human tissue. In the experiments described in more detail below an increased number of hemidesmosome-like structures were found in HaCaT cells treated with 804G growth media, as compared to control experiments wherein HaCaT cells were grown on rat tail collagen. Moreover, the majority of hemidesmosome-like structures in the treated cells contacted the cell-substrate and possessed basal dense plates. The basal dense plate structures are often used as indicators of mature or formed hemidesmosomes (Krawczyk and Wilgram, (1973) J. Ultrastruct. Res., 45:93-101).
Although methods relating to production and isolation of the 804G soluble factor are disclosed, it can be appreciated that any cell that secretes compounds transmitting the ability to support cell adhesion and hemidesmosome assembly in vitro is within the scope of the present invention. Soluble factors from other cell types, such as the murine bladder carcinoma cell line NBT-II (ATCC CRL 1655), also appear able to induce attachment and hemidesmosome assembly in vitro. The NBT-II cell line is also maintained as a Budapest Treaty patent deposit by the American Type Culture Collection (ATCC), Rockville, Md., under accession number ATCC CRL 11556 made Feb. 24, 1994. It should be noted that the term "804G Factor" is used herein to generically refer to any secreted cell factor with the ability to stimulate cell attachment and hemidesmosome formation.
One major use contemplated for the active components of the soluble material is in cell growth and attachment. A substrate upon which cells are to be grown is coated with a solution comprising the soluble factor. The cells to be grown are then plated or applied to the substrate. Such cells, including human cells in vitro and in Wvo, will grow in an organized fashion on the substrate and will form hemidesmosomes. Hemidesmosome formation is a major advantage, because it greatly enhances the attachment of the cells to the substrate. Furthermore, it appears that the organization of cells stimulated by the soluble factor is significantly more advanced, more tissue-like, than cells grown without stimulation by the active components in the secreted factors of the present invention.
The substrate used herein may be any desired substrate. For laboratory use, the substrate may be as simple as glass or plastic. For use in vivo, the substrate may be any biologically compatible material on which cells can grow. Suitable substrate materials may include shaped articles made of or coated with such materials as collagen; regenerated collagen; polylactic acid; biocompatible metals such as stainless steel and titanium; ceramic materials including prosthetic materials such as hydroxylapatite; synthetic polymers, including polyesters and nylons; and virtually any other material to which biological molecules can readily adhere.
One particular use of the present invention is to increase epidermal cell adhesion to target surfaces. For instance, prostheses for dental implantation may be coated with the 804G soluble factor to stimulate periodontal cell attachment. Existing teeth may similarly be coated as a treatment for gum (junctional epithelium) disease, such as gingivitis. Where a substrate is made of polymers of natural or synthetic bioerodible material in the form of a sheet or fabric, such as woven or bonded collagen, polylactic acid, lactide, glycolide, glutamic acid, collagen or albumin the matrix materials may be applied to the surface thereof or mixed in with the composition. Cells (such as epidermal cells) may then be grown on the matrix ex vivo to form transplantable or implantable materials; alternatively, the materials may be implanted and cells may be permitted to attach in vivo.
The 804G soluble factor will also be of great use in studies concerning hemidesmosome morphogenesis and α6 β4 integrin interactions with the epithelial extracellular matrix. Indeed, the active factors secreted by the 804G cells may prove to be a tool that allows definition of hemidesmosome-mediated interactions between epithelial cells and their underlying connective tissues at the molecular level.
In addition to the soluble factor and the active components thereof, the present invention also includes shaped articles coated with those materials. Preferably, those shaped articles are formed of materials other than glass, and include such forms as sheets, fabrics, prostheses, metal articles, bioerodible articles, and implantable articles.
Furthermore, pharmaceutical preparations having the soluble factor are contemplated. These preparations can be in any suitable form, and generally comprise the active ingredient in combination with any of the well known pharmaceutically acceptable carriers. The soluble factor may be isolated from the growth media in which appropriate cells have been grown. Alternatively, the soluble factor may be prepared synthetically or through recombinant DNA techniques, or through purification of isolated proteins from the growth media.
Carriers can include injectable carriers, topical carriers, transdermal carriers, and the like. The preparation may advantageously be in a form for topical administration, such as an ointment, gel, cream, spray, dispersion, suspension, or paste. The preparations may further advantageously include preservatives, antibacterials, antifungals, antioxidants, osmotic agents, and similar materials in composition and quantity as is conventional. For assistance in formulating the compositions of the present invention, one may refer to Remington's Pharmaceutical Sciences, 15th Ed., Mack Publishing Co., Easton Pa. (1975), the relevant disclosure of which is hereby incorporated by reference.
Finally, epithelial cells of various types may be grown in contact by the compositions contemplated herein.
As a first step in discovering the properties of the 804G soluble factor, HaCaT cells were treated with media from growing 804G cells.
The immortalized human keratinocyte cell line HaCaT, provided by Dr. Norbert Fusenig, Heidelberg, Germany (Boukamp et al., (1988) J. Cell Biol., 106:761-771), was cultured in DMEM medium (Bio-Whittaker, Walkersville, Md.) supplemented with 10% fetal calf serum (FCS, Bio-Whittaker) and antibiotics. The HaCaT cell line has normal keratinization properties in vitro, is positive for involucrin, filaggrin, cytokeratins 1, 10, 5, 6, 14, 16/17, 7, 8 and 19 and is negative for vimentin. Thus it has characteristics very similar to primary keratinocytes.
The rat bladder carcinoma cell line, 804G, and the human embryonic fibroblast cell line WI-38 (ATCC #CCL 75) were also cultured in DMEM medium with the same supplements.
Culture supernatant of 804G cells was collected from cultures that were approximately 70% confluent and reached confluence over a 48 hour period. At the end of this time 15 mls of supernatant was collected from a 75 cm2 culture flask. Supernatant of HaCaT and WI-38 cells were collected in the same manner over a 48 hour period.
HaCaT cells plated on tissue culture plastic in normal medium attach, spread very slowly, and still appear rounded 2 hours after seeding. In contrast, however, when the cells were seeded in the culture supernatant of 804G cells they attached to the growth substratum and acquired a flattened morphology within 30 minutes. After 24 hours, cells in normal medium formed epithelioid islands whereas cells seeded in supernatant from 804G cells exhibited a spread-out morphology and appeared to migrate so as to uniformly cover the growth substratum. The 804G culture supernatant effect was evident even if the cells were plated in a 1:1 dilution of the supernatant with normal medium. As a control, HaCaT cells were also plated in their own culture supernatant and in medium collected from cultures of human fibroblasts (WI-38). HaCaT cells plated in either their own medium or WI-38 medium did not exhibit the growth and morphology of those cells plated in 804G medium.
We then performed the following experiments to analyze the affect of the 804G supernatant on hemidesmosome elements in the HaCaT cells.
To study the effect of 804G culture supernatants on the distribution of α6 β4 integrins, HaCaT cells were grown on glass coverslips, fixed and immunolabeled for α6 and β4 integrin subunits, hemidesmosomal components, or epithelial matrix elements.
HaCaT cells were grown for 24 hours on glass coverslips for immunofluorescence microscopy either in normal medium, medium conditioned for 48 hours with 804G cells, or in co-culture (1:1) with 804G cells. The cells were fixed for 5 minutes in -20� C. methanol, washed in PBS and immunolabeled with the following antibodies:
(1) AA3; a mouse monoclonal antibody to the human integrin subunit (Tamura, et al., J. Cell Biol. 1990; 111:1593-1604). This antibody specifically binds to human integrin molecules.
(2) GOH3; a rat monoclonal antibody to the α6 integrin subunit (AMAC, Westbrook, Me.). This antibody reacts with human and mouse, but not rat integrin molecules.
(3) 6844; a rabbit polyclonal antiserum to the cytoplasmic terminal 15 amino acids of the α6 integrin subunit.
(4) J18; a rabbit antiserum to the solubilized matrix of 04G cells (Langhofer, et al., (1993) J. Cell Sci., 105:753-764.
(5) 5C5; a mouse monoclonal antibody the solubilized matrix of 804G cells.
(6) J17; a rabbit antiserum against the 180 kD hemidesmosomal protein (Riddelle et al., (1992) J. Cell Sci., 103:475-490).
(7) P1E1; a mouse monoclonal antibody to epiligrin (from Dr. William G. Carter, Fred Hutchinson Cancer Research Center, Seattle, Wash., Carter, et al. 1991).
(8) BM165; a mouse monoclonal antibody to kalinin (from Dr. Robert E. Burgeson, Oregon Health Sciences University, Portland, Oreg., Rousselle, et al., 1991, Marinkovich, et al. 1992) .
(9) GB3; a mouse monoclonal antibody to human basement membranes (from Accurate Chemical and Scientific Corporation, Westbury, N.Y., Verrando et al., (1987) Exp. Cell Res., 170:16-128; Verrando et al., (1988) Biochim. Biophys. Acta., 942:45-56.
Fluorescein isothiocyanate (FITC) or tetramethyl-rhodamine isothiocyanate (TRITC) conjugated anti-mouse and anti-rabbit antibodies were purchased from Jackson Immunoresearch Laboratories Inc. (West Grove, Pa.). FITC or TRITC conjugated anti-rat antibodies were from Sigma Chemical Corporation (St. Louis, Mo.).
After single or double immunolabeling the cells were studied under a Zeiss Axlophot microscope equipped with epifluorescence and phase contrast optics. Photographs were taken with a Leitz Orthomat E automatic camera system and Kodak TMY 400 film at EI 800.
The visualization and photography of living cells was performed with a Zeiss Axiovert microscope equipped with phase contrast optics and the same camera system as above.
In cells grown in normal medium, the α6 and β4 integrin subunits had a patchy, finely granular distribution most clearly visualized at the edges of cell islands. In cells grown in the 804G supernatant, the α6 and β4 subunits were reorganized into coarsely granular or "Swiss-cheese" type patterns. The same patterns were reflected in immunolabeling with any of the antibodies against epithelial matrix elements (P1E1, GB3, BM165) and with the antibody against the 180 kD hemidesmosomal protein (J17).
Polypeptide samples from the culture medium and solubilized matrix from 804G and HaCaT cells were analyzed by SDS-polyacrylamide gel electrophoresis on a 6% resolving gel (Laemmli 1974) with the NOVEX (Encinitas, Calif.) electrophoresis system. The separated polypeptides of culture medium, or purified rat laminin and fibronectin (as controls, Telios Pharmaceuticals/GIBCO, Grand Island, N.Y.) were electrophoretically transferred to Immobilon-P membranes (Millipore Corporation, Bedford, Mass.) and processed for immunoblotting with J18 and 5C5 antibodies. As controls, the polypeptides were immunoblotted with rabbit antiserum against rat laminin or rat fibronectin (Telios Pharmaceuticals). The Vectastain ABC immunoperoxidase or alkaline phosphatase were used to detect binding. (Vector Laboratories, Burlingame, Calif.).
The polyclonal antibody, J18 reacted with these polypeptides in immunoblotting experiments. Immunoblotting revealed further that the antibody did not cross-react with laminin or fibronectin, two common extracellular matrix molecules. However, the 804G cell matrix and medium do contain fibronectin, but only trace amounts of laminin-related material.
The monoclonal antibody 5C5 also reacts with the major polypeptides in 804G matrix. Immunoblotting of the HaCaT matrix reveals the same three 160-138 kD polypeptides as in the 804G matrix with an additional polypeptide having a molecular weight of approximately 130 kD. The 5C5 antibody is specific to rat proteins and therefore does not react with the HaCaT cell matrix grown in normal conditions.
In the culture medium of 804G cells the Mr 160-138 polypeptides are not clearly discernible by protein staining but can, instead, be identified by fluorography of metabolically S-labeled proteins.
Metabolic labeling with 35 S-methionine was performed by first incubating the cells for 30 minutes in starving medium (MEM-medium without methionine, supplemented with 1% dialyzed FCS, L-glutamine and antibiotics, GIBCO, Grand Island, N.Y.), and then replacing the medium with 3 ml of fresh MEM without methionine, supplemented with 1% dialyzed FCS and 250 μCi S-methionine (Tran S-Label ICN Biomedicals Inc., Costa Mesa, Calif.) for 10 hours.
Gels with radioactively labeled polypeptides were processed for fluorography according to Bonner and Laskey (1974) and exposed to Hyperfilm (Amersham Corporation, Arlington Heights, Ill.).
The incorporation of 35 S-methionine indicated that the polypeptides were synthesized by the cells and not simply deposited into the matrix from the culture medium. Moreover, the polypeptides can be visualized by immunoblotting with J18 and 5C5 antibodies. When HaCaT cells are grown in 804G Cell supernatant, and their matrix is processed for immunoblotting with 5C5, two reactive polypeptides can be identified indicating that the soluble immunoreactive material from 804G supernatant is able to bind to the matrix of HaCaT cells.
The effect of the soluble 804G cell components on HaCaT cells was also evident at the ultrastructural level. When HaCaT cells are grown on cell culture plastic in normal culture medium, they attach to their growth substratum by means of extracellular matrix contacts that resemble rudimentary hemidesmosomes. Alternatively, when the cells were grown in 804G cell supernatant, they formed adhesion complexes that are by morphological criteria mature hemidesmosomes.
When the cells were grown in 804G supernatant immunodepleted with J18 antibody, only occasional hemidesmosomes can be identified at the ventral plasma membrane. Immunodepletion of 804G supernatant was carried out by treating 1 ml of supernatant three consecutive times with the J18 antibody coupled to 50 μl protein A SEPHAROSE� beads (2 μg antiserum/10 μl packed beads, Sigma Chemical Corporation). As a control, 804G supernatant was also depleted with normal rabbit serum coupled to protein A coupled to crosslinked dextran (SEPHAROSE�). The cells were fixed in modified Karnowsky fixative (1% paraformaldehyde, 0.1M Na-cacodylate, 1.75% glutaraldehyde, 2.5 mM CaCl) and processed for electron microscopy by routine methods. Thin sections were cut perpendicular to the cell layer and studied at 75 kV in a Hitachi Hu-12A microscope. The specificity of this effect is verified by the fact that immunodepletion with normal rabbit serum does not affect the hemidesmosome inducing potential of 804G supernatant.
To facilitate purification of secreted proteins, the 804G cell line was adapted to grow under low serum conditions as described in the following example.
804G cells were gradually adapted to grow in 1:1 DMEM:OPTI-MEM (GIBCO, Grand Island, N.Y.) supplemented with 1% FCS, 2 mM glutamine, 100 μg/ml penicillin and 50 μg/ml streptomycin. The resulting 804G cell subpopulation was named 804GMH. According to the manufacturer, OPTI-MEM contains low amounts of transferrin and insulin, molecular weights 80 and 6 kDa, respectively, but no other proteins.
The virtual absence of serum proteins in the culture medium simplifies the purification of hemidesmosome-inducing soluble factors as described below.
For the collection of serum-free culture supernatant, confluent 804GMH cells grown under low serum conditions were removed by trypsinization (0.02%), washed once with DMEM containing 10% FCS and cultured in DMEM:OPTI-MEM with no added FCS at a split ratio of 1:6. Culture supernatant was collected when 804GMH cells had been confluent for 24 hours. The supernatant was centrifuged at 5,000�g for 10 minutes and stored at -20� C. until use. Secreted proteins were purified by precipitation with ammonium sulfate at 40% saturation. Culture supernatant (1 liter) was cleared of particulate material by centrifugation at 10,000�g for 30 minutes and transferred to another container on ice. Ammonium sulfate was slowly added, with stirring, to 30% saturation. The supernatant was then left at 4� C. overnight to allow complete precipitation. The sample was centrifuged for 30 min at 10,000�g and ammonium sulfate added to a final concentration of 40% saturation. After precipitation and centrifugation, the supernatant was discarded and the pellet resuspended in 1 ml PBS. The protein was dialyzed against PBS, the protein concentration estimated by absorbance at 280 nm, and an aliquot analyzed by SDS-PAGE. Bands of 240, 150 and 140 kDa were observed.
Thus, we have demonstrated that soluble factors produced by 804G cells are able to induce attachment and hemidesmosome assembly in mammalian cells and that the purification of these proteins from the culture medium is greatly facilitated by growing the cells under low serum conditions.
It should be noted that the present invention is not limited to only those embodiments described in the Detailed Description. Any embodiment which retains the spirit of the present invention should be considered to be within its scope. However, the invention is only limited by the scope of the following claims.
|Cited Patent
|Filing date
|Publication date
|Sep 12, 1991
|Dec 28, 1993
|The Regents Of The University Of California
|Method and apparatus for preparing composite skin replacement
|Mar 26, 1992
|Oct 15, 1992
|The State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The Oregon Health Sciences University
|Product and method for improving keratinocyte adhesion to the dermis
|Aug 27, 1993
|Mar 17, 1994
|The State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of Oregon Health Sciences University
|Products and methods for improving keratinocyte adhesion to the dermis
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|Jones, et al. "α6 β4 Integrins: Their Role in the Assembly of the Hemidesmosome (HD) and in Signal Transduction", J. Cell. Biochem., Supplement 16F: Apr., 1992.
|Jones, et al. 6 4 Integrins: Their Role in the Assembly of the Hemidesmosome (HD) and in Signal Transduction , J. Cell. Biochem., Supplement 16F: Apr., 1992.
|Jones, et al. Intermediate Filament Plasma Membrane Interactions , Curr. Opin. Cell Biology, 3: 127 132, 1991.
|Kurpakus, et al. "Integrins in the Hemidesmosome", J. Cell. Biol., 111: 402a, 1990.
|Kurpakus, et al. "Surface Relocation of α6 β4 Integrins and Assembly of Hemidesmosomes in an In Vitro Model of Wound Healing", The Journal of Cell Biology, 115:1737-1750, 1991.
|Kurpakus, et al. Integrins in the Hemidesmosome , J. Cell. Biol., 111: 402a, 1990.
|Kurpakus, et al. Surface Relocation of 6 4 Integrins and Assembly of Hemidesmosomes in an In Vitro Model of Wound Healing , The Journal of Cell Biology, 115:1737 1750, 1991.
|Langhofer, et al. "Matrix Signals Transduced by the α6 β4 Integrin Complex", Mol. Biol. Cell., 3, Supplement: 95a, Sep., 1992.
|Langhofer, et al. Matrix Signals Transduced by the 6 4 Integrin Complex , Mol. Biol. Cell., 3, Supplement: 95a, Sep., 1992.
|Langhofer, et al., "The Matrix Secreted by 804G Cells Contains Laminin-Related Components that Participate in Hemidesmosome assembly in Vitro", Journal of Cell Science, 105: 753-764, 1993.
|Langhofer, et al., The Matrix Secreted by 804G Cells Contains Laminin Related Components that Participate in Hemidesmosome assembly in Vitro , Journal of Cell Science, 105: 753 764, 1993.
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|Laminin 5 for growth of pancreatic islet cells
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|Mar 29, 1996
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|Northwestern University
|Human epithelial cell matrix and uses therefor
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|Mar 7, 2000
|Desmos, Incorporated
|Laminin 5 for periodontal treatment
|Jan 15, 1999
|Sep 25, 2001
|Northwestern University
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|C. R. Bard, Inc.
|Collagen hemostatic fibers
|Dec 11, 1998
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|Laminin 8 and methods for its use
|Apr 28, 2000
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|Aug 29, 2002
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|Human Mob-5 (IL-24) receptors and uses thereof
|Aug 29, 2002
|Apr 24, 2003
|Peng Liang
|Human Mob-5 (IL-24) receptors and uses thereof
|Jun 25, 2003
|Jan 22, 2004
|Ariel Boutaud
|Recombinant laminin 5
|Dec 17, 1998
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|U.S. Classification
|International Classification
|Cooperative Classification
|European Classification
|Jan 12, 1994
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In Huckleberry Finn, Mark Twain implements a wide range of humor composed of irony, satire, and exaggeration. He not only uses this humorous effect to make the book entertaining but also to accentuate his various themes and to help complement his main point, the cruelty of making slaves out of blacks.
Huckleberry Finn reeks of irony. Irony, according to the Oxford Dictionary, is defined as "use of language that has an inner meaning for a privileged audience and an outer meaning for the persons addressed or concerned.†The three main types of irony that are used throughout the story are verbal irony, situational irony, and dramatic irony. .
The verbal irony Mark Twain uses is close to sarcasm. Twain uses verbal irony by saying one thing but meaning something else. An example of verbal irony is given in this quote: "But Tom Sawyer he hunted me up and said he was going to start a band of robbers, and I might join if I would go back to the widow and be respectable.†A band of robbers is not supposed to be respectable, so therefore it is ironic that Huck could only join if he went back to the widow and became respectableOne very good example of verbal irony that Twain uses is in Chapter 31, where Twain writes "First they done a lecture on temperance; but they didn't make enough for them both to get drunk on.†Another good example of verbal irony is when Levi Bell the lawyer says "Set down, my boy; I wouldn't strain myself if I was you. I reckon you ain't used to lying, it don't seem to come handy; what you want is practice. You do it pretty awkward.†This is very ironic because Huck has survived throughout this whole story by lying and the lawyer seems to think that Huck has had no experience or exposure to the techniques of lying.
Another example of verbal irony that Twain uses in chapter 20 is when the duke and the king are interrogating Huck about their raft and Jim.


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You missed it This great deal for Carmello's is over. But don't despair, we've got more great deals every day.
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Dinner for Two with Appetizers, Salads, or Soups; Two Entrées; and Two Desserts from Pre-Set Menu
Forget about traversing the Atlantic; you can find an authentic blend of Italy and Portugal here in Old Town Manassas. With today's deal from Carmello's, give your standard dining routine the boot when you pay $35 dinner for two (a $70 value). You'll get a choice of two appetizers, soups, or salads; two entrées; and two desserts from a pre-set menu. Appetizers include bruschetta, mushroom caps, and a spinach salad. Then move on to entrées like a succulent veal tenderloin wrapped in bacon; homemade spinach-and-ricotta stuffed ravioli; a pork shank in rosemary demi glaze; or a traditional Portuguese recipe of pork cooked with clams, potatoes, garlic, and cilantro in a white wine sauce. End your meal with a mixed sorbet, fresh berries, creme brulee, or mascarpone cheese with a strawberry puree. This spot has been around for more than 20 years, and owner Alice Pires has created a space where dishes from her home in Portugal are perfectly paired with traditional and satisfying Italian cuisine. With dim lighting, white tablecloths, and exposed brick, this cozy space is the perfect destination for your next culinary adventure.
PAID VALUE EXPIRES ON January 17, 2022
PROMOTIONAL VALUE EXPIRES ON August 31, 2012


text:
Contact Us
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Since the year 2000, the citizens of one town have abandoned nearly fourteen hundred boys in the Utah Desert. Deemed the "Lost Boys of Utah", Exit 27 is based on their true stories. Merilo's play follows the journey of four of these "lost boys" to the edge of the desert. Exiled by their families from the only world they've known, the boys are forced to rebuild their lives as outsiders. Their only hope for survival? Each other.


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I'd say by the weight of this...it is quite heavy for size...that it is a spelter metal with a copperized finish. Measures 4 1/2" x 3 1/2". Nice decorative item...looks to be circa early 1900's..
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At Molloy's MEGA ANTIQUES CENTRE, Takapuna, Auckland, New Zealand
Specialists in Pacific Islands Items, Victoriana, And a Fascinating Range of General Collectibles
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Malarrimo Motel
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Best of Veracruz
... could enjoy it more. Just 1 group broke the boat after few meters and we had to wait until they got a new one. But then everything was fine and nobody fell off the boat (unintentionally), a great adventure!
Afterwards we just had a quick shower and lunch and then went towards the capital of Veracruz – Xalapa. On the way we stopped in Coatepec, a small colonial town which is famous for its coffee. Actually there is coffee in whole Veracruz. And ...
Creatures 7 -- Mind Officially Blown Now
... full" in a day. "They also enjoy sucking herring spawn of eel grass in the spring, a particularly nutritious meal after fasting down south (during calving season)."
Gray whales migrate from the Arctic Ocean, Bering and Chukchi Seas to the southernmost tip of Baja and sometimes even up into the Gulf of Mexico: a journey of at least five thousand miles in each direction. They feed enroute, but not in the calving lagoons. In ...
Guerrero Negro
... survive. Just outside of the Lagoon is a salt mine. The salt is what keep going almost the entire population of Guerrero Negro sustained . This is the largest evaporative salt production facility in the world. The operation produces close to six million tons of salt each year. There are thousands of evaporating ...
Confined to Quarters
... to feel a tad better, so maybe it's a sign of better things ahead, although I could have done without the nose bleed at around 5pm. Luckily, it didn't last long. I still have one more day of recovery at sea before Los Angeles, so tomorrow will be quiet, whatever happens. I have mentioned before that, whilst I hope this blog keeps my readers entertained, primarily it's for my benefit as a record of where I was, how I felt and what I ...
One Great Museum
... audio guide in English was available.
The building swoops down a hillside moving you down from the earlest history to the last stages of pre-Hispanic development. And many of the artefacts are breathaking in their beauty, in the miracle of their preservation (life-size pottery models of gods etc) and in the excellence of the preservation.
So that's it for this posting - a museum to be celebrated and lots of photos of exhibits.


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WDET's Discovery Series: Cabaret 313 Presents Norm Lewis
Friday, March 14, 2014 from 7:00 PM to 9:30 PM (EDT)
Who's Going
San Francisco, California
London, United Kingdom
Florence, Italy
Event Details
Tickets to the Early Performance and Late Performance have sold out.
We still invite members to attend Cabaret 313 presents Norm Lewis. You can find additional information and purchase tickets here. See you there!
Cabaret 313 is thrilled to present Norm Lewis for an intimate evening of song on March 14th at the Marlene Boll Theatre in Detroit.
Norm Lewis is a Tony-nominated actor, recognized for his leading roles in Broadway's Porgy & Bess and Les Mis. This handsome actor with a spectacularly beautiful voice is known to most for his role as Senator Edison Davis on ABC TV's hit series, Scandal. This show will be his Detroit debut!
Early performance - Reception: 6:00 pm | Show: 7:00 pm
Late performance - Reception: 9:00 pm | Show: 10:00 pm
*Reception includes complimentary hors d' oeuvres, beer and wine.
For more information or to purchase additional tickets visit cabaret313.org.
When & Where
Marlene Boll Theatre
1401 Broadway Street
Friday, March 14, 2014 from 7:00 PM to 9:30 PM (EDT)
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Best Buy, GameStop, Home Depot, Red Hat: U.S. Equity Movers
Shares of the following companies had unusual moves in U.S. trading. Stock symbols are in parentheses, and prices are as of 4 p.m. in New York.
Amazon.com Inc. (AMZN) rose 3.5 percent to $171.10 for the biggest gain since March 4. The largest e-commerce company was raised to "outperform" from "market perform" at William Blair & Co.
Arctic Cat Inc. (ACAT) rallied 4.2 percent to $14.55, the highest price since Feb. 11. The maker of all-terrain recreation vehicles was raised to "buy" from "hold" at KeyBanc Capital Markets Inc.
Bank of America Corp. (BAC US) had the second biggest decline in the Dow Jones Industrial Average, sinking 1.3 percent to $13.48. The biggest U.S. lender was cut to "market perform" from "outperform" at FBR Capital Markets. The bank may not have the earnings power to be in compliance with the new capital requirements, analyst Paul Miller said in a note to clients.
Best Buy Co. (BBY) slipped 5.4 percent, the most since Dec. 14, to $30.13. The world's largest consumer electronics retailer forecast full-year profit that trailed some analysts' projections, citing economic challenges.
Charming Shoppes Inc. (CHRS US) surged 17 percent to $3.47 and rose as much as 21 percent earlier, the most intraday since April 2009. The women's apparel retailer reported an adjusted fourth-quarter loss of 8 cents a share, beating the 20-cent loss average estimate of three analysts. Sales in the same period were $575.8 million, surpassing the prediction of $553 million by analysts.
Drugstore.com Inc. (DSCM US) rose the most since 1999, more than doubling to $3.82. Walgreen Co. (WAG) agreed to buy the online pharmacy for $3.80 a share, or about $409 million.
Ford Motor Co. (F) gained 5.1 percent, the most since Nov. 3, to $15.04. The second-largest U.S. automaker rose as consumer information firm J.D. Power and Associates upgraded its new-vehicle retail sales forecast for 2011 to 10.6 million from 10.5 million.
GameStop Corp. (GME) rose 2.9 percent to $21.73, the highest price since Jan. 5. The world's largest video-game retailer said it expects "solid growth" in 2011 and said it may conduct a share and debt repurchase program. The company earlier forecast first-quarter profit excluding some items of at least 53 cents a share, beating the average analyst estimate of 52 cents in a Bloomberg survey.
Harmony Gold Mining Ltd. (HMY) advanced 6.7 percent to $14.39, the highest price since March 2008. Africa's third-largest producer of the metal may become a takeover target, RBC Capital Markets said.
Harry Winston Diamond Corp. (HWD US) fell 3.8 percent to $14.36 for its biggest decline since Feb. 22. Kinross Gold Corp. sold 7.1 million shares in the mining company and jewelry retailer, representing a stake of about 8.5 percent, for about C$100 million ($102.7 million).
Imax Corp. (IMAX) rose 13 percent, the most since Oct. 28, to $29.99. The operator of large-screen movie theaters plans to open at least 100 theaters a year for two to three years, said Richard Gelfond, the company's chief executive officer.
Micron Technology Inc. (MU) advanced 8.4 percent, the most since June 15, to $11.50. The largest U.S. maker of computer-memory chips reported fiscal second-quarter sales of $2.26 billion, beating the average estimate of 2.06 billion by 19 analysts in a Bloomberg survey.
Nvidia Corp. (NVDA) , the maker of three dimensional graphics processors, rose 8 percent to $19.23. Atmel Corp. (ATML) , the maker of chips for household appliances, gained 6.6 percent to $13.23.
Omnova Solutions Inc. (OMN) surged 13 percent, the most since September 2009, to $7.65. The polymer maker posted first-quarter profit excluding some items of 14 cents a share, beating the average analyst estimate by 61 percent, according to Bloomberg data.
Paychex Inc. (PAYX) fell 4 percent to $30.96, the second-biggest decline in the Standard & Poor's 500 Index. Oppenheimer & Co. analyst Glenn Greene said that while the company's third-quarter earnings were "in line" with his estimate, new sales appear to have been "disappointing," citing comments from management at the seller of payroll and human-resource services to small- and medium-size businesses.
Powerwave Technologies Inc. (PWAVQ) climbed 3.8 percent to $4.66, the highest price since September 2008. The maker of wireless communications products was raised to "buy" from "neutral" at CL King & Associates Inc.
Red Hat Inc. (RHT) gained 18 percent to $47.26, the biggest gain in the S&P 500. The largest seller of the Linux operating system posted a profit of 26 cents a share excluding some items, beating the 22 cent average of 22 estimates in a Bloomberg survey.
Scholastic Corp. (SCHL) slid 11 percent, the most since September 2008, to $27.74. The children's publishing company cut its forecast for adjusted earnings in 2011 to no more than $1.40 a share, down from earlier guidance of at least $1.80 a share. The lowered forecast misses the average analyst estimate of $1.93.
Smithfield Foods Inc. (SFD US) rose 5.6 percent to $23.97 and rallied 6 percent earlier, the most intraday since Dec. 9. The world's largest pork producer was raised to "'buy" from "hold" at BB&T Capital Markets.
Steelcase Inc. (SCS) advanced 11 percent, the most since November 2009, to $10.58. The maker of office furniture reported fourth-quarter profit of 11 cents a share, excluding some items, beating the average analyst estimate by 41 percent, Bloomberg data show.
Talbots Inc. (TLB US) rallied 23 percent, the most since February 2009, to $5.89. The Hingham, Massachusetts-based retailer said its loss in the fourth-quarter was 14 cents a share, beating the 18-cent loss average estimate by analysts.
Trident Microsystems Inc. (TRID US) rose 29 percent, the most since 1992, to $1.15. The maker of graphic chips for digital televisions had a system-on-a-chip design incorporated into a new line of Hisense televisions for the Japanese market and said its chips are powering one of the first trial deployments of Next Generation Broadcasting in China.
To contact the reporter on this story: Whitney Kisling in New York at email@example.com
To contact the editor responsible for this story: Nick Baker at firstname.lastname@example.com


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On a small excursion to Greece.Rhodes to be exact!
... settled during the Bronze Age, 3000 BC. What remains today are ruins of a spiritual/cult community honoring a feminine deity, the temple to the goddess of witchcraft, Hecate.
There is a very nice description of this site:
http://turkeyfromtheinside.com/places-t o-go/l/1173-lagina.html
While it is off the main road and requires a bit of a trek it was a really beautiful site ...
Mosquitos and scorpions
Blue skies, no clouds and three mosquito bites. What a way to wake up. But I would rather wake up to a mosquito bite than being bitten by a scorpion. We killed a second one last night. Not quite an animal you want to have closer contact with. Karsten made menemen for breakfast and we were indecisive what we wanted to do. First we ...
Gyros and Jamming
... most of the evening jamming and forcing the other two surfers to sing to their dislike and our suffering ears. See the videos!!
It was a great experience and both Larissa and Victor are very generous and thoughtful people. They hosted over 150 people in the last 18 months and the guy leaving the day we arrived had been there for 20 days.
I signed up to a Greek train booking site before leaving the UK as you ...
A rainy afternoon in Rhodes town
Had a wonderful afternoon in the big city. Checked into our hotel, near both the port and the old city. Walked through Freedom Gate into the old city and had a delicious lunch at a place Mikhali Melenos had recommended. After lunch, Helen & Shelley went to the ...
The Old Town and its enchantment
... cheap. A bottle of water was €0.50 and a mixed grill sit in was €10. That a T-Shirt for €10 and the two attractions is all I've spent on what has been an incredible day. I headed back into the Old town in the evening and at 8pm the same stalls run by the same people were open. The Greeks really seem to put a days work in (much like me!!) and just like me!! They do it with a smile. I'm heading to Lindos tomorrow which is more of a beach like place to rest my weary ...


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Nutritional Information
To prepare dumplings, drop ginger and garlic through food chute with food processor on; process until minced. Add the asparagus, 1/4 cup green onion pieces, and water chestnuts; pulse 4 times or until chopped. Add mushrooms, 2 teaspoons sesame oil, 1 1/2 teaspoons soy sauce, and 1/4 teaspoon salt; pulse 4 times or until mixture is finely chopped.
Working with 1 wonton wrapper at a time (cover remaining wrappers with a damp towel to keep from drying), spoon about 2 teaspoons mushroom mixture in the center of wrapper. Moisten edges of wrapper with water. Bring 2 opposite corners together. Press edges together with fingertips to seal, forming a triangle. Place on a baking sheet coated with cooking spray while assembling; lightly cover with a towel or plastic wrap. Repeat procedure with remaining wonton wrappers and mushroom mixture.
Add water to a Dutch oven to a depth of 1 inch; bring to a boil. Coat a metal vegetable steamer with cooking spray. Arrange 9 dumplings in steamer, slightly overlapping. Steam dumplings, covered, 8 minutes or until tender. Remove dumplings from steamer; cover and keep warm. Repeat procedure with remaining dumplings.
To prepare sauce, combine 1/3 cup soy sauce and remaining ingredients. Drizzle over dumplings.


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HERMA Jewel Case Insert 5036 White 25
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Icecat: syndicator of product information via global open catalog with more than 3811026 data-sheets 14816 brands – register (free)
Add to compare Don't compare
The general trademark of a manufacturer by which the consumer knows its products. A manufacturer can have multiple brand names. Some manufacturers license their brand names to other producers.Sony
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Product name is a brand's identification of a product, often a model name, but not totally unique as it can include some product variants. Product name is a key part of the Icecat product title on a product data-sheet.ACCDVP2
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The brand's unique identifier for a product. Multiple product codes can be mapped to one mother product data-sheet if the specifications are identical. We map away wrong codes or sometimes logistic variants.ACCMVP2
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A camera kit contains all of the apparatus you need to build a camera.camera kits
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only logistic data imported: we have only basic data imported from a supplier, a data-sheet is not yet created by an editor.
created by Sony: a data-sheet is imported from an official source from a manufacturer. But the data-sheet is not yet standardized by an Icecat editor.
created/standardized by Icecat: the data-sheet is created or standardized by an Icecat editor.
only logistic data imported: we have only basic data imported from a supplier, a data-sheet is not yet created by an editor.
created by Sony: a data-sheet is imported from an official source from a manufacturer. But the data-sheet is not yet standardized by an Icecat editor.
created/standardized by Icecat: the data-sheet is created or standardized by an Icecat editor.
Included since: 08 Jun 2005
The date Icecat noticed for the first time that this product was included in the price list of a channel partner
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The short editorial description of Sony ACCDVP2Accy Kit For MiniDV-60min Tape,FP50,CASE ACCDVP2
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The official marketing text of Sony ACCDVP2 as supplied by the manufacturerNothing complements your new MiniDV camcorder like the ACCDVP Handycam Starter Kit. It contains all the necessary components to get you up and running in one convenient package.
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- Colour: Black.
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This short summary of the Sony ACCDVP2 data-sheet is auto-generated and uses the product title and the first six key specs.Sony ACCDVP2
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|The stupid question is the question not asked
Re^2: Hash slice from a hashrefby planetscape (Chancellor)
|on Sep 07, 2006 at 15:25 UTC ( #571714=note: print w/replies, xml )
|Need Help??
Indeed. :-) busunsl's intro to references is also quite useful.
IMHO, one of the best discussions of complex data structures, and how to access their innards (using slices and so on), is to be found in Chapter 3. References and complex data structures of perlinter.pdf, available from Perl Training Australia. The OP would probably want the most current version of the course notes, though, which have been reworked into progperl.pdf, available from the same page noted above.
In Section Seekers of Perl Wisdom


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Arkansas' first 10 home games of the 2015-16 season have seen an average attendance of 6,700 people. That's about 1/3 capacity of Bud Walton Arena.
Source: HogStats.com
In December, sports radio host John Nabors wrote that the crowds earlier this season haven't even been as good as in the mediocre John Pelphrey years:
I was the "leader" of the student section in my time at the University of Arkansas. I rocked my Hog headband and a Brandon Dean #13 jersey for every game I could. Most of the seasons where I was a student were pretty average. Minimal success. No postseason. No excitement. Nothing. But we in the Trough built up a pretty good group of kids that showed up and brought hell. The two front rows were filled every game except the ones where most of the kids went home for Winter Break. We were innovative, motivated, and dedicated to giving our team an edge over the opponent. The rest of the Bud Walton Arena spectators fed off of us.
It may not have been as impactful as the mythical mid 1990's Trough, but by God we did our best.
I thought about all of that during the Evansville game. So when I saw the pathetic excuse for a student section, it infuriated me.
Read the rest of Nabors' take on Bud Walton's plummeting attendance at Arkansas Fight.


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Community Development
The Department of Community Development acts as a liaison between the Planning Commission, Zoning Hearing Board and the Historical Commission.
EPA Announces Excellence in Site Reuse Award for Philadelphia Freedom Valley YMCA
Release Date: 10/09/2015
PHILADELPHIA (October 9, 2015) – Today, U.S. Environmental Protection Agency Mid-Atlantic Regional Administrator Shawn M. Garvin presented the Philadelphia Freedom Valley YMCA and its Vice President of Facilities, Michael Troupe, with a 2015 Excellence in Site Reuse Award.
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