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Publication number | US5856184 A |
Publication type | Grant |
Application number | US 08/800,965 |
Publication date | Jan 5, 1999 |
Filing date | Feb 18, 1997 |
Priority date | Jun 30, 1994 |
Fee status | Lapsed |
Also published as | DE69524094D1, DE69524094T2, DE69532835D1, DE69532835T2, EP0767676A1, EP0767676B1, EP1129721A2, EP1129721A3, EP1129721B1, US5567609, US5691182, US6384012, US6419924, US6448222, US6680293, US20030013194, WO1996000582A1 |
Publication number | 08800965, 800965, US 5856184 A, US 5856184A, US-A-5856184, US5856184 A, US5856184A |
Inventors | Michael P. Sarras, Jr., Billy G. Hudson |
Original Assignee | Yale University, University Of Kansas Medical Center |
Export Citation | BiBTeX, EndNote, RefMan |
Non-Patent Citations (32), Referenced by (25), Classifications (19), Legal Events (7) | |
External Links: USPTO, USPTO Assignment, Espacenet | |
The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grants No. 01-RR06500 and AM 18381 awarded by the National Institute of Health.
This is a continuation of application Ser. No. 08/497,206, filed Jun. 30, 1995 which is now U.S. Pat. No. 5,691,182, which is a continuation-in-part of application Ser. No. 08/268,969, filed Jun. 30, 1994 which is now U.S. Pat. No. 5,567,609.
1. Field of the Invention
This invention relates to methods for the manipulation of intercellular and intertissue interactions. The instant invention provides methods for the inhibition of cell adhesion to extracellular matrix components or the formation of functional basal lamina, and the manipulation of the results of such attachments. Thus the instant invention pertains to the modification of cellular interactions, with extracellular components, and methods for maintaining cell phenotype, developmental stage, and plasticity in vivo and in vitro.
2. Description of the Prior Art
The basement membrane (basal lamina) is a sheet-like extracellular matrix which is a basic component of all tissues. The basal lamina provides for the compartmentalization of tissues, and acts as a filter for substances traveling between tissue compartments. Typically, the basal lamina is found closely associated with an epithelium, or endothelium in all tissues of an animal including blood vessels and capillaries. The basal lamina components are secreted by cells, and then self assemble to form an intricate extracellular network. The formation of a biologically active basal lamina is important to the development and differentiation of the associated cells.
The Cnidarian, Hydra, is a simplified metazoan whose body wall is composed of an epithelial bilayer with an intervening extracellular matrix (ECM) termed the mesoglea. Hydra mesoglea have been shown to have a number of components seen in the ECM or basement membranes of higher invertebrates and vertebrates, these include: fibronectin, type IV collagen, laminin, and heparan sulfate proteoglycan. Hydra cell aggregation involves the complete morphogenesis of adult hydra from pellets of dissociated hydra cells. During this developmental process, cells segregate into an epithelial bilayer and then deposit a new extracellular matrix prior to the continuation of morphogenesis.
Extracellular matrix (ECM) components play a critical role in development through their affects on such cell processes as cell division, cell attachment, cell migration, and cell differentiation (reviewed by Timpl et al., 1989, Int. Rev. Exp. Pathol. 29:1-112; Damsky and Bernfield, 1991, Current Opn. in Cell Bio. 3:777-778; Hynes, 1992, Cell 69:11-25). It has been established that ECM/cell interactions are utilized by a wide range of vertebrate and invertebrate species to include such primitive organisms as the Cnidarian, Hydra. Hydra is particularly interesting in this regard because it represents one of the first animal phyla to develop defined tissue layers separated by an acellular extracellular matrix (Field et al., 1988, Science 239:748-752). Previous studies have shown that hydra ECM, termed mesoglea, contains type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycans (Sarras et al., 1991a, Dev. Biol. 148:481-494). These molecules are continuously synthesized and deposited into the mesoglea in adult hydra and during hydra head regeneration (Hausman et al., 1971, J. Exp. Zool. 177:435-446). Other studies have shown that developmental processes in hydra such as head regeneration are dependent on the normal formation of ECM. These studies have shown that head regeneration in hydra morphogenesis can be blocked by using drugs that perturb collagen cross linking or drugs that interfere with proteoglycan GAG chain extension (Sarras et al., 1991b, Dev. Biol. 148:495-500). These studies have most recently been extended to the hydra cell aggregate system. This system allows one to form a pellet with dissociated hydra cells and then observe the complete regeneration of the adult hydra body within 72-96 hours through the process of cytodifferentiation and morphogenesis (Gierer et al., 1972, Nature New Biol. 239:98-101; Sarras et al., 1993, Dev. Biol. 157:383-398). Such studies of hydra development and the role of the ECM have focused heavily on a chemical approach (Barzanski et al., 1974, Amer. Zool. 14:575-581; Sarras et al., 1991ab, supra). Hydra cell aggregates first form an epithelial bilayer and then deposit an ECM before morphogenesis proceeds. Hydra cell aggregate development is blocked by drugs that perturb ECM formation and by antibodies raised against isolated hydra mesoglea. These studies demonstrate that functional studies of ECM/cell interaction can be carried out under in vivo conditions with hydra.
Type IV collagen has been shown to be a major structural component of basement membranes and has also been shown to be present in hydra ECM. The protomeric form of type IV collagen is formed as a heterotrimer made up from a number of different subunit chains called α1(IV), α2(IV) etc. The type IV collagen heterotrimer is characterized by three distinct structural domains: the non-collagenous (NC1) domain at the carboxyl terminus; the triple helical collagenous domain in the middle region; and the 7S collagenous domain at the amino terminus (FIG. 1) (Martin et al., 1988, Adv. Protein Chem. 39:1-50; Gunwar et al., 1991, J. Biol. Chem. 266:14088-14094). Type IV collagen exists as a supramolecular structure in ECM and this structure is thought to serve as a framework which provides mechanical stability to ECM (Timpl et al., 1986, supra) and as a scaffolding for the binding and alignment of other ECM molecules such as fibronectin, laminin, eutectin, and heparan sulfate proteoglycans (Gunwar et al., 1991, supra). The biological function of type IV collagen is critically related to the formation of an intact ECM since disruption of collagen cross linking by β-aminopropionitrile interferes with the mesoglea formation and this leads to a blockage in normal hydra morphogenesis (Sarras et al., 1991b, 1993, supra).
Hydra cell aggregate development involves complete morphogenesis of adult hydra structures within 96 hr from pellets formed with dissociated hydra cells (Grierer et al., 1972, supra; Sato et al., 1992, Dev. Biol 151:111-116; Technau et al., 1992, Dev. Biol. 151: 117-127; Sarras et al., 1993, supra ). Morphologically, hydra cell aggregate development can be divided into two stages. The initial stage is from Time 0 to 24 hr when aggregates develop from a solid cell mass into a fluid-filled cyst where the outer wall is formed from an epithelial bilayer with an intervening ECM termed mesoglea. This stage involves active cell sorting between ectodermal and endodermal cells (Technau et al., 1992, supra) and subsequent mesoglea formation once the bilayer is established. The later developmental stages (24-96 hr) involve processes normally associated with tissue histogenesis; namely, alterations in the shape of epithelial layers, cell migration, cell differentiation, and other processes that result in morphogenesis of foot, head, and tentacle structures. In regard to the initial stages of hydra cell aggregate development, it has been shown that head regeneration in aggregates is not due to the clustering of cells from the original head regions. It has been suggested that head regeneration arises de novo (Gierer et al., 1972, supra ; Technau et al., 1992, supra ) from foci of developmental gradients established around the spherical aggregate. This indicates that cell differentiation or transdifferentiation into head region cells actively occurs during hydra cell aggregate development. In addition to positional information and possible activator influences, cells may differentiate or transdifferentiate under the influences of other developmental cues such as signals arising from the ECM.
Previous studies have shown that in vertebrates, fibronectin interacts with various collagens during matrix assembly, including type IV collagen (Carter, 1984, J. Cell Bio. 99:105-114). In addition, antibodies to the collagen binding domain of fibronectin had the ability to block ECM assembly by human lung fibroblasts. (McDonald, 1982, J. Cell Biol. 92:485-492). Other studies raised doubts as to the interaction, while polyclonal antibodies to the collagen binding domain blocked matrix assembly, purified collagen binding domains had no inhibitory effects in this assembly process (McDonald et al., 1987, J. Biol. Chem. 262:2957-2967; Hynes, 1990, Cell 48:549-554). In general fibronectin (FN) appears before collagen during assembly of vertebrate matrices, however, in the case of hydra ECM formation, FN and collagen appear in the mesoglea about the same time, based on immunofluorescent studies. Type IV collagen has been implicated as important in several human diseases (Hudson et al., 1993, J. Biol. Chem. 268:26033-26036). Basement membrane and its components have a role in lymphocyte adhesion, migration and proliferation (Li and Cheung, 1992, J. of Immunology 149:3174-3181).
The fundamental role ECM plays in tissue development and cell differentiation reverberates across phyla and kingdoms, to focus attention on the most basic elements that are required for all tissue interactions. The use of hydra as a model system for the study of basic elements of complex tissue interactions is a recognized approach. Instead of attempting to deduce the interaction between isolated tissues of higher order animals, the same mechanisms and phenomenon can be examined in vivo by using the complete animal, in hydra. This approach has led to the use of hydra to study the effects of glucose on tissue morphology, in an effort to understand the pathological effects of uncontrolled diabetes on kidney glomeruli, with excellent results (Zhang et al., 1990, Diabetologia 33:704-707).
Recently the β1-laminin gene has been cloned and sequenced in hydra, showing very high homology with the human counter part. The homologues of fibronectin and collagen are present as well. It is a reflection on the fundamental role ECM plays, that hydra and higher order animals show the same cell matrix interactions, with similar components, domain interactions, receptor molecules and response to extracellular signals. Mammalian, and even human hormones, when applied to hydra result in bioactivity and effects on cell behavior. It is possible to use human insulin to stimulate cell proliferation in hydra. Other such cross-phyla activities can be attributed to many growth factors as well, i.e. EGF (epidermal growth factor), TGF-β, FGF (fibroblast growth factor), PDGF, to name a few.
Specific methods for the manipulation of cell adhesion to ECM, basal lamina, or adjacent cells would be useful for the in vivo manipulation of tissues and cells. Methods which address the fundamental elements of basic cell and tissue interactions are applicable to all systems which exhibit similar characteristic features. Such in vivo uses include, and are not limited to, inhibition of basal lamina formation, inhibition of basal lamina/cell interactions, and to encourage cells to maintain phenotypic plasticity. Such methods will also be useful for the in vitro manipulation of cells and tissues, for instance in maintaining cell cultures in undifferentiated or homeostatic states, non-enzymatic dispersal of cells from attachments, or the maintenance of confluent cells in suspension for propagation, maintenance, or collection.
The instant invention provides methods for inhibiting basal lamina membrane formation, in cell or tissue development, comprising contacting the cell or tissue with one or more isolated domains of type IV collagen. The instant invention also provides methods for in vitro cultivation of cells comprising contacting the cells to be cultivated with an isolated domain of type IV collagen to disrupt the formation of basal lamina or extracellular matrix contacts. The instant invention further provides methods for disrupting basal lamina membrane formation in tissues comprising contacting the tissues with an isolated domain of type IV collagen. In a specific embodiment of the instant invention, the isolated domain of type IV collagen is the 7S or NC1 domain, or protein constructs having substantially the same structure as the active elements within the 7S or NC1 domain.
Thus the instant invention provides methods for the interference with cell interactions with basal lamina components which comprises contacting the cells or tissues with an isolated domain of type IV collagen, and in a preferred embodiment the isolated domain is either the 7S domain or the NC1 domain of type IV collagen or substantially homologous protein constructs thereof which contain the specific structural elements within the 7S and NC1 domain that convey activity.
FIG. 1 are two diagrams illustrating segments of type IV collagen (1A) and fibronectin proteolytic fragments (1B) used in blocking experiments.
FIG. 2 is a graph which illustrates results of the blockage of aggregate morphogenesis observed with NC1 domain monomer and 7S domain, when all type IV collagen fragments are compared on an equal molar basis (0.03 μM). From left to right: control group; NC1 monomer; NC1 hexamer, 80-kDa fragment of NC1; 7S domain; type I collagen. Results are reported as % of normal morphogenesis as described below.
FIG. 3 are photographs of representative Hydra cell aggregates at 96 hr of development. Control aggregates develop head and tentacle structures (3A) while blocked aggregates remain in the characteristic 24-hr cystic stage (3B). Bar=286 μm.
FIG. 4 is a graph which illustrates the concentration effect of the various blocking agents. At higher concentrations, NC1 domain hexamer also showed blocking effects, whereas no blockage was observed with the 80-kDa fragment of NC1. From left to right: control group; NC1 monomer (MN) at 0.03 μM; NC1 domain hexamer (HEX) at 0.1 μM; NC1 domain hexamer (HEX) at 0.03 μM; NC1 domain 80-kDa fragment (80K) at 0.3 μM; NC1 domain 80-kDa fragment (80K) at 0.03 μM; 7S domain (7 S) at 0.1 μM; and 7S domain (7 S) at 0.03 μM. Results are reported as % of normal morphogenesis as described below.
FIG. 5 are electron micrographs showing representative morphology of control (5A) and blocked (5B) aggregate cells.
FIG. 6 is a graph which illustrates the results of the blockage of aggregate formation using fibronectin and fibronectin derived proteolytic fragments. All samples were tested at 0.5 mg/ml. From left to right: BSA (Bovine Serum Albumin); FN (intact fibronectin); 120K (the 120-kDa fragment of FN); 45K (the 45-kDa fragment of FN); 40K (the 40-kDa fragment of FN); 30K (the 30-kDa fragment of FN); RGDS (RGDS peptide); RADS (RADS peptide). Results are reported as % of normal morphogenesis as described below. The effectiveness of the blocking agents was found to be concentration dependent.
FIG. 7 illustrates the effects of NC1 (Hexamer) and 7S domains of Type IV collagen at a 50 ug/ml concentration on angiogenesis from mouse thoracic aorta organ cultures.
FIG. 8 illustrates the effects of 7S domain of Type IV collagen on angiogenesis from mouse thoracic aorta organ cultures. The domain concentrations employed in this experiment were 0 ug/ml (control); 0.5 ug/ml; 5 ug/ml and 50 ug/ml.
FIG. 9 illustrates the effects of NC1 (Hexamer) domain of Type IV collagen on angiogenesis from mouse thoracic aorta organ cultures. The domain concentrations employed in this experiment were 0 ug/ml (control); 0.5 ug/ml; 5 ug/ml and 50 ug/ml.
FIG. 10 are photographs of mouse thoracic aorta segments embedded in Matrigel (EHS basement membrane matrix, Collaborative Biomedical Products, Bedford, Mass.) at 5 days of culture. Control specimen (0 ug/ml of NC1 (Hexamer) and 7S domains) exhibited growth of microvessels from the cultured tissue into the matrix (FIG. 10A). In contrast, angiogenesis was inhibited in specimens cultured with 50 ug/ml of 7S domain (FIG. 10B) and NC1 (Hexamer) domain (FIG. 10C).
Our studies indicate that the formation of an appropriate ECM structure involving fibronectin and type IV collagen is critical to cell aggregate development and that perturbation of ECM formation adversely affects cell division and cell differentiation during the formation of complex cell aggregations and tissues. This is illustrative of the critical role the ECM and cell-ECM interactions play in tissue development in all organisms that have differentiated tissues. Structural changes in mesoglea, inhibition of cell proliferation, and changes in cell differentiation patterns accompanied the blockage of cell aggregates. Thus type IV collagen is critical to the early stages of cell aggregate development when the mesoglea is initially formed and that perturbation of aggregate development by fragments of type IV collagen results in alterations in hydra cell division, cell differentiation, and morphogenesis. It is demonstrated that type IV collagen components are a critical element in the formation of any cell to ECM (basal lamina) contacts, and that by applying the proper methods, this interaction can be manipulated in a predictable fashion.
The following examples are meant to illustrate specific embodiments of the instant invention, and are in no way intended to limit the breadth or scope of the teachings embodied in the instant specification. One with ordinary skill in the art will be able to take the teachings of the instant specification and use the instant invention in other specific embodiments.
Disruption of Cell Aggregate Development
All experiments utilized Hydra vulgarie (previously named Hydra attenuata). Animals were cultured as described previously (Sarras et al., 1991a, supra) and were not fed for 24 hr prior to use.
Hydra cell aggregates were prepared according to Gierer et al., (1972, supra) with modifications described by Sarras et al. (1993, supra). They were incubated either in microtiter plates (NUNS, Denmark) with one aggregate/well/10 μl incubation solution or in 96-well plates (Falcon) with three to five aggregates/well/50 μl incubation solution. Antibiotics were used throughout all steps of aggregate preparation to assure that the aggregates used in this study were free of any symbionic bacterial populations (Sarra et al., 1993, supra). Various analysis indicated that aggregates were free of bacteria under these conditions. By 24 hr of cell pellet formation, hydra cell aggregates develop septate junctions between epithelial cells (Wood et al., 1980. J. Ultrastruc. Res. 70:104-117). These junctions prevent the introduction of macromolecules from medium into mesoglea. Therefore, exogenous matrix probes (e.g., fibronectin, type IV collagen, and type IV collagen fragments, peptides or antibodies) were added to the culture medium immediately after pellet formation (Time 0). Control hydra cell aggregates were cultured in hydra medium, bovine serum albumin (Sigma, St. Louis, Mo.), or nonimmune serum. After 24 hr of incubation, aggregates (both control and experimental groups) were transferred into fresh hydra medium and culture was continued until 96 hr.
Type IV collagen fragments used in blocking experiments are illustrated in FIG. 1 (A). The NC1 monomer--a mixture of α1(IV), α2(IV); NC1 hexamer, 80-kDa protomeric truncated fragment (NC1 domain with part of triple helix); and the 7S domain, were all obtained from enzymatic digestion and subsequent chromatographic purification of bovine kidney glomeruli basement membrane or bovine lens basement membrane (Langeveld et al., 1988, J. Biol. Chem. 263: 10481-10488; Gunwar et al., 1991, supra). Antibody to the NC1 (α1(IV)+α2(IV)) domain of type IV collagen was generated in rabbits immunized with monomeric subunits isolated from the globular domain of bovine kidney basement membrane type IV collagen (Langeveld et al., 1988, supra). The precise molar concentrations of these fragments was determined by spectrophotometry and amino acid composition analysis.
Criteria for morphogenesis was as follows. The morphological development of hydra cell aggregates was studied using a dissecting microscope (Wild, Herbrugg). Observations were carried out at Time 0, 24, 48, 72, and 96 hr. The normal morphogenesis of hydra cell aggregates between Time 0 and 96 hr has been described previously by Gierer et al., (1972, supra) and Sarras et al., (1993, supra). Abnormal development (blockage) of hydra cell aggregates was considered if an aggregate did not show head and tentacle structures at 96 hr. i.e., was retained in a cystic stage (FIG. 3). The percentage of aggregates with head and tentacle structures at 96 hr of development was calculated and data for each group from different experiments were pooled and plotted. A minimum of five aggregates were tested per group and all experiments were repeated at least three times except where indicated. Using an ANOVA statistical test, a P value <0.05 was taken as the level of significant difference in all groups analyzed. The ANOVA statistical test was used for all data analysis.
Transmission electron microscopy was used to examine the fine structure and morphology of control and treated aggregates. Cell aggregates were immersed in Karnovsky's fixative overnight at 4� C. and postfixed in 1% OsO4 for 1 hr. Samples were then stained en bloc in 0.5% uranyl magnesium acetate overnight at 4� C. After dehydration and infiltration, samples were embedded in Spurr's resin. Blocks were cut with a Reichert-Jung microtome, stained with uranyl acetate and lead citrate, and viewed using a JOEL 100S transmission electron microscope. Morphometry analysis was carried out as described previously (Zhang et al., 1990, supra).
Immunofluorescent screening for type IV collagen in hydra cell aggregates at various time points after pellet formation showed that signal was detected at 48 hr, and became progressively stronger and remained in the mesoglea throughout all later stages of aggregate development. FIG. 3 illustrates the typical appearance of control and blocked aggregates at 96 hr. Blocked aggregates failed to develop beyond the 24 hr stage, and either remaining in a cystic stage or disaggregating into dissociated cells by 96 hr.
As shown in FIG. 2, the 7S domain, and monomers of NC1 domain were most effective in blocking hydra cell aggregate morphogenesis when fragments were tested on an equal molar basis. This blockage was also concentration dependent. NC1 hexamer blocked aggregate development at higher concentrations whereas the 80-kDa fragment showed no effect (FIG. 4). Antibody to NC1 domain had a similar blocking effect (Table 1).
TABLE 1______________________________________Treatment with Antibody to ECM ComponentsTreatment % of normal morphogenesis______________________________________nonimmune serum 1:10-1:100 79 � 7.25 (54/68)aAnti-NC1 1:10b 0 (0/15)Anti-NC1 1:40 0 (0/6)Anti-NC1 1:80 100 (3/3)Anti-FN 1:10c 0 (0/15)Anti-FN 1:50 0 (0/12)Anti-FN 1:100 32 � 15.5 (10/30)Anti-FN 1:200 33 � 19.25 (5/15)______________________________________ a Data are presented as means � SEM (No. with normal morphogenesis/total No. treated). b Polyclonal Ab against NC1 (α1(IV) + α2(IV)) domain of bovine kidney type IV collagen. c Polyclonal Ab (ICN Biochemicals) against human plasma fibronectin.
Similar studies were carried out using fibronectin as the inhibitor. Intact fibronectin. proteolytic fragments, RGDS peptides, and antibody to fibronectin were tested to determine their effect on hydra cell aggregate development. FIG. 1(B) shows a diagram of the relationship of the various fragments to the intact fibronectin molecule. Of these, intact fibronectin and its 30-kDa gelatin binding domain were found to be effective at 0.5 mg/ml in blocking aggregate development (FIG. 6). Effectiveness was found to be concentration dependent. Antibodies against fibronectin also showed blocking effects (Table 1).
Transmission electron microscopy was used to study the ultrastructure of mesoglea under the influences of each exogenously introduced ECM molecule. Blocked aggregates were processed for TEM analysis at various time points of experiments and the ultrastructure of their mesoglea was compared to that of control aggregates fixed at the same time of development. As compared to control group specimens (FIG. 5A), the ultrastructure of mesoglea in blocked aggregates was reduced in thickness, irregular at its epithelial border, and appeared to have lost some of its normal ultrastructural organization (FIG. 5B). Morphometric analysis (Table 2) indicated that the mesoglea of blocked aggregates was reduced in thickness by approximately 50% compared to controls and this reduction was significant as determined using statistical analyses. This altered mesoglea ultrastructure was observed with all blocking reagents tested.
TABLE 2______________________________________Morphometric AnalysisTreatment Interepithelial width of mesoglea as reflected(from Time 0 to 24 hr) by area measurementsa______________________________________BSAb 1434 � 58230 kDac 724 � 245*Anti-FNd 969 � 458*______________________________________ a All EM micrographs of mesoglea were taken at 8,000� magnification and printed at the same size. An 80mm section was bracketed along mesoglea on each print and mesoglea within the bracketed area was measured by computer program, SigmaScan. The measuring units if mm2. Under these measuring conditions, area reflects interepithelial width changes as previously described (Zhang et al., 1990, supra). Data are represented as mean � SD. b Bovine serum albumin at 0.1 mg/ml concentration. c Fibronectin 30kDa gelatin binding fragment at 0.1 mg/ml. d Antibody to fibronectin at 1:100 dilution. *Statistically different from BSA group (P value <0.05).
To determine if inhibition of cell proliferation was responsible for the blockage of cell aggregate morphogenesis, aggregates were treated from Time 0 to 96 hr with 10 μM hydroxyurea (HU), which has been shown to inhibit DNA synthesis in hydra cells (Bode et al., 1976, J. Cell Sci. 20:29-46). Although HU completely blocked DNA synthesis, the drug did not inhibit morphogenesis of hydra cell aggregates.
A critical role for collagen in the process of matrix formation follows from previous pharmacological studies involving lanthrytic agents (Barzanski et al., 1974, supra; Sarras et al., 1993, supra). In the present demonstration, type IV collagen domains were effective in perturbing mesoglea formation and in blocking aggregate development. In this regard, the NC1 domain monomer and the 7S domain fragment were most effective in blocking aggregate development when all type IV collagen fragments were compared on an equal molar basis. Others have proposed that the NC1 domain and the 7S domain are the sites at which the type IV collagen protomer is involved in intermolecular bridging during formation of the supramolecular network (Martin et al., 1988, Adv. Protein Chem. 39:1-50).
It is not clear why the larger NC1 hexamer and the 80-kDa fragment were less effective than the smaller NC1 monomer in blocking development. The same phenomenon was seen with fibronectin, while the most effective blocker, the 7S fragment, has a mass greater than 100 kDa. On a total mass basis, the 7S domain has a relatively high proportion of carbohydrate residues associated with the polypeptide chain (15-18%), and further analysis of these carbohydrate residues indicate that they have unique structural features such as the presence of terminal α-D-Gal residues on N-linked oligosaccharide groups (Langenveld et al., 1991, supra; Nayak et al., 1991, J. Biol. Chem. 266:13978-13987). The inability of type I collagen to block development is consistent with the fact that it has not been detected in hydra mesoglea.
The effect of mesoglea components on hydra cell behavior may be viewed at two levels. At one level, mesoglea may be viewed as simply a structural entity which is required as a foundation for stability and maintenance of the epithelial bilayer. At another level, however, ECM components in mesoglea provide developmental cues which modulate such cell processes as cell division, migration, and differentiation during hydra morphogenesis. It is clear that ECM components are not simply structural molecules. Fibronectin and type IV collagen have been shown to control endothelial cell proliferation and differentiation (Tagami et al., 1992, Cell Tissue. Res. 268:225-232; Ingber, 1990, PNAS USA 87:3579-3583). It has been shown that the mechanochemical interactions between striated muscle cells in jellyfish and grafted mesoglea can induce or inhibit DNA replication and cell transdifferentiation. In the present examples, while cell proliferation is inhibited in morphologically blocked aggregates, it is also apparent that normal aggregate morphogenesis can occur even in the absence of cell proliferation. Therefore, a reduction of cell division in blocked aggregates can not account for the blockage observed in aggregate morphogenesis.
The homology between the hydra model system and what is known about the developmental and regulatory mechanisms of higher organisms makes the use of type IV collagen fragments in this system applicable to the same cell and tissue interactions in other organisms. The instant methods are applicable to, among other things, the inhibition of metastasis, control of cell division, reduction of scar tissue formation, intervention in epithelial tissue formation, inhibition of angiogenesis, reduction of complications due to cell adhesion in organ transplants, inhibition of angiogenic invasion of tissue, or the inhibition of lymphocyte adhesion and mobility.
Cell Culture Maintenance
The methods of the instant invention are quite useful for the maintenance of cells in culture, where such maintenance requires maintenance of cell phenotype and morphology with minimal adhesion. It is well recognized that there are many critical factors that contribute to the successful maintenance and propagation of cells in culture. Namely cell division can be anchorage dependent, and in some instances will show density-dependent inhibition. Generally most cultures made from cells dissociated from tissues, unlike bacteria, are not adapted to living in suspension and require a solid surface on which to grow and divide. Cells are now grown in cultures, so that they adhere to plastic. Cells can vary in the requirements of culture media, and the nature of the supports, and some cells will not grow without the proper ECM components coated on the plastic dish. Recognized methods for mammalian cell culture can be found in such general references as "Mammalian Cell Biotechnology," edited by M. Butler, Oxford University Press, 1991, and "Readings in Mammalian Cell Culture," 2nd Edition, edited by R. Pollack, Cold Spring Harbor Laboratory, 1981.
The methods of the instant invention will provide a means by which the requirement for adhesion is eliminated by providing the cells with an effective amount of the type IV collagen domains which will allow the cells to remain in solution while still appearing to be bound to ECM. This will greatly improve the use of cell cultures in bioreactors and other large scale commercial applications. The use of NC1 or 7 S domain in about the same effective concentration per cell as demonstrated in the previous example will keep cultures of cells effectively bound without adhesion. Further, isolation and manipulation of cells will not require the use of general pharmacological agents which effect many cellular function in an imprecise and non-specific manner.
Primary cultures, those isolated directly from animal tissues, can be used to form secondary cultures of specific cells. In this manner, the cells can be subcultured for several weeks or months, displaying the phenotype and morphology of the parent cells. Most vertebrate cells will die in culture after a finite number of divisions, for example human skin cells can typically last for several months dividing from 50 to 100 times before they die. Variant cell lines can arise, however, which are immortal in that they can be propagated in cell culture indefinitely. These cells usually grow best when attached to a solid support, and typically will cease to grow once they have formed a confluent layer on the surface, demonstrating contact inhibition. Cell lines prepared from cancer cells differ from those prepared from normal cells in many ways. Such cells tend to proliferate without solid support, and will grow to much greater density than normal cells.
Thus the methods of the instant invention can be used to affect primary and secondary cell cultures so that they behave as if in contact with a solid support, but without the effects of cellular contact inhibition. Thus parent cell lines can be maintained in vitro under conditions that will promote the maintenance of cell phenotype and morphology.
One area of great interest is the isolation and culture of embryonic or pluripotent stem cells in vitro for eventual manipulation and use in vivo. The isolation and maintenance of such relatively undifferentiated cells, and the propagation of such cells would allow for the production of vast amounts of specific cells which could form the basis of tissue regeneration or replacement. Thus the teachings of the instant invention can be applied to the maintenance of pluripotent cell isolates, and allow for the propagation of these cells while inhibiting morphological changes and differentiation in vitro.
In Vitro Effect on Angiogenesis
Angiogenesis, the process of formation of new blood vessels, plays an important role in physiological processes such as embryonic and postnatal development as well as in wound repair. Formation of blood vessels can also be induced by pathological processes involving inflammation (e.g., diabetic retinopathy and arthritis) or neoplasia (e.g., cancer) (Folkman, 1985, Perspect. Biol. Med., 29, 10). Neovascularization is regulated by angiogenic growth factors secreted by tumor or normal cells as well as the composition of the extracellular matrix and by the activity of endothelial enzmyes (Nicosia and Ottinetti, 1990, Lab. Invest., 63, 115).
During the intial stages of angiogenesis, endothelial cells sprouts appear through gaps in the basement membrane of preexisting blood vessels (Nicosia and Ottinetti, 1990, supra; Schoefl, 1963, Virehous Arch. Pathol. Anat. 337, 97-141; Ausprunk and Folkman, 1977, Microvasc. Res. 14, 53-65; Paku and Paweletz, 1991, Lab. Invest. 63, 334-346). As new vessels form, their basement membrane undergoes complex structural and compositional changes which are believed to affect the angiogenic response (Nicosia et al., 1994, Exp. Biology, 164, 197-206). Early planar culture models have shown that basement membrane molecules modulate the attachment, migration and proliferation and organizational behavior of endothelial cells (Nicosia et al., 1994, supra). More recent studies with three-dimensional aortic culture models which more closely simulate angiogenic conditions that occur during wound healing in vivo suggest that basement membrane is a dynamic regulator of angiogenesis whose function varies according to its molecular components (Nicosia, 1994, supra).
With modifications, the procedures of Nicosia and Ottinetti (1990), supra, and Nicosia et al (1994), supra, were utilized for experiments designed to test the effect of Type IV collagen on angiogenesis under in vitro conditions. The model has been used to study the effects of growth factors and extracellular matrix molecules on the angiogenic response and employs aortic rings cultured in three-dimensional collagen gels under serum-free conditions. These experiments are outlined below.
A. Methods
Experiments were performed with Swiss Webster male mice which were 1 to 3 months old. Following anesthesia, the thoracic aorta was excised under aseptic conditions and transferred to sterile MCDB 131 sterile growth medium (Clonctics, San Diego, Calif.) containing antibiotics. Fat was dissected away from the aorta and approximately six to eight 1 mm thoracic segments were obtained from each specimen. Segments were transferred to 48 well tissue culture plates. The wells of these plates were layered with 100 microliters of Matrigel (EHS basement membrane, Collaborative Biomedical Products, Bedford, Mass.) prior to transfer of the aortic segments. The Matrigel was diluted 1:1 with MCDB 131 growth medium prior to use. The segments were centered in the wells and an additional 100 microliters of Matrigel was then placed over the specimens. The aortic segments were therefore embedded in the basement membrane matrix. Each well then received 300 microliters of MCDB 131 growth medium. The plates were placed in an incubator maintained at 37� C. with 5% CO2. Specimens were observed daily over a 7 day period. Newly growing microvessels were counted using an inverted phase microscope at various times during the culture period, but data is expressed at 3 and 5 days of culture. To test for the effect of Type IV collagen on angiogenesis, domains at known concentrations are mixed with the Matrigel and with the MCDB 131 growth medium. Fresh MCDB 131 growth medium (plus and minus collagen domains) was changed every 3 days.
B. Results
After establishing the time course of angiogenesis under control conditions (Matrigel plus MCDB 131 growth medium), experiments were performed using various concentrations of Type IV collagen (isolated from bovine lens) NC1 (hexamer) and 7S domains. Data represents the analysis of at least 3 specimens per experimental condition. In the first experiment (FIG. 7), analysis indicated that a concentration of 50 micrograms/ml, NC1 domain and 7S domain significantly inhibited angiogenesis as monitored at 3 and 5 days of culture. In the second experiment, various concentrations of these domains were analyzed. As indicated in FIG. 8, 7S domain at 50 micrograms/ml again significantly inhibited angiogenesis at 3 and 5 days. Inhibition was reduced at 5 and 0.5 micrograms/ml concentrations. As indicated in FIG. 9, NC1 domain was less effective in blocking angiogenesis as compared to that observed in the first experiment (FIG. 7). In addition, as compared to the 7S domain, there was less of a correlation between concentration and inhibitory action.
FIG. 10 are photographs of mouse thoracic aorta segments embedded in Matrigel (EHS basement membrane matrix, Collaborative Biomedical Products, Bedford, Mass.) at 5 days of culture in the presence or absence of 50 ug/ml of Type IV collagen domains. The control specimen (no domains) exhibited growth of microvessels from the cultured tissue into the matrix (FIG. 10A). In contrast, angiogenesis inhibition was observed in tissues cultured in the presence of 50 ug/ml of 7S domain (FIG. 10B) and NC1 (Hexamer) domain (FIG. 10C).
The results of these experiments suggest that as observed previously with Hydra cell aggregates, the 7S domain of Type IV collagen is more effective as a blocking agent as compared to the NC1 domain.
In summary, the present invention is broadly applicable to a variety of in vivo and in vitro uses which include inhibition of metastasis, control of cell division, reduction of scar tissue formation, intervention in epithelial tissue formation, inhibition of angiogenesis, reduction of complications due to cell adhesion in organ transplants, inhibition of angiogenic invasion of tissue, the inhibition of lymphocyte adhesion and mobility, and the maintenance of pluripotent cell isolates in vitro while inhibiting morphological changes and differentiation.
In practicing the invention, the amount or dosage range of NC1 (Hexamer) and 7S domains of Type IV collagen employed is one that effectively inhibits or disrupts cell adhesion to extracellular matrix components or the formation of functional basal lamina. An inhibiting amount of Type IV collagen domain that can be employed ranges generally between about 0.1 and about 500 ug/ml, preferably ranging between about 0.5 and about 50 ug/ml.
While the fundamental novel features of the invention has been shown and described, it will be understood that various omissions, substitutions and changes in the form and details illustrated may be made by those skilled in the art without departing from the spirit of the invention. It is the intention, therefore, to be limited only as indicated by the scope of the following claims.
Reference | ||
1 | Fajardo et al., (1988) "Methods in Laboratory Investigation: The Disc Angiogenesis System" Laboratory Investigation 58(6):718-724. | |
2 | * | Fajardo et al., (1988) Methods in Laboratory Investigation: The Disc Angiogenesis System Laboratory Investigation 58(6):718 724. |
3 | * | Gunwar et al., J. Biol. Chem. (1991) 266:14088 14094, Properties of the collagenous domain of the .alpha.3(IV) chain, the Goodpasture antigen, of lens basement membrane collagen. |
4 | Gunwar et al., J. Biol. Chem. (1991) 266:14088-14094, "Properties of the collagenous domain of the α3(IV) chain, the Goodpasture antigen, of lens basement membrane collagen." | |
5 | * | Hudson et al., Collagen IV: Role in Goodpasture Syndrome, Alport Type Familial Nephritis, and DIabetic Nephropathy, in Collagen Volume V, Pathobiochemistry (ed. Kang and Nimni, CRC Press, Boca Raton, 1992). pp. 17 30. |
6 | Hudson et al., Collagen IV: Role in Goodpasture Syndrome, Alport-Type Familial Nephritis, and DIabetic Nephropathy, in Collagen Volume V, Pathobiochemistry (ed. Kang and Nimni, CRC Press, Boca Raton, 1992). pp. 17-30. | |
7 | * | Hudson et al., J. Biol. Chem. (1993) 268:26033 26036, Minireview: Type IV collagen:Struture, Gene Organization, and Role in Human Diseases . |
8 | Hudson et al., J. Biol. Chem. (1993) 268:26033-26036, "Minireview: Type IV collagen:Struture, Gene Organization, and Role in Human Diseases". | |
9 | * | Hynes, R.O., Cell (1992) 69:11 25, Integrins: Versatility, Modulation, and Signalling in Cell Adhesion . |
10 | Hynes, R.O., Cell (1992) 69:11-25, "Integrins: Versatility, Modulation, and Signalling in Cell Adhesion". | |
11 | * | Juliano et al., J. Cell Biol. (1993) 120:577 585, Minireview: Signal Transduction from the Extracellular Matrix . |
12 | Juliano et al., J. Cell Biol. (1993) 120:577-585, "Minireview: Signal Transduction from the Extracellular Matrix". | |
13 | * | Neilson et al., J. Biol. Chem. (1993) 268:8402 8405, Specificity on Goodpasture autoantiboides for the recombinant noncollagenous domains of human type IV collagen . |
14 | Neilson et al., J. Biol. Chem. (1993) 268:8402-8405, "Specificity on Goodpasture autoantiboides for the recombinant noncollagenous domains of human type IV collagen". | |
15 | Nicosia and Ottinetti, (1990) "Growth of Microvessels in Serum-Free Matrix Culture of Rat Aorta: A Quantitative Assay of Angiogenesis In vitro" Laboratory Investigation 63(1):115-122. | |
16 | * | Nicosia and Ottinetti, (1990) Growth of Microvessels in Serum Free Matrix Culture of Rat Aorta: A Quantitative Assay of Angiogenesis In vitro Laboratory Investigation 63(1):115 122. |
17 | Nicosia et al., (1994) "Modulation of Angiogenesis in Vitro by Laminin-Enactin Complex" Developmental Biology 164:197-206. | |
18 | * | Nicosia et al., (1994) Modulation of Angiogenesis in Vitro by Laminin Enactin Complex Developmental Biology 164:197 206. |
19 | * | Sarras et al., Dev. Bio. (1991a) 148:481 494, Extracellular matrix (Mesoglea) of Hydra vulgaris I. Isolation and Characterization . |
20 | Sarras et al., Dev. Bio. (1991a) 148:481-494, "Extracellular matrix (Mesoglea) of Hydra vulgaris I. Isolation and Characterization". | |
21 | * | Sarras et al., Dev. Bio. (1991b) 148;495 500, Extracellular matrix (Mesoglea) of Hydra vulgaris II. Influence of Collagen and Proteoglycan Components on Head Regeneration . |
22 | Sarras et al., Dev. Bio. (1991b) 148;495-500, "Extracellular matrix (Mesoglea) of Hydra vulgaris II. Influence of Collagen and Proteoglycan Components on Head Regeneration". | |
23 | * | Sarras et al., Dev. Bio. (1993) 157:393 398, Extracellular matrix (Mesoglea) of Hydra vulgaris III. Formation and function during morphogenesis of Hydra cell aggregates . |
24 | Sarras et al., Dev. Bio. (1993) 157:393-398, "Extracellular matrix (Mesoglea) of Hydra vulgaris III. Formation and function during morphogenesis of Hydra cell aggregates". | |
25 | * | Sato et al., Dev. Bio. (1992) 151:111 116, Patterning in Hydra cell aggregates without the sorting of cells from different axial origins . |
26 | Sato et al., Dev. Bio. (1992) 151:111-116, "Patterning in Hydra cell aggregates without the sorting of cells from different axial origins". | |
27 | * | Technau et al., Dev. Bio. (1992) 151:117 127, Cell sorting during the regeneration of Hydra from reaggregated cells . |
28 | Technau et al., Dev. Bio. (1992) 151:117-127, "Cell sorting during the regeneration of Hydra from reaggregated cells". | |
29 | * | Zhang et al., Dev. Bio. (1994, Jul.) 164:10 23, Hydra cell aggregates development is blocked by selective fragments of fibronectin and type IV collagen . |
30 | Zhang et al., Dev. Bio. (1994, Jul.) 164:10-23, "Hydra cell aggregates development is blocked by selective fragments of fibronectin and type IV collagen". | |
31 | * | Zhang et al., Diobetologia (1990) 33:704 707, A non mammalian in vivo model for cellular and molecular analysis of glucose mediatd thickening of basement membranes . |
32 | Zhang et al., Diobetologia (1990) 33:704-707, "A non-mammalian in vivo model for cellular and molecular analysis of glucose-mediatd thickening of basement membranes". |
Citing Patent | Filing date | Publication date | Applicant | Title |
US6358735 | Jul 18, 2000 | Mar 19, 2002 | University Of Kansas Medical Center | Method for inhibiting angiogenesis and tumors with the isolated NC1 α3 chain monomer of type IV collagen |
US6361994 | Jul 18, 2000 | Mar 26, 2002 | University Of Kansas Medical Center | Method for inhibiting angiogenesis and tumors with the isolated NC1 α1 chain monomer of type IV collagen |
US6384012 * | Oct 30, 1998 | May 7, 2002 | University Of Kansas Medical Center | Use of isolated domains of type IV collagen to modify cell and tissue interactions |
US6419924 | Nov 28, 2000 | Jul 16, 2002 | University Of Kansas Medical Center | Antibodies of isolated domains of type IV collagen modify cell interactions |
US6432706 | Jun 7, 2000 | Aug 13, 2002 | University Of Kansas Medical Center | Method for inhibiting angiogenesis and tumors with the isolated NC1 α6 chain monomer of type IV collagen |
US6448222 | Nov 28, 2000 | Sep 10, 2002 | University Of Kansas Medical Center | Use of isolated alpha 2 domain of type IV collagen to modify cell and tissue interactions |
US6498140 | Jun 7, 2000 | Dec 24, 2002 | University Of Kansas Medical Center | Use of isolated domains of type IV collagen to modify cell and tissue interactions |
US6680293 * | May 6, 2002 | Jan 20, 2004 | Kansas University Medical Center | Use of isolated domains of type IV collagen to modify cell and tissue interactions |
US6689746 * | Apr 29, 2002 | Feb 10, 2004 | Kansas University Medical Center | Use of isolated domains of type IV collagen to modify cell and tissue interactions |
US6759047 | Jun 17, 1999 | Jul 6, 2004 | Beth Israel Deaconess Hospital Corp. | Anti-angiogenic proteins and methods of use thereof |
US6893812 * | May 25, 2001 | May 17, 2005 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Three-dimensional ex vivo angiogenesis system |
US6962974 | Jul 21, 2000 | Nov 8, 2005 | Beth Israel Deaconess Medical Center | Anti-angiogenic proteins and fragments and methods of use thereof |
US7387779 | Dec 21, 2001 | Jun 17, 2008 | Beth Israel Deaconess Medical Center | Anti-angiogenic proteins and fragments and methods of use thereof |
US20020150879 * | Jun 17, 2002 | Oct 17, 2002 | Woltering Eugene A. | Three-demensional ex vivo angiogenesis system |
US20040106549 * | Mar 18, 2003 | Jun 3, 2004 | Beth Israel Deaconess Medical Center, Inc. | Anti-angiogenic proteins and methods of use thereof |
US20040132662 * | Dec 17, 2003 | Jul 8, 2004 | Kansas University Medical Center | Use of isolated domains of type IV collagen to modify cell and tissue interaction |
US20050004033 * | Jul 30, 2004 | Jan 6, 2005 | Kansas University Medical Center | Use of isolated domains of type IV collagen to modify cell and tissue interaction |
US20050100510 * | May 8, 2004 | May 12, 2005 | Frank Falco | Methods for using gadolinium as a contrast media |
US20050208476 * | May 16, 2005 | Sep 22, 2005 | Woltering Eugene A | Three-dimensional ex vivo angiogenesis system |
US20070042965 * | Sep 19, 2006 | Feb 22, 2007 | Kansas University Medical Center | Crystallized structure of type IV collagen NC1 domain hexamer |
US20090054344 * | May 27, 2008 | Feb 26, 2009 | Beth Israel Deaconess Medical Center | Anti-angiogenic proteins and fragments and methods of use thereof |
CN100447155C | May 23, 2001 | Dec 31, 2008 | 中国科学院上海上海生命科学研究院 | Recombinant human endothelial growth arrestin with activity of inhibiting endothelial cell growth |
WO2000059532A1 * | Mar 31, 2000 | Oct 12, 2000 | Biostratum, Inc. | The use of domains of type iv collagen t inhibit angiogenesis an tumour growth |
WO2004067762A2 * | Jan 27, 2004 | Aug 12, 2004 | University Of Kansas Medical Center | Crystallized structure of type iv collagen nc1 domain hexamer |
WO2004067762A3 * | Jan 27, 2004 | Nov 4, 2004 | Billy Hudson | Crystallized structure of type iv collagen nc1 domain hexamer |
U.S. Classification | 435/363, 530/356, 435/375, 435/325 |
International Classification | C12N5/00, A61M5/32, C12N5/08, A61P43/00, C07K16/18, A61K38/39 |
Cooperative Classification | Y10S514/801, C12N2501/998, A61K2039/505, C07K16/18, C12N5/0018, A61K38/39 |
European Classification | C12N5/00M, A61K38/39, C07K16/18 |
Date | Code | Event | Description |
Oct 19, 1999 | CC | Certificate of correction | |
Jul 3, 2002 | FPAY | Fee payment | Year of fee payment: 4 |
Jul 23, 2002 | REMI | Maintenance fee reminder mailed | |
Jul 5, 2006 | FPAY | Fee payment | Year of fee payment: 8 |
Aug 9, 2010 | REMI | Maintenance fee reminder mailed | |
Jan 5, 2011 | LAPS | Lapse for failure to pay maintenance fees | |
Feb 22, 2011 | FP | Expired due to failure to pay maintenance fee | Effective date: 20110105 |


text:
Efficent smooth GMM through dimension reduction
- Date: 11/23/2007
Pascal Lavergne (Simon Fraser University)
University of Victoria
We propose a new GMM criterion for models defined by conditional moment
restrictions that takes into account a number of moment conditions
which increases with the sample size. Our criterion allows us to reduce
the dimensionality of the conditioning variables, so that the resulting
estimator is not affected by this dimension. The two-step estimator
attains the semiparametric efficiency bound.
Other Information:
Special PIMS Lectures at UVIC 2007

subset: commoncrawl


text:
The Moorings Adds New Caribbean Destination
Explore the Caribbean with The Moorings newest charter destination in Puerto Rico. Boaters will be able to embark from Puerto Del Rey Marina on Puerto Rico's east coast and explore the surrounding Spanish Virgin Islands, including Culebra and Vieques. The Spanish Virgin Islands boast bea.... Read More


text: SI Publishes Disturbing Donald Sterling Anecdote It Spiked 14 Years AgoBarry Petchesky4/30/14 6:45pmFiled to: donald sterlinglos angeles clippersnbagettypic825EditPromoteShare to KinjaToggle Conversation toolsGo to permalink Here's longtime Sports Illustrated reporter Franz Lidz today, first writing something he tried—and failed—to publish 14 years ago. Advertisement Shortly after the Clippers made Danny Manning the top pick of the 1988 NBA draft, team owner Donald Sterling invited the player and his agent, Ron Grinker, to talk contract in Beverly Hills. It was recounted to me how Sterling lounged around his mansion in a bathrobe open to his navel, wearing nothing underneath. At one point Sterling's preteen son wandered in and was chastised for skipping Hebrew school. The owner commanded the boy, "Go to your room and get undressed." The child slouched upstairs. Sterling followed. The next thing Manning heard was a belt thrashing and the boy wailing, as Grinker bounded up the stairs yelling, "Stop! Stop! We'll sign." (This was almost certainly Scott Sterling, who wasn't charged with shooting a friend over a girl in 1999 thanks to his father's influence, and who died of a drug overdose last year.) Advertisement Lidz brings up the story not to pile on the deposed Clippers owner, but to illustrate the pass Sterling had long received for a series of awful things that lots of people knew about. Not just the awful things that found their way into print and were roundly ignored, but the open secrets among media members and NBA executives that for one reason or another never reached the page.Lidz's piece was an excoriating cover story that declared Sterling as unfit for franchise ownership way back in 2000. But this specific incident was excised from the magazine, and it was far from the only one. Today, Lidz writes:[S]o much of his behavior — extreme parsimony, discriminatory practices, wild sexual escapades — was deemed too weird, too cruel, too contemptible. An editor told me, "You've demonized him."If you've heard the anecdote about Sterling hitting his son before, it's because a version of it was published on this site in January 2013 after we received it from a former Clippers reporter. Influential and powerful people have known who Donald Sterling is for decades. Their biggest mistake was counting on him dying before it all came out. Sponsored [SI.com]Reply82 repliesLeave a reply


text:
Showing posts with label Week 181: Green. Show all posts
Showing posts with label Week 181: Green. Show all posts
Sunday, April 25, 2010
Moss Man
Jumbled by
Dave Pryor
Green sweet poison
Jumbled by
Jesus S
Just out from the oven. It's still fresh! This is the first piece I submit here and I hope it won't be the last :D tell me what you think!
Friday, April 23, 2010
Thursday, April 22, 2010
first post! green game designs
Jumbled by
Wendi Chen
so this is my first time ever posting here... i've been a member since forever but i've always felt very intimidated~ :< but this week's topic is my favorite color, so what better time to start posting? :)
here's a BG and a character sprite from a rhythm/RPG game i'm finishing up right now titled Sequence. hope you guys dig it!
here's a BG and a character sprite from a rhythm/RPG game i'm finishing up right now titled Sequence. hope you guys dig it!
Green foes
Jumbled by
Andry "Shango" Rajoelina
Honestly, just because he was red and blue, did they all really have to be green?
Wednesday, April 21, 2010
the sub-average dragon
Jumbled by
Jason Quinones
one teaser panel from my EPIC (WOW!) 4 panel (*SIGH*) comic strip titled: Les Manley: The Sub-Average Dragon. trust me....it's the tamest panel of the bunch and the most suitable for the jumble. i'll prob post the whole offensive thing on my blog soon. in the meantime, check out the other crap i got going on over there @ jasonsartblog.blogspot.com.
Green Legs and Ham
Jumbled by
© 2009 Barry/Right-Hemisphere Laboratory
Green Stuff
Jumbled by
Jordan Race
The green on this handsome fellow above is the only thing separating him from the animals!
Tuesday, April 20, 2010
Green Minder
Jumbled by
One older piece for this week theme,
I hope that human to be more friendly to green.
More green illustrations from my blog: House of Coffeebean
Jumbled by
Dave Pryor
Think Green On Apple
Jumbled by
Always think green, this Earth will be saved.
More illustrations from my blog: House of Coffeebean
And join the Green project at HERE.
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text:
Bacon Wrapped Trout Recipe
Trout is readily available but often overlooked. The bacon adds flavor to the fish while protecting it from the heat of the grill. Make sure the bacon...
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Herb, Garlic and Bacon Pork Loin
"Delicious pork loin roasted with bacon that the whole family will love. Serve with potatoes or any of your favorite veggies."
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Mustard-Roasted Chicken with Warm Frisée Salad and Fingerlings and Bacon
The chicken needs to marinate for at least four hours (or overnight), so be sure to start in advance. What to drink: Crisp, full-bodied white wine like...
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Bread Stuffing W/ Pears, Bacon, Pecans & Caramelized Onions
I found this recipe at the Pear Bureau Northwest sight. It's filled with Bread Stuffing w/ Pears, Bacon, Pecans or Chestnuts, Caramelized Onions, rosemary,...
rosemary, sage, +bacon, +bosc pears, +celery, +bread, +eggs, +black pepper, +parsley, +thyme, +pearl onions, +brown sugar, +chicken broth, +pecan, +salt, +butter
www.recipezaar.com - Similiar recipes
Bacon and Bourbon Thanksgiving Stuffing
"This recipe makes a HUGE amount of stuffing--enough to stuff a 24 lb. turkey--so adjust the ingredient amounts as needed. The flavors of bacon and bourbon...
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Interactive Seating Charts
Unfortunately, we do not currently have
a seating chart for Pomona Fairplex.
Pomona Fairplex
1101 West McKinley Ave.Pomona, CA 91768
Upcoming Events
The annual Day of the Dead Festival, put on by HARD Events, has a lineup for its 2014 showing. This year's addition will be held at The Fairplex in Pomona, CA (just outside of Los Angeles) on Saturd…
August 29, 2014
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We scour the web's major ticket sites and aggregate inventory into one place. Our Deal Score helps you identify the best ticket deals available for each event.
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From Wikipedia, the free encyclopedia
Solanaceae | |
A flowering Brugmansia x insignis from the US Botanic Garden |
Scientific classification | |
Kingdom: | Plantae |
Division: | Magnoliophyta |
Class: | Magnoliopsida |
Order: | Solanales |
Family: | Solanaceae Juss. |
The nightshades (Solanaceae) are a family of plants. All of them bear flowers. Many members of the family are edible, but some are poisonous. Very often, only certain parts of the plant are edible or poisonous. Well-known members of this family are chili peppers, petunias, deadly nightshade, mandrakes, potatoes, tomatoes, aubergines (eggplants) and tobacco.
Because it is thought that nightshades can cause or increase swelling and pain, many people do not eat them.


text:
10 CRPF commandos killed in Naxal IED blast in Bihar
Patna : At least ten CRPF (Central Reserve Police Force) commandos belonging to its elite COBRA battalion were killed and five others injured in a Naxal IED blast in the jungles of Bihar's Aurangabad district, officials said.
The jawans of the COBRA unit were ambushed in the IED blast after which an encounter started yesterday afternoon between the two resulting in the killing of three naxals by late night, they said.
"While eight personnel were killed on the spot, two others succumbed to their injuries when they were being evacuated," a senior official said.
The incident was reported from Chakarbanda-Dumarinala forests of the said district, bordering Gaya.
Some arms and ammunition, including an AK-47 rifle, INSAS rifle and under barrel grenade launcher were also recovered from the site.
Officials said five other personnel were critically injured in the fierce gun battle that ensued after the IED (Improvised Explosive Device) blast.
The jawans belonged to the 205th COBRA battalion and were deployed in the state for conducting anti-naxal operations.
This is one of the biggest casualties of the elite COBRA unit which has been raised by the CRPF for undertaking special jungle warfare operations.
Addition reinforcements of the state police and CRPF have reached the spot and a search operation has been launched.


text:
Want to Take Photos Like a Pro with your Nikon D5200? Now you have ready to go instruction of all Nikon D5200 camera functions where you need it and when you need it.
With Android Camera Guides from QuickPro, you'll never have to miss the shot of your life again. Whether it's that birthday surprise, graduation ceremony, or your daughters' wedding day, the ability to take a photo like a Pro is within reach.
This tutorial provides:
- Instructional video on all aspects of camera and photography.
- On the go video playback. No WIFI required after initial install for watching in the field.
Subjects Include:
- Introduction to the Nikon D5200
- Nikon D5200 Camera Tour
- Camera Tour: Top of Camera
- Camera Tour: Sides of Camera
- Camera Tour: Back of Camera
- Image Area, Quality, & Size Settings
- Image Quality & Size
- File Management
- Metering, Exposure & Release Modes
- Metering
- Shooting Modes
- Scene Modes
- Effects Mode
- Advanced Shooting Modes
- Using ISO
- Release Modes
- Live View & Movie Mode
- Live View
- Live View Displays
- Movie Recording
- Playback & Displays
- Playback Displays
- Processing Images In-Camera
- Focus Modes & Image Sharpness
- Choosing a Focus Mode
- Choosing an AF Area Mode
- Using Focus Modes & AF Area Modes
- Additional Focus Tips
- Menu System Tour
- Playback Menu
- Shooting Menu
- Setup Menu
- Controlling Color & Tone
- Picture Controls
- Active D Lighting
- Using the Built-In Flash
- Flash Modes
- Flash Compensation
This training content was produced by QuickPro, LLC. This product is not sponsored by Nikon, Corp. or its subsidiaries. Nikon Product Names and terminology are trademarks of Nikon, Corp.
Tags: d5200 quickpro .ipa , nikon d5200 wallpaper , nikon d5200 instructional video , nikon playback problems d5200 , d5200 , nikon d5200 by quickpro , nikon d5100 by , d5200 tutorial , app for nikon 5200
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text:
Kathryn Joosten has been cast in the fight of her life.
The character actress, 69, who played Karen McCluckey on ABC's 'Desperate Housewives," has been diagnosed with lung cancer, her publicist reported yesterday. A CAT-scan showed the cancer in her left lung, said Thomas DeLorenzo.
Catch up with 'Desperate Housewives'.
Joosten has been through this before. In 2001, cancer was found in her right lung and she had part of it out. A former nurse, she did not undergo chemotherapy and radiation, reported the NY Post.
'Desperate Housewives' producers said they will work around Joosten. She appears in the show's season opener on September 27th at 9 p.m.
The opinions expressed are solely those of the author and do not necessarily reflect the views of Comcast.


text:
Moto X Phone: Release Date, Price Rumors, Photos and Camera App Leak Ahead Of Google Aug. 1 NYC Event
Motorola Mobility, a subsidiary of Google (NASDAQ:GOOG), will officially announce the Moto X phone on Thursday in New York City, along with a release date and specifications for the device. Some details have leaked about the Moto X phone ahead of Google and Motorola's Aug. 1 event, including several photos and specs, as well as rumors regarding pricing for the phone.
One report priced the Moto X identically to the Nexus 4, which costs $299 for a 16GB model and $349 for 32GB. But since the device will be made in the U.S., unlike the Nexus 4 from LG, the Moto X price could be higher than the Nexus 4. Many consumers in the U.S. (more than 80 percent) as well as abroad are willing to pay more for devices considered made in America, according to a 2012 survey from the Boston Consulting Group.
The release date for the Moto X could be as early as the announcement this week. Taylor Wimberly, former owner of the Android and Me blog, reported that an unverified source said the Moto X was shipping to U.S. carriers on Monday. That would allow carriers to sell the device as soon as it is announced on Thursday, or shortly afterward.
A Moto X release date of Aug. 1 would follow the trend of Google releasing products the day that they are announced, much like the 35 Chromecast HDMI streaming dongle.
Twitter user and phone information leaker @evleaks also claimed in June to have the Moto X specs, which at the time seemed underwhelming: a 1.7GHz dual-core processor, 720p resolution OLED screen, 2GB of RAM and a 10-megapixel camera. The Moto X model that "evleaks" reported on at the time also had 16GB of storage and ran Android 4.2.2.
Motorola moto x: 1.7GHz dual-core MSM8960 Pro, 720p, 2GB/16GB, 10MP/2MP, Android 4.2.2
— @evleaks (@evleaks) June 15, 2013
More details regarding the processor and screen for the Moto X have emerged, telling a more intriguing story than another phone with a dual-core processor: the Moto X will likely incorporate Motorola's X8 Mobile Computing System, which means the phone will have eight processing cores.
The X8 system on a chip (SoC) was introduced last week with the new Motorola Droid lineup of phones exclusive to Verizon Wireless customers, including the Droid Ultra, Mini and Max. The Moto X will also incorporate the SoC with a 1.7GHz processor for applications, a quad-core GPU or Graphics Processing Unit, one core to handle language processing, and another for contextual computing.
That makes for a total of eight cores inside of the Moto X, likely what Motorola CEO Dennis Woodside was referring to when he said the device had "two processors" and was "aware" of its surroundings. The language processing core will tackle the Moto X's "always on" voice control, which listens for the command "OK Google Now" and then can give information and complete tasks hands free.
The "contextual computing" processing core will help the phone determine whether it is lying flat on a surface, in a user's pocket or on the move. Both the contextual computing and language processing cores will be low-power processors, to minimize battery drain or what Motorola CEO Dennis Woodside called the "huge problem of battery life."
The Moto X will run on the recently updated version of Android (4.3) with a gesture-based camera function that allows users to twist their wrist twice to turn it on, even from a lock screen. Several features have leaked online, including a partly functional version of the custom camera app.
Taylor Wimberly also released photos purported to be of the upcoming Moto X on his Google+ page. They show that the Moto X will boast a specially laminated Gorilla Glass called Moto Magic Glass. This specially made "continuous surface" will allow the Moto X screen to wrap around the edges of the phone.
The Moto X phone's carrier availability may have been revealed in filings with the U.S. Federal Communications Commission. The Moto X was previously approved with with radios compatible with AT&T, Verizon, Sprint and now T-Mobile can be added to the list.


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I drink the wine and am known to sit by stuff. Moscatasking is the ability to screw up several things all at the same time. On a side note, I find that "most" owls are pretty pissed off.
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AMD Athlon FX-57 - The Ultimate Gaming CPU AMD is also beating the crap out of Intel when it comes to power consumption and heat dissipation. Our review chip hovers around 41 degrees Celsius under normal operating modes and can jump up over 60 degrees, however, thats still a far cry from Intels 70 degrees plus we see on a regular basis. None of this may matter to you especially if you bought a pre-built PC where the system builder has more than likely taken the time to properly outfit the system with adequate power and cooling. http://www.gdhardware.com/hardware/c...4/fx57/001.htm |
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Throughout American history politicians have sought to gain favor with voters by promising material rewards in exchange for votes. In the days of Tammany Hall, there was a pretty clear quid pro quo in place where voting for the right candidate was directly linked to being rewarded with silver or perhaps a job. Later, these promises become more rhetorical in nature as was the case with Herbert Hoover's 1928 campaign slogan, "A chicken in every pot and a car in very garage." In recent years, it's usually been Democrats who have been more forthcoming with promises that putting them into office would improve the material well being of the voter and the ones who were then baffled as to why some voters would choose to vote against their apparent economic self interest.
A story in yesterday's WSJ explored how blatant vote buying is alive and well in the world's largest democracy:
Despite being one of India's better-performing states and its auto-industry hub, Tamil Nadu has its share of serious issues to debate this year, including how to halt corruption and fix electricity shortages. But the race between the two leading parties has boiled down mostly to a contest of who can offer a longer list of government-financed freebies.
The incumbent party Dravida Munnetra Kazhagam, or DMK, led by 86-year-old former screenwriter and current Chief Minister M. Karunanidhi, has promised blenders or grinders for poor families, laptops for engineering students and an insurance program for fishermen. The party is also promising washing machines and refrigerators for unspecified recipients.
The main opposition party, All India Anna Dravida Munnetra Kazhagam, or AIADMK, led by former film star J. Jayalalithaa, upped the ante with its list of handouts: a blender, a grinder and a fan for all women; four free sheep for poor families; four grams of gold for poor brides (for the necklaces brides wear on their wedding day); 60,000 cows for 6,000 villages and free cable-TV connections for all.
Free cable TV for all? Wonder if the AIADMK would be interested in a third party affiliation here...
The 60,000 cows for 6000 villages has a nice numbers ring to it too. Remindful of Clinton's "100,000 new cops" proposal.
As is often the case with government intervention in such matters, giving things away for free brings a host of unintended consequences.
There's so much on offer for free that retailers are feeling the pinch. At a local electronics outlet called "Beautiful Store," owner Ariya Palani says TV sales ground to a halt the past three years. He fears the same will happen to mixies and grinders. "If the government is giving them away, why would anyone buy from a store?" he said.
There really is no such thing as a free mixie.


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Toby Nathan
School: Boston University
Are you here to participate in one of our current school-based festivals? If so, choose your school and follow the instructions:
School: Boston University
One Night. One Party. One Epic Adventure...Well...Maybe Not So Much.
date posted: 2010-07-14 | views: 47 | type: Movie
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The following presentations, publications and accomplishments were submitted by UCA faculty and staff: Michael Rubach, Department of Marketing, Management and Information Systems, recently had an article titled ?Does Good Governance Matter to Institutional Investors? Evidence from the Enactment of Corporate Governance Guidelines? accepted for publication in the Journal of Business Ethics. The … [Read more...]
Honors College graduate earns Rotary scholarship
Rick White, University of Central Arkansas Honors College alumnus, has been awarded a 2006-07 Rotary Ambassadorial Scholarship. White was selected to represent Rotary District 6150 which includes Jonesboro, Little Rock, and most of eastern Arkansas.While serving as an Ambassadorial Scholar, he will obtain a postgraduate diploma in Social Studies for an academic year of graduate study at Lincoln … [Read more...]
Students earn SURF grants for research
Five University of Central Arkansas students recently earned Student Undergraduate Research Fellowship (SURF) grants from the Arkansas Department of Higher Education. The students will work with UCA faculty on the following research projects:Kyle Basham of Mountainburg will work with Biology Associate Professor Steve Runge and Chemistry Associate Professor Patrick Desrochers in a research project … [Read more...]
Religious studies professor to lecture at UCA
Dr. Judith Plaskow, professor of religious studies at Manhattan College, will give a public lecture at UCA on Friday, Feb. 17. The title of the lecture is "Religious Freedom and Gay and Lesbian Rights." Plaskow's lecture will explore the variety of religious positions on homosexuality and discuss the appropriate role of religious discourse in the making of public policy.The lecture will take … [Read more...]
UCA to host International Week, Feb. 13-17
The University of Central Arkansas will celebrate International Week 2006, Feb. 13-17. The theme for this year?s cultural celebration is ?Crossing Cultural Boundaries.? A number of events during the week will focus on international education at UCA.The highlight of the week will be the International Programs Banquet at 6:30 p.m. on Friday, Feb. 17. The dinner will be held in the Brewer-Hegeman … [Read more...]
Faculty Grants 2/3
The following faculty recently received grants to aid in various research projects and programs:Craig Oppel, a graduate student in the Department of Biology, and Dr. David Dussourd have been awarded a $1,000 grant in aid of research from Sigma Xi. The grant will partially fun efforts to adapt a microwave extraction technique for mapping the spatial distribution of alkaloids in leaves and … [Read more...]
Presentations and Publications 2/3
The following presentations, publications and accomplishments were submitted by UCA faculty and staff: Sandra Olson, Torreyson Library, attended the Arkansas Library Association Mid-Winter Meeting on Jan. 20 in San Antonio, Texas. She is also running for a councilor position with the association and for the position of vice president/president-elect of the library support staff interest roundtable … [Read more...]
UCA announces new chief technical officer
The University of Central Arkansas has named John A. Gale as its first Chief Technical Officer. The office of CTO will work to set and prioritize technology goals, to establish and enforce technology protocols and to support the university?s strategic plans. Before assuming the role of CTO, Gale has served as the Director of University College at UCA since 2003. Additionally, he was project … [Read more...]
Small Business program earns national awards
The University of Central Arkansas Small Business Institute Program and its Small Business Advancement National Center (SBANC) received the Showcase Award at the recent annual joint meeting between the Small Business Institute and the United State Association for Small Business and Entrepreneurship that was held in Tucson, Ariz. The Showcase Award recognizes SBI Programs and Directors for … [Read more...]
Adcock Study Abroad Scholarships
The Department of World Languages is accepting applications for the Robert and Carol Adcock scholarship program for study abroad. These awards are given to UCA students in foreign languages who plan to teach the language after graduation. Recipients must be enrolled in an academic course of study during the time of their travel. Scholarships may be awarded for the summer, semester or full … [Read more...]


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Get an overview of this hotel4-star hotel, walk to Alta Montana Archaeological Museum
Collect 200 Nectar points!
Simply enter your Nectar card number when you check out. Your points will be credited 35 days after you return from your trip.*Offers may apply
Situated in Salta, this hotel is within a 5-minute walk of Museum Alta Montana MAAM and Alta Montana Archaeological Museum. San Francisco Church and Convent is 0.5 mi (0.7 km) away.
Hotel Features
This hotel features a restaurant, an indoor pool, and a fitness center. Free buffet breakfast, free WiFi in public areas, and free self parking are also provided. Additionally, a bar/lounge, a spa tub, and a sauna are onsite.
Room Amenities
All 167 soundproofed rooms provide free WiFi, free wired Internet, and Tempur-Pedic beds. For a bit of entertainment, guests will find TVs with cable channels, and other amenities include 24-hour room service and minibars. Private bathrooms are not ensuite.
Information missing or incorrect? Tell us!Opens in a new windowAlejandro I Hotel
Renovations & Closures
- Fitness facilities
- Sauna
Hotel Amenities
In addition to an indoor pool, Alejandro I Hotel provides a spa tub, a sauna, and a fitness center. The hotel offers a restaurant. A bar/lounge is on site where guests can unwind with a drink. A complimentary breakfast is offered each morning. A computer station is located on site and high-speed wireless Internet access is complimentary.
Business-related amenities at this 4-star property consist of a business center and a meeting room. This business-friendly hotel also offers spa services, tour/ticket assistance, and multilingual staff. Complimentary self parking is available on site.
- 24-hour front desk
- Babysitting or childcare (surcharge)
- Total number of rooms - 167
- Bar/lounge
- Business center
- Free newspapers in lobby
- Free breakfast
- Concierge services
- Elevator/lift
- Fitness facilities
- Gift shops or newsstand
- Hair salon
- Computer station
- Tours/ticket assistance
- Free WiFi
- Turkish bath/Hammam
- Dry cleaning/laundry service
- Express check-in
- Luggage storage
- Laundry facilities
- Spa treatment room(s)
- One meeting room
- Multilingual staff
- Restaurant
- Free self parking
- Pool sun loungers
- Sauna
- Spa services on site
- Spa tub
- Steam room
- Indoor pool
Family Friendly Amenities
- Family Hotel
- Indoor pool
- Free Wi-Fi
- Soundproofed rooms
- Laundry facilities
- Babysitting or childcare (surcharge)
Available in all rooms: Free WiFi , Free wired high-speed Internet
Available in some public areas: Free WiFi
Free self parking
Room Amenities
- Premium bedding
- Soundproofed rooms
- Air conditioning
- In-room climate control (air conditioning)
- Phone
- Minibar
- Daily housekeeping
- Free toiletries
- Hair dryer
- Shower only
- Rainfall showerhead
- Television
- Desk
- Iron/ironing board
- Private bathroom (not in room)
- Cable TV service
- Free WiFi
- Free wired high-speed Internet
- In-room safe
- In-room massage available
- Room service (24 hours)
- Tempur-Pedic mattress
Where to Eat
A complimentary buffet breakfast is served each morning between 6:30 AM and 11:00 AM.
Alejandro I Hotel has a restaurant on site.
24-hour room service is available.
Nearby Things to Do
An indoor pool and a spa tub are on site. Other recreational amenities include a sauna and a fitness center.
Hotel Policies
Check-in time starts at 3 PM
Check-in time ends at 11:30 PM
Check-out time is noon
Payment types
Children and extra beds
- Children are welcome.
- Kids stay free! One child 12 years old or younger stays free when using existing bedding.
- Pets not allowed
You need to know
Extra-person charges may apply and vary depending on property policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
Hotel Name
- Alejandro Hotel
- Alejandro I Hotel
- Alejandro I Hotel Salta
- Alejandro I Salta
We should mention
One child 12 years old or younger stays free when occupying the parent or guardian's room, using existing bedding. Set meals are served in guestrooms only (no menus or public dining).


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Saturday, March 26, 2011
She unscrew the cap from a milk carton, and pour a second cup into her favorite green mug. She heats it in the microwave before adding the shot of coffee into it to make a latte. Now that there is no expensive coffee breaks with cherished pals, she sighs happily with these homemade concoctions free of worrisome gossips and back talks.
But the days are long, in solitude.
The sun rises with the same brilliance pouring through the window. Her morning routine of rushing through the first hours in complete hapless frenzy ends at nine. Then she sits, waiting for the day to end, moving through pages of books she must read, to seek comfort, to live in a fantasy, to rest in the dreams of others who she will never meet.
Sometimes she sheds a few tears, in trying to reach out for a world that is suddenly moving too fast for her, like touching a cascading water fall in an attempt to catch a particular drop. She typically fails at it, sighs some more, and sometimes, a few more tears.
It isn't unusual that she will go out and meet people, who sees nothing beneath her bubbly exterior, and greet her with warm hugs and chatty company. She thaws for them, even for the moments right after their departure, but then the same lone rider always return, wrapping her in his cape of darkness.
Afternoons bring solace to her melancholy. The sun being ever brighter, the day ever warmer, and the world ever louder. Through the window the new neighbor from somewhere in Europe, perhaps Russia, perhaps Spain, or somewhere in between, would play the same cheery tunes on his piano accompanied by harmonica. At first she was irritated by its monotonous melody, but after a few days she fell in love with it and would swing lightly with the music as do the leaves in the afternoon breeze. Children would run past the narrow walkway just beneath the window, chirping in incomprehensible tones that nonetheless livens up even the slow moving snails and caterpillars. Grandmas and housewives return from their grocery runs chatting about fluctuating prices and the latest choice vegetables; then there is always that one family who starts the cooking real early, fragrance of the spices culminating in the air, dancing to the tunes of exotic beats, and inspiring even tired nostrils to go for a different dream of culinary delights.
By then, all seems softer, and the slowness and solitude suddenly just right, for day dreams, for wanderings of the mind, for even tears and laughs, or for no reasons at all.
15 comments:
I love your writing style. I like this section of your work. "to rest in the dreams of others who she will never meet." I would like to hear more of this character. She lives in her own safe world, but can move outside it if needed. Thanks for sharing. Thank you for visiting my site. At the begining of Psychiatrist, I say that we all need to laugh at ourselves and the absurdity of the world around us. The post wasn't hard to write, because I do like to laugh at myself, and not take myself too seriously. Take care
ReplyDeleteI wonder if you built this around someone you actually know.
I don't know if this is fiction, of course, and you need not say whether it is. But recently I learned that one technique for novelists with great characters is to copy them from actual people they have known well enough, then change some details to protect the innocent victim!


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Wow. Can't believe I've been building and supporting IP Cam Viewer for over 7 years. If you have used it for many years, please consider becoming a patron to help keep IP Cam Viewer going. Thank you for your support!
Be a patron…
December 25th, 2016IP Cam Viewer tips for CCTV installers
October 15th, 2015If you're a CCTV installer and your clients are running IP Cam Viewer, then see this post on how to send cameras and update cameras for your customers.
Run IP Cam Viewer on Mac, Windows, Linux, Chromebook using Chrome and Arc Welder
April 8th, 2015You can run IP Cam Viewer on Mac, Windows, Linux and Chromebook via the Chrome browser! Until IP Cam Viewer is published in the Chrome App Store, use the following steps to run IP Cam Viewer via the Arc Welder Chrome App.
1. Install the Arc Welder app in Chrome.
2. Download the IP Cam Viewer Lite APK file.
3. Run Arc Welder, click "Add your APK", select the APK file.
4. On the configuration screen, use all defaults, except enter the following into the "Additional Metadata" field (last field on the bottom):
5. Click the "Launch App" button
When app is run for the first time, Chrome will ask you to select a folder (or create a new folder) on your computer. This folder is where IP Cam Viewer files will be stored. Anytime the app uses "/sdcard/…" or any other path, the files will be located under this folder.
CEL-iN1 Door Mount GSM Intercom
February 2nd, 2015Want to know who's ringing your doorbell? Want to let someone inside while away from home?
The Celius CEL-iN1 multifunction door bell will call you, let you open/close doors, and let you interact with visitors via cameras using IP Cam Viewer. Watch video.
Integrating IP Cam Viewer with third party apps and products is possible with the app's embedded web server API, Android intents and launch URLs. Device integration include Android Wear watches, Amazon Fire TV/Stick, Chromecast, HDMI sticks, Google TV, Android TV and SmartThings home automation hub.
IP Cam Viewer is available for Apple, Android and Blackberry devices. Windows Phone 8.1 release is targeted for summer 2015!
Android Wear support in v5.7
December 2nd, 2014The android version of IP Cam Viewer now supports Android Wear devices (see link).
In Gallery View, select More Actions then "Push Android Wear App". Once the wear app is deployed on the watch, you can launch the wear app on the watch and view your enabled cameras (including some basic functions like relay on/off and quick preset recall).
So now, IP Cam Viewer in one form or another supports Amazon Fire TV and Fire TV Stick, Android TV, Chromecast, Android Wear and stills works from Android 1.6-5.0!
Can't believe I got the Fire TV Stick for $19 bucks! Did you know it actually runs Android apps so it's basically the cheapest hdmi stick (unlike the dumb chromecast stream player).
More Integration: SmartThings Hub
August 22nd, 2014Good news! If you use IP Cam Viewer to record your cameras 24×7 and have a SmartThings hub, then you can automate motion notifications globally and vary camera groups based on the Away / Home modes in SmartThings.
Add presense detection in SmartThings to switch modes and you won't even have to push a button.
Finally, you can integrate any camera supported by IP Cam Viewer into SmartThings by using the app's embedded web server.
Remote Control of IP Cam Viewer and ChromeCast/FireTV Stick
April 10th, 2014Starting with v5.3.7, IP Cam Viewer now has an embedded HTTP server! This server can be used for remote control of record mode, tasker integration, chromecast, cam hosting and more.
IP Cam Viewer already has in-app motion detection with notifications in Record Mode. Adding remote control will allow you to change settings like turning in-app MD notification on/off while away from home, or recall a group to change recorded cameras. See this post for access information.
Many people have asked for tasker integration but I've held off in-order to find a lighter, simpler and more extensible solution (compared to their plugins). Adding commands to the embedded web server allows for tasker integration via it's HTTP automation features while enabling easy integration with other tools.
Want to share your cameras via a website but 1) don't want to share it directly, or 2) don't know it's API, or 3) it's has some custom binary protocol / video format? Now with the embedded web server, all 1900+ camera types supported by IP Cam Viewer can have it's own simple web page. Of course it's only available from the local network in record mode but you can choose to open it up (port forward) to the outside.
Chromecast support is now available in v5.4.3! In Gallery View, select More Actions and Show in Chromecast. IP Cam Viewer will send instructions to play (via it's web server) to a Chromecast device using a free app called IPC Cast Proxy. I published this app solely to support Chromecast integration (it does nothing else). The reason is that direct integration requires lots of libraries which I'd rather not burden IP Cam Viewer for performance, stability, size and portability reasons. Remember that IPC runs on pretty much all devices from Android 1.6+ and many third party Android platforms like Barnes & Nobel, Amazon, Blackberry, Microsoft, Intel, etc…
Backing up a bit, if you really want to see the display on a TV, you can do it today with a $39 Amazon Fire TV Stick, or $50 Android HDMI dongle like the MK908, or a sub $50 Walmart tablet. They all run full Android apps! Chromecast doesn't run android apps and require apps or android device to send it video using up bandwidth (why when you can run full android?) Future chromecast will have mirroring (should have come with it from beginning IMHO) so the proxy is no longer needed then (argh).
In summary, the embedded http server is here and will be built out over the coming releases.
Baby Mode and background audio
June 17th, 2013In the past, people have consistently emailed to confirm that the application does not run in the background (due to bandwidth concerns). In fact, the app does not run in the background unless you are using home screen widgets or record mode (rate is adjustable).
Because of this concern, the closest to a baby mode was to run in Gallery View with Auto Dimming turned on (see App Settings). This way, you can hear audio and have a dimmed screen (for night time). The screen will turn on if touched or motion is detected.
However, there's been an increasing email demand for another option to play audio even when app is not in the foreground and when screen is locked! This goes against the demand by other users to not run in background.
With Android v4.9.5, I've added a new Background Audio Mode option (in Gallery View, click Menu key). In this mode, and only in this mode, the app will continue to run in the background and play audio even when phone is locked (audio includes motion detection notification sounds if enabled).
In summary, the Android app will not run in the background except if you're using widgets, record mode or background audio mode.
ps. the iOS version never runs in the background.
Anyone can have a home surveillance system!
April 24th, 2013With the latest record on motion detection feature in IP Cam Viewer Pro, now anyone can have a home surveillance system for almost nothing. Motion detection reduces sdcard space usage dramatically. I'm getting weeks of recording time on an empty 8gb sdcard (app will automatically recycle old footage as needed).
Search, view, export past recordings and view live, all with no monthly fees. No middleman servers/services which can be a security/privacy issue, massive ISP bandwidth usage, exposure to monthly fee increases, slow network access and potential disaster if company goes belly up or get hacked.
Latest Android version now supports motion email notifications. iOS version will get this functionality soon. I switch between a 'record home' and a 'record away' group where the 'record away' group records more cameras and enables motion notifications for some cameras.
- old, left over android phone or tablet. Price: free
- cheap ip camera on amazon or ebay. Price: ~$50 each
- IP Cam Viewer Pro. Price: $3.99 one time cost
1) install IP Cam Viewer Pro on spare android device.
2) configure your cameras in IP Cam Viewer Pro (turn on record only when motion detected in Edit Camera -> More Options for each camera).
3) start recording, click matrix view button, hang on wall as a bonus display!
Finally, both Apple App Store and Android Play Store allows you to install IP Cam Viewer Pro on as many devices as you want (as long as you use the same account) so install it on your daily phone to get live viewing (use export/import or sharing to copy settings).
ps. for live viewing outside of your home network, you need to setup dynamic dns and port forwarding.
ps.ps. some users have asked to record to dropbox. See above on why I do not like to send private video to some cloud service. If you MUST sync to dropbox or google docs, use FolderSync Lite to sync the entire IP Cam Viewer Record folder.
ONVIF and Bitcoin
April 3rd, 2013I quietly added ONVIF support to recent versions. With your support, hopefully it'll get better over time.
Next, for those of you that download the Android app directly from my site, the next version will support upgrade via Bitcoin or Paypal. Bitcoin has really come a long way with tools and ease of use. See http://bitcoin.org for more information.


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Monday, 4 February 2013
1 comments:
- Le Sanglier said...
I am worried. Where did those hands come from? Are Linda's dolls safe? But, apart from my worry that there are mutilated dolls stuffed under a bed or in a closet, the machine IS quite soothing.
- 12:04 pm
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© 2017 Calgary Stampeders. All rights reserved.
Now Reading
We've reached Group C in the preliminary round of the 2012 Play of the Year Showdown.
Keenan MacDougall won Group A thanks to his fumble-return touchdown against the Montreal Alouettes (which just happened to be the first game of his CFL career) and Marquay McDaniel's dive for the pylon and touchdown was the most popular choice in Group C.
Group C features another McDaniel highlight — this time it's his sprint for the end-zone to set the tone on the West Division final at BC Place. Also featured this week are a great second effort and long run by Jon Cornish, an interception-return touchdown at a snowy McMahon Stadium by Keon Raymond and a clutch, long-distance field goal by Rene Paredes. Check out the video and cast your vote. The winner will be announced on Tuesday, Feb. 12.
>> Video: Group C candidates


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Collections are a JPG+ feature. You must be a JPG+ member to create new collections and to add photos to collections.
Sign up for JPG+ to start using collections now!
Photo license: © All rights reserved
4 responses
Carol Holmes gave props (4 Jan 2012):
wow! what is this?
MaYa Sinji Jung ì •ì‹ ì§€ said (6 Jan 2012):
Thanks Carol!! This is a family photo I found in some empty house. It was a wall decoration with dried flowers in it. Isn't it beautiful???
Maria L. Ferrari said (6 Jan 2012):
this is amazing
MaYa Sinji Jung ì •ì‹ ì§€ said (7 Jan 2012):
Thanks Gary Joseph Cohen כוהן for the nomination!
Also by MaYa Sinji Jung ì •ì‹ ì§€


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On arrival, each participant will be equipped with a harness and gloves. Additionally, a safety helmet will be provided for young climbers. All harnesses are double sling-style with a carabiner and pulley for zip-line crossings.
At our Gwinnett park, children from 4 to 6 years old experience the thrills of Treetop Quest on the Chick'Pea course. This level has a continuous lifeline; a belay system that is impossible to detach without a guide. Chick Pea obstacles are similar to our higher levels, but with equipment that is lighter and easier to use for our youngest adventurers. In Dunwoody, children age 4-6 participate in Kiddie Quest which consists of a net trampoline suspended in the trees, an on-ground obstacle course, and a harnessed obstacle course. All harnessed activities use a continuous belay system.
Older participants are led through a detailed briefing and practice session by Treetop Quest guides. Before beginning the course, we ensure all participants understand the mechanics, pulley and belay system that is impossible to detach without a guide. Ground guides will monitor climbers as they get the initial feel of the course on the practice route.
Once in the trees, guides will be present on the ground to ensure surveillance and assist participants if needed. All our instructors are specially trained and qualified professional guides in the industry. For more information, see our FAQ page.
For safety reasons, the weight limit is 250 lbs. All participants age 12+ are weighed at check in. Weights are kept confidential with our front desk staff and not communicated to others.