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We always feature what we feel like is the best value for the most affordable price. Our new homes that we offer are of the finest quality and are Fleetwood Homes and Cavalier Homes. Having been in the Manufactured Home business for over 25 years we feel that these two manufactures do an incredible job of offering the most home for the money. Their service is second to non and we have found both of them to offer the very best service after the sale. They both go the extra mile to make sure your buying decision and new home experience is stress free. With Fleetwood our company has earned the Customer Satisfaction Award for 16 years. We have always been at 97 to 100 percent with Fleetwoods program. Both manufactures have a variety of features to please all new home buyers.
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All prices on the homes include Delivery and Setup, Appliances, Central Heat and A/C, Skirting and two sets of steps.
New Doublewides
2016 Cavalier 32x72 4 Bed 2 Bath
This new floorplan has plenty of room and the comfort and style of the more expensive homes. The home features a separate living room and den and a huge island in the kitchen with maple cabinets. The utility room connects the kitchen and the master bath. The masterbath has a garden tub and separate shower with double sinks. The bedrooms are all nice and large and the den has a fireplace and a beamed ceiling.
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This fully loaded house is truly one of a kind. Cavalier has built this special edition house to honor our troops and for every home sold a donation will be made to the veterans relief fund. The house is fully loaded with thermo pane windows and pocket doors and a barn door covering the pantry. The island in the kitchen is granite and the kitchen has a side by side refrigerator and smooth top range and microwave as well as a dishwasher. The back side of the dining room and kitchen have a double set of windows and a sliding glass door. The utility room has a pocket door and has a counter to fold your clothes There is also a laundry shute between the 2nd bathroom and the utility room. The master suite has a huge closet and the masterbath has a claw tub and a huge huge shower stall. Even the outside of the home has unique exterior. This home has a special price and is a great deal
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This entry level house is a great buy It features a large living room and a well equipped kitchen with an island kitchen. The utility room is large enough for a washer and dryer and a freezer. All three bedrooms are large and there are two baths with a large master bath closet. Great house and it is well worth a look see.
2016 Cavalier 32x64 3 Bed 2 Bath
This house looks like a site built house and feels like home. It has a huge living room as you walk in and an even nicer kitchen with wood cabinets and a European range hood. The island features a large sink and the kitchen is equipped with a side by side refrigerator and smooth top sink and pot scrubber dishwasher. The whole wall behind the dining room and kitchen is thermopane windows from floor to ceiling to see everything in the back yard. The utility room has a counter for folding clothes and room for a washer and dryer and ironing board. The extra large master bedroom opens into a huge master bath with an over sized shower and double sinks and a linen cabinet. The kids rooms are over sized and each have a walk in closet with a hall bath that is also oversized. All and all a great floorplan with room for everyone
32x76 Cavalier 4 Bed 2 Bath
Awesome does not even describe this house. It has the look and feel of a high end site built home with many of the same amenities. You walk into the foyer and look to your right and see a spacious living room with a fireplace at the end with bookcases on both sides and a place for the TV on the mantle. The room also features a trey ceiling. The kitchen is on the opposite wall and has an island set up for bar stools and a shelf unit on both ends. There are drawers galore in this kitchen The appliances are high end and include a side by side refrigerator with ice and water in the door and a smooth top range with a microwave over head and of course a dishwasher. The master suite will knock your socks off. It has a bright master bedroom with a master bath that includes a center tub and double vanities and shelves everywhere. The walk in shower is huge and so is the master bath closet. Going towards the other end of the home there is a dining room and a bedroom and a nice sized utility room. The kids have their own Den area with a built in Entertainment Center. The other two bedrooms have a bath between them. This home also features tape and texture walls and 6 inch crown molding and baseboard trim It is a must see.
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This awesome seller is back in stock and has an entirely new look. It features a separate living room and den which both open into an Island Kitchen with an angle bar. The Den has a fireplace for those cold winters and has a large utilty room beyond it that has room for a washer dryer and freezer. The kids rooms are all together and have their own bath The living room is perfect for entertaining. The master bedroom is large and opens to a master bathroom complete with double sinks and a separate shower as well as a vanity area. Dont miss out on this great home. ]
2015 Clayton TRUMH 4 Bed 2 BAth
This newly arrived home has it all. A living a den a spacious island kitchen a huge utility room and a master suite that includes a huge bath and a huge walkin closet. Even the kids rooms are huge. The great thing is tho if you have a credit score of at least 575 we can put you in this house with only 10 percent down. It is great for starting over. Dont miss it.
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|Publication number
|Publication type
|Application number
|Publication date
|Apr 24, 1990
|Filing date
|Feb 6, 1987
|Priority date
|Jul 30, 1982
|Fee status
|Publication number
|Bharat B. Aggarwal
|Original Assignee
|Genentech, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (11), Non-Patent Citations (108), Referenced by (22), Classifications (19), Legal Events (3)
|External Links: USPTO, USPTO Assignment, Espacenet
This is a continuation of application Ser. No. 608,316, filed May 7, 1984, now abandoned, which is a continuation-in-part of U.S. Ser. No. 06/403,671 filed July 30, 1982 now abandoned.
The invention herein concerns lymphokines, a class of proteins originally extracted in impure form from lymphocytes, and believed to have anti-tumor activity. Specifically, the invention concerns a homogeneous preparation of the human lymphokine, lymphotoxin, and a process for preparing it.
Lymphokines are biologically active, hormone-like peptides or proteins produced by stimulated lymphocytes. The properties and functions of these lymphokines, including those exhibiting cytotoxic activity, have been studied extensively. Lymphotoxin is a type of lymphokine which is produced not only by mitogen or antigen stimulated lymphocytes, but also by cell lines which are grown in tissue culture derived therefrom.
Lymphotoxin has been implicated in the regulation of the immne system (1) and has been reported to inhibit tumor cell growth both in vivo (2-7) and in vitro (8-10). Under in vitro conditions, it is a more potent inhibitor of tumor cells than of normal ones from the same species (11-15). Also, lymphotoxin preparations have been shown to inhibit UV or chemical carcinogen induced cell transformations (16-17). The in vitro activity of lymphotoxin may be assayed by a number of methods as will be set forth hereinbelow and these methods provide convenient means for following the processes of purification of lymphotoxin from crude extracts. In vivo studies in human beings have also shown that crude preparations of lymphotoxin are effective in tumor regression (5,7).
Previous studies have been conducted with relatively impure fractions of cell supernatants prepared from lymphocytes or cell lines derived therefrom. A number of laboratories have attempted to prepare lymphotoxin in purified form, without conspicuous success (18-24). The present invention uniquely provides a homogeneous preparation of lymphotoxin proteins useful as an anti-tumor agents in mammals.
In one aspect, the invention is directed to lymphotoxin per se and its natural and otherwise equivalent pharmaceutically acceptable salts and pharmaceutically acceptable derivatives, including fragments thereof, in substantially homogeneous form. Homogeneity is defined in its classical sense including the substantial absence of other proteins of human origin. However, homogeneous lymphotoxin includes various species or derivatives of lymphotoxin such as lymphotoxin aggregates, lymphotoxin fragments as well as mixtures of such fragments and aggregates. Further, lymphotoxin and its fragments are characterized by their C-Terminal amino acid sequence, whereas various lymphotoxin species are characterized by different N-Terminal sequences. In an embodiment substantially homogeneous lymphotoxin is provided in nonaggregated form. The entire mature amino acid sequence for lymphotoxin, as well as a biologically active fragment thereof, is disclosed.
The invention further concerns pharmaceutical compositions containing the aforesaid lymphotoxin, and methods of using same for administration as an anti-tumor agent.
In still another aspect, the invention concerns processes for preparing homogeneous lymphotoxin. In particular, the process comprises contacting a nongelatinous hydrophobic substance and/or immobilized lentil lectin with an admixture of lymphotoxin and other protein to adsorb lymphotoxin, followed by elution of the lymphotoxin from the substance or lentil lectin.
FIG. 1 is a schematic representation of a preferred embodiment of the purification of lymphotoxin as practiced by the invention.
FIG. 2 shows the elution pattern fromm a DEAE cellulose purification step, specifically elution from DE-52 using 0-0.3M NaCl gradient. The activity elutes at approximately 0.1M, while large amounts of protein are retained.
FIG. 3 is the isoelectric focusing pattern obtained in a typical preparation, showing activity, protein and pH pattern.
FIG. 4 is the elution pattern from a lentil lectin sepharose purification step. More than 95% of the total protein with no lymphotoxin activity is filtered through the column prior to the elution of the lymphotoxin.
FIG. 5 depicts the elution profile of a lymphotoxin activity from Sephadex G-100. The insert shows the calibration curve for molecular size, using the known molecular weight proteins: soybean trypsin inhibitor, carbonic anhydrase, ovalbumin, bovine serum, and albumin. Blue dextran is used to determine a size maximum.
FIG. 6 shows the results of HPLC used to analyze a purified lymphotoxin fraction from the lentil lectin column.
FIG. 7 is the size distribution of a lentil lectin active fraction as determined by SDS-PAGE. A standard using the 20K fraction from the gel filtration step of FIG. 5 is also shown.
FIG. 8 is the elution profile of a lymphotoxin activity from SDS-PAGE. The upper panel shows the results using the gel in column C of FIG. 7. The lower panel is a control using only buffer (column E of FIG. 7). Slight activity appears to be an artifact associated with the dye front.
FIG. 9 shows an analysis of the Rf 0.33 fraction obtained by native-Page on a purified lymphotoxin by activity determination and by SDS-PAGE. This figure confirms a 20,000 MW at Rf 0.33 on native-PAGE. The lowest panel shows the origin of the gel slices from native-PAGE run on the purified lymphotoxin. These slices were assayed both for activity (upper panel) and size (middle panel).
FIG. 10 is the elution pattern from HPLC of a tryptic digest of a homogenous lymphotoxin.
FIG. 11 is the SDS-PAGE molecular weight analysis on the T1 and T2 fractions shown in FIG. 10.
FIG. 12 shows the results of amino acid sequencing of the fragments from trypsin-digest of the purified N-terminal histidine species of lymphotoxin. Those residues which are in parentheses are alternatives or uncertain determinations.
FIG. 13 is a diagrammatic representation of the amino acid sequencing strategy for determining the amino acid sequence of lymphotoxin.
FIG. 14 is the amino acid sequence for two lymphotoxin species, one of which commences with an N-terminal leucine and another of which begins with N-terminal histidine.
A. Definitions and Abbreviations
The sequence of amino acids in lymphotoxin will be designated by using the standard IUPAC three letter abbreviations for amino acid "residues". "Residue" has a functional meaning within the amino acid sequence. In the body of the sequence the residue and the abbreviation therefor denotes the amino acid less one N-hydrogen and the --OH from the carboxyl; at a terminal position, the designation denotes this residue with the appropriate hydrogen or hydroxide. Thus, in the sequence, Ala1 -Ala2 -Ala3, ##STR1## All chiral amino acid residues herein are of the natural or L-configuration unless otherwise noted. All peptide sequences are written according to the convention whereby the N-terminal acid is on the left, and the C-terminal on the right.
"Lymphotoxin" means polypeptides having detectable Specific Activity as defined below which contain regions homologous with the polypeptides set forth in FIG. 14. Thus, lymphotoxin as defined herein includes the two species in FIG. 14, hydrolytic fragments thereof, aggregates (including trimers) of such species with one another, and salts and derivatives of such species, all of which must exhibit detectable Specific Activity in order to fall within the scope of the term "lymphotoxin" as used herein.
"Peptide" and "protein" are used interchangeably herein to denote amino acid polymers; the conventional size distinctions wherein proteins are large and peptides small is not made, in view of the difficulty of finding agreement as to the boundary between the two.
"Amino acid residue" in general refers to a residue as defined hereinabove which is derived from one of the twenty amino acids coded for in proteins. This residue may however be modified so as to form a derivative as defined below.
As used herein the term "salts" refers to both salts of carboxyl groups of the polypeptide chain and to acid addition salts of amino groups of the polypeptide chain.
Salts of a carboxyl group may be formed with either inorganic or organic bases by means known in the art per se. Inorganic salts include, for example, sodium, calcium, ammonium, ferric or zinc, salts, and the like. Salts with organic bases include those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, caffeine, procaine, and the like.
Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid, or sulfuric acid, and salts with organic acids such as, for example, acetic acid, or oxalic acid.
Derivatives may also be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable (as defined below). These derivatives may, for example, include:
aliphatic esters of the carboxyl groups;
amides of the carboxyl groups by reaction with ammonia, or with primary or secondary amines;
N-acyl derivatives which are derivatives of an amino group of the polypeptide formed with acyl moieties (e.g. alkanoyl or carboxyclic aroyl groups);
or O-acyl derivatives which are derivatives of a hydroxyl group (for example that of seryl or threonyl residues) formed with acyl moieties.
Both the salts and the derivatives encompassed by this invention are those which are "pharmaceutically acceptable" i.e., which do not destroy the activity of the lymphotoxin and which do not confer toxic properties on compositions containing them.
A number of abbreviations with respect to techniques also will be used, which are well known to those skilled in the art. Specifically, "DEAE cellulose chromatograph" refers to chromatography using diethylaminoethyl cellulose, which provides an ion exchange support for packing into chromatography columns. At high pH values, the column is an anion exchanger and negative residues stick to column. Elution can be accomplished either by lowering the pH, or, more preferably, with a salt gradient at constant pH.
"PAGE" is electrophoresis as performed on a polyacrylamide gel and separates proteins or peptides on the basis of charge. If sodium dodecyl sulfate (SDS) is incorporated into the gel (SDS-PAGE), the surface active nature of SDS results in a uniform negative charge on the peptide or protein which is a function of size. The result is that separation is based on the molecular size. Native-PAGE denotes the employment of this technique without the presence of SDS and thus, proteins are separated on the basis of charge.
"Isoelectric focusing" can be performed as a form of native PAGE wherein a pH gradient is maintained across the electrodes, causing each protein to stop, or "focus" at its isoelectric point. However, other supports, preferably, for example, dextran, typically Ultrodex-LKB are also practical.
Other chromatography supports are also available which are of particular interest for this invention. A lectin isolated from lentils, "lentil lectin" is capable of retaining, selectively, glycoproteins which have galactosyl and mannosyl residues. Elution is accomplished by providing a sugar solution; in the case of this particular support, the solution must be of mannose or galactose.
Under suitable circumstances, chromatographic procedures may be carried out preferably in a narrow bore column containing a fine particle resin under increased pressure to enhance the effectiveness of separation, i.e., by high pressure liquid chromatography (HPLC).
Concentration and salt removal are commonly used precursors to certain chromatographic or separation techniques employed in the invention. Salt removal may be performed by, for example, dialysis or gel filtration, or by a relatively recently developed technique, controlled pore glass (CPG).
A number of gel filtration and concentration techniques are also used. Certain commercially available materials are especially useful. "Pellicon" membrane is a sheet like material composed of polysulfone manufactured by Millipore, Inc. Bedford; and "Amicon" membrane is a similar material also composed of polysulfone and manufactured by Amicon. These materials are capable of retaining large molecules while permitting passage of smaller ones. They thus operate in the opposite way to molecular seives, which allow large molecules to pass readily but retard the passage of smaller ones. Both Pellicon and Amicon are useful as concentration tools, permitting the smaller molecules to be "filtered" away from the desired macromolecular structures.
The extent to which concentration of a solution to be chromatographed is desirable is largely a matter of practicability of application. Salt removal is necessary if ion exchange or other techniques which depend on total ionic strength are employed. These preparation methods and the extent to which they are required for particular separation procedures are well known in the art.
The cells used as the source of lymphotoxin in the present invention are lymphocytes or their transformants. The literature shows lymphotoxin to be present in preparations of "buffy coat white blood cells" converted by mineral oil induction; golden Syrian hamster or guinea pig peritoneal leukocytes (17) and lymphoblasts such as the human lymphoid cell lines RPMI 1788, B-21 and MOLT (25).
"Specific Activity" refers to the activity of the protein in standard lymphotoxin assays as related to the amount of protein by weight in the sample. As specified in the current disclosure, the activity of lymphotoxin is measured in terms of "units" which refer to the amount of lymphotoxin required to cause 50 percent lysis in target cells according to the assay procedure set forth hereinbelow. Several standard assay procedures are available. The assay procedure described herein in detail is based on ability to mediate lysis of mitomycin C-treated mouse fibroblast cells, as measured by staining capability.
An additional procedure involves the release of the tritium from tritiated thymidine labeled murine alpha-L-929-fibroblast cells, a lymphotoxin sensitive cell strain (26). The "units" used to define specific activity may differ depending on assay procedure as does the mg protein determined. "Specific Activity" as defined herein is units/mg protein, where "units" are measured by the assay set forth below, and mg protein is measured by the method of Bradford (infra).
"Impurities" as they pertain to the lymphotoxin prepared by the method of this invention refers to those substances associated with lymphotoxin in its normal cellular environment or in a crude extract, filtrate, or centrifugate.
The overall process for preparation of homogeneous lymphotoxin is summarized in FIG. 1. The specific method for preparation of the various lymphotoxin species differs somewhat. The following Examples B through F deal primarily with the preparation and characterization of lymphotoxin having an N-terminal histidine residue. The method for the N-terminal leucyl species is described in Example G.
B. Purification of Lymphotoxin-Embodiment I
(1) Tissue Culture and Harvest
The human lymphoblastoid cell line RPMI 1788 was obtained from ATCC (No. CCL 156) and the seed culture was grown in 400 ml of the medium RPMI-1640, obtained from Irvine Scientific, Santa Ana, Calif., containing 10 mM HEPES and 5% fetal calf serum innoculated at a cell density of 6�104 cells/ml with 400 mls in 2 liter roller bottles. After 5 days at 37� C., when culture reaches a cell density of 2�106 ml, the cells were harvested and washed 2 times with a serum-free culture medium RPMI-1640. The cells were then transferred into a serum-free medium RPMI-1640 containing 10 mM HEPES and one percent Penicillin-Streptomycin at a cell density of 5�105 cells/ml. (The absence of serum in this medium is helpful in the purification of lymphotoxin, as the amount of contaminating protein is decreased). 400 ml of this cell suspension was placed in 2 liter roller bottles and also in 15 liter and 10 liter spinner flasks (Belco Glass Co., Vineland, N.J.), which were substituted for 2 liter roller bottles in some instances. After 65 hours the culture medium was harvested for the lymphotoxin activity.
The cell suspensions from both roller bottles and spinner flasks were pooled. The cells from this pool were removed by passing through a 3 uM Sealkleen filter supplied by Pall Trinity Micro Corp., Cortland, N.Y. (Step 1 of FIG. 1). The clear filtrate was further concentrated and dialyzed, as follows (Step 2).
(2) Concentration and Dialysis of Filtrates
The filtrates were concentrated approximately 20 fold at 4� C. on a Pellicon membrane (supplied by Millipore Company) with a molecular weight cut-off of 10,000. The concentrated samples were further dialyzed against 5 mM phosphate buffer, pH 7.8 using the same Pellicon, set up in its dialysis mode. When the conductivity of the sample reached that of the dialysis buffer, the Pellicon membrane was rinsed thoroughly with the buffer and all the protein recovered.
The resulting concentrate was assayed for lymphotoxin activity as were the resultants from Steps 3 through 6, in order to determine specific activity. The specific activity of the concentrate (I) FIG. 1 in a typical run was found to be of the order 2,000-3,000 units per mg of protein. Chromatography on a Sephacryl S-300 column to determine molecular size gave a single peak of activity at approximately 70,000 daltons.
(3) DEAE Cellulose Chromatography
The concentrate (I) was subjected to DEAE cellulose chromatography on a DEAE-52 cellulose column equilibrated in 5 mM phosphate buffer pH 7.8 (Step 3). After the sample was loaded, the column was washed with the equilibration buffer and thereafter it was eluted with a 0-0.3M linear sodium chloride gradient.
The active fraction was identified by selecting the fraction of the highest specific activity as defined above. A single sharp peak of activity eluted at around 0.1M NaCl as shown in FIG. 2. Typically, this active fraction (I) of FIG. 1 would have a specific activity of the order of 20,000-30,000 units per mg protein, a 10 fold purification.
(4) Isoelectric Focusing
The active fraction is then concentrated using an Amicon stir cell with 10,000 (PM-10) molecular weight cut-off membrane, (Step 4 of FIG. 1).
The concentrate (II) was dialyzed against 2.5 mM Tris and 20 mM glycine pH 8.2 and run on a dextran flat bed (Ultrodex-LKB) containing Ampholine carrier ampholytes in the pH range 5-8 (Step 5). The basic apparatus used for preparative isoelectric focusing was that supplied by LKB (2117 Multiphor). The sample was mixed with the whole gel bed before the pH gradient was formed. After placing the electrode strips on either side of the gel bed, the electrofocusing was run along its length with a constant power of 8W for 15-20 hours at 8� C. The focused zones were collected by sectioning the gel bed with a fractionating grid and then removing each section with a spatula. Elution of the protein from the respective gel section was achieved in several small disposable columns using 50 mM ammonium bicarbonate. The absorbance at 280 nm, lymphotoxin activity, and the pH of the eluted fractions were determined.
FIG. 3 shows the activity, protein and pH profile obtained. Though proteins were spread in the whole gel bed, more protein contaminants are observed in the acidic region as compared to basic pH range. The lymphotoxin activity was spread through the pH range of 5.5 to 6.5. Due to the presence of Ampholines it was difficult to determine accurately the protein concentration of the active fraction, thus it was not possible to calculate the degree of purification obtained at the isoelectric focusing step. The recovery of activity from this step was, however, fairly quantitative.
(5) Lentil Lectin Chromatography
The pool of the isoelectric focusing eluates containing lymphotoxin activity was applied on a Lentil Lectin (Pharmacia) column equilibrated in 10 mM phosphate buffer pH 7.8 (Step 6). After loading the sample the column was washed with the equilibration buffer and then eluted with 50 mM or 100 mM alpha-methylmannoside solution prepared in 10 mM phosphate buffer. The results are shown in FIG. 4. More than 95% of the protein, lacking in lymphotoxin activity, filtered through the column initially, and washing with column equilibration buffer (10 mM phosphate buffer pH 7.8) also did not elute the activity. When the column was exposed to 50-100 mM alpha-methylmannoside, a large peak of activity eluted with very little protein. The recovery of units of activity in this step was greater than 90 percent. The active fraction had specific activity on the order of 10 to 100 million units per mg.
C. Characterization of Purified Lymphotoxin
The active fraction eluted from the Lentil Lectin column was further subjected to the following analytical techniques and has the following properties:
(1) Gel Filtration
Gel permeation chromatography gave a single peak at 64,000 daltons, (as shown in FIG. 5). The active fraction from the lentil lectin column was applied to a Sephadex G-100 column (Pharmacia Fine Chemicals, Piscataway, N.J.), previously equilibrated with the eluting solvent, 10 mM phosphate buffer containing 0.5M in NaCl. The elution pattern was calibrated with bovine serum albumin, ovalbumin, carbonic anhydrase and soybean trypsin inhibitor; the elution of the lymphotoxin in the 64,000 dalton range indicates aggregation of individual peptides, as well be apparent from the results below.
(2) HPLC Chromatography
HPLC chromatography showed a substantially homogeneous preparation, as shown in FIG. 6.
The active fraction from the Lentil Lectin column was concentrated on a Amicon stir cell using PM-10 membrane and applied on a Synchropak RP-P column (25 cm�4.1 mm, Synchrom, Inc., Linden, Ind.). The effluent was monitored at 210 nm and at 280 nm absorbances using a Spectra Physics SP 8000 high pressure liquid chromatograph. The elution conditions consisted of a linear gradient from 0.1% trifluoroacetic acid to 70% acetonitrile in 0.1% trifluoroacetic acid at 25� C. and a flow rate of one ml per minute. A major protein peak which elutes at a concentration of about 50% acetonitrile is obtained (FIG. 6) and when run on 12.5% SDS-PAGE, this fraction contains protein with a molecular weight of 20,000. Besides this 20K peak on HPLC, there were also minor peaks which eluted at 43% and 51% acetonitrile with molecular weights of 15K and 70K respectively. The HPLC solvents appear to inactivate the lymphotoxin molecule, thus it was not possible to determine the activity of the 20,000 molecular weight band.
SDS-PAGE showed the majority of the protein to conform to a size estimate of approximately 20,000 daltons (FIG. 7). The lymphotoxin activity was associated with this 20,000 dalton fraction, as shown in FIG. 8. The SDS-PAGE was performed according to the method of Laemmli, et al. (27), which is incorporated herein by reference, using resolving acrylamide gel concentration of 12.5-15%.
(4) Native-PAGE
Native-PAGE also gave a peak of approximate MW of 20,000, as subsequently determined by SDS-PAGE.
The active fraction from the Lentil Lectin column was subjected to the native-PAGE procedure of Laemmli, et al. (27) with slight modifications. Both preparative (1.5-4 mm thick) and analytical (0.75 mm thick) gels consisted of 7.5% acrylamide for resolving gel and 4% for stacking gel. The running buffer used in both cases was Tris 25 mM pH 6.8. The analytical gel was run at constant current of 22 mA and preparative gel was run at 100 mA keeping the temperature constant.
At the end of the electrophoretic run, the protein on the gel was visualized by silver staining (28) incorporated herein by reference, to give a diffused band of Rf of 0.33. FIG. 9 gives a more detailed explanation of these results -as there shown, it was determined in a separate experiment that when gels are sliced and eluted by incubating the slices overnight at 4� C. in 50 mM ammonium carbonate buffer from the native-PAGE, the lymphotoxin activity is associated with a gel slice of Rf 0.33. The eluates from these gel slices were applied on SDS-PAGE, and the 20,000 MW band appeared only in those eluates which had lymphotoxin activity.
(5) Analysis by Tryptic Digestion and Isolation of Peptides
The purified lymphotoxin preparation was digested with trypsin-TPCK (Worthington) using 1 part trypsin, 20 parts protein in 50 mM ammonium bicarbonate buffer pH 8 for 24 hours at room temperature. The fragments have approximate molecular weights of 15,000 and 5,000.
At the end of hydrolysis, the tryptic peptides were chromotographed by HPLC on a Lichrosorb RP-18 column (25 cm�4.6 mm, EM reagents, Cincinnati, Ohio) at 25� C., using Spectra Physics SP-8000 chromatograph. The peaks were detected at 210 nm and 280 nm after elution with a linear gradient of 0.1% trifluoroacetic acid to 50% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of two ml per minute. (For later amino acid sequence analysis, each peak was collected, lyophilized and stored at -20� C.). As shown in FIG. 10, two peaks, T-1 and T-2, were observed eluting at 42.3% and 48.4% acetonitrile concentration respectively. The molecular size of each of these 2 peaks as determined by SDS-PAGE (FIG. 11) was 15,000 for T-1 and 5,000 for T-2. Both at 210 nm and 280 nm, the T-2 peak with relatively lower MW had higher absorbance than the T-1 peak.
The completeness of the trypsin hydrolysis was confirmed by applying a small aliquot of the hydrolyzed preparation to SDS-PAGE and showing that 20,000 dalton band was undetectable, and had been replaced by the 15,000 and 5,000 dalton bands.
Furthermore, the composition formed by the trypsin digestion as herein described exhibits lymphotoxin activity.
(6) Automated Amino Acid Sequence Analysis
Both intact and tryptic fragments were sequenced by automatic amino acid sequencing techniques by suspending the samples in 0.6 ml of 0.2M acetic acid and transferring the suspension into the cup of a Beckman 890C sequencer which had been previously coated with 4 mg polybrene and 100 nmoles norleucine. The phenyl thiohydanation (PTH) amino acids were identified using an ultrasphere ODS (25 cm�4.6 mm, 5 μM) column at a flow rate of one ml per minute with a program as described in, Kohr, W. et al (29) incorporated herein by reference.
The results obtained with both the intact protein and the tryptic fragments are shown in FIG. 12. As there seen, certain positions in the sequence could not be identified with certainty. However, sufficient identification was made to show that the 15K fragment (T-1) corresponds to the N-terminal portion of the intact lymphotoxin.
The above example is intended to illustrate rather than limit the invention. Other cell lines can be used as starting material; and equivalent chromatographic methods employed. The greatest purification appears to be achieved by the use of lentil lectin chromatography and as long as this step is included, a variety of preliminary steps remain satisfactory. Lentil lectin has not heretofore been used in the isolation of lymphotoxin. However, the integrated process as shown above provides a result which is substantially a pure protein.
D. Assay Procedure for Lymphotoxin Activity
The activity of lymphotoxin during purification was monitored by the modified cell-lytic assay of Spofford (30) which is incorporated herein by reference. Briefly, mouse L-929 fibroblast cells were grown in microtiter plates in the presence of mitomycin C. After 12-18 hours 0.125 ml of serially diluted sample to be assayed for lymphotoxin is added to each well. After 48 hours, the plates were washed and the lysis of the cells induced by lymphotoxin was detected as adhering to the plates by staining the plates with a 1% solution of crystal violet in methanol:water (1:4 v/v). The intensity of stain was observed both visually as well as spectrophotometrically at absorbance of 450 nm and 570 nm transmission using a Dynatech spectrophotometer. The cells plated in a microtiter well with culture medium alone were set at 0% lysis whereas those with 3M guanidine hydrochloride solution provided an end point for 100% lysis. One unit of lymphotoxin is defined as the amount required for 50% cell lysis out of 12,000 cells plated in each well.
E. Determination of Protein
The protein concentration throughout purification was determined by the method of Bradford (31) incorporated herein by reference. Briefly, 0.8 ml of the sample to be assayed was mixed with the 0.2 ml of the reagent concentrate solution from Bio-Rad Labs, Richmond, Calif. The reaction mixture was read at 595 nm in a Perkin Elmer Spectrophotometer Model 55B. This assay was linear between 2-20 μg of bovine serum albumin. During the final stages of purification, a rough estimate of protein was also made either from absorbance at 280 and 206 nm or by the silver staining of the SDS-polyacrylamide gels. This rough estimate was eventually confirmed by the nanomoles of various amino acid residues appearing at each cycle of the protein sequencing.
F. Utility and Administration
Administration of the lymphotoxin or its salts or derivatives or its active components can be via any of the accepted modes of administraton for agents which exhibit anti-tumor activity. These methods include oral, parenteral or topical administrations and otherwise systemic forms. Local or intravenous injection is preferred. The active components, for example, include the tryptic digest of lymphotoxin which has been shown to be active, and there is no reason it could not be used as the active ingredient in a pharmaceutical composition of this kind and for this purpose.
Depending on the intended mode of administration, the compositions used may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, pills, capsules, powders, liquids, suspensions or the like, preferably in unit dosage forms suitable for single administration of precise dosages. The compositions will include a conventional pharmaceutical carrier or excipient the lymphotoxin or the pharmaceutically acceptable salts or derivatives thereof and, in addition, may include other medicinal agents, pharmaceuticals, agents, carriers, adjuvants, etc. Such excipients may include other proteins, such as, for example, human serum albumin or plasma preparations.
The amount of active compound administered will of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. However, an effective dosage is in the range of 0.007-7 ng/kg/day, preferably 0.07-0.7 ng/kg/day. For solid compositions, conventional non-toxic solid carriers include, for example, pharmaceutical grades or mannitol, lactose starch, or magnesium stearate. Liquid pharmaceutically administerable compositions can, for example, be prepared by dissolving, dispersing, etc. the lymphotoxin as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a soluton or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975. The composition or formulation to be administered will, in any event, contain a quantity of the lymphotoxin in an amount effective to alleviate the symptoms of the subject being treated.
G. Purification of Lymphotoxin-Embodiment II
The human lymphoblastoid cell line RPMI-1788 was grown in 15L spinner flasks to a cell density of 4�105 cells per ml using a serum free culture medium as described previously above. Lymphotoxin was induced 10-20 fold over basal levels by the inclusion of 20 ng/ml of phorbol myristate acetate in the serum free RPMI-1640 medium. After 65 h of culture, the cells were harvested by filtration, and the lymphotoxin activity in the filtrate was absorbed to controlled pore glass beads (Electronucleonics) in a column (5 cm�20 cm), reequilibrated with 5 mM phosphate buffer (pH 7.4) and eluted with 50 percent ethylene glycol in 5 mM phosphate buffer (pH 7.4). Throughout the purification described in this Example G, 0.1 mM phenylmethyl sulfonyl fluoride (PMSF), a protease inhibitor, and 1 mM sodium azide, for inhibition of microbial growth, were included in all buffers. The eluate from glass beads contained 84,000 units of lymphotoxin/mg protein. This was followed by DEAE cellulose, Lentil Lectin Sepharose Chromatography, and preparative native PAGE as described above. Homogenity of the protein was determined by SDS-PAGE, reverse-phase HPLC on a LiChrosorb RP-18 column and by amino terminal sequencing, again substantially as set forth above.
The purification scheme of this example resulted in lymphotoxin yields of 20.1 percent based on the starting culture medium. The protein product was assayed at 40�15 million units of lymphotoxin/mg of protein. This lymphotoxin preparation contained greater than 95 percent by weight of a lymphotoxin species having N-Terminal leucine (FIG. 14) and an approximate molecular weight of 25,000 on SDS-PAGE. The theoreticl molecular weight of the protein component of the N-Terminal leucyl species is 18,664 daltons; the remaining approximately 6,500 daltons was attributed to a glycosyl side chain at Asn-62 (FIG. 14), and other O-linked sugar residues. The tissue culture supernatant contained putative trimers of this species (60,000 Da by TSK-HPLC or 64,000 Da by Sephadex G-100 chromatography). The isoelectric point was 5.8. Lymphotoxin lost about half of its control specific activity upon heating in aqueous solution for 60 min. at 60� C., and about 80 percent of its control specific activity at 80� C. for 5 min., as shown in the following table.
______________________________________TEMPERATURE (�C.) TIME (Min) BIOACTIVITY (Units)______________________________________Control -- 233,10060 5 259,70060 15 588,00060 30 334,10060 60 125,37080 5 33,190100 5 360______________________________________
Lymphotoxin (10 μg/ml) lost greater than about half of its specific activity at pH less than about 5 or greater than about 10 when stored for 24 hours at 4� C. in ammonium bicarbonate buffer; it was most stable at pH 8-9.
The remainder of the lymphotoxin mixture was the N-terminal histidyl species (FIG. 14) with a molecular weight of about 20,000 obtained in examples B-F above. The 25,000 Da species is full length mature lymphotoxin, while the 20,000 Da species is a fragment or "clip" believed to result from enzymatic hydrolysis during purification of the lymphotoxin. Both of these species exhibit the same specific activity, (within the variation imposed by the limits of the assay) and therefore are useful in the procedure of Example F.
Purified lymphotoxin exhibited tumor necrosis activity in vivo against Meth A sarcoma transplanted into (Balb/CXC57BC/b) F1 mice and cytotoxic or cytostatic activity in virto on human tumor lines ME-180 (cervix cancer), SK-Br3 and MCF-7 (breast cancer), SK-CO-1 (colon cancer), and SK-OV3 (ovary cancer). Lymphotoxin also activated human polymorphonuclear neutrophils in vitro to express increased antibody-dependent cellular cytotoxicity, superoxide anion production and phagocytic ability.
The use of a hydrophobic solid or substance to adsorb lymphotoxin is novel and a considerable improvement in ease of use compared to the concentration and dialysis step of Example B. Flow rates are superior to gelatinous adsorbents such as sepharose; the preparation of the adsorbent is facile and its cost is low. Other hydrophobic solids besides silicates (glass), such as polyolefins (polyvinyl or polystyrene beads), are suitable for use as well.
Elution of lymphotoxin from such absorbents is readily affected by polar solvents having hydrophobic domains or aprotic solvents, examples besides ethylene glycol being low molecular weight polyethylene glycol or lower nontoxic alkanols (propanol or butanol). Such solvents also are useful in stabilizing the lymphotoxin molecule if carried through the purification procedure and present during storage and/or lyophilization of the product.
The cleavage techniques used in sequencing all of this protein (except the designated residues in the C-terminal region) are described as follows:
Trypsin Cleavage. Lymphotoxin was digested with trypsin in 0.1M ammonium bicarbonate buffer pH 8.0 at an enzyme to substrate ratio of 1:20 for 24 h at 25� C. The reaction was stopped by lyophilization.
Cyanogen Bromide Cleavage. Peptide T4 (1.0 nmole) was dissolved in 70 percent formic acid and a small crystal of CNBr was added. The solution was flushed with nitrogen and then was allowed to stand for 17 h at 25� C., guanidine hydrochloride was added to 4M final concentration and the mixture was applied directly to an HPLC column for separation of the cleavage products. No guanidine hydrochloride was added to the peptide extracted from the sequencer cup prior to CNBr cleavage.
Acetic Acid Cleavage. A 3.0 nmole sample of intact lymphotoxin or its peptides was dissolved in 0.2 ml of 10 percent acetic acid, and incubated at 110� C. for 2 h in a nitrogen atmosphere and then applied directly to an HPLC column for separation of cleavage products.
Lysine-C Protease Cleavage. 1.1 nmole of tryptic peptide T4 was digested with Lysine-C peptidase in 0.1M ammonium bicarbonate at 37� C. for 24 h at an enzyme to substrate ratio of 1:20 (w/w). Two equal additions of enzyme were made at zero time and at 6 h. The reaction was stopped by lyophilization and the cleavage products were separated by HPLC.
Chymotrypsin Cleavage. Cyanogen bromide peptide CN4 was dissolved in 300 μl of 0.1M ammonium bicarbonate, pH 8.9, and incubated with chymotrypsin at an enzyme to substrate ratio of 1:12.5 (w/w) at 37� C. for 24 h. Two equal additions of enzyme were made at time zero and 6 h. The sample was then applied directly to an RP4 column (Synchrom, Linden, IN) for isolation of cleavage products.
Purifications of Peptide. Peptides were purified by reverse-phase HPLC using C4 Synchropak and Lichrosorb RP-C18 columns on a Spectra Physics SP-8000 chromatograph system as described previously (W. Kohr et al, 1982, Anal. Biochem. 122: 348-359). Peaks were detected at 210 nm and at 280 nm after elution with a linear gradient of 1 to 70 percent acetonitrile or 1-propanol in 0.1 percent trifluoroacetic acid at a flow rate of one ml per minute. Isolated peptides were dried under vacuum, and then processed for amino acid analysis and sequencing.
Amino Acid Analysis. Peptides were hydrolyzed for 24 h and the intact protein was hydrolyzed for 24, 48 and 72 h with constant boiling HCl in evacuated sealed tubes at 110� C. The hydrolysates were dried down under vacuum using a Savant Speed-Vac concentrator and analyzed on a Beckman 6300 amino acid analyzer equipped with ninhydrin detector using a 45 minute automated program.
Carboxypeptidase Digestion: 1.0 nmol of CNBr peptide CN4 was dissolved in 200 μl of pyridine acetate, pH 5.5, and digested with 0.5 μg carboxypeptidase P. Norleucine was included as an internal standad and aliquots were removed at 0, 3, 30 and 120 minutes. The samples were analyzed for free amino acids after removing the peptide on a C18 Sep-Pak column (Waters Associates, Milford, Ma.). The data was calculated as moles of amino acids released per mole of protein digested.
Amino Acid Sequence Analysis. Sequential Edman degradation was performed with Beckman sequencer models 890B and 890C equipped with cold traps. Polybrene (1.25 mg) was used as a carrier in the cup. Each PTH amino acid was identified as previously described (Kohr et al, Id.).
The nomenclature of peptide fragments of lymphotoxin is according to the cleavage procedure used, and they are numbered sequently from the NH2 -terminus. FIG. 13 summarizes the cleavage and overlap strategy used for determining the total amino acid sequence applicable in whole or part to both N-terminal species. Solid lines indicate the total length of a given peptide. Forward and backward arrows indicate amino acid sequence by Edman degradation and by carboxypeptidse digestion, respectively. Specific designations are summarized below:
T1 (1-15), T2 (16-19), T3 (2089), T3a (20-46), T3b (52-89), T4 (90-171), T4a (90-119) and T4b (120-171).
T3-KC1(20-39), T3-KC1b (29-39), T3-KC2 (40-46), T3-KC4 (52-84), T3-KC4a (52-74), T3-KC4b (75-84) and T3-KC5 (85-89).
T4-CN1 (90-120), T4 CN2 (121-133) and T4 CN3 (134-171).
CN1 (1-20), CN2 (21-120), CN3 (121-133) and CN4 (134-171).
T4-CN3-CY1 (134-139), T4,CN3-CY2 (140-159), T4-CN3-CY3 (160-165), T4-CN3-CY4 (166-169) and T4-CN3-CY5 (170-171).
The intact 25,000 Da lymphotoxin purified in accordance with this example was cleaved at three major sites when exposed to 5 percent (w/w) trypsin for 24 h. The three tryptic peptides T1, T3 and T4 which were fractionated by high performance liquid chromatography (HPLC) contain the entire sequence except for residues 16-19 (tryptic fragment T2), which was not recovered from HPLC. Tryptic fragments T3 and T4 described here correspond to peptide T1 and T2 respectively reported above. Edman degradation of the intact protein and tryptic fragments T1 and T3 established residues 1 through 41. In a later experiment, a tryptic digest of a larger amount of lymphotoxin (10 nmole) run on HPLC provided a minor fragment T3b which was not completely resolved from fragment T3. Peptides T3 and T3b were sequenced as a mixture through 35 cycles of Edman degradation and the amino acid sequence was extended up to residue 86. The tryptic fragment T3 was further digested with Lysine-C peptidase which generated peptides T3-KC1b, T3-KC4b, T3-KC5 and T3-KC4. This confirmed and extended the sequence to residue 89.
Tryptic fragment T4 of lymphotoxin was sequenced 40 cycles and thereafter the peptide was extracted from the sequencer with 70 percent formic acid. CNBr was added and the reaction was allowed to stand at room temperature for 40 h; then the mixture was reapplied to the sequencer. This resulted in two distinct sequences, which is consistant with the number of methionine residues present as indicated by amino acid composition. Although residues 32-40 of peptide T4 has already been sequenced, cleavage by CNBr at methionine residue 31 allowed these residues to be resequenced and confirmed in the mixture. The yield of valine at residue 32 was eightfold higher than originally seen during the initial sequencing of the intact T4 peptide. This is due to the partial blocking that occurs during the Edman degradation, allowing an accumulation of unsequenced peptide in the cup. In addition to reconfirming a portion of the sequence, cleavage by CNBr enabled 20 new residues to be identified. Further sequencing of the intact T4 peptide would have failed to reveal new sequence information due to the low signal (6 nmole) at cycle 40. Following CNBr cleavage, the yield was increased to 489 pmoles for tyrosine. This procedure should provide useful technique when a peptide is known to contain methionine residues and only a limited amount of material is available. Trypsin digestion of lymphotoxin also provided a fragment T4b which assisted in extending the sequence to residue 135. CNBr digestion of tryptic fragment T4 provided peptide T4-CN3 which extended the sequence to residue 155.
In order to obtain an overlap between peptides T3 and T4, intact lymphotoxin was cleaved with acetic acid and the fragments were separated by HPLC. Peptides HA2 and HA3 which could not be resolved by HPLC were sequenced as a mixture and provided an overlap between tryptic fragments T3 and T4. Carboxyl terminal sequencing of lymphotoxin was carried out on CNBr fragment CN4 and on the intact molecule using carboxypeptidase P. The results indicate that the amino acid sequence at the carboxyl terminus is -Phe-Ala-Leu.
H. References
The bibliography appended thereto, and referenced in the foregoing text is included to enhance the understanding of the invention. The subject matter so referenced is thereby incorporated herein.
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|Mar 10, 1992
|Sagami Chemical Research Center
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|Apr 7, 1995
|May 5, 1998
|Genentech, Inc.
|Cancer therapy using lymphotoxin
|Jun 7, 1995
|May 19, 1998
|Centocor, Inc.
|Peptide inhibitors of TNF containing predominantly D-amino acids
|May 16, 1994
|Jul 7, 1998
|Denki Kagaku Kogyo Kabushiki Kaisha
|Human lymphotoxin
|May 19, 1995
|Oct 20, 1998
|Genentech, Inc.
|Recombinant lymphotoxin cDNA and variants
|Mar 19, 1998
|Jan 23, 2001
|Genentech, Inc.
|Recombinant lymphotoxin
|Jan 21, 2005
|Feb 12, 2008
|Amgen Inc.
|LC/MS method of analyzing high molecular weight proteins
|Apr 12, 2011
|Genentech, Inc.
|Antibodies to lymphotoxin-alpha
|Jul 10, 2012
|Genentech, Inc.
|Antibodies to lymphotoxin-α
|Feb 11, 2011
|Sep 24, 2013
|Genetech, Inc.
|Antibodies to lymphotoxin-α
|Jul 6, 2012
|Feb 4, 2014
|Genentech, Inc.
|Antibodies to lymphotoxin-alpha
|Jan 21, 2005
|Jul 28, 2005
|Amgen Inc.
|LC/MS method of analyzing high molecular weight proteins
|Jun 23, 2011
|Genentech, Inc.
|Antibodies to lymphotoxin-alpha
|Aug 25, 2011
|Genentech, Inc.
|Antibodies to lymphotoxin-alpha
|Oct 7, 1999
|Apr 13, 2000
|Lt Technology Company, Llc.
|Methods for the treatment of cancer using cytokines in combination with low level doses of chemotherapy and/or radiotherapy
|Mar 2, 2010
|Sep 10, 2010
|Zymo Research Corporation
|Universal column
|U.S. Classification
|530/351, 435/70.2, 424/85.1, 530/418, 530/422, 530/412, 530/419, 530/417, 530/416, 530/415, 530/827, 435/70.4, 514/19.3
|International Classification
|Cooperative Classification
|European Classification
|Oct 4, 1993
|Fee payment
Year of fee payment: 4
|Sep 22, 1997
|Fee payment
Year of fee payment: 8
|Sep 26, 2001
|Fee payment
Year of fee payment: 12

subset: commoncrawl


text:
3 Reviews
Delicious. I used one fourth the oil and subbed more mustard seeds for the dry mustard (personal preference). It was a great dish!
My DH's favorite part of the meal. He likes his potatoes. We ended up running out of space on the stove and had to bake them instead of pan fry, and they still came out great. Thanks Dienia! Made for ZWT8 trip to India.
I really liked this, it was very spicy but the heat did not overwhelm the other flavors. I really like flavor that the mustard adds. The only thing I changed was using fresh cayenne pepper from my balcony. Thanks for sharing! Made for ZWT 8.


text:
T&T book place in final as Barbados flop again
GROS ISLET, St Lucia, CMC – Defending champions Trinidad and Tobago cruised into the final of the Caribbean Twenty20 after nonchalantly dismissing a timid challenge from Barbados here last night.
Set a woefully inadequate target of 102 for victory at the Beausejour Cricket Stadium, T&T wobbled briefly before getting home by five wickets and finishing their preliminary round campaign unbeaten.
Opener Lendl Simmons top scored with 36 from 23 balls to set up Trinidad's victory, staging a 45-run, first wicket stand off 32 balls with rookie Evin Lewis who stroked 24 in his first match of the tournament.
While T&T finished atop the standings with 21 points, the defeat Barbados left Barbados with only a slim chance of reaching the playoffs with their third straight loss. They lie fourth on eight points with one game left against Windward Islands on Thursday.
The Windwards and Guyana, who clash in the final game of the preliminaries on Friday, also have eight points but have two games remaining.
Winning the toss and batting, Barbados showed no serious attacking intent and meandered painfully to 101 for six off their 20 overs, with all-rounder Ashley Nurse punching an unbeaten 24 down the order while opener Justin Brathwaite (22) and Shane Dowrich (21) chipped in with useful scores.
Medium pacer Rayad Emrit claimed two for 14 from four frugal overs, with leg-spinner Samuel Badree also strangling the Barbadian batsmen with four mean overs that yielded just 15 runs.
A reshuffled batting order failed to improve Barbados' start as makeshift opener Carlos Brathwaite (2) edged Badree behind in the second over with the score on four for one.
Dowrich, promoted to number three, joined Justin Brathwaite to add 39 for the second wicket, with both batsmen looking to play positively. The left-handed Brathwaite hit three fours and a six over long-on off Sunil Narine in the off-spinner's first over while Dowrich counted two fours.
However, the partnership consumed 46 deliveries and built pressure, resulting in Brathwaite's run out in the ninth over looking for a non-existent single and with just eight runs added, Dowrich followed when he squirted Narine to Darren Bravo at point.
Captain Dwayne Smith dithered 14 balls over seven before providing Emrit with his first wicket, adjudged lbw in the 15th over at 59 for four while the left-handed Kyle Mayers skied to wicketkeeper Denesh Ramdin three balls later to fall for four, with five runs added.
With Barbados floundering, Nurse came to his side's aid, hitting two fours and a six in a brisk 19-ball knock, eking precious runs out at the death.
Barbados were always up against it, however, once Simmons and Lewis ensured there was no early loss of wickets. Simmons played with abandon and his 23-ball knock was laced with four fours and two sixes while the left-handed Lewis hit three fours off 27 balls.
Simmons punched left-arm spinner Sulieman Benn through cover and followed up with a six over long on in the second over that cost 17 runs, and also tore into speedster Fidel Edwards in the next over, which leaked 15 runs.
He was marching along merrily when he upper cut seamer Carlos Brathwaite in the sixth over for Mayers to take a well judged running catch at third man.
Darren Bravo, with unbeaten half-centuries in his three previous innings, stroked two typically elegant cover-driven boundaries but mis-timed a pull to mid-on off medium pacer Smith in the eighth over to fall for nine at 65 for two.
Smith then created some controversy when he ran out Dwayne Bravo (3) on delivery as the all-rounder backed up too far. Three balls later, Lewis failed to beat Edwards' throw from backward point and was also run out attempting a quick single.
Barbados scented a collapse but T&T never appeared flustered, and captain Ramdin (10 not out) and Kieron Pollard (15) calmly carried the score to within one of the target before being separated.
J Brathwaite run out 22
C Brathwaite c wkp Ramdin b Badree 2
+S Dowrich c DM Bravo b Narine 21
*D Smith lbw b Emrit 7
K Mayers c wkp Ramdin b Emrit 4
A Nurse not out 24
J Searles c Badree b DJ Bravo 11
S Brooks not out 5
Extras (lb3, w2) 5
TOTAL (6 wkts, 20 overs) 101
Did not bat: S Benn, T Best, F Edwards.
Fall of wickets: 1-4 (C Brathwaite, 1.2 overs), 2-43 (J Brathwaite, 9), 3-51 (Dowrich, 11.3), 4-59 (Smith, 14.1), 5-64 (Mayers, 14.4), 6-78 (Scantlebury-Searles, 17.2)
Bowling: Gabriel 3-0-13-0, Badree 4-0-15-1, Narine 4-0-22-1, DJ Bravo 4-0-30-1 (w2), Ottley 1-0-4-0, Emrit 4-0-14-2.
L Simmons c Mayers b C Brathwaite 36
E Lewis run out 24
DM Bravo c Best b Smith 9
DJ Bravo run out 3
*+D Ramdin not out 10
K Pollard c wkp Dowrich b Edwards 15
Y Ottley not out 0
Extras (w4, nb1) 5
TOTAL (5 wkts, 15.1 overs) 102
Did not bat: R Emrit, S Narine, S Badree, S Gabriel.
Fall of wickets: 1-45 (Simmons, 5.2 overs), 2-65 (DM Bravo, 8), 3-74 (DJ Bravo, 9.4), 4-77 (Lewis, 10.2), 5-100 (Pollard, 14.5)
Bowling: Edwards 4-0-27-1, Benn 1-0-17-0, C Brathwaite 2-0-14-1 (w1), Smith 3-0-14-1, Best 2-0-16-0 (w2, nb1), Nurse 1-0-10-0, Searles 2.1-1-4-0 (w1).
Result: T&T won by five wickets.
Points: T&T 4, Barbados 0.
Man-of-the-Match: Rayad Emrit.
Toss: Barbados.
Umpires: Patrick Gustard, Peter Nero; TV – Nigel Duguid.
Aussie Lynn upstages Munro as Amazon Warriors remain unbeaten
PORT OF SPAIN, Trinidad, CMC – Colin Munro smashed a sensational maiden Twenty20 hundred but it proved in vain as Guyana Amazon Warriors produced an audacious run chase to shock Trinbago Knight Riders by six wickets here Saturday night.
Will it be third time lucky for RCB?
The Indian Premier League will have a new champion either one of Royal Challengers Bangalore or Sunrisers Hyderabad, who will come up against each other in the Final of VIVO IPL 2016, will be crowned champions tonight.
Captain Smith takes Australia to victory with ton
(Reuters) – Steven Smith maintained his purple patch to hit a brilliant unbeaten 102 to secure Australia's three-wicket victory against England in a high-scoring tri-series match at Hobart on Friday.
Chanderpaul sparkles for Floodlights in win over Bel Air Rubis
Playing with a leather ball might be his forte but Guyana and West Indies middle order batsman Shivnarine Chanderpaul is also very adept at playing with a ball of a much softer variety.
Griffith hits maiden Inter County century
A maiden unbeaten century by Demerara opener Trevon Griffith left opponents Berbice facing a daunting 378 more runs for victory heading into today's fourth and final day in round one of the Guyana Cricket Board (GCB) Inter-County four-day tournament.
Development Team in six-wicket defeat of Essequibo
By Delvon Mc Ewan A dominant all-round performance by Steven Jacobs led his side to a convincing victory over Essequibo as the Guyana Cricket Board (GCB) Development Team got off to a winning start in their GCB Senior inter-county four-day campaign yesterday at the Demerara Cricket Club (DCC) ground.


text:
Hotels in Galway Bay
Search all hotels in Galway Bay
Last minute Galway deals
- Raheen Woods Hotelfrom $90
- Maldron Hotel Sandy Road Galwayfrom $135
- Claregalway Hotelfrom $105
- Imperial Hotelfrom $95
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- Most bookedGalway Bay Hotel
278 people looked at this hotel in the last hour
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"Very nice hotel. Free breakfast (although get there early), great bar, great location (a little far from bar area in galway but its quite). Very nice staff. Overall great stay and great experience. "
A Traveller, Jul 2016, US
259 people looked at this hotel in the last hour
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"Excellent place and perfect if you want to be close to city center but not directly in it. Clean, elegant and welcoming. Restaurant on property was a plus and food was fantastic. If I had to note a negative it would be that the beds and pillows were not very comfortable at all. I would definetly ..."
A Traveller, Jul 2016, US
193 people looked at this hotel in the last hour
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"Very good hotel just about 1.5 miles from Galway City The hotel is beautiful and located in a high end residential area. Very comfortable room and lovely staff."
A Traveller, Jun 2016, US
156 people looked at this hotel in the last hour
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"very nice hotel staff were friendly, very clean but not worth the money at all"
A Traveller, May 2016, CA
143 people looked at this hotel in the last hour
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"Beautiful upscale hotel with gorgeous lobby and in the best location: right on the famous Galway Promenade. We had a sea view room at the far end side of the building: no balcony, but you can still see and hear the ocean. The room was very quiet: this is a huge plus for a hotel which is a clear f..."
A Traveller, Jul 2016, US
129 people looked at this hotel in the last hour
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"Lovely hotel and the staff was exceptionally friendly and helpful. They were able to accommodate one extra person in our party and we enjoyed our stay. "
H, Jul 2016, US
63 people looked at this hotel in the last hour
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"Our apartment was spotlessly clean, only one bedroom but fully stocked with utensils and had an outside terrace with a view of the sea. The hosts were friendly and helpful without being intrusive. There is car-parking and it is a very short walk to the local neighbourhood which has a good selecti..."
A Traveller, Jul 2016, GB
29 people looked at this hotel in the last hour
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"We were very impressed from check in to check out! Beautiful flat with great decor and all we needed! Lovely balcony/terrace overlooking Galway Bay. Staff friendly, excellent location, gorgeous scenery, free parking, great reliable wifi! Will definitely return."
A Traveller, Jun 2016, US
21 people looked at this hotel in the last hour
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"WE attended a wedding so AH was very handy to attend both church and reception. The room was very quiet, clean and good value. Staff very pleasant and courteous. I didn't have breakfast. Didn't like the pillows."
A Traveller, May 2016, IE
21 people looked at this hotel in the last hour
Most recent review
"Pleasant staff, clean facilities. Great view of the bay."
A Traveller, Jul 2016, CA
Map of hotels in Galway Bay
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- National Aquarium of Galway
- Pearse Stadium
- Galway Bay Golf And Country Club
- Galway Shopping Center
- Silver Strand
- Galway Cathedral
- Oranmore Castle
- Galway Irish Crystal Heritage Centre
- Athenry Castle and Ennis Abbey
- Claddagh
- Galway City Museum
What you should know about Galway Bay
Are you looking for a cheap Galway Bay hotel, a 5 star Galway Bay hotel or a family friendly Galway Bay hotel? You just landed in the best site to find the best deals and offers on the most amazing accommodations for your stay.
When you search for hotels near Galway Bay with Hotels.com, you need to first check our online map and see the distance you will be from Galway Bay, Ireland.
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text:
Nationwide Health Information Network (NHIN)
The Nationwide Health Information Network (NHIN) is a set of standards, services and policies that enable secure health information exchange (HIE) over the Internet. The initiative is sponsored by the Office of the National Coordinator (ONC) for Health Information Technology (HIT), which began developing the NHIN in 2004.
Download Our Guide: mHealth Security Best Practices
There's no doubt that mobile health technology improves patient care, engagement, and physician productivity. But, the security risks are vast. Learn how to protect your mHealth devices and data in this exclusive e-guide.
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You also agree that your personal information may be transferred and processed in the United States, and that you have read and agree to the Terms of Use and the Privacy Policy.
The NHIN provides a common platform for nationwide health information exchange across diverse entities. NHIN standards and services will be used by federal agencies, health care providers, health information exchanges and consumers to securely exchange health information at both the national and local level. The NHIN is a fundamental requirement for achieving nationwide health information exchange, one of the primary goals of the Health Information Technology for Economic and Clinical Health (HITECH) Act.
A health IT policy committee called the NHIN Workgroup is responsible for providing a set of recommendations to the ONC to establish a policy and technical framework for the NHIN. Several components form the basis of the NHIN -- authentication and certification, vocabulary and message standards, delivery protocols, directories, a trust framework and security. A diverse group of organizations has been using the NHIN to demonstrate live health information exchange; this has resulted in the Nationwide Health Information Network Exchange.
Learn more:
NHIN standards come with some key benefits.
ONC turns its attention to NHIN governance.
Engineers of NHIN struggle to find common ground, tangle over policy.
The ONC's website explains more about the NHIN and provides an inventory of NHIN tools.
Continue Reading About Nationwide Health Information Network (NHIN)
Dig Deeper on Establishing interoperable electronic health systems
Find more PRO+ content and other member only offers, here.


text:
The FDA has added important new safety information to the labeling for zanamivir for inhalation (Relenza, Glaxo Wellcome Inc, Research Triangle Park, NC). Zanamivir was approved in July 1999 for treatment of uncomplicated acute illness due to influenza virus.
Original labeling for this drug included information about bronchospasm and decline in lung function and about lack of demonstrated efficacy in patients with underlying respiratory disease such as asthma or chronic obstructive pulmonary disease. Revised labeling adds a warning that zanamivir is not generally recommended for patients with underlying airways disease because of the risk of serious adverse events. Some deaths have been reported through the Adverse Events Reporting System among patients taking zanamivir, although the reasons have not been determined.
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Victory Nissan
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text: On 01/30/2009 03:44 AM, Tim Moore wrote: > Give the new version a try please. All better now. Thanks. VIRT = 460 peak, 440 steady state. Readers who aren't closely following the context should note that we are referring to the "next" branch of _simgear_. > Point of information: there are 2.6 million trees in the scene when starting at > KASE. That is an insane number! It is impressive that the ShaderGeometry > pseudo-instancing mechanism can handle this many models. Jeepers. So the bloat was only a couple hundred bytes per tree. It's impressive that it wasn't much, much worse, in terms of memory and/or CPU usage.


text:
I would like to make an html export of checkboxes lists with emacs with some sort of filter. For instance, I would like to export only the elements whose box is checked. How can this be achieved?
Add this hook:
Tick some checkboxes.
Create a list with one-line items in an .org file.
C-u C-c C-c on the first item of this list to add empty checkboxes to all items.
Put the list in an active region and do C-c C-e R.


text:
Role: Actor
Rishika Gandhi, a docile, middle class & Marathi girl from an orphanage in Kolhapur is a sort of prodigy in the field of computer ...
Pankaj Berry Filmography - lists out all of Pankaj Berry Movies including the story line as well as the cast and crew.
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text: This Robot Prefers Tecate to Bud LightMax Read7/30/11 2:10pmFiled to: BeerRobotsDarwinTecateBud LightAlcoholVideoTweetgTweetdTweetvFb15EditInvite manuallyPromoteDismissUndismissHideShare to KinjaToggle Conversation toolsGo to permalink Not all robots are bad! As you can see in the video above, Trossen Robotics' DARwIn-OP has okay taste in beer, even if it can't do anything except kick the can for now. Maybe it will even let you and your fellow humans keep some Tecate in your communal slave refrigerators, after the robot uprising. [via Popular Science]Reply15 repliesLeave a reply


text:
The official, revamped website for Fisker Automotive appears to be more of an homage to the company's focus on style rather than an accurate snapshot of the struggling automaker's actual troubles.
Congressional Hearing
3After outage, Fisker Automotive website hints at positive future
- Jon LeSage
- May 6, 2013
- 3
AddBob Lutz: GM deserved federal money, but Fisker, well, who knows?
- Jon LeSage
- Apr 29, 2013
- Add
Two days after a Congressional hearing about a US Department of Energy loan to Fisker Automotive, Bob Lutz wrote a column pondering what should be done to stop the financial hemorrhaging at the company that builds – well, used to build – the Karma extended-range sports car. When Lutz was vice chairman at General Motors, a federal bailout kept that company's factories open and helped bring the Chevrolet Volt to market. So, should the same sort of logic be applied here and Fisker given
29House committee releases Toyota execs' opening statements for tomorrow's hearing
- Jeremy Korzeniewski
- Feb 23, 2010
- 29
The Congressional hearings entitled "Response by Toyota and NHTSA to Incidents of Sudden Unintended Acceleration" may have started today, but the actual response by Toyoda – that's Akio Toyoda with a "D," head of the Japanese automaker – will come tomorrow.
63ABC News: Expert says electronic design flaw to blame in runaway Toyota models
- Frank Filipponio
- Feb 22, 2010
- 63
Southern Illinois University automotive technology professor David Gilbert and ABC News Brian Ross
- Volvo shoots for self-drivers by 2021
- Jeep spends $1 billion on factories
- Find Parts & Accessories for your vehicle!
- Obama rolls out new EV plan
- Infiniti dealers ranked best, Tesla worst
- Compare Volvo XC90 and Lincoln MKX


text:
In summary, traveling to Arizona on JetBlue was like travelling first class, almost. Coming home via American Airlines was like being luggage. But less comfortable.
It was a very long ride home, though shorter than flying westward. Eastbound, we had a tail wind that got us to Logan an hour early.
It seemed much longer. Not only were we starved — which I expected and for which I was prepared having brought a variety of semi-nutritious snack food (do salted peanuts and Fig Newtons count as nutritious?) and a large bottle of water. Bought at the airport because food for which you pay ten times the normal price is safe, while food bought in a grocery at normal prices will explode on impact.
I think we could have been dead in our seats on our return flight on American and only other passengers would notice. The flight attendants were in the back of the plane, playing cards. Having, as far as I can tell, a fine old time.
There was no entertainment. No television. The WiFi was not free. They wanted $12 (each) for 60 minutes (each) — and the Patriots-Kansas City game was on. Which was more than an hour. They had whacked us with a $25/per bag luggage fee … and wanted another $12 from each of us to use their WiFi? For an hour? It wasn't even unlimited WiFi. You had to watch one of their programs. Mean-spirited bastards run that airline.
As I told the attendant, "Your airline sucks." She agreed. They probably treat her like luggage too. Don't fly American Airlines.
We managed to get the score in real time on our smart phone. It somehow connected to the WiFi despite the firewall American Airlines erected. Let's hear it for Google. When the game ended, Garry and I had books on our Kindles, so we survived without WiFi …and those salted peanuts helped too.
When we got home, it was obvious no one had cleaned since we left. Talk about filthy. Wow. Two weeks of dog hair, sand, and odeur de canine. The Christmas tree is still up ("Don't worry about it, I'll take care of it and I'll put the wrapping paper away, I promise"). Right. Sure. Uh huh.
I swept three times before unpacking anything and washed the floors twice this morning, but it's going to take a lot more scrubbing before the place is habitable. I'm not a clean or neat freak, but I draw the line at genuine filth.
The kid's going to be 47 in May. You'd think he'd have a grip on "clean," wouldn't you? You'd be wrong.
We are home. No fresh food because we used it all before leaving. Today I threw away about five pounds of leftovers that had become lethal-looking science experiments during our absence. Garry made a very short trip to the grocery store. We needed half-and-half. That's not groceries. That's survival.
Tomorrow we'll deal with The Rest of the Story. Today, it's football and not being in transit. Sorry I missed your blogs today and yesterday. I'm surprised I'm awake and almost coherent.
While we were away, someone won the $1.5 billion PowerBall. Even after taxes and fees, it's still more money than I can imagine having. More money than Garry and I earned in our entire lives. Combined. Before taxes. More money than us and all our friends had or ever hope to have.
Someone won it. On a $1 lottery pick. Go figure.
NOTE: We have concluded that there is a secret interaction between hair gel and PowerZero so dangerous and explosive, it is banned from the air! That's the only sense I can make of it. Who knows what hidden dangers lie in your luggage?


text:
Prices, specials, features and availability subject to change.
Contact for details
Contact for details
Amenities abound at Saddle Brooke Apartments in Cockeysville, MD! Our one-, two-, and three-bedroom apartments for rent feature central air, an in-home washer and dryer, and separate dining room. Select apartments have renovated kitchens with rich maple cabinetry and brushed silver appliances, ceramic-tile baths, and a separate den or office. Bask in the sun at our swimming pool with deck, play a set of tennis, or relish quiet moments on your private balcony or patio. You'll also enjoy a playground, fitness center, and clubhouse. * Conveniently located near a commuter rail station, Saddle Brooke Apartments makes for a hassle-free commute into Baltimore, as well as to plentiful shopping, dining, and entertainment choices such as Wegmans, Hunt Valley Towne Centre, and Oregon Ridge Park. Food Lion of Cockeysville and Baltimore County Public Schools are nearby, too. Off-street parking is provided. Military clause honored. Cats and dogs are welcome.
Our lease terms are: 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months (Please note that lease terms may vary, are subject to change without notice, and are based on availability. Inquire with property staff for complete details).
Pet Policy: Cats and dogs are welcome. Maximum two pets per apartment. No weight limit. Monthly fees: $35 per pet. Refundable deposit: $300 (first pet only). Breed restrictions and other limitations apply.
Dogs Allowed. Cats Allowed. Pets Accepted. Call for service animal policy.
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There is a picture of my balcony on the home page . It was taken a few years ago. I doubt they would want to t Read Moreake another of my building. I take pride in the appearance of my balcony . It is a shame others in the building do not. Tom h
April 25, 2016
Hi Tom, Thank you for your feedback and letting me know about the appearance of the front of your building. I will be by today to take a look and see what we need to do to correct this issue. Please feel free to contact me in the office with any future concerns. Thanks, Caryn Ward, Property Manager, 410-667-0333
The facilities are very well-kept. Everything is clean and well-maintained. The in-unit washer+dryer is a defi Read Morenite plus. I appreciate the dual-layer security (main door requiring key access before getting to the individual apartments). There is adequate parking and I have never had to park outside on the main road, regardless of what time I got home. Staff is very friendly and helpful. Rent is a bit steep; however the cheaper apartments nearby are not all that much cheaper and the quality is not nearly as good, so the extra price is certainly worth it in my opinion.
April 25, 2016
Hello and thank you for your feedback! We're so glad you are happy here at Saddle Brooke and I'll pass your compliments along to the team, they'll be thrilled! Thanks, Caryn Ward, Property Manager
I like your location but I fear increases that will force me to leave. This place is very pricey. Don B
April 25, 2016
Hi Donald, Thank you so much for your feedback. Please feel free to contact me in the office and we can discuss your renewal. Thanks, Caryn Ward, Property Manager, 410-667-0333
I love out here .It's well maintained and secured.everything is fine with the apartment but only thing which b Read Moreothers me is that , there is always lots of noise when you are walking in your apartment which disturbs the people who live below you.
April 25, 2016
Thank you so much for your feedback! We are pleased to hear that you are enjoying your stay here at Saddle Brooke. Sincerely, Caryn Ward, Property Manager
Well kept community with excellent staff and service team.
April 25, 2016
Thank you for your feedback and we hope you continue to enjoy your home here at Saddle Brooke. Sincerely, Caryn Ward, Property Manager
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Saturday: 10:00am-5:00pm
Sunday: 11:00am-4:00pm
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text:
Tonight: The Strange Boys at the Hi-Dive
A rambunctious throwback to that time when the Kinks were first birthing some of the truest rock and roll in existence, the Strange Boys have been creating their own jangled jams for almost a decade. Guitarist and vocalist Ryan Sambol sounds slightly brattier than Ray Davies ever did (in a good way), as the poet and bandleader carries the quartet through rowdy three-minutes tracks.
With only two full-length albums and a handful of EPs under its belt, the band released Be Brave in 2010, slowly adding to the Texas group's rather small but intriguing repertoire of awesome rock songs. The Strange Boys come through Denver tonight with O'Death and Natural Child. This show is eighteen and up. Tickets are $12, doors open at 8 p.m., and the show begins at 9.
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7 S. Broadway
Denver, CO 80209
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