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German Bundesliga Opta Facts 18-20 Mar
FC Schalke 04 - Borussia Mönchengladbach
o Borussia have won five of the last six meetings between the two sides in all competitions. They have beaten Schalke twice already this term (3-1 in the league, 2-0 in the DFB Cup).
o Gladbach have won on two of their last three competitive visits to Gelsenkirchen, ending a run of 22 barren years (D6, L12 - 1992-2014).
o Schalke missed the chance to leap into third spot after going down 2-0 in Berlin. They are currently fifth - one place behind Borussia. Should S04 win this weekend they will overtake their rivals and climb back into the Champions League qualification places.
o Andre Breitenreiter's boys have won four of their last six at home (D1, L1). They have scored in each of their last nine in front of their own fans - their longest run for over three years (17, September 2012-August 2013).
o Five of Gladbach's last eight games have been on the road, where they are winless since the end of October (4-1 in Berlin, D2, L4 since then) - this is their longest wait for an away win in over three years (6, Nov 2012 - Feb 2013).
o S04 are unbeaten in 13 Bundesliga home matches to take place on a Friday (W12, D1). Their last defeat in this slot dates back to September 2009 (2-1 vs. Wolfsburg).
o Raffael has scored seven goals since the turn of the year - only Robert Lewandowski and Claudio Pizarro have bagged more than him in 2016 (nine each).
o Andre Schubert has beaten a S04 team on three occasions this term (twice with the first team and a 5-2 triumph when the clubs' reserves faced off in August).
o Andre Breitenreiter has never beaten Gladbach as a manager (L4 - two with SCP, two with S04).
o Roman Neustädter (2009-2012 - 59 Bundesliga appearances, one goal) and Raffael (2012/13 - 16 Bundesliga appearances, two goals) used to play for Schalke.
o S04 will be without Junior Caicara this weekend (after five yellow cards). Thorgan Hazard could make his 50th Bundesliga appearance in the game (all for Borussia).
1. FC Köln - FC Bayern München
o Bayern have won their last five league games against Köln - their longest victorious sequence in the history of this fixture. Their 4-0 triumph in the reverse fixture was their 100th win in the Bundesliga.
o The Bavarians have won all seven of their league games immediately following a Champions League fixture this term, scoring 22 goals and conceding just one in the process.
o Köln claimed their second win of 2016 last time out (2-0 vs. H96), having taken one point from the previous four. It was their first clean sheet since November 2015 (0-0 vs. SVD).
o FCB have kept 16 clean sheets in their 26 league game this season - a tally which has only been bettered on three occasions in the history of the Bundesliga (17 - Schalke in 1971/72, Bayern themselves in 2012/13 and 2014/15).
o The Billy Goats have won two of their last 10 home games, losing four of the last six on their own patch (W2). They have claimed more away points (17) than home ones this term (16).
o Köln have won one of their last 27 Bundesliga games against the side topping the pile (1-0 vs. Schalke in September 2008, otherwise D7, L19), including defeats in each of the last seven.
o Bayern are unbeaten in nine competitive away games (W6, D3). They kept six clean sheets in that timeframe and haven't conceded a goal on the road for 194 minutes.
o Franck Ribery is on the cusp of his 200th Bundesliga appearance. He has had a hand in 145 goals (69 goals, 76 assists) in his 199 to date (a record for a Frenchman).
o Leonardo Bittencourt has scored the Billy Goats' last three league goals and has been directly involved in more than any other Köln player in 2016 (4 - 3 goals, 1 assist). He had a hand in just two before Christmas.
o Robert Lewandowski has won on all seven of his Bundesliga appearances against the Billy Goats - only Nelson Valdez can top that tally (8/8). He has 24 goals to his name this term (equalling his personal best) - more than any other top-flight player.
o Lewandowski and Müller have scored 43 goals between them this term - the most potent duo after 26 games in the history of the Bundesliga. 14 sides competing in the division this season have scored fewer goals than the Bayern duo.
Hamburger SV - TSG 1899 Hoffenheim
o HSV have lost just one of their seven top-flight home games against TSG (W4, D2) but are without a win in two (D1, L1). Hoffenheim's only league win there came in August 2013 (5-1).
o Julian Nagelsmann's side have scored 26 Bundesliga goals against Hamburger SV (same as Wolfsburg) - they've only netted more against Hannover (32).
o Hoffenheim have only been ahead of Hamburger SV in the table once this season (after MD1). They have, however, performed better than the northern side so far in 2016.
o Only Dortmund (16) and Bayern (13) have claimed more points than TSG since Julian Nagelsmann took the job (10). He has claimed an average of 1.67 points-per-game - the highest tally among managers who have taken charge of Hoffenheim in the Bundesliga.
o Bruno Labbadia's men are unbeaten in four at home, taking eight points in the process - as many as they accumulated from their first nine of the campaign on their own patch combined. However, they are yet to win consecutive home matches this term.
o Hoffenheim are without a win in nine on their road (D3, L6). This is their longest run without an away victory in their Bundesliga history.
o Hamburger SV haven't won any of their last five home games against sides below them in the table (D1, L4), including defeats in each of the most recent three.
o Nicolai Müller, who has been directly involved in HSV's last four Bundesliga goals, will miss the game through suspension. The Hamburg side have lost all three of the competitive games that he has sat out in 2015/16.
o Andrej Kramaric has netted TSG's last two Bundesliga goals. He has had a hand in four strikes in 2016 - more than any other Hoffenheim player (3 goals, 1 assist).
o Aaron Hunt could make his 250th Bundesliga appearance this weekend. His 249 appearances to date have come for a combination of Bremen, Wolfsburg and HSV (48 goals, 34 assists). He has an excellent record against Hoffenheim (W6, D4, L1).
o HSV have scored just two goals in their last four league matches against TSG combined, both of which were netted by Pierre-Michel Lasogga. The striker is on a 567-minute drought though - his last goal came in November 2015's 3-1 win over Dortmund.
o BSC won the reverse fixture (1-0) and are unbeaten in five against FCI (W2, D3).
o Hertha have 45 points to their name - their best tally at this point of a season 2008/09 (49, same as 1969/70 -this is their club record for the 26-game mark).
o The Berlin side have finished in the top four on each occasion they've had at least 45 points to their name after 26 matches (5 previous occasions).
o Ingolstadt are on a club-record five match unbeaten run on the Bundesliga. They now have 33 points to their name and are nine clear of the play-off spot.
o Hertha are unbeaten in eight home league matches (W5, D3) - their longest such run since 2008/09 (10).
o Only Bayern (13) and Dortmund (25) have conceded fewer league goals than Hertha this season (26). Only FCB (16) have kept more clean sheets than Dardai's side (12).
o Since Pal Dardai took over in February 2015, BSC have kept 19 clean sheets in 41 Bundesliga matches - only FCB can top this figure (25).
o FCI haven't won on the road since matchday five (1-0 in Bremen). They have taken just five away points from the last 30 available, scoring four goals in the process.
o Matthew Leckie registered the highest speed of the season last weekend vs. VfB (35.18 km/h), taking the accolade from FCB's Kingsley Coman (35.06 km/h).
o Ten of Ingolstadt's last 12 Bundesliga goals have come from set pieces. They netted two goals from free kicks against Stuttgart last time out.
o FCI's Pascal Groß has completed his suspension so could return this weekend but colleague Benjamin Hübner will miss the match after picking up his fifth yellow of the season last time out.
SV Werder Bremen - 1. FSV Mainz 05
o This will be the 20th Bundesliga meeting between SVW and M05. Werder have the upper hand to date (W10, D4, L5) and are unbeaten in three against Mainz (W2, D1).
o However, Mainz have won three of the last five competitive meetings at the Weserstadion (D1, L1), scoring nine (1.8 per game) and conceding just four (0.8 per game) in the process.
o Mainz's heaviest Bundesliga defeat to date was administered by Werder (6-1, October 2006). Jürgen Klopp was in charge then. Current Bremen skipper Clemens Fritz featured.
o After picking up seven points from their previous three matches, Bremen's 5-0 loss to Bayern means they are now just three points clear of the relegation play-off spot.
o Despite losing to BVB last weekend (2-0), Mainz remain sixth, two points off the top four. Their tally of 40 points from 26 matches has only been higher on one occasion (43, 2010/11).
o Werder are unbeaten in five at home (W1, D4) - but have taken just 10 out of a possible 36 points on their own patch this term, their lowest ever return at this stage of a season!
o Mainz have picked up more points from their 13 away matches this season (17) than they did in the whole of the 2014/15 campaign (16), despite losing four of the last six (W2).
o SVW are on a run of 29 Bundesliga games without a clean sheet - the longest sequence in the league and a negative club record. They have conceded more goals than any other side this season (53).
o Clemens Fritz and Zlatko Junuzovic are once again available for selection after serving their bans. They were both fined by the DFB but their suspensions were not extended.
o Giulio Donati will miss the game in Bremen. He is still suspended having seen red against SVD on matchday 25.
VfL Wolfsburg - SV Darmstadt 98
o Saturday's game will be the fifth competitive meeting between VfL and SVD. The Wolves edge the head-to-head record with two victories (including a 1-0 win in Darmstadt in the reverse fixture) as well as triumphing on penalties in the 2014/15 DFB Cup first round (L1).
o Wolfsburg have won three of their last four games at the Volkswagen Arena (L1) and have taken 27 of their 37 points at home this season (73%). Proportionally speaking, only Mönchengladbach have taken more (74%).
o Only two newly-promoted sides have taken more points from the first 13 away fixtures than Darmstadt (19): Bayern in 1965/66 and Kaiserslautern in 1997/98 (both 23). The Lilies have collected 70% of their points on the road this season - proportionally speaking, a league high.
o VfL are unbeaten in their last nine Bundesliga games against newly-promoted sides (W5, D4) and haven't conceded a single goal in their three such games to date this term.
o Darmstadt are without a win in six and have picked up the fewest points in this time frame (3, the same as Frankfurt und Hannover). Only Frankfurt (8 games) are currently waiting longer for a victory. It is the Lilies' longest winless streak of the season.
o The Hesse-based club have dropped 22 points from leading positions this season - a league high (same as TSG). However, the Lilies have also already taken the lead on 11 occasions away from home this campaign - a feat no other team can match.
o The Wolves could claim their 250th Bundesliga victory this weekend; Darmstadt are on the verge of their 50th top-flight defeat.
o Only Stuttgart (17) have scored more goals from set pieces than the Lilies this season (16). However, Wolfsburg have conceded just once from a dead ball in 2015/16 - a league best.
o The Wolves have played almost twice as many successful passes (11,954) as Darmstadt (6,510) this season. The Lilies (56%) have the worst pass rate in the league, while Wolfsburg (84%) boast the second-best after Bayern (89%).
o Sandro Wagner leads the league in terms of headed goals (6), while the entire Wolfsburg squad have managed just four headed goals. The attacker has scored five times in his last six away appearances.
Eintracht Frankfurt - Hannover 96
o Frankfurt lie in 16th, Hannover 18th - the table also looked like this when they met on the final weekend of the 1988/89 season. The sides drew 1-1, meaning H96 were relegated - SGE finished 16th and stayed up thanks to relegation play-off success.
o 96 have beaten Eintracht on a club-record 19 occasions in the Bundesliga (also 19 vs. KOE and VfB). They have score more away goals against Frankfurt (33) than any other top-flight side.
o The Lower Saxony club have lost 11 of their last 12 league fixtures (W1).
o Eintracht Frankfurt are without a win in eight (D4, L4) - the longest ongoing run without a win in the division. SGE's last victory was a 3-2 triumph over Wolfsburg on matchday 18.
o Frankfurt (7 points) and Hannover (3) prop up the 2016 form table. Last week Schaaf's side lost 2-0 to Köln, who at the time were the second-weakest team since the winter break.
o Hannover have 17 points to their name after 26 games. All 26 of the sides who have had 19 points or fewer at this stage of a Bundesliga campaign have been relegated!
o With just 24 points after 26 games, this is SGE's third-worst Bundesliga season in the club's history. Back in 1998/99 and 1983/84 they had just 23 to their name - however, they avoided relegation on both occasions, coming through the playoffs in 1984.
o H96 are seven points adrift of the relegation play-off spot - no team has recovered from being so far behind at this late stage of a campaign since the introduction of the three-points-per-win rule in 1995/96. SGE, however, overcame a six-point gap after matchday 30 in 1998/99.
o Niko Kovac is the first SGE boss since Felix Magath in 1999/2000 (beaten 1-0 in Unterhaching) to lose his first competitive game in charge.
o Thomas Schaaf takes charge of his 523rd Bundesliga game this weekend at former club Frankfurt and overtakes Udo Lattek's tally. Only Otto Rehhagel (836), Jupp Heynckes (642) and Erich Ribbeck (568) have been in the dugout for more matches. Niko Kovac made his top-flight managerial debut last weekend.
VfB Stuttgart - Bayer 04 Leverkusen
o B04 are unbeaten in 11 games against Stuttgart (W8, D3). Stuttgart's last win in the fixture dates back to April 2010 (2-1).
o Kramny's side led 2-0 and 3-1 in Leverkusen but still lost the reverse fixture 4-3 (MD10) - Admir Mehmedi grabbed the winner but hasn't scored a league goal since.
o VfB have won four of their last five home matches (L1 - 2-1 vs. H96 on MD23), meaning they've equalled their tally of home points from the whole of last term (19).
o Bayer 04 beat HSV 1-0 last weekend to end a run of four Bundesliga games without a win. They are now just three points behind the Champions League spots.
o Roger Schmidt's side have lost just one of their last five away league matches (3-1 in Mainz). They overturned a three-goal deficit for the first time in the Bundesliga in their last away game (3-3 in Augsburg).
o Only BVB and FCB have claimed more points than VfB in 2016. They have taken more from their nine games since Christmas (17) than they did in their 17 before the winter break (15), scoring a league-high 21 goals in the process.
o Bayer 04 have benefited from five own goals this campaign - a record - including three in their last three (via HSV's Ekdal, SVW's Djilobodji and FCA's Verhaegh).
o Filip Kostic has had a hand in nine goals since Jürgen Kramny took over (5 goals, 4 assists) - a personal best. He wasn't involved in any under Alexander Zorniger.
o Stefan Kießling has scored more goals against Stuttgart than any other Bundesliga side (14 in 20 appearances).
o VfB sporting director Robin Dutt took charge of Bayer Leverkusen for 37 competitive fixtures (W14, D8, L15) between July 2011 and April 2012.
FC Augsburg - Borussia Dortmund
o Borussia Dortmund's competitive record against Augsburg is impressive (W7, D2, L1). That defeat came in 2015/15 when FCA won 1-0 in Dortmund.
o Three of Augsburg's four heaviest Bundesliga defeats have come at the hands of BVB (the other vs. Bayern). The 5-1 loss at Dortmund in the reverse fixture saw them concede five in the top flight for the first time.
o BVB have 61 points from 26 games - a joint club record (also 61 in 2010/11).
o FCA have taken eight points in 2016 - their lowest ever tally at this point of a calendar year (previously min. 11). Only SGE (7) and H96 (3) have fewer this year.
o Borussia have kept clean sheets in seven of their last eight league games and already have one more (11) than the whole of last season. They've conceded just two goals in 2016 - equaling Bayern's Bundesliga record from 2002/03.
o Markus Weinzierl's side have won just one of their last ten home Bundesliga games (2-1 vs. S04 on M16, otherwise D5, L4).
o Tuchel's men have won 10 of their 11 Bundesliga games immediately following a Europa League fixture this term (L1 - 5-1 vs. Bayern just days after drawing 1-1 with PAOK).
o Pierre-Emerick Aubameyang is on a run of three league games without a goal for the first time since November/December 2014. However, he has scored more goals against FCA than any other Bundesliga side (6 in 5 appearances).
o Marco Reus ended a five-game drought against M05 and has now reached 10 goals in five of the last six campaigns (last season being the only exception). He could make his 200th Bundesliga appearance this weekend (83 goals, 50 assists to date).
o Thomas Tuchel was a member of FCA's youth set-up (1988-1992). He coached their U19 and U23 sides (2005-2008) before moving to Mainz (summer 2008).


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112 E Orleans, Otsego MI, 49078
Sold Price:
3 beds , 1 Full/1 Half baths
1,880 sqft
Status: Closed
Last Updated: 11/19/2017 11:43 PM
Description for 112 E Orleans Otsego, MI 49078
Beautiful Victorian charmer! Open staircase in living room with 2 stained glass windows, fireplace and hardwood floors. Pillars and Victorian gingerbread scroll work between dining & living rooms. French doors to den, which has a wood-burning stove. Main floor bath and laundry with tin ceiling. Oak cabinetry & new counter tops in kitchen as well as a unique built in wine rack. Main floor bedroom off kitchen. Upstairs has 2 bedrooms with large closets and bathroom with claw foot tub, skylight & ceramic floors. Extra 500 Sq. ft. of space in partially finished basement; large windows make it seem very roomy. There is also a 3 season porch on the back with a slider to the patio & deck. New carpet & fresh paint. Seller is licensed Realtor in the state of Michigan.
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Sold Date:12/01/2015
Otsego City
1 Full/1 Half
1,880 (approx)
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Dishwasher, Garbage Disposal, Range, Refrigerator
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M89 into Otsego, South on Farmer to Orleans.
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This article describes the possible role of the endothelial cell-surface coat, containing proteoglycans (PGs) with connected glycosaminoglycans (GAGs), in maintaining glomerular permselectivity. Primary human glomerular endothelial cells (HGEC) in culture were treated with the nephrosis-inducing agent puromycin aminonucleoside (PAN). Analysis was made by TaqMan real-time PCR, Western blot analysis, and by metabolic labeling with [35S]sulfate. The HGECs express several PGs: syndecan, versican, glypican, perlecan, decorin, and biglycan, which may contribute to the glomerular charge barrier. PAN treatment downregulated both the protein expression (by 25%) and the mRNA expression (by 37 ± 6%, P < 0.001, n = 8) of versican compared with control. Transferases important for chondroitin and heparan sulfate biosynthesis were also significantly downregulated by PAN, resulting in less sulfate groups, shorter GAG chains, and reduced PG net-negative charge. Moreover, analysis of the cell media after PAN treatment revealed a reduced content of [35S]sulfate-labeled PGs (40% of control). We conclude that PAN may cause proteinuria by affecting the endothelial cell-surface layer and not only by disrupting the foot process arrangement of the podocytes. Thus the endothelium may be a more important component of the glomerular barrier than hitherto acknowledged.
- glomerular basal membrane
- glycosaminoglycan
- heparan sulfate
- chondroitin sulfate
- glycocalyx
- nephrotic syndrome
the permselectivity of the glomerular filtration barrier is pivotal for the electrolyte and fluid balance in the body. The barrier has both charge- and size-selective properties (35, 36) but the question is where the different components of the barrier are located. Considerable focus has been directed to the glomerular basal membrane (GBM), which is built up by a three-dimensional network of mainly collagen, laminin, nidogen, and proteoglycans (PGs). The GBM has been said to be responsible for both size and charge selectivity (8, 24, 31, 48), but the charge-selective component has been questioned (4). Several novel proteins important for permselectivity have recently been discovered in the podocyte slit diaphragm (15, 43, 48). This has led to the podocyte now being the favorite candidate for constituting the filtration barrier. The endothelial cells of the glomerulus (GEC) are highly fenestrated and have thus not been considered to have prominent restrictive properties, apart from a few observations in the literature (41). However, endothelial cells in general are covered with a cell-surface coat containing PGs (10, 14, 28, 32, 52). PGs are constituted of a core-protein to which one or more polyanionic galactose- or glucosaminoglycan (GAG) chains are attached, the latter is heparan sulfate and the former is chondroitin sulfate. Plasma proteins tend to bind to the GAG chains and together they construct an extracellular matrix with both size- and charge-selective properties. In particular, the plasma protein orosomucoid seems to be vital for maintaining normal capillary permeability (7, 16) possibly via effects on the endothelial structure. It also has been demonstrated that orosomucoid is produced by endothelial cells per se (46). Moreover, using a novel fixation method, Rostgaard and Qvortrup (40) visualized an almost 300-nm-thick filamentous surface coating that appeared to be present over both fenestral and interfenestral surfaces of the glomerular endothelia. In addition, similar observations have been made in other organs (52).
A recent study from our group showed that enzymatic cleavage of hyaluronic acid and chondroitin sulfate resulted in a decreased amount of negatively charged fibers in the barrier and a significant increase of fractional clearance for albumin in mice. When studying the barrier by electron microscopy after the enzyme treatment, no damage to the glomerular cell structures was seen, which is compatible with the hypothesis that the endothelial cell-surface coat was degraded by the enzymes (22). Also, we showed that bovine glomerular endothelial cells produce negatively charged PGs containing heparan, chondroitin, and dermatan sulfate and that their synthesis is significantly decreased when exposed to puromycin amino nucleoside (PAN; a nephrosis-inducing agent) (45). We and others demonstrated that the synthesis of sulfate-containing GAGs by bovine GEC could be induced after stimulation with IL-1β (45) or transforming growth factor (TGF)-β1 (25). This upregulation could be a part of the cell defense by maintaining glomerular permselective properties (24) and may also be of significance in glomerular disorders characterized by matrix expansion. The information of the composition of the endothelial surface coat is still limited. Thus the production of PGs in the glomerular barrier has mainly been studied with metabolic sulfate labeling and enzyme degradation of the GAG chains. The method can reveal what class of GAG the cells produce but it is not possible to tell exactly what sort of PGs they express. It is of interest to investigate which kind of core proteins that are expressed in this system. Different classes of PGs are found extracellularly, transmembranely intercalated in cell membranes or via GPI anchors, and also intracellularly in storage granules (Table 1). In the present study, we wanted to explore the details of the PG core proteins produced by the human GECs. We were also interested in the effects of PAN on the PG core proteins and on key transferases involved in the synthesis of PGs, galactose and GAGs.
Medium and Reagents
Glomerular endothelial cells were cultured in culture flasks (Costar, Cambridge, MA) in a humidified, 5% CO2 atmosphere at 37°C. The culture flasks were coated with attachment-factor (gelatin) from Cascade Biologics (Portland, OR). The medium used was RPMI 1640 with supplements as follows: penicillin, streptomycin, and amphotericin B (PSA solution, 1 ml) from Cascade Biologics, 10% human serum, bovine brain extract (BBE), 700 μl from stock solution (BioWhittaker, GTF, Västra Frölunda, Sweden) and 2 μg/ml heparin (Lövens, Malmö, Sweden). Collagenase type IV and V (Sigma, St. Louis, MO) were used to digest the whole glomeruli. Stainless steel sieves (Endecotts, London, UK) in the size of 180, 106, and 75 μm were used to extract the glomeruli.
Isolation of Glomeruli
Studies were performed on adult human kidney tissue removed by nephrectomy (due to stenosis or tumors). The study was approved by the local ethical committee at Göteborg University (R110–98). Glomeruli were isolated from healthy tissue under sterile conditions by a gradual sieving procedure done according to the protocol by Ballermann (2). The collected glomeruli were treated with 1 mg/ml collagenase type IV and V (Sigma). Single cells and cell clusters were plated onto gelatin-coated petri dishes. When the endothelial cell colonies had reached a size of ∼300 cells, they were transferred to 24-well plates by the use of cloning cylinders.
Outgrowths from collagenase-treated glomerular remnants and subcultures were examined with a phase-contrast microscope.
Acetylated low-density lipoprotein uptake.
The cells were seeded onto chamber-slides (Falcon, Becton Dickinson, Meylan Cedex, France) and allowed to grow under normal conditions until they reached 50% confluence. The medium was removed and Dil-Ac-LDL (10 μg/ml; Biomedical Technologies, Stroughton, MD) was added to each well. After incubation for 3 h at 37°C, the cells were washed with 0.01 M PBS, pH 7.4. After fixation with 4% paraformaldehyde for 15 min, the cells were rinsed, mounted, and examined by fluorescence microscopy at 570 nm.
Ulex europaeus agglutinin I staining.
The cells were seeded onto chamber-slides. When they reached 30–50% confluence, they were fixed with 4% paraformaldehyde for 15 min at room temperature (RT). After being rinsed with 0.01 M PBS, the cells were incubated with 2% BSA for 30 min at RT. Rhodamine Ulex europeaus agglutinin I (Vector Laboratories) diluted 1:200 in the blocking solution was applied and the cells were incubated for 30 min at RT. The cells were rinsed, mounted, and examined by fluorescence microscopy at 570 nm. Human dermal fibroblasts were used as negative control cells.
Expression of endothelin-1.
The RNA from the cells were analyzed by PCR for the specific sequence for endothelin-1. Sense primer was 5′-TGGACATCATTTGGGTCAACA-3′ and antisense primer was 5′-TCTCTTGGACCTAGGCTTCC-3′.
Cell Culture and Stimulation with PAN
Human glomerular endothelial cells (passage 4) originally derived from the healthy poles of two kidneys (one male patient born -53 and one female born -48) were seeded into eight 25-cm2 flasks or two six-well plates and allowed to grow to 70% confluence. Cells in four flasks and one six-well plate were stimulated with PAN (Sigma) at a concentration of 1 μM. As control we used four flasks and one six-well plate of nonstimulated cells grown under normal conditions (i.e., "starved" in standard media with 2% human serum and half the amount of BBE). Stimulation was performed for 48 h at 37°C in a humidified, 5% CO2 atmosphere. After the experiment, cells from two flasks and the six wells for each group were trypsinized and used to prepare total RNA according to the RNeasy Mini protocol (Qiagen, VWR, Stockholm, Sweden). The remaining cells (2 flasks per group) were washed with PBS and then lysed by a buffer containing 1% SDS, Complete mini protease inhibitor (Roche), and 10 mM Tris, pH 7.4.
Real-Time PCR
Reverse transcription of 1 μg of RNA from both PAN and control groups was performed in avian myeloblastosis virus reverse transcriptase (AMV RT) buffer with AMV RT, dNTP (deoxy-CTP, -GTP, -TTP, and -ATP), random hexamers, and RNase inhibitor to a final volume of 80 μl (all reagents from Roche). The reverse transcription reaction was carried out at 25°C for 5 min, 42°C for 50 min, and 70°C for 5 min. The mRNA level of each PG was quantified by real-time PCR on the ABI Prism 7700 Sequence Detection system (TaqMan, Applied Biosystems ABI, Foster City, CA). Briefly, this assay uses the 5′-nuclease activity of Taq polymerase to cleave a reporter dye from a nonextendable hybridization probe during the extension phase of the PCR reaction. This ends the activity of a quencher dye and the reporter dye starts to emit fluorescence which increases in each cycle proportional to the rate of probe cleavage and is monitored in real-time. The threshold cycle (CT) is defined as the fractional cycle number at which the reporter fluorescence reaches a certain level (i.e., usually 10 times the standard deviation of the baseline). Oligonucleotide primers and TaqMan probes were designed by using Primer Express version 1.5 (ABI), based on the GenBank database for syndecan, glypican, versican, decorin, biglycan, and perlecan (Table 2). Primers and probes for the enzymes for chondroitin sulfate biosynthesis: chondroitin 4-O-sulfotransferase (ST), accession no. NM_018413, chondroitin 6-O-ST (NM_004273), chondroitin synthase 1 (NM_014918) and for heparan sulfate biosynthesis: NDST (NM_001543), EXT-2 (NM_000127), HST-2 (NM_0012262), HS-3OST1 (NM_014918), HST-6 (NM_004807) were ordered as kits (Assays-on-Demand, ABI). PCR was carried out in a 30-μl reaction mix containing a 100-ng sample of cDNA, probe, primers, and 1× TaqMan universal PCR master mix (ABI; containing MgCl2, dUTP, dATP, dCTP, dGTP, Taq Gold polymerase, and AmpEraseUNG). The AmpEraseUNG was activated before the denaturation step by heating for 2 min at 50°C. Samples were denatured at 95°C for 10 min and then subjected to 50 cycles of two-step PCR (15 s at 95°C, 1 min at 60°C). All samples were amplified simultaneously in triplicate in one assay run. Standard curves were computed for all genes from a series of twofold serial template dilutions from 3,125 through 200 ng (7 concentrations). For each sample, the amount of each target gene and endogenous control was determined from the corresponding standard curves. Log input amount was calculated according to the formula: 10 − [(cell containing CT value) − b]/m; where b = y-intercept of standard curve line, and m = slope of standard curve line. Validation of amplification efficiency was made for every primer/probe set and was calculated for each run (Table 3). As endogenous control, we used β-actin (predesigned assay reagent applied by ABI, external run) to correct for potential variation in RNA loading or efficiency of the amplification reaction. The endogenous control gene was chosen after running eleven housekeeping genes to find the most stable for this material (human endogenous control plate, ABI). As negative control, we used mRNA samples without performing reverse transcription.
Western Blot Analysis
The protein concentration was determined by using the BCA-Protein Assay Reagent kit (Pierce, Rockford, IL). Half of the amounts from the protein samples were digested with enzymes: chondroitinase ABC (0.1 U/sample, Sigma), heparitinase I (10 U/sample, Sigma) and heparitinase III (1 U/sample, Sigma) in ABC-buffer (100 mM Tris-HAc, 10 mM EDTA, 3 mM CaCl2, pH 7.3) at 37°C for 7 h. Samples (∼10 μg of each) were separated on Novex (San Diego, CA) precast 4–12% Bis-Tris gels for syndecan or 5% Tris·HCl gels (Bio-Rad, Hercules, CA) for versican and perlecan. After separation, the proteins were transferred to either polyvinylidene difluoride membrane (syndecan) or nitrocellulose membranes (versican and perlecan). Membranes were blocked with 5% nonfat dry milk in TBS-T (30 mM Tris·HCl, pH 7.5, 100 mM NaCl, and 0.1% Tween 20). The following primary antibodies, dilutions, and incubation times were used: rabbit anti-syndecan-1 (2 μg/ml, 1 h at RT; Zymed, San Francisco, CA), mouse anti-perlecan (2 μg/ml, 2 h at RT; Zymed) and rabbit anti-versican [1:2,000, 2 h at RT; a kind gift from Dr. D. Zimmermann, Dept. of Pathology, Zurich, Switzerland (54)]. After being rinsed, membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Amersham Life Science, Amersham, UK). Immunoreactive bands were visualized using ECL+ (Amersham) and a CCD camera (LAS1,000; Fujifilm, Tokyo, Japan). The intensity of the bands were measured by densitometry (Image Gauge V.3.45) and compared between the groups.
Biolabeling of GAG
Sulfate labeling.
Human glomerular endothelial cells (passage 5) were allowed to grow to 70% confluence in 24-well plates before the media was changed. Cells in four wells were stimulated with PAN (1 μM) and as control we used four wells of nonstimulated cells grown under normal conditions (i.e., "starved" in standard media with 2% human serum and half the amount of BBE). Stimulation was performed for 48 h at 37°C in a humidified, 5% CO2 atmosphere. Metabolic labeling of GAGs were obtained by adding 10 μCi/ml of [35S]sodium sulfate (NEX041, New England Nuclear, Boston, MA) to the media. Only GAGs synthesized during the 48-h incubation period were [35S]sulfate labeled with this procedure.
Colabeling with sulfate and tritiated glucosamine.
Human glomerular endothelial cells (passage 8) were allowed to grow to 70% confluence in 24-well plates before the media was changed. The cells were starved and stimulated as described above. Metabolic colabeling with d-[6–3H]glucoseamine (20 μCi/ml) and sulfate-35 (30 μCi/ml) (Amersham Biosciences, Uppsala, Sweden) was made to measure both the sulfate content and the length of the GAG chains.
A time course study was previously done to determine the metabolic activity of glomerular endothelial cells (45). After the stimulation, conditioned media were collected for further analysis.
Analysis of PGs Synthesized and Released to the Media
Ion exchange and molecular sieve chromatography.
PGs in the media were separated on the basis of differences in charge density, by using an ion-exchange chromatography di-ethyl-amino-ethyl (DEAE)-Sephacel column (0.7 × 2.5 cm, Amersham Bioscience). Cold carrier (4-O-S-Chondroitin sulfate, 100 μg, Seikagaku, Tokyo, Japan) was added to each sample to decrease the loss of labeled PGs in the separation process (34). After application of media containing [35S]sulfate-labeled PGs from control and PAN-treated cells, unbound radioactivity was removed from the column by washing with urea buffer, and PGs were eluted with 4 M guanidine hydrochloride, 50 mM NaOAc, 0.2% Triton X-100, pH 5.8, with a flow rate of 1 ml/min. (33). Fractions containing [35S]sulfate were collected and quantified by scintillation counting and subsequently pooled. Pooled fractions were further analyzed by size exclusion chromatography on an S-200 column (10 × 300 mm, flow 0.4 ml/min, Superdex, Amersham Bioscience) in 4 M guanidine hydrochloride, 50 mM NaOAc, 0.2% Triton X-100, pH 5.8 to separate the PGs by their hydrodynamic size. Separated PGs were collected in 0.4-ml fractions and aliquots were quantified by scintillation counting.
The cell media was separated in a serial chromatography step. First, the samples were applied to ion-exchange chromatography (TSK gel, DEAE-5PW, 7.5 mm × 7.5 cm, Tosoh Bioscience, 50 mM phosphate buffer, pH 7.0), where the negatively charged PGs bind to the column and other molecules were wasted. After a 20-min wash period (in same buffer as above), the proteins bound to the ion-exchanger were eluted by a step increase of NaCl concentration from 0 to 1 M to a size-exclusion column (Bio Sep-SEC-S3000, 300 × 7.8 mm, in 50 mM phosphate buffer, pH 7.0) at a flow rate of 0.5 ml/min. Separated PGs were collected in 0.5-ml fractions and aliquots were quantified by scintillation counting.
The size-exclusion columns were calibrated by using the LMW and HMW kits from Amersham Bioscience.
Size Characterization of GAG Chains
We treated the colabeled PG fractions with alkali-borohydrate (1 M NaBH4 and 0.2 M NaOH) to cleave the binding between the GAG chains and the core proteins. The reaction was stopped by adding an equal volume of 0.2 M NaOH. GAG chains were precipitated (95% ethanol) and then separated on a size-exclusion column (Superdex 75, 10 mm × 30 cm, Pharmacia, in 4 M Guanidine/HCl, 0.2% Triton X-100, 50 mM NaOAc, pH 5.8) at flow rate of 0.4 ml/min. Separated GAG chains were collected in 0.4-ml fractions and quantified by scintillation counting.
Results are presented as means ± SE, and differences were tested using the Student's t-test pair design.
Glomerular Endothelial Cell Culture
The glomerular cells started to appear after approximately 1 wk, small colonies of different morphology of which about 1/5 was of endothelial origin. The cells were of endothelial origin as judged by light microscopy, by endocytosis of the endothelial cell-specific marker Dil-Ac-LDL, and by expression of the endothelial cell-specific lectin Ulex europaeus agglutinin as observed by fluorescence microscopy (Fig. 1). RT-PCR revealed that the cells expressed endothelin-1 (data not shown). No significant difference in number of cells or amount of RNA and protein was seen between the two groups after the experiment, confirming that there is no toxic effect by puromycin aminonucleoside at the concentration used (26).
Gene Expression Analysis
Core proteins.
The following PGs were tested in the TaqMan real-time PCR analysis system and were found to be expressed in the human glomerular endothelial cells: syndecan, versican, glypican, perlecan, decorin, and biglycan (Table 1). The expression of versican was downregulated by 37 ± 6% (means ± SE) in the PAN-treated group compared with the control (P < 0.001, n = 8). We could not see any significant difference in expression between the groups (control and PAN) for the other core proteins analyzed (Fig. 2A).
Enzymes important for the degree of sulfation and the length of the GAG chains were also analyzed by real-time PCR. In galactosaminoglycan assembly, chondroitin 4-O-sulfotransferase was downregulated by PAN by 34 ± 7% (P < 0.01, n = 8) but not the 6-O-sulfotransferase. In addition, PAN downregulated the chondroitin synthase 1 by 25 ± 6% (P < 0.05, n = 6). Two of the GAG enzymes important for the heparan sulfate (HS)-biosynthesis were also affected: HS-ST6 (68 ± 4.5% of control) and HS-3-OST1 (65.5 ± 5% of control, P < 0.01, n = 6; Fig. 2B). The other enzymes tested (CS-6-O-ST, NDST, EXT-2, and HST-2) seemed to be unaffected by the treatment.
Validation of amplification efficiency was made for every primer/probe set and was calculated for each run from the corresponding standard curve, see Table 3. By using the standard curve equation [instead of comparative CT (ΔΔCT) relative quantification method], we correlate for the differences in reaction efficiency between the target gene and the endogenous control gene.
Protein Expression by Western Blot Analysis
Syndecan, versican, and perlecan were expressed by the human glomerular endothelial cells as revealed by Western immunoblot analysis (Fig. 3). The versican antibody detects the V0 and V1 splice variants (∼370 and ∼260 kDa). When comparing the density of the enzyme-digested samples blotted for versican, we observed a 25% lower intensity in the PAN-treated cells compared with control. Syndecan (90 kDa) and perlecan (>400 kDa) were unaffected by the PAN treatment, a result in line with mRNA data.
PG Synthesis in the Culture Media
Sulfate labeling.
The extracellular PGs in the conditioned medium from both control and PAN-stimulated cells were separated from other extracellular matrix molecules by charge density on a DEAE column. As shown in Fig. 4A, the total amount of labeled sulfate-containing PGs was drastically lower in the PAN-treated group compared with control (reduced by 60%). Further analysis of the peak fractions by size exclusion chromatography revealed that the two groups contained similar type of molecules. The PGs in the media seemed to be very large, 250–450 kDa, which suggests versican and/or perlecan.
Colabeling with sulfate and tritiated glucosamine.
Extracellular PGs in the conditioned medium from both control and PAN-stimulated cells were separated by both charge (on a DEAE column)- and by size-exclusion chromatography. As shown in Fig. 4B, the amount of large (>55 Å), sulfate-containing PGs was lower in the PAN-treated group compared with control (reduced by 40%). When measuring 3H-labeled GAG chains, we saw a reduction of 50% after PAN treatment (Fig. 4B).
Size Characterization of the GAG Chains
When analyzing the size of the GAG chains by size-exclusion chromatography, we could see a difference in the elution profile after PAN treatment. For the control cells, 25% of the GAG chains were in the size range of ∼40 kDa or greater, whereas the activity in the same fractions for PAN-treated cells was almost undetectable. In the smaller size ranges (∼14 kDa), we saw a delayed peak for the PAN-treated cells compared with control cells, indicating a reduced length of the GAG chains (Fig. 5).
Our study is the first to describe that the human glomerular endothelial cells (HGEC) express several different classes of PG core proteins. We have found that 1) HGECs express mRNAs for several PGs: syndecan, versican, glypican, perlecan, decorin, and biglycan (both heparan and chondroitin sulfate containing). In addition, we showed at the protein level that HGECs synthesize and export syndecan, perlecan, and versican core proteins. The secreted PGs bind to structures and receptors situated on the cell-surface and they also interact with the GAG chains of other PGs (20, 51). Turley and Roth (49) showed in 1980 that there is an interaction between carbohydrate chains of hyaluronate and chondroitin sulfate. 2) The nephrosis-inducing agent PAN (18, 37) decreased both the mRNA expression (by 37%) and the protein expression (by 25%) of versican compared with control. Versican belongs to the family of hyaluronan-binding PGs that is collectively termed "hyalectans," and these PGs are extracellular matrix components (20, 30). Pericellular matrix expansion involves the interaction of versican with several binding proteins and can be stimulated by growth factors such as PDGF and TGF-β1 (51). A decrease in versican could therefore result in a decrease of the pericellular matrix hence reducing the net negative charge of the cell-surface coat and/or the glomerular basement membrane. 3) In addition, PAN affected the enzymatic machinery behind the biosynthesis of chondroitin sulfate along the galactosaminoglycan biosynthetic pathway. Among the effects seen was a downregulation of the 4-O-sulfotransferase (CSST4) by 34%. CSST4 adds sulfate to the fourth carbon position on GalNac residues. This downregulation will reduce the total negative charge of versican, as no compensatory upregulation by other sulfotransferases (i.e., CS-6-O-sulfotransferase) is seen. Because versican normally carries CS, sulfotransferases acting on dermatan sulfate GAGs can be disregarded. It should, however, be noted that 4-O-sulfation on GalNac is a prerequisite for dermatan sulfate stability by locking the l-iduronic acid in its energetically unfavorable L-form (29). This prevents the iduronic acid to switch back to its C-5 epimer in chondroitin sulfate, glucuronic acid (GlcA). With this in mind, it is also likely that decorin and biglycan (small PGs capable of carrying either dermatan or chondroitin sulfate) in this case mostly carry chondroitin sulfate chains (10, 20). Moreover, PAN downregulated the enzyme CS synthase, responsible for the elongation of the GAG chains, by 25%. Thus PAN induced a loss of negative charge by versican core protein downregulation, a loss of negative charge per versican molecule, and possibly a stereochemical alteration of the CS/DS content of the endothelial matrix. This indicates a total deorganization of the matrix, where the alteration of biological function can be expected to be severe in the context of tissue function. 4) Both syndecan and glypican are membrane bound and may be important for the structure of the GEC surface coat by acting as anchors for the pericellular gel structure (3). Glypican is heavily involved in cell-to-cell contact and morphogenesis (9). As described above, we could not detect changes in glypican expression, consistent with our observation that PAN (in this concentration) did not affect cell morphology. Syndecans have, in addition to many other functions, similar characteristics for cellular behavior including cell signaling (44). Similar to glypican, the syndecan expression was not affected on the core protein level. However, the observed downregulation of HS-ST6 and HS-ST3 could result in a decreased charge density on the immediate cell surface of the membrane bound PGs as well. It may also have biological importance for the binding of cytokines (such as FGF) in similar ways earlier reported (27). 5) The analysis of the cell media revealed that the glomerular endothelial cells produce a large amount of sulfate-containing GAGs. Versican and/or perlecan were the major core proteins found in the media. PAN reduced the total amount of sulfate-labeled PGs by 60%. When analyzing the larger molecules (>55 Å), we saw a 40% reduction of sulfate and a 50% decrease of tritiated glucosamine. This reduction is caused by loss in the amount of sulfate groups and by a reduced length of GAG chains due to downregulation of the enzymes mentioned above in combination with the decrease in versican expression. 6) Puromycin did not seem to affect the mRNA expression of the core proteins syndecan, glypican, decorin, biglycan, or perlecan. Neither did PAN affect the enzymes CS-6-O-ST, NDST, EXT-2, and HST-2.
A decreased synthesis of GAGs could increase the permeability of the layer covering the fenestrae and hence increase the sieving of macromolecules (17). In fact, effects of PAN on glomerular endothelial cell fenestrations have been described earlier by Avasti and Evan (1) but they reported the fenestrae to be decreased in size and number during PAN-induced nephrosis, as seen by TEM/SEM.
The role of glomerular endothelial cells for the charge and size selectivity of the filtration barrier in the kidney has been argued for several years. Recent functional studies from our laboratory (18, 22) are in qualitative agreement with the classical studies of a significant negative charge barrier, albeit with a lower charge density (50 vs. 100–150 meq/l) (47). However, when studying the charge barrier in vitro, experiments are conducted mainly on mesangial and epithelial cells to measure their production of negative charged molecules, i.e., PGs and glycoproteins (11, 13). Furthermore, isolated glomerular basement membranes showed some size but lacked charge selectivity in vitro (4), which has been taken as evidence of its limited role in the glomerular charge barrier. However, the isolation procedure may change the composition of the basement membranes and their permeabilities seem to partially depend on the hydrostatic pressures applied (38). In any case, knockout mice lacking the heparan sulfate chains of perlecan (the principal PG of the GBM) do not develop any kidney malfunction, such as proteinuria (39). There are other components, such as agrin, in the GBM which also might be of importance for the charge-selective properties of the barrier.
Anionic sites have also been demonstrated on glomerular epithelial cells (12). The importance of the GAGs is illustrated in a study where the gene for one of the enzymes responsible for site-specific sulfation of heparan sulfate (HS 2-O-sulfotransferase) was knocked out. The mice did not develop any kidneys, as a result from failure of ureteric bud branching and mesenchymal condensation, and they died shortly after birth (5).
It is already known that endothelial cells express different PG core proteins such as versican (6), perlecan (50), syndecan (3, 19), biglycan (23), glypican (3), and decorin (21). These studies were made in macrovascular endothelial cells, mainly from the aorta and the umbilical vein. However, there seem to be differences between endothelial cells originating from different vasculature (6, 53). The only study to describe human glomerular endothelial cell PG production is from Schaefer et al. (42) where they showed an expression of biglycan by using in situ hybridization. On the other hand, they did not find any expression of decorin. The present paper is therefore the first to demonstrate that glomerular endothelial cells produce several different classes of PGs.
We conclude that human GECs produce several different classes of PGs. The nephrosis inducing agent PAN downregulates the expression of only one core protein (versican) but has severe effects on foremost key galactosaminoglycan chondroitin sulfate transferases and some sulfation-linked effect on glucosaminglycan heparan sulfate biosynthesis. The overall effect in this context is less sulfate groups, shorter GAG chains, and reduced PG net negative charge. It is therefore conceivable that certain nephrotic conditions may be due to endothelial dysfunction with altered cell-surface coat composition.
This study was supported by Swedish Medical Research Council Grants 9898, 14764, and 4531, the IngaBritt and Arne Lundberg Research Foundation, the National Association for Kidney Diseases, Sahlgrenska University Hospital Grant LUA-S11733, Sahlgrenska University Hospital Foundation, The Swedish Heart and Lung Foundation, and AstraZeneca.
The Department of Urology at Sahlgrenska University Hospital supplied the tissue samples for which we are most grateful.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2005 the American Physiological Society


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Canny 'Ollie' hoping Blues leave chance for knockout
IAN HOLLOWAY evoked Henry Cooper, Sugar Ray Leonard and Herol 'Bomber' Graham as he promised to go toe-to-toe with Chelsea at Stamford Bridge tomorrow. It was vintage 'Ollie', colourful, quotable and thoughtful. Thoughtful? Oh yes.
With his vivid speech and strong Bristolian accent -- a rarity in football -- Holloway is regarded as a 'character'. He is, but such casting obscures the fact that he also has a very shrewd football brain honed by 14 years' experience in management.
This knowledge and intelligence has enabled him to steer Blackpool to an improbable fourth in the Premier League. They will not stay there -- he and we know that -- but they have a fighting chance of staying up, which is more than most thought at the start of the campaign.
On the face of it, tomorrow's match should be a ritual slaughter. Blackpool may have won at Wigan and Newcastle, but they shipped six at Arsenal, while Chelsea have scored 21 goals in five matches this season and 31 in their last six at home.
Would Holloway not be better off 'parking the bus' like Rangers did at Old Trafford?
That is not his style -- as a reference to admiring Leonard's 'showboating' underlined -- but, besides, he said: "I don't think my players could do it as effectively as Rangers did. I haven't got the people to do that. So, I'd be pretty stupid trying to."
With those words he challenged his players to prove him wrong.
He then added: "We are respectful of everyone and fearful of no one. We are taking on one of the giants, but we have always been good underdogs. It would have to be one of them unbelievably special days to get anywhere near them, but the object is to pick up three points.
"I believe you have to try to hit a champion; try to knock him out. Even Henry Cooper did that to Muhammad Ali and that shocked the world didn't it? So, why can't Blackpool score a goal against Chelsea?"
With those words he told his players to believe in themselves.
Holloway added: "I know the lads are up for it. It's a wonderful opportunity for us to have a free shot. We might get hurt, but will it hurt us that much? I don't think so. Most people go down there and get beat anyway."
With those words he took any pressure off his players. In terms of using the press to get a message across, Holloway is as smart as Jose Mourinho.
He knows his football too. There are two remarkable elements to Blackpool's breezy opening to the season. They have done it with an adventurous approach and a personnel which was still being assembled when the campaign began.
It is one thing to organise a group of players into a defensive framework, it is another entirely to mould them into a fluid creative force.
So far Blackpool have played a 4-3-3 formation, which involves a lot of interchanging of positions. That usually requires a significant amount of work on the training ground.
Due to the late recruitment, Holloway has not had that time. That he has integrated new players into this system so quickly speaks volumes for the clarity of his coaching. Holloway hinted that he might try a new approach at Chelsea, but only if he was sure his team had absorbed it.
"Players make excuses if you haven't got your tactics right. They don't feel comfortable. But I'm working on an option that, if Chelsea ever leave their chin open, means maybe I can land something on it."
Ducking and weaving to illustrate Holloway concluded: "Herol Graham was great defensively, the best defensive boxer around. But if you don't throw a punch, eventually you'll get knocked out."
Chelsea manager Carlo Ancelotti has seen his team score 21 goals so far this season, after managing 142 in all competitions last season on their way to winning the double.
Ancelotti, who has been making regular visits back to Italy to help care for his seriously ill father, admitted he has never managed another team who were able to score as freely.
Given that he has twice led Milan to the Champions League, and was also in charge of a great Juventus team that contained the likes of Zinedine Zidane, that is quite some compliment.
"There's no fear, now, which is good. The players are all involved in the team and there's a good atmosphere, Ancelotti said. "In the past, Chelsea played a different way. The players enjoy this kind of football because they're all involved. For this reason they can show their ability."
Since they lost 4-2 to Manchester City in February, at the height of the row concerning John Terry's alleged affair with Wayne Bridge's ex-girlfriend, Chelsea have won all eight Premier League games they have played at the Bridge, scoring 37 goals and conceding just two.
Ancelotti yesterday told the club's supporters to enjoy the fun while it lasts, and not to view a 2-0 victory as a "poor game, a s**t game."
He added: "I don't know if the fans expect us to score six. I'd say it's impossible. I'll say to my fans don't expect that, because it's not real football. Our moment is good for now, but this could change in a second.
"If you lose a game, maybe you can start to lose confidence. For this reason, we have to stay focused in training and in competition. My experience says that, when you're in a good moment, it's easy to lose this confidence." (© Independent News Service)
Chelsea v Blackpool,
Live, tomorrow, Sky Sports 1, 4.0
Irish Independent


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1Heat oven to 350°F. Spray 12x8-inch (2-quart) glass baking dish with cooking spray.
2Place bacon slices on cookie sheet with sides. Bake 10 to 15 minutes or until crisp. Remove bacon from sheet; place on paper towels to absorb excess drippings. When cool enough to handle, crumble bacon; set aside.
3Meanwhile, heat large stockpot of generously salted water to boiling. When water comes to a boil, add macaroni. Cook as directed on package to almost al dente. Drain; set aside.
4To make sauce, in 2-quart heavy saucepan, melt butter over medium heat. Using whisk, stir in flour until blended. Add milk; cook and stir until mixture comes to a boil. Stir in nutmeg and salt. Reduce heat to low; simmer about 15 minutes, stirring frequently, until mixture thickens enough to coat back of wooden spoon. Check seasoning level; add additional salt and nutmeg if necessary.
5Stir cheese, pumpkin and egg yolks into sauce until combined. Add cooked macaroni and crumbled bacon; stir to combine. Pour mixture into dish. Sprinkle bread crumbs over top.
6Bake 15 to 20 minutes or until top is golden brown.


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ST GEORGE Illawarra utility Nathan Fien will happily welcome star playmaker Jamie Soward back into the Dragons halves at his expense this weekend, believing his side is a stronger team when the five-eighth is on the park.
Following his side's finals-denting loss to the Sydney Roosters on Friday night, Fien said it was impossible to know if coach Steve Price had made a mistake by playing Soward in reserve grade on Saturday.
Having played just once for the Dragons since round 17 with back spasm injuries, Soward scored a try, set up two more and kicked five goals playing in the NSW Cup. However, Price was happy to nurse Soward in reserve grade, confident in his halves combination of Fien and captain Ben Hornby following their round 21 win over Melbourne.
Ahead of the Dragons' sudden-death Saturday blockbuster against the Tigers, Fien said the Dragons need Soward to play a part in their race for a spot in the finals.
"I said the other night he's just one of those players who can create things from nothing," Fien said.
"We've played together for a long time and I enjoy playing alongside him. I have no doubt he'll play a major role for us over the next month."
"There's no way we can do some of the things that Jamie Soward can do, but we do things a different way. That's what Pricey asks us to do."
Originally published as Soward's return is Fien by me


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Mumbai, Feb 4 (UNI) The Bombay High Court today rapped the Maharashtra government for failing to take any action against police officer Nikhil Kapse, indicted by Justice Srikrishna Commission, which enquired into the 1992-93 communal riots in Mumbai.
Kapse had allegedly opened 'unprovoked' firing in Hari Masjid, resulting in death of seven persons.
The court has asked the state to reply, by tomorrow, as to when all papers related to the case would be handed over to CBI.
One of the injured Farook Mohammed Kasim Mapkar moved the High Court, seeking criminal prosecution against the police party led by the then police Sub-Inspector Nikhil Kapse, who had opened fire on Namazis assembled in Hari Masjid.
Seven persons had died and seven others were injured in the January 10, 1993 police firing but despite numerous attempts on part of the petitioner, the police did not file FIR against the policemen.
The state government had, in October last year consented to hand over the case to the CBI.
Today, when Mapkar's petition came up for the hearing, a division bench comprising Justices S B Mhase and Amjad Sayyad questioned as to on what basis the state has decided to handover the case to CBI.
The Judges wanted to know whether congnisable offence was made out in the complaint filed by Mapkar and commented that ''you cannot pass the buck like this''.
The Judges also asked as to how Special Task Force, appointed by the Congress-led government in the state for implementing Justice Srikrishna Commission's recommendations, set aside judicial findings of the Commission, when Public Prosecutor Satish Borulkar pointed out towards STF's finding that there was no material for initiating criminal prosecution against Kapse and others, allegedly involved in Hari Masjid firing.
The Judges said it was not open for the STF to bypass findings of a Judicial Commission. The court also lamented that police had failed to prove any charge against Kapse in departmental disciplinary action observing it was just an eyewash, a camouflaging show for the public.
Referring to the incident of Hari Masjid police firing, Justice Srikrishna Committee has observed in its report that, ''the police version of the story was totally unbelievable and fabricated'' and also that, ''PSI Kapse, presently working with the economic offences wing of the Mumbai police, not only supressed facts but also fabricated records''.
UNI XR OBB KK HT2026 Reply Reply to all


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Talbot slams Ashton
By Paul Higham
Last Updated: 01/01/70 1:00am
Brian Talbot has slammed defender Jon Ashton's display against Stockport.
Oxford United manager Brian Talbot has slammed defender Jon Ashton for his two dreadful errors against Stockport County.
The U's late 2-1 defeat at resurgent Stockport saw The Hatters pull away from the drop zone while the visitors are just a point above the bottom two.
Ashton gifted Tes Bramble the opener at Edgeley Park with a weak backpass, although Tim Sills did manage to level for Oxford on the hour.
However, Ashton produced a reckless challenge on Bramble three minutes from time to hand County a penalty, which substitute Adam Le Fondre converted to hand the hosts all three points.
Talbot was bitterly disappointed with Ashton's performance, and was also unhappy at the defender for not apologising to his team-mates after the game.
"It was suicidal defending by Jon Ashton," said Talbot.
"After the game I left it for five minutes to see if he apologised to the lads.
"He didn't, and that disappoints me. The tackle and why he did it has no explanation."
Meanwhile, Oxford have extended the loan of defender Warren Goodhind from League Two rivals Rochdale.
The South African-born player initially joined The U's on a month-long loan deal and has now agreed to see out the season at The Kassam Stadium.


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My experience with Nazareth Housing has been one that I treasure. I was once homeless myself, many years ago and I know all too well the heartfelt despair that one feels when you do not have a home to call your own. However, Nazareth Housing is an organization that truly puts the needs of their clients first. They do not waste any time making sure that the needs of their clients are met. They go beyond their call to make sure that families gets food, shelter and human services on a daily basis. They are clean and productive and I am very happy to know that such an organization exists and that they are meeting the needs of the people they serve.
As a pastor in a busy parish with many needs I have found the director Fr Michael Callaghan unfailingly helpful and professional. Nazareth housing has with every referal made a significant and practical difference in the lives of some people who thought their was no one who cared and no help to be had.


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New superintendent for White River schools
Janel Keating is the new superintendent of the White River School District.
Keating, who has been the district's deputy superintendent for the past seven years and has worked for the district for 15 years, succeeds former Superintendent Tom Lockyer, who retired.
Keating became superintendent in the Buckley-based district July 1.
Keating has earned praise from both the school board and teachers in the district for building a culture of collaboration among staff members, boosting test scores and high school graduation rates, along with making academic improvements. They cite her hands-on style and accessibility as positives.
The school …


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Telephone Pendant Sweater Chain
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Balayya's Acts: Vulgar Or Childish
There is a certain aura that develops once an actor attains stardom. And then there are those actors who come with a legacy which builds an air of respect and high regard for them. So, when they want to be themselves, those around with lesser intellect interpret it in a rustic manner.
Nandamuri Natasimham Balakrishna has a very powerful image and status in Tollywood and even today, his films are a mania among the masses. Off the screen, he is known for his friendly banter and pun intended fun with the actresses. Given the straightforward and transparent nature he has, he doesn't mind that fun even in front of the media.
Recently, Balayya was seen as the chief guest for the trailer launch of 'Guntur Talkies' and he came up with few jovial lines addressing heroine Shraddha Das, anchor Shilpa Chakravarthy. Some media entities took this as the cue and started cooking up spicy gossip stuff but here is a truth no one can deny.
Balayya has got a child in him that surfaces now and then. Despite his aura and his background, Balayya doesn't mind showing the child in him in front of ordinary people. During Memu Saitham song, he did something similar with singers Kousalya and Malavika which was thoroughly enjoyed by all including the singers themselves. So, it is only those cheap minds interpreting as vulgar but those who are sensible know it is nothing but Balayya's Childlike nature and his clean heart.


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Squirrel Stole My Underpants by Bonnie Duncan
Sat. Jan 26, 1pm&3pm; Sun. Jan 27, 1pm&3pm; Thur. Jan 31, 10:30am; Fri. Feb 1, 10:30am; Sat. Feb 2, 1pm&3pm; Sun. Feb 3, 1pm&3pm. Sat, Jan 26 - Sun, Feb 3, 2013
Venue: Puppet Showplace Theatre
32 Station Street,, Brookline, MA 02445Contact:
Brenda Huggins
0 people saved this
Sylvie is sent to the backyard to hang up the laundry. The moment her back is turned, a mischievous squirrel steals her favorite piece of clothing and runs off. When Sylvie gives chase, an entire world emerges from her laundry basket, and curious characters show her the way through mysterious lands. Will our lonely heroine rescue her underpants and discover the magic within herself? Join us on this adventure to find out!
Trustees Literary Trail
In addition to the miles of beautiful wooded trails, open beaches, and green pastures found...
Search All Listings Here.


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We recommend upgrading to the latest Internet Explorer, Google Chrome, or Firefox.
If you are using IE 9 or later, make sure you turn off "Compatibility View".
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The view from the top of one trek is a good tease for the next: There will always be more to explore.


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- Gaines Investment Trust18.Dec 2015
- Employee
Workplace Safety
This is a family run business and is run this way. I have never worked for a less caring group in my life. One of the key corporate people had stated to the my boss that I was to old (at 57) and should fired


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Gochee's Garage Inc
Review for Gochee's Garage Inc
Kevin R. on 06/12/2015
Spoke with Nancy. She said this business did a great job for a great price.
Vehicle: Lincoln Town Car
Category: Service
Service Date: 06/10/2015
Review Created: 06/12/2015
This review was collected via phone interview by Customer Research Inc.


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The presence of God is a mysterious thing. I do not know if you can easily explain it or explain it at all for that matter.
What I am learning about the presence of God:
can't see this video because you are an apple lover and cannot rock flash videos, check out this link here
Would love for you to share if you feel comfortable to share a moment when you have felt the presence of God in the last month?


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A Practical Approach to Wireless 2.0
While all Wi-Fi vendors understand the need for and are taking steps in the directions of a fully-distributed architecture, crossing the bridge from controller-based to controller-less takes a significant amount of time and effort because all system features and the user interface must be re-architected for a fully-distributed platform. We invite you to take the road less traveled: controller-less. Read on.
Please supply your email address to receive this whitepaper


text:
Here's How Lamborghini Sold The Diablo In 1999
I don't know why the Lamborghini Diablo had any kind of advertising. The car sells itself. It has the doors. It has the sound. It has the looks. It has a V12 large enough to crush a small home. And yet, advertise it Lamborghini did.


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History is alive at Lothrop! Third graders examined aspects of our Sunshine Village in a year-long study which included collaboration with the Pittsford Historical Society and local resources. Students created many of the illustrations for their digital stories. Click on the links below to view our projects.
Project E.A.G.L.E. >


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VideoHive Walking Through the Woods 4559020
Stock Footage \ Nature
Videohive \ Projector \ Person \ Forest \ Memories \ Usefull \ Steady
Stock Footage - Walking Through the Woods 4559020 by Christian_Fletcher @ VideoHive
Stock Footage \ Nature
A walk throug the forest track, shooted from the person`s point of view steady.
Please rate if you like it from your purchased items page!
Also, check my best After Effects project- Film Projector Old Memories (usefull as romantic video introduction) if you like: http://videohive.net/item/film-projector-old-memories/4128455
or usefull RETRO TV with ALPHA CHANNEL http://videohive.net/item/retro-tv-with-alpha-channel/4686960
Find out about my new items first! Follow me on Videohive
Alpha Channel: No; Looped Video: No; Frame Rate: 25; Resolution: 1920x1080; Video Encoding: H.264; File Size: 351mb; Total Clip(s) Length: 0:39.
Beautiful Alpine Landscape; Forests and Grasslands in Summer; Butterfly in the Meadow; Wild Flowers and Meadow; Butterfly in the Meadow; Butterflies and Wild Flowers; In the Forest View.
Keywords: footpath, forest, leaf, path, person, point of view, steady, summer, track, trees, walking, way, wilderness, woods.


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Eric G. Dove performs Robert Gregory Browne's Negligence with a blue-collar, almost cocky swagger. His delivery fits this tale of treachery and "privileged idiots" at a Chicago prep school. Yes, this crime thriller pits the streetwise Matt Isaacs, crime reporter, and his pal, washed-up actor Hutch, against the mystery of a grisly killing and a prosecutor's shrewd case. Written in a tough-guy vernacular reminiscent of Richard Price and filled with curse words and slang, this audiobook is for those who like their crime with a little introspection and a lot of adrenaline.
She was found with a bullet in her head, and the kid she was last seen with woke up drugged and disoriented in a neighbor's backyard. Now the kid is awaiting trial for her murder, and crime reporter Matt Isaacs is convinced that there's more here than meets the eye.
But Matt's old college pal, Ethan "Hutch" Hutchinson - washed-up actor and newly-minted trial junkie - isn't so sure, until a violent attack propels Hutch and his friends into the middle of a conspiracy that takes them all the way from the courthouse gallery to the not so hallowed halls of an exclusive Chicago prep school... straight into the hands of a killer.
©2013 Robert Gregory Browne (P)2013 Audible, Inc.
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Mistress of Melody now on sale! The next book in my historical romance Music of the Heart series, Mistress of Melody is currently available in ebook format for a *limited time special offer* of only .99 cents (list price $4.99). Scoop up this … Continue reading
Category Archives: Music
I'm thrilled to announce that Sonata for a Scoundrel is going into audio! The incredibly talented Hollis McCarthy is doing the narration, and the audiobook should be for sale by mid October. Want to know the moment the title is available? … Continue reading
NOW AVAILABLE in e-book for only $4.99 at: AMAZON AMAZON UK iBOOKS B&N SMASHWORDS KOBO "Mesmerizing and addictive … this highly engrossing read will keep historical fiction fans clamoring for more." -Library Journal (starred review) The Muse… … Continue reading
Come and visit our newest blog stops! January 13 – Slip into Something Victorian where we have a post about the origins of the word posh. January 20 – To Be Read interview – more about our co-writing process. January … Continue reading
Check out Romance in the Back Seat! (ahem, that's romance writers giving interviews in cars, a fabulous project by Terry Kate, based on the Black Cab Sessions…) Anthea has an interview up, as well as a little teaser where she … Continue reading
Come join traditional Irish band FIDDLEHEAD for a St. Patrick's Day celebration on March 17th. Although we don't do any songs by Thomas Moore (see the Regency Parlor Music page!) we do perform songs and dance tunes that you'd find … Continue reading
Anthea & Lawson will be at Border's Books in Olympia this coming Friday, December 12, from 6-8 pm. Follow your ears to the performance of Victorian Holiday Music, chat with the authors and pick up a signed copy of PASSIONATE. … Continue reading


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Red Hat Bugzilla – Bug 489617
csh/tcsh : Unknown colorls variable `rs'.
Last modified: 2009-04-06 16:34:01 EDT
Description of problem:
when switching to csh/tcsh the error message is displayed.
Version-Release number of selected component (if applicable):
How reproducible:
Always (as far as I am concerned).repositories
Steps to Reproduce:
1. download and install tcsh from FC 10
2. type csh or tcsh
Actual results:
Unknown colorls variable `rs'.
Expected results:
to switch to csh or tcsh
Additional info:
Reassigning incorrectly assigned bug
This issue is already fixed in Rawhide (https://bugzilla.redhat.com/show_bug.cgi?id=487783).
It should not occur in F-10 and I'm also not able to reproduce it. New colorls variables (rs and hl) are not included in F-10 coreutils (latest version is coreutils-6.12-20.fc10).
Which coreutils do you use?
I am using coreutils-6.12-19.fc10. tcsh was working just fine a few days ago so I suspect there must be something wrong with the packages I installed in the interval.I noticed that I had also SELinux coreutils installed and tried to erase them from the package manager. The process took quite long and when it was about to finish the screen started blinking and the keyboard became irresponsive. I rebooted but couldn't restart, the process stucks when the progressbar becomes white!
tcsh-6.15-7.fc10 has been submitted as an update for Fedora 10.
Well, OK, I'll add these two colorls variables in F-10 too. It should help you
and nothing will break even if coreutils doesn't include them.
tcsh-6.15-7.fc10 has been pushed to the Fedora 10 testing repository. If problems still persist, please make note of it in this bug report.
If you want to test the update, you can install it with
su -c 'yum --enablerepo=updates-testing update tcsh'. You can provide feedback for this update here: http://admin.fedoraproject.org/updates/F10/FEDORA-2009-2685
Problem has been resolved, thank you!
tcsh-6.15-7.fc10 has been pushed to the Fedora 10 stable repository. If problems still persist, please make note of it in this bug report.


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Amnesty Intl. slams arrests for carrying Soviet symbols in Ukraine
Lena Savchuk
Press TV, Kiev
Amnesty International has criticized Ukraine for what it calls infringing on the freedom of speech and assembly of its citizens. The criticism follows the detention of demonstrators who carried Soviet symbols during a ceremony marking the anniversary of the victory over Nazism. The rights group says Kiev is intentionally trying to erase symbols of its Soviet past. Press TV's Lena Savchuk has the details.


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Sons das Zueiras 2 1.3.8
Install 100,000 - 500,000
Sons das Zueiras 2 apk info
Category: Comics
Publish Date: August 02, 2017
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Email: email@example.com
Top 5 User Reviews
May 30, 2015
May 30, 2015
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May 30, 2015
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Sons das Zueiras 2 has been downloaded by 100,000 - 500,000 people available all over the world, anyone can be easily downloaded from the wikinsta store. You could visit their official website http://www.escolhatecnologia.com.br to know more about the app or you can mail to email@example.com them if you have any query regarding the app. This is the top famous app available in Comics category. Average rating of the app is 4.6 which is a great indication of people trust and use the app frequently. To download the app you must have your android version 4.0 and up. The price of the app is 0. According to, the public data average daily active user (DAU) of the app is 10168. With Monthly active users (MAU) of 76260. The latest version of the app is which currently installed in 60% of the users, last version 1.3.8 was published on June 23, 2017, midnight.
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Hehe, you totally gotta keep up with her Tee, can't be letting her slip.
It's been awhile since I posted my ATC's and this being a fresh month and all I thought I might as well start it off right here, with a new ATC...of sorts.
Sorry about the ginormous size


text: I have a oppurtunity to put in a new lawn at a newly constructed daycare. only about 5000 sq. ft will be irrigated and the remaining 15,000 sq. ft will not. I was looking at various improved tall fescue blends: Defiance XRE Mow Less Rebel Supreme Does anyone have any experience with these blends. Can you folks recommend something else? Hydro-seeding is an option in the specs. I wanted to offer the client a better turf that will stand up to foot traffic and still look like a lawn should. I am NE Ohio so cool season grasses only, and their will be minimal shade. Obviously I will have to put some improved rye or Kbg in the blend as well. HELP??? Jeff


text:
- Please select a date to see the proper prices!
Description of the Accommodation
Rooms, services If you are planning a trip to Turkey and are just looking for a hotel to reserve a room at, we suggest to also consider the opportunities provided by Altes Hotel Antalya. The hotel is offering 30 nicely furnished rooms to stay in to those seeking accommodation in Antalya. The hotel is happy to have you in the restaurant for a hearty lunch or a romantic dinner. Should you be arriving by car, parking will be available nearby. The bar can be an ideal place for a pleasant evening talk or a meeting. You can browse the Internet via wireless connection (WiFi) available at the hotel. If you wish to learn more about the exact scope of services provided by Altes Hotel Antalya (including any available for an extra fee), you will find them on the information sheet below.
Payment Advance bank card payment or credit card guarantee may be required for the reservation, depending on the chosen date, room and price.
Arrival, opening hours The earliest arrival time is 14:00, the latest time to leave the rooms is 12:00. The personnel can speak Turkish and English languages.
Altes Hotel Antalya rooms Rooms
- Please select a date to see the proper prices!
Accommodation services
Free WiFi in shared places
Garden, Bar, Elevator
Own restaurant
Hairdryer, Desk in the room, Mini-bar


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Riverhead lost to Half Hollow Hills West 26-6 on Senior Day.
The first half was pretty evenly played by both sides, with Hills West leading 7-0 at halftime.
The steady rain got heavier in the second half and Hills West (4-2) tallied 19 more points before Riverhead was able to break through, and score on a pass from quarterback Kyle Kelly to wide receiver D.J. Chandler for six points. The field goal attempt was missed.
The Blue Waves (1-6) play their last game of the season next Saturday, Oct. 29 at Centereach High School, where they take on the Cougars (4-3) who are coming off a 26-0 win today against Smithtown East. Kickoff is at 2 p.m.
RiverheadLOCAL photos by George Faella


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