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All Stories
Plane crash investigation continues
COVINGTON - The Federal Aviation Administration's investigation into a single-engine plane crash in Porterdale is ongoing. However, information from the crash site Wednesday indicates that the plane may have run out of gas.
Child left in van spurs dad's arrest
COVINGTON - The father of a 2-year-old boy was arrested Tuesday afternoon in the Kroger parking lot on U.S. Highway 278 after a passerby found the child alone in a parked van with the windows partially down.
Jobless rate hits record County's 11.5 percent rate higher than state's
COVINGTON - Newton County's unemployment rate was at 11.5 percent in May, higher than Georgia's record rate of 9.7 percent.
Flowers in bloom
Photo by: Erin Evans
Robertson plans to keep busy
COVINGTON - Beginning Wednesday, Deborah Robertson, associate superintendent for administrative services at the Newton County School System, will have more time to sleep in and spend with her family.
Jack Simpson - 06/26/09 Household Pest
Regular readers of this column already know of my sympathy and love for animals. My views have resulted from the manner in which I was reared in the hills of Pennsylvania. Our family lived among animals, grandfather had a mink ranch, and we had animals for pets. I have long been compassionate about God's creatures.
Ruth and Richard Pendergrass of Conyers announce the engagement of their daughter, Kathryn Elizabeth Pendergrass, to Jonathan David Eveleth, son of Suzanne and Michael Eveleth of Lillian, Ala.
Hawks trade for Jamal Crawford
ATLANTA - The Hawks acquired guard Jamal Crawford from the Golden State Warriors on Thursday for guards Acie Law and Speedy Claxton, hours before the NBA draft.
Newton Blotter - 06/26/09
The following charges and arrests were reported by the Newton County Sheriff's Office, Covington Police Department, Oxford Police Department and Porterdale Police Department:
Darrell Huckaby - 06/26/09
It's one of those days. So much going on in the news.
Rockdale Blotter - 06/26/09
The following charges and arrests were reported by the Rockdale County Sheriff's Office, Conyers Police Department and Georgia State Patrol between June 16 and Monday:
Larry and Faye Lippard of Raeford, N.C., announce the engagement of Katie Lippard of Raleigh, N.C., to Bobby Watkins, Raleigh, N.C., son of Herb and Betty Watkins of Mansfield.
Mark Shields - 06/26/09 Health Care Reform: Partisan or Bipartisan?
All things being equal (which, as we all know, they never are), President Obama would rather, we are told, that the U.S. Senate pass 85 percent of his proposed health care reform with the backing of 70 senators than pass 100 percent of his plan with just 51 or 52 votes.
Venus victorious at Wimbledon Five-time champ wins despite knee trouble
WIMBLEDON, England - Even on one good leg, Venus Williams is tough to beat at Wimbledon.
No answers for young Newton All-Stars Team booted from tournament; coach seeking answers
COVINGTON - The 5-and-6-year-old Newton County All-Stars learned the hard way that not everyone likes winners after being kicked out of the Best of the Rest Tournament at Hunter Park in Douglas County on Saturday.
Community Calendar - 06/26/09
June 26
NCSO: 2 arrested during theft in progress
On the Beat is an occasional reporting of various crimes and complaints confronted by law enforcement personnel in Newton County in the course of their duties. It is compiled by News Editor Barbara Knowles, who can be reached at email@example.com.
New Level provides an opportunity Founder began program to help athletes play in college
CONYERS - Three years ago, Rick McCleskey came to Conyers with a vision: start a baseball training program that could help local players get an opportunity to play college baseball.
Trade joins Shaq, LeBron in Cleveland
CLEVELAND - One is the King, a reigning MVP who at age 24 needs only a championship to complete his resume. The other is a larger-than-life personality who may be past the prime of his career but remains an undeniable force and hungers for a fifth NBA title.
Tracy Lane Argo and Sgt. David Michael Sellars were married on June 13 in Newborn. The Rev. Deanna D. Lancaster officiated the ceremony.
Christina and Ray Prewitt of Newborn and Terry Bolden of Atlanta, announce the engagement of their daughter, Carrie Bolden, to David Long Jr., son of Kim Messier of Covington and David and Denise Long of Covington.


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Roasted Tomato Caprese
- Total Time:
- 1 hr 45 min
- Prep:
- 25 min
- Cook:
- 1 hr 20 min
- Yield:
- 6 servings
- Level:
- Easy
- Pesto:
- 4 cloves garlic
- 1 cup packed fresh basil leaves
- 1/2 cup freshly grated Parmesan
- 1 cup good olive oil
- 1/2 teaspoon kosher salt
- 1/2 teaspoon freshly ground black pepper
- 1 lemon, zested and juiced
- 4 large Roma tomatoes
- 4 tablespoons olive oil
- Salt and freshly ground black pepper
- 3 thin slices prosciutto, sliced into ribbons
- 2 large balls fresh buffalo mozzarella, sliced into 6 rounds
For the pesto: In a food processor chop the garlic and basil and puree until chopped finely. Add in the remaining ingredients and puree until smooth. Set aside until ready to use.
For the tomatoes: Preheat the oven to 375 degrees F. Slice the tomatoes into 3 pieces, each about 1/2-inch thick, being sure to slice off the tops and bottoms. Toss with olive oil, salt and pepper and then place onto a foil-lined baking sheet. Bake for 45 minutes to 1 hour. Set a side to cool.
For the prosciutto: Slice the prosciutto into ribbons and bake in a 350 degree F oven until crispy. About 20 minutes.
To plate: Place 2 slices roasted tomato caddy-wompus on your plate, top with fresh buffalo mozzarella, pesto and the crispy prosciutto.
Professional Recipe: This recipe was provided by a chef, restaurant or culinary professional and makes a large quantity. The Food Network Kitchens chefs have not tested this recipe in the proportions indicated and therefore cannot make any representation as to the results.
Recipe courtesy of Salt Kitchen & Fine Foods
Cooking Tips
More Recipes and Ideas
Stewed Scallions and Tomatoes
Recipe courtesy of Food Network Kitchen
Sliced Tomatoes with Honey-Balsamic Drizzle
Recipe courtesy of Robin Miller

subset: commoncrawl


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Around Americas non-stop..piracy report..world's largest yacht..more..
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26 Apr 2012
Around Americas non-stop..piracy report..world's largest yacht..more..
solo and non-stop around the Americas in a very ancient donated Albin Vega boat, so small he couldn't stand up in it, was the stuff of impractical dreamers.
Well, he did it, through the icy North West Passage, down the vast Pacific and around the dreaded Cape Horn. Both the boat and he were a little the worse for wear when he turned up in Annapolis, but it's not only an achievement it's heart-warming story of real grit and seamanship.
Talking of difficult sea routes, if you have a yen to explore where few have been before, Des Ryan tells us of
three possible sailing routes opening up through the Arctic
in the near future thanks to Global Warming.
annual piracy report
is out and makes interesting reading – it's also worth sparing a thought for the non-leisure sailors caught up in Somali piracy net. This is highlighted in a story this week of some
Yemeni sailors
on a traditional dhow who have just been released to the Yemeni coastguard thanks to EU naval forces patrolling the area.
Read about the
largest sailing yacht in the world, Dream Symphony
just being constructed in Turkey, made of wood! - and how the
oldest sailing junk
still in existence it finally going home to China.
Dream Symphony - artist's impression .. .
John Jamieson has some
Winch Wisdom
to spread around, there's a new
fuel purifier
(not a filter) on the market, and a
world-first personal device with AIS
is a big technological step up in MOB rescue. Of course, I have add there's no safety measure to beat just making sure you
stay on the boat
Sweet sailing!
Nancy Knudsen, Editor
Missing yacht Polska Miedz - call for help to cruising sailors
Sail-World Cruising,
On the 30th June 2011, Polish sailor Tomasz Chichocki departed on a solo sail around the world on his Delphia 40.3 yacht Polish Copper (Polska Miedz). It had taken eight years preparation, and he had a great following, particularly from his home country of Poland. Now he is missing somewhere in the Atlantic and all cruising sailors on his intended route are asked to assist.
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World-first AIS/GPS personal device approved for sale
David Pilvelait/Sail-World,
With the approval this week of the Safelink R10 SRS (Survivor Recovery System) by the USA's FCC (Federal Communications Commission), a new level of survivor recovery technology is reached. The small device, which combines GPS and AIS technology in a personal locator beacon, was the Overall Winner of the Pittman Innovation Awards, but had to wait approval before going on sale.
... [
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Sea Shepherd scout vessel Brigitte Bardot relaunched in Fremantle
Captain Locky Maclean,
The Sea Shepherd scout vessel Brigitte Bardot was relaunched in Fremantle near Perth in Western Australia on 16th April 2012 after three months of repairs needed from damage suffered by a rogue wave in the Southern Ocean. Ship's manager Simon Ager was euphoric, stating 'the last three and a half months of repairs by professionals and passionate volunteers alike have brought us to today,
... [
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Piracy down in Indian Ocean, but up in West Africa, Indonesia
International Chamber of Commerce,
Piracy in the waters east of Africa is slightly down, but it is still a no-go zone for yachts and a nervous crossing for ships. West African shores are experiencing increased piracy attacks, and there have been five kidnappings in Indonesia, according to the Global Piracy Report by the International Maritime Bureau.
... [
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Five simple steps to Winch Wisdom
John Jamieson,
Did you know that in just 15 knots of wind, a large 150% Genoa can exert over 750 pounds of pressure? Or that without the correct number of wraps around a winch, you will lose pulling and hauling power--and need to work harder than necessary?
... [
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Yacht Review: wooden Dream Symphony to be the largest yacht ever
Lee Mylchreest,
She purports to be the largest sailing boat ever built, yet, in a decision that classicists will love, she will be made of wood. The largest sailing boat in the world, a four-masted schooner, is currently being constructed near Marmaris in Turkey. She is such an amazing craft that she's our yacht of the week and she hasn't even touched the water yet.
... [
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Mystery Chinese sailing junk is finally going home
Sail-World Cruising round-up,
The Free China is an authentic Chinese sailing junk - the oldest surviving wooden Chinese sailing vessel in existence and the last of its kind. Its first fifty years are a mystery, but in 1955 the Free China made headlines when it beat all odds to cross the Pacific with a crew of five young Chinese men and one American diplomat. Now it is going home.
... [
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He's done it! Sailor sets Around-Americas solo sailing record
Nancy Knudsen,
He's done it! Matt Rutherford crossed the finish line at the Chesapeake Bay Tunnel Bridge in Norfolk, Va., at 10:48 a.m. Wednesday, becoming the first solo sailor to circumnavigate North and South America nonstop. Rutherford, who is sailing a 27-foot boat called the St. Brendan, steps on land for the first time today (Saturday) at noon during a public ceremony in Annapolis in Maryland, USA.
... [
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EU Warship Escorts Freed Dhow to Yemeni Coast
EU NAVFOR/Sail-World,
Dhows have for centuries been sailing the coasts of Africa and the Middle East, for fishing and for transport. But today's dhow crews are also vulnerable to the attacks of Somali pirates Unlike the rest of us, they depend completely on these waters. So there is always great joy when the crew of a dhow is freed by the efforts of the coalition and EU forces in the Indian Ocean.
... [
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Product of the Week: New MLS Fuel Purifier
Sail-World Cruising,
This week's product of the week addresses a major problem for cruising sailors, especially when they start to leave home waters, or even as a result of the use of marine fuel containing Ethanol, in jurisdictions where that applies. It's the MLS Fuel Purifier, which is NOT a filter.
... [
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Sailing the Arctic - the opening sea routes
Des Ryan,
Want to sail the Arctic? With global warming gradually changing the profile of the world's northern seaways, many of the world's seafaring nations are looking at the northern sea routes for alternative shipping routes. But where a ship may go, so may a yacht, so if you're after adventure, there are routes possible that one could have only dreamed of once.
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Insulin-like growth factor (IGF)-1 is a key factor in bone homeostasis and could be involved in bone tissue sclerosis as observed in osteoarthritis (OA). Here, we compare the key signaling pathways triggered in response to IGF-1 stimulation between normal and OA osteoblasts (Obs). Primary Obs were prepared from the subchondral bone of tibial plateaus of OA patients undergoing knee replacement or from normal individuals at autopsy. Phenotypic characterization of Obs was evaluated with alkaline phosphatase and osteocalcin release. The effect of IGF-1 on cell proliferation, alkaline phosphatase and collagen synthesis was evaluated in the presence or not of 50 ng/ml IGF-1, whereas signaling was studied with proteins separated by SDS-PAGE before western blot analysis. We also used immunoprecipitation followed by western blot analysis to detect interactions between key IGF-1 signaling elements. IGF-1 receptor (IGF-1R), Shc, Grb2, insulin receptor substrate (IRS)-1, and p42/44 mitogen-activated protein kinase (MAPK) levels were similar in normal and OA Obs in the presence or absence of IGF-1. After IGF-1 stimulation, the phosphorylation of IGF-1R in normal and OA Obs was similar; however, the phosphorylation of IRS-1 was reduced in OA Ob. In addition, the PI3K pathway was activated similarly in normal and OA Obs while that for p42/44 MAPK was higher in OA Obs compared to normal. p42/44 MAPK can be triggered via an IRS-1/Syp or Grb2/Shc interaction. Interestingly, Syp was poorly phosphorylated under basal conditions in normal Obs and was rapidly phosphorylated upon IGF-1 stimulation, yet Syp showed a poor interaction with IRS-1. In contrast, Syp was highly phosphorylated in OA Obs and its interaction with IRS-1 was very strong initially, yet rapidly dropped with IGF-1 treatments. The interaction of Grb2 with IRS-1 progressively increased in response to IGF-1 in OA Obs whereas this was absent in normal Ob. IGF-1 stimulation altered alkaline phosphatase in Ob, an effect reduced in the presence of PD98059, an inhibitor of p42/44 MAPK signaling, whereas neither IGF-1 nor PD98059 had any significant effect on collagen synthesis. In contrast, cell proliferation was higher in OA Obs compared to normal under basal conditions, and IGF-1 stimulated more cell proliferation in OA Obs than in normal Ob, an effect totally dependent on p42/44 MAPK activiy. The altered response of OA Obs to IGF-1 may be due to abnormal IGF-1 signaling in these cells. This is mostly linked with abnormal IRS-1/Syp and IRS-1/Grb2 interaction in these cells.
Osteoarthritis (OA) represents a major cause of disability, particularly among the aging population; indeed, it is the most common form of joint disease. OA is a multifactorial disease characterized by loss of articular cartilage and subchondral plate thickening [1]. As the loss of articular cartilage is believed to be the initial event responsible for joint destruction, numerous investigations have focused their efforts on understanding cartilage homeostasis. Therefore, biochemical analysis of the underlying bone tissue has received little attention, despite several reports of abnormal subchondral bone metabolism in OA.
Radin and coworkers were the first to study subchondral bone changes in patients with early degenerative joint disease, and to propose the participation of subchondral bone in the initiation and progression of cartilage damage [2,3]. They proposed that the thickening of the subchondral bone plate, resulting from repeated healing of microfractures, could be a key initiation factor in cartilage degradation. Other groups also reported abnormal cancellous bone collagen metabolism in OA, demonstrating that type I collagen, the most abundant bone matrix protein, is abnormal in OA [4,5]. Recently, we demonstrated that osteoblast (Ob) cells from human subchondral OA bone demonstrate an altered phenotype in vitro. Our results showed increased alkaline phosphatase activity, release of osteocalcin, an increased activity of urokinase plasminogen activator (uPA) with no changes in plasminogen activator inhibitor-1 (PAI-1) abundance, and increases in insulin-like growth factor (IGF)-1 release compared to normal Obs [6-8]. As IGF-1 production is increased in OA Obs compared to normal Obs, it is a likely candidate to promote bone remodeling and sclerosis in OA. Interestingly, our laboratory also demonstrated the presence of abnormal uPA regulation by IGF-1 in human OA Obs [6]. These results suggest that IGF-1 signaling could be altered in these cells [6]. The increased remodeling in OA bone could possibly account for the observation of hypomineralization of the subchondral bone tissues in established OA [9-11]. Not only the bone matrix is altered in OA but recent studies have demonstrated that a putative factor(s) produced by OA subchondral bone cells can influence cartilage metabolism [12]. This could possibly explain why increased subchondral bone activity can predict cartilage loss [13-15].
After binding of IGF-1 to its specific surface receptor, the IGF-1 receptor (IGF-1R) kinase undergoes tyrosine phosphorylation of its α-subunit and kinase activation. This involves the phosphorylation of tyrosine residues of substrate adaptor proteins, principally the insulin receptor substrate (IRS)-1. Other targets have also been identified, such as Shc, IRS-2, IRS-3 and IRS-4 and GAB1 [16,17]. These proteins contain insulin/IGF-1R-specific tyrosine phosphorylation sites responsible for their association with various SH2 domain-containing downstream effector molecules. In the case of IRS-1, these include binding sites for phosphatidylinositol 3-kinase (PI3K), protein tyrosine-specific phosphatase Syp, 14.3.3 proteins, and the small adaptor protein Grb2, which is responsible for the activation of Ras and the MAPK pathway [18,19].
Thus, as the response to IGF-1 in human OA Obs is abnormal, we investigated IGF-1 signaling in OA Obs. Data revelead an abnormal interaction of phospho-Syp with IRS-1, possibly leading to decreased IRS-1 activity. Moreover, the interaction of Grb2 with IRS-1 was abnormal in OA Obs, possibly leading to altered downstream signaling. These data suggest that an abnormal response to IGF-1 is linked to abnormal intracellular signaling, affecting multiple pathways in OA osteoblasts.
Materials and methods
Patients and clinical parameters
Subchondral bone was obtained from OA patients who had undergone total knee replacement surgery. Specifically, medial tibial plateaus were dissected away from the remaining cartilage and trabecular bone under sterile conditions as previously described [6-8,20]. Only the middle portion of the tibial plateaus was used to separate the subchondral bone plate specimens. A total of 40 patients (aged 70.8 ± 7.9 years (mean ± standard deviation (SD)); 18 males, 22 females) classified as having OA according to recognized American College of Rheumatology clinical criteria were included in this study [21]. None of the patients had received medication that would interfere with bone metabolism, including corticosteroids, for six months before surgery. A total of 16 bone specimens of medial tibial plateaus from normal individuals (aged 61.5 ± 15.6 years (mean ± SD); 9 males, 7 females) were collected at autopsy within 12 h of death. These were used following the establishment that the donors had not been on any medication that could interfere with bone metabolism or had any bone metabolic disease. Individuals showing cartilage deterioration and/or subchondral bone plate sclerosis were not included in the normal group. All human material was acquired following a signed agreement by patients undergoing knee surgery or their relatives for the specimens collected at autopsy following the Centre Hospitalier de l'Université de Montréal (CHUM) ethical committee guidelines.
Preparation of primary bone cell culture
The subchondral bone plate was dissected away from the remaining cartilage and trabecular bone under sterile conditions. Isolation of subchondral bone plate and the cell cultures were prepared as previously described by a collagenase digestion procedure [6,7,22,23]. At confluence, cells were passaged once at 25,000 cells/cm2 in 6 well plates and grown for 5 days in Ham F12/DMEM (Sigma, St-Louis, MO, USA) containing 10% FBS (Wisent Inc., St Bruno, Quebec, Canada) before specific assays. Under these culture conditions, Obs expressed bone-specific type I collagen without any contaminations with cartilage-specific type II collagen [23]. Conditioning was performed for an additional 24 h in serum free Ham F12/DMEM media. Confluent cells were incubated or not with human IGF-1 (50 ng/ml; Peninsula, Belmont, CA, USA) for different times as specified per individual experiments. In some experiments, PD98059 (Sigma-Aldrich), an inhibitor of the MAP-kinase/Erk kinase pathway, was used at a final concentration of 10 μM in the presence or absence of IGF-1, and controls were treated with the vehicle. Supernatants were collected at the end of the incubation and kept at -80°C prior to assays. Cells were lysed with RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.5% Na-deoxicholate, 0.1% SDS, 150 mM NaCl, with the inhibitors 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml O-phenatroline, 1 mM Na-orthovanadate, 1 mM dithiothreitol), and kept at -80°C prior to assays. Protein determination was performed by the bisinchoninic acid method [24].
Western immunoblotting and immunoprecipitation
The cell lysates were loaded on polyacrylamide gels and separated by SDS-PAGE under reducing conditions [25]. Loading was adjusted according to the cellular protein concentration of each specimen and western blotting of actin was performed to assess similar loading between samples. The proteins were electrophoretically transfered onto PVDF western blotting membranes (Boehringer Mannheim, Penzberg, Germany). Immunoblotting was performed as described in the ECL Plus Western blotting detection system's manual (Amersham Pharmacia Biotech, Baie d'Urfe, Québec, Canada) using a variety of primary and secondary antibodies. Primary antibodies included: IGF-1R α-subunit (ab-5, 1:10,000 dilution) from NeoMarkers (Fremont, CA, USA); phosphorylated IGF-1R (pY1158, 1:10,000 dilution) from Biosource International (Camarillo, CA, USA); IRS-1, IRS-2, Grb2 and PI3K (1:2,000 dilutions) from Upstate Biotechnology (Lake Placid, NY, USA); phospho IRS-1 (pS307, 1:1,000 dilution) from Upstate Biotechnology; phosphotyrosine Ab-1 (Clone PY-20, 1:15,000 dilution) from NeoMarkers; Shc, and Gab1 (1:5,000 dilutions) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); phosphorylated Shc (Tyr317, 1:10,000 dilution), protein kinase B (PKB), phosphorylated PKB (Ser473, 1:1,000 dilution), p42/44 and phosphorylated p42/44 (Thr202/Tyr204, 1:5,000 dilutions) from Cell Signaling Technology (Beverly, MA, USA); Syp from BD Transduction Laboratories (Mississauga, ON, Canada); and actin (1:10,000 dilution) from Sigma-Aldrich. Secondary antibodies included: goat anti-mouse IgG (1:100,000 dilution) from Pierce (Rockfort, IL, USA); rabbit anti-sheep IgG (1:20,000 dilution) and goat anti-rabbit IgG (1:10,000 dilution) from Upstate Biotechnology. The immunoprecipitation was performed with 175 μg of the solubilized cell extracts incubated with 4 μg of IRS-1 or 0.5 μg of IGF-1R α-subunit (Ab-4) antibody, overnight at 4°C. The resulting immunoprecipitates were subjected to SDS-PAGE as described above. Densitometry analysis of western blot films was performed on a Macintosh Mac OS 9.1 computer using the public domain NIH Image program developed at the US National Institutes of Health with the Scion Image 1.63 program [26].
Real-time RT-PCR quantification
Real-time quantification of Bax-α, Bcl2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA from normal and OA Obs treated for 6 h with 100 ng/ml IGF-1 was performed in the GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with the 2× Quantitect SYBR Green PCR Master Mix (Qiagen, Mississauga, ON, Canada) used according to the manufacturer's specifications. In brief, 100 ng of the cDNA obtained from the RT-PCR reactions were amplified in a total volume of 50 μl consisting of 1× Master Mix, 0.5 Unit uracil-N-glycosylase (UNG; Epicentre Technologies, Madison, WI, USA) and the gene-specific primers added at a final concentration of 200 nM. The specific primers were: for Bax-α, 5'-GGA TGC GTC CAC CAA GAA G-3' (sense) and 5'-CAC CAG TTT GCT GGC AAA G-3' (antisense); and for Bcl2, 5'-GGC ATC TTC TCC TCC CAG C-3' (sense) and 5'-GAA GGG CGT CAG GTG CA-3' (antisense); these generated fragments of 208 base-pairs (bp) and 202 bp, respectively. To ensure equivalent loading, GAPDH was amplified using 20 pmol of each of the primers 5'-CAG AAC ATC ATC CCT GCC TCT-3' (sense) and 5'-GCT TGA CAA AGT GGT CGT TGAG-3' (antisense) to generate a predicted amplified sequence of 318 bp [27]. The tubes were first incubated for 2 minutes at 50°C (UNG reaction), then at 95°C for 15 minutes (UNG inactivation and polymerase activation) followed by 40 cycles consisting each of denaturation (94°C for 15 s), annealing (60°C for 30 s), extension (72°C for 30 s) and data acquisition (77°C for 15 s) steps. The data were collected and processed with the GeneAmp 5700 SDS software (Applied Biosystems, Foster City, CA, USA) and given as threshold cycle (Ct), corresponding to the PCR cycle at which an increase in reporter fluorescence above baseline signal can first be detected. Plasmid DNAs containing the target gene sequences were used to generate the standard curves. When comparing normal and OA expression levels, the threshold cycle (Ct) was converted to number of molecules and the values for each sample calculated as the ratio of the number of molecules of the target gene/number of molecules of GAPDH.
Phenotypic characterization of human subchondral osteoblast cell cultures
Phenotypic features of Obs cultures were determined by evaluating 1,25(OH)2D3-dependent (50 nM) alkaline phosphatase activity and osteocalcin release, and by the production of collagen type 1. Alkaline phosphatase activity was determined on cell aliquots by substrate hydrolysis using p-nitrophenylphosphate, and osteocalcin release was determined in cell supernatants using an enzyme immunoassay as previously described [7,20]. Collagen synthesis was determined as the de novo release of the carboxy-terminal peptide fragment (CICP) of collagen type 1, reflecting true collagen synthesis. This fragment was determined using a selective enzyme-linked immunosorbent assay (ELISA; Quidel Corporation, Cederlane, Hornby, ON, Canada) in conditioned media from confluent Obs incubated in Ham F12/DMEM media containing 0.5% BSA. Cellular proliferation was assessed using the bromodeoxyuridine (BrdU) cell proliferation assay as described in the system's manual from Calbiochem (San Diego, CA, USA). Cells were plated at 10,000 cells/cm2 in 96-well plates in Ham F12/DMEM media containing 10% FBS. After overnight attachment, cells were serum-starved in Ham F12/DMEM media containing 0.5% BSA for 24 h prior to stimulation with or without 50 ng/ml IGF-1 in the presence or absence of 10 μM PD98059 in the same media.
Statistical analysis
All quantitative data are expressed as mean ± SEM. The data were analyzed by Student's t-test, and p values < 0.05 were considered statistically significant.
Phenotypic characteristics of normal and OA osteoblasts and response to IGF-1
The phenotypic characteristics of normal and OA Obs were first determined by the production of alkaline phosphatase and release of osteocalcin. Values for normal Obs were 575.6 ± 89.4 nmol/mg protein/30 minutes and 141.6 ± 15.5 ng/mg protein for alkaline phosphatase and osteocalcin, respectively. These values were increased in OA Obs and reached 1259.2 ± 158.0 nmol/mg protein/30 minutes (p < 0.01 versus normal) and 261.2 ± 23.5 nmol/mg protein (p < 0.005 versus normal) for alkaline phosphatase and osteocalcin, respectively, as previously described [6-8]. OA osteoblasts also synthesized more collagen type I than normal Obs (3254 ± 272 versus 4246 ± 189 for normal and OA, respectively, p < 0.01) whereas they failed to produce collagen type II as previously shown [20].
Cell proliferation was higher in OA Obs compared to normal Obs (p < 0.015), and IGF-1 promoted cell proliferation in both normal and OA Obs while the effect was more important in OA Obs than normal Obs (Figure 1). This effect of IGF-1 was blocked by PD98059, an inhibitor of the MAPK/Erk-kinase pathway, and cell proliferation rate returned to basal values. IGF-1 stimulation also increased alkaline phosphatase activity in both normal and OA Obs. In normal Obs, IGF-1 raised alkaline phosphatase activity from 504.9 ± 83 nmol/mgprotein/30 minutes to 625.4 ± 36.1 (23.9% increase) whereas this activity in OA Obs increased from 712.9 ± 154.9 to 1099.4 ± 346.6 nmol/mg protein/30 minutes (44.8% increase). This effect was blocked by PD98059 in OA Obs (Figure 2). However, IGF-1 failed to stimulate collagen type I synthesis in both normal and OA Obs, and PD98059 was without effect on this parameter (not illustrated). We also evaluated the expression of Bax-α and Bcl2 to evaluate the apoptotic potential of these cells [28-30]. Using quantitative RT-PCR we were able to demonstrate a reduction in the Bax-α to Bcl2 ratio in OA Obs compared to normal. Indeed, the ratio of Bax-α to Bcl2 was 22.6 ± 3.4 versus 12.2 ± 2.4 relative units in normal (n = 3) and OA Obs (n = 3), respectively (p < 0.05).
Figure 1. Cell proliferation of normal and osteoarthitis (OA) osteoblasts in response to insulin-like growth factor (IGF)-1 stimulation. Cells were plated at 10,000 cells/cm2 in 96-well plates and incubated overnight in Ham F12/DMEM media containing 10% FBS. Cells were then serum-starved for 24 hours in the same media containing 0.5% BSA then treated with or without 50 ng/ml IGF-1 in the presence or not of 10 μM PD98059 (PD) for 24 hours followed by incubation with bromodeoxyuridine (BrdU) for their last 24 hours of incubation in the same media. BrdU incorporation was then evaluated following the manufacturer's instructions. Results are expressed as mean OD units ± SEM for three normal and four OA osteoblast preparations.
Figure 2. Effect of insulin-like growth factor (IGF)-1 on alkaline phosphatase activity in osteoarthitis (OA) osteoblasts. Cells were grown to confluence and incubated overnight in serum free medium. Cells were then exposed to 50 nM 1,25(OH)2D3 in Ham F12/DMEM media containing 2% charcoal-treated FBS and in the presence or not of 50 ng/ml IGF-1 with or without 10 μM PD98059 (PD). Results are expressed as the mean ± SEM of control values without IGF-1 for 6 OA osteoblast preparations.
IGF-1R levels and activation are similar in normal and osteoarthitis osteoblasts
IGF-1R levels in normal and OA Obs were evaluated by western immunoblot analysis. As presented in Figure 3a, no significant difference could be observed between normal (n = 5) and OA (n = 5) Obs IGF-1R levels under basal conditions. The phosphorylation of IGF-1R following IGF-1 stimulation was very rapid and similar in normal (n = 3) and OA (n = 3) Obs and increased 2.86 ± 0.36 and 2.52 ± 0.06-fold, respectively, above basal levels after 30 s (Figure 3b), and phosphorylated IGF-1R levels remained similar until 5 minutes of stimulation with IGF-1 (data not shown).
Figure 3. Insulin-like growth factor (IGF)-1 receptor (IGF-1R) levels and activation in normal and osteoarthitis (OA) osteoblasts. Cells were grown to confluence and incubated overnight in serum free medium. (a) After cell lysis in RIPA buffer and immunoprecipitation with an anti-IGF-1R α-subunit antibody (ab 4), receptor levels were analyzed by western immunoblot with an anti-IGF-1R α-subunit antibody (ab 5). (b) After cell lysis in RIPA buffer, tyrosine phosphorylation of the receptor exposed to 50 ng/ml IGF-1 for 5 minutes was analyzed with anti-IGF-1R phosphotyrosine ab (PY-1158). Determination of actin was used as the control for loading. Representative data are shown in panel 1 (a,b). Results expressed as arbitrary scanning units are presented as the mean ± SEM (panel 2 (a,b)).
IRS-1 is underphosphorylated in osteoarthitis osteoblasts
Figure 4a shows that IRS-1 levels in normal (n = 4) and OA (n = 4) Obs were similar. However, the tyrosine phosphorylation of IRS-1 was significantly altered in OA Obs compared to normal. Under basal conditions, the tyrosine phosphorylation of IRS-1 in OA Obs was 4.65 ± 1.24-fold less phosphorylated than normal (p < 0.05), and after a 5 minute stimulation with IGF-1, this level was still about 3.52 ± 0.35-fold less phosphorylated (p < 0.005; Figure 4b). Serine phosphorylation of IRS-1 in OA Obs was similar to normal (data not shown). As we were unable to detect clear levels of IRS-2 in our Obs, we excluded the possibility that increasing IRS-2 activation could lead to downregulation of IRS-1 (data not shown). Moreover, 14.3.3 protein, which is known to bind IRS-1 and modulate its activation, was not significantly different between normal and OA Obs (data not shown).
Figure 4. Insulin receptor substrate (IRS)-1 levels and activation in normal and osteoarthitis (OA) osteoblasts. Cells were grown to confluence and incubated overnight in serum free medium. Cells were treated with 50 ng/ml insulin-like growth factor (IGF)-1 for 5 minutes. (a) After cell lysis in RIPA buffer and immunoprecipitation with anti-IRS-1 antibodies, IRS-1 levels were analyzed by immunoblotting with the same anti-IRS-1 antibody. (b) After cell lysis in RIPA buffer and immunoprecipitation with an anti-IRS-1 antibody, phosphorylated IRS-1 levels were analyzed by immunoblotting with an anti-phosphotyrosine antibody (PY-20). Determination of actin was used as the control for loading. Representative data are shown in panel 1 (a,b). Results expressed as arbitrary scanning units are presented as the mean ± SEM (panel 2 (a,b)).
p42/44 phosphorylation are upregulated while PI3K/PKB pathway is unaltered in OA Obs
Since IGF-1 signaling involves the downstream activation of two different pathways, namely the MAPK and the PI3K pathways, we next evaluated if these pathways were altered similarly to IRS-1. We first looked at the protein levels of p42/44. Data showed no differences between normal (n = 3) and OA (n = 4) Obs (Figure 5a). In contrast, the phosphorylation of p42/44 was different between normal and OA Obs. After a 5 minute IGF-1 treatment, both p42 and p44 phosphorylation levels in OA Obs were increased by about 1.53 ± 0.15 (p < 0.025) and 2.18 ± 0.37 (p < 0.05) fold, respectively, compared to normal (Figure 5b). This increase in p42/44 phosphorylation is consistent with the observation that OA Obs grow faster than normal Obs (this study and [7]) and show reduced apoptosis (see above). Phosphorylation of p42/44 was sustained until 15 minutes, when a difference between normal and OA Obs could still be observed (data not shown). PI3K protein levels were unaltered between normal (n = 3) and OA Obs (n = 3) (data not shown). Similarly, PKB protein levels in normal (n = 3) and OA (n = 3) Obs were similar. Moerover, although the phosphorylation of PKB was slightly upregulated in OA (19.2 ± 1.0 relative units, n = 3) compared to normal (14.1 ± 1.5 relative units, n = 3) after a 5 minute IGF-1 stimulation, this difference did not reach statistical significance.
Figure 5. p42/44 levels and activation in normal and osteoarthitis (OA) osteoblasts. Cells were grown to confluence and incubated overnight in serum free medium. Cells were then exposed to 50 ng/ml insulin-like growth factor (IGF)-1 for 5 minutes. (a) After cell lysis in RIPA buffer p42/44 levels were visualized by immunoblotting with an anti-p42/44 antibody. (b) After cell lysis in RIPA buffer phosphorylated p42/44 levels were visualized by immunoblotting with an anti-phospho p42/44 antibody. Determination of actin was used as the control for loading. Representative data are shown in panel 1 (a,b). Results expressed as arbitrary scanning units are presented as the mean ± SEM (panel 2 (a,b)).
Shc and Grb-2 protein levels and activation are similar in normal and osteoarthitis osteoblasts
As Shc can also be recruited by IGF-1R and activate downstream SH2-domain containing proteins, we also evaluated the activation of this alternative pathway. No differences were found between total Shc protein levels in normal (n = 4) and OA Obs (n = 4). The Shc phosphorylation levels were also not significantly different between normal and OA Obs after a 5 minute IGF-1 stimulation (data not shown). This suggests that this pathway was unaltered in OA Obs. We also examined the levels of the small adaptor protein Grb2. Data illustrated in Figure 6a showed no difference between normal and OA Obs under basal conditions. Upon IGF-1 stimulation, Grb2 levels did not change either. However, as Grb2 can be associated with either Shc or phosphorylated IRS-1, we next evaluated the levels of Grb2 associated with the IRS-1 pathway using a co-immunoprecipitation strategy. As shown in Figure 6b, co-immunoprecipitation with IRS-1 followed by western blot detection of Grb2 showed weak Grb2 levels in both normal (n = 4) and OA (n = 4), although higher levels could be detected in OA Obs (4.76 ± 1.44-fold increased levels in OA Obs compared to normal, p < 0.05) under basal conditions. Grb2 levels associated with IRS-1 did not change significantly in normal Obs in response to 30 s of IGF-1 stimulation while the levels decreased with a longer incubation in the presence of IGF-1. In OA Obs, co-immunoprecipitated Grb2 levels increased slowly in response to IGF-1 stimulation; however, Grb2 levels reached about a 2.6 ± 0.8-fold increase compared to basal levels after a 5 minute IGF-1 stimulation (p < 0.03 by ANOVA) in OA Obs. A significant difference was also noted between Grb2 levels upon a 5 minute IGF-1 stimulation in OA Obs compared to normal (p < 0.01).
Figure 6. Grb2 levels and co-immunoprecipitation with insulin receptor substrate (IRS)-1 in normal and osteoarthitis (OA) osteoblasts. Cells were grown to confluence and incubated overnight in serum free medium. (a) After cell lysis in RIPA buffer Grb2 levels were visualized by immunoblotting with an anti-Grb2 antibody. The data show a representative immunoblot for three normal and three OA osteoblast preparations. (b) In another set of experiments, cells were exposed to 50 ng/ml insulin-like growth factor (IGF)-1 for various length of time or its vehicle, as indicated. After cell lysis in RIPA buffer and immunoprecipitation with an anti-IRS-1 antibody, co-immunoprecipitated Grb2 levels were visualized by immunoblotting with an anti-Grb2 antibody. A representative immunoblotting of four different experiments is presented. Determination of actin was used as the control for loading.
Abnormal Syp modulation in OA Obs is implicated in underphosphorylation of IRS-1
Syp is a phosphatase known to bind IRS-1 and, once phosphorylated on its tyrosine residues, modulate its activity. We first compared Syp levels between normal and OA Obs. As observed with the other proteins, no differences were noted for Syp levels between normal (n = 3) and OA Obs (n = 4) as shown in Figure 7a. Surprisingly, time-dependent Syp phosphorylation in response to IGF-1 was clearly different between normal and OA Obs. As illustrated in Figure 7b, we observed the expected activation pattern with normal Obs (n = 3) in response to a 1 minute IGF-1 stimulation, increasing the phosphorylation levels of Syp by 2.7 ± 1.0-fold. On the other hand, in OA Obs (n = 5), basal Syp phosphorylation was already higher (12.23 ± 3.09 fold, p < 0.025) compared to normal. After a 1 minute IGF-1 stimulation, Syp phosphorylation in OA Obs decreased by 11.94 ± 5.93-fold, which reduced the phosphorylated Syp levels in OA Obs below those in normal Obs (p < 0.015). To demonstrate the implication of Syp phosphatase in the underphosphorylation of IRS-1 in the resting state, we next performed a series of co-immunoprecipitation assays to evaluate the interaction of Syp with IRS-1. As shown in Figure 8a, there was little Syp associated with IRS-1 in the basal state for normal Obs (n = 4). However, after a 30 s stimulation with IGF-1, there was a marked increased association of Syp with IRS-1 in normal Obs that returned to near basal state after 5 minutes. In OA Obs, the initial Syp levels associated with IRS-1 are higher than normal (approximately four-fold) and decreased rapidly to near undetectable levels upon a one minute IGF-1 stimulation (Figure 8b). These results suggest that the low IRS-1 phosphorylation levels observed in basal OA Obs could result from the increased association and activation of Syp phosphatase to IRS-1.
Figure 7. Syp activation in normal and osteoarthitis (OA) osteoblasts. Cells were grown to confluence and incubated overnight in serum free medium. Cells were then exposed to 50 ng/ml insulin-like growth factor (IGF)-1 for 5 minutes. (a) After cell lysis in RIPA buffer Syp levels were visualized by immunoblotting with an anti-Syp antibody. (b) After cell lysis in RIPA buffer and immunoprecipitation with an anti-phosphotyrosine antibody (PY-20), phosphorylated Syp levels were visualized by immunoblotting with an anti-Syp antibody. Determination of actin was used as the control for loading. Representative data are shown in panel 1 (a,b). Results expressed as arbitrary scanning units are presented as the mean ± SEM (panel 2 (a,b)).
Figure 8. Co-immunoprecipitation (Co IP) of Syp with insulin receptor substrate (IRS)-1 in normal and osteoarthitis (OA) osteoblasts. Cells were grown to confluence and incubated overnight in serum free medium. Cells were then exposed to 50 ng/ml insulin-like growth factor (IGF)-1 for various lengths of time, as indicated. After cell lysis in RIPA buffer and IP with an anti-IRS-1 antibody, co-immunoprecipitated Syp levels were visualized by immunoblotting with an anti-Syp antibody. A representative immunoblotting of three different experiments is presented. Determination of actin was used as the control for loading.
As an increasing amount of literature is demonstrating the involvement of bone tissue in the initiation/progression of OA, a better understanding of this tissue is clearly of utmost importance to better understand the etiology of this pathology. IGF-1 is one of the leading growth factors implicated in bone remodeling [31]. Interestingly, IGF-1 expression is increased in OA Obs [32] and these cells present an abnormal response to this growth factor [6,7]. Hence, we wanted to know if this is related to an alteration of its signaling pathway. We conducted a series of experiments to evaluate the IGF-1 signaling pathway in OA Obs and data revealed that the interaction of IRS-1 with Syp and Grb2 was modified in these cells in response to IGF-1 stimulation.
IGF-1R levels as well as phosphorylated IGF-1R levels following IGF-1 stimulation were similar in normal and OA Obs. However, IRS-1, the major IGF-1R docking protein, presented a reduced phosphorylation level in OA Obs, albeit the total protein level was similar between normal and OA Obs. In contrast, the IGF-1R docking protein Shc had similar protein and phosphorylation levels in normal and OA Obs. As IRS-1 was the only IGF-1R docking protein showing an abnormal modulation in OA Obs, we pursued our investigation with this factor and looked for molecules that could regulate IRS-1 phosphorylation.
First, 14.3.3 protein, which is known to bind IRS-1 and modulate its activation [19], was not significantly different between normal and OA Obs, and thus is unable to explain the underphosphorylation of IRS-1 in OA. Second, as the phosphorylation of both Shc and IRS-1 is linked to IGF-1R kinase activation and the phosphorylation of Shc was similar between normal and OA Obs, we can not conclude that an abnormal IGF-1R kinase activation explains the reduced phosphorylation of IRS-1. Third, we looked for phosphatases able to modulate IRS-1 activity. The best characterized phosphatase that binds to and modulates IRS-1 is Syp (or SHP-2) [33]. Syp protein levels were unaltered between normal and OA Obs. In contrast, its phosphorylation levels clearly demonstrated abnormal regulation in OA Obs. Indeed, OA Obs showed increased basal phosphorylation levels compared to normal, which was followed with a rapid decrease upon IGF-1 stimulation, in contrast to the situation for normal Obs. Moreover, the co-immunoprecipitation of Syp/IRS-1 also demonstrated an increased interaction in the basal state between IRS-1 and Syp in OA Obs, unlike in normal cells. Since there is an increase in IGF-1 production in OA Obs with a concomitant decrease of the major insulin-like growth factor binding proteins, namely BP-3, BP-4 and BP-5, OA Obs are likely to be more chronically stimulated by IGF-1 than normal Obs [32]. This suggests that the dowregulation of IRS-1 in OA Obs, the major IGF-1 signaling pathway, is a feedback response to increased exposure to elevated endogenous IGF-1 levels.
The observed increase in ERK1/2 phosphorylation, while there was no significant increase in PI3K activity measured by Akt/PKB phosphorylation, should promote an increase in cell proliferation. In this respect, it is noteworthy that primary OA Ob cell cultures grow faster than normal Obs [7], and we observed a reduction of the ratio of expression of Bax-α in OA Obs compared to normal. As Bax-α promotes apoptosis, a reduction in the Bax-α/Bcl2 ratio suggests an inhibition of apoptosis in OA Obs [30]. In the present study we also observed that OA Obs can grow faster than normal Obs and that they respond to IGF-1 stimulation with a greater cellular proliferation rate. This response to IGF-1 was strictly ERK1/2 dependent since PD98059 was able to fully inhibit the effect of IGF-1 on OA Obs. Such a situation could then lead to more cells being available and prolonged cell life that would possibly lead to the laying down of more extracellular matrix, as reported in OA subchondral bone tissue [4,5]. This also agrees with the recent demonstration that Obs from OA patients show enhanced proliferation and collagen type I expression in vitro compared to normal Obs [34]. However, addition of exogenous IGF-1 to OA Obs failed to increase collagen type I levels, which are already higher in these cells than normal. In contrast, as IGF-1 promoted alkaline phosphatase production by Obs, it is noteworthy that it stimulated this activity better in OA Obs than in normal Obs, and that this was also dependent on ERK1/2 activity. Similar observations were previously reported for both activities in Ob-like cells [35] and for cell proliferation alone in mesangial cells [36]. Overall, these data would then suggest that the activation of the ERK1/2 pathway in OA Obs in response to IGF-1 is important for cell proliferation, retards apoptosis and affects alkaline phosphatase. This could also promote the production of collagen type I overall as more cells would synthesize it, resulting in more collagen being laid down in vivo, although we could not show that more collagen was produced per cell in vitro in response to IGF-1. This is reminiscent of observations made in other tissues where IGF-1 alone could not promote collagen type I production but, in combination with other growth factors or high glucose levels, could do so [35-39].
One important question remains: if the main IGF-1R signaling pathway is dowregulated in OA Obs, how can we explain the increase in subchondral bone remodeling in OA bone tissue? Maybe the IGF-1 pathway is not implicated in this process. However, this seems unlikely since IGF-1 is a key regulator of bone remodeling and it increases uPA activity in OA Obs [6]. Once stimulated, the tyrosine kinase activity of IGF-1R leads to its autophosphorylation as well as the phosphorylation of a number of intracellular proteins, such as IRS-1, Shc and Gab1. This gives rise to the activation of Ras and PI3K, thus resulting in the activation of MAPK and PKB [40]. In our diseased cells, PI3K, MAPK and PKB protein levels were similar to normal, and we detected no significant increase in the activation of PKB while we observed a clear stimulation of the MAPK pathway. This implies the possibility of some complementary pathways for PKB activation that compensate for the lack of activation via IRS-1 in OA Obs. We recently demonstrated that transforming growth factor (TGF)-β production is increased in OA Obs [20] and, since this growth factor can activate PKB in arthritis, TGF-β stimulation could be one such compensatory mechanism activating PKB. Interestingly, IGF-1 dependent p42/44 stimulation was significantly increased in OA Obs. Since the activation of the Ras/MAPK pathway can modulate the production of uPA following growth factor stimulation, a situation we already observed in OA Obs [7], this suggests that the increased remodeling observed in OA subchondral bone could result from the upregulation of the p42/44 pathway following IGF-1 stimulation [41].
Increased activation of the p42/44 pathway concomitant with the dowregulation of IRS-1 in OA Obs may seem contradictory. One possible mechanism could be competition between IRS-1 and Shc for Grb2 [17]. In this model, IRS-1 and Shc compete for a limited cellular pool of Grb2, and the activation of the MAPK pathway would predominantly occur through the Shc-Grb2 signaling pathway. Grb2 is a small adaptor protein that can associate with IRS-1 and Shc via its SH2 domain and with the guanylnucleotide exchange factor for Ras, termed Son of Sevenless (SOS) via its SH3 domain. The association of the Grb2-SOS complex with tyrosine phosphorylated receptors and/or Shc have been directly implicated in the activation of the Ras signaling pathway. Since in OA Obs Grb2 levels were similar to normal, the downregulation of IRS-1 phosphorylation following IGF-1 stimulation in these diseased cells may result in an increased availability of Grb2 to the Shc pathway, leading to increased activity of the p42/44 pathway. However, as shown here, the interaction of Grb2 with IRS-1 was also increased in OA Obs, implying that the Grb2-Shc interaction should be reduced in these cells. On the other hand, the p42/44 kinase activity could also be activated directly by TGF-β via the Smad3 signaling pathway, as previously proposed by Sowa and colleagues [42], a situation that overules Grb2-Shc signaling. Indeed, as OA Obs have elevated TGF-β levels [20], this could directly activate the p42/44 pathway without the involvement of Grb2. Thus, the elevated endogenous TGF-β levels in OA Obs could then explain both the results for the PKB and p42/44 pathways observed here.
Taken together, these results could be interpreted as a general downregulation of the IGF-1R/IRS-1 pathways in OA Obs. However, dowstream signals were not actually reduced. The observed increase in Syp/IRS-1 interaction and increased Syp phosphorylation could actually promote IGF-1 signaling. A functional and highly phosphorylated SHP-2/Syp is necessary for sustained activation of ERK1/2 response to hepatocyte growth factor (HGF) stimulation in Madin-Darby canine kidney (MDCK) cells [43] and in rat fibroblasts in response to insulin, IGF-1 or epidermal growth factor [44]. Moreover, inactivating Syp antibodies [44] or expression of a mutant phosphatase [45] significantly reduces insulin, IGF-1 and epidermal growth factor signaling. Accordingly, as OA Obs showed high phosphorylated Syp levels and strong interaction with IRS-1, both under basal conditions and after IGF-1 stimulation, this could promote p42/44 activity in OA Obs, as observed in those studies. Moreover, a recent study indicated that functionally deficient SHP-1 mice are markedly glucose tolerant and insulin sensitive as a result of enhanced insulin receptor signaling to IRS-1 [46], which suggests that elevated activity of SHP-1/Syp could reduce IGF-1 signaling to IRS-1, as observed in the present study. This would also suggest that, although IGF-1R-dependent IRS-1 phosphorylation is reduced in OA Obs, Syp phosphorylation and activity could compensate for this reduction. As Syp is central to other growth factors, such as HGF and epidermal growth factor [43,44], and since we recently showed a key role for HGF in OA Obs [47] and possibly for the cross-talk between OA Obs and cartilage tissue [48], the present results suggest that the HGF-dependent pathway could also be altered in OA Obs, a situation not investigated at present.
This is the first study demonstrating abnormal IGF-1 cell signaling in human OA subchondral Obs that could explain the abnormal response of these cells to this growth factor. We demonstrated an altered IGF-1 pathway involving IRS-1 and Syp. Moreover, we also demonstrated increased MAPK activity in IGF-1 stimulated OA Obs, which could be implicated in the abnormal subchondral bone remodeling observed in OA. Since both Syp and Grb2 play key roles in the signaling pathways of other growth factors in Obs besides IGF-1, this may also suggest an abnormal response to these growth factors in OA Obs.
BrdU = bromodeoxyuridine; BSA = bovine serum albumin; FBS = fetal bovine serum; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; HGF = hepatocyte growth factor; IGF = insulin-like growth factor; IGF-1R = IGF-1 receptor; IRS = insulin receptor substrate; OA = osteoarthitis; Ob = osteoblast; PI3K = phosphatidylinositol 3-kinase; PKB = protein kinase B; SD = standard deviation; SEM = standard error of the mean; TGF = transforming growth factor; UNG = uracil-N-glycosylase; uPA = urokinase plasminogen activator.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
FM performed most of the experiments and wrote the first draft of the manuscript. IA performed the experiments shown in Figure 4 and contributed to writing the manuscript. JM-P and J-PP contributed to writing the manuscript and discussion of the results. JCF provided the OA knee samples and contributed to discussion of the results. DL proposed original concepts, planned and performed some of the experiments, performed the statistical analyses, participated in the discussion and wrote the final version of the manuscript.
Additional file 1. A figure showing p42/44 levels and activation in normal and osteoarthitis (OA) osteoblasts. Cells were grown to confluence and incubated overnight in serum free medium. Cells were then exposed to 50 ng/ml insulin-like growth factor (IGF)-1 for 15 minutes. Phospho p42/44 levels were detected by westen blot analysis. The figure shows a representative experiment with one normal and one OA osteoblast preparation. Similar assays were repeated with three different samples of normal and OA osteoblasts with similar results.
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Additional file 2. A figure showing phospho-protein kinase B (pPKB) and phosphatidylinositol 3-kinase (PI3K) levels and activation in normal and osteoarthitis (OA) osteoblasts. Cells were grown to confluence and incubated overnight in serum free medium. Cells were then exposed to 50 ng/ml insulin-like growth factor (IGF)-1 for 5 minutes. The top panel shows a representative western blot of pPKB and total PKB levels in one normal and one OA osteoblast preparation. Similar assays were repeated with four different samples of normal and OA osteoblasts with similar results. The bottom panel shows a representative western blot of PI3K levels for two normal and two OA osteoblast preparations. Actin levels were determined to ensure similar loading between samples. Similar assays were repeated with four different samples of normal and OA osteoblasts with similar results.
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We wish to thank Aline Delalandre for her technical expertise on this project. D Lajeunesse is a Chercheur National from the 'Fonds de la Recherche en Santé du Québec'. F Massicotte is the recipient of a PhD studenship from the 'Groupe de Recherche en Transport Membranaire' from the Université de Montréal. This study was supported by grants MOP-49501 from the Canadian Institutes for Health Research (CIHR) and TAS-0089 from the Arthrisis Society of Canada/CIHR to DL.
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Our Practice
Contact Us
Dealing with Oral Spirochetes – Treatment Information from Our Stockton Dentists
Oral Spirochete
First cure your mouth and then we will help you put it back together.
Please note: We do not place
dental implants
on patients infected with oral spirochetes.
At Martin Dentistry in Stockton, Drs. Douglas and Dax Martin understand that good oral hygiene is related to good overall health. As more and more studies are performed linking dental health with serious diseases like heart attack, stroke, and diabetes, our caring dentists take notice. Bacteria, including oral spirochete, can infect your mouth and cause harm to the rest of your body. Learn more about oral spirochetes and its treatment.
Dentistry Background
Technology for Bacteria Detection
Syphilis and Lyme Disease Spirochetes
Syphilis Spirochete
Lyme Disease Spirochete
Help Understanding of Oral Spirochete
Life Cycle of Oral Spirochetes
Oral Spirochetes and Other Diseases
Can Oral Spirochetes Be Defeated?
Combating Oral Spirochetes
Baking Soda
Clorox Bleach
Martin Dentistry
1310 East Swain Road. S-2
Stockton, California, 95210
Telephone: (209) 951-4251
Fax number: (209) 951-4822
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Douglas Martin , DDS - Martin Dentistry - 1310 East Swain Road S-2, Stockton, California, 95210 Telephone: (209) 951-4251 Fax number: (209) 951-4822
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Dentistry Background and How Bacteria Gets Involved
Drs. Dax and Douglas Martin mostly treat three things in dentistry: two bones and a seal. The first bone our implant dentists treat is what you usually think about when you go the dentist: the tooth bone. The tooth bone is the only bone in the skeleton which sticks out through the skin. The second bone they treat is called the alveolar bone and its job is to hold the teeth bones to the jaw bone. The alveolar bone goes away when the teeth are removed. If the alveolar bone deteriorates when teeth are still present, those teeth will fall out.
The third thing our dentists treat is a seal: the gum. The gum is specialized tissue that seals the skeleton where it sticks out through the skin. If the seal leaks, blood runs out when we brush our teeth. Worse yet, bacteria get into the body through the leaking seal and can destroy the alveolar bone. Modern medicine has been able to show that when the gum seals leak several other diseases are either caused or made much worse.
Using Technology to Detect Bacteria
Several years ago, Martin Dentistry purchased a research grade phase contrast microscope. This tool enables Drs. Dax and Douglas Martin to see the kinds of nasty creatures patients have in their mouths. There is one particular kind of nasty bug called a spirochete (spy-row-keet) which is very aggressive and can only be seen with the help of the phase contrast microscope.
Contact our Stockton implant dentistry practice
to learn more about our spirochetes screening process.
Spirochetes cause several diseases in man and animals, including syphilis, Lyme disease, stomach ulcers, cat scratch fever, trench mouth, pyorrhea, and necrotizing ulcerative gingivitis. This list is by no means exhaustive, as there are several diseases caused by these bugs. Take a look for yourself in this
oral spirochetosis video on YouTube
Spirochetes that Cause Syphilis and Lyme Disease
There are several types of spirochetes, which cause different types of diseases. By looking at spirochetes that cause syphilis and Lyme disease, you can get a better understanding of oral spirochetes.
Syphilis Spirochete
Syphilis starts as a sore on the mucosa of the genitals – then it seems to go away. Some unknown time later a red rash forms over the body, which also goes away. Many years later, very bad things begin to happen as the brain, blood vessels, and many other organs break down.
The syphilis spirochete is extremely aggressive bacteria, which is easily able to go through intact mucosa (the lining skin of the gastro-intestinal tract and genital urinary tract). Once in the body, it multiplies and causes syphilis.
Lyme Disease Spirochete
Lyme disease starts as a small sore after a tick bite. The person gets sick later with many bizarre symptoms. This cycle may go on for years. Doctors treat Lyme disease with every known kind of antibiotic, yet, it typically returns again. Lyme disease researchers believe the spirochete forms spores, or tiny seeds, which are not killed by any antibiotic. After the antibiotic treatment is ended the patient becomes sick again as the spirochetes begin to again multiply and flourish in the victim's body.
How Other Spirochetes Help in Understanding of Oral Spirochete
If we look at the spirochete which causes Lyme disease, we can learn a great deal about the connection between people with oral spirochetes and their deaths from heart disease, their onset of diabetes, and their development of Alzheimer's disease. (For an exhaustive library of systemic illnesses related to oral spirochetes, visit the
ZT4BG website
. This site contains many references showing how gum disease relates to systemic illnesses.)
Lyme disease researchers claim that spirochetes form spores. However, it is believed that oral spirochetes have a very interesting life cycle. This cycle is different from other spirochetes such as the syphilis spirochete or the Lyme disease spirochete, which are able to live inside the human body and multiply there.
It is believed that the oral varieties do not live and multiply in the human body (except perhaps in the brain). Instead, oral spirochetes live in the very special habitat: the crevice between the tooth and gum. This environment is unique because it is constantly fed by the filtrate of blood called extracellular fluid (ECF), but it is outside the usual bodily defenses. Even when the crevice is full of white blood cells (pus) the area is still safe from the body's natural defenses.
Once the spirochete from the crevice invades into the body, it can travel in the blood stream and destroy blood vessels. There is a very common disease—called by various names—which cause the gum to rot and disappear. The blood vessels of the gums are destroyed by the spirochetes of this illness. When the blood supply is destroyed the gums rot away and the characteristic grey chewed off gums of this disease is the result of the damage to the blood vessels. A very explicit depiction of this can be seen on a
graphic spirochetes video on YouTube
Life Cycle and Attack of Oral Spirochetes
Now, let's look at a model for oral spirochetes. These very aggressive creatures multiply by the trillions in the crevice between the tooth and gum, where they are fed constantly by the fluid coming from the blood inside the body which leaks out constantly due to improper oral hygiene. These bacteria can go through the skin of the gums just like the syphilis spirochete goes through the skin of the genitals. Once inside the body they can go through the blood stream to any part of the body where they can easily invade the blood vessels and damage them.
The attack of the blood vessels can account for the known facts that:
Not flossing is a major risk factor for heart attack
A small amount of alveolar bone loss (caused by oral spirochetes) raises the risk of stroke by 40 percent
Once inside the body these spirochetes do not multiply and divide or they would have been found to cause a disease such as syphilis or Lyme disease. However, if they form spores or seeds in the body they have succeeded in their life cycle. The seed waits until the animal dies when oxygen no longer circulates (due to lack of blood flow) and the tissue becomes anaerobic. The seed begin to grow. The next animal eating the flesh can ingest the seeds or the active spirochete and become infected. (This life cycle is used to account for the discovered fact that dinosaurs had gum disease.)
Although syphilis spirochetes can easily cross from the mother through the placenta and cause brain damage and nervous system problems in the child, oral spirochetes are not necessarily passed to children in that way. However, oral spirochetes can be passed through:
Food passed from parents' mouths to children
Eating meat infected with oral spirochete spores
Connection between Oral Spirochetes and Other Diseases
Dentistry has traditionally looked at red irritated gums as a local phenomenon, much like the athlete foot fungus. Dentists treated gum disease as an isolated phenomenon thinking, very foolishly, that sore rotting gums was not damaging the whole person. But we know very differently today. We know that gum disease is linked to these very common chronic illnesses and our dentists are dedicated to helping you fight this vicious cycle.
Studies have shown that approximately the same percentage of humans that do not have oral spirochetes have no heart disease, diabetes, or Alzheimer's disease. Over the last few years in our Stockton dental office, Drs. Dax and Douglas Martin have looked carefully at our older patients who have no chronic illnesses. Our dentists have found that they have no spirochetes in their mouths!
Every patient with heart disease, diabetes or Alzheimer's disease has oral spirochetes. Renowned spirochetes researcher, Dr. Nordquist has coined the term multiple missing tooth syndrome to describe those with oral spirochetosis. He has noted mental problems, heart disease, and diabetes as a part of oral spirochete infestation.
His book is available
for more information.
Can Oral Spirochetes Be Defeated?
What is the current level of knowledge as a result of our dentists' work and those who are very alarmed by these nasty creatures? At Martin Dentistry, we believe that oral spirochetes ARE the cause of many chronic problems found in humans.
The problem is that the life cycle of these spore-forming creatures can pass from generation to generation via the placenta in mammals or via spores from food, which may make it impossible to eradicate completely. However, our implant dentists hope that killing them in the gingival crevice where they can breed by the trillions and invade into the body by millions or billions over a life time can be a way to cut down their damage. Our dentists have devised ways to destroy them in their unique habitat the gingival crevice!
Combating Oral Spirochetes
You can defeat oral spirochetes with good oral hygiene and some additional steps. There are two ways to easily kill them with simple chemicals found in almost every home: baking soda and bleach.
Baking Soda
Baking soda is a salt. If you have ever seen someone pour salt on a snail or slug and watched the result, you have seen them shrivel up and die. Pushing the salt of baking soda into the crevices around your teeth with a tooth brush and floss does the same thing to spirochetes. THEY DIE!!
Clorox Bleach
Clorox bleach is also a great spirochete killer! By diluting one part of bleach with 20 parts of water, you create a safe dilution to use in the mouth. When diluted, the Clorox mixture is called Dakin's solution (which sounds a lot better than Clorox, if you are going to put it into your mouth, doesn't it?). The solution must be made fresh each time you use it.
When you use the solution in a battery operated Waterpik™ you have a formidable weapon against spirochetes. Keep a bottle of Clorox in the shower; dilute it 20 to 1; put the solution into the Waterpik™; and use it in the shower. Blast and kill the spirochetes in their homeland before they do the acts of terrorism in your body and form spores.
By brushing your teeth with baking soda and rinsing with the Dakin's solution, you can eradicate the oral spirochetes from your mouth and improve your overall health. We recommend the oral care regimen advocated by Jurgen Slots Ph.D., head of the Periodontics program at University of Southern California. Download this home care regimen .
Contact Our Stockton Practice for a Proper Dental Screening
Because oral spirochetes can cause some many other dental problems, it is important to check for these bacteria in your mouth.
Contact our Stockton dental practice
today to schedule a comprehensive oral exam. Take a stand against these invaders in your mouth and improve your health.
Science News Related to Your Oral Health
Below are news stories that show a correlation between the growth of bacteria, including oral spirochetes, in the mouth from lack of proper oral hygiene and the increased chance of being affected by heart disease, stroke, diabetes, and other diseases.
Trouble thinking? Better see the dentist
November 13, 2009 Reuters (New York)
Good oral care such as regular brushing, flossing and trips to the dentist, may help aging adults keep their thinking skills intact, according to a U.S. study.
Research has already established an association between poor oral health and heart disease, stroke and diabetes, as well as Alzheimer's disease.
But researchers from Columbia College of Physicians and Surgeons in New York found gum disease could also influence brain function through several mechanisms, such as causing inflammation throughout the body, a risk factor for loss of mental function.
The study based on adults aged 60 and older found those with the highest levels of the gum disease-causing pathogen Porphyromonas gingivalis were three times more likely to have trouble recalling a three-word sequence after a period of time.
The study, led by Dr. James Noble, also found that adults with the highest levels of this pathogen were two times more likely to fail three-digit reverse subtraction tests.
"Despite the association of periodontitis with stroke and shared risk factors between stroke and dementia, to our knowledge, no epidemiological studies have investigated periodontitis relative to cognition," the researchers wrote in their study.
"Although results presented here are preliminary and inconclusive, a growing body of evidence supports exploration of a possible association between poor oral health and incident dementia."
The study, reported in the Journal of Neurology, Neurosurgery, and Psychiatry, was based on more than 2,350 men and women who were tested for periodontitis and completed numerous thinking skills tests as part of a national survey.
Overall 5.7 percent of the adults had trouble completing certain memory tasks, 6.5 percent had impaired delayed recall, and 22.1 percent had trouble with serial subtractions.
But those with the levels of the pathogen were nearly three times more likely to struggle with the verbal memory tests, and twice as likely to fail on both delayed verbal recall and subtraction tests.
"Although our results are preliminary, they suggest that further exploration of relationships between oral health and cognition is warranted," they concluded.
(Reporting by Joene Hendry of Reuters Health, Editing by Belinda Goldsmith)
More Than 90 Percent of People with Gum Disease Are at Risk for Diabetes, Study Finds
December 15, 2009 Science Daily
An overwhelming majority of people who have periodontal (gum) disease are also at high risk for diabetes and should be screened for diabetes, a New York University nursing-dental research team has found. The researchers also determined that half of those at risk had seen a dentist in the previous year, concluded that dentists should consider offering diabetes screenings in their offices, and described practical approaches to conducting diabetes screenings in dental offices.
The study, led by Dr. Shiela Strauss, Associate Professor of Nursing and Co-Director of the Statistics and Data Management Core for NYU's Colleges of Dentistry and Nursing, examined data from 2,923 adult participants in the 2003-2004 National Health and Nutrition Examination Survey who had not been diagnosed with diabetes. The survey, conducted by the National Center for Health Statistics of the Centers for Disease Control and Prevention, was designed to assess the health and nutritional status of adults and children in the United States.
Using guidelines established by the American Diabetes Association, Dr. Strauss determined that 93 percent of subjects who had periodontal disease, compared to 63 percent of those without the disease, were considered to be at high risk for diabetes and should be screened for diabetes. The guidelines recommend diabetes screening for people at least 45 years of age with a body mass index (a comparative measure of weight and height) of 25 or more, as well as for those under 45 years of age with a BMI of 25 or more who also have at least one additional diabetes risk factor. In Dr. Strauss's study, two of those additional risk factors -- high blood pressure and a first-degree relative (a parent or sibling) with diabetes -- were reported in a significantly greater number of subjects with periodontal disease than in subjects without the disease. Dr. Strauss's findings, published today in the online edition of the Journal of Public Health Dentistry, add to a growing body of evidence linking periodontal infections to an increased risk for diabetes.
Dr. Strauss also examined how often those with gum disease and a risk for diabetes visit a dentist, finding that three in five reported a dental visit in the past two years; half in the past year; and a third in the past six months.
"In light of these findings, the dental visit could be a useful opportunity to conduct an initial diabetes screening -- an important first step in identifying those patients who need follow-up testing to diagnose the disease."
"It's been estimated that 5.7 million Americans with diabetes were undiagnosed in 2007," Dr. Strauss added, "with the number expected to increase dramatically in coming years. The issue of undiagnosed diabetes is especially critical because early treatment and secondary prevention efforts may help to prevent or delay the long-term complications of diabetes that are responsible for reduced quality of life and increased levels of mortality among these patients. Thus, there is a critical need to increase opportunities for diabetes screening and early diabetes detection."
Dr. Strauss said that dentists could screen patients for diabetes by evaluating them for risk factors such as being overweight; belonging to a high-risk ethnic group (African-American, Latino, Native American, Asian-American, or Pacific Islander); having high cholesterol; high blood pressure; a first-degree relative with diabetes; or gestational diabetes mellitus; or having given birth to a baby weighing more than nine pounds.
Alternatively, dentists could use a glucometer -- a diagnostic instrument for measuring blood glucose -- to analyze finger-stick blood samples, or use the glucometer to evaluate blood samples taken from pockets of inflammation in the gums.
"The oral blood sample would arguably be more acceptable to dentists because providers and patients anticipate oral intervention in the dental office," Dr. Strauss noted. In an earlier study involving 46 subjects with periodontal disease published in June 2009 by the Journal of Periodontology, an NYU nursing-dental research team led by Dr. Strauss determined that the glucometer can provide reliable glucose-level readings for blood samples drawn from deep pockets of gum inflammation, and that those readings were highly correlated with glucometer readings for finger-stick blood samples.
Dr. Strauss's coauthors on the study for the Journal of Public Health Dentistry include Ms. Alla Wheeler, Clinical Assistant Professor of Dental Hygiene; Dr. Stefanie Russell, a periodontist and Assistant Professor of Epidemiology & Health Promotion; and Dr. Robert Norman, Research Associate Professor of Epidemiology & Health Promotion, all of the NYU College of Dentistry; Dr. Luisa Borrell, an Associate Professor in the Department of Health Sciences at Lehman College of the City University of New York; and Dr. David Rindskopf, Distinguished Professor of Educational Psychology and Psychology at the City University of New York Graduate Center.
Periodontal Disease Independently Predicts New Onset Diabetes
August 8, 2008 Science Daily
Periodontal disease may be an independent predictor of incident Type 2 diabetes, according to a study by researchers at Columbia University Mailman School of Public Health. While diabetes has long been believed to be a risk factor for periodontal infections, this is the first study exploring whether the reverse might also be true, that is, if periodontal infections can contribute to the development of diabetes.
The Mailman School of Public Health researchers studied over 9,000 participants without diabetes from a nationally representative sample of the U.S. population, 817 of whom went on to develop diabetes. They then compared the risk of developing diabetes over the next 20 years between people with varying degrees of periodontal disease and found that individuals with elevated levels of periodontal disease were nearly twice as likely to become diabetic in that 20 year timeframe. These findings remained after extensive multivariable adjustment for potential confounders including, but not limited to, age, smoking, obesity, hypertension, and dietary patterns.
"These data add a new twist to the association and suggest that periodontal disease may be there before diabetes," said Ryan T. Demmer, PhD, MPH, associate research scientist in the Department of Epidemiology at the Mailman School of Public Health and lead author. "We found that over two decades of follow-up, individuals who had periodontal disease were more likely to develop Type 2 diabetes later in life when compared to individuals without periodontal disease."
Also of interest, the researchers found that those study participants who had lost all of their teeth were at intermediate risk for incident diabetes. "This could be suggestive that the people who lost all of their teeth had a history of infection at some point, but subsequently lost their teeth and removed the source of infection," noted Dr. Demmer. "This is particularly interesting as it supports previous research originating from The Oral Infections and Vascular Disease Epidemiology Study (INVEST) which has shown that individuals lacking teeth are at intermediate risk for cardiovascular disease" said Moïse Desvarieux, MD, PhD, director of INVEST, associate professor and Inserm Chair of Excellence in the Department of Epidemiology at the Mailman School and senior author of the paper.
The contributory role of periodontal disease in the development of Type 2 diabetes is potentially of public health importance because of the prevalence of treatable periodontal diseases in the population and the pervasiveness of diabetes-associated morbidity and mortality. However, observes Dr. Demmer, more studies are needed both to determine whether gum disease directly contributes to type 2 diabetes and, from there, that treating the dental problem can prevent diabetes. In addition to Dr. Desvarieux, David R. Jacobs Jr., PhD, professor in the Department of Epidemiology and Community Health at the University of Minnesota, also contributed to the research.
The full study findings are published in the July 2008 issue of Diabetes Care.
Contact Our Stockton Practice for a Proper Dental Screening
Because oral spirochetes can cause some many other dental problems, it is important to check for these bacteria in your mouth.
Contact our Stockton dental practice
today to schedule a comprehensive oral exam. Take a stand against these invaders in your mouth and improve your health.


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The Miami Dolphins are looking healthy as they head into their showdown with the Patriots.
The team released the list of inactives Sunday.
It did not include running back Lamar Miller.
The former Cane can return to action against the Patriots.
The inactive players are: QB Pat Devlin, S Jonathan Amaya, LB Josh Kaddu, T Will Yeatman, TE Michael Egnew, TE Kyle Miller, and DT Kheeston Randall.


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Human resource planning is concerned with the flow of people into, through and out of the organization. HR planning involves anticipating the need for labor and the supply of labor and then planning the programmes necessary to ensure that the organization will have the right mix of employees and skills when and where they are needed. Forecasting the future can be very inexact science, so human resource planning may also include multiple scenerios and contingency plans.
Some Other Definitions:
HRP has been defined by Geisler (1967),


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At this point in the baseball season, Conference USA is the main concern for Southern Miss.
But the Golden Eagles, who lead the conference standings, certainly didn't help build any down-the-stretch momentum with their performance Friday night at Taylor Park.
First baseman Joel Greatting had three hits and drove in three runs, including a two-run home run in the first inning off USM ace Andrew Pierce, and the visiting Cougars went on to a 6-5 victory over the Golden Eagles before 2,840. ...


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Free agent
US president George W Bush may not seem to be furthering the cause of independent political cinema, but according to Philippe Diaz, the French producer and director who heads US production, post-production, distribution and international sales operation Cinema Libre Studio, his election in 2000 gave political film-making a shot in the arm.
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