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Secondary Sources: Central Bank Limits, Housing Fever, Idle Youth
A roundup of economic news from around the Web.
–Central Bank Limits: Joseph E. Stiglitz says the Fed and ECB can't revive the economy on their own. "For both Europe and America, the danger now is that politicians and markets believe that monetary policy can revive the economy. Unfortunately, its main impact at this point is to distract attention from measures that would truly stimulate growth, including an expansionary fiscal policy and financial-sector reforms that boost lending. The current downturn, already a half-decade long, will not end any time soon. That, in a nutshell, is what the Fed and the ECB are saying. The sooner our leaders acknowledge it, the better."
–Housing Fever: Robert Shiller writes about the social epidemic behind housing. "People waiting to buy a home may be waiting for a sense that prices have a rosy long-term future. Home prices in the United States have been rising for several months, and that is generating some optimism that now is the time to buy. However, the social waves also carry other, less encouraging stories that compete with such optimism — for example, foreclosures, unemployment, Europe's troubles and the Asian slowdown. Will optimism about real estate emerge as a leading story? If this is a major upturning point for the housing market, it is still sociologically opaque."
–Idle Youth: Jacob Funk Kirkegaard examines the complications of youth unemployment in Europe. "To better calculate the true idleness and "wasted youth" phenomenon in advanced economies, the OECD calculates the share of youth "not in employment, education, or training" among the total in the 15- to 24-year-old age group. This is the so-called NEET ratio, which comprises "idle youth" in the labor force (looking for work, but unable to find it) and outside the labor force (inactive). Figure 2 shows the developments in the NEET ratio for available OECD countries from pre-crisis Q1 2007 to the latest available data from Q1 2011… Measured on this more genuine measure of "idle youth" with associated long-term "scarring effects"—the euro area peripheral countries look less bad than other OECD countries. Yes, there have been significant increases in Ireland and Spain during the crisis, but the Q1 2011 NEET ratio of these two countries was still only 2.8 percentage points higher at 17.6 percent than the corresponding 14.8 percent in the United States."

subset: commoncrawl


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OPN (osteopontin) is a highly phosphorylated glycoprotein present in many tissues and body fluids. In urine, OPN is a potent inhibitor of nucleation, growth and aggregation of calcium oxalate crystals, suggesting that it has a role in the prevention of renal stone formation. The role of OPN in nephrolithiasis is, however, somewhat unclear, as it may also be involved in urinary stone formation, and it has been identified among the major protein components of renal calculi. Most likely, the function of OPN in urine is dependent on the highly anionic character of the protein. Besides a very high content of aspartic and glutamic residues, OPN is subjected to significant PTM (post-translational modification), such as phosphorylation, sulfation and glycosylation, which may function as regulatory switches in promotion or inhibition of mineralization. In the present study, we have characterized the PTMs of intact human urinary OPN and N-terminal fragments thereof. MS analysis showed a mass of 37.7 kDa for the intact protein. Enzymatic dephosphorylation and peptide mass analyses demonstrated that the protein contains approximately eight phosphate groups distributed over 30 potential phosphorylation sites. In addition, one sulfated tyrosine and five O-linked glycosylations were identified in OPN, whereas no N-linked glycans were detected. Peptide mapping and immunoblotting using different monoclonal antibodies showed that the N-terminal fragments present in urine are generated by proteolytic cleavage at Arg228–Leu229 and Tyr230–Lys231.
- O-glycosylation
- osteopontin
- phosphorylation
- proteolytic processing
- sulfation
- uropontin
OPN (osteopontin) is a phosphorylated glycoprotein containing an integrin-binding RGD (Arg-Glu-Asp) sequence. The protein was originally purified from the mineralized matrix of bovine bone [1]; however, the expression of OPN is not limited to mineralized tissues, but extends to most of the tissues and body fluids, including milk, blood and urine [2]. OPN consists of approx. 300 amino acid residues with a very high content of aspartic and glutamic residues. The protein is involved in a broad range of functions in both physiological and pathological processes such as cytokine production, tumour growth, inflammation and mineralization [3–5]. Furthermore, OPN is a key molecule in bone remodelling [5,6] and it hinders ectopic calcification by inhibiting the formation of hydroxyapatite [7,8], which is the main mineral phase of bone and teeth, as well as calcium oxalate [9], the main mineral in renal calculi.
OPN is found at high levels in urine with an average of approx. 4 mg secreted into human urine daily [10]. In contrast, OPN is undetectable in normal kidney tissues [11], indicating that the OPN produced in the renal tubules is immediately secreted in the urine. OPN can inhibit nucleation, growth and aggregation of calcium oxalate crystals in vitro [9,12,13], and it blocks binding of the crystals to renal epithelial cells [14]. In addition, COM (calcium oxalate monohydrate) crystal growth and aggregation are inhibited by 50% at an OPN concentration between 16 and 28 nM, a concentration that is an order of magnitude lower than the OPN concentration of 131 nM found in normal urine [13]. OPN also favours the formation of COD (calcium oxalate dihydrate) over COM crystals [12]. The induction of COD by OPN may be protective against calcium oxalate nephrolithiasis, since COD has demonstrated less affinity for attachment to renal epithelial cells than COM [12]. Besides reducing crystal affinity for renal cells, OPN may act as an intracrystalline protein assisting stone prevention by rendering crystals more susceptible to intracellular degradation [15]. Furthermore, OPN-knockout mice fed with a crystal-inducing diet developed renal calcium oxalate stones, whereas wild-type mice were completely unaffected, perhaps due to an up-regulated OPN expression in response to the induced hyperoxaluria, suggesting a protective role of OPN [16]. Similarly, a recent study showed that 10% of mice lacking OPN spontaneously formed renal stones and 65% developed renal crystals under experimentally induced hyperoxaluria, whereas these events were not observed in wild-type mice [17].
There are many indications that OPN is an important inhibitor of renal stone formation; however, the protein has also been implicated as a promoter of stone formation. One study showed increased calcium oxalate crystal adherence to kidney cells when they were incubated with OPN [18]. Furthermore, inhibition of OPN synthesis has been shown to suppress the deposition and adhesion of calcium oxalate crystals to rat renal cells [19], and addition of polyclonal OPN antibodies reduced the deposition of crystals on the surface of kidney cells [20]. It has also been suggested that OPN is involved in urinary stone formation [21], and the protein has been identified as a prominent constituent of the major matrix of renal calculi [22]. Reduced OPN levels in stone formers compared with non-stone formers have been reported in one study [23], while no difference in urinary OPN levels has been reported in other studies [24,25].
OPN is extensively altered through PTM (post-translational modification), such as phosphorylation, sulfation, glycosylation and proteolytic processing, which have significant implications on the structure and the biological function of the protein [26]. The PTMs of OPN from bovine and human milk as well as rat bone have been thoroughly characterized [27–29]. Recently, a comparative study of OPN produced by murine ras-transformed fibroblasts and differentiating osteoblasts demonstrated remarkably different phosphorylation patterns between OPNs originating from the same species, which translated into significant functional differences in cellular adhesion [30].
The PTMs of OPN provide a basis for its regulatory functions in mineralization processes, as dephosphorylated OPN has been shown to lose the ability to inhibit hydroxyapatite formation [7,8]. Emphasizing the importance of the phosphorylations, it was recently shown that highly phosphorylated milk OPN promoted hydroxyapatite formation and growth, whereas the less phosphorylated bone OPN inhibited these processes [31]. Similarly, recombinant (non-phosphorylated) OPN increases mineralization of vascular smooth-muscle cells, whereas enzymatically phosphorylated OPN is a potent inhibitor of this process [32]. Recently, it was shown that only phosphorylated OPN dose-dependently inhibited mineralization of MC3T3-E1 osteoblast cell cultures [33]. Phosphorylation of OPN peptides also markedly enhanced the inhibition of hydroxyapatite and COM crystal growth [34,35], and C-terminal fragments of urinary OPN can inhibit COM growth as efficiently as the intact protein, suggesting that different parts of the protein can regulate crystal formation [36].
Western blotting of human urinary OPN has shown the presence of different forms in the 40–70 kDa range [15,24,25]. The structural variation underlying these differences in migration is unknown. Normal rat kidney cells have been shown to produce both phosphorylated and non-phosphorylated OPN, which also diverge in glycosylation and physiological properties [37]. Furthermore, low-molecular-mass OPN variants probably resulting from cleavage by serine proteases are present at a higher frequency in the urine from individuals with kidney stone disease than in the urine from healthy individuals [24]. Recently, a series of C-terminal OPN fragments were identified by two-dimensional electrophoresis and MS in patients with ovarian cancer, but not in healthy controls [38]. In the present study, we have separated and characterized different forms of human urinary OPN with regard to PTMs and proteolytic processing. These modifications are likely to be key factors in the regulatory roles of OPN in renal stone formation.
The monoclonal mouse antibodies 1H3 and 3D9 [39] were a gift from Dr David T. Denhardt (Rutgers University). Swine anti-rabbit and rabbit anti-mouse immunoglobulins conjugated to alkaline phosphatase were obtained from Dako (Glostrup, Denmark). Bovine pancreas trypsin and staphylococcal (Staphylococcus aureus) V8 protease were obtained from Worthington Biochemical (Freehold, NJ, U.S.A.). Thermolysin, ALP (bovine alkaline phosphatase), human plasma thrombin and arylsulfatase were from Sigma (St. Louis, MO, U.S.A.). The μRPC (μ reverse-phase chromatography) C2/C18 PC 2.1/10 column, Protein A–Sepharose CL-4B resin, DEAE-Sepharose material and PD-10 desalting columns were from GE Healthcare (Uppsala, Sweden). Vydac C4 and C18 reverse-phase resins were obtained from The Separations Group (Hesperia, CA, U.S.A.). Reagents used for sequencing were purchased from Applied Biosystems (Warrington, U.K.). 2,5-Dihydroxybenzoic acid was from LaserBio Labs (Sophia-Antipolis Cedex, France). The PNGase F (peptide N-glycosidase F) kit was obtained from New England Biolabs (Beverly, MA, U.S.A.). All other chemicals used were of analytical grade.
Polyclonal OPN antibodies
Polyclonal antiserum against human milk OPN was raised in rabbits at Dako. OPN-specific polyclonal antibodies were isolated from the rabbit serum on a Protein A–Sepharose column. The specificity of the antibodies was checked and verified by Western-blot analyses of milk samples and purified OPN.
Western blotting
OPN (50 ng/lane) and urine (10 μl) were loaded on to 16% Tris/Tricine gels, fractionated by SDS/PAGE and electrophoretically transferred on to Hybond-P PVDF membranes (GE Healthcare) for immunodetection. The membranes were blocked in 2% Tween in Tris-buffered saline before addition of either polyclonal rabbit OPN antibodies (5 μg/ml) or monoclonal mouse OPN antibodies 1H3 and 3D9 (1.5 μg/ml). OPN was detected with alkaline phosphatase-conjugated secondary immunoglobulins.
Purification of OPN
Urine samples were collected from seven healthy donors with normal renal function and no history of urinary disease and frozen at −80 °C. Thawed urine samples were centrifuged at 4500 g for 30 min and the resulting supernatant was incubated with DEAE-Sepharose at pH 8.1 for 2 h at room temperature (25 °C). Adsorbed proteins were batch eluted with stepwise increasing concentrations of ammonium bicarbonate (pH 8.1). The batch containing OPN was freeze-dried, followed by passage through a PD-10 column in 20% formic acid. The salt-free eluate was freeze-dried and subjected to further purification by RP-HPLC (reverse-phase HPLC) on a Vydac C4 column connected to a GE Healthcare LKB system. Separation was carried out at 40 °C in 0.1% TFA (trifluoroacetic acid) (buffer A) and the proteins were eluted with a gradient of 80% acetonitrile in 0.1% TFA (buffer B) developed over 50 min (0–5 min, 0% B; 5–40 min, 0–50% B; 40–50 min, 50–95% B) at a flow rate of 0.85 ml/min. The proteins were detected in the effluent by measuring the absorbance at 226 nm. The fraction containing OPN was identified by immunodetection and N-terminal sequencing. The full-length protein (OPN60) and N-terminal fragments (OPN45 and OPN50) were separated by RP-HPLC on a Vydac C18 column and eluted with stepwise increasing concentrations between 10 and 30% of 75% (v/v) propan-2-ol in 0.1% TFA. The fractions were analysed by N-terminal sequencing and SDS/PAGE, and proteins were visualized by CBB (Coomassie Brilliant Blue) staining or by Western blotting. The amount of purified OPN was determined by amino acid analysis.
Phosphate content of OPN
Native and dephosphorylated OPN isoforms were analysed by MS using a Voyager DE-PRO MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) mass spectrometer (Applied Biosystems). Samples for MS analyses were prepared by mixing the sample with a saturated solution of 2,5-dihydroxybenzoic acid in a 1:1 ratio directly on the MS-target probe. All spectra were obtained in positive linear-ion mode using a nitrogen laser at 337 nm and an acceleration voltage of 20 kV. Typically, 50–100 laser shots were added per spectrum and calibrated with external standards. The masses were assigned using the half-height width method. For dephosphorylation, 2.5 μg of OPN was incubated with 0.07 unit of ALP in 10 mM ammonium bicarbonate (pH 8.5) overnight at 37 °C. The average phosphate content estimated by MS was confirmed with a Phosphoprotein Phosphate Estimation Assay kit (Pierce, Rockford, IL, U.S.A.).
Carbohydrate analyses
Full-length OPN (OPN60) was quantified by amino acid analysis and hydrolysed in 2 M TFA for 4 h at 100 °C under oxygen-free conditions. α-L-Rhamnose was added as an internal standard. For quantifying the more stable amino sugars, GalNAc and GlcNAc, an additional OPN sample was hydrolysed in 4 M HCl for 6 h at 110 °C. For sialic acid analysis, the protein was hydrolysed in 0.1 M HCl for 60 min at 80 °C. Monosaccharides were separated by high-pH anion-exchange chromatography using a CarboPac PA1 column (4 mm×250 mm; Dionex, Sunnyvale, CA, U.S.A.) and monitored by pulsed electrochemical detection. The neutral monosaccharides were eluted isocratically with 16 mM NaOH, whereas sialic acid was eluted with 100 mM NaOH and 150 mM sodium acetate. Standard monosaccharide mixtures of α-L-fucose, D-galactosamine, D-glucosamine, D-galactose, D-glucose (all 0.1 mM), D-mannose (0.5 mM), α-L-rhamnose (0.2 mM) and NeuAc (0.02 mM) (all from Sigma) were used. Linearity was observed between the injected amounts of monosaccharide standards and the peak areas, and hence the amounts of the individual monosaccharides in the samples were deduced from standard curves.
Removal of potential N-linked glycosylations was performed as described by the manufacturer using a PNGase F deglycosylation kit. OPN was incubated with 500 units of PNGase F at 37 °C for 20 h. The reaction products from the enzymatic deglycosylation were analysed by SDS/PAGE.
Generation and separation of peptides
OPN was digested with trypsin by using an enzyme-to-substrate ratio of 1:30 (w/w) in 0.1 M ammonium bicarbonate at 37 °C for 6 h. Tryptic peptides were separated by RP-HPLC on a μRPC C2/C18 PC 2.1/10 column connected to a GE Healthcare SMART system. Separation was carried out in 0.1% TFA (buffer A) and eluted with a gradient of 60% acetonitrile in 0.1% TFA (buffer B) developed over 54 min (0–9 min, 0% B; 9–49 min, 0–50% B; 49–54 min, 50–100% B) at a flow rate of 0.15 ml/min. The peptides were detected in the effluent by measuring the absorbance at 214 nm. A fragment (residues Gln36/Ser62–Arg143) from the tryptic digest was further digested with thermolysin in 0.1 M pyridine acetate (pH 6.5) and 5 mM CaCl2 at 56 °C for 6 h. The resulting peptides were separated as described for the tryptic digest. A glycopeptide (residues Leu116–Thr139) from the thermolysin digest was further digested with staphylococcal (S. aureus) V8 protease in 0.1 M ammonium bicarbonate (pH 8.1) at 37 °C for 3.5 h.
OPN45/OPN50 and OPN60 were digested with thrombin (0.05 unit/μg of OPN) in 0.1 M ammonium bicarbonate at 37 °C for 1 h. The generated fragments were separated by RP-HPLC on a Vydac C18 column connected to a GE Healthcare LKB system. Separation was carried out in 0.1% TFA (buffer A) and proteins were eluted with a gradient of 75% propan-2-ol in 0.1% TFA (buffer B) developed over 80 min (0–5 min, 0% B; 5–70 min, 0–50% B; 70–80 min, 50–95% B) at a flow rate of 0.85 ml/min.
Characterization of peptides
Peptides and fragments were characterized by MS and amino acid sequence analyses. All MS spectra were obtained in both positive reflector-ion and positive linear-ion mode as described above. The theoretical peptide masses and sequence coverage were calculated using the GPMAW program (Lighthouse Data, Odense, Denmark). Amino acid sequence analyses were performed on an Applied Biosystems PROCISE HT protein sequencer with online identification of PTH (phenylthiohydantoin)-derivatives. Glycosylated threonine residues were identified by the lack of a PTH derivative in the cycles where these amino acids are modified.
Sulfate analysis
Modified peptides containing both potential phosphorylation (serine and threonine residues) and sulfation (tyrosine residues) sites were dephosphorylated with ALP as described above and analysed by MALDI–TOF-MS. The peptides Phe158–Lys187 and Arg160–Lys187 were further treated with 0.5 unit of arylsulfatase in 0.2 M sodium acetate (pH 5.0) at 37 °C for 2 h and then re-analysed by MS.
Purification of OPN from urine
Urine samples from seven healthy donors, with normal renal function and no history of urinary disease, were analysed by Western blotting to examine the molecular forms of OPN in urine (Figure 1). Urinary OPN was detected with variable intensity in all individuals by a polyclonal antibody. The observed OPN patterns show that all analysed urine samples contain OPN forms of relative homogeneous molecular mass in the range of 45–60 kDa. No low-molecular-mass OPN forms were observed in any of the tested individuals.
OPN was purified from human urine by anion exchange followed by RP-HPLC separation on a Vydac C4 column (Figure 2). Sequencing of the OPN-containing fraction showed two sequences: a major, I1PVKQA6, and a minor, V3KQADS8, both corresponding to the N-terminal part of human OPN. SDS/PAGE revealed the presence of three bands migrating at approx. 45, 50 and 60 kDa respectively and Western blotting confirmed their OPN nature (Figure 3A, lanes 1 and 2). In the following, these components will be referred to as OPN45, OPN50 and OPN60.
To separate the different forms, the OPNs were subjected to RP-HPLC on a Vydac C18 column and eluted with stepwise increasing concentrations of propan-2-ol. The elution profile revealed two fractions (inset in Figure 2). As judged by SDS/PAGE, the first fraction contained OPN45 and OPN50 and the second fraction contained OPN60 (Figure 3A, lanes 3 and 4). The monoclonal antibody 1H3 (epitope: S218AETHSH224 [39]) recognized all three OPN forms (Figure 3B, lanes 1 and 2), whereas Western blotting with the monoclonal antibody 3D9 (epitope: K283FRISHELDSASSEVN298 [39]) showed that only OPN60 contains the extreme C-terminal part of OPN (Figure 3B, lanes 3 and 4). This indicates that OPN60 represents the full-length OPN molecule from human urine, since it contains both the N- and C-termini, whereas OPN45 and OPN50 are truncated or cleaved forms lacking a C-terminal part of the protein.
Identification of cleavage sites in OPN
To identify the C-terminus and thereby the exact cleavage site that generates OPN45 and OPN50, these proteins were subjected to thrombin cleavage. RP separation of the products from the thrombin digest followed by sequence and MS analyses showed the presence of two N-terminal fragments, Ile1–Arg152 and Val3–Arg152, and two C-terminal fragments, Ser153–Arg228 and Ser153–Tyr230 (Table 1). This indicates that OPN45 and OPN50 are generated by cleavage at Arg228–Leu229 and/or Tyr230–Lys231 in human urine. In an additional experiment, digests of OPN45/OPN50 and OPN60 were compared by peptide mapping analysis. Peptides from the N-terminal part (Ile1–Leu229/Arg232) were found in all OPN isoforms, whereas peptides from the C-terminal part (Leu229–Asn298) only were identified in OPN60, further supporting that OPN45 and OPN50 are generated by cleavage in the region around Leu229–Arg232.
To test whether OPN45/OPN50 could be generated from OPN60 by thrombin cleavage, the full-length protein was incubated with this protease. This resulted in two major fractions in RP-HPLC containing the N-terminal part (Ile1–Arg152 and Val3–Arg152) and the expected C-terminal part of OPN (Ser153–Asn298). Furthermore, we observed a minor fraction representing a fragment corresponding to Ser153–Arg228/Tyr230. Judged by RP-HPLC and MS analyses, this fraction only represents a very minor portion of the cleavage products and, hence, we cannot, on this basis, conclude that thrombin would cleave OPN at this site in vivo.
Molecular masses
The masses of the OPNs were determined by linear MALDI–TOF-MS. MS analysis showed a mass of approx. 37.7 kDa for OPN60 (Figure 4A), whereas the sample containing OPN45 and OPN50 showed one broad mass peak with an average value of approx. 29.3 kDa (Figure 4B). To estimate the total number of phosphate groups present, OPN60 and the fragments OPN45/OPN50 were treated with ALP. The molecular mass of dephosphorylated OPN60 was 37.1 kDa, corresponding to a loss of approximately eight phosphate groups (Figure 4A). Phosphatase treatment of the N-terminal OPN fragments reduced the mass to 28.8 kDa, corresponding to a loss of approximately six phosphorylations (Figure 4B). This was confirmed by use of a Phosphoprotein Phosphate Estimation kit, revealing a total of 7.8 phosphate groups in OPN60 and 6.9 phosphate groups in the sample containing OPN45/OPN50. Subtraction of the theoretical mass of the human OPN polypeptide (33714 Da) from the observed average mass of dephosphorylated OPN60 (37.1 kDa) leaves approx. 3.4 kDa, which must then be accounted for by other PTMs.
To identify potential phosphorylation sites in urinary OPN, the OPN fraction (Figure 2) was digested with trypsin and the resulting peptides were separated by RP-HPLC (results not shown). A large acidic fragment of OPN (Gln36/Ser62–Arg143) not susceptible to tryptic cleavage was further digested with thermolysin and the resulting peptides were separated by RP-HPLC. All fractions from the RP-HPLC separations of peptides were analysed by MALDI–TOF-MS (Table 2; Supplementary Tables 1 and 2 at http://www.BiochemJ.org/bj/411/bj4110053add.htm). In total, the approximately eight phosphate groups in OPN60 were distributed over 30 potential phosphorylation sites. Some of the phosphopeptides contained more serine/threonine residues than the observed number of phosphorylations. In these cases, the phosphate groups were assigned to residues fitting the target sequence of the Golgi kinase/mammary-gland casein kinase [S/T-X-E/S(P)/D], based on the localization of phosphorylated residues in other OPN isoforms [27–29]. Data from the peptide phosphorylation analysis are summarized in Table 2 and the resulting map of PTMs is shown in Figure 5, which also contains a comparison with the PTMs of human milk OPN.
Sulfation of OPN
The linear MALDI–TOF-MS spectrum of a peptide covering R160PDIQYPDATDEDITSHMESEELNGAYK187 showed m/z values of 3307.18, 3387.53 and 3466.42 (Figure 6A). These masses correspond to Arg160–Lys187 with excess masses of ∼80, ∼160 and ∼240 Da respectively. These masses could correspond to different phosphorylation variants, or they could represent a combination of phosphate and sulfate groups on the peptide. After treatment with ALP, an excess mass of ∼80 Da remained associated with the peptide (Figure 6B), strongly indicating that Arg160–Lys187 is also sulfated. The presence of a sulfate group was verified by treatment of the dephosphorylated peptide with arylsulfatase, which removed the excess mass of 80 Da (Figure 6C). The peptide Arg160–Lys187 contains two potential sites for sulfation (Tyr165 and Tyr186). The residue corresponding to Tyr165 has been shown to be sulfated in OPN from rat bone [29], and it is therefore most likely that this residue is sulfated in urinary OPN. Two other tyrosine-containing peptides, Gln5–Lys35 and Gly205–Lys225, were observed with excess masses that could correspond to both phosphorylations and/or sulfate groups (Supplementary Table 1). Treatment of these peptides with ALP showed that they only contained phosphate groups. Peptides containing Tyr165 in human milk OPN were also analysed for sulfation. However, no indication of this modification was observed in the milk isoform (results not shown).
All O-glycosylated residues in OPN from urine were located in a single thermolytic peptide, Leu116–Thr139. This peptide was further digested with staphylococcal (S. aureus) V8 protease, resulting in the two peptides, L116VTDFPTDLPATE128 and V129FTPVVPTVDT139, which were separated by RP-HPLC. The peptide L116VTDFPTDLPATE128 was observed in linear MS with m/z values at 3679.83, 3970.97 and 4261.24 (Table 3). These masses correspond to Leu116–Glu128 with three GalNAc-galactose units and four to six sialic acid units attached. The lack of a PTH-threonine at the positions of Thr118, Thr122 and Thr127, when subjected to Edman sequencing, indicates that these threonine residues are glycosylated in the peptide. Similarly, MS analysis of the peptide V129FTPVVPTVDT139 showed that this peptide contained glycosylations composed of up to two GalNAc, one GlcNAc, two galactose and five sialic acid units (Table 3). Edman sequencing showed that Thr131 and Thr136 are modified. Collectively, these results show that Thr118, Thr122, Thr127, Thr131 and Thr136 are glycosylated in urinary OPN. The absence of any trace of PTH-amino acids in these positions indicates that they are fully glycosylated. The masses of the glycopeptides show that each glycosylated threonine residue can be modified by different glycan structures, but based on the major mass peaks for both Leu116–Glu128 and Val129–Thr139 they seem mainly to consist of the disialylated core-1 O-glycan sialic acid–galactose–[sialic acid]–GalNAc structure.
OPN60 was subjected to mild acid hydrolysis to determine the monosaccharide composition of the glycans on the protein and thereby evaluate the structures observed in MS. The obtained monosaccharides were separated by high-pH anion-exchange chromatography and subsequently monitored by pulsed electrochemical detection. The hydrolysates of OPN60 showed the presence of four types of monosaccharides: GalNAc, GlcNAc, galactose and sialic acid (Table 3). The detected amounts of monosaccharides give approx. 1 mol of GalNAc/galactose and 2 mol of sialic acid per glycosylated threonine on OPN, corresponding well to the glycans observed by MS.
The human OPN sequence contains two asparagine residues (Asn63 and Asn90) in putative N-glycosylation motifs. MS analysis of peptides containing these residues showed that none of these asparagine residues are glycosylated in urinary OPN. In addition, incubation of urinary OPN with PNGase F did not result in altered migration when subjected to SDS/PAGE (results not shown). Furthermore, no trace of mannose was detected in the analysis of OPN monosaccharides (Table 3), substantiating the absence of N-linked glycosylations in human urinary OPN.
The present study describes the purification and characterization of urinary OPN. Western blotting of urine obtained from seven healthy donors shows that urinary OPN exists in different forms migrating between 45 and 60 kDa (Figure 1). In the OPN peak eluted from the Vydac C4 column, three dominating forms were observed by CBB staining and immunoblotting (Figure 3A). The strongly staining top band observed by Western blotting may consist of more than one isoform. This assumption is supported by Western-blot detection of two bands in the samples containing OPN60 using the monoclonal antibodies 1H3 (faint) and 3D9 (Figure 3B).
The present study indicates that the N-terminal fragments OPN45 and OPN50 are generated from the full-length molecule by proteolytic cleavage at Arg228–Leu229 and/or Tyr230–Lys231 in urine. Although two bands are seen in SDS/PAGE, representing OPN45 and OPN50, only one broad peak is observed by MS, suggesting that the mass difference between the fragmented OPNs is not large enough to give separation in linear MALDI–TOF-MS. No heterogeneity at the identified O-glycosylation sites was observed. However, differences in the glycans at the individual sites might account for the observed migration of OPN45 and OPN50 during SDS/PAGE.
OPN is present in human urine in different fragmented forms, and it is therefore relevant to speculate on how these forms are generated. The protein has previously been shown to be a substrate for MMP-3 (matrix metalloproteinase-3) and MMP-7 [40] and thrombin [41]. In Figure 5, the cleavage sites of these proteolytic enzymes are compared with the observed fragmentation patterns of the characterized urinary OPN, showing that neither MMP-3, MMP-7 nor thrombin have been reported to cleave OPN at the sites reported in the present study. It is not clear whether OPN45/OPN50 is the result of cleavage by specific proteases in the urinary secretory system. The cleavage sites identified in the present study point in two directions. The cleavage of Arg228–Leu229 could be catalysed by a thrombin-type protease, whereas the cleavage site Tyr230–Lys231 fits the specificity of a chymotrypsin-type protease. However, it remains to be shown which proteases are actually responsible for the cleavage of OPN in urine. Kidney cells have been reported to produce an OPN peptide that is thought to represent tissue-specific intracellular processing of the intact protein [42], which supports the hypothesis that OPN45 and OPN50 are proteolytically derived fragments of the intact protein. Alternative mRNA splicing could also be speculated to be responsible for the presence of the truncated OPN forms. However, the region containing the cleavage sites is encoded by exon 7 of the OPN gene, and no intron–exon junction is located in the vicinity. It is therefore a much more plausible explanation that the N-terminal fragments are products of proteolytic activity rather than alternative mRNA splicing.
Interestingly, no C-terminal fragments corresponding to Leu229/Lys231–Asn298 were detected by the polyclonal antibody in the Western blot of urine (Figure 1). This indicates that the C-terminus of urinary OPN is completely degraded when OPN45 and OPN50 are generated from OPN60. In support of this, no low-molecular-mass C-terminal OPN fragments were detected in Western blots of human urine using different monoclonal OPN antibodies with epitopes mapped to the C-terminal part of the protein [39]. OPN forms below the 40 kDa electrophoretic mobility range have been observed in urine from patients with kidney stones [24], whereas normal individuals had OPN forms migrating between 55 and 66 kDa, as observed in the present study. These low-molecular-mass forms of OPN were generated by serine proteases and could reflect that augmented cleavage of urinary OPN could be a critical step in the formation of kidney stones.
OPN60 was shown to contain approximately eight phosphorylations and six phosphate groups were estimated to decorate OPN45/OPN50. This indicates that the region Leu229/Lys231–Asn298 lacking in these cleaved forms contains approx. two phosphate groups. It is not clear whether some serine or threonine residue in OPN are phosphorylated to a higher degree than others. However, the fragment Leu229/Lys231–Asn298 that is lacking in OPN45/OPN50 constitutes approximately one-fourth of the amino acids in OPN. So, the observation that this fragment contains two out of eight phosphorylations suggests that the phosphate groups decorating the intact molecule are evenly distributed. The degree of phosphorylation on urinary OPN is significantly lower than the levels observed in milk OPN [27,28], showing tissue-specific phosphorylation. A comparison of OPN from human milk and urine with regard to PTMs is shown in Figure 5. It is interesting to observe that of the 36 phosphorylation sites previously located in human milk OPN, 30 sites were also identified in human urinary OPN. However, the average phosphate content of the urinary isoform is only approximately one-third of the milk isoform. In both urine and milk, the highly anionic regions of OPN composed of multiple acidic amino acids and phosphorylations could constitute potential binding sites for minerals and calcium salts. These anionic regions could therefore enable OPN to form soluble complexes with calcium ions and thereby inhibit unintentional calcium precipitation and crystallization. It is not known whether the different degrees of phosphorylation of human milk and urinary OPNs are due to tissue-specific phosphorylation or dephosphorylation.
Urinary OPN was shown to be sulfated at Tyr165. This residue has recently been shown to be sulfated in rat bone OPN [29], and OPN produced from canine kidney cells has also been shown to be sulfated by metabolic labelling [42]. Tyr165 is conserved in mouse, rat and rabbit but not present in cow, sheep, pig and chicken, indicating that sulfation of the protein is species-dependent. No indication of a sulfate group on Tyr165 was observed in human milk OPN, showing that this modification is also tissue-specific.
In the present study, urinary OPN has been shown to contain five O-glycosylated threonine residue (Thr118, Thr122, Thr127, Thr131 and Thr136). As shown in Figure 5, all O-linked glycans identified in human urine OPN are also present in human milk OPN. It appears that the glycans in urinary OPN predominantly consist of a disialylated GalNAc-galactose core. This glycan structure has also been described in OPN from rat bone and mouse fibroblasts and osteoblasts [29,30]. No N-linked glycosylations were identified on urinary OPN at the two putative motifs (Asn63 and Asn90). This is consistent with data on milk, bone and fibroblast OPNs [27–30]. Normal rat kidney cells have been shown to produce both phosphorylated and non-phosphorylated forms of OPN in vitro, and the non-phosphorylated isoform contained N-linked carbohydrates [37]; whether this is also the case in vivo remains to be shown.
In summary, human urine contains different forms of OPN representing the full-length protein and truncated forms lacking the C-terminal fragment Leu229/Lys231–Asn298. The intact protein contains approximately eight phosphate groups distributed over 30 phosphorylation sites, whereas the fragments contain approximately six phosphate groups. In addition, one sulfate group and five O-glycosylated threonine residues were identified in urinary OPN. The PTMs of urinary OPN reported here are likely to play key roles in the protein's regulation of mineral crystallization in urine. Further studies are necessary to investigate whether different PTMs could account for the pleiotropic role of OPN as both an inhibitor and promoter of renal stone formation.
Abbreviations: ALP, bovine alkaline phosphatase; CBB, Coomassie Brilliant Blue; COD, calcium oxalate dihydrate; COM, calcium oxalate monohydrate; MALDI–TOF-MS, matrix-assisted laser-desorption ionization–time-of-flight MS; MMP, matrix metalloproteinase; OPN, osteopontin; PNGase F, peptide N-glycosidase F; PTH, phenylthiohydantoin; PTM, post-translational modification; μRPC, μ reverse-phase chromatography; RP-HPLC, reverse-phase HPLC; TFA, trifluoroacetic acid
- © The Authors Journal compilation © 2008 Biochemical Society


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Hampshire High School cheerleaders Corbin Hinson, Darian Doner, Hannah Gilliam, Austyn Earls-Fender, Alex Morrow, Savannah Young, Hannah Walker, Victoria Huitt, and Kaitlyn Devore participated in the pre-game show Sept. 8 of UT-Georgia State football game at Neyland Stadium in Knoxville with over 900 other cheerleaders from across the state of Tennessee.
Hampshire High School cheerleaders Corbin Hinson, Darian Doner, Hannah Gilliam, Austyn Earls-Fender, Alex Morrow, Savannah Young, Hannah Walker, Victoria Huitt, and Kaitlyn Devore participated in the pre-game show Sept. 8 of UT-Georgia State football game at Neyland Stadium in Knoxville with over 900 other cheerleaders from across the state of Tennessee.
Savannah Collie was among the Lewis County Cheerleaders participating in UT-Georgia State pre-game show September 8.
The unveiling of the statue of Pat Summit at UT-Martin Oct. 6 was witnessed by Jeanell Kutterer.
Exchanging marriage vows Oct. 6 were Stephanie Kennedy and Tucker Southall on the beach in Destin, Fla. Tucker is the son of Ronny and Gena Southall of Linden and the grandson of Helen and the late William Southall, also of Linden and previously of Hampshire.
Get-well wishes are extended to Harry Masterson, Gail Boyd, Frances Smith, Charlie and Ellen Anderson, Rick and Denise Gray, Hal Cathey, Walter Dycus, Gertha Mae Duncan, Martha Whiteside, Vera Grimes, Ruby Riley, Kathleen Morton, Christine Morgan, Annie Ruth Howard, J.D. Brooks, Ray Bassham, Kenneth Voss, Kay Baxter, Paul Sands, Joe Brooks, Sara Dale, Cecil Whiteside, and Paul Lusk (Richard and Laura Hayes� grandson-in-law).
On Oct. 12-13, Confederate Gray Chapter daughters Jeanell Kutterer, Renee McEndarfer, and Laura Hayes attended the 116th Annual Convention United Daughters of the Confederacy in Clarksville TN for �A Weekend with Mrs. Caroline.� Caroline Meriwether Goodlett, the founder of UDC, was born at Woodstock near Clarksville. Confederate Gray Chapter was awarded 31 certificates for their accomplishments, serving military and Lewis and Maury Counties� communities during 2011-12.
The Backpack Program is one aspect of the Hampshire Baptist church Food Ministry with the recognition that many children from Hampshire Unit School and Mt. Pleasant Elementary School are, in fact, hungry. Food is now being distributed to 66 children. The food supply here is diminishing with the government-provided food. Many families are depending on the supplemental assistance offered with the food distribution. The grant funding for the Backpack program will end in October. The cost to feed one child for a weekend is approximately $3.10. If you would like to become involved or have any ideas about funding this program contact Rhonda or Ron Malugin at (931) 379-0297 or firstname.lastname@example.com.
Oct. 16 was a Country Classroom day at UT Experiment and Research Station in Spring Hill for 3rd graders from Maury County public and private schools. Approximately 900 students enjoyed the warm, sunny day as they trekked about the farm, seeing cows, farm equipment, making butter and recycling farm wastes.
Sympathy is expressed to the families of Frances Woodall Fraser, Madelyn Olivia McKennon and Lina Harlan.
Hampshire School K-2 students enjoyed a day at Ring Farm�s pumpkin patch on Green Mills Road and touring the historic Pigg School House near Santa Fe in October.
The benefit concert, �A Night for Chris,� at Cherry Theater at Columbia State Community College was well attended. Wynn Varble, Joey and Rory, Leslie Satcher. Kerry Kurt Phillips, David Lee, Ruby Lovett, Georgia Claire Varble and Rock Jaggers, and Refried Howdy musicians Jonathan Harrison, Mel and Tommy. Chris is the nephew of Wynn and Marsha who suffered life-threatening injuries in an automobile accident. Jonathan returned from Afghanistan for a few days where he�s serving as a medic and quickly returned.
Laura Hayes grew up in Hampshire. She is a homemaker with two children and five grandchildren, a Sunday school teacher and volunteer with 4-H and the Hampshire Museum.


text: The GREE Game Studio develops AAA games for the iOS and Android platforms. The Studio builds games natively using the advantages of each operating system. To date, the Studio's published titles have all been in iTunes top 10 Free-to-Play apps (both in terms of downloads and revenue). Our game portfolio includes: Crime City, Modern War, Kingdom Age and Monster Quest. We have many new games in production.


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A classic cruising ketch with immense appearance, OCEAN WOLF offers all amenities for her new owner and guests to cruise the Med.
The semi-enclosed deckhouse hosts the control center and steering position, giving a commanding view over the whole yacht. A folding teak table and circumjacent seating makes it the place to be for owner and guests for informal meals, cocktails, reading or just relaxing and enjoying the surroundings.
Below deck the yacht shows the more the legendary comfort even of a 20m Jongert sailing yacht: The salon midships with an unfolding table for up to eight guests and integrated, fully equipped galley; the stylish owner's cabin to the aft offering a king-sized bed to port and a cosy seating to starboard, Jongert signature windows in the transom and an ensuite bathroom; two guest cabins located forward to the salon with berths for up to three guest each, as well ensuite, and a crew cabin in the bow.
OCEAN WOLF - an attractive cruising yacht for comfortable sailing with efficient handling!
Owners Cabin: 2 berths, bathroom en suite
Guest cabins: 2 x 2 berths, lower bed extendable to double, bathroom en suite each
Crew cabin: 1 berth, bathroom en suite
Rig type: Ketch
Reefing systems: RONDAL hydraulic, in-mast reefing system for main and mizzen, hydraulic RONDAL roller furling for foresails
Masts: 2 x RONDAL aluminium
Mast height: 25m
Booms: 2 x RONDAL aluminium
Main engine type: MERCEDES OM 407
Fuel tank: 2,400 l
Fresh water tank: 2,200 l
Waste water tank: 500 l
Waste treatment system: HAMANN Super Mini 7618
Batteries: SERVICE GEL 1,125 Ah, engines: 200Ah
Battery Charger: MASTERVOLT 24/100-3
Inverter: MASTERVOLT Mass 24V/2500W
Shore Power: Yes
Navigation system: B&G wind screen and direction
Autopilot: SEGATRON
Compass: SEGATRON
Gyro compass: B&G
GPS/plotter: RAYMARINE E120 and E80 (master an slave)
Radar: RAYMARINE, range 72'
Navtex: ICS Nav4 Navtex/NMEA
Echo sounder: B&G H3000 and sumlog, 2 x analogical batograph
Rudder angle indicator: VDO
TV: 1 x LG 21" (2008)
DVD/Video: 1 x SONY DVP-NS78H (2008)
Stereo system: 1 x KENWOOD KDV-5234 (2008)
refrigerator: Yes
Deep freezer: Yes
Stove: MIELE KM 6013 H5361 EP (2008)
Washing machine: MIELE NOVOTRONIC WT 946 (2008)
Dryer: MIELE T8623C (2008)
Sailmaker: HALSEY LIDGARD
Sail area: 305 sqm
Main: HALSEY LIDGARD
Mizzen: HALSEY LIDGARD
Stay: HALSEY LIDGARD
Spinnaker: HALSEY LIDGARD
Winches: LEWMAR: 2 x 65ST electric, 1 x 66ST electric, 3 x 55 ST, 3 x 52 ST, 3 x 46 ST
Windlass: LOFRANS Mod. Falkon
Anchor & chain: 2 x CQR 105 and 75 lbs, chain: 95m, 12mm
Deck shower: Yes
Deck wash system: Yes
Bathing ladder: Yes, 2
Canvas cover: Yes
Passerelle: Yes
Awning: Yes, awning and bimini
Sun matresses: Yes
Canvas cover: Yes
Air conditioning and heating: Domestic MARINE AIR SYSTEM +/- 6 kW
Water maker: DESALATOR 180 l/h, plus separate hot water heater
VHF-Radiotel: SAILOR Master RT5020 DSC duplex and slave CU 5000
SSB-Radiotel: SKANTI TRP 8000 250W
Satcom: SATCOM M and SATCOM B telephone and fax, INMARSAT C
Internet: outdoor antenna to connect computer to WIFI
Fax: Yes
Emergency transmitter: SAILOR INMARSAT
Tender: JOKER 315 (2012), engine TOHATSU 4T, 15 kW/20 hp (2012)
Diving equipment: Yes, only for emergency
Life rafts: ZODIAC 8-man
Life jackets: Yes
Life belts: Yes
Distress flares: Yes
Epirb: McMURDO
Bilge alarm: Yes
Fire extinguisher: Yes
MOB equipment: Yes
Refit works S/Y OCEAN WOLF
New teak deck.
New bars for stay sail sheet.
New air conditioning
New Intelligent Sewage Treatment Hamman
Painted and varnished superstructure
Installation of new navigation equipment, radar / plotter Raymarine with repeater at chart table, lifetag overboard system Raymarine, VHF Sailor, and Inmarsat-C Transceiver Sailor, AIS system and navigation equipment B & G H3000.
ISO9650 ZODIAC Life raft for 6 people
McMurdo EPIRB.
New whasing machine and dryer.
Review and fine tuning of the water treatment
New stove and oven combo
Review and fine tuning of the fridge and freezer.
Tuning main engine and generator, checking compressions.
New gel batteries for services and engine.
Reupholstering armchairs in master cabin and saloon.
New battery charger and converter 24-220 v
Renewal of carpet in cabin owner and guests
Review and fine tuning of toilets
New pump water and pipes for hot and cold water.
Checked the electrical installation. New navigation lights with LED lamps.
New wifi antenna.
Polished both sides (2013)
New water heater (2012)
Upholstered ceilings, saloon, owner and guest cabin. (2014)
Painted masts from deck to the first spreader (2012)
New dinghy and engine, 15 HP (2012)
New bowsprit (2013)
Replacement of some sections of the standing rigging (2014)
New cover entrance to the engine room from the cockpit(2014)
Reviewing and changing gaskets and seals of the hydraulic cylinders rudder(2015)
Scrape and paint treatment bilges master cabin, lounge, guest cabins and pit chains. Also parts of the room máquinas(2015)
Varnishing dog house (2015)
During the refit 2008 the mainsail traveller installation was relocated from the deck to the top of the deckhouse to enable unhindered maneuvers.
The Company offers the details of this vessel in good faith but cannot guarantee or warrant the accuracy of this information nor warrant the condition of the vessel. A buyer should instruct his agents, or his surveyors, to investigate such details as the buyer desires validated. This vessel is offered subject to prior sale, price change, or withdrawal without notice.
Club de Mar
Muelle Pelaires s/n
Palma de Mallorca


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Meg's done all of the research so you can find the very best serger for your sewing needs. Her article is coming up next… In the meantime, check out her Fashioned by Meg blog, Meg's etsy shop and her bio:
From Meg: Hi! I am Meg from Fashioned by Meg. I am a California girl currently living in Indiana- hopefully, we should be back in California by September! I am the wife of Kurt, my wonderful lawyer husband, and the mom to the two sweetest little girls, Juliana and Felicity. I like to sew everything! I learned to sew when I was a teenager, but only recently got back to sewing a couple of years ago. I finished my first quilt last month, and I can't wait to make more!
» Contributors » Introducing ~ Meg
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31 Responses to Introducing ~ Meg
Welcome Meg! What a cute first quilt you made!!
Great job on your first quilt Meg!
Hi and welcome Meg.
I love your market bags! Very cute!
Hi Meg
Its nice ot meet you
Your first quilt is great i cant wait to try my hand at quilting 🙂
Welcome Meg! LOVE your first quilt! How awesome!! Cute items in the shop too!
Hi Meg! I love your first quilt. I hope you're hooked, because it's fun!
What a great etsy shop!
Thanks for sharing with us! I love your quilt (beautiful fabrics!) and you have some great patterns in your shop!
I have been hoping you would talk about sergers this month! I am seriously thinking about buying one and I am so appreicative of folks who share their knowledge. Yea!
Oh this is wonderful. I am trying to learn everything i can about sergers because i just ordered one and have never used one. i havent read the story yet but i cant wait to check back when i have a little more time to really look at it!! thanks for this great subject.
Your first quilt it awesome and I love the colors. I have one started in my head that I hope to get to soon. It will be my first as well. Thank you also for the serger tute. It is very helpful (read that post first). ~Page
I've been following your blog for a while, glad to see you over here at SMS! Your serger info was great, thanks for researching and sharing!
I love your first quilt! Great job!
Cute etsy shop…..love the pattern for the owl purse!!
Great blog, congrats on your first (gorgeous) qulit! It's nice to see someone able to accomplish so much with little ones around. I'm still struggling to get anything done now that I have two…
Your little girls sure are cute. Congratulations on your first quilt. It looks great.
Welcome! I love your sense of style. I really love the kimono pjs, and the baby shoes. I can't wait to try to make some for my little ones.
I really like your first quilt. You did a great job. Also you have a lot of cute things in your shop.
Nice to meet Meg! And thanks for doing the hard work for us!
Great info there. I sure wish I had a serger. Well, maybe someday.
My Brother 1034 was just delivered today so I was so thrilled to visit your site today and see this article, and a picture of the serger I just bought. Thank you, it's timeliness is perfect for me. I'm just getting back into sewing and this is my first serger.
I love your blog! You may be as obsessed with sewing all of Oh, Fransson's patterns as I am 🙂 Your blog has given me a few new ideas! Thanks for sharing!
Meg has a great blog! I am on to learn about sergers and then I will spend some time exploring Meg's blog.
I love the blog and your first quilt looks great. I'm getting ready to quilt my first quilt and it's pretty exciting.
Welcome Meg! I love all the patterns that are in the Etsy shop.
Howdy Meg. great blog, lovely shop.
Hi! I visited your blog and the links for sergers have been very helpful. I am a little overwelmed with all that is out there, but info on your blog gives me some light 🙂 I am looking forward to your article tomorrow. Thank you for your time to bring this info to us. I have bookmarked your blog .
Hello from Southern Indiana!
Hi Meg! I'm looking forward to reading your serger research!
What a cool first quilt!


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ADM showcases the annual Top 40 Defence Contractors/Top 20 Defence SMEs.
Read about the DCNS offerings for projects various that Editor Katherine Ziesing got to see during her visit to France last year and Senior Correspondent Julian Kerr reviews maritime and joint projects during 2015 while Deputy Editor At Large Nigel Pittaway covers the air and land domains.
Online Editor Patrick Durrant takes a look at Army's Innovation Day where human factors were the theme of the event held late last year. The issue also presents a wrap on the expo success that was Pacific 2015.
And hear from our From the Source interviewee, BAE Systems Australia CEO Glynn Phillips, as he seeks to restructure Australia's number 1 defence contractor to meet the evolving needs of the Defence customer.
View the digital edition.


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Road 13 Vineyards Appoints Jean-Martin Bouchard as New Winemaker
As winemaker Michael Bartier transitions to a consultant role, Road 13 is excited to announce their new winemaker J-M Bouchard. Hailing from Montreal, Bouchard is a Canadian winemaker who has worked in cellars around the world perfecting his expertise. First enthralled by wine while working in hospitality roles, Bouchard worked in Australia and eventually studied wine science there as well. Through his work in Australia, Europe, and in the Niagara region and the Okanagan Valley, Bouchard is an international success. Road 13 Vineyards is very pleased to have him join their tight-knit team.
Owner Pam Luckhurst notes the staffing changes. "We are thrilled to have had a winemaker of Michael Bartier's calibre for the past five years, and are happy for him that he is working towards his personal goal of having his own winery," she says. "We wish him every success in his new venture and appreciate his assistance with our succession plan."
"And, we are excited to have J-M join us at Road 13," she continues. "We all know that he has big shoes to fill but we are confident that he will lead the winemaking team into the next chapter in Road 13's story."
Bouchard is enthusiastic about his new winemaking position at Road 13. "I am excited to join the team," says Bouchard "I had admired the work being done here, as the winery's approach is close to mine. Like Michael, I like to focus on non-interventionist and terroir-driven wines," he continues. "As I transition into my role and Michael leaves, we will work together to ensure that the wines that everyone loves from Road 13 are maintained."
About Road 13 Vineyards:
Owned by Pam and Mick Luckhurst since 2003, Road 13 Vineyards is an Okanagan sensation. The wines express the special character of the South Okanagan's renowned Golden Mile Bench. Their motto, "it's all about the dirt" reflects their belief that great wines start in the vineyard. The winery offers award-winning wines in three distinct tiers: a rose, and red and white blends called Honest John's; a varietal series of red and white wines under the Road 13 label; and a premium tier of small lots and blends called Jackpot. In 2010 two premium blended wines were added to great success: Rockpile, a blend of red varieties; and Stemwinder, a blend of white varieties. Road 13 is located south of Oliver, BC on Road 13 off Highway 97. Open year round, the wine shop offers small lots and back vintages in addition to current releases. To find out more, please visit www.road13vineyards.com.


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My colleague Vanitha Prabhakaran has written a fantastic article on Troubleshooting Direct Push to resolve issues if you are having trouble getting it working.
The major issue I've seen customers encounter is when they are using ISA 2000 rather than ISA 2004 and the client OPTIONS commands are not being passed. This article resolves those issues:


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Cameras catch every move, everywhere
Fri, 19 Apr 2013 22:26:24 GMT —
Everywhere you look in this digital age, there are cameras. The Glass City being no exception.
Toledo Police have about 70 cameras in place and being monitored right now, and they will continue to install these cameras through this fall.
The final number when finished will be around 150.
And if police cameras do not have eyes in a certain area, you're bound to find others that do.
Images from surveillance video at two seperate robberies on April 18th show thieves being caught in the act.
"If you walk downtown anywhere, you're gonna be on camera," says Kirk Davis, President of Continental Security.
He says his company's video footage is frequently used as an extended arm for law enforement.
"I can't think of any long period of time where we don't have a detective from the sheriffs deptartment or Toledo police contacting us about something that's gone on where our camera's were called on," he says.
While some people think cameras are an invasion of privacy, they've played a vital role in solving crime, including helping with the bombing in Boston.
And with more being installed daily, they're beginning to stop crime before it occurs.
"When you walk into an area and see a camera, you are less likely to steal something," says Davis.
For those with security issues, cameras can be very cost effective.
Davis says, "Once we put the cameras in, 99% of the time the problem stops."
As more cameras appear on buildings and neighborhood houses, police anticipate a decrease in crime. Because if someone breaks the law, they'll run the risk of giving authorities a front row seat via video surveillance.


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I've been talking a lot about Procter & Gamble (NYSE:PG) in my articles as of late, and thought I should balance it out by writing about one of their competitors. I decided to go with a company that recently reported fourth-quarter and full-year 2011 results, and is run by a man who spent 22 years as the president of European operations at P&G, as well as a short stint at Nestle before moving into his current role; his name is Paul Polman, and he is the CEO of Unilever.
In 2011, Unilever generated underlying sales growth of 6.5% (lapping 4.1% growth in 2010), with 4.8% coming from pricing and the remaining 1.6% coming from higher volume; in the words of CFO Jean Marc-Huët, "We would have preferred more volume and less price. But in a strong inflationary environment where significant pricing has been essential to protect profitability, we're pleased that the volumes have held up so well." After accounting for a 2.5% currency headwind and 1% of positive contribution from acquisitions/disposals, full-year sales came in at €46.5 billion, a 5% increase from 2010.
The company is split into four separate segments: Personal Care (33% of full year sales), Home Care (18%), Foods (30%), and Refreshments (19%); in regards to underlying sales growth for the year, Personal Care & Home Care both grew high single digits (8.2% and 8.1%, respectively), while Foods & Refreshments both expanded at a mid-single digit rate (4.9% for both segments).
On a regional basis, the company is split into three geographic regions: Asia Africa CEE (41% of full year sales), The Americas (33%), and Western Europe (26%). For the full year, underlying sales growth was up double digits (10.5%) in the company's Asia Africa CCE region, and mid-single digits (6.3%) in the Americas, with a 10.8% increase in Latin American sales being weighed down by 2.1% sales growth in North America; not surprisingly, Western Europe also struggled, and produced sales growth of just 0.7% for the full year.
Despite sales growth and price increases, €2.4 billion of commodity cost inflation for the year had its effect on the P&L, with gross margin dropping 180 basis points from 2010 as a result. At the bottom of the income statement, net income and diluted EPS were both 3% higher on a constant currency basis, at €4.62 billion and €1.46 per share, respectively.
Unilever is sometimes called "the emerging markets company," largely due to the concentration of business in Asia, Africa and Eastern Europe discussed above. Here is what Mr. Polman had to say about the emerging market results for 2011:
"Emerging markets continue to generate what we call outstanding top line performance. In fact, it's been an acceleration from recent years with underlying sales growth of 11.5% overall. Now many of our key countries achieved double-digit growth. Those include China, India, Turkey, South Africa and Mexico. For the full year, emerging markets, despite the Alberto Culver transaction, contributed 54% of total group turnover. That's a record high. Volume growth was again strong. Mid-single digit levels overall [in line with the past 20 years], but into double digits for key markets such as China as well as India.
Some of you naysayers out there were always worried about the emerging markets. I remember when I came in 2008, "Oh, you guys are so exposed to emerging markets." Then in 2009, "Oh, competition is coming in to the emerging markets." Then in 2010 or '11, "Oh, you got all the input cost in the emerging markets." Then I made a statement trying to be honest and say emerging markets are slowing down, "Oh, he's giving a warning about the emerging markets." I don't know what you guys do in your private time, but you're making your life incredibly miserable.
We see 11.5% as good growth, and the organization should be complimented on that. And we will continue to look for opportunities as 2 billion people come into this world and improve their standards of living to get more than a fair share of that."
Interestingly, Unilever's management specifically noted that 60% of their business is growing or maintaining share, compared to the 45% reported by P&G in the fourth quarter; while both parties pointed figures on the issue of pricing in the face of commodity cost inflation, one thing is clear in this chess match: Unilever has upped its game since Paul Polman took over, and P&G shouldn't overlook the experience he attained over the past 30 years (largely at their expense).
I've been talking a lot about Procter & Gamble (NYSE:PG) in my articles as of late, and thought I should balance it out by writing about one of their competitors. I decided to go with a company that recently reported fourth-quarter and full-year 2011 results, and is run by a man who spent 22 years as the president of European operations at P&G, as well as a short stint at Nestle before moving into his current role; his name is Paul Polman, and he is the CEO of Unilever.


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Courtesy of the Chinese forum xitek, come the first images of the Pentax K-01 with an exterior designed by Marc Newson. While specs are yet to be revealed about the camera itself, news has broke about their latest lens that is highly-recommended for the K-01. What follows is the detailed specification of the new PENTAX-DA 40mm f/2.8 XS lens.
Lens Mount: PENTAX KAF Mount
Focal Length: 40mm (Equivalent to 61mm in 35mm format)
Aperture: F2.8
Minimum Aperture: F22
Lens Construction: 5 elements in 4 groups
Angle of View: 39° (with PENTAX D-SLR camera body)
Minimum Focusing Distance: 0.4m
Maximum Magnification: 0.13x
Number of Diaphragm Blades: 9
Filter Diameter: 27mm
Maximum Diameter & Minimum Length: 62.9mm x 9.2mm
Weight: 52g
Source: Photo Rumors