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Angiotensin-converting enzyme-2 (ACE2) enhances the degradation of ANG II and its expression is altered in diabetic kidneys, but the regulation of this enzyme in the urine is unknown. Urinary ACE2 was studied in the db/db model of type 2 diabetes and stretozotocin (STZ)-induced type 1 diabetes during several physiological and pharmacological interventions. ACE2 activity in db/db mice was increased in the serum and to a much greater extent in the urine compared with db/m controls. Neither a specific ANG II blocker, telmisartan, nor an ACE inhibitor, captopril, altered the levels of urinary ACE2 in db/db or db/m control mice. High-salt diet (8%) increased whereas low-salt diet (0.1%) decreased urinary ACE2 activity in the urine of db/db mice. In STZ mice, urinary ACE2 was also increased, and insulin decreased it partly but significantly after several weeks of administration. The increase in urinary ACE2 activity in db/db mice reflected an increase in enzymatically active protein with two bands identified of molecular size at 110 and 75 kDa and was associated with an increase in kidney cortex ACE2 protein at 110 kDa but not at 75 kDa. ACE2 activity was increased in isolated tubular preparations but not in glomeruli from db/db mice. Administration of soluble recombinant ACE2 to db/m and db/db mice resulted in a marked increase in serum ACE2 activity, but no gain in ACE2 activity was detectable in the urine, further demonstrating that urinary ACE2 is of kidney origin. Increased urinary ACE2 was associated with more efficient degradation of exogenous ANG II (10−9 M) in urine from db/db compared with that from db/m mice. Urinary ACE2 could be a potential biomarker of increased metabolism of ANG II in diabetic kidney disease.
- angiotensin-converting enzyme 2
- biomarker
- diabetes
- nephropathy
- urinary
recent work within the renin-angiotensin system has focused on angiotensin-converting enzyme-2 (ACE2) (1, 3, 7, 8, 18, 19, 21, 25, 27, 35, 36, 38, 40, 43, 46–48). This enzyme, which is highly expressed in the kidney, efficiently metabolizes ANG II, an action that seems critically important in preventing the accumulation of ANG II, and promotes the formation of ANG-(1–7) (6, 28, 30, 37, 42).
Overactivity of the renin-angiotensin system (RAS) is felt to play an important role in the development of diabetic nephropathy. A line of evidence in support of the view that the RAS is overactive in diabetes comes from the effectiveness of ACE inhibitors and ANG II blockers in decreasing proteinuria and delaying the progression of kidney disease. The therapeutic response, however, is not homogenous with responders and nonresponders, consistent with the notion of different levels of kidney RAS activity interindividually (5).
The ultimate fate of whether there is ANG II overactivity in diabetic kidney disease and other cardiovascular pathologies may depend on genetic and acquired factors (23). For instance, changes in ACE activity which may be, in part, genetically determined may favor or attenuate ANG II formation and diabetic kidney disease in mice models (9). The degradation of ANG II is likewise critically important for the steady-state levels of this peptide. Measuring ANG II, however, is not the answer because the levels of ANG II in plasma are usually not elevated, and ANG II levels in the urine are highly variable and not very useful because this peptide is degraded rapidly intrarenally and later on in the urine which contains many peptidases (1).
We reasoned that urinary ACE2 could be a marker of the development of diabetic nephropathy and, moreover, could reflect the activity of RAS within the kidney. Recently urinary ACE2 has been found to be increased in urine of diabetic animals (2, 41) and diabetic patients (17, 44) but the mechanism of this increase is largely unknown. Moreover, very little is known regarding the physiological regulation of kidney ACE2 activity. Here we show that urinary ACE2 activity in db/db and streptozotocin (STZ)-induced model of diabetes is markedly increased and report the effect of various maneuvers on the activity and protein expression of this enzyme in the urine of control and diabetic mice.
Mouse animal models.
All studies were conducted with the review and approval of the Institutional Animal Care and Use Committee. As a model of type 2 diabetes female db/db mice (C57BLKS/JLepr) were used. Their lean age-matched littermates (db/m) served as nondiabetic controls.
As db/db mice are resistant to insulin (12, 31), streptozotocin (STZ)-treated mice were used to study the effect of glycemic control by insulin on urinary ACE2. Diabetes was induced in male C57BL/6J mice by intraperitoneal injections of STZ (Sigma-Aldrich, St. Louis, MO), at a dose of 150 mg/g body wt in sterile 0.05 M sodium citrate (pH 4.5) (32). STZ-treated mice were randomly divided into two groups: one group received insulin pellets for 12 wk (Linbit, LinShin, Canada; release rate 0.1–0.2 IU/mouse per day) and the other group of STZ mice did not receive insulin.
Blood was obtained from the tail vein and glycemia was assessed using One Touch Ultra Glucometer (LifeScan, Mountain View, CA). Urinary albumin and creatinine were measured using commercially available kits (Exocell, Philadelphia, PA).
Male wild-type (WT) and ACE2 knockout (ACE2KO) mice on C57BL/6J background (6) (breeding pairs donated by Drs. S. Gurley and T. Coffman, Duke University, Durham, NC) were used to obtain urine for validation of urinary ACE2 activity, to examine specificity of ACE2 immunoreactive protein bands in Western blot, and to perform ANG II-degradation studies.
Pharmacological agents and diets.
Starting at 7 wk of age, db/m (n = 10) and db/db mice (n = 10) were followed for several months to examine the effect of age on parameters such as urinary ACE2 and albumin/creatinine ratio. Another group of db/m and db/db mice were followed for 4 wk the same way, and then were assigned to drink tap water with an ACE inhibitor, captopril (n = 7–10 in each group), at a dose of 60 mg·kg−1·day−1 for several weeks. After 1 wk of washout, mice received tap water with an ANG II receptor antagonist, telmisartan (Boehringer Ingelheim), at a dose of 2 mg·kg−1·day−1 (n = 7) for several weeks. During the entire period, spot urine was collected repeatedly every 1–4 wk, so that under control conditions and at each treatment period at least four spot urine collections were obtained.
The effect of salt intake on urinary parameters was examined by providing high-salt (8% NaCl) and low-salt (0.1% NaCl) diets (Harland Teklad) for several weeks to female db/m and db/db mice.
Separation of the glomeruli from renal tubules using magnetic beads.
Mice were anesthetized by intraperitoneal injection of pentobarbital and perfused with 8 × 107 particles of Dynabeads M-450 (Invitrogen) diluted in 40 ml of PBS. Mouse kidneys were then removed and processed as described previously (34). Briefly, kidneys were minced into small pieces and digested in collagenase A (Roche) and deoxyribonuclease I (Sigma) solution at 37°C for 30 min with gentle agitation. The digested tissue was pressed through a 100-μm cell strainer using a flattened pestle and the cell strainer was then washed with 5 ml of PBS. The filtered material was passed again through a new cell strainer and the cell strainer was washed with 5 ml of HBSS. The resulting cell suspension was then centrifuged at 200 g for 5 min at 4°C. The glomeruli containing Dynabeads were collected by a magnetic particle concentrator (Dynal AS, Norway). The portion of digested kidney tissue deprived of glomeruli was considered to represent tubular fraction.
Isolated glomeruli in PBS suspension were viewed under the microscope and the purity of the glomeruli was established to be 95%. Glomeruli were homogenized in a buffer [(in mmol/l) 50 HEPES, pH 7.4, 150 NaCl, 0.5% Triton X-100, 0.025 ZnCl2, and 1.0 PMSF] for overnight at 4°C. The precipitate was removed by centrifugation at 6,000 g for 20 min in 4°C. ACE2 activity was measured in glomerular and tubular lysates corresponding to 1 μg of total protein.
ACE2 activity.
Urine, serum, and kidney ACE2 activity were determined following incubation with the intramolecularly quenched synthetic ACE2-specific substrate Mca-APK-Dnp (Anaspec). The measurements were performed in black microtiter plates with a 100 μl total volume. Briefly, 2 μl urine, 2 μl serum or 1 μg total protein from tissue homogenate (renal cortex, isolated glomeruli or tubular fraction) was added to wells containing a buffer (50 mmol/l 4-morpholineethanesulfonic acid, 300 mmol/l NaCl, 10 μmol/l ZnCl2, and 0.01% Triton-X-100, pH 6.5), containing EDTA-free tablets (Roche) and 10 μmol/l substrate. Reactions were in duplicates (one of two wells constituted a blank). Blank wells contained the same components, but 10 μmol/l of a specific ACE2 inhibitor, MLN-4760 (gift from Millenium Pharmaceuticals, Cambridge, MA), was also added.
After incubation at ambient temperature for 1 h (kidney cortex) or 16–24 h (serum and urine), fluorescence was measured using an FLX800 microplate fluorescence reader (BIOTEK Instruments, Winooski, VT) at 320 nm excitation and 420 nm emission wavelength. Total fluorescence was corrected for volume (in serum samples) or protein content (in tissue homogenates) after subtracting blank values.
At 420 nm, mouse urine by itself generates a detectable signal which may obscure fluorescence stemming from ACE2-specific substrate (Mca-APK-Dnp) hydrolysis. To account for this interference we used specific environment of the urine to examine detectability levels of mouse recombinant ACE2 protein in the ACE2 activity assay. Urine obtained from ACE2 knockout mice was spiked with increasing amounts of purified soluble mouse rACE2 which resulted in an increase in fluorescence signal in a dose-dependent manner with a linear relationship ranging from 15.6 to 1000 pg/ml. The range of ACE2 activity in urine from wild-type mice corresponded to concentrations of 454–1487 pg/ml urine by the comparison with purified recombinant enzyme under identical conditions. As little as 15.6 pg/ml of rACE2 was detectable in spiked urines from ACE2 knockout mice, suggesting that ACE2 protein levels in normal urines are far-off the detection limit of the ACE2 activity assay, which warrants its use for quantitative purposes.
Western blot.
Kidney cortex total and membrane protein fractions were extracted as previously described (43, 47).
For Western blot of urines, urine samples were concentrated on centrifugal concentrators of 5-kDa cut off. Proteins were separated by SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were blocked in nonfat dry milk solubilized in Tris-buffered saline solution containing 0.1% Tween 20 (7% wt/vol). The nitrocellulose membranes were incubated with primary anti-ACE2 antibody (R&D Systems or Abcam) and horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). Bands were visualized using chemiluminescence system (Super Signal Pico, Pierce). For comparisons of urinary ACE2 bands between control and diabetic mice, several gels were used in which a similar number of samples from each group was included. The average value of integrated density measured in db/m mice was set as 100% for each gel and the results for db/db mice were expressed as percentage of that measured in db/m controls. Variation from the mean was presented to reveal the level of variability in each group.
Exogenous ANG II degradation by the urine.
Urine from male ACE2KO and WT mice matched for creatinine concentrations and equal amounts of total protein (1 mg) were individually incubated with 10−9 M ANG II (Sigma) by constant shaking for up to 8 h. In similar experiments, urines collected from 16-wk-old female db/m and db/db mice representing equal creatinine concentration (10 μg/ml) were incubated with ANG II (10−9 M) for up to 4 h at 37° C in PBS pH 7.4 containing 5 μM ZnCl2. The incubation was performed with or without addition of the specific ACE2 inhibitor, MLN-4760 (10−6 M). Following the incubation, samples were diluted 1/5 in EDTA-containing buffer (0.1 M phosphate buffer, pH 7.4 containing 0.15 M NaCl, 1 mM EDTA, 0.1% BSA) and immediately frozen. The quantity of ANG II in the diluted samples was measured using a solid-phase immobilized epitope immunoassay kit (SPIBio, Cayman Chemical, Ann Arbor, MI). ANG II quantity in each sample was expressed as a percentage ratio of the ANG II remaining after a given incubation time to the initial ANG II load (100%). This EIA method shows less than 0.1% cross-reactivity with ANG-(1–7). However, ANG III, ANG IV, and ANG-(4–8) exhibit 36%, 33% and 41% cross-reactivity, respectively, expressed in terms of reactivity percent toward ANG II (39).
Administration of soluble recombinant ACE2.
Female db/m (n = 5) and db/db (n = 5) at 10 wk of age were used to study the effect of rACE2 administration on serum and urinary ACE2 activity. Before administration of rACE2, blood was collected from tail vein (around 15 μl). Immediately after voiding urine, mice were administered an intraperitoneal bolus of recombinant ACE2 (1 mg/kg). Urine was then collected in metabolic cages over 3 h after rACE2 intraperitoneal bolus and blood was taken from tail vein at 3 h after rACE2 administration.
Statistical analysis.
Changes over the course of the experiments between the groups were analyzed using general linear model (GLM) on the SPSS Statistics 20 software (IBM). Differences between two groups were analyzed using a two-tailed Student's t-test, and for nonnormally distributed data, a nonparametric Mann-Whitney test was used. For multiple comparisons ANOVA was used followed by LSD post hoc test. A Pearson's test was used for correlations between two variables. For data with normal distribution, values were expressed as means ± standard error of the mean (SE). For nonnormally distributed data, the median and interquartile range were used. For calculation of half-lives of ANG II one phase decay analysis was used (GraphPad Prism).
ACE2 activity in urine from diabetic (db/db mice) and controls (db/m mice).
At 7 wk of age, db/db mice have been diabetic for 2 wk (31). Urinary ACE2 activity at this early age was already twofold higher compared with db/m (n = 10) [23 ± 4 vs. 11 ± 0.5 relative fluorescence units (RFU)·μg creatinine−1·h−1, P < 0.01, respectively]. At this early age, the albumin/creatinine ratio, by contrast, was not significantly different between db/m (n = 10) and db/m (n = 10) (58 ± 12 vs. 45 ± 7 μg/mg, P = NS, respectively).
During several consecutive spot urine measurements from 7 to 25 wk of age, urinary ACE2 activity/creatinine ratio in db/db mice increased over time and was consistently and significantly higher than in db/m mice of the same age (Fig. 1). Albumin/creatinine ratio also increased with age in db/db but not in db/m mice (Fig. 1B). The increase in ACE2/creatinine ratio antedated the increase in albumin/creatinine ratio (compare Fig. 1, A and B). When two to three adjacent measurements were averaged at each time interval, the differences in urinary ACE2 activity/creatinine ratio (Fig. 1C) and albumin/creatinine ratio (Fig. 1D) became even more apparent.
When measured as excretion rate (12 h urine collection in mice housed in metabolic cages), urinary ACE2 activity was also increased in urines from db/db mice of about 18 wk of age compared with age-matched db/m mice (Fig. 2A). ACE2 activity/creatinine ratio was also increased to about the same extent (Fig. 2B). ACE2 activity/creatinine ratio correlated well with ACE2 activity expressed per 12-h time unit when data from db/m and db/db mice were pooled for regression analysis (R = 0.818, P < 0.01, n = 10) (Fig. 2C). Urinary ACE2 activity, either expressed as 12-h excretion rate or as urine ACE2/creatinine ratio, correlated positively with 12-h urine volume (R = 0.912, P < 0.01, and R = 0.858, P < 0.01, respectively) (Fig. 2, D and E). A significant positive correlation (R = 0.679, P < 0.01) was also observed between albumin/creatinine ratio and ACE2 activity measured from spot urines collected from db/m (n = 20) and db/db mice (n = 20) over a course of 20 wk (18–38 wk of age) (Fig. 2F).
Urinary ACE2 activity during changes in dietary salt intake.
Urinary ACE2 activity was measured at weekly intervals four consecutive times in mice on normal salt (0.49% NaCl), high salt (8% NaCl) and low salt (0.1% NaCl) salt in their diets (Fig. 3A). The values from four consecutive ACE2 activity measurements were also averaged for each experimental group (Fig. 3B). During high-salt diet urinary ACE2 activity increased in both db/m and db/db groups compared with respective groups receiving normal-salt diet (Fig. 3, A and B). Under all these dietary conditions, db/db had higher levels of urinary ACE2 activity than their age-matched db/m controls. The low-salt diet ingestion in db/db mice caused the urine ACE2 activity to fall below the level of the ACE2 activity measured at the time of normal salt intake (P < 0.05). In db/m mice, low salt also reduced urinary ACE2 activity compared with a high-salt diet (P < 0.05). The differences in ACE2 activity between control and diabetic mice persisted during changes in dietary salt intake (Fig. 3).
In a separate set of db/db mice that received high or low salt concurrently over several weeks, there was a significantly higher ACE2 activity in urines from mice kept on high-salt than those on low-salt diet (112 ± 30 vs. 23 ± 6 RFU·μg creatinine−1·h−1, respectively, P < 0.05). Kidney ACE2 activity, however, was not significantly different between high- and low-salt diets (126 ± 9 vs. 110 ± 15 RFU·μg creatinine−1·h−1, respectively, P = NS).
Urinary ACE2 activity during captopril and telmisartan administration.
Whether RAS inhibition affects urinary ACE2 activity was examined in db/m and db/db mice during the continuous administration of an ACE inhibitor, captopril, and a specific AT1 receptor blocker, telmisartan. In both db/m and db/db mice urinary ACE2 activity/creatinine ratio did not differ from ACE2 activity/creatinine ratio measured longitudinally in db/m and db/db mice before and during captopril administration daily (Fig. 4A). The AT1 receptor blocker, telmisartan, also did not alter significantly urinary ACE2 activity/creatinine ratio either in the db/m or the db/db mice (Fig. 4A). Accordingly, the difference between db/m and db/db persisted over the time course of the administration of captopril and telmisartan (Fig. 4A). The lack of a significant effect of these two RAS blocking agents on urinary ACE2 activity/creatinine ratio was also observed when the values from four consecutive measurements were averaged for each experimental condition (Fig. 4B). Thus neither form of RAS blockade altered urinary ACE2 activity and the large differences between db/m and db/db persisted throughout the control and treatment phases.
Urinary albumin/creatinine ratio was likewise significantly higher in db/db mice compared with db/m before (P < 0.01) and during captopril (P < 0.05) and telmisartan administration (P < 0.01) (Fig. 4C). When the values from 3–4 consecutive measurements were averaged for each experimental condition, however, in db/db mice the administration of both RAS blocking agents was associated with a significant decrease in albumin/creatinine ratio (P < 0.05) (Fig. 4D).
Kidney as a source of elevated urinary ACE2 in db/db mice.
It has been previously shown that kidney cortex ACE2 protein and ACE2 activity are increased in db/db mice of various age groups compared with db/m (22, 43, 47). Serum ACE2 activity in db/db mice was significantly increased compared with db/m in young 8-wk-old mice (3.7 ± 0.2 vs. 2.6 ± 0.1 RFU·μl−1·h−1, P < 0.01, respectively, n = 9 in each group) and in mice at 38 wk of age (1.2 ± 0.2 vs. 0.7 ± 0.1 RFU·μl−1·h−1, P < 0.05, n = 9 and n = 10, respectively).
We wanted to ascertain whether the increase in urinary ACE2 in db/db mice originates from an increase in renal or from an increase in circulatory ACE2. To examine this issue soluble rACE2 was administered to db/m mice at a dose of 1 mg/kg. Administration of rACE2 (1 mg·kg−1·day−1) has been previously shown to produce a marked and reproducible increase in serum but not kidney ACE2 activity in C57BL6 mice (42). In db/m mice (n = 5) that received soluble recombinant (r)ACE2 acutely (1 mg/kg ip), serum ACE2 activity was markedly increased compared with the ACE2 activity in serum collected before rACE2 administration (181 ± 12 vs. 1.9 ± 0.6 RFU·μl−1·h−1, P < 0.001) (Fig. 5). ACE2 activity in urine, however, did not increase significantly after rACE2 infusion to db/m mice (9.5 ± 2.9 vs. 5.7 ± 0.3, P = NS, respectively) (Fig. 5).
As in db/m mice, rACE2 (1 mg/kg) administration to db/db mice resulted in a marked increase in serum ACE2 activity compared with the ACE2 activity in serum collected before rACE2 administration (222 ± 11 vs. 2.7 ± 0.7 RFU·μl−1·h−1, P < 0.001), but no significant increase in urinary ACE2 activity was observed (44 ± 11 vs.75 ± 23 RFU·μg creatinine−1·h−1, P = NS, respectively) (Fig. 5).
Since a profound increase in serum ACE2 activity after rACE2 infusion does not translate into an increase in urinary ACE2 activity in control or diabetic mice with albuminuria, these results show that the primary source of ACE2 found in mouse urine is the kidney and not serum. This finding, moreover, suggests that the source of increased ACE2 in the urine of diabetic mice is of renal tubular origin rather than a consequence of altered glomerular permeability in diabetic mice.
ACE2 enzymatic activity in db/m and db/db mice in isolated kidney tubules and glomeruli.
In kidney cortex, at 32 wk of age, ACE2 activity was significantly higher than in the db/m (76 ± 8 vs. 53 ± 6 RFU·μg protein−1·h−1, respectively, P < 0.05). To study whether the augmented urinary ACE2 activity in db/db mice is of glomerular or tubular origin, we assessed ACE2 enzymatic activity in isolated glomeruli and kidney tubules from 32-wk-old db/db mice. ACE2 activity measured in isolated renal tubular fractions from db/db mice was significantly higher than in db/m mice (99 ± 8 vs. 58 ± 3 RFU·μg protein−1·h−1, respectively, P < 0.001) (Fig. 6A). ACE2 activity in isolated glomeruli was not significantly different between the control and diabetic mice (9.0± 4.6 vs. 4.8±1.6 RFU·μg protein−1·h−1, respectively, P = NS) (Fig. 6B). In tubular fractions, levels of ACE2 activity per microgram total protein were on average 10 times higher than in isolated glomeruli (compare Fig. 6, A and B).
Kidney tubular ACE2 activity plotted against ACE2 activity measured in urines from the same mice obtained at the date animal was euthanized showed a significant positive correlation (R = 0.639, P < 0.05, n = 13). In contrast, no correlation was found between glomerular and urinary ACE2 activity in 32-wk-old db/m and db/db mice (R = 0.079, P = NS, n = 14). Altogether these patterns of glomerular and tubular ACE2 expression in db/db mice suggest that the ACE2 activity increase seen in urines from diabetic mice is due to an increase in tubular rather than glomerular ACE2 because in db/db mice ACE2 in the kidney is increased in tubules but not in glomeruli. In fact, in glomeruli ACE2 has been shown to be downregulated by immunostaining in db/db mice (48) and patients with type 2 diabetes (18).
Urinary ACE2 activity in STZ-mice and effect of insulin.
To examine if the striking differences in urinary ACE2 in the db/db mice were found in other model of diabetes, mice with STZ-induced diabetes were studied as well. Moreover, because db/db mice are known to be insulin-resistant (12, 31), we used STZ-treated mice as a model to study whether insulin in itself, or by normalizing hyperglycemia or by improving kidney disease in diabetic mice, influences levels of urinary ACE2 activity.
Similar to db/db model, in the STZ-treated mice urinary ACE2 activity was significantly increased compared with vehicle-treated controls throughout the follow-up (Fig. 7). Glycemia was reasonably well controlled in a group of STZ-mice that received insulin compared with vehicle controls (Fig. 8A). Urinary ACE2 activity/creatinine ratio in STZ mice receiving insulin was not significantly different from STZ-treated mice without insulin administration in the initial 5–7 wk. After 8–12 wk of initiating the insulin administration urine ACE2 activity/creatinine ratio fell significantly in STZ-treated mice compared with STZ-treated mice not treated with insulin (Fig. 8B). Urinary albumin/creatinine ratio was lower in STZ-treated mice with insulin than in STZ mice without insulin administration but the difference did not reach statistical significance (Fig. 8C).
Urinary ACE2 increases ANG II degradation.
Urine from wild-type and ACE2KO mice matched for creatinine concentrations and representing equal amount of total protein (1 mg) were incubated with exogenous ANG II (10−9 M) to examine the contribution of urinary ACE2 to the degradation of this peptide. Urines from WT mice (n = 3) degraded ANG II more effectively than urines obtained from ACE2-deficient mice (n = 3) (Fig. 9A).
Since db/db mice consistently showed markedly elevated levels of urinary ACE2 activity than db/m controls, we sought to determine whether this increase in urinary ACE2 activity in the urines from db/db mice was associated with increased ANG II-degrading capacity. Urines from db/db mice (n = 11) degraded ANG II more rapidly than urines collected from db/m controls (n = 9) by general linear model (GLM) repeated-measures analysis (Fig. 9B). The half-life of ANG II calculated in urines from db/db mice was also significantly lower than in the db/m urines using a nonparametric test and the median (interquartile range) [0.8 (0.5–1.0) vs. 2.5 (1.6–3.8), P < 0.05, respectively]. When MLN-4760 (10−6 M), a specific ACE2 inhibitor was added to the urine there was no significant difference in the half-lives of ANG II between the urines from db/db (n = 11) and db/m mice (n = 9) [2.2 (0.9–3.16) vs. 5.95 (1.04–16)] and by the GLM analysis (Fig. 9C). Although not significant (P = 0.087), however, the half-life of ANG II in diabetic urine was reduced in the presence of MLN-4760, which suggests additional degradation of the peptide by peptidases other than ACE2. Altogether, these data show that mouse urine contains enzymatically active ACE2 that significantly influences urinary ANG II-degrading capability, which is increased in db/db urines.
Enzymatically active ACE2 protein in mouse urine.
In concentrated urines from db/m and db/db mice of 20–24 wk of age two ACE2 immunoreactive protein bands (75 and 110 kDa) were observed (Fig. 10). The relative abundance of the ACE2 protein band at 110 kDa was significantly increased in urine from db/db (n = 16) compared with urine from db/m mice (n = 14) (199 ± 25% of db/m control, P < 0.005). The relative abundance of the 75-kDa band was also higher in db/db than in db/m (259 ± 57% of db/m control, P < 0.05) (Fig. 10).
To examine whether both bands are also present in the kidney, Western blots were performed in kidneys from a group of 10- to 11-wk-old db/m and db/db mice (n = 5 in each group). As shown in older mice (Fig. 10), urines from these younger db/m and db/db mice also showed an ACE2 immunoreactive band at around 100–110 kDa and also a band at 75 kDa, which was variably seen (Fig. 11A).
Unlike in the urines, in isolated kidney cortex plasma membranes from these younger db/m and db/db mice, only the ∼100- to 110-kDa ACE2-immunoreactive band was detectable (Fig. 11). Moreover, in additional studies in whole cell lysates only the ∼100- to 110-kD band was found as well (Fig. 11). ACE2/GAPDH ratio for whole lysates and ACE2/B-actin ratio for membrane fractions were both significantly higher in db/db mice compared with db/m (302 ± 12% and 222 ± 25% of the db/m controls, respectively, P < 0.005). ACE2 activity measured in whole kidney cortex lysates from db/db mice was also significantly higher than in db/m (129.8 ± 9.8 vs. 50.3 ± 1.2 RFU·μg protein−1·h−1, respectively, P < 0.001).
Consistent with the Western blot results of the db/m and db/db mouse urines, in concentrated urines from a control wild-type mice (ace2+/y, C57BL/6 genetic background) Western blotting using a specific ACE2 antibody showed two main bands at ∼100–110 kDa and 75 kDa whereas in ACE2 knockout mice (ace2−/y,) neither band was found (Fig. 12). In the same concentrated urines, there was abundant ACE2 enzymatic activity detectable in WT but not in the ACE2KO (111.5 vs. −6.0 RFU·μl−1·h−1, respectively).
ACE2 is abundantly expressed in the kidney, mainly in tubular epithelial cells and to a lesser extent in glomeruli and the renal vasculature (47, 48). It is believed that the levels of ACE2 activity in the various nephron segments govern the local levels of ANG II in concert with the level of ACE and other enzymes involved in the formation and degradation of ANG II (1, 7, 8, 20, 30). The formation of ANG-(1–7), a peptide that opposes and counterbalances actions of ANG II (4), is also largely dependent on cleavage of ANG II by ACE2 (28, 37, 42, 46).
Alterations in ACE2 have been recognized both in animal models of diabetic kidney disease and humans with diabetic nephropathy (18, 21, 27, 36, 43, 47, 48). More recently, we and others have proposed that the level of ACE2 activity in the urine could potentially serve as a biomarker of diabetic kidney disease (2, 41, 44). Mizuiri et al. (17) recently reported that humans with chronic kidney disease have elevated urinary levels of ACE2 protein compared with control subjects (17). Of note, diabetic patients had higher urinary ACE2 levels than nondiabetics (17). Burns' group (44) likewise has reported increased levels of urinary ACE2 activity in renal transplant recipients with diabetes compared with nondiabetics.
The mechanism for increased urinary excretion of ACE2 in diabetic kidney disease, however, was not examined in these previous studies. Here we report that in urines from diabetic db/db mice and STZ-treated mice the levels of ACE2 activity are markedly increased compared with nondiabetic controls. This increase was observed in db/db mice already at 7 wk of age, before the development of albuminuria, and increased progressively over time as albuminuria increased (Fig. 1). Moreover, serum ACE2 activity in serum from diabetic mice was also increased, suggesting the possibility of a generalized compensatory mechanism that helps metabolize ANG II more efficiently in these models of diabetic kidney disease. To examine whether the increase in urinary ACE2 activity was of circulatory origin we infused recombinant ACE2 to control and diabetic mice. When db/m mice were infused with soluble recombinant ACE2, a marked increase in circulating (serum) ACE2 activity was observed but there was no effect whatsoever on urinary ACE2 activity (Fig. 5). This is in keeping with the high molecular weight of soluble ACE2, which renders it nonfilterable. Moreover, rACE2 administration to db/db mice also failed to increase urinary ACE2 activity despite a large increase in serum ACE2 activity. This shows that increased serum ACE2 activity in diabetic mice cannot account for the elevated urinary ACE2 activity. Accordingly, the source of increased urine ACE2 must be the kidney itself since ACE2, when infused, does not appear in the urine.
We also examined the effect of RAS inhibition on urinary ACE2 activity. Urinary ACE2 in db/m and db/db mice was unchanged by either chronic angiotensin type 1 receptor blocker (telmisartan, 2 mg·kg−1·day−1) or ACE inhibitor (captopril, 60 mg·kg−1·day−1) administration, and the large differences between db/m and db/db mice were not affected. One could speculate that the inability of telmisartan and captopril to reduce urinary ACE2 may be beneficial in terms of renal effects of these RAS blockers by not interfering with ACE2-driven ANG II degradation. In addition, the lack of effect would be advantageous for the potential use of urinary ACE2 as biomarker of kidney disease since most patients are currently receiving RAS blockers.
The potential effect of changes in dietary sodium intake on urinary ACE2 protein was also examined. Changing from a normal to a very high-salt diet resulted in a sharp increase in urinary ACE2 activity. This effect was noted both in diabetic and nondiabetic mice. Conversely, a low-salt diet resulted in a fall in the level of urinary ACE2 activity in both diabetic and nondiabetic mice. An increase in ACE2 with a high-salt diet would reduce ANG II levels and therefore promote natriuresis whereas a decrease in ACE2 would increase it and promote sodium retention. The observed changes in urinary ACE2 activity, however, did not follow the predicted changes in plasma ANG II levels (low in a high-salt diet and high in a low-salt diet) and therefore require other explanations. One possibility is that changes in urinary flow imposed by the extreme changes in dietary sodium intake in these experiments affected urinary ACE2 mechanical shedding. According with this possibility, urinary ACE2 activity had a highly significant positive correlation with urine flow (Fig. 2). The high urine flow associated with hyperglycemia in db/db mice could therefore contribute to the observed high levels of urine ACE2 protein. High levels of urinary ACE2 were also seen in a different model of diabetic kidney disease, the STZ-treated mice. In this model, insulin lowered urinary ACE2 activity but only after several weeks of continuous administration which is consistent with a recent study showing that administration of an insulin sensitizer, rosiglitazone, to db/db mice normalized hyperglycemia and decreased urinary ACE2 (2). An improvement in glycemic control and the attendant reduction in osmotic diuresis and urine flow does not seem therefore the only explanation for the lowering effect of insulin on urinary ACE2 which was observed only after several weeks of its administration.
ACE2 is primarily localized to the apical membrane of proximal tubular cells (48) but is also present in parietal and visceral epithelial glomerular cells (48). In previous studies (43, 47) it was found that ACE2 is increased in kidney cortex but decreased by immunostaining in glomeruli from young db/db mice (8 wk of age). In other models of diabetic kidney disease (Akita and in STZ-treated mice) kidney ACE2 protein or activity were also reported to be elevated in kidney cortex (33, 35, 40, 43). In the present study, we measured ACE2 activity in both glomerular and tubular kidney isolates from older db/db mice (32 wk of age) and found that tubular ACE2 activity was increased compared with db/m controls. Moreover, there was a positive correlation between tubular and urinary ACE2 but not between glomerular ACE2 activity and urinary ACE2 activity. These findings all together strongly suggest that the primary source of elevated urinary ACE2 in diabetic kidney disease is of tubular origin, the site of the nephron where this enzyme is more abundantly expressed. We found, moreover, that ACE2 protein was increased in kidney cortex membrane fractions from the db/db mice compared with db/m mice (Fig. 11). Therefore, the elevated levels of urinary ACE2 are likely a reflection of increased tubular ACE2 content in the apical plasma membrane. Further evidence for the notion that tubular levels of ACE2 determine urinary ACE2 activity comes from a study showing that urinary ACE2 was reduced along with reduced proximal tubule ACE2 in fetal programmed hypertension (29).
Apical ACE2 protein could be shed from the membrane of tubular epithelial cells, and therefore an increase in urinary ACE2 activity in diabetic kidneys could reflect a primary increase in tubular ACE2 protein, increased shedding from the apical membrane, or both. Studies in cultured human embryonic kidney cells and airway epithelial cells suggested ACE2 being proteolytically shed from the plasma membrane into the culture media at its carboxy terminus, by a process catalyzed by the enzyme "a disintegrin and metalloproteinase-17" (ADAM17) (10, 11, 13). Of note, renal ADAM17 is upregulated in ANG II-induced kidney injury (14), and de novo expression occurs in human kidney disease, in proximal tubule, podocytes, and mesangial cells (16). While ADAM17 is a recognized sheddase for TNF-α and ACE2 in vitro (26) its role in mediating shedding of soluble ACE2 into the urine from kidney epithelial cells in vivo is not known. It is possible that the increase in urinary ACE2 excretion in db/db mice may be due, in part, to ADAM17-mediated shedding. Because of the increased abundance of ACE2 protein in isolated tubules and in plasma membranes from the db/db mice, however, we think that the increase in urine ACE2 also must reflect augmented tubular ACE2 content. This increase in tubular ACE2 protein and activity would help foster ANG II degradation in a setting where there is RAS overactivity, such as diabetic kidney disease (see below). The increased urinary ACE2 could also be associated with tubular damage in db/db mice; however, renal tubules were reported to be relatively spared from injury in this animal model (31). Thus a correlation between urinary ACE2 and urinary albumin may reflect glomerular damage but the two may not be causally related.
We also assessed urinary ACE2 protein by immunobloting and found two bands in control and diabetic urine at a molecular mass of 100–110 and 75 kDa. Both these protein bands were absent in urines from ACE2-deficient mice (Fig. 12). The activity associated with the urinary 110-kDa band likely reflects ACE2 activity within the kidney which is where ACE2 resides in the plasma membrane (see Fig. 11). Mouse ACE2 has been shown to have slightly lower molecular mass (110 kDa) than human ACE2 (120 kDa) by two independent groups (19, 24). Therefore the different size of the bigger ACE2 immunoreactive band between human and mouse urinary samples may be a reflection of a difference in molecular weight of human and mouse ACE2 protein. The lower ACE2 band (75 kDa) may be the result of degradation of the soluble ACE2 protein. In support of this hypothesis are the data showing that in isolated kidney lysates the 100- to 110-kDa ACE2 band is only present whereas the 75-kDa ACE2 band appears in the urine with a varying frequency (Fig. 11). Lew et al. (15) reported two bands at approximately 110–115 kDa in healthy human urine samples. Another study showed ACE2 immunoreactive bands at approximately 120 and 90 kDa in healthy subjects and 120 and 75 kDa in patients with chronic kidney disease (17).
The ability of urinary ACE2 to metabolize exogenous ANG II was studied in control and diabetic mice. In a previous study, Lew et al. (15) could not ascertain a significant processing of angiotensin peptides by human urines. For their liquid chromatography-based angiotensin degradation studies, these authors employed amounts of ANG II substrate several thousand times higher (8 × 10−5 μM) than those normally present in the native urine, because of the relatively low sensitivity of this assay (15). We used ANG II, at much lower concentration (10−9 M) empirically arrived at by a careful titration, and this approach revealed ANG II degradation by mice urine as previously reported in sheep urine (30). We, moreover, found that ANG II degradation is increased in urine from diabetic mice and that this effect was largely ACE2-dependent because the differences between db/m and db/db urine degrading capacity were not significant in the presence of MLN-4760, a specific ACE2 inhibitor. It is important to acknowledge, however, that there was a component of ANG II degradation that was clearly ACE2-independent. Other peptidases, such as neprilysin, can also metabolize ANG II in the urine (30). Interestingly, in female diabetic rats Yamaleyeva et al. (45) have observed a marked reduction of kidney neprilysin activity, and it is possible that urinary neprilysin may explain why there is rapid degradation of ANG II in the first hour in urine of db/db and db/m mice that is apparently not blocked by the ACE2 inhibitor, MLN-4760.
Using micropuncture studies, ANG II levels in tubular fluid and urine have been reported to be severalfold higher than in plasma (20). Interestingly, we found that ACE2 activity in the urine is on average eightfold higher compared with serum. This would be consistent with the hypothesis that the increased ACE2 in urines from diabetic animals reflects kidney ANG II overactivity. That is, higher levels of ANG II within the kidney may require higher levels of ACE2 activity for its metabolism and this is accomplished by an increase in tubular plasma membrane ACE2 protein and therefore ACE2 activity. Possibly, the increase in ACE2 protein is compensatory to limit the accumulation of ANG II in diabetic kidneys. In any case, ANG II degradation in the native urine by ACE2 and other enzymes is of key importance in the interpretation of urinary peptide concentrations. This ongoing degradation renders the measurements of endogenous ANG II unreliable in terms of reflecting concentration of this peptide in the kidney prior to enzymatic degradation. Therefore, in this sense, urinary ACE2 could be used as a potential biomarker of RAS overactivity in diabetic kidney disease. This information could be valuable in providing guidance as to the timing of therapeutic intervention to block the renin-angiotensin system.
In summary, urinary ACE2 activity is increased in diabetic mice and the source of this increase is of tubular rather than glomerular origin. Salt intake, urine flow, and insulin can all affect urinary ACE2 activity. The activity of ACE2 in the urine significantly influences ANG II degrading capacity and probably reflects protein shed from the tubular plasma membrane. Overall, these findings suggest that increased urinary ACE2 in diabetic kidney disease could be a potential biomarker of increased metabolism of ANG II as a result of RAS overactivity.
D. Batlle has grant support from the National Institute of Diabetes and Digestive and Kidney Diseases (Grant 1R-01-DK-080089-01A2), the Juvenile Diabetes Research Foundation, and the Reuben Feinberg Foundation. J. Wysocki was supported by Postdoctoral Research Award F32-DK-079546 from the National Institute of Diabetes and Digestive and Kidney Diseases.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: J.W. and D.B. conception and design of research; J.W., L.G.-H., M.Y., C.M., and K.S. performed experiments; J.W., L.G.-H., M.Y., C.M., K.S., K.D.B., and D.B. analyzed data; J.W., L.G.-H., M.Y., C.M., K.S., and D.B. interpreted results of experiments; J.W., C.M., and D.B. prepared figures; J.W. and D.B. drafted manuscript; J.W., M.Y., K.D.B., and D.B. edited and revised manuscript; J.W., M.Y., K.D.B., and D.B. approved final version of manuscript.
- Copyright © 2013 the American Physiological Society


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Dennis Dodd (June 25, 2009): Q&A: Leader of the Pac, Hansen, looks back on his way out
Dennis Dodd (June 17, 2009): Higher standards? Title wins shouldn't exacerbate UF sins
Dennis Dodd (June 11, 2009): 'Bama offers textbook proof that rulebook no longer the rule
Dennis Dodd (June 11, 2009): Placing blame for Edwards injury a pain in itself
Dennis Dodd (June 5, 2009): 'Warding off pretenders? Former kicker aims to revamp trophy time
Dennis Dodd (June 20, 2009): Mailbag: Bands, 'Bama fans marching to similar beat
CBSSports.com Staff (June 3, 2009): In the Trenches: Who'll win SEC West? Best openers? Coach votes

subset: commoncrawl


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ST. HELENS, Ore. (KOIN) – Nearly eight months after a 30-year-old man died from an assault, a Columbia County grand jury indicted a 49-year-old man with murder.
Randall Lee Pritchard appeared in court on Tuesday and was arraigned on a grand jury indictment that charges him with the death of Christopher Earl Heller.
According to the sheriff's office, the investigation started on July 21, 2015 when deputies responded to a residence in the 68000 block of Whitney Road in the Goble area. Before paramedics arrived, Heller had been taken to a Longview hospital by private vehicle. On July 28, 2015, Heller died.
Details of the assault were not released. However, the South County Spotlight reported Heller suffered a skull fracture.
Pritchard turned himself into sheriff's deputies shortly before his court hearing. Defense attorney Jason Glenn Short declined to comment.
A not guilty plea was entered on Pritchard's behalf, who is being held without bail in Columbia County.
A trial date has not been set.


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Johnny Depp on Saturday Night Live
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Don't you think Johnny would be great on SNL? Then sign this petition Depp Lovers!!
Sign for Johnny Depp to be on SNL!
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The Johnny Depp on Saturday Night Live petition to Depp Lovers was written by Shelley Jackson and is in the category Television at GoPetition. Contact author here.


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In the event that you have a companion with a specific propensity for golf, instead of settling on the standard birthday present of a book or a few chocolates, why not give them a golf gift pack this year? A gift like this will be valued and they will have the capacity to receive hours of fun in return, while likewise enhancing their golfing abilities by no end. You can discover sites offering golf gift packs and vouchers by looking on the web now and can purchase ones that can be recovered at golf courses in their general vicinity. In the event that you know they would love to visit a golf course that is somewhat further abroad you can simply get them a gift pack for a golf course that would require a short break, with the goal that they can appreciate a little occasion in the meantime. You should ensure that your companion's disable is at the right level before you book them vouchers for any one course however, with the goal that they will really have the capacity to make utilization of your present.
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User talk:188.8.131.52
Hi, I've removed a lot of text from the Tangier page, because most of it was useless information that did not belong in the guide, however, I have not removed most of your contributions since your contributions seemed useful. Do you live in Tangier or have you visited? I'd like to suggest you get a user account to help us out. Thanks. -- Andrew Haggard (Sapphire) 20:13, 5 September 2006 (EDT)
This is the discussion page for an anonymous user who has not created an account yet, or who does not use it. We therefore have to use the numerical IP address to identify him/her. Such an IP address can be shared by several users. If you are an anonymous user and feel that irrelevant comments have been directed at you, please create an account or log in to avoid future confusion with other anonymous users.


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Item description for Corpus: Drawings 1981-2006 by Damien Hirst & Millicent Wilner...
This comprehensive monograph was produced to accompany the drawings retrospective 'Damien Hirst: Corpus: Drawings 1981-2006' held at Gagosian Gallery, Madison Avenue, New York in 2006. It features more than two hundred drawings which offer a historical insight into rarely seen aspects of the artist's work and process. Included are early drawings from Hirst's student days; pencil sketches for seminal sculptures such as 'The Physical Impossibility of Death in the Mind of Someone Living' 1991, 'A Thousand Years' 1991, 'The Acquired Inability to Escape' 1992, 'Away from the Flock' 1994 and 'The Hat Makes the Man' 2003; preparatory diagrams for early spot paintings and medicine cabinets; a large-scale series of fourteen drawings for The Stations of the Cross (2004); and proposals for unrealised and future projects. Accompanying the drawings is a conversation between the artist and political philosopher John Gray (author of Straw Dogs, False Dawn and Al Qaeda and What It Means to Be Modern), and an essay by British historian Simon Baker. Hardback/with 29 gatefolds and book ribbon
Promise Angels is dedicated to bringing you great books at great prices. Whether you read for entertainment, to learn, or for literacy - you will find what you want at promiseangels.com!
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GraphicRiver Summer Break Flyer 7752045
Photoshop \ Print Template \ Flyers \ Events
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Supplier Homepage Product Spectrophotometer Others Spectrophotometer High Stability Lab Instrument Ftir Spectrometer From Drawell Scientific
High Stability Lab Instrument Ftir Spectrometer From Drawell Scientific
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- Model NO.: DW-FTIR-510A
- Warranty: One Year, It′s Possible to Extension
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- Scanning Speed: 5-Step Adjustable for Different Applications
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- Origin: Shanghai, China
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Product Description
DW-FTIR-510A/520A FT-IR SpectrometerFEATURES:
- New type cube-corner Michelson interferometer features smaller size and more compact structure, providing higher stability and less sensitive to vibrations and thermal variations than conventional Michelson interferometer.
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- Spectral Range: 7800 to 350 cm-1
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- Fixed liquid cells and demountable liquid cells
- Gas cells with different pathlength


text:
USA American licence plate, number plate, LOUISIANA
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Genuine American licence plate (12 X 6 inch metal) fromLOUISIANA. This plate has the embossed digits. A valuable and attractive addition to your american licence plate collection. ____________________________________ UK postage is £1.75 recorded delivery plus £0.65 per extra plate. (Recorded/tracked mail is a requirement that Paypal demands) Europe Air Mail is £2.00 GBP, plus £0.65p per extra plate.
___________________________________________All licence plates I sell, are for collecting, souvenir or display, and must not be used on a motor vehicle on the road. These plates are all expired and over 3 years old. The American licence plates I sell, are genuine issue 12 X 6 inch metal licence plates. Most of these have been used on a motor vehicle of some kind, and will show some signs of this. These could be marks around the boltholes, scratches on the surface of the raised letters, road film, or minor dings. Some plates I sell are unissued, or unused, so can be in "as new" condition. ___________________________________________________________ These "embossed" plates will soon all be gone !!!!!!! The process of manufacturing licence plates is changing. Many states have already switched to ... read more


text: Human Rights Observatory China Aims to Break Foreign Influence on Religion Chinese President Xi Jinping this week asked religious groups to pledge their loyalty to the state and warned that religions in China must be independent of foreign influence. At a time when China's Christians now outnumber the membership of the Communist Party, some say there is an intensifying crackdown on religious groups. China's Communist Party is officially atheist and has long seen religion as a threat to its rule. Chinese citizens are barred from attending religious...Read complete article© Voice of America - Friday, May 22, 2015


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MWRInfoSecurity Talk
How Secure do you think your android phone is? Are only rooted android phones vulnerable to malware? Sai Sathya narayan Venkatraman came down from MWR InfoSecurity to share on how exactly an adversary can make use of a malicious application you have installed on your phone to extract passwords, make rogue phone calls on infected cellphones, overcome root detection.
During the 2 hour talk, participants got a very quick overview on how static malware analysis on android phone could be done and did a hands-on practical challenge at the end of the session where participants were given a T-shirt if they could solve the challenge!
Some of the tools that were discussed and used during the hands-on session were drozer, dex2jar and android phone emulator using ubuntu iso image. If you would like to know more feel free, please email WhiteHats Society to get more details!


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The Lewt Boys are back at it again with early impressions of Resident Evil 7, Overwatch's Year of the Rooster, best selling consoles games of 2016 and much more!
What Did We Talk About?
- Best Selling Console Games of 2016.
- Prey 2 Gets a Release Date.
- Battlefield 1's They Shall Not Pass DLC Details.
- Overwatch's Year of the Rooster Event.
- Obsidian Teases New Game.
- Playdead Tease New Game,
- Ubisoft Acquires FreeStyle.
- Injustice 2 Beta.
- Dead Cells.
- Resident Evil 7 DLC Details.
- Naughty Dog Devs Give Details on Uncharted's Lost Legacy DLC.
- Free Xbox Gold Games for Februrary/2016.
- Dev Gives Opinion on Project Scorpio.
- More Launch Games for the Nintendo Switch.
- No Titanfall for the Switch.
- Pokebank.
- and much more!
Podcast: Play in new window | Download (Duration: 1:51:05 — 50.9MB)


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Lewis Hamilton Tries Stand Up Comedy- And Fails!
An incident filled 2011 Monaco Grand Prix saw Lewis Hamilton penalised twice. In a post race interview he made a comment which suggests a career in stand up comedy is not for him…
Lewis Hamilton arrived on the Formula 1 scene with a bang. Well, quite a few bangs actually, as he brought a style with him that many described as dangerous. In the same way that Michael Schumacher had before him, Hamilton drives in way which expects other drivers to get out of his way.
It effectively bullies them, going for gaps which are too small, leaving the driver in front with a choice – hold the line and possibly go out of the race in a crash, or let Hamilton through.
It can be effective, but it does not win many friends. Results wise it can go either way, Hamilton has already won a world title, but has lost one as well.
Again, in the same way as Schumacher and Hamilton's hero Ayrton Senna, there is an evident arrogance about his attitude, that he does nothing wrong, and any problems are someone else's fault.
After the Monaco race, he was being interviewed by the BBC who asked him why he thought he was being brought before the race stewards so many times.
Here's what he should have said:
" I don't know, you'll have to ask them that"
Here's what he actually said:
"Maybe it's because I'm black?"
That was a reference to something the comedy character Ali G used to say. A comedy character who was based on saying outrageous things.
For a famous sporting name to say it, just comes across as a sore loser with people jumping on him for 'playing the race card'.
You would certainly expect Hamilton to take risks with his driving – that is what people at the top of his profession do after all. Plus, he is driving a slower car than his main rival, so needs to eek out any advantage he can get.
In that context, it is understandable that he will get frustrated, but he seems permanently unhappy, unable to appreciate the abundance he has.
He has his health, he is a multi millionaire, doing a job he loves, which brings him thrills and adulation.
Sounds pretty good, right? Yes, there are pressures, but they are pressures he chose, and he can similarly chose to stop them by simply stopping racing.
Really Lewis, give up the misguided attempts at comedy, and would it hurt to smile a bit more?
I enjoy sport, and I enjoy writing, so when I make money by writing about sport then yep you guessed it – I enjoy it! Discover the formula I use in my guide .
As for me suggesting Lewis Hamilton should appreciate his abundance a bit more, that's something I cover in my book 'Transform Your Life in 21 Days!'
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Discussion in 'Disney Weddings and Honeymoons' started by crista, Sep 30, 2005.
How may other Italy brides out there? Any Experienced Italy brides have any advice?
No advice, haven't had my wedding yet, but I found this thread useful:
I am also excited about the flower arrangement I am using for the Italy Isola - instead of an alter my fiance and I will be framed by two tall "marble" columns with the flower bouquet on top. Totally fits with the Italy look. If you want me to email you the pics the coordinator sent me, let me know
Separate names with a comma.


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Executive Director's video blog
At the 70th UN General Assembly
The Partnership's Robin Gorna explains why the UN General Assembly this week in New York is a game-changer for women's, children's and adolescents' health. Watch the video.
Together we can deliver for women, children and adolescents everywhere...
At the dawn of the new Sustainable Development Goals and Global Strategy for Women's, Children's and Adolescents' Health, The Partnership's Robin Gorna reflects on what this new ambitious agenda means for every woman, every child, everywhere.
Robin Gorna, 17th Meeting of the PMNCH Board
Robin Gorna reports from the PMNCH board meeting in Lusaka where some key decisions have been made on the Partnership's future.
Accountability for Results, Resources and Rights
Robin Gorna looks back at the recent Stakeholder Consultation on Accountability in Geneva, which charts the way forward for a new unified global accountability framework focused on Results, Resources and Rights.
A Special Year End Message
In this year-end video message, Robin Gorna highlights some of the key successes and remaining challenges in a 'landmark' year for women's, children's and adolescents' health.
Video: A year to prove women deliver, and not just babies
Its 2016, the beginning of the Sustainable Development Goals agenda and Robin Gorna, Executive Director of the Partnership, highlights some of the work that must be done to start delivering on the Global Goals and Global Strategy. Watch the video.
Joining together to step it up for gender equality
International Women's Day, the appointment of the Independent Accountability Panel led by Sania Nishtar, and what the Zika Virus means for women's rights are discussed in this new video blog from Robin Gorna. She highlights the important role of accountability in addressing gender equality and strengthening systems that give women and girls access to the SRHR services they need.
Translating evidence into practice for women, children and adolescents
Robin Gorna looks forward to a busy May and reflects on important events and opportunities for the Partnership's work at the upcoming 4th Women Deliver Conference, World Humanitarian Summit, and World Health Assembly. She also addresses some of the Partnership's primary focus as it moves from planning to action, including establishing better ways of working with its over 700 partners, taking forward its new responsibility around accountability and making sure work has an impact in countries.
The Partnership at the 4th Women Deliver Conference 16-19 May
Robin Gorna talks about the Women Deliver Conference, where PMNCH will join close to 6,000 young people, world leaders, advocates and policymakers in Copenhagen to advocate for the health, rights, and well-being of girls and women everywhere. The Global Strategy will be a big part of the conference discussions as well as gender equality, education, environment, and economic empowerment.


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Big Laxa (Arbot private beat)
Laxa in Adaldalur (The Big Laxa)
Laxa in Adaldalur is a world class brown trout and Salmon stream for fly fishermen only. Some people say it's the best brown trout river in Europe. The river is located in Adaldalur in northern Iceland, 480 km from the capital city Reykjavik. Big Laxa is a very productive river that keeps a huge stock of brown trout at the average size of 3-4 pounds. However, 6-8 pounders are not uncommon and even bigger trout are caught every season, the biggest brown being 10-13 pounds. This river is ideal for dry fly fishing but upstream fishing with strike indicators and nymphs can also be effective. Early season, streamers are often on the menu but you never know what the trout is up to here, so having all kinds of flies in the box is recommended.
Arbot beat
Arbot beat is an extraordinary beat with huge character and is an excellent option for those looking for bigger brown trout and salmon. It is 3,7km. Single bank beat limited with only two rods. Many beautiful great pools that holds both trout and Salmon. The season is from 1st of May to 20th of September. The peak time for the trout is usually from mid may or some years earlier, to the end of June. From early August you can be sure that there is Salmon in every pool. The river is well known for its huge salmoweighing up to and over thirty ponds. The beat is limited with three rods from May to 20th of June. And two rods only from 20th of June and till the end of the season.
The Comfortable Vorduholt Lodge.
Vorduholt is an exelent self-catering or full service lodge (up on request) set on a hillside overlooking the river, the spectacular snow capped (Kynnfjoll) mountains and the beautiful Adaldalur valley. There are three rooms with twin beds, kitchen, living room and dining room. Around the lodge is a veranda and a hot tub.
There are many activities and places of interest to visit for non-anglers, including: whale watching , Dettifoss Waterfall. Lake Myvatn. Horse riding and river rafting. This makes Arbot the perfect holiday for anglers brining their families to Iceland.
Good to know
Distance from Reykjavík: 442 km
Fishing season: May15th – September 20th
Average size: Brown trout 4 lbs.
Average size: Salmon 10 lbs.
Number of rods: 2-3
Tackle: Single-handed rods of weight # 3-8
Best flies: Streamers, nymphs and dry flies of assorted patterns
Access: 4x4
Accommodation: Vörðuholt lodge


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Difference between the hugesmp.s and huge.s kernel
What is the difference between the two kernels. The reason i am asking is because i generally like to compile my own kernels later on and in the past have had some problems with the hugesmp.s kernel that i have not had with the huge.s . Is there really any benefit to the hugesmp over the huge kernel if i am using a single core processor?
the difference is only the SMP support
even if you have single processor, using SMP will do just fine
OK but if i were to remove the SMP support when i recompile the kernel (i strip them down to only the things i need) will i get errors regarding it? (this has happened in the past with 12.0)
If it is any consolation, I have recompiled my SW 12.1 kernel without SMP for my single-core machine and have encountered no problems. I have, of course, kept the original kernels available in case I need them; and would add that I have only been using my custom kernel for a couple of weeks.
Just in case you missed this from the Changes & Hints text. I use the generic-smp kernel on two older non-smp and my smp machines with no problems. I never did like the huge because I always had the kobject errors and the initrd is eazy to build.
From Slackware 12.1
I understand that it can be used on the older single core processors, it was just something that i had come across that i was wondering about. And as i see in the last line of the 2nd paragraph, it wont matter as i custom build my kernels and new kernels.
Thanks for the clearing up though.
Wasn't sure which config you were speaking of in your second post since you had some problems. Thats the reason I posted the changes & hints. It was mostly because the Huge/Huge-smp was never designed for everyday use. The Generic/Generic-smp are the kernels for daily use because they are modular designed and require a initrd. I thought you may have used the config-huge instead of the config-generic since you had some problems.
I guess I should have made myself clear.
Have fun kernel building.
|All times are GMT -5. The time now is 12:55 AM.


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Family Swim
Children age 16 and under must be accompanied by an adult in the water or on the pool deck at all times
Ages: all
Times: Please see online schedule or pool schedule flyer from the Welcome Center
Fee: Included with membership
See the Pool Schedule
Mom's Network Pool Times
Join other Moms' Network members and their families for a swim at the YMCA Warm Pools. This is a great way to introduce your little one to water or practice swimming and water safety. For more information about the Moms' Network visit www.themomsnetworkww.com.
Ages: All ages welcome with moms or dads
Contact: Beth Swanson, firstname.lastname@example.com, 509-525-8863
Times: Please see online pool schedule or pool schedule flyer from the Welcome Center.
Fee: Included with Moms' Network membership


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Window Channel Balance Parts Replacement
A quick learner from Orange County, CA says:
May 23, 2015
Hello! I need to replace the channel balance hardware on a bedroom window. I have attached photos of the channel shaft (stamped 27 3 BSI OC) and both ends of the balance frame. The frame measures 1/2" wide and 28" long (metal edge-to-edge). I need to confirm which channel balance hardware is a compatible match, plus what mount hardware (clips, etc.) may be needed for installation. The existing channel balance and mounting pieces were damaged accidentally when dislodging the stuck window. Any info would be appreciated!
Paul from SWISCO responded:
May 27, 2015


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Exactly as I would have wished
My fiancée was overcome with the beauty of the arrangement and the quality of the blooms.
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The world is waiting in the coming year the new child of Chrysler, which will make people look different at the cars of business and luxury class. By the way, new 2016 Chrysler 100 is expected to get quite a lot of changes and become something new and different from the latest model.
The changes will concern both inner and outer parts of the car. The changes promise to make this car really great and unexpected. Producers and designers worked on the weight and size of the cabin, added new elements both outside and inside, tried to make the look and design more perfect and ideal. We will wait for it and hope that all the expectations will make the public happy.
2016 Chrysler 100 Expectations
The whole appearance of new 2016 Chrysler 100 is not known. We just know the separate details, which will be included in the concept. For example, the tail and front lights will be refreshed and now will get a new shape, which will underline the modernity and luxury of the car. Also an important feature is that there is plenty of space inside. Both passengers and driver will feel comfortable, because the space is enough for legs and head. Even several people can ride in the car and feel great and not bothering each other.
Engine of 2016 Chrysler 100
Actually this topic is poke about quite a lot, but nobody knows the information for sure. But producers promise to present something new and extraordinary. According to the rumors, the engine will be bigger than in the previous model with the engine of 2,4 l. It will be able to reach high speed easily by the shortest time than ever before this model could do.
All in all the work of producers and engineers must amaze their customers. As rumors tell us, the car will include so different peculiarities and features, that nobody thought they could be combined in such a way.
The release date and impressions
Sure many people are waiting for new 2016 Chrysler 100 every day. The rumors promise us to see one of the best model through the last years and car generations. We will hope that it will be the perfect reality and happy customers will be able to buy this luxury among the first one.
Nobody is convinced in the definite date, when the world could see the new car. Some people even talk that it may not be represented in 2016, but only in the beginning of 2017. The same situation concerns the price, which is not defined yet. So all we have to do is just to wait for concrete information and hope that producers will soon finish their work.


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|Half Baths
|4,400 sq. ft.
|Property type
|Minimum Stay
|7 nights
Just about one of the best spots in Jamestown RI to sit back and watch the boats sail by. The neighborhood is the 'Dumplings' which is all residential, great place to walk, run or bike. The Village of Jamestown is only about 1.5 miles away, the town beach less than 2 miles.
The main house has 4 bedrooms on the second floor, along with 2 baths. Bedrooms are configured with 1 king size, 1 queen size, a Jack and Jill bath with shower only. The other two bedrooms share a bath with tub/shower. One has 2 twins, the other a queen size. This part of the house sleeps 8 and has a washer and dryer. The main first floor has Kitchen, Dining, Living, family and sitting rooms as well as a level (half bath on first floor).
The third floor has 2 more bedrooms and a sitting area. One bedroom has 3 twins and the other one has 2 twins. Sleeps 5 more.
The lower apartment level has 2 more bedrooms, one with 2 twins, one with a full size. There's a bath with shower only, a kitchenette and a sitting room. This floor sleeps 4 more people.
* The main house, two floors, sleeps 8: $6.000/wk.
* The top three levels of the house, sleeps 13: $6500/wk
* Entire house all four floors, sleeps 17: $7,500/wk
Off Season nightly rate is $1000 per night, 3 night min.
Rates reflect full prime summer months (last 2 weeks of June, July and August, thru Labor day) Please inquire for off season rates.
* Winter/Off Season rate is $3,500 per month plus utilities.
Winter prices apply to a lease that is 6 to 9 months long, tenant pays utilities, trash removal and snow removal.
This is our family home, we want to share this beautiful setting with vacationers.
Liz Brazil, Broker Associate of Lila Delman Real Estate in Jamestown RI purchased this estate in 1971
Why not, water views, privacy, great family reunion setting. A wrap around porch that you wont feel the need to leave.
Water views, large private yard
sleeps 17 people or can sleep 8, 13 people at a different prices
See pricing and options
The house was exactly as advertised with amazing views of the surrounding water and across to Newport. Great wraparound porch. Lots of beds for a nice weekend get together. Well appointed with enough towels, linens, cleaning products etc. to make the weekend go smoothly. No problem during our stay as the coastal air and the fans worked well, but should be noted there is no A/C (at least that we found). Thanks for a great stay.
Classic Victorian home set on over two acres overlooking Narragansett bay and Newport, Rhode Island.
|security deposit
|cleaning fee
|$400 + Tax
|Refundable damage deposit
|Tax Rate
Cancellation: In the event of a cancellation by the Lessee, the Lessor reserves the right to retain any rent paid at the time the lease is cancelled. If the property is re-rented, the Lessor will return the rent paid minus a cancellation fee upon signing of the new lease. The Security Deposit will be refunded in full in either case.
We rent by the week from July to After Labor day. We do allow weekend rentals. Cleaning fees charged to the tenant, typically costs $400. The house is available all year long for short and long term rentals.


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I've purchased magic online from many different places but MJM is the BEST! Best service, best prices, best shipping. Keep up the good work guys!!!
Read More ->
I orderes the Masters Edition deck, a Tally Ho deck, Bobos Modern Coin Magic, and Expert at the Card Table. The prices are awesome. Very cheap. The...
Read More ->


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It might sound totally silly and, probably,
it's definitely totally silly,
but when I saw this picture I had a sudden impression of « deja vu »,
then I realized it reminded me of the Victorian statue of Peter Pan,
by Sir George Frampton, in Kensington Gardens, London.
A totally involuntary tribute,
which made me smile and suggested me some fancy thoughts too….
This photo is not isolated,
but part of a thematic gallery
all in green.
If you feel like,
you might click on these leaves
to see all the other related pictures.
Copyright © 2000-2017 Marisa Livet


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The Demon Castle
The Demon Castle
WAD Type
Read Me
=========================================================================== Advanced engine needed : No, but there's 4 torches that are not seen. But they cast lightning to environment in a port that supports dynamic lights Primary purpose : Single(+Coop play) =========================================================================== Title : The Demon Castle Filename : CASTLE.WAD Release date : Author : Jimi Email Address : <email removed> Other Files By Author : some maps I've done... Misc. Author Info : also known as Jow Description : Player escapes from The Demon Castle Additional Credits to : The demons and imps who built this castle. ========================================================================= * What is included * New levels : 1 Sounds : No Music : Use this cheat: IDMUS18 or IDMUS27 Graphics : No Dehacked/BEX Patch : No Demos : No Other : No Other files required : No, but a GWA file for gl ports is included. * Play Information * Game : DOOM2 Map # : MAP01 Single Player : Designed for Cooperative 2-4 Player : (Designed for) / Player starts only Deathmatch 2-4 Player : No Other game styles : No Difficulty Settings : Yes * Construction * Base : New from scratch Build Time : 24h Editor(s) used : Doom Builder Known Bugs : 141 on "Nightmare!" and "Ultra-Violence.", 105 on "Hurt me plenty.", 67 on "Hey, not too rough." and "I'm too young to die." May Not Run With... : DooM 1, Heretic and Hexen. * Copyright / Permissions * Authors may NOT use the contents of this file as a base for modification or reuse. Permissions have been obtained from original authors for any of their resources modified or included in this file. You MAY distribute this file, provided you include this text file, with no modifications. You may distribute this file in any electronic format (BBS, Diskette, CD, etc) as long as you include this file intact. I have received permission from the original authors of any modified or included content in this file to allow further distribution. * Where to get the file that this text file describes * The Usual: ftp://archives.3dgamers.com/pub/idgames/ and mirrors Web sites: http://zoowad.doomrepublic.com/files/castle.zip FTP sites: * Story * The fighting was hard. 8 of us were captured during the fight, they probably wants to transform us to some distorded zombies. Luckily they didn't find the pistol and 50 bullets that were hidden in my secret pocket. I must escape before I'm transformed into a zombie. I'll get out of this place, back to the outpost where I was before the demons captured me. I must not fail. * Tips * You shouldn't waste bullets to the first enemy, beat it with fists. The Demon Castle is dark, so try to hear where the monsters are and shoot where the sound comes from. Sometimes it's good to beat some imps or demons with fists. It's not really that difficult... In the end, you better save those cells you got... until you open the red door. Happy hunting.


text: Do we dare to pull for Texas on Sat to beat KU so we can gain a game on KU and get closer to first place? Or do we just pull for KU to win at home, and gain that game on TX and get solid position on second place and call it good? It would be nice to see TX lose to us, KU, and OU in a matter of a week or so too, but there is a slim chance we could get first if KU would somehow slip up...considering they only beat Tech by one...... What do ya think?


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Template >> Resume - The Smart CV Resume Set Template 141594 by SNIPESCIENTIST @ CreativeMarket
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" Staying Connected with our Chillers helps us not only bring down the cost of service and improve quality of services, but also gives us invaluable insights into the usage of the product by the customer enabling us to price and engage with our customers in significantly better ways" Leading Chiller OEM in APAC & MEA
" The cell tower monitoring solution enables real time monitoring of assets across various sites further optimizing asset utilization. The solution enables the global operators to achieve higher uptime with low cost of operations" Leading infrastructure companies in APAC & MEA
" Data centre monitoring solution provides real time monitoring of critical parameters to prevent system failures and data loss" Leading hosting organization in APAC
"Help you locate, track, and manage mobile assets. The application enables users to analyze the data gathered to achieve various benefits like improving operations efficiency, asset performance, utilization etc." Leading Companies in India
" The Building energy management solution (BEMS) provides measurable savings by monitoring and controlling the major energy consuming assets also supporting the fault management system enabling optimum utilization of assets" Leading IT companies in APAC


text:
I found a pill in my medicine cabinet and want to identify it. It is round, white, 1/4" across, 1/8" high, and just has a split line on one side. I thought maybe it was a generic claritin but don't want to take it if I can't be sure. If nobody is aware of such a thing, I'll probably just throw it out.
Are there pills with NO brand markings?
- Asked
- 22 Mar 2010 by jd1
- Active
- 22 Mar 2010
- Topics
- pill id
Answers (1)
Search for questions
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Animal books are great because they encourage speech and language development and babies love them! As you read animal books to baby, you can model animal names and sounds and leave space for baby to try to imitate. You can also show baby how to move like a certain animal as you make the animal's sound. Make it fun and silly!
As your baby gets older, they will likely start to imitate these sounds. Animal noises (also called onomatopoeias) are great for practicing early developing speech sounds ("m" in "moo, meow" "b" in "baa," "n" in "neigh") in a natural and fun way.
Check out this blog post with our favorite animal books!


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[kwi-noi-deen, -din] /kwɪˈnɔɪ din, -dɪn/
noun, Pharmacology.
a brownish-black, resinous substance consisting of a mixture of alkaloids, obtained as a by-product in the manufacture of and formerly used as a cheap substitute for it.
quinoidine qui·noi·dine (kwĭ-noi'dēn', -dĭn)
A brownish-black mixture of alkaloids remaining after extraction of crystalline alkaloids from cinchona bark, used as a quinine substitute.
Tagged: q
Read Also:
- Quinol
[kwin-awl, -ol] /ˈkwɪn ɔl, -ɒl/ noun, Chemistry. 1. . /ˈkwɪnɒl/ noun 1. another name for hydroquinone
- Quinoline
[kwin-l-een, -in] /ˈkwɪn lˌin, -ɪn/ noun, Chemistry. 1. a colorless, liquid, water-immiscible, nitrogenous base, C 9 H 7 N, having a disagreeable odor, occurring in coal tar, and usually prepared by oxidizing a mixture of glycerol and aniline: used as a solvent and reagent and to make dyes. /ˈkwɪnəˌliːn; -lɪn/ noun 1. an oily colourless […]
- Quinolone
/ˈkwɪnəˌləʊn/ noun 1. any of a group of synthetic antibiotics, including ciprofloxacin, that inactivate an enzyme required for the replication of certain microorganisms quinolone (kwĭn'ə-lōn') Any of a class of synthetic antibiotics that inhibit the replication of bacterial DNA.
- Quinone
[kwi-nohn, kwin-ohn] /kwɪˈnoʊn, ˈkwɪn oʊn/ noun, Chemistry. 1. a yellow, crystalline, cyclic unsaturated diketone, C 6 H 4 O 2 , formed by oxidizing aniline or : used chiefly in photography and in tanning leather. 2. any of a class of compounds of this type. /kwɪˈnəʊn; ˈkwɪnəʊn/ noun 1. another name for benzoquinone quinone qui·none […]


text:
In stock saddles drastically reduced..stop in and see what we have for you! Barrel, light weight Abetta's, trail, pony, all purpose english, dressage and more!
in stock items only
Look for our "red tags" on specially marked inventory for exceptional discounts on discon tinued/remaining styles/sizes of boots, clothing and accessories.
About The Riding Club
The Riding Club is located in Michigan's Beautiful Upper Peninsula. Whether you like to ride English or Western, show or trail we carry a wide variety of tack and equipment for all of your equestrian needs.
We carry Crosby, Wintec, Blyth Tait, Collegiate, Thornhill- Jorge Canaves, western saddles and strap goods by Weaver, Simco, Tahoe, and Big Horn, just to name a few.
We are the largest retailer of Breyer Figurines in the Upper Peninsula. We also have a great selection of books, and educational material, Western and English show clothes, gift items, and grooming supplies. Along with medicinal and supplement products.
Our selection of both English and Western boots are second to none. They include Durango, Georgia Boots, Ariat, Mountain Horse, Abilene, Sage, Laredo, Dan Post, Stetson, Redneck Rivera, Independent, Smoky Mountain and Acme, just to name a few.
You will find our staff knowledgeable, professional and willing to help you select the proper equipment from our in stock inventory. If the item is not available we can special order it just for you. If you find an in stock item elsewhere, we will price match it for you.
Hours: Monday thru Saturday 10 AM - 5 PM
Phone: 906-228-9033
Visit Our Partner Stores
Click the store front photo to enter websites
Thank You For Visiting


text:
A free, IAP-supported, WoW-like MMORPG for Android.
Ranked in these QuestionsQuestion Ranking
Pro Streamlined questing
Questing in Order and Chaos Online is done in such a way that it is not tedious and can be done quickly.
Pro Has mounts
In Order and Chaos (just like on World of Warcraft) the player can access a Mount at level 20. These are caught in the wild with what the game calls "bond stones."
Pro Large world
This game offers a large world that is similar to smaller PC MMO's.
Pro Free
This game that was once $6.99 became free on August 6th, 2014 and because of this, there are more players than ever.
Pro Many races and classes
5 Races and 4 Classes is a good amount for a mobile MMORPG and this is where Order & Chaos Online sets the standard.
Con Pay to win
Many feel that there are many elements that make Order and Chaos "pay-to-win."
The chat box does not have an option to turn off, and this can cause disruptions.


text: Ontology based digital libraryLalit S. Rajput, S. Shyam, Rekha Pandav, Bhavna Parmar, Archana ShirkeICWET2010In this paper, we describe the use of ontology for efficient searching in digital library. A digital library is a repository of digital documents of different files formats like.pdf, .doc, .ppt or even plain .txt which can be any books, magazines, instruction manuals, presentations etc. Ontology based digital library would have the additional features of… (More)


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I had an appointment today at 3:30 and around 6 i began to bleed. This has never happened before. I wont stop bleeding..i usually have some spotting on my third month right before im due for another shot and this time that happened as well..now im just worried because literally i havent had any bleeding since the 24th of February and now right after i recieved my shot i started bleeding again? Please help
Depo-Provera - why am I bleeding today if I just got my depo shot?
- Asked
- 10 Mar 2015 by volleyball06
- Active
- 10 Mar 2015
- Topics
- depo-provera, provera, abnormal uterine bleeding, bleeding disorder, birth control, depo injection, depo, bleeding, spotting
Responses (1)
It's pretty common on Depo. If you frequently have spotting during your 3rd month, then you health care provider needs to bring your next couple of shots forward to 8/9 weeks, & that will stop the bleeding near the time your shot is due. Then you can go back to having it at the normal interval.
You may find the bleeding is pretty unpredictable until this happens.
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