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Is there any way I can review my mother's power of attorney?
My mom has been living with me for the past 8 months due to her Alzheimer which is now in stage; starting stage 3. Starting severe Alzheimer. My older brother has always handled her finances, including her Government assistance which is Medicaid/Medicare. While I have taken care of our parents health and them as well. My father has passed away. My brother now gives me half of that and says the rest he is paying for my moms home expenses. My home is in foreclosure so I will be moving into my moms house with her soon. To make the story shorter; my brother went behind my back and got Power Of Attorney while he took my mom out for lunch; so he called it. He had her sign the papers without her knowledge as to what she was signing. He told me after that it needed to be done. However, I never saw the papers and he says it's not necessary for me to read them. My main concern is; I AM THE ONE THAT HAS TAKEN CARE OF MY MOM, SHE'S BEEN LIVING WITH ME, WHAT IS TO BECOME OF HER HOME AFTER SHE PASSES? I've always gotten along with my brother and that's why now his response is not acceptable to me. Is there a way that I can review the Power Of Attorney? I don't want to sound cold hearted but my mom loves her house still and I don't want any problems in the future. Thank You!
Expert Answers
There is no legal requirement that would give a person, even a close relative like you, the automatic right to review a power of attorney in which you are not named.
But in reality, that's probably not what you really want, anyway. It sounds as if you have some nagging doubts about whether your brother is acting on the up and up, and some fears that he may be squeezing you out of the picture in some way""and all the reviewing in the world wouldn't change that. About 99% of the powers of attorney for finances read the same way, anyway""just enabling the agent to manage money for another person's best interests while he or she is alive.
It sounds as if it's that "best interest" part that you and your brother need to get straight between you""and maybe, just maybe, that will be a little easier given that the two of you have had a good relationship in the past.
If you haven't done so yet, sit down with your brother and have a very focused and frank conversation. If you've tried this already, try it again. Explain that you realize that you and he want the same thing: to keep your mother as happy as possible and to avoid problems in the future. Without accusing, ask him to explain why he needed the power of attorney at this time; it might be something as simple as needing to write a check or transfer your mother's money between accounts.
Once you are living in your mom's home, the reality is that you and he will likely have to work together more closely in handling your mom's finances. So perhaps you could agree to be transparent with him by keeping a running log of her daily expenses if he will do the same for you with her home expenses that he is managing.
If this transparency idea doesn't work, there may be a legal solution: You could go to court and ask the court to require your brother to file a periodic accounting. While most courts will agree to require this, you can see that it can often have the effect of creating bad blood between the siblings involved. So probably better to try the informal approach""at least at first.
Finally, the issue of what will happen with your mom's house seemed to come up a few times, at least between the lines of your question. So that is another issue you and your brother will want resolved. Find out how the title to the home is held""and whether your mother has a will or trust that leaves it to others, as that may play a part in the complete picture.
Listen and convey the feelings involved in all this, too. If your brother is a straight numbers man, for example, he may not be aware that your mom's house provides her with a great deal of comfort. He may not be aware that getting her some supplemental in-home care if needed may be less expensive than moving her to a facility. He may even feel that since you're moving into the house, he needs to protect any interest he may have in it. Find out.
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Director Vasanth has already started shooting for his next film titled, Sivaranjaniyum Innum Sila Pengalum. We hear that the film has Padmapriya and actor Karunakaran in the lead. Actress Parvathy Menon and Remya Nambeesan have also been approached, but they are yet to sign up. Carnatic singer Sudha Ragunathan will be composing music for this film.
Story first published: Monday, January 19, 2015, 8:15 [IST]

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The American remake of ">REC, known as Quarantine, could go a long way if it stays as creepy as the original. It has a strong leading lady, and director John Erick Dowdle knows what he's doing when it comes to cinema vérité. You'll know what I am talking about whenever The Poughkeepsie Tapes is released.
Anyway, the Quarantine MySpace profile was updated this past weekend with a new pic of star Jennifer Carpenter. Sure, it's nothing amazing like the recent viral videos ("">Quarantine gets Viral" – March 2008), but something's better than nothing. And since I have nothing else to offer, let's bring newcomers to the film up-to-speed with an official synopsis:
Television reporter Angela Vidal (Jennifer Carpenter) and her cameraman (Steve Harris) are assigned to spend the night shift with a Los Angeles Fire Station. After a routine 911 call takes them to a small apartment building, they find police officers already on the scene in response to blood-curdling screams coming from one of the apartment units. They soon learn that a woman living in the building has been infected by something unknown. After a few of the residents are viciously attacked, they try to escape with the news crew in tow, only to find that the CDC has quarantined the building. Phones, internet, televisions and cell phone access have been cut off, and officials are not relaying information to those locked inside. When the quarantine is finally lifted, the only evidence of what took place is the news crew's videotape.
– Syxx
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In high glucose (HG), mesangial cells (MCs) lose their contractile response to endothelin-1 (ET-1) coincidently with filamentous (F)-actin disassembly. We postulated that these MC phenotypic changes are mediated by altered protein kinase C (PKC) isozyme activity, myosin light chain (MLC20) phosphorylation, or Ca2+ signaling. MCs were growth arrested for 24 h in 0.5% fetal bovine serum (FBS)-DMEM in 5.6 (normal glucose; NG) or 30 mM glucose (high glucose; HG). In HG, the planar area was reduced [2,608 ± 135 vs. 3,952 ± 225 (SE) μm2 in NG, P < 0.01, n = 31] with no contractile response to 0.1 μM ET-1. Mannitol did not affect cell size or ET-1 response. Confocal imaging of fluo 3- loaded cells revealed that the peak intensity of ET-1-induced Ca2+signaling was not altered in HG vs. NG. Immunoblotting of phosphorylated MLC20 showed that HG increased mono- and decreased unphosphorylated MLC20 (42 ± 16 and 49 ± 15 vs. 13 ± 3 and 80 ± 4% of total in NG,P < 0.05, n = 3), but the peak phosphorylation responses to ET-1 were identical in NG and HG. ET-1 stimulated translocation of PKC-δ and -ε from cytosolic to membrane and particulate fractions identically in NG and HG but did not cause PKC-ζ translocation. In HG, membrane accumulation of PKC-ζ was observed. Membrane PKC-ζ activity measured by immunoprecipitation and32P phosphorylation of PKC-ε pseudosubstrate peptide was 190 ± 18% of NG (P < 0.01,n = 4), which was completely inhibited by pretreatment with a myristoylated peptide inhibitor (ZI). In HG, pretreatment with ZI for 24 h restored normal MC size and contractile and F-actin disassembly responses to ET-1. In conclusion, in HG, decreased MC size is due to decreased F-actin assembly, and loss of contractile response to ET-1 occurs in the presence of normal Ca2+ signaling and normal MLC20 phosphorylation. In HG, altered F-actin and contractile functions in MCs are mediated by PKC-ζ.
- endothelin-1
- calcium signaling
- myosin light chain phosphorylation
- protein kinase C-ζ
endothelin-1(ET-1) is a potent vasoconstrictor that is synthesized by glomerular endothelial cells (33), epithelial cells (45), and mesangial cells (40). ET-1-stimulated mesangial cell contraction can regulate glomerular capillary surface area and filtration rate (3). However, mesangial cells cultured in high glucose (8, 17) and glomeruli isolated from streptozotocin (STZ)-diabetic rats fail to contract in response to ANG II, (24), ET-1, or raised intracellular Ca2+ (17). In diabetes, high glucose-induced loss of preglomerular afferent arteriolar contractile response to vasoconstrictors leads to elevated intraglomerular pressure, accelerating the progression of glomerulosclerosis (58).
The precise mechanisms by which high glucose alters the phenotype of mesangial cell and vascular smooth muscle cell (VSMC) responsiveness are not known. Reduced Ca2+ signaling mediated by either high glucose-induced protein kinase C (PKC) inhibition of inositol 1,4,5-trisphosphate (IP3) release (34) or via high glucose-induced inhibition of receptor-operated channels (35) have been proposed. Work from our laboratory has demonstrated that isolated glomeruli and mesangial cells cultured in 25 mM (high) glucose display normal Ca2+ signaling to ET-1, ANG II, and arginine vasopressin (AVP) as measured by45Ca2+ efflux and fura 2 spectrofluorimetry, respectively (53). The normal IP3 response observed in high glucose is attributed to upregulation ofmyo-inositol transport, which prevents mesangial cellmyo-inositol depletion (4).
The initial Ca2+ transient induced by vasoconstrictor peptides in VSMCs and mesangial cells is accompanied by Ca2+/calmodulin (CaM)-dependent activation of myosin light chain kinase (MLCK) and the phosphorylation of the regulatory 20-kDa myosin light chain (MLC20) at serine-19 and threonine-18. This leads to the activation of the actin-activated myosin ATPase, interaction of filamentous (F)-actin and myosin, and cellular contraction (42, 46). PKC, a ubiquitous serine-threonine protein kinase, is also implicated in the regulation of contraction in both smooth muscle and mesangial cells (8, 9, 50, 52). Activation of mesangial cell PKC isozymes by hyperglycemia in diabetes represents an important pathway potentially contributing to altered cytoskeletal responsiveness (2, 8, 30). PKC can directly phosphorylate MLC20 at serine-1, serine-2, and threonine-9 without activating contraction (52) and indirectly stimulates MLC20 phosphorylation at serine-19 and threonine-18 by inhibiting myosin light chain phosphatase (MLC-PP). PKC can also stimulate contraction, independent of MLC20phosphorylation, by phosphorylating the actin regulatory proteins calponin and caldesmon (15, 16).
Although we have previously shown that ET-1-stimulated mesangial cell contraction involves the activation of PKC-δ, -ε, and, to a lesser extent, -α in normal glucose (9), little is known about PKC isozyme regulation of mesangial cell MLC20phosphorylation. In high glucose, mesangial cell PKC isozyme expression patterns, subcellular distribution, and activity are altered (1,2, 20, 21, 23, 55). In mesangial cells, Kikkawa et al. (23) and, recently, Amiri and Garcia (1) demonstrated membrane translocation of PKC-ζ and -α after 72 and 120 h of high glucose. Therefore, high glucose-induced activation of selective PKC isozymes that mediate cytoskeletal restructuring may be implicated in the mechanism of reduced contractility. We have observed that mesangial cells in high glucose for 48 h demonstrate F-actin disassembly, which is reversed by inhibition of the polyol pathway and appears to be mediated through a PKC-dependent mechanism (61). Loss of mesangial cell contractility to ET-1 is also restored with aldose reductase inhibition (8).
In this study we postulated that high glucose may alter mesangial cell Ca2+ signaling, MLC20 phosphorylation, or PKC isozyme activity, mediating cytoskeletal dysfunction and hypocontractility. We examined the effects of high glucose on ET-1-stimulated mesangial cell planar area reduction (contraction), Ca2+ signaling, MLC20phosphorylation, PKC isozyme distribution, and F-actin disassembly. The use of a myristoylated PKC-ζ peptide inhibitor (myr-RRGARRWRK; ZI) was used to determine the role of the PKC-ζ isozyme in the regulation of contraction and F-actin assembly in high glucose.
DMEM, penicillin, streptomycin, and trypsin were purchased from GIBCO BRL Life Technologies (Burlington, ON). Fetal bovine serum (FBS) was purchased from Wisent (St. Bruno, PQ). 12 Phorbol-13 myristate (PMA), ET-1, leupeptin, pepstatin A, aprotinin, benzamidine, Tween 20, sodium orthovanadate, dithiothreitol (DTT), and polyclonal anti-PKC-α, -δ, -ε, and -γ antibodies were purchased from Sigma (St. Louis, MO). Polyclonal anti-PKC-βI and -ζ antibodies and protein G-Sepharose were from Santa Cruz Biotechnology (Santa Cruz, CA). Rhodamine-phalloidin and FITC-DNase 1 were from Molecular Probes (Eugene, OR). Horseradish peroxidase (HRP)-labeled goat anti-rabbit and goat anti-mouse secondary antibodies were from Bio-Rad (Hercules, CA) and Jackson Immunoresearch Laboratories (West Grove, PA), respectively. Polyclonal anti-MLC20 antibody and a monoclonal anti-phospho-Ser19-MLC20antibody were kindly supplied by Drs. Subah Packer (Indiana University, Indianapolis, IN) and Yasuharu Sasaki, (Asahi Chemical, Shizuoka, Japan), respectively. Dr. Michael P. Walsh (University of Calgary, Calgary, AB) generously provided purified chicken gizzard MLC20. The myristoylated PKC-ζ inhibitor ZI was synthesized by the Hospital for Sick Children Peptide Synthesis Laboratory (Toronto, ON). The purity of the peptide was >95% as determined by high-pressure liquid chromatography and mass spectroscopy. All other chemicals were of analytic or electrophoresis grade and purchased from BDH (Toronto, ON).
Mesangial Cell Planar Area Measurements
Mesangial cells (passages T5–T10) were cultured as previously described (18) in 20% FBS-DMEM in 5.6 mM (normal) glucose, pH 7.4, supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 mM HEPES.
Mesangial cells (100,000 cells/dish) were directly growth arrested for 24 h on 35-mm plastic dishes (Falcon, Becton-Dickinson Laboratories, Lincoln Park, NJ) in either 2 ml 0.5% FBS-DMEM with 5.6 or 30 mM (high) glucose. The dishes were transferred to the heated stage of an inverted phase-contrast light microscope (Bausch and Lomb, New York, NY) and maintained in 5% CO2 in air at 30°C. The images were captured by a Hitachi KP-113 solid-state television camera (Hitachi Denshi, Tokyo, Japan). After visualization, the image was digitized to represent time 0. To downregulate PKC in normal glucose, cells were pretreated for 24 h with 0.1 μM PMA. To inhibit PKC-ζ, cells were pretreated for 24 h with 10 μM of ZI. This peptide has been shown to inhibit PKC-ζ activity inXenopus laevis oocytes (10) and in pancreatic islet β-cells (47). ET-1 was added directly to the medium to achieve a final concentration of 0.1 μM for cells in normal or high glucose in the absence or presence of ZI. To control for osmolarity, cells were growth arrested in 0.5% FBS in normal glucose supplemented with 24.4 mM mannitol. The same group of cells (n = 10–20/group) was digitized serially for the next 60 min. Images were captured on a 486 DX PC and digitized with Sigma Scan morphometric software (Jandel Scientific, San Rafael, CA). Only those cells with a clearly defined border perimeter were used for planar surface area measurement. Changes in individual cell surface planar area were calculated for all of the cells at each time point.
Confocal Imaging of Intracellular Ca2+
Cells were sparsely plated on glass coverslips to minimize overlap of individual cells. With the use of our previously published methods (54), growth-arrested cells were loaded with 2.5 μM fluo 3 in DMEM (containing CaCl2) with 0.02% Pluronic F-127 and 1 mg/ml BSA for 30 min at 37°C. The cells were washed once in DMEM without fluo 3 and then incubated in the dark in DMEM for 30 min at room temperature (RT). The coverslip was mounted in a chamber on the stage of a Zeiss confocal microscope (LSM 410, Düsseldorf, Germany), and the cells were imaged at 30°C in response to 0.1 μM ET-1. An argon laser was focused with an inverted objective lens (Axiovert 100, ×10), and the pixel resolution was set at 512 × 512 with a gray-scale level of 0 (minimum) to 255 (maximum). A section through the maximum diameter of each cell was digitized at consecutive time points after the introduction of ET-1. To standardize the fluorescence intensity measurement between experiments, the image contrast and brightness levels were adjusted optimally and then kept constant for the experiments in the study. The pinhole (size = 25), scanning time (0.546 s), zoom (1), and magnification of the confocal scanning system were identical for each experiment. The digitized confocal images were analyzed using National Institutes of Health (NIH) Image software (version 1.62) for the Macintosh (NIH, Bethesda, MD), without enhancement.
Analysis of MLC20 Phosphorylation
Phosphorylation of the 20-kDa regulatory MLC20 was measured using a modified method from Persechini et al. (36). The separation of unphosphorylated, monophosphorylated, and diphosphorylated forms of MLC20 was accomplished with glycerol PAGE followed by electrophoretic transfer of protein to nitrocellulose membranes and immunoblot analysis.
Mesangial cells were growth arrested for 24 h on 100-mm plastic dishes in 0.5% FBS-DMEM containing 5.6 or 30 mM glucose. Cells were treated with either 0.1 μM ET-1, PMA, or ionomycin for 0, 2, 5, 10, 20, and 40 min at 30°C. To inhibit PKC-dependent MLC20phosphorylation, cells were exposed to 0.1 μM PMA for 24 h to downregulate PKC. Trypsin-detached cells served as a negative control. After treatment, cells were rinsed with PBS and cellular proteins were precipitated in 600 μl of 10% trichloroacetic acid containing 2 mM DTT at 4°C. Precipitates were scraped from the plates and centrifuged at 15,000 g for 15 min. The supernatant fraction was decanted, and the pellet was extracted three times in 6 ml of diethyl ether containing 2 mM DTT at 4°C for 2 min, followed by air drying at room temperature (RT) for 30 min to remove residual ether. The pellet was then dissolved in 100 μl of sample buffer containing 8 M urea, 20 mM Tris base, 23 mM glycine, and 2 mM DTT, pH 8.6. After centrifugation and protein determination, 100 μg of the urea-solubilized samples were electrophoresed at 400 V for 1.5 h at 10°C in 1.0-mm minigels containing 10% polyacrylamide, 0.5% bisacrylamide, 40% glycerol, 20 mM Tris base, and 23 mM glycine. The reservoir buffer contained (in mM) 20 Tris, 23 glycine, 1 sodium thioglycolate, and 1 DTT. Separated proteins were then electroblotted to 0.22-μm nitrocellulose (Bio-Rad) membranes at 24 V for 3 h, employing a 20 mM Tris-23 mM glycine-20% methanol transfer buffer, pH 7.6.
After transfer, membranes were blocked in 5% skim milk in Tris-buffered saline plus Tween 20 (TTBS) and then incubated for 1 h at RT in a rabbit polyclonal anti-MLC20 antibody (1:1,000) in 5% skim milk in TTBS. For chemiluminescent detection, the membranes were incubated with an HRP-conjugated anti-rabbit secondary antibody, dipped into luminol substrate solution for 1 min, and developed on film, as described above. Densitometry was performed using NIH Image 1.62 analysis software, and MLC20phosphorylation was expressed as the percentage of MLC20 in un-, mono-, or diphosphorylated form.
Immunoblotting of Ser19-MLC20 and the ETA Receptor
In a separate series of experiments, phosphorylation of Ser19-MLC20 and ETA receptor expression were analyzed in total mesangial cell lysates. Cells were growth arrested in six-well plates for 24 h in 0.5% FBS-DMEM in normal or high glucose. To determine if high glucose altered ETA receptor expression, cells were growth arrested in 0.5% FBS-DMEM in normal or high glucose for 1, 2, 3, 5, and 7 days and then lysed in 150 μl of 2× SDS sample buffer at 100°C. After protein determination, 15 μg of cellular protein were subjected to SDS-PAGE using 15 and 10% minigels for Ser19-MLC20 and the ETA receptor, respectively. The proteins were transferred to polyvinylidene difluoride (PVDF; Millipore, Bedford, MA) membranes, blocked as described above, and probed for 1 h at RT (1:1,000 in 5% skim milk in TTBS) with either a monoclonal anti-Ser19-MLC20 antibody or a sheep polyclonal anti-ETA receptor antibody (US Biologicals, Swampscott, MA). Chemiluminescent detection and densitometry were performed as described above.
Cell Fractionation and PKC Immunoblots
PKC isozymes were probed in cytosolic, membrane, and particulate fractions of 24-h-growth-arrested mesangial cells in normal and high glucose in the absence and presence of 0.1 μM ET for 10 min at 30°C. As described previously (9, 21), cellular fractions were obtained using sequential ultracentrifugation in the absence (cytosolic) or presence of the detergents 1% Triton X-100 (membrane) and 10% SDS (particulate). Total PKC isozyme expression was determined in cells lysed in 2× SDS sample buffer at 100°C. Protein content was measured using a Bio-Rad Dc Lowry protein assay (Bio-Rad). Fifteen microliters of protein were subjected to SDS-PAGE on 10% gels followed by overnight transfer onto PVDF membranes. Membranes were then exposed to polyclonal anti PKC-α, -βI, -δ, -ε, -γ, or -ζ antibodies for 1 h at 1:40,000 dilution for PKC-α; 1:15,000 for PKC-ζ; 1:10,000 for PKC-δ and -ε; 1:8,000 for PKC-γ; and 1:1,000 for PKC-βI. This was followed by a 20-min incubation with a HRP-conjugated affinity-purified goat anti-rabbit IgG antibody (Jackson Immunochemicals) diluted 1:5,000. The secondary antibody was detected by chemiluminescence (Kirkegaard & Perry Labs, Gaithersburg, MD), and the membranes were developed on Kodak X-Omat Blue film (Eastman Kodak, Rochester, NY). Densitometry was performed using NIH Image 1.62 analysis software. The specificity of primary antibody interaction with the sample was eliminated in the presence of the peptide that was used to generate the antibody.
Confocal Fluorescence Imaging
Mesangial cells were cultured on glass coverslips under conditions identical to those described above. The cells were fixed with 3.7% formaldehyde for 15 min at RT followed by plasma membrane and nuclear membrane permeabilization with 100% methanol at −20°C for 10 min, as previously published (61). After being washed three times with PBS, the cell proteins were blocked with 1% goat serum plus 0.1% BSA in PBS for 60 min at RT. For Ser19-MLC20, cells were permeabilized with 0.1% Triton X-100 for 30 min. Rabbit polyclonal anti-PKC-α, -δ, -ε, -ζ, and -γ and monoclonal anti-Ser19-MLC20 were diluted to 1:100 (1:10 PKC-βI) in blocking solution and added to each coverslip for 60 min at 37°C. After being washed three times with PBS, FITC-conjugated anti-rabbit or anti-mouse secondary antibodies were diluted 1:160 in blocking solution and added for 60 min at 37°C. The coverslips were mounted on glass slides with Aqua-Poly-mount (Polysciences, Warrington, PA) and SlowFade antifade reagent. The cells were visualized with an oil-immersion, inverted objective lens (Axiovert 100, ×63), and the image pixel resolution was 512 × 512 with a gray-scale level of 0 (minimum) to 255 (maximum) intensity. To standardize the fluorescence intensity for all the experimental preparations, the confocal image contrast and brightness levels were adjusted optimally for each PKC isozyme and then kept constant for all image analysis.
Mesangial cell F- and G-actin were simultaneously labeled with the fluorescent probes rhodamine-phalloidin (F-actin) and FITC-DNase 1 (G-actin) according to our previously published methods (60). Cells were cultured on glass coverslips under experimental conditions described above. After being washed in PBS (4°C), the cells were fixed in 3.7% formaldehyde in PBS for 15 min at RT and permeabilized with 0.1% Triton X-100 for 10 min. After additional washes in PBS, the cells were incubated simultaneously in the dark with 200 μl of 0.165 μM rhodamine-phalloidin and 0.3 μM FITC-DNase 1 for 20 min. Coverslips were mounted on glass slides and imaged with a dual-channel Zeiss LSM 410 confocal laser-scanning microscope, as described above (Confocal Imaging of Intracellular Ca 2+).
Measurement of PKC-ζ and PKC-δ Activity
PKC-ζ activity was determined in membrane fractions using immunoprecipitation and 32P phosphosphorylation of Ser159-PKC-ε pseudosubstrate peptide (44). To inhibit PKC-ζ, cells were pretreated with 10 μM ZI for 24 h. Cells cultured in normal glucose supplemented with 24.4 mM mannitol for 24 h served as an osmotic control. Membrane fractions were obtained as previously described (9, 21). The supernatant was precleared by incubation with protein G-Sepharose (100 μl/ml lysate) at 4°C for 20 min on a rocker, followed by centrifugation at 4°C at 15,000 g for 10 min. Samples of the lysate were removed for protein assay (Bio-Rad, Dcprotein assay), and 2 μg of rabbit polyclonal anti-PKC-ζ (Santa Cruz Biotechnology) were added to 300 μg of total cellular protein. The samples were allowed to rock at 4°C for 3 h, followed by immunoprecipitation with 40 μl of protein G-Sepharose for 1 h. The immunocomplexes were centrifuged at 15,000 gfor 10 min at 4°C and then washed three times with ice-cold lysis buffer, followed by three washes in kinase buffer. The kinase activity of immunoprecipitated PKC-ζ was assayed in 40 μl of kinase buffer that contained (in mM) 50 Tris, pH 7.5, 5 MgCl2, 2 NaF, 0.1 Na3VO4, and 0.1 Na2P2O7 · 10 H2O, as well as 5 μg of Ser159-PKC-149–164-ε pseudosubstrate peptide (Biomol Research Laboratories, Plymouth Meeting, PA), 40 μM ATP, 5 μCi of [γ-32P]ATP, and 10 nM microcystin. After 15 min at 30°C, the reaction was terminated by the addition of 25 μl of ice-cold quenching buffer containing 0.1 mM ATP and 100 mM EDTA, pH 8.0. Fifty microliters of the reaction mixture were spotted on P81 filter disks and washed four times in 75 mM phosphoric acid followed by a final wash in 80% ethanol. The disks were air dried and placed in 10 ml of Ready Protein scintillant (Beckman Industries); then,32P radioactivity was counted in a Beckman LSC5 scintillation counter.
PKC-δ activity was determined in total cell lysates using immunoprecipitation and 32P phosphorylation of Ser25-PKC-α pseudosubstrate peptide. To determine the specificity of the PKC-ζ inhibitor, cells were pretreated with 10 μM ZI for 24 h. Cells were lysed in 500 μl of lysis buffer containing (in mM) 150 NaCl, 25 HEPES, 1 EGTA, 2 EDTA, 10 NaF, 50 β-glycerophosphate, 1 Na3VO4, 2.5 bensamidine, and 1 phenylmethylsulfonyl fluoride, as well as 10 μg/ml leupeptin, pepstatin, and aprotinin, 10 nM microcystin, and 1% Triton-100, pH 7.5. Lysates were triturated five times through a 25-gauge needle, placed on ice for 30 min, and centrifuged at 15,000g for 5 min to remove cellular debris. Three hundred micrograms of total cellular protein were immunoprecipitated with 2 μg of rabbit polyclonal anti-PKC-δ (Santa Cruz Biotechnology) for 3 h, followed by the addition of 40 μl protein G-Sepharose for 1 h. The immunocomplexes were centrifuged, washed in lysis buffer, followed by three washes in kinase buffer. Immunoprecipitated PKC-δ was assayed in 40 μl of kinase buffer that contained 5 μg of Ser25-PKC-19–31-α pseudosubstrate peptide (GIBCO BRL Life Technologies) but was otherwise identical to that used for determining PKC-ζ activity. The reaction was terminated by the addition of quenching buffer, followed by spotting on P81 filter disks, subsequent washing in 75 mM phosphoric acid, and scintillation counting, as described above. Data for PKC-ζ and PKC-δ activities are expressed as the percentage of those observed in normal glucose.
Statistical Analyses
All results are expressed as means± SE. Statistical analysis was performed using InStat 2.01 statistics software (Graph Pad, Sacramento, CA). The means of two groups were compared using an unpaired Student's t-test. The means of three or more groups were compared by one-way ANOVA with Bonferroni's multiple comparison. To compare multiple groups with basal normal glucose, Dunnett's multiple comparison was utilized. Differences described as significant are P < 0.05 unless stated otherwise.
Effects of High Glucose and Chronic Phorbol Ester on Mesangial Cell Planar Area
Figure 1 demonstrates the change in planar surface area of mesangial cells growth arrested for 24 h in 5.6 (normal) and 30 mM (high) glucose and stimulated with 0.1 μM ET-1 at 30°C. Basal planar surface area in cells cultured in 5.6 mM glucose was 3,952 ± 225 μm2 (n = 53 cells). Cells incubated in 30 mM glucose displayed a smaller basal planar area of 2,608 ± 135 μm2 (n = 31 cells, P < 0.01 vs. normal glucose at time 0). This was not due to osmolarity as cells cultured in normal glucose supplemented with 24.4 mM mannitol were no different from cells in normal glucose alone (3,424 ± 238 μm2,n = 28 cells).
In normal glucose, administration of ET-1 for 60 min decreased planar area to 77 ± 3% of the original surface area (P< 0.05 vs. normal glucose at time 0) (Fig. 1 B). Mannitol in normal glucose did not alter mesangial cell responsiveness to ET-1 at 60 min (81 ± 2% of basal area).
Effects of High Glucose on ETA Receptor Expression
To determine whether the loss of ET-1 responsiveness in high glucose was due to reduced ETA receptor expression, mesangial cells were cultured in 0.5% FBS-DMEM in 5.6 or 30 mM glucose for up to 7 days, and total cell lysates were examined by immunoblot for the presence of the ETA receptor. In the representative immunoblot shown in Fig. 2, the antibody generated against a 39-kDa cytosolic fragment of the ETAreceptor reacted with cellular proteins of 64-, 50-, and 39-kDa molecular mass. The intensities of the three dominant bands were not altered by high glucose. Equal protein loading was confirmed by staining the PVDF membrane with Ponceau S.
Confocal Fluorescence Imaging of ET-1-Stimulated Intracellular Ca2+ Signaling
In normal glucose, fluo 3-loaded cells displayed perinuclear and cytosolic Ca2+ fluorescence staining at time 0(Fig. 3 A). The administration of 0.1 μM ET-1 stimulated an increase in cytosolic and nuclear Ca2+ beginning at 20 s and peaking at 40–60 s. Fluorescence intensity returned to basal levels at 80–100 s. This rapid Ca2+ response to ET-1 was seen in >90% of cells examined (n = 44 cells in 3 independent experiments).
In Fig. 3 B at time 0, cells in high glucose displayed a basal perinuclear and cytosolic staining pattern that was no different from that seen in normal glucose. However, an increase in cytosolic and nuclear Ca2+ did not occur until 40 s after the addition of 0.1 μM ET-1 and reached a peak at 70–80 s. Fluorescence intensity returned to basal levels at 120–200 s. Although the Ca2+ response was delayed compared with that for normal glucose, peak fluorescence pixel intensity (130 ± 13 in normal glucose vs. 117 ± 12 in high glucose) and the number of cells responding to ET-1 (>90%, n = 44 cells) were not different.
Phosphorylation of MLC20
Effects of ET-1, phorbol ester, and ionomycin on MLC20phosphorylation.
The change in ET-1-induced mesangial cell planar surface area was used as an indirect measurement of contraction. Glycerol-urea PAGE was used to measure MLC20 phosphorylation, the key regulatory step preceding smooth muscle cell contraction. As shown in Fig.4, at time 0 the majority of MLC20 was unphosphorylated [81 ± 5 (SE)%,n = 3], with 13 ± 4 and 6 ± 2% mono- and diphosphorylated, respectively. In comparison, trypsinized suspended cells contained 97 ± 5 un- and 3 ± 1% monophosphorylated MLC20. ET-1 at 2 min significantly stimulated MLC20 phosphorylation. Mono- and diphosphorylated MLC20 increased to 60 ± 9 and 24 ± 11% of total, respectively (P < 0.05 vs. time 0,n = 3), whereas unphosphorylated MLC20decreased to 16 ± 7% of total (P < 0.05 vs.time 0, n = 3). The effect of ET-1 was sustained at 40 min, with 29 ± 7, 40 ± 4, and 31 ± 5% un-, mono-, and diphosphorylated, respectively (P< 0.05 vs. time 0). Downregulation of PKC with 24-h PMA did not alter time 0 MLC20 phosphorylation (72 ± 4, 22 ± 10, 6 ± 2% un-, mono-, and diphosphorylated, respectively) and did not affect ET-1-stimulated phosphorylation at 10 min (22 ± 6, 40 ± 10, and 38 ± 13% un-, mono-, diphosphorylated, respectively, vs. 23 ± 8, 45 ± 2, and 32 ± 8% with no PMA).
As shown in the representative immunoblots, 0.1 μM acute PMA did not stimulate phosphorylation of MLC20, whereas 0.1 μM ionomycin stimulated phosphorylation to lower levels than seen with ET-1.
Effects of high glucose on ET-1-stimulated MLC20phosphorylation.
Densitometric analysis of ET-1-stimulated MLC20phosphorylation in high glucose is shown in Fig.5. At time 0, high glucose significantly increased monophosphorylated MLC20, whereas it decreased unphosphorylated MLC20 (42 ± 16 and 49 ± 15% of total, respectively, vs. 13 ± 3 and 80 ± 4% of total in normal glucose at time 0, P< 0.05, n = 3). ET-1 at 2 min significantly stimulated diphosphorylation of MLC20. Mono- and diphosphorylated MLC20 increased to 62 ± 20 and 28 ± 11% of total, respectively (P < 0.05 vs. time 0,n = 3), whereas unphosphorylated MLC20decreased to 10 ± 1% of total (P < 0.05 vs.time 0, n = 3). The effect of ET-1 on diphosphorylated MLC20 was sustained at 40 min, with 34 ± 20% of total MLC20 diphosphorylated (P < 0.05 vs. time 0). Downregulation of PKC with 24-h PMA did not alter basal MLC20 phosphorylation in high glucose (54 ± 20, 37 ± 19, and 9 ± 4% un-, mono-, di-phosphorylated, respectively) and did not affect ET-1-stimulated phosphorylation at 10 min (20 ± 10, 39 ± 13, and 41 ± 21% un-, mono-, diphosphorylated, respectively, vs. 24 ± 11, 40 ± 6, and 36 ± 15% with no PMA).
Effects of high glucose on phosphorylated Ser19-MLC20.
The use of glycerol-urea PAGE effectively separated the un-, mono-, and diphosphorylated MLC20. However, this method cannot distinguish which serine or threonine is phosphorylated. To determine whether Ser19-MLC20, the dominant site phosphorylated by MLCK and Rho kinase, was the monophosphorylated MLC20, we immunoblotted total cell lysates with a monoclonal phospho-specific Ser19-MLC20antibody. As shown in Fig. 6 A, basal levels of phospho-Ser19-MLC20 were not altered by high glucose (92 ± 8% of normal glucose,n = 8).
Confocal imaging of ET-1-stimulated phosphorylated Ser19-MLC20.
Basal phospho-Ser19-MLC20 cellular distribution in mesangial cells in normal glucose is shown in Fig. 6 B. Fluorescence was localized primarily to the cell membrane along with a fibrillar, cytosolic staining pattern. ET-1 administration failed to significantly alter the staining pattern. In high glucose, the intensity and staining pattern were not different from that in normal glucose. In high glucose, ET-1 also failed to change the distribution and fluorescence intensity of the membrane staining.
Mesangial Cell PKC Isozymes
Effects of high glucose on basal and ET-1-stimulated PKC isozyme distribution.
In normal glucose, ET-1 stimulates the translocation of PKC-α, -δ, and -ε isozymes, but not PKC-ζ, from cytosolic to membrane and particulate fractions (9). To determine whether high glucose alters ET-1-stimulated PKC isozyme translocation, we immunoblotted cytosolic, membrane, and particulate cellular fractions with PKC isozyme-specific antibodies. As shown in Fig.7, PKC-α, -δ, and -ε were present in all three fractions in both normal and high glucose and translocated in response to ET-1 from cytosolic to membrane and particulate fractions to a similar extent. In response to ET-1 in normal vs. high glucose, PKC-α distribution in membrane and particulate fractions, respectively, was 87 ± 17 and 133 ± 10% of that in basal normal glucose (P < 0.05) vs. in high glucose 92 ± 20 and 142 ± 6% of basal normal glucose levels (P < 0.05, n = 4). PKC-δ distribution in membrane and particulate fractions, respectively, was 300 ± 76 and 134 ± 3% of that in basal normal glucose (P < 0.01 and P < 0.05, respectively) vs. in high glucose 287 ± 74 and 137 ± 24% of that in basal normal glucose (P < 0.01 and P< 0.05, respectively). PKC-ε distribution in membrane and particulate fractions, respectively, was 205 ± 42 and 133 ± 16% of that in basal normal glucose (P < 0.01 andP < 0.05, respectively) vs. in high glucose 184 ± 52 and 190 ± 56% of that in basal normal glucose (P < 0.01 and P < 0.05, respectively,n = 4). Cells treated with 0.1 μM PMA for 10 min served as a positive control and demonstrated the disappearance of PKC-α, -δ, and -ε from the cytosolic fraction (12 ± 9, 2 ± 1, and 4 ± 1% of distribution in basal normal glucose, respectively, P < 0.01, n = 4). This was accompanied by enhanced recovery of isozymes in membrane (289 ± 97, 513 ± 90, and 267 ± 43% of basal normal glucose, respectively, P < 0.01, n = 4) and particulate fractions (376 ± 46, 205 ± 33, and 244 ± 70% of basal normal glucose, respectively, P < 0.01,n = 4).
Subcellular distribution of PKC-ζ and -βI are also shown in Fig. 7. Both isozymes were present in all three fractions but did not respond to ET-1 in either normal or high glucose. PKC-ζ was PMA unresponsive, whereas PKC-βI demonstrated translocation from cytosolic to membrane and particulate fractions (2.6 ± 1, 295 ± 117, and 322 ± 111% of that in cytosolic and membrane and particulate fractions with basal normal glucose, respectively, P < 0.01, n = 4). High glucose alone enhanced recovery of PKC-ζ in the membrane fraction (294 ± 106% of basal normal glucose, P< 0.01, n = 4) with no change in either the cytosolic or particulate fractions (96 ± 7 and 91 ± 7%, respectively, of basal normal glucose, n = 4).
Effects of high glucose on total PKC isozyme content.
In high glucose, it was postulated that the enhanced recovery of PKC-ζ in the membrane might be attributed to enhanced total PKC expression. Therefore, we determined the effects of 24-h high glucose on total PKC expression by immunoblotting total cell lysates (Fig.8). PKC-α, -δ, -ε, -ζ, and -βI were expressed as a single band in mesangial cells, but PKC-γ was not expressed. High glucose stimulated a significant 2.7-fold increase in PKC-δ (272 ± 75%, P < 0.05, vs. normal glucose, n = 6), whereas PKC-α (125 ± 12%, n = 6), -ε (90 ± 10%,n = 6), -ζ (102 ± 2%, n = 6), and -βI (90 ± 12%, n = 6) remained unchanged.
PKC-ζ and High Glucose-Induced Loss of Mesangial Cell Contraction to ET-1
Effects of high glucose on immunoprecipitated PKC-ζ and PKC-δ activity.
Figure 9 shows the effects of high glucose on membrane PKC-ζ activity in the absence and presence of the ZI. High glucose increased PKC-ζ activity to 190 ± 18% of normal glucose (P < 0.01, n = 4). Twenty-four-hour pretreatment with 10 μM ZI in either normal or high glucose significantly inhibited activity to 45 ± 10 and 73 ± 4% of normal glucose, respectively (P < 0.01,n = 4). Mannitol did not significantly affect PKC-ζ activity (99 ± 11% of normal glucose, n = 4).
In total cell lysates, high glucose increased PKC-δ activity to 241 ± 41% of normal glucose (P < 0.01,n = 3). Twenty-four-hour pretreatment with 10 μM ZI in either normal or high glucose did not alter total PKC-δ activity (110 ± 22 and 256 ± 37% of normal glucose, respectively,n = 3). Mannitol did not significantly affect PKC-δ activity (129 ± 12% of normal glucose, n = 4).
Effects of ZI on mesangial cell planar area and ET-1-stimulated contraction.
Basal planar surface area in cells cultured in normal glucose was 3,952 ± 225 μm2 (n = 53 cells) (Fig. 10 A). In comparison, cells incubated in 30 mM glucose displayed a smaller basal planar area of 2,608 ± 135 μm2 (n = 31 cells,P < 0.01 vs. normal glucose at time 0). This was not due to osmolarity as cells cultured in normal glucose supplemented with 24.4 mM mannitol were no different from cells in normal glucose (3,624 ± 238 μm2, n= 28 cells). Pretreatment of cells in high glucose with 10 μM ZI for 24 h restored the area to values seen in normal glucose (3,875 ± 213 μm2, n = 40 cells). In normal glucose, pretreatment with ZI did not alter basal planar area (3,871 ± 277 μm2, n = 23 cells). ZI pretreatment in normal glucose did not alter MC responsiveness to ET-1 at 60 min (69 ± 5 vs. 77 ± 3% of basal area in normal glucose) as shown in Fig. 10 B. Cells in high glucose did not respond to ET-1. In high glucose, pretreatment with ZI restored the ET-1 response, decreasing planar area to 80 ± 2% of basal area, similar to the change in normal glucose.
Effects of ZI on mesangial cell F-/G-actin in high glucose.
In Table 1, the average pixel intensity/cell values for F-actin and G-actin are presented. F-/G-actin ratio was calculated for each cell and the mean ± SE is reported for each group. In high glucose, the F-/G-actin ratio was significantly reduced to 1.5 ± 0.2 (P < 0.05 vs. normal glucose, n = 44 cells) from a value of 1.9 ± 0.1 in normal glucose. The addition of 0.1 μM ET-1 in normal glucose resulted in an F-/G-actin ratio that was not different from that seen in the basal high glucose state (1.5 ± 0.1, n = 44). In high glucose, ET-1 did not alter the mesangial cell F-/G-actin ratio (1.4 ± 0.1, n = 36). Mannitol (normal glucose+24.4 mM mannitol for 24 h) had no effect on the F- or G-actin content or F-/G-actin ratio (2.0 ± 0.2, n= 26 cells).
The lack of a contractile response to ET-1 in mesangial cells in high glucose may involve reorganization of the cellular cytoskeleton. In normal glucose, F-actin staining filled the cytoplasm and was organized in a bundled stress fiber pattern (Fig.11). G-actin was homogeneously distributed in the cytosol and followed a granular staining pattern. In high glucose, F-actin intensity was reduced, whereas cytosolic and perinuclear G-actin fluorescence intensity was increased. In normal glucose, ZI pretreatment had no affect on F-/G-actin staining pattern or intensity. However, pretreatment of cells in high glucose with ZI resulted in a staining pattern that was seen in normal glucose.
In this study, we examined the signaling events related to mesangial cell contractile and cytoskeletal dysfunction in high glucose. PKC has been implicated as an underlying mechanism of high glucose-induced VSMC dysfunction (25). In high glucose, lack of mesangial cell contractility in response to ET-1 and F-actin disassembly is not due to altered Ca2+ signaling or phosphorylation of MLC20 but is PKC-ζ mediated.
The observations that after exposure to 24-h high glucose mesangial cells are smaller in size, unresponsive to ET-1 (8), and display PKC-mediated F-actin disassembly (61) resemble our earlier studies in higher passage cells. We reported previously that mesangial cell reduced planar area, responsiveness to ET-1, and F-actin disassembly were reversed by tolrestat, an aldose reductase inhibitor (21). Aldose reductase inhibition of the polyol pathway prevented the de novo synthesis of diacylglycerol (DAG) (8, 29, 49) and thus activation of DAG-sensitive PKC isozymes at 48 h (21). At 48 h, high glucose stimulated the membrane translocation of PKC-δ and -ε (21). In the present study, total PKC-δ but not PKC-ε was increased by 24-h high glucose, whereas PKC-ζ was increased in the membrane fraction only. Although the exact mechanisms of PKC-ζ activation are not known, phosphatidic acid (PA), a precursor of DAG, is reported to activate PKC-ζ (32). In our earlier studies (21), tolrestat, through inhibition of de novo PA synthesis, may have reduced PKC-ζ activity concomitantly with either PKC-δ and/or -ε.
It is possible that the diminished responsiveness of mesangial cells to ET-1 in high glucose may be mediated by reduced endothelin-receptor A (ETA) expression (19). Hargrove et al. (14) demonstrated in cultured rat mesangial cells that high glucose for 24 h upregulates the production of ET-1 and ET-1 mRNA twofold. Increased ET-1 production may cause downregulation and decreased expression of mesangial cell ETA receptors (19). By contrast, glomerular ETA receptor expression was upregulated in glomeruli from alloxan-diabetic rabbits (22), whereas hyperglycemia did not alter kidney ETA receptor expression in humans or in STZ-diabetic rats (41). In our study, 1–7 days of high glucose did not alter mesangial cell ETA receptor expression. This suggests that reduced mesangial cell ETA receptor expression does not likely account for the high glucose-induced contractile dysfunction in response to ET-1.
As observed in Fig. 3, A and B, in high glucose ET-1 stimulated intracellular Ca2+ to similar levels observed in normal glucose. Although peak intensity of fluo 3-labeled release was not different, a time delay in the measured response in high glucose was consistently observed. We have previously shown that under the identical culture conditions used in this study (9), acute PMA does not elicit a Ca2+ signal. This suggests that PKC activation does not directly stimulate Ca2+ signaling in mesangial cells. However, others have shown that prior PKC activation may inhibit signaling. Interaction may be at the level of G protein coupling (57) or the cytoskeleton (37). Disruption of F-actin with cytochalasin D in platelets inhibits Ca2+ entry (39). Treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), also inhibits Ca2+ entry (39). PI 3-kinase is a potent activator of atypical PKC-ζ (59). In our hands, cytochalasin D pretreatment of mesangial cells did not affect either timing or intensity of ET-1-stimulated Ca2+ release (data not shown). This suggests that the time delay observed in Ca2+ signaling in response to ET-1 in high glucose is not attributable to mesangial cell F-actin disassembly. The potential role of PI 3-kinase in high glucose-induced activation of mesangial cell PKC-ζ and stress fiber disassembly was not explored.
Stress fibers are composed not only of actin but also contain myosin, tropomyosin, and α-actinin and require the small GTP-binding protein Rho for their formation (38). Stress fiber assembly is usually preceded by MLC20 phosphorylation, whereas stress fiber disassembly is a consequence of MLC20dephosphorylation (5). In mesangial cells, Kreisberg et al. (28) demonstrated that cAMP-mediated stress fiber disassembly preceded MLC20 dephosphorylation. In our study, basal MLC20 phosphorylation was markedly increased in high glucose, whereas F-actin was disassembled, resembling the effects of agonist stimulation. Fukuda et al. (11) reported increased basal MLC20 phosphorylation in platelets from type 2 diabetic patients, but the organization of actin was not studied. Because MLC20 phosphorylation is a balance between kinase and phosphatase activities (42), it is possible that high glucose may enhance MLCK activity or increase PKC- or Rho-mediated inhibition of phosphatase activity (31, 43). In this study, neither acute nor chronic PMA, in either normal or high glucose, affected basal MLC20 phosphorylation. The lack of MLC20 phosphorylation in response to acute PMA suggests that the PMA-induced mesangial cell contraction observed in our earlier studies (8, 9) must be MLC20 phosphorylation independent. In our earlier study, chronic PMA inhibited contraction by downregulating PKC-α, -δ, and -ε in the cytosolic, membrane, and particulate fractions (9), thus reducing DAG-sensitive PKC isozyme content. PKC isozymes may stimulate contraction by initiating a kinase cascade involving the phosphorylation of the actin regulatory proteins calponin, caldesmon (16), and/or myristoylated alanine-rich C-kinase substrate (48).
In high glucose, ET-1 stimulated phosphorylation of MLC20to levels seen in normal glucose, which was not accompanied by cell contraction. This might be explained by the preferential phosphorylation of PKC-specific sites serine-1, serine-2, or threonine-9 of the MLC20 by ET-1 in high glucose. Phosphorylation of MLC20 at these sites does not result in myosin-ATPase activation and contraction (52). If high glucose enhanced PKC-mediated inhibition of MLC-PP, phosphorylation of Ser19-MLC20 would increase. This was not detected by either immunoblotting or confocal imaging with a Ser19-MLC20 antibody. The effects of high glucose on either Rho expression or activity are not known. Therefore, in high glucose, MLC20 phosphorylation does not appear to correlate with either F-actin disassembly or lack of contraction in response to ET-1. High glucose must be exerting its actions at another cytoskeletal target.
In this study, high glucose for 24-h enhanced membrane accumulation of PKC-ζ. This was much earlier than the 5-day time point reported by others (1, 24). This was not accompanied by a concomitant decrease in cytosolic and/or particulate fraction and may be explained by the fact that the relative abundance of PKC-ζ in these fractions may hinder the detection of small compartmental changes in distribution. Immunocomplexed PKC-ζ membrane activity was significantly increased by high glucose and was normalized by pretreatment with the myristoylated ZI. ZI was specific for the PKC-ζ isozyme, as pretreatment with ZI did not affect immunoprecipitated PKC-δ activity in either normal or high glucose. Although total protein expression and activity of PKC-δ were increased by high glucose, this was not accompanied by changes in fractional distribution. PKC-δ may be activated by PKC-ζ-mediated Ser660 phosphorylation (62). Furthermore, overexpression of constitutively active PKC-ζ results in phosphorylation of PKC-δ coexpressed in HEK-293 cells (62). In our study, high-glucose-induced PKC-ζ-mediated activation of PKC-δ is unlikely, as pretreatment of cells in high glucose with the ZI, which inhibited PKC-ζ activity, did not inhibit PKC-δ activity. In COS-7 cells, H2O2 causes phosphorylation of Tyr502 and Tyr523, which results in prolonged activation of PKC-δ (26). Recent studies by Konishi et al. (27) using phosphorylation-site specific antibodies and mass spectrometric analyses have shown that H2O2-induced PKC-δ activation requires phosphorylation of Tyr311 and is not accompanied by membrane translocation. Mesangial cells cultured in 30 mM glucose for as little as 1 h generate H2O2, as detected by dichlorofluorescein (13), but it is not known whether this is associated with PKC isozyme tyrosine phosphorylation and activation.
The observations that 24-h high glucose-stimulated, PKC-ζ-dependent F-actin disassembly was reversed by ZI, which also reversed cell size and response to ET-1, suggest that the smaller mesangial cell planar area is the result of F-actin disassembly. The mechanism underlying PKC-ζ-mediated disassembly is not known. PKC-ζ can directly associate with the actin cytoskeleton in fibroblasts (12). In NIH-3T3 fibroblasts, atypical PKC-ζ and -λ are reported to mediate Cdc42-mediated stress fiber disassembly (6) and have also been implicated in regulating Ras-mediated stress fiber disassembly (51). In mesangial cells, high glucose for 48 h does not affect the expression of Ras but increases membrane associated Ras activity (56). It is not known whether high glucose-induced Ras activation results in increased PKC-ζ activity and F-actin disassembly. The recent findings of Cortes et al. (7) that perfused glomeruli from 9-mo-old STZ-diabetic rats display F-actin disassembly and loss of stress fibers strongly suggest that the F-actin disassembly we have observed in cultured mesangial cells in high glucose mimic the in vivo findings.
In summary, this study identifies the importance of PKC-ζ in the mediation of high glucose-induced loss of mesangial cell responsiveness to ET-1. Loss of the contractile response to ET-1 is not due to reduced Ca2+ signaling, phosphorylation of MLC20, or altered DAG-sensitive PKC isozyme activation but may be related to PKC-ζ-dependent F-actin disassembly. This cytoskeletal disorganization alters the mesangial cell phenotype and may account for the early effects of high glucose-induced cellular dysfunction.
The authors thank Hong Hua for technical assistance with confocal microscopy. We also thank Dr. C. S. Packer (Indiana University, Indianapolis, IN) and Dr. Yasuharu Sasaki (Asahi Chemical, Ltd., Shizuoka, Japan) for MLC20 and anti-phospho-Ser19-MLC20 antibodies, respectively.
This work was jointly funded by the Juvenile Diabetes Foundation International and Canadian Institutes of Health Research.
Address for reprint requests and other correspondence: C. I. Whiteside, Medical Sciences Bldg., Rm. 7302, 1 King's College Cir., University of Toronto, Toronto, Ontario, Canada M5S 1A8 (E-mail:catharine.whiteside{at}utoronto.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published August 15, 2001; 10.1152/ajprenal.00055.2001
- Copyright © 2002 the American Physiological Society


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Superfund Site: PETERSEN SAND & GRAVEL
Superfund Site Profile
The 120-acre Petersen Sand & Gravel site is located north of Libertyville in Lake County, Illinois. Raymond Petersen purchased land in 1952 to mine sand and gravel. The company later dumped non-hazardous wastes, solvents and paint wastes in buried drums at the site between 1955 and 1958. The Illinois Environmental Protection Agency inspected the site in 1971 and ordered it to close because of illegal dumping. The dumping contaminated groundwater, surface water and sediments with volatile organic compounds (VOCs), heavy metals, polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). Following cleanup, the Lake County Forest Preserve District (LCFPD) converted the quarry into a 115-acre recreational lake, which opened to the public in 2001.


text: RT Web Page DB /z-wcorg/ DS http://worldcat.org ID 55982726 LA English UL http://www.clir.org/pubs/reports/pub126/lavoie.html T1 Of mice and memory : economically sustainable preservation for the twenty-first century A1 Lavoie, Brian F., Council on Library and Information Resources., YR 2004 SN 193232609X AB From an economic perspective, many of the obstacles to preserving the scholarly and cultural record in the twenty-first century seem quite familiar. In the broadest sense, they can be distilled into the venerable economic problem of matching scarce means to competing ends. But the scale and scope of the current preservation challenge suggest the need to reexamine the mechanisms by which resources are channeled to preservation activities. Fundamental to the development of a new economic infrastructure for preservation is the recognition of an increasingly diverse set of decision makers associated with the preservation process; an understanding of the complex relationships that might arise between the need to preserve, the willingness to preserve, and the right to preserve; and a reevaluation of how to organize preservation resources to meet preservation objectives in economical ways. By engaging all stakeholders in the preservation process, ensuring that appropriate incentives to preserve emerge, and organizing preservation activities in ways that leverage resources and maximize efficiency, significant progress will be made toward preventing the twenty-first century equivalents of mice from eating around the edges of society's memory.


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Welcome to the ineedhits Search Engine Marketing blog, where we share the latest search engine and online marketing news, releases, industry trends and great DIY tips and advice.
Google has launched a new feature in its search engine results pages that will give users never before shared insights into its ranking process.
The "search insight" boxes will highlight whether geography, recent searches or the users' web history have been used to determine the rankings.
"As we continue to refine our search algorithms to deliver more relevant results, we strive to be as open as possible about how we use data to improve your search experience," shared Rachel Garb, a Google product manager.
Given Google's deliberate attempts to be more transparent, this could be viewed as a significant move in the right direction. That said, Google aren't quite ready to disclose the 12 secret "herbs and spices" that control the core of their ranking algorithm.
Rachel Grab went on to say: "This new feature doesn't change anything at all about how you search on Google and the results you get, it just gives you more of a behind-the-scenes look at how we customize your search experience."
As the new insights will be derived from users previous searches and web history, I'm fairly sure this new feature will be reserved for users who are logged into their Google accounts when searching.
I wonder if Google will permit users to change the search results by switching off filters and customization. If you're previous search history has no bearing on the current search, the new feature might create a less relevant search experience.
What do you think of the new search insights? Would you like to be able to switch the filters on and off to improve the relevance? share your thoughts via the comments below.
Switch the filters off? Definitely!
I share my computer and my interests certainly have no bearing on my husband's searches.
By Pat - August 5, 2008
Not sure if this is a good idea for Google's sake. I think a lot of marketers will be using this as a process for split testing with SERP manipulation. It will be interesting to see what happens.
By Gary Pollock - August 22, 2008
i think this may be a good thing!
By Tejas Vather - August 24, 2008
i have found the only thing that works for us is ineedhits when it comes to good google ranking. thanks ineedhits.
By Trisal - August 27, 2008
Keep up to date with the latest from our blogs.
Google Finally Gives Wikipedia a Run for It's Money!
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TSR Version of Lipton Diet Green Tea With Citrus by Todd Wilbur
Recipe by ColCadsMom
From www.TopSecretRecipes.com I LOVE Lipton Diet Green Tea with Citrus. I searched for a copycat recipe, and posted it here: Diet " Lipton " Green Tea With Citrus by Todd Wilbur. But I couldn't find the exact ingredients, so I modified what I could find and found that, although it doesn't taste exactly the same, it does have a similar taste and is equally delicious. The cost per serving is much less when you make it yourself, so I don't feel as badly about drinking as much of it as I do! I hope you'll try either this recipe, or the original one that was free on the Top Secret Web Site.
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- Cooking Time is steeping time.


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Imports$8.53 billion (2011 est.)
Exports$12.59 billion (2011 est.)
Inflation rate (consumer prices)13.5% (2011 est.)
Unemployment rate1% (2011 est.)
GDP - per capita (PPP)$3,300 (2011 est.)
National product real growth rate8.2% (2010 est.)
Internet users4.689 million (2009)
Telephones - mobile cellular20.952 million (2009)
Population growth rate0.94% (2012 est.)
Population28,394,180 (July 2012 est.)
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Historical background
Russia conquered the territory of present-day Uzbekistan in the late 19th century.
Stiff resistance to the Red Army after the Bolshevik Revolution was eventually suppressed and a socialist republic established in 1924.
During the Soviet era, intensive production of "white gold" (cotton) and grain led to overuse of agrochemicals and the depletion of water supplies, which have left the land degraded and the Aral Sea and certain rivers half dry....
Economic overview
Uzbekistan is a dry, landlocked country; 11% of the land is intensely cultivated, in irrigated river valleys.
More than 60% of the population lives in densely populated rural communities.
Export of hydrocarbons, including natural gas and petroleum, provided about 40% of foreign exchange earnings in 2009....
The Economist Intelligence Unit
This report contains detailed forecasts and analysis for Uzbekistan, including trade and investment analysis, economic forecasts, political risk assessments and demographic trend analysis. Each ISA Country ...
Uzbekistan: In-depth PEST insights Summary This PEST country analysis report on Uzbekistan provides a holistic view of the country, with insightful analysis of current and future issues, supplemented with ...
Oxford Economics
After a GDP rise of 7.8% in 2016, we forecast the pace of growth will slow to 6.3% this year, but the new president's push for infrastructure investment and trade expansion will keep growth above 6% in ...
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Need a Cholesterol Test in Alexandria, VA? Simply order online 24/7 and get a fast, affordable Cholesterol Test at a lab near you (Find More Locations):
What is a Cholesterol Test ?
A Cholesterol Test checks the cholesterol level through a lab test. People in Alexandria, VA order the Cholesterol Test for general screening or ongoing tracking.
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Our Cholesterol Test prices include the doctor's order that you need to get tested in Alexandria, VA, all lab fees and taxes, and a pdf copy of your results. For current prices, please CLICK HERE.
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Viewing 5 posts - 1 through 5 (of 5 total)
Viewing 5 posts - 1 through 5 (of 5 total)
I really like the features of plugPDF, but when I add the dependency plugpdf to my project, I almost takes a minute before I actually can debug/run my project to my device. I'm using Android Studio 1.3.2.
Any advice on how I can make the rendering less time expensive?
Thank for the advice!
Hello Joachim,
Thank you for the inquiry.
Actually we tried to build a project which has a plugpdf project. but build speed isn't slow.
I think that's a problem of android studio. Actually could you refer this two links?
Best Regards,
I also think the problem is related to Android Studio. :-/
I've tried to change a couple of settings but nothing really helped.
Can it be an option to include the libraries in my project immediately – not as dependency project?
Hi Joachim,
Thank you for the reply. Actually if you use all .so file for all ABI, your application will be increase the capacity. Then will take some time when install the application. So, you can to improve the speed by deleting the .so files not needed.
Thank you,
Dr. Plug
I've tried this, but it didn't help that much. I have to wait around one minute to start debug an app.


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Our Motto, Your Health. Your Rights is core to what we want to achieve. We aim to have a positive impact on the health and rights of hard to reach and vulnerable populations through advocacy, community empowerment projects, knowledge management, research and innovative technology.
To become a regional force in improving the health and lives of vulnerable and hard to reach populations by working with our partners to ensure all our programs have a human rights focused approach.
Cost Effectiveness
Zero stigma and Discrimination
Our growth strategy is based on our vision of becoming a regional force: Gateway believes in being visible and active. In doing and not only talking. In improving our own knowledge and skills and building the capacity of others.In forming coalitions and networks. In our values of Zero stigma and discrimination and fighting for equality. In being transparent and accountable. In being culturally and gender sensitive. In collecting evidence. And most of all by living up to our Motto: Your Health.Your Rights.


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Thanks for choosing the Creative Business Planning option. I'll be in touch soon via email with the guide you requested and your 30% off coupon.
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Boys in skirts?
I have looked on many websites to help me with this question, if anyone can help me thank you, but at what age should you let a boy start wearing a skirt? Also should one teach...
Started by Raye on 03/02/2013 in Welcome To Circle Of Moms!!
Last update on 03/02/2013 by Elfrieda


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Promote your brand with University Directory worldwide. Get your code - it's free!
58909 Companies worldwide » Companies in Russian Federation (Russia) » Uralelectro
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Orenburg region
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- "Professor McGonagall was shouting at someone who, by the sound of it, had turned his friend into a badger.'"
- —Card description[src]
The Badgered Boy transfiguration card is the ninety-third card of the Chamber of Secrets Expansion of the Harry Potter Trading Card Game.
This article or section is a stub. You can help by expanding it.


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I couldn't find this with a search.
Is there any sort of documentation on what needs to be enabled for the network map to work fully. I have a basic diagram of what my network looks like, however none of the bandwidth pieces have any data.
I have multiple vlans and they all say 0% out of 1gbps or 0% out of NA
However when I go to inventory and view my core switch i see data under the interfaces tab.
1 Reply
· · ·
Jun 16, 2009 at 10:14 UTC
I think this is connected with the issue I'm having here:


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The ANZ Fontan Registry – how you can help
One of the most significant areas of research HeartKids is currently supporting is the The ANZ Fontan Registry . Many of the leading Surgeons, Cardiologists and researchers in Australia and New Zealand have a huge interest in this project.
The Fontan completion is usually the third in a series of surgical procedures performed on children with some complex congenital heart defects, especially those with single pumping chambers. It was first described in 1971 by Dr Francis Fontan and carries his name.
The operation re-routes blood returning to the lungs and allows it to bypass the heart flowing passively into the lungs. The single functioning ventricle then pumps the oxygenated blood around the body.
The ANZ Fontan Registry is a secure database of information providing researchers and clinicians with valuable data to help improve treatment options and long -term health outcomes for patients. Under the guidance of Yves D'Udekem and team, it is promising to be the largest, and only population based database of its' kind. The information collated can enable discoveries to be made to benefit all Fontan recipients and provide valuable public health perspectives.
In 2012 we were delighted to award one of our first Grants In Aid to Associate Professor D'Udekem to help establish the Registry and we continue to support their incredible and vital work.
The focus on the whole patient, not just the heart, has prompted A/Prof D'Udekem to ask for assistance from Fontan patients. Medicare has a huge amount of information on the health care needs of the population. By completing a form, you allow your information to be used by the Registry team to establish other needs Fontan patients have.
Please see the videos below for more information on how you can help in the important work the team are doing.


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Estimating rural populations without access to electricity in developing countries through night-time light satellite imagery
A lack of access to energy and, in particular, electricity is a less obvious manifestation of poverty but arguably one of the most important. This paper investigates the extent to which electricity access can be investigated using night-time light satellite data and spatially explicit population datasets to compare electricity access between 1990 and 2000. We present here the first satellite derived estimates of rural population without access to electricity in developing countries to draw insights on issues surrounding the delivery of electricity to populations in rural areas. The paper provides additional evidence of the slow progress in expansion of energy access to households in Sub-Saharan Africa and shows how this might be ascribed in part due to the low population densities in rural areas. The fact that this is a continent with some of the lowest per-capita income levels aggravates the intrinsic difficulties associated with making the investments needed to supply electricity in areas with low population density and high dispersion. Clearly, these spatial dimensions of the distributions of the remaining unelectrified populations in the world have an impact on what options are considered the most appropriate in expanding access to these households and the relative attractiveness of decentralized options.
If you experience problems downloading a file, check if you have the proper application to view it first. In case of further problems read the IDEAS help page. Note that these files are not on the IDEAS site. Please be patient as the files may be large.
As the access to this document is restricted, you may want to look for a different version under "Related research" (further below) or search for a different version of it.
Volume (Year): 38 (2010)
Issue (Month): 10 (October)
Pages: 5661-5670
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| Web page: http://www.elsevier.com/locate/enpol
References listed on IDEAS
Please report citation or reference errors to , or , if you are the registered author of the cited work, log in to your RePEc Author Service profile, click on "citations" and make appropriate adjustments.:
- Nouni, M.R. & Mullick, S.C. & Kandpal, T.C., 2008. "Providing electricity access to remote areas in India: An approach towards identifying potential areas for decentralized electricity supply," Renewable and Sustainable Energy Reviews, Elsevier, vol. 12(5), pages 1187-1220, June.
- Pachauri, Shonali & Jiang, Leiwen, 2008. "The household energy transition in India and China," Energy Policy, Elsevier, vol. 36(11), pages 4022-4035, November.
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Ranchi, Aug. 16: In what is widely seen as a retaliatory move, the Assembly is all set to hold a debate on Jharkhand High Courts jurisdiction vis-à-vis the legislature.
The immediate provocation is the constitution of a full bench by the high court to decide the legality of a special Assembly committee, constituted to probe into the affairs of autonomous bodies like the Birsa Agriculture University (BAU). The high court had earlier commented adversely on another Assembly committee constituted to look into Rajendra Institute of Medical Sciences ( RIMS).
The on-going war of attrition shows no sign of abatement with the court issuing notices to the Speaker and the secretary of the Assembly to show cause. The Assembly secretariat has, however, refused to accept the notices. Now the Assembly is all set to debate the issue.
Legislators are vociferous in upholding their right to inquire into any body receiving government-grants, pointing out that the government is accountable to the Assembly, which approves the expenditure made by it. The critics argue that while Assembly committees can inquire into specific areas and submit their reports to the House, it is a grey area whether such committees can interfere in the administration.
Speaker Inder Singh Namdhari said he had received representations from at least 13 legislators, seeking a debate on the judiciarys frequent bids to encroach upon the jurisdiction of the legislature. I will place the letters before the business advisory committee for a decision, he said.
The Speakers attention was drawn to Article 212 of the Constitution, which appears to place a conditional restriction on the high court, which is debarred from questioning the validity of any proceeding in the House only on the ground of any alleged irregularity of procedure. The provision appears to suggest that the high court may look into the validity of proceedings on grounds other than procedural flaws.
The tug-of-war between the legislature and the judiciary is also over the interpretation of House proceedings. The grey area here is whether Assembly committees, which visit institutions outside the Assembly and seek to give directions, can be strictly held to be House proceedings.
But the Speaker cited conventions and spoke of the several occasions when the Bihar Assembly had formed several committees to look into bungling with government funds. The legislators seeking a debate, he pointed out, cut across party lines.
A court examines the constitutionality of a law. But if the high court can question the proceedings of the House, why cant we debate the courts jurisdiction' he asked.


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Back to TestGroup
Tet Source Build Instructions
This document has instructions on how to build the source for tet. (written based on a build on a Mandriva system, some variations expected on different distros)
- bzr is installed, 0.13 or better
- build tools are installed (make, gcc)
- LSB-build tools are installed (lsb-build-base lsb-build-cc, lsb-build-c++)
- Python development packages are installed (need Python.h - lib64python2.5-devel for Mandriva)
- lsb dynamic linker is in place (/%_lib/ld-lsb-%_arch.so.3)
- rpm-build is installed
- my 'pwd' is ~/LSB-work/doc-test
- all build work is done as a "normal" user, not root
- Distribution pre-requisites for running LSB tests are installed (package lsb-test on Mandriva)
Building Steps
Checkout a branch from Bazaar-NG
An initial branch will take some minutes to complete:
bzr branch http://bzr.freestandards.org/lsb/3.1/tet-harness tet-harness
or if you have an existing branch:
cd tet-harness bzr pull
Build the rpm packages
Source is pulled from http://www.opengroup.org/infosrv/TET/TET3/
I found the PYTHONPATH environment variable I had set for tet-devel interfered with the build:
unset PYTHONPATH
cd tet-harness make
You should find something like this at the end of the build:
lsb-tet3-lite-3.7-5.lsb3.src.rpm lsb-tet3-lite-3.7-5.lsb3.x86_64.rpm lsb-tet3-lite-devel-3.7-5.lsb3.x86_64.rpm
Troubleshooting Build Problems
If you find issues in following the above procedure, please feel free to fix it or add notes in this area.
If you happen to have the lsb-python binary on your PATH before the system one, you may see the build fail with something like this:
+ make make[1]: Entering directory `/var/tmp/rpm-build/BUILD/tet3-lite-3.7/contrib/pyth on_api' /opt/lsb/bin/lsbcc -D_POSIX_SOURCE -DTET_LITE -O -fPIC -I/usr/include/appbat -c pytet_wrap.c pytet_wrap.c:13:20: error: Python.h: No such file or directory
Where it should be more like:
+ make make[1]: Entering directory `/var/tmp/rpm-build/BUILD/tet3-lite-3.7/contrib/pyth on_api' /opt/lsb/bin/lsbcc -D_POSIX_SOURCE -DTET_LITE -O -fPIC -I/usr/include/python2.5 -c pytet_wrap.c
Some of the parsing for PYTHONLIB and friends in the spec file seems to fall apart in this scenario. This may be corrected soon.


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Louis Vuitton has covered the entire façade of the Fifth Avenue Maison with Ruben Toledo's artwork for the Louis Vuitton City Guides. In addition the special seasonal display windows, "Home for the Holidays" have been installed evoking the spirit of travel signature to Louis Vuitton, the excitement of exploration and the joy of spending the holiday season with family.
"We are thrilled to have renowned artist, Ruben Toledo working with us again," said Daniel Lalonde, President and CEO of Louis Vuitton North America. "Toledo's sketches and illustrations have been an integral part of our city guides for years and we are delighted that his artwork is the center of our Fifth Avenue Maison display for this holiday season."
Last year Louis Vuitton covered the façade of the Maison with Takashi Murakami's Monogram Multicolor print for the season. This year the store façade display is inspired by the city stamp artwork that Ruben Toledo designed for the Louis Vuitton City Guides for New York, Miami, Los Angeles, Paris, London, Tokyo, Rome and Mumbai. Painter, sculptor, illustrator, columnist and critic for the top fashion magazines, Ruben Toledo has had a long standing relationship with Louis Vuitton sketching each city for the City Guide and illustrating an entire New York City travel scrapbook published by Louis Vuitton.
Conceived by Naoemie Goudal and David Stienhorst, students of the central St. Martin's College of Art and Design in London, "Home for the Holidays" window displays feature authentic flapboards reminiscent of those found in train stations around the world. Departure and arrival cities, seasonal activities, inspired quotes regarding the marvel of travel, and personal sentiments will be featured on each flapboard throughout the duration of the season. Starting November 23rd, Louis Vuitton asks you, "Where will your journey take you?" Submissions are being collected at www.departures.vuitton.com and a selection of answers will be posted on the flapboards at the Fifth Avenue Maison throughout December.
Image via Louis Vuitton


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Meggitt (Booth No. 5189) is unveiling a new smart fuel probe that will give operators greater certainty over their aircraft's current fuel reserves, as well as offer better reliable and easier maintainability.
The company has developed a device that uses time domain reflectometry (TDR) to send fuel data to the cockpit via digital buses, a first for civil aviation, it claims. According to Meggitt Sensing Systems vice president Stuart Parker, the smart probe is a big leap forward from existing fuel probes, which he said are barely more sophisticated than a lavatory sistern float.
"We developed many of the current generation of float and capacitance-type sensors, so we know the limitations of these devices," he explained to NBAA Convention News. "What we found was that operators don't trust conventional fuel level gauging systems, so they don't use them. They fill up to the maximum and take care to track the distances they travel. What's more, many of today's level sensors can be used only with certain fuels and they deliver significant errors when fuels are changed."
Traditional fuel probes can also lead to major maintenance headaches, especially in larger business aircraft, which might have as many as 20 separate probes fitted throughout their fuel storage areas. The new smart probes are completely self-diagnostic, making it much easier to determine which probe is malfunctioning and to complete trouble-shooting.
The TDR sensors can work with any type of aircraft fuel, including jet-A, diesel, avgas or new bio fuels. They can recognize the differences between fuels and alert operators when the wrong fuel has been supplied.
The technology uses the electronic echoes from high-speed pulses fired down "waveguide" rods extending into the fuel tanks to locate the fuel-to-air interface and measure distance via the return time of the pulse. The gauging system includes probe antennas, signal processing and high-speed digital communications and an array of self-diagnostics. Fuel data is relayed to the cockpit avionics via digital buses.
The equipment has already been supplied for several commercial airliner applications, and at least one business aircraft manufacturer has agreed to start using them in its models. According to Meggitt, the new smart probes are easier to calibrate than traditional fuel probes and are also lighter.
"We have overcome a number of challenges to bring this next generation sensor to market, including developing the ultra-fast analog electronics you need to determine differences in echo signals that last for picoseconds, which is around a millionth of a millionth of a second. And we had to make the sensor immune to unwanted environmental influences such as temperature," explained Parker.
Meggitt Sensing Systems also provides advanced condition monitoring sensors for demanding environments such as inside the hot sections of engines, for example on the Rolls-Royce BR725 turbofan for Gulfstream's new G650. The company is providing this engine's ignition system and other Meggitt divisions are supplying pneumatic valves, fire detection equipment, and wheels and brakes for the aircraft.
Meggitt is making substantial investments in extending condition monitoring to comprehensive integrated vehicle health monitoring (IVHM). The aim is to take a more holistic approach that would deliver comprehensive knowledge about an aircraft's condition rather than just data.
In addition to the engine, this approach would analyze the fuel, as well as the structural condition of the airframe, wheels and brakes. Parker said that major manufacturers are now seriously considering this approach. He believes it could be in service in about 10 years and that it would be of particular interest to operators with large fleets.
Meggitt is working with Canada's AeroMechanical Services to improve the ability to report on maintenance issues and other aircraft data during flights. Data can be reported via satellite to ground stations where a secure Web-based server will relay it to operators and/or manufacturers.
The goal with IVHM is to make the technology scalable and affordable for business aviation applications. Separately, the group believes it has now achieved this for environmental control systems for cabins and cockpits, thanks to the incorporation of vapor-cycle technology provided by Keith Products, which the group acquired last year. According to Jim Duncan, president of Meggitt Thermal Systems, the company has been able to reduce the price point for ECS to the extent that it will be affordable as standard equipment on new aircraft, as well as available for retrofit.
Meggitt Thermal Systems is also engaged in R&D work on new electrothermal ice protection systems that are attracting interest from business aircraft OEMs. These will be able to deliver as much heat as is needed to where it is needed in a way that is more energy efficient than existing bleed-air pneumatic systems.


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What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
How to require multiples of 12 items on order formAsked by salamone on July 13, 2016 at 11:56 AM
I want to ensure that when ordering, the customer buys multiples of 12 bottles. How can I set this up? Don't see a calculation or condition for multiples.
firstname.lastname@example.com GBwines Order Form
Thank you.
JotForm Support
What is the next minimum orderable amount of bottles after the 12, is it a 13 or 24 value? The solutions are different depending on your answer.
Multiples of 12. So 12, 24, etc. What would be great would be to make the box only accept multiples of 12 but to have a little hover box that says call the winery if you have questions about this requirement. We can actually squeeze a 13th bottle into the box so they could call to negotiate this, but I want that to be a special option, not for everyone. I want everyone to order in multiples of 12.
JotForm Support
I have configured it on your form. The following actions were applied.
Added two Form Calculation widget fields, first to calculate the amount of ordered bottles.
And the second to divide it by 12.
Added a Text field (you can adjust the text) from Basic Fields.
The last steps is added 'UPDATE / CALCULATE FIELD' condition. To hide the form Submit button and show the Text field if the total amount of bottles / 12 is a number with decimal point (contains dot).
Please check the form.
You are a genius, that works perfectly. Thank you for your assistance.


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|Message view
|« Date » · « Thread »
|« Date » · « Thread »
|Rob Weir <robw...@apache.org>
|Re: [VOTE] Apache OpenOffice Community Graduation Vote
|Mon, 27 Aug 2012 13:38:36 GMT
On Mon, Aug 27, 2012 at 8:59 AM, Jim Jagielski <firstname.lastname@example.com> wrote: > > On Aug 27, 2012, at 8:56 AM, firstname.lastname@example.com wrote: >> >> Yes, that's what end users care about. But it's not sufficient for AOO >> since we are seeking alternative distribution channels. > > What does that mean? Can I grok "alternative distribution channels" > as "more mirrors" or something else? > You probably don't see this on the server yet, but end-user operating systems, both desktop and devices, both at OS level as well as in browsers and with antivirus software, are shifting over to excluding non-signed executable by default. This is equally true of software distributed on CD's, via downloads, or listed in OS-vendor "stores". That is the direction that the industry is going. Any desktop application that ignores this trend will become unusable by most users. Instead of detached digital signatures that Apache releases already carry, the OS vendors expect integrated signatures via code signing. Where I hear the churning is over whether the technological change - code signing rather than detached PGP/GPG signatures -- means anything different from a liability standpoint. One could argue that a signatures merely vouches for authentication, integrity and non-repudiation -- the classic guarantees of a digital signature. But I'm hearing others suggest that the move from one technology to another technology for signing suggests additional guarantees about the content of the signed artifact, above and beyond what the ASF normally offers. But of course, any additional liability is explicitly disclaimed by the Apache License. So given that other Apache projects distribute binaries that are.... 1) approved by the PMC's 2) distributed on Apache mirrors 3) linked to as ASF products by project websites 4) accompanied by PGP/GPG detached signatures ...what additional liability do we believe comes from the technological change from one signature mechanism to another? Or specifically, what liability is added that is not already explicitly disclaimed by ALv2? -Rob > --------------------------------------------------------------------- > To unsubscribe, e-mail: firstname.lastname@example.com > For additional commands, e-mail: firstname.lastname@example.com > --------------------------------------------------------------------- To unsubscribe, e-mail: email@example.com For additional commands, e-mail: email@example.com
|View raw message


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For a 3 star hotel this surpassed all expectations. Cracking location overlooking the sea and close enough to the lively central square but not too close for it to be noisy at night. Hotel and room decor fairly basic and functional but a very large bedroom, which was always immaculately clean each evening when I returned. Room also had a generous sized bathroom and a balcony looking down onto the beach. This is a small family hotel and the staff could not have been more accommodating, friendly and helpful. Breakfast very good; fresh fruit, fresh bread, a good selection of local sausage and ham and unlimited Italian coffee in whatever form I wanted it. I was on a business trip but would not hesitate to take the family (although the slightly low balcony rail might be a bit dodgy for younger kids who like to climb on things). Two slight criticisms: firstly, a lick of paint in the foyer, some more modern fixtures, a few pictures etc, would quickly give this place the feel of a 4 or 5 star. The only other drawback was that the air conditioning is controlled centrally and it had been switched off at this time of year. Only an irritation for one night when the weather was uncharacteristically hot and humid for late September.
- Reservation Options:
- TripAdvisor is proud to partner with Booking.com, Priceline, Hotels.com, Orbitz, Expedia, Travelocity, TripOnline SA, Hotwire, Cheap Tickets and Agoda so you can book your Hotel Byron reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.


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JACKSONVILLE, Fla. -- Richard Collier participated in the pregame coin toss ceremony at Jacksonville Municipal Stadium before the Jacksonville Jaguars's loss to the Indianapolis Colts on Thursday, an experience he said gave him chills.
Wednesday, Collier returned to the stadium, which also houses team headquarters, for the first time since he was shot 14 times on Sept. 2 while sitting in a car in an apartment parking lot. His left leg was amputated below the knee and he is paralyzed below the waist as a result of the wounds.
"Believe it or not, this is the happiest I've been my whole life," he said. "I've learned a lot, I've grown from this situation and I've surprised myself how I've come out of it, so I feel real good... Once I got here, man, I just felt normal. I was like, 'I'm back at home.' Everybody showed me love and it was good."
Wednesday he visited with the team for the first time since the shooting, encouraging players to enjoy life. Before he rolled out to midfield in a motorized wheelchair, fans chanted his last name after he appeared on the stadium's video screens.
"I never had my name chanted before, so that was real good," he said.
Collier left a rehabilitation center and went home last week but said he's still had plenty of physical therapy and that the people he works with are always "stretching me out."
Among the messages he took in was one from Jaguar quarterbacks coach Mike Shula.
"[He] told me his daughters prayed for me every night," Collier said. " ...I'll be around. I'm a Jag for life. I'll always come around and support the team."
Tyrone Romaro Hartsfield has been charged with attempted murder in Collier's shooting. He pleaded not guilty but remains jailed without bond. Authorities believe Hartsfield retaliated against Collier for an altercation they had earlier this year at a nightclub.
Paul Kuharsky covers the NFL for ESPN.com.