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Name of the Beach: Thurlestone Sands
City nearest to that beach: Kingsbridge
The state, country, or island where the beach is: Devon, England
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Port Vila, Vanuatu (PressExposure) January 07, 2010 -- China has made public what is said to be the fastest rail link in the world, AFP recently reported. A super high-speed bullet train, which is capable of reaching speeds of 400 kilometres per hour, now links the modern cities of Guangzhou and Wuhan.
The official Chinese news agency, Xinhua, reported that the ten and a half hour trip of 1,069 kilometres had been reduced to just three and a half hours.
The new link is aimed at ultimately joining Guangzhou, a business focal point in southern China near Hong Kong, with the capital Beijing. The project, which began in 2005, is part of a high-speed network of 42 lines that will be finished by 2012.
Zhang Shuguang, head of the transport bureau at the railways ministry advised Xinhua "The train can go 394.2 kilometres per hour; it's the fastest train in operation in the world".
By comparison, the average for high-speed trains in Japan was 243 kilometres per hour while in France it was 277 kilometres per hour, declared Xu Fangliang, general engineer in charge of designing the link. However, Franceâs TVG has the capacity of travelling at 515 kph, though it normally runs at around 300 kph.
In December 2003 the JR-Maglev MLX01, Japanese built train accomplished speeds of 581 kph. However, the Maglev line it needed to run on was only 30km long. The train arrived its destination in less than five minutes.
Some intercity flights in Europe have been cancelled because of the competition from high-speed rail travel. Nearly 90% of travelers choose to take the train between London and Paris. Flights between Paris and Brussels have been permanently cancelled.
"If you are travelling less than 15000 km, taking the train wins", said Rail Europe CEO Pierre-Stephane Austi. Railway stations are being redesigned as bustling centres in their own right. "They are modern, bigger, more beautiful, offering centres of real shopping and leisure".
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Riding your bike is cool. Uphill, downhill, it's all cool. That's why the Norco City Glide 2 Auto gives you options. The 2-speed automatic shifting hub picks up on the moments when you're almost spun out ripping through traffic, and the moments when you're pedaling squares up the largest hill in… [more]
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More Articles
- Do Retired People Pay Less on Property Taxes?
- How to Receive Temporary Social Security Benefits
- Do Married Couples Pay More or Less in Payroll or Social Security Taxes?
- Taxes Payable vs. Payroll Taxes
- How to File a Quarterly Federal Tax Return
- Does Social Security Withholding Figure Into My Tax Return?
When it comes to payroll taxes, the Internal Revenue Service generally treats churches as just another employer, particularly with respect to lay employees who handle a church's secular affairs such as building upkeep or bookkeeping. But there are a couple of unique tax wrinkles when applying payroll taxes on the clergy.
Lay Employees
In general, churches must withhold income tax and Social Security and Medicare taxes from full- or part-time employees other than their clergy, and provide a Form W-2 at the end of the year giving total compensation and taxes withheld. Churches must provide a Form 1099-MISC to self-employed persons who performed work for the church. A church's lay employees and self-employed workers must file tax returns. They are not exempt from taxes just because their wages are paid by a church.
Fringe Benefits
Fringe benefits such as health insurance, group life insurance or disability coverage a church provides to its employees are nontaxable to the employees. This is the same tax treatment generally accorded to fringe benefits provided to workers by non-church employers. The nontaxable status of fringe benefits applies to all church employees, whether clergy or lay employee.
The tax situation for the clergy is more complicated. Ministers have a dual tax status. Ministers are considered employees who must pay income taxes on their salary, but they are exempt from mandatory income tax withholding. A minister can elect voluntary withholding or make quarterly estimated income tax payments. Any minister housing allowance is subtracted from his pay for income tax purposes.
Self-Employment Tax
While ministers are employees for income tax purposes, they are considered self-employed for purposes of Social Security and Medicare taxes. That means a church does not withhold these taxes from the minister's salary. But the minister must pay self-employment tax in lieu of Social Security and Medicare taxes. The minister's housing allowance is added back to his pay when figuring his self-employment tax.
References (3)
Photo Credits
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Toyota of Puyallup
Review for Toyota of Puyallup
JULIA R C. on 07/15/2014
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All of my experiences with them have been great. They are quick and on top of it, which I really appreciate. They are always helpful and I love that there are free loaners. Their service is top notch! Everyone is very friendly.
Vehicle: TOYOTA 4RUNNER
Category: Service
Service Date: 07/14/2014
Review Created: 07/15/2014 02:23 PM


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The present study investigated the effects of diabetes and high glucose on GHRH receptor (GHRH-R) mRNA and protein levels in the pituitary of diabetic rats 2, 21, and 60 days post-streptozotocin (post-STZ) administration. Two days post-STZ, the 2.5-kb GHRH-R mRNA transcript was increased. Twenty-one days post-STZ, both the 2.5- and 4-kb transcripts and a 72-kDa 125I-GHRH-GHRH-R complex were elevated. Sixty days post-STZ, the 4-kb transcript remained increased and the 45-kDa 125I-GHRH-GHRH-R complex (functional receptor) was decreased. Hypothalamic GHRH mRNA and serum total IGF-I levels were reduced at all three time points. To better understand the role of high glucose on GHRH-R regulation, time-course effects of 33 compared with 6 mM d-glucose (DG) were examined in cultured anterior pituitary cells from 2-mo-old healthy rats. Membrane lipoperoxidation was present in 33 mM DG, and GHRH-R mRNA levels were diminished after 24 h, Fluo-GHRH internalization was marginal after 16–24 h, and GHRH-induced cAMP levels were decreased after 24 and 48 h. Altogether, these results indicate that the increase of the 2.5-kb GHRH-R mRNA transcript in vivo could be a consequence of a decrease of hypothalamic GHRH mRNA levels in STZ rats. Since it does not affect primarily functional GHRH-R levels, the initial diminution of circulating IGF-I levels could result from a decreased GHRH-R stimulation by GHRH. Thus, the effect of glucotoxicity would be related to a decrease of functional GHRH-R protein, as observed in rats 60 days post-STZ and in cultured pituitary cells from healthy rats exposed to a high-glucose environment.
- insulin-like growth factor I
- oxidative stress
- hypothalamus
- cyclic adenosine monophosphate
specific binding of hypothalamic growth hormone-releasing hormone (GHRH) to the anterior pituitary somatotroph GHRH receptor (GHRH-R) (1, 24, 30, 48) induces intracellular cAMP production (3), leading to GH secretion (2, 54), synthesis (2), and proliferation (4). In addition to protein kinase A (10, 58), protein kinase C (10), and mitogen-activated protein kinase (44, 59), pathways can be activated by GHRH. GHRH-R, a member of the subfamily B-III of G protein-coupled receptors, has been cloned in the anterior pituitary of several mammalian species (14, 17, 18, 19, 26, 30, 52). The presence of ≈2.5- and ≈4-kb GHRH-R mRNA transcripts has been reported (26, 30) in the rat and mouse pituitary. Although the short and most abundant transcript generates the functional 423-amino acid GHRH-R (30, 33), the structure of the 4-kb transcript remains to be elucidated. Correlative evidence (15) suggests that an increased ratio of 4/2.5-kb transcript levels may reflect a shift of high- to low-affinity GHRH binding sites. Chemical cross-linking with 125I-GHRH(1–44)NH2 identified the presence of specific ≈27- and ≈42-kDa (16) or ≈28-, ≈47-, and ≈65-kDa-labeled entities in the rat pituitary (6). The 47-kDa entity corresponds to the functional GHRH-R (30). The lowest molecular weight entity has been proposed to represent a degradation product of the 47-kDa entity, and the highest a glycosylated or dimeric form of GHRH-R (6, 7). The presence of GHRH and GHRH-R mRNAs has also been demonstrated in extrapituitary rat tissues (29), and the Henle's loop of the renal medulla contains the highest GHRH-R mRNA levels (5). Pituitary and renal GHRH-R mRNA levels are regulated in a complex manner according to physiological and hormonal status (5, 15, 20, 21, 23, 32, 34, 37, 42, 51).
Changes occurring in chronic pathologies, such as diabetes, induce disturbances of the somatotroph axis. In spontaneous diabetic rats and in the streptozotocin (STZ)-induced diabetic rat (STZ rat), a widely used model of type 1 diabetes, pulsatile GH secretion and GHRH-induced GH release are decreased (35, 38, 53, 55), and declines of pituitary GH and hypothalamic GHRH mRNA levels are observed (8). In addition, hypothalamic GHRH content rises 2–4 days post-STZ administration, returns to normal after 7 days, and declines thereafter (25). In these rats, pituitary GH content, serum GH, and insulin-like growth factor I (IGF-I) levels decreased over time (25). Other studies have shown a greater in vivo (11) or in vitro (49) sensitivity of GHRH to induce GH secretion in STZ rats. These discrepancies may originate from variations in the duration and severity of diabetes as well as STZ dosage and route of administration.
Studies in STZ rats have provided important information in identifying specific dysfunctions of the GHRH-GH-IGF-I axis. However, the time course of changes in both GHRH-R mRNA and protein levels has not yet been investigated. Therefore, the aims of the present study were to assess the dynamics of these changes in the anterior pituitary of 2-mo-old male rats 2, 21, and 60 days post-STZ administration and to determine whether or not glucotoxicity exerts direct effects on GHRH-R levels and functionality using cultured anterior pituitary cells from 2-mo-old healthy male rats.
Animal handling and STZ administration.
Two-mo-old male Sprague Dawley rats (Charles River Canada, St. Constant, QC, Canada; Tables 1 and 2) were kept in temperature- (22°C), humidity- (65%), and lighting-controlled (12:12-h light-dark cycle, lights on at 0700) rooms. They had free access to standard rat chow (2018-Teklad Global, 18% protein rodent diet) and water. They were either housed individually in metabolic cages for the duration of the short-term studies (2 and 21 days) or maintained in metabolic cages for the first 2 wk of the long-term study (60 days) and 1 day/wk subsequently. The rats were randomly assigned to either control or diabetic groups. Body weight (BW), food and water intakes, and urine volume were assessed before STZ administration to ensure that both groups were identical.
Diabetes was induced by a single intraperitoneal injection of STZ (Sigma-Aldrich Canada, Oakville, ON, Canada) freshly solubilized in 0.5 ml of citrate buffer, pH 4.5 (Sigma-Aldrich Canada) (8). A dosage of 100 and 65 mg STZ/kg BW was administered for the short- and long-term studies, respectively, in agreement with the protocol of Busiguina et al. (8). They were chosen to allow the correlation of our results with the published data and because serum glucose levels can be normalized in these conditions, upon insulin treatment. This intraperitoneal dose range has also been shown to cause no general anterior pituitary or renal toxicity (8, 12), whereas intravenous administration of 100–200 mg/kg BW STZ results in a GH secretory defect (27). Control rats were injected with 0.5 ml of citrate buffer. In the short-term studies, BW, food and water intakes, and urine volume were recorded every day. In the long-term study, they were assessed every 3 days of the first 2 wk and once/wk thereafter. Urine glucose and ketones were analyzed on 24-h samples with Chemstrip 10 (Roche Diagnostics, Laval, QC, Canada). Diabetes was defined by the appearance of glucosuria (≥1,000 mg/dl) and polyuria (≈70 ml/24 h) within 24 or 24–72 h in the short- and long-term studies, respectively.
Tissue handling.
The rats were killed by rapid decapitation 2, 21, or 60 days post-STZ administration or citrate buffer alone, in a block design fashion, between 0830 and 1130. Trunk blood was collected and blood glucose analyzed with a Glucometer Elite (Bayer Diagnostics, Toronto, ON, Canada), and tissues were rapidly dissected out. Macroscopic evaluation of the anterior pituitaries from STZ rats revealed an ≈50% reduction in size 21 and 60 days post-STZ administration compared with controls. For Northern blotting, anterior pituitaries and livers were snap-frozen in liquid nitrogen and stored at −80°C until RNA extraction (Table 1). For cross-linking, anterior pituitaries were rinsed and homogenized as previously described (Table 2) (6, 7). For cell culture, anterior pituitaries from 2-mo-old healthy male Sprague Dawley rats were cut into four to six pieces and rinsed in 25 mM HEPES collecting buffer (57). The protocol was approved by the Animal Care Committee of the Centre Hospitalier de l'Université de Montréal Research Center, in compliance with guidelines of the Canadian Council on Animal Care.
Isolation and culture of anterior pituitary cells.
Cells were isolated by enzymatic digestion (57), centrifuged (800 g, 4 min, 4°C), and washed three times in DMEM supplemented with 6 mM d-glucose (DG), 10% fetal bovine serum, 1% penicillin [50 U/ml streptomycin (1.25 μg/ml); Invitrogen Canada, Burlington, ON, Canada], and 0.1% amphotericin (Sigma-Aldrich Canada). They were cultured for 16 h in this medium and subsequently for 1, 4, 24, or 48 h in new medium containing 6 or 33 mM d- or l-glucose (37°C, 95% air-5% CO2; ≈1 × 106 cells/ml). The culture media were changed after 24 h. The experimental concentrations of glucose did not vary significantly over time (10 ± 2), as determined by the hexokinase assay (Sigma-Aldrich Canada). In some internalization experiments, cells were cultured for 4 h in 6 mM DG and 16 h in the medium containing 6 or 33 mM d- or l-glucose and detached by gentle manual scraping. They were centrifuged (800 g, 5 min, 4°C), washed 3 times in cold Tris-acetate buffer containing 50 mM sucrose, and suspended in the same buffer at a concentration of 0.5–0.8 × 106 cells/ml (57). Cell viability, assessed by the trypan blue exclusion method, was ≥91% after harvesting.
Detection of apoptosis and necrosis in cultured anterior pituitary cells.
Cells were plated on sterile glass coverslips in multiwell dishes at a density of ≈1 × 106 cells/ml culture medium, containing 6 mM DG, and cultured for 16 h. They were subsequently incubated for 4, 24, or 48 h in new medium containing 6 or 33 mM DG followed by a 20-min incubation at 4°C with 100 μl of YO-PRO-1 or propidium iodide solution to label apoptotic and necrotic cells, respectively (Vybrant Apoptosis Assay Kit No. 4; Molecular Probes, Eugene, OR). Cells were kept on ice in the dark for immediate fluorescence microscopy analysis with a Nikon Eclipse TE600 microscope (×20 objective) equipped with a Coolsnap camera (×0.45), a Nikon super high-pressure mercury lamp, and filters for excitation/emission of green (485/520 nm), red (595/660 nm), and blue fluorescence (360/460 nm) (Nikon Canada, Montreal, QC, Canada). Fluorescence microscopy image acquisition was performed on ≈240 individual cells from four independent experiments for each condition. Data were analyzed with MetaMorph 4.6 software (Universal Imaging; Canberra-Packard Canada, Mississauga, ON, Canada). The results were expressed as percentages, compared with the total number of cells, assessed in each condition by phase contrast microscopy.
Detection of membrane lipoperoxidation in cultured anterior pituitary cells.
Cells were plated as described above and cultured in 6 mM DG for 16 h. They were subsequently incubated for 1, 4, 24, or 48 h in medium containing 6 or 33 mM DG and then in medium containing 1 μM 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-S-indacene-3-undecanoic acid (C11-BODIPY 581/591; Molecular Probes) in a final concentration of 0.5% vol/vol DMSO for 30 min at 37°C (41). Incubations were also performed in medium containing 6 or 33 mM DG and 2.5 mM of the antioxidant Trolox (water-soluble analog of vitamin E) or 0.5 mM of the prooxidant cumene hydroperoxide (CHP) to determine the minimal and maximal level of oxidation in the cells. After being washed twice in cold PBS, the cells were mounted on glass slides and kept on ice in the dark for immediate fluorescence microscopy analysis. Ratios of green (presence of lipoperoxidation)/red fluorescence (absence of lipoperoxidation) (40) were determined by fluorescence imaging. The intra-assay coefficient of variation was ≤15% in all experiments. The results were expressed as percentages of the relative ratio of fluorescence intensities/cell.
Analysis of GHRH-R mRNA transcript levels in the anterior pituitary by Northern blotting.
Total RNA was extracted from the anterior pituitary of control and STZ rats with TRIzol (Invitrogen Canada). Northern blotting was performed on 12 μg of total RNA samples, as described previously (5), with minor modifications {overnight hybridization at 65°C in fresh Robbins solution with 10 × 106 counts·min [32P]RPR64−1·ml−1, membranes washed in standard saline citrate (SSC), pH 7.0 (2× SSC/0.1% SDS, 23°C, 2 × 10 min; 1× SSC-0.1% SDS, 65°C, 1–2 × 10 min; 0.5× SSC-0.1% SDS, 65°C, 1–2 × 10 min)}. Stripped membranes were rehybridized with rat GAPDH and 28S rRNA probes (American Type Culture Collection, Rockville, MD). Amounts of GHRH-R mRNA were normalized in each lane with GAPDH, after assessment of the stability of GAPDH mRNA compared with 28S rRNA, in our experimental conditions. Specificity of the [32P]RPR64 cDNA probe and linearity of the reaction were verified in each experiment's (5) GHRH-R mRNA transcripts, and GAPDH mRNA and 28S rRNA levels were quantified by densitometry using an IS1000 digital imaging system (Alpha InfoTech, Montreal, QC, Canada). The intra-assay coefficient of variation of normalized GHRH-R mRNA signals was ≤10% in all experiments. The results were expressed as percentages of relative densities to those of control groups, using a fixed amount of total RNA as well as total RNA from each anterior pituitary.
Analysis of GHRH mRNA levels in the hypothalamus by real-time RT-PCR.
Total RNA from the hypothalamus of control and STZ rats was extracted with TRIzol and resuspended in nuclease-free water (Ambion, Austin, TX). Quality control of RNA samples was assessed in a 2100 Bioanalyzer using the RNA 6000 Nano LabChip kit (Agilent Technologies, Mississauga, ON, Canada). The RNA integrity number of all samples was >9.0. Reverse transcription (RT) of 2 μg total RNA was performed with the SuperScript II RT kit and oligo(dT)12–18 primers according to the manufacturer's protocol (Invitrogen Canada), including RNase H treatment. Rat GHRH and GAPDH (internal control) mRNA levels were determined by real-time PCR in separate tubes at 1:50 (GHRH) and 1:100 (GAPDH) dilution of the RT product and reagents from the Quantitect SYBR Green PCR kit. Reactions were performed in triplicate (final volume 25 μl), in the presence of 300 nM sense and antisense primers, in a Rotor Gene 3000 real-time thermal cycler (Montreal Biotech, Montreal, QC, Canada). No template and no amplification controls were included in each experiment to confirm the specificity of the reactions. The parameters included a single cycle at 95°C for 15 min followed by 45 cycles at 94°C for 15 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, and a melting step from 72 to 99°C (ramping at 1°C/s). Specificity of the PCR products [GHRH forward: 5′-CATGCAGACGCCATCTTCAC-3′; GHRH reverse: 5′-AACCTGGATCTTTGTTCCTG G-3′ (nt 286-410, Genbank NM_031577); GAPDH forward: 5′-AGGGCTGCCTTCTCTTGTGAC-3′; GAPDH reverse: 5′-TGGGTAGAATCATACTGGAACATGTAG-3′ (nt 82-182, Genbank M17701)] was established by melting curve analysis and electrophoresis on 2% agarose gel containing 0.5 μg/ml of ethidium bromide, with a 100-bp molecular weight standard (Invitrogen Canada). The results were analyzed with the Rotor-Gene application software. A five-point standard curve was performed for each gene tested, using 1:5 serial dilutions (1:5 to 1:3,125) of hypothalamus total RNA from 2-mo-old healthy male rats. Hypothalamic GHRH mRNA relative levels were obtained in STZ rats by comparing with those of control rats, according to the Pfaffl (43) equation and using GAPDH as reference gene. The intra-assay coefficient of variation of GHRH and GAPDH cycle threshold (CT) values was ≤2.5% in all experiments.
Analysis of GHRH-R mRNA levels in cultured anterior pituitary cells by real-time RT-PCR.
Total RNA was extracted with TRIzol, and the samples were resuspended in nuclease-free water. Quality control of the RNA samples was assessed as above. The RNA integrity number of all samples was ≥9.8. RT of 2 μg total RNA and real-time PCR were also performed as in Analysis of GHRH mRNA levels in the hypothalamus by real-time RT-PCR, except that annealing was at 52°C for 30 s. Specificity of the PCR products [GHRH-R forward: 5′-CTGCTGTCTTCCAGGGTGAT-3′; GHRH-R reverse: 5′-TAGGAGATGTGGAGGCCAAC-3′ (nt 578-736, Genbank NM_012850); GAPDH forward: 5′-GGGTGTGAACCACGAGAAAT-3′; GAPDH reverse: 5′-ACTGTGGTCATGAGCCCT TC-3′ (nt 1242-1376, Genbank NM_017008)] was determined also as in Analysis of GHRH mRNA levels in the hypothalamus by real-time RT-PCR. A standard curve was performed for each gene tested using anterior pituitary total RNA from 2-mo-old male rats. Quantification of GHRH-R mRNA relative levels in cells cultured with 33 mM DG compared with those of cells cultured with 6 mM DG was performed as above. The intra-assay coefficient of variation of GHRH-R and GAPDH CT values was ≤2.5% in all experiments.
Analysis of GHRH-GHRH-R cross-linked complexes.
[125I-Tyr10] human (h)GHRH(1-44)NH2 (125I-GHRH, 2,000 Ci/mmol; Amersham Biosciences, Baie d'Urfé, QC, Canada) binding was carried out at equilibrium (23°C, 60 min) on rat anterior pituitary homogenates from STZ and control rats (250 μl, ≈375 μg of protein) in 50 mM Tris-acetate (pH 7.4) containing 5 mM MgCl2, 5 mM EDTA, 1 mM diprotin, 100 μM leupeptin (Sigma-Aldrich Canada), 0.42% BSA, and ≈175 pM 125I-GHRH in a final volume of 1.5 ml (6, 7). Some experiments were performed to compare cross-linking patterns with 100 and 500 pM 125I-GHRH in anterior pituitaries from 2-mo-old healthy rats. Nonspecific binding was determined in the presence of 1 μM rat (r)GHRH (1-29)NH2 (synthesized in our laboratory) (13). Incubation was stopped by centrifugation (12,000 g, 5 min, 4°C), and the pellets were resuspended in 25 mM HEPES (pH 8.0) containing 5 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Sigma-Aldrich Canada) (6, 7). Incubation (23°C, 30 min) was stopped in 50 mM glycine (7, 8). The final pellets were solubilized in 75 μl of Laemmli buffer (22) and processed for SDS-12% PAGE, blotting, and autoradiography (6, 7). Precision Plus protein standards (Bio-Rad) were run in each gel. 125I-GHRH-GHRH-R complex was quantified using an IS1000 digital imaging system. The intra-assay coefficient of variation was ≤10% in all experiments. The results were expressed in percentage of specific densities (specific binding: total − nonspecific) relative to those of controls, using a fixed amount of protein.
Deglycosylation of GHRH-GHRH-R cross-linked complexes.
125I-GHRH-GHRH-R complex was deglycosylated using a glycoprotein deglycosylation kit (Calbiochem, San Diego, CA) containing N-glycosidase F, endo-α-N-acetylgalactosaminidase, α2–3,6,8,9-neuraminidase, β1,4-galactosidase, and β-N-acetylglucosaminidase according to the manufacturer's protocol. Pellets with 200 μg of protein from rat anterior pituitary homogenates, resuspended in 30 μl pico-pure water, were used. In each experiment, as a negative control the tissue preparation was submitted to the deglycosylation protocol without enzymes. A standard of glycosylated proteins (phosphorylase B, BSA, aldolase, triose phosphate isomerase, trypsin inhibitor, lysozyme; all supplied with the kit) was used as positive control and detected with Ponceau red. The reactions were stopped on ice by acetone precipitation and centrifugation (12,000 g, 5 min, 4°C). Pellets were solubilized in Laemmli buffer and processed for SDS-PAGE.
Internalization studies with Fluo-GHRH in cultured anterior pituitary cells.
Cells were incubated in Tris-acetate buffer containing 50 mM sucrose and 10 nM of the fluorescent GHRH agonist [Nα-5-carboxyfluoresceinyl-d-Ala2,Ala8,Ala15,Lys22]hGHRH (1-29)NH2 (Fluo-GHRH) (58) at 4°C for 40 min. Nonspecific binding was determined in the presence of 1 μM rGHRH(1-29)NH2. In all experiments, the final incubation volume with Fluo-GHRH was 200 μl/assay point. After removal of the supernatant, the cells were washed once in cold Tris-sucrose buffer and either prepared for immediate visualization or warmed at 37°C for 90 min to allow Fluo-GHRH internalization. This reaction was stopped by washing in cold Tris-sucrose buffer and rapid cooling on ice (57). Cells cultured in petri dishes were cytocentrifuged onto glass slides, and coverslips were apposed for microscopic visualization (57), and those cultured on glass coverslips in multiwell dishes were mounted onto glass slides. Slides were kept on ice in the dark for immediate fluorescence microscopy analysis. Total fluorescence was quantified using the pseudo color/segmented histogram function of MetaMorph, and the results were expressed in intensity units (57).
Determination of immunoreactive cAMP levels in cultured anterior pituitary cells.
Anterior pituitary cells were cultured in multiwell plates (≈1.2 × 105 cells·well−1·150 μl−1) in 6 mM DG for 16 h and subsequently for 4, 24, or 48 h in new medium containing 6 or 33 mM DG. At the end of the culture period, the cells were washed twice with DMEM-0.2% BSA and equilibrated 30 min with this medium before a 10-min stimulation (37°C) with rGHRH(1-29)NH2 (0.1–100 nM) and 1 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich Canada). Basal levels were determined in DMEM-0.2% BSA-1 mM 3-isobutyl-1-methylxanthine alone, which was used for GHRH serial dilutions. Forskolin (10 μM, <0.01% vol/vol DMSO; Sigma-Aldrich Canada) was the positive control in each experiment. Reactions were stopped by adding the lysis buffer supplied with the enzyme immunoassay kit. Total cAMP was measured using the nonacetylation procedure of the cAMP Direct Biotrak enzyme immunoassay kit (Amersham Biosciences). Optical densities were measured at 450 nm in a Bio-Rad model 3550 microplate reader. The intra-assay coefficient of variation was ≤10% in all experiments. Total basal and GHRH-stimulated levels of immunoreactive cAMP were expressed in picomoles per well (36). Net GHRH-induced cAMP levels (minus basal cAMP levels) were expressed as percentages of relative levels compared with those obtained in the presence of 10 nM rGHRH for each culture period.
Serum IGF-I immunoassay.
Total IGF-I serum levels were determined with a commercial RIA kit (R&D Systems, Minneapolis, MN). All samples from each group were analyzed in duplicate in a single assay. The intra-assay coefficient of variation was 5.6%, and the lower sensitivity limit was 3.5 pg/ml.
Statistical analysis.
The results were expressed as means ± SE. Data from experimental (diabetes, deglycosylation, radioligand, and glucose concentrations) and control conditions were compared by unpaired Student's t-test, with Welch correction, when variances were significantly different. Changes in the level of lipoperoxidation over time in cells cultured with 33 mM DG as well as the combined effects of glucose concentration and Trolox or CHP on cell membrane lipoperoxidation were analyzed by ANOVA, followed by Dunnett's multiple comparison test.
Characteristics of STZ rats.
As shown in Tables 1 and 2, BW loss, hyperglycemia (>400 mg/dl), hyperglucosuria (>2,000 mg/dl), and polyuria characterized both short- and long-term diabetic status. At 2 days post-STZ administration, a few rats exhibited ketoneuria (40 mg/dl); however, at 21 and 60 days post-STZ, urinary ketone levels exceeded 60 mg/dl in all diabetic rats (data not shown). At 2, 21, and 60 days post-STZ, the BW of diabetic rats was decreased to 85–89 (P < 0.01 to P < 0.001), 67–70 (P < 0.001), and 60–73% (P < 0.001), respectively, of age-matched controls, and their right kidney wet weight/100 g BW was increased to 119–121, 132–151, and 170–171% (P < 0.01 to P < 0.001), respectively, of age-matched controls. Food intake per 24 h was unchanged at 2 days post-STZ but increased to 157–184% (P < 0.001) by 21 and 60 days postdiabetes induction compared with age-matched controls. Water intake and urine volume/24 h increased three to six times (P < 0.001) and to seven to 20 (P < 0.001) times, respectively, by 2–60 days post-STZ. No deaths occurred over the 2- to 60-day experimentation periods.
GHRH mRNA levels in the hypothalamus of STZ rats.
As shown in Fig. 1, hypothalamic GHRH mRNA levels decreased to 60, 49, and 67% of age-matched controls (P < 0.05 to P < 0.01) by 2, 21, and 60 days post-STZ, respectively.
GHRH-R mRNA transcript levels in the anterior pituitary of STZ rats.
GHRH-R mRNA levels were analyzed by Northern blotting, allowing the quantification of 2.5- and 4-kb transcripts. Both were present in the gland of control and diabetic rats 2, 21, and 60 days post-STZ, as reported previously in healthy young rats (15, 30). Levels of GHRH-R mRNA transcripts showed several changes according to the length and severity of diabetes (Fig. 2). When expressed per 12 μg total RNA, 2.5-kb GHRH-R mRNA transcript levels were increased to 140 and 150% of age-matched controls by 2 (Fig. 2, A and B) and 21 (Fig. 2, A and C) days post-STZ, respectively (P < 0.05). They returned to control levels by 60 days post-STZ (Fig. 2, A and D), indicating a decrease compared with shorter periods of diabetes. A significant increase of 4-kb transcript levels was observed at 21 and 60 days post-STZ compared with age-matched controls [280 (P < 0.001) and 170% (P < 0.05), respectively; Fig. 2, A, C, and D]. Combined levels of 2.5- and 4-kb transcripts were increased to 150 and 180% (P < 0.01) by 2 and 21 days post-STZ and returned to normal by 60 days postdiabetes induction (P < 0.01). Finally, the 4/2.5-kb ratio of transcripts was not modified by 2 days post-STZ but increased to 220 (P < 0.001) and 170% (P < 0.05) at 21 and 60 days post-STZ, respectively. Anterior pituitary total RNA content did not change by 2 days post-STZ but decreased to 40–43% of controls (P < 0.001) at 21 and 60 days postdiabetes, confirming our visual observation of a diminished anterior pituitary size at these time points. Therefore, when GHRH-R mRNA levels were expressed as a function of total RNA content per anterior pituitary, differences were seen mainly at the 2.5-kb GHRH-R mRNA transcript levels, which decreased to 63 and 52% (P < 0.05) at 21 and 60 days post-STZ, respectively, compared with age-matched controls (data not shown).
125I-GHRH-GHRH-R complex levels in the anterior pituitary of STZ rats.
The presence of 72 ± 2-, 45 ± 1-, and 24 ± 1-kDa 125I-GHRH-labeled complexes was observed in the gland of both control and diabetic rats. Specific binding, determined in the presence of 1 μM rGHRH(1–29)NH2, represented 60–75% of total binding for the 72- and 45-kDa entities and 40–55% of total binding for the 24-kDa entity. The level of complexes showed a number of changes during the evolution of diabetes (Fig. 3, A–D). The density of the 72-kDa complex increased to 260% (P < 0.05) by 21 days post-STZ (Fig. 3C). This increase did not remain significant (160%) at 60 days post-STZ (Fig. 3D) compared with age-matched controls. The density of the 45-kDa complex decreased to 45% of that in age-matched controls (P < 0.05) by 60 days post-STZ (Fig. 3, A and D). The level of the 24-kDa complex did not change in the course of diabetes. A low level of specific ≈150-kDa complex (3–7% of total densities) was also detected, but it did not change with diabetes. When complex levels were expressed as a function of total protein content per anterior pituitary, differences were seen mainly at the level of the 45-kDa complex. A decrease to 40–45% of age-matched control levels was observed at 21 and 60 days post-STZ (P < 0.05; data not shown).
Serum IGF-I levels in STZ rats.
Immunoreactive IGF-I levels declined to 71, 24, and 63% of age-matched controls 2 [STZ: 1,004 ± 99 ng/ml; controls: 1,410 ± 95 ng/ml (P < 0.05)], 21 [STZ: 539 ± 54 ng/ml; controls: 2,231 ± 135 (P < 0.001)], and 60 days [STZ: 1,339 ± 139 ng/ml; controls: 2,098 ± 146 (P < 0.01)] post-STZ, respectively.
Deglycosylated 125I-GHRH-GHRH-R complex levels in the anterior pituitary of normal rats.
Using a panel of enzymes, including N-glycosidase F, endo-α-N-acetylgalactosaminidase, and α2–3,6,8,9-neuraminidase, deglycosylation experiments in tissue preparations from 2-mo-old healthy rats showed that the low abundance of 153 ± 5-kDa 125I-GHRH-GHRH-R-specific complexes was reduced to 105 ± 5 kDa (P < 0.05; Fig. 4). No other change was seen in the profile of 125I-GHRH-GHRH-R cross-linked complexes in our experimental conditions, even when optimal enzyme concentrations were increased by twofold.
Effect of radioligand concentration on the density of 125I-GHRH-GHRH-R complexes in the anterior pituitary of normal rats.
Our preliminary data in baby hamster kidney cells, stably transfected with human or rat GHRH-R, revealed significant increases of ≈100 and ≈150-kDa-specific 125I-GHRH-GHRH-R complexes when 500 pM 125I-GHRH was used instead of 100 pM in cross-linking experiments (56). This approach was taken to induce changes of rat anterior pituitary 125I-GHRH-GHRH-R complexes, compatible with receptor dimerization/oligomerization. Cross-linking experiments, performed with 100 or 500 pM 125I-GHRH, revealed changes in the relative intensity of 25- and 69-kDa-specific complexes compared with the 48-kDa complex (Fig. 5, A and B). A decrease in the intensity of the 25 ± 1-kDa band to 63% (P < 0.02) and an increase in the density of the 69 ± 2-kDa band to 160% (P < 0.01) were observed without any significant change in the level of other molecular weight entities, suggesting a dimerization of the functional GHRH-R with a truncated form.
Effect of glucose concentration on apoptosis, necrosis, and membrane lipoperoxidation in cultured anterior pituitary cells from normal rats.
The percentages of fluorescent apoptotic cells after a 16- to 24-h culture period were 13 ± 1 and 9 ± 4% with 6 and 33 mM DG, respectively, whereas those of necrotic cells were 12 ± 1 and 10 ± 1%, respectively. The percentages of fluorescent apoptotic cells, after a 48-h culture period, were 5 ± 3 and 6 ± 1% with 6 and 33 mM DG, respectively, and that of necrotic cells was 6 ± 1% with both glucose concentrations. No significant variations occurred at any time when the cells were cultured in either 6 or 33 mM DG. The dynamics of anterior pituitary cell membrane lipoperoxidation were examined after a 1-, 4-, 24-, and 48-h culture in 6 or 33 mM DG. As shown in Fig. 6, A and C, top, the ratio of fluorescence intensity per cell was maximal after 1 h. It increased to 380% in cells cultured in 33 mM DG compared with those cultured in 6 mM (P < 0.001). This level of lipoperoxidation was not different from that seen in cells exposed to 33 mM DG for 24 h in the presence of 0.5 mM CHP [Fig. 6, B and C, bottom (d)]. It remained elevated to 250, 180, and 240%, when cells were exposed to 33 mM DG for 4, 24, and 48 h (P < 0.01), respectively (Fig. 6, A and C, top). The lipoperoxidation level was lower at 24 (P < 0.001) and 48 h (P < 0.01) compared with 1 and 4 h. Lipoperoxidation was prevented in anterior pituitary cells cultured in 33 mM DG for 24 h, when 2.5 mM Trolox was present [Fig. 6, B and C, bottom (b)].
Effect of glucose concentration on GHRH-R mRNA levels in cultured anterior pituitary cells from normal rats.
GHRH-R mRNA levels were quantified by real-time RT-PCR, allowing for high sensitivity with small amounts of cells. Normalized GHRH-R mRNA levels were not changed in cells incubated with 33 mM DG for 4 h (Fig. 7) compared with GHRH-R mRNA levels in cells incubated with 6 mM DG. They were decreased to 77 and 91% after 24- and 48-h incubation in the presence of 33 mM DG and returned to control levels after 72-h incubation (Fig. 7).
Effect of glucose concentration on Fluo-GHRH internalization in cultured anterior pituitary cells from normal rats.
After 40-min incubation at 4°C with Fluo-GHRH, a low level of fluorescence was observed at the surface of cells incubated with 6 or 33 mM DG (Fig. 8B). Because Fluo-GHRH internalization is time and temperature dependent, the incubation time of 90 min at 37°C was selected as optimal on the basis of our previous results in freshly dispersed anterior pituitary cells (57). When pituitary cells are warmed at 37°C for 90 min, a punctate pattern appears with moderate to high levels of fluorescence due to internalization and concentration of Fluo-GHRH in organelles (57). In pituitary cells cultured for 16–24 h in the presence of 6 mM DG, these changes were correlated with an increase in total fluorescence to 160% (P < 0.001) and Fluo-GHRH internalization [Fig. 8, A (a) and B]. In cells cultured in the presence of 33 mM DG, no significant increase of fluorescence and internalization was observed [Fig. 8A (b)], and the total fluorescence level was not different from that with 6 mM DG at 4°C (Fig. 8B).
Effect of glucose concentration on GHRH-induced cAMP levels in cultured anterior pituitary cells from normal rat.
Neither basal nor forskolin-stimulated levels of immunoreactive cAMP were altered after 4-, 24-, and 48-h culture in the presence of 33 mM DG compared with those in 6 mM DG (data not shown). The rGHRH(1-29)NH2-induced response was not affected after a 4-h culture in the presence of 33 mM DG (Fig. 9A). However, after 24-h culture in 33 mM DG, cAMP levels induced by 0.1, 1, 10, and 100 nM rGHRH(1-29)NH2 decreased to 47 (P < 0.05), 63 (P < 0.05), 59 (P < 0.01), and 55% (P < 0.01), respectively (Fig. 9B), compared with cells incubated with 6 mM DG. Similarly, after 48-h culture in 33 mM DG, cAMP levels induced by 1, 10, and 100 nM rGHRH(1-29)NH2 declined to 67 (P < 0.05), 63 (P < 0.01), and 48% (P < 0.01) of control values, respectively (Fig. 9C).
Type 1 diabetes, induced in rats by a single injection or multiple injections of STZ, has been employed in numerous studies to identify the in vivo effects of uncontrolled hyperglycemic states. Several perturbations of the GHRH-GH-IGF-I somatotroph axis have been identified in STZ rats. However, the regulation of anterior pituitary GHRH-R at both the mRNA and protein levels has not been investigated in relation to the duration and severity of diabetes and hyperglycemia. Moreover, these changes have not been compared with those obtained after the direct exposure of normal anterior pituitary cells to high glucose.
The hypothalamo-pituitary GHRH-GHRH-R system plays an important role in stimulating GH secretion and synthesis (2, 54) as well as somatotroph proliferation (4). In the pituitary of STZ rats, GHRH-R mRNA levels were submitted to a time-dependent regulation. At 2 days postdiabetes induction, a time at which no change in daily food intake was observed and a slight decrease of BW was present, hypothalamic GHRH mRNA levels were already greatly decreased. In parallel, the 2.5-kb anterior pituitary GHRH-R mRNA transcript, coding for the functional GHRH-R, was increased, but pituitary total RNA content was unchanged. Levels of the 45-kDa 125I-GHRH-GHRH-R complex, representing the functional GHRH-R (≈47 kDa in other studies) (6, 7) and that of other 125I-GHRH-GHRH-R complexes, were also unchanged. Since hypothalamic GHRH content has been reported to increase significantly only during the early stage of diabetes (2–4 days post-STZ) (26), enhanced GHRH tone could initially contribute to upregulate the 2.5-kb pituitary GHRH-R mRNA transcript (17). Alternatively, since hypothalamic functions are perturbed in STZ rats (20, 47), increased GHRH content could result from a decreased capacity of hypothalamic neurons to release the peptide, leading to compensatory upregulation of the 2.5-kb transcript. Because serum total IGF-I was already decreased, by 2-days post-STZ it may be proposed to result primarily from dampening of pituitary GHRH-R activation by GHRH.
Twenty-one days post-STZ, BW and food intakes were substantially modified and hypothalamic GHRH mRNA levels decreased, as reported previously by Busiguina et al. (8). Under the same conditions of STZ administration, we observed that pituitary GHRH-R mRNA transcripts and the 4/2.5-kb ratio were increased, suggesting a decrease in the number of high-affinity GHRH binding sites, as reported in aged rats (15). Although the structure of the 4-kb transcript is currently unknown, our preliminary evidence (56) suggests that it could represent a 2.5-kb GHRH-R mRNA transcript bound to an adapter protein, thus preventing optimal translation. In parallel, an increase in the 72-kDa 125I-GHRH-GHRH-R complex level could result from an elevated level of 24/45-kDa dimer formation in such a perturbed cellular environment. In the present study, we have shown that dimerization of GHRH-R entities can occur in the anterior pituitary cells of healthy rats. In addition, recent data from McElvaine and Mayo (31), showing a dimerization of the human GHRH-R with a COOH-terminal truncated variant, strengthen this hypothesis. Therefore, in the anterior pituitary of diabetic rats, dimerization of GHRH-R species could participate in the decreased responsiveness to GHRH, leading to a reduction of anterior pituitary size and a substantial decline of total IGF-I circulating levels.
Sixty days post-STZ, BW and food intakes remained altered, and hypothalamic GHRH mRNA levels were still reduced, but a trend toward a lower reduction than at 21 days post-STZ was observed. The pituitary 2.5-kb GHRH-R mRNA transcript was decreased and the 4-kb transcript remained elevated, resulting in an increase of the 4/2.5-kb ratio and a substantial diminution of the 45-kDa 125I-GHRH-GHRH-R complex. Interestingly, total IGF-I circulating levels were partly restored compared with those measured at 21 days post-STZ.
Our present results in a rat model of STZ-induced diabetes and weight loss do not entirely support the hypothesis that changes in anterior pituitary GHRH-R arise exclusively from nonfasting weight loss. Rather, it suggests that GHRH-R mRNA transcripts and functional protein are regulated in a complex manner in models of STZ-diabetic rats and that hyperglycemia plays a role in addition to that of other metabolic and hormonal factors. Apart from GHRH, a number of hormonal regulators could participate in the changes of GHRH-R levels in the pituitary. Thyroid hormones and glucocorticoids have been shown to increase GHRH-R mRNA levels (23, 32, 34, 37, 42, 51). In STZ rats, circulating levels of glucocorticoids and thyroid hormones are increased (9) and decreased (39, 46), respectively. Since physiological levels of both are required for optimal GHRH-R expression in the pituitary (23, 32, 34, 37, 42, 51), changes in their circulating levels with diabetes could contribute to some of the alterations observed in GHRH-R mRNA levels. However, the dynamics of changes in the concentration of these serum hormones will have to be correlated with binding proteins and nuclear receptors to clearly establish a causal role in pituitary GHRH-R dysregulation. Increased IGF-I (50) and insulin levels (28) have been shown to negatively regulate pituitary GHRH-R mRNA, and a diminution of their circulating levels in STZ rats could also be involved. However, Kim et al. (20) have proposed that the catabolic effect of weight loss, but not insulinopenia, reduced circulating levels of IGF-I, or hyperglycemia, is the primary regulator of pituitary GHRH-R mRNA levels in STZ-induced diabetes.
To more directly study the effect of a high-glucose environment on pituitary GHRH-R and exclude the confounding actions of regulators of the receptor (GHRH, steroid and thyroid hormones, IGF-I, insulin) present in vivo in STZ rats, studies in cultured rat anterior pituitary cells were performed. Exposure to a high glucose level did not promote the necrosis or apoptosis of anterior pituitary cells but increased oxidative stress, as measured by membrane lipoperoxidation. In these conditions, GHRH-R mRNA levels were decreased particularly after 24-h culture, Fluo-GHRH internalization was drastically reduced, and GHRH-induced cAMP production was diminished, indicating lowered levels of functional GHRH-R as seen in diabetic rats 60 days post-STZ. This could be related in part to a transient modification of GHRH-R mRNA levels and concurrent changes in functional receptor levels due to dimerization and/or other structural alterations occurring in a glucotoxic/prooxidant environment. These in vitro results are consistent with those of Renier and Serri (45) showing that GHRH-induced GH release was decreased after exposure of cultured normal rat anterior pituitary cells to high glucose (22 mM, 72 h).
Our study is the first report to show a direct effect of high glucose on GHRH-R, indicating that it can contribute to a regulation of GHRH-R expression and functionality and contribute to decrease GHRH sensitivity in somatotrophs, leading to a decline of GH secretion in vitro (45) and in vivo (35, 38, 53, 55). These results may reconcile some of the divergent reports on in vivo and in vitro somatotroph sensitivity to GHRH from a large variety of single time point evaluations, ranging from 2- to 60-day post-STZ administration and exclusively analyzing GHRH-R mRNA but not functional protein levels. The molecular and cellular events by which glucotoxicity-induced oxidative stress affects pituitary GHRH-R will now deserve investigation. Interestingly, in primary-cultured rabbit proximal tubule cells, the angiotensin type 1 receptor, another member of the G protein-coupled receptor family, is downregulated (mRNA and protein levels) by high glucose (25 mM) via a PKC-oxidative stress-transforming growth factor-β1 signaling cascade (41). A similar mechanism could be involved in GHRH-R regulation, affecting mRNA levels first and transiently. Alternatively, oxidative stress could damage GHRH-R protein structure and concurrently regulate GHRH-R mRNA levels. Therefore, this in vitro model should help to identify the mechanisms by which high glucose affects GHRH-R and develop strategies to counteract glucotoxicity.
In summary, when in vivo and in vitro data are analyzed together, it might be proposed that increase of the 2.5-kb pituitary GHRH-R mRNA transcript, seen 2 and 21 days post-STZ, could be related to the decrease of hypothalamic GHRH mRNA levels, leading to a reduced GHRH secretion. However, since it does not affect primarily functional GHRH-R levels, the initial diminution of circulating IGF-I could be considered, at least in part, as the result of a decreased GHRH-R stimulation by hypothalamic GHRH. Thus, the effect of glucotoxicity in somatotrophs would be related to a decrease of functional GHRH-R protein, such as that observed in diabetic rats 60 days post-STZ and in cultured anterior pituitary cells from healthy rats exposed to a high-glucose environment.
This research was supported by the Canadian Institutes of Health Research. K. Bédard was the recipient of a studentship from Centre hospitalier de l'Université de Montréal Research Center and presently holds a Fonds de la recherche en Santé du Québec (FRSQ) doctoral studentship. J. Strecko was the recipient of a studentship from the FRSQ and a summer studentship from Association Diabète Québec. P. Gaudreau was chercheur-boursier national from the FRSQ.
We are grateful to Dr. Kelly E. Mayo (Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL) for providing us with RPR64 rGHRH-R cDNA.
↵* These two authors contributed equally as first authors.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2008 by American Physiological Society


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National Training and Achievement Conference
Thank you for joining us in New Orleans for our 2017 National Training and Achievement Conference.
Attendees from all over the U.S. enjoyed in-depth educational track, panel discussions and networking events to exchange ideas with industry experts and colleagues. We look forward to seeing you at our 2018 conference in Indianapolis, Indiana, May 7-9.
Check back here for more information as we get closer to the 2018 conference or click below to view a gallery of images from this year's conference.
Conference Date:
May 1 - 3, 2017
Conference Location:
Hilton New Orleans Riverside
New Orleans, LA
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We believe that critical thinking is essential for learning. Given that there are seldom definite "correct" answers in the Social Sciences, our girls develop the independent intellectual skills which enable them to engage with complex real world issues and problems. The girls develop a curiosity for learning and an awareness of themselves and others and of how people function in widely varying groups, organisations and societies.
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The Social Sciences allow pupils opportunities to develop both their written and oral powers of expression. Emphasis is placed on discussions, debates and presentations, as well as more formal, written essays; these are essential skills for success at university and beyond.
The girls are encouraged to make the most of the many extra-curricular opportunities available both within and outside of school; there is an active Model United Nations Society, a Young Enterprise Group and a high-level debating club, amongst others.


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[UPDATE] Congratulation to George Kopp for being the winner of this giveaway! George, I sent you a Facebook message. Please just reply with your shipping address and I will send out the prize right away.
Thank you to everyone for your support and participation! Please go here to see the current running contest: https://mydrawingtutorials.com/giveaway
Lesson Detail
Step 1 – Draw 1st Impossible Triangle
This impossible star will be made up for 2 impossible triangles, so the first step is to draw the first impossible triangle.
By the way, I made a whole video on how to draw an impossible triangle. If you find yourself wanting more detailed instructions, click here to watch it.
OK, back to the drawing…
First, draw a simple triangle with all the sides being equal.
Next within the triangle you just drew, draw the 3 sides again to form a smaller triangle.
The gap between the larger and smaller triangle will determine the thickness of the shape. To keep the drawing from getting too overcrowded, I recommend keeping this gap relatively thin.
Repeat this process one more time to create a third (and even smaller) triangle.
Now go to each of 3 the diamond shapes at the outer corners of your drawing and put a short line through the middle of each.
Now erase the 3 corner tips.
Next, erase these lines here to complete the impossible triangle.
If you want a more detailed explanations of which lines to erase, check out my "Impossible Triangle" video.
Step 2 – Draw 2nd Impossible Triangle
Alright, we just finished the first impossible triangle. Now, let's draw another one, except this time, we'll flip it upside down.
I'll go about one-third of the way down and draw a horizontal line for the base of the new triangle. One key detail is that this line will interweave with the old triangle. In other words, the line will go under the old triangle and then over it.
And let's draw the other 2 sides of the triangle while still following this over-and-under pattern.
Next, just like last time, we'll draw the sides of the large triangle again to form a smaller triangle. And of course, you'll still want to follow the over-and-under pattern.
Repeat this process one more time to form a third triangle.
Now go to all the places where the second triangle overlaps the first and erase the lines of the first triangle.
You can see how this makes the two triangles look like they are interwoven with each others.
Next, close off the corners of the second triangle and erase the tips.
And lastly, erase these extra lines.
Step 3 – Inking & Shading Impossible Star
And that's it! We have our Impossible Star of David. Let me ink it in so you can see it more clearly.
Now I'll take a Sharpie marker and put a thick border on the outside of the shape to make it pop more.
Now for the shading. I'll take a 2B pencil and put a gradation at the three inner corners of the first triangle like so.
Then I'll put a gradation at the three outer corners.
And I'll repeat the same process for the second triangle.
And lastly, to make the drawing look even more three dimensional, I'll put a shadow in all the areas where the two triangle are overlapping each others.
And that's it, we're all done!


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CodePlexProject Hosting for Open Source Software
Codeplex SVN Bridge for TFS is not very good (while branching and merging specially). It also does not supports various svn properties.
So, the options available (that I can think of) are :
Although I'd work with the project no matter where it is hosted, here's what I think:
I haven't used either github nor bitbucket in projects. I've played around with both though and I think bitbucket UI is much neater, simpler and elegant. Most of the devs on those sites aren't .NET devs from what I can see which is good and bad at the same
time I think. Regarding SCM I have used Mercurial a bit for pet-hello-world projects and it's pretty cool. It even has a builtin http server (hgserve). I haven't used Git so I can't comment a lot on it but from what I read Git is the powerhouse of DVCS but
it's a bit harder to use. So my extremely subjective choice is... mmmm I think that although I don't see a problem with codeplex, If we are going to change hosting I prefer Bitbucket and Mercurial. But that's just my subjectivity talking. In the end what matters
is the result and that we all work as a team to achieve it.
On Codeplex, when you set-up a new project you can choose version control system between SVN (TFS) and Mercurial.
Maybe we can continue with Codeplex and (in the near future / when we fill confortable) ask the codeplex team to migrate our repository to Mercurial (other projects such as Umbraco have done that). If we want to do this, we should test (create a new project
and test the mercurial server) the performance of the branching/merging
What do you think?
PS: I added google code as an option
I think that's great a great idea.
Should we consider source safe? :-)
Don't kill me yet, I was joking hehe
XD Source safe reeeeaaally sucked!!
OK, I'll start contacting the codeplex team to migrate to mercurial in the future (after the next release?).
Way to go buddy! :-)
The migration was done, see the thread at http://nearforums.codeplex.com/discussions/246359 for more info.
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Scholarship Spotlight: Iris-Samuel Rothman Scholarship $10,000
The Iris-Samuel Rothman Scholarship is open to college bound, undergraduate, graduate, and post-graduate male students in all areas of study, who have lost their mother and have either lost their father, ...
Scholarship Shout-Out: Tylenol Scholarship
Since 1879, Tylenol has been providing treatment and support in an effort to bring relief to millions of people around the world as they've faced illness; and for the past 23 ...
The Fountainhead $10,000 Essay Contest
Are you familiar with Ayn Rand's The Fountainhead? If not, read up on it and enter this scholarship essay contest for your shot at $10,000. Ayn Rand was a novelist and ...
Scholarship Spotlight: Akash Kuruvilla Memorial Scholarship, $1,000
The Akash Kuruvilla Memorial Scholarship is available to full- time undergraduate students pursuing post- secondary study. You must submit a 750- word essay answering the question, "What does the phrase 'The ...
Scholarship Spotlight: Video Contest, $5,000 in Prizes
The Swackhamer Video Contest is open to students of all ages. To be eligible for this award, you must submit a video of no more than three minutes on the topic ...
The Gen and Kelly Tanabe Scholarship
Gen and Kelly Tanabe are considered experts when it comes to college planning. They are the award-winning authors of eleven books on admission and scholarships. The Gen and Kelly Tanabe Scholarship ...
Scholarship Spotlight: Edison Scholars Program
The Edison Scholars Program is open to high school students with a GPA of 2.8 or higher. Students must be planning to attend an accredited four-year institution of higher learning and ...
Scholarship Spotlight: Get Ready Scholarship, $500
The APHA Get Ready Scholarship is available to high school seniors, undergraduate, and graduate students. To be eligible for this award you must write an essay between 500 to 750 words ...
Adult Students in Scholastic Transition "ASIST" Scholarship
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College JumpStart Scholarship
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U.S. Bank Scholarship
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Scholarship Spotlight: 2400 Expert National Scholarship
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Healthy Lifestyles Scholarship
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"Hit the Books" Scholarship
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Discus Awards College Scholarship
The Discus Awards College Scholarship is available to high school students planning to attend college. You must choose three areas from the categories on the Web site and provide evidence of ...
Don Kuhn Memorial Scholarship
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MeetMyCollege $5,000 Scholarship
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Scholarship Spotlight: DCPS Urban Education Leaders Internship Program
The DCPS Urban Education Leaders Internship Program (UELIP) is an intensive multi-disciplinary internship program that runs during the academic school year, administered by the Office of the Chancellor. Organizers are recruiting ...
Scholarship Spotlight: Jimmy Rane Foundation Scholarship
The Jimmy Rane Foundation Scholarship is open to high school seniors and current college students in Alabama, Florida, Georgia, South Carolina, Mississippi, Tennessee, Louisiana, Texas, North Carolina, Kentucky, Oklahoma, Arkansas, Iowa, ...


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As usual, the long-awaited retreat began with a deluxe breakfast prepared by the House E-board. Residents spent the morning enjoying their favorite homemade breakfast foods while making introductions and conversation. When it was time to head out, House Vice President Yadirys Collado (CAS '14) finally revealed the retreat activity: pottery painting!
Together, we hopped onto the T, making quite a racket all the way to Arlington. From there, we found ourselves walking along a scenic bike path complete with a dog park, pond and lush greenery – the perfect place to take in the fresh autumn air. After arriving at the pottery place, everyone focused on painting their pieces to perfection while making the occasional chit-chat. Two hours later, we headed back toward BU, recommencing the conversation and laughter. By the time we arrived at those black gates of 191 Bay State Rd., we truly felt closer to one another. Here's looking toward a year filled with friendship, memories, and much more chatter.
"It is good to have an end to journey toward; but it is the journey that matters, in the end." – Ernest Hemingway
– Geraldine Lai, HER House President, COM 2014


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El Salvador: Viva Romero! Viva John Paul II!
A CAFOD delegation has just returned from El Salvador after what director Chris Bain describes as 'the Easter experience' of celebrating the life of Archbishop Oscar Romero. Tens of thousands of people packed the central square of the capital San Salvador on April 2 and turned the 'big day' of events for Romero into a double celebration. With news of the death of Pope Jean Paul II, crowds of young people in brightly coloured Romero T-shirts began shouting: "Viva, Oscar Romero! Viva, John Paul II!" It was the culmination of ten days of prayer, reflection and commemoration for the man who is already a saint to the peoples of Latin America. "Romero is truly resurrected in his people" said Mr Bain. Archbishop Romero - inspirational priest, martyr and hero - was murdered on March 24, 1980, a day after his defiant plea to the military: "I beg you, I beseech you, I order you, in the name of God, stop the repression! The actual day of his death fell on Maundy Thursday this year, but the official celebration came on April 2. CAFOD Latin American specialists Clare Dixon and Sarah Smith-Pearse accompanied Mr Bain to El Salvador. The Bishop of East Anglia Michael Evans, representing the Bishops, Conference, travelled with the group. Following a Mass on Maundy Thursday, a weeklong theology conference took place at the University of Central America (UCA), which sits on the slopes overlooking San Salvador. The university is integral to the mission of Romero. Six of its Jesuit priests and two women were murdered there in 1989 because of their commitment to his mission of social justice. At the conference, speeches by Bishop Kevin Dowling of Rustenberg, South Africa and Father Jean Floribert of the Democratic Republic of Congo evoked the spirit of Romero and roused a crowd that spilled out onto the grass and followed the proceedings on plasma screens. "Before Romero's death, El Salvador was described as 'the crucified country' but in these celebrations of his life, he was a symbol of hope - even to youngsters who were not born at the time of his assassination," said Ms Dixon. After the death of Romero, El Salvador suffered a decade-long civil war that claimed the lives of 60,000 civilians. Today the country remains wracked by violence, because of a failure to invest in the peace. A third of the population has fled and remittances from exiled Salvadorans are a lifeline to family members who remain in the country, said Ms Dixon. On his return, Mr Bain said: "It was important for CAFOD to be in El Salvador because of our close relationship with the church in Central America." "The legacy of Romero is as relevant now - with a whole new range of poverty and justice issues as it was 25 years ago."


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Claim: Warner Bros. will contribute money to the American Red Cross every time you forward an
Status: Not any more.
Example: [Collected on the Internet, 2000]
Warner Bros. would like to honor the real-life heroes of "The Perfect Storm," and we need your help. Every time you forward this
Origins: Finally, after all those bogus messages about how you can help raise money for the treatment of
You started by visiting the Warner Bros. web site for The Perfect Storm web site and clicking one of the
Warner Bros. said they would not be collecting
Last updated: 8 October 2007


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Join author Nancy J. Butler and the loveable Fred on his quest for fiery breath, and help him find out how to no longer be Fred the Fireless Dragon.
©2009 Nancy J. Butler; (P)2009 Tate
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Earth Day Generates Energy-Efficiency Efforts in the Home, With Lutron Leading the Way
Cut Costs, Boost Convenience With Such Smart-Home Devices as Occupancy Sensors, Dimmers
COOPERSBURG, Pa. --Earth Day is here - time to see the light of home energy-efficiency!
The average homeowner might not realize that lighting accounts for an average of 10 percent of his or her annual electric bill1. To celebrate Earth Day and practice saving energy, homeowners can install dimmers and sensors to help truly maximize energy efficiency and save up to 60 percent per year on utilities2.
"No matter what your budget or your needs, you can create a smart room or a smart home using such things as reasonably priced lighting sensors," said Caitlin Helterline, Lutron Sensors product manager. "In addition to the energy savings component, convenience plays a major role in smart homes: Imagine walking into a home carrying bags of groceries and turning on the lights without having to put down, or drop, a thing."
For those homeowners looking for ways to improve energy-efficiency and enhance convenience, all while using an environmentally conscious approach, the environmentalist and actor Ed Begley Jr. and spokesman for Lutron, offers 10 tips to coincide with Earth Day on April 22, 2015:
1. Install motion sensors that turn lights on when you enter a room, stairway or hallway, and off when motion is no longer detected. Forgetting to turn the lights off is a universal problem. Lutron, the inventor of the dimmer and leader in the lighting-control, remote-controlled shading and smart-home industry, offers the reasonably priced Maestro Occupancy Sensor, which automatically turns lights on or off when you enter and leave a space, making sensors a convenient way to save up to 50 percent annually on electricity3. They're ideal for rooms where lights are accidentally left on, such as an office, or rooms that you often enter with your hands full, such as a laundry room or supply closet.
2. Outdoor lighting is a necessity, but using electricity to light your driveway, patio and pathways is not. Install solar-powered outdoor light fixtures - they charge during the day and light up the night without using electricity. Installation is simple; most fixtures have a stake that simply needs to be pushed into the ground.
3. Unplug to halt the Phantom! Electronics and appliances can use juice even if they are turned off, raising your electricity bill by nearly 10 percent annually reports Energy.gov4. Plug gadgets (like phone chargers and gaming units) into power strips or surge protectors, and switch them off when you leave the house.
4. Almost everyone knows that weather stripping is a great way to insulate your home, but if you live in an older house, there's a good chance there might be cracks and it's time to replace windows. While you're at it, consider adding weather stripping to interior doors, especially ones that lead to uninsulated areas like garages, attics and basements.
5. Update to a programmable thermostat which make it easy to set back the temperature when the room is unoccupied, saving you an estimated 10% per year in energy savings5. A smart thermostat costs a little more but over time will save you even more money by learning your habits and keeping up with your schedule.
6. Keep your window blinds open during the day to let sun in and close them as the sun is setting to trap heat in. Interested in adding to your smart home capabilities, automated shades like Lutron's Serena battery powered shades, can do this for you.
7. Installing light dimmers can help lower your electricity needs, saving both money and energy. Lowering the lights by 25 percent can also cut your light energy usage by 20 percent while adding ambiance to any room6. Lutron's Maestro C.L Dimmer & Switch provides a range of designer, easy-to-install lighting and fan controls, sensors and timers through a high-performance switch that can also be adjusted manually to your preferred light setting with a simple tap on the touch rocker. Also available in the eco-dim feature, C.L models use HED technology to ensure reliable performance, and eliminate challenges of dimming CFL and LED bulbs.
8. Another bright idea is to replace old light bulbs with energy-efficient ones. LED light bulbs not only last longer but also use less energy when dimmed. An ENERGY STAR-qualified dimmable LED bulb has long-lasting, eco-friendly benefits and can last up to 25 times longer than traditional incandescent bulbs which minimizes maintenance costs7. Replacing just five frequently used light bulbs with ENERGY STAR models, you can save $75 each year8.
9. Being energy-efficient outside of the home is also important: Use public transportation whenever possible, and if you're going less than 10 miles, ride a bike; it's not only good for the environment, it's also good for you.
10. Eating more vegetables is always a good idea, eating locally grown ones is even better, but the best greens of all are the ones you grow yourself! If you want your garden to really thrive, composting is the only way to go. Get the whole neighborhood involved and plant a community garden.
Although going green may seem tough, small things can make a big difference. Installing sensors and recycling are smart ways to start. For more information on sensors, go to www.lutron.com
About Lutron Electronics (www.lutron.com) Founded in 1961, Lutron Electronics is headquartered in Coopersburg, Pennsylvania, in the heart of the Lehigh Valley. From dimmers for the home, to lighting management systems for entire buildings, the company offers more than 17,000 energy-saving products, sold in more than 100 countries around the world. In the US alone, Lutron products save an estimated 10 billion kWh of electricity, or approximately $1 billion in utility costs per year. The company's early inventions—including the first solid-state dimmer invented by Lutron's founder, Joel Spira—are now at the Smithsonian's National Museum of American History in Washington, DC.
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When I saw the incident between Texas Tech coach Bob Knight and his player, Michael Prince, it did not trouble me, nor did I believe that it crossed the line into the realm of unacceptable behavior. When I spoke to Bob Knight the day after the incident, and inquired as to the substance of what was said, I had no problem with it.
However, while I do not condemn the action, I do not feel that it is the best manner in which to get a point across to a player. I believe there was a better way, especially given how sensitive our society has become in recent years, and I do not feel that there was a need to touch the player on or near the face.
I do not feel that the incident merited such intense scrutiny and criticism by the media. I do not believe that Knight deserved to be so severely criticized for the manner in which he chose to make his point with his player. I believe the issue has been magnified, exaggerated, overstated and overhyped, and that Knight has been treated a distinctly different manner than any other coach would have been in a similar situation.


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- At the end of Leon's B path in Resident Evil 2, a supposedly dead ally throws down a rocket launcher to destroy a monster that's been hounding you the whole game— and it had already been pushed into a giant smelting vat in an attempt to kill it.
- At the end of the B scenario, when the Tyrant reappears outside the train, both characters have their own one-liners as they deliver the finishing blow:
- Leon: "Game over."
- Claire: "You lose, big guy!"
- The fact that Leon—21 years old, pre-government training, and what was to be his first day of work—continues kicking ass and killing monsters through the entire second half of the game with a bullet in his freaking shoulder. Considering why he was late to the massacre in the first place, we can probably add "hungover" to that list, at least in the early hours.
- The last line delivered in Leon B:
Leon: Hey, it's up to us to take out Umbrella. (cue the credits and a badass J-rock tune)
- The extended B scenario endings, in general. After going through both A games, first-time players were probably expecting a cut to the credit roll at the same point as in the ending cutscenes they've already watched. Nope! Cue an unexpected Final Boss battle, followed by the longest and most satisfying finale in the Resident Evil series to date.


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Samsung Portable 9000mAh Battery Charging Pack - Black
$69.99 for handy on-the-go charging!
Easily connects to smartphones, tablets, cameras & more via USB port!
Charge level indicator | Lightweight & compact design!
Please note: This item can only be shipped to a postal address, NOT a P.O box. Please ensure your address details, complete with contact number, are updated in your account.
Phone or tablet out of juice and no outlet available? Not to worry - the Samsung Portable Battery Charging Pack has got you covered! Compatible with almost any mobile device through the convenient USB port, this bad boy boasts an impressive 9000mAh capacity - powerful enough to fully charge an iPhone 5 six times over. Plus, the sleek, compact design means you can pop it into your backpack or handbag and take it with you anywhere.
- Samsung accessory
- Samsung Portable Battery Charging Pack
- Capacity: 9000mAh
- Compatibility: Universal
- Package contents:
- Battery charging pack
- USB to Micro USB cable
- User manual
- Ideal for smartphones, tablets, cameras & more
- LED charge level indicator
- Compact & lightweight design
- Cable length (approx, cm): 20
- Weight: 265g
- Dimensions (approx, mm): 23 x 65 x 94 (D x W x H)
- Input voltage: 5.0V
- Output voltage: 5.2V
- Output current: 1.8A
- Country of origin: China
- 1-Year Limited Manufacturer Warranty - full product warranty details inside the packaging.


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Wishing you all a very happy and blessed (Canadian) Thanksgiving Weekend!
To celebrate giving 25% off entire shop until Monday!
Coupon Code: "GIVETHANKS"
If you follow along on Instagram you'll know the last two days I spent styling and photographing some of my favourite fine art photographs in my old home! Here they all are :) Enjoy! XO
"Without a Care-ousel in the world" (in size 20x30 with 4" mat)
Join Me!


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