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Waterloo research: Lost medieval village holds climate change lessons
Coastal flooding forced medieval villagers to abandon English community 600 years ago
Coastal flooding forced medieval villagers to abandon English community 600 years agoBy Deepa Jolly St. Jerome's University
A Waterloo historian is leading a team of researchers to unearth the remains of a lost medieval village in the south of England, hoping to find lessons about climate change.
Villagers who lived near Herstmonceux, East Sussex abandoned their community 600 years ago when coastal floods, increased rainfall, and changing temperatures threatened their livelihood. Now, Steven Bednarski, a professor in the Department of History, is working with a team of historians, archaeologists and paleontologists to unearth the past.
“Climate change is a pressing global concern. Governments, researchers, and the public benefit from a more sophisticated appreciation of historical climate,” says Bednarski. “By contrasting a modern problem against an historical one, we see more readily how climate affects people.”
As a co-lead on the Herstmonceux Project, Bednarski hopes to offer alternative perspectives on our current climate crisis by revealing how people lived and left the pre-modern settlement of 1250-1440. Archaeological artifacts unearthed at Herstmonceaux will seed Canada’s first museum collection on medieval culture and environment, housed at St. Jerome’s.University, which is affiliated with the University of Waterloo.
“History demonstrates how nature plays an often dominant role in shaping everything from culture to politics and technological development,” says Bednarksi.
The project offers a rare humanities-based contribution to the growing discourse on global climate change, he adds. “While scientists can study climate change as an isolated and measurable phenomenon, a humanities approach underscores how nature, people, and culture are inextricably linked. To halt potentially harmful interactions with nature requires deep changes to culture and lifestyle.”
A recent Social Sciences and Humanities Research Council (SSHRC) grant led by Bednarski and his collaborator, Scott McLean, a history professor at the Bader International Study Centre, Herstmonceux, established a formal partnership between the University of Waterloo, St. Jerome’s University, the Bader International Study Centre, and Queen’s University. The partners share expertise, resources, and students. They provide training in archives, archaeology, conservation, and environment through an international setting.
Students who collaborate on the project become adaptive and competitive on the world stage while finding ways to contribute to the discourse of climate. This week is International Education Week at the University of Waterloo. Find out more about events happening on campus and how you can participate.


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From their ballot box this weekend and next will come the name of the candidate _ picked from among dozens _ that they want their mayor to endorse.
Normally, the choice would be Mayor Olivier Hoffmann's alone, under a right that, at election time, turns small-potato public office-holders into hot properties _ wooed by would-be candidates who need 500 endorsements from elected officials to get onto the April ballot.
But in an inflamed climate of election-time politics, and with fury among opponents of COVID-19 vaccinations increasingly bubbling over into violence directed at elected representatives, Sainte-Anastasie's staunchly apolitical mayor doesn't want to be seen taking sides.
Safer, he figures, to let the 2,000 villagers choose for him.
``I know lots and lots of people in the village, many are my friends, I don't want to create tensions,`` Hoffmann said in a phone interview. ``So no politics.''
``Politics,'' the mayor added, ``often do more harm than good.``
Even in a country with ingrained traditions of violent contestation, where the revolutionaries of 1789 guillotined King Louis XVI and Queen Marie-Antoinette, an upsurge of physical and verbal attacks and online torrents of hatred directed at public officials _ often, now, over COVID-19 policies _ are ringing alarm bells.
Violence hasn't approached the level of the storming of the U.S. Capitol by Donald Trump supporters in 2021. Nor have French lawmakers been killed like their counterparts in Britain. There, the fatal stabbing of a Member of Parliament in October prompted renewed national soul-searching about the safety of elected officials with a proud tradition of readily meeting voters.
Still, there's mounting disquiet in France in the wake of apparent arson attacks in December that targeted a lawmaker and a mayor, both aligned with President Emmanuel Macron, and other violence targeting elected officials as the government steadily increased pressure on the non-vaccinated to get COVID-19 jabs to curb the surge of infections fueled by the omicron variant.
The Interior Ministry recorded a year-on-year increase of 47% in acts of violence directed at elected officials through the first 11 months of 2021, with 162 lawmakers and 605 mayors or their deputies reporting attacks. Lawmakers say death threats have become everyday occurrences. Titled ``decapitation,'' an email received by lawmaker Ludovic Mendes in November read: ``That's how we dealt with tyrants during the French Revolution.``
This month, during protests against France's vaccine pass that bars the unvaccinated from cafes and other venues, about 30 angry people besieged the office of lawmaker Romain Grau, shoving him and yelling furiously.
``Death! We'll get you all!!'' shouted one man who launched a slap at the lawmaker's head. Grau later told broadcaster TF1 that he feared the confrontation would finish ``in a blood bath and a lynching.''
When lawmaker Pascal Bois' garage went up in flames in December, the words, ``Vote no'' and ``It's going to blow!'' were spray-painted on an outside wall, which he took as an intimidation attempt before parliamentary passage of the vaccine pass this month.
The National Assembly president, Richard Ferrand, says more than 540 of the 577 lawmakers have reported threats or verbal and physical attacks.
``France isn't bathed in fire and blood. These are acts of brutal minorities,`` Ferrand told the parliamentary TV channel this week. ``Still, it seems to me that we have ratcheted up a notch, expressing a rage that is new.''
Anti-vaccination sentiment is also dovetailing with residual anger among ``yellow vest'' protesters. Their frequently violent demonstrations against Macron rocked his government before the pandemic. Recent protests against COVID-19 measures have again seen some demonstrators wearing yellow vests.
When Bernard Denis was jolted awake by a loud boom in the middle of the night in December, the mayor of the Normandy village of Saint-Come-du-Mont discovered his cars on fire and the words, ``The mayor supports Macron,`` daubed in black on a wall.
Also written was ``Zemour president'' _ a misspelt apparent reference to presidential candidate Eric Zemmour, a far-right rabble-rouser with repeated hate-speech convictions.
Around 42,000 elected officials are empowered to sponsor a candidate for the presidential race. The bar of 500 endorsements is intended to whittle down the field. Endorsing a candidate doesn't require agreeing with their politics. Some sponsors simply want a politically broad election choice. But because endorsements are public, they're also not without potential consequences.
In Sainte-Anastasie, Hoffmann is keen to participate. But the mayor wants to avoid any risk of villagers turning on him if he decides alone, of them saying: ``'You endorsed him so you support him, you're this and that, you're red, yellow, green, blue, blue-white-and-red' or whatever.``
Hoffmann is instead pledging to endorse their choice, even if the winner of the ad hoc vote he's organizing isn't aligned with his own politics, which he keeps to himself. In the 2017 presidential run-off that Macron won, the village voted by a large majority for the loser, far-right leader Marine Le Pen, who is running again.
Villagers will choose from around 45 would-be candidates, including Macron, who Hoffmann assumes will seek re-election even though the president hasn't yet said so.
And thus, Hoffmann hopes, harmony will reign in what he calls ``the village of my heart.``
``I want to give it, my village, everything I have,`` he said, ``and I don't want politics to encroach on that.``

domain: news


“ Transcend JetFlash T5 - USB flash drive - 4 GB - Hi-Speed USB - white „
Transcend's T5 is incredibly compact and only weighs a mere 2g. Its non-breakable plastic casing is rugged enough to resist bending, stress, vibration, shock, and even full water submersion. The retractable connector design and eye-catching two-tone color schemes with intricate hexagon patterns make the drive easier to handle and use. With its convenient attachment loop, the ultra-light T5 can be used as a mobile phone dongle or a keychain fob. Transcend's T5 USB flash drive is fully High-Speed USB 2.0 compliant.


「Scratch that itch! All eyes on these clever parts for the 86 and BRZ.」User focus? You got it!
Published : 2021/01/14 22:42 Modified : 2021/01/14 22:42
Do NOT make the mistake of thinking that these are simply visual upgrades!
These high-grade parts are a must see for 86 and BRZ owners who place importance on functionality as well as form.
For stylish 2 door coupes like the 86 and BRZ, looks are important, so the existence of aftermarket accents to add a finishing touch should come in handy. YR Advance is one company that boasts a lineup of parts honed for just this purpose – with a twist in the tail.
YR Advance makes several items that make use of materials like carbon and aluminium, but in this article we’ll be focusing on these newly introduced paddle-shifter covers (8,800 yen). The anodized aluminium finish will liven up any cabin, and since they add surface area to the stock shifters it goes without saying that they are a level-up in terms of usability.
Variations are available for not only pre-facelift but also post-facelift models. These come with the same anodized aluminium finish, and are available in blue or red.
And there’s more: YR Advance has been busily releasing new carbon fiber exterior parts. The newest of these is this dry-carbon fender garnish, available for the post-facelift 86. As well as projecting a race-bred image, this is also a highly functional aerodynamic part.
Another popular item is this carbon fiber fuel filler cap cover (11,000 yen). Just stick it on to give your car a look of racing pedigree.
This ABS resin roof fin (24,000 yen) is available for both pre/post facelift models. In matte black, it can be installed with nothing more than double-sided tape.
With variants for the 86, the BRZ and a third, emblemless, version, this carbon fiber trunk garnish is 16,500 yen. Apparently the hole-less version is popular as a simple way to go emblem-free.
The wheels seen here are a YR Advance exclusive model of the Volk Racing TE37SAGA rim. These 18-inchers come with a 9J width and +39 offset for flush fitment on the 86 and BRZ.
The door handle cover (15,000 yen) and door handle protector (11,000 yen) adorning the side of the car are smash hits. You can apply these easily with double-sided tape alone.
Simply slip these clear-coated dry-carbon door mirror covers (16,500 yen) over your stock mirrors to add a touch of style.
A staple of the range are these number plate holders (from 8,000 yen to 12,000 yen). Moving the number plate up (15-30mm on the pre-facelift BRZ, 40-55mm on the pre-facelift 86 and 25-40mm on the latter models of both vehicles), they improve cooling efficiency. Buyers can choose between silver, red, blue or even cherry red accents and stripes.
Installed as easily as a standard tow-hook, both front and rear versions of this one cost 14,000 yen. They are available in 7 standard body colors, with cherry red as an extra addition.
“When it comes to body kits and larger tuning parts, I think the major parts manufacturers have already done most of it to death,” explains YR Advance’s representative, Mr. Yamashita. “So we’re focusing our efforts on fleshing out a range of items for perfecting the more intricate details that larger manufacturers don’t make.”
A look at the demo car reveals that it is decked out in a full ensemble of Varis aerodynamic parts. Mr. Yamashita takes things from there, adding flourishes where needed. The result is an even more satisfying end product.
- Thanks to: YR Advance, 3-3-19 Asahigaoka, Hino City, Tokyo


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Musician/actor/comedian/author (and probably some other things we're forgetting) Steve Martin is set to release his second full-length bluegrass album through Rounder Records on March 15.
The album, Rare Bird Alert, will be a collaboration with bluegrass band the Steep Canyon Rangers, who toured with Martin in 2010. It will also feature vocal appearances from Paul McCartney and the Dixie Chicks.
Martin's previous album, The Crow: New Songs for the Five-String Banjo, won a Grammy Award for best bluegrass album.
"Rare Bird Alert represents an intense, buy joyful period of songwriting and touring," Martin said in a statement. "My love of this music continues."
The new album includes 13 Martin-penned tracks, one of which is -- yes -- a live version of his comedy classic, "King Tut."

domain: artistic


text:
New audit report released
Audit report for Tarrawarra Abbey now available
Who we are
Find out about our purpose, our values and our commitment to keeping children and vulnerable adults safe.
Find out more about how we are helping to maintain a Church that keeps children and vulnerable people safe from abuse.
Report a concern
Report a concern for a child or vulnerable adult. Find out about support services.
Fr Hans Zollner SJ in discussion with CPSL
In June 2019 CPSL was fortunate enough to sit down for a conversation with Fr Hans Zollner SJ on the topic of child safeguarding in international contexts. Fr Hans is President of the Centre for Child Protection and Director and Professor of the Institute of Psychology at the Pontifical Gregorian University, Rome and one of the leading experts on sexual abuse working in the Catholic Church.
National Catholic Safeguarding Standards
The National Catholic Safeguarding Standards set out 10 Standards, providing the framework for Catholic Church entities to build child-safe cultures and to advance the safety of children across the Catholic Church in Australia.
The National Catholic Safeguarding Standards take into account the guidance of the Royal Commission into Institutional Responses to Child Sexual Abuse and the National Principles for Child Safe Organisations adopted by the Council of Australian Governments (Feb 2019).
Committed leadership, governance and culture
Children are safe, informed and participate
Partnering with families, carers and communities
Equity is promoted and diversity is respected
Robust human resource management
Effective complaints management
Ongoing education and training
Safe physical and online environments
Policies and procedures support child safety
Keep up to date
To stay informed about the development of the National Catholic Safeguarding Standards, publishing of reports, training opportunities and more of our work, subscribe to our newsletter.
CPSL NEWS editions in 2019 were full of information and resources as we worked progressively to roll out the National Catholic Safeguarding Standards and the Audit Program, so we thought this first edition for 2020 presented a good opportunity to recap in one edition all of the material and support available to help you develop and embed your safeguarding initiatives and processes.
CPSL CEO Sheree Limbrick explains how CPSL came to be established, the development of the National Catholic Safeguarding Standards, and the four main groupings of the 10 National Catholic Safeguarding Standards.
2019 training attendees from CPSL's NCSS Introductory Session for Leaders discuss what they learnt at the session, the key benefits of attending, and how they will bring what they have learnt back to their organisations.

domain: sociology


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domain: sports


text: Poxvirus virion biogenesis is a complex, multistep process, starting with the formation of crescent-shaped viral membranes, followed by their enclosure of the viral core to form spherical immature virions. at a reduced efficiency. Our results show that VACV-A6 has a two-domain architecture and functions in both crescent formation and its enclosure to form immature virions. While a cognate N area is not needed for crescent development, it is necessary for virion development, recommending that interactions from the N domain with cognate viral proteins may be crucial for purchase WIN 55,212-2 mesylate virion assembly. IMPORTANCE Poxviruses are exclusive among enveloped infections for the reason that they acquire their principal envelope not really through budding from mobile membranes but by developing and increasing crescent membranes. The crescents are extremely uncommon, open-ended membranes, and their origin and biogenesis have perplexed virologists for decades. A group of five viral proteins were recently identified as being essential for crescent formation, including the A6 protein of vaccinia computer virus. It is thus Rabbit Polyclonal to RPL39 important to understand the structure and function of A6 in order to solve the long-standing mystery of poxvirus membrane biogenesis. Here, we established an experimental system that allowed the genetic manipulation of the essential A6L gene. By studying A6 mutant viruses, we found that A6 plays an essential role not only in the formation of crescents but also in their subsequent enclosure to form immature virions. We described the area structures of A6 and recommended that among its two domains cooperates with cognate viral protein. -glucuronidase (GUS) (Fig. 2A). Infections that portrayed GUS had been plaque purified on HeLa-iA6 cells in the current presence of Tet (Fig. 2B). Recombinant infections that acquired GUS instead of the A6 ORF had been isolated and verified by PCR evaluation from the viral genome and Traditional western blotting for A6 (data not really proven). The recombinant infections (described right here as vA6) replicated in HeLa-iA6 cells just in the current presence of Tet, raising the titer a lot more than 50-fold after 48 h of infections (Fig. 2C). Open up in another screen FIG 2 Era and characterization of the A6 deletion mutant of VACV (vA6). (A) Schematic representation from the A6L locus of WT purchase WIN 55,212-2 mesylate VACV WR and vA6. The A6L ORF in WT VACV WR was changed using the GUS ORF governed with the improved VACV H5 promoter (PmH5) through homologous recombination, as well as the causing VACV vA6 mutant purchase WIN 55,212-2 mesylate was isolated from HeLa-iA6 cells. (B) Plaque morphology of vA6 on HeLa-iA6 cells which were harvested in the current presence of Tet. The cells had been stained with X-Gluc (5-bromo-4-chloro-3-indolyl–d-glucuronide) after infections with the infections for 48 h. The initial blue staining is certainly shown in dark. (C) Development curve of vA6 in HeLa-iA6 cells in the existence or lack of Tet. HeLa-iA6 cells had been contaminated with vA6 at an MOI of 0.5 PFU/cell. For infections with Tet, Tet was put into the cell lifestyle moderate 12 h before infections and preserved during infections. After 0, 24, and 48 hpi, the cells had been harvested, and trojan titers under purchase WIN 55,212-2 mesylate both circumstances had been dependant on a purchase WIN 55,212-2 mesylate plaque assay on HeLa-iA6 cells which were harvested with Tet-containing moderate. We used vA6 as the parental computer virus to construct VACVs with numerous mutations in A6 (Fig. 3A). The mutants were generated through homologous recombination with a plasmid made up of the.

domain: medical


text:
N‑Glycosylation plays a role in protein folding of human UGT1A9
著者 Nakajima Miki, Koga Toshihisa, Sakai Haruko, Yamanaka Hiroyuki, Fujiwara Ryoichi, Yokoi Tsuyoshi
page range 1165‑1172
N-Glycosylation plays a role in protein folding of human UGT1A9
Miki Nakajima, Toshihisa Koga, Haruko Sakai, Hiroyuki Yamanaka, Ryoichi Fujiwara, and Tsuyoshi Yokoi
Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920-1192, Japan
To whom all correspondence should be sent:
Miki Nakajima, Ph.D.
Drug Metabolism and Toxicology Faculty of Pharmaceutical Sciences Kanazawa University
Kakuma-machi, Kanazawa 920-1192, Japan Tel / Fax +81-76-234-4407
UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of a variety of xeno/endobiotics. UGTs are type I membrane proteins of the endoplasmic reticulum (ER) with a glycosylated luminal domain. In the present study, we investigated the role of N-glycosylation in the function of human UGT1A9. Mutation analysis at the potential N-glycosylation sites at residues 71, 292, and 344 (from asparagine to glutamine) revealed that all of them were glycosylated, but the extent of glycosylation and/or size of the glycan differed. In comparison with the wild type, these mutants showed decreased enzyme activities in parallel with the extent of the band shift in Western blot analysis. To evaluate the role of glycosylation in the enzyme activity, we produced unglycosylated UGT1A9 by treating HEK293 cells transiently transfected with expression plasmid with tunicamycin. The
unglycosylated UGT1A9 was almost inactive, which was not an indirect effect of ER stress.
To the contrary, the deglycosylated UGT1A9, which was produced by the treatment with Endo H under the non-denaturing condition, showed the same enzyme kinetics as the control.
These results suggest that the glycosylation that occurs during translation is important for the folding of UGT1A9. The thermal stability analysis of the mutated and deglycosylated
UGT1A9 proteins supported the findings. In conclusion, we found that the N-glycosylation has an important role in the folding of UGT1A9.
Keywords: UGT1A9, N-glycosylation, enzyme activity, protein folding
Over half of all proteins in nature are glycosylated, and more than three quarters of them contain N-linked glycan . N-Linked glycosylation occurs at the asparagine (N) residue of the NX(S/T) consensus sequence of a protein, where X is any amino acid except proline. It is estimated that approximately 70-90% of the consensus sequences are glycosylated .
N-Glycosylation is one of the most common and yet complex forms of post-translational modification. It is intricately involved in various cellular processes including protein folding, protein secretion, intracellular trafficking, stability, binding affinity, enzyme activity, and substrate specificity, enabling fine-tuning of a protein’s function .
UDP-Glucuronosyltransferases (UGTs) are a superfamily of enzymes that catalyze the glucuronidation of numerous xenobiotic and endobiotic compounds . The reaction results in the formation of hydrophilic glucuronides that are easily excreted into bile or urine. Thus, UGTs are centrally involved in the metabolism and detoxification of drugs, pollutants and toxic substances. Currently, 19 human UGTs have been identified and are classified into two families, UGT1 and UGT2. UGTs are type I endoplasmic reticulum (ER) resident membrane proteins with a single transmembrane domain near the carboxyl-terminus, and most parts including the catalytic domain are located on the luminal side [5,6]. Earlier studies have reported that several UGT isoforms such as human UGT1A6 , UGT2B7, UGT2B15, UGT2B17, monkey UGT2B20 , rat UGT2B1, and UGT2B2  are N-glycosylated.
Accumulating evidence suggests that some proteins are dependent on the glycosylation for biological activity, whereas the function of others is not altered by this post-translational modification. As for UGTs, it has been demonstrated that deglycosylation of the purified rabbit UGT1A6, UGT2B13, rat UGT1A6 and UGT2B2 proteins by endoglycosidase H (Endo H) did not decrease their enzyme activities . Mackenzie  has reported that rat UGT2B1 and UGT2B2 are N-glycosylated and the treatment of COS cells expressing these UGTs with tunicamycin, which inhibits the first step of N-glycosylation, i.e., translocation of
oligosaccharide core units to peptides, resulted in the production of less active enzymes.
However, the unglycosylated proteins showed similar substrate specificity and protein stability as the native proteins. Barbier et al.  have reported that human UGT2B15 and monkey UGT2B20, in which the glycosylation was abolished by the mutation of asparagine residues, showed decreased velocity of glucuronidation, but no changes in the Km values and substrate specificities. Additionally, it has been demonstrated that some other UGT isoforms including human UGT2B4, monkey UGT2B23, and rat UGT2B3 are not glycosylated [8,11].
Thus, the physiological and biological significance of the N-glycosylation of UGT proteins remains to be clarified. In this study, we evaluated the role of N-glycosylation of human UGT1A, focusing UGT1A9 because it catalyzes the glucuronidation of a variety of drugs, dietary constituents, steroids, and fatty acids [12-14] and there is no report for the
glycosylation of this enzyme. We performed a series of studies using Endo H and
tunicamycin as well as mutation analysis for recombinant UGT1A9 enzyme. Kinetic analysis, SDS-PAGE analysis under the non-reducing condition, and thermal stability analysis were conducted to elucidate the role of glycosylation of UGT1A9.
2. Materials and Methods
O-Glycosidase, tunicamycin, UDP-glucuronic acid (UDPGA), alamethicin 4-methylumbelliferone (4-MU), and 4-MU O-glucuronide were purchased from Sigma-Aldrich (St. Louis, MO). Endoglycosidase H (Endo H) was from New England Biolabs (Ipswich, MA). Castanospermin, 1-deoxynojirimycin, thapsigargin, G-418 and propofol were purchased from Wako Pure Chemicals (Osaka, Japan). Rabbit anti-human UGT1A antibodies were obtained from BD Gentest (Woburn, MA). Rabbit anti-human GAPDH antibodies and mouse anti-KDEL antibodies were from Imgenex (San Diego, CA) and Stressgen (Ann Arbor, MI), respectively. IRDye680-labeled goat anti-rabbit secondary antibody and Odyssey Blocking Buffer were from Li-COR Biosciences (Lincoln, NE).
Perfect NT Gel M was purchased from DRC (Tokyo, Japan). Taq DNA polymerase was
obtained from Greiner Japan (Tokyo, Japan). Primers were commercially synthesized at Hokkaido System Sciences (Sapporo, Japan). Restriction enzymes were purchased from Takara (Shiga, Japan), Toyobo (Osaka, Japan), Stratagene (La Jolla, CA), and New England Biolabs (Beverly, MA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA).
All other chemicals and solvents were of the highest or analytical grade commercially available.
2.2. Treatment of recombinant UGT1As with Endo H or O-glycosidase
HEK293 cells stably expressing UGT1A1, UGT1A4, UGT1A6, or UGT1A9 were previously established in our laboratory . The cells were grown in Dulbecco’s modified Eagle’s medium containing 4.5 g/l glucose, 10 mM HEPES, and 10% fetal bovine serum with 5% CO2 at 37°C. Total cell homogenates were prepared by a method described previously . Briefly, the cells were suspended in Tris-buffered saline [25 mM Tris-HCl buffer (pH 7.4), 138 mM NaCl, and 2.7 mM KCl] and disrupted by freeze-thawing three times. The suspensions were homogenized with a Teflon-glass homogenizer for 10 strokes. The protein concentrations of the homogenates were determined according to Bradford . Then, the homogenates were adjusted to a 2 mg/ml protein concentration with a denaturing buffer in a final concentration of 0.5% SDS and 40 mM DTT, and were denatured at 95°C for 10 min.
The denatured proteins (10 µg) were incubated with 250 U of Endo H or 2.5 mU of
O-glycosidase in 50 mM sodium citrate buffer (pH 5.5) in a final volume of 10 µl at 37°C for 1 h, and were subjected to SDS-PAGE and immunoblotting. For the evaluation of enzyme activity, the recombinant UGT1A9 was treated with Endo H under the non-denaturing condition as follows: the total cell homogenates (10 µg) were incubated with 250 U Endo H in 200 mM Tris-HCl buffer (pH 7.5) in a final volume of 20 µl at 37°C for 1 h.
2.3. SDS-PAGE and immunoblotting
The samples were boiled for 3 min in Laemmli sample buffer containing
2-mercaptoethanol and separated on a 10% SDS-polyacrylamide gel. The separated proteins
were electrotransferred onto PVDF membrane Immobilon-P (Millipore, Billerica, MA). The membrane was washed with phosphate buffered saline (PBS) two times and blocked with Odyssey Blocking Buffer (Li-COR Biosciences, Lincoln, NE) for 1 h. The membranes were incubated with rabbit anti-human UGT1A polyclonal antibody (1:500), rabbit anti-human GAPDH antibodies (1:1000), or mouse anti-KDEL antibodies (1:200) diluted with Odyssey Blocking Buffer containing 0.1% Tween-20 for 1 h. The membrane was washed with PBST (PBS containing 0.1% Tween-20) four times and incubated with IRDye680-labeled goat anti-rabbit or anti-mouse IgG secondary antibody diluted (1:5000) with PBST for 1 h. The densities of the bands were determined using an Odyssey infrared imaging system (Li-COR Biosciences).
2.4. Construction of expression plasmids of UGT1A9 mutants and transient transfection into HEK293 cells
Asparagine residues at the positions 71, 292, and 344 of UGT1A9 were mutated to glutamine using a QuickChange Site-Directed Mutagenesis Kit (Stratagene). The primer sets to construct the expression plasmids of single mutants N71Q, N292Q, and N344Q are shown in Table 1. The expression plasmids of double mutants N71Q/N292Q, N292Q/N344Q, and N71Q/N344Q and a triple mutant N71Q/N292Q/N344Q were constructed by digestion with appropriate restriction enzymes and ligation of the expression plasmids of single mutants.
Nucleotide sequences were confirmed by DNA sequence analysis. HEK293 cells (1 x 106 cells) seeded on 6-well plates were transfected with 4 µg of the expression plasmids using Lipofectamine 2000. After 48 h, the total cell homogenates were prepared as described above.
2.5. 4-MU O-glucuronosyltransferase activity
4-MU O-glucuronosyltransferase activity was determined as described previously .
Briefly, a typical incubation mixture (200 µl of total volume) contained 50 mM Tris-HCl buffer (pH 7.4), 10 mM MgCl2, 2.5 mM UDPGA, 25 µg/ml alamethicin, 0.1 mg/ml cell homogenate, and 50 µM 4-MU. The reaction was initiated by the addition of UDPGA
following a 3-min preincubation at 37°C. After incubation at 37°C for 30 min, the reaction was terminated by the addition of 100 µl of ice-cold methanol. After removal of the protein by centrifugation at 13,000 g for 5 min, a 50-µl portion of the sample was subjected to HPLC.
The analytical column was a CAPCEL PAK C18 UG120 (4.6 x 150 mm, 5 µm; Shiseido, Tokyo, Japan), and the mobile phase was 30% methanol/20 mM potassium phosphate buffer (pH 4.5). The eluent was monitored at 320 nm. The quantification of 4-MU O-glucuronide was performed by comparing the HPLC peak height to that of the authentic standard.
In kinetic analyses, the 4-MU concentrations varied from 0.5 µM to 50 µM when the UDPGA concentration was fixed at 2.5 mM; the UDPGA concentrations varied from 1 µM to 150 µM when the 4-MU concentration was fixed at 50 µM. Kinetic parameters were
estimated from the fitted curve using a computer program (KaleidaGraph, Synergy Software, Reading, PA) designed for non-linear regression analysis. The following equations were applied for Michaelis-Menten kinetics:
V = Vmax • S/ (Km + S)
where V is the velocity of the reaction, S is the substrate concentration, Km is the
Michaelis-Menten constant and Vmax is the maximum velocity. Data are expressed as the mean
± SD of triplicate determinations.
2.6. Treatment of HEK293 cells expressing wild-type UGT1A9 with glycosylation inhibitors The HEK293 cells were transiently transfected with 4 µg of the expression plasmid of the wild-type UGT1A9. After 5 h, the cells were treated with tunicamycin (0.025 – 1 µg/ml), castanospermine (100 µg/ml), or 1-deoxynojirimycin (40 µM) for 43 h. As a control
experiment to cause ER stress, the cells were treated with thapsigargin (0.5 µM) for 43 h. The total cell homogenates were prepared as described above.
2.7. SDS-PAGE under the non-reducing condition and immunoblotting
SDS-PAGE under the non-reducing condition and immunoblotting were performed as described previously  with slight modifications. The total cell homogenate was
solubilized with PBS containing 2% Triton X-100, 0.5% SDS, and 2 mM EDTA, shaking vigorously for 60 min at room temperature, and was centrifuged at 13,000 g for 30 min. To a 24-µl portion of supernatant, 1 µl of 60% glycerol containing 0.2% bromophenol blue was added. The sample (23 µg) was applied to Perfect NT Gel M (5-20% gradient, DRC, Tokyo, Japan). The electrophoresis was carried out at 10 mA for 5 h at 4 C in Tris-glycine
electrophoresis buffer (25 mM Tris base, 192 mM glycine, and 0.1% SDS). The separated proteins were transferred to a PVDF membrane Immobilon-P, and the membrane was treated with rabbit anti-human UGT1A polyclonal antibody as described above.
2.8. Examination of thermal instability of UGT1A9
To examine the effects of the deglycosylation on the thermal instability of UGT1A9, the reaction mixturesincluding the cell homogenate but excluding 4-MU were preincubated at 37, 42, 47, 52 and 57 °C for 15 min in the presence of 2.5 mM UDPGA. The enzyme activities were measured by adding 4-MU in a final concentration of 50 µM at 37°C for 20 min. The residual activity was determined by comparison with the control activity without the preincubation.
2.9. Statistical analyses
Data are expressed as the mean ± SD of three independent determinations. Statistical significance of the kinetic parameters was determined by two-tailed Student’s t test. A value of P < 0.05 was considered statistically significant.
3.1. SDS-PAGE analysis of Endo H- or O-glycosidase-treated recombinant human UGT1As In the SDS-PAGE analysis, human UGT1A1, UGT1A4, UGT1A6, and UGT1A9 stably expressed in HEK293 cells migrated differently (Fig. 1A), although they had quite similar numbers of amino acids (527 to 530 residues). To investigate whether the difference was due to the glycosylation, UGT1As were treated with Endo H and O-glycosidase, which cleave N-
and O-glycosylation sites, respectively. When the UGT1A1, UGT1A4, UGT1A6, and UGT1A9 treated with Endo H were separated, they showed the same migration patterns, which were faster than those of non-treated UGT1As. In contrast, the treatment with O-glycosidase did not affect the migration pattern of the original UGT1As. These results suggested that all of UGT1A1, UGT1A4, UGT1A6, and UGT1A9 were N-glycosylated. As deduced from the amino acid sequences, UGT1A1 and UGT1A9 have three potential N-linked glycosylation sites, NX(S/T) (Fig. 1B). UGT1A4 and UGT1A6 have five and two potential N-glycosylation sites, respectively. The differences in the migration of UGT1As on SDS-PAGE seemed to be in parallel with the differences in the number of the potential N-glycosylation sites. In the subsequent study, we focused on UGT1A9.
3.2. Effects of substitution of asparagine residues at the potential N-glycosylation sites of UGT1A9 on the migration on SDS-PAGE and the enzyme activity
To investigate the role of the glycosylation on the enzyme activity, three asparagine residues at 71, 292, and 344 of UGT1A9, the potential glycosylation sites, were substituted to glutamine. On the SDS-PAGE analysis, the single mutants N71Q and N344Q showed two bands that migrated faster than those of the wild type (Fig. 2A). The migration of two bands of the single mutants N292Q was slightly faster than those of the wild-type UGT1A9. The double mutants N71Q/N292Q and N292Q/N344Q showed a single band the migration of which was similar to the faster ones observed in the single mutants N71Q and N344Q, respectively. The double mutant N71Q/N344Q showed two bands, of which the band with faster migration seemed to be the same as the band of the triple mutant N71Q/N299Q/N344Q.
The migration of the triple mutant was the same as the band of the Endo H-treated wild type.
These results indicate that all of the three asparagine residues would be glycosylated, although the extent of glycosylation and/or size of the glycan at residue 292 would be lower than at the other residues.
The 4-MU O-glucuronosyltransferase activities by wild-type and mutants UGT1A9 were determined (Fig. 2B). The activities were normalized with the UGT1A9 protein levels
determined by immunoblotting. The kinetics of the activities by the wild-type UGT1A9 was fitted to Michaelis-Menten kinetics. With the varied concentrations of 4-MU and the
concentration of UDPGA fixed at 2.5 mM, the Km and Vmax values were 2.7 ± 0.8 µM and 2.33 ± 0.29 nmol/min/unit, respectively (Table 2). With the varied concentrations of UDPGA and the concentration of 4-MU fixed at 50 µM, the Km and Vmax values were 14.1 ± 1.4 µM and 1.23 ± 0.06 nmol/min/unit, respectively. The single mutants N71Q, N292Q, and N344Q showed lower Vmax values than that of the wild type, but no difference was observed in the Km values in comparison with the wild type. The order of intrinsic clearance was N292Q >
N344Q > N71Q. The double mutants N71Q/N292Q and N292Q/N344Q showed much lower activities, and the double mutant N71Q/N344Q and the triple mutant N71Q/N292Q/N344Q showed no activity. These results suggest that the substitution of asparagine residues resulted in the production of unglycosylated and inactive protein.
3.3. Effects of treatments with tunicamycin, castanospermine, or 1-deoxynojirimycin on the migration on SDS-PAGE and the enzyme activity of UGT1A9
In the mutation study, we could not exclude the possibility that the loss of enzyme activity may have been due to a change of the protein structure but not due to the lack of glycosylation. To investigate the effect of the glycosylation per se on the enzyme activity, we produced unglycosylated protein by treatment with tunicamycin, an inhibitor of
oligosaccharyltransferase, which catalyzes the translocation of oligosaccharide core units (Glc)3(Man)9(GlcNAc)2 to polypeptides (Fig. 3A). The treatment of the cells with
tunicamycin resulted in the production of faster migrating UGT1A9 protein in a
dose-dependent manner (Fig. 3B). The mobility of the UGT1A9 protein in the cells treated with 1 µg/ml tunicamycin was the same as that of the Endo H-treated UGT1A9 protein. Thus, we successfully produced unglycosylated UGT1A9 protein. The unglycosylated UGT1A9 protein showed a dose-dependent reduction in enzyme activity associated with the extent of unglycosylation (Fig. 3C). After the glycan core units are transferred to the peptides, the terminus glucoses are trimmed by !-glucosidases I and II in the ER and the Golgi apparatus
(Fig. 3A). To investigate whether the trimming of the terminus glucose may affect the enzyme activity, the cells were treated with castanospermine, an inhibitor of !-glucosidases I and II or 1-deoxynojirimycin, an inhibitor of !-glucosidases II. On the SDS-PAGE, the mobility of UGT1A9 from the cells treated with castanospermine was slightly lower than that of control (Fig. 3B). The effects of the treatment with castanospermine and 1-deoxynojirimycin on the enzyme activity were insignificant (77% and 80% of the control, respectively) (Fig. 3C).
These results suggest that the binding of core glycan rather than modification of the terminus would be important to exert the enzyme activity.
It is known that the treatment of cells with tunicamycin causes ER stress by the
accumulation of unfolded or misfolded proteins [18,19]. Actually, we observed that the levels of glucose-regulated protein 78 (GRP78) and GRP94 were increased in a dose-dependent manner by treatment with tunicamycin (Fig. 3B). To verify that the decrease of UGT activity would not be due to the secondary effects of ER stress, we determined the UGT activity under the treatment with thapsigargin, an inducer of ER stress inhibiting the Ca2+-ATPase on the ER membrane . The treatment with thapsigargin caused ER stress, demonstrated by the highly increased GRP78 and GRP94, but had little effect on the 4-MU
O-glucuronosyltransferase activity (81.0% of control) (Fig. 3C). In addition, the treatment with castanospermine or 1-deoxynojirimycin also caused ER stress, but their effects on UGT1A9 activity were insignificant. These results suggest that the dramatic decrease of UGT1A9 activity by tunicamycin treatment would be due to the inhibition of glycosylation during the translation.
3.4. Effects of deglycosylation on the enzyme activity of UGT1A9
Next, we investigated whether the digestion of glycan from the mature UGT1A9 protein might affect the enzyme activity. We measured the 4-MU glucuronosyltransferase activity of the wild-type UGT1A9 that was treated with Endo H under the non-denaturing condition. We first confirmed that UGT1A9 was completely deglycosylated by Endo H (Fig.
4A). The kinetics of 4-MU glucuronosyltransferase activity by deglycosylated UGT1A9 was
similar to that by the non-treated UGT1A9 (Fig. 4B). These results suggest that the glycan per se would not be a determinant of the enzyme kinetics.
3.5. Role of N-glycosylation on the protein folding of UGT1A9
It was surmised that the decreases of the enzyme activity of mutants UGT1A9 as well as the unglycosylated UGT1A9 produced by tunicamycin treatment might result from changes in the protein folding of UGT1A9. To investigate whether the inhibition of
N-glycosylation affects the higher structure of UGT1A9 protein, we performed SDS-PAGE under the non-reduced condition analysis. As shown in Fig. 5A, in control, two bands were observed at about 53 kDa and 110 kDa, corresponding to a monomer and dimer, respectively, as previously reported . The treatment with castanospermine or 1-deoxynojirimycin did not affect the higher structure of UGT1A9. Unexpectedly, no band was observed with the total cell homogenates from tunicamycin (1 µg/ml)-treated cells, though a clear band was observed in the SDS-PAGE analysis. This may imply that the epitope of UGT1A9 toward the anti-UGT1As antibody might be sterically distorted. In other words, the folding of UGT1A9 might be changed by the inhibition of N-glycosylation. To confirm this assumption, we also performed the SDS-PAGE under the non-reduced condition analysis for a series of mutant UGT1A9 proteins (Fig. 5B). Interestingly, the single mutants N71Q and N344Q showed two bands corresponding to a monomer and dimer that were weaker than those of the wild type, although the single mutant N292Q did not show a prominent difference in comparison with the wild type. Three double mutants barely showed the two monomer and dimer bands.
Finally, in triple mutants, no clear band was observed, supporting the hypothesis that unglycosylated protein would have aberrant protein folding. In contrast, Endo H-treated UGT1A9 clearly showed the two bands indicating that the deglycosylation from the correctly folded protein did not affect the higher structure of UGT1A9. These results suggest that impeding N-glycosylation would cause drastic changes in the folding of UGT1A9.
3.6. Thermally stable characteristics of UGT1A9: asparagine-substituted mutants UGT1A9
and Endo H-treated wild-type UGT1A9
In our previous study , we demonstrated that UGT1A9, among human UGT1A isoforms, is uniquely tolerable to heat treatment. The thermal stability of proteins largely depends on their three-dimensional structures. To investigate the role of the N-glycosylation on the protein folding of UGT1A9, we determined the thermal stability of single mutants of UGT1A9 or Endo H-treated wild-type UGT1A9. Double and triple mutants and the
tunicamycin-treated wild-type UGT1A9 were not used, since their enzyme activities were low.
The 4-MU O-glucuronosyltransferase activity by the wild-type UGT1A9 was almost completely retained by heat treatment up to 52°C (Fig. 5), showing thermal stability
consistent with our previous study . The activity was decreased by 80% of control by heat treatment at 57°C. The single mutants N71Q, N292Q, and N344Q were also stable in
response to the heat treatment up to 52°C, but treatment at 57°C decreased their activities (39%, 52%, and 57% of control, respectively), which were significantly lower than those of the wild type. In contrast, the Endo H-treated UGT1A9 showed thermal stability similar to that of the non-treated UGT1A9. These results suggest that N-glycosylation would be important for the protein folding of UGT1A9.
In the present study, we evaluated the role of N-glycosylation in human UGT1As and found that all of the UGT1A1, 1A4, 1A6, and 1A9 were N-glycosylated. It was not a specific phenomenon for the recombinant proteins, because we observed the Endo H-dependent shift of bands when human liver microsomes were applied for the SDS-PAGE analysis using isoform-specific antibodies against human UGT1A1, UGT1A6 (BD Gentest), and UGT1A9  (data not shown). The numbers of the potential N-glycosylation sites of UGT1A1, 1A4, 1A6, and 1A9 are 3, 5, 2, and 3, respectively (Fig. 1B), and seem to be in parallel with the molecular sizes of these glycoproteins (Fig. 1A). By the mutation analysis, we found that the three potential glycosylation sites at position 71, 292, and 344 in UGT1A9 were all
glycosylated, although the extent of glycosylation and/or size of the glycan at position 292
would be low. Previously, Ouzzine et al. , using mutation analysis of recombinant enzymes expressed in yeast, reported that human UGT1A6 is glycosylated at position 345, but not at 293. They concluded from the results of the SDS-PAGE and immunoblot analysis that there was no difference between the mobility of the mutant N293G and the wild-type UGT1A6.
Consistent with their finding, as shown in Fig. 2A, the difference in the mobility between the mutant N292Q and wild-type UGT1A9 was not large. However, analysis with the
combination mutants at three sites revealed that N292 would be N-glycosylated. The enzyme kinetic analysis also supported this (Fig. 2 and Table 2). Thus, it is conceivable that human UGT1As are commonly N-glycosylated at the sites corresponding to N292 and N344 of UGT1A9, which are encoded from common exons 2 and 5, although the extent of glycosylation and/or size of the glycan are different. The mutation of these asparagine
residues significantly decreased the Vmax values, but not the Km values (Table 2), consistent with a previous study of human UGT2B15 and monkey UGT2B20 . To investigate
whether the decreased velocity was due to the abolished glycosylation, not by the change in primary structure, we further studied the enzyme activity of unglycosylated and
deglycosylated UGT1A9 proteins.
The unglycosylated UGT1A9 protein produced by the treatment with tunicamycin was enzymatically inactive. In addition, the SDS-PAGE under the non-reduced condition analysis revealed that it had aberrant folding. Therefore, the decreased enzyme activity would result from the misfolding owing to the abolished glycosylation. The thermal stability assays supported the importance of glycosylation in the folding of UGT protein. Once UGTs are correctly folded in the presence of glycan, they are enzymatically active, because the
deglycosylation of UGT1A9 by Endo H did not affect the enzyme activity. These results also suggest that the N-glycosylation is important for the folding of UGT proteins. The C-terminal half including two N-glycosylation sites is common to UGT1A isoforms. Therefore, it would be reasonable to assume that the importance of N-glycosylation is common to UGT1As.
Supporting it, we found that unglycosylated UGT1A1 protein produced by the treatment with tunicamycin was enzymatically inactive, whereas the kinetics of 4-MU
glucuronosyltransferase activity by UGT1A6 was also not affected by deglycosylation with Endo H (data not shown). However, it should be pointed out that some UGT2B isoforms are not glycosylated. Barbier et al.  reported that human UGT2B4, monkey UGT2B18 and UGT2B23 are not N-glycosylated in spite of the presence of the potential N-glycosylation sites. Additionally, there is no potential N-glycosylation site for rat UGT2B3  and UGT2B6 . Although the precise mechanisms of the folding of UGTs are not known, glycosylated UGTs and non-glycosylated UGTs may be differently regulated. It would be of interest to determine whether such a difference affects the higher structures, stabilities, and membrane-traffic of UGTs in the future.
In the present study, we determined 4-MU glucuronosyltransferase activity to evaluate the effects of N-glycosylation on the enzyme activity of UGT1A9. Additionally, to investigate whether the effects of N-glycosylation might be different depending on substrates, we also determined propofol O-glucuronosyltransferase activities. The unglycosylated UGT1A9 produced by the treatment with tunicamycin was inactive for propofol O-glucuronidation, and the effects of the mutations of each N-glycosylation site on the kinetics of propofol
glucuronidation were similar with those observed in the 4-MU glucuronidation (data not shown). In addition, the kinetics of the deglycosylated UGT1A9 by Endo H was completely fitted with that by the non-treated UGT1A9 (data not shown). Thus, it is unlikely that the effects of N-glycosylation would be depending on substrates.
In conclusion, we found that human UGT1A9 was N-glycosylated, and impeding N-glycosylation resulted in the loss of function. This study clearly demonstrated that the N-glycosylation plays an important role in the protein folding of UGT1A9.
We acknowledge Mr. Brent Bell for reviewing the manuscript.
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Fig. 1. SDS-PAGE and immunoblotting of Endo H- or O-glycosidase-treated human UGT1A proteins and potential N-glycosylation site. (A) Total cell homogenates from HEK293 cells stably expressing UGT1A1, UGT1A4, UGT1A6, or UGT1A9 were treated with Endo H or O-glycosidase as described in Materials and Methods. The samples (10 µg) were subjected to 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was probed with an anti-human UGT1A antibody. (B) Schematic representation of UGT1A proteins and potential N-glycosylation sites. UGT1As are generated by alternative splicing of unique exon 1 to common exons 2-5, resulting in isoforms with an identical C-terminal half of protein and a unique N-terminal half.
Fig. 2. Molecular size and enzyme activity of UGT1A9 mutants. The expression plasmids of wild-type and mutants UGT1A9 were transiently transfected into the HEK293 cells. (A) Total cell homogenates (10 µg) were separated by 10% SDS-PAGE. Endo H-treated cell
homogenates (10 µg) expressing wild-type UGT1A9 was also separated to show the migration of non-glycosylated UGT1A9. The band density of the wild-type UGT1A9 was defined as 1 unit per 10 µg of protein. Values were the mean ± SD of three independent determinations. GAPDH levels were examined as a loading control. (B) Kinetic analyses of 4-MU O-glucuronosyltransferase activities were performed. Total cell homogenates were incubated with 0.5 to 50 µM 4-MU and UDPGA fixed at 2.5 mM (left panel), or with 1 - 150 µM UDPGA and 4-MU fixed at 50 µM (right panel). Data are the mean ± SD of triplicate determinations.
Fig. 3. Effects of the inhibitors of glycosylation or glycosidase on UGT1A9. (A) Schematic representation of biosynthesis of N-glycosylated protein in ER. Tunicamycin inhibits the first step of N-glycosylation, translocation of oligosaccharide core units to peptides.
Castanospermine inhibits !-glucosidases I and II and 1-deoxynojirimycin inhibits
!-glucosidase II (B) HEK293 cells were transiently transfected with the expression plasmid
of wild-type UGT1A9. After 5 h, the cells were treated with tunicamycin (0.025 to 1 µg/ml), an inhibitor of glycosylation, castanospermine (100 µg/ml) or 1-deoxynojirimycin (40 µM), inhibitors of glycosidase, for 43 h. To induce ER stress, the cells were treated with
thapsigargin (0.5 µM) for 24 h. SDS-PAGE and immunoblotting using anti-human UGT1A antibody were performed with 10 µg total cell homogenates. Endo H-treated cell homogenate expressing wild-type UGT1A9 was applied to show the migration of non-glycosylated UGT1A9. The band density of the control UGT1A9 was defined as 1 unit per 10 µg of protein. Values were the mean ± SD of three independent determinations. Immunoblotting using anti-KDEL antibody was performed with 40 µg total cell homogenates to determine the levels of GRP94 and GRP78. The band density of the non-treated cells (control) was defined as 1. GAPDH levels were examined as a loading control. (C) 4-MU
O-glucuronosyltransferase activities were determined with 50 µM 4-MU and 2.5 mM
UDPGA at 37°C for 20 min. Data are the mean ± SD of triplicate determinations. **P < 0.01 compared with control.
Fig. 4. Effects of deglycosylation on the enzyme activity of UGT1A9. The total cell
homogenates (10 µg) prepared from the HEK293 cells transiently expressing UGT1A9 were treated with 250 U Endo H under the non-denaturing condition (200 mM Tris-HCl buffer, pH 7.5) at 37°C for 1 h. (A) SDS-PAGE and immunoblotting were performed. (B) Kinetic analyses of 4-MU O-glucuronosyltransferase activities were performed with 1 to 50 µM 4-MU and UDPGA fixed at 2.5 mM (left panel) or with 2.5 to 150 µM UDPGA and 4-MU fixed at 50 µM (right panel).
Fig. 5. SDS-PAGE under the non-reduced condition and immunoblot analysis. Total cell homogenates from (A) tunicamycin (1 µg/mL)-, castanospermine (100 µg/ml)- or
1-deoxynojirimycin (40 µM)-treated HEK293 cells expressing wild-type UGT1A9, and (B) the HEK293 cells expressing mutants UGT1A9 were separated on Perfect NT Gel M (5-20%
gradient). (C) Endo H-treated homogenates from the HEK293 cells expressing wild-type UGT1A9 were also separated. Immunoblotting was performed as described in Materials and Methods.
Fig. 6. Thermal instability of wild-type and mutants UGT1A9 (A) as well as Endo H-treated UGT1A9 (B). The reaction mixturesincluding the cell homogenate but excluding 4-MU were preincubated at 37, 42, 47, 52and 57 °C for 15 min in the presence of 2.5 mM UDPGA. The enzyme activities were measured by adding 4-MU in a final concentration of 50 µM at 37°C for 20 min. The residual activity was determined by comparison with the control activity without the preincubation. Data are the mean ± SD of triplicate determinations. Control activities were as follows: (A) wild-type, 3.09 ± 0.11 nmol/min/unit; N71Q, 0.23 ± 0.01 nmol/min/unit;
N292Q, 1.69 ± 0.06 nmol/min/unit; N344Q, 0.40 ± 0.02 nmol/min/unit (B) non-treated, 2.53
± 0.14 nmol/min/unit; Endo H-treated, 2.48 ± 0.06 nmol/min/unit.
Non-treated Endo H O-Glycosidase
antibody 50 kDa
N292 N344 N71
N296 N348 N119 N142 N189
N295 N347 N102
Exon 1 Exon 2 - 5
Relative level (unit) 1.3 ± 0.5
1.3 ± 0.5 1.8 ± 0.5
2.9 ± 1.8 2.0 ± 1.2
2.7 ± 1.2 2.1 ± 1.0 1.0 ± 0.4
antibody 50 kDa
antibody 36 kDa
0 0.5 1.0 1.5 2.0 2.5
0 10 20 30 40 50 60
Wild N292Q N344Q N71Q/N292Q N71Q/N344Q N292Q/N344Q
0 0.5 1.0
0 30 60 90 120 150 180
Relative level (unit) 0.9 ± 0.1
0.8 ± 0.2 0.9 ± 0.2
0.9 ± 0.2 1.0 ± 0.1
0.6 ± 0.2 1.0 ± 0.2
50 kDa 53 kDa
GRP 94 GRP 78 Relative level
1.0 2.3 5.0 4.3 8.5 5.0 7.3
1.0 2.1 3.1 2.1 6.3 1.2 8.0
antibody 36 kDa
2 4-MU: 50 µM
Tunicamycin Endoplasmic reticulum
1-Deoxynojirimycin Castanospermine Glucose
Dol P P
1, 2-Glucosidase I 1, 3-Glucosidase II
50 kDa 53 kDa Anti-UGT1A
4-MU (µM) UDPGA (µM)
Control Endo H
(nmol/min/units) 5.7 ± 0.5
6.1 ± 1.7 1.45 ± 0.06
1.48 ± 0.05 Control
Endo H 27.3 ± 6.5
15.6 ± 3.0* 0.97 ± 0.07 0.87 ± 0.05 Km
0 0.5 1.0
0 30 60 90 120 150 180 0
0.5 1.0 1.5
0 10 20 30 40 50 60
0 25 50 75 100
37 42 47 52 57
Wild (Non-treated) Wild (Endo H)
0 25 50 75 100
37 42 47 52 57
Wild N71Q N292Q N344Q
Table 1. Primers used for site-direct mutagenesis of UGT1A9.
Primers ! Sequences
Table 2. Kinetic parameters for 4-MU O-glucuronidation by wild-type and mutants UGT1A9 expressed in HEK293 cells.
Wild/Mutants Km Vmax Vmax/Km
Varied concentration of 4-MU (µM) (nmol/min/unit) (µL/min/unit)
Wild 2.7 ± 0.8 2.33 ± 0.29 886 ± 142
N71Q 2.6 ± 0.1 0.17 ± 0.01** 66 ± 5**
N292Q 2.7 ± 0.2 1.09 ± 0.03** 413 ± 33**
N344Q 1.9 ± 0.4 0.31 ± 0.01** 164 ± 29**
N71Q/N292Q 3.6 ± 0.4 0.05 ± 0.00** 15 ± 1**
N71Q/N344Q ND ND ND
N292Q/N344Q 2.6 ± 0.4 0.05 ± 0.00** 18 ± 3**
N71Q/N292Q/N344Q ND ND ND
Varied concentration of UDPGA (µM) (nmol/min/unit) (µL/min/unit)
Wild 14.1 ± 1.4 1.23 ± 0.06 87 ± 5
N71Q 16.5 ± 5.6 0.11 ± 0.00** 7 ± 2**
N292Q 19.3 ± 3.9 0.97 ± 0.04** 52 ± 11*
N344Q 27.3 ± 8.6 0.22 ± 0.04** 8 ± 1**
N71Q/N292Q 20.2 ± 11.9 0.02 ± 0.00** 1 ± 1**
N71Q/N344Q ND ND ND
N292Q/N344Q 25.1 ± 2.9** 0.03 ± 0.00** 1 ± 0**
N71Q/N292Q/N344Q ND ND ND
Data are the mean ± SD. **P < 0.01, *P < 0.05 compared with control value.

domain: chemistry


text:
Doctor of Philosophy (PhD)
Social Work and Social Research
This qualitative exploratory study seeks to address gaps in the literature about the development of emerging adult siblings of people with schizophrenia. This study uses four sets of literature as a framework for this study, including emerging adult identity development, sibling identity development (and other psychodynamic theories), studies on siblings of people with chronic illnesses and studies that specifically focus on siblings of people with schizophrenia. This study consisted of a pilot study of three and 23 intensive interviews which were then coded using NVivo and grounded theory techniques. Key findings for the study included exploring how issues of self-regulation can influence the formation of relationships and support that in turn, supports greater self-regulation. As well, understanding how a siblings’ role shifts in the family system can often correlate to a subjective sense of well-being. Implications for future research, especially around identity formation and family roles were also postulated.
Graves, Jillian. "The Experiences of Emerging Adult Siblings of People with Schizophrenia: An Exploratory Qualitative Study." PhD diss., Bryn Mawr College, 2016.

domain: library


text:
Title: The Basics of Grilling
Discover Simple Recipes for Mouth Watering, Steaks, Chicken, Fall Off The Bone Ribs, Sides, Even Desserts So Tasty You'll Be the Envy of the Neighborhood...."
- Do you want to grill... but don't actually know much about it?
- Amazing grilling recipes so tasty your guests will line up for seconds and talk about it for weeks?
If so, then you've come to the right place!
Today I'm Going to Share With You my secrets to the simplest and tastiest Grill recipes. Here's just a glimpse of what you'll discover.......
- Grilled vegetarian main courses so everyone can enjoy.
- How to prepare and cook the perfect steak, ribs, & chicken.
- Grilled side dishes.
- Finally show off your new skills with grilled deserts. Your guest won't know what hit their taste buds.
- Drinks that complement grilled foods on a summer day.
This is Not Like Any Other Guide You've Read On Grilling It's taken my years of going to grilling classes reading grilling and BBQ cookbooks, trial and error to find the easy good recipes that will amaze you and your guests.
You can simply purchase "The Basics of Grilling" and learn in an hour or two how to grill like the pro's. I've Poured My Life into this Book and I Know You'll Love it...
Title: The Complete Beef Cookbook
Are you tired of the same old dinners each week?
Well, guess what? You can enjoy delicious new dinner recipes with this complete collection of mouth-watering beef recipes.
These easy-to-follow beef recipes are fool proof and are guaranteed to become dinner favorites!
When it comes to cooking, it is easy to fall into a rut. That is why I put together this collection of my favorite beef recipes.
With this collection of recipes, you can deliver great-tasting, healthy dishes to your dinner table on a regular basis. I have included everything from quick and easy dinners to special occasion entrees.
In this ebook you will find all of my favorite beef recipes, packed with over 150 of the best dishes. In The Complete Beef Cookbook you will find delicious recipes for any occasion. All the recipes are neatly organized so that you can easily browse for the perfect dish.
- Barbecue Ribs
- Perfect Beef Tenderloin
- Beef & Noodle Casserole
- Beef Stroganoff
- Beef Burgundy
- Beef Jerky
- Mexican Style Short Ribs
- Beef Fajitas
- Best Beef Gravy
- Beef & Broccoli
- Sesame Beef
- Vegetable Beef Soup
- Beef Curry
& Over 150 More Beef Recipes!
How would you like to come home this evening to a dinner of Perfectly Seasoned Porterhouse Steak? Or how about Teriyaki Beef, Beef
Ebook Type: PDF
Title: Caveman Paleo Recipes
How You Can Lose Lots Of Weight Very Quickly On The Paleo Diet WITHOUT Starving Yourself, Exercising Like Crazy, Feeling Deprived, Or Spending A Fortune!
I’ll tell you the basic idea behind the Paleo diet, the science behind why people lose so much weight on it, and why they’re able to KEEP the weight off.
See, the basic idea behind Paleo is simply this: our bodies are not designed to handle all the stuff we have in our modern diet. Modern man (homo sapiens) has been around for 200,000 years, and for almost all of that time, we didn’t even have farms, let alone grocery stores.
As I’m sure you can see by now,The Paleo diet isn’t just a great way to lose weight. It’s also a great way to ensure a proper balance of nutrients. Here are just a few of the health benefits you’ll get in addition to losing that all that fat:
- Better sleep
- More stamina
- Better mental health
- Better athletic performance
- Tons of energy
- Fewer allergies and colds
- Better mental performance
- Libido (not-fun fact for you men: the more grain you consume the smaller your testicle size— I’m not kidding, the University of Missouri in the U.S. did a study)
- Lower risk of diabetes
- MUCH lower risk of all other health problems associate with being overweight (stroke, heart disease, high blood pressure, and even
Title: Summer Eats - Paleo Recipes & Clean Cocktails
Paleo Bullies Look Away! We've got drinks!
Summer Eats is about more than just tasty recipes, though there are plenty within these pages. It was inspired by and created with friendship and fun in mind. In fact, this ebook was a collaboration with my best friends, each of us bringing a unique talent to the table. From the plate to the glass to the design, every page is filled with personality and of course a delicious offering. Based on the paleo diet, every recipe is soy, dairy, grain-free and gluten free, and avoids the use of processed ingredients without avoiding serious flavor and fun!
Summer Eats is a true labor of love and friendship. We took it so seriously that we all even got "HFF" tattoos! Celebrate with us WITHOUT giving a second thought about whether the ingredients on your plate or in your glass are deviating from your clean eating lifestyle. With Summer Eats you can eat clean, drink clarified and crush life. You know the motto, #eatplaycrush!
Title: Hangi Guide Official
Author: Neville Rangi-Tane Pettersson
How To Cook a Hangi the Traditional Maori Way.
Your search for quality Hangi information is over! You’ve found it! The Complete NZ Hangi Guide is the MOST comprehensive Hangi resource available.
The really good news is that I videoed the whole thing and took heaps of notes! Since then I’ve done loads of Hangi's on my own and I’ve learn from my own mistakes. So NOW I’ve written this Hangi Guide, especially for you guys! It includes all of the important information that you won’t find anywhere else, but you NEED to know! Such as:
- Maori Hangi - The size and shape of your Hangi basket is VERY important! All the info is in here plus I’ll also tell you the best places to buy them.
- Hangi Cooker - Not ALL soil is created equal! I’ll tell you how to carefully choose your Hangi spot and what type of earth is the best for putting down your Hangi.
- How To Make A Hangi - Hangi rocks are probably the most important factor in your Hangi success. I’ll tell you the geological classification of the rocks you’ll need including full high-resolution colour pictures so you can identify the right Hangi stones easily.
- Hangi Sacks, Cloths and Tools - An exact list of the necessary materials you’ll need including dimensions, how many you’ll need and where you can find them.
- Wood - You’ll discover which wood gives the best taste. Plus I’ll tell you the
Ebook Type: PDFVideo Included,
Title: Best Seafood Recipes Cookbook
Did you know that current advice from major health organizations recommends eating two seafood meals each week?
Seafood is nutrient rich and a great source of protein, vitamins and minerals.
We have compiled a collection of delicious and nutritious seafood recipes for you to enjoy in the Best Seafood Recipes cookbook...
- Baked Halibut
- Broiled Salmon
- Codfish Soup
- Fried Perch
- Fish Chowder
- Salmon Salad
- Baked Scallops
- Oyster Stew
- Steamed Clams
- Lobster Croquettes
- Lobster Newburg
- Soft Shell Crabs
- Prawn Salad
- Crawfish Stew
- Cocktail Sauce
& Over 100 More Seafood Recipes!
Ebook Type: PDF
Title: The Caveman Feast
Author: George Bryant, Abel James
Finally, it’s here! My cookbook, “The Caveman Feast,” features all of the best recipes I’ve ever made. It’s been a long-time coming – my buddy Abel James helped me put it all together in the past few months and we’re so happy it’s ready to share with you.
You get more than 200+ color photos of each recipe beautifully photographed by George himself. See what each meal will look like and how to best present them. All exclusive digital content delivered right through your computer!
Paleo recipes under the following categories will excite your taste buds and have you cooking easy but delicious meals in no time:
- Pork Entrees
- Beef Entrees
- Poultry Entrees
- Seafood Entrees
- Grain?Free Goodies
- Condiments, Sauces, Butters, Dressings, Rubs, & Blends
Every single recipe is 100% Paleo. They have:
- No Potatoes
- No Grains
- No Lentils
- No Processed Sugars
- No Dairy
- No Preservatives
Nothing but 100% real, fresh, simple ingredients. Our goal is to make every one of your meals as delicious as possible.
With clear, step-by-step instructions you will produce Paleo meals that will get rave reviews from friends and family every time!
Ebook Type: PDF
Title: The HCG Diet Recipe Handbook
Now You Can Enjoy Mouth-Watering, Simple, Low Calorie Meals And Lose Up To 1-2 Pounds Per Day!
200+ Scrumptious Low Calorie Recipes for the HCG Phase.
Here's What's Inside The HCG Diet Recipe Handbook:
- Elegant Fish & Seafood Entrees
- Savory Chicken Dishes
- Mouth-Watering Beef Dishes
- Refreshing Beverages
- Appetizing Salads and Soups
- Delicious Fresh Fruit Desserts
Each and every recipe has been perfectly constructed and designed to be 100% compatible with the very low calorie "HCG Phase" of the original HCG Diet protocol by Dr. Simeons. The HCG Diet Recipe Handbook is your perfect solution to add tons of variety, excitement, and an impact of exquisite cooking.
Mouth watering, exciting and easy recipes which make being on the diet and every meal an absolute pleasure. These recipes have been handpicked by top chefs, and restaurant owners.. meals so delicious that you won't want to share them with anyone who's tempted to have a taste!
Ebook Type: PDF
Title: Sensational Smoothies
Drink Your Way To Health Deliciously!
How to Create Amazingly Delicious Smoothies That Boost Your Long-Term Health, Energy, and Physique!
There are so many wonderful benefits to enjoy when you commit yourself to drinking smoothies. Benefits you can enjoy in no time! Smoothies can:
- Reduce your cravings for sweets and other fatty foods. (No more cheating on a diet then feeling bad about it!)
- Help you lose weight more easily than before!
- Give you better digestion. (No more constipation!)
- Give you the right vitamins and minerals. Plus the antioxidants from the fruit and vegetables will help you prevent major illnesses, like cancer.
- Give you your recommended fruit and vegetable requirements daily!
- Stabilize blood sugar and cholesterol levels.
- Lessen your intake of chemicals and preservatives, often found in other foods, and which may have been causing some of your health problems without you knowing it!
- Improve your dental health!
- Strengthen your immune system, lessening the number of times you get sick. (Now you don't have to worry about getting sick and how it may affect your work or other aspects of your life!)
- Give you more energy
- Lessen your aches and pains. (The healthy goodness can heal troubled areas in your body.)
- Sharpen your mental skills. (You can be on top of your game all the time, focusing and concentrating
Title: Great Juicing: Juicing for Health and Vitality
Author: Thomas Christopher
Do You Want To Have A Healthy And Vibrant Life with a Nutritious Diet without Spending a Lot of Your Precious Time - Guaranteed?
Here's what you'll discover in Great Juicing: Juicing for Health and Vitality eBook:
- Three little known, yet simple ways to take advantage of the power of juice...
- How to understand the power of home-made juice compared to bottled juice...
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- Three proven steps to understanding how juice can detoxify and cleanse your body...
- WARNING: 3 things you should never do when it comes to juicing...
- Six time tested and proven strategies to lower your blood pressure with the power of juice...
- You'll discover in just a few short minutes how to gain energy with juice...
- When to seek professional help when it comes to fasting with juice...
- A pennies on the dollar approach to seeking medical guidance for sciatic pain...
- Seven everyday but often overlooked tips and tricks for using juice to alleviate diabetes...
- How often to fast using juice...
- The once famous but forgotten secret that instantly allows you to have an overall healthier lifestyle with the power of juice...
- How to use the power of juice to alleviate asthma...
Ebook Type: PDF
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114 Cooking ebooks Click here to see the full list of these ebooks
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domain: food


text:
Are you ready for some football? I know I am! I cheer for my favorite college and pro teams, but really I love football season for the food. Chili, burgers, chili dip, pretzels ... I love it all. But, to me, no tailgate is complete without chicken wings.
Wings are my favorite. Drop 'em in the fryer, add some hot sauce and I'm happy (and don't forget the bleu cheese dressing on the side!). But, these days, chicken wings have gone to a whole new flavor level.
The wing sauce on Cynthia Sigler's Korean Chicken Wings is scrumptious. "Everyone loves these when I make them, they are usually the first to go," shares Cynthia. We loved this sauce so much so, in fact, that we decided to soak our second batch of wings directly in the sauce while they were still hot. It made them a lot messier, but oh-so-good! The soy sauce, garlic, ginger and scallions add great flavor, while the sugar adds a nice sweetness.
"These wings are delicious - a bit of heat and a bit of sweet," explains Teresa Clayton when describing her Wings Worth the Wait! recipe. "My husband talks me into making them every Sunday for the football games." These wings are fried and baked. It gives a nice crunch to the wings. The sauce is really good too. It has some heat, but not too much. Teresa mentions leftovers are yummy. These are so tasty, I don't think you'll have any left!
I love that Mary Ann Hanson's Slow Baked Chicken Wings are baked (making them a bit better for us!). The slow baking made the meat just fall off the bone. "My family has eaten these right out of the pan right from the oven because they are so good," says Mary Ann. The sauce won me over on these wings. It's really good, and different. Who would have thought to put french dressing in wing sauce? The Kitchen Crew gobbled these up and I'm sure your guests will too.
Why not throw wings onto the grill? Heather Johnson and Angela Gray do! Grilling adds a great extra flavor boost to their recipes.
"I made these marinated wings on the fly (pun intended) for a Sunday afternoon cookout," explains Heather. "I didn't have enough of the one type of marinade so I made my own." Well, the marinade she came up with for her Ginger Lime Chicken Wings makes this recipe. Ginger and lime are a wonderful combo and give these wings a ton of flavor!
Angela Gray's Orange Glazed Chicken Wings do not disappoint. "The orange marmalade and the lime juice are a tangy combo," says Angela. "And the hot sauce is the surprise kick!" The orange marinade and sticky orange glaze is unusual and made these wings so tasty. These wings are definitely gooey so make sure to have some wet naps on hand. These would be great at your next tailgate.
Kick-off a full-flavored football season with a few of these chicken wing recipes. You'll definitely earn extra points with the crowd! I'm curious ... what's your favorite tailgate food?
© 2011 Just A Pinch Recipe Club. Used by Permission.
Brought to you by American Hometown Media


text:
Today (30 March) is the start of World Autism Week, where autistic people and charities come together to raise awareness of autism and how we can all work together to support the autistic community.
Over half of people in the UK are connected to someone who is autistic; and it is estimated that 700,000 people are autistic. During World Autism Awareness Week (30 March to 5 April) autistic people and their loved ones share their experiences alongside tips on helping an autistic person.
WHAT IS AUTISM?
Autism is a lifelong, development disability which can mean autistic people are affected when communicating with people or relating to others. Autistic people will also see the world around them differently than others.
Being autistic is not a disease, and there is no cure, it simply means a person is different. The language used around autism is important in order to share knowledge, reduce stigma and discrimination.
With improved discussions around autism and the autistic spectrum, more people can learn about different forms of autism. Celebrities such as Christine and Paddy McGuinness are open and honest about parenting three autistic children, and environmental activist Greta Thunberg – with Asperger syndrome – has previously said that ‘being different is a superpower.’
Similarly, The Chase star Anne Hegerty shared that her life responds well to structure and overcrowded areas can be a source of stress. During Anne’s time on I’m A Celebrity… Get Me Out of Here! in 2018 awareness and understanding of Asperger syndrome and the autistic spectrum was improved.
And, Christine and Paddy McGuinness are set to further raise awareness of autism as they introduce a new character, Theo, to children’s show Daisy and Ollie.
Set to air on Thursday 2 April – on World Autism Awareness Day – Theo will become the first autistic character on the show, in the hopes it will help more children understand what makes us all unique.
Charities including the National Autistic Society have a range of stories, and experiences from autistic people. Their Stories from the Spectrum series is a fantastic tool in sharing what life is like on the autistic spectrum.
During these uncertain times, where routines are changed without notice and an overload of information, it can be challenging. Charities are coming together to share their advice on how to best support an autistic loved one, alongside tips for you, if you are on the spectrum.
We are working closely with these charities to bring you information on how to understand COVID-19 if you are autistic, or your loved one is autistic. What would you like to know?

domain: psychology


Sure, we've all seen "wireless" Bluetooth earbuds... tethered together by a wire with a bulky controller dangling on the side of your face. TheseTrue Wireless Earbudsfeature all the same great feature (and then some) without the restriction of a connective wire, giving you superior freedom of movement.
Stay active and comfortable with the True Wireless Earbuds, cutting the cord COMPLETELY! That's right, not only are these wireless in the sense that they connect to your phone or tablet via Bluetooth, but they have no connection cord between them. Use one earbud for hands-free talking and background music, or sync them both up for a stereo experience.
These are great for exercise and travel, especially on planes with their noise isolation capabilities. They are rechargeable and includes the handy charging case.
The True Wireless Earbuds feature built-in microphones and the power buttons doubles as a Call Answer buttons, so you can take calls without having to go for your phone.
Enjoy superior sound quality and a truly wireless experience with these sleek and sophisticated Earbuds!
- High quality chip: Super bass sound quality headset with a high quality chip for the best stereo sound and superb bass sound.
- Clear at high & low volumes make you enjoying the music true wirelessly!
- 40mah + 450mah: Built in 40 mAh lithium polymer battery and includes the intelligent charging box with a high capacity lithium battery.
- Easy to use: Easy to use technology with voice notification for incoming calls and phone numbers. The simple buttons allows /play/pause on all your music tracks as well as all phone calls without reaching for your phone.
- Super quick pairing with all Android and iOS devices.
- v4.1 + edr: A larger Bluetooth range makes these mini headphones extraordinary, up to 30ft.
- Ergonomic design: Optimized for extra comfort and a better fit in the ear.
- Sweatproof construction is perfect for sports,running,jogging & gym exercise,watching TV,or sleep.


text:
Waiting on Wednesday is a weekly meme hosted by Jill at Breaking The Spine. It features each blogger’s most anticipated books.
This week, my pick is Onyx (Lux #2)
” Being connected to Daemon Black sucks…
Daemon’s determined to prove what he feels for me is more than a product of our bizarre connection. I’ve sworn him off even though he’s running more hot than cold these days. But against all common sense, I’m falling for him. Hard.
Our relationship issues aren’t out biggest problem…
The Department of Defense is here. If they ever find out what Daemon can do and that we’re linked, I’m a goner. So is he. And when a new boy shows up a school with a secret of his own, things get complicated fast. I need to choose between my own instincts and Daemon’s.
But then everything changes…
I’ve seen someone who shouldn’t be alive. Daemon’s never going to stop searching until gets the truth. What happened to his brother? Who betrayed him? And what does the DOD want from them—from me?
No one is who they seem. And not everyone will survive the lies.”
Why I Can’t Wait:
I’ve just read the first book, Obsidian, last week in a buddy read in one of my goodreads group. I freakingly loved it! It’s so awesome! But I didn’t make a review of it because I’m so overwhelmed with how great the book is. I know it has some flaws, but I couldn’t remember them because of Daemon! He’s a hot guy and gorgeous, and now I understand all the girl readers that are so crazy about him!
August 14th 2012 by Entangled Teen

domain: literature


What does Plan Change 6 cover?
Want to know more?
How do I make a submission?
Where can I view Plan Change 6?
The easiest way to view the proposed provisions is by clicking on the links in the below table. The amendments are shown in green type.
In addition to the documents below, Proposed Plan Change 6 has also been integrated into the relevant chapters of the Operative District Plan- Proposed Plan Changes.


text:
- CPD Notices
- HUD Guidance
Date Published: April 2003Print ShareThis
This Notice establishes requirements, procedures and deadlines to be followed in the urban county qualification process for Fiscal Years 2004-2006. Information concerning specific considerations and responsibilities for urban counties is also provided. A listing of any newly identified potential urban counties for this qualification period based on 2002 population estimates from the U.S. Census Bureau, if any, will be provided electronically at a later time. As in the past, any delay in establishing the participating population in just one urban county may result in delays in determining the final allocations of CDBG funds for all entitlement and State CDBG grantees. Therefore, HUD Field Offices and urban counties are expected to adhere to the deadlines in This Notice. This Notice provides guidance for counties wishing to qualify or requalify for entitlement status as urban counties, as well as for existing urban counties that wish to include previously nonparticipating communities. This Notice includes five attachments which contain listings of all currently qualified urban counties (Attachment A); counties that can potentially qualify for the first time or requalify this qualification period (Attachment B); counties scheduled to qualify or requalify in FY 2004 for FY 2005 - 2007 (Attachment C); counties scheduled to qualify or requalify in FY 2005 for FY 2006 - 2008 (Attachment D); and currently qualified urban counties that can add nonparticipating units of government for the remaining one or two years of their qualification period (Attachment E). As noted above, additions to Attachment B may be provided separately.

domain: geography


text:
Boulder, CO -- (SBWIRE) -- 02/10/2014 -- When it comes to the important matter of investing into real estate, individuals are always recommended to be rather careful in the process. Acquiring the services of real estate agents and companies is one of the most commonly used ways of buying real estate properties and saving money too, throughout the whole procedure. However, not all real estate companies and agents work in the best interests of home buyers and it is entirely too often that people end up losing truckloads of money in a short period of time.
In order to avoid this from happening in the near future, individuals are highly recommended to go for Boulder real estate as the Colorado based real estate company is excessively well-known and has been helping home buyers in making the best possible decisions in buying real estate properties of their dreams. Not only do they help home buyers to buy the perfect real estate properties but they are also known to help them build houses in the long run, which is quite impressive and exactly why the reason behind both the local and international success of Boulder realty.
The city of Boulder is surely a beautiful one and is considered to be one of the best locations for investing in real estate, which is exactly where the company comes in for the purpose of helping people save money in the process. Colorado home buyers can now sit back and enjoy the thousands of dollars after closing. This way, home buyers can keep their homes and also gain more money in the entire procedure.
Buyers’ Slice Realty has managed to acquire countless clients over the past couple of years and the number only seems to be increasing. This is not only because of the excellent services they have to offer but it is also because of the fact that these real estate agents believe in presenting cheques to the average home buyers of the Boulder, Colorado area. Investing in the area allows people to benefit from all the places there such as schools, hospitals, cafes, bus stations, malls, supermarkets, parks and many more, which is why individuals are highly recommended to go for it.
In order to gain more information regarding how Buyers’ Slice Realty clients can acquire added value along with the homes of their dreams, all the interested individuals are recommended to visit coloradocommissionrebate.com at the earliest convenience in the near future.
For more information, please visit: http://www.coloradocommissionrebate.com/boulder-real-estate.ctrl

domain: finance


Small 1hp 2hp 5hp Single Phase Solar Pump Inverter Variable Frequency Drive
|Place of Origin:||Guangdong,China|
Payment & Shipping Terms:
|Minimum Order Quantity:||1pcs|
|Packaging Details:||Wooden case,Paper box|
|Delivery Time:||3-5 working days|
|Payment Terms:||T/T, , Western Union,Paypal|
|Supply Ability:||3000pcs per week|
|Protection Function:||20 Kinds Of Motor Protection||Input Voltage:||150-450VDC Or 220VAC|
|Output Voltage:||Single Phase 220VAC Output||Product Name:||Variable Frequency Drive Solar Inverter|
|Output Current:||10A||Overload Protection:||Yes|
VFD Single Phase Solar Pump Inverter,
5hp Single Phase Solar Pump Inverter,
2hp VFD Solar Inverter
Small Power 1hp 2hp 3hp 5hp Variable Frequency Drive Solar Inverter Single Phase Inverter Drive Ac Pump
ZK SG600 Solar pumping system description
ZK SG600 series products are suitable for solar photovoltaic water pump systems, and are positioned in the environmentally friendly and economical photovoltaic product market, replacing electricity storage with water storage without battery components. The direct current generated by the solar modules is input to the photovoltaic water pump controller, converted into alternating current, and directly drives various water pumps.
1. Adopt advanced MPPT technology to give full play to the power generation efficiency of the solar cell array;
2. Automatically adjust the motor speed and the water output of the water pump according to the change of sunlight intensity;
3. The inverter automatically sleeps when the water level of the reservoir is high, and restarts automatically when the water level is low, realizing automatic water level control;
4. It can prevent the water pump from dry pumping without water when the water source is dry;
ZK Solar Pump Inverter Features
1.Energy saving and environmental protection.
2.LED display operating panel and support remote control
3.Low water probe sensor, and water level control function
Strong lightning protection
4.Bulit -in MPPT function and efficiency up to 99.6%
5.PV working voltage 150VDC to 450VDC
6.Can dive both pmsm and im pump
SG600 solar pump inverter voltage range
|SG600-2T||For 200V AC||150V-450V||450V||100V||310VDC||380VDC|
|SG600-4T||For 400V AC||250V-800V||800V||200V||520VDC||650VDC|
|Recommended MPPT voltage range||Vmpp 131 to 350 VDC for 1S (80V to 350VDC input, 3PH 110 to 220VAC output)
Vmpp 280 to 375VDC for 2T ( 150V to 350VDC input, 3PH 220 to 240VAC output)
Vmpp 486 to 800 VDC for 4T ( 250V to 900VDC input, 3PH 380 to 460VAC output)
|Recommended input voltage (Voc and Vmmp)||Voc 180(VDC), Vmpp 155(VDC) for 1S model or 110V AC pumps
Voc 355(VDC), Vmpp 310(VDC) for 2T model or 220V AC pumps
Voc 620(VDC), Vmpp 540(VDC) for 4T model or 380V AC pump
|Motor type||Control for permanent magnet servo motor and asynchronous motor pumps.|
|Input power||DC power from solar arrays or AC grid power|
|Maximum DC power input||450VDC for 220-240VAC output /900VDC for 380-460VAC output|
|Rated output voltage||3-phase , 110V/160V/220V. 3-phase, 220V/240V/380V/460V/480V|
|Output frequency range||0~50/60Hz|
|Ambient temperature range||(G-type inverter with submersible pumps, and P type for general pumps.|
|Solar pump control special performance||MPPT ( maximum power point tracking), CVT (constant voltage tracking), auto/manual operation,
dry run protection, low stop frequency protection, minimum power input, motor maximum
current protection, flow calculating, energy generated calculating
|Protection function||Phase loss protection, phase short circuit protection , ground to phase circuit protection , input
and output short circuit protection. Stall protection
|Protection degree||IP20, Air force cooling|
|Running mode||MPPT , CVT, variable frequency mode|
|Altitude||Below 1000m; above 1000m, derated 1% for every additional 100m.|
Q1: What’s your trade terms?
A: Term of price: FOB, EXW, CIF Term of payment: By T/T, Paypal, western union etc
Q2: How to ship the order?
A: By sea, by air or by express, any way is ok for us,we have very professional forwarder to provide the best shipping cost, service and guarantee.
Q3:What is your main market?
A:Our brands are very famous in Africa, Middle East, Southeast Asia and South America.
Q4: Do you accept OEM ?
A:Yes, we accept OEM,we can do your logo in the device.