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Welcome to the Science Department
The implementation of the Next Generation Science Standards offers us an opportunity to bring together science and engineering practices in a way that has previously eluded science education. From the understanding of known science concepts we will use this knowledge to identify and solve problems through the application of scientific and engineering practices through authentic labs and investigations. The Grades 6-12 science program will transition to the Next Generation Science Standards and students will notice an immediate change in their science courses. Using a storyline that threads through units of study, students will work through problems and be confronted with questions that can only be answered through research and investigation. Students will engage in problem solving, critical thinking, and data analysis more so than ever before. From Pre-K through Grade 12, science is an integral part of each child's experience in Irvington Public Schools. We continue to partner with STEM organizations that are also devoted to inspiring young minds such as The New Jersey Center for Teaching and Learning (NJCTL) and Students 2 Science (S2S) in helping us develop tomorrow's 21st Century workforce. Irvington High School offers advanced elective courses such as Forensic Science and Advanced Placement courses in Biology, Chemistry, Physics '1', and Environmental Science. We congratulate our high school students that are accepted into Project Seed through the American Chemical Society and always make us proud. Most importantly we are steadfast in our commitment to providing a quality STEM education for ALL of our students.
"Scientists investigate that which already is; Engineers create that which has never been… It is the supreme art of the teacher to awaken joy in creative expression and knowledge." ~Albert Einstein
You Should Know
For up-to-date information on science events at colleges, science scholarships, and other STEM-related contests and opportunities visit our "Colleges & Contests" page
The Science Department of the Irvington Public School District is committed to fostering an environment whereby students experience a comprehensive science program that is supportive, engaging, and challenging. Students will develop the knowledge and skills to confidently go forward as productive and scientifically literate members of society. In support of its mission, the Science Department is dedicated to:
- Developing the knowledge and skill sets necessary for a STEM career and for life-long learning.
- Promoting instruction that fosters the skills of observation, inquiry, analysis and reflection.
- Achieving a climate in which students of varying abilities and backgrounds are encouraged to fully participate in the science program.
- Creating an environment that is conducive for learning through the integration of technology and applying current research to curriculum and instruction.
- Fostering a learning community comprised of staff and students that promotes learning, respect, honesty, and civility.
- High student achievement through a rigorous curriculum and high expectations.
Kindergarten – Grade 8 Science Program
In grades K-8, students participate in an integrated science program that exposes students to topics within the physical sciences, life sciences, and earth and space sciences each year. At the elementary level, our dedicated and creative teaching staff has successfully provided Irvington students with the skills and knowledge they need to pass the NJ ASK4 Science test with over 75% proficiency (district average) for the past seven years. The Progressive Science Initiative serves as the primary resource for students and teachers. This innovative, technology-driven course sequence from the New Jersey Center For Teaching & Learning offers students greater opportunity to participate in science because of the social constructivist approach that fosters peer interactions under the guidance of the teacher. Teachers benefit from being a part of a broad professional learning network in which resource and professional development are on-going. Students benefit from having broad access to resources, smooth transitions between grade levels, a rigorous curriculum, and research-based practices that will help them construct knowledge for greater understanding and retention. For more information on this program, visit www.njctl.org.
The K-8 program Is aligned with the Next Generation Science Standards (NGSS). These new, national set of standards for science was adopted for implementation In New Jersey In July 2014. The standards focus on moving students toward a deeper and richer understanding of scientific concepts and their application. The NGSS focus on fewer topics and are aligned K-12 to offer a comprehensive, aligned, and developmentally-appropriate approach to learning science
High School Science Program
At the high school, several laboratory/Inquiry-based courses are offered depending on previous student performance and interest. Courses available include Environmental Science, Biology, Chemistry, Physics, Forensic Science and Earth & Physical Science. Honors designations are offered in most of these courses in addition to AP Physics 1, AP Biology, AP Chemistry, and AP Environmental Science for students electing to enroll In advanced science courses. The methodology behind the Progressive Science Initiative (PSI) continues into high school. Most students will follow a sequence of courses that begins with physics. Following physics, students will enroll in chemistry and then biology. Advanced Placement (AP) courses in physics, chemistry, and biology are available to students following the successful completion of the pre-requisite course(s). Senior students also have the option of taking Forensic Science as an elective.
IMPORTANT: All New Jersey students, REGARDLESS OF GRADE LEVEL, enrolled in a Biology course must take the BCT.
To prepare for success on this test, Biology students must prepare the entire year. Teachers are carrying out instruction with the purpose of making certain their students have the knowledge and skills necessary to be proficient and successful.
High School Graduation requirements mandate that students take at least 15 credits of science including laboratory biology; chemistry, environmental science, or physics; and an additional laboratory/inquiry-based science.
Next Generation Science Standards Parent Q&A
A parent's guide from the National Science Teachers Association. Click here to view
New Jersey Center for Teaching and Learning
The primary resource used in courses K-12 – http://www.njctl.org
NJ ASK4; NJ ASK8; Biology Competency Test:
For Information regarding state examinations in science visit https://www.measinc.com/nj/default.aspx
Students 2 Science:
Field trip destination and provider of Virtual lab (V-lab) experiences for students. The mission of S2S is to inspire students to pursue STEM careers, increase their aptitude in science, and expose them to the laboratory skills and technology that are used in today's high-tech laboratories. Visit their homepage at http://www.students2science.org or visit their Facebook page at https://www.facebook.com/pages/Students-2-Science
Africam – remote streaming video from locations in Africa showing wildlife in natural surroundings – www.africam.com
American Museum of Natural History – www.amnh.org
Animal Genome Size Database – comprehensive catalog of animal genome size data – www.genomesize.com
The Center for Innovation In Engineering and Science Education – inter-disciplinary, technology-based projects using real-time data hosted by Stevens Institute of Technology- www.ciese.org
Discovery Education – host for teacher STEM resources – www.discoveryeducation.com
Earth Day Network – lesson plans, school greening ideas, and grants – www.earthday.org/education
Environmental Science – online resources for the Pearson textbook http://www.myenvironmentalscience.com
National Geographic – print-rich, reliable site for accessing information on animals and the environment – www.nationalgeographic.com
National Geographic for Kids – portal for accessing kid-friendly science videos and information – www.kids.nationalgeographic.com/kids
National Oceanographic Data Center – national environmental data centers operated by the National Oceanic and Atmospheric Administration – www.nodc.noaa.gov
PBS – Public Service Broadcasting website offering teacher resources by grade span – www.pbs.org/teachers
We Believe that our students are unique individuals, each capable of achieving greatness. That we will guide, challenge and support our students to one day become scientifically literate citizens. That we will Inspire our students to pursue careers in STEM and become life-long learners. That our students will become effective problem solvers and capable of learning independently. That we will guide students to construct their own knowledge and use it in authentic situations.
We Believe scientific literacy will only be achieved through a carefully planned educational program from Pre-K through grade 12 and beyond. That providing a quality science education requires many stakeholders to share responsibility, collaborate, and act with good judgment and ethical intentions. That technology is a tool that offers innumerable possibilities for teaching and learning. That professional development will be used to maintain a quality instructional program and be adapted to changing needs.
We Believe that science involves the study and exploration of the natural and designed world. The Supernatural and subjective opinions have no place in the formation of scientific knowledge. That science begins with observation and inquiry and proceeds through experimentation, analysis and reflection. Science is constantly changing, producing more questions than answers. That a scientific mind is skeptical and accepts that nothing is infallible or forever.
We Believe that we must uphold the highest professional standards as science educators. That we must take responsibility for and promote our own professional learning and growth. That we must advocate for the resources needed to provide a quality science education. That we will be sensitive to the unique learning needs of our students and that we will hold high expectations for their achievement. That we will guide our students to understanding and proficiency. That we will encourage their curiosity and rectify their misconceptions.
We Believe that to achieve scientific literacy one must be observant and curious enough to ask questions and inquisitive enough to seek answers to those questions. That conclusions are supported by scientifically-derived evidence. That scientific laws and theories can be explained thoroughly and applied to authentic situations. That information is evaluated before being accepted.
Michelle Tofel – Grades 6,7
Leah Thornton – Grades 6,7
Kwesi Sarabo – Grades 6-8
Corey Olumbe – Grade 7
Clifford Smith – Grades 7, 8
Njideka Emele – Grades 7, 8
University Middle School
Dr. Sidney Zelin – Grade 6
Deoborah Ervin – Grades 6-8
Michael Guernsey – Grade 7
Terry Johnson – Grade 8
Andrew Obssuth – Grades 7, 8
Irvington High School
Christiana Amadi
Natalie Amores
Theresa Bennin
Gale Bohnarczyk
Dr. Pierette Charles
Erin Bontempo
Gisha Anie George
Ariusz Kornacki
Randall Post
Muhammad Rahman
Christina Rishiy
Ezzard Wilson


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Agent Behaviour, Financial Market and Welfare Theory
An important literature has pointed out the coordination problems faced by the agents, in particular the financial one when they have to manage risk and their portfolio. If we follow Kaldor and its definition of speculation, then we could point out that in this case agents are short term oriented because they have to face to an uncertain reality: uncertainty about the behaviour of their competitors at present time and in the future, and uncertainty about the future reality which will be built by their own decision and action. Then each agent tries to anticipate the behaviour of the others, on one hand to do the same (then in average it is a way to avoid lost), on another hand to try to find an opportunity which has not been seen by the other (the way to earn money, doing what the other can't or may not do), that means mimetic versus opportunism (or free rider behaviour). In both case, we have a kind of reproduction of habits without any collective perspective. The latent hypothesis is that individual decision produces the people satisfaction, the social welfare. We thinks there are three reasons to disagree with this hypothesis ( cancelling that it does not work in reality): a lack of specific tool allowing us to anticipate change and communicate about it; a lack of understanding "what is the common reality"; a lack of an agreement on "what and how can we do together". That means that we need to understand that rules are not a constraint like they could be in the contract theory but more a through out line to help us to coordinate a collective action
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|9 pages
|Date of creation:
|01 Aug 2005
|Date of revision:
|02 Aug 2005
|Type of Document - pdf; pages: 9. Presented at the 4th Global Conference on Business & Economics, Sponsored by Association for Business & Economic Research and International Journal of Busines s & Economics - June 26-28, 2005 - Oxford University, Oxford, UK
|Contact details of provider:
| Web page: http://econwpa.repec.org
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subset: commoncrawl


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Other name: いとしの猫っ毛 小樽篇, The Long Way Home, Itoshi no Nekokke - Otaru Hen, Itoshi no Nekokke Otaru Hen
Status: Completed
Viewed: 294
Summary: Shy Mi-kun and lively Kei-chan have been friends ever since their first meeting in the snowy countryside. The two are inseparable - that is until Kei-chan gets a girlfriend. Mi-kun, who is secretly carrying a torch for his best friend, is devastated. How will their relationship change? Sequel: http://www.mangahere.co/manga/itoshi_no_nekokke_sequel/


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2017-01-06 / Sports
Soccer madness
BOYS' SOCCER /// Coastal Canyon League Royal, Simi Valley boys' teams to battle for glory in league
Rain, rain, go away . . . Coastal Canyon League boys' soccer action is on the way.
Squads at Camarillo, Oak Park, Royal and Simi Valley high schools kick off league action on Jan. 11. Moorpark's league slate begins Jan. 13.
Every team is trying to find the formula to put an end to Royal's streak of five straight league titles, including three Coastal Canyon crowns since the league was formed.
It'll be a dogfight for first place this year.
"I can't remember a single game that's been easy," Royal head coach Ignacio Saldain said. "Even Oak Park can give you a battle. It's a coin toss with these teams. Anyone that thinks there will be an easy game is delirious."
Royal will battle for a sixth straight league title this winter.
The Highlanders, a Division 1 team, are off to a hot start, plowing to a 14-3-1 overall record. They took third at the Burroughs Tournament and at the Crescenta Valley Classic. Royal won its own Royal Showcase on Dec. 17.
Head coach Ignacio Saldain said he hopes the early success translates to league and postseason glory.
"Even though they want a CIF title, they don't think by any means that league is in the bag," Saldain said. "We know the other teams are going to be fierce. It would make their season to beat us."
Royal's potent offense is tough to slow down.
Alexis Garcia, a senior star forward, has 13 goals and five assists.
Jose Torres, also a senior forward, gives Royal another lethal scoring threat after moving from Oregon in the summer.
"He's probably, in the past six games, our best player on the pitch," the coach said of Torres.
Angel Perez, a four-year varsity senior, controls the pace at midfield.
The Highlander defense, led by senior Mike Ramos, is solid in front of senior goalkeeper Nick Lapinid, who has 40 saves in 17 games.
Royal returns to the pitch at 6:45 p.m. Jan. 11 at Oak Park.
The Pioneers must be tired of watching the rival Highlanders hoist all the hardware.
Simi Valley (5-4-2 overall) finished second to Royal last winter.
Pioneer head coach Tim Clunen believes the league will boil down to the same teams again this year.
"Moorpark will give people problems, but at the end of the day, it's going to be Simi and Royal," Clunen said.
He said the key to beating Royal is stopping superstar striker Alexis Garcia.
"There's nobody else on Royal that scares me, but Alexis is a good player," the coach said. "He could win a title for them by himself if no one accounts for him."
Simi Valley has its own ace in the hole. Caleb Malabo, a senior striker, already has 12 goals and two assists this season.
"He doesn't need a lot of space to finish," the coach said of Malabo. "He's probably one of the best in the area in terms of finishing."
Midfielders Blake Ferry, a senior; Ryan Fraye, a junior; and Nicholas Villamizar, a sophomore, push the tempo.
Freshman midfielder Dylan Studer is a star in the making.
Lucas Malabo, Caleb's twin, stars as a senior sweeper on defense.
Simi Valley plays at 6:45 p.m. Jan. 11 at Camarillo.
Manny Galvez, an assistant coach last season, has taken over the program at Moorpark.
Galvez, a firefighter, likes what he sees from his young lineup.
"They get along really well," he said. "They're all working for the same goal. It's just a matter of getting those finishing touches before league."
Moorpark (2-5-3 overall) has a final tuneup at noon today at Saugus, then the Musketeers play host to Oak Park at 6:45 p.m. Jan. 13 in Moorpark's Coastal Canyon opener.
Moorpark's defense is a consistent unit.
Seniors Trevor Hadju, a captain, and Richy Garcia join forces with junior Carson Goodwin and sophomore Johnny Morales on defense.
Manny Martinez, a junior, is a starting goalie.
The Musketeer midfield, which includes senior Cameron Nieven and juniors Justin Magaña and Isaac Hurtado, is feisty.
"They hunt like a pack of wolves," the coach said.
Juan Garcia, a senior captain, junior Ryan Galvez, the coach's son, and sophomore Zayne Elnaka power the offense.
"We'll be able to hold teams off with our midfield and defensive strength," Manny Galvez said. "It's a matter of capitalizing on the opportunities we're given."
Camarillo trots out a stout, physical defense.
The back line helped the Scorpions, a Division 3 team, start this season 5-2-2 overall, including two scoreless draws against Division 1 Santa Barbara, ranked No. 6 in the coaches' poll.
"I'm really happy with the way we're playing as a group," head coach Lonnie Sizemore said. "We're never going to be the technical
Santa Barbara or Pacifica, but we're always going to be scrappy."
Returning defensive starters Kris Hotchkiss, Carlos Alfaro and Alex Bibian, all seniors, solidify the back row.
Jacob Cuccia, a senior captain who roams the midfield, is one of Camarillo's top contributors with two goals and two assists.
"He's really been a great leader for us, making sacrifices," the coach said of Cuccia's versatility. "He can play the wing or up top. If I asked him to play keeper, he would go in goal for me."
Senior forward Matthew Alcozar, senior midfielder Matias Montory and junior midfielders Alex Medina and Brandon Duran are also playing well.
Camarillo is next in action at 6 tonight at home against Oxnard.
The Scorpions open league play at 6:45 p.m. Jan. 11 at home against Simi Valley.
Oak Park's found some consistency on the field.
The Eagles (3-2-2 overall) are unbeaten in their past four games, picking up wins against Calabasas and Westlake.
"We're looking good," head coach Kai Werring said. "We've had kind of an up-and-down preseason, but we're coming into league with positive wins."
Oak Park opens Coastal Canyon play at 6:45 p.m. Jan. 11 against Royal at home.
The Eagles are playing smart, controlled soccer.
"They're picking and choosing the best times to move the ball forward and keep possession of the ball," Werring said.
Danny Taras, a senior who has three goals, thrives at forward.
"He's really buying into our possession-based philosophy," the coach said of Taras.
Fellow senior forwards Lars Defty and Kaya Sunguroglu are also playing well.
Seniors Cole Spooner and Garrett Naylor control the midfield. Daniel Glickstein and Alec Greenbaum, both senior defenders, keep opposing scorers at bay.
"My expectations are very high," Werring said. "It's up to the boys to prove it. We have to do our best at being patient, trying to wear teams down."


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TEKJournalism UK is now an archived website. For the latest news and opinion from the team drop by NEWTEKWORLDNEWS here
Organisers of protests though stressed they wanted to make it plain that UKIP is not welcome in Scotland but foreigners are.
In spite of Scotland UKIP continues to garner support. UKIP's gains are others losses.
Should David Cameron worry about defections to UKIP? Hell we should all worry about that one.
Voters should not forget that UKIP are simply Tories in other clothing. If no party wins a political majority in 2015 UKIP may do a deal. Coalitions could become a permanent fixture in British politics as people remain divided. If you think a Lib Dem Tory alliance is bad how about a Tory UKIP one? It does not bear thinking about.
Your vote and political support is precious. Use both wisely.
5/21/2013 10:59:20 pm
How derailed can you get? The EU has a budget with 84billion Euro unaccountable for and David Cameron still wants to stick to it. What the reasons are one ponders? If he thinks he can fob the people again with a promise about a referendum he really needs to think again. It cost again a £19billion for membership, £6billion for a new fund to help bankrupt countries to get a membership in the EU, endless bail-outs and the man is still not willing to call it a day. This is incredible.
5/22/2013 02:10:32 am
It is my friend but remember that UKIP are Tories just anti EU.
Leave a Reply.
TEKJournalism UK is now an archived website. For the latest news and opinion from the team drop by NEWTEKWORLDNEWS here


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We have dated for 2 months and it ended up not good, she rejected me. She said she doesn't feel the same and she ended it because it is too unfair for me. It just makes me confuse, she keeps saying she miss me and wants to see me, I thougth everything was fine.
Now I am trying contact her but I am not sure if it is worth it or just totally move on.
Most Helpful Girl
If she doesn't feel the same then let her go. What's the point of reaching out to someone who admits they don't feel the same way.
Hi. I know this sounds stupid. But is there a possibility the she might have changed her mind?
- Show AllShow Less
Thanks for the MHO
You are welcome. :)


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Category: casting calls
I would like to send out a big THANK YOU to all of my wonderful clients and Facebook fans, I can't thank you all enough for helping me live my dream of capturing beautiful memories for a living. That said, I have decided a contest was in order, just in time for the upcoming holiday season!! This is the largest contest I have ran as of today and I am extremely excited to announce it!! So . . . I know I said on my Facebook page that a free photo session and holiday cards were going to be included, however, what I didn't tell you is that the winner will receive an ALL INCLUSIVE PHOTO SESSION!
~ 2 hour portrait session with Christy (Family Session, Just the Kids or Beach Session)
~ Entire Gallery of artistically edited high resolution digital images by Christy
(20-30 images total will be provided on the CD with a limited copyright release)
~ (1) 5×7 professional print of each image that is provided on the CD
~ A set of our Holiday Cards/Birth Announcement/Personalized Card, etc. (24 cards/set)
~ A SURPRISE that I'm not going to tell you about 🙂
This is an extremely awesome giveaway
Valued at $750.00!!!
That said, I believe that a little work is in order to earn it!!
There are three ways to win:
1. Leave a comment on this blog post and tell me why
you would like to win a photography session with Christy Marrone Photography.
2. "Like" me on Facebook.
Just follow the link below and click "Like" to become a Facebook Fan.
Come back here and leave me a comment on this post to let me know you have done so.
(If you are already a fan, you can just leave a comment that you are already a fan on this post for your entry).
Christy Marrone Photography Facebook Page – CLICK HERE!
3. Share this post
Share my contest on your Facebook page to tell your friends and family about our giveaway!
Come back here and leave a comment on this blog post saying that you have done so.
You may enter the contest 3 times total (not every day) by completing the 3 steps listed above.
Make sure you leave a separate comment when you complete each of the items above so they will be counted as separate entries. All entries must be in by Monday, October 29th at 9:00 am, any comments made after this time will be disqualified. I will announce the winner by luck of random draw on Monday, October 29th by 7:00 pm.
Please email me at firstname.lastname@example.com if you have any questions, please do not post your questions on the blog! Also, note that I will have to "approve" your comment before it is posted on the blog post. If you do not see your comment right away, please know that I will approve it as soon as possible.
You must live in the Tampa/St. Petersburg and surrounding areas to enter!
~~ Christy ~~
I finally photographed a baby boy and they came out so well! He is such a sweetie and was a joy to photograph. Here are a few so far, enjoy!
I am currently building up my newborn and maternity portfolio and the Bump to Baby and Baby of Mine Session Plans will be 30% off until September 30th!
Even if your due in October and beyond you can still get this discount as long as you contact me to set up your appointment and pay your retainer fee before September 30th!
Maternity (must be between 25-35 weeks of pregnancy)
Newborn (1-10 Days Old)
Signed Model Release Required
Retainer Fee: $200 (due at time of booking, goes towards session package price)
Remaining session balance is due one week after the session takes place
Bump to Baby Session
Whats included:
Mini Maternity Session
Newborn Session
25 Birth Announcements
(2) 8×10 Prints (one from each session)
8×8 Custom Art Book
1 set of 4×6 Keepsake Prints from "bump" session
1 set of 4×6 Keepsake Prints from "baby" session
1 Custom Photo Box
Baby of Mine Session
Whats included:
Newborn Session
50 Birth Announcements
1 Custom Photo Box
1 set of 4×6 Keepsake Prints
8×8 Custom Art Book
Contact me at email@example.com with any questions or to book a Baby Plan Session!
Christy Marrone Photography is holding a casting call for newborns! If you are expecting in the next month or two please contact Christy at email@example.com and learn how you can get a FREE session for your newborn! Just type "Newborn Casting Call" in the subject line. Be sure to let me know your due date and if you know your baby's gender please add that as well. Space is limited, so act fast!


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|Home | About | Journals | Submit | Contact Us | Français
Animal cells control their membrane lipid composition within narrow limits, but the sensing mechanisms underlying this control are largely unknown. Recent studies disclosed a protein network that controls the level of one lipid – cholesterol. This network resides in the endoplasmic reticulum (ER). A key component is Scap, a tetrameric ER membrane protein that binds cholesterol. Cholesterol binding prevents Scap from transporting SREBPs to the Golgi for activation. Using a new method to purify ER membranes from cultured cells, we here show that Scap responds cooperatively to ER cholesterol levels. When ER cholesterol exceeds 5% of total ER lipids, SREBP-2 transport/activation are abruptly blocked. They resume when ER cholesterol falls below the 5% threshold. The 5% threshold is lowered to 3% when cells overexpress Insig-1, a Scap-binding protein. Cooperative interactions between cholesterol, Scap, and Insig create a sensitive switch that controls the cholesterol composition of cell membranes with remarkable precision.
The endoplasmic reticulum (ER) of mammalian cells contains an elaborate feedback system that senses the level of membrane cholesterol and modulates the transcription of genes that mediate cholesterol synthesis and uptake. This system maintains cholesterol homeostasis by increasing cholesterol synthesis and uptake when membrane cholesterol falls and by down-regulating these processes when excess cholesterol accumulates. The regulatory system revolves around a family of membrane bound transcription factors called Sterol Regulatory Element-binding Proteins (SREBPs), and it has been called the SREBP pathway (Brown and Goldstein, 1997).
Central to the SREBP pathway is a polytopic membrane protein called Scap. Scap forms a complex with SREBPs, and it also binds CopII proteins. The latter cluster the Scap/SREBP complex into coated vesicles that bud from the ER and travel to the Golgi complex (Sun et al., 2007). There the SREBP is processed by two proteases, liberating the active transcription factor domain which enters the nucleus and activates the genes for cholesterol synthesis and uptake. When excess cholesterol accumulates, the Scap/SREBP complex binds to a resident ER protein, designated Insig. This binding precludes the binding of CopII proteins. As a result, the Scap/SREBP complex remains in the ER, transcription of the target genes declines, and cholesterol synthesis and uptake fall (Goldstein et al., 2006).
The mechanism by which Scap senses cholesterol has been elucidated through cholesterol-binding studies conducted with the purified octahelical membrane domain of Scap which forms tetramers in detergent solution (Radhakrishnan et al., 2004). This domain of Scap binds cholesterol with saturation kinetics and with stereospecificity. When studied in membrane vesicles, cholesterol binding leads to a conformational change in the membrane domain of Scap that can be monitored by the appearance of a new cleavage site that is accessible to trypsin (Brown et al., 2002; Adams et al., 2004). This conformational change promotes the binding of Scap to Insig. The Scap:Insig ratio is crucial for determining the sensitivity of the cellular response to cholesterol. Overexpression of Insig lowers the threshold for suppression of SREBP cleavage by cholesterol in intact cells (Yang et al., 2002).
A major unanswered question relates to the level of cholesterol in ER membranes that is necessary in order to inhibit SREBP processing. It has long been known that the concentration of cholesterol in ER membranes is much lower than the concentration of cholesterol in plasma membranes when expressed as the molar ratio of cholesterol to phospholipids (Bergstrand and Dallner, 1969; Zambrano et al., 1975). However, there have been no measurements of the changes in ER cholesterol that are necessary to regulate SREBP processing. In part, this lack of information is attributable to the difficulty in attempting to obtain a pure population of ER membranes that would permit direct measurements of cholesterol levels. Lange and Steck (Lange and Steck, 1997; Lange et al., 1999) approached the problem indirectly by measuring the percentage of total cholesterol in human fibroblasts that could be esterified in vitro by the resident ER enzyme acyl-CoA:cholesterol acyltransferase (ACAT). From these data, they concluded that the ER contained 0.5% of total cholesterol when cells were depleted of sterols. This value rose to 5% when the total cellular cholesterol content was increased 2-fold by addition of cholesterol complexed to hydroxypropyl-cyclodextrin (Lange et al., 1999).
In the current studies, we have developed a procedure for purification of ER membranes from cultured Chinese hamster ovary (CHO) cells, which allows direct measurement of the ER cholesterol content by mass spectrometry. With this assay, we demonstrate that the suppression of SREBP cleavage by cholesterol exhibits a cooperative threshold response. Suppression occurs abruptly when the molar cholesterol level in ER membranes reaches 4–5% of total lipids on a molar basis. Through such cooperativity, cells are able to respond with great precision to changes in membrane cholesterol, and thereby to fine-tune their cholesterol content.
We developed a fractionation scheme to isolate pure ER membranes from CHO-K1 cells as outlined in Figure 1 (left panel). Efficient and reproducible cell homogenization was achieved through the use of a ball-bearing homogenizer (Balch and Rothman, 1985). The cell homogenate (designated Fraction A in Figure 1) was subjected to three centrifugation steps, which progressively separated ER membranes from other organelles. The results of the first two centrifugation steps are summarized in Figure 1 (right panel). Centrifugation of the homogenate at 3000g eliminates unbroken cells and the heaviest membranes. As assessed by immunoblot analysis of the fractions, a soluble nuclear protein marker (CREB) and a mitochondrial membrane protein marker (Prohibitin-1) are mostly retained in the 3000g pellet (Fraction B), whereas significant amounts of all other membrane markers escape into the 3000g supernatant (Fraction C). Fraction C was then subjected to centrifugation through a discontinuous sucrose gradient and yielded two distinct bands of membranes (Fractions D and E). The small amount of CREB in Fraction C is absent in Fractions D or E and likely represents a portion that was released into the cytosol, owing to rupture of the nuclear membranes during the fractionation procedure. The Golgi membrane proteins (GM130 and GRASP65), plasma membrane proteins (transferrin receptor, caveolin-1, and Na+/K+ ATPase), and the early endosomal membrane protein (EEA1) are found in the light membrane Fraction D and are almost completely absent from the heavy membrane Fraction E. The peroxisomal membrane protein (PMP70) is localized predominantly in Fraction E, and a lysosomal membrane protein (LAMP1) is present in both Fractions D and E. ER-resident membrane proteins (Sec61α and ACAT-1) are localized almost completely to Fraction E. Scap, a membrane protein that cycles between ER and Golgi membranes (Goldstein et al., 2006), is found in both Fractions D and E. The two forms of Site-1 protease, the inactive precursor form and the processed active form (Espenshade et al., 1999), are separated into Fraction E and Fraction D, respectively.
The aforementioned data suggest that Fraction E contains partially purified ER membranes devoid of membranes from the nucleus, mitochondria, plasma membrane, Golgi and early endosomes, but still contaminated with lysosomal and peroxisomal membranes.
In order to further purify ER membranes, we subjected Fraction E to an additional centrifugation step through a continuous iodixanol gradient (van Veldhoven et al., 1996). As shown in Figure 2A, the lysosomal and peroxisomal membrane proteins, LAMP1 and PMP70, are exclusively localized to a floating membrane layer (tube 13, which is designated as Fraction F). On the other hand, ER membrane proteins (Sec61α, ACAT-1, and Scap) divide into two portions. A portion of all three of these proteins float (tube 13, Fraction F), but the majority of all three are found in denser regions of the gradient (tubes 2–7), which were pooled and designated Fraction G. Fraction G is devoid of the lysosomal protein LAMP1 and the peroxisomal protein PMP70. Moreover, fraction G did not show any detectable signal for plasma membrane markers (Na+/K+ ATPase and transferrin receptor) even when we analyzed aliquots that were 10-fold higher than those shown in Figure 2.
We further characterized the iodixanol gradient fractions with enzyme assays as shown in Figures 2B and 2C. Consistent with the immunoblot results of Figure 2A, the activities of two lysosomal enzymes (acid phosphatase and β-hexosaminidase) and a peroxisomal enzyme (catalase) were localized exclusively to Fraction F (Figure 2B), whereas the activities of ER enzymes (glucose-6-phosphatase and cytochrome c reductase) were present in both Fractions F and G (Figure 2C). The activities of a Golgi enzyme (mannosidase II) and a plasma membrane enzyme (alkaline phosphodiesterase I) were localized to Fraction D. Neither of these enzyme activities were detected in any fractions from the iodixanol gradient (data not shown). Based on both the immunoblot and enzymatic analysis, we conclude that Fraction G contains ER membranes and contains less than 10% contamination with lysosomal, peroxisomal, plasma, or other membranes. Fraction G is hereafter designated as "purified ER membranes."
We examined the morphology of membranes from Fractions F and G by electron microscopy. Figures 2D and 2E show images representative of more than 50 different sections examined. Fraction F is a heterogeneous mixture, containing vesicles 50–200 nm in diameter, large structures with intense electron staining (characteristic of lysosomes), and other irregularly shaped structures (Figure 2E). Fraction G is more homogeneous, containing primarily spherical vesicles 50–200 nm in diameter (Figure 2D). Since we did not take measures to preserve ribosome attachment, the ER vesicles are not decorated by ribosomes. Immunoblot analysis of ribosomal subunit S6 showed that it did not localize with the membranes of Fractions D or E and was likely dissociated into the cytosol (data not shown).
The availability of purified ER membranes allowed us to study how changes in cell lipids alter the ER cholesterol content and how these alterations regulate the exit of the Scap/SREBP complex. We first sought to measure the effects of cyclodextrin-induced cholesterol depletion on cholesterol concentrations in both the whole cell and ER and on proteolytic processing of SREBP-2. Cholesterol percentages are expressed as moles of cholesterol divided by moles of total lipid × 100. The lipids are defined as the measured moles of sterols plus phospholipids. As shown in Figure 3A, switching cells from medium containing 5% FCS to a lipid-poor medium containing 1% hydroxypropyl cyclodextrin (HPCD) triggered the conversion of SREBP-2 from an inactive precursor form to an active cleaved nuclear form in 0.5 to 1 hr (top panel). This increase in SREBP-2 processing was accompanied by a decrease in the ER cholesterol content from ~7% to ~2% of the total lipid in the ER (middle panel) and a decrease in whole cell cholesterol from ~35% to ~10% of whole cell total lipids (bottom panel). We then analyzed the consequences of adding back either cholesterol or 25-hydroxycholesterol (25-HC). Both sterols were added in complexes with methyl-β-cyclodextrin (MCD). After the initial cholesterol depletion, all of the visible SREBP-2 was found in the cleaved nuclear form (zero time in Figures 3B and 3C, top panels). After addition of 50 μM cholesterol/MCD, the nuclear form declined and the uncleaved membrane precursor form of SREBP-2 became visible at 2 hr (Figure 3B). By 4 hr the nuclear form was barely visible, indicating that SREBP-2 processing had been inhibited nearly completely. Over the 4-hr time period, the addition of 50 μM cholesterol/MCD increased ER cholesterol content from ~0.13 to ~1.3 nmol/dish (representing 1% and 10% of total ER lipids, respectively) and whole cell cholesterol content from ~40 to ~90 nmol/dish (representing 13% and 35% of total cellular lipids, respectively) (Figure 3B, middle and bottom panels). In the sterol-depleted cells, the content of 25-HC was below the detectable limit (~25 fmol) in both the ER and whole cell fractions (Figure 3C). Four hr after addition of 10 μM 25-HC to the culture medium, SREBP-2 cleavage was suppressed to the same degree as seen with cholesterol addition. This treatment did not alter cholesterol levels significantly in either the whole cell or the ER (Figure 3C, middle and bottom panels). On the other hand, the level of 25-HC increased from below the detectable limit to ~5 pmol/dish in the ER (representing 0.042% of total lipids) and to ~4 nmol/dish in the whole cell (representing ~1.5% of total lipids). Thus, a relatively small amount of 25-HC in the ER was able to inhibit SREBP-2 cleavage without causing a measurable change in ER cholesterol. In experiments not shown, we verified that the ER purification procedure is not altered at the extreme conditions of lipid depletion or addition used in these studies.
We next sought to determine whether a threshold exists between ER cholesterol levels and SREBP-2 processing. We again depleted cellular cholesterol by treament with 1% HPCD for 1 hr. We then added varying levels of cholesterol in MCD for various times. As shown in Figure 4A, addition of 10 μM cholesterol/MCD had no effect on SREBP-2 processing even after 4 hr, whereas 50 μM cholesterol/MCD suppressed SREBP-2 processing after 2 hr and 250 μM cholesterol/MCD suppressed SREBP-2 processing even earlier than 1 hr. Figure 4B (top panel) shows a quantification of the immunoblot from Figure 4A by densitometry, where the active nuclear form of SREBP-2 is expressed as a fraction of the total amount of SREBP-2 (precursor form plus nuclear cleaved form). The bottom panel of Figure 4B shows the cholesterol content of purified ER membranes at each level of added cholesterol/MCD. To gain additional data, we carried out two other similar experiments where cells were first depleted of cholesterol by treatment with 1% HPCD for 1 hr and then with different concentrations of cholesterol (in MCD) for various times. In a fourth experiment, we varied membrane cholesterol in the other direction, i.e., by first incubating cells with 50 μM cholesterol/MCD for 4 hr and then depleting cholesterol with 1% HPCD for various times (open squares in Figure 4C). We then plotted nuclear SREBP-2 as a function of ER cholesterol using the data from all four experiments. This analysis revealed a threshold effect. When the mole fraction of cholesterol in the ER was less than 2%, nuclear SREBP-2 levels were maximal. As the ER cholesterol rose above 2%, there was a sharp drop in SREBP-2 processing with 50% inhibition occurring at about 4.5% cholesterol. The sigmoidal nature of this curve was confirmed by subjecting the data to analysis by the Hill equation, which revealed a high degree of positive cooperativity with a Hill coefficient of 3.7.
In addition to delivery in MCD complexes, cholesterol can be delivered to cells by receptor-mediated endocytosis of lipoprotein particles that bind to LDL receptors. One of these lipoproteins, β-VLDL, is isolated from plasma of cholesterol-fed rabbits (Kovanen et al., 1981) and is highly enriched in cholesteryl esters. These esters are hydrolyzed in lysosomes and the free cholesterol is transported to the ER. We used β-VLDL to deliver cholesterol to lysosomes in CHO-K1 cells and monitored SREBP-2 processing and ER cholesterol as in Figure 4. We first treated the cells with 1% HPCD for 1 hr to deplete cholesterol and then incubated them with increasing levels of β-VLDL for various times. As shown in Figure 5A, β-VLDL at 3 μg protein/ml showed maximal suppression of SREBP-2 cleavage at 2 hr. Increasing the β-VLDL concentration to 10 μg/ml did not shorten this time. The top panel of Figure 5B shows a densitometric quantification of the immunoblot from Figure 5A, and the bottom panel of Figure 5B shows the cholesterol content from purified ER membranes after each treatment. The data suggest that cholesterol from β-VLDL requires more than 1 hr to reach the ER, whereas cyclodextrin-delivered cholesterol reaches the ER in less than 1 hr (compare Figures 5 and and44).
To quantify the correlation between ER cholesterol and SREBP-2 processing after β-VLDL treatment, we carried out five additional experiments using different concentrations of β-VLDL for various times. The combined analysis is shown in Figure 5C and again there is a clear threshold effect. We observed 50% inhibition of SREBP-2 cleavage at mole fraction of ER cholesterol of about 5.7%. This is slightly higher than the 4.5% value obtained with cholesterol/MCD (Figure 4). The significance of this difference is uncertain. Once again, analyzing data using the Hill equation reveals a high degree of positive cooperativity with a Hill coefficient of 3.5, similar to that observed with cholesterol/MCD.
We next sought to determine whether an increase in Insig-1 in the ER membrane would lower the threshold for inhibition of SREBP-2 processing. In order to test this hypothesis, we took advantage of a CHO-7 cell line that stably overexpresses human Insig-1 (designated CHO/pInsig1-Myc cells) (Yang et al., 2002). Immunoblotting and densitometry show that the total amount of Insig-1 is elevated ~6.2-fold in CHO/pInsig1-Myc cells compared to the parental CHO-7 cells (Figure 6A). (The two bands of Insig-1-Myc6 in the immunoblots of the transfected cells represent versions with alternative initiator methionines, both of which are equally active (Yang et al., 2002)). Cholesterol levels in both cell lines were manipulated by first treating them with 1% HPCD for 1 hr to deplete cholesterol and then with increasing levels of cholesterol/MCD or β-VLDL for various times. We then assayed SREBP-2 processing and measured cholesterol levels in purified ER membranes as described for Figures 4 and and5.5. In experiments not shown, we verified that the ER purification procedure developed for CHO-K1 cells applies equally well to these two cell lines. The combined analysis from three different experiments (Figure 6B) shows clear threshold effects, just as in Figures 4 and and5.5. The mole fraction of cholesterol in the ER at which the processing of SREBP-2 is blocked by more than 50% is shifted to 1.8-fold lower values in the cells expressing increased vs. normal Insig-1 levels (i.e., from 5.5% to 3.1%). Analysis of the data by the Hill equation shows that the additional Insig increased the Hill coefficient from 5.1 to 6.9, sharpening the threshold for inhibition of SREBP-2 processing.
Together with previous data, the current studies reveal a delicate balance that controls the level of membrane cholesterol in mammalian cells (Figure 7). When cultured cells are depleted of cholesterol, the cholesterol content of ER membranes falls below 5 mol%. Under these conditions, the MELADL sequence in Scap binds CopII proteins which cluster the Scap/SREBP complex into transport vesicles that move to the Golgi where SREBPs are processed (Sun et al., 2007). When ER cholesterol rises above 5 mol%, the cholesterol binds to Scap, inducing Scap to bind Insig. As a result, CopII proteins no longer bind to Scap, and the Scap/SREBP complex is not transported to the Golgi. Inasmuch as Golgi processing is essential for SREBP activation, the net effect of cholesterol accumulation is to reduce transcription of genes required for cholesterol biosynthesis and uptake, thereby lowering membrane cholesterol and restoring cholesterol balance (Sun et al., 2007; Goldstein et al., 2006). Previous studies have shown that sterols regulate the SREBP pathway at the ER-to-Golgi transport step (which produces the active nuclear form of SREBPs) and not at other points such as degradation of the nuclear form (Yang et al., 1994; Yang et al., 1995).
The current study was made possible by obtaining highly purified ER membranes, which allowed us to accurately measure changes in ER cholesterol using mass spectrometry. We established a three-step protocol, beginning with gentle but efficient disruption of cells with a ball-bearing homogenizer followed by two gradient centrifugation steps that efficiently separate ER membranes from those of other organelles. The method was shown to work successfully in three CHO cell lines – CHO-K1, CHO-7, and a line of CHO-7 cells that are stably transfected with Insig-1. In studies to be reported elsewhere, we found that the method also works for cultured human fibroblasts (L. Abi-Mosleh, A. Radhakrishnan, M. S. Brown, and J. L. Goldstein, unpublished). We were especially careful to eliminate plasma membranes, lysosomes, and other membranes of the endocytic pathway as these have the highest concentrations of cholesterol (Figures 1 and and22).
Despite its virtual insolubility, cholesterol can be transported between various organelle membranes. Cholesterol depletion from the plasma membrane results in a drop in ER cholesterol from a resting value of ~7 mol% to ~2 mol% in 0.5 hr, triggering the processing of SREBP-2 to its active nuclear form (Figure 3A). This pathway is reversible: replenishing the plasma membrane cholesterol pool by the delivery of cholesterol from MCD complexes or from β-VLDL particles returns ER cholesterol to its original resting value, stopping SREBP-2 processing (Figures 3B, ,4,4, and and5).5). When cholesterol is delivered in MCD, the kinetics are dominated by mass action as higher concentrations of cholesterol/MCD increase the speed with which cholesterol accumulates in the ER (Figures 4A and 4B). The kinetics differ when cholesterol is delivered in β-VLDL. Under these conditions, ER cholesterol does not increase until 1 hr even at the highest level of β-VLDL tested (Figure 5). This difference in kinetics is likely attributable to the distinct pathways taken by MCD- or β-VLDL-derived cholesterol to reach the ER. MCD-cholesterol likely partitions into the plasma membrane, from which it is transferred rapidly to the ER by vesicular or non-vesicular carriers (Prinz, 2007). On the other hand, β-VLDL must be taken up by endocytosis, its cholesteryl esters must be hydrolyzed, and the resultant cholesterol must be exported from lysosomes. One or more of these steps constitutes a rate-limiting barrier.
The pathways that transport cholesterol from one organelle to another allowing the ER to efficiently monitor cellular cholesterol levels are largely unknown. In studies by Xu and Tabas (1991) and Lange, et al. (Lange et al., 1999), ER cholesterol levels (as estimated by ACAT activity) rose only after total cell cholesterol crossed a threshold value, suggesting that the step(s) mediating transport between different cellular organelles may also be subject to regulation. Moreover, studies in artificial membranes have suggested a model whereby the chemical activity of cholesterol in the plasma membrane, by being poised near a threshold point, could serve as a regulatory sensor (Radhakrishnan and McConnell, 2000).
It has long been known that oxysterols like 25-HC can block cholesterol synthesis when added to cultured cells (Kandutsch and Chen, 1974; Brown and Goldstein, 1974). Once the SREBP pathway was discovered, it became clear that 25-HC acted through the same mechanism as cholesterol, i.e., by sequestering the Scap/SREBP complex in the ER (Brown and Goldstein, 1997). Yet, 25-HC was shown not to bind to Scap (Radhakrishnan et al., 2004). Rather, it binds to the other member of the Scap/Insig complex, i.e., Insig (Radhakrishnan et al., 2007). 25-HC binding to Insig promotes the same reaction as cholesterol binding to Scap, i.e., formation of the Scap/Insig complex (Sun et al., 2007). The current results support this explanation. Thus, addition of 10 μM 25-HC/MCD to cells caused a reduction in SREBP-2 processing at a time when the ER cholesterol level had not risen measurably (Figure 3C). When added at higher concentrations for longer periods of time, 25-HC can increase ER cholesterol as evidenced by an increase in the rate at which endogenous cholesterol is esterified by the ER resident enzyme ACAT (Brown et al., 1975). Consistent with this conclusion, Lange and Steck (1997) used their indirect method to demonstrate an increase in ER cholesterol 18–48 hr after adding 50 μM 25-HC to cells. The current results show that SREBP-2 cleavage is suppressed as early as 2 hr when no increase in ER cholesterol occurs.
Detailed analysis of the ER cholesterol concentration required to block SREBP-2 processing revealed a sharp, switch-like response at 4.5–5.7 mol% cholesterol in both CHO-K1 and CHO-7 cells (Figures 4–6). The threshold could be reached in either direction, i.e., whether cholesterol was being removed or added. In an initial attempt to model this response curve, we performed a Hill analysis, which assumes that a single reaction governs the inhibition of SREBP-2 cleavage. This is an oversimplification of the complexity of the SREBP pathway, but it provides us with a convenient starting point to quantify the sigmoidal behavior. The analysis suggested a high degree of positive cooperativity (best-fit Hill coefficient of 3.5–5.1 in Figures 4–6). Since the Hill coefficient is close to 4 and since Scap is a tetramer (Radhakrishnan et al., 2004), it is tempting to speculate that, analogous to the binding of oxygen to tetrameric hemoglobin (Koshland et al., 1966), the binding of cholesterol to one of the four binding sites in the Scap tetramer increases the affinity of the other sites for cholesterol, resulting in sharp cooperativity. Unfortunately, this hypothesis is difficult to test in our in vitro binding assay which contains purified Scap and cholesterol in detergent solutions (Radhakrishnan et al., 2004). In this system the rate-limiting step is transfer of cholesterol from detergent micelles to micelles that contain Scap. Because of this detergent effect, we are unable to measure cooperativity in the binding reaction (Radhakrishnan et al., 2004).
In CHO-7 cells with increased Insig-1 levels, the ER cholesterol that caused 50% inhibition of SREBP-2 processing was shifted from 5.5 mol% to 3.1 mol% (Figure 6), and the Hill coefficient increased from 5.1 to 6.9. The mechanism for this enhanced sensitivity is currently not known. It is possible that the interaction of Insig with Scap enhances the affinity of Scap for cholesterol, providing yet another source of cooperativity. The dependence of the cholesterol threshold on the Insig/Scap ratio increases the importance of the relative levels of these two proteins in controlling cholesterol metabolism in cells and in the body. In the liver, Insig levels are under exquisite control by insulin, a phenomenon that may play a role in regulating lipid synthesis in that crucial organ (Engelking et al., 2005; Yabe et al., 2003).
We obtained hydroxypropyl-β-cyclodextrin (HPCD) and methyl-β-cyclodextrin (MCD) from Cyclodextrin Technologies (High Springs, FL); 25-hydroxycholesterol (25-HC) from Steraloids, Inc.; cholesterol and iodixanol (Optiprep density gradient medium) from Sigma; monoclonal anti-CREB and anti-transferrin receptor antibodies from Invitrogen; monoclonal anti-caveolin-1 antibody from BD Biosciences; polyclonal anti-prohibitin-1 antibody from Abcam (Cambridge, MA); polyclonal anti-Na+/K+ ATPase antibody from Cell Signaling Technology (Danvers, MA); polyclonal anti-EEA1 and anti-PMP70 antibodies from Affinity BioReagents (Golden, CO); polyclonal anti-Sec61α from Millipore; and horseradish peroxidase-conjugated, affinity-purified donkey anti-mouse and anti-rabbit IgGs from Jackson ImmunoResearch Laboratories. Rabbit polyclonal antibody IgG-R139 raised against hamster Scap (amino acids 54–277 and 540–707) (Sakai et al., 1997); rabbit polyclonal antibody U1683 against hamster S1P (amino acids 1023–1052) (Espenshade et al., 1999); rabbit polyclonal antibody 81-2 against hamster ACAT-1 (amino acids 1–140) (Cao et al., 1996); and monoclonal antibody IgG-7D4 against hamster SREBP-2 (amino acids 32–350) (Yang et al., 1995) have been described in the indicated references. Other antibodies used in this study were generous gifts from J. Seemann, (polyclonal anti-GM130, anti-GRASP65); W. Dunn, (polyclonal anti-LAMP1); and from Y. K. Ho and Linda Donnelly (anti-Insig-1, hybridoma culture medium). Solutions of compactin and sodium mevalonate were prepared as described (Brown et al., 1978). Rabbit β-migrating very low density lipoproteins (β-VLDL, d <1.006 g/ml) (Kovanen et al., 1981) and newborn calf lipoprotein-deficient serum (Goldstein et al., 1983) were prepared by ultracentrifugation as described in the indicated reference. Stock solutions of complexes of cholesterol/MCD or 25-HC/MCD were prepared at a final sterol concentration of 2.5 mM and a sterol/MCD ratio of 1:10 as described (Brown et al., 2002). From many experiments in our laboratory, we have consistently found that HPCD is more effective than MCD in removing cholesterol from cells and MCD is more effective in delivering sterols in cells.
Buffer A contains 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl. Buffer B is Buffer A supplemented with a protease inhibitor mixture (50 μg/ml leupeptin, 25 μg/ml pepstatin A, 10 μg/ml aprotinin, 25 μg/ml phenylmethylsulfonylfluoride, and 1/100 tablet/ml of complete EDTA-free protease inhibitor cocktail (Roche)). Medium A is a 1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium containing 100 units/ml penicillin and 100 μg/ml streptomycin sulfate. Medium B is Medium A supplemented with 5% (v/v) fetal calf serum. Medium C is Medium A supplemented with 5% (v/v) newborn calf lipoprotein-deficient serum, 50 μM sodium compactin, and 50μM sodium mevalonate.
Cells were maintained in monolayer culture at 37°C in 8–9% CO2. CHO-7 cells are a clone of CHO-K1 cells selected for growth in lipoprotein-deficient serum (Metherall et al., 1989). CHO/pInsig1-Myc cells are a clone of CHO-7 cells stably transfected with pCMV-Insig-1-Myc6 (Yang et al., 2002).
On day 0, cells were set up in medium B at 700,000 cells/10-cm dish. On day 3, the cells were subjected to the indicated treatments, then washed twice with phosphate-buffered saline, and then scraped into 50-ml tubes. All further operations were carried out at 4°C. The cell pellets were collected after centrifugation at 500g for 10 min and resuspended in 1 ml of ice-cold Buffer B containing 15% (w/v) sucrose. Cells were then disrupted by passage 13 times through a ball bearing-homogenizer with a 10 μm clearance (Balch and Rothman, 1985). The homogenized cells (Fraction A, see fractionation scheme in Figure 1) were centrifuged at 3000g for 10 min to yield a pellet (Fraction B) and a supernatant (Fraction C). The supernatant was diluted to a total volume of 3 ml using Buffer A containing 15% sucrose. A discontinuous sucrose gradient was generated in a SW41 tube (Beckman Instruments) by overlaying the following sucrose solutions all in Buffer A: 2 ml 45%, 4 ml 30%, 3 ml of the diluted supernatant in 15% sucrose, and 1 ml 7.5%. The gradient was centrifuged at 100,000g in a SW40Ti rotor (Beckman) for 1 hr and allowed to slow down without the application of a brake, after which two bands of membranes were clearly visible and collected using a Pasteur pipet. The sucrose concentration in the light membrane (Fraction D) ranged from 14–18% and that in the heavy membrane fraction (Fraction E) ranged from 34–38%, as measured using a refractometer (model ABBE-3L, Milton Roy Co., Rochester, NY). A discontinuous iodixanol gradient was generated by underlaying, in succession, 2.25 ml of 19%, 21%, 23%, and 25% (v/v) iodixanol, all in buffer A. This discontinuous gradient was allowed to stand for 1–2 hr, allowing for diffusion across the interfaces to form a continuous gradient. The heavy membrane fraction from above (E) was loaded at the bottom of this gradient, which was then centrifuged for 2 hr at 110,000g in a SW40Ti rotor and allowed to slow down without the application of a brake. Fractions (0.8 ml each) were collected from the bottom. The entire fractionation process was completed in ~ 5 hr. Aliquots of Fraction (A) containing equal protein amounts were subjected to immunoblot analysis to monitor SREBP-2 processing. Protein concentrations were measured with a BCA kit (Pierce). Organelle enrichment was assessed by assaying equal volumes of all fractions for either enzyme activity or presence of organelle markers by immunoblot analysis.
Enzyme assays for acid phosphatase, catalase, and cytochrome c reductase were carried out with assay kits (Sigma). Glucose-6-phosphatase activity was measured as described (Nordlie and Arion, 1966). β-Hexosaminidase activity was measured as described (Green et al., 1987).
Samples were mixed with 5x SDS loading buffer, heated for 10 min at 95°C, and then subjected to 10% SDS-PAGE. After SDS-PAGE, the proteins were transferred to Hybond-C extra nitrocellulose filters (Amersham) and then incubated for 1 hr at room temperature with the indicated antibodies at the following concentrations: anti-CREB, 0.5 μg/ml; anti-transferrin receptor, 0.5 μg/ml; anti-caveolin-1, 0.25 μg/ml; anti-prohibitin-1, 1 μg/ml; anti-Na+/K+ ATPase, 1:1000 dilution; anti-EEA1, 2 μg/ml; anti-PMP70, 2 μg/ml; anti-Sec61α, 1:1000 dilution; anti-GM130, 1:1000 dilution; anti-GRASP65, 1:1000 dilution; anti-LAMP1, 1:5000 dilution; anti-SCAP IgG-R139, 10 μg/ml; anti-S1P U1683, 1:1000 dilution; anti-ACAT-1 81-2, 1:500 dilution; anti-SREBP-2 IgG-7D4, 3 μg/ml; anti-mouse IgG, 1:5000 dilution; anti-Insig-1, undiluted hybridoma culture medium; anti-rabbit IgG, 1:5000 dilution. Bound antibodies were visualized by chemiluminescence (Super Signal Substrate, Pierce). Filters were exposed to Kodak X-Omat Blue XB-1 film at room temperature for 1–120 sec.
Membranes were fixed with 2% (v/v) gluteraldehyde in 100 mM cacodylate (pH 7.4) containing 200 mM sucrose at room temperature for 30 min, washed with 100 mM cacodylate (pH 7.4), embedded in 2.5% low melt agarose, and postfixed for 1 hr with 1% (w/v) osmium tetroxide, 1.5% (w/v) potassium cyanoferrate in 100 mM cacodylate (pH 7.4). The membranes were then washed with water, stained with 2% (w/v) aqueous uranyl acetate, dehydrated by washing with increasing concentrations of ethanol, and embedded in EPON 812 according to the manufacturer's instructions (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections (70–90 nm) were cut by using a Leica Ultracut E ultramicrotome, placed on Formvar coated grids, and post-stained with 2% aqueous uranyl acetate (10 min) and lead citrate (5 min). Images were taken on a JEOL 1200EXII transmission electron microscope operated at 120 kV, with a Sis Morada 11 Mpixel CCD camera.
Lipids were extracted from cellular fractions using a chloroform/methanol (1:1, v/v) mixture (Bligh and Dyer, 1959). The organic solvent was evaporated under a gentle stream of nitrogen, and the dried lipid extracts were reconstituted in 95% methanol. Free unesterified cholesterol levels were measured with isotope dilution mass spectrometry using a deuterated analog of cholesterol (d7) (Avanti Polar Lipids) added to the sample prior to extraction as a standard for quantification. Lipid extracts were resolved and detected using high-performance liquid chromatography (HPLC) coupled to a triple quadrupole mass spectrometer (MS) through an electrospray ionization interface (McDonald et al., 2007). Total phospholipid levels were determined by a colorimetric assay that measures inorganic phosphate released after acidic digestion (detection limit ~ 1 μg) (Chalvardjian and Rudnicki, 1970). In estimating cholesterol:phospholipid molar ratios, we assumed that the mean molecular mass of the phospholipids (~800 Da) was twice that of cholesterol (388 Da).
The dependence of SREBP-2 processing on cholesterol levels in the ER was analyzed by a least squares fit of the Hill equation: Y = 100 (1 − Xn/(C + Xn)), where Y is the fraction of SREBP-2 in the processed nuclear form (expressed as % of control as described in the legends to Figures 4–6) and X is the mol% of free cholesterol in ER. The quantity 1/C can be considered as a cumulative binding constant for the species containing n ligands, assuming that this species dominates over all other liganded forms; n is regarded as a measure of positive cooperativity and is usually referred to as the Hill coefficient (Dahlquist, 1978; Wyman, Jr., 1964). Curve fitting and statistical analysis were performed with Mathematica (Wolfram Research, Inc.).
We thank our colleague Joachim Seemann for helpful discussion; Lisa Beatty, Shomanike Head, and Ly Le for invaluable help with tissue culture; Oliver Ritim and Matthew Francis for excellent technical assistance; and the UT Southwestern Live Cell Imaging Facility for electron microscopy. This research was supported by grants from the NIH (HL20948 and GM069338) and the Perot Family Foundation.
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It is now confirmed that 11th of November 2016 would be very special day in Gujarati film industry. The film titled ''Kaik Karne Yaar' directed by Kabir Jani would be hitting the movie halls on that day. The great news about this film is it is supported by three main entities of Bollywood film world now. It is well known that for a film to become highly successful it must have superb music ,marketing and distribution. These three aspects are covered in great extent for this Gujarati film'' Kaik Karne Yaar''.
T- series that has created superb impact for many years is giving its support for this film plus trigger Max that was responsible for many super hit Hindi films like Ki & Ka, Dishoom etc are taking care of marketing part and AA films of Anil Thadani that has distributed some of blockbusters like Baahubali etc would be doing its part in distributing of this film.
This Gujarati film would be super special for the reason that actress Aruna Irani who last acted 13 years before would be doing a nice role in this film. Others like evergreen Tiku Talsania, Yatin Parmar, Raj Jatania plus others provide good star cast. It must be noted that this film has music composed by Lavan and Veera. This film is produced by trio Darshan Shah, Rishi Doshi and Varun Jhaveri of Kriya Films.
It is said that this film is about a strict businessman Mehta who is living in USA with his cute daughter Jia. He wants to marry his daughter to Indian man and what happens next forms the rest of the story. The expectations of this Gujarati film from people is huge now. Interesting days ahead!!


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Daryl Hannah
April 9, 2011 by Post Team
Daryl Hannah, Daryl Hannah, star of "Splash" and "Kill Bill", and 62,622 owed in state taxes in arrears, according to the Los Angeles County Recorder of Deeds.
The state of California filed a lien against the actress for 50 years, but the actress's manager apparently does not care.
"I just made a deal for the week so any kind of things you should, if you do should get clean to come," said ana representation of The Detroit News.
The actress is reportedly worth more and 15 million after starring in 64 films as "Blade Runner", "Roxanne" and two "Kill Bill movies." Hannah also plans to resume her villain eye patch to wear, Elle Driver in "Kill Bill: Vol. 3, which was released in 2014.
This is not the first legal issue of Ana, in; the environmental activist was arrested after participating in a protest against the mountain top coal mining. Hannah and thirty other protesters blocked a road out of West Virginia plant Massey Energy Company. Everyone involved was cited for impeding traffic and obstructing an officer, but were later released.
Several other players have gotten in trouble for tax. Wesley Snipes was sentenced to three years in prison for tax evasion and Nicolas Cage reportedly owes as much as to 14 million dollars to the IRS.
Please feel free to send if you have any questions regarding this post , you can contact on
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I created forms but all of them is now showing as disabled?Asked by Taxrek8652 on May 05, 2016 at 05:08 AM
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Adam Faith - Someone Else's Baby "Live" 1960 MP3
Adam Faith sings his 3rd hit single called "Someone Else's Baby" which hit No2 in the British Hit Parade on the 14th April 1960.
Will Hoge - "Somebody Else's Baby" | Live at the Grand Ole Opry | Opry MP3
Will Hoge performs "Somebody Else's Baby" live at the Grand Ole Opry. Visit www.opry.com to find out more! © ℗ 2012 Grand Ole Opry, LLC Subscribe: ...
murder city devils - Somebody else's baby MP3
merder city devils Somebody else's baby lyrics: I heard and read The only love is Lost love If it's true then People like me and you We get our share of love We ...
Will Hoge - Someone else's baby (Studio version) MP3
Someone else's baby by Will Hoge I do not own any of the rights of this song.
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By: Luke Bryan!!!
ADAM FAITH - What Do You Want & Someone Else's Baby (1969) MP3
Adam Faith Singing "What Do You Want" & "Someone Else's Baby" From 1969.
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Terence "Terry" Nelhams-Wright, known as Adam Faith (23 June 1940 – 8 March 2003), was a British teen idol, singer, actor, and financial journalist. He was ...
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Wednesday, February 29, 2012
Welcome to Another Heartfelt Creations Wednesday......
Since it is Wednesday, I'm here to share another project I did for Heartfelt Creations. Today it is a tag I made using purples and greens. For all the products I used and the instructions for this tag, just head on over to the Heartfelt Creations blog. Everything is listed there along with the instructions for this project. While you are there you can see all the beautiful submissions from the other design team members. You can always visit each individual team member's blog by clicking on the names on the sidebar of my blog. You will truly be inspired when you visit.
I know this is short and sweet this morning, but I have a ton of stuff to do today and I need to get to moving! So have a wonderful day and I hope you enjoy my tag!
29 comments:
- JoZart said...
Gorgeous tag and the seam binding as ribbon sets it off perfectly.
JoZarty x
- February 29, 2012 at 6:00 AM
- Spyder said...
This is lovely!!
- February 29, 2012 at 6:04 AM
- Becky said...
Glenda, this is gorgeous! That flower looks almost real!!!
- February 29, 2012 at 6:06 AM
- Olga said...
WOWZERS!!!! This is a traffic stopper!!! Absolutley gorgeous!
- February 29, 2012 at 6:22 AM
- Emmy said...
What an amazing tag, Glenda! Love your colour combination!
- February 29, 2012 at 6:44 AM
- Rita said...
Absolutely Stunning Glenda. I am having a bash at my very first tag to-day. hugs Rita xxx
- February 29, 2012 at 7:21 AM
- Beate said...
Gorgeous flowers and tag!
Hugs and smiles
- February 29, 2012 at 7:25 AM
- Janett said...
Wow, Beautiful Tag. Love your flowers, they are amazing.
- February 29, 2012 at 8:14 AM
- Selma said...
Glenda you are a master at creating gorgeous tags. This is fabulous!
- February 29, 2012 at 8:16 AM
- LISA said...
I am always inspired when I see your creations Glenda!!! This is absolutely gorgeous girl!!! Love love love it!! I am inspired!! ♥
- February 29, 2012 at 8:27 AM
- The Other Patti Sue said...
Pure gorgeousness! Super pretty!
- February 29, 2012 at 8:32 AM
- Broni said...
Another gorgeous work of art from you, Glenda! Love it!
- February 29, 2012 at 8:58 AM
- Elly said...
Gorgeous creation Glenda!!!
- February 29, 2012 at 9:04 AM
- Carla S. said...
Stunning tag, Glenda! Wonderful coloring of the gorgeous flowers!
- February 29, 2012 at 9:09 AM
- Lisa Lynne said...
Gorgeous tag! Love the purples.
- February 29, 2012 at 10:01 AM
- Sylvia said...
Beautiful tag, Glenda, I love that color! Your flowers are fabulous as always, have a fun Leap Day!
- February 29, 2012 at 11:23 AM
- L00py's Craft Creations said...
Glenda, there is something special on my blog for you, if you wouldnt mind going to collect it, xxx
- February 29, 2012 at 1:41 PM
- Joelle said...
Absolutely gorgeous tag!! Love the colors and the seam binding is just yum!!
- February 29, 2012 at 2:51 PM
- Sylvia/LittleTreasures said...
Oh this Tag is perfect. I love tags and the color here is so vibrant, looks great with the aged, distressing. Enjoy
- February 29, 2012 at 7:35 PM
- Dawn B. said...
This is so pretty. Your coloring is amazing. I love the depth of color. Great job.
- February 29, 2012 at 8:45 PM
- Lisa aka Raining said...
Totally stunning!!!!
- February 29, 2012 at 8:55 PM
- Sue from Oregon said...
This is pure gorgeousness!
- February 29, 2012 at 9:01 PM
- Angelica said...
So beautiful Glenda, love the colors.
- February 29, 2012 at 10:29 PM
- Starla said...
I think I would love to have a garden full of all your flower creations!!
- February 29, 2012 at 11:05 PM
- Candy said...
Beautiful tag! Your coloring is gorgeous!
- February 29, 2012 at 11:55 PM
- Marie-Louise said...
Beautiful tag.
- March 1, 2012 at 4:39 AM
- Carol (Gingerbeary8) said...
This is just beautiful Glenda. I love the color combination and those exquisite flowers. Very elegant and inspiring in your signature style.
- March 1, 2012 at 9:16 PM
- huda said...
Nice post dear
Good blog
I am following your blog with GFC
Now follow back my blog with GFC too
- March 2, 2012 at 1:44 AM
- Leslie Miller said...
You've colored and arranged those flowers so beautifully this tag rivals those other gorgeous flowers you use so well. Love the colors!
- March 3, 2012 at 8:47 PM


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Supervisors Role as Trainer & Coach
The Supervisors Role as Trainer & Coach, organized by the National Seminars Training will take place on 1st August 2016 at the Fairbridge Hotel & Conf. Ctr. in Morristown, US. The conference will cover areas like Know when training is the answer and when it is not Follow up with employees to make sure new knowledge is being applied.
Venue -:
1st August 2016
Fairbridge Hotel & Conf. Ctr.
130 NJ-10, East Hanover, NJ 07936, United States Morristown, USA


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List all words that contain eirwb
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Variation in carbon isotope discrimination and its relationship with harvest index in the reference collection of chickpea germplasm
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Terminal drought is a major constraint to chickpea productivity. Carbon isotope discrimination (Δ13C), an integrator of plant behaviour influencing transpiration efficiency (TE), is an important component of yield under drought. The variation in Δ13C and its association with yield was assessed in the reference collection of chickpea germplasm. Drought stress reduced shoot biomass by 36–39% and grain yield by 23%. Mean Δ13C was low and the range of genetic variation was high under drought stress. Largely, high Δ13C accessions were early in flowering (40–50 days), moderate in shoot biomass, high in seed yields and high in harvest index (HI). Δ13C was positively correlated with seed yield in both the years under drought stress, only in 2008–09 under optimal irrigation. This positive association was very close with HI. Among the yield components, Δ13C was closely associated with pod numbers per unit area and seed size under drought stress. Path coefficients showed no direct association of Δ13C with grain yield but an indirect negative association through shoot biomass at maturity and a close positive association through HI. The closest association of HI or shoot biomass was seen in the maturity group of accessions that experienced the optimum terminal drought stress.


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Om Sai Ram Do something Good Today. Don't shine just to outshine someone else. Shine as a blessing to others. Be the ray behind their dreams!. Om Sai Ram Good Morning Have a Good Day Jab Teri Inayat Par Meri Nazar Jaati Hai, Mere SATGURU Meri Aankh Bhar Aati Hai, Tu De raha Hai Mujhe is Kadar, Ki Hath …Read More »
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True Life Shayari in Hindi Language – Hindi Shayari Picture Messages
Nikle the issi aas pe, Kisi ko bana lenge apna…. Ek khwahish ne umar bhar ka musafir bana diya…..!! terms:anmol vachan in gujaratianmol bachan image sayariAnmol bachhon good night Hindi messages text big messagessubichar by school life More posts: Maa Baap ke Suvichar – Hindi Inspiring Quotes Pictures Unka Kandha Prabhu Ne Kitna Mazboot Banaya Hai Meri Khwahishon Ko Uthate …Read More »


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that would help get rid of blackheads? maybe not even face wash, just anything to get rid of them. they're horrible, I get them on my chin and nose. they aren't visible unless you're up really close, but my school is all windows and its right on the water (which means its EXTREMELY bright) which brings them out.
i'm open to anything that will help, thanks!
Most Helpful Girl
I know of this scrub, St. Ives Swiss Formula Apricot Scrub Blemish & Blackhead Control. I've heard a lot (and I mean A LOT) of girls say it's really good, plus it's super cheap (about 3$) so you might wanna try that out. As for other methods, this website has tips on how you can do it yourself: link
Ooooor, the way I heard you could do it is, you take a hot shower to open up your pores or you use a hot compress over the area with the blackheads. Now your skin has to be clean for this, of course. So what you need is this professional blackhead remover that basically every beauty supply store has, it's that thingy that has a scoop with a hole in the middle of it on one end, and the other end is a lance; all you have to do is you put the scoop over the blackhead, and apply pressure. Don't apply too much pressure, just a little should work just fine! If you don't find the tool, you could do the same thing with a disinfected bobby pin.
Hope this helps!
Thanks, the apricot face scrub works well I forgot about that, but could you explain how to do this bobby pin thing? haha
I completely agree. I used to have a lot of acne but not blackheads, and I started using proactive. It got rid of my acne, but my blackheads started to appear. I started using St. Ives Swiss Formula as it is stated above me. It got rid of my blackheads.


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Soldier Stands 9 Hours In Rain With Flag, What Happens Next Will Leave You In Tears
A disabled veteran just taught every American how to give our troops the respect they deserve. This is so inspiring!
What this disabled veteran did to honor the men and women who died while serving in the U.S. military is still captivating Americans.
For many of us, Memorial Day isn't just a day off from work — it's a time filled with tears and visiting cemeteries or memorials.
So when this disabled veteran found out the flagpole outside a nearby nursing home was knocked down over the winter, he took it upon himself to create a patriotic display this past Memorial Day.
In the midst of pouring rain, Army Specialist Darren Swallow stood for more than 9 hours outside of Wingate of Wilbraham in Massachusetts, an assisted living and rehab facility, and held a flag as a tribute to those who served and made the ultimate sacrifice. According to 22News, the veteran, who is disabled following multiple tours in Iraq, arrived at Wingate at around 3:30 A.M. and remained there holding the flag until 1:00 P.M.
"My girlfriend told me yesterday the flagpole behind us was taken down either during a storm or from a plow truck, so I thought I'd come down and honor our veterans, and let them know they have not been forgotten," Swallow said.
Anara Dupont, Swallow's girlfriend, said, "He was very passionate about it when I told him we didn't have a flag here, he is an angel, he is, he really is one of the best people I've ever met in my whole life."
When I saw how people reacted to Swallow's act of patriotism, my eyes instantly filled with tears.
Swallow's patriotic gesture especially touched the heart of Wingate employee Susan Huart, whose two children and a son-in-law are all in the service.
"Pride. I mean it's a feel-good story, he's celebrating the veterans because we didn't have a flagpole, and he came to celebrate the veterans everywhere," Huart said.
The community was so touched by Swallow's gesture that some parents even drove to the facility to simply thank him and to show their children his inspirational patriotism.
"Absolutely inspired, we came here just to thank him for his service. It's phenomenal that he's standing out here for the residents and for our country," said Chris White of Monson.
In addition to holding the flag in the rain for hours, Swallow went room to room in the nursing home to shake hands with residents that are veterans. Eighty-five-year-old Korean War Veteran, Paul King Sr., became emotional when Swallow visited his room.
"My father sincerely appreciated the visit. My father was tearful when he heard what the soldier was doing and was even more tearful when the soldier came to his room for a visit. God bless America!" King's daughter, Laura Hendrickson said.
Watch the incredible story right now:
God bless Darren Swallow! With all the negative news surrounding us lately, stories like this are truly uplifting.
Share this article to show Americans how to RESPECT our troops!


text: Home Recipes Course Appetizers Hot Appetizer Recipes Stuffed Mushrooms Share: Stuffed Mushrooms Looking for recipes for stuffed mushrooms? Taste of Home has the best stuffed mushrooms recipes from real cooks like you, featuring reviews, ratings, how-to videos and tips. Recipe Spotlight Healthy Cheese Stuffed Mushrooms "If you like mushrooms, you'll love this yummy broiled appetizer," assures Kelli Egbert of Idaho Falls, Idaho. A tasty cheese filling is easy to combine and tuck into large mushroom caps. Get Recipe More Stuffed Mushrooms Sort: Most Relevant Highest Rated Previous Page ... 4 5 6 ... 7 Next Page Cheese-Stuffed Mushrooms Cheese-Stuffed Mushrooms "There are never leftovers when I serve these! This recipe is from a friend, but I modified it for a slimmer version that tastes as good as the original." Cathy Barger - Clarksville, Michigan View Recipe Spinach-Stuffed Portobellos Spinach-Stuffed Portobellos "The meaty texture of portobello mushrooms will make you think you're eating steak," says Diane Lombardo of New Castle, Pennsylvania. "With a cheesy spinach filling, this is a marvelous main dish. Pepper flakes add color and a kick." View Recipe Artichoke Mushroom Caps Artichoke Mushroom Caps These crumb-topped appetizers never last long at parties. The rich filling of cream cheese, artichoke hearts, Parmesan cheese and green onion is terrific. You can broil them in your oven to enjoy any time of year. —Ruth Lewis, West Newton, Pennsylvania View Recipe Advertise with us ADVERTISEMENT Spinach Dip-Stuffed Mushrooms Spinach Dip-Stuffed Mushrooms I use a melon baller to hollow out the mushroom caps to make them easier to stuff. They also fit neatly into muffin tins or a deviled egg tray for traveling. —Ashley Pierce, Brantford, Ontario View Recipe Stuffed Asiago-Basil Mushrooms Stuffed Asiago-Basil Mushrooms Don't like mushrooms? Guess again…you will now! These pretty appetizers taste divine. For a main dish, double the filling and use large portobellos. (This recipe was first place winner in the Mushrooms Every Day, Every Way contest "Inspiring Appetizers" category.) —Lorraine Caland, Thunder Bay, Ontario View Recipe Portobello Mushroom Pizza Cups Portobello Mushroom Pizza Cups Making stuffed portobellos for guys watching a hockey game was quite an endeavor. I made two dozen for six guys, and as the game got more exciting, they screamed for more. Scraping the gills from the underside of the mushroom is easy if you use a small teaspoon, gently, so as not to crack the cap. The amount of cheese, pepperoni and spices is entirely up to whatever satisfies your team. —Lorraine Caland, Shuniah, Ontario View Recipe Spinach-Cheese Mushroom Caps Spinach-Cheese Mushroom Caps Dainty finger foods like these mushrooms are a nice way to welcome guests into your home. A hearty spinach filling will tide folks over until the meal is served. —Sandy Herman, Marietta, Georgia View Recipe Low-Fat Stuffed Mushrooms Low-Fat Stuffed Mushrooms "I first tasted these fun mushroom appetizers at a support group meeting for diabetics," says Beth Ann Howard of Verona, Pennsylvania. "I couldn't believe how yummy they were. Since then, I've shared the recipe with friends and co-workers." View Recipe Prosciutto-Stuffed Mushrooms Prosciutto-Stuffed Mushrooms Prosciutto and oregano give these stuffed mushrooms a bit of an Italian twist. To make them ahead, cover with plastic wrap and refrigerate until ready to bake. View Recipe Appetizer Stuffed Mushrooms Appetizer Stuffed Mushrooms Heat-and-serve convenience foods are forbidden in the House of Loreto Kitchen, and residents appreciate the "real" food we serve. Everyone reaches for seconds of these tasty morsels. View Recipe Crab-Stuffed Mushrooms Crab-Stuffed Mushrooms "Mushroom caps plump with crabmeat make a wonderful appetizer for entertaining," suggests Tonya Farmer, Iowa City, Iowa. She suggests using the recipe as a light entree, served on salad greens or with pasta. View Recipe Previous Page ... 4 5 6 ... 7 Next Page Advertise with us ADVERTISEMENT Advertise with us ADVERTISEMENT