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Patient Resources
ASK Dr. LUNEBURG
We are always here to answer your questions.
Common Chiropractic Questions
Yes. A Doctor of Chiropractic (DC) will care for patients of all ages, with a variety of health conditions. These Doctors are especially well known for their expertise in caring for patients with spinal disorders, including back pain, neck pain and headaches. Chiropractors also achieve excellent results with conditions and disorders of the musculoskeletal system that involve muscles, ligaments and joints. Dr. Luneburg has years of experience treating patients that are dealing with many different conditions and would be honored to be your family's Chiropractor. If you would like to find out more, contact our office for a free consultation. If we feel your condition could be improved outside the scope of our practice, we would be happy to make the appropriate referral to best fit your needs. Your health is our goal.
If you are unsure whether your condition could be treated by Chiropractic, please reference our list of conditions commonly seen in our office, or contact us for more information.
Chiropractic removes pressure on irritated spinal nerves. When these nerves are irritated or "pinched", nerve impulses are blocked and a host of symptoms can occur. With proper detection and location of these pinched nerves, a gentle adjustment of the spine will help to relieve the discomfort, allow the nerves to flow freely and your body to heal properly.
No. Dr. Luneburg makes sure his treatments are adjusted to your comfort level. He always asks patients to notify him if they are experiencing any discomfort or if there is too much pressure. The experience of having nerve compression removed often times results in greater range of motion, less pain, a reduction in symptoms and a feeling of relaxation..
Absolutely! We treat newborns, infants and kids of all ages. Many parents will testify to the numerous health benefits they have seen in their own children with Chiropractic care, including ear infections, colic, reflux and digestive issues. Adjustments on little ones are extremely gentle and are most often associated with a light pressure. Yes, even little ones can have misalignments, and if left untreated they can leave parents wondering why their children are struggling with health issues.
Yes. We accept most insurance plans, including Medicare and Medicaid. We also treat personal injury cases, such as worker's compensation and motor vehicle accident claims. Our staff is happy to contact your insurance carrier on your behalf to let you know your benefits. We also offer affordable payment options and cash plans to fit your budget.


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From Barbados-
After two sittings separated by 19 days and 16 hours of voting, the Anglican Church here is no closer to electing a bishop to lead the Barbadian flock.
The House of Clergy and the House of Laity aborted yet another attempt today, having failed to reach consensus after six hours of voting.
They ended today in the same position as they did on April 25 after ten hours of voting and four ballots at the Ivan Harewood Centre of the Christ Church Parish Church to choose a successor to the retired John Holder.
As it did back then, the laity continued to throw its support behind youthful candidate Rev John Rogers, 45, rector of St George Parish Church, while the clergy backed his senior, Dr Jeffrey Gibson, 61, dean of The Cathedral of St Michael's and All Angels.
More here-
Diakonia: The Rev Courtney Jones
13 hours ago

subset: commoncrawl


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SPRINGFIELD, Mass. – Sophomore Alec Brunelle (Salem, N.H.) and freshman Eddie Morin (East Longmeadow, Mass.) of the Saint Anselm College men's golf team led the Hawks to a sixth place finish to conclude the opening day of competition at the American International College Invitational.
The Hawks sit in sixth place out of 10 teams, carding a combined 314 (+26) through the par-72 course. Saint Anselm will return to action Sunday, Sept. 23 to conclude play at the two-day invitational.
Brunelle and Morin each shot a first-round 75 (+3) with the latter doing so in his collegiate debut for the Hawks. Freshman Peter Hudner (Canton, Mass.) turned in a score of 81 for Saint Anselm with classmates James Henry (Reading, Mass.) and Jared Carr (Milton, Mass.) producing first-day scores of 83 and 87, respectively.
Saint Thomas Aquinas leads with a team score of 296 (+8) through the first day of competition. Bentley's Tommy Ethier is three-under 69 through the first 18 holes at the Veterans Memorial Golf Course.


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The Right is getting all apoplectic about comedian Michelle Wolf's treatment of Trump, Sarah Huckabee Sanders, and Mike Pence at the White House Correspondence Dinner. It's fine to be upset, but let's not pretend that we on this side of the political aisle care about free speech if we're calling for Wolf's firing from Comedy Central.
She's a writer for the Daily Show with Trevor Noah... what did you expect? And that's the crux of the matter. Comedians are becoming more socially conscious and thus are trying to set the agenda of what's fair game for ridicule. This is something that comedy has never attempted to tackle, because everything was fair game. If you didn't like a particular comedian, the door was open for you to exit.
I've listened to some comedians who have been brutal in who or what they'll make fun of and it wasn't because they were necessarily mean. For some, making fun of something that's taboo was a way of dealing with the discomfort that comes with taboos. Comedian Joan Rivers made 9/11 jokes! Does that mean she's uncaring... maybe? Or maybe making obscene jokes about sensitive subjects is a way of cauterizing the wound of reality. It isn't a way to numb the pain, it's a way of being shocked and laughing through the pain. I've heard some awful 9/11 jokes that made me laugh, yet I still cry when I see the Twin Towers fall. This might not be everyone's bag, so this isn't an attempt to get you to laugh at something you don't want to laugh at. That's the genius of comedy.
That being said, Michelle Wolf's jokes about the media were spot on... but isn't that a problem? Her jokes about Trump were spot on in some places, but isn't that part of the problem? The result of being woke in comedy is mean only attacking those who you perceive as the oppressors. This leaves comedy in a perpetual feedback loop where comedians are more concerned about scoring woke points with their contemporaries and like-minded peers and if the audience laughs, then that's icing on the cake.
Michelle Wolf's performance is another example of agenda comedy where attitude and political ideology are wrapped up in a smug joke. It's not to say that comedians back in the day were right-wingers. Most artists are not right-wing but what made comedians of the past unique is that their main objective was to be objectively funny. That meant that either side of the political spectrum open for ridicule, even if it was the comedian's own side. Now, comedians tend to protect their side and attack only in one direction. The result is that jokes are predictable and bland. There was nothing Michelle Wolf said that couldn't be said about any past Republican politician. That's a sign that comedy might be dying on the vine.
Sorry for the long preamble... watch the video please.


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Academic Job Opportunities
We are seeking applicants for three lectureships in Development and Social Anthropology
The School of Social and Political Science is advertising for three open-ended lectureships that relate to the Centre of African Studies. We are advertising for a lecturer in International Development, a lecturer in the Anthropology of Development and a lecturer/senior lecturer in Social Anthropology.
While these jobs are not all Africa-specific we would like to encourage applications from people working on development and anthropology in Africa.
For more information: www.jobs.ed.ac.uk


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Allah Gave Me Two Eyes To See (allah The Maker)
- Number of Pages: 24
- Languages:
- English (Unknown)
- English (Published)
© 2019 By Islam Children Books - All Rights Reserved.
Islamic Foundation
Author: Fatima M. D'Oyen
Allah Gave Me Two Eyes To See written by Fatima M. D'Oyen is an excellent child's book. The author is Fatima M. D'Oyen and it was published on the 1st of January, 2002 by Islamic Foundation. The book has 24 pages. To buy a copy at the cheapest price, click on our store button on this site.ASIN: 0860373665


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Got a coupon from one of the dealsXXX.com website. $12.90 instead of $19.90.
It was a kiosk at the basement of a shopping mall – no atmosphere, no decor, which is fine. Then the chocolate fondue came. There is a reason why Haagen dazs's fondue costs $60 and this one costs $12.90. The chocolate was probably hershey's liquid syrup, and it wasn't even hot. The ice cream was horibble, marigold-standard ones, which wasn't freeze to rock-hard. (Fondue ice cream has to be rock hard because the chocolate is hot.) The waffle was flat, and dry and not hot. It was made some time ago and the server heated it in a microwave. The fruits were browning.
14K left it all to me because he's so picky with food. Goodness, I need to find a date who eats everything.
I don't understand the coupon thing. If you're offering a discount, it means that your business is bad. And you should take the chance to attract customers but instead, the place serves inferior food. Sooner or later, it will collapse.
Cottage Waffle Place
City Square Mall
Rating: 1.013/5 stars
Categories: $0-$20, Dessert, Little India


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Published on September 23rd, 2018 by Scuttlebutt Sailing News
The 2018 edition of the J/70 World Championship has attracted 90+ entrants to compete for Open and Corinthian Class titles September 25-29 in Marblehead, MA.
Among the team may represent the most unprecedented assemblage of world-class talent ever gathered together in a single one-design regatta in history – keelboat or dinghy.
Teams have come from as far away as Australia, Germany, Mexico, Canada, Italy, Great Britain, Japan, Monaco, Russia, Spain, Norway, Switzerland, Sweden and Turkey. Additionally, South America is well represented: Argentina, Brazil, Chile, and Uruguay.
To appreciate the relative "fire power" and depth of talent that some J/70 teams bring to the table, here are the notable combinations of the top USA skippers and pro-sailors on the leading teams:
• Jud Smith's AFRICA: 3x Etchells 22 World Champion, 2017 J/70 World Champion/ Lucas Calabrese- Olympics 470 Bronze Medallist for Argentina
• Tim Healy's USA 2: first J/70 World Champion, 3x J/70 Midwinter Champion, 2x J/24 World Champion/ John Mollicone- Brown University Coach and 2x J/24 World Champion
• Joel Ronning's CATAPULT: J/70 World Champion, Melges 32 World Champion/ John Kostecki- J/24 World Champion/ Offshore World Champion, America's Cup Champion
• Peter Duncan's RELATIVE OBSCURITY: J/70 World Champion/ Willem van Waay and Victor Diaz de Leon- 2x J/70 World Champion crew
• Glenn Darden's HOSS: J/105 North American Champion, J/80 North American & World Champion/ Jonathan McKee- Olympic Gold Medallist
• Brian Keane's SAVASANA: 3x College All-American & J/80 North American Champion/ Thomas Barrows was College Sailor of the Year at Yale.
• Bruce Golison's MIDLIFE CRISIS: Etchells 22 World Champion/ J/24 North American Champion
• Martie Kullman's HYDRA: J/22 World Champion
• John Brim's RIMETTE: Storm Trysail Offshore Champion on Farr 60 RIMA/ Taylor Canfield- 3x World Match Racing Champion/ 2x Congressional Cup Match Race Champion
• Will Welles' SCAMP: 2x J/24 World Champion/ J/24 North American Champion
• Jack Franco's 3 BALL JT: 2x College All-American/ Bill Hardesty- Etchells 2x World Champion & J/70 World Champion/ Allen Terhune- J/22 World Champion.
• Pat Toole's 3 BIG DOGS: J/24 North American Champion
• Chris Snow's COOL STORY BRO: J/24 North American Champion & J/70 West Coast Champion
• Bill Lynn's FLYING NONE: 2x Sonar World Champion
• Jenn & Ray Wulff's JOINT CUSTODY: J/70 North American Corinthian Champion
• Tom Bowen's REACH AROUND: J/70 North American Champion
• Bruno Pasquinelli's STAMPEDE: J/70 Midwinter Series Champion/ Eric Doyle- Star World Champion
• John & Molly Baxter's TEAM VINEYARD VINES: J/70 Midwinter Series Champion
• Mark Foster's WINTERWIND: J/22 World Champion and J/24 North American Champion
Read the full story here...
Request a quote for your sails or simply get in touch with the Doyle Sails Team by clicking below
Keep up to date with the latest Doyle Sails news on products, projects and people by subscribing here for our regular newsletter


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A large front covered porch welcomes you to this beautiful traditional style house. Immediately inside the house you will find a formal dining room with a built in hutch. The amazing great room is warmed by a fireplace that is flanked by built-in bookshelves. The large kitchen comes complete with a large island with a breakfast bar and a huge walk-in pantry. The well lit breakfast nook has a cathedral ceiling and provides great views of the back yard.
Upstairs you will find all 4 bedrooms. The fantastic master suite includes a bathroom with his/her vanities, a corner soaking tub, an enclosed toilet and a massive walk-in closet. Bedrooms 2 and 3 each have their own walk-in closet and all three bedrooms share a centrally located bathroom in the hallway.
Back on the main floor, the large 3 car garage enters the home through a convenient mud room and also leads to a large laundry room with a soaking sink and a large folding counter.
As always any Advanced House Plans home plan can be customized to fit your needs with our alteration department. Whether you need to add another garage stall, change the front elevation, stretch the home larger or just make the home plan more affordable for your budget we can do that and more for you at Ahp.
Unless you purchase an unlimited use license or a multi-use license you may only build one home from a set of plans. Plans cannot be re-sold.
|Car Bays
|1255 Sq. Ft.
|1325 Sq. Ft.
|Total Finished
|2580 Sq. Ft.
|Garage Area
|767 Sq. Ft.
|Ridge Height
|Foundation Type
|Exterior Walls
|Roof Pitch
|7/12 Primary, 10/12 Secondary
|Basement Wall Height
|Main Wall Height
|Second Wall Height
Each set of home plans that we offer will provide you with the necessary information to build the home. There may be some adjustments necessary to the home plans or garage plans in order to comply with your state or county building codes. The following list shows what is included within each set of home plans that we sell. Depending on the home designer there might be more home building information provided.
Our blueprints include:


text: I'm a 19 year old freshman at MATC working to become an LPN (licensed practical nurse). I jumped at the opportunity to take the free CNA (certified nursing assistant) course that my high school offered because my passion lies in medicine and being able to help others. I am the middle child of three. Both my mother and father work hard in order to give us all what we need. My father works two jobs as a special Ed teacher as well as a soccer coach and my mother a receptionist, they are both working long hours in order to put both me and my older brother through college and pay for all my younger sisters soccer tournaments and club fees so she can keep up her passion of sports. Im asking for your help so I can continue my goal of becoming a nurse and lift some of the burden and stress away from my prarents.


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[note color="#FFE4C4″]The winners of the #ClearlyKiehls Give back contest for Week 2 are :
Premium Package : Samyukta Mkeup
Regular Package : Anupama, Hasna Hena Hasan, Swati Murti, Sophia S and Shruti Shetty.
Congratulations Girls!
The contest is on for another week and I'll be picking winners at the end of the week. Click HERE to enter![/note] [smartads]


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Thank you for visiting Fallon Family Dental Care online! Our talented dentists, Dr. Cary Jaques and Dr. Chad Shepphird, are dedicated to providing comprehensive dentistry, including restorative, cosmetic, and preventive dental care. Our dental team is friendly, experienced, and knowledgeable, and we work hard to ensure that you have a pleasant, positive dental experience. We are proud to serve Fallon and the surrounding areas of Nevada, including Fernley, Lovelock, Austin, Round Mountain, and Hawthorne. We welcome you to contact us today to learn more about our dental treatments and to schedule your individual consultation with our experienced dentists. We look forward to caring for your smile!
Monday-Friday: 8:00 a.m.-5:00 p.m.
Closed for lunch 1:00-2:00 p.m.
Fallon Family Dental Care
300 W. A Street
Fallon, NV 89406


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Breathe. Just…breathe. And prepare for the most unexpected Star Wars film yet.
Tag: Mark Hamill
Star Wars: The Last Jedi Trailer – "It's Time For The Jedi To End."
"It's time for the Jedi to end."
How To Survive A Star Wars Movie Marathon
Seven films, 906 minutes, over 15 hours…this is the movie marathon you are looking for.
Star Wars: The Force Awakens Review
"The Force, it's calling to you. Just let it in."
Star Wars: The Force Awakens Trailer Breakdown
Best trailer ever? Best trailer ever!


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Published by Sam Swyer on Fri, 31 Aug 2018 11:00
We are excited to announce that we are hosting a three-session Kingdom Essentials course on three consecutive Wednesday evenings this autumn. 26th September, 3rd & 10th October from 7.30-9pm at Ringwood Parish Church.
This three session course explores how to pray for one another and for healing.
Kingdom Essentials is open to Christians from other churches outside Ringwood Benefice, so please do read more about the course and sign up online.


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Activists of Defend Europe have been patrolling the French-Italian borders for almost a month now.
According to a new administrative circular issued on May 4th by the minister of justice, discovered by Mediapart, they will now face arrest if they continue their actions.
Defend Europe militants arrested a lot of migrants when they crossed the French-Italian border.


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Alessandra Ambrosio's Michael Kors Dress at CFDA Awards 2016
Alessandra Ambrosio's Purple Gown at the CFDA Awards Screams "Fashion Royalty"
Alessandra Ambrosio's Purple Gown at the CFDA Awards Screams "Fashion Royalty"
Alessandra Ambrosio looked drop-dead gorgeous at the CFDA Awards at the Hammerstein Ballroom in New York City, wearing a sequined curve-hugging, one-shouldered Michael Kors Collection gown. This dress marked yet another collaboration between the Brazilian Victoria's Secret Angel, who has stolen every single red carpet she's stepped on this year, and the American designer who dressed Alessandra at the Cannes Film Festival.
At one of the most fashionable red carpets of the year, it was hard to keep your eyes off Alessandra's vibrant purple dress. Paired with superstraight hair and bronzed skin, the model was glowing as she made her way down the red carpet to the award ceremony. Keep reading to see more more angles of her sexy look.


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The Amherst-Pelham Regional Middle School Grade Span Advisory Board had it's second meeting of the school year. Here is a photo showing some of the work that was discussed.
2016-2017 School Calendar
The 2018-2019 School Calendar has been Approved by the School Committee.
Key information about the 2018-2019 School Year calendar:
School will start on Wednesday, August 29, 2018 for students in grades 1-12. Preschool and Kindergarten start on Tuesday, September 2, 2018.
There will be no school on the day before Thanksgiving.
Winter break will begin with an early release day on Friday, December 21, 2018. School will reopen on Wednesday, January 2, 2019.
The projected last day of school, including five snow days, is June 21, 2018. One weekday is deducted for each snow day not used, with the earliest possible last day of school being June 13, 2019.
Please note that schools are required to provide 180 days of instruction. While the five snow days built into the calendar are typically adequate to cover closures, any days schools must be closed beyond that must be made-up per Massachusetts Department of Education regulations. Whenever possible, such days are added to the end of the school year.


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|Substrates for HIV protease
|Heath, Jr. et al.
|Peptide substrates for the detection, characterization and purification of protein kinase C
This is a continuation of co-pending application Ser. No. 08/208,573, filed Mar. 4, 1994.
a. providing a biotinylated peptide substrate containing a biotin moiety, wherein the biotin moiety is conjugated to the peptide substrate at its N-terminal amino group via a linker at least six carbon atoms in length;
b. adding a sufficient quantity of the biotinylated peptide substrate to a solution containing the selected protein kinase;
c. incubating the selected protein kinase with the biotinylated peptide substrate under conditions where the selected protein kinase is active for a time sufficient to form a phosphorylated, biotinylated peptide product;
d. binding the biotinylated peptide product to a biotin-binding matrix, wherein the matrix has a sufficient binding capacity and specificity for the biotinylated peptide product to capture essentially all of the biotinylated peptide substrate and biotinylated peptide product from solution; and
e. detecting the presence and measuring the activity of the selected protein kinase.
a. a container containing a biotinylated peptide substrate containing a biotin moiety; wherein the biotin moiety does not result in a steric hindrance of the biotinylated peptide substrates accessibility to the protein kinase, the biotinylated peptide substrate further having specific reactivity to the selected protein kinase and modified by chemical reaction to allow quantitation; and
b. instructions for use.
a. a container containing a biotinylated peptide substrate as shown in SEQ. ID. NO: 7;
b. a container containing a biotin-binding matrix, wherein the biotin-binding matrix has a sufficient binding capacity and specificity for the tyrosine kinase-biotinylated peptide substrate; and
c. instructions for use.
a. a container containing a biotinylated peptide substrate which can be any one of biotinylated SEQ. ID. NOS: 1 through 8;
b. a container holding a biotin-binding matrix, wherein the biotin-binding matrix has a sufficient binding capacity and specificity for the biotinylated peptide substrate; and
c. instructions for use.
The invention is directed to a process for providing an assay protocol that measures enzymatic activity. More particularly, the invention is directed to a process to precisely and conveniently quantitate enzymatic activity of protein kinases and further to provide an assay specific for individual protein kinases in the presence of other protein. kinases.
A bibliography of the references cited in this application can be found in the section preceding the claims.
Enzymes are large proteins that catalyze reactions in living cells. Enzymes build up or tear down other molecules. For example, enzymes catalyze the synthesis of fat from fatty acids, form complex sugars from glucose and fructose, and aid in the formation of other proteins from amino acids. Enzymes also reverse the build-up process by breaking down more complex structures. Enzymes are generally specific to certain substrates for their reactions. For example, an individual enzyme may catalyze the reaction where only one substrate is involved or it may act on a group of related substrates.
In healthy persons, most enzymes are found within cells. Some diseases however cause the release of enzymes from dying cells into the blood. The increased levels of enzymes can then be measured. An abnormal level of enzymes in the blood characterizes certain medical conditions. For example, an enzyme assay for abnormal levels of the enzyme creatine kinase in the blood is useful as a diagnostic measure of heart disease. In like manner, bone or liver diseases can be diagnosed by observing increasing levels of alkaline phosphatase in the blood stream. Prostate cancer is diagnosed by increased levels of acid phosphatase in the blood stream.
Enzymes are classified into groups according to the general kind of reaction they catalyze. The present invention refers to a specific group of enzymes called transferases, which catalyze the transfer of a group from one substrate to another. The present invention is specifically directed to the transferase subgroup called protein kinases.
Protein kinase is a generic name for all enzymes that transfer a phosphate to a protein. About three to four percent of the human genome contains transcription information for the formation of protein kinases. Currently, there are up to 200 known different protein kinases. However, because three to four percent of the human genome is a code for the formation of protein kinases, there may be many thousands of distinct and separate kinases in the human body.
Protein kinases are enzymes which catalyze the transfer of phosphorous from adenosine triphosphate (ATP), or guanosine triphosphate (GTP) to the targeted protein to yield a phosphorylated protein and adenosine diphosphate (ADP) or guanosine diphosphate (GDP), respectively. ATP or GTP is first hydrolyzed to form ADP or GDP and inorganic phosphate. The inorganic phosphate is then attached to the targeted protein. The protein substrate which is targeted by kinases may be a structural protein, found in membrane material such as a cell wall, or another enzyme which is a functional protein.
Due to their physiological relevance, variety and ubiquitousness, protein kinases have become one of the most important and widely studied family of enzymes in biochemical and medical research. Studies have shown that protein kinases are key regulators of many cell functions, including signal transduction, transcriptional regulation, cell motility, and cell division. Several oncogenes have also been shown to encode protein kinases, suggesting that kinases play a role in oncogenesis.
Protein kinases are often divided into two groups based on the amino acid residue they phosphorylate. The first group, called serine/threonine kinases, includes cyclic AMP and cyclic GMP dependent protein kinases, calcium and phospholipid dependent protein kinase, calcium and calmodulin-dependent protein kinases, casein kinases, cell division cycle protein kinases and others. These kinases are usually cytoplasmic or associated with the particulate fractions of cells, possibly by anchoring proteins.
The second group of kinases, called tyrosine kinases phosphorylate tyrosine residues. They are present in much smaller quantities but play an equally important role in cell regulation. These kinases include several receptors for molecules such as growth factors and hormones, including epidermal growth factor receptor, insulin receptor, platelet derived growth factor receptor and others. Studies have indicated that many tyrosine kinases are transmembrane proteins with their receptor domains located on the outside of the cell and their kinase domains on the inside.
Phosphorylation of serine-, threonine- and tyrosine-containing proteins by kinases is important because the phosphorylated protein products have been implicated in a variety of cellular processes including oncogenesis, cellular transformation, cellular growth and exocytosis. Currently, much experimentation is performed involving kinases which may inhibit cancer growth or promote cancer cell death. Determining the specific kinase involved in inhibiting cancer growth or promoting cell death is important to society. Therefore, advances in recognizing kinase activity levels are extremely important.
Reference is made to Robyt and White (1990) which is incorporated herein by reference, for a general description of methods for determining the activity of an enzyme. Robyt and White defines the activity of an enzyme as the amount of reaction that a certain amount of enzyme will produce in a specified period of time. The activity is determined by measuring the amount of product produced or the amount of substrate used up per unit of time under high concentrations or saturating conditions of substrate. This is usually accomplished by performing a chemical analysis for the product or substrate.
Substrates that are typically used in an assay for specific kinase activity include casein, which is isolated from milk; histones, isolated from calves; phosphovitin, isolated from egg yolks; and myelin basic proteins, isolated from bovine spinal cords. These substrates can be phosphorylated in an assay, assuming that the correct kinase has been chosen. Assays utilizing these substrates to determine kinase activity are well known in the prior art.
Most current methods of measuring protein kinase activity are based on the radioactive detection method. In these methods, a sample containing the kinase of interest is incubated with activators and a substrate in the presence of γ-32 P-ATP or γ-32 P-GTP. Often, a general and inexpensive substrate such as histone or casein is used. After a suitable incubation period, the reaction is stopped and an aliquot of the reaction mixture is placed directly onto a filter which binds the substrate. The filter is then washed several times to remove excess radioactively-labeled free ATP, and the amount of radio-labeled phosphate incorporated into the substrate is measured by scintillation counting. This method is widely used and provides an accurate method for determining protein kinase activity in both crude and purified samples.
Babcook et al. (1991) also describe an assay using monoclonal antibodies and immunofluorescence technology for the determination of protein-tyrosine kinase and protein-tyrosine phosphatase activities. The method was performed utilizing p561ck or p60src.
Budde et al. (1991) disclose assay techniques utilizing acidic peptide substrates of protein kinases. This technology uses radioactive phosphorous placed in a substrate to be studied. After kinase activity, the phosphopeptide is eluted while the individual radioactive phosphorus, ATP and protein are impeded.
Gopalakrishna et al. (1992) disclose a method which utilizes the conventional approaches to measure protein kinase activity. The method combines the incubations and filtrations necessary to determine the protein kinase activity using multi-well plates with fitted filtration disks. In a related reference by Chakravarthy et al. (1990), kinase C activity is measured by using protein kinase C (PKC) selective peptide substrates by incorporating radioactive phosphorous into the substrate. The radioactivity is measured by liquid scintillation.
A non-radioactive method of detecting kinase activity has been developed in which tyrosine phosphorylation is detected by using anti-phosphotyrosine antibodies (Rijksen et al., 1991). After incubation of the tyrosine kinase with unlabeled ATP and a suitable substrate, the reaction mixture is subjected to a dot blot assay on a polyvinylidene diflouride (PVDF) membrane. The extent of phosphorylation is determined by reaction with anti-phosphotyrosine antibody, followed by detection with an immunogold staining procedure. The amount of phosphotyrosine present is detected with a densitometer.
A disadvantage of the dot blot method is that it is limited to detecting tyrosine kinases. Antibodies to phosphotyrosine can be produced due to the size of the antigen. Attempts to produce similar antibodies to phosphoserine and phosphothreonine have not been successfully employed to assay for phosphoserine- and phosphothreonine-containing proteins. In addition, the assay requires several incubation and washing steps, each taking a considerable amount of time, which results in a greatly extended assay completion time. The result of the assay is a colored dot on the blot. The colored dot may limit the effective sample range of the assay and may require the user to quantitate the final results using a scanning densitometer, which is an expensive piece of equipment not available in all laboratories. The densitometer must have beam dimensions covering at least the targeted cross section of the dots generated.
The present invention provides for a method of determining the presence or activity of a selected protein kinase, comprising conjugating a binding compound to a peptide substrate forming a modified peptide substrate; adding a sufficient quantity of the modified peptide substrate to a solution containing the selected protein kinase; incubating the protein kinase with the modified peptide substrate under conditions where the protein kinase is active for a time sufficient to form a modified peptide product; and measuring the activity of the protein kinase.
The present invention is further directed to a kit for determining the presence or activity of a selected protein kinase comprising a container containing a modified peptide substrate having specific reactivity to the protein kinase and modified by chemical reaction to allow quantitation; and instructions for use.
The present invention is also directed to a kit for determining the presence or activity of a tyrosine kinase comprising a container containing a modified peptide substrate selected from the group consisting of biotinylated Promega peptide G (SEQ. ID. NO: 7) and analogs and combinations thereof; a container containing a biotin-binding matrix; and instructions for use.
The present invention is also directed to a kit for determining the presence or activity of serine-threonine kinases comprising a container holding a modified peptide substrate selected from the group consisting of biotinylated Promega peptide A (SEQ. ID. NO: 1), biotinylated Promega peptide B (SEQ. ID. NO: 2), biotinylated Promega peptide C (SEQ. ID. NO: 3), biotinylated Promega peptide D (SEQ. ID. NO: 4), biotinylated Promega peptide E (SEQ. ID. NO: 5), biotinylated Promega peptide F (SEQ. ID. NO: 6), biotinylated Promega peptide G (SEQ. ID. NO: 7), biotinylated Promega peptide H (SEQ. ID. NO: 8) and analogs and combinations thereof; a container holding a biotin-binding matrix; and instructions for use.
Further, the present invention is directed to a method for detecting the presence or activity of a selected protein kinase in a body fluid comprising reacting the body fluid with a sufficient amount of a biotinylated peptide substrate forming a modified peptide product under conditions where the protein kinase is active for a time sufficient to form the modified peptide product in an amount such that the modified peptide product may be detected and measuring the amount of modified peptide product.
The present invention makes it possible to assay for a specific protein kinase in the presence of several other proteins kinases in a tissue extract. This type of assay is highly desirable to investigators because the purification of the specific protein kinase under study is not required. Conventionally, purification is required to remove other protein kinases in the extract. This lengthy approach may be circumvented by using the specific biotinylated peptide substrate approach.
In addition, the investigator is aided in determining the expression level of the specific kinase under various physiological conditions with minimal losses in the enzyme, since purification is obviated. This means that the activity assayed is an accurate estimate of the total active enzyme expressed.
Peptide substrates for several protein kinases such as cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), Ca2+ /phospholipid-dependent protein kinases (PKC), casein kinases 1 and 2 (CK-1 and CK-2), growth factor receptors, non-growth factor receptors and soluble proteins that contain an active tyrosine kinase, cell cycle-dependent protein kinase (p34cdc2 protein kinase), S6 protein kinase, Ca2+ /calmodulin-dependent or multifunctional (CAM) protein kinases, DNA-dependent protein kinase, and carboxyl-terminal domain (CDT) kinases are custom synthesized using a peptide synthesis protocol that incorporates a biotinylated amino acid at the N-terminus of the peptide. Thus, there are several protein kinases that can be assayed by the present procedure providing for the ability to make several kits four use with the various protein kinases.
The assay is very fast and may typically be completed in less than 10 minutes after termination of the reaction. This is important since current protocols require approximately 2 hours to obtain the same objectives.
No special equipment is required other than standard equipment that is currently available in most laboratories. The procedure does not require special inorganic or organic solvents such as phosphoric acid, acetic acid, acetone or ethanol, found in prior art protocols.
The present invention is amenable to be adapted to a large scale assay which may be demanded by large laboratories or pharmaceutical laboratories engaged in drug research.
The cost of the present invention is similar to the cost of prior art assays. The projected increased cost of avidin and biotinylating reagent may be offset by the elimination of expensive solvents required by other assays.
Further, the present invention eliminates the need for the use of an assay system requiring gel electrophoresis to separate the exogenous peptide from endogenous substrates. Gel electrophoresis separates the various mono- and phosphorylated forms of the substrate that may be useful. However, this method further complicates the quantitation of total phosphate trsfer.
Problems are often encountered when utilizing the prior art filter binding paper assay to bind strongly positive molecules that are present in the tissue of cellular extract and are phosphorylated. The present invention overcomes the problems because only the substrate and product are bound since they are the only components that are biotinylated.
The present invention eliminates the need to change the primary structure of the consensus sequence, which is the primary amino acid sequence target of the enzyme being analyzed, by adding additional arginine residues in order to maintain the primary sequence for the enzyme that is present in vivo. Prior art assays utilizing filter binding require the substrates to contain multiple arginine residues which may alter the specificity of the substrate such that the substrate may become suitable for another kinase. An example of this alteration is given by the sequence Arg-Arg-Arg-Tyr-Ser (SEQ. ID. NO: 9) (a key to amino acid abbreviations is found in Table 1) that is present in the CDT kinase peptide substrate Arg-Arg-Arg(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4 (SEQ. ID. NO: 10) which might be recognized by cAMP-dependent protein kinase and other kinases recognizing this sequence.
The present invention yields results with a greater probability of accuracy since virtually all of the phosphorylated peptide molecules are captured by the strong binding avidin or streptavidin-coated matrix. Conversely, the phosphorylated proteins aliquoted onto the phosphocellulous paper are bound by an electrostatic interaction that is not strong. Therefore, loss of phosphorylated peptide occurs during the washing procedure.
Reference is now made in detail to the preferred embodiments of the invention, examples of which are illustrated in the accompanying figures.
FIG. 1 is a graph illustrating the effect of enzyme concentration on the kinase activity of PKA using the modified Kemptide substrate. The peptide was present at 100 μM, and the enzyme was assayed as described in example 1.
FIG. 2 is a graph illustrating the effect of increasing the concentration of the biotinylated Kemptide substrate on the protein kinase activity of PKA. Two Kemptide units of PKA were present in the reaction. The remaining reaction components were present as described in example 1.
FIG. 3 is a graph illustrating the kinetic analysis of PKA with Kemptide as a substrate at 2 units per reaction as described in example 1.
FIG. 4 is a graph illustrating the effect of the peptide inhibitor of PKA on its protein kinase activity as described in example 1.
FIG. 5 is a graph illustrating the protein kinase activity of PKA in various rat tissues as described in example 2.
FIG. 6 is a graph illustrating the effect of enzyme concentration on the kinase activity of PKC using the biotinylated neurogranin (28-43) as substrate as described in example 3.
FIG. 7 is a graph illustrating the effect of increasing the concentration of the biotinylated neurogranin (28-43) substrate on the protein kinase activity of PKC as described in example 3.
FIG. 8 is a graph illustrating the effect of the PKC inhibitor on the activity of PKC using the biotinylated neurogranin (28-43) as a substrate as described in example 3.
FIG. 9 is a graph illustrating the effect of protein kinase activity of PKC in rat brain extract and of a partially pure PKC as described in example 4 .
For purposes of the present invention, the following definitions apply:
Activity of an enzyme: The amount of reaction product(s) that a certain amount of enzyme acting on a substrate will produce in a specified period of time.
Modified peptide: A chemical species composed of a peptide which has been subsequently reacted with a second chemical moiety, in this case biotin, which allows the peptide to be monitored by virtue of the properties of the second chemical moiety. The moiety may be referred to as a detector segment or modification tag.
Peptide: A compound generally consisting of 2-30 naturally occurring or synthetic amino acids which can also be further modified including covalently linking the peptide bonds of the alpha carboxyl group of a first amino acid and the alpha amino group of a second amino acid by eliminating a molecule of water. The amino acids can be either those naturally occurring amino acids or chemically synthesized variants of such amino acids or modified forms of these amino acids which can be altered from their basic chemical structure by addition of other chemical groups which can be found to be covalently attached to them in naturally occurring compounds.
Phosphorylation: The addition of a phosphate group to a substrate.
Modified peptide product: A chemical species which is formed by the action of an enzyme on a modified peptide substrate, which has been altered from the original bonding pattern. Such alterations can include the addition or removal of new chemical species to the modified peptide substrate. The particular changes made in the modified peptide substrate will depend upon the particular enzyme which has been involved in the alteration of the modified peptide substrate. Examples of a modified peptide product may include, but are not limited to, a product, formed by incubation of a modified peptide substrate with a protein kinase under conditions where the kinase has altered the modified peptide substrate by addition of a phosphate group to the peptide from a phosphate donor such as ATP or GTP.
Sequence requirement: The property of an enzyme to recognize and catalyze a reaction due to the peptide sequence of the substrate.
Substrate: The substance on which an enzyme acts.
Modified peptide substrate: A modified peptide which can be changed by the action of an enzyme. Such changes can include the addition or removal of chemical species to the modified peptide. Often it would be desirable to design a particular modified peptide substrate for assay of a particular enzyme of interest. A potential modified peptide substrate for protein kinase must have an amino acid which can act as a phosphate acceptor, such as serine. The utility of a potential modified peptide substrate for assay of a particular enzymatic activity can be determined by incubating the potential modified peptide substrate with the enzyme under conditions where the enzyme is known to be active and observing the rate with which a modified peptide product is generated.
The present invention quantitates the activities of kinase enzymes by measuring the amount of a specific modified peptide substrate that has undergone a reaction by the enzyme.
The kinases quantified in the present invention preferably use ATP or GTP as the phosphate donor and transfer the gamma phosphate from the molecule to a serine, threonine or tyrosine amino acid. Protein kinases are distinguished by their ability to phosphorylate substrates on discrete sequences. These sequences have been determined by sequencing the amino acids around the phosphorylation sites and are usually distinct for each protein kinase. The recognition sequence on each substrate is for a specific kinase catalyst.
The substrate binding site on the kinase is believed to exist in the enzyme's catalytic domain. This domain is essential to all protein kinases. It typically contains over 240 residues and also contains the kinase's ATP or GTP binding site.
Measurement of Activity
The activity of an enzyme is determined by measuring the amount of product produced, i.e., modified peptide product. The measurement of the activity of the enzymes can be performed by quantitation of the amount of the tagged product of the reaction. The activity of protein kinases can be measured because of their capability to modify specific peptide substrates by addition of phosphate groups to the peptide substrates of the invention.
Incubation of Enzyme
The enzyme is incubated under conditions known to the art with the modified peptide substrate to form modified peptide products under conditions where the enzyme is active. Each of the enzymes is usually assayed at its optimum pH, temperature and other conditions.
Assay Protocol
An assay protocol has been developed to determine enzymatic activity of various protein kinases in a specific, fast and convenient manner. The assay protocol is intended to assay for individual protein kinases in the presence of a mixture of other protein kinases. The enzyme assay may be packaged in a kit format and separate kits can be provided for the specific protein kinase required. In addition, this assay may be scaled to accommodate a high through-put format.
Peptide Substrates
A number of specialized peptide substrates, including analogs and combinations of these peptide substrates, have been developed for use with the process, described in the following table:
|Promega SEQ. ID. Peptide Sequence NUMBER
A *-Leu-Arg-Arg-Ala-Ser-Leu-Gly
B *-Ala-Ala-Lys-Ile-Gln-Ala-Ser-Phe-Arg- 2
C *-Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu 3
D *-Pro-Lys-Thr-Pro-Lys-Lys-Ala-Lys-Lys-Leu 4
E *-Asp-Asp-Asp-Glu-Glu-Ser-Ile-Thr-Arg-Arg 5
F *-Glu-Pro-Pro-Leu-Ser-Gln-Glu-Ala-Phe-Ala- 6
G *-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe 7
H *-Arg-Lys-Ile-Ser-Ala-Ser-Glu-Phe 8
*Refers to Biotin C6 moietyKey: Abbreviation Amino Acid Ala Alanine Cys Cysteine Asp Aspartic Acid Glu Glutamic Acid Phe Phenylalanine Gly Glycine His Histidine Ile Isoleucine Lys Lysine Leu Leucine Met Methionine Asn Asparagine Pro Proline Gln Glutamine Arg Arginine Ser Serine Thr Threonine Val Valine Trp Tryptophan Tyr Tyrosine
Promega peptides A-C contain a favorable sequence specific for serine-threonine kinases. Promega peptide G forms a favorable substrate for the tyrosine kinase.
While the material presented takes advantage of peptides which are chemically synthesized in vitro and some of which are commercially available, it should be noted that the invention can be performed by isolation of a peptide from a natural source including digestion of a larger precursor protein or peptide and isolation of the proper resulting fragment by methods well known in the art followed by its attachment to a detector segment as defined.
The assay utilizes a biotinylated labeled peptide that is required for the protein kinase under consideration. In contrast to protein substrates often used for protein kinase assays, peptides can be designed to be a selective substrate for a particular kinase by synthesizing a peptide that mimics the consensus sequence recognized by the enzyme of interest. The biotinylated peptide can be phosphorylated by the respective protein kinase under optimal phosphotransferase conditions for that enzyme in the presence of γ-32 P-ATP. Since both the phosphorylated and unphosphorylated peptides are biotinylated, they can be captured on a biotin-binding matrix and the remaining free γ-32 P-ATP can be removed by several washings.
The biotin-binding matrix can be any matrix that is covalently linked to avidin or streptavidin molecules such as filter disks, beads or a soluble matrix. FMC Corporation (Pine Brook, N.J.) is an example of a manufacturer of avidin and streptavidin in matrices such as Streptavidin-Linked Polyvinylcarbonate (PVC) Silica, Microporous Plastic Sheets or 12.5 mm Disks. The captured peptides may be placed in a liquid scintillation spectrometer to determine the 32 P incorporated into the peptide and hence the protein kinase activity of the enzyme under consideration.
The basic principle of the biotinylated peptide approach is to conjugate the peptide substrates for a specific protein kinase with biotin from which the biotinylated peptide can be used as a substrate to assay for the enzyme in question. After completion of the reaction, the phosphorylated and unphosphorylated peptides are aliquoted onto streptavidin- or avidin-coated filter membranes, ELISA-formatted plates or avidin-coated magnetic particles, where the peptides bind. The free ATP can be removed by a washing procedure and the ATP-free peptides which are bound to the filters, particles or plates can then be counted in a liquid scintillation spectrometer, and the activity of the enzyme can be determined from the 32 P incorporated. A biotin group may be successfully attached to a peptide to be used as a substrate for protein kinase activity measurement if the following criteria are met:
1. The biotin group must-be covalently linked to the N-terminal amino group and not to any other amino group present internally or C-terminally to the peptide substrate. This is essential since linking a biotin molecule covalently to an ε amino group of an internal lysine will result in an alteration of the overall peptide charge. Hence, the effectiveness of the peptide as a substrate for some protein kinases such as PKA, PKC and CAM kinases may be altered.
2. The addition of the biotin moiety to the peptide should not result in a steric hindrance of the peptide substrate's accessibility to the enzyme under investigation. Preferably, six carbon atoms link the biotin to the substrate to minimize steric hindrance.
3. A matrix that can bind the biotinylated phosphorylated peptide must be made available so that the activity of the protein kinase in question may be determined by measuring the amount of radio-labeled peptide product. The binding should be specific for the modified phosphopeptide and any phosphorylated proteins or other peptide present in a sample should not bind to the matrix. Thus, the activity is proportional to the amount of bound phosphorylated, biotinylated peptide.
4. The excess γ-32 P-ATP that was not used in the reaction should be completely removed. The objective is to promote a low background noise level and further, to provide a quick method to assay for the enzyme in question.
5. The binding capacity of the matrix to the biotinylated peptide should be high enough such that a concentration several times the Km value for the peptide substrate is used. This requirement will ensure that the initial rate of reaction is linear within a reasonable time. Further, the initial rate of reaction should be proportional to the amount of enzyme in the reaction.
For small scale experiments, a 25 mm diameter avidin-coated membrane disk can be numbered with a pencil. Alternatively, a known volume of avidin-coated magnetic particles in an Eppendorf tube may be used.
Kinase Assay
The kinase assay is carried out as described using purified enzymes or tissue extracts as an enzyme source and the biotinylated peptide as a substrate in the presence of other reagents that are required for the reaction such as buffer, divalent cations such as Mn2+, Ca2+, and Mg2+, and γ-32 P-ATP. For some protein kinases, other cofactors may be required such as phospholipids and calmodulin. After completion of the reaction, an aliquot is withdrawn and applied directly to the avidin-coated disks. The disks are placed in a beaker containing 2M NaCl to wash the free γ-32 P-ATP. Alternatively, the aliquots are pipetted into the particles-containing tube and mixed well. The NaCl in the beaker can be changed 5 times each for 1 minute, which completes the operation in 5 minutes. When particles are used they may be washed by conventional means to remove free soluble reaction components including ATP and endogenous proteins.
The assay may be scaled up to a high through-put assay by slight modification of the matrix. Twenty-four or 96 wells containing streptavidin-coated plates may be used. Aliquots, withdrawn after completion of the assay reaction, are pipetted directly into individual wells rather than onto individual disks. Subsequently, the whole plate is washed by rinsing the wells with 2M NaCl. It is noted that prior art devices are available to apply the completed reaction mixture to the individual wells. Washing of the wells may be accomplished with an automated system. The plates may then be counted by using a liquid scintillation spectrometer such as a Top Count Microplate Scintillation and Luminescence Counter manufactured by Packard Corporation in Meriden, Connecticut and Wallac, Inc., in Gaithersburg, Maryland. Non-limiting examples of assays utilizing the present invention follow.
cAMP-Dependent Protein Kinase (PKA)
When cAMP-dependent protein kinase (PKA) is under investigation, a peptide substrate that is reported to be specific for this enzyme is biotinylated on its N-terminal group. A modified peptide *-Lys-Arg-Arg-Ala-Ser-Leu-Gly (Promega Peptide A; SEQ. ID. NO: 1) is synthesized using a peptide synthesizer. The peptide is tested by methods known to the art for being a substrate for PKA at different concentrations; namely, 0, 10, 20, 50 and 100 μM with purified PKA at several dilutions. The enzyme used in the studies is the catalytic subunit of PKA (#V5161, Promega Corporation, Madison, Wis.) with a specific activity of 50 pmol 32 P transferred to casein per μg enzyme in 1 minute at 37° C. or 10,000 pmol 32 P transferred to the Kemptide substrate Lys-Arg-Arg-Ala-Ser-Leu-Gly (Promega Peptide A, SEQ. ID. NO: 1) per μg enzyme per minute at 37° C.
Phosphotransferase Activity of Protein Kinases
This assay tests for determining the phosphotransferase activity of other protein kinases using the corresponding biotinylated peptide substrates. For example, to assay for the kinase activity of Ca2+ and phospholipid-dependent protein kinase (PKC), biotinylated neurogranin-derived peptide *-Ala-Ala-Lys-Ile-Gln-Ala-Ser-Phe-Arg-Gly-His-Met-Ala-Arg-Lys-Lys (Promega Peptide B; SEQ. ID. NO: 2) is used as a substrate under optimal condition for PKC protein kinase activity.
Other Assays
Other protein kinases can be assayed as described above for PKA and PKC but employing the appropriate peptide substrates and assayed under the corresponding optimal assay conditions. For example, the peptide substrate, *-Asp-Asp-Asp-Glu-Glu-Ser-Ile-Thr-Arg-Arg (Promega Peptide E, SEQ. ID. NO: 5) is used to assay for casein kinase I (CK-1). The peptide *-Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu (CK-2) (Promega Peptide C, SEQ. ID. NO: 3) is used to assay for casein kinase II (CK-2). The peptide, *-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (Promega Peptide G, SEQ. ID. NO: 7) is used to assay for the tyrosine kinase activity of activated epidermal growth factor receptor (EGFR).
In each case, the enzyme activity of the protein kinase under study is assayed using the method that was developed using the biotinylated derivative of the appropriate peptide substrate and the streptavidin-linked matrix, and then by the phosphocellulose disk method using an unmodified peptide substrate.
Protein kinases are assayed under their optimal conditions as described below with the exception of using the unmodified peptide substrates in the reaction and the recommended processing procedure described in Casnellie, J. E., 1991. Briefly, the reaction is terminated by withdrawing 40 μL aliquots and spotting them onto P81 paper (Whatman, Clifton, N.J.). The filters are dropped immediately into a beaker containing 0.5% phosphoric acid, and they are left with occasional swirling of the beaker for 5 minutes. The phosphoric acid solution is decanted and a fresh solution is added. This step is repeated again for a total of 4 washes, each for 5 minutes. The filters are dried and placed in scintillation vials and 5 ml of scintillation fluid was added. Enzyme activity is determined as described above in the assay system.
To determine the protein kinase activity for various kinases, certain criteria have to be met as follows:
1. The biotinylated derivative of the peptide should serve as a specific and good substrate for the enzyme in question as the unmodified peptide substrate.
2. The phosphorylation of the biotinylated peptide should not affect its binding to the avidin-linked matrix or to the streptavidin-linked matrix.
3. The phosphorylation of the biotinylated peptide should be proportional to the amount of the enzyme added and to certain time periods of the assay.
4. The phosphorylated proteins should not bind to the matrix unless they are biotinylated, i.e., only the phosphorylated and biotinylated peptide should be bound to the matrix.
5. The avidin-linked or streptavidin-linked matrix should have enough binding capacity for the biotinylated peptide added to the matrix.
6. There should be a means to remove the free, excess γ-32 P-ATP after completion of the reaction.
To achieve these criteria, peptides are synthesized that are specific substrates for several protein kinases (Kemp, B. E. and Pearson, R. B., 1991). Each of these peptides contain the consensus sequence required for phosphorylation by the corresponding enzyme. It is usually necessary to have these peptides linked to basic amino acids to impart a positive charge(s) on the peptide so that it may bind to the negatively charged phosphocellulose paper.
In the present invention, the peptides are modified in a manner that makes them tightly bound to the matrix. The biotinylated moiety is linked to the NH2 terminal of the peptide substrate via a 6-carbon atom linker.
The biotin-modified peptides are synthesized on a peptide synthesizer using established procedures of solid-phase peptide synthesis (Nova Biochem/Calbiochem, San Diego, Calif.). The biotin group is added before cleavage of the peptide from the resin. The identity and purity of the biotinylated peptides are confirmed by quantitative amino acid analysis, 2 high pressure liquid chromatography (HPLC) solvent systems, and fast atom bombardment (FAB) mass spectrometry.
Several parameters are investigated that may have influenced or affected the results of this assay. These are the diameter of the disks, the density of streptavidin per disk, the level of drying of the disks, the concentration of the modified peptide substrate and the amount of enzyme that can be used.
The assay is carried out at 37° C. for 5 minutes. A proportional increase in the kinase-activity to the increase in the amount of enzyme present is observed.
Silica-based filter disks are manufactured that are linked to avidin or streptavidin. Two different sizes are made. One is 25 mm (i.d.) and the other was 12.5 mm (i.d.). The filters are linked to 0.5, 1.0, or 2.0 mg of streptavidin or avidin. The 12.5 mm diameter disks with 0.5 mg streptavidin per disk give the best results. In addition, several levels of dryness are tested such as wet, 1 hour dried disks, 2 hour dried disks, and 2 week dried disks for their suitability in this assay. This is examined to ensure that the assay is user friendly. Results indicate that semi-wet as well as dry disks can be used successfully in this assay.
The specificity of this assay is further examined by using tissue extract as a source of PKA. The linearity of 32 P incorporation into the biotinylated peptide is also checked by increasing the substrate concentration.
Experimental results indicate that the biotinylation of the peptide substrate does not affect the suitability of the peptide as substrate. One mg of avidin (or streptavidin) per disk of 25 mm (i.d.) (0.5 mg/disk of 12.5 mm (i.d.) is sufficient for the binding of up to 5 nmol of biotinylated peptide which gives a final concentration of 200 μM (reaction vol of 25 μL). This peptide concentration is sufficient for most of the purposes required. Both avidin and streptavidin can be used for this assay, and due to the lower cost of streptavidin, most of the studies are performed with streptavidin.
Finally, all wash solutions are tested to remove the free ATP and the best background is obtained with 2M NaCl. Thus, this solution was used as the wash solution.
The following conditions were carried out to generate data:
1. Reactions were terminated with the termination solution (2M urea, 125 mM EDTA, and 1% SDS), and 25 μL aliquots were spotted onto 12.5 mm streptavidin-linked disks (0.5 mg of streptavidin) or into wells of the 24-well plate.
2. The disks or wells were rinsed or washed five times each for one minute with 2M NaCl.
3. The disks or plates were dried and counted using a liquid scintillation counter.
The present invention is also directed to kits that utilize the process described. A basic kit for quantitating the activity of an enzyme includes a container containing a bioreagent, which is a modified peptide substrate having specific reactivity to the enzyme, modified by chemical reaction to allow quantitation, and instructions for use.
The modified peptide substrate supplied in the kit includes the binding compound biotin. The kit also contains at least one buffer which is compatible with the enzyme selected.
A kit is directed to specifically detect activity from enzymes selected from the group of serine-threonine kinases and tyrosine kinases. The kit includes a container holding a modified peptide substrate with the binding compound biotin attached. The substrate modified peptide is specific for the selected enzyme.
A kit is directed to quantitating the activity of an enzyme which is selected from the group consisting of kinases. The basic kit includes at least one container with a modified peptide substrate including the binding compound biotin. The substrate modified peptide is specific for the selected enzyme.
The modified peptide substrate in the kit may be one of the following peptides, depending upon the type of enzyme to be assayed as explained: Promega Peptide A (SEQ. ID. NO: 1), Promega Peptide B (SEQ. ID. NO: 2), Promega Peptide C (SEQ. ID. NO: 3), Promega Peptide D (SEQ. ID. NO: 4), Promega Peptide E (SEQ. ID. NO: 5), Promega Peptide F (SEQ. ID. NO: 6), Promega Peptide G (SEQ. ID. NO: 7) and Promega Peptide H (SEQ. ID. NO: 8). Reference is made to Table 1 indicating modified peptide substrates specific for selected enzymes.
Instructions for use are also included. "Instructions for use," is a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amount of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.
The kit may also include a biotin-binding matrix linked to avidin or streptavidin molecules such as filter disks, beads, soluble matrix or plates. The biotin-binding matrix may be utilized to bind the modified peptide substrate and modified peptide product containing biotin.
The amounts of the various reagents in the kits can be varied depending on a number of factors, such as the optimum sensitivity of the assay. The instructions for use are suitable to enable an analyst to carry out the desired assay.
It is within the scope of this invention to provide manual test kits or test kits for use in automated analyzers.
The following examples are presented to illustrate the advantages of the present invention and to assist one of ordinary skill of making and using the same. The examples are not intended in any way to otherwise limit the scope of the disclosure or protection granted by the patent. While precise descriptions are given for assays of protein kinases, it should be clear to one well versed in the art that the results of the present invention can be applied to the assay of a wide variety of selected enzymes.
While the material presented takes advantage of peptides which are chemically synthesized in vitro, and some of which are commercially available, it should be noted that the invention can be performed by isolation of a peptide from a natural source.
The assays in these examples are based on the use of biotinylated peptide substrates that are specific for certain enzymes.
Kinase Activity of cAMP-Dependent Protein Kinase
The kinase activity of cAMP-dependent protein kinase (PKA) was carried out in duplicates at each of the various peptide substrate concentrations, in a reaction volume (50 μL) containing 40 mM Tris HCl, pH 7.4, 20 mM MgCl2, 100 μM γ-32 P-ATP (sp.act. of 100-200 cpm/pmol), 100 μg/ml of bovine serum albumin (BSA), and the appropriate biotinylated peptide substrate: *-Leu-Arg-Arg-Ala-Ser-Leu-Gly (Promega Peptide A, SEQ. ID. NO: 1, M.Wt of 998.4).
The reaction was started by addition of the appropriate amount of enzyme (Promega #V5221) to give 2 Kemptide units and incubated for 0, 5, and 10 minutes at 37° C. (one Kemptide unit is defined as the amount of enzyme required to catalyze the incorporation of 1 pmol of phosphate into Kemptide substrate in 1 minute). At specified periods of time at the appropriate temperature, the reaction was terminated with the addition of 10 μL of terminating solution as described above.
Twenty-five μL aliquots were withdrawn and spotted in a 12.5 mm (i.d.) streptavidin-linked disk (0.5 mg of streptavidin/disk) and placed in 2M NaCl-containing beaker. After one minute of gently swirling the beaker at room temperature, fresh NaCl was added. This washing procedure was repeated 5 more times at one minute each. The disks were then washed once more with water, dried and placed in scintillation vials. Five ml of scintillation fluid was then added.
Enzyme activity was calculated by determining the radioactivity via liquid scintillation spectrometry. To confirm that the phosphorylation was specifically catalyzed by PKA, a specific and potent inhibitor of PKA (#V5681, Promega Corporation, Madison, Wis.) was included in the reaction. All PKA phosphotransferase activity should have been inhibited by the addition of inhibitor.
Results Using Measured PKA
The relationship between rate of reaction (initial velocity of PKA) as determined by the protocol and under the condition described above, and the amount of enzyme in the reaction is shown in FIG. 1. Using 100 μM of modified peptide substrate in the reaction, a linear response in the activity of PKA was obtained with increasing the amount of enzyme in the reaction. The activity of the enzyme was also investigated in relation to the concentration of the biotinylated peptide substrate.
As shown in FIG. 2, the kinetic plot displays typical Michaelis-Menten saturation behavior (hyperbolic curve). The transformation of the same data resulted in a Km Value for the substrate of 33 μM, and a Vmax value of 1.45 pmol/min as illustrated in FIG. 3. Similar values were obtained with the phosphocellulose paper assay using the Kemptide as substrate, and a Km value for the unmodified substrate was not affected by biotinylation as illustrated in FIG. 4. Kemp, B. E. and Pearson, R. B. 1991. The protein kinase activity was specific to PKA since the addition of 50 μM of the PKA inhibitor-(PKI) drastically reduced the enzyme activity.
Kinase Activity of PKA in Rat Tissues
The kinase activity of PKA in various rat tissues was also investigated using the modified peptide substrate and assay protocol as described above.
As illustrated in FIG. 5, the protein kinase activity of PKA was 2.2, 1.75, 3.2, and 1.9 nmol 32 P/min/mg of rat liver, ovary, heart, and brain, respectively, and was comparable to those obtained with unmodified peptide and the phosphocellulose paper method. The phosphate incorporation into the modified substrate was completely abolished when 100 μM of PKA inhibitor (PKI) was included in the reaction. Thus, the phosphorylation of the modified peptide substrate, under the conditions described above and assayed with the protocol, is catalyzed by PKA and not by other protein kinases present in the tissue extract.
Any protein(s) that is phosphorylated in the reaction and has a basic charge will not bind to the matrix. Only the phosphorylated modified peptide substrate is bound to the matrix. Thus, the phosphate incorporation using the assay protocol reflects the true 32 P incorporation into the peptide substrate. This is not the case with the phosphocellulose paper assay method where any basic protein in the extract that is phosphorylated will bind to the paper and results in overestimation of the kinase activity of the enzyme.
Protein Kinase Activity of Ca2+ and Phospholipid Dependent Protein Kinase
The protein kinase activity of Ca2+ and phospholipid dependent protein kinase activity (PKC) was determined in a similar manner to that of PKA described above, except that the biotinylated peptide substrate was a derivative of neurogranin; namely, biotinylated neurogranin (28-43) Chen, S-J, et al., 1993.
The peptide was modified by the addition of biotin containing moiety: *-Ala-Ala-Lys-Ile-Gln-Ala-Ser-Phe-Arg-Gly-His-Met-Ala-Arg-Lys-Lys (Promega Peptide B, SEQ. ID. NO: 2, Mol.Wt. is 2139.6), and was used at a concentration of 100 μM. The enzyme (#V5261, Promega Corporation) was diluted 5× in 100 μg/ml of BSA.
The kinase activity of the PKA was determined in a reaction volume of 50 μL consisting of 40 mM Tris. HCl, pH 7.4, 10 mM MgCl2, BSA at 100 μg/ml, and 100 μM of γ-32 P-ATP (sp.act. of 100 cpm/pmol). The activity was determined in the presence and absence of 100 μg/ml of phosphatidylserine, 10 μg/ml of diacylglycerol and 0.4 mM calcium chloride. Reaction was started with the addition of 5 μL of the enzyme and incubated at 25° C. After specified periods of time, reactions were terminated by the addition of 10 μL of terminating solution (see above). Twenty-five μL aliquots were aliquoted on disks and the disks were processed as described above for PKA.
Results Using Measured PKC
The activity of PKC obtained was also compared with that obtained using the unmodified peptide substrate and assayed by the phosphocellulose paper method. The results shown in FIG. 6, using a modified peptide substrate concentration of 100 μM, indicate that the PKC activity was linear with increasing the amount of enzyme protein in the reaction. Similar to the work presented with PKA, the effect of substrate concentration on the kinase activity of PKC was also investigated.
A Michaelis-Menten kinetics was obtained, i.e., hyperbolic curve as illustrated in FIG. 7, and the activity of the enzyme reached a maximum around 100 μM of modified substrate. However, the activity of PKC was about 6-7 fold higher than its activity when assayed with the phosphocellulose paper method. The increase in activity of PKC in the assay system may be due to the enhanced and specific binding of the phosphorylated peptide and also to the small loss of the phosphorylated peptide product from the matrix during the washing procedure. This demonstrates that the kinase activity of the enzyme can be determined accurately by using the assay system with the biotinylated neurogranin (28-43).
The activity was fully inhibited by the specific myristoylated PKC peptide inhibitor as illustrated in FIG. 8. Therefore, 10-20 μM of the inhibitor was sufficient to inhibit the kinase activity of PKC, indicating that all incorporated 32 P into the peptide substrate was catalyzed via PKC.
Kinase Activity of PKC Using Rat Tissue
The kinase activity of PKC in the rat brain was also studied utilizing the materials and methods of Example 3.
As illustrated in FIG. 9, the activity of PKC in an enriched fraction of a high spin supernatant of rat brain extract was about 500 pmol 32 P/mg/min, and that of partially purified PKC was 2200 pmol 32 P/mg/min.
It is understood that the invention is not confined to the particular construction and arrangements herein illustrated and described, but embraces such modified forms thereof and comes within the scope of the claims following the bibliography.
Babcook, John, Julian Watts, Ruedi Aebersold and Hermann J. Ziltener, 1991, "Automated Nonisotopic Assay for Protein-Tyrosine Kinase and Protein-Tyrosine Phosphatase Activities." Anal. Biochem., 196, 245-251.
Budde, Raymond J. A., John S. McMurray and Donald A. Tinker, 1992, "An Assay for Acidic Peptide Substrates of Protein Kinases." Anal. Biochem., 200, 347-351.
Casnellie, J. E., 1991. "Assay of Protein Kinases Using Peptides with Basic Residues for Phosphocellulose Binding." Meth. Enzymol., 200:115-120.
Chakravarthy, Balu R., Alexandra Bussey, James F. Whitfield, Marianna Sikorska, Ross E. Williams and Jon P. Durkin, 1991, "The Direct Measurement of Protein Kinase C (PKC) Activity in Isolated Membranes-Using a Selective Peptide Substrate." Anal. Biochem., 196, 144-150.
Chen, S-J, Klann, E., Gower, M. C., Powell, C. M., Sessoms, J. S., and Sweatt, J. D., 1993. "Studies with Synthetic Peptide Substrates Derived from the Neuronal Protein Neurogranin Reveal Structural Determinants of Potency and Selectivity for Protein-Kinase." C. Biochem., 32:1032-1039.
Gopalakrishna, Rayudu, Zhen H. Chen, Usha Gundimeda, John C. Wilson and Wayne B. Anderson, 1992, "Rapid Filtration Assays for Protein Kinase C Activity and Phorbol Ester Binding Using Multiwell Plates with Fitted Filtration Disks." Anal. Biochem., 206, 24-35.
Kemp, B. E. and Pearson, R. B., 1991. "Design and Use of Peptide Substrates for Protein Kinases." Meth. Enzymol., 200:121-134.
Rijksen, Gert, Brigit A. van Oirschot and Gerard E. J. Staal, 1991, "Nonradioactive Assays of Protein-Tyrosine Kinase Activity Using Anti-phosphotyrosine Antibodies." Methods in Enzymology, 200:98-107.
Robyt, John F. and Bernard J. White, 1990, Biochemical Techniques--Theory and Practice, Waveland Press, Inc., Prospect Heights, Ill., pp. 291-320.
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Today we'd like to introduce you to Joe Rindone.
So, before we jump into specific questions about the business, why don't you give us some details about you and your story.
I started Personal Training in 2007, while living in Boston. I went to a vocational school for Personal Training and added on many specialty certifications throughout my five few years of training.
After renting space from various locations to train my clients, in 2012 I decided to open Back Bay Fit on Comm Ave in the Charlesgate community. I created the gym to offer the best private training experience in Boston. Not only did my clientele grow but there are now seven of us training at the facility today.
In addition, I grew up in Arlington MA (about 7 miles west of Boston) and from social media / word of mouth my Arlington clientele was growing quickly. So in 2015 I decided to open another Back Bay Fit in east Arlington on Mass Ave. near the Cambridge line. We currently have five trainers at the studio and also run some Small group Training at that facility as well.
Overall, has it been relatively smooth? If not, what were some of the struggles along the way?
I definitely had my share of obstacles to hurdle. Social Media is a great marketing tool but to get to the next level, you definitely need a good website and presence on review sites. Finding the right teams to know your business and work on this was a good obstacle that we still sometimes fight with today.
In addition, as trainers we are always so busy with clients and the other day to day responsibilities of running two gyms, that trying to work Reviews/Testimonials can be a complete afterthought. Scheduling time to work on a little extra day to day things is very important!
Alright – so let's talk business. Tell us about Back Bay Fit – what should we know?
Back Bay Fit is a small studio, where we make sure each client is a name and not a number. The trainers that work at Back Bay Fit all work independently and are true veterans in the industry. What we offer in comparison to any other studio cannot be matched. Every trainer truly cares and thinks about their clients daily. We take focus on dedication and reaching goals week to week.
In addition, the facility itself is just a fun, great atmosphere. We keep the gym extremely clean, equipped with the best equipment and make it the premiere private training facilities we claim them to be!
Any shoutouts? Who else deserves credit in this story – who has played a meaningful role?
A lot of credit definitely goes to the trainers at both Boston and Arlington locations. Without their daily commitment, Back Bay Fit does not exist. When I first got started my teacher at my Personal Training School, Graeme Prue was very motivating and supportive. Training clients came naturally right out of school, yet there were always a few curve balls thrown. Graeme was a major help my first year in the field.
- Personal Training starts at $70 per hour for both locations
- Group Ex classes in Arlington starts at $10 per class
Contact Info:
- Address: Boston: 617-450-0001
Arlington: 781-643-0002
- Website: www.BackBayFit.com
- Phone: 617-450-0001
- Email: email@example.com
- Instagram: BackBayFit100
- Facebook: https://www.facebook.com/backbayfitarlington/
- Yelp: https://www.yelp.com/biz/back-bay-fit-arlington
- Other: Back Bay Fit


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Tuesday, February 28, 2012
Going Social
As a mom, time is very precious to me. Even now, I'm finding it a constant challenge to juggle the many roles I play - wife, working mom, daughter... you get the drift. So for a long time, I resisted getting involved with social media because I don't want to stretch myself thin with too many activities on top of family and work commitments. After all, I'm not someone who's crazy about chasing trends (among my friends, I was probably one of the last to change to a smartphone).
Yet I really didn't want to be left out. I'm curious about what's so exciting about Twitter or LinkedIn (I have a friend who raved about how LinkedIn has helped to bring in lots of freelance projects for her). At the very least, I want to know how these social networking sites work because I enjoy the challenge of learning something new.
So after much deliberation, I decided to jump on the bandwagon. I've got a Twitter account that had been dormant for a while. I revived that and am still learning how to navigate my way around the world of tweets. Sometimes, the number of messages are just too overwhelming for me. I can't figure out how some people can follow so many others and keep up with the avalanche of tweets!
Next, I got myself a Pinterest account and now I'm hooked! I love this very visual networking tool and have so much fun creating boards and pinning photos of things that inspire me or brilliant ideas for home organising, toddler activities etc. I'm loving it!
Of course, I guess for me as a blogger, starting a Facebook page is another milestone for me. I look forward to sharing with you interesting stuff or useful information that I come across; I also hope to be able to interact more with you this platform.
So let's get connected!
Please visit the Mommy Cafe Facebook page here (please like my page, thanks!)
If you are interested to see my Pinterest boards, you can go here.
I'm also on LinkedIn over here, let's link up!
Going Social
Mommy Musings|


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At the top or the world you can find Topper's Mountain … a scenic paradise that is home to some of the most amazing wines New England has to offer … Mark Kirby introduces us to Topper's Mountain Wines; with 14 (soon to be 18!) varieties of grapes grown on site, there's bound to be a wine that suits your palate. Give the team at Topper's a heads up before you visit, so they'll be on hand to provide you with a wine tasting and fabulous charcuterie plate at the cellar door …
Hi Mark. Where did your history/interest in the wine industry begin?
From the "draining the bottle" end of the industry, my involvement began at uni, but this time wasn't a very cerebral approach to the wonders of the world of wine!
My involvement from the "filling the bottle" end, i.e. the viticulture and oenology end of the industry, began in the late 1990s, when we bought Topper's Mountain.
Prior to this, a partnership from Moree of my two brothers' families and Peter and Jenni Birch decided in the early 1990s to shift focus from annual broadacre crops such as cotton and wheat on the Moree Plains to horticulture – Gwydir Grove was the first blossoming of this move, and Topper's Mountain the second.
But my family's history in the NENW goes back a long way before this – our great grandfather moved to Moree in the 1860s, and my brother and I still own his son's original selection, Glen Prairie, from 1874.
When did you establish Topper's Mountain Vineyard – and why did you choose this location?
The partnership mentioned above was the Thunderbolt's Rock Partnership, and it purchased Topper's Mountain in 1998.
Peter Birch is an agronomist who consulted all over the NSW North West, and he also has his own light 'plane. From his bird's-eye view while in the air, Peter knew all the good bits of dirt in the region. He knew Topper's Mountain is a degraded basalt cap and that such soils are excellent deep, free-draining horticultural soils. When Topper's Mountain came on the market in early 1998, on Peter's recommendation we all had a look at it and bought it.
Describe the property … how many hectares do you currently have planted with grapes, and what's the overall terrain like?
Topper's is on the north side Tingha at the very viticulturally cool-climate altitude of 900 m. It is open and undulating Tablelands rangeland with a wonderful view on the watershed between the Gwydir and MacIntyre Rivers.
The vineyard is 10 Ha and nurtures far too many grape varieties – the count is currently 14 and about to rise to 18, with more grafting happening this coming spring.
Of the wide variety of grapes grown at your vineyard, there are some our readers may never have heard about! Why is your vineyard particularly suited to growing such a large selection?
It's not so much that Topper's is suited to growing such a large selection of varieties, as I've found it hard to separate those really kicking goals from those merely doing well.
It has taken the Europeans and other old world wine regions centuries to home in on the few varieties that are best suited to each of their regions, so this process requires time, patience and loving attention to detail.
Having said that, it is quite remarkable that Topper's can produce excellent and acclaimed wines from such a range of varieties. For instance, early ripening Gewurztraminer hails from Alto Adige in north-eastern Italy and Alsace in France, and late ripening Nebbiolo comes from much warmer Piemonte in western Italy – these two varieties would and could never be found in the same vineyard in the old world.
Who is responsible for making your wines, and how do they come up with new blends?
Mike Hayes is our winemaker – he's based in Stanthorpe in our neighbouring wine region, The Granite Belt. Mike was the 2017 ASVO Winemaker of the Year.
This is the plus side of being where we are – a young winery in an unknown wine region. This allows us to do what we like, unconstrained by history.
The wines and blends we make come from a fusion of Mike's decades of experience making wines, in emerging varieties, knowledge and local markets and my penchant to do things differently – mostly sitting around the winery tasting bench with a table full of samples in front of us! My family says, "Why do things the easy way, when you can do them the Kirkby way?"
Recently a young fella, Jan Taborsky, has joined the team, and he's moving us into some new wine styles influenced by his European heritage – he's Czech – including a skin contact Gewurz, a traditional Portuguese blend of Touriga, Tinta Cao and Tinta Roriz fermented in Georgian qvervi (large earthenware vessels that are buried in the ground to control temperature) and a Pet Nat (Petillant Naturale – the original winemaking technique that results in lightly sparkling wines).
What's your wine making philosophy?
Mike and I focus on making wines that sing of their home and heritage – their unique Topper's terroir. We do this by unrelenting focus on quality in the vineyard, use of wild yeast from the vineyard for fermentation and minimal winemaking adjustments and additions.
Your wines have won so many awards, it'd be impossible to list them all here. You were also a finalist in this year's Armidale Regional Business Awards for Customer Service – Hospitality. What does it mean to you and your team to be acknowledged in this way?
It makes us proud and humbled in equal measure. We were only involved in the Regional Business Awards because some of our customers nominated us for the awards, so it's very comforting to know that our efforts to show cellar door visitors an enjoyable and instructive peek into the production end of the wine industry are appreciated and hitting their intended mark.
Terrible drought has affected so many areas of our country this year. Has your area been largely affected – and what impact has this had (if any) on your production?
The drought is terrible in our little corner of heaven and the broader region at the moment. However, our vines are dormant now and have low soil moisture requirements. Additionally, we have plenty of water from a large dam and a very productive underground bore.
In fact, we are due to give the vines a little drink in the next few days to start to help prepare them up for spring – which is going to be upon us before we know, it I suspect.
Probably our biggest fear resulting from the dry period is the increased likelihood of spring frost, which can be devastating for grapes. Budburst will be about the third week of September, but we can get frosts into late October or even early November, so a lot of pondering BOM forecasts and our weather station output is on the cards until mid-November!
What's the best way for us to try your wines?
The best way to try our wines are
- Local outlets such as the Royal, White Bull, Railway and St. Kilda Hotels, Red Grapevine and Charlie's Last Stand in Armidale,
- The Visitors' Info Centre, the General Merchant, the Inverell Club and the Australian Hotel in Inverell
- The Rest Point Motor Inn and Great Central Hotel in Glen Innes
- New England Brewing in Uralla
We'd love to see you at our Cellar Door for tastings with a charcuterie plate. Being off the beaten track, it's only open by appointment – call Colin 0420 902 554 or Mark 0411 880 580 or visit www.toppers.com.au for details.
Our website www.toppers.com.au is also where you can join our Top100 Wine Club, to make sure you don't run out!
I'm sure you love all your wines … but if you had to recommend something special that's ready to drink now, and also something that would be terrific to cellar for a few years – what would be your top picks?
Ready to enjoy now:
- 2015 Gewurztraminer, fantastic with Asian curries while it's still cool, then with Asian stir-fries, fruity salads and strong cheeses once the weather warms.
One to cellar:
- 2016 wild ferment Petit Manseng – a white wine that thinks it's a red as it relaxes into middle age. Great with chicken stews, duck and pork dishes.
Thanks Mark.
Interview: Jo Robinson.


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Yes, you did read that correctly. Colouring in. An activity that is typically reserved for kids and involves a set of colored pencils or markers and some black and white illustrations.
It sounds strange at first, but it turns out that adult colouring has become an incredibly popular activity over the last year or so. Just type "coloring" into Amazon and you'll see the enormous amount of adult colouring books available. When I first started researching this topic earlier this year, 2 of the 3 top bestselling books on Amazon were adult colouring books created by Johanna Basford. Now, over 2 million copies of her books have been sold, which isn't surprising given that her illustrations are quite magnificent.
So what is this new trend all about?
So why are adults spending time performing an activity that many would feel is not a particularly 'grown up' thing to do?
For a few really good reasons, it seems. Research suggests that for many, the process of colouring in helps to combat stress, reduce anxiety, and 'quieten' the mind, with some psychologists also reporting that colouring results in an increased ability to think creatively. Colouring also seems to have become an alternative practice for those who struggle with meditation, providing the same kind of calm, focused attention and relaxing effect.
Anecdotally it seems that many adults are simply finding the activity to be quite enjoyable once they've tried it, often describing the experience as one which helps to provide a 'mental break'. On her website Basford comments that most people haven't engaged in colouring activities since childhood, and that "coloring seems to help people think about a time when life was simpler and more carefree".
Colorful Workplaces
The trend has also been picked up in business, with some of Australia's largest organisations purchasing colouring books as a way of helping employees to more effectively handle stress and improve wellbeing.
Indeed, a 2014 study reported in the Journal of Occupational and Organizational Psychology identified a link between engaging in creative activity and demonstrating improved stress tolerance and work performance.
The authors encouraged organisations to consider professional development opportunities for employees that involve creative activities, and to promote creative activity in the same way that healthy eating and exercise are emphasised.
The Science behind Colouring
An explanation of the brain science behind colouring can be found on the Colourtation website, with Stan Rodski (a Melbourne-based Psychologist and Neuroscientist) explaining that colouring helps the brain to "manually change gears" from a stressed and pressured state to a more relaxed, calm state.
To get a little bit more scientific with that explanation, this shift in 'gears' occurs when brainwave patterns change from 'beta' (pressured and stressed) to 'alpha' (more relaxed) waves. The same state of brain activity is achieved from a range of other relaxing activities that adults regularly engage in – although not typically within the workplace (e.g. listening to music, meditating, watching TV).
Given the growing focus on mindfulness and meditation within leadership and employee populations, this trend will be an interesting trend to watch.
My Colouring Experience
When I first started researching this topic, I decided to try it out for myself of course – and quickly became hooked. Personally, adult colouring gets a big 'thumbs up' from me.
For me the experience is exactly as others described it – within the first few minutes of colouring I usually start to feel extremely relaxed and calm, and often find myself thinking a bit more creatively. Now I regularly use colouring as a break when completing mentally challenging tasks, especially if I find myself a bit 'stuck'. I find that the process of colouring allows my mind to just 'wander' by itself, often resulting in some new or alternative ideas surfacing.
The other reason I enjoy colouring so much? Because it creates an opportunity for both my mind and my body to be simultaneously quiet – a rare occurrence for me given the constant juggle of being a professional working mother. So in my house and office, the 'research' will continue – and my collection of adult colouring books will keep on growing.


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No, this isn't a doomsday post. My summer class ends TOMORROW and I'm excited!!! I love my teacher, and the class (or else I wouldn't be pursuing this particular path) but I am ready for a damn break!
Between celebrating our 4 year anniversary and going to the ER to have my lip glued back together, July has been a crazy busy month, not to mention all the homework/studying/tests that have been happening on my end.
Once Wednesday is over and done with, I have quite the to-do list. I am going to finish up crocheting a baby blanket a friend requested for her son (born in June…oops!), spend time with my baby Bailey (she was born 7/17…only twelve days late!), hopefully swim if the weather warms itself up, take a trip to Green Bay, and just enjoy myself before the fall semester begins.
I'm 95% sure I'm going to attend Valparaiso University after I finish up at Ivy Tech, doing their accelerated program for nursing, as long as I can swing the loans and aid and whatnot. If I can't, I'll go the Purdue route (against my better judgement as I'm an IU girl at heart). Praying that VU works out because I'll be there with my new bff (she might not agree to this term though). Since I've changed my…final outcome, if you will, my classes are different. This fall I'm taking chemistry (with two friends), algebra (solo, boo!), lifespan development (solo but with my psychology teacher from spring teaching it), and advanced physiology (with two friends). In the spring I'm taking philosophy, an English class required at those two universities, and statistics (with a friend) and communications (solo and again because THIS one is what the other schools want, not the one I took, which would have been GRAND to know prior to taking that class with the worst teacher ever), and microbiology (with another friend). I'm pretty excited about this all, and then NEXT fall (2014) I'll hopefully be taking a semester at VU, followed by the 15-month accelerated program.
In other exciting news, I've sold two things on my Etsy store, and two things to a friend's mother. Sweet! ♥


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New Delhi [India], Feb 3 (ANI): The team of 'Race 3' has wrapped up the first schedule of the highly anticipated film.
Filmmaker Remo D'Souza took to social media to announce that the shooting schedule of the action-packed thriller starring Salman Khan in Mumbai has been completed.
"It's a wrap #mumbaischedule. #race3. #racetofinish," he wrote alongside a picture of Salman and Jacqueline Fernandez.
It's a wrap #mumbaischedule. #race3. #racetofinish. pic.twitter.com/cvPAyDon4i— Remo D'souza (@remodsouza) February 2, 2018
Also starring Bobby Deol, Saqib Saleem, Daisy Shah and Anil Kapoor, 'Race 3' will hit theatres on Eid 2018. (ANI)


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