text:
My how time flies, it was just 5 years ago that my lovely Bella came into this world! It feels like it was just the other day she was a tiny little fluff ball, running and pouncing around the house in a way only a spunky little kitten can.
Nowadays you're more likely to find her sunning herself in a warm sunbeam or passed out cold, belly up in the air, with her arms stretched out over her head.
She still has her spunk though, which is evidenced by her zoomie fueled chases of the other kitties, when I swear it sounds as if I have a heard of elephants stampeding through the house. But more than anything she has turned out to be the sweetest, most docile, happy cats with a kind soul that I've had the pleasure of sharing my life with. Happy Birthday Sweet Bella! And to many more!


text:
In the Sweet Kitchen
Baker Heaven!
Regan Daley
Are you a baker or want to be? Read on.
Cartoon: Sign in bakery window in a small town.
"Pies like your mother made: $8.00.
Pies like your mother thought she made: $23.00″
Regan Daley is my daughter. She wrote this book, In The Sweet Kitchen, which became the No 1 cookbook in the world at the time.
Most hardcover books go to paper with 18 months of publication.
This one took ten years.
Why? Because it never went out of print and sold so well.
She got a $100,000 contract and a 23 city US and Canada book tour.
This is one fine book. When we're all dead, people will still be using it.
These desserts are like the store's above.
Only better.
- Utensils
- Recipies (tested and perfected by Regan)
- Ingredient substitutes
- Detailed instructions for each recipe
- Chemistry
- Fine, elegant writing
- Desserts to kill for.
- Pictures, in case you have no imagination.
Note: this links to Amazon.ca.
Every time I try to link to Amazon.com they send me here.
'Cus I live in Canada, eh?
But in the States, just go to the regular site.
You will be famous in your circle of friends.
Oh, and your circle will grow.
Here are some reviews.
"Regan Daley's grasp of which flavours complement each other, and which methods show them to their best advantage, is amazing."— Alison Fryer, The Cookbook Store
"[Regan Daley's] goal was to compile a definitive guide to all the baker's ingredients and tools…And oh my, does she succeed! Blowtorches, kumquats, dental floss (unflavoured, please for cutting cheesecake), gas ranges, butter, eggs, flours, ice-cream makers, Chartreuse, knives, partridgeberries — you name it, it's here. Daley…has written a baker's encyclopedia that is so arrestingly comprehensive that it redefines definitive with regard to baking cookbooks. And…that is only the book's first half. The second half, the recipe half, is where Daley really shines. Page after page, Daley's brilliant recipes…make one want to run to the kitchen to raid that Platonic pantry of the book's first half….Profuse with information, clearly written, wholly unaffected and masterfully artistic, In the Sweet Kitchen will almost undoubtedly become an instant classic. Like Julia Child's Baking with Julia and Rose Levy Berenbaum's The Cake Bible, no cookbook shelf will be complete without it." — Quill & Quire (starred review)
"Don't be fooled by her sweet expression. Regan Daley is very serious when it comes to recipe writing and educating people about how to bake…In the Sweet Kitchen is destined to become a baking classic." — Homemaker's
"This whopping cookbook is one of the best investments anyone interested in baking could make. From tools for the baker, to key ingredient guidelines, this book is packed with ideas from this pastry chef, along with pages upon pages of her favourite recipes." — Calgary Sun
"Daley knows and cherishes ingredients the way grammarians know and cherish language… In the Sweet Kitchen has the makings of a classic." — Georgia Straight
"The book is an outstanding reference, packed with tips and recipes, for both novice home cooks and professional bakers…Daley's no-nonsense approach is easy and entertaining to read." — Edmonton Sun
"Dessert lovers take note: Regan Daley's In the Sweet Kitchen will send your senses into overdrive. An insistence on the finest ingredients, careful instructions and an exuberant love of food add up to a cookbook that's inspiring, not intimidating. You do want to know how to make the World's Sexiest Sundae, don't you?" — Chatelaine
"In the Sweet Kitchen is a complete baking guide full of yummy recipes." — Bonnie Stern, National Post
"[I]t's the utter thoroughness with which [Daley] covers every aspect of the pastry kitchen that makes this book the ultimate reference volume for the dedicated baker. Aspiring pastry chefs and professionals could well choose this to be their Bible — full of invaluable tables of ingredient substitutions, sugar cookery stages and troubleshooting charts…Daley's grasp of which flavours belong together…is astounding…these are downright sensual desserts which live up to their enticing names….Daley has assembled a wonderful, definitive and delicious work that deserves to stay in print for decades." — Canadian Living (Editors' Choice)
"In her excellent 'guide to the baker's pantry'…Regan Daley devotes four dense pages to the various types of vanilla, where they're grown, the pitfalls in shopping for them and the joys of finding supple ones full of aromatic beans. Her hardcover is titled In the Sweet Kitchen, and in my view, worth every cent of its cover price, especially if you're a sweet-tooth." — Vancouver Sun
"With such traditional baked sweets as angel-food cake surfacing on the menus of cutting-edge restaurants alongside rosemary crème brûlée, Daley's book seems well-timed for a back-to-basics revival. The 692-page volume is crammed with rules, tips and secrets. Besides detailing the behaviour of flours, the temperament of sugars and the many facets of fats, Daley's book provides just the information and creative inspiration bakers need." — Globe and Mail
"In the Sweet Kitchen contains everything a person could ever want to know about baking. Daley walks the reader through section after section of accurate and useful information…then Daley tops it off with a section of more than 140 sweet recipes. This fabulous book is an essential reference to be treasured by anyone who bakes." — Edmonton Journal
There are more like this on the Amazon.com site.
Your soul needs this book.
Just get the book.
If you don't bake, you'll start.
If you don't start, give it to a friend. Eat at her place.

subset: commoncrawl


text:
Kohl's 20% off In-Store or On-Line
It is that time of year — clearance deals a plenty!!! If you are heading out to Kohl's today, they have holiday items deeply discounted and you can save an additional 20% off with a new printable savings pass!!!
Of course, if you like me (and have 3 kids at home on holiday break and don't want to go out), you can still find great deals online. You can use the code NEWYEAR20 at checkout and you will instantly save 20% off of your entire purchase!! Shipping is free on your purchase of $50. Here are a few ideas:
Holiday Stockings as low as $1.50 each
Holiday Ornaments as low as $1.50 each


text:
How to Get Muscular and Toned Most Efficiently
You can get muscular and toned so long as they're willing to strength-train, perform regular cardiovascular exercise and eat a diet that supports their training efforts. However, if you want to get muscular and toned as quickly and efficiently as possible, you'll need to amp up your strength-training schedule, understand the crucial importance of rest, and make specific dietary choices that take into account exactly what you're eating and when you eat it.
Step 1
Engage in upper-body strength-training exercises at least two days per week. Work all of your major upper-body muscle groups, including the abs, forearms, biceps, chest, shoulders, traps, back and lats, over the course of the week. Ideal upper-body exercises include dumbbell shrugs, dumbbell bench presses, incline barbell bench presses, military presses, concentration curls, preacher curls, lat pulldowns and triceps pushdowns.
Step 2
Do lower-body strength-training exercises at least two days per week. Work all of your major lower-body muscle groups, including your calves, quads, hamstrings and glutes, over the course of the week. Ideal lower-body weight-training exercises include squats, lunges, deadlifts, seated and standing calf raises, leg curls, leg extensions and leg presses.
Step 3
Perform at least two sets of 10 to 12 reps for each exercise, with six to 10 exercises per session depending on your fitness level. An initial schedule might include upper-body training on Monday and Thursday, lower-body training on Tuesday and Friday, and rest on Wednesday and the weekend. Add more exercises per session and more sessions per week as your fitness level improves, with at least 24 to 48 hours of rest in between workouts that target the same muscle group. Work new exercises into your sessions and swap out old ones every four to six weeks to avoid exercise boredom and work the same muscle groups in different ways.
Step 4
Eat a nutritious, balanced diet focused on protein, complex carbohydrates, unsaturated fats, vitamins and minerals. Consume four to six meals per day instead of the usual three to stabilize your energy levels and supply your body with a continuous stream of nutrition. Eat carbohydrate-rich foods before workouts for energy, and eat a high-protein meal within one hour of each workout to fuel your muscles for rapid growth.
Step 5
Perform cardiovascular exercise at least two to three times each week for no less than 30 minutes per session. This burns away any excess fat you accumulate through your diet, making visible your gains in muscular mass, definition, strength and density.
Step 6
Get at least eight hours of sleep every night. People who spent two weeks sleeping for less than 5.5 hours per night lost 60 percent more muscle tissue and 55 percent less fat tissue than people who spent two weeks sleeping for more than 8.5 hours per night, according to a 2010 study published in the "Annals of Internal Medicine."
- Drink a protein shake made with whey protein powder shortly after your workouts. This supplies your body with plenty of protein without excess fat.
Photo Credits
- Photodisc/Photodisc/Getty Images


text:
We continue our Legions of Agora campaign this week and the story begins to unfold. Our legionnaire, chosen and plainsman fight for their lives and begin to get a grip on what is happening around them.
We would love to hear your feedback on the podcast at firstname.lastname@example.com which we will either answer via email or on our podcast. If you know an artist that would be willing to help us out with our maps and character portraits feel free to let us know about that as well. If you leave us great reviews on iTunes (which we appreciate greatly) we might read those as well. Thanks for listening.
Podcast: Play in new window | Download


text:
I've said it before and I will continue to say it again and again - I LOVE the bounty of fresh produce available at farmer's markets during the summer! Today I bought so many beautiful things...heirloom tomatoes, peaches, peppers, apricots...and of course the 2-in-1 treat of radishes with the greens still attached. Many people toss the leaves out not realizing they are edible and insanely delicious too!
Last weekend was one of my favorites of the year - greens are back at the farmers market! A true sign of spring and summer vegetable bounty. I bought some baby collards and baby kale and of course I got some ramps!
I have missed fresh greens so much. I had to have them raw in a salad. I bought some good quality steak and decided to make my own dressing. If you haven't made your own salad dressing before then this delicious eye-opener is for you - I only use dressing I've made at home. It's healthier, prettier, and yummier!
delish!: versatile (raw) green sauce
You ever feel like you just need more greens in your life and there isn't enough ways to get them? After all, you can only eat so many salads. This calls for getting creative. If there is one thing I love to finish to the last drop it is sauce of any kind. I will lick a plate/bowl clean to get it all. This green sauce is PACKED with good for you ingredients plus it's raw so no cooking is involved!
It's also super versatile. You can use it as a sauce, a dip, a dressing, a marinade - you name it! Plus the color is so amazing and vibrant...it is a great way to get kids and picky eaters alike to eat their greens.
There are so many reasons why I always make my own tomato sauce. One simply being comfort - after all, the aroma that fills your home is amazing. Another being flavor - the focus is on the tomato much more than that grocery store jar stuff. And another, perhaps the best reason, is health - you know exactly what is going into it without the preservatives or added sugars of most pre-made tomato sauces.
If you have never made your own tomato sauce then what better time is there than now to try it? It is SO simple and once you get the basics down (as this recipe is) you can play around and add all sorts of flavors (like oregano, basil, mushroom, etc). I promise you'll find yourself making sauce all the time!
I'm Ali! Living it up in Brooklyn. I love cooking, baking, crafting, crocheting - you name it! Most of what I cook is healthy for you (and equally delicious) with the occasional "not-healthy" meal (but so worth it). My passion truly lies with inspiring others not only to cook at home but to try new foods in the process!


text:
Some answers to frequently asked questions...
How do you build your Wee Planets?
Please read the description of my Wee Planets set. It has many links and explanations.
What kind of equipement do you use?
Most of my pictures were taken with a Pentax K10D, or (since October 2009) with a Pentax K-7.
For panoramic shots I use a Pentax 10-17mm fisheye zoom. I also use a
Manfrotto panoramic head (303SPH), but with the fisheye lens it's
often easy enough to shoot handheld.
What software do you use?
I use only free software. I've been an exclusive GNU/Linux user for about 15 years now. I use tools like The Gimp, enblend, hugin, and mathmap.
What are the licenses of your pictures?
Many of my pictures are licensed under one of the following two
Creative Commons License in order to ease sharing.
2. Attribution Share-alike Non-Commercial usage
Beware that not ALL pictures are under a Creative Commons. You should be able to tell which license is used by searching for these symbols in the "additional info" block at the bottom of each picture's page.
The Creative Commons license allows you to display, print, and generally reuse my work as long as you play by the rules of the license. The abridged rules are :
1. that any use should be duly credited, and the license of the picture should always be indicated
2. any derivative work should be distributed under the same license
3. for some pictures (), you may not use them for any commercial purpose
Derivative work is when you build upon my work to create another work. For instance if you use my pictures as textures in a composite, it's a derivative. Silhouetting a picture to create a sticker, also creates derivate work. You may only distribute your derivation under the same license.
Simply reproducing the picture (for instance on the web or in print) does not constitute a derivative work. In other words a blog or a journal that publishes one of my pictures does not need to be distributed under the same license... However they have to credit me and state that the picture is under a Creative Commons license.
Someone who alters the picture to turn in into an advertisement is obviously both making a derivative work and having a commercial usage of the picture.
For credits, I prefer my full name to be used (it's Alexandre Duret-Lutz) along with a mention of the license. For websites, it's OK if you replace this credit with an obvious link to the picture's page on flickr.
Please take the time to actually read the text of the licenses before using my pictures, these explanations are by no way a substitute.
Where can I contact you?
Mail me at email@example.com.
Can I use your pictures on my web site?
I receive too many such questions my mail. Please read the previous text and get yourself familiar with the Creative Commons licenses. If your intended use is compatible with the license you do not need my permission to exercize a right that I've already granted you. If it's not compatible, you should tell me how before I can give (or sell) you my permission.
Can I print your picture for private use?
For any of my picture that is under a Creative Commons license, you can download the full size picture and have it printed for your personal use, without bothering about the obligation to credit and display the license of the picture. (Any distribution of the resulting print should however respect these obligations, because it's not longer private use.)
If you hang the picture in your house, I would really appreciate if you could send me a picture of the installation (or better: post that picture as a flickr comment to the picture you printed).
Some of my pictures are on sell on ImageKind. If you buy here, I'll earn a small commission. However you should feel free to have the picture printed elsewhere if you want.
Can I sell postcards, or mugs showing your pictures?
If the license allows commercial uses, and if you respect the rules of the license, the short answer is yes. For postcards, it's easy to credit the author and display the license on the back. For mug, you might decide (that's a hypothesis) to modifiy the picture to overlay it with credit and license, in that case you have built a derived picture that you should make available using the same license as the original picture. The idea is that anybody should be able to print the same mug without buying yours.
Regardless of these licensing issues, the reason I distribute some pictures using a license that allow commercial uses (like printing in a journal) is not so that other people make money on my back by selling derived products on a day-to-day basis. If you want to start such a commerce, I'd appreciate if you could be fair and contact me to buy reproduction rights.
- JoinedDecember 2005
- OccupationComputer Science
- Current cityParis
- CountryFrance
Most popular photos
Photos of Alexandre Duret-Lutz
TestimonialsWrite a testimonial
Always on the lookout for a new and creative way to entertain us all, Alexandre has one of those few photostreams that makes people click on every new photo, in case there is something more interesting and fun than what the thumbnail shows -and there always is!
Alexandre's photos are consistently creative. The often keep me looking a little longer than the average photo, and often require a second or third visit. He certainly has a wonderful style with a mathematical / geeky tilt (in a good way). And having his photo's in my contacts' stream has paid off again and again.


text:
- Research article
- Open Access
Epigarcinol and isogarcinol isolated from the root of Garcinia ovalifolia induce apoptosis of human promyelocytic leukemia (HL-60 cells)
- Constant Anatole Pieme1, 2Email author,
- Pathaleon Ambassa2, 3,
- Emmanuel Yankep3 and
- Ajit Kumar Saxena2Email author
© Pieme et al. 2015
- Received: 18 July 2014
- Accepted: 19 October 2015
- Published: 23 November 2015
Plants from garcinia genus have been used for centuries against several diseases.
This study aimed to investigate the mechanism of apoptosis induced by epigarcinol and isogarcinol isolated from the root of Garcinia ovalifolia (Clusiaceae) on human promyelocytic leukemia (HL-60 cells).
Epigarcinol and isogarcinol were isolated from the root of G. ovalifolia by using column chromatography method. The antiproliferative property of these molecules and fractions were assessed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The light fluorescence microscope was utilized to observe the morphological changes of HL-60 cells after 24 h treatment. Early apoptosis and cell cycle distribution were analyzed by using flow cytometry (FCM).
The results showed that epigarcinol and isogarcinol inhibited the proliferation of HL-60 and PC-3 cells in a concentration-dependent manner with IC50 varying between 4 and 76 µg/mL depending on the cell line and the molecule. The apoptosis rate and the number of apoptotic cells significantly increased with the augmentation of the concentration of the molecules. The results of flow cytometry (FCM) indicated that epigarcinol and isogarcinol induced significant G2/S arrest of HL-60 cells, the disruption of mitochondrial membrane potential and reactive oxygen species (ROS) generation.
These results indicated that epigarcinol and isogarcinol demonstrated in vitro antiproliferative properties and induce apoptosis of HL-60 cells which is related to the G2/S arrest, and it exerts its apoptotic effect through the loosing of mitochondrial membrane potential.
- Apoptosis
- Cytotoxicity
- 7-epigarcinol
- Isogarcinol
- Antiproliferative
- Garcinia ovalifolia
The global burden of cancer continues to increase largely because of the aging and growth of the world population alongside an increasing adoption of cancer-causing behaviors in economically developing countries. Based on the GLOBOCAN 2012 estimations, about 14.1 million cancer cases and 8.2 million cancer deaths are recorded in 2012. More than half of all cancers (56.8 %) and cancer deaths (64.9 %) in 2012 occurred in less developed regions of the world, and these proportions will increase further by 2025 [1]. Given the fact that the incidence rate of cancer disease is continuously increasing in Africa, a number of effective prevention measures have been introduced in order to substantially reduce the incidence and the mortality. Of all the efforts that have been made, the search for new anticancer compounds in foods or plant medicines is a realistic and promising approach to the prevention and the treatment of cancer [2]. Natural products, including plants, microorganisms and marines, have been considered as valuable sources for anticancer drug discovery [3].With the high speed advances in biomedical research, there has been a growing interest in screening natural products for their potential use in disease prevention and treatment. These natural substances are of interest, as they are potential sources of anticancer compounds with minimal debilitating toxicity and side effects [4]. Because of the different components, herbs and plants may have synergistic activities or buffering toxic effects, mixtures or extracts of herbs might have more therapeutic or preventive activity than alone [5, 6]. Several chemopreventive properties have long been attributed to polyphenolic compounds present in the human diet. One remarkable compound in this list is resveratrol, which possesses a wide range of pharmacological properties, including antiinflammatory, antioxidant, and antiplatelet effects and has been proposed as a cancer chemotherapeutic agent [7, 8].
Garcinia ovalifolia (G. ovalifolia) which belongs to Clusiaceae family is growing in lowland forests tropical of Africa, Asia, America and Australia [9]. G. ovalifolia is a tree of 10–15 m high, with yellow sticky latex, generally distributed in fringing forests and riverbanks in West and central Africa [10]. The genus Garcinia includes some 200 species found in the tropics, especially Asia and Africa. Plants from Garcinia genus demonstrated several pharmacological proprieties including anti-HIV [10, 11], antioxidant, antibacterial [12], cytotoxic [11, 12], anticancer and antimalarial [13]. The literature review shows that G. ovalifolia contains several molecules among which, isoxanthochimol [12, 14], endodesmiadol, canophyllol, canophyllal [12, 15], gallic acid, garcinane [16], 3-methylcheffouxanthone, two new friedelane triterpene derivatives ovalifolone A and B [12]. Epi-garcinol, iso-garcinol and manniflavanone isolated from our plant (G. ovalifolia) have also been discovered in other plants extract such as Garcinia preussii [17] Hypericum lanceolatum [18] Moronobea coccinea [19] Symphonia globulifera [20] and Garcinia bancana [21]. Several biological properties of epi-garcinol, iso-garcinol and manniflavanone have been investigated which included antiplasmodial [20–23], antibacterial [17, 18] and immunosuppressant effects [24].Several biological properties of these molecules have been investigated such as antibacterial, cytotoxicity activity on Artemia salina [13] and anti-HIV [25]. However, G. ovalifolia has not been studied for its anticancer effects. Therefore, we attempted to investigate the growth-inhibitory and apoptotic effects of G. ovalifolia extract and fractions against human cancer cells (HL-60 cells and PC-3).
Collection of plant material
The Stem bark of Garcinia ovalifolia (Clusiaceae), was collected in Makenene (Centre region of Cameroon) in December 2010 and identified by Victor NANA of the National Herbarium Cameroon and a sample specimen is deposited on the voucher no. 20854/SRFCam.
Extraction and isolation of compounds
Air-dried and powdered stem bark of G. ovalifolia (2.5 kg) were macerated in methanol (5 L) for 48 h at room temperature. The solution obtained was then filtered through Whatman No. 1 filter paper. The filtrate solution was concentrated under vacuum into a paste to give a dark brown crude extract (150 g). The slurry was made of crude extract (100 g) by dissolving in MeOH, adsorbed on 120 g of silica gel (60–120 mesh) which was subjected to Vacuum Liquid Chromatography (VLC) column packed with 800 g of silica gel (120–200 mesh). Elution was carried out using using hexane/ethyl acetate and ethyl acetate/methanol gradients as eluents at a flow rate of 2 mL/min. Fractions (250 mL each) were collected as follows: pure hexane (fractions 1–5), hexane/ethyl acetate 75/25 (fractions 6–12), hexane/ethyl acetate 50/50 (fractions 13–25),ethyl acetate (fractions 26–33), acetate/methanol 90/10 (fractions 116–125) and methanol (fractions 126–132). These fractions were pooled on the basis of the thin layer chromatography analysis on seven sub-fractions from A to G respectively.
Further chemical investigation of sub- fractions B, C and D was carried out using column chromatography, preparative tin layer chromatography and recrystallization in different solvent yielded three compounds: 7-epigarcinol (250 mg); isogarcinol (25 mg) manniflavanone (40 mg) respectively. All these structures were obtained by the means of spectroscopic analysis including 1D and 2D NMR and mass spectra.
Cell culture
Human promyelocytic leukemia (HL-60 cells) and prostate cancer (PC-3 cells) were obtained from European Collection of Cells Culture (ECCC), Sigma–Aldrich, India. They were grown in RPMI-1640 medium containing 10 % Fetal bovine serum (FBS),penicillin (100 IU/mL) and streptomycin (100 µg/mL medium).The cells suspension was kept in the incubator (Thermocom Electron Corporation, USA) at 37 °C, 5 % CO2; 98 % humidity. Cells were used for different assays during logarithmic growth phase while the untreated control cultures received only the vehicle (DMSO < 0.1 %).
Cells viability and treatments
The human promyelocytic leukemia (HL-60 cells) and prostate cancer (PC-3 cells) were seeded in 96 different well plates containing 15 × 103 and 6 × 103 cells/100 µL/well, respectively. The cultured cells were then treated (triplicate wells per condition) by adding 100 µL of serial dilutions of the three molecules (7-epigarcinol, isogarcinol and manniflavanone) in DMSO to give a final concentration of 100, 30, 10 and 1 µg/mL. The HL-60 treated cells were incubated immediately while for PC-3 cells, the molecules were added after 24 h of incubation. In addition, the DMSO alone was added to another set of cells as the solvent control (DMSO < 0.1 %). The cells were then incubated for another 48 h prior to the addition of 20 µL of 2.5 mg/mL solution of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) into each well. The incubation was continued for 3 h before the media was removed. A mixture of DMSO (150 µL) was added to each well and mixed to ensure dissolving of the crystal formazan before the absorbance at 570 nm was measured. Three replications of each experiment were performed and fifty percent of inhibitory concentration (IC50) of each extract was calculated.
DNA content and cell cycle phase distribution
HL-60 cells (1 × 106 cells/2 mL/well) were treated with molecules at 20, 50, 100 µg/mL for 24 h. They were harvested and washed with 1 mL of PBS, then centrifuged at 400 g for 5 min at 4 °C. The pellet was suspended in 100 µL of PBS and 900 µL of hypertonic citrate buffer (PI-25 µg/mL, RNAase- 40 µg/mL, sodium citrate-0.1 % and Triton-100X-0.03 %) and incubated at 37 °C in dark for 20 min. Finally, cells were analyzed immediately on flow cytometer FACS Calibur (Becton–Dickinson, USA). The data were collected in list mode on 10,000 events and illustrated in a histogram, where the number of cells (counts) is plotted against the relative fluorescence intensity of PI (FL-2; λem: 585 nm; red fluorescence). The resulting DNA distributions were analyzed by Modfit (Verity Software House Inc., Topsham, ME) for the proportions of cells in G0–G1, S- phase, and G2-M phases of the cell cycle [26].
Hoechst 33258 staining of cells for nuclear morphology
HL-60 cells (2 × 106 cells/3 mL/well) were treated with isogarcinol and 7-epigarcinol at different concentrations of extract for 24 h. They were collected, centrifuged at 400 g and washed once with PBS. A solution of Hoechst (Hoechst, 10 µg/mL; citric 10 mM; Na2HPO4 0.45 M; Tween-20 0.05 %) was added in each tube and kept in the dark at room temperature for 30 min. The mixture was washed with PBS and the pellet suspended in 100 µL of PBS/glycerol (1:1). The solution (10 µL) was poured into the slide and nuclear morphology alterations observed under fluorescence microscope (Olympus ×70, magnification ×20) [26].
Mitochondrial membrane potential (MMP) assay
HL-60 cells (1 × 106 cells/2 mL/well) were treated with isogarcinol and 7-epigarcinol at different concentrations for 24 h. Thirty minutes before the end of the experiment, the cell culture was treated with Rhodamine-123 (200 nM) and kept in the dark for 30 mn. Cells were collected, centrifuged (400 g; 4 °C; 5 min), the pellet was washed with 1 mL of PBS and centrifuged as mentioned earlier. The fluorescence intensity of 10,000 events was analyzed in FL-1 channel on BD FACSCalibur (Becton–Dickinson, USA) flow cytometer. The decrease in fluorescence intensity because of membrane mitochondrial potential loss was analyzed in FL-1 channel and the change of potential membrane (∆ψm) was assessed by comparing fluorescence.
Reactive oxygen species (ROS) assay
ROS production was monitored by flow cytometry using 2′,7′-dichlorodihydrofluorescin diacetate (DCFH2-DA). This dye is a stable non polar compound that readily diffuses into cells and is hydrolyzed by intracellular esterase to yield 2′,7′-dichlorodihydrofluorescin (DCFH2), which is trapped within the cells. Hydrogen peroxide or low molecular weight peroxides produced by the cells oxidizes DCFH2 to a highly fluorescent compound 2′,7′-dichlorofluorescein (DCF). Thus, the fluorescence intensity was proportional to the amount of hydrogen peroxide produced by the cells. Briefly, HL-60 cells (1 × 106 cells/2 mL/well) were treated with isogarcinol and epigarcinol at different concentrations for 24 h. Thirty minutes before the end of the experiment, the cell culture was treated with DCFH2-DA (50 µM) and kept in the dark. Cells were then collected, centrifuged (200 g; 4 °C; 5 min) and the pellet was washed with 1 mL of PBS and centrifuged as mentioned earlier. The pellet was suspended in 500 µL of PBS and the fluorescence was assessed by comparing two fluorescence emission 480 nm/530 nm using a flow-cytometer (BD-LSR).
Statistical analysis
The viability experiments were done in triplicates and each data point represents the average of at least 3 independent experiments. For other assays, three independent experiments were made and one of them had been chosen as result to post in this study.
Cytotoxic effect of G. ovalifolia fractions on HL-60 and PC-3 cells
Fifty percent inhibition of molecules isolated from G. ovalifolia
IC50 (µg/ml)
7 ± 1.9
8 ± 2.6
12 ± 3.4
7 ± 1.3
4 ± 2.1
76 ± 2.3
Morphology changes of HL-60 cells
Effect of B and C fractions on intracellular reactive oxygen species in HL-60 cells
Mitochondrial membrane potential (ΔΨm)
Effect of fractions of G. ovalifolia on cell cycle distribution
Different types of cancer chemopreventive agents, including natural substances and pharmaceutical compounds, have been investigated for efficacy and efficiency in vitro and in vivo. The induction of apoptosis is known to be an efficient strategy for cancer therapy. Recently, extracts and molecules prepared from a variety of plants were demonstrated to possess the ability in triggering the apoptotic pathway [28]. Our study investigated the in vitro effects of epigarcinol and isogarcinol from G. ovalifolia extract on cell growth, morphology, cell cycle of HL-60 cancer cell line and the mechanism of apoptosis induced these two molecules.
As commonly known, naturally polyphenols such as garcinol and isogarcinol exhibited cytotoxic activity against HL-60 and PC-3 cells. The two molecules inhibited the proliferation of these two cells in a dose-dependent manner (Fig. 1) with the IC50 varying between 5 and 12 μg/mL (Table 1). Several studies demonstrated the growth inhibition properties of garcinol on different cells cancer including breast, lung, kidney, hepatocellular carcinoma, pancreatic [29–31]. The antiproliferative effects of isogarcinol was shown on HCT116 and CCRF-CEM cell lines [32].
It is also well known that in apoptosis, the earliest recognized morphological changes are compaction and segregation of the nuclear chromatin, with the result of chromatin margination and condensation of the cytoplasm. Our study demonstrated that epigarcinol and isogarcinol induced apoptosis of HL-60 cells as evidenced of alteration of cell morphology, DNA fragmentation, mitochondrial depolarization. After treatment of HL-60 cells with epigarcinol and isogarcinol for 24 h, a representative population of apoptotic cells was also observed mainly at 10 and 20 µg/mL, suggesting a rapid mechanism of apoptose induction that is gradually overwhelmed by the activation of apoptotic pathways. In the present study, the treated cells displayed significant apoptosis-related morphological alterations, such as apoptotic body formation and chromatin condensation. The effects of garcinol in tumor cells have been studied; its mechanism of action is elucidated for several cancer cells lines [29, 33]. The mechanistic cytotoxic activity of garcinol similar to isogarcinol which may include downregulation of MMP-9, IL-8, PGE-2, and VEGF, markers of angiogenesis and metastasis in pancreatic cancer cell lines, Panc 1 and BxPC3 and induction of ROS through death receptor, mitochondrial and modulating GADD153 [34, 35].
ROS are involved in several different cellular processes ranging from apoptosis and necrosis to cell proliferation and carcinogenesis [36, 37], and the mitochondrion is a very important site of ROS production. Mitochondria are a source of ROS during apoptosis and reduced mitochondrial membrane potential leads to increase generation of ROS and apoptosis [38] ROS can induce and maintain the collapse of Δψm, leading to cellular damage through the oxidation of lipids and proteins, resulting in apoptotic cell death [39]. Controversially, other studies estimated that both mitochondria and ROS production seem to be involved in necrotic cell death [37, 40]. Whether the treated cells undergo apoptosis or necrosis depend on the concentration of ROS and the time of exposure to ROS [41]. Our results showed that epigarcinol and isogarcinol inhibited growth of HL-60 cells and induced apoptosis and ROS generation not in a dose-dependent manner. Because cell cycle and apoptosis are involved in the regulation of cell growth, they were detected with a flow cytometer 24 h after the treatment with epigarcinol and isogarcinol in our study. The results showed that after treated with the two molecules for 24 h, the cells arrested at Go/G1 and G2/M phases decreased respectively and the increase of sub-G1, suggesting that these molecules induce significant apoptosis.
Even though apoptosis involves both intrinsic and extrinsic pathways, tumors arise more frequently through the intrinsic pathway than the extrinsic pathway because of its sensitivity [42]. The main intrinsic pathway is characterized by mitochondrial dysfunction, with the release of cytochrome c. Since mitochondria have the ability to activate the cellular apoptotic program directly, they are considered the major cellular component in the intrinsic way of apoptosis [43]. The mitochondria play an important role in the progression of apoptosis. In the study, the decrease of Δψm was enhanced by increasing concentration of epigarcinol and isogarcinol. These results suggest that the two molecules indeed induced apoptosis in HL-60 cells A decrease in Δψm disrupts the outer mitochondrial membrane and causes the opening of the permeability transition pore, followed by the lease of pro-apoptotic factors, and resulting in subsequent apoptosis [44–47]. Other studies demonstrated the inhibition of CBP/p300 acetyltransferase activity by garcinol and several multiple biological effects in cancer cells, including the activation of DNA damage signaling and the induction of chromatin regulators such as TIP60 and SUV420 [47].
The bioassay-guided approach to the processing of G. ovalifolia led to the isolation and purification of epigarcinol and isogarcinol. We demonstrated that epigarcinol and isogarcinol from G. ovalifolia can inhibit the proliferation of HL-60 cells and this inhibitory effect was related to G2/M phase arrest and apoptosis the mitochondrial pathway. The cytotoxic results obtained from our work represent new insight into the traditional use of G. ovalifolia as an anticancer plant. Epigarcinol and isogarcinol may thus warrant further in vivo studies so as to develop it as a successful natural compound that could be used for the treatment in leukemia after determine the entire mechanism of its cytotoxicity.
Authors' contributions
PCA design, perform the study and write the manuscript; AP and YE carried out the purification of molecule, AKS provided all the reagent and chemical. All authors read and approved the final manuscript.
The authors wish to thank the Federation of Indian Chambers of Commerce and Industry (FICCI), who provided scholarships to Dr. PIEME C.A. through CV Raman program to conduct this research in the Indian Institute of Integrative Medicine (CSIR).
Competing interests
The author(s) declared no potential conflicts of interest with respect to the research authorship and/or publication of this article.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors' Affiliations
- Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. Cancer Incidence and Mortality Worldwide: IARC Cancer Base 2013 No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer. Available from http://globocan.iarc.fr.
- Xia D, Fang-Shi Z, Min L, Si-Guo G. Induction of apoptosis in human hepatoma SMMC-7721 cells by solamargine from Solanum nigrum L. J Ethnopharmacol. 2012;139:599–600.View ArticleGoogle Scholar
- Schwartsmann G, Ratain MJ, Cragg GM, Wong JE, Saijo N, Parkinson DR, Fujiwara Y, Pazdur R, Newman DJ, Dagher R, Di Leone L. Anticancer drug discovery and development throughout the world. J Clin Oncol. 2002;20(18):47S–59S.PubMedGoogle Scholar
- Tan ML, Sulaiman SF, Najimuddin N, Samian MR. Tengku Muhammad TS: methanolic extract of Pereskia bleo (Kunth) DC. (Cactaceae) induces apoptosis in breast carcinoma, T47-D cell line. J Ethnopharmacol. 2005;96:287–94.View ArticlePubMedGoogle Scholar
- Li XK, Motwani M, Tong W, Bornmann W, Schwartz GK. Huanglian a Chinese herbal extract, inhibits cell growth by suppressing the expression of cyclin B1 and inhibiting CDC2 kinase activity in human cancer cells. Mol Pharmacol. 2000;58:1287–93.PubMedGoogle Scholar
- Vickers A. Botanical medicines for the treatment of cancer: rationale, overview of current data, and methodological considerations for phase I and II trials. Cancer Invest. 2002;20:1069–79.View ArticlePubMedGoogle Scholar
- Orallo F. Comparative studies of the antioxidant effects of cis- and transresveratrol. Current Med Chem. 2006;13(1):87–98.View ArticleGoogle Scholar
- Nigro P, Bloise E, Turco MC, Skhirtladze A, Montoro P, Pizza C, Piacente S, Belisario MA. Antiproliferative and pro-apoptotic activity of novel phenolic derivatives of resveratrol. Life Sci. 2007;81(11):873–83.View ArticlePubMedGoogle Scholar
- Magadula JJ. Bioactive benzophenone from stem bark and fruit hull from Garcina Senseii Verdc. Int J Phytochem Pharmacol. 2012;2(1):16–20.Google Scholar
- Gustafson KR, Blunt JW, Munro MHG, Fuller RW, McKee TC, Cardellina IIJH, McMahon JB, Cragg GM, Boyd MR. The guttiferones, HIY-inhibitory Benzophenones from Svmuhonia alobulifera, w livinastonei, Garcinia ovalifolia and Clusia rosea. Telrahedron. 1992;46:10093–102.View ArticleGoogle Scholar
- Magadula JJ, Suleimani HO. Cytotoxic and anti-HIV activities of some Tanzanian Garcinia species. Tanz J Health Res. 2010;12(2):1–7.Google Scholar
- Lannang AM, Noudou BS, Sewald N. Ovalifolone A and B: new friedelane derivatives from Garcinia ovalifolia. Phytochem Lett. 2013;6:157–61.View ArticleGoogle Scholar
- Magadula JJ. Bioactive mammea type coumarins and benzophenone from two Clusiaceae plants. J Pharmaceut Sci Innov. 2012;1(5):31–3.Google Scholar
- Louh NG, Meli LA, Mbazoa DC, Tangmouo JG, Komguem J, Castillo J, Mofo NF, Naz Q, Lontsi D, Iqbal MC, Sondengam BL. Polyanxanthone A, B and C, three xanthones from the wood trunk of Garcinia polyantha Oliv. Phytochem. 2008;69:1013–7.View ArticleGoogle Scholar
- Ngouamegne ET, Fongang RS, Ngouela S, Boyom FF, Rohmer M, Tsamo E, Gut J, Rosenthal P. Endodesmiadiol, a friedelane triterpenoid, and other antiplasmodial compounds from Endodesmia calophylloides. J Chem Pharm Bul. 2008;56:374–7.View ArticleGoogle Scholar
- Meli Lannang A, Louh NG, Lontsi D, Specht S, Sarite SR, Florke U, Hussain H, Hoerauf A, Krohn K. Antimalarial compounds from the root bark of Garcinia polyantha Oliv. J Antibiotic 2008;61:518–523.Google Scholar
- Biloa MB, Ndjoko IK, Hertlein AB, Lannang AM, Nkengfack EA, Wolfender JL, Hostettmann K, Bringmann G. Preussianone, a New Flavanone-Chromone Biflavonoid from Garcinia preussii Engl. Molecules. 2012;17:6114–25. doi:10.3390/molecules17056114.View ArticleGoogle Scholar
- Tchakam PD, Lunga KP, Kowa KT, Lonfouo NAH, Wabo KH, Tapondjou AL, Tane P, Kuiate JR: Antimicrobial and antioxidant activities of the extracts and compounds from the leaves of Psorospermum aurantiacum Engl. and Hypericum lanceolatum Lam. BMC Compl Altern Med 2012;12(136):1–8.Google Scholar
- Kim JJ, Chung IM, Jung JC, Kim MY, Moon HI. In vivo antiplasmodial activity of 11(13) -dehydroivaxillin from Carpesium ceruum. J Enzym Inhib Med Chem. 2009;24:247–50.View ArticleGoogle Scholar
- Marti G, Eparvier V, Moretti C, Prado S, Grellier P, Hue N, Thoison O, Delpech B, Guéritte F, Litaudon M. Antiplasmodial benzophenone derivatives from the root barks of Symphonia globulifera (Clusiaceae). Phytochem. 2010;71:964–74.View ArticleGoogle Scholar
- Vatcharin R, Wanpen N, Yaowapa S, Souwalak P. An Antibacterial Biphenyl Derivative from Garcinia bancana MIQ. Chem Pharm Bull. 2005;53(3):342–3.View ArticleGoogle Scholar
- Noungoue DT, Chaabi M, Ngouela S, Antheaume C, Boyom FF, Gut J, Rosenthal PJ, Lobstein A, Tsamo E. Antimalarial compounds from the stem bark of Vismia laurentii. Z Naturforsch (C). 2009;64:210–4.Google Scholar
- Bero J, Quetin-Leclercq J. Natural products published in 2009 from plants traditionally used to treat malaria. Planta Med. 2011;77:631–40.View ArticlePubMedGoogle Scholar
- Cen J, Shi M, Yang Y, Fu Y, Zhou H, Wang M, Su Z, Qun W. Isogarcinol is a new immunosuppressant. PLoS ONE. 2013;8(6):e66503. doi:10.1371/journal.pone.0066503.PubMed CentralView ArticlePubMedGoogle Scholar
- Kirk RG, Blunt JW, Murray HGM, Fuller RW, McKee TC, Cardellina IIJH, McMahon JB, Cragg GM, Boyd MR. The guttiferones, HIY-inhibitory Benzophenones from Svmuhonia alobulifera, w livinastonei, Garcinia ovalifolia and Clusia rosea. Tetrahedron. 1992;48(46):10093–102. doi:10.1016/S0040-4020(01)89039-6.View ArticleGoogle Scholar
- Bhushan S, Kakkar V, Pal HC, Guru SK, Kumar A, Mondhe DM, Sharma PR, Taneja SC, Kaur IP, Singh J, Saxena AK. Enhanced anticancer potential of encapsulated solid lipid nanoparticles of TPD: a novel triterpenediol from Boswellia serrata. Mol Pharmarcol. 2013;10:225–35.View ArticleGoogle Scholar
- Standera A, Marais S, Stivaktas V, Vorster C, Albrecht C, Lottering ML, Joubert AM. In vitro effects of Sutherlandia frutescens water extracts on cell numbers, morphology, cell cycle progression and cell death in a tumorigenic and a non-tumorigenic epithelial breast cell line. J Ethnopharmacol. 2009;124:45–60.View ArticleGoogle Scholar
- Shu-Jing W, Lean-Teik N, Ching-Hsein C, Doung-Liang L, Shyh-Shyan W, Chun-Ching L. Antihepatoma activity of Physalis angulata and P. peruviana extracts and their effects on apoptosis in human Hep G2 cells. Life Sci. 2004;74(16):2061–73.View ArticleGoogle Scholar
- Prasad S, Ravindran J, Sung B, Pandey MK, Aggarwal BB. Garcinol potentiates TRAIL-induced apoptosis through modulation of death receptors and antiapoptotic proteins. Mol Cancer Therap. 2010;9(4):856–68.View ArticleGoogle Scholar
- Subhash P, Aamir A, Nikhil O. Fazlul HS. Emerging role of Garcinol, the antioxidant chalcone from Garcinia indica Choisy and its synthetic analogs. J Hematol Oncol. 2009;2(38):1–13.Google Scholar
- Nadia S, Gupta SV: Potential Role of Garcinol as an Anticancer Agent. J Oncol 2012 ID 647206. doi:10.1155/2012/647206.
- Kuete V, Tchakam PD, Wiench B, Ngameni B, Wabo HK, Tala M, Moungang ML, Ngadjui BT, Murayama T, Efferth T. Cytotoxicity and modes of action of four naturally occuring benzophenones: 2,2′,5,6′ tetrahydroxybenzophenone, guttiferone E, isogarcinol and isoxanthochymol. Phytomed. 2013;20(6):528–36.View ArticleGoogle Scholar
- Ahmad A, Sarkar SH, Bitar B, Ali S, Aboukameel A, Sethi S, Li Y, Bao B, Kong D, Banerjee S, Padhye SB, Sarkar FH. Garcinol Regulates EMT and Wnt Signaling Pathways In Vitro and In Vivo, Leading to Anticancer Activity against Breast Cancer Cells. Mol Cancer Therap. 2012;11(10):2193–201.View ArticleGoogle Scholar
- Ohnish H, Asamoto M, Tujimura K, Hokaiwado N, Takahashi S, Ogawa K, Kuribayashi M, Ogiso T, Okuyama H, Shirai T. Inhibition of cell proliferation by nobiletin, a dietary phytochemical, associated with apoptosis and characteristic gene expression, but lack of effect on early rat hepatocarcinogenesis in vivo. Cancer Sci. 2004;95(12):936–42.View ArticleGoogle Scholar
- Cheng AC, Tsai ML, Liu CM, Lee MF, Nagabhushanam K, Ho CT, Pan MH. Garcinol inhibits cell growth in hepatocellular carcinoma Hep3B cells through induction of ROS-dependent apoptosis. Food Funct. 2010;1(3):301–7.View ArticlePubMedGoogle Scholar
- Matés JM, Segura JA, Alonso FJ, Márquez J. Intracellular redox status and oxidative stress: implications for cell proliferation, apoptosis, and carcinogenesis. Arch Toxicol. 2008;82:273–99.View ArticlePubMedGoogle Scholar
- Marinho-Filho JDB, Bezerrab DP, Araújo AJ, Montenegro RC, Pessoa C, Diniz JC, Viana FA, Pesso ODL, Silveira ER, De Moraes MO, Costa-Lotufo LV. Oxidative stress induction by (+)-cordiaquinone J triggers both mitochondria-dependent apoptosis and necrosis in leukemia cells. Chem Biol Interact. 2010;183(3):369–79.View ArticlePubMedGoogle Scholar
- Pieme Constant Anatole, Santoh Kumar G, Ngamegni B., Ngogang J, Shashi B, Tetsuya M, Ajit Kumar S. Ethyl acetate fraction of Garcina epunctata inducesapoptosis in human promyelocytic cells (HL-60) through the ROS generation and G0/G1 cell cycle arrest: A bioassay-guided approach. Environ Toxicol Pharmacol. 2013;865–874.Google Scholar
- Castello-Branco MVS, Tavares JF, Silva MS, Barbosa-Filho JM, Anazetti MC, Frungillo L, Haun M, Melo-Diniz MFF, Melo PS. Xylodiol from Xylopia langsdorfiana induces apoptosis in HL60 cells. Revista Brasileira de Farmacognosia Braz J Pharmacog. 2011;21(6):1035–42.View ArticleGoogle Scholar
- Hillard EA, Abreu FC, Ferreira CCM, Jaouen G, Goulart MOF, Amatore C. Electrochemical parameters and techniques in drug development, with an emphasis on quinone and related compounds. Chemical Comm 2008;2612–2628.Google Scholar
- Bartosz G. Reactive oxygen species: destroyers or messengers? Bioch Pharmacol. 2009;77:1303–5.View ArticleGoogle Scholar
- Mohan S, Abdelwahab SI, Kamalidehghan B, Syam S, May KS, Harmal NSM, Shafifiyaz N, Hadi AHA, Hashim NM, Rahmani M. Involvement of NF-kB and Bcl2/Bax signaling pathways in the apoptosis of MCF7 cells induced by axanthone compound Pyranocycloartobiloxanthone A. Phytomed. 2012;19:1007–15.View ArticleGoogle Scholar
- Ismail AA, Ahmad BA, Mohd AS, Rasedee A, Suvitha S, Behnam K, Mohamed Y, Manal-Mohamed ET, Siddig IA, Hapipah MA, Syam M. Dentatin isolated from Clausena excavata induces apoptosis in MCF-7cells through the intrinsic pathway with involvement of NF-kB signaling and G0/G1 cell cycle arrest: a bioassay-guided approach. J Ethnopharmacol. 2013;145:343–54.View ArticleGoogle Scholar
- Zimmermann KC, Green DR. How cells die: apoptosis pathways. J Allergy Clin. Immunol. 2001;108:S99–103.View ArticlePubMedGoogle Scholar
- Bouchier-Hayes L, Lartigue L, Newmeyer DD. Mitochondria: pharmaco- logical manipulation of cell death. J Clin Invest. 2005;115:2640–7.PubMed CentralView ArticlePubMedGoogle Scholar
- Hong-xia L, Pan W, Quanhong L, Xiaoxia C, Yuetao Z, Yaping X. Cell cycle arrest and cell apoptosis induced by Equisetum hyemale extract in murine leukemia L1210 cells. J Ethnopharmacol. 2012;144:322–7.View ArticleGoogle Scholar
- Collins HM, Abdelghany MK, Messmer M, Baigong Y, Deeves SE, Kindle KB, Kempegowda M, Akhmed A, Winkler GS, Kundu TK, Heery DM. Differential effects of garcinol and curcumin on histone and p53 modifications in tumour cells. BMC Cancer 2013;13(37): doi:10.1186/1471-2407-13-37.


text:
"We are deeply saddened to inform, our beloved mother Sumedha passed away peacefully in sleep last night."
42 years of a friendship had come to an abrupt end. Just like that. Another piece of me fallen and turned to ashes. If I was sad for her or for me, I couldn't tell the difference. The mark of this date was etched for all of my remaining time. This loss was so permanent.
I had woken up from a deep sleep, my morning cup of coffee had turned out perfect, I managed maintaining my resolution of not checking the phone for the first hour of waking up, the newspaper was already at my doorstep – neatly stacked, just like I prefer. Everything was in harmony and the thought of this being a great morning lingered in my head as I went through the science section of the newspaper. People do not get me when they discover that I only read one page of my 50 + pages edition. They tell me how more I can find on the internet without wasting a dime. Thing is, I know all that. I am well-versed with the internet-thing. But I am also an oldschool person. I love print more than screen. The feel of paper and ink against my fingers is my daily sensual satisfaction. Also, I don't have to lose time and energy to pick topics to browse, the newspaper serves it like a buffet. I only ate desserts.
This morning, I read about the last position of Voyager II in space, a blind female astronomer who listens to stars, a 10mg pill that can combat 5 different infections and how having close friends can elongate life by almost 8 years!
Do you know that feeling of discovering something amazing and sharing it with the one that you first miss? My first thought was to take a snapshot and send it to her. We always shared interesting things. She and I, were not everyone's regular, middle-aged girlfriends who discussed detached children, scheming in-laws, lack of sex and dying relatives. I could never put up with such discussions and that's why I only had a single girlfriend and my need to keep her happy was natural and reciprocated.
So much is felt for the family when someone dies. But how many families have members who really know one-another and are completely at peace with their personality? The obligation of behaving properly is so great. There is love and care, no doubt about that, but the weight of responsiblity and also, criticism take away people's individual beauty. But friends? They know us. There are no obligations. We are chosen, loved by choice. Is there anything more aesthetic? Or any loss greater than the loss of a true friend?
She and I, we were girls on fire! Even at 56, we were creative, knowledge-loving, self-assured women who had visited every little and big café in our city. We had traveled places, went on adventures like paragliding and scuba-diving with pounding hearts. We had been two spirits with the sole purpose of making most of our time after children had fled to their own worlds and husbands had left us behind. We had been soul-sisters, who opened a whole new dimension into each-other's mind. Together, we were brave. We exchanged exuberant compliments even before any of us could feel useless or lonely. With her by my side, the world had been my friend.
Until the moment I unlocked my phone, I had it all going.
Recent Posts
Recent Comments
RJ Talespeare on Self-Righteousness & other… akank5ha on Self-Righteousness & other… RJ Talespeare on Self-Righteousness & other… Akanksha krishnatre on Haunted akank5ha on Haunted
Flickr Photos


text:
Welcome to Syrus Wills
At Syrus Wills, ethical values and a responsible approach are at the heart of what we aim to provide.
Our client's satisfaction is the way we measure the success of the company and Syrus Wills has the ambitious goal of providing the best service in the Manchester area.
Do you need a Will?
Last year, approximately 70% of the people in the UK did not leave a Will, potentially reducing the value of their families inheritance, and causing untold stress, problems and additional costs at a time when the last thing they needed was difficulty and worry.
The client's feedback is crucial to the future development of the service offered to clients.
Syrus Wills can work in partnership to provide the perfect solutions.


text:
Tributes paid to Sinn Féin activist killed in road crash
January 30, 2017
Tributes have been paid to the political activist who died in a car crash near Keady on Saturday afternoon.
Tim Woods had spent the day electioneering for Sinn Féin when he was tragically killed in a single-vehicle collision on the Castleblayney Road around 4.20pm.
An active Sinn Féin worker, the tragedy has shocked and saddened his fellow party members and has cast a cloud over preparations for the upcoming election.
The area's Assembly Member, Cathal Boylan, led tributes to the 52-year-old father-of-two, describing him as "a valued friend".
Extending his condolences to Mr Woods' family, he said: "I am shocked and deeply saddened at the news. My thoughts and prayers are with Tim's family as they come to terms with the news of his tragic death and I extend my condolences to them on behalf of myself and the party locally.
"Tim was a valued friend and party activist. He was a man of great wit and charm and will be sadly missed by all who knew him and it's still hard to take in that he has been so tragically taken from us."
The main Keady to Castleblayney Road was closed for some time after the accident and police have appealed for anyone with information to contact them.


text:
Primary tabs
The Department of Pediatric Dentistry provides dental care to children and adolescents up to 21 years of age with special attention to patients with special health care needs on a referral basis.
Patient care is provided in several different clinics. All treatment locations participate in student and resident training and fees are adjusted according to the type of provider and payment plan. A limited number of emergency appointments are available.
About Our Location
The newly renovated Robert E. Primosch Clinical Education Center includes 11 private operatories, a four-chair open bay, two radiology rooms, a simulation station with two pediatric dental simulators, and two sedation suites and a recovery room for patients who need extensive dental work done or who have high anxiety or other issues with dental treatment.
Frequently Asked Questions
Parents often have many concerns about proper oral health care for their children. The American Academy of Pediatric Dentistry has developed a website called Pediatric Oral Health Information for Parents that will answer many of your questions.
- Raenie Lynn Alim
- Lindsey B Borgner
- Jessica Marie Sanchez
- Susan D Tuten
- Karen S Owens
- Jennifer M Parsons
Reduced-Fee Dental Care
UF College of Dentistry students and faculty participate in programs such as the We Care Physicians Network (352-334-7926).Such programs provide limited services for qualified individuals that may be reduced in fees.
- Clinical Trials
- Search engine of "safety net" clinics
- Federally funded clinics that provide primary care, prescriptions and other health care; some have dental clinics
- Fees are charged based on your ability to pay
Medical Records
To obtain a copy of your medical records from UF Health Pediatric Dentistry, please contact 352-273-7643, 1-800-633-3953 ext 3.
Pay your bill
Please have your billing statement along with your debit / credit card or bank account information. Your account number is located in the top righthand corner of your statement.
Accepted Insurance
We accept Assurant and the Eagles dental plan now offered to UF and Shands employees. If you have different dental insurance that can be used with any provider, you will pay us at the time of service and file with your insurance company for reimbursement. We can help you with that.
UF Health is tobacco-free
Help us maintain our strong commitment to patients and to promoting good health in our communities: please don't smoke, chew tobacco or use electronic cigarettes on UF Health-owned or -operated properties. Thank you.


text:
The main reason why you need hire an experienced and capable worker's comp attorney in San Marino is so that they can bridge the gap between your employer obtain compensation for your injury or loss. There are different kinds of injuries and incidents that may be sustained at your workplace and the following is just an example. Disability, slips and falls, work vehicle accidents and even wrongful death. All these are issues that can find the justice they deserve.
The Best Injury Lawyer in California
Before you retain an experienced compensation attorney in California to help you, it is important for you to consider some factors. You need to consider the extent of your injury and ask yourself whether there is need for compensation. Severe injuries will stand a much better ground with this regard. Minor issues may not be enough cause to lay these kinds of claims.
Before you proceed with a case, it is vital that you sort out the percentage fee that your workers compensation attorney is going to get. Once you are clear, you can proceed. There are many conflicts that present themselves with lawyers. Different states will have a certain percentage that lawyers are entitled to.
The Advantages of Working With a Workers Compensation Lawyer
Workman's compensation, or as it is often called, "Worker's Comp", is the name that refer to a system of laws that offers protection to injured workers. Its intended goal is to make sure that anyone who is injured at work receives medical care appropriate to their injury. The laws also provide for recovery of lost wages. When necessary, it provides for the retraining and rehabilitation necessary to return to the workforce. In the event a worker is killed on the job, benefits would extend under most circumstances to the worker's family.
Although the system of laws governing Worker's Comp makes recovery of benefits easier than in normal personal injury cases, it is not unusual to need the services of an attorney to protect your rights and to insure that you receive all the benefits to which you are entitled. There are attorneys that specialize in Worker's Comp laws, and it is advisable to seek one of them. Not only do laws vary from state to state, but often courts vary from city to city, so by all means, if your injury occurs in, say, Boston, than be sure to hire a Boston personal injury attorney to represent you.
There are a few things that could exclude you from coverage under Worker's Comp laws. Although this does depend on the jurisdiction, employees may lose their benefits if their injuries or death results from either willful misconduct or from intoxication.
In addition to normal Worker's Comp benefits there are some special federal laws which provide additional protection to certain classes of workers.
-----The Jones Act. Intended for seaman on U.S. flagged vessels
----The Federal Employment Liability Act (FELA). For employees of railroads engaged in interstate commerce.
----The Longshore and Harbor Worker's Compensation Act (LHWCA). For employees of certain classes of private maritime employers
----The Black Lung Benefits Act. For miners suffering from black lung disease.
It is not unusual for an injured employee to have some difficulty with either their employer or with the Worker's Compensation system. The employee usually has little knowledge of his rights although the employer is usually pretty sure about them. The employer also has some little tricks up their sleeves for minimizing their liability. The worker is walking through a minefield here, and this is where that Boston personal injury lawyer comes into the picture.
It is hard to even trust the doctor in these cases. Employees are often told they are fine to return to work by a doctor whose loyalty is much stronger to the employer than the employee. Another practice that is sometimes used by employers to limit Workman's Comp liability is to return the employee to work at a different position. This special position is a promotion that involves much less physical exertion, and the employee accepts it, and then in a couple of weeks he finds himself laid off and the position eliminated. Once he returned to work his Workman's Comp benefits ceased, and now he has lost them.
The attorney can guide you, and make sure you receive what you have coming to you under the law. These cases are procedural, and are not lawsuits as in non-work related personal injury. However, an exception is made when the injury is caused by a deliberate action of the employer, or when the employer fails to carry Workman's Comp Insurance even though required by law to do so. You then have the right to sue the employer outside the Workman's Comp system.
What is Workmans Comp Lawsuit Loan?
Injury compensation is a way to reclaim the losses you've suffered after an injury or accident because of another's negligence. These could be financial, for example loss of earnings because you've taken time off work, or they could be physical, where your injury has resulted in considerable pain and suffering. Once you are informed about the process of claiming compensation you can make a decision as to whether you want to make a claim. This simple guide should help clear up any confusion over the legal aspects of what's involved.
What do people claim compensation for?
Injury compensation is the term used to describe the financial payout awarded to a person who has suffered an accident that could have been prevented or avoided by another party. For example, a car accident victim who suffers whiplash could potentially make a claim against the person who caused the incident. A person who has been injured as a result of using a faulty product might be able to claim compensation from the manufacturers of that product, if they are found to be at fault. Someone who has been in a work accident because of poor health and safety standards might think about making a claim against their employer.
There are many examples of cases where people choose to make a claim against the party responsible for their injuries, and generally each case is different to the next. What they all have in common is that there is a requirement to prove that someone was at fault or negligent, and this is what makes it possible to claim compensation.
Why people claim injury compensation
Injury compensation is a route that some accident victims might take in order to reclaim some of the losses they have suffered. In some cases, where a person has sustained severe injuries, they may have been out of work for long periods and unable to earn a living as a result. It could be that expensive medical treatment is required, or even extra help to look after their family. In these cases, injury compensation not only means that they can get back on their feet financially, but they might also be able to pay for any treatment necessary to speed up their recovery, such as physiotherapy or nursing care.
Where the injuries have been psychological, compensation will not take away a person's suffering but it may help to ease some of the stress they feel following an accident. Finally, making a claim can help prevent similar accidents in the future, as it might raise awareness about what causes such accidents to occur. For example, a claim for a car accident caused by a pothole in the road could result in those responsible repairing the road and improving safety for the drivers who use it.
How much claiming injury compensation might cost
Claiming compensation does not have to cost anything at all. After all, you may have already suffered enough financial losses because of your injuries. A no win no fee service is often possible whereby the solicitor that handles the claim simply doesn't get paid unless the claimant is awarded compensation. Also, the recipient of the compensation will usually get to keep 100% of the payout awarded to them, and the legal fees are paid by the party responsible for their injuries.
Now that you know a little more about injury compensation you should be less confused about making a claim and what's involved. Finding a reputable company to deal with your claim could be your next logical step.
San Marino
Workers' Compensation - Settling Vs Trial


text:
Welcome to our official Forum, you can post topics here about Praise and Worship, Sunday Service Preaching, Testimonies, Line-ups, and all Spiritual Topics. To GOD be all the Glory.
Search Query
Display results as :PostsTopics
Advanced Search
Current date/time is Mon Jun 24, 2019 6:49 pm
Jump to: Select a forum||--ANNOUNCEMENTS| |--Registration| |--General Announcements & Guidelines| |--TESTIMONY| |--Introduction About Yourself| |--WORD OF GOD |--Quote(s) |--Bible Verse |--Your Favorite Verse |--Your Life/Memory Verse |--Your Verse for the Day


text:
- Research article
- Open Access
Immunization of mice with YscF provides protection from Yersinia pestis infections
- Jyl S Matson1,
- Kelly A Durick1,
- David S Bradley1 and
- Matthew L Nilles1Email author
© Matson et al; licensee BioMed Central Ltd. 2005
- Received: 26 March 2005
- Accepted: 24 June 2005
- Published: 24 June 2005
Yersinia pestis, the causative agent of plague, is a pathogen with a tremendous ability to cause harm and panic in populations. Due to the severity of plague and its potential for use as a bioweapon, better preventatives and therapeutics for plague are desirable. Subunit vaccines directed against the F1 capsular antigen and the V antigen (also known as LcrV) of Y. pestis are under development. However, these new vaccine formulations have some possible limitations. The F1 antigen is not required for full virulence of Y. pestis and LcrV has a demonstrated immunosuppressive effect. These limitations could damper the ability of F1/LcrV based vaccines to protect against F1-minus Y. pestis strains and could lead to a high rate of undesired side effects in vaccinated populations. For these reasons, the use of other antigens in a plague vaccine formulation may be advantageous.
Desired features in vaccine candidates would be antigens that are conserved, essential for virulence and accessible to circulating antibody. Several of the proteins required for the construction or function of the type III secretion system (TTSS) complex could be ideal contenders to meet the desired features of a vaccine candidate. Accordingly, the TTSS needle complex protein, YscF, was selected to investigate its potential as a protective antigen. In this study we describe the overexpression, purification and use of YscF as a protective antigen. YscF immunization triggers a robust antibody response to YscF and that antibody response is able to afford significant protection to immunized mice following challenge with Y. pestis. Additionally, evidence is presented that suggests antibody to YscF is likely not protective by blocking the activity of the TTSS.
In this study we investigated YscF, a surface-expressed protein of the Yersinia pestis type III secretion complex, as a protective antigen against experimental plague infection. Immunization of mice with YscF resulted in a high anti-YscF titer and provided protection against i.v. challenge with Y. pestis. This is the first report to our knowledge utilizing a conserved protein from the type III secretion complex of a gram-negative pathogen as a candidate for vaccine development.
- Plague
- Subunit Vaccine
- Protective Antigen
- Yersinia Pestis
- Strong Antibody Response
Yersinia pestis, the causative agent of plague, causes rapidly progressing disease in humans with a high mortality rate, especially in the pneumonic form of the disease. Due to the severe nature of plague, its ability for aerosol transmission, and the potential for human to human transmission plague is considered to be a disease of high concern as an agent of biological warfare or biological terrorism [1]. For this reason, an improved vaccine for plague is desirable. Current efforts for vaccine development have focused on two proteins: LcrV (also known as the V antigen) and the capsular F1 antigen [2]. The best results to date have been obtained by using a combination of recombinant LcrV and F1 subunits [3] separately or as a fusion protein [4, 5]. These subunit vaccines demonstrate very good protection against both pneumonic and systemic forms of plague [2] in mouse models. One of the potential limitations of these subunit vaccines is that F1 is not required for full virulence of Y. pestis, as F1-negative strains have the same LD50 value as F1-positive strains [6–9]. A second limitation that could result in undesired side-effects in immunized individuals is the demonstrated immunosuppressive effect of LcrV [10–13]. Additionally, serologic diversity of LcrV has been reported, in Yersinia species other than Y. pestis, that could theoretically limit the usefulness of an LcrV based vaccine. While the recombinant subunit vaccines are very effective in experimental animals and offer protection against F1 minus strains of Y. pestis [2], the inclusion of other antigens with the LcrV/F1 subunit vaccine candidates could improve the ability of the resulting vaccine to offer protection against multiple Y. pestis strains, or the new antigens could be developed as separate vaccine candidates.
The type III secretion apparatus encoded on the low-calcium response (LCR) virulence plasmid, pCD1 in strain KIM [14], of Y. pestis is a conserved virulence mechanism that is absolutely required for virulence of Y. pestis [15]. YscF is a surface localized protein that is required both to secrete Yops and to translocate toxins into eukaryotic cells [16–19]. One report speculates that YscF polymerization is required for a YscF needle to puncture eukaryotic cell membranes [18]. Other researchers suggest that YscF and its homologs function to provide a base that a translocon complex is built upon, or that YscF builds a conduit from the bacterium to the eukaryotic membrane [20]. This suggestion seems more likely given that other proteins such as YopB, YopD, and LcrV are also required for translocation into eukaryotic cells [21–28]. Additionally, YscF needle producing Y. enterocolitica deficient in production of the translocators (LcrV, YopB, and YopD) do not translocate Yops into macrophages, demonstrating that the YscF-needle is not sufficient for translocation [19].
Most currently described pathogenesis-related type III secretion systems possess homologs to YscF. In pathogenic Salmonella and Shigella, the YscF homologs (PrgI and MxiH, respectively) have been demonstrated to form a needle structure that protrudes from the surface of bacterial cells [29–31]. The best-characterized homolog of YscF is EscF of enteropathogenic E. coli (EPEC). EscF is required for "attaching and effacing" (A/E) lesion formation on the intestinal mucosa and for type III secretion of effector proteins [32–34]. EscF is thought to be a structural component of the needle complex on the bacterial surface as it binds EspA, the major component of a filamentous surface organelle, and is required for formation of the EspA filaments. [32–34] However, this surface localization has never been visualized directly, as the only EscF antiserum generated was unable to recognize the native protein [33].
Based on the fact that YscF is thought to be a surface-expressed protein in Y. pestis and is required for virulence, we sought to determine if YscF could serve as a protective antigen against experimental plague infection. Immunization of mice with His-tagged YscF resulted in a high anti-YscF titer and significant protection against i.v. challenge with Y. pestis. The findings of this study suggest that YscF may be a potential plague vaccine candidate that could be used in conjunction with other plague antigens, or possibly alone if its efficacy can be improved by alternative delivery methods.
Results and discussion
Expression and purification of HT-YscF
Specificity of the antibody response to YscF
Active immunization of outbred mice followed by challenge with Y. pestis KIM5
IgG response to HT-YscF vaccination and LD50 determination.
anti-YscF GMT*
Fold increase in survival
< 1:400
Characterization of the antibody response to HT-YscF
Antibody isotype titers from mice* immunized with HT-YscF.
Antibody isotype
Total Ig
< 1:800
> 1:100,000
< 1:800
Ability of α-YscF to effect Yop translocation
To examine one possible method that anti-YscF could be functioning to provide protection in immunized animals the ability to translocate Yops in the presence of anti-YscF antiserum was examined. Antibody to the surface-localized LcrV has been shown to block the ability of the TTSS in Y. pestis to translocate Yops into cultured macrophages [41, 42] but anti-LcrV was unable to block translocation into HeLa cells [21].
However, anti-LcrV was able to block Yops translocation by Y. pseduotuberculosis into HeLa cells [26]. Since YscF is also surface-localized the ability of anti-HT-YscF to block Yop translocation into HeLa cells was tested. Day et al have described an elegant methodology to follow the translocation of Yop effector by fusing them to a Elk reporter [43]. Elk, a eukaryotic transcriptional activator, becomes phosphorylated only after entering the nucleus, providing a reporter for translocation into eukaryotic cells [43, 44]. This methodology has the advantage of not requiring cell fractionation and protease protection assays to establish the intracellular localization of translocated proteins. To test the ability of anti-YscF to block translocation Y. pestis KIM8-3002 was transformed with plasmid pYopE129-Elk [43]. HeLa cells were infected at an MOI of 10 and infection was allowed to progress for 4 h. After the 4 h incubation infected HeLa cells were harvested and immunoblotted to analyze YopE, Elk and PO4-Elk production. Y. pestis KIM8-3002 (wt; [24]) and an isogenic translocation defective strain containing a yopD deletion (KIM8-3002.2, ΔyopD; [45]) both containing pYopE129-Elk were used as positive and negative translocation controls, respectively. Immunoblots of HeLa cells infected with the wt and the ΔyopD strains showed that only the wt strain elicited the production of PO4-Elk while the ΔyopD strain had no production of PO4-Elk. The wt and the ΔyopD strains were used to infect HeLa cells in the presence of a 1:10 or a 1:25 dilution of anti-YscF (titer for HT-YscF, 1:100,000) or in the presence of anti-PcrG (a Yersinia non-reactive antibody control, titer for PcrG, 1:20,000). The wt strain was capable of translocating YopE-Elk in presence of both anti-sera, demonstrating that anti-YscF was not capable of blocking Yops translocation and expectedly the ΔyopD strain was still defective for translocation. The experiment likely contained sufficient antibody against YscF to block translocation. In a previous report Pettersson et al used as low as a 1:100 dilution of an anti-LcrV anti-sera with a titer of 1:20,000 for LcrV and in that experiment translocation of Yops into HeLa cells was blocked [26]. These results suggest that antibody to YscF may not exert its protective effect by blocking Yops translocation. The results also suggest that while YscF is surface-exposed in the yersinae, antibody directed against YscF, unlike, anti-LcrV cannot block translocation. This may imply that YscF activity is shielded from neutralization by antibody, unlike LcrV activity that is blocked by antibody in some cases. However, since anti-LcrV was unable to block Yops translocation into by Y. pestis into HeLa cells [21] but could block translocation into cultured macrophages [41, 42], the possibility remains that anti-YscF also display this type of differential blockage.
Cloning of yscF for overexpression and HT-YscF purification
Plasmid pJM119 was constructed by cloning a Bam HI-and Xho I-cleaved PCR product into pET24b (Novagen, Madison, WI). The primers used to amplify yscF were HT-YscF Start (5' CGG GAT CCG ATG AGT AAC TTC TCT GGA TTT 3') and HT-YscF Stop (5' CCG CTC GAG TGG GAA CTT CTG TAG GAT GCC 3'). E. coli BL21(DE3) (Novagen) harboring pJM119 was used for HT-YscF overpexpression according to the manufacturer's suggestions. HT-YscF was purified using Talon resin (BD Clontech, Palo Alto, CA) according the manufacturer's directions.
Immunization of mice and infection with Y. pestis KIM5
For primary immunization 6-to 8-week-old female Swiss-Webster mice were immunized i.p. with 40 μg/mouse His-tagged YscF or phosphate-buffered saline [37] PBS (control mice) emulsified 1:1 with complete Freund's adjuvant (CFA). Booster immunizations were performed the same as the primary immunization with the substitution of Incomplete Freund's Adjuvant for CFA. Mice were challenged with Y. pestis via the retro-orbital sinus using blunt-feeding needles. Y. pestis used to infect mice was grown overnight in HIB broth, sub-cultured to an A620 of 0.2 absorbance units and grown with shaking to an A620 of 1.0 absorbance unit. Y. pestis cells for infection were harvested by centrifugation and resuspended in PBS. Plate counts were performed to verify CFUs for the infectious doses. Infected animals were monitored for death for up to 19 days, after which survivors were euthanized by CO2 inhalation, according to the guidelines of the Panel on Euthanasia of the American Veterinary Medical Association. All animal work for this project was reviewed and approved by UND's IACUC.
Bacterial strains, growth and fractionation
Bacterial strains used were KIM8-3002 [24], ΔyscF an isogenic in-frame deletion of yscF (G. Plano, University of Miami), and KIM8.3002.2 ΔyopD [45]. Y. pestis strains were grown in heart infusion broth or on tryptose blood agar base medium (Difco Laboratories, Detroit, MI) at 26°C for genetic manipulations. For physiological studies, growth of Y. pestis was conducted in a defined medium, TMH, as previously described [46]. Bacterial cells were fractionated as previously described [24]. Briefly, bacterial cells were chilled on ice after growth, harvested by centrifugation, and washed in cold phosphate-buffered saline (PBS; [37]). Bacterial whole cell fractions were prepared by resuspending the washed cells in cold PBS and precipitating total proteins with 10% (vol/vol) trichloroacetic acid (TCA) on ice overnight. Secreted proteins were recovered from the bacterial growth medium by centrifuging the spent medium a second time, transferring the supernatant to a clean tube, and precipitating with 10% (vol/vol) TCA on ice overnight. The TCA-precipitated proteins were pelleted by centrifugation (20,800 × g at 4°C) for 20 min and resuspended in 2X sodium dodecyl sulfate (SDS) sample buffer [37].
Protein electrophoresis, visualization and immunodetection
Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), using 12.5 % or 15 % (wt/vol) polyacrylamide gels according to the method of Laemmli [47]. Samples were boiled 3–5 min before loading on the gels. Samples were loaded such that lanes containing different culture fractions represented equivalent amounts of the original cultures. Proteins were visualized in gels using GelCode Blue stain (Pierce Chemical, Rockford, IL) according to directions. For immunoblots, proteins resolved by SDS-PAGE were transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA) using carbonate transfer buffer (pH 9.9) [48]. Specific proteins were visualized using mouse or rabbit polyclonal antibodies specific for YopE (rabbit α-YopE; gift from G. Plano, University of Miami, Miami, FL), YscF (mouse α-YscF, this study), Elk (rabbit α-Elk, Cell Signaling Technology, Beverley, MA) and PO4-Elk (rabbit α-PO4-Elk, Cell Signaling Technology). Hexahistidine tagged YscF was visualized using a penta-histidine specific antibody (mouse α-Penta-His, Qiagen, Valencia, CA) Alkaline phosphatase-conjugated secondary antibody (goat anti-rabbit immunoglobulin G or goat anti-mouse immunoglobulin G; Pierce) was used to visualize proteins by development with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP; Fisher Scientific, Fair Lawn, NJ).
Antibody characterization and isotyping
Flat-bottom, 96-well Nunc Maxisorp immunoplates (Fisher Scientific, Pittsburgh, PA) were coated with 100 μl of HT-YscF solution (4 μg/ml in Binding solution (0.1 M NaH2PO4, ph 9.0) at room temperature for 2 h (or overnight at 4°C). The wells were blocked with 200 μl/well blocking buffer (1% bovine serum albumin in TTBS (tris-buffered saline [37] + 0.5% Tween 20) and washed with TTBS. Test sera were serially diluted in blocking buffer and 100 μl of each dilution was added to duplicate wells that were incubated for 2 h at RT (or overnight at 4°C). The plates were washed and incubated for 2 h at RT with alkaline-phosphatase-conjugated anti-mouse secondary antibody. For quantitation of YscF-specific immunoglobulin isotypes and subclasses the plates were coated with alkaline-phosphatase-labeled anti-mouse isotype-specific antibody (1:400 in blocking buffer; Southern Biotech, Birmingham, AL). The wells were washed and 75 μl 3 mM para-nitro phenyl phosphate (p-NPP) was added to each well. The plates were incubated for 15 min at RT and the reaction was stopped by the addition of 50 μl of 1.5 M NaOH to each well. A405 was measured to monitor the cleavage of p-NPP. Antibody titers were determined as reciprocal numbers of the highest serum dilution that displayed values for optical density twofold higher than the value of the control serum.
Infection assays
Infection of eukaryotic cells was performed as described previously [24]. Prior to infection, eukaryotic cells were subcultured into 35-mm-diameter six-well tissue culture plates in RPMI-FBS and incubated at 37°C under 5% CO2 for 48 to 72 h to a density of 5 × 105 to 8 × 105 cells per well. Cells were washed twice with warm L15 lacking FBS immediately prior to infection. Bacteria were cultivated at 26°C in HIB and used at an OD620 of ~1.0 for tissue culture infections. Bacteria were added (at a multiplicity of infection (MOI) of 5 to 10) directly to prewarmed medium in the wells of the six-well plates. Plates were then centrifuged at 200 × g at RT for 5 min to achieve contact between the bacteria and the target cells and incubated at 37°C for 4 h.
Translocation of YopE
Detection of Elk-tagged YopE from pYopE129-Elk was performed as described [43]. Y. pestis strains carrying plasmid pYopE129-Elk were used to infect HeLa cells. After 4 h, the culture supernatants were removed, and the infected adherent cells were lysed by the addition of 100 μl of 2X SDS-PAGE lysis buffer containing Pefabloc (Roche Molecular Biochemicals, Indianapoli, IN) and phosphatase inhibitor (P-2850) cocktail (Sigma, St. Louis, MO). Samples were boiled for 5 min and loaded onto 12.5 % SDS-PAGE gels, immunoblotted to PVDF membranes and probed with Elk-1 (#9182) or phosphospecific Elk-1 (#9181) antibody preparations (Cell Signaling Technology). Anti-sera specific for HT-YscF (titer of 1:100,000, this study) or the Pseudomonas aeruginosa LcrG homolog, PcrG (titer of 1:20,000, Matson and Nilles, unpublished), were used at dilutions of 1:10 or 1:25 in the infection medium to assess the ability of α-YscF to effect YopE translocation.
The author's would like to thank Deanna O'Bryant and Jennifer Lamoureux for assistance with mouse experiments, Jennifer Miller for help with antibody ELISAs and Gregory Plano (University of Miami, Miami, FL) for YopE antiserum and the ΔyscF strain of Y. pestis. This work was supported by the UND Faculty Research Seed Money program. J. S. M was supported by a pre-doctoral fellowship from ND-EPSCoR. Work in M. L. N.'s laboratory is supported by NIAID grants R01-AI051520 and U01-AI54815.
Authors' Affiliations
- Inglesby TV, Dennis DT, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, Fine AD, Friedlander AM, Hauer J, Koerner JF, Layton M, McDade J, Osterholm MT, O'Toole T, Parker G, Perl TM, Russell PK, Schoch-Spana M, Tonat K: Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. Jama. 2000, 283 (17): 2281-2290. 10.1001/jama.283.17.2281.View ArticlePubMedGoogle Scholar
- Titball RW, Williamson ED: Yersinia pestis (plague) vaccines. Expert Opin Biol Ther. 2004, 4 (6): 965-973. 10.1517/147125184.108.40.2065.View ArticlePubMedGoogle Scholar
- Titball RW, Williamson ED: Vaccination against bubonic and pneumonic plague. Vaccine. 2001, 19 (30): 4175-4184. 10.1016/S0264-410X(01)00163-3.View ArticlePubMedGoogle Scholar
- Anderson GWJ, Heath DG, Bolt CR, Welkos SL, Friedlander AM: Short- and long-term efficacy of single-dose subunit vaccines against Yersinia pestis in mice. Am J Trop Med Hyg. 1998, 58 (6): 793-799.PubMedGoogle Scholar
- Heath DG, Anderson GWJ, Mauro JM, Welkos SL, Andrews GP, Adamovicz J, Friedlander AM: Protection against experimental bubonic and pneumonic plague by a recombinant capsular F1-V antigen fusion protein vaccine. Vaccine. 1998, 16 (11-12): 1131-1137. 10.1016/S0264-410X(98)80110-2.View ArticlePubMedGoogle Scholar
- Davis KJ, Fritz DL, Pitt ML, Welkos SL, Worsham PL, Friedlander AM: Pathology of experimental pneumonic plague produced by fraction 1-positive and fraction 1-negative Yersinia pestis in African green monkeys (Cercopithecus aethiops). Arch Pathol Lab Med. 1996, 120 (2): 156-163.PubMedGoogle Scholar
- Drozdov IG, Anisimov AP, Samoilova SV, Yezhov IN, Yeremin SA, Karlyshev AV, Krasilnikova VM, Kravchenko VI: Virulent non-capsulate Yersinia pestis variants constructed by insertion mutagenesis. J Med Microbiol. 1995, 42 (4): 264-268.View ArticlePubMedGoogle Scholar
- Du Y, Galyov E, Forsberg A: Genetic analysis of virulence determinants unique to Yersinia pestis. Contrib Microbiol Immunol. 1995, 13: 321-324.PubMedGoogle Scholar
- Welkos SL, Davis KM, Pitt LM, Worsham PL, Freidlander AM: Studies on the contribution of the F1 capsule-associated plasmid pFra to the virulence of Yersinia pestis. Contrib Microbiol Immunol. 1995, 13: 299-305.PubMedGoogle Scholar
- Nakajima R, Brubaker RR: Association between virulence of Yersinia pestis and suppression of gamma interferon and tumor necrosis factor alpha. Infection and Immunity. 1993, 61: 23-31.PubMed CentralPubMedGoogle Scholar
- Nakajima R, Motin VL, Brubaker RR: Suppression of cytokines in mice by protein A-V antigen fusion peptide and restoration of synthesis by active immunization. Infection and Immunity. 1995, 63: 3021-3029.PubMed CentralPubMedGoogle Scholar
- Nedialkov YA, Motin VL, Brubaker RR: Resistance to lipopolysaccharide mediated by the Yersinia pestis V antigen-polyhistidine fusion peptide: amplification of interleukin-10. Infection and Immunity. 1997, 65: 1196-1203.PubMed CentralPubMedGoogle Scholar
- Sing A, Rost D, Tvardovskaia N, Roggenkamp A, Wiedemann A, Kirschning CJ, Aepfelbacher M, Heesemann J: Yersinia V-antigen exploits toll-like receptor 2 and CD14 for interleukin 10-mediated immunosuppression. J Exp Med. 2002, 196 (8): 1017-1024. 10.1084/jem.20020908.PubMed CentralView ArticlePubMedGoogle Scholar
- Perry RD, Straley SC, Fetherston JD, Rose DJ, Gregor J, Blattner FR: DNA sequencing and analysis of the low-Ca2+-response plasmid pCD1 of Yersinia pestis KIM5. Infection and Immunity. 1998, 66: 4611-4623.PubMed CentralPubMedGoogle Scholar
- Perry RD, Fetherson JD: Yersinia pestis — etiologic agent of plague. Clinical Microbiology Reviews. 1997, 10: 35-66.PubMed CentralPubMedGoogle Scholar
- Allaoui A, Schulte R, Cornelis GR: Mutational analysis of the Yersinia enterocolitica virC operon: characterization of yscE, F, G, I, J, K required for Yop secretion and yscH encoding YopR. Mol Microbiol. 1995, 18 (2): 343-355. 10.1111/j.1365-2958.1995.mmi_18020343.x.View ArticlePubMedGoogle Scholar
- Haddix PL, Straley SC: Structure and regulation of the Yersinia pestis yscBCDEF operon. J Bacteriol. 1992, 174 (14): 4820-4828.PubMed CentralPubMedGoogle Scholar
- Hoiczyk E, Blobel G: Polymerization of a single protein of the pathogen Yersinia enterocolitica into needles punctures eukaryotic cells. Proc Natl Acad Sci U S A. 2001, 98 (8): 4669-4674. 10.1073/pnas.071065798.PubMed CentralView ArticlePubMedGoogle Scholar
- Marenne MN, Journet L, Mota LJ, Cornelis GR: Genetic analysis of the formation of the Ysc-Yop translocation pore in macrophages by Yersinia enterocolitica: role of LcrV, YscF and YopN. Microb Pathog. 2003, 35 (6): 243-258. 10.1016/S0882-4010(03)00154-2.View ArticlePubMedGoogle Scholar
- Cornelis GR: The Yersinia Ysc-Yop 'type III' weaponry. Nat Rev Mol Cell Biol. 2002, 3 (10): 742-752. 10.1038/nrm932.View ArticlePubMedGoogle Scholar
- Fields KA, Nilles ML, Cowan C, Straley SC: Virulence role of V antigen of Yersinia pestis at the bacterial surface. Infection and Immunity. 1999, 67 (10): 5395-5408.PubMed CentralPubMedGoogle Scholar
- Holmström A, Olsson J, Cherepanov P, Maier E, Nordfelth R, Pettersson J, Benz R, Wolf-Watz H, Forsberg AA: LcrV is a channel size-determining component of the Yop effector translocon of Yersinia. Mol Microbiol. 2001, 39 (3): 620-632. 10.1046/j.1365-2958.2001.02259.x.View ArticlePubMedGoogle Scholar
- Håkansson S, Schesser K, Persson C, Galyov EE, Rosqvist R, Homblé F, Wolf-Watz H: The YopB protein of Yersinia pseudotuberculosis is essential for the translocation of Yop effector proteins across the target cell plasma membrane and displays a contact-dependent membrane disrupting activity. EMBO Journal. 1996, 15: 5812-5823.PubMed CentralPubMedGoogle Scholar
- Nilles ML, Fields KA, Straley SC: The V antigen of Yersinia pestis regulates Yop vectorial targeting as well as Yop secretion through effects on YopB and LcrG. Journal of Bacteriology. 1998, 180 (13): 3410-3420.PubMed CentralPubMedGoogle Scholar
- Nordfelth R, Wolf-Watz H: YopB of Yersinia enterocolitica Is Essential for YopE Translocation. Infect Immun. 2001, 69 (5): 3516-3518. 10.1128/IAI.69.5.3516-3518.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Pettersson J, Holmström A, Hill J, Leary S, Frithz-Lindsten E, von Euler-Matell A, Carlsson E, Titball R, Forsberg , Wolf-Watz H: The V-antigen of Yersinia is surface-exposed before targent cell contact and involved in virulence protein translocation. Molecular Microbiology. 1999, 32 (5): 961-976. 10.1046/j.1365-2958.1999.01408.x.View ArticlePubMedGoogle Scholar
- Sory MP, Cornelis GR: Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells. Molecular Microbiology. 1994, 14: 583-594.View ArticlePubMedGoogle Scholar
- Tardy F, Homblé F, Neyt C, Wattiez R, Cornelis GR, Ruysschaert JM, Cabiaux V: Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops. EMBO Journal. 1999, 18 (23): 6793-6799. 10.1093/emboj/18.23.6793.PubMed CentralView ArticlePubMedGoogle Scholar
- Blocker A, Jouihri N, Larquet E, Gounon P, Ebel F, Parsot C, Sansonetti P, Allaoui A: Structure and composition of the Shigella flexneri "needle complex", a part of its type III secreton. Mol Microbiol. 2001, 39 (3): 652-663. 10.1046/j.1365-2958.2001.02200.x.View ArticlePubMedGoogle Scholar
- Kubori T, Sukhan A, Aizawa SI, Galan JE: Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system. Proc Natl Acad Sci U S A. 2000, 97 (18): 10225-10230. 10.1073/pnas.170128997.PubMed CentralView ArticlePubMedGoogle Scholar
- Kubori T, Matsushima Y, Nakamura D, Uralil J, Lara-Tejero M, Sukhan A, Galan JE, Aizawa SI: Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science. 1998, 280: 602-605. 10.1126/science.280.5363.602.View ArticlePubMedGoogle Scholar
- Sekiya K, Ohishi M, Ogino T, Tamano K, Sasakawa C, Abe A: Supermolecular structure of the enteropathogenic Escherichia coli type III secretion system and its direct interaction with the EspA-sheath-like structure. Proc Natl Acad Sci U S A. 2001, 98 (20): 11638-11643. 10.1073/pnas.191378598.PubMed CentralView ArticlePubMedGoogle Scholar
- Wilson RK, Shaw RK, Daniell S, Knutton S, Frankel G: Role of EscF, a putative needle complex protein, in the type III protein translocation system of enteropathogenic Escherichia coli. Cell Microbiol. 2001, 3 (11): 753-762. 10.1046/j.1462-5822.2001.00159.x.View ArticlePubMedGoogle Scholar
- Daniell SJ, Takahashi N, Wilson R, Friedberg D, Rosenshine I, Booy FP, Shaw RK, Knutton S, Frankel G, Aizawa S: The filamentous type III secretion translocon of enteropathogenic Escherichia coli. Cell Microbiol. 2001, 3 (12): 865-871. 10.1046/j.1462-5822.2001.00168.x.View ArticlePubMedGoogle Scholar
- Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology. 1998, New York , John Wiley & SonsGoogle Scholar
- Cordes FS, Komoriya K, Larquet E, Yang S, Egelman EH, Blocker A, Lea SM: Helical structure of the needle of the type III secretion system of Shigella flexneri. J Biol Chem. 2003, 278 (19): 17103-17107. 10.1074/jbc.M300091200.View ArticlePubMedGoogle Scholar
- Coligan JE, Dunn BM, Speicher DW, Wingfield PT: Current protocols in protein science. 1998, New York , John Wiley & SonsGoogle Scholar
- Welkos S, O'Brien A: Determination of median lethal and infectious doses in animal model systems. Methods Enzymol. 1994, 235: 29-39.View ArticlePubMedGoogle Scholar
- Andrews GP, Strachan ST, Benner GE, Sample AK, Anderson GWJ, Adamovicz JJ, Welkos SL, Pullen JK, Friedlander AM: Protective efficacy of recombinant Yersinia outer proteins against bubonic plague caused by encapsulated and nonencapsulated Yersinia pestis. Infect Immun. 1999, 67 (3): 1533-1537.PubMed CentralPubMedGoogle Scholar
- Williamson ED, Vesey PM, Gillhespy KJ, Eley SM, Green M, Titball RW: An IgG1 titre to the F1 and V antigens correlates with protection against plague in the mouse model. Clin Exp Immunol. 1999, 116 (1): 107-114. 10.1046/j.1365-2249.1999.00859.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Philipovskiy AV, Cowan C, Wulff-Strobel CR, Burnett SH, Kerschen EJ, Cohen DA, Kaplan AM, Straley SC: Antibody against V antigen prevents Yop-dependent growth of Yersinia pestis. Infect Immun. 2005, 73 (3): 1532-1542. 10.1128/IAI.73.3.1532-1542.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Weeks S, Hill J, Friedlander A, Welkos S: Anti-V antigen antibody protects macrophages from Yersinia pestis -induced cell death and promotes phagocytosis. Microb Pathog. 2002, 32 (5): 227-237. 10.1006/mpat.2002.0498.View ArticlePubMedGoogle Scholar
- Day JB, Ferracci F, Plano GV: Translocation of YopE and YopN into eukaryotic cells by Yersinia pestis yopN, tyeA, sycN, yscB and lcrG deletion mutants measured using a phosphorylatable peptide tag and phosphospecific antibodies. Mol Microbiol. 2003, 47 (3): 807-823. 10.1046/j.1365-2958.2003.03343.x.View ArticlePubMedGoogle Scholar
- Day JB, Lee CA: Secretion of the orgC gene product by Salmonella enterica serovar Typhimurium. Infect Immun. 2003, 71 (11): 6680-6685. 10.1128/IAI.71.11.6680-6685.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Williams AW, Straley SC: YopD of Yersinia pestis plays a role in the negative regulation of the low-calcium response in addition to its role in the translocation of Yops. Journal of Bacteriology. 1998, 180: 350-358.PubMed CentralPubMedGoogle Scholar
- Straley SC, Bowmer WS: Virulence genes regulated at the transcriptional level by Ca2+ in Yersinia pestis include structural genes for outer membrane proteins. Infection and Immunity. 1986, 51: 445-454.PubMed CentralPubMedGoogle Scholar
- Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 227: 680-685.View ArticlePubMedGoogle Scholar
- Skrzypek E, Straley SC: LcrG, a secreted protein involved in negative regulation of the low-calcium response in Yersinia pestis. Journal of Bacteriology. 1993, 175: 3520-3528.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.