text:
You should send congratulations for the 6th Birthday of a boy or a girl you know. Here are some sayings and wishes, messages and quotes for the sixth birthday for children with whom you can congratulate via SMS or in form of a birthday card. There are free messages, sweet and funny birthday wishes. They are suitable for a godchild, for the offspring of relatives or friends, for nephew and niece and befriended children.
Congratulations to the 6th Birthday - you are now a year older and a year cooler! I wish you an additional 365 days of fun!
I wish you all the best for your sixth birthday and that all your dreams and wishes come true in the next few years.
6 candles a waiting for you to blow them out. They are on the tastiest birthday cake ever. Have fun!
Happy Birthday to the Princess, who is 6 years old today! I think that you deserve a royal treatment today and wish you a great day.
I do not know if you will be a vet, a astrophysicist or the next president, but I know that you're already the cutest girl in the world. Congratulations on your 6th birthday.
A birthday is the day to dream new dreams and to face new challenges. I wish you lots of fun for your 6th birthday.
Congratulations for the 6th Birthday of the center forward of the national football team in 2018!
Free Children Birthday Cards to print
Children Birthday Cards to print. Free Wird 2010 template for funny colorful birthday cards with illustration and text. Cards for boys and girs.
4th Birthday Congratulations Happy 4th Birthday for boys and girls. Congratulation messages for birthday cards for kids 4th birthday. Sayings, quotes and wordings.
5th Birthday Messages Happy 5th Birthday wishes and messages for children birthday cards and SMS congratulations. Free 5th birthday sayings for girls and boys.
6th Birthday Wishes and Messages for Kids
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text:
Help! My edible forest garden is getting the better of me.
Hi, all! Late winter, a year and a half ago, I converted my yard into an edible forest garden. We laid down cardboard three layers thick, planted trees, shrubs, and ground-level edible plants all according to edible forest garden principles, (in fact, I hired an expert to do all the planning and purchasing), and put down mulch. It is hog fuel, with no cedar. We got it 4 inches deep in most places, perhaps a little shallower in some areas. This is the second summer since we put in the garden, and it has been a real struggle keeping it looking decent. I have spent every spare moment pulling weeds. I know dandelions are supposed to be good for the soil, but I'm not enough of a convert to want to have a yard that is completely carpeted with them. If it weren't for pulling them out, all of the ground level edible plants I put in would be completely hidden by them. I love the ideal behind edible landscaping, but the reality has not lived up to that. Does anyone have any advice on what I can do to make my garden more manageable?
Have you thought about using edible ground cover instead of mulch to compete with the weeds? Some examples are: clover, mint, sweet potato vine, creeping thyme, cover crops, etc. There are some that even produce pretty flowers (maybe nasturtiums).
One thing to keep in mind is that an edible food forest is still a forest, and the forest is naturally unkempt. It is that way on purpose, not accident. It creates a better ecosystem and naturally healthier plants.
Edible ground level plants are always a lot of work. Plants that puts a lot of energy into creating things we can eat are at a bit of a competitive disadvantage as compared to small plants that put that energy into fast growth (aka "weeds"). And ground-level edible plants are easily overshadowed.
Growing edible ground level plants without resorting to monoculture, machinery and pesticides is a lot of work in most climates...it's why farming is a full-time job and most farms are monoculture.
Dandelions are edible...you could let them take over and declare victory? JK
You could scale back the edible ground level plants into a small patch, and focus on the trees and bushes that produce fruit.
most weeds are and medicinal, if you start a foodforest there is not way around it - you will get dandelions and other weeds. the only way around is is heavy mulching with mulch made from bark ( large particles ) like pine bark mulch. OR you can plant groundcovers that supresses weed..MOST groundcovers are invasive.
when you choose a groundcover choose one that is really good looking and reliable, do not get groundcovers that become very high or bushy. Alot of groundcovers have medicinal properties and alot of them are edible.
pachysandra terminalis looks natural and is easy to pull
Ajuga reptans
lysimachia nummelaria ''aurea'' ( less invasive cultivar )
some bergenia cordifolia cultivars do not get too large and are evergreen, will grow in any type of condition.
Fragaria 'Lipstick' for example is edible one ( doesn't taste like normal strawberry tho). you also have creeping chamomille which has edible foliage ( for herbal tea), Creeping thyme, Creeping prostrate rosemary ( just layer all branches, rinse and repeat ). if your zone isn't too cold you may consider growing gotu kola.
Cornus canadensisis a edible creeping groundcover, there are 2 cultivars that stay smaller than one foot.
you also have small shrubs like Gaultheria procumbens, certain edible dwarf mahonia species, Certain grass species like acorus gramineus '' licorice'' has sweet tasting foliage that is edible , its low creeping evergreen species that can be easily propagated.
2 links :
just check the edible rating and medicinal rating and what parts are edible from each plant. Just make sure that you get the right plant for proper locations, in edible / foodforest gardens it's often shade tolerant species.
This post was edited by Lodewijkp on Mon, Aug 25, 14 at 16:13
there is a new kind of micro-clover bred esp. for low-maintenance lawns. Look at the lawns on YouTube. I want it. I have something like it a co-worker gave me the seed of. It is super low. There are varying reports about its ability to withstand a lot of walking on, though. Guess it needs a lot of sun, though. Ajuga and creeping jenny and vinca minor are super low and take shade and creep/spread fairly well.

subset: commoncrawl


text:
Drug Induced Peripheral Neuropathy
Here's a taste of what TheBody.com has to offer on this topic:
Ask the Experts
POSITIVE AND ALIVEDoc,I am on trimune 30 and my health is just fine. The only problem is I get tickling sensations in my legs. I am a living proof because my CD4 was 15 but I was up and about though I had just lost weight. That was 6 months ago, now my CD4 is 110. ...
Response from Eric Daar, M.D.
UCLA School of Medicine...combination pill of stavudine (D4T) with 3TC and nevirapine (viramune). What you are experiencing may be consistent with drug-induced peripheral neuropathy which is a common side effect of stavudine. I would encourage you to meet with your... Read more »
K Pax vitamin?Hi,I've tried to do research on K Pax vitamins and found very little information besides on the... Read more »
Hip and finger tips (PERIPHERAL NEUROPATHY)Dear Doctor:I have been positive for only 3 years and I have never been on any HIV meds yet, but I... Read more »
Peripheral Neuropathy... What Is Peripheral Neuropathy? What Causes PN? How Is PN Diagnosed? How Is It Treated? Other Resources The Bottom Line What Is Peripheral Read more »
Understanding and Managing Peripheral Neuropathy... Table of Contents Introduction A Snapshot of the Nervous System Clinical Features of Peripheral Neuropathy Development of DSPN Diagnosing DSPN Risk Factors for DSPN Neuropathy Management DSPN Therapies Conclusion Read more »
Peripheral Neuropathy and HIV/AIDS... Table of Contents What Is Peripheral Neuropathy? What Causes PN? Who Is at Risk of Developing PN? How Do You Know if You Have PN? PN Treatments Finding Support Read more »
Peripheral Neuropathy...Measures Resources Selected Sources Introduction Of the many symptoms associated with HIV/AIDS and its treatment, peripheral neuropathy (PN) can be among the most painful and debilitating. The most common estimate is that about... Read more »
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Peripheral Neuropathy - L-Carnetine
Posted by Anonymous, 3 RepliesGreetings! Just thought I would post the following info, it is based strictly on my personal experience and is not a recommendation. I am a long-term survivor(21 years) and in general good health. Last year I successfully complete a course of... Read more »
Excessive Mastubation
Posted by Anonymous, 5 RepliesEver since I found out that I'm poz, I've been spending a lot more time mastubating. Is that normal? Or could it be because of the amount of time I've been spending reading posts about sexual issues? Pleasing myself has become an every day thing... Read more »
Seroconversion and Peripheral Neuropathy
Posted by Anonymous, 2 RepliesThis is a question for anyone who is living with HIV or who has recently been diagnosed as HIV positive: Did any of you experience the numbness/tingling in the feet, legs, hands and arms during your seroconversion? If so . . . for what period of... Read more »
Peripheral Neuropathy
Posted by Anonymous, 1 Reply...dear jackie blue, my doctor says that i have Peripheral Neuropathy,but my elizer test is still neg at 8-9months. should i need to keep testing until one year.... as im very stressed out.i recently Read more »
Resource Centers
Guides for People With HIV


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Hiv Positive African American Men And Women And The Media
Here's a taste of what TheBody.com has to offer on this topic:
Get the Facts
HIV Among African Americans... African Americans face the most severe burden of HIV in the United States (US). At the end of 2007, blacks accounted for almost half (46%) of people living with a diagnosis of HIV infection in the 37 states and 5 US dependent areas with long-term,
Ask the Experts
Do HIV carrirers test negative (HIV CARRIERS) (HIV BASICS)DO HIV carriers test negative? (I did an HIV 1&2 test which came back negative. But someone told me that I could still be a carrier. The test was done six (6) months after my last possible exposure.
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation...people who are still engaging in high-risk behaviors, and infection rates remain alarmingly high among young people, women, African Americans, and Hispanics. Are women especially vulnerable to HIV? In Western countries, women are four times more... Read more »
its for you ( Update on AIDS Vaccine Research and Advocacy 2009good morning siri would like to draw your kind attention over this recent research for an effective vaccinelots of love one of your lovers kualalumpur
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation...a vector, developed from an adenovirus type 5 (Ad5) that causes the common cold. The vaccine was designed to stimulate cell-mediated immunity, in which T cellsmajor players in immune functiontarget and kill cells that are already infected with... Read more »
when we can expect first therupetic vaccine to come in market?i have been on meds for almost 2 weeks and feel as if its already been decade popping up that poison every day,it makes me feel depressed and sick, recently i read about developements in field of immunotherapy and therupetic vaccines of different...
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation...a vector, developed from an adenovirus type 5 (Ad5) that causes the common cold. The vaccine was designed to stimulate cell-mediated immunity, in which T cells -- major players in immune function -- target and kill cells that are already infected... Read more »
31 billion! And angels our watchingIf we can as a society work to help improve this world and make it better for future generations we have succeeded in the most difficult and most important job known to man....
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation...Blessing ... after all. Plus, why disregard or disrespect my email message, thus allow me, a monetary lacking-little lower-class African American Black man with spiritually rich ideas, be your Rock of Offense & BIG STUMBLING BLOCK? Trust me, the... Read more »
HIV/AIDS in Black America: The Uphill Battle...ages 24-35. Black men who have sex with men (MSM) have the highest HIV rate among all racial groups of MSM. Overall, while African Americans make up a mere 14 percent of the overall U.S. population, they account for more than half of all new... Read more »
The Heightened National Response to the HIV/AIDS Crisis Among African Americans... As the impact of HIV/AIDS on African Americans has grown over time, so have the Centers for Disease Control and Prevention's (CDC) efforts to address it. We are highly committed Read more »
Fact Sheet: Refocusing National Attention on the HIV Crisis in the United States...which are scheduled to run concurrently. Each phase, with its own unique objectives and target audiences, will utilize mass media and direct-to-consumer communication channels to deliver important HIV prevention messages in a manner... Read more »
Cleared for Takeoff: What Ending the HIV Travel Ban Means for Black Americans...someone dies of AIDS, they're really dying of poverty, racism, homo-hatred, stigma around substance abuse and no health care. African Americans make up 13 percent of the U.S. population and [almost] 50 percent of the AIDS population. Lifting the... Read more »
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time line
Posted by Anonymous...44. By 1996, it would no longer be the leading cause of death for all Americans in this age group, but would rank first among African Americans and third among women aged 25 to 44. Rapper Eazy-E dies of AIDS A member of the seminal gangsta... Read more »
new documentary on HIV and African American youths
Posted by bartleby, 1 Reply...Hey all, Have you heard about this new documentary by Claudia Pryor entitled "Why Us? Left Behind and Dying"? Even though African Americans between the ages of 11 and 25 account for 65% of new infections, HIV is still not discussed enough (or at... Read more »
gay men
Posted by Anonymous, 13 RepliesI asked the forum to take off the gay men and just put men so people will stop thinking about people being gay. we need to stop this when people use gay some people are judgemental and turn away when we need to educate people this is not a gay... Read more »
Question for African American Women
Posted by sadiesmom, 2 Replies...Do you feel you have good support from your church? My daughter is living with HIV and although the members of my congregation have been supportive, I have not been satisfied with the spiritual leaders. I just Read more »
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text:
The Iraqi Red crescent
The Iraqi Red crescent by Jamal Al-Karboli The Iraqi Red crescent society (IRcs) is among the services. We have a staff of 3,200 and 100,000 volunteers – 10,000 of few organisations working to assist displaced persons whom are active. The society owns throughout Iraq. four hospitals – including maternity, surgery and children's hospitals in The IRCS is an independent national discrimination on grounds of religion, Baghdad – with a total of 300 beds humanitarian society. One of the denomination or political belief. and runs an artificial limb centre oldest Red Crescent societies in the in Mosul and two centres for the region, it was founded in 1932 and The IRCS has 15 administrative, rehabilitation of war casualties in recognised by the International financial or operational divisions Basra. Storage facilities are available Federation in 1934. We uphold and branches in each of Iraq's 18 in all governorates and we have three international humanitarian principles governorates. There are 135 branch large strategic warehouses. The IRCS of neutrality, impartiality and non- offices offering humanitarian works in close cooperation with the International Federation of Red Cross Family from and Red Crescent Societies (IFRC) Fallujah at and with UN agencies, especially Al-Khadraa' WHO, UNICEF and UNHCR. displaced persons camp (established Our activities, like those of other by the National Societies, are, of course, not Iraqi Red confined to responding to wars or Crescent), natural disasters. At times of peace we near Baghdad. focus on such activities as attracting young volunteers, youth camps, first aid training, seminars to increase awareness of how to reduce road accidents, vaccination campaigns, blood donations, water and sanitation and provision of medical supplies and services in remote areas of Iraq. Sadly, as a result of the tragic circumstances in Iraq, most of our CICR/AL-RAWI Ahmad activities, attention and resources are today directed towards emergency response to the needs of Iraqis at home or in neighbouring countries. FMR IRAQ specIAl Issue Among the activities we undertake n food assistance packages: with and terrorists. Our aid convoys have are: support from the International been attacked on 30 occasions and Committee of the Red Cross (ICRC) 13 Red Crescent facilities have been n evacuation and rescue services and the Red Crescent Societies of attacked – and damaged or destroyed and first aid to victims of Kuwait, Iran and the United Arab – by occupation forces. We have violence and explosions Emirates we have distributed food problems with some Iraqi insurgents, to over 700,000 vulnerable families. but in general most of them respect n establishing emergency camps our work and understand our role. for those displaced by conflict or n provision of drinking water We face far greater problems with whose houses have been destroyed to displaced people the American forces as they, often and providing them with food aggressively, search our premises parcels, hygiene kits, blankets, n liaison between families and and damage our facilities. We have kitchen sets and jerrycans the estimated 18,000 prisoners to spend much time explaining about currently being held by the the Red Crescent. Many US soldiers n organising blood donation occupation forces and the roughly fail to realise that we are recognised campaigns to support 34,000 detained by the Iraqi members of the international acute-care hospitals authorities: we have facilitated humanitarian movement, despite exchanges of tens of thousand the fact we use the Muslim Red n exhuming and identifying bodies of letters and arranged phone Crescent symbol. In spite of these discovered in mass graves calls, thus reassuring relatives difficult circumstances, however, the – in Iraq and abroad – that their Iraqi Red Crescent Society remains n providing artificial limbs, loved ones were alive and well. committed to assisting our fellow wheelchairs and other equipment Iraqis to the best of our abilities. for victims of conflict Red Crescent workers and volunteers have paid a high price as they Dr Jamal N Al-Karboli is the n provision of emergency first carry out their duties. They have vice-president of the Iraqi Red aid kits, blood bags, body bags, been exposed to death, injury, Crescent Society. For information blankets, bed sheets, and stretchers, kidnapping and imprisonment at about the work of IRCS, email: painkillers and anaesthetics the hands of armed men, militias email@example.com
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text:
When PC Ports of Console Games Go Wrong 398
from the in-other-words-always dept.
We've improved Slashdot's video section; now you can view our video interviews, product close-ups and site visits with all the usual Slashdot options to comment, share, etc. No more walled garden! It's a work in progress -- we hope you'll check it out (Learn more about the recent updates).
After the outcry over the adblock plus filter snafu, NoScript has stopped adding any filters to adblocking extensions.
IIRC, there was a movement to vote NoScript down so that it would be required to undergo a full code review of each update. The author apologised and removed the offending code.
You show me a better protection from JS and other plugins and I'll install it. But you can't, because there isn't anything in NoScript's league.
Promising costs nothing, it's the delivering that kills you.


text:
|UniProt functional annotation for P04650
|UniProt code: P04650.
|Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast).
|Eukaryota; Fungi; Dikarya; Ascomycota; Saccharomycotina; Saccharomycetes; Saccharomycetales; Saccharomycetaceae; Saccharomyces.
|Component of the large ribosomal subunit. Mature ribosomes consist of a small (40S) and a large (60S) subunit. The 40S subunit contains 32 different proteins (encoded by 56 genes) and 1 molecule of RNA (18S). The 60S subunit contains 46 different proteins (encoded by 81 genes) and 3 molecules of RNA (25S, 5.8S and 5S). {ECO:0000269|PubMed:9559554}.
|Subcellular location:
|Cytoplasm {ECO:0000250}.
|Belongs to the ribosomal protein L39e family. {ECO:0000305}.
Annotations taken from UniProtKB at the EBI.


text:
6. Mayr Anton Dross Grüner Veltliner Nussbaum (2009)
Gewurztraminer has become on of my favorite white wines. Its floral, spicy tones on the nose are truly seductive, while the palate offers a lush mouthfeel contrasted by zippy acidity. It's a perfect white for a holiday meal, because it complements everything else around it with its gorgeous aromatics.


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Order by 3/30 to beat the Easter Bunny. LEARN MORE
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bucket of joy & loveby Eric Hendrick and Noelle VanHendrick
- the story
To Have And To Hold
Carry love and joy wherever you go with these hand-etched ceramic jars, meant for holding fresh flowers, inspired love notes or special somethings. Keep these ceramic buckets on hand as an inspiring pick-me-up to grow old with. One-of-a-kind and made with love by husband-and-wife team Eric and Noelle Van Hendrick. Handmade in Vermont.
Sold separately.
Unfortunately, this item cannot ship outside of the United States at this time.
- details
- Item ID
- 19968
- Made from
- clay, stainless steel
- Measurements
- Approx. 10" H x 5" dia.
- the maker
Eric Hendrick and Noelle VanHendrick
Noelle VanHendrick and Eric Hendrick are a husband and wife team who have been creating together since the year 2000. They met in pottery class at West Virginia University and where good friends for a good while. They courted candling wood kilns and playing chess. Now, from their home in southern Vermont, they make it all happen. They homeschool their three sons and create art for the world to enjoy. "It is poetic, I have raised my sons on buckets of love and cups of beautiful…", says Noelle. " I intend for it all only to get better and better…." There is an underlying mission to the work. A mission of cultivating world peace beginning at home, in the body, mind, and spirit. Eric and Noelle create and design goods that align with this mission. collection
You might also like…
Himalayan Salt Tequila Glasses- Set of 4 $45.00
Scratch Map $20.00
Smartphone Projector $27.00
What I Love About You by Me Book $10.00
Love Is Art Kit $60.00
Wishing Ball $32.00
Stone Drink Dispenser $125.00
Personalized Whiskey Barrel $200.00
Define Bottle $25.00
Aquafarm $60.00
- reviews
- q & a


text:
Page 1
Isolation, characterization and total regioselective synthesis of the novel
mO-conotoxin MfVIA from Conus magnificus that targets voltage-gated
sodium channels
Irina Vettera,1, Zoltan Dekana,1, Oliver Knappb, David J. Adamsb, Paul F. Alewooda, Richard J. Lewisa,*
aDivision of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Australia
bHealth Innovations Research Institute, RMIT University, Melbourne, Australia
1. Introduction
Voltage-gated sodium channels (Nav) are primarily found in
excitable cells, including central and peripheral neurons as well as
in skeletal and cardiac muscle, where they play a pivotal role in the
initiation and propagation of action potentials. The nine pore-
forming a subunits, termed Nav1.1–Nav1.9, are differentially
expressed in various tissues. Nav1.4 expression is restricted to
skeletal muscle and Nav1.5 is found primarily in cardiac muscle
[1,2]. All other isoforms are expressed, in a temporally and spatially
restricted manner, in central or peripheral neurons, where they
have been proposed as pharmacological targets for a variety of
neurological diseases including epilepsy and pain [2–4].
The tetrodotoxin-resistant (TTX-R) isoform Nav1.8 is expressed
predominantly in small nociceptive afferent fibres, where it
contributes to action potential initiation and repetitive firing
[2,5–7]. In addition, Nav1.8 is essential for maintaining excitability
at noxious cold temperatures and the development of cold pain [8].
A variety of inflammatory mediators have been shown to increase
Nav1.8 activity in sensory neurons by increasing protein expres-
sion and/or modulating channel kinetics [7]. Similarly, Nav1.8
protein expression is increased locally after nerve injury [9,10],
Biochemical Pharmacology 84 (2012) 540–548
Article history:
Received 6 March 2012
Accepted 9 May 2012
Available online 16 May 2012
Regioselective disulfide bond synthesis
Voltage-gated sodium channels
The mO-conotoxins are notable for their unique selectivity for Nav1.8 over other sodium channel
isoforms, making them attractive drug leads for the treatment of neuropathic pain. We describe the
discovery of a novel mO-conotoxin, MfVIA, from the venom of Conus magnificus using high-throughput
screening approaches. MfVIA was found to be a hydrophobic 32-residue peptide (amino acid sequence
RDCQEKWEYCIVPILGFVYCCPGLICGPFVCV) with highest sequence homology to mO-conotoxin MrVIB. To
overcome the synthetic challenges posed by mO-conotoxins due to their hydrophobic nature and
difficult folding, we developed a novel regioselective approach for the synthesis of mO-conotoxins.
Performing selective oxidative deprotections of the cysteine side-chain protecting groups of the fully
protected peptide allowed manipulations in organic solvents with no chromatography required between
steps. Using this approach, we obtained correctly folded MfVIA with increased synthetic yields.
Biological activity of MfVIA was assessed using membrane potential-sensitive dyes and electrophysio-
logical recording techniques. MfVIA preferentially inhibits Nav1.8 (IC5095.9 ? 74.3 nM) and Nav1.4 (IC50
81 ? 16 nM),
Nav1.5 > 1.6 ? 1.7 ? 1.3 ? 1.1 ? 1.2). This improved approach to mO-conotoxin synthesis will facilitate
the optimization of mO-conotoxins as novel analgesic molecules to improve pain management.
Crown Copyright ? 2012 Published by Elsevier Inc. All rights reserved.
431–6203 nM;
Abbreviations: Nav, voltage-gated sodium channel; Acm, acetamidomethyl; Boc,
tertbutyloxycarbonyl; CHO, Chinese hamster ovarian; DCM, dichloromethane;
DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DRG, dorsal root
ganglion; HEK, human embryonic kidney; HEPES, 4-(2-hydroxyethyl)-1-piperazi-
neethanesulfonic acid; HF, anhydrous hydrogen fluoride; HFIP, 1,1,1,3,3,3-
hexafluoroisopropanol; Meb, 4-methylbenzyl; Pbf, 2,2,4,6,7-pentamethyldihydro-
benzofuran-5-sulfonyl; RP-HPLC, reversed-phase high-performance liquid chro-
matography; tBu, tertbutyl; TFA, trifluoroacetic acid; TFE, trifluoroethanol; Trt,
triphenylmethyl; TTX-R, tetrodotoxin-resistant; TTX-S, tetrodotoxin-sensitive; ESI-
MS, electrospray ionization mass spectrometry; FBS, foetal bovine serum; RPMI,
Roswell Park Memorial Institute; DMEM, Dulbecco's Modified Eagle's Medium;
MALDI-TOF, Matrix-assisted laser desorption/ionization-time of flight mass
spectrometry; CHCA, a-cyano-4-hydroxycinnamic acid; HBTU, 2-(1H-Benzotria-
zole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; FLIPR, fluorescent
imaging plate reader; EDTA, ethylenediaminetetraacetic acid; HBSS, Hank's
balanced salt solution; DMSO, dimethyl sulfoxide; SEM, standard error of the mean.
* Corresponding author at: Institute for Molecular Bioscience, The University of
Queensland, St Lucia QLD 4072, Australia. Tel.: +61 7 3346 2374;
fax: +61 7 3346 2984.
E-mail address: email@example.com (R.J. Lewis).
1These authors contributed equally to this research.
Contents lists available at SciVerse ScienceDirect
Biochemical Pharmacology
jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/bio c hem p har m
0006-2952/$ – see front matter. Crown Copyright ? 2012 Published by Elsevier Inc. All rights reserved.
Page 2
suggesting a prominent role of this Navsubtype in neuropathic
pain. Indeed, knockout and gene deletion studies have confirmed
Nav1.8 as an attractive analgesic target in inflammatory and
neuropathic pain [9,11,12].
While peptides have yielded some of the most subtype-
selective Navinhibitors known, few Nav1.8-selective modulators
have been described to date. The mO-conotoxins MrVIA and
MrVIB, from the venom of Conus marmoreus, are of particular
interest because they potently inhibit Nav1.8 in mammalian
sensory neurons with >10-fold selectivity over tetrodotoxin-
sensitive (TTX-S) Navisoforms [13,14]. Accordingly, both peptides
display analgesic activity in a variety of animal models of pain and
decrease mechanical and thermal pain in inflammatory and
neuropathic pain models [14,15]. Importantly, selectivity over
motor side effects was more than 30-fold greater than for
lidocaine [14], making the mO-conotoxins promising leads for the
development of novel, Nav1.8-selective molecules with analgesic
Here, we report the isolation, synthesis and pharmacological
characterization of a novel mO-conotoxin, MfVIA, isolated from the
venom of Conus magnificus. MfVIA is selective for Nav1.8 and
Nav1.4 over other mammalian Navsubtypes and displays weak
activity at the centrally expressed Nav1.2. To facilitate the study of
mO-conotoxins, we have developed a novel approach to synthe-
sizing MfVIA, resulting in improved yields of the correctly folded
2. Experimental
2.1. Isolation of MfVIA
Crude venom was isolated from two specimens of Conus
magnificus by stripping the venom duct contents. Stripped venom
was dissolved in 30% acetonitrile/0.1% formic acid, vortexed and
centrifuged at 10,000 g for 5 min to remove insoluble components.
Crude venom (500 mg) was fractionated into 60 ? 1 min fractions
using a Vydac 218TP C18 column (250 ? 4.6 mm, 5 mm) eluted at a
flow rate of 0.7 mL/min with 5–100% solvent B over 60 min
(solvent A, H2O/0.1% formic acid; solvent B, 90% acetonitrile/0.1%
formic acid).
Native peptide purified by reversed-phase high-performance
liquid chromatography (RP-HPLC) was analyzed on a MALDI-TOF
mass spectrometer (4700 Proteomics Analyzer, Applied Biosys-
tems, Mulgrave, VIC) using a-cyano-4-hydroxycinnamic acid
(CHCA) (5 mg/mL) as the matrix. The sequence of the purified
peptide was determined by Edman degradation at the Australian
Proteome Research Facility. In brief, the peptide was dissolved in
urea (4 M) in ammonium bicarbonate (50 mM) and reduced
with dithiothreitol (100 mM) at 56 8C for 1 h under argon.
The sample was then alkylated using acrylamide (220 mM)
for 0.5 h in the dark. The reaction was quenched by the addition
of excess dithiothreitol. After desalting by RP-HPLC, the
collected fraction was loaded onto pre-cycled bioprene discs
and subjected to 35 cycles of Edman N-terminal sequencing
using an Applied Biosystems 494 Procise Protein Sequencing
2.2. Peptide synthesis
MfVIA was assembled using standard Fmoc protocols on
preloaded H-Val-2-chlorotrityl resin on a 0.1 mmol scale
(0.61 mmol/g resin loading) on a Symphony (Protein Technologies
Inc.) automated peptide synthesizer. Couplings were performed
using 5 equivalents (relative to resin loading) of amino
acid:HBTU:DIEA (1:1:1) for 2 ? 20 min. Fmoc deprotections
were accomplished using 30% piperidine/dimethylformamide
(DMF) (1 ? 1 min then 1 ? 3 min). The cysteine side-chain
protecting groups used were 4-methylbenzyl (Meb) for Cys 1
and 4, acetamidomethyl (Acm) for Cys 2 and 5, and triphenyl-
methyl (Trt) for Cys 3 and 6. The remaining side-chain
functionalities were protected as Asp(OtBu), Glu(OtBu), Lys(Boc),
Gln(Trt), Arg(Pbf), Trp(Boc) and Tyr(tBu). After removal of the
final Fmoc group, the fully side-chain protected peptide was
cleaved from the resin using 10% AcOH/10% trifluoroethanol (TFE)
in dichloromethane (DCM) for 1 h. The resin was then filtered off,
the solvent evaporated under vacuum and triturated with hexane
(3x) to remove traces of AcOH giving a white powder (386 mg).
ESI-MS (m/z) calculated (average) 1822.6 [M + 3H]3+, observed
Selective oxidative deprotection of the Cys 3–6 (Trt) groups was
based on the method described by Kamber et al. [16]. The fully
protected peptide (386 mg) was dissolved in neat 1,1,1,3,3,3-
hexafluoroisopropanol (HFIP) (5 mL) and added drop-wise over
5 min to a solution of I2(8 eq) in 20 mL of 10% HFIP/DCM, then
stirred at room temperature for an additional 5 min. The solution
was then poured into 0.2 M ascorbic acid/0.5 M NaOAc. The
aqueous layer was extracted with 10% MeOH/CHCl3(2x) then DCM
(1x). The organic layer was washed with H2O (1x) and dried over
MgSO4. The solvent was removed to obtain a sticky solid (357 mg).
ESI-MS (m/z) calculated (average) 1660.7 [M + 3H]3+, observed
To oxidise the Cys 2–5 (Acm) pair, the obtained product was
redissolved in DMF (10 mL) and added drop-wise to a stirred
solution of I2(8 eq) in DMF (20 mL) at room temperature over
20 min. After stirring for another 20 min, the solution was diluted
with DCM (150 mL) and washed with 0.2 M ascorbic acid/0.5 M
NaOAc. The aqueous layer was extracted with 10% MeOH/CHCl3
(2x) and DCM (1x), then the organic layer was washed with H2O
(1x) and dried over MgSO4. Most of the solvent was removed under
vacuum and the product was precipitated with cold Et2O and
hexane. The precipitate was collected by filtration and dried to give
an off-white powder (295 mg). ESI-MS (m/z) calculated (average)
1612.4 [M + 3H]3+, observed 1612.5.
To remove the remaining protecting groups (except Cys(Meb)),
the peptide was treated with 95% trifluoroacetic acid (TFA)/2.5%
triisopropylsilane/2.5% H2O (20 mL) for 2 h at room temperature.
Most of the solvent was removed under vacuum. The product was
precipitated with Et2O, collected by filtration and lyophilized from
0.05% TFA in MeCN/H2O (1:1) to give a white powder (230 mg).
ESI-MS (m/z) calculated (average) 1287.2 [M + 3H]3+, observed
1287.1 (Fig. 3A).
The Cys 1–4 (Meb) groups were removed using 90% hydrogen
fluoride (HF)/10% p-cresol at 0 8C for 1 h. The product was
precipitated with Et2O and lyophilized from 0.05% TFA in MeCN/
H2O (1:1) to give a white powder (180 mg). ESI-MS (m/z)
calculated (average) 1217.8 [M + 3H]3+, observed 1217.7 (Fig. 3B).
To oxidize the obtained Cys 1–4 free thiols, the crude product
(180 mg) was redissolved in 0.1% TFA in MeCN/H2O (1:1) at a
concentration of ?0.3 mg/mL. A solution of I2(4 eq) in MeCN was
added and stirred for 20 min before the addition of ascorbic acid to
obtain a colorless solution (Fig. 3C). This solution was purified by
preparative HPLC (6 separate runs) on a Vydac C18 218TP1022
column, running at 16 mL/min using a gradient of 30–70% B over
60 min. The combined fractions were lyophilized to give a total of
15 mg white powder. The overall yield was 4%, as calculated from
the initial resin loading. ESI-MS (m/z) calculated 1217.1 [M + 3H]3+,
observed 1217.2.
Analytical HPLC of the synthetic intermediates was performed
on a Shimadzu LC-20AT system using a Thermo Hypersil GOLD C18
2.1 ? 100 mm column at a flow rate of 0.3 mL/min with a gradient
of 25 to 70% B over 30 min. Solvent A consisted of 0.05% TFA/H2O,
solvent B contained 0.043% TFA in 90% MeCN/H2O.
I. Vetter et al. / Biochemical Pharmacology 84 (2012) 540–548
Page 3
2.3. Biological activity
2.3.1. Cell culture and transfection
Activity-guided isolation of MfVIA was carried out using a FLIPR
high-throughput Ca2+
assay assessing veratridine-stimulated
activity of Navendogenously expressed in the human neuroblas-
toma cell line SH-SY5Y [17]. SH-SY5Y human neuroblastoma cells
were a gift from Victor Diaz (Max Planck Institute for Experimental
Medicine, Goettingen, Germany). Cells were maintained at 37 8C/
5% CO2 in Roswell Park Memorial Institute (RPMI) medium
containing 15% foetal bovine serum (FBS) and 2 mM L-glutamine.
Human embryonic kidney 293 (HEK293) cells and ND7/23 cells (all
American Tissue Culture Collection, Manassas, VA, USA) were
maintained at 37 8C in a 5% humidified CO2incubator in Dulbecco's
Modified Eagle's Medium (DMEM) containing 10% FBS, 2 mM L-
glutamine, pyridoxine and 110 mg/mL sodium pyruvate (Invitro-
gen). Chinese hamster ovarian (CHO) cells stably expressing
hNav1.4, hNav1.6, and hNav1.7 a-subunits (Genionics, Schlieren,
Switzerland) were grown in F-12 Medium (Invitrogen, Mulgrave,
VIC), supplemented with 9% fetal bovine serum (Lonza, Mt
Waverley, VIC), 0.9% penicillin/streptomycin (Invitrogen) and
100 mg/mL hygromycin B (Invitrogen). HEK293 cells stably
transfected with the hNav1.5 a-subunit (Genionics) were grown
in Dulbecco's Modified Eagle Medium (Invitrogen), 0.9% penicillin/
streptomycin and 100 mg/mL zeocin (Invitrogen). All cells were
split every 3–6 days in a ratio of 1:5, using 0.25% trypsin/EDTA
(ethylenediaminetetraacetic acid) (Invitrogen).
ND7/23 cells were plated on T75 tissue culture flasks 24 h prior to
transfection with plasmid DNA of rNav1.8 cloned into pCMV-Script
(K. Zimmermann, University of Erlangen) using Fugene (Roche, Dee
Why, NSW). Nav1.2 and Nav1.5 were transfected in HEK293 cells
using a commercial BacMam transfection system (Invitrogen),
following the manufacturer's instructions. In brief, cells were
incubated with virus solution in phosphate-buffered saline for 3 h
at room temperature, then incubated with enhancer solution in
complete medium for 2 h at 37 8C. BacMam-transfected cells were
plated on 384-well plates after an overnight incubation in normal
complete growth medium at 37 8C in a 5% humidified CO2incubator.
They were then assayed after incubation for another 24 h. hNav1.1,
hNav1.3, hNav1.5, hNav1.6 and hNav1.7 EZCells were purchased from
ChanTest Corp (Cleveland, OH, USA), plated on 384-well plates
immediately after thawing, and assayed after incubation for another
24 h at 37 8C in a 5% humidified CO2incubator.
2.4. FLIPR Ca2+and membrane potential assays
Ca2+responses were assessed using the FLIPRTETRA(Molecular
Devices, Sunnyvale, CA) plate reader, as described previously [18].
SH-SY5Y cells were plated at a density of 50,000 cells/well on 384-
well black-walled imaging plates (Corning), 48 h before the assay.
Cells were loaded with the Calcium 4 No-wash dye (Molecular
Devices) by diluting the lyophilized dye in physiological salt
solution, composition: 140 mM NaCl, 11.5 mM glucose, 5.9 mM
KCl, 1.4 mM MgCl2, 1.2 mM NaH2PO4, 5 mM NaHCO3, 1.8 mM
CaCl2, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic ac-
id (HEPES), pH 7.4, and incubated for 30 min at 37 8C in a 5%
humidified CO2incubator. Fluorescence responses were recorded
every second for 300 s after adding crude venom or venom
fractions (excitation, 470–495 nm; emission, 515–575 nm). A
further 300 fluorescence reads were recorded at 1 s intervals after
adding veratridine (50–100 mM).
To assess changes in membrane potential, HEK293, ND7/23 and
CHO cells expressing Nav1.1–Nav1.8 isoforms were loaded with
membrane potential dye (red, proprietary composition; Molecular
Devices) for 30 min at 37 8C in a 5% humidified CO2incubator. After
addition of MfVIA using the FLIPRTETRA, cells were incubated a
further 30 min before stimulating Navsubtypes using veratridine
(Nav1.1–Nav1.7) or deltamethrin (100 mM, Nav1.8) [19]. Fluores-
cence reads (excitation 510–545 nm; emission 565–625 nm) were
acquired every second for 300 s after adding agonists.
2.5. Electrophysiology
Sub-confluent HEK293 and CHO cells were detached from the
cell culture flask with 0.25% trypsin (Invitrogen), washed with
extracellular solution and pelleted by centrifugation at 300 g for
5 min. After agitating the cells to break cell clusters and an
additional centrifugation step, we resuspended the pellet in
extracellular solution to get optimal concentration of 5 ? 105–
5 ? 107cells/mL for electrophysiological experiments.
Depolarization-activated Na+currents from Nav1.4, Nav1.5,
Nav1.6 and Nav1.7 were recorded using a Port-a-Patch system
(Nanion, Munich, Germany) in the whole-cell mode. Cells were
held at ?80 mV in the whole-cell recording configuration and
depolarized every 20 s–0 mV. The experiments were carried out at
room temperature (22–23 8C). Concentration-response curves
were obtained by adding an external solution containing various
concentrations of MfVIA to the recording chamber and Na+
currents were recorded using a HEKA EPC10 patch-clamp amplifier
(HEKA Elektronik GmbH, Lambrecht/Pfalz, Germany). The external
solution contained 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM
CaCl2, 5 mM D-glucose and 10 mM HEPES, pH 7.4 (NaOH). The
internal solution contained 50 mM CsCl, 10 mM NaCl, 60 mM CsF,
2 mM MgCl2, 20 mM EGTA, and 10 mM HEPES, pH 7.2 (CsOH).
2.6. Oocyte preparation, RNA injection and oocyte electrophysiology
Stage V–VI oocytes were harvested from sexually mature
female Xenopus laevis anesthetized with 0.1% tricaine (3-amino-
benzoic acid ethyl ester), following protocols approved by the
RMIT University Animal Ethics Committee. The oocytes were
defolliculated in collagenase (1.5 mg/mL) and dissolved in calci-
um-free solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and
5 mM HEPES-NaOH, at pH 7.4) before RNA injection. The
constructs encoding rNav1.2 and hNav1.8 (cloned into pcDNA3.1)
were linearized and cRNA was synthesized using a T7 RNA
polymerase in vitro transcription kit (mMESSAGE mMachine;
Ambion, Foster City, CA). cRNA concentration was measured
spectrophotometrically and adjusted to 2.5 ng rNav1.2 and 5 ng
hNav1.8 for injection in Xenopus laevis oocytes. Injected oocytes
were incubated for 2–5 days at 18 8C in ND98 solution containing
96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2and 5 mM
HEPES-NaOH, at pH 7.4 supplemented with pyruvate (5 mM), and
gentamicin (50 mg/ml).
Oocytes were transferred to a 50 mL volume recording chamber
and gravity perfused at 1.5–2.5 mL/min with ND96 solution
(96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2and 5 mM
HEPES, at pH 7.4 (NaOH)). Two-electrode (virtual ground circuit)
voltage-clamp experiments were performed at room temperature
(21–23 8C) using a GeneClamp 500B amplifier (Molecular Devices)
and pCLAMP 8 software (Molecular Devices). Peak Na+current was
elicited at a test potential of +10 mV for Nav1.8 and ?10 mV for
Nav1.2. Micropipettes pulled from glass capillaries (3-000-203 GX,
Drummond Scientific Co.) and filled with 3 M KCl had open-tip
resistances of 0.3–1.5 M V. Whole-cell Na+currents were low-pass
filtered at 1 kHz and digitized at 10 kHz. Leak currents were
subtracted online using a-P/6 protocol.
2.7. Dorsal root ganglion (DRG) neuron preparation
DRG neurons were enzymatically dissociated from ganglia of 6
to 14-day-old Wistar rats according to standard protocols. Rats
I. Vetter et al. / Biochemical Pharmacology 84 (2012) 540–548
Page 4
were killed by cervical dislocation, as approved by the RMIT
University Animal Ethics Committee. Ganglia of all areas of the
spinal cord were collected in ice cold Hank's Balanced Salt Solution
(HBSS; Invitrogen) and incubated in 100 mg/mL collagenase
(Sigma Aldrich) in HBBS for 30 min at 37 8C. Ganglia were
triturated with a fine bore glass pipette and resuspended in warm
Neurobasal media containing B27 supplement (both Invitrogen),
0.5 mM L-glutamine and 1% Penicillin/Streptomycin. They were
then seeded onto poly-D-lysine-coated multi-well plates or glass
cover slips. The cells were incubated at 37 8C in a 5% humidified
CO2incubator and used after 2-4 days.
Depolarization-activated Na+currents were recorded using the
whole-cell patch-clamp technique with an Axopatch 200B amplifier
(Molecular Devices). The patch recording electrodes had resistances
of 1.5–3 M V. Under the voltage-clamp recording configuration,
cells were depolarized to +10 mV from a holding potential of
?80 mV every 20 s to elicit voltage-gated Na+channel currents.
Membrane currents were generated using pClamp 9.2 software
(Molecular Devices), filtered at 2 kHz and sampled at 10 kHz by a
Digidata 1322A interface (Molecular Devices). Sampled data was
stored digitally on a personal computer for analysis. Na+currents
were recorded in the presence of TTX (300 nM) using patch pipettes
filled with an internal solution containing 10 mM NaCl, 130 mM CsF,
10 mM CsCl, 10 mM EGTA, 10 mM HEPES, pH 7.2 (CsOH). The bath
solution contained 50 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM
CaCl2, 10 mM glucose, 90 mM tetraethylammonium (TEA)-Cl,
10 mM HEPES, pH 7.3 (TEA-OH). Series resistance was routinely
compensated by 70–80%. Capacitive and leakage currents were
digitally subtracted using a-P/6 pulse protocol.
MfVIA was diluted to the appropriate final concentration and
applied via perfusion in the bath solution. The toxin solution
contained ?0.01% dimethyl sulfoxide (DMSO). DMSO at this
concentration had no effect on Na+currents. Peak current amplitude
in response to the depolarizing pulse was acquired once we achieved
a steady-state current (?5–10 min). The current amplitudes
recorded in the presence of the drug were normalized by dividing
by the current amplitude from control conditions (I/Imax).
2.8. Data analysis
Unless otherwise stated, all data are expressed as the mean
? standard error of the mean (SEM) determined from at least n = 3
replicates and are representative of at least 3–5 independent
experiments. For measurement of membrane potential, responses
after addition of veratridine or deltamethrin were plotted against
MfVIA concentration. A 4-parameter Hill equation with variable Hill
slope was fitted to the data using GraphPad Prism (Version 4.00, San
Diego, California). Potency of MfVIA is reported as the mean ? SEM of
3–5 separate experiments. Electrophysiological data were analyzed
using PatchMaster v2.20 (HEKA Elektronik GmbH, Lambrecht/Pfalz,
Germany). Concentration-response curves were analyzed using
SigmaPlot 10 (Systat, Chicago USA) and fitted with a 4-parameter
logistic curve. Oocyte electrophysiological data were analyzed with
Clampfit (v 10.2, MDS Analytical Technologies) and Sigma Plot (v 11,
Systat Software Inc.) software. Hill slopes were determined to be
statistically significantly different from ?1 if the 95% confidence
intervals calculated using GraphPad Prism (Version 4.00, San Diego,
California) did not include ?1.
For other data, statistical significance was determined using a
one-way ANOVA with Tukey's post hoc test and defined as p < 0.05
unless otherwise stated.
2.9. Chemicals and reagents
We sourced veratridine from Ascent Scientific (Bristol, UK).
Fmoc amino acids, resins and HBTU were obtained from Iris
Biotech GmbH (Marktredwitz, Germany). All other reagents, unless
otherwise stated, were sourced from Sigma–Aldrich (Castle Hill,
NSW, Australia).
3. Results
3.1. Isolation of a novel mO-conotoxin from Conus magnificus venom
Crude venom isolated from Conus magnificus inhibited veratri-
dine-induced Nav responses in SH-SY5Y cells at ?300 mg/mL.
Activity-guided fractionation revealed activity corresponding to a
single peak eluting at ?64% solvent B (Fig. 1A). N-terminal Edman
degradation of the purified native peptide corresponding to this
activity revealed a novel 32-residue mO-conotoxin homologous to
MrVIA and MrVIB (Table 1) with the following sequence:
RDCQEKWEYCIVPILGFVYCCPGLICGPFVCV (Table 1).
Compared with MrVIB, this novel peptide has an extended
N-terminus and the residue substitutions A1D, S3Q and K4E
Fig. 1. Isolation of MfVIA from Conus magnificus crude venom. (A) Crude venom isolated from Conus magnificus (500 mg) was fractionated on a Vydac 218TP C18 column with a
two-step linear acetonitrile/0.1% formic acid gradient (5–50% B/10 min, 50–100% B/50 min; blue line). Green circles: activity of 1 min fractions on Navresponses in SH-SY5Y
cells. The fractions containing MfVIA (arrow; elution at ?64% B) fully inhibited veratridine-induced Navresponses. Inset; Conus magnificus shell. (B) Native peptide purified by
RP-HPLC was analyzed on a MALDI-TOF mass spectrometer. The observed mass (M + H, 3646.7 m/z) was consistent with the predicted mass of MfVIA. (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of the article).
I. Vetter et al. / Biochemical Pharmacology 84 (2012) 540–548
Page 5
(Table 1). The observed mass for MfVIA (M + H 3646.7 Da) was
consistent with the predicted mass of the peptide sequence
obtained by Edman degradation (Fig. 1B). Identity of the peptide
was further confirmed by co-elution of the native and synthetic
peptide (Fig. 3D).
3.2. An improved approach for regioselective synthesis of mO-
mO-conotoxins like MfVIA have until now posed significant
synthetic challenges. Their highly hydrophobic nature leads to
aggregation in solution and poor recovery. In addition, difficulties
with oxidative folding of the three disulfide bonds to the native
isomer have hindered the efficient synthesis, structure–activity
studies and further optimization of these molecules. Thus, we
developed a simple and more efficient, fully regioselective protocol
for the synthesis of MfVIA requiring only a single HPLC purification
step (Fig. 2).
Peptide assembly was performed using Fmoc chemistry on the
highly acid-labile 2-chlorotrityl resin, with cysteine side-chains
protected as 1,4(Meb), 2,5(Acm) and 3,6(Trt). Cleavage from the
resin using AcOH left all side-chain protecting groups intact,
enabling subsequent manipulations to be performed in organic
solvents. This circumvented the poor aqueous solubility and
chromatographic problems we previously encountered with
MrVIB. Based on the solvent-dependent rate difference between
the reactions of S-Trt and S-Acm groups towards iodine [16], the
Cys3–6 and Cys2–5 disulfide bonds were formed selectively by
iodine oxidation of the 3,6(Trt) cysteine pair in a HFIP/DCM
mixture, followed by oxidation of the 2,5(Acm) pair by a second
treatment with iodine in DMF [16,20]. The third protecting group
for the Cys1-4 pair required more optimization. Our initial trials
with the p-methoxybenzyl group, which was expected to be stable
to iodine treatment [21] and is removable by other oxidative
methods [21,22], resulted in loss of regioselectivity at the iodine-
DMF oxidation step. This was most likely because of the reaction of
p-methoxybenzyl thioether with the sulfenyl iodide intermediates
[16,23] formed during deprotection of the Acm groups. The p-
methylbenzyl (Meb) group proved to be completely stable under
these conditions and was used to complete the synthesis. The
remaining side-chain protecting groups, except Cys 1–4 (Meb),
were removed by treating the Cys 3–6 and Cys 2–5 oxidized
peptides with TFA (Fig. 3A). The Meb groups were cleaved using HF
to obtain the free thiols in the 1–4 positions (Fig. 3B), which were
then oxidized by iodine treatment in acidic aqueous acetonitrile
solution (Fig. 3C) to give the fully folded product following
purification by HPLC. Identity of the product was confirmed by co-
elution with the native peptide (Fig. 3D).
Fig. 2. An improved approach for regioselective synthesis of mO-conotoxins. Schematic representation of the approach for regioselective synthesis of the disulfide bonds of
Table 1
Peptide sequences of mO-conotoxins.
Conus sp.
Conus marmoreus
Conus marmoreus
Conus magnificus
Conus litteratus
Conus litteratus
This study
I. Vetter et al. / Biochemical Pharmacology 84 (2012) 540–548
Page 6
3.3. Navsubtype selectivity of MfVIA
The biological activity of synthetic MfVIA was assessed using a
membrane potential assay in mammalian cells over-expressing
Navisoforms. Consistent with previously reported activity of mO-
expressed Nav1.8 (IC50529 ? 50 nM, Hill slope ?2.9 ? 0.37) over
the TTX-S and TTX-R Navisoforms Nav1.3 (IC502.2 ? 0.3 mM, Hill
slope ?1.7 ? 0.23), Nav1.5 (IC503.9 ? 0.9 mM, Hill slope ?1.5 ? 0.42),
Nav1.6 (IC504.6 ? 0.5 mM, Hill slope ?1.2 ? 0.12) and Nav1.1 (IC50
3.3 ? 0.1 mM, Hill slope ?1.6 ? 0.27) (Fig. 4A and B). Nav1.7 and
Nav1.2 were relatively resistant to inhibition by MfVIA (IC50
5.5 ? 0.9 mM, Hill slope ?2.3 ? 1.0 and 6.3 ? 0.7 mM, Hill slope
?4.4 ? 1.36, respectively) (Fig. 4A and B).
These results were confirmed using Xenopus oocytes expressing
Nav1.2 and Nav1.8 (Fig. 5) and cell lines (HEK and CHO)
heterologously expressing Nav1.4, Nav1.5, Nav1.6 and Nav1.7 a-
subunits (Fig. 6). MfVIA inhibited depolarization-activated Nav1.4
currents with an IC50 of 81 ? 16 nM, consistent with previous
reports that mO-conotoxins display relatively high affinity for the
skeletal muscle Navsubtype. Heterologously expressed Nav1.8 was
also potently inhibited with an IC50of 157.6 ? 63.8 nM. In contrast,
(IC50= 431 ? 146 nM), Nav1.6
Nav1.7 (IC50= 2.3 ? 0.3 mM) were much less sensitive to inhibition
by MfVIA. Similar to the results using membrane potential
measurement, Nav1.2 was the subtype most resistant to block by
MfVIA (IC50= 5.1 ? 0.5 mM). With the exception of Nav1.2 (Hill slope
?2.3 ? 0.3), the Hill slopes of MfVIA-mediated inhibition of Nav1.4
(IC50= 1.2 ? 0.2 mM)
Fig. 3. Analytical HPLC traces of synthetic MfVIA and intermediates. (A) Analytical HPLC traces of crude product obtained after TFA cleavage with oxidized Cys3–6 and Cys2–5
and Meb protected Cys 1 and 4 (B) crude product obtained after HF cleavage of the Meb groups to give the free thiols of Cys 1 and 4, and (C) crude product obtained after iodine
oxidation of Cys1-4. The retention time of the major peak corresponds to that of native MfVIA. (D) Co-elution of native and synthetic MfVIA. Analytical HPLC traces of native
and synthetic MfVIA show identical retention time and peak width.
Fig. 4. Navsubtype selectivity of MfVIA assessed using membrane potential assays. (A and B) Biological activity of synthetic MfVIA on veratridine-evoked Navresponses was
assessed using a membrane potential assay in mammalian cells overexpressing Nav1.1 (light blue; IC50= 3.3 ? 0.1 mM), Nav1.2 (purple; IC50= 6.3 ? 0.7 mM), Nav1.3 (light
green; IC50= 2.2 ? 0.3 mM), Nav1.5 (magenta, IC50= 3.9 ? 0.9 mM), Nav1.6 (dark green; IC50= 4.67 ? 0.5 mM), Nav1.7 (dark blue; IC50= 5.5 ? 0.9 mM) and Nav1.8 (orange;
IC50= 529 ? 50 nM). (B) MfVIA was significantly (p < 0.05) more potent at heterologously expressed Nav1.8 (pIC506.28 ? 0.05) than the TTX-S and TTX-R Navisoforms Nav1.3 (pIC50
5.68 ? 0.09), Nav1.5 (pIC505.55 ? 0.14), Nav1.6 (pIC505.65 ? 0.04) and Nav1.1 (pIC505.47 ? 0.02). Nav1.7 and Nav1.2 were relatively resistant to inhibition by MfVIA (pIC50
5.36 ? 0.13 and 5.21 ? 0.05, respectively). Data are presented as mean ? SEM from at least 3 independent experiments. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of the article).
I. Vetter et al. / Biochemical Pharmacology 84 (2012) 540–548
Page 7
(Hill slope ?0.8 ? 0.11), Nav1.5 (Hill slope ?1.0 ? 0.3), Nav1.6 (Hill
slope ?3.3 ? 1.98), Nav1.7 (Hill slope ?1.9 ? 0.6) and Nav1.8 (Hill
slope ?1.2 ? 0.28) were not statistically different from ?1. Finally,
the effects of MfVIA on whole-cell TTX-R currents in rat DRG neurons
were assessed in the presence of TTX under conditions that inactivate
Nav1.9, but not Nav1.8. In this assay, MfVIA (1 mM) potently inhibited
native rat Nav1.8 (IC5095 ? 74 nM) (Fig. 7).
4. Discussion
mO-Conotoxins belong to the O superfamily of conopeptides
that inhibit Navby interfering with the voltage sensors in domain II
of Nav[24,25]. Despite their selectivity for Nav1.8 over other TTX-S
isoforms, and resultant analgesic activity in a variety of animal
models of pain [13–15], little is known of the structure-activity
relationships that define their subtype specificity. In addition, only
four native mO-conotoxins have been described to date, including
the recently identified LtVIIA and LtVIC [26,27] which were
classified as mO-conotoxins based on sequence homology and
Fig. 5. Inhibition of depolarization-activated Nav1.2 and Nav1.8 currents by
synthetic MfVIA. (A) Voltage protocols and superimposed inward Na+currents
evoked by a voltage step are shown in the absence (control) and presence of MfVIA
(1 mM) for Nav1.2 and Nav1.8 expressed in Xenopus oocytes. For ease of comparison,
data are presented as I/Imax. Peak current amplitudes were ?2.0 nA (Nav1.2, 10%
inhibition by 1 mM MfVIA to ?1.8 nA) and ?0.4 nA (Nav1.8, 63% inhibition by 1 mM
MfVIA to ?0.15 nA). (B) Concentration-response relationships obtained for MfVIA
inhibition of Na+currents mediated by Nav1.2 (closed circle; IC50= 5.1 ? 0.5 mM)
and Nav1.8 (open circle; IC50= 157.6 ? 63.8 nM).
Fig. 6. Inhibition of depolarization-activated Nav1.4, Nav1.5, Nav1.6 and Nav1.7
currents by synthetic MfVIA. (A) Voltage protocols and superimposed inward Na+
currents evoked by a voltage step are shown in the absence (control) and presence
of MfVIA (1 mM) for Nav1.4, Nav1.5, Nav1.6 and Nav1.7 a-subunits expressed in
mammalian cell lines (HEK and CHO). For ease of comparison, data are presented as
I/Imax. Peak current amplitudes were ?5.1 nA (Nav1.4, 79% inhibition by 1 mM
MfVIA to ?1.1 nA); ?5.0 nA (Nav1.5, 76% inhibition by 1 mM MfVIA to ?1.2 nA);
?1.0 nA (Nav1.6, 24% inhibition by 1 mM MfVIA to ?0.76 nA) and ?1.6 nA (Nav1.7,
1 mM
relationships obtained for MfVIA inhibition of Na+currents mediated by Nav1.4
(closed circle; IC50= 81 ? 16 nM), Nav1.5 (open square; IC50= 431 ? 146 nM), Nav1.6
(closed triangle; IC50= 1.2 ? 0.2 mM) and Nav1.7 (open rhombus; IC50= 2.3 ? 0.3 mM).
?1.1 nA).
I. Vetter et al. / Biochemical Pharmacology 84 (2012) 540–548
Page 8
pharmacological activity. However, MrVIA and MrVIB remain the
only mO-conotoxins for which mammalian Navsubtype selectivity
data is available, albeit not across all Nav subtypes. Here, we
describe the isolation of the novel mO-conotoxin MfVIA from the
venom of the molluscivorous Conus magnificus using a high-
throughput Navassay [17]. MfVIA is a hydrophobic 32-residue
GLICGPFVCV) with highest sequence homology to mO-conotoxin
The pharmacological activity of synthetic mO-conotoxin MfVIA
was characterized across mammalian Nav1.1–Nav1.8 subtypes.
Despite relatively few changes in the sequence of MfVIA compared
with MrVIA and MrVIB, we found surprising changes in potency
and mammalian Nav subtype selectivity. Relative to Nav1.8
potency, MfVIA was found to have 3-fold enhanced Nav1.4 potency
and 5-fold reduced Nav1.2 potency (see Table 2). Interestingly,
MfVIA was ?5-fold more potent at native rNav1.8 expressed in
DRG neurons than rNav1.8 heterologously expressed in ND7/23
cells. This observation is consistent with the decreased affinity of
other mO-conotoxins, in particular MrVIB, for heterologously
expressed Nav1.8 compared with native Nav1.8-mediated TTX-R
currents in DRG neurons (Table 2). This effect has been suggested
to occur due to the presence of auxiliary b subunits, and perhaps
other accessory proteins, in neurons, which particularly affect the
on-rate of MrVIB and can thus profoundly affect the pharmacology
of mO-conotoxins [28].
The difference in potency between native and heterologous
expression systems was less pronounced for MfVIA than the ?15-
fold difference in potency reported for MrVIB [28,29]. This may be
due to the presence of endogenous b subunits in the ND7/23 cell
line we used for heterologous expression, as b subunits have been
reported to affect the Kdof mO-conotoxins at Nav1.8 [28,29]. The
potency of MfVIA may also be enhanced by the prolonged pre-
incubation protocol used in the present study, as b subunits have
been reported to particularly affect the on-rate of inhibition by
mO-conotoxins [28] and electrophysiological experiments gen-
erally preclude prolonged pre-incubation protocols. With the
exception of Nav1.4 and Nav1.8, MfVIA inhibited all other Nav
subtypes with significantly lower affinity (IC50 431–6203 nM;
Nav1.5 > 1.6 ? 1.7 ? 1.3 ? 1.1 ? 1.2). This includes Nav1.1 and
Nav1.6, for which mO-conotoxin activity has not previously been
The simplest model of mO-conotoxin-mediated inhibition of
Navchannels, where a single molecule of MfVIA is sufficient for full
channel block, is consistent with MfVIA block of all sodium channel
subtypes except Nav1.2. At this subtype, the concentration-
dependence of inhibition was signficantly steeper than a Hill
slope of ?1, indicative of positive cooperativity which may be more
apparent at Nav1.2. This might arise if mO-conotoxins have more
than a single binding site on sodium channels, bind to the channel
after first partitioning into the plasma membrane, or have slow on-
rates that make it difficult to reach equilibrium at low concentra-
tions of peptide. Although not directly assessed in this study, the
effects of membrane potential (Vm) on the interaction of mO-
conotoxins with Nav have been assessed systematically in the
literature [24,28]. These studies have found that inhibition of Nav
currents by MrVIA was voltage-dependent and strongly dimin-
ished after depolarizing voltage steps, consistent with mO-
conotoxins acting as gating modifiers. While MrVIA and MrVIB
are analgesic in various animal models of pain, the analgesic
activity of MfVIA remains to be determined.
The mO-conotoxins MfVIA, MrVIA and MrVIB are notable for
their unique selectivity profile for blocking Nav channels [14],
making these peptides attractive drug leads for development of
improved analgesic molecules [14,30]. Unfortunately, these
peptides are also notable for the synthesis challenges they have
posed since their discovery [31,32]. These challenges arise due to
their highly hydrophobic nature and the difficulties of forming
their three disulfide bonds to the native isomer, which to date
could not be done through the oxidative folding of fully reduced
precursors. These properties have largely hindered the efficient
synthesis, structure-activity studies and further optimization of
these molecules.
Fig. 7. Effect of mO-conotoxin MfVIA on transient Na+currents in rat DRG neurons in
the presence of 300 nM TTX. (A) Voltage protocol and superimposed inward Na+
currents evoked by a voltage step to +10 mV from a holding potential of–80 mV are
shown in the absence (control) and presence of MfVIA (1 mM). (B) Concentration-
response relationships obtained for MfVIA inhibition of Na+currents mediated by
TTX-R currents in rat DRG neurons. MfVIA inhibition of native TTX-R currents in
DRG neurons gave an IC50of 95.9 ? 74.3 nM.
Table 2
Navsubtype selectivity of mO-conotoxins MrVIA, MrVIB and MfVIA.
TTX-R (nM)
TTX-S (nM)
This study
nr – not reported; nd – not determined.
I. Vetter et al. / Biochemical Pharmacology 84 (2012) 540–548
Page 9
While replacement of cysteines with selenocysteines has been
used to assist with the folding of MrVIB [29], the synthesis of the
native molecule currently relies on a semi-selective approach
[15,31] with a low overall yield (? 1%) and possibly requiring
labour-intensive re-optimizations for the preparation of non-
native analogues. Fully regioselective strategies that involve
chromatographic purifications between disulfide forming steps
are not well suited to hydrophobic peptides. In addition to being
time consuming, these approaches tend to produce even lower
yields of the mO-conotoxins due to poor recovery from RP-HPLC
purification steps. In these cases, an efficient one-pot oxidation
strategy would be ideal. However, using the described methods
[33] failed to produce correctly folded mO-conotoxins during our
To facilitate the study of mO-conotoxins, we developed an
improved, fully regioselective protocol for the synthesis of the novel
mO-conotoxin MfVIA. To confirm its usability, we successfully
applied this approach to the synthesis of MrVIB, which also required
only a single HPLC purification step (data not shown). Our approach
resulted in sufficient yields to enable the production of toxin for
detailed subtype selectivity analysis. This new synthetic approach is
expected to facilitate the first structure-activity relationship studies
and pharmacophore mapping of mO-conotoxins.
This research was facilitated by access to the Australian
Proteome Analysis Facility, supported under the Australian
Government's National Collaborative Research Infrastructure
Strategy (NCRIS). We thank the Australian Proteome Research
Facility staff, in particular Bernie McInerney, for N-terminal
sequencing. This research was funded by a National Health and
Medical Research Council (NHMRC) Australian Postdoctoral
Fellowship (IV), NHMRC Research Fellowship (RJL), ARC Australian
Professorial Fellowship (DJA) and NHMRC Program grant (RJL, DJA
and PFA). The FLIPR was supported by an Australian Research
Council LIEF equipment grant.
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I. Vetter et al. / Biochemical Pharmacology 84 (2012) 540–548


text:
Maricopa Master Gardener
The University of Arizona Cooperative Extension in Maricopa County has a vibrant and active Master Gardener program and volunteer force. There are three parts to becoming and staying a Certified Master Gardener: 1) attending the training, which provides novice and experienced gardeners alike with the opportunity to improve their horticultural knowledge and skills, and 2) sharing at least 25 hours of expertise and time with the public through organized volunteer activities, and 3) earning at least 12 hours of personal continuing education.
If you are interested in learning more about the Master Gardener program or applying for the upcoming class, please visit the "How do I become a Master Gardener?" page.
What's Happening
Given the complications of growing gardens in various regions of Arizona, University of Arizona students and staff have developed short films to te
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Faith in Syria's rebel army moves President Obama a step closer to giving arms
President seeks proof of how chemical weapons were used in Syria before taking major decision
New York
Wednesday 01 May 2013
The Obama administration is reported to be considering supplying lethal weapons to the Syrian rebel forces.
The US has thus far held back from supplying military hardware to the rebels for fear that they could end up in the hands of radical groups. But recent contacts with the Syrian opposition have made the White House more confident about certain rebel factions, and preliminary evidence of the use of chemical weapons inside the country have reportedly led Washington to consider changing its policy.
While it remains unclear what kind of equipment the US might provide, President Barack Obama is likely to make a decision on supplying arms ahead of a June meeting with Russia's President Vladimir Putin, a key supporter of the Assad regime, according to a report in The Washington Post.
The suggestion of a shift in US policy came as President Bashar al-Assad visited a Damascus power station, a rare public appearance that came just a day after the Syrian capital was rocked by a bomb. The Associated Press said that at least 14 people were killed in the explosion – the second devastating explosion to hit central Damascus in as many days.
In Washington the growing violence is pressuring the government into considering policy changes that would see it play a more active role in the conflict. Supplying arms would bring the US in line with allies such as Saudi Arabia and Qatar. But the Obama administration remains cautious and continues to pursue diplomatic avenues, with the country's top diplomat, Secretary of State John Kerry, due to travel to Moscow early next week as the Americans look to bring the Russian government on to their side. Mr Kerry is expected to meet both his Russian counterpart Sergei Lavrov and President Putin.
Evidence of chemical weapons use is likely to be a key plank of the case that the US makes to the Russians in order to convince them to withdraw their support for the Syrian government. Although the American officials have been cautious about blaming the Assad regime for using such weapons, it is known to have stockpiled chemical arms. Speaking at the White House earlier this week, President Obama said that "if we end up rushing to judgment without hard, effective evidence, then we can find ourselves in the position where we can't mobilise the international community to support what we do.
"What we now have is evidence that chemical weapons have been used inside of Syria, but we don't know how they were used, when they were used, [or] who used them. We don't have chain of custody that establishes what exactly happened," he said, adding that he had to make sure that he had all the facts before considering "the potential for taking additional action in response."
"If I can establish, in a way that [makes] the United States [and] the international community feel confident, the use of chemical weapons by the Assad regime, then that is a game-changer," the President said, citing the possibility then of "even more devastating attacks on civilians" and the risk of those weapons falling into the wrong hands.
Despite the caveats, a senior US official told The Washington Post that "we're clearly on an upward trajectory. We've moved over to assistance that has a direct military purpose."
Hezbollah leader vows to stop rebel forces taking over Syria
The Hezbollah leader Sayyed Hassan Nasrallah has said that Syria's allies in the region and the world would not allow it to fall "into the hands of Americans, Israel and [Sunni] extremists".
Mr Nasrallah, a staunch ally of President Bashar al-Assad, said in a televised speech from an undisclosed location that Syrian rebels fighting the regime's forces would not be able to topple him using force.
"The battle is long … We tell you [Syrian rebels] that you will not be able to bring this regime down through military means," he said.
Rebels have long accused Hezbollah of fighting alongside Mr Assad's forces. The group has formally denied this, and said it was only fighting to protect Shia villages along the border with Lebanon. However on Tuesday Mr Nasrallah admitted that Hezbollah fighters had been killed in Syria. But reports of the death toll were "exaggerated", he added.
He also left the door open to greater participation in the conflict "if the situation escalates more".
Fighting between Hezbollah-backed Shia militants and Syrian rebels has raged in border villages in recent weeks, threatening to drag Lebanon into the conflict. The border area is a key supply line for the rebels, making it important for Hezbollah to stop them taking over Shia villages there that would bring them close to Hermel, one of the group's strongholds.
From Theme Park to Prison Architect: A clever way of exploring how the penal system is run or an ethical offence?
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Win a trip to Tobago for two
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BBQ Smells
Thinking about anneliese's comment about BBQ smells on the 4th.
And also thinking about how things change in small ways.
Prophane super grills v the old weber.It is my observation that the propane grills do not give the satisfying smell of the grease hitting the coals on the old Weber and permiate the air.
A small change makes a difference.
There was a certain ritual to the old-time Weber, the coals had to be just so and timing was important, but living in a high fire danger area I like the idea that most everyone is using propane grills, much safer.
I had to move my grill to it's summer location, it was too close to the back-door and set off both smoke alarms in the house, so I think there was enough BBQ smell emanating from it.
Personally, I think propane grills shouldn't be compared to the wood or charcoal grills ~ the gas may be easier to use and the food may look the same, but imo, nothing tastes better than real coal-grilled! However, I'm sufficiently odd enough to think that a teeny hibachi does just as good [or better!] than any big grill. -Of course, grilling for two is different from grilling for ten or twenty.
We won't ever have a gas grill. Robert had one before we got married, but he got hooked on my Weber! With a chimney starter and one sheet of newspaper, coals start very easily with very little waiting time. We love the Kingsford Competition briquettes ....super fast and zero chemical smell so common with most briquettes.


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Am I the only one who bawls???/
My DS, who is only five, has discovered the Christmas movie "Home Alone". Am I the only one who bawls at the final scene with the old man has called his son and they come over for Christmas day?? I mean, I know I am a sap who cried at "Benji" but seriously, does anyone else think this reunion is REALLY touching????
Ivette (who, perhaps, has had a little too much Christmas spirit today).
Are you kidding? I was a sniffing mess over the American Girl Samantha Christmas movie yesterday. Has anyone seen that? Dead parents all over the place, plucky little orphan girls, unkind orphanage mistresses, child factory labor...OY. Yes, the problems were solved by the likes of folks named the VanDerGelds LOL, but the girls were plucky and that's what matters.
Although I must say, there was a "Christmas" movie with Gary Sinise I watched while baking just before that and it was *horrible*. I expected better from him, having seen him in Grapes of Wrath, etc. It was pretty easy to keep my cynicism muscle flexed through that whole pitiful thing. I could envision Gary, and Joely Richardson, of all people, both of whom I usually respect, practically calculating how much they would bank per sap-filled line. It must have been excruciating. And imagine the off-screen scenes--oh my goodness in a sick way it would have been fun to have been there during filming. I hope they really like whatever new toys they bought with the $$$.
Omygosh, I even cry every year when Peter comes home and makes Folgers coffee! (How many years has this commercial been running???)
I just saw The Family Stone, and cried lots through it. It's a Christmas movie, and no one told me it was a SAD comedy!
When my kids were infants, I bought them a CD called Peter, Paul and Mommy, where Peter, Paul and Mary sing a bunch of kids songs. The last song on it is Puff the Magic Dragon, and my kids love it and ask me to sing it for them all the time (for the lyrics, surely, because this momma can NOT sing). They are 3 years old now and I still cry every time I sing or hear the song, in the last verse, when they sing "A dragon lives forever but not so little boys; Painted wings and giant rings make way for other toys" and on to the end of the verse. It makes me teary now just thinking about it. It reminds me that time is so fleeting and some day they won't want me to sing to them anymore, let alone the cuddles and kisses.
Boy, am I getting sappy. Once we were on a transatlantic flight with them, at 11 months, and me singing this song was the only thing that would settle them. And there I am, walking the aisles of the plane, singing as quietly as I could into the ear of whomever I was holding (and it was always someone!) crying and trying to get through the verse without them noticing. I wonder what the other passengers were thinking. . .
atsmith, if you haven't already, look at "Zits", the comic strip, for Sunday. OH yeah, you'll cry...
I cried over a movie yesterday. My sister ordered a DVD with the cartoon character Arthur in it that is personalized for my son. So she can never wait to give gifts and brings it over so she can watch Andrew watch himself in Arthur. She sent the company a pic of Andrew and his picture was on the cover with Arthur and then they stick his head on another character so it looks like HE is interacted. So the whole thing was already creeping me out. We make a big show of putting it on and......the superimposed head wad not Andrew - it was Nathan! We laughed so hard we cried. I wonder if Nathan's mom has meet Andrew yet? lol
pamela - lol - Peter!!!! Oh, and I really want to see that movie
flyleft - I'm glad you don't want gary sinise - he's mine. You have jon stewart.
I'm a total sap these days, too. Music does it for me more than movies. I posted on another thread that I can't make it through the John Denver and the Muppets Christmas cd with dry eyes. And atsmith, I have never been able to listen to "Puff" without crying. Nor can I listen to (or, apparently, think about) our old Pete Seeger cd, Children's Concert at Town Hall, because it was DS' favorite and we had little routines worked out to many of the songs on our long, long trip between CT and where I went to law school. I buy that CD now for all of the people I know who are having babies. Sniff.
Oh my...you don't know how much better you have all made me feel. I had to turn off the car radio today because they started playing "Silent Night", sung by the Mormon Tabernacle Choir and I started to cry in the car.
I also can't get through It's a Wonderful Life, A Christmas Carol, Oliver, or Little Lord Fauntelroy without kleenex..just to name a few.
I cry at just about every movie. I drag my dd to any kids film that comes out, the sappier the better, because I love them!
I also tear up at parades when the old veterans march by in uniform. I was at a funeral a few years back and they played Puff the Magic Dragon during the service.
FlyLeft - I just checked it out and I am all teary!
But I also discovered this site that posts all the comics. . .excellent. . .
Here is a link that might be useful: comic archive
atsmith, I think that Zits comic is a keeper for the ages. They got it *right*...DD's baby years seem so accessible to me, but not to her at all, evidently. She's forgotten a lot of it. She's really growing and becoming a person and it was all *just a short while ago*...
Ivette, anything my kid does that is a ritual or right of passage or maybe just the essence of normal can make me tear up. I often wonder why this isn't true for everyone. Childhood is fleeting and any of its moments make me cry simply because the moment will never return and it's all so wonderful.
I'm never without tissue, seriously.
When my daughter was 4 she handed me a favorite picture book and said "We can read this one but no crying!" Even that brought tears!
I'm not usually a weeper about handling my own life except in times of frustrations, for the most part. But put me in front of a movie like old Cary Grant movie The Bishop's Wife where we can see a real life example of how angels walk in and out of our lives, it gives me a quickening that can warm my soul. I've watched it three times already this week. Last night, I was watching it again with my 14 year old son....finishing at 2 AM!!!
Just about anything does it for me - movies, television shows, music. You would think I was the one having the baby on the TLC series, "Baby Story" because I'm crying along with the new parents.
I, too, cry with Christmas songs. Two, especially, do it to me: Kenny Rogers, "A Child Is Born" and Nat King Cole's, "A Christmas Song". I also bawl during the Nativity scene at the "Radio City Christmas Show". When I see the Macy's Thanksgiving Day Parade, I choke up. I really am hopeless.


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Mizuno Mens Premier Pro Piped Pant Closeouts 13 13
Really like these pants. Fit well and wash up nice. April 7, 2014
Love these pants, they wash well which is a mom's dream! I wish I had bought several pairs for my son. March 11, 2014
Bought these for my nephew plays college baseball He loves them and really lie Mizuno overall. With the savings from Baseball Savings I was able to afford 2 pair. Thanks! March 10, 2014
Great price but... The price on these pants was solid, much less than what I would have paid in ANY store. For me, though, I am thicker around the waist so these pants were snug in that area. From the thighs down they fit very well. So if you are slender top to bottom you will like these pants. If you carry a little too much around the waist they will be uncomfortable. March 5, 2014


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Earlier this week, my sister Kathy called me, "Am I correct in thinking that Mom used to send us little kid Valentine's like the ones school children use?"
We are more than a third of the way through the legislative session, and it seems like we've passed very little legislation. Some might say that is a good thing … the less laws we pass, the less trouble we can make for the people of Georgia. In any case, we have 25 days left, with the session scheduled to end on the April 2.
Wisconsin Governor Scott Walker has emerged as a serious contender for the 2016 Republican presidential nomination. In response, the Washington Post researched and published a lengthy article on the "mystery" of why Walker dropped out of college.
Oxfam reports that the richest 1 percent of people in the world own 48 percent of the world's wealth. Many claim that we should be alarmed by income inequality because it hampers upward mobility. Others argue that because income is distributed so unevenly, justice and fairness require income redistribution. Let's look at fairness and justice.
The Education Committee will be the busiest it has been in 22 years. I'll take some of these bills in turn.
Recently, while responding to a question about how to get young people involved in politics, President Obama expressed fears that they see politics as a "sideshow in Washington" and should be taught that "government is not something separate from you - it is you."
In grammar school, Valentine's Day meant wrapping a shoe box with brown craft paper, cutting a slot in the top for cards to drop in and decorating the outside of the box with hearts and cupids. Store-bought Valentines were labeled the night before and carefully taken to school to be given away. When the big day came, it wasn't only if you received Valentines that counted, it was from whom, and if they gave you your card first that mattered.
This week, the House passed the 2015 supplementary budget, but still has to work on the 2016 budget. As I said before, revenues were up by $276 million, and we directed most of that increase to education, as well as Medicaid growth and mandatory compliance with Obamacare. The vote was bipartisan and unanimous.
Let's get off the backs of law enforcement, shall we? Most of us couldn't do their job or wouldn't do it if we had the chance.
President Obama's proposed federal budget for 2016 envisions never-ending growth of federal spending.
This week budget committees in the General Assembly have been working on a balanced budget. Believe it or not a balanced budget is the only Constitutional requirement of Georgia's General Assembly.
When gasoline sold at record prices, Sen. Bernie Sanders, I-Vt., said, "I think it's time to say to these people, 'Stop ripping off the American people.'" When the average price of regular gas was close to $4 a gallon, Sen. Chuck Schumer, D-N.Y., called for Congress to look into breaking up giant oil companies. The claim was that "Wall Street greed (was) fueling high gas prices."
On Monday, President Obama will unveil his proposed federal budget for 2016. Voters should be warned that virtually all the numbers reported in news coverage of the federal budget will be misleading at best.
This week marks the 50th anniversary of the death of Sir Winston Churchill, Britain's prime minister during World War II. Churchill took up painting as a hobby in 1915, after the Gallipoli debacle, where 46,000 allied lives were lost over nine months of the campaign. He went on to paint more than 500 paintings during his lifetime. As wartime prime minister, he took a break from painting, focusing instead on the task at hand - winning the war, no matter the cost.
Last week I attended two informal meetings of citizens and two Chamber of Commerce moderated meetings on the 2050 Plan and a meeting on the Highway 278 Community Improvement District. I came away with an appreciation of just how similar is the end result most of us want for Newton County and yet how distant are the means that we would employ to accomplish that end.
I wrote this some time ago. With so many folks in the same boat as I was in those early days of my adulthood, I thought you might get a chuckle from my young eager mistakes:
This summer has served as a reminder to me about the virtue of virtues, specifically hard work and perseverance. Last winter, our 12-year-old son, Robert, was accepted into an honors performance group as a string bass player, based on his teacher's recommendation and an MP3 submission of his playing. In May, he was sent four pieces of music to master by late June, when he was to perform them in New York.
During last year's budget negotiation meetings, President Barack Obama told House Speaker John Boehner, "We don't have a spending problem." When Boehner responded with "But, Mr. President, we have a very serious spending problem," Obama replied, "I'm getting tired of hearing you say that." In one sense, the president is right. What's being called a spending problem is really a symptom of an unappreciated deep-seated national moral rot. Let's examine it with a few questions.
They had a term for her, but I've forgotten it. It was a name applied to a person who could not say no to a door-to-door salesman. The one I remember from my brief career selling magazines was totally upfront about her intentions. "I'll buy whatever you're selling," she said. I sold her Esquire and two other subscriptions. Salesmen back then had a name for such people. Today, I would call them conservatives. They, too, will buy anything.
"I have gotten bad news and am much the worse for it.
In his weekly column for CNN.com, Julian Zelizer makes a reasonable case that "Distrustful Americans still live in age of Watergate." In his eyes, this helps explain why the president's health care law and other initiatives have encountered so much resistance.
One of the advantages of being an older baby boomer is that your mind can easily wander back to days of your youth and every detail of those experiences can be seen as clearly as if you were still that age.
Based on this fiscal year's eight-and-a-half months of activity so far, the number of unaccompanied alien children from Honduras apprehended by the U.S. Border Patrol will increase 22 times from what it was in 2009.
In a piece titled, "They're Not Telling Us The Truth," I wrote: "Clinton, Bush and Obama, et al, have positioned us in harm's way by providing an accommodating environment for these illegal disease carriers. It is not my contention that everyone who crosses the border illegally is diseased or a disease carrier. I am saying, the fact that we do not know which ones are and which ones are not puts us in peril...I confess that when I walk into an uptown restaurant and see illegals in the kitchen or busing tables, I am concerned." (mychal-massie.com ...
With the July 22 runoff elections fast approaching, I called Junior E. Lee, general manager of the Yarbrough Worldwide Media and Pest Control Company, located in Greater Garfield, Georgia, to get his thoughts on the various races and to see who he thinks will make it to the finals of the November general election and who will be eliminated this round.
FADE IN: Michael Corleone's den.
Happy Fourth of July!
I would like to provide a brief history on the intent and origin of the 2050 Plan that I think is important as we begin the public input sessions on the Base Line Ordinances (BLO).
I wonder how Leroy Jones, the black Chairman of the Essex County Democrat Committee, looks at himself in the mirror. I wonder how he looks at his family and the members of his community. I wonder how he feels as he sits in the pews of the black church he attends, and how he feels while sitting in the black barbershop. More specifically, I wonder how the people of Essex County, New Jersey as a whole, feel about Jones.


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I'm a long-time Brooklyn resident and a big believer in the AirBnB way of traveling. It's the best way to experience a new destination, meet and connect with people on equal footing and discover the more subtle and surprising aspects of a neighborhood and its neighbors.
I make my living as a consultant, working in the nonprofit field. I'm respectful, clean and accommodating.