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Welcome to BioMax Rubber Industries SDN BHD
The Company does not itself engage in any substantive business other than acting as the ultimate parent company for the Group. Currently, the only direct wholly‐owned subsidiary of the Company is Biomax Rubber Industries Sdn Bhd.
Today, Biomax Rubber Industries` products are exported to more than 20 countries worldwide to countries such as United State of America, United Kingdom, Japan, Vietnam, Europe, China, Middle East, India and Australia with its strong reputation of good product quality, timely delivery and excellent customer service. There are multinational companies in developed countries, who have made Biomax Rubber Industries as their preferred OEM manufacturer.
Currently, Biomax Malaysia employs seventy eight (78) full time employees at its factory in Malaysia, and produces approximately 384 million pieces per annum of ME gloves per annum and the factory has four (4) operating production lines. The production lines produce Latex ME gloves ‐ powdered (smooth, white) in all glove sizes.


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Tired of coloring your own rolled fondant? Well color no more! FondX is now available pre-colored with the same great taste, consistency, and workability as FondX Virgin White. With its vibrant color and yummy vanilla (marshmallow middle of an oreo cookie) taste, FondX Colors are a must have for any cake designer.
FondX is the best premium rolled fondant to use for professionals and beginners alike. Its very easy to knead, rolls out to an 1/8and it is the most forgiving fondant to use.
It can also easily be firmed up and made into a yummy gumpaste with use of CMC or Tylose.
1lb can cover up to an 8" cake.
To learn more about how to use Fondx fondant HERE
Fondx red fondant is also available in 10 lbs pails, click here.
Ingredients: Sugar, Corn Syrup, Water, Interesterified Soybean Oil, Food Color (Water, Sugar, Red 40, Red 3, Yellow 6, Modified Corn Starch, Vegetable Gum, Citric Acid, Sodium Benzoate, Potassium Sorbate), Gum Blend (Sodium Alginate, Cellulose Gum, Xanthan Gum, Propylene Glycol Alginate, Guar Gum), Natural and Artificial Flavors, Potassium Sorbate, Cellulose Gum, Glycerin.
Gluten Free
Nut (including Tree Nut) Free
Kosher Pareve - contains NO dairy
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Aluminium Decorating Turntable by Ateco
Edgers Sharp Top Smoother
Aluminum Rolling Pin 19" Long - 3.5" Diameter
Confectioners Choice White Gumpaste 4 lbs
Gold 0.045" Round Scallop Thin Board - 8"
Strawberry Emulsion - 4 oz by Lorann Oils
Cinnamon Spice Bakery Emulsion - 4 oz by Lorann Oils
Velvet Hi Ratio Cake Flour - 20kg by Robin Hood
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Rebecca Lawson
Hi everyone, I am Rebecca Lawson and am the new licensed massage therapist working at Sharon's and Aspiring Health Chiropractic. I grew up in Mt. Morris and Oregon; when I finished my time in the military I chose to come back to this area because of the friendliness and comfort of a small town. I have a five year old daughter and this is a great place for her to grow up. I graduated from Rockford Career College with an associate's degree in massage therapy and was blessed to discover that Aspiring Health was looking for a massage therapist. What a great place to work, with good people to meet. I have met some of you already and am looking forward to meeting everyone else!


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The Three Best Exercises For Building Your Abs
"How would I best function my abs?" is a standout amongst the most made inquiries I get as a wellness coach.
Since I get this question a LOT... in this article I'm going to impart to you my three undisputed top choice (since they work) and straightforward floor practices for working your abs.
1) Full Vertical Crunch
This one is incredible on the grounds that it enlists all your stomach muscle muscles by getting your abdominal area and your lower body into the activity. Get on your back and bring your legs up so they're indicating the roof. Review CRAZY bulk Put your hands behind your head (or simply hold your ears) and raise your shoulder bones off the floor - this will get your abs. While you're doing this, extend your legs upward as though you're attempting to get your heels on the roof (point with your heels not your toes).
The thought here is to make your body into a U shape. To end it: lower again into the beginning position.
You can go at your own particular level, yet go for 10-15 reps with this one.
2) "Long Arm Crunch"
This simple to perform activity will concentrate on your upper abs. Begin by Lying on your back (on a mat in the event that you have one) with your arms extended in a straight line behind your head. In the event that your arms are touching or close to your ears, you're doing it right! Flex your abs to lift your shoulder bones off the floor. The perfect reach is 10-15 reps for each set.
3) Vertical Leg Crunch
Here's a decent one for your obliques. Begin by Lying face up on the floor, legs straight up and cross your knees. Lift your shoulder bones off the floor and get your abs - in this activity, envision that you're attempting to touch your mid-section to your feet. Do a reversal to beginning position and rehash 10-15 times for each set.


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Taslima Nasreen, a writer from Bangladesh who stays in controversies, who never had "high hopes with Hindi movies" has been left "speechless" after watching the internationally-acclaimed Indian Hindi movie 'Masaan'.
The movie Masaan has been starred by the actress Richa Chadha and is basically a women-oriented film.
Taslmia Nasreen said, "I did not have high hopes about Hindi movies as most of them were musical melodrama. But 'Masaan' has made me speechless. I salute Neeraj Ghaywan".Watch movie online A Cure for Wellness (2017)
She tweeted this compliment about the movie.
Neeraj Ghaywan is the director of film Masaan and it marked the debut for him in the field of directing the movies.
The story of the movie revolves around four lives which intersect along the Ganga Ghats in Varansasi. The plot continues as a low-caste boy falls hopelessly in love; a hapless father with fading morality; a spirited child yearning for a family; a daughter ridden with guilt of sexual encounter ending in a tragedy, long to escape the moral constructs of a small town.
The 'overwhelmed debut director replied to the compliment by saying, "Overwhelmed! Thank you so much!".
The movie has already won two awards at the 68th Cannes International Film Festival. It bagged the 'Promising Future Prize' and the 'International Federation of Film Critics (Fipresci) Award' at the prestigious fest.
The other lead stars in the movie along with Richa Chadha are Sanjay Mishra, Shweta Tripathi and Vicky Kaushal.
The film has been jointly produced by Drishyam Films, Phantom Films, Macassar Productions and Sikhya Entertainment.
The film Masaan hit the Indian screens of July 24th and got positive reviews from critics as well as audience.
Bollywood star, Ayushmann Khurrana also expressed his delight after watching the film Masaan and said that he is really happy as more women-oriented movies are coming.
He also appreciated the work of Richa Chadha in the movie.


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Love, relationship, courting…… all these phrases are fairly simple as well difficult for some individuals. If you wish to make everything smooth in your love life, then always make your partner blissful by spending time with him/her, giving items, and yet another factor could be attainable. There is not any point powering a tv if you cannot hear the television over the noise of the distant generator. El funcionamiento de la aplicación es muy simple. Tan solo tendremos que escoger un tipo de marco para nuestras fotografías, teniendo en cuenta el número de imágenes que queremos meter en su inside. Podremos coger un marco para dos fotos… uno para siete, depende de nosotros.
Spite houses generally are born out of petty feuds and personal grievances, or self centred ambitions to out-do one's neighbours. However Aaron Jackson's is a tremendous instance of a more wholesome intent. Yes, it was painted to 'spite' the Westboro Church. Sure, it's intentionally provocative. But it surely's a delight to suppose that every morning when the members of that church wake up, they get to take a look at a home painted within the rainbow colours which are pure anathema to them!
He appears okay to me ProCw. I really we not argue with him if he thinks I'm cute :). Template-Hiasi foto kamu dengan format majalah lucu dan keren dan template untuk menjadi orang yang populer di sampul majalah. As we move into the colder months here in Australia, I am about to start making newspaper bricks once more. I've a pile of newspapers ready and waiting. Making a paper brick is a lot simpler than chopping wood.
Tip: Contemplate choosing up a few and saving them for holiday items; they make terrific stocking stuffers. Allows to access precise location from location sources equivalent to GPS, cell towers and Wi-Fi. Nonetheless, the Venus actually is a thing of magnificence. UtechSmart has saved the fee down in comparison with different comparable mice on the market at around $forty while giving it similar to higher high quality than those choices.
All newly issued U.S. passports and plenty of credit cards include an RFID chip (Radio Frequency Identification Data) that shops private data about the proprietor as a security measure. Criminals typically use RFID readers to steal necessary private information from RFID chips embedded in passports and credit cards to entry the proprietor' accounts, so it is important for travelers — particularly business vacationers who carry company as well as private credit cards — to guard themselves by carrying their passports and bank cards in wallets and passport circumstances that block RFID readers successfully.


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Conference Tools for 2018 TMS Annual Meeting & Exhibition
Register as a New User
Submit An Abstract
Propose A Symposium
Presenter/Author Tools
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About this Symposium
2018 TMS Annual Meeting & Exhibition
Perfluorocarbon Generation and Emissions from Industrial Processes
TMS Light Metals Division
TMS: Aluminum Committee
Pascal Lavoie
, Alcoa
David S. Wong
, University of Auckland
Pernelle Nunez
, International Aluminium Institute
Perfluorinated carbon compounds (PFCs) are potent greenhouse gases that are generated from multiple industrial processes, including the production of aluminum, rare-earth metals, semi-conductors and fluorinated gases.
This symposium aims at:
1. Quantifying emissions including measurement techniques and accounting within and between industries
2. Understanding generation mechanisms
3. Providing emission reduction and mitigation techniques
Abstracts Due
Proceedings Plan
Planned: Light Metals
An Estimation of PFC Emission by Rare Earth Electrolysis
Attributing PFC Emissions to Different Industries: How Bottom-up Trends Can Complicate Top-down Analyses
Challenges in Estimating Global CF4 and C2F6 Emissions
Conditions and Mechanisms of Gas Emissions from Didymium Electrolysis and Its Process Control
Evaluation of Time Consistency when Quantifying Emissions of Perfluorocarbons Resulting from Low Voltage Anode Effects
Fluorinated Gas Production: Underestimated Source of PFCs?
Low Voltage PFC Measurements and Potential Alternative to Reduce Them at Alcoa Smelters
Methodologies to Measure Greenhouse Gas (GHG) Emissions from Industrial Processes and Determine the GHG Emission Factors
New Algorithm for Calculating CF4 Emissions from High Voltage Anode Effects
New Approach for Quantification of Perfluorocarbons Resulting from High Voltage Anode Effects
Perfluorocarbon Formation during Rare Earth Electrolysis
PFC Emission Reduction in the Semiconductor Industry
PFC Evolution Characteristics during Aluminium and Rare Earth Electrolysis
PFCs from the Chinese Aluminium Sector – Challenges in Emissions Accounting and Further Characteristics
Updated Factors for Calculating PFC Emissions from Primary Aluminum Production
Validation of Online Monitoring of PFC by QCL with FTIR Spectroscopy
Questions about ProgramMaster? Contact


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Thursday, January 19, 2012
What's going to happen along Granville Street?
Over lunch Faaiza and I were discussing various new projects around the region and the subject of Granville Street came up. Not the downtown portion, but the area south of 41st where a parade of 'For Sale' signs has recently sprouted up. "What do you think should happen there?" she asked.
Rather than spend our remaining minutes focussing on this one topic, I promised to post my thoughts on my blog. So here they are!
For decades I have argued that Vancouver's arterial roads should be rezoned and redeveloped with higher density forms of housing. As some readers know, I have twice put my money where my mouth is, resulting in the redevelopment of Oak Street between West 42 and West 43 and West 41st between Balaclava and Carnarvon Streets. It has been gratifying to see a couple of new projects follow suit, although I can't say the same for the redevelopment on Oak Street immediately south of Oak Gardens, which to my mind is an awful development that should never have been approved by the City. My biggest concern with this project is its relationship to the street and the almost complete absence of vegetation between the buildings and the curb. (Why no oak trees along Oak Street?)This brings me to Granville Street. I cannot forget the visitor who once commented to me how exceptional he thought it was driving into Vancouver along Granville Street. What stuck him was the lush green lawns and hedges lining the street, especially in Februrary when the rest of Canada was white. He noted that the vistas along Granville were so very different from what what one usually found driving from the airport into the downtown of most other major cities.Recently there have been a couple of redevelopments along Granville. Granville Mews south of West 49th is currently under construction and replaces three single family homes on large lots with two rows of townhouses. Designed by Formwerks, the architectural firm I am working with in West Vancouver, the units have a traditional look and would not be out of place in nearby Shaughnessey. Most people are not even aware of this development that is offering a housing choice generally not found in the area. And there seems to be a strong demand with prices starting above $1million and good sales activity.
Nearby, Wall Financial recently received Council approval for what will be a very significant redevelopment of Shannon Mews. The Arthur Erickson townhouses will be replaced by a variety of apartment buildings, some of which are well above the three storey height of surrounding buildings. While I have seen the plans, I will wait to see how this will look. To my mind, the key will be to maintain the trees along the street, and to add new vegetation along the perimeter of the development.
Last year, at 49th and Granville, NSDA Architects received approval for a three or four storey seniors complex. I have worked with this firm on many occasions and will watch with interest to see how their development will transform the property owned by Ben Wosk for many decades. My hope is that the greenery at this major intersection will either be maintained or replaced.
At 16th and Granville another controversial project is currently under construction. Also designed by Formwerks, I think we have to wait until it's finished before casting final judgement. However, there is no doubt that much of the greenery that used to be at this important intersection has disappeared and we can only hope that new trees will be planted so that the building edge is not as severe as it appears today.
And this brings me to how I feel about the rest of the street. I still believe that many of the single family properties along Granville Street south of Shaughnessey should be redeveloped over time. But rather than continue with ad-hoc spot rezonings, I would like to see the city put in place a comprehensive plan and design guidelines that provide for variety along the street including semi-detached/duplex units, street row housing, and some low-rise apartments in appropriate locations.
However, whatever the building form, I think it is important that the sense of greenery be maintained. This might be accomplished through a greater landscaped setback than generally required, and specifically guidelines mandating a double row of trees, lawns and extensive planted areas, etc.
In this way we might increase the density along this well serviced transit arterial, while maintaining the 'green aesthetic' that both visitors and residents have come to appreciate along the street.
That Faaiza, is what I'm currently thinking. Now I'd like to hear from you and others....knowing that it will likely be a while before the City gets around to rezoning this important city street.
7 comments:
- jesse said...
Interesting, though redeveloping these thoroughfares as you suggest will not add significant supply relative to the city at large. The overwhelming majority of dwellings are off major streets, obviously.
- January 20, 2012 at 2:45 AM
The development on Oak that you dislike so much is reminiscent of older cities where row houses open directly onto the street and any vegetation exists out back. I only know the development from driving by, but I suspect there's a second row of dwellings behind the first leaving no room whatsoever for green. While that provides maximum density it hardens the environment both literally and figuratively. People need vegetation for both physical and mental health and a rainy city needs porous ground to absorb moisture.
I agree with you that the city needs to open up these new developments a bit by requiring permeable surfaces, preferably filled with trees/shrubs/flowers/grass.
- January 20, 2012 at 11:18 AM
Ever since you spoke of your frustration with "spot rezoning" it is gaining relevance for me. I was reading France Bula's blog about the problem with lights on BC Place Stadium being a problem for nearby condo dwellers, and also the art gallery is becoming a question around a cultural precinct downtown. It seems that larger scale planning than what is being practiced today has potential for better planning solutions, such as better higher density solutions that you advocate for streets like Granville and Oak.
- January 20, 2012 at 9:19 PM
- Michael Geller said...
Thanks for your comments...David, you are right about the building form...however, look at the Carrington Mews development further to the south...it has two rows of units AND some greenery!
- January 21, 2012 at 10:41 AM
- Dave said...
Interesting to see an article about my 'hood. Couldn't agree more about the "ugly" development on Oak. It manages to avoid greenery completely. Similar developments b/w 45/46 on Oak, b/w 45/49 on Ash do the two-row townhouse much better. I'm looking forward to see what happens on Granville where those for sale signs you spoke of were. Seems to be two big chunks on the market right now.
I'd like to see some street-front retail activity near the major intersections (41, 49, 57) since Granville S of 41st is basically a dead zone until Marpole. Pedestrian activity would help slow traffic speeds to a more urban pace, since it functions as a bit of a highway right now with few traffic lights to get in the way.
- January 21, 2012 at 11:04 AM
It looks like there are some short trees/bushes in front of those rowhouses that have yet to fill out. It is likely that the underghound parkade prevents planting of larger trees. Plantings don't generally lend themselves to instant gratification (just look at the new saplings on Granville St. downtown).
One question - why are there no street trees in front of those rowhouses? Perhpas a letter to the City may fix that.
- January 26, 2012 at 10:00 PM
Talk about an intersection ripe for development, try 41st at Knight. I hope the City planners put into practice lessons learned(?) from Granville St. What seems to be lacking is an overall, big picture plan for our City.
- March 14, 2014 at 7:20 AM


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Recognizing the best petrol hedge trimmers is no more a hustle. Just read our petrol hedge trimmer reviews and you will learn how to choose it. A petrol hedge trimmer and a petrol hedge cutter are relatively simple grass trimming, shrub pruning, and hedge trimming machines. The devices perform a number of useful and common yard work chores.
The size of the best petrol hedge trimmer or petrol hedge cutter varies greatly depending on the intended use of the device. Also a key factor is the size of the hedge or yard the device will work. Similarly the volume of the petrol tank varies between devices for home gardens and those for large scale trimming operation like for a city or business.
Best Petrol Hedge Trimmers Key Points Review
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Engine housing.
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Petrol long reach hedge trimmers are combination of petrol and long reach cutters. No doubt, they are very handy. Petrol long reach hedge trimmer is the ideal machine for big jobs and tall hedges. These cutters are powerful and long enough to do even with the toughest jobs. Therefore the petrol long reach hedge trimmers save money and time. Our professionals advice to think wise and if needed take the dimensions of your hedges. It they are tall enough, do not wonder, buying a petrol long reach hedge trimmer is a good move.
Best Petrol Hedge Trimmers Pros Review:
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The Best Petrol Hedge Trimmer Reviews
The best petrol hedge trimmer should be defined first by the applicability of the trimmer to the job. Therefor larger models with longer shafts are much more applicable to trimming large areas.
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Petrol hedge trimmers could require assembly or can come as an assembled unit. The best petrol hedge trimmer for you depends on your level of mechanical ability and the tools that you have.
Finally, this is by no means all that there is to know about the best petrol hedge trimmer. Our experts at hedge trimmer reviews advice not to buy a cheap petrol hedge trimmers. It means low class and low quality materials. Cheap petrol hedge trimmers will become expensive during the time.
Below you can read reviews for the best petrol hedge trimmers on the market.


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|This article is about 'the magazine story'. You may be looking for 'the episode or the DVD'.
Toby's Triumph is a magazine story illustrated using photographs from the television series.
The Fat Controller comes to the shed with news that Allicia Botti is arriving on the Island. He then gives Toby the job of collecting her from the airport. Thomas is happy for his friend, but Toby is worried that he may make a mistake. Before Toby leaves the station, the refreshment lady prepares a special tea in Henrietta. Toby is worrying so much that he sets off too fast. He sends the lovely tea flying into the air, ignores some children and takes the wrong track. He finds himself on an old, disused line where he gets covered in leaves and branches. Soon, he finds himself in a muddy pond; he is in a right mess! At the airport, Allicia Botti refuses to get in such a filthy train, so Sir Topham Hatt sends Toby straight to the wash down. Soon, Allicia Botti is satisfied with Toby's cleanliness and is delivered to the picnic fields. The Fat Controller is very pleased and Toby decides that next time he has to take an important passenger, he won't worry so much.
- Toby
- Sir Topham Hatt
- Allicia Botti (speaks in speech bubble only)
- The Refreshment Lady (speaks in speech bubble only)
- Thomas (does not speak)
- Henrietta (does not speak)
- Percy (cameo)
- Lady Hatt (cameo)
- The Mayor of Sodor (cameo)
- This story is based on the eleventh season episode of the same name.


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What is your favorite team to use when you are playing NCAA Football?
"Florida or Alabama. I use Alabama because they run the football and they are one of the best teams on the game."
Area team with the worst looking uniforms?
"Bell. I don't like purple."
What football player did you pretend to be when you were younger?
"Fred Taylor. I was always a big Jacksonville fan and he played at Florida, so I liked him because of that. He was a running back, and I wanted to be a running back when I was little."
What kind of rookie season will Tim Tebow have in the NFL?
"I don't think he is going to play too much, honestly. I think he'll sit at second string for a year or two and then I think he'll probably get moved into the starting job."
Least favorite part of practice?
"The conditioning, definitely, of course. I hate gassers. We run down and back the width of the field."
- Cliff Olsen
Q and A with Trenton RB/LB Corbyn Pugh
What is your favorite team to use when you are playing NCAA Football?


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Book Buyback
Through the NPC Online Bookstore, students can sell back their textbooks 24 hours a day, 7 days a week. Click the "Sell Your Books" button; enter the ISBN number from the back of the textbook and an instant quote for the textbook will appear.
- Buyback shipping is FREE! Print a prepaid shipping label, send your textbooks to eCampus and receive a check in the mail! It's that easy.
- Students will also be able to receive cash for books during on-site buyback days, which will be held at each of the 4 campuses, and Whiteriver at the end of the semester.
- Students who sell back books either online and on-site will receive premium buyback prices for any book being reused by NPC the next semester.
Bring back your Rentals textbooks, too!
Textbook Buyback Schedule –
sell your books back for cash at these NPC locations!
Tuesday, May 7, 2019
- Winslow • 9 a.m. to Noon
- Holbrook • 2 to 5 p.m.
Wednesday, May 8, 2019
- Show Low • 9 a.m. to Noon
- Whiteriver • 2 to 5 p.m.
Thursday, May 9, 2019
- Snowflake • 9 a.m. to Noon
- Show Low • 2 to 5 p.m.


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[accordion sync="true"]
[accordion_item title="Linkedin" open="true"]
Linkedin is the b2b social network. you can use it for engaging with customers and prospects, finding new ones or creating a network of
[accordion_item title="Facebook"]
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[…] alternative ways to drive traffic. Blogging, social media, content marketing, inbound marketing all point to the future of marketing. If you don't […]


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I've been hosting the Bandolier evidence-based medicine site for Oxford pain researcher Andrew Moore. I'd previously collaborated with him on analysing drug-trial data, but this was the first time we'd worked together on a website. One thing we thought we might want to add was a WordPress blog; and since Bandolier has a very distinctive style, I needed to make a WordPress theme that would give the blog the same style. This sort of thing is quite common in computing: you've got two completely different pieces of software, and you have to make one match the other, even though they're written by different people with different tools. Luckily, I found Tania Rascia's "Developing a WordPress Theme from Scratch".
To appreciate the style I needed to emulate, look at the screenshot below of an existing Bandolier page. The site is about evidence-based medicine and pain, so the pages explain such things as the evidence for ways of easing back pain, safety in acupuncture, and the placebo effect. The page shown here is about clinical trials.
Before I could style a blog, I had to set one up. This meant installing WordPress, which I did as described at WordPress Codex's "Installing WordPress". I put it into a subdirectory called
blog under the main Bandolier site.
I then joined Tania's page at the section headed "Creating your custom theme", and made a subdirectory called
blog/wp-content/themes/bandolier. Themes always live in
.../wp-content/themes. So, for example, when I was styling AudioTheme's Promenade theme for a customer, I had a subdirectory called
promenade holding the original theme, and another called
promenade-child which contained my adaptations.
The first file I had to create in
blog/wp-content/themes/bandolier was a stylesheet,
style.css. As the WordPress Codex explains on its "Theme Development" page, this provides not only the kind of visual styling information that you get in any CSS stylesheet, but also information about the theme. This is essential for WordPress to use the theme properly, and goes in the comments at the top:
/* Theme Name: Bandolier Author: Jocelyn Ireson-Paine Description: Bandolier site templates converted to WordPress Version: 0.0.1 */
Actually, for these experiments, this was the only stuff in the stylesheet. I let the blog's styling depend on default styles, e.g. for paragraph fonts, set up in other stylesheets, the existing Bandolier ones.
The next thing was to start styling, and decide what a list of blog posts should look like. On my site, for example, that's
http://www.j-paine.org/blog, and opening it in my browser gives me a complete list of posts, each with its title, date, and text. Where could I fit these on the Bandolier site? Well, there's an obvious content area — starting at the heading "Clinical trial" in the above screenshot, and ending at a horizontal line. So I took an existing page, and played around with the page source and the Firefox Inspector, looking for the relevant HTML elements:


text:
In our recent study, we have demonstrated that short carbon chain n-alcohols (up to octanol) stimulated while long carbon chain n-alcohols inhibited the conductance of connexin (Cx) 36 (Cx36) gap junction (GJ) channels. In contrast, GJ channels composed of other types of Cxs all were inhibited by n-alcohols independent of their carbon chain length. To identify the putative structural domains of Cx36, responsible for the dual effect of n-alcohols, we performed structural modeling of Cx36 protein docking with hexanol and isoflurane that stimulated as well as nonanol and carbenoxolone that inhibited the conductance of Cx36 GJs and revealed their multiple common docking sites and a single pocket accessible only to hexanol and isoflurane. The pocket is located in the vicinity of three unique cysteine residues, namely C264 in the fourth, and C92 and C87 in the second transmembrane domain of the neighboring Cx36 subunits. To examine the hypothesis that disulphide bonding might be involved in the stimulatory effect of hexanol and isoflurane, we generated cysteine substitutions in Cx36 and demonstrated by a dual whole-cell patch-clamp technique that in HeLa (human cervix carcinoma cell line) and N2A (mouse neuroblastoma cell line) cells these mutations reversed the stimulatory effect of hexanol and isoflurane to inhibitory one, typical of other Cxs that lack respective cysteines and a specific docking pocket for these compounds. Our findings suggest that the stimulatory effect of hexanol and isoflurane on Cx36 GJ conductance could be achieved by re-shuffling of the inter-subunit disulphide bond between C264 and C92 to the intra-subunit one between C264 and C87.
Gap junction (GJ) channels (Figure 1A,B) are composed of two apposed hemichannels in the contiguous cells and provide a direct pathway for electrical and metabolic intercellular communication. Each hemichannel consists of six connexin (Cx) subunits. The family of Cx genes comprises 21 members in the human genome. Various tissues express different Cxs that form GJs with unique single channel properties and responses to transjunctional voltage (Vj), intracellular Ca2+, pHi, phosphorylation, and chemical reagents (reviewed in references [7,35,37,45]). Recently we have demonstrated that Cx36 distinguishes from other Cxs by its opposite response to widely used GJ uncouplers, such as n-alcohols or general anesthetics. For instance, while these agents inhibit Cx45 GJs [40], they strongly potentiate Cx36 GJs [28]. Importantly, the effect of n-alcohols is carbon chain length dependent. Short carbon chain n-alcohols (up to octanol) stimulate, and long carbon chain n-alcohols inhibit the conductance of Cx36 GJs [28]. We hypothesized that stimulatory and inhibitory effects of n-alcohols may originate from their interaction with different domains of Cx36 protein as, for instance it has been shown in nicotinic acetylcholine receptor channels [49]. Structural modeling of Cx36 protein docking with hexanol that stimulates and nonanol that inhibits the conductance of Cx36 GJs revealed multiple common docking sites for hexanol and nonanol and a single pocket accessible only to hexanol. The pocket is located in the vicinity of two unique cysteine residues, C264 in the fourth and C92 in the second transmembrane domain (transmembrane domain (TM) TM 4 and TM2, respectively) of the neighboring Cx36 subunits. The presumptive proximity of C92 and C264 suggested that putative disulphide bonding between Cx36 subunits might modulate GJ conductance as it has been demonstrated, for instance in acid-sensing ion channels [50]. In addition, we identified C87 in TM2 as a possible candidate for disulphide bond re-shuffling. Cysteines in the extracellular loops that are involved in hemichannel docking and form intra-connexin disulphide bonds are not accessible to external reducing agents [10]. The same may apply to the cysteines residing in the TM domains of Cx36 because the pilot experiments in which we tested the effect of reducing agent DTT on Cx36 GJ conductance did not provide an unambiguous answer. Therefore, we generated single and double cysteine substitution(s) in Cx36 by replacing C87 and C264 with serine and C92 with valine to minimize the local volume changes. C92V mutation was chosen following the analysis of the Cx36 structure. C92 faces the membrane; therefore, it was reasonable to substitute it by valine, a hydrophobic amino acid, because serine could cause larger changes in local conformation. We demonstrate by dual whole-cell patch-clamp experiments that these mutations reversed the stimulatory effect of hexanol and isoflurane to inhibitory one, typical to other Cxs that lack respective cysteines or/and a specific docking pocket for these compounds.
Cx36 contains multiple docking sites for both hexanol and nonanol and a unique docking pocket for hexanol
Materials and methods
Cell lines and culture conditions
Experiments were performed on HeLa (human cervix carcinoma cell line, ATCC CCL-2) and N2A (mouse neuroblastoma cell line, ATCC CCL-131) cells transfected with Cx36 tagged with the GFP (Cx36-EGFP). The HeLa cell line and the pIRESpuro2_mCx36-EGFP vector were obtained from the laboratory of Dr F. Bukauskas (Albert Einstein College of Medicine, New York, U.S.A.). N2A (purchased from Sigma–Aldrich, Germany) or HeLa cells were transiently transfected with Cx36-EGFP or its mutants using Lipofectamine 2000 (Invitrogen, U.S.A.) and following the manufacturer's transfection protocol. Cells were grown in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco Laboratories). Typically, the experiments were performed on the second day after cell passage.
Construction of Cx36-EGFP mutants
In order to generate point mutants, we used a site-directed mutagenesis kit (QuickChange Lighting Site-Directed Mutagenesis Kit, Agilent Technologies, Santa Clara, CA, U.S.A.). The kit utilizes the three-step procedure protocol comprising the following: (i) PCR with mutagenic primers, (ii) digestion of template following on PCR with DpnI restriction endonuclease, and (iii) the transformation of XL10-Gold ultracompetent cells. Mutagenic primers containing desired mutation(s) are equal in length and anneal to the same sequence on opposite DNA strands. In Table 1, underlined nucleotides of primers indicate introduced codons of amino acids.
|Number .
|Primer names .
|Forward and reverse primers .
|Length, bp .
|Number .
|Primer names .
|Forward and reverse primers .
|Length, bp .
For the introduction of two point mutations in the same vector of DNA we performed double-stage PCR using templates from the previous mutagenesis, i.e. the single point mutant template was utilized for construction of double point mutant. Agarose gels were captured using the Herolab gel documentation system, Biometra TI 1, UV transilluminator, and BioDoc Analyze 2.0 software. Clones were tested using restriction endonucleases and afterward potentially positive clones were sequenced for the final confirmation of introduced mutation(s) (Sequencing Centre of the Institute of Biotechnology, Vilnius University, Lithuania).
Structural modeling
Cxs (Cx30.2, Cx36, Cx43, Cx45, and Cx47) homology modeling was carried out with MODELLER (version 9.10) [47], using a Cx26 crystal structure [26] as a template. Computational modeling of molecular docking was performed using the Vdock program [20]. Proteins and small molecules were modeled using CHARMM22 (Chemistry at Harvard Macromolecular Mechanics, version 22) [25] and CHARMm with Momany–Rone charges [30], respectively. The ligand parameters were generated using Discovery Studio Visualizer (version 3.5, Accelrys Software Inc., San Diego, U.S.A.). Distance-dependent dielectric approximation was employed to model solvent effects [23]. Hexanol, nonanol, R-isoflurane, S-isoflurane, and carbenoxolone were selected as docking ligands. All molecular structures were created as the Brookhaven Protein Data Bank 'pdb' or MDL Molfile 'mol' molecular data format files and converted into Charmm 'crd' and molecular topology 'top' files using the Discovery Studio 3.5 Visualizer, UCSF Chimera (version 1.8.1, University of California, San Francisco, U.S.A.) [36] and VMD (Visual Molecular Dynamics, version 1.9.2) [17] programs and custom scripts for docking with Vdock graphical user interface. Most docking settings were left as default as provided in the Vdock program except 300 docking conformations were saved instead of usual 20, and the genetic algorithm for the conformational search was disabled. Such settings enabled more thorough and unbiased exploration of multiple binding pockets on the surface of the receptor, especially if the lowest energy minima were narrow in the conformational space [21]. The docking box was set up in such way that docking explored the entire surface of two neighboring Cx36 subunits. All modeling results were visualized with the UCSF Chimera system or PyMOL (Molecular Graphics System, version 1.8, Schrödinger, LLC, U.S.A.).
All molecular dynamics (MD) calculations were performed with the GROMACS (Groningen Machine for Chemical Simulations, version 2016.2) [2]. Time steps of 2-fs duration were used for all MD simulations. PLUMED (version 2.3.0) [44] was used for steered MD (SMD). We used the Amber99 SB-ILDN [24] force field for the protein and the SPCE [5] for all water molecules of the system. The electroneutrality of the system was achieved by adding Cl− ions. Prior to MD simulations, the steepest descent minimization with up to 50000 steps was being carried out until maximum force declined below 1 MJ.mol–1.nm–1. Then, two 100-ps equilibrations (isothermal–isochoric and isothermal–isobaric) with constrained with protein heavy atoms and 1-ns SMD production run were carried out with a force constant between C92 and C264 sulphur atoms set to 1 Mcal.mol–1.nm–2 for an initial approach into contact range. During the production of SMD runs, all position restraints were released. An additional 100-ps SMD run with a force constant set to 1 Gcal.mol−1.nm−2 was used to overcome steric repulsion between the sulphur atoms and to model a covalent bond. The ligands were treated with the general AMBER force field (GAFF) [42] for organic molecules using the ACPYPE program (version 01/17/2017) that is based on the Antechamber software [41,46]. RMSD was calculated with GROMACS between starting point of SMD run and all other points after protein superposition.
Electrophysiological measurements
For electrophysiological recordings, the cells grown on to glass coverslips were transferred to the experimental chamber with a constant flow through perfusion system mounted on the stage of the inverted microscope Olympus IX81 equipped with the Orca-R2 cooled digital camera, fluorescence excitation system MT10 (Olympus Life Science Europa Gmbh, Hamburg, Germany), and fluorescence imaging system XCELLENCE. Appropriate excitation and emission filters (Chroma Technology, Brattleboro, VT, U.S.A.) were used to image Cx36-EGFP GJs. Junctional conductance between cells was measured in selected cell pairs using the dual whole-cell patch-clamp technique (MultiClamp 700B with digitizer Digidata 1440A; Molecular Devices, Inc., U.S.A.). Cell-1 and cell-2 of a cell pair were voltage clamped independently at the same holding potential. By applying voltage ramp (ΔVj) from –10 to 10 mV in cell-1 and keeping the other constant, junctional current ΔIj (GJ current) was measured as the change in current in cell-2. Thus, gap junction conductance (gj) was obtained from the ratio −ΔIj/ΔVj, and the negative sign indicates that junctional current measured in cell-2 is oppositely oriented to the one measured in cell-1. The experiments were performed at room temperature in a modified external Krebs–Ringer solution: 140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 2 mM pyruvate, 5 mM HEPES (pH 7.4). Patch pipettes were pulled from borosilicate glass capillary tubes with filaments and filled with internal solution: 130 mM KCl, 10 mM Na aspartate, 2 mM MgATP, 1 mM MgCl2, 0.2 mM CaCl2, 2 mM EGTA, 5 mM HEPES (pH =7.3). To minimize the effect of series resistance on the measurements of gj, we maintained pipette resistances below 3 MOhms.
Statistical analysis
Data are expressed as mean of at least four independent experiments ± S.E.M. The unpaired Student's t test was used for quantitative evaluation. Differences were considered statistically significant at P<0.05.
Docking sites of hexanol and nonanol on the Cx36 protein
To identify the putative structural domains of Cx36, responsible for a dual effect of n-alcohols, we performed molecular docking of hexanol that stimulated and nonanol that inhibited the conductance of Cx36 GJ channels. The docking revealed multiple common docking sites for hexanol and nonanol, and a single pocket accessible only to hexanol (Figure 1C,c).
We assessed the stability of hexanol binding to this pocket by MD simulations. As can be seen in Supplementary Figure S1 (additional file 1), stabilization of conformation was achieved during the first 5 ns of simulation, and later on the complex structure was stable and no dissociation occurred during the entire 100-ns simulation. Such validation also was used by other authors [43]. The pocket is located in the vicinity of two cysteine residues, C264 in the TM4 and C92 in the TM2, of the neighboring Cx36 subunits, respectively. Notably, hexanol molecules but not nonanols also docked into an analogous pocket of Cx47 (Figure 2). However, the amino acid sequence in TM2 and TM4 is unique to Cx36 compared with Cx47 and other tested Cxs because of the presence of cysteines that presumably could form the inter-subunit disulphide bonds. This could explain the observation that except for Cx36, all other tested Cxs isoforms (Cx26, Cx30.2, Cx43, Cx45, and Cx47) did not exhibit a dual, carbon chain-dependent response to n-alcohols [28].
Docking of hexanol (orange) and nonanol (green) to different Cxs
Isoflurane is a racemic mixture of (R)- and (S)-optical isomers [12]; however, only R-isoflurane was able to dock in the same hexanol-specific pocket of Cx36 (Figure 1D).
Differences in the specific isoflurane docking pocket in the examined Cxs are shown in Supplementary Figure S2 (additional file 2). All Cxs were aligned, and isoflurane was placed inside the unique docking pocket in the same conformation as it docks into Cx36. Side chains in a close range with isoflurane are shown as sticks for every Cx for comparison. For docking of the specific compound, the dimensions of the pocket should be of an appropriate size. Larger dimensions make it nonspecific while lower dimensions create significant Van der Waals atom surface overlaps between amino acid residues and the ligand. Only Cx36 and Cx47 possess a specific docking pocket for isoflurane or hexanol (Figure 2). In both, the docking pocket is composed of nine amino acids (Supplementary Figure S2, additional file 2). The positions of Gln212, Tyr91, Glu11, Glu12, Lys22, His16, Tyr209, and Arg216 in Cx47, and Gln202, Phe93, Glu12, Ala13, Arg24, His18, Tyr199, and Arg206 in Cx36 are homologous. As determined by the docking procedure, access of hexanol and isoflurane to the analogous site is blocked by Arg143 and Tyr136 in Cx30.2, by Tyr17 and Lys162 in Cx43, and by Tyr91 and Arg184 in Cx45. In Cx26, the docking pocket is not sterically blocked as in these Cxs, but it lacks an aromatic ring as Phe93 inside Cx36 or Tyr91 inside Cx47, and therefore, it cannot accommodate hexanol or isoflurane.
The estimated disulphide bond length is ~2 Å [32]. It is important to stress that a distance between C264 and C92 of the neighboring Cx36 subunits provided by a homology model was >10 Å (Figure 3A); however, this model was developed using the structure of Cx26 [26] that differs from Cx36 by its amino acid sequence and electrophysiological properties. In addition, in contrast with Cx26 and all other Cx isoforms, Cx36 has two cysteines in TM2. We presumed that real distances between C264 and C92 might be much shorter. To examine a possibility that the molecular structure of Cx36 subunits in general can allow their conformation bringing cysteines together, we performed SMD. C264 and C92 approached each other into contact range (3.6 Å) during an initial SMD run, and as it is shown in Figure 3B, a distance between them could be reduced to 2.0 Å during the additional SMD run.
Putative disulphide bond formation between neighboring subunits of Cx36
During SMD simulation, work increased from 0 to 334 kJ/mol within the first 100 ps and remained constant over the remaining simulation time. Supplementary Figure S3A (Additional file 3) shows that 334 kJ/mol of energy was sufficient to overcome structural hindrances precluding the disulphide bond formation. Such an energy value is comparable with single covalent bond strength.
Thus, these results suggested that formation of disulphide bonds between C264 and C92 was likely, and to further examine this possibility, we constructed C264S and C92V single and double mutants of Cx36-EGFP, and examined their properties in HeLa and N2A cells by the dual whole-cell patch-clamp technique.
The effects of hexanol and nonanol on GJ conductance of Cx36-EGFP and its mutants
In our earlier experiments, we used HeLa cells exogenously expressing Cx36 or Cx36-EGFP [28]. HeLa cells express low levels of endogenous Cx45 that occasionally forms low transjunctional conductance GJs [9]. Therefore, in addition to HeLa cells we used other communication-deficient N2A cell line. Junctional conductance was measured between the selected Cx36-EGFP-expressing cell pairs (Figure 4A). Under control conditions, gj varied from 1 to 15 nS depending on a GJ plaque size. A typical experiment (Figure 4B) demonstrates that while hexanol (5 mM) induced ~2.5-fold stimulation of Cx36-EGFP GJ conductance, it was almost completely blocked by nonanol (0.5 mM). The stimulatory effect of hexanol on Cx36 GJ conductance was not affected by insertion of EGFP in the C-terminus of Cx36 since hexanol stimulated to the similar extent the gj of exogenous Cx36 GJ channels in HeLa cells (2.7 ± 0.4-fold; n 4) and endogenous Cx36 GJ channels in mouse pancreatic β-cells (2.2 ± 0.6-fold; n 7) [28] expressing solely Cx36 [31,39]. In contrast, the stimulatory effect of hexanol was absent from cells expressing Cx36C264S-EGFP (Figure 4C). In these cells both hexanol and nonanol inhibited gj presumably through a common docking site(s) on Cx36. Similar results were obtained in both HeLa and N2A cells (summary is presented in Figure 4D) just in HeLa cells the inhibitory effect of hexanol was slightly stronger suggesting that endogenous Cx45 GJs may have manifested. These differences could be reduced using siRNA against endogenous Cx45 (data not shown). The stimulatory effect of hexanol was absent also from both types of cells expressing Cx36C92V-EGFP or double-mutant Cx36C92V/C264S-EGFP (Figure 4D).
The effect of hexanol on GJ conductance of Cx36-EGFP and its mutants
The effect of isoflurane on GJ conductance of Cx36-EGFP and its mutants
General anesthetics such as isoflurane and halothane uncouple cells expressing GJs at concentrations used for inhalation anesthesia [27]. However, isoflurane stimulated the Cx36-EGFP GJ conductance in HeLa and N2A cells (Figure 5A) suggesting that it may act through the same mechanism as hexanol. Structural modeling revealed that R-isoflurane (but not S-isoflurane) is capable of docking to Cx36 in the same unique pocket as hexanol and at multiple other docking sites as nonanol (Figure 1C). This suggested that stimulatory effect of isoflurane on Cx36 GJ conductance also could be exerted by counteracting the inter-subunit disulphide bond formation between C92 and C264. Indeed, the stimulatory effect of isoflurane was absent from cells expressing single mutants Cx36C92V-EGFP or Cx36C264S-EGFP, or double mutant Cx36C92V/C264S-EGFP (see Figure 5B–D).
The effect of isoflurane on GJ conductance of Cx36-EGFP and its mutants
Importantly, another nonselective inhibitor of GJ conductance carbenoxolone, a derivative of glycyrrhetinic acid, that did not fit to the hexanol docking pocket (Figure 1E), also did not stimulate but, like nonanol, reversibly inhibited gj of Cx36-EGFP GJ channels (Figure 5E). Gj was inhibited to 40 ± 4% (n=7) of control with 5 µM and was completely blocked with 100 µM of carbenoxolone (not shown).
The effect of
n-alcohols and isoflurane on GJ conductance of Cx31-EGFP
Some other Cxs, such as Cx30.3, Cx31, and Cx31.1, possess cysteines in TM2 and TM4 at similar positions as Cx36. Therefore, we chose human Cx31 to compare its responses to n-alcohols and isoflurane with those of Cx36-EGFP. As determined by homology modeling, an estimated distance between C86 and C198 of Cx31 is 11.48 Å, and it can be reduced by SMD; however, the access of n-alcohols and isoflurane to the analogous site on Cx31 is occluded by Tyr16 (Supplementary Figure S2, Additional file 2). Consequently, short C-chain n-alcohols and isoflurane had no stimulatory effect on its gj (Figure 6A). Surprisingly, they also had no inhibitory effect while longer C-chain n-alcohols completely blocked Cx31 GJ conduction (Figure 6B). This suggests that the accessibility of n-alcohols to the inhibitory site(s) on Cx31-EGFP may depend on their C-chain length; however, confirmation of this hypothesis requires detailed examination and is within scope of our future investigations.
The effect of
n-alcohols and isoflurane on GJ conductance of Cx31-EGFP
C87 as a candidate for disulphide bond re-shuffling
Recent evidences suggest that disulphide bonds in proteins are highly dynamic (reviewed in [34]). Disulphide bonds may react with thiols within proteins even in the absence of enzymes and re-shuffle spontaneously or on an external stimulus, inducing changes in protein conformation and possibly function [3,22]. To examine this possibility, we performed homology modeling search for possible candidate cysteines. Cx36 has one more cysteine in its TM2 domain, and quite unexpectedly to us, we found that it was located even closer to C264 of the same subunit (11.2 Ǻ) than C92 of the neighboring subunit (Supplementary Figure S3B, additional file 3). C264 and C87 approached each other into a contact range (3.6 Ǻ) during an initial SMD run (Supplementary Figure S3A), as in the case of C264 and C92, and a distance between them could be reduced to 2.0 Ǻ during the additional SMD run. Moreover, the stimulatory effect of pentanol, hexanol, and heptanol was abolished in the Cx36C87S-EGFP mutant while the inhibitory effect of nonanol remained unchanged (Figure 6C).
These observations support our hypothesis that the potentiation of Cx36-EGFP GJ conductance by n-alcohols and anesthetics involves the specific binding site and re-shuffling of the inter-subunit disulphide bond between C264 and C92 to the intra-subunit one between C264 and C87. Other possibilities of re-shuffling (intra-subunit bonding of C87 and C92 or inter-subunit bonding of C264 and C87) are hardly possible due to unfavorable orientations of cysteine residues.
Neurological actions of alcohols and anesthetics are linked with pentameric ligand-gated ion channels (pLGICs) such as γ-aminobutyric acid receptor type A (GABA(A)), glycine, and nicotinic acetylcholine receptors [8,11,13,51]. Alcohols and anesthetics evoke their effects by binding to the specific residues in the transmembrane domain that lines the ion channel pore [13,38], and it is proposed that pLGICs contain intrasubunit-inhibitory and inter-subunit-potentiating sites [16,33]. GJs or, in other words, electrical synapses in the CNS can be inhibited by general anesthetics suggesting that they may play a role in anesthesia [19]. Cx36 that is a dominant neuronal GJ protein also has been shown to be implicated in mechanisms of general anesthetic action [18,48]. For instance, isoflurane or propofol was more potent in Cx36 knockout animals [18], and the authors suggested that Cx36 GJs may act as buffer against effects of anesthetic drugs. This observation may harmonize with our recent discovery [28] that in contrast with other Cxs, the activity of Cx36 GJ channels is not inhibited but can be several times potentiated by n-alcohols up to octanol and, as we demonstrate in the present study, by isoflurane. This property is particularly interesting because n-alcohols with a longer carbon chain such as nonanol inhibited GJs composed of Cx36 suggesting that like pLGICs it may have inhibitory site(s) accessible to all n-alcohols and a potentiating site accessible only to short carbon chain alcohols. In the present study, we demonstrate by structural modeling that such putative stimulatory site may exist in the TM4 of Cx36 (Figure 1). Moreover, we noticed that C264 of the TM4 is located in the proximity to C92 of the TM2 of the neighboring Cx36 subunit suggesting that disulphide bonds may form between them. As it has been shown with other ion channels [6,15], such disulphide bonding resulted in decreased channel activity and could be abolished by reducing agents. Therefore, we hypothesized that n-alcohols and anesthetics may potentiate Cx36 GJ channels by initiating disulphide bond re-arrangement. Indeed, mutation(s) of C92 or C264 completely abolished the stimulatory effect of both hexanol, a representative of short carbon chain alcohols, and isoflurane, and did not change the effect of nonanol, a representative of long carbon chain alcohols. As a possible mechanism, we proposed that potentiation may be achieved due to re-shuffling of the inter-subunit disulphide bond between C264 and C92 to the intra-subunit one between C264 and C87. We supported this presumption by demonstrating that even though Cx31 with single cysteine in its TM2 (C86) and TM4 (C198) (see below) might form putative disulphide bonds, it has no options for disulphide re-shuffling as a response to external stimulus and consequently is not potentiated by n-alcohols and isoflurane.
It is worth noting that predicted distances (>10 Å) between C264 and C92 of the neighboring Cx subunits exceed the estimated length of S–S bonds (~2 Å). However, as a template for structural modeling of Cx36, we used the only determined so far structure of Cx26 [26]. TM4 and TM2 of Cx36 differs in the amino acid sequence from those of Cx26 and contains two cysteines in TM2 while Cx26 has no cysteines in its TM2 [4]:
TM2 of mCx26: LWALQLIMVSTPALLVAM
TM2 of mCx36: WVFQIIMVCTPSLCFITYSV
TM2 of hCx31: LWALQLIFVTCPSLLVIL
TM4 of mCx26: VFTVFMISVSGICILLNIT
TM4 of mCx36: VFLVFMFAVSGICVVLNLA
TM4 of hCx31: IFTYFMVGASAVCIVLTIC
For that reason, we made a presumption that in reality Cys264 and Cys92 may stand closer and demonstrated that by SMD applying the reasonable energy. Alternatively, hexanol and isoflurane may act through another mechanism involving cysteines. For instance, alcohols and anesthetics may potentiate glycine or GABA(A) receptors by binding at the conserved inter-subunit cavity [38] or they, covalently binding to cysteine residues introduced into TM2 of glycine and GABA(A) receptor subunits, irreversibly enhance a receptor function [29]. In addition, it is possible that a unique disulphide bond has to exist for the potentiation of Cx36 GJ conductance by n-alcohols and anesthetics.
General anesthetics have minimal effects on bilayer properties at clinically relevant concentrations indicating that their effects rather involve direct protein interactions, but higher concentrations of certain anesthetic agents can alter lipid bilayer properties [14]. However, short carbon chain alcohols (up to hexanol) cause an increase in free (interstitial) volume of phospholipid membranes, whereas longer alcohols penetrating into the membrane core cause a decrease in it [1]. Consequently, membranous proteins can be either stimulated or inhibited what is in agreement with our results. Our hypothesis is supported by additional observations: (i) unique docking pocket of hexanol is absent from other types of tested Cxs (Cx26, Cx30.2, Cx43, Cx45) that did not exhibit a dual, carbon chain-dependent response to alcohols [28]; (ii) even though Cx47 possesses a similar hexanol-docking pocket, it has no cysteines in its TM2 (as well as all other tested Cxs) that could form intra- or inter-subunit disulphide bonds, and consequently is not stimulated by hexanol or isoflurane [28]; (iii) Cx31 with single cysteine in its TM2 and TM4 does not possess a docking pocket for hexanol and isoflurane, and consequently is not stimulated by these compounds; (iv) carbenoxolone, another nonselective inhibitor of GJ conductance, that did not fit to the hexanol docking pocket of Cx36-EGFP also did not stimulate but, like nonanol, reversibly inhibited gj of Cx36-EGFP GJ channels.
The specific amino acid distribution may at least in part explain the preferences of the specific docking site for hexanol and isoflurane (Supplementary Figure S2, additional file 2); however, further investigations are necessary to relate the docking sites of used compounds to their effects on the Cx36 GJ channel function. First, determination of the Cx36 structure is necessary instead of using the Cx26 homology model. Second, MD would help determine the putative docking sites of compounds, evoking the observed phenomenon, more precisely. Finally, mutagenesis of these sites and patch-clamp experiments will plausibly link the structural domains with responses of Cx36 GJs to n-alcohols and anesthetics.
Our findings propose a novel mechanism by which n-alcohols and general anesthetics may potentiate Cx36 GJ conductance by binding to the specific site, located at the TM4 domain and inducing disulphide bond re-arrangement (putative model is shown in Supplementary Figures S3C,D, additional file 3).
We thank late Professor Feliksas Bukauskas for providing Cx36-EGFP and Cx31-EGFP constructs.
Author contribution
V.A.S. and V.K. designed the study, performed, analyzed, and interpreted the experiments, and drafted the manuscript. V.R. performed the structural modeling experiments. V.J., L.R., and M.K. performed the electrophysiological experiments. A.M. constructed the Cx36 mutants.
Competing interests
The authors declare that there are no competing interests associated with the manuscript.
This work was supported by the Research Fund of Lithuanian University of Health Sciences .
- Cx
γ-aminobutyric acid receptor type A
gap junction
- gj
GJ conductance
Groningen Machine for Chemical Simulations
- HeLa
human cervix carcinoma cell line
- Ij
GJ current
molecular dynamics
mouse neuroblastoma cell line
- pLGIC
pentameric ligand-gated ion channel
steered MD
transmembrane domain
- Vj
transjunctional voltage
Author notes
These authors contributed equally to this work.


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I admit it, I haven't crossed many finish lines as of lately. Heck I am not even signed up for an upcoming race. Usually I am scheduled a year in advance on my race calendar. ( Yes, runners generally have a separate calendar)
What I have been doing is hitting my local gym 4-5 times a week and LOVING it! To be back in a group environment again doing Spin, Tabata or even good ole treadmill ( better known as the dread mill) to work on incline training and it has been MARVELOUS! For me it is a boredom buster to throw in something different. I can disconnect my brain and just follow the instructor. Plus it helps to improve my overall fitness such as core work to ward off those pesky injuries like my I.T. band or weak hips.
Am I getting the itch to sign up for a fall Half Marathon? YEP! Have I picked one yet? Nope! but its coming….In the mean time I will be at the gym because sweat is sweat no matter if its on the roads or off. Just enjoy !
Cue song 🙂


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NBA Law
Kobe Bryant, Chris Webber, Jerry Stackhouse, Allen Iverson, Damon Stoudamire, you would think I'm putting together a list of NBA All-Stars, but no, unfortunately it's the NBA's most wanted list.
Whether, drugs, or assault charges, or lying to a grand jury, all your favorite NBA superstars seem to be getting more publicity for their time in court then their play on the court. With the highly talked about Kobe situation still making headlines, of course Kobe denies all charges and vows to clear his name, just seeing the poster boy for the NBA being dragged through this must send chills down David Sterns' spine. When the NBA's biggest star, Michael Jordan, got in the headlines for reasons other then winning titles, they seemed to be less aggressive charges. Problems with his wife, what marriage doesn't have these problems, betting on golf, he's a competitive guy with alot of money to spend. The new breed of NBA stars seem to be a little more harsh, and more visible in the public eye. Iverson who's well known for his problems with the law has actually been keeping himself out of the police blodder, and had a quiet summer. The same can't be said for Kobe and his sexual assault allegations, Jerry Stackhouse assault charges, Chris Webber and his problems stemming from receiving money from a U. of Michigan booster, and Stoudamire again being arrested for possession of marijuana. Stoudamire may be the most troubling considering this is the third time in 2 years he's been arrested on possession charges. The former rookie-of-the-year, had a promising career that started in Toronto, then after demanding a trade to a contender, he landed in Portland and quickly declined in production. Portland Jailblazers as many have deemed them have had problems on and off the court, in addition to Stoudamire, Rasheed Wallace, Bonzi Wells,Ruben Patterson and Qyntel Woods have all been suspended for various drug, assault charges, or on court behavior.
Why are so many players getting in trouble? It seems to be the more money these players get the more they like to experiment, the more people hang around them, and sometimes they just make really bad decisions. When your young and rich, as most NBA stars are, decisions making is not always a top priority, making fast women, fast cars, and a fast high seem like a much more enjoyable route for them. Obviously not all young NBA players are getting in trouble but until things change, the media will continue to give extra coverage to this type of behavior and more scrutiny on a league that is based on it's players marketability.
What can be done about all this, well I think it's up to the players. You can fine and suspend them all you want but until these guys accept the fact that they are not above the law and that just because you play in the NBA you can get in trouble. The real disappointing part is that these are big name guys who are role models (like it or not) to millions of kids and while no one expects them to be perfect, their clearly not sending the right message to the young NBA fans.


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[A little background here – after I posted my first review of Andrew Root's book, Baker Academic "tweeted" a link to it. I was mortified. It's one thing to opine about someone's work if you are, like, 99.999% sure the author will never read your critique. With the surprise advertisement, I was suddenly faced with the fact that Dr. Root might read my review. He did. And he responded. In some of the most gracious words imaginable, no less. I emailed back and forth with him a couple of times, and I am deeply touched by his willingness to discuss his book, and what he saw as legitimate critiques that I made. His correspondence made my week.]
Okay, its been a while in coming, but I think I am finally ready to share my last (maybe, hopefully) review of Andrew Root's book, Faith Formation in a Secular Age. As I have hopefully already made clear, this book has deeply cut into what I consider to be "faith development," and I eagerly await the next two volumes in this series. If you have not read my earlier posts, the bottom line is I highly recommend this book – even with the caveat that there is some technical language used, so parts of the book may be daunting.
Now I want to share my main "quibble" with the book. I have a real hesitancy in doing this for one huge reason. My quibble is not so much with something Root said, but with what he did not say. My hesitancy is that I have a real issue with people criticizing what does not exist in a work. I have two master's degrees and a doctorate, and the one thing that always irritated me the most in getting a graded paper back was when the professor said something like, "…but you did not address 'x'" (what ever 'x' might have been.) The one time I really deserved this critique was in my Master of Divinity comprehensive exams. I totally forgot to answer half a question. I thought I was going to get "pass plus" and I think I got a pass-minus. Oops.
In regard to Dr. Root's book, the essence of what he identifies as "faith" centers in the Pauline expression, "in Christ." My ears perked up. He went on to discuss Saul of Tarsus' conversion in Acts 9, and the transformation that took place in Saul's life. I grew more interested. Root emphatically defended his contention that faith in the New Testament occurs when a person symbolically dies, has someone come and minister the death of Christ to them, and then comes to a new life consisting in service and ministry to others. I was totally captivated. And, with bated breath, I kept turning pages waiting for the ultimate hammer to fall – the hammer that would locate this death, burial, and resurrection to a new life in the physical moment of baptism.
It never fell.
And, as I mentioned above, it is not appropriate to overly critique the omission of a topic in someone's else's work – so in terms of reviewing Faith Formation in a Secular Age, I will leave you with this simple observation. The omission in no way minimizes the value of this book – I recommend it highly. But – if the book had been mine to write, this is the section I would add regarding baptism.
Ever since the days of Barton W. Stone and Alexander Campbell, the heirs of the Restoration Movement (especially the Churches of Christ) have been accused of overly emphasizing the physical and spiritual act of baptism. Too many times, I fear, this accusation has been all too accurate. Maybe "overly emphasizing" is overly critical, but we have taken one aspect of conversion and, hopefully not to generalize too much, have turned it into the only aspect of conversion. Just as an example, when someone comes to one of our congregations and seeks membership, the primary question asked of them is not, "do you have the gifts of the Spirit active in your life?" but, "have you been baptized?" (And often included in that question, ". . . by the right person in the right church. . . ")
The end result of this emphasis is that now two hundred years (give or take a few) from the writings of Stone and Campbell, I honestly believe that many members of the Churches of Christ have a totally sacramental view of baptism. Conversion and transformation have been replaced with a magical view of baptism that is utterly absent from the New Testament. What I hear from all too many members of the Church is a curious mixture of evangelicalism (you must be 'born again') and Roman Catholicism (it's just 'one and done, baptized you're in, unwashed you're out'). Talk about your unholy marriages.
Don't believe me? Just look at the practice of baptism in so many churches. We say that we do not believe in infant baptism, that we are "credo-baptists," that we only baptize believing adults, or, at the very least, those who have reached the "age of accountability." [WOULD SOMEONE PLEASE SHOW ME WHERE THAT PHRASE IS USED IN THE NEW TESTAMENT?] And, yet, I see and hear of younger and younger children being baptized – as young as 8 or 9, in some cases probably younger.
We do not allow adolescents to drive until they reach an age twice what we gloriously accept as a "believing adult." We do not allow anyone to join the military until a couple of years past that. We do not allow anyone to purchase or legally consume alcohol until a couple of years past that. Yet, we trumpet the decision of a child barely into elementary school as a great transformation of life and character. And, we come up with some of the most specious arguments to defend that practice. "But, they believe in Christ." Um, yeah, so do the demons, according to James 2:19, and it does them no good. "But, what happens if we tell them 'no' and they leave the church?" Well, why do we tell our children they can't have a driver's license until a certain age, that they cannot marry until a certain age, that they cannot join the military or drink a beer until a certain age? Why does our judicial system protect minors who are not capable of making adult decisions and being responsible for those decisions until they reach a certain age? And, just to respond to an absurd argument with one equally absurd, what happens if we DO baptize them and then they leave the church – as is happening by the hundreds if not thousands? What then? Do we become crypto-Calvinists and whisper, "Once saved, always saved" as our young adults stream out the back door?
[If you have never sat and answered the anguished questions of one who was baptized as an infant/child, and had them question their faith, their beliefs, their actions, then I suggest that you do sometime. You will not be so quick to dismiss their hopelessness. If they ask for baptism again they feel like they are rejecting the approval of their loved ones years ago. But to continue on living in the doubts and fears of realizing they simply "got wet" in order to please parents or to succumb to peer pressure is spiritually paralyzing.]
You see, as a group of biblical reformers – whose sincere and genuine desire was to restore baptism to a foundational place in the conversion process – we have come full circle to simply practicing baptism as a quaint "right of passage" that doubles as an entry into the membership of a congregation and provides legitimacy to partaking of the Lord's Supper.
Which brings me right back to Dr. Root's book. If he is correct in his definition of faith (and I think that if not fully correct, his view points us in a bright and helpful direction), then we must, repeat must, restore the biblical view of baptism to that picture of faith. And, I emphasize this, not just as a brief, momentary photo opportunity, but as a life-long, unending commitment to following in the life, death, burial, and resurrection of Jesus.
After examining Saul of Tarsus' conversion in Acts 9, the passage that was most lacking in Dr. Root's book is Romans 6:1-14. But, hear me on this – it has also been lacking in my preaching and in my life as well. I too have fallen into the "one-and-done, baptized you're in, unwashed you're out" mentality. I too have been caught up in the baptism of infant/children. Mea culpa, mea culpa, mea maxima culpa.
I think it is time, past time actually, that the heirs of the Restoration Movement do some actual restoring again. I think we need to restore baptism to its rightful place, not just in the event of a new birth, but in Root's words, to a lifelong commitment to the death of Jesus – as lived out in service and ministry to others.
In other words, we need to restore the biblical view of faith back into our vocabulary and our practice.


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Here's the latest cinematic view of Isaac and Johnny and all those other guys who go by the name Modest Mouse. At least a cinematic view of one of the songs from their last album, We Were Dead Before The Ship Even Sank. Check out the band's upcoming tour dates here.
Technorati Tags: Modest Mouse | Video | Tour Dates


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Dennerby release 30 player preliminary WWC squad
Kristin Hammarström (Örebro), Hedvig Lindahl (Linköping), Sofia Lundgren (AIK), Carola Söberg (Umeå).
Kristin Bengtsson (Djurgården), Maria Bergkvist (Umeå), Sara Larsson (Linköping), Hanna Marklund (Sunnanå), Anna Paulsson (Umeå), Charlotte Rohlin (Linköping), Stina Segerström (Örebro), Marlene Sjöberg (Kopparbergs/Göteborg), Sara Thunebro (Djurgården), Jane Törnqvist (Djurgården), Karolina Westberg (Umeå), Frida Östberg (Linköping)
Midfielders and forwards:
Maria Aronsson (Linköping), Madelaine Edlund (Umeå), Nilla Fischer (Malmö), Sara Johansson (Hammarby), Linda Forsberg (Djurgården), Hanna Ljungberg (Umeå), Therese Lundin (Malmö), Maria Nordbrandt (Sunnanå), Frida Nordin (Malmö), Salina Olsson (Kopparbergs/Göteborg), Lotta Schelin (Kopparbergs/Göteborg), Caroline Seger (Linköping), Therese Sjögran (Malmö), Victoria Svensson (Djurgården)
Aftonbladet: Första truppen till fotbolls-VM presenterad
Svensk Fotboll: Dennerbys 30-trupp lämnad till FIFA
i'm crossing my fingers for bengtsson and törnqvist! oldies=goodies
There's a lot of defenders there, are Bengtsson and Tornqvist really needed?!
törnqvist deserves to go.
she has played so fantastic all year.
and bengtsson...she is the most consistant player in the league.
you can always count on her.
she makes you feel safe.
please...take them.
what is the bulk charge, brothers give me flat! Immediately rushed to five or six big man, with bare hands, there to take chains, there are numerous pipe get,,, vol XII School Commander (b)
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This is worth to read,everyone enjoy it.


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Inaugural Crossroads International Celtic Festival to Be Held in Western Maine
Just in case you need another excuse to visit the glorious mountains and large lakes of western Maine, the region is holding its first Crossroads International Celtic Festival September 11-15. More than 100 entertainers including musicians, singers, dancers and storytellers from all over the Celtic world will descend on the region for a series of performances. The unique choice of venues include Stratton Brook, the newest hut in the Maine Huts and Trails system, and Church of the Good Shepherd in one of my favorite towns in the state, Rangeley. One performance that should not be missed is the "Fiddles on the Frontier" show Thursday, September 12th at 7 pm at the Eustis Community Building in Stratton. The list of impressive fiddlers includes Cape Breton's Dwayne Cote and Rachel Davis. Better be ready to dance!