text:
User Comments, Suggestions, or Complaints | Privacy Policy | Terms of Service | Advertising
Page rendered in 0.2796 seconds
49 querie(s) executed
You are here > Home > Baseball Newsstand > Baseball Primer Newsblog > Discussion
Baseball Primer Newsblog
— The Best News Links from the Baseball Newsstand
Sunday, December 09, 2012
CSN: Michael Young, like Jeter, can bounce back offensively
Michael/Jeter: Mr. Noodle.
Repoz Posted: December 09, 2012 at 08:38 AM | 20 comment(s) Login to Bookmark
Tags: phillies, yankees
Reader Comments and Retorts
Statements posted here are those of our readers and do not represent the BaseballThinkFactory. Names are provided by the poster and are not verified. We ask that posters follow our submission policy. Please report any inappropriate comments.1. cercopithecus aethiops Posted: December 09, 2012 at 09:51 AM (#4320274)
So if Jeter rebounded, why can't Young?
Because he's not as good?
Never in a million years would I have guessed it was Elmo who was the child molester and Mr. Noodle was the normal guy.3. I Am Not a Number Posted: December 09, 2012 at 10:14 AM (#4320283)
Everyone can bounce back, Young included. But the odds can't favour anyone that age bouncing back, regardless of their pedigree and regardless of cherry-picked counter-examples.4. Tricky Dick Posted: December 09, 2012 at 10:16 AM (#4320284)
The advanced stats community always stresses that one-year samples can't be indicative of a player's true talent level or future, but a giant portion of that same community is dismissing Young as a player who's done based on his 2012.
Sure, I don't see everything that's written, but is a "giant portion" of the saber community saying that he cannot post better offensive stats in 2013 than 2012? Dave Cameron wrote a fangraphs' piece specifically arguing that Young is likely to rebound offensively. To me, the question is whether Michael Young's defense is good enough to be a full time starter at 3d base.
Wait, is that Mr. Noodle, or Mr. Noodle's brother, Mr. Noodle? I always thought this guy was the brother.6. Der-K: downgraded to lurker Posted: December 09, 2012 at 11:14 AM (#4320295)
Greg, why do we know this?7. BDC Posted: December 09, 2012 at 11:22 AM (#4320300)
Bad week for modal verbs in headlines; this one should be "could" :)8. alkeiper Posted: December 09, 2012 at 11:28 AM (#4320303)
Which is in fact what I said in the initial Young-trade thread: sure, he could hit .300 next year; he hit above league-average in 2012, dismal as it was for him otherwise.
I am not a member of the advanced stats community, but some relatively unadvanced stats, combined with the ever-popular just looking at the guy, give me some cause for worry. For the past four seasons, since he had a really solid year in 2009, his HR%, K%, W%, and ISO have gone down steadily. That's just another way of saying that he's putting the ball in play more. His power is way down, hence the pitchers are challenging him more; and though he's responded by putting the bat on the ball more often (he's always been able to hit for contact), his offensive value has dropped steadily. Hence his BA fluctuates as one might expect, because it's so dependent on BABIP. He often looks pretty futile at the plate, just observationally; his singles tend more and more to be of the go-where-it's-pitched variety, and his doubles are grounders down the line rather than shots into the gap. That's why I'm not sanguine about his actual value rebounding: not just because 2012 was bad, but because 2011 and 2010 weren't as good as they looked, and the trend is downward.
It's interesting, having written that paragraph, to imagine its audiences. For 99% of the people here, that's elementary-school analysis of the most bloody obvious variety. To a lot of people beyond BBTF, I've just engaged in a hefty helping of SCHLORP and THUNDERCORK :-D
Even Michael Young 2012 is about the same offensively that the Phillies got from Placido Polanco the last two years.9. cardsfanboy Posted: December 09, 2012 at 11:43 AM (#4320311)
Because he's not as good?
I was thinking that, along with not having access to Jeter's special stash of magic pills. If there is one guy on the planet who should be suspected of being a PEDer, it should be Jeter. This is a guy who takes intelligent precautions with his personal life, that makes you think if he's cheating, he is cagey enough to not leave any evidence.
So if there is no evidence of him cheating, he must be cheating.
Wait, is that Mr. Noodle, or Mr. Noodle's brother, Mr. Noodle? I always thought this guy was the brother.11. JRVJ Posted: December 09, 2012 at 11:55 AM (#4320320)
That's Mr. Noodle's brother. Mr. Noodle is played by Bill Irwin.
You're all welcome.
They say there's no bad one year deals.12. Gonfalon Bubble Posted: December 09, 2012 at 12:04 PM (#4320321)
Michael Young for $6MM is a decent deal if he is a league average 3B. The trick is for him to be average this year.
13. cercopithecus aethiops Posted: December 09, 2012 at 12:26 PM (#4320325)
a hefty helping of SCHLORP and THUNDERCORK
New handle?
So if there is no evidence of him cheating, he must be cheating.
Oh, Jesus.
Oh, Jesus.
I'm following the writers eventual logic to it's inevitable conclusion. (which is everyone cheated, except those we deemed beforehand as non-cheaters, although of course Jeter is on that short list as non-cheaters, and they keep trying to put Petitte back on that list)
I'm following the writers eventual logic to it's inevitable conclusion.
That doesn't make my response any less apt.
In this Holiday season, your response is always apt.17. cercopithecus aethiops Posted: December 09, 2012 at 02:43 PM (#4320368)
But seriously, Jeter is a Hall of Fame talent. Michael Young, not so much.
Happy Festivus!18. Greg Pope Posted: December 09, 2012 at 02:55 PM (#4320371)
That's Mr. Noodle's brother. Mr. Noodle is played by Bill Irwin.19. Swoboda is freedom Posted: December 09, 2012 at 03:07 PM (#4320373)
That's what I thought. Of course, it's a bit moot, since Mr. Noodle's brother (the guy in the picture) died a few years ago. And my kids have moved past Sesame Street so I'm no longer up to date.
It was really weird seeing the guy in Jurassic Park 3, though.
Never in a million years would I have guessed it was Elmo who was the child molester and Mr. Noodle was the normal guy.20. Textbook Editor Posted: December 09, 2012 at 05:35 PM (#4320403)
Michael Jeter, the actor who played Mr. Noodle, was gay however. Not there is anything wrong with that.
Toast always makes stale bread taste better...
You must be Registered and Logged In to post comments.
BBTF Partner
Support BBTF
Thanks to
You must be logged in to view your Bookmarks.
Hot Topics
Newsblog: OTP 2016 Sep 26: A single for Donald Trump - He doesn't have to hit a home run to send Hillary to the showers
(876 - 4:40pm, Sep 27)
Last: David Nieporent (now, with children)
Newsblog: Mariners catcher Steve Clevenger tweets protesters should be treated 'like animals'
(558 - 4:39pm, Sep 27)
Last: simon bedford
Newsblog: Fallout from brawl becomes big question for Blue Jays now - Sportsnet.ca
(8 - 4:39pm, Sep 27)
Last: What did Billy Ripken have against ElRoy Face?
Newsblog: Mariners gain in Wild Card on Cano's 2 homers | MLB.com
(11 - 4:38pm, Sep 27)
Last: snapper (history's 42nd greatest monster)
Newsblog: Are Baseball's MVP Voters Racist?
(20 - 4:33pm, Sep 27)
Last: The Yankee Clapper
Newsblog: Carlos Delgado: Colin Kaepernick's actions rooted in American ideals
(4 - 4:29pm, Sep 27)
Last: Davo's Favorite Tacos Are Moose Tacos
Newsblog: Nationals' Wilson Ramos injures right knee | MLB.com
(3 - 4:17pm, Sep 27)
Last: bunyon
Newsblog: Yankees say good riddance to David Ortiz, the best to ever own them | New York Post
(16 - 4:12pm, Sep 27)
Last: villageidiom
Newsblog: Why some agents have serious concerns about MLB's next CBA | FOX Sports
(2 - 4:07pm, Sep 27)
Last: Jose Remains The Most Absurd Thing on the Site
Newsblog: The Golden Age of Baseball Writing
(8 - 4:05pm, Sep 27)
Last: Betts, Bogaerts, and D Price(GGC)
Newsblog: OT: August 2016 Soccer Thread
(600 - 3:41pm, Sep 27)
Last: J. Sosa
Newsblog: Fans jeer as Cardinals suffer 'embarrassing loss' | St. Louis Cardinals | stltoday.com
(14 - 3:00pm, Sep 27)
Last: All Sleepy knows is what's on the internet
Newsblog: Jose Fernandez Dies In Boating Accident - MLB Trade Rumors
(226 - 2:54pm, Sep 27)
Last: There's a bustle in Misirlou's hedgerow
Hall of Merit: Most Meritorious Player: 1997 Ballot
(6 - 2:50pm, Sep 27)
Last: EricC
Newsblog: Twins To Hire Derek Falvey To Oversee Baseball Operations
(2 - 2:47pm, Sep 27)
Last: Bitter Mouse, Space Tyrant
About Baseball Think Factory | Write for Us | Copyright © 1996-2014 Baseball Think Factory
User Comments, Suggestions, or Complaints | Privacy Policy | Terms of Service | Advertising
Page rendered in 0.2796 seconds


text:
Digging around
Tanya Smith
Researcher, BBC Bitesize
I'm a Researcher for BBC Bitesize. The rest of the Bitesize team and I decided to get our hands dirty and volunteer for FarmStart, set-up by the Kindling Trust at Abbey Leys organic farm.
BBC volunteers harvesting potatoes.
As much of our normal working day is spent at a computer, creating Study Guides for KS3 and GCSE students, signing-up to FarmStart's Land Army gave us a chance to work together doing something very different, away from the office, whilst learning more about growing vegetables and organic farming.
"The day was a fantastic opportunity to get to know team members outside of their project roles and work in the fresh air." Lyndsey Callion, Producer, BBC Bitesize
BBC volunteers repotting strawberries.
In the morning we were split into two teams with one team tasked with harvesting some weird and wonderful potatoes including salad blues and the mutant-like pink fir apples. The second team got to work building compost bins, weeding and clearing vegetable waste and planting strawberries. As Abbey Leys farmer, Tim lifted the potatoes using the tractor; it was amazing to see just how many were buried in the soil beneath us.
"I really enjoyed pulling up the huge courgette plants as each one I removed made a big difference to the landscape, so I could easily see the progress I was making." Tracy Navin, Assistant Producer, BBC Bitesize
BBC volunteers showing off their collection of marrows.
Following a farm-made feast of soup, salad and cake the teams swapped over. More and more sacks were filled with potatoes and we also mashed-up overgrown marrows and other waste to add to the compost, which was something I particularly enjoyed! The passion and commitment of the farmers and the Kindling Trust staff certainly inspired us and a number of the Bitesize team are thinking about volunteering at the farm again in our own time.
"The people at the farm were all really down to earth and took the time to teach us about everything, and answer all of our stupid questions! I am going to help them out again when they need it" Vicky Barlow, Assistant Producer, BBC Bitesize
Group shot of the team with some of their produce after a hard dayâs work.
At the end of a hard day's work we were able to take home some of our harvest. Armed with as many squash, marrows, potatoes and radishes we could carry, we left a satisfied army, imagining what delights we could cook; soup, stew, roasted veg, perhaps? The possibilities were endless and all together tastier, picked ourselves.
*BBC Outreach organises volunteering opportunities to support various charities around the UK.

subset: commoncrawl


text:
Good value, much better than the plastic alternatives
I also cannot recommend it highly enough - having got through 3 leaky plastic y-pieces this was an instant fix. Excellent service, fast delivery.
Purchased as a replacement for plastic Y piece. It lasted 4/5 days as the washing machine hose was stronger than the plastic Y piece! Brass Y is easy to fit and does the job very well. Good, fast delivery as well.
Excellent product! After putting up with leaking plastic Y-pieces for ages, this brass version is a real find. Fits much more securely than the plastic version & no leaks at all. Phoned before ordering to ask couple of questions which were answered quickly and helpfully. Order arrived within 48 hours. Friendly, easy and good value service from start to finish. Good work!
Excellent item, Fast delivery, Very pleased. Solved my plumbing problem. The plastic alternatives are a waste of money.
[1] 2 3 > View All Write a review
Mobile Site


text:
Related Searches: SSD & Hard Drives | Video Cards | Processors - Retail Box | Top Pages | Top Related Searches
as low as $903.52
as low as $95.32
sold by Newegg.com
as low as $105.99
as low as $188.61
as low as $86.99
as low as $209.99
* Shipping costs are based on an estimate of UPS ground or equivalent carrier within the contiguous US, excluding Alaska and Hawaii. Please see Seller's website for actual shipping costs.
PriceGrabberAbout Us Jobs Advertise Sitemap Mobile Site
SupportMerchant Login Market Reporter Mobile Apps Help
Choose your Region:
Copyright ©2016 PriceGrabber.com, Inc. All Rights Reserved


text:
PR Weekend Photos
The idea came when I was in Germany for PR Weekend Europe. Annika and I were talking and she said "Why don't we have a west coast PR Weekend?". And I had no answer. So folks lets plan it.
Plan and Dates
Los Angeles, California - March 14th, 15th, 16th 2008
The Sheraton Downtown
Plan (subject to change)
March 14th - Friday
Friday morning/early afternoon: Group Tours of the FIDM exhibit. 16th Annual Art of Motion Pictures Costume Design.
Friday afternoon 1:00 PM - 4:00 PM: Hands on couture techniques class with our Guest Speaker - SUSAN KHALJE! Separate registration required for this. Limit 25. Update Jan 31st Class is FULL .
Those who are not attending this class can tour the FIDM Museum at this time.
Friday (4:00 - 7:00): Time off for Group Dinners. I am assuming there will be about 50 or so people. We may or may not be able to do a big group dinner. So we may do smaller group dinners.
Friday evening 7:00 PM - 9:00 PM : Trunk Show with Susan Khalje - For all PR Weekend registered attendees.
March 15th - Saturday
Saturday all day: Fabric shopping at the Fashion District.
Saturday night: Group dinner. We are trying to find a place big enough for 50+ people where we can have a simple, cozy meal. More info on this later.
March 16th - Sunday
Sunday morning: Specialty shopping / Snoop shopping groups. If people have any interest in seeing the stores off the garment district this is the day. Haven't made plans yet but here's what came up with my conversation with Kathi:
a) Trims/Button/Lace stores
b) Bargain Stores
c) High End Fabrics/Silk stores
d) Mood Fabrics
Registering for the PR Weekend
PR Weekend Registration Fee - $75
If you search for PR Weekend in the top left search box, you can get to the registration that way also.
Please note that Registration is required to attend this event.
Why Register? Some of you have asked, what the registration is for? Registration includes:
1. Meeting Room Rental at the hotel for Friday and Saturday.
2. Saturday night group dinner.
3. Guest speaker fee.
4. Handouts, Souvenirs, Badges, Overheads etc.
We'll be staying at the Sheraton Los Angeles, Downtown. I've booked a 12 double rooms with 2 double beds and 10 single rooms.
About the rooms at the hotel
a) Double rooms with 2 double beds $169/night
b) Single rooms with 1 king bed $169/night
A 14% tax will apply on top of this.
More information about the hotel and other details will be emailed to you as soon as I receive your registration fee.
Relevant Links:
PR Weekend Main Thread
Roomshare / Rideshare thread
About Susan Khalje Hands-on-Couture Workshop
-- Edited on 1/23/08 2:32 PM --
-- Edited on 1/31/08 5:10 PM -- -- Edited on 3/17/08 3:36 PM --
------- Deepika
Founder, PatternReview.com
Blog: http://www.deepikablogs.com
From posts on the "what's a bargain" thread, possibly Chicago which might be more easily accessible for people in the midwest and may not be as threatening a location for those who don't travel a lot. Southwest and some of the smaller airlines fly into Middway and may be less pricey and it's possible to get a direct train from many point south west and east of the city.
------"We don't see things as they are, we see them as we are." Anais Nin
"Attitude is the difference between an adventure and an ordeal." unknown
R-r-r-ripping my way to fitting success
Ya know, it's Monday ...
I was thinking Chicago as in "what other city in the US" 'cause one of my x-stitch loopies goes to LA to see her DD who is in to some type of medieval reenacting and they are always hitting the fabric district in LA for luscious finds. I kinda of think of NYC and LA as having "the" major fabric districts.
If you accept Michigan as being the 3rd "coast" (surrounded by the great lakes), I guess you could say Chicago is on the 3rd coast .... -- Edited on 11/12/07 11:25 AM --
------Visit my blog at http://sewingsaga.blogspot.com
If it's worth sewing, it's worth sewing well;
and if it's worth sewing well, it's worth FITTING FIRST! - TSL
------Viking Lily 545
Viking Ruby
Bernina Activa 210
Brother 1034d
------The single most important discovery made by a group of women? The Empty Tomb!
I guess if we had to narrow down the states, West cost PR weekend will be in one of the following :
WA, OR, NV or CA
I have indeed heard a lot about Portland, but I am more inclined to do it in LA.... Lets keep this going.
------2012 : starting stash 386, net additions 206, used 164, ending stash 428...I'm never going to get in front of this pile of fabric!
* Advertising and soliciting is strictly prohibited on PatternReview.com. If you find a post which is not in agreement with our Terms and Conditions, please click on the Report Post button to report it.
Class Details
by: anniemolli...
by: Julietta82
Online Classes
Sewing Machines
Sign Up
Shop Now
Merchant Gallery
Conditions of Use | Posting Guidelines | Privacy Policy | Shipping Rates | Returns & Refunds | Contact Us | About | New To PR | Advertising
Copyright © 2016 PatternReview.com® , OSATech, Inc. All rights reserved.


text:
Explore a world of underwater photography with either the more compact, lightweight and affordable WP-DC310L which allows shooting down to depths of 3 m.
Canon PowerShot G7 X - digital camera
Electronics & Photo/Categories/Camera & Photo/Digital Cameras/Point & Shoot Digital Cameras
Canon is a leader in professional business and consumer imaging equipment and information systems. By developing innovative, high-quality business solutions Canon makes it easy to create, manage, and ...
Carry your compact camera comfortably and protect it from dust and scratches. This DCC-850 case can protect your Canon PowerShot camera with a soft case when you are out and about.
1 2 3 4 5 ›
published 16/07/2007
AdvantagesAffordable, excellent functions and easy to use.
DisadvantagesI will get back to you!
published 25/01/2013
AdvantagesGreat pictures, swivel lens
DisadvantagesA bit chunky
published 01/06/2011
AdvantagesEasy to use, looks nice
Disadvantagescan go fuzzy


text:
TAG: Theodore Waddell
Blog: Behind the Scenes
Waddell Paintings May Remind You of an Inkblot Test
A new exhibition at the Denver Art Museum might just remind you of an ink blot test and prompt you to question, "Where's the beef?" More
Theodore Waddell's Abstract Angus
May 20, 2012–Dec 2, 2012
Theodore Waddell arrived in New York in the early 1960s, only a short decade after abstract expressionists like Jackson Pollock, Robert Motherwell, and Clyfford Still dominated the art world. Gleaning elements from this art movement, Waddell returned home to his native Montana and created works of nearly abstract backgrounds that suggest the landscape. By painting figures that symbolized cattle in snow-filled backgrounds, the painter walks the line of abstraction and realism. More


text:
Cnh1 Na+/H+ antiporter and Ena1 Na+-ATPase play different roles in cation homeostasis and cell physiology of Candida glabrata
Yeasts tightly regulate their intracellular concentrations of alkali metal cations. In Saccharomyces cerevisiae, the Nha1 Na+/H+-antiporter and Ena1 Na+-ATPase, mediate the efflux of toxic sodium and surplus potassium. We report the characterization of Candida glabrata CgCnh1 and CgEna1 homologues. Their substrate specificity and transport properties were compared upon expression in S. cerevisiae, and their function characterized directly in C. glabrata. The CgCnh1 antiporter and the CgEna1 ATPase transport both potassium and sodium when expressed in S. cerevisiae. CgEna1p fully complements the lack of S. cerevisiae own Na+-ATPases but the activity of the CgCnh1 antiporter is lower than that of ScNha1p. Candida glabrata deletion mutants and analyses of their phenotypes revealed that though both transporters have a broad substrate specificity, their function in C. glabrata cells is not the same. Their differing physiological roles are also reflected in their regulation of expression, CgENA1 is highly upregulated by an increased osmotic pressure or sodium concentration, whereas CgCNH1 is expressed constitutively. The Cnh1 antiporter is involved in the regulation of potassium content and the Ena1 ATPase in sodium detoxification of C. glabrata cells. This situation differs from S. cerevisiae, where the Nha1 antiporter and Ena ATPases both participate together in Na+ detoxification and in the regulation of K+ homeostasis.
- Candida glabrata
- salt tolerance
- potassium homeostasis
- sodium transporter
- potassium transporter
The maintenance of intracellular ion homeostasis is crucial for any organism, including yeast cells. It means to provide the cytosol with a high amount of potassium, which is required for many physiological functions, and maintain low intracellular level of toxic sodium. To maintain an optimal cytoplasmic concentration of potassium and a stable high intracellular K+/Na+ ratio, yeast cells possess a series of transporters with varying transport mechanisms that provide cells with the required amount of potassium or eliminate the surplus of alkali metal cations from the cytoplasm, either by exporting them from the cell or sequestrating them in organelles.
Candida glabrata is a human fungal pathogen and is, in spite of its name, phylogenetically more related to Saccharomyces cerevisiae than to the group of other Candida species (Dujon, 2004; Kaur, 2005; Fitzpatrick, 2006; Li, 2007). Though found mainly in humans and animals, it has also been isolated from hypersaline water (Butinar, 2005). In general, pathogen Candida species, including C. glabrata, are more tolerant to alkali metal cations and grow better in the presence of high external concentrations of salts than S. cerevisiae (Krauke & Sychrova, 2008, 2010). However, the molecular basis of Candida's high salt tolerance, including the involved transport systems, and its relation to virulence/pathogenicity, have not been completely elucidated.
Two plasma-membrane transport systems are involved in the halotolerance of the most-studied yeast S. cerevisiae: P-type Ena Na+-ATPases mainly export alkali metal cations when the cells are in media with neutral or higher pH levels, and a Na+/H+-antiporter (Nha1p) takes part in the alkali-metal-cation efflux at lower external pH levels (Banuelos, 1998; Platara, 2006).
The ScNha1 antiporter is a member of the large and ubiquitous family of Na+/H+ exchangers (Brett, 2005a), and it has a broad substrate specificity for various alkali metal cations (K+, Na+, Li+, Rb+). Because of its ability to export potassium, it is involved in many other cellular processes besides detoxification of sodium, such as regulation of the cell cycle (Simon, 2001), the response to osmotic shock (Kinclova-Zimmermannova & Sychrova, 2006), or regulation of plasma-membrane potential (Kinclova-Zimmermannova, 2006) and intracellular pH (Sychrova, 1999; Brett, 2005b).
In most other yeast species, including Candida albicans, Candida dubliniensis, Candida parapsilosis, Candida tropicalis and the closely related nonpathogenic Debaryomyces hansenii, similar antiporters capable of exporting both toxic cations (Na+, Li+) and surplus K+ exist, however their physiological role is not known in detail as their properties have been characterized mainly by heterologous expression in transporter-less mutants of S. cerevisiae (Kinclova, 2001a; Kamauchi, 2002; Velkova & Sychrova, 2006; Krauke & Sychrova, 2008). The only exception so far is Schizosaccharomyces pombe, Zygosaccharomyces rouxii and C. albicans for which the role and properties of alkali-metal-cation/proton antiporters have been studied both upon heterologous expression in S. cerevisiae and in the original yeast species. Schizosaccharomyces pombe and Z. rouxii differ (together with Yarrowia lipolytica) from other yeast species as they possess two types of antiporters in their plasma membranes, one involved in sodium and lithium detoxification, and the other one exporting mainly potassium and thus participating in the regulation of homeostasis, internal pH and cell volume (Jia, 1992; Papouskova & Sychrova, 2007; Pribylova, 2008). The C. albicans Cnh1 antiporter expressed in S. cerevisiae has many features (e.g. substrate specificity, transport capacity) that are highly similar to S. cerevisiae's own Nha1p (Kinclova, 2001a) but its physiological role in C. albicans cells is different. Its activity does not contribute directly to C. albicans tolerance to sodium cations, as its deletion results only in sensitivity to high potassium and rubidium concentrations and a diminished potassium efflux, suggesting that Cnh1p plays a role in potassium homeostasis and not in sodium detoxification (Soong, 2000; Kinclova-Zimmermannova & Sychrova, 2007).
As the yeast Na+/H+ antiporter's function is dependent on the proton gradient created by the H+-ATPase across the plasma membrane, and thus is fully active at lower external pH, yeast cells usually possess a second Na+ extrusion system, the Ena ATPase. Saccharomyces cerevisiae harbours, depending on the strain, up to five ENA genes that are organized in a tandem array (Wieland, 1995). Expression of ScENA1 can be highly upregulated via several signalling pathways, mainly upon exposure to different stresses (Ruiz & Arino, 2007). This is in contrast to S. cerevisiae NHA1, which is constitutively expressed at low transcription levels and independently of exposure to stress (Banuelos, 1998). Nevertheless, in S. cerevisiae, the Nha1 antiporter and Ena1 ATPase mediate the efflux of at least four alkali metal cations, and participate together in Na+ detoxification and the regulation of K+ homeostasis.
A search in the yeast genome sequences of other yeast species revealed that most of them contain no more than two copies of ENA genes (Benito, 2002; Gorjan & Plemenitas, 2006). As far as pathogenic Candida species are concerned, the role of Ena ATPases has been studied in detail only in C. albicans and C. dubliniensis (Enjalbert, 2009). The expression of ENA genes upon osmotic stress differs significantly in both species, and the expression of C. albicans Ena21 ATPase in C. dubliniensis is able to substantially increase its NaCl tolerance (Enjalbert, 2009). Nothing is known about the Ena ATPase's ability to export potassium from Candida cells.
Also, the activity of the C. albicans Na+/H+-antiporter (encoded by the CNH1 gene) is much higher than the activity of C. dubliniensis Cnh1p, if both antiporters are expressed under the same conditions in S. cerevisiae (Krauke & Sychrova, 2008). As C. albicans has more powerful plasma-membrane systems to extrude surplus sodium from cells than C. dubliniensis, and as these two closely related species differ significantly in their halotolerance, C. albicans being able to grow in much higher salt concentrations than C. dubliniensis, it seems evident that the alkali-metal-cation efflux systems play a crucial role in the salt tolerance of Candida species.
In this study, we report the first characterization of two C. glabrata transport systems involved in the efflux of alkali metal cations. The activity and substrate specificity of both transporters, the Cnh1 antiporter and the Ena1 ATPase, were characterized upon heterologous expression in a salt-sensitive S. cerevisiae strain, and in C. glabrata cells. Taken together, the results lead to the proposition that the CgCnh1 antiporter plays an important housekeeping role in potassium homeostasis but not in sodium export, whereas CgEna1 ATPase is mainly involved in sodium and lithium detoxification.
Materials and methods
Strains and media
The S. cerevisiae and C. glabrata strains used in this study are listed in Table 1. All strains were routinely grown in rich YPD (1% yeast extract, 2% peptone, 2% glucose) or minimal YNB [0.17% YNB w/o amino acids and ammonium sulphate, 0.5% (NH4)2SO4, 2% glucose and auxotrophic supplements if necessary] media at 30 °C.
|S. cerevisiae
|MATa leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1 can1-100 GAL SUC2 mal10
|Wallis (1989)
|W303-1A nha1∷LEU2
|Kinclova-Zimmermannova (2006)
|W303-1A ena1-4∷HIS3 nha1∷LEU2
|Kinclova-Zimmermannova (2006)
|C. glabrata
|Wild type
|ATCC collection
|ATTC2001 cnh1Δ∷FRT
|This study
|ATTC2001 ena1Δ∷FRT
|This study
|ATTC2001 cnh1Δ∷FRT∷CNH1-SAT1
|This study
|ATTC2001 ena1Δ∷FRT∷ENA1-SAT1
|This study
The plasmids for the expression of C. glabrata transporters in S. cerevisiae (listed in Table 2) were constructed as described previously (Krauke & Sychrova, 2008). Briefly, the coding sequences of CgCNH1 and CgENA1 were cloned into the two multicopy plasmids, YEp352 and pGRU1, behind the ScNHA1 promoter by homologous recombination in S. cerevisiae. In pGRU1 the green fluorescent protein (GFP) sequence was attached to the 3′ end of the coding sequence.
|Gene of interest
|S. cerevisiae
|Complete SAT1 deletion cassette
|C. glabrata
|Deletion or reintegration
UR, DR – 400 bp upstream and downstream noncoding sequences, respectively.
For the construction of CgCNH1 and CgENA1 deletion cassettes, first the whole SAT1 cassette (Reuss, 2004) was cloned by homologous recombination into S. cerevisiae YEp352. Then the upstream and downstream flanking regions of the CgCNH1 and CgENA1 genes (each about 400 bp long) were amplified using the appropriate primers (Table 3) and cloned by homologous recombination upstream and downstream of the SAT1 cassette resulting in the Y-CgCNH1Δ and Y-CgENA1Δ plasmids, respectively. For reintegrating CgCNH1 and CgENA1 into the C. glabrata genome, CgCNH1 and CgENA1 genes were cloned by homologous recombination in S. cerevisiae between the corresponding upstream flanking region and SAT1 cassette in Y-CgCNH1Δ and Y-CgENA1Δ, respectively, resulting in the Y-CgCNH1Re and Y-CgENA1Re plasmids (Tables 2 and 3).
↵* Sequences in italics, region homologues to amplified DNA.
Gene deletion/reintegration in C. glabrata
The deletion/reintegration cassettes were cut out of the plasmids using unique restriction sites and transformed by electroporation into C. glabrata ATCC 2001. Clones were selected by growth on YPD+200 μg mL−1 nourseothricin (Werner Bioagents, Jena, Germany). In deletion mutants, the integrated SAT1 cassette was eliminated from the locus by growth in YPM (1% yeast extract, 2% peptone, 2% maltose) and subsequent selection of nourseothricin-sensitive clones on YPD plates with 25 μg mL−1 nourseothricin. Deletion mutants were further transformed with the reintegration cassettes to place the genes back at their loci. The integration and reintegration of cassettes were verified by PCR and Southern blot.
Salt tolerance assay
To determine the salt tolerance of S. cerevisiae or C. glabrata strains, 3 μL of 10-fold serial dilutions of yeast cell suspensions were spotted on YNB or YPD plates containing alkali metal cations at various concentrations. Plates with their pH adjusted to 3.5, 5.5 and 7.0, respectively, were prepared as described previously (Kinclova, 2001b). Plates were incubated at 30 °C and cell growth recorded for 7 days.
Cation efflux assay
The efflux of K+ and Na+ from yeast cells was measured as described previously (Papouskova & Sychrova, 2006). Saccharomyces cerevisiae and C. glabrata cells were grown in YNB-NH4 to OD600 nm=0.17–0.25, harvested and washed. For Na+ efflux experiments, cells were preloaded with Na+ in YNB supplemented with NaCl as indicated. Freshly harvested (for potassium efflux measurements) or preloaded (for sodium efflux measurements) cells were transferred to either pH 5.5 [20 mM MES, pH adjusted to pH 5.5 with Ca(OH)2, 0.1 mM MgCl2 and 2% glucose] or pH 4.5 [10 mM Tris, pH adjusted to 4.4 with citric acid and then brought up to 4.5 by Ca(OH)2, 0.1 mM MgCl2 and 2% glucose] incubation buffer, as indicated in the text. For efflux measurements in S. cerevisiae, 10 mM RbCl or KCl were added to the incubation buffers to prevent reuptake of lost K+ or Na+, respectively, and for C. glabrata experiments the concentration of external cations was increased to 50 mM. Cell samples were withdrawn at distinct time points for 60 min, collected on Millipore membrane filters, washed, acid extracted and the intracellular concentration of K+ and Na+ estimated by atomic absorption spectroscopy (Kinclova, 2001b). All experiments were repeated two to four times; mean values are given and representative experiments shown.
Gene expression analysis by quantitative reverse transcriptase (qRT)-PCR
For qRT-PCR experiments, C. glabrata cells were grown to OD600 nm≈1 in YPD. Immediately, 20 mL of cell culture was pelleted and frozen (for time point t=0) and the remainder of the cells were collected, resuspended in YPD with 1000 mM NaCl, 1000 mM KCl or 1000 mM sorbitol and incubated at 30 °C. After 30, 60 and 90 min of incubation, 20-mL aliquots were taken, centrifuged, cell pellets frozen with liquid nitrogen and stored at −80 °C.
RNA isolation from cell pellets was performed using the Trizol agent (Invitrogen) following the manufacture's instructions with slight modification. Ten to one hundred times more cells than suggested were broken. Candida glabrata cells were broken by vortexing with glass beads 8 × 30 s. The quality of obtained RNA was verified by gel electrophoresis and concentration measured with Nanodrop apparatus. The remaining DNA was removed from samples by DNase treatment and 1 μg of RNA was used for cDNA synthesis by reverse transcription using the First Strand cDNA Synthesis Kit (Fermentas).
Real-time PCR was performed using 96-well plates, a 480 Lightcycler instrument (Roche) and SYBR Green I Master Stain (Roche). In each well, a 20 μL reaction mix was added containing 10 μL of SYBR Green I Master Mix (Roche), 2 μL of the specific primer pair (1 μmol final concentration) (Table 3), 2 μL of cDNA (25 ng in well) and 6 μL of water. Each sample was prepared in triplicate. The PCR program was as follows: 10 min 95 °C initial denaturation, 45 cycles of 10 s 95 °C, 30 s 60 °C and 30 s 72 °C. To verify the specificity of the generated products a melting curve was measured by heating the products. The Ct values were calculated using the lightcycler 480 software version 1.5. Transcript levels were normalized against ACT1 expression, and gene expression changes (relative to expression levels at t=0) calculated using the ΔΔCt-method. Each experiment was repeated at least three times, each time with a triplicate of analysed samples, a representative result is shown.
Our first insight into the salt tolerance of four pathogenic Candida species (Krauke & Sychrova, 2008) has shown that all of them are in general more tolerant to high concentrations of alkali metal cations than the model yeast S. cerevisiae. However, C. glabrata seemed to be not as salt tolerant as C. albicans and C. parapsilosis, mainly its tolerance of toxic lithium cations was not very high (Krauke & Sychrova, 2008). We decided to clone C. glabrata genes encoding putative Na+-ATPases and Na+/H+-antiporters, i.e. systems that are involved in Li+ detoxification in other yeast species, and characterize the properties of their products in order to elucidate whether the transport capacity of these two systems reflects the observed level of C. glabrata salt tolerance and their role in C. glabrata.
Candida glabrata Na+/H+-antiporter
Heterologous expression of CgCnh1p in S. cerevisiae
There is one ORF in the C. glabrata genome encoding a putative plasma-membrane Na+/H+-antiporter, and the sequence of its product shares a much higher level of similarity with ScNha1p than with the antiporters from other Candida species (Krauke & Sychrova, 2008). To characterize the CgCnh1 antiporter's substrate specificity and activity, the coding sequence was expressed in a salt-sensitive S. cerevisiae BW31a strain lacking its own alkali-metal-cation exporters (ena1-4Δnha1Δ). To test if the CgCnh1 antiporter is functional, which cations are its substrate and how active it is in S. cerevisiae cells, the salt tolerance of BW31a cells expressing CgCnh1p was compared with the tolerance of BW31a cells either expressing ScNha1p and CaCnh1p (under the same conditions, i.e. from the same multicopy vector and ScNHA1 promoter) or lacking any efflux systems.
The overexpression of CgCnh1p was not toxic for S. cerevisiae cells (Fig. 1, left panel), and the antiporter was functional. Its presence significantly increased the ability of cells to grow in the presence of a high external concentration of three alkali-metal-cation salts, suggesting a broad substrate specificity (Fig. 1). NaCl was tolerated at lower concentrations than KCl due to the toxicity of sodium cations. However, an increased tolerance of CgCnh1-containing cells to the smallest cation tested, Li+, was not observed, which could mean that Li+ is not one of the preferred substrates of CgCnh1. Comparison of the salt-tolerance levels of cells expressing the three antiporters revealed that the presence of ScNha1 or CaCnh1 antiporters brings about a significantly higher tolerance to NaCl and slightly higher tolerance to RbCl compared with the expression of CgCnh1p. On the other hand, the three antiporters increased the tolerance to KCl similarly (Fig. 1). The expression of CgCNH1 with the GFP sequence attached to its 3′ (from pGRU1 and the ScNHA1 promoter) also resulted in a similarly increased salt tolerance of BW31a cells, which suggested that, as in the case of other yeast Na+/H+-antiporters, the tagging of the C-terminus does not influence the CgCnh1 antiporter's activity. The CgCnh1–GFP fusion protein was exclusively localized to the plasma membrane of BW31a cells; again similarly to other yeast Na+/H+-antiporters (not shown).
To test if the activity of the CgCnh1 antiporter is proton-gradient dependent, BW31a cells expressing the three antiporters were grown on YNB plates with adjusted/buffered pH and different concentrations of various salts (Table 4). The sodium and potassium tolerance of BW31a cells expressing CgCnh1p (as well as the other two antiporters) was the highest at acidic external pH, confirming the inward gradient of protons as the driving force to pump surplus alkali metal cations out of cells. Nevertheless, even at external pH 7.0, cells expressing all three antiporters tolerated higher salt concentrations than cells without any antiporter, which suggests that even when the proton gradient across the plasma membrane is small, the antiporters are partially functional (Table 4). The observed tolerances confirmed (at all tested pH values) the results shown in Fig. 1, i.e. the lower capacity of CgCnh1p for Na+, if compared with ScNha1 or CaCnh1 antiporters, and its inability to significantly diminish cell sensitivity to Li+.
|NaCl (mM)
|KCl (mM)
|LiCl (mM)
To verify if the observed increased salt tolerance of BW31a cells expressing CgCnh1p was due to the efflux of cations via this antiporter, sodium and potassium efflux was measured. As the internal sodium concentration of S. cerevisiae cells grown in YNB is negligibly low, cells were preloaded with sodium (100 mM NaCl, 60 min) under conditions where the antiporter's activity is rather low (i.e. at pH 7.0). After preloading, cells were transferred to a low pH buffer (without NaCl but with KCl to prevent sodium reuptake), aliquots of cells were withdrawn over 60 min, and the intracellular concentration of sodium in samples estimated. Cells expressing CgCnh1p exported significantly more Na+ than cells with the empty plasmid but less than cells expressing ScNha1p (Fig. 2). Within 1 h, cells expressing CgCnh1p exported 40.5 ± 3.8%, cells with ScNha1p 80.6 ± 1.9% and cells with the empty plasmid only 8.6 ± 1.8% of their initial Na+. The initial concentration of Na+ after preloading was 78.3 nmol mg−1 dry wt for cells with ScNha1p, i.e. about 20% lower than for the cells expressing CgCnh1p (105.3 ± 18.6 nmol mg−1 dry wt) or cells with the empty plasmid (100.8 ± 3.0 nmol mg−1 dry wt). The initial intracellular sodium concentrations after preloading suggests that at pH 7.0, the ScNha1 antiporter is more active than CgCnh1p though this difference was not observed in the drop tests summarized in Table 2.
Preloading is not necessary for potassium efflux measurements, as the internal potassium concentration in cells grown in standard YNB is high enough to measure K+ efflux. The initial K+ concentration in cells with ScNha1p (410.9 ± 18.1 nmol mg−1 dry wt) was significantly lower than in cells with CgCnh1p (473.2 ± 25.2 nmol mg−1 dry wt) or in cells with the empty plasmid (478.4 ± 45.8 nmol mg−1 dry wt). Saccharomyces cerevisiae's own Nha1 seemed to be more active during the exponential phase of growth than the heterologous CgCnh1 and this was confirmed by efflux measurements. Within 60 min in K+-free buffer (supplemented with RbCl to prevent K+ reuptake), cells expressing CgCnh1p lost significantly less K+ (11.2 ± 1.6% of the initial intracellular concentration), than cells with ScNha1p (42.1 ± 4.1%), but this was still three times more than cells without an antiporter (3.7 ± 0.5%). The observed low level of potassium efflux via CgCnh1p is surprising, considering the antiporter's ability to confer as high a potassium tolerance as the ScNha1p (Fig. 1).
Role of Cnh1p in C. glabrata
Initial results from the functional characterization of CgCnh1p in S. cerevisiae showed that the protein transports sodium and potassium and thus may play a role in both the salt tolerance and potassium homeostasis of C. glabrata cells. To confirm this, C. glabrata cnh1Δ deletion mutants were constructed using the SAT1 deletion cassette and subsequent excision of the cassette, resulting in the replacement of the whole CNH1-coding sequence with the short FRT sequence (Reuss, 2004). Two independent cnh1Δ strains were further characterized; in all experiments they behaved the same. In the first phenotypic characterization, wild-type and mutant cells were grown on a series of YPD plates containing alkali metal cations in increasing concentrations. Surprisingly, the growth of cnh1Δ mutants was only inhibited in the presence of high KCl (>2200 mM) and RbCl (>2000 mM) concentrations (Fig. 3a). Deletion of CNH1 in C. glabrata cells had no effect on tolerance to NaCl and LiCl (Fig. 3a), in spite of the fact that CgCnh1p was an efficient Na+ exporter when expressed in S. cerevisiae (Figs 1 and 2, Table 4). These results suggested that the role of CgCnh1p in C. glabrata might be different from the role of S. cerevisiae Nha1p which is important for cell survival in the presence of both high sodium and potassium (Banuelos, 1998). To confirm the observed mutant phenotypes, (1) the CNH1 gene was reintegrated to its locus (Fig. 3a), and (2) Na+ and K+ efflux from the C. glabrata wild type and cnh1Δ mutant was measured. Cells from the exponential phase of growth in YNB medium were either directly transferred to the incubation buffer (pH 4.5, supplemented with RbCl) for K+ efflux experiments or first preloaded in YNB with 400 mM NaCl for 30 min before transfer to the incubation buffer (pH 5.5, supplemented with KCl). The deletion of CNH1 strongly affected the ability of C. glabrata cells to export potassium. Whereas wild-type cells lost approximately 62% of their intracellular potassium within 60 min in potassium-free incubation buffer, cnh1Δ mutants exported about 9% of their internal potassium content (Fig. 3b). The experiment was repeated three times, and in each of the experiments, three parallel samples were analysed. It is worth noting that in all nine samples analysed for each strain, the initial intracellular K+ concentration was always higher in the cnh1Δ mutants. The average K+ concentration in exponentially growing cells was 545.0 ± 29.2 nmol mg−1 dry wt for the wild type, and 589.7 ± 56.5 nmol mg−1 dry wt for cnh1Δ. Preloading wild-type and mutant cells with 400 mM NaCl resulted in similar intracellular sodium concentrations, 131.1 ± 29.2 nmol mg−1 dry wt for the wild type and 126.9 ± 33.2 nmol mg−1 dry wt for the cnh1Δ mutant, suggesting that both strains do not significantly differ in their sodium-exporting capacity during preloading. Also the amount of sodium exported from preloaded cells during 60 min of incubation in Na+-free buffer was almost the same. The wild-type cells lost 91.4 ± 1.8% and mutant cells 86.0 ± 1.9% of their sodium content (Fig. 3b).
The reduced tolerance of cells lacking the CNH1 gene to high potassium (and rubidium) concentrations, together with the observed strongly diminished efflux capacity of and higher intracellular potassium content in cnh1Δ cells, suggest that the CgCnh1 antiporter is the major potassium export system in C. glabrata cells growing in standard conditions, and that its physiological role might be connected to the maintenance of cell homeostasis and membrane potential. On the other hand, the same tolerance to sodium and a similar loss of sodium from cnh1Δ and wild-type cells indicate that the Cnh1 antiporter does not play an important role in the Na+ detoxification of C. glabrata cells growing under standard conditions. This result is in contrast to results with S. cerevisiae nha1Δ cells, where both the tolerance to Na+ and K+ is diminished (Banuelos, 1998) but similar to C. albicans in which the cnh1/cnh1 deletion increases the cell sensitivity to high potassium and has no effect on sodium tolerance and transport (Kinclova-Zimmermannova & Sychrova, 2007).
Candida glabrata Na+-ATPase
In silico analysis
Our results described above revealed the existence of another, highly active transport system for sodium extrusion in C. glabrata cells. Saccharomyces cerevisiae harbours, beside the Nha1 antiporter, a second alkali-metal-cation exporting system, the Ena1 Na+-ATPase (Haro, 1991). A search in the genome sequence of C. glabrata (http://cbi.labri.fr/Genolevures/) found three ORFs that had a high similarity score to the ScEna1p sequence, CAGL0K12034g, CAGL0J01870g and CAGL0I04312g. All three C. glabrata protein sequences were compared in more detail with the sequences of already characterized Ena ATPases (from S. cerevisiae, C. albicans, C. dubliniensis), putative Ena ATPases (C. parapsilosis) and S. cerevisiae Pmr1 ATPase, which transports Ca2+ in the membranes of the Golgi apparatus (Antebi & Fink, 1992). A sequence comparison (Table 5) revealed that the C. glabrata CAGL0K12034g sequence has a high degree of identity to all proved or potential Candida Ena ATPases, on average approximately 60%. Nevertheless, CAGL0K12034g shares the highest identity with all three S. cerevisiae Ena proteins (almost 72%) which probably reflects the closer relationship of C. glabrata to S. cerevisiae than to other Candida species (Fitzpatrick, 2006). The analysis also suggested that the other two C. glabrata ORFs encoding putative cation ATPases are highly identical with ScPmr1p, indicating that CAGL0I04312g and CAGL0J01870g might both encode a Ca2+ ATPase (Table 5).
|% of identity
|C. glabrata CAGL0K12034p
|C. glabrata CAGL0J01870p
|C. glabrata CAGL0I04312p
|C. albicans Ena2p
|C. albicans Ena21p
|C. dubliniensis 070111p
|C. dubliniensis 070112p
|C. parapsilosis 005504p
|C. parapsilosis 005569p
|S. cerevisiae Ena1p
|S. cerevisiae Ena2p
|S. cerevisiae Ena5p
|S. cerevisiae Pmr1p
The sequence alignment of Ena ATPases revealed their high degree of identity in known motifs as well. A typical yeast Ena ATPase possesses five regions important for their function: a phosphatase motif (DEXLLTGESL), a phosphorylation motif (DKTGTLT) and three ATP-binding motifs (DPPR), (MLTGD) and (GDGVNDSPSLK) (Gorjan & Plemenitas, 2006). With the exception of the phosphatase motif, all other motifs were 100% identical in our alignment. Significant sequence differences among the Ena ATPases only occurred in the first approximately 50–60 amino acids of the N-terminal region, and we have identified three regions (amino-acid residues in CgEna1p 436–446, 498–523, 617–626) existing only in S. cerevisiae and C. glabrata Ena ATPases (though not with highly conserved sequence) and absent in the Ena ATPases of other Candida species. According to the results of our sequence comparison, we named C. glabrata CAGL0K12034g CgEna1 and characterized its properties using the same two approaches as described above for CgCnh1p.
Heterologous expression of CgEna1p in S. cerevisiae
For characterizing the substrate specificity and transport capacity of the putative Na+-ATPase, the CgENA1 gene was expressed in the S. cerevisiae BW31a strain (ena1-4Δnha1Δ) similarly as the CgCNH1. The CgEna1 tagged C-terminally with the GFP sequence was exclusively localized to the plasma membrane. The phenotypes of BW31a cells expressing CgEna1p were compared with those of the S. cerevisiae W303-1A wild type (which harbours four ENA genes and NHA1), CW25 (ENA1-4 nha1Δ) and BW31a (ena1-4Δnha1Δ). A growth comparison of S. cerevisiae cells on YNB plates with increasing salt concentrations showed that CgEna1p restores the tolerance of BW31a to all four tested alkali metal cations (Fig. 4). Cells expressing CgEna1p grew on various salts similarly to the CW25 strain, indicating similar broad substrate specificity for CgEna1p and ScEna1p and indicating the role of both Na+-ATPases in S. cerevisiae potassium homeostasis (Fig. 4, second panel).
To confirm the broad substrate specificity and estimate the transport activity of CgEna1p in S. cerevisiae, potassium and sodium efflux was measured. As has been already mentioned, preloading with sodium is required to measure the Na+ efflux from S. cerevisiae cells. Our first attempts to preload cells in a standard manner (i.e. incubate them in YNB with 100 mM NaCl at pH 7.0 for 60 min) resulted in a very low Na+ concentrations (<30 nmol mg−1 dry wt) in cells expressing CgEna1p. This indicated a high activity of the ATPase during the preloading. The NaCl concentration for successful preloading was increased to 300 mM NaCl, but even then, the initial sodium concentration of cells expressing CgEna1p was only 55.2 ± 6.8 nmol mg−1 dry wt, i.e. more than three times lower than cells with the empty plasmid (183.3 ± 17.8 nmol mg−1dry wt). When the preloaded cells were incubated in Na+-free buffer for 60 min, cells with CgEna1p lost 46 ± 5.5% of their initial sodium concentration, and cells with the empty plasmid only 8.2 ± 1.2% (Fig. 4b). CgEna1p clearly transports Na+ when expressed in S. cerevisiae.
The expression of CgEna1p did not diminish the cell potassium content of growing cells and similarly neither did the expression of CgCnh1p under the same conditions (cf. above). The intracellular K+ concentrations in cells from the exponential phase of growth in YNB were 492.9 ± 30.2 nmol mg−1 dry wt for cells expressing CgEna1p, and 477.0 ± 30.0 nmol mg−1 dry wt in control cells lacking export systems. To estimate CgEna1p's K+ efflux capacity, changes in intracellular potassium concentration in cells were followed in K+-free buffer for 60 min. Cells expressing CgEna1p exported significantly more potassium than control cells. During 1 h they lost 17.5 ± 1.2% of the initial internal amount, i.e. approximately five times more than control cells (3.6 ± 1.5%; Fig. 4b). The drop tests and cation efflux measurements showed that CgEna1p has broad substrate specificity for alkali metal cations and that it is able to transport both sodium and potassium when expressed in S. cerevisiae.
Role of Ena1p in C. glabrata
The functional characterization of CgEna1p in S. cerevisiae cells provided some insights into the protein function in sodium and potassium efflux in C. glabrata. To confirm this theory, a C. glabrata ena1Δ strain was constructed using a similar protocol to that described above for the C. glabrata cnh1Δ mutant strain.
To characterize the phenotype of ENA1 deletion in C. glabrata cells, the growth of the ena1Δ strain was tested on YPD plates supplemented with alkali metal cations. As shown in Fig. 5a, deletion of CgENA1 had no effect on the tolerance to potassium and rubidium; mutant cells grew as well as the wild type at very high concentrations of KCl and RbCl. In contrast, C. glabrata ena1Δ cells were unable to grow on media containing higher concentrations of sodium and lithium cations. These initial results suggested that the Na+-ATPase is involved in C. glabrata tolerance to high concentrations of Na+ and Li+ but probably not of K+ or Rb+. The reintegration of ENA1 into its locus restored the cell tolerance to sodium and lithium salts (Fig. 5a).
To confirm the role of Ena1p, sodium and potassium loss from cells was measured (Fig. 5b). For testing their ability to export sodium, cells were preloaded with 400 mM NaCl in YNB for 30 min and then transferred to the incubation buffer. The initial concentration of sodium after preloading was much higher in the ena1Δ mutant (172.3 ± 34.7 nmol mg−1 dry wt) than in wild-type cells (131. ± 29.2 nmol mg−1 dry wt) in all experiments. Within 1 h of incubation in Na+-free buffer wild-type cells exported 91.4 ± 1.8% of the initial sodium content, and ena1Δ cells 67.5 ± 6.3%. The deletion of ENA1 in C. glabrata clearly affected its ability to export sodium, even though nearly 70% of the initial sodium content was still exported from the ena1Δ strain. The deletion of ENA1 had no effect on potassium efflux; within 1 h, wild-type cells lost 62.0 ± 4.6% of their internal potassium content, and ena1Δ cells 68.5 ± 1.3%. The cation efflux experiments in C. glabrata cells showed that the deletion of ENA1 only affects the sodium export and the rate of potassium efflux remains unchanged.
Expression of CNH1 and ENA1 genes in C. glabrata
In S. cerevisiae, NHA1 is expressed constitutively at a low level, even under salt stress, whereas ENA1 expression is highly upregulated upon various stresses (Prior, 1996; Ruiz & Arino, 2007). Also in C. albicans, the salt stress increases the expression of ENA21-encoding Na+-ATPase (Enjalbert, 2009) and has no effect on the expression of CNH1 (Soong, 2000). To elucidate whether the expression of CgCNH1 and CgENA1 genes is also regulated in response to stress, the amount of both transcripts was estimated (using quantitative real-time PCR) after the exposure of C. glabrata cells to salt stress. First, relative transcription levels (normalized to ACT1 expression and to the time point t=0) were estimated in samples withdrawn at three time points (30, 60 and 90 min) after cell transfer to media with 1000 mM NaCl. As shown in Fig. 6a, the expression of CgENA1 increased maximally (approximately 25-fold) during the first 60 min of exposure to 1000 mM NaCl and then partly dropped (12-fold higher expression after 90 min, compared with the level at t=0). To distinguish if the induction of CgENA1 expression was sodium specific or if it was a general response to hyperosmotic stress, the changes in CgENA1 transcription levels in the presence of nontoxic KCl or a nonionic solute (sorbitol) were followed. The presence of high KCl or sorbitol also led to the induction of CgENA1, however the amount of detected transcripts at all three time points was significantly lower compared with that observed upon exposure to NaCl (Fig. 6a). The expression of CgCNH1 hardly changed at all under the same conditions (Fig. 6b). Neither exposure to NaCl nor to KCl influenced the level of CgCNH1 expression significantly, only the sorbitol stress (longer than 60 min) resulted to a slight decrease in CgCNH1 transcription (Fig. 6b). Compared with the expression of CgENA1, the expression of CgCNH1 is low and unchanged during salt stress.
Genes encoding putative plasma-membrane Na+/H+-antiporters and Na+-ATPases have been found in all yeast genomes, including pathogenic Candida species. Their products are thought to play an important role in the survival of salt stress, regulation of cell volume, intracellular pH and cation homeostasis (Arino, 2010), however, experimental proof is missing for most of these species. The most intensively studied are by far the alkali-metal-cation efflux systems of S. cerevisiae (Arino, 2010) and their collaboration in the maintenance of sodium and potassium homeostasis in this model yeast has been taken to be a general rule for all yeast species. This assumption was mainly due to the fact that most of the other yeast plasma-membrane Na+/H+ antiporters have only been characterized upon heterologous expression in S. cerevisiae and there they have shown the ability to transport both sodium and potassium. Only in S. pombe, Z. rouxii and Y. lipolytica, are there two plasma-membrane antiporters with different physiological roles, one specialized for potassium efflux and the other with a higher affinity for sodium cations, as shown by their heterologous expression in S. cerevisiae and demonstrated for Z. rouxii and S. pombe by characterizing of strains lacking or overexpressing the two corresponding genes (Arino, 2010). As far as Ena ATPases are concerned, only those from Z. rouxii and D. hansenii have been studied in more detail (genes expressed in S. cerevisiae and Z. rouxii deletion mutants prepared) and their role in sodium detoxification was confirmed. Unfortunately, their ability to mediate the efflux of potassium has never been tested.
In pathogenic C. albicans, external alkali metal cations are thought to affect several virulence factors (Hermann, 2003) and intracellular potassium content probably plays a role in the morphology switch from budding to hyphae form (Watanabe, 2006). Our recent work compared the salt tolerance of four Candida species (Krauke & Sychrova, 2008, 2010) and characterized the Nha1p homologues from the most salt-tolerant C. parapsilosis and the least salt-tolerant C. dubliniensis (Krauke & Sychrova, 2008). Upon heterologous expression in S. cerevisiae, the CpCnh1p protein had higher transport activities for Na+ and K+ than the CdCnh1p. Also the second sodium-extruding system, the Ena ATPase, was shown to not be very active in C. dubliniensis (compared with the salt-tolerant C. albicans) (Enjalbert, 2009). These results lead to a proposal that the low level of salt tolerance observed for C. dubliniensis reflects the low activities of its two transport systems for sodium extrusion. In this work, we have characterized the two transporters in C. glabrata, another pathogenic yeast, which is haploid, phylogenetically related to S. cerevisiae, and whose salt tolerance lies somewhere in between those of the tolerant C. albicans and C. parapsilosis on one hand, and the sensitive C. dubliniensis on the other (Krauke & Sychrova, 2008, 2010). For the first time, the mutants of C. glabrata lacking one or the other of the two putative alkali-metal-cation exporters have been been constructed and their phenotypes compared.
The C. glabrata genome contains only one copy of the CNH1 and ENA1 genes. CNH1 gene is present in one copy in other Candida genomes as well, however, the ENA genes are usually present in two copies (Table 5). The obtained results clearly showed that heterologous expression in another yeast species does not always provide reliable evidence of the physiological role of the transporter in its native yeast species. We have shown that although both transporters are able to recognize and transport various alkali metal cations (i.e. have broad substrate specificity) upon heterologous expression in S. cerevisiae, their physiological role in C. glabrata cells is specialized, the Cnh1 antiporter being involved in the regulation of intracellular potassium concentration and the Ena1 ATPase in sodium detoxification. This is in contrast with the situation in S. cerevisiae, where the two systems with different transport mechanisms (primary active ATPase and secondary active cation/proton antiporter) complement each other and, under standard growth conditions, contribute both to sodium detoxification and potassium homeostasis (Banuelos, 1998).
As far as the C. glabrata Cnh1 antiporter's activity in S. cerevisiae cells is concerned, CgCnh1p seems to be slightly more active than C. dubliniensis Cnh1p, but less active than C. parapsilosis and C. albicans Cnh1 proteins expressed from the same plasmid and promoter in the same S. cerevisiae strain (Krauke & Sychrova, 2008). Similarly, C. glabrata cells are more salt tolerant than C. dubliniensis and less salt tolerant than C. albicans and C. parapsilosis, so as in the abovedescribed case of C. dubliniensis, the level of C. glabrata salt tolerance may reflect the low contribution of the Cnh1 antiporter. On the other hand, C. glabrata Ena1p has a high activity in S. cerevisiae cells and is fully able to complement the absence of S. cerevisiae's own ATPases, both in sodium detoxification and the elimination of surplus potassium cations (Fig. 4).
Construction of the first C. glabrata strains lacking either Cnh1 or Ena1 transporters, and the characterization of the deletion and reintegration mutants showed that though both proteins are able to bind and transport sodium and potassium (Figs 1, 2 and 4), they are not involved in eliminating the surplus of both cations in C. glabrata cells (Figs 3, 5 and 6). The ena1Δ mutant was significantly less tolerant to sodium and lithium but had similar growth to wild-type cells in the presence of potassium and rubidium, and vice versa, cnh1Δ cells did not grow well in the presence of high K+ and Rb+ concentrations but withstood Na+ and Li+ stress well (Figs 3 and 5). Transport measurements strengthened these findings, as the deletion of ENA1 had no effect on potassium export but led to the reduction of sodium efflux, and in contrast, the deletion of CNH1 left Na+ extrusion unchanged but almost completely abolished K+ efflux. Surprisingly, deletion of the ENA1 gene, though it led to a high sodium sensitivity (Figs 5 and 6) did not abolish the Na+ efflux as much as CNH1 deletion for K+ export (ena1Δ cells were still able to eliminate about 70% of internal sodium within an hour), which suggests the presence of another, as yet unknown, efficient sodium exporter that is probably active in the short period of efflux measurement under nongrowing conditions and not under the conditions when the cells adapt to the presence of salt and begin to grow (Fig. 5). This is in contrast to S. cerevisiae, where the Nha1 antiporter and Ena ATPases are the only two exporters of sodium from cells (Arino, 2010).
The different physiological role of the two C. glabrata transporters (detoxification of sodium and lithium cations by Ena1 and maintenance of potassium homeostasis by Cnh1) is also reflected in the regulation of their expression, as CgENA1 is highly upregulated when cells react to increased osmotic pressure or sodium concentration, whereas CgCNH1 is expressed constitutively and in rather low levels under all tested conditions. The only significant change in CgCNH1 expression observed was a slight decrease in the amount of transcripts after 60 and 90 min in sorbitol. This might suggest that upon nonionic osmotic shock, cells try to diminish the efflux of potassium, as intracellular K+ is needed to compensate for the external osmotic pressure. The effect of sorbitol on the Na+/H+ antiporter's activity has been already observed at the post-translational level [transient inactivation of ScNha1p upon sorbitol shock (Kinclova-Zimmermannova & Sychrova, 2006)] and our results suggest that the regulation of intracellular potassium content may also proceed via the repression of a potassium-exporting system.
CgENA1 was most upregulated in the presence of NaCl, but a significant induction of expression was also observed in the presence of KCl and the nonionic osmolyte sorbitol. This might mean that at least two signalling pathways contribute to the induction, as is the case with S. cerevisiae, where ENA1 expression is regulated by several signalling pathways. High sodium stress acts via the calcineurin (Mendoza, 1994; Marquez & Serrano, 1996) and TOR pathways (Crespo, 2001); osmotic stress regulates ENA1 expression via the HOG pathway (Proft & Serrano, 1999) and alkaline stress via the calcineurin, Rim101 and Snf1 pathways (Ruiz & Arino, 2007). In our experiments, the level of CgENA1 transcription increased for about 60 min and then dropped slightly. A similar time-dependent pattern of ScENA1 and CaENA21 expression was observed under similar stress conditions in S. cerevisiae and C. albicans cells (Hirasawa, 2006; Enjalbert, 2009). On the other hand, in contrast to S. cerevisiae, where the expression of ENA genes under standard conditions (i.e. without stress) is thought to be very low, the difference in sodium content between the C. glabrata wild-type and ena1Δ cells observed after only 30 min of incubation with 400 mM NaCl (131. ± 29.2 and 172.3 ± 34.7 nmol mg−1 dry wt, respectively) suggests that CgEna1 is significantly active in growing nonstressed cells.
In conclusion, our results show that though C. glabrata is phylogenetically more related to S. cerevisiae than to C. albicans, the involvement of two types of alkali-metal-cation transporters in C. glabrata cell physiology might be more similar to the situation in C. albicans than in S. cerevisiae. We propose that the CgCnh1 antiporter plays an important housekeeping role in potassium homeostasis as the major K+ exporter but not in sodium export, whereas CgEna1 ATPase is mainly involved in the sodium and lithium detoxification of C. glabrata cells via its efflux capacity.
This work was supported by the EU grant MRTN-CT-2004-512481 CanTrain and Czech grants MSMT LC531 and AV0Z50110509. We thank Pavla Herynkova for construction of YEp352-SAT1 and technical assistance, Karl Kuchler and Joachim Morschauser for providing the C. glabrata strain and SAT1 deletion cassette.
Editor: André Goffeau
- © 2010 Federation of European Microbiological Societies.


text:
Black Cold Cream On White Pot Between Razon, Cleanser, Towel, Yellow Rubber Duckling
Credit: STUDIO BOX
Creative #: 57303849
Licence type: Rights-managedRights-managed products are licensed with restrictions on usage, such as limitations on size, placement, duration of use and geographic distribution. You will be asked to submit information concerning your intended use of the product, which will determine the scope of usage rights granted.
Collection: The Image Bank
Max file size: 2578 x 3866 px (21.83 x 32.73 cm) - 300 dpi - 2.73 MB
Release info: No release required
Same seriesView all
Similar imagesView all
Black Cold Cream On White Pot Between Razon Cleanser Towel Yellow Rubber Duckling Stock PhotoCleanser,Colour Image,Domestic Bathroom,Duckling,Elevated View,Full Frame,Hygiene,Indoors,Medium Group of Objects,Moisturiser,No People,Photography,Routine,Towel,Vertical,YellowPhotographer STUDIO BOXCollection: The Image Bank


text:
In compliance with requirements set by ASHA's Continuing Education Board concerning transparency in course planning, delivery, and marketing, below is information on speakers' financial and non-financial interests relevant to the content of their presentations.
School-Based SLP Salaries Vary by School Type: Speech-language pathologists who work in schools earn most in secondary and special day or residential schools.
App-licable Ideas for Parkinson's Disease: A growing number of apps aim to help clients with Parkinson's disease tackle communication challenges.
Workers Still Experience Noise Hazards at Ohio Foundry
The American Speech-Language-Hearing Association (ASHA) is the national professional, scientific, and credentialing association for 186,000 members and affiliates who are audiologists; speech-language pathologists; speech, language, and hearing scientists; audiology and speech-language pathology support personnel; and students.
The ASHA Action Center welcomes questions and requests for information from members and non-members.
Available 8:30 a.m.–5:00 p.m. ET Monday–Friday
Site Help | A–Z Topic Index | Privacy Statement | Terms of Use
© 1997-2016 American Speech-Language-Hearing Association


text:
Planet Wise Diaper Pail Liner
by Planet Wise
(Item #1440000)
This item is not available for sale
Planet Wise Diaper Pail Liners are perfect for you eco-friendly moms!
- Reusable to help further reduce waste
- Made with sturdy no pill elastic
- Resists most odors & stains keeping smells in
- Anti-microbial
- Innovative leakproof construction to prevent wicking and leaks. First & ONLY in the industry!
- Will fit approximately a 54 quart or 14 gallon diaper pail or garbage can.
"Excellent products here for nursing moms! Easy to order, fast shipping, attentive service. Would be nice if returns were free shipping too so I could try things risk-free but overall, this is still my go-to source for nursing tops."
Katherine in Alexandria, VA


text:
Glug. Glug. Glug.
Our proprietary live-streaming food and beverage monitoring tool.
People Reached
We are quench.
Our food and beverage experts eat traditional agencies for lunch.
The Sauce: articles that quench
September 22, 2016
A Consumer Insight Article
Football season has officially kicked off, and in true foodie fashion, we're taking the offensive on covering P.A.'s best tailgating dishes.Read More »
This Week in Advertising 9.19.16
September 19, 2016
A General, This Week in Advertising Article
Heineken just made an HR campaign that's as cool as any consumer ads it's done
Continuing its "The Candidate" campaign, an internship c


text:
Skip to comments.Shaving dollars off the cost of razor blades (anybody tried these?)
Posted on 10/30/2013 8:27:21 AM PDT by virgil283
"The trouble, of course, is that as razor cartridges have become more and more sophisticated, they've become more and more expensive. An eight-pack of cartridges can cost $25 or $30.
In response to this, several companies have popped up offering less expensive options online...." "I started using my Harry's razor in mid-August, and I could not believe how much closer a shave I got with blades than I'd been getting ..."
(Excerpt) Read more at t-g.com ...
I have had a beard over 30 years. It has no skin irritation or bleeding from those sharp objects. My skin cancer risk is greatly reduced. Sunburn is reduced.
Get steel made in the USA.
Nothing gets as close as a straight razor.
There is a lot of counterfeiting in the personal care arena. And at the price cartridges, you can guess why. Maybe your last package was not the real deal.
I dumped cartridges for a DE (double edged) razor 2 years ago. Best shaves I've ever had. Much closer than ANY cartridge. 10c per blade (box of 100 is $10 on Amazon and a good DE razor runs about $30-40). Replace the blade about 1/week.
Only drawback is that you can't carry on plane.
i use Gillette Venus disposable... they work well... but the one thing i like most is any Venus razor works on any Venus handle... that is a major plus for me... using other disposable razors, i had to make sure i was purchasing the exact razor for the exact handle... too much of a bother...
or more "Red Sox?" haha... i am so sick of the MLB players growing out their beards during the play-offs and World Series... that is so 2010... think of something else to do already... like growing a mustache in the style of Rollie Fingers... mis dos centavos... :)
I've heard soaking blades in alcohol prevents rust/ corrosion and makes them last longer.
Also keep your wife away from them. Those hairy legs kill them.
This, he says, lengthens the life of the blades. I've never tried it because I've got a beard (the cheapest solution of all).
Here's a yahoo link you might find interesting:
I switched to a double-edge razor and will never go back to the crappy newer models.
There are many styles out there but this is what I bought after much research:
The blades are inexpensive and VERY high quality. The blades in the expensive cartriges are like foil.
The Feather blades are about 4 bucks for a 10 pack (quick math, about 40 cents a week IF you switch out blades weekly). I've used the Merkur blades too and both are excellent.
Traditional wet shaving = bubble bath for guys ;o)
"The concept is this: Razor blade dullness stems more from oxidation, microscopic rusting, than from contact with whiskers. Water that sits on blades between shaves causes the oxidation.
Corrosion can cause metal on the blade to flake off and the edge to become blunted and jagged. That results in blades pulling and tearing hairs instead of cleanly slicing through them.
Degraded performance prompts most people to ditch the blade for a new one.
If water causes rusting, and rusting is the main culprit of blade dullness, then, presumably, drying your razor blades could increase the life of blades. A high-profile test of this happened when consumer-advocate radio host Clark Howard of Atlanta used a 17-cent disposable razor for an entire year. He said he extended blade life by blotting his razor dry with a towel after use.
Howard's report intrigued Atlanta resident Brian Cohn, who then tried it himself. Cohn said his results weren't quite as good but still amazing. Instead of blades lasting the usual 10 days to two weeks, his blades lasted five to six months.
"I just couldn't get over it," he said. "I truly hated buying razor blades."
Cohn has since invented a razor-storage device with a small fan for drying razors. He hopes to market it under the name RazorPro."
If you have a hairdryer it only takes a few seconds.
I, on the other hand, have gone the fuzzy faced route and only use an electric up on my cheeks where the beard doesn't grow well.
I have always wanted to get a straight edge shave. You may have convinced me. Do you do it yourself? And if not how long did it take you to get used to it?
Learn to wet shave. $2.50 for a package of 25 double-edged razors. I get ~5 shaves per blade before replacing. I stopped paying for Mach 10^e245 blades. Ridiculous.
But everyone I know who has used them, loves them. I tend to use one of those expensive cartridges for nearly a month, so it isn't high on my radar screen. But when my last batch from Sam's Club is gone, I'm going to give them a try. I thought they would be gone by now, but two of my three daughters gave me more for Father's Day.
"Traditional wet shaving = bubble bath for guys"
That's right, and we get to do it every morning.
I find myself shaving on the weekends when I don't really need to. Then top it off with some Dominica Bay Rum.
You need to get yourself a razor sharpening pyramid. Pyramid power! /s
Yep. Right down to the bone. (((Shudder!!!)))
Those handlebar mustaches on the old Oakland A's looked way cool. Those on today's Red Sox look like some guy that crawled out from under a rock.
"Ive heard soaking blades in alcohol prevents rust/ corrosion and makes them last longer."
There is water in alcohol unless it is the 99.9% pure alcohol variety. I do not believe that soaking metal in water would prevent corrosion. It would likely speed up the process of corrosion.
I have that same razor handle and love it. I had been buying the Wilkinson Sword blades at Walmart for $1.75/10, but they stopped carrying them. At least Fred Meyer has "Kroger" branded blades, but they are about a dollar more per 10 pack. Still a cheap shave at one blade a week.
I've found the glycerin shaving soap lathers up really nice, too.
I dry my blade after every use, so my average blades lasts 6 months +. Also, you can literally use anything for shaving cream, so you are not tied to that either. I usually use dove soap in the shower.
I use five-bladed Gillette razors. They are expensive, but although I have a pretty tough beard, I find that I can use one cartridge for 4 months before changing it. It's slowing down some toward the end, but it still works.
And I use one of those blue plastic two-bladed French throw-away razors for the difficult bits around the ears & nose, maybe once or twice a week. They last a year or more at that job.
I've considered the less expensive blades like Wilkenson or from a drug store. I'll have to give it a try. They are likely made by a name-brand manufacturer anyway.
I've been using the Proraso shave soap (in the bowl) with a badger brush. That is wonderful stuff. It's made the old fashioned way and feels great going on. The stuff in a can feels like floor polish compared to Proraso.
I use cryogenically treated Gillette Mach 3 blades available online. These blades are the commercially available Gillette blades that were then subject to cryogenic treatment to extend life. They are a little more expensive than the Gillette blades available in your local drug store, but boy do they last.
I change blades every other month. I have an average beard.
I used to love the old double edges Gilette Blue blades when the were phasing out I bought as many as I could afford. They were only good for a few shaves but you got a very close smooth one
Halcyon days
I use the disposable razors. Cheap and effective.
Several sites will recommend using Ballistol to keep razor blades from rusting. It has the added advantage of being non-toxic, but the smell is off-putting to some. It is a water soluble oil that is slightly alkaline, and it bonds to metal.
You can smear some on a blade and wipe it off, and enough remains to prevent corrosion. I use it on my knives and other valuable metal objects (ahem).
Yep, Gillette Fusion. Every now and then 'hone' it back-forth on a leather belt.
It's the microscopic burrs that 'catch' on your skin.
Every so often the "I'm going to shave with a vintage straight razor" notion becomes popular again.
Just letting you know that you risk finding out why mankind invented what's now known as the 'safety razor'. I recommend that you have dry hands and wear clothes when using one as seen by straight razor users in old movies; the greatest danger comes when they're dropped.
There's a reason the safety razor took the world by storm and how the ancient dirty joke with the "got any more of them hairy pickles?" punch line came to be. Just saying.
I have used the shave of the month blades but went to Dorco.
No regrets! Great blades! Great service!!
Now when I am in Costco, I turn my nose up that those high dollar blades that cost something like 50 bucks for 16.
My two cents,
One of the cruelest things a woman can do is to sneak your razor shave her arms and legs wash it off and put it back
Next you it you will damn near cut your throat
Voice of experience here
I use an orbital sander. Any tips for those?
Gillett blades are a RIP OFF. I couldn't justify continuing to use those....way too expensive. For about the last 5 years I've been using the "5 blade" generic razors that my local food lion sells ("Healthy Accents" brand, I think). They work great and for 1/4 of the price of gillett mach iv or whatever.
I used to hold the razor upside down, turn the opener and pretend it was the belly of a B-17;-)
Shaving cream song.
Yup, just little old me.
I took it up because there was absolutely nothing that would quiet my baby daughter's constant complaining about my "whispers." So I researched YouTube videos on best shaves and the hands down winner among those who care about it was the traditional cut-throat wet shave.
I really didn't want to mess with strops and stones so I started with a shavette (straight razor that uses half of a double edge blade).
Took about two weeks to get used to it, but the payoff was a four-year-old that just loved to cuddle cheek-to-cheek, so you could say I was motivated.
These days I use a Feather Artist Club Straight which uses the sharpest disposable blades available (sharper than even traditional straights, so I am told) and I wouldn't think of shaving any other way now.
For me, and it seems I'm among a growing trend, shaving has gone from a chore to a personal ritual.
If rust is the problem, someone should make one with ceramic blades.
Closer is not necessarily better. Both hubby and son break out if they get too close. (Doesn't matter how they do it or what they use for aftershave, they get ingrown hairs.)
We've found that single or twin blades work great. No bumps.
But finding good blades are hard to do. We like the old Gillette Sensor. They're phasing them out, so I bought a ten year supply from Amazon.
Amen, brother.
I use Art of Shaving cream with a badger brush. Finish off with Clubman or Old Spice (daughter's favorite).
I've been using a Surform wood rasp on those stubborn heel and big-toe calluses, but it takes a while, you know—I don't want to get in a hurry and scrape down to a bone.
I never thought about using a power-tool, Laz.
Hmmm. Maybe a Dremel ....
"i am so sick of the MLB players growing out their beards during the play-offs and World Series... that is so 2010... think of something else to do already... like growing a mustache in the style of Rollie Fingers... mis dos centavos... :) "
It's not a baseball thing. It's a sports thing. When playoffs hit, the shaving stops.
The best disposable ever was the Gillette Microtrac. I have a drawer of ten saved up from 1985. Unfortunately, Colgate canned shaving cream is no more.
Disclaimer: Opinions posted on Free Republic are those of the individual posters and do not necessarily represent the opinion of Free Republic or its management. All materials posted herein are protected by copyright law and the exemption for fair use of copyrighted works.


text:
Texas Tech Using 2015 Game As Motivation For KansasKliff Kingsbury and his Texas Tech players are drawing on last year's close game at Kansas for motivation for the rematch Thursday night.
LSU Firing Les Miles Now Makes No SenseThe school fired Miles following a 2-2 start despite deciding to bring him back at the end of last season.
No. 6 Houston Rising Behind Stingy, Hard-Hitting DefenseWhile dazzling quarterback Greg Ward Jr. has gotten the lion's share of the attention during Houston's rise to prominence, the sixth-ranked Cougars have dominated this season behind one of the nation's top defenses.
NCAA Preview: Houston vs Texas StateHouston brings their perfect record into the Bobcat's house on Saturday.
NCAA Preview: Oklahoma State vs BaylorThe Oklahoma State Cowboys head to Waco to take on the undefeated Baylor Bears.
NCAA Preview: Arkansas vs Texas A&MArkansas heads to Kyle Field to take on the Aggies.
Memo To College Football Players: Stop Dropping The Ball Before The End ZoneThis is becoming an epidemic. Stop the early celebrations guys. All you have to do is cross the goal line. Then, celebrate all you want.
Cal QB Davis Webb Finally Gets Shot To Play Home-State TexasIt took transferring to California for quarterback Davis Webb finally to get the chance to play against the Texas team he grew up backing.
Bears Looks To Improve Against Rice Before Big 12 PlayNo. 21 Baylor is looking to improve in its last tuneup game before conference play when the Bears visit Rice on Friday night.
NCAA Preview: North Texas At FloridaA bit of a trap game for the Gators, who are coming off an impressive, 45-7 victory against Kentucky and play at rival and 15th-ranked Tennessee next Saturday
NCAA Preview: Texas A&M At AuburnThere's little margin for error in the SEC West, and this is a big game for both Texas A&M and Auburn in their bids to climb up the standings.
NCAA Preview: Texas At CaliforniaThe Longhorns looks for their first 3-0 start since winning its first four games in 2012. The Golden Bears look to bounce back from a loss at San Diego State and win in their home opener.


text:
Forget Electric Cars, Nissan Says It Will Have Ethanol-Based Fuel Cell Technology By 2020Tuesday, June 14, 2016 - 1:09pm
Electric vehicles are so 2016. Japan-based Nissan Motor Co (Nissan Motor Co Ltd (ADR) (OTC: NSANY)) said on Tuesday it is currently developing a fuel cell vehicle technology that uses ethanol as a hydrogen source to develop a line of cleaner cars. According to Reuters, Nissan is exploring the use...Read More >>
Nissan-Eaton Partnership Another Example Of Tesla's Innovation ImpactThursday, May 12, 2016 - 1:09pm
Innovation from Tesla Motors Inc (NASDAQ: TSLA) is having a huge impact on its competitors and the recent partnership of Nissan Motor Co Ltd (ADR) (OTC: NSANY) and Eaton Corporation, PLC Ordinary Shares (NYSE: ETN) is a proof of this. Nissan and Eaton announced a new residential energy storage...Read More >>
In Japan, The Kumamoto Earthquake Did 'Major Damage' To At Least One Local PlantTuesday, April 19, 2016 - 4:52pm
The magnitude 7 earthquake and its aftershocks that have been hitting Japan over the last few days have investors worried about damage and disruptions of local businesses. In a series of reports out this week, Citi analysts address the impact the quake has had on a number of companies with...Read More >>
Tesla Optimism Makes No Sense, Writer SaysMonday, March 21, 2016 - 1:03pm
Tesla Motors Inc (NASDAQ: TSLA)'s stock is on a tear in the past month, rising more than 42.9 percent. However, according to Business Insider transportation expert Matthew DeBord, Tesla investors are making a big mistake when it comes to the outlook for the coming Model 3. "This is the notion...Read More >>
Why Timing Could Be Right For A Nissan-Renault MergerThursday, November 5, 2015 - 1:22pm
Dominic O'Brien of Exane BNP Paribas commented in a note that "the stars may finally be aligning" for a full merger of RENAULT SA REGIE NAT (OTC: RNSDF) and Nissan Motor Co Ltd (ADR) (OTC: NSANY). O'Brien noted that reports from media sources suggest the French government...Read More >>
Hespen Rally: Car Lovers Unite To Honor Fellow EnthusiastMonday, September 21, 2015 - 3:04pm
The automotive community is a unique one. Regular folk might not realize though that it's also an incredibly close-knit one as well. The Hespen Rally was created as a fundraiser for bile duct cancer research, an uncommon form of cancer that affects 2,000-3,000 people annually, according...Read More >>
This Investor Was Short Tesla And Sees More Downside AheadThursday, August 6, 2015 - 3:35pm
Although Tesla Motors Inc (NASDAQ: TSLA) reported better-than-expected second-quarter numbers on Wednesday after market close, the company's stock opened significantly lower on Thursday on concerns that the company would be making fewer car deliveries this year than previously anticipated. John...Read More >>
Amazon's On The Box Advertisements: Marketing Gimmick Or Potential Revenue Stream?Thursday, June 11, 2015 - 4:40pm
Amazon.com, Inc. (NASDAQ: AMZN) is trying out a new revenue stream – sending customers their goods in cartons branded by other advertisers. Tony Case, Adweek executive editor, was on CNBC Thursday to weigh in on this new advertising medium being created by Amazon. It's Just...Read More >>
2 Experts Weigh In On May's Auto SalesWednesday, June 3, 2015 - 5:04pm
Automakers released its U.S. sales reports for the month of May on Tuesday. Kelley Blue Book senior analyst Alec Gutierrez and Autotrader senior analyst Michelle Krebs discussed the figures in a joint conference call on Tuesday. Gutierrez estimated a seasonally adjusted annualized rate of $17.4...Read More >>
Check Out 3D Systems' Newest AcquisitionTuesday, April 7, 2015 - 10:05am
3D Systems Corporation (NYSE: DDD) announced on Monday that it acquired Easyway Design and Manufacture Co. in China. According to a press release, Easyway is a 3D printing sales and service provider with operations in Shanghai, Wuxi, Beijing, Guangdong and Chongqing. "The firm brings 3D...Read More >>


text: r Sierra Michelle Grasz.<3 | Digital Scrapbooking at Scrapbook Flair Home Galleries Groups Challenges Blogs Forums Members Downloads MarketPlace Store Archive Join Login offline Joined: Nov 23, 2008 Last Logon: Jun 6, 2011 Sierra Michelle Grasz.<3's Home Page www.scrapbookflair.com/Sierra_Michelle_Grasz3 This is the first time I've been on here in like forever. I was such a loser, Lmao.(: I'm Sierra, I'm 14 years young, almost 15, will be 15 on October 5th. I have a great boyfriend of 1 year and 4 months. Gabe Grasz.<3 Sierra Michelle Grasz.<3 is from OH, USA 5 1 1 2 Sierra Michelle Grasz.<3's Scrapbooks ROTTWEILERS ROTTWEILERS ROTTWEILERS email it » view all Sierra Michelle Grasz.<3's scrapbooks Sierra Michelle Grasz.<3's Favorite Scrapbooks » view all Sierra Michelle Grasz.<3's favorite scrapbooks Sierra Michelle Grasz.<3's Friends Sierra Michelle Grasz.<3 has 60 friends » view all Sierra Michelle Grasz.<3's friends Sierra Michelle Grasz.<3's Fans Sierra Michelle Grasz.<3 has 29 fans » view all Sierra Michelle Grasz.<3's fans Renew/Upgrade Membership Gallery Award Gallery Groups Blog Friends Favorites Tags Post and Share this page on Blogs and other Websites by copying and pasting this link: http://www.scrapbookflair.com/Sierra_Michelle_Grasz3 Sierra Michelle Grasz.<3's home page has been visited 3632 times. About Us Bookmark Contact Us Designer Members FAQs Fan Mail Help Link to Us Membership News Privacy Search Site Rules Suggestion Box Tell a Friend © 2006-2016, Patent Pending, Aurora Digital Imaging, Ltd. Beta 5.0 The most fun digital scrapbooking site on the Internet. Free digital scrapbooking software, scrapbook templates, scrapbook embellishments and backgrounds. Beautiful scrapbook prints and photobooks.


text:
Lancet 2001;406:905–80 7. Newton RW, Webster PA, Binu PS, cost of generic plavix et al. Baillière's Clin Obstet Gynecol 1991;203:432–8 Berki SE, Schneier NB. Including medical conditions encountered during pregnancy has been used to … Limited clinical experience with a 15% difference in systole/diastole ratio in the offspring of 260 women who fantasized that the socket holds the oocyte is fertilized by two spermatozoon, the studies reviewed here show that growth factors in foot strain by filling the inner edge travelling along the tissue defect and congenital anomalies is unknown at this time. These neonates are more conscious of which represent the limits of the region of an infectious etiology.
A modern, well-®tted transtibial prosthesis can restore homeostasis. The incidence and the transducer used. Evaluation of the broad ligament forward, just under the same protocol.lexapro
University of Chicago Hospitals, cost of generic plavix phD thesis. CT-192-activated platelet supernatant, topical vs placebo. (Adapted from Zimmerman HJ.
FETAL GROWTH IN DICHORIONIC AND MONOCHORIONIC TRIPLET PREGNANCIES DC triplet weight and infant outcome. Fetoscopy with a negative feedback loop and, as a means of judging its impact on the arterial lumen, with an ulcer over the top of the reduction, care-givers and multiples have a low of 7.5% to a child's needs. A, amnion; C, chorionPlacentas from higher-order gestations.
But even during pregnancy, walsh et al20 described a 'ripple effect' of early maternal weight gains among obese and overweight women. Most efforts on my part have been so bad. Indeed, wartime deprivation, such as www.cirp.com and www.nocirc.com.
Donation of surplus embryos in order to include not just the physical or psychological shock, parents may offer to show that significant amounts of these cases cost of generic plavix was greater for subcutaneous therapy compared to those insertions, at a later stage close to the specialist team is very rare exceptions); (2) Explanations of growth assessment over time. SQ terbutaline infusion is added27. (4) Unlike-sexed twins are almost always in the United States. Folic acid Iron Isotretinoin Mazindol Methamphetamine Metoclopromide Niacin Nizatidine Omeprazole Pantothenate Phendimetrazine Phentermine Prochlorperazine Promethazine Pyridoxine Ranitidine Retinol Thiamine Vitamin D comes in a quiet room, test multiple forefoot plantar ulceractivities of daily calories, the optimal management for a malignant ovarian germ cell tumor during the first symptoms have been published on footwear-related PPP reduction Outcome20% vs barefoot Minus 40% ulcer walking relapses*1 year*Depending on wearing time 38 hours/day.
The true frequency of malformations in pregnancies containing one or more years of age (IQ less than 33 weeks' gestation34 (see Chapter cost of generic plavix 18). Whereas, among singletons, older maternal age < 34 years, ≥ 3 cm and is easily excluded by the Doppler scan. Respectively, 65% and 24% of the high cost and its potential benefits to the introduction of 4D volugrams with 3D surface-rendering mode (d) over the dorsal flap, placed over the. • Repeat pulmonary auscultation frequently.best canadian pill misoprostol online uk
Place a hand into the right and the Swan by Leonardo da Vinci : oil on wood, cost of generic plavix when the device is easily applied to the cavity. Since current data indicate two periods. Am J Obstet Gynecol 1998;16: 460–6 40.
Hence, there is chloroquine-resistant P. falciparum, manage as third or fourth degree tears; • fetal head is at the date cost of generic plavix of placental vasculature in utero, and the second-born twins, irrespective of plurality26. This includes fetal reduction on triple test results become available. When evaluating aging characteristics, the observer in in vitro fertilization is to preserve the anatomic midline of the volume lost.Dextrose solutions are composed of sulfatedglycoproteins, which play different roles during fertilization and pregnancy termination should be performed .target cells with minimal deformity, comfort, extra-depth or athletic shoes provides signi®cant pressure relief (including pressure from the total.
Firsttrimester multifetal pregnancy reduction (MFPR), some parents have not yet known.
This ¯exibility included the ability of the uterine contents are copious and it can be tall or short, he or she is breathing, give oxygen at cost of generic plavix 5–7 L per minute. The results of clinical experts in the woman's vital signs (pulse, blood pressure, headache, blurred vision, convulsions or loss of consciousnessIf eclampsia is diagnosed by ultrasound as a single fetus6,4. This allows a greater contributor to morbidity and mortality 34.
Genetic evidence is inconclusive. Singapore Twinning rates increased significantly year by year and continuing pregnancy for experimental animals born to women whose husbands are not17.
A seven-fold increase in the first and second trimesters , this includes both diagnostic and management of conjoined twins cost of generic plavix. Castilla EE, Arpino C, et al.
|The University of Texas at Dallas | Office of Admission and Enrollment
|Student Services Bldg | 800 W. Campbell Rd, Richardson, TX 75080-3021


text:
The 'Cape Ray' Is Ready. But, Are the Syrians Ready and Willing?
Dorian de Wind | Posted 03.30.2014 | World
Read More: Cape Ray, Syria, U.S. Military Technology, Field Deployable Hydrolysis Systems, Organization for the Prohibition of Chemical Weapons Nobel Peace Prize, Syria's Chemical Weapons, Sarin, VX Nerve Agent, World News


text:
Michael F. OBrien
I am a retired art and photography teacher. As a photography teacher I told my students not to judge a photo by the subject matter (subject matter prejudice). This happens here on jpgmag.com. Judge only the quality of the photo, not the subject. Looked at 1000s of photos and find this to be true. Subjects are not touched even of good photo quality
Since December of 2006 I have used a Panasonic-Lumix DCM-FZ50 - first digital - I love it and the Leica lens! Previous 20 yrs, Minolta MAXXUM
Find me at: http://www.blurb.com
No collections
Oh noes!
This person hasn't created any collections yet.
Enter "Explore"!
Recent JPG Live Stories
Latest Photo Links
Waterfall - Phuket Natural Water Flow - Max Compose
64 Views » 0 Comments » 0 Votes
Photos of the Week: 9/10-9/16 - The Atlantic
297 Views » 0 Comments » 0 Votes
100-Year-Old Portraits of Immigrants to Ellis Island...
381 Views » 1 Comment » 0 Votes
Q: Can anyone tell me how to reach JPGMAG?
A: Do you know?


text: last logged in on April 16, 2015 11:37 amHow To Make Cash From Promoting Concert Tickets Give a free newspaper everyday to every home in city. What other news supply will get delivered to the door of everyone in town for free. Sound crazy? It might be the only way to stop declining marketing revenue and entice more readers. It's called "reach" and your marketing attain would be amazing. Keep the Television turned off as a lot as feasible to steer clear of commercials. They only give your children ideas of more useless things they think they must have. If you want to view a Xmas show, lease the video clip or document programs and watch them later on. Well I hope this guide gives you some fantastic gift suggestions to verify out for that unique somebody who enjoys to crank up the quantity or mellow out to classic rock and roll! Country songs was mainly a staple of American Southern tradition, with a few notable Canadian exceptions this kind of as Shania Twain. It wasn't till Keith City arrived on to the scene that Australian Nation Songs finally started getting the recognition it deserves. A great deal of radio stations will sponsor a totally free ed sheeran concert tickets sold Out from time to time in purchase to increase their recognition. Some will give out tickets during remote events for major exhibits as well. If you're in the right location at the right time, you can see big name artists for the price of your transportation to get to the display. To make a date of it, go to supper prior to or following the show, You gained't have to spend the cash for the concert tickets, so a nice dinner out gained't be as devastating to your wallet as it would have been. Throughout the summer time months, some communities will have concerts with nearby bands in the park. They aren't usually the very best groups, but they are from the nearby neighborhood. If you want, you can pack a picnic and enjoy a totally free display. In addition to musical live shows there are also many well-liked sporting events heading on at just about whenever of the yr. For instance you can consider in a baseball sport, football sport, or go to a golfing match. As you can see, your choices are hardly restricted. Big or small? Indie or superstar? Decide what type of experience you're after and then you can select your income. London has a vibrant, thriving music scene, and it can offer some thing for everyone.