text:
How will we characterise our age? By the birth of the internet? The rise of China? The first black US president? Perhaps in all those ways. But we could also say, less obviously but perhaps more fundamentally, that ours is the age when the insects disappeared.
Edward O Wilson, America's greatest naturalist, called invertebrates – the insects, the spiders, the worms, the snails and all their fellows – "the little things that run the world". He meant that these tiny creatures were at the very base of much of life. For example, in the case of pollination, where bees and other insect pollinators fertilise plants, and enable them to produce fruit and seeds, by transferring pollen between flowers.
In the past five years or so, pollinators, honeybees in particular, have started to vanish in many places, and governments have woken up to the problem, as pollination is worth billions.
In fact, insects such as butterflies, moths, bumblebees and mayflies have been disappearing for a long time, although hardly anyone except specialists has noticed or cared.
Their decline began half a century ago with the introduction of pesticides and other agricultural chemicals. But the decline has gathered pace over the past decade with the introduction of systemic insecticides such as the neonicotinoids, which are absorbed into every part of the plant, including the pollen and nectar which pollinating insects collect.
It is too simple to say that one has caused the other, but the link is being made. In his book The Systemic Insecticides – A Disaster In The Making, the Dutch toxicologist Henk Tennekes argues that neonicotinoids are now present in much of Holland's surface water, killing off aquatic insects and leading to a decline in insect-eating birds across the country.
If we care about the little things that run the world, we must wake up to what could be their biggest threat yet.
- More about:
- Animals
- Butterflies
- Insects
- Pesticide


text:
Brunton Boatyard Package ( MAP ) - 3N
Brunton Boatyard3N Kochi
Book Online Get upto Rs. 5,000 off
What's Included
Hotel DetailsHotel options available in the package
|Hotel and Room
|3 night(s)
Brunton Boatyard
Non Sea Facing Standard Room
|Room only
Select Departure Airport:
Prices shown include two way flights, stay and all taxes
* Prices mentioned are for per person on twin sharing basis (minimum 2 people staying together)
Hotel Overview
Brunton is build on the old Portuguese architecture and is steeped in luxury and hospitality. Situated at the Harbour mouth overlooking the busy ship channel all rooms boast of a magnificent view of the sea and the Delta. There is never a dull moment out there for boats of every size and colour constantly ply these waters not forgetting the mammoth Ocean liners which silently glide past just outside the window. Its large harbor facing rooms is a favorite destination for overseas tourists and Indian Corporate Traveler.
Hotel Highlights
- 42 km from the airport
- 15 km from city center
- 0 km to the beach
Facilities at hotel
- Golf
- Children's Club
- Room Service
- Restaurant
- Pets Allowed
- Spa
- Bar
- Concierge Services
- Wi-Fi
- Air Conditioning
- Airport Transfer
- Swimming Pool
- Tennis Courts
- Baby Sitting
- Conference Facility
- Gym
- Casino
- Coffee Shop
Hotel Rooms
Sea Facing Standard Room
Non Sea Facing Standard Room
Standard Room
Cancellation Policy
- The package once booked is refundable and cancellable.
- Flight cancellation will be as per airline cancellation policy.
- Date of booking to 30 days before travel the cancellation charges will be 10% of the tour cost
- 30 to 15 days before travel - cancellation charges will be 50% of the tour cost
- 15 to 7 days before travel - cancellation charges will be 75% of the tour cost
- 0 to 7 days before travel - cancellation charges will be 100% of the tour cost. No refund shall be given
- Please note cancellation policy is subject to change. It depends on the hotel policy. In peak season, some hotels may charge 100% cancellation.
Additional Information
- Check-in at hotel starts from 14:00 hrs.
- Check-out time is until 12:00 hrs.
- Early check-in or late check-out is subject to availability and may be chargeable by the hotel.
- Once your payment has been processed, we will send you an email confirmation.
- You will receive all travel vouchers required for your flights and accommodation shortly afterwards.

subset: commoncrawl


text:
Buy.com milestones
Buy.com milestones
November 1997: Buycomp.com launches with 30,000 high-tech products.
November 1998: Name changes to Buy.com.
October 1999: Buy.com files for IPO.
November 2001: Founder Scott Blum purchases Buy.com and takes company private.
May 2010: Rakuten acquires Buy.com for $250 million.
January 2013: Site is rebranded as Rakuten.com Shopping. The Buy.com URL will remain, but consumers who go to the site will be redirected to Rakuten.com starting Jan. 31.
Source: Buy.com
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10 Best Songs
Album Of The Week
Band To Watch
Best Covers
But Who's Buying?
Cover Story
Counting Down
Premature Evaluation
Shut Up Dude
Sounding Board
Status Ain't Hood
Straight To Video
The 5 Best Songs Of The Week
The Anniversary
The Black Market
The Week In Pop
Ultimate Playlist
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the latest //
The 5 Best Songs Of The Week
The 5 Best Songs Of The Week
Steven Meli
Comments from Steven Meli
Perfectly agree with Amilcar Schell... this album is NOT Two Suns and can never be... They are two excellent bodies of work... One can't top the other as they're excellent at what they are in their own different territories... You must not compare them.... This album has got it's instant moments "Lillies" hit me on first listen although upon more listens it revealed it's mystical sheen... I'm still puzzled how you didn't even mention 2 of this album's masterpieces... That's Oh Yeah & Winter Fields.... The latter as well as the title track are two of the most captivating BFL moments to date... Add that with Laura and you've got a shivering centerpiece to this album... Oh Yeah reveals more on the sensual side of Natasha Khan while Marilyn is possibly the boldest most confident song I've ever heard from them... I agree with the fact that there's no "Daniel" in here but it would sound out of place in this tighter body of work... The Haunted Man stands on it's own with so many stand out songs, each one complimenting the other to form one of the best albums of the year! *****
November 10, 2012
Premature Evaluation: Bat For Lashes
The Haunted Man
What an awesome song... you can say that this is Ladytron but the sound is very different... It's amazing how they always seem to re-invent themselves!!! 10/10
May 19, 2011
Ladytron – "White Elephant"
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Re: Left Luggage in Exeter
Due of the spate of terrorist bombings around, most stations no longer offer left-luggage facilities and the same is true in Exeter.
However the offices of the Capitol Taxi firm, which is just across the road to the left of the main station exit, do have a secure luggage room and charge 1.50 GBP per item.
As it's a taxi office it is manned 24 hours and so ideal if you're just in Exeter for the day and don't want to haul your kit around.
Good luck, problem solved :-)
Be the first to rate this answer!Was this helpful? Quote & Answer
Re: Left Luggage in Exeter
Yep Durfun's answer is correct and up-to-date.
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The Wave (Die Welle) (2008)
Directed by
Starring (View all)
Jürgen Vogel
Rainer Wenger
Rainer Wenger
Frederick Lau
Tim Stoltefuss
Tim Stoltefuss
Max Riemelt
Jennifer Ulrich
Christiane Paul
Anke Wenger
Anke Wenger
Jacob Matschenz
Cristina do Rego
Elyas M'Barek
Written by (View all)
Johnny Dawkins
125 votesAmazing Underrated Obscure Bizarre Films PART 9 (50 items)
list by JayTrotter
Published 4 years, 8 months ago 16 comments
98 votesClever movies that make you think *updated* (34 items)
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Published 5 years, 5 months ago 23 comments
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list by Coby
Published 4 years, 1 month ago 5 comments
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Published 3 years, 1 month ago 12 comments
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4 years, 2 months ago at Dec 5 18:01
3 votesThe scary message about this movie is that when people say it would never happen to us, it can. It starts with minor things like excluding people for small so called minor Infractions. This progressively builds to the most important of things that has us questioning again, how did it happen? Before you know it occurred, things are getting out of hand and then an escalation that happens which is b... read more
cuidado con las masas
8 years, 1 month ago at Jan 9 8:45
3 votesCuando se dan ciertas condiciones sociales, es posible perder el sentido común convertirnos en demonios para nosotros mismos.
6 years, 1 month ago at Jan 20 16:18
3 votesKlasse.
Because history is never the past.
A high school teacher's unusual experiment to demonstrate to his students what life is like under a dictatorship spins horribly out of control when he forms a social unit with a life of its own.
Release date: 27 May 2011
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text:
My husband looked into the bowl and groaned. 'I'm fed up with frilly salads that look as if they have come from a French nightclub. What on earth is wrong with real lettuce?' The children continued the attack. 'We like salads with green lettuce, tomato and cucumber and we like ordinary vinegar, the sort you put on chips,' the eldest said. My seven- year-old added wistfully, 'for school dinners we have real salad cream with our salad.'
When a state-of-the-art Sainsbury's store opened at the end of my road more than a year ago I was delighted. I too could make those effortlessly superior meals advertised by the stars on television. Visiting friends who shopped at Sainsbury's convinced me they enjoyed superior perks: their barbecues had fragrant hickory chips thrown upon them. They bought balsamic vinegar, dark and mysterious, by the cut-price pint, vacuum-packed ready-to-eat chestnuts, high-juice natural squashes for their kids.
But the reality of shopping at Sainsbury's proved disappointing, compared with the smaller supermarkets and local shops I had used before.
First, it took so much longer, at least 40 minutes more per week. This is because the entire neighbourhood flocks there; first you hunt for a parking space, then you face queues at the checkouts - even on Sundays.
The distance you are expected to trundle your trolley is equivalent to a good hike. I had not expected to encounter the Sainsbury's exhaustion factor but it is horribly real. I arrived there at 9.30am on Friday morning and was still there at 11.45am. One day I returned home, sat on the sofa and woke at 2.30pm. It was time to pick the children up from school. Sainsbury's was taking the best part of the day.
Second, despite all those cut- price offers they advertise, I persistently spent more than I wanted to: perhaps pounds 30 a week - even when I tried steeling myself against temptation. Another drawback was that I kept running into people I knew. A good friend said it was the only supermarket she knew that she put lipstick on for. I found myself reluctantly accepting supper invitations and discussing teachers while pondering the packs of chicken breasts.
In order to avoid all this, and save time, I even experimented by going at 8.30am. But what shop is worth doing that for? It was just at this point that the salad revolt occurred, a month ago. It hit a raw nerve. I was secretly raring for change. There was something relaxing about standing in a local shop watching the assistants weigh the exact produce you wanted. Local traders, who estimate they have lost 30 per cent of their business since the Sainsbury's store opened, welcomed me back. They even carried my bags to the car.
The specialist cheese shop two doors up now sells better unpasteurised cheese than Sainsbury's: even Lenny Henry's chef would be impressed.
Three out of the four butchers around me have closed in recent months. But the one that remains sells fine meat. The children's favourite lunch, believe it or not, is roast beef with Yorkshire pudding. They also love the shop's sausages made into toad-in-the- hole. These were children recently toying with vegetarianism. They also prefer traditional gammon to the slender, sweet-cure packs I bought at the Sainsbury's store. I have also started to make real rice pudding (1 pint of milk, 1 1/2 tablespoons of pudding rice, 1 tablespoon of sugar), at a fraction of the cost of those fiddly yoghurt desserts. It goes down famously.
In truth, I never once made a Sainsbury's celebrity recipe. There was something too manipulative about it all. My final discovery is that the smaller local supermarket I used to despise is a joy to visit. It is deserted. I go there every fortnight for boring basics and there are always plenty of people available to help me pack.
So, until Sainsbury's makes the experience less exhausting and cheaper I will remain a satisfied post-Sainsbury's person. I've been there and I've done it.
And I've been testing out my experience against friends. They too have been put off. We say long live local shops. But my small daughter has yet to amend her version of the famous nursery rhyme. It runs: 'This little piggy went to the supermarket . . .'
- More about:
- Butchers
- Retail
- Sainsbury (j)
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text:
Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function as well as susceptibility to various diseases such as Parkinson disease, Alzheimer disease, bipolar disorder, and cancer. However, it is unclear whether and how mtDNA polymorphisms affect intracellular function, such as calcium signaling or pH regulation. Here we searched for mtDNA polymorphisms that have intracellular functional significance using transmitochondrial hybrid cells (cybrids) carrying ratiometric Pericam (RP), a fluorescent calcium indicator, targeted to the mitochondria and nucleus. By analyzing the entire mtDNA sequence in 35 cybrid lines, we found that two closely linked nonsynonymous polymorphisms, 8701A and 10398A, increased the basal fluorescence ratio of mitochondria-targeted RP. Mitochondrial matrix pH was lower in the cybrids with 8701A/10398A than it was in those with 8701G/10398G, suggesting that the difference observed by RP was mainly caused by alterations in mitochondrial calcium levels. Cytosolic calcium response to histamine also tended to be higher in the cybrids with 8701A/10398A. It has previously been reported that 10398A is associated with an increased risk of Parkinson disease, Alzheimer disease, bipolar disorder, and cancer, whereas 10398G associates with longevity. Our findings suggest that these mtDNA polymorphisms may play a role in the pathophysiology of these complex diseases by affecting mitochondrial matrix pH and intracellular calcium dynamics.
Mitochondria play important roles in energy production and regulation of intracellular calcium levels. Mitochondria have their own genetic material, mitochondrial DNA (mtDNA). In spite of its short length (16 kbp), mtDNA is highly variable among individuals and is thought to contribute to interindividual functional variability in energy-requiring activities such as intelligence and athletic performance. However, it is unclear whether mtDNA polymorphisms affect intracellular function and condition. Using transmitochondrial hybrid cells, the authors found two closely linked mtDNA polymorphisms, 10398A/G and 8701A/G, which cause alterations in mitochondrial pH and calcium concentration. Cytosolic calcium response to histamine tended to be different between transmitochondrial hybrid cells carrying these two mtDNA polymorphisms. It has been reported that the 10398A mtDNA polymorphism is a risk factor for Parkinson disease, Alzheimer disease, cancer, and bipolar disorder, whereas 10398G is associated with longevity. The present findings suggest that these mtDNA polymorphisms may play a role in the pathophysiology of these complex diseases by affecting mitochondrial matrix pH and intracellular calcium dynamics.
Citation: Kazuno A-a, Munakata K, Nagai T, Shimozono S, Tanaka M, Yoneda M, et al. (2006) Identification of Mitochondrial DNA Polymorphisms That Alter Mitochondrial Matrix pH and Intracellular Calcium Dynamics. PLoS Genet 2(8): e128. doi:10.1371/journal.pgen.0020128
Editor: Harry Orr, University of Minnesota, United States of America
Received: June 17, 2005; Accepted: June 28, 2006; Published: August 11, 2006
Copyright: © 2006 Kazuno et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by an Independent Investigator Award from the National Alliance for Research on Schizophrenia and Depression (NARSAD) to T.K., a grant from the Japanese Ministry of Health, Labour and Welfare, and a grant from Japanese Ministry of Education, Culture, Sports, Science and Technology.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: cybrid, transmitochondrial hybrid cell; mt DsRed, mitochondria-targeted red fluorescent protein from Discosoma; mt pH-GFP, mitochondria-targeted pH-sensitive green fluorescent protein; mtDNA, mitochondrial DNA; mtRP, ratiometric Pericam targeted to mitochondria; mtSNPs, mitochondrial DNA single nucleotide polymorphisms; nucRP, ratiometric Pericam targeted to nucleus; RP, ratiometric Pericam; rRNA, ribosomal RNA; tRNA, transfer RNA
The central importance of mitochondria in ATP production is well established [1,2]. The pH gradient across the mitochondrial membrane and the inner mitochondrial membrane potential make up the electrochemical gradient, which regulates the efficiency of ATP synthesis and other mitochondrial activity. Recent studies are also focusing on the roles of mitochondria in regulation of intracellular calcium dynamics. Mitochondrial calcium uptake affects various important cellular processes such as apoptosis [3–5], exocytosis [6,7], synaptic plasticity [8], and possibly spine dynamics [9].
Mitochondria have their own DNA, mitochondrial DNA (mtDNA), which encodes the genes of 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and 13 subunits of enzymes related to oxidative phosphorylation [10]. Other subunits of mitochondrial proteins are encoded in the nuclear genome.
Mutations in mtDNA are known to cause various mitochondrial diseases such as mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), which is caused by the 3243A/G mutation in mtDNA [11,12]. The mechanisms by which these mtDNA mutations cause functional impairment are well studied [13,14]. On the other hand, mtDNA is highly polymorphic, and certain polymorphisms are thought to be risk factors in complex diseases such as diabetes mellitus [15], Alzheimer disease [16,17], Parkinson disease [18–21], bipolar disorder [22,23], and some kinds of cancer [24,25]. It has also been reported that mtDNA polymorphisms are related to interindividual functional variability in human cognition [26], personality [27], athletic performance [28], and longevity [29]. However, these associations are solely dependent on population genetics. It is difficult to draw a definite conclusion from genetic association analyses alone because the high variability of mtDNA among individuals makes such analysis susceptible to confounding effects of population stratification; in addition, the effects of other polymorphisms in mitochondrial or nuclear genes are difficult to control. Although functional analyses of these polymorphisms are needed, to date there are few reports that identify functional effects of mtDNA polymorphisms. This is mainly due to methodological difficulties, as conventional molecular biological techniques are not readily applicable to mtDNA.
We examined the phenotypic effect of mitochondrial DNA without interference from nuclear genes by analysing transmitochondrial hybrid cells (cybrids), which were made by fusing a cell line lacking mtDNA, called ρ0 (rho zero) cells [30], with platelets from humans. Although the consequences of pathogenic mtDNA mutations have been examined using this technique, the functional significance of mtDNA polymorphisms has not been well investigated yet.
In this study, we searched for functional mtDNA polymorphisms using the following strategies: (1) by targeting a calcium indicator, ratiometric Pericam (RP) [31], to mitochondria and the nucleus in the same cell, mitochondrial and cytosolic calcium levels were monitored simultaneously; (2) to reduce cellular variability, a ρ0 cell line was subcloned before generation of cybrids; (3) by sequencing the whole mtDNA genome in 35 cybrids, functional mtDNA polymorphisms were comprehensively analyzed, and two nonsynonymous mtDNA polymorphisms, 10398A/G and 8701A/G, were identified; and (4) using a pH indicator, we confirmed that these mtDNA polymorphisms altered both mitochondrial matrix pH and intracellular calcium levels.
Generation and Confirmation of Cybrid Cell Lines
First, we established a 143B.TK− ρ0206 cell line that stably expresses two RPs. RP is a ratiometric fluorescent protein developed as a calcium indicator [31]. The fluorescence ratio of 510 nm emission at 480 nm excitation to that at 410 nm excitation is reported to reflect calcium concentration at a constant pH. Mitochondria-targeted RP is also sensitive to pH because there is a higher pH [32,33] in the mitochondrial matrix (approximately 7.7–8.0) [34,35] than in the cytosol. In this study, two RPs, one targeted to mitochondria (mtRP) and the other to the nucleus (nucRP), were expressed in the same living cell. Thus, we were able to simultaneously monitor mitochondrial and cytosolic calcium concentrations [31]. Because calcium levels in the nucleus were reported to be similar to those in cytosol, nuclear calcium levels were used to indicate cytosolic calcium level.
We subcloned several ρ0 cell lines carrying RPs and selected one that showed a reproducible calcium response. We confirmed by mtDNA-specific PCR and Southern blot analysis that this cell line lacked mtDNA. We fused this cell line with platelets taken from 35 human volunteers and confirmed the integration of mtDNA from the volunteers by Southern blot analysis.
Measurements of Fluorescence Ratio of mtRP in all Cybrid Cell Lines
Using these cybrids, we measured fluorescence ratios of mtRP and nucRP. In fluorescent images of cybrids, mitochondria had a higher 480 nm/410 nm ratio than the nucleus (Figure 1A), possibly reflecting higher calcium levels and pH in mitochondrial matrix than in cytosol. This result is consistent with previous observations in HeLa cells [32]. At first, baseline fluorescence ratios were recorded for 2 minutes. Subsequently, cells were stimulated by 10 μM histamine, which elicits inositol trisphosphate–mediated calcium release from the endoplasmic reticulum [36]. In the first 60 s following stimulation, the fluorescence ratio of mtRP and nucRP initially increased and then returned to basal levels (Figure 1B). The mtRP fluorescence ratio was measured in 11–42 cells for each of the 35 cell lines by fluorescent microscopy.
(A) Image of a typical cybrid showing fluorescence ratios of excitation at 480 nm to 410 nm in pseudocolors. Mitochondria had a higher 480 nm/410 nm ratio than the nucleus, possibly reflecting higher calcium levels and pH in mitochondrial matrix than in cytosol. Scale bar, 10 μm.
(B) Representative fluorescence ratio responses of 8701A/10398A cybrids (left) and 8701G/10398G cybrids (right) evoked by 10 μM histamine. The y axis shows the ratio of the 525 nm fluorescence at 480 nm to that at 410 nm. Blue traces show the ratio in the nucleus and red traces show the ratio in mitochondria. Histamine (10 μM) was added at time 0 s and was not washed out during the experiment.
Using a one-way analysis of variance, we tested whether or not interindividual variation of mtDNA causes differences in basal mtRP fluorescence ratios. A statistically significant variation of basal mtRP fluorescence ratios was found among these cybrid lines (p < 0.001, F = 6.328).
Entire Sequences of mtDNA in All Cybrid Cell Lines
To identify the mtDNA polymorphisms that cause heterogeneity among cybrids, we determined the entire 16.5-kbp sequence of mtDNA in each of the 35 cybrids. mtDNA polymorphisms were selected in comparison with the revised Cambridge Reference Sequence [10,37]. A total of 216 polymorphic sites, including 13 novel polymorphisms, were identified. All cybrids had different sequences.
Identification of mtDNA Polymorphisms Altering the Fluorescence Ratio of mtRP
To identify the polymorphisms with functional significance, all nonsynonymous polymorphisms or polymorphisms in tRNA or rRNA found in more than three cybrids (n = 16) were selected (Table 1). For each polymorphism, the average mtRP fluorescence ratios for two genotypes at the particular polymorphic site were compared (Table 1). The polymorphisms at only two positions, 8701 and 10398, showed nominally significant differences of basal mtRP fluorescence ratios. Basal mtRP fluorescence ratios in cybrids with mtDNA 10398A were significantly higher than those with 10398G (10398A, 1.67 ± 0.09 [mean ± SD], n = 11; 10398G, 1.59 ± 0.12 [mean ± SD], n = 24; p < 0.05, Mann-Whitney U test). Basal mtRP fluorescence ratios in the cybrids with 8701A were also significantly higher than those with 8701G (8701A, 1.67 ± 0.1 [mean ± SD], n = 13; 8701G, 1.59 ± 0.11 [mean ± SD], n = 22; p < 0.05). Because 8701A was linked to 10398A in all but two cybrids, it was difficult to assess the independent effects of these polymorphisms. Since these two cybrids showed intermediate values (average fluorescence ratio, 1.64), it suggests that both of these polymorphisms may contribute to the observed difference. Because there were only two subjects with 8701G/10398A, further analyses were performed using only 8701A/10398A and 8701G/10398G cybrids.
The mtDNA Polymorphisms in 35 Cybrids
Basal mtRP fluorescence ratios in cybrids with 8701A/10398A mtDNA were significantly higher than those with 8701G/10398G mtDNA (8701A/10398A, 1.67 ± 0.09 [mean ± SD], n = 11; 8701G/10398G, 1.59 ± 0.11 [mean ± SD], n = 22; p < 0.05, Mann-Whitney U test; Figures 1 and 2). Peak mtRP fluorescence ratios after the histamine stimulation also showed a similar but nonsignificant trend (8701A/10398A, 1.86 ± 0.11 [mean ± SD]; 8701G/10398G, 1.78 ± 0.14 [mean ± SD]; p = 0.05).
Differences between the basal fluorescence ratios in 10398A and 10398G cybrids are shown. All cybrids with 8701A have 10398A (left). While most cybrids with 8701G had 10398G (right), two cybrids had 8701A and 10398G (the data of these two cybrids were 1.52 and 1.77). For each cybrid, data taken from 11–42 cells (average, 26 cells) were averaged. The y axis shows the ratio of the 525 nm fluorescence at 480 nm to that at 410 nm. Horizontal bars indicate the mean values for each type of cybrid. *p < 0.05 (Mann-Whitney U test).
Replication Study Using Cybrid Cell Lines Stably Expressing RP
Because the nominally significant effects of polymorphisms at positions 10398 and 8701 on basal mtRP fluorescence ratio were found after multiple statistical analyses, we performed a second study to ensure it was not a false-positive finding. Three cybrid lines with 8701A/10398A mtDNA and three with 8701G/10398G mtDNA were chosen from among the 35 cybrids for the replication study (Table S1). The results confirmed our initial observation of a higher basal mtRP fluorescence ratio in cybrids with mtDNA 8701A/10398A (8701A/10398A, 1.78 ± 0.04 [mean ± SD], three cell lines; 8701G/10398G, 1.67 ± 0.01 [mean ± SD], three cell lines). Peak mtRP fluorescence ratios after the histamine stimulation also showed a similar trend (8701A/10398A, 1.99 ± 0.04 [mean ± SD], three cell lines; 8701G/10398G, 1.88 ± 0.02 [mean ± SD], three cell lines).
Replication Study Using Independently Established Cybrid Cell Lines
To further confirm that the observed difference is caused by the difference of mtDNA sequence, we performed another replication study using independently established cybrid cell lines. In this experiment, the native 143B.TK− ρ0206 cell line was used without any further subcloning. We established independent cybrid cell lines by using platelets derived from a subgroup of the original 35 donors (Table S1). By transiently transfecting the cybrids with ratiometric coxIV-Pericam cDNA, fluorescence imaging was performed. Moreover, cells were stimulated by 10 μM histamine, and in vivo calibration was performed using calibration buffers with ionomycin, which equilibrate intramitochondrial and extracellular calcium. To minimize the cell-to-cell variability, an index for calcium level, (R − Rmin) / (Rmax − R) (see Materials and Methods), was analyzed in cells responded to histamine. As a result, our initial observation in cybrids with mtDNA 8701A/10398A was confirmed (8701A/10398A, 1.07 ± 0.40 [mean ± SD], three cell lines; 8701G/10398G, 0.51 ± 0.09 [mean ± SD], three cell lines; Figure S1).
Generation of pH Indicator for Measurements of Mitochondrial Matrix pH
It has been reported that the fluorescence ratio of RP is dependent not only on calcium levels but also on pH [32,33], especially at the higher pH range found in the mitochondrial matrix. To test the relative contributions of these factors to the observed finding, we attempted to measure mitochondrial pH using mitochondria-targeted, pH-sensitive green fluorescent protein (mt pH-GFP) [38,39] and mitochondria-targeted red fluorescent protein from Discosoma (mt DsRed) expressed under the control of a bidirectional promoter [40–42]. Because the pKa of pH-GFP is approximately 8.0, it is useful for measuring mitochondrial matrix pH. Since DsRed is a pH-insensitive protein, it can be used as a reference [43].
We confirmed that this pH indicator, mt pH-GFP/DsRed, could be successfully used to measure mitochondrial pH.
Measurements of Mitochondrial Matrix pH in Cybrids with mtDNA 8701A/10398A and 8701G/10398G
For the measurements of mitochondrial matrix pH, we used the above-mentioned independently established lines of cybrids generated from the native 143B.TK− ρ0 206 cell line that does not carry RPs (four with 8701A/10398A and four with 8701G/10398G). By transiently cotransfecting the mt pH-GFP/DsRed and Tet-Off vectors, basal mitochondrial matrix pH was recorded for several minutes in each cybrid cell line. We performed in vivo calibration of mitochondrial basal matrix pH in each cell by using four calibration buffers (pH 7.0, 7.5, 8.0, and 8.5) with the ionophores nigericin and monensin, which equilibrate intramitochondrial and extracellular pH. The calculated basal mitochondrial matrix pH was about 8.0, which is consistent with previous reports using HeLa cells and ECV304 cells [34,35]. Compared with the 8701G/10398G cybrids, the basal mitochondrial matrix pH was significantly lower in the 8701A/10398A cybrids (8701A/10398A, 8.03 ± 0.03 [mean ± SD], four cell lines; 8701G/10398G, 8.13 ± 0.07 [mean ± SD], four cell lines; p < 0.05, t test; Figure 3).
Data from 11–18 cells in a triplicate experiment were averaged for each of four cell lines in each group. The y axis shows mitochondrial matrix pH calculated by calibration in vivo. Horizontal bars indicate the mean values for each type of cybrid. *p < 0.05 (t test).
Cytosolic Calcium Dynamics
To test whether or not these mtDNA polymorphisms affect cytosolic calcium dynamics, we examined the effects of 8701A/10398A and 8701G/10398G genotypes on the cytosolic calcium levels.
The nucRP data from the first experiments indicated that the 8701A/10398A cybrids tended to have higher peak cytosolic calcium levels after the histamine stimulation compared with the 8701G/10398G cybrids (8701A/10398A, 2.10 ± 0.14 [mean ± SD], 11 cell lines; 8701G/10398G, 2.02 ± 0.15, 22 cell lines, [mean ± SD]; p = 0.08, Mann-Whitney U test; Figure 4). There was no significant difference of basal cytosolic calcium level (8701A/10398A, 1.29 ± 0.07 [mean ± SD], 11 cell lines; 8701G/10398G, 1.25 ± 0.07 [mean ± SD], 22 cell lines; p > 0.10, Mann-Whitney U test). This result suggests that cytosolic calcium response may be enhanced in the 8701A/10398A cybrids.
The values of two cybrids with 8701A/10398G were 1.92 and 2.20. For each cybrid, data taken from 11–42 cells (average, 26 cells) were averaged. The y axis shows the ratio of the 525 nm fluorescence at 480 nm to that at 410 nm. Horizontal bars indicate the mean values for each type of cybrid. +p = 0.08 (Mann-Whitney U test).
The nucRP data from the replication study using cybrid cell lines stably expressing RP confirmed that peak cytosolic calcium levels after histamine stimulation were higher in the 8701A/10398A cybrids than in 8701G/10398G cybrids (8701A/10398A, 2.56 ± 0.03 [mean ± SD], three cell lines; 8701G/10398G, 2.26 ± 0.16 [mean ± SD], three cell lines).
In the present study, we comprehensively searched for mtDNA polymorphisms that alter the fluorescence ratios of mtRP, and we identified two mtDNA single nucleotide polymorphisms (mtSNPs), 10398A and 8701A, that increase the basal fluorescence ratios of mtRP.
In the 10398A/G mtSNP, threonine is substituted by alanine at the C terminus of ND3, a subunit of complex I (NADH: ubiquinone oxidoreductase) [10]. In the 8701A/G mtSNP, there is an amino acid substitution from threonine to alanine at ATPase6, the F0 subunit 6 of complex V (ATP synthase) [10]. Complex I generates a proton gradient across mitochondrial inner membrane by exporting protons, whereas ATP synthase produces ATP by using this proton gradient. The proton gradient is also the driving force of calcium uptake by mitochondria. Therefore, both polymorphisms can affect the proton gradient across mitochondrial inner membrane and calcium levels.
During the course of our study, it was reported that the fluorescence ratio of RP is dependent not only on calcium levels but also on pH at the higher pH ranges in mitochondrial matrix [32,33]. Thus, we measured basal mitochondrial matrix pH in cybrids with 8701A/10398A and 8701G/10398G. Basal mitochondrial matrix pH was significantly lower in cybrids with 8701A/10398A than in those with 8701G/10398G. Alteration in mitochondrial pH has also been hypothesized to modulate ATP production, apoptosis, and the opening of the mitochondrial membrane permeability transition pore [44]. Transport of protons is coupled with electron transport by a respiratory chain consisting of complexes I, III, and IV. As described above, the mtDNA 10398 polymorphism affects the amino acid composition in a subunit of complex I. When the activity of complex I in the electron-transport chain was measured using citrate synthase activity as a reference [45], the activity of 8701A/10398A cybrids tended to be lower than that of 8701G/10398G cybrids (8701A/10398A, 5.77 ± 1.41 [mean ± SD], four cell lines; 8701G/10398G, 7.58 ± 1.27 [mean ± SD], four cell lines; p = 0.10, t test). This finding supports the above-mentioned difference in mitochondrial matrix pH.
The difference in basal mitochondrial matrix pH could not contribute to the initial finding of higher mtRP fluorescence ratio in the cybrids with 8701A/10398A because lower pH could only decrease the mtRP fluorescence rate, not increase it. Therefore, the most probable interpretation for the difference in basal mtRP fluorescence ratio is higher mitochondrial calcium levels in cybrids carrying 8701A/10398A. In addition, the calcium responses to histamine stimulation tended to be higher in the cytosol of 8701A/10398A cybrids than 8701G/10398G cybrids, both in the initial experiment and the replication study using cybrid cell lines stably expressing RP. These findings suggest that alterations in mitochondrial function cause the enhanced cytosolic calcium response and higher basal mitochondrial calcium levels in cybrids with 8701A/10398A.
Mitochondria have several calcium transport mechanisms. Calcium uptake is attributable to a mitochondrial calcium uniporter in inner mitochondrial membrane. Calcium efflux from mitochondria is mediated by the Na+/Ca2+ exchanger and the H+/Ca2+ antiporter. The opening of the membrane permeability transition pore also allows efflux of calcium from mitochondria. Clarification of mitochondrial calcium transport systems at the molecular level would facilitate the understanding of biochemical mechanism of how the 10398 polymorphism alters mitochondrial calcium dynamics. Further study is needed to reveal how these two mtSNPs alter mitochondrial matrix pH and calcium levels.
Among the two polymorphisms we found to have functional significance, 10398A/G has previously been associated with human health and diseases. The 10398G polymorphism is reportedly associated with a reduced risk of Parkinson disease [19], whereas 10398A was reported to increase the risk of Alzheimer disease in men [17], invasive breast cancer in African-American women [24], and prostate cancer in African-American men [25]. In addition, 10398A is reportedly associated with an increased risk of bipolar disorder [22,23,46]. According to the mtSNP database (http://www.giib.or.jp/mtsnp/index_e.html), most Europeans have 8701A, but it is polymorphic in the Japanese population. In contrast, 10398A is polymorphic both in Europeans and Japanese. The 10398A/G characterizes the European haplogroup I, J, and K [17], and Asian-specific super haplogroup M [47]. Thus, the present findings can be translated that Asian-specific super haplogroup M is associated with different mitochondrial pH and calcium from other Asian-specific haplogroups.
The frequency of 10398A is much smaller in the Japanese population (approximately 29% in the mtSNP database) compared with Europeans (approximately 74% in the mtSNP database). The average life expectancy in Japan is the highest in the world, and its biological basis is not yet well understood. Tanaka et al. sequenced the whole mtDNA sequence in 11 Japanese centenarians and reported that a subset of haplogroup M characterized by several mtDNA polymorphisms is more frequently seen in centenarians [48]. Among these polymorphisms, they have mainly focused on the 5178A polymorphism that characterizes haplogroup D in super haplogroup M. The 5178C/A is associated with several common diseases such as Parkinson disease [28], atherosclerosis in patients with diabetes mellitus [49], and myocardial infarction [50]. A recent study, however, suggested that association of 5178A with longevity is ethnicity dependent [51]. Therefore, it might be possible that these apparent effects of 5178C/A in the Japanese population may be mediated by the other linked polymorphisms that cause functional change. Although 5178A did not affect the fluorescence ratio of mtRP in this study, most subjects with 5178A have 10398G. Thus, it could be possible that 10398G mediates these apparent associations of 5178C/A with complex diseases and longevity. In fact, based on the mtSNP database, 10398G was significantly more frequently seen in centenarians (76 of 96 [79%]) compared with young Japanese (129 of 192 [67%]; p = 0.03). A recent study by Niemi et al. also suggested that 10398G affects longevity [52].
To date, there has been no study on the association of 8701A/G with health and disease, and information on 8701A/G is limited. Most of the above-noted associations with the 5178 polymorphism observed in the Japanese population could also be mediated by 8701, since it is closely linked with 10398 and 5178. In fact, 8701G is also more frequency seen in centenarians (68 of 96 [71%]) compared with young Japanese (111 of 192 [58%]; p = 0.03).
It was difficult to assess the functional effects of these two mtSNPs separately, because there were only two subjects carrying 8701A/10398G in our sample set. Although it is still an open question whether or not 8701A/G causes some functional change, at least 10398A/G seems to have some functional significance. The tendency of altered complex I activity between the 8701A/10398A and 8701G/10398G cybrids suggest that the observed difference between 8701A/10398A and 8701G/10398G is mediated at least partly by the functional alteration of complex I by 10398A/G.
Because there was considerable overlap, including an outlier, with regard to the basal mtRP fluorescence between 8701A/10398A and 8701G/10398G, there might be a concern that the observed difference may be caused by those outliers. Thus, we applied the Smirnov-Grubbs test for the detection of outliers. One cell line, having the lowest value in the 8701G/10398G group, was found to be an outlier (T = 3.17; p < 0.05). Even after this cell line was omitted, basal mtRP fluorescence ratios in cybrids with 8701A/10398A were significantly higher than those with 8701G/10398G (8701A/10398A, 1.67 ± 0.09 [mean ± SD], n = 11; 8701G/10398G, 1.60 ± 0.09 [mean ± SD], n = 21; p < 0.05, Mann-Whitney U test). Thus, the observed difference was not due to the effect of the outlier. One might suspect that observed variation of basal mtRP fluorescence ratios might not be caused by mtDNA sequence variation but resulted from an experimental variation. However, the interassay variance within one cybrid cell line having the same mtDNA sequence was significantly smaller than the variation among cybrid cell lines having different mtDNA sequences. This indicates that observed variation of mtRP fluorescence ratios among cybrid cell lines were not caused by experimental variation but caused by the difference of mtDNA sequence.
There might be the other concern that the present finding is somehow affected by the process of subcloning, and such a finding cannot be generalized. However, in our replication study using independently established cybrid cell lines, it was confirmed that the observed finding was not specific to this particular subcloned cybrids, but was actually caused by differences of mtDNA sequences.
In summary, we identified two mtSNPs that were suggested to alter mitochondrial matrix pH and intracellular calcium dynamics. To our knowledge, this is the first report of mtDNA polymorphisms affecting these intracellular functions. Among the two mtSNPs examined, the 10398A/G polymorphism was previously reported to be associated with Parkinson disease, Alzheimer disease, some kinds of cancer, bipolar disorder, and longevity. A drug that affects mitochondrial matrix pH or calcium levels might be a promising strategy for health and disease.
Materials and Methods
Cybrids were generated from platelets derived from 35 volunteers, 17 patients with bipolar disorder (mean age, 48 years; 9 men and 8 women) and 18 healthy controls (mean age, 48 years; 10 men and 8 women). All were Japanese. Patients with bipolar disorder were included in this study because these blood samples were collected as a part of a project to study the genetics of bipolar disorder. None of the experimental parameters reported in this paper showed a significant difference between patients and controls. Diagnoses were made by the consensus of two senior psychiatrists using the DSM-IV criteria. Written informed consent was obtained from all volunteers. The Ethics Committees of the RIKEN Brain Science Institute and other participating institutes approved the study. For the replication study using cybrid cell lines stably expressing RP, we selected cybrids from three subjects with 8701A/10398A mtDNA (mean age, 48 years; 1 man and 2 women) and three subjects with 8701G/10398G mtDNA (mean age, 51 years; 1 man and 2 women). Cybrids for replication studies using independently established cybrid cell lines and measurements of mitochondrial matrix pH were generated using platelets from four 8701A/10398A subjects (mean age, 52 years; 2 men and 2 women) and four 8701G/10398G subjects (mean age, 56 years; 2 men and 2 women). Cybrids for replication studies and the measurement of mitochondrial matrix pH were derived from healthy controls. Although these cell lines have several mtDNA polymorphisms in addition to 8701 and 10398, they were carefully selected so that they have minimum difference in other positions of mtDNA.
Generation of the 143B.TK− ρ0206 cell line stably carrying calcium-sensitive probes.
The 143B.TK− ρ0206 cell line derived from the osteosarcoma cell line (143B.TK−) was established by Attardi and King [30]. The 143B.TK− ρ0206 cells were grown in DMEM (Invitrogen, Carlsbad, California, United States), supplemented with 10% FBS, 50 U penicillin, 50 μg/ml streptomycin, and 50 μg/ml uridine. The cells were grown on dishes 35 mm in diameter to 50% confluence. The cells were cotransfected with Lipofect AMINE PLUS (Invitrogen) with ratiometric Pericam-nuc (0.25 μg DNA/dish) and ratiometric coxIV-Pericam (0.75 μg DNA/dish) cDNAs. After 2 d, these cells were replated onto coverslips 14 mm in diameter and allowed to grow to 2.5% confluence with selection medium, supplemented with 10% FBS, 1,000 μg/ml geneticin (Invitrogen), and 50 μg/ml uridine. After 2 wk, a single colony with fluorescence in nuclei and mitochondria was picked up under a fluorescent microscope. mtDNA-specific PCR and Southern blot analysis were used to confirm that the cells lacked mtDNA. The cell lines were replated onto coverslips and stimulated by histamine (Sigma-Aldrich, Saint Louis, Missouri, United States) as described below. One of the ten cell lines had a reproducible calcium response without spontaneous calcium oscillation. This line was selected for further examination.
Generation of cybrid cell lines.
Peripheral blood was drawn into a 10 ml Vactainer tube containing 1.5 ml acid citrate dextrose (BD Biosciences, Palo Alto, California, United States). Platelets were separated by centrifugation at a speed of 1,200 rpm for 20 min. The 143B.TK− ρ0206 cells that had been stably expressing RPs were fused with platelets from individuals using a polyethylene glycol solution [53]. After 3 d of fusion, the medium was replaced by a selection medium without uridine. After 2 wk, surviving cells were collected. The integration of mtDNA was verified by Southern blot analysis as previously described with some modifications [54]. Cybrids for the replication study using independently established cybrid cell lines and measurements of mitochondrial matrix pH were generated by fusing the native 143B.TK− ρ0206 cells and platelets from individuals as described above.
Measurements of fluorescence ratios of RP.
Cybrids in modified Krebs R buffer (125 mM NaCl, 5 mM KCl, 5.5 mM glucose, 20 mM HEPES, and 1 mM MgCl2 [pH 7.4]) were imaged at room temperature on an Olympus IX-70 (Olympus, Tokyo, Japan) with a CoolSNAPHQ CCD camera (Roper Scientific, Tucson, Arizona, United States) controlled by Universal Imaging Meta series 4.5/4.6 (Universal Imaging, Media, Pennsylvania, United States). Dual-excitation imaging with RP used two excitation filters (480DF10 and 410DF10), alternated by a Lambda 10–2 filter exchanger (Sutter Instruments, Novato, California, United States), a 505DRLP-XR dichroic mirror, and a 525AF45 emission filter. The cybrids were stimulated by bath-application of 10 μM histamine applied to the coverslip. The ratio of the 525 nm fluorescence at 480 nm to that at 410 nm was used for further analysis. The regions of interest for measuring calcium response in each cell were the mitochondria and nucleus.
For each cell line, 11–42 cells were measured. At first, the basal fluorescence ratios of mtRP obtained from each cell were used for statistical analysis by one-way analysis of variance. Using one-way analysis of variance (df = 34), we tested whether or not the variation among 35 cybrid cell lines having different mtDNA was larger than the variation within one cell line. Then, the 11–42 data points in each cell line were averaged to calculate the representative value for each cell line.
The coefficient of variation in fluorescence ratio of mtRP in each cell line was 12.2% ± 3.2% (mean ± SD in 35 cybrids).
For the replication study using eight independently established cybrid cell lines (four with 8701A/10398A mtDNA and four with 8701G/10398G mtDNA), cybrids were transiently transfected with ratiometric coxIV-Pericam (1 μg DNA/dish) cDNA using Lipofect AMINE PLUS. After 2 d, measurements were performed in these cell lines. Different from the case in the cells stably expressing RP, there is no assurance that the expression level of RP is constant across cells in this experiment, which may potentially affect the stability of fluorescence ratio. To avoid the effect of variability of expression levels of RP among cells, we performed in vivo calibration for each cell. To exactly estimate calcium levels, we applied histamine stimulation to the cells, and only those responded to histamine were further analyzed. Cells responded to calcium could be found in three of four cell lines for each group. Cells were stimulated by 10 μM histamine, and calibration was performed using calibration buffers. Calcium-free buffer consisted of modified Krebs R buffer with 10 μM ionomycin (Calbiochem, San Diego, California, United States), 50 μM BAPTA-AM (Dojindo Laboratories, Kumamoto, Japan), and 10 mM EGTA (Dojindo Laboratories). Calcium saturation buffer was made of modified Krebs R buffer with 10 μM ionomycin and 10 mM CaCl2. [Ca2+] measured by in situ calibration was calculated by the equation [Ca2+] = K′d [(R − Rmin) / (Rmax − R)](1/n), where K′d is the apparent dissociation constant corresponding to the calcium concentration and n is the Hill coefficient [55]. Because K′d and n are constants but K′d of RP in mitochondria is not known, we simply used the value of (R − Rmin) / (Rmax − R) for the assessment of mitochondrial calcium level. The condition for imaging is basically similar to those described above.
Analysis of entire sequences of mtDNA.
Total DNA was extracted from each cybrid using standard protocols. The entire mtDNA was sequenced as described [56]. A computer program, Sequencher version 4.1.1 (Gene Codes, Ann Arbor, Michigan, United States), was used to indicate possible SNP loci. For verification, visual inspection of each candidate SNP was carried out. At least two overlapping DNA templates amplified with different primer pairs were used for identification of each SNP. mtSNPs were identified by comparison with the revised Cambridge sequence (GI1944628) reported by Andrews et al. [37]. For all polymorphisms that resulted in changed amino acid sequence or those within rRNA or tRNA regions, averaged fluorescence ratios of mtRP were compared between genotypes by Mann-Whitney U test. This analysis was performed only when three or more cybrids had the same polymorphism. For statistical analysis, Mann-Whitney U tests were applied using SPSS software (SPSS, Tokyo, Japan).
Construction of pH indicator for measurements of mitochondrial matrix pH.
GFP variants were prepared as described [57]. As a template, the original GFP mutation was employed. This template incorporates the mutation [38,58]. Mutations were verified by sequencing the entire gene. The pH-GFP [39] with polyhistidine tag at the N terminus was expressed in Escherichia coli JM109 (DE3) (Promega, Madison, Wisconsin, United States), purified, and spectroscopically characterized as described [55]. The gene for mt pH-GFP was amplified by PCR using the GFP variants as a template, with a forward primer containing complex IV mitochondrial target sequences and an MluI site, and a reverse primer containing an EcoRV site. The gene for mt DsRed was amplified by PCR using pDsRed1–1 Mito as a template with a forward primer containing a NotI site and a reverse primer containing a SalI site. The restricted mt pH-GFP product was subcloned in-frame into multiple cloning site I, and mt DsRed was subcloned into multiple cloning site II of the pBI bidirectional Tet vector (Clontech Laboratories, Mountain View, California, United States).
Transient expression of pH indicator in cybrid cell lines using the Tet system.
To ensure the same expression levels of two genes, mt pH-GFP and mt DsRed, the bidirectional Tet expression vector was used in Tet-Off Gene expression systems [40,41]. The pBI bidirectional Tet expression vector contains a "bidirectional" promoter composed of a tetracycline-response element flanked by two minimal cytomegalovirus promoters in opposite orientations [42]. To induce these two genes, the other vector expressing the tetracycline-controlled transactivator under the cytomegalovirus promoter control was cotransfected.
Cybrids were grown in DMEM (Sigma-Aldrich) supplemented with 10% Tet system-approved FBS (Clontech), 100 U penicillin, and 100 μg/ml streptomycin. The cells were grown on dishes 35 mm in diameter to about 80% confluence. The cells were cotransfected with Tet-Off vector (Clontech) and pBI–(mt pH-GFP)–(mt DsRed) (total 1 μg DNA/dish) using Lipofect AMINE PLUS (Invitrogen). After 2 d, mitochondrial matrix pH was measured in these cells.
Mitochondrial matrix pH measurements and in vivo calibration.
Cybrids in modified Krebs R buffer were imaged at room temperature. The imaging system is described above. Imaging with mt pH-GFP/DsRed used two excitation filters (480DF10 and S565/25), a dichroic mirror for GFP/DsRed, and emission filters (525AF45 and S620/60).
It was confirmed that the fluorescence ratio (525 nm/620 nm) decreased after the treatment with FCCP (Sigma-Aldrich), suggesting that this set of fluorescent indicators is sensitive to the mitochondrial matrix pH.
For in situ calibration, cybrids were perfused with the following pH titration buffer (125 mM KCl, 20 mM NaCl, 0.5mM CaCl2, 0.5 mM MgCl2, and 25 mM pH buffer [MOPS was used for pH 7.0, HEPES for pH 7.5 and 8.0, and glycylglycine for pH 8.5]) [59]. All calibration experiments were carried out in the presence of 10 μM nigericin and 10 μM monensin (Sigma-Aldrich). After baseline measurements were recorded for several minutes, calibration was performed in vivo. The fluorescence ratio of 525 nm emission at 480 nm excitation to 620 nm emission at 565 nm excitation reflected the dynamics of mitochondrial matrix pH. Mitochondrial matrix pH was calculated by plotting the calibration curve of each cell.
Supporting Information
Figure S1. Effects of 8701A/10398A and 8701G/10398G on Basal Mitochondrial Calcium Levels Measured by mtRP
The y axis shows the basal mitochondrial calcium levels indicated by the value of (R − Rmin) / (Rmax − R) measured by mtRP. In situ calibration for [Ca2+] uses the equation [Ca2+] = K′d [(R − Rmin) / (Rmax − R)](1/n). R is the basal ratio of the 525 nm fluorescence at 480 nm to that at 410 nm. Rmin is the ratio at free calcium. Rmax is the ratio at saturating calcium. K′d is the apparent dissociation constant. n is the Hill coefficient. Bars indicate the standard error of mean for each cybrid cell line.
Table S1. mtDNA Polymorphisms in Eight Cybrids
We sincerely thank the members of the Laboratory for Cell Function and Dynamics at the RIKEN Brain Science Institute. We thank the members of the Research Resource Center at the RIKEN Brain Science Institute, especially Mr. Miyazaki, for technical assistance. We thank Ms. Bonnie Lee La Madeleine and Dr. S. Takeda, and Dr. T. Yoshida for valuable suggestions on the manuscript.
Author Contributions
TK conceived and designed the experiments. AK and KM performed the experiments. AK and TK analyzed the data. AK, KM, TN, SS, MT, MY, NK, AM, and TK contributed reagents/materials/analysis tools. AK and TK wrote the paper.
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|Medicare Prescription Drug Plan Benefit Details in Plain Text
|The following Medicare Prescription Drug plan (PDP) benefits apply to the United American - Select (PDP) plan (S5755 - 073) in CMS Region 1, which includes all counties in ME NH.
This plan is administered by UNITED AMERICAN INSURANCE COMPANY, a national plan provider. This means that the insurance carrier offers Medicare Part D plan in most every state. To switch to a different Medicare Prescription Drug plan or to change your location, click here.
Click here to see this information for the United American - Select (PDP) plan in a chart format along with the plan enrollment options. We will also send a copy of the plan benefit details chart to your email account.
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|The United American - Select (PDP) plan has a monthly premium of $40.20. That is $482.40 for 12 months. There are a few factors that can increase or decrease this premium. If you qualify for full or partial extra help, your premium will be lower. If you have a premium penalty, your premium will be higher.
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|The United American - Select (PDP) plan a new plan in county, therefore we do not yet have membership figures. The Centers for Medicare and Medicaid Services (CMS) has has given this plan carrier a summary rating of 2.50 stars. The detail CMS plan carrier ratings are as follows: a Customer Service Rating of 3 out of 5 stars , a Member Experience Rating of 3 out of 5 stars, and a Drug Cost Information Accuracy Rating of 3 out of 5 stars.
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|The Coverage Gap, which is also known as the Donut (Doughnut) Hole is the phase of your Medicare Part D plan where you are responsible for 100% of your medication costs. Healthcare Reform mandates that the insurance carrier pay 14% of your generic drug prescription costs in the donut hole on your behalf. The brand-name drug pharmceutical company will pay 50% of the cost of your brand-name drugs pruchased in the donut hole on your behalf. Since the brand-name drug manufacturer pays on your behalf, the portion that they pay counts toward your TrOOP (or True Out of Pocket) costs. Some Medicare Part D plans offer coverage during the Coverage Gap that is beyond the mandated discounts. Any drug not covered by the plan's Gap Coverage will still receive the discounts noted above -- even if the plan has "No Gap Coverage". This plan (United American - Select (PDP)) offers No Coverage during the Coverage Gap phase.
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The Restoration Gameby Ken MacLeod
There is no such place as Krassnia. Lucy Stone should know—she was born there. In that tiny, troubled region of the former Soviet Union, revolution is brewing. Its organizers need a safe place to meet, and where better than the virtual spaces of an online game? Lucy, who works for a start-up games company in Edinburgh, has a project that almost seems made for
There is no such place as Krassnia. Lucy Stone should know—she was born there. In that tiny, troubled region of the former Soviet Union, revolution is brewing. Its organizers need a safe place to meet, and where better than the virtual spaces of an online game? Lucy, who works for a start-up games company in Edinburgh, has a project that almost seems made for the job: a game inspired by The Krassniad, an epic folk tale concocted by Lucy's mother, Amanda, who studied there in the 1980s. Lucy knows Amanda is a spook. She knows her great-grandmother Eugenie also visited the country in the 1930s and met the man who originally collected Krassnian folklore, and who perished in Stalin's terror. As Lucy digs up details about her birthplace to slot into the game, she finds the open secrets of her family's past, the darker secrets of Krassnia's past—and hints about the crucial role she is destined to play in The Restoration Game.
Combining international intrigue with cutting-edge philosophical speculation, romance with adventure, and online gaming with real-life consequences, this book delivers as science fiction and as a sharp take on our present world from the viewpoint of a complex, engaging heroine who has to fight her way through a maze of political and family manipulation to take control of her own life.
Editorial Reviews
"In this brash, odd tour-de-force, he succeeds partly by taking chances many authors would balk at and partly by virtue of some solid craftsmanship. The Restoration Game is the first Ken MacLeod novel I've read. It won't be the last."
-Green Man Review
"This is a writer at the peak of his powers."
Product Details
- 9781616145255
- Publisher:
- Prometheus Books
- Publication date:
- 09/20/2011
- Pages:
- 240
- Sales rank:
- 896,322
- Product dimensions:
- 6.00(w) x 8.90(h) x 0.60(d)
Read an Excerpt
By KEN MACLEOD
Prometheus BooksCopyright © 2011 Ken MacLeod
All right reserved.
Chapter OnePAGING LUCY STONE
I was just out of Arrivals and eyeballing the Remarkables above the Queenstown terminal roof when the PA system called my name.
"Paging Lucy Stone ... Passenger Lucy Stone, just off Flight NZ03 from Auckland, please go to the Airport Information desk."
I stopped dead. My rolling case, in accordance with Newton's first law of motion, kept rolling and collided painfully with my calf muscles. It then rebounded (third law), toppled over (second law), and made me stumble (Murphy's law). As I squatted to recover the case I wondered wildly what I'd done wrong. My passport was in date, my card hadn't coughed at the ridiculously cheap internal flight ticket, and I didn't have any drugs or weapons (apart from the Leatherman Juice ladylike, natch) or animal or plant matter. Could it have been the shower gel? New Zealand's a war-on-terror state but didn't, as far as I knew, share the British and American exploding shampoo bottle and nitroglycerine-in-the-Evian paranoia du jour.
Speaking of paranoia, it didn't so much as flit across my mind that this might have anything to do with the Other Thing. I'd put two oceans between me and the Other Thing.
There was a ball of nickel-iron bigger than the moon between me and the Other Thing. There was a whole planet between me and the Other Thing. No, the Other Thing was definitely partitioned off elsewhere on the hard disk of my spinning mind as I rocked the case back on its wheels and stood up.
Another name and my name sounded across the parking lot, from different loudspeakers like an echo: "Alexander Hamilton ... message for Lucy Stone ... Alexander Hamilton ..."
I straightened, clear in a swift conviction of what was going on. Alec Hamilton. Love of my life had stood me up. So much for the hair down and the pretty skirt. I scrunchied a severe ponytail and marched, case on its leash, back through the sliding doors and over to the Information desk.
"Hi," I said, slapping down my open passport. "Lucy Stone. There's a message for me?"
Black eyebrows arched to a black hairline; slate-grey uniform shoulders shrugged. Over the top of the desk I could see her graphite-pencil skirt. Neat, neat. Maori stewardess, cuter than me.
"Oh yay," she said. An opal oval fingernail swivelled a scrap of paper. "Ms. Stone. For you."
I stared down at it, read it, then picked it up, holding hard on my hand's tremor.
"Thank you," I managed to say, from a dry mouth.
She bestowed me a smile and I gave her a rictus back. I turned around and walked out again, knees shaky as a newborn lamb's. I sat down on a bench facing the parking lot and the not-Remarkable mountain straight ahead. I looked down at the paper again. NZ Airlines headed notepaper with a message in a careful hand that belied the words:
Please tell Lucy Stone that Alexander Hamilton has been unavoidably detained with friends from the East.
That, and a telephone number.
The pretty skirt's lap was vibrating like a drum. I clutched my knees. The shaking spread to my elbows. Coming here and finding Alec had been for me an idea of escape. It had been in a different world entirely from all that other stuff. Alec had been in a different world. He'd known nothing, nothing at all, of my other life. I'd thought I'd been shielding him from it. I had been shielding me. All the partitions in my mind went down. The Other Thing was not on the other side of the world. It had followed me. It was here, and it was coming for me through Alec.
There is no such place as Krassnia. If you were to draw it on a map, right where the borders of Russia, Abkhazia, and Georgia meet, and then fill it in, you'd need a fifth colour. On the other hand, Krassnia is a real place. I know, because I've been there; heck, I was born there. It has an official name, for the day when everyone's embassy recognises it (they won't): the Former Soviet Autonomous Region of Krassnia. FSARK. Look familiar? It should. Walk down any high street in Europe and you'll see these letters in black lowercase: fsark; on red plastic shopping bags, distributed free by the million in a rare fit of marketing nous by the Popular Department Store. All that the Store (Krasnorglav) needs to do now is get people to actually put its wares in the bags; which, since its bestselling lines are pirated CDs and Chinese and Vietnamese fakes of big-name luxury brands, could prove tricky. (There's also the fact that the bags themselves were the result of an accidental five extra zeroes in an order placed with the recently privatised plastics factory, KrasNorPlasKom.)
More about Krassnia later. For now, you only need to remember two things. First, you won't find it on the map, except on very detailed old maps of the SU and maps made by the Krassnian Ministry of Information (Kraskomfakt). Second, I was there when I was very young, and I've been back. Oh yes, I've been back. But when Amanda called, I hadn't been back. Except in dreams. The dreams mattered, as it turned out.
Home, late. Me on the sofa, laptop on my knees. Thai takeaway half eaten, remainder fit only for the fridge and the microwave if tomorrow was the same as today. Cherry smoothie likewise congealing. I was test-playing the raw version of the gory first-person slicer (Dark Britannia, sword and sorcery, barbarian-Arthurian grail quest with Roman-legionary revenants and Pictish zombies) that we hoped would make our fortune, and ticking boxes and noting glitches when the Skype icon winked. The caller ID was Mom.
I saved the action midchop (blue skin splits! green blood splatters!) and opened the speaker.
"Hi, Mom."
"Oh hello, Lucy. Everything's all right."
(Amanda always says that. I appreciate it.)
"I'm fine too."
"It's late where you are."
"It was you who phoned, Mom."
"Oh! Yes. Well." She made one of her us-girls-together noises, which I think is achieved by a light, throaty laugh while rubbing the phone through the hair behind her ear. It's usually a bad sign. "Do you remember Krassnia?"
"Of course I do. We left when I was what? Seven?"
"Seven, yes. So it was. But do you remember the language?"
"I'm not sure."
"Do you ever dream that you're there?"
I stared at the screen as if the Eye of Sauron had just opened and closed on it. "What?"
I closed my eyes, leaned back on the sofa, and thought about it. "Yes, I suppose I do."
I leaned closer again. "Well Mom, yeah, I don't pay much attention to dreams, but as it happens, yes, whenever my unconscious or my brain's offline processing—or whatever it's supposed to be these days—wants me to dream about dark valleys or endless mountain slopes or long corridors where something really scary and official is waiting for me and I've missed my appointment and ... yes, I guess I do go back there."
"Great!" she said. "And what language do you dream all that in?"
"I don't ... Wait. Russian, I think."
"Think harder."
I blinked hard. Amanda not being an early adopter, of video or anything else, the gesture was of course wasted.
"It's a goddamn dialect, Mom!"
"Language!" she chided, ambiguously. "Anyway, not to worry. You'll pick it up again in no time."
"Why," I asked, "would I want to pick it up?"
"I want you to write—is that the word?—a game scenario in it. Written in Krassnian and based on Krassnian legends. You know, like in The Krassniad."
The Krassniad is Amanda's one and only commercial success. After she'd completed her PhD ("Myth, Memory and Ideology in the Krassnian Autonomous Region: An Investigation") and failed to get it published even as an academic book, she'd had the bright idea of doing for Krassnia what James MacPherson had done for Scotland a couple of centuries earlier with the poems of Ossian. She'd taken the raw material of her notes: snatches of poetry still mumbled from the gap-toothed gobs of mountain bards who claimed to have been born in the reign of Tsar Alexander II; such fragments of illuminated manuscript as remained in Krasnod's Museum of the Peoples and in the two Orthodox monasteries that hadn't been turned into Houses of Atheism; and in the one surviving copy of Life and Legends of the Krasnar, compiled in the 1920s by Krassnia's leading Bolshevik ethnologist (shot in 1937)—all that and more she'd cobbled together and "freely translated" into English as ancient lays, into even what you might call a national epic, which was instantly banned in Krassnia (even in English, even under democracy) and enjoyed a brief vogue with the Mind, Body, Spirit crowd for its ancient wisdom and shamanic spirituality and among Hell's Angels and gaming geek-boys for the sword fights and the sex bits.
"You mean that hasn't been done already?" I asked.
(Not the most cogent of questions, but the one at the top of the stack.)
"No, it hasn't," said Amanda, sounding exasperated. "The book's out of print in the US, and it's still banned in Krassnia, not that that makes much difference these days.... Anyway, nobody outside of Krassnia is interested in writing a game for it—the market's too small—and nobody inside of Krassnia is interested in anything but Western and Japanese games. So whoever developed it would have that market all to themselves."
The cat ambled in, stretched himself onto the coffee table, and nosed at the foil containers. I swatted him away. He glared at me from under the stereo.
"Amarket you've just said isn't big enough. The game wouldn't break even."
"I happen to know it would," Amanda said.
I recognised that tone. It wasn't one that expected to be questioned.
"If you say so," I said. "But who would actually develop it?"
"That company you work for," she said. "Digital ... Fist, yeah?"
"Digital Damage Productions," I corrected, abstractedly. "The fist is the logo."
Then I caught up with myself. "But we're just starting! Why not—?"
"Go to one of the big-name companies? You know why not."
I did, too. "OK," I said. "I can see that. But—"
"Look, Lucy, give me a minute to explain how this is gonna work. You told me ages ago that Digital ... whatever was working on some kind of dark fantasy fighting game, yeah? Heroes with swords, craggy landscape, gloomy ruins, spectres and slime, right?"
"Yeah," I said. "Dark Britannia."
"Right!" she said. "All you'd have to do is change the landscape map, tweak the costumes, plug in Krassnian dialogue and prompts, rename and rejig your demons and dwarfs and so forth, and there you have it, tah-dah!—Dark Krassnia!"
Now this was actually not a bad idea, and one very much along the lines that Sean Garrett, the PTB (Pony-Tailed Boss), founder and genius and hard taskmaster of DDP, had been thinking aloud about for months. Aloud is more or less how he does all his thinking, and you can occasionally interrupt this stream or rather torrent of consciousness and break it up into something that could from a distance be mistaken for a conversation (another word for which—here's a clue, Sean—is dialogue) and one component of such sequences that you could have drag-and-dropped into more or less any of his recent rants and rambles was: You know, when this thing takes off we can franchise it out for local adaptation in every fucking country in the world that has a Dark Age heroic mythology and you know what countries that leaves out? Only the ones that are still in the fucking Dark Ages! Ha ha ha!
"That's by no means all," I said. "And I don't see us doing it while we still don't have Dark Britannia done and dusted. When do you need it?"
"Middle of June," she said.
"Four months? No way!"
"It can be as quick and dirty as you like," Amanda said. "We're not talking a flagship release here. Digital could even claim it had been pirated. Come to think of it, that's probably ... hmm ..."
"The money would have to be good," I said. "If I'm going to pitch it to the lads."
"Fifty thousand on agreement, hundred thousand on delivery."
"Pounds? Euros?"
Oh well. Still in not-to-be-sneezed-at territory, for a company as small as ours.
"I think I can make that fly," I said.
"Oh, don't you try selling this to the team," said Amanda. "My people will talk to your people, OK?"
A couple of questions will have occurred to you. One: how is this lady professor of cultural anthropology or whatever going to come up with a hundred and fifty thousand dollars? Two: what does she want with a multiplayer online role-playing game in Krassnian in the first place?
They occur to you, but they didn't occur to me.
The only question that occurred to me was: hmm, so what's behind the CIA's sudden interest in Krassnia?
I already knew the answers to the other two, because I already knew my mother was a spook.
Not that it hadn't been a bit traumatic finding out, at the age of thirteen, right in the middle of my rebellious, weed-smoking, body-piercing, diary-keeping, two-fingers-down-the-throat puking, hormone-churned huff at the world. If this scene was in a movie they'd need to cast a different actress, who'd be in the credits as Teenage Lucy, and rummage up a roomful of late-nineties kipple. So you imagine me sitting on my bed, chin on knees poking through the cultivated distress of my jeans, leaning on a big batik cushion and facing a Kurt Cobain poster on the wall opposite. The Cranberries are messing with my head through earphones the size of earmuffs. I'm reading a thick Guy Gavriel Kay paperback. Scene set? Good. Enter Amanda, after a token knock.
I scowled at her and saw her lips move. I stuck a thumb in the book and reached around the back of my head and prised away Dolores's dolorous lyrics.
"There's something I've been meaning to tell you," Amanda said, looking awkward.
"I know," I said. "Don't smoke. Do my homework. Use a condom. Eric isn't my father."
Wow, that one worked. I could see her flinch. I almost felt sorry for her. Almost. But I was cruel then.
"That isn't ..." she said.
"That wasn't ..." she added.
"How do you know?" she got her act together enough to ask.
"Mom, I'm not stupid," I said.
She didn't inquire further on that point. She came and sat down on the only other seating in the room, an old beanbag opposite the bed, sinking so far that I had to lean forward to see her face.
"What I've been meaning to tell you," she said, "is, um ... it's about Krassnia. We had some good times there, didn't we?"
"Yes, Mom, I had a very happy childhood there. Until you took us away from it."
She winced again. "It wasn't my choice. The place looked like it was about to blow. All US citizens were advised to leave."
"And nothing happened."
(Apart from the scariest day of my life, which rather undercut my point, but Amanda ignored that opening.)
"We didn't know that then," she said. "Anyway"—she chopped with her hand, looking impatient—"that's all beside the point. I'm not going to let you rake all that up again. This is about something that really does concern you. It's about what I was doing in Krassnia in the first place."
She leaned back farther into the beanbag, as if to make sure that if I were to make some sudden movement, she would be out of range.
"Your research?"
"Kind of," she said. "Um, well. My research wasn't just for my thesis, and it wasn't just about, you know, all that ancient stuff. I was sending a lot of it to, well, someone at the US embassy in Moscow. Someone who sent it all back to, um, to Langley, Virginia."
"You were a CIA agent?" I shrieked.
"The correct term is 'asset,'" she quibbled. "But, yes, that's about the size of it."
The implications weren't really sinking in yet.
"Why are you telling me now?" I asked.
"I've been exposed," she said. "A guy at Langley has been arrested for working for the Russians, and for the Soviets before that, and he's confessed. He gave the Agency a list of the people he exposed."
"And your name's on the list?"
Amanda nodded. "Uh-uh. None of this is public, OK?"
"So why are you tellingme?" I demanded. "AmI on it? Are you in danger?"
"No," she said. "People I worked with, yeah . . ." She chewed her lip, looking up to a corner of the ceiling, and sighed. "Some of them have been arrested already."
"But Krassnia broke away, didn't it? Why should they care about anyone's spying on the Soviet Union?"
Excerpted from THE RESTORATION GAME by KEN MACLEOD Copyright © 2011 by Ken MacLeod. Excerpted by permission of Prometheus Books. All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.
Meet the Author
Ken MacLeod was born in Stornoway, Isle of Lewis, Scotland, on August 2, 1954. He is married with two grown-up children and lives in West Lothian. He has an honours and a master's degree in biological subjects and worked for some years in the IT industry. Since 1997 he has been a full-time writer, and in 2009 was Writer in Residence at the ESRC Genomics Policy and Research Forum at Edinburgh University. He is the author of twelve novels, from The Star Fraction (1995) to The Restoration Game (2010), and many articles and short stories. His novels have received two BSFA Awards and three Prometheus Awards, and several have been short-listed for the Clarke and Hugo Awards. Ken MacLeod's weblog is The Early Days of a Better Nation: http://kenmacleod.blogspot.com.
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Part science-fiction (only a small part) mostly international intrigue and spy thriller. If you are intrigued by the Matrix and really like the Davinci Code and spy thrillers/mysteries you will really enjoy this. The main focal point is a country that doesn't exist, so don't bother looking for it on a map. The only place it exists is in our minds. Like many other real countries, the country of Krassnia is trying to find an identity. The conflicts this involves seem to be something we would be hearing about in the news today. These issues, which are shown to be deep reaching and long lasting (as with many countries today) greatly complicate the missions and the search for the truth. The main character is someone we can all empathize with. A young lady searching for her truth and finding out **********spoiler*********. And then ***********spoiler***********. Sorry, I can't tell you any more, or it will really ruin the enjoyment of a very well written story by an award winning, greatly respected author. If this is your first introduction to Ken Macleod, you will really enjoy the visit. If you have tried some of his work before, you will not be disappointed. Get the book, enjoy the story and wear a warm coat. © Night Owl Reviews
Her CIA asset mom Amanda who wrote the definitive mythology on Krassnia asks her game designer daughter Lucy Stone to create a special interactive multiplayer game based on their heritage. Lucy who has not been back to Krassnia, the former Soviet Autonomous Region since they left when she was a small child, looks forward to going "home". Amanda's objective is to enable the country's revolutionaries to use the game clandestinely to communicate and cohesively organize. As she readjusts to the locality's language, Lucy hears tales about a power source high in the mountains that the Soviets never could replicate, but has renewed interests from outside parties not concerned about collateral damage. Starting with Newton's Laws of Motion combined with Murphy's Law, The Restoration Game is an insightful satirical espionage thriller. The story line contains an incredible amount of information on Krassnia such as how the Soviets converted a nation filled with literates into illiterates with a stroke of a pen and the Post-Soviet government turned a nation filled with literates into illiterates with a stroke of a keyboard. Although there is so much going on in terms of culture, mythology and the power source, the story line rarely accelerates. Readers who appreciate a deep cerebral thriller will enjoy a visit to Krassnia. Harriet Klausner