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Period due and as usual just before, feeling whistful, feminine, and very very horny. I tend to regress at this time of my cycle, although I'm only 19, I guess there's not too far to regress to, but that's what I feel like, anyway. I tend to dress either 'young teen' or sensually. Never a mix of the two. I wan...» Read more
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Search Results for: Sweat
Posted by: Pilotssister Age: 19 Posted on: 23 Apr 2017 2 comments
3 likes 390 views Category: Female Solo Tags: masturbating, mood, dressing up,
Posted by: Pilotssister Age: 19 Posted on: 20 Apr 2017 1 comments
2 likes 333 views Category: Female Solo Tags: fantasy, masturbation, watched,
Another lovely day. Brighton beckons!
Another sunny warm day, and I've just showered and picked out a linen summer dress. It's white and I know that with the sun behind me, it's almost transparent. This one needs a careful choice of panties. Almost everything shows through, and I've even worn black one... » Read more
Posted by: footlover28 Age: 15 then Posted on: 18 Apr 2017 4 comments
10 likes 1307 views Category: Male-Male Tags: friends, naked outside, masturbating, helping, streaking
After finishing the last one, I thought I might write another story, this time with a different style
Back when I was in high school, I was friends with a guy I had known for many years, since around kindergarten. The summer after our freshman year, I talked my parents into dropping me off at his house one Frid... » Read more
Posted by: Menace1 Age: 30 Something Posted on: 18 Apr 2017 2 comments
5 likes 774 views Category: Male-Female Tags: Outdoors, fingering, wet, fit
This incident surprised the hell out of me whilst I was enjoying some fresh air and exercise last week.
I recently decided to take a short break from work. One of my aims was to have a bit of time for myself and in particular, I wanted to take a bit of exercise and get myself back in a slightly healthier ... » Read more
Posted by: Maturebator Age: 67 Posted on: 12 Apr 2017 2 comments
6 likes 602 views Category: Male Solo Tags: introduction, first time, masturbate
My introduction to masturbation
It all began one sultry August afternoon 56 years ago, as I was sweeping out the garage. It was just a hot, sticky day and I was wearing just sneakers and cut-off shorts. And all of a sudden, in walks the kid from next door who was a few years older than I, and a... » Read more
Posted by: Pilotssister Age: 19 Posted on: 01 Apr 2017 2 comments
7 likes 2002 views Category: Female-Female Tags: first time, masturbation, video,
As I told you I've been scoping out my close friends about their sexual emergence. Now to give this some context, remember, initially at least I was brought up Catholic. I met a lot of my early friends through church, and Anna was one of them. While I got less and less interested in church, Anna gor more and more in...» Read more
Posted by: shashtzoh Age: 18 Posted on: 15 Mar 2017 3 comments
6 likes 1790 views Category: Female-Male Tags: Rafting, Sauna, Sweat, Cold, moms friend, fart
When I was a senior in high school, I was invited to participate in a 5 day-long science field trip. While this was pretty special, something unexpected formed a lifelong sexual memory for me.
Every day was spent on the wild and natural river, riding down in big rubber rafts or boats that were from WW II, the h... » Read more
Posted by: Pilotssister Age: 18 till romorrow Posted on: 27 Feb 2017 0 comments
4 likes 820 views Category: Female Solo Tags: sweat, scent, hair,
This isn't a dirty story as such, but it does have a few questions, and a few references to my early masturbatory experiences.
In my early teens I was always very sensitive between my legs, and also very wet at times. I do mean very wet too. This was not just some cream in my panties, this was wetness enough to... » Read more
Posted by: polywheels Age: 25 then, 34 now Posted on: 24 Feb 2017 1 comments
3 likes 729 views Category: Male Solo Tags: Mormon, BYU, summer, cerebral palsy
I'm sorry for the LONG-TELLING of this story...
From my previous story: �My First Handjob�, you learned that I have a disability called: cerebral palsy. And what that means: Is all is well with my mind, it is just my body/muscles are different than the �normal�. There are things that I CAN and CAN�T DO but I kn... » Read more
Posted by: Pilotssister Age: 18 Posted on: 23 Feb 2017 1 comments
7 likes 1437 views Category: Men with Women Tags: brother, fantasy, role play,Huge storm and a tree down at the start of the long lane down which we live. My parents phoned and said they would book a motel for the night and the next day arranged to get the tree cleared. Almost as soon as the call was over, the power went out. This left me alone with Chris for the night.....well.....what's a girl... » Read more
Posted by: shashtzoh Age: 28 Posted on: 02 Feb 2017 0 comments
2 likes 1108 views Category: Female-Male Tags: Fingering, Precum, Wet,
This is another episode in a relationship with Barbara that started right after my divorce. She was still married but was on the way to filing for a divorce.
One afternoon Barbara called me and said she was alone for awhile because her husband had taken her children to the movies. This may not sound like ... » Read more
Posted by: SoloTouchArchive Author: Michael Age: 19 Posted on: 22 Jan 2017 1 comments
7 likes 2434 views Category: Male-Male Tags: gay, sucking, oral
Fun time with a friend!
My friend Willy was 17 years old nearly four years ago. I had a crush on him since before we got to know each other after a summer camp in school when we had nights chatting about friends and girls. He was a member of the swimming team of our college and many gays in our campus admire hi... » Read more
Posted by: qwickh1 Age: 19 Posted on: 16 Jan 2017 1 comments
1 likes 874 views Category: Male Solo Tags: lonley, student, college, bathroom, toilet
This story is for all of the hardworking students out there that can�t seem to find love no matter how hard they try�
Perhaps one of the most distressing aspects of being a student is finding love. Not our grades, not our financial responsibilities, and not the superficial titles we are trying to earn.  ... » Read more
Posted by: Runnerdude2 Age: 32 Posted on: 16 Jan 2017 3 comments
4 likes 2031 views Category: Male-Male Tags: frot, m2m, massage, oil, cock, runner
A good massage.
When he flipped over, I was pretty impressed by his body. The abs were like a washboard and the cock stood straight up pointing at his chin. I strattled him as before. This time I was riding my ass on top of his cock. I could feel the pulsating, and it was nice. I liked how his cock ... » Read more
Posted by: prestonjesquire Age: 19 then Posted on: 03 Jan 2017 3 comments
10 likes 2045 views Category: Male-Female Tags: caught, neighbor, redhead, tits, cum, panties, mutual
My first encounter with my neighbor Amanda
I had finished up my Freshman year in college and was back home for the summer. I hadn�t found a job yet and didn�t plan to for a little while as I felt like I needed a break. I slept in late most days and, being the only one home, usually spent a good port... » Read more
Posted by: shashtzoh Age: 42 Posted on: 26 Dec 2016 0 comments
2 likes 875 views Category: Male Stories Tags: Showers, Caught, Gym
After a long work out at the gym, I went to shower up in the communal shower room.
My evening exercise happened to end up later than usual that night because of work. The gym was largely empty except for a several women and two other guys. It happened that one of the women came and got on the treadmill ri... » Read more
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subset: commoncrawl


text:
Natural brick is preferred by many homeowners, so you may be dismayed to find that a previous owner decided to paint the brick in your home. Paint, once applied, is very difficult to remove from brick. However, if you have the time and patience, and a little bit of luck, you may be able to remove the paint.
Before beginning, you should consider that whoever painted the brick had a very good reason to do so. You may find mismatched brick, discolored brick, or stained brick underneath the paint. If possible, try to find out why the paint was applied in the first place. If you are removing the paint from the brick without knowing what is beneath it, be prepared to paint the brick once more if you find something you don't like. If you must repaint, use a latex or lime based paint for best results.
Step 1: Generously apply a gel based paint remover to the surface of the brick.
TIP: It is best to spot test the remover to make sure it will not discolor the surface of the brick. Choose an out of the way area for the spot test. If the paint remover works as expected, continue using it on the rest of the brick.
Step 2: Place a piece of cheap fabric over the surface that has just been covered with paint remover. With a gloved hand, press the fabric into place, into the paint remover.
Step 3: Wait the recommended time for the paint remover to sit on the brick. Then, wearing gloves, carefully remove the piece of fabric. Often the old paint will have adhered to the fabric. The fabric also prevents the paint remover from drying out too quickly.
Step 4: Use a stiff bristled nylon brush to remove the rest of the paint that did not adhere to the fabric. If necessary, apply a second coat of the paint remover, then use the nylon brush to remove the paint that is softened.
TIP: It is important to have a disposal plan already in place for the used paint. Many areas have local laws governing how this paint and paint remover should be discarded to minimize the environmental impact of the waste.
Step 5: Once all of the paint is removed from a specific area, clean the surface of the brick with water and a sponge. Allow the brick to dry completely before repainting or further cleaning the brick.


text:
Today's Devotional
Representing JesusIn what practical ways can you effectively represent Christ?
Top Music Videos
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I'm really popular and rich. i am into all kinds of music except contry music.i love to dance and sing!!Durring the summer i do VBS and i spend every day at the pool hangen out with my b#$@!$ and my bf and during the school year i spend every day after school with my friends hangen out in town. oh by the way i have 1 older brother and 2 sisters.


text:
The roles of Bcl-xL in modulating apoptosis during development of Xenopus laevis
© Johnston et al; licensee BioMed Central Ltd. 2005
Received: 25 February 2005
Accepted: 26 September 2005
Published: 26 September 2005
Apoptosis is a common and essential aspect of development. It is particularly prevalent in the central nervous system and during remodelling processes such as formation of the digits and in amphibian metamorphosis. Apoptosis, which is dependent upon a balance between pro- and anti-apoptotic factors, also enables the embryo to rid itself of cells damaged by gamma irradiation. In this study, the roles of the anti-apoptotic factor Bcl-xL in protecting cells from apoptosis were examined in Xenopus laevis embryos using transgenesis to overexpress the XR11 gene, which encodes Bcl-xL. The effects on developmental, thyroid hormone-induced and γ-radiation-induced apoptosis in embryos were examined in these transgenic animals.
Apoptosis was abrogated in XR11 transgenic embryos. However, the transgene did not prevent the apoptotic response of tadpoles to thyroid hormone during metamorphosis. Post-metamorphic XR11 frogs were reared to sexual maturity, thus allowing us to produce second-generation embryos and enabling us to distinguish between the maternal and zygotic contributions of Bcl-xL to the γ-radiation apoptotic response. Wild-type embryos irradiated before the mid-blastula transition (MBT) underwent normal cell division until reaching the MBT, after which they underwent massive, catastrophic apoptosis. Over-expression of Bcl-xL derived from XR11 females, but not males, provided partial protection from apoptosis. Maternal expression of XR11 was also sufficient to abrogate apoptosis triggered by post-MBT γ-radiation. Tolerance to post-MBT γ-radiation from zygotically-derived XR11 was acquired gradually after the MBT in spite of abundant XR11 protein synthesis.
Our data suggest that Bcl-xL is an effective counterbalance to proapoptotic factors during embryonic development but has no apparent effect on the thyroid hormone-induced apoptosis that occurs during metamorphosis. Furthermore, post-MBT apoptosis triggered by irradiation before the MBT could only be restrained by maternal expression of Bcl-x L . Although maternal expression of XR11 was sufficient to abrogate apoptosis triggered by post-MBT γ-radiation, radiation tolerance from zygotically-derived XR11 was acquired gradually, indicating that synthesis of XR11 protein is not sufficient to prevent apoptosis. Thus, repression of radiation-induced apoptosis by overexpression of Bcl-xL during embryonic development depends upon the timing of its expression and post-translational events that enable the protein to become effective.
Cell death is an essential and integral aspect of embryonic development (for reviews, see [1–3]). The genome is programmed to eliminate certain cells or groups of cells at particular times during development by a process called "programmed cell death" (PCD) [4–6]. Well-known examples of PCD during vertebrate development include interdigital cell death and regression of the tail and gills during amphibian metamorphosis [3]. Considerable PCD also occurs in the central nervous system (CNS; for review, see [7]), where as much as 85% of some neuronal populations may be lost by PCD during development [8–11]. One form of PCD is apoptosis, which is characterized by membrane blebbing, nuclear and cytoplasmic shrinkage, chromatin condensation and DNA fragmentation [12]. The detection of DNA fragmentation in dying cells utilizing the TUNEL (TdT-mediated dUTP nick end labeling) assay provides the basis for a sensitive and specific in situ measure of apoptosis.
The frog Xenopus laevis provides an excellent model system for studying apoptosis during embryonic development. The TUNEL assay has been used on whole-mounts and tissue sections of Xenopus embryos to monitor spontaneous developmental apoptosis [13, 14]. Apoptosis also provides the means for the embryo to rid itself of cells damaged by DNA-damaging agents, such as gamma radiation [15]. Gamma radiation during embryonic development is a threat to the viability of the embryo and to its potential to develop into a healthy, functional adult organism. Embryos have two alternative mechanisms to prevent developmental abnormalities caused by irradiation: elimination of damaged cells through apoptosis or repair of the damaged DNA. Xenopus embryos undergo a dramatic transition in their response to ionizing radiation coincident with the onset of gastrulation. Embryos irradiated before gastrulation continue to cleave normally and undergo an abrupt, catastrophic, comprehensive and synchronous apoptosis of DNA-damaged cells after the mid-blastula transition (MBT) using a maternally-derived mechanism that does not require transcription [15–20]. In contrast, cells in embryos irradiated after gastrulation reportedly do not undergo apoptosis [17, 19, 20].
The induction of apoptosis is subject to both pro- and anti-apoptotic cellular factors and is executed by cysteine proteases called caspases after their activation from the proenzyme state. The activation of caspases can be triggered by cytochrome c release from the intermembrane spaces of mitochondria through mitochondrial outer membrane permeabilization [21–24]. Release of cytochrome c, in turn, is regulated by members of the Bcl-2 family (for reviews, see [23, 25, 26]). Members of the Bcl-2 family serve a key regulatory role in apoptosis, acting as either pro- or anti-apoptotic factors. Thus, the relative levels of members with opposing functions can determine whether cells live or die [27] (for review, see [26]). One of the anti-apoptotic members of the Bcl-2 family is Bcl-xL, which functions to inhibit apoptosis in situations in which Bcl-2 does not suffice [28]. For example, bcl-2 knockout mice are viable as embryos [29, 30] whereas bcl-x L knockout mice die as embryos that exhibit massive cell death in the nervous and hematopoietic systems [31]. Over-expression studies conducted both in vitro and in vivo in mice indicate that Bcl-xL is also sufficient to promote survival of embryonic neurons [32–34]. Similar results have been obtained with chick embryonic neurons in vitro [35]. Members of the Bcl family also modulate the apoptogenic effects of DNA-damaging agents. For example, lymphoid cells from bcl-2 null mice are hypersensitive to irradiation in vitro [29, 30]. Conversely, over-expression of bcl-2 reduces the rate of apoptosis of γ-irradiated hematopoietic cells both in vitro and in vivo [36, 37].
Cruz-Reyes and Tata [38]cloned two bcl-related genes in Xenopus laevis, which they designated as XR1 and XR11. XR11 [GenBank:X82461], which most closely resembles Bcl-xL, is expressed as early as stage 12 (gastrula) and continues to be expressed throughout embryonic, larval and adult life. Over-expression of XR11 in Rat-1 fibroblasts protected the cells from apoptosis, thus demonstrating that XR11 is an anti-apoptotic member of the Bcl family. Misexpression of human Bcl-2 or XR11 in Xenopus embryos has been shown to impede developmental- or chemically-induced apoptosis, respectively, during early Xenopus laevis embryonic development [17, 39]. The transgenesis technique developed by Kroll and Amaya [40] provides the means to study the effects of genes on later developmental events and to examine the effects of the transgenes on subsequent generations. For example, Coen et al. [41] employed a neuron-specific promoter driving XR11 over-expression to demonstrate selective protection of Rohon-Beard cells during metamorphosis. In this report, we have used a constitutive promoter to examine the effects of generalized XR11 over-expression throughout Xenopus development and to examine the respective roles of maternal and zygotic XR11 in protection against γ-radiation-induced apoptosis during development.
Over-expression of XR11 dramatically reduced spontaneous developmental apoptosis in embryos
Over-expression of XR11 did not impede apoptosis that occurs during normal or thyroid hormone-induced metamorphosis
To address more specifically whether apoptosis can be triggered by thyroid hormone in XR11 transgenics, we used the metamorphosis induction assay described by Tata [43] and Huang et al. [44]. One-week tadpoles were exposed to triiodothyronine (T3) for five days. As shown in Figure 3C–J, tadpoles transgenic for XR11 displayed the same gross responses to thyroid hormone, including gill resorption, as shown by the GFP transgenics with no discernable delay. The gill apparatus, which is visible in the absence of T3 (arrow in H), regresses after T3 treatment in both cases. Meckel's cartilage (the lower jaw) shows a dramatic beak-like reshaping (arrows in F and J).
To determine whether apoptosis was occurring equally in the GFP and XR11 transgenic tadpoles, we focused on the ventral velum, a portion of the larval filter feeding apparatus that has a chevron shape in cross section before it dissociates through apoptosis during metamorphosis [45]. As shown in Figure 3K–R, TUNEL-positive nuclei, which are rare in the velums in the absence of T3, were prevalent in the velums of both T3-treated GFP and XR11 transgenic tadpoles. These results indicate that apoptosis that occurred in response to thyroid hormone was unaffected by over-expression of XR11. One possible explanation for the extensive apoptosis in the XR11 transgenic tadpoles is that the XR11 protein was no longer over-expressed. However, as shown in Figure 3S, immunoblotting with an antibody to XR11 confirmed that the 22 kDa XR11 protein was over-expressed in the transgenic tadpoles. Proteins were extracted from individual tadpoles at approximately stage 45, separated by electrophoresis, blotted and probed as described in Methods. These tadpoles had been treated with triiodothyronine (T3). Similar results were obtained with samples from untreated tadpoles (data not shown). The variation in the GFP signal reflected the intensity of GFP fluorescence in the live tadpoles, presumably reflecting differences in the number of integrated copies of GFP. The 22 kDa XR11 signal is considerably stronger in the XR11 transgenic sample than in the GFP transgenic sample, reflecting XR11 transgene expression.
XR11 transgenic frogs are fertile and transmit the transgene
Representative Crosses Illustrating Inheritance of the XR11 Transgene
GFP positive
GFP negative
Ratio (χ2)
XR11-01 male × WT-01 female
1:1 (p < 0.9)
XR11-01 male × XR11-01 female
3:1 (p < 0.1)
XR11-01 male × XR11-02 female
7:1 (p < 0.9)
Over-expression of XR11 curtailed apoptosis induced by irradiation
In contrast to the results with wild-type embryos, embryos produced by fertilization of eggs from XR11 females with wild-type sperm showed evidence of partial protection from γ-radiation (Fig. 4E, F). Reduced damage from radiation was always observed after exposure to 10 Gy, but protection from 20 Gy was variable from experiment to experiment, suggesting that overexpression of XR11 was not always sufficient to overcome the effects of 20 Gy.
To investigate whether protection from radiation-induced damage was due to maternal or zygotic expression of the transgene, we irradiated embryos derived from fertilizing eggs of wild-type females with sperm from XR11 males and compared them to wild-type embryos. If zygotic expression of the transgene could partially overcome the effects of pre-MBT γ-radiation, we would have expected to see a subset of embryos with few or no TUNEL-positive nuclei that resembled embryos derived from eggs of XR11 females. Instead, all embryos resembled irradiated wild-type embryos (compare Fig. 4J–L to Fig. 4G–I). Extensive TUNEL-positive nuclei are seen in both groups at both doses. Furthermore, the embryos exposed to 20 Gy fragmented during the TUNEL procedure. These results are consistent with a requirement for a maternal source of radiation protection at these early stages.
The TUNEL data presented above indicate that: (1) maternal expression of the XR11 transgene reduces the amount of apoptosis induced by γ-radiation during pre-MBT and early post-MBT stages, (2) protection from apoptosis by maternal transgene expression is sustained in the tail tip after tissues in the head become susceptible to apoptosis (irradiation in the mid-30 stages) and (3) radiation tolerance is acquired gradually after the MBT and is sufficient to provide complete protection from apoptosis to embryos irradiated in the mid-30 stages of development.
The application of technology to modify Xenopus genetically has created opportunities to conduct functional analyses that are not readily achieved by other means. In this study, we have used XR11 transgenics to examine the role of Bcl-xL in suppressing apoptosis during embryonic development, thyroid-hormone-induced metamorphosis and in response to γ-radiation. Our ability to raise XR11 transgenic frogs to maturity has also given us the opportunity to distinguish between maternal and zygotic contributions of Bcl-xL to radiation protection.
This study supports the growing evidence in the literature that Bcl-xL is sufficient to promote neuronal survival in mice and chicken embryos [32–35]. Our experiments showed that the XR11 transgene could eliminate most apoptosis in the developing Xenopus nervous system, including the brain and eyes. This result is consistent with a model in which the execution of developmental apoptosis is dependent upon a balance between pro-and anti-apoptotic factors with Bcl-xL being one of the anti-apoptotic factors. In spite of the abatement of developmental apoptosis in XR11 transgenic embryos, there were no apparent gross developmental consequences; the embryos developed into phenotypically normal tadpoles. This result suggests that developmental apoptosis is not essential for development of vital organ systems, notably the central nervous system, and that embryos can adapt to an overabundance of neurons. However, detailed morphometric studies would be necessary to confirm this conclusion. Yeo and Gautier [39] previously showed that injection of human bcl-2 mRNA abrogated programmed cell death during early Xenopus development, resulting in an expansion of neural tissue and variations in the patterns of expression of a number of genes expressed during early neurogenesis. We did not examine gene expression patterns in our embryos. However, if such alterations did occur, they had no apparent functional consequences.
Contrary to expectation, metamorphosis of XR11 transgenic tadpoles was normal, as was the response to exogenous thyroid hormone. If over-expression of Bcl-xL had hindered metamorphosis, it would have indicated that thyroid hormone triggers apoptosis by shifting the balance between pro-apoptotic factors and Bcl-xL. However, this proved not to be the case, even though we found that Bcl-xL was still misexpressed at metamorphosis. One possibility is that other members of the Bcl-2 family counter apoptosis during these later stages. Another possibility for the failure of XR11 transgenesis to prevent apoptosis during metamorphosis is that expression of the transgene was insufficient to shift the balance between pro-and anti-apoptotic factors sufficiently to prevent apoptosis. It is also possible that subtle changes in the timing, rate or cellular selectivity of metamorphosis did occur that we failed to detect. For example, Coen et al. [41] demonstrated selective protection of Rohon-Beard neurons in XR11 over-expressing tadpoles during metamorphosis. We did not assess cell-specific neoronal survival during metamorphosis.
Upon reaching sexual maturity, both male and female frogs were fertile. Crosses of transgenic frogs indicated that most had small numbers of transgene integration sites. This result was consistent with a previous transgene transmission study [46]. The ability to produce second generation transgenic XR11 embryos provided us with the opportunity to examine both the maternal and zygotic contributions of Bcl-xL to the apoptotic response to γ-radiation. This provided evidence for a significant role for apoptotic factor balance in determining whether cells that had received damaging dosages of radiation during development were eliminated. Other Bcl-2 family members may carry out similar roles in that microinjection of human Bcl-2 mRNA into Xenopus embryos is effective in inhibiting irradiation-induced apoptosis [16].
Expression of the XR11 transgene had a profound effect on whether cells of irradiated embryos underwent apoptosis. Furthermore, the application of transgenics allowed us to demonstrate that this effect depended upon the source of the over-expressed protein. Over-expression of Bcl-xL derived from XR11 females provided partial protection from the normal massive apoptosis that occurs in irradiated pre-MBT wild-type embryos after reaching the MBT. However, if the XR11 gene was derived from XR11 males, no protection was seen. This result demonstrates conclusively that apoptosis triggered by irradiation before the MBT can be restrained by maternal expression of Bcl-xL and that zygotic expression of Bcl-xL is ineffective in overcoming the effects of prior irradiation.
Irradiation soon after the MBT (at stage 11.5) had much more subtle effects on embryos. In wild-type embryos, apoptosis was enhanced in the brain, eyes, pharynx and tail tip. However, maternal expression of XR11 was sufficient to abrogate apoptosis. Interestingly, zygotic expression of XR11 at this time was not effective in preventing apoptosis even though XR11 protein was abundant at the time of irradiation. The failure of zygotically-derived XR11 to provide protection from apoptosis implies that synthesis of XR11 protein is not sufficient to prevent apoptosis.
Embryos irradiated during the mid-30 stages of development revealed that the maternal effect conferring protection from radiation-induced apoptosis had diminished significantly in the head and pharynx but persisted in the tails. It will be interesting to learn whether the difference between the heads and tails is due to preferential distribution or stability of maternal XR11 protein itself or is due to auxiliary factors that influence XR11 protein function. Bcl-xL and other members of the Bcl-2 family are known to be subject to post-translational regulation by such events as phosphorylation, proteolysis, cleavage, protein-protein interactions and subcellular localization [47, 48]. Intriguingly, at these later stages, zygotic XR11 protein conferred global radiation protection. Thus, the initial lack of functionality of zygotic XR11 protein that we observed after the MBT had been overcome.
Biologically, the responses to irradiation after the MBT are probably more significant than the universal, catastrophic apoptosis that occurs in embryos irradiated before the MBT. Failure to eliminate cells mutagenized by irradiation would result in retention of cells that may have functional deficiencies and have the potential to become malignant due to genetic damage. Thus, the mechanisms that control apoptosis in response to DNA damage have important adaptive significance. Radiotherapy and a number of chemicals have been shown to damage DNA and induce apoptosis in mammals; this property has been exploited in the clinic to induce apoptosis of cancer cells. Resistance to apoptosis has important implications for the efficacy of chemotherapy and radiotherapy because tumor cells that overexpress either bcl-2 or bcl-x L may be resistant to either mode of cancer therapy [37, 49–52]. On the other hand, their over-expression in non-tumor cells could enhance the ability of these cells to survive the otherwise lethal effects of radiation and chemotherapy.
Our results indicate that synthesis of Bcl-xL is not sufficient to confer radiation protection; rather, it belatedly acquires functionality. Possible explanations for the failure of Bcl-xL synthesized after the MBT to protect against apoptosis could include the absence of posttranslational modifications to the protein or the lack of a cooperative factor that is necessary for the protein to function effectively. The presence of this mechanism that confers functionality on Bcl-xL protein before the MBT and its absence soon after the MBT may provide an assay system that would facilitate its identification. The discovery of a mechanism that could control whether cells undergo apoptosis after exposure to an apoptogenic stimulus could have important implications for improving the efficacy of chemotherapy and radiotherapy.
Our results are consistent with a model in which the execution of developmental apoptosis in embryos is dependent upon a balance between pro-and anti-apoptotic factors with Bcl-xL being one of the anti-apoptotic factors. The drastic reduction of apoptosis in XR11 transgenic embryos had no apparent functional consequences. Metamorphosis of transgenic tadpoles was apparently normal, as was the response to exogenous thyroid hormone. The ability to produce second-generation transgenic XR11 embryos enabled us to examine both the maternal and zygotic contributions of Bcl-xL to the apoptotic response to γ-radiation. Post-MBT apoptosis that was triggered by irradiation before the MBT was restrained by maternal expression of Bcl-xL. However, zygotic expression of Bcl-xL was ineffective in overcoming the effects of prior irradiation. Maternal expression of XR11 was also sufficient to abrogate apoptosis triggered by post-MBT γ-radiation. However, zygotic expression of XR11 at this time was not effective in preventing apoptosis even though XR11 protein was abundant.
Adult Xenopus laevis were maintained in compliance with the University of Calgary guidelines for animal care. Our transgenesis protocol is based upon the technique described by Sparrow[53], which is a simplification of the procedure described by Kroll and Amaya [40]. The simplified technique eliminates the need to use egg extract and restriction enzyme to facilitate transgenesis. We have found no significant difference in the frequencies of either normal development, transgenesis or viability of embryos, tadpoles or frogs using these two techniques (data not shown). The simplified transgenic reaction involves mixing 2.5 × 105 nuclei in 2.5 μl of sperm storage buffer, 100 ng linearized DNA in 2.5 μl of water. The components are mixed and left for 10 minutes at room temperature (~20–23°C). At this point, 495 μl of sperm dilution buffer is added, and the suspension is mixed gently and thoroughly before loading into microinjection needles for nuclear transplantation. Nuclear transplant embryos were screened for expression of the transgene by detecting GFP fluorescence using an Olympus SZX9 fluorescent stereomicroscope.
Tadpole husbandry
Tadpoles were fed Sera Micron http://www.sera.de/english/produkte/micron.htm, which is a fine granulated tropical fish food. This proved to be an excellent diet for tadpoles, which were maintained on it until metamorphosis. After metamorphosis, froglets were fed progressively larger forms of NASCO Frog Brittle. Tadpoles were either maintained in plastic food containers in which water was changed manually as needed or in continuously circulating Z-MOD tanks (Marine Biotech, Beverly, MA). After metamorphosis, frogs were maintained in Z-MOD tanks until they became large enough to be transferred to larger-sized X-MOD tanks.
RNA purification
Whole tadpoles were homogenized in 100 μl TRIZOL (Invitrogen), and 400μl of the reagent were added to the homogenate. After incubation for 5 min at room temperature, 100μl of chloroform were added, the mixture was shaken vigorously for 15 seconds, incubated for 2–3 minutes at room temperature and centrifuged at 12,000 × g for 15 minutes at 4°C. 250 μl of isopropanol were added to the supernatant, which was then incubated for 10 minutes at room temperature and centrifuged at 12,000 × g for 10 minutes at 4°C. The supernatant was removed, and 500 μl of 75% ethanol were added to the pellet. After mixing well, the mixture was centrifuged at 7,500 × g for 5 minutes at 4°C. The supernatant was discarded, and the pellet was air-dried. The dried pellet was rehydrated in RNase-free water and stored at -80°C. Any contaminating DNA was removed with DNA-free (Ambion) according to the manufacturer's protocol. RNA concentration was determined by spectrophotometry at 260 nm.
Ready-to-Go RT-PCR beads (Amersham Pharmacia) were used according to the manufacturer's instructions for one-step reactions. RT-PCR reactions for XR11 and EF1α were run in tandem. 500 ng total RNA and 10 pmoles XR11 primers or 5 pmoles EF1α primers were used in each 50 ml reaction. The first strand reaction ran for 15 minutes followed by a 5 minute incubation at 95°C The PCR step ran for 35 cycles for the XR11 product or 30 cycles for the EF1α product. Each cycle was 94°C for 1 minute, followed by 55°C for 1 minute and 72°C for 1 minute. The reaction was stopped by reducing the temperature to 4°C. If necessary, the reaction products could be stored at -20°C until analyzed. The PCR products were analyzed by applying 10 μl samples to a 1.5%agarose/TAE gel containing 0.25 μg ethidium bromide (Sigma-Aldrich) in parallel with a 100 bp ladder (Amersham Pharmacia).
EF1α-downstream primer: ACTGCCTTGATGACTCCTAG
EF1α-upstream primer: CAGATTGGTGCTGGATATGC
This primer pair will generate a 270 bp band.
XR11-downstream primer: GGCATTCTTTCCATACAGGC
XR11-upstream primer: CCTTCTACTTCAGAGCGCCC
This primer pair will generate a 400 bp band.
Antiserum to XR11 was prepared by Alpha Diagnostics International, Inc. (San Antonio TX) in rabbit against a synthetic peptide encompassing amino acids 11–30 (KFV SKK LSQ NEA CRK FSN NP). This antiserum was used for immunoblotting at a dilution of 1:1000. Secondary antibody was goat anti-rabbit conjugated to alkaline phosphatase (Jackson ImmunoResearch), which was used at a dilution of 1:1000.
Individual embryos were homogenized so as to correlate immunoblot results with GFP expression levels. Proteins in tissue homogenates were separated using 15% SDS-polyacrylamide gel electrophoresis and blotted onto PVDF. Blots were blocked either overnight at 4°C or for 1.5 hours at room temperature in 5% skim milk powder in TBS containing 0.1% Tween 20. The blots were then incubated with the primary antibody in TBS containing 0.1% Tween 20 for 1 to 2.5 hr at room temperature, washed 1 × 15 min and 3 × 5 min in TBS containing 0.1%Tween 20. The blots were then incubated with the conjugated secondary antibody in TBS containing 0.1% Tween 20 for 1 hr at room temperature, washed as described above, rinsed with TBS, then with staining buffer without stain reagents, followed by staining with NBT/BCIP until developed.
Embryo whole-mount TUNEL assays
Pigmented embryos were collected at stages 28, 33, 37, and 41 and processed for TUNEL assays as described by Hensey and Gautier [13]. After staining, the embryos were fixed while rocking overnight at room temperature in Bouin's solution (70 ml water, 25 ml 37% formaldehyde, 5 ml glacial acetic acid). Embryos were then washed (four times for 15 minutes each) in TE buffered ethanol. Embryos were then bleached for 1 hour at room temperature in 0.5 × SSC, 5% formamide, 1%H2O2 in a Petri dish on a piece of aluminum foil on a shaker under a fluorescent lamp, washed twice for 5 min each in methanol and cleared in BB/BA (2:1 benzylbenzoate:benzyl alcohol). Photographs were taken of embryos representative of the extremes of TUNEL-positive nuclei.
Samples were collected at the appropriate stages and fixed overnight at 4°C in MEMFA (0.1 M MOPS pH 7.4, 2 mM EGTA, 1 mM MgSO4, 3.7 % formaldehyde). Samples were then embedded in OCT Compound (Tissue Tek Cat #4583). The embedded samples could then either be stored at -80°C or sectioned immediately. 14 μm sections were made using a Microm cryostat with cabin temperature set at -12°C. Sections were stored at -20°C if not processed immediately. Sections were processed using the ApoTagRed in situ Apoptosis Detection Kit, catalogue #7165 according to the manufacturer's protocol (Intergen Co., Purchase NY). Sections were visualized using a Zeiss Axioplan 2 microscope with Spot camera and software.
Induced metamorphosis
3,3',5-Triiodo-L-thyronine (T3; Sigma Cat #T2752) was added one week after fertilization to a final concentration of 10 nM to tadpole water and changed daily for the duration of the experiment [44](Huang et al., 1999). Before and during treatment, tadpoles were maintained in 0.1 × MMR (for description, see Kroll and Amaya [40]) in the absence of food. Tadpoles responded uniformly to the hormone.
Cartilage staining
Cartilage was visualized in tadpoles according to the procedure described by Klymkowsky and Hanken [54]. Tadpoles were anesthetized in 0.02% benzocaine and immersed in 0.02% Alcian blue 8GX in 70% ethanol/30% glacial acetic acid. After staining, tadpoles were passed through an ethanol series (100, 100, 95, 70, 40, 15%) and then into distilled water. They were then washed in two changes of methanol and cleared in BB/BA.
Irradiation of embryos
Embryos to be irradiated were placed in ~2.5 ml 0.1 × MMR in 35 × 10 mm plastic Petri dishes (approx. 20/dish) and exposed to either 10 Gy or 20 Gy from a Cesium 137 source in a Gammacell 1000 irradiation chamber (MDS Nordion International Inc., Ottawa, ON Canada). After irradiation, embryos were transferred to 60 × 10 mm plastic Petri dishes containing 0.1 × MMR and allowed to recover for 12 hours at room temperature. Unless noted otherwise, each experiment was repeated at least once.
List of abbreviations
4',6-diamidino-2-phenylindole dihydrochloride
green fluorescent protein
Mid-blastula transition
Nitroblue tetrazolium
programmed cell death
sodium chloride/sodium citrate
Tris-buffered saline
40 mM Tris-acetate and 1 mM EDTA
10 mM Tris-HCl and 1 mM EDTA
TdT-mediated dUTP digoxigenin nick end labeling
Supported by grants to LWB and SMcF from the Natural Sciences and Engineering Research Council of Canada and to LWB from the Alberta Cancer Board. Plasmids containing the CMV-GFP-SV40 and CMV cassettes for construction of the plasmid containing co-linear CMVXR11 and CARGFP genes were generous gifts from Dr. Enrique Amaya. Dr. James Maller generously supplied the XR11 gene, which was originally cloned by Cruz-Reyes and Tata.
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Meet Dr. Farias
Chicago area physician, Dr Carlos Farias, has transformed the lives of patients with natural results for over a decade. As the co-founder of InsideLook MD, Dr. Farias was one of the first physicians to perform minimally invasive facial surgery.
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Vitals Compassionate Doctor, 2014
Professional Recognition:
Medical Director at National Cosmetic Surgery practice, 2010 - 2014
Undergraduate & Medical SchoolUndergraduate: University of Texas at Austin
Medical: MD, University of Texas Health Sciences Center – San Antonio
Post-Medical School TrainingResidency: Surgery, University of Missouri, Columbia
Fellowship: Plastic Surgery, University of Missouri, Columbia
Treatments offered at InsideLook MD
Most popular answers from Carlos Farias, MD
Can you have Smart Lipo done on your abs if you have stretch marks and will the result be good or will the skin hang?
Good morning Sharon! Thanks for the question. Unfortunately, stretch marks are still one of the most problematic issues that people have as far as... READ MORE
What is a more permanent fix to these under eye bags when I smile? I'm 23 years old. (Photo)
Good morning Ariel.You have beautiful eyes that most people would love to have! When you smile, your orbicularis oculi muscle contracts (everyone's... READ MORE
I would like to know the price to remove the eye bags? (Photo)
Good morning Yuso from a former Austinite,Bags under the eyes such as yours come from fat that exists in everyone's orbits slowly pushing forward from... READ MORE
Is laser lipo a good instrument too smooth out liposuction irregularities? Anyone here with experience? (photos)
Hello,Laser lipolysis would be a part of the treatment regimen for smoothing the contour irregularities in your photo, along with conservative... READ MORE
I have dark spots under my eyes; will laser help remove my dark spots under my eyes? (Photo)
Hello Claudia,"Dark Circles" can be due to so many things, that it's imperative to have a consultation to determine what is best for you, Even... READ MORE
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