text:
In mammalian embryos, zygotic gene transcription initiates after a limited number of cell divisions through a two‐step process termed the zygotic gene activation (ZGA). Here we report that RNA polymerase II undergoes major changes in mouse and rabbit preimplantation embryos during the ZGA. In transcriptionally inactive unfertilized oocytes, the RNA polymerase II largest subunit is predominantly hyperphosphorylated on its carboxy‐terminal domain (CTD). The CTD is markedly dephosphorylated several hours after fertilization, before the onset of a period characterized by a weak transcriptional activity. The largest subunit of RNA polymerase II then lacks immunological and drug‐sensitivity characteristics related to its phosphorylation by the TFIIH‐associated kinase and gradually translocates into the nuclei independently of DNA replication and mitosis. A phosphorylation pattern of the largest subunit, close to that observed in somatic cells, is established in both mouse and rabbit embryos at the stage when transcription becomes a requirement for further development (respectively at the 2‐ and 8/16‐cell stage). As these events occurred in the presence of actinomycin D, the nuclear translocation of RNA polymerase II and the phosphorylation of the CTD might be major determinants of ZGA.
In many animals, shortly after fertilization, the embryo is transcriptionally inactive and the onset of development does not require RNA synthesis (reviewed in Davidson, 1986; Yasuda and Schubiger, 1992). Transcription resumes and becomes an absolute requirement for development after a number of cell cycles, this number being a characteristic of each species. In mammals, zygotic gene activation (ZGA) involves a two‐step process. This process has been particularly well defined in mice (reviewed in Telford et al., 1990; Schultz, 1993). In this species, a 'minor' ZGA phase initiates at the late 1‐cell stage (G2 phase) with a very weak transcriptional activity (Latham et al., 1992; Ram and Schultz, 1993; Matsumoto et al., 1994; Temeles et al., 1994; Bouniol et al., 1995; Christians et al., 1995; Aoki et al., 1997). As a consequence, a small set of proteins are synthesized at the early 2‐cell stage (G1/S phase) (Flach et al., 1982; Latham et al., 1991): the 70 kDa heat‐shock proteins (Bensaude et al., 1983), the TRCs (transcription‐requiring complexes) (Conover et al., 1991), the U2afbp‐rs splicing factor (Latham et al., 1995) and the translation initiation factor, eIF‐4C (Davis et al., 1996). Reporter genes microinjected into the pronuclei of a 1‐cell mouse embryo can be transcribed during the minor ZGA phase (for reviews, see Schultz, 1993; Majumder and DePamphilis, 1995; Nothias et al., 1995; see also Christians et al., 1995; Henery et al., 1995). However, this transcription is repressed after the first mitosis unless the microinjected genes possess appropriate enhancers. At the late 2‐cell stage (G2 phase), a sharp transcriptional activation and increase of translational activity characterize the 'major' ZGA and result in a complete change in the pattern of protein synthesis (Van Blerkom and Brockway, 1975; Flach et al., 1982; Howlett and Bolton, 1985; Taylor and Piko, 1987; Latham et al., 1991; Nothias et al., 1996). In summary, three transcriptional transitions have been characterized in the early mouse embryo: (i) the onset of a minor ZGA phase at the late 1‐cell stage; (ii) the repression of enhancer‐less promoters after the first mitosis; and (iii) the occurrence of a major ZGA at the late 2‐cell stage after the second round of DNA replication.
In other mammalian species, the ZGA spans a longer developmental period, postponing the requirement in transcription. Depending on the species, the major ZGA generally occurs after two or three cleavage divisions (Telford et al., 1990) and up to four in the rabbit embryo (Manes, 1977; Delouis et al., 1992). In this species, although zygotic transcripts have not yet been detected before the 2‐cell stage (Cotton et al., 1980; Kanka, 1993), the minor ZGA phase may begin at the end of the 1‐cell stage, as in the mouse (Christians et al., 1994). The same study indicated that microinjected enhancer‐less genes are repressed after the first mitosis.
Conserved mechanisms appear to be involved in the control of ZGA in groups as distinct as amphibians and mammals. In Xenopus laevis, the major ZGA occurs at the mid‐blastula transition (MBT) and might rely on titration of inhibitory factors by the replicating genomes (Kirschner et al., 1985); the large excess of maternal histones stored in the oocyte of that species might compete for the recruitment of the basal transcription machinery (Prioleau et al., 1995). A deficiency in the activity of transcriptional activators has also been suggested to contribute to the transcriptional inactivity before MBT (Almouzni and Wolffe, 1995). In the mouse, the first cell cycles are characterized by extensive chromatin rearrangements and changes in nuclear protein composition which are likely to control the ZGA (Debey et al., 1989; Adenot et al., 1991; Clarke et al., 1992; Prather and Schatten, 1992; Vautier et al., 1994; Worrad et al., 1994, 1995; Thompson et al., 1995). Taken together, these studies suggest that the accessibility of the transcription machinery to DNA is controlled by the extensive chromatin modifications taking place during ZGA (reviewed in Patterton and Wolffe, 1996). In the mouse, a 'zygotic clock' which might involve protein kinases, has also been proposed to determine the time‐lapse separating fertilization from the beginning of the minor ZGA phase (Schultz, 1993).
Oocytes from X.laevis (Roeder, 1974; Toyoda and Wolffe, 1992; Bellier et al., 1997) and from the mouse (Latham et al., 1992) contain large amounts of functional RNA polymerase II. The onset of embryonic development must use this maternal store. The present study was initiated to examine the possibility that phosphorylation of RNA polymerase II largest subunit (RPB1 subunit) might be involved in ZGA. Indeed, both in vitro and in somatic cells, transcription involves a cycle of phosphorylation/dephosphorylation of the carboxy‐terminal domain (CTD) of this subunit (reviewed in Emili and Ingles, 1995; Dahmus, 1996). Moreover, phosphorylation of the CTD is altered in response to stimuli which have a general influence on the transcriptional activity of the cells, such as viral infection (Rangel et al., 1987; Rice et al., 1994), growth‐factor release from quiescence (Dubois et al., 1994b), heat‐shock (Venetianer et al., 1995) and meiotic maturation (Bellier et al., 1997).
Mouse and rabbit embryos were used as model systems because they differ markedly in timing and number of cell cycles delimiting the ZGA. In both species, we show that in addition to sharp and specific changes in the pattern of RPB1 phosphorylation, the RPB1 subunit translocates into the nucleus during the period of zygotic gene activation. These time‐controlled events which provide a molecular support to define the minor ZGA phase, might control the onset of zygotic gene activation.
Dephosphorylation of RPB1 after fertilization
The POL3/3 antibody which reacts with an internal epitope distinct from the CTD, recognized two forms of RPB1 on a Western blot from murine fibroblast lysates (Figure 1, lane 1). A hypophosphorylated form, IIa, migrated at a position similar to that of a 210×103 Mr protein and a hyperphosphorylated form, IIo, to that of a 240×103 Mr protein (Dubois et al., 1994c; Dahmus, 1995). Both forms gave signals of similar intensity. This pattern remained constant when synchronized fibroblasts were investigated at different stages of the cell cycle (Kim et al., 1997 and our data not shown). In contrast, a form co‐migrating with the IIo form predominated in metaphase‐arrested mouse oocytes (Figure 1, lane 2) and several hours after fertilization (Figure 1, lanes 3–4). A transition then occurred, at 2–3 h after the appearance of the pronuclei (22 h post‐hCG), with the decrease in the IIo‐like form and a concomitant sharp increase in the IIa form (Figure 1, lane 5). Forms with intermediate electrophoretic mobilities were observed during a short period at the 1‐cell stage, at ∼24 h post‐hCG (Figure 1, lane 6). A two‐band pattern similar to that of transcriptionally active somatic cells was observed at the end of the 1‐cell stage (30 h post‐hCG) (Figure 1, lane 7). This pattern did not change significantly at the 2‐cell stage (lane 8) and throughout further development (not shown). The above‐described dephosphorylation was not linked to RPB1 neosynthesis since cycloheximide added to mouse embryos just after the formation of the pronuclei did not prevent the appearance of the hypophosphorylated form of RPB1 (Figure 2, lane 2). However, in the presence of cycloheximide, the hyperphosphorylated forms of RPB1 were no longer detectable. Inhibition of DNA replication with aphidicolin did not affect the phosphorylation status of RPB1 (Figure 2, lane 3).
From these results, we conclude that in the mouse embryo, two sharp time‐controlled transitions in the phosphorylation status of the RPB1 subunit take place during interphase of the first zygotic cycle and are completed at the onset of the major ZGA phase.
An embryonic form of RPB1 in preimplantation embryos
Experiments were next designed to establish whether the RPB1 phosphorylation transitions were related to ZGA or were dependent on events occurring during the first cycle. As a first investigation of embryonic events, a comparison between mouse and rabbit embryos was undertaken because in the latter species, the ZGA spans over five cell cycles instead of two. In rabbit fibroblasts, as in mouse fibroblasts, the POL3/3 antibody recognized two bands which corresponded to the IIa and IIo forms of the RPB1 subunit (Figure 3A, lane 1). In rabbit metaphase‐arrested oocytes, a RPB1 form co‐migrating with the IIo form predominated (Figure 3A, lane 2) as already seen in mouse oocytes. This phosphorylated form dominated for several hours following fertilization (Figure 3A, lanes 3 and 4). A first transition was observed at 18 h post‐coitum (hpc) (2–3 h after the appearance of the pronuclei), with the increase in the hypophosphorylated IIa form (Figure 3A, lane 6). The mobility of the hyperphosphorylated form increased gradually and stabilized into a form with an intermediate electrophoretic mobility (Figure 3A, lanes 5–7). This form, which was designated as IIe (embryonic), predominated from the 2‐cell to the 8‐cell stage (Figure 3B, lanes 1–5). Meanwhile, a faint band co‐migrating with the IIo form could be seen on overexposed autoradiograms. The RPB1 subunit dephosphorylated normally in rabbit embryos enucleated just after the formation of the pronuclei and analysed at the late 1‐cell stage (data not shown). Thus, in both mouse and rabbit 1‐cell stage embryos the increase in hypophosphorylated form, IIa, of RPB1 and the replacement of the oocyte form IIo by an embryonic RPB1 form, IIe, are linked to the onset of the minor ZGA.
Actinomycin D does not prevent a new RPB1 phosphorylation pattern to be established at the onset of the major ZGA
In rabbit embryos, a second transition in the RPB1 phosphorylation state initiated at the 6/8‐cell stage (the beginning of the fourth cell cycle), when a form co‐migrating with the IIo form gradually replaced the IIe form (Figure 3B, lanes 4–6). At the late 8/16‐cell stage, the IIe form was no longer detectable and the RPB1 phosphorylation pattern was very similar to that observed at the morula stage (Figure 3B, lane 7) and in fibroblasts (lane 8). The mouse intermediate form observed during a short period at the 1‐cell stage (Figure 1, lane 6) may be the counterpart of the rabbit IIe form.
Since transcription involves a cycle of CTD phosphorylation and dephosphorylation, we next examined the influence of transcription inhibitors on the evolution of the RPB1 phosphorylation pattern at the major ZGA. The non‐specific transcription inhibitor, actinomycin D was used because the RNA polymerase II‐specific inhibitor, α‐amanitin promoted the degradation of RPB1 (Nguyen et al., 1996). Actinomycin D was efficient in inhibiting luciferase expression driven by an HSP70 promoter after microinjection of the N3Luc plasmid (Christians et al., 1994) into the rabbit pronuclei at the 1‐cell stage (data not shown). In embryos left in actinomycin D from 29 hpc (2‐cell stage) to 55 hpc, the content in RPB1 was significantly lower than that in controls (Figure 3C). However, in the actinomycin‐treated embryos the embryonic form of RPB1, IIe, disappeared completely and the phosphorylation pattern was close to that observed in 16‐cell embryos. Thus, a new RPB1 phosphorylation pattern is established at the onset of the major ZGA and is unlikely to be a consequence of transcriptional activation.
Embryonic forms of RPB1 insensitive to DRB, a CTD‐kinase inhibitor
Several serine/threonine kinases have been demonstrated to phosphorylate the CTD in vitro (for a review, see Dahmus, 1996). To characterize the phosphorylation status of the RPB1, embryos were exposed to 5,6‐dichloro‐1‐β‐d‐ribofuranosylbenzimidazole (DRB). DRB is a CTD‐kinase inhibitor (Yankulov et al., 1995; Herrmann et al., 1996; Marshall et al., 1996) which promotes RPB1 dephosphorylation in fibroblasts (Dubois et al., 1994c).
In mouse embryos, DRB also promoted the dephosphorylation of RPB1 at the 4‐cell stage, after the onset of the major ZGA (Figure 4A, lanes 7 and 8). Although the phosphorylation profiles remained identical in untreated embryos from the late 1‐cell to the late 2‐cell embryos, the DRB effects were markedly different. DRB had no effect on RPB1 phosphorylation in late 1‐cell embryos (G2 phase) (Figure 4A, lanes 1 and 2), led to a partial dephosphorylation in early 2‐cell embryos (G1/S phases) (lanes 3 and 4) and to a complete dephosphorylation in the late 2‐cell embryos (G2 phase) (lanes 5 and 6) at the onset of the major ZGA.
In rabbit embryos, DRB also promoted the dephosphorylation of the RPB1 subunit at the morula stage, long after the onset of the major ZGA (Figure 4B, lanes 5 and 6). Furthermore, DRB provoked the disappearance of a band co‐migrating with the IIo form and detectable at the 4‐cell or at the 8/16‐cell stages (Figure 4B, lanes 1–4). However, it did not affect the IIe form present in the embryos either at the 4‐cell or at the 8/16‐cell stage.
Our observations show that the predominant slow‐migrating forms of RPB1 present during the minor ZGA phase are not DRB‐sensitive. Strikingly, the changes in DRB sensitivity are linked to the onset of the major ZGA in both rabbit and mouse embryos.
The embryonic RPB1 form IIe lacks a phosphoepitope generated by TFIIH‐associated kinase phosphorylation
DRB is an inhibitor of the TFIIH‐associated kinase (Yankulov et al., 1995) which phosphorylates the CTD within preinitiation complexes of transcription (reviewed in Hoeijmakers et al., 1996; Svejstrup et al., 1996). To characterize further the RPB1 form, IIe, present in the early rabbit embryo, we investigated its recognition by the monoclonal antibody, CC‐3. Indeed, this antibody, which is directed against a phosphorylated epitope of the CTD, binds to RPB1 phosphorylated in vitro by the TFIIH‐associated kinase (Dubois et al., 1997). In rabbit fibroblasts, the CC‐3 antibody reacted with a major band which co‐migrated with the IIo form (Figure 5A) and a non‐identified protein of 180 kDa (Vincent et al., 1996). In 8/16‐cell stage embryos, the two RPB1 forms, IIo and IIe, were detected in equivalent amounts with the POL 3/3 antibody, but only the slower‐migrating one, IIo, was stained with the CC‐3 antibody. In 16‐cell embryos and in embryos treated with actinomycin D (from 29 to 55 hpc), the CC‐3 antibody clearly reacted with both the non‐identified 180 kDa protein and the slow‐migrating form of RPB1 (Figure 5B). Thus, in contrast to the IIo form, the IIe form of RPB1 present from the 1‐cell to the 8/16‐cell rabbit embryo lacks a TFIIH‐generated phosphoepitope on the CTD.
The embryonic form IIe of RPB1 is not bound to nuclear structures
Since transcribing RNA polymerase is firmly bound to chromatin (Jackson and Cook, 1985; Razin et al., 1985), we next investigated the association of RPB1 with embryonic nuclear structures. Rabbit embryos at the 8/16‐cell stage were used because they contained similar amounts of IIe and IIo forms. When embryonic lysates were fractionated, most of the IIe form remained in the cytosolic fraction (Figure 6, lane 6); the hypophosphorylated IIa form was clearly present in the nuclear fraction but recovered mainly in the cytosolic fractions obtained from embryos whereas the major part of the hyperphosphorylated IIo form was associated with the nuclear fraction (Figure 6, lanes 5 and 6, respectively). When lysates from rabbit fibroblasts were fractionated using the same procedure, the hypophosphorylated IIa form distributed equally between the cytosolic and nuclear fractions, whereas the hyperphosphorylated IIo form remained almost totally in the nuclear fraction (Figure 6, lanes 2 and 3). As a whole, most of the RPB1 is linked to the nuclear fraction in fibroblasts but remains in the cytosolic fraction in the 8/16‐cell embryos.
Gradual nuclear accumulation of the RPB1 subunit before the major ZGA
The presence of RPB1 in the cytosolic fraction does not preclude its compartmentalization and might be due to its leaking from the nucleus. Indeed, several nuclear proteins have been found to be extracted in low‐salt buffers (Krek et al., 1992; Michels et al., 1995). Therefore, to investigate further the localization of RPB1 in the rabbit embryos, an immunofluorescence investigation was undertaken using the POL3/3 antibody, visualizing the nuclei with propidium iodide. No staining was evident in metaphase II oocytes and 1‐cell stage embryos in which the pronuclei had just formed (Figure 7A, panels 1 and 2). A very faint nuclear staining was visible at the late 1‐cell stage (panel 3), increasing slightly at the 2‐cell stage (panel 4), more pronounced at the 4‐cell stage (panel 5) and still increasing up to the 8/16‐cell stage (panel 6) which corresponds to the onset of the major ZGA. The POL3/3 antibody is directed against an RPB1 epitope located outside the CTD (Krämer et al., 1980) and might have been masked in the early stages. Therefore, the immunofluorescence study was repeated using another antibody, 8WG16, which binds an epitope on the CTD (Thompson et al., 1989). Again, the nuclear staining was faint at the 1‐cell stage (Figure 7B, panel 1), weak but clearly visible at the 2‐cell stage (panel 2), stronger at the 4‐cell stage (panel 3) and more intense at the 8/16‐cell stage (panel 4). From these concordant results we conclude that, in the rabbit embryo, the RPB1 subunit accumulates gradually in the nuclei throughout the first four cell cycles.
To question whether RPB1 localization would rely on cell cycling, the immunocytology was repeated with mouse embryos. In metaphase II‐arrested oocytes (Figure 8, panel 1) and 1‐cell embryos within 1 h after formation of the pronuclei (Figure 8, panel 2), the POL 3/3 anti‐RPB1 antibody stained the cytoplasm and the pronuclei very weakly but uniformly; however, the nucleoli (arrowheads) did not stain. Weak staining of the pronuclei (excluding the nucleoli) became visible only 3–4 h following their formation (Figure 8, panel 3). It was more pronounced 6–7 h later (at ∼10 h after formation of the pronuclei) corresponding to the end of the first cell cycle (panel 4). At the 2‐cell stage, the nuclear staining continued to increase (Figure 8, panels 5 and 6) and became comparable with that found in fibroblasts (data not shown). In embryos and fibroblasts, the RPB1 staining was excluded from the nucleoli. Interestingly, the polar bodies which have been reported to be transcriptionally active (Bouniol et al., 1995) were also heavily stained for RPB1 (arrows) (Figure 8, panels 3 and 5).
Hence in both species, RPB1 accumulates into the nuclei along the ZGA period. The accumulation occurs more abruptly in the mouse than in the rabbit, in agreement with the respective ZGA chronologies.
Nuclear translocation of RPB1 does not require either protein or nucleic acid synthesis
It has been reported that protein synthesis and DNA replication are required for the increase in nuclear concentration of the general transcription factors, SP1 and the TATA box binding protein (TBP), that occurs during the first cell cycle of the 1‐cell embryo (Worrad et al., 1994). To document this requirement for RPB1 accumulation, 1‐cell mouse embryos that had recently formed a pronucleus were cultured in the presence of cycloheximide. Inhibition of protein synthesis did not prevent the nuclear accumulation of RPB1 at the 1‐cell stage (Figure 9). Inhibiting DNA replication with aphidicolin, or RNA synthesis with actinomycin D, did not prevent the nuclear translocation of RPB1 either (data not shown). These observations demonstrate that the nuclear accumulation of RPB1 does not result from neosynthesis but is due to a translocation process which does not require transcription, DNA replication or mitosis.
Based on the results presented in this study, we propose that compartmentalization of the RPB1 subunit, together with phosphorylation of the CTD, might control ZGA in mammalian embryos. In two different species, mouse and rabbit, with different ZGA chronologies, four major changes in RNA polymerase II properties were closely linked to the minor and major ZGA transitions: (i) the RPB1 subunit translocated gradually into the nucleus throughout the minor ZGA phase; (ii) the RPB1 subunit was dephosphorylated during the first cell cycle before the onset of the minor ZGA phase; (iii) throughout the minor ZGA phase, the RPB1 subunit did not show the characteristics of TFIIH phosphorylation; and (iv) at the onset of the major ZGA, the profile of RPB1 hyperphosphorylated forms evolved into that observed in somatic cells.
Nuclear translocation of the RPB1 subunit along the minor ZGA phase
Our results demonstrate that in mammals the nuclear accumulation in RPB1 can be assigned to a gradual translocation of RPB1 from the cytoplasm to the nuclei. This translocation spans over a 24 h period corresponding to one cell cycle in the mouse and throughout at least four cell cycles in the rabbit. In fibroblasts, RNA polymerase II is spread throughout the cytoplasm during mitosis from late prophase to anaphase and concentrates abruptly within minutes into the nucleus during telophase (Warren et al., 1992; Bregman et al., 1995; also our unpublished data). In contrast, RPB1 translocation clearly occurs along interphase in the 1‐cell mouse embryo. We also show, using cycloheximide, that the translocation of RPB1 in mouse embryos does not require protein synthesis. It has been suggested that in somatic cells, specific CTD kinase(s) may influence the subnuclear localization of RPB1 (Bregman et al., 1995). Since cycloheximide prevented RPB1 phosphorylation, it can be inferred that its translocation into the nucleus does not require CTD phosphorylation. Since evidence for translocation of RPB1 coincides temporally with the appearance of a transcriptionally permissive phase at the late 1‐cell stage, and since in the rabbit it proceeds up to the major ZGA, translocation of RPB1 might be considered as delineating and controlling the minor ZGA phase. It has been proposed that the first round of DNA replication governs the onset of the minor ZGA phase by providing an opportunity for the transcription machinery to gain access to promoters after disruption of the assembled nucleosomes (Davis et al., 1996). In support of this hypothesis, the TBP as well as transcription factors have been shown to translocate into the pronuclei along the first cell cycle in the mouse embryo (Worrad et al., 1994). However, in contrast to TBP, the translocation of RPB1 was not under the control of DNA synthesis. Thus, the nuclear translocation of RPB1 and TBP may not rely on connected molecular events. The RNA polymerase II core enzyme is an assembly of several subunits (Sawadogo and Sentenac, 1990; Young, 1991; Shpakovski et al., 1995), and further studies will examine whether it translocates as such, or whether the various subunits translocate individually and assemble within the nucleus.
RPB1 dephosphorylation at the 1‐cell stage: a requirement to turn on the minor ZGA?
In unfertilized mouse and rabbit oocytes, when most of the RPB1 subunit is hyperphosphorylated, an increase in the hypophosphorylated form, IIa, can be detected only several hours after fertilization, when the pronuclei have formed. The increase in the IIa form of RPB1 does not result from de novo synthesis as it also appeared in cycloheximide‐treated embryos; it is very likely the consequence of a dephosphorylation process. In X.laevis, RPB1 phosphorylation has been attributed to MAP kinase during oocyte maturation; the RPB1 subunit is dephosphorylated after fertilization coincidentally with MAP kinase deactivation (Bellier et al., 1997). In parthenogenetically activated and in fertilized mouse embryos, the decline in MAP kinase activity correlates with the formation of the pronuclei (Kalab et al., 1996; Moos et al., 1996). Thus, in the 1‐cell mouse embryo, the dephosphorylation of RPB1 also follows MAP kinase inactivation.
In the mouse, the hypophosphorylated form, IIa, increases from 2–3 h after the appearance of the pronuclei, that is before class II gene transcription, which only initiates 5–6 h after formation of the pronuclei (Bouniol et al., 1995; Aoki et al., 1997). In the rabbit, where transcription from a microinjected plasmid cannot be detected until 24 hpc (Christians et al., 1994), the IIa form also increases from 2 h after formation of the pronuclei (18 hpc). As the CTD was also dephosphorylated in rabbit embryos enucleated just after formation of the pronuclei (data not shown), RPB1 subunit dephosphorylation at this stage relies neither on its nuclear localization nor on transcriptional activation. In both species, dephosphorylation of RPB1 precedes the minor ZGA. This event might be a requirement for the onset of the minor ZGA phase since the hyperphosphorylated RPB1 form, IIo, is reported to be unable to initiate transcription (Dahmus, 1996).
Embryonic forms of RPB1 insensitive to DRB are present during the minor ZGA phase
A previously undescribed slow‐migrating form of RPB1, IIe, is now reported to be present at the onset of embryonic development. The IIe form is mainly found in a cytosolic fraction which strongly suggests that it is not engaged in transcription. Indeed, the transcribing RNA polymerase molecules remain bound to the chromatin in low‐salt buffers similar to that used for fractionation (Jackson and Cook, 1985; Linial et al., 1985; Razin et al., 1985). The period during which this IIe form is detected, closely coincides with the minor ZGA phase in the rabbit, up to the fifth cell cycle (8/16‐cell stage). In the mouse, a similar IIe form is observed but very transiently and not as clearly as in the rabbit. In both species, the bulk of the slow‐migrating forms of RPB1 is not affected by DRB during the minor ZGA phase. The differences in DRB susceptibilities between the early and late 2‐cell stage mouse embryos are particularly striking. Indeed, in the mouse, numerous studies have pointed out that the major ZGA initiates precisely at the late 2‐cell stage (Flach et al., 1982; Howlett and Bolton, 1985; Taylor and Piko, 1987; Latham et al., 1991). Hence in both species, the establishment of the major ZGA is linked to the complete dephosphorylation of RPB1 in the presence of DRB.
Inefficient phosphorylation of the RPB1 subunit by the TFIIH‐associated kinase throughout the minor ZGA phase
In yeast, inactivation of the TFIIH‐associated kinase results in the rapid disappearance in vivo of the hyperphosphorylated form, IIo, of RPB1 (Valay et al., 1995, 1996). Other CTD‐kinases have been detected under stressful or developmental conditions (Dubois et al., 1994b; Venetianer et al., 1995; Baskaran et al., 1996; Bellier et al., 1997). The rabbit IIe form of RPB1 is unlikely to be derived from phosphorylation by the mammalian TFIIH‐kinase. Indeed, the IIe form lacks the CC‐3 phosphoepitope which was shown to be generated in vitro by phosphorylation with the TFIIH‐associated kinase (Dubois et al., 1997). Furthermore, both the rabbit IIe form and the slow‐migrating RPB1 forms found in the 1‐cell mouse embryo do not respond to DRB, a TFIIH‐kinase inhibitor (Yankulov et al., 1995). The phosphorylation of the CTD by the TFIIH‐associated kinase occurs within the preinitiation complex of transcription and the formation of such a complex indeed requires a DNA template plus a transactivator‐assisted recognition of promoters by the general transcription factors (reviewed in Hoeijmakers et al., 1996; Svejstrup et al., 1996). Several CTD kinases have been described in eukaryotes (reviewed in Dahmus, 1996) and the involvement of kinase(s) specific to the early embryos might be considered. Alternatively, the gradual increase in the electrophoretic migration of the phosphorylated RPB1 subunit observed in rabbit embryos at the 1‐cell stage, may suggest that the IIe form directly derives from the oocyte hyperphosphorylated form through a partial dephosphorylation.
The embryonic characteristics of RPB1 phosphorylation are compatible with transcription of the HSP70.1 gene, a landmark of early zygotic gene activity
The CTD mediates the interaction of RNA polymerase II with transcriptional activators (Gerber et al., 1995; Koleske and Young, 1995; Björklund and Kim, 1996) and the pre‐mRNA processing components (Greenleaf, 1993; Mortillaro et al., 1996; Yuryev et al., 1996; Du and Warren, 1997; McCracken et al., 1997). Phosphorylation of the CTD releases the interaction of the 'core' RNA polymerase II with TBP (Usheva et al., 1992), TFIIE (Maxon et al., 1994), and is involved in pre‐mRNA processing (Greenleaf, 1993; Mortillaro et al., 1996; Kim et al., 1997). In yeast, phosphorylation of the CTD by the TFIIH‐associated kinase is necessary for the polymerase to carry out transcription elongation (Cismowski et al., 1995; Valay et al., 1995, 1996; Akhtar et al., 1996). The hyperphosphorylated form of RPB1 recognized by the CC‐3 antibody is generated in vitro by phosphorylation with the TFIIH‐associated kinase (Dubois et al., 1997) and associates with spliceosomes (Bisotto et al., 1995; Chabot et al., 1995; Vincent et al., 1996). It is therefore intriguing to note that the heat‐shock‐inducible HSP70.1 gene (which has no introns) is transcribed during the minor ZGA phase in the mouse embryo, from the late 1‐cell to the early 2‐cell stage (Christians et al., 1995). In heat‐shocked cells, although DRB inhibits heat‐shock gene expression (data not shown), the bulk of the phosphorylated RPB1 is insensitive to DRB and the CC‐3 epitope is also lost (Dubois et al., 1994a, 1997).
DRB‐sensitive hyperphosphorylated form of RPB1: a requirement to turn on the major ZGA?
The comparative study of mouse and rabbit embryos shows that the onset of the major ZGA is linked to the disappearance of the embryonic IIe form and to the marked increase in a IIo form associated with the predominance of a DRB‐sensitive turnover of RPB1 phosphorylation. This new phosphorylation pattern is unlikely to be a consequence of transcriptional activation. First, a RPB1 hyperphosphorylated form very similar to the IIo form was generated in the rabbit embryo, even in the presence of actinomycin D which inhibited transcription. Although this form showed CC‐3 immunoreactivity, it was unlikely to be generated by the TFIIH‐associated kinase as its CTD‐kinase activity is exerted within a preinitiation complex of transcription and actinomycin D prevents the formation of such complexes (J.‐M.Egly, personal communication). However, transcription‐independent kinases such as p34cdc2 may also generate the CC‐3 epitope on the CTD (Dubois et al., 1997). Second, the number of polymerase molecules present in the rabbit embryos during the ZGA period extends far beyond the fraction likely to be involved in transcription as, on average, a single rabbit embryo contains as much RPB1 as 650 rabbit fibroblasts and a single mouse embryo contains as much RPB1 as 80 mouse fibroblasts (data not shown). Taking into account the number of cells at the ZGA (8/16 in the rabbit, two in the mouse), the number of polymerase molecules per genome is ∼40‐ to 80‐fold higher in mammalian embryonic cells than in fibroblasts. Therefore, a fast turnover of the bulk RPB1 phosphorylation due to transcriptional activity is improbable, as a very minor fraction of the RPB1 molecules is likely to be involved in transcription in these early stages.
Thus, a progressive translocation of RPB1 combined with low levels in DRB‐sensitive hyperphosphorylated IIo form may account for the time‐controlled restricted zygotic gene activity that follows fertilization. The onset of zygotic transcription is also delayed in non‐mammalian metazoans such as Caenorhabditis elegans and Drosophila melanogaster (Yasuda and Schubiger, 1992). In these species, transcription is initiated in the presomatic cells first, and in the primordial germ cells afterwards. It has recently been shown that in the transcriptionally inactive primordial germ cells, the CTD lacks the H5 phosphoepitope which is present in the transcriptionally active presomatic cell nuclei (Seydoux and Dunn, 1997); the H5 phosphoepitope is associated with transcription foci (Zeng et al., 1997). Therefore, we propose that deficient phosphorylation of the CTD is linked to the transcriptional repression which characterizes early embryos from different phyla.
Materials and methods
Oocyte and embryo collection
F1 hybrid (C57BL/6×CBA) mice females, superovulated by injection of 10 IU pregnant mare serum gonadotrophin (PMSG, Intervet) followed 46–48 h later by injection of 5 IU of human chorionic gonadotrophin (hCG), were mated or not with F1 hybrid males. Fertilization occurs at ∼12 h post‐hCG injection. Unfertilized oocytes or 1‐cell zygotes were collected 16 h post‐hCG injection and cultured in an incubator in 8% CO2 at 37°C, in M16 medium under oil (light paraffin, BDH) (Christians et al., 1995). The appearance of the pronuclei was followed by visual inspection every 30 min and allowed batches of synchronized embryos to be collected.
New Zealand White rabbits were mated after superovulation and sacrificed 14 hpc for the experiment reported in Figure 3A or 19 hpc for all other experiments. Embryos were recovered as described previously (Christians et al., 1994) and cultured in 199 medium supplemented with 10% fetal calf serum in a 5% CO2 atmosphere at 39°C. In this species, fertilization occurs at ∼12 hpc. Fertilization was assessed by the presence of two polar bodies. Asynchronous cell division is a general feature of rabbit embryonic development after the second cleavage division (4‐cell stage) leading to an 8/16‐cell stage and a 16/32‐cell stage occurring at a relatively precise timing from mating (Ménézo and Renard, 1993).
Monoclonal antibody POL3/3 (a generous gift of Prof. E.K.Bautz) was directed against an epitope located in the core of the RPB1 subunit (Krämer et al., 1980). The 8WG16 monoclonal antibody (a generous gift of Dr N.Thompson) was targeted against the CTD (Thompson et al., 1989). The CC‐3 monoclonal antibody was directed against a hyperphosphorylated form of RPB1 (Vincent et al., 1996; Dubois et al., 1997).
Lysis, fractionation and Western blot analysis
Whole lysates prepared from batches of five rabbit embryos or 50 mouse embryos in denaturing buffer L [20 μl of 50 mM Tris–HCl, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate (SDS), 1% 2‐mercaptoethanol] were electrophoresed in denaturing 5% polyacrylamide–SDS gels and blotted onto nitrocellulose. For fractionation, batches of 15 rabbit embryos were depellucidated in pronase, lysed in 20 μl of buffer F (20 mM sodium β‐glycerophosphate, pH 7.4, 1 mM orthovanadate, 0.2 mM EGTA, 2 mM MgCl2, 0.5% Nonidet P40, 0.1% 2‐mercaptoethanol) and the lysate was loaded on a 20 μl cushion consisting of buffer F supplemented with sucrose (20%) and stained with bromophenol blue. After 5 min of centrifugation at 1000 g, the supernatant which remained above the cushion was designated as the 'cytosolic' fraction and was supplemented with buffer L. The nuclear pellet was dissolved in buffer L. Immunodetection of the RPB1 subunit involved the use of either the POL3/3 or the CC‐3 antibody followed by a peroxidase‐labelled anti‐mouse immunoglobulin antiserum and enhanced chemiluminescence (New England Nuclear). All Western blot experiments were repeated at least three times and showed essentially the same results on each occasion.
Immunofluorescence studies
For immunofluorescence studies with the POL3/3 monoclonal antibody, oocytes and embryos were fixed in phosphate‐buffered saline (PBS), 4% paraformaldehyde for 15 min, permeabilized in PBS, 0.2% Triton X‐100 for 15 min, blocked in PBS, 3% bovine serum albumin (BSA) for 30 min and incubated with the POL3/3 antibody in PBS, 0.1% Triton X‐100, 0.3% BSA for 1 h, then washed three times in PBS, 0.1% Triton X‐100 followed by incubation with fluorescein (FITC)‐labelled anti‐mouse antibody. Nucleic acids were counterstained with 10 μg/ml propidium iodide in PBS for 15 min. The fluorescence was detected with a Zeiss laser‐scanning confocal microscope LSM‐310 using a Zeiss plan neofluor 100× (NA 1.3) oil immersion objective and 1 μm sections were made through the embryos.
For immunofluorescence studies with the 8WG16 antibody, embryos were treated as above except that: (i) the paraformaldehyde fixation step lasted 18 h; (ii) an RNase step was included as described (Worrad et al., 1995); and (iii) the fluorescence was detected by a Molecular Dynamics laser‐scanning confocal microscope with a Zeiss Plan‐Apochromat 63×/1.40 oil immersion objective.
Series of embryos corresponding to different stages of development were collected, processed and analysed together under the same staining conditions and using the same settings (filter, gain, background levels). Image processing did not involve either background or contrast adjustments. All the embryos presented in a given figure were selected from a single experiment, which was repeated at least three times and showed essentially the same results on each occasion.
This work was supported by grants from the Association pour la Recherche sur le Cancer (ARC 6250), and ACC SV4 (contract No. 504152). We are much indebted to Evelyne Campion and Patrick Chesné for their help in experiments, to Geneviève Almouzni, Michael Dahmus and Marie‐Françoise Dubois for their interest in this work and for critical reading of the manuscript, and to Susan Joyce for English proof‐reading.
↵† S.Bellier and S.Chastant contributed equally to this work
- Copyright © 1997 European Molecular Biology Organization


text:
I must say I'm quite thrilled with the new album. After feeling just a hair let down by Hey Venus!, their ninth studio album is adventurous in all the right places. It hop scotches from psyched-out jams to krautrock to funk-laced grooves to foot-pounding stadium rock. Best from SFA in a long while.
Moped Eyes
Inaugural Trams
The Very Best Of Neil Diamond

subset: commoncrawl


text:
I can't believe we made it this far! I feel so blessed and am truly loving being pregnant. I have my next doctor appointment on Thursday and I can't wait!!! For now here is the newest belly shot!
October 27, 2009
12 weeks!!!
Posted by Faith Bud at 1:32 PM
Labels: 12 Weeks, Bump pictures, Faith Bud, success after loss
2 comments:
- bethierose said...
your belly is too too cute!
- October 27, 2009 at 7:10 PM
- Faith Bud said...
Thank you!!!! It has grown SO much since just last week! lol
- October 28, 2009 at 5:35 PM
Subscribe to: Post Comments (Atom)


text:
When someone shares with you something of value, you have an obligation to share it with others.
Thursday, 25 February 2010
Sovereign debt crisis on the cards
1 comment:
- Guanyu 道 said...
Sovereign debt crisis on the cards
Harvard's Rogoff warns that history will repeat itself with global defaults
Bloomberg in Tokyo
24 February 2010
Ballooning public debt is likely to force several countries to default and the United States to slash spending, according to Harvard University Professor Kenneth Rogoff, who in 2008 predicted the failure of big US banks.
Following banking crises, "we usually see a bunch of sovereign defaults, say in a few years. I predict we will again", Rogoff, a former chief economist at the International Monetary Fund, said at a forum in Tokyo yesterday.
He said financial markets would eventually drive bond yields higher, and European countries such as Greece and Portugal would "have a lot of troubles".
Global scrutiny of sovereign debt has risen as nations including Greece reveal fiscal deficits that have swollen in the wake of the worst global financial crisis since the Depression.
"It's very, very hard to call the timing, but it will happen," Rogoff, a co-author of a history on financial calamities, said in the speech. "In rich countries - Germany, the United States and maybe Japan - we are going to see slow growth.
"They will tighten their belts when the problem hits with interest rates. They will deal with it."
Concern about Greece's ability to fund its debt have roiled financial markets since the government said it had a budget shortfall of 12.7 per cent last year, the highest ratio in the 27-member European Union.
Greece's debt totalled €298.5 billion (HK$3.16 trillion) at the end of last year. That is about five times more than Russia owed when it defaulted in 1998 and Argentina when it missed payments in 2001.
The cost of protecting Greek sovereign debt from default surged last month, then fell this month as concern eased over the country's creditworthiness. Credit-default swaps on Greek sovereign debt have fallen to 356 basis points from 428 last month, according to CMA DataVision. That is up from 171 at the start of December.
"Greece just highlights that one of those risks is sovereign default," said Naomi Fink, a strategist at Bank of Tokyo-Mitsubishi UFJ.
Still, "it doesn't justify the situation where we're all in a panic and are going back to cash in the post-Lehman shock", she said, referring to the global credit freeze following the September 2008 collapse of Lehman Brothers Holdings.
In an August 2008 interview, Rogoff said "the worst is yet to come in the US" and he predicted the collapse of major investment banks.
Meanwhile, Japan has the largest debt of all, with the finance ministry estimating borrowings of 973 trillion yen (HK$83 trillion) by March next year, more than the economic output of Britain, France and Italy combined.
Japanese fiscal policy is "out of control", said Rogoff, who is a member of the Group of 30, a panel of central bankers, finance officials and academics headed by former Federal Reserve chairman Paul Volcker.
Standard & Poor's last month warned that it may downgrade Japan's AA credit rating unless the government comes up with a plan to reduce the debt burden.
Bank of Japan governor Masaaki Shirakawa last week urged the government to show how it plans to repair its finances, and Finance Minister Naoto Kan aims to release a fiscal strategy by June.
Fink said Japan's debt was sustainable because more than 90 per cent of the country's bonds were held by domestic investors, reducing the risk of capital flight.
"For Japanese investors, JGBs are risk-free assets no matter what S&P ranks them," she said.
Rogoff's 2009 book This Time is Different, co-written with Carmen Reinhart, charts the history of financial crises in 66 countries.
- 25 Feb 2010, 01:46:00


text: Beware the big bear hug. Bewear-gx can smush opposing Pokémon with its powerful bear hug, or Chuck them out of a battle entirely with the big throw-gx attack! it's an amazing demonstration of the raw strength that makes bewear-gx more than just another charming face. Add this astonishing Pokémon-gx to your collection give your opponents new respect for this strong-and-cuddly Pokémon.


text:
Tomorrow you will be 1! But you are already so much fun!
Tuesday, September 7, 2010
Happy Birthday baby boy!
Penguin suit
Gavin was a ring bearer in Paul's wedding Aug 28th. Here are a couple pictures to show him trying it on. I didn't get any of the ceremony but hopefully someone can share.
Posted by The Wiechman's at 9/07/2010 No comments:
Mom and Boys
Gavins Birthday
Yes he was born in July. Yes I know its September. Enjoy the pictures! We went to a Saltdogs game and celebrated with the Wiechman clan.
Subscribe to: Posts (Atom)


text:
Day 21.
The boxes are slowly dwindling and the rooms are starting to resemble actual rooms.
We still have no central heating or hot water – the engineer is due Monday so fingers crossed for then. We have been given electric heaters but I'm reluctant to use them because of the cost. But actually. this place isn't that cold. I'm making sure doors are closed and have hung fleeces on the bedroom windows. Tucking the kids up with a hot water bottle has always been a winter ritual anyway.
I was talking to my daughter about how we used to chip the ice off the inside of our bedroom windows as kids! My mum still refuses to have central heating fitted and has only ever used a gas fire in the lounge for heat. Anyway, I digress…
It is starting to feel like home. It's starting to sink in that we can settle here for a long while now. It is worth buying curtains and silly things for each room. I'm off tomorrow to buy some furniture from a great second-hand charity warehouse. Just drawers and wardrobes and maybe even a bookshelf or two. Then the last few boxes can be unpacked and It really will be home.
But for now I am scratching my head at the world of curtains. I'm so indecisive sometimes!


text:
Managen Sie das Citrix-Produktportfolio bequem über die Cloud. Vereinfachen Sie die Kontrolle über Ihre Apps, Desktops, Inhalte, Geräte und Netzwerke.
Israel Electric Corporation is the primary electric utility for the State of Israel, generating most of the energy and transmitting essentially all electricity used in the country. Headquartered in Haifa, it's one of Israel's largest industrial companies, with total assets of $21,6 billion. The company designs, builds, maintains and operates 17 power-generation stations, along with all the nation's electric transmission and distribution networks. Its 13.500 employees serve more than 2,5 million residential, commercial and industrial customers.
Israel Electric was planning to migrate 8.000 of its employees to Microsoft Windows 7 and Office 2010 before Microsoft withdrew support for Windows XP—which meant the utility had to upgrade 80 percent of its desktops to run the new operating system. Not only that, the company had more than 4.000 applications in its environment, and there was no consistency because desktops were maintained by administrators in each local office. IT management was cumbersome and inefficient, and desktop performance was poor. What's more, it was nearly impossible to keep track of security patches for desktop applications, which put corporate data at risk.
The utility turned to Citrix for a virtual desktop infrastructure (VDI). The goal was to enable the Windows 7 upgrade in the short term and to improve IT management, desktop performance and service to customers in the long term. Citrix XenDesktop delivers a consistent, stable desktop image to employees. Citrix XenApp provides access to a standardized set of centrally managed applications. Citrix Receiver allows employees to access their desktop environments at home and on the road from their devices of choice, whether a smartphone, tablet or personal laptop. "In the past we supported the PC," says Shai Levi, manager of the desktop virtualization project for Israel Electric. "With Citrix, we can support the employee. It's the user that's important."
The Citrix solutions made it straightforward for Israel Electric to migrate employees to Windows 7 and Office 2010. Rather than upgrade physical PCs and then manually migrate each desktop, IT staff can now manage that effort centrally. "The entire VDI project, including the migration to Windows 7, cost less and took less time than when we upgraded from Windows 95 to Windows XP," says Yosi Shneck, senior vice president of information and communication for Israel Electric. The utility also reduced the number of applications in its environment from 4.000 to about 700. As a result, IT management is more efficient and cost-effective; users have access to the software they need to do their jobs; and a stable, high-performance desktop environment enables employees to be more productive.
With a virtualized desktop environment, Israel Electric no longer relies on inexperienced administrators in local offices to manage individual PCs. Now IT staff can centrally manage standardized desktop images to support the requirements of each user role. Just as important, IT technicians have the confidence that all applications are updated with the latest security patches, helping ensure the protection of sensitive corporate data. Overall, the IT environment benefits from greater stability and performance. "The Citrix environment has meant huge efficiency gains for our IT technicians in the field. In fact, the time it takes IT field staff to serve employees has decreased by 50 percent," Shneck reports.
Empowering employees to work from anywhere and deliver better service to customers
Israel Electric management and staff increasingly work from home and on the road. The Citrix solutions allow them to access their desktop environments wherever they need to, from smartphones, tablets or their personal laptops. That flexible connectivity proved vital in December 2013 when much of Israel was hit with the worst winter weather in decades. "We set up command-and-control centers around the country," Levi explains. "With Citrix, those users could get immediate access to the same corporate workspace they use every day." Users are equally pleased with the Citrix environment on an ongoing basis. "I've been working in IT for 40 years," Shneck says. "This was the first project where users actually came to us asking to be switched to the new environment because they saw how happy other employees were with VDI and wanted it for themselves."
Israel Electric is now migrating its computer-aided design (CAD) users to the Citrix virtual desktop environment. The CAD users include the engineers who design, plan and construct the power plants, transmission lines and other infrastructure that's core to the utility's mission. Going forward, the company will move users from 32-bit to 64-bit desktop images. "We simply create two application catalogs, one for 32 bits and one for 64 bits," Levi explains. When it's time to move the user, IT technicians just assign the new catalog. "The Citrix environment means any future upgrades will be far easier," Shneck concludes. "We can change the operating system without the need to replace PCs, and employees will continue to see their familiar environment and applications."
zu Citrix
Citrix (NASDAQ:CTXS) is a leader in mobile workspaces, providing virtualisation, mobility management, networking and cloud services to enable new ways to work better. Citrix-Lösungen ermöglichen Business Mobility durch sichere, persönliche Arbeitsplätze, die Mitarbeitern von jedem Gerät aus über beliebige Netzwerke und Clouds einen sofortigen Zugriff auf Apps, Desktops, Daten und Kommunikationen bietet. Bereits seit 25 Jahren gestaltet Citrix mit innovativen Produkten IT einfacher und Arbeitsweisen effektiver. With annual revenue in 2013 of $2,9 billion, Citrix solutions are in use at more than 330.000 organisations and by over 100 million users globally. Weitere Informationen unter www.citrix.de.
Copyright © 2014 Citrix Systems Inc. All rights reserved. Citrix, XenDesktop, XenApp and Receiver are trademarks of Citrix Systems Inc., or a subsidiary thereof, and are or may be registered in the U.S. Patent and Trademark Office and other countries. All other trademarks are the property of their respective owners.
Die wichtigsten Vorteile


text:
We are headed to Hong Kong for 4 days the first weekend of April.
Our now world famous winners: Renee Lim and Bryan Chang, both WAA coworkers in Malaysia which will make delivery easy for their oh-so-exciting prizes, which will be purchased in Hong Kong.
Thanks everyone for playing. Hope you enjoyed the game. Great guesses–most of which are on our list of travel destinations for the next 2 years. It was really fun for us to see where you guessed. As we book future plane tickets, we'll play again!
Some of you need to go find a globe or make friends with Google maps (sorry Lo, but KL is not an Island, but at least you were correct that it's north of Singapore). Others need to Wikipedia geographic terms so they know what an Island is or is not (yes Japan is an Island, technically it's a grouping of islands). I told you this was going to jog your 4th grade memory!! Learn something new every day.
Please remember, only one guess per person per day, and that also means each guess can only include one destination. Renee had actually guessed two, but as Hong Kong was her first one, and I hadn't been specific with the directions, I gave it to her.
Several asked how specific you had to be: generally speaking, it depends on the trip and the country. If our trip is only going to be a single city (usually a very well known city like Hong Kong) then I'd think you need to name the city and a guess of China would not count. I'll make note of how specific you need to be in the header of future games. Also, we might factor in the vagueness of the hint at the time you guess. ex: if we said North of Singapore and you said Japan and the answer was Tokyo, we'd probably give it to you.


text:
Actions to protect road bridges and tunnels against terrorist attacks are rarely cost-effective, an EU-funded project has concluded.
Either the cost of protection must be very low or the piece of infrastructure must be very important for measures to be worthwhile on the grounds of attack resilience alone, according to the team working on the SeRoN (Security of Road Transport Networks) project. However, additional safety or traffic management benefits may tip the economic balance.
The three-year project, which is just ending, set out to study the regional and wider impacts of possible manmade attacks on the transport network. Seven partners were involved from six European countries, led by Germany's PTV Group.
They developed a four-step methodology and toolset that: identifies and prioritises 'infrastructure objects' on the road network; evaluates the importance of the asset to the network; assesses the risk for potentially critical objects; and gauges the effectiveness of protective measures.
Results and conclusions from the project were presented this month at a summit in London on infrastructure risk and resilience, organised by team member Parsons Brinckerhoff and the IET.
SeRoN focused on bridges and tunnels because they act as bottlenecks and attacks have the potential to cause structural damage and substantial human casualties, as well as having economic, political and environmental effects. Dr-Ing Georg Mayer of PTV said that one problem was the huge number of infrastructure objects on a road corridor, forcing the team to modify their pre-selection method.
Selection criteria were based on traffic volume including HGV traffic, damage potential, reconstruction time and symbolic value. Assets identified as potentially critical were ranked according to their importance to the network by considering the consequences of non-availability, using traffic simulations. Then the most critical structures were assessed 'as is' and with possible protective measures. From this the effectiveness and cost-effectiveness of the measures can be established.
Mayer pointed out that detailed and comprehensive analysis of this type may not be affordable for most road operators. However, he said, "step 1 is easy, fast and reliable" and can be followed by further analysis for specific objects.
"One lesson learned," Mayer continued, "is that from the engineering point of view there are hardly any cost-effective security measures. But it's one view. If you take social and political views into account it may be different."


text:
|Dictionary of Law
memorandum :: legal definition
Memorandum - n. 1) a brief writing, note, summary or outline. 2) A "memorandum of decision," or "memorandum opinion," is a brief statement by a judge announcing his/her ruling without detail or giving extensive reasons, which may or may not be followed by a more comprehensive written decision. Such memoranda (plural) are issued by appeals courts in language such as: "The petition of appellant is denied for the reasons stated in Albini v. Younger," or "The decision below is affirmed."
Related Law Termsappeal :: 1) v. to ask a higher court to reverse the decisio ... ng, as in "he has filed an appeal."
authorities :: n. 1) previous decisions by courts of appeal which ... also: brief cite decision precedent
brief :: 1) n. a written legal argument, usually in a forma ... another person. See also: precedent
constitution :: n. the fundamental, underlying document which esta ... ts common law constitutional rights
law book :: n. any of numerous volumes dealing with law, inclu ... : common law law Shepardize statute
living will :: n. also called "a durable power of attorney," it i ... upport. See also: power of attorney
not guilty :: n. 1) plea of a person who claims not to have comm ... t guilty by reason of insanity plea
objection :: n. a lawyer's protest about the legal propriety of ... ng question object overrule sustain
proof :: n. confirmation of a fact by evidence. In a trial, ... doubt preponderance of the evidence
review :: n. the judicial consideration of a lower court jud ... ate court reversal reversible error
Popular Law Searcheslegal definition ministerial act :: n. an act, particularly of a governmental employee ... exercising any individual judgment.
chancery legal definition :: n. a court that can order acts performed. Today ch ... ts in most states. See also: equity
testamentary disposition legal definition :: n. how the terms of a will divide the testator's ( ... named beneficiaries. See also: will
contract of sufferance :: n. a "hold-over" tenancy after a lease has expired ... y tenancy at will unlawful detainer
forthwith legal definition :: adv. a term found in contracts, court orders and s ... mediacy, with no excuses for delay.
forthwith definition legal :: adv. a term found in contracts, court orders and s ... mediacy, with no excuses for delay.
what is to wit in a legal contract :: prep. that is to say. Example: "the passengers in ... tty Bumgartner and Sherry Younger."
mirror wills legal definition :: n. the wills of a husband and wife which are ident ... d each names the other as executor.
clean hands doctrine latin :: n. a rule of law that a person coming to court wit ... ices. See also: affirmative defense
sine qua non only relates to contract law :: (see-nay kwah nahn) prep. Latin for "without which ... qua non of the injury to Playmate.
Other Law Terms - MM. O. :: n. slang for modus operandi, the way or pattern in ... her crime. See also: modus operandi
magistrate :: n. 1) a generic term for any judge of a court, or ... ce of the peace preliminary hearing
Magna Carta :: n. Latin for "Great Charter," it was a document de ... s. It is also spelled Magna Charta.
mail box rule :: n. in contract law, making a written offer or acce ... nse in hand when the time runs out.
maim :: v. to inflict a serious bodily injury, including m ... to a prison term. See also: mayhem
majority :: n. 1) the age when a person can exercise all norma ... t. See also: child infancy minority
make :: v. 1) to create something. 2) to sign a check, pro ... xchange check maker promissory note
make one whole :: v. to pay or award damages sufficient to put the p ... fault of another. See also: damages
maker :: n. 1) the person who signs a check or promissory n ... : check payee payor promissory note
malfeasance :: n. intentionally doing something either legally or ... sponsibility. See also: misfeasance
malice :: n. a conscious, intentional wrongdoing either of a ... cution murder public figure slander
malice aforethought :: n. 1) the conscious intent to cause death or great ... : first degree murder malice murder
malicious prosecution :: n. filing a lawsuit with the intention of creating ... vor of the victim. See also: malice
malpractice :: n. An act or continuing conduct of a professional ... eed. See also: errors and omissions
malum in se :: (mal-uhm in say) adv. Latin referring to an act th ... hibitum. See also: malum prohibitum
malum prohibitum :: (mal-uhm prohibit-uhm) adj. Latin meaning "wrong d ... lso: malum in se white collar crime
mandamus :: (man-dame-us) n. Latin for "we order," a writ (mor ... mandamus. See also: writ of mandate
mandate :: n. 1) any mandatory order or requirement under sta ... See also: mandamus writ of mandate
mandatory :: adj., adv. absolutely demanded or required. ... v. absolutely demanded or required.
mandatory joinder :: n. the required inclusion of a party in a lawsuit ... sues in the case. See also: joinder


text:
Another video
Well, I do not know if you guys liked my last video or not. I only had a few comments about it. But I have one more for you and yes I am in this one too. I actually like being behind the scenes but we had a little technical difficulties and had to shoot the opening scene again. The guy who we used for the first scene could not come back and do it again so I was the lucky one to take his place.
This is probably my favorite video we have ever done. It is funny AND it has a message. I know when we have a good idea for a video because God gives us an idea and it just seems to go so smoothly. This is one such video. Our youth pastor said the high school retreat was going to be call "A Reflective Life." God put the idea for this video in my head as soon as he said the name of the retreat. Some one had just given me a bedroom suit and it came with this big mirror. It was like it was all planned out for us. It is so cool how God works!
Anyway, Josh over at Youth Group videoes. com has put this video up on his website for all to see. Go by and check it out. It is the video called A Reflective Life. If you are a person who will cry at the drop of a hat you may want some tissues. I hope ya'll will like it.
God bless, Joe Another video


text:
Performance Details
Event 5000 Meter Run
Mark 17:22.00
Place 32nd
Race/Results Page Varsity Results
Round Finals
Team Arapahoe High School
Native Country USA
Season XC 2013
Level HS
Meet Date Aug 30, 2013
Date Added Aug 31, 2013
Grade 2016
Timing FAT
Personal Best? No
Season Best? No
Venue deKoevend Park XC Course, Littleton, CO, USA
No comments on this performance. Add a comment


text:
Thursday, June 28, 2007
Dust to Dust
New Scientist Environment has this report on a microwave technology to turn plastics back into Diesel oil and gas. Could be the way to handle these mucky plasitcs. I like the idea of stuffing the car tank with plastic bags and driving to work.
A US company is taking plastics recycling to another level – turning them back into the oil they were made from, and gas.
All that is needed, claims Global Resource Corporation (GRC), is a finely tuned microwave and – hey presto! – a mix of materials that were made from oil can be reduced back to oil and combustible gas (and a few leftovers).
Key to GRC's process is a machine that uses 1200 different frequencies within the microwave range, which act on specific hydrocarbon materials. As the material is zapped at the appropriate wavelength, part of the hydrocarbons that make up the plastic and rubber in the material are broken down into diesel oil and combustible gas.
GRC's machine is called the Hawk-10. Its smaller incarnations look just like an industrial microwave with bits of machinery attached to it. Larger versions resemble a concrete mixer.
"Anything that has a hydrocarbon base will be affected by our process," says Jerry Meddick, director of business development at GRC, based in New Jersey. "We release those hydrocarbon molecules from the material and it then becomes gas and oil."
Whatever does not have a hydrocarbon base is left behind, minus any water it contained as this gets evaporated in the microwave.
Subscribe to: Post Comments (Atom)


text:
">Yet again, in comparison with units that experienced no unfastened gear (other than plates remaining within the trays though unused), this aspect of the Ironmasters manufactured them a bit unwieldy.This minimal bit listed here will let you justify the price you'll have paying for a fresh set of adjustable dumbbells. Let's assume which the likel… Read More
The CAP brand name is a popular one particular In terms of Exercise gear. It's no surprise that the Barbell Black A Frame Dumbbell Rack is one of the better-sellers on our listing. You can use this A-frame rack to carry 5 pairs of cast iron, neoprene and rubber hex dumbbells nearly two hundred lbs.When you're buying a dumbbell rack, there are m… Read More
Biceps are uncomplicated to operate with curls of any sort but some strike different parts of the bicep based on form. Be sure to go gradual up and down and squeeze at the very best. To work your inner bicep far more, convey the DB up and out.The previous leg exercise routines ought to have worked your hamstrings and finishing with This could be en… Read More
Inside the Amazon description from the product or service you'll find a complete bullet listing of exercises you could experiment with.If you're looking for the spending budget possibility we might propose Yes4All. It really is Establish quality is outstanding for these types of rate and gives threaded grip which happens to be ergonomic and easy to… Read More


text: Sue talks with Chuck Colby from "Three Men and a Tenor" about 25 years with the group and the 25 years of MFEA. They touch on some of the good times, and a bit of music from the group. They also talk about the importance of festivals and events to our communities.


text:
A poster bearing the words "Europe's shame" was hanging in the heart of the EU quarter in Brussels on Wednesday as a European summit got underway.
The idea of Europe is drowning in the Mediterranean: Mayor of Lampedusa warns in Brussels
The idea of Europe is drowning in the Mediterranean: Mayor of Lampedusa warns in Brussels
Due to police restrictions, protesters were not allowed stand beside their banners.
Meanwhile, the President of the European Parliament Martin Schulz hosted the Mayor of Lampedusa and the President of Sicily to discuss the EU's migration policy.
Earlier this week in Strasbourg, a European Parliament resolution said the drownings on Europe's shores should mark a turning point for the EU and that members states had a legal obligation to do their bit.
For his part, Martin Schulz said the ball was in the court of the member states: "The European Parliament has a clear line, but the opinions between the 28 member states are very different on temporary protection, on legal immigration and on political asylum."
The Mayor of Lampedusa Maria Nicolini said the whole idea of Europe was drowning with the deaths in the Mediterranean. "Now that you have all seen the boats, she said, "something has to change so please help us and do not disappoint us."
While the 28 European heads of state discussed how the EU's migration policy should be reformed, 127 migrants were rescued by the Italian Coast Guard seven miles from Lampedusa on Thursday, among them 63 women and 19 children.


text:
Aircraft Parts Stores near:
About Aircraft Parts Stores Near Me
Aircraft Parts Stores Near Me
Find the best Aircraft Parts Stores near you
Sponsored link
Info about Aircraft Parts Stores in Adair, Oklahoma, OK
There are 14 Aircraft Parts Stores in or near
Adair, Oklahoma OK
AVIATION CONTROLS INC is located approximately
57 miles
from Adair. If you want to pay them a visit, go to 101 FREEDOM DR. Their current phone number is (620) 331-8180.
Read more about
AVIATION CONTROLS INC in INDEPENDENCE, KS
Avia Plan
Avia Plan is located approximately
46 miles
from Adair. They are regarded as one of the best Aircraft Parts Stores in Adair area. Their phone number is (479) 549-3313.
Read more about
Avia Plan in Siloam Springs, AR
Aviaplan Inc
Aviaplan Inc is located approximately
45 miles
from Adair. Visit Aviaplan Inc at 751 Highway 16 E Apt C7. Their current phone number is (479) 685-3542.
Read more about
Aviaplan Inc in Siloam Springs, AR
Bemco Inc Aircraft Equipment
Bemco Inc Aircraft Equipment is located approximately
55 miles
from Adair. Visit Bemco Inc Aircraft Equipment at 233 Main St. If you need more information, call them: (479) 795-2121.
Read more about
Bemco Inc Aircraft Equipment in Centerton, AR
Camtronics Llc
Camtronics Llc is located approximately
49 miles
from Adair. A friendly Aircraft Parts Store. Their current phone number is (918) 299-5148.
Read more about
Camtronics Llc in Tulsa, OK
Sponsored link
Coffeyville Sektam Inc
Coffeyville Sektam Inc is located approximately
48 miles
from Adair. Why don't you give them a try?. Contact them at (620) 251-3880.
Read more about
Coffeyville Sektam Inc in Coffeyville, KS
Controls Components Ltd
Controls Components Ltd is located approximately
33 miles
from Adair. Customers have good opinions about Controls Components Ltd. Need to give Controls Components Ltd a call? (918) 739-4400.
Read more about
Controls Components Ltd in Catoosa, OK
Eagle Aircraft Supply
Eagle Aircraft Supply is located approximately
47 miles
from Adair. They're a really good Aircraft Parts Store. Call them at (918) 369-1693.
Read more about
Eagle Aircraft Supply in Bixby, OK
Kansas Aviation-Independence
Kansas Aviation-Independence is located approximately
57 miles
from Adair. Kansas Aviation-Independence is very popular place in this area. Contact them at (620) 331-7716.
Read more about
Kansas Aviation-Independence in Independence, KS
Mid-America Parts Intl
Mid-America Parts Intl is located approximately
10 miles
from Adair. They're a decent Aircraft Parts Store in Adair. Contact them at (918) 825-7898.
Read more about
Mid-America Parts Intl in Pryor, OK
Sabreliner Corp
Sabreliner Corp is located approximately
57 miles
from Adair. Their exact address is: 101 Freedom Dr. Phone number: (620) 331-8180.
Read more about
Sabreliner Corp in Independence, KS
Simair is located approximately
49 miles
from Adair. A decent Aircraft Parts Store, they're located at 613 W Needles Ave. Phone number: (918) 366-6670.
Read more about
Simair in Bixby, OK
Southwestern Aero Exch
Southwestern Aero Exch is located approximately
33 miles
from Adair. Join the group of happy customers of Southwestern Aero Exch!. Their phone number is (918) 272-9815.
Read more about
Southwestern Aero Exch in Owasso, OK
Spec Tech Usa Inc
Spec Tech Usa Inc is located approximately
50 miles
from Adair. A decent Aircraft Parts Store, they're located at 6203 S 39th West Ave. Their phone number is (918) 794-8282.
Read more about
Spec Tech Usa Inc in Tulsa, OK
Sponsored link


text:
Lately, I have been doing some shopping at Furniture From Home. I think the prices are really good and I love that they have free shipping! I also like that the site shows several different photos of the different pieces of furniture. It's very easy to navigate…and did I tell you that the prices are great?!
After I replace the couch, I'm going to head over to the furniture home office section to look for a computer desk and a chair. I really need a good chair so I can blog in comfort!! After that, I'm going to check out the bedroom furniture for my bedroom….it's so time for a bedroom makeover!


text:
The guide also mentions the egg->chicken money making strategy I learned. and it suggests doing the 2nd melody after rescuing Paula, like I thought...
Stopped reading after the page that tells you how to get to Threed.
- Threed part 1 -
Someone at the hotel warns you about the monsters outside... it's especially dangerous at the graveyard.
Someone outside the hotel:
There's a rumor that there is an underground path at the edge of the graveyard that takes you somewhere. When you get through the path, you'll meet a dirty, stinky, ugly powerful monster.Everyone is hiding out here at the center of town.. near the circus tent that is the HQ of zombie relief corps.
lol this guy in this one house... a double agent for the zombies, gives lore/spoilers on this zombie creator named Belch, who is a follower of Giygas, who "is female, I think."
Hmm, exploring the town is the obvious next priority here... I checked out a bit of the south graveyard, fought some zombies, found a photo...
Then went around the upper right gated graveyard and found a hint house... and a teddy bear in a trashcan... score!
The hospital has a sign saying the cure to being bitten by a zombie is to bite back... but this theory hasn't been tested.
lmao I love this dialog this game is amazing


text:
It's been so long since I had an entry for this. Maybe it was because I do not have much problems with things I often thought I had.
Now that I am sitting here again, typing the story of my life out for non-existant audiences. But it feels good, it actually feels good that things are out of my mind, thoughts flowing into fingers and fingers onto the keyboard to this anonymous space where no one knows me.
News from abroad had been bad. Not coming back, might be staying on.. things like that. Not something very happy when all you were thinking about was how exciting things will be when this year and a certain month arrives. Oh well.. I know what G means when she says 'You do know that he doesn't feel the same way about you right?' Because I had been telling a few people about this too and I am totally ignorant about it. Or maybe I just didn't want to accept the fact. Maybe. Definitely. Ohwell..
So what? So what? Questions run through my mind and hits the side of my brain with a loud thud. Things never really had worked out for me in a long long time and maybe just one fine day, hopefully, with fingers crossed thing could finally work out the way I would want it to be.
Starting to sound selfish here. Things should always be my way. Of course. I am only human. Selfish little human me says, ' Come back you fool! Even though you'd be here, so near yet so far, come back..'
Friday, March 04, 2005
No comments:
Subscribe to: Post Comments (Atom)


text:
In this issue:
CRY Freedom 2013: Music for a cause»
Groupon partners with GiveIndia to change a life»
Donors Meet at Banglore»
Change a life:
Educate a disabled child for a year»Rs. 5,000»
Sponsor education & development needs of a child for a year» Rs. 4,160»
Support medical diagnosis and treatment cost for a poor person» Rs. 3,600»
Every cause needs a voice, with CRY Freedom 2013, music gives a voice to women/girls who have faced abuse. GiveIndia is proud to be the philanthrophy partner for the event to enable donations being channeled to NGO's in this domain.
The event comprises of two concerts to be held in Mumbai & Delhi on the 9th and 26th of August 2013 respectively. Various popular artists will be performing on songs based on the theme.
CRY Freedom 2013 aims to help 51 women/girls from 2 NGO's,you can support the cause by donating on www.GiveIndia.org
We're happy to inform you that Groupon has partnered with GiveIndia and Sankara Nethralaya, a non profit for a charitable cause. This tie up will help 2 underprivileged patients get a set of spectacles each. For the poor, eyeglasses often are either inaccessible or unaffordable, forcing millions of people to live below their full potential.
You can give the gift of sight to 2 underprivileged patients by providing for spectacles worth Rs 550.This deal can be bought over the next 21 days.You can be the one to help them see the world better!! For details please visit: www.Groupon.co.in
Our mission this month is toprovide school supplies to 200 children in July 2013. We've been able to help 190 children with your help.
Thank you very much!! We know we can do a lot more! Do pitch in:
Sponsor text books and uniform for 2 poor children pursuing quality education for a year - Rs. 4,800»
Sponsor clothing and school supplies for 2 poor children studying in a primary or a secondary school -Rs. 3,520»
The 5 NGOs to receive the maximum donations were:
1. The Akshaya Patra Foundation - Rs. 2,50,335
2. Deepalaya - Rs. 2,14,552
3.Priyadarshini Seva Mandali - Rs. 1,70,800
4 Dr. Shroff's Charity Eye Hospital - Rs. 1,68,996
5. The Association of People with Disability - Rs.1,48,788
The 5 NGOs to receive the minimum donations were:
1. Sankalp Rehabilitation Trust - Rs. 1,700
2. Hariksha People's Welfare Trust - Rs. 1,666
3. Bhumi - Rs. 1,250
4. Basic Research Education And Development (BREAD) Society - Rs. 750
5.Ashadeep - Rs. 250


text:
Tuesday, May 27, 2014
2 comments:
- Terri Cote said...
I am so happy to hear that I am not the only one who believes in dressing modestly. God will honor it and I know teaching your girls to dress modest must be a challenge (I don't have any girls) but it will be worth it. The Bible says:
Exodus 28:42 "And thou shalt make them linen breeches to cover their nakedness; from the loins even unto the thighs they shall reach"
That verse referrs to thigh as nakedness.
Deuteronomy 22:5 "The woman shall not wear that which pertaineth unto a man, neither shall a man put on a woman's garment: for all that do so are abomination unto the Lord thy God."
That verse couldn't be any plainer. I am pretty sure this is referring to pants.
Modesty is very important and I commend you. From one mother to another, GOOD JOB!!!
- May 27, 2014 at 5:10 PM
- Courtney @ LAAC said...
- June 2, 2014 at 10:32 PM


text:
FILM DETAILS Added 3 months ago
Release: 13 April 2018 (Canada)
Info IMDB: https://www.imdb.com/title/tt5085924/?ref_=nv_sr_1
Rating: 5.7/10
Stars: Bel Powley, Liv Tyler, Brad Dourif
Director: he dark secret behind her traumatic childhood. Director:
Decription: A blossoming teenager uncovers the dark secret behind her traumatic childhood.
7 views
18 views
14 views
30 views
COMMENTS[fbcomments url="http://peadig.com/wordpress-plugins/facebook-comments/" width="375" count="off" num="3" countmsg="wonderful comments!"]


text:
Aristides first came to notice as strategos in command of his native tribe Antiochis at the Battle of Marathon, and it was no doubt in consequence of the distinction which he then achieved that he was elected archon for the ensuing year (489—488). In pursuance of a conservative policy which aimed at maintaining Athens as a land power, he was one of the chief opponents of the naval policy proposed by Themistocles. The conflict between the two leaders ended in the ostracism of Aristides at a date variously given between 485 and 482.
Aristides in "Counting Potsherds" Edit
While Mithredath and Polydoros carried out a circuit of the walls of the Akropolis, they came to a section with a long inscription carved into it. It stated in part that Aristides proposed these things concerning the words of the prophetess of Delphi and the Persians: let the Athenians fortify the citadel with beams of wood as well as stone to meet the Persians, as was bade by the prophetess." This led Mithredath to ask if Aristides was the king at the time but Polydoros replied that the inscription did not call him basileus, the usual Yauna term for king.[1]
- ↑ See, e.g. Departures, pgs. 15-16, pb.


text:
Rogue adj: no longer obedient, belonging, or accepted and hence not controllable or answerable; deviating, renegade
(Disclaimer: We are not an official affiliate of CrossFit. The purpose of this blog is for tracking our own progress in our CrossFit-inspired workouts, and to encourage open dialogue among those involved in the CrossFit culture.)
Over & Under
Overhead Squats x10
Knees to Elbows x10
Handstand Push Ups (or modified) x5
Double Unders (or Box Jumps) x20
Post time & weight to comments
Dani pumping out some modified Handstand Push Ups!
Nicole preparing for Overhead Squats...3-2-1-Go!
CrossFit "abs"
Our 'boy wonder' Eric rockin' some nice Overhead Squats!
8 comments:
are you sure about that?
berto xxx
- June 25, 2008 at 7:25 PM
- welcometothegunshow said...
Time...17:32....I won! Except for the whole weight trumping time thing :) And I FINALLY fell into place with my overhead squats today, last 2 rounds felt good!!
I did 65# overhead squats, and made it through 3 rounds of real handstand pushups before switching to modified for the last 2 rounds.
...and Berto, I am sure about that. But who are you???
- June 25, 2008 at 8:56 PM
Meeks comes in with 18:40, yeah who the hell is berto?
- June 25, 2008 at 10:19 PM
- TheRevoltingBlob said...
Minturn pulled out a 18:20 time.
- June 26, 2008 at 9:03 AM
- wannabeamilf said...
Nissen came in at 18:26.
What's next? Thrusters and snatches?
Who is Berto? And are we sure about what?
- June 26, 2008 at 11:51 AM
- wannabeamilf said...
Oh, yeah. I forgot to blog my weights so I can refer to this next time. Overhead squats were 55# and Mod. Handstand Push-ups were 45#. I think I can try moving up on the handstand push-ups. If I ever want to be as strong as Nicole, I better get to work!
- June 26, 2008 at 11:54 AM
- lotto numbers said...
What a great moment of reading blogs.
- June 26, 2008 at 7:38 PM
- lottery results said...
Katon, Goukakyu no jutsu.
- June 26, 2008 at 7:38 PM


text:
neway, i was reading my former kadramahan when isaw this posted last january 18, 2005.. before my open-close brain surgery.. wala lang.. i just wanna point that i got the best present last Christmas of 2004.. STRENGTH! ayun..
here it it..
im scheduled for surgery on wednesday (january 19) already.. that soon huh?! scary?! im not sure.. actually, i still feel normal.. like it's just gonna be another day tomorrow.. or another minor operation.. i was in deep thinking the other night.. but not that deep.. haha! i was just thinking, how i felt when i first had my operation.. it was my very first..my cyst removal.. i was in grade 6 then.. i wasn't that scared though two cyst were removed.. imagine this: my left arm is being stitched while the cyst at my back is being cut.. o dba?! san ka?! i felt everything.. i was awake.. but that's nothing.. hehe! N.R. i've undergone a lot of operation.. cysts operation.. in my left arm.. my back.. my forehead.. my left ring finger.. actually, i got lots of it pa.. if i'll enumerate where each so-called cysts are located.. you might searched for it when when we saw it other.. haha! newei, it was only last year when we learned that these so-called cysts are not normal cyst at lall like what we used to know.. these are neurofibromas.. tumours of the nerve.. yup! it grows like mushrooms.. anywhere.. everywhere.. wherever.. whenever.. so.. yun.. anyway, this is it.. i'll gonna be in the hospital tomorrow.. and the day after that.. the operation.. haha! i know naman from the start that there's a reason behind everything.. even this.. dba?! God Bless as people say.. but i know God Blesses us more than we know.. ryt?!
newei, i was chatting with a friend a week ago i think.. he asked me what was the greatest gift i received last christmas.. ya, he's question was weird but that made me think.. ano nga kaya.. and it popped into my mind.. STRENGTH! ;)
i waana post this din..
just finished having a heart-to-heart talk with tita angie.. grabeh.. i never thought we could talk about things like that.. basically about life.. it started with a small talk regarding what happend here in our house last night.. aba! tito jim's friend though toti jim's not friend with him anymore after what happened) stole my moms phone inside our house ha! haha! bsta un! he got what he deserves naman.. he's in jail right now. nako! then i told her what happened to me.. i told her that we're friends with madie again.. (yup, lam nya rin.. hehe) galign nga eh.. ganda ng timing.. kokie's here in the philippines din.. la lang.. 6 kme.. just like before.. we we're all immature lang tlga before.. pero we should be proud of ourselves ha.. coz that just mean we handled it like a mature ladies.. haha! (sabi ni tita angie yan.. hehe) then we we're chatting and chatting.. girl talk.. i told her my outlook in life.. how i really think that everything has a reason.. how everything person that enters in our life helps us grow.. inspires us in different ways.. and makes us learn.. yes, even those who hurt us and made us cry.. kaaliw tlga! as in! i told her everything.. even the people i met.. new found friends.. (hmmm?! hehe), my friends.. they all shared a part in my of what i am today.. ano ba yan.. ka-shock naman tong mga pinagsasabi ko.. hehe! grabeh! we talked about her life.. her family.. grabeh! i didn't think na magkakasundo kme knowing she's a born again.. la lang.. kc i got a lot of contradictions regarding they're beliefs.. not in everything naman pla.. galing! super aliw.. then she told me.. she's asking the Lord nga, why should i have this.. imagine 1 out of 10,000 people.. she said that God has a purpose for it.. then i told her.. "kung wla akong sakit, nde tau makakapag-usap ng ganito.." hehe! totoo naman dba?! ;P i told her why i think i got sick.. it's still my 'everything has a reason'.. ano ba yan.. a walk to remember ha.. hehe! then she asked me what i really feel daw.. i told her.. "tanggap ko" im not trying to hide my feelings.. whatever i'm showing.. yun na un.. why should i cry? why should i get lonely? ano magagawa non dba?! i shouldn't stop living life the way i want it to be just because i'm sick.. lalo lang ako magkakasakit.. (depression) mind over matter which i learned from ysiad ha! hehe! there's a reason behind this.. i know there is.. o dba?! is this really me?! im not showing you that i'm a strong person even if i'm scared.. i need to be strong.. but i'm not really that scared.. ganito lng tlga ako.. even at the hardest situation.. i still try to look on the positive side of things.. cguro naman maniniwala na kau that i'm really sick and i got a tumor in my brain.. hehe! nde me nagpapaka-manhid.. la lang.. this is how i take it.. smile nlng.. i don't really want people pity me.. though i'm really touched.. as in! promise.. pero u don't have to think about it a lot of times.. cge na nga, d naman mapipigilan mag-worry.. thank you. pero nalulungkot lang me.. not because i'm sick.. kc nakikita ko kayong ganyan.. hehe! bsta u'll wear bandana if ever you're planning to visit me after my operation ha! hehe! ok na un! ;P o dba?! another side of tack na naman.. drama! hehe!
newei, grabeh pla.. mga friends.. touch tlga me sa inyo ha.. as in! ysiad.. when i told u about it, kahit d mo lam kung maniniwala ka or nde.. then u hagged me.. grabeh! thanks! thank you din coz u asked utchie's mom pa how we could get some money from pcso (ung nga ba?! hehe).. michelle, thanks for worrying.. kaw ang super worried.. as in! every hug u gave me.. i feel the comfort.. touched din me sa mom mo when she can't sleep the night after she knew about it.. ;) lheeyah! thank you thank you thank you! touch me sa mom mo.. and when u said you'll be there waiting outside the operating room while my operation is undergoing.. grabeh! c pau.. na ang puro question.. bkt?! bkt?! bkt?! may reason naman yan eh.. kahit nung nalaman nya.. parang mommy pa rin.. touched tlga me sa inyo! mei.. i never expected wat you texted me ha.. "a spacial person lyk you shouldn't be sick lyk dat" la lang.. simple words lang pero promise.. iba! hehe! special child ba?! hehe! cathy, 1 text lang.. aba reply kagad.. i felt u even sa txt lang.. buti nlng may load ka.. himala! wahahaha! and all my friends.. dami2.. im touched! promise.. i told tita angie nga eh.. im so grateful for having friends like you.. saya! oist! walang iiyak! wahaha! ay huhuhu pla.. kuha muna tissue.. punas luha.. haha! and syempre may fam.. bsta un na un.. hehe! you're all the reason kaya me nagiging strong.. hehe! o dba!? serious na tlga! hehe! minsan lang to.. hirap pla pag wlang tulog.. kung ano2ng naiicp.. haha! sabi nga ni warren (di ko sya boylet).. God blessed us more than we know.. hehe! share ko lang.. sabi nya yan eh.. ;P
tama na drama! *singhot* wahahaha! o, throw the tissue na.. nyahahahaha!
posted by tacK @ 10/17/2004 08:45:00 AM
i just wanna share my kadramahan lang.. i've been reading old posts kasi from my other (private) blog eh.. i had it since may 2003.. so u figure out what's posted there.. lots of memories.. hahaha! so, who knows about it? ewan ko na.. a few friends siguro.. o baka they forgot about it na.. eh di ako nalang! pero if u wanna read my kadramahan.. just ask me about the link.. and we'll see.. ayos!


text:
New items on display at fine arts center
The Breckenridge Fine Arts Center has acquired a new festival dress and head-dress to be added to their permanent collection. The BFAC now has 30 very colorful festival dresses on display in that permanent exhibit.
The festival dresses in the BFAC collection represents the three festivals in Texas, which includes the Tyler Rose Festival, the San Antonio Fiesta Festival and the Corpus Christi Buccaneer Days.
The new acquisition is from the 2015 Tyler Rose Festival, worn by Madison Tierney Jones of Albany, who was Duchess of Albany. The dress is titled 'Octopus,' which includes a very elaborate jeweled black dress and headdress.
Among the collection is one worn by Courtney Carrie Clark, daughter of David and Lynn Clark of Breckenridge. She participated in the 1994 San Antonio Fiesta Festival in the Court of International Celebrations as Duchess of Carnival.
The girls who participate in the festivals and wear the elaborate dresses are usually in their sophomore year of college. The value of the dresses can range from $4,000 to as much as $48,000. The oldest dress in the BFAC collection is the 1951 dress that was worn in the San Antonio Fiesta Festival.


text:
We've designed two amazing routes that allow riders of all abilities to take on the gravel challenge and enjoy the amazing scenery of the Haute Savoie. The full La Resistance route has been designed for the experienced rider whilst La Petite gives you a taste of the big event and more time to party at La Guinguette!
Both La Resistance and La Petite routes feature the HC climb of Col de l'Arpettaz and the "HC Gravel" of Route de la Soif from which you will enjoy an uninterrupted panorama of Mont Blanc. La Petite turns left just before the ski station of La Clusaz to ascend the Cat 3 Col de la Croix Fry, while La Resistance continues on up to the Plateau des Glieres via a ferocious 6.8km, Cat 2 climb at an average gradient of over 10%.
La Resistance route will take participants to the National Monument to La Resistance on Plateau des Glieres. The routes converge just before La Resistance Museum and Necropolis with around 10km left to ride. All riders arrive back at the beach in Talloires, with the finish line in the middle of the festivities at La Guinguette. Your choice of route is completely open so you can change your mind on the day to suit the strength of your legs !
Arnaud Mignotte +33 (0)6 79 71 84 41
From 70 €
Point of interest
74290 Talloires - Montmin


text:
- Description
SnoreStop® FastTabs™ quick dissolving chewable tablets 'designed to improve upper airway muscle tone, regulate nasal secretions as well as decreasing nasal allergic reactions'- Dr. Derek Lipman, M.D. Author of 'Snoring from A to Zzzz'.
Chew or suck one tablet 30 minutes before bedtime and repeat at bedtime. For best results, we recommend repeated use of the product for 3 to 5 consecutive nights. When continuous improvement is noticed, you may start using every other night until no longer needed. In some cases, regular use may be more effective. For Maximum Results, use in conjunction with SnoreStop® NasoSpray®. Please discard 6 months after first use. Store product at room temperature.
* Individual results may vary. If you do not see improvement you may have a better result using our SnoreStop Extinguisher.
Please view our guarantees.
- Ingredients
SnoreStop® FastTabs™ formula is prepared in accordance with the Homeopathic Pharmacopoeia of the United States (HPUS). All ingredients in SnoreStop® FastTabs™ formula are recognized as an official collection of accepted drug ingredients by the Federal Food, Drug and Cosmetic Act and are manufactured in FDA-approved laboratories. Each natural ingredient comes either from plant, mineral or protein sources and is present only in harmless microscopic amounts in each dose.
None of our ingredients contain any alkaloids, ephedrine or pseudo - ephedrine. SnoreStop is a natural homeopathic over-the-counter medicine regulated by the FDA.
Nux Vomica 4x
Belladonna 6x
Ephedra vulgaris 6x (there is no measureable amount of Ephedra or pseudoephedrine in our formula)
Hydrastis Canadensis 6x
Kali Bichromicum 6x
Teucrium marum 6x
Histaminum hydrochloricum 12x
Inactive ingredients:
Anhydrous Lactose (3mg/tab), Croscamellose sodium, Dextrose (45 mg/tab), Lactose (180 mg/tab) Magnesium Stearate, Microcrystalline Cellulose. Lactose is a sugar from milk. Dextrose is a simple sugar (glucose). If you do not count lactose as a simple sugar then you only have 0.003 g of sugar per tablet. If you count lactose as sugar than you have 0.18 g of sugar per tablet (minute amount).
For ingredient details, please click (here)


text: Strap On Dildo Xxx Tube can give you the incredible thrill that you are looking for on the 24/7 basis. This huge free Strapons Sex Tube has the hottest Strapon. hidaya.se strap on videos, free sex videos. Girlfriend Fucks Boyfriend with Strap-on - Live (7 min) sex rated Guy with lesbians and strap on fucking in bed. hidaya.se 'strapon xxx pegging' Search, free sex videos.


text:
Natalie recovered in time to go to church!*, hunt for eggs, eat more chocolate than we'd have liked, and generally have a great time with family. More Easter pics on flickr.*Natalie was so excited to be going to church! that it almost made me feel bad that we don't take her more often. Almost.
posted by Jen at
Post a Comment
<< Home
We have both known for as long as we can remember that we wanted to have kids. We decided to start down the road to parenthood about three years ago, beginning with research. In May of 2004 we began inseminating, and in July, we hit the jackpot. Unfortunately, it turned out to be a partial molar pregnancy, and we endured a miscarriage, followed by months of medical treatment, monitoring, and frustration. We began trying to conceive again in May 2005 (against our doctor's advice). We were lucky to get pregnant again in July. Despite our fears and worries, this pregnancy went smoothly and ended with a wonderful home birth and a beautiful baby girl. A new chapter begins....
View Jen's complete profile
View Cait's complete profile


text:
Far beyond our teenage productions on Microsoft paint, advances in technology have given birth to creativity beyond our pre-dial-up internet dreams. Phones at the ready, take a trip through a world of pure imagination as Neighbourhood selects the digital Instagram artists you should be following.
2. @Slimesunday
Hi! *attention please read* in case you missed it, my laptop has broke! which means no more art or projects for a while (hence why I haven't responded to any of your inquires *plz stay aware instead of sending me multiple messages, I'm sry ) I started a gofundme last week and wanted to thank every1 & any1 who has donated because I'M SO CLOSE to my goal!!! so thank you thank you thank you. if you want to help, link is in bio <3 #cgi #digitalart #3dart
"The maximalists are coming" ~ My hotel was featured on @nytimes today thru an article about @decorhardcore and how she wishes to steer the design world into the direction of appreciating design excess rather than the over celebrated "minimalism" ❤ I'm not gonna stop maximizing material and color content in spaces as long as I'm alive and I invite anyone and everyone to jump on the wagon! https://mobile.nytimes.com/2017/10/04/fashion/ugly-design-decor-hardcore.html?referer=
Header image with thanks to digital Instagram artist blakekathryn.


text:
Here are some pictures of the 1920s wedding dresses. Now, we want to try to share this some galleries to give you inspiration, we found these are amazing photos. We like them, maybe you were too. We got information from each image that we get, including set size and resolution. Wedding dress dead sea years, Wedding dress recreation gown worn local actress whose name unknown seems like random thing but considering her eerie inspiration choice has chilling meaning salt bride.
If you like these picture, you must click the picture to see the large or full size image. If you like and want to share please click like/share button, so other people can get these collection too. Right here, you can see one of our 1920s wedding dresses gallery, there are many picture that you can surf, do not miss them.
Below are 5 top images from 12 best pictures collection of 1920s wedding dresses photo in high resolution. Click the image for larger image size and more details.