Millisecond Photon Lifetime in a Slow-Light Microcavity.

Optical microcavities with ultralong photon storage times are of central importance for integrated nanophotonics. To date, record quality (Q) factors up to 10^{11} have been measured in millimetric-size single-crystal whispering-gallery-mode (WGM) resonators, and 10^{10} in silica or glass microresonators. We show that, by introducing slow-light effects in an active WGM microresonator, it is possible to enhance the photon lifetime by several orders of magnitude, thus circumventing both fabrication imperfections and residual absorption. The slow-light effect is obtained from coherent population oscillations in an erbium-doped fluoride glass microsphere, producing strong dispersion of the WGM (group index n_{g}∼10^{6}). As a result, a photon lifetime up to 2.5 ms at room temperature has been measured, corresponding to a Q factor of 3×10^{12} at 1530 nm. This system could yield a new type of optical memory microarray with ultralong storage times.

A variant form of the nuclear triiodothyronine receptor c-ErbAalpha1 plays a direct role in regulation of mitochondrial RNA synthesis.

In earlier research, we identified a 43-kDa c-ErbAalpha1 protein (p43) in the mitochondrial matrix of rat liver. In the present work, binding experiments indicate that p43 displays an affinity for triiodothyronine (T3) similar to that of the T3 nuclear receptor. Using in organello import experiments, we found that p43 is targeted to the organelle by an unusual process similar to that previously reported for MTF1, a yeast mitochondrial transcription factor. DNA-binding experiments demonstrated that p43 specifically binds to four mitochondrial DNA sequences with a high similarity to nuclear T3 response elements (mt-T3REs). Using in organello transcription experiments, we observed that p43 increases the levels of both precursor and mature mitochondrial transcripts and the ratio of mRNA to rRNA in a T3-dependent manner. These events lead to stimulation of mitochondrial protein synthesis. In transient-transfection assays with reporter genes driven by the mitochondrial D loop or two mt-T3REs located in the D loop, p43 stimulated reporter gene activity only in the presence of T3. All these effects were abolished by deletion of the DNA-binding domain of p43. Finally, p43 overexpression in QM7 cells increased the levels of mitochondrial mRNAs, thus indicating that the in organello influence of p43 was physiologically relevant. These data reveal a novel hormonal pathway functioning within the mitochondrion, involving a truncated form of a nuclear receptor acting as a potent mitochondrial T3-dependent transcription factor.

Molecular basis of the cell-specific activity of v-erb A in quail myoblasts.

We have previously shown that v-erb A expression strongly stimulates quail myoblast proliferation and differentiation without alteration of the triiodothyronine (T3) influence in this cell type. In order to understand the molecular basis of v-erb A action in myoblasts, we have studied the influence of this oncoprotein on c-erb A alpha1 encoded T3 nuclear receptor (TR alpha) activity. In transfection experiments, v-erb A did not inhibit the T3-dependent c-erb A alpha1 transcriptional activity in QM7 myoblasts in contrast to its action in HeLa cells. However, it repressed the retinoic acid receptor RAR alpha activity in both cell-types, indicating that v-erb A interactions with T3 or RA mediated transcription significantly differs. In EMSA experiments using a TREpa1 probe, T3R alpha binds as three complexes in HeLa cells. We have previously identified the slow migrating complex, undetectable in QM7 myoblasts, as a T3R/RXR heterodimer. Interestingly, v-erb A inhibited binding of this complex in HeLa cells, but did not affect binding of the two other complexes in QM7 myoblasts. Expression of RXR (gamma isoform), the TR alpha dimerization partner absent in proliferating QM7 cells, restored inhibition of c-erb A alpha1 transcriptional activity in these cells and abrogated the v-erb A myogenic influence. Lastly, v-erb A induced a T3-independent c-erb A alpha1 activity in QM7 cells when cotransfected in equimolar ratio with the receptor, by inhibiting AP-1 activity and stimulating transcription of a reporter gene driven by a TRE sequence.

Identification of functional domains involved in BTG1 cell localization.

We have previously shown that BTG1 stimulates myoblast differentiation. In addition, this protein displays a major nuclear localization in confluent myoblasts, decreasing during the early steps of differentiation, and is essentially detected in the cytoplasm of mature myotubes. To identify the domains involved in the cellular trafficking of BTG1, we observed the localization of several BTG1 sequences fused to betaGalactosidase. The highly conserved B box among all members of the BTG family induces a significant nuclear localization of the betaGal moiety, enhanced by presence of the BTG1 carboxy-terminal sequence. In addition, a functional Nuclear Export Signal (NES) overlaps the B box. Moreover, presence of the first 43 NH(2)-terminal amino acids reduced the nuclear localization of each chimeric protein tested. Last, the BTG1 amino-terminal domain bears an LxxLL motif favouring nuclear accumulation, and another region encompassing the A box inhibiting nuclear localization. In contrast to a BTG1 mutant exclusively localized in the cytoplasm, transient expression of a mutant displaying a nuclear localization enhanced myoblasts withdrawal from the cell cycle and terminal differentiation, thus mimicking the myogenic influence of BTG1. In conclusion, several regions of BTG1 are implicated in its cellular localization, and BTG1 myogenic activity is induced at the nuclear level.

New molecular aspects of regulation of mitochondrial activity by fenofibrate and fasting.

Fenofibrate and fasting are known to regulate several genes involved in lipid metabolism in a similar way. In this study measuring several mitochondrial enzyme activities, we demonstrate that, in contrast to citrate synthase and complex II, cytochrome c oxidase (COX) is a specific target of these two treatments. In mouse liver organelles, Western blot experiments indicated that mitochondrial levels of p43, a mitochondrial T3 receptor, and mitochondrial peroxisome proliferator activated receptor (mt-PPAR), previously described as a dimeric partner of p43 in the organelle, are increased by both fenofibrate and fasting. In addition, in PPAR alpha-deficient mice, this influence was abolished for mt-PPAR but not for p43, whereas the increase in COX activity was not altered. These data indicate that: (1) PPAR alpha is involved in specific regulation of mt-PPAR expression by both treatments; (2) fenofibrate and fasting regulate the mitochondrial levels of p43 and thus affect the efficiency of the direct T3 mitochondrial pathway.

A 45 kDa protein related to PPARgamma2, induced by peroxisome proliferators, is located in the mitochondrial matrix.

Besides their involvement in the control of nuclear gene expression by activating several peroxisome proliferator-activated receptors (PPARs), peroxisome proliferators influence mitochondrial activity. By analogy with the previous characterization of a mitochondrial T3 receptor (p43), we searched for the presence of a peroxisome proliferator target in the organelle. Using several antisera raised against different domains of PPARs, we demonstrated by Western blotting, immunoprecipitation and electron microscopy experiments, that a 45 kDa protein related to PPARgamma2 (mt-PPAR) is located in the matrix of rat liver mitochondria. In addition, we found that the amounts of mt-PPAR are increased by clofibrate treatment. Moreover, in EMSA experiments mt-PPAR bound to a DR2 sequence located in the mitochondrial D-loop, by forming a complex with p43. Last, studies of tissue-specific expression indicated that mt-PPAR is detected in mitochondria of all tissues tested except the brain in amounts positively related to p43 abundance. Besides their involvement in the control of nuclear gene expression by activating several peroxisome proliferator-activated receptors (PPARs), peroxisome proliferators influence mitochondrial activity. By analogy with the previous characterization of a mitochondrial T3 receptor (p43), we searched for the presence of a peroxisome proliferator target in the organelle. Using several antisera raised against different domains of PPARs, we demonstrated by Western blotting, immunoprecipitation and electron microscopy experiments, that a 45 kDa protein related to PPARgamma2 (mt-PPAR) is located in the matrix of rat liver mitochondria. In addition, we found that the amounts of mt-PPAR are increased by clofibrate treatment. Moreover, in EMSA experiments mt-PPAR bound to a DR2 sequence located in the mitochondrial D-loop, by forming a complex with p43. Last, studies of tissue-specific expression indicated that mt-PPAR is detected in mitochondria of all tissues tested except the brain in amounts positively related to p43 abundance.

v-erb A and v-erb B do not cooperate in quail myoblasts.

We have previously shown that v-erb A stimulates quail myoblast differentiation in a T3 independent, cell-specific manner. In this work, we have studied the influence of v-erb B (the second oncogene carried in the AEV genome) upon quail myoblast proliferation and differentiation. v-erb B expression,moderately stimulates myoblast proliferation, and inhibits differentiation. Moreover, this oncoprotein fully inhibits the v-erb A myogenic influence. These data provide evidence that these two oncogenes do not cooperate in avian myoblasts. Consequently, in contrast to results obtained in other cell-types, coexpression of both oncogenes does not transform quail myoblasts.

Changes in mitochondrial activity during avian myoblast differentiation: influence of triiodothyronine or v-erb A expression.

Numerous data suggest that mitochondrial activity is involved in the regulation of cell growth and differentiation. Therefore, we have studied the changes in mitochondrial activity in avian myoblast cultures (QM7 line) undergoing differentiation or in BrdU-treated, differentiation-deficient cells. As we have previously shown that triiodothyronine and v-erb A expression stimulate myogenic differentiation, we have also observed their influence upon mitochondrial activity. Comparison of control and BrdU-treated myoblasts indicated that precocious differentiation events were associated with a stimulation of citrate synthase and cytochrome oxidase activities. They also induced a transient decrease in mitochondrial membrane potential assessed by rhodamine 123 uptake. In control myoblasts, a general stimulation of mitochondrial activity was recorded at cell confluence, prior to terminal differentiation. These events did not occur in BrdU-treated myoblasts, thus indicating that they were tightly linked to myoblast commitment. Whereas no significant triiodothyronine influence could be detected upon mitochondrial activity, we observed that v-erb A expression significantly depresses the mitochondrial membrane potential in control myoblasts. This action was not observed in BrdU-treated myoblasts, thus suggesting that it involves an indirect pathway linked to differentiation. Moreover, the oncoprotein abrogated the decrease in E2-PDH subunit level observed at cell confluence. These data underline that changes in mitochondrial activity occurred prior to myoblast terminal differentiation and could be involved in the processes regulating myogenesis. In addition, they provide the first evidence that the v-erb A oncoprotein influences mitochondrial activity.

Quantitative variation and selection of esterase gene amplification in Culex pipiens.

Although descriptions of evolutionary mechanisms are common in the literature, very few studies focus on the possible evolution of the adaptive genes themselves, i.e. their quantitative and qualitative changes. Evolution of insecticide resistance in Culex pipiens is a suitable model for studying such processes. In this species, organophosphorous insecticide resistance can be achieved through the overproduction of esterases that sequester the insecticide, and this overproduction can be caused by gene amplification. It is generally assumed, but never verified, that esterase activity, and therefore resistance, is monotonically related to gene amplification. We have analysed resistance, esterase activity and gene amplification in different laboratory strains and natural populations in order to detect variability and to infer effects of selection on these factors. We have shown that resistance, esterase activity and amplification covary, that insecticide selection is able to increase amplification levels, and that a fitness cost is probably attached to the amplification in laboratory strains, related to the level of amplification. The importance of variation in gene amplification level is discussed and some evolutionary implications are proposed.

Mitochondrial activity is involved in the regulation of myoblast differentiation through myogenin expression and activity of myogenic factors.

To characterize the regulatory pathways involved in the inhibition of cell differentiation induced by the impairment of mitochondrial activity, we investigated the relationships occurring between organelle activity and myogenesis using an avian myoblast cell line (QM7). The inhibition of mitochondrial translation by chloramphenicol led to a potent block of myoblast differentiation. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone and oligomycin, which affect the organelle at different levels, exerted a similar influence. In addition, we provided evidence that this phenomenon was not the result of an alteration in cell viability. Conversely, overexpression of the mitochondrial T3 receptor (p43) stimulated organelle activity and strongly potentiated myoblast differentiation. The involvement of mitochondrial activity in an actual regulation of myogenesis is further supported by results demonstrating that the muscle regulatory gene myogenin, in contrast to CMD1 (chicken MyoD) and myf5, is a specific transcriptional target of mitochondrial activity. Whereas myogenin mRNA and protein levels were down-regulated by chloramphenicol treatment, they were up-regulated by p43 overexpression, in a positive relationship with the expression level of the transgene. We also found that myogenin or CMD1 overexpression in chloramphenicol-treated myoblasts did not restore differentiation, thus indicating that an alteration in mitochondrial activity interferes with the ability of myogenic factors to induce terminal differentiation.

Induction of c-Erb A-AP-1 interactions and c-Erb A transcriptional activity in myoblasts by RXR. Consequences for muscle differentiation.

We have previously shown that c-Erb A and v-Erb A display a cell-specific activity in avian myoblasts. In this work, we have compared the molecular basis of thyroid hormone action in HeLa cells and in QM7 myoblasts. The transcriptional activity of c-Erb A alpha 1 through a palindromic thyroid hormone response element (TRE) was similar in both cell types. However, c-Erb A did not activate gene transcription through a direct repeat sequence (DR) 4 TRE in myoblasts in contrast to results obtained in HeLa cells. Moreover, whereas retinoic acid receptor-AP-1 interactions were functional in both cell types, thyroid hormone receptor (T3R)-AP-1 interactions were only functional in HeLa cells. Using electrophoretic mobility shift assays, functional tests, and Northern blot experiments, we observed that RXR isoforms are not expressed in proliferating myoblasts. Expression of RXR gamma in these cells did not influence T3R transcriptional activity through a palindromic TRE but induced such an activity through a DR4 TRE. Moreover, it restored c-Erb A-AP-1 functionality in QM7 myoblasts and enhanced the myogenic influence of T3. We also observed that c-Jun overexpression in proliferating QM7 cells restored T3R transcriptional activity through a DR4 TRE. Therefore, alternative mechanisms are involved in the induction of T3R transcriptional activity according to the cell status (proliferation: c-Jun; differentiation: RXR). In addition we provide the first evidence that RXR is required to allow inhibition of AP-1 activity by ligand-activated T3R. Lastly, we demonstrate the importance of RXR in the regulation of myoblast differentiation by T3.

BTG1: a triiodothyronine target involved in the myogenic influence of the hormone.

The product of the B-cell translocation gene 1 (BTG1), a member of an antiproliferative protein family including Tis-21/PC3 and Tob, is thought to play an important role in the regulation of cell cycle progression. We have shown in a previous work that triiodothyronine (T3) stimulates quail myoblast differentiation, partly through a cAMP-dependent mechanism involved in the stimulation of cell cycle withdrawal. Furthermore, we found that T3 or 8-Br-cAMP increases BTG1 nuclear accumulation in confluent myoblast cultures. In this study, we report that BTG1 is essentially expressed at cell confluence and in differentiated myotubes. Whereas neither T3 nor cAMP exerted a direct transcriptional control upon BTG1 expression, we found that AP-1 activity, a crucial target involved in the triiodothyronine myogenic influence, repressed BTG1 expression, thus probably explaining the low BTG1 expression level in proliferating myoblasts. In transient transfection studies, we demonstrated that an AP-1-like sequence located in the BTG1 promoter was involved in this negative regulation. Our present data also bring evidence that the stimulation of BTG1 nuclear accumulation by T3 or 8-Br-cAMP probably results from an increased nuclear import or retention in the nucleus. Lastly, BTG1 overexpression in quail myoblasts mimicked the T3 or 8-Br-cAMP myogenic influence: (i) inhibition of myoblast proliferation due to an increased rate of myoblast withdrawal from the cell cycle; and (ii) stimulation of terminal differentiation. These data suggest that BTG1 is probably involved in T3 and cAMP myogenic influences. In conclusion, BTG1 is a T3 target involved in the regulation of myoblast differentiation.