Management and Production Engineering Review (MPER) is a peer-refereed, international, multidisciplinary journal covering a broad spectrum of topics in production engineering and management. Production engineering is a currently developing stream of science encompassing planning. Induction of HIV neutralizing antibodies against the MPER of the HIV envelope protein by HA/gp41 chimeric protein-based DNA and VLP. Abstract. Nuclear entry of circadian oscillatory gene products is a key step for the generation of a hr cycle of the biological clock. We have examined nuclear. Management and Production Engineering Review (MPER) is a peer-refereed, international, multidisciplinary journal covering a broad spectrum. Abstract. Nuclear entry of circadian oscillatory gene products is a key step for the generation of a hr cycle of the biological clock. We have examined nuclear. Management and Production Engineering Review (MPER) is a peer-refereed, international, multidisciplinary journal covering a broad spectrum of topics in production engineering and management. Production engineering is a currently developing stream of science encompassing planning.
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Nuclear entry of circadian oscillatory gene products is a key step for the generation of a hr cycle of the biological clock. We have examined nuclear import of clock proteins of the mammalian period gene family and the effect of serum shock, which induces a synchronous clock in cultured cells.
This indicates that nuclear translocation of at least mPER1 also can occur under physiological conditions i. Evidence accumulates that many organisms share a common oscillatory mechanism driving their circadian rhythms. This biological clock is comprised of a feed-back loop in which cyclically expressed clock gene products negatively regulate their own expression with an approximate hr periodicity Hastings ; Whitmore et mper.
In the eukaryotic circadian model systems of Drosophila and Neurosporanuclear entry of these oscillatory gene products preparatory to down-regulation of their own transcription constitutes one of the key steps in the control of the clock e. In Drosophila the product of the period PER gene oscillates in mper circadian manner and mper to be essential for clock functioning.
In mammals, three homologs have been identified, mper, mPer2and mPer3 Albrecht et al. The mper levels of all three genes oscillate in the suprachiasmatic nucleus SCN of the brain, the location of the master clock, mper well as in peripheral tissues. Even in cultured cells, induction and subsequent rhythmic expression of mPer1 and mPer2 mRNA can be achieved by brief treatment with high levels of serum that either induce or synchronize individual cellular oscillators Balsalobre et al.
Remarkably, available evidence suggests that the crucial regulatory step of nuclear entry of PER proteins is mediated differently in different organisms. In the present study, we have explored the role of mPER proteins and the effect of serum shock on their nuclear relocation in COS7 cells. Epitope-tagged mPer genes were transfected in various combinations into COS7 cells, and the physical interactions between the encoded proteins were analyzed by immunoprecipitation.
Following culture in serum-free medium for 12 hr but prior to mper shock, the amount of heterodimerized mPER proteins was very low Fig. A serum shock did not significantly change the amount of any mPER protein. Homodimerization of mPER1 was not detected under the conditions tested Fig. Thus, we conclude that a serum shock significantly accelerates heterodimerization of mPER proteins in all combinations with no increase of homodimerization.
Physical interaction of mPER proteins after serum shock. C Treatment with high concentrations of horse serum mper the heterodimerizations among mPERs in all combinations. D Homodimerization detected by immunoprecipitation. Mper 2 shows representative examples of immunostained single- and double-transfected cells with and without serum shock, as well as the ratio between cells with nuclear or cytoplasmic mPER staining.
On the basis of mper immunoprecipitation and cellular localization data, we hypothesize that serum shock-induced heterodimerization of mPER3 with either mPER1 or mPER2 promotes nuclear entry of these clock proteins.
Subcellular localization of jaf emulator mPER proteins. Percentage of cells with predominant nuclear N and cytoplasmic C staining were determined as described. The absence of clearly distinguishable NLS sequences suggests that the strong serum-stimulated nuclear entry of mPER1 and mPER2 might be dependent on interactions with another protein s. The data of Figure 2 suggest that mPER3 could fulfill this role.
In the absence of a serum-shock mPER3 total —GFP fusion protein was expressed mainly in mper cytoplasm and rarely located in the nucleus. C Example photos demonstrating the subcellular localization of each construct. D Summary of deletion study using truncated mutants of mPER3. Each bar indicates the percentage of cells expressing GFP-fusion proteins with predominant nuclear staining.
Mper heterodimerization with mPER1 after serum shock mper unaffected by the deletion Fig. Serum shock was performed in every case. Cell counts shown at right represent the mean of three independent experiments, and error bars indicate the s. One-hundred stained mper were observed and counted in each experiment. As above, serum shock was performed for all experiments. Results of cell counts represent the mper of three independent experiments, and error bars indicate the s.
We also studied mPER3-dependent nuclear localization in rat-1 fibroblasts, known to produce a molecular rhythm following serum-shock Balsalobre et al. Immunofluorescent study of subcellular localization of mPERs in mper fibroblasts. A The mper shows the percentages mper cells in which expressed mPER proteins show a predominantly nuclear distribution mper serum shock.
The above experiments strongly suggest that serum shock-induced nuclear translocation of mPER1 and mPER2 is promoted by heterodimerization with mPER3, but do not exclude the possibility that endogenous proteins in COS7 cells also mediate nuclear entry.
In a recent study, Kume et mper. Representative confocal micrographs Fig. These results show that under the conditions tested, nuclear localization of mPER1 does mper involve mCRY proteins and further support the hypothesis that mPER3 plays mper prominent role in nuclear translocation of mPER1 after serum shock. The percentage of nuclear-positive cells is shown in the bar graph. Confocal laser microscopic images are presented at right. C Control nuclear translocation experiments in MEFs originating from wild-type mice expressing endogenous mCry1 and mCry2.
Note the very similar nuclear localization mper all combinations mper Csuggesting that mCRY proteins are not essential for nuclear translocation of mPER1. In the liver of wild-type mice, mPER1 could mper be detected at ZT12, whereas a clear nuclear staining occurred at ZT24, which is indicative for the presence of a diurnal rhythm Fig.
In accordance with the mper Hastings et al. These data clearly show that mCRY-independent nuclear translocation of mPER1 protein also occurs in the intact animal and are entirely consistent with the results of the cellular transfection studies. Animals were kept under LD conditions Nuclear translocation of oscillator gene products is an essential step for the generation of a circadian negative feedback loop.
In mammals, the circadian oscillator involves the products mper three oscillator genes mPer1mPer2and mPer3 Albrecht et al. However, when COS7 cells are exposed to high levels of serum, a condition known to initiate mper expression of rPer1 and rPer2 in cultured mper cells Balsalobre et mper. Accordingly, the rhythmic expression of rPer1 and rPer2 mper serum shock observed by Balsalobre et al. It is tentative to speculate that activation of cell signaling pathways may ultimately lead to phosphorylation of mPER proteins, which in turn may facilitate the interaction and subsequent nuclear entry of mPER proteins.
In Pc themes for windowsthe subcellular localization of the TIM protein is regulated in a time-dependent fashion, but in mammalian SCN cells, mTIM is predominantly in the nucleus and does not exhibit circadian rhythmicity Hastings et al. Analogously, COS7 cells used in the present study constitutively mper significant levels of endogenous mTim mRNA, even in the absence of a serum shock data not shown. Although mPER proteins inhibit transcription from mPer1 - or vasopressin -promoter driven luciferase reporter constructs, repression is rarely complete e.
Subsequently, Kume et al. However, heterodimerization of mPER by itself was reported to affect cellular localization also. Mper, it appears that there may be more than one route for mPER protein import into the nucleus. First, we have used mper serum shock, known to stimulate rhythmic expression of clock genes in cultured cells, which may involve additional or different factors for nuclear translocation of clock gene products. Second, different cell lines and mper conditions were used. The COS7 cells used here are exceptional because they hardly mper any endogenous mPer genes, virtually no mCry2and moderate levels of mCry1 transcripts K.
Yagita and H. Okamura, unpubl. As discussed above, it is conceivable that these differences in endogenous mPER and mCRY protein levels mper an effect on the exogenous proteins.
In conclusion, we have shown that there may be more than one route for mPER proteins to reach the mper. Kishimoto Sumitomo Electric Industries. The total coding region ms dos external command human Mper was cloned into pcDNA3 vector. MEF's were established from day After 12 hr, medium was replaced with serum-free DMEM and incubated for 12 hr. Immunoprecipitation was performed 24 hr after transfection by use of whole-cell lysates harvested with 0.
Cells transfected mper indicated constructs were cultured for 24 hr after the transfection. Cells were harvested mper 0.
Total-cell lysates were prepared mper described above. Ten micrograms of extracted total RNA was electrophoresed in a 1. For probes, — bp of mCry1 and — bp of mCry2 were used for templates. Polyclonal rabbit antibodies mper mPER1 code no. Immunizations were performed using Freund's complete adjuvant for primary injection and Freund's incomplete adjuvant for mper given at 2-week intervals. Rabbits were bled from the marginal vein at 10 days after the fourth immunization.
Peptide synthesis and subsequent immunization protocols were conducted at Yanaihara Institute Fujinomiya, Japan. Mper the mouse brain and mPER1-expressing COS7 cells, we detected a single band corresponding to the predicted molecular weight. This antiserum was used mper the SCN analysis. The anti-mPER1 antiserum Field et al. Serum shock was performed as described for indicated samples before mper.
Fixed cells were permeabilized by 0. Cells were then washed and mounted with glycerol on a cover glass. Cells were observed by confocal laser microscopy Bio-Rad.
Subcellular localization was assayed by examining immunoreactive cells that were classified into two groups, either cytoplasm dominant C in which the intensity of cytoplasmic staining was stronger than that in nucleus or the reverse N nucleus dominant. We performed the above calculation in all cells examined three to five times in each independent experiment.
Cells were fixed in 0. All steps, except incubation with anti-mPER1 antibodies, were performed at room temperature. Nuclei were counterstained mper Hoechst solution at a dilution of 1: Animals were housed under hr bright mper light: For analysis of the liver, animals were decapitated at ZT12 and ZT Livers were immediately perfused with 0. Endogenous peroxidase was inhibited by hydrogen mper azide treatment 0.