Mathematical modeling and summary.

<p>(A) Numerical simulation of the model describing the circadian PER1 protein expression. The solid and dotted curves indicate the level of mPER1 protein treated with Con_si and hnQ_si for HNRNPQ knockdown, respectively. (B) The relation between the amplitude of mPER1 protein and the level of HNRNPQ was obtained by numerical simulation using the model. (C) Numerical simulation of the model describing the circadian mPER1 protein expression with the assumption that mPER1 protein stability was influenced by the level of HNRNPQ. The solid and dotted curves indicate the level of mPER1 protein treated with Con_si and HNRNPQ-specific hnQ_si, respectively. (D) The model described mPER1 protein stability as a function of HNRNPQ and predicted the effect of HNRNPQ on both the amplitude and phase of the mPER1 protein oscillation. (E) The amplitude of mPER1 protein was described as a function of HNRNPQ levels. However, the relationship was not linear; mPER1 protein became saturated when HNRNPQ was abundant. (F) The proposed model for rhythmic translation of m<i>Per1</i> as a key regulatory mechanism of circadian mPER1 expression.</p

IRES activity of m<i>Per1</i> 5′UTRs.

<p>(A) Schematic diagram of bicistronic reporter plasmids. 5′UTRs were inserted into intergenic region between Rluc and Fluc. Bicistronic reporter plasmid (pRF), <i>Renilla</i> luciferase (Rluc), and <i>firefly</i> luciferase (Fluc). (B) NIH 3T3 cells were transiently transfected with bicistronic reporters that harbor 5′UTRs of <i>Per1</i>, <i>Bip</i>, and <i>c-Myc</i>. After 24 h incubation, cells were subjected to luciferase assay. The results are expressed as the mean ± SEM. (C) Bicistronic reporters that harbor 5′UTRs were transfected to HEK 293A cells. After 24 h, cells were harvested, and total RNAs were prepared and subjected to Northern blotting. Total RNA (2.5 µg) was hybridized with a specific probe for the <i>Fluc</i> coding region. 18S and 28S RNAs are shown as controls. The data was quantified by measuring the ratio of Fluc/28S.</p

Rhythmic phosphorylation of HNRNPQ.

<p>(A) Dexamethasone-treated NIH 3T3 cells were harvested at the indicated time points, and then proteins were prepared under a phosphatase-free condition. Extracts were used for immunoprecipitation with HNRNPQ-specific antibody or IgG; then immonoblotting was performed with pTy- or HNRNPQ- specific antibodies. The blot for detection of HNRNPQ was stripped, and pTy bands were detected by pTy-specific antibody. (B) The one to fifth of the samples immunoprecipitated by HNRNPQ in panel A were subjected to total RNA preparation, then real-time PCR was performed.</p

Cap-independent translation of m<i>Per1</i>.

<p>(A) Rapamycin (Rapa) or cycloheximide (CHX)-treated NIH 3T3 cells were harvested at indicated time points; then the protein levels were checked by immunoblotting. (B, C, D and E) Vehicle (DMSO)-, rapamycin (Rapa)-, or cycloheximide (CHX)-treated NIH 3T3 cells were harvested at the indicated time points; then mRNA levels were checked by real-time PCR with specific primers, (B) m<i>Per1</i>, (C) m<i>Actb</i>, (D) m<i>Gapdh</i> and (E) m<i>Rpl32</i>. mRNA levels were shown as cycle threshold (Ct) value.</p

HNRNPQ binding site and m<i>Per1</i> IRES activity.

<p>(A) Schematic diagram of serially deleted mutation strategy and design of competitive oligonucleotides to perform UV cross-linking with oligonucleotide competition. (B and C) Radiolabeled <i>in vitro</i> transcribed RNAs were incubated with cytoplasmic extracts and competitive oligonucleotide for in vitro binding. Then UV cross-linking was performed. (D) Radiolabeled deletion mutants RNAs, e1BΔ51 and e1BΔ89, were subjected to UV cross-linking. (E) Schematic diagram of bicistronic mRNA reporter of m<i>Per1</i> 5′UTRs; 7-methyl-guanosine (m⁷G) and 20-nt-long poly(A) tail [poly(A)20]. (F) <i>In vitro</i> transcribed reporter mRNAs of 5′UTRs were transfected, then a luciferase assay was performed. The activity of the mock was set to 1 (n = 3). (G) Bicistronic mRNA reporters, e1A and e1AΔ51, were transfected into synchronized cells. After 6 h, cells were harvested at indicated time points; and then luciferase activity was checked. The activity of e1A at ∼20–26 time point was set to 1 (n = 3).</p

A Reaction-Based Sensing Scheme for Gold Species: Introduction of a (2-Ethynyl)benzoate Reactive Moiety

To alleviate side reactions identified in an <i>N</i>-propargyl-rhodamine lactam sensing system, we devised the novel reaction-based sensing scheme for gold species based on the alkynophilicity. A fluorescein (2-ethynyl)benzoate underwent Au(III)-promoted ester hydrolysis selectively over other metal ions with high sensitivity, which accompanies a turn-on fluorescence change in pH 7.4 HEPES buffer. The work offers a versatile reactive moiety for the development of gold probes with improved sensing properties

Ground-State Elevation Approach To Suppress Side Reactions in Gold-Sensing Systems Based on Alkyne Activation

A novel approach to suppress the side reactions observed in the reaction-based gold-sensing systems based on the alkyne activation is disclosed. By elevating steric strain around the reaction site, the gold ion promoted ring-opening process in rhodamine-lactam probes is significantly accelerated, which also leads to suppression of those possible side reactions. As a result, the probes show very high sensitivity in addition to excellent selectivity toward gold species. Furthermore, bioimaging of gold species in live cells was demonstrated with a FRET version