Piperonylic acid stimulates keratinocyte growth and survival by activating epidermal growth factor receptor (EGFR).

Epidermal growth factor (EGF) stimulates cell growth, proliferation, and survival. The biological benefits of EGF have been utilized in medical uses for improving wound healing as well as in today's skin cosmetics. EGF has been found in urine, saliva, milk, and plasma, but its efficient isolation remains a difficult task. With technical advances, recombinant protein purification technique has been used for EGF production. However, the recombinant EGF is still expensive and keeping it with stable activity is difficult to be used widely. Thus, a molecule that can mimic the EGF activity would be a useful alternative of EGF. Herein, we have discovered that a natural small molecule piperonylic acid shows EGF-like activity in HaCaT keratinocytes. Piperonylic acid induced EGF receptor (EGFR) activation and resulted in serial activation of the downstream modulators. The activated signaling pathway eventually up-regulated gene expression of egr-1, c-fos, c-jun, and c-myc, which are involved in cell growth and survival. Moreover, piperonylic acid showed promoting role in keratinocyte growth and survival from UVB-induced cellular damages. This study has revealed the EGF-like activity of piperonylic acid and proposed that the piperonylic acid could be a promising component for skin wound healing agents or cosmetic ingredient.

Heterogeneous ribonucleoprotein R regulates arylalkylamine N-acetyltransferase synthesis via internal ribosomal entry site-mediated translation in a circadian manner.

Rhythmic arylalkylamine N-acetyltransferase (AANAT) synthesis is a prominent circadian-controlled response that occurs in most mammals. AANAT is the core enzyme in melatonin production; because melatonin participates in many physiological processes, the regulation of AANAT is an important research topic. In this study, we focused on the role of heterogeneous ribonucleoprotein R (hnRNP R) in the translation of AANAT. A novel RNA-binding protein hnRNP R widely interacted with the 5' untranslated region (UTR) of AANAT mRNA and contributed to translation through an internal ribosomal entry site (IRES). Fine-tuning of AANAT protein synthesis occurred in response to knockdown and overexpression of hnRNP R. Nocturnal elevation of AANAT protein was dependent on the rhythmic changes of hnRNP R, whose levels are elevated in the pineal gland during nighttime. Increases in hnRNP R additionally improved AANAT production in rat pinealocytes under norepinephrine (NE) treatment. These results suggest that cap-independent translation of AANAT mRNA plays a role in the rhythmic synthesis of melatonin through the recruitment of translational machinery to hnRNP R-bound AANAT mRNA.

A microfluidic device for label-free detection of Escherichia coli in drinking water using positive dielectrophoretic focusing, capturing, and impedance measurement.

While sensors that allow for high-throughput enumeration of microorganisms within drinking water are useful for water quality monitoring, it is particularly challenging to accurately quantify microorganisms that are present in low numbers (<100 CFU/mL) in a high-throughput manner. Negative dielectrophoresis (nDEP) is typically utilized in DEP-based cell focusing methods; however, due to its low conductivity, drinking water cannot be analyzed by this approach. Here, we report a positive DEP (pDEP)-based Escherichia coli detection system that is integrated with a focusing and sensing electrode. By incorporating a passivation layer, we avoided issues with adhesion of E. coli to the electrode, and achieved efficient cell focusing under high flow rate conditions (1500 µL/h). The resulting focused E. coli cells were then trapped on the sensor electrode, resulting in changes in impedance. The proposed system was evaluated using four different E. coli populations (150-1500 CFU/mL). We successfully enumerated populations as low as 300 CFU/mL within 1 min, and the signal variation was 1.13±0.37%. The device introduced in this study provides the basis for the development of portable, highly sensitive microorganism sensors that enable rapid detection of bacteria in drinking water.

Rhythmic interaction between Period1 mRNA and hnRNP Q leads to circadian time-dependent translation.

The mouse PERIOD1 (mPER1) protein, along with other clock proteins, plays a crucial role in the maintenance of circadian rhythms. mPER1 also provides an important link between the circadian system and the cell cycle system. Here we show that the circadian expression of mPER1 is regulated by rhythmic translational control of mPer1 mRNA together with transcriptional modulation. This time-dependent translation was controlled by an internal ribosomal entry site (IRES) element in the 5' untranslated region (5'-UTR) of mPer1 mRNA along with the trans-acting factor mouse heterogeneous nuclear ribonucleoprotein Q (mhnRNP Q). Knockdown of mhnRNP Q caused a decrease in mPER1 levels and a slight delay in mPER1 expression without changing mRNA levels. The rate of IRES-mediated translation exhibits phase-dependent characteristics through rhythmic interactions between mPer1 mRNA and mhnRNP Q. Here, we demonstrate 5'-UTR-mediated rhythmic mPer1 translation and provide evidence for posttranscriptional regulation of the circadian rhythmicity of core clock genes.

hnRNP Q mediates a phase-dependent translation-coupled mRNA decay of mouse Period3.

Daily mRNA oscillations of circadian clock genes largely depend on transcriptional regulation. However, several lines of evidence highlight the critical role of post-transcriptional regulation in the oscillations of circadian mRNA oscillations. Clearly, variations in the mRNA decay rate lead to changes in the cycling profiles. However, the mechanisms controlling the mRNA stability of clock genes are not fully understood. Here we demonstrate that the turnover rate of mouse Period3 (mPer3) mRNA is dramatically changed in a circadian phase-dependent manner. Furthermore, the circadian regulation of mPer3 mRNA stability requires the cooperative function of 5'- and 3'-untranslated regions (UTRs). Heterogeneous nuclear ribonucleoprotein Q (hnRNP Q) binds to both 5'- and 3'-UTR and triggers enhancement of translation and acceleration of mRNA decay. We propose the phase-dependent translation coupled mRNA decay mediated by hnRNP Q as a new regulatory mechanism of the rhythmically regulated decay of mPer3 mRNA.

Modulation of exosome-mediated mRNA turnover by interaction of GTP-binding protein 1 (GTPBP1) with its target mRNAs.

Eukaryotic mRNA turnover is among most critical mechanisms that affect mRNA abundance and are regulated by mRNA-binding proteins and the cytoplasmic exosome. A functional protein, guanosine-triphosphate-binding protein 1 (GTPBP1), which associates with both the exosome and target mRNAs, was identified. The overexpression of GTPBP1 accelerated the target mRNA decay, whereas the reduction of the GTPBP1 expression with RNA interference stabilized the target mRNA. GTPBP1 has a putative guanosine-triphosphate (GTP)-binding domain, which is found in members of the G-protein family and Ski7p, a well-known core factor of the exosome-mediated mRNA turnover pathway in yeast. Analyses of protein interactions and mRNA decay demonstrated that GTPBP1 modulates mRNA degradation via GTP-binding-dependent target loading. Moreover, GTPBP1-knockout models displayed multiple mRNA decay defects, including elevated nocturnal levels of Aanat mRNA in pineal glands, and retarded degradation of TNF-α mRNA in lipopolysaccharide-treated splenocytes. The results of this study suggest that GTPBP1 is a regulator and adaptor of the exosome-mediated mRNA turnover pathway.

hnRNP Q and PTB modulate the circadian oscillation of mouse Rev-erb alpha via IRES-mediated translation.

The physiological and behavioral circadian rhythms of most creatures are controlled by a harmony of functional relationships between clock genes. In mammals, several core clock genes show rhythmic profiles of their mRNA and protein expression. Among them, Rev-erb α functions as a transcriptional repressor, affecting expression patterns of other clock genes. For the continuous and robust oscillation of the molecular clock system, the levels of Rev-erb α protein are expected to be tightly regulated with the correct timing. Here, we demonstrate that Rev-erb α has an internal ribosomal entry site (IRES) in its 5' untranslated region. Furthermore, we demonstrate that heterogeneous nuclear ribonucleoprotein Q and polypyrimidine tract-binding protein (PTB) modulate the IRES-mediated translation of Rev-erb α. We suggest that the rhythmic binding affinity of hnRNP Q to the Rev-erb α IRES and the change in PTB cytosolic levels lead to maintenance of the oscillation profile of the Rev-erb α protein.

Circadian amplitude of cryptochrome 1 is modulated by mRNA stability regulation via cytoplasmic hnRNP D oscillation.

The mammalian circadian rhythm is observed not only at the suprachiasmatic nucleus, a master pacemaker, but also throughout the peripheral tissues. Its conserved molecular basis has been thought to consist of intracellular transcriptional feedback loops of key clock genes. However, little is known about posttranscriptional regulation of these genes. In the present study, we investigated the role of the 3'-untranslated region (3'UTR) of the mouse cryptochrome 1 (mcry1) gene at the posttranscriptional level. Mature mcry1 mRNA has a 610-nucleotide 3'UTR and mediates its own degradation. The middle part of the 3'UTR contains a destabilizing cis-acting element. The deletion of this element led to a dramatic increase in mRNA stability, and heterogeneous nuclear ribonucleoprotein D (hnRNP D) was identified as an RNA binding protein responsible for this effect. Cytoplasmic hnRNP D levels displayed a pattern that was reciprocal to the mcry1 oscillation. Knockdown of hnRNP D stabilized mcry1 mRNA and resulted in enhancement of the oscillation amplitude and a slight delay of the phase. Our results suggest that hnRNP D plays a role as a fine regulator contributing to the mcry1 mRNA turnover rate and the modulation of circadian rhythm.

Mouse period 2 mRNA circadian oscillation is modulated by PTB-mediated rhythmic mRNA degradation.

Circadian mRNA oscillations are the main feature of core clock genes. Among them, period 2 is a key component in negative-feedback regulation, showing robust diurnal oscillations. Moreover, period 2 has been found to have a physiological role in the cell cycle or the tumor suppression. The present study reports that 3'-untranslated region (UTR)-dependent mRNA decay is involved in the regulation of circadian oscillation of period 2 mRNA. Within the mper2 3'UTR, both the CU-rich region and polypyrimidine tract-binding protein (PTB) are more responsible for mRNA stability and degradation kinetics than are other factors. Depletion of PTB with RNAi results in mper2 mRNA stabilization. During the circadian oscillations of mper2, cytoplasmic PTB showed a reciprocal expression profile compared with mper2 mRNA and its peak amplitude was increased when PTB was depleted. This report on the regulation of mper2 proposes that post-transcriptional mRNA decay mediated by PTB is a fine-tuned regulatory mechanism that includes dampening-down effects during circadian mRNA oscillations.

Rhythmic control of AANAT translation by hnRNP Q in circadian melatonin production.

The circadian rhythm of pineal melatonin requires the nocturnal increment of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]) protein. To date, only limited information is available in the critical issue of how AANAT protein expression is up-regulated exclusively at night regardless of its species-specific mRNA profiles. Here we show that the circadian timing of AANAT protein expression is regulated by rhythmic translation of AANAT mRNA. This rhythmic control is mediated by both a highly conserved IRES (internal ribosome entry site) element within the AANAT 5' untranslated region and its partner hnRNP Q (heterogeneous nuclear ribonucleoprotein Q) with a peak in the middle of the night. Consistent with the enhancing role of hnRNP Q in AANAT IRES activities, knockdown of the hnRNP Q level elicited a dramatic decrease of peak amplitude in the AANAT protein profile parallel to reduced melatonin production in pinealocytes. This translational regulation of AANAT mRNA provides a novel aspect for achieving the circadian rhythmicity of vertebrate melatonin.

Junctional membrane inositol 1,4,5-trisphosphate receptor complex coordinates sensitization of the silent EGF-induced Ca2+ signaling.

Ca(2+) is a highly versatile intracellular signal that regulates many different cellular processes, and cells have developed mechanisms to have exquisite control over Ca(2+) signaling. Epidermal growth factor (EGF), which fails to mobilize intracellular Ca(2+) when administrated alone, becomes capable of evoking [Ca(2+)](i) increase and exocytosis after bradykinin (BK) stimulation in chromaffin cells. Here, we provide evidence that this sensitization process is coordinated by a macromolecular signaling complex comprised of inositol 1,4,5-trisphosphate receptor type I (IP(3)R1), cAMP-dependent protein kinase (PKA), EGF receptor (EGFR), and an A-kinase anchoring protein, yotiao. The IP(3)R complex functions as a focal point to promote Ca(2+) release in two ways: (1) it facilitates PKA-dependent phosphorylation of IP(3)R1 in response to BK-induced elevation of cAMP, and (2) it couples the plasmalemmal EGFR with IP(3)R1 at the Ca(2+) store located juxtaposed to the plasma membrane. Our study illustrates how the junctional membrane IP(3)R complex connects different signaling pathways to define the fidelity and specificity of Ca(2+) signaling.

Rhythmic serotonin N-acetyltransferase mRNA degradation is essential for the maintenance of its circadian oscillation.

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]) is the key enzyme in melatonin synthesis regulated by circadian rhythm. To date, our understanding of the oscillatory mechanism of melatonin has been limited to autoregulatory transcriptional and posttranslational regulations of AANAT mRNA. In this study, we identify three proteins from pineal glands that associate with cis-acting elements within species-specific AANAT 3' untranslated regions to mediate mRNA degradation. These proteins include heterogeneous nuclear ribonucleoprotein R (hnRNP R), hnRNP Q, and hnRNP L. Their RNA-destabilizing function was determined by RNA interference and overexpression approaches. Expression patterns of these factors in pineal glands display robust circadian rhythm. The enhanced levels detected after midnight correlate with an abrupt decline in AANAT mRNA level. A mathematical model for the AANAT mRNA profile and its experimental evidence with rat pinealocytes indicates that rhythmic AANAT mRNA degradation mediated by hnRNP R, hnRNP Q, and hnRNP L is a key process in the regulation of its circadian oscillation.

Phytoestrogen cimicifugoside-mediated inhibition of catecholamine secretion by blocking nicotinic acetylcholine receptor in bovine adrenal chromaffin cells.

We investigated the effect of the phytoestrogen cimicifugoside, one of the pharmacologically active ingredients of the medicinal plant Cimicifuga racemosa (black cohosh) that has been used to treat many kinds of neuronal and menopausal symptoms, such as arthritis, menopausal depression, and nerve pain. Cimicifugoside inhibited calcium increase induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), a nicotinic acetylcholine receptor (nAChR) agonist in bovine adrenal chromaffin cells with a half-maximal inhibitory concentration (IC(50)) of 18 +/- 2 microM. In contrast, cimicifugoside did not affect the calcium increases evoked by high K(+), veratridine, and bradykinin. The DMPP-induced sodium increase was also inhibited by cimicifugoside with an IC(50) of 2 +/- 0.3 microM, suggesting that the activity of nAChRs is inhibited by cimicifugoside. Cimicifugoside did not affect the KCl-induced secretion but markedly inhibited the DMPP-induced catecholamine secretion that was monitored by carbon-fiber amperometry in real time and high-performance liquid chromatography through electrochemical detection. The results suggest that cimicifugoside selectively inhibits nAChR-mediated response in bovine chromaffin cells.