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PC12 cells, which are derived from rat adrenal pheochromocytoma cells, are widely used for the study of neuronal differentiation. NGF induces neuronal differentiation in PC12 cells by activating intracellular pathways via the TrkA receptor, which results in elongated neurites and neuron-like characteristics. Moreover, the differentiation requires both the ERK1/2 and p38 MAPK pathways. In addition to NGF, BMPs can also induce neuronal differentiation in PC12 cells. BMPs are part of the TGF-β cytokine superfamily and activate signaling pathways such as p38 MAPK and Smad. However, the brief lifespan of NGF and BMPs may limit their effectiveness in living organisms. Although PC12 cells are used to study the effects of various physical stimuli on neuronal differentiation, the development of new methods and an understanding of the molecular mechanisms are ongoing. In this comprehensive review, we discuss the induction of neuronal differentiation in PC12 cells without relying on NGF, which is already established for electrical, electromagnetic, and thermal stimulation but poses a challenge for mechanical, ultrasound, and light stimulation. Furthermore, the mechanisms underlying neuronal differentiation induced by physical stimuli remain largely unknown. Elucidating these mechanisms holds promise for developing new methods for neural regeneration and advancing neuroregenerative medical technologies using neural stem cells. [ABSTRACT FROM AUTHOR]

This study evaluated the mechanism of temperature-controlled repeated thermal stimulation (TRTS)-mediated neuronal differentiation. We assessed the effect of SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, on neuronal differentiation of rat PC12-P1F1 cells, which can differentiate into neuron-like cells by exposure to TRTS or neurotrophic factors, including bone morphogenetic protein (BMP) 4. We evaluated neuritogenesis by incubating the cells under conditions of TRTS and/or SP600125. Cotreatment with SP600125 significantly enhanced TRTS-mediated neuritogenesis, whereas that with other selective mitogen-activated protein kinase (MAPK) inhibitors did not—e.g., extracellular signal-regulated kinase (ERK)1/2 inhibitor U0126, and p38 MAPK inhibitor SB203580. We tried to clarify the mechanism of SP600125 action by testing the effect of U0126 and the BMP receptor inhibitor LDN193189 on the SP600125-mediated enhancement of intracellular signaling. SP600125-enhanced TRTS-induced neuritogenesis was significantly inhibited by U0126 or LDN193189. Gene expression analysis revealed that TRTS significantly increased β3-Tubulin, MKK3, and Smad7 gene expressions. Additionally, Smad6 and Smad7 gene expressions were substantially attenuated through SP600125 co-treatment during TRTS. Therefore, SP600125 may partly enhance TRTS-induced neuritogenesis by attenuating the negative feedback loop of BMP signaling. Further investigation of the mechanisms underlying the effect of SP600125 during TRTS-mediated neuritogenesis may contribute to the future development of regenerative neuromedicine. [ABSTRACT FROM AUTHOR]

Genetically encoded sensor proteins provide unique opportunities to advance the understanding of complex cellular interactions in physiologically relevant contexts; however, previously described sensors have proved to be of limited use to report cell signaling in vivo in mammals. Here, we describe an improved [Ca.sup.2+] sensor, GCaMP2, its inducible expression in the mouse heart, and its use to examine signaling in heart cells in vivo. The high brightness and stability of GCaMP2 enable the measurement of myocyte [Ca.sup.2+] transients in all regions of the beating mouse heart and prolonged pacing and mapping studies in isolated, perfused hearts. Transgene expression is efficiently temporally regulated in cardiomyocyte GCaMP2 mice, allowing recording of in vivo signals 4 weeks after transgene induction. High-resolution imaging of [Ca.sup.2+] waves in GCaMP2-expressing embryos revealed key aspects of electrical conduction in the preseptated heart. At embryonic day (e.d.) 10.5, atrial and ventricular conduction occur rapidly, consistent with the early formation of specialized conduction pathways. However, conduction is markedly slowed through the atrioventricular canal in the e.d. 10.5 heart, forming the basis for an effective atrioventricular delay before development of the AV node, as rapid ventricular activation occurs after activation of the distal AV canal tissue. Consistent with the elimination of the inner AV canal muscle layer at e.d. 13.5, atrioventricular conduction through the canal was abolished at this stage. These studies demonstrate that GCaMP2 will have broad utility in the dissection of numerous complex cellular interactions in mammals, in vivo. atrioventricular node | [Ca.sup.2+] imaging | genetic sensor | heart development | fluorescent [Ca.sup.2+] sensor

G-CaMP is a [Ca.sup.2+] probe based on a single green fluorescent protein (GFP). G-CaMP shows a large fluorescence increase upon [Ca.sup.2+] binding, but its fluorescence is dim and pH sensitive, similar to other single GFP-based probes. Here we report an improved G-CaMP, named G-CaMP1.6, which enables easier detection of intracellular [Ca.sup.2+] signals. G-CaMP1.6 was ~40 times more fluorescent than G-CaMP, mainly due to an increase in quantum yield. Furthermore, compared with G-CaMP, G-CaMP1.6 had not only a lower pH sensitivity but also a higher selectivity for divalent cations having an ionic radius similar to [Ca.sup.2+]. [Ca.sup.2+] sensitivity of G-CaMP1.6 ([K.sub.d] = 146 nM, Hill coefficient = 3.8, [F.sub.max]/[F.sub.min] = 4.9) was slightly shifted toward higher affinity compared with that of G-CaMP. When expressed in mammalian cells, G-CAMP1.6 showed large fluorescence changes with drug applications. Notably, local [Ca.sup.2+] changes in such tiny structures as dendritic spines of neurons were successfully observed with G-CaMP1.6, this being the first observation using a GFP-based probe. Additional mutations in [Ca.sup.2+]-binding sites of G-CaMP1.6 shifted the affinity for [Ca.sup.2+] and reduced the [Ca.sup.2+]-buffering effect. G-CaMP1.6-CaM(E140K), which has a mutation in the [Ca.sup.2+] binding site, is an improved probe with its increased brightness and reduced [Ca.sup.2+]-buffering capacity.

The silkworm Bombyx mori is one of the most well-studied insects in terms of both genetics and physiology and is recognized as the model lepidopteran insect. To develop an efficient system for analyzing gene function in the silkworm, we investigated the feasibility of using the GAL4/UAS system in conjunction with piggyBac vector-mediated germ-line transformation for targeted gene expression. To drive the GAL4 gene, we used two endogenous promoters that originated from the B. mori actin A3 (BmA3) and fibroin lightchain (FiL) genes and the artificial promoter 3xP3. GFP was used as the reporter. In initial tests of the function of the GAL4/UAS system, we generated transgenic animals that carried the UAS-GFP construct plus either BmA3-GAL4 or 3xP3-GAL4. GFP fluorescence was observed in the tissues of GFP-positive animals, in which both promoters drove GAL4 gene expression. Animals that possessed only the GAL4 gene or UAS-GFP construct did not show GFP fluorescence. In addition, as a further test of the ability of the GAL4/UAS system to drive tissue-specific expression we constructed FiL-GAL4 lines with 3xP3-CFP as the transformation marker. FiL-GAL4 X UAS-GFP crosses showed GFP expression in the posterior silk gland, in which the endogenous FiL gene is normally expressed. These results show that the GAL4/UAS system is applicable to B. mori and emphasize the potential of this system for controlled analyses of B. mori gene function.

The Gly16Arg polymorphism results in a G to C nucleotide mutation in the human beta 2-adrenergic receptor (ADRB2) gene and has a relationship with obesity; however, this substitution's effects on food preferences are unclear. Therefore, we determined this relationship among healthy young adults (mean age, 23.4; n = 52). To evaluate food preferences, four categories of food (sweet, salty, sour, and bitter) along with high-fat foods were evaluated using a self-reporting questionnaire. Male (n = 26) and female subjects (n = 26) were genotyped for the polymorphism and further divided into three groups (two homozygous groups, GG, CC; and a heterozygous group, GC). Preference for sour foods in the GG group was higher compared with that in the CC group in females (p < 0.05). When sweet foods were classified into low- and high-fat subgroups, preference for high-fat sweet foods in the GG group was higher than that for low-fat sweet foods in all subjects (p < 0.05). The degree of preference for high-fat foods in the GG group was higher than other groups for males (p < 0.05). These results suggest that ADRB2 polymorphism is associated with food preference. Understanding the relationship of ADRB2 substitution to food preference will be valuable for designing individualized anti-obesity strategies. [ABSTRACT FROM AUTHOR]

Author(s): Yuji Shiba (corresponding author) [1, 2]; Toshihito Gomibuchi [3]; Tatsuichiro Seto [3]; Yuko Wada [3]; Hajime Ichimura [3]; Yuki Tanaka [3]; Tatsuki Ogasawara [3]; Kenji Okada [3]; Naoko Shiba [...]

The effect of ryanodine receptor (RyR-1) in dihydropyridine channel activity is determined using dyspedic mice myotubes as a destroyed RyR-1 gene. There was no excitation-contraction coupling recorded in dyspedic myotubes. L-type Ca2+-current density was reduced 30 times than the original. The application of of RyR-1 complementary DNA to dyspedic myotubes caused normal excitation-contraction coupling activity and normalized L-type Ca2+ currents. The enhanced L-type current was a result of the skeletal isoform of the dihydropyridine receptor (DHPR) as a calcium channel enhanced by a retrograde signal of RyR-1.