Nodal/Bozozok-independent induction of the dorsal organizer by zebrafish cell lines

Formation of the dorsal organizer (Spemann organizer) is an important process in early vertebrate development. In zebrafish, two molecular cascades—Bozozok/Dharma (Boz) and Nodal signaling—act in parallel to induce the dorsal organizer. However, the complete molecular mechanism regulating this event remains unclear. Here we report that zebrafish cell lines derived from various developmental stages can induce a secondary axis when they are implanted into the mid-blastula but not the early gastrula. The implanted cellsthemselves did not differentiate, but instead induced ectopic expression of dorsal organizer markers incells around the implanted cells and induced notochord formation in the secondary axis. These results indicate that cultured cell lines have the ability to induce a secondary axis through the initiation of dorsal organizer activity. However, ectopic expression of boz and sqt were not observed in cultured cells. In addition, implanted cell lines could induce the dorsal organizer even in maternal-zygotic one-eyed pinhead mutants, which are not responsive to Nodal signaling. Finally, the Nodal signaling pathway was not activatedfollowing implantation of cultured cells. Collectively, these data suggest that zebrafish cell lines induce the dorsal organizer independent of the boz and Nodal signaling pathways.     

Brain-derived neurotrophic factor stimulates bone/cementum-related protein gene expression in cementoblasts

Brain-derived neurotrophic factor (BDNF), recognized as essential in the developing nervous system, is involved in differentiation and proliferation in non-neuronal cells, such as endothelial cells, osteoblasts, and periodontal ligament cells. We have focused on the application of BDNF to the regeneration of periodontal tissue and indicated that BDNF promotes the regeneration of experimentally created periodontal defects. Cementoblasts form cementum, mineralized tissue, which is key to establishing a functional periodontium. The application of BDNF to the regeneration of periodontal tissue requires elucidation of the mechanism by which BDNF regulates the functions of cementoblasts. In this study, we examined how BDNF regulates the mRNA expression of bone/cementum-related proteins (alkaline phosphatase (ALP), osteopontin (OPN), and bone morphogenetic protein-2 (BMP-2)) in cultures of immortalized human cementoblast-like (HCEM) cells. BDNF elevated the mRNA levels of ALP, OPN, and BMP-2 in HCEM cells. Small interfering RNA (siRNA) for TRKB, a high affinity receptor of BDNF, siRNA for ELK-1, which is a downstream target of ERK1/2, and PD98059, an ERK inhibitor, obviated the increase in the mRNA levels. BDNF increased the levels of phosphorylated ERK1/2 and Elk-1, and the blocking of BDNF signaling by treatment with siRNA for TRKB and PD98059 suppressed the phosphorylation of ERK1/2 and Elk-1. Furthermore, BDNF increased the levels of phosphorylated c-Raf, which activates the ERK signaling pathway. These findings provide the first evidence that the TrkB-c-Raf-ERK1/2-Elk-1 signaling pathway is required for the BDNF-induced mRNA expression of ALP, OPN, and BMP-2 in HCEM cells.     

Phosphorylation of Streptomycin at C6-OH of Streptidine Moiety by an Intracellular Enzyme of Streptomyces griseus

In streptomycin-producing Streptomyces griseus HUT 6037, an enzyme which phosphorylated streptomycin appeared in old mycelium at stationary to autolyzing stage. This enzyme phosphorylated streptomycin with equimolar ATP at C₆-OH in the streptidine moiety. This phosphomonoester of streptomycin was identified with the phosphorylated streptomycin (referred to as L compound) which was previously reported to accumulate in the culture broth when the pH was controlled below neutral.     

Calcitonin gene-related peptide is an important regulator of cutaneous immunity: effect on dendritic cell and T cell functions

Some cutaneous inflammations are induced by percutaneous exposure to foreign Ags, and many chemical mediators regulate this inflammation process. One of these mediators, calcitonin gene-related peptide (CGRP), is a neuropeptide released from nerve endings in the skin. CGRP binds to its receptors composed of receptor activity-modifying protein 1 and calcitonin receptor-like receptor to modulate immune cell function. We show that CGRP regulates skin inflammation under physiological conditions, using contact hypersensitivity (CHS) models of receptor activity-modifying protein 1-deficient mice. CGRP has different functions in CHS responses mediated by Th1 or Th2 cells; it inhibits Th1-type CHS, such as 2,4,6-trinitrochlorobenzene-induced CHS, but promotes Th2-type CHS, such as FITC-induced CHS. CGRP inhibits the migration of Langerin(+) dermal dendritic cells to the lymph nodes in 2,4,6-trinitrochlorobenzene-induced CHS, and upregulates IL-4 production of T cells in the draining lymph nodes in FITC-CHS. These findings suggest that CGRP regulates several types of CHS reactions under physiological conditions and plays an important role in cutaneous immunity.     

Induced Differentiation of Human Myeloid Leukemia Cells into M2 Macrophages by Combined Treatment with Retinoic Acid and 1α,25-Dihydroxyvitamin D3

Retinoids and 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) induce differentiation of myeloid leukemia cells into granulocyte and macrophage lineages, respectively. All-trans retinoic acid (ATRA), which is effective in the treatment of acute promyelocytic leukemia, can induce differentiation of other types of myeloid leukemia cells, and combined treatment with retinoid and 1,25(OH)₂D₃ effectively enhances the differentiation of leukemia cells into macrophage-like cells. Recent work has classified macrophages into M1 and M2 types. In this study, we investigated the effect of combined treatment with retinoid and 1,25(OH)₂D₃ on differentiation of myeloid leukemia THP-1 and HL60 cells. 9-cis Retinoic acid (9cRA) plus 1,25(OH)₂D₃ inhibited proliferation of THP-1 and HL60 cells and increased myeloid differentiation markers including nitroblue tetrazolium reducing activity and expression of CD14 and CD11b. ATRA and the synthetic retinoic acid receptor agonist Am80 exhibited similar effects in combination with 1,25(OH)₂D₃ but less effectively than 9cRA, while the retinoid X receptor agonist HX630 was not effective. 9cRA plus 1,25(OH)₂D₃ effectively increased expression of M2 macrophage marker genes, such as CD163, ARG1 and IL10, increased surface CD163 expression, and induced interleukin-10 secretion in myeloid leukemia cells, while 9cRA alone had weaker effects on these phenotypes and 1,25(OH)₂D₃ was not effective. Taken together, our results demonstrate selective induction of M2 macrophage markers in human myeloid leukemia cells by combined treatment with 9cRA and 1,25(OH)₂D₃.     

Germ cell–specific expression of Venus by Tol2-mediated transgenesis in endangered endemic cyprinid Honmoroko (Gnathopogon caerulescens)

Fishes expressing a fluorescent protein in germ cells are useful to perform germ cell transfer experiments for conservation study. Nonetheless, no such fish has been generated in endangered endemic fishes. In this study, we tried to produce a fish expressing Venus fluorescent protein in germ cells using Honmoroko (Gnathopogon caerulescens), which is one of the threatened small cyprinid endemic to the ancient Lake Biwa in Japan. To achieve germ cell-specific expression of Venus, we used piwil1 (formally known as ziwi) promoter and Tol2 transposon system. Following the co-injection of the piwil1-Venus expression vector and the Tol2 transposase mRNA into fertilized eggs, presumptive transgenic fish were reared. At 7 months of post-fertilization, about 19% (10/52) of the examined larvae showed Venus fluorescence in their gonad specifically. Immunohistological staining and in vitro spermatogenesis using gonads of the juvenile founder fish revealed that Venus expression was detected in spermatogonia and spermatocyte in male, and oogonia and stage I and II oocytes in female. These results indicate that the Tol2 transposon and zebrafish piwil1 promoter enabled gene transfer and germ cell-specific expression of Venus in G. caerulescens. In addition, in vitro culture of juvenile spermatogonia enables the rapid validation of temporal expression of transgene during spermatogenesis.     

Improvement of heme oxygenase-1-based heme sensor for quantifying free heme in biological samples

We recently reported a novel heme sensor using fluorescently labeled heme oxygenase-1; however, its inherent enzyme activity would be a potential obstacle in quantifying heme in biological samples. Here, we found that mutation of the catalytically important residue, Asp140, with histidine in the sensor not only diminished the heme degradation activity but also increased heme binding affinity. The sensor with a visible fluorophore was also found to be beneficial to avoid background emission from endogenous substance in biological samples. By using the improved heme sensor, we succeeded in quantifying free heme in rat hepatic samples for the first time.     

Role of motoneuron-derived neurotrophin 3 in survival and axonal projection of sensory neurons during neural circuit formation

Sensory neurons possess the central and peripheral branches and they form unique spinal neural circuits with motoneurons during development. Peripheral branches of sensory axons fasciculate with the motor axons that extend toward the peripheral muscles from the central nervous system (CNS), whereas the central branches of proprioceptive sensory neurons directly innervate motoneurons. Although anatomically well documented, the molecular mechanism underlying sensory-motor interaction during neural circuit formation is not fully understood. To investigate the role of motoneuron on sensory neuron development, we analyzed sensory neuron phenotypes in the dorsal root ganglia (DRG) of Olig2 knockout (KO) mouse embryos, which lack motoneurons. We found an increased number of apoptotic cells in the DRG of Olig2 KO embryos at embryonic day (E) 10.5. Furthermore, abnormal axonal projections of sensory neurons were observed in both the peripheral branches at E10.5 and central branches at E15.5. To understand the motoneuron-derived factor that regulates sensory neuron development, we focused on neurotrophin 3 (Ntf3; NT-3), because Ntf3 and its receptors (Trk) are strongly expressed in motoneurons and sensory neurons, respectively. The significance of motoneuron-derived Ntf3 was analyzed using Ntf3 conditional knockout (cKO) embryos, in which we observed increased apoptosis and abnormal projection of the central branch innervating motoneuron, the phenotypes being apparently comparable with that of Olig2 KO embryos. Taken together, we show that the motoneuron is a functional source of Ntf3 and motoneuron-derived Ntf3 is an essential pre-target neurotrophin for survival and axonal projection of sensory neurons.     

Non-antibiotic antibacterial activity of dodecyl gallate

Dodecyl (C(12)) gallate (3,4,5-trihydroxybenzoate) (1) was found to possess antibacterial activity specifically against Gram-positive bacteria, in addition to its potent antioxidant activity. The time-kill curve study indicates that this amphipathic gallate exhibits bactericidal activity against methicillin resistant Staphylococcus aureus (MRSA) strains. Dodecyl (lauryl) gallate inhibited oxygen consumption in whole cells and oxidation of NADH in membrane preparation. The antibacterial activity of this gallate comes in part from its ability to inhibit the membrane respiratory chain. As far as alkyl gallates are concerned, their antimicrobial spectra and potency depend in part on the hydrophobic portion of the molecule.