The transcription factor p53 has been shown to mediate cellular responses to diverse stresses such as DNA damage. However, the function of p53 in cellular differentiation in response to growth factor stimulations has remained obscure. We present evidence that p53 regulates cellular differentiation by modulating signaling of the TGFβ family of growth factors during early Xenopus embryogenesis. We show that p53 functionally and physically interacts with the activin and bone morphogenetic protein pathways to directly induce the expression of the homeobox genes Xhox3 and Mix.1/2. Furthermore, functional knockdown of p53 in embryos by an antisense morpholino oligonucleotide reveals that p53 is required for the development of dorsal and ventral mesoderm. Our data illustrate a pivotal role of interplay between the p53 and TGFβ pathways in cell fate determination during early vertebrate embryogenesis.
During left-right (L-R) axis formation, Nodal is expressed in the node and has a central role in the transfer of L-R information in the vertebrate embryo. Bone morphogenetic protein (BMP) signaling also has an important role for maintenance of gene expression around the node. Several members of the Cerberus/Dan family act on L-R patterning by regulating activity of the transforming growth factor-β (TGF-β) family. We demonstrate here that chicken Dan plays a critical role in L-R axis formation. Chicken Dan is expressed in the left side of the node shortly after left-handed Shh expression and before the appearance of asymmetrically expressed genes in the lateral plate mesoderm (LPM). In vitro experiments revealed that DAN inhibited BMP signaling but not NODAL signaling. SHH had a positive regulatory effect on Dan expression while BMP4 had a negative effect. Using overexpression and RNA interference-mediated knockdown strategies, we demonstrate that Dan is indispensable for Nodal expression in the LPM and for Lefty-1 expression in the notochord. In the perinodal region, expression of Dan and Nodal was independent of each other. Nodal up-regulation by DAN required NODAL signaling, suggesting that DAN might act synergistically with NODAL. Our data indicate that Dan plays an essential role in the establishment of the L-R axis by inhibiting BMP signaling around the node.
In the ciliated protozoan Blepharisma, step-up photophobic response is believed to be mediated by a novel type of photosensory pigment known as "blepharismins" (BL) that are contained in the pigment granules located just beneath the plasma membrane. We examined the ultrastructure of the pigment granules by freeze-fracture and thin-section electron microscopy and proposed a schematic diagram showing the granules' three-dimensional inner membranous structure. Some of the BL are suggested to be associated with 200 kDa membrane protein. High-pressure liquid chromatography analysis of pigment species associated with 200 kDa protein obtained from blue forms of Blepharisma (oxyblepharisma) revealed that the 200 kDa protein was associated with five types of oxyblepharismin. The fluorescence intensity was increased when the pigments were dissociated from the 200 kDa protein. The result supports the hypothesis that the pigment-200 kDa complex is able to transduce light energy into signals mediating the photobehavior of Blepharisma.
In the ciliated protozoan Blepharisma, step‐up photophobic response is believed to be mediated by a novel type of photosensory pigment known as “blepharismins” (BL) that are contained in the pigment granules located just beneath the plasma membrane. We examined the ultrastructure of the pigment granules by freeze‐fracture and thin‐section electron microscopy and proposed a schematic diagram showing the granules' three‐dimensional inner membranous structure. Some of the BL are suggested to be associated with 200 kDa membrane protein. High‐pressure liquid chromatography analysis of pigment species associated with 200 kDa protein obtained from blue forms of Blepharisma (oxyblepharisma) revealed that the 200 kDa protein was associated with five types of oxyblepharismin. The fluorescence intensity was increased when the pigments were dissociated from the 200 kDa protein. The result supports the hypothesis that the pigment–200 kDa complex is able to transduce light energy into signals mediating the photobehavior of Blepharisma.
Hypercholesterolemia is a major risk factor for cardiovascular events. Among them, abnormal VSM contractions such as vasospasm are caused by Ca 2þ -sensitization of VSM contraction. However, the relationship between cholesterol (CHOL) and the VSM Ca 2þ -sensitization has not been clarified yet. Our recent studies showed that a sphingosylphosphorylcholine (SPC)/Src family tyrosine kinase (Src-TK) / Rho-kinase (ROK) pathway mediates the Ca 2þ -sensitization, and SPC indeed induces severe vasospasm in vivo. We found that serum CHOL potentiates the SPC/Src-TK/ROK pathway leading to Ca 2þ -sensitization in both human and rabbit: the extent of SPC-induced Ca 2þ -sensitization correlated well with total CHOL and LDL-CHOL, but inversely correlated with HDL-CHOL. Moreover, the depletion of CHOL by b-cyclodextrin destroyed CHOL-enriched membrane lipid rafts and abolished the SPC-induced ROK translocation and Ca 2þ -sensitization, suggesting the SPC-induced translocation of ROK to lipid rafts. Taken together, we suggested that not only CHOL, but also lipid rafts mediates the SPC-induced Ca 2þ -sensitization. Understanding the mechanism(s) by which lipid rafts promotes Ca 2þ -sensitization in human VSM requires the elucidation of lipid raft protein composition. As a first step, we succeeded for the first time in purifying lipid rafts from human VSM by sucrose density gradient ultracentrifugation, which were confirmed by western blot of raft marker proteins such as caveolin-1 and flotillin-1. Subsequently, using mass spectrometry (MALDI TOF-MS), proteomic analysis was performed to compare the protein compositions between lipid raft and non-lipid raft membrane fractions. The ongoing studies have identified so far previously unreported novel raft-localized proteins, in addition to the known proteins, including lipid-or GPI-anchored proteins and membrane proteins. We are attempting to accumulate functional data to suggest that some novel signaling molecules contribute to an SPC/Src-TK/ROK pathway leading to the VSM Ca 2þ -sensitization.
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