Identification of a 100 kDa Microtubule-Associated Protein from Xenopus Eggs

A 100 kDa protein was identified by a combination of microtubule affinity chromatography and microtubule co-sedimentation from Xenopus egg extracts. The 100 kDa protein was expressed in Stage VI oocytes and early embryos, and then decreased at tailbud stage. The 100 kDa protein was found in adult organs such as brain and liver. Immunofluorescent microscopy revealed that the 100 kDa protein was mainly detected in spinal cord, notochordal sheath, optic cup, lens and cement gland in tailbud embryos. In Xenopus A6 cells, the 100 kDa protein showed filamentous networks in the peripheral cytoplasm and uniform distribution around the nucleus. During cell division, the 100 kDa protein was localized to the mitotic apparatus. The 100 kDa protein may have some roles in microtubule dynamics, organization of the mitotic apparatus and maintenance of cell shape

Acetyl-L-carnitine suppresses thyroid hormone-induced and spontaneous anuran tadpole tail shortening

Mitochondrial membrane permeability transition (MPT) plays a crucial role in apoptotic tail shortening during anuran metamor phosis. L-carnitine is known to shuttle free fatty acids (FFAs) from the cytosol into mitochondria matrix for -oxidation and energy production, and in a previous study we found that treatment with L-carnitine suppresses 3, 3', 5-triiodothyronine (T3) and FFA-induced MPT by reducing the level of FFAs. In the present study we focus on acetyl-L-carnitine, which is also involved in fatty acid oxidation, to determine its effect on T3-induced tail regression in Rana rugosa tadpoles and spontaneous tail regression in Xenopus laevis tadpoles. The ladder-like DNA profile and increases in caspase-3 and caspase-9 indicative of apoptosis in the tails of T3-treated tadpoles were found to be suppressed by the addition of acetyl-L-carnitine. Likewise, acetyl-L-carnitine was found to inhibit thyroid hormone regulated spontaneous metamorphosis in X. laevis tadpoles, accompanied by decreases in caspase and phospholipase A2 activity, as well as non-ladder-like DNA profiles. These findings support our previous conclusion that elevated levels of FFAs initiate MPT and activate the signaling pathway controlling apoptotic cell death in tadpole tails during anuran metamorphosis

A Secreted BMP Antagonist, Cer1, Fine Tunes the Spatial Organization of the Ureteric Bud Tree during Mouse Kidney Development

The epithelial ureteric bud is critical for mammalian kidney development as it generates the ureter and the collecting duct system that induces nephrogenesis in dicrete locations in the kidney mesenchyme during its emergence. We show that a secreted Bmp antagonist Cerberus homologue (Cer1) fine tunes the organization of the ureteric tree during organogenesis in the mouse embryo. Both enhanced ureteric expression of Cer1 and Cer1 knock out enlarge kidney size, and these changes are associated with an altered three-dimensional structure of the ureteric tree as revealed by optical projection tomography. Enhanced Cer1 expression changes the ureteric bud branching programme so that more trifid and lateral branches rather than bifid ones develop, as seen in time-lapse organ culture. These changes may be the reasons for the modified spatial arrangement of the ureteric tree in the kidneys of Cer1+ embryos. Cer1 gain of function is associated with moderately elevated expression of Gdnf and Wnt11, which is also induced in the case of Cer1 deficiency, where Bmp4 expression is reduced, indicating the dependence of Bmp expression on Cer1. Cer1 binds at least Bmp2/4 and antagonizes Bmp signalling in cell culture. In line with this, supplementation of Bmp4 restored the ureteric bud tip number, which was reduced by Cer1+ to bring it closer to the normal, consistent with models suggesting that Bmp signalling inhibits ureteric bud development. Genetic reduction of Wnt11 inhibited the Cer1-stimulated kidney development, but Cer1 did not influence Wnt11 signalling in cell culture, although it did inhibit the Wnt3a-induced canonical Top Flash reporter to some extent. We conclude that Cer1 fine tunes the spatial organization of the ureteric tree by coordinating the activities of the growth-promoting ureteric bud signals Gndf and Wnt11 via Bmp-mediated antagonism and to some degree via the canonical Wnt signalling involved in branching

A Cytoplasmic Factor Required for Contraction of the Cleavage Furrow in af Mutant Eggs of Xenopus laevis

The abnormal furrow (af) gene was identified through a maternal effect mutation that causes a defect in cell division in Xenopus embryos. In eggs obtained from mutant females (af eggs), polar body formation and cytokinesis are completely inhibited while nuclear division continues uninterrupted. Cleavage furrows are recognized as unpigmented narrow bands on the egg surface but they do not contract. Transfer of cytoplasm from wild-type eggs into af eggs partially rescues contraction of the cleavage furrow. The factor responsible for contraction promoting activity was present in wild-type eggs throughout the first cell division cycle and induced contraction in a dose dependent manner. The factor was characterized as a high molecular weight protein complex that was associated with particulate fraction in the extract. The recovery of contraction was associated with accumulation of filamentous actin and WGA-binding sites within the cleavage furrow of the af eggs which were normally not enriched for these components. Formation and contraction of a filamentous actin ring during cortical wound healing in af eggs was indistinguishable from that of wild-type eggs. From these results, the af gene product may be specifically required for reorganization of the actin filaments and WGA-binding sites in the cleavage furrow and contraction through these structures during cytokinesis

Breeding of Buckwheat for Usage of Sprout and Pre-Harvest Sprouting Resistance

Buckwheat is recognized as an important traditional crop and supports local economies in several regions around the world. Buckwheat is used, for example, as a cereal grain, noodle and bread. In addition, buckwheat is also used as a sprout or a young seedling. For these foods, sprouting is an important characteristic that affects food quality. For foods made from buckwheat flour, pre-harvest sprouting may decrease yield, which also leads to the deterioration of noodle quality. Breeding buckwheat that is resistant to pre-harvest sprouting is therefore required. Germination and subsequent growth are also important characteristics of the quality of sprouts. Although buckwheat sprouts are the focus because they contain many functional compounds, such as rutin, several problems have been noted, such as thin hypocotyls and husks remaining on sprouts. To date, several new varieties have been developed to resolve these quality issues. In this review, we summarize and introduce research on the breeding of buckwheat related to quality, sprouting and subsequent sprout growth

Multi-modal effects of BMP signaling on Nodal expression in the lateral plate mesoderm during left–right axis formation in the chick embryo

AbstractDuring development of left–right asymmetry in the vertebrate embryo, Nodal plays a central role for determination of left-handedness. Bone morphogenetic protein (BMP) signaling has an important role for regulation of Nodal expression, although there is controversy over whether BMP signaling has a positive or negative effect on Nodal expression in the chick embryo. As BMP is a morphogen, we speculated that different concentrations might induce different responses in the cells of the lateral plate mesoderm (LPM). To test this hypothesis, we analyzed the effects of various concentrations of BMP4 and NOGGIN on Nodal expression in the LPM. We found that the effect on Nodal expression varied in a complex fashion with the concentration of BMP. In agreement with previous reports, we found that a high level of BMP signaling induced Nodal expression in the LPM, whereas a low level inhibited expression. However, a high intermediate level of BMP signaling was found to suppress Nodal expression in the left LPM, whereas a low intermediate level induced Nodal expression in the right LPM. Thus, the high and the low intermediate levels of BMP signaling up-regulated Nodal expression, but the high intermediate and low levels of BMP signaling down-regulated Nodal expression. Next, we sought to identify the mechanisms of this complex regulation of Nodal expression by BMP signaling. At the low intermediate level of BMP signaling, regulation depended on a NODAL positive-feedback loop suggesting the possibility of crosstalk between BMP and NODAL signaling. Overexpression of a constitutively active BMP receptor, a constitutively active ACTIVIN/NODAL receptor and SMAD4 indicated that SMAD1 and SMAD2 competed for binding to SMAD4 in the cells of the LPM. Nodal regulation by the high and low levels of BMP signaling was dependent on Cfc up-regulation or down-regulation, respectively. We propose a model for the variable effects of BMP signaling on Nodal expression in which different levels of BMP signaling regulate Nodal expression by a balance between BMP-pSMAD1/4 signaling and NODAL-pSMAD2/4 signaling

Breeding of Buckwheat to Reduce Bitterness and Rutin Hydrolysis

Buckwheat (Fagopyrum esculentum) is recognized as an important traditional crop in some regions, and its taste is an important characteristic. Of the three cultivated buckwheat species, Tartary buckwheat (Fagopyrum tataricum) and perennial buckwheat (Fagopyrum cymosum) have strong bitterness in their seeds, which has prevented the wider use of the seeds of these varieties. In Tartary buckwheat, some studies have focused on the cause of strong bitterness generation. Tartary buckwheat seeds contain large amounts of the functional compounds rutin and rutinosidase, and rutin hydrolysis by rutinosidase has been found to be the trigger of rutin hydrolysis. Therefore, a variety with only a trace of rutinosidase and with reduced bitterness is required. The rutinosidase in Tartary buckwheat seeds consists of two major isozymes with very similar enzymatic characteristics, which can hydrolyze flour rutin within several minutes after the addition of water. Recently, the trace-rutinosidase variety Manten-Kirari in Tartary buckwheat was developed. The trace-rutinosidase characteristics were dominated by a single recessive gene. In ‘Manten-Kirari’ dough and foods, such as breads, confectionaries, and noodles, the rutin residual ratio was higher and bitterness was reduced compared to that of the normal-rutinosidase variety. In this review, we summarize the detailed research on the breeding of buckwheat related to reducing bitterness and rutin hydrolysis

Additional file 1: of A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity

Primer sets used for PCR. (PDF 92 kb

Additional file 2: of A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity

Genotyping of FeDFR1a and FeDFR2. (A) Genotyping of FeDFR1a using PCR-amplified genomic DNA. A1 and A2 indicate the two alleles, and arrowheads indicate their positions. (B) Genotyping of FeDFR2 using the HincII digest of PCR-amplified genomic DNA. B1 and B2 indicate the two alleles, and arrowheads indicate their positions. (C) Sequence analysis of the FeDFR2 genomic DNA. An A to G transition at nucleotide 370, which results in a HincII restriction site in the FeDFR2 genome, is boxed in red. (D) Partial sequence alignment of FeDFR1aA1 and FeDFR1aA2. Identical nucleotides are highlighted in black. (PDF 68 kb