Lamin B receptor-mediated chromatin tethering to the nuclear envelope is detrimental to the Xenopus blastula.

In the nucleus of eukaryotic cells, chromatin is tethered to the nuclear envelope (NE), wherein inner nuclear membrane proteins (INMPs) play major roles. However, in Xenopus blastula, chromatin tethering to the NE depends on nuclear filamentous actin that develops in a blastula-specific manner. To investigate whether chromatin tethering operates in the blastula through INMPs, we experimentally introduced INMPs into Xenopus egg extracts that recapitulate nuclear formation in fertilized eggs. When expressed in extracts in which polymerization of actin is inhibited, only lamin B receptor (LBR), among the five INMPs tested, tethered chromatin to the NE, depending on its N2 and N3 domains responsible for chromatin-protein binding. N2-3-deleted LBR did not tether chromatin, although it was localized in the nuclei. We subsequently found that the LBR level was very low in the Xenopus blastula but was elevated after the blastula stage. When the LBR level was precociously elevated in the blastula by injecting LBR mRNA, it induced alterations in nuclear lamina architecture and nuclear morphology and caused DNA damage and abnormal mitotic spindles, depending on the N2-3 domains. These results suggest that LBR-mediated chromatin tethering is circumvented in the Xenopus blastula, as it is detrimental to embryonic development.

The WD40 Domain of HIRA Is Essential for RI-nucleosome Assembly in Xenopus Egg Extracts.

Histone chaperones are a group of histone-binding proteins that facilitate the assembly of nucleosomes, the fundamental structural units of chromatin in eukaryotes. In nucleosome assembly, deposition of a histone H3-H4 tetramer onto DNA is the first and critical step, which is mediated by the histone chaperones HIRA and CAF-1. HIRA and CAF-1 are reportedly involved in DNA replication independent (RI) and replication coupled nucleosome assembly, respectively. However, the mechanisms by which they mediate histone deposition remain unclear. In this study, we focused on the mechanism by which HIRA induces RI-nucleosome assembly. We looked for HIRA domains that are required for nucleosome assembly and its localization to chromatin. We used cell-free extracts from Xenopus eggs that carry out RI-nucleosome assembly of plasmid DNA. We confirmed that HIRA formed stable complexes with Asf1, another histone H3-H4 chaperone, and the HIRA-Asf1 complex was solely responsible for RI-nucleosome assembly in egg extracts. We further demonstrated that the HIRA N-terminus containing the WD40 domain, which comprises seven WD40 repeats, and the B domain, to which Asf1 binds, were essential for RI-nucleosome assembly; the three WD40 repeats from the N-terminus were especially critical. Using egg extracts that reproduce nuclear formation accompanying the duplication of chromatin, we also demonstrated that the Hir domain was indispensable for the binding of HIRA to chromatin. Thus, the WD40 and B domains are the core elements for inducing RI-nucleosome assembly. Hir domain regulates the binding to chromatin. Based on these findings, similarities and differences between HIRA and CAF-1 are discussed.

Chromatin tethering to the nuclear envelope by nuclear actin filaments: a novel role of the actin cytoskeleton in the Xenopus blastula.

The Xenopus oocyte is known to accumulate filamentous or F-actin in the nucleus, but it is currently unknown whether F-actin also accumulates in embryo nuclei. Using fluorescence-labeled actin reporters, we examined the actin distribution in Xenopus embryonic cells and found that F-actin accumulates in nuclei during the blastula stage but not during the gastrula stage. To further investigate nuclear F-actin, we devised a Xenopus egg extract that reproduces the formation of nuclei in which F-actin accumulates. Using this extract, we found that F-actin accumulates primarily at the subnuclear membranous region and is essential to maintain chromatin binding to the nuclear envelope in well-developed nuclei. We also provide evidence that nuclear F-actin increases the structural stability of nuclei and contributes to chromosome alignment on the mitotic spindle at the following M phase. These results suggest the physiological importance of nuclear F-actin accumulation in rapidly dividing large Xenopus blastula cells.

Stilbene analogs of resveratrol improve insulin resistance through activation of AMPK.

Resveratrol (RSV), 3,5,4'-trihydroxy-trans-stilbene, is known to have many beneficial physiological activities. We have synthesized several stilbene analogues and have reported that the hydroxyl group in the 4' position of RSV exhibited strong radical scavenging action. Using stilbene analogs, we investigated the structure of RSV to explain its protective effect against obesity and type 2 diabetes. All six analogs used in this study inhibited the differentiation of 3T3-L1 adipocytes. 3-Hydroxy-trans stilbene (3(OH)ST), and 3,4'-dihydroxy-trans stilbene (3,4'(OH)2ST) increased glucose uptake and induced adenosine monophosphate kinase (AMPK) phosphorylation in C2C12 myotubes independently of insulin. An in vivo study using mice fed high-fat diets indicated that 3(OH)ST was more effective than RSV in improving insulin resistance. In conclusion, RSV and its derivatives, particularly 3(OH)ST, inhibited adipocyte differentiation and enhanced glucose uptake in the myotubes, resulting in a reduction of obesity and an improvement in glucose tolerance in vivo.

Intranuclear DNA density affects chromosome condensation in metazoans.

Chromosome condensation is critical for accurate inheritance of genetic information. The degree of condensation, which is reflected in the size of the condensed chromosomes during mitosis, is not constant. It is differentially regulated in embryonic and somatic cells. In addition to the developmentally programmed regulation of chromosome condensation, there may be adaptive regulation based on spatial parameters such as genomic length or cell size. We propose that chromosome condensation is affected by a spatial parameter called the chromosome amount per nuclear space, or "intranuclear DNA density." Using Caenorhabditis elegans embryos, we show that condensed chromosome sizes vary during early embryogenesis. Of importance, changing DNA content to haploid or polyploid changes the condensed chromosome size, even at the same developmental stage. Condensed chromosome size correlates with interphase nuclear size. Finally, a reduction in nuclear size in a cell-free system from Xenopus laevis eggs resulted in reduced condensed chromosome sizes. These data support the hypothesis that intranuclear DNA density regulates chromosome condensation. This suggests an adaptive mode of chromosome condensation regulation in metazoans.

Kendrin is a novel substrate for separase involved in the licensing of centriole duplication.

The centrosome, consisting of a pair of centrioles surrounded by pericentriolar material, directs the formation of bipolar spindles during mitosis. Aberrant centrosome number can promote chromosome instability, which is implicated in tumorigenesis. Thus, centrosome duplication needs to be tightly regulated to occur only once per cell cycle. Separase, a cysteine protease that triggers sister chromatid separation, is involved in centriole disengagement, which licenses centrosomes for the next round of duplication. However, at least two questions remain unsolved: what is the substrate relevant to the disengagement, and how does separase, activated at anaphase onset, act on the disengagement that occurs during late mitosis. Here, we show that kendrin, also named pericentrin, is cleaved by activated separase at a consensus site in vivo and in vitro, and this leads to the delayed release of kendrin from the centrosome later in mitosis. Furthermore, we demonstrate that expression of a noncleavable kendrin mutant suppresses centriole disengagement and subsequent centriole duplication. Based on these results, we propose that kendrin is a novel and crucial substrate for separase at the centrosome, protecting the engaged centrioles from premature disengagement and thereby blocking reduplication until the cell passes through mitosis.

Thyroid hormone-regulated expression of nuclear lamins correlates with dedifferentiation of intestinal epithelial cells during Xenopus laevis metamorphosis.

In the Xenopus laevis intestine during metamorphosis, which is triggered by thyroid hormone (TH), the adult epithelium develops and replaces the larval one undergoing apoptosis. We have previously shown that progenitor/stem cells of the adult epithelium originate from some differentiated larval epithelial cells. To investigate molecular mechanisms underlying larval epithelial dedifferentiation into the adult progenitor/stem cells, we here focused on nuclear lamin A (LA) and lamin LIII (LIII), whose expression is generally known to be correlated with the state of cell differentiation. We analyzed the spatiotemporal expression of LA and LIII during X. laevis intestinal remodeling by reverse transcription PCR, Western blotting, and immunohistochemistry. At the onset of natural metamorphosis, when the adult epithelial progenitor cells appear as small islets, the expression of LA is down-regulated, but that of LIII is up-regulated only in the islets. Then, as the adult progenitor cells differentiate, the expression of LA is up-regulated, whereas that of LIII is down-regulated in the adult cells. As multiple intestinal folds form, adult epithelial cells positive for LIII become restricted only to the troughs of the folds. In addition, we have shown that TH up- or down-regulates the expression of these lamins in the premetamorphic intestine as during natural metamorphosis. These results indicate that TH-regulated expression of LA and LIII closely correlates with dedifferentiation of the epithelial cells in the X. laevis intestine, suggesting the involvement of the lamins in the process of dedifferentiation during amphibian metamorphosis.

Genetically modified rapeseed oil containing cis-9,trans-11,cis-13-octadecatrienoic acid affects body fat mass and lipid metabolism in mice.

Punicic acid, one of the conjugated linolenic acid (CLN) isomers, exerts a body-fat reducing effect. Although punicic acid is found in pomegranate and Tricosanthes kirilowii seeds, the amount of this fatty acid is very low in nature. The goal of this study was to produce a transgenic oil containing punicic acid. A cDNA encoding conjugase that converts linoleic acid to punicic acid was isolated from T. kirilowii, and the plant expression vector, pKN-TkFac, was generated. The pKN-TkFac was introduced into Brassica napus by Agrobacterium-mediated transformation. As a result, a genetically modified rapeseed oil (GMRO) containing punicic acid was obtained, although its proportion to the total fatty acids was very low (approximately 2.5%). The effects of feeding GMRO in ICR CD-1 male mice were then examined. Wild-type rapeseed (B. napus) oil (RSO) containing no CLN was used as a control oil. For reference oils, RSO-based blended oils were prepared by mixing with different levels of pomegranate oil (PO), either 2.5% (RSO + PO) or 5.0% (RSO + 2PO) punicic acid. Mice were fed purified diets containing 10% of either RSO, RSO + PO, RSO + 2PO, or GMRO for 4 weeks, and dietary PO dose-dependently reduced perirenal adipose tissue weight with a significant difference between the RSO group and the RSO + 2PO group. GMRO, as compared to RSO, lowered the adipose tissue weight to the levels observed with RSO + 2PO. The liver triglyceride level of the RSO + 2PO and GMRO groups but not that of the RSO + PO group was lower than that of the RSO group. The RSO + 2PO and GMRO groups, but not the RSO + PO group, had increased carnitine-palmitoyltransferase activity in the liver and brown adipose tissue. These results showed that dietary GMRO, even at a dietary punicic acid level as low as 0.25 wt % of diet, reduced body fat mass and altered liver lipid metabolism in mice and was more effective than an equal amount of punicic acid from PO.

Oocyte extracts for the study of meiotic M-M transition.

In meiotic cell cycles, meiosis I (MI) is followed by meiosis II (MII) without an intervening S phase, whereas in mitotic cell cycles, an S phase necessarily alternates with an M phase. For the study of mitotic cell cycles, extracts prepared from unfertilized and parthenogenetically activated Xenopus eggs have been very useful as they can perform the progression of mitotic cycles in vitro. To establish a cell-free system to study the regulatory mechanisms of meiotic transition from MI to MII, extracts have been prepared from maturing Xenopus oocytes isolated from ovaries, stimulated with progesterone to induce the resumption of meiosis, and arrested at meiotic metaphase I by cold treatment. In oocyte extracts, the activity of cyclin B-Cdc2 complexes, the M phase inducer, fluctuates in the same manner as it does in maturing oocytes during the MI to MII transition period. By the use of oocyte extracts, it has been found that incomplete inactivation of Cdc2 at the end of MI is required for meiotic M-M transition. The meiotic extract should provide a useful tool to elucidate molecular mechanisms of meiotic M to M transition, including a role of Mos/mitogen-activated protein kinase cascade in the suppression of S phase entry after MI exit. In this chapter, we describe methods for the preparation and the uses of meiotic extracts. As a comparison, we also include a protocol for the preparation of mitotic extracts.

Production of trans-10, cis-12 conjugated linoleic acid in rice.

Conjugated linoleic acid (CLA) has anti-carcinogenic and anti-atherosclerosis activity, and modulatory effects on the immune system and lipid metabolism. To produce a transgenic rice plant that can accumulate CLA, a linoleate isomerase gene that can convert linoleic acid to trans-10, cis-12 CLA was introduced and expressed under the control of seed-specific promoters from the oleosin and globulin genes. The fatty acid composition of the transgenic rice grain was analyzed by gas chromatography. Although there was no clear difference in the fatty acid composition between seeds from transformed versus untransformed plants, a peak of trans-10, cis-12 CLA methyl ester, which was not present in seeds from untransformed plants, was found in transformed plants. The trans-10, cis-12 CLA comprised an average of 1.3% (w/w) of the total fatty acids in seeds carrying the oleosin promoter in comparison to 0.01% (w/w) in seeds carrying the globulin promoter. In addition, approximately 70 and 28% of the total amount of the CLA isomer were present in the triacylglycerol and free fatty acid fractions, respectively. These results demonstrate the ability to produce fatty acid components of vegetable oils with novel physiological activities in crops.

Nucleosome assembly protein-1 is a linker histone chaperone in Xenopus eggs.

In eukaryotic cells, genomic DNA is primarily packaged into nucleosomes through sequential ordered binding of the core and linker histone proteins. The acidic proteins termed histone chaperones are known to bind to core histones to neutralize their positive charges, thereby facilitating their proper deposition onto DNA to assemble the core of nucleosomes. For linker histones, however, little has been known about the regulatory mechanism for deposition of linker histones onto the linker DNA. Here we report that, in Xenopus eggs, the linker histone is associated with the Xenopus homologue of nucleosome assembly protein-1 (NAP-1), which is known to be a chaperone for the core histones H2A and H2B in Drosophila and mammalian cells [Ito, T., Bulger, M., Kobayashi, R. & Kadonaga, J. T. (1996) Mol. Cell Biol. 16, 3112-3124; Chang, L., Loranger, S. S., Mizzen, C., Ernst, S. G., Allis, C. D. & Annunziato, A. T. (1997) Biochemistry 36, 469-480]. We show that NAP-1 acts as the chaperone for the linker histone in both sperm chromatin remodeling into nucleosomes and linker histone binding to nucleosome core dimers. In the presence of NAP-1, the linker histone is properly deposited onto linker DNA at physiological ionic strength, without formation of nonspecific aggregates. These results strongly suggest that NAP-1 functions as a chaperone for the linker histone in Xenopus eggs.

APC/C-Cdc20-mediated degradation of cyclin B participates in CSF arrest in unfertilized Xenopus eggs.

In vertebrates, unfertilized eggs are arrested at meiotic metaphase II (meta-II) by cytostatic factor (CSF), with Cdc2 activity maintained at a constant, high level. CSF is thought to suppress cyclin B degradation through the inhibition of the anaphase-promoting complex/cyclosome (APC/C)-Cdc20 while cyclin B synthesis continues in unfertilized eggs. Thus, it is a mystery how Cdc2 activity is kept constant during CSF arrest. Here, we show that the APC/C-Cdc20 can mediate cyclin B degradation in CSF-arrested Xenopus eggs and extracts, in such a way that when Cdc2 activity is elevated beyond a critical level, APC/C-Cdc20-dependent cyclin B degradation is activated and Cdc2 activity consequently declines to the critical level. This feedback control of Cdc2 activity is shown to be required for keeping Cdc2 activity constant during meta-II arrest. We have also shown that Mos/MAPK pathway is essential for preventing the cyclin B degradation from inactivating Cdc2 below the critical level required to sustain meta-II arrest. Our results indicate that under CSF arrest, Mos/MAPK activity suppresses cyclin B degradation, preventing Cdc2 activity from falling below normal meta-II levels, whereas activation of APC/C-Cdc20-mediated cyclin B degradation at elevated levels of Cdc2 activity prevents Cdc2 activity from reaching excessively high levels.

Delta 12-oleate desaturase-related enzymes associated with formation of conjugated trans-delta 11, cis-delta 13 double bonds.

Conjugated linolenic acids are present as major seed oils in several plant species. Punicic acid (or trichosanic acid) is a conjugated linolenic acid isomer containing cis-delta9, trans-delta11, cis-delta13 double bonds in the C(18) carbon chain. Here we report cDNAs, TkFac and PgFac, isolated from Trichosanthes kirilowii and Punica granatum, that encode a class of conjugases associated with the formation of trans-delta11, cis-delta13 double bonds. Expression of TkFac and PgFac in Arabidopsis seeds under transcriptional control of the seed-specific napin promoter resulted in accumulation of punicic acid up to approximately 10% (w/w) of the total seed oils. In contrast, no punicic acid was found in lipids from leaves even when the conjugases were driven under control of the cauliflower mosaic virus 35S promoter. In yeast cells grown without exogenous fatty acids in the culture medium, TkFac and PgFac expression resulted in punicic acid accumulation accompanied by 16:2delta(9cis, 12cis) and 18:2delta(9cis, 12cis) production. Thus, TkFac and PgFac are defined as bifunctional enzymes having both conjugase and delta12-oleate desaturase activity. Furthermore, we demonstrate that 16:2delta(9cis, 12cis) and 18:3delta(9cis, 12cis, 15cis) as well as 18:2delta(9cis, 12cis) are potential substrates for the conjugases to form trans-delta11 and cis-delta13 double bonds.

Coordinated regulation of M phase exit and S phase entry by the Cdc2 activity level in the early embryonic cell cycle.

In the early embryonic cell cycle, exit from M phase is immediately followed by entry into S phase without an intervening gap phase. To understand the regulatory mechanisms for the cell cycle transition from M to S phase, we examined dependence on Cdc2 inactivation of cell-cycle events occurring during the M-S transition period, using Xenopus egg extracts in which the extent of Cdc2 inactivation at M phase exit was quantitatively controlled. The result demonstrated that MCM binding to and the initiation of DNA replication of nuclear chromatin occurred depending on the decrease of Cdc2 activity to critical levels. Similarly, we found that Cdc2 inhibitory phosphorylation and cyclin B degradation were turned on and off, respectively, depending on the decrease in Cdc2 activity. However, their sensitivity to Cdc2 activity was different, with the turning-on of Cdc2 inhibitory phosphorylation occurring at higher Cdc2 activity levels than the turning-off of cyclin B degradation. This means that, when cyclin B degradation ceases at M phase exit, Cdc2 inhibitory phosphorylation is necessarily activated. In the presence of constitutive synthesis of cyclin B, this condition favors the occurrence of the Cdc2 inactivation period after M phase exit, thereby ensuring progression through S phase. Thus, M phase exit and S phase entry are coordinately regulated by the Cdc2 activity level in the early embryonic cell cycle.