HuR Regulates Alternative Splicing of the TRA2β Gene in Human Colon Cancer Cells under Oxidative Stress

Hu antigen R (HuR) regulates stress responses through stabilizing and/or facilitating the translation of target mRNAs. The human TRA2β gene encodes splicing factor transformer 2β (Tra2β) and generates 5 mRNA isoforms (TRA2β1 to -5) through alternative splicing. Exposure of HCT116 colon cancer cells to sodium arsenite stimulated checkpoint kinase 2 (Chk2)- and mitogen-activated protein kinase p38 (p38^MAPK)-mediated phosphorylation of HuR at positions S88 and T118. This induced an association between HuR and the 39-nucleotide (nt) proximal region of TRA2β exon 2, generating a TRA2β4 mRNA that includes exon 2, which has multiple premature stop codons. HuR knockdown or Chk2/p38^MAPK double knockdown inhibited the arsenite-stimulated production of TRA2β4 and increased Tra2β protein, facilitating Tra2β-dependent inclusion of exons in target pre-mRNAs. The effects of HuR knockdown or Chk2/p38^MAPK double knockdown were also confirmed using a TRA2β minigene spanning exons 1 to 4, and the effects disappeared when the 39-nt region was deleted from the minigene. In endogenous HuR knockdown cells, the overexpression of a HuR mutant that could not be phosphorylated (with changes of serine to alanine at position 88 [S88A], S100A, and T118A) blocked the associated TRA2β4 interaction and TRA2β4 generation, while the overexpression of a phosphomimetic HuR (with mutations S88D, S100D, and T118D) restored the TRA2β4-related activities. Our findings revealed the potential role of nuclear HuR in the regulation of alternative splicing programs under oxidative stress.     

Long-Distance Dispersal by Sea-Drifted Seeds Has Maintained the Global Distribution of Ipomoea pes-caprae subsp. brasiliensis (Convolvulaceae)

Ipomoea pes-caprae (Convolvulaceae), a pantropical plant with sea-drifted seeds, is found globally in the littoral areas of tropical and subtropical regions. Unusual long-distance seed dispersal has been believed to be responsible for its extraordinarily wide distribution; however, the actual level of inter-population migration has never been studied. To clarify the level of migration among populations of I. pes-caprae across its range, we investigated nucleotide sequence variations by using seven low-copy nuclear markers and 272 samples collected from 34 populations that cover the range of the species. We applied coalescent-based approaches using Bayesian and maximum likelihood methods to assess migration rates, direction of migration, and genetic diversity among five regional populations. Our results showed a high number of migrants among the regional populations of I. pes-caprae subsp. brasiliensis, which suggests that migration among distant populations was maintained by long-distance seed dispersal across its global range. These results also provide strong evidence for recent trans-oceanic seed dispersal by ocean currents in all three oceanic regions. We also found migration crossing the American continents. Although this is an apparent land barrier for sea-dispersal, migration between populations of the East Pacific and West Atlantic regions was high, perhaps because of trans-isthmus migration via pollen dispersal. Therefore, the migration and gene flow among populations across the vast range of I. pes-caprae is maintained not only by seed dispersal by sea-drifted seeds, but also by pollen flow over the American continents. On the other hand, populations of subsp. pes-caprae that are restricted to only the northern part of the Indian Ocean region were highly differentiated from subsp. brasiliensis. Cryptic barriers that prevented migration by sea dispersal between the ranges of the two subspecies and/or historical differentiation that caused local adaptation to different environmental factors in each region could explain the genetic differentiation between the subspecies.     

Overexpression of DnaK from a halotolerant cyanobacterium Aphanothece halophytica enhances growth rate as well as abiotic stress tolerance of poplar plants

The DnaK/Hsp70 family is a molecular chaperone that binds non-native states of other proteins, and concerns to various physiological processes in the bacterial, plant and animal cells. Previously, we showed that overexpression of DnaK from a halotolerant cyanobacterium Aphanothece halophytica (ApDnaK) enhances tolerance to abiotic stresses such as high salinity and high temperature in tobacco plants. Here, we tested the transformation of poplar (Populus alba) with ApDnaK for enhancing the growth of transformed poplar plants. Under control growth conditions, transgenic poplar plants exhibited similar growth rates with the wild-type plants during young seedlings under low light intensity, whereas they showed faster growth, larger plant size, and higher cellulose contents when poplar plants were grown under high light intensity. Transgenic young poplar plants exhibited more rapid recovery from the stresses of high salinity, drought, and low temperature compared with those of the wild type plants when poplar plants were grown under low light intensity. These results suggest that ApDnaK could be useful to enhance the growth rate as well as to increase the stress tolerance.     

Isolation and characterization of microsatellite loci in the red mangrove Rhizophora mangle (Rhizophoraceae) and its related species

Fourteen microsatellite markers were isolated from the red mangrove Rhizophora mangle (Rhizophoraceae), a widely distributed mangrove plant in the New World and West Africa. The range of expected heterozygosity of these markers was 0.000–0.672 in the two populations of R. mangle. Cross-species testing was examined for five other species of Rhizophora, and Kandelia candel and Bruguiera gymnorrhiza. All 14 markers could be amplified in R. samoensis, thirteen in R. racemosa, and six markers in all other species of Rhizophora. Our findings greatly increase the utility of these markers.     

Rapid evolution of a divergent ecogeographic cline in introduced lady beetles

The evolution of ecogeographic clines in recently introduced species is often rapid and predictable, as introduced populations often converge on the same or similar clines found among native populations. In contrast, we demonstrate the rapid evolution of a divergent ecogeographic cline among introduced populations of the seven-spotted lady beetle, Coccinella septempunctata. In the native range of Eurasia, this species follows Gloger’s rule—a positive correlation between melanin and precipitation. However, over several decades, introduced populations in North America have developed a divergent ecogeographic cline, Bogert’s rule, in which melanin is negatively correlated with temperature. Our common garden experiment indicates that this new pattern is not simply a plastic response to temperature, but likely reflects evolutionary change in North America. Furthermore, this new cline is not correlated with colonization history or sample collection time, but is convergent on a pattern found in other species of insects including other lady beetles. The rate of melanin evolution in North America was rapid (6897–10,000 darwins and 0.051–0.077 haldanes) and among the highest rates found in introduced insects. We propose that high levels of genetic diversity from human mediated admixture combined with altered selection pressures may have contributed to the rapid evolution of this unpredicted ecogeographic cline.     

A theoretical study on the optical absorption of green fluorescent protein chromophore in solutions

Ethyl 4-(4-hydroxyphenyl) methylidene- 2-methyl-5-oxoimidazolacetate (HBMIA) is a model chromophore of green fluorescent protein. The electronic structure of HBMIA in aqueous solution phase is studied using a hybrid method of quantum chemistry and statistical mechanics, RISM-SCF-SEDD. The solvatochromic shift is correctly reproduced by the present computations.     

Clinical study on increased serum thyroxine-binding globulin in cancerous state

Serum thyroxine-binding globulin (TBG) in 169 patients with various cancers was determined by radioimmunoassay (RIA). Eleven patients showed a high serum TBG level (greater than 35 micrograms/ml). Two of them had been treated with estrogen for prostate cancer. One patient had high serum TBG with serum hepatitis. Another 8 cases had normal liver function and also normal levels serum estrogen. Thus, about 4.7% (8/169) of the cancer patients had high serum TBG and mild hyperthyroxinemia caused by unknown mechanisms. The high TBG level in these patients continued until just before death, or in some cases decreased to normal after removal of cancer tumors by operation. Cancer is occasionally associated with an increase in serum TBG. Although the mechanism is not clear, the increased TBG in the cancerous state in interesting and has significance as a tumor marker.     

The Native Form and Maturation Process of Hepatitis C Virus Core Protein

The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles.     

Alterations in the Biosynthesis of Lignin in Transgenic Plants with Chimeric Genes for 4-Coumarate: Coenzyme A Ligase

The introduction of chimeric sense and antisense gene constructsfor 4-coumarate:coenzyme A ligase into tobacco plants causedthe reduction of the 4CL activity in the transgenic plants.In the transgenic plants, the cell walls of the xylem tissuein stems were brown and the molecular structure of lignin inthe colored cell walls was dramatically different from thatin the control plants. Analysis with different types of stainrevealed that levels of cinnamyl aldehyde residues and syringylunits in lignin were depressed in the brownish cell walls. Furthermore,the lignin content in colored tissue was lower than that inthe normal tissue. Our results indicate that 4CL has importantroles in the determination of the composition and the amountof lignin in tobacco plants. (Received December 27, 1995; Accepted July 23, 1996)     

Oxygen exchange reaction during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments

The oxygen exchange during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments was studied from the distribution of the [18O]Pi species produced by the hydrolysis of [gamma-18O]ATP. The products were mixtures of two species, one with a low extent of oxygen exchange and the other with a high extent. The low and high extents of oxygen exchange in these two Pi species were the same as those of the acto-S-1 ATPase reaction through the routes with and without the dissociation of actomyosin, respectively (Yasui, M., Ohe, M., Kajita, A., Arata, T., & Inoue, A. [1988] J. Biochem. 104, 550-559). During isometric contraction of glycerinated muscle fibers at 20 degrees C, the fraction of ATP hydrolysis with low extent of oxygen exchange was 0.83 and 0.70, respectively, in 0 and 120 mM KCl. In myofibrils, the fraction of ATP hydrolysis with a low extent of oxygen exchange was 0.72-0.88 in 0-120 mM KCl at 20 degrees C. Therefore, in glycerinated muscle fibers and myofibrils ATP seems to be mainly hydrolyzed through a route without the dissociation of actomyosin, especially at low ionic strength and at room temperature when the tension development is high. ATP hydrolysis through this route may be coupled with muscle contraction.