Integration of Linkage and Chromosome Maps of Red Clover ( Trifolium pratense L.)

Red clover (Trifolium pratense L.) is a forage legume and an allogamous diploid plant (2n = 14; 440 Mb). Here, we examine the 7 prometaphase chromosomes of red clover using fluorescence in situ hybridization (FISH) with ribosomal RNA sequences, pericentromeric and telomeric repeats, as well as bacterial artificial chromosome (BAC) clones. Position of hybridization signals and chromosome condensation patterns were quantified by the help of the chromosome image analysis system ver. 4.0 (CHIAS IV). Fourteen BAC clones belonging to linkage groups (LG) 1-7 hybridized to individual chromosomes 4, 2, 6, 5, 1, 7, and 3, respectively. Quantitative analysis of FISH mapping and chromosome analysis using CHIAS IV allowed us to construct a quantitative idiogram that constitutes the comprehensive chromosome map of red clover. Chromosomal positions of the 26S rDNA locus were detected at a heterozygous locus on chromosome 6 in the variety HR, and polymorphisms of rDNA loci were observed in other varieties, although chromosomal positions of some BAC clones did not vary among HR and other varieties. These results demonstrate chromosomal collinearity among allogamous red clover varieties. This integration of genetic linkage and quantitative chromosome maps should provide valuable insight into allogamous legume genetics.     

New systematic assignments in Gonyleptoidea (Arachnida, Opiliones, Laniatores)

As part of an ongoing revision of the family Gonyleptidae, we have identified many species that are synonyms of previously described species or misplaced in this family. This article summarizes these findings, adding previously unavailable information or correcting imprecise observations to justify the presented taxonomic changes. The following new familial or subfamilial assignments are proposed: Nemastygnus Roewer, 1929 and Taulisa Roewer, 1956 are transferred to Agoristenidae, Agoristeninae; Napostygnus Roewer, 1929 to Cranaidae; Ceropachylinus peruvianus Roewer, 1956 and Pirunipygus Roewer, 1936 are transferred to Gonyleptidae, Ampycinae; Gyndesops Roewer, 1943, Haversia Roewer, 1913 and Oxapampeus Roewer, 1963 are transferred to Gonyleptidae, Pachylinae. The following generic synonymies are proposed for the family Gonyleptidae: Acanthogonyleptes Mello-Leitão, 1922 = Centroleptes Roewer, 1943; Acrographinotus Roewer, 1929 = Unduavius Roewer, 1929; Gonyleptes Kirby, 1819 = Collonychium Bertkau, 1880; Mischonyx Bertkau, 1880 = Eugonyleptes Roewer, 1913 and Gonazula Roewer, 1930; Parampheres Roewer, 1913 = Metapachyloides Roewer, 1917; Pseudopucrolia Roewer, 1912 = Meteusarcus Roewer, 1913; Haversia Roewer, 1913 = Hoggellula Roewer, 1930. The following specific synonymies are proposed for the family Gonyleptidae: Acanthogonyleptes singularis (Mello-Leitão, 1935) = Centroleptes flavus Roewer, 1943, syn. n.; Geraeocormobius sylvarum Holmberg, 1887 = Discocyrtus serrifemur Roewer, 1943, syn. n.; Gonyleptellus bimaculatus (Sørensen, 1884) = Gonyleptes cancellatus Roewer,1917, syn. n.; Gonyleptes atrus Mello-Leitão, 1923 = Weyhia brieni Giltay, 1928, syn. n.; Gonyleptes fragilis Mello-Leitão, 1923 = Gonyleptes banana Kury, 2003, syn. n.; Gonyleptes horridus Kirby, 1819 = Collonychium bicuspidatum Bertkau, 1880, syn. n., Gonyleptes borgmeyeri Mello-Leitão, 1932, syn. n., Gonyleptes curvicornis Mello-Leitão, 1932, syn. n., Metagonyleptes hamatus Roewer, 1913, syn. n. and Paragonyleptes simoni Roewer, 1930, syn. n.; Gonyleptes pustulatus Sørensen, 1884 = Gonyleptes guttatus Roewer, 1917, syn. n.; Haversia defensa (Butler, 1876) = Sadocus vallentini Hogg, 1913, syn. n.; Liogonyleptoides minensis (Piza, 1946) = Currala bahiensis Soares, 1972, syn. n.; Megapachylus grandis Roewer, 1913 = Metapachyloides almeidai Soares & Soares, 1946, syn. n.; Mischonyx cuspidatus (Roewer, 1913) = Gonazula gibbosa Roewer, 1930 syn. n.; Mischonyx scaber (Kirby, 1819) = Xundarava holacantha Mello-Leitão, 1927, syn. n.; Parampheres tibialis Roewer, 1917 = Metapachyloides rugosus Roewer, 1917, syn. n.; Parapachyloides uncinatus (Sørensen, 1879) = Goyazella armata Mello-Leitão, 1931, syn. n.; Pseudopucrolia mutica (Perty, 1833) = Meteusarcus armatus Roewer, 1913, syn. n. The following new combinations are proposed: Acrographinotus ornatus (Roewer, 1929), comb. n. (ex Unduavius); Gonyleptellus bimaculatus (Sørensen, 1884),comb. n. (ex Gonyleptes);Gonyleptes perlatus (Mello-Leitão, 1935), comb. n. (exMoojenia);Mischonyx scaber (Kirby, 1819), comb. n. (ex Gonyleptes); and Neopachyloides peruvianus (Roewer, 1956), comb. n. (ex Ceropachylus). The following species of Gonyleptidae, Gonyleptinae are revalidated: Gonyleptes atrus Mello-Leitão, 1923 and Gonyleptes curvicornis (Roewer, 1913).     

Genetic and ecophysiological diversity of Cladophora (Cladophorales,Ulvophyceae) in various salinity regimes

Although Cladophora species frequently appear in brackish environments, their genetic diversity, phenological patterns and physiological properties have not been well investigated in these environments. Cladophora is distributed throughout Mikata‐goko, an area consisting of five coastal lakes that are directly or indirectly connected to the sea, resulting in a salinity gradient ranging from fully marine to freshwater. To elucidate genetic and ecological variation in Cladophora, we monthly compared ribosomal internal transcribed spacer sequences of Cladophora specimens across six study sites characterized by different salinity regimes for a year. A total of 12 ribotypes were detected and assigned to six species, four of which were restricted to the marine habitat. Ribotype I of C. vagabunda (L.) Hoek was distributed in high‐salinity brackish waters (mean salinity ≤13 psu; maximum salinity ≤31 psu), whereas C. vagabunda ribotype II and C. glomerata were abundant in low‐salinity brackish waters (mean salinity ≤5 psu; maximum salinity ≤10 psu). Although Cladophora albida (Nees) Kütz. and C. glomerata (L.) Kütz. were collected during all four seasons, C. laetevirens (Dillwyn) Kütz., C. oligocladoidea Hoek and Chihara, C. opaca Sakai and C. vagabunda displayed marked seasonal variation. Culture experiments revealed that optimal salinity conditions for vegetative growth vary among ribotypes collected from different salinity regimes, suggesting that Cladophora distributions are controlled by ribotype‐specific ecophysiological adaptations. In contrast, temperature optima and tolerance were similar among ribotypes showing different seasonalities, and thus their phenologies may be controlled by other environmental factors or biotic conditions, such as reproductive maturity and spore germination.     

Unique distribution of epiphytic Neosiphonia harveyi (Rhodomelaceae,Rhodophyta) along sargassacean hosts

Although epiphytism is generally heaviest on older portions of the host thallus and both variation and abundance of epiphytes decrease with proximity to meristematic apices, Neosiphonia harveyi (Bailey) Kim, Choi, Guiry and Saunders was frequently found on upper parts of sargassacean hosts. This study compared density, thallus length and fertility of epiphytic N. harveyi among different regions of Sargassum patens C. Agardh thalli to reveal a unique distribution pattern of this epiphyte. The majority of epiphytic N. harveyi was observed on the upper part of host thalli and was seldom seen near the basal part. Over a 3‐month period, mean epiphyte density was greater on host apices while thallus length and fertility were greater on subapical portions of host thalli, suggesting the colonization by this epiphyte near the apical part of the host. Distribution patterns of this epiphyte were similar among S. patens growing at different depths. Apical portions of S. patens appear to be more suitable substrata for N. harveyi settlement and colonization compared with other portions of the host thallus, regardless of depth.     

9,10-Phenanthrenequinone promotes secretion of pulmonary aldo-keto reductases with surfactant

9,10-Phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust particles, induces apoptosis via the generation of reactive oxygen species (ROS) because of 9,10-PQ redox cycling. We have found that intratracheal infusion of 9,10-PQ facilitates the secretion of surfactant into rat alveolus. In the cultured rat lung, treatment with 9,10-PQ results in an increase in a lower-density surfactant by ROS generation through redox cycling of the quinone. The surfactant contains aldo-keto reductase (AKR) 1C15, which reduces 9,10-PQ and the enzyme level in the surfactant increases on treatment with 9,10-PQ suggesting an involvement of AKR1C15 in the redox cycling of the quinone. In six human cell types (A549, MKN45, Caco2, Hela, Molt4 and U937) only type II epithelial A549 cells secrete three human AKR1C subfamily members (AKR1C1, AKR1C2 and AKR1C3) with the surfactant into the medium; this secretion is highly increased by 9,10-PQ treatment. Using in vitro enzyme inhibition analysis, we have identified AKR1C3 as the most abundantly secreted AKR1C member. The AKR1C enzymes in the medium efficiently reduce 9,10-PQ and initiate its redox cycling accompanied by ROS production. The exposure of A549 cells to 9,10-PQ provokes viability loss, which is significantly protected by the addition of the AKR1C3 inhibitor and antioxidant enzyme and by the removal of the surfactants from the culture medium. Thus, the AKR1C enzymes secreted in pulmonary surfactants probably participate in the toxic mechanism triggered by 9,10-PQ.     

Sex hormones induce a gender-related difference in renal expression of a novel prostaglandin transporter, OAT-PG, influencing basal PGE2 concentration

Based on the nucleotide sequence of a mouse prostaglandin-specific transporter (mOAT-PG), we identified a rat homolog (rOAT-PG) which shares 80% identity with mOAT-PG in a deduced amino acid sequence. rOAT-PG transports PGE(2) and colocalizes with 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a metabolic enzyme for PGs, in proximal tubules, suggesting that rOAT-PG is involved in PGE(2) clearance to regulate its physiological function in the renal cortex. We found that the expression level of rOAT-PG in the renal cortex was much higher in male rats than in female rats whereas there was no gender difference in the expression level of cyclooxygenase-2, a key enzyme producing PGE(2), and 15-PGDH in the renal cortex. Tissue PGE(2) concentration in the renal cortex was lower in male rats than in female rats, suggesting that renocortical PGE(2) concentration is primarily determined by the expression level of OAT-PG, which is regulated differently between male and female rats. Castration of male rat led to a remarkable reduction in OAT-PG expression and a significant increase in renocortical PGE(2) concentration. These alterations were recovered by testosterone supplementation. These results suggest that OAT-PG is involved in local PGE(2) clearance in the renal cortex. Although the physiological importance of the gender difference in local PGE(2) clearance is still unclear, these findings might be a key to clarifying the physiological roles of PGE(2) in the kidney.     

Bone morphogenetic protein 4 promotes vascular smooth muscle contractility by activating microRNA-21 (miR-21), which down-regulates expression of family of dedicator of cytokinesis (DOCK) proteins

The bone morphogenetic protein 4 (BMP4) signaling pathway plays a critical role in the promotion and maintenance of the contractile phenotype in vascular smooth muscle cell (vSMC). Misexpression or inactivating mutations of the BMP receptor gene can lead to dedifferentiation of vSMC characterized by increased migration and proliferation that is linked to vascular proliferative disorders. Previously we demonstrated that vSMCs increase microRNA-21 (miR-21) biogenesis upon BMP4 treatment, which induces contractile gene expression by targeting programmed cell death 4 (PDCD4). To identify novel targets of miR-21 that are critical for induction of the contractile phenotype by BMP4, biotinylated miR-21 was expressed in vSMCs followed by an affinity purification of mRNAs associated with miR-21. Nearly all members of the dedicator of cytokinesis (DOCK) 180-related protein superfamily were identified as targets of miR-21. Down-regulation of DOCK4, -5, and -7 by miR-21 inhibited cell migration and promoted cytoskeletal organization by modulating an activity of small GTPase. Thus, this study uncovers a regulatory mechanism of the vSMC phenotype by the BMP4-miR-21 axis through DOCK family proteins.     

Spatial arrangement of rhodopsin in retinal rod outer segment membranes studied by spin-labeling and pulsed electron double resonance

We have determined the spatial arrangement of rhodopsin in the retinal rod outer segment (ROS) membrane by measuring the distances between rhodopsin molecules in which native cysteines were spin-labeled at ～1.0mol/mol rhodopsin. The echo modulation decay of pulsed electron double resonance (PELDOR) from spin-labeled ROS curved slightly with strong background decay. This indicated that the rhodopsin was densely packed in the retina and that the rhodopsin molecules were not aligned well. The curve was simulated by a model in which rhodopsin is distributed randomly as monomers in a planar membrane.     

High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization

In vitro-matured bovine oocytes do not tolerate vitrification as well as mature murine or human oocytes. Delayed first cleavage in vitrified and in vitro-fertilized bovine oocytes may be responsible for the decreased yield of blastocysts in vitro. Because formation of sperm-aster and the subsequent assembly of microtubule network play an important role for migration and fusion of both pronuclei, aster formation in vitrified-warmed oocytes was analyzed by confocal laser-scanning microscopy. At 10 h post-insemination (hpi), proportions of oocytes fertilized normally were comparable between the vitrified and fresh control groups (67 and 70%, respectively). Proportions of oocytes that exhibited microtubule assembly were similar between the two groups (95% each), but the proportion of oocytes with multiple asters was higher in the vitrified group when compared with the fresh control group (68 vs 29%, P < 0.05). Both migration and development of two pronuclei were adversely affected by multiple aster formation. In the next experiment, multiple asters observed in 5.5 vs 8 hpi pronuclear zygotes were located near the male pronucleus, suggesting that those multiple asters were not the cytoplasmic asters of maternal origin. In conclusion, multiple aster formation frequently observed in vitrified-warmed bovine oocytes may be related to loss of ooplasmic function responsible for normal microtubule assembly from the sperm-aster.     

The juvenile myoclonic epilepsy-related protein EFHC1 interacts with the redox-sensitive TRPM2 channel linked to cell death

The transient receptor potential M2 channel (TRPM2) is the Ca(2+)-permeable cation channel controlled by cellular redox status via β-NAD(+) and ADP-ribose (ADPR). TRPM2 activity has been reported to underlie susceptibility to cell death and biological processes such as inflammatory cell migration and insulin secretion. However, little is known about the intracellular mechanisms that regulate oxidative stress-induced cell death via TRPM2. We report here a molecular and functional interaction between the TRPM2 channel and EF-hand motif-containing protein EFHC1, whose mutation causes juvenile myoclonic epilepsy (JME) via mechanisms including neuronal apoptosis. In situ hybridization analysis demonstrates TRPM2 and EFHC1 are coexpressed in hippocampal neurons and ventricle cells, while immunoprecipitation analysis demonstrates physical interaction of the N- and C-terminal cytoplasmic regions of TRPM2 with the EFHC1 protein. Coexpression of EFHC1 significantly potentiates hydrogen peroxide (H(2)O(2))- and ADPR-induced Ca(2+) responses and cationic currents via recombinant TRPM2 in HEK293 cells. Furthermore, EFHC1 enhances TRPM2-conferred susceptibility of HEK293 cells to H(2)O(2)-induced cell death, which is reversed by JME mutations. These results reveal a positive regulatory action of EFHC1 on TRPM2 activity, suggesting that TRPM2 contributes to the expression of JME phenotypes by mediating disruptive effects of JME mutations of EFHC1 on biological processes including cell death.