Diversity in the Integrase Coding Domain of a gypsy-like Retrotransposon Among Wild Relatives of Rice in the Oryza officinalis Complex

The Oryza officinalis complex is a genetically diverse, tertiary genepool of rice. We analyzed part of the primary structure of the integrase coding domain (ICD) of a gypsy-like retrotransposon from species of the O. officinalis species complex. PCR was performed with degenerate primers that hybridized to conserved sequences in the integrase genes of gypsy-type retrotransposons, using total DNA from different species of the O. officinalis complex as templates. Cloning and sequencing of the PCR products showed that the amplified fragments are highly homologous to each other (75–90%) and belong to one family of retrotransposons that is related to the previously studied RIRE-2 element from rice. Two main subfamilies of 292 and 351 bp were distinguished. Analysis of primary sequence data supports previous reports that sequence divergence during vertical transmission has been the major influence on the evolution of gypsy-type retrotransposons in Oryza species. Based on sequence data phylogenetic relationships among species of the O. officinalis complex were estimated. The data suggests that O. eichingeri is more closely related to the ancestral species of the complex. This revised version was published online in July 2006 with corrections to the Cover Date.     

Heterogeneity of high density lipoprotein generated by ABCA1 and ABCA7

The assembly of HDL by helical apolipoprotein and cellular lipid was studied using HEK293 cells to which ecdysone-inducible human ABCA1 or human ABCA7 was transfected. Expression of both ABCA1 and ABCA7 was induced linearly proportional to ponasterone A concentration in the medium. In the experimental conditions used, the ABC protein expression levels limited the rate of lipid release when the apolipoprotein concentration was high, and the apolipoprotein concentration was rate-limiting when the ABC protein expression levels were high. When ABCA1 expression increased in conditions in which it was rate-limiting, relative cholesterol content to phospholipid increased in the HDL produced. In contrast, it was constant when ABCA7 expression increased. To investigate the background mechanism, the HDL particles were analyzed by density gradient ultracentrifugation and high performance lipid chromatography. The ABCA1-mediated reaction produced two distinct HDLs, large cholesterol-rich and small cholesterol-poor particles, and the ABCA7-mediated reaction generated mostly small cholesterol-poor particles. The increase of HDL assembly with the increase of ABCA1 expression was predominant in large cholesterol-rich particles, whereas only small cholesterol-poor HDL increased as ABCA7 expression increased. We conclude that ABCA1 generates cholesterol-rich and cholesterol-poor HDL and that the former is more prominently dependent on the increase of ABCA1 expression. ABCA7 produces this HDL subfraction only as a very minor component.     

Impaired fertility in female mice lacking urinary trypsin inhibitor

Urinary trypsin inhibitor (UTI) is a serine proteinase inhibitor that is found in blood and urine. To investigate the physiological functions of UTI in vivo, we generated UTI-deficient mice by gene targeting. The mice showed no obvious abnormalities and appeared healthy. However, the females displayed a severe reduction in fertility. Wild-type embryos developed normally when transplanted into UTI-deficient female mice, suggesting that UTI-deficient females have a normal ability to maintain pregnancy. The number of naturally ovulated oocytes from UTI-deficient mice was greatly reduced compared with that from wild-type mice. Histologically, oocytes with disorganized corona radiata were frequently seen in the ovaries of UTI-deficient mice after hormonal stimulation. When ovaries from UTI-deficient mice were transplanted into wild-type mice, pups derived from the transplanted ovaries were obtained, suggesting that the ovary of UTI-deficient mice functions normally if UTI is supplied from the systemic circulation. These results demonstrate that UTI plays an important role in the formation of the stable cumulus-oocyte complex that is essential for oocyte maturation and ovulation.     

Analysis of the Genetic Diversity in the Vigna minima Complex and Related Species in East Asia

Vigna subgenus Ceratotropis (V. angularis, V. minima, V. nakashimae, V. reflexo-pilosa, V. riukiuensis and V. umbellata) was investigated using AFLP methodology. Species in the V. minima complex, V. minima, V. nakashimae and V. riukiuensis, had greater intra-specific polymorphism than the other species analyzed. Vigna species from East Asia analysed could be clearly distinguished. The relationship between species was consistent using different analyses and showed V. riukiuensis and V. nakashimae are closely related. The phenetic distance between species (1-Jaccard's similarity coefficient) ranged from 0.279 between V. riukiuensis and V. nakashimae to 0.688 between V. reflexo-pilosa and V. minima. Genetic distance based on inferred nucleotide diversity (π) ranged from 0.012 between V. riukiuensis and V. nakashimae and 0.053 between both V. riukiuensis and V. nakashimae and V. reflexo-pilosa. The diversity (Ha) within species, based on Shannon's information index of phenotypic diversity, was lowest in V. reflexo-pilosa (0.006) and highest in V. minima (0.095). Korean and Japanese populations of V. nakashimae had distinct polymorphism. Vigna riukiuensis populations from Miyako island were genetically different from populations of other islands of southern Okinawa. Received 21 April 2000/ Accepted in revised form 5 September 2000     

Development and Evaluation of a Parenting Resilience Elements Questionnaire (PREQ) Measuring Resiliency in Rearing Children with Developmental Disorders

We developed a parenting resilience elements questionnaire (PREQ) measuring the degree to which mothers possess elements that aid in adapting to challenges and difficulties related to children with developmental disorders (DD). A total of 424 parents of children with DD were recruited from five medical institutes. Psychometric properties of PREQ were evaluated using data of 363 mothers of children with DD. Furthermore, multiple regression analysis was performed, predicting depressive symptoms and parenting behavior with PREQ subscales, a general health questionnaire, and the total difficulties score of a strength and difficulties questionnaire. Factor analysis revealed three reliable factors: “knowledge of the child’s characteristics,” “perceived social supports,” and “positive perceptions of parenting.” Moreover, multiple regression analysis showed that “knowledge of the child’s characteristics” was associated with parenting behavior, whereas “perceived social supports” predicted depressive symptoms; “positive perceptions of parenting” influenced both parenting behavior and depressive symptoms. These findings indicated that the PREQ may be used as a scale measuring resiliency in mothers of children with DD and is useful for evaluating their parenting ability in clinical interventions.     

Nucleosides and Nucleotides. 232. Synthesis of 2′-C-Methyl-4′-thiocytidine: Unexpected Anomerization of the 2′-Keto-4′-thionucleoside Precursor

The synthesis of 2′-C-methyl-4′-thiocytidine (16) is described. Since the 2′-keto-4′-thiocytidine derivative 2β unexpectedly isomerized to 2α and the methylation of 2β proceeded predominantly from the less hindered α-face to give 7, the desired product 16 was synthesized via the Pummerer reaction of the sulfoxide 14 and N ⁴ -benzoylcytosine.     

Selective synthesis of 1,6-anhydro-β-d-mannopyranose and -mannofuranose using microwave-assisted heating

The dehydration of d-mannose and the demethanolization of methyl-α-d-mannopyranoside (MαMP) or methyl-α-d-mannofuranoside (MαMF) were examined using microwave-assisted heating for a 3-min irradiation at temperature from 120 to 280 °C in ordinary or dry sulfolane without any catalyst. The microwave-assisted heating of MαMP and MαMF smoothly proceeded to selectively afford the anhydromannoses, 1,6-anhydro-β-d-mannopyranose (AMP) and 1,6-anhydro-β-d-mannofuranose (AMF), respectively, in high yields. For MαMP in ordinary sulfolane at 240 °C, AMP was selectively obtained in the AMF:AMP ratio of 4:96, whereas AMF was the major product at the AMF:AMP ratio of 97:3 from MαMF in dry sulfolane at 220 °C.     

Identification of radiation-induced expression changes in nonimmortalized human T cells

In the event of a radiation accident or attack, it will be imperative to quickly assess the amount of radiation exposure to accurately triage victims for appropriate care. RNA-based radiation dosimetry assays offer the potential to rapidly screen thousands of individuals in an efficient and cost-effective manner. However, prior to the development of these assays, it will be critical to identify those genes that will be most useful to delineate different radiation doses. Using global expression profiling, we examined expression changes in nonimmortalized T cells across a wide range of doses (0.15-12 Gy). Because many radiation responses are highly dependent on time, expression changes were examined at three different times (3, 8, and 24 h). Analyses identified 61, 512 and 1310 genes with significant linear dose-dependent expression changes at 3, 8 and 24 h, respectively. Using a stepwise regression procedure, a model was developed to estimate in vitro radiation exposures using the expression of three genes (CDKN1A, PSRC1 and TNFSF4) and validated in an independent test set with 86% accuracy. These findings suggest that RNA-based expression assays for a small subset of genes can be employed to develop clinical biodosimetry assays to be used in assessments of radiation exposure and toxicity.     

CD81 expression is important for the permissiveness of Huh7 cell clones for heterogeneous hepatitis C virus infection

Huh7 cells constitute a permissive cell line for cell culture of hepatitis C virus (HCV) particles. However, our Huh7 line shows limited permissiveness for HCV. Thus, in this study we set out to determine which host factors are important for conferring permissiveness. To analyze the limited permissiveness of our Huh7 cells, 70 clones were obtained after single-cell cloning of parental Huh7 cells. The cloned Huh7 cells exhibited various levels of HCV pseudoparticles and JFH-1 virus infection efficiency, and some clones were not permissive. A subgenomic replicon was then transfected into the cloned Huh7 cells. While the replication efficiencies differed among the cloned Huh7 cells, these efficiencies did not correlate with infectious permissibility. Flow cytometry showed that CD81, scavenger receptor class B type I, and low-density-lipoprotein receptor expression on the cell surfaces of the Huh7 clones differed among the clones. Interestingly, we found that all of the permissive cell clones expressed CD81 while the nonpermissive cell clones did not. To confirm the importance of CD81 expression for HCV permissiveness, CD81 was then transiently and stably expressed on a nonpermissive Huh7 cell clone, which was consequently restored to HCV infection permissiveness. Furthermore, permissiveness was down-regulated upon transfection of CD81 silencing RNA into a CD81-positive cell clone. In conclusion, CD81 expression is an important determinant of HCV permissiveness of Huh7 cell clones harboring different characteristics.     

Screening of a novel octamer peptide, CNSCWSKD, that induces caspase-dependent cell death

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to induce apoptosis to various tumor cells but not in normal cells. We have screened cell death-inducing peptides from the extracellular domain sequence of TRAIL, using a peptide array. Peptides of higher activity were found through amino acid substitution, and the CNSCWSKD peptide induced >90% cell death in treated Jurkat cells. Features of apoptosis, such as DNA fragmentation, activation of caspase, phosphatidylserine externalization, chromatin condensation, and competition with TRAIL for binding to the death receptor (DR) 4 or DR5 were observed, suggesting that this peptide is a TRAIL mimic. Caspase-3 activation was observed in various tumor cells treated with this peptide as well as with TRAIL, while no activation was observed in human normal fibroblasts. The CNSCWSKD peptide is a potential candidate for use in cancer therapy.