Comparative Analysis of Disease-Linked Single Nucleotide Polymorphic Markers from Brassica rapa for Their Applicability to Brassica oleracea

Numerous studies using single nucleotide polymorphisms (SNPs) have been conducted in humans, and other animals, and in major crops, including rice, soybean, and Chinese cabbage. However, the number of SNP studies in cabbage is limited. In this present study, we evaluated whether 7,645 SNPs previously identified as molecular markers linked to disease resistance in the Brassica rapa genome could be applied to B. oleracea. In a BLAST analysis using the SNP sequences of B. rapa and B. oleracea genomic sequence data registered in the NCBI database, 256 genes for which SNPs had been identified in B. rapa were found in B. oleracea. These genes were classified into three functional groups: molecular function (64 genes), biological process (96 genes), and cellular component (96 genes). A total of 693 SNP markers, including 145 SNP markers [BRH—developed from the B. rapa genome for high-resolution melt (HRM) analysis], 425 SNP markers (BRP—based on the B. rapa genome that could be applied to B. oleracea), and 123 new SNP markers (BRS—derived from BRP and designed for HRM analysis), were investigated for their ability to amplify sequences from cabbage genomic DNA. In total, 425 of the SNP markers (BRP-based on B. rapa genome), selected from 7,645 SNPs, were successfully applied to B. oleracea. Using PCR, 108 of 145 BRH (74.5%), 415 of 425 BRP (97.6%), and 118 of 123 BRS (95.9%) showed amplification, suggesting that it is possible to apply SNP markers developed based on the B. rapa genome to B. oleracea. These results provide valuable information that can be utilized in cabbage genetics and breeding programs using molecular markers derived from other Brassica species.     

64Cu-ATSM Hypoxia Positron Emission Tomography for Detection of Conduit Ischemia in an Experimental Rat Esophagectomy Model

Background

We designed a hypoxia-imaging modality to detect ischemia of the gastric conduit after esophagectomy.

Materials and Methods

A rat esophagectomy model was created using 12-16-week-old, 300-350 g male Sprague-Dawley rats. In the operation group (n=6), partial gastric devascularization was performed by ligating the left gastric artery and the short gastric arteries and an esophagogastric anastomosis was performed. In the control group (n=6), the esophageal-gastric junction was incised and suturing was performed without gastric devascularization. Positron emission tomography (PET) images were taken using a microPET rodent model scanner, 24 h after the initial operation, after injection of 200 μCi 64Cu-diacetyl-bis (N4-methylsemicarbazone) (64Cu-ATSM) and pimonidazole 120 mg/kg. After microPET imaging, autoradiography and immunohistochemistry were performed.

Results

The PET image revealed 64Cu-ATSM uptake at the fundus in the operation group 3 h after 64Cu-ATSM injection. The maximum percentage of the injected dose per gram of tissue was higher in the operation group (0.047±0.015 vs. 0.026±0.006, p=0.021). The fundus/liver ratio was also higher in the operation group (0.541±0.126 vs. 0.278±0.049, p=0.002). Upon autoradiography, 64Cu-ATSM uptake was observed in the fundus in the operation group, and was well-correlated to that observed on the PET image. Upon immunohistochemistry, expression of hypoxia-inducible factor 1a and pimonidazole were significantly increased at the fundus and lesser curvature compared to the greater curvature in the operation group.

Conclusion

Hypoxia PET imaging with 64Cu-ATSM can detect ischemia in a rat esophagectomy model. Further clinical studies are needed to verify whether hypoxia imaging may be useful in humans.     

Anion Transport or Nucleotide Binding by Ucp2 Is Indispensable for Ucp2-Mediated Efferocytosis

Rapid and efficient engulfment of apoptotic cells is an essential property of phagocytes for removal of the large number of apoptotic cells generated in multicellular organisms. To achieve this, phagocytes need to be able to continuously uptake apoptotic cells. It was recently reported that uncoupling protein 2 (Ucp2) promotes engulfment of apoptotic cells by increasing the phagocytic capacity, thereby allowing cells to continuously ingest apoptotic cells. However, the functions of Ucp2, beyond its possible role in dissipating the mitochondrial membrane potential, that contribute to elevation of the phagocytic capacity have not been determined. Here, we report that the anion transfer or nucleotide binding activity of Ucp2, as well as its dissipation of the mitochondrial membrane potential, is necessary for Ucp2-mediated engulfment of apoptotic cells. To study these properties, we generated Ucp2 mutations that affected three different functions of Ucp2, namely, dissipation of the mitochondrial membrane potential, transfer of anions, and binding of purine nucleotides. Mutations of Ucp2 that affected the proton leak did not enhance the engulfment of apoptotic cells. Although anion transfer and nucleotide binding mutations did not affect the mitochondrial membrane potential, they exerted a dominant-negative effect on Ucp2-mediated engulfment. Furthermore, none of our Ucp2 mutations increased the phagocytic capacity. We conclude that dissipation of the proton gradient by Ucp2 is not the only determinant of the phagocytic capacity and that anion transfer or nucleotide binding by Ucp2 is also essential for Ucp2-mediated engulfment of apoptotic cells.     

Exploring the Relationships among Epistemological Beliefs, Nature of Science, and Conceptual Change in the Learning of Evolutionary Theory

We explored the relationship between epistemological beliefs and nature of science in a college biology course. One hundred thirty-three college students participated in the research. Exploratory factor analysis with 29 Nature of Science (NOS) items yielded three aspects of NOS: empirical, tentative, and sociocultural nature of scientific knowledge. Pearson r correlations suggested that students who have immature epistemological beliefs are more likely to also have immature beliefs of nature of science. In addition, students’ epistemological beliefs significantly correlate with their conceptual change but their beliefs about nature of science did not. The research is significant in that it provides empirical evidence explaining the relationship between students’ epistemological beliefs and nature of science as well as the relationships between epistemological beliefs and conceptual change in evolution theory.     

The Arabidopsis RING E3 ubiquitin ligase AtAIRP2 plays combinatory roles with AtAIRP1 in abscisic acid-mediated drought stress responses

The ubiquitin (Ub)-26S proteasome pathway is implicated in various cellular processes in higher plants. AtAIRP1, a C3H2C3-type RING (for Really Interesting New Gene) E3 Ub ligase, is a positive regulator in the Arabidopsis (Arabidopsis thaliana) abscisic acid (ABA)-dependent drought response. Here, the AtAIRP2 (for Arabidopsis ABA-insensitive RING protein 2) gene was identified and characterized. AtAIRP2 encodes a cytosolic C3HC4-type RING E3 Ub ligase whose expression was markedly induced by ABA and dehydration stress. Thus, AtAIRP2 belongs to a different RING subclass than AtAIRP1 with a limited sequence identity. AtAIRP2-overexpressing transgenic (35S:AtAIRP2-sGFP) and atairp2 loss-of-function mutant plants exhibited hypersensitive and hyposensitive phenotypes, respectively, to ABA in terms of seed germination, root growth, and stomatal movement. 35S:AtAIRP2-sGFP plants were highly tolerant to severe drought stress, and atairp2 alleles were more susceptible to water stress than were wild-type plants. Higher levels of drought-induced hydrogen peroxide production were detected in 35S:AtAIRP2-sGFP as compared with atairp2 plants. ABA-inducible drought-related genes were up-regulated in 35S:AtAIRP2-sGFP and down-regulated in atairp2 progeny. The positive effects of AtAIRP2 on ABA-induced stress genes were dependent on SNF1-related protein kinases, key components of the ABA signaling pathway. Therefore, AtAIRP2 is involved in positive regulation of ABA-dependent drought stress responses. To address the functional relationship between AtAIRP1 and AtAIRP2, FLAG-AtAIRP1 and AtAIRP2-sGFP genes were ectopically expressed in atairp2-2 and atairp1 plants, respectively. Constitutive expression of FLAG-AtAIRP1 and AtAIRP2-sGFP in atairp2-2 and atairp1 plants, respectively, reciprocally rescued the loss-of-function ABA-insensitive phenotypes during germination. Additionally, atairp1/35S:AtAIRP2-sGFP and atairp2-2/35S:FLAG-AtAIRP1 complementation lines were more tolerant to dehydration stress relative to atairp1 and atairp2-2 single knockout plants. Overall, these results suggest that AtAIRP2 plays combinatory roles with AtAIRP1 in Arabidopsis ABA-mediated drought stress responses.     

The effect of paternal investment on female fertility intention in South Korea

Life history theory views reproduction as an outcome of resource allocation. The allocation of resources such as parental investments of time, energy and material resources involves trade-offs between number of offspring and timing of reproduction. Within the framework of mammalian parental investment, the outstanding feature of human reproduction is the high level of paternal care. Although empirical evidence suggests that human paternal investment may have evolved as a reproductive strategy to reduce infant and child mortality rates, the effects of actual paternal investment, including allocating time to child care, on female reproductive decisions have received relatively little attention. We examined the trade-off from two perspectives using a representative sample of married South Korean women aged 20–44 in 2005 (n=977). First, paternal investment in domestic labor, including child care and housework, was expected to be associated with women's preference regarding future reproduction. Second, relative paternal investment was expected to increase women's preference for future reproduction, especially among employed women. We found that increased paternal investment in child care and housework remarkably enhanced women's intention to have a second child, especially among employed women. In addition, although family members provide a low percentage of child care in South Korea, such help is likely to be a useful resource for second childbirth among employed women. Somewhat expectedly, older age and longer time since first birth had negative effects on women's second-child intention. There is growing evidence that, in the lowest fertility societies, paternal investment may be an essential resource for promoting future reproductive behavior of women, especially employed women.