Repeated copulations benefit of the female inLethocerus deyrollei vuillefroy (Heteroptera: Belostomatidae)

TheLethocerus deyrollei male copulates repeatedly with the same female before and between ovipositions. Females stopped oviposition and waited for the next copulation when males were experimentally removed. This result suggests that females need to copulate before each oviposition bout. Since eggs fail to hatch without care of males, females have to detain males until the end of sequential ovipositions. Males assure their paternity with repeated copulations, and females can thus detain males.     

Restoration of Mismatch Repair Functions in Human Cell Line Nalm-6, Which Has High Efficiency for Gene Targeting

Gene targeting is a powerful approach in reverse genetics. The approach has been hampered in most of human cell lines, however, by the poor targeting efficiency. Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, exhibits exceptionally high gene targeting efficiency and is used in DNA repair and the related research fields. Nonetheless, usage of the cell line is still limited partly because it lacks expression of MSH2, a component of mismatch repair complex, which leads to increased genome instability. Here, we report successful restoration of MSH2 expression in Nalm-6 cells and demonstrate that the recovery does not affect the high targeting efficiency. We recovered the expression by introduction of cDNA sequences corresponding to exons 9 to 16 at downstream of exon 8 of the MSH2 gene. Endogenous exons 9 to 16 were deleted in the cell line. The MSH2 expression substantially reduced spontaneous HPRT mutation frequency. Moreover, gene targeting efficiency in the MSH2-expressing cells was similar to that in the MSH2-lacking cells. In fact, we generated heterozygously REV3L knockout and the catalytically dead mutants in the MSH2-proficient Nalm-6 cells with efficiency of 20–30%. The established cell line, Nalm-6-MSH+, is useful for reverse genetics in human cells.     

Transferrin receptor-1 suppresses neurite outgrowth in neuroblastoma Neuro2A cells

Transferrin receptor-1 (TfR1) is a cell membrane-associated glycoprotein responsible for incorporation of the iron bound to transferrin through an endocytotic process from the circulating blood. Iron is believed to play a dual role as an active center of the electron transfer system in mitochondria and as an endogenous cytotoxin through promoted generation of reactive oxygen species in different eukaryotic cells. In this study, we evaluated expression profiles of different genes related to iron mobilization across plasma membranes in neuronal cells. Marked mRNA expression was seen for various iron-related genes such as TfR1 in cultured mouse neocortical neurons, while TfR1 mRNA levels were more than doubled during culture from 3 to 6days. In mouse embryonal carcinoma P19 cells endowed to differentiate into neuronal and astroglial lineages, a transient increase was seen in both mRNA and corresponding protein for TfR1 in association with neuronal marker expression during culture with all-trans retinoic acid (ATRA). In neuronal Neuro2A cells cultured with ATRA, moreover, neurite was elongated together with increased expression of both mRNA and protein for TfR1. Overexpression of TfR1 significantly decreased the length of neurite elongated, however, while significant promotion was invariably seen in the neurite elongation in Neuro2A cells transfected with TfR1 siRNA as well as in Neuro2A cells cultured with an iron chelator. These results suggest that TfR1 would be highly expressed by neurons rather than astroglia to play a negative role in the neurite outgrowth after the incorporation of circulating transferrin in the brain.     

Probing dynamics and conformational change of the GroEL-GroES complex by 13C NMR spectroscopy

Bacterial chaperonin GroEL with a molecular mass of 800 kDa was studied by (13)C NMR spectroscopy. Carbonyl carbons of GroEL were labeled with (13)C in an amino acid specific manner in order to reduce the number of signals to be observed in the spectrum. Combination of selective labeling and site-directed mutagenesis enabled us to establish the sequence specific assignment of the (13)C resonances from GroEL. ADP-binding induced a chemical shift change of Tyr478 in the equatorial domain and His401 in the intermediate domain, but little of Tyr203 in the apical domain. Upon complex formation with co-chaperonin GroES in the presence of ADP, Tyr478 exhibits two peaks that would originate from the cis and trans rings of the asymmetric GroEL-GroES complex. Comparison between the line width of the GroEL resonances and those from GroES in complex with GroEL revealed broadening disproportionate to the size of GroEL, implying the existence of conformational fluctuations which may be pertinent to the chaperone activity. Based on these results, we concluded that (13)C NMR observation in combination with selective labeling and site-directed mutagenesis can be utilized for probing the conformational change and dynamics of the extremely large molecules that are inaccessible with current NMR methods.     

Topoisomerase IIα inhibition following DNA transfection greatly enhances random integration in a human pre-B lymphocyte cell line

DNA transfection can be too inefficient to establish a desired number of stable transfectants, particularly in lymphocytes; however, this could be circumvented by increasing the absolute frequency of random integration. In this paper, we show that treating cells with topoisomerase II inhibitor following electroporation greatly (∼10- to 20-fold) enhances random integration of input DNA in a human pre-B lymphocyte cell line, Nalm-6. With the use of various kinds of topoisomerase II-targeting agents, we also present evidence that topoisomerase IIα inhibition is critical for the enhancement of random integration, while the contribution of topoisomerase IIβ may be negligible. As topoisomerase IIα is highly expressed in vigorously growing cells, our results show that topoisomerase IIα inhibition provides a promising way of enhancing random integration in virtually all cultured cell lines.     

Isolated tumor endothelial cells maintain specific character during long-term culture

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells.In this study, we have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research.     

Chromosomal Manipulation by Site-Specific Recombinases and Fluorescent Protein-Based Vectors

Feasibility of chromosomal manipulation in mammalian cells was first reported 15 years ago. Although this technique is useful for precise understanding of gene regulation in the chromosomal context, a limited number of laboratories have used it in actual practice because of associated technical difficulties. To overcome the practical hurdles, we developed a Cre-mediated chromosomal recombination system using fluorescent proteins and various site-specific recombinases. These techniques enabled quick construction of targeting vectors, easy identification of chromosome-rearranged cells, and rearrangement leaving minimum artificial elements at junctions. Applying this system to a human cell line, we successfully recapitulated two types of pathogenic chromosomal translocations in human diseases: MYC/IgH and BCR/ABL1. By inducing recombination between two loxP sites targeted into the same chromosome, we could mark cells harboring deletion or duplication of the inter-loxP segments with different colors of fluorescence. In addition, we demonstrated that the intrachromosomal recombination frequency is inversely proportional to the distance between two recombination sites, implicating a future application of this frequency as a proximity sensor. Our method of chromosomal manipulation can be employed for particular cell types in which gene targeting is possible (e.g. embryonic stem cells). Experimental use of this system would open up new horizons in genome biology, including the establishment of cellular and animal models of diseases caused by translocations and copy-number variations.     

Growth-related changes in the mechanical properties of collagen fascicles from rabbit patellar tendons

Growth-related changes in the mechanical properties of collagen fascicles (approximately 300 microm in diameter) were studied using patellar tendons obtained from skeletally immature 1 and 2 months old and matured 6 months old rabbits. Tensile properties were determined using a specially designed micro-tensile tester. In each age group, there were no significant differences in the properties among cross-sectional locations in the tendon. Tangent modulus and tensile strength significantly increased with age; the rates of their increases between 1 and 2 months were higher than those between 2 and 6 months. The tangent modulus and tensile strength were positively correlated with the body weight of animals. However, growth-related changes in the mechanical properties were different between collagen fascicles and bulk patellar tendons, which may be attributable to such non-collagenous components as ground substances and also to mechanical interactions between collagen fascicles.     

Structure and serological characterization of 5,7-diamino-3,5,7,9-tetradeoxy-non-2-ulosonic acid isolated from lipopolysaccharides of Vibrio parahaemolyticus O2 and O-untypable strain KX-V212

Lipopolysaccharides (LPS) of Vibrio parahaemolyticus O2 and O-untypable (OUT) strain (KX-V212) isolated from an individual patient were shown to contain 5,7-diamino-3,5,7,9-tetradeoxy-non-2-ulosonic acid (NonlA), which was readily released from LPS by mild acid hydrolysis. In the present study, we investigated the chemical and serological properties of NonlA isolated from LPS of V. parahaemolyticus O2 and OUT KX-V212. GC-MS and NMR analysis identified the NonlA from LPS of O2 to be 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5NAc7NAcNonlA) and that from LPS of KX-V212 to be 5-acetamido-7-(N-acetyl-D-alanyl)amido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5NAc7NAlaNAcNonlA). In ELISA inhibition analysis, 5NAc7NAcNonlA inhibited the O2 LPS/anti-O2 antiserum system, whereas, 5NAc7NAlaNAcNonlA did not show any inhibitory activity. However, after N-deacylation of 5NAc7NAlaNAcNonlA followed by N-acetylation, the product (5NAc7NAcNonlA) inhibited the O2 LPS/anti-O2 antiserum system to the same extent as that of 5NAc7NAcNonlA obtained from O2 LPS. These results suggest that 5NAc7NAcNonlA might be related to the serological specificity of O2 LPS as one of main epitope(s) involved in O2 LPS.