Transgenic-cloned pigs systemically expressing red fluorescent protein, Kusabira-Orange

Genetically engineered pigs with cell markers such as fluorescent proteins are highly useful in lines of research that include the tracking of transplanted cells or tissues. In this study, we produced transgenic-cloned pigs carrying a gene for the newly developed red fluorescent protein, humanized Kusabira-Orange (huKO), which was cloned from the coral stone Fungia concinna. The nuclear transfer embryos, reconstructed with fetal fibroblast cells that had been transduced with huKO cDNA using retroviral vector DDeltaNsap, developed efficiently in vitro into blastocysts (28.0%, 37/132). Nearly all (94.6%, 35/37) of the cloned blastocysts derived from the transduced cells exhibited clear huKO gene expression. A total of 429 nuclear transfer embryos were transferred to four recipients, all of which became pregnant and gave birth to 18 transgenic-cloned offspring in total. All of the pigs highly expressed huKO fluorescence in all of the 23 organs and tissues analyzed, including the brain, eyes, intestinal and reproductive organs, skeletal muscle, bone, skin, and hoof. Furthermore, such expression was also confirmed by histological analyses of various tissues such as pancreatic islets, renal corpuscles, neuronal and glial cells, the retina, chondrocytes, and hematopoietic cells. These data demonstrate that transgenic-cloned pigs exhibiting systemic red fluorescence expression can be efficiently produced by nuclear transfer of somatic cells retrovirally transduced with huKO gene.     

Conformational significance of EH21A1-A4, phenolic derivatives of geldanamycin, for Hsp90 inhibitory activity

Hsp90 is an attractive chemotherapeutic target because it is essential to maturation of multiple oncogenes. We describe the conformational significance of EH21A1-A4, phenolic derivatives of geldanamycin isolated from Streptomyces sp. Their native free structures are similar to the active form of geldanamycin bound to Hsp90 protein. Their conformational character is a probable reason for their high-affinity binding. Lack of toxic benzoquinone in EH21A1-A4 also adds to their potential as lead compounds for anti-tumor drugs.     

Distinct immunohistochemical localization in Kuru plaques using novel anti-prion protein antibodies

By immunizing Prnp-knockout mice with synthetic polypeptides, a panel of mAbs directed to bovine PrP(C) was obtained. The mAb panel was characterized by the ELISA method, where synthetic polypeptides were used for epitope mapping. Different reactivity patterns were identified. The ability of these mAbs to detect abnormal PrP(Sc) in CJD cases was studied by immunohistochemistry. All mAbs were tested for PrP(Sc) in murine, bovine, monkey and human brain tissues. Three mAbs recognized the fragmented PrP epitope in our ELISA. Antibody 1D12 was strongly reactive to ovine and squirrel monkey tissues infected with a scrapie agent, although non-reactive to scrapie-infected mouse tissues. Antibody 2D8 was clearly reactive to type-2 but not type-1 CJD human tissues. Of particular interest was the reactivity of mAb 6C4 with the inner structure of Kuru plaques (peripheral pattern) in a type-2 CJD case and mAb T2, 1D12, 2B11, 2D8, 4B5 and 6G3-2 with the central area (central pattern). The fact that different anti-PrP mAbs possess distinct staining properties suggests that the PrP(c) to PrP(Sc) conversion might involve a multiple-step process.     

Syntheses, structures and antimicrobial activities of various metal complexes of hinokitiol

Metal-oxygen bonding complexes (M = Mg^II, Mn^II, Ni^II, Mo^VI, W^VI, Pd^II, Sb^III, Bi^III, Fe^III, Ti^IV, K^I, Ba^II, Zr^IV and Hf^IV) with a hinokitiol (Hhino; 2-hydroxy-4-isopropylcyclohepta-2,4,6-trienone or β-thujaplicin) ligand, which has two unequivalent oxygen donor atoms, were synthesized and characterized by elemental analysis, TG/DTA, FT-IR and solution (¹H and ¹³C) NMR spectroscopy. Single-crystal X-ray structure analysis revealed various molecular structures for the complexes, which were classified into several families of family, i.e. type A [M^II(hino)₂(L)]₂ (M = Mg^II, Mn^II, Ni^II; L = EtOH or MeOH), with a dimeric structure consisting of one bridging hino⁻ anion, one chelating hino⁻ anion and one alcohol or water molecule, type B, with the octahedral, cis-dioxo, bis-chelate complexes cis-[M^VIO₂(hino)₂] (M = Mo^VI, W^VI), type C, with square planar complex [M^II(hino)₂] (M = Pd^II), type D, with tris-chelate, 7-coordinate complexes with one inert electron pair [M^III(hino)₃] (M = Sb^III, Bi^III), type D′, with the bis-chelate, pseudo-6-coordinate complexes with one inert electron pair [M^III(hino)₂X] (M = Sb^III, X = Br), type E, with tris-chelate, 6-coordinate complexes with Δ and Λ isomers [M^III(hino)₃] (M = Fe^III), type E′ of bis-chelate, 6-coordinate complex [M^IV(hino)₂X₂] (M = Ti^IV, X = Cl), type F, with water-soluble alkali metal salts [M^I(hino)] (M = K^I), and type H, with tetrakis-chelate, 8-coordinate complexes [M^IV(hino)₄](M = Zr^IV, Hf^IV). These structural features were compared with those of metal complexes with a related ligand, tropolone (Htrop). The antimicrobial activities of these complexes, evaluated in terms of minimum inhibitory concentration (MIC; μg mL⁻¹) in two systems, were compared to elucidate the relationship between structure and antimicrobial activity.     

Localization of insulinoma associated protein 2, IA-2 in mouse neuroendocrine tissues using two novel monoclonal antibodies

AimsInsulinoma-associated protein 2 (IA-2) is a member of the protein tyrosine phosphatase family that is localized on the insulin granule membrane. IA-2 is also well known as one of the major autoantigens in Type 1 diabetes mellitus. IA-2 gene deficient mice were recently established and showed abnormalities in insulin secretion. Thus, detailed localization of IA-2 was studied using wild-type and IA-2 gene deficient mice.Main methodsTo localize IA-2 expression in mouse neuroendocrine tissues, monoclonal antibodies were generated against IA-2 and western blot and immunohistochemical analyses were carried out in IA-2^+/+ mice. IA-2^?/? mice served as a negative control.Key findingsWestern blot analysis revealed that the 65 kDa form of IA-2 was observed in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, pituitary gland, muscular layers of the stomach, small intestine, and colon. By immunohistochemical analysis, IA-2 was produced in endocrine cells in pancreatic islets, adrenal medullary cells, thyroid C-cells, Kulchitsky cells, and anterior, intermediate, and posterior pituitary cells. In addition, IA-2 was found in somatostatin-producing D-cells and other small populations of cells were scattered in the gastric corpus. IA-2 expression in neurites was confirmed by the immunostaining of IA-2 using primary cultured neurons from the small intestine and nerve growth factor (NGF)-differentiated PC12 cells.SignificanceThe IA-2 distribution in peripheral neurons appeared more intensely in neurites rather than in the cell bodies.     

Nucleomorph genome diversity and its phylogenetic implications in cryptomonad algae

The relationship between phylogeny and nucleomorph genome size was examined in 16 strains of cryptomonad algae using pulsed‐field gel electrophoresis, Southern hybridization and phylogenetic analyses. Our results suggest that all cryptomonads examined in this study contain three nucleomorph chromosomes and their total genome size ranges from 495 to 750 kb. In addition, we estimated the plastid genome size of the respective organisms. The plastid genomes of photosynthetic strains were approximately 120–160 kb in size, whereas the non‐photosynthetic Cryptomonas paramecium NIES715 possesses a genome of approximately 70 kb. Phylogenetic analysis of the nuclear small subunit ribosomal DNA (SSU rDNA) gene showed that nucleomorph genome size varies considerably within closely related strains. This result indicates that the reduction of nucleomorph genomes is a rapid phenomenon that occurred multiple times independently during cryptomonad evolution. The nucleomorph genome sizes of Cryptomonas rostratiformis NIES277 appeared to be approximately 495 kb. This is smaller than that of Guillardia theta CCMP327, which until now was thought to have the smallest known nucleomorph genome size among photosynthetic cryptomonads.     

ArfGAP family proteins in cell adhesion, migration and tumor invasion

The identification of several ArfGAP proteins as binding partners of paxillin, an integrin signaling and scaffolding protein, has suggested the existence of molecular links between integrin functions and intracellular traffic, as proposed by MS Bretscher long ago. Among the paxillin-binding ArfGAPs, AMAP1 has recently been strongly implicated in tumor invasion as well as malignancy, owing to its highly augmented expression in tumors and its direct involvement in invasive activities. Another ArfGAP, Git2, was found to be a component of the Gbetagamma-mediated directional sensing machinery, while simultaneously playing an essential role in the suppressive control of superoxide production, which is mediated by vesicle transport in GPCR-stimulated neutrophils. These emerging molecular mechanisms may further delineate key processes regulating intracellular traffic as principal controls of cell motility and invasive activities.     

A crucial role for matrix metalloproteinase 2 in osteocytic canalicular formation and bone metabolism

Extracellular matrix production and degradation by bone cells are critical steps in bone metabolism. Mutations of the gene encoding MMP-2, an extracellular matrix-degrading enzyme, are associated with a human genetic disorder characterized by subcutaneous nodules, arthropathy, and focal osteolysis. It is not known how the loss of MMP-2 function results in the pathology. Here, we show that Mmp2(-/-) mice exhibited opposing bone phenotypes caused by an impaired osteocytic canalicular network. Mmp2(-/-) mice showed decreased bone mineral density in the limb and trunk bones but increased bone volume in the calvariae. In the long bones, there was moderate disruption of the osteocytic networks and reduced bone density throughout life, whereas osteoblast and osteoclast function was normal. In contrast, aged but not young Mmp2(-/-) mice had calvarial sclerosis with osteocyte death. Severe disruption of the osteocytic networks preceded osteocyte loss in Mmp2(-/-) calvariae. Successful transplantation of wild-type periosteum restored the osteocytic canalicular networks in the Mmp2(-/-) calvariae, suggesting local roles of MMP-2 in determining bone phenotypes. Our results indicate that MMP-2 plays a crucial role in forming and maintaining the osteocytic canalicular network, and we propose that osteocytic network formation is a determinant of bone remodeling and mineralization.     

Effects of nutrient contents and defense compounds on herbivory in reproductive organs and leaves of Iris gracilipes

Optimal defense theory (ODT) states that the plant tissue with the highest value to fitness will receive the most protection compared with other plant parts. ODT can be applied to the differences in defenses among floral organs, although most studies have concentrated on the comparison between leaves and flowers. Using Iris gracilipes, we investigated whether ODT is supported when primary and accessory floral organs and leaves are distinguished. We found that anthers and perianths tended to be attacked more severely than ovaries and leaves in the bud and flower stages and that anthers contained the highest nitrogen and phosphorus concentrations. Although ovaries were also found to contain high nitrogen and phosphorus concentrations, they were less severely attacked by herbivores than anthers, perhaps because ovaries contained the highest condensed tannins concentrations among the floral organs except for perianths in the flower stage. Thus, noting that the number of ovules is very much smaller than that of pollen grains, we concluded that ovaries are the most intensively protected, consistent with the prediction of ODT as applied to floral organs. ODT is applicable to the difference in defense allocation among floral organs.