Developmental competence of morphologically poor oocytes in relation to follicular size and oocyte diameter in the pig

The objective of this study was to assess the practical usefulness of morphologically poor oocytes (MPCOCs) in relation to follicular size and oocyte diameter. Oocytes collected from medium (3–8 mm in diameter) and small (<3 mm) follicles were classified into five categories of morphologically good oocytes (MGCOCs) from medium follicles (MA, control), MPCOCs with larger and smaller diameters from medium follicles (ML and MS, respectively), and those from small follicles (SL and SS, respectively). The oocytes were examined for maturation and developmental competence after parthenogenesis and somatic cell nuclear transfer (SCNT). Nuclear maturation of ML oocytes (91%) was similar to that of control oocytes (94%), but higher than MS (80%), SL (79%), and SS (63%) oocytes. This pattern was also observed in the intracellular glutathione level, p34^cdc2 kinase activity, and gene (CDK1, PCNA, and ERK2) expression levels in in vitro‐matured oocytes. ML oocytes showed a similar proportion of blastocyst formation (20%) after SCNT to control oocytes (21%). In addition, the use of ML oocytes resulted in a 50% farrowing rate with 1.8% efficiency of piglet production after SCNT embryo transfer, while control oocytes showed a 60% farrowing rate with 2.4% production efficiency. Our results demonstrate that MPCOCs, if appropriately selected, have a comparable ability to MGCOCs in supporting not only in vitro blastocyst formation, but also development to term in vivo after SCNT. These oocytes can be used as a source for in vitro production of embryos with normal in vivo viability in pigs. Mol. Reprod. Dev. 77: 330–339, 2010. © 2009 Wiley‐Liss, Inc.     

Ala99ser mutation in RIα Regulatory Subunit of protein kinase A causes reduced kinase activation by cAMP and arrest of hormone-dependent breast cancer cell growth

Expression of the RI regulatory subunit of protein kinase A type I is increased in human cancer cell lines, in primary tumors, in cells after transformation, and in cells upon stimulation of growth. Ala99 (the pseudophosphorylation site) of human RI was replaced with Ser (RI-p) for the structure-function analysis of RI. MCF-7 hormone- dependent breast cancer cells were transfected with an expression vector for the wild-type RI or mutant RI-p. Overexpression of RI-P resulted in suppression of protein kinase A type II, the isozyme of type I kinase, production of kinase exhibiting reduced cAMP activation, and inhibition of cell growth showing an increase in G0/G1 phase of the cell cycle and apoptosis. The wild-type RI overexpression had no effect on protein kinase A isozyme distribution or cell growth. Overexpression of protein kinase A type II regulatory subunit, RII, suppressed RI and protein kinase A type I and inhibited cell growth. These results show that the growth of hormone-dependent breast cancer cells is dependent on the functional protein kinase A type I.     

Photosynthetic eukaryotes unite: endosymbiosis connects the dots

The photosynthetic organelle of algae and plants (the plastid) traces its origin to a primary endosymbiotic event in which a previously non-photosynthetic protist engulfed and enslaved a cyanobacterium. This eukaryote then gave rise to the red, green and glaucophyte algae. However, many algal lineages, such as the chlorophyll c-containing chromists, have a more complicated evolutionary history involving a secondary endosymbiotic event, in which a protist engulfed an existing eukaryotic alga (in this case, a red alga). Chromists such as diatoms and kelps then rose to great importance in aquatic habitats. Another algal group, the dinoflagellates, has undergone tertiary (engulfment of a secondary plastid) and even quaternary endosymbioses. In this review, we examine algal diversity and show endosymbiosis to be a major force in algal evolution. This area of research has advanced rapidly and long-standing issues such as the chromalveolate hypothesis and the extent of endosymbiotic gene transfer have recently been clarified.     

Influence of liquid medium and surface morphology on the response of QCM during immobilization and hybridization of short oligonucleotides

With the goal of developing a quartz crystal microbalance (QCM)-based DNA sensor, we have conducted an in situ QCM study along with fluorescence measurements using oligonucleotides (15-mer) as a model single-stranded DNA (ss-DNA) in two different aqueous buffer solutions; the sequence of 15-mer is a part of iduronate-2-sulphate exon whose mutation is known to cause Hunter syndrome, and the 15-mer is thiolated to be immobilized on the Au-coated quartz substrate. The fluorescence data indicate that the initial immobilization as well as the subsequent hybridization with a complementary strand is hardly dependent on the kind of buffer solution. In contrast, the mass increases deducible from the decrease of QCM frequency via the Sauerbrey equation are 2.7-6.2 and 3.0-4.4 times larger than the actual mass increases, as reflected in the fluorescence measurements, for the immobilization and the subsequent hybridization processes, respectively. Such an overestimation is attributed to the trapping of solvent as well as the formation of quite a rigid hydration layer associated with the higher viscosities and/or densities of the buffer solutions. Another noteworthy observation is the excessively large frequency change that occurs when the gold electrode is deposited in advance with Au nanoparticles. This clearly illustrates that the QCM detection of DNA hybridization is also affected greatly by the surface morphology of the electrode. These enlarged signals are altogether presumed to be advantageous when using a QCM system as an in situ probing device in DNA sensors.     

Effects of green tea polyphenol on preservation of human saphenous vein

The potential role of green tea polyphenol (GtPP) in preserving the human saphenous vein was investigated under physiological conditions. The vein segments were incubated for 1, 3, 5, 7 and 14 days, either after 4h of treatment with 1.0mg/ml GtPP or in the presence of GtPP at the same concentration. After incubation, the endothelial cell viability, endothelial nitric oxide synthase (eNOS) expression and the vein histology were evaluated. When the veins were not treated with GtPP, the viability of the endothelial cells was significantly reduced with the progress in the culture time, and none of the cells expressed eNOS after 5 days. Furthermore, severe histological changes and structural damage were observed in the non-treated veins. In contrast, incubating the veins after 4h of GtPP treatment significantly prevented these phenomena. The cellular viability of the GtPP-treated vein was approximately 64% after 7 days, and eNOS expression was maintained up to 40%, compared to that of the fresh vein. The histological observations showed that the vasculature was quite similar to that of the fresh vein. When incubated with GtPP, the vein could also be preserved for 1 week under physiological conditions retaining both its cellular viability (61%) and eNOS expression level (45%) and maintaining its venous structure without any morphological changes. These results demonstrate that GtPP treatment may be a useful method for preserving the HSV.     

Identification and characterization of a novel angiostatin-binding protein by the display cloning method

Angiostatin is a potent anti-angiogenic protein. To examine the angiostatin-interacting proteins, we used the display-cloning method with a T7 phage library presenting human cDNAs. The specific T7 phage clone that bound to the immobilized angiostatin was isolated, and a novel gene encoding the displayed polypeptide on the isolated T7 phage was identified. The displayed angiostatin-binding sequence was expressed in E. coli as a soluble protein and purified to homogeneity. This novel angiostatin-binding region interacted specifically to angiostatin with a dissociation constant of 3.4 x 10(-7) M. A sequence analysis showed that the identified sequence was a part of the large ORF of 1,998 amino acids, whose function has not yet been characterized. A Northern analysis indicated that the gene containing the angiostatin-binding sequence was expressed differentially in the developmental stages or cell types.     

<Emphasis Type="Italic">Prorocentrum vietnamensis</Emphasis> sp. nov. (Prorocentraceae Dinophyta): A new armored dinoflagellate from Vietnam coastal waters

TheProrocentrum species, which are primarily marine, are distributed worldwide and occur in ocean, neritic, and littoral environments.Prorocentrum is either a planktonic or benthic genus, with some species possibly being both. Until now, about 35 species had been reported including two that are fresh-water. Some species contain toxic substances. As one of many currently active studies, we collectedP. vietnamensis sp. nov. from Hon Mé, Gulf of Tongkin, Vietnam. Based on its cell shape, smooth valve surface, arrangement of valves and marginal pores, plus characteristics of the central pyrenoid, we propose thatP. vietnamensis is a new species. Its attributes include straight cell sides that are parallel to each other, as well as anterior and posterior areas with rounded ends that form a short, rod-shaped cell that is unique to this species.     

Hidden biodiversity of the extremophilic Cyanidiales red algae

The Cyanidiales is a group of asexual, unicellular red algae, which thrive in acidic and high temperature conditions around hot springs. These unicellular taxa have a relatively simple morphology and are currently classified into three genera, Cyanidium, Cyanidioschyzon and Galdieria. Little is known, however, about the biodiversity of Cyanidiales, their population structure and their phylogenetic relationships. Here we used a taxonomically broadly sampled three-gene data set of plastid sequences to infer a robust phylogenetic framework for the Cyanidiales. The phylogenetic analyses support the existence of at least four distinct Cyanidiales lineages: the Galdieria spp. lineage (excluding Galdieria maxima), the Cyanidium caldarium lineage, a novel monophyletic lineage of mesophilic Cyanidium spp. and the Cyanidioschyzon merolae plus Galdieria maxima lineage. Our analyses do not support the notion of a mesophilic ancestry of the Cyanidiales and suggest that these algae were ancestrally thermo-acidotolerant. We also used environmental polymerase chain reaction (PCR) for the rbcL gene to sample Cyanidiales biodiversity at five ecologically distinct sites at Pisciarelli in the Phlegrean Fields in Italy. This analysis showed a high level of sequence divergence among Cyanidiales species and the partitioning of taxa based on environmental conditions. Our research revealed an unexpected level of genetic diversity among Cyanidiales that revises current thinking about the phylogeny and biodiversity of this group. We predict that future environmental PCR studies will significantly augment known biodiversity that we have discovered and demonstrate the Cyanidiales to be a species-rich branch of red algal evolution.     

Anti-tumor activity of the farnesyl-protein transferase inhibitors arteminolides, isolated from Artemisa

Members of the Artemisia genus are important medicinal plants found throughout the world. Arteminolides A-D (1-4), isolated from the aerial parts of Artemisia, have an inhibitory activity on farnesyl-protein transferase (FPTase; EC 2.5.1.29) in in vitro assay. This study was carried out with the purpose of validating anti-tumor effects of the compounds in human tumor cells and mouse xenograft model. The arteminolides inhibited tumor cell growth in a dose-dependent manner. Furthermore, arteminolide C (3) blocked in vivo growth of human colon and lung tumor xenograft without the loss of body weight in nude mice.     

Effects of Aroclor 1254 on the expression of rat placental PRL-family genes

This study was performed to investigate the effects of Aroclor 1254 (A1254), a commercial polychlorinated biphenyl mixture, on the expression of rat placental prolactin (PRL) family genes and reproductive activity. Placental lactogen-Iv and -II, and prolactin-like protein-A and -C mRNA levels were significantly decreased in the placentas of A1254-treated rats in a dose-dependent manner. The mRNA levels of Pit-1alpha and beta isotypes, which are involved in the regulation of PRL family gene expression, were also decreased in the A1254-treated rat placenta. In the rat placental junctional zone, high-dose A1254 (25 mg/kg B.W.) treatment reduced the number of spongiotrophoblasts, cells in which the PRL family genes are expressed. Finally, maternal exposure to A1254 was shown to have significant toxic effects on reproductive activity, including embryonic and placental growth retardation, delay of parturition, and reduction of the number of pups per litter. The results of the present study indicated that A1254 has an inhibitory effect on PRL family, Pit-1alpha, and beta gene expression in the rat placenta, leading to significant toxic effects on reproductive activity in rats.