Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1-2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.     

Global structure analysis of acid-unfolded myoglobin with consideration to effects of intermolecular coulomb repulsion on solution X-ray scattering

To obtain information on the global structure of protein in the acid-unfolded (AU) state, the structure of apomyoglobin (apoMb) was analyzed by using the solution X-ray scattering (SXS) method. SXS profiles were obtained over a wide range of protein concentrations, 1-18 mg mL-1, under strongly acidic conditions. From analysis of the SXS profile extrapolated to a zero protein concentration, the mean square radius, Rsq, of AU-apoMb at 20 mM HCl was estimated to be 4.81 +/- 0.31 nm. This estimate is more than 1.3 nm larger than those of 3.0-3.5 nm reported thus far. The difference originates from the fact that effects of Coulomb repulsive forces acting between AU-apoMb molecules have not been correctly taken into account in the conventional analysis. In fact, even at a low protein concentration of 1 mg mL-1 close to the limit of measurement in the present SXS method, the solution condition applicable to estimating accurately structural parameters of AU-apoMb is very limited. At HCl concentrations lower than 10 mM, the scattering intensity at a small scattering vector decreases remarkably through the effect of intermolecular repulsive forces and the forward scattering intensity is significantly lower than the estimate from the partial specific volume of protein. On the other hand, at HCl concentrations higher than 50 mM, some compact molten-globule-like structures emerge. As a result, the intermediate concentration of 20 mM HCl is the best choice of the solution condition for determining Rsq of AU-apoMb. The effect of intermolecular Coulomb repulsion on the SXS profile of AU-apoMb is at its maximum for forward scattering and decreases monotonously with an increase in the scattering angle to be virtually negligible at K approximately 0.63 nm(-1). Whereas urea-denatured apoMb shows a SXS profile typical of Gaussian chains, the intrinsic SXS profile of AU-apoMb differs significantly from those of Gaussian chains.     

Protective Effect of Oren-gedoku-to Against Induction of Neuronal Death by Transient Cerebral Ischemia in the C57BL/6 Mouse

We examined the neuroprotective effects of oren-gedoku-to (TJ15), a herbal medicine, after transient forebrain ischemia. Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 15 min in C57BL/6 mice treated with TJ15. In the control ischemic group without TJ15 treatment, histologic examination of brain tissue collected seven days after reperfusion showed death of pyramidal cells in CA2-3 area of the hippocampus, unilaterally or bilaterally. In mice treated with oral TJ15 (845 mg/kg/day) for five weeks, the frequency of ischemic neuronal death was significantly lower. Immunohistochemistry for Cu/Zn-superoxide dismutase (Cu/Zn-SOD) showed strongly reactive astrocytes in the hippocampus of ischemic mice treated with TJ15. Damage to nerve cells by free radicals plays an important role in the induction of neuronal death by ischemia-reperfusion injury. Our results suggest that TJ15 protects against ischemic neuronal death by increasing the expression of Cu/Zn-SOD and suggest that oren-gedoku-to reduces the exposure of hippocampal neurons to oxidative stress.     

Establishment of a swine monocyte cell line

A swine monocyte cell line was established from peripheral blood sample collected from a healthy adult male pig. The cloned cells grow actively in forming monolayers in both glass and plastic cell culture flasks with the growth medium reported previously (Kadoi, 2000) at 36.5 degrees C incubation. The plating efficiency is more than 95%. Densely grown cells in flasks show an epithelioid morphology. The fundamental properties of the cells were examined for cytological definition as monocytes. A positive property detected was guinea pig complement receptor, porcine IgG receptor, non-specific esterase, and acid phosphatase. A significant phagocytic activity proved by the inoculation of Saccharomyces cerevisiae is also one of the characteristics observed in the LPS-activated cells.     

Genetic isolation between the Western and Eastern Pacific populations of pronghorn spiny lobster Panulirus penicillatus

The pronghorn spiny lobster, Panulirus penicillatus, is a circumtropical species which has the widest global distribution among all the species of spiny lobster, ranging throughout the entire Indo-Pacific region. Partial nucleotide sequences of mitochondrial DNA COI (1,142-1,207 bp) and 16S rDNA (535-546 bp) regions were determined for adult and phyllosoma larval samples collected from the Eastern Pacific (EP)(Galápagos Islands and its adjacent water), Central Pacific (CP)(Hawaii and Tuamotu) and the Western Pacific (WP)(Japan, Indonesia, Fiji, New Caledonia and Australia). Phylogenetic analyses revealed two distinct large clades corresponding to the geographic origin of samples (EP and CP+WP). No haplotype was shared between the two regional samples, and average nucleotide sequence divergence (Kimura's two parameter distance) between EP and CP+WP samples was 3.8±0.5% for COI and 1.0±0.4% for 16S rDNA, both of which were much larger than those within samples. The present results indicate that the Pacific population of the pronghorn spiny lobster is subdivided into two distinct populations (Eastern Pacific and Central to Western Pacific), with no gene flow between them. Although the pronghorn spiny lobster have long-lived teleplanic larvae, the vast expanse of Pacific Ocean with no islands and no shallow substrate which is known as the East Pacific Barrier appears to have isolated these two populations for a long time (c.a. 1MY).     

Coenzyme Q10 as a potent compound that inhibits Cdt1–geminin interaction

A human replication initiation protein Cdt1 is a very central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by directly binding to it. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1–geminin imbalance, for example by geminin silencing with siRNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. In the present study, we first established a high throughput screening system based on modified ELISA (enzyme linked immunosorbent assay) to identify compounds that interfere with human Cdt1–geminin binding. Using this system, we found that coenzyme Q₁₀ (CoQ₁₀) can inhibit Cdt1–geminin interaction in vitro. CoQ compound is an isoprenoid quinine that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. CoQ₁₀, having a longer isoprenoid chain, was the strongest inhibitor of Cdt1–geminin binding in the tested CoQs, with 50% inhibition observed at concentrations of 16.2 μM. Surface plasmon resonance analysis demonstrated that CoQ₁₀ bound selectively to Cdt1, but did not interact with geminin. Moreover, CoQ₁₀ had no influence on the interaction between Cdt1 and mini-chromosome maintenance (MCM)4/6/7 complexes. These results suggested that CoQ₁₀ inhibits Cdt1–geminin complex formation by binding to Cdt1 and thereby could liberate Cdt1 from inhibition by geminin. Using three-dimensional computer modeling analysis, CoQ₁₀ was considered to interact with the geminin interaction interface on Cdt1, and was assumed to make hydrogen bonds with the residue of Arg243 of Cdt1. CoQ₁₀ could prevent the growth of human cancer cells, although only at high concentrations, and it remains unclear whether such an inhibitory effect is associated with the interference with Cdt1–geminin binding. The application of inhibitors for the formation of Cdt1–geminin complex is discussed.     

Saccharomyces cerevisiae Bor1p is a boron exporter and a key determinant of boron tolerance

Boron is toxic to living organisms when present in excess. Saccharomyces cerevisiae Bor1p is a plasma membrane protein that decreases the intracellular concentration of boron and confers boron tolerance in yeasts. We investigated the detailed characteristics of boron transport by Bor1p and its roles in boron tolerance. Boron transport assays showed that the bor1 deletion mutant (bor1Delta) accumulates higher intracellular concentrations of boron and has a lower rate of boron export. The bor1Delta showed greater susceptibility to high concentrations of boron than the wild-type strain, and the growth rates of both strains were negatively correlated with the intracellular concentrations of boron. With normal to toxic levels of external boron, green fluorescent protein (GFP)-tagged Bor1p localized to the plasma membrane irrespective of the concentration of boron in the medium. Taken together, these results establish Bor1p as a plasma membrane boron exporter and a key determinant of boron tolerance.     

Mechanism for oxidation of high-molecular-weight substrates by a fungal versatile peroxidase, MnP2

Unlike general peroxidases, Pleurotus ostreatus MnP2 was reported to have a unique property of direct oxidization of high-molecular-weight compounds, such as Poly R-478 and RNase A. To elucidate the mechanism for oxidation of polymeric substrates by MnP2, a series of mutant enzymes were produced by using a homologous gene expression system, and their reactivities were characterized. A mutant enzyme with an Ala substituting for an exposing Trp (W170A) drastically lost oxidation activity for veratryl alcohol (VA), Poly R-478, and RNase A, whereas the kinetic properties for Mn(2+) and H(2)O(2) were substantially unchanged. These results demonstrated that, in addition to VA, the high-molecular-weight substrates are directly oxidized by MnP2 at W170. Moreover, in the mutants Q266F and V166/168L, amino acid substitution(s) around W170 resulted in a decreased activity only for the high-molecular-weight substrates. These results, along with the three-dimensional modeling of the mutants, suggested that the mutations caused a steric hindrance to access of the polymeric substrates to W170. Another mutant, R263N, contained a newly generated N glycosylation site and showed a higher molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Interestingly, the R263N mutant exhibited an increased reactivity with VA and high-molecular-weight substrates. The existence of an additional carbohydrate modification and the catalytic properties in this mutant are discussed. This is the first study of a direct mechanism for oxidation of high-molecular-weight substrates by a fungal peroxidase using a homologous gene expression system.