The parthenogenetic activation of canine oocytes with Ca-EDTA by various culture periods and concentrations

In the present study, canine oocytes were exposed to various concentrations of and durations of exposure to EDTA saturated with Ca(2+) (Ca-EDTA), a cell membrane-impermeable metal ion chelator, to determine if parthenogenetic activation could be induced. When oocytes were cultured for 48 or 72 h in parthenogenetic activation medium (PAM) without Ca-EDTA (control) or PAM supplemented with 1 or 5mM Ca-EDTA, the highest rate of pronuclear formation (PN) was obtained in oocytes cultured in 1mM Ca-EDTA for 48 h (8.0%; P<0.05). There was no pronuclear formation in the control group (PAM without Ca-EDTA). Oocytes treated with 5mM Ca-EDTA for 48 h or 1mM Ca-EDTA for 72 h formed a parthenogenetic pronucleus (3.1 and 4.5, respectively). However, there was no pronuclear formation in oocytes treated with 5mM Ca-EDTA for 72 h. In summary, exposure to Ca-EDTA can induce pronuclear formation in canine oocytes.     

Physicochemical and biological characteristics of DEAE-derivatized PS7 biopolymer of Beijerinckia indica

Physicochemical and biological characteristics of the exopolysaccharide, PS7, produced from Beijerinckia indica were investigated. The PS7 weight fractions of Glc and GlcUA were 0.45 and 0.25, respectively, and the molar ratio of Glc:Rha:GalUA was approximately 5:1:1.3. The PS7 was chemically derivatized with diethylaminoethyl chloride-HCl (DEAE-HCl), and the resulting modified PS7 contained both positive and negative charges. The elemental and IR analyses were conducted to confirm the successful incorporation of DEAE groups into PS7. Large increase in nitrogen fraction was observed from the derivatized PS7 by elemental analysis. The characteristic CH(3) and CH(2) peaks originated from DEAE group were detected in (1)H NMR spectrum of the derivatized PS7 as well. Solubility of native PS7 was improved almost twice from 40 to 75% after DEAE-derivatization, while water holding capacity (WHC) drastically decreased from 10,026 to 245%. Oil binding capacity (OBC) of PS7 also significantly dropped from 1528 to 331% after the derivatization. The [eta] values of native and derivatized PS7 were 27.6 and 0.31 dL/g at 25 degrees C, respectively, which means that the DEAE-derivatization significantly decreased the [eta] of PS7. The bile acid binding capacity of PS7 was indirectly determined by measuring the holding capability of cholic acid inside the dialysis membrane. When PS7 was DEAE-derivatized, there was substantial decrease in the cholic acid retardation index (CRI). Up to 8-9h of dialysis, the derivatized PS7 hold 8.6% less of cholic acid compared to native one.     

Differences in Doublecortin Immunoreactivity and Protein Levels in the Hippocampal Dentate Gyrus Between Adult and Aged Dogs

Doublecortin (DCX), a microtubule-associated protein, specifically expresses in neuronal precursors. This protein has been used as a marker for neuronal precursors and neurogenesis. In the present study, we observed differences in DCX immunoreactivity and its protein levels in the hippocampal dentate gyrus between adult and aged dogs. In the adult dog, DCX immunoreactive cells with well-stained processes were detected in the subgranular zone of the dentate gyrus. Numbers of DCX immunoreactive cells in the dentate gyrus of the aged dog were significantly decreased compared to those in the adult dog. DCX immunoreactive cells in both adult and aged dog did not show NeuN (a marker for mature neurons) immunoreactivity. NeuN immunoreactivity in the aged dog was poor compared to that in the adult dog. DCX protein level in the aged dentate gyrus was decreased by 80% compared to that in the adult dog. These results suggest that the reduction of DCX in the aged hippocampal dentate gyrus may be involved in some neural deficits related to the hippocampus.     

Phylogeny of Carpinus and subfamily Coryloideae (Betulaceae) based on chloroplast and nuclear ribosomal sequence data

Phylogenetic studies were conducted for Carpinus and the subfamily Coryloideae (Betulaceae) using sequences of the chloroplast matK gene, the trnL-trnF region (trnL intron, and trnL [UAA] 3' exon-trnF [GAA] intergenic spacer) and the psbA-trnH intergenic spacer, and the nuclear ribosomal ITS regions. The combined analyses of the three chloroplast regions suggest that Coryloideae is monophyletic; Ostryopsis is sister to the Carpinus - Ostrya clade; Corylus is monophyletic and sister to the Ostrya - Carpinus - Ostryopsis clade; Ostrya is paraphyletic; and within Carpinus, species of sect. Carpinus from eastern Asia form a monophyletic group, whereas the positions of C. betulus from Europe and C. caroliniana from eastern North America are unresolved within the Carpinus clade. The cpDNA tree generated in this study is largely congruent with the previously published ITS results, but the ITS tree places Carpinus sect. Distegocarpus as sister to the Ostrya - Carpinus sect. Carpinus clade. Future work is needed to examine the relationships within the Ostrya - Carpinus clade, evaluate the generic status of Ostrya, and test the phylogenetic position of Ostryopsis.     

Heavy water labeling method for measuring the effect of genistein on mammary gland carcinogenesis

Heavy water labeling method was applied to measure the effect of genistein on mammary gland carcinogenesis by incoporating ²H from ²H₂O into the deoxyribose (dR) moiety of purine deoxyribonucleotides in dividing cells. In the present study, we followed the study design of Lamartiniere group to evaluate the efficacy of ²H₂O labeling on the measurement of mammary gland carconogenesis. Female Sprague–Dawley rats were fed estrogen-free AIN-93G diet starting 1 week before breeding and continuing through pregnancy and lactation. Female pups were assigned to the following groups on postnatal day 16 and fed AIN-93G diet: vehicle (dimethylsulfoxide) (DMSO), genistein, and estradiol benzoate (EB). On postnatal days 16, 18, and 20, female pups were injected subcutaneously with 500 μg genistein/g body wt, 500 ng EB/g body wt, or an equivalent volume of the vehicle. At day 50 postpartum, half of each group were gavaged with 60 mg dimethylbenz[a]anthracene (DMBA) in perila oil. After 1 week of DMBA treatment, all animals were labeled with ²H₂O by administration of 4% ²H₂O in drinking water after single intraperitonial bolus injection with 99.9% ²H₂O until sacrifice on postnatal day 81. The time-dependent weight gains were observed in all groups throughout the experimental period. The enrichment of body ²H₂O was attained at 1.84–2.47% through oral administration of ²H₂O. Mammary epithelial cell proliferation was measured by enrichment (EM1) of dA from rats. DMBA-treated groups showed higher fractional synthesis than DMBA non-treated groups. The group exposed only to genistein showed significantly lower EM1 (1.46 ± 0.87%) than those of control groups, i.e., the DMBA non-treated group (2.28 ± 0.29%) and the DMBA-treated group (2.32 ± 0.28%). Bromodeoxyuridine (BrdU) immunostaining of mammary tissue revealed that genistein reduced proliferation of the mammary epithelial, and the number of cells stained positive for BrdU both in DMBA-treated groups and DMBA non-treated groups. H&E staining of mammary epithelium also showed that the exposure to genistein decreased proliferation of the mammary epithelium. The epithelium in the rats treated with DMBA showed mostly multiple cell layers, in contrast to the mostly double layer shown in the DMBA non-treated rats. The exposure to genistein in the prepubertal period inhibited mammary epithelial cell proliferation. In conclusion, the ²H₂O labeling results were in good agreement with the results of BrdU incorporation and histomorphometry, which demonstrates that ²H₂O labeling can be used as a tool to measure carcinogenesis.     

Effect of hyperthermia on TRAIL-induced apoptotic death in human colon cancer cells: development of a novel strategy for regional therapy

Approximately 25% of patients with colorectal cancer will develop metastatic disease exclusively or largely confined to the liver, and the vast majority of these cases are not amenable to surgical resection. These unresectable cases of liver metastatic disease can be treated with isolated hepatic perfusion (IHP), which involves a method of complete vascular isolation of the liver to allow treatment of liver tumors with toxic systemic doses of chemotherapeutic agents. To improve the efficacy of IHP, hyperthermia and biological agents have been applied along with the chemotherapeutic agents. In this study, we investigated whether hyperthermia in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) enhances mortality in human colorectal carcinoma CX-1 cells. Cells were treated with various concentrations of TRAIL (0-200 ng/ml) at various temperatures (40-46 degrees C) for 1 h and further incubated at 37 degrees C in the presence of TRAIL. We observed that hyperthermia at 42-43 degrees C effectively promoted TRAIL-induced apoptosis, as indicated by cell death, poly (ADP-ribose) polymerase (PARP) cleavage, and activation of caspase-8, -9, and -3. In contrast, hyperthermia at 45-46 degrees C suppressed TRAIL-induced apoptosis. We also observed that mild hyperthermia, but not acute hyperthermia, promoted cytochrome c release during treatment with TRAIL. Our data suggest that promotion of cytochrome c release during mild hyperthermia is responsible for the enhancement of TRAIL cytotoxicity.     

Sulfated polysaccharide purified from <Emphasis Type="BoldItalic">Ecklonia cava</Emphasis> accelerates antithrombin III-mediated plasma proteinase inhibition

Surface plasmon resonance is an important technique for studying molecular interactions and was used to investigate the molecular interaction of anticoagulant sulfated polysaccharides purified from an enzymatic hydrolysate of the brown alga Ecklonia cava (ECA) with blood coagulation factors. In a direct binding assay, binding affinity between ECA/antithrombin III (ATIII) and activated blood coagulation factors was in the order: factor VIIa (FVIIa) > factor Xa (FXa) > thrombin (FIIa); kinetic analysis determined K _D values of ECA for FVIIa, FXa, and FIIa of 15.1, 45.0 and 65.0 nM, respectively. Therefore, ECA strongly and selectively (FVII, FX, and FII) enhanced ATIII-mediated coagulation factor inhibition in both the extrinsic and common coagulation pathways. This may contribute to its high anticoagulant activity in vitro. The low cytotoxicity of ECA to venous endothelial cell line (ECV-304) also expands its value in future in vivo studies. However, to utilize it as a model for novel anticoagulant agents, its possible interference with other anticoagulant mechanisms must be addressed.     

Amadori-glycated albumin-induced vascular smooth muscle cell proliferation and expression of inhibitor of apoptosis protein-1 and nerve growth factor-gamma

We investigated the effects of Amadori-glycated serum albumin (GSA) on cell proliferation as well as expressions of antioxidant enzyme genes and marker genes associated with signal transduction pathways in rat aortic vascular smooth muscle cells (VSMCs). Quiescent VSMCs treated with GSA (0-500 microg/mL, 48 h) exhibited a dose-dependent increase in proliferation that was prevented by PD98059 (25 microM), suggesting a MAPK-dependent signaling pathway. Compared with bovine serum albumin (BSA)-treated cells, the GSA (500 microg/mL, 24~h)-treated VSMCs showed a higher superoxide dismutase 2 gene expression in quantitative RT-PCR, suggesting the involvement of oxidative stress. In a focused oligonucleotide array containing 96 signal transduction-related genes, expression of inhibitor of apoptosis protein-1 (IAP-1), nerve growth factor-gamma (NGF-gamma), and c-jun genes was significantly higher in the GSA-treated VSMCs. These results suggest that induction of antiapoptotic proteins like IAP-1 and strong mitogens like NGF-gamma by GSA might further contribute to the VSMC proliferation and accelerated vascular remodeling in diabetes.