Antibabesial activity of protoberberine alkaloids and 20-hydroxyecdysone from Arcangelisia flava against Babesia gibsoni in culture

Bioassay-guided fractionation of the boiled extract from the stems of Arcangelisia flava led to the isolation of palmatine (1), berberine (2), jatrorrhizine (3), dihydroberberine (4) and 20-hydroxyecdysone (5). The chemical structures of these compounds were elucidated on the basis of their chemical and spectral evidence. The isolated compounds were evaluated for their growth inhibiting effects on Babesia gibsoni in culture for a week. Compounds (1-4) showed significant inhibitions at concentrations from 100 to 1.0 microg/ml, while compound 5 at a concentration of 100 microg/ml, only.     

Nuclear transfer preserves the nuclear genome of freeze-dried mouse cells

Mouse spermatozoa can be freeze dried without losing genetic integrity and reproductive potential. However, it is not known if freeze-dried mouse cells similarly maintain their genetic integrity and developmental potential following nuclear transfer. Here, we investigated the developmental capacity and embryonic stem (ES) cell derivation of reconstructed oocytes by nuclear transfer using freeze-dried cumulus or ES cells. Cumulus and ES cells were lyophilized overnight and stored at 4 C for up to 1 week. After rehydration, all cells showed membrane damage and were unviable. However, following nuclear transfer, 1-4% of the reconstructed oocytes developed to the blastocyst stage. A total of five nuclear transfer ES (ntES) cell lines were generated from blastocysts and morulae. All ntES cell lines had normal karyotypes and were positive for the ES-cell-specific markers (alkaline phosphatase, Oct3/4 and Nanog). After aggregation of ntES cells with fertilized embryos, chimeric mice with a high level of coat color chimerism were generated. Our findings show that the genomic integrity of cells can be maintained after freeze-drying and that it is possible to produce offspring from the cells using nuclear transfer techniques.     

Successful mouse cloning of an outbred strain by trichostatin A treatment after somatic nuclear transfer

Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, extremely low success rates have plagued this technique for a decade. Further in mice, the "clonable" strains have been limited to mainly hybrid F1 strains such as B6D2F1. Recently, we established a new efficient cloning technique using trichostatin A (TSA) which leads to a 2-5 fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. To further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4-5% success rates, which is comparable to 5-7% of B6D2F1. Thus, the TSA treatment is the first cloning technique to allow us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from hybrid strains, but also enables mouse cloning from normally "unclonable" strains.     

Improved bioassay method for Spodoptera litura chitinase inhibitors using a colloidal chitin powder with a uniform particle size as substrate

A previously reported bioassay method for Spodoptera litura chitinase inhibitors has been improved by use of colloidal chitin powder with a uniform particle size. This improvement made the assay four times more sensitive. Detection of three active supernatants by screening of supernatants and cell extracts from 135 fermentation broths has proved the efficiency of this improved method. © 1999 Society of Chemical Industry     

Changes in peripheral leukocytes enzymes activity and plasma metabolite concentrations in growing Holstein calves

Plasma metabolite and immunoreactive insulin concentrations and activities of enzymes related to energy metabolism in peripheral leukocytes were measured in growing Holstein calves. A ratio of girth of abdomen divided by girth of thorax (A/T ratio) of calves was significantly elevated after weaning, and the A/T ratio maybe a good indicator to evaluate rumen development. Plasma glucose and free fatty acid concentrations were changed in calves accompanying change in feeding. Activities of lactate dehydrogenase with pyruvate as substrate (LDH-P) and hexokinase (HK) in cytosolic fractions of peripheral leukocytes decreased significantly after weaning the calves reflecting the change of energy source from milk replacer with high percentages of fat and glucose and lactose as absorbable carbohydrate to pelleted feed containing starch as less absorbable carbohydrate and roughage. Some peripheral leukocyte enzymes such as LDH and HK may be good indicators to evaluate changes in energy metabolism of growing calves.     

Effects of Central Kalimantan plant extracts on intraerythrocytic Babesia gibsoni in culture

The inhibitory effects of 45 plant extracts selected from Central Kalimantan, Indonesia on Babesia gibsoni in vitro and their acute toxicity to mice were evaluated. Of these plant extracts studied, Arcangelisia flava, Curcuma zedoaria, Garcinia benthamiana, Lansium domesticum and Peronema canescens were found to have appreciable antibabesial activity with IC50 values from 5.3 to 49.3 microg/ml without acute toxicity in mice at the intraperitoneal dose of 0.7 g/kg of body weight.     

Long-term preservation of mouse spermatozoa as frozen testicular sections

We previously demonstrated that testicular spermatozoa can be preserved as frozen testicular sections, allowing us to preserve male gametes in less space than conventional methods. However, it remains unclear whether the testicular spermatozoa can be preserved for a long period using this procedure. In this study, we examined the function of testicular spermatozoa preserved as frozen testicular sections for l year at -30 or -80 C. Testicular spermatozoa were successfully retrieved from frozen testicular sections preserved at either -30 or -80 C, and their function was assessed using intracytoplasmic sperm injection (ICSI). Over 90% of the oocytes injected with long-term preserved testicular spermatozoa formed pronuclei, which was a frequency similar to that obtained with spermatozoa preserved for a short term, indicating that the testicular spermatozoa retained oocyte activation factor(s). Approximately 70% of the fertilized oocytes developed to 2-cell stage embryos, and 9.3 to 12.8% of the embryos developed to term after transfer into pseudopregnant females, regardless of the preservation temperatures examined. These results indicate that the birthrates of progeny did not differ between the preservation temperatures examined. They also indicate that male gametes can be preserved in testicular frozen sections for at least 1 year without loss of function.