Birth of common marmoset (Callithrix jacchus) offspring derived from in vitro-matured oocytes in chemically defined medium.

Optimization of oocyte culture conditions is a crucial aspect of reproductive biology and technology. In the present study, maturation of germinal vesicle-stage marmoset oocytes were evaluated in the following media: Waymouth medium, Waymouth medium containing porcine follicular fluid (pFF) (Waymouth-pFF medium), and porcine oocyte medium (POM). Oocytes cultured in Waymouth-pFF medium had higher maturation rates to the metaphase II stage than those cultured in Waymouth medium (36.1% vs. 24.8%, respectively, P < 0.05), indicating the suitability of this medium for culturing marmoset oocytes. Hence, maturation of marmoset oocytes cultured in POM was subsequently evaluated. The rate of maturation to the metaphase I stage was significantly higher and degradation rates were significantly lower in oocytes cultured in POM than those cultured in Waymouth medium. In addition, three offspring were successfully obtained after transfer of embryos matured in chemically defined medium. Therefore, we concluded that POM was suitable for marmoset oocyte culture. Furthermore, this was apparently the first report of marmoset offspring derived from oocytes cultured in chemically defined medium.

Development of rat tetraploid and chimeric embryos aggregated with diploid cells.

In the present study, we examined the preimplantation and postimplantation development of rat tetraploid embryos produced by electrofusion of 2-cell-stage embryos. Developmental rate of tetraploid embryos to morula or blastocyst stage was 93% (56/60) and similar to that found in diploid embryos (95%, 55/58). After embryo transfer, rat tetraploid embryos showed implantation and survived until day 8 of pregnancy, however the conceptuses were aberrant on day 9. In mouse, tetraploid embryos have the ability to support the development of blastomeres that cannot develop independently. As shown in the present study, a pair of diploid blastomeres from the rat 8-cell-stage embryo degenerated immediately after implantation. Therefore, we examined whether rat tetraploid embryos have the ability to support the development of 2/8 blastomeres. We produced chimeric rat embryos in which a pair of diploid blastomeres from an 8-cell-stage green fluorescent protein negative (GFP-) embryo was aggregated with three tetraploid blastomeres from 4-cell GFP-positive (GFP+) embryos. The developmental rate of rat 2n(GFP-) <--> 4n(GFP+) embryos to the morula or blastocyst stages was 93% (109/117) and was similar to that found for 2n(GFP-) <--> 2n(GFP+) embryos (100%, 51/51). After embryo transfer, 2n(GFP-) <--> 4n(GFP+) conceptuses were examined on day 14 of pregnancy, the developmental rate to fetus was quite low (4%, 4/109) and they were all aberrant and smaller than 2n(GFP-) <--> 2n(GFP+) conceptuses, whereas immunohistochemical analysis showed no staining for GFP in fetuses. Our results suggest that rat tetraploid embryos are able to prolong the development of diploid blastomeres that cannot develop independently, although postimplantation development was incomplete.

[Problems of cancer patient home therapy in our clinic].

We studied the problems of cancer patient home therapy in our clinic over the last 4 years. Cancer patients tend to be younger than other home patients, and their home therapy period is shorter than others. Therefore, hospital doctors should decide the local home doctor of the patients at an early stage. The frequency of emergency calls is higher than with other patients, so cooperation between hospital and clinic is very important.

Synthesis of P1,P4-di(adenosine 5'-) tetraphosphate by leucyl-tRNA synthetase, coupled with ATP regeneration.

A simple and practical procedure for the synthesis of P1,P4-di(adenosine 5'-) tetraphosphate from ATP by the catalysis of leucyl-tRNA synthetase from Bacillus stearothermophilus is described. Km for leucine was 6.7 microM and for ATP was 3.3 mM. The reaction yielded not only diadenosine tetraphosphate, but various byproducts such as P1,P3-(diadenosine 5'-) triphosphate, ADP and AMP. By coupling the reaction with an ATP regeneration system by acetate kinase and adenylate kinase with acetylphosphate as a phosphate donor, diadenosine tetraphosphate was prepared as a sole product at a high yield (96%).

Dipeptide synthesis catalyzed by aminoacyl-tRNA synthetases from Bacillus stearothermophilus.

A new approach to enzymatic peptide synthesis by using aminoacyl-tRNA synthetase (ARS) as a catalyst has been investigated. Four ARSs (AspRS, HisRS, LeuRS and TyrRS) have been purified from a thermophilic bacterium, Bacillus stearothermophilus. By using TyrRS as a catalyst, tyrosine and leucinamide were shown to be condensed in the presence of ATP to give tyrosylleucinamide. In this manner, all of the ARSs investigated catalyzed the peptide synthesis reactions. TyrRS did not have strict specificity for the amino acid derivatives used as substrates and even D-amino acids were incorporated into peptides fairly easily in this enzymatic reaction. Preparative scale synthesis of L-Tyr-L-LeuNH2 was carried out and from this the scope and limitation of this new enzymatic reaction as a tool to the peptide synthesis has been described.

Reactions of the aminoacyl-tRNA synthetase-aminoacyl adenylate complex and amino acid derivatives. A new approach to peptide synthesis.

Several kinds of dipeptide derivative were shown to be formed by the reactions of the aminoacyl adenylate-aminoacyl-tRNA synthetase complex and amino acid ester or amide. It was shown that the peptide bond could be formed by aminoacyl-tRNA synthetases even in the absence of the ribosome.

Construction of a system for the regeneration of adenosine 5'-triphosphate, which supplies energy to bioreactor.

An engineering model was successfully developed for an ATP regeneration system by using two enzymes, acetate kinase (AK) and adenylate kinase (AdK), both obtained from the thermophile Bacillus stearothermophilus. This model is composed of five units: a substrate unit consisting of substrate solutions--AMP, ATP, and acetyl phosphate (AcOP)--an enzymatic reactor unit consisting of AK and AdK immobilized to Sepharose 4B, an auto sampler unit, an analytical unit made up of high-performance liquid chromatography, and a control unit made up of a microcomputer. Operation of the four units could be systematically controlled by the microcomputer. Fundamental, operational conditions were examined using this engineering model. The conversion of AMP to ATP concentration and space velocity (SV). The minimum amount of ATP, which is required to obtain the 100% conversion of AMP to ATP, was determined to be about 4% of AMP concentration. The conversion of AMP to ATP was controlled effectively by changing the SV value. Based on the above experimental data, the continuous operation of an ATP regeneration system was tested at pH 7.5 and 30 degrees C under the conditions of 1.59 mM AMP, 0.084 mM ATP, and 5.0 mM AcOP. It was found that the conversion of AMP to ATP was more than 99% over a period of 6 days without changing SV.