leupeptins and scriptaid

Although improving, the efficiency of producing offspring by somatic cell nuclear transfer (SCNT) is still low (<1.5%). Our laboratory has demonstrated that histone deacetylase inhibitor (Scriptaid) treatment of reconstructed embryos enhances blastocyst formation and cloning efficiency in pigs. It has also been shown that proteasomal inhibitor MG132 treatment for 2 h after activation of oocytes increases blastocyst rate and pregnancy rate. The current experiment was carried out to determine the effects of combined MG132 and Scriptaid treatment on early embryo development in vitro and on term development in vivo. Immediately after electrofusion and activation, SCNT oocytes were treated with 0, 1, or 10 μM MG132 for 2 h in the presence of 500 nM Scriptaid, washed and treated with Scriptaid for an additional 14 to 15 h, then cultured in porcine zygote medium 3 (PZM3) until day 6. There was no difference in percent cleavage (58.1 ± 7.2%, 62.7 ± 7.2%, and 62.5 ± 7.2%) on day 2, or total cell number (23.1 ± 2.2, 24.0 ± 2.0, and 24.5 ± 2.3 for the 0, 1, and 10 μM MG132 groups, respectively) on day 6 among the three groups. Interestingly, there was no difference in percentage of blastocysts between the 0 (18.5±4.7%) and 1 (25.1 ± 4.7%) μM MG132 treatment groups; however, compared with the 10 μM MG132 group (14.0 ± 4.7%), more embryos from the 1 μM MG132 group developed into blastocysts (p<0.05). To determine the effects on term development in vivo, two MG132 groups were included (0 and 1 μM MG132), and embryos were treated as above and transferred into synchronized surrogates after treatment. There was no difference in the oocyte-donor cell fusion rate, number of embryos transferred, pregnancy rate at days 28, 60, and at term, pigs delivered per embryo transfer, litter size, body weight at birth, nor cloning efficiency between the Scriptaid-alone control and MG132+Scriptaid combined groups. In summary, the combined treatment of MG132 and Scriptaid did not improve term development compared to Scriptaid treatment alone.

Topics: Animals; Cells, Cultured; Dose-Response Relationship, Drug; Embryo Transfer; Histone Deacetylase Inhibitors; Hydroxylamines; Leupeptins; Nuclear Transfer Techniques; Proteasome Inhibitors; Quinolines; Swine

Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs. TPMT genetic polymorphisms represent a striking example of the potential clinical value of pharmacogenetics. Subjects homozygous for TPMT*3A, the most common variant allele for low activity, an allele that encodes a protein with two changes in amino acid sequence, are at greatly increased risk for life-threatening toxicity when treated with standard doses of thiopurines. These subjects have virtually undetectable levels of TPMT protein. In this study, we tested the hypothesis that TPMT*3A might result in protein misfolding and aggregation. We observed that TPMT*3A forms aggresomes in cultured cells and that it aggregates in vitro, functional mechanisms not previously described in pharmacogenetics. Furthermore, there was a correlation among TPMT half-life values in rabbit reticulocyte lysate, aggresome formation in COS-1 cells, and protein aggregation in vitro for the three variant allozymes encoded by alleles that include the two TPMT*3A single-nucleotide polymorphisms. These observations were compatible with a common structural explanation for all of these effects, a conclusion supported by size-exclusion chromatography and CD spectroscopy. The results of these experiments provide insight into a unique pharmacogenetic mechanism by which common polymorphisms affect TPMT protein function and, as a result, therapeutic response to thiopurine drugs.

Topics: Alleles; Animals; Bacterial Proteins; Chromatography; Circular Dichroism; COS Cells; Cysteine Proteinase Inhibitors; Escherichia coli; Genetic Variation; Histone Deacetylase 6; Histone Deacetylases; Homozygote; Humans; Hydroxylamines; Kinetics; Leupeptins; Methyltransferases; Microscopy, Fluorescence; Microtubules; Pharmacogenetics; Polymorphism, Genetic; Protein Folding; Protein Structure, Tertiary; Proteins; Purines; Quinolines; Recombinant Proteins; Temperature; Transfection; Ubiquitin; Vinblastine