Hiroshi Araya

Freeze-dried mouse spermatozoa are capable of participating in normal embryonic development after injection into oocytes. When the freeze-dried spermatozoa are used as a method for storage of genetic materials, however, it is essential to assure the relevance of long-term preservation over several decades or centuries. Thus, we applied the theory of accelerated degradation kinetics to freeze-dried mouse spermatozoa. Thermal denaturation kinetics were determined based on Arrhenius plots derived from transition-state theory analysis at three elevated temperatures: 30, 40, and 50 degrees C. Accelerated degradation kinetics were calculated by extrapolation of Arrhenius plots. This theory also is being applied to the long-term stability of drugs. The estimated rate of development to the blastocyst stage at 3 and 6 mo and at 1, 10, and 100 yr of sperm storage at 4 degrees C were 21.60%, 7.91%, 1.00%, 0%, and 0%, respectively. At -80 degrees C, estimated development rates to the blastocyst stage that would be expected after 100 yr of storage did not decline significantly. In addition, after 3 or 6 mo of storage at 4 or -80 degrees C, preimplantation development of the embryos derived from intracytoplasmic sperm injection (ICSI) was examined. The actual developmental rates to the blastocyst stage from ICSI by freeze-dried sperm stored for 3 mo at 4 and -80 degrees C were 21% and 62%, respectively, and the rates for such sperm stored for 6 mo were 13% and 59%, respectively. These results indicate that the determination of accelerated degradation kinetics can be applied to the preservation of freeze-dried mouse spermatozoa. Furthermore, for long-term preservation, freeze-dried mouse spermatozoa appear to require being kept at lower than -80 degrees C.

Synopsis A genetic linkage map of guinea grass ( Panicum maximum Jacq.) was generated with nine of the AFLP markers found to be associated with apospory. These apospory‐associated markers were assigned to a linkage group having previous association with microsporogenesis of the aposporous guineagrass cultivar ‘Natsukaze’. An aposporous linkage group was constructed utilizing 38 AFLP markers. Embryo sac analysis revealed that sexual and apomictic embryo sacs occurred at a frequency of 1 : 1, indicating simple inheritance of a single major gene controlling apospory in guineagrass. In addition, utilizing 56 AFLP primer combinations and 41 RAPD primers, 39 linkage groups and 360 simplex marker loci were assigned to the genetic map of the ‘Natsukaze’ cultivar. These markers covered 1703.5 cM of the autotetraploid guineagrass genome (2n = 4x = 32), with an average spacing of 4.7 cM. These tightly linked markers to apospory locus in guineagrass could be a powerful tool for marker‐assisted selection of apospory and map‐based cloning of the apospory gene.

Context: Pancreatic α-amylase and α-glucosidase inhibitors serve as important strategies in the management of blood glucose. Even though Syzygium cumini (L.) Skeels (Myrtaceae) (SC) is used extensively to treat diabetes; scientific evidence on antidiabetic effects of SC leaves is scarce.Objective: SC leaf extract was investigated for α-amylase inhibitory effect and continued with isolation and identification of α-amylase inhibitors.Materials and methods: Bioassay-guided fractionation was conducted using in vitro α-amylase inhibitory assay (with 20–1000 μg/mL test material) to isolate the inhibitory compounds from ethyl acetate extract of SC leaves. Structures of the isolated inhibitory compounds were elucidated using 1H NMR and 13C NMR spectroscopic analysis and direct TLC and HPLC comparison with authentic samples. Study period was from October 2013 to October 2015.Results: An active fraction obtained with chromatographic separation of the extract inhibited porcine pancreatic α-amylase with an IC50 of 39.9 μg/mL. Furthermore, it showed a strong inhibition on α-glucosidase with an IC50 of 28.2 μg/mL. The active fraction was determined to be a 3:1 mixture of ursolic acid and oleanolic acid. Pure ursolic acid and oleanolic acid showed IC50 values of 6.7 and 57.4 μg/mL, respectively, against α-amylase and 3.1 and 44.1 μg/mL respectively, against α-glucosidase.Discussion and conclusions: The present study revealed strong α-amylase and α-glucosidase inhibitory effects of ursolic acid and oleanolic acid isolated from SC leaves for the first time validating the use of SC leaves in antidiabetic therapy.