Kimie Yamazaki

Quin2 protects against neuronal cell death due to Ca2+ overload

Quin2-acetoxymethylester (quin2/AM) (50 microM), administered directly to the motoneuronal pool of the frog spinal cord, could be loaded into the motoneuron as well as the other cells in the lumbar region. Depolarizing responses of the ventral root to L-glutamate in the quin2-loaded side persisted even after prolonged exposure to A23187 (2.0 microM), while the responses in the unloaded side were markedly reduced. Histologically confirmed neuronal cell loss from the motoneuronal pool induced by A23187 (2.0 microM) or by a high concentration of L-glutamate (10 mM) was prevented by pretreatment with quin2/AM. A23187- and L-glutamate-induced histological and functional damage in neuronal cells and the protective effects of quin2 on them provide further evidence for cell death due to Ca2+ overloading.

CELL CYCLE STUDY UP TO THE TIME OF HATCHING IN THE EMBRYOS OF THE SEA URCHIN, HEMICENTROTUS PULCHERRIMUS*

Cell cycles have been analyzed in 10 divisions up to the time of hatching in the embryos of the sea urchin, Hemicentrotus pulcherrimus. In the first 5 cleavages, division synchrony is very high. On the average, TGC= 55.4 min, TG1= 0 min, Ts= 12 min, TG2=±0 min, TM= 42 min. In the remaining 5 cleavages, TGC becomes longer: 70 min for the 7th to 246 min for the 10th cleavage. G1 and G2 become definitely recognizable and become longer along with Ts. TM stays more or less constant. Plots of the changing lengths of the four compartments (G1, S, G2, M) on the Y‐axis against TGC (X‐axis) can be fitted to the following 4 regression equations; TG1= 0.28TGC ‐ 19.7, Ts= 0.609TGC ‐ 15.2, TG2= 0.104TGC ‐ 4.72 and TM= 0.007TGC+ 39.6.

Implanting mouse embryo stain with a LNF-I bearing fluorescent probe at their mural trophectodermal side

Mouse embryos at implantation stage were stained successfully with lacto-N-fucopentaose I (LNF-I) bearing neoglycoprotein labeled with rhodamine synthesized by us for the first time. The fluorescent neoglycoproteins carrying LNF-II, -III, LND-I, or LNT failed to stain the embryos. The embryo was stained only at the cell surface of trophectoderm at the mural side. Since the attachment of the mouse embryo to the uteric epithelium occurs at its mural side trophectoderm and LNF-I is the key substance in mouse implantation (Lindenberg, S. et al, (1988) J. Reprod. Fert. 83, 149-158), the material stained with the probe carrying LNF-I appears to be the molecule responsive to attachment to the endometrium surface and leading to implantation.

Kinetics of DNA replication in the Indian muntjac chromosomes as studied by quantitative autoradiography

DNA replication patterns of individual chromosomes and their various euchromatic and heterochromatic regions were analyzed by means of quantitative autoradiography. The cultured cells of the skin fibroblast of a male Indian muntjac were pulse labeled with 3H-thymidine and chromosome samples were prepared for the next 32 h at 1–2 h intervals. A typical late replication pattern widely observed in heterochromatin was not found in the muntjac chromosomes. The following points make the DNA replication of the muntjac chromosomes characteristics: (1) Heterochromatin replicated its DNA in a shorter period with a higher rate than euchromatin. (2) Two small euchromatic regions adjacent to centromeric heterochromatin behaved differently from other portions of euchromatin, possessing shorter Ts, higher DNA synthetic rates and starting much later and ending earlier their DNA replication. (3) Segmental replication patterns were observed in the chromosomes 2 and 3 during the entire S phase. (4) Both homologues of the chromosome 3 showed a synchronous DNA replication pattern throughout the S phase except in the distal portion of the long arms during the mid-S phase.

Trypsin-like Hatching Enzyme of Mouse Blastocysts: Evidence for Its Participation in Hatching Process before Zona Shedding of Embryos'

Effects of twelve protease inhibitors on hatching of mouse embryos were investigated. Mouse hatching was strongly or moderately inhibited by trypsin inhibitors including p‐toluenesulfonyl‐Lys‐CH2Cl (TLCK) and chicken ovomucoid, while inhibitors for chymotrypsin and elastase showed weak or no inhibition. These results indicate the participation of a trypsin‐like protease in the hatching of mouse embryos as a hatching enzyme., Since TLCK is the strongest and an irreversible inhibitor for the enzyme, timing of the participation of the hatching enzyme in the hatching process was examined by pulse treatment of embryos with TLCK before and during the zona shedding. The results indicated that a trypsin‐like hatching enzyme functions before, but not during, the zona shedding of embryos, especially during a 15 h period immediately before the beginning of the shedding.

CHANGES OF CHROMOSOMES DURING THE EARLY NEURAL DEVELOPMENT OF A JAPANESE NEWT, CYNOPS PYRRHOGASTER

The karyotype of Cynops pyrrhogaster was determined on the mitotic chromosomes in the presumptive neural area of an early gastrula. 24 chromosomes of a diploid set consisted of 8 metacentric and 4 submetacentric pairs. Individual chromosomes were identified on the basis of their morphology and characteristic C‐binding patterns. Sex chromosomes were not identified. Total length of the haploid chromosome set in the presumptive neural area decreased remarkably from morulae to gastrulae, further continued to decrease up to neurulae and thereafter remained unchanged till tail‐buds. Chromosome shortening occurring from morulae to gastrulae was accompanied with a prominent decrease in chromosome volume, keeping chromosome width constant. Shortening took place evenly along the longitudinal axis of a chromosome. When gastrulae and neurulae were compared concerning their positions of the appearance of the C‐bands, the basic pattern remained unchanged. In certain chromosomes, the number of C‐bands decreased as the result of their fusion, as gastrulae proceeded to neurulae.

Inhibition of Mouse Blastocyst Hatching by Subsite-Specific Trypsin Inhibitors, Peptidyl Argininals1

To explore the substrate or subsite specificity of a mouse hatching enzyme, effects of leupeptin [acetyl(P4)‐Leu(P3)‐Leu(P2)‐argininal(P1)] and its analogs (peptidyl argininals) on mouse blastocyst hatching were investigated. The compounds containing benzyloxycarbonyl group (Z) in the P4 position inhibited the hatching more strongly than those containing acetyl group or unprotected N‐terminal amino acid. Among five Z‐Leu‐P2‐argininals, a derivative containing a P2 Ser residue was the most potent inhibitor, and the derivatives containing Leu, Thr, Pro, and Gly in the P2 position followed in this order. Then, we synthesized four Z‐P3‐Ser‐argininals and tested their effects on hatching. The result indicated that the compound with Phe residue in the P3 position was the strongest inhibitor, and the Leu‐, Pro‐, and Ala‐containing derivatives were ranked in this order. Thus, among Z‐dipeptidyl‐argininals tested, Z‐Phe‐Ser‐argininal most potently inhibited the mouse embryonic hatching, suggesting the preference of the mouse hatching enzyme for Phe(P3)‐Ser(P2)‐Arg(P1) sequence as a substrate.