The chromosome numbers of two sea anemones, Anthopleura midori and A. kurogane, were determined to be 2n=18 in both by the air-drying method. The chromosomes of A, midori are all telocentric, while the first pair of chromosomes of A. kurogane is subtelocentric and the remainders are all telocentric.

Cytogenetic features of three ascidian species were studied with specimens obtained from Korean coastal areas. The gonadal tissue was processed in order to obtain mitotic and meiotic chromosomes. The chromosome numbers of Amaroucium pliciferum turned out to be 2n=26 and n=l3 with 2sm, 3st, 8t and Halocynthia roretzi to be 2n=16 and n=8 with 6m, 2t. Diploid number of chromosomes in Dendrodoa aggregata was 64 with 32t. The karyotypes of these Korean ascidians were reported for the first time.

To clarify taxonomic status of the two sibling species, Moroco oxycephalus and M. lagowskii reproductive isolation mechanisms were investigated at sympatric area located in Kansung-up, Kosung-gun, Kangwon-do, Korea. Genetic analysis was performed to reveal mating system and intensity of Hybridization between the two species. The frequencies of hybrids were increased since 1989, and then the observed hybrid frequencies ($H_O$) did not significantly differ from the expected hybrid ($H_E$) in 1998 and 1999. However, based on histological analysis of two parents and their hybrid s gonads, the hybridizations between M. oxycephalus and M. lagowskii produced mostly fertile females but sterile males in accordance with Haldane s rule. Although it was suspected that pre- and postmating isolation mechanisms were affected between the two species, M. oxycephalus and M. lagowskii seemed to be strongly isolated with microhabitat at sympatry until 1997. Since 1998, hybrid frequencies were increased by habitat disturbance. However, their hybrid frequencies would be reduced by postmating isolation mechanisms. Therefore, the two species are considered to be distinct species recently diverged.

Genetic relationships among six species of the genus Rana from Korea were investigated by complete nucleotide sequence analyses of mitochondrial cytochrome b gene (1143 bp). Based on Kimura-2-parameter distance, the interspecific sequence differences of cytochrome b gene within the genus Rana were ranged from 7.83% to 25.00%. The genetic distances were 7.83% between R. nigromaculata and R. plancyi, 8.47% between two types of R. rugosa (type A and B), 10.42% between the brown frogs (R. amurensis and R. dybowskii), 16.11% between R. dybowskii types 1 and 2 and 12.36% between pond frogs (R. nigromaculata and R. plancyi) and R. catesbeiana. In the neighbor-joining and parsimony trees, R. catesbeiana was more closely related to pond frogs than brown frogs. R. dybowskii types 1 and 2 were considered to be at a distinct and specific level of differentiation (16.11%), while two types of R. rugosa were suspected to be at a subspecific level (8.47%).

Lynceid conchostracans of the order Laevicaudata in East Asia are reviewed. Validity of the three older species, Lynceus mandsuricus Daday, Lynceus biformis (Ishikawa), and Lynceus dauricus Thiele, is examined and discussed. Two species among them were acknowledged but L. dauricus was synonymized to L. biformis and redescribed and illustrated in the present paper, based on the materials collected from the rice-fields at 15 localities in Korea.

The pattern of variations in cone sex ratio and pollen traits such as pollen number and size was examined for two years in populations of red pines In Korea. We evaluated; (1) the relationship between tree size and cone sex ratio, (2) the relationship between year and cone sex ratio, and (3) the relationship between cone sex ratio and pollen traits. Larger trees in height and circumference at breast height produced more male as well as female cones. However, cone sex ratio was independent of these plant sizes. Across the two populations, both female cone numbers per tree and male cone numbers per new shoot increased 2.2 and 1.2 times, respectively, in 1999 compared to 1998, while the yearly pattern of male cone-bearing shoots per tree differed significantly between populations. Thus, cone sex ratio varied significantly between years in one of the two populations. Pollen traits such as the number of pollen grains and diameter did not vary significantly among populations. Pollen diameter and grain numbers per male cone were significantly negatively correlated with the cone sex ratio in two populations and one population, respectively. Trees which stopped female cone production in the first year and those which produced female cones in both years differed in the cone sex ratio and pollen size in the second year. The long duration of one reproductive episode and the potential of masting in red pines emphasize the need to conduct long-term studies on the dynamics of cone production, cone sex ratio, and simultaneous changes in pollen traits in red pines.

Effects of deamido-$\textrm{NAD}^{+}$on self-splicing of primary transcripts of the phage T4 thymidylate synthase gene (td) was investigated. The self-splicing was not affected by deamido-$\textrm{NAD}^{+}$- at concentrations up to 2 mM. However, it began to decrease at 5 mM and the formation of splicing products such as the linear intron, intron-exon 2 and exon 1-exon 2, was slightly reduced. At 20 mM the self-splicing activity was almost completely abolished. This analog of the coenzyme $\textrm{NAD}^{+}$- inhibits the self-splicing of td intron RNA although it does not possess a guanidine group in its structure. The analysis of inhibitory concentrations and structural examination suggests that the key structural features of deamido-$\textrm{NAD}^{+}$ responsible for the inhibition of splicing may be the ADP-ribose moiety.

Retinoic acid (RA) evokes pattern duplication in the regenerating salamander limb. Interestingly, it also enhances dedifferentiation in the regenerate by the morphological, histological and biochemical criteria. To examine whether there is any correlation between the RA-evoked pattern duplication and de novo protein synthetic profile in the regenerating salamander limb, especially during dedifferentiation, we analyzed stage-specific protein synthesis pattern in the normal and RA-treated regenerating limbs by metabolic labeling followed by two-dimensional gel electrophoresis. In the regenerating limbs without RA treatment, a few hundred kinds of proteins were found to be synthesized at the stage of wound healing and the total number of protein synthesized increased greatly as regeneration proceeded. The same trend was also observed in the RA-treated regenerating limbs. Interestingly, some protein spots were noted to be either newly synthesized or highly expressed by the RA treatment especially at the stage of dedifferentiation. The results shows that the enhancement of dedifferentiation state after the RA treatment correlates well with the protein synthesis profile, and suggest that those proteins are important for the RA-evoked pattern duplication in the regenerating limbs of salamander.

Nitric oxide (NO) is a reactive free radical which plays important roles in animal physiology. To investigate involvement of NO in acrosome reaction (AR), effects of drugs which modulate the intracellular NO level were examined in mouse spermatozoa. N (G)-nitro-L-arginine (L-NA), a potent inhibitor of NO synthesis, decreased AR in a reversible manner, On the other hand, sodium nitroprusside (SNP), an NO generating agent, increased spontaneous AR. Preincubation of sperm in the presence of L-NA potentiated AR after sperm transfer into plain- or SNP-media. Methylene blue, a NO scavenging agent, decreased spontaneous AR. Taken together, it is concluded that NO positively controls AR.

Yeast pheromone a-factor is a 13-amino acid peptide hormone that is synthesized as a part of a larger precursor, prepro-$\alpha$-factor, consisting of a signal peptide and a proregion of 64 amino acids. The carboxy-terminal half of the precursor contains four tandem copies of mature $\alpha$-factor. To investigate the molecular basis of intracellular sorting, proteolytic processing, and storage of the peptide hormone, yeast prepro-$\alpha$-factor precursors were heterologously expressed in rat pituitary $GH_3 cells. When cells harboring the precursor were metabolically labeled, a species of approximately 27 kD appeared inside the cells. Digestion with peptide: N-glycosidase F (PNG-F) shifted the molecular mass to a 19 kD, suggesting that the 27 kD protein was the glycosylated form as in yeast cells. The nascent polypeptide is efficiently targeted to the ER in the $GH_3 cells, where it undergoes cleavage of its signal peptide and core glycosylation to generate glycosylated pro-a-factor. To look at the post ER intracellular processing, the pulse-labelled cells were chased up to 2 hrs. The nascent propeptides disappeared from the cells at a half life of 30 min and only 10-25% of the newly synthesized, unprocessed precursors were stored intracellularly after the 2 h chase. However, about 20% of the pulse-labeled pro-$\alpha$-factor precursors were secreted into the medium in the pro-hormone form. With increasing chase time, the intracellular level of propeptide decreased, but the amount of secreted propeptide could not account for the disappearance of intracellular propeptide completely. This disappearance was insensitive to lysosomotropic agents, but was inhibited at $16^{circ}C or 20^{\circ}C$, suggesting that the turnover of the precursors was not occurring in the secretory pathway to trans Golgi network (TGN) or dependent on acidic compartments. From these results, it is concluded that a pan of these heterologous precursors may be processed at its paired dibasic sites by prohormone processing enzymes located in TGN/secretpry vesicles producing small peptides, and that the residual unprocessed precursors may be secreted into the medium rather than degraded intracellularly.

Ubiquitinated proteins in lysosomes were characterized by using two monoclonal antibodies (mAbs): LYS8-1, a mAb to lysosomal proteins, and NYA124, a mAb to ubiquitin. LYS8-1 stained lysosome-like vesicles in immunofluorescence microscopy of Amoeba proteus and Acanthamoeba castellanii. In immunoblotting, LYS8-1's antigens (LYS proteins) were detected as 68-kDa and 77-kDa proteins in A. proteus, and as 30-kDa and 39-kDa proteins in A. castellanii. In immunoprecipitation of A. castellanii, at least four distinct LYS proteins, LVS35p, LyS39p, LyS42p, and LYS46p, were detected and accumulated upon inhibition of lysosome functions but not upon that of 26S proteasome functions. They were all found to be ubiquitinated, and were recovered in the lysosome fractions in subcellular fractionation experiments. In chemical fractionation analyses, LYS35p and LYS39p were demonstrated to be peripherally associated with lysosome membrane, while LYS42p and LYS46p tightly bound to the membrane. These results suggest that the LYS proteins become associated to lysosomal membrane upon ubiquitination.

Evidence suggested that atypical antipsychotics (APs) such as clozapine show less side effects than those of typical APs such as haloperidol and sulpiride. However, little is known about chronic effects of these drugs on changes in gonadotropin releasing hormone (GnRH) mRNA expression and luteinizing hormone (LH) immunoreactivity. Male rats were divided into water-, haloperidol-, sulpiride-, and clozapine-treated groups, and these drugs were administered orally for 4 weeks. The changes in the expression of GnRH mRNA and the LH immunoreactivity were determined in the hypothalamus and pituitary, respectively, using in situ hybridization and immunohistochemistry. GnRH mRNAs were clearly expressed in the water-treated control vats. This was significantly reduced by the chronic treatments with the typical APs, especially with haloperidol, but not with atypical APs clozapine. Likewise, LH immunoreactivity was clearly stained in the control group. While its immunoreativity was significantly reduced by the chronic APs treatments, clozapine treatment showed only slight attenuation. The results show that the atypical APs clozapine has less side effects in the gonadal function than the typical APs haloperidol and the sulpiride. These results suggest that clozapine is a safer drug than the typical APs, at least in the reproductive system.

The expression of $p21^{WAF1/ClP1/SDl1}$, one of the cyclin-dependent kinase inhibitors, is regulated by a variety of transcription factors including p53 and STAT. Heat shock induces the expression of p21 in a temperature- and time-dependent manner. Although the p21 induction by heat shock has been reported to be controlled by p53, a p53-independent mechanism Is also involved. To understand the p53-independent regulation of heat shock-induced p21 expression, we searched the promoter region of p21 gene and found one or two heat shock element (HSE)-like sequences in human, rat, and mouse. Electromobility shift assay (EMSA) showed that heat shock factor (HSF) could bind to these HSE-like sequences In response to heat shock, even though to a lesser extent than to HSE. In addition, p21 promoter deletion analysis revealed that heat shock activated a p21 deletion promoter construct containing the HSE-like sequences but lacking p53-binding sites, but not a promoter construct containing neither HSE-like sequences nor the p53-responsive element. Furthermore, the p21 induction by heat shook was significantly inhibited in confluent cells in which heat shock-induced HSF activation was reduced. These results suggest that the transcriptional regulation of p21 by heat shock may be mediated through activation and binding to HSE-like sequences of HSF.

Regional distribution and relative frequency of endocrine cells in the pancreas of the red-eared slider, Trachemys scripta elegans, were investigated by immunohistochemical methods. Chromogranin (Cg) A-, serotonin-, insulin-, glucagon-, somatostatin-, bovine pancreatic polypeptide (BPP)- and human pancreatic polypeptede (HPP)-immunoreactive cells were identified in this study. Most of immunoreactive cells in the exocrine and endocrine pancreas (Langerhans islet) were generally spherical or spindle-shaped (open-typed cell), while occasionally cells round in shape (close-typed cell) were found in the basal portion or interepithelial regions of the pancreatic duct. These immunoreactive cells were located in the exocrine, endocrine pancreas and/or basal or interepithelial portion of the pancreatic duct. Serotonin-immunoreactive cells were found in the basal portion of epithelia of the pancreatic duct at a low frequency and interacinar region of the exocrine at a moderate frequency. Insulin-immunoreactive cells were found in the central portion of the endocrine pancreas, interacinar regions of the exocrine pancreas and basal portion of the epithelia of the pancreatic duct at high, moderate and low frequencies, respectively. Glucagon-immunoreactive cells were detected in the periphery of the endocrine pancreas, interacinar region of the exocrine pancreas and basal portion of the epithelia or interepithelia of the pancreatic duct at high, moderate and moderate frequencies, respectively. Somatostatin-immunoreactive cells were dispersed in the whole area of the endocrine pancreas, interacinar regions of exocrine pancreas and basal portion of the epithelia or interepithelia of the pancreatic duct at a moderate frequency. BPP- and HPP-immunoreactive cells were detected in the iinteracinar region of the exocrine pancreas at moderate and hige frequencies, respectively. However, no Cg A- and motilin-immunoreactive cells were detected in this study.