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We attempted to identify the total proteome in sesame lipid droplets. Results from two-dimensional electrophoresis showed 139 protein spots in lipid droplet samples. Each spot was isolated, digested with trypsin, and applied to liquid chromatography–tandem mass spectrometry (Q-Tof Premier). As a result, 103 spots were identified. Although oleosin, caleosin, and steroleosin are known major components of the lipid droplet, many other proteins were also found in the lipid droplet. In addition to the three major proteins, TAG factor protein, glyceraldehyde-3-phosphate dehydrogenase, F1 ATPase, 70-kDa heat shock protein, seed maturation protein PM24, and 11S globulin precursor isoforms 3 and 4 were found in the lipid droplet. Three types of oleosins, 15-, 15.5-, and 17-kDa were present in the sesame lipid droplet, and the 15.5-kDa oleosin had high homology with oleosin from Coffea canephora. It has been shown by acid phosphatase treatment that oleosin proteins contain phosphate groups. Protein disulfide-isomerase 2 precursor, calreticulin-1, and BiP, which are known as marker proteins of the endoplasmic reticulum, were found as the components of the lipid droplet. Immunoconfocal microscopy was used to show that 11S globulin precursor isoform 3 and 4 were indeed localized in the lipid droplet. The presence of 11S globulin in the lipid droplets suggested a new mechanism for the lipid droplet formation.

The cDNA library prepared from Lentinula edodes, Hokken 600 (H600), primordia was screened using cDNA expressed specifically in Dictyostelium discoideum prestalk as a probe. Twenty-one clones, Le-Dd1 ~ 21, were isolated from the L. edodes primordia cDNA library. Functional analysis of each gene was carried out by transformation into protoplast cells from L. edodes Mori 252 (M252) mycelia with the overexpression vector pLG-RasF1 of each gene because M252 protoplast cells were transformed with an 11-fold higher efficiency than H600 cells. Transformants with the overexpression vector of Le-Dd10 formed a fruiting body at almost the same time as H600, a positive control, although M252, a negative control, did not form a fruiting body under culture conditions. This suggested that Le-Dd10 is involved in the formation of fruiting bodies. Single-strand conformation polymorphism analysis revealed that Le-Dd10 is located on No. 4 linkage group of L. edodes. The properties of Le-Dd10 products were investigated by Western blotting analysis using polyclonal antibodies against GST:Le-Dd10 fusion proteins. As a result, 56-kDa, 27-kDa, and 14-kDa protein bands appeared in primordial and fruiting body stages, although the expected molecular weight of the Le-Dd10 product was 50 kDa.

In this study a detection method for 2S albumin (14-kDa albumin) among the sesame allergens was developed. Polyclonal antibodies against 14-kDa albumin from sesame seeds (anti 14-kDa albumin antibody) were prepared. The anti 14-kDa albumin antibody absorbed with acetone powder from soybean seeds specifically reacted with sesame albumin, and did not react with albumins from soybean, corn, Arabidopsis. The absorbed anti 14-kDa albumin antibody was used as a probe for screening sesame varieties with scant albumins by indirect enzyme-immunosorbent assay (ELISA). We isolated accession number (Acc. No.) 526 with one-third of albumin amounts compared to standard variety Acc. No. 800. This method will be useful in breeding sesame cultivars with low sesame allergens

A cDNA clone SSJ337 (accession no. AF161253) of 1230 bp, encoding a catalytic subunit of protein phosphatase 4, was selected as one of the clones expressed specifically in prestalk cells from a cDNA library of D. discoideum slugs. Cells transformed with a knockout construct of SSJ337 showed an aberrant and tiny fruiting-body formation with a short stalk. A knockout mutant, SSJ337KO was allowed to develop much slower than a wild-type AX2 after the post-aggregation stage. This suggested that the SSJ337 cDNA clone has played an important role especially in the later development of Dictyostelium discoideum. Results from Northern blotting, analysis showed that transcripts for SSJ337 were accumulated at 16 h to 24 h after starvation began.

Sesamum mulayanum is a wild relative of cultivated sesame, Sesamum indicum, and sometimes grows in sesame crop fields as an associated weed. This species shows deep seed dormancy and is characterized by conspicuous purple pigmentation on the lower lip of the corolla. The present study examined the inheritance mode of seed dormancy by using reciprocal progeny from crosses between the two species. The seeds of S. indicum and F1 (S. indicum×S. mulayanum) showed good germination, but those of S. mulayanum and F1 (S. mulayanum×S. indicum) showed deep dormancy. The F2 seeds from both reciprocal crosses showed deep dormancy. These results, combined with the maternal inheritance of seed-coat characteristics, indicated that the seed dormancy of S. mulayanum can be attributed to its seed-coat structure (coat-enhanced dormancy). The F3 (S. indicum×S. mulayanum) seeds varied in their depth of seed dormancy and those seeds with deep dormancy (

Electrophoretic karyotype analyses of Flammulina velutipes FSB and its monokaryotic progeny, omFSB1 and omFSB2, obtained from oidia were performed by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. At least 11 chromosome-sized DNA bands (CB 1 through CB 11) for FSB, 6 bands for omFSB1, and 7 bands for omFSB2, respectively, were resolved on a CHEF gel. Southern hybridization analysis on CHEF-separated chromosomal DNA of FSB was carried out using RAPD marker probes prepared from each of the 12 linkage groups. The bands CB 1, 2, and 4 each hybridized to two or three probes for different linkage groups. The bands CB 5 and 6 both hybridized to a common probe. The bands CB 3, 7, 8, and 9 each hybridized to a single specific probe for different linkage groups. The two smallest bands (CB 10 and 11) did not hybridize with any probes.

A cell adhesion molecule, 80-kDa csA, is involved in EDTA-resistant cell contact at the aggregation stage of Dictyostelium discoideum. A 31-kDa csA was isolated from the 80-kDa csA by treatment with Achromobacter protease I. Results from thin-layer chromatography and MALDI-TOF MS analysis indicated that the 31-kDa csA contains ceramide as a component of glycosylphosphatidyl-inositol (GPI). Comparison between the 80-kDa csA and the 31-kDa csA treated with phosphatidylinositol-specific phospholipase C (PI-PLC) or GPI-specific phospholipase D (GPI-PLD) was carried out. Our results indicated that the GPI-anchor of the 31-kDa csA was more sensitive to PI-PLC treatment than that of the 80-kDa csA, and that the anchor in both was easily cleaved by GPI-PLD treatment. They suggested that the resistance of 80-kDa csA to PI-PLC treatment was due to steric hindrance and myo-inositol modification. The results of the 80-kDa csA and the 31-kDa csA treated with sphingomyelinase were similar to those with PI-PLC treatment. In the presence of 1,10-phenanthroline, a GPI-PLD inhibitor, development of Dictyostelium was markedly inhibited, suggesting that GPI-PLD is functional in developmental regulation through cell adhesion.

Megalin, a family of endocytic receptors related to the low-density lipoprotein (LDL) receptor, is a major pathway for proximal tubular aminoglycoside accumulation. We previously reported that aminoglycoside antibiotics reduce SGLT1-dependent glucose transport in pig proximal tubular epithelial LLC-PK1 cells in parallel with the order of their nephrotoxicity. In this study, using a model of gentamicin C (GMC)-induced reduction in SGLT1 activity, we examined whether ligands for megalin protect LLC-PK1 cells from the GMC-induced reduction in SGLT1 activity. We employed apolipoprotein E3 (apoE3) and lactoferrin as ligands for megalin. Then the cells were treated with various concentrations of apoE3, lactoferrin and bovine serum albumin with or without 100 μg/ml of GMC, and the SGLT1-dependent methyl α- d -glucopyranoside (AMG) uptake and levels of SGLT1 expression were determined. As a result, we demonstrated that the apoE3 significantly protects these cells from GMC-induced reduction in AMG uptake, but neither lactoferrin nor albumin does. In accord with a rise in AMG uptake activity, the mRNA and protein levels of SGLT1 were apparently up-regulated in the presence of apoE3. Furthermore, we found that the uptake of [3H] gentamicin is decreased by apoE3, and that apoE3 showed obvious protection against the GMC-dependent N-acetyl-β- d -glucosamidase (NAG) release from LLC-PK1 cells. Thus, these results indicate that apoE3 could be a valuable tool for the prevention of aminoglycoside nephrotoxicity.

The nucleotide sequence reported for the Dictyostelium discoideum ceramidase is available on the DNA Data Bank of Japan (DDBJ). Ceramidases (CDases) are currently classified into three categories (acid, neutral and alkaline) based on their optimal pHs and primary structures. Here, we report the first exception to this rule. We cloned the CDase cDNA, consisting of 2142 nucleotides encoding 714 amino-acid residues, from the slime mould, Dictyostelium discoideum. The putative amino-acid sequence indicates 32-42% identity with various neutral CDases, but does not show any similarity to the acid and alkaline CDases, indicating the enzyme should be classified as a neutral CDase. However, overexpression of the cDNA in D. discoideum resulted in increased CDase activity at an acidic, but not a neutral pH range. Knockout of the gene in slime mould eliminated CDase activity at acidic pH. The recombinant enzyme expressed in the slime mould was purified and then characterized. Consequently, the purified CDase was found to exhibit the maximal activity at approx. pH 3.0. The singular pH dependency of slime mould CDase is not derived from the specific post-translational modification in the slime mould, because the enzyme showed an acidic pH optimum even when expressed in Chinese hamster ovary cells, whereas rat neutral-CDase exhibited a neutral pH optimum when expressed in slime mould.

The cell adhesion molecule, csA, treated with lysyl endopeptidase led to a peptide fragment with a molecular weight of 31 kDa (31-kDa csA). It was shown after the treatment of the 31-kDa csA with phosphatidylinositol-specific phospholipase C and following extraction with chloroform using TLC that the 31-kDa csA contained a component of glycosylphosphatidylinositol (GPI), and it was also confirmed that the cell adhesion molecule, csA, consisted of ceramide instead of diacylglycerol. The results from GC-MS suggested that hydrocarbons of C16- C33 interacted with the GPI-anchor region. 1. Presented at the XVIII th Intenrarional Carbohydrate Symposium, Milan, Italy, July 21-26, 1996.

This paper presents further investigation of the properties of carbohydrate II in the cell adhesion molecule, contact site A, from Dictyostelium discoideum. A purified contact site A was digested with Achromobacter protease I to produce a 31-kDa fragment to which carbohydrate II was mainly bound and a 21-kDa fragment containing the NH2 terminus of contact site A, which was identified as Ala-Pro-Thr-Ile-Thr-Ala. The NH2 terminus of the 31-kDa fragment was Thr-Glu-Ala-Thr-Thr-Ser. It was estimated from the cDNA sequence data of contact site A that more than 20 Ser/Thr residues exist as target sites for the O-linked oligosaccharides in the 31-kDa fragment, but not for the N-linked oligosaccharides. These results suggest that carbohydrate II exists as clustered O-linked oligosaccharides in the COOH terminus of contact site A. The results of two-dimensional electrophoresis confirm that oligosaccharides of contact site A contain sialic acids. Immunoelectron microscopy was carried out to define the organelle in which O-glycosylation by carbohydrate II occurs and how carbohydrate II antigens are distributed on the cell surface. The results show that O-glycosylation can occur in the Golgi apparatus in D. discoideum as observed in other cells, although this O-glycosylation was inhibited by tunicamycin. Furthermore, gold particles were densely concentrated in cell-cell contact regions but sparsely distributed in noncontact regions.

This study was conducted to evaluate protective efficiency of three different protocols for vaccination in canine heartworm infection. To evaluate the three protocols of immunization, dogs were separately immunized with living larvae; 1) immunization with gamma-attenuated infective larvae, 2) with 50 micrograms/kg ivermectin-abbreviation, and 3) with chemical abbreviation plus Freund's complete adjuvant (FCA). Each group was composed of two dogs. All dogs used for this study were subcutaneously challenged with 100 intact third-stage larvae (L3) various days after the last immunization, and the worms in the pulmonary arteries and the right ventricle of the heart were recovered 17 to 25 weeks post-infection. The numbers and the sexes of the worms were determined. A mean of 38 worms was burdened in the group immunized with irradiated L3, 36 worms in the chemically-abbreviated group, but 15.5 worms in the group with chemical abbreviation plus FCA. The percentages of the protection in the former two groups were nearly 50%, but 72.3% in the group with ivermectin plus FCA. The adjuvant enhanced the protective immunity against L3 challenge. Obvious eosinophilia was observed in both immunized and control dogs except for two dogs. There was no correlation between the suppression of eosinophilia and the protective immunity in the present study.

This study examined how the addition of a sawdust extract from Castanopsis cuspidata, several aromatic compounds, and copper affected the expression of a phenol-oxidizing enzyme in the white-rot basidiomycete, Lentinula edodes. Compared to liquid media that had not been supplemented with sawdust extract (MYPG), MYPG containing low (MYPG-S100) or high (MYPG-S500) concentrations of sawdust extract had a marked effect on the promotion of mycelial growth. No manganese peroxidase (MnP) production was observed in either MYPG or MYPG-S100 media until 35 days after inoculation. However, MnP production was enhanced by culture in MYPG-S500, with a marked increase observed suddenly at 14 days after inoculation. Northern blot analysis revealed that the transcription of the lemnp2 gene coding extracellular MnP was initially observed at detectable levels at day 10 after the initial inoculation of MYPG-S500, increasing gradually thereafter until days 22-25. However, laccase (Lcc) production was not observed in any of the media until 35 days after inoculation. Addition of 10 mM aromatic compounds - 1,2-benzenediol, 2-methoxyphenol, hydroquinone, and 4-anisidine--into the MYPG-S500 medium completely inhibited MnP production and did not enhance any Lcc production. While the addition of 1 or 2 mM Cu2+ (CuSO4 x 5H2O) to MYPG-S500 medium completely inhibited MnP production, this Cu2+ addition caused a marked increase in Lcc production at 17 and 6 days after the addition, respectively.

The karyotype of Flammulina velutipes (Curt. : Fr.) Sing. was investigated using contour-clamped homogeneous electric fields (CHEF) gel electrophoresis. A parental dikaryotic stock, JA, was resolved into at least eight chromosomal DNA bands ranging from 1.4- to 4.9-megabase (Mb) pairs. Overall, little size variation was found among monokaryotic strains with a few major exceptions. Among 13 monokaryotic progenies examined, 11 strains were resolved into at least eight chromosomal DNA bands in a manner similar to the parent dikaryon, whereas the other 2 were resolved into at least seven chromosomes lacking the 2.1-Mb chromosome possessed in the former. A slightly larger size variation was found in a chromosome carrying ribosomal DNA. An estimated haploid genome size of this stock was 24.0Mb or more.

Seven differentially-expressed cDNA clones were isolated by using an mRNA differential display between a Dictyostelium wild-type AX2 and a mutant HG794 defective in O-glycosylation. Transcript levels for the seven clones were reduced or not detectable in the mutant HG794. Homology search showed that the four cDNA clones, DD-3 and DD-7~9 are novel and that three cDNA clones, DD-4 and DD-5, -6 encode an actin-bundling protein and phosphodiesterase inhibitors, respectively. Full-length cDNAs for DD-3 and -8 were isolated and labeled DD3-3 and DD8-14, respectively. DD3-3 consists of 2,166 bp and DD8-14 of 2,084 bp. DD3-3 was preliminarily reported in a previous paper [1]. SSL850 was named a clone by the “Dictyostelium cDNA Project in Japan”, containing a fulllength cDNA for DD-7 and was labeled DD7-1 of 902 bp. It has 60% homology with discoidin Ia. DD8-14 most likely has no direct role in glycosylation, while DD3-3 and DD7-1 very likely are involved in some aspect of recognition of glycosylation.

It has been believed that Dictyostelium discoideum cell membranes contain no sialic acid. In this study, however, we found that contact site A, the cell adhesion molecule of D. discoideum, is a major glycoprotein containing sialic acids. This suggests that sialic acid in non-reducing terminal plays an important role in the cell adhesion in which contact site A is involved.

Antisera against native yeast of 6 species, Candida intermedia, C. parapsilosis, C. guilliermondii, C. Iambica, Crytococcus lamentii, and Rhodotorula rubra, were prepared for use as probes in an enzyme-linked immunosorbent assay (ELISA). These antisera reacted with yeast cell surface antigens that are thought to consist of protein moieties. The qross-reactivities between the antisera and yeast of 11 species were investigated by immunofluorescent and immunoblotting methods. It was shown that ELISA using the antiserum against C. intermedia or C. parapsilosis, is a valuable means of detecting yeast in orange juice. Yeast of more than 103 were capable of being detected by ELISA.

Mutants of Dictyostelium discoideum were isolated and found to be defective in the epitope recognized by the monoclonal antibody 120 against the carbohydrate moieties of an integral membrane glycoprotein, contact site A, with the apparent molecular mass of 80 x 10(3). One mutant, HG764, did not express any contact site A and had lost cell contact resistant to EDTA. The others, including HG794, expressed a 68-kDa form of contact site A. In comparison with the parental strain HG592, HG794 showed weaker EDTA-resistant cell contact and the same degree of EDTA-sensitive cell contact. This suggested that the moieties which HG794 lacked were involved in EDTA-resistant cell contact. The 68-kDa contact site A in HG794 could be labeled with wheat germ agglutinin and incorporated [35S] sulfate. The modB mutant HL220 also expresses 68-kDa contact site A, although it cannot be labeled with wheat germ agglutinin. Therefore, the mutants HG794 and HL220 were compared by a complementation test. The diploid strain DG701 expressed 80-kDa contact site A and showed the same degree of EDTA-resistant cell contact as strain HG592. In its EDTA-resistant cell contact, HG794 was stronger than HL220. These results suggest that HG794 is a new mutant, and that there might be at least two processes in the glycosylation of 68-kDa contact site A to the 80-kDa form. The carbohydrate moieties recognized by monoclonal antibody 120 and by wheat germ agglutinin might be involved in EDTA-resistant cell contact.

Five raccoon dogs (Nyctereutes procyonoides) and two domestic dogs (Canis familiaris) were subcutaneously infected with 100 infective larvae (L3) of Dirofilaria immitis. Two and five worms, respectively, were collected from two of three raccoon dogs. Villous endarteritis was found in the raccoon dog with five worms and two dogs at 116 days after infection. The number of recovered worms in the raccoon dogs was significantly smaller than that of the domestic dogs having 22 and 29 worms, while histopathological features and the severity of the lesions in the raccoon dogs were similar to those in the domestic dogs. The vascular lesions in two chronically-infected raccoon dogs turned into much severe at 565 and 590 days after inoculation.

A novel gene, DD3-3, from Dictyostelium discoideum has been isolated by an mRNA differential display between a wild-type strain AX2 and a mutant HG794 which is defective in O-glycosylation. Functional analysis of the novel gene, DD3-3, was conducted by preparing a knockout mutant, DD3-3KO, and a GST:DD3-3 fusion protein. The mutant DD3-3KO cells were allowed to develop about 1.5 h earlier than the wild-type strain AX2 cells. Northern blotting analysis of the knockout mutant cells showed a remarkable downregulation of Reg A, cAMP-dependent phosphodiesterase, and overexpression of protein tyrosine kinase (PTK) during early development and its shutdown during late development. The relationship between O-glycosylation and phosphorylation involving Reg A gene is discussed.

The prevalence of Toxoplasma gondii was surveyed in wild boars (Sus scrofa leucomystax) and domiciled cats obtained in various areas of Amakusa Island, Kumamoto Prefecture, Japan. The antibody titers against T. gondii were measured with a latex agglutination test. Among specimens taken from 90 wild boars, 1 (1.1%) was positive and 3 (3.3%)were doubtfully positive. Among the specimens from 50 cats, none were positive and 1 (3.3%) was doubtfully positive. These results suggest that the wild boars and cats on Amakusa Island have quite low prevalence of the T. gondii infection. Continuous surveys will be needed to monitor the prevalence of Toxoplasmosis and other zoonoses in game animals.

Three differential cDNAs of Dictyostelium, not detected in the mod B mutant defective in O-glycosylation, were isolated by using an mRNA differential display. These cDNAs encode a protein tyrosine kinase, an adenylyl cyclase and a putative protein kinase C inhibitor whose expression is developmentally regulated.

Nucleotide sequences surrounding the trans-spliced leader SL1 exon in the 5S rRNA gene spacer regions of Dirofilaria immitis, Brugia malayi, and B. pahangi were determined after PCR amplification, aligned with the genus Onchocerca for comparison, and used for the prediction of secondary structures. The nucleotide sequence of this region in B. pahangi was first shown in the present study. Hypothetical secondary structures of the spacer region suggested that the SL1 transcript is capable to form a stable stem-loop structure which may render transposition of the SL1 sequence to mRNA molecules. A homologous sequence to Sm-binding site was assigned on a bulge loop. No significant difference was observed in adult worms of D. immitis irrespective of sex or location. No difference was apparent between the two species in genus Brugia.

1. 1. The relationship between glycosylation of contact site A (csA) of 80 kDa with two types of N-linked carbohydrates, I and II, and EDTA-resistant cell contact of Dictyostelium was investigated by tunicamycin treatment. 2. 2. Carbohydrate I glycosylation, involved in a shift of csA from 66 to 80 kDa, was more sensitive to tunicamycin than carbohydrate II glycosylation in its shift from 53 to 66 kDa. 3. 3. The appearance of csA of 80 kDa corresponded to that of EDTA-resistant cell contact. Carbohydrate I may be essential for EDTA-resistant cell contact. 4. 4. In starved cells treated with tunicamycin, only 4–8% of moieties labeled with wheat germ agglutinin in carbohydrate II were modified.

We examined immunological methods for the detection of microbial contaminants in fruit juice. Polyclonal antibodies against native yeast of 7 species, including Candida, Cryptococcus, Rhodotorula and Saccharomyces, and the monoclonal antibody against native C. intermedia, which was named mAb44 (IgG2a subclass), were prepared for use as probes in enzyme-linked immunosorbent assay (ELISA). These antibodies reacted with yeast cell-surface antigens that are thought to consist of protein moieties. The cross-reactivities between the antibodies and yeast antigens of 12 species were investigated by immunofluorescence and immunoblotting methods. C. intermedia polyclonal antibody showed extensive cross-reactivities with yeast antigens of 11 species. ELISA was carried out using C. intermedia polyclonal antibody and mAb44. Yeast cells (104-107) suspended per 100μl of fruit juice or PBS could be quantitatively measured using polyclonal antibody, although quantitative values were not obtained in the case of mAb44. When more than 103 per 100μl yeast cells were present, they were detectable by using mAb44 as well as polyclonal antibody. These results show that ELISA is a valuable method to detect microbial contaminants in fruit juice.

Univalent antibody (Fab fragments) against sperm-binding factor inhibits the fertilization of eggs species-specifically. The sperm-binding factor was partially purified from unfertilized eggs of the sea urchin Anthocidaris crassispina by monitoring its neutralizing effect on fertilization inhibiting Fab fragments. It formed two species-specific precipitin lines by the double-immunodiffusion test and gave three main bands of protein with apparent molecular weights of 80,000, 87,000 and 225,000, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate was detected in the first and third of these protein bands.

Antisera against purified contact site A glycoprotein, with an apparent molecular weight of 80 X 10(3) (80 kDa), from Dictyostelium discoideum were raised by using Freund's adjuvant (antiserum-A) and by using Alu-Gel-S (antiserum-B) as immunoadjuvants. They were converted into Fab fragments for the cell agglutination assay. Fab fragments of antiserum-B inhibited only EDTA-stable cell contact, whereas Fab fragments of antiserum-A (Fab-A) inhibited EDTA-sensitive cell contact as well as EDTA-stable cell contact. We prepared several cell types in order to identify target antigens for the adhesion-blocking Fab-A in EDTA-sensitive cell contact or EDTA-stable cell contact. One of these cell types produced contact site A without N-glycosidically-linked carbohydrate chains. It is known that contact site A contains two kinds of N-glycosidically-linked carbohydrate chains (carbohydrates I and II, Yoshida, M., Stadler, J., Bertholdt, G., and Gerisch, G. (1984) EMBO J. 3, 2653-2670). When growth-phase cells were treated with tunicamycin (TM) at a final concentration of 2 micrograms/ml in nutrient medium (TM-pretreated cells), the cells produced contact site A without N-glycosidically-linked carbohydrate chains (53 kDa) at the normal developmental stage. These cells lacked EDTA-sensitive cell contact as well as EDTA-stable cell contact. The neutralization of the adhesion-blocking Fab-A was done by using particulate fractions from each cell type. The blocking activity in EDTA-stable cell contact was neutralized by the cell type with carbohydrate II. Taking these results into consideration, EDTA-stable cell contact may be formed by the interaction between protein moieties of contact site A and carbohydrate II. Concerning EDTA-sensitive cell contact, the blocking activity was neutralized by each cell type irrespective of TM treatment. This suggests that O-glycosidically-linked carbohydrate chains play a role in EDTA-sensitive cell contact. Moreover, the biological activity in EDTA-sensitive cell contact of TM-pretreated cells suggests that N-glycosidically-linked carbohydrate chains may also be involved in this contact.

Fab fragments against 225 000 D glycoprotein (225 K), 87 000 D protein (87 K) and 80 000 D glycoprotein (80 K) of partially purified sperm-binding factor of Anthocidaris crassispina were prepared, and their effects upon fertilizability of dejellied homologous and heterologous eggs examined. Only the 225 K Fab impaired the fertilizability of homologous, not heterologous eggs by decreasing their sperm-binding capacity. It was concluded that 225 K glycoprotein is the active core structure of the sperm-binding factor of this species. The possible participation of the other two proteins as residual ingredients of the sperm-binding factor was also discussed. Immunofluorescence studies showed that 225 K core protein is localized on the whole surface of unfertilized eggs and of fully-grown oocytes. The fluorescence disappeared following fertilization.

A substance was isolated from sperm of the sea urchin, Hemicentrotus pulcherrimus . When unfertilized eggs of homologous species were pre-treated with this substance, they rapidly lost the fertilizability due to loss of the sperm-binding capacity. Such an effect was not exerted upon eggs either in CaMg free sea water or of heterologous species. This substance caused neither iso-agglutination of eggs nor precipitation of jelly coat. When it was pre-incubated with the sperm-binding factor purified from eggs of homologous species, it lost the fertilization-inhibiting effect on eggs. It seems very likely that a complementary relationship exists between the present substance and the sperm-binding factor.

Papain-digested, anti-sperm-binding factor serum which has only a species-specific antibody, deprives the egg of fertilizability as well as does undigested serum. This effect is shown to be exerted by direct masking of species-specific sperm-binding sites on the vitelline layer by univalent antibodies.

We isolated mutants defective in aggregation (aggregation-less) by mutagenizing the "double-bypass" mutant HG592 of Dictyostelium discoideum as the parental strain. One of the mutants expressed the contact site A glycoprotein with an apparent molecular weight of 80 X 10(3) on the cell surface in the normal developmental stage and retained EDTA-stable cell contact as well as EDTA-sensitive cell contact. However, the mutant failed to aggregate on agar plates with bacteria. This mutant was designated HG700. We could not identify any differences between this mutant and the parental strain in levels of adenylate cyclase or extracellular phosphodiesterase activity, or in its chemotaxis toward cAMP. The mutant had greatly decreased the incorporation of [35S] sulfate into the particulate fractions of the cells starved for 6 h. This suggests that the modification by sulfation may crucially affect the mechanism of cell aggregation.

We determined the carbohydrate structures of contact site A from Dictyostelium discoideum. The carbohydrate moieties of contact site A were released by hydrazinolysis. Fractionation of the deacidified oligosaccharide mixture by Bio-Gel P-4 column chromatography revealed that it was composed of four major oligosaccharides. Their respective structures were determined by sequential exoglycosidase digestion. It is known that contact site A consists of two kinds of carbohydrates, I and II. Taking together the previous and the present results, it was deduced that carbohydrate I comprises N-linked oligosaccharides and carbohydrate II O-linked ones. Furthermore, the relative molar contents of GalNAc and GlcNAc in reducing terminal suggested that contact site A contains 67% of N-linked and 33% of O-linked oligosaccharides.

A sperm-binding factor, which seems to have a primary role in binding sperm to the egg, was isolated from the egg surface of Hemicentrotus pulcherrimus and purified by monitoring the neutralization of the fertilization inhibition exerted by Fab fragments against crude sperm-binding factor. An improved purification for this sperm-binding factor is described in the present paper. The preparation of purified sperm-binding factor revealed one major protein band with an apparent molecular weight of 61,000 after sodium dodecylsulfate-polyacrylamide gel electrophoresis. A substance with the fertilization inhibitory effect on sperm, was isolated by diethylaminoethyl (DEAE)-Sephadex column chromatography in the course of purification of the sperm-binding factor and termed "sperm attack molecule." One precipitin line was formed between the sperm attack molecule and anti-crude sperm-binding factor serum in a double-immunodiffusion test. Fab fragments were prepared against partially purified sperm-binding factor or sperm attack molecule, and the effect of these Fab fragments on eggs was investigated. Anti-sperm-binding factor Fab fragments inhibited the fertilizability of eggs, whereas anti-sperm attack molecule Fab fragments did not. However, anti-sperm attack molecule Fab fragments impaired elevation of the vitelline layer. It is possible that the sperm attack molecule prevents polyspermy. Sperm attack molecule contains 3.7% neutral sugars. Its inhibitory effect was cancelled by trypsin or heat.

The functions of type 1 and 2 carbohydrates of the contact site A (csA) glycoprotein of Dictyostelium discoideum have been investigated using mutants lacking type 2 carbohydrate. In two mutant strains, HG220 and HG701, a 68-kd glycoprotein was synthesized as the final product of csA biosynthesis. This glycoprotein accumulated to a much lower extent on the surfaces of mutant cells than the mature 80-kd glycoprotein did in wild-type cells. There was also no accumulation of the 68-kd glycoprotein observed within the mutant cells nor was a precursor of lower molecular mass detected, in accordance with previous findings that indicated cotranslational linkage of type 1 carbohydrate by N-glycosylation. Pulse-chase labelling showed that a 50-kd glycopeptide was cleaved off from the mutant 68-kd glycoprotein and released into the medium, while the fully glycosylated 80-kd glycoprotein of the wild type was stable. These results assign a function to type 2 carbohydrate in protecting the cell-surface-exposed csA glycoprotein against proteolytic cleavage. HG220 cells were still capable of forming EDTA-stable contacts to a reduced extent, consistent with the low amounts of the 68-kd glycoprotein present on their surfaces. Thus type 1 rather than type 2 carbohydrate appears to be directly involved in intercellular adhesion that is mediated by the csA glycoprotein. Tunicamycin-treated wild-type and mutant cells produce a 53-kd protein that lacks both type 1 and 2 carbohydrates. While this protein is stable and not transported to the cell surface in the wild type, it is cleaved in the mutants and fragments of it are released into the extracellular medium. These results suggest that the primary defect in the two mutants studied is relief from a restriction in protein transport to the cell surface, and that the defect in type 2 glycosylation is secondary.

In an effort to identify and characterize genes expressed during multicellular development ill Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism.

Japans social security expenditure has been increasing rapidly. Its ratio against the national income reached to as high as 19.6% in fiscal year 1999. The interdependency between Japans macro-economy and her social security system is now obvious and cannot be neglected when we discuss possible reform plans of the system. To take it into account empirically, we have built a macro-economic model of the Japanese economy that places special emphasis on the social security system, which consists of public pension, medical care, social welfare and long-term care. In this paper, we have conducted several policy simulations of pension reform plans by using the model. Main conclusions are as follows. a. The Employees Pension ( Kosei Nenkin ) is considered to be sustainable if the 1999 Pension Reform is fully realized as scheduled. b. On the other hand, the sustainability of the National Pension ( Kokumin Nenkin ) remains doubtful even if the 1999 reform is fulfilled. c. Switching the revenue source of the Basic Pension (=National Pension) from a mixture of taxes and social security contributions to taxes only by increasing the consumption tax, may raise Japans potential economic growth rate. It is because that the change would make it possible to reduce the contribution rates and to raise the real wage, which would result in increasing the labor supply especially of women and elders. This proposal would deserve full consideration at least from the standpoint of the potential growth rate.