Analysis of genes specific to the early maturation stage of Sesamum indicum seeds by subtraction method*,*.

The mechanisms regulating the content ratio of unsaturated fatty acid in sesame oil need to be clarified in order to breed novel varieties with high contents of unsaturated fatty acids. Full-length cDNA libraries prepared from sesame seeds 1 to 3 weeks after flowering were subtracted with cDNAs from plantlets of 4 weeks after germination. A total of 1545 cDNA clones was sequenced. The functions of novel genes expressed specifically during the early maturation of sesame seeds were investigated by the transformation of Arabidopsis thaliana. Thirteen genes for a transcription factor were identified, four of which were involved in ethylene signaling. Fifty-nine genes, including those for the aquaporin-like protein and ethylene response factor, were analyzed by overexpression in A. thaliana. The overexpression of novel genes and the aquaporin-like protein gene in A. thaliana increased the content of unsaturated fatty acids. The localization of these products was investigated by the induction of the expression vectors for the GFP fusion protein into onion epidermal cells and sesame root cells with a particle gun. As a result, two cDNA clones were identified as good candidate genes to clarify the regulation in the yield and the ratio of unsaturated fatty acids in sesame seeds. Sein60414 (Accession No. LC603128), an intrinsic membrane protein, may be involved in the increase of unsaturated fatty acids, and Sein61074 (Accession No. LC709278) MAP3K δ-1 protein kinase in the regulation of the total ratio of unsaturated fatty acids in sesame seeds.

A novel gene, Le-Dd10, is involved in fruiting body formation of Lentinula edodes.

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.

Proteomic Analysis of Lipid Droplets in Sesamum indicum.

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.

Histopathological comparison of pulmonary artery lesions between raccoon dogs (Nyctereutes procyonoides) and domestic dogs experimentally infected with Dirofilaria immitis.

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.

Apolipoprotein E3 (apoE3) safeguards pig proximal tubular LLC-PK1 cells against reduction in SGLT1 activity induced by gentamicin C.

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 microg/ml of GMC, and the SGLT1-dependent methyl alpha-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-beta-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.

Cleavage with phospholipase of the lipid anchor in the cell adhesion molecule, csA, from Dictyostelium discoideum.

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.

A neutral ceramidase homologue from Dictyostelium discoideum exhibits an acidic pH optimum.

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.

Partial Purification of the Sperm-binding Factor from the Egg of the Sea Urchin, Anthocidaris Crassispina, Followed by an Immunological Method: (sperm-binding factor/sea urchin egg/Fab fragments/neutralizing activity/purification).

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.

EFFECT OF PAPAIN-DIGESTED, UNIVALENT ANTIBODY AGAINST SPERM-BINDING FACTOR ON THE FERTILIZABILITY OF SEA URCHIN EGGS.

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.

Prevalence of anti-Toxoplasma gondii antibody in hunter-killed wild boars, Sus scrofa leucomystax, on Amakusa Island, Kumamoto Prefecture, Japan.

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.

Phenol-oxidizing enzyme expression in Lentinula edodes by the addition of sawdust extract, aromatic compounds, or copper in liquid culture media.

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.