The endosome-lysosome system in the absorptive cells of goldfish hindgut

Summary The vacuolar system in the absorptive cells of the goldfish hindgut was studied by rapid freeze-substituted and cytochemical techniques. The apical cytoplasm of the absorptive cells contained two types of vacuoles: endosomes and lysosomes. The former were characterized by an absence of acid phosphatase activity, a dot-like distribution of material at the peripheral rim, the labelling of the inner surface with horseradish peroxidase (HRP), and by frequent connections to cytoplasmic tubules (CT), which were also free of acid phosphatase activity. The latter vacuole was preferentially located in the deeper cytoplasm and was characterized by the presence of acid phosphatase activity, an electron-dense interior matrix, a peripheral electron-lucent region (a halo), and by the detachment of HRP from the inner surface. Connections between CTs and these latter vacuoles were rarely seen. In the deeper cytoplasm, fusion between endosomes and lysosomes was sometimes observed. These results suggest that the vacuoles which are associated with CTs are endosomes, but not lysosomes, and that internalized materials are transported through the endosome-lysosome system to a giant food vacuole in the cell.     

Batchwise assessment of porcine embryos for cryotolerance

The viability or developmental ability of porcine embryos after slow-freezing and thawing differs depending on the embryonic stage or the batch, which is defined as a group of embryos obtained from one donor at one time. We froze porcine blastocysts in batches and assessed their cryotolerance by using two expanded blastocysts (EBs) as samples to predict the developmental potential of other blastocysts from the same batch at different stages. Two EBs from the same batch that had been separately frozen were thawed and cultured in vitro for 48 h to examine their in vitro ability to develop to the hatched blastocyst stage. Thereafter, each batch was assigned to Grade A, B, or C according to the viability of the two EBs, i.e., 100% viability (2/2: number of hatched blastocysts/number of cultured EBs) was Grade A; 50% (1/2) was Grade B; and 0% (0/2) was Grade C. The viability of EBs after freeze-thawing and in vitro culture varied depending on the batch and was lower (31.0+/-10.2%, mean+/-S.E.M.; P<0.01) than that of unfrozen controls (96.8+/-2.3%). The viability of frozen-thawed hatched blastocysts (HBs) did not differ among the graded batches, but the blastocyst diameter decreased (from 409 to 326 microm) as the batch grade decreased (from A to C). When both EBs and HBs from batches of the same grade were transferred to recipients (average 11.7 EBs and 16.0 HBs per recipient), the rate of pregnancy and farrowing in recipients decreased (from 77.8% to 0%) and the number of piglets obtained decreased (from 15.3 to 0) as the batch grade decreased. However, when not only frozen-thawed EBs from Grade B or C batches, but also four helper embryos at the morula to early blastocyst stage (which were expected to support the pregnancy) were transferred, the number of piglets generated was higher from EBs from Grade B batches (16.0) than from EBs from Grade C batches (0.0). When frozen-thawed HBs and helper embryos were transferred, the number of piglets generated was higher from HBs from Grade B batches (12.7) than that from HBs from Grade C batches (1.9). After slow-freezing of porcine blastocysts, their rate of survival to the piglet stage differs batchwise, and in vitro viability assessment of sample EBs after freezing and thawing may help in assessing the post-freezing and post-thawing developmental potential of other blastocysts at different stages from the same batch.     

Net release of dissolved organic matter by the scleractinian coral Acropora pulchra

The net production of dissolved organic matter (DOM) and dissolved combined and free amino acids (DCAA and DFAA, respectively) by the hermatypic coral Acropora pulchra was measured in the submerged condition, and the production rates were normalized to the coral surface area, tissue biomass, and net photosynthetic rates by zooxanthellae. When normalized to the unit surface area, the production rates of dissolved organic carbon and nitrogen (DOC and DON, respectively) were 37 and 4.4 nmol cm^− 2 h^− 1, respectively. Comparing with the photosynthetic rate by zooxanthellae, which was measured by ¹³C-tracer accumulation in the soft tissue of the coral colony, the release rate of DOC corresponded to 5.4% of the daily net photosynthetic production. The tissue biomass of the coral colony was 178 µmol C cm^− 2 and 23 µmol N cm^− 2, indicating that the release of DOC and DON accounted for 0.021% h^− 1 and 0.019% h^− 1 of the tissue C and N, respectively. The C:N ratios of the released DOM (average 8.4) were not significantly different from those of the soft tissue of the coral colonies (average 7.7). While DFAA did almost not accumulate in the incubated seawater, DCAA was considerably released by the coral colonies at the rate of 2.1 nmol cm^− 2 h^− 1 on average. Calculating C and N contents of the hydrolyzable DCAA, it was revealed that about 20% and 50%–60% of the released bulk DOC and DON, respectively, were composed of DCAA.     

Highly pathogenic H5N1 influenza virus causes coagulopathy in chickens

Severe hemorrhage at multiple organs is frequently observed in chickens infected with highly pathogenic avian influenza (HPAI) A viruses. In this study we examined whether HPAI virus infection leads to coagulation disorder in chickens. Pathological examinations showed that the fibrin thrombi were formed in arterioles at the lung, associated with the viral antigens in endothelial cells of chickens infected intravenously with HPAI virus. Hematological analyses of peripheral blood collected from the chickens revealed that coagulopathy was initiated at early stage of infection when viral antigens were detected only in the endothelial cells and monocytes/macrophages. Furthermore, gene expression of the tissue factor, the main initiator of blood coagulation, was upregulated in the spleen, lung, and brain of HPAI virus-infected chickens. These results suggest that dysfunction of endothelial cells and monocytes/macrophages upon HPAI virus infection may induce hemostasis abnormalities represented by the excessive blood coagulation and consumptive coagulopathy in chickens.     

Lysis of Yeast Cell Wall by Enzymes from Streptomycetes

This paper deals with yeast cell-wall lytic enzymes formed by Streptomyces with regard to the connection with the cell-wall structure.

In the first place, 29 organisms of β-glucanase-producing Streptomycetes were selected among 777 strains belonging to genus Streptomyces by means of a cylinder-plate method employing the yeast glucan as a substrate. As for these organisms, the depolymerizing activity against the yeast glucan was considered to be mainly due to β-1,3-glucanase activity. Against the heat-treated cell of bakers’ yeast, the crude enzymes merely showed poor lytic activities, however, in the combined employment with some protease preparations, especially with an alkaline protease from St. satsumaensis nov. sp., a remarkable increase of the lytic activities was demonstrated. On the other hand, the intact cell wall of bakers’ yeast, or both the heat-treated and the intact cells of Sacch. cerevisiae 18.29 strain were dissolved very easily by a sole action of β-glucanase or of protease, respectively. In consequence, it seemed that the lysis occurred with different mechanisms in response to differences of substrates. On this subject, the results of investigations and discussions were described in special measure. In addition, the possibility, that some other enzymes than β-glucanase or protease might concern to the lysis of the cell wall, was also investigated and discussed.     

Absolute configuration of ceriporic acids, the iron redox-silencing metabolites produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora

Ceriporic acids are a class of alk(en)ylitaconic acids produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora. The unique function of alkylitaconic acid is the redox silencing of the Fenton reaction system by inhibiting reduction of Fe³⁺. Ceriporic acids have an asymmetric centre at carbon-3, but absolute configuration has not been determined. We have isolated a series of ceriporic acids from the cultures of C. subvermispora, and measured their NMR spectra using a chiral shift reagent. In comparison with NMR spectra of (R)-(−)- and (S)-(+)-methylsuccinic acid and those of natural and chemically synthesized racemic mixtures of ceriporic acids, we have determined the absolute configuration of ceriporic acids as (R)-3-tetradecylitaconic acid (ceriporic acid A), (R)-3-hexadecylitaconic acid (ceriporic acid B) and (R,Z)-2-(hexadec-7-enyl)-3-itaconic acid (ceriporic acid C). We herein discuss their stereoselective biosynthetic pathway and the structural diversity of fungal secondary metabolites.