Skin test-active substance prepared from culture filtrate of Fonsecaea pedrosoi

Ethanol-precipitated substance (EP) was prepared from culture filtrate of Fonsecaea pedrosoi. EP was separated into two components by passing through a Sephadex G-50 column; the faster passing component was referred to as EP-1, the slower as EP-2. EP-1 and EP-2 were evaluated as an antigen for detecting cutaneous delayed hypersensitivity in patients with chromomycosis. EP-1 elicited positive delayed skin reactions in all of 8 patients with chromomycosis, of which 7 caused by F. pedrosoi and one by Exophiala jeanselmei. Healthy subjects, patients with sporotrichosis and patients with tinea barbae failed to react to EP-1. These results indicate that EP-1 is a useful tool for detecting cutaneous delayed hypersensitivity in patients with chromomycosis caused by F. pedrosoi. It was found that precipitin test using EP-1 as an antigen had little diagnostic value in chromomycosis. EP-2 did not show antigenic activity in both skin and precipitin reactions.     

Rapid identification of yeasts by serological methods: a combined serological and biological method.

A total of 387 yeasts from the contents of the digestive tracts of domestic animals and poultry were identified by slide agglutination tests using factor antisera and urease tests. The results of this serological test were very satisfactory with respect to accuracy and rapidity, particularly when performed in combination with concomitant physiological tests only for assimilation of inositol and potassium nitrate. It may be concluded that such a combination of serological and biological tests is very useful for identifying yeast strains from various sources.     

Phospholipase C from Clostridium novyi type A. II. Factors influencing the enzyme activity

The apparent activity of phospholipase C[EC 3.1.4.3] of Clostridium novyi type A toward phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine increased in the presence of sodium deoxycholate (SDC). The effects of divalent cations on phospholipase C activity were examined in detail at various concentrations of these cations. These effects varied with substrate. Hydrolysis of phosphatidylcholine by this enzyme significantly increased in the presence of Mg2+ or Ca2+. Hydrolysis of sphingomyelin was inhibited by Ca2+, but increased in the presence of Mg2+. Phosphatidylethanolamine-hydrolyzing activity increased only slightly in the presence of Mg2+ and Ca2+. Zn2+ rather inhibited hydrolysis of these substrates. The effects of divalent cations and detergent appear to be directly related to the physical state of the phospholipid micelles used as substrates. When phosphatidylcholine, sphingomyelin, or phosphatidylethanolamine was used as a substrate, phospholipase C activity was completely inhibited by 2.5 mM EDTA or o-phenanthroline (concentration in the final incubation mixture: 0.5 mM), and was fully restored by Zn2+ alone. Both Ca2+ and Mg2+ were ineffective for reactivation. The isoelectric point of the enzyme was 7.1 +/- 0.1.     

The regeneration of pioneer tree species under browsing pressure of Sika deer in an evergreen oak forest

The effects of Sika deer (Cervus nippon) browsing on the regeneration of pioneer species were studied in relation to canopy gaps in a warm temperate evergreen oak forest in Kasugayama, Nara City. Four study sites, three in canopy gaps and one under a closed canopy, were selected and each divided into fenced and unfenced plots. Under the closed canopy, seedlings of all the pioneer species died irrespective of browsing pressure. However, in the canopy gap sites, seedlings of the pioneer species could establish and grow well. The seedling survival ratio in the fenced plots in the canopy gaps was >60% 1 year after germination. However, in the unfenced plots, only <20% of the seedlings survived 1 year, with all dying within 3 years after germination. Thus, the regeneration of pioneer trees in this forest was strongly inhibited by deer browsing. Successful regeneration of a pioneer,Zanthoxylum ailanthoides, occurred for several years even after two major wind disturbances during the past 90 years. This may be due to less browsing pressure from the deer.     

Changes in molecular species profiles of glycosylphosphatidylinositol anchor precursors in early stages of biosynthesis

Glycosylphosphatidylinositol (GPI) anchor is a major lipidation in posttranslational modification. GPI anchor precursors are biosynthesized from endogenous phosphatidylinositols (PIs) and attached to proteins in the endoplasmic reticulum. Endogenous PIs are characterized by domination of diacyl species and the presence of polyunsaturated fatty acyl chain, such as 18:0-20:4, at the sn-2 position. In contrast, the features of mammalian glycosylphosphatidylinositol-anchored proteins (GPI-APs) are domination of alkyl/acyl PI species and the presence of saturated fatty acyl chains at the sn-2 position, the latter being consistent with association with lipid rafts. Recent studies showed that saturated fatty acyl chain at sn-2 is introduced by fatty acid remodeling that occurs in GPI-APs. To gain insight into the former feature, we analyzed the molecular species of several different GPI precursors derived from various mammalian mutant cell lines. Here, we show that the PI species profile greatly changed in the precursor glucosamine (GlcN)-acyl-PI and became very similar to that of GPI-APs before fatty acid remodeling. They had alkyl (or alkenyl)/acyl types with unsaturated acyl chain as the major PI species. Therefore, a specific feature of the PI moieties of mature GPI-APs, domination of alkyl (or alkenyl)/acyl type species over diacyl types, is established at the stage of GlcN-acyl-PI.     

DNA probes for identifying chromosomes 5, 6, and 7 of Schistosoma mansoni

Schistosoma mansoni has a genome of 270 Mb contained on 8 pairs of chromosomes. C-banding has been a useful technique in identifying the 7 autosomal and sex chromosomes. However, even with C-banding, S. mansoni chromosomes 5, 6, and 7 are difficult to discriminate from each other, because of their small sizes, morphological similarity, and poor banding patterns. We have identified probes that specifically paint chromosomes 5, 6, and 7 of S. mansoni with the use of chromosome microdissection and the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). Exact chromosome identification is required for accurate chromosome mapping of genomic clones and genetic elements, which is an essential component of the schistosome genome project.     

Transposon-tagged mutagenesis in the rat

Although the laboratory rat (Rattus norvegicus) is an indispensable experimental animal for biomedical research and drug development, the lack of embryonic stem cell lines hampers gene-knockout studies. Here we report the successful generation of insertional mutant rats using the Sleeping Beauty (SB) transposon system. This would benefit a variety of biomedical research fields for which the rat model is better suited than the mouse model.     

Myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction prepared from rat pituitary

Pituitary gland is a well-known endocrine tissue. The hypothalamo-neurohypophysial system, containing arginine vasopressin and oxytocin, shows a reversible morphological reorganization of both neurons and glial cells during chronic physiological stimulations. Since many signal transducing and cell adhesion molecules (CAMs) are recovered in membrane microdomain (MD) fractions, MDs are considered as signaling platforms of cells. In order to know the molecular background for these endocrine systems, we characterized MD-components derived from rat pituitary and found specific enrichment of several proteins in the fraction. One of them was identified as myelin protein zero (P0) with mass analysis and this result was further confirmed by a result that a specific antibody to this protein reacted to the authentic P0 protein in the myelin fraction of rat sciatic nerve. P0 is one of type-I transmembrane CAMs and a major structural component of mammalian peripheral nerve myelin. In mammals, expression of P0 has been considered to be restricted to peripheral nervous system. This result however indicates that P0 expresses more widely and its enrichment in the MD-fraction from rat pituitary suggests the participation in cell-cell communications.     

Design and synthesis of potent myostatin inhibitory cyclic peptides

Myostatin is a negative regulator of skeletal muscle growth and myostatin inhibitors are promising lead compounds against muscle atrophic disorders such as muscular dystrophy. Previously, we published the first report of synthetic myostatin inhibitory 23-mer peptide 1, which was identified from a myostatin precursor-derived prodomain protein. Our structure-activity relationship study afforded the potent inhibitory peptide 3. In this paper, we report an investigation of the synthesis of conformationally-constrained cyclic peptide based on the linear peptide 3. To examine the potency of side chain-to-side chain cyclized peptides, a series of disulfide-, lactam- and diester-bridged derivatives were designed and synthesized, and their myostatin inhibitory activities were evaluated. The diester-bridged peptide (11) displayed potent inhibitory activity with an in vitro IC₅₀ value of 0.26?µM, suggesting that it could serve as a new platform for development of cyclic peptide inhibitors.