Ultrasonic degradation of schizophyllan, an antitumor polysaccharide produced by Schizophyllum commune Fries

Schizophyllan, a water-soluble beta-D-glucan elaborated by Schizophyllum commune Fries, was partially depolymerized by ultrasonic irradiation to a low-molecular-weight polysaccharide, designated "sonic-degraded schizophyllan". Both native and degraded polysaccharides exhibited essentially the same antitumor activities against Sarcoma-180 ascites. Both glucans are comprised solely of D-glucose residues and have a main chain of (1 leads to 3)-beta-D-glucopyranosyl residues, one out of three glucose residues being attached as single, (1 leads to 6)-beta-D-glucopyranosyl groups. Although both glucans have similar structural features, significant differences are observed in such physical properties as molecular weight and intrinsic viscosity. End-group analysis by using radioisotope-labeled glucans suggests that ultrasonic degradation occurs mainly by cleavage of glycosidic bonds of the main chain of schizophyllan. The molecular weights of the native and sonic-degraded schizophyllan were shown to be 75% of those of corresponding, original schizophyllan preparations, suggesting that there is no anomalous linkage sensitive to periodate oxidation, and ultrasonic irradiation may cause random hydrolysis of (1 leads to 3)-beta-D-glucosidic linkages in the main chain.     

Formation of Isoamylase by Pseudomonas

We have isolated a Pseudomonas sp. (strain SB15) which produces an isoamylase (EC 3.2.1.9). Highest yields of this enzyme were obtained when the bacterium was grown in shaken culture in a medium containing maltose, dextrin, starch, or isomaltose. Specific carbon and nitrogen sources were required for growth. The most satisfactory medium consisted of 2% maltose, 0.4% sodium glutamate, 0.3% diammonium hydrogen phosphate, and other inorganic salts. The optimal pH for enzyme production was 5 to 6. The enzyme is stable between pH 3 and 6 but is extremely labile above pH 7. It splits amylopectin completely by combined action with beta-amylase but does not attack pullulan.     

Mast cells are important in the development of hypersensitivity pneumonitis. A study with mast-cell-deficient mice

We have previously reported that C57B1/6 mice develop lung lesions similar to human hypersensitivity pneumonitis (HP) by repeated transnasal administration of Thermoactinomyces vulgaris antigen. Since the HP-like lesions were induced via respiratory route and by the causative antigen in human HP (farmer's lung), it seems that this murine model is useful for investigating the cell-to-cell interactions in human HP. To clarify the involvement of mast cells (MC) in the development of HP, T. vulgaris (90 micrograms/day) was transnasally administered to MC-deficient WBB6F1-W/Wv mice (W/Wv) and their littermates (+/+) five times a wk for 3 wk. When the lungs were examined by scoring pathological findings and lung indexes, HP-like lesions were significantly less severe in W/Wv than in +/+, whose lesions were equivalent to those of C57B1/6. Bone-marrow-derived cultured MC from +/+ mice (98% purity) were obtained by in vitro culture mixed with WEHI-3B-derived conditioned medium which contained IL-3. When these MC were adoptively transferred to W/Wv mice (10(7) cells/mouse), the HP-like lesions in W/Wv mice were enhanced to be as severe as those in +/+. Importantly, significant numbers of MC were found in the lungs of MC-transferred W/Wv mice. These results suggest that MC play an important role in the development of the murine experimental HP.     

Immunochemical studies on dextrans: Microheterogeneity of dextran NRRL B1397 demonstrated by precipitation with concanavalin A

Dextran NRRL B1397 containing C-2 and C-3 branching points was fractionated by successive additions of concanavalin A (Con A). Quantitative experiments of the precipitations of the fractionates with Con A and homologous rabbit anti-dextran, and on inhibition of the precipitin reaction showed that the original dextran sample was composed of a heterogeneous population of dextran molecules. This heterogeneity was confirmed by methylation studies. The fractions were found to have almost the same contents of 1→3 branches but different contents of 1→2 branches. Fractions rich in 1→2 branches reacted well with both a homologous anti-dextran serum and Con A, whereas fractions poor in 1→2 branches either reacted little or not at all with the antiserum or Con A.     

Evidence for lysophosphatidic acid 1 receptor signaling in the early phase of neuropathic pain mechanisms in experiments using Ki-16425, a lysophosphatidic acid 1 receptor antagonist

Lysophosphatidic acid is a bioactive lipid mediator with neuronal activities. We previously reported a crucial role for lysophosphatidic acid 1 receptor-mediated signaling in neuropathic pain mechanisms. Intrathecal administration of lysophosphatidic acid (1 nmol) induced abnormal pain behaviors, such as thermal hyperalgesia, mechanical allodynia, A-fiber hypersensitization, and C-fiber hyposensitization, all of which were also observed in partial sciatic nerve injury-induced neuropathic pain. Ki-16425 (30 mg/kg, i.p.), a lysophosphatidic acid 1 receptor antagonist, completely blocked lysophosphatidic acid-induced neuropathic pain-like behaviors, when administered 30 min but not 90 min before lysophosphatidic acid injection, suggesting that Ki-16425 is a short-lived inhibitor. The blockade of nerve injury-induced neuropathic pain by Ki-16425 was maximum as late as 3 h after the injury but not after this critical period. The administration of Ki-16425 at 3 h but not at 6 h after injury also blocked neurochemical changes, including up-regulation of voltage-gated calcium channel α₂δ-1 subunit expression in dorsal root ganglion and reduction of substance P expression in the spinal dorsal horn. All of these results using Ki-16425 suggest that lysophosphatidic acid 1 receptor-mediated signaling which underlies the development of neuropathic pain works at an early stage of the critical period after nerve injury.     

The preprophase band is a localized center of clathrin-mediated endocytosis in late prophase cells of the onion cotyledon epidermis

The preprophase band (PPB) marks the site on the plant cell cortex where the cell plate will fuse during the final stage of cytokinesis. Recent studies have shown that several cytoskeletal proteins are depleted at the PPB site, but the processes that bring about these changes are still unknown. We have investigated the membrane systems associated with the PPB regions of epidermal cells of onion cotyledons by means of serial thin sections and electron tomograms. In contrast with specimens preserved by chemical fixatives, our high-pressure frozen cells demonstrated the presence of large numbers of clathrin-coated pits and vesicles in the PPB regions. The vesicles were of two types: clathrin-coated and structurally related, non-coated vesicles. Quantitative analysis of the data revealed that the number of clathrin-coated pits and vesicles is higher in the PPB regions than outside of these regions. Immunofluorescent microscopy using anti-plant clathrin-antibody confirmed this result. In contrast, no differences in secretory activities were observed. We postulate that the removal of membrane proteins by endocytosis plays a role in the formation of PPB 'memory' structures.     

Mammalian Fat and Dachsous cadherins regulate apical membrane organization in the embryonic cerebral cortex

Compartmentalization of the plasma membrane in a cell is fundamental for its proper functions. In this study, we present evidence that mammalian Fat4 and Dachsous1 cadherins regulate the apical plasma membrane organization in the embryonic cerebral cortex. In neural progenitor cells of the cortex, Fat4 and Dachsous1 were concentrated together in a cell–cell contact area positioned more apically than the adherens junction (AJ). These molecules interacted in a heterophilic fashion, affecting their respective protein levels. We further found that Fat4 associated and colocalized with the Pals1 complex. Ultrastructurally, the apical junctions of the progenitor cells comprised the AJ and a stretch of plasma membrane apposition extending apically from the AJ, which positionally corresponded to the Fat4–Dachsous1-positive zone. Depletion of Fat4 or Pals1 abolished this membrane apposition. These results highlight the importance of the Fat4–Dachsous1–Pals1 complex in organizing the apical membrane architecture of neural progenitor cells.     

Palmitoylated Ras proteins traffic through recycling endosomes to the plasma membrane during exocytosis

Ras proteins regulate cell growth, death, and differentiation, and it is well established that this functional versatility is accomplished through their different subcellular localizations. Palmitoylated H- and N-Ras are believed to localize at the perinuclear Golgi and plasma membrane (PM). Notably, however, recycling endosomes (REs) also localize to a perinuclear region, which is often indistinguishable from the Golgi. In this study, we show that active palmitoylated Ras proteins mainly localize intracellularly at REs and that REs act as a way station along the post-Golgi exocytic pathway to the PM. H-Ras requires two palmitoyl groups for RE targeting. The lack of either or both palmitoyl groups leads to the mislocalization of the mutant proteins to the endoplasmic reticulum, Golgi apparatus, or the PM. Therefore, we demonstrate that palmitoylation directs Ras proteins to the correct intracellular organelles for trafficking and activity.     

N-linked glycan structures of human lactoferrin produced by transgenic rice

Human lactoferrin was produced in genetically engineered rice. N-linked glycan structures of recombinant human lactoferrin were determined. The oligosaccharides liberated by hydrazinolysis were labeled with 2-aminopyridine (PA). The PA-labeled glycans were purified by reverse-phase and size-fractionation HPLCs. The structures of these glycans were identified by HPLC, exoglycosidase digestion, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The glycan structures determined were ManFucXylGlcNAc(2) (3.4%), Man(2)FucGlcNAc(2) (2.1%), Man(3)FucGlcNAc(2) (2.5%), Man(3)FucXylGlcNAc(2) (42.5%), two isomers of Man(2)FucXylGlcNAc(2) (39.1%), Man(3)XylGlcNAc(2) (6.5%), and Man(2)XylGlcNAc(2) (3.9%).