Induction of Apoptosis and Tyrosine Phosphorylation of Cellular Proteins in T Cells and Non-T Cells by Stimulation with Concanavalin A

A high concentration (30 μg/ml or more) of Con A caused the death of not only thymocytes but also splenic cells of BALB/c mice, whereas a moderate concentration (3 μg/ml) of Con A induced proliferation of these cells. A high concentration of Con A also induced the death of splenic cells of athymic BALB/c-nu/nu mice and the bone marrow cells of BALB/c mice which mainly consist of non-T cells. However, any concentration (1-30 μg/ml) of Con A failed to induce the proliferation of these cells. Specific binding of tetrameric Con A to mannose-containing receptors was required for the induction of cell death. DNA fragmentation was observed by both laser flow cytometry and electrophoresis in Con A-stimulated T cells and non-T cells. This indicated that the mechanism of induction of apoptosis with Con A is not necessarily TCR-dependent. Con A induced tyrosine phosphorylation of a number of proteins in various types of cells. Interestingly, phosphorylation of the 40 kDa protein developed only in the thymocytes and spleen cells that contain T cells, whereas phosphorylation of the 80 and 120 kDa proteins appeared in both T cells and non-T cells. These results suggested that the Con A-induced apoptosis of T cells and non-T cells involves different but possibly mutually related protein tyrosine phosphorylation-linked signals.     

Microglia with an Endothelin ETB Receptor Aggregate in Rat Hippocampus CA1 Subfields Following Transient Forebrain Ischemia

Abstract: We examined endothelin (ET) receptors in the hippocampus CA1 subfields of stroke-prone spontaneously hypertensive rats subjected to a 10-min bilateral carotid occlusion and reperfusion. When delayed neuronal death had occurred in the pyramidal cell layer at 7 days after transient forebrain ischemia, the quantitative receptor autoradiographic method we used revealed a dramatic increase in number of ¹²⁵I-ET-1 binding sites in the hippocampus CA1 subfields. The highest number of de novo binding sites appeared in the area corresponding anatomically to the pyramidal cell layer with neuronal death. These binding sites were characteristically the ET_B receptor. The de novo ¹²⁵I-ET-1 binding was mainly present on microglia aggregating with a high density in the damaged pyramidal cell layer. As ET-1- and ET-3-like immunoreactivities were highly expressed within astrocytes in damaged neural tissue, the possibility that microglia with the ET_B receptor are activated to participate in the pathophysiology of ischemia-related neural tissue damage by astrocytic ET-1 and ET-3 produced in response to transient forebrain ischemia would have to be considered.     

Effects of cumulus cells on male pronuclear formation and subsequent early development of bovine oocytes in vitro

Bovine oocytes were matured in culture with and without cumulus cells. The proportion of oocytes matured to metaphase-II 24 h after culture was not different between those matured with and without cumulus cells. When cultured oocytes were inseminated in vitro, high proportions (84 to 87%) of oocytes were penetrated, with no difference between those matured with and without cumulus cells. However, the proportion of oocytes penetrated at the male pronuclear stage was significantly higher in cumulus-intact than in cumulus-free oocytes (90 vs 31%). The proportion of oocytes cleaved beyond the 8 approximately 32-cell stage was also significantly higher in cumulus-intact than in cumulus-free oocytes. It is concluded that the cumulus cells may have an important function for normal cytoplasmic maturation of bovine oocytes in vitro.     

Methylation imprinting was observed of mouse mo-2 macrosatellite on the pseudoautosomal region but not on chromosome 9

Mouse mo-2 macrosatellites consisting of 31-bp tandem repeat units are mainly located at two loci in the C57BL/6 genome, one being at the centromere-distal telomeric region of chromosome 9 and the other at the pseudoautosomal (PA) region of chromosomes X and Y. The two clustes constitute approximately 300 kb and 150 kb, respectively. Southern analysis of a methylation-sensitive enzyme, HpaII-digested DNA showed that the mo-2 macrosatellites are detected as more than 30 polymorphic bands. Comparison of those bands between reciprocally crossed F₁ mice revealed that approximately 20% of the allele-specific fragments exhibit different band intensities depending on the sex of the parent of origin. The differential methylation is observed in the mo-2 macrosatellite on the PA region but not in that on chromosome 9. Several fragments including the 3.4-kb fragment without internal HpaII site are more clearly detected when paternally derived, suggesting that the male-derived macrosatellite is undermethylated. Interestingly the difference is much more remarkable in inter-subspecific F₁ mice between C57BL/6 and MSM than F₁ between C57BL/6 and C3H/He. This suggests the presence of a modifier(s) that affect(s) the methylation of mo-2 in the MSM genome.     

Rapid Free Radical Reduction in the Perfused Rat Liver

The reduction of nitroxide free radicals was investigated in detail by Electron Paramagnetic Resonance (EPR) spectroscopy in perfused liver. The nitroxide free radical was rapidly reduced to the corresponding hydroxylamine more efficiently at the lower flow rate of 8 [ml/min], while at higher flow rates, the amount of reduced nitroxide showed a significant decrease. Oxidation of hydroxylamine using hydrogen peroxide provided dynamic information concerning the reduction of the free radical within the liver. In addition, liver homogenates were also investigated to determine the level of nitroxide uptake. The results suggested that a portion of the infused nitroxide was taken up by the liver and cleared from the circulation.     

Cooperative Interactions of Oligosaccharide and Peptide Moieties of a Glycopeptide Derived from IgE with Galectin-9

We previously showed that galectin-9 suppresses degranulation of mast cells through protein-glycan interaction with IgE. To elucidate the mechanism of the interaction in detail, we focused on identification and structural analysis of IgE glycans responsible for the galectin-9-induced suppression using mouse monoclonal IgE (TIB-141). TIB-141 in combination with the antigen induced degranulation of RBL-2H3 cells, which was almost completely inhibited by human and mouse galectin-9. Sequential digestion of TIB-141 with lysyl endopeptidase and trypsin resulted in the identification of a glycopeptide (H-Lys13-Try3; 48 amino acid residues) with a single N-linked oligosaccharide near the N terminus capable of neutralizing the effect of galectin-9 and another glycopeptide with two N-linked oligosaccharides (H-Lys13-Try1; 16 amino acid residues) having lower activity. Enzymatic elimination of the oligosaccharide chain from H-Lys13-Try3 and H-Lys13-Try1 completely abolished the activity. Removal of the C-terminal 38 amino acid residues of H-Lys13-Try3 with glutamyl endopeptidase, however, also resulted in loss of the activity. We determined the structures of N-linked oligosaccharides of H-Lys13-Try1. The galectin-9-binding fraction of pyridylaminated oligosaccharides contained asialo- and monosialylated bi/tri-antennary complex type oligosaccharides with a core fucose residue. The structures of the oligosaccharides were consistent with the sugar-binding specificity of galectin-9, whereas the nonbinding fraction contained monosialylated and disialylated biantennary complex type oligosaccharides with a core fucose residue. Although the oligosaccharides linked to H-Lys13-Try3 could not be fully characterized, these results indicate the possibility that cooperative binding of oligosaccharide and neighboring polypeptide structures of TIB-141 to galectin-9 affects the overall affinity and specificity of the IgE-lectin interaction.     

Protective Effect of Silibinin on Learning and Memory Impairment in LPS-Treated Rats via ROS–BDNF–TrkB Pathway

Silibinin, a flavonoid derived from the herb milk thistle (Silybum marianum), has been used as a hepato-protectant in the clinical treatment of liver disease. In the present study, the effect of silibinin on lipopolysaccharide (LPS)-induced neuroinflammatory impairment in rats is investigated. Injection of LPS into lateral ventricle caused learning and memory impairment. Rats were treated with silibinin to see the effect in comparison with resveratrol as a positive control. Y-maze and Morris water maze tests showed that silibinin significantly attenuated memory damage caused by LPS treatment. At the molecular analysis, the levels of IL-1β and of IL-4 in the hippocampus were decreased and enhanced, respectively, by the treatment with silibinin. NF-κB expression was attenuated by silibinin treatment. Furthermore, generation of total reactive oxygen species (ROS) in the hippocampus was elevated in silibinin-treated groups, and so were the expressions of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB). At the same time, LPS-induced reduction of neurons in hippocampus was reversed by silibinin. In conclusion, silibinin ameliorated the impairment of learning and memory of LPS-injection rats, possibly due to the activation of ROS–BDNF–TrkB pathway in the hippocampus as well as the suppression of inflammatory response. This study gives an insight on the beneficial consequences of ROS in central nervous system. Silibinin might be a potential candidate drug for neurodegenerative diseases.     

Creation of mouse TNFR2-selective agonistic TNF mutants using a phage display technique

Tumor necrosis factor-α (TNF), which is an immuno-modulatory cytokine, has been suggested to cause inflammatory responses as well as protection against tissue dysfunction by binding two types of TNF receptor (TNFR1/TNFR2). However, the physiological effects of TNFR2-specific activation remain unclear. We therefore aimed to generate a TNF mutant with full TNFR2-selective agonist activity as a functional analytical tool. In this study, we utilized a phage display technique to create mouse TNFR2 (mTNFR2)-selective TNF mutants that bind specifically to mTNFR2 and show full bioactivity compared with wild-type TNF. A new phage library displaying TNF mutants was created, in which nine amino acid residues at the predicted receptor-binding site were randomized. From this library, an agonistic TNF mutant exhibiting high binding selectivity and bioactivity to mTNFR2 was isolated. We propose that this TNF mutant would be a powerful tool with which to elucidate the functional roles of mTNFR2.     

Molecular analysis of mutant ompR genes exhibiting different phenotypes as to osmoregulation of the ompF and ompC genes of Escherichia coli

Summary Expression of the ompF and ompC genes coding for major outer membrane proteins is osmoregulated by solutes, such as sucrose and NaCl, in the growth medium. The OmpR protein, a positive regulator of these genes, is involved in the osmoregulation (Dairi et al. 1985; Nara et al. 1984). In the present work, five mutant ompR genes exhibiting different phenotypes of osmoregulation were cloned and sequenced. Three of them, ompR1, ompR2 and ompR20, were previously isolated mutants. The others, ompR3 and ompR4, were isolated in the present work. The ompR1 mutation resulted in the deletion of 19 amino acids near the C-terminus of the OmpR protein. The ompR3 and ompR4 mutations resulted in Arg¹⁵ to Cys and Arg⁷¹ to Thr conversions, respectively, at the N-terminal portion, whereas the ompR20 and ompR2 mutations resulted in Arg¹⁵⁰ to Cys and Val²⁰⁷ to Met conversions, respectively, at the C-terminal portion. Based on these results, the structure and function of the OmpR protein are discussed in relation to the mechanism of osmoregulation.Abbreviations Tc^r tetracycline resistance - Sm^r streptomycin resistance - Cm^r chloramphenicol resistance - Km^r kanamycin resistance - SDS sodium dodecyl sulphate