Increased binding and chemotactic capacities of PDGF-BB on fibroblasts in radiation pneumonitis

Although pulmonary fibrosis is a frequent and serious consequence of radiotherapy for thoracic malignant diseases such as lung cancer, the pathogenesis of this radiation-induced lung disorder remains unclear. To clarify the mechanisms underlying radiation pneumonitis and pulmonary fibrosis, we investigated the expression of platelet-derived growth factor receptor (PDGFR) on fibroblasts obtained from irradiated rat lungs and on control fibroblasts. Whole lungs of male Wistar rats were irradiated with a single dose of 15 Gy, and lung fibroblasts were isolated at 4 weeks after the irradiation. The chemotactic response of irradiated lung fibroblasts to PDGF-BB was significantly higher than that of control lung fibroblasts, whereas there was no significant difference between irradiated lung fibroblasts and control lung fibroblasts in the response to PDGF-AA. Receptor binding assay showed more specific binding sites for PDGF-BB on irradiated lung fibroblasts than on control lung fibroblasts, and the displacement of (125)I-labeled PDGF binding to fibroblasts by unlabeled PDGF showed that (125)I-labeled PDGF-BB was displaced by PDGF-BB but not by PDGF-AA. These results suggest that the increased binding sites for PDGF-BB on irradiated lung fibroblasts correspond mainly to PDGFRB. Scatchard analysis of the saturation data demonstrated an approximately twofold increase both in the number of PDGF-BB binding sites and in the binding affinity in irradiated lung fibroblasts compared to that in control lung fibroblasts. Those results suggest that the increased chemotactic response of irradiated lung fibroblasts to PDGF-BB is related to the overexpression of PDGFRB, which may have an important role in the pathogenesis of radiation-induced pneumonitis and pulmonary fibrosis.     

Possibility of cytoplasmic pre-tRNA splicing: the yeast tRNA splicing endonuclease mainly localizes on the mitochondria

Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.     

Induced expression of sarcotoxin IA enhanced host resistance against both bacterial and fungal pathogens in transgenic tobacco

We demonstrate here that induced expression of sarcotoxin IA, a bactericidal peptide from Sarcophaga peregrina, enhanced the resistance of transgenic tobacco plants to both bacterial and fungal pathogens. The peptide was produced with a modified PR1a promoter, which is further activated by salicylic acid treatment and necrotic lesion formation by pathogen infection. Host resistance to infection of bacteria Erwinia carotovora subsp. carotovora and Pseudomonas syringae pv. tabaci was shown to be dependent on the amounts of sarcotoxin IA expressed. Since we found antifungal activity of the peptide in vitro, transgenic seedlings were also inoculated with fungal pathogens Rhizoctonia solani and Pythium aphanidermatum. Transgenic plants expressing higher levels of sarcotoxin were able to withstand fungal infection and remained healthy even after 4 weeks, while control plants were dead by fungal infection after 2 weeks.     

Glutamate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1: enzymatic characterization, identification of the encoding gene, and phylogenetic implications

NADP-dependent glutamate dehydrogenase (l-glutamate: NADP oxidoreductase, deaminating, EC 1.4.1.4) from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) was purified to homogeneity for characterization. The enzyme retained its full activity on heating at 95°C for 30 min, and the maximum activity in l-glutamate deamination was obtained around 100°C. The enzyme showed a strict specificity for l-glutamate and NADP on oxidative deamination and for 2-oxoglutarate and NADPH on reductive amination. The K _m values for NADP, l-glutamate, NADPH, 2-oxoglutarate, and ammonia were 0.039, 3.3, 0.022, 1.7, and 83 mM, respectively. On the basis of the N-terminal amino acid sequence, the encoding gene was identified in the A. pernix K1 genome, cloned, and expressed in Escherichia coli. Analysis of the nucleotide sequence revealed an open reading frame of 1257 bp starting with a minor TTG codon and encoding a protein of 418 amino acids with a molecular weight of 46 170. Phylogenetic analysis revealed that the glutamate dehydrogenase from A. pernix K1 clustered with those from aerobic Sulfolobus solfataricus, Sulfolobus shibatae, and anaerobic Pyrobaculum islandicum in Crenarchaeota, and it separated from another cluster of the enzyme from Thermococcales in Euryarchaeota. The branching pattern of the enzymes from A. pernix K1, S. solfataricus, S. shibatae, and Pb. islandicum in the phylogenetic tree coincided with that of 16S rDNAs obtained from the same organisms. Received: April 24, 2000 / Accepted: August 10, 2000     

Responses of immunocompetent cells in the dental pulp to replantation during the regeneration process in rat molars

Responses of immunocompetent cells to tooth replantation during the regeneration process of the dental pulp in rat molars were investigated by immunocytochemistry using antibodies to class II major histocompatibility complex (MHC) molecules (OX6 antibody), monocyte/macrophage lineage cells (ED1 antibody) and protein gene product 9.5 (PGP 9.5), as well as by histochemical reaction for periodic acid-Schiff (PAS). Tooth replantation caused an increase in both the number of OX6- and ED1-positive cells and their immunointensity in the replanted pulp, but almost all PGP 9.5-immunoreactive nerves diminished in the initial stages. By postoperative day 3, many OX6- and ED -immunopositive cells had accumulated along the pulp-dentin border to extend their cytoplasmic processes into the dentinal tubules in successful cases. Once reparative dentin formation had begun after postoperative day 7, OX6- and ED1-immmunopositive cells became scattered in the odontoblast layer, while reinnervation was found in the coronal pulp. The temporal appearance of these immunocompetent cells at the pulp-dentin border suggests their participation in odontoblast differentiation as well as in initial defense reactions during the pulpal regeneration process. On postoperative day 14, the replanted pulp showed three regeneration patterns: (1) reparative dentin, (2) bone-like tissue formation, and (3) an intermediate form between these. In all cases, PAS-reactive cells such as polymorphonuclear leukocytes (PML) and mesenchymal cells occurred in the pulp space. However, the prolonged stagnation of inflammatory cells was also discernible in the latter two cases. Thus, the findings on PAS reaction suggest that the migration of the dental follicle-derived cells into the pulp space and the subsequent total death of the proper pulpal cells are decisive factors for eliciting bone-like tissue formation in the replanted pulp.     

Nitric oxide nitrates tyrosine residues of tumor-suppressor p53 protein in MCF-7 cells

It has been reported that mammalian cells incubated with excess nitric oxide (NO) accumulate p53 protein but concomitantly this p53 loses its capacity for binding to its DNA consensus sequence. As nitration of tyrosine residues in various proteins has been shown to inhibit their functions, we examined whether NO nitrates tyrosine residues in p53 protein. MCF-7 cells expressing wild-type p53 were incubated with S-nitrosoglutathione for 4 h and cellular extracts were immunoprecipitated with an anti-p53 antibody. Western blot analyses of immunoprecipitates for p53 or for nitrotyrosine revealed low levels of nitrotyrosine in p53 from untreated cells. Incubation with 2 mM S-nitrosoglutathione induced a significant increase in the nitrotyrosine level in p53 protein compared to nontreated cells. These results suggest that excess NO produced in inflamed tissues could nitrate p53 protein, playing a role in carcinogenesis by impairing functions of this tumor-suppressor protein.     

Characteristics and Efficiency of Glutamine Production by Coupling of a Bacterial Glutamine Synthetase Reaction with the Alcoholic Fermentation System of Baker’s Yeast

Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker’s yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain.