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.     

Polymorphism, Heteroplasmy, Mitochondrial Fusion and Diabetes

Mitochondrial DNA (mtDNA) is highly susceptible to mutations that result in polymorphisms and diseases including diabetes. We analyzed heteroplasmy, polymorphisms related to diabetes, and complementation by fusogenic proteins. Cytoplast fusion and microinjection allow, defects in mutated mtDNA inside a heteroplasmic cell to be complemented by fusing two mitochondria via human fusogenic proteins. We characterized three hfzos as well as two OPAls that prevent apoptosis. Two coiled coil domains and GTPase domains in these fusogenic proteins regulate membrane fusion. The hfzo genes were expressed mainly in the brain and in muscle that are postmitotic, but not in the pancreas. Under the influence of polymorphisms of mtDNA and nDNA, the vicious circle of reactive oxygen species and mutations in cell can be alleviated by mitochondrial fusion.     

Crystal structures of bacterial lipoprotein localization factors,LolA and LolB

Lipoproteins having a lipid-modified cysteine at the N-terminus are localized on either the inner or the outer membrane of Escherichia coli depending on the residue at position 2. Five Lol proteins involved in the sorting and membrane localization of lipoprotein are highly conserved in Gram-negative bacteria. We determined the crystal structures of a periplasmic chaperone, LolA, and an outer membrane lipoprotein receptor, LolB. Despite their dissimilar amino acid sequences, the structures of LolA and LolB are strikingly similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta barrel and an alpha-helical lid. The cavity represents a possible binding site for the lipid moiety of lipoproteins. Detailed structural differences between the two proteins provide significant insights into the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB and from LolB to the outer membrane. Furthermore, the structures of both LolA and LolB determined from different crystal forms revealed the distinct structural dynamics regarding the association and dissociation of lipoproteins. The results are discussed in the context of the current model for the lipoprotein transfer from the inner to the outer membrane through a hydrophilic environment.     

Energy-yielding properties of SoxB-type cytochrome bo(3) terminal oxidase: analyses involving Bacillus stearothermophilus K1041 and its mutant strains

We isolated a K17q8 mutant from K17 mutant cells of Bacillus stearothermophilus which contain SoxB-type cytochrome bo(3) as well as cytochrome bd but not SoxM-type cytochrome caa(3), which is the main terminal oxidase in B. stearothermophilus K1041. The respiration of K17q8 was highly sensitive to as little as 10 microM cyanide, indicating that the main terminal oxidase is cytochrome bo(3). The aerobic growth yield of K17q8 was lower than that of wild-type K1041, but higher than that of parental K17. The H(+)/O ratio of K17q8 was about 5, i.e. a little lower than the 6.1-6.5 of K1041, but higher than the 2.9-3.1 of K17 [Sone et al. (1999) J. Biosci. Bioeng. 87, 495-499]. Analyses of membrane fragments indicated that K17q8 contains about 0.2 nmol cytochrome bo(3) per mg membrane protein, and scarcely any subunits of cytochromes caa(3) and bd. From the membrane fraction of K17q8, cytochrome bo(3) was purified and shown to be composed of two subunits with apparent molecular masses of 56 and 19 kDa. The enzyme contained protoheme IX and heme O, as the main low-spin heme and high-spin heme. Analysis of the substrate specificity indicated that the high-affinity site is very specific to cytochrome c-551, a cytochrome c which is a membrane-bound lipoprotein of thermophilic Bacillus. The I(50) of purified cytochrome bo(3) was determined to be 4 microM, indicating that cytochrome bo(3) among the three terminal oxidases in B. stearothermophilus was most susceptible to cyanide. The respiration of K17q8 was mostly inhibited by the addition of cyanide at this concentration.     

Purification and characterization of cytochrome c-553 from Helicobacter pylori

Helicobacter pylori, a microaerophilic Gram-negative spiral bacterium residing in the human stomach, contains a small size soluble cytochrome c. This cytochrome c was purified from the soluble fraction of H. pylori by conventional chromatographies involving octyl-cellulose and CM-Toyopearl. Its reduced form gave an alpha absorption band at 553 nm, and thus the cytochrome was named H. pylori cytochrome c-553. The cytochrome, giving a band below 10,000 Da upon SDS-PAGE, was determined to have a mass of 8,998 by time of flight mass spectroscopy. Its N-terminal peptide sequence was TDVKALAKS---, indicating that the nascent polypeptide was cleaved to produce a signal peptide of 19 amino acid residues and a mature protein composed of 77 amino acid residues. The cb-type cytochrome c oxidase oxidized ferrocytochrome c-553 of this bacterium actively (V(max) of about 250 s(-1)) with a small K(m) (0.9 microM). Analysis of the effect of the salt concentration on the oxidase activity indicated that oxidation of cytochrome c-553 is highly inhibited under high ionic conditions. The amino acid sequence of H. pylori cytochrome c-553 showed the closest similarity to that of Desulfovibrio vulgaris cytochrome c-553, and these sequences showed a weak relationship to that of the cytochrome c(8)-group among class I cytochromes c.     

Biologically active oligodeoxyribonucleotides. Part 12: N2-methylation of 2'-deoxyguanosines enhances stability of parallel G-quadruplex and anti-HIV-1 activity

2'-Deoxyguanosine residues of a 3',5'-end-modified hexadeoxyribonucleotide (R-95288) with anti-HIV-1 activity were substituted with N2-methyl-2'-deoxyguanosine (m2dG). These modified oligodeoxyribonucleotides (ODNs) showed a 2-fold higher activity than R-95288. Also, the CD spectra of these ODNs indicated that the m2dG modification stabilized the tertiary structure of the G-quadruplex.     

Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts

For introducing regions of human chromosomes greater than a megabase into cells or animals, we have developed a chromosome-cloning system in which defined regions of human chromosomes can be cloned into a stable human minichromosome vector in homologous recombination-proficient chicken DT40 cells. The stable minichromosome vector allowed a 10 Mb-sized region of the mitotically unstable human chromosome 22 to be stably maintained in mouse embryonic stem (ES) cells, and in mice. Furthermore, we demonstrated functional expression of human genes from the HAC in mice. This study describes a stable cloning and expression system for greater than megabase-sized regions of human chromosomes.     

Localization of the Xyloglucan in Cell Walls in a Suspension Culture of Tobacco by Rapid-Freezing and Deep-Etching Techniques Coupled with Immunogold Labelling

Localization of xyloglucan in cell walls regenerated from tobaccoprotoplasts (Nicotiana tabacum L.; cv. BY-2) is visualized byrapid-freezing and deep-etching (RFDE) electron microscopy coupledwith immunogold electron microscopy. Xyloglucan was alreadydeposited in the cell wall 3 h after culture initiation. Xyloglucanwas mainly localized along microfibrils with a lesser amountin intersections between two crossed microfibrils in 120-hour-oldcells. These data support the previous hypothesis of Keegstraet al. (1973) that propose an interconnection between xyloglucanand cellulose. (Received May 22, 1998; Accepted July 13, 1998)