Newly identified Chinese hamster ovary cell mutants are defective in biogenesis of peroxisomal membrane vesicles (Peroxisomal ghosts), representing a novel complementation group in mammals

We isolated peroxisome biogenesis-defective mutants from Chinese hamster ovary cells by the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) method. Seven cell mutants, ZP116, ZP119, ZP160, ZP161, ZP162, ZP164, and ZP165, of 11 P9OH/UV-resistant cell clones showed cytosolic localization of catalase, a peroxisomal matrix enzyme, apparently indicating a defect of peroxisome biogenesis. By transfection of PEX cDNAs and cell fusion analysis, mutants ZP119 and ZP165 were found to belong to a novel complementation group (CG), distinct from earlier mutants. CG analysis by cell fusion with fibroblasts from patients with peroxisome biogenesis disorders such as Zellweger syndrome indicated that ZP119 and ZP165 were in the same CG as the most recently identified human CG-J. The peroxisomal matrix proteins examined, including PTS1 proteins as well as a PTS2 protein, 3-ketoacyl-CoA thiolase, were also found in the cytosol in ZP119 and ZP165. Furthermore, these mutants showed typical peroxisome assembly-defective phenotype such as severe loss of resistance to 12-(1'-pyrene)dodecanoic acid/UV treatment. Most strikingly, peroxisomal reminiscent vesicular structures, so-called peroxisomal ghosts noted in all CGs of earlier Chinese hamster ovary cell mutants as well as in eight CGs of patients' fibroblasts, were not discernible in ZP119 and ZP165, despite normal synthesis of peroxisomal membrane proteins. Accordingly, ZP119 and ZP165 are the first cell mutants defective in import of both soluble and membrane proteins, representing the 14th peroxisome-deficient CG in mammals, including humans.     

Selection system for genes encoding nuclear-targeted proteins

Nuclear proteins have essential roles in cell proliferation and differentiation. We have developed a yeast selection system-the nuclear transportation trap (NTT)-to identify genes encoding nuclear transport signals. Both unknown and previously identified nuclear localization signals were identified from a human fetal brain cDNA library. The majority (75%) of the unknown proteins examined were exclusively localized to the nucleus in COS-7 cells. We propose that NTT is an efficient method for isolating cDNAs that encode nuclear targeted proteins that can be applied to the retrieval of novel nuclear proteins and to annotate gene function.     

Core-shell microbioreactor microencapsulated denitrifying bacteria for nitrate-nitrogen treatment

Summary Droplets of water-in-oil-in-water (w/o/w) emulsion were prepared using sodium alginate solution with denitrifying bacteria (Paracoccus denitrificans IFO13301) and dichloromethane (DCM) with polymethylmethacrylate (PMMA). By phase-separation and solvent-evaporation these droplets could be formed into core-shell PMMA microencapsulated denitrifying bacteria (PMMA-MC) possessing a large single core and a highly porous wall. The average thickness of the PMMA shell was 30 μm and the denitrifying bacteria were incorporated in the inner core at a high density. The PMMA-MC completely reduced 20 mg/L nitrate-nitrogen (NO₃-N) and the intermediate product, nitrite-nitrogen (NO₂-N), to N₂ in the presence of H₂ using a batchwise method. Thus, incorporated denitrifying bacteria can be used to treat water polluted with NO₃-N. The PMMA-MC can be used repeatedly, and the third denitrification experiment directly denitrified (NO₃-N → N₂) without the intermediate step (NO₃-N → NO₂-N). In addition, the PMMA-MC with decreased activity could be reactivated by incubating in a culture medium.     

Glial cell line-derived neurotrophic factor/neurturin-induced differentiation and its enhancement by retinoic acid in primary human neuroblastomas expressing c-Ret, GFR alpha-1, and GFR alpha-2

Neuroblastomas often undergo spontaneous differentiation and/or regression in vivo, which is at least partly regulated by the signals through neurotrophins and their receptors. Recently, glial cell line-derived neurotrophic factor (GDNF) and a second family member, neurturin (NTN), have been found to mediate their signals by binding to a heterotetrameric complex of c-Ret tyrosine kinase receptors and glycosylphosphatidylinositol-linked proteins, GFR alpha-1 (GDNFR-alpha) or GFR alpha-2 (TrnR2/GDNFR-beta/NTNR-alpha/RETL2). Here, we studied the effect of GDNF and NTN on human neuroblastomas in the short-term primary culture system, as well as the expression of c-Ret, GFR alpha-1, GFR alpha-2, GDNF, and NTN. GDNF (1-100 ng/ml) induced morphological differentiation in 34 of 38 primary neuroblastomas and an accompanying increase in c-Fos induction. These effects were markedly enhanced by treatment with 5 microM all-trans-retinoic acid. Although GDNF alone induced a rather weak differentiation independent of the disease stages, the enhancement of neurite outgrowth induced by treatment with both GDNF and all-trans-retinoic acid was significantly correlated with younger age (less than 1 year; P = 0.0039), non-stage 4 diseases (P = 0.0023), a single copy of N-myc (P = 0.027), and high levels of TRK-A expression (P = 0.0062). To examine the expression levels of GFR alpha-1, we cloned a short form of the human GFR alpha-1 gene with a 15-bp deletion by screening a human adult substantia nigra cDNA library. Many primary neuroblastomas expressed c-Ret, GFR alpha-1, and GFR alpha-2 as well as their ligands, GDNF and NTN, suggesting the presence of a paracrine or autocrine signaling system within the tumor tissue. The effect of NTN on primary culture cells of neuroblastoma was similar to that of GDNF. These imply that the GDNF(NTN)/c-Ret/GFR alpha-1(GFR alpha-2) signaling may have an important role in regulating the growth, differentiation, and cell death of neuroblastomas.     

A new antithrombogenic RV-PA valved conduit

A new antithrombogenic right ventricular (RV)-pulmonary artery (PA) valved conduit was developed using a bovine jugular vein containing a natural valve. To maintain the natural and mechanical properties of the venous tissue, a hydrophilic cross-linking reagent, glycerol polyglycidyl ether polyepoxy compound (PC) was used instead of glutaraldehyde (GA). Moreover, to induce antithrombogenicity, heparin was bonded to the inner surface of the bovine jugular vein cross-linked with PC. Conduits of 18 to 20 mm inner diameter (ID) were implanted between the RV and PA in nine dogs weighing 7-17 kg, with the native main PA being ligated proximally. The handling and suturing of the graft was easy and adaptable, and the anastomosis was completed with excellent coaptation and no blood leakage at the suture lines. All animals were chronic survivors, but one animal died of hematemesis on the 438th postoperative day. Grafts were explanted from 182 to 385 days after implantation. The luminal surface of the conduits were white, glistening, and smooth with good coaptation of the cusps, without calcification or degenerative changes except for one cusp that showed a minimal deformation with a small thrombus. Macroscopic and microscopic observation showed that there were no thrombi at the anastomotic lines, but small thrombi on the luminal surface of the conduits near the cusps and in some cusps. Endothelium-like cells were noticed on the luminal surface of the graft, except in the area near the cusps, and on one cusp at 196 days after surgery. These results indicated that the new RV-PA valved conduit provided adequate antithrombogenicity by temporary slow heparin release, followed later by endothelialization of the graft in a low pressure system at 1 year after implantation.     

Application of the distinct element method to the analysis of the concrete members under impact

In this paper, the distinct element method is applied to the analysis of the behavior of a structure under impact. At first, this method is applied to the one-dimensional wave propagation problem by comparing with the experimental results and the theoretical results. The effectiveness of this method is confirmed by including not only elastic behavior but also the fracture of a structural member. Second, this method is developed to two-dimensional problems to simulate the behavior of a simply supported beam under an impact load. Finally, it could be shown that this method is effective to simulate wide phenomena from elastic behavior to a fracture under impact.     

Investigation of pre- and post-dryout heat transfer of steam-water two-phase flow in a rod bundle

Pre- and post-dryout heat transfer experiments were performed for steam-water two-phase flow in a 5 × 5 rod bundle under conditions of total mass fluxes from 80 to 320 kg/m²s, inlet qualities from 0.1 to 0.8, heat fluxes from 3 to 26 W/cm² and a pressure of 3 MPa. Heater rod surface temperatures or heat transfer coefficients predicted by several correlations were compared with experimental data with emphasis on the applicability of the correlations to the present experimental conditions which were pertinent to thermal-hydraulic conditions during a LOCA in a nuclear reactor. The Chen and Biorge et al. correlations underestimated heat transfer coefficients in the pre-dryout region. The Varone-Rohsenow prediction which accounted for the thermal nonequilibrium effect, calculated heater rod surface temperatures relatively well in the post-dryout region over the whole region of the present experimental conditions. The Dittus-Boelter and Groeneveld correlations predicted heater rod surface temperatures relatively well in the post-dryout region under high total mass flux conditions, but underestimated considerably under low total mass flux conditions.     

Genomic structure and chromosomal localization of processed pseudogenes for human RBP-Jk

The functional gene for human recombination signal sequence-binding protein (RBP-Jk) and corresponding processed psudogenes have been isolated from various species, such as Drosophila, Xenopus, mouse, and human. Here we report the isolation of another two genomic pseudogenes of human RBP-Jk, named K2 and K7, from a cosmid library of Hela cells. The nucleotide sequences of both genes exhibited more than 95% homology to the functional human gene for RBP-Jk. Moreover, they did not contain any intron sequences and were interrupted by several stop codons in all frames. In situ hybridization demonstrated that the pseudogenes, K2 and K7, were localized at chromosomes 9p13 and 9q13, respectively. Their physical maps differed from those of the true functional gene and of the pseudogenes reported previously by Amakawa et al. (1993).