Role for laminin-alpha5 chain LG4 module in epithelial branching morphogenesis

Laminin-alpha5 chain was localized in all epithelial basement membranes (BMs) of mouse submandibular gland (SMG) from the onset of branching morphogenesis and became restricted to BMs of epithelial ducts in the adult. To investigate whether the laminin-alpha5 chain plays a role in branching morphogenesis, a set of cell-adhesive peptides from the C-terminal globular domains (LG1-5) was tested for their effects in SMG organ cultures. One peptide, LVLFLNHGH (A5G77f), which represents a sequence located in the connecting loop between strands E and F of LG4, perturbed branching morphogenesis and resulted in irregularities in the contours of epithelial structures, with formation of deep clefts. The data suggest a role for the laminin-alpha5 LG4 module in the development of the duct system, rather than in the bifurcation of epithelial clusters. The epithelial BM of A5G77f-peptide-treated explants was continuous, which was in contrast to our previous finding of impaired epithelial BM assembly in explants treated with the laminin-alpha1 LG4 module peptide, or with a monoclonal antibody against this domain. A5G77f also perturbed in vitro development of lung and kidney. These results suggest a crucial role for the LG4 module of laminin-alpha5 in epithelial morphogenesis that is distinct from that of the laminin-alpha1 LG4.     

Expression and characterization of recombinant human antithrombin III in Pichia pastoris

Antithrombin III (ATIII) is a member of the serpin superfamily and a major regulator of the blood coagulation cascade. To express recombinant human ATIII (rATIII) in the methylotrophic yeast Pichia pastoris, we constructed an rATIII expression plasmid which contained the ATIII cDNA encoding mature protein region connected with the truncated mAOX2 promoter and the SUC2 secretion signal, introduced it into the P. pastoris genome, and screened for a single copy transformant. The secretion of rATIII from the transformant reached a level of 320 IU/L in the culture broth at 169 h. From the culture-supernatant, rATIII was purified to over 99% by heparin-affinity chromatography and other column chromatography methods. We characterized rATIII and compared it with human plasma-derived ATIII (pATIII). The purified rATIII possessed correct N-terminal amino acid sequence, and its molecular weight by SDS-PAGE of 56,000 Da was slightly different from the 58,000 Da of pATIII. Sequence and mass spectrometry analysis of BrCN fragments revealed that posttranslational modifications had occurred in rATIII. O-linked mannosylation was found at Ser 3 and Thr 9, and in some rATIII molecules, modification with O-linked mannosyl-mannose had probably occurred at Thr 386, close to the reactive center. Although the heparin-binding affinity of rATIII was 10-fold higher than that of pATIII, its inhibitory activity against thrombin was only half. As the conformation of rATIII and pATIII by circular dichroism spectroscopy was similar, O-glycosylation in the reactive center loop was assumed to be mainly responsible for the decreased inhibitory activity. pATIII can inactivate thrombin through formation of a stable thrombin-ATIII complex, but rATIII modified with O-glycosylation in the reactive center loop may act as a substrate rather than an inhibitor of thrombin.     

Adjustment function among antioxidant substances in acatalasemic mouse brain and its enhancement by low-dose X-ray irradiation

The catalase activities in blood and organs of the acatalasemic (C3H/AnLCsbCsb) mouse of the C3H strain are lower than those of the normal (C3H/AnLCsaCsa) mouse. We conducted a study to examine changes in the activities of antioxidant enzymes, such as catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX), the total gluathione content, and the lipid peroxide level in the brain, which is more sensitive to oxidative stress than other organs, at 3, 6, or 24 hr following X-ray irradiation at doses of 0.25, 0.5, or 5.0 Gy to the acatalasemic and the normal mice. No significant change in the lipid peroxide level in the acatalasemic mouse brain was seen under non-irradiation conditions. However, the acatalasemic mouse brain was more damaged than the normal mouse brain by excessive oxygen stress, such as a high-dose (5.0 Gy) X-ray. On the other hand, we found that, unlike 5.0 Gy X-ray, a relatively low-dose (0.5 Gy) irradiation specifically increased the activities of both catalase and GPX in the acatalasemic mouse brain making the activities closer to those in the normal mouse brain. These findings may indicate that the free radical reaction induced by the lack of catalase is more properly neutralized by low dose irradiation.     

Suppression of antibody production by TNF-related apoptosis-inducing ligand (TRAIL)

TNF-related apoptosis-inducing ligand (TRAIL), a type II membrane protein belonging to the TNF family, induces apoptotic cell death in various types of tumor cells. However, little is known about its pathological and physiological functions in the immune system. In this study, we showed that administration of neutralizing anti-TRAIL mAb markedly increased serum auto-Ab levels, particularly of IgG1 subclass, in autoimmune-prone C3H/HeJ gld/gld mice without affecting lymphocytosis and lymphocytes populations. In an experimental system where TNP-specific Ab production was induced by immunization with TNP-modified syngeneic B lymphoma cells, expression of TRAIL on these cells significantly reduced TNP-specific Ab production, especially of IgG1 subclass, without affecting T cell priming. These results suggest a new role for TRAIL in the suppression of Ab production.     

Photosynthesis–nitrogen relationships in species at different altitudes on Mount Kinabalu, Malaysia

In situ photosynthetic nitrogen-use efficiency (PNUE, photosynthetic capacity per unit leaf nitrogen) was investigated in species that commonly distributed at different altitudes (600–3700m above sea level) on Mount Kinabalu. Photosynthetic nitrogen-use efficiency was lower in species at higher altitudes, with a mean PNUE at 3700m being one-third as large as that at 600m. This difference in PNUE was larger than that explained by the biochemical response to lower air pressures only. Across altitudes a negative correlation between ¹³C abundance (¹³C) and PNUE was found. Species at higher altitudes tended to have higher ¹³C, suggesting that they had a lower conductance for CO₂ diffusion from the air to chloroplasts. The lower conductance might be responsible for the lower PNUE in species at higher altitudes. Although leaf nitrogen content per unit area tended to be higher at higher altitudes, it did not seem to contribute to increasing photosynthetic rates. Thus, the idea that a higher nitrogen content at higher altitudes is a compensation for a lower PNUE was not supported. In contrast to the large difference in PNUE among altitudes, PNUE tended to converge within a narrow range among species growing at the same altitude.     

Quantitative determination of glufosinate in biological samples by liquid chromatography with ultraviolet detection after p-nitrobenzoyl derivatization

We have established a new HPLC method for derivatizing and quantifying glufosinate (GLUF) in human serum and urine using p-nitrobenzoyl chloride (PNBC). The p-nitrobenzoyl derivative of GLUF (PNB-GLUF) was produced quantitatively over 10 min at room temperature. PNB-GLUF possesses the property of ultraviolet (UV) light absorption with a lambda(max) of 272.8 nm, and was isolated from biological specimens by reversed-phase chromatography using Inertsil Ph-3. In experiments at a UV wavelength of 273 nm, GLUF has a quantitative detection limit of 0.005 microg/ml, and when it was added to both serum and urine to yield concentrations of 0.1-1000 microg/ml, its recovery rate was quite satisfactory: at least 93.8% in all cases. Further, the measured amounts of GLUF in 23 serum samples from patients intoxicated by ingestion of GLUF compared favorably with those obtained by fluorescence derivatization-HPLC using 9-fluorenylmethyl chloroformate (R=0.998). This technique of analysis is, in addition, applicable for Glyphosat, which possesses a chemical structure resembling that of GLUF, and it will be of great use in the determination of these two compounds.     

Enantioselective analysis of glufosinate using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate and reversed-phase liquid chromatography

We have developed a new analytical method to quantify the DL-homoalanine-4-yl(methyl)phosphinate (DL-GLUF) enantiomers in biological specimens using a reversed-phase high-performance liquid chromatography system with a fluorescence detection system. The derivatization of DL-GLUF enantiomers with (+)-1-(9-fluorenyl)ethyl chloroformate was carried out under mild conditions (40 degrees C for 30 min) without inducing racemization. The lower limit of quantitation was 0.01 microg/ml for both D-GLUF and L-GLUF, and the detection limit was 5 ng/ml. When DL-GLUF enantiomers were added to serum to produce concentrations between 0.1 and 100 microg/ml, the mean recovery rate was at least 93.8%. The recovery rate from urine was also satisfactory.