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.     

Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.     

Developmental potential of cumulus cell-derived culture frozen in a quiescent state after nucleus transfer

An efficient method for freezing donor cells is necessary when using nucleus transfer of somatic cells for large-scale cloning. In the present study, we developed a method for freezing and thawing bovine cumulus cell-derived cultured cells to be used as nucleus donors. Cumulus cells were obtained from ovaries of living and slaughtered bovine and cultured in vitro. Cumulus cell-derived cultured cells were serum-starved for several days to induce a quiescent state and then frozen at -70 degrees C for at least 2 d. Immediately thereafter or 2 h after thawing, the cells were used as donor cells for nuclear transfer without additional in vitro culture. The fusion rate with recipient cytoplasts was not affected by the cumulus cell source (slaughtered or living) or time after thawing (0 and 2 h). The cleavage rate of frozen-thawed cumulus cell-derived cultured cells from slaughtered cows immediately after thawing (0 h) was highest (97%) and was significantly higher than that of controls (85%) or cells transferred 2 h after thawing (85%). There were no significant differences among any of the groups in the potential of the nuclear transfer embryos to develop into blastocysts (34 vs 44 and 44%, 39 vs 45 and 46%). Thus, storage of bovine cumulus cell-derived cultured cells in the quiescent state at -70 degrees C is effective and might be useful and convenient for large-scale cloning. The maximum storage periods and developmental potential of embryos after such nucleus transfers requires further examination.     

Development of a Direct In Situ PCR Method for Detection of Specific Bacteria in Natural Environments

We applied HNPP (2-hydroxy-3-naphthoic acid-2′-phenylanilide phosphate) to direct in situ PCR for the routine detection of specific bacterial cells at the single-cell level. PCR was performed on glass slides with digoxigenin-labeled dUTP. The digoxigenin-labeled PCR products were detected with alkaline phosphatase-labeled antidigoxigenin antibody and HNPP which was combined with Fast Red TR. A bright red fluorescent signal was produced from conversion to HNP (dephosphorylated form) by alkaline phosphatase. We used the ECOL DNA primer set for amplification of ribosomal DNA of Escherichia coli to identify cells specifically at the single-cell level in a bacterial mixture. High-contrast images were obtained under an epifluorescence microscope with in situ PCR. By image analysis, E. coli cells in polluted river water also were detected.     

Albutensin A, an ileum-contracting peptide derived from serum albumin, acts through both receptors for complements C3a and C5a

Albutensin A is an ileum-contracting peptide derived from serum albumin. The sequences of bovine, human and porcine albutensin A are ALKAWSVAR, AFKAWAVAR, and AFKAWSLAR, respectively. These albutensin A homologs all exhibited biphasic ileal contractions in the longitudinal strips of guinea pig ileum. The order of potency in the contraction was porcine > bovine > human homologs. The ileal contraction profiles were similar to those of oryzatensin and casoxin C, agonist peptides for complement C3a receptors derived from rice albumin and bovine -casein, respectively. All three homologs of albutensin A have homology with the COOH-terminal sequences of complements C3a and C5a, which are essential for their activities; porcine albutensin A showed the highest homology. Indeed, porcine albutensin A was confirmed to act through both C3a and C5a receptors by a radioreceptor assay and cross-desensitization in the ileal contraction. In addition, bovine and human homologs also showed affinity for both receptors. This study suggests that a bioactive peptide acting through both C3a and C5a receptors is released by the proteolytic cleavage of serum proteins other than complement components.     

Change in bacterial community during biodegradation of aniline

The response of river water microbial communities to chemical compounds was monitored under laboratory conditions using aniline as a model. Bacteria were collected from unpolluted and polluted sites. Bacterial abundance (plate and total direct counting) and its relation to aniline biodegradation was examined. Colony hybridization with 16S rRNA oligonucleotide probes was used to study the changes in microbial community structure during biodegradation of aniline. The changes in bacterial abundance and community structure were related to biodegradation of aniline. Burkholderia–Pseudomonas (rRNA group III), an authentic Alcaligenes group became dominant despite the initial differences in the microbial communities, suggesting that these genera are the main aniline degraders in the aquatic environment.     

Developmental Competence of Vitrified-Warmed Bovine Oocytes at the Germinal-Vesicle Stage is Improved by Cyclic Adenosine Monophosphate Modulators during In Vitro Maturation

Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.