Concentrations of thioredoxin,a redox-regulating protein,in umbilical cord blood and breast milk

Growing evidence indicates that oxidative stress occurs during the fetal-to-neonatal transition. Such stress plays an important role in the pathogenesis of many neonatal diseases. Thioredoxin (TRX), a redox-regulating protein with antioxidant activity, is induced in various cells against oxidative stress and is secreted extracellularly. This study was undertaken to examine the clinical and biological importance of TRX in the perinatal setting. We measured concentrations of TRX in umbilical cord blood and breast milk using a sandwich ELISA. Our study demonstrated that concentrations of TRX in umbilical cord blood were six to seven times higher than those in blood of healthy adults. This study also showed that umbilical concentrations of TRX were correlated significantly with the extent of prematurity of the newborn, and that they were elevated significantly in newborns of mothers with preeclampsia compared to those of mothers without preeclampsia. In contrast, concentrations of coenzyme Q₁₀ and vitamin E in umbilical blood were lower than adult blood levels. Breast milk concentrations of TRX during the early postpartum period were seven to eight times higher than those in blood of lactating women. Those of the coenzyme Q₁₀ were lower than adult blood levels, while those of vitamin E were comparable to adult blood levels. Our findings suggest that the systemic release of TRX is enhanced at birth, and that early breast milk is a rich source of this protein. Consequent high levels of TRX in newborns may provide a unique protective mechanism that allows the maintenance of redox balance during the fetal-to-neonatal transition.     

The effects of cell adhesion on the growth and protein productivity of animal cells

We investigated the effect of cell adhesion on cellgrowth and productivity of recombinant protein inChinese hamster ovary (CHO) cells. Cells cultured innormal tissue culture dishes attached to the dishsurfaces and grew as a monolayer, while cells culturedin non-treated dishes proliferated in suspension assingle cells without adhering to the dish surfaces. On an agarose-coated dish surface, cell aggregatesformed without attaching to the dish. Growth rates inboth suspension cultures were slightly lower thanthose in monolayer culture. Cell cycle analysisindicated that the duration of the G₁ phase insuspension cultures was longer than that in monolayerculture, suggesting that attachment to the substratummainly affected the transition from the G₁ to theS phase. Consistent with this, CDK inhibitor p27,that inhibits the G₁S transition, was induced inthe cells cultured in suspension.To assess the productivity of recombinant proteins,CHO cells were transfected with a plasmid containingmurine interferon (mIFN-) under thecontrol of the cytomegalovirus promoter. Insuspension culture, mIFN- productivity wasslightly lower than that in the monolayer culture. When protein kinase C was activated by phorbol ester,mIFN- production was enhanced in both themonolayer and suspension cultures. However, theproductivity in the suspension culture was lower thanthat in the adherent culture even in the presence ofhigh concentrations of phorbol ester. These resultssuggested that cell adhesion to the substratum affectsvarious features of CHO cells.     

Q-FISH Measurement of Hepatocyte Telomere Lengths in Donor Liver and Graft after Pediatric Living-Donor Liver Transplantation: Donor Age Affects Telomere Length Sustainability

Along with the increasing need for living-donor liver transplantation (LDLT), the issue of organ shortage has become a serious problem. Therefore, the use of organs from elderly donors has been increasing. While the short-term results of LDLT have greatly improved, problems affecting the long-term outcome of transplant patients remain unsolved. Furthermore, since contradictory data have been reported with regard to the relationship between donor age and LT/LDLT outcome, the question of whether the use of elderly donors influences the long-term outcome of a graft after LT/LDLT remains unsettled. To address whether hepatocyte telomere length reflects the outcome of LDLT, we analyzed the telomere lengths of hepatocytes in informative biopsy samples from 12 paired donors and recipients (grafts) of pediatric LDLT more than 5 years after adult-to-child LDLT because of primary biliary atresia, using quantitative fluorescence in situ hybridization (Q-FISH). The telomere lengths in the paired samples showed a robust relationship between the donor and grafted hepatocytes (r = 0.765, p = 0.0038), demonstrating the feasibility of our Q-FISH method for cell-specific evaluation. While 8 pairs showed no significant difference between the telomere lengths for the donor and the recipient, the other 4 pairs showed significantly shorter telomeres in the recipient than in the donor. Multiple regression analysis revealed that the donors in the latter group were older than those in the former (p = 0.001). Despite the small number of subjects, this pilot study indicates that donor age is a crucial factor affecting telomere length sustainability in hepatocytes after pediatric LDLT, and that the telomeres in grafted livers may be elongated somewhat longer when the grafts are immunologically well controlled.     

Overproduction of human epidermal growth factor/urogastrone in Escherichia coli and demonstration of its full biological activities

A man-made gene coding for [21-Leu] human epidermal growth factor (hEGF)/β-urogastrone was constructed, inserted into a lacZ fusion-gene expression vector, and cloned into Escherichia coli. In the cloned cells the β-galactosidase/hEGF fusion gene was efficiently expressed and the yield of the hybrid protein reached 10% of the total cellular protein. The [21-Leu] hEGF synthesized in the bacterial cells was proved to be as functional as the natural hEGF or urogastrone and mouse EGF by the following criteria: (1) stimulation of cell proliferation, (2) stimulation of thymidine incorporation into cultured cells, (3) competition with mouse EGF for binding sites on the plasma membrane of human KB cells, and (4) stimulation of phosphorylation of a membrane-bound protein of human KB cell, which has an apparent molecular weight of 150 000 to 170 000 and is indistinguishable from the protein phosphorylated in the presence of mouse EGF in the sodium dodecyl sulfate—polyacrylamide electrophoretic gel. The hEGF produced in the bacterial cells also resulted in precocious eyelid-opening by the daily subcutaneous injection into newborn mice and in accelerated incisor eruption in the animals as mouse EGF did, indicating that the hEGF is also fully active in vivo or physiologically.     

Raptor,a binding partner of target of rapamycin (TOR), mediates TOR action

mTOR controls cell growth, in part by regulating p70 S6 kinase alpha (p70alpha) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). Raptor is a 150 kDa mTOR binding protein that also binds 4EBP1 and p70alpha. The binding of raptor to mTOR is necessary for the mTOR-catalyzed phosphorylation of 4EBP1 in vitro, and it strongly enhances the mTOR kinase activity toward p70alpha. Rapamycin or amino acid withdrawal increases, whereas insulin strongly inhibits, the recovery of 4EBP1 and raptor on 7-methyl-GTP Sepharose. Partial inhibition of raptor expression by RNA interference (RNAi) reduces mTOR-catalyzed 4EBP1 phosphorylation in vitro. RNAi of C. elegans raptor yields an array of phenotypes that closely resemble those produced by inactivation of Ce-TOR. Thus, raptor is an essential scaffold for the mTOR-catalyzed phosphorylation of 4EBP1 and mediates TOR action in vivo.     

Induction of cytotoxicity of peritoneal exudate cells by agrimoniin,a novel immunomodulatory tannin of Agrimonia pilosa Ledeb

Summary The cytotoxic activities of the PEC after an i.p. injection of agrimoniin, a tannin contained in Agrimonia pilosa Ledeb. were studied. The plastic nonadherent PEC had significantly higher NK cell activity than the untreated control, and the adherent PEC were cytostatic toward MM2 and MH134 cells. The adherent PEC did not cause tumor cell lysis by themselves, but were cytolytic against MM2 cells in the presence of anti-MM2 sera. In the course of these effects of PEC after the i.p. injection of agrimoniin, the augmentation of NK cell activity was the earliest reaction, reaching a peak at 2 days after the injection; then, cytostatic activity increased. The induction of antibody-dependent cell lytic activity was a later reaction, which reached a peak at 6 days after the injection. Abbreviations used: PEC, peritoneal exudate cells; NK cell, natural killer cell; ADCC, antibody-dependent cell-mediated cytotoxicity; PMN, polymorphonuclear leukocytes     

Naloxone inhibits prolactin and growth hormone release induced by intracellular glucopenia in the rats

Intraventricular administration of 2-deoxy-D-glucose (2DG), which causes intracellular glucopenia in the central nervous system, increased plasma prolactin and growth hormone levels in the urethane anesthetized male rats. Naloxone, an opiate antagonist, inhibited the 2DG-induced prolactin and growth hormone release. Apomorphine, a dopaminergic agonist, also inhibited the release of these hormones induced by 2DG. These results suggest that endorphins play a role in hypoglycemia-induced prolactin and growth hormone release and that the dopaminergic mechanism may be involved in this phenomenon.