Proceedings of the Society for Experimental Biology and Medicine最新文献

The role of NO and superoxide (O(2)(-)) in tissue injury during cardiac allograft rejection was investigated by using a rat ex vivo organ perfusion system. Excessive NO production and inducible NO synthase (iNOS) expression were observed in cardiac allografts at 5 days after cardiac transplantation, but not in cardiac isografts, as identified by electron spin resonance spectroscopy and Northern blotting. Cardiac isografts or allografts obtained on Day 5 after transplantation were perfused with Krebs bicarbonate buffer with or without various antidotes for NO or O(2)-, including N(omega)-monomethyl-L-arginine (L-NMMA; 1 mM), 2-phenyl-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO; 100 microM), 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP; a xanthine oxidase inhibitor; 100 microM), and superoxide dismutase (SOD; 100 units/ml). Treatment of the cardiac allografts with PTIO showed most remarkable improvement of the cardiac injury as revealed by significant reduction in aspartate transaminase, lactate dehydrogenase, and creatine phosphokinase concentrations in the perfusate. Similar but less potent protective effect on the allograft injury was observed by treatment with L-NMMA, AHPP, and SOD. Immunohistochemical analyses for iNOS and nitrotyrosine indicated that iNOS is mainly expressed by macrophages infiltrating the allograft tissues, and nitrotyrosine formation was demonstrated not only in macrophages but also in cardiac myocytes of the allografts, providing indirect evidence for the generation of peroxynitrite during allograft rejection. Our results suggest that tissue injury in rat cardiac allografts during acute rejection is mediated by both NO and O(2)(-), possibly through peroxynitrite formation.

In this report, we have characterized the upregulation of glucose transport in two different respiration-deficient fibroblast cell cultures. We have demonstrated that glucose transport increases in respiration-deficient cells as measured by 2 deoxy D-glucose transport and is readily observed in both the WG750 human and G14 Chinese hamster fibroblast respiration-deficient cell lines when compared with the MCH55 normal human and V79 parental Chinese hamster cell lines, respectively. Using subcellular fractionation techniques, the GLUT 1 glucose transporter was found located predominantly in the plasma membrane-enriched fraction of the human and hamster cell lines. In human cells, the expression of the GLUT 1 glucose transporter was elevated three-fold in the plasma membrane-enriched fraction of the WG750 respiration-deficient mutant cells. In the Chinese hamster cell lines, the respiration-deficient G14 cells exhibited no such GLUT 1 glucose transporter elevation in the plasma membrane-enriched fraction, yet expressed a >2-fold increase in glucose transport. Furthermore, the G14 cells had a similar content of GLUT 1 glucose transporter in the plasma membrane fraction when compared with the V79 parental cell line. Using Western blot analysis, the GLUT 1 glucose transporter in G14 cells exhibited a different mobility on a polyacrylamide gel when compared with the mobility of the GLUT 1 glucose transporter of the V79 cell line. This differential mobility of the glucose transporters in the hamster cells appeared to be related to glycosylation differences of the glucose transporters. Although normal human and hamster cell lines exhibited significant increases in insulin-stimulated sugar transport (P < 0.05), the two respective respiration-deficient cell lines exhibited no significant increases in insulin-stimulated sugar transport (P > 0.05). Additionally, the expression of the GLUT 1 mRNA in the human WG750 mutant cells was elevated when compared with GLUT 1 mRNA in normal cells. Insulin exposure significantly increased GLUT 1 mRNA in human cells (P < 0.05). No differences in the GLUT 1 mRNA were observed between both hamster cell lines. Thus, both respiration-deficient cell lines are insulin resistant (i.e., regarding their insulin-stimulated sugar transport). The respiration-deficient mutation results in an increased sugar transport in the human and hamster cells; however, the human cells adapt to the mutation by increasing their levels of GLUT 1 mRNA and eventually membrane-located glucose transporters. On the other hand, the hamster cells adapt by apparently modifying their glucose transporters' intrinsic activity via glycosylation. We feel that these cell systems can be effective models to study the multiple factors involved in sugar transport regulation in vertebrate cells.

The insulin-like growth factors (IGFs) are mitogenic polypeptides that have been linked to a variety of normal physiological processes as well as neoplasia. Overexpression of several components of the IGF system is associated with hepatocarcinogenesis in humans and rodents. In rat liver, diets rich in n-6 polyunsaturated fatty acid (PUFA) enhance the development of preneoplastic lesions and tumors. Therefore, the objective of this study was to determine the effect of these dietary fatty acids on the hepatic expression of the various components of the IGF system. The mRNA levels of IGF-1 and the type 1 receptor were not different in livers of rats fed a diet containing 20% corn oil (CO) compared with those fed 5% CO. Analysis of the IGF binding proteins revealed that insulin-like growth factor binding protein-1 (IGFBP-1) levels were altered by the amount and type of dietary fat. A 2.5-fold induction of IGFBP-1 mRNA occurred within 1 week after the animals were fed the 20% corn oil diet compared with those fed 5% CO and was further enhanced to over 6-fold after 1 month. Furthermore, IGFBP-1 protein was only detectable in the livers of animals fed the 20% CO diet. Induction of IGFBP-1 mRNA (4.5-fold) also occurred in rats fed a high-fat diet containing safflower (rich in n-6 PUFAs) compared with those fed a high-fat diet containing menhaden oil (rich in n-3 PUFAs). The induction of IGFBP-1 mRNA was independent of serum insulin levels and the development of insulin resistance. Since IGFBP-1 mRNA is upregulated in regenerating liver, we reasoned that the induction of IGFBP-1 mRNA may be associated with an increase in cell proliferation; however, no difference was observed in the hepatic labeling index of rats fed the 20% CO compared with the 5% CO diet. In summary, these studies show a striking induction by dietary n-6 PUFAs of hepatic IGFBP-1, a protein that has been implicated in liver cancer development.

Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in the synthesis of long-chain fatty acids. Since aging influences adiposity, we studied the activity of ACC and its mRNA content in livers of 4-, 12-, and 24-month-old male Fischer 344 rats. The mean (+/- SEM) activity of ACC (mU/mg protein) in liver homogenates from 4-month-old rats was 1.01 +/- 0.14. There was an 80% increase in activity (1.83 +/- 0.27) in 12-month-old rats (P < 0.01). However, there was significantly less activity (0.46 +/- 0.06) in livers of 24-month-old rats (P < 0.001). The total activity of ACC (per g liver) followed the same trend. The enzyme from all age groups was purified by avidin-affinity chromatography. The purified preparation migrated as a major protein band (M(r) 262,000) on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The specific activity of the purified preparation was 1.5, 1.8, and 1.8 U/mg for 4-, 12-, and 24-month-old rats, respectively. The alkali-labile phosphate content was 5.66 +/- 0.17, 5.64 +/- 0.21, and 6.21 +/- 0.35 mols P(i)/mole subunit for 4-, 12-, and 24-month-old rats, respectively. These age-related differences were not significant. The hepatic ACC mRNA measured by ribonuclease protection assay when corrected for G3PDH mRNA was significantly reduced in 24-month-old rats (0.24 +/- 0.03) compared with 12-month-old (0.58 +/- 0.04) or 4-month-old rats (0.43 +/- 0.007) P < 0.01. In summary: (i) Aging in rats is associated with significant changes in ACC activity; (ii) the purified ACC preparations from the three age groups had similar specific activity and similar phosphate content; and (iii) the changes in ACC mRNA content of the liver paralleled the changes in total enzyme activity when 12-month-old rats were compared with 24-month-old rats whereas the increase in ACC activity in 12-month-old rats compared with 4-month-old rats could not be ascribed to changes in hepatic mRNA levels. These results indicate that the age-related changes in hepatic ACC occur at a post-translational level during early years of aging and at a pretranslational level at late states of senescence. These changes may contribute to the age-related alterations in body adiposity.

Cell death induction by cytotoxic T lymphocytes (CTLs) is an important thesis for the understanding of tumor immunotherapy. In the current study we investigated the molecular machinery of CTL-induced cell death in human hepatocellular carcinoma cell lines (HCC lines). CTLs prepared from human peripheral blood induced cell death in all tested HCC lines. As the CTL-induced death system, the effectiveness of Fas ligand/Fas and/or Perforin/Granzyme B systems has been suggested, whereas cell death induction by CTLs was shown independently on Fas expression in the current study. Using various tetrapeptide inhibitors for caspase and its associated factor, we additionally demonstrated that inhibitors for caspase 3 (Ac-DEVD-CHO) and caspase 8/granzyme B (Ac-IETD-CHO) suppressed CTL-induced cell death, but an inhibitor for Fas-activated serine proteinase, which acts for the caspase 3 activator, did not, suggesting that CTL-induced cell death was initiated by the Perforin/Granzyme B system, rather than the Fas ligand/Fas system. On the basis of our current results, we report here that the Perforin/Granzyme B system acts dominantly for the cell death induction of HCC lines.

This review examines experimental models of Sertoli cell injury resulting in germ cell apoptosis. Since germ cells exist in an environment created by Sertoli cells, paracrine signaling between these intimately associated cells must regulate the process of germ cell death. Germ cell apoptosis may be signaled by a decrease in Sertoli cell pro-survival factors, an increase in Sertoli cell pro-apoptotic factors, or both. The different models of Sertoli cell injury indicate that spermatogenesis is susceptible to disruption, and that targeting critical Sertoli cell functions can lead to rapid and massive germ cell death.

The objective of this experiment was to investigate whether the ergot alkaloid, ergotamine (ET), an alkaloid used to model fescue toxicosis in cattle, modifies the response of cattle to endotoxin (LPS) challenge. Steers (n = 16) were divided into the following treatment groups: control (C), ergotamine (ET), endotoxin (LPS), and ET + LPS. ET and ET + LPS groups received a single bolus intravenous injection of ET (40 microg. kg. body wt(-1)), whereas C and LPS steers received a single bolus injection of sterile vehicle. Thirty minutes after ET/vehicle administration, a single bolus intravenous injection of LPS (0.2 microg. kg. body wt(-1)) was given. Blood was collected at various time points for 48 hr post. Endotoxin increased rectal temperature (RT) and the circulating levels of tumor necrosis factor-alpha (TNF-alpha), cortisol, haptoglobin (Hp), thromboxane B(2) (TXB(2)). The circulating Hp, TNF-alpha, and TXB(2) increases were blunted by pretreatment with ET compared with ET + LPS. Ergotamine by itself increased circulating cortisol and RT, whereas it decreased serum prolactin (PRL). Therefore, whereas administration of LPS at 0.2 microg/kg to steers resulted in an expected response, the combination of ET + LPS attenuated major effects of LPS alone. Thus, acute administration of ET appeared to be anti-inflammatory as it decreased the inflammatory response to LPS, an effect likely driven at least in part by the ET-caused cortisol increase.

Previously we demonstrated that proliferation of peripheral blood mononuclear cells (PBMC) causes a five-fold increase in cellular uptake of biotin; this increase is mediated by an increased number of biotin transporters on the PBMC surface. In the present study, we investigated the specificity of this phenomenon by determining whether the cellular uptake of riboflavin also increases in proliferating PBMC and whether the increase is also mediated by an increased number of transporters per cell. We characterized [3H]riboflavin uptake in both quiescent and proliferating PBMC. In quiescent PBMC, [3H]riboflavin uptake exhibited saturation kinetics and was reduced by addition of unlabeled riboflavin (P < 0.05) or lumichrome (P < 0.01). These observations are consistent with transporter-mediated uptake. [3H]Riboflavin uptake was reduced at 4 degrees C compared with 37 degrees C (P < 0.01) and by 2, 4-dinitrophenol (P < 0.05) but not by ouabain or incubation in sodium-free medium. These data provide evidence for an energy-dependent but sodium-independent transporter. Proliferating PBMC accumulated approximately four times more [3H]riboflavin than quiescent PBMC (P < 0.05). Because both transporter affinity and transporter number per cell (as judged by maximal transport rate) were similar in quiescent and proliferating PBMC, we hypothesize that the increased riboflavin uptake by proliferating PBMC reflects only increased cellular volume. To test this hypothesis, PBMC volume was reduced using hyperosmolar medium; [3H]riboflavin uptake decreased to about 50% of isotonic controls (P < 0.01). Thus we conclude that proliferating PBMC increase cellular content of riboflavin and biotin by two different mechanisms.

We examined the effects of the transparent fibroin film (silk film) on full-thickness skin wounds. Full-thickness dermatotomies (15 mm x 9 mm) were prepared on the dorsal wall of CRJ:CD-1 nu/nu (ICR nu/nu) mice. The area of the wounds dressed with silk film was reduced to 10% of that made by the dermatotomy 14 days after the dermatotomy and were covered with regenerated epidermis 21 days after the dermatotomy. In contrast, less recovery and epidermal regeneration were found 14 days after dermatotomy in the wounds dressed with a conventional hydrocolloid dressing (Duro Active). Furthermore, only partial incomplete epidemal growth was obtained 21 days after dermatotomy. Most importantly, the healing time of wounds dressed with silk film was 7 days shorter than those dressed with DuoActive dressing. The silk film showed an almost similar or slightly better promotive effect as the lyophilized porcine dermis (Alloask D), which is used as a dressing for burns, ulcers, and decubitis. Histologic findings revealed that there was greater collagen regeneration and less inflammation and neutrophil-lymphocyte infiltration of the wounds dressed with silk film than with DuoActive dressing. It is clear that regeneration of the epidermis and dermis of the wound beds covered with silk film was faster than with DuoActive dressing. Finally, silk film is easily obtainable, sterilizable, and transparent, and it allows easy observation of tissue recovery. Therefore, silk film offers advantages over other dressings and may be clinically useful for wound treatment.