The sensitivity of three genetic lines of Japanese quail to polybrominated biphenyls (PBBs) was evaluated using criteria of egg production, reproduction, and induction of the hepatic microsomal mixed‐function oxidase (MFO) system. Two genetic lines of quail, developed to diverge in their plasma cholesterol response to exogenous adrenocorticotropin (ACTH) (a “Low” line and a “High” line), were compared to a random‐bred line (“Random”). ACTH administration caused increases in plasma cholesterol in the Low line that were 15 and 39% below the Random‐line values in males and females, respectively, while High‐line values were 31% higher in males and 36% higher in females when compared to the respective Random‐line values. Hepatic activities of aryl hydrocarbon hydroxylase (AHH) and hexobarbital hydroxylase (HxH) were not significantly influenced by ACTH administration or by genetic line in either sex. PBBs fed at 40 or 80 mg/kg diet for 5 wk resulted in significant increases in hepatic AHH and aminopyrine N‐demethylase (APND) activities and cytochrome P‐450 concentrations. The induction of AHH, APND, and cytochrome P‐450 was significantly less in Low‐line males in comparison to Random‐ and High‐line males, while the induction of AHH was less in Low‐line females when compared to females from the other two lines, based on covariance analysis. In terms of reproductive parameters, there was a greater adverse effect on egg production at 80ppm PBBs in Low‐line females when compared to the Random and High lines. These data indicate an example in which the biological toxicity of a compound and the induction of a 3‐methylcholanthrene‐type hepatic enzyme are not directly correlated.
Abstract. Differential display PCR was used to identify alternate expression of serum glucocorticoid-regulated kinase (Sgk) mRNA in diabetes-induced renal disease. Differential expression of Sgk mRNA was identified in the kidneys of normal and obese db/db mice, a model of select aspects of human diabetic nephropathy. Sgk mRNA was selectively increased in diabetic mouse kidneys. The Sgk mRNA levels remained constant in other tissues from obese db/db mice. An increase in Sgk mRNA was also observed in the human diabetic kidney. In addition, thrombin, which may play a role in the progression of renal disease, increased Sgk message in cell culture. Because the diabetes-induced increase in Sgk was only observed in the kidney, which is particularly susceptible to diabetes-induced damage, Sgk may play a role in diabetic nephropathy.
Transforming growth factor beta1 (TGF-beta1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF-beta1 acts through the TGF-beta type I and type II receptors to activate intracellular mediators, such as Smad proteins, the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGF-beta type I receptor [activin receptor-like kinase (ALK)5] and the substrate, Smad3, and determined that SB-431542 is a selective inhibitor of Smad3 phosphorylation with an IC50 of 94 nM. It inhibited TGF-beta1-induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC50 values of 6 and 3 microM, respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 microM to selectively address p38 MAPK mechanisms. However, the p38 MAPK inhibitor SB-242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF-beta1-induced matrix production, the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF-beta1-induced fibronectin (FN) mRNA, indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast, SB-431542, but not the selective p38 MAPK inhibitor SB-242235, inhibited TGF-beta1-induced collagen Ialpha1 (col Ialpha1). These data indicate that some matrix markers that are stimulated by TGF-beta1 are mediated via the p38 MAPK pathway (i.e., FN), whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e., col Ialpha1).
The poor absorption of organophosphate delayed neurotoxins through the gastrointestinal tract has been suggested as a reason why young chickens are not susceptible to organophosphate-induced delayed neurotoxicity (OPIDN). In the present study, 4-wk-old White Leghorn chickens were administered a single dose of 500 mg tri-o-tolyl phosphate (TOTP)/kg body weight or 100 mg o-tolyl saligenin phosphate (TSP)/kg body weight via the oral, intramuscular, or intraperitoneal route. In addition, TOTP TSP were administered intravenously at 250 and 50 mg/kg body weight, respectively. Forty-eight hours after dosing, half the birds in each group were killed for subsequent determination of whole-brain and sciatic nerve neurotoxic esterase (NTE) activity while the remaining 5 birds per group were observed daily from d 7 through d 21 for development of OPIDN clinical signs. TOTP administered by the 4 routes generally resulted in whole-brain and sciatic nerve NTE inhibition in excess of 85%. TSP given via the different routes resulted in 75-84% inhibition of whole-brain NTE activity and 66-79% inhibition of sciatic nerve NTE activity. No birds displayed clinical signs typical of OPIDN during the 21-d test. Thus, the resistance of the young chicken to the delayed effects of organophosphate compounds is due to factors other than the poor absorption of the compound through the gastrointestinal tract or the inability of the bird to convert TOTP to its neuroactive metabolite, TSP.
Abstract Immune cells naturally secrete extracellular antigen-presenting vesicles (APVs) displaying peptide:MHC complexes to facilitate the initiation, expansion, maintenance, or silencing of immune responses. Previous work has sought to manufacture and purify these vesicles for cell-free immunotherapies. In this study, APV assembly and release is achieved in non-immune cells by transfecting HEK293T or Expi293F cells with a single-chain heterotrimer (SCT) peptide/major histocompatibility complex I (pMHCI) construct containing an ESCRT- and ALIX-binding region (EABR) sequence appended to the cytoplasmic tail; this EABR sequence recruits ESCRT proteins to induce the budding of APVs displaying SCT pMHCI. A comparison of multiple pMHCI constructs shows that inducing the release of APVs by the addition of an EABR sequence generalizes across SCT pMHCI constructs. Purified pMHCI/EABR APVs selectively stimulate IFN-γ release from T cells presenting their cognate T cell receptor, demonstrating the potential use of these vesicles as a form of cell-free immunotherapy. Significance Statement Immune cells are known to naturally release pMHC-displaying extracellular vesicles (EVs), called antigen-presenting vesicles (APVs), which can orchestrate immune responses either directly or with the aid of antigen-presenting cells (APCs). For decades, researchers have pursued ways to replicate these APVs for immunotherapy by using chemically modified nanoparticles or by engineering the increased expression of APVs from immune cells which are typically low yield. Here we presents a broadly applicable platform for generating high concentrations of pMHCI-displaying APVs that can selectively modulate T cells, demonstrating a significant advance in the engineering of APVs for cell-free immunotherapy. The APVs presented here, and related APVs, could be translated into clinical therapies for modulating cancer progression or regulating autoimmunity in addition to their use as a tool to help characterize how endogenous extracellular vesicles influence the immune system.
Young chickens fed hexachlorobenzene (HCB) or pentachlorophenol (PCP) for 14 d at 10 ppm in the diet contained body burdens of 573 or 362 μg, respectively. These diets were withdrawn (d 0) and replaced for 21 d with diets containing 5% mineral oil (MO), or 5% (CO), a bile‐acid‐binding resin, or the chickens were restricted in feed intake to 50% of controls (50‐RF), fed MO plus 50‐RF, or CO plus 50‐RF. Without any treatment during withdrawal, body burdens were reduced to 63% and 70% of the d 0 values for HCB and PCP, respectively. MO, CO, or 50‐RF reduced body burdens of HCB to 37% of d 0 burdens, but the combination treatments with 50‐RF reduced body burdens to 19% of d 0 values. PCP was at 35% of the d 0 burdens from 50‐RF, while all other treatments had reduced body burdens to nondetectable amounts of less than 0.7 μglbird by d 21 of withdrawal. Body fat was not reduced by mineral oil, but was reduced to some extent by CO, and was markedly reduced by 50‐RF. 50‐RF always reduced body burdens of PCP or HCB alone or in combination with MO or CO. These data are discussed in relationship to the nonbiliary excretion of xenobiotics.
In an attempt to improve the nutritional value of animal fats (including milkfat and lard), two technological approaches (i.e., cholesterol removal by steam distillation and linoleic acid enrichment by addition of safflower oil) were tested for cholesterolemic effects in a cohort of 29 older women (age 68 +/- 7 years).Test fat sources were incorporated into crackers, cookies, cheese, ice cream, whipped topping, sour cream, baking shortening, and table spreads. Subjects were permanent residents of a convent where meals were prepared in a centralized kitchen, allowing test fats to be provided in daily food menu items. The foods containing test fats were introduced into three sequential dietary treatment periods, each lasting 4 weeks, in the following order: cholesterol-reduced animal fat (CRAF): fatty-acid modified, cholesterol-reduced animal fat (FAMCRAF); and-unaltered animal fat (AF). Subjects were offered menu items cafeteria style and encouraged to make food selections consistent with their habitual diets, which were recorded daily.Fasted blood lipid profiles determined at the end of each treatment period showed that FAMCRAF reduced mean plasma total cholesterol, LDL cholesterol, and apolipoprotein B concentrations relative to AF (p < 0.05). Mean HDL cholesterol concentrations were not influenced by diet.Relative to native products, animal fats modified by cholesterol removal and linoleic acid enrichment reduced plasma total and LDL cholesterol concentrations in a predictable manner similar to that based on studies of men.
Flaxseed, the richest known source of plant lignans, has been shown to have chemoprotective effects in animal and cell studies. Some of its effects may be mediated through its influence on endogenous hormone production and metabolism. Two competing pathways in estrogen metabolism involve production of the 2-hydroxylated and 16 alpha-hydroxylated metabolites. Because of the proposed differences in biological activities of these metabolites, the balance of the two pathways has been used as a biomarker for breast cancer risk. We examined the effects of flaxseed consumption on urinary estrogen metabolite excretion in postmenopausal women. Twenty-eight postmenopausal women were studied for three seven-week feeding periods in a randomized crossover design. During the feeding periods, subjects consumed their usual diets plus ground flaxseed (0, 5, or 10 g/day). Urinary excretion of the estrogen metabolites 2-hydroxyestrogen (2-OHEstrogen) and 16 alpha-hydroxyestrone (16 alpha-OHE1) as well as their ratio, 2/16 alpha-OHE1, was measured by enzyme immunoassay. Flaxseed supplementation significantly increased urinary 2-OHEstrogen excretion (p < 0.0005) and the urinary 2/16 alpha-OHE1 ratio (p < 0.05) in a linear, dose-response fashion. There were no significant differences in urinary 16 alpha-OHE1 excretion. These results suggest that flaxseed may have chemoprotective effects in postmenopausal women.
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