Lower levels of F2-isoprostanes in serum and livers of long-lived Ames dwarf mice

F₂-isoprostanes (IsoPs), lipid peroxidation products, are markers that quantitatively measure levels of oxidative stress. IsoP levels increase in tissues and serum of aging animals suggesting an increase in oxidative stress. This supports the Free Radical Theory of Aging, which proposes that elevated levels of reactive oxygen species (ROS) cause macromolecular damage, and is a factor in the age-associated decline in tissue function. Numerous studies have shown that the longevity of long-lived mutant mice correlates with their resistance to oxidative stress. However, although the Ames dwarf (DW) mice show resistance to oxidative stress, it has not been shown that these mice have inherently lower levels of ROS. Our results show that the serum and liver IsoP levels in DW mice are lower at all ages suggesting that the lower levels of endogenous ROS production in DW mice may be a factor in their resistance to oxidative stress and longevity.     

Evidence that Ames dwarf mice age differently from their normal siblings in behavioral and learning and memory parameters

There is strong evidence supporting the deleterious effects of aging on learning and memory and behavioral parameters in normal mice. However, little is known about the Ames dwarf mouse, which has a Prop-1 gene mutation resulting in deficiencies in growth hormone, thyroid-stimulating hormone, and prolactin. These mice are much smaller and live significantly longer than their normal siblings. Using the elevated plus-maze, locomotor activity meters, and an inhibitory avoidance learning task, the present study compared Ames dwarf mice to their normal siblings. Results showed that Ames dwarf mice did not experience an age-related decline in locomotor activity when compared to their young counterparts. Furthermore, old dwarf mice did not differ from the young groups in inhibitory avoidance retention, while old normal animals performed more poorly than both young groups on this test. Elevated plus-maze behavior did not differ in the old normal versus dwarf groups, but the old groups did differ from the young. Results indicate that both old groups experienced a significant decline in anxiety with age. Taken together, these results indicate that multiple hormone deficiencies resulting from a lack of primary pituitary function have beneficial effects on cognitive function and locomotor behavior in advanced age. In fact, the Ames dwarf mouse may provide a model for studies of delayed mental as well as physical aging.     

Elements in autopsy liver tissue samples from Greenlandic inuit and Danes. V. Selenium measured by X-ray fluorescence spectrometry

The content of selenium in normal liver tissue samples from Greenlandic Inuit was measured and the results compared with those obtained in normal liver tissue samples from Danes. Normal liver tissue samples were obtained at autopsy from 50 Greenlandic Inuit (27 men, 23 women) with a median age of 61 years (range 23–83) and from 74 Danes (44 men, 30 women) with a median age of 60 years (range 15–87). Total liver selenium content was measured by X-ray fluorescence spectrometry. The content of selenium (median) was in Inuit 26.6 mol/kg dry liver (5–95 percentile: 15.2–49.4) and in Danes 17.7 mol/kg dry liver (5–95 percentile: < 3.8–36.5) (p < 0.0001). Liver selenium content displayed no significant gender difference, either in Inuit or Danes. In Inuit men, there was a negative correlation between liver selenium content and age (r_s = −0.39, p < 0.05), whereas Danish men displayed a positive correlation between liver selenium content and age (r_s = 0.37, p = 0.02). There was no correlation in Inuit or Danish women. In Inuit, the median hepatic selenium index (liver selenium content divided by age) was 0.48 and in Danes 0.33 (p = 0.001). There was an inverse correlation between hepatic selenium index and age both in Inuit (r_s = −0.77, p < 0.0001) and in Danes (r_s = −0.47, p < 0.0001). In conclusion, Inuit had a higher liver content of selenium and a higher hepatic selenium index compared with Danes. The more favourable selenium status is due to a higher nutritional selenium intake with fish and meat from sea mammals.     

Isolation and characterization of dihydromaleimide and its glucoside as growth inhibitors from dwarf pea

Two new growth inhibitors, R-dihydromaleimide and R-dihydromaleimide β-d-glucoside, were isolated from 2-week-old pea shoots.     

Comparison of growth and gibberellin concentrations in shoots from orchard-grown standard and thermosensitive dwarf apple trees

Apple (Malus domestica Borkh.) trees were propagated by budding from selected fully grown hybrids that ranged in height from 1.5 to 8 m. The growth and development of the selected budded trees after 7 years in the orchard was similar to that of the parent trees. Additional grafting studies showed that the dwarfism was not associated with the roots. Differences in photosynthetic activity and associated processes were not related to the size difference between tissue culture-propagated orchard-grown standard cv. Golden Delicious and dwarf hybrid trees. Applications of GA₃ did not stimulate elongation of shoots of dwarf trees. Shoots of both standard and dwarf trees started to develop in mid-April when they contained nearly the same amounts of GA₁, GA₃ and GA₈, but standard shoots contained higher concentrations of GA₁₉, GA₂₀ and GA₂₉. On 2 June standard shoots were almost three times the length of dwarf shoots, but the number of leaves and area per leaf were nearly the same. The relative amounts of GAs on 12 May and 2 June for both plant types were similar to those on 20 April, except that GA₁₉, GA₂₀, GA₁ and GA₂₉ levels had declined. Gibberellin levels in standard shoots declined further between 2 and 22 June, after which there was no further shoot elongation or production of new leaves. Between 2 June and the end of the growing season, when summer temperatures were high, dwarf shoots continued to elongate slowly and to develop new leaves, which expanded little. During this time, the GA₁₉ content of dwarf shoots nearly doubled, whereas the amounts of GA₂₀, GA₁, GA₂₉ and GA₈ declined. By the end of the season, standard shoots were 40 cm in length with 20 leaves and dwarf shoots were 28 cm in length, but with 36 leaves. High summer temperatures appear to induce loss of GA-responsiveness in orchard-grown dwarf trees and to cause a reduced rate of conversion of GA₁₉ to GA₂₀ in these genotypes.     

Response of normal and reperfused livers to glucagon stimulation: NMR detection of blood flow and high-energy phosphates

The effects of glucagon on blood flow and high-energy phosphates in control and in rat livers damaged by ischemia were studied using in vivo nuclear magnetic resonance (NMR) spectroscopy. Normal livers and livers which had been made ischemic for 20, 40, and 60 min followed by 60 min of reperfusion were studied. Ischemia led to a loss in adenosine triphosphate (ATP) within 30 min. Reperfusion after 20 min of ischemia led to complete recovery of ATP. 60 min of reperfusion after 40 or 60 min of ischemia led to only a 76% and 48% recovery of ATP, respectively. Glucagon, at doses up to 2.5 mg/kg body weight, caused no changes in the inorganic phosphate (Pi) to ATP ratio in normal livers as measured by ³¹P-NMR spectroscopy. In livers which had been made ischemic for 20, 40, or 60 min, glucagon caused an increase in the Pi/ATP ratio of 18%, 40%, and 40%, respectively. ¹⁹F-NMR detection of the washout of trifluoromethane from liver was used to measure blood flow. Glucagon-stimulated flow in the normal liver in a dose-dependent manner, with 2.5 mg glucagon/kg body weight leading to a 95% increase in flow. Ischemia for 20, 40, and 60 min followed by 60 min of reperfusion led to hepatic blood flows which were 63%, 68%, and 58% lower than control liver. In reperfused livers, blood flow after glucagon-stimulation was reduced to 56%, 43%, and 48% of control glucagon-stimulated flow after 20, 40, and 60 min of ischemia. These results indicate that ischemia followed by reperfusion leads to deceases in hepatic blood flow prior to alterations in ATP and the response of the liver to glucagon is altered in the reperfused liver.     

Selenium uptake by plants as a function of soil type,organic matter content and pH

In pots containing sandy soils at two levels (pH 5 and 7) to which 0.5 mg Se L^-1 soil had been added, an increase in the proportion of clay soil or peat soil led to a decrease in the uptake of Se by spring wheat grain (Triticum aestivum L., var. Drabant) and winter rape plants (Brassica napus L., var. Emil). The effect was most pronounced for the smallest additions of clay and peat soils. Differences in Se uptake between the two pH levels were greatest in treatments where the additions of clay and peat soils were small. At the high pH, an increase in clay content from 7% to 39% resulted in a decrease in Se uptake of 79% for wheat and 70% for rape. At the low pH, the uptake decreased by 72% and 77%, respectively. At the higher pH, an increase in the content of organic matter from 1.4% to 39% resulted in decreases in Se uptake of 88% for wheat grain and 69% for rape. At the low pH, Se uptake decreased by 63% and 48%, respectively. Adding peat soil to clay soil had little effect on Se uptake. Among the limed, unmixed clay, sand and peat soils to which Se had not been added, uptake was highest from the sandy soil, i.e. 8.3 ng Se/g wheat grain and 42 ng Se/g rape. The lowest uptake rates were obtained in the clay soil, i.e. 3.0 ng Se/g for wheat grain and 9.0 ng Se/g for rape.     

Selenium content of Spanish infant formulae and human milk: Influence of protein matrix, interactions with other trace elements and estimation of dietary intake by infants

The selenium content of infant formulae varies as a result of differences in the amount of intrinsic selenium compounds. Manufacturers have been gradually changing the protein profile of infant formulae to reflect human milk contents more closely. Because of these variations in infant formula composition and their potential impact on selenium content, this trace element was analysed with regard to the different protein sources.

The aims of this study were to determine the selenium content in infant formulae sold commercially in Spain, to estimate a daily dietary intake for infants fed on formulae and to compare with the selenium provided by Spanish breast milk samples used as a reference. We have also identified certain trace elements added to formulae which interact with selenium according to the type and protein matrix of the infant formulae.

Selenium concentration was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) with a hydride generator.

The selenium concentrations in human milk and infant formulae determined in this study are similar to those found by other researchers in different countries. The daily selenium intake from the formulae studied was estimated according to the recommended doses from the manufacturers. The theoretical selenium intake of nursed infants has been studied in relation to the Recommended Dietary Allowance (RDA: 10 μg Se/day) and the specific recommendations for infant formula nutrient contents (10–35 μg Se/L) set by the Expert Panel of Life Sciences Research Office (LSRO) of the American Society for Nutritional Sciences.

According to our results, on an overall view, infants fed on the studied infant formulae have an intake between basal and normative requirements. This might be considered as providing an adequate selenium supply. However, the intake of selenium provided by several formulae included in this research did not reach the RDA for the first month of neonate life.     

An evaluation study of trace element content in colorectal liver metastases and surrounding normal livers by X-ray fluorescence

Background Trace elements are involved in many key pathways involving cell cycle control. The levels of trace metals such as iron, copper, and zinc in colorectal liver metastases have not previously been assessed. Methods The trace element content in snap-frozen cancerous liver tissue from patients who underwent liver resection for colorectal liver metastases was compared with the normal surrounding liver (distant from the cancer) using X-ray fluorescence (XRF). Results X-ray fluorescence was performed on a total of 60 samples from 30 patients. Of these 29 matched pairs (of cancer and normal liver distant from cancer from the same patient) were eligible for univariate analysis. Iron (0.00598 vs. 0.02306), copper (0.00541 vs. 0.00786) and zinc (0.01790 vs. 0.04873) were statistically significantly lower in the cancer tissue than the normal liver. Iron, copper, and zinc were lower in the cancer tissue than in the normal liver in 24/29 (82.8%), 23/29 (79.3%), and 28/29 (96.6%) of cases respectively. Multivariate analysis of the 60 samples revealed that zinc was the only trace element decreased in the cancer tissue after adjusting for the other elements. Zinc levels were not affected by any of the histopathological variables. Conclusion Iron, copper, and zinc are lower in colorectal liver metastases than normal liver. An investigation into the pathways underlying these differences may provide a new understanding of cancer development and possible novel therapeutic targets.     

Influence of season and diet on liver and kidney content of essential elements and heavy metals in svalbard reindeer

Samples of liver (n=78) and kidney (n=60) from Svalbard reindeer (Rangifer tarandus platyrhynchus Vrolik) collected at four different seasons in Svalbard were analysed for their content of Cd, Pb, Cu, Zn, Mn, and Se. The study shows that when animals are exposed to large seasonal variations in both the quality and quantity of food, it is crucial to relate element concentrations to the physiological condition of the animal, e.g., to look at seasonal fluctuations in the total element content of the different organs. Deceased January 9, 1991.     

Distributional pattern of zinc,cadmium, mercury,and selenium in livers of Hooded Seal (Cystophora cristata)

The liver of Hooded Seal (Cystophora cristata) consists of six lobes of unequal sizes. Extensive sampling by means of cutout samples covering the depth horizon 2–30 mm below liver surface indicates that the liver may be considered homogeneous as to concentrations of zinc, cadmium, mercury, and selenium (residuals of overall mean compatible with normality, the all-organ variance negligibly in excess of within-lobe variance). Mercury and selenium are present in equimolar concentrations. Cutout samples have mercury and selenium concentrations respectively c. 17 and 8% (dry weight basis) above those of homogenates of the same lobe. The apparent dilution of mercury and selenium is tentatively attributed to coarse blood vessels and bile ducts abundantly present in the homogenate but almost absent from the cutouts.     

Growth requirements and neoplastic transformation of two types of normal human breast epithelial cells derived from reduction mammoplasty

Summary A chemically defined culture medium was developed to support the growth of two distinctly different types of normal human breast epithelial cells (HBEC) derived from reduction mammoplasty. Type I cells expressed luminal epithelial cell markers and were deficient in gap junctional intercellular communication (GJIC), whereas Type II cells expressed basal epithelial cell markers and were efficient in GJIC. In this study, we examined and compared the growth factor and hormone requirements of these two types of cells and a series of cell lines that were obtained by sequential transfection with SV40 DNA (extended lifespan, nontumorigenic), treatment with 5-bromodeoxyuridine (BrdU)/black light (immortal and weakly tumorigenic), and infection of a virus carrying the neu oncogene (highly tumorigenic). Growth of Type I cells was inhibited by withdrawing epidermal growth factor (EGF), hydrocortisone (HC), or insulin (INS) from the culture media, but was enhanced by fetal bovine serum (FBS) supplementation. Growth of Type II cells was inhibited by withdrawal of EGF, HC, or INS from the media, and was inhibited by FBS supplementation. Withdrawal of human transferrin (HT) or 17β-estradiol (E₂) from the media did not alter the growth of Type I or Type II cells. SV40 transfected Type I cell lines still required EGF, HC, or INS for optimal growth. However, the highly tumorigenic cell line did not show a growth dependence on EGF, HC, or INS but did appear to require HT and 3,3′,5-triiodo-D.L. thyronine (T₃) for optimal growth. In addition, FBS stimulated the growth of these cell lines. Thus, this study shows that Type I HBEC are distinctly different from Type II HBEC in growth response to FBS and that neoplastically transformed Type I cells could become growth factor and hormone independent.     

Features of insulin receptor interaction in placenta from normal,overt and poorly controlled gestational diabetic patients

Features of insulin binding to trophoblast plasma membranes were studied in six normal pregnant women (NP), six overt diabetes (ODP) and six poorly controlled glycemic gestational patients (PCDP) i.e. women who did not strictly follow the management of diabetes mellitus during pregnancy. A decreased maximum specific insulin receptor binding per 0.1 mg membrane protein in placenta from PCDP (12%) was found comparing with that from ODP or NP (17.5% and 36.2%, respectively, P<0.01), The insulin binding in PCDP declined at a faster rate until it reached minimum when studied at a higher temperature (25–37°C). The binding equilibrium was likewise attained faster at this temperature than that at lower temperature of 4°C for all studied groups.The insulin receptor binding in all studied groups was pH dependent. The maximum binding in ODP and PCDP groups was attained at pH 7.8 while for NP maximum binding was at pH 7.4. The competitive dinding assay was carried out with 14 concentrations of unlabelled insulin and the half maximal displacement of¹²⁵I-insulin was at 8×10^–9 M, 6×10^–9 M and 4×10^–9 M for NP, ODP and PCDP, respectively (P<0.05) suggesting the differences in the effect of glycemic control on the insulin binding. Furthermore the binding yielded curvilinear Scatchard plots with the apparent affinity of the receptors being affected in the ODP and PCDP groups.The molecular characteristics of the receptors in the diabetic patients as revealed by the cross-linking technique used in this study did not reveal any changes in the subunit structures when compared with normals except that the¹²⁵I-insulin bound as shown by the band intensity was much less in PCDP. These findings indicate that control of hyperglycemia could optimize the outcome of insulin receptor function during diabetic pregnancy.     

The relation between inhibition of glycolysis and stimulation of oxygen uptake due to glucagon in livers from rats in different metabolic conditions

The relation between the effects of glucagon on oxygen consumption and glycolysis in livers from rats under different metabolic conditions was examined. Respiration of substrate-free perfused livers with different glycolytic fluxes, induced by changes in the pattern of food intake, responds differently to the infusion of 1 nM glucagon. The increases in oxygen uptake caused by 1 nM glucagon correlate reasonably well with the absolute decreases in glycolysis. The degree of inhibition of glycolysis is approximately constant (58 per cent) for all metabolic conditions. When no recovery of glycolysis occurs upon cessation of glucagon infusion, the same happens with oxygen consumption, which remains stimulated. It is concluded that in livers with no appreciable biosynthetic activities, the action of glucagon on respiration and glycolysis may be interpreted in terms of an interaction of interpreted in terms of an interaction of cytosolic and mitochondrial ATP generating processes.     

Mercury and Selenium in Blood and Epidermis of Bottlenose Dolphins (Tursiops truncatus) from Sarasota Bay, FL: Interaction and Relevance to Life History and Hematologic Parameters

Blood and epidermal biopsies from free-ranging Tursiops truncatus captured and released during either summer or winter health assessments in Sarasota Bay, FL, were evaluated for concentrations of mercury, selenium, stable isotopes (δ¹³C and δ¹⁵N), and blood glutathione peroxidase activity in conjunction with routine hematology and serum chemistry panels. Major objectives were to: 1) quantify and describe relationships among mercury, selenium, glutathione peroxidase, and stable isotopes of C and N in blood and epidermis; 2) elucidate major parameters that influence blood mercury and glutathione peroxidase activity; 3) relate measures of tissue mercury, selenium, and glutathione peroxidase to specific ecological, hematological, morphological, or life history parameters, including season, sex, age, and trophic level. Mercury in both tissues examined is almost exclusively methylmercury. Epidermal concentrations of mercury and selenium reflect their respective amounts in blood, albeit at several times blood concentrations of mercury. The strong association between blood mercury and serum selenium, in conjunction with a lack of significant correlation between blood mercury and glutathione peroxidase, implies that a substantial proportion of blood mercury is affiliated with another selenium-containing moiety or is related to recent dietary intakes (e.g., trophic level, intensive fish consumption). Circulating blood mercury may be described in terms of serum selenium concentration, along with interaction terms among serum selenium, blood δ¹⁵N, and age. Current selenium concentrations in Sarasota Bay dolphins appear adequate for maintenance of blood glutathione peroxidase activity. However, dolphins evidently are subject to seasonal exacerbation of oxidative stress, which might render them more vulnerable to toxic effects of mercury. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Three-dimensional aspects of blood vessels in thyroids from normal,low iodine diet-treated,TSH-treated,and PTU-treated rats

Summary Three-dimensional images of blood vessels in thyroid glands from normal, low iodine diet-treated, thyroid-stimulating hormone (TSH)-treated and propylthiouracil (PTU)-treated rats were investigated by use of the corrosion-cast method. The vascular casts made by the injection of methacrylate resin were observed with the scanning electron microscope. In normal animals, each follicle is surrounded by a clearly defined basket-like capillary network, which is generally independent of adjacent networks, though a few anastomoses or common capillaries are sometimes seen. In low iodine diet-treated or TSH-treated animals, the capillaries in the basket-like network become markedly dilated and fuse with one another. Though the vascular casts of PTU-treated animals are similar to those of low iodine diet-treated or TSH-treated ones in some aspects, most basket-like networks become distorted and irregular in shape, and the capillaries are heterogeneously dilated and show many buds, branches and anastomoses. We consider that these peculiar changes in the thyroid of the PTU-treated animals are due not only to the elevation of serum TSH but also to other unknown factors. It is clear that the distribution and morphology of the thyroid capillaries are extremely affected and changed by functional states of the gland.This study was supported in part by grant from the Research Fund of the Ministry of Education, Science, and Culture, Japan     

Reperated establishment of diploid epithelial cell cultures from normal and partially hepatectomized rats

Summary A number of liver epithelial cell cultures were established from 10 to 12-d-old sucklings, 6 to 8-wk-old young adults, or from 2 to 18-month-old partially hepatectomized rats. Improvements in the methods for cell isolation and culture yielded replicating cells from every experiment and they were maintained for different periods with regular passages. The proliferative potential in vitro of adult rat liver cells could be increased if the rats were subjected to partial hepatectomy before cell isolation. In the early passages, the majority of the cells were found to have a true diploid karyotype as studied by the Giemsa-banding technique. Under the culture conditions described, a very high percentage of cells remained in the diploid range for, in most cases, at least 4 months and in some cases for up to 6 months. Afterward, the karyotype was unstable, but no “crisis” period was seen before the cells became aneuploid. Until this time, the growth characteristics of the cells also followed a normal pattern showing density dependent inhibition of division and a lack of markers associated with malignancy. The cultured liver cells exhibited a number of liver specific properties when they were maintained as a confluent monolayer. The early passages of diploid epithelial cell cultures derived from normal and regenerating rat liver are good models for studies of the regulation of cell division and the changes that are related to carcinogenesis. Research Sponsored by the National Cancer Institute, DHHS, under Contract N01-CO-23909 with Litton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsment by the U.S. Government.     

Spatiotemporal distribution of insulin-like growth factor receptors during nephrogenesis in fetuses from normal and diabetic rats

Exposure to hyperglycemia in utero impairs rat nephrogenesis. The effect of maternal diabetes on insulin-like growth factors and their receptors in the fetal kidney is associated with an increase in both mRNA and protein of the insulin-like growth factor II/mannose 6-phosphate receptor. However, this receptor has never been localized in the fetal kidney. The spatial and temporal distribution of the three insulin-like growth factor receptors (insulin-like growth factor I receptor, insulin-like growth factor II/mannose 6-phosphate receptor and insulin receptor) in rat metanephros during both normal and streptozotocin-induced diabetic renal development was investigated using in situ hybridization and immunohistochemistry. All receptors were found in the fetal kidney from the start of nephrogenesis. Insulin-like growth factor I receptor expression was ubiquitous and continuously present during metanephric development. Insulin receptor expression was developmentally regulated during kidney maturation with an enhanced expression in proximal tubules at the late stages of development. Insulin-like growth factor II/mannose 6-phosphate receptor expression was ubiquitous in the early stages of development and was dramatically decreased at the late stages of normal kidney development. Insulin receptor and insulin-like growth factor I receptor expressions were unchanged in diabetic metanephroi. Although the spatial expression of insulin-like growth factor II/mannose 6-phosphate receptor was unaffected by hyperglycemia, its expression was not downregulated in the mesenchyme of the nephrogenic zone of diabetic fetuses on gestational day 20. This study suggests a crucial role of insulin-like growth factor II/mannose 6-phosphate receptor in the pathogenesis of the impaired nephrogenesis in fetuses of diabetic mothers.