Development of chicken embryos in a pulsed magnetic field

Six independent experiments of common design were performed in laboratories in Canada, Spain, Sweden, and the United States of America. Fertilized eggs of domestic chickens were incubated as controls or in a pulsed magnetic field (PMF); embryos were then examined for developmental anomalies. Identical equipment in each laboratory consisted of two incubators, each containing a Helmholtz coil and electronic devices to develop, control, and monitor the pulsed field and to monitor temperature, relative humidity, and vibrations. A unipolar, pulsed, magnetic field (500-microseconds pulse duration, 100 pulses per s, 1-microT peak density, and 2-microseconds rise and fall time) was applied to experimental eggs during 48 h of incubation. In each laboratory, ten eggs were simultaneously sham exposed in a control incubator (pulse generator not activated) while the PMF was applied to ten eggs in the other incubator. The procedure was repeated ten times in each laboratory, and incubators were alternately used as a control device or as an active source of the PMF. After a 48-h exposure, the eggs were evaluated for fertility. All embryos were then assayed in the blind for development, morphology, and stage of maturity. In five of six laboratories, more exposed embryos exhibited structural anomalies than did controls, although putatively significant differences were observed in only two laboratories (two-tailed Ps of .03 and less than .001), and the significance of the difference in a third laboratory was only marginal (two-tailed P = .08). When the data from all six laboratories are pooled, the difference in incidence of abnormalities in PMF-exposed embryos (approximately 25 percent) and that of controls (approximately 19 percent), although small, is highly significant, as is the interaction between incidence of abnormalities and laboratory site (both Ps less than .001). The factor or factors responsible for the marked variability of inter-laboratory differences are unknown.     

Determination of halothane ED-50 in Mongolian gerbils (Meriones unguiculatus)

The effective dose-50 (ED-50) for halothane anesthesia was studied in 45 male Mongolian gerbils at age 12 weeks. Close control of circadian variation and animal temperature was maintained. Regression analysis for the percent of animals moving in response to a standard stimulus revealed an ED-50 of 1.32 volumes %, a concentration that approximates the ED-50 of the rat.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Response of soil microbial biomass and activity in early restored lands in the northeastern Brazilian Atlantic Forest

Intensive land use of the Brazilian Atlantic Forest accelerated with the rise of sugar cane plantations in the northeastern part of Brazil. Consequently, many ecosystems were destroyed, including riparian forests. The number of studies of riparian restoration has increased but comparative studies on the belowground effects of common reforestation strategies are rare. Here, we compared soil microbial properties among four different land use types: native rainforest, sugar cane plantation, single species reforestation, and mixed species reforestation, each replicated at two spatially independent sites. Soil samples were taken in 2013 and 2014, that is 2 and 3 years after reforestation, respectively. In both years, land use types had a significant effect on basal respiration, microbial biomass, and specific respiration (whereas specific respiration was marginally affected in 2014). In 2013, basal respiration in sugar cane plantations was significantly lower (?65%) when compared to native forests. In 2014, basal respiration (+60%) and soil microbial biomass (+90%) were significantly higher in mixed species reforestation compared to sugar cane, whereas single species reforestation had comparable values as in sugar cane plantations. Our results indicate that soil microbial biomass and activity respond rapidly to land use change when mixed species reforestation is used. Thus, using mixed species reforestation may enhance the provisioning of ecosystem services already in the short term.     

Beta-carotene storage, conversion to retinoic acid, and induction of the lipocyte phenotype in hepatic stellate cells

Hepatic stellate cells (HSCs) are the major site of retinol (ROH) metabolism and storage. GRX is a permanent murine myofibroblastic cell line, derived from HSCs, which can be induced to display the fat-storing phenotype by treatment with retinoids. Little is known about hepatic or serum homeostasis of beta-carotene and retinoic acid (RA), although the direct biogenesis of RA from beta-carotene has been described in enterocytes. The aim of this study was to identify the uptake, metabolism, storage, and release of beta-carotene in HSCs. GRX cells were plated in 25 cm(2) tissue culture flasks, treated during 10 days with 3 micromol/L beta-carotene and subsequently transferred into the standard culture medium. beta-Carotene induced a full cell conversion into the fat-storing phenotype after 10 days. The total cell extracts, cell fractions, and culture medium were analyzed by reverse phase high-performance liquid chromatography for beta-carotene and retinoids. Cells accumulated 27.48 +/- 6.5 pmol/L beta-carotene/10(6) cells, but could not convert it to ROH nor produced retinyl esters (RE). beta-Carotene was directly converted to RA, which was found in total cell extracts and in the nuclear fraction (10.15 +/- 1.23 pmol/L/10(6) cells), promoting the phenotype conversion. After 24-h chase, cells contained 20.15 +/- 1.12 pmol/L beta-carotene/10(6) cells and steadily released beta-carotene into the medium (6.69 +/- 1.75 pmol/ml). We conclude that HSC are the site of the liver beta-carotene storage and release, which can be used for RA production as well as for maintenance of the homeostasis of circulating carotenoids in periods of low dietary uptake.     

Hepatic stellate cells uptake of retinol associated with retinol-binding protein or with bovine serum albumin

Retinol is stored in liver, and the dynamic balance between its accumulation and mobilization is regulated by hepatic stellate cells (HSC). Representing less than 1% total liver protein, HSC can reach a very high intracellular retinoid (vitamin-A and its metabolites) concentration, which elicits their conversion from the myofibroblast to the fat-storing lipocyte phenotype. Circulating retinol is associated with plasma retinol-binding protein (RBP) or bovine serum albumin (BSA). Here we have used the in vitro model of GRX cells to compare incorporation and metabolism of BSA versus RBP associated [(3)H]retinol in HSC. We have found that lipocytes, but not myofibroblasts, expressed a high-affinity membrane receptor for RBP-retinol complex (KD = 4.93 nM), and both cell types expressed a low-affinity one (KD = 234 nM). The RBP-retinol complex, but not the BSA-delivered retinol, could be dislodged from membranes by treatments that specifically disturb protein-protein interactions (high RBP concentrations). Under both conditions, treatments that disturb the membrane lipid layer (detergent, cyclodextrin) released the membrane-bound retinol. RBP-delivered retinol was found in cytosol, microsomal fraction and, as retinyl esters, in lipid droplets, while albumin-delivered retinol was mainly associated with membranes. Disturbing the clathrin-mediated endocytosis did not interfere with retinol uptake. Retinol derived from the holo-RBP complex was differentially incorporated in lipocytes and preferentially reached esterification sites close to lipid droplets through a specific intracellular traffic route. This direct influx pathway facilitates the retinol uptake into HSC against the concentration gradients, and possibly protects cell membranes from undesirable and potentially noxious high retinol concentrations.     

Blood gas and catecholamine levels in capture stressed desert bighorn sheep.

Forty-seven bighorn sheep (Ovis canadensis nelsoni) were captured within a 3-day period in December, 1989 as part of a California Department of Fish and Game effort to repopulate historic ranges in California. They were captured on the Mojave Desert in the Kelso Mountains near Old Dad Peak, San Bernardino County, California. Venous blood gases measured at the site of capture demonstrated a severe metabolic acidosis (base deficit, 23 mEq/liter), with no evidence of respiratory acidosis. There were moderately elevated plasma epinephrine (1.25 ng/ml), norepinephrine (2.60 ng/ml), and dopamine (114 pg/ml) levels. These data appear to reflect animals that have been moderately stressed. These acid-base-catecholamine values differ from values in resting domestic sheep, and are similar to those reported in greyhounds after brief strenuous exercise.