Effects of dietary cobalt on testicular structure

Adult male rats were maintained on a diet containing 265 ppm cobalt for up to 98 days. Three rats were sacrificed weekly and assayed for testicular damage by light and electron microscopy. Testicular damage was first apparent after 70 days of treatment, followed by a progressive deterioration of cell architecture and decrease in testicular volume. The degenerative changes were of a very general nature; e.g., thickening of basal lamina and basement membranes, increased packing of red blood cells in veins and arteries, formation of "giant" cells, loss of sperm tail filaments, and degeneration of sperm mitochondria. No cobalt residues could be detected by energy dispersive x-ray microanalysis. These data indicate that testicular degeneration was not a primary response to cobalt and suggest that the testes become hypoxic due both to blockage of veins and arteries by red blood cells and to changes in permeability caused by thickening of basal lamina and basement membranes.     

Surface-exposed proteins of 3T3-L1 adipocytes: identification of phosphorylated, insulin-translocated, and recycling proteins.

Twenty-seven adipocyte-specific, cell surface-exposed proteins were detected by derivatizing undifferentiated and differentiated 3T3-L1 cells with membrane impermeant sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate at 0 degrees C. Biotinylated proteins were adsorbed onto streptavidin-agarose, resolved on two-dimensional polyacrylamide gels, and detected by autoradiography or silver staining. Of the surface-exposed proteins specific to adipocytes, three were phosphorylated and seven were glycoproteins that bound to wheat germ agglutinin and eluted with N-acetylglucosamine. Eleven of the adipocyte-specific proteins were bound to streptavidin-agarose after the cells were biotinylated at 20 degrees C and then stripped with glutathione at 0 degrees C to isolate plasma membrane proteins that localize to recycling endosomes as well as the cell surface. When insulin-deprived cells were acutely treated with insulin, only a few proteins, including one protein tentatively identified as the GLUT4 glucose transporter, were found to increase in concentration at the cell surface. These latter results imply that up-regulation of glucose transport by the translocation of GLUT4 to the cell surface in response to insulin occurs by exocytic fusion of an intracellular compartment having a limited number of proteins.     

Insulin-like growth factor I levels decrease in the development of diabetes inMacaca nigra

Members of the monkey speciesMacaca nigra spontaneously develop impairments in insulin secretion and glucose clearance, and eventually become overtly diabetic. Changes in certain metabolic signals such as clearance of glucose and insulin increment secreted in an intravenous glucose tolerance test have allowed the identification of four stages in the progression from non-diabetes to diabetes in monkeys — non-diabetic, hormonally impaired, borderline diabetic, and diabetic. Recently, another metabolic stage, hyperinsulinemic, was also identified in these animals. In recent years, other factors besides those listed above have been implicated to be correlated with the metabolic progression from a nondiabetic to a diabetic state. One of these factors, is insulin like growth factor I (IGF-I). In diabetic humans who are in poor metabolic control, and in rats with streptozotocin induced ketotic diabetes, serum levels of IGF-I are lowered by as much as 40–50% of control non-diabetics. If indeed decreased IGF-I levels are correlated with the onset of diabetes then changes in IGF-I concentrations prior to the clinically diagnosed disease state would be expected. Using serum samples collected from different animals in a colony ofMacaca nigra in a variety of metabolic states, we have found that IGF-I and insulin levels decrease in each defined metabolic state as the animals progress from nondiabetic to diabetic. Since IGF-I and insulin levels decrease in a similar fashion in the progression of this disease then this maybe indicative of the coordinate expression of these two factors.     

Chromatographic analysis of amino acids and primary amines with o-phthalaldehyde detection

The applicability of the o-phthalaldehyde reaction with fluorometric detection to the simultaneous chromatographic analysis of amines and nonprotein amino acids is demonstrated. The majority of the compounds tested could be quantitatively determined with high sensitivity. The method is particularly well suited for the analysis of β-amino acids. Amino compounds lacking an α-hydrogen atom are detected with lower sensitivity, due to a lower relative fluorescence. Secondary amino compounds are undetectable with this system.     

Analysis of nuclear sugar-binding components in undifferentiated and in vitro differentiated human promyelocytic leukemia cells (HL60)

The nuclear sugar-binding components (i.e., lectinlike molecules) were analyzed using isolated and membrane-depleted nuclei after incubation in the presence of fluorescein-labeled neoglycoproteins. This analysis was performed before and during the in vitro differentiation of HL60 cells into monocytes by PMA treatment and into granulocytes by DMSO treatment. The nucleoli of undifferentiated and differentiated HL60 cells were not labeled, unlike the nucleoli of other mammalian cells studied so far. This peculiarity allowed us to quantitatively analyze by flow cytometry the changes in the lectin activity associated with the extranucleolar territories enriched in ribonucleoprotein complexes. The neoglycoprotein binding was found to be significantly lower in differentiated than in undifferentiated cells. The decrease in neoglycoprotein binding was observed within the first 24 h of DMSO or PMA treatment, just before the arrest of DNA synthesis. Taking into account that the granulocytic differentiation required 72 h of chemical treatment, the extra-nucleolar lectins might be involved in modulation of the DNA synthesis rather than in phenotypic differentiation. These data are discussed in an attempt to reconcile the association of lectins with RNP complexes and their possible involvement in modulation of HL60 cell proliferation.     

Differential inactivation of the “L” and “Ly+” amino acid transport systems by a sulfhydryl reagent and a photoaffinity probe

Using a substrate-stimulated amino acid efflux system, it has been shown that the “Ly⁺” and “L” amino acid transport systems of mouse embryo cells in culture are differentially inhibited by parachloromercuribenzene sulfonate (PCMB-S) and the photoaffinity probe 4-fluoro-3-nitrophenylazide (FNPA). Three types of evidence support the conclusion that these transport systems are mediated by separate carrier proteins. (1) The specificity of substrate-stimulated efflux is high for each system; (2) PCMB-S inhibits l-phenylalanine and l-leucine stimulated l-[³H]phenylalanine efflux with no effect on l-lysine stimulated l-[³H]lysine efflux, and (3) the photoaffinity probe FNPA inhibits l-lysine efflux with little effect on the l-phenylalanine-stimulated efflux.     

Intron-derived small RNAs for silencing viral RNAs in mosquito cells

Aedes aegypti and Ae. albopictus are the main vectors of mosquito-borne viruses of medical and veterinary significance. Many of these viruses have RNA genomes. Exogenously provided, e.g. transgene encoded, small RNAs could be used to inhibit virus replication, breaking the transmission cycle. We tested, in Ae. aegypti and Ae. albopictus cell lines, reporter-based strategies for assessing the ability of two types of small RNAs to inhibit a chikungunya virus (CHIKV) derived target. Both types of small RNAs use a Drosophila melanogaster pre-miRNA-1 based hairpin for their expression, either with perfect base-pairing in the stem region (shRNA-like) or containing two mismatches (miRNA-like). The pre-miRNA-1 stem loop structure was encoded within an intron; this allows co-expression of one or more proteins, e.g. a fluorescent protein marker tracking the temporal and spatial expression of the small RNAs in vivo. Three reporter-based systems were used to assess the relative silencing efficiency of ten shRNA-like siRNAs and corresponding miRNA-like designs. Two systems used a luciferase reporter RNA with CHIKV RNA inserted either in the coding sequence or within the 3’ UTR. A third reporter used a CHIKV derived split replication system. All three reporters demonstrated that while silencing could be achieved with both miRNA-like and shRNA-like designs, the latter were substantially more effective. Dcr-2 was required for the shRNA-like siRNAs as demonstrated by loss of inhibition of the reporters in Dcr-2 deficient cell lines. These positive results in cell culture are encouraging for the potential use of this pre-miRNA-1-based system in transgenic mosquitoes.     

Myeloid-derived suppressor cells are associated with disease progression and decreased overall survival in advanced-stage melanoma patients

Myeloid-derived suppressor cells are increased in the peripheral blood of advanced-stage cancer patients; however, no studies have shown a correlation of these immunosuppressive cells with clinical outcomes in melanoma patients. We characterized the frequency and suppressive function of multiple subsets of myeloid-derived suppressor cells in the peripheral blood of 34 patients with Stage IV melanoma, 20 patients with Stage I melanoma, and 15 healthy donors. The frequency of CD14⁺ MDSCs (Lin^? CD11b⁺ HLA-DR^? CD14⁺ CD33⁺) and CD14^? MDSCs (Lin^? CD11b⁺ HLA-DR^? CD14^? CD33⁺) was increased in the peripheral blood of Stage IV melanoma patients relative to healthy donors. The frequency of CD14⁺ and CD14^? MDSCs correlated with each other and with the increased frequency of regulatory T cells, but not with classically defined monocytes. CD14^? MDSCs isolated from the peripheral blood of Stage IV melanoma patients suppressed T cell activation more than those isolated from healthy donors, and the frequency of these cells correlated with disease progression and decreased overall survival. Our study provides the first evidence that the frequency of CD14^? MDSCs negatively correlates with clinical outcomes in advanced-stage melanoma patients. These data indicate that suppressive MDSCs should be considered as targets for future immunotherapies.     

Reliance on head versus eyes in the gaze following of great apes and human infants: the cooperative eye hypothesis

As compared with other primates, humans have especially visible eyes (e.g., white sclera). One hypothesis is that this feature of human eyes evolved to make it easier for conspecifics to follow an individual's gaze direction in close-range joint attentional and communicative interactions, which would seem to imply especially cooperative (mututalistic) conspecifics. In the current study, we tested one aspect of this cooperative eye hypothesis by comparing the gaze following behavior of great apes to that of human infants. A human experimenter "looked" to the ceiling either with his eyes only, head only (eyes closed), both head and eyes, or neither. Great apes followed gaze to the ceiling based mainly on the human's head direction (although eye direction played some role as well). In contrast, human infants relied almost exclusively on eye direction in these same situations. These results demonstrate that humans are especially reliant on eyes in gaze following situations, and thus, suggest that eyes evolved a new social function in human evolution, most likely to support cooperative (mututalistic) social interactions.