The spatial arrangement of germ line cells in ovarian follicles of the mutantdicephalic inDrosophila melanogaster

Summary The pattern of intercellular connections between germ line cells has been studied in follicles of the mutantdicephalic (dic), which possess nurse cell clusters at both poles. Staining of follicles with a fluorescent rhodamine conjugate of phalloidin reveals ring canals and cell membranes and thus allows us to reconstruct the spatial organization of the follicle. Each germ line cell can be identified by the pattern of cell-cell connections which reflect the mitotic history of individual cells in the 16-cell cluster. The results indicate that in both wild-type anddicephalic cystocyte clusters one of the two cells with four ring canals normally becomes the pro-oocyte. However, in some follicles (dicephalic and wild-type) oocytes were found with fewer or more than four ring canals. Indic follicles, one or several nurse cells may become disconnected from the other cells during oocyte growth at stage 9–10. Such disconnected cells cannot later on empty their cytoplasm into the oocyte. This, in turn, might be of consequence for the determination of axial polarity of the embryo.     

Intracellular transport of endocytosed chylomicron [3H]retinyl ester in rat liver parenchymal cells. Evidence for translocation of a [3H]retinoid from endosomes to endoplasmic reticulum

The intracellular transport of chylomicron remnants labeled with [3H]retinyl ester was studied in rat liver parenchymal cells by means of subcellular fractionation in Nycodenz and sucrose density gradients. The data presented indicate that endocytosed chylomicron remnant [3H]retinyl ester initially is located in low density endosomes. Radioactivity is subsequently transferred to a denser vesicle. Equilibrium as well as rate zonal centrifugation suggest that this denser [3H] retinoid-containing vesicle may represent endoplasmic reticulum. We have compared the intracellular transport of chylomicron remnant [3H]retinyl ester and 125I-asialofetuin. The receptor-mediated endocytosis of asialoglycoproteins in rat liver parenchymal cells is a thoroughly studied system. Our results suggest that the [3H] retinoid and 125I-asialofetuin follow the same path initially to the endosomes. After transit in endosomes, the intracellular transport differs. While asialofetuin is transported to the lysosomes, the retinoid is probably transferred to the endoplasmic reticulum.     

Effect of Feed Restriction on Performance and Postprandial Nutrient Metabolism in Pigs Co-Infected with Mycoplasma hyopneumoniae and Swine Influenza Virus

As nutritional status and inflammation are strongly connected, feeding and nutritional strategies could be effective to improve the ability of pigs to cope with disease. The aims of this study were to investigate the impact of a feed restriction on the ability of pigs to resist and be tolerant to a coinfection with Mycoplasma hyopneumoniae (Mhp) and the European H1N1 swine influenza virus, and the consequences for nutrient metabolism, with a focus on amino acids. Two groups of specific pathogen-free pigs were inoculated with Mhp and H1N1 21 days apart. One group was fed ad libitum, the other group was subjected to a two-week 40% feed restriction starting one week before H1N1 infection. The two respective mock control groups were included. Three days post-H1N1 infection, 200 g of feed was given to pigs previously fasted overnight and serial blood samples were taken over 4 hours to measure plasma nutrient concentrations. Throughout the study, clinical signs were observed and pathogens were detected in nasal swabs and lung tissues. Feed-restricted pigs presented shorter hyperthermia and a positive mean weight gain over the 3 days post-H1N1 infection whereas animals fed ad libitum lost weight. Both infection and feed restriction reduced postprandial glucose concentrations, indicating changes in glucose metabolism. Post-prandial plasma concentrations of the essential amino acids histidine, arginine and threonine were lower in co-infected pigs suggesting a greater use of those amino acids for metabolic purposes associated with the immune response. Altogether, these results indicate that modifying feeding practices could help to prepare animals to overcome an influenza infection. Connections with metabolism changes are discussed.     

Photolabeling of brain membrane proteins by lysergic acid diethylamide

³H-Lysergic acid diethylamide (³H-LSD) is irreversibly incorporated into bovine caudate membranes during ultraviolet light illumination. The incorporated radioligand apparently forms a covalent bond with a sub-population of the membrane proteins. Although the photolabeling pattern differs significantly from the Coomassie blue staining pattern on SDS gels, the photolabeling is apparently not specific for LSD binding sites associated with neurotransmitter receptors. ³H-LSD photolabeling can occur during prolonged exposure of membrane samples to room lighting and thus may introduce artifacts into receptor binding assays.     

Influence of Organic-Phosphates on 3-Phosphoglycerate Dependent O2 Evolution in C3 and C4 Mesophyll Chloroplasts

In C₄ plants phosphoenolpyruvate (PEP) of the C₄ cycle may betransported on a chloroplast transporter which also transports3-phosphoglycerate (3-PGA) and triosephosphates. In C₃ plantsPEP is not considered to be effectively transported on the chloroplastphosphate translocator. The influences of certain organic phosphates,having a similar structure to either PEP or triose-phosphates,on 3-PGA dependent O₂ evolution by C₄ (Digitaria sanquinalisL. Scop.) and C₃ (Hordeum vulgare L.) mesophyll chloroplastswere investigated. In the C₄ mesophyll chloroplasts phosphoglycolatewas a competitive inhibitor (K_i = 2.1 mM) of 3-PGA dependentO₂ evolution, and was as effective as previously reported forPEP. 2-Phosphoglycerate was also a competitive inhibitor (K_t= 8.6 mM) of O₂ evolution in the C₄ mesophyll chloroplasts with3-PGA as substrate, while phospholactate was a weak inhibitorand glyphosate had no effect. Neither PEP, phosphoglycolatenor 2-phosphoglycerate were effective inhibitors of 3- PGA dependentO₂ evolution in the C₃ chloroplasts. Phosphohydroxypyruvatewas a competitive inhibitor of 3-PGA dependent O₂2 evolutionin both chloroplast types. The selectivity in inhibition ofO₂ evolution with 3-PGA as substrate suggests that the C₄ mesophyllchloroplasts can recognize certain organic phosphates with thephosphate in the C-2 or C-3 position but that the C₄ mesophyllchloroplasts can only effectively recognize certain organicphosphates with the phosphate in the C-3 position. The resultsalso support the view that 3-PGA and PEP are transported onthe same phosphate translocator in C₄ mesophyll chloroplasts. ¹ Current address: Department of Horticulture, 2001 Fyffe Court,The Ohio State University, Columbus, Ohio 43210-1096. (Received March 24, 1987; Accepted April 16, 1987)     

Intensive RNAi with lentiviral vectors in mammalian cells

RNAi is a powerful technology for analyzing gene function in human cells. However, its utility can be compromised by inadequate knockdown of the target mRNA or by interpretation of effects without rigorous controls. We review lentiviral vector-based methods that enable transient or stable knockdowns to trace mRNA levels in human CD4+ T cell lines and other targets. Critical controls are reviewed, including rescue of the pre-knockdown phenotype by re-expression of the targeted gene. The time from thinking about a potential knockdown target to analysis of phenotypes can be as short as a few weeks.     

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [³H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [³H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.