Reorganization of the Endoplasmic Reticulum during Meiotic Maturation of the Mouse Oocyte

The endoplasmic reticulum (ER) of live metaphase II mouse eggs and prophase I-arrested oocytes was compared using the fluorescent, lipophilic dicarbocyanine dye, DiI. DiI, dissolved in soybean oil, was microinjected into oocytes and eggs; the dye diffused throughout the cytoplasm to label the ER, which was imaged by confocal microscopy. The mature egg had a fine reticular network of ER throughout the cell and numerous dense accumulations of membrane in the cortex. These ER accumulations, 1-2 μm in diameter, were generally absent deeper in the cytoplasm. A similar staining pattern was observed when the eggs were fixed within 1 min of injection, providing evidence that the cortical accumulations of membrane are part of a continuous ER membrane system, since membrane trafficking could not occur in a fixed egg. Cortical ER accumulations were localized to the same region of the egg as the cortical granules and were not observed in the cortical granule-free region adjacent to the meiotic spindle. In contrast, ER accumulations were rarely found in the cortex of the immature, prophase I-arrested oocyte, but larger and less well-defined membrane clusters were found throughout the deeper cytoplasm of the oocyte. The appearance of ER clusters in the egg cortex following oocyte maturation correlates with an increased ability of the mature egg to release calcium at fertilization. Since the ER is a calcium store, structural reorganization of the ER may be necessary to permit the large release of calcium and resulting cortical granule exocytosis at fertilization.     

Effects of prostaglandins on the affinity of hemoglobin for oxygen in human whole blood in vitro

The effect of prostaglandin on the affinity of hemoglobin for oxygen was tested on human blood $\text{in vitro}$, using six different prostaglandins at several dosage levels in fresh heparinized blood from normal donors and in stored citrated blood, and using prostaglandin E₂ on the blood from four seriously ill patients. No significant alterations in the affinity of hemoglobin for oxygen were dtected. A very small decrease in oxygen affinity in stored blood with high doses of prostaglandin was not statistically significant and would be of no physiologic significance even if real.We conclude that under the circumstances of this experiment prostaglandins do not alter the affinity of hemoglobin for oxygen in human whole blood $\text{in vitro}$.     

Role of subdermal current shunts in the failure of frogs to regenerate.

Large, uniform, skin-driven currents (20-40 muamp/cm2) leave the ends of limb stumps of post-metamorphic frogs (Rana pipiens) from about the first through the tenth day after amputation. However, right after amputation, while currents of comparable density may leave the periphery of the cut surface, current densities are greatly depressed in the center of this surface. We suggest that this depression is brought about by shunting through the subdermal lymph space (characteristic of anurans but not urodeles); continues in covert form after formation of a wound epithelium; and helps explain the ability of small, imposed currents to initiate frog limb regeneration.     

A caterpillar (Callopistria floridensis G. (Lepidoptera: Noctuidae)) accumulates arsenic from an arsenic‐hyperaccumulating fern (Pteris vittata L.).

1. The consumption of arsenic is toxic to most biota. However, a noctuid caterpillar was recently reported feeding on a plant known to hyperaccumulate arsenic. 2. The aim of this study was to investigate the effects of arsenic‐rich Pteris vittata L. consumption by Callopistria floridensis G., and measure differences in arsenic concentrations at various stages of development (larval and adult), and associated with exuviae and frass. 3. Callopistria floridensis accumulated extraordinary concentrations of arsenic. The relative accumulation of arsenic was highest in exuviae and larvae. Larvae invariably preferred P. vittata grown on low arsenic soil to P. vittata grown on higher soil arsenic concentrations, and appeared able to selectively forage on lower arsenic concentrations within each treatment. 4. These findings show that C. floridensis is tolerant of arsenic, and successfully develops to adulthood containing elevated concentrations of arsenic. Callopistria floridensis represents the only known terrestrial animal capable of accumulating arsenic, and may have developed novel physiological and behavioural adaptations to regulate the negative effects of arsenic.     

Antigen-stimulated lymphokines from patients with cutaneous leishmaniasis induce monocyte killing of Leishmania major intracellular amastigotes

The specific immune response of patients with cutaneous leishmaniasis including the ability of their lymphokines to enhance the monocytes' leishmaniacidal activity was studied. In 16 patients with cutaneous leishmaniasis, their concanavalin A-induced lymphocyte proliferative responses, interferon-gamma and interleukin 2 activities and the ability of their concanavalin A-induced lymphokines to kill monocyte intracellular amastigotes were not different from normal controls. Antigen-stimulated lymphocyte cultures showed that 13 of 13 patients had an increased lymphocyte proliferative response; 11 of 16 produced interleukin 2 and 12 of 13 produced interferon-gamma; in addition, 10 of 11 of these antigen-induced supernatants increased the monocytes' killing of Leishmania major amastigotes. Antibody levels to parasite membrane antigens determined by radioimmunoassay showed that 8 of 13 titers were greater than 10 and 4 of 13 titers were 2 to 10 times higher than control. Our findings demonstrate that patients with cutaneous leishmaniasis elicit a specific immune response to L. major antigens and part of this response is the production of lymphokine capable of promoting monocyte killing of intracellular amastigotes.     

A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

PGE restores the immune response in chemotherapy-treated,tumor-bearing mice

Two hundred seventy-one B-16 melanoma-bearing mice were randomized and treated for 4 days with either control diluent, 10 μg of 16, 16-dimethyl-PGE₂-methyl-ester (di-M-PGE₂), chemotherapy, or chemotherapy plus di-M-PGE₂. The chemotherapeutic regimens included adriamycin (7.5 mg/kg), 5-fluorouracil (250 mg/kg), nitrogen mustard (5 mg/kg), and vincristine (0.5 mg/kg). The number of plaque-forming cells and hemagglutinin titers in response to sheep erythrocytes were used as measures of humoral immunity while cellular immunity was assessed by evaluation of delayed hypersensitivity. As we previously reported, the presence of subcutaneous B-16 tumors induced substantial immunosuppression and this suppression was reversed by treatment with di-M-PGE₂. Treatment with all four chemotherapeutic agents induced profound immunosuppression. Similarly, the addition of di-M-PGE₂ to the chemotherapy protocols resulted in significant augmentation of cellular and humoral immunity.     

Modulation of potassium transport in cultured retinal pigment epithelium and retinal glial cells by serum and epidermal growth factor.

The ionic environment of retinal photoreceptors is partially controlled by potassium transporters on retinal glial and retinal pigment epithelial cells (RPE). In this study, serum and epidermal growth factor (EGF) were examined as modulators of potassium transport in confluent cultures of human RPE and rabbit retinal glia. EGF is a known mitogen for confluent RPE cultures and was shown here to also stimulate [3H]thymidine incorporation in cultures of retinal glia. For potassium transport studies 86Rb was used as a tracer during a 17-min incubation. For both retinal cell types the mean total 86Rb uptake in 10% serum was approximately 60% above basal, serum-free controls. For EGF, tested in several experiments in a concentration range from 1 to 100 ng/ml, maximal total uptake was 33 and 24% above controls for RPE and glia, respectively. Inhibitor studies suggested that basal and serum-stimulated uptake for both cell types occurred by the ouabain-sensitive Na-K ATPase pump and by the furosemide- or bumetanide-sensitive Na-K-Cl cotransporter. EGF-stimulated uptake appeared to be due predominantly to the cotransporter. The data suggest that serum components and EGF, which may be available to retina-derived cells under pathologic conditions, may not only stimulate proliferation but may also act as short-term modulators of potassium ion movement and thus affect physiologic processes that are sensitive to ion homeostasis.     

Expression of apo-aequorin during embryonic development; how much is needed for calcium imaging?

Aequorin is a bioluminescent calcium indicator consisting of a 21 kDa protein (apo-aequorin) that is covalently linked to a lipophilic cofactor (coelenterazine). The aequorin gene can be expressed in a variety of cell lines and tissues, allowing non-invasive calcium imaging of specific cell types. In the present paper, we describe the possibilities and limitations of calcium imaging with genetically introduced apo-aequorin during embryonic development. By injecting aequorin into sea urchin, Drosophila and zebrafish eggs, we found that higher aequorin concentrations are needed in smaller eggs. Our results suggest that for measuring resting levels of free cytosolic calcium, one needs aequorin concentrations of at least 40 μM in sea urchin eggs, 2 μM in Drosophila eggs, and only 0.11 μM in zebrafish eggs. A simple assay was used to determine the absolute concentrations of expressed apo-aequorin and the percentage of aequorin formation in vivo. The use of this assay is illustrated by expression of the aequorin gene in Drosophila oocytes. These oocytes form up to 1 μM apo-aequorin. In our hands, only 0.3% of this apo-aequorin combined with coelenterazine entering from the medium to form aequorin, which was not enough for calcium imaging of the oocytes, but did allow in vivo imaging of the ovaries. From these studies, we conclude that coelenterazine entry into the cell is the rate limiting step in aequorin formation. Based on the rate of coelenterazine uptake in Drosophila, we estimate that complete conversion of 1 μM apo-aequorin would take 50 days in zebrafish eggs, 19 days in Drosophila eggs, 7 days in sea urchin eggs or 18 h in a 10 gm tissue culture cell. Our results suggest that work based on genetically introduced apo-aequorin will be most successful when large amounts of small cells can be incubated in coelenterazine. During embryonic development this would involve introducing coelenterazine into the circulatory system of late stage embryos. Calcium imaging in early stage embryos may be best done by injecting aequorin, which circumvents the slow process of coelenterazine entry.