The effects of ultrarapid freezing on meiotic and mitotic spindles of mouse oocytes and embryos

Preovulatory mouse oocytes and 2-cell embryos were frozen with dimethyl sulfoxide and propanediol by an ultrarapid method. The survival of frozen oocytes was low (33–34%) compared to that of 2-cell embryos (78–79%) with either cryoprotectant. Development to blastocysts after postthaw culture was about 7–15% for oocytes and 79–80% for the embryos. Ultrarapid freezing preserves cell structure quite well as revealed by electron microscopy, but meiotic oocytes and late 2-cell embryos undergoing mitosis showed evidence of spindle disorganization involving loss or clumping of microtubules resulting in some scattering of chromosomes. Embryos developed from frozen eggs showed clear evidence of micronuclear formation and incomplete incorporation of chromosomal material into main nuclei. These experiments confirm our observations on freezing of human oocytes and show that spindle microtubules are sensitive to freeze-thawing and that cryopreservation could cause chromosomal aberrations during early development. A cautious approach to the introduction of oocyte freezing in human in vitro fertilization (IVF) programs is advocated.     

Characterization and in vitro selection for iron efficiency inPyrus andCydonia

Summary Responses to low Fe were characterized in tissue cultures ofPyrus amygdaliformis andCydonia oblonga (quince), two species used as rootstocks for pear. Cultured shoots and plantlets ofP. amygdaliformis had a higher chlorophyll concentration and Fe²⁺/total Fe ratio than those ofC. oblonga when grown under low Fe conditions. This tolerance to low Fe was correlated with high Fe³⁺-reducing ability and medium acidification. The adaptive responses were manifested in roots of plantlets, shoot bases, root cultures, and cell suspension cultures. Shoots were regenerated from leaves of quince and subjected to Fe-deficient conditions. Two somaclonal variants (IE-1 and IE-2) were recovered; each displayed higher ability to reduce Fe³⁺ and acidify the medium. These variants may be useful as rootstocks for regions with calcareous soils, which limit Fe availability.     

Evaluation of the Enhanced Antioxidant Activity of Curcumin within Exosomes by Fluorescence Monitoring

In this study, we compared the antioxidant activities of curcumin (Cur) and a Cur formulation using a fluorescence analysis assay. The Cur formulation was prepared by a simple incorporation of Cur into exosomes (EXO) to produce Cur/EXOs. Free Cur had a low fluorescence intensity in aqueous solution because of its poor stability as a result of its autoxidation, whereas a significantly higher fluorescence intensity was observed for Cur/EXOs. Compared to free Cur, the increased level of intact Cur in EXOs allowed for enhanced antioxidant activity in H₂O₂ scavenging activity and DPPH assays. Compared to Cur at high concentration (200 μM), Cur/ EXOs were significantly less cytotoxic. The antioxidant activity of Cur or Cur/EXOs in cells could be easily demonstrated by monitoring decreases in their fluorescence intensity. Following subcutaneous injection, the fluorescence intensities of Cur/EXOs were much higher than that of Cur, suggesting that Cur/EXOs improve Cur stability in vivo. Taken together, we have demonstrated the superiority of Cur/EXOs over free Cur in terms of aqueous stability and antioxidant activity using fluorescence monitoring both in vitro and in vivo.     

Development of Electrochemiluminescent Serology Assays to Measure the Humoral Response to Antigens of Respiratory Syncytial Virus

Sensitive and precise serology assays are needed to measure the humoral response to antigens of respiratory syncytial virus (RSV) following natural infection or vaccination. We developed and evaluated a collection of electrochemiluminescent (ECL) serology assays using four RSV antigens (F, N, Ga and Gb). To assess the merits of ECL technology, the four ECL serology assays were evaluated using a well-characterized “gold standard” panel of acute and convalescent serum samples from fifty-nine RSV-positive and thirty RSV-negative elderly subjects (≥65 years old). The combined results from the four ECL assays demonstrated good concordance to the “gold standard” diagnosis, reaching 95% diagnostic sensitivity and 100% diagnostic specificity. Additionally, a combination of ECL assays provided higher diagnostic sensitivity than a commercially available diagnostic ELISA or cell-based microneutralization assay. In summary, these data demonstrate the advantages of using ECL-based serology assays and highlight their use as a sensitive diagnostic approach to detect recent RSV infection in an elderly population.     

Examination of the potential role of the glycerophosphorylcholine (GPC) pathway in the biosynthesis of phosphatidylcholine by liver and lung

The potential involvement of the glycerophosphorylcholine (GPC) pathway for the synthesis of phosphatidylcholine (PC) has been examined in rat liver and lung and in a human line, the A549 cell which possesses characteristics representative of mature alveolar type II epithelial cells. Although mitochondrial and microsomal fractions from the above sources readily incorporated radioactive glycerophosphate into lipids, the only incorporation observed with radioactive GPC was a small variable labelling with the mitochondrial and microsomal fractions from rat lung. Even with these fractions, no radioactivity from GPC was incorporated into PC or lysoPC. Attempts to increase the incorporation of GPC into lipids by manipulating the incubation conditions were unsuccessful. It was concluded that the occurrence of the GPC pathway in liver and lung is unlikely.     

Effects of sulfur dioxide on pore populations of canine tracheal epithelium

The bioelectric and barrier properties of the tracheal epithelium in nose-breathing dogs and in dogs that had been exposed for 75 min to compressed air or to two high concentrations of SO2 were measured and compared. We also studied tissues that had been treated with chloroform. Based on a model of restrictive diffusion we demonstrated heteropores (6 and 250 A) in the control tissues. Bioelectric changes due to 100-ppm SO2 were minimal. After exposure to 500 ppm SO2, adverse changes in the bioelectric properties were focal; they were marked in 8 out of 12 animals but were less striking in the other 4. Nonelectrolyte permeability increased with an increase in SO2 concentrations. Small pores were still present in the tissues severely affected by SO2 but they were absent in chloroform-treated tissues. Scanning electron microscopy of tissues from animals exposed to 500 ppm SO2 showed that in the same dog tissue appearance varied from normal to one of repair (normal bioelectric properties) or one of marked exfoliation of ciliated cells (abnormal bioelectric measurements).     

The effects of glucose and oxygen on the cytochromes and metabolic activity of yeast batch cultures. II. Candida utilis

Candida utilis was grown in batch culture with and without oxygen control. The concentrations of A-, B-, and C-type cytochromes were found to vary with the initial glucose concentration, with the dissolved oxygen concentration, and with time. A-type was the most sensitive. After glucose was essentially exhausted, the yeast catabolized ethanol, if it had been growing in a relatively low initial glucose concentration, or non-glucose carbohydrate, including some of that previously accumulated within the cell, if it had been growing in a high initial glucose concentration. This difference in metabolic pattern could explain why cytochrome derepression was initiated soon after glucose uptake ceased only if initial glucose had been relatively low. The effects of glucose and dissolved oxygen concentrations on yeast cytochromes and respiratory activity are discussed.     

Phorbol ester dissociates endothelin-stimulated phosphoinositide hydrolysis and arachidonic acid release in vascular smooth muscle cells

Endothelin-stimulated [3H]-inositol phosphate formation and [3H]-arachidonic acid release were measured in cultured vascular smooth muscle cells from rabbit renal artery. Both responses were partially inhibited by pretreatment with pertussis toxin, indicating the involvement of pertussis toxin-sensitive guanine nucleotide binding regulatory proteins in the coupling processes. Pretreatment with the phorbol ester PMA inhibited endothelin-stimulated [3H]-inositol phosphate formation, but potentiated endothelin-stimulated [3H]-arachidonic acid release, suggesting that these two coupling processes occur in a parallel and independent manner in vascular smooth muscle cells.     

Immunochemistry and localization of the enzyme disaggregatase in Methanosarcina mazei.

The enzyme disaggregatase (Dag) from Methanosarcina mazei was studied immunochemically. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified Dag under reducing and nonreducing conditions revealed a single band with a 94-kDa molecular mass. Dag was found to be immunogenic in rabbits; a polyclonal antibody probe was prepared and used to detect the enzyme by slide immunoenzymatic assay, immunofluorescence, and immunoblotting in various species of Methanosarcina known to convert from packets to single cells, including M. mazei. The enzyme could not be detected in other members of the family Methanosarcinaceae that do not convert. By immunogold electron microscopy, Dag was mapped to the cell wall of packets and to the cell membrane of single cells of two M. mazei strains.