Liposome-nucleus interactions. Flow cytometric study on the role of the nuclear surface

The water-soluble probe carboxyfluorescein (CF), contained in the internal aqueous phase of liposomes, was used to investigate the interaction of phospholipid vesicles with isolated nuclei. Ultrastructural analysis indicated that adherent liposomes coated the nuclear surface, and fluorescence microscopy showed that they contained quenching concentrations of the dye. Flow cytometry revealed that the transfer of the entrapped dye from the adhering liposomes to nuclei was blocked by chilling at 0 degrees C. Chase experiments demonstrated that the most reliable mechanism of dye transfer involved fusion phenomena between the liposomal and the nuclear membranes. After the release of the fluorophore into the nucleus, empty liposomes could withdraw the intranuclear soluble fraction of the dye.     

Characterization of liposomes prepared using a microemulsifier

A new type of device can prepare liposomes continuously, in large quantities and with excellent aqueous space capture efficiency. At initial lipid concentration of 300 μmol/ml these liposomes capture approx. 75% of cytosine arabinoside used as an aqueous space marker. Liposome size can be reduced by increasing the number of times the preparations are recycled through the microemulsifier. Liposomes less than 0.1 μm in diameter, as shown by electron microscopy, can be made easily. Liposomes prepared at 300 μmol/ml, composed of phosphatidylglycerol/phosphatidylcholine/cholesterol in a 0.1:0.4:0.5 molar ratio leaked less than 1% of entrapped cytosine arabinoside (Ara-C) at 4°C, and less than 10% Ara-C at 37°C plus serum, over a 48 h period. These liposomes could be useful for a number of applications including diagnostics, therapeutics and model membrane studies.     

SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines

Proteinaceous cysteine residues act as privileged sensors of oxidative stress. As reactive oxygen and nitrogen species have been implicated in numerous pathophysiological processes, deciphering which cysteines are sensitive to oxidative modification and the specific nature of these modifications is essential to understanding protein and cellular function in health and disease. While established mass spectrometry-based proteomic platforms have improved our understanding of the redox proteome, the widespread adoption of these methods is often hindered by complex sample preparation workflows, prohibitive cost of isotopic labeling reagents, and requirements for custom data analysis workflows. Here, we present the SP3-Rox redox proteomics method that combines tailored low cost isotopically labeled capture reagents with SP3 sample cleanup to achieve high throughput and high coverage proteome-wide identification of redox-sensitive cysteines. By implementing a customized workflow in the free FragPipe computational pipeline, we achieve accurate MS1-based quantitation, including for peptides containing multiple cysteine residues. Application of the SP3-Rox method to cellular proteomes identified cysteines sensitive to the oxidative stressor GSNO and cysteine oxidation state changes that occur during T cell activation.     

Cyclic Nucleotides and the Release of Vasopressin from the Rat Posterior Pituitary Gland

Abstract: The effect of ATP, Mg²⁺, or MgATP on the release of luteinizing hormone-releasing hormone (LH-RH) from hypothalamic granules was examined under in vitro conditions. Granules, isolated from adult male hypothalami, were incubated at 37°C in a buffered (pH 7.8) medium containing 0.15 m -KCl. The addition of ATP to the incubation mixture did not stimulate the release of LH-RH. In contrast, the addition of MgATP stimulated the release of LH-RH, the release being 62% greater than control. The addition of Mg²⁺ to the incubated granules also stimulated the release of LH-RH. However, the magnitude of this Mg²⁺-stimulated release of LH–RH was significantly ( P < 0.01) lower than that of the MgATP-stimulated release, indicating that ATP stimulates LH-RH release in a Mg²⁺-dependent manner. As both MgATP and Mg²⁺ alone stimulated LH-RH release, we characterized further these two release processes by incubating the granules under one of the following conditions: incubation at 4°C in a buffered medium containing 0.15 m -KCl or incubation at 37°C in a medium that does not contain KCl. Under these two incubation conditions, the MgATP-stimulated release of LH-RH was not manifested, whereas the Mg²⁺-stimulated release of LH-RH was manifested. On the basis of these differences, we propose that two different processes can lead to the release of LH-RH from isolated hypothalamic granules: one process involves ATP and Mg²⁺ (MgATP) and another process involves Mg²⁺ alone.     

Nothia ex gr. excelsa (Grzybowski, 1898), ‘flysch-type’ agglutinated foraminifera from the Karpatian (Early-Miocene) of Hungary

Abstract

Nothia ex gr. excelsa (Grzybowski, 1898) branched, agglutinated, tubular foraminifera is documented for the first time from the Karpatian (latest Burdigalian) molasse sediments of the Paratethys. Excellently preserved specimens allowed the study of the macro- and microstructure of the test using reflected and polarized light microscopes, SEM and Raman spectroscopy. The mineralogical components of the agglutinated grains, the appearance of the protruded aperture with a scalloped-edge, the microstructure of the bilamellar wall, the presence of the calcite microgranular cement could all be described in detail. Based on autecology, the test morphology and the associated fauna indicate that Nothia ex gr. excelsa was a surface-dwelling detritivore with a seasonal-phytophagous mode of life.     

The value of paddock trees for regional conservation in an agricultural landscape

Summary Isolated trees and small patches of trees – paddock trees – are a prominent feature of agricultural landscapes in Australia, but are declining in many areas due to natural senescence, clearing, dieback and the general absence of recruitment. We assessed the importance of paddock trees for woodland conservation in a 30 000 ha sample of the New South Wales (NSW) South‐west Slopes using Satellite Pour l’Observation de la Terre (SPOT) panchromatic satellite imagery combined with models predicting the original distribution of vegetation communities. Tree‐cover occurred over 12% of the study area. The patch‐size distribution of vegetation in the study area varied between woodland types. For woodland communities that were confined to hills and ridges, most tree‐cover occurred as few, large remnants. For woodland communities of the foothills and plains (Blakely's Red Gum, Eucalyptus blakelyi and Yellow Box, Eucalyptus melliodora, or White Box, Eucalyptus albens and Red Stringybark), 54% of remnant tree‐cover occurred as patches < 1 ha. The loss of paddock trees will cause substantial reductions to some woodland communities. For example, the loss of patches < 1 ha in woodlands dominated by Blakely's Red Gum and Yellow Box would reduce this association from 7.4% to 3.4% of its predicted pre‐1750 distribution. Mean distance to tree‐cover across the study area increased almost fourfold if patches < 1 ha were removed from the landscape, which may have consequences for movements of some flora and fauna. Failure to protect and perpetuate paddock trees will diminish the likelihood of achieving the conservation objectives of comprehensiveness, adequacy and representativeness in agricultural landscapes.     

The antibody preparation and expression of human Pescadillo

To explore the biological roles of human Pescadillo and investigate its potential effect on tumorigene sis, the eDNA of Pescadillo was fused with that of GST. After purification and elution, the purified GST-Pescadillo fusion protein was obtained, and the antibody against the fusion protein was generated.Endogenous Pescadillo protein was observed to be remarkably induced by estrogen. It was mainly distributed in the tissues such as breast, ovary and intestine, all of which contain proliferating cells,and was also detected in many cell lines of human cancer: renal carcinoma, hepatoma, ovarian cancer,colon carcinoma, and breast cancer. The expression level of Pescadillo was increased significantly in breast cancer tissues compared with their paired margin tissues. Taken together, these data suggest that Pescadillo may play important roles in the initiation and development of cancer and may be a potential target in cancer diagnosis and therapy.