Improved separation of integral membrane proteins by continuous elution electrophoresis with simultaneous detergent exchange: application to the purification of the fusion protein of the human immunodeficiency virus type 1

We describe a protocol for preparative-scale purification of the fusion protein of the human immunodeficiency virus type 1 (HIV-1), gp41, from cells overexpressing the viral envelope proteins and from HIV-1 isolates. In the first step, the proteins were extracted from the membrane in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The extract was then subjected to separation by continuous elution electrophoresis using a nonionic or zwitterionic detergent in the mobile elution buffer, which results in the simultaneous exchange of SDS with that detergent. The separated proteins were obtained in an SDS-free buffer containing either Brij, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Triton X-100 and could then be subjected to subsequent purification steps like isoelectric focusing in the second dimension or immunoaffinity chromatography. The dilute protein fraction was concentrated and applied on a 10 mL immunoaffinity column packed with anti-gp41 monoclonal antibody immobilized on protein-G sepharose. The protein was eluted from the column at pH 2.7 and obtained in pure form in amounts of 30-50 micrograms that constituted a yield of 1%. The pure gp41 could not be sustained in solution in the absence of detergent and was not susceptible to proteolytic digestion by trypsin. The identification of the protein and the degree of purity was confirmed indirectly using surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS). The possible application of this approach for the isolation of integral membrane proteins with the propensity to undergo spontaneous folding and aggregation is being discussed.     

32SF6 dissociation rate during irradiation by a tea CO2 laser

A new experimental method is presented for measuring the multiple-photon dissociation rate of SF₆. It appears that there is a reverse process which associates the photofragments into SF₆. The measured dependence of the dissociation probability versus laser flux seems to agree qualitatively with theoretical models.     

Evaluating microbial activity in composts using microcalorimetry

Microcalorimetric isothermal monitoring was carried out for compost and compost-containing growing medium, to provide fingerprints of compost microbial activity at different conditions. Microbial activity is a key property of composts used in agricultural practice. Two aspects are addressed in this study: (1) microcalorimetric evaluation of compost response to glucose as a model stimulating agent; (2) examination of the impact of compost pre-drying and re-wetting on its biological activity.

Addition of glucose solution to the compost involved a strong increase in microbial activity, which was associated with a significant heat evolution without a lag period. In certain cases, this heat evolution was of complicated shape thus manifesting the heterogeneity of microbial populations in composts. Relation between cumulative energy evolved and the amount of added glucose was found to be helpful in distinguishing between aerobic and anaerobic regime of compost microbial activity. As distinct from non-dried compost or growing mixture, glucose addition to samples pre-dried at 65 °C resulted in delayed heat response with initial exponential-like heat evolution. This delay in heat evolution suggests that biological activity was significantly suppressed upon compost drying. Such a temperature-induced inactivation process might also result in dominance of a relatively homogeneous microbial population which survived the heating, thus involving a smooth, exponential-like initial step of the heat evolution. Noteworthy, addition of water (without glucose) to pre-dried compost results in an outburst of activity as compared with non-dried compost. This heat evolution outburst was also characterized by a lag period and an initial exponential-like phase. The heat evolution obtained with pre-dried compost upon water addition was not related to the compost wetting energy but rather reflected the formation of new carbon source due to the changes in compost organic matter upon heating or to the dead biomass of those microbial populations that did not resist the compost heating/drying.     

Identification of new proteins in follicular fluid of mature human follicles

Proteins present in human follicular fluid (HFF) have been poorly characterized to date. The purpose of our study was to analyse the protein content and identify new proteins originating from fluid of mature human follicles. A total of six females from infertile couples referred for in vitro fertilization (IVF) were stimulated and 44 follicular fluid samples from mature follicles yielding an oocyte were collected 34-36 h after human chorionic gonadotropin administration. HFF samples were processed for high-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Comparative analysis of the 2-D gels revealed up to 600 spots, of which four were selected because of variations in their expression level. Using direct sequencing procedures (Edman degradation) or matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS), these four spots were identified as three new proteins: thioredoxin peroxydase 1 (TDPX1), transthyretin (TTR) and retinol-binding protein (RBP). The proteins identified here may emerge as potential candidates for specific functions during folliculogenesis and may prove useful as biomedical markers for follicle and/or oocyte maturation.     

Highly Stable,Amide‐Bridged Autoinducing Peptide Analogues that Strongly Inhibit the AgrC Quorum Sensing Receptor in Staphylococcus aureus

Blocking quorum sensing (QS) pathways has attracted considerable interest as an approach to suppress virulence in bacterial pathogens. Toward this goal, we recently developed analogues of a native autoinducing peptide (AIP‐III) signal that can inhibit AgrC‐type QS receptors and attenuate virulence phenotypes in Staphylococcus aureus. Application of these compounds is limited, however, as they contain hydrolytically unstable thioester linkages and have only low aqueous solubilities. Herein, we report amide‐linked AIP analogues with greatly enhanced hydrolytic stabilities and solubilities relative to our prior analogues, whilst maintaining strong potencies as AgrC receptor inhibitors in S. aureus. These compounds represent powerful tools for the study of QS.     

Polariton Dynamics under Strong Light–Molecule Coupling

We present a comprehensive experimental study of the photophysical properties of a molecule–cavity system under strong coupling conditions, using steady‐state and femtosecond time‐resolved emission and absorption techniques to selectively excite the lower and upper polaritons as well as the reservoir of uncoupled molecules. Our results demonstrate the complex decay routes in such hybrid systems and that, contrary to expectations, the lower polariton is intrinsically long‐lived.     

New developments in the spatial encoding of spin interactions for single‐scan 2D NMR

Single‐scan 2D NMR relies on a spatial axis for encoding the indirect‐domain internal spin interactions. Various strategies have been demonstrated for fulfilling the needs underlying this procedure. All such schemes use gradient‐echoed sequences that leave at their conclusion solely the effects of the internal interactions along the indirect domain; they also include a real‐time scheme that though simple, yields in general mixed‐phase line shapes. The present paper introduces two new proposals geared up for easing the spatial encoding underlying single‐scan 2D NMR methodologies. One of these is capable of delivering dispersive‐free peaks along the indirect domain, and thereby purely‐absorptive 2D line shapes, in amplitude‐encoded experiments. The other demonstrates for the first time, the possibility to obtain single‐scan 2D spectra without echoing the effects of the encoding gradient–simply by applying a single‐pulse frequency sweep to encode the interactions. Both of these modes are compatible with homo‐ and heteronuclear correlations, and exhibit a number of complementary features vis‐à‐vis encoding alternatives that have so far been presented. The overall principles underlying these new spatially encoding protocols are derived, and their performance demonstrated with single‐scan 2D NMR TOCSY and HSQC experiments on model compounds. Copyright © 2009 John Wiley & Sons, Ltd.     

Nachweis und Bestimmung des Aluminiums

Ohne Zusammenfassung     

Characterization of the Effects of Aryl-azido Compounds and UVA Irradiation on the Viral Proteins and Infectivity of Human Immunodeficiency Virus Type 1

Hydrophobic UV-activatable compounds have been shown to partition into the hydrophobic region of biological membranes to selectively label transmembrane proteins, and to inactivate enveloped viruses. Here, we analyze various UV-activatable azido- and iodo-based hydrophobic compounds for their ability to inactivate a model-enveloped virus, human immunodeficiency virus (HIV-1 MN). Treatment of HIV-1 with 1,5-diazidonapthalene (DAN), 1-iodo, 5-azidonaphthalene (INA), 1-azidonaphthalene (AzNAP) or 4,4′-diazidobiphenyl (DABIPH) followed by UVA irradiation for 2 min resulted in complete viral inactivation, whereas treatment using analogous non–azido-containing controls had no effect. Incorporation of an azido moiety within these hydrophobic compounds to promote photoinduced covalent reactions with proteins was found to be the primary mechanism of viral inactivation for this class of compounds. Prolonged UVA irradiation of the virus in the presence of these azido compounds resulted in further modifications of viral proteins, due to the generation of reactive oxygen species, leading to aggregation as visualized via Western blot analysis, providing additional viral modifications that may inhibit viral infectivity. Furthermore, inactivation using these compounds resulted in the preservation of surface antigenic structures (recognized by neutralizing antibodies b12, 2g12 and 4e10), which is favorable for the creation of vaccines from these inactivated virus preparations.