Effects of lipid environment on the conformational changes of an ABC importer

In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.     

Tricarbocyanine cholesteryl laurates labeled LDL: new near infrared fluorescent probes (NIRFs) for monitoring tumors and gene therapy of familial hypercholesterolemia

For monitoring low-density lipoprotein receptors (LDLr) in tumors and in livers of patients with familial hypercholesterolemia (FH) treated with gene therapy, a series of tricarbocyanine cholesteryl laurates were synthesized with the cholesteryl laurate moiety serving as the lipid-chelating anchor for low-density lipoprotein (LDL). One of these conjugates, TCL17, was successfully used to label LDL to give a new NIRF, TCL17-LDL. Ex vivo biological studies on an LDLr overexpressing tumor model, human hepatoblastoma G(2) (HepG(2)), confirmed that this NIRF were internalized selectively by the tumor and detected with high sensitivity by a low-temperature 3-D redox scanner.     

A protease-activatable luminescent biosensor and reporter cell line for authentic SARS-CoV-2 infection

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.     

Functional genomics of the yeast DNA-damage response

High-throughput approaches are beginning to have an impact on many areas of yeast biology. Two recent studies, using different experimental platforms, provide insight into new pathways involved in the response of yeast to DNA damage.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Proton nuclear magnetic resonances study of bleomycin in aqueous solution. Assignment of resonances.

The 1H NMR spectrum of the glycopeptide antineoplastic antibiotic bleomycin has been examined in D2O solution (Fourier transform nuclear magnetic resonance, 270 MHZ) and in H2O solution (correlation nuclear magnetic resonance, 250 MHZ). Resonances have been assigned to specific hydrogens of the two most abundant congeners, bleomycin-A2 (BLM-A2) and bleomycin-B2 (BLM-B2), on the basis of (1) homonuclear spin decoupling, (2) comparison of the spectra of BLM-A2, BLM-B2, fragments of these antibiotics, and the related antibiotic phleomycin, and (3) the pH dependence of chemical shifts. Resonance assignments are presented for all the CH protons of BLM-A2 and BLM-B2 except for the saccharide groups, for which only the anomeric proton assignments are given. All of the NH protons have been identified with specific resonances except for the two primary amide groups, which yield four well-resolved peaks, whose specific assignment was not attempted. This study serves as a basis for future investigations of the conformation of bleomycin and its interaction with metals and nucleic acids.     

Models of bleomycin interactions with poly(deoxyadenylylthymidylic acid). Fluorescence and proton nuclear magnetic resonance studies of cationic thiazole amides related to bleomycin A2

The interaction of eight 2-substituted thiazole-4-carboxamides, structurally related to cationic terminus of bleomycin A2, with poly(deoxyadenylylthymidylic acid) [poly(dA-dT)] has been studied by using proton nuclear magnetic resonance and fluorescence spectroscopy. These analogues have been used as probes of the complex formed between the parent drug molecule and poly(dA-dT). Aliphatic substituents on the 2' position of 2,4'-bithiazole derivatives restrict the ability of the aromatic ring system to intercalate in the double-helical form of the polynucleotide. Absence or partial removal of the 2' substituent enhances intercalation of the bithiazole system. The cationic side chain does not appear to be involved in the stabilization of any of these complexes, although it may be necessary for their formation. A 2,4':2',4"-terthiazole derivative shows a substantial degree of intercalation which is accompanied by extensive immobilization of the cationic side chain. This suggests that insertion of the aromatic system into the nucleic acid causes the cationic side chain to be pulled in also. Monothiazole analogues do not appear to bind, indicating that at least two thiazole rings are necessary for binding or that proper spacing between the two side chains on either side of the thiazole system is important for binding. The relation of the interactions of these analogues to the biochemical and biological properties of the parent bleomycins is discussed as is the possible use of these data in the design of synthetic bleomycin derivatives having varying affinities and specificities for DNA.