No abstract

The pathway of cell membrane lysis by the peptide antibiotic cecropin B (CB), which contains both a hydrophobic and an amphipathic alpha-helix, was analysed by assessing the morphological changes of Escherichia coli following treatment with the peptide. Exposure of green fluorescent protein (GFP)-expressing E. coli to CB does not lead to an efflux of GFP. Moreover, transmission electron microscopic (TEM) examination of cecropin B-treated cells showed that severe swelling precedes cell death and that the outer membrane becomes distended away from the plasma membrane. Using immuno-gold staining and TEM of E. coli expressing the maltose-binding protein in the cytoplasm, it was apparent that the protein remains restricted to the cytoplasmic compartment. These observations suggest that CB causes gross disruption of the outer membrane of Gram-negative bacteria. Circular dichroism measurements of CB in the presence of cell membrane-mimicking liposomes showed that CB forms secondary structure dependent on the ratio of [lipid]/[peptide]. These observations from this study are important for the future design of custom antimicrobial peptides.

The tumor suppressor gene p53 plays an important role in guarding genomic integrity. When induced in response to environmental results, the gene product of p53 functions as a transcription factor to transactivate genes involved in arresting the cell cycle and as a facilitator of DNA repair. In contrast, the status of p53 in Chinese hamster ovary (CHO) cells, commonly used as a model system for various studies including those involving the cell cycle and transformation, remains an enigma. In this study, the function and sequence of p53 in CHO.K1 cells were investigated. The level of p53 proteins was elevated on ultraviolet (UV) irradiation of the cells, and the proteins formed specific complexes as probed with DNA containing p53-binding sequences. Its activities toward responsive promoters were inducible by UV in a dose-dependent manner. Although p53 in CHO.K1 contained a single missense mutation at codon 211, the mutation apparently had no effect on the functional properties of the protein. The CHO.K1 cells on X-ray irradiation failed to arrest at G1 phase even when the cells were transfected with a wildtype human p53 gene, indicating that the failure probably was not caused by dysfunction of its p53, but by some other mechanism. This result is consistent with the finding that p21(Waf1/Cip1) is undetectable in UV-treated CHO.K1 cells, whereas Gadd45 is induced by UV light in the cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carrying the D614G mutation on the spike protein is the predominant circulating variant and is associated with enhanced infectivity. However, whether this dominant variant can potentially spread through the cold chain and whether the spike protein affects virus stability after cold storage remain unclear. To compare the infectivity of two SARS-CoV-2 variants, namely, SARS-CoV-2 variants with spike protein with the D614 mutation (S-D614) and G614 mutation (S-G614), after different periods of refrigeration (4°C) and freezing (−20°C). We also determined the integrity of the viral RNA and the ability of the spike protein to bind angiotensin-converting enzyme 2 (ACE2) after storage at these conditions. The results showed that SARS-CoV-2 was more stable and infectious after storage at −20°C than at 4°C. Particularly, the S-G614 variant was found to be more stable than the S-D614 variant. The spike protein of the S-G614 variant had better binding ability with the ACE2 receptor than that of the S-D614 variant after storage at −20°C for up to 30 days. Our findings revealed that SARS-CoV-2 remains stable and infectious after refrigeration or freezing, and their stability and infectivity up to 30 days depends on the spike variant. Stability and infectivity are related to each other, and the higher stability of S-G614 compared to that of S-D614 may contribute to rapid viral spread of the S-G614 variant. IMPORTANCE It has been observed that variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more stable and infectious after storage at −20°C than at 4°C. A SARS-CoV-2 S-D614G variant is currently the most dominant variant in circulation and is associated with enhanced infectivity. We compared the stability of two SARS-CoV-2 variants: the early S-D614 variant carrying the D614 spike protein and the new S-G614 variant carrying the G614 spike protein, stored at both 4°C and −20°C for different periods. We observed that SARS-CoV-2 remains stable and infectious after refrigeration or freezing, which further depends on the spike variant, that is, the ability of the spike protein to bind with the ACE2 receptor with higher efficiency. The high stability of the S-G614 variant also explains its rapid spread and infectivity. Therefore, precautions should be taken during and after handling food preserved under cold conditions.