Formation Of Two-dimensional Arrays Research Articles

Electron microscopic evidence for the transmembrane displacement of calcium ATPase.

Incubation of the Ca2+-ATPase in vanadate solutions leads to the formation of two-dimensional arrays in the sarcoplasmic reticulum membrane. Electron micrographic freeze fracture replicas show depressions on the inner leaflet for the first time. This indicates that the ATPase has moved perpendicular to the plane of the membrane. Our results also suggest that aggregation of the Ca2+-ATPase into the two-dimensional arrays occurs before they move into the membrane. These phenomena were observed as soon as 15 minutes after vanadate was added. The effects of vanadate appear to be completely reversible. When SR was incubated in the vanadate solutions and was then diluted into a buffer containing Ca2+ and ATP, the ATPase activity was normal for up to several hours of incubation and only somewhat reduced after 3 days.

The formation of two-dimensional arrays of isometric plant viruses in the presence of polyethylene glycol

The formation of two-dimensional arrays of isometric plant viruses using the negative staining carbon-film technique has been extended to include experiments on the addition of polyethylene glycol (PEG). The original negative staining carbon-film method depended on the availability of freshly prepared highly concentrated plant virus suspensions, as material stored at 4°C from 24hr to several weeks resulted in a considerable reduction in the areas of crystalline arrays. The addition of PEG, mol. wt. 6000, to stored plant virus suspensions containing cowpea chlorotic mottle virus, broad bean mottle virus, turnip rosette virus and southern bean mosaic virus has resulted in the formation of extensive continuous areas of crystalline arrays.

The formation of two-dimensional arrays of isometric plant viruses in the presence of polyethylene glycol

Polyethylene glycol (PEG) precipitation is one of the conventional methods for virus concentration. This technique has been used to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. The procedures and seeded surrogate viruses were different among implementers; thus, the reported whole process recovery efficiencies considerably varied among studies. The present study compared five PEG precipitation procedures, with different operational parameters, for the RT-qPCR-based whole process recovery efficiency of murine hepatitis virus (MHV), bacteriophage phi6, and pepper mild mottle virus (PMMoV), and molecular process recovery efficiency of murine norovirus using 34 raw wastewater samples collected in Japan. The five procedures yielded significantly different whole process recovery efficiency of MHV (0.070%–2.6%) and phi6 (0.071%–0.51%). The observed concentration of indigenous PMMoV ranged from 8.9 to 9.7 log (8.2 × 108 to 5.6 × 109) copies/L. Interestingly, PEG precipitation with 2-h incubation outperformed that with overnight incubation partially due to the difference in molecular process recovery efficiency. The recovery load of MHV exhibited a positive correlation (r = 0.70) with that of PMMoV, suggesting that PMMoV is the potential indicator of the recovery efficiency of SARS-CoV-2. In addition, we reviewed 13 published studies and found considerable variability between different studies in the whole process recovery efficiency of enveloped viruses by PEG precipitation. This was due to the differences in operational parameters and surrogate viruses as well as the differences in wastewater quality and bias in the measurement of the seeded load of surrogate viruses, resulting from the use of different analytes and RNA extraction methods. Overall, the operational parameters (e.g., incubation time and pretreatment) should be optimized for PEG precipitation. Co-quantification of PMMoV may allow for the normalization of SARS-CoV-2 RNA concentration by correcting for the differences in whole process recovery efficiency and fecal load among samples.