Three-dimensional structures of bacteriophage neck subunits are shared in Podoviridae, Siphoviridae and Myoviridae.

Tailed bacteriophages (Caudovirales) are divided into three families: Myoviridae with long contractile tails, Siphoviridae with long noncontractile tails and Podoviridae with short noncontractile tails. All have an icosahedral head with a portal vertex connected to a neck structure followed by a tail. Bacteriophage Mu belongs to the Myoviridae family. Herein, the gp29 portal subunit and neck subunits gp35, gp36 and gp37 of the Mu phage were purified to elucidate their arrangement in the neck. Both gp29 and gp36 were monomeric in solution, like the corresponding subunits of Podoviridae P22 and Siphoviridae SPP1. X-ray crystal structure of gp36 showed structural similarity to neck subunits of Siphoviridae and Podoviridae. The gp36 structure has a characteristic aromatic hydrophobic core, and the structure of the ring form of the Mu phage connector deduced from the Siphoviridae and Podoviridae connector showed that this feature builds the contact surface between gp36 subunits. Structural comparison with the neck of Siphoviridae and Podoviridae also implies direct interaction between gp36 and gp29. Because gp35 and gp36 form a stable complex, we predict that the head-portal ring (gp29), the connector complex (gp36 and gp35), the tail terminator (gp37) and the tube (gp40) are arranged in the Mu phage neck in this order.

Size-based analysis of virus removal filter fouling using fractionated protein aggregates.

Fouling by protein aggregates reduces virus removal filter performance. In the present study, we investigated the effects of different-sized protein aggregates on fouling and aggregate retention in order to better understand the fouling mechanisms. Human immunoglobulin G was denatured by heating to produce aggregates of various sizes and then fractionated by size exclusion chromatography into different-sized aggregates with a narrow size distribution. The fractionated aggregates were filtered on Planova 20N, a virus removal filter known for its stable filtration capability. Analysis of flux behavior demonstrated different flux decrease patterns for different-sized aggregates. Observation of aggregate retention by staining revealed that larger aggregates were captured closer to the inner surface of the membrane while smaller aggregates penetrated farther into the membrane. These findings demonstrate that Planova 20N has a gradient structure with decreasing pore size from the inner to the outer surface of the membrane. This structure minimizes fouling and enables stable filtration by protecting the smaller pores located closer to the outer surface from clogging by large aggregates. Applying the predominant clogging models to the present filtrations revealed that clogging behavior transitioned from complete blocking to cake filtration as filtration progressed. In this combination model, after a certain number of pores are blocked by complete blocking, newly arrived aggregates begin to accumulate on previously captured aggregates, generating cake between capture layers within the membrane. Application of the approaches described here will facilitate elucidation of membrane fouling and virus removal mechanisms.

Bongkrekic acid facilitates glycolysis in cultured cells and induces cell death under low glucose conditions.

Bongkrekic acid (BKA) inhibits adenine nucleotide translocator (ANT) and suppresses ADP/ATP exchange in the mitochondrial inner membrane. Previously, we demonstrated that BKA exhibited cytotoxic effects on 4T1 tumor cells, depending on the cell number in the culture, but not on NIH3T3 cells. However, the cause of this differential sensitivity was unelucidated. Here we demonstrate that BKA reduced the O2 consumption in both cell lines and increased the mitochondrial membrane potential, thereby facilitating glucose consumption. BKA reduced cellular ATP in 4T1 cells in a dose-dependent manner but not in NIH3T3 cells. The cellular ATP of 4T1 cells was decreased with a reduced glucose concentration in the media, but that of NIH3T3 cells remained constant. We also demonstrated that BKA-induced cell death in both cell lines in low glucose media; however, the susceptibility to the reduced glucose concentration was slightly higher in 4T1 cells, which may be attributed to the difference in the dependency on glycolysis as their energy source. These results indicate that 4T1 tumor cells rely heavily on glucose for energy production. Our data demonstrate that BKA disturbs ATP production in mitochondria and increases the susceptibility to a low glucose condition.