Purification and properties of the Bombyx mori nuclear polyhedrosis virus liberated from polyhedra by dissolution with silkworm gut juice

Occluded virions of the Bombyx mori nuclear polyhedrosis virus were efficiently liberated from polyhedra by dissolution with the silkworm gut juice. The liberated virions were purified by sucrose density gradient centrifugation and the bands of enveloped virions were observed in the gradients. There was no functional difference between the gut juice-liberated and the carbonate-liberated virions. Disruption of enveloped virions by the gut juice was observed, but the formation of nucleocapsids from the degradation of the occluded virions was not detected. High yields of the enveloped virions from the polyhedra dissolved by the gut juice was obtained by separating the virions through sucrose density gradient centrifugation immediately after the dissolution of the polyhedra. Many factors, e.g., rearing seasons, silkworm strains, and rearing conditions, affect the polyhedra-dissolving property of the larval gut juice.     

Properties of crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase from bovine adrenocortical mitochondria. II. Essential histidyl and cysteinyl residues at the NADPH binding site of NADPH-adrenodoxin reductase.

The binding site of NADPH in NADPH-adrenodoxin reductase was examined using crystalline enzyme from bovine adrenocortical mitochondria by studies on the effects of photooxidation and chemical modifications of amino acid residues in the reductase. (1) Photoxication decreased the enzymatic activity of NADPH-adrenodoxin reductase. Photooxidation of the reductase was prevented by NADP+, adrenodoxin, or reduced glutathione, but not NAD+. Photoinactivation caused loss of a histidyl residue, but not of tyrosyl, tryptophanyl, cysteinyl, or methionyl residues of the reductase. It did not affect the circular dichroism spectrum of the reductase appreciably. (2) NADPH-adrenodoxin reductase activity was inhibited by diethyl pyrocarbonate and the inhibition was partially reversed by addition of hydroxylamine. The inhibition was prevented by NADP+, but not NAD+. (3) NADPH-adrenodoxin reductase activity was inhibited by 5,5'-dithiobis(2-nitrobenzoate) and the inhibition was reversed by reduced glutathione. It was also protected by NADP+, but not NAD+. The results indicate that a histidyl residue and a cysteinyl residue of NADPH-adrenodoxin reductase are essential for the binding of NADPH by the reductase.     

Structural Change of DNA Induced by Nucleoid Proteins: Growth Phase-Specific Fis and Stationary Phase-Specific Dps

The effects of nucleoid proteins Fis and Dps of Escherichia coli on the higher order structure of a giant DNA were studied, in which Fis and Dps are known to be expressed mainly in the exponential growth phase and stationary phase, respectively. Fis causes loose shrinking of the higher order structure of a genome-sized DNA, T4 DNA (166 kbp), in a cooperative manner, that is, the DNA conformational transition proceeds through the appearance of a bimodal size distribution or the coexistence of elongated coil and shrunken globular states. The effective volume of the loosely shrunken state induced by Fis is 30–60 times larger than that of the compact state induced by spermidine, suggesting that cellular enzymes can access for DNA with the shrunken state but cannot for the compact state. Interestingly, Dps tends to inhibit the Fis-induced shrinkage of DNA, but promotes DNA compaction in the presence of spermidine. These characteristic effects of nucleotide proteins on a giant DNA are discussed by adopting a simple theoretical model with a mean-field approximation.     

Alteration of division polarity and preprophase band orientation in stomatogenesis by light

We have developed an experimental system in which the irradiation with a red light pulse induces stomatal disorientation in the hypocotyl epidermis ofCucumis sativus L. In this system, the orientation of the division plane in guard mother cells was not defined correctly. Preprophase bands formed in these cells but their orientation was abnormal.     

Impact of Noncovalent Sulfur–Fluorine Interaction Position on Properties,Structures, and Photovoltaic Performance in Naphthobisthiadiazole‐Based Semiconducting Polymers

Controlling the energetics and backbone order of semiconducting polymers is essential for the performance improvement of polymer‐based solar cells. The use of fluorine as the substituent for the backbone is known to effectively deepen the molecular orbital energy levels and coplanarize the backbone by noncovalent interactions with sulfur of the thiophene ring. In this work, novel semiconducting polymers are designed and synthesized based on difluoronaphthobisthiadiazole (FNTz) as a new family of naphthobisthiadiazole (NTz)–quaterthiophene copolymer systems, which are one of the highest performing polymers in solar cells. The effect of the fluorination position on the energetics and backbone order is systematically studied. It is found that the dependence of the solar cell fill factor on the active layer thickness is very sensitive to the fluorination position. It is thus further investigated and discussed how the structural features of the polymers influence the photovoltaic parameters as well as the diode characteristics and bimolecular recombination. Further, the polymer with fluorine on both the naphthobisthiadiazole and quaterthiophene moieties exhibits a quite high power conversion efficiency of 10.8% in solar cells in combination with a fullerene. It is believed that the results would offer new insights into the development of semiconducting polymers.     

Transient rho-associated coiled-coil containing kinase (ROCK) inhibition on human retinal pigment epithelium results in persistent Rho/ROCK downregulation

Retinal pigment epithelium (RPE) cells is the outermost layer of the retina and RPE dysfunction is a key factor in the disease pathogenesis of age-related macular degeneration (AMD). Transplantation therapy using induced pluripotent stem cell (iPSC)-derived RPEs has recently received much attention as a treatment for AMD. Preserving these cells under the best possible conditions is important, and preservation methods using Y-27632 have been reported. Rho-associated coiled-coil containing kinase (ROCK) inhibitors are known to inhibit cell death, emerging as important drug candidates for stem cell differentiation and regenerative medicine. However, it has recently been shown that ROCK inhibitors may have a vasodilatory effect on human retinal arterioles, a side effect that should ideally be avoided in RPE transplantation. Although ROCK inhibitors hold great potential, optimizing efficacy while minimizing adverse reactions is critical for translation into a clinical treatment. We examined the effect of transient exposure of RPE cells to ROCK inhibitor Y-27632 to determine whether the extracellular presence of the drug is necessary for ongoing Rho/ROCK downregulation. Human RPE cells were subcultured as a suspension for 4 h in drug-free medium following exposure to Y-27632 for 2 h. A Y-27632 concentration of >10 μM improved cell survival beyond 4 h and cell proliferation in recovery culture medium. ROCK2 expression levels were specifically downregulated by Y-27632 in the Rho/ROCK signaling pathway. In conclusion, we demonstrated that the effect of Y-27632 is not dependent on its extracellular availability and can last beyond the 2 h of exposure. The lasting Rho/ROCK signaling pathway downregulation by Y-27632 suggests that RPE cell transplantation with ROCK inhibitor-free media is possible, which can minimize side effects to host tissue and have wider implications for transplantation methods requiring ROCK inhibition.     

Urinary and plasma proteomics to discover biomarkers for diagnosing between diabetic nephropathy and minimal change nephrotic syndrome or membranous nephropathy

The choice of treatment for primary nephrotic syndrome depends on the pathologic type of the disorder. Renal biopsy is necessary for a definitive diagnosis, but it is burdensome for the patients, and can be avoided if tests could be performed using urine or plasma. In this study, we analyzed 100 urinary proteins, 141 plasma proteins, and 57 urine/plasma ratios in cases of diabetic nephropathy (DN; n = 11), minimal change nephrotic syndrome (MCNS; n = 14), and membranous nephropathy (MN; n = 23). We found that the combination of urinary retinol-binding protein 4 and SH3 domain-binding glutamic acid-rich-like protein 3 could distinguish between MCNS and DN, with an area under the curve (AUC) of 0.9740. On the other hand, a selectivity index (SI) based on serotransferrin and immunoglobulin G, which is often used in clinical practice, distinguished them with an AUC of 0.9091. Similarly, the combination of urinary afamin and complement C3 urine/plasma ratio could distinguish between MN and DN with an AUC of 0.9842, while SI distinguished them with an AUC of 0.8538. Evidently, the candidates identified in this study were superior to the SI method. Thus, the aim was to test these biomarkers for accurate diagnosis and to greatly reduce the burden on patients.     

An ESR contrast agent is transported to rat liver through organic anion transporter

Carboxy PROXYL is a useful extracellular paramagnetic contrast reagent in electron spin resonance (ESR) and magnetic resonance imaging (MRI). Active transfer of the probe was investigated using an in situ liver model in rats. Carboxy PROXYL, a nitroxyl spin probe, was perfused into in situ liver perfusion system from Wistar rats. Concentration of nitroxyl form of the spin probe in effluent increased gradually after introducing perfusate with the spin probe and reached a plateau. The disappearance of Carboxy PROXYL from the perfusate was 40%, which could not be explained with its partition coefficient. Administration of non-selective inhibitors of organic anion transporters, p-aminohippuric acid and penicillin G, inhibited competitively and in a dose dependent manner the transfer of Carboxy PROXYL into rat liver in situ, resulting in increases of Carboxy PROXYL in the effluent. The results demonstrate that there is an active transfer system of an ESR contrast reagent into in situ rat liver through organic anion transporters.