Structural and functional analysis of the baculovirus single-stranded DNA-binding protein LEF-3

AbstractThe single-stranded DNA-binding protein LEF-3 of Autographa californica multinucleocapsid nucleopolyhedrovirus consists of 385 amino acid residues, forms oligomers, and promotes Mg2+-independent unwinding of DNA duplexes and annealing of complementary DNA strands. Partial proteolysis revealed that the DNA-binding domain of LEF-3 is located within a central region (residues 28 to 326) that is relatively resistant to proteolysis. In contrast, the N-terminus (27 residues) and C-terminal portion (59 residues) are not involved in interaction with DNA and are readily accessible to proteolytic digestion. Circular dichroism analyses showed that LEF-3 is a folded protein with an estimated α-helix content of more than 40%, but it is structurally unstable and undergoes unfolding in aqueous solutions at temperatures near 50 °C. Unfolding eliminated the LEF-3 domains that are resistant to proteolysis and randomized the digestion pattern by trypsin. The structural transition was irreversible and was accompanied by the generation of high molecular weight (MW) complexes. The thermal treatment inhibited DNA-binding and unwinding activity of LEF-3 but markedly stimulated its annealing activity. We propose that the shift in LEF-3 activities resulted from the generation of the high MW protein complexes, that specifically stimulate the annealing of complementary DNA strands by providing multiple DNA-binding sites and bringing into close proximity the interacting strands. The unfolded LEF-3 was active in a strand exchange reaction suggesting that it could be involved in the production of recombination intermediates

Characterization of the replication of a baculovirus mutant lacking the DNA polymerase gene

AbstractIn a previous study, the DNA polymerase gene (dnapol) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was identified as one of six genes required for plasmid replication in a transient replication assay (M. Kool, C. Ahrens, R.W. Goldbach, G.F. Rohrmann, J.M. Vlak, Identification of genes involved in DNA replication of the Autographa californica, Proc. Natl. Acad. Sci. U.S.A. 91, (1994) 11212–11216); however, another study based on a similar approach reported that the virally encoded polymerase was only stimulatory (A. Lu, L.K. Miller, The roles of 18 baculovirus late expression factor genes in transcription and DNA replication, J. Virol. 69, (1995) 975–982). To reconcile the conflicting data and determine if the AcMNPV DNA polymerase is required for viral DNA replication during the course of an infection, a dnapol-null virus was generated using bacmid technology. To detect viral DNA replication, a highly sensitive assay was designed based on real-time PCR and SYBR green chemistry. Our results indicate that a bacmid in which the dnapol ORF was deleted is unable to replicate its DNA when transfected into Spodoptera frugiperda (Sf-9) cells, although when the dnapol ORF was introduced into the polyhedrin (polh) locus, this repaired virus could propagate at levels similar to the control virus. These results confirm that the AcMNPV-encoded DNA polymerase is required for viral DNA replication and the host DNA polymerases cannot substitute for the viral enzyme in this process

Homologous p35 proteins of baculoviruses show distinctive anti-apoptotic activities which correlate with the apoptosis-inducing activity of each virus1The last author, Professor Maeda, has passed away after the acceptance of the paper. The rest of the authors would like this paper to be a memorial to him.1

AbstractThe anti-apoptotic activity of p35s from two baculoviruses, Autographa californica nucleopolyhedrovirus (AcNPV) and Bombyx mori NPV (BmNPV), was compared in mammalian cells. AcNPV p35 efficiently blocked apoptosis induced by caspase overexpression, but BmNPV p35 did so very poorly. Analysis of chimeric p35s and in vitro cleavage of wild type p35s suggest that the cleavage efficiency of p35 correlates with the blocking activity. Single amino acid substitutions of BmNPV p35 with those observed in AcNPV p35, however, resulted in significant loss of its anti-apoptotic activity. We speculate that sequences flanking the cleavage site have uniquely evolved during baculovirus evolution

サメ類の研究-20 : ホシザメとシロザメ胎児の腸管上皮細胞について

Electron microscopic observations were made on the epithelial cells of the intestines in Mustelus manazo and M. griseus embryos in order to examine their structure and function. The intestinal epithelial cells in the embryos of both species are characterized by microvilli on the free surface, intermicrovillous invaginations of the plasma membrane, and a number of vesicles, granules, and vacuoles. These characteristics suggest that the intestinal epithelial cells in M. manazo and M. griseus embryos may be engaged in absorption. It has been clear that interdigitations of the lateral plasma membrane at the intercellur junctions, which are not seen in the intestine of teleostean fish, are observed to occur in the M. manazo and M. griseus embryos.ホシザメとシロザメ胎児を用いて,それらの腸管上皮細胞の構造と機能を電子顕微鏡的手法により究明した.両種の胎児の腸管上皮細胞は遊離表面上の微絨毛,微絨毛間の細胞膜の陥入,多数の小胞,空胞,顆粒等により特徴づけられる.これらの特徴により,ホシザメとシロザメ胎児の腸管上皮細胞は吸収細胞として機能すると考えられる.また,硬骨魚類の腸管上皮には見られない隣接細胞の側面および基底面の細胞膜のかみ合いの形成がホシザメとシロザメ胎児において観察された

Pemafibrate Dramatically Ameliorated the Values of Liver Function Tests and Fibrosis Marker in Patients with Non-Alcoholic Fatty Liver Disease

[Background] Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease related to metabolic syndrome, which can progress to liver cirrhosis. Standard medication has not been established. Pemafibrate is a selective peroxisome proliferatoractivated receptor (PPAR) α modulator. We retrospectively evaluated the efficacy of pemafibrate in patients with NAFLD. [Methods] We retrospectively enrolled 17 patients (ten men, seven women; median age, 63 years; range, 27?81 years). They were all proven to have fatty liver through imaging and had little or no history of drinking (ethanol consumption of < 20 g/day for women and < 30 g/day for men). They were administered pemafibrate from October 2018 to June 2020. [Results] After administration, serum triglyceride (TG) tended to be decreased (300.5 ± 22.5 to 239.5 ± 34.3 mg/dL, P = 0.06). Serum high-density lipoprotein (HDL) cholesterol and low density lipoprotein (LDL) cholesterol levels did not change. ALT was significantly decreased (-47.4%) for six months (57.5 ± 8.8 to 30.3 ± 5.8 U/L, P < 0.01). The values of serum GGT significantly decreased (-48.7%) for sixth months (63.9 ± 10.3 to 32.8 ± 6.6 U/L, P < 0.01). Aspartate aminotransferase (AST) to platelet ratio (APRI), a fibrosis marker, also was significantly decreased in the sixth month (0.7 ± 0.1 to 0.4 ± 0.1, P < 0.05). Body mass index (BMI) and hemoglobin A1c (HbA1c) showed no significant change. [Conclusion] Pemafibrate dramatically ameliorated the values of liver function tests and APRI in patients with NAFLD

Role of the silkworm argonaute2 homolog gene in double-strand break repair of extrachromosomal DNA

The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. Here, we isolated, from a Bombyx mori cell, a cDNA clone named BmAGO2, which is homologous to Drosophila ARGONAUTE2, the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increased homologous recombination (HR) repair of DSBs in episomal DNA, but had no effect on that in chromosomes. Moreover, we found that RNAi for BmAGO2 enhanced the integration of linearized DNA into a silkworm chromosome via HR. These results suggested that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair

Liver-specific γ-glutamyl carboxylase-deficient mice display bleeding diathesis and short life span

Liver-Specific γ-Glutamyl Carboxylase-Deficient Mice Display Bleeding Diathesis and Short Life Span. Azuma K, Tsukui T, Ikeda K, Shiba S, Nakagawa K, et al. PLOS ONE. 2014. 9(2) doi:10.1371/journal.pone.008864

Differential gene expression profiles in neurons generated from lymphoblastoid B-cell line-derived iPS cells from monozygotic twin cases with treatment-resistant schizophrenia and discordant responses to clozapine

Schizophrenia is a chronic psychiatric disorder with complex genetic and environmental origins. While many antipsychotics have been demonstrated as effective in the treatment of schizophrenia, a substantial number of schizophrenia patients are partially or fully unresponsive to the treatment. Clozapine is the most effective antipsychotic drug for treatment-resistant schizophrenia; however, clozapine has rare but serious side-effects. Furthermore, there is inter-individual variability in the drug response to clozapine treatment. Therefore, the identification of the molecular mechanisms underlying the action of clozapine and drug response predictors is imperative. In the present study, we focused on a pair of monozygotic twin cases with treatment-resistant schizophrenia, in which one twin responded well to clozapine treatment and the other twin did not. Using induced pluripotent stem (iPS) cell-based technology, we generated neurons from iPS cells derived from these patients and subsequently performed RNA-sequencing to compare the transcriptome profiles of the mock or clozapine-treated neurons. Although, these iPS cells similarly differentiated into neurons, several genes encoding homophilic cell adhesion molecules, such as protocadherin genes, showed differential expression patterns between these two patients. These results, which contribute to the current understanding of the molecular mechanisms of clozapine action, establish a new strategy for the use of monozygotic twin studies in schizophrenia research