Identification of a highly immunogenic mouse breast cancer sub cell line, 4T1-S

Cancer vaccines serve as a promising clinical immunotherapeutic strategy that help to trigger an effective and specific antitumor immune response compared to conventional therapies. However, poor immunogenicity of tumor cells remains a major obstacle for clinical application, and developing new methods to modify the immunogenicity of tumor cells may help to improve the clinical outcome of cancer vaccines. 4T1 mouse breast cancer cell line has been known as poorly immunogenic and highly metastatic cell line. Using this model, we identified a sub cell line of 4T1—designated as 4T1-Sapporo (4T1-S)—which shows immunogenic properties when used as a vaccine against the same line. In 4T1-S-vaccinated mice, subcutaneous injection of 4T1-S resulted in an antitumor inflammatory response represented by significant enlargement of draining lymph nodes, accompanied with increased frequencies of activated CD8 T cells and a subpopulation of myeloid cells. Additionally, 4T1-S vaccine was ineffective to induce tumor rejection in nude mice, which importantly indicate that 4T1-S vaccine rely on T cell response to induce tumor rejection. Further analysis to identify mechanisms that control tumor immunogenicity in this model may help to develop new methods for improving the efficacies of clinical cancer vaccines.     

A single amino acid in the PSPG-box plays an important role in the catalytic function of CaUGT2 (Curcumin glucosyltransferase), a Group D Family 1 glucosyltransferase from Catharanthus roseus

Curcumin glucosyltransferase (CaUGT2) isolated from cell cultures of Catharanthus roseus exhibits unique substrate specificity. To identify amino acids involved in substrate recognition and catalytic activity of CaUGT2, a combination of domain swapping and site-directed mutagenesis was carried out. Exchange of the PSPG-box of CaUGT2 with that of NtGT1b (a phenolic glucosyltransferase from tobacco) led to complete loss of enzyme activity in the resulting recombinant protein. However, replacement of Arg378 of the NtGT1b PSPG-box with cysteine, the corresponding amino acid in CaUGT2, restored the catalytic activity of the chimeric enzyme. Further site-directed mutagenesis revealed that the size of the amino acid side-chain in that particular site is critical to the catalytic activity of CaUGT2.     

Cloning and characterization of ferredoxin and ferredoxin-NADP+ reductase from human malaria parasite

The human malaria parasite (Plasmodium falciparum) possesses a plastid-derived organelle called the apicoplast, which is believed to employ metabolisms crucial for the parasite's survival. We cloned and studied the biochemical properties of plant-type ferredoxin (Fd) and Fd-NADP+ reductase (FNR), a redox system that potentially supplies reducing power to Fd-dependent metabolic pathways in malaria parasite apicoplasts. The recombinant P. falciparum Fd and FNR proteins were produced by synthetic genes with altered codon usages preferred in Escherichia coli. The redox potential of the Fd was shown to be considerably more positive than those of leaf-type and root-type Fds from plants, which is favourable for a presumed direction of electron flow from catabolically generated NADPH to Fd in the apicoplast. The backbone structure of P. falciparum Fd, as solved by X-ray crystallography, closely resembles those of Fds from plants, and the surface-charge distribution shows several acidic regions in common with plant Fds and some basic regions unique to this Fd. P. falciparum FNR was able to transfer electrons selectively to P. falciparum Fd in a reconstituted system of NADPH-dependent cytochrome c reduction. These results indicate that an NADPH-FNR-Fd cascade is operative in the apicoplast of human malaria parasites.     

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.     

The Gln27Glu beta2-adrenergic receptor variant is associated with obesity due to subcutaneous fat accumulation in Japanese men

The Trp64Arg beta3-adrenergic receptor (AR) variant is associated with visceral obesity probably due to decreased lipolysis in visceral fat (H. Kim-Motoyama et al., Diabetologia 40, 469-472, 1997). Functional alteration of beta2AR may also change fat distribution. We investigated the influence of the Gln27Glu beta2AR variant upon obesity and fat distribution. We screened 278 unrelated Japanese men and detected 249 wild-type Gln27 homozygotes, 28 Gln27/Glu27 heterozygotes, and one mutant Glu27 homozygote. The frequency of mutant Glu27 allele was significantly higher in obese subjects than in nonobese/intermediate subjects (0.11 vs 0.04, P = 0. 004). The Gln27/Glu27 heterozygotes had a significantly higher mean age-adjusted body-mass index (BMI) and mean age-adjusted subcutaneous fat area assessed by CT scan than the wild-type homozygotes but not the mean age-adjusted visceral fat areas. In summary, we have found that in Japanese men the Gln27Glu beta2AR variant is associated with obesity due to subcutaneous fat accumulation.     

The complete nucleotide sequence of wild rice (Oryza nivara) chloroplast genome: first genome wide comparative sequence analysis of wild and cultivated rice

We determined the complete nucleotide sequence of the chloroplast genome of wild rice, Oryza nivara and compared it with the corresponding published sequence of relative cultivated rice, Oryza sativa. The genome was 134,494 bp long with a large single-copy region of 80,544 bp, a small single-copy region of 12,346 bp and two inverted repeats of 20,802 bp each. The overall A+T content was 61.0%. The O. nivara chloroplast genome encoded identical functional genes to O. sativa in the same order along the genome. On the other hand, detailed analysis revealed 57 insertion, 61 deletion and 159 base substitution events in the entire chloroplast genome of O. nivara. Among substitutions, transversions were much higher than transitions with the former even more frequent than the latter in the coding region. Most of the insertions/deletions were single-base but a few large length mutations were also detected. The frequency of insertion/deletion events was more in the coding region within inverted repeats. In contrast, a very few substitution events were identified in the coding region. Polymorphism was observed among rice cultivars at loci of large insertion/deletion events. This is the first report describing comparative and genome wide chloroplast analysis between a wild and cultivated crop.     

Phytotoxic cis-cinnamoyl glucosides from Spiraea thunbergii

Spiraea thunbergii Sieb. was found to contain 1-O-cis-cinnamoyl-beta-D-glucopyranose and 6-O-(4'-hydroxy-2'-methylene-butyroyl)-1-O-cis-cinnamoyl-beta-D-glucopyranose as major plant growth inhibitory constituents along with related compounds of lower phytotoxicity including 6-O-(trans-cinnamoyl)-1-O-(4"-hydroxy-3"-methyl-furan-2"-one)-beta-D-glucopyranose, 6-O-(4'-hydroxy-2'-methylene-butyroyl)-1-O-trans-cinnamoyl-beta-D-glucopyranose, and 1-O-trans-cinnamoyl-beta-D-glucopyranose. The former three compounds were cinnamoyl glucosides.     

Generation of reactive oxygen species undergoing redox cycle of nostocine A: a cytotoxic violet pigment produced by freshwater cyanobacterium Nostoc spongiaeforme

Nostocine A (1) is an extracellular cytotoxic violet pigment produced by the freshwater cyanobacterium, Nostoc spongiaeforme TISTR 8169. Treatment with 1 was found to accelerate the generation of reactive oxygen species (ROS) in the green alga, Chlamydomonas reinhardtii, in the light. In vitro analysis revealed that 1 specifically eliminated superoxide radical anion (O(2)(-)) among several ROS tested. During the course of the reaction, oxygen (O(2)) was simultaneously synthesized and the O(2) synthesizing rate increased with the amount of 1 added. In contrast, O(2)(-) generation occurred when NADPH or NADH was added to a solution of 1 under aerobic condition. The reduction potential of 1 is very similar to that of O(2) indicating that 1 and O(2) can easily exchange electrons depending on the mass balance between their oxidized and reduced forms. Based on these results, the following hypothesis is formulated for the mechanism of intracellular ROS generation by treatment with 1: 1 taken into the target cells is reduced specifically by intracellular reductants such as NAD(P)H. When the O(2) level is sufficiently higher than that of 1, the reduced product of 1 is immediately oxidized by O(2). This is accompanied by the synthesis of O(2)(-) from O(2). The generation of O(2)(-) successively occurs, undergoing repeated redox cycles of 1, when the levels of the reductant and O(2) are still dominant to promote these reactions. This similar intracellular ROS generation mechanism to that of paraquat may cause the cytotoxicity.