Functional expression of thiocyanate hydrolase is promoted by its activator protein, P15K

AbstractThiocyanate hydrolase (SCNase) is a cobalt-containing enzyme with a post-translationally modified cysteine ligand, γCys131-SO2H. When the SCNase α, β and γ subunits were expressed in Escherichia coli, the subunits assembled to form a hetero-dodecamer, (αβγ)4, like native SCNase but exhibited no catalytic activity. Metal analysis indicated that SCNase was expressed as an apo-form irrespective of the presence of cobalt in the medium. On the contrary, SCNase co-expressed with P15K, encoded just downstream of SCNase genes, in cobalt-enriched medium under the optimized condition (SCNase(+P15K)) possessed 0.86 Co atom/αβγ trimer and exhibited 78% of the activity of native SCNase. SCNase(+P15K) showed a UV–Vis absorption peak characteristic of the SCNase cobalt center. About 70% of SCNase(+P15K) had the γCys131-SO2H modification. These results indicate that SCNase(+P15K) is the active holo-SCNase. P15K is likely to promote the functional expression of SCNase probably by assisting the incorporation of cobalt ion

Tetrodotoxin functions as a stress relieving substance in juvenile tiger puffer Takifugu rubripes

We tested whether tetrodotoxin (TTX) functions as a stress relieving substance in puffer fish. We orally administered TTX to the juveniles of hatchery-reared non-toxic tiger puffer Takifugu rubripes and measured the effects of TTX on brain corticotropin-releasing hormone (CRH) mRNA expression and plasma cortisol levels in comparison with effects in non-toxic juveniles. Firstly, the reciprocal connections of CRH and adrenocorticotropic hormone (ACTH) were confirmed by dual-label immunohistochemistry. CRH-immunoreactive (ir) cell bodies were detected in the hypothalamus and CRH-ir fibers were observed to project to ACTH-ir cells in the rostral pars distalis of the pituitary. Next, a TTX-containing diet (2.35 mouse units (517?ng)/g diet) or a non-toxic diet were fed to the fish for 28 days under a recirculating system. Standard length and body weight became significantly larger in the TTX-treated group. The degree of loss of the caudal fin, which is an indicator of the degree of agonistic interactions, where high values show a higher loss of caudal fin of a fish due to nipping by other individuals, was significantly lower in the TTX-treated group. Relative CRH mRNA expression levels in the brain and cortisol levels in the plasma were significantly lower in the TTX-treated group. These results indicate that TTX functions as a stress relieving substance by affecting the CRH-ACTH-cortisol axis and reducing agonistic interactions in tiger puffer juveniles

タネコウジ セイゾウ オヨビ セイキク ニ オケル コウジキン Aspergillus oryzae ノ セイギョインシ KpeA ノ キノウ カイセキ

麹菌の転写因子KpeAは平板培養でのコウジ酸生産の制御因子として見出されたが，その後，広く二次代謝と分生子形成の制御に関わることが明らかとなった。種麹製造及び製麹は分生子形成の制御を伴う醸造工程であり，また，米麹中の二次代謝物は醸造物の品質や安全性に影響する。そこで本研究では，実際の醸造における培養工程である種麹製造と製麹において，KpeAの遺伝子破壊株と高発現株の形質を調べることでKpeAの醸造における役割を明らかにすることを目的とした。製造した種麹の状貌と遺伝子解析から，KpeAは，分生子形成の主要因子であるBrlAの遺伝子発現制御を介して，分生子形成を促進する役割を果たしていることが示された。製麹においても米麹に着生する分生子に差が生じたことから，KpeAは醸造工程においても分生子形成に重要な転写因子であることが分かった。一方，米麹中のコウジ酸量が破壊株で顕著に増加した。コウジ酸の推定生合成酵素KojAの遺伝子発現解析から，KpeAは平板培養と同様に，製麹においてもコウジ酸生産を転写レベルで制御することが確認された。さらに破壊株の麹抽出液は，コウジ酸が多く含まれるために褐変しにくいことが明らかとなったことから，KpeAの遺伝子破壊によるコウジ酸生産の増加が，米麹の褐変抑制に効果があることが示された。以上よりKpeAは種麹製造と製麹において，分生子形成やコウジ酸生産を制御しており，実際の醸造においても重要な役割を果たす転写因子であることが確認された。KpeAの遺伝子破壊株は，米麹の酵素活性が若干低下するものの，製麹において分生子が形成しにくく，かつ米麹の褐変性が低いといった清酒醸造に使用する麹菌として好ましい特性をもつため，KpeAは醸造における麹菌の育種ターゲットの新しい候補として期待できる。We previously identified a novel transcription factor, KpeA, in Aspergillus oryzae. KpeA is involved in secondary metabolism and conidiation when A. oryzae is cultured in liquid or agar medium. To clarify the role of KpeA in sake brewing, we analyzed the functionality of KpeA in rice koji making and seed koji making. In seed koji making, the kpeA disruption (ΔkpeA) strain showed longer aerial hyphae and the number of conidia was one-third that of the control strain. In accordance with conidiation, expression of brlA, a pivotal gene for conidiation, was also decreased in the ΔkpeA strain, whereas it was increased in the kpeA overexpression (OE) strain. These results suggested that, similar to the phenomenon that occurs in agar culture, KpeA induced conidiation via positive regulation of brlA expression in seed koji making.Rice koji samples made with ΔkpeA strain, OE strain, and control strain were analyzed for conidiation, enzyme activity, and secondary metabolites. The OE strain and the control strain formed conidia after 46 and 72 h of inoculation, respectively, whereas the ΔkpeA strain did not. In contrast, activities of representative hydrolytic enzymes in rice koji did not differ among the strains. Regarding secondary metabolism, we confirmed that kpeA is responsible for the enhanced kojic acid production via inducing the expression of the putative biosynthesis gene, kojA. Notably, the browning of rice koji extract was not observed in the ΔkpeA strain sample. We confirmed that additional dosage of kojic acid at the level of the rice koji of the ΔkpeA strain prevented the browning of rice koji extract of the control sample. As a result, kpeA disruption led to increased kojic acid amount in rice koji, which consequently prevented the browning of rice koji.Taken together, our results showed that KpeA regulates secondary metabolism and conidiation of A. oryzae during rice koji making and seed koji making. Although enzyme activities of the ΔkpeA strain were reduced slightly, we consider that the observed phenotypes, such as suppressed conidiation or repression of the browning of rice koji, are beneficial for rice koji or rice koji making. Thus, we propose that kpeA could be a new target for the breeding of A. oryzae for traditional brewing

Analysis of Genetic Variation and Phylogeny of the Predatory Bug, Pilophorus typicus, in Japan using Mitochondrial Gene Sequences

Pilophorus typicus (Distant) (Heteroptera: Miridae) is a predatory bug occurring in East, Southeast, and South Asia. Because the active stages of P. typicus prey on various agricultural pest insects and mites, this species is a candidate insect as an indigenous natural enemy for use in biological control programs. However, the mass releasing of introduced natural enemies into agricultural fields may incur the risk of affecting the genetic integrity of species through hybridization with a local population. To clarify the genetic characteristics of the Japanese populations of P. typicus two portions of the mitochondrial DNA, the cytochrome oxidase subunit I (COI) (534 bp) and the cytochrome B (cytB) (217 bp) genes, were sequenced for 64 individuals collected from 55 localities in a wide range of Japan. Totals of 18 and 10 haplotypes were identified for the COI and cytB sequences, respectively (25 haplotypes over regions). Phylogenetic analysis using the maximum likelihood method revealed the existence of two genetically distinct groups in P. typicus in Japan. These groups were distributed in different geographic ranges: one occurred mainly from the Pacific coastal areas of the Kii Peninsula, the Shikoku Island, and the Ryukyu Islands; whereas the other occurred from the northern Kyushu district to the Kanto and Hokuriku districts of mainland Japan. However, both haplotypes were found in a single locality of the southern coast of the Shikoku Island. COI phylogeny incorporating other Pilophorus species revealed that these groups were only recently differentiated. Therefore, use of a certain population of P. typicus across its distribution range should be done with caution because genetic hybridization may occur

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target