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Termite queens and kings live longer than nonreproductive workers. Several molecular mechanisms contributing to their long lifespan have been investigated; however, the underlying biochemical explanation remains unclear. Coenzyme Q (CoQ), a component of the mitochondrial electron transport chain, plays an essential role in the lipophilic antioxidant defense system. Its beneficial effects on health and longevity have been well studied in several organisms. Herein, we demonstrated that long-lived termite queens have significantly higher levels of the lipophilic antioxidant CoQ 10 than workers. Liquid chromatography analysis revealed that the levels of the reduced form of CoQ 10 were 4 fold higher in the queen's body than in the worker's body. In addition, queens showed 7 fold higher levels of vitamin E, which plays a role in antilipid peroxidation along with CoQ, than workers. Furthermore, the oral administration of CoQ 10 to termites increased the CoQ 10 redox state in the body and their survival rate under oxidative stress. These findings suggest that CoQ 10 acts as an efficient lipophilic antioxidant along with vitamin E in long-lived termite queens. This study provides essential biochemical and evolutionary insights into the relationship between CoQ 10 concentrations and termite lifespan extension., (© 2023 Institute of Zoology, Chinese Academy of Sciences.)

Tissue-specific endopolyploidy is widespread among plants and animals and its role in organ development and function has long been investigated. In insects, the fat body cells of sexually mature females produce substantial amounts of egg yolk precursor proteins (vitellogenins) and exhibit high polyploid levels, which is considered crucial for boosting egg production. Termites are social insects with a reproductive division of labor, and the fat bodies of mature termite queens exhibit higher ploidy levels than those of other females. The fat bodies of mature termite queens are known to be histologically and cytologically specialized in protein synthesis. However, the relationship between such modifications and polyploidization remains unknown. In this study, we investigated the relationship among cell type, queen maturation, and ploidy levels in the fat body of the termite Reticulitermes speratus. We first confirmed that the termite fat body consists of two types of cells, that is, adipocytes, metabolically active cells, and urocytes, urate-storing cells. Our ploidy analysis using flow cytometry has shown that the fat bodies of actively reproducing queens had more polyploid cells than those of newly emerged and pre-reproductive queens, regardless of the queen phenotype (adult or neotenic type). Using image-based analysis, we found that not urocytes, but adipocytes became polyploid during queen differentiation and subsequent sexual maturation. These results suggest that polyploidization in the termite queen fat body is associated with sexual maturation and is regulated in a cell type-specific manner. Our study findings have provided novel insights into the development of insect fat bodies and provide a basis for future studies to understand the functional importance of polyploidy in the fat bodies of termite queens., (© 2023. The Zoological Society of Japan.)

Division of labor is a prominent feature of social insect societies, where different castes engage in different specialized tasks. As brain differences are associated with behavioral differences, brain anatomy may be linked to caste polymorphism. Here, we show that termite brain morphology changes markedly with caste differentiation and age in the termite, Reticulitermes speratus. Brain morphology was shown to be associated with reproductive division of labor, with reproductive individuals (alates and neotenic reproductives) having larger brains than nonreproductives (workers and soldiers). Micro-computed tomography (CT) imaging and dissection observations showed that the king's brain morphology changed markedly with shrinkage of the optic lobes during their long life in the dark. Behavioral experiments showed that mature primary kings lose visual function as a result of optic lobe shrinkage. These results suggested that termites restructure their nervous systems to perform necessary tasks as they undergo caste differentiation, and that they also show flexible changes in brain morphology even after the final molt. This study showed that brain morphology in social insects is linked to caste and aging, and that the evolution of the division of labor is underpinned by the development of diverse neural systems for specialized tasks., (© 2023 The Authors. Development, Growth & Differentiation published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Developmental Biologists.)

Colony size in social insects is one of the most important factors in shaping their self-organized system. It affects a wide variety of traits such as foraging and defense strategies, social immune responses, the degree of polymorphism, and reproductive output. However, colony size estimation of subterranean termites in the field has been challenging, due to their extremely cryptic biology and multiple site-nesting behavior. Since natural selection favors workers that maximize the number of their siblings, the amount of egg production may reflect the number of workers in the colony. Here, we report a method for inferring colony size in the field using total egg production in each colony from a subterranean termite, Reticulitermes speratus. Our investigation of field colonies revealed that the body weight of queens reaches a peak and had the largest variance in June and July and accurately predicts the number of eggs laid by the queen per 24 h. Using laboratory-reared colonies, we found that the total egg production in each colony is proportional to the number of workers. We also estimated the colony size of 198 field colonies and found that the median and maximum colony size was 24,500 and 451,800 workers per colony. The method for inferring colony size presented here may also be applicable to termite species with a clear seasonality in egg production. The colony size estimate will contribute to understanding the life history strategies and social systems of termites., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Society in eusocial insects is based on the reproductive division of labor, with a small number of reproductive individuals supported by a large number of nonreproductive individuals. Because inclusive fitness of all colony members depends on the survival and fertility of reproductive members, sterile members provide royals with special treatment. Here, we show that termite kings and queens each receive special food of a different composition from workers. Sequential analysis of feeding processes demonstrated that workers exhibit discriminative trophallaxis, indicating their decision-making capacity in allocating food to the kings and queens. Liquid chromatography tandem-mass spectrometry analyses of the stomodeal food and midgut contents revealed king- and queen-specific compounds, including diacylglycerols and short-chain peptides. Desorption electrospray ionization mass spectrometry imaging analyses of 13 C-labeled termites identified phosphatidylinositol and acetyl-l-carnitine in the royal food. Comparison of the digestive tract structure showed remarkable differences in the volume ratio of the midgut-to-hindgut among castes, indicating that digestive division of labor underlies reproductive division of labor. Our demonstration of king- and queen-specific foods in termites provides insight into the nutritional system that underpins the extraordinary reproduction and longevity of royals in eusocial insects., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Overwintering is a critical part of the annual cycle for species that live in temperate, polar, and alpine regions. Consequently, low-temperature biology is a key determinant of temperate species distribution. Termites are distributed predominantly in tropical regions, and a limited number of species are found in the temperate zone. Here, in the termite Reticulitermes speratus, we report the discovery of an underground chamber that protects kings and queens to survive the winter, which is separate from the one they used during the warmer breeding season. In the spring, the royals inhabited decayed logs on the ground, then moved to their underground chamber located in the roots of stumps in the fall. The winter minimum temperature measured in the royal chamber was higher than that in the logs on the ground. In overwintering termites, the kings and queens had higher cold tolerance than workers and soldiers. Air temperatures dropped below the critical temperature multiple times, as evidenced from the past 140 years of weather records in Kyoto. These results demonstrated the survival strategies of reproductives to overcome the environment at the latitudinal limits. This study helps further the understanding of the termite's seasonal phenology, long-term survivorship, and life cycle., (© 2023. The Author(s).)

The ecological success of social insects is attributed to the division of labor, where newly hatched offspring differentiate into either fertile progeny or functionally sterile worker castes. There is growing evidence for the heritable (genetic or epigenetic) effects on caste determination based on laboratory experiments. Here, we indirectly demonstrate that heritable factors have the principal role in caste determination and strongly affect colony-level production of both sexes of fertile dispersers (i.e., alates) in field colonies of the termite Reticulitermes speratus . An egg-fostering experiment suggests that the colony-dependent sex-specific caste fates were almost entirely determined before oviposition. Our investigation of field colonies revealed that such colony-dependent sex-specific caste fates result in the intercolonial variation in the numerical sex ratio of differentiated fertile offspring and, eventually, that of alates. This study contributes to better understanding the mechanisms underlying the division of labor and life-history traits in social insects., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Caste-based reproductive division of labour in social insects is built on asymmetries in resource allocation within colonies. Kings and queens dominantly consume limited resources for reproduction, while non-reproductive castes such as workers and soldiers help reproductive castes. Studying the regulation of such asymmetries in resource allocation is crucial for understanding the maintenance of sociality in insects, although the molecular background is poorly understood. We focused on uric acid, which is reserved and used as a valuable nitrogen source in wood-eating termites. We found that king- and queen-specific degradation of uric acid contributes to reproduction in the subterranean termite Reticulitermes speratus . The urate oxidase gene ( RsUAOX ), which catalyses the first step of nitrogen recycling from stored uric acid, was highly expressed in mature kings and queens, and upregulated with differentiation into neotenic kings/queens. Suppression of uric acid degradation decreased the number of eggs laid per queen. Uric acid was shown to be provided by workers to reproductive castes. Our results suggest that the capacity to use nitrogen, which is essential for the protein synthesis required for reproduction, maintains colony cohesion expressed as the reproductive monopoly held by kings and queens.

Lifespan varies greatly across the tree of life. Of the various explanations for this phenomenon, those that involve trade-offs between reproduction and longevity have gained considerable support. There is an important exception: social insect reproductives (queens and in termites, also kings) exhibit both high reproductive outputs and extraordinarily long lives. As both the ultimate and proximate mechanisms underlying the absence of the fecundity/longevity trade-off could shed light on the unexpected dynamics and molecular mechanisms of extended longevity, reproductives of social insects have attracted much attention in the field of ageing research. Here, we highlight current ecological and physiological studies on ageing and discuss the various possible evolutionary and molecular explanations of the extended lifespans of termite reproductives. We integrate these findings into a coherent framework revealing the evolution of longevity in these reproductives. Studies on termites may explain why and how ageing is shaped by natural selection. This article is part of the theme issue 'Ageing and sociality: why, when and how does sociality change ageing patterns?'

Eusocial insects display a caste system in which different castes are morphologically and physiologically specialized for different tasks. Recent studies have revealed that epigenetic modifications, including DNA methylation and histone modification, mediate caste determination and differentiation, longevity, and polyethism in eusocial insects. Although there has been a growing interest in the relationship between epigenetic mechanisms and phenotypic plasticity in termites, there is little information about differential expression levels among castes and expression sites for these genes in termites. Here we show royal-tissue-specific expression of epigenetic modification genes in the termite Reticulitermes speratus. Using RNA-seq, we identified 74 genes, including three DNA methyltransferases, seven sirtuins, 48 Trithorax group proteins, and 16 Polycomb group proteins. Among these genes, 15 showed king-specific expression, and 52 showed age-dependent differential expression in kings and queens. Quantitative real-time PCR revealed that DNA methyltransferase 3 is expressed specifically in the king's testis and fat body, whereas some histone modification genes are remarkably expressed in the king's testis and queen's ovary. These findings imply that epigenetic modification plays important roles in the gamete production process in termite kings and queens., (© 2018 Institute of Zoology, Chinese Academy of Sciences.)

Extreme conditions are normal for animals living in harsh environments. These animals adapt to their habitats and can use difficult conditions by default. Organisms living in enclosed spaces, notably termites in decaying wood, experience low O 2 and high CO 2 gas conditions due to limited gas exchange and high insect density. Termite queens, in particular, reproduce in royal chambers deep inside the wood, wherein tens of thousands of individuals engage in social labour. Here, we demonstrate that royal chambers in termite nests have low O 2 and high CO 2 gas concentrations, which enhance egg production by queens. We identified a unique gas condition of royal chambers in the nest of the subterranean termite Reticulitermes speratus , which is characterized by low O 2 (15.75%) and high CO 2 (4.99%) concentrations. Queens showed significantly greater fecundity under the low O 2 and high CO 2 gas conditions in the royal chambers than under ambient gas conditions. Quantitative PCR analysis revealed that the royal chamber gas conditions significantly promoted the expression levels of the vitellogenin genes RsVg1 , RsVg2 and RsVg3 in queens compared with ambient gas conditions. This study highlights the adaptation of animals that live in closed habitats, which are hypoxic and hypercapnic as the result of their own metabolism, so as to have a high fitness in such environmental conditions.

Termite royals (queen and king) exhibit extraordinary longevity without sacrificing reproductive performance, unlike most animals, in whom lifespan is generally negatively associated with reproduction. Therefore, the regulatory mechanisms underlying longevity have attracted much attention. Although the ageing process is influenced by environmental factors in many insects during their life cycle, it remains unclear whether any factors have an effect on the extended survival and high reproductive capacity of termite royals. Here, we show that hypoxia, possibly an important environmental factor in the nests, enhances survival and reproductive activity in incipient royals of the subterranean termite Reticulitermes speratus compared with those in control conditions. Quantitative real-time PCR analysis revealed that the expression levels of the vitellogenin gene in queens are maintained to a greater extent under hypoxic conditions than under control conditions. The expression levels of the antioxidant enzyme genes RsCAT1 and RsPHGPX are also significantly promoted by hypoxia in queens and kings respectively. These results suggest that hypoxic exposure can contribute in part to achieving high reproductive output by altering gene expression after founding of colonies in the royals. Our study provides novel insights into the effect of a nest environment on the reproductive characteristics in termite royals., (© 2018 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Aging is associated with the accumulation of DNA damage. High expression of DNA repair genes has been suggested to contribute to prolonged lifespan in several organisms. However, the crucial DNA repair genes contributing to longevity remain unknown. Termite kings have an extraordinary long lifespan compared with that of non-reproductive individuals such as workers despite being derived from the same genome, thus providing a singular model for identifying longevity-related genes. In this study, we demonstrated that termite kings express higher levels of the breast cancer susceptibility gene BRCA1 than other castes. Using RNA sequencing, we identified 21 king-specific genes among 127 newly annotated DNA repair genes in the termite Reticulitermes speratus . Using quantitative PCR, we revealed that some of the highly expressed king-specific genes were significantly upregulated in reproductive tissue (testis) compared to their expression in somatic tissue (fat body). Notably, BRCA1 gene expression in the fat body was more than 4-fold higher in kings than in workers. These results suggest that BRCA1 partly contributes to DNA repair in somatic and reproductive tissues in termite kings. These findings provide important insights into the linkage between BRCA1 gene expression and the extraordinary lifespan of termite kings.

In most organisms, superoxide dismutases (SODs) are among the most effective antioxidant enzymes that regulate the reactive oxygen species (ROS) generated by oxidative energy metabolism. ROS are considered main proximate causes of aging. However, it remains unclear if SOD activities are associated with organismal longevity. The queens of eusocial insects, such as termites, ants, and honeybees, exhibit extraordinary longevity in comparison with the nonreproductive castes, such as workers. Therefore, the queens are promising candidates to study the underlying mechanisms of aging. Here, we found that queens have higher Cu/Zn-SOD activity than nonreproductive individuals of the termite Reticulitermes speratus . We identified three Cu/Zn-SOD sequences and one Mn-SOD sequence by RNA sequencing in R. speratus . Although the queens showed higher Cu/Zn-SOD activity than the nonreproductive individuals, there were no differences in their expression levels of the Cu/Zn-SOD genes RsSOD1 and RsSOD3A . Copper (Cu 2+ and Cu + ) is an essential cofactor for Cu/Zn-SOD enzyme activity, and the queens had higher concentrations of copper than the workers. These results suggest that the high Cu/Zn-SOD activity of termite queens is related to their high levels of the cofactor rather than gene expression. This study highlights that Cu/Zn-SOD activity contributes to extraordinary longevity in termites.

Reactive oxygen species (ROS) are generated spontaneously in all organisms and cause oxidative damage to biomolecules when present in excess. Accumulated oxidative damage accelerates aging; enhanced antioxidant capacity may be a positive factor for longevity. Recently, numerous studies of aging and longevity have been performed using short-lived animals, however, longevity mechanisms remain unknown. Here we show that a termite Reticulitermes speratus that is thought to be long-lived eusocial insect than other solitary insects uses large quantities of uric acid as an antioxidant against ROS. We demonstrated that the accumulation of uric acid considerably increases the free radical-scavenging activity and resistance against ultraviolet-induced oxidative stress in laboratory-maintained termites. In addition, we found that externally administered uric acid aided termite survival under highly oxidative conditions. The present data demonstrates that in addition to nutritional and metabolic roles, uric acid is an essential antioxidant for survival and contributes significantly to longevity. Uric acid also plays important roles in primates but causes gout when present in excess in humans. Further longevity studies of long-lived organisms may provide important breakthroughs with human health applications.

Peroxiredoxin (PRDX), a newly discovered antioxidant enzyme, has an important role in hydrogen peroxide reduction. Among six PRDX genes (PRDX1-6) in mammals, PRDX4 gene is alternatively spliced to produce the somatic cell form (PRDX4) and the testis specific form (PRDX4t). In our previous study, PRDX4 knockout mice displayed testicular atrophy with an increase in cell death due to oxidative stress. However, the antioxidant function of PRDX4t is unknown. In this study, we demonstrate that PRDX4t plays a protective role against oxidative stress in the mammalian cell line HEK293T. The PRDX4t-EGFP plasmid was transferred into HEK293T cells; protein expression was confirmed in the cytoplasm. To determine the protective role of PRDX4t in cells, we performed image-based analysis of PRDX4t-EGFP expressed cells exposed to UV irradiation and hydrogen peroxide using fluorescent probe CellROX. Our results suggested that PRDX4t-EGFP expressed cells had reduced levels of oxidative stress compared with cells that express only EGFP. This study highlights that PRDX4t plays an important role in cellular antioxidant defense., Competing Interests: No potential conflicts of interest were disclosed.

The trade-off between reproduction and longevity is known in wide variety of animals. Social insect queens are rare organisms that can achieve a long lifespan without sacrificing fecundity. The extended longevity of social insect queens, which contradicts the trade-off, has attracted much attention because it implies the existence of an extraordinary anti-aging mechanism. Here, we show that queens of the termite Reticulitermes speratus incur significantly lower oxidative damage to DNA, protein and lipid and have higher activity of antioxidant enzymes than non-reproductive individuals (workers and soldiers). The levels of 8-hydroxy-2'-deoxyguanosine (oxidative damage marker of DNA) were lower in queens than in workers after UV irradiation. Queens also showed lower levels of protein carbonyls and malondialdehyde (oxidative damage markers of protein and lipid, respectively). The antioxidant enzymes of insects are generally composed of catalase (CAT) and peroxiredoxin (Prx). Queens showed more than two times higher CAT activity and more than seven times higher expression levels of the CAT gene RsCAT1 than workers. The CAT activity of termite queens was also markedly higher in comparison with other solitary insects and the queens of eusocial Hymenoptera. In addition, queens showed higher expression levels of the Prx gene RsPRX6. These results suggested that this efficient antioxidant system can partly explain why termite queens achieve long life. This study provides important insights into the evolutionary linkage of reproductive division of labor and the development of queens' oxidative stress resistance in social insects., Competing Interests: The authors have declared that no competing interests exist.

Insulin-like growth factor-1 (IGF-1) receptors play a crucial role in the biology of human cancer, making them an attractive target for anti-cancer agents. We previously designed oligopeptides containing the amino-acid sequences surrounding the autophosphorylation sites of the insulin receptor and found that two of them, namely, Ac-DIYET-NH2 and Ac-DYYRK-NH2, suppressed phosphorylation of purified insulin receptors in a non-ATP-competitive manner, whereas Ac-NIYQT-NH2 and Ac-NYYRK-NH2 suppressed in an ATP-competitive manner. Because the IGF-1 receptor is closely related to the insulin receptor, the aim of this study was to observe the effects of these peptides, which correspond to the amino-acid sequences of the autophosphorylation sites of the IGF-1 receptor, on the activity of the human breast cancer cell lines MCF-7, T47D, MDA-MB-231, and MDA-MB-453. To facilitate peptide delivery into breast cancer cells, the cell-penetrating peptide, human immunodeficiency virus type 1-transactivator of transcription (Tat), was linked to these peptides. When breast cancer cells were treated with each of these synthetic Tat-conjugated peptides, the conjugated peptides penetrated into the cells and suppressed cell proliferation. An inhibitory effect of Tat-conjugated peptides against IGF-1-stimulated phosphorylation of IGF-1 receptors was observed. In addition, we found that combinations of these peptides suppressed phosphorylation of IGF-1 receptors to a greater extent than the peptides did individually. In conclusion, IGF-1 receptor autophosphorylation site-derived membrane-permeable peptides have the potential to suppress IGF-1 receptor function in breast cancer cells and to be developed into novel and useful agents for cancer therapy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Epidermal growth factor (EGF) receptor plays a crucial role in the biology of human cancer, and is a highly appropriate target for anticancer agents. We have previously designed oligopeptides containing the amino acid sequences around autophosphorylation sites of EGF receptor to identify a specific inhibitor of this receptor. We found that Ac-ENAEYLR-NH(2) and Ac-NYQQN-NH(2) suppressed phosphorylation of purified EGF receptor in a non-ATP-competitive manner whereas Ac-QNAQYLR-NH(2) and Ac-DYQQD-NH(2) caused inhibition in an ATP-competitive manner. The aim of this study was to observe the effects of these peptides on the proliferation, cell death, and apoptosis of human lung carcinoma A549 cells. To facilitate transfer of these inhibitory peptides into A549 cells, the cell-penetrating peptide, human immunodeficiency virus type 1-transactivator of transcription (Tat), was linked to the peptides. When A549 cells were treated with each Tat-conjugated peptide, the peptides penetrated the cells and EGF-stimulated tyrosine phosphorylation of EGF receptor was significantly suppressed. These Tat-conjugated peptides played a suppressive role in EGF-stimulated A549 cell responses. In particular, Tat-epsilon-aminocaproic acid (acp)-ENAEYLR-NH(2) significantly inhibited proliferation and showed cytotoxicity, while Tat-acp-NYQQN-NH(2) and Tat-acp-DYQQD-NH(2) suppressed the anti-apoptotic effect of EGF. In addition, we found that Tat-acp-ENAEYLR-NH(2) also inhibited the phosphorylation of epidermal growth factor receptor 2 (ErbB2) as well as EGF receptor in A549 cells. In conclusion, membrane-permeable synthetic peptides derived from EGF receptor autophosphorylation sites have the potential to suppress EGF receptor function in A549 cells and to be developed into novel and useful agents for cancer therapy., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We aimed to clarify if Campylobacter lari exerts a cytolethal distending toxin (CDT) effect on HeLa cells. Campylobacter cell lysates (CCLys) from C. jejuni 81-176 and urease-positive thermophilic Campylobacter (UPTC) CF89-12 and UPTC NCTC12893 isolates were shown to exert a CDT effect on HeLa cells with morphological changes examined by Giemsa staining and microscopy. However, Campylobacter lari JCM2530(T) isolate showed no effect. In addition, Campylobacter cell culture supernatant wash gave low or absent toxic effects with both C. jejuni and C. lari organisms. When western blot analysis was carried out to clarify if there was a CDTB effect in the CCLys and soluble fractions from Campylobacter isolates, which had a CDT effect on HeLa cells or did not have any effect, anti-recombinant CjCDTB antibodies identified an immunoreactively positive signal at around approximately 25 kDa on all the C. lari isolates examined, as well as the C. jejuni 81116 strain. Thus, all the Campylobacter isolates including those without any CDT effect were shown to express CDTB at the translational level., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A total of 57 Taylorella equigenitalis (n=22) and Taylorella asinigenitalis (n=35) isolates was shown not to carry any intervening sequences (IVSs) within 16S rRNA gene sequences. By contrast, we have already shown the genus Taylorella group to carry several kinds of IVSs within the 23S rRNA gene sequences., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The pentatricopeptide repeat (PPR) protein family is involved in various steps of RNA metabolism in plastids and mitochondria. To investigate the function of a DYW sub-class PPR protein in the moss Physcomitrella patens, we constructed and characterized knockout mutants of the PpPPR_43 gene, which encodes a mitochondrial localized PPR protein with a C-terminal DYW domain. The disruptants showed poor growth of moss protonemata. To investigate whether mitochondrial transcripts were affected by disruption of PpPPR_43, we sequenced the cDNA to detect RNA editing events and performed RT-PCR analyses to measure steady-state mitochondrial transcript levels. Disruption of PpPPR_43 did not result in defective RNA editing, but a substantial reduction in the level of mature cox1 transcript was observed in the disruptants. RT-PCR analysis showed that the 3rd intron of cox1 pre-mRNA was not spliced out in the disruptants, but the 1st, 2nd and 4th introns were efficiently spliced out. This suggests that PpPPR_43 is an intron 3-specific splicing factor. The role of the C-terminal domains of PpPPR_43 in intron 3 splicing was analyzed by complementation experiments with truncated constructs lacking the DYW domain or both the E and DYW domains. Both truncated genes completely restored splicing in the PpPPR_43 knockout mutant. This indicates that the E and DYW domains of PpPPR_43 are not required for splicing, and can be deleted without loss of cox1 intron 3 splicing., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Novel clustered regularly-interspaced short palindromic repeats (CRISPRs) locus [7,500 base pairs (bp) in length] occurred in the urease-positive thermophilic Campylobacter (UPTC) Japanese isolate, CF89-12. The 7,500 bp gene loci consisted of the 5'-methylaminomethyl-2-thiouridylate methyltransferase gene, putative (P) CRISPR associated (p-Cas), putative open reading frames, Cas1 and Cas2, leader sequence region (146 bp), 12 CRISPRs consensus sequence repeats (each 36 bp) separated by a non-repetitive unique spacer region of similar length (26-31 bp) and the phosphatidyl glycerophosphatase A gene. When the CRISPRs loci in the UPTC CF89-12 and five C. jejuni isolates were compared with one another, these six isolates contained p-Cas, Cas1 and Cas2 within the loci. Four to 12 CRISPRs consensus sequence repeats separated by a non-repetitive unique spacer region occurred in six isolates and the nucleotide sequences of those repeats gave approximately 92-100% similarity with each other. However, no sequence similarity occurred in the unique spacer regions among these isolates. The putative σ(70) transcriptional promoter and the hypothetical ρ-independent terminator structures for the CRISPRs and Cas were detected. No in vivo transcription of p-Cas, Cas1 and Cas2 was confirmed in the UPTC cells.

This study aims to characterise biochemically urease from an atypical Campylobacter lari, namely urease-positive thermophilic Campylobacter (UPTC). Urease was purified from cells of a Japanese UPTC isolate (CF89-12) using phenyl-Sepharose chromatography. Two protein components (estimates molecular masses 24 kDa and 61 kDa) were obtained that appeared to be structural proteins of urease (subunits A and B), and these were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE). The native molecular weight for the final purified UPTC urease was estimated to be approximately 186,000 Da which is close to the calculated molecular weight (182,738 Da) based on all six open reading frames of UPTC CF89-12 urease genes (ureA, B, E, F, G and H), as described previously. Moreover, an active band was observed on phenol red staining after a nondenaturing native PAGE of the crude extract from the UPTC cells. In addition, the purified urease of UPTC CF8912 showed enzyme activity over a broad pH range (pH 6-10), with maximal activity at pH 8.0. The urease was also stable against heat treatment, with almost no loss of enzyme activity seen following 60-min incubation at temperatures of 20-60 degrees C. Urease subunits A and B were identified immunologically by Western blot analysis with rabbit anti-urease alpha (A) and beta (B) raised against Helicobacter pylori.

The present study examines the expression of cytolethal distending toxin (cdt) gene encoding a cytotoxin in Campylobacter lari (n=6 urease-negative [UN] C. lari; n=4 urease-positive thermophilic Campylobacter [UPTC]). When reverse transcription polymerase chain reaction (RT-PCR) was carried out with 10 C. lari isolates using a primer pair to amplify the cdtB gene transcript segment, an approximate 260 bp RT-PCR amplicon was generated with all the isolates. In addition, cdtA, cdtB and cdtC gene operon was identified to be polycistronicly transcribed in the C. lari cells. The cdtB gene translation in the C. lari cells was also confirmed by Western blot analysis. Thus, the cdt gene operon in C. lari organisms, including UN C. lari and UPTC, was expressed at the transcriptional and translational levels in the cells. The present results suggest that all three cdt genes may be functional in the cells.

Molecular cloning, nucleotide sequencing, and characterization of the flaA gene from additional isolates of urease-positive thermophilic Campylobacter (UPTC) were performed. These isolates were obtained from the natural environment in Northern Ireland (n = 9 from mussels) and in England (n = 1 from sea water). All isolates carried the shorter flaA gene, [open reading frames (ORFs), 1,461 to 1,503 base pairs], without any internal termination codons, and did not carry any flaA pseudogenes. The UPTC isolates were well discriminated by the neighbor joining (NJ) phylogenetic tree constructed based on the putative flaA genes ORFs nucleotide sequence information. In addition, the NJ tree constructed based on the flaA-short variable region sequence information discriminated the Campylobacter lari isolates with a similar degree of discrimination power.

In the present study, the reliability of full-length gene sequence information for several genes including 16S rRNA was examined, for the discrimination of the two representative Campylobacter lari taxa, namely urease-negative (UN) C. lari and urease-positive thermophilic Campylobacter (UPTC). As previously described, 16S rRNA gene sequence are not reliable for the molecular discrimination of UN C. lari from UPTC organisms employing both the unweighted pair group method using arithmetic means analysis (UPGMA) and neighbor joining (NJ) methods. In addition, three composite full-length gene sequences (ciaB, flaC and vacJ) out of seven gene loci examined were reliable for discrimination employing dendrograms constructed by the UPGMA method. In addition, all the dendrograms of the NJ phylogenetic trees constructed based on the nine gene information were not reliable for the discrimination. Three composite full-length gene sequences (ciaB, flaC and vacJ) were reliable for the molecular discrimination between UN C. lari and UPTC organisms employing the UPGMA method, as well as among four thermophilic Campylobacter species., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We aimed to clarify if the thermophilic Campylobacter lari organisms including urease-negative (UN) C. lari and urease-positive thermophilic Campylobacter (UPTC) can be differentiated at the species and/or subspecies levels by employing the full-length flaA gene and flaA short variable region (SVR) nucleotide sequence information or not. Thermophilic Campylobacter isolates (n = 45) including UN C. lari (n = 17), UPTC (n = 18), and Campylobacter jejuni (n = 10) were well discriminated at the isolate level by the unweighted pair group method using arithmetic means analysis and neighbor joining procedures constructed based on the full-length flaA gene and flaA SVR nucleotide sequence information. Thus, these procedures may possibly be useful for epidemimological studies for C. lari and C. jejuni.

In plant organelles, RNA editing frequently occurs in many transcripts, but little is known about its molecular mechanism. Eleven RNA editing sites are present in the moss Physcomitrella patens mitochondria. Recently PpPPR_71, one member of 10 DYW-subclass pentatricopeptide repeat (PPR-DYW) proteins, has been identified as a site-specific recognition factor for RNA editing in the mitochondrial transcript. In this study, we disrupted three genes encoding a PPR-DYW protein-PpPPR_56, PpPPR_77, and PpPPR_91-to investigate whether they are involved in RNA editing. Transient expression of an N-terminal amino acid sequence fused to the green fluorescent protein (GFP) suggests that the three PPR-DYW proteins are targeted to mitochondria. Disruption of each gene by homologous recombination revealed that PpPPR_56 was involved in RNA editing at the nad3 and nad4 sites, PpPPR_77 at the cox2 and cox3 sites, and PpPPR_91 at the nad5-2 site in the mitochondrial transcripts. The nucleotide sequences surrounding the two editing sites targeted by a single PPR-DYW protein share 42 to 56% of their identities. Thus, moss PPR-DYW proteins seem to be site-specific factors for RNA editing in mitochondrial transcripts.

RNA editing is an enigmatic phenomenon in which specific cytidines (C) in the transcripts are changed to uridines (U). In flowering plants, over 500 editing sites have been identified in the mitochondria and plastids. By contrast, in a moss Physcomitrella patens, only 12 editing sites are found in both organelles. Recent extensive genetics studies have revealed involvement of the pentatricopeptide repeat (PPR) proteins with a C-terminal DYW domain (PPR-DYW) in site-specific RNA editing events. Flowering plants have ~100 PPR-DYW genes while P. patens has only 10 PPR-DYW genes. Thus, the number of PPR-DYW genes is somewhat related to the number of RNA editing sites. The P. patens gametophyte, the haploid phase of the life cycle, is dominant, making it possible to study the phenotype of knockouts directly after transformation for gene-targeting. This makes it easier to identify the PPR-DYW proteins required for RNA editing. Recently, we have shown that one of 10 PPR-DYW proteins, PpPPR_71, was responsible for RNA editing in the mitochondrial ccmFc mRNA. In this study, we propose a working model of PpPPR_71 function on RNA editing.

In most land plants RNA editing frequently occurs in many organelle transcripts, but little is known about the molecular mechanisms of the organelle RNA editing process. In this study, we have characterized the Physcomitrella patens PpPPR_71 gene that is required for RNA editing of the ccmFc transcript. This transcript harbors two RNA editing sites, ccmF-1 and ccmF-2, that are separated by 18 nucleotides. Complementary DNA sequence analysis of ccmFc suggested that RNA editing at the ccmF-1 site occurred before ccmF-2 editing. RNA editing of the ccmF-2 downstream site was specifically impaired by disruption of the PpPPR_71 gene that encodes a polypeptide with 17 pentatricopeptide repeat motifs and a C-terminal DYW domain. The recombinant PpPPR_71 protein expressed in Escherichia coli specifically bound to the 46-nucleotide sequence containing the ccmF-2 editing site. The binding affinity of the recombinant PpPPR_71 was strongest when using the edited RNA at ccmF-1. In addition, the DYW domain also binds to the surrounding ccmF-2 editing site. We conclude that PpPPR_71 is an RNA-binding protein that acts as a site recognition factor in mitochondrial RNA editing.

To more clearly understand the molecular mechanisms involved in synergistic enhancement of cancer preventive activity with the green tea polyphenol (-)-epigallocatechin gallate (EGCG), we examined the effects of cotreatment with EGCG plus celecoxib, a cyclooxygenase-2 selective inhibitor. We specifically looked for induction of apoptosis and expression of apoptosis related genes, with emphasis on growth arrest and DNA damage-inducible 153 (GADD153) gene, in human lung cancer cell line PC-9: Cotreatment with EGCG plus celecoxib strongly induced the expression of both GADD153 mRNA level and protein in PC-9 cells, while neither EGCG nor celecoxib alone did. However, cotreatment did not induce expression of other apoptosis related genes, p21(WAF1) and GADD45. Judging by upregulation of GADD153, only cotreatment with EGCG plus celecoxib synergistically induced apoptosis of PC-9 cells. Synergistic effects with the combination were also observed in 2 other lung cancer cell lines, A549 and ChaGo K-1. Furthermore, EGCG did not enhance GADD153 gene expression or apoptosis induction in PC-9 cells in combination with N-(4-hydroxyphenyl)retinamide or with aspirin. Thus, upregulation of GADD153 is closely correlated with synergistic enhancement of apoptosis with EGCG. Cotreatment also activated the mitogen-activated protein kinases (MAPKs), such as ERK1/2 and p38 MAPK: Preteatment with PD98059 (ERK1/2 inhibitor) and UO126 (selective MEK inhibitor) abrogated both upregulation of GADD153 and synergistic induction of apoptosis of PC-9 cells, while SB203580 (p38 MAPK inhibitor) did not do so, indicating that GADD153 expression was mediated through the ERK signaling pathway. These findings indicate that high upregulation of GADD153 is a key requirement for cancer prevention in combination with EGCG.