Effect of chlorpyrifos-exposure on the expression levels of CYP genes in Daphnia magna and examination of a possibility that an up-regulated clan 3 CYP, CYP360A8, reacts with pesticides.

Daphnia magna is a test organism used for ecological risk assessments of pesticides, but little is known about the expression levels of cytochrome P450s (CYP)s and their changes after pesticide exposure in the less than 24-h-olds used for ecotoxicity tests. In this study, D. magna juveniles were exposed to 0.2 µg/L of chlorpyrifos under the conditions for acute immobilization test as specified by the OECD test guideline for 24 h, and then the gene expression was compared between the control and chlorpyrifos-exposure groups by RNA-sequencing analysis, with a focus on CYP genes. Among 38 CYP genes expressed in the control group, seven were significantly up-regulated while two were significantly down-regulated in the chlorpyrifos-exposure group. Although the sublethal concentration of chlorpyrifos did not change their expression levels so drastically (0.8 < fold change < 2.6), CY360A8 of D. magna (DmCYP360A8), which had been proposed to be responsible for metabolism of xenobiotics, was abundantly expressed in controls yet up-regulated by chlorpyrifos. Therefore, homology modeling of DmCYP360A8 was performed based on the amino acid sequence, and then molecular docking simulations with the insecticides that were indicated to be metabolized by CYPs in D. magna were conducted. The results indicated that DmCYP360A8 could contribute to the metabolism of diazinon and chlorfenapyr but not chlorpyrifos. These findings suggest that chlorpyrifos is probably detoxified by other CYP(s) including up-regulated and/or constitutively expressed one(s).

Utilization of piperonyl butoxide and 1-aminobenzotriazole for metabolic studies of toxic chemicals in Daphnia magna and Chironomus yoshimatsui.

Daphnids and chironomids have been used to assess the ecological effects of chemicals released into water bodies; however, the toxicity mechanisms in organisms are generally difficult to identify. Here, we developed a system capable of estimating the contribution of cytochrome P450 (CYP) to the metabolism of test substances in Daphnia magna and Chironomus yoshimatsui based on toxicity differences in the absence and presence of the CYP inhibitors piperonyl butoxide (PBO) and 1-aminobenzotriazole (ABT). The optimum concentrations of PBO and ABT that could effectively reduce the toxicity of diazinon, which is toxic after oxidative metabolism in vivo, were determined as 0.5 and 0.6 mg/L for D. magna, and 2.0 and 40.0 mg/L for C. yoshimatsui, respectively. Acute immobilization tests of 15 insecticides were conducted for D. magna and C. yoshimatsui, with and without the optimum concentrations of PBO or ABT. In the presence of either inhibitor, chlorpyrifos and chlorfenapyr toxicity was reduced in both organisms, whereas those of thiocyclam, nereistoxin, and silafluofen were enhanced in C. yoshimatsui. Liquid chromatography-mass spectrometry analysis of D. magna and C. yoshimatsui samples exposed to chlorfenapyr confirmed that the level of the active metabolite produced by CYP was decreased by PBO or ABT in both organisms. The system to which the test substance was co-exposed to PBO or ABT will be valuable for estimating the contribution of CYPs to metabolism and elucidating the toxicity mechanism in daphnids and chironomids.

Host-specific activation of a pathogen effector Aave_4606 from Acidovorax citrulli, the causal agent for bacterial fruit blotch.

RipAY, an effector protein from the plant bacterial pathogen Ralstonia solanacearum, exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the host cellular glutathione (GSH) when stimulated by host eukaryotic-type thioredoxins (Trxs). Aave_4606 from Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbit plants, shows significant homology to RipAY. Based on its homology, it was predicted that the GGCT activity of Aave_4606 is also stimulated by host Trxs. The GGCT activity of a recombinant Aave_4606 protein was investigated in the presence of various Trxs, such as yeast (ScTrx1), Arabidopsis thaliana (AtTrx-h1, AtTrx-h2, AtTrx-h3, and AtTrx-h5), or watermelon (Cla022460/ClTrx). Unlike RipAY, the GGCT activity of Aave_4606 is stimulated only by AtTrx-h1, AtTrx-h3, AtTrx-h5 and ClTrx from a watermelon, the primary host of A. citrulli, but not by ScTrx1, AtTrx-h2. Interestingly, GGCT activity of Aave_4606 is more efficiently stimulated by AtTrx-h1 and ClTrx than AtTrx-h5. These results suggested that Aave_4606 recognizes host-specific Trxs, which specifically activates the GGCT activity of Aave_4606 to decrease the host cellular GSH. These findings provide new insights into that effector is one of the host-range determinants for pathogenic bacteria via its host-dependent activation.

Requirement of the exopolyphosphatase gene for cellular acclimation to phosphorus starvation in a cyanobacterium, Synechocystis sp. PCC 6803.

Polyphosphate, which is ubiquitous in cells in nature, is involved in a myriad of cellular functions, and has been recently focused on its metabolism related with microbial acclimation to phosphorus-source fluctuation. In view of the ecological importance of cyanobacteria as the primary producers, this study investigated the responsibility of polyphosphate metabolism for cellular acclimation to phosphorus starvation in a cyanobacterium, Synechocystis sp. PCC 6803, with the use of a disruptant (Δppx) as to the gene of exopolyphosphatase that is responsible for polyphosphate degradation. Δppx was similar to the wild type in the cellular content of polyphosphate to show no defect in cell growth under phosphorus-replete conditions. However, under phosphorus-starved conditions, Δppx cells were defective in a phosphorus-starvation dependent decrease of polyphosphate to show deleterious phenotypes as to their survival and the stabilization of the photosystem complexes. These results demonstrated some crucial role of exopolyphosphatase to degrade polyP in the acclimation of cyanobacterial cells to phosphorus-starved conditions. Besides, it was found that ppx expression is induced in Synechocystis cells in response to phosphorus starvation through the action of the two-component system, SphS and SphR, in the phosphate regulon. The information will be a foundation for a fuller understanding of the process of cyanobacterial acclimation to phosphorus fluctuation.

Characterization of the mechanism of thioredoxin-dependent activation of γ-glutamylcyclotransferase, RipAY, from Ralstonia solanacearum.

A class II ChaC protein, RipAY, from phytopathogenic bacterium, Ralstonia solanacearum exhibits γ-glutamylcyclotransferase (GGCT) activity to degrade intracellular glutathione in host cells upon its interaction with host thioredoxins (Trxs). To understand the Trx-dependent activation of RipAY, we constructed various deletion mutants of RipAY and found the determinant region for GGCT activation in the N- and C-terminal sequences of RipAY by analyzing their yeast growth inhibition activity and the interaction with Trxs. Mutational analysis of the active site cysteine residues of Arabidopsis thaliana Trx-h5 (AtTrx-h5), one of the most efficiently stimulating Trxs, revealed that each active site cysteine residue of AtTrx-h5 contributes to efficient RipAY-binding and -activation activity. We also estimated that RipAY and AtTrx-h5 form a complex at a 1:2 M ratio. Furthermore, we found that the constitutive GGCT activity of Gcg1, a yeast class I ChaC protein, is also stimulated by yeast Trx1. These results indicate that class I ChaC proteins can sense the intracellular redox state and interact with Trxs to promote more efficient degradation of glutathione and regulate intracellular redox homeostasis. We hypothesize that RipAY acquired a more efficient and specific Trx-dependent activation mechanism to activate its GGCT activity only in the host eukaryotic cells during the evolution.

RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins.

The plant pathogenic bacterium Ralstonia solanacearum injects more than 70 effector proteins (virulence factors) into the host plant cells via the needle-like structure of a type III secretion system. The type III secretion system effector proteins manipulate host regulatory networks to suppress defense responses with diverse molecular activities. Uncovering the molecular function of these effectors is essential for a mechanistic understanding of R. solanacearum pathogenicity. However, few of the effectors from R. solanacearum have been functionally characterized, and their plant targets remain largely unknown. Here, we show that the ChaC domain-containing effector RipAY/RSp1022 from R. solanacearum exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the major intracellular redox buffer, glutathione. Heterologous expression of RipAY, but not other ChaC family proteins conserved in various organisms, caused growth inhibition of yeast Saccharomyces cerevisiae, and the intracellular glutathione level was decreased to ∼30% of the normal level following expression of RipAY in yeast. Although active site mutants of GGCT activity were non-toxic, the addition of glutathione did not reverse the toxicity, suggesting that the toxicity might be a consequence of activity against other γ-glutamyl compounds. Intriguingly, RipAY protein purified from a bacterial expression system did not exhibit any GGCT activity, whereas it exhibited robust GGCT activity upon its interaction with eukaryotic thioredoxins, which are important for intracellular redox homeostasis during bacterial infection in plants. Our results suggest that RipAY has evolved to sense the host intracellular redox environment, which triggers its enzymatic activity to create a favorable environment for R. solanacearum infection.

Extending magnifying NBI diagnosis of intestinal metaplasia in the stomach: the white opaque substance marker.

Background and aims Intestinal metaplasia (IM) of the stomach is associated with an increased risk of differentiated gastric cancer. While it is important to diagnose IM endoscopically, it can be difficult to observe by white-light endoscopy. In magnifying endoscopy with narrow-band imaging (M-NBI) of the stomach, a light-blue crest (LBC) is widely known to be a useful marker in the endoscopic diagnosis of IM. However, IM that exhibits only white opaque substance (WOS) without an LBC can also occur. The aim of this study was to elucidate whether the presence of WOS on M-NBI of the stomach could serve as a marker of IM in the same way that an LBC does. Methods The subjects were 40 consecutive patients who underwent M-NBI between July and December 2014. The primary endpoint in this study was to evaluate the diagnostic performance of M-NBI for histologically observed IM in WOS- and LBC-positive mucosa. Results The sensitivity and specificity of WOS for histologically diagnosed IM were 50.0 % (95 % confidence interval [CI] 40.0 % - 50.0 %) and 100.0 % (95 %CI 85.0 % - 100.0 %), respectively. Meanwhile, the sensitivity and specificity of LBC were 62.5 % (95 %CI 51.1 % - 65.9 %) and 93.8 % (95 %CI 76.7 % - 98.9 %), respectively. The sensitivity and specificity of WOS and/or LBC (WOS positive and LBC positive, WOS positive and LBC negative, or WOS negative and LBC positive) for histologically diagnosed IM were 87.5 % (95 %CI 76.9 % - 90.9 %) and 93.8 % (95 %CI 77.9 % - 98.9 %), respectively. Conclusions LBC and WOS are both useful markers for endoscopic diagnosis of IM. Combining both markers improves the sensitivity.Clinical trial number: UMINCTR000014453.

Pleurochrysome: A Web Database of Pleurochrysis Transcripts and Orthologs Among Heterogeneous Algae.

Pleurochrysis is a coccolithophorid genus, which belongs to the Coccolithales in the Haptophyta. The genus has been used extensively for biological research, together with Emiliania in the Isochrysidales, to understand distinctive features between the two coccolithophorid-including orders. However, molecular biological research on Pleurochrysis such as elucidation of the molecular mechanism behind coccolith formation has not made great progress at least in part because of lack of comprehensive gene information. To provide such information to the research community, we built an open web database, the Pleurochrysome (http://bioinf.mind.meiji.ac.jp/phapt/), which currently stores 9,023 unique gene sequences (designated as UNIGENEs) assembled from expressed sequence tag sequences of P. haptonemofera as core information. The UNIGENEs were annotated with gene sequences sharing significant homology, conserved domains, Gene Ontology, KEGG Orthology, predicted subcellular localization, open reading frames and orthologous relationship with genes of 10 other algal species, a cyanobacterium and the yeast Saccharomyces cerevisiae. This sequence and annotation information can be easily accessed via several search functions. Besides fundamental functions such as BLAST and keyword searches, this database also offers search functions to explore orthologous genes in the 12 organisms and to seek novel genes. The Pleurochrysome will promote molecular biological and phylogenetic research on coccolithophorids and other haptophytes by helping scientists mine data from the primary transcriptome of P. haptonemofera.

Genes for a series of proteins that are involved in glucose catabolism are upregulated by the Hik8-cascade in Synechocystis sp. PCC 6803.

MAIN CONCLUSION: In summary, we could show the involvement of a Hik8-cascade in the expression of genes involved in the glycolytic and OPP pathways induced by GPL, and another signal pathway under photosynthetic conditions in Synechocystis . The Hik8-cascade under GPL conditions may regulate glucose degradation to produce some energy and carbon compounds. This cascade might be important for the supply of organic materials such as amino acids and nucleotides through enhancement of the rates of the glycolysis and OPP pathways. Histidine kinase Hik8 upregulates the expression of one of the important glycolytic genes, fbaA, via sll1330 under heterotrophic growth conditions (i.e., in the presence of glucose with an indispensable short period of light) in Synechocystis sp. PCC 6803. In this study, expression of the genes for the glycolytic and OPP pathways was investigated using the wild type, and disruption mutants of Hik8 and sll1330, to determine whether or not the Hik8-involving signal transduction system generally regulates glucose catabolism. In the wild type, all the genes for the glycolytic and OPP pathways were upregulated under the same conditions as for fbaA. Analyses of the disruption mutants suggested that the signal transduction system involving Hik8 and Sll1330 plays a key role in the upregulation of genes such as pfkA, pgmB, and glk, and also that Hik8 induces genes including gap1 and pgk independently of Sll1330. This complicated signal transduction cascade, designated as the Hik8-cascade, occurs under heterotrophic growth with light pulses. In addition, a disruption mutant of a putative histidine kinase, sll1334, exhibited growth and gene expression patterns that suggested it to be a negative regulator in the cascade. Possible histidine kinases and response regulators as candidates for other components in the cascade are discussed.

Can we accurately diagnose minute gastric cancers (≤5 mm)? Chromoendoscopy (CE) vs magnifying endoscopy with narrow band imaging (M-NBI).

BACKGROUND: Chromoendoscopy (CE) is relatively ineffective at identifying the cancer-specific morphological characteristics of minute gastric cancers less than or equal to 5 mm in diameter, and on its own is insufficient to make an accurate diagnosis. The aim of this study is to assess the diagnostic performance of magnifying endoscopy with narrow band imaging (M-NBI) for minute gastric cancers. METHODS: The minute cancer group comprised consecutive endoscopic submucosal dissection-resected minute gastric cancers histologically measured as no larger than 5 mm in diameter. The non-cancer group comprised consecutive non-cancer lesions no larger than 5 mm in diameter. The two groups were subject to retrospective analysis to evaluate the diagnostic ability (sensitivity, specificity, and diagnostic accuracy) and reproducibility of CE and M-NBI. RESULTS: The results for CE versus M-NBI were as follows: sensitivity 43.7 % (95 % CI, 26.5-61.0 %) versus 78.0 % (95 % CI, 64.0-92.0 %); specificity 81.6 % (95 % CI, 72.6-90.6 %) versus 92.9 % (95 % CI, 87.0-98.9 %); and diagnostic accuracy 69.9 % (95 % CI, 61.0-78.6 %) versus 88.3 % (95 % CI, 82.0-94.5 %). The sensitivity and diagnostic accuracy were, therefore, significantly higher for M-NBI than for CE. The inter-observer variability was κ = 0.08 for CE and κ = 0.56 for M-NBI, while the intra-observer variability was κ = 0.38 and κ = 0.65, respectively. CONCLUSIONS: M-NBI has greater sensitivity and reproducibility than CE for the diagnosis of minute gastric cancers.

Diversity of reaction characteristics of glucan branching enzymes and the fine structure of α-glucan from various sources.

To investigate the functional properties of 10 α-glucan branching enzymes (BEs) from various sources, we determined the chain-length distribution of BE enzymatic products and their phosphorylase-limit dextrins (Φ-LD). All BEs could be classified into either of the three rice BE isozymes: OsBEI, OsBEIIa, or OsBEIIb. Escherichia coli BE (EcoBE) had the same enzymatic properties as OsBEI, while Synechococcus elongatus BE (ScoBE) and Chlorella kessleri BE (ChlBE) had BEIIb-type properties. Human BE (HosBE), yeast BE (SacBE), and two Porphyridium purpureum BEs (PopBE1 and PopBE2) exhibited the OsBEIIa-type properties. Analysis of chain-length profile of Φ-LD of the BE reaction products revealed that EcoBE, ScoBE, PopBE1, and PopBE2 preferred A-chains as acceptors, while OsBEIIb used B-chains more frequently than A-chains. Both EcoBE and ScoBE specifically formed the branch linkages at the third glucose residue from the reducing end of the acceptor chain. The present results provide evidence for the first time that great variation exists as to the preference of BEs for their acceptor chain, either A-chain or B-chain. In addition, EcoBE and ScoBE recognize the location of branching points in their acceptor chain during their branching reaction. Nevertheless, no correlation exists between the primary structure of BE proteins and their enzymatic characteristics.

The vessels within epithelial circle (VEC) pattern as visualized by magnifying endoscopy with narrow-band imaging (ME-NBI) is a useful marker for the diagnosis of papillary adenocarcinoma: a case-controlled study.

BACKGROUND AND AIMS: Pathological studies indicate papillary adenocarcinomas are more aggressive than tubular adenocarcinomas, but a definitive diagnosis is difficult using conventional endoscopy alone. The vessels within an epithelial circle (VEC) pattern, visualized using magnifying endoscopy with narrow-band imaging (ME-NBI), may be a feature of papillary adenocarcinoma. The aims of our study were to investigate whether the VEC pattern is useful in the preoperative diagnosis of papillary adenocarcinoma and to determine whether VEC-positive adenocarcinomas are more malignant than VEC-negative lesions. PATIENTS AND MATERIALS: From 395 consecutive early gastric cancers resected using the endoscopic submucosal dissection method, we analyzed 35 VEC-positive lesions and 70 VEC-negative control lesions matched for size and macroscopic type. We evaluated (1) the correlation between the incidence of VEC-positive cancers and the histological papillary structure and (2) differences in the incidence of coexisting undifferentiated carcinoma in VEC-positive and VEC-negative cancers and the incidence of submucosal and vascular invasion. RESULTS: Histological papillary structure was seen in 94 % (33/35) of VEC-positive and 9 % (6/70) of VEC-negative cancers, a significant difference (P < 0.001). The incidence of coexisting undifferentiated carcinoma was 23 % (8/35) in VEC-positive and 3 % (2/70) in VEC-negative cancers (P = 0.002). The incidence of submucosal invasion by the carcinoma was 26 % (9/35) in VEC-positive cancers and 10 % (7/70) in VEC-negative cancers (P = 0.045). CONCLUSIONS: The VEC pattern as visualized using ME-NBI is a promising preoperative diagnostic marker of papillary adenocarcinoma. Coexisting undifferentiated carcinoma and submucosal invasion were each seen in approximately one fourth of VEC-positive cancers.

Incidence, clinical characteristics, long-term course, and comparison of progressive and nonprogressive cases of aphthous-type Crohn's disease: a single-center cohort study.

BACKGROUND/AIMS: In Japan, aphthous-type Crohn's disease (type A CD) is thought to represent an early phase of Crohn's disease (CD), and diagnosis of type A CD is possible in the diagnostic criteria for CD in Japan. However, the details of type A CD are not well understood. METHODS: Subjects comprised 649 CD patients diagnosed between 1985 and 2011. The incidence of type A CD over time was clarified in two periods (1985-2004 and 2005-2011). The course of type A CD was also investigated, and cases that did and did not progress to typical CD were compared. RESULTS: No significant difference was seen in the incidence of type A CD between the two periods (5.2 vs. 8.5%, p = 0.125). Type A CD patients followed at our hospital progressed to typical CD at a rate of 59.3%. In comparing progressive and nonprogressive cases, the frequency of large, densely distributed aphthous lesions in the small intestine was higher among progressive cases (p = 0.018). CONCLUSION: Type A CD is an early phase of CD, and CD diagnostic criteria including early cases are valid in Japan.

Changes in suicide-related indices at a student counseling center at a Japanese University before and after COVID-19.

Suicides by university students in Japan have increased with the onset of the COVID- 19 pandemic. Changes in the suicidality of clients in student counseling centers are unknown. We compared OQ 45.2, suicidal risk, and suicidality before and after the pandemic, testing 1801 clients at the Student Counseling Center of University A in Japan. The post-group exhibited significantly decreases in all suicide-related indices. Results reveal that fewer students may feel suicidal and that the number of consultations with suicidal students may have decreased. It is important to build support systems easier for students to access in crises and alone.

Kre6 (yeast 1,6-ß-transglycosylase) homolog, PhTGS, is essential for ß-glucan synthesis in the haptophyte Pleurochrysis haptonemofera.

Haptophytes synthesize unique ß-glucans containing more ß-1,6-linkages than ß-1,3 linkages, as a storage polysaccharide. To understand the mechanism of the synthesis, we investigated the roles of Kre6 (yeast 1,6-ß-transglycosylase) homologs, PhTGS, in the haptophyte Pleurochrysis haptonemofera. RNAi of PhTGS repressed ß-glucan accumulation and simultaneously induced lipid production, suggesting that PhTGS is involved in ß-glucan synthesis and that the knockdown leads to the alteration of the carbon metabolic flow. PhTGS was expressed more in light, where ß-glucan was actively produced by photosynthesis, than in the dark. The crude extract of E. coli expressing PhKre6 demonstrated its activity to incorporate 14C-UDP-glucose into ß-glucan of P. haptonemofera. These findings suggest that PhTGS functions in storage ß-glucan synthesis specifically in light, probably by producing the ß-1,6-branch.

Cellular response of Parachlorella kessleri to a solid surface culture environment.

Attached culture allows high biomass productivity and is a promising biomass cultivating system because neither a huge facility area nor a large volume of culture medium are needed. This study investigates photosynthetic and transcriptomic behaviors in Parachlorella kessleri cells on a solid surface after their transfer from liquid culture to elucidate the physiological and gene-expression regulatory mechanisms that underlie their vigorous proliferation. The chlorophyll content shows a decrease at 12 h after the transfer; however, it has fully recovered at 24 h, suggesting temporary decreases in the amounts of light harvesting complexes. On PAM analysis, it is demonstrated that the effective quantum yield of PSII decreases at 0 h right after the transfer, followed by its recovery in the next 24 h. A similar changing pattern is observed for the photochemical quenching, with the PSII maximum quantum yield remaining at an almost unaltered level. Non-photochemical quenching was increased at both 0 h and 12 h after the transfer. These observations suggest that electron transfer downstream of PSII but not PSII itself is only temporarily damaged in solid-surface cells just after the transfer, with light energy in excess being dissipated as heat for PSII protection. It thus seems that the photosynthetic machinery acclimates to high-light and/or dehydration stresses through its temporal size-down and functional regulation that start right after the transfer. Meanwhile, transcriptomic analysis by RNA-Seq demonstrates temporary upregulation at 12 h after the transfer as to the expression levels of many genes for photosynthesis, amino acid synthesis, general stress response, and ribosomal subunit proteins. These findings suggest that cells transferred to a solid surface become stressed immediately after transfer but can recover their high photosynthetic activity through adaptation of photosynthetic machinery and metabolic flow as well as induction of general stress response mechanisms within 24 h.

Two regulatory networks mediated by light and glucose involved in glycolytic gene expression in cyanobacteria.

Fructose 1,6-bisphosphate aldolase (FBA) is an enzyme involved in both glycolytic and photosynthetic reactions in photosynthetic organisms. In prokaryotes, the bidirectional reaction proceeds in the same cellular compartment, i.e. the cytoplasm. Expression of the FBA gene, fbaA, is induced through two independent pathways, stimulated by continuous light and by glucose plus pulsed light (GPL), in a cyanobactrium, Synechocystis sp. PCC 6803. Under GPL conditions, glucose can be replaced by glucose analogs that are not even metabolized in a cell. Analyses of transcripts in deletion mutants suggested that both a histidine kinase, Hik8, and a response regulator, Sll1330, played important roles as signal components in fbaA expression under GPL conditions, but not under photosynthetic conditions. Analysis of a transformant in which sll1330 expression was enhanced demonstrated that fbaA expression was induced at least partially even without glucose, but for its further induction a pulsed light stimulus was required. These results substantiated that there are two light-dependent regulatory pathways for aldolase gene expression in this cyanobacterium.

Arsenic tolerance in a Chlamydomonas photosynthetic mutant is due to reduced arsenic uptake even in light conditions.

Arsenate resistance has been used for screening for photosynthetic mutants of Chlamydomonas, since photosynthetic mutants, such as CC981 defective in phosphoribulokinase, were shown to have arsenate resistance. Also, another type of arsenate-resistant mutants, including AR3 that lacks a homolog of a phosphate (Pi) transporter, PTB1, has been isolated. We investigated the uptake of Pi and arsenate, and the gene expression of Pi transporters, which are involved in both Pi and arsenate transport, in mutants CC981 and AR3. In the wild type, both Pi and arsenate uptake were initially high, but were inactivated in the presence of arsenate with time, especially in the dark. In contrast, both mutants were shown to exhibit higher Pi uptake, but lower arsenate uptake than the wild type, regardless of the presence or absence of light. Then, the gene expression of Pi transporters in the cells used for the uptake measurements was investigated and compared between the mutants and the wild type. In CC981, the mRNA levels of PTA2 and PTA4 were higher, while those of PTB3 and PTB5 were lower, as compared with in the wild type. In AR3, those of PTA2 and PTB2 were higher, but that of PTB5 was lower than in the wild type. These findings suggest that the arsenate resistance shown by the mutants in light is due to reduction of arsenate uptake probably through the down-regulation of some Pi transporter expression, while the Pi uptake maintained even in the dark is possibly related to higher expression of other Pi transporter(s) than in the wild type.

Clinicopathological characteristics of abnormal micro-lesions at the oro-hypopharynx detected by a magnifying narrow band imaging system.

BACKGROUND: Narrow band imaging (NBI) with magnifying endoscopy (NBI-ME) allows the detection of abnormal micro-lesions smaller than 5 mm in diameter in the oro-hypopharynx that could not be visualized previously. The purpose of the present study was to clarify the clinicopathological characteristics of abnormal micro-lesions of the oro-hypopharynx detected by NBI-ME. METHODS: Of the 62 lesions detected by NBI-ME, 40 abnormal micro-lesions in 37 patients were removed by endoscopic treatment and were pathologically evaluated. We reviewed the medical records of patients with these lesions and investigated the relationship between NBI-ME findings and pathological findings. RESULTS: Pathological examination revealed the following: high-grade intraepithelial neoplasia (HGIN) in nine (23%) lesions, low-grade intraepithelial neoplasia (LGIN) in 22 (55%), pharyngitis in seven (18%) and papilloma in two (5%). Two NBI-ME findings, high microvascular density (MVD) and a brownish area (BA), were recognized more frequently as the grade of malignancy advanced. The likelihood ratio (confidential interval) for having HGIN in the patients with both MVD and BA was 13 (3.62-127). CONCLUSIONS: The pathological diagnosis of abnormal micro-lesions ranged from pharyngitis to HGIN. High MVD and BA may be important findings for grading the malignancy of abnormal micro-lesions.

Diacylglyceryl-N,N,N-trimethylhomoserine-dependent lipid remodeling in a green alga, Chlorella kessleri.

Membrane lipid remodeling contributes to the environmental acclimation of plants. In the green lineage, a betaine lipid, diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), is included exclusively among green algae and nonflowering plants. Here, we show that the green alga Chlorella kessleri synthesizes DGTS under phosphorus-deficient conditions through the eukaryotic pathway via the ER. Simultaneously, phosphatidylcholine and phosphatidylethanolamine, which are similar to DGTS in their zwitterionic properties, are almost completely degraded to release 18.1% cellular phosphorus, and to provide diacylglycerol moieties for a part of DGTS synthesis. This lipid remodeling system that substitutes DGTS for extrachloroplast phospholipids to lower the P-quota operates through the expression induction of the BTA1 gene. Investigation of this lipid remodeling system is necessary in a wide range of lower green plants for a comprehensive understanding of their phosphorus deficiency acclimation strategies.