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Gametocytes play key roles in the Plasmodium lifecycle. They are essential for sexual reproduction as precursors of the gametes. They also play an essential role in parasite transmission to mosquitoes. Elucidation of the gene regulation at this stage is essential for understanding these two processes at the molecular level and for developing new strategies to break the parasite life cycle. We identified a novel Plasmodium transcription factor (TF), designated as a partner of AP2-FG or PFG. In this paper, we report that this TF regulates the gene expression in female gametocytes in concert with another female-specific TF AP2-FG. Upon the disruption of PFG, majority of female-specific genes were significantly downregulated, and female gametocyte lost the ability to produce ookinetes. ChIP-seq analysis showed that it was located in the same position as AP2-FG, indicating that these two TFs form a complex. ChIP-seq analysis of PFG in AP2-FG-disrupted parasites and ChIP-seq analysis of AP2-FG in PFG-disrupted parasites demonstrated that PFG mediates the binding of AP2-FG to the ten-base motif and that AP2-FG binds another motif, GCTCA, in the absence of PFG. In promoter assays, this five-base motif was identified as another female-specific cis-acting element. Genes under the control of the two forms of AP2-FG, with or without PFG, partly overlapped; however, each form had target preferences. These results suggested that combinations of these two forms generate various expression patterns among the extensive genes expressed in female gametocytes.

Plasmodium parasites, the causative agents of malaria, possess a complex lifecycle; however, the mechanisms of gene regulation involved in the cell-type changes remain unknown. Here, we report that gametocyte sucrose nonfermentable 2 (gSNF2), an SNF2-like chromatin remodeling ATPase, plays an essential role in the differentiation of male gametocytes. Upon disruption of gSNF2 , male gametocytes lost the capacity to develop into gametes. ChIP-seq analyses revealed that gSNF2 is widely recruited upstream of male-specific genes through a five-base, male-specific cis-acting element. In gSNF2 -disrupted parasites, expression of over a hundred target genes was significantly decreased. ATAC-seq analysis demonstrated that decreased expression of these genes correlated with a decrease of the nucleosome-free region upstream of these genes. These results suggest that global changes induced in the chromatin landscape by gSNF2 are the initial step in male differentiation from early gametocytes. This study provides the possibility that chromatin remodeling is responsible for cell-type changes in the Plasmodium lifecycle.

Gametocytes play key roles in thePlasmodiumlifecycle. They are essential for sexual reproduction as precursors of the gametes. They also play an essential role in parasite transmission to mosquitoes. Elucidation of the gene regulation at this stage is essential for understanding these two processes at the molecular level and for developing new strategies to break the parasite life cycle. We identified a novelPlasmodiumtranscription factor (TF), designated as a partner of AP2-FG or PFG. In this paper, we report that this TF regulates the gene expression in female gametocytes in concert with another female-specific TF AP2-FG. Upon the disruption ofPFG, majority of female-specific genes were significantly downregulated, and female gametocyte lost the ability to produce ookinetes. ChIP-seq analysis showed that it was located in the same position as AP2-FG, indicating that these two TFs form a complex. ChIP-seq analysis of PFG inAP2-FG-disrupted parasites and ChIP-seq analysis of AP2-FG inPFG-disrupted parasites demonstrated that PFG mediates the binding of AP2-FG to the ten-base motif and that AP2-FG binds another motif, GCTCA, in the absence of PFG. In promoter assays, this five-base motif was identified as another female-specific cis-acting element. Genes under the control of the two forms of AP2-FG, with or without PFG, partly overlapped; however, each form had target preferences. These results suggested that combinations of these two forms generate various expression patterns among the extensive genes expressed in female gametocytes.

Gametocyte development is a critical step in the life cycle of Plasmodium. Despite that numbers of studies in the gametocyte development have been conducted, the molecular mechanisms regulating this process remains to be fully understood. This study investigates the functional roles of two female-specific transcriptional regulators, PbAP2-FG2 and AP2R-2, in P. berghei. Knockout of pbp2-fg2 or ap2r-2 impairs female gametocyte development, resulting in developmental arrest during ookinete development. ChIP-seq analyses of these two factors indicated their colocalization on the genome, suggesting they function as a complex. These analyses also revealed that their target genes contained a variety of genes, including both male and female-enriched genes. Moreover, differential expression analyses showed that these target genes were upregulated through the disruption of pbp2-fg2 or ap2r-2, indicating that these two factors function as a transcriptional repressor complex in female gametocytes. Further target analysis demonstrated a significant overlap between the target genes of PbAP2-FG2 and AP2-G, suggesting that repression of early gametocyte genes activated by AP2-G is one of the key roles for this female transcriptional repressor complex. Our results indicate that the PbAP2-FG2-AP2-R2 complex-mediated repression of the target genes supports the female differentiation from early gametocytes.Author SummaryGametocyte development in Plasmodium parasites, a causative agent of malaria, is an essential step for their transmission from vertebrate hosts to mosquitoes. Gametocytes are sexual precursor cells produced from a subpopulation of asexual blood-stage parasites. Upon uptake by mosquitoes through blood feeding, the male and female gametocytes become microgametes and macrogametes, respectively, and then they fertilize and develop into the mosquito midgut invasive stage, called ookinete. Therefore, it is crucial to understand the underlying mechanisms regulating this developmental process. This study revealed that the two female transcriptional regulators, PbAP2-FG2 and AP2R-2, function together as an essential transcriptional repressor complex in P. berghei, the target genes of which include male, female, and early gametocyte genes activated by AP2-G. Our findings suggest that PbAP2-FG2 and AP2R-2 play multiple roles in supporting the development of female gametocytes from early gametocytes.

Plasmodium sporozoites travel a long way from the site where they are released by a mosquito bite to the liver, where they infect hepatocytes and develop into erythrocyte-invasive forms. The success of this infection depends on the ability of the sporozoites to correctly recognize the hepatocyte as a target and change their behavior from migration to infection. However, how this change is accomplished remains incompletely understood. In this paper, we report that 6-cysteine protein family members expressed in sporozoites including B9 are responsible for this ability. Experiments on parasites using double knockouts of B9 and SPECT2, which is essential for sporozoite to migrate through the hepatocyte, showed that the parasites lacked the capacity to stop migration. This finding suggests that interactions between these parasite proteins and hepatocyte-specific cell surface ligands mediate correct recognition of hepatocytes by sporozoites, which is an essential step in malaria transmission to humans.

The transcription factor (TF) AP2-G is essential for gametocytogenesis in the malaria parasite; however, it remains unclear if AP2-G determines commitment to sexual stage development fate in the schizont stage, or whether AP2-G directly initiates sexual stage differentiation and development beginning in the late-trophozoite stage. In this study, we addressed this issue by investigating the expression profile of AP2-G and determining genome-wide target genes in Plasmodium berghei. Fluorescence microscopy showed that AP2-G expression was first observed in the parasite 12 h after erythrocyte invasion and peaked at 18 h when sexual features were first manifested in early gametocytes. Expression of AP2-G decreased with manifestation of sex-specific features. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed at peak AP2-G expression and identified over 1000 binding sites in the genome. The main binding motif of the TF predicted from the binding sites was GTACNY. Predicted targets contained a number of genes related to protein biogenesis, suggesting that AP2-G plays a role in establishing a cellular basis required for sexual differentiation. AP2-G binding sites also existed upstream of gametocyte-specific TFs, namely AP2-G2, AP2-FG, and AP2-G itself. Furthermore, the target contained two AP2 TF-related genes. Disruption of these genes resulted in the arrest of ookinete development. These results suggest another role of AP2-G: activating a transcriptional cascade to promote conversion into early gametocytes. Taken together, AP2-G is involved not in establishing sexual commitment of schizonts, but rather in triggering the initiation of differentiation and the early development of gametocytes in the late trophozoite stage.

The malaria gametocyte, the gamete precursor, is the essential stage for malaria transmission to the mosquito vector. In the vertebrate host's blood, it develops into a mature male or female capable of transforming into a gamete in the mosquito blood meal. Despite the importance of this stage in the malaria life cycle, the genetic regulation of gametocyte development is poorly understood. In particular, transcription factors involved in sex-specific gene expression have not been identified. In this paper, we report that an AP2-family transcription factor, AP2-FG, is responsible for female-specific gene regulation. AP2-FG expression in Plasmodium berghei was observed exclusively in female gametocytes, in the beginning of 4-6 h before sexual dimorphism manifests in developing gametocytes. AP2-FG disruption resulted in the arrest of female maturation, but did not affect the development of males. Chromatin immunoprecipitation sequencing analysis suggested that AP2-FG directly regulates over 700 genes. Its targets include genes for female gametocyte-specific functions, such as gametogenesis, fertilization and zygote development. AP2-FG binding to target gene promoters was associated with a 10 bp sequence motif. These results indicate that AP2-FG plays a role in the differentiation of early gametocytes to mature females by governing a female-specific gene expression repertoire.

Gametocytes are nonreplicative sexual forms that mediate malaria transmission to a mosquito vector. They are generated from asexual blood-stage parasites that proliferate in the circulation. However, little is known about how this transition is genetically regulated. Here, we report that an Apetala2 (AP2) family transcription factor, AP2-G2, regulates this transition as a transcriptional repressor. Disruption of AP2-G2 in the rodent malaria parasite Plasmodium berghei did not prevent commitment to the sexual stage but did halt development before the appearance of sex-specific morphologies. ChIP-seq analysis revealed that AP2-G2 targeted ∼1,500 genes and recognized a five-base motif in their promoters. Most of these target genes are required for asexual proliferation of the parasites in the blood, suggesting that AP2-G2 blocks the program that precedes asexual replication to promote conversion to the sexual stage. Microarray analysis showed that the identified targets constituted ∼70% of the up-regulated genes in AP2-G2-depleted parasites, suggesting that AP2-G2 actually functions as a repressor in gametocytes. A promoter assay using a centromere plasmid demonstrated that the binding motif functions as a cis-acting negative regulatory element. These results suggest that global transcriptional repression, which occurs during the initial phase of gametocytogenesis, is an essential step in Plasmodium sexual development.

This study quantitatively examines the behavior of pedestrians moving close to a person with a trolley bag or baby stroller. The data will prove useful in estimating how much extra space needs to be allowed for those with such gear, for example in a railroad station or other transport facility. The experiment was conducted on a crowded street in a Tokyo business district during the morning commute. A male assistant hired for the experiment was asked to walk in three directions with respect to pedestrian flow in four conditions for a total of 12 experimental situations. The study revealed how large evasive space is required in each situation. Language: ja

To maintain sterility of PET drug is the most important for in-house positron emission tomography (PET) drug manufacturing, and sanitary control of the laboratory to perform aseptic procedure is the key point for the sterility of PET drugs. However, rigorous sanitary control affects both the high cost and the low efficiency. To conquer those, we developed an isolator system especially for PET drug compounding including sterilization and dispensing units. This system consists of a HEPA unit for inlet and outlet, positive regulation of the ear inside isolator, a sterilizer with vapored hydrogen peroxide and a dispenser with self-shield for radiation. We set the materials for the dispenser through gloves, and the compounding such as sterilization and dispensing PET drugs to the containers is performed automatically without radiation. High level assurance of PET drug sterility is expected to be accomplished in the PET centers of the hospitals without high level sanitary control.

A CD1d-binding invariant natural killer T (iNKT)-cell stimulatory glycolipid, namely 7DW8-5, is shown to enhance the efficacy of radiation-attenuated sporozoites (RAS)-based malaria vaccine in mice. In the current study, we aim to determine whether 7DW8-5 can display a potent adjuvant effect in human immune system (HIS) mice.HIS-A2/hCD1d mice, which possess both functional human iNKT cells and CD8+ T cells, were generated by the transduction of NSG mice with adeno-associated virus serotype 9 expressing genes that encode human CD1d molecules and HLA-A*0201, followed by the engraftment of human hematopoietic stem cells. The magnitudes of human iNKT-cell response against 7DW8-5 and HLA-A*0201-restricted human CD8+ T-cell response against a human malaria antigen in HIS-A2/hCD1d mice were determined by using human CD1d tetramer and human HLA-A*0201 tetramer, respectively.We found that 7DW8-5 stimulates human iNKT cells in HIS-A2/hCD1d mice, as well as those derived from HIS-A2/hCD1d mice in vitro. We also found that 7DW8-5 significantly increases the level of a human malarial antigen-specific HLA-A*0201-restricted human CD8+ T-cell response in HIS-A2/hCD1d mice.Our study indicates that 7DW8-5 can display a potent adjuvant effect on RAS vaccine-induced anti-malarial immunity by augmenting malaria-specific human CD8+ T-cell response.

Protozoan parasites rely on purine nucleosides supplied by the host because they are unable to synthesise purine rings denovo. Nucleoside transporter 1 (NT1) and purine nucleoside phosphorylase (PNP) play an essential role in purine salvage in Plasmodium. It is unclear whether severe pathology, such as cerebral malaria (CM), develops in hosts infected with Plasmodium parasites that lack activity of NT1 or PNP. Plasmodium berghei (Pb) ANKA-infected mice show features similar to human CM, such as cerebral paralysis and cerebral haemorrhage. Therefore, Pb ANKA infection in mice is a good experimental model of CM. In this study, we generated pbnt1-disrupted Pb ANKA (Δpbnt1 parasites) and pbpnp-disrupted Pb ANKA (Δpbpnp parasites), and investigated the effect of pbnt1 or pbpnp disruption on the outcome of infection with Pb ANKA. We showed that the rapid increase of wild-type Pb ANKA (WT parasites) in mice early in infection was significantly inhibited by disruption of pbnt1. Moreover, Δpbnt1 parasite-infected mice showed neither cerebral paralysis nor cerebral haemorrhage, and all mice spontaneously recovered from infection. By contrast, mice infected with Δpbpnp parasites showed features similar to those of mice infected with WT parasites. In this study, we demonstrated that the high virulence of Pb ANKA in the asexual phase is suppressed by disruption of pbnt1 but not pbpnp.

The liver stage is the first stage of the malaria parasite that replicates in the vertebrate host. However, little is known about the interplay between the parasite liver stage and its host cell, the hepatocyte. In this study, we identified an exported protein that has a critical role in parasite development in host hepatocytes. Expressed sequence tag analysis of Plasmodium berghei liver-stage parasites indicated that transcripts encoding a protein with an N-terminal signal peptide, designated liver-specific protein 2 (LISP2), are highly expressed in this stage. Expression of LISP2 was first observed 24 h after infection and rapidly increased during the liver-stage schizogony. Immunofluorescent staining with anti-LSP2 antibodies showed that LISP2 was carried to the parasitophorous vacuole and subsequently transported to the cytoplasm and nucleus of host hepatocytes. Gene targeting experiments demonstrated that majority of the LISP2-mutant liver-stage parasites arrested their development during formation of merozoites. These results indicate that exported LISP2 is involved in parasite-host interactions required for the development of liver-stage parasites inside hepatocytes. This study demonstrated that mid-to-late liver-stage malarial parasites have a system for exporting proteins to the host cell as intraerythrocytic stages do and presumably to use the proteins to modify the host cell and improve the environment.

The emergence and spread of drug-resistant parasites are major problems that prevent the control of malaria. Parasite resistance to most anti-malarial drugs, such as chloroquine, sulphadoxine-pyrimethamine, quinine, and mefloquine, has increased globally, rendering these drugs useless in most malaria-endemic areas (Wongsrichanalai et al. 2002). The World Health Organization currently recommends artemisinin-based combination therapy (ACT) as the first-line treatment for malaria; however, a decline in the effectiveness of ACT was recently reported in the border region between Thailand and Cambodia, indicating the emergence of an artemisinin-resistant parasite (Dondorp et al. 2009; World Health Organization 2010). If such drug-resistant parasites become widely distributed, global malaria control efforts will be significantly hampered. The identification of genes responsible for drug resistance is crucial for combating drug-resistant parasites because the mutations found in these genes are useful molecular markers for the surveillance of the emergence and spread of drug-resistant parasites. In the past, the identification of resistance genes was carried out by restriction fragment length polymorphism (RFLP) analysis of the progeny generated by genetic crosses between drug-resistant and drug-sensitive parasites using primates and mosquitoes. The chloroquine-resistance gene (pfcrt, MAL7P1.27) of Plasmodium falciparum was identified by this approach and facilitated the elucidation of the mechanisms of chloroquine resistance and the global surveillance of this resistant parasite (Wellems et al. 1991; Su et al. 1997; Djimde et al. 2001a,b). However, because this approach requires a large amount of time and effort, resistance genes for other anti-malarial drugs, such as mefloquine and quinine, have not yet been identified. Moreover, unknown resistance genes are thought to be present even in the chloroquine-resistant parasite (Valderramos et al. 2010). Now, whole-genome sequencing and microarray technologies are used for the identification of single nucleotide polymorphisms (SNPs) involved in the drug resistance of parasites (Hunt et al. 2010; Rottmann et al. 2011). However, it may be difficult to identify the drug-resistance genes from field-isolated parasites using these two technologies because these parasites have more than 10,000 SNPs (Dharia et al. 2010). Therefore, a more effective method for the identification of drug-resistance genes in parasites is required. The artificial chromosome—which consists of three essential elements, the centromere, the telomere, and the replication origin—is an attractive genetic tool for molecular biology–based studies in eukaryotes. The first eukaryotic artificial chromosome was developed in the budding yeast Saccharomyces cerevisiae and is known as the yeast artificial chromosome (YAC) (Murray and Szostak 1983). The YAC can be stably maintained throughout mitosis and meiosis and has been widely used for cloning large DNA fragments. The development of artificial chromosomes in other eukaryotes, including humans, has been attempted by combining these three essential elements but has not yet been successful. Human artificial chromosomes are integrated at the centromere or the telomere of the original chromosome during cell division and are therefore not episomally maintained (Ikeno et al. 1998). In a previous study, we made a Plasmodium artificial chromosome (PAC) from the small centromere (1189 bp) and the short telomeric fragment ( 99.9% efficiency during multiple rounds of cell divisions. After cell division, it is stably maintained as a single copy without integration throughout the parasite life cycle. Notably, the transfection efficiency of the PAC is significantly higher than that of a normal plasmid. These unique features of the PAC overcome the current technical limitations of parasite genetic modification technology and could be applied in various parasite research fields. In the present study, we demonstrate that the PAC can be utilized for the identification of drug-resistance genes from parasites. Finally, we discuss the advantages of the method for identifying drug-resistance genes.

SummaryThe artificial chromosome represents a useful tool for gene transfer, both as cloning vectors and in chromosome biology research. To generate a Plasmodium artificial chromosome (PAC), we had to first functionally identify and characterize the parasite's centromere. A putative centromere (pbcen5) was cloned from chromosome 5 of the rodent parasite P. berghei based on a Plasmodium gene-synteny map. Plasmids containing pbcen5 were stably maintained in parasites during a blood-stage infection with high segregation efficiency, without drug pressure. pbcen5-containing plasmids were also stably maintained during parasite meiosis and mitosis in the mosquito. A linear PAC (L-PAC) was generated by integrating pbcen5 and telomere into a plasmid. The L-PAC segregated with a high efficiency and was stably maintained throughout the parasite's life cycle, as either one or two copies. These results suggest that L-PAC behaves like a Plasmodium chromosome, which can be exploited as an experimental research tool.

The malarial sporozoite is the stage that infects the liver, and genes expressed in this stage are potential targets for vaccine development. Here, we demonstrate that specific gene expression in this stage is regulated by an AP2-related transcription factor, designated AP2-Sp (APETALA2 in sporozoites), that is expressed from the late oocyst to the salivary gland sporozoite. Disruption of the AP2-Sp gene did not affect parasite replication in the erythrocyte but resulted in loss of sporozoite formation. The electrophoretic mobility-shift assay showed that the DNA-binding domain of AP2-Sp recognizes specific eight-base sequences, beginning with TGCATG, which are present in the proximal promoter region of all known sporozoite-specific genes. Promoter assays demonstrated that these sequences act as cis-acting elements and are critical for the expression of sporozoite-specific genes with different expression profiles. In transgenic parasites that express endogenous AP2-O (APETALA2 in ookinetes), but whose AP2 domain had been swapped with that of AP2-Sp, several target genes of AP2-Sp were induced in the ookinete stage. These results indicate that AP2-Sp is a major transcription factor that regulates gene expression in the sporozoite stage.

Problem statement: Mast cells develop from bone marrow-derived progenitor cells and are distributed in the skin or mucosa where they play proinflammatory roles in the first line of defense. Since some tumors in humans and experimental animals exhibited infiltration of increased mast cells, we investigated the contribution of mast cells to the override of tumor rejection. Approach: MRL/N-1 cells are malignant fibrous histiocytoma-like cells established from the spleen of a Fas ligand (FasL)-deficient MRL/Mp-FasLgld/gld (MRL/gld) mouse and are implantable in Fas-deficient MRL/Mp-Faslpr/lpr (MRL/lpr) mice. MRL/N-1 cells were implanted in MRL/gld, MRL/lpr and MRL/+mice after antibody treatments or with mast cells or macrophages and the tumor growth was observed. Results: MRL/N-1 cells were rejected by Fas-intact syngeneic MRL/+ mice in CD8+ T cell-mediated manner. This rejection was inhibited by the co-implanted mast cells. MRL/N-1 cells transfected with FasL were rejected by MRL/+ and MRL/gld mice. Conclusion: Mast cells abrogate the rejection of MRL/N-1 tumor cells and that this tumor rejection is mediated by CD8+ T cells and dependent on host Fas-FasL axis.

The cysteinyl leukotrienes (LTs), LTC(4), LTD(4), and LTE(4), are potent inflammatory mediators and are involved in allergic reactions, such as bronchoconstriction, eosinophilic inflammation, and allergic cell proliferation. The present study aimed to elucidate the role of constitutively produced cysteinyl LTs in mast cell activation. We used a newly developed quantification method based on mass spectrometry to detect cysteinyl LTs in the cultured medium of mouse bone marrow-derived mast cells (BMMCs), which were obtained by interleukin (IL)-3-conditioned culture of mouse bone marrow. BMMCs were stimulated with immunoglobulin (Ig) E and antigen (IgE/Ag) or lipopolysaccharide for 1 or 24 h. This new quantification method revealed that unstimulated BMMCs produced and secreted LTB4 and LTE4 after 24 h of incubation. The treatment of unstimulated BMMCs for 2 h with montelukast, an antagonist of a cysteinyl LT receptor, CysLT1, resulted in the suppression of a downstream signaling event of this receptor, i.e., the decrease in phosphorylation of extracellular responsive kinases. Thus, cysteinyl LTs constitutively simulate BMMCs through the CysLT1 receptor in an autocrine manner. Treatment of BMMCs for 3 weeks with montelukast, which caused long-term inhibition of the autocrine cyteinyl LT-derived signal, significantly attenuated the IgE/Ag-dependent degranulation, as judged by the decrease in the release of beta-hexosaminidase, an enzyme contained in the granules, whereas the production of cytokines, such as IL-6 and tumor necrosis factor-alpha, were largely unaffected. In conclusion, an autocrine signal derived from constitutively produced cysteinyl LTs predisposes mast cells to the degranulation upon allergic stimulation.

There has been increasing evidence for the involvement of fatty acid-binding proteins (FABPs) in the cytokine production of macrophages and dendritic cells probably through the control of cellular lipid metabolism and signal transduction. Since mast cells (MCs) are recently shown to be involved in immune response through modification of cytokine production, it is possible that some FABPs could also be involved in the immune response of MCs. In this study, we found that epidermal-type FABP (E-FABP) was expressed in murine bone marrow-derived MCs (BMMCs). Using BMMCs from genetically E-FABP-null mutated mice, we demonstrated that E-FABP in BMMCs plays a key role in the production of TNF-alpha following lipopolysaccharide (LPS) stimulation. In the in vivo septic peritonitis model (cecal ligation and puncture model), E-FABP-null mice showed a significantly increased mortality compared to wild-type mice. However, no significant difference in antigen-induced cytokine production was observed between wild-type and E-FABP-null BMMCs, and systemic anaphylaxis was equally induced in vivo in both wild-type and E-FABP-null mice. These results suggest that E-FABP is specifically involved in the LPS-induced cytokine production of MCs, and could play a role in the host-defense against bacterial infection, possibly through regulation of TNF-alpha production.

We have developed a method for the simultaneous estimation of the levels of the prostanoids 6-keto prostaglandin (PG) Flalpha, PGB2, PGD2, PGE2, PGF2(alpha), PGJ2, and thromboxane (TX) B2 in blood- or serum-containing medium using liquid chromatography-tandem mass spectrometry. These prostanoids and their deuterium derivatives, which were used as internal standards, were subjected to solid-phase extraction using Empore C18 HD disk cartridges and analyzed in the selected reaction-monitoring mode. A linear response curve starting at 10 pg of prostanoid/tube was observed for each prostanoid. The accuracy of the method was demonstrated with samples containing known amounts of the prostanoids. Furthermore, we used this method to analyze the prostanoids produced in mouse bone marrow-derived mast cells stimulated with arachidonic acid, which resulted in the production of PGD2, PGE2, PGF2alpha, and TXB2. The results suggest that this simultaneous quantification method is useful for the analysis of the production of biomedically important prostanoids.

Gametogenesis of Porphyra yezoensis thalli is induced by ageing as well as by changing water temperature and photoperiod. Under laboratory conditions, thalli cultivated at 10 °C with a 10:14 h (light: dark) cycle develop vegetatively to adult form without gametogenesis. On the other hand, sexual reproduction, which involves differentiation of vegetative cells and subsequent gametogenesis, is induced by culturing at 15 °C with a 16: 8 h (light: dark) cycle. We have constructed subtracted cDNA libraries enriched for differentially expressed transcripts in vegetative and reproductive thalli, and randomly selected 1,152 cDNAs from each subtracted library. Results of the dot blot analyses used for identification of differentially expressed cDNAs indicated that mRNA levels of 176 and 138 cDNAs tended to increase in the vegetative and reproductive thalli, respectively. BLAST analysis of nucleotide and deduced amino acid sequences showed that the cDNAs represented 63 and 59 unique clones for the vegetative and reproductive cDNA enriched subtracted libraries, respectively. Interestingly, some of the cDNAs isolated from the reproductive subtracted library were homologous to genes encoding protein kinases, GTP-binding protein, and heat shock proteins involved in signal transduction and the molecular chaperon system.

We evaluated renal functions by urinary biochemical parameters in normotensive pregnancy and preeclampsia. The parameters are expected to be altered resulting from different abnormalities of renal glomeruli and tubules. We chose N-acetyl-beta-d-glucosaminidase (NAG), beta2-microglobulin (beta2MG), total protein (TP), albumin (Alb), urea nitrogen (UN), uric acid (UA), and creatinine (Cr). Urinary excretion of these biochemical parameter concentrations (relative to Cr) was measured simultaneously in first morning fasting urine samples from 27 healthy nonpregnant women (group 1), 32 women with normotensive pregnancies (group 2), and 26 women with preeclampsia (group 3). The average gestational age at entry was 36 weeks. Serum UN and serum UA also were measured. All the ratios were significantly higher in group 2 than in group 1. The NAG-to-Cr, TP-to-Cr, and Alb-to-Cr ratios were significantly higher in group 3 than in group 2. In contrast, the UN-to-Cr and UA-to-Cr ratios were significantly lower in group 3 than in group 2. The percent increase in the beta2MG-to-Cr ratio in group 2 relative to that in group 1 was the highest, followed by percent increases in the NAG-to-Cr, TP-to-Cr, Alb-to-Cr, UA-to-Cr, and UN-to-Cr ratios. In contrast, the percent increase in the Alb-to-Cr ratio in group 3 relative to that in group 2 was the highest, followed by percent increases in the TP-to-Cr, NAG-to-Cr, beta2MG-to-Cr, UA-to-Cr, and UN-to-Cr ratios. The percent increases in the NAG-to-Cr and beta2MG-to-Cr ratios rose markedly in normotensive pregnancy, whereas percent increases of the Alb-to-Cr and TP-to-Cr ratios were far greater in preeclampsia than in normotensive pregnancy. Renal tubular damage and reabsorption dysfunction may be impaired markedly even in normotensive pregnancy, and further deterioration in reabsorption dysfunction may be slight in preeclampsia. Renal glomerular permeability of TP and Alb may be enhanced in normotensive pregnancy and markedly enhanced in preeclampsia.

Stage-specific transcription is a fundamental biological process in the life cycle of the Plasmodium parasite. Proteins containing the AP2 DNA-binding domain are responsible for stage-specific transcriptional regulation and belong to the only known family of transcription factors in Plasmodium parasites. Comprehensive identification of their target genes will advance our understanding of the molecular basis of stage-specific transcriptional regulation and stage-specific parasite development. AP2-O is an AP2 family transcription factor that is expressed in the mosquito midgut-invading stage, called the ookinete, and is essential for normal morphogenesis of this stage. In this study, we identified the genome-wide target genes of AP2-O by chromatin immunoprecipitation-sequencing and elucidate how this AP2 family transcription factor contributes to the formation of this motile stage. The analysis revealed that AP2-O binds specifically to the upstream genomic regions of more than 500 genes, suggesting that approximately 10% of the parasite genome is directly regulated by AP2-O. These genes are involved in distinct biological processes such as morphogenesis, locomotion, midgut penetration, protection against mosquito immunity and preparation for subsequent oocyst development. This direct and global regulation by AP2-O provides a model for gene regulation in Plasmodium parasites and may explain how these parasites manage to control their complex life cycle using a small number of sequence-specific AP2 transcription factors., Author Summary Although malarial parasites have a complex life cycle, they harbor only 30 transcription factors in their genome. The majority of these transcription factors belong to a single family referred to as the AP2 family. Our previous study suggested that stage-specific AP2 family transcription factors have critical roles in maintaining the Plasmodium parasite life cycle. However, it remains fairly elusive as to how these transcription factors regulate each stage. AP2-O is an AP2 family transcription factor that is expressed during the mosquito midgut-invading stage, the ookinete, and is essential for normal development of this stage. In the present study, we identified the entire set of AP2-O target genes to elucidate how this AP2 family transcription factor contributes to the formation of this stage. Our results showed that AP2-O directly regulates 10% of the parasite genome and is involved in the whole process of mosquito midgut-invasion by ookinetes. The global and comprehensive regulation by the AP2 family transcription factor that we revealed provides a model for transcriptional regulation of this parasite and may explain how malarial parasites regulate their complex life cycle using a small number of sequence-specific transcription factors.

Liver-stage malaria parasites are a promising target for drugs and vaccines against malaria infection. However, little is currently known about gene regulation in this stage. In this study, we used the rodent malaria parasite Plasmodium berghei and showed that an AP2-family transcription factor, designated AP2-L, plays a critical role in the liver-stage development of the parasite. AP2-L-depleted parasites proliferated normally in blood and in mosquitoes. However, the ability of these parasites to infect the liver was approximately 10,000 times lower than that of wild-type parasites. In vitro assays showed that the sporozoites of these parasites invaded hepatocytes normally but that their development stopped in the middle of the liver schizont stage. Expression profiling using transgenic P. berghei showed that fluorescent protein-tagged AP2-L increased rapidly during the liver schizont stage but suddenly disappeared with the formation of the mature liver schizont. DNA microarray analysis showed that the expression of several genes, including those of parasitophorous vacuole membrane proteins, was significantly decreased in the early liver stage of AP2-L-depleted parasites. Investigation of the targets of this transcription factor should greatly promote the exploration of liver-stage antigens and the elucidation of the mechanisms of hepatocyte infection by malaria parasites.

Concanavalin A (Con A)-induced hepatitis is a T-cell-mediated murine experimental model of autoimmune hepatitis. Mice lacking Valpha14 NKT cells were found to be less sensitive to this hepatitis and the MRL/Mp-Fas(lpr/lpr) (MRL/lpr; i.e. Fas deficient) mice were also less sensitive. We report herein that MRL/Mp-Fas(lpr/lpr)-Sap(rpl/-) (MRL/lpr/rpl) mice lack Valpha14 NKT cells and are deficient in the Fas antigen but sensitive to Con A-induced hepatitis. The signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) is an adaptor molecule containing a Src homology 2 (SH2) domain. We previously reported new mutant mice found among MRL/lpr mice and revealed that SAP deficiency led to the regression of autoimmune phenotypes in mutant MRL/lpr/rpl mice. It was also revealed that CD4(+) and CD8(+) T cells were effector cells and that blockade of 2B4, one of the SLAM family receptors, inhibited the induction of hepatitis in MRL/lpr/rpl mice. These data suggest that signals mediated by molecules other than SAP from 2B4 in T cells played important roles in the induction of hepatitis in MRL/lpr/rpl mice.

There is an increasing body of evidence that prostanoids modulate mast cell functions and contribute to the development of allergic inflammation. The present study aimed to identify an undetermined function of prostaglandin (PG) F(2alpha) in mast cell activation and the signaling mechanism involved in it. Simultaneous quantification of prostanoids by liquid chromatography/tandem mass spectrometry revealed the constitutive release of PGF(2alpha), thromboxane B(2), and 6-keto-PGF(1alpha) from bone marrow-derived mast cells (BMMCs). Upon activation of BMMCs by lipopolysaccharide, the cytokine production in BMMCs was enhanced when the culture was supplemented with PGF(2alpha). However, F prostanoid receptor-a selective receptor for PGF(2alpha)-was not detected in BMMCs. Further investigations performed using prostanoid receptor antagonists revealed an alternative mechanism wherein the receptors for PGE species-E prostanoid receptors-mediated the PGF(2alpha) signal in BMMCs. The present study provides an insight into a novel function of PGF(2alpha), i.e., an autocrine accelerator for mast cell activation.

The introduction of transgenes into Plasmodium falciparum, a highly virulent human malaria parasite, has been conducted either by single crossover recombination or by using episomal plasmids. However, these techniques remain insufficient because of the low transfection efficiency and the low frequency of recombination. To improve the genetic manipulation of P. falciparum, we developed the centromere plasmid as a new genetic tool. First, we attempted to clone all of the predicted centromeres from P. falciparum into E. coli cells but failed because of the high A/T contents of these sequences. To overcome this difficulty, we identified the common sequence features of the centromere of Plasmodium spp. and designed a small centromere that retained those features. The centromere plasmid constructed with the small centromere sequence, pFCEN, segregated into daughter parasites with approximately 99% efficiency, resulting in the stable maintenance of this plasmid in P. falciparum even in the absence of drug selection. This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum. In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence. Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions. These unique properties of the pFCEN plasmid will solve the current technical limitations of the genetic manipulation of P. falciparum, and thus, this plasmid will become a standard genetic tool for the study of this parasite.

喀痰細胞診により発見し切除した, 気管支原発早期扁平上皮癌25例の喀痰細胞所見について検討した. 全例胸部X線写真上無所見で, 4例は上皮内癌, 21例は浸潤が気管支壁内に限局していた.浸潤の著明な11例, および喀痰細胞診疑陽性で扁平上皮癌の発見されない境界症例12例と所見を対比し, 計量可能な所見については数量的に扱った.その結果, 早期扁平上皮癌においては, 異様な形の扁平上皮癌細胞は少なく, 細胞異型は概して軽度で, 扁平上皮化生細胞と鑑別困難な異型細胞も少なからず出現していた. 全体として, 小型類円型のオレンジGに好染するN/C比のやや高いものから, 多辺形でやや大型のオレンジGに淡染するN/C比の低いものまで, 角化した異型細胞が優勢で, ライトグリーン好性のいわゆる旁基底型細胞は少なかった. また, 多核細胞の出現が目立った.これらの細胞所見に影響を与える因子として, 表層へ向かっての細胞の分化・成熟傾向と, 表面における一部の細胞の変性・膨化・扁平化, および異常な細胞分裂が重要であると考えた.