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Objectives As the prognosis of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains poor, novel treatment strategies are urgently needed. Clinical trials have shown that chimeric antigen receptor (CAR)‐T cells for AML are more challenging than those targeting CD19 in B‐cell malignancies. We recently developed piggyBac‐modified ligand‐based CAR‐T cells that target CD116/CD131 complexes, also known as the GM‐CSF receptor (GMR), for the treatment of juvenile myelomonocytic leukaemia. This study therefore aimed to develop a novel therapeutic method for R/R AML using GMR CAR‐T cells. Methods To further improve the efficacy of the original GMR CAR‐T cells, we have developed novel GMR CAR vectors incorporating a mutated GM‐CSF for the antigen‐binding domain and G4S spacer. All GMR CAR‐T cells were generated using a piggyBac‐based gene transfer system. The anti‐tumor effect of GMR CAR‐T cells was tested in mouse AML xenograft models. Results Nearly 80% of the AML cells predominant in myelomonocytic leukaemia were found to express CD116. GMR CAR‐T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. Furthermore, GMR CAR‐T cells incorporating a G4S spacer significantly improved long‐term in vitro and in vivo anti‐tumor effects. By employing a mutated GM‐CSF at residue 21 (E21K), the anti‐tumor effects of GMR CAR‐T cells were also improved especially in long‐term in vitro settings. Although GMR CAR‐T cells exerted cytotoxic effects on normal monocytes, their lethality on normal neutrophils, T cells, B cells and NK cells was minimal. Conclusions GMR CAR‐T cell therapy represents a promising strategy for CD116+ R/R AML., This study introduces a new CAR‐T therapy for acute myeloid leukaemia (AML) that targets the GM‐CSF receptor complex. GM‐CSF receptors are a novel target for AML treatment in the CAR‐T field. Furthermore, by modifying the spacer (G4S) and employing a mutated GM‐CSF construct (E21K), we could remarkably improve the anti‐tumor effects of CAR‐T cells in multiple mouse xenograft models.

Background: The prognosis of relapsed/refractory (R/R) myeloid malignancies remains poor, and the development of novel treatment strategies is crucial. Although chimeric antigen receptor (CAR)-modified T cell therapy for B cell malignancies has shown excellent clinical efficacy, the use of CAR-T cells for myeloid malignancies has been more challenging, partly due to the heterogeneous expression of candidate target antigens in leukemia cells and shared expression of those antigens in normal myeloid cells or progenitor cells. We previously developed the piggyBac-modified chimeric antigen receptor (CAR)-T cells targeting CD116, also known as GM-CSF receptor alpha chain (GMR) (Nakazawa Y, et al. J Hematol Oncol. 2016 and Hasegawa A, et al. Clin Transl Immunology. 2021). GMR CAR-T cells showed substantial antitumor effects against both acute myeloid leukemia and juvenile myelomonocytic leukemia. Moreover, modulation of the spacer and antigen recognition site of the CAR vector further enhanced the anti-tumor effects of GMR CAR-T cells. GMR CAR-T cells showed an acceptable safety profile with limited cytotoxicity on normal hematopoietic cells except monocytes. Based on these results, we have initiated a first-in-human clinical trial of GMR CAR-T cell therapy in March 2021 in Japan. Study Design and Methods: The study is a phase I/II, single-center, dose-escalation study with a traditional 3+3 dose-escalation design (Table 1). Maximum of 18 patients will be recruited. Primary objectives of this study are to determine the safety and severe adverse events of piggyBac-modified GMR CAR-T cells for CD116 + relapsed/refractory myeloid malignancies by assessing the dose-limiting toxicity within 28 days from the single infusion of GMR CAR-T cells. The patients with CD116 + myeloid malignancies aged more than 1 year with myeloid malignancies who experienced an induction failure or a relapse after hematopoietic stem cell transplantation (HSCT) will be recruited. CD116 is defined as positive when a CD116 relative mean fluorescence (RFI) in leukemia cells ≥ 2. RFI was calculated by dividing the MFI of samples with that of the isotype control. Major exclusion criteria are as follows, acute promyelocytic leukemia, acute graft-versus-host disease (GVHD) (Grade ≥ 2), extensive chronic GVHD, and concurrent treatment with corticosteroid (≥ 6 mg/m2). Statistical analysis will be performed when the data will be fixed. Peripheral blood mononuclear cells will be harvested from the patient by leukapheresis and then will be transduced with GMR CAR vector by piggyBac transposon system. All manufacturing process of GMR CAR-T cells is performed in Cell Processing Center in Shinshu University Hospital under good manufacturing practice conditions. The patient will be treated with lymphodepleting chemotherapy consisting of fludarabine and cyclophosphamide, followed by CAR-T cell infusion. The dose of CAR-T cells will be 3 x 10 5 and 1 x 10 6 in cohorts 1 & 2 and cohort 3, respectively (Table 1). Kinetics of GMR CAR-T cells will be determined by quantifying the GMR CAR gene in the peripheral blood using real-time PCR after the CAR-T cell infusion. All the patients will be required to receive HSCT by day 56 following CAR-T cell infusion. The primary endpoint of the study is the safety, pharmacokinetics, and efficacy of GMR CAR-T therapy (Trial registration: jRCT2033210029). Conclusion: We herein described the protocol of first-in-human GMR CAR-T cells for relapsed/refractory myeloid malignancies. By employing the optimized CAR vector and production protocol, the safety and efficacy of GMR CAR-T cells will be evaluated in this study. Figure 1 Figure 1. Disclosures Saito: Toshiba corporation: Research Funding. Yagyu: AGC Inc.: Research Funding. Nakazawa: AGC Inc.: Research Funding; Toshiba Corporation: Research Funding.

Background: The prognosis of relapsed/refractory (R/R) acute myeloid leukemia (AML) remains poor; therefore, novel treatment strategies are required urgently. Meanwhile, recent clinical trials have demonstrated that CAR-T cells for AML have been less successful than those targeting CD19 for B cell malignancies. Recently, we developed piggyBac-modified ligand-based CAR-T cells that target CD116, also called granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) α chain, for treating juvenile myelomonocytic leukemia (Nakazawa, et al. J Hematol Oncol. 2016). Since CD116 is overexpressed in 60%-80% of AML cases, the present study aimed to develop a novel therapeutic method for R/R AML using GMR CAR-T cells. Methods: CD116 expression in AML cell lines or primary leukemia cells were examined using flow cytometry. The original piggyBac transposon plasmid for GMR CAR comprises GM-CSF as an antigen recognition site, IgG1 CH2CH3 hinge region, CD28 costimulatory domain, and CD3ζ chain. To improve the in vivo persistency and anti-tumor effects, two types of spacer (∆CH2H3 and G4S) that lack CH2CH3 lesion were newly constructed. In order to modulate the antigen recognition ability, mutated ligand-based GMR CAR vectors were constructed with a mutation at residue 21 of GM-CSF that is reported to play a critical role in its biological activity (Lopez, et al. Embo j. 1992). All the GMR CAR-T cells were generated with piggyBac gene modification. To investigate the in vitro anti-tumor activity, GMR CAR-T cells were co-cultured with AML cell lines. In order to evaluate the in vivo anti-tumor effects, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were intravenously injected with THP-1, THP1-ffLuc, or MV4-11 and then treated with GMR CAR-T cells. To characterize the safety profile of GMR CAR-T cells, peripheral blood mononuclear cells or polymorphonuclear cells were co-cultured with GMR CAR-T cells at an effector:target ratio of 1:1 for 3 days. Thereafter, B cells, NK cells, neutrophils, and monocytes were quantified using flow cytometry using counting beads. Results: Approximately 80% of the AML cells predominant in myelomonocytic leukemia expressed CD116. PiggyBac-modified GMR CAR-T cells displayed a favorable CD45RA+CCR7+-dominant phenotype, consistent with our previous findings. GMR CAR-T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. GMR CAR-T cells incorporating a G4S spacer significantly improved the long-term in vitro and in vivo anti-tumor effects as compared to those incorporating a ∆CH2CH3 spacer. Furthermore, by employing a mutated GM-CSF at residue 21 (E21K and E21R) as an antigen recognition site, the in vivo anti-tumor effects were also substantially improved along with prolonged survival (Figure 1) over controls (PBS or CD19.CAR-T cells) (all, p < 0.01) as well as over GMR CAR-T cells with a wild-type GM-CSF ligand (E21R: p < 0.01; E21K: p = 0.02), with 4 out of 5 mice surviving for > 150 days. Safety tests revealed that the toxicity of GMR CAR-T cells was restricted to normal monocytes. It is noteworthy that the cytotoxic effects of GMR CAR-T cells on normal neutrophils, T cells, B cells, and NK cells were minimal. Conclusions: GMR CAR-T cell therapy appears to be a potentially useful strategy for CD116+ R/R AML. Based on the promising results, we plan to perform the first-in-human clinical trial of GMR CAR-T cells. Disclosures Saito: Toshiba Corporation: Research Funding. Hasegawa:Toshiba Corporation: Research Funding. Inada:Kissei Pharmaceuticals: Ended employment in the past 24 months. Nakashima:Toshiba Corporation: Research Funding. Yagyu:Toshiba Corporation: Research Funding. Nakazawa:Toshiba Corporation: Research Funding.

SUMMARY Under a nonuniform field in an atmospheric short gap, a branched pulse streamer corona changes to a single filamentary discharge (SFD) for a split second just before sparkover occurs. This SFD is a pretransitional phenomenon of sparkover. Therefore, it is very important to study SFD for an understanding of the sparkover mechanism. In this work, the SFD was recreated well by applying a highly repetitive impulse voltage to a needle-plane gap in synthetic air with dilute CO2. The characteristics of the SFD, including the channel temperature, the propagation of the streamer head, and the formation process, were investigated by means of an intensified charge coupled-device (ICCD) camera and a spectroscopic measurement system. It was found that the transition to SFD progresses with localized gas heating along the discharge channel. Furthermore, it was revealed that the SFD is a nonbranched positive streamer.

The antihypertensive effect of dipotassium (Z)-2-[[5-ethyl-3-[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl - -1,3,4- thiadiazoline- 2 - ylidene] aminocarbonyl] - I - cyclopentenecarboxylate (CAS 169328-25-0, KRH-594), a new angiotensin II type 1 (AT 1 ) receptor antagonist, was studied in several experimental hypertensive models. The effects of KRH-594 on the circulating renin-angiotensin system and on renal function were also investigated. Oral administration of KRH-594 (0.3 or I mg/kg) dose-dependently inhibited the angiotensin II-induced pressor response in common marmosets. KRH-594 (1, 3, or 10 mg/kg p.o,) dose-dependently exerted a long-lasting antihypertensive effect in spontaneously hypertensive rats (SHRs) and in 2-kidney 1-clip renal hypertensive rats (RHRs). Furthermore, repeated oral administration of KRH-594 (3 or 10 mg/kg/d) reduced blood pressure dose-dependently in SHRs, RHRs, and renal hypertensive dogs without tachycardia and with no evidence of a rebound phenomenon following drug withdrawal. On the other hand, in deoxycorticosterone acetate salt rats and normotensive rats, KRH-594 (10 or 30 mg/kg p.o.) did not have significant effects on systolic blood pressure. In SHRs, KRH-594 (3 or 10 mg/kg/d p.o. for 2 weeks) dose-dependently increased both plasma renin activity and the plasma angiotcnsin I concentration, but had no effect on the urinary excretion of sodium, potassium, and chloride or on creatinine clearance. These results suggest that KRH-594 should be effective in patients with essential or renal hypertension.

We evaluated the effects of bezafibrate, a peroxisome proliferator-activated receptor (PPAR) pan-agonist, and GW501516, a PPARdelta agonist, on mice fed a methionine- and choline-deficient (MCD) diet, a model of non-alcholic steatohepatitis (NASH), to investigate (a) the efficacy of bezafibrate against non-alcholic steatohepatitis and (b) the relation between non-alcholic steatohepatitis and the functional role of PPARdelta. Bezafibrate (50 or 100 mg/kg/day) and GW501516 (10 mg/kg/day) were administered by gavage once a day for 5 weeks. Hepatic lipid contents, plasma triglyceride, high density lipoprotein (HDL)-cholesterol and alanine aminotransferase (ALT) concentrations were evaluated, as were histopathological changes in the liver and hepatic mRNA expression levels. Bezafibrate and GW501516 inhibited the MCD-diet-induced elevations of hepatic triglyceride and thiobarbituric acid-reactants contents and the histopathological increases in fatty droplets within hepatocytes, liver inflammation and number of activated hepatic stellate cells. In this model, bezafibrate and GW501516 increased the levels of hepatic mRNAs associated with fatty acid beta-oxidation [acyl-CoA oxidase (ACO), carnitine palmitoyltransferase-1 (CPT-1), liver-fatty acid binding protein (L-FABP) and peroxisomal ketothiolase], and reduced the levels of those associated with inflammatory cytokines or chemokine [transforming growth factor (TGF)-beta1, interleukin (IL)-6, IL-1beta, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF) alpha and nuclear factor (NF)-kappaB1]. In addition, bezafibrate characteristically reduced the elevation in the level of plasma ALT, but enhanced that in plasma adiponectin and increased the mRNA expression levels of its receptors (adiponectin receptors 1 and 2). These results suggest that (a) bezafibrate (especially) and GW501516 might improve hepatic steatosis via an improvement in fatty acid beta-oxidation and a direct prevention of inflammation, (b) treatment with a PPARdelta agonist might improve non-alcholic steatohepatitis, (c) bezafibrate may improve non-alcholic steatohepatitis via activation not only of PPARalpha but also of PPARdelta, because bezafibrate is a PPAR pan-agonist.

An in vitro system for liver organogenesis from murine embryonic stem (ES) cells has been recently established. This system is expected to be applied to the development of a new drug metabolism assay system that uses ES cells as a substitute for animal experiments. The objective of this study was to elucidate the drug metabolism profiles of the murine ES cell-derived hepatic tissue system compared with those of primary cultures of murine adult and fetal hepatocytes. The expression of the genes of the cytochrome P450 (P450) family, such as Cyp2a5, Cyp2b10, Cyp2c29, Cyp2d9, Cyp3a11, and Cyp7a1, was observed in the murine ES cell-derived hepatic tissue system at 16 days and 18 days after plating (A16 and A18). To investigate the activities of these P450 family enzymes in the murine ES cell-derived hepatic tissue system at A16 and A18, testosterone metabolism in this system was analyzed. Testosterone was hydroxylated to 6beta-hydroxytestosterone (6beta-OHT), 16alpha-OHT, 2alpha-OHT, and 2beta-OHT in this system, and was not hydroxylated to 15alpha-OHT, 7alpha-OHT, and 16beta-OHT. This metabolism profile was similar to that of fetal hepatocytes and different from that of adult hepatocytes. Furthermore, pretreatment with phenobarbital resulted in a 2.5- and 2.6-fold increase in the production of 6beta-OHT and 16beta-OHT. Thus, evidence for drug metabolic activities in relation to P450s has been demonstrated in this system. These results in this system would be a stepping stone of the research on the development and differentiation to adult liver.

Fibrates, including bezafibrate (BF), upregulate the expression of ATP binding cassette protein B4 (ABCB4) through gene transcription in mice. To determine the effects of BF on the expression levels of ABCB4 and on the stimulation of biliary phosphatidylcholine (PC) transport in human HepG2 hepatoblastoma cells, mRNA and protein levels as well as subcellular localization were investigated in the cells treated with BF. The canalicular accumulation of a fluorescent PC was assessed by confocal laser scanning microscopy. Treatment with 300 micromol/l BF for 24 h increased levels of ABCB4 mRNA but not protein by up to 151%. BF caused redistribution of ABCB4 into pseudocanaliculi formed between cells. In association with this redistribution, BF accelerated the accumulation of fluorescent PC in bile canaliculi (up to 163% of that in nontreated cells). Suppression of peroxisome proliferator-activated receptor alpha (PPARalpha) expression by either a small interfering RNA duplex or morpholino antisense oligonucleotide attenuated the BF-induced redistribution of ABCB4. These findings suggest that BF may enhance the capacity of human hepatocytes to direct PC into bile canaliculi via PPARalpha-mediated redistribution of ABCB4 to the canalicular membrane. This provides a rationale for the use of BF to improve cholestasis and/or cholangitis that is attributable to hypofunction of ABCB4.

We isolated a novel leucine-rich repeat protein (LRRP) cDNA from E13 mouse embryos by the in silico approach. The cDNA encoded a protein of 274 amino acids having 7 leucine-rich repeat motifs at the center of the protein. An in vitro transcription/translation study showed that the cDNA coded for a peptide of approximately 31 kDa. Northern blot analysis suggested that the mRNA of this novel LRRP was expressed only in the heart, although RT-PCR indicated slight expression in skeletal muscle as well. The transcripts of this gene and Nkx-2.5/Csx were detected in the early stage of cardiac differentiation of P19CL6 embryonal carcinoma cells treated with 1% dimethyl sulfoxide. The fusion protein made between it and GFP was detected at a high level in mitochondria and a low level in the nuclei of COS7 cells. The nuclei of the adult mouse heart were strongly stained with the antibody raised against the synthetic peptide of the protein. Therefore, we designated the gene as heart-restricted leucine-rich repeat protein (HRLRRP) and assume that mouse HRLRRP may play important roles in cardiac development and/or cardiac function.

SUMMARY 1. The pacemaker site is known to shift away from the sinoatrial (SA) node in response to autonomic stimuli. 2. To test whether the shifting of the impulse-initiation site that occurs during autonomic nerve stimulation can be explained by the spatial distributions of β-adrenoceptor and muscarinic receptors, we obtained densities (Bmax) and dissociation constants (Kd) for these receptors in three regions along the sulcus terminalis (the SA node itself and regions superior and inferior to it) and a region of the right atrial appendage in the dog. 3. The Bmax values for [125I]-iodocyanopindolol binding to β-adrenoceptors were not different among the four regions. The Bmax value for [3H]-quinuclidinyl benzilate binding to muscarinic receptors was significantly higher in the SA node area than in any other region (P < 0.01), although there was no difference among the other three regions. 4. For each receptor, Kd values were similar among the four regions. 5. These results suggest that spatial variations in the densities of β-adrenoceptors and muscarinic receptors are not important for shifting the impulse-initiation site and that other factors, such as non-uniform innervation by autonomic nerve fibres, may be mainly responsible for the pacemaker shift induced by autonomic nerve stimulation.

We investigated the relationship between the menstrual cycle and hormone levels in cynomolgus monkeys, and developed a sulpiride-induced hyperprolactinemic anovulation model. On this study, we demonstrated the usefulness of the commercial human prolactin immunoradiometric assay kit for the measurement of cynomolgus monkey serum samples. In the normal menstrual cycle of the cynomolgus monkey, serum prolactin concentrations were not significantly different between luteal and follicular phases. However, the serum prolactin concentration tended to elevate at the ovulation stage. And serum progesterone began to increase after an estradiol surge, and then declined before the ensuing preovulatory rise in estradiol. During the luteal phase, the serum concentration of progesterone was elevated. Moreover, we aimed to develop an anovulation model, using sulpiride-induced hyperprolactinemia in the cynomolgus monkey. The serum prolactin level gradually increased during the twice-daily administration of sulpiride, and the drug produced as big a response at 5 mg/kg. In this study, the length of the menstrual cycle was approximately 29 days in normal cynomolgus monkeys. When treatment with sulpiride had been continued for more than one month, serum progesterone and estradiol levels fell to within the range seen in the follicular phase of the normal cycle, and the absence of ovulation was recognized by laparoscopy. Moreover, in this period we found that amenorrhea or anovulatory menstruation in the experimental animals. We could produce an anovulatory model induced by sulpiride repeatedly administered over a long time period. Our findings suggest that the cynomolgus monkey is useful as a endocrinological model that uses prolactin as a parameter and as an anovulatory model; thus, it could be a useful model for the hyperprolactinemic amenorrhea and/or anovulation seen in humans.

Complementary DNA of mouse deafness dystonia peptide 1 (DDP1) was isolated from adipocyte cDNA library and expressed in mammalian cells. The sequence shares homology of 92 and 97% on the nucleic acid and the amino acid levels with human DDP1. In comparison to mouse Bruton's tyrosine kinase (Btk) locus, the coding region spans 2 exons and the splice point is the same as human DDP1. Northern blot analysis suggests that mouse DDP1 expresses ubiquitously. In vitro transcription/translation study showed that the cDNA of mouse DDP1 codes about 11 kDa peptide. DDP1 tagged with FLAG localized in mitochondria and cytoplasm of COS7 cells. P19 embryonal carcinoma cells transfected with anti sense DDP1 cDNA were frequently dead after subculture and all the survivals expressed endogenous DDP1 mRNA. Therefore, mouse DDP1 may play an important role to survive in contrast to Tim8p, a yeast homologue, which was unnecessary in yeast.

We aimed to develop an anovulation model, using sulpiride-induced hyperprolactinemia in common marmosets. The serum prolactin level gradually increased during the twice-daily administration of sulpiride and reached a plateau after 4 days. Sulpiride produced as big a response at 10 mg kg −1 as at 50 mg kg −1 . In this study, the length of the ovarian cycle was approximately 30 days in normal common marmosets. Serum progesterone and estradiol levels showed no consistent change during the first 2 months of treatment with sulpiride. When treatment with sulpiride had been continued for more than 2 months, serum progesterone and estradiol levels fell to within the range seen in the follicular phase of the normal cycle and absence of ovulation was recognized by laparoscopy. A single oral administration of cabergoline (at doses between 0.01 and 0.1 mg kg −1 ) dose dependently reduced the elevated serum prolactin level. Bromocriptine (at an oral dose of 10 mg kg −1 ) also reduced the serum prolactin level at 4 and 8 h after its administration. With bromocriptine, the prolactin level had recovered at 24 h, but with cabergoline at doses of 0.05 mg kg −1 or more, it had still not recovered at 48 h. In anovulatory common marmosets, oral administration of cabergoline at a daily dose of 0.05 mg kg −1 restored ovarian function and resulted in ovulation in 100% of the group (following a reduction in the serum prolactin level). Bromocriptine at a daily oral dose of 10 mg kg −1 resulted in ovulation in 67% of the group, but this dose was about 200 times higher than the dose of cabergoline. We could produce an anovulatory model induced by sulpiride repeatedly administered over a long time period. It is suggested that, in this anovulatory model in common marmosets, cabergoline has a potent and long-lasting action as a dopamine D 2 receptor agonist, and thus could be a useful drug for the treatment of galactorrhea and hyperprolactinemic amenorrhea and/or anovulation.

A thermoelectric device consisting of porous and bulk parts was proposed. Conception, generated power and effectiveness of the device are discussed in comparison with the conventional bulk one. Heat exchange between the device and heating/cooling fluids occurs by using a porous medium. The partially porous device enhances generated power per unit area, although porous medium increases electric resistivity of the partially porous device. The partially porous device will be of advantage when gas is used as heating/cooling sources because of its low thermal conductivity. An estimation based on physical properties of FeSi 2 suggests that generated power of the partially porous device can be several times higher than that of the bulk one in the case of gas heating/cooling system.

Influences of gas heating and product molecules on discharge characteristics in dry-air-fed ozonizer was investigated. Especially, the dependence of gas temperature in streamer channel on occurrence frequency of streamer discharge was investigated in detail. In the experiment, repetitive positive or negative impulse streamer was generated respectively between needle-plane electrodes in synthetic air at atmospheric pressure. The repetition rate of the applied pulse voltage was controlled from 0.1 to 7.0 kilo-pulse/sec (kpps). The temperature of streamer channel was estimated from the emission spectrum of N2 2nd positive system band (0-0). As a result, the gas temperature increased with increasing the pulse repetition rate in both polarities. In the case of positive polarity, the gas temperature increased from 341 to 383 K in response to the increase of repetition rate (0.1 to 6.0 kpps). Eventually, sparkover occurred at 7.0 kpps due to the increase of reduced electric field (E/N) caused by gas heating. The product ozone concentration was saturated with increasing the pulse repetition.

A homologous double-antibody radioimmunoassay developed for humans was used to measure serum prolactin, progesterone, and estradiol in common marmosets. In the ovarian cycle of common marmosets, serum progesterone began to increase after an estradiol surge, attained a peak level, and then declined before the ensuing pre-ovulatory rise in estradiol. During the luteal phase, the change in serum concentrations of estradiol was synchronized with that of progesterone. During the ovarian cycle there was no consistent change in serum prolactin concentrations. During the last 75 days of pregnancy the prolactin level was higher than during the ovarian cycle and the first 70 days of pregnancy. Moreover, during lactation, mothers with suckling twin infants had a higher prolactin level than during the final stage of pregnancy.

In order to elucidate the structure-function relationship of RNases belonging to the RNase T2 family (base non-specific and adenylic acid-preferential RNase), an RNase of this family was purified from Trichoderma viride (RNase Trv) to give three closely adjacent bands with RNase activity on slab-gel electrophoresis in a yield of 20%. The three RNases gave single band with the same mobility on slab-gel electrophoresis after endoglycosidase F digestion. The enzymatic properties including base specificity of RNase Trv were very similar to those of typical T2-family RNases such as RNase T2 from Aspergillus oryzae and RNase M from A. saitoi. The specific activity of RNase Trv towards yeast RNA was about 13-fold higher than that of RNase M. The complete primary structure of RNase Trv was determined by analyses of the peptides generated by digestion of reduced and carboxymethylated RNase Trv with Staphylococcus aureus V8 protease, lysylendopeptidase and alpha-chymotrypsin. The molecular weight of the protein moiety deduced from the sequence was 25,883. The locations of 10 half-cystine residues were almost superimposable upon those of other RNases of this family. The homologies between RNase Trv and RNase T2, RNase M, and RNase Rh (Rhizopus niveus) were 124, 132, and 92 residues, respectively. The sequences around three histidine residues, His52, His109, and His114, were highly conserved in these 4 RNases.

Aim The aim of this study was to investigate the preventive actions of bezafibrate against non-alcoholic steatohepatitis (NASH), the activation of hepatic stellate cells (HSC), and fibrogenesis by using a model of NASH and an in vitro model. Methods Male KK-A(y)/TaJcl (KK-A(y)) mice were fed a methionine and choline-deficient (MCD) diet or a MCD diet containing bezafibrate or pioglitazone for 7 weeks, after which biochemical parameters, pathological changes, and hepatic mRNA levels were assessed. An in vitro HSC model was designed by using a previously described RI-T cell line stimulated by transforming growth factor-beta1 (TGF-beta1). Results MCD diet-fed KK-A(y) mice developed hepatic steatosis, oxidative stress, inflammation, and hepatic fibrosis. Bezafibrate markedly decreased the hepatic content of triglyceride accumulation of fatty droplets within hepatocytes, and increased the expression of hepatic fatty acid beta-oxidative genes in MCD diet-fed KK-A(y) mice. Bezafibrate markedly inhibited the increases in the plasma alanine aminotransferase level and hepatic content of thiobarbituric acid-reactive substances in this model. Moreover, it dramatically reduced hepatic inflammatory changes and fibrosis concomitantly with marked reductions in the mRNA levels for inflammatory cytokine, chemokine, and profibrogenic genes. Importantly, both bezafibrate and pioglitazone markedly reduced the mRNA levels of profibrogenic and fibrogenic genes in TGF-beta1-stimulated cells. Conclusion Bezafibrate improved hepatic steatosis and potently prevented inflammation, oxidative stress, HSC activation, and fibrogenesis in the liver. Moreover, this study was the first to demonstrate that bezafibrate directly inhibits hepatic fibrogenic response induced by TGF-beta1 in vitro. Hence bezafibrate may be a new therapeutic strategy against NASH and hepatic fibrosis.

OBJECTIVE. The purpose of this study was to use synchrotron radiation imaging with 6-μm resolution to evaluate amorphous and pleomorphic breast tissue microcalcifications.CONCLUSION. Synchrotron radiation imaging depicted microcalcifications as small as 24 μm. Imaging with this technique revealed that most amorphous and pleomorphic calcifications on conventional mammograms are clusters of fine specks and that in addition to the shape or density of a speck, the distribution density of clustered specks is a factor determining the apparent shape.

Aim and Methods: A decreased function of multidrug-resistance 3 P-glycoprotein (MDR3), limiting the rate of biliary phospholipid secretion, predisposes individuals to cholestasis and/or cholangitis. Fibrates induce the expression of mdr2 (homolog of human MDR3) in rodents. To investigate the effects of bezafibrate (BF) on the expression levels of MDR3 in cultured human hepatocytes and human livers, the amount of protein and subcellular localization of MDR3 was assessed in HepG2 cells treated with BF and humanized livers of BF-treated chimeric mice. Results: In HepG2 cells, while treatment with BF did not increase the protein levels of MDR3, the treatment caused a redistribution of MDR3 in the bile canaliculi. In humanized livers of chimeric mice, oral administration of BF induced a large increase in the protein amount of MDR3 and its redistribution in the bile canaliculi. Moreover, the modulatory effects of BF on key factors involved in hepatic cholesterol and bile acid metabolism in human subjects were traced in the humanized livers of BF-treated chimeric mice. Conclusion: BF causes an induction of MDR3 expression in human livers. This provides a rationale for the beneficial role of BF in improving cholestasis and/or cholangitis associated with defective MDR3 expression and function in various types of cholestatic hepatobiliary diseases.

We characterized the alpha(1)-adrenoceptor subtypes in hamster ureters according to gene and protein expressions and contractile function. Real-time quantitative reverse-transcription polymerase chain reaction and immunohistochemical analysis were performed to determine mRNA levels and receptor protein expressions respectively, for alpha(1A)-, alpha(1B)- and alpha(1D)-adrenoceptors in hamster ureteral smooth muscle. alpha(1)-Adrenoceptor antagonists were tested against the phenylephrine (alpha(1)-adrenoceptor agonist)-induced contraction in isolated hamster ureteral preparations using a functional experimental approach. In the smooth muscle, relative mRNA expression levels for alpha(1a)-, alpha(1b)- and alpha(1d)-adrenoceptors were 10.7%, 1.2% and 88.1%, respectively, and protein expressions were identified for alpha(1A)- and alpha(1D)-adrenoceptors immunohistochemically. Noradrenaline and phenylephrine (alpha(1)-adrenoceptor agonist) each produced a concentration-dependent tonic contraction, their pD(2) values being 6.87+/-0.08 and 6.10+/-0.05, respectively. Prazosin (nonselective alpha(1)-adrenoceptor antagonist), silodosin (selective alpha(1A)-adrenoceptor antagonist) and BMY-7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride) (selective alpha(1D)-adrenoceptor antagonist) competitively antagonized the phenylephrine-induced contraction (pA(2) values, 8.60+/-0.07, 9.44+/-0.06 and 5.75+/-0.07, respectively). Chloroethylclonidine (3x10(-6) mol/L or more) produced a rightward shift in the concentration-response curve for phenylephrine. Thus, in hamster ureters, alpha(1A)- and alpha(1D)-adrenoceptors were more prevalent than the alpha(1B)-adrenoceptor, with contraction being mediated mainly via alpha(1A)-adrenoceptors. If these findings hold true for humans, alpha(1A)-adrenoceptor antagonists could become useful medication for stone passage in urolithiasis patients.

A clinically employed antihyperlipidemic drug, bezafibrate, has been characterized as a PPAR(α, -γ, and -δ) pan-agonist in vitro. Recent extended trials have highlighted its antidiabetic properties in humans. However, the underlying molecular mechanism is not fully elucidated. The present study was designed to explore potential regulatory mechanisms of intracellular glucocorticoid reactivating enzyme, 11β-HSD1 and anti-diabetic hormone, adiponectin by bezafibrate in murine adipose tissue, and cultured adipocytes. Treatment of db/db mice with bezafibrate significantly ameliorated hyperglycemia and insulin resistance, accompanied by a marked reduction of triglyceride and nonesterified fatty acids. Despite equipotent in lipid-lowering effects, another fibrate, fenofibrate, did not show such beneficial effects on glycemic control. Treatment of bezafibrate caused a marked decrease in the mRNA level of 11β-HSD1 preferentially in adipose tissue of db/db mice (−47%, P < 0.05), concomitant with a significant increase in plasma adiponectin level (+37%, P < 0.01). Notably, treatment of bezafibrate caused a marked decrease in the mRNA level (−34%, P < 0.01) and enzyme activity (−32%, P < 0.01) of 11β-HSD1, whereas the treatment substantially augmented the expression (+71%, P < 0.01) and secretion (+27%, P < 0.01) of adiponectin in 3T3-L1 adipocytes. Knockdown of 11β-HSD1 by siRNA confirmed that 11β-HSD1 acts as a distinct oxoreductase in adipocytes and validated the enzyme activity assays in the present study. Effects of bezafibrate on regulation of 11β-HSD1 and adiponectin in murine adipocytes were comparable with those in thiazolidinediones. This is the first demonstration that bezafibrate directly regulates 11β-HSD1 and adiponectin in murine adipocytes, both of which may contribute to metabolically-beneficial effects by bezafibrate.

An experimental method of determining a qualitative two-dimensional image of the gas temperature in stationary atmospheric nonthermal plasma by spectral image processing was presented. In the experiment, a steady-state glow corona discharge was generated by applying a positive DC voltage to a rod-plane electrode in synthetic air. The changes in the gas temperature distribution due to the amplitude of applied voltage and the ambient gas pressure were investigated. Spectral images of a positive DC glow corona were taken using a gated ICCD camera with ultranarrow band-pass filters, corresponding to the head and tail of a N2 second positive system band (0–2). The qualitative gas temperature was obtained from the emission intensity ratio between the head and tail of the N2 second positive system band (0–2). From the results, we confirmed that the gas temperature and its distribution of a positive DC glow corona increased with increasing applied voltage. In particular, just before the sparkover voltage, a distinctly high temperature region was formed in the positive DC glow at the tip of the rod electrode. In addition, the gas temperature decreased and its distribution spread diffusely with decreasing ambient gas pressure.

Hepatolithiasis is prevalent in East Asia, including Japan, but occurs much less frequently in Western countries. Hepatolithiasis appears mostly as brown pigment stones (calcium bilirubinate stones). The disease is characterized by its intractable nature and frequent recurrence, requiring multiple endoscopic or operative interventions, in distinct contrast to gallbladder cholesterol or black pigment stones. In view of the lack of information on the pathogenesis, a multidisciplinary approach has been carried out through the Hepatolithiasis Research Group organized by the Ministry of Health, Labor and Welfare of Japan. In this review, the up-to-date data on the molecular pathogenesis of hepatolithiasis with special reference to a defect in phospholipid metabolism are introduced and discussed. Furthermore, a potential medical treatment targeting hepatic phospholipid transporters is proposed as a future therapeutic option for the disease.

Synchrotron radiation imaging with the refraction-enhancement mode visualized structural inhomogeneities in phantoms used for image quality control of mammography. Eight phantoms were examined, all of which were manufactured in the United States and approved by the American College of Radiology as dedicated phantoms. In addition to fiber- and mass-mimicking test objects, each phantom has 5 groups of calcification specks of various sizes. Synchrotron radiation (SR) imaging was performed at Spring-8, a synchrotron radiation facility in Japan. Images were obtained with monochromatic 20-keV x-ray beams, a radiation field of 15 mm X 26 mm at a sample plane, a CCD camera with a resolution of 6 micrometers as a detector, and a sample-to-detector distance of 10 to 11 m. Two hundred and forty specks were evaluated in total in the SR images, and the surrounding area of each speck was also included. Evaluation of the images showed that 14 crack-like structures were depicted near specks, and there were 62 specks with attached void(s) or air bubble(s). Refraction-enhanced SR imaging sensitively detected structural inhomogeneities and abnormalities in phantoms which were implicitly agreed to have a homogeneous matrix and test objects without foreign substances. A possible manufacturing-dependent quality issue was identified. The effect of inhomogeneities detected by SR imaging on visual scoring of specks could not be identified in the tested phantoms; this should be assessed on images of other phantoms in a future study.

The YIPP (tyrosine-isoleucine-proline-proline, amino acids 319–322) motif within the C-terminal part of the human AT 1 receptor is associated with angiotensin II (AII)-induced activation of the Jak-STAT pathway and phospholipase Cγ1 phosphorylation. We report here that mutations of the YIPP motif strongly affect ligand-binding to the receptor. We analysed AT 1 receptors of the wild type (WT) and 11 mutants with a FLAG-epitope-tag within their C-terminal portion. Mutations of the “P–P” amino acid sequence of this motif decreased both AII binding and the AII-induced intracellular Ca 2+ transients. Mutant and WT receptors were expressed equally in the cell membrane and were localized within the plasma membrane. These results suggest that the “P–P” amino acid sequence within the YIPP motif is important for AII binding to the AT 1 receptor.

SUMMARY 1. In the present study, we investigated the effects of hyperprolactinaemia, induced by transplantation of anterior pituitary glands under the kidney capsule in female rats, on the relationship between serum and pituitary concentrations of the gonadotropins and on the oestrous cycle. 2. Rats with pituitary transplants showed increased serum prolactin concentrations and decreased serum concentrations of gonadotropins and increased pituitary concentrations of gonadotropins. Moreover, these rats showed persistent dioestrous and anovulation from 3 to 6 days after transplantation. 3. A single oral administration of cabergoline (at doses between 0.001 and 0.1 mg/kg) dose-dependently inhibited the elevated serum prolactin concentrations in hyperprolactinaemic rats. At 0.1 mg/kg, cabergoline induced a continuous reduction in serum prolactin concentrations for 5 days after administration. Terguride (0.1 mg/kg) and bromocriptine (10 mg/kg) also reduced serum prolactin concentrations at 1 and 3 days after administration. All three dopamine D2 receptor agonists increased serum gonadotropin concentrations and ovarian weight at 3 days after administration. 4. In rats exhibiting anovulation, a single oral administration of any one of the three dopamine D2 receptor agonists dose-dependently restored ovulation and a normal oestrous cycle appeared. Oral administration of cabergoline (0.03 mg/kg) or terguride (0.1 mg/kg) restored ovarian function and abolished the anovulation following a reduction in serum prolactin concentrations. However, bromocriptine (10 mg/kg) did not completely abolish anovulation. Following administration of terguride (0.3 mg/kg) or bromocriptine (30 mg/kg), only one normal oestrous cycle appeared; however, following cabergoline (0.1 mg/kg), two normal oestrous cycles appeared. 5. These results suggest that cabergoline has a potent and long-lasting action as a dopamine D2 receptor agonist and, thus, should be a useful drug for the treatment of galactorrhoea and hyperprolactinaemic amenorrhoea and/or anovulation in humans.

The rat homologue of the human M r 110 000 antigen, which cross-reacts with anti-carcinoembryonic antigen antibodies, was isolated from a rat lung cDNA library. The deduced amino acid sequence revealed a signal peptide, cysteine-rich and immunoglobulin-like region, serine-threonine region, and N -glycosylation sites in the extracellular portion. Northern blot analysis demonstrated a wide distribution of the mRNA in adult rat tissues and A10 rat vascular smooth muscle cells. Therefore, the rat homologue of the human M r 110 000 antigen may be a receptor or a cell adhesion molecule rather than a specific carcinogenic antigen.

SUMMARY 1. We examined whether KRH-594, a new angiotensin AT1 receptor antagonist, ameliorates the progression of diabetic nephropathy and hyperlipidaemia in streptozotocin (STZ)-induced diabetic unilateral nephrectomized spontaneously hypertensive rats (DM-1K-SHR) or not. 2. The oral administration of KRH-594 (3 and 10 mg/kg per day) and candesartan cilexetil (1 mg/kg per day) for 16 weeks significantly reduced systolic blood pressure, urinary albumin and urinary total protein in DM-1K-SHR. 3. In a histological study, KRH-594 (3 and 10 mg/kg per day) and candesartan cilexetil (0.3 and 1 mg/kg per day) dose-dependently improved glomerulosclerosis and the hyalin cast of tubules in DM-1K-SHR kidneys. Both KRH-594 (10 mg/kg per day) and candesartan cilexetil (0.3 and 1 mg/kg per day) dose-dependently inhibited cardiac hypertrophy. 4. KRH-594 (3 and 10 mg/kg per day), but not candesartan cilexetil, dose-dependently reduced the levels of triglyceride, total cholesterol and phospholipids in DM-1K-SHR. 5. These results suggest that KRH-594 improves diabetic complications, such as nephropathy and hyperlipidaemia, with hypertension.

ABASTRACT:A thermoelectric device consisting of the porous part and the bulk part was proposed. Increase of heat exchange area due to porous medium will improve the efficiency of heat exchange between heating/cooling sources and the device. Estimation based on physical properties of FeSi2 indicated that generated power of the partially porous device per unit area can be several times higher than that of the bulk one in case of gas heating / cooling system. The partially porous thermoelectric devices were produced. In measurement of power, it has been confirmed that generated power per unit area of the partially porous FeSi2 devices was roughly 10 times higher than that of the conventional device. The proposed porous device will exhibit its advantage in the case of low heat transfer coefficient between the device and the heating / cooling sources.