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PCSK9 (proprotein convertase subtilisin-like/kexin type 9) is an emerging target for pharmaceutical intervention. This multidomain protein interacts with the LDL receptor (LDLR), promoting receptor degradation. Insofar as PCSK9 inhibition induces a decrease in plasma cholesterol levels, understanding the nature of the binding interaction between PCSK9 and the LDLR is of critical importance. In this study, the ability of PCSK9 to compete with apoE3 N-terminal domain-containing reconstituted HDL for receptor binding was examined. Whereas full-length PCSK9 was an effective competitor, the N-terminal domain (composed of the prodomain and catalytic domain) was not. Surprisingly, the C-terminal domain (CT domain) of PCSK9 was able to compete. Using a direct binding interaction assay, we show that the PCSK9 CT domain bound to the LDLR in a calcium-dependent manner and that co-incubation with the prodomain and catalytic domain had no effect on this binding. To further characterize this interaction, two LDLR fragments, the classical ligand-binding domain (LBD) and the EGF precursor homology domain, were expressed in stably transfected HEK 293 cells and isolated. Binding assays showed that the PCSK9 CT domain bound to the LBD at pH 5.4. Thus, CT domain interaction with the LBD of the LDLR at endosomal pH constitutes a second step in the PCSK9-mediated LDLR binding that leads to receptor degradation. [ABSTRACT FROM AUTHOR]

Abstract: A swim-bed reactor for partial nitritation with polymeric coagulant treatment and an UASB reactor for anammox were applied to the treatment of livestock manure digester liquor. The partial nitritation was maintained for 32days under a 1.6kgN/m3/d nitrogen loading rate (NLR) with an average conversion efficiency of 51%, and achieved 1.65kgN/m3/d of the maximum nitrite production rate under 2.58kgN/m3/d of NLR. Although 200mg/L of TOC remained in the effluent of the partial nitritation reactor, the anammox nitrogen removal rate was not significantly decreased and a relatively high rate of 2.0kgN/m3/d was obtained under a NLR of 2.2kgN/m3/d. 16S rRNA gene analysis showed that Nitrosomonas and KSU-1 were dominant in the partial nitritation and anammox reactor, respectively. The results of this study demonstrated that the partial nitritation–anammox process has possibility of applying to the nitrogen removal of livestock manure digester liquor. [ABSTRACT FROM AUTHOR]

The low density lipoprotein receptor (LDLR) plays a key role in plasma cholesterol homeostasis by binding and internalizing lipoprotein ligands. Studies have revealed that one or more of the seven LDL type A repeats (LA1-LA7) in the receptor are responsible for apolipoprotein binding. In the present study, protein engineering was performed to swap or replace key LA repeats in a recombinant soluble LDLR (sLDLR). Although wild type sLDLR showed strong ligand binding activity, an sLDLR variant in which LA repeat 5 was replaced by a second copy of LA repeat 2 showed low binding activity. Likewise, a variant wherein LA repeats 2 and 5 were swapped displayed low binding activity. At the same time, substitution of LA repeat 2 with a second a copy of repeat 5 resulted in a receptor with ligand binding activity similar to wild type LDLR. When binding assays were conducted with human low density lipoprotein as ligand, LA repeat order was a less important determinant of binding activity. Taken together, the data indicate that the sequential order of LA repeats plays a key role in ligand binding properties of LDLR. [ABSTRACT FROM AUTHOR]

Abstract: Effect of high salt concentration on the anammox treatment was investigated to establish an acclimation strategy under high salt concentration conditions. An anammox fixed-bed reactor with non-woven biomass carrier was used and the salt concentration was gradually increased from 2.5 g L−1 to 33 g L−1. The anammox reactor demonstrated stable nitrogen removal rate (NRR) of 1.7 kg-N m−3 d−1 for 65 days under a salt concentration of 30 g L−1. However, the NRR sharply declined at a salt concentration of greater than 30 g L−1. The bacterial community was examined by 16S rRNA gene analysis and DGGE after the acclimation of the anammox sludge to high salt conditions. Although the salt concentration was almost sea level, the freshwater anammox bacteria, KU2, were detected. In addition, the unidentified bacteria which perhaps belong to candidate division OP10 and Lysobacter sp. were found to coexist with anammox bacteria at a salt concentration of 30 g L−1. [Copyright &y& Elsevier]

The release of ligand from the low-density lipoprotein receptor (LDLR) has been postulated to involve a "histidine switch"-induced intramolecular rearrangement that discharges bound ligand. A recombinant soluble low-density lipoprotein receptor (sLDLR) was employed in ligand binding experiments with a fluorescently tagged variant apolipoprotein E N-terminal domain (apoE-NT). Binding was monitored as a function of fluorescence resonance energy transfer (FRET) from excited Trp residues in sLDLR to an extrinsic fluorophore covalently attached to Trp-nulI apoE3-NT. In binding experiments with wild- type (WT) sLDLR, FRET-dependent AEDANS fluorescence decreased as the pH was lowered. To investigate the role of His190, His562, and His586 in sLDLR in pH-dependent ligand binding and discharge, site-directed mutagenesis studies were performed. Compared to WT sLDLR, triple His → Ala mutant sLDLR displayed attenuated pH-dependent ligand binding and a decreased level of ligand release as a function of low pH. When these His residues were substituted for Lys, the positively charged side chain of which does not ionize over this pH range, ligand binding was nearly abolished at all pH values. When sequential His to Lys mutants were examined, the evidence suggested that His562 and His586 function cooperatively. Whereas the sedimentation coefficient for WT sLDLR increased when the pH was reduced from 7 to 5, no such change occurred in the case of the triple Lys mutant receptor or a His562Lys/His586Lys double mutant receptor. The data support the existence of a cryptic, histidine side chain ionization-dependent alternative ligand that modulates ligand discharge via conformational reorganization. [ABSTRACT FROM AUTHOR]

Partial nitritation using inhibition of free ammonia and free nitric acid is an effective technique for the treatment of high concentrations of ammonium in wastewaters. This technique was applied to the digester liquor of swine wastewater and the stability of its long-term operation was investigated. Partial nitritation was successfully maintained at a nitrogen loading rate (NLR) of 1.0kgNm−3 d−1 for 120 days without acclimatization of nitrite oxidizing bacteria (NOB) to the inhibitory compounds (free ammonia and free nitric acid). The conversion efficiencies of NH4–N to NO2–N and to NO3–N were determined to be around 58% and <5%, respectively. After the establishment of partial nitritation, the influence of swine wastewater on the Anammox reaction was examined using continuous flow treatment experiments. Consistent nitrogen removal was achieved for 70 days at a nitrogen removal rate (NRR) of 0.22kgNm−3 d−1 and the color of Anammox bacteria changed from red to greyish black. The NO2–N consumption and the NO3–N production increased concurrently and the Anammox reaction ratio was estimated to be 1:1.67:0.53, which is different from that reported previously (1:1.32:0.26). [Copyright &y& Elsevier]

Abstract: Apolipoprotein E (apoE) is a ligand for members of the low-density lipoprotein receptor (LDLR) family and functions in plasma cholesterol homeostasis. A fluorescence-based assay has been employed in molecular studies of receptor–ligand interactions. Competition experiments revealed isoform-specific differences in binding of lipid-associated apoE N terminal (NT) domain to a recombinant soluble LDLR (sLDLR). In a similar manner, lipid-associated—but not lipid-free—full-length apoE3 showed binding activity to sLDLR. The molecular chaperone, receptor-associated protein, inhibited apoE3–NT–phospholipid complex binding to sLDLR. Kinetic studies of apoE3–NT–phospholipid complex interaction with sLDLR revealed time-dependent effects of apoE–NT isoform binding to sLDLR. The results reveal a discerning method for study of the molecular basis of ligand interactions that likely influence receptor function in maintenance of whole body cholesterol homeostasis. [Copyright &y& Elsevier]

Abstract: Apolipoprotein E (apoE) is a ligand for members of the low-density lipoprotein receptor (LDLR) family. Lipid-free apoE is not recognized by LDLR, yet interaction with lipid confers receptor recognition properties. Although lipid interaction is known to induce a conformational change in apoE, it is not known if the lipid composition of the resulting complex influences binding. Using reconstituted lipoprotein particles of apoE3 N-terminal (NT) domain and dimyristoylphosphatidylcholine (DMPC), maximal LDLR binding was observed at DMPC:apoE3-NT ratios >2.5:1 (w/w). ApoE3-NT lipid particles prepared with egg sphingomyelin were functional as LDLR ligands while complexes formed with the anionic phospholipids dimyristoylphosphatidylglycerol or dimyristoylphosphatidylserine (DMPS) were not. In the case of apoE3-NT, lipid particles comprised of a mixture of DMPC and DMPS, a DMPS concentration dependent inhibition of LDLR binding activity was observed. Thus, in addition to affecting apoE conformational status, the lipid composition of ligand particles can modulate LDLR binding activity. [Copyright &y& Elsevier]

A swim-bed reactor using the biofringe acryl-fiber biomass carrier was used for partial nitritation treatment for anaerobic digestion liquor of swine wastewater. The sludge in the reactor demonstrated excellent settling properties, and the sludge volumetric index (SVI) was always about 50 ml g−1. The mixed liquor suspended solids (MLSS) concentration was maintained above 10,000 mg l −1 with a maximum of 16,800 mg l −1. Satisfactory and stable partial nitritation was obtained at a nitrogen loading rate (NLR) of 1.9 kg-N m−3 d−1 without any operational control. Only a little nitrate was produced almost during the whole operational period and the nitrite to total oxidized nitrogen ratio (NO2-N/(NO2-N+NO3-N)) was always above 95%. In addition, the influence of temperature on partial nitritation efficiencies was also investigated and non-controlled efficiencies were maintained stably between 15°C and 30°C at an NLR of 1.9 kg-N m−3 d−1, but suddenly deteriorated when the temperature fell below 15°C. Nitrite oxidizing bacteria were inhibited by free ammonia and free nitric acid, which prevented the conversion of nitrite to nitrate and the inhibition due to free nitric acid weaken with a decrease in temperature. It was apparent that these phenomena were crucial to the control of partial nitritation treatment. [Copyright &y& Elsevier]

Abstract: The N terminal domain of human apolipoprotein E3 (apoE3-NT) functions as a ligand for members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid-free apoE3-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational change is required for LDLR recognition. To investigate the role of a leucine zipper motif identified in the helix bundle on lipid binding activity, three leucine residues in helix 2 (Leu63, Leu71 and Leu78) were replaced by alanine. Recombinant “leucine to alanine” (LA) apoE3-NT was produced in E. coli, isolated and characterized. Stability studies revealed a transition midpoint of guanidine hydrochloride induced denaturation of 2.7 M and 2.1 M for wild type (WT) and LA apoE3-NT, respectively. Results from fluorescent dye binding assays revealed that, compared to WT apoE3-NT, LA apoE3-NT has an increased content of solvent exposed hydrophobic surfaces. In phospholipid vesicle solubilization assays, LA apoE3-NT was more effective than WT apoE3-NT at inducing a time-dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. Likewise, in lipoprotein binding assays, LA apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent than WT apoE3-NT. On the other hand, LA apoE3-NT and WT apoE3-NT were equivalent in terms of their ability to bind a soluble LDLR fragment. The results suggest that the leucine zipper motif confers stability to the apoE3-NT helix bundle state and may serve to modulate lipid binding activity of this domain and, thereby, influence the conformational transition associated with manifestation of LDLR binding activity. [Copyright &y& Elsevier]

Abstract: The homeodomain protein Hex can function either as a transcriptional repressor or activator in animals. Recent reports have indicated that Hex is involved in liver development. However, its target genes and interacting proteins are largely unknown. We found that Hex functionally interacted with hepatocyte nuclear factor (HNF) 1α to further stimulate its activity using reporter gene containing multiple copies of HNF1α-binding site of the L-type pyruvate kinase (L-PK) gene promoter or natural L-PK promoter. This stimulation required the homeodomain and the acidic carboxyl-terminal region of Hex. Over-expression of Hex in primary cultured hepatocytes resulted in stimulation of the L-PK gene expression. Glutathione S-transferase pull-down assay and co-immunoprecipitation revealed that Hex physically interacted with HNF1α in mammalian cells through the homeodomain of Hex and POU-homeodomain of HNF1α. Since HNF1α is an important liver-enriched transcription factor involved in liver differentiation, Hex may contribute to liver differentiation through interaction with HNF1α. [Copyright &y& Elsevier]

Abstract: The origin recognition complex (ORC) protein plays a critical role in DNA replication through binding to sites (origins) where replication commences. The protein is composed of six subunits (ORC1 to 6) in animals and yeasts. Our knowledge of the ORC protein in plants is, however, much less complete. We have performed cDNA cloning and characterization of ORC subunits in rice (Oryza sativa L. cv. Nipponbare) in order to facilitate study of plant DNA replication mechanisms. Our previous report provided a description of a gene, ORC1 (OsORC1), that encodes one of the protein subunits. The present report extends this initial analysis to include the genes that encode four other rice ORC subunits, OsORC2, 3, 4 and 5. Northern hybridization analyses demonstrated the presence of abundant transcripts for all OsORC subunits in shoot apical meristems (SAM) and cultured cells, but not in mature leaves. Interestingly, only OsORC5 showed high levels of expression in organs in which cell proliferation is not active, such as flag leaves, the ears and the non-tip roots. The pattern of expression of OsORC2 also differed from other OsORC subunits. When cell proliferation was temporarily halted for 6–10 days by removal of sucrose from the growth medium, expression of OsORC1, OsORC3, OsORC4 and OsORC5 was substantially reduced. However, the level of expression of OsORC2 remained constant. We suggest from these results that expression of OsORC1, 3, 4 and 5 are correlated with cell proliferation, but the expression of OsORC2 is not. [Copyright &y& Elsevier]

Proliferating cell nuclear antigen (PCNA) is an essential protein for both DNA replication and DNA repair. In the present study using two-hybrid analysis with PCNA from rice, Oryza sativa L. cv. Nipponbare (OsPCNA), we found that OsPCNA interacted with rice DnaJ protein. We have identified DnaJ and designated it as OsDnaJ. OsDnaJ was able to bind to OsPCNA in vitro. Transcripts of OsDnaJ were found to be strongly expressed in the proliferating cells. mRNA of DnaJ was induced by UV and DNA-damaging agents such as H2O2. The expression patterns of OsPCNA were almost the same as OsDnaJ. The relationship between OsPCNA and OsDnaJ is discussed. [ABSTRACT FROM AUTHOR]

Studies the anti-allergic activity of Naringenin Chalcone from a tomato skin extract using an in vitro histamine-release assay. Inhibitory effects of the polyphenols of the tomato skin extract on the histamine release from rat peritoneal mast cells; Purification and identification of the active compound in tomato skin.

We report here the existence of interactions between a ubiquitin-conjugating enzyme, Rad6, from rice, Oryza sativa L. cv. Nipponbare (OsRad6), and Sgt1 (OsSgt1), a novel subunit of the SCF ubiquitin ligase complex. Rad6 is not only related to post-replicational repair but also to the proteasome system, while Sgt1 has a function in kinetochore assembly. The relationship between the two is unexpected, but of great interest. The open reading frames of OsRad6 and OsSgt1 encode predicted products of 152 and 367 amino acid residues, respectively, with molecular weights of 17.3 and 40.9 kDa. Two-hybrid and pull-down analyses indicated that OsRad6 binds to OsSgt1, and transcripts of both OsRad6 and OsSgt1 were found to be strongly expressed only in the proliferating tissues such as the shoot apical meristem, suggesting that their expression is cell cycle-dependent. The amount of the Rad6 mRNA in cultured cells increased rapidly after division was halted, and mRNA levels of Rad6 and Sgt1 were induced by UV- and DNA-damaging agents such as MMS or H2O2. The Rad6 pathway for repair or the proteasome system may thus require Sgt1 as ubiquitin-conjugating enzyme. [Copyright &y& Elsevier]

The deletion of the Hhex (Hematopoietically expressed homeobox) gene causes agenesis of the liver and polycystic liver disease depending on its timing. The present study was undertaken to determine the role of the Hhex gene in not only signaling cascades to cyst and abnormal bile duct formation but also the liver progenitor contribution to cystic development. Liver-specific Hhex knockout mice (Alb - Cre / Hhex loxP / loxP ) in adult stages were used. Wild-type and conditional knockout (cKO) livers were immunohistologically compared for cell growth, and gene expression of liver functions, biliary markers and cystic markers. In Hhex cKO livers, cyst formation and dilated intrahepatic bile ducts were noted, which resembled the histology of the von Meyenburg complex. Ki67 immunohistochemistry showed that the growth activity in bile ducts and cysts of cKO livers was elevated compared with that of wild-type livers. There were far fewer liver progenitor cells or bile ductule cells around portal veins of cKO livers than in wild-type livers. Several liver-enriched transcription factors, including Foxa1 and Foxa2, were heterogeneously expressed in bile ducts and cysts of cKO livers whereas their expression in wild-type bile ducts was comparatively homogeneous. PC1 and PC2 immunohistochemistry revealed their up-regulation in cysts of cKO livers. These data indicate that Hhex is not only required for proper bile duct morphogenesis, but is also involved in cyst formation through promoted cell growth. Liver progenitor cells may form cysts. Unbalanced expression of liver-enriched transcription factors might be involved in cyst formation. Hhex cKO mice may be a good animal model for hepatic cystic diseases. • Hhex is involved in bile duct development and cyst formation through growth control. • Liver progenitor cells may participate in cyst formation. • Cyst cells have unbalanced expression of liver-enriched transcription factors. • Hhex knockout mice may be a good animal model for hepatic cystic disease. [ABSTRACT FROM AUTHOR]

Abstract: Underground brine waste containing high concentrations of ammonium and with a salinity of 3% is usually generated during the production of methane gas and iodine in the gas field of Chiba Prefecture, Japan. In this study, one swim-bed reactor, packed with a novel acrylic fiber biomass carrier (Biofringe), was applied to the partial nitritation treatment of this kind of underground brine waste. A stable nitrite production rate of 1.6 kg NO2-N m−3 d−1 was obtained under a nitrogen loading rate of 3.0 kg-N m−3 d−1, at a pH of 7.5 and a temperature of 25 °C. Nitrate production was negligible and the effluent NO2-N/NO x -N ratio was above 98% due to the successful inhibition of nitrite-oxidizing bacterial activity. Free ammonia was considered to be the main factor for inhibiting the activity of nitrite-oxidizing bacteria. A microbial community shift was demonstrated by 16S rRNA analysis, and it was shown that the ammonium-oxidizing bacteria became the predominant species after successful nitrite accumulation was observed. [Copyright &y& Elsevier]

Little is known about the functions of DNA polymerase λ (Pol λ) recently identified in mammals. From the genomic sequence information of rice and Arabidopsis, we found that Pol λ may be the only member of the X-family in higher plants. We have succeeded in isolating the cDNA and recombinant protein of Pol λ in a higher plant, rice ( Oryza sativa L. cv. Nipponbare) ( OsPol λ). OsPol λ had activities of DNA polymerase, terminal deoxyribonucleotidyl transferase and deoxyribose phosphate lyase, a marker enzyme for base excision repair. It also interacted with rice proliferating cell nuclear antigen (OsPCNA) in a pull-down assay. OsPCNA increased the processivity of OsPol λ. Northern blot analysis showed that the level of OsPol λ expression correlated with cell proliferation in meristematic and meiotic tissues, and was induced by DNA-damaging treatments. These properties suggest that plant Pol λ is a DNA repair enzyme which functions in plant meristematic and meiotic tissues, and that it can substitute for Pol β and terminal deoxyribonucleotidyl transferase. [ABSTRACT FROM AUTHOR]

Hex is one of the homeobox genes suggested to be important for hematopoietic cell differentiation. However, its biological function and mechanism of transcriptional regulation in hematopoietic cells remain elusive. We have identified the regulatory region necessary for transcription of the mouse Hex gene in K562 leukemia cells through transient reporter assays involving various deletion mutants. This region, comprising +775 to +1177 in the first intron, had enhancer-like properties and showed high activity in other hematopoietic cell lines such as U937, HEL, and RAW264.7, but little activity in other Hex-expressing cell lines such as MH1C1 and H4IIE hepatoma cells, suggesting that this region functions as a hematopoietic cell-specific enhancer-like element. Binding site mutation of hematopoietic transcription factors, such as GATAs and c-Myb present in the enhancer-like element, significantly decreased the luciferase reporter gene expression in K562 cells. Electrophoretic mobility shift assays showed that GATA-1, GATA-2, or c-Myb actually binds to three of these putative binding sites, and also suggested that several unidentified factors might interact with the enhancer-like element. Overexpression of GATA-1, GATA-2, or c-Myb stimulated the enhancer-like activity via these three binding sites. Thus, we conclude that Hex expression in hematopoietic cells is mainly regulated by GATA-1, GATA-2, and c-Myb via this intronic enhancer-like element. [ABSTRACT FROM PUBLISHER]

The homeodomain-containing protein Hex acts as an activator as well as a repressor of transcription in animals. While its repression domain has been mapped to the amino-terminal region, the activation domain has never been identified. Here, we show that the homeodomain and the acidic carboxyl-terminal region are necessary for full activation of the sodium-dependent bile acid cotransporter gene promoter in a cell type-independent manner, suggesting that the carboxyl-terminal region comprising residues 197 to 271 functions as the activation domain. In addition, we observed that a Hex mutant without this activation domain acts as a dominant-negative mutant as to the transactivating function of Hex. [ABSTRACT FROM PUBLISHER]

Ultraviolet-damaged DNA binding protein (UV-DDB) is an important factor involved in DNA repair. To study the role of UV-DDB, we attempted to obtain the cDNA and the protein of a plant UV-DDB. We succeeded in isolating both genes for UV-DDB subunits from rice (Oryza sativa cv. Nipponbare), designated as OsUV-DDB1 and OsUV-DDB2. OsUV-DDB2 (65 kDa) was much larger than human UV-DDB2, but immunoprecipitation and gel mobility shift assay suggested that OsUV-DDB2 is a plant counterpart of UV-DDB2. The transcripts were expressed in proliferating tissues such as the meristem, but were detected at only low levels in the mature leaves, although the leaves are strongly exposed to UV. These transcripts were induced in the meristem after UV-irradiation. The expression levels of OsUV-DDB were significantly reduced when cell proliferation was temporarily halted. These results indicated that the level of OsUV-DDB expression is correlated with cell proliferation, and its expression may be required mostly for DNA repair in DNA replication. [Copyright &y& Elsevier]

Chimeric mice are immunodeficient mice in which the majority of the hepatic parenchymal cells are replaced with human hepatocytes. Following intravenous administration of 24 model compounds to control and chimeric mice, human hepatic clearance (CLh) was predicted using the single-species allometric scaling (SSS) method. Predictability of the chimeric mice was better than that of the control mice. Human CLh was predicted by the physiologically based scaling (PBS) method, wherein observed CLh in chimeric mice was first converted to intrinsic CLh (CLh,int). As the liver of chimeric mice contains remaining mouse hepatocytes, CLh,int was corrected by in vitro CLh ratios of the mouse to human hepatocytes according to their hepatocyte replacement index. Further, predicted human CLh was calculated based on an assumption that CLh,int in chimeric mice normalised for their liver weight was equal to CLh,int per liver weight in humans. Consequently, better prediction performance was observed with the use of the PBS method than the SSS method. SSS method is an empirical method, and the effects of coexisting mouse metabolism cannot be avoided. However, the PBS method with in vitro CLh correction might be a potential solution and may expand the application of chimeric mice in new drug development. [ABSTRACT FROM AUTHOR]

• Alternative NOG MHC double KO (dKO) mouse has been generated for the human PBMC-engrafted humanized mice lacking xeno-GVHD. • Breeding efficiency was improved in new version of dKO mice compared to conventional dKO mice. • Significant engraftment of human cells was observed in new version of dKO mice after human PBMC transplantation. Humanized mice are widely used to study the human immune system in vivo and develop therapies for various human diseases. Human peripheral blood mononuclear cells (PBMC)-engrafted NOD/Shi- scid IL2rγ null (NOG) mice are useful models for characterization of human T cells. However, the development of graft-versus-host disease (GVHD) limits the use of NOG PBMC models. We previously established a NOG-major histocompatibility complex class I/II double knockout (dKO) mouse model. Although humanized dKO mice do not develop severe GVHD, they have impaired reproductive performance and reduced chimerism of human cells. In this study, we established a novel beta-2 microglobulin (B2m) KO mouse model using CRISPR/Cas9. By crossing B2m KO mice with I-Ab KO mice, we established a modified dKO (dKO-em) mouse model. Reproductivity was slightly improved in dKO-em mice, compared with conventional dKO (dKO-tm) mice. dKO-em mice showed no signs of GVHD after the transfer of human PBMCs; they also exhibited high engraftment efficiency. Engrafted human PBMCs survived significantly longer in the peripheral blood and spleens of dKO-em mice, compared with dKO-tm mice. In conclusion, dKO-em mice might constitute a promising PBMC-based humanized mouse model for the development and preclinical testing of novel therapeutics for human diseases. [ABSTRACT FROM AUTHOR]

Discusses the ability of enterostatin and its peptide fragment DPR to reduce serum cholesterol levels after oral administration in mice. Drug dosage; Inhibition of the secretion of insulin after peripheral administration; Time course to reduce serum cholesterol levels; Mechanism for hypocholesterolemic effect.

Aquaporin-4 (AQP4) has been suggested to be involved in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD), which may be due to the modulation of neuroinflammation or the impairment of interstitial fluid bulk flow system in the central nervous system. Here, we show an age-dependent impairment of several behavioral outcomes in 5xFAD AQP4 null mice. Twenty-four-hour video recordings and computational analyses of their movement revealed that the nighttime motion of AQP4-deficient 5xFAD mice was progressively reduced between 20 and 36 weeks of age, with a sharp deterioration occurring between 30 and 32 weeks. This reduction in nighttime motion was accompanied by motor dysfunction and epileptiform neuronal activities, demonstrated by increased abnormal spikes by electroencephalography. In addition, all AQP4-deficient 5xFAD mice exhibited convulsions at least once during the period of the analysis. Interestingly, despite such obvious phenotypes, parenchymal amyloid β (Aβ) deposition, reactive astrocytosis, and activated microgliosis surrounding amyloid plaques were unchanged in the AQP4-deficient 5xFAD mice relative to 5xFAD mice. Taken together, our data indicate that AQP4 deficiency greatly accelerates an age-dependent deterioration of neuronal function in 5xFAD mice associated with epileptiform neuronal activity without significantly altering Aβ deposition or neuroinflammation in this mouse model. We therefore propose that there exists another pathophysiological phase in AD which follows amyloid plaque deposition and neuroinflammation and is sensitive to AQP4 deficiency. [ABSTRACT FROM AUTHOR]

Abstract: Small compounds that activate the insulin-dependent signaling pathway have potential therapeutic applications in controlling type 2 diabetes mellitus. The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor that decreases expression of the phosphoenolpyruvate carboxykinase gene, a gluconeogenic enzyme gene. In this study, we screened for soybean isoflavones that can induce the rat SHARP-2 gene expression and analyzed their mechanism(s). Genistein and (S)-Equol, a metabolite of daidzein, induced rat SHARP-2 gene expression in H4IIE rat hepatoma cells. The (S)-Equol induction was mediated by both the phosphoinositide 3-kinase- and protein kinase C (PKC)-pathways. When a dominant negative form of atypical PKC lambda (aPKCλ) was expressed, the induction of SHARP-2 mRNA level by (S)-Equol was inhibited. In addition, Western blot analyses showed that (S)-Equol rapidly activated both aPKCλ and classical PKC alpha. Furthermore, the (S)-Equol induction was inhibited by treatment with a RNA polymerase inhibitor or a protein synthesis inhibitor. Finally, a reporter gene assay revealed that the transcriptional stimulation by (S)-Equol was mediated by nucleotide sequences located between −4687 and −4133 of the rat SHARP-2 gene. Thus, we conclude that (S)-Equol is an useful dietary supplement to control type 2 diabetes mellitus. [Copyright &y& Elsevier]

Abstract: This study demonstrated that partial nitritation using nitrifying activated sludge entrapped in a polyethylene glycol (PEG) gel carrier, as a pretreatment to anammox process, could be successfully applied to digester liquor of biogas plant at a nitrogen loading rate of 3.0kg-N/m3/d. The nitritation process produced an effluent with a NO2–N/NH4–N ratio between 1.0 and 1.4, which was found to be suitable for the subsequent anammox process. A high SS concentration (2000–3000mg/l) in the digester liquor did not affect partial nitritation treatment performances. Effluent from this partial nitritation reactor was successfully treated in the anammox reactor using anammox sludge entrapped in the PEG gel carrier with T-N removal rates of greater than 4.0kg-N/m3/d. Influent BOD and SS contents did not inhibit anammox activity of the anammox gel carrier. The combination of partial nitritation and anammox reactors using PEG entrapped nitrifying and anammox bacteria was shown to be effective for the removal of high concentration ammonium in the digester liquor of a biogas plant. [Copyright &y& Elsevier]

Abstract: Carbohydrate response element binding protein (ChREBP) is a transcription factor that activates liver glycolytic and lipogenetic enzyme genes in response to high carbohydrate diet. Here we report the transcriptional regulatory mechanisms for the rat ChREBP gene. Firstly, we determined the transcription initiation site and the nucleotide sequences of the rat ChREBP promoter region encompassing approximately 900bp from the ATG initiation codon. Reporter gene assays demonstrated that the major positive regulatory region exists in the nucleotide sequence between −163 and −32 of the ChREBP gene. This region contains a cluster of putative transcription factor binding elements that consist of two specificity protein 1 (Sp1) binding sites (−66 to −50 and −93 to −78), a sterol regulatory element (−101 to −110), and two nuclear factor-Y (NF-Y) binding sites (−23 to −19 and −131 to −127). Mutations introduced into these sites caused marked reduction of ChREBP promoter activities. Functional synergisms were observed between Sp1/NF-Y and Sp1/sterol regulatory element-binding protein. Additionally, electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that these factors bound to these elements. Thus, we conclude that functional synergisms between these transcription factors are critical for ChREBP gene transcription. [Copyright &y& Elsevier]

Abstract: A novel RecA-like protein, differing from Dmc1 and Rad51, was characterized in Oryza sativa L. cv. Nipponbare. Because the protein is homologous to bacterial RadA, the gene was designated OsRadA. The open reading frame was predicted to encode a 66kDa protein of 619 amino acid residues and was found in plants but not animals or yeast. OsRadA showed D-loop and single-stranded DNA-dependent ATPase activities. Gene expression was found to be high in meristematic tissues, and was localized in the nucleus. An RNAi mutant of Arabidopsis thaliana RadA (AtRadA) was sensitive to mutagenic agents such as UV and MMC, suggesting that RadA functions in DNA repair. [Copyright &y& Elsevier]

Abstract: Previously, we described a novel DNA polymerase, designated as OsPolI-like, from rice. The OsPolI-like showed a high degree of sequence homology with the DNA polymerase I of cyanobacteria and was localized in the plastid. Here, we describe two PolI-like polymerases, designated as AtPolI-like A and AtPolI-like B, from Arabidopsis thaliana. In situ hybridization analysis demonstrated expression of both mRNAs in proliferating tissues such as the shoot apical meristem. Analysis of the localizations of GFP fusion proteins showed that AtPolI-like A and AtPolI-like B were localized to plastids. AtPolI-like B expression could be induced by exposure to the mutagen H2O2. These results suggested that AtPolI-like B has a role in the repair of oxidation-induced DNA damage. Our data indicate that higher plants possess two plastid DNA polymerases that are not found in animals and yeasts. [Copyright &y& Elsevier]

The biochemical and cell cycle-dependent properties of proliferating cell nuclear antigen (OsPCNA) and flap endonuclease-1 (OsFEN-1) were characterized from rice (Oryza sativa). OsPCNA was physically associated with OsFEN-1 and increased the flap-endonuclease activity of OsFEN-1 by 2.5-fold. Northern and Western blotting analysis revealed that OsPCNA and OsFEN-1 were present in meristematic tissues such as cultured cells, shoot apical meristem and root apical meristem. No expression was detected in the mature leaves, although they were exposed to UV. Both of these proteins were localized in the nuclei of the interphase cells including G1, S and G[sub 2], and in the nuclear region at telophase. The distribution patterns of plant PCNA and FEN-1 in meiotic cell progression were investigated using microsporocytes of lily (Lilium longiflorum cv. Hinomoto). During the leptotene to pachytene stages, PCNA and FEN-1 were localized in the nuclear region. The florescence gradually disappeared from diplotene to metaphase I. Interestingly, signals for PCNA formed 10-20 intense spots at leptotene. The number of spots decreased to 1-5 at zygotene and finally to I at pachytene. The roles of OsPCNA and OsFEN-1 in mitotic and meiotic cell cycles are discussed. [ABSTRACT FROM AUTHOR]

Abstract. Many kinds of bioactive peptides which might prevent lifestyle-related diseases are released from food proteins after enzymatic digestion. Inhibitory peptides for angiotensin I-converting enzyme (ACE) having anti-hypertensive effect have been isolated from enzymatic digests of various food proteins. LKPNM, which was isolated from the thermolysin digest of dried bonito was activated 8-fold by ACE itself and showed a prolonged effect after oral administration. Two vasorelaxing peptides, ovokinin and ovokinin(2-7), showing antihypertensive effect after oral administration were obtained from ovalbumin digests. We found that low molecular weight peptides derived from food proteins lowered serum cholesterol without increasing excretion of cholesterol and bile acids. An immunostimulating peptide isolated from an enzymatic digest of soybean protein prevented alopecia induced by cancer chemotherapy. [ABSTRACT FROM AUTHOR]