Search

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]

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: 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 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]

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]

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]

• 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]

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]