Attempt to simultaneously generate three chiral centers in 4-hydroxyisoleucine with microbial carbonyl reductases

A panel of microorganisms was screened for selective reduction ability towards a racemic mixture of prochiral 2-amino-3-methyl-4-ketopentanoate (rac-AMKP). Several of the microorganisms tested produced greater than 0.5 mM 4-hydroxyisoleucine (HIL) from rac-AMKP, and the stereoselectivity of HIL formation was found to depend on the taxonomic category to which the microorganism belonged. The enzymes responsible for the AMKP-reducing activity, ApAR and FsAR, were identified from two of these microorganisms, Aureobasidium pullulans NBRC 4466 and Fusarium solani TG-2, respectively. Three AMKP reducing enzymes, ApAR, FsAR, and the previously reported BtHILDH, were reacted with rac-AMKP, and each enzyme selectively produced a specific composition of HIL stereoisomers. The enzymes appeared to have different characteristics in recognition of the stereostructure of the substrate AMKP and in control of the 4-hydroxyl group configuration in the HIL product.     

Genetic improvement of heavy metal tolerance in plants by transfer of the yeast metallothionein gene (CUP1)

Cauliflower (Brassica oleracea var. botrytis) tolerates treatment with 25 µM CdCl2 for eight days, but is killed by that with a 50 µM concentration. However, even 15 µM CdCl2 is toxic in the presence of 1 mM L-buthionine sulfoximine (BSO), suggesting the presence of a Cd-inducible phytochelatin and its involvement in Cd-tolerance in cauliflower. To develop heavy metal-tolerant transgenic plants, we ligated the structural gene of yeast metallothionein gene (CUP1) downstream of CaMV35S promoter and introduced the fused gene into cauliflower. A Cd-tolerant transgenic cauliflower was selected, which grew well in the presence of 400 µM or less Cd, whereas the non-transformed cauliflower tolerated only up to 25 µM Cd. The transgenic cauliflower accumulated more Cd, especially in the upper leaves, than the non-transformed plant.In conclusion, by transfer of the yeast metallothionein gene into cauliflower increased Cd-tolerance and Cd-accumulating ability can be conferred to the plant.     

Thyroid hormone response to thyrotrophin releasing hormone (TRH) in the sex-linked dwarf chicken

The effect of an injection of thyrotrophin releasing hormone (TRH) on plasma levels of thyroid hormones was studied in dwarf and normal Rhode Island Red chickens with similar genotypes other than for the sex-linked dwarf gene dw. The sex-linked dwarf chickens had different plasma iodothyronine levels from control normal chickens: high thyroxine (T4), low triiodothyronine (T3) and similar reverse T3 (rT3) levels. The injection of TRH (10 micrograms/kg) in 5-day- and 5-week-old normal chickens increased the plasma T4 within 30 min without a significant increase in T3, whereas the injection of TRH in 11-and 26-week-old normal chickens increased plasma T3 60 min later. In dwarfs the response of T4 to TRH was the same as that in normals but no increased T3 response was observed. The plasma level of rT3 was not influenced by the TRH injection in either strain. These results suggest that although in the sex-linked dwarfs thyroidal response to exogenous TRH is similar to that of normals, the dwarf gene dw inhibits the conversion of T4 to T3 in peripheral tissues without any inhibitory effect on rT3 production.     

Identification of 4-hydroxy-2-nonenal-histidine adducts that serve as ligands for human lectin-like oxidized LDL receptor-1

LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) is an endothelial scavenger receptor that is important for the uptake of OxLDL (oxidized low-density lipoprotein) and contributes to the pathogenesis of atherosclerosis. However, the precise structural motifs of OxLDL that are recognized by LOX-1 are unknown. In the present study, we have identified products of lipid peroxidation of OxLDL that serve as ligands for LOX-1. We used CHO (Chinese-hamster ovary) cells that stably express LOX-1 to evaluate the ability of BSA modified by lipid peroxidation to compete with AcLDL (acetylated low-density lipoprotein). We found that HNE (4-hydroxy-2-nonenal)-modified proteins most potently inhibited the uptake of AcLDL. On the basis of the findings that HNE-modified BSA and oxidation of LDL resulted in the formation of HNE-histidine Michael adducts, we examined whether the HNE-histidine adducts could serve as ligands for LOX-1. The authentic HNE-histidine adduct inhibited the uptake of AcLDL in a dose-dependent manner. Furthermore, we found the interaction of LOX-1 with the HNE-histidine adduct to have a dissociation constant of 1.22×10(-8) M using a surface plasmon resonance assay. Finally, we showed that the HNE-histidine adduct stimulated the formation of reactive oxygen species and activated extracellular-signal-regulated kinase 1/2 and NF-κB (nuclear factor κB) in HAECs (human aortic endothelial cells); these signals initiate endothelial dysfunction and lead to atherosclerosis. The present study provides intriguing insights into the molecular details of LOX-1 recognition of OxLDL.     

Arsenic Trioxide Inhibits Hepatitis C Virus RNA Replication through Modulation of the Glutathione Redox System and Oxidative Stress

Arsenic trioxide (ATO), a therapeutic reagent used for the treatment of acute promyelocytic leukemia, has recently been reported to increase human immunodeficiency virus type 1 infectivity. However, in this study, we have demonstrated that replication of genome-length hepatitis C virus (HCV) RNA (O strain of genotype 1b) was notably inhibited by ATO at submicromolar concentrations without cell toxicity. RNA replication of HCV-JFH1 (genotype 2a) and the release of core protein into the culture supernatants were also inhibited by ATO after the HCV infection. To clarify the mechanism of the anti-HCV activity of ATO, we examined whether or not PML is associated with this anti-HCV activity, since PML is known to be a target of ATO. Interestingly, we observed the cytoplasmic translocation of PML after treatment with ATO. However, ATO still inhibited the HCV RNA replication even in the PML knockdown cells, suggesting that PML is dispensable for the anti-HCV activity of ATO. In contrast, we found that N-acetyl-cysteine, an antioxidant and glutathione precursor, completely and partially eliminated the anti-HCV activity of ATO after 24 h and 72 h of treatment, respectively. In this context, it is worth noting that we found an elevation of intracellular superoxide anion radical, but not hydrogen peroxide, and the depletion of intracellular glutathione in the ATO-treated cells. Taken together, these findings suggest that ATO inhibits the HCV RNA replication through modulation of the glutathione redox system and oxidative stress.Hepatitis C virus (HCV) is the causative agent of chronic hepatitis, which progresses to liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive single-stranded 9.6-kb RNA genome, which encodes a large polyprotein precursor of approximately 3,000 amino acid residues. This polyprotein is cleaved by a combination of the host and viral proteases into at least 10 proteins in the following order: core, envelope 1 (E1), E2, p7, nonstructural 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B (30).Alpha interferon has been used as an effective anti-HCV reagent in clinical therapy for patients with chronic hepatitis C. The current combination treatment with pegylated alpha interferon and ribavirin, a nucleoside analogue, has been shown to improve the sustained virological response rate to more than 50% (15). However, the adverse effects of the combination therapy and the limited efficacy against genotype 1b warrant the development of new anti-HCV reagents.Arsenic trioxide (ATO) (As₂O₃, arsenite) has been used as a therapeutic reagent in acute promyelocytic leukemia, which bears an oncogenic PML-retinoic acid receptor alpha fusion protein resulting from chromosomal translocation (51, 52, 68, 70). The ATO treatment induces complete remission through degradation of the aberrant PML-retinoic acid receptor α (70). The PML tumor suppressor protein is required for formation of the PML nuclear body (PML-NB), also known as nuclear dot 10 or the PML oncogenic domain, which is often disrupted by infection with DNA viruses, such as herpes simplex virus type 1, human cytomegalovirus, and Epstein-Barr virus (17). The treatment with ATO results in degradation of the PML protein and disruption of the PML-NB (70). Therefore, ATO has been become a useful probe for investigating the functions of the PML-NB, including cell growth, apoptosis, stress response, and viral infection. Indeed, ATO has been shown to increase retroviral infectivity, such as human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus infectivity, but the mechanisms of this change are not well understood (5, 6, 32, 44, 47, 50, 57). In contrast, ATO was recently reported to inhibit the replication of HCV subgenomic replicon RNA (24). However, it also remains unclear how ATO inhibits the HCV RNA replication. In this study, using genome-length HCV RNA replication systems, we investigated the molecular mechanism(s) of the anti-HCV activity of ATO, and we provide evidence that ATO inhibits HCV RNA replication through modulation of the glutathione redox system and oxidative stress.     

Proteasomal Turnover of Hepatitis C Virus Core Protein Is Regulated by Two Distinct Mechanisms: a Ubiquitin-Dependent Mechanism and a Ubiquitin-Independent but PA28γ-Dependent Mechanism

We have previously reported on the ubiquitylation and degradation of hepatitis C virus core protein. Here we demonstrate that proteasomal degradation of the core protein is mediated by two distinct mechanisms. One leads to polyubiquitylation, in which lysine residues in the N-terminal region are preferential ubiquitylation sites. The other is independent of the presence of ubiquitin. Gain- and loss-of-function analyses using lysineless mutants substantiate the hypothesis that the proteasome activator PA28γ, a binding partner of the core, is involved in the ubiquitin-independent degradation of the core protein. Our results suggest that turnover of this multifunctional viral protein can be tightly controlled via dual ubiquitin-dependent and -independent proteasomal pathways.Hepatitis C virus (HCV) core protein, whose amino acid sequence is highly conserved among different HCV strains, not only is involved in the formation of the HCV virion but also has a number of regulatory functions, including modulation of signaling pathways, cellular and viral gene expression, cell transformation, apoptosis, and lipid metabolism (reviewed in references 9 and 15). We have previously reported that the E6AP E3 ubiquitin (Ub) ligase binds to the core protein and plays an important role in polyubiquitylation and proteasomal degradation of the core protein (22). Another study from our group identified the proteasome activator PA28γ/REG-γ as an HCV core-binding partner, demonstrating degradation of the core protein via a PA28γ-dependent pathway (16, 17). In this work, we further investigated the molecular mechanisms underlying proteasomal degradation of the core protein and found that in addition to regulation by the Ub-mediated pathway, the turnover of the core protein is also regulated by PA28γ in a Ub-independent manner.Although ubiquitylation of substrates generally requires at least one Lys residue to serve as a Ub acceptor site (5), there is no consensus as to the specificity of the Lys targeted by Ub (4, 8). To determine the sites of Ub conjugation in the core protein, we used site-directed mutagenesis to replace individual Lys residues or clusters of Lys residues with Arg residues in the N-terminal 152 amino acids (aa) of the core (C152), within which is contained all seven Lys residues (Fig. ​(Fig.1A).1A). Plasmids expressing a variety of mutated core proteins were generated by PCR and inserted into the pCAGGS (18). Each core-expressing construct was transfected into human embryonic kidney 293T cells along with the pMT107 (25) encoding a Ub moiety tagged with six His residues (His₆). Transfected cells were treated with the proteasome inhibitor MG132 for 14 h to maximize the level of Ub-conjugated core intermediates by blocking the proteasome pathway and were harvested 48 h posttransfection. His₆-tagged proteins were purified from the extracts by Ni²⁺-chelation chromatography. Eluted protein and whole lysates of transfected cells before purification were analyzed by Western blotting using anticore antibodies (Fig. ​(Fig.1B).1B). Mutations replacing one or two Lys residues with Arg in the core protein did not affect the efficiency of ubiquitylation: detection of multiple Ub-conjugated core intermediates was observed in the mutant core proteins comparable to the results seen with the wild-type core protein as previously reported (23). In contrast, a substitution of four N-terminal Lys residues (C152K6-23R) caused a significant reduction in ubiquitylation (Fig. ​(Fig.1B,1B, lane 9). Multiple Ub-conjugated core intermediates were not detected in the Lys-less mutant (C152KR), in which all seven Lys residues were replaced with Arg (Fig. ​(Fig.1B,1B, lane 11). These results suggest that there is not a particular Lys residue in the core protein to act as the Ub acceptor but that more than one Lys located in its N-terminal region can serve as the preferential ubiquitylation site. In rare cases, Ub is known to be conjugated to the N terminus of proteins; however, these results indicate that this does not occur within the core protein.Open in a separate windowFIG. 1.In vivo ubiquitylation of HCV core protein. (A) The HCV core protein (N-terminal 152 aa) is represented on the top. The positions of the amino acid residues of the core protein are indicated above the bold lines. The positions of the seven Lys residues in the core are marked by vertical ticks. Substitution of Lys with Arg (R) is schematically depicted. (B) Detection of ubiquitylated forms of the core proteins. The transfected cells with core expression plasmids and pMT107 were treated with the proteasome inhibitor MG132 and harvested 48 h after transfection. His₆-tagged proteins were purified and subsequently analyzed by Western blot analysis using anticore antibody (upper panel). Core proteins conjugated to a number of His₆-Ub are denoted with asterisks. Whole lysates of transfected cells before purification were also analyzed (lower panel). Lanes 1 to 11, C152 to C152KR, as indicated for panel A. Lane 12; empty vector.To investigate how polyubiquitylation correlates with proteasome degradation of the core protein, we performed kinetic analysis of the wild-type and mutated core proteins by use of the Ub protein reference (UPR) technique, which can compensate for data scatter of sample-to-sample variations such as levels of expression (10, 24). Fusion proteins expressed from UPR-based constructs (Fig. ​(Fig.2A)2A) were cotranslationally cleaved by deubiquitylating enzymes, thereby generating equimolar quantities of the core proteins and the reference protein, dihydrofolate reductase-hemagglutinin (DHFR-HA) tag-modified Ub, in which the Lys at aa 48 was replaced by Arg to prevent its polyubiquitylation (Ub^R48). After 24 h of transfection with UPR constructs, cells were treated with cycloheximide and the amounts of core proteins and DHFR-HA-Ub^R48 at the indicated time points were determined by Western blot analysis using anticore and anti-HA antibodies. The mature form of the core protein, aa 1 to 173 (C173) (13, 20), and C152 were degraded with first-order kinetics (Fig. 2B and D). MG132 completely blocked the degradation of C173 and C152 (Fig. ​(Fig.2B),2B), and C152K6-23R and C152KR were markedly stabilized (Fig. ​(Fig.2C).2C). The half-lives of C173 and C152 were calculated to be 5 to 6 h, whereas those of C152K6-23R and C152KR were calculated to be 22 to 24 h (Fig. ​(Fig.2D),2D), confirming that the Ub plays an important role in regulating degradation of the core protein. Nevertheless, these results also suggest possible involvement of the Ub-independent pathway in the turnover of the core protein, as C152KR is more destabilized than the reference protein (Fig. ​(Fig.2C2C and ​and2D2D).Open in a separate windowFIG. 2.Kinetic analysis of degradation of HCV core proteins. (A) The fusion constructs used in the UPR technique. Open boxes indicate the DHFR sequence, which is extended at the C terminus by a sequence containing the HA epitope (hatched boxes). Ub^R48 moieties bearing the Lys-Arg substitution at aa 48 are represented by open ellipses. Bold lines indicate the regions of the core protein. The amino acid positions of the core protein are indicated above the bold lines. The arrows indicate the sites of in vivo cleavage by deubiquitylating enzymes. (B and C) Turnover of the core proteins. After a 24-h transfection with each UPR construct, cells were treated with 50 μg of cycloheximide/ml in the presence or absence of 10 μM MG132 for the different time periods indicated. Cells were lysed at the different time points indicated, followed by evaluation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using antibodies against the core protein and HA. (D) Quantitation of the data shown in panels B and C. At each time point, the ratio of band intensity of the core protein relative to the reference DHFR-HA-Ub^R48 was determined by densitometry and is plotted as a percentage of the ratio at time zero.We have shown that PA28γ specifically binds to the core protein and is involved in its degradation (16, 17). Recent studies demonstrated that PA28γ is responsible for Ub-independent degradation of the steroid receptor coactivator SRC-3 and cell cycle inhibitors such as p21 (3, 11, 12). Thus, we next investigated the possibility of PA28γ involvement in the degradation of either C152KR or C152. Since C152KR carries two amino acid substitutions in the PA28γ-binding region (aa 44 to 71) (17), we tested the influence of the mutations of C152KR on the interaction with PA28γ by use of a coimmunoprecipitation assay. When Flag-tagged PA28γ (F-PA28γ) was expressed in cells along with C152 or C152KR, F-PA28γ precipitated along with both C152 and C152KR, indicating that PA28γ interacts with both core proteins (Fig. ​(Fig.3A).3A). Figure ​Figure3B3B reveals the effect of exogenous expression of F-PA28γ on the steady-state levels of C152 and C152KR. Consistent with previous data (17), the expression level of C152 was decreased to a nearly undetectable level in the presence of PA28γ (Fig. ​(Fig.3B,3B, lanes 1 and 3). Interestingly, exogenous expression of PA28γ led to a marked reduction in the amount of C152KR expressed (Fig. ​(Fig.3B,3B, lanes 5 and 7). Treatment with MG132 increased the steady-state level of the C152KR in the presence of F-PA28γ as well as the level of C152 (Fig. ​(Fig.3B,3B, lanes 4 and 8).Open in a separate windowFIG. 3.PA28γ-dependent degradation of the core protein. (A) Interaction of the core protein with PA28γ. Cells were cotransfected with the wild-type (C152) or Lys-less (C152KR) core expression plasmid in the presence of a Flag-PA28γ (F-PA28γ) expression plasmid or an empty vector. The transfected cells were treated with MG132. After 48 h, the cell lysates were immunoprecipitated with anti-Flag antibody and visualized by Western blotting with anticore antibodies. Western blot analysis of whole cell lysates was also performed. (B) Degradation of the wild-type and Lys-less core proteins via the PA28γ-dependent pathway. Cells were transfected with the UPR construct with or without F-PA28γ. In some cases, cells were treated with 10 μM MG132 for 14 h before harvesting. Western blot analysis was performed using anticore, anti-HA, and anti-Flag antibodies. (C) After 24 h of transfection with UPR-C152KR and UPR-C191KR with or without F-PA28γ (an empty vector), cells were treated with 50 μg of cycloheximide/ml for different time periods as indicated (chase time). Western blot analysis was performed using anticore and anti-HA antibodies. The precursor core protein and the core that was processed, presumably by signal peptide peptidase, are denoted by open and closed triangles, respectively.We further investigated whether PA28γ affects the turnover of Lys-less core protein through time course experiments. C152KR was rapidly destabilized and almost completely degraded in a 3-h chase experiment using cells overexpressing F-PA28γ (Fig. ​(Fig.3C,3C, left panels). A similar result was obtained using an analogous Lys-less mutant of the full-length core protein C191KR (Fig. ​(Fig.3C,3C, right panels), thus demonstrating that the Lys-less core protein undergoes proteasomal degradation in a PA28γ-dependent manner. These results suggest that PA28γ may play a role in accelerating the turnover of the HCV core protein that is independent of ubiquitylation.Finally, we examined gain- and loss-of-function of PA28γ with respect to degradation of full-length wild-type (C191) and mutated (C191KR) core proteins in human hepatoma Huh-7 cells. As expected, exogenous expression of PA28γ or E6AP caused a decrease in the C191 steady-state levels (Fig. ​(Fig.4A).4A). In contrast, the C191KR level was decreased with expression of PA28γ but not of E6AP. We further used RNA interference to inhibit expression of PA28γ or E6AP. An increase in the abundance of C191KR was observed with PA28γ small interfering RNA (siRNA) but not with E6AP siRNA (Fig. ​(Fig.4B).4B). An increase in the C191 level caused by the activity of siRNA against PA28γ or E6AP was confirmed as well.Open in a separate windowFIG. 4.Ub-dependent and Ub-independent degradation of the full-length core protein in hepatic cells. (A) Huh-7 cells were cotransfected with plasmids for the full-length core protein (C191) or its Lys-less mutant (C191KR) in the presence of F-PA28γ or HA-tagged-E6AP expression plasmid (HA-E6AP). After 48 h, cells were lysed and Western blot analysis was performed using anticore, anti-HA, anti-Flag, or anti-GAPDH. (B) Huh-7 cells were cotransfected with core expression plasmids along with siRNA against PA28γ or E6AP or with negative control siRNA. Cells were harvested 72 h after transfection and subjected to Western blot analysis.Taking these results together, we conclude that turnover of the core protein is regulated by both Ub-dependent and Ub-independent pathways and that PA28γ is possibly involved in Ub-independent proteasomal degradation of the core protein. PA28 is known to specifically bind and activate the 20S proteasome (19). Thus, PA28γ may function by facilitating the delivery of the core protein to the proteasome in a Ub-independent manner.Accumulating evidence suggests the existence of proteasome-dependent but Ub-independent pathways for protein degradation, and several important molecules, such as p53, p73, Rb, SRC-3, and the hepatitis B virus X protein, have two distinct degradation pathways that function in a Ub-dependent and Ub-independent manner (1, 2, 6, 7, 14, 21, 27). Recently, critical roles for PA28γ in the Ub-independent pathway have been demonstrated; SRC-3 and p21 can be recognized by the 20S proteasome independently of ubiquitylation through their interaction with PA28γ (3, 11, 12). It has also been reported that phosphorylation-dependent ubiquitylation mediated by GSK3 and SCF is important for SRC-3 turnover (26). Nevertheless, the precise mechanisms underlying turnover of most of the proteasome substrates that are regulated in both Ub-dependent and Ub-independent manners are not well understood. To our knowledge, the HCV core protein is the first viral protein studied that has led to identification of key cellular factors responsible for proteasomal degradation via dual distinct mechanisms. Although the question remains whether there is a physiological significance of the Ub-dependent and Ub-independent degradation of the core protein, it is reasonable to consider that tight control over cellular levels of the core protein, which is multifunctional and essential for viral replication, maturation, and pathogenesis, may play an important role in representing the potential for its functional activity.     

Replication of a hepatitis C virus replicon clone in mouse cells

Background

The duration of treatment for HCV infection is partly indicated by the genotype of the virus. For studies of disease transmission, vaccine design, and surveillance for novel variants, subtype-level classification is also needed. This study used the Shimodaira-Hasegawa test and related statistical techniques to compare phylogenetic trees obtained from coding and non-coding regions of a whole-genome alignment for the reliability of subtyping in different regions.

Results

Different regions of the HCV genome yield inconsistent phylogenies, which can lead to erroneous conclusions about classification of a given infection. In particular, the highly conserved 5' untranslated region (UTR) yields phylogenetic trees with topologies that differ from the HCV polyprotein and complete genome phylogenies. Phylogenetic trees from the NS5B gene reliably cluster related subtypes, and yield topologies consistent with those of the whole genome and polyprotein.

Conclusion

These results extend those from previous studies and indicate that, unlike the NS5B gene, the 5' UTR contains insufficient variation to resolve HCV classifications to the level of viral subtype, and fails to distinguish genotypes reliably. Use of the 5' UTR for clinical tests to characterize HCV infection should be replaced by a subtype-informative test.     

Effects of electric cell fusion treatment among leaf protoplasts of Populus alba and alnus firma on growth, leaf morphology, and rapd pattern of eleven acclimatized plants

Summary This study reports the characterization of 11 plants regenerated from electrically fused protoplasts between Populus alba and Alnus firma. Growth characteristics of five regenerated plants (AP-1-AP-5) in terms of shoot height and leaf color showed small differences compared with those of P. alba grown, in pots, and showed no difference in shoot height and diameter compared with those grown in nursery field. There was also no difference in the RAPD pattern between the plants regenerated from interfamilial protoplast fusion and P. alba. In contrast, the lately regenerated plants (AP-6-AP-11) grown in pots showed a marked difference in leaf morphology and RAPD pattern. There was a variation in the ratio of longitudinal to transverse length of leaves among the 11 plants from interfamilial fusions compared with that of protoclones and intraspecific fused protoplasts of P. alba.