Enantioselective 2-hydroxylation of RS-8359, a selective and reversible MAO-A inhibitor, by cytochrome P450 in mouse and rat liver microsomes

RS-8359, (+/-)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]-pyrimidine is a racemic compound with a selective and reversible monoamine oxidase A (MAO-A) inhibition activity. The substrate and product enantioselectivity with respect to 2-hydroxylation of RS-8359 enantiomers was studied using mouse and rat liver microsomes. In mice, the (S)-enantiomer was transformed to the cis-diol metabolite, whereas the (R)-enantiomer to the trans-diol metabolite. The Vmax/Km value for the formation of the cis-diol metabolite from the (S)-enantiomer was sevenfold greater than that for the formation of the trans-diol metabolite from the (R)-enantiomer. The greater Vmax/Km value for the (S)-enantiomer was due to the tenfold smaller Km value compared to that for the (R)-enantiomer. The results were in fair agreement with the previously reported low plasma concentrations of the (S)-enantiomer and the high recovery of the cis-diol metabolite derived from the (S)-enantiomer in urine after oral administration of RS-8359 to mice. Similarly to mice, in rats the (R)-enantiomer was transformed to the trans-diol metabolite, whereas the (S)-enantiomer yielded the cis-diol and trans-diol metabolites. The Vmax/Km value for the (R)-enantiomer was larger than that for the (S)-enantiomer in rats, indicating that the low plasma concentration of the (S)-enantiomer in rats might be caused by a metabolic reaction other than P450-dependent hydroxylation. CYP3A was shown to be responsible for the trans-diol formation from the (R)-enantiomer.     

Determination of eumelanin in human urine

Normal and malignant melanocytes produce melanins and melanin-related metabolites, most of which are retained in the cells but some are secreted into the blood and then excreted in the urine. In this study, we developed a method to measure levels of eumelanin in urine samples and evaluated its clinical significance in comparison with the melanin-related metabolites 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C) and 5-S-cysteinyldopa (5-S-CD), and with pheomelanin, measured after degradation as 4-amino-3-hydroxyphenylalanine (4-AHP). The method is based on the production of pyrrole-2,3,5-tricarboxylic acid (PTCA) on permanganate oxidation of eumelanin, followed by quantification by liquid chromatography. For 118 urine samples from 10 control subjects, mean urinary excretions of PTCA, 6H5MI2C, 5-S-CD and 4-AHP were 19, 67, 37 and 59 micromol/mol creatinine respectively. In melanoma patients (n = 45), the mean urinary excretions of PTCA, 6H5MI2C, 5-S-CD, and 4-AHP were 91, 926, 4070 and 3530 micromol/mol creatinine respectively. Median level of PTCA in melanoma patients was elevated 2.1-fold compared with control subjects. The degrees of elevation for 6H5MI2C, 5-S-CD, and 4-AHP were 1.8-, 22- and 6.2-fold respectively. Thus, although urinary PTCA is of little clinical value in following the progression of melanoma, urinary 4-AHP appears to be of considerable value in this respect.     

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.     

Effects of the number of amino acid residues in the signal segment upstream or downstream of the NS2B-3 cleavage site on production and secretion of prM/M-E virus-like particles of West Nile virus

Expression of genes for precursor M (prM) and envelope (E) proteins of West Nile virus (WNV) leads to the production of small, capsidless, and non-infectious virus-like particles (VLPs) possessing the E antigen which is responsible for viral entry and immune protection. It has been reported that processing of the secretion signal affects viral release. We examined the secretion efficiency of VLPs into the culture medium from RK13 or 293 T cells transfected with expression vectors for prM and E proteins of WNV which were constructed to comprise different lengths of signal peptides upstream of the prM-E domain. The number of amino acid residues present in the segment markedly affected the production, processing, and secretion of VLPs. Secreted VLPs possessed both the processed M protein and the glycosylated E protein. In addition, immunization with VLPs induced neutralizing antibodies in C3H/HeN mice. These results indicate that the number of amino acid residues comprising the N-terminus of the signal segment controls the efficiency of assembly, maturation, and release of VLPs in the absence of viral protease, which in turn indicates the potential of VLPs as a candidate for an effective WNV subunit vaccine.     

Association of TNFAIP3 interacting protein 1, TNIP1 with systemic lupus erythematosus in a Japanese population: a case-control association study

Introduction

TNFAIP3 interacting protein 1, TNIP1 (ABIN-1) is involved in inhibition of nuclear factor-κB (NF-κB) activation by interacting with TNF alpha-induced protein 3, A20 (TNFAIP3), an established susceptibility gene to systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Recent genome-wide association studies revealed association of TNIP1 with SLE in the Caucasian and Chinese populations. In this study, we investigated whether the association of TNIP1 with SLE was replicated in a Japanese population. In addition, association of TNIP1 with RA was also examined.

Methods

A case-control association study was conducted on the TNIP1 single nucleotide polymorphism (SNP) rs7708392 in 364 Japanese SLE patients, 553 RA patients and 513 healthy controls.

Results

Association of TNIP1 rs7708392C was replicated in Japanese SLE (allele frequency in SLE: 76.5%, control: 69.9%, P = 0.0022, odds ratio [OR] 1.40, 95% confidence interval [CI] 1.13-1.74). Notably, the risk allele frequency in the healthy controls was considerably greater in Japanese (69.9%) than in Caucasians (24.3%). A tendency of stronger association was observed in the SLE patients with renal disorder (P = 0.00065, OR 1.60 [95%CI 1.22-2.10]) than in all SLE patients (P = 0.0022, OR 1.40 [95%CI 1.13-1.74]). Significant association with RA was not observed, regardless of the carriage of human leukocyte antigen DR β1 (HLA-DRB1) shared epitope. Significant gene-gene interaction between TNIP1 and TNFAIP3 was detected neither in SLE nor RA.

Conclusions

Association of TNIP1 with SLE was confirmed in a Japanese population. TNIP1 is a shared SLE susceptibility gene in the Caucasian and Asian populations, but the genetic contribution appeared to be greater in the Japanese and Chinese populations because of the higher risk allele frequency. Taken together with the association of TNFAIP3, these observations underscore the crucial role of NF-κB regulation in the pathogenesis of SLE.     

Micelles as intermediates in the preparation of protein-liposome conjugates

A method for preparing protein-liposome conjugates based on micelles as intermediates was developed. Ovalbumin was thiolated with 2-IT and conjugated to the surface of micelles composed of a maleimide-derivatized active lipid and a micelle-forming lipid. These micelles were then incubated with liposomes, allowing the micelle components to exchange into the liposome bilayers. Using this technique we were able to demonstrate that it was possible to saturate the surface of the micelle with protein and use this property to control the level of conjugation. Titration of these protein-micelle conjugates into liposome solutions resulted in reproducible batches of protein-liposome conjugates. Chemical cross-linking could be observed in some cases; however, this was controllable through selection of reagent concentrations. The effects of parameters such as thiolation levels, micelle lipid composition, active lipid structure, micelle-forming lipid structure, and micelle/liposome/protein ratios were examined. The method represents a general approach to the preparation of well defined and reproducible protein-liposome-based drug formulations.     

Overexpression of heat shock protein 70 restores the structural stability and functional defects of temperature-sensitive mutant of large T antigen at nonpermissive temperature

The effects of heat shock protein 70 (Hsp70), a molecular chaperone, on the degradation and functional alterations of a mutant large T antigen induced by a nonpermissive temperature were examined. In this study, mouse tracheal epithelial TM02-3 cells harboring temperature-sensitive simian virus 40 large T antigen and stable TM02-3 cells overexpressing human Hsp70 and/or Hsp40 were used. Although the temperature shift from 33 degrees C (permissive temperature) to 39 degrees C (nonpermissive temperature) induced increases in the endogenous chaperones including Hsp70 and Hsp40, degradation of the T antigen, activation of the p53-p21(waf1) pathway, and an arrest of cell growth were observed in the mock cells. In contrast, these changes induced by the temperature shift were partially but significantly prevented in stable cells overexpressing human Hsp70 and/or Hsp40. A combination of Hsp70 and Hsp40 was the most effective, suggesting that Hsp40 may cooperate with Hsp70. Moreover, immunocytochemical observation indicated that human Hsp70 was expressed in the cytoplasm at 33 degrees C, but it colocalized with T antigen in the nucleus at 39 degrees C. These results suggest that overexpressed Hsp70 translocates from the cytoplasm to nucleus, and significantly restores the structural stability and functional defects of mutant large T antigen in the cells.     

Fungal ammonia fermentation, a novel metabolic mechanism that couples the dissimilatory and assimilatory pathways of both nitrate and ethanol. Role of acetyl CoA synthetase in anaerobic ATP synthesis

Fungal ammonia fermentation is a novel dissimilatory metabolic mechanism that supplies energy under anoxic conditions. The fungus Fusarium oxysporum reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP (Zhou, Z., Takaya, N., Nakamura, A., Yamaguchi, M., Takeo, K., and Shoun, H. (2002) J. Biol. Chem. 277, 1892-1896). We identified the Aspergillus nidulans genes involved in ammonia fermentation by analyzing fungal mutants. The results showed that assimilatory nitrate and nitrite reductases (the gene products of niaD and niiA) were essential for reducing nitrate and for anaerobic cell growth during ammonia fermentation. We also found that ethanol oxidation is coupled with nitrate reduction and catalyzed by alcohol dehydrogenase, coenzyme A (CoA)-acylating aldehyde dehydrogenase, and acetyl-CoA synthetase (Acs). This is similar to the mechanism suggested in F. oxysporum except A. nidulans uses Acs to produce ATP instead of the ADP-dependent acetate kinase of F. oxysporum. The production of Acs requires a functional facA gene that encodes Acs and that is involved in ethanol assimilation and other metabolic processes. We purified the gene product of facA (FacA) from the fungus to show that the fungus acetylates FacA on its lysine residue(s) specifically under conditions of ammonia fermentation to regulate its substrate affinity. Acetylated FacA had higher affinity for acetyl-CoA than for acetate, whereas non-acetylated FacA had more affinity for acetate. Thus, the acetylated variant of the FacA protein is responsible for ATP synthesis during fungal ammonia fermentation. These results showed that the fungus ferments ammonium via coupled dissimilatory and assimilatory mechanisms.     

The X-ray structure of human mannan-binding lectin-associated protein 19 (MAp19) and its interaction site with mannan-binding lectin and L-ficolin

MAp19 is an alternative splicing product of the MASP-2 gene comprising the N-terminal CUB1-epidermal growth factor (EGF) segment of MASP-2, plus four additional residues at its C-terminal end. Like full-length MASP-2, it forms Ca(2+)-dependent complexes with mannan-binding lectin (MBL) and L-ficolin. The x-ray structure of human MAp19 was solved to a resolution of 2.5 A. It shows a head to tail homodimer held together by interactions between the CUB1 module of one monomer and the EGF module of its counterpart. A Ca(2+) ion bound to each EGF module stabilizes the dimer interfaces. A second Ca(2+) ion is bound to the distal end of each CUB1 module, through six ligands contributed by Glu(52), Asp(60), Asp(105), Ser(107), Asn(108), and a water molecule. Compared with its counterpart in human C1s, the N-terminal end of the MAp19 CUB1 module contains a 7-residue extension that forms additional inter-monomer contacts. To identify the residues involved in the interaction of MAp19 with MBL and L-ficolin, point mutants were generated and their binding ability was determined using surface plasmon resonance spectroscopy. Six mutations at Tyr(59), Asp(60), Glu(83), Asp(105), Tyr(106), and Glu(109) either strongly decreased or abolished interaction with both MBL and L-ficolin. These mutations map a common binding site for these proteins located at the distal end of each CUB1 module and stabilized by the Ca(2+) ion.