Amino acid positions 69-132 of UGT1A9 are involved in the C-glucuronidation of phenylbutazone

Phenylbutazone (PB) is known to be biotransformed to its O- and C-glucuronide. Recently, we reported that PB C-glucuronide formation is catalyzed by UGT1A9. Interestingly, despite UGT1A8 sharing high amino acid sequence identity with UGT1A9, UGT1A8 had no PB C-glucuronidating activity. In the present study, we constructed eight UGT1A9/UGT1A8 chimeras and evaluated which region is important for PB C-glucuronide formation. All of the chimeras and UGT1A8 and UGT1A9 had 7-hydroxy-(4-trifluoromethyl)coumarin (HFC) O-glucuronidating activity. The K_m values for HFC glucuronidation of UGT1A8, UGT1A9 and their chimeras were divided into two types, UGT1A8 type (high K_m) and UGT1A9 type (low K_m), and these types were determined according to whether their amino acids at positions 69-132 were those of UGT1A8 or UGT1A9. Likewise, PB O-glucuronidating activity was also detected by all of the chimeras, and their K_m values were divided into two types. On the contrary, PB C-glucuronidating activity was detected by UGT1A9_(1-132)/1A8_(133-286), UGT1A9_(1-212)/1A8_(213-286), UGT1A8_(1-68)/1A9_(69-286), and UGT1A8_(1-68)/1A9_(69-132)/1A8_(133-286) chimeras. The region 1A9_(69-132) was common among chimeras having PB C-glucuronidating activity. Of interest is that UGT1A9_(1-68)/1A8_(69-132)/1A9_(133-286) had lost PB C-glucuronidation activity, but retained activities of PB and HFC O-glucuronidation. These results strongly suggested that amino acid positions 69-132 of UGT1A9 are responsible for chemoselectivity for PB and affinity to substrates such as PB and HFC.     

The methylation status of FBXW7 beta-form correlates with histological subtype in human thymoma

FBXW7 is reported to be a tumor suppressor gene, and the functional inactivation of FBXW7 has been reported in various human tumors. In this study, we investigated the FBXW7 gene in human thymoma; although no mutations were evident, a significantly high frequency of methylation in the FBXW7 β-form promoter was observed in types B1 or higher (P = 0.014). We propose a novel mechanism for the pathogenesis of thymoma by FBXW7 β-form and hypothesize that expressional suppression plays an important role in the malignant potential of thymoma.     

Effect of reversed heme orientation on circular dichroism and cooperative oxygen binding of human adult hemoglobin

We found that recombinant human adult hemoglobin (rHb A) expressed in Escherichia coli showed heterogeneity of components with the intensity of a positive CD band at 260 nm and that it could be resolved into three components (SP-1, SP-2, and SP-3) by SP-Sepharose column chromatography. 1H NMR revealed that SP-1 is identical with native Hb A, while SP-2 and SP-3 largely contain the reversed heme isomer in both the alpha and beta subunits, with contents of approximately 50 and >80% in SP-2 and SP-3, respectively. Rotation of the heme 180 degrees about the 5,15-meso axis (reversed heme) causes an interexchange of the methyl groups at positions 2 and 7 with the vinyl groups at positions 8 and 3, respectively. To examine the effect of the modification of the heme-protein contact on the structure and function of Hb A, we compared the 1H NMR, CD, and oxygen binding properties of the three components with those of native Hb A. Native Hb A exhibits a distinct positive CD band in both the near-UV and Soret regions, but rHb A with reversed heme exhibits a very weak positive CD band at 260 nm and a prominent negative CD band in the Soret region. Cooperativity, as measured by Hill's n value, decreased from 3.18 (SP-1) to 2.94 (SP-2) to 2.63 (SP-3) with an increase in the reversed heme orientation. The effect of an allosteric effector, inositol hexaphosphate (IHP), on the oxygen binding properties was also reduced in rHb A with reversed heme. These results indicate that changes in the heme-globin contact exert a discernible influence on CD spectra and cooperative oxygen binding.     

Structures and functions of the extrinsic proteins of photosystem II from different species

This minireview presents a summary of information available on the variety and binding properties of extrinsic proteins that form the oxygen-evolving complex of photosystem II (PSII) of cyanobacteria, red alga, diatom, green alga, euglena, and higher plants. In addition, the structure and function of extrinsic PsbO, PsbV, and PsbU proteins are summarized based on the crystal structure of thermophilic cyanobacterial PSII together with biochemical and genetic studies from various organisms.     

4-O-acetylation and 3-O-acetylation of trichothecenes by trichothecene 15-O-acetyltransferase encoded by Fusarium Tri3

In the biosynthesis of Fusarium trichothecenes, the C-3 hydroxyl group of isotrichodermol must be acetylated by TRI101 for subsequent pathway genes to function. Despite the importance of this 3-O-acetylation step in biosynthesis, Tri101 is both physically and evolutionarily unrelated to other Tri genes in the trichothecene gene cluster. To gain insight into the evolutionary history of the cluster, we purified recombinant TRI3 (rTRI3), one of the two cluster gene-encoded trichothecene O-acetyltransferases, and examined to determine whether this 15-O-acetyltransferase can add an acetyl to the C-3 hydroxyl group of isotrichodermol. When a high concentration of rTRI3 was used in the assay (final concentration, 50 microM), we observed 3-O-acetylation activity against isotrichodermol that was more than 10(5) times less efficient than the known 15-O-acetylation activity against 15-deacetylcalonectrin. The rTRI3 protein also exhibited 4-O-acetylation activity when nivalenol was used as a substrate; in addition to 15-acetylnivalenol, di-acetylated derivatives, 4,15-diacetylnivalenol, and, to a lesser extent, 3,15-diacetylnivalenol, were also detected at high enzyme concentrations. The significance of the trace trichothecene 3-O-acetyltransferase activity detected in rTRI3 is discussed in relation to the evolution of the trichothecene gene cluster.     

Loss of Borealin/DasraB leads to defective cell proliferation, p53 accumulation and early embryonic lethality

Borealin/DasraB is a member of the chromosomal passenger protein complex (CPC) required for proper segregation of chromosomes during mitosis. In Drosophila melanogaster, inactivation of Borealin/DasraB results in polyploidy, delayed mitosis and abnormal tissue development, indicating its critical role for cell proliferation. However, the in vivo role of mammalian Borealin/DasraB remains unclear. Here, we analyzed the expression of Borealin/DasraB and found that borealin is widely expressed in embryonic tissues and later restricted to adult tissues which relies on rapid cell proliferation. To determine the role of borealin during mouse development, we generated borealin-null mice through targeted disruption. While heterozygous mice developed normally, disruption of both borealin alleles resulted in early embryonic lethality by 5.5 dpc (days postcoitus) due to mitotic defects and apoptosis in blastocyst cells that showed microtubule disorganization and no CPC enrichment. At 5.5 dpc, borealin-null embryos exhibited excessive apoptosis and elevated expression of p53. However, loss of p53 did not abrogate or delay embryonic lethality, revealing that Borealin/DasraB inactivation triggered impaired mitosis and apoptosis though p53-independent mechanisms. Our data show that Borealin/DasraB is essential for cell proliferation during early embryonic development, and its early embryonic lethality cannot be rescued by the loss of p53.     

Selectable tolerance to herbicides by mutated acetolactate synthase genes integrated into the chloroplast genome of tobacco

Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicide-resistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation. To this end, we developed a novel vector system for chloroplast transformation with acetolactate synthase (ALS). ALS catalyzes the first step in the biosynthesis of the branched amino acids, and its enzymatic activity is inhibited by certain classes of herbicides. We generated a series of Arabidopsis (Arabidopsis thaliana) mutated ALS (mALS) genes and introduced constructs with mALS and the aminoglycoside 3'-adenyltransferase gene (aadA) into the tobacco (Nicotiana tabacum) chloroplast genome by particle bombardment. Transplastomic plants were selected using their resistance to spectinomycin. The effects of herbicides on transplastomic mALS activity were examined by a colorimetric assay using the leaves of transplastomic plants. We found that transplastomic G121A, A122V, and P197S plants were specifically tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides, respectively. Transplastomic plants possessing mALSs were able to grow in the presence of various herbicides, thus affirming the relationship between mALSs and the associated resistance to herbicides. Our results show that mALS genes integrated into the chloroplast genome are useful sustainable markers that function to exclude plants other than those that are GM while maintaining transplastomic crops. This investigation suggests that the resistance management of weeds in the field amid growing GM crops is possible using (1) a series of mALSs that confer specific resistance to herbicides and (2) a strategy that employs herbicide rotation.     

MgLig4, a homolog of Neurospora crassa Mus-53 (DNA ligase IV), is involved in, but not essential for, non-homologous end-joining events in Magnaporthe grisea

In many eukaryotic organisms, the non-homologous end-joining (NHEJ) system is a major pathway for the repair of DNA double-strand breaks (DSBs). DNA ligase IV is a component of the NHEJ system and is strictly required for the NHEJ system in Saccharomyces cerevisiae and in Neurospora crassa. To investigate the functions of DNA Ligase IV in Magnaporthe grisea, we generated deletion mutants of MGLIG4, which encodes a homolog of N. crassa DNA Ligase IV. Mutants (mglig4) showed no defects in asexual or sexual growth, and were fully pathogenic. Compared to the wild-type, mglig4 exhibited weak sensitivity to a DNA-damaging agent, camptothecin. In addition, the frequency of targeted-gene replacement was relatively elevated in mglig4, although this varied in a gene-dependent manner. Surprisingly, non-homologous integration of DNA was frequently observed in mglig4 transformants. Our results demonstrate that MgLig4 is involved in, but not essential for, the NHEJ system in M. grisea.     

Role of HIV-1 Vpu protein for virus spread and pathogenesis

Vpu is an accessory viral protein almost unique to HIV-1 among primate immunodeficiency viruses, and has two major functions: degradation of the CD4 molecule in endoplasmic reticulum and enhancement of virion release from cells. Recent identification of a novel host restriction factor, tetherin, as a Vpu-antagonist suggests that Vpu contributes to virus spread by facilitating progeny virion production. This review focuses on the two distinct functions of Vpu and summarizes current knowledge on its virological role in the HIV-1 life cycle.