Zinc-induced G2/M blockage is p53 and p21 dependent in normal human bronchial epithelial cells

The involvement of p53 and p21 signal pathway in the G2/M cell cycle progression of zinc-supplemented normal human bronchial epithelial (NHBE) cells was examined using the small interferring RNA (siRNA) approach. Cells were cultured for one passage in a different concentration of zinc: <0.4 microM (ZD) as zinc deficient; 4 microM as normal zinc level (ZN) in culture medium; 16 microM (ZA) as normal human plasma zinc level; and 32 microM (ZS) as the high end of plasma zinc attainable by oral supplementation. Nuclear p21 protein and mRNA levels as well as promoter activity in ZS cells, but not in ZD cells, were markedly elevated to almost twofold compared with ZN control cells. G2/M blockage in ZS cells was coupled with the observation of elevated p21 gene expression. In ZS cells, the abrogation of p21 protein induction by the transfection of p21 siRNA was shown to alleviate the G2/M blockage, demonstrating the positive linkage of p21 elevation and G2/M blockage. Abolishment of the increase in p53 protein in ZS cells with transfection of p53 siRNA normalized the elevated p21 protein to a similar level as in ZN control cells, which demonstrated that the p21 induction is p53 dependent. Furthermore, the normalization of p53 protein by siRNA treatment in ZS cells alleviated cell growth depression and G2/M blockage, which demonstrated that p53 was involved in the high zinc status-induced G2/M blockage and growth depression. Thus high zinc status in NHBE cells upregulates p53 expression which in turn elevates p21 that eventually induces G2/M blockage.     

Synthesis, cytostatic and anti-HCV activity of 6-(N-substituted aminomethyl)-, 6-(O-substituted hydroxymethyl)- and 6-(S-substituted sulfanylmethyl)purine nucleosides

An efficient and facile synthesis of a large series of diverse 6-(N-substituted aminomethyl)-, 6-(O-substituted hydroxymethyl)- and 6-(S-substituted sulfanylmethyl)purine nucleosides (55 examples of both ribo- and 2'-deoxyribonucleosides), aimed at identifying novel homologues of natural nucleosides, was developed. The key transformation involved nucleophilic substitutions of Tol-protected 6-(mesyloxymethyl)purine nucleosides by primary or secondary amines, alcoholates or thiolates. While the 2'-deoxyribonucleosides were inactive, the ribonucleosides exerted considerable cytostatic effects and some anti-HCV activity with low selectivity.     

Functional characterization of key structural genes in rice flavonoid biosynthesis

Rice is a model system for monocot but the molecular features of rice flavonoid biosynthesis have not been extensively characterized. Rice structural gene homologs encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) were identified by homology searches. Unique differential expression of OsF3H, OsDFR, and OsANS1 controlled by the Pl ^w locus, which contains the R/B-type regulatory genes OSB1 and OSB2, was demonstrated during light-induced anthocyanin accumulation in T65-Plw seedlings. Previously, F3H genes were often considered as early genes co-regulated with CHS and CHI genes in other plants. In selected non-pigmented rice lines, OSB2 is not expressed following illumination while their expressed OSB1sequences all contain the same nucleotide change leading to the T⁶⁴ M substitution within the conserved N-terminal interacting domain. Furthermore, the biochemical roles of the expressed rice structural genes (OsCHS1, OsCHI, OsF3H, and OsF3′H) were established in planta for the first time by complementation in the appropriate Arabidopsis transparent testa mutants. Using yeast two-hybrid analysis, OsCHS1 was demonstrated to interact physically with OsF3H, OsF3′H, OsDFR, and OsANS1, suggesting the existence of a macromolecular complex for anthocyanin biosynthesis in rice. Finally, flavones were identified as the major flavonoid class in the non-pigmented T65 seedlings in which the single-copy OsF3H gene was not expressed. Competition between flavone and anthocyanin pathways was evidenced by the significant reduction of tricin accumulation in the T65-Plw seedlings. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hepatitis B surface antigen levels and sequences of natural hepatitis B virus variants influence the assembly and secretion of hepatitis d virus

Various domains of hepatitis B surface antigen (HBsAg) are essential for the assembly and secretion of hepatitis D virus (HDV). This study investigated the influences of the levels and sequences of HBsAg of naturally occurring HBV variants on the assembly and secretion of HDV. Six hepatitis B virus (HBV)-producing plasmids (three genotype B and three genotype C) and six HBsAg expression plasmids that expressed various HBsAg levels were constructed from the sera of HDV-infected patients. These plasmids were cotransfected with six expression plasmids of HDV of genotype 1, 2, or 4 into the Huh-7 hepatoma cell line. Serum HBsAg and HBV DNA levels were correlated with HDV RNA levels and outcomes of chronic hepatitis D (CHD) patients. The secretion of genotype 1, 2, or 4 HDV generally correlated with HBsAg levels but not with HBV genotypes or HBV DNA levels. Swapping and residue mutagenesis experiments of HBsAg-coding sequences revealed that the residue Pro-62 in the cytosolic domain-I affects the assembly and secretion of genotype 2 and 4 HDV and not those of genotype 1. The pre-S2 N-terminal deletion HBV mutant adversely affects secretion of the three HDV genotypes. In patients, serum HDV RNA levels correlated with HBsAg levels but not with HBV DNA levels. Viremia of HDV or HBV correlated with poor outcomes. In conclusion, the assembly and secretion of HDV were influenced by the amounts and sequences of HBsAg. For an effective treatment of CHD, reduction of HBsAg production in addition to the suppression of HBV and HDV replication might be crucial.     

Prognostic correlations with the microbiome of breast cancer subtypes

Alterations to the natural microbiome are linked to different diseases, and the presence or absence of specific microbes is directly related to disease outcomes. We performed a comprehensive analysis with unique cohorts of the four subtypes of breast cancer (BC) characterized by their microbial signatures, using a pan-pathogen microarray strategy. The signature (includes viruses, bacteria, fungi, and parasites) of each tumor subtype was correlated with clinical data to identify microbes with prognostic potential. The subtypes of BC had specific viromes and microbiomes, with ER+ and TN tumors showing the most and least diverse microbiome, respectively. The specific microbial signatures allowed discrimination between different BC subtypes. Furthermore, we demonstrated correlations between the presence and absence of specific microbes in BC subtypes with the clinical outcomes. This study provides a comprehensive map of the oncobiome of BC subtypes, with insights into disease prognosis that can be critical for precision therapeutic intervention strategies.Subject terms: Cancer, Prognostic markers     

The Helicase-Like Domain of Plant Potexvirus Replicase Participates in Formation of RNA 5′ Cap Structure by Exhibiting RNA 5′-Triphosphatase Activity

Open reading frame 1 (ORF1) of potexviruses encodes a viral replicase comprising three functional domains: a capping enzyme at the N terminus, a putative helicase in the middle, and a polymerase at the C terminus. To verify the enzymatic activities associated with the putative helicase domain, the corresponding cDNA fragment from bamboo mosaic virus (BaMV) was cloned into vector pET32 and the protein was expressed in Escherichia coli and purified by metal affinity chromatography. An activity assay confirmed that the putative helicase domain has nucleoside triphosphatase activity. We found that it also possesses an RNA 5'-triphosphatase activity that specifically removes the gamma phosphate from the 5' end of RNA. Both enzymatic activities were abolished by the mutation of the nucleoside triphosphate-binding motif (GKS), suggesting that they have a common catalytic site. A typical m(7)GpppG cap structure was formed at the 5' end of the RNA substrate when the substrate was treated sequentially with the putative helicase domain and the N-terminal capping enzyme, indicating that the putative helicase domain is truly involved in the process of cap formation by exhibiting its RNA 5'-triphosphatase activity.     

Effects of carboxyl-terminal truncations on the activity and solubility of human monoamine oxidase B

Monoamine oxidase is an outer mitochondrial membrane protein that catalyzes the deamination of a number of neurotransmitters and dietary amines. To determine the roles of the carboxyl-terminal amino acids on the activity and solubility of human monoamine oxidase (MAO B), 10 sequential mutants were made with stop codons at amino acid positions 511, 504, 498, 492, 486, 481, 476, 467, 417, and 397, respectively. All truncated mutants were expressed in Sf21 insect cells using baculovirus, and the enzyme kinetic parameters were determined. Truncations at amino acid positions 511, 504, and 498 slightly decreased MAO B catalytic activity and had no significant changes on deprenyl inhibition. Further deletions up to amino acid 417 decreased the specific activity 10--100-fold without significant changes of the K(m) for phenylethylamine or dopamine or the IC(50) for deprenyl and clorgyline. The truncation mutant C397, which lacks covalently attached FAD, was inactive. Progressive carboxyl-terminal truncations up to position 481 were correlated with increased solubility of MAO B mutants. 47% of the activity of the truncated C481 was found in the 105,000 x g supernatant in the absence of detergent. However, further truncated mutants, C476, C467, and C417, remained associated with the membrane fraction. In contrast to crude homogenate, the water-soluble C481 mutant was rapidly inactivated at 4 degrees C and 37 degrees C, which indicates that the membrane environment is required for the stability of MAO B. Expression of the green fluorescent protein-MAO B C481 fusion protein revealed that this mutant was located in the cytoplasm, whereas its counterpart in MAO A, truncated mutant C490, was located on the mitochondria. These results suggest that the carboxyl-terminal amino acid residues 417--520 of MAO B are not directly involved in the active site but are required for maintaining the appropriate conformation and interaction with the outer mitochondrial membrane. The different solubilities of the various carboxyl-terminal truncation mutants indicate that the interaction of MAO B with mitochondrial membrane is not simply anchoring through the carboxyl-terminal hydrophobic tail. Further, our results suggest that the carboxyl-terminal of MAO A and B plays different roles in mitochondrial attachment.     

Reactive oxygen species modulate angiotensin II-induced beta-myosin heavy chain gene expression via Ras/Raf/extracellular signal-regulated kinase pathway in neonatal rat cardiomyocytes

Angiotensin II (Ang II) causes cardiomyocytes hypertrophy. Cardiac beta-myosin heavy chain (beta-MyHC) gene expression can be altered by Ang II. The molecular mechanisms are not completely known. Reactive oxygen species (ROS) are involved in signal transduction pathways of Ang II. However, the role of ROS on Ang II-induced beta-MyHC gene expression remains unclear. Here we found that Ang II increased beta-MyHC promoter activity and it was blocked by Ang II type 1 receptor antagonist losartan. Ang II dose-dependently increased the intracellular ROS. Cardiomyocytes cotransfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf301), or a catalytically inactive mutant of extracellular signal regulated kinase (mERK2) inhibited Ang II-induced beta-MyHC promoter activity, indicating Ras/Raf/ERK pathway was involved. Antioxidants such as catalase or N-acetyl-cysteine decreased Ang II-activated ERK phosphorylation and inhibited Ang II-induced beta-MyHC promoter activity. These data indicate that Ang II increases beta-MyHC gene expression in part via the generation of ROS.     

xCt cystine transporter expression in HEK293 cells: pharmacology and localization

xCT, the core subunit of the system x(c)(-) high affinity cystine transporter, belongs to a superfamily of glycoprotein-associated amino acid transporters. Although xCT was shown to promote cystine transport in Xenopus oocytes, little work has been done with mammalian cells (Sato, H., Tamba, M., Ishii, T., and Bannai, S. J. Biol. Chem. 274, 11455-11458, 1999). Therefore, we have constructed mammalian expression vectors for murine xCT and its accessory subunit 4F2hc and transfected them into HEK293 cells. We report that this transporter binds cystine with high affinity (81 microM) and displays a pharmacological profile expected for system x(c)(-). Surprisingly, xCT transport activity in HEK293 cells is not dependent on the co-expression of the exogenous 4F2hc. Expression of GFP-tagged xCT indicated a highly clustered plasma membrane and intracellular distribution suggesting the presence of subcellular domains associated with combating oxidative stress. Our results indicate that HEK293 cells transfected with the xCT subunit would be a useful vehicle for future structure-function and pharmacology experiments involving system x(c)(-).     

Preparation of novel aza-1,7-annulated indoles and their conversion to potent indolocarbazole kinase inhibitors

The synthesis of novel aza-1,7-annulated indoles was achieved and these were converted to indolocarbazoles that proved to be potent kinase inhibitors. These compounds were also evaluated in a human colon carcinoma cell line and proved to be good antiproliferative agents.