Glycosidic Bond Conformation Preference Plays a Pivotal Role in Catalysis of RNA Pseudouridylation: A Combined Simulation and Structural Study

The most abundant chemical modification on RNA is isomerization of uridine (or pseudouridylation) catalyzed by pseudouridine synthases. The catalytic mechanism of this essential process remains largely speculative, partly due to lack of knowledge of the pre-reactive state that is important to the identification of reactive chemical moieties. In the present study, we showed, using orthogonal space random-walk free-energy simulation, that the pre-reactive states of uridine and its reactive derivative 5-fluorouridine, bound to a ribonucleoprotein particle pseudouridine synthase, strongly prefer the syn glycosidic bond conformation, while that of the nonreactive 5-bromouridine-containing substrate is largely populated in the anti conformation state. A high-resolution crystal structure of the 5-bromouridine-containing substrate bound to the ribonucleoprotein particle pseudouridine synthase and enzyme activity assay confirmed the anti nonreactive conformation and provided the molecular basis for its confinement. The observed preference for the syn pre-reactive state by the enzyme-bound uridine may help to distinguish among currently proposed mechanisms.     

Damage and recovery of the bone growth mechanism in young rats following 5-fluorouracil acute chemotherapy

Chemotherapy-induced bone growth arrest and osteoporosis are significant problems in paediatric cancer patients, and yet how chemotherapy affects bone growth remains unclear. This study characterised development and resolution of damage caused by acute chemotherapy with antimetabolite 5-fluorouracil (5-FU) in young rats in the growth plate cartilage and metaphyseal bone, two important tissues responsible for bone lengthening. In metaphysis, 5-FU induced apoptosis among osteoblasts and preosteoblasts on days 1-2. In growth plate, chondrocyte apoptosis appeared on days 5-10. Interestingly, Bax was induced prior to apoptosis and Bcl-2 was upregulated during recovery. 5-FU also suppressed cell proliferation on days 1-2. While proliferation returned to normal by day 3 in metaphysis, it recovered partially on day 3, overshot on days 5-7 and normalised by day 10 in growth plate. Histologically, growth plate heights decreased by days 4-5 and returned normal by day 10. In metaphysis, primary spongiosa height was also reduced, mirroring changes in growth plate thickness. In metaphyseal secondary spongiosa, a reduced bone volume was observed on days 7-10 as there were fewer but more separated trabeculae. Starting from day 4, expression of some cartilage/bone matrix proteins and growth factors (TGF-beta1 and IGF-I) was increased. By day 14, cellular activity, histological structure and gene expression had returned normal in both tissues. Therefore, 5-FU chemotherapy affects bone growth directly by inducing apoptosis and inhibiting proliferation at growth plate cartilage and metaphyseal bone; after the acute damage, bone growth mechanism can recover, which is associated with upregulated expression of matrix proteins and growth factors.     

Plasmodium vivax: microsatellite analysis of multiple-clone infections

We used mixtures of genomic DNA from two genetically distinct isolates from Brazil, 42M and 312M, to investigate how accurately 12-locus microsatellite typing describes the overall genetic diversity and characterizes multilocus haplotypes in multiple-clone Plasmodium vivax infections. We found varying PCR amplification efficiencies of microsatellite alleles; for example, from the same 1:1 mixture of 42M and 312M DNA we amplified predominantly 312M-type alleles at 10 loci and 42M-type alleles at 2 loci. All microsatellite alleles were accurately scored in 1:0.5 and 1:0.25 312M:42M DNA mixtures, even when minor peak heights did not meet previously suggested criteria for minor allele detection in multiple-clone infections. Relative proportions of major and minor alleles were unaffected by multiple displacement amplification of template DNA prior to PCR-based microsatellite typing. Although microsatellite typing may detect minor alleles in clone mixtures, amplification biases may lead to inaccurate assignment of predominant haplotypes in multiple-clone P. vivax infections.     

8-Oxoguanine DNA glycosylase-1-mediated DNA repair is associated with Rho GTPase activation and α-smooth muscle actin polymerization

Reactive oxygen species (ROS) are activators of cell signaling and modify cellular molecules, including DNA. 8-Oxo-7,8-dihydroguanine (8-oxoG) is one of the prominent lesions in oxidatively damaged DNA, whose accumulation is causally linked to various diseases and aging processes, whereas its etiological relevance is unclear. 8-OxoG is repaired by the 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated DNA base excision repair (BER) pathway. OGG1 binds free 8-oxoG and this complex functions as an activator of Ras family GTPases. Here we examined whether OGG1-initiated BER is associated with the activation of Rho GTPase and mediates changes in the cytoskeleton. To test this possibility, we induced OGG1-initiated BER in cultured cells and mouse lungs and used molecular approaches such as active Rho pull-down assays, siRNA ablation of gene expression, immune blotting, and microscopic imaging. We found that OGG1 physically interacts with Rho GTPase and, in the presence of 8-oxoG base, increases Rho–GTP levels in cultured cells and lungs, which mediates α-smooth muscle actin (α-SMA) polymerization into stress fibers and increases the level of α-SMA in insoluble cellular/tissue fractions. These changes were absent in cells lacking OGG1. These unexpected data and those showing that 8-oxoG repair is a lifetime process suggest that, via Rho GTPase, OGG1 could be involved in the cytoskeletal changes and organ remodeling observed in various chronic diseases.     

Globotriaosylceramide is correlated with oxidative stress and inflammation in Fabry patients treated with enzyme replacement therapy

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism due to deficient activity of α-galactosidase A that leads to accumulation of the enzyme substrates, mainly globotriaosylceramide (Gb3), in body fluids and lysosomes of many cell types. Some pathophysiology hypotheses are intimately linked to reactive species production and inflammation, but until this moment there is no in vivo study about it. Hence, the aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokines and Gb3 levels in Fabry patients under treatment with enzyme replacement therapy (ERT) and finally to establish a possible relation between them. We analyzed urine and blood samples of patients under ERT (n = 14) and healthy age-matched controls (n = 14). Patients presented decreased levels of antioxidant defenses, assessed by reduced glutathione (GSH), glutathione peroxidase (GPx) activity and increased superoxide dismutase/catalase (SOD/CAT) ratio in erythrocytes. Concerning to the damage to biomolecules (lipids and proteins), we found that plasma levels of malondialdehyde (MDA) and protein carbonyl groups and di-tyrosine (di-Tyr) in urine were increased in patients. The pro-inflammatory cytokines IL-6 and TNF-α were also increased in patients. Urinary Gb3 levels were positively correlated with the plasma levels of IL-6, carbonyl groups and MDA. IL-6 levels were directly correlated with di-Tyr and inversely correlated with GPx activity. This data suggest that pro-inflammatory and pro-oxidant states occur, are correlated and seem to be induced by Gb3 in Fabry patients.     

Protective effect of an acidic glycoprotein obtained from culture of Chlorella vulgaris against myelosuppression by 5-fluorouracil

 An acidic glycoprotein prepared from a culture of Chlorella vulgaris (CVS) was examined for its protective effect on 5-fluorouracil(5FU)-induced myelosuppression and indigenous infection in mice. Subcutaneous administration of CVS greatly reduced the mortality of non-tumor-bearing mice given a high dose of 5FU, and could increase the LD₅₀ value of 5FU for these mice. After 5FU treatment, indigenous infection developed probably as a result of the impairment of the host defense system. CVS reduced the incidence of indigenous infections and this effect was attributable to the acceleration of recovery from 5FU-induced myelosuppression. Early recovery of hematopoietic stem cells, or cells responding to interleukin-3 or granulocyte/macrophage-colony-stimulating factor, was especially observed in the bone marrow of CVS-treated mice on days 4 – 9 after the injection of 5FU. When tumor-bearing mice were given CVS during treatment with 5FU, CVS prolonged the survival of mice without affecting the antitumor activity of 5FU. In addition, CVS was itself shown to exert an antitumor effect. These results suggested that CVS may be beneficial for the alleviation of side-effects in cancer chemotherapy without affecting the antitumor activity of the chemotherapeutic agent. Received: 15 August 1995 / Accepted: 23 April 1996     

Bystander killing of breast cancer MCF-7 cells by MDA-MB-231 cells exposed to 5-fluorouracil is mediated via Fas

The major drawback with cancer therapy is the development of resistant cells within tumors due to their heterogeneous nature and due to inadequate drug delivery during chemotherapy. Therefore, the propagation of injury ("bystander effect" (BE)) from directly damaged cells to other cells may have great implications in cancer chemotherapy. The general advantage of the bystander cell killing phenomenon is the large therapeutic index that can be achieved. Experiments suggest that this phenomenon is detected in radiation therapy as well as in gene therapy in conjunction with chemotherapy. In the present study, we developed an original in vitro model dedicated to the exploration of bystander cytotoxicity induced during breast carcinoma chemotherapy. In brief, we investigated this perpetuation of injury on untreated bystander MCF-7 breast cancer cells which were coplated with 5-fluorouracil (5-FU)-treated MDA-MB-231 breast cancer cells. To achieve this goal, a specific in vitro coculture model which involved mixing of aggressive MDA-MB-231 breast cancer cells with enhanced green fluorescent protein (EGFP) expressing stable clone of non-metastatic MCF-7 breast cancer cells (MCF-EGFP), was used. A bystander killing effect was observed in MCF-EGFP cells cocultured with MDA-MB-231 cells pretreated with 5-FU. The striking decrease in MCF-EGFP cells, as detected by assaying for total GFP intensity, is mediated by activation of Fas/FasL system. The implication of Fas in MCF-EGFP cell death was confirmed by using antagonistic anti-FasL antibody that reverses bystander cell death by blocking FasL on MDA-MB-231 cells. In addition, inhibition of CD95/Fas receptor on the cell surface of MCF-EGFP cells by treatment with Pifithrin-alpha, a p53 specific transactivation inhibitor, partially abrogated the sensitivity of bystander MCF-EGFP cells. Our data, therefore, demonstrates that the Fas/FasL system could be considered as a new determinant for chemotherapy-induced bystander cell death in breast cancers.     

Keyhole Limpet Hemocyanin: 9-Å CryoEM Structure and Molecular Model of the KLH1 Didecamer Reveal the Interfaces and Intricate Topology of the 160 Functional Units

Hemocyanins are blue copper-containing respiratory proteins in the hemolymph of many arthropods and molluscs. Molluscan hemocyanins are decamers, didecamers, or multidecamers of a 340- to 400-kDa polypeptide subunit containing seven or eight globular functional units (FUs; FU-a to FU-h), each with an oxygen-binding site. The decamers are short 35-nm hollow cylinders, with their lumen narrowed by a collar complex. Our recently published 9-Å cryo-electron microscopy/crystal structure hybrid model of a 3.4-MDa cephalopod hemocyanin decamer [Nautilus pompilius hemocyanin (NpH)] revealed the pathway of the seven-FU subunit (340 kDa), 15 types of inter-FU interface, and an asymmetric collar consisting of five “arcs” (FU-g pairs). We now present a comparable hybrid model of an 8-MDa gastropod hemocyanin didecamer assembled from two asymmetric decamers [isoform keyhole limpet hemocyanin (KLH) 1 of the established immunogen KLH]. Compared to NpH, the KLH1 subunit (400 kDa) is C-terminally elongated by FU-h, which is further extended by a unique tail domain. We have found that the wall-and-arc structure of the KLH1 decamer is very similar to that of NpH. We have traced the subunit pathway and how it continues from KLH1-g to KLH1-h to form an annulus of five “slabs” (FU-h pairs) at one cylinder edge. The 15 types of inter-FU interface detected in NpH are also present in KLH1. Moreover, we have identified one arc/slab interface, two slab/slab interfaces, five slab/wall interfaces, and four decamer/decamer interfaces. The 27 interfaces are described on the basis of two subunit conformers, yielding an asymmetric homodimer. Six protrusions from the cryo-electron microscopy structure per subunit are associated with putative attachment sites for N-linked glycans, indicating a total of 120 sugar trees in KLH1. Also, putative binding sites for divalent cations have been detected. In conclusion, the present 9-Å data on KLH1 confirm and substantially broaden our recent analysis of the smaller cephalopod hemocyanin and essentially solve the gastropod hemocyanin structure.