Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Control of Cytomegalovirus in Bone Marrow Transplantation Chimeras Lacking the Prevailing Antigen-Presenting Molecule in Recipient Tissues Rests Primarily on Recipient-Derived CD8 T Cells

Cytomegalovirus (CMV) infection during the transient immunodeficiency after bone marrow transplantation (BMT) develops into disease unless antiviral CD8 T cells are restored in due course. Histoincompatibility between donor and recipient is associated with increased risk. Complications may include a rejection response against the foreign major histocompatibility complex (MHC) antigens and a lack of antiviral control resulting from a misfit between donor-derived T cells and the antigenic viral peptides presented in recipient tissues. Here we have established a murine model of CMV disease after experimental BMT performed across a single MHC class I disparity. Specifically, BALB/c bone marrow cells expressing the prevailing antigen-presenting molecule L^d were transplanted into the L^d gene deletion mutant BALB/c-H-2^dm2, an experimental setting that entails a selective risk of host-versus-graft but not graft-versus-host response. The reconstituted T-cell population proved to be chimeric in that it consisted of L^d-positive donor-derived and L^d-negative recipient-derived cells. Pulmonary infiltrates did not include cytolytic T cells directed against L^d. This finding implies that the infection did not trigger a host-versus-graft response. Notably, upon adoptive transfer, donor-derived CD8 T cells preferentially protected tissues of donor genotype, whereas recipient-derived CD8 T cells protected tissues of either genotype. We infer from these data that the focus on immunodominant antigens presented by L^d within the donor cell population distracted the donor T cells from protecting recipient tissues and that protection in the chimeras was therefore primarily based on recipient T cells. As a consequence, T-cell chimerism after BMT should give a positive prognosis with respect to control of CMV.Cytomegaloviruses (CMV) are kept under tight immune control (for reviews, see references 22 and 23). As a consequence, acute CMV infection is resolved rapidly and does not result in disease unless the host is immunologically immature or immunocompromised. Bone marrow (BM) transplantation (BMT) as a therapy of hematological malignancies is associated with a transient immunodeficiency. Accordingly, during the period of immunocompromise, transmission of donor-type CMV with the transplant as well as recurrence of CMV from latency established within the organs of the transplantation recipient both entail a risk for destructive virus replication in tissues resulting in multiple-organ CMV disease (16). In BMT recipients, CMV-induced interstitial pneumonia is a frequent and endangering manifestation of CMV disease (11, 27). However, CMV infection does not inevitably result in fatal disease. It appears that CD8 T-cell reconstitution is the decisive parameter in the control of CMV after BMT. Clinical data have shown that both efficient reconstitution of CD8 T cells (41) and supplementation of antiviral CD8 T cells by preemptive cytoimmunotherapy with T-cell lines (42, 50) correlate with a reduced risk of human CMV disease, whereas combined in vivo-ex vivo T-cell depletion, intended as a prophylaxis against graft-versus-host (GvH) disease, accidentally resulted in an increased incidence of CMV infections in BMT patients (14). Aspects of these clinical problems can be approached experimentally in a murine model of BMT and concurrent infection with murine CMV (for an overview, see reference 35). Specifically, depletion of CD8 T cells, but not of CD4 T cells, performed in vivo during the phase of reconstitution after BMT abolished the development of protective antiviral immunity, with an inevitably lethal outcome (34, 47) resulting from multiple-organ pathology (34), including BM aplasia (29, 30). Likewise, an insufficient endogenous reconstitution was successfully supplemented by experimental adoptive cytoimmunotherapy with antiviral CD8 T cells. Again, CD4 T cells were not effective (36, 37, 39, 47). Altogether, clinical data on human CMV infection and experimental data from the murine model have so far been concordant and have identified CD8 T cells as the principal effectors controlling CMV infections after BMT.These findings imply that all conditions which lower the efficacy of CD8 T-cell reconstitution will increase the risk for progression of asymptomatic CMV infection to fatal CMV disease. Histoincompatibility between graft and recipient is a factor likely to negatively influence the restoration of antiviral immunity. Accordingly, even though cases of severe human CMV disease have been reported also after autologous BMT (27, 40), the incidence of CMV-related complications is generally higher after histoincompatible BMT (51). In clinical BMT, donor and recipient are usually matched in major histocompatibility complex (MHC) class II molecules, whereas differences in minor histocompatibility loci and in MHC class I loci are tolerated if unavoidable. Complications caused in the CMV-infected recipient by histoincompatibility may include (i) an impaired engraftment of transplanted cells in the recipient BM stroma, (ii) an immunological GvH response as well as a host-versus-graft (HvG) response directed against the foreign minor or major histocompatibility molecules, and (iii) a lack of antiviral T-cell control resulting from an inappropriate repertoire of viral antigenic peptides presented by infected tissue cells of the transplantation recipient.In a first attempt to dissect these possibilities, we have established a murine model of experimental BMT performed across a single MHC class I disparity, namely, the presence and absence of the L^d molecule in BALB/c mice (MHC class I molecules K^d, D^d, and L^d) and the L^d gene deletion mutant BALB/c-H-2^dm2 (44), respectively. Depending on the choice of donor and recipient for the BMT, immunogenetical GvH and HvG conditions can be studied separately (35). Work presented herein focuses on the HvG setting with BALB/c as the donor strain and the mutant as the recipient. Hence, after incomplete depletion of hematopoietic cells of the recipients, this model entails a risk for graft rejection caused by a recipient response directed against the donor MHC class I molecule L^d. In addition, presentation of viral peptides by L^d, including the immunodominant IE1 nonapeptide of murine CMV (18, 38), is confined to donor-derived hematopoietic cells and their progeny, whereas the parenchymal and stromal sites of cytocidal infection (34) lack L^d as the prevailing peptide presenter. The aim of the study was to investigate the influence of this particular MHC class I disparity on the control of murine CMV after BMT.     

Distinct Pathways for Tumor Necrosis Factor Alpha and Ceramides in Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) infection can be fatal to immunocompromised individuals. We have previously reported that gamma interferon and tumor necrosis factor alpha (TNF-α) synergistically inhibit HCMV replication in vitro. Ceramides have been described as second messengers induced by TNF-α. To investigate the mechanisms involved in the inhibition of HCMV by TNF-α, in the present study we have analyzed ceramide production by U373 MG astrocytoma cells and the effects of TNF-α versus ceramides on HCMV replication. Our results show that U373 MG cells did not produce ceramides upon incubation with TNF-α. Moreover, long-chain ceramides induced by treatment with exogenous bacterial sphingomyelinase inhibited HCMV replication in synergy with TNF-α. Surprisingly, short-chain permeant C6-ceramide increased viral replication. Our results show that the anti-HCMV activity of TNF-α is independent of ceramides. In addition, our results suggest that TNF-α and endogenous long-chain ceramides use separate pathways of cell signalling to inhibit HCMV replication, while permeant C6-ceramide appears to activate a third pathway leading to an opposite effect.Human cytomegalovirus (HCMV) infections are well controlled in the immunocompetent host. Cellular immune responses (CD4⁺ and CD8⁺ T cells and NK cells) which accompany both acute and latent infections (for a review, see reference 4) are thought to be the main components of this control. HCMV infection during immunosuppression such as in cancer, transplantations, or AIDS results in severe pathology (4). We have previously shown that tumor necrosis factor alpha (TNF-α), in synergy with gamma interferon (IFN-γ), inhibits the replication of HCMV (7). In mice, TNF-α is involved in the clearance of CMV infection (25). TNF-α is a cytokine with multiple effects which is produced by many cell types, including macrophages and CD8⁺ and CD4⁺ T lymphocytes (for a review, see reference 40), and is known to possess antiviral effects (20, 47). The molecular mechanisms involved in the signalling by TNF-α depend on the type of receptor, p55 (TNF-R1) or p75 (TNF-R2) (5), to which it binds. Some cells express only one type of TNF-α receptor; however, expression of these receptors is not always mutually exclusive (5). The cytotoxicity of TNF-α has been reported to be mediated by TNF-R1 (38), whose intracellular region carries a death domain which signals for programmed cell death (39). Signalling through TNF-R1 with specific antibodies can also protect Hep-G2 cells from vesicular stomatitis virus-mediated cytopathic effects (48). Ceramide production after TNF-α treatment has been widely reported (16, 19, 31) and has also been shown to depend on signalling through the TNF-R1 receptor (45). In these experiments concerning myeloid cells, TNF-α induced the activation of a sphingomyelinase, which cleaved sphingomyelin to release ceramide and phosphocholine. The production of ceramides can lead to cell apoptosis (11, 14, 23) or cell cycle arrest (13). Induction of apoptosis by TNF-α has been mimicked by exogenous sphingomyelinase and by synthetic, short-chain, permeant ceramides, which suggests that ceramides, as second messengers, are sufficient to induce the cytotoxic effects of TNF-α (11, 23). Acidic and neutral sphingomyelinases activated in different cell compartments may be responsible for the diverse effects of TNF-α (46), with the former being involved in signalling through NF-κB (34) and the latter being involved in signalling through a ceramide-activated protein kinase and phospholipase A₂ (46).One of the characteristics of HCMV infection is the increase in the content of intracellular DNA, reported to be of viral (3, 8, 18) or cellular (12, 37) origin. Since TNF-α has been known to display antiproliferative properties and to block cells in the G₁ phase (29), we initially tested its effects on the cell cycle of infected cells. Then, based on studies reporting that TNF-α induces ceramides in cells (16, 19, 31) and on a study showing the role of ceramide in cell cycle blockade (13), we originally postulated that ceramide was responsible for the antiviral effect of TNF-α. In the present study, we used astrocytoma cells (U373 MG) as a model for brain cells, which are important targets of HCMV in vivo (22). In contrast to fibroblasts, infected U373 MG cells release smaller quantities of virus particles even though all the cells were infected in our experiments. We believe that the U373 MG model is closer to HCMV infection in vivo. We show that ceramides are not produced by U373 MG cells upon incubation with even high concentrations of TNF-α. In addition, we demonstrate that exogenously added sphingomyelinase induces anti-HCMV effects whereas permeant C6-ceramide increases HCMV proliferation in U373 MG cells. This suggests that lipid second messengers can modulate HCMV infection and that TNF-α and ceramides use distinct signalling pathways in the control of HCMV infection. This is supported by our observation that the protein kinase JNK1 is activated exclusively by TNF-α in U373 MG cells and that TNF-α and exogenous sphingomyelinase act in synergy on HCMV infection.