Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6

We have characterized HsCdc6, a human protein homologous to the budding yeast Cdc6p that is essential for DNA replication. We show that, unlike Cdc6p, the levels of HsCdc6 protein remain constant throughout the cell cycle in human cells. However, phosphorylation of HsCdc6 is regulated during the cell cycle. HsCdc6 is an excellent substrate for Cdk2 in vitro and is phosphorylated in vivo at three sites (Ser-54, Ser-74, and Ser-106) that are phosphorylated by Cdk2 in vitro, strongly suggesting that HsCdc6 is an in vivo Cdk substrate. HsCdc6 is nuclear in G1, but translocates to the cytoplasm at the start of S phase via Crm1-dependent export. An HsCdc6A1A2A3 mutant, which mimics unphosphorylated HsCdc6, is exclusively nuclear, and its expression inhibits initiation of DNA replication. An HsCdc6E1E2E3 mutant, which mimics phosphorylated HsCdc6, is exclusively cytoplasmic and is not associated with the chromatin/nuclear matrix fraction. Based on these results, we propose that phosphorylation of HsCdc6 by Cdks regulates DNA replication of at least two steps: first, by promoting initiation of DNA replication and, second, through nuclear exclusion preventing DNA rereplication.

Human cytomegalovirus inhibits antigen presentation by a sequential multistep process.

The human cytomegalovirus (HCMV) genomic unique short (US) region encodes a family of homologous genes essential for the inhibition of major histocompatibility complex (MHC) class I-mediated antigen presentation during viral infection. Here we show that US3, the only immediate early (IE) gene within the US region, encodes an endoplasmic reticulum-resident glycoprotein that prevents intracellular transport of MHC class I molecules. In contrast to the rapid degradation of newly synthesized MHC class I heavy chains mediated by the early gene product US11, we found that US3 retains stable MHC class I heterodimers in the endoplasmic reticulum that are loaded with peptides while retained in the ER. Consistent with the expression pattern of US3 and US11, MHC class I molecules are retained but not degraded during the IE period of infection. Our data identify the first nonregulatory role of an IE protein of HCMV and suggest that HCMV uses different T-cell escape strategies at different times during the infectious cycle.

Cell-cell communication regulates the effects of protein aspartate phosphatases on the phosphorelay controlling development in Bacillus subtilis.

Rap phosphatases are a recently discovered family of protein aspartate phosphatases that dephosphorylate the Spo0F--P intermediate of the phosphorelay, thus preventing sporulation of Bacillus subtilis. They are regulators induced by physiological processes that are antithetical to sporulation. The RapA phosphatase is induced by the ComP-ComA two-component signal transduction system responsible for initiating competence. RapA phosphatase activity was found to be controlled by a small protein, PhrA, encoded on the same transcript as RapA. PhrA resembles secreted proteins and the evidence suggests that it is cleaved by signal peptidase I and a 19-residue C-terminal domain is secreted from the cell. The sporulation deficiency caused by the uncontrolled RapA activity of a phrA mutant can be complemented by synthetic peptides comprising the last six or more of the C-terminal residues of PhrA. Whether the peptide controls RapA activity directly or by regulating its synthesis remains to be determined. Complementation of the phrA mutant can also be obtained in mixed cultures with a wild-type strain, suggesting the peptide may serve as a means of communication between cells. Importation of the secreted peptide required the oligopeptide transport system. The sporulation deficiency of oligopeptide transport mutants can be suppressed by mutating the rapA and rapB genes or by introduction of a spo0F mutation Y13S that renders the protein insensitive to Rap phosphatases. The data indicate that the sporulation deficiency of oligopeptide transport mutants is due to their inability to import the peptides controlling Rap phosphatases.

Stability and convergence of general multistep and multivalue methods with variable step size.

Thesis--University of Illinois.

The effect of variable mesh size on the stability of multistep methods /

Bibliography: leaf 33.

Stability and convergence of variable order multistep methods,

Includes bibliographical references.

Equivalent forms of multistep formulas /

"UIUL-ENG 78 1730."

Blended linear multistep methods /

Bibliography: p. 64-65.

Runge-Kutta starters for multistep methods /

"Supported in part by the U.S. Department of Energy under contract US ENERGY/EY-76-S-02-2383."

A search for better linear multistep methods for stiff problems /

Thesis--Illinois.

Determination of the Pu autoionizing level angular momenta by a multistep excitation with linearly polarized laser light

The modified polarization spectroscopy method was applied for determination of angular momenta of autoionizing states of Pu in multistep resonance ionization processes. In comparison with the known one, our method does not require circular polarization at all, only linear polarizations are needed. This simplicity was reached using a three-dimensional excitation geometry. Angular momenta of nine new autoionizing ²⁴²Pu states were determined. The method suggested could be applied for efficiency improvement in multistep RIMS applications as well as for the odd-even isotope separation for elements with a J = 0 ground state (Pu, Yb, Sm etc.).

EpiJen:a server for multistep T cell epitope prediction

Background - The main processing pathway for MHC class I ligands involves degradation of proteins by the proteasome, followed by transport of products by the transporter associated with antigen processing (TAP) to the endoplasmic reticulum (ER), where peptides are bound by MHC class I molecules, and then presented on the cell surface by MHCs. The whole process is modeled here using an integrated approach, which we call EpiJen. EpiJen is based on quantitative matrices, derived by the additive method, and applied successively to select epitopes. EpiJen is available free online. Results - To identify epitopes, a source protein is passed through four steps: proteasome cleavage, TAP transport, MHC binding and epitope selection. At each stage, different proportions of non-epitopes are eliminated. The final set of peptides represents no more than 5% of the whole protein sequence and will contain 85% of the true epitopes, as indicated by external validation. Compared to other integrated methods (NetCTL, WAPP and SMM), EpiJen performs best, predicting 61 of the 99 HIV epitopes used in this study. Conclusion - EpiJen is a reliable multi-step algorithm for T cell epitope prediction, which belongs to the next generation of in silico T cell epitope identification methods. These methods aim to reduce subsequent experimental work by improving the success rate of epitope prediction.

Insights into the multistep transformation of MGUS to myeloma using microarray expression analysis.

To define specific pathways important in the multistep transformation process of normal plasma cells (PCs) to monoclonal gammopathy of uncertain significance (MGUS) and multiple myeloma (MM), we have applied microarray analysis to PCs from 5 healthy donors (N), 7 patients with MGUS, and 24 patients with newly diagnosed MM. Unsupervised hierarchical clustering using 125 genes with a large variation across all samples defined 2 groups: N and MGUS/MM. Supervised analysis identified 263 genes differentially expressed between N and MGUS and 380 genes differentially expressed between N and MM, 197 of which were also differentially regulated between N and MGUS. Only 74 genes were differentially expressed between MGUS and MM samples, indicating that the differences between MGUS and MM are smaller than those between N and MM or N and MGUS. Differentially expressed genes included oncogenes/tumor-suppressor genes (LAF4, RB1, and disabled homolog 2), cell-signaling genes (RAS family members, B-cell signaling and NF-kappaB genes), DNA-binding and transcription-factor genes (XBP1, zinc finger proteins, forkhead box, and ring finger proteins), and developmental genes (WNT and SHH pathways). Understanding the molecular pathogenesis of MM by gene expression profiling has demonstrated sequential genetic changes from N to malignant PCs and highlighted important pathways involved in the transformation of MGUS to MM.

Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases

The cysteine protease cathepsin C (CatC) activates granule-associated proinflammatory serine proteases in hematopoietic precursor cells. Its early inhibition in the bone marrow is regarded as a new therapeutic strategy for treating proteolysis-driven chronic inflammatory diseases, but its complete inhibition is elusive in vivo Controlling the activity of CatC may be achieved by directly inhibiting its activity with a specific inhibitor or/and by preventing its maturation. We have investigated immunochemically and kinetically the occurrence of CatC and its proform in human hematopoietic precursor cells and in differentiated mature immune cells in lung secretions. The maturation of proCatC obeys a multistep mechanism that can be entirely managed by CatS in neutrophilic precursor cells. CatS inhibition by a cell-permeable inhibitor abrogated the release of the heavy and light chains from proCatC and blocked ∼80% of CatC activity. Under these conditions the activity of neutrophil serine proteases, however, was not abolished in precursor cell cultures. In patients with neutrophilic lung inflammation, mature CatC is found in large amounts in sputa. It is secreted by activated neutrophils as confirmed through lipopolysaccharide administration in a nonhuman primate model. CatS inhibitors currently in clinical trials are expected to decrease the activity of neutrophilic CatC without affecting those of elastase-like serine proteases.

Abstracting and correlating heterogeneous events to detect complex scenarios

The research presented in this thesis addresses inherent problems in signaturebased intrusion detection systems (IDSs) operating in heterogeneous environments. The research proposes a solution to address the difficulties associated with multistep attack scenario specification and detection for such environments. The research has focused on two distinct problems: the representation of events derived from heterogeneous sources and multi-step attack specification and detection. The first part of the research investigates the application of an event abstraction model to event logs collected from a heterogeneous environment. The event abstraction model comprises a hierarchy of events derived from different log sources such as system audit data, application logs, captured network traffic, and intrusion detection system alerts. Unlike existing event abstraction models where low-level information may be discarded during the abstraction process, the event abstraction model presented in this work preserves all low-level information as well as providing high-level information in the form of abstract events. The event abstraction model presented in this work was designed independently of any particular IDS and thus may be used by any IDS, intrusion forensic tools, or monitoring tools. The second part of the research investigates the use of unification for multi-step attack scenario specification and detection. Multi-step attack scenarios are hard to specify and detect as they often involve the correlation of events from multiple sources which may be affected by time uncertainty. The unification algorithm provides a simple and straightforward scenario matching mechanism by using variable instantiation where variables represent events as defined in the event abstraction model. The third part of the research looks into the solution to address time uncertainty. Clock synchronisation is crucial for detecting multi-step attack scenarios which involve logs from multiple hosts. Issues involving time uncertainty have been largely neglected by intrusion detection research. The system presented in this research introduces two techniques for addressing time uncertainty issues: clock skew compensation and clock drift modelling using linear regression. An off-line IDS prototype for detecting multi-step attacks has been implemented. The prototype comprises two modules: implementation of the abstract event system architecture (AESA) and of the scenario detection module. The scenario detection module implements our signature language developed based on the Python programming language syntax and the unification-based scenario detection engine. The prototype has been evaluated using a publicly available dataset of real attack traffic and event logs and a synthetic dataset. The distinct features of the public dataset are the fact that it contains multi-step attacks which involve multiple hosts with clock skew and clock drift. These features allow us to demonstrate the application and the advantages of the contributions of this research. All instances of multi-step attacks in the dataset have been correctly identified even though there exists a significant clock skew and drift in the dataset. Future work identified by this research would be to develop a refined unification algorithm suitable for processing streams of events to enable an on-line detection. In terms of time uncertainty, identified future work would be to develop mechanisms which allows automatic clock skew and clock drift identification and correction. The immediate application of the research presented in this thesis is the framework of an off-line IDS which processes events from heterogeneous sources using abstraction and which can detect multi-step attack scenarios which may involve time uncertainty.