NRP/B, a Novel Nuclear Matrix Protein, Associates With p110RB and Is Involved in Neuronal Differentiation

The nuclear matrix is defined as the insoluble framework of the nucleus and has been implicated in the regulation of gene expression, the cell cycle, and nuclear structural integrity via linkage to intermediate filaments of the cytoskeleton. We have discovered a novel nuclear matrix protein, NRP/B (nuclear restricted protein/brain), which contains two major structural elements: a BTB domain–like structure in the predicted NH₂ terminus, and a “kelch motif” in the predicted COOH-terminal domain. NRP/B mRNA (5.5 kb) is predominantly expressed in human fetal and adult brain with minor expression in kidney and pancreas. During mouse embryogenesis, NRP/B mRNA expression is upregulated in the nervous system. The NRP/B protein is expressed in rat primary hippocampal neurons, but not in primary astrocytes. NRP/B expression was upregulated during the differentiation of murine Neuro 2A and human SH-SY5Y neuroblastoma cells. Overexpression of NRP/B in these cells augmented neuronal process formation. Treatment with antisense NRP/B oligodeoxynucleotides inhibited the neurite development of rat primary hippocampal neurons as well as the neuronal process formation during neuronal differentiation of PC-12 cells. Since the hypophosphorylated form of retinoblastoma protein (p110^RB) is found to be associated with the nuclear matrix and overexpression of p110^RB induces neuronal differentiation, we investigated whether NRP/B is associated with p110^RB. Both in vivo and in vitro experiments demonstrate that NRP/B can be phosphorylated and can bind to the functionally active hypophosphorylated form of the p110^RB during neuronal differentiation of SH-SY5Y neuroblastoma cells induced by retinoic acid. Our studies indicate that NRP/B is a novel nuclear matrix protein, specifically expressed in primary neurons, that interacts with p110^RB and participates in the regulation of neuronal process formation.     

Serum apolipoproteins C-I and C-III are reduced in stomach cancer patients: results from MALDI-based peptidome and immuno-based clinical assays

Finding new peptide biomarkers for stomach cancer in human sera that can be implemented into a clinically practicable prediction method for monitoring of stomach cancer. We studied the serum peptidome from two different biorepositories. We first employed a C8-reverse phase liquid chromatography approach for sample purification, followed by mass-spectrometry analysis. These were applied onto serum samples from cancer-free controls and stomach cancer patients at various clinical stages. We then created a bioinformatics analysis pipeline and identified peptide signature discriminating stomach adenocarcinoma patients from cancer-free controls. Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) results from 103 samples revealed 9 signature peptides; with prediction accuracy of 89% in the training set and 88% in the validation set. Three of the discriminating peptides discovered were fragments of Apolipoproteins C-I and C-III (apoC-I and C-III); we further quantified their serum levels, as well as CA19-9 and CRP, employing quantitative commercial-clinical assays in 142 samples. ApoC-I and apoC-III quantitative results correlated with the MS results. We then employed apoB-100-normalized apoC-I and apoC-III, CA19-9 and CRP levels to generate rules set for stomach cancer prediction. For training, we used sera from one repository, and for validation, we used sera from the second repository. Prediction accuracies of 88.4% and 74.4% were obtained in the training and validation sets, respectively. Serum levels of apoC-I and apoC-III combined with other clinical parameters can serve as a basis for the formulation of a diagnostic score for stomach cancer patients.     

Dynamics of a multistage circadian system

Tissues throughout the body exhibit circadian rhythms, forming a multioscillatory system whose disruption results in jet lag and other health problems in travelers and rotational shift workers. The authors' simulations of the dynamics of a multistage circadian system (based on experimental results for nocturnal rodents) reveal the flexibility and stability inherent in a multistage system, as well as potential pitfalls. The modeling predicts that jet lag tends to be most severe following an eastward change of 5 to 8 time zones due to prolonged desynchrony of the system. This desynchrony is partly due to differing reentrainment rates among components, but a much greater source of desynchrony is the antidromic reentrainment of some but not all components (reentrainment by partition), triggered by the overshoot of the master pacemaker's phase in response to these advances. Based on the multistage system dynamics, the authors design a simple protocol that results in a more orderly transition that avoids antidromic reentrainment in all components, thereby reducing the reentrainment time from nearly 2 weeks to just a few days for the most difficult shifts. The authors compare the predicted behavior of self-sustaining versus damped oscillatory components in the system as well as the effect of weak versus strong coupling from the master pacemaker to the peripheral components.     

Flexible Kernel Memory

This paper introduces a new model of associative memory, capable of both binary and continuous-valued inputs. Based on kernel theory, the memory model is on one hand a generalization of Radial Basis Function networks and, on the other, is in feature space, analogous to a Hopfield network. Attractors can be added, deleted, and updated on-line simply, without harming existing memories, and the number of attractors is independent of input dimension. Input vectors do not have to adhere to a fixed or bounded dimensionality; they can increase and decrease it without relearning previous memories. A memory consolidation process enables the network to generalize concepts and form clusters of input data, which outperforms many unsupervised clustering techniques; this process is demonstrated on handwritten digits from MNIST. Another process, reminiscent of memory reconsolidation is introduced, in which existing memories are refreshed and tuned with new inputs; this process is demonstrated on series of morphed faces.     

Antibody-mediated targeting of human single-chain class I MHC with covalently linked peptides induces efficient killing of tumor cells by tumor or viral-specific cytotoxic T lymphocytes

Soluble forms of human MHC class I HLA-A2 were produced in which the peptide binding groove was uniformly occupied by a single tumor or viral-derived peptides attached via a covalent flexible peptide linker to the N terminus of a single-chain -2-microglobulin-HLA-A2 heavy chain fusion protein. A tetravalent version of this molecule with various peptides was found to be functional. It could stimulate T cells specifically as well as bind them with high avidity. The covalently linked single chain peptide-HLA-A2 construct was next fused at its C-terminal end to a scFv antibody fragment derived from the variable domains of an anti-IL-2R subunit-specific humanized antibody, anti-Tac. The scFv–MHC fusion was thus encoded by a single gene and produced in E. coli as a single polypeptide chain. Binding studies revealed its ability to decorate Ag-positive human tumor cells with covalent peptide single-chain HLA-A2 (scHLA-A2) molecules in a manner that was entirely dependent upon the specificity of the targeting Antibody fragment. Most importantly, the covalent scHLA-A2 molecule, when bound to the target tumor cells, could induce efficient and specific HLA-A2-restricted, peptide-specific CTL-mediated lysis. These results demonstrate the ability to generate soluble, stable, and functional single-chain HLA-A2 molecules with covalently linked peptides, which when fused to targeting antibodies, potentiate CTL killing. This new approach may open the way for the development of new immunotherapeutic strategies based on antibody targeting of natural cognate MHC ligands and CTL-based cytotoxic mechanisms.Kfir Oved and Avital Lev contributed equally to this work     

Blm10 protein promotes proteasomal substrate turnover by an active gating mechanism

For optimal proteolytic function, the central core of the proteasome (core particle (CP) or 20S) has to associate with activators. We investigated the impact of the yeast activator Blm10 on proteasomal peptide and protein degradation. We found enhanced degradation of peptide substrates in the presence of Blm10 and demonstrated that Blm10 has the capacity to accelerate proteasomal turnover of the unstructured protein tau-441 in vitro. Mechanistically, proteasome activation requires the opening of a closed gate, which allows passage of unfolded proteins into the catalytic chamber. Our data indicate that gate opening by Blm10 is achieved via engagement of its C-terminal segment with the CP. Crucial for this activity is a conserved C-terminal YYX motif, with the penultimate tyrosine playing a preeminent role. Thus, Blm10 utilizes a gate opening strategy analogous to the proteasomal ATPases HbYX-dependent mechanism. Because gating incompetent Blm10 C-terminal point mutants confers a loss of function phenotype, we propose that the cellular function of Blm10 is based on CP association and activation to promote the degradation of proteasome substrates.     

ATM-mediated phosphorylation of polynucleotide kinase/phosphatase is required for effective DNA double-strand break repair

The cellular response to double-strand breaks (DSBs) in DNA is a complex signalling network, mobilized by the nuclear protein kinase ataxia-telangiectasia mutated (ATM), which phosphorylates many factors in the various branches of this network. A main question is how ATM regulates DSB repair. Here, we identify the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) as an ATM target. PNKP phosphorylates 5'-OH and dephosphorylates 3'-phosphate DNA ends that are formed at DSB termini caused by DNA-damaging agents, thereby regenerating legitimate ends for further processing. We establish that the ATM phosphorylation targets on human PNKP-Ser 114 and Ser 126-are crucial for cellular survival following DSB induction and for effective DSB repair, being essential for damage-induced enhancement of the activity of PNKP and its proper accumulation at the sites of DNA damage. These findings show a direct functional link between ATM and the DSB-repair machinery.     

Persistence of apoptosis and inflammatory responses in the heart and bone marrow of mice following whole-body exposure to 28Silicon (28Si) ions

It has been well established that the bone marrow (BM) is a radiosensitive tissue, but the radiosensitivity of the heart is poorly understood. In this study, we investigated the comparative effects of ²⁸Silicon (²⁸Si) ions (one type of heavy ion found in space) on tissue from the heart and the BM of exposed mice. We gave adult male CBA/CaJ mice a whole-body exposure to a total dose of 0, 0.1, 0.25, or 0.5 Gy of 300 MeV/nucleon (n) ²⁸Si ions, using a fractionated schedule (two exposures, 15 days apart that totaled each selected dose). The heart and BM were collected from 5 mice per treatment group at various times up to 6 months post-irradiation. In each mouse, we obtained tissue lysates from the heart and from the total population of BM cells for measuring the levels of cleaved poly (ADP-ribose) polymerase (cleaved PARP, a marker of apoptotic cell death) and the levels of activated nuclear factor-kappa B (NF-κB) and selected NF-κB-regulated cytokines known to be involved in inflammatory responses. Our data showed that, up to 6 months post-irradiation, the levels of apoptotic cell death and inflammatory responses in tissues from the heart and BM collected from exposed mice were statistically higher than those in sham controls. Hence, these findings are suggestive of chronic apoptotic cell death and inflammation in both tissues after exposure to ²⁸Si ions. In summary, our data are indicative of a possible association between exposure to ²⁸Si ions during space flight and long-term health risk.