Theoretical treatment of helix–coil transition of complexes DNA with two different ligands having different binding parameters

The melting transition of DNA–ligand complexes, allowing for two binding mechanisms to different DNA conformations is treated theoretically. The obtained results express the behavior of the experimentally measurable quantities, degree of denaturation, and concentrations of bound ligands on the temperature. The range of binding parameters is obtained, where denaturation curves become multiphasic. The possible application to the nanocomposites crystallization is discussed.     

Characterization and Drug Resistance Patterns of Ewing's Sarcoma Family Tumor Cell Lines

Despite intensive treatment with chemotherapy, radiotherapy and surgery, over 70% of patients with metastatic Ewing''s Sarcoma Family of Tumors (EFT) will die of their disease. We hypothesize that properly characterized laboratory models reflecting the drug resistance of clinical tumors will facilitate the application of new therapeutic agents to EFT. To determine resistance patterns, we studied newly established EFT cell lines derived from different points in therapy: two established at diagnosis (CHLA-9, CHLA-32), two after chemotherapy and progressive disease (CHLA-10, CHLA-25), and two at relapse after myeloablative therapy and autologous bone marrow transplantation (post-ABMT) (CHLA-258, COG-E-352). The new lines were compared to widely studied EFT lines TC-71, TC-32, SK-N-MC, and A-673. These lines were extensively characterized with regard to identity (short tandem repeat (STR) analysis), p53, p16/14 status, and EWS/ETS breakpoint and target gene expression profile. The DIMSCAN cytotoxicity assay was used to assess in vitro drug sensitivity to standard chemotherapy agents. No association was found between drug resistance and the expression of EWS/ETS regulated genes in the EFT cell lines. No consistent association was observed between drug sensitivity and p53 functionality or between drug sensitivity and p16/14 functionality across the cell lines. Exposure to chemotherapy prior to cell line initiation correlated with drug resistance of EFT cell lines in 5/8 tested agents at clinically achievable concentrations (CAC) or the lower tested concentration (LTC): (cyclophosphamide (as 4-HC) and doxorubicin at CAC, etoposide, irinotecan (as SN-38) and melphalan at LTC; P<0.1 for one agent, and P<0.05 for four agents. This panel of well-characterized drug-sensitive and drug-resistant cell lines will facilitate in vitro preclinical testing of new agents for EFT.     

Sample Limited Characterization of a Novel Disulfide-Rich Venom Peptide Toxin from Terebrid Marine Snail Terebra variegata

Disulfide-rich peptide toxins found in the secretions of venomous organisms such as snakes, spiders, scorpions, leeches, and marine snails are highly efficient and effective tools for novel therapeutic drug development. Venom peptide toxins have been used extensively to characterize ion channels in the nervous system and platelet aggregation in haemostatic systems. A significant hurdle in characterizing disulfide-rich peptide toxins from venomous animals is obtaining significant quantities needed for sequence and structural analyses. Presented here is a strategy for the structural characterization of venom peptide toxins from sample limited (4 ng) specimens via direct mass spectrometry sequencing, chemical synthesis and NMR structure elucidation. Using this integrated approach, venom peptide Tv1 from Terebra variegata was discovered. Tv1 displays a unique fold not witnessed in prior snail neuropeptides. The novel structural features found for Tv1 suggest that the terebrid pool of peptide toxins may target different neuronal agents with varying specificities compared to previously characterized snail neuropeptides.     

An extremely acidic copper-containing protein from chromaffin granules

The contribution to magnetic shieldings of the anisotropy of the atomic susceptibility tensors is calculated and added to the ring current effect for nuclei located in a plane at z = 0.35 nm above the surface of the conjugated rings of the aromatic amino acids of proteins and of porphyrin. The variation of this contribution with the value of z is compared to the variation of the ring current effect.     

Mass spectral characterization of organophosphate-labeled,tyrosine-containing peptides: Characteristic mass fragments and a new binding motif for organophosphates

We have identified organophosphorus agent (OP)-tyrosine adducts on 12 different proteins labeled with six different OP. Labeling was achieved by treating pure proteins with up to 40-fold molar excess of OP at pH 8–8.6. OP-treated proteins were digested with trypsin, and peptides were separated by HPLC. Fragmentation patterns for 100 OP-peptides labeled on tyrosine were determined in the mass spectrometer. The goals of the present work were (1) to determine the common features of the OP-reactive tyrosines, and (2) to describe non-sequence MSMS fragments characteristic of OP-tyrosine peptides. Characteristic ions at 272 and 244 amu for tyrosine-OP immonium ions were nearly always present in the MSMS spectrum of peptides labeled on tyrosine by chlorpyrifos-oxon. Characteristic fragments also appeared from the parent ions that had been labeled with diisopropylfluorophosphate (216 amu), sarin (214 amu), soman (214 amu) or FP-biotin (227, 312, 329, 691 and 708 amu). In contrast to OP-reactive serines, which lie in the consensus sequence GXSXG, the OP-reactive tyrosines have no consensus sequence. Their common feature is the presence of nearby positively charged residues that activate the phenolic hydroxyl group. The significance of these findings is the recognition of a new binding motif for OP to proteins that have no active site serine. Modified peptides are difficult to find when the OP bears no radiolabel and no tag. The characteristic MSMS fragment ions are valuable because they are identifiers for OP-tyrosine, independent of the peptide.     

Characterization of Conformation-Sensitive Antibodies to ADAMTS13, the von Willebrand Cleavage Protease

Background

The zinc metalloprotease ADAMTS13 is a multidomain protein that cleaves von Willebrand Factor (VWF) and is implicated in Thrombotic Thrombocytopenic Purpura (TTP) pathogenesis. Understanding the mechanism of this protein is an important goal. Conformation sensitive antibodies have been used to monitor protein conformation and to decipher the molecular mechanism of proteins as well as to distinguish functional and non-functional mutants.

Methodology/Principal Findings

We have characterized several antibodies against ADAMTS13, both monoclonal and polyclonal. We have used flow cytometry to estimate the binding of these antibodies to ADAMTS13 and demonstrate that antibodies raised against the TSP and disintegrin domains detect conformation changes in the ADAMTS13. Thus for example, increased binding of these antibodies was detected in the presence of the substrate (VWF), mainly at 37°C and not at 4°C. These antibodies could also detect differences between wild-type ADAMTS13 and the catalytically deficient mutant (P475S). The flow cytometry approach also allows us to estimate the reactivity of the antibody as well as its apparent affinity.

Conclusions/Significance

Our results suggest that these antibodies may serve as useful reagents to distinguish functional and non-functional ADAMTS13 and analyze conformational transitions to understand the catalytic mechanism.     

Murine embryonic stem cells as a model for human embryonic stem-cell research

Over the last several decades, murine embryonic stem cells (mESCs) have been used as a model for human embryonic stem cell (hESC) research. The relevance of this approach has not yet been proven. There is a great deal of evidence that is indicative of substantial differences between these two cell types. An analysis of the literature shows that the differences concern ESC proliferation, self-renewal, and differentiation. Consequently, mESC may be considered as a model object for hESC studies only for some aspects of their biology. The alternative model objects, such as primate ESC, are also discussed briefly in this review.     

“Regular” visual receptive fields of neurons of the cat lateral geniculate body

The spatial organization of receptive fields (RF) of neurons was studied in the lateral geniculate body (LGB) of cats with pretrigeminal transection of the brainstem (without general anesthesia). Using systematic point testing of the entire RF area and adjacent regions, the RF configuration and distribution of the response types for a stable flickering stimulus throughout the RF area were determined. Only 40% (64 units of 160 studied) LGB neurons had simple RF configuration. Such RF of ellipsoid or round shape were called regular receptive fields, RRF. Most RRF (51, or about 80%) demonstrated spatially homogeneous organization with similar-type (on, off, oron-off) responses to stimulation of the entire RF area. The RRF of 13 neurons, i.e., about 20%, included subfields with qualitatively different responses to application of a stable flickering light spot. The position of subfields was asymmetrical in 8 neurons (13%), while a nearly concentric RF arrangement, with the center surrounded by an antagonistic area, was found only in 5 units (7%) with RRF. Nearly all neurons with heterogeneous RRF demonstrated directional selectivity to moving stimuli.Neirofiziologiya/Neurophysiology, Vol. 27, No. 5/6, pp. 413–424, September–December, 1995.